# Patent

## Title
Aphid resistant soybean plants

## Abstract

The present invention relates to compositions and methods for providing and using markers of aphid resistant germplasm, particularly for identifying aphid resistance germplasm in soybean plants. Specifically, the inventions related to providing aphid resistant germplasm identified by markers associated with decreased damage from aphid feeding, as well as enhanced tolerance to aphid infestation of soybean plants. More particularly, the invention relates to compositions and methods for using genetic markers in breeding methods for producing plants with increased resistance to aphid damage and tolerance, while retaining and acquiring desired agronomic traits. Additionally, markers were developed for fine mapping of aphid resistance genes including allele specific contributions to plants for breeding soybean plants with increased aphid resistance. Furthermore, the invention relates to plants produced by these breeding programs, including plants with having aphid resistant genes between the molecular markers identifying aphid resistant genes, for use in commercial soybean production.

## Background
This Continuation-In-Part application claims priority to a U.S. Non-Provisional Divisional patent application Ser. No. 12/324,331, filed Nov. 26, 2008 now U.S. Pat. No. 8,237,022 which claims priority to U.S. Non-Provisional patent application Ser. No. 11/436,262, filed May 18, 2006 now U.S. Pat. No. 7,781,648, which claims priority to U.S. Provisional Patent Application Ser. No. 60/682,583, filed 18 May 2005, all of which is herein incorporated by reference in their entirety.


GOVERNMENT LICENSE RIGHTS
This invention was made with government support under United Soybean Board grant RC064560, awarded by the United States Department of Agriculture—Agricultural Research Service (USDA-ARS). The government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates to compositions and methods for providing and using markers of aphid resistant germplasm, particularly for identifying aphid resistance germplasm in soybean plants. Specifically, the invention relates to providing aphid resistant germplasm identified by markers associated with decreased damage from aphid feeding, as well as enhanced tolerance to aphid infestation of soybean plants. More particularly, the invention relates to compositions and methods for using genetic markers in breeding methods for producing plants with increased resistance to aphid damage and tolerance, while retaining and acquiring desired agronomic traits. Additionally, markers were developed for fine mapping of aphid resistance genes including allele specific contributions to plants for breeding soybean plants with increased aphid resistance. Furthermore, the invention relates to plants produced by these breeding programs, including plants with having aphid resistant genes between the molecular markers identifying aphid resistant genes, for use in commercial soybean production.
BACKGROUND OF THE INVENTION
Soybean is the leading oilseed crop produced and consumed worldwide (Wilcox World distribution and trade of soybean, 2004, Soybeans: Improvement, production, and Uses, 3rd ed., Agron. Monogr. 16, ASA, CSSA, and SSSA, Madison, Wis. p: 1-14; Hymowitz, 2004, Speciation and Cytogenetics, p. 97-136. In H. R. Boerma and J. E. Specht (ed) Soybeans: Improvement, production, and Uses. 3rd ed. Agron. Monogr. 16. ASA, CSSA, and SSSA). In the past half century, the USA has been the world's leading producer. In 2003, the USA produced 35% (65.8 million metric tons (MT)) of the world's total soybean (FAOSTAT, 2004, Production Crops). Soybean has many insect pests limiting its production in other parts of the world, including the soybean aphid.
Around 2000 A.D., the soybean aphid (Aphis glycines Matsumura) a native to eastern Asia, became a major sucking pest of soybean [Glycine max (L.) Merr.] in North America. Since then, this insect pest has rapidly spread to the major soybean production areas in the USA and Canada (Plant Health Initiative, 2004, Soybean Aphids Research Update “Aphids discovered in Wisconsin” from the North Central Soybean Research Program (NCSRP) published online by the Plant Health Initiative Available at planthealth.info/soyaphid.htm (verified Oct. 5, 2004). Aphid outbreaks have been severe in the northern part of the Midwestern USA and in Ontario, particularly in years 2001 and 2003.
Several factors affect soybean aphid outbreaks, including environmental conditions, over-wintering success, cultural practices, natural enemies, and the synchronization of soybean and aphid development (Wu et al., 1999, How Peasants Can Increase Wealth [Nongmin zhifu zhiyou] 6:20). The soybean aphid develops large colonies on soybean plants. Plant damage occurs when large numbers of aphids remove significant amounts of water and nutrients as they feed (suck) on leaves and stems, causing leaves to wilt, curl, yellow, and even drop off. Other symptoms of direct feeding damage include plant stunting, poor pod fill, reduced pod and seed counts, smaller seed size, and nutrient deficiencies resulting in overall yield and quality reduction (DiFonzo and Hines, 2002, Michigan State University Extension Bulletin E-2746). Significant yield loss (8-25%) occurs when the aphid heavily infests the soybean plants during the early reproductive stage (DiFonzo and Hines, 2002, Michigan State University Extension Bulletin E-2746). Honeydew, a sticky substance excreted by soybean aphids onto the leaves, leads to the development of sooty mold, which affects photosynthesis and results in yield loss (Baute, 2004, (Soybean Aphid Fact sheet and Soybean Webpage sponsored by the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), published online). During the feeding process, soybean aphids are capable of transmitting viruses including alfalfa mosaic virus, soybean mosaic virus, and bean yellow mosaic virus. These viruses commonly occur together and form a disease complex that leads to plant stunting, leaf distortion and leaf and stem mottling, reduced pod numbers, and seed discoloration (Glogoza, 2002, North Dakota State University Extension Bulletin E-1232).
Aphids are particularly difficult to control because of their rapid reproduction rates and ability to disperse over wide areas. Populations build rapidly (females give live birth, young mature in 3-7 days, doubling time 2-5 days under favorable conditions). Winged forms appear and disperse to other fields under high insect densities and when infested plants are stressed. Since aphids are relatively weak fliers, long-distance dispersal is often at the mercy of prevailing winds.
Aerial applicators frequently report having to stop to clean their windshields from flying into clouds of these aphids above heavily infested fields. In 2001, the influx of winged soybean aphids into the open dome of the Toronto Blue Jays even caused an early end to a Toronto Blue Jays game. It's these flights that lead to rapid, progressive colonization of soybean, almost like a wave moving across the countryside. Under favorable conditions for aphid infestations, the settling of winged aphids into uninfested fields has been described as “aphid rain.” (Ostlie, Soybean Aphid Pages published online by Just for Growers, MN (University of Minnesota) Soybean Production, published online by the University of Minnesota, the University of Minnesota Extension Service, and the MN Soybean Research and Promotion Council Jul. 6, 2004).
Insecticides are the primary available method of controlling soybean aphids in the USA. Although the use of insecticides can be a quick and easy way to control aphids, the ideal time to spray is not well defined. Insecticides also have many adverse effects such as killing beneficial insects, environmental contamination, and increased production costs (Sun et al., 1991, Soybean Sci. 10(2):98-103). Aphid populations may resurge when applications of insecticides are poorly timed or applied.
In the USA, there are currently no commercial soybean cultivars with aphid resistance and there are no reported resistance sources in early maturing soybean germplasm. Although there have been recent reports of aphid resistant soybean plants, (Hill et al., J. of Econ. Entomol. 97:1071-1077 (2004); Hill et al., Crop Sci. 44:98-106 (2004); Mueller, et al., The 2003 Entomological Society of America Annual Meeting and Exhibition Cincinnati, Ohio, October 2003, all of which are herein incorporated by reference), with further reports showing the results of studies on their effectiveness, (Li et al., J Econ Entomol. 2004 June; 97(3):1106-1111). These soybean plants are late maturing and not well suited for commercial development.
Currently none of the commercial soybean varieties show resistance to the aphid and further there are no sources of resistance reported in early maturing soybean germplasm in the USA. Therefore, developing soybean varieties that are resistant to aphids is a long-term solution to the aphid problem. Furthermore, there is a need for soybean plant varieties with engineered resistance to specific aphid biotypes in addition to the desirability of having commercially available soybean plants with a broad resistance to aphids.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for providing and using markers of aphid resistant germplasm, particularly for identifying aphid resistance germplasm in soybean plants. Specifically, the invention relates to providing aphid resistant germplasm identified by markers associated with decreased damage from aphid feeding, as well as enhanced tolerance to aphid infestation of soybean plants. More particularly, the invention relates to compositions and methods for using genetic markers in breeding methods for producing plants with increased resistance to aphid damage and tolerance, while retaining and acquiring desired agronomic traits. Additionally, markers were developed for fine mapping of aphid resistance genes including allele specific contributions to plants for breeding soybean plants with increased aphid resistance. Furthermore, the invention relates to plants produced by these breeding programs, including plants with having aphid resistant genes between the molecular markers identifying aphid resistant genes, for use in commercial soybean production.
The present invention relates to compositions and methods for providing aphid resistance in plants. More particularly, the invention relates to compositions and methods for using aphid resistant germplasm for breeding soybean aphid resistant soybean plants, including but not limited to cultivars, varieties, lines and methods of breeding the same for commercial use, the breeding methods further involving identifying and using genetic markers for aphid resistant traits.
The present invention also relates to the field of plant breeding, specifically to methods of soybean breeding and the resulting soybean plants and soybean lines for commercial distribution. The soybean breeding methods include but are not limited to natural breeding, artificial breeding, molecular marker selection, commercial breeding, and transgenics. More particularly, the invention relates to producing soybean aphid-resistant plants, populations, cultivars, varieties, lines and methods of breeding the same, the methods further involving DNA molecular marker analysis.
The invention further relates to soybeans that are resistant to aphids, and in particular to germplasm that was identified as providing aphid resistance, methods of breeding aphid resistant soybean plants, and the resulting new aphid resistant soybean plant varieties, lines and cultivars developed through traditional plant breeding methods that provide for successful commercialization of the aphid resistant soybean germplasm. The present invention is not limited to any particular soybean variety, line, and cultivar having aphid resistance activities.
In some embodiments, the aphid resistant germplasm in the form of seeds is deposited under the terms of the Budapest Treaty at the American Type Culture Collection (A.T.C.C./ATCC) 10801 University Boulevard, Manassas, Va., 20110-2209, The United States of America.
In some embodiments, the invention provides a soybean plant comprising aphid resistant germplasm, wherein said aphid resistant germplasm has a 5′ end and a 3′ end having a 5′ molecular marker identifying said 5′ end and a 3′ molecular marker identifying said 3′ end, wherein an aphid resistant gene is located in between said molecular markers. In some embodiments the plant is selected from the group consisting of accession PI567598B, accession PI567543C, accession PI567541B, accession PI567597C, line E06902 deposited under ATCC accession No: PTA-8794, and progeny thereof. In some embodiments the aphid resistant gene is selected from the group consisting of rag1b, rag1c, rag3, Rag3 and rag4. In some embodiments the aphid resistant germplasm having said rag1b gene is in between markers selected from the group consisting of a 5′ marker Satt567 and 3′ marker Satt435; 5′ marker 5 BARCSOYSSR07—0295 and 3′ marker BARCSOYSSR07—0309; 5′ marker Satt435 and 3′ marker BARCSOYSSR07—0295; SNP07—420 and 3′ marker Satt435. In some embodiments the aphid resistant germplasm having said rag1c gene is in between a 5′ marker MSUSNP7-19 and a 3′ marker MSUSNP7-10. In some embodiments the aphid resistant germplasm having said rag3 gene is in between a 5′ marker MSUSNP16-10 and 3′ marker MSUSNP16-6424. In some embodiments the aphid resistant germplasm having said rag3 gene is selected from the group consisting of Glyma16g06940 and Glyma16g07200. In some embodiments the aphid resistant germplasm having said Rag3 gene is in between a 5′ Gm16—6262227_C_T and 3′ Gm16—6469551_A_C; 5′ Gm16—6262227_C_T and 3′ Gm16—6423098_G_A. In some embodiments the aphid resistant germplasm having said rag4 gene is in between markers selected from the group consisting of 5′ MSUSNP13-29 and 3′ MSUSNP13-31; 5′ MSUSNP13-29 and 3′ MSUSNP13-31; 5′ MSUSNP13-5 and 3′MSUSNP13-6; 5′ MSUSNP13-5 (7766353) and 3′ ss247923149. In some embodiments the aphid resistant germplasm comprises germplasm located between markers for identifying rag1b and rag3. In some embodiments the aphid resistant germplasm comprises germplasm located between markers for identifying rag1c and rag4.
In some embodiments, the invention provides a polymerase reaction primer for isolating aphid resistant germplasm, wherein said primer comprises a fragment of a MSUSNP sequence having a polymorphism for amplifying an aphid resistant gene.
In some embodiments, the invention provides an isolated aphid resistant gene, wherein said germplasm has the presence of a molecular marker identifying an aphid resistance gene selected from the group consisting of rag1b, rag1c, rag3, Rag3 and rag4. In some embodiments, said isolated aphid resistant gene has a 5′ end and a 3′ end, herein a 5′ molecular marker is on the 5′ end and a 3′ molecular marker is on the 3′ end. In some embodiments, said aphid resistant gene comprises a single nucleotide polymorphism.
In some embodiments, the invention provides an isolated aphid resistant gene, located in between a 5′ molecular marker and a 3′ molecular marker.
In some embodiments, the invention provides a method for producing a progeny soybean plant having increased aphid resistance, comprising, a) providing, i) a soybean plant cell, wherein said soybean plant cell was obtained from a soybean plant showing little aphid resistance, ii) an isolated aphid resistant gene, wherein said gene is selected from the group consisting of rag1b, rag1c, rag3, Rag3 and rag4, and b) transfecting said soybean plant cell with said isolated gene, and c) growing said soybean cell into said progeny soybean plant having increased aphid resistance. The method further comprises using a molecular marker for identifying said aphid resistant gene in said progeny soybean plant.
In some embodiments, the invention provides a seed of said progeny plant having increased aphid resistance.
In some embodiments, the invention provides a soybean cultivar comprising aphid resistant germplasm, wherein said soybean cultivar is selected from a soybean maturity group consisting of 000, 00, 0, I, II, and III. In other embodiments, the present invention provides a soybean cultivar from an early maturing soybean maturity group of at least 000.1, 000.9, 00.1, 00.9, 0.1, 0.9, I.1 (1.1), I.9 (1.9), II.1 (2.1), II.9 (2.9), III.1 (3.1), and III.9 (3.9), wherein said soybean cultivar is an early maturing soybean. In some embodiments the soybean cultivar is from soybean maturity group III. In some embodiments, the present invention provides a soybean cultivar from soybean maturity group III of at least III (3.0), III.1 (3.1), III.2 (3.2), 111.3 (3.3), III.4 (3.4), III.5 (3.5), III.6 (3.6), III.7 (3.7), III.8 (3.8), III.9 and (3.9). The present invention is not limited to any particular type of aphid resistance germplasm. Indeed, a variety of aphid resistance germplasm traits are contemplated, including, but not limited to antibiosis resistance toxicity to aphids, antixenosis resistance, and repellency to aphids. In some embodiments the soybean cultivar comprises aphid resistance germplasm wherein the aphid resistance is antibiosis resistance. In some embodiments the aphid resistance germplasm provides antibiosis resistance toxicity to aphids. In some embodiments the aphid resistance germplasm provides an aphid toxin. In some embodiments the soybean cultivar comprises aphid resistance germplasm wherein the aphid resistance is antixenosis resistance. In some embodiments the aphid resistance germplasm provides repellency to aphids. In some embodiments the aphid resistance germplasm provides an aphid repellant. In some embodiments the soybean cultivar comprises aphid resistant germplasm, wherein aphid resistant germplasm derives from an Asian soybean cultivar. In some embodiments the soybean cultivar is a Glycine max subsp max. In some embodiments the soybean cultivar further comprises introgressed germplasm for at least one desired trait. The present invention is not limited to any particular type of trait. Indeed a variety of traits are contemplated including but not limited to tolerance to an herbicide, resistance to an arthropod, resistance to a microorganism, resistance to a fungus, and an agronomic trait. In some embodiments the soybean cultivar further comprises introgressed germplasm for resistance to arthropods in addition to aphid resistance. The present invention is not limited to any particular type of arthropod. Indeed, a variety of arthropods are contemplated, including, but not limited to herbivore arthropods. In some embodiments the soybean cultivar further comprises introgressed germplasm for arthropod resistance to one or more of insecta including but not limited to Coleopteran, for example, Matsumuraeses sp., for example, bean pod worm (Matsumuraeses phaseoli), Anthomomus sp., such as a cotton boll weevil (Anthomomus grandis), Cerotoma sp., such as Bean Leaf Beetle (Cerotoma trifurcate), Epilachna sp. such as Mexican Bean Beetle (Epilachna varivestris), etc., Melanoplus sp., for example, red-legged grasshopper (Melanoplus femurrubrum), differential grasshopper (Melanoplus differentialis), American bird grasshopper or American grasshopper or American locust or American locust grasshopper (Shistocerca Americana), etc., and two-spotted spider mite (Tetranychus urticae Koch), etc.; Lepidopteran, such as Anticarsia sp. for example, Velvetbean Caterpillar (Anticarsia gemmatalis), Pseudoplusia sp., for example, Soybean Looper (Pseudoplusia includens), soybean pod borer (Leguminivora glycinivorella), Plathypena sp., green cloverworm (Plathypena scabra (F.)), Heliothis sp. for example, Tobacco budworm (Heliothis virescens (Fabricius)), cotton bollworm or corn earworm or soybean podworm (Heliothis (Helicoverpa) zea), etc.; Spodoptera sp., for example, fall armyworm (Spodoptera frugiperda), common cutworm (Spodoptera litura Fabricius), etc.; Hemiptera, for example, alfalfa hopper (Spissistilus festinus, Say), Pentatominae, such as green stink bug (Clemson) (Acrosternum hilare (Say)); brown stink bug (Euschistus servus (Say)); and southern green stink bug (Nezara viridula (L.)), East Asian stink bug or yellow-brown stink bug (Pentatomidae: Halyomorpha halys), etc.; and Cicadellidae, such as leafhoppers, for example, a potato leafhopper, a soybean leafhopper, for example, Empoasca decipiens Paoli, Macrosteles quadripunctulatus (Kirschbaum), M. laevis (Rib). Psammotettix alienus (Dahlbom), P. Striatus (Linne), and Neoaliturus tenellus (Baker).
In some embodiments the soybean cultivar further comprises introgressed germplasm for nematode resistance, for example, resistance to soybean cyst nematode (Heterodera glycines) and root knot nematode (Meloidogyne sp.).
In some embodiments the soybean cultivar further comprises introgressed germplasm for resistance to microorganisms and diseases caused by microorganisms. The present invention is not limited to any particular microorganism or disease. Indeed, a variety of microorganisms and diseases are contemplated, including, but not limited to microorganisms such as bacteria, viruses, fungi, and the like, and diseases thereof. In some embodiments the soybean cultivar further comprises introgressed germplasm for resistance to one or more of microorganisms such as fungi, including but not limited to Phytophthora sp., Sclerotinia sp., Phytophthora sp., Fusarium sp., Phialophora sp., Peronospora sp., Cercospora sp., Diaporthe sp., Pythium sp., soybean rust or Asian soybean rust fungus (Phakopsora pachyrhizi); bacteria, including but not limited to Xanthomonas sp.; virus including but not limited to Soybean mosaic virus, Bean Pod Mottle Virus, Peanut Mottle Virus, Soja virus, et cetera. In some embodiments the soybean cultivar further comprises introgressed germplasm for disease resistance to one or more diseases of leaf rot, brown leaf spot, frogeye leaf spot, stem rot, brown stem rot, stem canker, root rot, pod rot, powdery mildew, sudden death syndrome, bacterial pustule, reaction to bacterial pustule, bacterial blight, seedling blight, pod blight, stem blight, purple seed stain, mottling, stem mottling, pod mottling, leaf mottling, rust, soybean rust, rust, Asian soybean rust fungus a viral infection, a bacterial infection, a fungal infection, a nematode infection, insect feeding, and the like.
In some embodiments the aphid resistant soybean cultivar further comprises a selected agronomic trait. The present invention is not limited to any particular type of agronomic trait. Indeed, a variety of agronomic traits are contemplated, including, but not limited to a preferred oil content, protein content, seed protein content, seed size, seed color, seed coat thickness, seed sugar content, seed free amino acid content, seed germination rate, seed texture, seed fiber content, food-grade quality, hilium color, seed yield, maturity group, plant type, drought resistance, water resistance, cold weather resistance, hot weather resistance, and growth in a hardiness zone. In some embodiments the aphid resistant soybean plant comprises an agronomic trait comprising a seed trait, including, but not limited to a soybean seed with altered fatty acid content, such as altered linoleic acid content, altered polyunsaturated fat content, altered lipoxygenase activity, and the like. In some embodiments the soybean cultivar further comprises a plant part. The present invention is not limited to any particular type of plant part. Indeed, a variety of soybean plant parts are contemplated, including, but not limited to pollen, an ovule, a tissue, a pod, a seed, and a cell. In some embodiments the soybean cultivar further comprises an introgressed heterologous gene. The present invention is not limited to any particular type of heterologous gene. Indeed a variety of heterologous genes are contemplated, including, but not limited to a gene encoding an insecticidal protein, herbicide tolerance, and agronomic trait. In some embodiments the heterologous gene comprises one or more of a gene encoding an insecticidal protein, herbicide tolerance, and agronomic trait. In some embodiments the heterologous gene is a transgene. In some embodiments the transgene comprises one or more of a gene encoding an insecticidal protein, herbicide tolerance, and agronomic trait. In some embodiments the heterologous gene comprises one or more of a gene encoding a modified phosphinothricin acetyltransferase (PAT) from the soil bacterium Streptomyces viridochromogenes, fatty acid desaturase (GmFad2-1) from soybean, a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from the soil bacterium Agrobacterium tumefaciens, one or more of a Bacillus thuringiensis (Bt) insecticidal crystal protein tIC851, Bt Δ-endotoxin with insecticidal activity, mutant Bt Δ-endotoxins with insecticidal activity, crystal protein (Cry) Bt toxins with insecticidal activity, for example, a cryIIIC toxin, cryET1 toxin, PS63B, PS176 toxin, NRRL B-1 8721 toxin, Bt protease resistant toxins such as BTS02618Aa or BTS02618Ab Bt nematode-active toxins, an enzyme for altering a fatty acid, Δ-12 desaturase, plant acyl-ACP thioesterase, FAN1 protein for altering seed linolenic acid content, a palmitoyl-ACP thioesterase, an enzyme for reducing linolenic acid, an enzyme for reducing palmitic acid, an enzyme for increasing protein in a soybean seed, a protein for modifying an agronomic trait, a protein for providing an agronomic trait, and the like. In some embodiments, the invention provides an aphid resistant germplasm, wherein said aphid resistant germplasm is selected from an early maturing soybean cultivar. In other embodiments, the early maturing soybean cultivar is selected from the maturity group consisting of 000, 00, 0, I, II, and III. In other embodiments, the present invention provides aphid resistant germplasm from an early maturing soybean maturity group of at least 000.1, 000.9, 00.1, 00.9, 0.1, 0.9, I.11 (1.1), I.9 (1.9), II.1 (2.1), II.9 (2.9), III.1 (3.1), III.9 (3.9), wherein said aphid resistant germplasm is an early maturing soybean. In other embodiments, the aphid resistant germplasm derives from a soybean cultivar of the soybean maturity group III. In some embodiments, the present invention provides aphid resistance germplasm from soybean maturity group III is of at least III (3.0), III.1 (3.1), III, 2 (3.2), III.3 (3.3), III.4 (3.4), III.5 (3.5), III.6 (3.6), III.7 (3.7), III.8 (3.8), III.9 and (3.9). In other embodiments, the aphid resistant germplasm provides antibiosis resistance. In other embodiments, the aphid resistance germplasm provides an aphid toxin. In other embodiments, the aphid resistance germplasm provides antixenosis resistance. In other embodiments, the aphid resistance germplasm provides an aphid repellant. In other embodiments, the aphid resistant germplasm derives from an Asian soybean cultivar. In other embodiments, the aphid resistant germplasm derives from a Glycine max subsp max. 
In some embodiments, the invention provides a transgenic aphid resistant soybean plant. The present invention is not limited to any particular transgene of a transgenic aphid resistant soybean plant. Indeed, a variety of transgenes are contemplated, including, but not limited to a transgene encoding an insecticidal protein, herbicide tolerance, and an agronomic trait. In some embodiments the transgene comprises one or more of a gene encoding a modified phosphinothricin acetyltransferase (PAT) from the soil bacterium Streptomyces viridochromogenes, fatty acid desaturase (GmFad2-I) from soybean, a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from the soil bacterium Agrobacterium tumefaciens, one or more of a Bacillus thuringiensis (Bt) insecticidal crystal protein tIC851, Bt A-endotoxin with insecticidal activity, mutant Bt A-endotoxins with insecticidal activity, crystal protein (Cry) Bt toxins with insecticidal activity, for example, a cryIIIC toxin, cryET1 toxin, PS63B, PS176 toxin, NRRL B-I 8721 toxin, Bt protease resistant toxins such as BTS02618Aa or BTS02618Ab Bt nematode-active toxins, an enzyme for altering a fatty acid, Δ-12 desaturase, plant acyl-ACP thioesterase, FAN1 protein for altering seed linolenic acid content, a palmitoyl-ACP thioesterase, an enzyme for reducing linolenic acid, an enzyme for reducing palmitic acid, an enzyme for increasing protein in a soybean seed, a protein for modifying an agronomic trait, a protein for providing an agronomic trait, and the like. The present invention is not limited to any particular transgenic aphid resistant soybean plant. Indeed, a variety of transgenic aphid resistant soybean plants are contemplated, including, but not limited to an aphid resistant soybean plant comprising aphid resistant germplasm derived from one or more of soybean cultivars or lines designated PI567598B, PI567543C, PI567541B, PI567597C, line E06906, line E06902 deposited under ATCC accession No: PTA-8794, line E06907, line E06901, and line E06904.
In some embodiments, the invention provides a method for producing a transgenic aphid resistant plant, comprising: an aphid resistant soybean plant, a transgene, and introducing the transgene into the aphid resistant soybean plant. The present invention is not limited to any particular aphid resistant soybean plant. Indeed, a variety of aphid resistant soybean plants are contemplated, including, but not limited to an aphid resistant soybean plant comprising aphid resistant germplasm derived from one or more of soybean cultivars or lines designated PI567598B, PI567543C, PI567541B, PI567597C, line E06906, line E06902 deposited under ATCC accession No: PTA-8794, line E06907, line E06901, and line E06904. The present invention is not limited to any particular transgene for producing a transgenic aphid resistant plant. Indeed, a variety of transgenes are contemplated, including, but not limited to a transgene encoding an insecticidal protein, herbicide tolerance, and an agronomic trait. In some embodiments the transgene comprises one or more of a gene encoding a modified phosphinothricin acetyltransferase (PAT) from the soil bacterium Streptomyces viridochromogenes, fatty acid desaturase (GmFad2-I) from soybean, a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from the soil bacterium Agrobacterium tumefaciens, one or more of a Bacillus thuringiensis (Bt) insecticidal crystal protein tlC851, Bt Δ-endotoxin with insecticidal activity, mutant Bt Δ-endotoxins with insecticidal activity, crystal protein (Cry) Bt toxins with insecticidal activity, for example, a cryIIIC toxin, cryET1 toxin, PS63B, PS176 toxin, NRRL B-I 8721 toxin, Bt protease resistant toxins such as BTS02618Aa or BTS02618Ab Bt nematode-active toxins, an enzyme for altering a fatty acid, Δ-12 desaturase, plant acyl-ACP thioesterase, FAN1 protein for altering seed linolenic acid content, a palmitoyl-ACP thioesterase, an enzyme for reducing linolenic acid, an enzyme for reducing palmitic acid, an enzyme for increasing protein in a soybean seed, a protein for modifying an agronomic trait, a protein for providing an agronomic trait, and the like. The present invention is not limited to any particular method for introducing a transgene into an aphid resistant soybean plant. Indeed, a variety of introduction methods are contemplated, including, but not limited to particle acceleration mediated transformation, biolistic transformation, Agrobacterium-mediated transformation, incubation in transformation medium, electroporation, microinjection, protoplast fusion, viral infection, and the like.
In some embodiments, the invention provides a method for producing an aphid resistant plant line, comprising: identifying germplasm conferring aphid resistance, wherein the aphid resistant germplasm derives from an aphid resistant soybean cultivar from the maturity group consisting of 000, 00, 0, I, II, and III; and introducing said germplasm into an elite soybean cultivar. In some embodiments the identifying aphid resistant germplasm conferring aphid resistance comprises molecular marker analysis of DNA samples isolated from one or more of an aphid resistant soybean plant, an aphid resistant soybean cultivar, a non-aphid resistant soybean plant, and a non-aphid resistant soybean cultivar, wherein said analysis identifies DNA molecules associated with aphid resistance. The present invention is not limited to any particular type of molecular marker. Indeed, a variety of molecular markers are contemplated, including, but not limited to a simple sequence repeat (SSR) analysis, a single nucleotide polymorphism analysis (SNP), a random amplified polymorphic DNA analysis (RAPD), and an amplified fragment length polymorphism analysis (AFLP). In some embodiments the identifying aphid resistant germplasm conferring aphid resistance comprises identifying linkage groups associated with aphid resistant germplasm. In some embodiments the identifying aphid resistant germplasm conferring aphid resistance comprises using simple sequence repeat markers for identifying linkage groups comprising aphid resistant germplasm. In some embodiments, a simple sequence repeat marker is selected from one or more of soybean “Satt,” “Sat,” “Sctt,” “Satgt,” “Scaa,” “Staga,” or “Sct” markers. The present invention is not limited to any particular type of Satt marker. Indeed, a variety of simple sequence repeat markers are contemplated, including, but not limited to a Satt271, Satt280, Satt304, Satt439, Satt468, Satt529, Satt686, and Satt628 marker and their PCR primer pairs. In some embodiments, a Satt marker is selected from one or more of a PCR primer pair of Satt271 (SEQ ID NO:01 Forward primer and SEQ ID NO:02 Reverse primer), Satt280 (SEQ ID NO:03 Forward primer and SEQ ID NO:04 Reverse primer), Satt304 (SEQ ID NO:05 Forward primer and SEQ ID NO:06 Reverse primer), Satt439 (SEQ ID NO:07 Forward primer and SEQ ID NO:08 Reverse primer), Satt468 (SEQ ID NO:09 Forward primer and SEQ ID NO:10 Reverse primer), Satt529 (SEQ ID NO:11 Forward primer and SEQ ID NO:12 Reverse primer), Satt628 (SEQ ID NO:13 Forward primer and SEQ ID NO:14 Reverse primer), and Satt686 (SEQ ID NO:15 Forward primer and SEQ ID NO:16 Reverse primer). In some embodiments, the association of a Satt marker to a linkage group comprising aphid resistant germplasm is demonstrated by using one or more of a PCR primer pair of Satt271 (SEQ ID NO:01 Forward primer and SEQ ID NO:02 Reverse primer), Satt280 (SEQ ID NO:03 Forward primer and SEQ ID NO:04 Reverse primer), Satt304 (SEQ ID NO:05 Forward primer and SEQ ID NO:06 Reverse primer), Satt439 (SEQ ID NO:07 Forward primer and SEQ ID NO:08 Reverse primer), Satt468 (SEQ ID NO:09 Forward primer and SEQ ID NO:10 Reverse primer), Satt529 (SEQ ID NO:11 Forward primer and SEQ ID NO:12 Reverse primer), Satt628 (SEQ ID NO:13 Forward primer and SEQ ID NO:14 Reverse primer), and Satt686 (SEQ ID NO:15 Forward primer and SEQ ID NO:16 Reverse primer). In some embodiments the molecular marker analysis provides a DNA fingerprint of aphid resistant germplasm. In some embodiments the DNA molecule is a marker for an allele of a quantitative trait locus associated with aphid resistant germplasm. In some embodiments the allele provides enhanced aphid resistance. In some embodiments, the invention provides an isolated DNA molecule associated with germplasm conferring aphid resistance. The present invention is not limited to any particular elite soybean cultivars or varieties or maturity group. Indeed, a variety of elite soybean cultivars are contemplated, including, but not limited to PI257345 and its progeny S1346, PI71506, Hutcheson, Resnik, Lincoln, Richland, Patoka, PI 81041, Illini, PI 54610, PI 88788, Mukden, Palmetto, Haberlandt No. 171, PI 257345, PI 71506, Lincoln, Mandarin (Ottawa), PI 90763, CNS, PI 209332, Richland, Tokyo, S-100, Minsoy, Ogden, Noir 1, A.K. (Harrow), Archer, Dunfield, Evans, Mukden, Clark, Jackson, Harosoy, Illini, Essex, Roanoke, PI 88788, Peking, Asgrow AG4201, Asgrow AG3703, Croplan Genetics RC4432, A2704-12, A2704-21, A5547-35 (Aventis Crop Science), A5547-127, GU262, W62, W98, (Bayer Crop Science (Aventis Crop Science(AgrEvo))), G94-1, G94-19, G168 (DuPont Canada Agricultural Products), GTS 40-3-2 (Monsanto Company), OT96-15 (Agriculture & Agri-Food Canada), Maple Glen, PI361088B and Roundup Ready Soybeans. In some embodiments the aphid resistant soybean cultivar is an Asian soybean cultivar. In some embodiments the aphid resistant soybean cultivar is a Glycine max subsp. Max. In some embodiments the aphid resistant germplasm comprises soybean germplasm derived from one or more of PI 567543C, PI 567597C, PI 567541B, and PI 567598B cultivars obtained from the USDA Soybean Germplasm. In some embodiments, the aphid resistant soybean cultivar derives from one or more of soybean line E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06907, and E06904, a soybean progeny plant from crossing PI 567598B×PI 567597C and soybean plants of population Identification (ID) numbers 020138-1, 030100-1, 030100-2, 030100-3, and 030100-4. In some embodiments producing an aphid resistant plant line further comprises crossing a first soybean plant, wherein said first soybean plant provides aphid resistant germplasm, with a second soybean plant and harvesting the resultant hybrid soybean seed. In some embodiments the first soybean plant comprises aphid resistant germplasm from one or more of PI 567543C, PI 567597C, PI 567541B, and PI 567598B. In some embodiments the first soybean plant comprises aphid resistant germplasm from one or more of soybean line E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06907, and E06904.
In some embodiments the second soybean plant is one or more of accession PI 567598B, accession PI 567543C, accession PI 567541B, accession PI 567597C, a line E06906, a line E06902, a line E06907, a line E06901, a line E06904, a variety Titan, a variety Loda, a line E00075 and a line E99034. In some embodiments the second soybean plant is one or more of an elite soybean plant. In some embodiments the crossing further comprises introgressing aphid resistance into hybrid soybean seed. In some embodiments the crossing further comprises one or more of a backcrossing, an outcrossing, and a self-crossing. In some embodiments the identifying aphid resistant germplasm further comprises molecular marker analysis of DNA samples isolated from one or more of a progeny plant, a second soybean plant, an aphid resistant donor soybean cultivar, a parental aphid resistant soybean cultivar, and a non-aphid resistant soybean cultivar, wherein said analysis identifies DNA molecules associated with aphid resistance. The present invention is not limited to any particular type of molecular marker. Indeed, a variety of molecular markers are contemplated, including, but not limited to a simple sequence repeat (SSR) analysis, a single nucleotide polymorphism analysis (SNP), a random amplified polymorphic DNA analysis (RAPD), and an amplified fragment length polymorphism analysis (AFLP). In some embodiments, the molecular marker analysis is selected from one or more of a simple sequence repeat (SSR) analysis, a single nucleotide polymorphism analysis (SNP), a random amplified polymorphic DNA analysis (RAPD), and an amplified fragment length polymorphism analysis (AFLP). In some embodiments, an SSR marker is selected from one or more of a “Satt,” “Sat,” “Sctt,” “Satgt,” “Scaa,” “Staga,” or “Sct” marker. The present invention is not limited to any particular type of Satt marker. Indeed, a variety of molecular markers are contemplated, including, but not limited to a Satt271, Satt280, Satt304, Satt439, Satt468, Satt529, Satt686, and Satt628 marker. In some embodiments, a Satt marker is selected from one or more of a Satt271 (SEQ ID NO:01 Forward primer and SEQ ID NO:02 Reverse primer), Satt280 (SEQ ID NO:03 Forward primer and SEQ ID NO:04 Reverse primer), Satt304 (SEQ ID NO:05 Forward primer and SEQ ID NO:06 Reverse primer), Satt439 (SEQ ID NO:07 Forward primer and SEQ ID NO:08 Reverse primer), Satt468 (SEQ ID NO:09 Forward primer and SEQ ID NO:10 Reverse primer), Satt529 (SEQ ID NO:11 Forward primer and SEQ ID NO:12 Reverse primer), Satt628 (SEQ ID NO:13 Forward primer and SEQ ID NO:14 Reverse primer), and Satt686 (SEQ ID NO:15 Forward primer and SEQ ID NO:16 Reverse primer). In some embodiments the molecular marker analysis provides a DNA fingerprint of aphid resistant germplasm. In some embodiments the DNA molecule is a marker for an allele of a quantitative trait locus. In some embodiments the allele provides enhanced aphid resistance. In some embodiments, the invention provides a method for isolating an aphid resistant DNA molecule, comprising, providing, a soybean genomic DNA library selected from germplasm of one or more of soybean populations designated 030100-1, 030100-2, 030100-3, and 030100-4 and isolating said DNA molecule from said library.
In some embodiments, the invention provides a method for isolating an aphid resistant DNA molecule, comprising, providing, a soybean linkage group germplasm selected from germplasm of one or more of soybean populations designated 030100-1, 030100-2, 030100-3, and 030100-4 and isolating said DNA molecule from said library.
In some embodiments, the invention provides a method for isolating an aphid resistant DNA molecule, comprising, providing, a soybean linkage group germplasm selected from germplasm of one or more of linkage groups J, K, B2, D1a, and D1b and isolating said DNA molecule from said linkage group.
In some embodiments the aphid resistant germplasm comprises a linkage group selected from one of more of linkage groups J, K, B2, D1a, and D1b.
In some embodiments, the invention provides an isolated DNA molecule associated with aphid resistant germplasm of a soybean plant, wherein said soybean plant is a cultivar selected from the soybean maturity group consisting of 000, 00, 0, I, II, and III. In some embodiments the soybean cultivar is an Asian soybean cultivar. In some embodiments the soybean cultivar is a Glycine max subsp. Max. 
In some embodiments, the invention provides a soybean cultivar, wherein at least one ancestor of the soybean cultivar comprises aphid resistant germplasm of one or more of a soybean cultivar designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B. In some embodiments, the invention provides a soybean cultivar, wherein at least one ancestor of the soybean cultivar comprises aphid resistant germplasm of one or more of a soybean line E06906, line E06902 deposited under ATCC accession No: PTA-8794, line E06907, line E06901, and line E06904. In some embodiments, the invention provides a soybean cultivar with resistance to a soybean aphid comprising germplasm of one or more of a soybean cultivar designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B. In some embodiments, the invention provides a soybean cultivar with resistance to a soybean aphid comprising germplasm of one or more of a soybean line designated E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06901, and E06904. In some embodiments, the invention provides a soybean line with resistance to a soybean aphid comprising germplasm of one or more of a soybean line designated E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06901, and E06904. In some embodiments the resistant germplasm comprises antibiosis resistance germplasm. In some embodiments the resistant germplasm comprises antixenosis resistance. In some embodiments the soybean cultivar is selected from the soybean maturity group comprising 000, 00, 0, I, II, III, IV, V, VI, VII, VIII, IX, and X. In other embodiments, the present invention provides the soybean cultivar is selected from the soybean maturity group of at least 000.1, 000.9, 00.1, 00.9, 0.1, 0.9, I.11 (1.1), I.9 (1.9), II.1 (2.1), II.9 (2.9), III.1 (3.1), III.9 (3.9), IV.1 (4.1), IV.9 (4.9), V.1 (5.1), V.9 (5.9), VI.1 (6.1), VI.9 (6.9), VII.1 (7.1), VII.9 (7.9), VIII.1 (8.1), VIII.9 (8.9), IX.1 (9.1), IX.9 (9.9), X.1 (10.1), and X.9 (10.9). In some embodiments the soybean cultivar further comprises a selected agronomic trait. In some embodiments the agronomic trait comprises one or more of a preferred oil content, protein content, seed protein content, seed size, seed color, seed coat thickness, seed sugar content, seed free amino acid content, seed germination rate, seed texture, seed fiber content, food-grade quality, hilium color, seed yield, maturity group, plant type, drought resistance, water resistance, cold weather resistance, hot weather resistance, and growth in a hardiness zone. In some embodiments the aphid resistant soybean plant comprises an agronomic trait comprising a seed trait, including, but not limited to a soybean seed with altered fatty acid content, such as altered linoleic acid content, altered polyunsaturated fat content, altered lipoxygenase, and the like.
In some embodiments, the invention provides an aphid toxin, comprising germplasm from one or more of a soybean cultivar designated PI 567541B and PI 567598B. In some embodiments, the invention provides an aphid toxin, comprising germplasm from one or more of a soybean line designated E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06901, and E06904.
In some embodiments, the invention provides a soybean cultivar with resistance to a soybean aphid comprising germplasm designated PI 567543C and PI 567597C, wherein said resistance is an antixenosis resistance.
In some embodiments, the invention provides an aphid repellant, comprising germplasm from one or more of a soybean cultivar designated PI 567543C and PI 567597C.
In some embodiments, the invention provides an aphid repellant, comprising germplasm from one or more of a soybean line designated E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06901, and E06904.
In some embodiments, the invention provides a soybean plant part comprising aphid resistant germplasm of one or more of a soybean cultivar designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B.
In some embodiments, the invention provides a soybean plant part comprising aphid resistant germplasm of one or more of a soybean line designated E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06901, and E06904.
In some embodiments the soybean plant part is one or more of a pollen grain, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides a breeding method for producing a commercial variety of an aphid resistant soybean plant, wherein said aphid resistant soybean plant comprises crossing a first hybrid plant comprising aphid resistant germplasm from one or more of a soybean cultivar germplasm designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B with a second soybean plant.
In some embodiments, the invention provides a breeding method for producing a commercial variety of an aphid resistant soybean plant, wherein said aphid resistant soybean plant comprises crossing a first hybrid plant comprising aphid resistant germplasm from one or more of soybean germplasm designated line designated E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06901, and E06904 with a second soybean plant. In some embodiments the second soybean plant comprises aphid resistant germplasm from one or more of a soybean cultivar germplasm designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B. In some embodiments the second soybean plant comprises aphid resistant germplasm from one or more of a soybean line germplasm designated E06906, E06902 deposited under ATCC accession No: PTA-8794, E06907, E06901, and E06904 with a second soybean plant. In some embodiments the first soybean plant and said second soybean plant are different soybean plants. In some embodiments the first soybean plant and said second soybean plant are unrelated soybean plants. In some embodiments the first soybean plant and said second soybean plant are the same soybean plant. In some embodiments the first soybean plant and said second soybean plant are related soybean plants. In some embodiments the crossing comprises one or more a backcrossing, an outcrossing, and a self-crossing. In some embodiments the producing further comprises using a molecular marker for identifying a gene associated with aphid resistance in a first soybean plant. In some embodiments the commercial variety of an aphid resistant soybean plant further comprises a selected agronomic trait. In some embodiments the agronomic trait further comprises one or more of a preferred oil content, protein content, seed protein content, seed size, seed color, seed coat thickness, seed sugar content, seed free amino acid content, seed germination rate, seed texture, seed fiber content, food-grade quality, clear hilium, seed yield, drought resistance, water resistance, cold weather resistance, hot weather resistance, and growth in a hardiness zone.
In some embodiments, the invention provides a DNA molecular marker associated with aphid resistance of a soybean plant comprising germplasm from one or more of a soybean cultivar designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B, wherein the DNA molecule provides aphid resistance in a soybean plant.
In some embodiments, the invention provides an isolated DNA molecule associated with aphid resistance of a soybean plant comprising germplasm from one or more of a soybean cultivar designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B, wherein the DNA molecule provides enhanced aphid resistance in a soybean plant.
In some embodiments, the invention provides a DNA molecule associated with aphid resistance of a soybean plant comprising germplasm from one or more of a soybean cultivar designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B, wherein said DNA molecule is a marker for an allele of a quantitative trait locus. In some embodiments the allele provides enhanced aphid resistance in a soybean plant. In some embodiments the quantitative trait locus provides enhanced aphid resistance in a soybean plant.
In some embodiments, the invention provides a method for isolating an aphid resistant DNA molecule, comprising, providing, a soybean genomic DNA library selected from germplasm of one or more of soybean cultivars designated PI 567543C, PI 567597C, PI 567541B, and PI 567598B and isolating said DNA molecule from said library.
In some embodiments, the invention provides a soybean plant of cultivar PI 567543C.
In some embodiments, the invention provides a soybean plant part of cultivar PI 567543C, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides a soybean plant of cultivar PI 5567597C.
In some embodiments, the invention provides a soybean plant part of cultivar PI 567597C, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides a soybean plant of cultivar PI 567541B.
In some embodiments, the invention provides a soybean plant part of cultivar PI 567541 and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides a soybean plant of cultivar PI 567598B.
In some embodiments, the invention provides a soybean plant part of cultivar PI 567598B, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of cultivar PI 567541B, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of cultivar PI 567541B.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of cultivar PI 567597C, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of cultivar PI 567597C.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of cultivar PI 567543C, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of cultivar PI 567543C.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of cultivar PI 567598B, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of cultivar PI 567598B.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of line E06906, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of line E06906.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of line E06902, wherein seed of said cultivar having been deposited under ATCC accession No: PTA-8794, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of line E06902, wherein seed of said cultivar having been deposited under ATCC accession No: PTA-8794.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of line E06907, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of line E06907.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of line E06901, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of line E06901.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant part of line E06904, and said soybean plant part is one or more of a pollen, an ovule, a tissue, a pod, a seed, and a cell.
In some embodiments, the invention provides an aphid resistant germplasm of a soybean plant of line E06904.
The present inventions provide a composition, comprising, aphid resistant plant germplasm and a molecular marker selected from the linkage groups consisting of F, M, and J. In some embodiments the marker is selected from the group consisting of simple sequence repeat (SSR), single nucleotide polymorphism (SNP), random amplified polymorphic DNA (RAPID), and amplified fragment length polymorphism (AFLP). In some embodiments the marker is a polymerase chain reaction primer sequence selected from the group consisting of SEQ ID NOs: 1-90 and at least one primer which is a variant 20 of any one of said SEQ ID NOs: 1-90. In some embodiments the germplasm is isolated from a soybean plant selected from the group consisting of accession PI567598B, accession PI567543C, accession PI567541B, accession PI567597C, line E06902 deposited under ATCC accession No: PTA-8794, hybrids thereof and progeny thereof. In some embodiments the composition is created from the group consisting of a polymerase chain reaction product, a restriction fragment length polymorphism analysis, amplification fragment length polymorphism analysis, a microsatellite amplification, hybridization, and a southern blot.
The present inventions provide a method of identifying a marker for aphid resistance, comprising, a) providing, i) a molecular marker, and ii) a first soybean plant germplasm, wherein said germplasm comprises nucleic acid sequences isolated from a soybean plant having a phenotype of aphid resistance, and iii) a second soybean plant germplasm, wherein said germplasm comprises nucleic acid sequences isolated from a soybean plant having less aphid resistance than the first soybean plant, and b) detecting a product that is different from the second soybean plant germplasm. In one embodiment, the method further comprises hybridizing the molecular marker to said nucleic acid sequences from said first soybean plant and said second soybean plant, wherein said detecting is the presence and absence of a hybridization product. In one embodiment, the method further provides a second molecular marker and comprising hybridizing said second molecular marker to said nucleic acid sequences from said first and said second soybean plant, wherein said detecting is the presence and absence of a hybridization product. In one embodiment, the method further comprises nucleic acid amplification. In some embodiments the molecular marker comprises a primer set consisting of a forward and reverse primer to replicate at least a portion of the nucleic acid sequences. In some embodiments the primer set is selected from the group consisting of SEQ ID NOs: 1-90 and at least one primer sequence which is a variant of any one of said SEQ ID NOs: 1-90. In some embodiments the said detecting a product is an observation of a band present in germplasm from an aphid resistant soybean plant that is not present in the germplasm from a soybean plant with less aphid resistance. In some embodiments the said observation of a band is an observation of a band of a certain molecular weight that is not present in the germplasm from a soybean plant with less aphid resistance. In some embodiments the said band is an allele present in germplasm from an aphid resistant soybean plant that is not present in the germplasm from a soybean plant with less aphid resistance. In some embodiments the said observation of a band is an observation of a band pattern. In some embodiments the said detecting is using a technique selected from the group consisting of restriction fragment length polymorphism analysis, amplification fragment length polymorphism analysis, microsatellite amplification, southern blotting, and nucleic acid sequencing. In some embodiments the detected allele is present in a soybean plant having a phenotype of enhanced aphid resistance.
The present inventions provides a method of breeding, comprising, a) providing, i) a first soybean plant comprising aphid resistant germplasm, ii) second soybean plant, and iii) a molecular marker, and b) crossing said first soybean plant with said second soybean plant, and using said molecular marker for identifying germplasm associated with aphid resistance. In some embodiments the first soybean plant is selected from the group consisting of accession PI567598B, accession PI567543C, accession PI567541B, accession PI567597C, line E06902 deposited under ATCC accession No: PTA-8794, hybrids thereof and progeny thereof. In some embodiments the marker identifies a region of a linkage group selected from the group consisting of F, M, and J. In some embodiments the marker identifies a marker selected from the group consisting of Satt343, Satt193. Satt649, Satt150, Satt435, Sat—299, Sat—339, Satt414, Satt654, Satt596, Satt686, Satt622, Satt380, Satt596, Satt456, Satt529, Satt183, Satt215, Sat—366, Sat—350, Satt380, Satt596, Satt456, Satt529, Satt183, Satt215, Sat—366, and Sat—350. In some embodiments the first soybean plant is selected from the group consisting of accession PI 567541B, line E06902 deposited under ATCC accession No: PTA-8794, hybrids thereof and progeny thereof. In some embodiments the marker is selected from a linkage group selected from the group consisting of F and M. In some embodiments the marker for linkage group F is selected from the group consisting of Satt343, Satt193, and Satt649. In some embodiments the marker for linkage group M is selected from the group consisting of Satt150, Satt435, and Sat—299. In some embodiments the second soybean plant is selected from the group consisting of aphid resistant soybean plants and susceptible soybean plants. In some embodiments the crossing further comprises one or more of a backcrossing, an outcrossing, and a self-crossing. In some embodiments the crossing creates soybean seed. In some embodiments the seed is germinated and grown into progeny soybean plants. In some embodiments the progeny soybean plants provide germplasm for use with said markers. In some embodiments the using said markers is identifying the aphid resistant germplasm in the first soybean plants.
The present inventions provides a method, comprising, a) providing, ii) a soybean plant comprising aphid resistant germplasm, iii) a molecular marker, and b) using said molecular marker for identifying aphid resistant germplasm in a soybean plant. In one embodiment, the method further comprises testing a line of said soybean plants for an aphid resistant phenotype.
The present invention comprises a method, comprising, providing, a first aphid resistant soybean plant, wherein said plant aphid resistant germplasm comprises antibiosis aphid resistant germplasm and a second soybean plant, wherein said plant comprises antixenosis aphid resistance germplasm, and growing said first soybean plant until aphids overcome resistance then growing said second soybean plants.
The present invention comprises a method, comprising, providing, a first aphid resistant soybean plant, wherein said plant comprises aphid resistant germplasm derived from accession PI567598B, accession PI567541B, and line E06902 deposited under ATCC accession No: PTA-8794, hybrids thereof and progeny thereof and a second soybean plant wherein said plant is a resistant soybean plant, wherein said plant comprises aphid resistant germplasm derived from accession PI 567543C, accession PI 567597C, PI567598B, accession PI567541B, and line E06902
DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and phrases are defined below:
The use of the article “a” or “an” is intended to include one or more.
As used herein, the term “aphid” refers to any of various small plant-sucking insects with or without wings of the order Homoptera, further of the family Aphididae, wherein examples of Aphididae include but are not limited to a genius of Aphis, Acyrthosiphum, Brevicoryne, Cavariella, Chaitophorus, Cinara, Diuraphis, Drepanosiphum, Elatobium, Macrosiphum, Megoura, Myzus, Phorodon, Rhopalosiphum, Sitobion, Toxoptera, Therioaphis, Tuberocephalus, etc. while even further any one or more of the following genus-species of Aphis, for example, soybean aphid Aphis glycines Matsumura, Black Bean Aphid Aphis faba, Groundnut Aphid Aphis craccivora Cotton Aphid Aphis gossypii cotton/melon aphid, Aphis gossypii, Acyrthosiphum, for example, Pea Aphid Acyrthosiphum pisum, Brevicoryne, for example, Cabbage Aphid Brevicoryne brassicae, Cavariella, for example, Carrot Aphid Cavariella aegopodii Willow Aphid Cavariella spp. Chaitophorus, for example, Willow Leaf Aphids Chaitophorus spp., Cinara, for example, Black Pine Aphids Cinara spp., Diuraphis, for example, Russian wheat aphid Diuraphis noxia, Drepanosiphum, for example, Sycamore Aphid Drepanosiphum platanoides, Elatobium, for example, Spruce Aphids Elatobium spp., Macrosiphum, for example, English Grain Aphid Macrosiphum avenae, Megoura, for example, Vetch aphid Megoura viciae, Myzus, for example, Peach-Potato Myzus persicae, Phorodon, for example, Damson hop aphid Phorodon humuli, Sitobion, for example, Grain Aphid Sitobion avenae, Rhopalosiphum for example, Corn Leaf Aphid Rhopalosiphum maidis, the Oat Bird-Cherry Aphid Rhopalosiphum padi Toxoptera, for example, Black Citrus Aphid Toxoptera auranti, Therioaphis, for example, spotted alfalfa aphid Therioaphis maculata, Tuberocephalus, for example, peach aphid Tuberocephalus momonis, Giant Willow aphid Tuberolachnus salignus (aka Lachnus salignus) Gmellin and the like. For the purposes of the present invention, an aphid is a pest.
As used herein, the terms “soybean aphid” and “Aphis glycines” and “Aphis glycines Matsamura” refers to an aphid that feeds on soybeans, for example, an aphid that derived from an eastern Asian soybean aphid. However for the compositions and methods of the present invention, any aphid that may be found on and thus potentially feed on a soybean plant, such as a cotton/melon aphid, Aphis gossypii Glover, is an aphid target for aphid soybean resistance.
As used herein, the term “biotype” in reference to an aphid refers to an aphid population having different characteristics from another aphid population, for example, see, Kim K, Hill C B, Hartman G L, Mian M R, Diers B W (2008) Discovery of soybean aphid biotypes. Crop Sci 48:923-928.
As used herein, the terms “arthropoda” and “arthropods” refer to a branch (phylum) of the animal kingdom whose members have jointed legs and are also made up of rings or segments, for example, Insecta, crustaceans, spiders, and the like. As used herein, some arthropod larvae (for example, grubs and maggots) are legless while spiders and ticks have four pairs of jointed legs.
As used herein, the terms “insect” and “Insecta” refer to a Class of Arthropoda whose members have a body with distinct head, thorax and abdomen; the head bears one pair of antennae and paired mouthparts; the thorax bears three pairs of legs and one or two pairs of wings in winged insects (Pterygota) and none in primarily wingless insects (Apterygota); the abdomen bears no legs but other appendages might be present with three pairs of jointed legs and one pair of antennae, at least in the adult phase, for example, aphids, Lepidoptera, such as butterflies and moths, Coleoptera, such as Beetles, have this arrangement in the adult phase. As used herein, some insect larvae (for example, grubs) are legless.
As used herein, the terms “Nematoda” or “nemathelminths” refer to a branch (phylum) of the animal kingdom whose members include “nematode” and “roundworm” organisms that are bilaterally symmetrical and surrounded by a strong and flexible noncellular layer called a cuticle, such as a Heterodera glycines soybean cyst nematode.
As used herein, the terms “Sudden Death Syndrome” or “SDS” refer to a fungal disease of soybeans caused by a fungus, such as Fusarium solani fungus.
As used herein, the terms “Sclerotinia Stem Rot,” “SSR” or “white mold” refer to a soilborne disease caused by a fungus Sclerotinia sclerotiorum. 
As used herein, the term “Rhizoctonia Root Rot” refers to a soil borne disease resulting in root rot and stunting of plant growth caused by a fungus Rhizoctonia solani. 
As used herein, the terms “Phytophthora rot” in reference to a plant part, such as Phytophthora seed rot, Phytophthora stem rot or Phytophthora root rot, refers to a disease caused by a Phytophthora fungus.
As used herein, the term “damping-off” refers to a fungal disease in the soil causing seedlings to wilt and die, such as caused by Pythium ultimum. 
As used herein, the terms “Pythium rot” in reference to a plant part, such as a Pythium seed rot, Pythium stem rot or Pythium root rot or Pythium seed decay, refers to a disease caused by a fungus Pythium ultimum. 
As used herein, the terms “Phomopsis seed rot” refers to a disease caused by seed-borne fungi, Phomopsis longicolla, Diaporthe phaseolorum var. sojae, and D. phaseolorum var. caulivora. 
As used herein, the term “powdery mildew” refers to fungal growth that appears as a white fuzzy coating on the upper leaves.
As used herein, the term “seedling blight” refers to a disease causing weakened or killed seedlings.
As used herein, the term “mottling” refers to a discoloration of a plant part, such as seed mottling, which is not fungal in origin. Mottling of soybean seed is caused by viruses such as Bean Pod Mottle Virus (BPMV) and Soybean Mosaic Virus (SMV).
As used herein, the term “Bean pod mottle virus” and “BPMV” refers to a virus with small isometric particles and a single-stranded RNA genome that is beetle-transmitted, such as Leaf-feeding beetles (Coleoptera) belonging to Cerotoma trifurcata, Colaspis brunnea, C. lata, Diabrotica balteata, D. undecimpunctata howardi, Epicauta vittata, and Epilachna varivestis, to soybean and causes a mottling of soybean leaves.
As used herein, the term “Soybean Mosaic Virus” and “SMV” refers to a flexuous rod consisting of positive-sense, single-stranded RNA infected cultivars are slightly stunted with fewer pods that are sometimes dwarfed and flattened, without hairs, and without seeds. At least 32 aphid species, belonging to at least 15 different genera, transmit SMV in a nonpersistent manner.
As used herein, the term “Tobacco ringspot virus” and “TRSV” refers to a bud blight causing nepovirus group of plant viruses with two single-stranded positive sense polyadenylated RNA molecules transmitted by nymphs of Thrips tabaci. 
As used herein, the term “bacterial pustule” refers to an undesired physical condition, primarily of leaves and pods as the result of an infection, primarily a disease of leaves and pods of a plant [caused by Xanthomonas campestris pv. Glycinea. 
As used herein, the term “bacterial blight” refers to a disease caused by bacteria, such as Pseudomonas savastonoi pv. Glycinea. 
As used herein, the terms “rust” or “soybean rust” or “Leaf Rust” or “Asian soybean rust” refer to a fungal disease, such as that caused by fungi such as Phakopsora pachyrhizi. 
As used herein, the terms “Bacillus thuringiensis” and “Bt” in reference to a toxin refers to insecticidal compounds, such as crystals and proteins, naturally produced by a Bacillus thuringiensis bacterium and modified by man for agricultural use.
As used herein, the term “host” refers to any organism (animal or plant) fed upon by a parasite or parasitoid. As used herein, when insects or nematodes feed upon plants they are considered parasites of those plants, and the plants are then referred to as “host plants.”
As used herein, the term “plant” is used in its broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable, fruit plant or vegetable plant, flower or tree, macroalga or microalga, phytoplankton and photosynthetic algae (e.g., green algae Chlamydomonas reinhardtii). A plant also refers to a unicellular plant (e.g. microalga) and a plurality of plant cells that are largely differentiated into a colony (e.g. volvox) or a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, a seed, a shoot, a stem, a leaf, a flower petal, etc.
As used herein, the term “soybean plant” refers to a legume plant of the family Fabaceae, herein used in its broadest sense and includes but is not limited to any species of soybean, for example, a Glycine species. A soybean plant may be a Glycine max, such as a cultivated soybean plant, a Glycine soia [Sieb. & Zucc.], such as a wild form of soybean, and a Glycine gracilis Skvortz, such as a weedy form of soybean. The present invention is not meant to limit the type of soybean plant. Indeed numerous varieties of aphid resistant soybean plants are contemplated. In some embodiments, an aphid resistant soybean plant provides human food-grade soybeans, such as for soymilk, soynuts, whole soybeans, miso, tofu (such as soybean curd), tempeh, soy sauce (such as shoyu, tamari and teriyaki sauce), soybean oil, margarine, salad oil, and the like. In some embodiments, a human food-grade aphid resistant soybean provides pharmaceutical products, such as for cancer prevention, for example, providing genistein.
As used herein, the term “soybean” refers to a seed of a soybean plant.
As used herein, the term “seed” refers to a fertilized and ripened ovule of a plant, consisting of an embryo and a casing, such as a bean and a soybean, for example, a soybean is a seed.
As used herein, the term “pod” refers to a seed of a soybean plant.
As used herein, the term “hybrid” in reference to a seed or plant is produced as the result of controlled cross-pollination as opposed to a non-hybrid seed produced as the result of natural pollination, as in a “hybrid soybean seed” produced by breeding methods of the present invention.
The terms “leaf” and “leaves” refer to a usually flat, green structure attached to a stem or branch of a plant wherein photosynthesis and transpiration take place.
The term “stem” refers to a main ascending axis of a plant.
The term “node” refers to the joint of a stem and the region of attachment of leaves on a stem.
As used herein, the term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture.
As used herein, the term “plant part” as used herein refers to a plant structure or a plant tissue, for example, pollen, an ovule, a tissue, a pod, a seed, and a cell. In some embodiments of the present invention transgenic plants are crop plants.
As used herein, the terms “crop” and “crop plant” is used in its broadest sense. The term includes, but is not limited to, any species of plant or alga edible by humans or used as a feed for animals or fish or marine animals, or consumed by humans, or used by humans (natural pesticides), or viewed by humans (flowers) or any plant or alga used in industry or commerce or education.
As used herein, the term “host plant resistance” refers to any one of the preferred methods for minimizing the damage caused by aphids, insects, pests, bacteria, virus, fungi and the like.
As used herein, the terms “resistant” and “resistance” refer to aphid resistance, arthropod resistance, nematode resistance, such as resistance to a soybean cyst nematode pathogen resistance or disease resistance, such as resistance to Seed Mottling caused by viruses, such as Bean Pod Mottle Virus (BPMV) and Soybean Mosaic Virus (SMV), Sudden Death Syndrome (SDS) caused by a fungus Fusarium solani, bacterial pustule caused by Xanthomonas campestris pv. Glycines, etc., fungus resistance, such as soybean rust resistance, and the like.
As used herein, the term “increasing resistance” refers to increasing the ability of a host plant to repel an insect, such as an aphid, nematode, etc., pathogen, fungus, virus, disease, and the like, including by decreasing the physical impact on or damage to the plant of the particular insect, pathogen, disease, and/or nematode attack on a host plant, such as reducing the feeding activity of an aphid, reducing the feeding activity of an insect, reducing the feeding activity of an insect larvae, reducing the number of parasitic nematodes on a plant, reducing the number of parasitic nematodes on a plant, reducing egg laying activity of an insect, reducing the symptoms of infection such as stem rot, root rot, seed mottling, and the like. Increasing resistance also refers to increasing the ability of the host plant to diminished and/or entirely avoid infestation and damage by an aphid, an insect, a bacterium, a fungi, a virus, and a parasitic organism, for example, increasing soybean cyst nematode resistance in a soybean line see, U.S. Pat. No. 6,096,944, herein incorporated by reference, an infection, a disease, a fungus, and the like.
As used herein, the terms “Soybean Cyst Nematode” or “SCN” refer to small roundworms, such as Heterodera glycines, that cause root damage and subsequent above-ground disease symptoms to soybeans. At least sixteen physiological races of the SCN have been identified.
As used herein, the term “resistance” in reference to a plant, means a situation wherein insects and/or pathogens are prevented and/or inhibited from causing plant damage and/or diseases and the associated disease symptoms, or alternatively, some or all of the disease symptoms caused by the pathogen are minimized or lessened. This includes but is not limited to the ability of a host to prevent aphid and/or nematode reproduction.
As used herein, the terms “resistant” and “resistance” in reference to insect resistance, refers to aphid resistance or arthropod resistance.
As used herein, the terms “aphid resistant” and “aphid resistance” refers to a capacity of a host plant to prevent and/or reduce the ability of an aphid to damage a host plant, such as reducing feeding, reducing development and the like, when an aphid is in contact with an aphid resistant plant.
As used herein, the term “antibiosis” “antibiosis resistance” and “antibiosis resistance toxicity” in relation to aphid resistance refers to a trait for preventing the aphids from reproducing on plants, for example, as shown in a no-choice study of the present invention, see, EXAMPLE 5. In some embodiments, aphid resistance in a cultivar is indicated by antibiosis resistance preventing the aphids from reproducing on the plants in a no-choice study as compared to a non-resistant cultivar. Examples of soybean cultivars of the present invention showing antibiosis resistance are PI 567541B and PI 567598B. Antibiosis further refers to adverse effects on an insect's life history after a resistant host plant has been used for food (for example, in Painter, Insect Resistance in Crop Plants, Macmillan, New York (1951)).
As used herein, the term “toxin” refers to any substance (usually a protein or conjugated protein) that is detrimental (i.e., poisonous) to cells and/or organisms, in particular an insect organism, i.e. an aphid insect as in an insecticidal substance. In particularly preferred embodiments, the term refers to extracellular toxins and intracellular toxins produced by various plant species, including, but not limited to a soybean plant toxin that provides toxicity activity toward members of the genus Glycine. However, it is not intended that the present invention be limited to any particular toxin or plant species. Indeed, it is intended that the term encompass toxins produced by any organism. In one embodiment, an aphid toxin results in the death of an aphid. In one embodiment, an insecticidal protein results in the death of an insect.
As used herein, the terms “antixenosis” and “antixenosis resistance” in relation to aphid resistance refer to a trait for nonpreference of insects for a host plant (for example, Kogan and Ortman, (1978) Bull. Entomol. Soc. Am. 24:175-176), for example, “repellency to aphids” and “aphid repellent” in reference to soybean cultivars of the present invention demonstrating antixenosis resistance are PI 567543C and PI 567597C.
As used herein, the term “repellent” such as an “insect repellent” and an “aphid repellent” refers to a substance, such as a protein, that will ward off and/or keep away and insect, for example, an aphid, as to “repel” as in “repelling an aphid.”
As used herein, the term “agronomic trait” and “economically significant trait” refers to any selected trait that increases the commercial value of a plant part, for example, a preferred oil content, protein content, seed protein content, seed fatty acid content, seed size, seed color, hilium color, seed coat thickness, seed sugar content, seed free amino acid content, seed germination rate, seed texture, seed fiber content, seed Vitamin E content, seed isoflavone content, seed phytate content, seed phytosterol content, seed isoflavone content, lecithin content, food-grade quality, hilium color, seed yield, plant type, plant height, lodging, shatter, herbicide resistance, disease resistance, insect resistance, nematode resistance, drought tolerance, drought resistance, water tolerance, water resistance, temperature tolerance, such as cold weather resistance, hot weather resistance, and the like, growth habit, maturity group, field tolerance, and growth in a hardiness zone.
As used herein, the term “fatty acid” refers to a chemical unit occurring either as a single molecule or a molecule of at least 2 or more combined fatty acid units, wherein a fatty acid unit comprises any number of carbon (C), hydrogen (H), and oxygen (O) atoms arranged as a carbon chain skeleton with a carboxyl group (—COOH) at one end. A fatty acid may be a saturated fatty acid or an unsaturated fatty acid. Examples of fatty acids found in soybeans include but are not limited to palmitic, stearic, oleic, linoleic, and linolenic.
As used herein, the terms “saturated fatty acid,” “SFAs,” “hydrogenated fatty acid” refer to fatty acid molecules or chains of fatty acid molecules without double bonds between the carbon atoms for example, palmitic acid.
As used herein, the term “monounsaturated fatty acids” “MUFAs” refers to a fatty acid molecule with no more than one double bond, for example, oleic acid.
As used herein, the term “polyunsaturated fatty acids” “PUFAs” refers to a fatty acid molecule having more than one double bond, for example, linoleic acid, and linolenic acid found in soybean oil, wherein linolenic acid is an omega-3 polyunsaturated fatty acid that under certain conditions causes soybean oil to become rancid.
As used herein, the term “lecithin” refers to a naturally occurring emulsifier extracted from crude soybean oil.
As used herein, the terms “isoflavone” and “isoflavonoid” refer to a polyphenol molecule or phytoestrogen molecule or estrogen-like molecule found in soybeans, for example, genistein (genistin) a 4′,5,7-trihydroxy-isoflavone or a 5,7-dihydroxy-3-(4-hydroxyphenyl)-4-benzopyrone of molecular formula C15-H10-O5 and CAS Registry Number 446-72-0; daidzein (daidzin) 4′,7-dihydroxy-(8CI) Isoflavone or 4′,7-dihydroxyisoflavone of molecular formula C15-H10-O4 and CAS Registry Number 486-66-8; glycinin of CAS Registry Number 9007-93-6; and glycitein a 7-hydroxy-3-(4-hydroxyphenyl)-6-methoxy-4H-1-benzopyran-4-one of molecular formula C16-H12-05 and CAS Registry Number 40957-83-3.
As used herein, the term “shatter” in reference to shattering refers to a percentage of open pods determined at the time of harvest.
As used herein, the term “lodging” refers to measurement of soybean plants leaning or having fallen or laying on the ground at harvest.
As used herein, the term “growth habit” refers to indeterminate growth habit or determinate growth habit of a soybean plant, in particular, to a growth habit of a variety of soybean plant. For example, indeterminate soybean plant varieties are adapted to maturity group IV and earlier (northern U.S.) have overlapping vegetative and reproductive growth periods. On the other hand, determinate soybean plant varieties with a determinate growth habit are adapted to maturity group V and later (southern U.S.) having distinct vegetative and reproductive development periods.
As used herein, the term “plant type” refers to a physical characteristic of a plant ranging from highly branching types to thin-line types that produce a single main stem.
As used herein, the term “subgenus” in reference to a soybean plant refers to one or more of a “soja” and a “soia,” a “max” and a “glycine,” wherein a soja and a soia refer to a wild-type soybean plant and a reseeding soybean plant while max and glycine refer to a cultivated plant.
As used herein, the term “soybean maturity group” refers to an industry division of groups of varieties based on the zones in which the varieties are adapted. Soybean maturity groups range from 000-X, wherein 000 represents the earliest and X the latest. Plants adapted to northern day-lengths are classified as early-maturing; those adapted to the southern regions are classified as late-maturing. Maturity groups may include very long day length varieties (000, 00, 0) and extend to very short day length varieties (VII, VII, IX, X). For example, maturity group I soybean cultivars are typically grown in southern Minnesota, whereas maturity group IV soybean cultivars are typically grown in southern Illinois.
As used herein, the term “early maturing” or “early maturity group” in reference to a variety, line or cultivar of a soybean plant refers to soybean plants assigned to a maturity group ranging from 000 to III.
As used herein, the term “early season” or early season variety” in reference to a U.S. variety refers to a variety, line or cultivar of a soybean plant assigned to a maturity group ranging from 000 to IV.
As used herein, the term “relative maturity” when used in reference to a soybean plant maturity group subdivides a maturity group into tenths and provides a more precise maturity assignment, for example, a relative maturity of 3.3 is assigned to a later maturing early maturity group III soybean cultivar than a 3.0 soybean cultivar. The number following the decimal point refers to the relative earliness or lateness within each maturity group, for example, a 3.0 is an early group III variety, while a 3.9 is a late group III variety.
As used herein, the term “line” refers to a nursery term to describe a group of individuals from similar parentage with similar traits; for example, E98076 is a soybean line developed at Michigan State University from the cross DSC Dairyland 217×30 Northrup King S19-90 and lines E06906, E06902, E06907, E06901, and E06904 are soybean lines developed at Michigan State University from crossing Titan×PI 567598B.
As used herein, the term “cultivar” refers to an unvarying variety of plant propagated by man using selective hybridization and maintained by vegetative propagation or by inbred seed.
As used herein, the term “soybean cultivar” is used in its broadest sense and includes but is not limited to any species of soybean that is cultivated by man.
As used herein, the term “cultivated” in reference to a plant includes any plant or plant part grown and maintained by man for use in food compositions or in nonfood compositions.
As used herein, the term “group” in reference to a plant refers to an artificial category between species and cultivar used to designate a collection of cultivars with similar parentage.
As used herein, the terms “variety” and “varietas” and “var” refer to a rank of taxa below subspecies but above form a for example a plant which retains most of the characteristics of the species, but differs in some way such as seed oil content, seed color, seed size, insect resistance, soybean aphid resistance, and the like.
As used herein, the terms “F-generation” and “filial generation” refers to any of the consecutive generations of cells, tissues or organisms after a biparental cross. The generation resulting from a mating of the a biparental cross (i.e. parents) is the first filial generation (designated as “F1” and “F1”) in reference to a seed and it's plant, while that resulting from crossing of F1 individuals is the second filial generation (designated as “F2” or “F2”) in reference to a seed and it's plant. For example, an F2 seed and a resulting plant are produced by self-pollination of F1, while later F generations are produced from self-pollination of the immediate prior generation.
As used herein, the terms “plant introductions” and “PI” refers to a plant accession number that can be assigned by the USDA Plant Introduction Office, for example, PI 567597C; PI 567543C; PI 567598B; and PI 567541B.
As used herein, the terms “germplasm” refers to any genetic material of plants, animals or other organisms containing functional units of heredity, in other words germplasm comprises nucleic acid molecules.
As used herein, the term “germplasm” in reference to “aphid resistant germplasm” and “aphid resistance germplasm” refers to and encompasses hereditary material that provides resistance to aphids, in particular resistance to soybean aphids.
As used herein, the term “aphid resistant gene” or “rag” refers to an expressed DNA sequence found within aphid resistant germplasm, for example, rag1b, rag1c, rag3, Rag3 and rag4 are aphid resistant genes of the present inventions located in between molecular markers of the present inventions. The presence of an aphid resistant gene may also be identified by markers found within the aphid resistant germplasm in regions of aphid resistant soybean plants that are absent from regions of germplasm located in similar areas of matching chromosome in susceptible soybean plants. When lower case letters for rag are used the gene is generally considered recessive.
The term “transfection” as in “transfecting a plant cell” or “transfecting a plant tissue,” refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment) and the like.
The term “isolated” when used in relation to a nucleic acid or polypeptide sequence, as in “isolated aphid resistance gene” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a particular protein includes, by way of example, such nucleic acid in cells ordinarily expressing the protein, where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).
The term “purified” refers to molecules, either nucleic or amino acid sequences that are removed from their natural environment isolated or separated. An “isolated nucleic acid sequence” is therefore a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.
As used herein, the term “presence” in relation to an aphid resistant gene refers to when a marker associated with that gene hybridizes or amplifies an aphid resistant gene resulting in a DNA sequence of the appropriate size or sequence. Conversely, the term “absent” in relation to an aphid resistant gene refers to when a marker associated with that gene does not hybridize or does not result in amplification of a fragment of an appropriate size or sequence using PCR primers for said marker.
As used herein, the term “elite germplasm” in reference to a soybean cultivar or line refers to soybean plant hereditary material of proven genetic superiority, for example, a commercial cultivar of a soybean plant, such as a “Round-up ReadyTM” soybean plant, a soybean plant having a desired agronomic trait, such as lodging, oil content, nematode resistance, et cetera, and for the purposes of the present inventions, elite germplasm includes aphid resistant germplasm of the present inventions.
As used herein, the term “elite plant,” “elite soybean plant,” “elite soybean plant line,” or “elite soybean plant cultivar” refers to any plant, plant line or plant cultivar, respectively, that has resulted from breeding and selection for superior agronomic performance. For example, elite soybean cultivar and elite soybean germplasm refer to isolated soybean cultivars comprising a transgene that provide resistance to herbicides, nematodes, fungi, or an agronomic trait including but not limited to PI257345 and its progeny S1346, PI71506, Hutcheson, Resnik, Lincoln, Richland, Patoka, PI 81041, Illini, PI 54610, PI 88788, Mukden, Palmetto, Haberlandt No. 171, PI 257345, PI 71506, Lincoln, Mandarin (Ottawa), PI 90763, CNS, PI 209332, Richland, Tokyo, S-100, Minsoy, Ogden, Noir 1, A.K. (Harrow), Archer, Dunfield, Evans, Mukden, Clark, Jackson, Harosoy, Illini, Essex, Roanoke, PI 88788, Peking, Asgrow AG4201, Asgrow AG3703, Croplan Genetics RC4432, FFR RT446, HARTZTM variety H4994, HARTZTM variety H5218, HARTZTM variety H5350, HARTZTM variety H5545, HARTZTM variety H5050, HARTZTM variety H5454, HARTZTM variety H5233, HARTZTM variety H5488, HARTZTM variety HLA572, HARTZTM variety H6200, HARTZTM variety H6104, HARTZTM variety H6255, HARTZTM variety H6586, HARTZTM variety H6191, HARTZTM variety H7440, HARTZTM variety H4452 Roundup ReadyTM, HARTZTM variety H4994 Roundup ReadyTM, HARTZTM variety H4988 Roundup ReadyTM, HARTZTM variety H5000 Roundup ReadyTM, HARTZTM variety H5147 Roundup ReadyTM HARTZTM variety H5247 Roundup ReadyTM, HARTZTM variety H5350 Roundup ReadyTM, HARTZTM variety H5545 Roundup ReadyTM, HARTZTM variety H5855 Roundup ReadyTM, HARTZTM variety H5088 Roundup ReadyTM, HARTZTM variety H5164 Roundup ReadyTM, HARTZTM variety H5361 Roundup ReadyTM, HARTZTM variety H5566 Roundup ReadyTM, HARTZTM variety H5181 Roundup ReadyTM HARTZTM variety H5889 Roundup ReadyTM, HARTZTM variety H5999 Roundup ReadyTM, HARTZTM variety H6013 Roundup ReadyTM, HARTZTM variety H6255 Roundup ReadyTM, HARTZTM variety H6454 Roundup ReadyTM, HARTZTM variety H6686 Roundup ReadyTM, HARTZTM variety H7152 Roundup ReadyTM HARTZTM variety H7550 Roundup ReadyTM, HARTZTM variety H8001 Roundup ReadyTM (HARTZ SEED, Stuttgart, Ark., USA); A0868, AG0901, A1553, A1900, AG1901, A1923, A2069, AG2101, AG2201, A2247, AG2301, A2304, A2396, AG2401, AG2501, A2506, A2553, AG2701, AG2702, AG2703, A2704, A2833, A2869, AG2901, AG2902, AG2905, AG3001, AG3002, A3204, A3237, A3244, AG3301, AG3302, A3404, A3469, AG3502, AG3503, A3559, AG3601, AG3701, AG3704, AG3750, A3834, AG3901, A3904, A4045 AG4301, A4341, AG4401, AG4501, AG4601, AG4602, A4604, AG4702, AG4901, A4922, AG5401, A5547, AG5602, A5704, AG5801, AG5901, A5944, A5959, AG6101, AJW2600COR, FPG26932, QR4459 and QP4544 (Asgrow Seeds, Des Moines, Iowa, USA); DKB26-52, DKB28-51, DKB32-52, DKB35-51 and DeKalb variety CX445 (DeKalb, Ill., USA); 91B91, 92B24, 92B37, 92B63, 92B71, 92B74, 92B75, 92B91, 93B01, 93B11, 93B26, 93B34, 93B35, 93B41, 93B45, 93B51, 93B53, 93B66, 93B81, 93B82, 93B84, 94B01, 94B32, 94B53, 95B71, 95B95, 9306, 9294, and 9344 (Pioneer Hi-bred International, Johnstonville, Iowa, USA), A2704-12, A2704-21, A5547-35 (Aventis CropScience), A5547-127, GU262, W62, W98, (Bayer CropScience (Aventis CropScience (AgrEvo))), G94-1, G94-19, G168 (DuPont Canada Agricultural Products), GTS 40-3-2 (Monsanto Company), OT96-15 (Agriculture & Agri-Food Canada), Maple Glen, PI361088B, Ohio FG1, Agracola Farms AF271, Burtch Seed BBF44, H.A.P.I. Ohio GL2930, LG Seed EX230FG, Wellman WFG268, Line Trelay 230 (comprising Phytophthora resistance germplasm), Trelay 271 (comprising Phytophthora resistance germplasm), and the like. For the purposes of the present inventions, an elite soybean plant also refers to plants related to accessions PI567598B, PI567543C, PI567541B, and PI567597C comprising aphid resistant germplasm of the present invention.
As used herein, the term “hybrid” refers to a seed and a plant produced as the result of controlled pollination as opposed to a seed and a plant produced as the result of natural pollination.
As used herein, the term “trait” refers to an observable and/measurable characteristics of an organism, such as a trait of a plant, for example, resistance to a soybean aphid, tolerance to an herbicide, an agronomic trait, insect, and microbe.
As used herein, the terms “marker” and “DNA marker” and “molecular marker” in reference to a “selectable marker” refers to a physiological or morphological trait which may be determined as marker for its own selection or for selection of other traits closely linked to that marker, for example, a gene or trait that associates with aphid resistance, such as a marker, such as a DNA marker including but not limited to simple sequence repeat (SSR), single nucleotide polymorphism analysis (SNP), random amplified polymorphic DNA analysis (RAPID), amplified fragment length polymorphism analysis (AFLP), and the like that will link phenotype information, such as aphid resistance to a QTL locus, to provide a genomic map, for example a fingerprint map, and chromosome location and/or map. Examples of SSR markers include but are not limited to “Satt” markers.
As used herein, “Satt” markers refer to forward and reverse PCR primers used for amplifying a genomic marker fragment, in particular for identifying a “linkage group.”
As used herein, the term “linkage group” refers to a group of two or more genetically or physically mapped loci with observed linkage to a trait, for example, one or more of a SSR, SNP, AFLP, and RAPD marker of the present invention that may map to aphid resistant germplasm. Examples of soybean linkages groups that are associated with aphid resistant germplasm comprise, for example, J, B2, D1a, D1b and K.
As used herein, the term “selection” as used herein refers to the process of determining the relative aphid resistance of a soybean cultivar.
As used herein, the term “introgress” and “introgressing” refers to incorporating a genetic substance, such as germplasm, loci, allele, gene, DNA, and the like for introducing a trait into an organism, such as a plant, a soybean cultivar and the like, for example, incorporating aphid resistant germplasm into a previously aphid susceptible plant variety. Introgression may refer to a breeding method for a incorporating a genetic trait, such as aphid resistance, including compositions and methods for using QTL, DNA markers including but not limited to simple sequence repeat (SSR), single nucleotide polymorphism analysis (SNP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism analysis (AFLP), DNA fingerprinting, and the like for incorporating aphid resistant germplasm into a formerly aphid-susceptible plant variety.
As used herein, the terms “quantitative trait locus” and “QTL” refer to a genomic region including a gene underlying a trait on which many genes act, in other words a genetic locus that affects a quantitative trait, for example, a QTL is associated with soybean cyst nematode resistance as shown herein and in U.S. Pat. No. 6,538,175, herein incorporated by reference.
As used herein, the term “QTL” in relation to a linkage, such as “QTL J aphid resistant germplasm and the like” refers to a polymorphic location of a banding pattern of a marker unique to the soybean plants showing a specific phenotypic trait, such as an aphid resistant phenotype, and other phenotypes as described herein.
As used herein, the terms “associated region gene” or “ARG” in reference to a mapped QTL refers to the actual gene controlling or influencing a phenotype, in other words the “true QTL”
As used herein, the terms “simple sequence repeat” and “SSR” refer to short, tandem repeat nucleotide sequences that are useful as genetic markers, for example, microsatellite DNA is a highly polymorphic DNA marker comprised of mononucleotides, dinucleotides, trinucleotides or tetra-nucleotides that are repeated in tandem arrays and distributed throughout the genome, such as CA (alternatively GT) dinucleotide repeats.
As used herein, the terms “single nucleotide polymorphism” and “SNP” refer to a single base difference between two DNA sequences.
As used herein, the terms “random amplified polymorphic DNA” and “RAPD” refer to a common technique for amplifying anonymous stretches of DNA using PCR with arbitrary primers, for example, using random PCR primers used to amplify genomic DNA to provide a pattern of bands, such that one pattern of bands may be different between individuals in a population, such as between aphid resistant and aphid susceptible plants or show germplasm differences between closely related plants.
As used herein, the terms “restriction fragment length polymorphism” and “RFLP” refer to genetic variation between individuals such that DNA fragment sizes resulting from a difference in DNA sequence that affects the recognition sequence for restriction enzymes when cut by specific restriction enzymes. When a particular enzyme digests DNA the fragment sizes will differ depending on the presence or absence of the proper recognition sequence for the enzyme. Polymorphic sequences that result in RFLPs are used as markers on both physical maps and genetic linkage maps. RFLPs can be caused by a change in at least one nucleotide at a cutting site.
As used herein, the terms “amplified fragment length polymorphism” and “AFLP” refer to a highly sensitive method for detecting polymorphisms in DNA. Following restriction enzyme digestion of DNA, a subset of DNA fragments is selected for PCR amplification and visualization.
As used herein, the term “DNA fingerprinting” refers to techniques for uniquely identifying an individual among a population based on one's DNA. This type of method of isolating and visualizing sequences of DNA may show a unique pattern of DNA fragments revealed by Southern hybridization or by a polymerase chain reaction (PCR) analysis.
As used herein, the term “polymerase chain reaction” and “PCR” refer to the method of K. B. Mullis (U.S. Pat. Nos. 4,683,195 4,683,202, and 4,965,188, hereby incorporated by reference), which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing an excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.”
As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
As used herein, the term plant cell “compartments or organelles” is used in its broadest sense. As used herein, the term includes but is not limited to, the endoplasmic reticulum, Golgi apparatus, trans Golgi network, plastids, sarcoplasmic reticulum, glyoxysomes, mitochondrial, chloroplast, thylakoid membranes and nuclear membranes, and the like.
As used herein, the term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of RNA, or a polypeptide or its precursor (e.g., proinsulin). A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained.
As used herein, the term “portion” when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide.
As used herein, the term “a nucleotide comprising at least a portion of a gene” may comprise fragments of the gene or the entire gene.
As used herein, the term “gene” encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
As used herein, the terms “allele” and “alleles” refer to each version of a gene for a same locus that has more than one sequence. For example, there are multiple alleles for eye color at the same locus.
As used herein, the terms “recessive,” “recessive gene,” and “recessive phenotype” refers to an allele that has a phenotype when two alleles for a certain locus are the same as in “homozygous” or as in “homozygote” and then partially or fully loses that phenotype when paired with a more dominant allele as when two alleles for a certain locus are different as in “heterozygous” or in “heterozygote.”
As used herein, the terms “dominant,” “dominant,” and “dominant phenotype” refers to an allele that has an effect to suppress the expression of the other allele in a heterozygous (having one dominant and one recessive allele) condition.
As used herein, the term “heterologous” when used in reference to a gene or nucleic acid refers to a gene that has been manipulated in some way. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Examples of a heterologous gene includes a gene encoding an insecticidal protein, an herbicide resistant protein, or for providing an agronomic trait. Heterologous genes may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous genes are distinguished from endogenous plant genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
As used herein, the term “nucleic acid sequence,” “nucleotide sequence of interest” or “nucleic acid sequence of interest” refers to any nucleotide sequence (e.g., RNA or DNA), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, etc.), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
As used herein, the term “structural” when used in reference to a gene or to a nucleotide or nucleic acid sequence refers to a gene or a nucleotide or nucleic acid sequence whose ultimate expression product is a protein (such as an enzyme or a structural protein), an rRNA, an sRNA, a tRNA, etc.
As used herein, the term “cDNA” refers to a nucleotide copy of the “messenger RNA” or “mRNA” for a gene. In some embodiments, cDNA is derived from the mRNA. In some embodiments, cDNA is derived from genomic sequences.
As used herein, the term “oligonucleotide” refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
As used herein, the term “polynucleotide” refers to refers to a molecule comprised of several deoxyribonucleotides or ribonucleotides, and is used interchangeably with oligonucleotide. Typically, oligonucleotide refers to shorter lengths, and polynucleotide refers to longer lengths, of nucleic acid sequences.
As used herein, the term “an oligonucleotide having a nucleotide sequence encoding a gene” or “a nucleic acid sequence encoding” a specified polypeptide refers to a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence that encodes a gene product. The coding region may be present either in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers, exogenous promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
As used herein, the term “exogenous promoter” refers to a promoter in operable combination with a coding region wherein the promoter is not the promoter naturally associated with the coding region in the genome of an organism. The promoter which is naturally associated or linked to a coding region in the genome is referred to as the “endogenous promoter” for that coding region.
As used herein, the term “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
As used herein, the term “expression” when used in reference to a nucleic acid sequence, such as a gene, refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein where applicable (as when a gene encodes a protein), through “translation” of mRNA. Gene expression can be regulated at many stages in the process. “Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called “activators” and “repressors,” respectively.
As used herein, the terms “in operable combination” and “in operable order” and “operably linked” refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. As used herein, the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
As used herein, the term “regulatory element” refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237, (1987), herein incorporated by reference). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect, mammalian and plant cells. Promoter and enhancer elements have also been isolated from viruses and analogous control elements, such as promoters, are also found in prokaryotes. The selection of a particular promoter and enhancer depends on the cell type used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review, see Maniatis, et al., supra (1987), herein incorporated by reference).
As used herein, the terms “promoter element,” “promoter,” or “promoter sequence” refer to a DNA sequence that is located at the 5′ end (i.e. proceeds) of the coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.
As used herein, the term “regulatory region” refers to a gene's 5′ transcribed but untranslated regions, located immediately downstream from the promoter and ending just prior to the translational start of the gene.
As used herein, the term “promoter region” refers to the region immediately upstream of the coding region of a DNA polymer, and is typically between about 500 bp and 4 kb in length, and is preferably about 1 to 1.5 kb in length.
Promoters may be “constitutive” or “inducible.” As used herein, the term “constitutive” when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue. Exemplary constitutive plant promoters include, but are not limited to SD Cauliflower Mosaic Virus (CaMV SD; see e.g., U.S. Pat. No. 5,352,605, incorporated herein by reference), mannopine synthase, octopine synthase (ocs), superpromoter (see e.g., WO 95/14098, herein incorporated by reference), and ubi3 promoters (see e.g., Garbarino and Belknap, Plant Mol. Biol. 24:119-127 (1994), herein incorporated by reference). Such promoters have been used successfully to direct the expression of heterologous nucleic acid sequences in transformed plant tissue.
In contrast, an “inducible” promoter is one that is capable of directing a level of transcription of an operably linked nucleic acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.) that is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus.
As used herein, the term “regulatory element” refers to a genetic element that controls some aspect of the expression of nucleic acid sequence(s). For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, et cetera.
The enhancer and/or promoter may be “endogenous” or “exogenous” or “heterologous.” An “endogenous” enhancer or promoter is one that is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer or promoter is one that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of the gene is directed by the linked enhancer or promoter. For example, an endogenous promoter in operable combination with a first gene can be isolated, removed, and placed in operable combination with a second gene, thereby making it a “heterologous promoter” in operable combination with the second gene. A variety of such combinations are contemplated (e.g., the first and second genes can be from the same species or from different species).
As used herein, the term “naturally linked” or “naturally located” when used in reference to the relative positions of nucleic acid sequences means that the nucleic acid sequences exist in nature in the relative positions.
As used herein, the term “vector” refers to nucleic acid molecules that transfer DNA segment(s). Transfer can be into a cell, cell-to-cell, etc.
As used herein, the term “vehicle” is sometimes used interchangeably with “vector.”
As used herein, the term “transfection” refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment), Agrobacterium infection, and the like.
As used herein, the term “stable transfection” or “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell. As used herein, the term “stable transfectant” refers to a cell that has stably integrated foreign DNA into the genomic DNA.
As used herein, the term “transient transfection” or “transiently transfected” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell. The foreign DNA persists in the nucleus of the transfected cell for several days. During this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes.
As used herein, the term “transient transfectant” refers to cells that have taken up foreign DNA but have failed to integrate this DNA.
As used herein, the term “calcium phosphate co-precipitation” refers to a technique for the introduction of nucleic acids into a cell. The uptake of nucleic acids by cells is enhanced when the nucleic acid is presented as a calcium phosphate-nucleic acid co-precipitate. The original technique of Graham and van der Eb (Graham and van der Eb, Virol., 52:456 (1973), herein incorporated by reference) has been modified by several groups to optimize conditions for particular types of cells. The art is well aware of these numerous modifications.
As used herein, the terms “bombarding, “bombardment,” and “biolistic bombardment” refer to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample. Methods for biolistic bombardment are known in the art (e.g., U.S. Pat. No. 5,584,807, herein incorporated by reference), and are commercially available (e.g., the helium gas-driven microprojectile accelerator (PDS-1000/He, BioRad).
As used herein, the term “transgene” refers to a foreign gene that is placed into an organism by the process of transfection.
As used herein, the term “foreign gene” refers to any nucleic acid (e.g., gene sequence) that is introduced into the genome of an organism by experimental manipulations and may include gene sequences found in that organism so long as the introduced gene does not reside in the same location as does the naturally-occurring gene.
As used herein, the terms “transformants” or “transformed cells” include the primary transformed cell and cultures derived from that cell without regard to the number of transfers. Resulting progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants.
As used herein, the term “wild-type” when made in reference to a gene refers to a functional gene common throughout an outbred population. As used herein, the term “wild-type” when made in reference to a gene product refers to a functional gene product common throughout an outbred population. A functional wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene.
As used herein, the terms “modified” or “mutant” when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product which displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Thus, the terms “variant” and “mutant” when used in reference to a nucleotide sequence refer to an nucleic acid sequence that differs by one or more nucleotides from another, usually related nucleotide acid sequence. A “variation” is a difference between two different nucleotide sequences; typically, one sequence is a reference sequence.
As used herein, the term “polymorphic locus” refers to a genetic locus present in a population that shows variation between members of the population (i.e., the most common allele has a frequency of less than 0.95). Thus, “polymorphism” refers to the existence of a character in two or more variant forms in a population. A “single nucleotide polymorphism” and “SNP” refers a genetic locus of a single base that may be occupied by one of at least two different nucleotides. In contrast, a “monomorphic locus” refers to a genetic locus at which little or no variations are seen between members of the population (generally taken to be a locus at which the most common allele exceeds a frequency of 0.95 in the gene pool of the population).
As used herein, the terms “Southern blot analysis” and “Southern blot” and “Southern” refer to the analysis of DNA on agarose or acrylamide gels in which DNA is separated or fragmented according to size followed by transfer of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then exposed to a labeled probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists (J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58, herein incorporated by reference).
As used herein, the term “Northern blot analysis” and “Northern blot” and “Northern” refer to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists (J. Sambrook, et al. supra, pp 7.39-7.52, (1989), herein incorporated by reference).
As used herein, the terms “protein,” “polypeptide,” “peptide,” “encoded product,” “amino acid sequence,” are used interchangeably to refer to compounds comprising amino acids joined via peptide bonds and. A “protein” encoded by a gene is not limited to the amino acid sequence encoded by the gene, but includes post-translational modifications of the protein. Where the term “amino acid sequence” is recited herein to refer to an amino acid sequence of a protein molecule, the term “amino acid sequence” and like terms, such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an “amino acid sequence” can be deduced from the nucleic acid sequence encoding the protein. The deduced amino acid sequence from a coding nucleic acid sequence includes sequences which are derived from the deduced amino acid sequence and modified by post-translational processing, where modifications include but not limited to glycosylation, hydroxylations, phosphorylations, and amino acid deletions, substitutions, and additions. Thus, an amino acid sequence comprising a deduced amino acid sequence is understood to include post-translational modifications of the encoded and deduced amino acid sequence.
As used herein, the term “isolated” when used in relation to a nucleic acid such as an isolated DNA molecule or polypeptide, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a particular protein includes, by way of example, such nucleic acid in cells ordinarily expressing the protein, where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded).
As used herein, the term “purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated. An “isolated nucleic acid sequence” is therefore a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the term “purified” or “to purify” also refer to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of polypeptide of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.
As used herein, an “isolated soybean cultivar” refers to a soybean cultivar of the present invention removed from a Soybean Germplasm Collection, isolated or separated, and are at least 0.1% free, preferably 0.01% free, and most preferably 0.002% free from other soybean cultivars in a collection.
As used herein, an “Asian soybean cultivar” refers to a cultivar developed in and originating from soybean plants from an Asian country, for example, China and Japan.
As used herein, the term “portion” when used in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino sequence minus one amino acid.
As used herein, the term “sample” is used in its broadest sense. In one sense it can refer to a plant cell or tissue. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.
As used herein, the term “positional cloning” refers to an identification of a gene based on its physical location in the genome.
As used herein, the term “NB-ARC gene” refers to a gene having a NB-ARC homologous domain sequence containing a nucleotide-binding adaptor sequence homologous to domains in genes such as Mi gene in tomato (Milligan et al. 1998) or RPM1 gene in Arabidopsis thaliana (Grant et al. 1995).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an exemplary illustration of the visual rating scale used to establish the Damage Index (DI). 0=No aphids, plant appears normal and healthy; 1=Less than 100 aphids per plant, plant appears normal and healthy; 2=101-300 aphids per plant, mostly on the young leaves and the tender stem at top of plant, plant appears normal and healthy; 3=301-800 aphids per plant, leaves slightly curly and shiny, young leaves and stems covered with aphids; 4=More than 800 aphids per plant, plants stunted, leaves severely curled, yellow, covered with sooty mold and cast skins.
FIG. 2 shows an exemplary illustration of a general method to transfer the aphid resistance from the aphid resistant PIs to elite soybean germplasm.
FIG. 3 shows exemplary results of choice testing soybean aphid resistance in progeny of aphid resistant cultivars. These data were obtained in 2004 in a choice test in a field cage. The test procedure was described in Mensah, et al., 2005, Resistance to soybean aphid in early maturing soybean germplasm. Crop Sci. 45:2228-2233 (2005) and published online 23 Sep. 2005; herein incorporated by reference. Week 3=3 weeks after inoculation; Week 4=4 weeks after inoculation.
FIG. 4 shows exemplary resource information for a soybean aphid resistant soybean cultivar accessions PI 567597C, PI 567543C, PI 567598B, and PI 567541B at the time these accessions were obtained from the USDA Soybean Germplasm Database.
FIG. 5 shows exemplary Satt304 marker information of Linkage Group B2 in association to aphid resistant germplasm.
FIG. 6 shows exemplary Satt271 marker information associating Linkage Group D1b in association to aphid resistant germplasm.
FIG. 7 shows exemplary Satt280 marker information associating Linkage Group J with aphid resistant germplasm.
FIG. 8 shows exemplary Satt529 marker information associating Linkage Group J with aphid resistant germplasm.
FIG. 9 shows exemplary rSatt686 marker information associating Linkage Group J with aphid resistant germplasm.
FIG. 10 shows exemplary Satt628 marker information associating Linkage Group K with aphid resistant germplasm.
FIG. 11 shows exemplary Satt468 marker information associating Linkage Group D1a with aphid resistant germplasm.
FIG. 12 shows exemplary Satt439 marker information associating Linkage Group D1a with aphid resistant germplasm.
FIG. 13 shows an exemplary frequency distribution of aphid resistance for 228 recombinant inbred lines derived from a cross of PI 567541B×Skylla. FIG. 13A shows Aphid damage indices where parents are shown by arrows. AA: 3-week rating in the Weld trial, AB: 4-week rating in the Weld trial, AC: 3-week rating in the greenhouse trial, AD: 4-week rating in the greenhouse trial and FIG. 13 shows aphid ratings. BA: three-week rating in the field trial; BB: four-week rating in the field trial; BC: three-week rating in the greenhouse trial; BD: four-week rating in the greenhouse trial.
FIG. 14 shows exemplary locations of soybean aphid resistance regions in QTLs F (A-C) and M (E-F) using composite interval mapping method. 1-LOD and 2-20 LOD support intervals of each QTL are marked by thick and thin bars, respectively. Unfilled bars represent QTLs for the three-week rating in the field cage trial (Cage3WK). Black bars represent QTLs for the four-week rating in the field cage trial (Cage4WK). Bars filled with hatch lines represent QTLs for the three-week rating in the greenhouse trial (GH3WK). Bars filled with cross lines represent QTLs for the four-week rating in the greenhouse trial (GH4WK). A) A and E show maps for linkage groups F and M using a subset of 94 lines selected from within the mapping population PI 567541B×Skylla. B and F: maps for linkage groups F and M using the whole mapping population PI 567541B×Skylla. The QTL positions based on this map are listed at its right side. C and G: maps for linkage groups F and M using the validation population PI 567541B×E00003. The QTL positions based on this map are listed at its right side.
FIG. 15 shows exemplary PCR products amplified by SSR markers Satt299, Satt435, Satt343 and Satt649 within E00003 (E) aphid resistant soybean plants, Skylla (S) susceptible plants, PI567541B (P) aphid resistant soybean plants, Jackson (J), and Dowling (D) soybean plants. M=123 bp PCR marker. Arrows in the figure point to specific bands, collectively a pattern, for differentiating the soybean resistant plants (parents and progeny) of the present inventions from other soybean cultivars (lines) plants. PCR products amplified by SSR markers Satt299, Satt435, Satt649 and Satt343 for E00003 (E), Skylla (S), PI567541B (P), Jackson (J), and Dowling (D). M=123 bp PCR marker. Arrows in the figure points to the particular bands for differentiating among the populations of aphid resistant phenotypes and susceptible phenotypes present in the soybean lines, plants.
FIG. 16A-16F show exemplary QTL locations for some embodiments of aphid resistant germplasm.
FIG. 17 shows an exemplary scheme for breeding (producing) fine mapping populations of soybean plants for use in the present inventions. Populations 080107 and 080111 were produced as segregating plant populations for mapping rag1c and rag4. HRH lines were used for genotyping rag1 containing soybean plant lines. E03=susceptible parent soybean plant line E00003, E075=susceptible parent soybean plant line E00075, PI41B=PI 567541B.
FIG. 18 shows PCR products for two candidate genes (I & II) identified from Bulk Segregant Analysis (BSA) that were differentially expressed in a simple RT-PCR (A) and regular PCR (B). The different amplicons were obtained when the genomic DNAs of these cultivars were amplified by the same primers used in the RT-PCR in gene II but not in gene I. G: genomic DNA. The remaining templates were cDNA.
FIG. 19 shows exemplary results from SNP and heterozygosis of the rag1c locus.
FIG. 19A: Table 1A shows polymorphic SNPs and their genome positions on soybean chromosome 7 as identified by the BSA approach. PI 41B (PI567541B) and P1 (E07906-2) were soybean aphid resistant genotypes and P2 (Skylla) was an aphid susceptible variety. R represented aphid resistant progenies and S represented aphid susceptible progenies derived from P1×P2.
FIG. 19 B: Table 1B shows screening of 52kBeadChip SNP markers and construction of physical contig around the rag1c locus. More than four HRLs from PI567541B×E00003 were chosen. The recombinant event was captured in a RHL05-153-3 line and three RHLs of 05-383-AR6 using the 52K BeadChip to conduct the genotyping. The interval is also about 80 kbp. These RHLs displayed heterozygosis near rag1c locus.
FIG. 19 C: Table 1C shows more than five HRLs from E07906-2×Skylla population were selected. One recombination event was identified among the lines in RHL 07-757-1 using the 52K BeadChip to perform the genotyping. The interval was about 100 kbp. RHL07-757-1 showed that the heterozygosis was near rag1c locus.
FIG. 20: Table 2 shows exemplary selected candidate genes in the interval identified by the BSA as well as their Arabidopsis and rice orthologs annotated using Phytochrome. The sequence similarities among them were between 15.8-89%. TA: transcript abundance.
FIG. 21: Table 3 shows exemplary screening recombinants in the RHL fine mapping populations using TAQMAN® and KASPAR® SNPs. Recombination events were highlighted as green color for a susceptible genotype, red as a resistance genotype and yellow as a heterozygote.
FIG. 22: Table 4 shows exemplary Pearson correlations of BC3F3 fine-mapping population 3 between molecular marker MSUSNP7-18 with the trait.
FIG. 23 shows an exemplary QTL position of rag1c interval mapped using Illumina 6K HD beadchip and Taqman® and Kaspar® arrays of the present inventions. The peak of LOD score in this QTL is between MSUSNP7-4 and MSUSNP7-15. The QTL explained 19.9% phenotypic variation (a field trial). R2=19.9 Additive effect −0.58.
FIG. 24 shows an exemplary preliminary gene expression study on a rag1c candidate gene Glyma07g07110 which includes molecular marker MSUSNP7-18 A) A single amplicon was obtained in the cDNA of resistant genotype PI 567541B using the primers specific for Glyma07g07110 gene B) no significant change was observed in an adjacent NB-ARC gene (Glyma07g07150) using primers derived from the neighbor candidate gene C) amplicons from these varieties distinguished showing that the resistance gene in PI 567541B is different from the gene in Dowling, which carries Rag1 (dominant) gene. The amplicon of Dowling is similar to that of line E00003. The TA levels of the reference gene tubulin4 in these samples were similar (FIG. 1C). PI41B and PI98B are different from Dowling.
FIG. 25: Table 1 shows exemplary Polymorphic SNPs and their genomic position on soybean chromosome 13. PI41B (PI567541B) and P1 (E07906-2) were soybean aphid resistant genotypes and P2 (Skylla) was aphid susceptible variety. R represented aphid resistant progenies and S represented aphid susceptible progenies derived from P1×P2.
FIG. 26: Table 2 shows exemplary Recombination events in the RIL lines around rag4 interval. Recombinants were highlighted as green color for a susceptible genotype, red for a resistance genotype and yellow for a heterozygote. Pearson correlation value (r, P>|r|) is in the cells underneath each group of the breakpoints for the recombination. Note that 070070-17, 329 and 757AR were eliminated in the correlation analysis due to the confounded aphid resistant effect from rag1c.
FIG. 27 shows an exemplary QTL position of rag4 interval mapped using Illumina 6K HD beadchip and Taqman® and Kaspar® arrays as developed for use in the present inventions. The QTL explained 17.2% phenotypic variation (a field trial, Summer 2011) with −0.46 additive effect.
FIG. 28: Table 1 shows exemplary aphid damage index (DI) in the greenhouse and in the field cage in summer for the parents, PI 567543C and E00003, and 249 F 4 derived lines of the mapping population.
FIG. 29: Table 2 shows an exemplary summary for aphid resistance locus detected in the mapping population PI 567543C×E00003 and in the validation population PI 567543C×Skylla with aphid damage index data using the composite interval mapping method.
FIG. 30: Table 3 shows an exemplary Average aphid damage index for different genotypes of marker Satt414 in the mapping population PI 567543C×E00003.
FIG. 31 shows an exemplary frequency distribution of soybean aphid damage index for 249 lines derived from the PI 567541B×E00003 cross. Parents are shown by arrows, a Week-3 rating in the greenhouse trial; b week-4 rating in the greenhouse trial; c week-3 rating in the field trial; d week-4 rating in the field trial
FIG. 32 shows exemplary locations of soybean aphid resistance loci as determined using the composite interval mapping method. 1-LOD and 2-LOD support intervals of each locus are marked by thick and thin bars, respectively. Bars filled with hatch lines represent loci for the week-3 rating in the greenhouse trial (GH3WK). Bars filled with cross lines represent loci for the week-4 rating in the greenhouse trial (GH4WK). Unfilled bars represent loci for the week-3 rating in the field cage trial (Cage3WK). Black bars represent loci for the week-4 rating in the field cage trial (Cage4WK). a Map of chromosome 16 (linkage group J) in mapping population PI 567543C×E00003 with the aphid resistance locus shown on the left; b map of chromosome 16 (linkage group J) on the consensus map (Song et al. 2004); c map of chromosome 16 (linkage group J) in validation population PI 567543C×Skylla with the aphid resistance locus shown on the right.
FIG. 33 shows an exemplary QTL mapping of Rag3 using 249 F 4 derived lines in PI 567543C. Rag 3: PI 567543C.
FIG. 34 shows exemplary Rag3 Fine Mapping Populations.
FIG. 35 shows an exemplary phenotype histogram of soybean aphid rating score of F2 plants from bi-parental cross E07048×E10902, where E07048 is susceptible to aphid and E10902 is resistant. A. phenotype distribution of all 1889 F2 plants with y-axis being the percentage of total plants for each score; B. phenotype of a subset of 376 F2 plants from the same population with y-axis being the number of F2 plants for each score.
FIG. 36 shows an exemplary validation of the location of Rag3 on Chromosome 16 using a subset of 376 F 2 plants from cross E07048×E10902, with E07048 and E10902 being susceptible and resistant parent. QTL analysis was conducted with both composite interval mapping and multiple interval mapping methods with P<0.0001. MSUSNP16-14 (Gm16—6164774), MSUSNP16-10 (Gm16—6262227), MSUSNP16-11 (Gm16—6413214), MSUSNP16-12 (Gm16—6423098) and MSUSNP16-15 (Gm16—8051585) are SNP markers with physical position indicated in the middle of the marker names. The black bar next to the linkage map indicates the location of Rag3 with composite interval mapping method; the grey bar on the right shows that with multiple-interval mapping method.
FIG. 37: Table 1 shows exemplary SNP genotypes of selected F3 and F4 lines on the 52K SNP Beadchip.
FIG. 38: Table 2 shows exemplary Correlations of SNPs with phenotypes in 376 F2 lines from cross E07048×E10902 and all 983 F3 progeny of 102 F2 recombinants. MSUSNP16-10 (Gm16—6262227), MSUSNP16-11 (Gm16—6413214) and MSUSNP16-12 (Gm16—6423098) were developed from 52K SNP Beadchip (Song et al., 2011).
FIG. 39 shows exemplary rag3 fine mapping populations.
FIG. 40: Table 1 shows exemplary Populations derived from crosses with PI 567598B that were used for screening of recombinants to delimit the location of rag3.S=Parent susceptible to soybean aphid; R=Parent resistant to soybean aphid (parentheses show pedigree information).
A Genomic position of single nucleotide polymorphism on the Williams 82 genome assembly, Glyma1 (Schmutz et al. 2010).
B Target sequence for TaqMan custom design with 60 bp upstream and downstream of the single nucleotide polymorphism. SNPs in corresponding wild-type and mutant-alleles are in brackets [ ].
FIG. 41: Table 2 shows exemplary TaqMan SNP assay information designed from SoySNP50 iSelect BeadChips. SoySNP50 Infinium assay Gm16—6050 targets SEQ ID NO: 121, SoySNP50 Infinium assay Gm16—626222 targets SEQ ID NO: 122, SoySNP50 Infinium assay Gm16—641321 targets SEQ ID NO:123, SoySNP50 Infinium assay Gm16—642309 targets SEQ ID NO:124, SoySNP50 Infinium assay GM16—642406 targets SEQ ID NO: 125.
FIG. 42: Table 3 shows exemplary recombination breakpoints among identified recombinants that mapped the position rag3 on Chromosome 16. Bold letters represent the breakpoints and italicized letters are loci with TaqMan SNP assays used for marker association with progeny phenotype.
a Phenotype of the F2 or F 7:8 recombinant lines based on soybean aphid evaluation of progenies.
b SNPs from SoySNP50 iSelect BeadChips converted to TaqMan SNP assays,
c Polymorphic SNP markers from SoySNP50 iSelect Beadchip.
d Physical position of SNP markers in SoySNP50 based on Glyma1 assembly of soybean Williams 82 (www.phytozome.com).
e R2 value of the marker association.
f Level of significance of the marker association.
FIG. 43 Fine mapping the location of rag3 on chromosome 16. A Genetic map of chromosome 16 with SNP markers appended from previous patent application. Genetic positions are in centimorgans (cM). B Recombination breakpoints of recombinant lines from SNP genotyping by Infinium and TaqMan assays. Genomic physical positions are in (Mbp) on the Williams 82 genome assembly, Glyma1 (Schmutz et al. 2010). White bars represent homozygote genotype for the allele from the resistant parent; gray bars represent heterozygote genotype; black bars represent homozygote genotype for the allele from the susceptible parent; patterned bar represent the position of rag3; dotted horizontal lines separate recombination bins. Phenotype of recombinants as confirmed by progeny test: Seg, segregating phenotype; R, resistant phenotype.
FIG. 44A: Table 1. Shows exemplary populations derived from crosses with PI 567598B that were used for screening of recombinants to delimit the location of rag3.
FIG. 44B: Table 2. Shows exemplary TaqMan SNP assay information designed from SoySNP50 iSelect BeadChips.
FIG. 45: Table 1. Phenotypic summary of the F 4-derived main mapping population and its parental lines and grandparent, PI567598B, for the mean soybean aphid ratings in a summer field trial and greenhouse trials.
FIG. 46: Table 2. Shows an exemplary phenotypic summary of the F 2 validation population and its parents PI 567598B and Titan for the soybean aphid damage index in the greenhouse trials.
FIG. 47: Table 3. Shows an exemplary summary of QTLs for soybean aphid resistance detected in the main mapping population (IA2070×E06902) and validation population (PI 567598B× Titan) using the composite interval mapping method.
aChromosome/Linkage group. The chromosome number and linkage group name are according to the SoyBase (Grant et al. 2010)
bQTL peak position is expressed in cM
cMarkers flanking the peak position
dThe LOD threshold for the field 2009 rating is 3.89. TheLOD threshold for the three week rating in 2010 is 3.48. The LOD threshold for the four-week rating in 2009 is 1.78. The LOD threshold for the three week rating in 2008 is 2.3. The LOD threshold for the four week rating in 2008 is 2.4. The LOD threshold for the 2009 rating is 5.1.

eR2, percentage of phenotypic variation explained by a QTL

fAdditive effect. The negative value implies that the IA2070 allele increases the phenotypic value

gAdditive effect. The positive value implies that the PI 567598B allele decreases the phenotypic value

FIG. 48: Table 4. Summary of QTLs for soybean aphid resistance detected in the main mapping population (IA2070×E06902) and validation population (PI 567598B× Titan) using the multiple interval mapping method.
aChromosome/Linkage group. The chromosome number and linkage group name are according to the SoyBase (Grant et al. 2010)
bQTL peak position is expressed in cM
cMarkers flanking the peak position or the marker at the peak position
dUsing the same LOD thresholds as in the composite interval mapping method (FIG. 48: Table 2).
eR2, percentage of phenotypic variation explained by a QTL.
fAdditive effect. The negative value implies that the IA2070 allele increases the phenotypic value
fAdditive effect. The positive value implies that the PI 567598B allele decreased the phenotypic value
FIG. 49: Table 5. Shows exemplary genotypic groups of 139 F4-derived lines from the mapping population IA207×E06902 containing alternative alleles from associated markers on chromosome 7 (QTL M) and 16 (QTL J).
FIG. 50 shows exemplary QTL mapping of rag1b and rag3 using 282 F4-derived lines.
FIG. 51 shows exemplary frequency distribution of soybean aphid ratings taken from F4-derived lines of the cross IA2070×E06902. Parental lines, PI567598B, and Dowling ratings are shown by arrows, a Three-week rating in the field trial in summer 2009, b Three-week rating in the greenhouse trial in fall 2010, c Four-week rating in the greenhouse trial.
FIG. 52 shows exemplary locations of soybean aphid resistance QTLs using multiple interval mapping method. Bars filled with hatch lines represent QTLs for the three-week rating in the 2010 trial (2010Wk3). Bars filled with cross lines represent QTLs for the four-week rating in a trial (2010Wk4). Black bars represent QTLs for the three-week rating in a field trial.
a and d Maps of chromosome 16 (LG J) and 7 (LG M) in the mapping population, the QTL positions are listed at its left side; b and e Consensus maps of chromosome 16 (LG J) and 7 (LG M) (Song et al. 2004); c and f Maps of chromosome 16 (LG J) and 7 (LG M) in the validation population, the QTL positions are listed at its right side.



GENERAL DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for providing aphid resistance in plants. More particularly, the invention relates to compositions and methods for using aphid resistant germplasm for breeding soybean aphid resistant soybean plants, including but not limited to cultivars, varieties, lines and methods of breeding the same for commercial use, the breeding methods further involving identifying and using genetic markers for aphid resistant traits.
The present invention relates to compositions and methods for providing and using markers of aphid resistant germplasm, particularly for identifying aphid resistance germplasm in soybean plants. Specifically, the invention relates to providing aphid resistant germplasm identified by markers associated with decreased damage from aphid feeding, as well as enhanced tolerance to aphid infestation of soybean plants. More particularly, the invention relates to compositions and methods for using genetic markers in breeding methods for producing plants with increased resistance to aphid damage and tolerance, while retaining and acquiring desired agronomic traits. Additionally, markers were developed for fine mapping of aphid resistance genes including allele specific contributions to plants for breeding soybean plants with increased aphid resistance. Furthermore, the invention relates to plants produced by these breeding programs, including plants with having aphid resistant genes between the molecular markers identifying aphid resistant genes, for use in commercial soybean production.
Advantages of growing and using an aphid resistant soybean plant includes increased effectiveness since the plant produced toxin would be readily available to any aphid feeding anywhere on the resistant plant, increased safety to people by replacing harmful synthetic insecticides with specially bred resistant plants and economical since the plant provides its own constant insect protection. The potential market is substantial because of the recent uncontrolled introduction of aphid pests into the Midwest, which is a major source of soybean products.
At the time the research of the present invention was initiated in 2002, there were no known sources of host plant resistance in the USA. The objectives of this study were to: (i) screen soybean germplasm, originally imported from northern China where soybean aphids are natural pests, where screened plants were publicly available in the USDA Soybean Germplasm Collection but whose soybean aphid resistance was unknown in order to identify sources of germplasm resistance to soybean aphids in early maturity groups; and (ii) determine the resistance type of the identified sources. Unlike the references that describe late maturing aphid resistant soybean plants discussed below the soybean plants of the present invention are an early maturing variety of soybean plant. Further, the source of aphid resistant germplasm of the present invention is originally from soybean plants of China wherein soybean aphids are natural pests. The objective of this study was to identify sources of resistance to soybean aphids from early maturing soybean germplasm and to determine the type of resistance they possess.
Over a two-year period, 2,147 early maturing soybean accessions, obtained from the USDA public soybean germplasm database, from maturity group (MG) 0 to III, originally from northern China, were screened for aphid resistance in the greenhouse and in field cages. The plants were hand-inoculated and aphid populations were evaluated 10 days after inoculation. A damage index (0-100%) was calculated for each accession. After two years of screening and confirmation in choice tests, four accessions from Shandong province, PI 567543C, PI 567597C, PII 567541B, and PI 567598B, in MG III were found to be resistant to the soybean aphid. Two of these accessions, PI 567541B and PI 567598B, possessed antibiosis resistance preventing the aphids from reproducing on the plants in a no-choice study. These resistant sources can be used to develop commercial varieties with aphid resistance for the North Central States of America where soybean varieties of MG 0 to III are cultivated and other areas of the world.
1. Plants 
The discovery and isolation of an early maturing aphid resistant soybean cultivar is disclosed herein. Specifically, soybean plant cultivars corresponding to PI 567543C, PI 567597C, PI 567541B, and PI 567598B were isolated from over 2,000 tested soybean cultivars. Further, novel soybean groups, from crosses of another soybean variety with PI 567543C, PI 567597C, PI 567541B, and PI 567598B, are also disclosed. The invention relates to a seed of one or more of a soybean cultivar PI 567543C, PI 567597C, PI 567541B, and PI 567598B, to the plants, i.e. comprising aphid resistant germplasm, of one or more of a soybean PI 567543C, PI 567597C, PI 567541B, and PI 567598B, and to methods for producing a soybean seed and plant produced by crossing any one of a cultivar of PI 567543C, PI 567597C, PI 567541B, and PI 567598B, with itself or another soybean variety, and further to provide offspring comprising the aphid resistant germplasm of the present invention. The invention further relates to an aphid resistant soybean plant and seed from that plant comprising germplasm of any one of a soybean cultivar PI 567543C, PI 567597C, PI 567541B, and PI 567598B. Examples of offspring comprising the aphid resistant germplasm of the present invention include the soybean lines E06906, E06902, E06907, E06901, and E06904.
The methods of the present invention are not limited to the use of any particular plant. Indeed, a variety of plants are contemplated for introducing aphid resistance, including but not limited to soybean, beans, tomato, pepper, cotton, barley, sorghum, sunflowers, rice, corn, wheat, Brassica, and flowers.
In some embodiments, aphid resistant germplasm is introgressed into a food-grade soybean plant that includes but is not limited to Ohio FG1, Agracola Farms AF271, Burtch Seed BBF44, H.A.P.I. Ohio GL2930, LG Seed EX230FG, Wellman WFG268, and the like. In some embodiments an aphid resistant a food-grade soybean plant is a specialty soybean plant, for example, provides Edamame soybeans, and the like. In some embodiments, aphid resistant germplasm is introgressed into a soybean plant that provides food for livestock, poultry, cattle and swine, for example, a conventional soybean plant that includes but is not limited to Asgrow AG2905, Pioneer 93B01, and Public Sandusky. In some embodiments a soybean plant provides a non-food product, for example, a fuel additive, such as a diesel fuel additive, soy biodiesel, soybean ink, soy crayons, soybean based wood adhesive, soybean based lubricants, and the like.
2. Vectors
The methods of the present invention contemplate the use of a heterologous gene such as a gene encoding an insect resistant protein, an herbicide resistant protein, a gene for providing a selected agronomic trait, or more than one gene, such as a linkage group for providing a selected agronomic trait (such as aphid resistant germplasm or germplasm comprising an integrated transgene).
Heterologous genes intended for expression in plants are first assembled in expression cassettes comprising a promoter. Methods which are well known to or developed by those skilled in the art may be used to construct expression vectors containing a heterologous gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Exemplary techniques are widely described in the art (See e.g., Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., herein incorporated by reference).
In general, these vectors comprise a nucleic acid sequence encoding a heterologous gene, or encoding a sequence designed to decrease endogenous gene expression, (as described above) operably linked to a promoter and other regulatory sequences (e.g., enhancers, polyadenylation signals, etc.) required for expression in a plant.
Promoters include but are not limited to constitutive promoters, tissue-, organ-, and developmentally-specific promoters, and inducible promoters. Examples of promoters include but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine amino peptidase (“LAP,” Chao et al., Plant Physiol 120:979-992 (1999), herein incorporated by reference); a chemically-inducible promoter from tobacco, Pathogenesis-Related 1 (PRO (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); a heat shock promoter (U.S. Pat. No. 5,187,267, herein incorporated by reference); a tetracycline-inducible promoter (U.S. Pat. No. 5,057,422, herein incorporated by reference); and seed-specific promoters, such as those for seed storage proteins (e.g., phaseolin, napin, oleosin, and a promoter for soybean beta conglycin (Beachy et al., (1985) EMBO J. 4: 3047-3053, herein incorporated by reference). All references cited herein are incorporated in their entirety.
The expression cassettes may further comprise any sequences required for expression of mRNA. Such sequences include, but are not limited to transcription terminators, enhancers such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
A variety of transcriptional terminators are available for use in expression of sequences using the promoters such as those disclosed herein. Transcriptional terminators are responsible for the termination of transcription beyond the transcript and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include, but are not limited to, the CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminator (See e.g., Odell et al., Nature 313:810 (1985); Rosenberg et al., Gene, 56:125 (1987); Guerineau et al., Mol. Gen. Genet. 262:141 (1991); Proudfoot, Cell, 64:671 (1991); Sanfacon et al., Genes Dev., 5:141; Mogen et al., Plant Cell, 2:1261 (1990); Munroe et al., Gene, 91:151 (1990); Ballas et al., Nucleic Acids Res. 17:7891 (1989); Joshi et al., Nucleic Acid Res., 15:9627 (1987), all of which are incorporated herein by reference).
In addition, in some embodiments, constructs for expression of the heterologous gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
In some embodiments of the present invention, a construct for expression of the heterologous nucleic acid sequence of interest also includes a regulator such as a nuclear localization signal (Kalderon et al., Cell 39:499 (1984); Lassner et al., Plant Molecular Biology 17:229 (1991)), a plant translational consensus sequence (Joshi, Nucleic Acids Research 15:6643 (1987)), an intron (Luehrsen and Walbot, Mol. Gen. Genet. 225:81 (1991)), and the like, operably linked to the nucleic acid sequence encoding an heterologous gene.
In preparing the construct comprising the nucleic acid sequence encoding an heterologous gene, or encoding a sequence designed to decrease heterologous gene expression, various DNA fragments can be manipulated, so as to provide for the DNA sequences in the desired orientation (e.g., sense or antisense) orientation and, as appropriate, in the desired reading frame. For example, adapters or linkers can be employed to join the DNA fragments or other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like is preferably employed, where insertions, deletions or substitutions (e.g., transitions and transversions) are involved.
Numerous transformation vectors are available for plant transformation. The selection of a vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the nptII gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra, Gene 19: 259 (1982); Bevan et al., Nature 304:184 (1983), all of which are incorporated herein by reference), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., Nucl Acids Res. 18:1062 (1990); Spencer et al., Theor. Appl. Genet. 79:625 (1990), all of which are incorporated herein by reference), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann, Mol. Cell. Biol. 4:2929 (1984), incorporated herein by reference)), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J., 2:1099 (1983), incorporated herein by reference).
In some preferred embodiments, the Ti (T-DNA) plasmid vector is adapted for use in an Agrobacterium mediated transfection process (See e.g., U.S. Pat. Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all of which are herein incorporated by reference). Construction of recombinant Ti and Ri plasmids in general follows methods typically used with the more common vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the native plasmids and sometimes constructed from foreign sequences. These may include but are not limited to structural genes for antibiotic resistance as selection genes.
There are two systems of recombinant Ti and Ri plasmid vector systems now in use. The first system is called the “cointegrate” system. In this system, the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJ1 shuttle vector and the non-oncogenic Ti plasmid pGV3850. The use of T-DNA as a flanking region in a construct for integration into a Ti- or R1-plasmid has been described in EPO No. 116,718 and PCT Application Nos. WO 84/02913, 02919 and 02920 all of which are herein incorporated by reference). See also Herrera-Estrella, Nature 303:209-213 (1983); Fraley et al., Proc. Natl. Acad. Sci, USA 80:4803-4807 (1983); Horsch et al, Science 223:496-498 (1984); and DeBlock et al., EMBO J. 3:1681-1689 (1984), all of which are herein incorporated by reference).
The second system is called the “binary” system in which two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some of these vectors are commercially available. In other embodiments of the invention, the nucleic acid sequence of interest is targeted to a particular locus on the plant genome. Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium-derived sequences. Generally, plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (U.S. Pat. No. 5,501,967 herein incorporated by reference). One of skill in the art knows that homologous recombination may be achieved using targeting vectors that contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known. Agrobacterium tumefaciens is a common soil bacterium that causes crown gall disease by transferring some of its DNA to the plant host. The transferred DNA (T-DNA) is stably integrated into the plant genome, where its expression leads to the synthesis of plant hormones and thus to the tumorous growth of the cells. A putative macromolecular complex forms in the process of T-DNA transfer out of the bacterial cell into the plant cell.
In yet other embodiments, the nucleic acids such as those disclosed herein is utilized to construct vectors derived from plant (+) RNA viruses (e.g., brome mosaic virus, tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, tomato mosaic virus, and combinations and hybrids thereof). Generally, the inserted heterologous polynucleotide can be expressed from these vectors as a fusion protein (e.g., coat protein fusion protein) or from its own subgenomic promoter or other promoter. Methods for the construction and use of such viruses are described in U.S. Pat. Nos. 5,846,795; 5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785, all of which are incorporated herein by reference.
In some embodiments of the present invention, where a heterologous nucleic acid sequence of interest is introduced directly into a plant. One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (WO 93/07278, herein incorporated by reference).
3. Transformation Techniques 
Once a nucleic acid sequence encoding an heterologous gene is operatively linked to an appropriate promoter and inserted into a suitable vector for the particular transformation technique utilized (e.g., one of the vectors described above), the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation. In some embodiments, the vector is maintained episomally. In other embodiments, the vector is integrated into the genome.
In some embodiments, direct transformation in the plastid genome is used to introduce the vector into the plant cell (See e.g., U.S. Pat. Nos. 5,451,513; 5,545,817; 5,545,818; PCT application WO 95/16783 all of which are incorporated herein by reference). The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleic acid encoding the RNA sequences of interest into a suitable target tissue (e.g., using biolistics or protoplast transformation with calcium chloride or PEG). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., PNAS, 87:8526 (1990); Staub and Maliga, Plant Cell, 4:39 (1992), all of which are incorporated herein by reference). The presence of cloning sites between these markers allowed creation of a plastid targeting vector introduction of foreign DNA molecules (Staub and Maliga, EMBO J., 12:601 (1993)). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab and Maliga, PNAS, 90:913 (1993)). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the present invention. Plants homoplasmic for plastid genomes containing the two nucleic acid sequences separated by a promoter of the present invention are obtained, and are preferentially capable of high expression of the RNAs encoded by the DNA molecule.
In other embodiments, vectors useful in the practice of the present invention are microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (Crossway, Mol. Gen. Genet, 202:179 (1985)). In still other embodiments, the vector is transferred into the plant cell by using polyethylene glycol (Krens et al., Nature, 296:72 (1982); Crossway et al., BioTechniques, 4:320 (1986)); fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley et al., Proc. Natl. Acad. Sci., USA, 79:1859 (1982)); protoplast transformation (EP 0 292 435); direct gene transfer (Paszkowski et al., EMBO J., 3:2717 (1984); Hayashimoto et al., Plant Physiol. 93:857 (1990)).
In still further embodiments, the vector may also be introduced into the plant cells by electroporation. (Fromm, et al., Pro. Natl Acad. Sci. USA 82:5824 (1985); Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602 (1986)). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
In yet other embodiments, the vector is introduced through ballistic particle acceleration using devices (e.g., available from Agracetus, Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del.), see e.g., U.S. Pat. No. 4,945,050; and McCabe et al., Biotechnology 6:923 (1988), all of which are incorporated herein by reference). Examples of methods for transforming crop plants are provided for soybean plants in U.S. Pat. No. 5,015,580, herein incorporated by reference, Christou et al., Plant Physiol., 87:671 (1988) (soybean); McCabe et al., Bio/Technology 6:923 (1988) (soybean); and other plants such as Weissinger et al., Annual Rev. Genet. 22:421 (1988); Sanford et al., Particulate Science and Technology, 5:27 (1987) (onion); Svab et al., Proc. Natl. Acad. Sci. USA, 87:8526 (1990) (tobacco chloroplast); Klein et al., Proc. Natl. Acad. Sci. USA, 85:4305 (1988) (maize); Klein et al., Bio/Technology, 6:559 (1988) (maize); Klein et al., Plant Physiol., 91:4404 (1988) (maize); Fromm et al., Bio/Technology, 8:833 (1990); and Gordon-Kamm et al., Plant Cell, 2:603 (1990) (maize); Koziel et al., Biotechnology, 11:194 (1993) (maize); Hill et al., Euphytica, 85:119 (1995) and Koziel et al., Annals of the New York Academy of Sciences 792:164 (1996); Shimamoto et al., Nature 338: 274 (1989) (rice); Christou et al., Biotechnology, 9:957 (1991) (rice); Datta et al., Bio/Technology 8:736 (1990) (rice); European Application EP 0 332 581 (orchardgrass and other Poaceae); Vasil et al., Biotechnology, 11: 1553 (1993) (wheat); Weeks et al., Plant Physiol., 102: 1077 (1993) (wheat); Wan et al., Plant Physiol. 104: 37 (1994) (barley); Jahne et al., Theor. Appl. Genet. 89:525 (1994) (barley); Knudsen and Muller, Planta, 185:330 (1991) (barley); Umbeck et al., Bio/Technology 5: 263 (1987) (cotton); Casas et al., Proc. Natl. Acad. Sci. USA 90:11212 (1993) (sorghum); Somers et al., Bio/Technology 10:1589 (1992) (oat); Torbert et al., Plant Cell Reports, 14:635 (1995) (oat); Weeks et al., Plant Physiol., 102:1077 (1993) (wheat); Chang et al., WO 94/13822 (wheat) and Nehra et al., The Plant Journal, 5:285 (1994) (wheat), all of which are herein incorporated by reference.
In addition to direct transformation, in some embodiments, the vectors comprising a nucleic acid sequence encoding a heterologous gene are transferred using Agrobacterium-mediated transformation (Hinchee et al., Biotechnology, 6:915 (1988); Ishida et al., Nature Biotechnology 14:745 (1996), all of which are herein incorporated by reference). Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for plant tumors such as crown gall and hairy root disease. In the dedifferentiated tissue characteristic of the tumors, amino acid derivatives known as opines are produced and catabolized. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. Heterologous genetic sequences (e.g., nucleic acid sequences operatively linked to a promoter of the present invention) can be introduced into appropriate plant cells, by means of the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Schell, (1987) Science, 237:1176). Species which are susceptible infection by Agrobacterium may be transformed in vitro. Transformation methods for producing transgenic soybean plants using Agrobacterium-mediated transformation are provided in U.S. Patent Appln. No. 20020157139, U.S. Pat. Nos. 6,384,301, 5,416,011, 5,569,834, and 5,824,877, all of which are herein incorporated by reference.
4. Regeneration
After selecting for transformed plant material that can express a heterologous gene encoding a heterologous gene or variant thereof, whole plants are regenerated, for example methods for regenerating transformed soybean plants are provided in U.S. Pat. No. 5,015,580, herein incorporated by reference. Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co. New York, (1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, (1984) and Vol. III, (1986), herein incorporated by reference. It is known that many plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables, and monocots (e.g., the plants described above). Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted.
Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate and form mature plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. The reproducibility of regeneration depends on the control of these variables.
5. Generation of Transgenic Aphid Resistant Soybean Lines
Transgenic lines are established from transgenic plants by tissue culture propagation. The presence of nucleic acid sequences encoding a heterologous gene or mutants or variants thereof in a transgenic plant line may be introgressed into aphid resistant plants for providing transgenic aphid resistant plants using traditional plant breeding techniques. Transgenic lines of aphid resistant soybean cultivars may be utilized for evaluation of aphid resistant activity, insect resistance ratios, phenotype, pathogen resistance and other agronomic traits, such as agronomic shown for transgenic soybean plants in European Patent No. 301,749, herein incorporated by reference, in the presence of an introgressed transgene.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for providing aphid resistant in plants. More particularly, the invention relates to compositions and methods for using aphid resistant germplasm for breeding soybean aphid resistant soybean plants, including but not limited to cultivars, varieties, lines and methods of breeding the same for commercial use, the breeding methods further involving identifying and using genetic markers for aphid resistant traits.
The present invention relates to compositions and methods for providing and using markers of aphid resistant germplasm, particularly for identifying aphid resistance germplasm in soybean plants. Specifically, the invention relates to providing aphid resistant germplasm identified by markers associated with decreased damage from aphid feeding, as well as enhanced tolerance to aphid infestation of soybean plants. More particularly, the invention relates to compositions and methods for using genetic markers in breeding methods for producing plants with increased resistance to aphid damage and tolerance, while retaining and acquiring desired agronomic traits. Additionally, markers were developed for fine mapping of aphid resistance genes including allele specific contributions to plants for breeding soybean plants with increased aphid resistance. Furthermore, the invention relates to plants produced by these breeding programs, including plants with stacked aphid resistant genes, for use in commercial soybean production.
Specifically, this invention discloses novel soybean lines and cultivars including transgenic, hybrid, outcrossed, backcrossed, inbred and self-fertilized progeny comprising soybean aphid resistant soybean germplasm, specifically founder soybean cultivars of Glycine max (L.) Merr. max, designated accessions PI 567543C, PI 567597C, PI 567541B, and PI 567598B and their progeny, such as lines, E06902, E06907, E06901, and E06904 are disclosed. The invention relates to the seeds and plants of novel aphid resistant lines and cultivars, to the groups of plants comprising aphid resistant lines and cultivars and to methods for producing an aphid resistant soybean plant obtained by crossing the founder cultivars (i.e. accessions PI 567543C, PI 567597C, PI 567541B, and PI 567598B) with another soybean variety (preferably elite soybean varieties), including backcrosses with the founder cultivars, backcrosses with the original soybean variety, and further, crosses within and between a filial generation (F), for example, one or more of an F1-F7, including but not limited to inbreeding using self-pollination. The present invention further relates to the generation of a commercially viable aphid resistant early maturing soybean seed and plant produced by the compositions and methods of the present invention. Additionally, the present invention relates to the generation of molecular markers, including SSR and other DNA markers for identifying linkage groups comprising aphid resistant germplasm, for example, sequences for PCR primers used to amplify SSR loci in Soybean, Zhu et al. Genetics 2003 March; 163(3): 1123-34, for genes relating to aphid resistance and using molecular marker analysis for identifying and using genes relating to aphid resistance.
In experiments conducted during the course of the present invention, sources and types of resistance to soybean aphid from early maturing soybean germplasm were identified. Over a two-year period, 2,147 soybean accessions from maturity group (MG) 0 to III, originally from northern China, were evaluated for aphid resistance in a greenhouse and in field cages. The plants were hand-inoculated and aphid populations were evaluated 10 days after inoculation. A damage index (0-100%) was calculated for each accession. After two years of evaluation and confirmation in choice tests, four MG III accessions from Shandong province, accessions PI 567543C, PI 567597C, PI 567541B, and PI 567598B, were found to be resistant to the soybean aphid. Two of these 20 accessions, PI 567541B and PI 567598B, possessed antibiosis resistance, preventing the aphids from reproducing on the plants in a no-choice study. Two additional accessions PI 567543C and PI 567597C possessed antixenosis resistance. These resistant sources can be used to develop commercial cultivars with aphid resistance for the North Central States of America and other areas of the world.
In order to develop aphid resistant varieties, sources of germplasm comprising aphid resistance must be identified. Sources of resistance to the soybean aphid are reported in China. In the late 1980's, two highly resistant varieties were found among 181 varieties evaluated (Yi-heng, (1988) Soybean Science. 7(2):167-169; Fan, (1988) Soybean Sci. 7:152; herein incorporated by reference). In 1991, resistance was also reported in soybean germplasm in China (Sun et al., (1991) Soybean Sci. 10(2): 98-103; Gao, et al., (1991) Chin. J. Biol. Control 7:95; Sun, et al., 1991, Study on the resistance in wild soybean to soybean mosaic potyvirus. Soybean Science 10:212-216; herein incorporated by reference). The type of resistance, antixenosis or antibiosis, was not indicated in these studies. Antixenosis is nonpreference of insects for a host plant (Kogan and Ortman, (1978) Bull. Entomol. Soc. Am. 24:175-176). Antibiosis includes all adverse effects on an insect's life history after a resistant host plant has been used for food (Painter, 1951, Insect Resistance in Crop Plants, Macmillan). Knowing the type of resistance is important to fully understand and utilize resistant accessions in a breeding program. Hill et al. (Hill et al., (2004) Crop Sci. 44: 98-106) recently reported three lines with resistance to soybean aphid. PI 71506 (MG IV) has antixenosis and the cultivars Dowling (MG VIII) and Jackson (MG VII) are reported to have antibiosis resistance.
In 2002, there were no known sources of host plant resistance to soybean aphid in the United States of America. Hill (Hill et al., (2004) Crop Sci. 44: 98-106) evaluated 1,542 soybean genotypes, mostly current North American soybean cultivars, and found resistance in three North American soybean ancestral lines: Dowling, Jackson, and PI 71506. These resistant genotypes, which belong to MG IV to VIII, are not well adapted to the northern U.S. where soybean aphids are most prevalent. In experiments conducted during the course of the present invention, four resistant accessions (PI 567543C, PI 567597C, PI 567541B, and PI 567598B) belonging to MG III after evaluating 2,147 soybean accessions in MG 0 to III were identified. These primitive Chinese cultivars originated from Shandong province, but their resistance to the soybean aphid has never been reported.
In experiments conducted during the course of the present invention, the aphid resistance germplasm of the original four accessions, PI 567543C, PI 567597C, PI 567541B, and PI 567598B, are incorporated into elite soybean germplasm of soybean plants grown in the U.S. and Canada. In particular, incorporating the aphid resistance germplasm of these accessions into elite soybean germplasm and the progeny of elite soybean germplasm of soybean plants grown in north central regions and southern regions of the United States is contemplated. Incorporating aphid resistance germplasm into elite soybean plants grown in South America, including Brazil and Argentina, Indonesia, China and other countries where soybean plants are grown is also contemplated.
I. Aphid Resistant Transgenic Soybean Plants.
The present invention contemplates providing commercial lines of transgenic aphid resistant soybean plants by introgressing the aphid resistance germplasm of the present invention into commercially established transgenic soybean lines. In addition, introgressing the germplasm comprising a preferred transgene into aphid resistant soybean plants for developing commercial lines of aphid resistant transgenic soybean plants is contemplated.
Numerous cultivars and lines of transgenic soybean plants have been and are being developed as commercial varieties for use by growers and breeders for providing preferred agronomic traits including such traits as a preferred herbicide resistance, a preferred insect resistance, a preferred nematode resistance, a preferred microorganism, such as fungi or bacterial resistance, a preferred soybean seed oil content and the like. Therefore, one contemplated aspect of the present invention is for providing an aphid resistant transgenic plant by introgressing aphid resistant germplasm of the present invention into a transgenic variety. In one embodiment, the germplasm of a transgenic plant comprising an integrated transgene is used for introgressing said transgene into an aphid resistant soybean plant, for example, transgenic plants comprising a transgenes providing one or more of herbicide resistance, insect resistance, nematode resistance, fungal resistance, bacterial resistance, an agronomic trait and the like. Examples of transgenic plants for providing herbicide resistance transgenes include but are not limited to transgenic soybean lines such as lines A2704-12 (U.S. Pat. No. 4,940,835, herein incorporated by reference), A2704-21, A5547-35 (Aventis CropScience) developed tolerate the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Ignite®, Rely®, Liberty®, Harvest®, and Finale®) as a weed control option and lines A5547-127 (Bayer Crop Science (Aventis Crop Science (AgrEvo))) developed for tolerating the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Ignite®, Rely®, Liberty®, Harvest®, and Finale®) as a weed control option, GU262, genetically engineered to express tolerance to glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Finale®, and Liberty®) www.agbios.com/dbase.php?action=ShowProd&data=W62%2C+W98 (Bayer Crop Science (Aventis Crop Science (AgrEvo))) W62, W98 (Bayer Crop Science (Aventis Crop Science (AgrEvo))) genetically engineered to express tolerance to glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Finale®, and Liberty®); GTS 40-3-2 (Monsanto Company) developed for tolerating glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option by inserting a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens, all of which are herein incorporated by reference. Other glyphosate-resistant plants are provided by U.S. Pat. No. 4,940,835, herein incorporated by reference.
As used herein, the terms “Roundup Ready” and “RR” refer to a registered trademark of Monsanto Chemical Company. The present invention contemplates the use of experimental and commercial Roundup Ready tolerant transgenic soybean lines in compositions and methods of the present invention for providing herbicide tolerance to ROUNDUP, glyphosate-isopropylammonium, MON-0573 in aphid resistant plants. In addition, the present invention provides methods for introgressing aphid resistant germplasm into Roundup Ready soybean plants for providing aphid resistant ROUNDUP tolerant soybean plants for experimental use and commercial development. Numerous varieties of Roundup Ready soybean plants are contemplated for use in the present invention, including, but not limited to, Roundup Ready (RR) soybeans for early maturity varieties of soybeans in maturity group I, Mars 618RR, and High Cycle Roundup Ready soybeans in Maturity/Group 0-1.7, 2111RR, 2133RR, 2143RR, 2154RR, 2162RR, 2163RR, 2174RR, 2175RR; GROUP II, Munsee IVRR, Mohegan 624RR, Apache 626RR, Sioux IIRR, Shawnee 527RR, and Shawnee 527RR, High Cycle Roundup Ready soybeans in maturity/group 1.8-2.4 2183RR, 2184RR, 2194RR, 2202RR, 2213RR, 2222RR, 2223RR, 2224RR, 2232RR, 2245RR; GROUP III Jefferson 630RR, Grant IIIRR, Truman 636RR, Kennedy 538RR, Washington IXRR, AG 3702, AG 3902, DPX 3919RR, DPX 3761RR, DPX 3940RR, Asgrow 3906, Delta King 3968, DPL 3861, Progeny 3900, Dyna-Gro 31J39, Mor Soy 3883N; High Cycle Roundup Ready soybeans, maturity/group 2.5-3 Line High Cycle 2274 (further comprising germplasm conferring white mold tolerances, Phytophthora tolerance and Brown stem Rot (BSR) resistance), Line High Cycle 2274 (further comprising germplasm conferring excellent Phytophthora field tolerance), Line High Cycle 2293 (further comprising germplasm conferring excellent Phytophthora tolerance, Soybean Cyst Nematode (SCN) resistant (Race 3, MR14), all of which are herein incorporated by reference. Examples of early season roundup resistant soybean lines for use in the present invention in maturity group III include, but are not limited to, AG 3901, HTS 3600RR, 3902-4 8390 RR, HTS 3600RR, CX 383RR, H 3090RR and maturity group IV Manokin DP, 4344RR, AP 4602RR, DP 4750RR, CX 444cRR, H 4252RR, 8411 RR, 4001-4, CX 414cRR, CX 433RR, AP 4888RR, and AP 4980RR lines.
In some embodiments, aphid resistant germplasm is for introgressing into Roundup Ready soybean lines. In some embodiments, germplasm comprising the Roundup Ready gene is used for introgressing into aphid resistant plants. In some embodiments, the Roundup Ready gene is used for inserting into an aphid resistant soybean plant part so as to provide ROUNDUP tolerant aphid resistant soybean seeds and plants. The present invention contemplates the use of disclosed transgenic plants comprising heterologous transgenes for providing insect resistance, including but not limited to, Bt derived transgenes (e.g., a gene encoding a Coleopteran inhibitory insecticidal crystal protein tIC851 as described in U.S. Patent Application. Nos. 20020103362, 0030229919 and U.S. Pat. No. 6,541,448); genes and their encoded crystal proteins that exhibit insecticidal activity against lepidopteran insects (see, e.g., U.S. Patent Application. No. 20030237111); genes encoding novel crystal Δ-endotoxin proteins which exhibit insecticidal activity against lepidopteran insects (see, e.g., U.S. Pat. No. 6,593,293); genes encoding Δ-endotoxins, mutant endotoxins and endotoxin derived proteins having pesticidal activity against pests of the order Coleoptera as described in U.S. Patent Application. Nos. 20020151709 and 20030177528; genes encoding Δ-endotoxins such as for Cry9 and derived proteins for having pesticidal activity against insect pests, including but not limited to Lepidoptera (see, e.g., U.S. Patent Application. No. 20050138685); Bt genes encoding Δ-endotoxins having pesticidal activity against insect pests (see, e.g., U.S. Patent Application. No. 20040091505, 20050261188, and 20050261483; genes encoding proteins with toxicity to Coleopteran insects (see, e.g., U.S. Pat. No. 5,763,241); genes encoding synthetic insecticidal crystal protein gene derived from Bt (see, e.g., U.S. Pat. Nos. 5,380,831 5,567,862); Bt genes encoding protease resistant toxins BTS02618Aa or BTS02618Ab (see, e.g., U.S. Pat. Nos. 5,861,543 and 6,143,550) (all references are herein incorporated by reference).
The present invention contemplates the use of transgenic plants comprising a heterologous transgene for providing nematode resistance and pest resistance, in particular Soybean cyst nematode, as described in International patent application nos. 20020144310, 20030005491, 20060095987, WO96/30517, and WO93/19181, and U.S. Pat. Nos. 6,538,175, and 6,096,944, all of which are herein incorporated by reference in their entireties. In some embodiments, the present invention provides plants comprising transgenes that provide resistance for a variety of diseases and pathogens. The present invention is not limited to any particular resistance gene. Those known and later discovered resistance genes will find use in the present invention (see, e.g., U.S. Patent Application Nos. 20060059580 and 20060041954; each of which are incorporated by reference in their entireties). Examples of transgenic plants used for providing germplasm providing an agronomic trait, such as a preferred oil content, include but are not limited to lines G94-1, G94-19, G168 (DuPont Canada Agricultural Products). The present invention further contemplates the use of methods and compositions for identifying soybean plants that are tolerant, have improved tolerance or are susceptible to iron deficient growth conditions (see, e.g., U.S. Patent Application Nos. 20060041951 and 20060005276). Providing experimental transgenic aphid resistant soybean plants for identifying any loss of desirable traits by inserting a particular transgene into an aphid resistant soybean plant is also contemplated.
Another aspect of the present invention is to provide aphid resistant transgenic plants by introgressing the aphid resistant germplasm into transgenic soybean plants comprising a transgene (e.g., a transgene providing for preferred agronomic traits and preferred economic traits, preferred herbicide resistance, preferred insect resistance, preferred nematode resistance, preferred microorganism, such as fungi or bacterial resistance).
A. Maturity Group Identification.
QTL region identification for use in breeding methods and developing commercial cultivars of aphid resistant soybean plants of the present inventions. Q QTL mapping study is to map QTLs underlying a trait of interest on a genetic linkage map, which is a linear map showing the relative positions of genetic markers. Therefore, genetic markers and linkage maps are essential for any QTL mapping study. Several types of genetic markers are available in soybean, including classical markers, isozyme markers, restriction fragment length polymorphism (RFLP) markers, random amplified polymorphic DNA (RAPD) markers, amplification fragment length polymorphism (AFLP) markers, simple sequence repeat (SSR) markers, and single nucleotide polymorphism (SNP) markers. The most abundant markers developed for soybean are RFLP markers (Apuya et al. 1988; Keim et al. 1989), SSR markers (Akkaya et al. 1995), AFLP markers (Keim et al. 1997), and SNP markers (Choi et al. 2007). The first soybean molecular linkage map was published in 1990 by Keim et al. (1990a). This map contained 150 RFLP markers and three classical markers. The map was further expanded to include 355 RFLP markers and 16 other types of markers by 1993 (Shoemaker and Olson 1993). By 1999, the map was expanded to have 501 RFLPs markers, 486 SSRs, and 27 markers of other types (Cregan et al. 1999). This map was integrated with two maps developed with two additional mapping populations (Cregan et al. 1999). The integrated map had 689 RFLPs, 606 SSRs, 79 RAPDs, and 47 markers of other types. Using the three mapping populations and two additional mapping populations, a new version of the integrated map was constructed in 2004 (Song et al. 2004). The new integrated map contained 1015 SSRs, 709 RFLPs, 73 RAPDs, and 52 markers of other types, with a total map length of 2523.6 cM (Song et al. 2004). The most recent version of the integrated map was published in 2007 (Choi et al. 2007). This map contains 2,989 markers, including 1141 SNPs, 1014 SSRs, 709 RFLPs, and 125 other types of markers. The map consists of 20 linkage groups with a total map length of 2550.3 cM (Choi et al. 2007). The molecular markers, especially the SSR markers, from the integrated maps have been widely used in QTL mapping studies in soybean (Diers et al. 1992; Keim et al. 1990a; Neto et al. 2007; Wang et al. 2004a; Zhu et al. 2006).
In addition to the integrated linkage maps, several other molecular linkage maps were developed for soybean. A map with over 600 RFLP markers was developed by the DuPont Corporation (Rafalski and Tingey 1993). A map with 132 RFLP markers and 8 other types of markers was developed by Lark et al. (Lark et al. 1993). A map with 650 AFLPs, 165 RFLPs, and 25 RAPDs was developed by Keim et al. (1997). Liu et al. (2000) developed a map containing 100 RFLPs, 62 RAPDs, 42 AFLPs, 33 SSRs, and three other types of markers. Matthews et al. (2001) developed a map with 105 AFLPs, 39 RFLPs, 25 SSRs, 17 RAPDs, and four classical markers. Yamanaka et al. (2001) developed a map with 401 RFLPs, 96 SSRs, and six other types of markers. Wu et al. (2001) constructed a map with 486 AFLPs, 196 RFLPs, 87 SSRs, 18 RAPDs, and five other types of markers. This map and the mapping population have been used in several QTL mapping studies in China (Fu et al. 2007; Wang et al. 2004b, 2004c; Wang et al. 2004d).
B. Maturity Group Markers.
QTLs for maturity were found in eight consensus regions on six LGs: C1, C2, D1a, I, L, and M (Tables A-G and FIG. 14). The regions 53-66 cM on LG C1, 37-49 cM on LG D1a, 31-36 cM on LG I, 54-68 cM on LG L, and near 18-20 cM on LG M were each found containing QTLs for maturity in two different mapping populations with three or four mapping parents (Tables A-G). The 111-125 cM region on LG C2 was found with five different mapping populations developed from eight mapping parents (Tables A-G). The region 88-96 cM on LG L and the region 32-40 cM on LG M were each found with three mapping populations developed from five or six mapping parents (Table 1).







TABLE A







Consensus regions for Maturity Groups.















Start
End
Mapping
Mapping



Trait/QTL
LGa
positionb
positionb
parent 1
parent 2
Reference










Maturity













Pod mat 1-1
Cl
53
55
A81356022
PI
Keim et







468916
al.








(1990a)


Pod mat 8-5
Cl
64
66
Archer
Minsoy
Orf et al.








(1999b)


Pod mat 8-1
C2
111
113
Archer
Minsoy
Orf et al.








(1999b)


Pod mat 13-4
C2
111
113
Minsoy
Noir 1
Specht et








al. (2001)


Pod mat 14-3
C2
112
114
IA2008
PI
Wang et







468916
al.








(2004a)


Pod mat 4-1
C2
117
119
PI 27890
PI
Mansur et







290136
al. (1996)


Pod mat 1-5
C2
123
125
A81356022
PI
Keim et







468916
al.








(1990a)


Pod mat 1-2
D1a
37
39
A81356022
PI
Keim et







468916
al.








(1990a)


Pod mat 13-2
D1a
47
49
Minsoy
Noir 1
Specht et








al. (2001)


Pod mat 12-1
I
31
33
Parker
PI
Yuan et







468916
al. (2002)


Pod mat 11-1
I
34
36
A81356022
PI
Yuan et







468916
al. (2002)


Pod mat 14-1
L
54
56
IA2008
PI
Wang et







468916
al.








(2004a)


Pod mat 8-4
L
66
68
Archer
Minsoy
Orf et al.








(1999b)


Pod mat 13-6
L
88
90
Minsoy
Noir 1
Specht et








al. (2001)


Pod mat 4-3
L
91
93
PI 27890
PI
Mansur et







290136
al. (1996)


Pod mat 9-2
L
94
96
Archer
Noir 1
Orf et al.








(1999b)


Pod mat 8-2
M
18
20
Archer
Minsoy
Orf et al.








(1999b)


Pod mat 13-7
M
18
20
Minsoy
Noir 1
Specht et








al. (2001)


Pod mat 14-4
M
32
34
IA2008
PI
Wang et







468916
al.








(2004a)


Pod mat 10-2
M
33
35
Minsoy
Noir 1
Orf et al.








(1999b)


Pod mat 7-1
M
38
40
PI 27890
PI
Lark et







290136
al. (1994)









Uniform tests are planted in multiple-row plots with three or four replications, and the center rows are harvested for yield and seed quality determinations. Preliminary Tests are multiple-row plots with two replications. Usually 15 to 20 feet of row are planted and 12 to 16 feet harvested, to eliminate end-of-row effects. Coefficients of variability are included with all replicated test data. Discretion is used in including data with high CVs in the regional means. If the CV is greater than 15, participants should include the reason, such as disease or environmental conditions. Lines may be heterogeneous for morphological traits the first year in the Uniform Tests but must be pure lines the second year of testing. It is the responsibility of the breeder to purify heterogeneous lines. Generation ComDosited is the generation after the final single-plant selection, when seeds from plants or rows are composited. Previous Testing is the number of previous years in the same Uniform Test or, in the case of new entries, a reference to the previous year's test, abbreviated to PT IIA for Preliminary Test IIA, for example. Yield is measured after the seeds have been dried to uniform moisture content and is recorded in bushels (60 pounds) per acre. To convert to kilograms/hectare multiply by 67.25. Maturity is the date when 95% of the pods have ripened, as indicated by their mature pod color. Delayed leaf drop and green stems are not considered in assigning maturity. Maturity is expressed as days earlier (−) of later (+) than the average date of the reference variety. To aid in maturity group classification, one earlier (E) and one later (L) check variety are given in the maturity column for each test, or a maturity check from an earlier or later maturity group is included. Current reference and check varieties and the maturity group limits relative to the reference varieties are:














Group
Reference:
Range
Early check
Late check







00
MN0071
−7 to +5
Jim
Trail (L)


0
Lambert
−6 to +2
Traill (E)
Parker (L)


I
MN1410
−4 to +4
Lambert (0)
IA1008 (SCN)


II
IA 2068
−3 to +5
IA1021 (I)
IA3024 (L)


III
IA3023
−6 to +2
IA3024
Macon (L)


IV
LD00-3309
−4 to +7
Macon (III)


00RR
RG700RR


AG0202


0RR
RG200RR

RG200RR
SD1111RR(L)


IRR
SD1611RR

SD1111RR (E)
AG2002


IIRR
AG2403

AG2002
NEX2905A0R






(L)


IIIRR
AG3505

NEX2905A0R (E)
DKB3852


IVRR
AG4103

DKB38-52
AG4403









These maturity group ranges are based on long-term means over many locations.
C. Agronomic Traits Including Lodging, Height and Seed Size.
When using data from other environments, the interval between reference varieties may vary, and the division between maturity groups should be estimated in proportion to the above figures. Additional check varieties may be included in specific tests such as IA 1022 (SCN) for resistance to the soybean cyst nematode in UT I, or IA3024 as a 1% linolenic check in PTII, and PTIII.
Lodging is rated at maturity according to the following scores:
1. Almost all plants erect
2 All plants leaning slightly or a few plants down.
3 All plants leaning moderately (45 degrees), or 25% to 50% of the plants down.
4 All plants leaning considerably, or 50% to 80% of the plants down.
5 Almost all plants down.
Height is the average length in inches of mature plants from the ground to the tip of the main stem. To convert to centimeters, multiply by 2.54.
Seed Size (i.e. weight per seed) is recorded in grams per 100 seeds based on a 100- or 200-seed sample. To convert to seeds per pound, divide this into 45,359.2.
QTLs for lodging were found in three consensus regions on two LGs: C2 and L (Tables A-G and FIG. 14). All three regions, 107-116 cM on LG C2, 3-11 cM on LG L, and 68-101 cM on LG L were each found in three mapping populations developed from five or six mapping parents (Tables A-G).







TABLE B







Consensus regions for Lodging.















Start








posi-
End
Mapping
Mapping



Trait/QTL
LGa
tionb
positionb
parent 1
parent 2
Reference










Lodging













Ldge 6-1
C2
107
109
Archer
Minsoy
Orf et al.








(1999b)


Ldge 9-1
C2
111
113
Minsoy
Noir 1
Specht et








al. (2001)


Ldge 3-2
C2
114
116
PI 27890
PI
Orf et al.







290136
(1999b)


Ldge 5-11
L
3
5
PI
Young
Lee et al.






416937

(1996a)


Ldge 3-3
L
8
10
PI 27890
PI
Mansur et







290136
al. (1996)


Ldge 9-3
L
9
11
Minsoy
Noir 1
Specht et








al. (2001)


Ldge 1-1
L
68
87
PI 27890
PI
Mansur et







290136
al. (1996)


Ldge 8-4
L
88
90
Minsoy
Noir 1
Orf et al.








(1999b)


Ldge 4-2
L
88
90
Coker237
PI 97100
Lee et al.








(1996c)


Ldge 9-5
L
88
90
Minsoy
Noir 1
Specht et








al. (2001)


Ldge 4-3
L
89
101
Coker237
PI 97100
Lee et al.








(1996c)


Ldge 3-1
L
91
93
PI 27890
PI
Mansur et







290136
al. (1996)









QTLs for plant height were found in 10 consensus regions on eight LGs: C2, D1b, F, I, J, K, L, and M (Tables A-G and FIG. 14). The 107-118 cM region on LG C2 was found in five mapping populations developed from nine mapping parents (Tables A-G The regions 120-133 cM on LG D1b, 66-69 cM on LG F, 34-38 cM on LG I, 36-48 cM on LG K, 8-15 cM on LG L, and 34-44 cM on LG L were each found in two mapping populations developed from four mapping parents (Table 1). The regions 11-29 cM on LG J and 32-40 cM on LG M were each found in three mapping populations developed from six mapping parents (Table 3). The 68-114 cM region on LG L was found in six mapping populations developed from nine mapping parents (Tables A-G).







TABLE C







Consensus regions for Plant height.















Start
End







posi-
po-
Mapping
Mapping



Trait/QTL
LGa
tionb
sitionb
parent 1
parent 2
Reference










Plant height













Pl ht 8-1
C2
107
109
Archer
Minsoy
Orf et al.








(1999b)


Pl ht 18-4
C2
112
114
IA2008
PI
Wang et







468916
al. 2004a)


Pl ht 13-2
C2
112
114
Minsoy
Noir 1
Specht et








al. (2001)


Pl ht 11-1
C2
116
118
S100
Tokyo
Mian et








al. (1998)


Pl ht 6-3
C2
116
118
PI 27890
PI
Lark et







290136
al. (1995)


Pl ht 6-12
D1b
120
122
PI 27890
PI
Lark et







290136
al. (1995)


Pl ht 5-5
D1b
131
133
PI 416937
Young
Lee et al.








(1996a)


Pl ht 5-8
F
66
68
PI 416937
Young
Lee et al.








(1996a)


Pl ht 11-3
F
67
69
S100
Tokyo
Mian et








al. (1998)


Pl ht 12-1
I
34
36
A81356022
PI
Yuan et







468916
al. (2002)


Pl ht 16-1
I
36
38
Essex
Williams
Chapman








et al.








(2003)


Pl ht 13-5
J
11
13
Minsoy
Noir 1
Specht et








al. (2001)


Pl ht 6-6
J
20
22
PI 27890
PI
Lark et







290136
al. (1995)


Pl ht 5-9
J
27
29
PI 416937
Young
Lee et al.








(1996a)


Pl ht 18-3
K
36
38
IA2008
PI
Wang et







468916
al.








(2004a)


Pl ht 15-1
K
46
48
Flyer
Hartwig
Yuan et








al. (2002)


Pl ht 6-7
L
8
10
PI 27890
PI
Lark et







290136
al. (1995)


Pl ht 13-7
L
13
15
Minsoy
Noir 1
Specht et








al. (2001)


Pl ht 5-12
L
34
36
PI 416937
Young
Lee et al.








(1996a)


Pl ht 6-4
L
42
44
PI 27890
PI
Lark et







290136
al. (1995)


Pl ht 8-4
L
66
68
Archer
Minsoy
Orf et al.








(1999b)


Pl ht 1-1
L
68
87
PI 27890
PI
Mansur et







290136
al. (1993)


Pl ht 8-3
L
69
71
Archer
Minsoy
Orf et al.








(1999b)


Pl ht 6-1
L
86
88
PI 27890
PI
Lark et







290136
al. (1995)


Pl ht 4-2
L
88
90
Coker237
PI 97100
Lee et al.








(1996c)


Pl ht 13-8
L
88
90
Minsoy
Noir 1
Specht et








al. (2001)


Pl ht 4-4
L
89
101
Coker237
PI 97100
Lee et al.








(1996c)


Pl ht 3-1
L
91
93
PI 27890
PI
Mansur et







290136
al. (1996)


Pl ht 5-10
L
100
102
PI 416937
Young
Lee et al.








(1996a)


Pl ht 9-2
L
106
108
Archer
Noir 1
Orf et al.








(1999b)


Pl ht 6-2
L
112
114
PI 27890
PI
Lark et







290136
al. (1995)


Pl ht 18-6
M
32
34
IA2008
PI
Wang et







468916
al.








(2004a)


Pl ht 13-9
M
33
35
Minsoy
Noir 1
Specht et








al. (2001)


Pl ht 6-5
M
38
40
PI 27890
PI
Lark et







290136
al. 1995)









D. Regions Controlling Argonomic Traits in QTL Mapping Studies in Soybean Plants.
Based on the data collected in the SoyBase database (Grant et al. 2008), at least 85 traits within over 1,100 QTLs were identified. These argonomic traits were mapped to at least one QTL controlling region for each trait including abnormal seedling, acidic protein fraction, alpha prime conglycinin protein fraction, aluminum tolerance, arabinose, arabinose-galactose basic protein fraction, beginning maturity, beginning pod, beta conglycinin protein fraction, brown stem rot resistance, canopy height, canopy width, carbon isotope discrimination, cell wall polysaccharide, chlorimuron ethyl sensitivity, common cutworm resistance, conglycinin protein fraction, corn earworm resistance, daidzein content, first flower, flooding tolerance, flowering time, fructose content, galactose content glycinin protein fraction, glycitein content, height/lodging, hypocotyl length, iron efficiency, javanese root-knot nematode resistance, leaf area, leaf ash, leaf chlorosis leaf length, leaf phosphorus content, leaf width, leaflet area, leaflet shape, linoleic acid content, linolenic acid content, lodging, nitrogen accumulation at growth stage R5, oil content, oil/protein ratio, oleic acid concentration, palmitic acid concentration, peanut root-knot nematode resistance, pectin concentration, phomopsis seed decay, photoperiod insensitivity, phytophthora sojae partial resistance, plant height, pod dehiscence, pod maturity date, protein concentration, reproductive period, rhizoctonia rot and hypocotyl rot, root necrosis, salt tolerance, sclerotinia stem rot, seed abortion, seed coat hardness, seed filling period, seed number, seed set, seed weight, southern root-knot nematode resistance, soybean cyst nematode resistance, soybean looper resistance (229-M), specific leaf weight, sprout yield, stearic acid concentration, stem diameter, stem length, sucrose concentration, sudden death syndrome resistance, tobacco budworm resistance (229-M), tobacco ringspot virus resistance, trigonelline concentration (dry weight), trigonelline concentration (fresh weight), water use efficiency, and yield, such as Yield/Height,Yield/Seed weight, SoyBase (Grant et al. 2008),
Some of the QTLs listed as separate QTLs in SoyBase appear to be the same QTL identified in the same population (e.g. SCN 29-1, SCN 29-4, and SCN 29-8 on linkage group G) while some other QTLs might be the same QTL identified in different populations (see the “consensus QTL regions” section below). The amount of phenotypic variation accounted for by a single QTL varied from 1% to 97% (Table 2). The majority of the QTLs listed in SoyBase were not confirmed by separate studies. However, independent mapping studies with populations developed from different parents frequently identified QTLs for the same trait in a similar region on the integrated linkage map. The consistence of the QTL locations found in independent studies with different mapping populations indicates the existence of a real QTL in the region. For example, a QTL for SCN resistance was identified in the 0-37 cM region on linkage group G in 14 mapping populations. The major SCN resistance gene rhg1 was found in this region (Concibido et al. 2004). Thus the inventors contemplate identifying specific genes contributing to aphid resistance within the QTLs identified during the development of the present inventions. SCN resistance, 99 QTLs were identified (Tables A-G).







TABLE D







Consensus regions for QTLs for soybean cyst nematode resistance listed in SoyBase by Grant et al. (2008)



















LOD


Start
End

Mapping
Mapping



QTL
R2 (%)a
score
P-value
LGb
positionc
positionc
Peak marker/interval
parent 1
parent 2
Reference




















SCN
1.0

0.0008
Al
7.8
9.8
A487_1
Hartwig
Williams 82
Vierling et


2-3









al. (1996)


SCN
7.4
2.78
0.0010
A1
30.9
53.4
A262_1, Satt300
PI
Hamilton
Yue et al.


18-1







438489B

(2001a)


SCN


0.0015
A2
31.2
33.2
A085_1
M85-
M83-15
Concibido


1-1







1430

et al.












(1994)


SCN
19.1
7.00
0.0010
A2
45.6
53.1
K400_2, T155_2
PI
Hamilton
Yue et al.


19-1







438489B

(2001a)


SCN
26.2
5.20

A2
47.6
49.6
E(CCG)M(AAC)405
Essex
Forrest
Meksem


27-2









et al.












(2001)


SCN
25.0


A2
47.8
49.8
I
Peking
Essex
Mahalingam


9-2









et al.












(1995)


SCN
9.0
5.80

A2
47.8
49.8
I
PI
BSR101
Webb et


3-1







437654

al. (1995)


SCN
17.7
14.50

A2
49.4
60.6
Sat_400, Satt424
Hamilton
PI 90763
Guo et al.


29-5









(2006)


SCN
23.2
5.10

A2
53.2
55.2
BLT065_1
Essex
Forrest
Chang et


8-5









al. (1997)


SCN
40.0

0.6400
A2
53.2
55.2
BLT065_1
Flyer
Hartwig
Prabhu et


13-2









al. (1999)


SCN
8.0


A2
56.9
58.9
S07a
Peking
Essex
Mahalingam


9-3









et al.












(1995)


SCN
29.0

0.0005
A2
59.6
61.6
Satt424
PI
Bell
Brucker et


30-3







437654

al. (2005)


SCN
15.1
2.80

A2
65.8
67.8
OW15_400
Essex
Forrest
Chang et


8-4









al. (1997)


SCN
12.5


A2
70.4
72.4
A136_1
Peking
Essex
Mahalingam


9-1









et al.












(1995)


SCN
9.5
3.71
0.0011
B1
58.9
64.8
A118_1, A006_1
PI 89772
Hamilton
Yue et al.


26-1









(2001)


SCN
91.0

0.0001
B1
63.8
65.8
A006_1
Hartwig
Williams 82
Vierling et


2-1









al. (1996)


SCN2
16.6
6.83
0.0001
B1
64.8
84.2
A006_1, Satt583
PI 89772
Hamilton
Yue et al.


3-1









(2001b)


SCN2
6.8
2.78
0.0035
B1
64.8
84.2
A006_1, Satt583
PI 89772
Hamilton
Yue et al.


4-1









(2001b)


SCN
12.7
4.20
0.0010
B1
84.2
101.0
Satt583, Sat_123
PI
Hamilton
Yue et al.


17-1







438489B

(2001a)


SCN
7.4
2.79
0.0010
B1
84.2
101.0
Satt583, Sat_123
PI
Hamilton
Yue et al.


18-2







438489B

(2001a)


SCN
11.0
2.70
0.0010
B1
84.2
101.0
Satt583, Sat_123
PI
Hamilton
Yue et al.


20-1







438489B

(2001a)


SCN
11.2
6.00

B1
102.6
124.0
Satt359, Satt453
Hamilton
PI 90763
Guo et al.


29-10









(2006)


SCN
1.0

0.0001
B1
125.0
127.0
A567_1
Hartwig
Williams 82
Vierling et


2-2









al. (1996)


SCN
11.7
2.75
0.0010
B2
55.2
62.7
A329_1, Satt168
PI
Hamilton
Yue et al.


17-2







438489B

(2001a)


SCN
8.1
2.56
0.0010
B2
55.2
62.7
A329 1, Satt168
PI
Hamilton
Yue et al.


19-2







438489B

(2001a)


SCN
21.0


B2
97.5
99.5
A593_1
Peking
Essex
Qiu et al.


10-1









(1999)


SCN
15.0


B2
117.7
119.7
T005_1
Peking
Essex
Qiu et al.


10-3









(1999)


SCN
9.0


B2
117.7
119.7
T005_1
Peking
Essex
Qiu et al.


11-3









(1999)


SCN
11.1
3.61
0.0010
C1
18.6
21.0
A059_1, A463_1
PI
Hamilton
Yue et al.


21-1







438489B

(2001a)


SCN
10.2
2.56
0.0010
C1
21.0
24.1
A463_1, Satt396
PI
Hamilton
Yue et al.


18-3







438489B

(2001a)


SCN
5.0
4.40

C2
1.0
1.0
A121_1
PI
A81356022
Wang et


22-1







468916

al. (2001)


SCN
8.0


C2
94.6
96.6
A635_1
Peking
Essex
Mahalingam


9-6









et al.












(1995)


SCN
7.1
6.80
0.0010
C2
126.2
145.5
Satt202, Satt371
PI
Hamilton
Yue et al.


17-3







438489B

(2001a)


SCN
8.3
3.05
0.0010
C2
126.2
145.5
Satt202, Satt371
PI
Hamilton
Yue et al.


20-2







438489B

(2001a)


SCN
10.7
5.47
0.0010
D1a
6.4
34.9
A398_1, K478_1
PI
Hamilton
Yue et al.


19-3







438489B

(2001a)


SCN
9.4
4.17
0.0010
D1a
6.4
34.9
A398_1, K478_1
PI
Hamilton
Yue et al.


20-3







438489B

(2001a)


SCN
7.4
4.14
0.0010
D1a
6.4
34.9
A398_1, K478_1
PI
Hamilton
Yue et al.


21-2







438489B

(2001a)


SCN
7.8
3.30
0.0015
D1a
43.8
48.1
Satt342, Satt368
PI 89772
Hamilton
Yue et al.


26-2









(2001b)


SCN
9.7
4.59
0.0014
D2
15.0
39.4
B132_4, Satt372
PI 89772
Hamilton
Yue et al.


23-2









(2001b)


SCN
41.0

0.0010
D2
86.3
88.3
Satt082
Hartwig
BR92-31983
Schuster


16-1









et al.












(2001)


SCN
9.0


E
16.1
18.1
A963_1
Peking
Essex
Qiu et al.


12-2









(1999)


SCN
23.0
3.50

E
33.2
35.2
Satt598
PI 46891
A81356022
Wang et


22-3









al. (2001)


SCN
12.5
7.20

E
35.8
43.1
Satt573, Satt204
Hamilton
PI 90763
Guo et al.


29-9









(2006)


SCN
8.0
2.57
0.0010
E
37.3
45.1
A656_1, Satt452
PI
Hamilton
Yue et al.


18-4







438489B

(2001a)


SCN
18.7
5.01
0.0010
E
37.3
45.1
A656_1, Satt452
PI
Hamilton
Yue et al.


21-3







438489B

(2001a)


SCN
15.7
3.56
0.0053
E
51.0
70.2
A135_3, Satt231
PI 89772
Hamilton
Yue et al.


25-1









(2001b)


SCN
14.7
7.90

G
0.0
12.5
Satt163, Satt688
Hamilton
PI 90763
Guo et al.


29-1









(2006)


SCN
28.1
22.10

G
0.0
12.5
Satt163, Satt688
Hamilton
PI 90763
Guo et al.


29-4









(2006)


SCN
13.0
7.10

G
0.0
12.5
Satt163, Satt688
Hamilton
PI 90763
Guo et al.


29-8









(2006)


SCN
26.2

0.0001
G
0.8
2.8
C006_1
Evans
Peking
Concibido


4-1









et al.












(1997)


SCN
44.8

0.0001
G
0.8
2.8
C006_1
Evans
PI 90763
Concibido


5-1









et al.












(1997)


SCN
36.3

0.0001
G
0.8
2.8
C006_1
Evans
PI 88788
Concibido


6-1









et al.












(1997)


SCN
6.4

0.0760
G
0.8
2.8
Satt038
Flyer
Hartwig
Prabhu et


13-1









al. (1999)


SCN
12.9
2.70

G
1.0
3.0
OI03_450
Essex
Forrest
Chang et


8-3









al. (1997)


SCN
28.1


G
1.8
8.6
C006_1, Bng122_1
Evans
Peking
Concibido


4-4









et al.












(1996)


SCN
52.7


G
1.8
8.6
C006_1, Bng122_1
Evans
PI 90763
Concibido


5-3









et al.












(1996)


SCN
40.0


G
1.8
8.6
C006_1, Bng122_1
Evans
PI 88788
Concibido


6-2









et al.












(1996)


SCN
51.4


G
1.8
8.6
C006_1, Bng122_1
Evans
PI 209332
Concibido


7-1









et al.












(1996)


SCN
14.0

0.0400
G
3.5
5.5
Satt309
PI
Bell
Brucker et


30-1







437654

al. (2005)


SCN
32.0

0.0001
G
3.5
5.5
Satt309
PI
Bell
Brucker et


30-2







437654

al. (2005)


SCN
97.0

0.0001
G
3.5
5.5
Satt309
Essex
Forrest
Meksem


14-2









et al.












(1999)


SCN
26.6
13.67
0.0001
G
4.5
5.8
B053_1, Satt309
PI 89772
Hamilton
Yue et al.


23-3









(2001b)


SCN
4.6
2.53
0.0095
G
4.5
5.8
B053_1, Satt309
PI 89772
Hamilton
Yue et al.


24-2









(2001b)


SCN
23.0
12.65
0.0001
G
4.5
5.8
B053_1, Satt309
PI 89772
Hamilton
Yue et al.


25-2









(2001b)


SCN
10.0
5.02
0.0001
G
4.5
5.8
B053_1, Satt309
PI 89772
Hamilton
Yue et al.


26-3









(2001b)


SCN
87.0
40.60

G
4.5
8.6
Satt309, Bng122
Bell
Colfax
Glover et


28-1









al. (2004)


SCN
64.0
17.70

G
4.5
8.6
Satt309, Bng122
Bell
Colfax
Glover et


28-3









al. (2004)


SCN
24.1
5.10

G
4.8
6.8
E(ATG)M(CGA)87
Essex
Forrest
Meksem


27-1









et al.












(2001)


SCN



G
5.8
35.5
B053_1, A112_1
PI 88287
PI 89008
Vaghchhipawal


15-1









et












al. (2001)


SCN
11.3
2.50

G
7.6
9.6
Bng122_1
Essex
Forrest
Chang et


8-2









al. (1997)


SCN
19.0

0.0730
G
7.6
9.6
Bng122_1
Essex
Forrest
Meksem


14-1









et al.












(1999)


SCN
4.2
1.20

G
10.6
12.6
OG13_490
Essex
Forrest
Chang et


8-1









al. (1997)


SCN
22.0
15.40

G
11.0
13.0
PHP05354a, PHP05219a
PI
BSR101
Webb et


3-2







437654

al. (1995)


SCN
36.0

0.0001
G
23.1
25.1
K069_1
M85-
M83-15
Concibido


1-3







1430

et al.












(1994)


SCN
15.8
9.08
0.0010
G
23.1
54.7
A096_3, Satt130
PI
Hamilton
Yue et al.


17-4







438489B

(2001a)


SCN
12.8
7.52
0.0010
G
23.1
54.7
A096_3, Satt130
PI
Hamilton
Yue et al.


18-5







438489B

(2001a)


SCN
13.6
4.46
0.0010
G
23.1
66.6
Satt012, Satt130
PI
Hamilton
Yue et al.


19-4







438489B

(2001a)


SCN
1.0

0.0018
G
34.5
36.5
A112_1
Hartwig
Williams 82
Vierling et


2-4









al. (1996)


SCN
5.8
2.03
0.0010
G
62.2
66.6
Satt012, Satt199
PI
Hamilton
Yue et al.


20-4







438489B

(2001a)


SCN
27.0
4.80

G
89.0
91.0
A245_2
PI
A81356022
Wang et


22-2







468916

al. (2001)


SCN
6.7
3.00

G
102.6
124.0
Satt453, Satt359
Hamilton
PI 90763
Guo et al.


29-3









(2006)


SCN
17.6

0.0002
G
108.5
110.5
A3781
Evans
Peking
Concibido


4-2









et al.












(1997)


SCN
12.0


H
120.3
122.3
K014_1
Peking
Essex
Qiu et al.


10-5









(1999)


SCN
9.0


H
120.3
122.3
K014_1
Peking
Essex
Qiu et al.


11-2









(1999)


SCN
13.0


H
123.1
125.1
B072_1
Peking
Essex
Qiu et al.


10-4









(1999)


SCN
13.0


H
123.1
125.1
B072_1
Peking
Essex
Qiu et al.


11-1









(1999)


SCN
11.0


I
37.1
39.1
K011_1
Peking
Essex
Qiu et al.


12-1









(1999)


SCN
2.0
2.50

J
65.0
67.8
Satt244, Satt547
Bell
Colfax
Glover et


28-2









al. (2004)


SCN
7.0
3.40

J
65.0
78.6
Satt244, Satt431
Bell
Colfax
Glover et


28-4









al. (2004)


SCN
7.8
4.60

J
67.8
75.1
Satt547, Sat_224
Hamilton
PI 90763
Guo et al.


29-2









(2006)


SCN
4.2
13.90

J
67.8
75.1
Satt547, Sat_224
Hamilton
PI 90763
Guo et al.


29-6









(2006)


SCN


0.0001
J
73.0
75.0
B032_1
M85-
M83-15
Concibido


1-2







1430

et al.












(1994)


SCN
18.8

0.0001
J
73.0
75.0
B032_1
Evans
PI 90763
Concibido


5-2









et al.












(1997)


SCN
4.0
3.00

L
87.4
93.9
Sat_286, Satt229
Hamilton
PI 90763
Guo et al.


29-7









(2006)


SCN
7.0
4.80

M
74.0
76.0
PHP02275a, PHP02301a
PI
BSR101
Webb et


3-3







437654

al. (1995)


SCN
14.3

0.0001
N
33.9
35.9
A280_1
Evans
Peking
Concibido


4-3









et al.












(1997)


SCN
16.0





A018_3
Peking
Essex
Qiu et al.


10-2









(1999)


SCN
6.0





E01c
Peking
Essex
Mahalingam


9-4









et al.












(1995)


SCN
6.0





G15d
Peking
Essex
Mahalingam


9-5









et al.












(1995)





aR2 = Phenotypic variation explained by a QTL


bLG = linkage group. The linkage group names are from the integrated map by Song et al. (2004).


cThe start positions and end positions are from the integrated map by Song et al. (2004). When a single marker that was associated with a QTL in a published study could be placed on the consensus map, an arbitrary 2 cM interval with 1 cM on either side of the marker was defined as the QTL region in SoyBase













TABLE E







Consensus regions for QTLs for nematode resistance and earworm resistance.











Name
PI
Reference











Soybean cyst nematode resistance.













SCN 19-1
A2
46
53
Hamilton
PI 438489B
Yue et al. (2001)


SCN 3-1
A2
48
50
BSR101
PI 437654
Webb et al. (1995)


SCN 9-2
A2
48
50
Essex
Peking
Mahalingam et al. (1995)


SCN 29-4
A2
49
61
Hamilton
PI 90763
Guo et al. (2006)


SCN 8-5
A2
53
55
Essex
Forrest
Chang et al. (1997)


SCN 13-2
A2
53
55
Flyer
Hartwig
Prabhu et al. (1999)


SCN 30-3
A2
60
62
Bell
PI 437654
Brucker et al. (2005)


SCN 2-1
B1
64
66
Hartwig
Williams 82
Vierling et al. (1996)


SCN 24-1
B1
65
84
Hamilton
PI 89772
Yue et al. (2001)


SCN 20-1
B1
84
101
Hamilton
PI438489B
Yue et al. (2001)


SCN 29-7
B1
103
124
Hamilton
PI 90763
Guo et al. (2006)


SCN 22-3
E
33
35
A81356022
PI 468916
Wang et al. (2001)


SCN 29-6
E
36
43
Hamilton
PI 90763
Guo et al. (2006)


SCN 21-3
E
37
45
Hamilton
PI 438489B
Yue et al. (2001)


SCN 25-1
E
51
70
Hamilton
PI 89772
Yue et al. (2001)


SCN 29-5
G
0
13
Hamilton
PI 90763
Guo et al. (2006)


SCN 4-1
G
1
3
Evans
Peking
Concibido et al. (1997)


SCN 5-1
G
1
3
Evans
PI 90763
Concibido et al. (1997)


SCN 6-1
G
1
3
Evans
PI 88788
Concibido et al. (1997)


SCN 13-1
G
1
3
Flyer
Hartwig
Prabhu et al. (1999)


SCN 8-3
G
1
3
Essex
Forrest
Chang et al. (1997)


SCN 7-1
G
2
9
Evans
PI 209332
Concibido et al. (1996)


SCN 30-2
G
4
6
Bell
PI 437654
Brucker et al. (2005)


SCN 23-3
G
5
6
Hamilton
PI 89772
Yue et al. (2001)


SCN 15-1
G
6
36
PI 88287
PI 89008
Vaghchhipawala et al. (2001)


SCN 3-2
G
11
13
BSR101
PI 437654
Webb et al. (1995)


SCN 19-4
G
23
67
Hamilton
PI438489B
Yue et al. (2001)


SCN 1-3
G
23
25
M83-15
M85-1430
Concibido et al. (1994)


SCN 2-4
G
35
37
Hartwig
Williams 82
Vierling et al. (1996)


SCN 28-4
J
65
79
Bell
Colfax
Glover et al. (2004)


SCN 29-2
J
68
75
Hamilton
PI 90763
Guo et al. (2006)


SCN 5-2
J
73
75
Evans
PI 90763
Concibido et al. (1997)


SCN 1-2
J
73
75
M83-15
M85-1430
Concibido et al. (1994)







Corn earworm resistance













CEW 3-1
C2
90
102
Cobb
PI
Rector et







227687
al. (1999)


CEW 8-3
C2
111
113
Archer
Minsoy
Terry et al. (2000)


CEW 8-1
E
2
4
Archer
Minsoy
Terry et al. (2000)


CEW 7-1
E
8
10
Minsoy
Noir 1
Terry et al. (2000)


CEW 2-2
H
49
62
Cobb
PI
Rector et







171451
al. (1999)


CEW 3-2
H
49
62
Cobb
PI
Rector et







227687
al. (1999)


CEW 9-3
H
53
61
Cobb
PI
Narvel et







229358
al. (2001)


CEW 6-2
J
15
17
Cobb
PI
Rector et







229358
al. (2000)


CEW 7-4
J
20
22
Minsoy
Noir 1
Terry et








al. (2000)


CEW 6-3
M
59
71
Cobb
PI
Rector et







229358
al. (2000)


CEW 4-1
M
59
71
Cobb
PI
Rector et







171451
al. (2000)
















TABLE F





Consensus regions for QTLs for disease resistance.







Phytophthora resistance













Phyto 1-la
F
16
18
Conrad
Sloan
Burnham et al. (2003)


Phyto 1-lb
F
16
18
Conrad
Williams
Burnham et al. (2003)


Phyto 1-lc
F
16
21
Conrad
Harosoy
Burnham et al. (2003)







Sudden death syndrome resistance













SDS 8-2
C2
120
122
Douglas
Pyramid
Njiti et al. (2002)


SDS 2-6
C2
131
133
Essex
Forrest
Chang et al. (1996)


SDS 8-1
G
0
1
Douglas
Pyramid
Njiti et al. (2002)


SDS 3-2
G
1
3
Essex
Forrest
Chang et al. (1996)







Brown stem rot resistance













BSR 1-1
J
67
69
BSR101
PI 437654
Lewers et al. (1999)


BSR 4-1
J
78
80
Century
PI 437833
Bachman et al. (2001)


BSR 3-1
J
78
80
Century84
L78-4094
Bachman et al. (2001)







Sclerotinia stem rot resistance













Sclero 5-1
A2
60
62
S1 9-90
Williams82
Arahana et al.








(2001)


Sclero 6-2
A2
60
62
Vinton81
Williams82
Arahana et al.








(2001)


Sclero 2-2
A2
60
62
Corsoy79
Williams82
Arahana et al.








(2001)


Sclero 4-1
D1a
109
110
DSR173
Williams82
Arahana et al.








(2001)


Sclero 5-3
D1a
109
110
S1 9-90
Williams82
Arahana et al.








(2001)


Sclero 3-5
D1b
118
120
Dassel
Williams82
Arahana et al.








(2001)


Sclero 2-7
D1b
118
120
Corsoy79
Williams82
Arahana et al.








(2001)


Sclero 2-12
F
63
65
Corsoy79
Williams82
Arahana et al.








(2001)


Sclero 5-6
F
63
65
S1 9-90
Williams82
Arahana et al.








(2001)


Sclero 5-9
G
85
97
S1 9-90
Williams82
Arahana et al.








(2001)


Sclero 6-7
G
96
98
Vinton81
Williams82
Arahana et al.








(2001)


Sclero 2-20
L
54
56
Corsoy79
Williams82
Arahana et al.








(2001)


Sclero 3-14
L
54
56
Dassel
Williams82
Arahana et al.








(2001)


Sclero 6-13
O
120
129
Vinton81
Williams82
Arahana et al.








(2001)


Sclero 3-19
O
120
129
Dassel
Williams82
Arahana et al.








(2001)


Sclero 4-11
O
127
129
DSR173
Williams82
Arahana et al.








(2001)









E. Consensus QTL Regions for Seed Protein and Oil Contents
QTLs for protein content were found in 13 consensus regions on 11 LGs: A1, A2, B1, B2, C1, C2, E, G, I, K, and M (Tables A-G and FIG. 14). The regions 93-96 cM on LG A1, 145-151 cM on LG A2, 28-37 cM on LG B1, 28-46 cM on LG B2, 9-34 cM on LG C1, 90-98 cM on LG C1, 123-128 cM on LG C1, 117-123 cM on LG C2, 89-98 cM on LG G, and 31-42 cM on LG K were each found in two mapping populations developed from four mapping parents (Table 3). The 26-32 cM region on LG E and the 33-40 cM region on LG M were each found in three mapping populations developed from five or six mapping parents (Table 3). The 31-40 cM region on LG I was found in four mapping populations developed from seven mapping parents (Table 3).
QTLs for oil content were found in nine consensus regions on eight LGs: A1, C1, E, H, I, K, L, and M (Tables A-G and FIG. 14). The 88-96 cM region on LG A1 was found in four mapping populations developed from seven mapping parents (Table 3). The regions near 9-11 cM on LG C1, 23-36 cM on LG E, 86-91 cM on LG H, 98-106 cM on LG K, 34-38 cM on LG L, and 35-40 cM on LG M were each found in two mapping populations developed from three or four mapping parents (Table 3). The 22-40 cM region on LG I was found in five mapping populations developed from nine mapping parents (Tables A-G). The 91-96 cM region on LG L was found in three mapping populations developed from six mapping parents (Tables A-G).
F. Consensus QTL Regions for Disease and Insect Resistance.
QTLs for resistance to soybean cyst nematode (SCN) were found in five consensus regions on five LGs: A2, B1, E, G, and J (Tables A-G and FIG. 14). The 46-72 cM region on LG A2 was found in seven mapping populations derived from 11 mapping 25 parents (Table 3). This region contains the Rhg4 SCN resistance gene. The regions 64-124 cM on LG B1 and 33-70 cM on LG E were each found in four mapping populations derived from six mapping parents (Table 3). The 0-37 cM region on LG G was found in 14 mapping populations derived from 20 mapping parents (Tables A-G). This region contains the rhg1 SCN resistance gene. The 65-79 cM region on LG J was found in four mapping populations derived from seven mapping parents (Tables A-G).
One consensus region, 16-21 cM on LG F, was found to contain a QTL for partial resistance to Phytophthora root rot in three mapping populations developed from four mapping parents (Tables A-G and FIG. 14). Two consensus regions, 120-133 cM on LG C2 and 0-3 cM on LG G, were found to contain QTLs for resistance to sudden death syndrome in two mapping populations developed from four mapping parents (Table 1 and FIG. 14). A consensus region, 67-80 cM on LG J, was found to contain a QTL for resistance to brown stem rot in three mapping populations developed from six mapping parents (Tables A-G and FIG. 14).
QTLs for Sclerotinia stem rot resistance were found in seven consensus regions on seven LGs: A2, D1a, D1b, F, G, L, and O (Tables A-G and FIG. 14). The near 60-62 cM region on LG A2 and the 120-129 cM region on LG 0 were each identified in three mapping populations developed from four mapping parents (Tables A-G). The regions near 109-110 cM on LG D1a, near 118-120 cM on LG D1b, near 63-65 cM on LG F, 85-98 cM on LG G, and near 54-56 cM on LG L were each identified in two mapping population developed from three mapping parents (Tables A-G).
QTLs for corn earworm resistance were found in five consensus regions on five LGs: C2, E, H, J and M (Tables A-G and FIG. 14). The regions 90-113 cM on LG C2, 2-10 cM on LG E, 15-22 cM on LG J, and 59-71 cM on LG M were each identified in two mapping populations developed from three or four mapping parents (Tables A-G). The 49-61 cM region on LG H was identified in three mapping populations developed from four mapping parents (Tables A-G).
G. Consensus QTL Regions Containing QTLs for Multiple Agronomic Traits
In QTL mapping studies, it is common to find QTLs for different traits mapped to a common genomic region. In many cases, the traits affected by the co-localized QTLs are correlated, while in other cases, the trait correlation may not be obvious. While it remains to be resolved whether the co-localized QTLs are tightly linked QTLs or the same QTL with pleiotropic effects, it is useful to document the genomic regions containing QTLs for multiple traits. When the consensus QTL regions for each trait were compared, over 20 genomic regions were found containing QTLs for multiple traits (Table 1 and FIG. 14). Five genomic regions, 88-96 cM on LG A1, near 9-11 cM on LG C1, 26-36 cM on LG E, 31-40 cM on LG I, and 35-40 cM on LG M contain QTLs for both seed protein and seed oil contents (FIG. 14). Soybean protein content and oil content are highly negatively correlated with a correlation coefficient as high as −0.98 (P<0.001) (Mansur et al. 1996). It is, therefore, expected to have some QTLs for the two traits mapped to common genomic regions. Four genomic regions, near 34-36 cM on LG I, 15-10 22 cM on LG J, 36-42 cM on LG K, and 35-40 cM on LG M, contain QTLs for both yield and plant height (FIG. 2). Yield was correlated (r=0.59, P<0.001) with plant height in the study by Mansur et al. (1996). Yield was also correlated (r=0.48, P<0.001) with maturity (Mansur et al. 1996). Three genomic regions, near 34-36 cM on LG I, near 18-20 cM on LG M, and 35-40 cM on LG M, contain QTLs for both yield and maturity (FIG. 2). Yield was negatively correlated (r=−0.58, P<0.001) with protein content and positively correlated (r=0.60, P<0.001) with oil content (Mansur et al. 1996). Two genomic regions, near 34-36 cM on LG I and 35-40 cM on LG M, contain QTLs for yield, protein content, and oil content. Another region, 36-42 cM on LG K, contains QTLs for yield and protein content (FIG. 2). Lodging was highly correlated with plant height (r=0.84, P<0.001) (Mansur et al. 1996). Three genomic regions, 107-113 cM on LG C2, 8-11 cM on LG L, and 68-101 cM on LG L, contain QTLs for these two traits (FIG. 2).
QTLs for Sclerotinia stem rot resistance were co-localized with QTLs for plant height near 120 cM on LG D1b and near 65 cM on LG F, for maturity near 54-56 cM on LG L, for SCN resistance near 60-62 cM on LG A2, and for protein content in 89-98 cM on LG G (FIG. 2). Sclerotinia stem rot disease severity index was correlated with plant height (r=0.54, P<0.001) and maturity (r=0.67, P<0.001) in the study by Kim and Diers (2000). Shorter plant and earlier maturity were associated with greater resistance to Sclerotinia stem rot, which was considered an escape mechanism (Kim and Diers 2000). However, the QTLs co-localized with plant height on LG D1b and LG F and with maturity on LG L were for physiological resistance to Sclerotinia stem rot (Arahana et al. 2001). Therefore, the resistance associated with shorter plant and earlier maturity may also involve physiological resistance. A significant correlation (r=0.40, P<0.05) between Sclerotinia stem rot disease severity index and protein content was reported by Hoffman et al. (1998). There is no report of any correlation between Sclerotinia stem rot resistance and SCN resistance.
QTLs for SCN resistance were co-localized with QTLs for sudden death syndrome (SDS) near 4-6 cM on LG G, for brown stem rot resistance in 67-79 cM on LG J, for oil content in 33-36 cM on LG E, and for Sclerotinia stem rot resistance as described above. Coinheritance of SDS resistance with SCN resistance was reported by Chang et al. (1997) and the locus underlying the coinheritance was assigned to the region on LG G where the major SCN resistance was located (Chang et al. 1997). The region on LG J is known to contain multiple resistance genes to different pathogens (Bachman et al. 2001). Correlation of SCN resistance with oil content was not reported in the literature. Qiu et al. (1999) carried out a QTL mapping study in a population that was segregating for both SCN resistance and oil content. A marker on LG H was found to be associated with both SCN resistance and oil content.
QTLs for corn earworm resistance were co-localized with QTLs for plant height in 107-113 cM on LG C2 and in 15-22 cM on LG J. There is no known correlation between corn earworm resistance and plant height in soybean.
H. Future Opportunities for QTL Discovery in Soybean Germplasm.
Over a thousand SNP markers were recently added to the integrated soybean linkage map (Choi et al. 2007) and research is ongoing to add several more thousands of SNP markers to the map. High-throughput SNP genotyping systems have been developed and are commercially available. For example, the Illumina BeadStation 500 (Shen et al. 2005) can analyze 1536 SNP loci in parallel in 192 DNA samples in three days. The addition of thousands of SNP markers to the integrated linkage map and the availability of high-throughput SNP genotyping systems will significantly reduce the time needed to genotype mapping populations and accelerate QTL discovery in soybean.
The first draft sequence of the whole soybean genome was released in 2008 (JGI 2008). The availability of a whole genome sequence will allow scientists to fine-map QTLs and eventually pinpoint the specific mutations that cause the phenotypic variations.
The development of new statistical approaches and computer software that allow joint analysis of multiple populations will also increase the power to identify real QTLs, especially QTLs with small effects. Bink et al. (2008) developed a pedigree-based approach that jointly analyzes the data from multiple populations that are related through their common ancestors in the pedigree. This approach is currently implemented in the computer software FlexQTLTM. Jourjon et al. (2005) developed a computer software package, MCQTL, that can perform QTL mapping in multi-cross designs.
The major limitation to QTL discovery in soybean is the difficulty in obtaining accurate measurement of the traits with low heritability. For certain traits such as field resistance to Sclerotinia stem rot, reliable measurement is difficult to obtain even with efforts to provide the optimum conditions to induce the disease. Large experiments with multiple locations in multiple years are often required to obtain the phenotypic data.
I. Consensus QTL Regions in Soybean.
Using the integrated linkage map developed by Song et al. (2004) as a reference map, when QTLs identified in different mapping populations for the same trait are less than 10 cM from one another, the region containing these QTLs can be considered a consensus QTL region for the trait. These consensus QTL regions are used to determine the true location of the QTL. Tables A-G and FIG. 14 summarize the consensus QTL regions for each trait based on the QTL data collected in SoyBase. The chromosome length in cm (centimorgan) generally covered by a single marker associated with a QTL on the consensus map, is contemplated as an arbitrary 2 cM interval with 1 cM on either side of the marker defined as the QTL region, for example, as shown for QTL mapping in SoyBase. Thus the true QTL position (i.e. the actual gene (DNA sequence) controlling the mapped trait) may be outside the arbitrary 2 cM region. For use in the present inventions the arbitrary 2 cM region was used for each marker, such that the actual region identified by the markers of the present inventions may extend at least 1 cM before and after the most distant areas identified, for example, in Tables 9-10, FIG. 14, preferably 10 cM before and after the most distant areas identified herein. Another example of determining the location of a true QTL position is shown in Table 18 and in the text for other Soybean QTLs, for example, the true QTL may be in a position at least 1 cM before the start position and 1cM after the end position.







TABLE G







Consensus regions for QTLs Soybean QTLs that were less than 10 cM


apart but were identified in different populations for the same trait.















Start
End
Mapping
Mapping



Trait/QTL
LGa
positionb
positionb
parent 1
parent 2
Reference










Yield













Sd yld 11-1
C2
39
41
Minsoy
Noir 1
Specht et al.








(2001)


Yld/SW
C2
45
47
Archer
Noir 1
Orf et al.


2-2





(1999b)


Sd yld 15-1
C2
97
99
BSR101
LG82-
Kabelka







8379
et al.








(2004)


Sd yld 5-1
C2
107
109
Archer
Noir 1
Orf et al.








(1999b)


Yld/Ht 2-1
C2
107
109
Archer
Minsoy
Orf et al.








(1999b)


Sd yld 16-3
C2
112
114
IA2008
PI 468916
Wang et








al.








(2004a)


Sd yld 3-2
C2
117
119
PI 27890
PI 290136
Mansur et








al. (1996)


Yld/Ht 4-2
C2
117
119
Minsoy
Noir 1
Orf et al.








(1999b)


Sd yld 5-2
D2
47
49
Archer
Noir 1
Orf et al.








(1999b)


Yld/Ht 2-4
D2
53
55
Archer
Minsoy
Orf et al.








(1999b)


Sd yld 10-1
I
31
33
Parker
PI 468916
Yuan et








al. (2002)


Sd yld 9-1
I
34
36
A81356022
PI 468916
Yuan et








al. (2002)


Sd yld 14-1
I
36
37
A3733
PI437088A
Chung et








al. (2003)


Yld/Ht 4-1
J
11
13
Minsoy
Noir 1
Orf et al.








(1999b)


Yld/Ht 1-3
J
20
22
PI 27890
PI 290136
Mansur et








al. (1996)


Sd yld 16-1
K
36
38
IA2008
PI 468916
Wang et








al.








(2004a)


Sd yld 12-1
K
46
47
Essex
Forrest
Yuan et








al. (2002)


Sd yld 13-1
K
47
50
Flyer
Hartwig
Yuan et








al. (2002)


Sd yld 8-1
L
70
72
Archer
Minsoy
Orf et al.








(1999a)


Sd yld 11-6
L
88
90
Minsoy
Noir 1
Specht et








al. (2001)


Yld/Ht 1-1
L
91
93
PI 27890
PI 290136
Mansur et








al. (1996)


Yld/Ht 3-1
L
94
96
Archer
Noir 1
Orf et al.








(1999b)


Sd yld 6-1
M
18
20
Minsoy
Noir 1
Orf et al.








(1999b)


Yld/Ht 2-2
M
18
20
Archer
Minsoy
Orf et al.








(1999b)


Yld/SW
M
35
37
Archer
Minsoy
Orf et al.


1-1





(1999b)


Sd yld 3-1
M
38
40
PI 27890
PI 290136
Mansur et








al. (1996)







Protein content













Prot 2-1
Al
93
95
PI 27890
PI 290136
Mansur et








al. (1996)


Prot 12-1
Al
94
96
Minsoy
Noir 1
Specht et








al. (2001)


Prot 21-1
A2
145
147
BSR 101
LG82-
Kabelka







8379
et al.








(2004)


Prot 14-1
A2
149
151
M91-
SZG9652
Vollmann






212006

et al.








(2002)


Prot 3-2
B1
28
30
A87296011
C1763
Brummer








et al.








(1997)


Prot 16-1
B1
35
37
Essex
Williams
Chapman








et al.








(2003)


Prot 4-11
B2
28
30
PI 416937
Young
Lee et al.








(1996b)


Prot 1-6
B2
32
34
A81356022
PI 468916
Diers et








al. (1992)


Prot 4-10
B2
43
46
PI 416937
Young
Lee et al.








(1996b)


Prot 9-2
Cl
9
11
Minsoy
Noir 1
Orf et al.








(1999b)


Prot 4-4
Cl
20
22
PI 416937
Young
Lee et al.








(1996b)


Prot 12-2
Cl
32
34
Minsoy
Noir 1
Specht et








al. (2001)


Prot 3-3
Cl
90
92
A87296011
C1763
Brummer








et al.








(1997)


Prot 4-3
Cl
96
98
PI 416937
Young
Lee et al.








(1996b)


Prot 21-2
Cl
123
125
BSR 101
LG82-
Kabelka







8379
et al.








(2004)


Prot 4-2
Cl
126
128
PI 416937
Young
Lee et al.








(1996b)


Prot 17-1
C2
117
119
Essex
Williams
Hyten et








al. (2004)


Prot 13-2
C2
121
123
Ma.Belle
Proto
Csanadi








et al.








(2001)


Prot 4-6
E
26
28
PI 416937
Young
Lee et al.








(1996b)


Prot 3-6
E
30
32
A87296011
C1763
Brummer








et al.








(1997)


Prot 18-1
E
30
32
Coker 237
PI 97100
Fasoula








et al.








(2004)


Prot 1-8
G
89
91
A81356022
PI 468916
Diers et








al. (1992)


Prot 3-10
G
96
98
A87296011
C1763
Brummer








et al.








(1997)


Prot 11-1
I
31
33
Parker
PI 468916
Yuan et








al. (2002)


Prot 1-3
I
31
33
A81356022
PI 468916
Diers et








al. (1992)


Prot 3-12
I
31
33
A87296011
C1763
Brummer








et al.








(1997)


Prot 10-1
I
34
36
A81356022
PI 468916
Yuan et








al. (2002)


Prot 15-1
I
36
37
A3733
PI
Chung et







437088A
al. (2003)


Prot 1-2
I
38
40
A81356022
PI 468916
Lark et








al. (1994)


Prot 5-4
K
31
33
Coker237
PI 97100
Lee et al.








(1996b)


Prot 12-3
K
40
42
Minsoy
Noir 1
Specht et








al. (2001)


Prot 12-4
M
33
35
Minsoy
Noir 1
Specht et








al. (2001)


Prot 13-3
M
33
35
Ma.Belle
Proto
Csanadi








et al.








(2001)


Prot 7-1
M
38
40
Archer
Minsoy
Orf et al.








(1999b)







Oil content













Oil 8-1
A1
88
90
Archer
Minsoy
Orf et al.








(1999b)


Oil 4-3
A1
91
93
A87296011
C1763
Brummer








et al.








(1997)


Oil 3-2
A1
93
95
PI 27890
PI
Mansur et







290136
al. (1996)


Oil 13-1
A1
94
96
Minsoy
Noir 1
Specht et








al. (2001)


Oil 9-1
C1
9
11
Archer
Noir 1
Orf et al.








(1999b)


Oil 8-2
C1
9
11
Archer
Minsoy
Orf et al.








(1999b)


Oil 5-1
E
23
25
PI 416937
Young
Lee et al.








(1996b)


Oil 2-9
E
34
36
A81356022
PI
Diers et







468916
al. (1992)


Oil 17-2
H
86
88
Coker 237
PI 97100
Fasoula








et al.








(2004)


Oil 19-2
H
89
91
N87-984-
TN93-99
Panthee






16

et al.








(2005)


Oil 14-3
I
22
24
Ma.Belle
Proto
Csanadi








et al.








(2001)


Oil 12-1
I
31
33
Parker
PI
Yuan et







468916
al. (2002)


Oil 11-1
I
31
33
A81356022
PI
Yuan et







468916
al. (2002)


Oil 13-4
I
34
36
Minsoy
Noir 1
Specht et








al. (2001)


Oil 15-1
I
36
37
A3733
PI
Chung et







437088A
al. (2003)


Oil 2-2
I
38
40
A81356022
PI
Diers et







468916
al. (1992)


Oil 4-11
K
98
100
A87296011
C1763
Brummer








et al.








(1997)


Oil 14-2
K
104
106
Ma.Belle
Proto
Csanadi








et al.








(2001)


Oil 18-2
L
34
36
PI 416937
Young
Fasoula








et al.








(2004)


Oil 2-7
L
36
38
A81356022
PI
Diers et







468916
al. (1992)


Oil 5-3
L
36
38
PI 416937
Young
Lee et al.








(1996b)


Oil 3-1
L
91
93
PI 27890
PI
Mansur et







290136
al. (1996)


Oil 16-1
L
93
95
Essex
Williams
Hyten et








al. (2004)


Oil 9-3
L
94
96
Archer
Noir 1
Orf et al.








(1999b)


Oil 16-2
M
35
37
Essex
Williams
Hyten et








al. (2004)


Oil/Prot
M
38
40
PI 27890
PI
Lark et


1-2




290136
al. (1994)





aLG = linkage group. The linkage group names are from the integrated map by Song et al. (2004).


bThe start positions and end positions are from the integrated map by Song et al. (2004). When a single marker that was associated with a QTL in a published study could be placed on the consensus map, an arbitrary 2 cM interval with 1 cM on either side of the marker was defined as the QTL region in SoyBase






Summary of QTL Locations of Aphid Resistant Germplasm.

The inventors used 123 SSR markers and a subset of 94 soybean lines, for the construction of a linkage map of 25 linkage groups, which covered the majority of the soybean genome except for some regions where no polymorphic markers were found in this study. For LGs A2, B2, F, L, M, each LG was consisting of two unlinked segments. Based on the consensus map (Song et al. 2004); the intervals between two unlinked segments were about 30 cM or more. These intervals are contemplated to be exaggerated due to the small size of the subset. Since there were no polymorphic SSR loci in these intervals for this study, it was difficult to connect these unlinked segments. Three markers, Satt194 (LG C1), Sat—130 (LG C2), and Satt353 (LG H), were unlinked because of the lack of polymorphic loci around them. These three markers are about 46 cM, 59 cM, and 56 cM away from the rest of mapped markers in this study based on the soybean consensus map (Song et al. 2004), respectively. Additionally, the top half of the LG E had no polymorphic markers (about 40 cM). In this study, a subset of lines was used to perform the whole genome scan First, which saved us a large amount of time and resources. The maps for LGs F and M constructed using the whole population were similar to the maps using the subset of 94 lines (FIG. 14), which indicates the effectiveness of the subset of lines used in this study.
In this study, two QTLs for controlling the aphid resistance in PI 567541B were identified using a mapping population that was inoculated with either the natural mixed aphids in the Field trial or the single-clone aphid in the green house two unlinked segments. Based on the consensus map (Song et al. 2004); the intervals between two unlinked segments were about 30 cM or more. These intervals could be exaggerated due to the small size of the subset. Since there were no polymorphic SSR loci in these intervals for this study, it was difficult to connect these unlinked segments. Three markers, Satt194 (LG C1), Sat—130 (LG C2), and Satt353 (LG H), were unlinked because of the lack of polymorphic loci around them. These three markers are about 46 cM, 59 cM, and 56 cM away from the rest of mapped markers in this study based on the soybean consensus map (Song et al. 2004), respectively. Additionally, the top half of the LG E had no polymorphic markers (about 40 cM). In this study, a subset of lines was used to perform the whole genome scan First, which saved us a large amount of time and resources. The maps for LGs F and M constructed using the whole population were similar to the maps using the subset of 94 lines (FIG. 14), which indicates the effectiveness of the subset of lines used in this study.
In this study, two QTLs for controlling the aphid resistance in PI 567541B were identified using a mapping population that was inoculated with either the natural mixed aphids in the Field trial or the single-clone aphid in the explained a large portion of the phenotypic variation. This major QTL was tightly linked to marker Satt299, which has not been mapped before. In this study, Satt299 was about 3 cM away from Satt435, which was the closely linked marker for the aphid resistance genes identified in Dowling and Jackson (Li et al. 2007). Thus, the major QTL identified in this study is coincidentally located in a similar genomic region as the resistance genes in Dowling and Jackson, which indicates that they are either allelic at the same locus or different genes, but tightly linked to each other. Kim et al. (2008) recently found that PI 567541B was resistant to the aphids from Ohio while both Dowling and Jackson were susceptible. These results indicated that the better resistance of PI 567541B compared with Dowling and Jackson could be due to one or more of the following three factors: (1) the existence of the other resistance gene, (2) a different resistance allele conferring better resistance at the same locus on LG M as the resistance genes in Dowling and Jackson, and (3) a new resistance locus conferring better resistance on LG M that is closely linked to the resistance genes in Dowling and Jackson. Understanding the allelic relationship between the major QTL in PI 567541B and the resistance genes found in Dowling and Jackson could be important for soybean breeders to determine if these genes from different resistance sources can be pyramided. However, an allelic test using progenies from the cross of PI 567541B by Dowling or Jackson might not resolve the question because of the confounding effects of the additional resistance gene in PI 567541B and its interaction with the major gene. Therefore, fine mapping or gene cloning might be necessary to determine their allelic relationship.
The other QTL was identified on LG F in this study and had a smaller effect than the one on LG M. Aphid or other insect resistance genes have not been reported in that region yet, but a QTL for leaf phosphorus content was reported at a similar region (Li et al. 2005). The relationship between leaf phosphorus content and soybean aphid resistance is unknown. However, potassium content in leaf has been related to the soybean aphid resistance in several studies (Myers et al. 2005; Myers and Gratton 2006; Walter and Difonzo 2007), which concluded that the deficiency of potassium could increase the reproduction of the soybean aphid. Phosphorus is an essential element for all the living cells; therefore, it is possible that the deficiency of phosphorus in leaf tissues might also affect the soybean aphid. Surprisingly, in the greenhouse trial, the QTL on LG F explained very little phenotypic variation while the major QTL on LG M explained the majority of the genetic variation (over 94%). Most likely the aphid resistance in PI 567541B in the greenhouse trial is mainly controlled by a single gene, the major QTL on LG M. Although the QTL on LG F had very little effect in the greenhouse trial, it was detected and validated in the Field trials. This QTL even explained a relatively large portion of the phenotypic variation in the Field trial, over 25% for the 4-week rating in either mapping or validation population. This indicates that the QTL on LG F might be critical in the Field resistance. The two QTLs combined with their interaction explained the majority of the phenotypic variation in the Field trial. Thus, the aphid resistance in PI 567541B might be mainly controlled by these two genes under the Field conditions. Therefore, this study demonstrates that the two resistance genes in PI 567541B were expressed differently in the Field and greenhouse trials. The major gene on LG M was needed for providing aphid resistance in the greenhouse trial while both genes were required in the Field trial. This difference might be due to the different inoculum used in the trials. The aphids used in the greenhouse trial were a single clone aphid, which was collected from the Field in 2002 and maintained in the greenhouse thereafter. In contrast, the aphids used in the Field trial were a mixture of the natural aphids collected from the infested Fields during the year of the Field trial. It is possible that the aphids used in the Field trials had a different biotype, which caused different reactions of the resistance genes in PI 567541B.
Recently, Kim et al. (2008) reported a new soybean aphid biotype in Ohio, which has overcome the resistance genes in Dowling and Jackson. In 2006, Dowling was also found susceptible to aphids in the Field while PI 567541B was resistant (Mensah et al. 2007). This might be evidence of a new soybean aphid biotype occurrence in the Fields of Michigan. This study was the First to map genes conferring resistance to mixed natural aphids using a Field trial. Results indicated that the QTL on LG F might have played an important role in providing resistance to an unknown new aphid biotype in Michigan. However, further investigation is warranted to determine the role of the QTL on LG F in the Field aphid resistance.
Evaluation of aphid resistance usually requires artificial infestation, which is laborious and time consuming. The infestation of aphid might also be complicated by the environmental conditions, such as heavy rainfalls and strong winds. Marker-assisted selection (MAS) can be a useful and powerful tool for breeders to select aphid resistance lines even without the aphid infestation. The two aphid resistance QTLs identified in PI 567541B could be ready for MAS since they have been validated in different genetic backgrounds in this study. Moreover, the resistance genes found in PI 567541B may confer broader resistance to various biotypes of aphids than the ones in Dowling and Jackson because PI 567541B provides resistance to some new aphid biotypes that have overcome the resistance in Dowling and Jackson (Kim et al. 2008; Mensah et al. 2007).
The following references are herein incorporated by reference.


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II. Fine Mapping of Aphid Resistant Genes in Aphid Resistant Soybean Germplasm.



The germplasm providing aphid resistance was fine mapped for additional genetic markers and for identifying specific genes within the germplasm for use in the present inventions. Thus, genomic regions containing aphid resistant genes in PI 567541B, PI567543C, and PI 567598B were determined in intervals of 231 kb or less. Genes underlying aphid resistance were identified with new markers and assigned specific names. Genes were named using the following criteria.
A recessive gene mapping near or allelic to a dominant Rag1 gene on chromosome 7 was named and rag1c (obtained (derived) from PI 567541B), a partially dominant gene on chromosome 16 was named Rag3 (obtained (derived) from PI567543C), while a recessive gene on chromosome 13 was named rag4 (obtained (derived) from PI 567541B).
Further, the present invention is contemplated for use to expedite the development of soybean varieties with resistance to soybean aphids, increase the accuracy of selection for breeding progenies carrying the aphid resistant genes, and therefore reduce the time and cost for the development of aphid resistant soybean varieties. The discovery and use of DNA markers that more accurately map a functional gene or is more tightly linked to a functional genes for aphid resistance are contemplated to increase the accuracy of marker-assisted selection for plant progeny of cultivars PI 567541B, PI 567543C and PI 567598B carrying functional genes. Using markers identified during the fine mapping of genes is contemplated up to increase accuracy of identifying aphid resistance germplasm up to 90%, in some cases up to 95%, and contemplated up to 99%. The DNA markers for aphid resistance genes developed in this invention are within the candidate genes and tightly linked to the genes (less than 126 kb).
A. Methods for Fine Mapping Genes in Aphid Resistant Germplasm from PI 567541B, PI 567543C and PI 567598B.
The following basic methods were used in order to accurately identify progeny plants that inherited the aphid resistant genes from a resistant parent soybean plant having aphid resistance gene(s) (as determined by methods as described herein) from cultivars named PI 567541B, PI 567543C, and PI 567598B. After producing plants for Bulk segregant analysis, QTL map using RILs the following steps. Step 1: use multiple DNA markers, such as SNPs (see, Song et al, 2004 and 2010 for at least 1,015 and 33,065 markers respectively), to test the two parents of progeny plants for identifying at least one DNA marker that is polymorphic between the two parents, i.e. find at least one marker that identifies each of the two parents in the corresponding region, i.e. at least one marker that identifies the parent contributing aphid resistant germplasm and at least one marker that identifies the parent not having aphid resistant germplasm in order to differentiate the two parents. Step 2: use the identified polymorphic DNA marker(s) (from step 1) to test the DNA of the progeny plants (at least F2 and progeny including back crossed progeny) to determine whether the progeny plants have a resistance gene. Thus, when the progeny plants have the same DNA allele markers as the resistant parent, the progeny will be determined as having inherited at least one copy of a resistant gene from the resistant parent. When the progeny has the same DNA allele markers from the susceptible parent, the progeny does not inherit a resistant gene from the resistant parent for at least one of the alleles at that loci and has at least one susceptible allele. Therefore, when the progeny plants have both sets of markers for the same loci then the progeny plants are heterozygous for resistant and susceptible alleles at that genetic loci. Alternatively, when the progeny plants do not have DNA markers from the susceptible parent at that loci then the progeny plants are considered to be homozygous for the resistant allele. In some embodiments, a SoySNP50 iSelects SNP Chip was used for genotyping, including BSA and fine mapping. In some embodiments, a TaqMan® Assay was used, including in endpoint genotyping for use in QTL mapping and screening for recombinants. The methods, plants, cells, pollen and seeds of the present invention are contemplated to be readily used in soybean plant variety development.
In one embodiment, SNPs (i.e. markers) are used as DNA allele markers. Single nucleotide polymorphism (SNP) is widely distributed throughout a plant genome. However, SNPs are dispersed unevenly on the genome and the distribution of SNPs shows different frequency of occurrence between intron and exon regions (Edwards et al., 2007). Thus selection, either natural or through breeding leads to increased abundance of beneficial (or desired) genetic sequences while deleterious sequences are often purged from the population. Hence, selection of target genomic regions in breeding programs often reduce genetic diversity and therefore, fewer SNPs are observed. Hyten et al. (2006) showed that cultivated soybean [Glycine max (L.) Merr.] has an average SNP abundance of 1/1000 bp compared to 1/425 bp of its wild counterpart [Glycine soja (Sieb and Zucc.)]. Generally, SNPs show higher frequency in the non-coding regions of the genome than that of coding sequences. SNPs and sequence structural variants are the root of genetic variation among individuals and populations. The presence of these variations influences pathogen accessibility and host resistance during the pathogenesis of pathogens. Because of low mutation rate and evolutionary stability from generation to generation for SNPs they were chosen as markers for genetic polymorphism (for reviews, see, Rafalski 2002; Edwards et al., 2007).
Therefore, high fidelity SNPs associated with soybean aphid resistance germplasm were sought, especially SNPs within the resistance genes. These SNPs are contemplated as valuable molecular markers for fine-mapping and identification of functional resistance genes for use in marker-assisted selections (MAS) for resistant soybean plants.
In a high generation inbred line (multiple generations of breeding), most of the loci in the genome are fixed (i.e. homozygous for either of the parental alleles) but a small percentage of genetic loci remained heterozygous. This type of heterozygosity is referred to as residual hetorozygosity. The progeny plants of a high generation inbred line with residual heterozygosity will segregate for residual heterozygous loci in a relatively uniform genetic background due to the predominant homozigosity in the rest of the genome. When the residual heterozygous locus has a major effect on a phenotype, such as aphid resistance, the phenotypic differences of the progenies in high generation residual heterozygous lines (RHLs) will be mainly due to the difference of alleles carried by the progeny plants when the non-genetic effects on the phenotype are minimized. Thus bulk segregant analysis (BSA) was used as an efficient platform to identify the target genomic regions underlying the phenotype of interest.
Bulk segregant analysis (BSA) as used herein, was used to identify individuals with the opposite phenotypes within a segregating population. Thus the segregating population was separated and used to form two bulk populations with opposite phenotypes. Equal amounts of DNA was pooled from each individual in the same bulk to create a bulked DNA sample. The two bulked DNA samples were tested with numerous DNA markers in order to identify markers that were polymorphic between these two bulked DNA samples. Markers closely linked to the gene underlying the phenotype were expected to be polymorphic between the two bulked DNA samples.
As an example, residual heterozygous plant lines (RHLs) were used for mapping other genes for mapping loci underlying SDS (soybean sudden death syndrome) and SCN (soybean cyst nematode) resistance in soybean (Njiti et al. 1998; Meksem et al. 1999; Triwitayakorn et al. 2005), in order to identify the QTL associated with seed weight in sorghum (Tuinstra et al. 1997) and to perform map-based cloning (Watanabe et al., 2011). However, there are relatively few studies on the use of a combination of the BSA and RHL methods for fine-mapping a soybean disease resistance gene in a relatively short time. Thus in one embodiment, a combination of the BSA and RHL methods were used for fine-mapping a soybean aphid resistant gene.
1. A Rapid Method to Fine-Map and Identify a Soybean Aphid Resistance Gene, Rag1c and the SNP Markers Derived from PI 567541B Soybean (Glycine max L.) Plant Lines.
During the development of the present inventions, aphid resistance in PI 567541B was found to be controlled by two recessive genes (Mensah et al., 2008) that were mapped to the soybean chromosome 7 (LG M) and chromosome 13 (LG F). These two areas of germplasm explained 31-88% of the phenotypic variations in aphid resistant plant trials described herein and were named rag1c and rag4, respectively.
In order to fine-map these areas of aphid resistant germplasm, two recombinant inbred line (RIL) populations derived from crosses of PI567541B (Resistant)×E00003 (Susceptible) and E07906-2 (Resistant)×Skylla (Susceptible) with population sizes of 215, 210 lines, respectively, were produced. FIG. 17. These two populations were selected from the previous existing populations (F 4:8 and F 2:6) by a single seed decent method. E07906-2 was derived from PI567541B. A residual heterozygosis (5%) was also captured in the RILs. Some parts of the chromosomes in these lines are still heterozygous while most of the chromosomes sections are fixed with either parental type. These high generation heterozygous lines (HRLs) were used for fine-mapping soybean aphid resistance genes.
Novel SNP Genotyping was Conducted Using an Illumina Infinium® Bead Chip is Described Herein.
A SNP genotyping approach was conducted using the Illumina Infinium® Genotyping HD 52 K and 6 K BeadChips (Illumina, San Diego, Calif.). The laboratory analysis was performed on the Illumina iScan platform at Michigan State University (East Lansing, Mich.). Each DNA sample required not less than 200 ng genomic DNA in a 4 uL volume. Intensities of the beads fluorescence were detected using the Illumina iScan Reader, and genotypes were called using Illumina's BeadStudio software (Illumina, San Diego, Calif., v3.2.23) following Illumina standard protocol. 96 RILs from E07906-2×Skylla were genotyped with 6K BeadChip array. The SNP validation was carried out by re-sequencing target SNPs in both aphid resistant and susceptible parental lines. The objective here was to verify genetic association between SNP genotypes in the region of interest and the resistance trait phenotypic values.
Bulk Segregant Analysis (BSA) with 52 K Chip.
The 10 most consistently aphid resistant and susceptible lines were pooled to make resistant and susceptible DNA pools. The bulks were genotyped using 52 K beadchip. The SNPs produced different allele calls, i.e. showed that different alleles were present at the same or similar genetic loci were used in further detailed analysis.
TaqMan® SNP Genotyping
Based on BSA genotyping results, target flanking region was sequenced in both resistant and susceptible parents. SNPs were developed by submitting the sequence information to Customer Taqman® Assay Design Tools of the Applied Biosystems (ABI, Foster City, Calif., USA) to obtain the allele specific primers and probes (assays). Taqman® SNP PCR reactions were carried out on 384-well plates with a total volume of 3 uL/well on the LightCycler 480 instrument (Roche Applied Science, Indianapolis, Ind., USA). Genotyping analysis was performed using the Roche Applied Science software version 1.5.0.
Expression Assisted Fine-Mapping Strategy for Identifying More Accurate Markers.
In order to narrow down the target region identified in BSA, 30 candidate genes were selected among 110 genes in the genomic region based on sequence information in Soybase for RT-PCR (real time PCR). Differential expression of candidate genes in resistant and susceptible parental lines suggested association with the resistance. Two candidate genes (I & II) identified from BSA were differentially expressed in a simple RT-PCR. The different amplicons were obtained when the genomicDNAs of these cultivars were amplified by the same primers used in the RT-PCR in gene II but not in gene I. G: genomic DNA. The remaining templates were cDNA. FIG. 18.
Because one of the candidate genes showed association with the Rag1 location, one of the Rag1 candidate genes, Glyma07g06920 in the Rag1 locus was also tested using cDNA samples from the plant introductions PI 567541B and PI 567598B. Screening of 52k BeadChip, TaqMan® and Kaspar®SNP markers and construction of a physical contig around the rag1c locus also showed that rag1c (partial dominant) was different than Rag1 (dominant) found in Dowling plants.
For this screen, more than five HRLs from E07906-2×Skylla populations were selected for use in mapping. One recombination event was identified among the lines in RHL 07-757-1 using the 52K BeadChip to perform the genotyping. The interval was about 100 kbp. RHL07-757-1 showed heterozygosis near rag1c locus.
Additionally, more than four HRLs from PI567541B×E00003 progeny plants were chosen for use in mapping. The recombinant event was captured in a RHL05-153-3 line and in three RHLs of plant line 05-383-AR6 using the 52K BeadChip TaqMan® and Kaspar® to conduct the genotyping. The interval containing the aphid resistant gene was about 80 kbp. These RHLs displayed heterozygosis near rag1c locus. F 9:10 plants were used in screening methods for determining soybean aphid resistance in the greenhouse,
a. Fine Mapping the Rag1c Region
During the development of the present inventions, the QTL underlaying the rag1c region was found positioned between the SSR markers Satt299 and Sat—244 on soybean chromosome 7. However, this QTL spanned more than 10 cM (>2.5 million base pairs) on soybean genome and the location of the specific resistance gene was not known. To rapidly confirm and narrow down the genomic interval of the rag1c, Bulk segregant analysis (BSA) methods was used to define a gene within the rag1c region.
Eight RILs with residual heterozygosity in the rag1c regions were selected from the two RIL populations. The eight RILs were selfed in order to develop eight RHL populations segregating for soybean aphid resistance. These eight RHL populations were genotyped using SNPs in the flanking region of rag1c locus identified by the BSA approach. In turn, rag1c was positioned in a 221548 bp interval flanked by a chip designed for use in the development of the present inventions, i.e. Kaspar® SNP MSUSNP7-19 (Chromosome 7: 5650536 and MSUSNP7-10 (Chromosome7: 5882084) based on the recombination events that resulted in the phenotypic variation for the aphid resistance in the RHL populations. Furthermore, a Kaspar® SNP MSUSNP7-18 (Chromosome 7: 5762798) was discovered located between these adjacent SNPs and the genotypes highly correlate to the phenotypes.
The result was further confirmed in at least a 467 soybean plant BC3F3 fine-mapping population segregating for soybean aphid resistance. Using the marker MSUSNP7-18, the correlation between genotypes of the BC3F3 plants with the phenotypes was found to be significant (P<0.0001). The SNP MSUSNP7-18 was anchored in the first intron of a NB-ARC gene, Glyma07g07110. The transcript abundance level of the gene was distinguished to the adjacent candidate genes. Thus, these data described above indicated that the NB-ARC is the rag1c and Kaspar® MSUSNP7-18 was a SNP derived from the NB-ARC2 gene. Specifically, fine-mapping of the rag1c gene is described in Example 13.
The following references are herein incorporated by reference in their entirety:

    1. Edwards, et al., 2007. What are SNPs? in Association Mapping in Plants (C. N. Oraguzie, A. H. E. Rikkerink, E. S. Gardiner, and N. H. De Silva, eds.), Springer, NY 41-52.
    2. Rafalski. 2002. Applications of single nucleotide polymorphisms in crop genetics. Current Opin. Plant Biol. 5: 94-100.
    3. Syvanen. 2001. Genotyping single nucleotide polymorphisms. Nat. Rev. Genet. 2: 930-942.
    4. Hyten, et al., 2006. Impacts of genetic bottlenecks on soybean genome diversity. Proc. Natl. Acad. Sci. USA 103: 16666-16671.
    5. Zhang, et al., 2009. Molecular mapping of soybean aphid resistance genes in PI 567541B. Theor. Appl. Genet. 118: 473-482.
    6. Mensah, et al., 2008. Inheritance of soybean aphid resistance in PI 567541B and PI 567598B. Crop Sci 48:1759-1763.
    7. Michelmore, et al., 1991. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. 88:9828-9832.
    8. Kim, et al., 2010. Fine mapping the soybean aphid resistance gene Rag1 in soybean. Theor. Appl. Genet. 120:1063-1071.


b. Rag4 Region
Resistance in PI 567541B was found to be controlled by two genes, mapped to the soybean chromosome 7 (LG M) and chromosome 13 (LG F), that were later named rag1c and rag4, respectively. These two genes explained 31-88% of the phenotypic variations in aphid resistance trials. The following describes the fine mapping of rag4, using SNPs and sequence structural variants for use in marker-assisted selection (MAS) programs.
During the development of the present inventions, the QTL identified as containing the rag4 region was positioned between the SSR markers Satt348 (5491250) 20 and Satt649 (12953321) on soybean chromosome 13. The QTL on LG 13 was closely linked to marker Satt649 or Satt343. Its peak position was located at Satt649 or 2 cM below. The QTL spanned more than 7.5 million base pairs.
To rapidly confirm and narrow down the genomic interval of the rag4 and identify more specific markers for the rag4 region, the BSA approach was employed to define the rag4 region in this study. Two recombinant inbred lines (RILs) populations were derived (artificially bred) from crosses of soybean aphid resistant variety PI567541B (Resistant)×E00003 (Susceptible) lines and E07906-2 (Resistant)×Skylla (Susceptible) with population size of 215, 210 lines, respectively were developed. The Illumina® 6k HD beadchip and Taqman® and Kaspar® SNP markers specifically designed for use in these inventions were used to map the resistance QTL. In turn, the QTL containing rag4 was positioned between MSUSNP13-5 (7766353) and ss247923149 (8293174). Furthermore, several recombination breakpoints were identified and these recombination events displayed a significant segregation of the soybean aphid resistant trait. Based on these recombination events in the RIL lines, the rag4 gene was fine-mapped between SNP marker MSUSNP13-29 and MSUSNP13-31 within a 162,135 5 bp interval.
Thus in one embodiment, the gene for rag4 is located between SNP markers MSUSNP13-29 and MSUSNP13-31. In another embodiment, plants having a rag4 gene are identified having approximately a 162,135 bp fragment between markers MSUSNP13-29 and MSUSNP13-31.
The following references are herein incorporated by reference in their entirety:

    1. D. Edwards, W. J. Forster, D. Chagné, and J. Batley. 2007. What are SNPs? in Association Mapping in Plants (C. N. Oraguzie, A. H. E. Rikkerink, E. S. Gardiner, and N. H. De Silva, eds.), Springer, NY 41-52.
    2. J. A. Rafalski. 2002. Applications of single nucleotide polymorphisms in crop genetics. Current Opin. Plant Biol. 5: 94-100.
    3. A. C. Syvanen. 2001. Genotyping single nucleotide polymorphisms. Nat. Rev. Genet. 2: 930-942.
    4. Hyten, D. L., Q. Song, Y. Zhu, I. Y. Choi, R. L. Nelson, J. M. Costa, J. E. Specht, R. C. Shoemaker, and P. B. Cregan. 2006. Impacts of genetic bottlenecks on soybean genome diversity. Proc. Natl. Acad. Sci. USA 103: 16666-16671.
    5. G. Zhang, C. Gu, and D. Wang. 2009. Molecular mapping of soybean aphid resistance genes in PI 567541B. Theor. Appl. Genet. 118: 473-482.
    6. Mensah, C. DiFonzo, and D. Wang. 2008. Inheritance of soybean aphid resistance in PI 567541B and PI 567598B. Crop Sci 48:1759-1763.
    7. R. Michelmore, I. Paran, and R. V. Kesseli. 1991. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. 88:9828-9832.
    8. K. Kim, C. Hill, G. Hartman, D. Hyten, M. Hudson and B. Diers. 2010. Fine mapping the soybean aphid resistance gene Rag 1 in soybean. Theor. Appl. Genet. 120:1063-1071.


2. Soybean Aphid Resistance Genes in PI 567543C, Fine-Mapping Rag3
Resistance genes were identified in PI 567543C with molecular markers and then these markers were used to validate their use in a different genetic background. PI 567543C showed resistance to soybean aphids from both Michigan and Ohio A partially dominant locus Rag3 was identified from PI 567543C and mapped to a 10 cM interval between simple sequence repeat (SSR) markers Sat—339 and Satt414 on chromosome 16 (linkage group J), see, section a below. Further objectives of this example are to show the results of fine mapping Rag3 in bi-parental F2 populations in order to identify markers with greater accuracy for identifying the Rag 3 gene, i.e. closer SNP markers for marker-assisted selection, see, section b, below.
Advanced breeding lines E10902 and E10905 were derived from PI 567543C which is resistant to soybean aphid, while line E07048 soybean plants were susceptible to aphids, to achieve higher mapping resolution, using high density SNPs is not sufficient. In this example, two large F2 populations were developed with nearly 4000 plants to increase the number of recombinants within the region of interest.
a. A Genomic Region was Mapped within the QTL
A mapping population of 249 F4 derived plant lines were produced from a cross between PI 567543C and a susceptible parent plant for identifying aphid resistant plants in both the green-house and the field. The broad sense heritability of aphid resistance in the field trial was over 0.95. The segregation of aphid resistance in this population indicated that at least one major gene controlled the resistance. Bulked segregant analysis with molecular markers revealed a potential genomic region for the location of this gene.
After saturating this putative region with additional markers, a genetic locus was mapped in an interval between Sat—339 and Satt414 on chromosome 16 (linkage group J) using the composite interval mapping method. This locus explained the majority of the phenotypic variation ranging from 84.7% in the field trial to 90.4% in the greenhouse trial. Therefore, the aphid resistance in PI 567543C was considered as mainly controlled by this gene. This aphid resistance gene was mapped on a different chromosome than the other resistance genes reported previously from other resistant germplasms. The action of this gene appears to be additive based on the aphid resistance of the heterozygous lines at this locus.
Specifically, Phenotypic analysis for mapping population The phenotypic data of the mapping population and its parents are summarized in FIG. 28: Table 1. In the greenhouse, susceptible parent E00003 was heavily infested by soybean aphids, while resistance parent PI 567543C had relatively lower DI than E00003 for the week-4 rating. Similarly, PI 567543C had a significantly lower DI than E00003 in the field (P\0.05). Highly significant variations (P\0.0001) were observed among the population lines for both week-3 and week-4 ratings in the field trial. The aphid infestation in the greenhouse was similar to that in the field. The correlation between the greenhouse and field data was strong (0.91 for both week-3 and week-4 ratings, P\0.0001). The DI frequency distributions were similar between greenhouse and field trials, and both distributions appeared bimodal with a ratio of 1:1 (FIG. 31). However, there was no clear-cut separation between the resistant and susceptible plants. The broad-sense heritability for the field DI was over 0.95 (FIG. 28: Table 1).
A total of 223 SSR markers, were used for aphid resistance loci mapping. These markers were distributed throughout the soybean genome based on the consensus map (Song et al. 2004), and selected for testing the two DNA pools. Several markers on chromosomes 1, 6, 9, 13, 16, and 19 (LGs D1a, K, C2, F, J, and L) showed polymorphism between the two DNA pools. Of the markers tested, Satt686 on chromosome 16 (LG J) was associated with the aphid resistance when the individual lines from the DNA pools were genotyped and compared to the parents' genotypes. Therefore, Satt686 was further genotyped on the rest of the population, which confirmed its association with aphid resistance. Then, SSR markers within ±20 cM of this marker were screened for parental poly-morphism. Seven additional polymorphic markers in that region were further genotyped on the whole population. For consideration as a marker it had to fit a 7:2:7 (homozygous female:heterozy-gotes:homozygous male) segregation ratio (P [0.001) except Satt596 (P=0.0009), Satt622 (P=0.0003), and Sat—339 (P=0.0000002). The distorted ratios of these three markers were due to fewer heterozygotes than expected.
These eight markers formed one linkage group, and the linkage map spanned a total distance of 36.6 cM (FIG. 32a), which was about 12 cM larger than the corresponding map distance of 24.8 cM in the consensus map of Song et al. (2004) (FIG. 32b). The main inflated interval on the map was between Sat—339 and Satt414 (10 cM larger than expected). However, the marker order in this study was highly comparable with the consensus map (Song et al. 2004) except that the order of Satt686 and Satt596 was inverted (FIG. 32a). The linkage map was then used in QTL analysis with the CIM method.
One QTL was consistently identified in the interval between Sat—339 and Satt414 for both ratings in each trial and was located closer to Satt414 (FIG. 29: Table 2; FIG. 32a). The PI 567543C allele at this locus conferred aphid resistance. This QTL explained the majority of the phenotypic variation ranging from 84.7% for the week-3 rating in the field trial to 90.4% in the greenhouse trial. Considering the high percentage of phenotypic variation explained by this QTL and the high heritability of this trait, this single gene, Rag3, was determined to control aphid resistance in PI 567543C. To determine this resistance gene's action, the DI value for each geno-type class of Satt414 was estimated (FIG. 30: Table 3) and compared statistically. The DI average for the heterozygous class was significantly (P\0.05) lower than the susceptible class (populations), higher than the resistant class, and not significantly different from the average of the two homozygous classes, indicating that this resistance gene has an additive gene action. This gene's location was different from those of genes that were previously identified for the other three resistant germplasms, including Rag1, Rag2, and rag4 (Li et al. 2007; Mian et al. 2008b; Zhang et al. 2009).
For aphid resistance locus validation, a total of eight polymorphic SSR markers in the region containing Rag3 were chosen to genotype the validation population. The eight markers formed one linkage group. A distorted segregation ratio (7:2:7) was observed for marker Satt674 (P=0.0000007), which had a higher number of homozygous PI 567543C genotypes (67 lines) than expected (41 lines). The order of these eight markers was similar to that on the consensus map (Song et al. 2004) except that the order of Satt622 and Satt215 was inverted (FIG. 32c). However, the map of the validation population was expanded about 19 cM more compared with the consensus map (Song et al. 2004) (FIG. 32b, c). In the QTL analysis with the CIM method, one QTL was detected in the interval between Satt674 and Satt414 at a position of about 6 or 10 cM above Satt414 (FIG. 32c), which was the same as the QTL detected in the mapping population. Similarly, the QTL identified in the validation population explained the majority of the phenotypic variation (75.3 and 85.4% for week-3 and week-4 ratings, respectively) in the field trial (FIG. 29: Table 2). Hence, the results from the validation population confirmed the location of the Rag3 locus identified in the mapping population.
Thus, a new symbol Rag3 was used to designate this antixenosis gene according to the conventions of Soybean Genetics Committee. Moreover, Rag3 was confirmed in a validation population. The aphid resistance gene Rag3 mapped in this study was located in the interval between Sat—339 and Satt414 in the mapping population, and in the interval between Satt674 and Satt414 in the validation population. This locus was located at the same position in both maps and was more closely linked to Satt414. There were a few polymorphic SSR markers in the Rag3 region, which resulted in a relatively large interval for this resistance locus. Thus additional polymorphic markers, especially single nucleotide polymorphism markers in this region are contemplated for use.
Other genes on chromosome 16 (LG J). Two QTLs (CEW6-2 and CEW7-4) for corn earworm resistance were positioned in a genomic region (Grant et al. 2009) close to Rag3. However, Rag3 and these corn earworm QTLs were about 15 cM apart and the corm earworm resistance parents, Minsoy and PI 229358 (Parrott et al. 2008), were aphid susceptible. Therefore, Rag3 and the corn earworm QTLs on chromosome 16 (LG J) were not the same locus. Surprisingly, a large cluster of disease resistance gene analogs [RGAs, encoding the nucleotide binding site, i.e. leucine rich repeat (NBS-LRR) resistance proteins] representing five different classes were mapped on chromosome 16 (LG J), one of which (RGA6) was close to Rag3 and located a few cM down from Sat—339 (Kanazin et al. 1996). Additionally, Klingler et al. (2005) mapped an antixenosis aphid resistance gene to a region flanked by RGAs in barrel medic (Medicago truncatula Gaert.). The cloned genes Mi-1 in tomato (Lycopersicon esculentum) and vat in melon (Cucumis melo L.) encoded specific NBS-LRR proteins conferring aphid resistance (Gregg and Jander 2008). However Rag3 is different from these known genes.
In summary, soybean plants with a Rag 1 aphid resistance gene, were found susceptible to the Ohio aphid biotype (Kim et al. 2008) and aphids from Michigan. Another gene, Rag2, also did not provide resistance to plants infested with the aphids from Michigan. Coincidentally, Rag1 was found close to RGA5b and RGA2b on chromosome 7 (LG M) (Li et al. 2007), and Rag2 was located in a genomic region on chromosome 13 (LG F) where a cluster of R-genes resides (Mian et al. 2008b). During the development of the present inventions, two different isolates of aphids were used for infestations in the greenhouse and field experiments, respectively. These two isolates of aphids are contemplated to belong to different biotypes.
Surprisingly, PI 567543C germplasm in soybean plants showed resistance to both isolates of aphids. Moreover, PI 567543C also showed resistance to the Ohio aphid biotype (Mian et al. 2008a). These facts indicated that PI 567543C has a broad resistance to soybean aphids. Therefore, this novel aphid-resistant locus and the linked molecular markers found in this study could be valuable in breeding aphid-resistant soybean cultivars.
FIG. 28: Table 1. Aphid damage index (DI) in the greenhouse in spring 2008 and in the field cage in summer 2008 for the parents, PI 567543C and E00003, and 249 F4 derived lines of the mapping population.
FIG. 29: Table 2. Summary for aphid resistance locus detected in the mapping population PI 567543C×E00003 and in the validation population PI567543C×Skylla with aphid damage index data using the composite interval mapping method.
FIG. 30: Table 3. Average aphid damage index for different genotypes of marker Satt414 in the mapping population PI 567543C×E00003.
The following are exemplary materials and methods used for mapping Rag3. Aphid resistance loci mapping. Plant materials and aphid resistance evaluation in a population of 249 F4-derived lines produced from the cross of PI 567543C×E00003 by single seed descent and used for genetic mapping. PI 567543C has antixenosis resistance to the soybean aphid (Mensah et al. 2005), while E00003 was an advanced breeding line developed at Michigan State University (MSU) that was susceptible to the soybean aphid. Aphid resistance was evaluated in choice tests in both greenhouse and field trials. Since aphid resistance in soybean has a high heritability (around 0.90) in greenhouse experiments, tests were replicated for field trials.
A greenhouse trial was performed in the Plant Science Greenhouse at MSU in East Lansing, Mich. on the PI 567543C progeny F4-derived lines. In this trial, eight seeds per line were planted in a plastic pot. The pot size was 105 mm in diameter and 125 mm deep. The population (F 4:5) and parent lines were randomly laid out on the bench without replications. The greenhouse was maintained at 26/15° C. day/night temperature, and sodium vapor lights were used to supplement light intensity during the day (14 h).
A field trial was performed on the Agronomy Farm of MSU. The population (F 4:6) and parent lines were arranged in a randomized complete block design with two replications in an aphid- and predator-proof polypropylene cage with 0.49-mm size mesh (Redwood Empire Awning Co., Santa Rosa, Calif., USA). In each replication, eight seeds per line were planted in a single plot that was 30 cm long, with a row spacing of 60 cm.
In both greenhouse and field trials each plant was infested with two wingless aphids at the V1 stage (Fehr and Caviness 1977). The aphid biotype used to infest plants in the greenhouse trial was a clone from a single insect that was collected from a naturally infested field on the MSU Agronomy Farm, and which was maintained for at least 6 years in the greenhouse. The aphids used to infest plants in the field trial were collected from the naturally infested field on the Agronomy Farm of MSU during the year of testing.
Aphid resistance was visually rated for each plant 3 and 4 weeks after infestation using a scale of 0-4 developed by Mensah et al. (2005, 2008), where 0=no aphids; 0.5=fewer than 10 aphids per plant, no colony formed; 1=11-100 aphids per plant, plants appear healthy; 1.5=101-150 aphids per plant, plants appear healthy; 2=151-300 aphids per plant, mostly on the young leaves or tender stems, plants appear healthy; 2.5=301-500 aphids per plant, plants appear healthy; 3=501-800 aphids per plant, young leaves and tender stems are covered with aphids, leaves appear slightly curly and shiny; 3.5=more than 800 aphids per plant, plants appear stunted, leaves appear curled and slightly yellow, no sooty mold and few cast skins; 4=more than 800 aphids per plant, plants appear stunted, leaves appear severely curled and yellow and are covered with sooty mold and cast skins.
An aphid damage index (DI) for each line was calculated by the following formula: DI=P (scale value 9 no. of plants in the category)/(4/9 total no. of plants) 9 100. The DI ranges between 0 for no infestation and 100 for the most severe damage (Mensah et al. 2005). The DI was used as an indicator of aphid resistance and was applied in the analysis. DNA extraction and marker analysis Six plants for each line (F 4:5) and their parents were grown in the greenhouse for DNA extraction in 2008. The non-expanded trifoliates from each line were bulk harvested for isolating the genomic DNA. The DNA was extracted with the CTAB (hexadecyltrimethyl ammonium bromide) method as described by Kisha et al. (1997), and the concentration was determined with a ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Del., USA). The PCR was performed using the genomic DNA with simple sequence repeat (SSR) markers as described by Cregan and Quigley (1997) and was run on an MJ TetradTM thermal cycler (MJ Research, Waltham, Mass., USA). The SSR primer sequences were provided by Dr. Perry Cregan of the USDA-ARS, Beltsville, Md. The PCR products were separated on 6% non-denaturing polyacrylamide gels using an electrophoresis unit DASG-400-50 (C.B.S. Scientific Co., Del Mar, Calif., USA) as described by Wang et al. (2003). Gels were stained with ethidium bromide, visualized under UV light, and photographed. In order to accelerate the identification of genomic regions associated with aphid resistance, the bulked segregant analysis method described by Michelmore et al. (1991) was used in this study. Twelve resistant lines with the lowest DI values and 12 susceptible lines with the highest DI values were selected to form a resistant pool and a susceptible pool, respectively. SSR markers at approximately every 10 cM on the integrated soybean map of Song et al. (2004) were selected to test the bulked DNA of each pool. The polymorphic markers between the two pools were selected to genotype the individual lines in the two pools together with the two parents. The markers that appeared to be associated with the aphid resistance were used to genotype the remaining lines of the whole mapping population. The genomic regions associated with the aphid resistance were then saturated with additional markers.
Statistical and mapping analysis. The DI data from the greenhouse and field trials were analyzed separately, as their experimental designs and infested aphids differed. Analysis of variance (ANOVA) was performed for the field data using the GLM procedure of SAS Institute (1999). The broad-sense heritability of DI in the field trial was estimated based on entry means according to Fehr (1987). Pearson correlations for the aphid resistance between trials were estimated with the CORR procedure of SAS Institute (1999). A linkage map was constructed with JoinMap 3.0 using the Kosambi function and a LOD score of 3 (Van Ooijen and Voorrips 2001). Composite interval mapping (CIM) was performed to locate aphid resistance loci using QTL Cartographer V2.5 with a standard model Zmapqtl 6 (Wang et al. 2008). The forward and backward regression method was used to select markers as cofactors to control the genetic back-ground (Zeng 1994). The walking speed chosen for CIM was 2 cM. The empirical LOD threshold at the 5% probability level was determined by a 1,000-permutation test (Churchill and Doerge 1994). Entry means were used in the analysis for the field trial. The maps and the locus positions were drawn using MapChart (Voorrips 2002).
Resistance locus validation. A population of 96 F 4:5 lines was used to validate the resistance loci identified in the mapping population. The validation population was developed by single seed descent from a cross between PI 567543C and ‘Skylla’, where Skylla is a cultivar developed by MSU and is aphid-susceptible (Wang et al. 2006). In the summer, aphid resistance was evaluated for the validation population together with its parents in a field trial, which was conducted in the same way as for the mapping population without replications. The fresh leaf tissues in the field trial were collected and placed into a 96-well plate. Due to the limited space in each well, one leaflet tip was randomly sampled from each line. DNA was then extracted with the quick method as described by Bell-Johnson et al. (1998). Polymorphic markers in the region containing the aphid resistance locus were selected to genotype the validation population. Linkage map construction and mapping analysis were performed in the same way as for the mapping population.
b. Fine Mapping of Aphid Resistance Gene Rag3 in Soybean PI 567543C with Bi-Parental F2 Populations
As described above, partially dominant Rag3 was mapped to 10 cM interval between SSR markers Sat—339 and Satt414 on chromosome 16 (linkage group J). However, to achieve a higher mapping resolution using more specific markers, high density SNPs was not sufficient. Thus, two large F2 populations were developed with nearly 4000 plants to increase the number of recombinants within the region of interest. The objective of this study were to fine map partially dominant Rag3 with closely linked SNP markers for MAS, and to fine locate the locus for gene cloning and further genetic study.
Advanced breeding lines E10902 and E10905 were derived from PI 567543C and resistant to soybean aphid, while E07048 was susceptible to aphid. 1,889 F2 lines were developed from cross E07048×E10902 and 1,913 F2 lines from E10905×E07048. At least 889 F2 lines from E07048×E10902 were phenotyped for aphid resistance in the greenhouse along with a subset of 376 F2 lines genotyped with TaqMan® SNP markers at 6.16, 6.26, 6.41, 6.42 and 8.05 mb on chr.16. With QTL analysis of these 376 lines, a locus explaining 44.7% of the phenotype variance was detected between markers MSUSNP16-10 (Gm16—6262227) and MSUSNP16-12 (Gm16—6423098) on chr.16. Then, 3802 F2 were genotyped with SNPs at 6.16 and 8.05 mb to select recombinants. 983 F3 progeny of 102 F2 recombinants were planted in the greenhouse and tested for aphid resistance evaluation and genotyped with SNPs at 6.16, 6.26, 6.41, 6.423 and 6.424 mb on chr.16. Sixteen susceptible, heterozygous and recombinant F3 lines were selected and applied onto Infinium® SNP assays. Two F3 recombinants with break points between 6.26 and 6.47 mb were identified and confirmed according to their F4 progeny phenotypes and genotypes from the SNP Beadchip.
In conclusion, Rag3 was fine mapped to 207 kb between TaqMan® SNPs MSUSNP16-10 (Gm16—6262227) and Gm16—6469551_A_C with serine-threonine protein kinase coding genes as candidate functional genes. Additionally, TaqMan® SNPs MSUSNP16-10 (Gm16—6262227), MSUSNP16-11 (Gm16—6413214) and MSUSNP16-12 (Gm16—6423098) is contemplated for use in MAS for aphid resistance of Rag3 gene. In other embodiments, MSUSNP16-10 (Gm16—6262227) and Gm16—6469551_A_C are contemplated for use in identifying the Rag3 gene in soybean plants for identifying plants having aphid resistance and for use in breeding aphid resistant soybean plants for commercial use.
The following references are herein incorporated by reference in their entirety.

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3. Soybean Aphid Resistance Genes in PI 567598B
Aphid resistance in PI 567598B was discovered as being controlled by two recessive genes. The objective of this study was to locate these two genes on the soybean genetic linkage map and define the aphid resistance loci in PI 567598B with molecular markers.
A mapping population of 282 F4:5 lines was evaluated for aphid resistance in field cage trial in 2009 and greenhouse trials. Validation of QTLs was evaluated using a population consisting of 94 F2:5 lines in at least two greenhouse trials. Two quantitative trait loci (QTLs) were found using the composite and multiple interval mapping method in three years. These two QTLs were mapped on chromosome 7 (linkage group M) and 16 (linkage group J). PI 567598B conferred resistance at both loci. In one of the field trials, 56.1% of the phenotypic variation was explained by the QTL on chromosome 16. No significant interaction was found between the two QTLs detected in one of the greenhouse trials, which explained 37.6-44% of the total phenotypic variation. In the validation population, these two loci together explained about 50% and 80.4% of the total phenotypic variations, respectively. An additive×additive interaction between these two QTLs was identified in the validation population using the multiple interval mapping method. The soybean aphid isolates used in one of the field trials defeated the germplasm identified in the QTL found on chromosome 7 from PI 567598B and Rag1 from Dowling. Surprisingly, the presence of both QTLs conferred durable resistance against soybean aphids compared to when one QTL was present. The markers linked to the aphid resistance QTLs in PI 567598B are contemplated for use in marker assisted breeding for aphid resistance when using PI 567598B as the aphid resistance source.
In this study, two QTLs for controlling the aphid resistance in PI 567598B were consistently detected over three years of testing plants during a growing season. These two QTLs explained most of the phenotypic variation, indicating that two major genes controlled the aphid resistance in PI 567598B. This finding was consistent with the conclusion of Mensah et al. (2008), who conducted a genetic study and suggested a two-gene model for the aphid resistance in PI 567598B. However, no interaction was detected between these two QTLs and their genetic effects appeared relatively small in the greenhouse trials compared to the field trial when one of these genes controlled aphid resistance.
The QTL detected on chromosome 7 in this study was a few cM on the 5′ end of Satt435 or at Satt435. In an earlier study, Rag1 was mapped between Satt435 and Satt463 on chromosome 7 (Li et al. 2007). Most recently, a fine mapping study further narrowed down this genomic region to an interval of 0.6 to 1.8 cM down from Satt435 (Kim et al. 2010). The 1-LOD support intervals for the QTL on LG M in described herein overlapped with this fine mapped region of Rag1 (FIG. 45d, f). Thus the QTL on chromosome 7 identified in this study was located in a similar genomic region as Rag1. Similarly, the QTL detected on chromosome 16 in this study was located in a similar genomic region as Rag3 (Zhang et al. 2010). Although the two resistant genes in PI 567598B were considered recessive (Mensah et al. 2008), and Rag1 or Rag3 were considered dominant or co-dominant (Hill et al. 2006a; Zhang et al. 2010), Rag1 and Rag3 might have been the same genes since the susceptible parent in this study was different from the ones used for characterizing Rag1 and Rag3. It was also possible that the two genes discovered in this study were allelic to Rag1 or Rag3, or different genes, but tightly linked to Rag1 or Rag3. Rag1 in Dowling was overcome by the aphids in Michigan and Rag3 in PI 567543C did not provide antibiosis resistance (Mensah et al. 2005). However, this study revealed a relatively lower DI value in PI 567598B than PI 567543C.
The better resistance of PI 567598B compared with Dowling and PI 567543C might be due to one or more of the following factors: (1) the stacking of resistant genes rag1 and rag3; (2) different resistant alleles at rag1, rag3, or both loci conferring better resistance than the alleles in Dowling and PI 567543C; (3) one or two new genes closely linked to Rag1 and/or Rag3 and have better resistance than Rag1 and Rag3. Further investigations, such as fine mapping or gene cloning, might be necessary to elucidate their relationships with Rag1 and Rag3.
The inventors discovered that the interactions of different sources of aphids and their biotypes affected the resistant reaction of a soybean plant at some of the Rag_genes. For one example, QTL analysis revealed that rag3 was detected in a field trial. However it was also determined that two resistance genes from PI 567598B parents were expressed differently in the field and greenhouse trials. Since the same F4-derived lines were evaluated in these trials, the aphid isolate used in the field trial was able to overcome rag1b while the aphid isolate in the greenhouses was affected by the plants. Phenotypic data of Dowling from the same trial verified that the aphid isolate defeated Rag1, i.e. there were no differences between aphid infestation between Dowling and susceptible plants. The soybean aphid isolates used in the field trial may have the same biotype as those found in Ohio (Kim et al. 2008). This provided additional information that the QTL on chromosome 7 (rag1b) may have the same gene action as Rag1. The present study demonstrated that the presence of both rag1b and rag3 conferred better aphid resistance than when one QTL was present. This supports that stacking by selection for more than one aphid resistance gene will provide durable resistance against soybean aphids.
PI 567598B possessed strong and broad resistance to soybean aphids; therefore, it was used as a resistance source of germplasm for improving soybean for aphid resistance. However, evaluation of aphid resistance usually requires artificial infestation, which is quite laborious and time consuming. The infestation of aphids might also be complicated by the environmental conditions, such as heavy rainfalls and strong winds. The localization of the two resistance genes in PI 567598B using molecular markers in this study are contemplated for use in marker-assisted selection for breeders to select aphid resistance lines. The use of these markers for identifying specific areas having a gene in the aphid resistant germplasm should result in faster breeding methods for developing cultivars for commercial use by bypassing progeny tests for aphid infestation.
Phenotypic analysis was done as follows. Phenotypic values of the 282 F4-derived RILs and its parents, grandparent PI 567598B were summarized in FIG. 45: Table 1. In both field and greenhouse trials, the susceptible parent, IA2070, was severely damaged by the aphids compared to the resistant parent E06902 and grandparent PI567598B, respectively. There is no significant difference in the aphid resistance between E06902 and PI567598B. Correlation between the three and four week ratings from the 2010 greenhouse trial was strong (r=0.88, P<0.0001). However, ratings from 2010 greenhouse trial is not strongly correlated with the 2009 field ratings (0.37 and 0.44 for the week three and four ratings, respectively, P<0.0001). The distributions for the population ratings in both field and greenhouse trials were continuous, but not normal and the distribution in the field trial appeared bimodal (FIG. 51: a, b, c). This indicates that major genes might control the aphid resistance in PI 567598B.
QTL analysis. Among 1056 SSR markers, 38 markers revealed polymorphism between the resistant bulk DNA sample and the susceptible bulk DNA sample. These 38 markers were from chromosomes 1, 3, 7, 13, 16 and 18 (LGs D1a, N, M, F, J and G). Satt654 and Sct—001 on chromosome 16 (LG J) and Satt435 on chromosome 7 (LG M) appeared to be associated with aphid resistance when the individual lines from the DNA pools were genotyped. Therefore, these two regions were saturated with parental polymorphic markers within ±20cM in the consensus map (Song et al., 2004) and genotyped with the whole population. Using BARCSOYSSR 1.0 markers (Song et al., 2010), 48 additional markers were screened for polymorphism within the identified intervals. BARCSOYSSR160366 on chromosome 16 was found to be associated with aphid resistance while four other BARCSOYSRR07 markers between Satt435 and 5 Satt323 in the chromosome 7 interval were found to be polymorphic. SNP markers in these two interested intervals were also designed from the SoySNP50 iSelect Infinium assay (Song et al., 2011) for Taqman endpoint genotyping.
At least eight SSR and four SNP markers were mapped for the interval on chromosome 16 spanning a total of 43.5 cM (FIG. 52A) while at least seven SSR and at least one SNP markers were mapped for the interval on chromosome 7 spanning a total of 45.9 cM (FIG. 52D).
The QTL analysis detected two QTLs for the greenhouse trial while the one on chromosome 16 is significant in the field trial. In both trials, the allele from E06902 (PI 567598B derived line) conferred resistance against soybean aphids at the identified QTLs. Using the CIM method, the QTL on chromosome 16 was consistently mapped between SNP16-10 and SNP16-6424 and explained 30.7-45.8% of the phenotypic variation, with the field trial having the highest percentage (FIG. 47: Table 3 and FIG. 52A). The QTL on chromosome 7 was detected in the greenhouse trials and located between Satt435 and BARCSOYSSR07—0309, explaining over 30% of the phenotypic variation (FIG. 47: Table 3 and FIG. 52D).
The QTL analysis detected two QTLs for the greenhouse trial while one on chromosome 16 was significant in the field trial. In both trials, the allele from E06902 (PI 567598B derived line) conferred resistance against soybean aphids at the identified QTLs. Using the CIM method, the QTL on chromosome 16 was consistently mapped between SNP16-10 and SNP16-6424 and explained 30.7-45.8% of the phenotypic variation, with the field trial having the highest percentage (FIG. 47: Table 3 and FIG. 52A). The QTL on chromosome 7 was detected in the greenhouse trials and located between Satt435 and BARCSOYSSR07—0309, explaining over 30% of the phenotypic variation (FIG. 47: Table 3 and FIG. 52D).
The MIM method was further conducted to determine whether there is significant QTL interaction. The MIM results are presented in FIG. 48: Table 4. The MIM method detected same QTLs as CIM method with two QTLs in the greenhouse trial and one QTL in the field trial. For the week four ratings in the greenhouse trial, MIM method detected a significant additive×additive interaction between the two QTLs located on chromosome 7 and 16, but not for the week three ratings. The LOD score of the QTL interaction is 3.4 and it explained 1.2% of the total phenotypic variations. The two QTLs together with their interaction explained 41.7% of the total phenotypic variation. For the week three ratings, these two QTLs together explained 33.6% of the phenotypic variation. The QTL on chromosome 16 detected in the field trial explained the highest phenotypic variation, 56.1%.
QTL validation. For the validation population, a dense aphid population developed on the susceptible parent Titan while resistant parent PI 567598B had very few aphids in both 2008 and 2009 trials (FIG. 46: Table 2). A total of four markers on chromosome 7 and four markers on chromosome 16 were genotyped. The marker orders were highly comparable with the consensus map (Song et al. 2004).
With the CIM method. One QTL was detected on each linkage group in both trials (FIG. 52: C, F). The QTLs were located at similar regions between trials. The QTL on chromosome 7 was located between Satt567 and Satt435, explaining about 15% and 20% of the total phenotypic variation in the 2008 and 2009 trials, respectively (FIG. 47: Table 3). The QTL on chromosome 16 was located between Satt285 (or Sct—046) and Satt414 and explained about 30% and 40% of the total phenotypic variation in the 2008 and 2009 trials, respectively. The PI 567598B allele at both loci conferred aphid resistance.
The MIM analysis was performed to detect the epistatic effect and results are presented in Table 4. The two QTLs identified with the CIM method were also found using the MIM method in each trial. No QTL interactions were found in the 2008 trial. However, a significant additive×additive interaction between the two QTLs was detected in the 2009 trial. The LOD score of the QTL interaction was 6.0 and it explained 9.2% of the total phenotypic variation. The two QTLs together with their interaction explained 80.4% of the total phenotypic variation. Results presented from the validation population confirmed the QTLs found from the mapping population. Since the QTLs from this study were mapped to same region as Rag1 (Li et al., 2007) and Rag3 (Zhang, et al., 2009), we named the locus on chromosome 7 as rag1b and the locus on chromosome 16 as rag3, according to the conventions of the Soybean Genetics Committee.
Effect of the combination of QTL alternative alleles. The F4-derived lines from the mapping population were classified based on the E06902 alleles at the QTLs identified. Four distinct genotypes were defined by the presence or absence of the allele from E06902 for those QTL associated markers on chromosomes 7 and 16 (FIG. 49: Table 5). Individual lines with complete and unambiguous genotype data for all loci were grouped into the defined genotypes and a total of 139 lines were grouped. Mean soybean aphid rating for all lines within each genotypic group was obtained for each of the trials over two years. In a greenhouse trial, the presence of E06902 alleles at both QTL M and QTL J gave the lowest soybean aphid rating while absence of alleles at both QTLs made lines very susceptible. The absence of E06902 allele at one QTL (either rag1b or rag3) gave intermediate reaction against soybean aphids. However, in a field cage trial, the lines absence of rag3 were as susceptible as those without any of the two QTL alleles from E06902. On the other hand, genotypes containing rag3 gave resistant phenotypes that were comparable to the lines that had both resistant alleles. It seems that the QTL on chromosome 7 (rag1b) was defeated in the field trial while the QTL on chromosome 16 (rag3) conferred resistance. This shows that the QTLs identified in this study confer differential reactions against the soybean aphids in the field and greenhouse trails.
Fine mapping of aphid resistance gene rag3 in soybean PI 567598B. Plant materials and methods for screening recombinants are described in brief. Two backcross populations were first used to determine recombinants that delimit the position of rag3 in PI 567598B. The first population was composed of 2,214 BC1F2 lines from a cross between E00003 and PI 567598B, where E00003 was the recurrent parent. E00003 was a cultivar having resistance against soybean root rot caused by Phytophthora sojae but susceptible to soybean aphid. The second population consisted of 1,827 BC1F2 lines from a cross between Skylla and PI 567598B. Skylla was the recurrent parent and soybean aphid-susceptible cultivar. The recurrent parents were cultivars developed at Michigan State University (Wang et al. 2006).
4,041 F2 plants from the two populations were screened for recombination breakpoints near rag3 using TaqMan single nucleotide (SNP) assays: MSU16-4 and MSU16-10 identified from the previous genetic QTL mapping study (Zhang et al., in preparation) in the spring of 2011. Seeds from identified recombinants were harvested and planted in the field in summer of 2011 for aphid resistance test. The F2-derived lines were analyzed for marker association with segregation of the trait.
Additional screening was done using advanced lines obtained from three F7 lines from a cross between IA2064 and E06906. E06906 was an elite line developed from a cross between Titan and PI 567598B and thus, inherited the resistant allele from PI 567598B (Mensah et al. 2005). Three F7 lines were found to possess residual heterozygosity at the region where rag3 was mapped by initially screening 864 F4:7 plants with the identified flanking markers. Seeds (F8) from these three lines were evaluated for soybean aphid resistance and genotyped for residual heterozygosity.
Evaluation for soybean aphid resistance. A greenhouse trial was conducted for aphid resistance evaluation in the Plant Science Greenhouse on the MSU campus in the spring. The condition in the greenhouse was optimized at 26/15° C. day/night temperature and light intensity was extended during the day (14 hr) using sodium vapor bulbs. BC1F2 lines from the two populations were planted at eight seeds per pot. The pot size was 85×85×120 mm. The corresponding parents for each population and resistant checks were replicated three times and arranged randomly. Two wingless soybean aphids were inoculated into the soybean plants at V2 stage. Soybean aphids used for infestation were obtained from a single clone maintained in the greenhouse that was collected from the field at the MSU Agronomy Farm.
Resistance evaluation was conducted in the field located at the Michigan State University (MSU) Agronomy Farm in the summer of 2011 to assess the phenotype of the progenies of each of the F2 recombinants. Single row plots were set up inside aphid- and predator-proof cages (Redwood Empire Awning Co., Santa Rosa, Calif.) 12.8×19.5 m in dimension for the field evaluation. Each F2:3 line was planted in one plot having at least 12 plants per line. The same procedure was done for the aphid infestation as did in the greenhouse trials. The soybean aphid source was from a naturally infested field at the MSU Agronomy Farm.
For both trials, visual ratings and calculation of damage index (% DI) were done after four weeks of aphid infestation as previously described by Mensah et al. (2008; 2005).
DNA extraction and marker analysis are described as follows. A quick DNA extraction method was employed to screen the genotype of all F2 lines at a rapid rate. The youngest non-expanding trifoliate leaves (5-8 mm long) were collected for extraction before soybean aphid infestation. Tissues from each F2 plant were placed in individual wells of a 96-well PCR plate and added with 100 ul 1×TE buffer (10 mM Tris-HCl and 0.5M EDTA, pH 7.5). The plate was sealed with foil seal (3MTM) using a heat sealer to keep the moisture in. The plate was placed in a pre-heated oven to boil at 94° C. for 30 mins. The plate with lid was centrifuged at 1500 rpm for 3 min to condense the leaf tissues to the bottom of the well. After centrifugation, the plate was stored in 4° C. for at least 30 min to overnight. Lysate was aspirated from the plate and diluted ten times with 0.1×TE buffer (pH 7.5) prior to genotyping.
More than 52,000 SNP markers on the SoySNP50 iSelect Infinium assay (Song et al. 2011) for Illumina BeadChip arrays (www.illumina.com) were screened for polymorphism between the parental lines for each of the fine mapping populations. Custom primers and hybridization probes for TaqMan SNP genotyping assays were designed through the Custom Taqman® Assays Design tool (www.appliedbiosystems.com). From the Williams 82 genome assembly (Glyma1) available at www.phytozome.net (Schmutz et al. 2010), 60 bp upstream (5′ end) and downstream (3′ end) of the identified SNP position were used as target sequences for custom design. This was based on the genomic physical position of the SNP screened for polymorphism among parents (FIG. 38: Table 2). Endpoint genotyping was performed on the LightCycler® 480 system (Roche Applied Science) For one DNA sample, a total of 3 ul reaction volume was analyzed that comprised 1.50 ul of 2× TaqMan Universal PCR Master Mix, 0.15 ul of 10× working stock of SNP genotyping assay and 1.35 ul of DNA sample. The parameters used to perform PCR were as follows: 95° C. for 10 mins to activate enzyme followed by 45 cycles of denaturation at 92° C. for 15s; and annealing and extension at 60° C. for 1 min. Genotype calling was performed using the Endpoint Analysis module of LightCycler® 480 Software version1.5.
For the identified recombinants, good quality DNA was needed to run whole genome SNP genotyping analysis with the SoySNP50 iSelect Inifinium assay. The CTAB (hexadecyltrimethyl ammonium bromide) method was used to extract DNA from F2:3 samples as described by Kisha et al. (1997). Determination of DNA concentration was done using the Quant-iTTM Picogreen® dsDNA Assay Kit (Invitrogen, USA) and quantified using BioTek Multi-Detection Microplate Reader (Biotek, USA). Each DNA sample was normalized to 50 ng/ul for Infinium assay and performed following manufacturer's protocol. Infinium BeadChip data analysis for SoySNP50 iSelect was performed using the GenomeStudio Genotyping module.
Statistical analysis was done by Pearson correlation computation and one-way analysis of variance for the phenotype and genotype of the progenies of recombinants was calculated with the R Statistical package (R Development Core Team, 2011).
Screening for recombination breakpoints was done using 4,104 F2 plants in the recombinant screening, 107 (from population 090004) and 94 lines (from population 090068) were selected for planting in the field and soybean aphid evaluation of progenies. These were lines that had breakpoints between MSU16-04 and MSU16-10 SNP markers using the quick DNA extraction method and assayed with TaqMan. The quick DNA extraction method in the study proved to be a good protocol to screen thousands of lines but was not robust enough for SSR markers and Infinium assay analyses. Thus, a DNA pool of F3 progenies for each F2 line was collected in the field for CTAB extraction and re-analyzed with TaqMan assay. Confirmed genotypes were subsequently used for SoySNP50 Infinium assay. Out of the 107 and 94 lines, 34 and 21 F2:3 lines, respectively, were selected for SoySNP50 Infinium assay. Among the F2 lines genotyped with the Infinium assay, five lines from the 090004 population had recombination events in the rag3 region and three lines from the 090068 population were also selected (FIG. 42: Table 3). Soybean aphid evaluation in the greenhouse was done for F3:4 plants derived from these eight lines in the spring of 2012. A summary of the rag3 fine mapping populations is shown in FIG. 39.
Fine mapping of rag3. Results from the Infinium whole genome SNP genotyping revealed SNPs that were polymorphic within the region of interest and the density was sufficient enough to be able to delimit the position of rag3 into a 231-kb interval (FIG. 4 and FIG. 42: Table 3). The leftmost (towards the telomere) border of the rag3 region was first defined by the line 04-2-653. Marker test revealed association of the trait with segregation of the marker (italicized in FIG. 42: Table 3) and thus, rag3 was positioned at the right side of Gm16—605831_T_C. The rightmost (towards the centromere) border of the rag3 region was position by the lines 04-2-471, 04-2-742 and 04-2-466 with a breakpoint between Gm16—6680549_G_A and Gm16—6713173_T_G. Marker association with the trait revealed that all F3 lines were resistant and this positioned rag3 at the left side of Gm16—6713173_T_G. The next lines that narrowed the position of rag3 on the left border were 04-2-229, 04-2-471 and 04-742. Marker association at the recombination breakpoint between Gm16—6079769_A_G and Gm16—61339859_A_G revealed that rag3 is located at the right side of Gm16—6079769_A_G. Two more lines that narrowed the position of rag3 at the right border were 68-1rem-39 and 68-5-146. These lines were segregating for the trait and marker association test revealed that there is significant association on the phenotype and segregation of the marker. This demonstrates that rag3 may be located at the left side of Gm16—6469551_A_C. The line 04-2-466 revealed a breakpoint between Gm16—6179363_T_G and Gm16—6184915_A_G. The marker test did not reveal association of the trait with the segregating marker since the line has a resistant phenotype. This revealed that rag3 is located on the right side of Gm16—6179363_T_G. The lines, 68-1rem-168 and 68-5-146, narrowed the closest SNP marker border at the left side of rag3 gene. Marker association of both lines showed that the gene is on the right side of Gm16—6192576_T_G. The same line, 68-5-146, had a breakpoint towards the centromere side that defined the right border of rag3, which is Gm16—6469551_A_C. This now delimits rag3 at an interval of 277 kbp.
A F7:8 line from a parallel experiment utilizing residual heterozygosity of advance lines for fine mapping contained a breakpoint that further narrowed the position of rag3 gene at the centromere side. Residual heterozygosity of 11-831-7 revealed marker association for segregation of the SNP marker MSUSNP16-11. This then delimits the position of rag3 at a 231-kb interval. The SNP markers from SoySNP50 that bordered the genomic position of rag3 were Gm16—6192576_T_G (left) and Gm16—6423098_G_A/MSUSNP16-12 (right).
Candidate genes are contemplated for use in breeding aphid resistant soybean plants. The location between the genomic region 6,192,576 bp-6,423,098 bp of chromosome 16 has 18 annotated genes based on the Williams 82 Glyma1 annotation (www.phytozome.net/soybean). Out of the 18 annotated genes, 11 were identified to be candidate genes that may contribute to soybean aphid resistance based on literature search. Of the 11 candidate genes, eight were annotated to encode serine-threonine protein kinase and/or NBS-LRR tandem repeat genes. The eight tandem kinase genes that were annotated were named Glyma16g06940 to Glyma16g07200. These genes span a 160-kb interval within the recombination bin containing MSUSNP16-10 and MSUSNP16-11 (FIG. 43). Recombinant screening of F3:4 lines that were still segregating phenotypically within the interval are contemplated for use in revealing breakpoints within the 231-kb region for more specific identification of a rag3 gene.
The following references are herein incorporated by reference in their entirety.

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EXPERIMENTAL
The following examples are provided in order to demonstrate and further Illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C. (degrees Centigrade/Celsius).
Example 1
Materials and Methods 
Soybean plant introductions (PI) from MG 0 to III were obtained from the USDA Soybean Germplasm Collection in Urbana, Ill. (Table 1). A total of 2,147 PI were evaluated in 2002 and 2003, including 5 MG 0 accessions (PI 468920 to PI 597467), 530 MG I accessions (FC 03609 to PI 612761E), 979 MG II accessions (PI 253650A to PI 612758E), and 633 MG III accessions (FC 02108 to PI 612759D). Accessions originally from northern China were selected, as the climatic conditions are similar to those in the northern USA, where the soybean aphid over-winters, and because soybeans in that region have been exposed to aphids over the years. ‘Williams 82’ was included as a susceptible check in these experiments and one or more of the three resistant genotypes, ‘Dowling’, ‘Jackson’, and PI 71506 (Hill et al., (2004) Crop Sci. 44: 98-106), were included as resistant checks. Both the susceptible and resistant checks were obtained from Dr. Glen Hartman, USDA-ARS at Urbana, Ill., United States.







TABLE 1







Total number of plant introductions (FC and PI numbers)


evaluated in each maturity group (MG) in 2002 and 2003.










Range from which evaluated



MG
Accessions where selected
Total Number












0
PI 468920 to PI 597467
5


I
FC 03609 to PI 612761E
530


II
PI 253650A to PI 612758E
979


III
FC 02108 to PI 612759D
633


Total

2147









PIs and checks were first evaluated in choice tests (Davis, (1985) Insect Sci Appl 6:391-400), in which the aphids colonized genotypes they preferred, to identify PIs with either antixenosis or antibiosis. The resistant PIs were then re-evaluated in no-choice tests (Davis, (1985) Insect Sci Appl 6:391-400), in which aphids were confined on plants of one genotype, to identify PIs with antibiosis resistance. The insects have no choice but to feed on the genotype on which they are confined. The no-choice test is also conducted to overcome the uneven distribution of insects, which normally occurs in choice tests, resulting in escapes (Saxena and Khan, (1984) Crop Sci. 24:1204-1206).
Experiments were carried out on the campus of Michigan State University (MSU), East Lansing, Mich. Soybean aphids were obtained from nearby naturally infested soybean fields for summer fieldwork, and from a colony maintained in growth chambers at the Field Crops Entomology Laboratory at MSU for winter greenhouse work. The experiments were set up as a randomized complete block design with two replications.
During vegetative growth of soybean, aphid colonies were usually found at the growing points e.g., partially expanded young trifoliate, petioles, and stems. At the reproductive stage the aphids became more widely dispersed on the plant and could be found on the underside of mature leaves, on lower stems, lateral branches, petioles, and pods (Ragsdale et al., (2004) Ann. Entomol. Soc. Am. 97:204-208). Based on experiments conducted during the course of the present invention, most aphid colonies stayed on inoculated trifoliates for more than 10 days after inoculation, with the inoculated leaves still not overcrowded. Therefore, an estimate of the increase of the aphid population in the first 10 days can be obtained by counting aphids on the inoculated trifoliate 10 days after inoculation.
Five seeds per accession were planted in the field in a 0.30m long plot (Greenhouse: 3 seeds, gallon-sized plastic pots). Inoculation of plants was 2 weeks after planting (early vegetative stage), two plants per accession inoculated with 2 aphids on the new trifoliate. Counting of aphid population was done 10 days after inoculation using a hand counter. Four weeks after inoculation the plants were rated visually using the rating method of Zhuang (Zhuang, (1999) Biological studies of Chinese wild soybean. 1st ed., Science Publisher, Beijing, China). Weekly visual ratings using the method of Zhuang (1999), supra, showed that there was a clear difference in susceptibility or resistance among accessions four weeks after inoculation when aphid densities reached their peaky. Thus DI values four weeks after inoculation were used to determine susceptibility of the PIs. Visual rating data two weeks after inoculation were not used because of low aphid populations. Two weeks after inoculation, the method of Zhuang (supra) categorizes the plants as either a ‘1’ or ‘2’ and the results are similar to counting aphids 10 days after inoculation. On the other hand, five weeks after inoculation, the aphid populations started to decline due to overcrowding and development of winged aphids, which left the plants. Therefore, visual rating data five weeks after inoculation were not used in the analysis.
Lin et al. (Lin et al., (1992) Soybean Science, 11(4):318-321) showed that the soybean aphid colonizes soybeans in China at the early vegetative stage. Aphid populations increase gradually and reach a 10 to 15 day exponential growth phase coinciding with late vegetative to early reproductive stage of the plants. Ten days after inoculation, at the early vegetative stage, a high percentage of test plants had very few aphids per leaflet. Correlations were low between the numbers of aphids per leaflet 10 days after inoculation and the DI four weeks after inoculation in the first and the second years of evaluation (r=0.16 and r=0.20, respectively). These low correlation values indicate that counting aphids on the inoculated trifoliate 10 days after infestation in the early vegetative stage is not an optimal method for determining the resistance or susceptibility of an accession. Counting the total number of aphids on the whole plant 10 days after inoculation would also not have helped to separate resistant from susceptible accessions because most aphid colonies did not move away from the inoculated trifoliate during the first 10 days after inoculation. It is advisable to count aphids on the whole plants in the late vegetative or early reproductive stage in order to identify truly resistant accessions. However, counting aphids is very tedious and time consuming. For further large-scale evaluation of aphid resistance such as progeny evaluation in a breeding program, the preferred method is described in Zhuang (Zhuang, 1999, Biological studies of Chinese wild soybean. 1st ed., Science Publisher, Beijing, China).
Summer Field Evaluation
Choice Test
Two experiments were carried out in the summers of 2002 and 2003 to evaluate soybean germplasm for aphid resistance. Summer plantings were done at the Agronomy Farm, Michigan State University (MSU), in 12.2.times.18.3 m (40×60 feet seed cage) polypropylene cages with a 0.49 mm mesh size (Redwood Empire Awning Co., Santa Rosa, Calif.) that are aphid- and predator-proof. Results showed Low correlations (r=0.16, n=1017, p<0.0001) in 2002 and (r=0.20, n=1108, p<0.0001) in 2003 between the average number of aphids per leaflet and the DI of an accession. Specifically, 6 PIs—Aphid 1, Aphid 2, Aphid 3, Aphid 4, Aphid 5, and Aphid 6 Aphid 5 and Aphid 6 showed some resistance to the soybean aphid (SBA).
In 2002, 1,043 PIs, the susceptible check (Williams 82), and a resistant check (Jackson) were evaluated in the field cage. The PIs and checks were planted on 26 June and each check was treated as an accession in the test. Five seeds per accession were planted in a plot 0.3 m long and with a row spacing of 0.3 meter. Each accession was planted in a single plot without replication. At the VI stage (Fehr and Caviness, 1977, Iowa State University, No. 80), two plants per accession were inoculated with two wingless aphids each on the partially expanded trifoliate, using a camel-hair brush. Aphids were obtained from naturally infested fields on the Agronomy Farm, MSU. The aphids were left to multiply and move among plants.
In 2003, a new set of 1,103 PIs, the resistant checks (Dowling, Jackson, and PI 71506), and the susceptible check (Williams 82), were evaluated in two field cages. In each cage, a complete set of the PIs plus the checks were planted as a randomized complete block. Each check was treated as an accession in the test. The lines were sown 20 on 30 May in one cage (planting 1) and on 6 June in the second cage (planting 2). The methods of inoculation plot sizes, and evaluation procedures were the same as for the 2002 field evaluation.
Winter Greenhouse Evaluation
Choice and No-Choice Tests
A winter evaluation was carried out in a large greenhouse with temperatures between 22 and 25° C. to verify the results obtained in the field in 2002. The PIs planted in the field in 2002 were evaluated. Seeds were planted on 21 Nov. 2002 in the greenhouses at the Horticulture Research Farm at MSU. Three seeds of each genotype were planted in a plastic pot 22 cm in diameter and 23 cm deep. Each genotype was planted in a single pot without replication and the pots of genotypes were randomly laid out on the benches in the greenhouse. The soil used in greenhouse tests was Baccto High Porosity Professional Planting mix (Michigan Peat Company. Houston, Tex.). Two of the three plants were inoculated at the V1 stage (Fehr and Caviness, 1977, Iowa State University, No. 80) with two wingless aphids each on the partially expanded trifoliate.
A no-choice test was carried out in the greenhouse from December 2003 to February 2004, to determine the type of resistance of each resistant source. Each pot was set up as described for the 2002 greenhouse plantings with two replications and in a randomized complete block design. Each pot was isolated by the use of a no-see-um mesh cage (Venture Textiles, Inc. Braintree, Mass.). The entries in the no-choice test were the resistant PIs identified in the 2002 and 2003 evaluation in choice tests, the resistant check (Jackson), the susceptible check (Williams 82), and two soybean varieties (cultivars), Titan and Loda.
Confirmation of Resistance
In the summer of 2004, PIs previously identified as potentially aphid resistant after two years of evaluation, and Williams 82, were evaluated in the field to confirm the resistance found in previous tests. The experiment was set up as a randomized complete block design with three replications. Ten seeds were planted in each 0.6 m plot. Ten plants were inoculated at the V1 stage (Fehr and Caviness, 1977, Iowa State University, No. 80) with wingless aphids as described earlier.
Data Collection
In these studies, except the confirmation of resistance test, aphid populations on inoculated trifoliate were counted 10 days after inoculation when the plants were at the V3 stage (Fehr and Caviness, 1977, Iowa State University, No. 80). Four weeks after inoculation, the plants in each accession were visually rated for susceptibility to soybean aphid using the rating scale shown in FIG. 1 (Zhuang, 1999, Biological studies of Chinese wild soybean. 1st ed., Science Publisher, Beijing, China). A damage index (DI) for each accession was calculated using the following formula (Zhuang, 1999, Biological studies of Chinese wild soybean. 1st ed., Science Publisher, Beijing, China):
DI═.SIGMA. (Scale value.times.No. of plants in the category)/(4.times. Total no. of plants evaluated).times.100. The DI ranges between 0% for no infestation and 100% for the most severe damage. In early studies a DI was the susceptibility of the plant to aphid infestation expressed as a percentage, with 25% or less as resistant and greater than 25% to 100% as susceptible. However in later studies a DI of 30% or less was classified as resistant, whereas a DI of 30% or more was classified as susceptible. The 30% break point was chosen based on the observation that a soybean genotype with a DI value less than 30% never showed symptoms of damage under high aphid pressure until the end of the season. In the second year of field evaluation, the plants were visually rated weekly from the second week through the fifth week after inoculation to determine and confirm the best time to carry out the visual rating.
Statistical Analysis
The data for each year were analyzed using the PROC GLM procedure in the SAS statistical package V8 (SAS Institute, 1999, Software release 8, SAS Institute, Inc. Cary, N.C.). Means were separated by least significant difference (LSD) at the 5% probability level. Linear correlations between the average number of aphids per leaflet ten days after inoculation and the DI were calculated with PROC CORR.
Genetic Studies of Aphid Resistance for Linkage Group Analysis
Linkage group analysis was used for identifying the linkage groups comprising aphid resistance germplasm. F2 populations from crosses between aphid resistant soybean and aphid susceptible soybean were evaluated for aphid resistance then tested with simple sequence repeat (SSR) DNA markers for identifying J, K, B2, D1a and D1b Linkage Groups comprising aphid resistant germplasm.
Evaluation of soybean plants for resistance to soybean aphids was carried out as described in Mensah, et al. 2005 (Crop Sci. 45:2228-2233) as described herein. Aphid damage data were collected weekly two weeks after inoculation until the fourth week. Data collected at weeks 3 and 4 were used to identify DNA markers associated with aphid resistance. Data collected at week 4 were used to test the segregation ratios. Chi-square tests were performed to test the goodness-of-fit of observed segregations among the seven F2 populations with different genetic ratios.
A SOYBASE website hosted by Iowa State University was used for providing PCR sequences, forward and reverse, for amplifying Satt SSR markers and for providing linkage group identification using Satt SSR marker information, (Tables 5-11 and FIGS. 9-16).
Evaluation of the soybean plants with SSR markers:
PCR amplification of SSR markers was carried out as described in Cornelious, et al. ((2005) (Mol. Breed. 16:103-112)). The PCR products were analyzed in a 6% non-denaturing polyacrylamide gel system as described by Wang, et al. ((2003) (Crop Sci. 43:1828-1832)).
Sequences for Satt PCR Primers Used to Amplify SSR Loci in Soybean are described in Zhu et al. Single-nucleotide polymorphisms in soybean, Genetics 2003 March; 163(3):1123-34. Sequences for Satt PCR Primers Used to Amplify SSR markers for identifying associations with aphid resistant germplasm are as follows: Satt271 (SEQ ID NO:01 Forward primer: GTT GCA GTT GTG CGT GGG AGA GAG and SEQ ID NO:02 Reverse primer: GCG ACA TAG CTA ATT AAG TAA GTT), Satt280 (SEQ ID NO:03 Forward primer GCG GAA TCT GCT TAT TCA TTG TGT G and SEQ ID NO:04 Reverse primer GCG CCA TGC TGT AAC ACG TCA AT), Satt304 (SEQ ID NO:05 Forward primer GGG TAG TGA CGT ATT TCA TGG TC and SEQ ID NO:06 Reverse primer GCG TAA AAA CAT TCG TTG ACT ACA TAA), Satt439 (SEQ ID NO:07 Forward primer GCG AAA ATG ATT AAA TTG TTT TCT CAA G and SEQ ID NO:08 Reverse primer GCG GCA CGT TGC CAT ATA AGA TAA AGG), Satt468 (SEQ ID NO:09 Forward primer GCG TCT CTT ATT TTG ACC TTT TTA ACT T and SEQ ID NO:10 Reverse primer GCG TTT TGT ATT TGG TCT ATC TGC TTA G), Satt529 (SEQ ID NO: 11 Forward primer GCG CAT TAA GGC ATA AAA AAG GAT A and SEQ ID NO:12 Reverse primer GCA CAA TGA CAA TCA CAT ACA), Satt628 (SEQ ID NO:13 Forward primer CTA CCT TTA AGG TCG TTT TCA AGT and SEQ ID NO:14 Reverse primer GCA TGC TCC TTT TAT GCT CCT TTT), and Satt686 (SEQ ID NO:15 Forward primer ACG GAA AAT AAA TGA AAC TAA GA and SEQ ID NO:16 Reverse primer: GCG CTA TCA GAT AGA GAA GCA GAA GAA T).
A method of PCR amplification using Satt primers PCR Reagents for Soybean SSR Amplification is provided as follows: a PCR reaction mixture is provided comprising 30 ng genomic soybean DNA, buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), and 0.1% Triton X-100) 1.5 mM MgCl2, 0.15 mM for each of the NTPs, and 1 unit Taq DNA Polymerase. Thermocycling Profile for Amplification of Soybean SSRs is 1 cycle of 2 min at 95° C., 33 cycles of: Denaturation: 92° C. then annealing (optimum temperature or 47° C.) then extension at 68° C.
Associations of SSR markers with resistance to soybean aphids were determined with the single marker analysis method in WinQTLcart Version 2.5 (Wang, et al. (2005) Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, N.C.).
Example 2
Illustration of the Visual Rating Scale Used to Establish the Damage Index (DI)
The following index was used for establishing a Damage Index rating. 0=No aphids, plant appears normal and healthy; 1=Less than 100 aphids per plant, plant appears normal and healthy; 2=101-300 aphids per plant, mostly on the young leaves and the tender stem at top of plant, plant appears normal and healthy; 3=301-800 aphids per plant, leaves slightly curly and shiny, young leaves and stems covered with aphids; 4=More than 800 aphids per plant, plants stunted, leaves severely curled, yellow, covered with sooty mold and cast skins. (FIG. 1).
Example 3
Choice Tests in 2002
In the first year of evaluation in the field cage, the average number of aphids per leaflet ranged from 0 to 500. In the greenhouse, the average number of aphids per leaflet ranged from 0 to 170. Results from the visual rating and calculation of the DI showed that 1008 and 973 of the accessions evaluated in the field and greenhouse, respectively, were susceptible to the soybean aphid (DI>30%). Twenty-eight and 62 accessions did not germinate in the field and the greenhouse, respectively. The correlation between the average number of aphids per leaflet 10 days after inoculation and the DI of an accession was low (r=0.16, n=1043, p<0.0001). Seven of the 1043 accessions appeared to be resistant (DI<30%) to the aphid in the field cage, while eight accessions showed resistance in the greenhouse (Table 2).
Three of these accessions were resistant in both the field and greenhouse evaluations. The accessions that showed resistance in one test, field or greenhouse, were replanted in the greenhouse in the spring of 2003 and found to be susceptible. The resistant check, Jackson, had a DI of 55% in the field and 25% in the greenhouse. After the first year of evaluation, accessions PI 567543C, PI 567597C, PI 567541B and PI 567598B appeared to be resistant to the soybean aphid.







TABLE 2







Number of accessions in Each DI category


For 2002 (n = 1043) and 2003 (n = 1108)











Year and
Damage Index














Location/replication
<30%
31 to 50%
51 to 75%
>75%

















Field (2002)
7
10
56
942



Greenhouse (2002)
8
27
200
746



Field Rep. 1 (2003)
12
14
164
753



Field Rep. 2 (2003)
10
12
127
856










Example 4
Choice Tests in 2003
In the second year of evaluation, the number of aphids per leaflet ranged from 0 to 326 for the first planting and 0 to 244 for the second planting. Based on DI, 931 and 995 of the plants were found to be susceptible (DI>30%) in plantings 1 and 2 respectively. As in the previous year, the DI value four weeks after inoculation did not reflect the aphid population 10 days after inoculation. The correlation between the average number of aphids per leaflet 10 days after inoculation and the DI value 4 weeks after inoculation was low (r=0.20, n=1103, p<0.0001) in 2003. (Table 3).
Eight accessions were rated as resistant in planting 1 and ten accessions were rated as resistant in planting 2. The difference in numbers of accessions rated as resistant was due to some accessions failing to germinate in both plantings. However there were two accessions, PI 603392 and PI 603418C, which had a DI of <30% in both plantings. Resistant checks had a DI of 25% in both plantings. In cases where germination did not occur in both plantings, the accessions were replanted in the greenhouse in the winter of 2003 and found to be susceptible.







TABLE 3







Results of the no-choice test for the six resistant accessions,


Resistant and susceptible checks, and varieties in 2003.











Maturity
Average No. of
Damage index


Entry
group
aphids per leaflet1
(%)2





PI 567543C: Aphid-R1
III
 8 ab
 56 b3


PI 567597C: Aphid-R2
III
 1 a
 62 b


PI 567541B: Anhid-R3
III
 1 a
 25 a


PI 567598B: Aphid-R4
III
11 bc
 25 a


PI 603392: Aphid-R5
III
 5 a
 81 c


PI 603418C: Aphid-R6
III
12 c
 77 c


Jackson
VII
 2 a
 25 a


Titan
I
17 c
 71 c


Loda
II
19 c
 83 c


Williams 82
III
19 c
100 d


Mean

 9.4
 60.5





1The data are the averages of 12 leaflets from two replications with two plants per replication and three leaflets per plant taken 10 days after inoculation.


2Averages of two replications.


3Means followed by the same letters are not significantly different by the least significant difference test (p = 0.05).






A brief summary of 2003 No-Choice results reveals that soybean plants Aphid-R3 and Aphid-R4 showed antibiosis while Aphid-R1, Aphid-R2, Aphid-R5 and Aphid-R6 demonstrated antixenotic properties.
Example 5
No-Choice Test
The six MG III accessions classified as resistant in evaluation trials, PI 567543C, PI 567597C, PI 567541B, PI 567598B, PI 603392 and PI 603418C, were identified in field and greenhouse choice tests. The no-choice test showed that PI 567541B and PI 567598B had adverse effects on the aphid and thus possessed antibiosis as defined by Painter (Painter, 1951, Insect Resistance in Crop Plants, Macmillan). The high DIs obtained in no-choice test for PI 567543C and PI 567597C (which were classified resistant in choice tests) is likely due to the change in feeding response of the aphid in choice and no-choice tests as found by Smith et al. (Smith, et al., 1994, Techniques for evaluating insect resistance in crop plants, CRC Press, Inc.). Also, it is possible for a genotype classified as resistant in a choice test to be declared susceptible in a no-choice test (Tingey, 1986, Techniques for evaluating plant resistance to insects, in insect-plant interactions, Springer-Verlag, New York). Soybean plants PI 567543C and PI 567597C, while having lower DI values than Williams 82, are not resistant (Table 1). The high (r=0.63, p=0.048) correlation between the average number of aphids per leaflet 10 days after inoculation and the DI of an entry in the no-choice test is attributed to the fact that the entries chosen for this test were truly susceptible or resistant as found in previous evaluations. The inconsistent average numbers of aphids per leaflet for PI 567598B and PI 603392 (Table 1) strengthens the fact that counting of aphids 10 days after inoculation is not optimal for selecting aphid resistant plants. The method of Zhuang (Zhuang, 1999, Biological studies of Chinese wild soybean. 1st ed., Science Publisher, Beijing, China) would still be the best to use in experiments with few entries.
The test conducted to confirm the resistance after two years of evaluation revealed that PI 603392 and PI 603418C, both from Liaoning province, were not resistant to the soybean aphid. These plants, when evaluated in 2003 in the field cages, did not show symptoms of severe aphid infestation. According to Painter (Painter, 1951, Insect Resistance in Crop Plants, Macmillan), the type of resistance that enables a host plant to withstand infestation by insects without suffering severe damage is tolerance. PI 603392 and PI 603418C plants might be tolerant, but tolerance can be confirmed with further yield and dry matter studies. These two accessions were not considered resistant after their poor performance in the confirmation test. Smith (Smith, 1989, Plant resistance to insects: A fundamental approach, Wiley, New York) also observed that pseudo-resistance or false resistance may occur in normally susceptible plants. Resistance may have been induced temporarily by variations in temperature, day length, soil chemistry, plant or soil water content, or internal plant metabolism. Susceptible plants may simply escape damage because of incomplete infestation.
Example 6
Confirmation of Resistance
Resistance in the four accessions (PI 567543C, PI 567597C, PI 567541B, and PI 567598B) identified in the choice tests in 2002 was confirmed in 2004 (Table 2). At three and four weeks after inoculation, highly significant differences (p<0.0001) were found between the DIs for these four accessions and the DIs for PI 603392 and PI 603418C, identified in choice tests in 2003. The amount of damage to the plant as a result of aphid feeding was greater on the susceptible check than on PI 603392 or PI 603418C four weeks after inoculation. The susceptible check appeared stunted, and its leaves were curled and covered with black sooty mold, while PI 603392 and PI 603418C showed none of these symptoms. (Table 4).







TABLE 4







Damage Index (DI) based on three replications in 2004 for six putative


resistant accessions, identified after two years of screening, and a


susceptible check three and four weeks after inoculation.









Damage Index (%)












Three weeks after
Four weeks after



Entry
Inoculation
Inoculation







PI 567543C: Aphid-R1
25 a1
 25 a



PI 567597C: Aphid-R2
26 a
 26 a



PI 567541B: Aphid-R3
25 a
 25 a



PI 567598B: Aphid-R4
26 a
 26 a



PI 603392: Aphid-R5
75 b
 79 b



PI 603418C: Aphid-R6
75 b
 79 b



Williams 82
83 c
100 c



Mean
46.86
 51.43







1Mean of three replications of a maximum of 10 plants each. Means followed by the same letters are not significantly different by the least significant difference test (P >= 0.05).






A brief summary of 2004 results supports the resistance of Aphid-R1, Aphid-R2, Aphid-R3 and Aphid-R4 to SBAs while Aphid-R5 and Aphid-R6 showed low level resistance as tolerance.
Therefore the inventors discovered four resistant PI s in MG III found (2 have antibiotic and 2 have antixenotic properties). Further refinements of the methods were made for subsequent studies such that before inoculation aphids maintained in growth chamber were acclimated to field or greenhouse conditions, counting of aphid populations were made at the late vegetative to early reproductive stage due to low correlation between the average number of aphids per leaflet and the DI of an accession at the early vegetative stage, and the rating method of Zhuang (1999), supra, used at 3, 4, 5 weeks after inoculation were continued.
Example 7
Transfer Aphid Resistance from the Aphid Resistant Germplasm to Elite Soybean Germplasm
Inventors' preliminary data showed that aphid resistance in their elite soybean cultivars is a dominant trait. A backcross method as shown in FIG. 3 will be an efficient method to transfer the resistant gene(s) from the aphid resistant PIs (plant introductions) to elite soybean germplasm. The aphid resistant accession PIs are: PI 567543C, PI 567597C, PI 567541B, and PI 567598B. The elite variety can be any soybean varieties. To shorten the total time needed for the transfer process, greenhouses or winter nurseries can be used to carry out the activities of any season in FIG. 3. Progress for up to three seasons per year can be made. To minimize the transfer of undesirable genes from the PIs to the elite germplasm, DNA markers can be used to select progenies with minimum proportion of the genome from the PIs. Forty to eighty simple sequence repeat (SSR) DNA markers evenly spaced on the soybean linkage map can be used to assist the selection. Computer simulation showed that 93% of the genome of the recurrent parent can be recovered in two cycles of backcrosses if DNA markers are used to assist the selection (Frisch et al., (1999) Crop Science 39:1295-1301.
Evaluation of progenies for aphid resistance can be carried out as described by Mensah, et al. 2005 (Crop Sci. 45:2228-2233). Evaluation of progenies for their genome compositions using SSR DNA markers can be carried out as described by Wang et al., (2003) Crop Sci. 43: 1828-1832, herein incorporated by reference.
Variations of the Method Described Above:
The method outlined in FIG. 3 can be modified. The following are examples of modifications:
Modification 1:
In season 3, self-pollinate the selected BC1F1 (BC=backcross) to obtain BC1F2. In season 4, select BC1F2 individuals that are aphid resistant and have the highest percentage of elite genome based on DNA fingerprinting data. In season 5 and after, evaluate progenies of selected individuals for aphid resistance and agronomic performance and release the lines that are homozygous for aphid resistance and acceptable in agronomic performance as new varieties or germplasm.
Modification 2:
In season 2, self-pollinate the F1 to obtain F2. In season 3, select F2 individuals that are aphid resistant and have the highest percentage of elite genome based on DNA fingerprinting data. In season 4 and after, evaluate progenies of selected individuals for aphid resistance and agronomic performance and release the lines that are homozygous for aphid resistance and acceptable in agronomic performance as new varieties or germplasm.
Modification 3:
Use the method outlined in FIG. 3 with the modifications 1 and 2 described above without fingerprinting with SSR DNA markers and/or without selection based on DNA fingerprinting data, an example of soybean SSR mapping is provided in U.S. Patent Appln. No. 20020133852, herein incorporated by reference. Marker-assisted selection is generally described in the following U.S. Pat. Nos. 5,536,901, 5,612,191, 5,606,823, 5,574,210, 5,492,547, 5,491,081, 5,476,524, and 5,385,835, the entire contents of each of which are herein incorporated by reference.
Example 8
Genetics of Aphid Resistance
Crosses of an aphid susceptible parent with an aphid resistant parent (PI 567541B or PI 567598B) were done for determining whether aphid resistance segregated as a Medelian dominant or recessive trait. Following crosses, the No. of resistant F1 vs. No. of susceptible F1 progeny plants were identified and counted.
The results in Table 5 show that antibiosis resistance in PI 567541B and PI 567598B is recessive.







TABLE 5







F1 plants from the crosses between aphid resistant parents (PI 567541B


and PI 567598B) and an aphid a susceptible parent (E00075) were shown


to be susceptible to soybean aphids, which is the expected result for a


recessive trait.














No. of
No. of





resistant F1
susceptible F1


Cross ID
Parents
No. of F1 plant
plant
plant














040129
E00075x PI
6
0
6



567541B


040130
E00075x PI
12
0
12



567598B









Further segregation breeding studies, as described below, were done in order to identify the number of recessive genes contributing to aphid resistance. These results show that aphid resistance in both PI 567541B and PI 567598B appeared to be controlled by two recessive genes (see, Table 6).







TABLE 6







Segregation of aphid resistance in F2 populations derived from susceptible


x resistant crosses. The segregation data were tested for goodness of fit to a 15:1


(Susceptible:Resistant) ratio, which is the expected ratio for a trait controlled by


two recessive genes. The observed ratios for the six populations did not deviate


from the expected ratio.












Population
Susceptible
Resistant
Observed*
Expected*
P value















ID
parent
parent
Total
R
S
R
S
of X2 test


















040129-1
E00075
PI567541B
155
5
150
9.7
145.3
0.120


040129-2
E00075
PI567541B
98
5
93
6.1
91.9
0.639


040130-1
E00075
PI567598B
100
7
93
6.3
93.8
0.757


040130-2
E00075
PI567598B
126
8
118
7.9
118.1
0.963


030104-3
Titan
PI567598B
415
26
389
25.9
389.1
0.990


030104-10
Titan
PI567598B
416
26
390
26.0
390.0
1.000





*R = resistant, S = Susceptible






The following breeding study and analysis described below for determining a resistant:susceptible ratio in F2:3 lines was done in order to determine whether the two recessive genes are the same or different genes. The results show that resistant loci in PI 567541B and PI 567598B appeared to be two different sets of resistance genes (see, Table 7).







TABLE 7







The progenies from the cross between the two aphid resistant soybean


genotypes (PI 567541B and PI 567598B) were segregating for aphid


resistance at a 47:209 (resistant:susceptible) ratio, indicating the two


resistance sources have two different sets of resistance genes.


The 47:209 ratio was the expected results of the following genotypic


configuration: aabbCCDD x AABBccdd 47:209 (Resistant:Susceptible).


Resistant: aabb---, ---ccdd, aa--cc--. Susceptible: A-B-C-D-, aaB-


C---, A-bb---, A---ccD-, ---C-dd. PI 567597C and PI 567598B appeared


to share resistant loci but with different alleles (see, Table 8).














No.







of
No. of
No. of
P value of


Population

F2:3
resistant
susceptible
X2 test


ID
Parents
line
F2:3 line
F2:3 line
(47R:209S)





020138-1
PI567598B x
193
34
159
0.79



PI567541B
















TABLE 8







Progenies from the cross between PI 567597C and PI 567598B were


resistant to soybean aphids indicating they have the same resistant loci.


However, the resistant alleles are different because PI 567597C has


antixenosis resistance while PI 567598B has antibiosis resistance.














No. of
No. of




No. of F2
resistant F2
susceptible F2


Population ID
Parents
plant
Plant
Plant





030100-1
PI567598B x
541
541
0



PI567597C


030100-2
PI567598B x
322
322
0



PI567597C


030100-3
PI567598B x
356
356
0



PI567597C


030100-4
PI567598B x
596
596
0



PI567597C









Example 9
Molecular markers were found linked to genes conferring resistance to soybean aphids in PI 567598B and PI 567541B (Tables 9 and 10).
Linkage group analysis was used for identifying the linkage groups comprising aphid resistance germplasm. F2 populations from crosses between aphid resistant soybean and aphid susceptible soybean were evaluated for aphid resistance then tested with simple sequence repeat (SSR) DNA markers for identifying J, K, B2, D1a and D1b Linkage Groups comprising aphid resistant germplasm.







TABLE 9







Markers associated with aphid resistance in PI 567598B in single marker


analysis. P-value less than or equal to 0.05 and 0.01 are indicated by * and


** respectively. Linkage group names and marker positions were obtained


from the soybean composite map (Song, et al. (2004) Theor. Appl.


Genet. 109: 122-128).














Linkage
Position
Week 3
Week 4



Marker
Group
(cM)
P-value
P-value

















Satt304
B2
65.55
0.049*
0.012*



Satt271
D1b
137.05
0.076
0.024*



Satt280
J
38.70
0.019*
0.053



Satt686
J
40.50
0.016*
0.007**



Satt529
J
41.29
0.004**
0.002**



Satt628
K
49.59
0.122
0.012*










These results show that in particular, linkage group J showed the closest association with aphid resistance germplasm in PI 567598B plants.







TABLE 10







Markers associated with aphid resistance in PI 567541B in single marker


analysis. P-value less than or equal to 0.05 and 0.01 are indicated by * and


** respectively. Linkage group names and marker positions were obtained


from the soybean composite map (Song, et al. (2004) Theor. Appl.


Genet. 109: 122-128).














Linkage

Week 3
Week 4



Marker
Group
Position (cM)
P-value
P-value

















Satt468
D1a
69.91
0.118
0.030*



Satt439
D1a
72.26
0.023*
0.089










These results show that in addition to the above linkage groups, linkage groupD1a showed an association with aphid resistance germplasm in PI 567541B plants.
Example 10
The inventors developed soybean breeding lines comprising economic and agronomic desirable traits for commercial development. The following Table 11 shows the parents, generation and preference order for development as a commercial soybean plant with aphid resistance.







TABLE 11







Breeding lines with antibiosis resistance to soybean aphids











Parents (Female x
Current



Line ID
Male)
generations
Preference order





E06906
Titan x PI
F4 derived F5 and
1



567598B
F6


E06902
Titan x PI
F3 derived F4 and
2



567598B
F5


E06907
E99034x PI
F4 derived F5 and
3



567598B
F6


E06901
Titan x PI
F3 derived F4 and
4



567598B
F5


E06904
Titan x PI
F3 derived F4 and
5



567598B
F5









Example 11
This Example shows exemplary mapping of aphid resistance genes in soybean cultivar PI 567541B using molecular markers. Accession PI 567541B soybean plants were discovered during the course of developing the present inventions, as described herein, as containing aphid resistance germplasm with a plant phenotype that showed early maturity characteristics. Therefore, hundreds of PI 567541B plants, parental susceptible plants, and progeny plants thereof, were used for screening hundreds of markers for identifying regions of DNA in specific linkage groups for identifying polymorphic regions present in aphid resistant soybean plants that were not present in plants susceptible to aphid damage.
During the identification and development of sets of molecular markers useful to methods of the present inventions, a mapping population of at least 228 F3 derived soybean plant lines was investigated for aphid resistance in both field and greenhouse trials. After testing hundreds of markers, the inventors discovered an exemplary set of markers showing unique banding patterns on regions within two quantitative trait loci (QTLs) associated with an aphid resistance phenotype using a composite interval mapping method. These two QTL regions were localized on soybean plant genetic linkage groups (LGs) F and M. After further testing, cultivars of PI 567541B plants were shown to have transferred resistant alleles at both QTL loci to their progeny aphid resistant plants. Therefore, in addition to identifying regions within at least 2 linkage groups associated with an aphid resistant phenotype, an additive X (times) additive interaction between these two QTL regions was identified using this multiple interval mapping method.
The presence of unique banding patterns within QTLs M and F, as a combination in soybean plants with a higher level of resistance than in soybean plants with either one alone, showed their additive interaction. This additive interaction is contemplated to explain the majority of the phenotypic variation in aphid resistance observed and measured in both field and greenhouse trials.
In general, the QTL region on LG F had less effect, less aphid resistance, than the one on LG M, especially in the greenhouse trial. These two QTLs were further validated using an additional independent population (a population of plants different than the mapping population. The effects of these two QTLs were also confirmed using 50 advanced breeding lines, which were all derived from PI 567541B and had various genetic backgrounds.
QTL Mapping.
Plant Materials and Aphid Resistance Evaluation
A population of 228 F3-derived F4 lines was developed from the cross of PI 567541B X Skylla by single seed descent and was used for QTL detection. PI 567541B possesses antibiosis resistance to the soybean aphid (Mensah et al. 2005) while ‘Skylla’ (Wang et al. 2006) is an aphidsusceptible cultivar.
One Field and one greenhouse trial were conducted for aphid resistance evaluation. In the summer of 2007, a Field trial was performed on the Agronomy Farm of Michigan State University (MSU). A polypropylene cage with the 0.49-mm size mesh (Redwood Empire Awning Co., Santa Rosa, Calif.), which was aphid- and predator-proof, was constructed over the Field experiment to create conditions for an artificial aphid infestation. The whole population (F3:4 generation) and its parents were randomly arranged in the Field plots without replication. Depending on the seed availability, two to twenty seeds per line were seeded in a single row plot, 60 cm long with a row spacing of 60 cm. The average number of plants per line was about 11 with most plots having at least ten plants. In the spring of 2008, a greenhouse trial was performed in the Plant Science Greenhouse on the MSU campus. In this trial, the whole population (F3:5 generation) and its parents were arranged in a randomized complete block design with two replications. In each replication, six seeds per line were seeded in a plastic pot. The pot size was 105 mm wide×105 mm long×125 mm deep. The greenhouse was maintained at a temperature of 26° C. by day, 15° C. by night, and sodium vapor lights were used to supplement light intensity during the day (14 h).
Both trials were choice tests for aphid resistance evaluation, which identifies resistance genotypes with either antibiosis or antixenosis. Each plant was inoculated with two wingless aphids at the V1 stage. The aphids inoculated in the Field trial were collected from the naturally infested Field on the Agronomy Farm of MSU during that year. The aphid inoculated in the greenhouse trial was a single clone, which was collected from the naturally infested Field on the Agronomy Farm of MSU in 2002 and has been maintained in the greenhouse ever since. Aphid resistance was visually rated for each plant 3 and 4 weeks after inoculation using a scale of 0-4 developed by Mensah et al. (2005, 2008), where 0=no aphids; 0.5=less than 10 aphids per plant, no colony formed; 1=11-100 aphids per plant, plant appears healthy; 1.5=101-150 aphids per plant, plant appears healthy; 2=151-300 aphids per plant, mostly on the young leaves or tender stems, plant appears healthy; 2.5=301-500 aphids per plant, plant appears healthy; 3=501-800 aphids per plant, young leaves and tender stems covered with aphids, leaves slightly curly and shiny; 3.5=more than 800 aphids per plant, plants stunted, leaves curled and slightly yellow, no sooty mold and few cast skins; 4=more than 800 aphids per plant, plant stunted, leaves severely curled and yellow, covered with sooty mold and cast skins. A damage index (DI) for each line was calculated by the following formula (Mensah et al. 2005): DI=(scale value×no. of plants in the category)/(4×total no. of plants)×100. The DI ranges between 0 for no infestation and 100 for the most severe damage. The DI was used as an indicator of aphid resistance and was applied in the following analysis. DNA extraction and marker analysis
At least ten plants for each line (F3:4 generation) and their parents were grown in the greenhouse for DNA extraction in 2007. The non-expanded trifoliates from each line were bulk-harvested for isolating the genomic DNA. The DNA was extracted with the CTAB (hexadecyltrimethyl ammonium bromide) method as described by Kisha et al. (1997) and the concentration was determined with a ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Del.). The PCR was performed using the genomic DNA with SSR markers as described by Cregan and Quigley (1997) and run on a MJ TetradTM thermal cycler (MJ Research, Waltham, Mass.). The PCR products were separated on 6% non-denaturing polyacrylamide gels using an electrophoresis unit DASG-400-50 (C.B.S. Scientific Co., Del Mar, Calif.) as described by Wang et al. (2003). Gels were stained with ethidium bromide, visualized under UV light, and photographed. A total of 1,056 SSR markers were screened for the parental polymorphism, of which 329 markers showed polymorphism. Based on the soybean consensus map (Song et al. 2004); 10 these polymorphic markers covered most of the genomic regions. The SSR primer sequences were provided by Dr. Perry Cregan at USDA-ARS, Beltsville, Md. In order to accelerate the identification of genomic regions for aphid resistance, markers at an approximate distance of 20 cM were used to Wrst genotype a subset of 94 lines, which were randomly selected from the whole population. The markers in regions potentially associated with aphid resistance were genotyped on the remaining lines and these regions were further saturated with more markers.
Statistical and QTL Analysis
The DI data from the Field and greenhouse trials were analyzed separately since their experimental designs and inoculums differed. Analysis of variance (ANOVA) was performed for the greenhouse data using the GLM procedure of SAS (1999). The broad sense heritability of DI in the greenhouse trial was calculated based on entry means according to Fehr (1987). Pearson correlation for the aphid resistance between trials was calculated with the CORR procedure of SAS (1999). Linkage map was constructed with the Kosambi function and a LOD score of 3 or lower (to force some distantly located markers to be linked) using Map Manager QTXb20 (Manly et al. 2001). The maps were drawn using MapChart (Voorrips 2002). Assignment of linkage groups to the specific linkage groups was based on the soybean consensus map (Song et al. 2004). Composite interval mapping (CIM) was performed to detect aphid resistance QTLs using QTL Cartographer V2.5 with the standard model Zmapqtl 6 (Wang et al. 2008). Entry means were used in the analysis for the greenhouse trial data. The CIM analysis uses markers other than the interval being tested as cofactors to control the genetic background (Zeng 1994). The forward and backward regression method was used to select markers as cofactors. The walking speed chosen for CIM was 2 cM. The empirical LOD threshold at 5% probability level was determined by a 1,000-permutation test (Churchill and Doerge 1994). The QTL X QTL interaction was further determined using the multiple interval mapping (MIM) method of QTL Cartographer. The whole genome scan was conducted on the subset of 94 lines.
QTL validation A population of 51 F3-derived lines was developed by single-seed descent from a cross between PI 567541B and E00003, where E00003 is an elite advanced breeding line and is susceptible to the soybean aphid. This population and another 50 advanced breeding lines were used for the QTL validation. The 50 advanced breeding lines (F4 generation in the 2007 Field trial and F5 in the 2008 greenhouse trial) pre-selected for agronomic traits were derived from five different crosses, where the male parent is PI 567541B or F1 progeny derived from PI 567541B, and the female parent is an aphid-susceptible cultivar or breeding line (Table 12). The same type of trials conducted for the mapping population were performed for the validation population and the advanced breeding lines in the summer of 2007 and spring of 2008. However, there was no replication in the greenhouse trial and the aphid resistance was only rated 4 weeks after inoculation in the Field trial. The DNA was extracted with a quick-extraction method (Bell-Johnson et al. 1998). Several markers in the QTL associated regions were genotyped for the validation population. Linkage maps were constructed and QTL analysis was performed in the same way as in the mapping population. For the advanced breeding lines, only markers closely linked to the identified QTLs were genotyped and their allele effects were calculated and compared using at test (P=0.05).
Phenotypic analysis The phenotypic values of the mapping population and its parents are summarized in Table 13. In the Field cage, susceptible parent Skylla was severely damaged by the aphid infestation while resistant parent PI 567541B had relatively lower DI than Skylla for both 3- and 4-week ratings.







TABLE 12







Crosses used for deriving the 50 advanced breeding lines










CrossIDa
Male
Female
Line no.













050016
PI567541B
E00003
2


050023
(PI) 567541B X SDx00R-
E00003
9



39-42) F1


050027
PI567541B
E01260
7


050098
PI567541B
SDx00R-39-42
9


050105
PI567541B
Skylla
23





aCrosses 050016 and 050105 have the same parents as in the mapping and validation population, but they were made independently in a different year






Thus, in one embodiment, the two QTLs, LG F and LG M, identified and validated herein are contemplated for use in improving soybean aphid resistance by marker-assisted selection for these QTLs. In another embodiment, the marker-assisted selection for identifying the presence of aphid resistant germplasm in LG F and LG M








TABLE 13A







Phenotypic summery of PI 567541B, Skylla, and 228 recombinant


inbred lines (RILs) for soybean aphid resistance investigated in the


field cage in summer 2007 and in the greenhouse in spring 2008










Parentsa
RILs population













Trials
PI567541B
Skylla
Mean
Range
SE
H2b





Field Cage








3-week rating
2.4a
3.8b
3.3
1.3
4.0
—


4-week rating
3.0a
4.0b
3.7
2.0
4.0
—


Greenhouse


3-week rating
1.0a
2.3b
1.8
0.7
3.2
0.90


4-week rating
1.0a
3.3b
2.2
0.9
3.5
0.94





aMeans followed different letters within the same row are significantly different at P < 0.05


bBroad sense heritability. Unavailable heritability is marked with ‘—’













TABLE 13B







Phenotypic summery of PI 567541B, Skylla, and 228 recombinant


inbred lines (RILs) for soybean aphid resistance investigated in the


field cage in summer 2007 and in the greenhouse in spring 2008










Parentsa
RILs population













Trials
PI567541B
Skylla
Mean
Range
SE
H2b
















Field Cage








3-week
60.0
95.8
82.2
32.5-100.0
16.3
—


rating


4-week
75.0
100.0
91.4
50.0-100.0
13.2
—


rating


Greenhouse


3-week
26.8a
56.1b
45.9
18.5-77.9
17.5
0.89


rating


4-week
25.0a
81.3b
55.7
22.3-87.9
24.1
0.93


rating





aMeans followed different letters within the same row are significantly different at P < 0.05


bBroad sense heritability. Unavailable heritability is marked with ‘—’






PI 567541B in the greenhouse trial had a significantly (P<0.05) lower DI than Skylla. Highly significant variation (P<0.0001) was observed among the population lines for both 3- and 4-week ratings in the greenhouse trial. The aphid infestation in the Field cage was generally more severe than in the greenhouse, which might be because the Field environment was more favorable for the aphid development. However, correlation coefficients between the Field and greenhouse data were significant (0.68 and 0.66 for the 3- and 4-week ratings, respectively, P<0.0001). The frequency distributions of the Field DI were continuous, but not normal and skewed to the susceptible parent (FIG. 13 Aa, Ab), indicating that more than one recessive gene might control the aphid resistance. However, the frequency distributions of the greenhouse DI appeared more bimodal with a ratio of 1:1 (FIG. 13 Ac, Ad). Additionally, the broad sense heritability for the greenhouse DI was high (0.89 and 0.93 for the 3- and 4-week ratings, respectively) (Table 13). These might indicate that only one gene controls the aphid resistance in the greenhouse trial.

QTL Mapping Using CIM.

A total of 123 SSR markers, which distribute throughout the soybean genome based on the consensus map (Song et al. 2004), were genotyped on the subset of 94 lines. These markers generated 131 loci, of which 124 loci were mapped into 25 linkage groups that were segments of the 20 linkage groups on the consensus map. The linkage map spanned 1,703 cM with an average interval length of 13.7 cM. This map was used to conduct the whole genome scan to identify aphid resistance QTLs with the subset of 94 lines. Two QTLs were detected, which were located on LGs F and M (Table 14). The PI 567541B allele conferred aphid resistance at both loci. The QTL on LG M was consistently detected for both 3- and 4-week ratings in each trial and explained a large portion of phenotypic variations ranging from 43.6 to 85.2%. The QTL on LG F was only associated with the 3-week rating in the Field trial and had much less effect (explained 9.1% of the phenotypic variation) than the one on LG M. However, using the whole population of 228 lines, these two QTLs were both consistently detected for both 3- and 4-week ratings in each trial (Table 14, FIG. 14). The QTL on LG M was closely linked to marker Satt299 or Satt435, which was only 3.6 cM away from Satt299. Its peak position was located at Satt299 or 2 cM below in most cases, but it shifted about 10 cM above for the 4-week rating in the Field trial, which might be due to the limited marker saturation in the region above Satt229. However, the genomic region around Satt299 could be conservatively declared as a major QTL region. The QTL on LG F was closely linked to marker Satt649 or Satt343, which was only 1.8 cM away from Satt649. Its peak position was located at Satt649 or 2 cM below. Although the QTL on LG F was significant in the greenhouse trial, it explained very little phenotypic variation (1.5 and 0.9% for the 3- and 4-week ratings, respectively) (Table 14). QTL mapping using MIM







TABLE 14A







Summery of QTLs for soybean aphid resistance detected in the mapping


population PI 56754IB x Skylla using the composite interval mapping method












Peak

94 RILsd
228 RILs
















Trials
LGa
Pos.b
Flanking markersc
LOD
R2e
af
LOD
R2
a



















Field Cage











3-week
M
33.4
Satt299
14.8
40.9
0.47
29.7
43.8
0.43


rating
F
5.1
Satt649
 4.9
10.3
0.24
8.2
9.5
0.14


4-week
M
23.5
Satt150-Satt435
25.9
77.6
0.54
34.1
79.0
0.51


rating
F
5.1
Satt649
—
—
—
5.5
6.7
0.20


Greenhouse


3-week
M
35.4
Satt299-
33.2
81.7
0.65
62.0
77.2
0.61


rating


Sat_244


4-week
F
3.3
Satt343
—
—
—
2.8
1.7
0.10


rating
M
35.4
Satt299-
33.2
83.2
0.73
64.1
79.6
0.70





Sat_244





aLinkage group


bQTL Peak position is expressed in cM and based on the analysis from the whole population (228 lines)


cMarkers flanking the peak position or the marker at the peak position based on the analysis from the whole population (228 lines)


dA subset of 94 recombinant inbred lines. QTL not significant in the subset is marked with ‘—’


eR2, percentage of phenotypic variation explained by QTL


fAdditive effect. The positive value implies that PI 567541B allele decrease the phenotypic value.













TABLE 14B







Summery of QTLs for soybean aphid resistance detected in the mapping


population PI 567541B x Skylla using the composite interval


mapping method with additional markers.












Peak

94 RILsd
228 RILs



Pos.b

94 RILsd
228 RILs
















Trials
LGa
Pos.b
Flanking markersc
LOD
R2e
af
LOD
R2
a



















Field Cage
M
33.4
Satt299
15.4
43.6
12.4
30.4
44.7
10.9


Trials
F
5.1
Satt649
 4.4
 9.1
 5.6
8.0
9.2
4.9



M
23.5
Satt150-Satt435
26.1
76.8
13.4
35.0
79.6
12.8



F
5.1
Satt649
—
—
—
5.2
6.3
3.3


Greenhouse
M
35.4
Satt299-Sat_244
36.2
82.9
16.3
76.2
83.2
16.1



F
7.1
Satt649-Satt348
—
—
—
2.1
1.5
2.1


4-
M
35.4
Satt299-Sat_244
40.8
85.2
22.4
87.9
87.7
22.5


week
F
7.1
Satt649-Satt348
—
—
—
1.8
0.9
2.3


rating





aLinkage group


bQTL Peak position is expressed in cM and based on the analysis from the whole population (228 lines)


cMarkers flanking the peak position or the marker at the peak position based on the analysis from the whole population (228 lines)


dA subset of 94 lines. The LOD threshold for this subset of lines is 2.8. The LOD threshold for the whole population (228 lines) is 1.8. QTL not significant in the subset is marked with ‘—’


eR2, percentage of phenotypic variation explained by QTL


fAdditive effect. The positive value implies that PI 567541B allele decrease the phenotypic value.






The data were also subjected to MIM analysis and the MIM results using the whole mapping population are presented in Table 15. The two QTLs identified with the CIM method were also found using the MIM method. Additionally, a significant additive £ additive interaction between these two QTLs was detected using the MIM method. For the 3-week rating in the Field trial, the LOD score of the QTL interaction was 10.3 and it explained 6.5% of the phenotypic variation. The two QTLs combined with their interaction explained 67.4% of the phenotypic variation. For the 4-week rating in the 15 Field trial, the two QTLs combined with their interaction explained 87.2% of the phenotypic variation, of which the interaction explained 24.7%. The QTL position on LG M was refined to 38.4 cM. For the 3-week rating in the greenhouse trial, the two QTLs combined with their interaction explained 85.6% of the phenotypic variation, of which the major QTL on LG M explained 83.8% of the phenotypic variation while the QTL on LG F and the interaction explained a very small portion (1.6 and 0.2%).
For the 4-week rating in the greenhouse trial, both QTLs were detected, but their interaction was not significant. These two QTLs explained 88.7% of the phenotypic variation, of which the major QTL on LG M explained 88.0%. Given the broad sense heritability of 0.89 and 0.93 for the 3- and 4-week ratings in the greenhouse trial, the major QTL on LG M accounted for 94.2 and 94.6% of the genetic variation, respectively.







TABLE 15A







Summery of QTLs and interactions for soybean aphid resistance


detected in the whole mapping population PI 567541B x Skylla and validation


population PI 567541B x E00003 using the multiple interval mapping method.










Flanking
Genetic effect














Population
Trials
LGa
Peak Pos.b
markersc
LOD
R2d
ae

















PI
Field Cage








567541B X
3-week
M
33.5
Satt299-
38.8
49.6
0.47


Skylla
rating


Sat_244




F
8.1
Satt649-
12.5
10.9
0.23






Satt348




Interaction


10.4
6.5
0.21




Total



67.0



4-weak
M
33.5
Satt299-
38.5
46.1
0.37



rating


Sat_244




F
10.1
Satt649-
13.9
14.1
0.21






Satt348




Interaction


12.9
11.0
0.20




Total



71.2



Greenhouse



3-week
M
35.4
Satt299-
82.1
85.3
0.66



rating


Sat_244




F
6.1
Satt649-
5.4
1.4
0.10






Satt348




Interaction


1.8
0.2
0.06




Total



86.9



4-weak
M
35.4
Satt299-
95.7
89.4
0.92



rating


Sat_244


PI
Field Cage


567541B
4-week
M
17.4
Satt435-
3.0
18.6
0.19


X E00003
rating


Satt245




F
1.0
Satt343
2.4
14.8
0.17




Interaction


2.1
14.6
0.16




Total



48.0



Greenhouse



3-week
M
20.4
Satt435-
4.9
43.4
0.40



rating


Satt245



4-weak
M
13.0
Satt150-
14.4
81.4
0.68



rating


Satt299





aLinkage group


bQTL Peak position is expressed in cM


cMarkers flanking the peak position or the marker at the peak position


dR2, percentage of phenotypic variation explained by QTL


eAdditive effect. The positive value implies that PI 567541B allele decrease the phenotypic value.













TABLE 15B







Summary of QTLs and their interactions for soybean aphid resistance


detected in the whole mapping population derived from PI 567541B X


Skylla and in the validation population derived from PI 567541B X


E00003 using the multiple interval mapping method.










Flanking
Genetic effect














Population
Trials
LGa
Peak pos.b
markersc
LODd
R2e
af

















PI
Field cage








567541B X


Skylla
3-week
M
33.4
Satt299
39.6
50.3
12.0



rating




F
8.1
Satt649-
12.2
10.6
5.7






Satt348




Interaction


10.3
6.5
5.3




Total



67.4



4-week
M
38.4
Satt299-
43.8
36.7
7.5



rating


Sat_244




F
10.1
Satt649-
22.2
25.8
6.4






Satt348




Interaction


21.3
24.7
6.4




Total



87.2



Greenhouse



3-week
M
35.4
Satt299-
78.2
83.8
16.2



rating


Sat_244




F
5.2
Satt649-
5.7
1.6
2.6






Satt348




Interaction


1.8
0.2
1.4




Total



85.6



4-week
M
35.4
Satt299-
91.8
88.0
22.6



rating


Sat_244




F
6.1
Satt649-
3.8
0.7
2.6






Satt348


PI
Field cage


567541B X


E00003
4-week
M
8.1
Satt150-
21.2
30.9
6.8



rating


Satt299




F
4.1
Satt343
21.5
29.5
7.0




Interaction


20.4
34.7
6.8




Total



95.2



Greenhouse



3-week
M
20.4
Satt435-
4.9
43.7
9.9



rating


Satt245



4-week
M
13.0
Satt150-
14.4
81.4
17.0



rating


Satt299





aLinkage group


bQTL peak position is expressed in cM


cMarkers Xanking the peak position or the marker at the peak position


dUsing the same LOD threshold as in the composite interval mapping method. The LOD threshold for the mapping population is 1.8. The LOD threshold for the validation population is 1.7


ePercentage of phenotypic variation explained by a QTL


fAdditive effect. The positive value implies that the PI 567541B allele decreases the phenotypic value






QTL Validation.

Four and five markers around the QTLs identified on LGs F and M using the mapping population were genotyped on the validation population, respectively. The linkage maps for this validation population were similar as the mapping population except that the orders of a few tightly linked markers were switched (FIG. 14), which might be due to the small size of the validation population. Using both CIM and MIM methods, two QTLs were detected at similar regions as in the mapping population (Tables 15, 16; FIG. 14). Both QTLs were detected in the Field trial while the major QTL on LG M was significant in the greenhouse trial. The interaction between these two QTLs was also significant in the Field trial (Table 16). The two QTLs combined with their interaction explained 95.2% of the phenotypic variation in the Field trial. Hence, the results from the validation population further confirmed the QTLs identified in the mapping population. The 50 advanced breeding lines were genotyped with the markers closely linked to the QTLs identified in the mapping population, which were Satt299, Satt435, Satt649, and Satt343. Table 17 summarized these allele effects for the aphid resistance. The DI for the breeding lines with the resistant allele (PI 567541B allele) from either Satt435 or Satt299 was significantly (P<0.05) lower than the ones with the corresponding susceptible allele in either Field or greenhouse trials. However, the resistant allele from Satt649 or Satt343 only had effects in the Field trial and for the 3-week rating in the greenhouse trial. Mapping results were consistent, where the QTL on LG F explained the least phenotypic variation for the 4-week rating in the greenhouse trial. Therefore, the allele effects in these advanced breeding lines further validated the QTLs identified in the mapping population. Band pattern analysis of markers linked to QTLs for aphid resistance
Three parental lines, PI 567541B, Skylla, and E00003, together with two other aphid resistance germplasms, Dowling and Jackson, were genotyped using markers Satt299, Satt435, Satt649, and Satt343 that were tightly linked with the QTLs identified in this study. The band patterns of the PCR products from PI 567541B were different from those in Dowling and Jackson for all the four markers (FIG. 15), indicating that PI 567541B is a different resistance source than Dowling and Jackson. However, the band patterns between Dowling and Jackson were the same for each of the four markers. None of the band patterns in the susceptible genotypes Skylla and E00003 were the same as the ones in the three resistant genotypes.







TABLE 16A







Summary of QTLs for soybean aphid resistance detected in the


validation population PI 567541B x E00003 using the


composite interval mapping method.











Peak
Flanking
Genetic effect













Trials
LGa
Pos.b
markersc
LOD
R2d
ae
















Field Cage








4-week rating
M
16.4
Satt435
2.5
16.6
0.18



F
1.0
Satt343
2.3
15.1
0.17


Greenhouse


3-week rating
M
20.4
Satt435-Satt245
5.1
40.1
0.38


4-week rating
M
12.0
Satt150-Satt299
15.6
80.5
0.68





aLinkage group


bQTL Peak position is expressed in cM


cMarkers flanking the peak position or the marker at the peak position


dR2, percentage of phenotypic variation explained by QTL


eAdditive effect. The positive value implies that PI 567541B allele decrease the phenotypic value













TABLE 16B







Effects of alternative alleles at four soybean aphid resistance associated


markers among the 50 advanced breeding lines, which were all derived


from PI 567541B.









Average phenotypic valuec












LGa
Marker
Alleleb
Field cage
GH3WK
GH4WK





M
Satt299
R (26)
2.2a
2.1a
2.1a




S (17)
2.8b
2.6b
3.0b



Satt435
R (33)
2.0a
2.0a
2.0a




S (12)
3.0b
2.8b
3.3b


F
Satt343
R (23)
1.9a
2.1a
2.2a




S (24)
2.8b
2.4b
2.5a



Satt649
R (27)
1.9a
2.1a
2.2a




S (23)
2.8b
2.4b
2.5a





aLinkage group


bAlternate alleles for each marker. R implies allele from resistant parent PI 567541B. S implies susceptible allele from another parent. The number in the parenthesis indicate the number of the lines, which had the allele


cAverage phenotypic values from the lines with same allele. Field cage: four-week rating in the field cage trial. GH3WK: three-week rating in the greenhouse trial. GH4WK: four-week rating in the greenhouse trial. Means from each pair of alleles followed same letter are not significant at P = 0.05.













TABLE 17A







Summary of QTLs for soybean aphid resistance detected in the


validation population PI 567541B X E00003 using the


composite interval mapping method.











Peak

Genetic effect













Trials
LGa
Pos.b
Flanking markersc
R2d
R2d
R2d
















Field cage








4-week rating
M
16.4
Satt435
2.5
16.6
4.6



F
1.0
Satt343
2.3
15.1
4.4


Greenhouse


3-week rating
M
20.4
Satt435-Satt245
5.1
40.0
9.6


4-week rating
M
12.0
Satt150-Satt299
15.6
80.4
17.1
















TABLE 17B







Effects of alternative alleles at four soybean aphid resistance associated


markers among the 50 advanced breeding lines, which were all derived


from PI 567541B.









Average Phenotypic valuec












LGa
Marker
Alleleb
Field cage
GH3WK
GH4WK





M
Satt299
R (26)
54.5a
52.1a
51.4a




S (17)
69.1b
64.7b
75.0b



Satt435
R (33)
49.1a
51.7a
50.3a




S (12)
75.0b
69.0b
82.2b


F
Satt649
R (27)
46.8a
52.7a
55.3a




S (23)
68.8b
59.7b
64.2a



Satt343
R (23)
46.8a
52.1a
55.7a




S (24)
67.3b
59.7b
63.3a





aLinkage group


bAlternate alleles for each marker. R implies allele from resistant parent PI 567541B. S implies susceptible allele from another parent. The number in the parentheses indicates the number of the lines that had the allele


cAverage soybean aphid damage index for the lines with the same allele. Field cage: 4-week rating in the Weld cage trial. GH3WK: 3-week rating in the greenhouse trial. GH4WK: 4-week rating in the greenhouse trial. Means from each pair of alleles followed by the same letter are not significant at P = 0.05






Example 12 
This example shows the linkage group makers discovered by the inventors during the development of the present inventions. for identifying the aphid resistant germplasm from each founder aphid resistant cultivar. These linkage groups and markers were used in some embodiments and contemplated for use in other embodiments in compositions and methods of the present inventions.







TABLE 18







Markers linked to aphid resistance genes in soybean plant cultivars


PI 567541B.












Linkage






Group on
Position on
P value in
P value in



consensus map
consensus map
single
composite



by Song et al.
by Song et al.
marker
interval


Marker name
2004*.
2004. cm
analysis
mapping














Satt343
F
 3.04 cm
<0.001
<0.0001


Satt193
F
 3.42 cm
<0.004
<0.0001


Satt649
F
 5.36 cm
<0.0001
<0.0001


Satt150
M
18.58 cm
<0.002
<0.001


Satt435
M
38.94 cm
<0.0001
<0.0001


Satt299
M
42.23** cm 
<0.0001
<0.0001





*Song, et al., (2004) Theor Appl Genet 109: 122-128, herein incorporated by reference.


**Estimated position on consensus map by Song et al. 2004, supra.













TABLE 19







Markers linked to aphid resistance genes in soybean plant cultivars


PI 567543C.












Linkage






Group on
Position on
P value in
P value in



consensus map
consensus map
single
composite



by Song et al.
by Song et al.
marker
interval


Marker name
2004.
2004. cm
analysis
mapping





Sat_339
J
27.97 cm
<0.0001
<0.0001


Satt414
J
37.04 cm
<0.0001
<0.0001


Satt654
J
 38.1 cm
<0.0001
<0.0001


Satt596
J
39.64 cm
<0.0001
<0.0001


Satt686
J
 40.5 cm
<0.0001
<0.0001


Satt622
J
42.25 cm
<0.0001
<0.0001





*Song, et al., (2004) Theor Appl Genet 109: 122-128, herein incorporated by reference.


**Estimated position on consensus map by Song et al. 2004, supra.













TABLE 20







Markers linked to aphid resistance genes in soybean plant cultivars


PI 567597C.












Linkage






Group on
Position on
P value in
P value in



consensus map
consensus map
single
composite



by Song et al.
by Song et al.
marker
interval


Marker name
2004.
2004. cm
analysis
mapping





Satt380
J
38.70 cm
<0.0001
na


Satt596
J
39.64 cm
<0.0001
<0.0001


Satt456
J
40.83 cm
<0.0001
<0.0001


Satt529
J
41.90 cm
<0.0001
<0.003 


Satt183
J
42.51 cm
<0.0001
<0.0001


Satt215
J
44.08 cm
<0.0001
na


Sat_366
J
52.84 cm
<0.0001
na


Sat_350
J
55.73 cm
<0.0001
na
















TABLE 21







Markers linked to aphid resistance genes in soybean plant cultivars


PI 567598B.












Linkage






Group on
Position on
P value in
P value in



consensus map
consensus map
single
composite



by Song et al.
by Song et al.
marker
interval


Marker name
2004.
2004. cm
analysis
mapping














Sct_046
J
 23.8 cm
<0.005
na


Sat_228
J
 24.1 cm
0.159
na


Satt285
J
25.51 cm
<0.0001
<0.003


Satt414
J
37.04 cm
<0.006
na


Set 065
J
39.81 cm
<0.001
na


Satt406
J
41.05 cm
<0.0001
na


Satt596
J
42.59 cm
<0.0001
<0.003


Satt529
J
44.59 cm
<0.0001
 <0.0001


Satt380
J
45.41 cm
<0.0001
<0.042


Sat_255
J
46.03 cm
<0.0001
na


Satt215
J
46.43 cm
<0.0001
na


Satt280
J
49.14 cm
<0.0001
<0.004


Sat_366
J
55.73 cm
<0.0001
na


Satt540
M
32.17 cm
<0.0001
na


Satt567
M
33.47 cm
<0.003
<0.037


Satt435
M
38.94 cm
<0.006
<0.041


satt463
M
47.45 cm
<0.0001
na





*Song, et al., (2004) Theor Appl Genet 109: 122-128, herein incorporated by reference.


**Estimated position on consensus map by Song et al. 2004, supra.













TABLE 22







Sequence of PCR Primers for Markers linked to 


aphid resistance genes in PI 567541B.











Marker
Linkage
Forward 
Reverse 
SEQ ID


name*
Group*
primer (F)*
primer (R)*
NOs: F-R





Satt343
F
CATGGCGGAAA
TCCCAATTCACC
SEQ ID




GCGAAACA
TCTTCA
NOs:






17-18


 


Satt193
F
GCGTTTCGATA
TGTTCGCATTAT
SEQ ID




AAAATGTTACA
TGATCAAAAAT
NOs:




CCTC

19-20


 


Satt649
F
TTACTGGCCGTG
GCGGACGTTAT
SEQ ID




TTTACCCGTGTA
AAGATTTTTTTA
NOs:




A
TCATG
21-22


 


Satt150
M
AAGCTTGAGGT
TGCCATCAGGTT
SEQ ID




TATTCGAAAAT
GTGTAAGTGT
NOs:




GAC

23-24


 


Satt435
M
GCGGTGAAACG
GCGTTGGATTA
SEQ ID




GCTCTCTTTGAT
ATTAATTAAATT
NOs:




AGTGA
ATTTT
25-26


 


Satt299
M
GCGACAAGGCA
GCGCTACCCAT
SEQ ID


Sat_299

CTCACATCTCTT
AACAAAAAGTT
NOs:




CTC
CAAATC
27-28





*Grant et al., (2008) SoyBase, The USDA-ARS Soybean Genome Database. (soybase.agron.iastate.edu.) Cited Oct. 30, 2008; Cregan, et al., Crop Sci 39: 1464-1490 (1999), (soybase.org/resources/ssr.php#misc); all of which is herein incorporated by reference in their entirety.


crop.scijournals.org/cgi/content/full/39/5/1464?ijkey=3943de8a8b441cf631d216d7f70b4497e2c65ec3.













TABLE 23





Sequence of PCR Primers for Markers linked to 


aphid resistance genes in PI 567543C.



















Sat_339
J
GCGTTCATCGAAATG
GCGACTGATATGCACC
SEQ ID




CGTTTAGGATA
TCTAAGTCTCAA
NOs: 






29-30


 


Satt414
J
GCGTATTCCTAGTCA
GCGTCATAATAATGCC
SEQ ID




CATGCTATTTCA
TAGAACATAAA
NOs: 






31-32


 


Satt654
J
AGACGCGCACACAAG
GCTGGGACTCCTCATG
SEQ ID




CATATA
TC
NOs:






33-34


 


Satt596
J
TCCCTTCGTCCACCAA
CCGTCGATTCCGTACA
SEQ ID




AT
A
NOs: 






35-36


Satt686
J
ACGGAAAATAAATGA
GCGCT AT C AG AT 
SEQ ID




AACTAAGA
AG AGAA GCAGAAGAAT
NOs: 






37-38


 


Satt622
J
GCGGTGTAGGTAATA
GCGGTGT AGGTTTC
SEQ ID




ATTTTAATTCTCAT
AC AC TTCATTCAC
NOs: 






39-40





*Grant et al., (2008) SoyBase, The USDA-ARS Soybean Genome Database. (soybase.agron.iastate.edu.) Cited Oct. 30, 2008; Cregan, et al., Crop Sci 39: 1464-1490 (1999), (soybase.org/resources/ssr.php#misc); all of which is herein incorporated by reference in their entirety.













TABLE 24





Sequence of PCR Primers for Markers linked to


aphid resistance genes in PI 567597C.



















Satt380
J
GCGAGTAACGGTCTT
GCGTGCCCTTACTCTC
SEQ ID




CTAACAAGGAAAG
AAAAAAAAA
NOs:






41-42


 


Satt596
J
TCCCTTCGTCCACCAA
CCGTCGATTCCGTACA
SEQ ID




AT
A
NOs:






43-44


 


Satt456
J
GGGCCTTCGTTTGAGT
GGGATCATTGGTTAAT
SEQ ID




TCATAG
TGTTGTAAGA
NOs:






45-46


 


Satt529
J
GCGCATTAAGGCATA
GCACAATGACAATCAC
SEQ ID




AAAAAGGATA
ATACA
NOs:






47-48


 


Satt183
J
TAGGTCCCAGAATTT
CACCAACCAGCACAA
SEQ ID




CATTG
AA
NOs:






49-50


 


Satt215
J
GCGCCTTCTTCTGCTA
CCCATTCAATTGAGAT
SEQ ID




AATCA
CCAAAATTAC
NOs:






51-52


 


Sat_366
J
GCGGCACAAGAACAG
GCGGACATGGTACATC
SEQ ID




AGGAAACTATT
TATATTACGAGTATT
NOs:






53-54


 


Sat_350
J
GCGTAAGAGCATCTC
GCGATTT ATT AC
SEQ ID




CAAACCATCAAACTC
ATTTAACAATTGTTAT
NOs:




A
TTA
55-56





*Grant et al., (2008) SoyBase, The USDA-ARS Soybean Genome Database. (soybase.agron.iastate.edu.) Cited Oct. 30, 2008; Cregan, et al., Crop Sci 39: 1464-1490 (1999), (s0ybase.org/resources/ssr.php#misc); all of which is herein incorporated by reference in their entirety.













TABLE 25





Sequence of PCR Primers for Markers linked to 


aphid resistance genes in PI567598B.



















Sct_046
J
AAAATAACTAAAA
TTGGTCAGATTATTAT
SEQ ID


Satt046

TGTCTTCTCA
AAGATTG
NOs:






57-58


 


Sat_228
J
GCGTGACTACGGG
GCGTTGGCGGTAAGA
SEQ ID




AAGTTGGAAC
GCACTATA
NOs:






59-60


 


Satt285
J
GCGACATATTGCA
GCGGACTAATTCTATT
SEQ ID




TTAAAAACATACT
TTACACCAACAAC
NOs:




T

61-62


 


Satt414
J
GCGTATTCCTAGT
GCGTCATAATAATGC
SEQ ID




CACATGCTATTTC
CTAGAACATAAA
NOs:




A

63-64


 


Sct_065
J
CCCTGTGTTTCCCT
GAAAAGTTTTATGTTC
SEQ ID




CT
TGAGTG
NOs:






65-66


 


Satt406
J
GCGTGAGCATTTT
TGACGGGTTTAATAG
SEQ ID




TGTTT
CAT
NOs:






67-68


 


Satt596
J
TCCCTTCGTCCAC
CCGTCGATTCCGTAC
SEQ ID




CAAAT
AA
NOs:






69-70


 


Satt529
J
GCGCATTAAGGCA
GCACAATGACAATCA
SEQ ID




TAAAAAAGGATA
CATACA
NOs:






71-72


 


Satt380
J
GCGAGTAACGGTC
GCGTGCCCTTACTCTC
SEQ ID




TTCTAACAAGGAA
AAAAAAAAA
NOs:




AG

73-74


 


Sat_255
J
GCGGCATGTCATG
GCGCAACTGAAGCAA
SEQ ID




GTATACGTAACTT
GAAAAGAAACCT
NOs:




TAGA

75-76


 


Satt215
J
GCGTAGCAACAAA
GCGTCCCATTTTATTC
SEQ ID




GCAATCTACAG
CACACTATGTAAT
NOs:






77-78


 


Satt280
J
GGCGGTGGATATG
GGCGGGCTTCAAATA
SEQ ID




AAACTTCAATAAC
ATTACTATAAAACTA
NOs:




TACAA
CGG
79-80


 


Sat_366
J
GCGGCACAAGAAC
GCGGACATGGTACAT
SEQ ID




AGAGGAAACTATT
CTATATTACGAGTATT
NOs:






81-82


 


Satt540
M
CTGGCGAATCAAG
CCGTGATTGCGAAGA
SEQ ID




CTTTGTAAC
GGATATT
NOs:






83-84


 


Satt567
M
GGCTAACCCGCTC
GGGCCATGCACCTGC
SEQ ID




TATGT
TACT
NOs:






85-86


 


Satt435
M
CAAGCTCAAGCCT
TGACCAGAGTCCAAA
SEQ ID




CACACAT
GTTCATC
NOs:






87-88


 


satt463
M
TTGGATCTCATAT
CTGCAAATTTGATGC
SEQ ID




TCAAACTTTCAAG
ACATGTGTCTA
NOs:






89-90





*Grant et al., (2008) SoyBase, The USDA-ARS Soybean Genome Database. (soybase.agron.iastate.edu.) Cited Oct. 30, 2008; Cregan, et al., Crop Sci 39: 1464-1490 (1999), (s0ybase.0rg/res0urces/ssr.php#misc); all of which is herein incorporated by reference in their entirety.






Example 13
This example describes fine-mapping the rag1c gene. Bulk segregant analysis (BSA) and SNP genotyping were used to identify a rag1c region then it was fine-mapped to a smaller genetic interval.
In order to quickly confirm and narrow down the genomic intervals that were associated with soybean aphid resistance in the soybean mapping populations, the BSA approach was applied. Genomic DNA from 10 aphid resistant lines showing high levels of aphid resistance were pooled to form the resistant pool while DNA from 10 extremely aphid susceptible lines were pooled to form the susceptible DNA pool. These two pools were then subjected to the SNP genotyping using the Illumina® iSelect HD BeadChip (illumine.com) that contains over 52,000 soybean SNP markers (Song et al., 2011, a-c-s.confex.com/crops/2011am/webprogram/Paper65642.html). The genotyping approach was conducted on the Illumina iScan platform at Michigan State University (MSU, East Lansing, Mich., USA). Each DNA sample required not less than 200 ng genomic DNA in a 4 uL volume. Intensities of the beads fluorescence were detected using an Illumina iScan Reader, and genotypes were called using Illumina's BeadStudio software (Illumina, San Diego, Calif., v3.2.23) following the company's standard protocol. The polymorphic SNPs between bulks and individual lines to form the bulks were selected. These target SNPs were subjected to the Sanger based re-sequencing at MSU core facility to validate the SNPs. The allele specific array (probe plus primers) were designed and sent to be synthesized from the Applied Biosystems (ABI, Foster City, Calif., USA) and Kapsar® (Kbioscence.co.uk) for mapping and fine-mapping projects.
The BSA approach defined a target region in the soybean chromosome 7 encompassing 1.4 mbp (5152231-6531559 bp) and a total 143 SNPs were identified in this region and 30 in-silico candidate resistance genes annotated within the interval (FIG. 19: Table 1). Out of 143 SNPs, 17 high quality SNPs were polymorphic between two bulks and homozygous parental lines (FIG. 19: Table 1). These thirty candidate genes were selected based on their function characteristics. Significant numbers of transcript abundances (TAs) were obtained from 26 genes in the soybean genome using oiligos based on sequence information of these genes (FIG. 20: Table 2). These 26 genes were functionally annotated because they were likely to be associated with aphid resistance. The orthologous genes in the BSA defined region showed similar function conservation and displayed level of colinearity between the soybean, rice and Arabidopsis. The nearly 1 to 1 ratio of the orthologous function relationship was found among those three species, indicating that these gene groups were descended from a common ancestor and corresponded to well-conserved functions. Nine of the 26 candidate genes were transcription factors.
Eight candidate genes were predicted as NBS-LRR or LRR disease resistance genes. The NBS-LRR genes were categorized into CC- and TIR-NBS-LRR protein families, respectively based on the presence of signature domains/motifs of a particular protein family as provided in Phytozome (www.phytozome.net). Six genes belonged to protein kinase families. Remaining genes possessed various functions that should potentially related to disease resistance directly or indirectly (FIG. 20: Table 2). These candidate genes were named following the same manner as that of Phytozome.
Fine-Mapping the Rag1c Interval.
Two recombinant inbred line (RIL) populations derived from crosses of soybean aphid resistant variety PI567541B (Resistant)×E00003 (Susceptible) lines and E07906-2 (Resistant)×Skylla (Susceptible) were selected from the previous existing populations (F4:8 and F2:6, respectively by the single seed decent method. Combining Illumina® 6K HD beadchip (illumine.com) with the SNP markers identified in the BSA, the QTL in rag1c interval was remapped between SNP markers MSUSNP7-5 and MSUSNP7-15 and explained 19.9% phenotypic variation with −0.58 additive effect (FIG. 23). Among 425 RILs, total eight RHL populations were selected based on the segregation of soybean aphid resistance. These RHLs (15-50 lines/population) were genotyped with both TaqMan® and Kaspar® SNP molecular markers flanked the rag1c locus using the end-point detection system with some modification on Roche LightCycler® 480 (Roche Diagnostics, Indianapolis, Ind.). The genotypes were analyzed using the Roche Gene Scanning software version 1.5.0 (Roche Diagnostics, Mannheim, Germany). Among them, RHL 050016-383-6 has 4 sub-populations and total 24 recombinants surround the rag1c interval were identified (FIG. 20: Table 3) near SNP marker SNP7-2. These RHLs were selected for further aphid resistance testing based on the presence of unique recombination events in the rag1c region. However, no phenotypic variation was observed due to the recombination events, suggesting that the rag1c gene is positioned leftward (upstream) the SNP.
Surprisingly the results of genotyping for the remaining RHLs using these SNP markers suggest that rag1c was anchored within a 231548 bp interval between Kaspar® SNP MSUSNP7-19 (5650536) and MSUSNP7-10 (5882084) interval (FIG. 20: Table 3). The left most boundary of the gene was defined through genotyping of soybean plant line RHL 070070-5-3-3 (F7:8) using SNP marker MSUSNP7-19 and the right most border that was marked by SNP marker MSUSNP7-10 in RHL 070070-613-3 (F7:8). These two lines displayed the opposite phenotypes for the aphid resistance due to the recombination recombinant events indicating that rag1c should be located between these two SNP markers. SNP marker MSUSNP7-18 (5762798) was found to be positioned in the middle of the genomic interval flanked by MSUSNP7-19 and MSUSNP7-10 and showed a highly significant Pearson correlation (r=0.96753, P<0.0001, PROC CORR, SAS 9.3) with soybean aphid resistance in the four populations of RHL050016-383-6 (F9:10) soybean lines derived from RIL PI567541B×E00003 (FIG. 20: Table 3).
To further verify whether these recombination events were phenotypically segregated in F2 soybean plant populations, at least 2540 BC3F2 lines developed from crossing E8929 (E00003×(SD×00R-039-42×PI567541B)×E00003 were subjected to the first round of recombinant selection. Overall, 467 lines (BC3F3) of the BC3F2 recombinants were put into the second round of selection. The results showed correlations (0.48-0.70, P<0.0001) between MSUSNP7-18 with soybean aphid phenotypes due to the complex of the genetic background in these populations (FIG. 22: Table 4) but as long as the genotype of the lines was susceptible in the locus, the phenotype was susceptible to soybean aphis suggesting that there is a aphid resistance gene underlain molecular marker MSUSNP7-18.
After annotated with the soybean Phytozome, the SNP MSUSNP7-18 was anchored in the first intron of a NB-ARC gene, Glyma07g07110 based on the reference sequence information of soybean Williams 82 (www.phytozome.net/soybean). Total genomic sequence of the gene is about 19575 bps. It had three isoforms and possessed about 7827 bp transcript and 7389 coding sequences. The NB-ARC gene belongs to the coiled-coil (CC) NBS-LRR disease resistance gene family. Several orthologs of the gene exist in planta and shares a high similarity with a soybean rust resistance gene, Rpp4 (Meyer et al., 2009) and a disease resistance candidate gene in Medicago truncatula, MTR—7g037880.
Note that genomic positions of the SNPs in the Illumina beadchip platform were based on the genome position of Williams 82. The physical positions of the SNP markers described herein are consistent with their genetic positions in the map of the rag1c in these mapping lines when compared to the Williams 82 soybean line suggesting that there is no large scale sequence alteration that occurred between the two genomes within this region.
Expression of Rag1c.
Because there were differences in transcript abundances (TAs) between a soybean aphid resistant variety and a susceptible line, RT-PCR was done on the RNA samples extracted from leaf tissues of the plant three weeks after soybean aphid inoculation under greenhouse conditions in order to obtain a direct comparison of gene expression. The resistant varieties used in this study included PI 567541B, E07906-2, PI 567598B and Dowling whereas the susceptible varieties were E00003, Skylla and RR Titan. Leaf tissue was collected and immediately frozen in liquid nitrogen and stored at −80° C. RNA samples were extracted from leaf tissue of susceptible (E00003, Skylla, RR Titan) and resistant (PI 567541B, E07906-2, PI 567598B, Dowling) plants three weeks after soybean aphid infection and non-infection control with three biological replications. RNA was extracted with an RNeasy Plant Mini Kit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. RNA samples were treated with DNase in order to remove any residual DNA using the RNase-free DNase kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. After DNase treatment, RNA was purified on RNeasy mini spin columns (Qiagen, Valencia, Calif.). The quantity of RNA was determined by RiboGreen RNA quantification kit (Molecular Probes, Eugene, Oreg.) to normalize quantity of each RNA sample prior to perform RT-PCR. Reverse transcription was carried out with 1 ug of total RNA with the Retroscript kit at 50° C. for one hour and 92° C. for 14 5 min, following manufacturer's instruction (Ambion, Austin, Tex.). The PCR was performed at MJ Research Tetrad Thermal Cycler (MJ Research, Boston Mass.) and confirmed on Roche Lightcycler 480 with the relative quantification platform (Roche Diagnostics, Indianapolis, Ind.). RT-PCR primers for NB-ARC [NB-ARC-F (5′-ATGTAACCTATCAAGCATCCAGCA-3′; SEQ ID NO:91) and NB-ARC-R (5′-ATTGGATGCTTCACAGTGTTGCT-3′; SEQ ID NO:92)] were designed based on exon 3 and exon 4 sequences, respectively, of the NB-ARC gene. RT-PCR primers for Rag1 [Rag1-F (5′-ATGGGATAGTCACAAGTAATTAAAGA-3′; SEQ ID NO:93) and Rag1-R (5′-ACGAACTAAGAA TGAGGAAAGCT-3′; SEQ ID NO:94)] were designed based on sequence information of Glyma07g06920. The internal standard used to normalize the results was a fragment amplified with primers derived from the soybean tublin4 (Tub4-F: 5′-GGCGTCCACATTCATTGGA-3′; SEQ ID NO:95; Tub4-R: 5′-CCGGTGTACCAATGCAAGAA-3′; SEQ ID NO:96). To distinguish non-specific amplification from target amplicons, melting curve analyses were performed in which the samples were heated from 50° C. to 85° C. at a heating rate of 0.5° C./s. 8 Three biological samples for each time interval per genotype were run in 30 triplicates, including no template controls. In addition, the amplicons amplified from both cDNA and genomic DNA were sequenced and BLASTed against the soybean genome to verify the physical positions of the DNA fragments.
The relative expression levels of the NB-ARC gene in the resistant variety PI 567541B and susceptible variety E00003 were significantly different three weeks after soybean aphid infection based on expression data (FIG. 24). A single amplicon was obtained in the cDNA of resistant genotype PI567541B using the primers specific for Glyma07g07110 gene (FIG. 24A). However, similar amplicon could not be amplified in susceptible genotype E00003 at this stage even if the plant was still relatively healthy. At the meantime, no significant change was observed in an adjacent NB-ARC gene (Glyma07g07150) using primers derived from the neighbor candidate gene (FIG. 24B). The level of transcript abundance (TA) in resistant variety PI 567541B is slightly higher than that of susceptible variety E00003.
Since another fine-mapped soybean aphid resistance gene named Rag1 in soybean variety Dowling (Kim et al., 2010, Glyma07g06920, 5570161-5578474) was 200 kb upstream of Gylymag07110, RT-PCR was used to determine the distance between these genes. RT-PCR using primers derived from the candidate gene for Rag1, Glyma07g06920 were tested on aphid resistant progeny of PI 567541B and Dowling. The results showed that amplicons from these varieties were different further confirming that the resistance gene in PI 567541B was different from the gene in Dowling, which carries Rag1 gene (FIG. 24C). Surprisingly, the amplicon of Dowling was similar to that of the susceptible E00003. For use in comparing loading levels, the TA levels of a reference (control) gene tubulin4 in these samples were similar (FIG. 24C).
In brief, the rapid method to fine-map and identify a soybean aphid resistance gene described herein, for example, rag1c, and the SNP markers derived from the gene in soybean (Glycine max L.). The Kaspar® molecular marker MSUSNP7-18 identified the resistance gene rag1c based on the evidence of the genetic study and preliminary gene expression analysis, rag1c is a NB-ARC (NBS-LRR) gene with 19 kb genomic DNA and 7 Kb coding sequence. The inventors contemplate that the use of this gene has great practical value for use in a soybean breeding program as well as marker assisted-selection of aphid resistant soybean plants.
FIG. 19: Table 1. Polymorphic SNPs and their genome position on soybean chromosome 7 identified by the BSA approach. PI41B (PI567541B) and P1 (E07906-2) were soybean aphid resistant genotypes and P2 (Skylla) was aphid susceptible variety. R represented aphid resistant progenies and S represented aphid susceptible progenies derived from P1×P2.

FIG. 20: Table 2. Selected Candidate genes in the interval identified by the BSA as well as their Arabidopsis and rice orthologs annotated using Phytochrome. Sequence similarities among them were between 15.8-89%. TA: transcript abundance.

FIG. 21: Table 3: Screening recombinants in the RHL fine mapping soybean plant populations using Taqman® and Kaspar® SNPs. Recombination events were highlighted as green color for a susceptible genotype, red as a resistance genotype and yellow as a heterozygote.

FIG. 22: Table 4. Pearson correlation of BC3F3 fine-mapping population3 associating a molecular marker MSUSNP7-18 with an aphid resistant trait.

Exemplary Genotyping Methods of TaqMan® and Kaspar®:

Allele specific arrays (probe plus primers) were designed and sent for synthesized at the Applied Biosystems (ABI, Foster City, Calif., USA) and Kaspar® (Kbioscence.co.uk) for mapping and fine-mapping projects. Based on genotyping information of the Illumina HD beadchip analysis, the target sequences where the SNP anchored were selected in homozygous parental genotypes. The sequences were then subjected to the Customer Taqman® and Kaspar® Assay Design Tools of Applied Biosystems to obtain the allele specific primers and probes (assays). The allele specific Taqman® assays were synthesized by ABI. Taqman® SNP PCR reactions were carried out on 384-well plates with a total volume of 3 uL/well on the LightCycler 480 instrument (Roche Applied Science, Indianapolis, Ind., USA). The PCR reaction mixture for the Taqman® assay consisted of 1-20 ng of genomic DNA, 0.15 uL of 10× Taqman® Assay, and 1.5 uL of 2×ABI Genotyping Master mix containing a modified Taq DNA polymerase, reaction buffer, MgCl2, and dNTPs (ABI, Foster City, Calif., USA). After 10 min pre-incubation at 95° C., 45 PCR cycles were conducted with 10 s of denaturation at 95° C., 30 s of annealing at 60° C., and 10 s extension at 72° C. A final melting cycle for nonspecific amplicon screening was performed by raising the temperature to 95° C. for 10 s, lowering the temperature to 40° C. for 30 s, and increasing the temperature to 83° C. with continuous fluorescent acquisition followed by a cool down to 40° C. on the LightCycler 480. Genotyping analysis was performed using the Roche Applied Science software version 1.5.0.
The allele specific Kaspar® assays were synthesized by Kbioscience genotyping service. Kaspar® SNP PCR reactions were performed on 384-well plates with a total volume of 4 uL/well on the LightCycler 480 instrument. The PCR reaction mixture for the Kaspar® assay contained 5-30 ng of genomic DNA, 2 uL of 2× Kaspar® reaction mix containing a modified Taq DNA polymerase, reaction buffer, MgCl2, and dNTPs (kbioscience.co.uk). After 15 min pre-incubation at 95° C., 12 PCR cycles were conducted with 20 s of denaturation at 95° C., 60 s of annealing at 65° C. for the amplification-1 and 28 PCR cycles were required with 20 s of denaturation at 95° C., 60 s of annealing at 57° C. for the amplification-II. A quantification step was programmed by hold temperature to 37° C. for 5 s with continuous fluorescent acquisition on the LightCycler 480. Genotyping analysis was also analyzed using the Roche Applied Science software version 1.5.0.







TABLE 26







TaqMan ® and Kaspar ® SNP assay sequence 


information for raglc locus on soybean 


chromosome 7. Note that the genomic position  


of SNP is based on the sequenced reference   


genome of Williams 82 in Phytozome  


(phytozome.net/soybean.php). SNPs in 


corresponding resistant/susceptible alleles were


in brackets [ ]. None-target SNPs were masked as


N. Target sequence for TaqMan ® and Kaspar ®


type Position assays











SNP
Pos-
Target sequence for 


Marker
type
ition
TaqMan ® and Kaspar ® assays





MSUSNP7-
_A_G
5152231
GCTGCTGTGACAATGTTTTTTACTCTTC


4 (SEQ


TTTTGGGAGTCAATTAGAAATAGCAG


ID NO:


GGTTGGACGAACACTCTATTTCTGTTT


97)


TGCTAGCCCATTGAAATGT[G/A]GCGA





ACACCTTNTCTGTCCATTTATGCTCAA





CTGAATTTTGTTTCCCAAACTATTCAG





CATTCCAAGTCAAAGCTAGAACCATC





AAGTTGAAACTATTT


 


MSUSNP7-
_T_C
5636973
GATAAGAAAAATTCACTAAGTTCAAA


1 (SEQ


AAGGTCAAAGTGGTTTGAAATCCTAGT


ID NO:


TGCAAAGAGTAAAGGAATGCAGATAG


98)


AACATTTTT[T/C]GGGGAGAGTCACTC





AGTAATATATTGGTCTGTATGGGCCTG





GTTGTGATATCCTTGTTGCAAAGAGGA





AAGGAATGCATCCTTTGTCACAGAAG





AG


 


MSUSNP7-
_C_T
5650536
CTATTTCAAATACTTCATACCATGTCT


19 (SEQ 


ATTCAGTTATTGAACGTTCTTTTAATG


ID NO:


ATTTTGATATTTACAAAATTACTAACA


99)


GTGTACTTTTTGGAGAATG[C/T]TAAT





AAATTCAATTTTAAACAAACAATTAACA





ATTATAATTTAAAGTTATTAATATAAG





AAGGATTACTATATATGTTTCAAACTT





TATAAGGATTATT


 


MSUSNP7-
_C_T
5762798
ACAAGAGCTTTTCTTAGTTTATCTCAC


18 (SEQ


AAGGAAGGTAAGATCCTTAACACCCT


ID NO:


TACCGCAGTTACTTGCCGCCTCAATCC


100)


ATTGCTGAAATGAATGCTCA[C/T]AAA





TAAATATATTTGCAGGCTACGAATCTC





TGCTCCTAGATTTTTGTTTCTTAATTAT





TTAGTCTCTAAATTATATTAAAAGATT





TAATTTGATTTCAA


 


MSUSNP7-
_C_T
5961174
TATACTCAGGTTGTAGTGGCAGACATG


2 (SEQ


CGGAGGATCGTAACCCTAAATATTAG


ID NO:


CATAAAGAATTGATCTGTCTTGCATGA


101)


GACAAACTGATTTGAGTCGANGAAAT





TACTCACAAGAT[T/C]TCCTTTTTTTA





ATCATTTAAAGTAGTTATATGTCTAATA





CTAAACTTAAAATTACTCCTTAGAACA





ATATAATAATTAATT


 


MSUSNP7-
_G_A
5882084
AAACGTCACTCTTTTCATGGACAAGAT


10 (SEQ


TTAATTTAGGTGTTATTTTCCAATTCTC


ID NO:


TAGAAAATAATCAACTTCAATGCATTA


102)


TTTCCTAAATCGGTGTATGAACATGGG





TGATCAGGCTATATAATTAATAACAAT





AAATCATAAAAAGGAACAATAAAATT





GAAATTACATTAAATAGATAGTAAAA





AAGAGTTACATTACAAGAGTTTATCTT





GTCAGACTCTTAACAATGCTATTTAGT





CTCTCATTATTATCAGAGGCTTTACAT





TTTATAAGTTGATAGAGATAGAAGAA





GAAAA[G/A]TGATGGATAAAGNAAGA





GAAGGAAAGAATTTTTATAGAAAAAG





GGTCTCTCGTATTAAAGATGCTCAGAC





TTTGGGTGTATCCAACAGAAAGTTTTG





AGTATCGGTGTATTTTTCTCCTTTGTTT





TCTAATTATTTTATAAGCTTAAGATAG





TTTAAAATTCACGCGAACTCGCGTCAA





ACGCACCCTTTTGGGCTTAGCAGGTAT





AATGACATGTTGAACACGAGATTCAC





GTTAAGTGTGTCTTTATGCTTCTTCAC





GTTAAGTTTATGCTGACTGCTGAG





CGAGTTGACACATTAG


 


MSUSNP7-
_A_G
5900018
CAGAACCAAACTATCAAACAAGAATT


11 (SEQ


CTCTTGGCCAGGAAACAACTTGAAGA


ID NO:


GTTTATTTAATCAATGTCAATCATGGC


103)


AAAGGGTGAGGAATGGCTTGG[A/G]TT





AAATGGGATTTGGTTGCTTCACCCATT





GACATGGGGGGTTGGGGATTAAAAAT





TTAGAAACATTTAACAAAACTTTGTTG





GGGAAATGGATGTGGAG


 


MSUSNP7-
_G_A
6253050
TACCACTACGTAAAAGCCACACTTGTC


15 (SEQ


TAACCCCTGTTACCGTGAAGAGAAAA


ID NO:


TCTGCGTAGCTATTGGAGGTAACTTGG


104)


TAACNTTCTATAAACAGGAT[G/A]GAA





TTTAGAACAAGACATATAATATATAT





GTTTGGTGTATTCACCATGAATATT





CAGAACAAGACACATATTCTGACGTT





GTAGA









Example 14
Fine-Mapping the Rag4 Interval
In order to quickly confirm and narrow down the genomic intervals that were associated with soybean aphid resistance in these soybean-mapping populations, bulk segregant analysis (BSA) was used to identify a rag4 interval. Briefly, genomic DNA was isolated as described for rag1c mapping, for providing a susceptible pool and resistant pool of DNA from the respective plants. These two pools were then subjected to the SNP genotyping using the Illumina® 52K iSelect HD beadchip BeadChip (illumine.com) that contained over 52,000 soybean SNP markers (Song et al., 2011, a-c-s.confex.com/crops/2011 am/webprogram/Paper65642.html). The genotyping approach was conducted on the Illumina iScan platform at Michigan State University (MSU, East Lansing, Mich. (MI)). Each DNA sample required not less than 200 ng genomic DNA in a 4 uL volume. Intensities of the beads fluorescence were detected using the Illumina iScan Reader, and genotypes were called using Illumina's BeadStudio software (Illumina, San Diego, Calif., v3.2.23) following the company's standard protocol. The polymorphic SNPs between bulks and individual lines to form the bulks were selected. These target SNPs were subjected to the Sanger based re-sequencing at MSU core facility to validate the SNPs. The allele specific array (probe plus primers) were designed and sent to be synthesized from the Applied Biosystems (ABI, Foster City, Calif., USA) and Kapsar® (Kbioscence.co.uk) for mapping and fine-mapping projects. The BSA approach defined a target region in the soybean chromosome 13 encompassing 4.9 mbp (4892419 bp) and 21 high quality SNPs were identified (FIG. 25: Table 1).
Methods and Materials for Fine-Mapping Rag4 Gene.
Two recombinant inbred lines (RILs) populations derived from crosses of soybean aphid resistant variety PI567541B (Resistant)×E00003 (Susceptible) lines (050016) and E07906-2 (Resistant)×Skylla (Susceptible) lines (070070) were selected from the previous existing populations (F4:8 and F2:6, respectively Zhang et al. 2009) by the single seed decent method. Combining Illumina® 6K HD beadchip (illumine.com) with the SNP markers identified in the BSA, the QTL in rag4 interval was remapped between SNP markers MSUSNP13-5 (7766353) and ss247923149 (8293174) and explained 17.2% phenotypic variation with −0.46 additive effect (FIG. 1). Among 425 RILs, a total of 11 recombinant events were discovered based on the genotypes and phenotypic segregation within these lines. These RIL were genotyped with both TaqMan® and Kaspar® SNP molecular markers flanked the rag4 locus using the end-point detection system with some modification on Roche LightCycler® 480 (Roche Diagnostics, Indianapolis, Ind.). The genotypes were then analyzed using the Roche Gene Scanning software version 1.5.0 (Roche Diagnostics, Mannheim, Germany).
The results of genotyping for the RILs using these SNP markers suggest that rag4 is anchored within a 162135 bp interval between SNP MSUSNP13-29 (7756558) and MSUSNP13-31 (7918693) interval (Table 2). The left most boundary of the gene was defined through genotyping of lines 070070-27, 52, 69 and 109 (F7:8) using SNP marker MSUSNP13-29 and the right most border that was marked by SNP marker MSUSNP13-31 in line 070070-66 (F7:8) following by SNP marker MSUSNP13-13 (7971079). These valuable lines displayed the opposite phenotypes for the aphid resistance due to the recombination events indicating that rag4 is anchored between SNP markers MSUSNP13-29 and MSUSNP13-31. In left side of the boundaries, SNP marker MSUSNP13-29 (7756558) showed significant Pearson correlation (r=0.89872, P<0.0004, PROC CORR, SAS 9.3) with soybean aphid resistance in the RIL recombinants (F7:8) derived from E07906-2×Skylla (FIG. 25: Table). However, the leftward adjacent SNP marker MSUSNP13-28 (6402610) did not showed any positive correlation with the trait suggesting that the rag4 is located on the rightward of the molecular marker MSUSNP13-29. In the right side of the borders, SNP marker MSUSNP13-31 (7918693) also demonstrated a significant correlation between genotype with the trait (r=0.89872, P<0.0004) but rightward adjacent SNP marker 7935638 (7935638) showed slightly less significance of the correlation (r=0.89066, P<0.0036) indicating that rag4 is anchored between SNP marker MSUSNP13-29 and MSUSNP13-31. The other breakpoints on the further rightward are between SNP marker MSUSNP13-13 (7971079, r=0.89066, P<0.0036) and MSUSNP13-33 (8067378). However, the SNP marker MSUSNP13-33 (r=0.32026, P<0.367) did not show significant correlation between the genotype and soybean aphid resistance as an additional evidence that rag4 gene is in the leftward of the marker (FIG. 26: Table 2). Consequently, the genomic interval containing rag4 is positioned between SNP marker MSUSNP13-29 and MSUSNP13-31 with a 162135 base pair region. The results also indicate that rag4 gene is a partially dominant resistance gene. Note that the Pearson correlation was obtained using lines without rag1c background in susceptible genotypes (070070-17, 329 and 757AR) so that the confound effect can be eliminated due to soybean aphid resistant effect of rag1c in these lines.
To further verify if these recombination events could also results in the phenotypic segregation in F2 populations, total 2540 BC3F2 lines developed from crossing E8928 [E00003×(PI567541B×E00075)]×E00003 with E00003 were subjected to the first round of recombinant selection. Overall, 684 lines (BC3F3) of the BC3F2 recombinants were put into the second round of selection. The results also showed various correlation values between marker MSUSNP13-29 and MSUSNP13-31 with soybean aphid phenotypes due to the complex of the genetic background in these populations. However, if the confound effect of rag1c in the susceptible genotypes at rag4 locus was eliminated, the Pearson correlation value between the genotype and the aphid resistant trait was greatly enhanced.
After annotated with the soybean Phytozome (www.phytozome net/soybean.php), the rag4 interval containing several candidate resistance genes including cytochrome P450 Glyma13g07580 (7774966-7780224), a candidate aphid resistance gene based on the RNA-seq data. The gene is located between MSUSNP13-5 and MSUSNP13-6 with 5259 bp genomic sequence and 1539 bp coding sequence. The function of cytochrome P450 is involved in catalyzing the oxidation of organic substances as well as plant hormones and defensive compounds biosynthetic reactions.
The genomic positions of the SNPs in the Illumina beadchip platform were based on the in-silico position of Williams 82. The physical positions of SNP markers are consistent with their genetic positions in the map of the rag1c compared that of mapping lines described herein and Williams 82 indicating that a large scale sequence alteration did not occur between the two genomes within this region.
FIG. 25: Table 1. Polymorphic SNPs and their genome position on soybean chromosome 13. PI41B (PI567541B) and P1 (E07906-2) were soybean aphid resistant genotypes and P2 (Skylla) was aphid susceptible variety. R represented aphid resistant progenies and S represented aphid susceptible progenies derived from P1×P2.

FIG. 26: Table 2: Recombination events in the RIL lines around rag4 interval. Recombinants were highlighted as green color for a susceptible genotype, red for a resistance genotype and yellow for a heterozygote. Pearson correlation value (r, P>|r|) is in the cells underneath each group of the breakpoints for the recombination. Note that 070070-17, 329 and 757AR were eliminated in the correlation analysis due to the confounded aphid resistant effect from rag1c.








TABLE 27







TaqMan ® and Kaspar ® SNP assay sequence information for rag4


locus on soybean chromosome 13. Note that the genomic position 


of SNP is based on the reference genome of Williams 82 in 


Phytozome(www.phyto zome.net/soybean.php). SNPs 


in corresponding resistant/susceptible alleles were 


in brackets [ ]. None-target SNPs were masked as N.













Target sequence for TaqMan ® 


Marker
SNP type
Position
and Kaspar ® assays





MSUSNP13-5.89
_A_C
 5899475
ACGAGCTTTTTTCTTAGCGAGCACATAT


(SEQ ID NO:


ATTTTGTGTTGCCTCTATAAATGCTTGT


105)


ATCG[A/C]AAATATATTTTGTGTTAATT





AATAGAAACAATATTTATAATGAATGA





TTTAGGATAAGAA


 


MSUSNP13-25
_A_G
 6390360
TTCTGCTTGCAAGACCACCAAGATATC


(SEQ ID NO:


AAGTTTGGACCAAGAAAGATGGCAGCC


106)


CCTGAGGTGGAACGCCTATCATCCACA





TCAGATGCCCAATCAGCATCACAGAAT





GCATAAAAAGCCAAAGGTTCCGCAATA





GAAGCAGACTAAAGA[G/A]GTACCCCA





TGAGACAAGGTACCCTTCAGATATCTC





AAAATCCTTTTAACCACTGTCCAATGA





GAGTCCAGAGGACTAGCCATAAACTGA





CAAACTTTATTGACAGCAAAACAGATT





TCAGGTCTAGTGAGAGTAGCATACTGT





AGAGCA


 


MSUSNP13-28
_T_C
 6402610
GACGTGTGAAGGAAGAGCATAGTGCAT


(SEQ ID NO:


TGTGGTTAGGGATTTTAAGGGTAAATA


107)


AGAGAAAGAAGGTGGTTGTTGAGCCAA





TGGGGGAAGAAGATGCTCAAATTGGTG





AAAAATATTAAGGAGAATTACTCTTTA





AACCAAGATGATTTT[C/T]GAATTTTAT





TCCTCCATGGTCGCACGACTTAGTTTGT





CATTCATTCTTGTGATTGTCTTTCGACT





TTTCTCCAGACAGACCTGATTTGTCGAT





TCTGACCAATTTTATCAGGTTGGTTGAT





TTTTAACCAGCTTTATGTGGGTGAATTG


 


SNPSNP13-29
_A_G
 7756588
TTATTAAGTTTTTGAAGGAAAAACCGC


(SEQ ID NO:


TTCTACGTTATGGCTCAAGTTGAAGAA


108)


ACTCTTCATGATGAAATTAATTTGCAAC





AAGTTATTGTTGAAACGACATCTGTTTG





ACCTCCGTGAAGGAAGTTACGCCTTTG





AAAGATCATCTGG[G/A]CGAGTTAAACT





CTGTCTTGTTGGAGCTTAGTGGCATTGA





TGCCAAATTAGAAGATGTAGATCTTGC





AATGATTTTGTTAGCCTCTCTCCCCCCT





TCATACAAGAATTTTGTTTATTCTCTAA





GTGTTGGAAAGGATTGCATTATGCTTG


 


MSUSNP13-5
_A_G
 7766353
TGGTCAGGACGACATACTTAATACTGT


(SEQ ID NO:


TATTGGGCGACCGAAGCATCCAGGTCA


109)


TGTTCGTGTAGCGGGGTCTAGTATGAT





GATCAGTCNNTATTATGGCA[G/A]TTCC





TCTACATCAATAACCCAACAACAATTG





GCTGACATAATTGGCAGCCTTAAGGAA





GAGTGGAGGAATGAAATTATCAAAAAC





CTCAAGGAAGAAGT


 


MSUSNP13-7
_T_C
 7805648
ATAGAATGGATAACTCAAGTGCTGGTT


SEQ ID NO:


CCATTACCAAATTTCAGTGGTTTTGGTT


110)


GAATATGGAATTCAGTGGAAGTGGTGC





AGAAGAGGGGAGGGAATG[T/C]TTGGG





GTACATGATTNGAAAAAAGGGGAGAAT





GATTAGAAGAGATAGAAAATTGGCAGA





CCAACATTGAAGGATTCATAGGAATGT





TGTGTGTTAGT


 


MSUSNP13-8
_T_G
 7809048
TGATTCTATTGTTTGTAACTTGATAGTT


(SEQ ID NO:


ATGGATTAGCAACTGATAGATAAATCA


111)


TGGTTCTAAGTTGTGAGTAGTTATAGGT





AACTTATCTCGACACCGTTGTTAGTAGG





TTGCATTAATATTTAGATTGGACCCTTT





CGATATAACAA[G/T]GATACAATAAGAT





AAAGAAATAGATAAGTTTCACCTCCTT





TACTCCCATGACCACATCATGACATATT





CAAAGTCAATGGTGAATACACAAATCA





AAGGATTTTTTTTCCAAATATTTATTTG





TGGATGTGAATATACTCATCCTAGAA


 


MSUSNP13-10
_C_T
 7812489
TCAAAGTGTGCTTTATTCGAGGGTGAA


(SEQ ID NO:


CATCGGTCGCCACTATGGGTCTTCGTTG


112)


GTTGCCATCGTGCCATGCTTATCCTACG





CACCCTTGGACCCATTA[T/C]TTGTTTCT





ATTTAGTATACCACATTTGCAAGCTTTA





TTCGCATGACATATATGTTCCAACTTGA





AGCAGTGCGTGAAATATATGATTTGAT





ATG


 


MSUSNP13-11
_C_T
 7842688
TCAAATCGAAGTAAGTGTGTGTAGCGT


(SEQ ID NO:


GGCGCAAATCACGAACACGAATTCATG


113)


ACTGACTGGTTGGTTTCTTCGATCAGAC





ACAAGAAGAGAGGGTCTTGTTTTTCTCT





TCTTCCTCAAGTTTCAGTTCGTCGTCTT





TGGGTTCGGTGA[T/C]ATGGGCTCTGGG





AAAATTTTGGGCCCTCGTTCCAATTTGT





TTATTTTAGGAAAATTAGGGGGCGGGG





AGAGACCAAAAATTAAAAATAAATAA





ATTACATGCATGTTCTTATAAGTAGGTA





AACTTTTTTTATAAATATATAAATAATA


 


MSUSNP13-7.86
_A_G
 7865557
CAAATTTAAAATTCAACTTGTAACAGA


(SEQ ID NO:


AGTTGCAAGGATTGAACTCAAGCCAAG


114)


CCACTT[A/G]GTTGAAAAGGCTATACAT





GATATCATTTCATGAACTAACTATTTCA





AAATTAGAAATATT


 


MSUSNP13-13
_A_G
 7971079
ACTTCTTATTTTTGCTTCATTTTGATGTC


(SEQ ID NO:


GATACTCAAGAATATTATAAAGAAGTC


115)


GTTATATTGCCTAGGACCCACAAAATTT





ACATGGCGTCCTACTTGTCACTTAGAG





ATACATGTCAATCCTCCATGTCAGTCTT





GCTACAGAAGC[G/A]CGTAACCTAACTA





ATATTCAAATGTATTTGAATATCTTATC





CATCGGTAGGTAATTAATTATCTACAA





CGTCACATTATCTACAAGGTACGATTAT





CTTCTACCTTTTATCTATAGGCAAATGT





TTATCTCTAACGTTATCTATAAGCT


 


MSUSNP13-33
_A_G
 8067378
CAATGGACTTTTTGAAAGGATACATTTT


(SEQ ID NO:


TTAGGGTGTTACACATGAGCACGCCAA


116)


ACGGTGAGGTTCCCCTACACATGTTCTT





TTGAACTCTCTTTGATCGCTATTTTTAT





GGAGCACTGGACAAAGTCAGAGGCGA





GATCTTGAGGTCG[A/G]GGCCATCGTCG





GTGTAGAATTGCAACATGTTGCTTCAG





GCGTTGAAACCTCCATCGGGTTTTCGGT





TCGCCGATGCTTGCACAATTCTAGAGA





TGAGTTTTTTTTCATAGATCTAAGTTTT





TTTTAGATCTAAGATTGGTCAGAAAAC





G


 


MSUSNP13-34
_C_T
 8106523
CACACTGGACTCAATCTTCGTATAACAT


(SEQ ID NO:


AAAACTCAACTACAAAATAAGATAACA


117)


TAATTTATCTATATAGTATTATTATGAA





ACAAGAAAAAAATTATCAATGGATTTT





GCTTCTAGGAAATGTCACCAATTATTCC





AAAAATCCAATA[T/C]TTAGGAATCAAA





TATGAAGCAACTAATTGCATGGCAATA





ATCTTTAGGAATTAGGAAGCACATACA





AATTATAGCAACAGCATCATAAAATAC





ATTTTCTGAAACTCGTACTAGATATATC





TTGTGATAAAAAAATATAACACAAATC





T


 


MSUSNP13-1
_A_C
 8264628
GCATGGTGCAACAGCCATTAAGATTTC


(SEQ ID NO:


GGTCATGAGCAATTTTTCTGCAGGGGG


118)


TTTTGAATCAGTTCTCATTTTCTTCCCAT





TTCCGTGGCCAAATAAG[C/A]TTGAAAG





AAACTGCATGACACTGATACCAATCAA





GAGTTGTGTGCAGCAAGCTTACCTCAT





CAATCTCAGGTCAGGAGATGGTTCCAC





TATCTAGGCAC


 


MSUSNP13-2
_A_G
 9483078
TACATTTGGCCCTTACCATGGGGCCATG


(SEQ ID NO:


GTAAAACATATCCTATGATCTCTGGTCA


119)


ACTCTCTCGGCAACTGCATCATGAGAG





ATTTCATGAAGGGATCT[G/A]CAGTCTC





CACACTAGAGGACACTCCCTAGCCACC





AAAGCGGTACGAGCTGGCTACTATTGG





CCAACACTCAGGAAAAATGCCCTCAAC





TTTACCAGGAA


 


MSUSNP13-4
_A_G
11040574
TCTTAAGTCAAGCCTGGCTTGATAGGC


(SEQ ID NO:


CCATCAAGCTGGTCCAAATAGGCCCGA


120)


TTCTATTTGGGCCAATATTTTTTAGTCC





AATTCAACTCAATCTGGT[G/A]GGCTAA





TGGGTCAACNTNACAAGCCCGATCCAT





TTTACTANATCTACATATGAAGAAAGT





ACAAAAGATAATTTTCAATCGTTTGAA





TTTTGAAAGAAT





NOTE:


bases in red [B/B] show alternative bases at that position.






Example 15
This example further describes fine mapping of aphid resistance gene Rag3 in soybean PI 567543C with bi-parental F2 populations.
Phenotype of Soybean Aphid Resistance.
The phenotypes of at least 1889 F2 plants and its subset 376 plants from cross E07048×E10902 were plotted as histograms in FIG. 1. The ratio for the three scores among all F2 plants was 5.06:8.48:4.72, which is approximately 1:2:1. The ratio of the F2 plant subset was 1.24:1.64:0.88.
Validation of Rag3 in F2 Population.
With the genotypic data of 376 F2 plants from the cross E07048×E10902, a 18.7 cM long linkage map was constructed with five SNPs, MSUSNP16-14 (Gm16—6164774), MSUSNP16-10 (Gm16—6262227), MSUSNP16-11 (Gm16—6413214), MSUSNP16-12 (Gm16—6423098) and MSUSNP16-15 (Gm16—8051585) (FIG. 36). The marker order was consistent with their physical positions (Song et al., 2011). The slight inflation of the linkage map was caused by genotyping errors from SNP TaqMan® assay. One QTL was detected for both CIM and MIM methods between SNPs MSUSNP16-10 (Gm16—6262227) and MSUSNP16-12 (Gm16—6423098) (FIG. 36). The resistance parent E10902 was derived from PI 567543C with no other aphid resistance source integrated; therefore, the QTL for PI 567543C was indeed Rag3. The LOD scores were 38.7 for CIM and 44.2 for MIM. R2 estimated approximately as 0.447 in both CIM and MIM, suggesting that Rag3 can explain about 44.7% of the phenotype variance among these 376 F2 plants.
Fine Mapping Rag3 with Recombinant Lines
After validation of Rag3 between 6.1 and 8.0 mb of genomic DNA, the following SNPs were used for candidate regions designated MSU SNP16-14 (Gm16—6164774) and MSUSNP16-15 (Gm16—8051585). These SNPs were used to select recombinants within 6.1 and 8.0 mb conservatively. In total, 102 F2 plants were identified as recombinants within this region. After genotyping 983 F3 progeny of these F2 plants with SNPs, MSUSNP16-14 (Gm16—6164774) and MSUSNP16-15 (Gm16—8051585), 16 F3 lines were selected further genotyping with 52K SNP Beadchip. The genotypes of these 16 F3 plants on 19 polymorphic SNPs between 6.185 to 6.522 mb are listed in FIG. 37: Table 1. First three lines with markers and genotypes being homozygous at the susceptible allele showed susceptible phenotype in the greenhouse aphid resistance test. Nine segregating lines of these SNPs showed intermediate resistance and resistance phenotypes, and the following two homozygous lines of the resistance allele showed resistance phenotype. This evidence is consistent with the result from the QTL analysis above, suggesting that Rag3 locates at least within 6.1.85 and 6.522 mb on Chr. 16. Most surprisingly, two aphid resistant F3 lines, 2-499-2 and 2-277-6 were observed with break points within this region (FIG. 37: Table 1). For line 2-499-2, SNPs within 6.185 and 6.262 were homozygous susceptible type and the remaining SNPs to the right were alleles from resistant parent, indicating the genotype on the left of 6.262 mb did not affect phenotype. Similarly for line 2-277-6, SNPs on the right to 6.470 mb did not affect the resistance phenotype. These two recombinants within 6.262 and 6.470 narrowed the Rag3 region down to a 207 kb length on Chr. 16. To further confirm this finding, two F4 progeny of 2-499-2 and 14 F4 progeny of 2-277-6 were phenotyped and genotyped with 52K SNP Beadchip.
As shown in FIG. 37: Table 1, the phenotypes and genotypes of 16 F4 progeny are highly consistent as their F3 parents, confirming the 207 kb region of Rag3. The distance between SNPs on the inner side of the breaking points is actually 17 kb. To be conservative, we picked one SNP more on each side with a total distance of 207 kb to make sure Rag3 gene is covered. The limitation to further narrow this region down is the lack of recombination events and polymorphic SNP markers within this interval.
Association of SNPs MSUSNP16-10 (Gm16—6262227), MSUSNP16-11 (Gm16—6413214) and MSUSNP16-12 (Gm16—6423098) between genotypes and the phenotypes. Correlations of SNPs MSUSNP16-10 (Gm16—6262227), MSUSNP16-11 (Gm16—6413214) and MSUSNP16-12 (Gm16—6423098) to genotypes and aphid resistance phenotypes in 376 F2 subset of E07048 by E10902 and all F3 progeny of 102 F2 recombinants are summarized in FIG. 38: Table 2. Among the subset of 376 F2 lines, the correlation coefficients of SNP genotype and aphid resistance phenotype ranged from 0.52 to 0.68 with P<0.0001. These three SNPs can explain 27 to 47% of the phenotype variance. Among all 983 F3 progeny, the correlation coefficients ranged from 0.60 to 0.66 with P<0.0001, explaining 36 to 43% of the phenotypic variance. Therefore, these three TaqMan® SNPs, MSUSNP16-10 (Gm16—6262227), MSUSNP16-11 (Gm16—6413214) and MSUSNP16-12 (Gm16—6423098), can be effective in MAS for aphid resistance.
Example 16
The following example describes the unexpected discovery of recessive genes allelic rag1b and rag3 genes from PI567598B.
Phenotypic analysis. The phenotypic values of the 282 F4-derived RILs and its parents, grandparent PI 567598B were summarized in FIG. 45: Table 1. In both field and greenhouse trials, the susceptible parent, IA2070, was severely damaged by the aphids compared to the resistant parent E06902 and grandparent PI567598B, respectively. There is no significant difference in the aphid resistance between E06902 and PI567598B. Correlation between the three and four week ratings from the greenhouse trial was strong (r=0.88, P<0.0001). However, ratings from the greenhouse trial is not strongly correlated with the 2009 field ratings (0.37 and 0.44 for the week three and four ratings, respectively, P<0.0001). The distributions for the population ratings in both field and greenhouse trials were continuous, but not normal and the distribution in the field trial appeared bimodal (FIG. 51: a, b, c). This indicates that major genes might control the aphid resistance in PI 567598B.
QTL analysis. Among 1056 SSR markers, 38 markers revealed polymorphism between the resistant bulk DNA sample and the susceptible bulk DNA sample. These 38 markers were from chromosomes 1, 3, 7, 13, 16 and 18 (LGs D1a, N, M, F, J and G). Satt654 and Sct—001 on chromosome 16 (LG J) and Satt435 on chromosome 7 (LG M) appeared to be associated with aphid resistance when the individual lines from the DNA pools were genotyped. Therefore, these two regions were saturated with parental polymorphic markers within ±20cM in the consensus map (Song et al., 2004) and genotyped with the whole population. Using BARCSOYSSR 1.0 markers (Song et al., 2010), 48 additional markers were screened for polymorphism within the identified intervals. BARCSOYSSR16—0366 on chromosome 16 was found to be associated with aphid resistance while four other BARCSOYSRR07 markers between Satt435 and Satt323 in the chromosome 7 interval were found to be polymorphic. SNP markers in these two interested intervals were also designed from the SoySNP50 iSelect Infinium assay (Song et al., 2011) for Taqman endpoint genotyping.
A total of eight SSR and four SNP markers were mapped for the interval on chromosome 16 spanning a total of 43.5 cM (FIG. 52: FIG. 2A) while seven SSR and one SNP markers were mapped for the interval on chromosome 7 spanning a total of 45.9 cM (FIG. 52: FIG. 2D).
The QTL analysis detected two QTLs for the greenhouse trial while the one on chromosome 16 is significant in the field trial. In both trials, the allele from E06902 (PI 567598B derived line) conferred resistance against soybean aphids at the identified QTLs. Using the CIM method, the QTL on chromosome 16 was consistently mapped between SNP16-10 and SNP16-6424 and explained 30.7-45.8% of the phenotypic variation, with the field trial having the highest percentage (FIG. 47: Table 3 and FIG. 52: FIG. 2A). The QTL on chromosome 7 was detected in the greenhouse trials and located between Satt435 and BARCSOYSSR07—0309, explaining over 30% of the phenotypic variation (FIG. 47: Table 3 and FIG. 52: FIG. 2D).
The QTL analysis detected two QTLs for the greenhouse trial while only the one on chromosome 16 is significant in the field trial. In both trials, the allele from E06902 (PI 567598B derived line) conferred resistance against soybean aphids at the identified QTLs. Using the CIM method, the QTL on chromosome 16 was consistently mapped between SNP16-10 and SNP16-6424 and explained 30.7-45.8% of the phenotypic variation, with the field trial having the highest percentage (FIG. 47: Table 3 and FIG. 52: FIG. 2A). The QTL on chromosome 7 was only detected in the greenhouse trials and located between Satt435 and BARCSOYSSR07—0309, explaining over 30% of the phenotypic variation (FIG. 47: Table 3 and FIG. 52: FIG. 2D).
The MIM method was further conducted to determine whether there is significant QTL interaction. The MIM results are presented in Table 4. The MIM method detected same QTLs as CIM method with two QTLs in the greenhouse trial and one QTL in the field trial. For the week four ratings in the greenhouse trial, MIM method detected a significant additive×additive interaction between the two QTLs located on chromosome 7 and 16, but not for the week three ratings. The LOD score of the QTL interaction is 3.4 and it explained 1.2% of the total phenotypic variations. The two QTLs together with their interaction explained 41.7% of the total phenotypic variation. For the week three ratings, these two QTLs together explained 33.6% of the phenotypic variation. The QTL on chromosome 16 detected in the field trial explained the highest phenotypic variation, 56.1%.
QTL validation. For the validation population, a dense aphid population developed on the susceptible parent Titan while resistant parent PI 567598B had very few aphids in both 2008 and 2009 trials (FIG. 46: Table 2). A total of four markers on chromosome 7 and four markers on chromosome 16 were genotyped. The marker orders were highly comparable with the consensus map (Song et al. 2004).
With the CIM method. One QTL was detected on each linkage group in both trials (FIG. 52: FIG. 2C, F). The QTLs were located at similar regions between trials. The QTL on chromosome 7 was located between Satt567 and Satt435, explaining about 15% and 20% of the total phenotypic variation in the 2008 and 2009 trials, respectively (FIG. 47: Table 3). The QTL on chromosome 16 was located between Satt285 (or Sct—046) and Satt414 and explained about 30% and 40% of the total phenotypic variation in the 2008 and 2009 trials, respectively. The PI 567598B allele at both loci conferred aphid resistance.
The MIM analysis was performed to detect the epistatic effect and results are presented in Table 4. The two QTLs identified with the CIM method were also found using the MIM method in each trial. No QTL interactions were found in the 2008 trial. However, a significant additive×additive interaction between the two QTLs was detected in the 2009 trial. The LOD score of the QTL interaction was 6.0 and it explained 9.2% of the total phenotypic variation. The two QTLs together with their interaction explained 80.4% of the total phenotypic variation. Results presented from the validation population confirmed the QTLs found from the mapping population. Since the QTLs from this study were mapped to same region as Rag1 (Li et al., 2007) and Rag3 (Zhang, et al., 2009), we named the locus on chromosome 7 as rag1b and the locus on chromosome 16 as rag3, according to the conventions of the Soybean Genetics Committee.
Effect of the combination of QTL alternative alleles was discovered. The F4-derived lines from the mapping population were classified based on the E06902 alleles at the QTLs identified. Four distinct genotypes were defined by the presence or absence of the allele from E06902 for those QTL associated markers on chromosomes 7 and 16 (FIG. 49: Table 5). Only individual lines with complete and unambiguous genotype data for all loci were grouped into the defined genotypes and a total of 139 lines were grouped. Mean soybean aphid rating for all lines within each genotypic group was obtained for each of the trials in 2009 and 2010. In the 2010 greenhouse trial, the presence of E06902 alleles at both QTL M and QTL J gave the lowest soybean aphid rating while absence of alleles at both QTLs made lines very susceptible (FIG. 53: FIG. 3). The absence of E06902 allele at one QTL (either rag1b or rag3) gave intermediate reaction against soybean aphids. However, in the 2009 field cage trial, the lines absence of rag3 were as susceptible as those without any of the two QTL alleles from E06902. On the other hand, genotypes containing only rag3 gave resistant phenotypes that were comparable to the lines that had both resistant alleles. It seems that the QTL on chromosome 7 (rag1b) was defeated in the field trial while only QTL on chromosome 16 (rag3) conferred resistance. This shows that the QTLs identified in this study confer differential reactions against the soybean aphids in the field and greenhouse trails.
The following are exemplary materials and methods for QTL Mapping.
Plant Materials.
Two hundred eighty-two F4:5 recombinant inbred line (RIL) population was used for the mapping study and was developed from a cross between E06902 and IA2070. E06902 (PI 567598B× Titan) is an elite advanced breeding line derived from the original PI 567598B resistance source that possessed resistance levels similar to PI 567598B in field evaluations during 2006 (Dr. Dechun Wang, unpublished data). E06902 was crossed to the susceptible soybean accession IA2070 in 2007, followed by selling, to generate segregating F2 population. Single seed descent was applied to reach F4 generation.
Aphid Resistance Evaluation.
The F4:5 RIL population and parental plants were evaluated for aphid damage without replication in the field in summer 2009. The field evaluation of soybean aphid resistance was carried out in a 12.2×18.3m aphid- and predator-proof cage (Redwood Empire Awning Co., Santa Rosa, Calif.) on the Agronomy Farm of Michigan State University (MSU). The parental plants were planted randomly in the field, 5.1 cm apart. Each RIL was planted in a single row plot, 60 cm long with a row spacing of 60 cm. The average number of plants per line was around 10 with most plots having at least 12 plants. A similar complete randomized design (CRD) was used to arrange each population and its parents in the field plots.
Greenhouse evaluations were done for the F4:6 population in the fall without replication while the parental lines and PI 567598B were replicated three times. The greenhouse was maintained at 26/15° C. day/night temperature and sodium vapor lights were used to supplement light intensity during the day (14 h). Eight seeds per line were seeded in a large plastic pot. The pot size was 105 mm in diameter and 125 mm deep.
In both field and greenhouse trials, each plant was inoculated at the V2 stage with two wingless aphids. All aphid resistance evaluation trials were choice tests, which identifies resistance genotypes with either antibiosis or antixenosis resistance. The soybean aphids used for inoculation in the field trial were collected from a naturally infested field on the MSU Agronomy Farm. A single aphid clone was collected from a naturally infested field at the MSU Agronomy Farm, and maintained in an isolation chamber in the greenhouse for the inoculation of plants in the greenhouse trial in the same year.
Aphid resistance was visually rated for each plant three weeks after infestation in the summer and three and four weeks after infestation in the fall using a scale of 0 to 4 developed by Mensah et al. (2005, 2008), where 0=no aphids; 0.5=less than 10 aphids per plant, no colony formed; 1=11 to 100 aphids per plant, plant appears healthy; 1.5=101 to 150 aphids per plant, plant appears healthy; 2=151-300 aphids per plant, mostly on the young leaves or tender stems, plant appears healthy; 2.5=301-500 aphids per plant, plant appears healthy; 3=501-800 aphids per plant, young leaves and tender stems covered with aphids, leaves slightly curly and shiny; 3.5=More than 800 aphids per plant, plants stunted, leaves curled and slightly yellow, no sooty mold and few cast skins; 4=more than 800 aphids per plant, plant stunted, leaves severely curled and yellow, covered with sooty mold and cast skins.
DNA Extraction and Marker Analysis
Before infestation, the non-expanded trifoliates from each line were bulk harvested for genomic DNA isolation. The DNA was extracted with the CTAB (hexadecyltrimethyl ammonium bromide) method as described by Kisha et al. (1997) and the concentration was determined with a ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Del.). SSR markers (Song et al., 2004) were used to amplify the genomic DNA according to the PCR protocol described by Cregan and Quigley (1997) using a MJ TetradTM thermal cycler (MJ Research, Waltham, Mass.). PCR products were detected on 6% non-denaturing polyacrylamide gels by using a DASG-400-50 electrophoresis system (C.B.S. Scientific Co., Del Mar, Calif.) as described by Wang et al., (2003). Gels stained with ethidium bromide were photographed and scored under UV light.
In order to accelerate the location of the loci associated with the aphid resistance, the bulked segregant analysis method described by Michelmore et al. (1991) was used in this study. Based on the 2009 phenotypic data, 10 resistant lines with the lowest DI values and 10 susceptible lines with the highest DI values were selected to form a resistant pool and a susceptible pool, respectively. Parental polymorphic SSR markers at approximately every 15 cM of the integrated soybean map of Song et al. (2004) were selected to test the polymorphism between the two bulked DNA pools. The polymorphic markers between the two pools were chosen to genotype the individual lines in the two pools together with the two parents. The markers that appeared to be associated with the aphid resistance were genotyped on the remaining lines of the whole mapping population. The genomic regions associated with the aphid resistance were then saturated with additional markers. Additional SSR markers from 33,065 BARCSOYSSR—1.0 markers (Song et al., 2010) were also screened within the associated region to saturate more microsatellites within the interval of the associated polymorphic SSR markers from the first screen. Primers and hybridization probes for single nucleotide polymorphic (SNP) markers were developed for TaqMan® endpoint genotyping assay (ABI Life Sciences, USA) performed using Lightcycler® 480 (Roche Diagnostics). The SNP markers were designed based on the genomic physical position obtained from about 52,000 SNP markers on SoySNP50 iSelect Infinium assay (Song et al., 2011) for Illumina BeadChip arrays (www.illumina.com).
Genotyping Methods of TaqMan® and Kaspar®:
The allele specific array (probe plus primers) were designed and sent to be synthesized from the Applied Biosystems (ABI, Foster City, Calif., USA) and Kaspar® (Kbioscence.co.uk) for mapping and fine-mapping projects. Based on genotyping information of the Illumina HD beadchip analysis, the target sequences where the SNP anchored were selected in homozygous parental genotypes. The sequences were then subjected to the Customer Taqman® and Kaspar® Assay Design Tools of Applied Biosystems to obtain the allele specific primers and probes (assays). The allele specific Taqman® assays were synthesized by ABI. Taqman® SNP PCR reactions were carried out on 384-well plates with a total volume of 3 uL/well on the LightCycler 480 instrument (Roche Applied Science, Indianapolis, Ind., USA). The PCR reaction mixture for the Taqman® assay consisted of 1-20 ng of genomic DNA, 0.15 uL of 10× Taqman® Assay, and 1.5 uL of 2×ABI Genotyping Master mix containing a modified 30 Taq DNA polymerase, reaction buffer, MgCl2, and dNTPs (ABI, Foster City, Calif., USA). After 10 min pre-incubation at 95° C., 45 PCR cycles were conducted with 10 s of denaturation at 95° C., 30 s of annealing at 60° C., and 10 s extension at 72° C. A final melting cycle for nonspecific amplicon screening was performed by raising the temperature to 95° C. for 10 s, lowering the temperature to 40° C. for 30 s, and increasing the temperature to 83° C. with continuous fluorescent acquisition followed by a cool down to 40° C. on the LightCycler 480. Genotyping analysis was performed using the Roche Applied Science software version 1.5.0.
The allele specific Kaspar® assays were synthesized by Kbioscience genotyping service. Kaspar® SNP PCR reactions were performed on 384-well plates with a total volume of 4 uL/well on the LightCycler 480 instrument. The PCR reaction mixture for the Kaspar® assay contained 5-30 ng of genomic DNA, 2 uL of 2× Kaspar® reaction mix containing a modified Taq DNA polymerase, reaction buffer, MgCl2, and dNTPs (kbioscience.co.uk). After 15 min pre-incubation at 95° C., 12 PCR cycles were conducted with 20 s of denaturation at 95° C., 60 s of annealing at 65° C. for the amplification-1 and 28 PCR cycles were required with 20 s of denaturation at 95° C., 60 s of annealing at 57° C. for the amplification-II. A quantification step was programmed by hold temperature to 37° C. for 5 s with continuous fluorescent acquisition on the LightCycler 480. Genotyping analysis was also analyzed using the Roche Applied Science software version 1.5.0.
Statistical and QTL Analysis.
Pearson correlation for the aphid resistance between trials was calculated using R statistical software (R Development Core Team, 2005). Linkage map was constructed with the Kosambi function and a LOD score of 3 using JoinMap 4.0 (Manly et al. 2001). Then linkage groups were assigned to specific chromosomes according to the soybean consensus map (Song et al., 2004). The maps and QTL intervals were drawn using MapChart (Voorrips 2002). Composite interval mapping (CIM) was performed to locate the aphid resistance QTLs using QTL Cartographer V2.5 with the standard model Zmapqtl 6 (Wang et al. 2008). The CIM analysis uses markers other than the interval being tested as cofactors to control the genetic background (Zeng 1994). The forward and backward regression method was used to select markers as cofactors. The walking speed chosen for CIM was 1 cM. The empirical LOD threshold at 5% probability level was determined by a 1,000-permutation test (Churchill and Doerge 1994). The QTL×QTL interaction was further determined using the multiple interval mapping (MIM) method of QTL Cartographer.
Validation of QTLs. A population of 94 F2-derived lines was developed from a cross of PI 567598B× Titan and was used for validation of the mapped QTLs from the mapping population. PI 567598B possesses antibiosis resistance to the soybean aphid (Mensah et al. 2005) while ‘Titan’ is an aphid-susceptible cultivar developed at Michigan State University (Diers et al. 1999).
Two greenhouse trials were conducted for aphid resistance evaluation in the Plant Science Greenhouse on the MSU campus in 2008 and 2009 using the same procedure as described for the main population. Six seeds per line were seeded in a small plastic pot. Same aphid infestation and damage rating methods were used as described before. The aphid resistance score was determined as the mean of the rated plants in each line for each replication.
A damage index (DI) for each line was calculated by the following formula (Mensah et al. 2005): DI=Σ(Scale value×No. of plants in the category)/(4×Total no. of plants)×100. The DI ranges between 0 for no infestation and 100 for the most severe damage. The DI was used as an indicator of aphid resistance and was applied in the following analyses.
Separate DNA extractions were done on the plants used for each trial. FIG. 39: Table 1 Phenotypic summary of the F4-derived main mapping population and its parental lines and grandparent, PI567598B, for the mean soybean aphid ratings in a field trial and greenhouse trials.
FIG. 40: Table 2 Phenotypic summary of the F2 validation population and its parents PI 567598B and Titan for the soybean aphid damage index in the greenhouse trials.
FIG. 41: Table 3 Summary of QTLs for soybean aphid resistance detected in the main mapping population (IA2070×E06902) and validation population (PI 567598B× Titan) using the composite interval mapping method.
FIG. 42: Table 4 Summary of QTLs for soybean aphid resistance detected in the main mapping population (IA2070×E06902) and validation population (PI 567598B× Titan) using the multiple interval mapping method.
FIG. 43: Table 5 Genotypic groups of 139 F4-derived lines from the mapping population IA207×E06902 containing alternative alleles from associated markers on chromosome 7 (QTL M) and 16 (QTL J).
All publications and patents mentioned in the above specification are herein Incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

## Claims

1. A soybean plant comprising heterologous aphid resistant germplasm with an aphid resistant gene selected from the group rag1b, rag1c, rag3, Rag3 and rag4 is located in between two molecular markers, and wherein:
the rag1b gene is between two markers selected from the group: marker Satt567 (SEQ ID NO:85) and marker Satt435 (SEQ ID NO:88); marker BARCSOYSSR—07—0295 and marker BARCSOYSSR—07—0309; marker Satt435 (SEQ ID NO:87) and marker BARCSOYSSR—07—0295; and marker SNP07—420 and marker Satt435 (SEQ ID NO:88) on chromosome 7 of a soybean plant with accession number PI 567598B;
the rag1c gene is between marker MSUSNP7-19 (SEQ ID NO:99) and marker MSUSNP7-10 (SEQ ID NO:102) on chromosome 7 of a soybean plant with accession number PI 567541B;
the rag3 gene is between marker MSUSNP16-10 (SEQ ID NO:122) and marker MSUSNP16-6424 (SEQ ID NO:125) on chromosome 16 of a soybean plant with accession number PI 567598B;
the Rag3 gene is between marker Gm16—6262227_C_T (MSUSNP16-10; SEQ ID NO:122) and a marker Gm16—6469551_A_C; between marker Gm16—6262227_C_T (MSUSNP16-10; SEQ ID NO:122) and marker Gm16—6423098_G_A (MSUSNP16-12; SEQ ID NO:124) on chromosome 16 of a soybean plant with accession number PI 567543C; and
the rag4 gene is between two markers selected from the group: MSUSNP13-29 (position 7756558; SEQ ID NO:108) and MSUSNP13-31 (position 7918693); MSUSNP13-5 (SEQ ID NO:109) and MSUSNP13-6 (position 7786145); MSUSNP13-5 (position 7766353; SEQ ID NO:109)) and ss247923149 (position 8293174) on chromosome 13 of a soybean plant with accession number PI 567541B.

2. The plant of claim 1 wherein said aphid resistant germplasm comprises germplasm for both rag1b and rag3.

3. The plant of claim 1 wherein said aphid resistant germplasm comprises germplasm for both rag1c and rag4.

4. A seed from the soybean plant of claim 1, said seed comprises the aphid resistance gene.

5. The soybean plant of claim 1, wherein said aphid resistant germplasm having said rag3 gene is in between a marker MSUSNP16-10 ((Gm16—6262227; SEQ ID NO:122) and marker MSUSNP16-11 (Gm16 6413214; SEQ ID NO:123) on chromosome 16 of a plant with accession number PI 567598B.

6. The soybean plant of claim 1, wherein the aphid resistant germplasm having said rag4 gene is in between markers MSUSNP13-29 (position 7756558; SEQ ID NO:108) and MSUSNP13-33 (position 8067378; SEQ ID NO:116).

7. A method, comprising:
a) providing,
i) a first soybean plant comprising aphid resistant germplasm linked to a molecular marker,
ii) second soybean plant, and

b) crossing said first soybean plant with said second soybean plant, and using said molecular marker for identifying germplasm associated with aphid resistance;
wherein the aphid resistant germplasm is selected from the group consisting of:
a rag1b gene between two markers selected from the group: marker Satt567 (SEQ ID NO:85) and marker Satt435 (SEQ ID NO:88); marker BARCSOYSSR—07—0295 and marker BARCSOYSSR—07—0309; marker Satt435 (SEQ ID NO:87) and marker BARCSOYSSR—07—0295; and marker SNP07 420 and marker Satt435 (SEQ ID NO:88) on chromosome 7 of a soybean plant with accession number PI 567598B;
a rag1c gene between marker MSUSNP7-19 (SEQ ID NO:99) and marker MSUSNP7-10 (SEQ ID NO:102) on chromosome 7 of a soybean plant with accession number PI 567541B;
a rag3 gene between marker MSUSNP16-10 (SEQ ID NO:122) and marker MSUSNP16-6424 (SEQ ID NO:125) on chromosome 16 of a soybean plant with accession number PI 567598B;
a Rag3 gene between marker Gm16—6262227_C_T (MSUSNP16-10; SEQ ID NO:122) and a marker Gm16—6469551_A_C; between marker Gm16—6262227_C_T (MSUSNP16-10; SEQ ID NO:122) and marker Gm16—6423098_G_A (MSUSNP16-12; SEQ ID NO:124) on chromosome 16 of a soybean plant with accession number PI 567543C;
a rag4 gene between two markers selected from the group: MSUSNP13-29 (position 7756558; SEQ ID NO:108) and MSUSNP13-31 (position 7918693); MSUSNP13-5 (SEQ ID NO:109) and MSUSNP13-6 (position 7786145); MSUSNP13-5 (position 7766353; SEQ ID NO:109)) and ss247923149 (position 8293174) on chromosome 13 of a soybean plant with accession number PI 567541B; and
a combination thereof;

wherein.

8. A method of generating an aphid resistant plant, comprising:
(a) introducing at least one heterologous aphid resistant gene into a plant cell to generate a transformed cell;
(b) generating a plant from the transformed cell;

wherein the aphid resistant gene is selected from the group consisting of:
a rag1b gene between two markers selected from the group: marker Satt567 (SEQ ID NO:85) and marker Satt435 (SEQ ID NO:88); marker BARCSOYSSR—07—0295 and marker BARCSOYSSR—07—0309; marker Satt435 (SEQ ID NO:87) and marker BARCSOYSSR—07—0295; and marker SNP07—420 and marker Satt435 (SEQ ID NO:88) on chromosome 7 of a soybean plant with accession number PI 567598B;
a rag1c gene between marker MSUSNP7-19 (SEQ ID NO:99) and marker MSUSNP7-10 (SEQ ID NO:102) on chromosome 7 of a soybean plant with accession number PI 567541B;
a rag3 gene between marker MSUSNP16-10 (SEQ ID NO:122) and marker MSUSNP16-6424 (SEQ ID NO:125) on chromosome 16 of a soybean plant with accession number PI 567598B;
a Rag3 gene between marker Gm16—6262227_C_T (MSUSNP16-10; SEQ ID NO:122) and a marker Gm16—6469551_A_C; between marker Gm16—6262227_C_T (MSUSNP16-10; SEQ ID NO:122) and marker Gm16—6423098_G_A (MSUSNP16-12; SEQ ID NO:124) on chromosome 16 of a soybean plant with accession number PI 567543C;
a rag4 gene between two markers selected from the group: MSUSNP13-29 (position 7756558; SEQ ID NO:108) and MSUSNP13-31 (position 7918693); MSUSNP13-5 (SEQ ID NO:109) and MSUSNP13-6 (position 7786145); MSUSNP13-5 (position 7766353; SEQ ID NO:109)) and ss247923149 (position 8293174) on chromosome 13 of a soybean plant with accession number PI 567541B; and
a combination thereof.

9. The soybean plant of claim 1, wherein the rag3 gene is between marker MSUSNP16-10 ((Gm16—6262227; SEQ ID NO:122) and marker MSUSNP16-12 (Gm16—642309; SEQ ID NO:124) on chromosome 16 of a plant with accession number PI 567598B; and/or wherein said aphid resistant germplasm having said rag3 gene is between marker MSUSNP16-10 ((Gm16—6262227; SEQ ID NO:122) and marker MSUSNP16-13 (Gm16—6424067; SEQ ID NO:125) on chromosome 16 of a plant with accession number PI 567598B.

10. The soybean plant of claim 1, wherein said rag1c gene comprises MSUSNP7-18 (SEQ ID NO:100).