Novel genetic loci associated with disease resistance in soybean

Abstract

The present invention relates to methods and compositions for identifying, selecting and / or producing disease resistant soybean plants or germplasm using markers, genes and chromosomal intervals derived from Glycine tomentella PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224. Also provided are soybean plants or germplasm identified, selected and / or produced by any of the methods of the present invention. Disease resistant soybean seeds, plants and germplasm are also provided.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 347,945, filed June 9, 2016, the contents of which are incorporated herein by reference.

[0003] Declaration regarding the electronic submission of the sequence list

[0004] A sequence listing, named 81045SEQ LIST_ST25.txt and 6.90 MB in size, generated on June 9, 2017, is submitted in ASCII text format and is filed with this application. This sequence listing is hereby incorporated by reference in this specification with respect to its disclosure. Technical Field

[0005] This invention relates to compositions and methods for identifying, selecting, and producing enhanced disease and / or pathogen resistant soybean plants. Background Technology

[0006] Plant pathogens are known to cause considerable damage to important crops, resulting in significant agricultural losses, with widespread consequences for food supplies and other industries that depend on plant materials. Similarly, there has long been a need to reduce the incidence and / or impact of agricultural pathogens on crops.

[0007] Several pathogens are associated with damage to soybeans, which can individually and collectively cause significant yield losses in the United States and worldwide. Exemplary pathogens include, but are not limited to, fungi (e.g., Phytophthora and Asian soybean rust fungus), nematodes (e.g., root-knot nematodes, particularly Javan root-knot nematode), and soybean stem canker. Given the significant threat these pathogens pose to global food supplies and the time and expense associated with treating soybean crops to prevent yield losses, new methods are needed for generating pathogen-resistant soybean cultivars. What is needed is a novel resistance gene (“R gene” in this paper) that can be introduced into commercial soybean plants to control soybean pathogens. Summary of the Invention

[0008] This overview sets forth several embodiments of the subject matter disclosed herein, and in many cases, variations and arrangements of these embodiments are shown.

[0009] Therefore, one object of this disclosure is to provide a method for transferring pathogen resistance to non-resistant soybean germplasm or plant lines. Additionally, this disclosure provides novel soybean (Glycine max) lines that contain chromosomal regions, loci, and / or genes in their genomes derived from Glycine tomentella and further confer resistance to Asian soybean rust (hereinafter, “ASR”).

[0010] A brief explanation of sequence listings

[0011] SEQ ID NO:1 and 2 are chromosomal regions derived from the short-fiber wild soybean cultivar registration PI441001, referred to herein as “scaffold 46840” and “scaffold 49652”, respectively. Scaffold 49652 has been mapped to chromosome 5 of short-fiber wild soybean, and scaffold 46840 has also been mapped to chromosome 5 of short-fiber wild soybean. SEQ ID NO:3 is a chromosomal region derived from the short-fiber wild soybean cultivar registration PI 483224, referred to herein as “scaffold 002687F”. Scaffold 002687F has been mapped to chromosome 5 of short-fiber wild soybean. SEQ ID NO:4 is a chromosomal region derived from the short-fiber wild soybean cultivar registration PI 583970, referred to herein as “scaffold 001084F”. Scaffold 001084F has been mapped to chromosome 5 of short-fiber wild soybean. SEQ ID NO:5 is a chromosomal region derived from the short-fiber wild soybean cultivar registration PI 583970, referred to herein as “scaffold 000819F”. Scaffold 000819F has been mapped to chromosome 5 of short-fibered wild soybean. Genetic population mapping studies of PI441001, PI483224, and PI583970 indicate that chromosome 5 of short-fibered wild soybean contains chromosomal regions highly associated with ASR resistance (e.g., as corresponding to SEQ ID NO: 1-5). Experimental data, not limited by theory, also indicate that short-fibered wild soybean registrations PI446958 and / or PI483224 can be used as sources of the chromosomal regions corresponding to SEQ ID NO: 1-5. These chromosomal regions or portions thereof can be introduced (i.e., genetic introgression using embryo rescue and marker-assisted breeding (MAB)) into soybean lines to produce soybean lines resistant to various diseases such as ASR. Tables 1-5 show the single nucleotide polymorphisms (SNPs) associated with ASR resistance in SEQ ID NO: 1-5. Table 1 shows the SNP associations for SEQ ID NO:1 (stent 46840), and Table 2 shows the SNP associations for SEQ ID NO:2 (stent 49652). Table 3 shows the SNP associations for SEQ ID NO:3 (stent 002687F). Table 4 shows the SNP associations for SEQ ID NO:4 (stent 001084F). Table 5 shows the SNP associations for SEQ ID NO:5 (stent 000819F). All alleles of the SNPs identified in Tables 1-5 were significantly associated with resistance or susceptibility to ASR (p<0.05).

[0012] Table 1: SNP positions in SEQ ID NO:1 associated with increased resistance to ASR

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[0021] Table 2: In SEQ SNP position in ID NO:2 associated with increased resistance to ASR

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[0064] Table 3: SNP positions in SEQ ID NO: 3 associated with increased resistance to ASR

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[0069] Table 4: SNP positions in SEQ ID NO: 4 associated with increased resistance to ASR

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[0082] Table 5: SNP positions in SEQ ID NO: 5 associated with increased resistance to ASR

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[0087] Oligonucleotide primers (“primers”) can be developed and used to identify plants carrying any of the chromosomal regions described in SEQ ID NO:1, 2, 3, 4 and / or 5 that are found to be highly associated with ASR resistance. Specifically, for the identification or generation of soybean lines having any or a portion of any of the chromosomal regions described in SEQ ID NO:1, 2, 3, 4 or 5 that are associated with ASR resistance, those skilled in the art can develop primers to detect any single nucleotide polymorphisms (“SNPs”) identified as any of those in Tables 1-5. Assays (e.g., typically two-step allele identification assays or similar assays), KASP TM Assays (typically a one-step allele identification assay or similar assay as defined below), or both, can be used to identify SNPs associated with increased ASR resistance as disclosed herein (e.g., favorable alleles as described in Tables 1-5). In an exemplary two-step assay, forward primers, reverse primers, and two assay probes (or hybridization oligonucleotides) are used. The forward and reverse primers are used to amplify a genetic locus containing an SNP associated with an ASR resistance locus (e.g., any favorable alleles shown in Tables 1-5). Assay primers (in some embodiments, these assay primers are differentially labeled with, for example, fluorophores to allow differentiation of the two assay probes in a single reaction) are then used to determine the specific nucleotide present at the SNP location. These assay primers are different from each other in each pair for the nucleotide present at the SNP location (although it is noted that in any given pair, the probes may differ at their 5' or 3' ends without affecting their ability to distinguish the nucleotide present at the respective SNP location). In some embodiments, the assay primers and reaction conditions are designed such that the assay primers will hybridize only with the reverse complement of a 100% perfectly matching sequence, thereby allowing hybridization-based detection to identify which or which alleles are present.

[0088] Compositions and methods are provided for identifying, selecting, and producing Glycine plants (including wild Glycine (e.g., short-haired wild soybean) and soybean lines) with enhanced disease resistance. Disease-resistant soybean plants and germplasm are also provided.

[0089] In some embodiments, methods are provided for identifying disease-resistant soybean plants or germplasm. Such methods may include detecting genetic loci or molecular markers (e.g., SNPs or quantitative trait loci (QTLs)) in soybean plants or germplasm that are associated with enhanced disease resistance, particularly ASR resistance. In some embodiments, the genetic loci or molecular markers are associated with the presence of a chromosomal region comprising a nucleotide sequence or a portion thereof of SEQ ID NO 1, 2, 3, 4, or 5, wherein a portion thereof is associated with ASR resistance.

[0090] In some embodiments, methods for generating disease-resistant soybean plants are provided. Such methods may include detecting the presence of genetic loci and / or genetic markers associated with enhanced pathogen (e.g., ASR) resistance in soybean plants or germplasm, and generating progeny plants from said soybean germplasm.

[0091] In some embodiments, a method for selecting disease-resistant soybean plants or germplasm is provided. Such a method may include hybridizing a first soybean plant or germplasm with a second soybean plant or germplasm, wherein the first soybean plant or germplasm comprises a genetic locus derived from any one of plant registrations PI441001, PI441008, PI446958, PI509501, PI583970, PI483224 or their progeny plants that contain any one of these locus and are associated with enhanced disease and / or ASR resistance and / or tolerance, and selecting progeny plants or germplasm having the genetic locus.

[0092] In some embodiments, a method is provided to infiltrate genes from soybean registration loci PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224 that are associated with enhanced pathogen resistance into soybean plants or germplasm. Such methods may include crossing a first soybean plant or germplasm (which contains a chromosomal region (e.g., SEQ ID No: 1, 2, 3, 4, or 5) derived from soybean registrations PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224) associated with enhanced pathogen resistance) with a second soybean plant or germplasm (which lacks the said locus), and optionally repeatedly backcrossing the progeny containing the said genetic alleles with the second soybean plant or germplasm to produce soybean plants (e.g., soybeans) or germplasm with enhanced pathogen resistance, which contain a chromosomal region derived from soybean registrations PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224) associated with enhanced ASR resistance. Progeny containing chromosomal regions associated with enhanced pathogen resistance can be identified by detecting the presence in their genome a marker associated with or genetically linked to the chromosomal regions derived from soybean accession numbers PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions contain SEQ ID NO 1, 2, 3, 4, or 5 or portions thereof, and the marker can be any of the favorable alleles described in Tables 1-5. R-genes from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224 can be introduced into soybean lines using embryo rescue methods known to those skilled in the art (e.g., as disclosed in U.S. Patent 7,842,850, which is incorporated herein by reference) and by methods described in the working examples herein (which describe alternative methods of embryo rescue).

[0093] Soybean plants and / or germplasm identified, produced, or selected by the methods of the present invention are also provided, as well as any progeny or seeds derived from soybean plants or germplasm identified, produced, or selected by these methods. In one embodiment, a molecular marker associated with the presence of a chromosomal region described in any one of SEQ ID NO 1, 2, 3, 4, or 5 can be used to identify or select plant lines resistant to ASR. Additional said molecular markers may be located within 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, and 1 cM of said chromosomal region, or derived from any corresponding favorable allele associated with ASR resistance as described in any one of Tables 1-5. In another embodiment, said molecular markers may be located within 20 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, and 1 cM of any SNP marker associated with ASR as described in any one of Tables 1-5.

[0094] Also provided are non-naturally occurring soybean seeds, plants, and / or germplasm containing one or more genetic loci derived from Plant Registry Numbers PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224 (hereinafter referred to as "PI441001", "PI441008", "PI446958", "PI509501", "PI583970", or "PI483224") that are associated with enhanced resistance to pathogens such as ASR, cyst nematodes, Phytophthora, etc. In specific embodiments, the genetic loci contain SEQ ID NO 1, 2, 3, 4, or 5 and / or any favorable alleles as described in Tables 1-5.

[0095] A marker associated with enhanced pathogen (more specifically, ASR) resistance may comprise, consist essentially of, or consist of a single allele associated with enhanced pathogen (ASR) resistance or a combination of alleles derived from one or more loci of PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224. In one embodiment, the marker is located within a chromosomal region as described in SEQ ID NO 1, 2, 3, 4, or 5. In another embodiment, the marker is any of the favorable alleles described in Tables 1-5.

[0096] The above-described objects and other aspects of the invention will be explained in detail in the accompanying drawings and descriptions set forth below. Attached Figure Description

[0097] Figure 1 shows a description of the short-fiber soybean SNPs associated with ASR resistance on the short-fiber soybean genome scaffold located on chromosome 5.

[0098] Figure 2 shows the positioning range of 0.023–1.16 Mb on stent 49652 associated with ASR resistance.

[0099] Figure 3 shows the positioning range of 0.02–1.19 Mb on stent 46840 associated with ASR resistance.

[0100] Figure 4 is a marker association diagram of short-fibered wild soybean (PI441001), where the bands indicate the corresponding chromosomal regions / intervals associated with ASR resistance.

[0101] Figure 5 shows the rust assessment scale used to measure plant phenotype.

[0102] Figure 6 shows the chromosomal localization of the ASR resistance QTL in short-fibered wild soybean PI583970.

[0103] Figure 7 is a marker association diagram of short-fibered wild soybean (PI583970), where the bands indicate the corresponding chromosomal regions / intervals associated with ASR resistance.

[0104] Figure 8 shows the positioning range on stent 000819F associated with ASR resistance.

[0105] Figure 9 shows the chromosomal localization of the ASR resistance QTL in short-fibered wild soybean PI483224.

[0106] Figure 10 is a marker association diagram of short-fibered wild soybean (PI483224), where the bands indicate the corresponding chromosomal regions / intervals associated with ASR resistance.

[0107] Figure 11 shows the positioning range on stent 002687F associated with ASR resistance. Detailed Implementation

[0108] This disclosure relates, at least in part, to the identification of genomic regions (i.e., one or more chromosomal regions on chromosome 5 of soybean PI441001, PI441008, PI446958, PI509501, PI583970, PI483224, PI441008 or their progeny) derived from soybean PI441001, PI441008 or their progeny that are associated with enhanced ASR resistance. Similarly, the chromosomal regions from PI441001, PI441008, PI446958, PI509501, PI583970, PI483224, PI441008, or their progeny can be infiltrated into soybean lines via somatic embryo rescue (see, for example, U.S. Patent Publication 2007 / 0261139 and examples of alternative methods of embryo rescue described herein) or by using soybean donor lines whose genomes have been genetically infiltrated into genetic regions from PI441001, PI441008, PI583970, or PI483224, wherein the region comprises any one or a portion of SEQ ID NO: 1-5, and the presence of said genetic region is associated with increased or enhanced disease resistance to, for example, ASR, SCN, stem termination, stem canker, bacterial pustule, root-knot nematode, brown stem rot, soybean frogeye leafspot, or Phytophthora. In another embodiment, a chromosomal region derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224 is introduced into a soybean line that does not contain the chromosomal region, wherein the introduction confers increased resistance to diseases (e.g., ASR) in the soybean line or its progeny, wherein the chromosomal region is derived from chromosome 5 of short-haired wild soybean, and further wherein the chromosomal region contains at least one allele associated with the trait of increased disease (e.g., ASR) resistance, wherein the allele is any one of the alleles selected from any one of those described in Tables 1-5.

[0109] This description is not intended to be a detailed list of all the different ways in which the invention can be practiced, or of all the features that can be added to the invention. For example, a feature described with respect to one embodiment may be incorporated into other embodiments, and a feature described with respect to a particular embodiment may be removed from that embodiment. Therefore, the invention is contemplated that, in some embodiments of the invention, any feature or combination of features stated herein may be excluded or omitted. Furthermore, given this disclosure, numerous variations and additions to the different embodiments suggested herein will be apparent to those skilled in the art without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention and is not an exhaustive description of all permutations, combinations, and variations thereof.

[0110] All references listed below, and all references cited in this immediate disclosure, including but not limited to all patents, patent applications and their publications, articles in scientific journals, and database entries (e.g., Database entries and all available annotations therein are incorporated herein in their entirety by reference to the extent that they supplement, explain, or provide context for the methods, techniques, and / or compositions employed herein.

[0111] definition

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0113] Although the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to facilitate understanding of the subject matter of this disclosure.

[0114] As used herein, the terms "a / an" or "the" can refer to one or more than one. For example, "a / an" sign can refer to one or more signs.

[0115] As used herein, the term “and / or” means and covers any and all possible combinations of one or more associated listed items, together with the absence of a combination when interpreted in terms of alternatives (“or”).

[0116] As used herein, the term “about” when referring to a measurable value (such as a quantity of mass, dose, time, temperature, etc.) means covering up to 10% of the variation in the specified quantity.

[0117] The term "substantially composed of" (and its grammatical variations) when applied to the polynucleotide sequences of the present invention means a polynucleotide sequence consisting of both the enumerated sequence (e.g., SEQ ID number) and all ten or fewer additional nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the 5' and / or 3' ends of the enumerated sequence, such that the function of the polynucleotide sequence is not substantially altered. The total of ten or fewer additional nucleotides includes all additional nucleotides added together at both ends. The term "substantially altered," when applied to the polynucleotides of the present invention, means that the ability to express the polynucleotide sequence is increased or decreased by at least about 50% or more compared to the expression level of the polynucleotide sequence consisting of the enumerated sequence.

[0118] As used herein, the term “introduction” in relation to plants means that it is accomplished in any manner, including but not limited to: gene introgression, transgenic, regularly clustered short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs) (Feng et al. 2013, Joung and Sander 2013), meganucleases, or zinc finger nucleases (ZFNs).

[0119] As used in this article, the term "wild soybean" refers to perennial plants of the genus *Glycine*, such as *Glycine canescens*, *Glycine argyrea*, *Glycine clandestine*, *Glycine latrobeana*, *Glycine albicans*, *Glycine aphyonota*, *Glycine areenaria*, *Glycine curvata*, *Glycine cyrtoloba*, *Glycine dolichocarpa*, *Glycine falcate*, and *Glycine spp.* Any one of the following: gracei, hairy soybean (G. hirticaulis), milky green soybean (G. lactovirens), broadleaf soybean (G. latifolia), small-leaf soybean (G. microphylla), Monti-Douglas soybean (G. montis-douglas), G. peratosa, G. pescadrensis, G. pindanica, G. pullenii, yellow-purple soybean (G. rubiginosa), G. stenophita, G. syndetika, or short-haired wild soybean.

[0120] As used herein, the term "allelic gene" refers to one of two or more distinct nucleotides or nucleotide sequences that are present at a particular locus.

[0121] A marker is “associated” with a trait when its presence indicates whether and / or to what extent a desired trait or phenotype will occur in the plant / germplasm containing the marker. Similarly, a marker is “associated” with an allele when its presence indicates the presence of the allele in the plant / germplasm containing the marker. For example, “a marker associated with enhanced pathogen resistance” refers to a marker whose presence or absence can be used to predict whether and / or to what extent a plant will exhibit a pathogen resistance phenotype (e.g., any favorable SNP alleles as described in Tables 1-5 that are “associated” with ASR resistance in soybean plants).

[0122] As used herein, the terms “backcross” and “backcrossed” refer to a method by which progeny plants are repeatedly backcrossed with one of their parents. In a backcross scheme, the “donor” parent is a parent plant that possesses the desired gene or locus to be introgressed. The “recipient” parent (used once or multiple times) or “recurrent” parent (used twice or more) is a parent plant in which the gene or locus is 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. 41-53 (1994). The initial cross produces 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.

[0123] A centimolar ("cM") is a unit of measurement for recombination frequency. One cM equals a 1% chance that a marker at one locus will separate from a marker at a second locus due to crossover in a single generation.

[0124] As used herein, the term “chromosomal region defined by and including therein” with respect to a particular locus and / or allele means the chromosomal region defined by and encompassing the locus / allele.

[0125] As used herein, the term "cross" or "crossed" refers to the fusion of gametes through pollination to produce offspring (e.g., cells, seeds, or plants). This term includes both sexual hybridization (one plant being pollinated by another) and self-pollination (self-pollination, such as when pollen and ovules are from the same plant). The term "crossing" refers to the act of fusing gametes through pollination to produce offspring.

[0126] As used herein, the terms “cultivar” and “variety” refer to a group of similar plants that can be distinguished from other varieties within the same species by structural or genetic characteristics and / or phenotypes.

[0127] As used herein, the terms “desired allele,” “favorable allele,” and “target allele” are used interchangeably to refer to the allele associated with the desired trait (e.g., ASR resistance).

[0128] As used herein, the terms “enhanced pathogen resistance” or “enhanced disease resistance” refer to an improvement, enhancement, or increase in a plant’s tolerance and / or reproductive capacity despite infection with a disease (e.g., Asian soybean rust) compared to one or more control plants (e.g., one or two parents, or plants lacking markers associated with enhanced pathogen resistance to the corresponding pathogen / disease). Enhanced disease resistance includes any mechanism (other than whole-plant immunity or resistance) that reduces the expression of symptoms indicating infection with the corresponding disease (e.g., Asian soybean rust, soybean cyst nematode, Phytophthora, etc.).

[0129] A “superior line” or “superior variety” is an agronomically superior line that has been developed through numerous cycles of selection targeting superior agronomic performance. Numerous superior lines are available and known to those of ordinary skill in the field of soybean breeding. A “superior population” is a class of superior individuals or lines that can be used to represent existing technology regarding genotypes that are agronomically superior in a given crop species (such as soybean). Similarly, a “superior germplasm” or superior variety of germplasm is agronomically superior germplasm, typically derived from and / or capable of producing plants with superior agronomic performance, such as existing or newly developed superior soybean lines.

[0130] "Superior" plants are any plants that come from superior strains; therefore, superior plants are representative plants that come from superior varieties. Non-restricted examples of commercially available superior soybean varieties for farmers or soybean breeders include: AG00802, A0868, AG0902, A1923, AG2403, A2824, A3704, A4324, A5404, AG5903, AG6202, AG0934; AG1435; AG2031; AG2035; AG2433; AG2733; AG2933; AG3334; AG3832; AG4135; AG4632; AG4934; AG5831; AG6534; and AG7231 (Asgrow Seeds, Des Moines, Iowa, USA); BPR0144RR, BPR 4077NRR, and BPR 4390NRR (Bio Plant Research, Camp Point, Illinois, USA); DKB17-51 and DKB37-51 (DeKalb Genetics, DeKalb, Illinois, USA); DP 4546RR and DP 7870RR (Delta & Pine Land Company, Lubbock, Texas, USA); JG 03R501, JG 32R606C ADD and JG 55R503C (JGL Inc., Greencastle, Indiana, USA).NKS 13-K2 (NK Division of Syngenta Seeds, Golden Valley, Minnesota, USA); 90M01, 91M30, 92M33, 93M11, 94M30, 95M30, 97B52, P008T22R2; P16T17R2; P22T69R; P25T51R; P34T07R2; P35T58R; P39T67R; P47T36R; P46T21R; and P56T03R2 (Pioneer Hi-Bred International Ltd.) International), Johnston, Iowa, USA; SG4771NRR and SG5161NRR / STS (Soygenetics, LLC, Lafayette, Indiana, USA); S00-K5, S11-L2, S28-Y2, S43-B1, S53-A1, S 76-L9, S78-G6, S0009-M2; S007-Y4; S04-D3; S14-A6; S20-T6; S21-M7; S26-P3; S28-N6; S30-V6; S35-C3; S36-Y6; S39-C4; S47-K5; S48-D9; S52-Y2; S58-Z4; S67-R6; S73-S8; and S78-G6 (Syngenta Seed Company) Seeds (Henderson, Kentucky, USA); Richer (Northstar Seed Ltd., Alberta, Canada); 14RD62 (Stine Seed Co., Iowa, USA); or Armor 4744 (Armor Seed, LLC, Alaska, USA).

[0131] As used in this article, the term "agronomically superior" refers to a genotype with many distinguishable traits (such as emergence, vigor, nutritional vigor, disease resistance, seed set, standing ability, yield, and threshing ability) that allows producers to harvest commercially viable products.

[0132] As used herein, the terms “commercially significant yield” or “agronomically acceptable yield” refer to at least 100% of the grain yield of a commercially comparable (check) variety such as AG2703 or DKB23-51.

[0133] As used herein, the terms “exotic,” “exotic strain,” and “exotic germplasm” refer to any plant, strain, or germplasm that is not superior. Typically, an exotic plant / germplasm is not derived from any known superior plant or germplasm, but is selected to introduce one or more desired genetic elements into a breeding program (e.g., introducing novel alleles into a breeding program).

[0134] A genetic map is a description of genetic linkages between loci on one or more chromosomes within a given species, typically depicted as a graph or table. For each genetic map, the distance between loci is measured by the frequency of recombination between them. Recombination between loci can be detected using various markers. A genetic map is the product of the localized population, the types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distance between loci on one genetic map can differ from those on another.

[0135] As used herein, the term "genotype" refers to the genetic makeup of an individual (or group of individuals) at one or more genetic loci, as opposed to observable and / or detectable and / or expressed traits (phenotypes). A genotype is defined by one or more alleles inherited by an individual from one or more known loci of its parents. The term genotype can be used to refer to the genetic makeup of an individual at a single locus, multiple loci, or more generally, to refer to the genetic makeup of an individual across all genes in its genome. Genotypes can be characterized indirectly, for example, using markers, and / or directly, through nucleic acid sequencing.

[0136] As used herein, the term "germplasm" refers to genetic material belonging to or derived from an individual (e.g., a plant), a group of individuals (e.g., a plant strain, variety, or family), or a clone derived from a strain, variety, species, or culture. Germplasm can be a part of an organism or cell, or can be isolated from that organism or cell. Typically, germplasm provides genetic material with a specific molecular structure that provides the physical basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm can refer to seeds, cells (including protoplasts and callus), or tissues from which new plants can grow, as well as plant parts (e.g., stems, buds, roots, leaves, etc.) that can be cultured into a whole plant.

[0137] A "haplotype" is the genotype of an individual at multiple genetic loci, that is, a combination of alleles. Typically, the genetic loci that define a haplotype are physically and genetically linked, that is, located on the same segment of a chromosome. The term "haplotype" can refer to polymorphism at a specific locus (e.g., a single marker locus) or polymorphism at multiple loci along a segment of a chromosome.

[0138] As used herein, the term "heterozygous" refers to a genetic state in which different alleles are located at corresponding loci on homologous chromosomes.

[0139] As used herein, the term “homozygous” refers to a genetic state in which identical alleles are located at corresponding loci on homologous chromosomes.

[0140] As used herein, the term "hybrid" refers to the seeds and / or plants produced when at least two genetically dissimilar parents are crossed.

[0141] As used herein, the term "inbreeding" refers to a plant or variety that is substantially homozygous. The term can refer to a plant or variety that is substantially homozygous throughout its genome, or a plant or plant variety that is substantially homozygous relative to a portion of the genome of particular interest.

[0142] As used herein, the term "indel" refers to an insertion or deletion in a pair of nucleotide sequences, wherein the first sequence may be referred to as having an insertion relative to the second sequence, or the second sequence may be referred to as having a deletion relative to the first sequence.

[0143] As used herein, the terms “introgression,” “introgressing,” and “introgressed” refer to the natural and artificial transfer of a desired allele or combination of desired alleles at one or more genetic loci from one genetic background to another. For example, a desired allele at a designated locus can be transferred to at least one offspring through sexual hybridization between two parents of the same species, where at least one of these parents carries the desired allele in its genome. Alternatively, for example, allele transfer can occur through recombination between two donor genomes, such as in fused protoplasts, where at least one donor protoplast carries the desired allele in its genome. The desired allele can be a marker-selected allele, a QTL, a transgene, etc. Progeny containing the desired allele can be repeatedly backcrossed with a line having a desired genetic background, and selection can be performed on the desired allele, whereby the desired allele becomes fixed in a desired genetic background. For example, a marker associated with enhanced ASR tolerance can be introgressed from a donor gene into a recurrent parent that does not have disease resistance. The resulting offspring can then be repeatedly backcrossed and selected until the offspring have one or more ASR tolerance alleles in the recurrent parental background.

[0144] As used herein, the term "linkage" refers to the degree to which one marker locus is associated with another marker locus or some other locus (e.g., the ASR tolerance locus). Linkage between a molecular marker and a phenotype can 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 genetically and physically linked to any other marker when it is separated by less than about 50, 40, 30, 25, 20, or 15 map distance units (or cM). For example, embodiments of the invention provide marker loci tightly linked to the ASR resistance chromosomal region (which contains the nucleotide sequence of any one of SEQ ID NO 1-5).

[0145] In some aspects of the invention, defining the linkage range (e.g., from about 10 cM and about 20 cM, from about 10 cM and about 30 cM, or from about 10 cM and about 40 cM) is advantageous. The closer the linkage between the marker and the second locus, the better the marker's indicative effect on the second locus. Thus, "closely linked loci" (such as the marker locus and the second locus) exhibit recombination frequencies of about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% or less. In some embodiments, the associated locus exhibits a recombination frequency of about 1% or less (e.g., about 0.75%, 0.5%, 0.25% or less). Two loci located on the same chromosome and having a distance between them such that recombination between the two loci occurs at a frequency of less than about 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25% or less) can also be considered “proximate” to each other. Since one cM is the distance between two markers that indicates a 1% recombination frequency, any marker is closely linked (genetically and physically) to any other marker that is closely adjacent (e.g., at a distance equal to or less than about 10 cM). Two closely linked markers on the same chromosome can be located relative to each other at a distance of about 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM, or 0.25 cM or less.

[0146] As used herein, the term “linkage disequilibrium” refers to the non-random segregation of a genetic locus or trait (or both). In either case, linkage disequilibrium means that associated loci are physically close enough along a segment of chromosome that they segregate together at a frequency higher than random (i.e., non-random) (in the case of co-segregating traits, the loci controlling those traits are close enough to each other). Markers exhibiting 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 co-segregating markers have a recombination frequency of less than 50% (and, by definition, less than 50 cM segregation on the same chromosome). As used herein, linkage can exist between two markers, or alternatively, between a marker and a phenotype. Marker loci can be “associated” (linked) with a trait (e.g., Asian soybean rust). For example, the degree of linkage between a molecular marker and a target trait is measured as the statistical probability of co-segregation of that molecular marker with that phenotype.

[0147] The most common measure of chain imbalance is r. 2 The assessment, the measure r 2Calculate using the formula described in the following literature: Hill and Robertson, Theor. Appl. Genet. [Theoretical and Applied Genetics] 38:226 (1968). When r 2 When r = 1, there is complete linkage disequilibrium between the two marker loci, meaning that the markers have not yet undergone recombination and have the same allele frequency. 2 Values ​​greater than 1 / 3 indicate sufficiently strong linkage disequilibrium that is useful for localization. Ardlie et al., Nature Reviews Genetics 3:299 (2002). Therefore, when r between paired marker loci... 2 When the value is greater than or equal to approximately 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, the allele is in linkage disequilibrium.

[0148] As used herein, the term “linkage equilibrium” describes a situation where two markers separate independently, i.e., are randomly assigned in offspring. Markers showing linkage equilibrium are considered unlinked (regardless of whether they are located on the same chromosome).

[0149] A "locus" is the position on a chromosome where a gene, marker, or allele is located. In some embodiments, a locus may encompass one or more nucleotides.

[0150] As used herein, the terms “marker” and “genetic marker” are used interchangeably to refer to nucleotides and / or nucleotide sequences that have been associated with a phenotype, trait, or form of trait. In some embodiments, a marker may be associated with an allele or a target allele and may indicate the presence or absence of an allele or target allele in a cell or organism. Markers may be, but are not limited to, alleles, genes, haplotypes, restriction fragment length polymorphisms (RFLPs), simple repeat sequences (SSRs), random amplified polymorphic DNA (RAPDs), enzyme-digested amplified polymorphic sequences (CAPS) (Rafalski and Tingey, Trends in Genetics 9:275 (1993)), and amplified fragment length polymorphisms (AFLPs) (Vos et al., Nucleic Acids). Res. [Nucleic Acid Research] 23:4407 (1995)), Single Nucleotide Polymorphism (SNP) (Brookes, Gene [Gene] 234:177 (1993)), Sequence Characteristic Amplified Region (SCAR) (Paran and Michelmore, Theor. Appl. Genet. [Theoretical and Applied Genetics] 85:985 (1993)), Sequence Tagged Site (STS) (Onozaki et al., Euphaitica [Dutch Journal of Plant Breeding] 138:255 (2004)), Single Strand Conformation Polymorphism (SSCP) (Orita et al., Pr The term "marker" can refer to: simple repeat sequence intervals (ISSRs) (Blair et al., Theor. Appl. Genet. 98:780 (1999)), intertransferotropic amplification polymorphism (IRAP), retrotransferotropic microsatellite amplification polymorphism (REMAP) (Kalendar et al., Theor. Appl. Genet. 98:704 (1999)), chromosomal regions, or RNA cleavage products (e.g., Lynx tags). Markers can be present in genomic nucleic acids or expressed nucleic acids (e.g., ESTs). The term "marker" can also refer to nucleic acids used as probes or primers (e.g., primer pairs) for amplification, hybridization, and / or detection of nucleic acid molecules according to methods well known in the art. A large number of soybean molecular markers are known in the art and are either publicly available or available from various sources such as the SoyBase Internet resource.

[0151] Markers corresponding to genetic polymorphisms among population members can be detected using methods recognized in the art. These methods include, for example, nucleic acid sequencing, hybridization methods, amplification methods (e.g., PCR-based sequence-specific amplification), restriction fragment length polymorphism detection (RFLP), isoenzyme marker detection, polynucleotide polymorphism detection via allele-specific hybridization (ASH), amplified variable sequence detection in plant genomes, autonomous sequence replication detection, simple repeat sequence detection (SSR), single nucleotide polymorphism detection (SNP), and / or amplified fragment length polymorphism detection (AFLP). Recognized methods are also known for detecting expressed sequence tags (ESTs) and SSR markers derived from EST sequences, as well as randomly amplified polymorphic DNA (RAPD).

[0152] A “marker allele,” also described as “an allele of a marker locus,” can refer to one of several polymorphic nucleotide sequences found at a marker locus that are polymorphic to that locus in a population.

[0153] "Marker-assisted selection" (MAS) is a method for selecting phenotypes based on marker genotypes. In some embodiments, marker genotypes are used to identify plants that will be selected for breeding programs or for planting. In some embodiments, marker genotypes are used to identify plants that will not be selected for breeding programs or for planting (i.e., counter-selected plants), allowing them to be removed from the breeding / planting population.

[0154] As used herein, the terms "marker locus" and "marker loci" refer to one or more specific chromosomal locations in the genome of an organism in which one or more specific markers can be found. Marker loci can be used to track the presence of second-linked loci (e.g., linked loci that encode or contribute to the expression of phenotypic traits). For example, marker loci can be used to monitor the segregation of alleles at loci (such as QTLs or single genes) that are genetically or physically linked to that marker locus.

[0155] 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 specific alleles within a marker locus (e.g., a nucleic acid probe that is complementary to all or part of the marker or marker locus via nucleic acid hybridization). Marker probes containing about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive nucleotides can be used for nucleic acid hybridization. Alternatively, in some respects, a marker probe refers to any type of probe capable of distinguishing (i.e., genotyping) specific alleles present at a marker locus.

[0156] As used herein, when identifying linked loci, the terms "molecular marker" or "genetic marker" can be used to refer to a genetic marker as defined above, or its coding product (e.g., a protein) used as a reference point. Molecular markers can originate from genomic nucleotide sequences or expressed nucleotide sequences (e.g., from spliced ​​RNA, cDNA, etc.). The term also refers to nucleotide sequences complementary to or flanked by the marker sequence, such as nucleotide sequences used as probes and / or primers capable of amplifying the marker sequence. These nucleotide sequences are "complementary" (e.g., according to the Watson-Crick base pairing principle) when they specifically hybridize in solution. Some of the markers described herein are also referred to as hybridization markers when located on indel regions. This is because, by definition, the insertion region is a polymorphism concerning the absence of the insertion in plants. Therefore, the marker only needs to indicate the presence of the indel region. Such hybridization markers can be identified using any suitable marker detection technique, such as the SNP technique used in the examples provided herein.

[0157] A “non-naturally occurring soybean variety” is any soybean variety that does not exist in nature. A “non-naturally occurring soybean variety” can be produced by any method known in the art, including but not limited to transforming soybean plants or germplasm, transfecting soybean plants or germplasm, and hybridizing naturally occurring soybean varieties with non-naturally occurring soybean varieties. In some embodiments, a “non-naturally occurring soybean variety” may contain one or more heterologous nucleotide sequences. In some embodiments, a “non-naturally occurring soybean variety” may contain one or more non-naturally occurring copies of naturally occurring nucleotide sequences (i.e., foreign copies of genes naturally occurring in soybeans). In some embodiments, a “non-naturally occurring soybean variety” may contain a non-natural combination of two or more naturally occurring nucleotide sequences (i.e., two or more naturally occurring genes not naturally occurring in the same soybean, such as genes not found in soybean lines).

[0158] As used herein, the term "primer" refers to an oligonucleotide that, when placed under conditions for inducing the synthesis of primer extension products (e.g., in the presence of nucleotides and reagents for polymerization (e.g., DNA polymerase) and at suitable temperature and pH), can anneal to a nucleic acid target and serve as an initiation site for DNA synthesis. To obtain maximum efficiency in extension and / or amplification, in some embodiments, primers (extension primers in some embodiments and amplification primers in some embodiments) are single-stranded. In some embodiments, primers are oligodeoxynucleotides. Primers are generally long enough to initiate the synthesis of extension and / or amplification products in the presence of reagents for polymerization. The minimum length of a primer can depend on many factors, including but not limited to the temperature and composition of the primer (A / T versus G / C content). In the case of amplification primers, these amplification primers are typically provided as a pair of bidirectional primers consisting of one forward and one reverse primer, or as a pair of forward primers commonly used in the field of DNA amplification (e.g., in PCR amplification). Thus, it should be understood that the term "primer" as used herein can refer to more than one primer, particularly where there is some ambiguity in the information regarding one or more terminal sequences of the target region to be amplified. Therefore, "primer" can include a collection of primer oligonucleotides containing sequences representing possible variations in that sequence, or nucleotides that allow typical base pairing. Primers can be prepared by any suitable method known in the art. Methods for preparing oligonucleotides with specific sequences are known in the art and include, for example, cloning and restriction of suitable sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, the phosphodiester or triester method, diethylaminophosphate method, and solid-phase support method disclosed in U.S. Patent No. 4,458,066. If desired, primers can be labeled by incorporating a detectable portion, such as a spectral portion, a fluorescent portion, a photochemical portion, a biochemical portion, an immunochemical portion, or a chemical portion. Primers for diagnosing ASR resistance (i.e., the ability to identify or select based on the presence of ASR resistance alleles) can be generated for any favorable SNP described in any of Tables 1-5. PCR methods are well described in the manual and are known to those skilled in the art. Following PCR amplification, the target polynucleotide can be detected by hybridization with a probe polynucleotide that forms a stable hybrid with the target sequence under stringent to moderately stringent hybridization and washing conditions. Stringent conditions can be used if the probe is expected to be substantially perfectly complementary to the target sequence (i.e., about 99% or more). If some mismatches are expected, for example if variant varieties are expected to result in imperfect probe complementarity, the stringency of the hybridization can be reduced. In some embodiments, conditions are selected to exclude nonspecific / accidental bindings.Conditions affecting hybridization and conditions for nonspecific binding selection are known in the art and described, for example, in the following literature: Sambrook and Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. Generally, lower salt concentrations and higher temperatures for hybridization and / or washing increase the stringency of hybridization conditions.

[0159] As used herein, the terms “phenotype,” “phenotypic trait,” or “trait” refer to one or more characteristics and / or expressions of an organism. A phenotype is a manifestation that can be observed with the naked eye or by any other evaluative means known in the art (e.g., microscopy, biochemical analysis, or electromechanical assay). In some cases, a phenotype or trait is directly controlled by a single gene or gene locus, i.e., a “monogenous trait.” In other cases, a phenotype or trait is the result of multiple genes. It should be noted that, as used herein, the term “disease resistance phenotype” takes into account environmental conditions that may affect the corresponding disease, thus making the effect real and reproducible.

[0160] As used herein, the term "plant" can refer to the whole plant, any part thereof, or a cell or tissue culture derived from a plant. Therefore, the term "plant" can refer to any of the following: the whole plant, plant components or organs (e.g., roots, stems, leaves, buds, flowers, pods, etc.), plant tissues, seeds, and / or plant cells. Plant cells are plant cells obtained from a plant or plant cells derived from cells taken from a plant through culture. Therefore, the term "soybean plant" can refer to the whole soybean plant, one or more parts of a soybean plant (e.g., roots, root tips, stems, leaves, buds, flowers, pods, seeds, cotyledons, etc.), soybean plant cells, soybean plant protoplasts, and / or soybean plant callus.

[0161] As used herein, the term "plant part" includes, but is not limited to, embryo, pollen, seed, leaf, flower (including but not limited to anther, ovule, etc.), fruit, stem or branch, root, root tip, cell (including intact cells in a plant and / or plant part), protoplast, plant cell tissue culture, plant callus, plant mass, etc. Therefore, plant part includes soybean tissue culture capable of regenerating soybean plants. Additionally, as used herein, "plant cell" refers to the structural and physiological unit of a plant, including the cell wall, and may also refer to a protoplast. The plant cells of the present invention can be in the form of isolated single cells, or can be cultured cells, or can be part of a higher-level tissue unit (e.g., plant tissue or plant organ).

[0162] As used herein, the term "polymorphism" refers to a variation in the nucleotide sequence at a locus that is so common as to be more than just a result of spontaneous mutation. Polymorphisms can be single nucleotide polymorphisms (SNPs) or insertion / deletion polymorphisms (also referred to herein as "indels"). Additionally, the variation can be in transcriptional profiles or methylation patterns. One or more polymorphic sites in the nucleotide sequence can be identified by comparing the nucleotide sequences at one or more loci in two or more germplasm entries.

[0163] As used herein, the term “tightly linked” means that a linked marker exhibits a crossing frequency of approximately 10% or less for a given marker (e.g., the given marker is within approximately 10 cM of a tightly linked ASR marker). In other words, tightly linked sites co-segregate at least approximately 90% of the time. Regarding their physical location on the chromosome, tightly linked markers can be separated by, for example, approximately 1 megabase (Mb; 1 million nucleotides), approximately 500 kilobases (Kb; 1000 nucleotides), approximately 400 Kb, approximately 300 Kb, approximately 200 Kb, approximately 100 Kb, approximately 50 Kb, approximately 25 Kb, approximately 10 Kb, approximately 5 Kb, approximately 4 Kb, approximately 3 Kb, approximately 2 Kb, approximately 1 Kb, approximately 500 nucleotides, approximately 250 nucleotides, or less.

[0164] As used in this article, the term "population" refers to a genetically heterogeneous collection of plants that share a common genetic derivation.

[0165] As used herein, the terms "offspring" and "offspring plant" refer to plants produced by asexual or sexual reproduction of one or more parent plants. Offspring plants can be obtained through cloning or self-pollination of a single parent plant, or through hybridization of two parent plants.

[0166] As used herein, the term "reference sequence" refers to a defined nucleotide sequence used as the basis for nucleotide sequence comparisons. A reference sequence can be obtained, for example, by genotyping multiple lines at one or more target loci, comparing these nucleotide sequences in a sequence alignment program, and then obtaining a shared sequence from the alignments. Thus, the reference sequence identifies polymorphisms in alleles at a locus. The reference sequence may not be a copy of the actual nucleic acid sequence from any particular organism; however, it is useful for designing primers and probes targeting actual polymorphisms at one or more loci.

[0167] As used herein, the terms "disease tolerance" and "disease resistance" refer to the tolerance and / or reproductive capacity of a plant despite infection with a corresponding disease. When used with respect to germplasm, these terms refer to the tolerance and / or reproductive capacity of plants produced from that germplasm despite infection with a corresponding disease. In some embodiments, infected disease-resistant soybean plants may be produced in the same (or nearly the same) manner as uninfected soybean plants. Typically, if a plant or germplasm exhibits "enhanced pathogen resistance," it is labeled "disease resistance."

[0168] The marker "unfavorable allele" is a marker allele that segregates from the said unfavorable plant phenotype, thus providing the benefit of identifying plants that can be removed from breeding programs or cultivation. For example, one can eliminate plant lines carrying unfavorable alleles against ASR resistance from plant breeding program programs.

[0169] "PI441001, PI441008, PI583970 or PI483224" refers to the registered names of the short-haired wild soybean plant PI441001, PI441008, PI583970 or PI483224.

[0170] Genetic mapping

[0171] Genetic loci associated with specific phenotypes (such as disease resistance) can be located within an organism's genome. By identifying markers or marker clusters that cosegregate with the desired trait, breeders can rapidly select for the desired phenotype by choosing appropriate markers (a method known as marker-assisted selection or "MAS"). Breeders can also use such markers to simulate genotype design on computers and implement genome-wide selection.

[0172] This invention provides markers associated with enhanced disease resistance. These markers and / or the detection of other linked markers can be used to identify, select, and / or produce disease-resistant (more specifically, ASR-resistant) plants and / or eliminate disease-insensitive plants from breeding programs or planting.

[0173] Short-fibered soybean locus associated with enhanced disease resistance

[0174] This article identifies markers associated with enhanced disease resistance (see Tables 1-5, indicating favorable markers associated with enhanced ASR resistance). The markers of this invention may comprise a single allele or a combination of alleles at one or more genetic loci (e.g., any combination of favorable markers from Tables 1-5). For example, a marker may comprise one or more marker alleles located in a first chromosomal region (e.g., SEQ ID NO:1) and one or more marker alleles located in a second chromosomal region (e.g., SEQ ID NO:2).

[0175] Logo-assisted selection

[0176] Markers can be used in a variety of plant breeding applications. See, for example, Staub et al., Hortscience 31:729 (1996); Tanksley, Plant Molecular Biology Reporter 1:3 (1983). One of the main areas of application is using marker-assisted selection (MAS) to increase the efficiency of backcrossing and gene introgression. Typically, MAS utilizes genetic markers that have been identified as having a significant probability of cosegregation with the desired trait. It is presumed that such markers are located in / near the genes that produce the desired phenotype, and their presence indicates that the plant will possess the desired trait. It is expected that plants possessing the marker will transfer the desired phenotype to their offspring.

[0177] Markers exhibiting linkage to loci influencing desired phenotypic traits provide a useful tool for selecting traits in plant populations. This is especially true when phenotypes are difficult to determine or occur later in plant development. Because DNA marker assays are less labor-intensive and require less physical space than field phenotypic analysis, they can be used to measure larger populations, increasing the probability of finding recombinants with target segments that have migrated from donor to recipient lines. The tighter the linkage, the more useful the marker, because recombination is less likely to occur between the marker and the gene that causes or induces the trait. Using flanking markers reduces the chance of false positive selection. Ideally, the pathogenic gene itself contains the marker, preventing recombination between the marker and the gene. Such markers are called "perfect markers."

[0178] When genes are introduced via MAS, not only the gene itself but also lateral regions are introduced. Gepts, Crop Sci 42:1780 (2002). This is called “linkage slack.” In cases where the donor and recipient plants are highly unrelated, these lateral regions carry additional genes that can encode traits that are agronomically unwanted. Even after multiple backcrosses with superior soybean lines, this “linkage slack” can lead to reduced yield or other negative agronomic traits. This is sometimes also called “yield slack.” The size of lateral regions can be reduced through additional backcrosses, although this is not always successful because breeders cannot control the size of the region or the recombination breakpoint. Young et al., Genetics 120:579 (1998). In classical breeding, recombinations that contribute to reducing the size of donor segments are often selected, usually by chance. Tanksley et al., Biotechnology 7:257 (1989). Even after 20 backcrosses, it is possible to find fairly large donor chromosome fragments that are still linked to the gene being selected. However, using markers allows for the selection of rare individuals that have undergone recombination near the target gene. In 150 backcrossed plants, there is a 95% chance that at least one plant will undergo hybridization within 1 cM (based on the single meiotic map distance) of the gene. Markers allow for the definitive identification of these individuals. Using a single additional backcross of 300 plants, there is a 95% probability of hybridization within 1 cM of the single meiotic map distance on the other side of the gene, resulting in a segment near the target gene within less than 2 cM based on the single meiotic map distance. This can be achieved in two generations with markers, compared to an average of 100 generations without them. See Tanksley et al., ibid. When the exact location of a gene is known, flanking markers around that gene can be used to select for recombination at different population sizes. For example, in smaller populations, recombination is expected to be further away from the gene, thus requiring more distant flanking markers to detect it.

[0179] The availability of integrated linkage maps of the soybean genome containing publicly available soybean markers with enhanced density has facilitated soybean genetic mapping and MAS.

[0180] Of all molecular marker types, SNPs are the most abundant and have the potential to provide the highest resolution genetic mapping. (Bhattramakki et al., Plant Molec. Biol. [Plant Molecular Biology] 48:539 (2002)). SNPs can be determined in a so-called "ultra-high throughput" manner because they do not require large amounts of nucleic acids, and the automation of the assay is straightforward. SNPs also have the advantage of being a relatively low-cost system. These three factors together make the use of SNPs in MAS highly attractive. Several methods can be used for SNP genotyping, including but not limited to: hybridization, primer extension, oligonucleotide ligation, nuclease cutting, microsequencing, and coded spheres. Such methods have been reviewed in various publications: Gut, Hum. Mutat. [Human Mutation] 17:475 (2001); Shi, Clin. Chem. [Clinical Chemistry] 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 query SNPs, including Masscode. TM (Qiagen, Germantown, MD) (Hologic, Madison, WI) (Applied Biosystems, Foster City, CA) (Applied Biosystems, Foster City, CA) and Beadarrays TM (Illumina, San Diego, California).

[0181] Haplotypes of any particular genotype can be described using multiple SNP alleles, either within a sequence or across linked sequences. (Ching et al., BMC Genet. 3:19 (2002); Gupta et al., (2001), Rafalski, Plant Science 162:329 (2002b).) Haplotypes can be more informative than a single SNP and can describe any particular genotype in more detail. For example, for a particular disease-resistant line or variety, a single SNP might be the allele “T,” but the allele “T” can also appear in soybean breeding populations used for recurrent parents. In this case, a combination of alleles of linked SNPs can be more informative. Once a unique haplotype is assigned to a donor chromosomal region, that haplotype can be used in that population or any subpopulation to determine whether an individual possesses the specific gene. The use of automated, high-throughput marker detection platforms known to those skilled in the art makes this method efficient and effective.

[0182] The marker of this invention can be used in marker-assisted selection schemes to identify and / or select progeny with enhanced resistance to Asian soybean rust. Such methods may include, substantially consist of, or consist of: crossing a first soybean plant or germplasm with a second soybean plant or germplasm, wherein the first soybean plant or germplasm contains chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein said chromosomal regions correspond to nucleotide bases 1 to 1251375 of SEQ ID NO:1, or wherein said chromosomal regions correspond to nucleotide bases 1 to 2515428 of SEQ ID NO:2, and selecting progeny plants having the marker. One or both of the first and second soybean plants may be non-naturally occurring soybean varieties. In some embodiments, the second soybean plant or germplasm is a second soybean plant or germplasm of a superior soybean variety. 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 the genome of a superior soybean variety. In another embodiment, the first soybean plant comprises chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal region corresponds to nucleotide bases 1 to 1251375 of SEQ ID NO:1, or wherein the chromosomal region corresponds to nucleotide bases 1 to 2515428 of SEQ ID NO:2, wherein the chromosomal region further comprises at least one allele as described in any one of Tables 1-5.

[0183] Methods for identifying and / or selecting disease-resistant soybean plants or germplasm may include, substantially consist of, or consist of the following: detecting the presence of markers associated with enhanced ASR tolerance. These markers can be detected in any sample obtained from the plant or germplasm, including but not limited to the whole plant or germplasm, a portion of the plant or germplasm (e.g., seed slices, leaf punch disks, or cells from the plant or germplasm), or a nucleotide sequence from the plant or germplasm. Such samples can be obtained from the plant or germplasm using any existing or future methods known in the art (including but not limited to automated methods: removing a portion of the endosperm with a sharp blade, drilling a small hole in the seed and collecting the resulting powder, laser-cutting the seeds, and punching leaf discs). The soybean plant may be a non-naturally occurring soybean variety. In some embodiments, the genome of 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 the genome of a superior soybean variety.

[0184] In some embodiments, the markers detected in a sample may comprise, consist substantially of, or consist of one or more marker alleles located within a chromosomal region selected from the group consisting of:

[0185] 1) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 1251375 of SEQ ID NO:1; or

[0186] 2) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 2515428 of SEQ ID NO:2; or

[0187] 3) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 365842 of SEQ ID NO:3; or

[0188] 4) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to 646429 of SEQ ID NO:4.

[0189] 5) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 750080 of SEQ ID NO:5; or

[0190] 6) SNP markers in soybean that are associated with increased ASR resistance spanning chromosomal intervals of 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, and 0.5 cM, wherein the SNP markers are selected from the following group, which consists of any favorable SNP markers shown in Tables 1-5.

[0191] Methods for producing disease-resistant soybean plants may include, substantially consist of, or consist of: detecting in germplasm a marker associated with enhanced disease (e.g., ASR) resistance, wherein said marker is selected from Tables 1-5, or wherein the marker is a locus closely linked to any marker described in any of Tables 1-5 and from which soybean plants are produced. The marker can be detected in any sample obtained from germplasm, including but not limited to a portion of said germplasm (e.g., seed slices or cells from said germplasm) or a nucleotide sequence from said germplasm. Such samples can be obtained from germplasm using any existing or future methods known in the art (including but not limited to automated methods: removing a portion of the endosperm with a sharp blade, drilling a small hole in the seed and collecting the resulting powder, laser-cutting the seeds, and stamping leaf discs). Germplasm may be a non-naturally occurring soybean variety. In some embodiments, the genome of the germplasm is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the genome of a superior soybean variety. Disease-resistant soybean plants are then produced from germplasm identified as having markers associated with enhanced disease (e.g., ASR) resistance, according to methods well known in the art for breeding and producing plants from germplasm.

[0192] In some embodiments, the markers detected in germplasm may comprise, consist substantially of, or consist of one or more marker alleles located within a chromosomal region selected from the group consisting of:

[0193] 1) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 1251375 of SEQ ID NO:1; or

[0194] 2) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 2515428 of SEQ ID NO:2; or

[0195] 3) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 365842 of SEQ ID NO:3; or

[0196] 4) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to 646429 of SEQ ID NO:4.

[0197] 5) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 750080 of SEQ ID NO:5; or

[0198] 6) SNP markers in soybean that are associated with increased ASR resistance spanning chromosomal intervals of 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, and 0.5 cM, wherein the SNP markers are selected from the following group, which consists of any favorable SNP markers shown in Tables 1-5.

[0199] In some embodiments, the markers detected in the germplasm may comprise, consist substantially of, or consist of one or more marker alleles selected from any one of Tables 1-5.

[0200] Methods for generating and / or selecting soybean plants or germplasm resistant to / tolerant to Asian soybean rust may include crossing a first soybean plant or germplasm with a second soybean plant or germplasm, wherein the first soybean plant or germplasm contains chromosomal regions selected from the group consisting of:

[0201] 1) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 1251375 of SEQ ID NO:1; or

[0202] 2) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 2515428 of SEQ ID NO:2; or

[0203] 3) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 365842 of SEQ ID NO:3; or

[0204] 4) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 646429 of SEQ ID NO:4; or

[0205] 5) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 750080 of SEQ ID NO:5; or

[0206] 6) A chromosomal region in soybean from which increased ASR resistance is associated with a SNP marker spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, or 0.5 cM, wherein the SNP marker is selected from the group consisting of any favorable SNP markers shown in Tables 1-5; and is crossed with a second soybean plant that does not contain this chromosomal region, thereby producing progeny plants with increased ASR resistance. One or both of the first or second soybean plant or germplasm may be a non-naturally occurring soybean variety. In some embodiments, the second soybean plant or germplasm is a second soybean plant or germplasm of a superior soybean variety. 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 the genome of the superior soybean variety.

[0207] This article also provides a method for introgression of alleles associated with enhanced disease resistance / tolerance (e.g., ASR, SCN, SDS, RKN, Phytophthora, etc.) into soybean plants. Such a method for introgression of alleles associated with enhanced disease resistance / tolerance (e.g., ASR, SCN, SDS, RKN, Phytophthora, etc.) into soybean plants or germplasm may include, substantially consist of, or consist of the following: crossing a first soybean plant or germplasm containing the allele (donor) (wherein the allele is selected from any allele listed in Table 1 or Table 2) or a marker “closely similar” to the markers listed in Tables 1-5 with a second soybean plant or germplasm lacking the allele (recurrent parent), and repeatedly backcrossing the progeny containing the allele with the recurrent parent. Progeny containing the allele can be identified by detecting the presence of markers associated with enhanced disease resistance / tolerance (e.g., ASR, SCN, SDS, RKN, Phytophthora, etc.) in their genome. The marker can be detected in any sample obtained from the progeny, including but not limited to a portion of the progeny (e.g., seed slices, leaf pellets, or cells from the plant or germplasm) or a nucleotide sequence from the progeny. Such a sample can be obtained from the progeny using any existing or future methods known in the art (including but not limited to automated methods: removing a portion of the endosperm with a sharp blade, drilling a small hole in the seed and collecting the resulting powder, laser-cutting the seeds, and punching leaf discs). One or both of the donor or recurrent parent can be a non-naturally occurring soybean variety. In some embodiments, the recurrent parent is a superior soybean variety. In some embodiments, the genome of the recurrent parent is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the genome of the superior soybean variety.

[0208] In some embodiments, a marker for identifying offspring containing alleles associated with enhanced disease (e.g., ASR, SCN, SDS, RKN, Phytophthora, etc.) resistance / tolerance may comprise, consist substantially of, or consist of one or more alleles located within a chromosomal region selected from the group consisting of:

[0209] 1) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 1251375 of SEQ ID NO:1; or

[0210] 2) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to nucleotide bases 2515428 of SEQ ID NO:2.

[0211] 3) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 365842 of SEQ ID NO:3; or

[0212] 4) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to 646429 of SEQ ID NO:4.

[0213] 5) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 750080 of SEQ ID NO:5; or

[0214] 6) SNP markers in soybeans that are associated with increased ASR resistance spanning chromosomal intervals of 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, and 0.5 cM, wherein the SNP markers are selected from the following group, which consists of any SNP markers shown in Tables 1-5 or any closely linked markers that are closely adjacent to the intervals 1)-5).

[0215] In some embodiments, a marker may comprise, consist substantially of, or consist of marker alleles located in at least two distinct chromosomal regions. For example, a marker may comprise one or more alleles located in a chromosomal region defined and included by any two markers in Table 1, any two markers in Table 2, any two markers in Table 3, or any two markers in Table 4 and / or Table 5.

[0216] The following are all embodiments considered and covered by this invention:

[0217] 1. A superior soybean plant having a chromosomal region in its genome derived from a wild soybean species, wherein the chromosomal region contains any one of SEQ ID NO: 1-5 or a functional portion thereof that confers ASR resistance in said plant; in some cases, the wild soybean species is short-haired wild soybean, and in other cases, the wild soybean species is selected from plant registrations PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal region confers increased resistance to Asian soybean rust (ASR) compared to a control plant that does not contain said chromosomal region; additionally, in a variation of Example 1, said superior soybean plant is produced by: a) a non-natural exchange between soybean plants and short-haired wild soybean plants. Hybridization, where “non-natural hybridization” means hybridization between two species that would not produce viable offspring without further intervention (e.g., embryo rescue as described in Example 3), such as: a) performing embryo rescue substantially as described in Example 3 to produce a double diploid plant; b) backcrossing the double diploid plant of b) with a soybean plant for at least one backcross (preferably BC4), resulting in the production of a superior soybean plant with genes infiltrated into one or more of the said chromosomal regions in its genome; additionally, the superior soybean plant may have 20 chromosome pairs or may maintain chromosome pairs from short-haired wild soybean and inherit the said regions to confer ASR resistance in the superior soybean plant.

[0218] 2. The plant as described in Example 1, wherein the chromosome region comprises SEQ ID NO: 1, 2, 3, 4, 5 or any part thereof, wherein the portion confers superior ASR resistance in soybean plants.

[0219] 3. The plant as described in Examples 1-2, wherein the chromosome region contains SNP markers associated with increased ASR resistance, wherein the SNP markers are consistent with any of the favorable SNP markers listed in Tables 1-5; or in another variation, the haplotype contains at least two SNP markers associated with ASR resistance, and furthermore, wherein the at least two SNP markers are selected from any of the favorable SNP markers listed in Tables 1-5.

[0220] 4. The plant as described in Examples 1-3, wherein the chromosome region is derived from the short-haired wild soybean chromosome 5 in the approximate location region of 0.02-1.19 Mb.

[0221] 5. The plant as described in Examples 1-5, wherein the chromosomal region corresponds to a location within the genome of wild soybean and / or short-haired wild soybean containing SEQ ID NO:1-5 or a portion thereof, wherein the portion confers increased ASR resistance in the superior soybean plant.

[0222] 6. The plant as described in Examples 1-5, wherein the superior soybean plant further exhibits resistance to any of the following stresses: diseases (e.g., powdery mildew, Pythium spp., Phytophthora root rot, leaf spot, rice blast, brown spot, root-knot nematodes, soybean cyst nematodes, soybean vein necrosis virus, soybean stem canker, soybean sudden death syndrome, leaf blast and ear blast, rust, frog-eye leaf spot, brown stem rot, Fusarium wilt, or sheath blight); insect pests (e.g., whiteflies, aphids, wheat slugs, sugarcane borers, green aphids, or aphids); abiotic stresses (e.g., drought tolerance, flooding, high salinity, heavy metals, aluminum, manganese, cadmium, zinc, UV-B, boron, iron deficiency chlorosis, or cold tolerance (i.e., extreme temperatures)), and additionally, wherein the resistance to the stress is conferred from the said chromosomal region.

[0223] 7. The plant as described in Examples 1-6, wherein at least one parental line of the plant is selected or identified by molecular marker selection, wherein the parental line is selected or identified based on the presence of a molecular marker located within or closely associated with the chromosomal region, the chromosomal region corresponding to any one of SEQ ID NO: 1-5.

[0224] 8. The plant as described in Example 7, wherein the molecular marker is a single nucleotide polymorphism (SNP), a quantitative trait locus (QTL), an amplified fragment length polymorphism (AFLP), a random amplified polymorphic DNA (RAPD), a restriction fragment length polymorphism (RFLP), or a microsatellite.

[0225] 9. The plant as described in Examples 7-8, wherein the molecular marker is an SNP marker and the molecular marker is any advantageous marker as shown in Tables 1-5.

[0226] 10. The plant as described in any one of Examples 1-9, wherein the plant is an agronomically superior soybean plant having commercially significant yields and commercially susceptible vigor, seed setting, standing ability, or threshing ability.

[0227] 11. The plant as described in any one of Examples 1-10, wherein the region is introduced into the plant genome by “non-natural” distant hybridization between soybean and short-haired wild soybean lines, followed by subsequent embryo rescue (e.g., as described in Example 3) and then the resulting plant is backcrossed at least once, more preferably at least four times (BC4), with a soybean line (i.e., in some cases a recurrent soybean line) to produce a superior or agronomically superior soybean line.

[0228] 12. The plant as described in any one of Examples 1-10, wherein the interval is introduced into the plant genome by transgenic expression of the sequence or genome editing, the sequence corresponding to and comprising any one or a portion of SEQ ID NO: 1-5, wherein the portion retains ASR resistance when introduced into a susceptible line.

[0229] 13. The plant as described in Example 12, wherein the region is introduced by: genome editing of a soybean genome region homologous to any of the regions corresponding to SEQ ID NO: 1-5 or an ortholog of any of the regions corresponding to SEQ ID NO: 1-5, and further performing at least one gene edit on the soybean genome region to include at least one allelic alteration corresponding to any favorable allele described in any of Tables 1-5, wherein the soybean genome region did not contain the allelic alteration prior to the genome editing, and the genome editing conferred increased ASR resistance in the plant compared to the control plant.

[0230] 14. The plant as described in Example 13, wherein the gene editing is accomplished by CRISPR, TALEN, meganuclease or by modification of genomic nucleic acids.

[0231] 15. The plant as described in Examples 1-14, wherein the chromosome region contains any one or a portion of the following nucleotide base pairs (wherein the portion retains ASR resistance): SEQ ID NO:1 1-1251375; SEQ ID NO:2 1-2515428; SEQ ID NO:3 1-365842; SEQ ID NO:4 1-646429 or SEQ ID NO:5 1-750080.

[0232] 16. A commercially significant yield of ASR-resistant agronomically superior soybean plant, wherein the plant comprises a gene introgression from a chromosomal region of a wild soybean genus, wherein the chromosomal region corresponds to and / or comprises any one of SEQ ID NO: 1-5; in some cases, the wild soybean genus is *Glycine max*, and in other cases, the wild soybean genus is any one of registered PI441001, PI441008, PI446958, PI509501, PI583970, PI483224 or its progeny, wherein the gene introgression comprises conferring ASR resistance to at least one QTL linked to a marker located on chromosome 5 equivalent to *Glycine max*.

[0233] 17. The ASR-resistant soybean plant as described in Example 16, wherein the QTL is homozygous.

[0234] 18. The plant as described in Examples 16-17, wherein the parent plant of the ASR-resistant agronomically superior soybean plant is a distant hybrid between soybean and short-fibered wild soybean parent lines; in a variation, the parent line is a double diploid hybrid between soybean and short-fibered wild soybean parent lines (“double diploid hybrid” herein).

[0235] 19. A commercially significant agronomically superior soybean plant comprising an ASR resistance allele conferring increased resistance to ASR, wherein the ASR allele comprises at least one single nucleotide polymorphism (SNP) selected from the group consisting of any of the favorable SNPs described in Tables 1-5, and further wherein the plant contains chromosomal regions (i.e., any of SEQ ID NO: 1-5) from short-haired wild soybean or its progeny (e.g., double diploid hybrids) in its genome.

[0236] 20. An agronomically superior soybean plant with commercially significant yield, comprising a chromosomal region from chromosome 5 of short-haired wild soybean, the chromosomal region containing at least one favorable SNP marker selected from any one of Tables 1-5; in one variation, the chromosomal region is any one of SEQ ID NO: 1-5.

[0237] 21. The plant as described in Example 20, wherein the chromosomal region from chromosome 5 of short-haired wild soybean is derived from any one of plant registrations PI441001, PI441008, PI446958, PI509501, PI583970, PI483224 or their progeny (e.g., like a double diploid hybrid).

[0238] 22. The plant as described in Examples 20-21, wherein the chromosome region comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or a portion thereof, wherein the chromosome region confers increased ASR resistance in the plant compared to a control plant that does not contain the chromosome region.

[0239] 23. A plant cell or plant part derived from the plant or seed described in Examples 1-22; in some cases, the plant cell or plant part cannot be used to regenerate the plant (e.g., therefore the plant cell or plant part cannot be used to regenerate the plant or seed from Examples 1-22).

[0240] 24. A progeny plant or seed from any one of Examples 1-22.

[0241] 25. The soybean plant as described in Examples 1-22, wherein the plant has 20 chromosome pairs and / or 40 paired chromosomes, or in some cases, the plant maintains chromosome pairs derived from wild soybean strains.

[0242] 26. The soybean plant as described in Examples 1-25, wherein there is no yield burden or negative agronomic effects compared with the control plant.

[0243] 27. A method for producing soybean plants (in some cases, “double diploid hybrids”) with increased resistance to Asian soybean rust (ASR), the method comprising the steps of:

[0244] a. Cross soybean plants with ASR-resistant wild soybean species (in some cases, short-haired wild soybean plants);

[0245] b. Soybean pods are produced from the hybridization of a);

[0246] c. Separate the embryos from the pods of b) and place them on embryo rescue medium (in some cases, the embryo rescue medium contains or has a composition substantially equivalent to the following: 3.1 g B5 basal salt, 1 ml Gamborg B5 vitamin 1000X, 40 g sucrose [C12H22O11], 0.25 g casein hydrolysate, 0.25 mg BAP, 0.75 g MgCl2*6H2O, 20 ml glutamine 25 mg / ml, 0.1 g serine [C3H7NO3], 4 ml asparagine 25 mg / ml, 0.05 ml IBA 1 mg / ml, and 2.0 g Gelzan); or Murashige and Skog medium (MS) or Gamborg B-5 medium (Bridgen, 1994)), wherein no callus induction was induced or initiated in step c).

[0247] d. Transfer the embryos from c) to germination medium or elongation medium (in some cases, the germination medium contains or has a composition substantially equivalent to the following: 3.2 g Schenk and Hilderbrandt basal salt mixture, 1 g inositol [C6H12O6], 5 ml thiamine 1 mg / ml, 0.5 ml pyridoxine 1 mg / ml, 10 g sucrose [C12H22O11], and 7.5 g purified agar), or elongation medium (which contains or has a composition substantially equivalent to the following: 4.3 g MS basal salt mixture [MSPO1], 5 ml MS iron 200X, 30 g sucrose [C12H22O11], 1 g MES [C6H13NO4S], 8 g purified agar, 1 ml vitamin B5 100X, 2 ml glutamine 25 mg / ml, 0.50 ml zeatin nucleoside, trans isomer 1 mg / ml, 0.1 ml IAA 1 mg / ml, 0.2 ml GA3); and collecting branches from the embryo;

[0248] e. Collected branches from d) grown on germination or elongation media; and

[0249] f. Transfer the established e) branches into the soil to produce soybean plants with increased resistance to ASR (in the preferred case, the branches are rooted on an elongation or germination medium to form plantlets and then harden).

[0250] 28. The method as described in Example 27, wherein the wild soybean plant or short-haired wild soybean plant of step a) contains in its genome a chromosomal region corresponding to or defined by any one or a portion of SEQ ID NO: 1-5 (in this document, "chromosomal region defined by" means the span of nucleotide bases from the first nucleotide to the final nucleotide of the corresponding SEQ ID NO:), wherein said portion confers increased ASR resistance in the plant.

[0251] 29. The method of Examples 27-28, wherein the short-haired wild soybean plant contains alleles associated with ASR resistance in its genome, wherein the alleles correspond to any favorable alleles listed in Tables 1-5 or alleles or SNPs within any chromosomal region containing any of SEQ ID NO: 1-5, wherein the alleles or SNPs are associated with or closely linked to ASR resistance in the plant.

[0252] 30. The method as described in Examples 27-29, wherein the wild soybean or short-haired wild soybean plant is any one of PI441001, PI441008, PI446958, PI509501, PI583970, PI483224 or its progeny (in some cases, the progeny is a double diploid hybrid).

[0253] 31. The method as described in Example 30, wherein the offspring plant is a distant hybrid between soybean and short-haired wild soybean parent lines.

[0254] 32. The method of Examples 27-31, further comprising a molecular marker selection step, wherein isolated genomic nucleic acids from any of the following: a) the parent plant, c) the embryo, or f) the shoot or resulting plant are isolated and analyzed (e.g., genotyping) for the presence of alleles associated with increased ASR resistance, and additionally, wherein said alleles are closely associated with chromosomal regions corresponding to and defined by SEQ ID NO: 1-5 (e.g., any favorable alleles described in any of Tables 1-5).

[0255] 33. The method as described in Example 32, wherein the allele corresponds to any of the favorable alleles listed in Tables 1-5.

[0256] 34. The method as described in Examples 27-33, wherein the soybean plants in a) have a relative maturity of 3.7-4.8, or in other variations, the soybean plants may be selected from soybean maturity group 000, X.

[0257] 35. The method as described in Examples 27-34, wherein the soybean plant of a) is used as the female plant and the wild soybean or short-haired wild soybean plant is the pollen donor; alternatively, in some cases, the soybean plant of a) may be used as the male pollen donor plant and the wild soybean or short-haired wild soybean plant is used as the female plant.

[0258] 36. The method as described in Examples 27-35, wherein the pods of b) are treated with a hormone mixture comprising GA3, NAA and kinetin (in some examples, 100 mg GA3, 25 mg NAA and 5 mg kinetin / L, and in some cases, the hormone treatment is sprayed onto the pods daily until harvest).

[0259] 37. The method as described in Examples 27-36, wherein the pods are collected before 19 days after pollination.

[0260] 38. The method as described in Examples 27-37, wherein in any of steps e)-h), embryos and / or shoots are treated with a chromosome doubling agent to produce a double diploid plant (or double diploid hybrid) capable of being backcrossed with a non-wild (domestic) annual soybean cultivar to produce at least one backcross generation (in some cases, BC4 generation), wherein the double diploid plant also contains favorable agronomic traits in its genome, such as ASR resistance inherited from wild soybean or short-haired wild soybean parents; in other cases, these double diploid plants are backcrossed with a recurrent soybean parent line, followed by embryo rescue (e.g., as described in Examples 65-83) to produce BC1 and repeated multiple times to produce BC4 generation, wherein the BC4 generation is fertile (i.e., does not require embryo rescue to produce plants) and capable of producing ASR-resistant soybean progeny plants.

[0261] 39. The method as described in Example 38, wherein the chromosome doubling agent is colchicine or trifluralin; additionally, the intended chromosome doubling agent that can be used in Example 38 may contain sulfamethoxazole.

[0262] 40. The method as described in Examples 27-39, wherein the embryo in c) is retained on embryo rescue medium at about 24°C for at least about 20 days.

[0263] 41. The method as described in Examples 27-39, wherein the embryo of d) is kept at about 24°C on a germination or elongation medium for at least about 20 days.

[0264] 42. The method as described in Example 41, wherein the embryo rescue medium is soybean ER1-1, wherein soybean ER1-1 comprises the following: 3.1 g B5 basal salt, 1 ml Gamborg B5 vitamin 1000X, 40 g sucrose [C12H22O11], 0.25 g casein hydrolysate, 0.25 ml BAP, 0.75 g MgCl2*6H2O, 20 ml glutamine 25 mg / ml, 0.1 g serine [C3H7NO3], 4 ml asparagine 25 mg / ml and 0.05 ml IBA 1 mg / ml (hereinafter, "soybean ER1-1").

[0265] 43. A method for producing soybean plants with increased resistance to Asian soybean rust (ASR), the method comprising the steps of:

[0266] a. Providing a first soybean plant having a genome containing a chromosomal region corresponding to any one of SEQ ID NO:1-5, wherein the first soybean plant has increased resistance to ASR;

[0267] b. Cross the soybean plant from a) with a second soybean plant that does not contain the said chromosome region;

[0268] c. (In some embodiments, this step is omitted) Selecting progeny plants from the hybridization in b) by isolating nucleic acids from the progeny plants and detecting alleles associated with increased ASR resistance within the nucleic acids, wherein the alleles are tightly linked to chromosomal regions corresponding to SEQ ID NO: 1-5.

[0269] d. This results in soybean plants with increased resistance to ASR.

[0270] 44. The method as described in Example 43, wherein the allele in c) corresponds to any favorable allele described in any of Tables 1-5.

[0271] 45. The method as described in Examples 43-44, wherein the first or second soybean plant is a superior soybean plant; alternatively, the first or second soybean plant may also be a superior soybean plant with commercially significant yields, and in other embodiments, agronomically superior soybean plants.

[0272] 46. ​​The method as described in Examples 43-45, wherein the progeny plant undergoes one or more backcrosses, in some cases at least four backcrosses (BC4); in other cases, after BC1, BC2, BC3 generations, there is embryo rescue as described in Example 3 or Examples 65-82 below.

[0273] 47. The method as described in Examples 27-46, wherein the chromosome region corresponds to the short-haired soybean chromosome 5 located in the approximate positioning range of 0.02-1.19 Mb, and additionally includes any one or a portion of SEQ ID NO: 1-5, wherein the portion confers or is associated with ASR resistance.

[0274] 48. The method as described in Example 47, wherein the short-haired wild soybean chromosome region is derived from any of the following plant registrations: PI441001, PI441008, PI446958, PI509501, PI583970, PI483224 or their progeny, and in some cases, the progeny comprises a double diploid hybrid.

[0275] 49. The method as described in Example 48, wherein the offspring plant is a distant hybrid between soybean and short-haired wild soybean parent lines.

[0276] 50. A method for producing soybean plants with increased ASR resistance, the method comprising the steps of:

[0277] a) Isolating nucleic acids from soybean plants;

[0278] b) Detect at least one molecular marker associated with increased ASR in the nucleic acid of a), wherein the molecular marker is located within 20 cM, 10 cM, 5 cM, 1 cM, 0.5 cM, or is closely linked to a chromosomal region corresponding to a genomic region from a wild soybean plant or short-haired wild soybean plant containing any one or a portion of SEQ ID NO: 1-5.

[0279] c) Selecting plants based on the presence of the molecular markers detected in b), and further hybridizing the selected plants with a second plant (e.g., a soybean plant) that does not contain the said chromosomal region; and

[0280] d) Produce soybean progeny plants from plants identified as having the alleles associated with increased ASR resistance and / or from plants crossed with a second plant (c).

[0281] 51. The method as described in Example 50, wherein the molecular marker is closely linked to or corresponds to any of the favorable alleles described in Tables 1-5, and / or the molecular marker is located within the chromosomal region from short-haired wild soybean that corresponds to and / or is defined by SEQ ID NO: 1-5.

[0282] 52. A method for identifying and / or selecting plants resistant to ASR (in some embodiments, soybean or short-haired wild soybean plants), the method comprising the following steps:

[0283] a. Isolating nucleic acids from soybean plants or plant parts;

[0284] b. Detect the presence in the nucleic acid of a molecular marker associated with increased ASR resistance, wherein the molecular marker is located within 20 cM, 10 cM, 5 cM, 1 cM, or 0.5 cM of a marker as described in any of Tables 1-5, or in another embodiment, the molecular marker is closely linked to, closely proximates to, closely associated with, or within a chromosomal region corresponding to and / or containing any of SEQ ID NO: 1-5; and

[0285] c. Identify or select soybean plants with increased ASR resistance based on the molecular markers detected in b), thereby identifying and / or selecting plants with ASR resistance.

[0286] 53. The method as described in Example 52, wherein the molecular marker detected in b) consists of any favorable marker described in any of Tables 1-5 or an allele or molecular marker associated with ASR, wherein the allele or molecular marker is located within a chromosomal region containing or defined by any of SEQ ID NO: 1-5.

[0287] 54. The method as described in Examples 50-53, wherein the molecular marker is a single nucleotide polymorphism (SNP), a quantitative trait locus (QTL), an amplified fragment length polymorphism (AFLP), a random amplified polymorphic DNA (RAPD), a restriction fragment length polymorphism (RFLP), or a microsatellite.

[0288] 55. The method of any one of Examples 50-54, wherein the detection comprises amplifying a marker locus or a portion thereof and detecting the resulting amplified marker amplicon (in some embodiments, the amplicon contains a nucleotide sequence as shown in any one of SEQ ID NO 1-5; in another embodiment, primers capable of annealing to and amplifying the marker locus to allow detection of the resulting amplified marker amplicon).

[0289] 56. The method of Example 55, wherein the amplification comprises: a) mixing an amplification primer or primer pair with nucleic acid isolated from a first soybean plant or germplasm, wherein the primer or primer pair is complementary or partially complementary to at least a portion of a marker locus and is capable of using the soybean nucleic acid as a template to initiate DNA polymerization by a DNA polymerase; and b) extending the primer or primer pair in a DNA polymerization reaction comprising a DNA polymerase and a template nucleic acid to produce at least one amplicon; further embodiments include primer pairs used in the DNA polymerization reaction and / or compositions comprising the primer pairs.

[0290] 57. The method as described in Examples 32-56, wherein the nucleic acid is selected from DNA or RNA, and in some examples from isolated genomic DNA and / or RNA.

[0291] 58. The method as described in Examples 55-57, wherein the amplification includes using polymerase chain reaction (PCR) or ligase chain reaction (LCR), using nucleic acid isolated from soybean plants or germplasm as a template in the PCR or LCR, and in another embodiment, wherein the soybean plant is soybean or short-haired wild soybean.

[0292] 59. A primer for diagnosing ASR resistance, wherein the primer can be used in a PCR reaction to indicate the presence of an allele associated with ASR resistance, wherein the allele is any favorable allele as described in Tables 1-5 and / or the primer comprises a nucleotide sequence (the nucleotide sequence includes any one of SEQ ID NO: 1-5), wherein the primer is used in a PCR reaction to produce an amplicon for diagnosing at least one allele associated with ASR resistance; further, a composition comprising the primer.

[0293] 60. The method as described in Examples 27-58, wherein the plants produced or identified by the method are further conferred with increased resistance to any of the following: soybean cyst nematode, bacterial pustule, IDC, root-knot nematode, soybean gray spot, Phytophthora, brown stem rot, nematode, rust, smut, Diplosporium powdery mildew, Asteraceae powdery mildew, wheat powdery mildew, Cucurbita scabra, Cucumber powdery mildew, Pythium cerevisiae, Gracilaria spp., Pea spores, Rice blast fungus, Rice blast fungus, Rhizoctonia solani, Soybean Phytophthora, Wheat aphid, Tobacco whitefly, Corn aphid, Reticulated slug, Sugarcane borer, Wheat aphid and Prunus persica, or combinations thereof.

[0294] 61. A plant, seed, or plant part produced by the method as described in Examples 27-58 or 60.

[0295] 62. The plant as described in Example 61, wherein the plant is a superior soybean plant, and in some embodiments, the plant is a superior soybean plant with commercially significant yields.

[0296] 63. The plant as described in Example 62, wherein the plant is an agronomically superior soybean plant, and in some cases, the agronomically superior soybean plant exhibits commercially significant yields.

[0297] 64. The plants described in Examples 61-63, wherein the plants show no yield burden or negative agronomic effects compared to control and / or comparison plants; or otherwise exhibit susceptible commercial performance compared to commercial comparisons.

[0298] 65. A method for producing at least one hybrid between a first parent plant of a wild perennial *Glycine* species and a second parent plant of a non-wild annual *Glycine* cultivar, wherein the *Glycine* cultivar is capable of backcrossing with a non-wild annual *Glycine* cultivar to produce at least one plant of a first backcross or its fertile offspring, the method comprising the steps of:

[0299] a. Provide at least one wild soybean species (wg) and at least one non-wild annual soybean parent species (dg);

[0300] b. Allow the parent plants to flower;

[0301] c. Cross the wg plant with the dg plant (in most embodiments, this cross is a “non-natural cross” and therefore cannot produce living / fertile offspring);

[0302] d. Pods are produced from the hybridization of c);

[0303] e. Separate the embryo from the pod of d) and place the embryo on an embryo rescue medium in which no callus induction occurs;

[0304] f. Transfer the embryos from e) to a germination medium or elongation medium and collect the cuttings from the embryos;

[0305] g. Collected branches grown on germination or elongation medium; and

[0306] h. The established e) branch is transferred into the soil to produce a hybrid between a first parent plant of a wild perennial soybean species and a second parent plant of a non-wild annual soybean species; in some embodiments, the hybrid is a double diploid hybrid capable of backcrossing with soybean plants for at least one generation.

[0307] 66. The method as described in Example 65, wherein the wild soybean strains are selected from the group consisting of: grey-haired soybean, silver-haired soybean, Penghu soybean, G. latrobeana, white soybean, G. aphyonota, sand soybean, curved soybean, curved-lobed soybean, flat-pod soybean, sickle soybean, G. gracei, densely hairy soybean, milky green soybean, broadleaf soybean, small-leaf soybean, Monty-Douglas soybean, G. peratosa, G. pescadrensis, G. pindanica, G. pullenii, yellow-purple soybean, G. stenophita, G. syndetika, and short-haired wild soybean.

[0308] 67. The method as described in Examples 65-66, wherein the parent plant of the wild soybean genus is short-haired wild soybean.

[0309] 68. The method as described in Examples 65-66, wherein the wild perennial soybean carries the desired agronomic traits, such as disease (e.g., ASR) resistance and / or abiotic stress resistance (e.g., drought resistance) and / or insect resistance (e.g., aphid resistance) and / or higher yield.

[0310] 69. The method as described in Example 68, wherein the desired trait is selected from the group consisting of: disease resistance, including but not limited to resistance to: soybean cyst nematode, bacterial pustule, root-knot nematode, soybean gray spot, Phytophthora, brown stem rot, nematodes, rust, smut, Erythrina variegata, Asteraceae powdery mildew, wheat powdery mildew, Cucurbita scabra, Cucumber powdery mildew, Pythium cerevisiae, Gracilaria spp., Pea spores, Rice blast fungus, Rice blast fungus, Rhizoctonia solani, Soybean Phytophthora, Wheat aphid, Tobacco whitefly, Corn aphid, Reticulated field slug, Sugarcane borer, Wheat aphid, or Prunus persica aphid; increased yield, increased resistance or tolerance to iron deficiency chlorosis, and increased drought or heat tolerance.

[0311] 70. The method as described in Examples 65-69, wherein the non-wild annual soybean genus is soybean.

[0312] 71. The method as described in Examples 65-70, wherein the dg plant is used as a female plant and the wg plant is a pollen donor, or alternatively, the dg plant may be used as a pollen donor (i.e., a male strain) and the wg plant may be used as a female strain.

[0313] 72. The method as described in Examples 65-71, wherein the pods of d) are treated with a hormone mixture comprising GA3, NAA and kinetin (in some examples, 100 mg GA3, 25 mg NAA and 5 mg kinetin / L, and additionally, in some cases, the hormone treatment is sprayed onto the pods daily until harvest).

[0314] 73. The method as described in Examples 65-72, wherein the pods are collected before 19 days after pollination.

[0315] 74. The method as described in Examples 65-73, wherein in any one of steps e)-h) the embryos and / or branches are treated with a chromosome doubling agent to produce a double diploid plant capable of being backcrossed with a non-wild annual soybean cultivar to produce at least one backcross generation.

[0316] 75. The method as described in Example 74, wherein the chromosome doubling agent is colchicine, sulfamethoxazole, or trifluralin.

[0317] 76. The method as described in Examples 65-75, wherein the embryo of e) is retained on embryo rescue medium at about 24°C for at least about 20 days.

[0318] 77. The method as described in Examples 65-76, wherein the embryo of f) is held at about 24°C on a germination or elongation medium for at least about 20 days.

[0319] 78. The method as described in Example 76, wherein the embryo rescue medium is soybean ER1-1.

[0320] 79. The method as described in Examples 65-78, wherein the hybridization in c) does not require emasculation of the flowers.

[0321] 80. The method as described in Examples 65-79, wherein the hybridization in c) is a non-natural hybridization, wherein no living and / or fertile offspring are produced without further intervention, as in, for example, steps d)-h).

[0322] 81. The method as described in Examples 65-80, wherein the method further comprises step i), wherein the hybrid plant of h) is backcrossed with a soybean plant at least once, in most embodiments, the backcross retains the favorable genetic agronomic traits from said wg, for example, the favorable agronomic traits may be disease (e.g., ASR) resistance and / or abiotic stress resistance (e.g., drought resistance) and / or insect resistance (e.g., aphid resistance) and / or higher yield; in another instance, the backcross is carried out for four generations (BC4) to produce fertile dg progeny plants containing said favorable genetic agronomic traits, wherein the backcross progress follows distant hybridization between wg and dg plants, and then embryo rescue is performed as in Example 65, thereby producing backcross plants that repeat continuously for BC2, BC3 and, preferably, BC4, to produce fertile progeny plants containing said favorable genetic agronomic traits.

[0323] 82. The method as described in Examples 65-81, wherein the method further comprises step j), wherein the hybrid plants of h) are backcrossed multiple times, and additionally, wherein each backcross (BC) generation is selected based on favorable agronomic traits derived from the wg line; in some cases, after (ah) each backcross four generations (BC4), thereby producing fertile hybrid offspring / progeny plants.

[0324] 83. The method as described in Examples 65-82, wherein the method further comprises step k) counting the number of chromosomes in at least one BC generation and selecting a line having 40 paired chromosomes, 20 chromosome pairs, or no unpaired chromosomes, or in some cases, the plant having more than 20 chromosome pairs.

[0325] 84. The method as described in Examples 65-82, wherein the method further includes step k) counting the number of chromosomes in at least one BC generation, selecting lines that do not have unpaired chromosomes and further breeding these lines.

[0326] 85. A double diploid plant produced by the method as described in Examples 65-85.

[0327] 86. A non-wild annual soybean plant derived from the plant described in Example 86.

[0328] 87. A non-wild annual soybean plant as described in Example 87, wherein the soybean plant has inherited desired agronomic traits from a perennial wild soybean parent, such as disease (e.g., ASR) resistance and / or abiotic stress resistance (e.g., drought resistance) and / or insect resistance (e.g., aphid resistance) and / or higher yield.

[0329] 88. A non-wild annual soybean plant as described in Example 88, wherein the desired agronomic trait is increased resistance to any of the following diseases: soybean cyst nematode, bacterial pustule, root-knot nematode, soybean gray spot, Phytophthora, brown stem rot, nematode, rust, smut, Erythrophagus dispora, Asteraceae powdery mildew, wheat powdery mildew, Cucurbita scabra, Cucumber powdery mildew, Pythium cerevisiae, Gracilaria spp., Pea spores, Rice blast fungus, Rice blast fungus, Rhizoctonia solani, Soybean Phytophthora, Wheat aphid, Tobacco whitefly, Corn aphid, Reticulated slug, Sugarcane borer, Wheat aphid, or Peach aphid.

[0330] 89. A non-wild annual soybean plant as described in Examples 88-89, wherein the desired agronomic traits have better field or commercial performance relative to any of the following: increased yield, lodging, plant height, field emergence, resistance or tolerance to herbicides, bacteria, fungi, viruses and nematodes, drought tolerance, heat tolerance, cold tolerance or chilling tolerance, excess water, salt stress, oxidative stress, food content and composition, physical appearance, male sterility, lodging, standability, fertility, sugar properties, biomass, oil quality or production, protein quality and overall quality.

[0331] 90. A plant cell or plant part from any plant of Examples 85-90; in some embodiments, the plant cell cannot produce a plant.

[0332] 91. A method for controlling ASR in the field, the method comprising the step of planting seeds from any one of the plants described in Examples 1-21; 24-26; 61-64 or 85-89.

[0333] Disease-resistant soybean plants and germplasm

[0334] This invention provides disease-resistant soybean plants and germplasm. As described above, the methods of this invention can be used to identify, produce, and / or select disease-resistant soybean plants or germplasm (e.g., soybean plants resistant or with increased tolerance to Asian soybean rust). In addition to the methods described above, disease-resistant soybean plants or germplasm can be produced by any method that introduces markers associated with enhanced disease tolerance into the soybean plants or germplasm. These methods include, but are not limited to, transformation, protoplast transformation, or fusion, double haploid technology, embryo rescue, gene editing, and / or production via any other nucleic acid transfer system.

[0335] In some embodiments, the soybean plant or germplasm includes a non-naturally occurring soybean variety. In some embodiments, the genome of 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 the genome of a superior soybean variety.

[0336] Disease-resistant soybean plants or germplasm can be the progeny of a cross between a superior soybean variety and a soybean variety containing an allele associated with enhanced disease (e.g., ASR) tolerance, wherein the allele is located on a chromosome selected from the following group:

[0337] 1) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 1251375 of SEQ ID NO:1; or

[0338] 2) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to nucleotide bases 2515428 of SEQ ID NO:2.

[0339] 3) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 365842 of SEQ ID NO:3; or

[0340] 4) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to 646429 of SEQ ID NO:4.

[0341] 5) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 750080 of SEQ ID NO:5; or

[0342] 6) SNP markers in soybeans that are associated with increased ASR resistance spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM or very close chromosomal intervals, wherein the SNP marker is selected from the following group, which consists of any favorable SNP markers shown in Tables 1-5.

[0343] Disease-resistant soybean plants or germplasm may be offspring of gene introgression, wherein the recurrent parent is a superior soybean variety and the donor contains alleles associated with enhanced disease tolerance and / or resistance, wherein the individual carries a chromosomal region or portion thereof comprising any one of SEQ ID NO: 1-4, and wherein the chromosomal region contains at least one allele selected from Tables 1-5 respectively.

[0344] Disease-resistant soybean plants or germplasm can be the offspring of a cross between a first superior soybean variety (e.g., a test line) or between a second superior soybean variety (e.g., a recurrent parent) and a soybean variety (e.g., a donor) that contains alleles associated with enhanced ASR tolerance.

[0345] Disease-resistant soybean plants or germplasm can be offspring of a first superior soybean variety and a genetically introgressed offspring, wherein the recurrent parent is a second superior soybean variety and the donor contains alleles associated with enhanced ASR tolerance.

[0346] The disease-resistant soybean plants and germplasm of the present invention may contain one or more of the marks of the present invention (e.g., any mark described in Tables 1-5; or any mark closely related to any mark described in Tables 1-5).

[0347] In some embodiments, disease-resistant soybean plants or germplasm may include markers associated with enhanced ASR tolerance within their genome, wherein said markers are located in chromosomal regions selected from the following group:

[0348] 1) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 1251375 of SEQ ID NO:1; or

[0349] 2) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to nucleotide bases 2515428 of SEQ ID NO:2.

[0350] 3) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 365842 of SEQ ID NO:3; or

[0351] 4) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to 646429 of SEQ ID NO:4.

[0352] 5) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 750080 of SEQ ID NO:5; or

[0353] 6) SNP markers in soybeans that are associated with increased ASR resistance spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM or very close chromosomal intervals, wherein the SNP marker is selected from the following group, which consists of any favorable SNP markers shown in Tables 1-5.

[0354] In some embodiments, disease-resistant soybean plants or germplasm may include markers within their genomes that comprise, consist substantially of, or consist of marker alleles located in at least two distinct chromosomal regions. For example, a marker may comprise one or more alleles located in a chromosomal region defined and including any combination of the two markers in Table 1, and one or more alleles located in a chromosomal region defined and including any combination of the two markers in Table 2.

[0355] Disease-resistant soybean seeds

[0356] This invention provides disease-resistant soybean seeds. As described above, the methods of this invention can be used to identify, produce, and / or select disease-resistant soybean seeds. In addition to the methods described above, disease-resistant soybean seeds can be produced by any method that introduces markers associated with enhanced ASR tolerance into the soybean seeds. These methods include, but are not limited to, transformation, protoplast transformation or fusion, double haploid technology, collagen rescue, gene editing (e.g., CRISPR, TALEN, or megnuclease), and / or production via any other nucleic acid transfer system.

[0357] In some embodiments, disease-resistant soybean seeds comprise non-naturally occurring soybean varieties. In some embodiments, the soybean seeds share at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% genomic identity with superior soybean varieties.

[0358] Disease-resistant soybean seeds can be produced from disease-resistant soybean plants identified, generated, or selected by the methods of the present invention. In some embodiments, disease-resistant soybean seeds are produced from disease-resistant soybean or wild soybean plants (e.g., *Glycine max*) containing any of the chromosome intervals corresponding to SEQ ID NO: 1-5.

[0359] The disease-resistant soybean seeds of the present invention may contain one or more markers from Tables 1-5 of the present invention, selected by use or generated by use.

[0360] In some embodiments, disease-resistant soybean seeds may include markers associated with enhanced ASR tolerance within their genome, wherein said markers are located in chromosomal regions selected from the following group:

[0361] 1) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 1251375 of SEQ ID NO:1; or

[0362] 2) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to nucleotide bases 2515428 of SEQ ID NO:2.

[0363] 3) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions correspond to nucleotide bases 1 to 365842 of SEQ ID NO:3; or

[0364] 4) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to 646429 of SEQ ID NO:4.

[0365] 5) Chromosomal regions derived from PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224, wherein the chromosomal regions are consistent with nucleotide bases 1 to nucleotide bases 750080 of SEQ ID NO:5.

[0366] 6) SNP markers in soybeans that are associated with increased ASR resistance spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM or very close chromosomal intervals, wherein the SNP marker is selected from the following group, which consists of any favorable SNP markers shown in Tables 1-5.

[0367] Example

[0368] The following examples are not intended to be a detailed list of all different ways in which this invention may be implemented or all features that may be incorporated into this invention. Those skilled in the art will understand that numerous variations and additions can be made to different embodiments without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention and is not an exhaustive description of all its permutations, combinations, and variations.

[0369] Example 1. Identification of ASR-resistant wild soybean strains

[0370] To assess ASR resistance against sixteen rust strains collected from various environments, multiple wild soybean (Glycine max) lines were evaluated. Rust data were generated using isolates derived from single pustules from USDA-ARS (FL Q09, FL Q12, LABR13, FLQ11) and field populations (FL Q15, FLQ16, RTP1, RTP2, Vero, GA15, UBL, BR south, and BR central) and screened in a closed facility. Glycine max cultivars were screened for ASR resistance; the following Glycine max cultivars showed broad resistance against all tested ASR strains: PI441001, PI441008, PI446958, PI509501, PI583970, and PI483224.

[0371] Each short-haired wild soybean line was evaluated during the multi-day infection process and rated at different time points using a rust rating scale based on the groupings modified by Burdon and Speer, TAG, 1984 (see Figure 6). Each short-haired wild soybean line was registered and screened >2 times, with approximately 4 plants each time, using a large number of different groups of rust isolates in North and South America.

[0372] Example 2. For PI441001, PI441008, PI446958, PI509501, PI583970, or PI483224ASR Allelic mining and association at loci

[0373] Resistant parental lines (i.e., PI441001, PI441008, PI446958, PI509501, PI583970, and PI483224) were crossed with susceptible short-haired wild soybean lines to produce F1 plants (see Table 5). F1 plants were self-fertilized to produce F2 seeds. F2 seeds were harvested from the self-pollinated F1 plants. Approximately 200 F2 seeds were sown, and leaf tissue from each plant was collected for genotyping studies. Each line was incubated with *Gnaphalium affine* to determine the resistance / susceptibility phenotype of each F2 individual. Tissue from 50 resistant F2 and 50 susceptible F2 individuals was combined in separate libraries, and genomic DNA was prepared from each library. For each of these libraries, Illumina sequencing libraries were prepared from the DNA, and each library was sequenced in two Illumina HiSeq2000 2x100bp paired end (PE) lanes. The average yield per sample was 383 million read pairs, which is equivalent to 77 gigabase of sequence per library. Sequencing reads were adjusted to remove bases with a PHRED quality fraction <15.

[0374] Using GSNAP (WU and NACU 2010) as paired end fragments, quality-adjusted reads were aligned with reference genome sequences PI441001, PI441008, PI583970, and PI483224. If a pair of reads could not be aligned together, they were treated as single units for alignment. If a read uniquely located to a reference, it was used for subsequent analysis (≤2 mismatches per 36 bp and less than 5 bases per 75 bp as tails).

[0375] Based on read depth, SNPs were filtered prior to BSA analysis, retaining those with read depths ranging from 40 to 200x. A chi-square test was used to select SNPs with significantly different reads between two alleles in both libraries. An empirical Bayesian method (LIU et al., 2012) was used to estimate the conditional probability of no recombination between each SNP marker and the causal locus in both the resistance and susceptibility libraries. The probability of linkage between the SNP and the causal locus was the geometric mean of these two conditional probabilities. Approximately 1000 SNPs were found to be potentially linked to the target locus. Using phenotypic F2 individuals, a subset of these hypothetically linked SNPs was used for fine-tuning the locus. See references: LIU, S., C.-T. YEH, HMTANG, D. NETTLETON and PSSCHNABLE, 2012 Gene Mapping via Bulked Segregant RNA-Seq (BSR-Seq). PLoS ONE 7:e36406 and Wu, TD and S. Nacu, 2010 Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26:873-881.

[0376] Table 5: Plant Hybridization and Research Types

[0377]

[0378] 1. PI441001 Data2Bio LLC (Ames, Iowa) for tetraploid soybeans Laboratory methods for gBSA-Seq analysis.

[0379] Chromosomal discovery of causal loci in a tetraploid soybean population (PI441001) was performed using Data2Bio's gBSA (genomic population segregation analysis) technology. Theoretically, ASR resistance is controlled by a single dominant allele. Four libraries were generated from DNA samples extracted from two susceptible tissue libraries and two resistant tissue libraries. These libraries were then sequenced in eight (8) Illumina HiSeq2000 2x100bp paired end (PE) lanes. An overview of the reference genome used for subsequent analysis, from raw data to quality-adjusted read processing, alignment, SNP discovery, and SNP effects is shown in Figures 1–5 for population 441001. Following various filtering steps, 110,503 informative SNPs significantly associated with ASR resistance were identified in the PI441001 genome. The trait association probabilities were then calculated using Bayesian methods. Next, a physical map of trait-associated SNPs (probability cutoff of 0.01) in the top contig was created (Figure 1). Two scaffolds, 46840 and 49652 (SEQ ID NO: 1 and 2, respectively), were identified in PI441001. SNPs from these enriched scaffolds were localized to the published soybean genome. In both populations, most SNPs from the top scaffold clustered on small subsets of Chr05 and Chr08 (see Figure 4).

[0380] 2.PI583970 Data2Bio LLC (Ames, Iowa) for tetraploid soybeans Laboratory methods for gBSA-Seq analysis

[0381] Chromosomal loci identification in a tetraploid soybean population (PI583970) was performed using Data2Bio's gBSA (gGenome Population Segregation Analysis) technique. Two libraries were generated from RNA samples extracted from a susceptible tissue library and a resistant tissue RNA library. After various filtering steps, 59,014 informative SNPs significantly associated with ASR resistance were identified in the PI583970 genome. The trait association probabilities were then calculated using Bayesian methods. Next, a physical map of the trait-associated SNPs on contigs was created. Clustering of these SNPs indicated that the resistance locus was located in or near scaffold 000819F (see Figure 7; scaffold 001084F is SEQ ID NO:4). Background sequences associated with these SNPs were also aligned to the published soybean genome to provide an understanding of the chromosomal level of the localization regions. The chromosomal locations of the trait (ASR resistance)-associated SNPs were then graphically displayed. Most SNPs from scaffold 001084F are localized and clustered in a small region of Chr05 (see Figure 8). The data indicate that the loci responsible for ASR resistance are located in or near the 0.11 to 0.30 Mbp region on scaffold 001084F (Figure 9).

[0382] 3.PI483224 Data2Bio LLC (Ames, Iowa) for tetraploid soybeans Laboratory methods for gBSA-Seq analysis

[0383] Chromosomal discovery of causal loci in a tetraploid soybean population (PI483224) was performed using Data2Bio's gBSA (genomic population segregation analysis) technique. Two libraries were generated from DNA samples extracted from a susceptible tissue library and a resistant tissue library (PI483224). After various filtering steps, 428,263 informative SNPs significantly associated with ASR resistance were identified in the PI483224 genome. The probability of trait association was then calculated using Bayesian methods. Next, a physical map of trait-associated SNPs on contigs was created. The clustering of these SNPs indicated that the ASR resistance loci were located on or near scaffold 002687F (see Figure 10). Background sequences associated with these SNPs were also aligned to the published soybean genome to provide an understanding of the chromosomal level of the localization regions. The chromosomal locations of the trait (ASR resistance)-associated SNPs are graphically displayed. Most SNPs from scaffold 002687F are localized to a small region of Chr05 (see Figure 11). Data indicate that the ASR locus can be located on or near the 0.17 to 0.36 MB region on scaffold 002687F (see Figure 12 and SEQ ID NO:3).

[0384] Example 3. Embryo rescue and introgression of R gene regions into soybean lines

[0385] Embryo rescue (as described below) is performed, followed by chemical treatment to induce chromosome doubling and produce double diploid shoots. If these double diploid plants are fertile, they are used for backcrossing with soybean. Backcrossing with soybean followed by embryo rescue is required for several generations to gradually eliminate chromosomes in perennial short-haired wild soybean, ultimately resulting in ASR-resistant soybean plants.

[0386] Outcrossing was performed using superior Elite Syngenta soybean (soybean) lines (RM 3.7 to 4.8). Superior soybean lines were used as females (pollen donors), and multiple registrations of short-haired wild soybean were used as males or pollen donors. It is important to select flowers from short-haired wild soybean plants containing anthers at the appropriate developmental stage. Fresh, fully open, brightly colored flowers should retain anthers with mature pollen. The pollen should appear as loose, yellowish dust. These flowers were removed from the short-haired wild soybean plants and crossed with superior soybean plants for pollination. Pollen from short-haired wild soybean plants should be used within 30 minutes of flower removal. Identifying and selecting high-quality soybean flower buds suitable for pollination is also important. When soybean flower buds are larger in size compared to immature buds, they are generally considered ready for use. The sepals of soybean flowers are lighter in color, and the petals are just beginning to emerge. First, carefully separate the sepals from the flower bud using a pair of fine-tipped tweezers to expose the outer petals. Then, gently grasp and remove the petals (5 in total) from the flower to expose the annulus stamens surrounding the pistil. It is important to recognize the developmental stage where the stigma is pollinated one day before the anthers begin to shed pollen, indicating that the female is ready but the male is not. When pollinating soybean flowers at this developmental stage, it is not necessary to remove the female flowers. Locate the stigma on a good soybean flower. Then, using one male flower, carefully peel off the petals to expose the anthers and gently sprinkle the pollen grains onto the stigma of the soybean flower. During this process, care should be taken not to damage the stigma. Starting the day after pollination, spray the hormone mixture onto the pollinated flower and continue spraying until the F1 pods develop 1X per day until harvest. The pollinated flower or pod is filled with a light mist of the hormone mixture; care should be taken not to allow the flower / pod to fall off the plant prematurely. The mixture contains 100 mg GA3, 25 mg 1-naphthaleneacetic acid (NAA), and 5 mg kinetin / L distilled water. The application of these hormones helps maintain pod development and increase pod growth. The aforementioned distant hybridization method resulted in a significantly higher success rate than reported in the literature. Furthermore, female flowers do not require emasculation, which saves time and reduces the risk of stigma damage.

[0387] Harvesting: Pods from distant hybrids are harvested approximately 14 to 16 days after pollination. (Literature suggests harvest dates of 19 to 21 days; however, the method described above allows for faster harvest times and stronger pods). Pods are collected and counted according to the distant hybrid combinations to determine hybridization success. The average hybridization success rate for multiple female soybeans and five different registrations of short-haired wild soybean is approximately 40%. Distant hybrid pods may contain 1 to 3 seeds, but typically 2 seeds are found in each F1 pod. The method described above allows for pod harvesting 14 to 16 days after pollination, approximately 5 days earlier than described in the literature.

[0388] Embryo rescue: Harvested pods are collected and brought back to the laboratory for sterilization. First, the pods are rinsed with 70% EtOH for 2 to 3 minutes, then placed in 10% Clorox bleach at approximately 130 RPM on a platform shaker for another 30 minutes. Finally, the pods are rinsed several times with sterile water to remove any residual bleach. Embryo separation can begin immediately after pod sterilization, or the pods can be stored at 4°C for up to 24 hours before embryo separation. Next, the sterilized pods are placed in a laminar flow hood where the embryo can be rescued. Individual pods are placed in sterile petri dishes and opened using a scalpel and forceps. A cut is made along the length of the distantly hybridized pod, away from the seed. The pod can then be easily opened to expose the seed. Alternatively, two pairs of forceps can be used to separate the pod shell. The seed is carefully removed from the pod and placed in a sterile petri dish under a dissecting microscope. Very fine forceps are required to separate the embryo from the seed. Holding the tweezers in one hand, gently move one side of the seed away from the embryo, so that the umbilicus is facing upwards. Using another pair of tweezers in the other hand, remove the seed coat from the side of the seed containing the embryo. Peel off the membrane around the embryo and push the embryo upwards from the bottom. The embryo should have gone through the globular development stage and preferably through the early heart development stage (mid to late heart stage, cotyledon stage, and early maturity stage embryos are desired). Transfer the isolated embryo to embryo rescue medium, such as soybean ER1-1 (i.e., 3.1g B5 basal salt, 1ml Gamborg B5 vitamin 1000X, 40g sucrose [C12H22O11], 0.25g casein hydrolysate, 0.25ml BAP, 0.75g MgCl2*6H2O, 20ml glutamine 25mg / ml, 0.1g serine [C3H7NO3], 4ml asparagine 25mg / ml, and 0.05ml IBA 1mg / ml). Murashige and Skog medium (MS) and Gamborg's B-5 medium (Bridgen, 1994) can also be used as embryo rescue media. Embryos can then be treated to induce chromosome doubling. (See below for details on chromosome doubling.) Isolated embryos are held at 24°C on embryo rescue medium for 21 to 30 days. Embryos can be kept in darkness for the entire incubation of ER1-1, or they can be incubated in darkness and then completed under light, or they can be incubated entirely under light. There is no callus induction phase in this protocol; shoots develop directly from the embryo, allowing for faster turnaround times, smaller plantlet survival, and better quality results. The above embryo rescue method involves direct shoot regeneration from the embryo, rather than regeneration through embryogenesis, resulting in faster plant recovery (shoots recover in approximately 2–3 months compared to the up to 1 year timeline reported in the literature). Additionally, the following protocols do not require incubation in darkness after transfer to germination medium, and the above protocols do not require transfer to rooting medium.

[0389] Chromosome doubling treatment: Colchicine or trifluralin can be used to induce chromosome doubling. Ideally, late-heart stage distant hybrid embryos (or larger) are chemically treated to induce chromosome doubling at any time immediately following separation, up to one week later. The doubling agent can be mixed in solid or liquid culture medium and applied for several hours or up to several days. Trifluralin is used at a concentration of 10–40 μM in solid or liquid culture medium. Alternatively, colchicine is used at a concentration of 0.4–1 mg / ml in solid or liquid culture medium. After chemical treatment, the embryos are transferred to fresh embryo rescue medium.

[0390] Branch regeneration: The developing embryos were transferred from the rescue medium to the germination medium, such as SoyER GSMv2 (i.e., 3.2g Schenk and Hilderbrandt basic salt mixture, 1g inositol [C6H12O6], 5ml thiamine 1mg / ml, 0.5ml pyridoxine 1mg / ml, 10g sucrose [C12H22O11] and 7.5g purified agar), and kept under 24°C light for about 3 to 5 weeks. Alternatively, developing embryos can be transferred from rescue medium to elongation medium, such as soybean E1 0No TCV (i.e., 4.3g MS basal salt mixture [MSPO1], 5ml MS iron 200X, 30g sucrose [C12H22O11], 1g MES [C6H13NO4S], 8g purified agar, 1ml vitamin B5 100X, 2ml glutamine 25mg / ml, 0.50ml zeatin nucleoside, trans isomer 1mg / ml, 0.1ml IAA 1mg / ml, 0.2ml GA3 5mg / ml, 1.5ml termethin 100mg / ml, 0.3ml cefotaxime 250mg / ml, 0.5ml vancomycin 100mg / ml). Under 24°C light, the cuttings can be maintained on the medium for approximately 3 to 5 weeks. Developing shoots can be transferred from culture plates to Phytocons containing germination or elongation media for further shoot development. Established shoots with suitable roots are then transplanted into the soil.

[0391] ploidy analysis Ploidy analysis was performed using flow cytometry. Leaf tissues for ploidy analysis were collected from cultures or from shoots established in soil. Tissues were collected on dry ice and stored at -80°C until analysis, or collected on wet ice and analyzed on the same day. 0.5 cm 2 The sample size was sufficient. Samples were prepared according to the instructions in the Sysmex kit (Sysmex Inc., Kobe, Japan). Each sample group contained untreated F1 plants (untreated to induce chromosome doubling) as a control.

[0392] Example 4. Introgression of ASR resistance trait genes

[0393] The double diploid lines produced by distant hybridization (i.e., cross between short-haired wild soybean and soybean) and subsequent embryo rescue as described in Example 3 are backcrossed multiple times with recurrent superior soybean lines. It is known in the art that multiple backcrosses are necessary to produce fertile hybrid lines, and in particular, the literature indicates that generation BC3 is essential. In this case, it was determined that additional backcrosses were needed, specifically BC4, in the case of short-haired wild soybean x soybean to produce fertile hybrids. The F1 hybrids produced by the method described above are generated from distant hybridizations comprising PI441001, PI441008, PI446958, PI509501, PI583970, and PI483224. The F1 plants are then crossed as female to male recurrent soybean plants for the first backcross (BC1 progeny). The BC1 progeny are further backcrossed multiple times (e.g., BC2). Assess ASR resistance and chromosome number in BC plants, and in some cases, genotype the lines using molecular markers as described herein to detect the presence of chromosomal regions corresponding to SEQ ID NO 1-5 or any markers identified in Tables 1-5.

[0394] The above examples clearly illustrate the advantages of the present invention. Although the invention has been described in detail with reference to certain embodiments, such details are not intended to be construed as limiting the scope of the invention unless they are included in or to the extent that they are included in the appended claims.

[0395] Throughout this application, various patents, patent publications, and non-patent publications are cited. The disclosures of these patents, patent publications, and non-patent publications are incorporated herein by reference in their entirety in order to provide a more comprehensive description of the current state of the art to which this invention pertains.

Claims

1. A method for producing soybean plants with increased resistance to Asian soybean rust (ASR), the method comprising the following steps: a. Widely cross soybean plants with ASR-resistant short-fibered wild soybean plants of the registered lines PI446958, PI509501 or PI483224; b. Soybean pods are produced from the hybridization of a); c. Separate the embryo from the pod of b) and place the embryo on an embryo rescue medium in which no callus induction is performed; d. Transfer the embryos from c) to a germination medium or elongation medium and collect the cuttings from the embryos; e. Collected branches from d) grown on germination or elongation medium; as well as f. Transferring established e) branches into soil to produce soybean plants with increased resistance to ASR. The short-haired wild soybean plant of step a) contains a chromosomal region corresponding to SEQ ID NO: 3 in its genome, and the embryo of c) or the branch of f) or the resulting plant contains an allele in its genome that is associated with increased ASR resistance and is closely linked to the chromosomal region.

2. The method of claim 1, wherein the short-haired wild soybean plant containing the chromosome region corresponding to SEQ ID NO: 3 is derived from the registered strain PI483224 or its progeny or double diploid hybrid.

3. The method of claim 1, further comprising a molecular marker selection step, wherein isolated genomic nucleic acids from the parental plant of a), the embryo of c), or the branch of f), or the resulting plant are analyzed for the presence of an allele associated with increased ASR resistance and tightly linked to a chromosomal region corresponding to SEQ ID NO: 3, wherein the molecular marker is the marker described in Table 3.

4. The method of claim 1, wherein the pods of b) are treated with a hormone mixture containing GA3, NAA and kinetin.

5. The method of claim 1, wherein the pods are collected no later than 19 days after pollination.

6. The method of claim 1, wherein in any one of steps c)-f) the embryos and / or branches are treated with a chromosome doubling agent to produce a double diploid plant capable of being backcrossed with a non-wild annual soybean cultivar to produce at least one backcross (BC1).

7. The method of claim 6, wherein the chromosome doubling agent is colchicine or trifluralin.

8. A method for producing soybean plants with increased resistance to Asian soybean rust (ASR), the method comprising the following steps: a. Providing a first soybean plant having a chromosomal region corresponding to SEQ ID NO: 3 in its genome, wherein the first soybean plant has increased resistance to ASR; b. Cross the soybean plant of a) with a second soybean plant that does not contain the said chromosomal region; and c. Selecting progeny plants from the hybridization of b) by isolating nucleic acids from the progeny plants and detecting alleles associated with increased ASR resistance within the nucleic acids, wherein the alleles are tightly linked to the chromosomal region corresponding to SEQ ID NO: 3 and are favorable alleles in Table 3, thereby producing soybean plants with increased resistance to ASR.

9. The method of claim 8, wherein the short-haired wild soybean chromosome region of SEQ ID NO: 3 is derived from any one of plant registrations PI446958, PI509501, PI483224 or their progeny.

10. The method of claim 8, wherein the first or second soybean plant is a superior soybean plant.

11. A method for producing soybean plants with increased ASR resistance, the method comprising the steps of: a) Isolating nucleic acids from soybean plants; b) Detect molecular markers associated with increased ASR resistance in the nucleic acids of a), wherein the molecular markers are the favorable alleles in Table 3, which are closely linked to the chromosomal region containing SEQ ID NO: 3, wherein the chromosomal region confers increased ASR resistance to the plant, and wherein the chromosomal region is derived from chromosome 5 of plant registration PI446958, PI509501, PI483224 or any of their progeny; c) Selecting plants based on the presence of the molecular markers detected in b); and d) Produce soybean progeny plants from plants identified as having the alleles of c) that are associated with increased ASR resistance.

12. A primer for diagnosing ASR resistance in soybean plants, wherein the primer can be used in a PCR reaction to indicate the presence of an allele in the genome of the plant that is associated with ASR resistance and is tightly linked to a chromosomal region corresponding to SEQ ID NO: 3, wherein the allele is a favorable allele described in Table 3.

13. A method for identifying or selecting soybean plants resistant to ASR, the method comprising the following steps: a. Isolating nucleic acids from soybean plants; b. Detect the presence of a molecular marker associated with increased ASR resistance in the nucleic acid, wherein the molecular marker is located within the chromosomal region corresponding to SEQ ID NO: 3, and the molecular marker is a favorable marker described in Table 3; and c. Identify or select soybean plants with increased ASR resistance based on the molecular markers detected in b).

14. The method of claim 13, wherein the molecular marker is a single nucleotide polymorphism (SNP), a quantitative trait locus (QTL), an amplified fragment length polymorphism (AFLP), a random amplified polymorphic DNA (RAPD), a restriction fragment length polymorphism (RFLP), or a microsatellite.

15. The method of claim 13, wherein the detection comprises amplifying a marker locus or a portion thereof and detecting the resulting amplified marker amplicon.

16. The method of claim 15, wherein the amplification comprises: a) mixing an amplification primer or primer pair with nucleic acid isolated from a first soybean plant or germplasm, wherein the primer or primer pair is complementary or partially complementary to at least a portion of a marker locus and is capable of using the soybean nucleic acid as a template to initiate DNA polymerization by a DNA polymerase; and b) extending the primer or primer pair in a DNA polymerization reaction comprising a DNA polymerase and a template nucleic acid to produce at least one amplicon.

17. The method of claim 15, wherein the amplification comprises employing polymerase chain reaction (PCR) or ligase chain reaction (LCR), using nucleic acids isolated from soybean plants or germplasm as templates in the PCR or LCR.

18. The method of claim 13, wherein the nucleic acid is selected from DNA or RNA.

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