# Patent

## Title
Molecular markers asscociated with soy iron deficiency chlorosis

## Abstract

The present invention relates to methods and compositions for identifying, selecting and/or producing a soybean plant or germplasm having iron deficiency chlorosis tolerance. A soybean plant, part thereof and/or germplasm, including any progeny and/or seeds derived from a soybean plant or germplasm identified, selected and/or produced by any of the methods of the present invention is also provided.

## Background
RELATED APPLICATION INFORMATION
This Application claims the benefit of U.S. Provisional Application No. 62/086,834, filed 3 Dec. 2014, the contents of which are incorporated herein by reference.


STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 80663_ST25.txt, 57 kilobytes in size, generated on Nov. 24, 2015 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
FIELD OF THE INVENTION
The present invention relates to compositions and methods for identifying, selecting and/or producing soybean plants having tolerance to iron deficiency chlorosis (IDC).
BACKGROUND
Soybean (Glycine max L. Merr) is a major cash crop and investment commodity in North America and elsewhere. Soybean oil is one of the most widely used edible oils, and soybeans are used worldwide both in animal feed and in human food production. Iron deficiency chlorosis (IDC) in soybeans is a widespread problem in the Upper Midwest (North Central region) of the United States and is the result of reduced availability of iron and therefore, reduced iron levels in the plant. High pH in the soil, high water tables, too much rainfall, salinity in the soil, calcium carbonate in the topsoil, and elevated soil nitrate levels all contribute to the problem. The symptoms include interveinal chlorosis (the leaves turn yellow while the veins remain green) and stunting of plant growth. If the youngest leaves and growing points are damaged due to iron deficiency, growth of the plant will be stunted and yields are reduced substantially.
Different varieties of soybean vary in their sensitivity or tolerance to iron deficiency. Therefore, one of the most effective control measures is planting IDC tolerant soybean varieties, and thus varietal selection is important for the management of IDC. However, currently, determining whether a soybean cultivar might have tolerance to IDC typically involves testing each cultivar in the field or greenhouse under conditions that typically produce IDC. Thus, the present invention overcomes the shortcomings in the art by providing markers associated with tolerance to IDC, thereby allowing the characterization of soybean cultivars for IDC tolerance by molecular analysis rather than phenotypic analysis.
SUMMARY OF THE INVENTION
Compositions and methods for identifying, selecting and/or producing soybean plants with tolerance to iron deficiency chlorosis (IDC) are provided. As described herein, a marker associated with enhanced IDC tolerance may comprise, consist essentially of or consist of a single allele or a combination of alleles at one or more genetic loci.
Accordingly, in one aspect of the present invention, a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof is provided, the method comprising: detecting, in said soybean plant or part thereof, the presence of a marker associated with IDC tolerance in a soybean plant, wherein said marker is located within a chromosomal interval on chromosome 5 defined by and including a T allele at IIY12124, a G allele at IIY12130, a A allele at IIY12132 SEQ ID NO 3 or any combination thereof; (b) a chromosomal interval on chromosome 7 defined by and including a A allele at IIY13279, a G allele at SY0106A, a G allele at SY0642A, or any combination thereof; (c) a chromosomal interval on chromosome 13 defined by and including a C allele at SY0132A, a A allele at SY1091A, or any combination thereof; and (k) any combination of (a) through (c) and selecting a progeny soybean plant or germplasm that possesses said marker within its genome, thereby selecting an IDC tolerant soybean plant or germplasm.
In another aspect, the present invention provides a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof, comprising: detecting, in said soybean plant or part thereof, the presence of a marker associated with IDC tolerance, wherein said marker is selected from the group consisting of: (a) a A allele at IIY712; (b) a A allele at SY0622A; (c) a G allele at SY0023A; (d) an T allele at IIY9605; (e) a T allele at IIY12124; (f) a G allele at IIY12132; (g) a G allele at IIY12130; (h) a A allele at IIY13279; (i) a G allele at SY0106A; (j) a G allele at SY0642A; (k) a C allele at SY0132A; (l) a A allele at SY1091A; (m) a A allele at IIY16255; (n) a A allele at IIY6614; (o) a A allele at IIY6608; (p) a G allele at IIY6613; and any combination of (a) through (p) above, thereby identifying and/or selecting an IDC tolerant soybean plant or part thereof.
In an additional aspect of the present invention, a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof is provided, the method comprising: detecting, in said soybean plant or part thereof, the presence of a combination of genetic markers (haplotype) associated with IDC tolerance in a soybean plant, the combination of genetic markers selected from the group consisting of: (a) a A allele at IIY712; (b) a A allele at SY0622A; (c) a G allele at SY0023A; (d) an T allele at IIY9605; (e) a T allele at IIY12124; (f) a G allele at IIY12132; (g) a G allele at IIY12130; (h) a A allele at IIY13279; (i) a G allele at SY0106A; (j) a G allele at SY0642A; (k) a C allele at SY0132A; (l) a A allele at SY1091A; (m) a A allele at IIY16255; (n) a A allele at IIY6614; (o) a A allele at IIY6608; (p) a G allele at IIY6613; and any combination of (a) through (p) above, thereby identifying and/or selecting an IDC tolerant soybean plant or part thereof.
In other aspects, the present invention provides a method of producing an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof, comprising: detecting, in a soybean germplasm, the presence of a marker associated with IDC tolerance in a soybean plant, wherein said marker is located within a chromosomal interval selected from the group consisting of: (a) a chromosomal interval on chromosome 5 defined by and including a T allele at IIY12124, a G allele at IIY12130, a A allele at IIY12132 or any combination thereof; (b) a chromosomal interval on chromosome 7 defined by and including a A allele at IIY13279, a G allele at SY0106A, a G allele at SY0642A, or any combination thereof; (c) a chromosomal interval on chromosome 13 defined by and including a C allele at SY0132A, a A allele at SY1091A, or any combination thereof; and (k) any combination of (a) through (c) above, and producing a soybean plant from said soybean germplasm, thereby producing an IDC tolerant soybean plant or part thereof.
In further aspects of the invention, a method of producing an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof is provided, the method comprising: detecting, in a soybean germplasm, the presence of a marker associated with IDC tolerance, wherein said marker is selected from the group consisting of: (a) a A allele at IIY712; (b) a A allele at SY0622A; (c) a G allele at SY0023A; (d) an T allele at IIY9605; (e) a T allele at IIY12124; (f) a G allele at IIY12132; (g) a G allele at IIY12130; (h) a A allele at IIY13279; (i) a G allele at SY0106A; (j) a G allele at SY0642A; (k) a C allele at SY0132A; (l) a A allele at SY1091A; (m) a A allele at IIY16255; (n) a A allele at IIY6614; (o) a A allele at IIY6608; (p) a G allele at IIY6613; and any combination of (a) through (p) above, thereby identifying and/or selecting an IDC tolerant soybean plant or part thereof.
In additional aspects, a method of selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or germplasm is provided, the method comprising: crossing a first soybean plant or germplasm with a second soybean plant or germplasm, wherein said first soybean plant or germplasm comprises within its genome a marker associated with IDC tolerance in a soybean plant, wherein said marker is located within a chromosomal interval selected from the group consisting of (a) a chromosomal interval on chromosome 5 defined by and including a T allele at IIY12124, a G allele at IIY12130, a A allele at IIY12132 or any combination thereof; (b) a chromosomal interval on chromosome 7 defined by and including a A allele at IIY13279, a G allele at SY0106A, a G allele at SY0642A, or any combination thereof; (c) a chromosomal interval on chromosome 13 defined by and including a C allele at SY0132A, a A allele at SY1091A, or any combination thereof; and (k) any combination of (a) through (c) and selecting a progeny soybean plant or germplasm that possesses said marker within its genome, thereby selecting an IDC tolerant soybean plant or germplasm.
Other aspects of the present invention provide a method of selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or germplasm, comprising: crossing a first soybean plant or germplasm with a second soybean plant or germplasm, wherein said first soybean plant or germplasm comprises within its genome a marker associated with IDC tolerance in a soybean plant, wherein said marker is selected from the group consisting of: (a) a A allele at IIY712; (b) a A allele at SY0622A; (c) a G allele at SY0023A; (d) an T allele at IIY9605; (e) a T allele at IIY12124; (f) a G allele at IIY12132; (g) a G allele at IIY12130; (h) a A allele at IIY13279; (i) a G allele at SY0106A; (j) a G allele at SY0642A; (k) a C allele at SY0132A; (l) a A allele at SY1091A; (m) a A allele at IIY16255; (n) a A allele at IIY6614; (o) a A allele at IIY6608; (p) a G allele at IIY6613; and any combination of (a) through (p) above, thereby identifying and/or selecting an IDC tolerant soybean plant or part thereof.
In other embodiments, the present invention provides a method of selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or germplasm is provided, the method comprising: crossing a first soybean plant or germplasm with a second soybean plant or germplasm, wherein said first soybean plant or germplasm comprises within its genome a combination of genetic markers (haplotype) associated with IDC tolerance in a soybean plant, the combination of genetic markers selected from the group consisting of: (a) a A allele at IIY712; (b) a A allele at SY0622A; (c) a G allele at SY0023A; (d) an T allele at IIY9605; (e) a T allele at IIY12124; (f) a G allele at IIY12132; (g) a G allele at IIY12130; (h) a A allele at IIY13279; (i) a G allele at SY0106A; (j) a G allele at SY0642A; (k) a C allele at SY0132A; (l) a A allele at SY1091A; (m) a A allele at IIY16255; (n) a A allele at IIY6614; (o) a A allele at IIY6608; (p) a G allele at IIY6613; and any combination of (a) through (p) above; and selecting a progeny soybean plant or germplasm that possesses said marker within its genome, thereby selecting an IDC tolerant soybean plant or germplasm.
Another aspect of the invention is that some markers have been surprisingly associated with IDC tolerance in both soil type 1 and soil type 2. Soil type 1 includes U.S. soils found Southern Minnesota and Northern Iowa often referred to as glacial deposits. Soil Type 2 refers to soil found in the U.S. Red River Valley area that includes most of North Dakota and Northern Minnesota often referred to as lake bed over glacial deposits. The following markers have been identified to work in a wider geographical area including both soil types 1 and 2 comprising (a) a A allele at IIY712; (b) a A allele at SY0622A; (c) a G allele at SY0023A; (d) an T allele at IIY9605; (e) a T allele at IIY12124; (f) a G allele at IIY12132; (g) a G allele at IIY12130; (h) a A allele at IIY13279; (i) a G allele at SY0106A; (j) a G allele at SY0642A, (k) a G allele at SY0781AQ, (l) a G allele at SY0152AQ, (m) a G allele at SY1069AQ, (n) a G allele at SY2140AQ, (o) a A allele at SY0632AQ and any combination of (a) through (o) above, thereby identifying and/or selecting an IDC tolerant soybean plant or part to be grown in either soil type 1, soil type 2 or both.
Another aspect of the invention provides for markers and/or genes within a soybean chromosomal interval that associate with IDC tolerance wherein said soybean chromosomal interval is: on soybean chromosome 2 wherein the interval is defined by at least two of SY0781AQ, SY0322AQ, or SY1300AQ; on soybean chromosome 3 wherein the interval is defined by at least two of SY0673AQ and SY0674AQ; on soybean chromosome 5 wherein the interval is defined by at least two of SY3925, IIY12130, IIY12132, or SY0152AQ; on soybean chromosome 7 wherein the interval is defined by at least two of SY0106A, SY0642A, or IIY13279; on soybean chromosome 9 wherein the interval is defined by at least two of IIY712 and SY1069AQ; on soybean chromosome 13 wherein the interval is defined by at least two of SY3929, SY1258AQ, SY1091A, SY0132A, SY0023A, or SY3931; on soybean chromosome 14 wherein the interval is defined by at least two of SY0121AQ and SY0224AQ; on soybean chromosome 15 wherein the interval is defined by at least two of SY0386AQ, SY0952AQ or SY0808AQ; on soybean chromosome 16 wherein the interval is defined by at least two of IIY6608, IIY16255, IIY6613, or IIY6614; on soybean chromosome 19 wherein the interval is defined by at least two of SY0622A, SY0066AQ, or SY0632AQ.
Another aspect of the invention provides for methods of identifying, selecting or producing IDC tolerant soybean plants through the use of markers as shown in Table 1. Further the invention provides for methods of introducing IDC tolerant soybean plants wherein introducing encompasses introgression of chromosome interval comprising a marker and/or gene that confers IDC tolerance into a plant. Additionally, introducing can mean the use of gene editing tools (e.g. TALEN, CRISPR, etc.) to create allelic variants having favorable genotypes as described herein (e.g. as shown in Table 1). The term introducing can mean the introduction of genes within close proximity of the markers listed in Table 1 wherein close proximity can be for example 10 cM, ScM, 2 cM, 1 cM, 10000 nucleotide base-pairs (bp), 5000 bp, 2500 (bp), 1000 (bp), 500 bp or 250 bp within the physical position of a marker described in Table 1. Finally, the term introducing can mean the heterologous expression of a gene in close proximity to any marker in Table 1 in a plant to create a IDC tolerant plant.
Soybean plants and/or germplasms identified, produced or selected by the methods of this invention are also provided, as are any progeny and/or seeds derived from a soybean plant or germplasm identified, produced or selected by these methods.
These and other aspects of the invention are set forth in more detail in the description of the invention below.


DETAILED DESCRIPTION
The present invention provides compositions and methods for identifying, selecting and/or producing soybean plants having iron deficiency tolerance, as well as soybean plants and parts thereof, including but not limited to seeds, that are identified, selected and/or produced by a method of this invention. The present invention further provides an assay for the detection of IDC in a soybean plant. In addition, the present invention provides soybean plants and/or soybean germplasm having within their genomes one or more SNP or QTL markers associated with tolerance to iron deficiency chlorosis.
All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.
All patents, patent publications, non-patent publications and sequences referenced herein are incorporated by reference in their entireties.
Definitions
Although the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate understanding of the presently disclosed subject matter.
As used herein, the terms “a” or “an” or “the” may refer to one or more than one. For example, “a” marker (e.g., SNP, QTL, haplotype) can mean one marker or a plurality of markers (e.g., 2, 3, 4, 5, 6, and the like).
As used herein, the term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
As used herein, the term “about,” when used in reference to a measurable value such as an amount of mass, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.
As used herein, the transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”
As used herein, the term “allele” refers to one of two or more different nucleotides or nucleotide sequences that occur at a specific locus.
A “locus” is a position on a chromosome where a gene or marker or allele is located. In some embodiments, a locus may encompass one or more nucleotides.
As used herein, the terms “desired allele,” “target allele” and/or “allele of interest” are used interchangeably to refer to an allele associated with a desired trait. In some embodiments, a desired allele may be associated with either an increase or a decrease (relative to a control) of or in a given trait, depending on the nature of the desired phenotype. In some embodiments of this invention, the phrase “desired allele,” “target allele” or “allele of interest” refers to an allele(s) that is associated with tolerance to iron deficiency chlorosis in a soybean plant relative to a control soybean plant not having the target allele or alleles.
A marker is “associated with” a trait when said trait is linked to it and when the presence of the marker is an indicator of whether and/or to what extent the desired trait or trait form will occur in a plant/germplasm comprising the marker. Similarly, a marker is “associated with” an allele or chromosome interval when it is linked to it and when the presence of the marker is an indicator of whether the allele or chromosome interval is present in a plant/germplasm comprising the marker. For example, “a marker associated with an IDC tolerance allele” refers to a marker whose presence or absence can be used to predict whether a plant will display tolerance to iron deficiency chlorosis.
Iron deficiency chlorosis (IDC) is a physiological disease in soybean plants that is caused by a lack of iron in the plant. Most soils contain sufficient iron. However, in some soils the iron is insoluble and thus unavailable to the plants. As a result of the unavailability of the iron in the soil, plants grown in such soil lack iron. It is also known in the art that IDC can be the result of any one or combination of a) the plant's inability to uptake iron from the soil (e.g. iron insolubility, or root uptake hindered), b) the inability of the plant to transport the iron to the leaf and c) the inability of the plant to activate the iron in the leaf. Any one of these (a-c) scenarios can lead to the symptoms that are indicative of IDC. Herein, the terms “Iron deficiency chlorosis” or “IDC” interchangeably represent a physiological disease in any plant that is caused by the lack of iron whether that lack of iron is due to the plant's inability to uptake the iron; a plant's inability to transport the iron or thirdly the plant's inability to activate the iron in the leaf tissue.
As used herein, the terms “low iron,” “low iron conditions,” “low iron growth conditions,” “low iron availability” or “iron deficiency” or the like refer to conditions where iron availability is less than optimal for soybean growth and can cause physiological disease, e.g., iron deficiency chlorosis, due to the lack of soluble or available iron in the growth medium (e.g., soil). While the absolute level of iron may be sufficient, the form of the iron, which is affected by various environmental factors, may make the iron that is present unavailable for plant use (cannot be taken up by the plant's roots). See, Dahiya and Singh, Plant and Soil 51:13-18 (1979). For example, high carbonate levels, high pH, high salt content (high salinity; e.g., phosphorus, manganese and zinc), saturated soils (and/or poor drainage) and/or other environmental factors can result in lower iron solubility; thereby, reducing the solubilized forms of iron that are necessary for plant uptake. Thus, soils having low available iron include, but are not limited to, those that are calcareous (i.e., high in calcium carbonate) and have a high pH (greater than 7.5). Iron levels in soil that are optimal/not optimal for plant growth are well known in the art as are methods for measuring iron content.
The initial symptoms of iron deficiency chlorosis include interveinal chlorosis in the newly developing trifoliate leaves. Interveinal chlorosis can be described as a contrast of the inter-vein tissue color, which turns yellow, as compared to the vein color, which remains green. The interveinal chlorosis is referred to as “yellow flash.” Yellow flash occurs at about 21 days after planting or at the V2 stage of growth. Eventually, the leaves of symptomatic plants may develop necrotic spots that coalesce and then, finally the leaves may fall off. Tolerant varieties may express more normal leaf color and little contrast between inter-vein tissue color and vein color. Intolerant varieties express greenish-yellow or yellow or yellowish-white colored inter-vein tissue while the vein remains green which produces relatively greater and greater contrast. Intolerant varieties are also slow in vegetative growth and biomass compared to tolerant varieties. Extremely intolerant varieties produce white trifoliate leaves that quickly decline and become necrotic. Extremely intolerant plants essentially stop growing vegetatively, producing maximum contrast compared to tolerant varieties.
The term “recovery” as used herein refers to the extent of iron deficiency chlorosis symptoms as measured in newly developed leaves or about 14 days after the initial yellow flash. Tolerant varieties signal recovery by producing a more normal green color in the new leaves (i.e., little contrast between leaf tissue and veinal tissue) as compared to the initial yellow flash response measured earlier in that same plant. Intolerant varieties continue to produce yellow flash symptoms in the new leaves resulting in a continuing contrast between interveinal tissue and the veins, as discussed herein.
As used herein, the term “iron deficiency tolerance” or “iron deficiency chlorosis tolerance” refers to a plant's ability to have increased efficiency in uptake of, transporting and activating iron as compared to one or more control plants not tolerant to IDC (e.g., a plant lacking a marker associated with iron deficiency tolerance). In some cases an iron deficiency tolerant plant can uptake iron, transport iron or activate iron once in the leaf tissue at an increased or more efficient rate than a control plant not tolerant to iron deficiency chlorosis grown in the same or similar environment
Thus, “tolerance” in a soybean plant to iron deficient or low iron growth conditions is an indication that the soybean plant is less affected by the low iron growth conditions with respect to yield, survivability and/or other relevant agronomic measures, compared to a less tolerant, more “susceptible” plant. Tolerance is a relative term, indicating that a “tolerant” soybean plant survives and/or produces a better yield in iron deficient growth conditions when compared to a different (less tolerant) soybean plant (e.g., a different soybean strain or variety) grown in similar conditions of low iron availability. That is, under iron deficient growth conditions a tolerant plant can have a greater survival rate and/or yield, as compared to a soybean plant that is susceptible or intolerant to these low iron growth conditions. Iron deficiency “tolerance” sometimes can be used interchangeably with iron deficiency “resistance.” Iron deficiency chlorosis intolerant soybean varieties and cultivars are well known in the art. A non-limiting example of an IDC intolerant soybean cultivar is soybean cultivar M08851 (U.S. Pat. No. 7,126,047).
In some embodiments, a plant of this invention that is iron deficiency tolerant or iron deficiency chlorosis tolerant includes a plant that exhibits reduced yellow flash symptoms as compared to a plant not having in its genome the genetic markers described herein as associated with IDC tolerance. In other embodiments, a plant of this invention that is IDC tolerant also includes a plant that exhibits recovery from yellow flash as compared to a plant not having in its genome the genetic markers described herein as associated with IDC tolerance. In still other embodiments, a plant of this invention that is iron deficiency tolerant includes a plant that exhibits both reduced yellow flash symptoms and recovery from yellow flash as compared to a plant not having in its genome the marker(s) described herein as associated with IDC tolerance.
As is understood by the skilled artisan, soybean plant tolerance to low-available iron conditions varies widely, and can represent a range of more tolerant to less-tolerant phenotypes. Non-limiting examples of methods for determining the relative tolerance or susceptibility of different plants, plant lines or plant families under low-available iron conditions include visual observation (e.g., visual chlorosis scoring system) (See, Helms et al. Agronomy J 102:492-498 (2010)) and/or electronic scanning using a Greenseeker® RT100 radiometer (See, PCT/US10/46303; WO/2011/022719). Other methods for determining IDC tolerance include but are not limited to the use of hydroponics (See, Niebur and Fehr, Crop Sci. 21:551-554 (1981)).
In the case of a visual chlorosis scoring system, a plant that is grown in soil having low available iron, or in low available iron experimental conditions, can be assigned a tolerance rating of between 1 (highly tolerant; yield and survivability not significantly affected; all plants normal green color) to 9 (highly susceptible; most or all plants dead; those that live are stunted and have little living tissue) based on visual observation of the level of chlorosis in the plant.
In a further example, a radiometer can be used to take electronic measurements. In this case, a plant that is grown in a known low available iron soil, or in low available iron experimental conditions, is assigned a tolerance rating of between 1 (highly tolerant; yield and survivability not significantly affected; all plants normal green color) to 0 (highly susceptible; most or all plants dead; those that live are stunted and have little living tissue) based on the reading provided by scanning the foliage with the radiometer.
As used herein, the terms “backcross” and “backcrossing” refer to the process whereby a progeny plant is crossed back to one of its parents one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.). In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. Marker-assisted Backcrossing: A Practical Example, in TECHNIQUES ET UTILISATIONS DES MARQUEURS MOLECULAIRES LES COLLOQUES, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, in PROCEEDINGS OF THE SYMPOSIUM “ANALYSIS OF MOLECULAR MARKER DATA,” pp. 41-43 (1994). The initial cross gives rise to the F1 generation. The term “BC1” refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on.
As used herein, the terms “cross” or “crossed” refer to the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant). The term “crossing” refers to the act of fusing gametes via pollination to produce progeny.
As used herein, the terms “cultivar” and “variety” refer to a group of similar plants that by structural or genetic features and/or performance can be distinguished from other varieties within the same species.
As used herein, the terms “elite” and/or “elite line” refer to any line that is substantially homozygous and has resulted from breeding and selection for desirable agronomic performance.
As used herein, the terms “exotic,” “exotic line” and “exotic germplasm” refer to any plant, line or germplasm that is not elite. In general, exotic plants/germplasms are not derived from any known elite plant or germplasm, but rather are selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce novel alleles into a breeding program).
A “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by the recombination frequencies between them. Recombination between loci can be detected using a variety of markers. A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distances between loci can differ from one genetic map to another.
As used herein, the term “genotype” refers to the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable and/or detectable and/or manifested trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple loci, or more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome. Genotypes can be indirectly characterized, e.g., using markers and/or directly characterized by nucleic acid sequencing.
As used herein, the term “germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific genetic makeup that provides a foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, as well as plant parts that can be cultured into a whole plant (e.g., leaves, stems, buds, roots, pollen, cells, etc.).
A “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e., a combination of alleles. Typically, the genetic loci that define a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term “haplotype” can refer to polymorphisms at a particular locus, such as a single marker locus, or polymorphisms at multiple loci along a chromosomal segment.
As used herein, the term “heterozygous” refers to a genetic status wherein different alleles reside at corresponding loci on homologous chromosomes.
As used herein, the term “homozygous” refers to a genetic status wherein identical alleles reside at corresponding loci on homologous chromosomes.
As used herein, the term “hybrid” in the context of plant breeding refers to a plant that is the offspring of genetically dissimilar parents produced by crossing plants of different lines or breeds or species, including but not limited to the cross between two inbred lines.
As used herein, the term “inbred” refers to a substantially homozygous plant or variety. The term may refer to a plant or plant variety that is substantially homozygous throughout the entire genome or that is substantially homozygous with respect to a portion of the genome that is of particular interest.
As used herein, the term “indel” refers to an insertion or deletion in a pair of nucleotide sequences, wherein a first sequence may be referred to as having an insertion relative to a second sequence or the second sequence may be referred to as having a deletion relative to the first sequence.
As used herein, the terms “introgression,” “introgressing” and “introgressed” refer to both the natural and artificial transmission of a desired allele or combination of desired alleles of a genetic locus or genetic loci from one genetic background to another. For example, a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele may be a selected allele of a marker, a QTL, a transgene, or the like. Offspring comprising the desired allele can be backcrossed one or more times (e.g., 1, 2, 3, 4, or more times) to a line having a desired genetic background, selecting for the desired allele, with the result being that the desired allele becomes fixed in the desired genetic background. For example, a marker associated with IDC tolerance may be introgressed from a donor into a recurrent parent that is IDC intolerant. The resulting offspring could then be backcrossed one or more times and selected until the progeny possess the genetic marker(s) associated with iron deficiency chlorosis tolerance in the recurrent parent background.
As used herein, the term “linkage” refers to the degree with which one marker locus is associated with another marker locus or some other locus (for example, an IDC tolerance locus). The linkage relationship between a genetic marker and a phenotype may be given as a “probability” or “adjusted probability.” Linkage can be expressed as a desired limit or range. For example, in some embodiments, any marker is linked (genetically and physically) to any other marker when the markers are separated by less than about 50, 40, 30, 25, 20, or 15 map units (or cM).
A centimorgan (“cM”) or a genetic map unit (m.u.) is a unit of measure of recombination frequency and is defined as the distance between genes for which one product of meiosis in 100 is recombinant. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation. Thus, a recombinant frequency (RF) of 1% is equivalent to 1 m.u.
As used herein, the phrase “linkage group” refers to all of the genes or genetic traits that are located on the same chromosome. Within the linkage group, those loci that are close enough together can exhibit linkage in genetic crosses. Since the probability of crossover increases with the physical distance between loci on a chromosome, loci for which the locations are far removed from each other within a linkage group might not exhibit any detectable linkage in direct genetic tests. The term “linkage group” is mostly used to refer to genetic loci that exhibit linked behavior in genetic systems where chromosomal assignments have not yet been made. Thus, the term “linkage group” is synonymous with the physical entity of a chromosome, although one of ordinary skill in the art will understand that a linkage group can also be defined as corresponding to a region of (i.e., less than the entirety) of a given chromosome.
As used herein, the term “linkage disequilibrium” refers to a non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency (in the case of co-segregating traits, the loci that underlie the traits are in sufficient proximity to each other). Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51% to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and, by definition, are separated by less than 50 cM on the same chromosome). As used herein, linkage can be between two markers, or alternatively between a marker and a phenotype. A marker locus can be “associated with” (linked to) a trait, e.g., IDC tolerance. The degree of linkage of a genetic marker to a phenotypic trait is measured, e.g., as a statistical probability of co-segregation of that marker with the phenotype.
Linkage disequilibrium is most commonly assessed using the measure r2, which is calculated using the formula described by Hill and Robertson, Theor. Appl. Genet. 38:226 (1968). When r2=1, complete linkage disequilibrium exists between the two marker loci, meaning that the markers have not been separated by recombination and have the same allele frequency. Values for r2 above 1⁄3 indicate sufficiently strong linkage disequilibrium to be useful for mapping. Ardlie et al., Nature Reviews Genetics 3:299 (2002). Hence, alleles are in linkage disequilibrium when r2 values between pairwise marker loci are greater than or equal to about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.
As used herein, the term “linkage equilibrium” describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome).
As used herein, the terms “marker” and “genetic marker” are used interchangeably to refer to a nucleotide and/or a nucleotide sequence that has been associated with a phenotype and/or trait. A marker may be, but is not limited to, an allele, a gene, a haplotype, a chromosome interval, a restriction fragment length polymorphism (RFLP), a simple sequence repeat (SSR), a random amplified polymorphic DNA (RAPD), a cleaved amplified polymorphic sequence (CAPS) (Rafalski and Tingey, Trends in Genetics 9:275 (1993)), an amplified fragment length polymorphism (AFLP) (Vos et al., Nucleic Acids Res. 23:4407 (1995)), a single nucleotide polymorphism (SNP) (Brookes, Gene 234:177 (1993)), a sequence-characterized amplified region (SCAR) (Paran and Michelmore, Theor. Appl. Genet. 85:985 (1993)), a sequence-tagged site (STS) (Onozaki et al., Euphytica 138:255 (2004)), a single-stranded conformation polymorphism (SSCP) (Orita et al., Proc. Natl. Acad. Sci. USA 86:2766 (1989)), an inter-simple sequence repeat (ISSR) (Blair et al., Theor. Appl. Genet. 98:780 (1999)), an inter-retrotransposon amplified polymorphism (IRAP), a retrotransposon-microsatellite amplified polymorphism (REMAP) (Kalendar et al., Theor. Appl. Genet. 98:704 (1999)), an isozyme marker, an RNA cleavage product (such as a Lynx tag) or any combination of the markers described herein. A marker may be present in genomic or expressed nucleic acids (e.g., ESTs). A large number of soybean genetic markers are known in the art, and are published or available from various sources, such as the SoyBase internet resource (soybase.org). In some embodiments, a genetic marker of this invention is an SNP allele, a SNP allele located in a chromosome interval and/or a haplotype (combination of SNP alleles) each of which is associated with IDC tolerance.
Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well-established in the art. These include, but are not limited to, nucleic acid sequencing, hybridization methods, amplification methods (e.g., PCR-based sequence specific amplification methods), detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of randomly amplified polymorphic DNA (RAPD), detection of single nucleotide polymorphisms (SNPs), and/or detection of amplified fragment length polymorphisms (AFLPs). Thus, in some embodiments of this invention, such well known methods can be used to detect the SNP alleles as defined herein (See, e.g., Table 2)
Accordingly, in some embodiments of this invention, a marker is detected by amplifying a Glycine sp. nucleic acid with two oligonucleotide primers by, for example, the polymerase chain reaction (PCR).
A “marker allele,” also described as an “allele of a marker locus,” can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population that is polymorphic for the marker locus.
“Marker-assisted selection” (MAS) is a process by which phenotypes are selected based on marker genotypes. Marker assisted selection includes the use of marker genotypes for identifying plants for inclusion in and/or removal from a breeding program or planting.
As used herein, the terms “marker locus” and “marker loci” refer to a specific chromosome location or locations in the genome of an organism where a specific marker or markers can be found. A marker locus can be used to track the presence of a second linked locus, e.g., a linked locus that encodes or contributes to expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a locus, such as a QTL or single gene, that are genetically or physically linked to the marker locus.
As used herein, the terms “marker probe” and “probe” refer to a nucleotide sequence or nucleic acid molecule that can be used to detect the presence of one or more particular alleles within a marker locus (e.g., a nucleic acid probe that is complementary to all of or a portion of the marker or marker locus, through nucleic acid hybridization). Marker probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more contiguous nucleotides may be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Non-limiting examples of probes of this invention include SEQ ID NOs:19-54 and 137-300.
As used herein, the term “molecular marker” may be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus. A molecular marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.). The term also refers to nucleotide sequences complementary to or flanking the marker sequences, such as nucleotide sequences used as probes and/or primers capable of amplifying the marker sequence. Nucleotide sequences are “complementary” when they specifically hybridize in solution, e.g., according to Watson-Crick base pairing rules. Some of the markers described herein can also be referred to as hybridization markers when located on an indel region. This is because the insertion region is, by definition, a polymorphism vis-ã-vis a plant without the insertion. Thus, the marker need only indicate whether the indel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g., SNP technology.
As used herein, the term “primer” refers to an oligonucleotide which is capable of annealing to a nucleic acid target and serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product is induced (e.g., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH). A primer (in some embodiments an extension primer and in some embodiments an amplification primer) is in some embodiments single stranded for maximum efficiency in extension and/or amplification. In some embodiments, the primer is an oligodeoxyribonucleotide. A primer is typically sufficiently long to prime the synthesis of extension and/or amplification products in the presence of the agent for polymerization. The minimum lengths of the primers can depend on many factors, including, but not limited to temperature and composition (A/T vs. G/C content) of the primer. In the context of amplification primers, these are typically provided as a pair of bi-directional primers consisting of one forward and one reverse primer or provided as a pair of forward primers as commonly used in the art of DNA amplification such as in PCR amplification. As such, it will be understood that the term “primer”, as used herein, can refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the target region to be amplified. Hence, a “primer” can include a collection of primer oligonucleotides containing sequences representing the possible variations in the sequence or includes nucleotides which allow a typical base pairing.
Primers can be prepared by any suitable method. Methods for preparing oligonucleotides of specific sequence are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, the phospho di- or tri-ester method, the diethylphosphoramidate method and the solid support method disclosed in U.S. Pat. No. 4,458,066.
Primers can be labeled, if desired, by incorporating detectable moieties by for instance spectroscopic, fluorescence, photochemical, biochemical, immunochemical, or chemical moieties.
The PCR method is well described in handbooks and known to the skilled person. After amplification by PCR, target polynucleotides can be detected by hybridization with a probe polynucleotide which forms a stable hybrid with that of the target sequence under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes are essentially completely complementary (i.e., about 99% or greater) to the target sequence, stringent conditions can be used. If some mismatching is expected, for example if variant strains are expected with the result that the probe will not be completely complementary, the stringency of hybridization can be reduced. In some embodiments, conditions are chosen to rule out non-specific/adventitious binding. Conditions that affect hybridization, and that select against non-specific binding are known in the art, and are described in, for example, Sambrook & Russell (2001). Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., United States of America. Generally, lower salt concentration and higher temperature hybridization and/or washes increase the stringency of hybridization conditions.
As used herein, the term “probe” refers to a single-stranded oligonucleotide sequence that will form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative.
Different nucleotide sequences or polypeptide sequences having homology are referred to herein as “homologues.” The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleotide sequences and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids, amino acids, and/or proteins.
As used herein, the phrase “nucleotide sequence homology” refers to the presence of homology between two polynucleotides. Polynucleotides have “homologous” sequences if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence. The “percentage of sequence homology” for polynucleotides, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100 percent sequence homology, can be determined by comparing two optimally aligned sequences over a comparison window (e.g., about 20-200 contiguous nucleotides), wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., gaps) as compared to a reference sequence for optimal alignment of the two sequences. Optimal alignment of sequences for comparison can be conducted by computerized implementations of known algorithms, or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST®; Altschul et al. (1990) J Mol Biol 215:403-10; Altschul et al. (1997) Nucleic Acids Res 25:3389-3402) and ClustalX (Chenna et al. (2003) Nucleic Acids Res 31:3497-3500) programs, both available on the Internet. Other suitable programs include, but are not limited to, GAP, BestFit, PlotSimilarity, and FASTA, which are part of the Accelrys GCG Package available from Accelrys Software, Inc. of San Diego, Calif., United States of America.
As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).
As used herein, the term “substantially identical” or “corresponding to” means that two nucleotide sequences have at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity. In some embodiments, the two nucleotide sequences can have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. As used herein, the term “percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). In some embodiments, “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.
Optimal alignment of sequences for aligning a comparison window is well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, Mass.). The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this invention “percent identity” may also be determined using BLAST®X version 2.0 for translated nucleotide sequences and BLAST®N version 2.0 for polynucleotide sequences.
The percent of sequence identity can be determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software PackageTM (Version 10; Genetics Computer Group, Inc., Madison, Wis.). “Gap” utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol. 48:443-453, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. “BestFit” performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482-489, 1981, Smith et al., Nucleic Acids Res. 11:2205-2220, 1983).
Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo et al. (Applied Math 48:1073(1988)). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST®) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST® Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); version 2.0 or higher of BLAST® programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLAST®X can be used to determine sequence identity; and for polynucleotide sequence BLAST®N can be used to determine sequence identity.
As used herein, the terms “phenotype,” “phenotypic trait” or “trait” refer to one or more traits of an organism. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, or an electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait.” In other cases, a phenotype is the result of several genes.
As used herein, the term “polymorphism” refers to a variation in the nucleotide sequence at a locus, where said variation is too common to be due merely to a spontaneous mutation. A polymorphism must have a frequency of at least about 1% in a population. A polymorphism can be a single nucleotide polymorphism (SNP), or an insertion/deletion polymorphism, also referred to herein as an “indel.” Additionally, the variation can be in a transcriptional profile or a methylation pattern. The polymorphic site or sites of a nucleotide sequence can be determined by comparing the nucleotide sequences at one or more loci in two or more germplasm entries.
As used herein, the term “plant” can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term “plant” can refer to a whole plant, a plant component or a plant organ (e.g., leaves, stems, roots, etc.), a plant tissue, a seed and/or a plant cell. A plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant.
As used herein, the term “soybean” refers to a plant, and any part thereof, of the genus Glycine including, but not limited to Glycine max. 
As used herein, the term “plant part” includes but is not limited to embryos, pollen, seeds, leaves, flowers (including but not limited to anthers, ovules and the like), fruit, stems or branches, roots, root tips, cells including cells that are intact in plants and/or parts of plants, protoplasts, plant cell tissue cultures, plant calli, plant clumps, and the like. Thus, a plant part includes soybean tissue culture from which soybean plants can be regenerated. Further, as used herein, “plant cell” refers to a structural and physiological unit of the plant, which comprises a cell wall and also may refer to a protoplast. A plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue or a plant organ.
As used herein, the term “population” refers to a genetically heterogeneous collection of plants sharing a common genetic derivation.
As used herein, the terms “progeny”, “progeny plant,” and/or “offspring” refer to a plant generated from a vegetative or sexual reproduction from one or more parent plants. A progeny plant may be obtained by cloning or selfing a single parent plant, or by crossing two parental plants and includes selfings as well as the F1 or F2 or still further generations. An F1 is a first-generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offspring of second generation (F2) or subsequent generations (F3, F4, and the like) are specimens produced from selfings or crossings of F1s, F2s and the like. An F1 can thus be (and in some embodiments is) a hybrid resulting from a cross between two true breeding parents (the phrase “true-breeding” refers to an individual that is homozygous for one or more traits), while an F2 can be (and in some embodiments is) an offspring resulting from self-pollination of the F1 hybrids.
As used herein, the term “reference sequence” refers to a defined nucleotide sequence used as a basis for nucleotide sequence comparison (e.g., Chromosome 3 of Glycine max cultivar Williams 82). The reference sequence for a marker, for example, can be obtained by genotyping a number of lines at the locus or loci of interest, aligning the nucleotide sequences in a sequence alignment program, and then obtaining the consensus sequence of the alignment. Hence, a reference sequence identifies the polymorphisms in alleles at a locus. A reference sequence may not be a copy of an actual nucleic acid sequence from any particular organism; however, it is useful for designing primers and probes for actual polymorphisms in the locus or loci.
Genetic Mapping
Genetic loci correlating with particular phenotypes, such as tolerance to iron deficiency chlorosis, can be mapped in an organism's genome. By identifying a marker or cluster of markers that co-segregate with a trait of interest, the breeder is able to rapidly select a desired phenotype by selecting for the proper marker (a process called marker-assisted selection, or MAS). Such markers may also be used by breeders to design genotypes in silico and to practice whole genome selection.
The present invention provides markers associated with tolerance to iron deficiency chlorosis in soybean. Detection of these markers and/or other linked markers can be used to identify, select and/or produce soybean plants having IDC tolerance and/or to eliminate soybean plants from breeding programs or from planting that do not have IDC tolerance
Markers Associated with Tolerance to Iron Deficiency Chlorosis
Molecular markers are used for the visualization of differences in nucleic acid sequences. This visualization can be due to DNA-DNA hybridization techniques after digestion with a restriction enzyme (e.g., an RFLP) and/or due to techniques using the polymerase chain reaction (e.g., SNP, STS, SSR/microsatellites, AFLP, and the like). In some embodiments, all differences between two parental genotypes segregate in a mapping population based on the cross of these parental genotypes. The segregation of the different markers can be compared and recombination frequencies can be calculated. Methods for mapping markers in plants are disclosed in, for example, Glick & Thompson (1993) Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., United States of America; Zietkiewicz et al. (1994) Genomics 20:176-183.
Table 1 provides a summary of markers associated with IDC tolerance in soybean, their corresponding name, the physical location of the marker on the respective soybean chromosome, and the target allele that is associated with IDC tolerance.
Markers of the present invention are described herein with respect to the positions of marker loci in the 8X public build of the Williams82 soybean genome at the SoyBase internet resource (soybase.org/SequenceIntro.php) or USDA at (bfgl.anri.barc.usda.gov/cgi-bin/soybean/Linkage.pl). See Table 1 below.







TABLE 1







Summary of genetic regions associated with IDC.

















SEQ ID NO 






Physical

for DNA 
SEQ ID NOs 
Favorable 



Soybean
Position in

fragment
for exemplary 
Allele/



Chro-
Williams82

comprising SNP
Primer/
Unfavorable


Assay Name
mosome
Genome
Traits
(SNP position)
Probe sequences
Allele
















SY0781AQ
2
2850183
IC_N
 1(255)
 36-37/38-39
G/A


SY0322AQ
2
3091839
IC_N
 2(330)
 40-41/42/43
T/A


SY1300AQ
2
4189924
IC_N
 (61)
 44-45/46-47
C/A


SY0673AQ
3
45098253
ICFLR; IC_N
 4(424)
 48-49/50-51
A/C


SY0674AQ
3
45416367
ICFLR; IC_N
 5(229)
 52-53/54-55
A/G


SY3925
5
8817107
YVA (ICFAR)
 6(201)
 56-57/58-59
T/A


IIY12130
5
8937527
YVA (ICFAR)
 7(201)
 60
G/A


IIY12132
5
8965937
YVA (ICFAR)
 8(201)
 61
G/A


SY0152AQ
5
1035989
IC_R; IC_N
 9(125)
 62-63/64-65
G/A


SY0106A
7
5227284
YVA (ICFAR)
10(191)
 66-67/68-69
G/C


SY0642A
7
5227284
YVA (ICFAR)
11(191)
 70-71/72-73
G/C


IIY13279
7
7735885
YVA (ICFAR)
12(201)
 74-75
A/T


IIY712
9
38965186
YVA (ICFAR)
13(434)
 76-77
A/T


SY1069AQ
9
40300598
YVA (ICFAR)
14(117)
 78-79/80-81
G/A


SY2140AQ
10
44378814
ICFLR; IC_N
15(251)
 82-83/84-85
G/A


SY0498AQ
12
36574820
IC_N
16(477)
 86-87/88-89
G/A


SY3929
13
28861276
ICFLR; IC_N
17(501)
 90-91/92-93
G/A


SY1258AQ
13
29310338
ICFLR; IC_N
18(327)
 94-95/96-97
G/A


SY1091A
13
29702280
YVA (ICFAR)
19(90) 
 98-99/100-101
A/G


SY0132A
13
29825027
YVA (ICFAR)
20(131)
102-103/104-105
C/A


SY0023A
13
30771524
YVA (ICFAR)
21(381)
106-107/108-109
G/A


SY3931
13
31004768
ICFAR
22(501)
110-111/112-113
A/T


SY0121AQ
14
1359785
ICFLR; IC_N
23(619)
114-115/116-117
A/C


SY0224AQ
14
4305821
ICFLR; IC_N
24(187)
118-119/120-121
A/T


SY0386AQ
15
5897794
IC_R; IC_N
25(465)
122-123/124-125
A/G


SY0952AQ
15
7030013
YVA (ICFAR)
26(311)
126-127/128-129
G/A


SY0808AQ
15
32474587
ICFLR
27(292)
130-131/132-133
G/A


IIY6608
16
7372457
YVA (ICFAR)
28(201)
134
A/G


IIY16255
16
7421364
YVA (ICFAR)
29(251)
135
A/G


IIY6613
16
7421475
YVA (ICFAR)
30(201)
136
G/A


IIY6614
16
7441547
YVA (ICFAR)
31(201)
137-138
A/T





YVA (ICFAR; ICRAR; ICFAN





SY0622A
19
40201168
RVA; ICRAN)
32(434)
139-140/141-142
A/C


SY0066AQ
19
40773864
ICFAN
33(568)
143-144/145-146
G/A


SY0632AQ
19
48091800
ICFLR; IC_N
34(81) 
147-148/149-150
A/G





ICFAR; ICRAR; ICRAN;





IIY9605
20
35291114
ICRAN
35(201)
151-152
T/A









Table 2 provides a summary of marker positions that are statistically associated with IDC tolerance. Markers from Table 2 may be used to select, identify or produce soybean plants that are IDC tolerant.







TABLE 2







Summary of marker positions that statistically associate with IDC 


tolerance in soybean. Chromosome physical position points correspond 


with Williams 82 reference sequence (i.e. 8X public build of the 


Williams82 soybean genome at the SoyBase internet resource 


(soybase.org/SequenceIntro.php) or USDA at 


(bfgl.anri.barc.usda.gov/cgi-bin/soybean/Linkage.pl).









Marker Name
Chromosome #
Physical Position












IIY1731
1
2696842


IIY1794
1
3361120


IIY1987
1
48174959


IIY1840
1
39251460


IIY1846
1
39598517


IIY17813
1
41992944


IGGY2483
1
39249687


IIY17388
1
44787967


IIY1935
1
44365728


IIY17427
1
38704520


IIY17110
1
43075379


IGGY2445
1
43025867


IIY2007
1
48469424


IIY381
1
45165995


IIY8883
2
3571878


IIY17463
2
3535661


IIY268
2
2381400


IIY17262
2
3334278


IIY201
2
2144787


IIY8885
2
3574125


IIY8900
2
3798059


IIY8907
2
3826932


IIY8930
2
4130147


IIY17269
2
4152757


IIY8953
2
4225831


IIY15005
2
4333374


IIY18
2
4344854


IIY19423
5
464085


IIY12065
5
528958


IIY12107
5
8590432


IIY14727
5
8680438


IGGY2242
5
8680438


IIY12119
5
8690578


IIY19434
5
8711271


IIY12122
5
8793760


IIY12124
5
8817107


IIY12132
5
8965937


IIY714
5
9097414


IGGY2577
5
24873202


IIY12773
6
9187422


IIY1159
6
9621882


IIY14865
6
10054383


IIY12149
6
10122552


IIY1217
7
4835105


IIY17933
7
5014857


IIY1219
7
5150928


IIY13170
7
5158922


IGGY3022
7
5227284


IIY13183
7
5366035


IIY13185
7
5484851


IIY13189
7
5603441


IIY13191
7
5636973


IIY13197
7
5902793


IIY13206
7
5964630


IIY13209
7
5983847


IIY13221
7
6370540


IGGY821
7
6404645


IGGY1808
7
6404690


IIY13248
7
6775437


IIY1221
7
6850608


IIY13279
7
7735885


IIY13284
7
7807344


IIY1226
7
7813163


IIY18900
7
8346282


IIY328
7
8426971


IIY15798
7
8427812


IIY13323
7
8481040


IIY13334
7
8596256


IIY13340
7
8683036


IIY1260
8
12458183


IIY807
8
12971581


IIY13425
8
13896731


IIY1286
9
3110521


IIY14156
9
3399524


IIY625
9
3447521


IIY18960
9
3822389


IIY14596
9
5464507


IIY18894
9
5568002


IIY14600
9
5569484


IIY20441
12
7941435


IIY20488
12
8011912


IIY3994
12
9772990


IIY600
12
10342367


IIY3377
12
10990890


IIY20714
12
11193280


IGGY2960
12
13635572


IIY4052
13
13666288


IIY4055
13
14552235


IIY4064
13
17544665


IIY4069
13
20039573


IIY141
13
20365685


IIY4072
13
20419690


IIY4073
13
20423675


IIY20140
13
20805842


IIY20141
13
20828611


IIY21977
13
20989581


IIY20302
13
21511291


IIY20413
13
23107929


IIY116
13
23209914


IIY20038
14
2269239


IIY4907
14
2322359


IIY4908
14
2329015


IIY4938
14
2771681


IIY4953
14
2926741


IIY20325
14
3106964


IIY4978
14
3127412


IIY4994
14
3412862


IIY5036
14
3825644


IIY15710
14
4236984


IIY5126
14
4655438


IIY5180
14
4857870


IIY5182
14
4861117


IIY5223
14
5001537


IIY428
14
5028572


IIY5226
14
5040236


IIY19806
14
17652349


IIY19935
14
19494194


IIY4944
14
28146341


IIY20580
15
2316022


IIY5554
15
2805463


IIY20067
15
3796702


IIY5625
15
4108526


IIY20290
15
4174719


IIY6133
16
25570


IIY6561
16
60477


IIY6594
16
68850


IIY6642
16
95735


IIY6112
16
231562


IIY6131
16
253889


IIY6376
16
348886


IIY6536
16
465392


IIY6050
16
1312414


IIY6053
16
1346152


IIY15765
16
1358540


IIY6226
16
3057779


SY2898
16
3560231


IIY6446
16
3648660


IIY6512
16
3887097


IIY18435
16
3964335


IIY18546
16
4825835


IIY14795
16
5384269


IIY18907
16
6125865


IIY6564
16
6247202


IIY6567
16
6411631


IIY6603
16
7287728


IIY18918
16
18715395


IIY6989
17
414639


IGGY1878
17
691058


IIY20836
17
1132147


IGGY131
17
5141900


IIY7134
17
8109126


IIY8052
18
926063


IIY16693
18
993080


IIY7218
18
1087302


IIY21953
18
1107973


IIY21085
18
1140731


IIY7234
18
1156406


IGGY444
18
1229025


IIY7269
18
1376711


IIY7292
18
1541073


IIY7294
18
1544945


IIY22231
18
1638718


IIY7315
18
1686815


IIY7317
18
1690011


IGGY2342
18
1712035


IIY14734
18
1736100


IIY7321
18
1769041


IIY7373
18
2154488


IIY8133
19
2130765


IIY1577
19
4141465


IIY21405
19
7897176


IIY21406
19
8023757


IIY8166
19
31003233


IIY8278
19
39440215


IIY21959
19
39676454


IIY8287
19
39733215


IIY1594
19
39921349


IIY14726
19
40774016


IIY8326
19
40779456


IIY8337
19
41332455


IIY8339
19
41341024


IIY8342
19
41722212


IIY857
19
42821675


IIY9566
20
34401254


IIY9631
20
35770280


IIY9645
20
35929462


IIY21710
20
36062846


IIY9685
20
36462133


IIY15282
15
9652022


IIY15157
15
9777403


IIY6009
15
9779127


IIY6017
15
9860854


IIY6020
15
9907375


IIY5335
15
10437176


IIY5359
15
11416165


IIY3392
12
115957


IIY3538
12
297849


IIY3998
12
994443


IIY3382
12
1112602


IIY3450
12
1827361


IIY3495
12
2385732


IIY151
12
2681306


IIY3532
12
2888033


IGGY174
12
3143935


IIY3571
12
3203743


IIY1395
12
4647654









In some embodiments, any one of the marker allele(s) associated with iron deficiency chlorosis are as set forth in Tables 1 or 2 may be used to identify, select or produce a plant having tolerance to iron deficiency chlorosis.
As would be understood by one of skill in the art, additional chromosomal intervals can be defined by the SNP markers provided herein in Tables 1 or 2.
This invention further provides methods of identifying, selection, and/or producing an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof, comprising: detecting, in said soybean plant or part thereof, the presence of any combination of genetic markers associated with IDC tolerance in a soybean plant, as described in Tables 1 or 2.
In further embodiments, the marker can comprise, consist essentially of or consist of any marker linked to the aforementioned markers. That is, any genetic marker that is in linkage disequilibrium with any of the aforementioned markers, as described in Table. SNPs, chromosome intervals and/or combinations of markers (haplotypes)) may also be used to identify, select and/or produce a soybean plant having IDC tolerance. Linked markers may be determined, for example, by using resources available on the SoyBase website (soybase.org).
The present invention further provides that the detecting of a molecular marker can comprise the use of a nucleic acid probe having a nucleotide base sequence that is substantially complementary to the nucleic acid sequence defining the genetic marker and which nucleic acid probe specifically hybridizes under stringent conditions with a nucleic acid sequence defining the genetic marker. A suitable nucleic acid probe can for instance be a single strand of the amplification product corresponding to the marker. In some embodiments, the detecting of a marker is designed to determine whether a particular allele of an SNP is present or absent in a particular plant.
Additionally, the methods of this invention include detecting an amplified DNA fragment associated with the presence of a particular allele of an SNP, for example as those SNP allele markers identified in Table 1. In some embodiments, the amplified fragment associated with a particular allele of a SNP has a predicted length or nucleic acid sequence, and detecting an amplified DNA fragment having the predicted length or the predicted nucleic acid sequence is performed such that the amplified DNA fragment has a length that corresponds (plus or minus a few bases; e.g., a length of one, two or three bases more or less) to the expected length based on a similar reaction with the same primers with the DNA from the plant in which the marker was first detected or the nucleic acid sequence that corresponds (i.e., has a homology of in some embodiments more than 80%, in some embodiments more than 90%, in some embodiments more than 95%, in some embodiments more than 97%, and in some embodiments more than 98% or 99%) to the expected sequence based on the sequence of the marker associated with that SNP in the plant in which the marker was first detected.
The detection of an amplified DNA fragment having the predicted length or the predicted nucleic acid sequence can be performed by any of a number or techniques, including, but not limited to, standard gel-electrophoresis techniques or by using automated DNA sequencers. These methods are not described here in detail as they are well known to those of ordinary skill in the art, although exemplary approaches are set forth in the Examples.
As shown in Table 1, the SNP markers of this invention are associated with IDC tolerance. In some embodiments, as described herein, one marker or a combination of markers can be used to detect the presence of an IDC tolerant plant. In some embodiments, a marker can be located within a chromosomal interval (e.g. QTL) or be present in the genome of the plant as a haplotype as defined herein.
Thus, methods for identifying and/or selecting a soybean plant or germplasm comprising IDC tolerance comprise detecting the presence of a genetic marker (e.g., SNP, SNP located in chromosomal interval (QTL) and/or combination of SNPs) associated with IDC tolerance in a soybean plant or part thereof. Thus, the genetic marker can be detected in any sample taken from the soybean plant or from a soybean germplasm, including, but not limited to, the whole plant or germplasm or any part thereof (e.g., a seed, a leaf, a tissue culture, a cell, etc.).
In another aspect of the invention, a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof is provided, the method comprising: detecting, in said soybean plant or part thereof, the presence of a marker associated with IDC tolerance (e.g as shown in Table 1) in a soybean plant, wherein the IDC tolerance is exhibited as reduced yellow flash symptoms.
In other embodiments of this invention, a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof is provided, the method comprising: detecting, in said soybean plant or part thereof, the presence of a marker associated with IDC tolerance in a soybean plant (e.g. such as those described in Table 1), wherein the IDC tolerance is exhibited as reduced yellow flash symptoms, and the marker is associated with reduced yellow flash symptoms in a soybean plant.
In a further aspect, a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof is provided, the method comprising: detecting, in said soybean plant or part thereof, the presence of a marker associated with IDC tolerance in a soybean plant, wherein the IDC tolerance is exhibited as recovery from yellow flash, and the marker is associated with recovery from yellow flash in a soybean plant and is located within a chromosomal interval of: chromosomal interval on chromosome 5 defined by and including a T allele at IIY12124, a G allele at IIY12130, a A allele at IIY12132 or any combination thereof; (b) a chromosomal interval on chromosome 7 defined by and including a A allele at IIY13279, a G allele at SY0106A, a G allele at SY0642A, or any combination thereof; (c) a chromosomal interval on chromosome 13 defined by and including a C allele at SY0132A, a A allele at SY1091A, or any combination thereof; and (k) any combination of (a) through (c) and selecting a progeny soybean plant or germplasm that possesses said marker within its genome, thereby identifying and/or selecting an IDC tolerant soybean plant or part thereof.
In a further aspect, a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof is provided, the method comprising: detecting, in said soybean plant or part thereof, the presence of a marker associated with IDC tolerance in a soybean plant, wherein the IDC tolerance is exhibited as recovery from yellow flash, and the marker is associated with recovery from yellow flash in a soybean plant and is located within a chromosomal interval of as indicated by any combination of one or more SNP markers as indicated in Tables 1 or 2.
The present invention additionally provides a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof, the method comprising: detecting, in said soybean plant or part thereof, the presence of a marker associated with IDC tolerance in a soybean plant, wherein the IDC tolerance is exhibited as recovery from yellow flash, and the marker is associated with recovery from yellow flash in a soybean plant.
Another embodiment of the invention comprises the use of one or more markers to identify, select or create a soybean plant that are tolerant or nontolerant (listed respectfully “tolerant allele or intolerant allele) to IDC the one or more markers selected from the group consisting of the following alleles: (a) a A allele at IIY712; (b) a A allele at SY0622A; (c) a G allele at SY0023A; (d) an T allele at IIY9605; (e) a T allele at IIY12124; (f) a G allele at IIY12132; (g) a G allele at IIY12130; (h) a A allele at IIY13279; (i) a G allele at SY0106A; (j) a G allele at SY0642A; (k) a C allele at SY0132A; (l) a A allele at SY1091A; (m) a A allele at IIY16255; (n) a A allele at IIY6614; (o) a A allele at IIY6608; (p) a G allele at IIY6613; and any combination of (a) through (p) above
In another aspect of the invention a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant or part thereof is provided, the method comprising: detecting, in said soybean plant or part thereof, the presence of a marker associated with IDC tolerance in a soybean plant, wherein the IDC tolerance is exhibited as reduced yellow flash and recovery from yellow flash, and the marker is associated with reduced yellow flash and recovery from yellow flash in a soybean plant and is associated with an allele comprising: (a) a A allele at IIY712; (b) a A allele at SY0622A; (c) a G allele at SY0023A; (d) an T allele at IIY9605; (e) a T allele at IIY12124; (f) a G allele at IIY12132; (g) a G allele at IIY12130; (h) a A allele at IIY13279; (i) a G allele at SY0106A; (j) a G allele at SY0642A; (k) a C allele at SY0132A; (l) a A allele at SY1091A; (m) a A allele at IIY16255; (n) a A allele at IIY6614; (o) a A allele at IIY6608; (p) a G allele at IIY6613; and any combination of (a) through (p) above.
In one embodiment, one may select for IDC markers within specific regions of the Soybean genome these regions comprise (+/−10-20 nucleotides from each relative position within said interval) (a) a chromosomal interval consisting of positions 4.94 to 6.15 on Soybean chromosome 5; (b) a chromosomal interval consisting of positions 7.04-26.97 on Soybean chromosome 14; (c) a chromosomal interval consisting of positions 32.70-36.69 on Soybean chromosome 6; (d) a chromosomal interval consisting of positions 72.72 or 93.69 on Soybean chromosome 1; (e) a chromosomal interval consisting of positions 22.10-54.83 on Soybean chromosome 2; (f) a chromosomal interval consisting of positions 99.70-132.16 on Soybean chromosome 17; (g) a chromosomal interval consisting of positions 31.99-95.92 on Soybean chromosome 15; (h) a chromosomal interval consisting of positions 77.49-92.55 on Soybean chromosome 13; (i) a chromosomal interval consisting of positions 127.05-129.01 on Soybean chromosome 18; (j) a chromosomal interval consisting of positions 91.92-117.61 on Soybean chromosome 12; (k) a chromosomal interval consisting of positions 69.90-75.67 on Soybean chromosome 9; (l) a chromosomal interval consisting of positions 65.52-69.09 on Soybean chromosome 19; (m) a chromosomal interval consisting of positions 105.76-113.05 on Soybean chromosome 3; (n) a chromosomal interval consisting of position 116.87 on Soybean chromosome 10 and (o) any combination of markers selected from the chromosome intervals as stated in (a)-(n) above.
As described herein, methods for identifying and/or selecting a soybean plant or germplasm having IDC tolerance can comprise detecting the presence of a marker or a combination of markers associated with IDC tolerance. Any combination of the genetic markers of this invention can be used to identify and/or select a soybean plant or germplasm having IDC tolerance.
As described herein, in some aspects of this invention, the reduced yellow flash symptoms and/or recovery from yellow flash are exhibited by the soybean plant when the soybean plant is grown in calcareous soil having a pH greater than 7.5 and the marker is associated with reduced yellow flash symptoms and/or recovery from yellow flash in a soybean plant when the soybean plant is grown in calcareous soil having a pH greater than 7.5. In other embodiments the markers may be used to select, identify, and introgress IDC tolerant soy line in either soil type 1 or soil type 2.
Accordingly, some embodiments of the present invention provide a method of identifying and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant, wherein the IDC tolerance is exhibited as reduced yellow flash symptoms and/or recovery from yellow flash when the plant is grown calcareous soil having a pH greater than 7.5, and the marker (e.g., SNP allele, combination of SNP alleles and/or SNP allele located in a chromosome interval) is associated with reduced yellow flash symptoms and/or recovery from yellow flash in a soybean plant grown in calcareous soil having a pH greater than 7.5.
Marker-Assisted Selection
The subject matter disclosed herein also relates to methods for producing IDC tolerant soybean plants comprising detecting the presence of an allele associated with IDC tolerance in a donor soybean plant according to the methods as described herein and transferring a nucleic acid sequence comprising at least one allele thus detected from the donor plant to an IDC intolerant recipient soybean plant. The transfer of the nucleic acid sequence can be performed by any of the methods described herein.
Thus, the present invention encompasses methods of plant breeding and methods of selecting/identifying plants, in particular soybean plants, particularly cultivated soybean plants as breeder plants for use in breeding programs or cultivated soybean plants having desired genotypic or potential phenotypic properties, in particular related to producing valuable soybeans, also referred to herein as commercially valuable plants. Herein, a cultivated plant is defined as a plant being purposely selected or having been derived from a plant having been purposely selected in agricultural or horticultural practice for having desired genotypic or potential phenotypic properties, for example, a plant obtained by inbreeding. It is also understood by those skilled in the art that it is of equal value to be able to select for plants that are not tolerant to IDC in for example, a Soybean plant breeding program.
The presently disclosed subject matter thus also provides methods for selecting a plant of the genus Glycine exhibiting tolerance to iron deficiency chlorosis (IDC) comprising detecting in the plant the presence of one or more genetic markers associated with IDC tolerance as defined herein. In an exemplary embodiment of the presently disclosed methods for selecting such a plant, the method comprises providing a sample of genomic DNA from a soybean plant; and (b) detecting in the sample of genomic DNA at least one genetic marker associated with IDC tolerance. In some embodiments, the detecting can comprise detecting one or more SNPs, a combination of SNPs (haplotype), and/or SNPs located in chromosomal intervals that are associated with IDC tolerance.
The providing of a sample of genomic DNA from a soybean plant can be performed by standard DNA isolation methods well known in the art.
As is well known in the art, the detecting of a genetic marker can in some embodiments comprise the use of one or more sets of primer pairs that can be used to produce one or more amplification products that are suitable for identifying, for example, a SNP. In exemplary embodiments of this invention, the nucleotide sequences comprising the genetic markers (SNPs) and probes for the detection of respective markers are provided in Table 1.
In some embodiments of this invention, a method is provided, said method comprising the transfer by introgression of the nucleic acid sequence from an IDC tolerant donor soybean plant into an IDC intolerant recipient soybean plant by crossing the plants. This transfer can be accomplished by using traditional breeding techniques. IDC tolerant loci are introgressed in some embodiments into commercial soybean varieties using marker-assisted selection (MAS) or marker-assisted breeding (MAB). MAS and MAB involves the use of one or more of the molecular markers, identified as having a significant likelihood of co-segregation with a desired trait, and used for the identification and selection of those offspring plants that contain one or more of the genes that encode for the desired trait. As disclosed herein, such identification and selection is based on selection of one or more SNP alleles of this invention or markers associated therewith. MAB can also be used to develop near-isogenic lines (NIL) harboring one or more IDC tolerance alleles of interest, allowing a more detailed study of an effect of such allele(s), and is also an effective method for development of backcross inbred line (BIL) populations. Soybean plants developed according to these embodiments can in some embodiments derive a majority of their traits from the recipient plant and derive IDC tolerance from the donor plant. MAB/MAS techniques increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS) or marker-assisted breeding (MAB).
Thus, traditional breeding techniques can be used to introgress a nucleic acid sequence associated with IDC tolerance into an IDC intolerant recipient soybean plant. For example, inbred IDC tolerant soybean plant lines can be developed using the techniques of recurrent selection and backcrossing, selfing, and/or dihaploids, or any other technique used to make parental lines. In a method of recurrent selection and backcrossing, IDC tolerance can be introgressed into a target recipient plant (the recurrent parent) by crossing the recurrent parent with a first donor plant, which differs from the recurrent parent and is referred to herein as the “non-recurrent parent.” The recurrent parent is a plant that is IDC intolerant or has a low level of IDC tolerance and, in some embodiments, possesses commercially desirable characteristics, such as, but not limited to disease and/or insect resistance, valuable nutritional characteristics, valuable abiotic stress tolerance (including, but not limited to, drought tolerance, salt tolerance), and the like. In some embodiments, the non-recurrent parent exhibits IDC tolerance and comprises a nucleic acid sequence that is associated with IDC tolerance. The non-recurrent parent can be any plant variety or inbred line that is cross-fertile with the recurrent parent.
In some embodiments, the progeny resulting from a cross between the recurrent parent and non-recurrent parent are backcrossed to the recurrent parent. The resulting plant population is then screened for the desired characteristics, which screening can occur in a number of different ways. For instance, the population can be screened using phenotypic pathology screens or quantitative bioassays as known in the art. Alternatively, instead of using bioassays, MAB can be performed using one or more of the hereinbefore described molecular markers, hybridization probes, or polynucleotides to identify those progeny that comprise a nucleic acid sequence associated with IDC tolerance. Also, MAB can be used to confirm the results obtained from the quantitative bioassays. In some embodiments, the markers defined herein are suitable to select proper offspring plants by genotypic screening.
Following screening, the F1 hybrid plants that exhibit an IDC tolerance phenotype or, in some embodiments, the genotype, and thus comprise the requisite nucleic acid sequence associated with IDC tolerance, can be then selected and backcrossed to the recurrent parent for one or more generations in order to allow for the soybean plant to become increasingly inbred. This process can be performed for one, two, three, four, five, six, seven, eight, or more generations.
Thus, a marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay or occurs at a late stage in plant development. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant plant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene that causes or imparts the trait. In addition, having flanking markers can decrease the chance that false positive selection will occur. Ideally, a marker is in the gene itself, so that recombination cannot occur between the marker and the gene. Such a marker is called a “perfect marker.”
The availability of integrated linkage maps of the soybean genome containing increasing densities of public soybean markers has facilitated soybean genetic mapping and MAS. See, e.g. soybeanbreederstoolbox.org, which can be found on the SoyBase website (soybase.org).
Of all the molecular marker types, SNPs are the most abundant and have the potential to provide the highest genetic map resolution (Bhattramakki et al., Plant Molec. Biol. 48:539 (2002)). SNPs can be assayed in a so-called “ultra-high-throughput” fashion because they do not require large amounts of nucleic acid and automation of the assay is straight-forward. SNPs also have the benefit of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing and coded spheres. Such methods have been reviewed in various publications: Gut, Hum. Mutat. 17:475 (2001); Shi, Clin. Chem. 47:164 (2001); Kwok, Pharmacogenomics 1:95 (2000); Bhattramakki and Rafalski, Discovery and application of single nucleotide polymorphism markers in plants, in PLANT GENOTYPING: THE DNA FINGERPRINTING OF PLANTS, CABI Publishing, Wallingford (2001). A wide range of commercially available technologies utilize these and other methods to interrogate SNPs, including MasscodeTM (Qiagen, Germantown, Md.), Invader® (Hologic, Madison, Wis.), SnapShot® (Applied Biosystems, Foster City, Calif.), Taqman® (Applied Biosystems, Foster City, Calif.) and BeadarraysTM (Illumina, San Diego, Calif.).
Accordingly, the markers of the present invention can be used in marker-assisted selection methods to identify and/or select and/or produce progeny having a genetic marker associated with IDC tolerance. Thus, in some embodiments, the present invention relates to methods for producing soybean plants having an IDC tolerance associated allele comprising detecting the presence of at least one allele associated with IDC tolerance in a donor soybean plant as described herein, crossing the donor soybean plant with a second soybean plant or germplasm, and detecting in the progeny plant(s) the presence of said at least one allele, thereby transferring the at least one allele thus detected from the donor plant to the second soybean plant and thus producing a soybean plant having IDC tolerance. In some embodiments, the second plant is IDC intolerant. The transfer of the allele can be performed by any of the methods described herein.
Embodiments of the invention provides a method of identifying, selecting or producing an iron deficiency chlorosis (IDC) tolerant soybean plant through any one or a combination of the markers as set forth in Table 2.
One embodiment of the invention is the use of at least one marker from Table 1 associated with IDC in a soybean plant breeding program.
In some embodiments, the second soybean plant or germplasm of this invention is of an elite variety of soybean. In some embodiments, the genome of the second soybean plant or germplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that of an elite variety of soybean.
In other embodiments, the present invention provides a method of introgressing a combination of genetic markers associated with iron deficiency chlorosis (IDC) tolerance into a genetic background lacking said combination of markers, comprising: crossing a donor comprising said combination of markers with a recurrent parent that lacks said combination of markers; and backcrossing progeny comprising said combination of markers with the recurrent parent, wherein said progeny are identified by detecting, in their genomes, the presence of said combination of markers associated with IDC tolerance in a soybean plant, wherein said combination of genetic markers comprises any marker shown in Tables 1 or 2 and any combination thereof, thereby producing an IDC tolerant soybean plant or germplasm comprising said combination of markers associated with IDC tolerance in the genetic background of the recurrent parent, thereby introgressing the combination of markers associated with IDC tolerance into a genetic background lacking said combination of markers.
Accordingly, some embodiments of the present invention provide a method of producing and/or selecting an iron deficiency chlorosis (IDC) tolerant soybean plant, wherein the IDC tolerance is exhibited as reduced yellow flash symptoms and/or recovery from yellow flash when the plant is grown in calcareous soil having a pH greater than 7.5, and the marker (e.g., SNP allele, combination of SNP alleles, SNP allele located in a chromosome interval) is associated with reduced yellow flash symptoms and/or recovery from yellow flash in a soybean plant grown in calcareous soil having a pH greater than 7.5. In other aspects the markers may be used to identify, create or select soy lines that may be grown on soil type 1, soil type 2 or both.
The present invention provides soybean plants and germplasms having IDC tolerance. As discussed above, the methods of the present invention can be utilized to identify, produce and/or select a soybean plant or germplasm having IDC tolerance. In addition to the methods described above, a soybean plant or germplasm having IDC tolerance may be produced by any method whereby a marker associated with IDC tolerance (for exemplary one or more of the markers identified in Table 1) is introduced into the soybean plant or germplasm by such methods that include, but are not limited to, transformation (including, but not limited to, bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria)), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, microparticle bombardment, electroporation, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into the plant cell, or any combination thereof), protoplast transformation or fusion, a double haploid technique, embryo rescue, or by any other nucleic acid transfer system.
“Introducing” in the context of a plant cell, plant and/or plant part means contacting a nucleic acid molecule with the plant, plant part, and/or plant cell in such a manner that the nucleic acid molecule gains access to the interior of the plant cell and/or a cell of the plant and/or plant part (“Introducing” encompasses introgression, gene editing and/or transgenic expression). Where more than one nucleic acid molecule is to be introduced, these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, e.g., as part of a breeding protocol. Thus, the term “transformation” as used herein refers to the introduction of a heterologous nucleic acid into a cell.
Thus, a soybean plant, or part thereof, having a genetic marker associated with IDC tolerance, obtainable by the methods of the presently disclosed subject matter, are aspects of the presently disclosed subject matter. The soybean plant, or part thereof, or soybean germplasm of this invention having a genetic marker associated with IDC tolerance can be heterozygous or homozygous for the genetic marker.
In some embodiments, the soybean plant or germplasm may be the progeny of a cross between an elite variety of soybean and a variety of soybean that comprises an allele associated with IDC tolerance. In some embodiments, the soybean plant or germplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to that of an elite variety of soybean.
The soybean plant or germplasm may be the progeny of an introgression wherein the recurrent parent is an elite variety of soybean and the donor comprises a genetic marker associated (e.g., SNP, combination of SNPs, SNP located in a chromosome interval) with IDC tolerance as described herein (e.g. such as markers disclosed in Table 1).
The soybean plant or germplasm may be the progeny of a cross between a first elite variety of soybean (e.g., a tester line) and the progeny of a cross between a second elite variety of soybean (e.g., a recurrent parent) and a variety of soybean that comprises a genetic marker associated with IDC tolerance as described herein (e.g., a donor).
The soybean plant or germplasm may be the progeny of a cross between a first elite variety of soybean and the progeny of an introgression wherein the recurrent parent is a second elite variety of soybean and the donor comprises a genetic marker associated with IDC tolerance.
Another aspect of the presently disclosed subject matter relates to a method of producing seeds that can be grown into IDC tolerant soybean plants. In some embodiments, the method comprises providing an IDC tolerant soybean plant of this invention (e.g. via use of IDC markers as disclosed in Table 2), crossing the IDC tolerant soybean plant with another soybean plant, and collecting seeds resulting from the cross, which when planted, produce IDC tolerant soybean plants.
Accordingly, the present invention provides improved soybean plants, seeds, and/or soybean tissue culture produced by the methods described herein.
In some embodiments, the presently disclosed subject matter provides methods for analyzing the genomes of soybean plants/germplasms to identify those that include desired markers associated with IDC tolerance. In some embodiments, the methods of analysis comprise amplifying subsequences of the genomes of the soybean plants/germplasms and determining the nucleotides present in one, some, or all positions of the amplified subsequences.
Thus, in some embodiments, the present invention provides compositions comprising one or more amplification primer pairs capable of initiating DNA polymerization by a DNA polymerase on a Glycine max nucleic acid template to generate a Glycine max amplicon. In some embodiments, the Glycine max marker amplicon corresponds to Glycine max marker comprising a nucleotide sequence of any of SEQ ID NOs: 1-16. In view of the disclosure of SEQ ID NOs: 1-16 being linked to IDC tolerance loci, one of ordinary skill in the art would be aware of various techniques that could be employed to analyze the sequences of the corresponding soybean nucleic acids.
The following examples are included to demonstrate various embodiments of the invention and are not intended to be a detailed catalog of all the different ways in which the present invention may be implemented or of all the features that may be added to the present invention. Persons skilled in the art will appreciate that numerous variations and additions to the various embodiments may be made without departing from the present invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.
EXAMPLES
Example 1. QTL Mapping and Phenotyping Soybean Plant Material
Syngenta soybean plant materials were used to develop the iron deficiency chlorosis (IDC) quantitative trait loci (QTL) mapping populations. The parent populations were either IDC tolerant or IDC intolerant soybean materials based upon phenotyping of the population and knowledge of the germplasm. The parent materials classifications are provided in Table 2.
A connected structure of populations was fashioned from the parent materials. Tables 3a and 3b shows the generation, harvest method, timeline, and nursery location of the QTL population. Finally, checks were chosen based upon breeding experience and product knowledge. The phenotyping check populations are listed in Table 4.







TABLE 3a







Parent materials classifications










Parental
IDC Tolerance



Material
Classification







03DL052038
Tolerant



04KL108888
Tolerant



9378
Intolerant



1162
Intolerant



9428
Intolerant



5763
Intolerant



1519
Intolerant



1531
Intolerant

















TABLE 3b







Population development












Harvest




Generation
Method
Timeline







Crossing
Bulk
Summer Year 1



F1 Plants
Bulk
Fall Year 1 - Winter





Year 2



F2 Plants
SSD*
Spring Year 2



F3 Plants
SSD*
Summer Year 2



F4 Plants
Plant pull
Fall Year 2 - Winter





Year 3







*SSD = Single Seed Descent













TABLE 4







IDC phenotyping of check populations.










Tolerant Checks
Intolerant Checks







03DL052038
1107



2251
8295



4015
8413



8047
8851



0011
1285










Example 2. Experiment Design and Phenotyping
The eleven F4 populations were arranged into eleven—two replicate, three location, IDC phenotyping experiments. The same ten phenotyping checks/controls were used in all experiments. The experimental design was Randomized Complete Block (RCB), which also included a repeating intolerant check (material 8314) occurring every 10th hill.
The three planting locations were used: Truman, Minn.; Ogden, Iowa; and Fort Dodge, Iowa. The field area at each site was prepared with a 48 inch wide rotary tiller just prior to planting to remove compaction.
The plots were kept weed free throughout the life of the experiment; however, no Post-Emergence herbicide was used. The planter's four row units were spaced 10 inches apart and the hills were placed every 15 inches down the row to minimize the field size needed. Six seeds per hill (replicates) were planted. The 12 experiments were contiguously arranged in a block. Experimental replicates were blocked and mapped adjacent to each other. The hills within replicates were arranged in a serpentine fashion
Plants were evaluated for IDC visually and by electronic scanning (radiometer). Table 5, below, summarizes the trait codes, description, type, minimum and maximum values for each type of measurement, and the calculation (formula) when applicable that were used in the evaluations. At approximately the V2 stage of growth, the hills were visually rated and canopy reflectance measured (or NDVI (Normalized Difference Vegetation Index)) with a Greenseeker® RT100 radiometer. The visual rating and NDVI measurement were repeated 14 days later. These times, V2 stage and 14 days later, correspond to IDC yellow flash symptom and recovery reaction times, respectively.
As shown in Table 5, ICFLR and ICFLN are codes that identify the Yellow Flash ratings for visual and radiometer, respectively. Likewise, ICR_R and ICR_N are codes that identify the Recovery for the visual ratings and the radiometer number, respectively. IC_R and IC_N are codes that identify the mean of the yellow flash and recovery data for the visual ratings and the radiometer number, respectively. The visual ratings scale was 1-9 with 1 being the best (no chlorosis) and 9 being the worst (plant death). Arithmetic averages of the visual and radiometer traits were calculated. Table 6 shows the results of a single experiment.







TABLE 5







Phenotyping Traits













Trait

Type of
Type of
Minimum
Maximum



Code
Description
Measurement
Measurement*
Value
Value
Calculation





IC_N
Mean of Flash
Radiometry
Measured
0
1
ICFLN + 



and Recovery




ICR_N)/2


IC_R
Mean of Flash
Visual
Measured
1
9
ICFLR + 



and Recovery




ICR_R)/2


IC_AN
Mean of Flash
Radiometry
Adjusted
0
1
ICFAN + 



and Recovery




ICR_AN)/2


IC_AR
Mean of Flash
Visual
Adjusted
1
9
ICFAR + 



and Recovery




ICR_AR)/2


ICFAN
Flash
Radiometry
Adjusted
0
1



ICFAR
Flash
Visual
Adjusted
1
9



ICFLN
Flash
Radiometry
Measured
0
1



ICFLR
Flash
Visual
Measured
1
9



ICR_N
Recovery
Radiometry
Measured
0
1



ICR_R
Recovery
Visual
Measured
1
9



ICRAN
Recovery
Radiometry
Adjusted
0
1



ICRAR
Recovery
Visual
Adjusted
1
9





*Indicates whether the phenotypic data was adjusted by the surface analysis utility as discussed in Example 5.













TABLE 6







Phenotyping results from a single experiment (sorted by IC_R).












Visual
Radiometer














ENTRY
Material
IC_R
ICFLR
ICR_R
IC_N
ICFLN
ICR_N

















42
03DL052038
1.7
2.2
0.6
0.474
0.441
0.514



Tolerant Control








21

2
2.5
0.9
0.473
0.466
0.509


3

2.2
2.7
0.9
0.455
0.399
0.514


20

2.2
2.9
0.6
0.415
0.408
0.452


37
2251 Tolerant Control
2.2
2.5
1.3
0.469
0.447
0.507


16

2.7
2.4
2.3
0.454
0.45
0.495


44
4015 Tolerant Control
3
3
1.9
0.415
0.398
0.49


8

3
3.7
1.3
0.447
0.453
0.475


39
8047 Tolerant Control
3
3.5
1.6
0.389
0.376
0.457


31

3.5
4
1.9
0.433
0.369
0.495


14

3.7
4.4
1.6
0.448
0.444
0.48


33
0011 Tolerant Control
3.7
3.1
3.6
0.171
0.177
0.164


13

3.8
4.5
1.9
0.397
0.392
0.445


30

3.8
4.2
2.6
0.308
0.382
0.276


22

4
4.3
2.6
0.398
0.404
0.437


35

4
4.1
2.6
0.462
0.469
0.508


7

4.3
3.7
3.9
0.435
0.468
0.455


5

4.3
4.2
3.3
0.379
0.417
0.38


10

4.3
4.5
3.3
0.395
0.417
0.409


15

4.5
4.4
3.3
0.417
0.435
0.461


19

4.5
4.8
2.9
0.383
0.388
0.425


4

4.7
4.8
3.6
0.37
0.368
0.397


6

4.7
5
2.9
0.275
0.43
0.173


24

4.7
4.2
3.9
0.319
0.382
0.351


18

5
5.2
3.6
0.322
0.351
0.337


2

5.2
5.2
3.9
0.355
0.383
0.408


32

5.3
4.7
4.6
0.319
0.378
0.345


36

5.3
5.2
4.3
0.23
0.283
0.2


9

5.5
5
4.6
0.397
0.365
0.461


17

5.5
4.5
5.3
0.336
0.352
0.371


27

5.5
4.7
4.9
0.38
0.41
0.417


25

5.8
5.5
4.6
0.335
0.378
0.359


28

5.8
5
5.3
0.351
0.381
0.385


40
1107 Intolerant Control
5.8
5.7
4.9
0.331
0.355
0.353


29

6
6.2
4.6
0.34
0.367
0.389


11

6.2
4.9
6.3
0.235
0.291
0.301


41
8295 Intolerant Control
6.2
5.8
5.6
0.325
0.376
0.352


1
8413 Intolerant Control
6.2
5.1
5.5
0.345
0.392
0.37


26

6.3
5.2
5.9
0.224
0.31
0.255


43
8851 Intolerant Control
6.3
5.9
5.9
0.271
0.322
0.295


38
1285 Intolerant Control
6.7
5.5
6.6
0.238
0.248
0.257



Mean General
4.6
4.4
3.6
0.362
0.384
0.391



Mean Control
2.7
2.9
1.8
0.383
0.368
0.426



Trials w/data
2
3
2
2
3
2



Entries w/data
41
41
41
41
41
41



LSD General (5%) EE
2
1.5
2.8
0.161
0.106




LSD* Control (5%) EC
1.5
1.1
2.1
0.125
0.082




CV** (Effective) %
22
19.1
39.4
22.175
16.957
31.129





*LSD = Least significant different;


**CV = Coefficient of Variation






The IDC phenotyping results in Table 6 indicate that at 95% confidence level, significant differences were detected between materials/entries. LSD General (5%) EE and LSD Control (5%) EC statistics allow entry to entry or entry to control comparisons, respectively. The results also indicate that significant differences are detected in the traits within visual and radiometer phenotyping.
Example 3. Classification of IDC Prone Soils
Soil samples were collected from eight IDC phenotyping locations in Nebraska, Iowa, Minnesota and North Dakota. These samples were collected from field spots in which non-IDC tolerant soybean plants show IDC symptoms. These soils samples were analyzed for standard soil nutrients, salts, and pH at Mid-West Laboratories, Omaha NB. The data from these soil samples was analyzed for Principal Component Analysis (PCA). PCA is a multivariate analysis which can be used to reveal patterns or clusters in multivariate data. Principal component 1 and Principal component 2 revealed two main distinct clusters for these soil samples. Soils samples collected from Iowa-Southern Minnesota and North Dakota-Northern Minnesota were grouped in two distinct clusters (soil type 1 and soil type 2 respectively). A location from Nebraska did not group with any of these two clusters. This analysis indicated that soil conditions and their properties which cause IDC can be grouped into three classes-Iowa-Southern Minnesota type soils, North Dakota-Northern Minnesota type soils and Nebraska type soils.
Example 4. Genotyping of the IDC QTL Population
All parents of the populations identified in Example 1 were fingerprinted with genome wide SNP markers. The fingerprinting data on the parents was used to determine polymorphic SNPs for each population. Only suitable polymorphic SNPs were genotyped for each population. Table 7 provides the number of markers used to genotype each population.







TABLE 7







The number of genotyping markers.










Population 

Number of recombinant 
Number of 


Number
Pedigree
inbred lines (RILs)
SNPs













1
04KL108888/9428
60
193


2
04KL108888/1162
29
195


3
9378/03DL052038
80
192


4
5763/03DL052038
81
202


5
1519/03DL052038
53
147


6
9428/03DL052038
52
183


7
1531/03DL052038
64
153


8
1162/1519
85
199


9
9378/9428
45
 54


10
1531/9378
83
132


11
1531/1162
41
132









The tissue of recombinant inbred lines (RILs) was obtained by growing them in the field or greenhouse. DNA was extracted from the leaf tissue of 7-10 day old seedlings (7-10 days after planting). DNA can be extracted from plant tissue in any way known in the art, including the CTAB (hexadecyltrimethylammonium bromide) method (See, e.g., Stewart et al., BioTechniques 14(5):748-749 (1993)), sodium hydroxide, and the Dellaporta method (Dellaporta et al., Plant Mol. Biol. Rep. 1:19-21 (1983)). See also, Sambrook & Russell Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., United States of America (2001)) for additional DNA extraction methods. DNA is diluted in TE buffer and stored at 4° C. until used in PCR reactions as described below in Table 8.







TABLE 8







PCR was set up in 5 μl final volumes according to the following formula.












Stock con-
Per reaction 
For 96 
Final con-


Reagent
centration
(μl)
samples (μl)
centration














2× Master Mix 
2×
2.5
296.88
1×


(JumpStart TM






Taq ReadyMix TM)






AbD primer/
40×
.0625
6
0.5×


probe mix (80×)






PCR-quality H2O
—
2.44
234.24
—


DNA (dried in 384)
4.5 ng/μl
4
—
3.6 ng/μl 






(18 ng)


Final Volume (ul)
—
5.00
357.44
—









The Master Mix is JumpStartTM Taq ReadyMixTM (Sigma Catalogue No. 2893; Sigma Chemical Co., St. Louis, Mo., United States of America), a premix of all the components, including nucleotides and Taq polymerase (but not primers and/or probes) necessary to perform a 5′-nuclease assay. Before use, 1375 μl of 1.0 M MgCl2 (Sigma Catalogue No. M1028) and 250 μl of 300 μM Sulforhodamine 101 (Sigma Catalogue No. S7635), also known as ROX, are added to a 125 mL bottle of JumpStartTM Taq ReadyMixTM. PCR plates were placed in an ABI 9700 thermal cycler and the program set forth in Table 9 was run:







TABLE 9







PCR program.










Task
SNP1







Initial denaturation
50° C. for 2 mm; followed 




by 95° C. for 10 min



Cycles
95° C. for 15 sec




60° C. for 1 min



Number of cycles
40



Final elongation
72° C. for 5 min



Hold
Hold at 4° C.










The ABI 7900 Sequence Detection System (or Taqman®) was used to visualize the results of an allelic discrimination (SNP) assay. Using the Sequence Detection System (SDS) software, allele calls were made based on the fluorescence for the two dyes measured in each sample.
Example 5. Phenotypic Data Analysis
The raw data was analyzed using fixed effects analysis of variance (ANOVA), with the traits and populations kept separate. Populations were phenotyped with two replicates at two locations in Iowa. The model below was used, allowing testing for material ID*location interactions. Least square means within and across locations used as phenotype variables for Quantitative Trait Locus (QTL) analysis.

IDC trait=location+material ID+material ID*location+error.

Since the potential severity of IDC is related to spatially variable soil properties, statistical methods that can reduce the effects of this variability are important to increase the ability to detect QTL. Software containing a surface analysis utility was used to perform spatial adjustments based on the phenotype of a repeated check planted throughout the evaluation trial. This tool was used as a way to reduce spatial effects caused by differing potentials for IDC development across different areas of the phenotyping locations. If surface analysis could not detect the spatial patterns in phenotypic data, it returned the original, measured values. This leads to high correlations between the original measured and surface adjusted values. Therefore, comparisons between measured and surface analysis adjusted phenotype data were performed using pair-wise correlations of means across locations in the statistical analysis software package, JMP.
Across the mapping populations, 62 out of 66 comparisons (representing different combinations of IDC trait (e.g., yellow flash, recovery and mean)) had correlations of 0.98, 0.99, or 1.0. The remaining four comparisons were all from one mapping population. They had correlation coefficients ranging from 0.29 to 0.94. Regardless of the level of correlation, all traits whether surface-analyzed or from ANOVA were used in the QTL analysis.
Example 6. QTL Analysis Using Network Population Mapping (NPM)
To detect QTLs for IDC tolerance, Network Population Mapping analysis was performed using Syngenta software and analysis method (See, US Patent Publication No. 20100269216). This method is superior to standard bi-parental QTL mapping in that it uses multiple mapping populations (termed connected networks) that are designed so that the mapping parents are used in multiple populations. This design results in greater statistical power to detect QTL, since individuals across all populations are used for testing for the presence of QTL.
The population network was analyzed using the NPM method, with 1000 permutations performed to empirically determine a 0.05 significance threshold for every trait, rather than arbitrarily choosing a significance threshold. In the final analysis, trait-location combinations with very low heritability of 0.2 or less were excluded from some populations, which increased the number and significance of detected QTL.
The raw results from NPM analysis were processed using an internally developed SAS script. The output from the script was used to create summarized reports for QTL that passed the permutation test.
The network detected multiple QTL across the soy genome. Two important values in QTL studies are the LOD (logarithm of odds) and R2. A higher LOD value represents greater statistical evidence for the presence of a QTL, and a higher R2 indicates that the particular QTL has more effect on the trait of interest. The maximum LOD was 20.3, and the maximum R2 was 0.65.
Example 7. Selecting QTL of High Confidence
From the large number of QTL observed, a subset of high confidence QTL was selected. For example, in one case, QTL could be found for only one trait-location combination at a marginal significance level, and would thus be of limited utility for marker-assisted breeding. Thus, this QTL was not included in the high confidence subset. In other cases, QTL were found that had a marginal LOD score but a suspiciously high R2 value.
Thus, the following criteria were used to prioritize QTL regions and QTL were retained if:
(1) they had a LOD score of 2.7 or greater with a reasonable R2,
(2) were observed in more than one phenotyping location, or
(3) were observed for multiple correlated traits in the same genomic region at one or more phenotyping locations.
Based on the criteria outlined above, only those QTL that were of a high confidence were considered further.
Example 8. Validation of the Utility of the QTLs Associated with IDC
Eighteen candidate validation populations were made between soybean varieties to determine the utility of these QTLs in improving the tolerance to iron deficiency chlorosis in soybean.
Out of these 18 populations, 12 were selected for validation based on their relationship to the parents of the discovery populations and numbers of segregating QTL. F3 progenies of the 12 populations were genotyped as described in Example 4 using marker assays flanking QTL (only QTL of very high confidence identified in Example 7 were used).
For each population, 1380 F3 progenies were genotyped. Out of these 1380 progenies some were selected based on their QTL status. Selected progenies are evaluated for IDC at four locations in Iowa and Minnesota as per the Example 2.
Standard statistical analyses are conducted to determine the performance of the QTL in selection for progeny having tolerance to IDC.
The list of SNP markers comprising the QTL of the present invention is provided in Table 1, above. Further analysis indicated that the markers in Table 2 are further examples of markers that may associate with increased IDC tolerance.
The above examples clearly illustrate the advantages of the invention. Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.
Throughout this application, various patents, patent publications and non-patent publications are referenced. The disclosures of these patents, patent publications and non-patent publications in their entireties are incorporated by reference herein into this application in order to more fully describe the state of the art to which this invention pertains.

## Claims

1. A method of producing a soybean plant with increased tolerance to iron deficiency chlorosis (IDC), the method comprising the steps of:
a. isolating at least one nucleic acid from a population of soybean plants;
b. detecting in said nucleic acid a marker located within a chromosomal interval corresponding to Glycine max Chromosome 2 at physical positions 2850183-4189924, wherein said marker is within 5 cM from a locus associated with increased IDC tolerance, wherein said locus comprises SEQ ID NO:1 and wherein said locus comprises a “G” allele corresponding to position 255 or SEQ ID NO:1;
c. selecting a first soybean plant from the population of (a) based of the presence of the marker detected in (b);
d. crossing the first soybean plant of (c) with a second soybean plant not comprising in its genome the marker detected in (b);
e. collecting seed from the cross of (d); and
f. growing a progeny soybean plant from the seed of (e), wherein said progeny soybean plant comprises in its genome said locus associated with increased IDC tolerance, thereby producing a plant with increased IDC tolerance.

2. The method of claim 1, wherein the marker is detected through the use of a nucleotide probes comprising a nucleotide sequence as depicted in any one of SEQ ID NOs: 38 or 39.

3. The method of claim 1, wherein the marker is detected through use of a PCR primer pair that anneals to said chromosome interval, wherein the primer pair is capable of initiating DNA polymerization by a DNA polymerase on a Glycine max nucleic acid template to generate a amplicon determinative for the presence of said marker associated with increased IDC tolerance.

4. The method of claim 3, wherein a resulting amplicon comprises a nucleotide sequence comprising SEQ ID NO: 1.

5. The method of claim 1, wherein the progeny soybean plant is an elite soybean plant.

6. The method of claim 3, wherein the PCR primer pair comprises SEQ ID NOs: 36 and 37.