Patent Publication Number: US-2015074858-A1

Title: Lolium multiflorum line inducing genome loss

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 13/692,080 filed on Dec. 3, 2012 (allowed), which is a divisional of U.S. patent application Ser. No. 12/565,350, filed on Sep. 23, 2009 and is now abandoned, which claims the benefit under 35 U.S.C. 1.19(e) of U.S. Provisional Application 61/101,218, filed Sep. 30, 2008, the contents of which are incorporated by reference herein. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a novel diploid  Lolium multiflorum  line and a method of using the same to produce dihaploid homozygous  Lolium  and  Festuca  species. 
     2. Description of the Prior Art 
     Turf and forage grass cultivars are essentially germplasm synthetics segregating for a multitude of genes. Most if not all forage grass cultivars are developed from traditional mass, phenotypic recurrent or half-sib selection methods. However, the availability of homozygous lines, also called inbred lines, can provide completely new approaches for breeding and grass cultivar development. In one application, such dihaploid lines could be utilized for F1 hybrid development, taking advantage of the power of heterosis to increase biomass production, increase stress resistance, increase insect and disease resistance, etc. 
     However, the successful, reproducible development of such hybrids would require the availability of true breeding, homozygous dihaploid parental lines. Currently, the only techniques for generating homozygous dihaploid  Fescue  is through the use of anther-microspore culture, followed by genome doubling techniques. These techniques are time consuming, labor intensive, and unpredictable due to the unknown genetic predisposition that a segregating germplasm may exhibit toward such culture and genome doubling techniques. Essentially, this is an unpredictable approach for the generation of homozygous lines since only trial and error, and experience can be used to determine which particular cultivars are amendable to such a technique. 
     SUMMARY OF THE INVENTION 
     Novel ryegrass,  Lolium multiflorum,  lines, referred to herein as inducer lines, have been discovered which may be crossed with species representing the genus of  Fescue, Festuca,  with the interspecific hybrid subsequently incurring genome loss to produce  Fescue  and  Lolium  (ryegrass) dihaploid individuals that are homozygous for all loci across each species respective genomes. In addition, dihaploid amphidiploid  Lolium - Fescue  lines also are a result of the original hybridizations. These  L. multiflorum  inducer lines are either fully pollen fertile, partially sterile (leaky) or completely pollen sterile, and are characterized by the property that upon interspecific hybridization as the maternal parent using a  Festuca  species as the male parent, confer to the resultant sterile F1 interspecific hybrids, the ability to lose either the  Lolium  or  Festuca  genome via somatic loss during megaspore (egg) development; followed by a low level of a parthenogenic seed development response, that results in the production of some seed in the panicle which exhibits a viable embryo and the lack of a normally developed endosperm. When sown, seedlings originating from these parthenogenic seed produce an F2 generation where at least some of which are dihaploid homozygous  Festuca  lines comprising a karyotype exhibiting a balanced genome of  Festuca  chromosomes, equivalent to the chromosome number of their  Festuca  parent, with a complete absence of all  Lolium  chromosomes. Other F2 plants are produced as well, including dihaploid homozygous  Lolium  having a complete loss of the  Festuca  genome; and homozygous amphidiploids which have no loss in either the  Lolium  or  Fescue  genome. 
     Dihaploid homozygous  Festuca  species may also be generated directly from these same F1 interspecific hybrids without the need for parthenogenic seed development and the subsequent F2 generation. During the growth cycle of the F1 interspecific hybrids, many of the F1 plants eventually form somatic sectors that result from the complete loss of all their ryegrass genome and appear phenotypically as  Fescue  growing shoots. Sectors having only this haploid Fescue chromosome constitution spontaneously double, resulting in a dihaploid  fescue  sector possessing a full complement of the parental  fescue  genome. These  Fescue  somatic sectors can be retained, and will develop into mature, dihaploid homozygous  Festuca  plants possessing a balanced genome of  Festuca  chromosomes equivalent to the chromosome number of the  Festuca  paternal parent with a complete absence of  Lolium  chromosomes. 
     Mature dihaploid homozygous  Festuca  plants produced from the interspecific hybridization using either of the above-mentioned techniques are fertile, and may be utilized in crosses with other dihaploid  Festuca  recoveries to produce true F1 hybrid  Festuca  or can be combined in a series of hybridizations to develop polycross hybrid populations. 
     In accordance with this discovery, it is an object of this invention to provide a novel ryegrass,  Lolium multiflorum,  line that may be used to produce dihaploid homozygous  Festuca  species. 
     Another object of this invention to provide novel ryegrass,  Lolium multiflorum,  lines that may be used to produce not only dihaploid homozygous  Festuca  species but also dihaploid homozygous  Lolium  species and/or homozygous  Lolium - Fescue  amphidiploids. 
     A further object of this invention is to provide a method for producing dihaploid homozygous  Festuca  lines which are effective as inbred lines and may be crossed with any other dihaploid Fescue recovery to produce true F1 hybrid  Fescue.    
     Yet another object of this invention to provide a simple, reproducible, and relatively fast method for producing dihaploid homozygous  Festuca  species. 
     Other objects and advantages of this invention will become readily apparent from the ensuing description. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a diagram of dihaploid generation via parthogenesis in accordance with a first embodiment of the invention. 
         FIG. 2  shows a diagram of dihaploid generation via chimeral sectors in the F1 or F2 generations in accordance with a second embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A sample of at least 2,500 seeds of each of the preferred novel ryegrass,  Lolium multiflorum,  lines of this invention, referred to herein as inducer line IL-1 and inducer line IL-2, and which may be used to produce dihaploid homozygous  Festuca  species, have been deposited under the conditions of the Budapest Treaty with the American Type Culture Collection (10801 University Blvd, Manassas, Va., 20110-2209, USA) on Jul. 22, 2009, and Aug. 28, 2009, respectively, and have been assigned deposit accession nos. ATCC PTA-10229 and ATCC PTA-10315, respectively. 
     The inducer lines of this invention are  Lolium multiflorum  (Italian ryegrass or annual ryegrass) a diploid (2n=2x=14), herbaceous annual grass, exhibiting phenotypic traits typical of this species as described by Wheeler (Forage and Pasture Crops, A Handbook of Information about the Grasses and Legumes Grown for Forage in the United States. Van Nostrand Company. New York. 1950. pp. 537-541) and Quattrocchi (CRC World Dictionary of Grasses: Common Names, Scientific Names, Eponyms, Synonyms, and Etymology. CRC Press. Boca Raton, Fla., USA. 2006. p. 2408), the contents of which are incorporated by reference herein. The inducer line IL-1 is a leaky pollen sterile line that is uniquely characterized by its ability, upon interspecific hybridization as a maternal parent with a  Festuca  species being used as the paternal parent to 1) confer to the resultant sterile F1 interspecific hybrids the ability to generate a low level of a parthenogenic response and produce some seed in the panicle (i.e., seed heads) that possess a viable embryo; and 2) induce genome loss following the interspecific hybridization as described herein. 
     Induction of genome loss following interspecific hybridization generates dihaploid homozygous  Festuca  species comprising a karyotype (nuclear genome) of  Festuca  chromosomes equal to the chromosome number of the  Festuca  male parent (i.e., they retain a full complement of  Festuca  chromosomes with their chromosome number being the same as the  Festuca  male parent) with no  Lolium  chromosomes. Possessing a full complement of  Festuca  chromosomes and no  Lolium  chromosomes, these dihaploid homozygous  Festuca  species appear as normal  Fescue  plants. For example, in a preferred embodiment using Tall Fescue,  Festuca arundinaceae  (also referred to as  Lolium arundinaceae ), as the male parent, the resultant dihaploid homozygous  Festuca  comprise a karyotype of 42  Festuca  chromosomes. Alternatively, using Meadow  Fescue, F. pratensis,  as the male parent, the resultant dihaploid homozygous  Festuca  would comprise a karyotype of 14  Festuca  chromosomes. As described in detail herein below, depending upon the particular technique used, other dihaploid homozygous plants, including dihaploid homozygous  Lolium multiflorum,  as well as dihaploid homozygous amphidiploids may also be recovered. The dihaploid homozygous  Lolium multiflorum  plants comprise a karyotype of 14  Lolium multiflorum  chromosomes and appear as normal ryegrass plants, while the generation of amphidiploids do not undergo genome loss, but result only from spontaneous genome doubling, and comprise a karyotype of 14  Lolium multiflorum  chromosomes (representative of the maternal parent) and  Festuca  chromosomes equal to the chromosome number of the  Festuca  male parent (e.g., 42  Festuca  chromosomes when using Tall  Fescue ). Significantly, these dihaploid homozygous  Lolium multiflorum  may be used as new inducer lines for interspecific hybridization with  Fescue  as described herein. Although any of these dihaploid homozygous  Lolium  may be recovered and used as inducers as described herein, it is preferable to utilize those recoveries that appear healthy and vigorous. Moreover, these dihaploid homozygous  Lolium  may be used directly as inducers in crosses without requiring a generation of seed production or offspring from such seed. 
     For the purpose of this invention, any fully pollen fertile, completely pollen sterile or partially pollen sterile  Lolium multiflorum,  having the identifying characteristics of line IL-1, including progeny or variants thereof which retain the ability, upon interspecific hybridization as a maternal parent with a  Festuca  species as the paternal parent, to 1) confer to the resultant sterile F1 interspecific hybrids the ability to generate a low level of a parthenogenic response and produce some seed in the panicle that possess a viable embryo; and 2) induce genome loss following the interspecific hybridization, are effective as inducers. The term variant is defined herein to include transformants and mutants of line IL-1. For example, in accordance with this invention, the IL-1 line may be modified to include heterologous genes and/or mutations in the genes therein. Transformation techniques which may be used herein include but are not limited to microparticle gene transfer such as described by Lowe et al. (1995, Bio/Technology, 13:677-682), pollen transformation techniques such as described by Ohta (1986, Proc. Nat&#39;l Acad. Sci. USA, 83:715-719), Smith et al. (1994, Plant Science, 104:49-58), and deWet (International Patent Application WO 85/01856), electroporation techniques such as described by Rhodes (1988, Science, 240:204-207), Krzyzk et al. (U.S. Pat. No. 5,384,253), and viral transformation such as described by Langenberg et al. (U.S. Pat. No. 5,416,010). Techniques for introducing mutations include but are not limited to conventional chemical or irradiation techniques for random mutagenesis, or insertional or site directed point mutagenesis processes such as described by Neuffer [“Induction of Genetic Variability”, In: Maize Breeding and Genetics, Walden (ed.), 1978, pp. 579-600]. The contents of each of the above-mentioned references are incorporated by reference herein. 
     As noted above, additional inducer lines may be developed from the inducer line IL-1. Two of these new lines, referred to herein as IL-2 and IL-3, were each derived from single plant selections from the original IL-1 population. Because IL-1 is a “leaky” pollen sterile population that segregates a range of sterility levels, additional inducer lines can be generated from IL-1 through typical plant selection procedures. One such method involves examination of individual plants within the IL-1 population, with inducer lines IL-2 and IL-3 being selected from IL1 on the basis of their higher level of pollen sterility and improved vigor, both in comparison to the overall or mean of the IL-1 population. Thus, both IL-2 and IL-3 have a narrower genetic base (defined herein as reduced or limited genetic variation) than their IL-1 parent). However, since 112 and IL3 represent two different, individual plant selections, they will possess differing genotypes from IL1 and each other. Both IL-2 and IL-3 exhibit a significantly increased level of pollen sterility, increased plant height, and shorter time to flower, than IL-1. Of these, IL-2 is particularly preferred as it exhibits the greatest increase in the level of pollen sterility and plant height, and the shortest time to flower. Each of IL-2 and IL-3 are  Lolium multiflorum  (Italian ryegrass or annual ryegrass) a diploid (2n=2x=14), herbaceous annual grass, exhibiting phenotypic traits typical of this species as described by Wheeler (ibid) and Quattrocchi (ibid). Each of IL-2 and IL-3 are also effective inducer lines, and as such are characterized by their ability, upon interspecific hybridization as a maternal parent with a  Festuca  species being used as the paternal parent to 1) confer to the resultant sterile F1 interspecific hybrids the ability to generate a low level of a parthenogenic response and produce some seed in the panicle (i.e., seed heads) that possess a viable embryo; and 2) induce genome loss following the interspecific hybridization as described herein. In addition, both IL2 and IL3 also provide for the allowance of somatic sectors in the generated F1 hybrids which can result in the further generation of DH  Lolium  lines. 
     Another aspect of this invention is to provide for cells which upon growth and differentiation produce  Lolium multiflorum  plants which are effective as inducer lines, such that upon interspecific hybridization as a maternal parent with a  Festuca  species as the paternal parent, they 1) confer to the resultant sterile F1 interspecific hybrids the ability to generate a low level of a parthenogenic response and produce some seed in the panicle that possess a viable embryo; and 2) induce genome loss following the interspecific hybridization. Thus, as used herein, the term “ L. multiflorum  plant or parts thereof” includes plant cells, plant protoplasts, plant cells of tissue culture from which  L. multiflorum  plants can be regenerated, plant calli, plant clumps, as well as plant cells that are intact in plants or parts of plants such as, seed, flowers, embryos, ovules, roots, root tips, pollen, leaves, stalks, and the like. 
     The  L. multiflorum  inducer lines of this invention are stably maintained by conventional intercrossing or selfing. 
     In a first preferred embodiment shown in  FIG. 1 , the  L. multiflorum  inducer line is crossed as a maternal parent with any desired  Festuca  species being utilized as the paternal parent, and the resultant F1 interspecific hybrid seed is sown and allowed to grow into F1 plants, whereby they can mature and flower. The F1 plants comprise a karyotype of 7  Lolium multiflorum  chromosomes plus a contributed haploid chromosome set from the paternal  Festuca  parent (e.g. 21  Festuca  chromosomes when using  Festuca arundinaceae,  7 chromosomes when using  Festuca pratensis,  etc.) As is typical for most wide cross or interspecific hybrids with unbalanced genomes or differing chromosome number contributions, the F1 plants are typically both pollen and seed sterile. However, despite this sterility, a low level of parthenogenesis produces some seed in the panicle that possess a viable embryo; and lacks an obvious endosperm. The seed heads from the F1 plants are collected, preferably rubbed to remove seed husks surrounding the parthenogenic seed, then sown and allowed to grow, such that any of those of seeds that have developed embryos via parthenogenesis will develop into F2 plants. The F2 plants that are recovered are primarily (nearly 50% each) diploid homozygous  Fescue  with no  Lolium  chromosomes (comprising a karyotype of a balanced genome of  Festuca  chromosomes equal to the chromosome number of the  Festuca  paternal parent, 42 when using Tall  Fescue ) and diploid homozygous ryegrass with no  Festuca  chromosomes (comprising a karyotype of 14  Lolium multiflorum  chromosomes representative of the maternal parent). A low frequency of mixed genotypes are also produced, most notably amphidiploids which comprise a karyotype of 14  Lolium multiflorum  chromosomes (representative of the maternal parent) plus  Festuca  chromosomes equal to the chromosome number of the  Festuca  paternal parent (42 when using Tall  Fescue , providing a total chromosome number of 56). 
     The recovered F1 or F2 dihaploid homozygous  Festuca &#39;s and  Lolium &#39;s are not genetically identical to their parents since each interspecific hybrid represents a randomly generated haploid gamete/egg provided from each parent. In fact, each dihaploid  Lolium  or  Festuca  recovery represents only a single possible gene combination out of billions of possibilities. Essentially, the generation of the interspecific hybrids is a form of gamete selection through the pollen (in the case of the  Festuca  parent) or through the egg (in the case of the  Lolium  parent). The dihaploid  Lolium  and  Festuca  and amphidiploid recoveries are both male and female fertile and maintain their dihaploid genotypes by selfing or sibbing; they remain fixed at all loci (dihaploid), and are suitable for potential use as inbred lines for mapping studies, population development, selection, and breeding. Moreover, since the dihaploids are generated utilizing the  Lolium  as the maternal parent in the interspecific cross, all of the progeny, including the F2 dihaploid homozygous  Festuca  and homozygous amphidiploids, will retain the extranuclear genes and cytoplasm (including mitochondrial or cytoplasm and chloroplasts) of the  Lolium multiflorum  inducer parent. The presence of  Lolium  extranuclear genes in the dihaploid homozygous  Festuca  allows these plants to be readily distinguished from any dihaploid homozygous  Festuca  which might be produced using conventional techniques, such as use of anther-microspore culture followed by chromosome doubling. Extranuclear  Lolium  genes may be detected, for example, by conventional mitochondrial DNA or cytoplasmic analysis techniques [McGrath, Hodkinson, Salamin and Barth, 2006; Development and testing of novel chloroplast microsatellite markers for  Lolium perenne  and other grasses (Poaceae) from de novo sequencing and in silico sequences. Molecular Ecology Notes 6:449-452] 
     In an alternative preferred embodiment shown in  FIG. 2 , the dihaploid homozygous  Festuca  species may be produced directly from the same F1 interspecific hybrids described above, without the need for generating an F2 generation. In this embodiment, the  L. multiflorum  inducer line is crossed as the maternal parent with a  Festuca  species as the paternal parent to generate sterile F1 interspecific hybrid plants in the same manner as described above. Again, these F1 hybrids initially comprise a karyotype of 7  Lolium multiflorum  chromosomes plus 21  Festuca  chromosomes if, as example, an  F. arundinaceae  is utilized as the paternal parent. During the growth stage of the F1 interspecific hybrids, many of the F1 hybrids eventually form somatic (chimeral) sectors that have lost all of their ryegrass genome (7R) and retain only the  Fescue  chromosomes. The occurrence of this event is random and can occur early on; such as the seedling stage or very late in the plants growth phase such as the flowering stage. These chimeral sectors appear as  Fescue  growing shoots (i.e., apical meristems) from the F1 plant. Loss of the ryegrass genome is due to somatic loss or ryegrass genome instability in the interspecific hybrid condition. These  Fescue  somatic sectors will have their  Festuca  genome double spontaneously, without induction, generating a dihaploid homozygous  Festuca  sector (tiller) possessing a full balanced complement of  Festuca  chromosomes and no  Lolium  chromosomes. The  Fescue  somatic sectors can be readily identified and distinguished from ryegrass sectors and the ryegrass- Fescue  hybrid by their appearance as normal  Fescue , that is, exhibiting often wider leaves, rough leaf texture, alteration in leaf color and at flowering maturity, and a typical  Fescue  inflorescence/panicle. 
     These  Fescue  somatic sector regions may be removed and transferred to another pot, and will develop into mature, fertile dihaploid homozygous  Festuca  individuals, fixed for all loci, and comprising a karyotype of a balanced genome of  Festuca  chromosomes equal to the chromosome number of the  Festuca  paternal parent (42 when using Tall  Fescue ) with no  Lolium  chromosomes. The  Fescue  somatic sectors may be removed from the interspecific hybrid and transferred to pots or field nursery. The fescue sectors are perennial and can be maintained indefinitely by vegetative propagation or via seed. As in the previous embodiment, the resultant F2 dihaploid homozygous  Festuca  plants will retain the extranuclear genes (mitochondrial DNA and cytoplasm) of  Lolium multiflorum.  Although the process has been described as identifying and retaining  Fescue  somatic sectors from the F1 generation of plants, it is understood that  Fescue  somatic sectors can also develop in any subsequent generation of hybrid plants that may possess a haploid set of chromosomes from the  Lolium  or  Festuca  parents (e.g., 28 chromosomes in the case of  Festuca arundinaceae  (7  Lolium  +21  F. arundinaceae )) such as the F2, F3, or later generations, which are produced from seed harvested from the panicles in the same manner as the previous embodiment. 
     Interspecific hybridization of the  L. multiflorum  inducer lines to generate dihaploid homozygous  Festuca  may be practiced with any  Fescue  species, provided that the  Fescue  is used as the paternal parent. Without being limited thereto, preferred  Fescue  species include Meadow  Fescue, F. pratensis,  and particularly Tall  Fescue, F. arundinaceae.    
     The dihaploid homozygous  Festuca  and dihaploid homozygous  Lolium  plants produced from the interspecific hybridization using either of the above mentioned techniques are fertile, and are inbred lines. Therefore, the plants may be used in the production of true F1 hybrids with any dihaploid line obtained from a like  Fescue  or  Lolium  species. Preferably, for the generation of true F1 hybrids, the dihaploid homozygous  Festuca  should be crossed with a dihaploid line representing the same  Festuca  species of interest. 
     The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention that is defined by the claims. 
     EXAMPLE 1 
     Dihaploid Tall Fescue plants were produced using the process of the invention and examined to confirm that they were homozygous/inbred lines. 
     In brief, the  L. multiflorum  inducer line IL-1 was crossed as the maternal parent with Tall  Fescue, F. arundinaceae,  as the paternal parent (pollen source) grown in a greenhouse. F1 interspecific hybrid seed is collected and planted into individual pots, and the F1 plants were allowed to grow, mature, and flower. After the panicle or seed heads matured, the seed heads were collected, gently ground, and sown in fresh potting soil. F2 plants were allowed to grow and plants appearing as normal  Fescue  by their broad leaves were examined and confirmed to be dihaploid Tall  Fescue , containing 42  Fescue  chromosomes with no  Lolium  chromosomes. Four of the dihaploid  Fescue  plants were retained for further analysis. 
     The four retained dihaploid  Fescue  plants were each selfed, and the seed was collected, planted, and allowed to grow. The offspring derived from the selfing were analyzed by PCR-EST-SSR using eight highly discriminatory Tall  Fescue  markers. All offspring from each selfing were confirmed to be identical to each other by molecular analysis and were phenotypically identical to each other and their F2 dihaploid  Fescue  parent. 
     The identical molecular marker patterns exhibited across the offspring; and, their identical pattern to their F2 dihaploid parent, indicate a genetic uniformity and genetic homozygosity of the lines, allowing for the usage of the descriptive term ‘dihaploid’ to the F2 parent and its selfed generated offspring. These data also indicate the ability of the dihaploids to breed true to type by sibbing or selfing (i.e., uniformity with no genetic segregation). 
     It is understood that the foregoing detailed description is given merely by way of illustration and that modifications and variations may be made therein without departing from the spirit and scope of the invention.