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 germplasms using markers, genes and chromosome intervals derived from Penghu soybeans, such as PI339656 or its progeny. Also provided are soybean plants or germplasms identified, selected and / or produced by any method of the present invention. Also provided are disease-resistant soybean seeds, plants and germplasms.

Description

Technical Field

[0001] The present invention relates to compositions and methods for identifying, selecting and producing soybean plants with enhanced disease and / or pathogen resistance.

[0002] Statement Concerning Electronic Submission of Sequence Listings

[0003] A sequence listing in ASCII text format is provided as an alternative to a paper copy, which is submitted pursuant to 37 C.FR §1.821, entitled "81923-WO-REG-ORG-NAT-1_SeqList_ST25", generated on July 31, 2019 and submitted via EFS-Web. This sequence listing is hereby incorporated by reference into this specification for its disclosure. Background Art

[0004] Plant pathogens are known to cause considerable damage to important crops, which results in significant agricultural losses with wide-ranging consequences for the food supply and other industries that rely on plant materials. Likewise, there has long been a need to reduce the incidence and / or impact of agricultural pathogens on crops.

[0005] Several pathogens are associated with damage to soybean, which may cause significant yield losses individually and collectively in the U.S. and worldwide. Exemplary pathogens include, but are not limited to, fungi (e.g., Phytophthora and Asian soybean rust Phakopsora pachyrhizi), nematodes (e.g., Root-knot nematodes, particularly javanic root-knot nematodes), and soybean stem canker. In view of the significant threat these pathogens present to global food supply and the time and expense associated with processing soybean crops to prevent losses, new methods for producing pathogen-resistant soybean cultivars are needed. What is needed is a novel resistance gene (herein, "R gene") that can be introduced into commercial soybean plants to control soybean pathogens. Summary of the invention

[0006] This summary lists several embodiments of the presently disclosed subject matter and, in many cases, lists variations of those embodiments.

[0007]

[0013] Accordingly, one object of the presently disclosed subject matter is to provide methods for transferring pathogen resistance to non-resistant soybean germplasm or plant lines, which object is achieved in whole or in part by the presently disclosed subject matter.

[0008] Brief Description of Sequence Listing

[0009] SEQ ID NO: 1 is a chromosomal interval derived from the Penghu soybean (Glycine clandestina) line accession PI339656, which is referred to herein as "scaffold 14851". Scaffold 14851 has been mapped to an interval of approximately 3.18 to 3.50 MB on chromosome 14 of Penghu soybean with scaffold 14851. This chromosomal interval or part thereof can be introduced (i.e., by using embryo rescue and / or marker assisted breeding (MAB) or transgenic introgression) into soybean lines to produce soybean lines resistant to various diseases (e.g., Asian soybean rust (ASR)). In some embodiments, it is expected that the genes from this interval encode one or more polypeptides that can be transgenically expressed in plants or genetically modified (i.e., genes edited by TALEN or CRISPR) to confer disease resistance (e.g., Asian soybean rust (ASR) resistance). Table 1 represents single nucleotide polymorphisms (SNPs) associated with ASR resistance in SEQ ID NO: 1. All alleles of the SNPs identified in Table 1 were determined to be significantly associated with resistance or susceptibility (p<0.05).

[0010] Table 1: SNP positions associated with increased resistance to ASR in SEQ ID NO: 1

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[0275] It is well known in the art that given the sequences and SNP alleles associated with a given trait (eg, ASR resistance), one of ordinary skill in the art can develop oligonucleotide primers and use the primers to identify plants carrying any of the chromosomal intervals described in SEQ ID NO:1. Assay (e.g., typically a two-step allele discrimination assay or similar assay), KASP TMDetermination (usually a one-step allele discrimination assay or similar assay defined below), or both can be used to determine SNPs as disclosed herein. In an exemplary two-step assay, a forward primer, a reverse primer and two assay probes (or hybridization oligonucleotides) are used. Forward and reverse primers are used to amplify the genetic locus containing the SNP associated with the ASR resistance locus. Then assay primers (in some embodiments, these assay primers are differentially labeled with, for example, fluorophores to allow two assay probes to be distinguished in a single reaction) are used to determine the specific nucleotides present at the SNP position, and these assay primers are different from each other in each pair for the nucleotides present at the SNP position (although it is noted that in any given pair, the probes can be different at their 5' or 3' ends without affecting their ability to distinguish the nucleotides present at the corresponding SNP position). In some embodiments, the assay primers and reaction conditions are designed so that the assay primers will hybridize only with the reverse complement of the sequence of 100% complete matching, thereby allowing hybridization-based detection to identify which allele or alleles are present.

[0276] Compositions and methods for identifying, selecting and producing Glycine max plants (including wild Glycine max and soybean lines) with enhanced disease resistance are provided. Disease resistant soybean plants and germplasm are also provided.

[0277] In some embodiments, methods for identifying disease-resistant soybean plants or germplasm are provided. Such methods may include detecting genetic loci or molecular markers (e.g., SNPs or quantitative trait loci (QTLs)) associated with enhanced disease resistance (particularly ASR resistance) in soybean plants or germplasm. In some embodiments, the genetic loci or molecular markers are associated with the presence of a chromosomal interval comprising the nucleotide sequence of SEQ ID NO: 1, or a portion thereof.

[0278] In some embodiments, methods of producing 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 germplasm, and producing progeny plants from the soybean germplasm.

[0279] In some embodiments, a method for selecting disease-resistant soybean plants or germplasm is provided. Such methods 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 soybean Penghu soybean accession PI339656 or its progeny comprising any one or part of SEQ ID NO:1 associated with enhanced disease resistance and / or tolerance, and selecting a progeny plant or germplasm with the genetic locus. In a further embodiment, the first soybean plant or germplasm comprises a genetic locus derived from soybean Penghu soybean accession PI591576.

[0280] In some embodiments, methods of introgressing a genetic locus associated with enhanced pathogen resistance, derived from soybean accession number PI339656, PI591576, or a progeny thereof, into a soybean plant or germplasm are provided. Such methods may include crossing a first soybean plant or germplasm (the soybean plant or germplasm comprising a chromosome interval associated with enhanced pathogen resistance, derived from soybean accession number PI339656, PI591576, or a progeny thereof) with a second soybean plant or germplasm (the soybean plant or germplasm lacks the locus), and optionally repeatedly backcrossing a progeny plant comprising the genetic allele with a second soybean plant or germplasm to produce a soybean plant (e.g., soybean) or germplasm with enhanced pathogen resistance, the soybean plant or germplasm comprising a chromosome interval associated with enhanced pathogen resistance, derived from soybean accession number PI339656, PI591576, or a progeny thereof. Progeny comprising a chromosomal interval associated with enhanced pathogen resistance can be identified by detecting in their genome the presence of a marker associated with the chromosomal interval derived from soybean accession number PI339656, PI591576, or a progeny thereof, wherein the chromosomal interval comprises SEQ ID NO: 1 or a portion thereof. The R-gene from PI339656, PI591576, or a progeny thereof can be introgressed into soybean lines using embryo rescue methods known to those skilled in the art (e.g., as disclosed in U.S. Pat. No. 7,842,850, which is incorporated herein by reference) and by methods as described in the working examples herein.

[0281] In some embodiments, a plant, progeny plant, or plant part of a soybean species is provided, wherein a portion of the plant's genome is obtained from a wild Glycine max species by using chemically induced chromosome doubling, and wherein the portion confers increased ASR resistance to the plant compared to a control plant not comprising the portion.

[0282] In some embodiments, a plant, progeny plant, or plant part of a soybean species is provided, wherein the genome of the plant comprises a chromosomal interval that confers increased resistance to ASR compared to a control plant not comprising the interval, wherein the chromosomal interval is introduced into the plant from a wild Glycine max species, and wherein the plant having the chromosomal interval is obtained by chemically induced chromosome doubling.

[0283] Also provided are soybean plants and / or germplasms identified, produced or selected by the methods of the present invention, as well as any progeny or seeds derived from soybean plants or germplasms identified, produced or selected by these methods. In one embodiment, the molecular marker associated with the presence of the chromosome interval described in SEQ ID NO: 1 can be used to identify or select plant lines resistant to ASR. In addition, the molecular marker may be located within 20cM, 10cM, 5cM, 4cM, 3cM, 2cM, 1cM of the chromosome interval. In another embodiment, the molecular marker may be located within 20cM, 10cM, 5cM, 4cM, 3cM, 2cM, 1cM of any SNP marker associated with ASR as described in Table 1.

[0284] Also provided are non-naturally occurring soybean seeds, plants and / or germplasm comprising one or more genetic loci associated with enhanced resistance to pathogens (e.g., ASR, cyst nematodes, Phytophthora, brown stem rot, etc.) derived from soybean plant accession number PI339656, PI591576, or progeny thereof.

[0285] The marker associated with enhanced pathogen resistance can comprise, consist essentially of, or consist of a single allele associated with enhanced pathogen resistance or a combination of alleles at one or more genetic loci derived from PI339656, PI591576, or a progeny thereof. In one embodiment, the marker is within a chromosomal interval as described in SEQ ID NO: 1.

[0286] The above and other objects and aspects of the present invention will be explained in detail in the drawings and description set forth below. BRIEF DESCRIPTION OF THE DRAWINGS

[0287] Figure 1 Shown is the screening of wild Glycine max lines for rust resistance against a panel of 16 rust isolates.

[0288] Figure 2 The rust rating scale used to measure plant phenotypes is illustrated.

[0289] Figure 3 Delineation of the Penghu soybean (PI339656) SNPs associated with ASR resistance on scaffolds 14851 and scaffold 9588 is shown.

[0290] Figure 4 It is the mapping interval of the flags associated with ASR in bracket 14851.

[0291] Figure 5It is a marker association map of Penghu soybean (PI339656), in which the bands indicate the corresponding chromosomal regions / intervals associated with ASR resistance. DETAILED DESCRIPTION

[0292] The presently disclosed subject matter relates, at least in part, to the identification of genomic regions (i.e., chromosomal intervals) associated with enhanced pathogen resistance (e.g., Asian soybean rust or soybean cyst nematode) derived from the Penghu soybean accession lines PI339656, PI591576, or progeny thereof. Likewise, the chromosomal intervals from PI339656, PI591576, or progeny thereof can be introgressed into soybean lines via somatic embryo rescue (see, e.g., U.S. Patent Publication 2007 / 0261139 and Examples herein) or by using soybean donor lines that have been genetically introgressed into their genomes a genetic region from PI339656, PI591576, or progeny thereof, wherein the region comprises SEQ ID NO: 1 or a portion thereof, wherein the presence of the genetic region is associated with increased soybean disease resistance to, e.g., ASR, SCN, stem termination, stem canker, bacterial pustule, root-knot nematode, brown stem rot, frogeye leaf spot, or phytophthora. In another embodiment, a chromosomal interval derived from PI339656, PI591576 or their progeny is introduced into a soybean line that does not contain the chromosomal interval, wherein the introduction confers increased resistance to disease (e.g., ASR) in the soybean line or its progeny, wherein the chromosomal interval is derived from Chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or Chromosome 20 of Penghu soybean, and in addition, wherein the chromosomal interval contains at least one allele associated with the trait of increased disease resistance, wherein the allele is any one of the following alleles, respectively, which are respectively selected from any one described in Table 1.

[0293] All references listed below, as well as all references cited in the instant disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (e.g., database entries and all annotations available therein), the entire contents of which are incorporated herein by reference to the extent they supplement, explain, provide a context for (or impart) the methods, techniques and / or compositions employed herein.

[0294] definition

[0295] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs.

[0296] Although it is believed that the following terms are well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate understanding of the disclosed subject matter.

[0297] As used herein, the term "a / an" or "the" may refer to one or more than one. For example, "a / an" sign may refer to one sign or multiple signs.

[0298] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0299] As used herein, the term "about" when used with respect to a measurable value (such as an amount of mass, dose, time, temperature, etc.) is meant to encompass variations of up to 20%, 10%, 5%, 1%, 0.5% or even 0.1% of the specified amount.

[0300] The term "consisting essentially of..." (and grammatical variations thereof), when applied to the polynucleotide sequences of the present invention, means a polynucleotide sequence consisting of both the recited sequence (e.g., SEQ ID NO: 1) and all ten or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides at the 5' and / or 3' ends of the recited sequence, such that the function of the polynucleotide sequence is not substantially altered. The total of ten or fewer additional nucleotides includes the total number of additional nucleotides at both ends added together. 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 recited sequence.

[0301] As used herein, the term "Glycine max" refers to the Pescadores soybean plant.

[0302] As used herein, the term "allele" refers to one of two or more different nucleotides or nucleotide sequences occurring at a particular locus.

[0303] A marker is "associated" with a trait when it is linked to the trait and when the presence of the marker indicates whether and / or to what extent a desired trait or trait form will occur in a plant / germplasm containing the marker. Similarly, a marker is "associated" with an allele when it is linked to the allele and when the presence of the marker indicates whether the allele is present 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 display a pathogen resistance phenotype.

[0304] As used herein, the terms "backcross" and "backcrossed" refer to a process whereby a progeny plant is repeatedly crossed back to one of its parents. In a backcrossing protocol, the "donor" parent refers to the parent plant that has the desired allele or locus to be introgressed. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is introgressed. See, for example, Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques [Marker-assisted backcrossing: A practical example, a symposium on molecular marker technology and applications] Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, in Proceedings of the Symposium "Analysis of Molecular Marker Data," pp. 41-43 (1994). The initial cross 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.

[0305] Centimorgan ("cM") is a unit of measure of recombination frequency. One cM equals a 1% chance that a marker at one genetic locus will segregate from a marker at a second locus due to crossover in a single generation.

[0306] As used herein, the term "chromosome interval defined by and including" used with respect to a particular locus and / or allele refers to the chromosomal interval defined by and including said locus / allele.

[0307] As used herein, the term "cross" or "crossed" refers to the fusion of gametes through pollination to produce progeny (e.g., cells, seeds, or plants). The term includes both sexual crossing (one plant being pollinated by another) and selfing (self-pollination, e.g., when the pollen and ovules are from the same plant). The term "crossing" refers to the act of fusing gametes through pollination to produce progeny.

[0308] 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 performance.

[0309] As used herein, the terms "desired allele" and "target allele" are used interchangeably to refer to an allele associated with a desired trait. In some embodiments, "desired allele" and / or "target allele" can be associated with an increase or decrease in a given trait, depending on the nature of the desired phenotype. In some embodiments, "desired allele" and / or "target allele" can be associated with changes in morphology, color, etc.

[0310] As used herein, the term "enhanced pathogen resistance" or "enhanced disease resistance" refers to an improvement, enhancement or increase in a plant's tolerance and / or reproductive ability despite infection with a disease (e.g., Asian soybean rust) compared to one or more control plants (e.g., one or both of the parents, or a plant lacking a marker 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.).

[0311] As used herein, the terms "excellent" and "elite lines" refer to any lines resulting from breeding and selection for desired agronomic performance. Elite lines can be substantially homozygous. Numerous superior lines are available and known to those skilled in the art.

[0312] As used herein, the term "elite germplasm" refers to any germplasm derived from or capable of producing superior plants.

[0313] As used herein, the terms "exotic," "exotic lines," and "exotic germplasm" refer to any plant, line, or germplasm that is not superior. Typically, the 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., to introduce novel alleles into a breeding program).

[0314] A "genetic map" is a description of the genetic linkage relationships between loci on one or more chromosomes within a given species, usually depicted in a graphical or tabular form. For each genetic map, the distances between loci are measured by the frequencies of recombination between them. Recombination between loci can be detected using various markers. A genetic map is the product of the mapping population, the types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distances between loci can differ from one genetic map to another.

[0315] As used herein, the term " genotype " refers to the genetic composition of the individual (or individual group) at one or more genetic loci in contrast to observable and / or detectable and / or demonstrated proterties (phenotype). Genotype is defined by one or more alleles of one or more known loci of its parent by individual inheritance. The term genotype can be used to refer to the genetic composition of the individual at a single locus, a plurality of loci, or more generally, the term genotype can be used to refer to the individual inheritance of all genes in its genome. Genotype can be, for example, indirectly characterized using a mark and / or directly characterized by nucleic acid sequencing.

[0316] As used herein, the term "germplasm" refers to the genetic material belonging to or coming from an individual (e.g., plant), an individual population (e.g., plant strain, variety or family) or a clone derived from a strain, variety, species or culture. Germplasm can be the part of an organism or cell, or can be separated from the organism or cell. Typically, germplasm provides the genetic material with a specific molecular makeup 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 the seed, cell (including protoplasts and callus) or tissue from which new plants can be grown, and plant parts (e.g., stems, buds, roots, leaves, etc.) that can be cultured into whole-strain plants.

[0317] A "haplotype" is the genotype of an individual at multiple genetic loci, i.e., a combination of alleles. Typically, the genetic loci defining a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term "haplotype" can refer to a polymorphism at a specific locus (e.g., a single marker locus), or a polymorphism at multiple loci along a chromosome segment.

[0318] As used herein, the term "heterozygous" refers to a genetic state in which different alleles are located at corresponding loci on homologous chromosomes.

[0319] As used herein, the term "homozygous" refers to a genetic state in which identical alleles are located at corresponding loci on homologous chromosomes.

[0320] As used herein, the term "hybrid" refers to a seed and / or plant produced when at least two genetically non-identical parents are crossed.

[0321] As used herein, the term "inbred" refers to a substantially homozygous plant or variety. The term can refer to a plant or variety that is substantially homozygous throughout the genome, or a plant or plant variety that is substantially homozygous for a particular portion of the genome of interest.

[0322] As used herein, the term "indel" refers to an insertion or deletion in a pair of nucleotide sequences, wherein a first sequence may be referred to as having an insertion relative to a second sequence, or the second sequence may be referred to as having a deletion relative to the first sequence.

[0323] As used herein, the terms "introgression", "introgressing" and "introgressed" refer to the natural and artificial transmission of a desired allele or combination of desired alleles of one or more genetic loci from one genetic background to another. For example, a desired allele at a given locus can be transmitted to at least one progeny by sexual hybridization between two parents of the same species, wherein at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of the allele can occur by recombination between two donor genomes, such as in fused protoplasts, wherein at least one donor protoplast has the desired allele in its genome. The desired allele can be a selected allele of a marker, a QTL, a transgenic, etc. The offspring comprising the desired allele can be repeatedly backcrossed with a strain having a desired genetic background, and selection is performed for the desired allele, wherein the result is that 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 progeny can then be repeatedly backcrossed and selected until the progeny has one or more ASR tolerance alleles in the recurrent parent background.

[0324] As used herein, the term "linkage" refers to the degree of association of a marker locus with another marker locus or some other locus (e.g., ASR tolerance locus). The linkage relationship between a molecular marker and a phenotype can be given as a "probability" or an "adjusted probability". Linkage can be expressed as a desired limit or range. For example, in some embodiments, when the markers are separated by less than about 50, 40, 30, 25, 20, or 15 map distance units (or cM), any marker is linked to any other marker (genetically and physically).

[0325] In some aspects of the invention, it is advantageous to define a range of linkage (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). The tighter the linkage of the marker to the second locus, the better the indication effect of the marker on the second locus. Therefore, a "tightly linked locus" (e.g., a marker locus and a second locus) exhibits a recombination frequency between loci of about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% or less. In some embodiments, the related loci exhibit a recombination frequency of about 1% or less (e.g., about 0.75%, 0.5%, 0.25% or less). Two loci that are located on the same chromosome and have a distance 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 to be "adjacent" to each other. Because one cM is the distance between two markers that show a recombination frequency of 1%, any marker is closely linked (genetically and physically) to any other marker that is in close proximity (e.g., at a distance equal to or less than about 10 cM). Two closely linked markers on the same chromosome can be located 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 from each other.

[0326] As used herein, the term "linkage disequilibrium" refers to the non-random separation of genetic loci or proterties (or both). In either case, linkage disequilibrium means that the associated loci are physically close enough along a section of chromosome so that they are separated together with a frequency higher than random (i.e. non-random) (in the case of co-segregated proterties, the loci controlling these proterties are close enough to each other). Marks showing linkage disequilibrium are considered to be linked. The linked loci are co-segregated more than 50% of the time (e.g., from about 51% to about 100% of the time). In other words, the two co-segregated marks have a recombination frequency less than 50% (and, by definition, separated less than 50cM on the same chromosome). As used herein, linkage can be present between two marks, or alternatively, between a mark and a phenotype. The marker locus can be "associated" (linkage) with a proterties (e.g., Asian soybean rust (here "ASR")). For example, the degree of linkage of a molecular marker with a target proterties is measured as the statistical probability of the co-segregation of the molecular marker with the phenotype.

[0327] Linkage disequilibrium is most commonly measured using the measure r 2 Evaluation, the measure r 2The calculation was performed using the formula described in Hill and Robertson, Theor. Appl. Genet. 38:226 (1968). 2 =1, there is complete linkage disequilibrium between the two marker loci, meaning that the markers have not yet undergone recombination segregation and have the same allele frequency. 2 Values ​​greater than 1 / 3 indicate sufficiently strong linkage disequilibrium to be useful for mapping. Ardlie et al., Nature Reviews Genetics 3:299 (2002). Thus, when r between paired marker loci is 2 When the value is greater than or equal to about 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, the alleles are in linkage disequilibrium.

[0328] As used herein, the term "linkage equilibrium" describes a situation in which two markers segregate independently, ie, randomly distribute among progeny. Markers that exhibit linkage equilibrium are considered unlinked (regardless of whether they are located on the same chromosome or not).

[0329] A "locus" is a position on a chromosome where a gene or marker or allele is located. In some embodiments, a locus may encompass one or more nucleotides.

[0330] 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, a trait, or a trait form. In some embodiments, a marker can be associated with an allele or a target allele and can indicate the presence or absence of an allele or a target allele in a cell or organism. The marker can be, but is not limited to, an allele, a gene, a haplotype, a restriction fragment length polymorphism (RFLP), a simple sequence repeat (SSR), a randomly amplified polymorphic DNA (RAPD), an enzyme-cut amplified polymorphic sequence (CAPS) (Rafalski and Tingey, Trends in Genetics [Genetics Trends] 9:275 (1993)), an amplified fragment length polymorphism (AFLP) (Vos et al., Nucleic Acids Res. [Nucleic Acids Research] 23:4407 (1995)), single nucleotide polymorphism (SNP) (Brookes, Gene [Gene] 234:177 (1993)), sequence-characterized amplified region (SCAR) (Paran and Michelmore, Theor. Appl. Genet. [Theoretical and Applied Genetics] 85:985 (1993)), sequence tagged site (STS) (Onozaki et al., Euphytica [Netherlands Plant Breeding Journal] 138:255 (2004)), single strand conformation polymorphism (SSCP) (Orita et al., Pr USA [Proceedings of the National Academy of Sciences of the United States of America] 86:2766 (1989)), simple sequence repeat interval (ISSR) (Blair et al., Theor. Appl. Genet. [Theoretical and Applied Genetics] 98:780 (1999)), retrotransposon inter-amplified polymorphism (IRAP), retrotransposon microsatellite amplified polymorphism (REMAP) (Kalendar et al., Theor. Appl. Genet. [Theoretical and Applied Genetics] 98:704 (1999)), chromosomal interval or RNA cleavage product (e.g., Lynx tag). Markers can be present in genomic nucleic acid or expressed nucleic acid (e.g., EST). The term marker can also refer to a nucleic acid used as a probe or primer (e.g., primer pair) 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 published or can be obtained from various sources such as the SoyBase Internet resource.

[0331] The mark corresponding to the genetic polymorphism between the members of the colony can be detected by methods generally recognized in the art. These methods include, for example, nucleic acid sequence, hybridization method, amplification method (for example, sequence-specific amplification method based on PCR), restriction fragment length polymorphism detection (RFLP), isozyme marker detection, polynucleotide polymorphism detection by allele-specific hybridization (ASH), amplification variable sequence detection of plant genome, autonomous sequence replication detection, simple repeat sequence detection (SSR), single nucleotide polymorphism detection (SNP), and / or amplified fragment length polymorphism detection (AFLP). Known generally recognized methods are also used to detect expressed sequence tags (ESTs) and SSR marks derived from EST sequences, as well as randomly amplified polymorphic DNA (RAPD).

[0332] A "marker allele," also described as an "allele of a marker locus," may refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus that are polymorphic for the marker locus in a population.

[0333] "Marker assisted selection" (MAS) is a method of selecting a phenotype based on a marker genotype. In some embodiments, the marker genotype is used to identify plants that will be selected for a breeding program or for planting. In some embodiments, the marker genotype is used to identify plants that will not be selected for a breeding program or for planting (i.e., counter-selected plants), allowing them to be removed from the breeding / planting population.

[0334] As used herein, the terms "marker locus and marker loci" refer to one or more specific chromosomal locations in an organism's genome where one or more specific markers can be found. Marker loci can be used to track the presence of a second linked locus (e.g., a linked locus that encodes or contributes to the expression of a phenotypic trait). For example, a marker locus can be used to monitor the separation of alleles at a locus (e.g., a QTL or a single gene) that are genetically or physically linked to the marker locus.

[0335] As used herein, the terms "marker probe" and "probe" refer to nucleotide sequences or nucleic acid molecules that can be used to detect the presence of one or more specific alleles in a marker locus (e.g., a nucleic acid probe that is complementary to all or part of the marker or marker locus by nucleic acid hybridization). Marker probes comprising 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 aspects, a marker probe refers to any type of probe that can distinguish (i.e., genotype) a specific allele present at a marker locus.

[0336] As used herein, when identifying linked loci, the term "molecular marker" or "gene marker" can be used to refer to a genetic marker as defined above, or a coded product (e.g., protein) thereof as a reference point. Molecular markers can be derived from a genomic nucleotide sequence or an expressed nucleotide sequence (e.g., RNA, cDNA, etc. from splicing). The term also refers to a nucleotide sequence complementary to a marker sequence or to a nucleotide sequence located on its flank, for example, a nucleotide sequence used as a probe and / or primer capable of amplifying a marker sequence. For example, according to the Watson-Crick base pairing principle, when a nucleotide sequence specifically hybridizes in a solution, the nucleotide sequence is "complementary". When located on an indel region, some of the markers described herein are also referred to as hybrid markers. This is because, by definition, the insertion region is a polymorphism about a plant that does not have the insertion. Therefore, the marker only needs to indicate whether the indel region exists. Any suitable marker detection technology can be used to identify this hybrid marker, for example, for the SNP technology in the examples provided herein.

[0337] "Non-naturally occurring soybean varieties" are soybeans of any variety that do not exist in nature. "Non-naturally occurring soybean varieties" 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, "non-naturally occurring soybean varieties" may include one or more heterologous nucleotide sequences. In some embodiments, "non-naturally occurring soybean varieties" may include 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, "non-naturally occurring soybean varieties" may include non-natural combinations of two or more naturally occurring nucleotide sequences (i.e., two or more naturally occurring genes that do not naturally occur in the same soybean, such as genes not found in soybean lines).

[0338] As used herein, the terms "phenotype", "phenotypic trait" or "trait" refer to one or more traits and / or performance of an organism. A phenotype is a performance observable to the naked eye or by any other evaluation means known in the art (e.g., microscopy, biochemical analysis, or electromechanical determination). In some cases, a phenotype or trait is directly controlled by a single gene or genetic locus, i.e., a "monogenic 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, so that the effect is real and repeatable.

[0339] As used herein, the term "plant" can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term "plant" can refer to any of the following: a whole plant, a plant component or organ (e.g., root, stem, leaf, bud, flower, pod, etc.), a plant tissue, a seed, and / or a plant cell. A plant cell is a plant cell obtained from a plant, or a plant cell derived from a cell taken from a plant by culturing. Thus, the term "soybean plant" can refer to a whole soybean plant, one or more parts of a soybean plant (e.g., root, root tip, stem, leaf, bud, flower, pod, seed, cotyledon, etc.), a soybean plant cell, a soybean plant protoplast, and / or a soybean plant callus.

[0340] As used herein, the term "polymorphism" refers to the variation in the nucleotide sequence at the locus, wherein the variation is too common, and is not merely due to spontaneous mutation. Polymorphism can be a single nucleotide polymorphism (SNP) or an insertion / deletion polymorphism (also referred to as "indel" herein). In addition, this variation can be in transcriptional profile or methylation pattern. One or more polymorphic sites of nucleotide sequence can be determined by carrying out nucleotide sequence comparison at one or more loci in two or more germplasm entries.

[0341] As used herein, the term "population" refers to a genetically heterogeneous collection of plants sharing a common genetic derivation.

[0342] As used herein, the terms "progeny" and "progeny plants" refer to plants produced by asexual or sexual reproduction from one or more parent plants. Progeny plants can be obtained by cloning or selfing of a single parent plant, or by crossing two parent plants.

[0343] As used herein, the term "reference sequence" refers to a determined nucleotide sequence used as the basis for nucleotide sequence comparison. For example, a reference sequence of a marker is obtained by genotyping multiple strains at one or more target loci, comparing these nucleotide sequences in a sequence alignment program, and then obtaining a consensus sequence for the comparison. Therefore, the reference sequence identifies the polymorphism in the allele at the 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 for the actual polymorphism in one or more loci.

[0344] As used herein, the terms "disease tolerance" and "disease resistance" refer to the tolerance and / or reproductive ability of a plant despite infection with a corresponding disease. When used with respect to germplasm, these terms refer to the tolerance and / or reproductive ability of a plant produced by the germplasm despite infection with a corresponding disease. In some embodiments, infected disease-resistant soybean plants can be produced the same (or nearly the same) as uninfected soybean plants. Typically, if a plant or germplasm shows "enhanced pathogen resistance," it is labeled "disease resistance."

[0345] An "unfavorable allele" of a marker is a marker allele that segregates with the unfavorable plant phenotype, thus providing the benefit of identifying plants that can be removed from a breeding program or planting.

[0346] “PI339656” refers to Penghu soybean registration number PI339656, PI446934, PI483193, PI505154, PI595799 or PI591576.

[0347] Genetic mapping

[0348] Genetic loci associated with specific phenotypes (such as disease resistance) can be mapped to the genome of an organism. By identifying markers or marker clusters that co-segregate with the desired trait, breeders can quickly select the desired phenotype by selecting appropriate markers (a method called marker-assisted selection or MAS). Breeders can also use such markers to simulate design genotypes on a computer and implement whole genome selection.

[0349] The present invention provides markers associated with enhanced disease resistance. These markers and / or detection of other linked markers can be used to identify, select and / or produce disease-resistant plants and / or eliminate non-disease-resistant plants from breeding programs or plantings.

[0350] Penghu soybean genetic loci associated with enhanced disease resistance

[0351] Markers associated with enhanced disease resistance are identified herein (see Table 1). Markers of the invention can comprise a single allele or a combination of alleles at one or more genetic loci (e.g., any marker combination from Table 1. For example, a marker can comprise one or more marker alleles located within a first chromosome interval (e.g., SEQ ID NO: 1).

[0352] The markers of the present invention are described herein with respect to the location of the marker locus within a chromosomal interval including the sequenced genomic DNA of PI339656 or a progeny thereof as depicted in any one of SEQ ID NO: 1 and as shown in Table 1.

[0353] Logo assisted selection

[0354] Markers can be used in a variety of plant breeding applications. See, e.g., Staub et al., Hortscience 31:729 (1996); Tanksley, Plant Molecular Biology Reporter 1:3 (1983). One of the main areas of interest is the use of 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 likelihood of co-segregating with a desired trait. Such markers are presumed to be located in / near one or more genes that produce the desired phenotype, and their presence indicates that the plant will have the desired trait. Plants with the marker are expected to transfer the desired phenotype to their progeny.

[0355] Markers that exhibit linkage with loci that affect desired phenotypic traits provide useful tools for selecting traits in plant populations. This is especially true when the phenotype is difficult to measure or occurs in the later stages of plant development. Since DNA marker determination is more labor-saving than field phenotyping and the physical space occupied is smaller, larger populations can be measured, increasing the probability of finding recombinants with target segments that move from donor strains to recipient strains. The tighter the linkage, the more useful the marker, because recombination is unlikely to occur between the marker and the gene that causes or causes the trait. Using flanking markers reduces the probability of false positive selection. Ideally, the pathogenic gene itself has a marker so that the recombination between the marker and the gene cannot occur. Such markers are called "perfect markers".

[0356] When genes are introgressed by MAS, not only the genes but also the flanking regions are introduced. Gepts, Crop Sci [Crop Science] 42: 1780 (2002). This is called "linkage drag". In the case where the donor plant is very unrelated to the recipient plant, these flanking regions carry additional genes that can encode agronomically undesirable traits. Even after multiple cycles of backcrossing with elite soybean lines, this "linkage drag" can also lead to reduced yields or other negative agronomic characteristics. This is sometimes also called "yield drag". The size of the flanking regions can be reduced by additional backcrossing, although this is not always successful because breeders cannot control the size of the region or the recombination breakpoints. Young et al., Genetics [Genetics] 120: 579 (1998). In classical breeding, it is usually only by chance that recombination that helps reduce the size of the donor segment is selected. Tanksley et al., Biotechnology [Biotechnology] 7: 257 (1989). Even after 20 backcrosses, it is possible to find a fairly large donor chromosome fragment that is still linked to the gene and is selected. However, if markers are used, rare individuals that have undergone recombination near the target gene can be selected. In 150 backcross plants, there is a 95% chance that at least one plant will undergo hybridization within 1cM (based on a single meiotic map distance) of the gene. Markers allow clear identification of these individuals. Using an additional backcross of 300 plants, there is a 95% probability of hybridization within a 1cM single meiotic map distance on the other side of the gene, thereby producing a segment near the target gene less than 2cM based on a single meiotic map distance. This can be achieved in two generations with markers, while an average of 100 generations is required without markers. See Tanksley et al., supra. When the exact location of a gene is known, the side-joining markers around the gene can be used to select recombination in different population sizes. For example, in smaller populations, it is expected that recombination can be further away from the gene, so more distal side-joining markers are needed to detect the recombination.

[0357] The availability of an integrated linkage map of the soybean genome containing an enhanced density of public soybean markers has facilitated soybean genetic mapping and MAS.

[0358] Of all the types of molecular markers, SNPs are the most abundant and have the potential to provide the highest genetic map resolution. Bhattramakki et al., Plant Molec. Biol. 48:539 (2002). SNPs can be determined in a so-called "ultra-high throughput" manner because they do not require large amounts of nucleic acid and automation of the determination is straightforward. SNPs also have the benefit 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 cleavage, 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 [Pharmacogenomics] 1:95 (2000); Bhattramakki and Rafalski, Discovery and application of single nucleotide polymorphism markers in plants, in Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford (2001). A wide range of commercially available technologies utilize these and other methods to interrogate SNPs, including Masscode TM (Qiagen, Germantown, MD), (Hologic, Madison, WI), (Applied Biosystems, Foster City, CA), (Applied Biosystems, Foster City, CA) and Beadarrays TM (Illumina, San Diego, CA).

[0359] Many SNP alleles within a sequence or across linked sequences can be used together to describe the haplotype of any particular genotype. Ching et al., BMC Genet. [BMC Genetics] 3: 19 (2002); Gupta et al., (2001), Rafalski, Plant Sci [Plant Science] 162: 329 (2002b). Haplotypes can be more informative than single SNPs and can describe any particular genotype in more detail. For example, for a particular disease resistant strain or variety, a single SNP may be the allele "T", but the allele "T" may also appear in a soybean breeding population for a recurrent parent. In this case, a combination of alleles of linked SNPs may be more informative. Once a unique haplotype is assigned to a donor chromosome region, the haplotype can be used in the population or any of its subpopulations to determine whether an individual has a particular gene. The use of an automated high-throughput marker detection platform known to those of ordinary skill in the art makes this method efficient and effective.

[0360] The marker of the present invention can be used in a marker-assisted selection scheme to identify and / or select a progeny with enhanced tolerance to Asian soybean rust. Such methods may include, consist essentially of, or consist of: hybridizing a first soybean plant or germplasm with a second soybean plant or germplasm, wherein the first soybean plant or germplasm comprises a chromosome interval derived from PI339656 or its progeny, wherein the chromosome interval is consistent with nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO:1, and selecting a progeny plant with the marker. One or both of the first and second soybean plants may be a non-natural soybean variety. In certain embodiments, the second soybean plant or germplasm is a second soybean plant or germplasm of an excellent soybean variety. In certain 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 an excellent soybean variety. In another embodiment, the first soybean plant comprises a chromosomal interval derived from PI339656 or a progeny thereof, wherein the chromosomal interval is identical to nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO: 1; wherein the chromosomal interval further comprises at least one allele as described in any one of the corresponding Table 1.

[0361] Methods for identifying and / or selecting disease-resistant soybean plants or germplasm may comprise, consist essentially of, or consist of detecting the presence of a marker associated with enhanced ASR tolerance. The marker may be detected in any sample obtained from a plant or germplasm, including but not limited to the entire plant or germplasm, a portion of the plant or germplasm (e.g., a seed slice, leaf punch disk, or cell from the plant or germplasm), or a nucleotide sequence from the plant or germplasm. Such a sample may be obtained from a plant or germplasm using any existing or future method 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, cutting the seeds with a laser and punching leaf disks). 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 an elite soybean variety.

[0362] In some embodiments, the marker detected in the sample may comprise, consist essentially of, or consist of: one or more marker alleles located in a chromosomal interval selected from the group consisting of:

[0363] 1) a chromosomal interval derived from PI339656 or a progeny thereof, wherein the chromosomal interval is identical to nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO: 1; or

[0364] 2) a chromosome interval derived from PI339656 or a progeny thereof, wherein the chromosome interval comprises a portion of any of the chromosome intervals described in 1), and wherein the chromosome interval comprises at least one SNP marker as shown in any of Table 1, respectively; or

[0365] 3) A chromosomal interval spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM from a SNP marker associated with increased ASR resistance in soybean, wherein the SNP marker is selected from the group consisting of any SNP marker shown in any one of Table 1.

[0366] The method for producing a disease resistant soybean plant may comprise, consist essentially of, or consist of detecting a marker associated with enhanced disease (e.g., ASR) resistance in a germplasm, wherein the marker is selected from any one of Table 1, or wherein the marker is a locus that is tightly linked to any marker described in any one of Table 1 and from which a soybean plant is produced. The marker may be detected in any sample taken from the germplasm, including but not limited to a portion of the germplasm (e.g., a seed slice or cell from the germplasm) or a nucleotide sequence from the germplasm. Such a sample may be taken from the germplasm using any existing or future method 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, cutting the seeds with a laser and punching leaf discs. The 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 an elite 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.

[0367] In some embodiments, the marker detected in the germplasm may comprise, consist essentially of, or consist of: one or more marker alleles located in a chromosomal interval selected from the group consisting of:

[0368] 1) a chromosomal interval derived from PI339656 or a progeny thereof, wherein the chromosomal interval is identical to nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO: 1; or

[0369] 2) a chromosome interval derived from PI339656 or a progeny thereof, wherein the chromosome interval comprises a portion of any of the chromosome intervals described in 1), and wherein the chromosome interval comprises at least one SNP marker as shown in any of Table 1, respectively; or

[0370] 3) A chromosomal interval spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM from a SNP marker associated with increased ASR resistance in soybean, wherein the SNP marker is selected from the group consisting of any SNP marker shown in any one of Table 1.

[0371] In some embodiments, the markers detected in the germplasm can comprise, consist essentially of, or consist of one or more marker alleles selected from any one of Table 1.

[0372] Methods for producing and / or selecting for an Asian soybean rust resistant / tolerant soybean plant or germplasm may comprise crossing a first soybean plant or germplasm with a second soybean plant or germplasm, wherein the first soybean plant or germplasm comprises a chromosome interval selected from the group consisting of:

[0373] 1) a chromosomal interval derived from PI339656 or a progeny thereof, wherein the chromosomal interval is identical to nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO: 1; or

[0374] 2) a chromosome interval derived from PI339656 or a progeny thereof, wherein the chromosome interval comprises a portion of any of the chromosome intervals described in 1), and wherein the chromosome interval comprises at least one SNP marker as shown in any of Table 1, respectively; or

[0375] 3) A chromosomal interval spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM from a SNP marker associated with increased ASR resistance in soybean, wherein the SNP marker is selected from the group consisting of any SNP marker shown in any one of Table 1.

[0376] Also provided herein are methods of introgressing alleles associated with enhanced disease (e.g., ASR, SCN, SDS, RKN, Phytophthora, etc.) resistance / tolerance (e.g., ASR, SCN, SDS, RKN, Phytophthora, etc.) into soybean plants. Such methods for introgressing alleles associated with enhanced disease (e.g., ASR, SCN, SDS, RKN, Phytophthora, etc.) resistance / tolerance into soybean plants or germplasm may comprise, consist essentially of, or consist of crossing a first soybean plant or germplasm comprising the allele (donor) (wherein the allele is selected from any of the alleles listed in Table 1) or a marker that is "closely proximal" to a marker listed in Table 1 with a second soybean plant or germplasm lacking the allele (recurrent parent), and repeatedly backcrossing the progeny comprising the allele to the recurrent parent. Progeny containing the allele can be identified by detecting the presence of a marker 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 taken from the progeny, including but not limited to a portion of the progeny (e.g., a seed slice, leaf punch, or cell from the plant or germplasm) or a nucleotide sequence from the progeny. Such samples can be taken from the progeny using any existing or future method 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, cutting the seeds with a laser 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 an elite 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 elite soybean variety.

[0377] In some embodiments, markers for identifying progeny comprising alleles associated with enhanced disease (e.g., ASR, SCN, SDS, RKN, Phytophthora, brown stem rot, etc.) resistance / tolerance may comprise, consist essentially of, or consist of one or more alleles located within a chromosomal interval selected from the group consisting of:

[0378] 1) a chromosomal interval derived from PI339656 or a progeny thereof, wherein the chromosomal interval is identical to nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO: 1; or

[0379] 2) a chromosome interval derived from PI339656 or a progeny thereof, wherein the chromosome interval comprises a portion of any of the chromosome intervals described in 1), and wherein the chromosome interval comprises at least one SNP marker as shown in any of Table 1, respectively; or

[0380] 3) A chromosomal interval spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM from a SNP marker associated with increased ASR resistance in soybean, wherein the SNP marker is selected from the group consisting of any SNP marker shown in any one of Table 1.

[0381] In some embodiments, the marker may comprise, consist essentially of, or consist of marker alleles located in at least two different chromosome intervals. For example, the marker may comprise one or more alleles located in a chromosome interval defined by and including any two markers in Table 1.

[0382] Disease-resistant soybean plants and germplasm

[0383] The present invention provides disease-resistant soybean plants and germplasm. As described above, the methods of the present invention can be used to identify, produce and / or select disease-resistant soybean plants or germplasm (e.g., soybean plants with resistance or increased tolerance to Asian soybean rust). In addition to the above methods, disease-resistant soybean plants or germplasm can be produced by any method, by which markers associated with enhanced disease tolerance are introduced into soybean plants or germplasm, including but not limited to transformation, protoplast transformation, or fusion, double haploid technology, embryo rescue, gene editing, and / or produced by any other nucleic acid transfer system.

[0384] In some embodiments, the soybean plant or germplasm comprises 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 an elite soybean variety.

[0385] The disease resistant soybean plant or germplasm can be the progeny of a cross between an elite soybean variety and a soybean variety comprising an allele associated with enhanced disease (e.g., ASR) tolerance, wherein the allele is within a chromosomal interval selected from the group consisting of:

[0386] 1) a chromosomal interval derived from PI339656 or a progeny thereof, wherein the chromosomal interval is identical to nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO: 1; or

[0387] 2) a chromosome interval derived from PI339656 or a progeny thereof, wherein the chromosome interval comprises a portion of any of the chromosome intervals described in 1), and wherein the chromosome interval comprises at least one SNP marker as shown in any of Table 1, respectively; or

[0388] 3) A chromosomal interval spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM from a SNP marker associated with increased ASR resistance in soybean, wherein the SNP marker is selected from the group consisting of any SNP marker shown in any one of Table 1.

[0389] The disease-resistant soybean plant or germplasm can be the progeny of a genetic introgression, wherein the recurrent parent is an elite soybean variety and the donor comprises alleles associated with enhanced disease tolerance and / or resistance, wherein the individual carries a chromosomal interval or portion thereof comprising any one of SEQ ID NO: 1, and wherein the chromosomal interval comprises at least one allele selected from Table 1, respectively.

[0390] The disease resistant soybean plant or germplasm can be the progeny of a cross between a first elite soybean variety (e.g., a test line), and the progeny of a cross between a second elite soybean variety (e.g., a recurrent parent) and a soybean variety (e.g., a donor) containing alleles associated with enhanced ASR tolerance.

[0391] The disease resistant soybean plant or germplasm can be the progeny of a cross between a first elite soybean variety and a progeny of a gene introgression, wherein the recurrent parent is a second elite soybean variety and the donor comprises an allele associated with enhanced ASR tolerance.

[0392] Disease-resistant soybean plants and germplasm of the invention can comprise one or more markers of the invention (eg, any marker described in Table 1; or any marker that is closely associated with any marker described in Table 1).

[0393] In some embodiments, the disease resistant soybean plant or germplasm may comprise within its genome a marker associated with enhanced ASR tolerance, wherein the marker is located within a chromosomal interval selected from the group consisting of:

[0394] 1) a chromosomal interval derived from PI339656 or a progeny thereof, wherein the chromosomal interval is identical to nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO: 1; or

[0395] 2) a chromosome interval derived from PI339656 or a progeny thereof, wherein the chromosome interval comprises a portion of any of the chromosome intervals described in 1), and wherein the chromosome interval comprises at least one SNP marker as shown in any of Table 1, respectively; or

[0396] 3) A chromosomal interval spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM from a SNP marker associated with increased ASR resistance in soybean, wherein the SNP marker is selected from the group consisting of any SNP marker shown in any one of Table 1.

[0397] In some embodiments, the disease resistant soybean plant or germplasm may comprise within its genome a marker comprising, consisting essentially of, or consisting of marker alleles located in at least two different chromosomal intervals. For example, a marker may comprise one or more alleles located in a chromosomal interval defined by and including any combination of two markers in Table 1.

[0398] Disease-resistant soybean seeds

[0399] The present invention provides disease-resistant soybean seeds. As described above, the methods of the present invention can be used to identify, produce and / or select disease-resistant soybean seeds. In addition to the above methods, disease-resistant soybean seeds can be produced by any method, whereby markers associated with enhanced ASR tolerance are introduced into soybean seeds, including but not limited to transformation, protoplast transformation or fusion, double haploid technology, collagen rescue, gene editing (e.g., CRISPR or TALEN or large-range nuclease), and / or produced by any other nucleic acid transfer system.

[0400] In some embodiments, the disease resistant soybean seed comprises a non-naturally occurring soybean variety. In some embodiments, the soybean seed is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to the genome of an elite soybean variety.

[0401] Disease resistant soybean seeds can be produced from disease resistant soybean plants identified, produced or selected by the methods of the present invention. In some embodiments, disease resistant soybean seeds are produced from disease resistant soybean plants of the present invention.

[0402] The disease resistant soybean seeds of the present invention may comprise one or more markers from Table 1 of the present invention.

[0403] In some embodiments, the disease resistant soybean seed may comprise within its genome a marker associated with enhanced ASR tolerance, wherein the marker is located within a chromosomal interval selected from the group consisting of:

[0404] 1) a chromosomal interval derived from PI339656 or a progeny thereof, wherein the chromosomal interval is identical to nucleotide base 1 to nucleotide base 5166731 of SEQ ID NO: 1; or

[0405] 2) a chromosome interval derived from PI339656 or a progeny thereof, wherein the chromosome interval comprises a portion of any of the chromosome intervals described in 1), and wherein the chromosome interval comprises at least one SNP marker as shown in any of Table 1, respectively; or

[0406] 3) A chromosomal interval spanning 20 cM, 15 cM, 10 cM, 5 cM, 1 cM, 0.5 cM from a SNP marker associated with increased ASR resistance in soybean, wherein the SNP marker is selected from the group consisting of any SNP marker shown in any one of Table 1.

[0407] Examples

[0408] The following examples are not intended to be an exhaustive list of all the different ways in which the present invention can be implemented or all the features that can be added to the present invention. Those skilled in the art will appreciate that numerous changes and additions can be made to the different embodiments without departing from the present invention. Therefore, the following description is intended to set forth some specific embodiments of the present invention and does not exhaustively describe all permutations, combinations, and variations thereof.

[0409] Example 1: Identification of ASR-resistant wild soybean lines

[0410] Wild soybean lines were evaluated for rust resistance to sixteen rust strains (see Figure 1 Rust data were generated using single pustule-derived isolates from USDA-ARS (FL Q09, FL Q12, LABR13, FLQ11) and single pustule-derived isolates from field populations (FL Q15, NC06, Vero, GLC15, UBL, BR south, and BR central), screened in a closed facility.

[0411] Rust grades are classified according to the grouping modified by Burdon and Speer, TAG, 1984 (see Figure 2 ). In North and South America, approximately 4 plants were used to screen each accession >2 times.

[0412] Example 2: Allele mining and association to the PI339656 ASR locus

[0413] The resistant parent is crossed with susceptible Penghu soybean strain and produces F1 plant (see Table 2). F1 plant is self-fertilized and gathers F2 seed from self-fertilized F1 plant. About 200 F2 seeds are sown, and the leaf tissue of each plant is collected for DNA preparation, and then the plant is inoculated with pyrifos to determine the resistance / susceptibility phenotype of each F2 individual. The tissue from 50 resistant F2 and 50 susceptible F2 is combined in a separate library, and genomic DNA is prepared from each library. For each in these libraries, Illumina sequencing libraries are prepared from DNA, and each library is sequenced in two Illumina HiSeq2000 2x 100bp paired end (PE) swimming lanes. The sequencing reads are adjusted to remove the bases with PHRED quality score <15.

[0414] As a non-limiting example, an ASR resistance phenotype may include the formation of lesions without spores on leaves of plants exposed to ASR, while an ASR susceptible phenotype includes the formation of spores on leaves of plants exposed to ASR. Still other phenotypes can be used to distinguish ASR resistant plants from susceptible plants.

[0415] Quality-adjusted reads were aligned to the PI339656 reference genome sequence using GSNAP (WU and NACU 2010) as paired-end fragments. If a pair of reads could not be aligned together, they were treated as singletons for alignment. If reads mapped uniquely to the reference, they were used for subsequent analysis (≤2 mismatches per 36 bp and less than 5 bases per 75 bp as tails).

[0416] Based on the read depth, SNPs were filtered before BSA analysis, wherein SNPs with 40 to 200x read depth were retained. Chi-square test (Chi-square) was used to select SNPs with significantly different reads between two alleles in two libraries. The conditional probability that there was no recombination between each SNP mark and the causal locus (causal locus) in both the resistance library and the susceptible library was estimated using the empirical Bayesian method (LIU et al., 2012). The probability of linkage between SNP and causal gene is the geometric mean of these two conditional probabilities. It was found that approximately 780 SNPs may be linked to the target locus. Using phenotype F2 individuals, a subset of these assumed linked SNPs was used for fine positioning of the locus.

[0417] 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.

[0418] Table 8: Plant hybridization and research type

[0419]

[0420] 1. PI339656 Data2Bio LLC (Ames, Iowa) for tetraploid Laboratory methods for gBSA-Seq analysis of soybeans.

[0421] Chromosomal discovery of causal loci in a tetraploid soybean population,

[0422] Chromosomal discovery of the causal locus in PI339656 was performed using Data2Bio genomic segregating population group analysis (gBSA) technology. In theory, resistance is controlled by a single dominant allele. Data2Bio generated several libraries from DNA samples extracted in two susceptible tissue pools and two resistant tissue pools and sequenced them in eight (8) Illumina HiSeq20002x 100bp paired-end (PE) lanes. The reference genome summary, read processing from raw data to quality trimming,

[0423] Alignment, SNP discovery and SNP impact are shown in Figure 3-5 After various filtering steps, 4,610,234 informative SNPs were identified in the PI339656 genome. The trait association probability was then calculated using a Bayesian approach. Next, a physical map of the trait-associated SNPs (with a probability cutoff of 0.01) of the top contigs was created ( Figure 3-5). One scaffold, scaffold 14851, was identified and mapped to chromosome 14 of PI339656 (SEQ ID NO: 1). SNPs from this enriched scaffold were mapped to the public soybean genome. The data suggest that the causal R gene may be located in or near the interval of 3.18 to 3.5 MB on chromosome 14.

[0424] Example 3: Embryo rescue and introgression of the R gene interval into soybean lines

[0425] Embryo rescue is performed and chemical treatments are applied to produce amphidiploid shoots. If the amphidiploid plants are fertile, they will be used for backcrossing with soybean. Backcrossing with soybean and subsequent embryo rescue will need to be performed for several generations to gradually eliminate the perennial soybean chromosomes.

[0426] Distant hybridization: Elite Syngenta soybean lines (RM 3.7 to 4.8) were used as females (pollen inoculators), and multiple accessions of Penghu soybean were used as males or pollen donors. It is important to select flowers from Glycine max plants that contain anthers at the appropriate developmental stage. Fresh, fully open, bright flowers retain anthers with mature pollen. The pollen should appear loose and dusty yellow. These flowers are taken from Glycine max plants and brought to soybean plants for pollination. Pollen from Glycine max plants should be used within 30 minutes of flower removal. It is also important to identify and select soybean flower buds that are available for pollination. When the size of soybean flower buds is larger when compared to immature buds, they are usually ready for use. The sepals of soybean flowers are lighter in color, and the petals are just beginning to appear. First, use a pair of fine-tipped tweezers to carefully separate the sepals from the flower buds, exposing the outer petals. Then, gently grasp and remove the petals (5 pieces in total) on the flower, exposing the stamen ring around the pistil. Since the stigma is pollinated 1 day before the anthers begin to shed pollen, it is important to recognize the stage of sexual development when "females are available and males are not ready". When pollinating soybean flowers at this stage of development, it is not necessary to remove the female flowers. Find the stigma on the soybean flower. Then use 1 male flower, carefully peel off the petals to expose the anthers, and gently sprinkle the pollen grains on the stigma of the soybean flower. In this process, care should be taken not to damage the stigma at any time. Starting the second day after pollination, the hormone mixture is sprayed on the pollinated flowers, and eventually F1 pods are developed 1X every day until harvest. The pollinated flowers or pods are filled with a light mist of the hormone mixture, taking care not to cause the flowers / pods to fall off the plants prematurely. The mixture contains 100mg GA3, 25mg NAA and 5mg kinetin / L distilled water. These hormones help maintain pod development and increased pod growth.

[0427] Harvest: Pods from distant crosses were harvested approximately 14 to 16 days after pollination. Harvest dates in the literature suggest 19 to 21 days. Harvesting of pods 14 to 16 days after pollination was approximately 5 days earlier than suggested in the literature, shortening the timeline. Before selecting individual pods for harvest, confirm that sepals are removed (indicating an distant cross attempt) and that seed size is as expected for a distant cross. Pods are collected and counted based on the distant cross combination to determine hybrid success. The average hybridization success rate for multiple soybean females and 5 different accessions of Pescador soybean is approximately 40%. Distant hybrid pods can contain 1 to 3 seeds, but 2 seeds are typically found in each F1 pod.

[0428] Embryo rescue: Our embryo rescue protocol involves direct shoot regeneration from embryos, rather than regeneration through embryogenesis, resulting in faster plant recovery (shoots recover in approximately 2-3 months compared to up to 1 year timeline). Our protocol does not include incubation in the dark after transfer to germination medium. Our protocol does not require transfer to rooting medium.

[0429] The harvested pods are collected and brought back to the laboratory for sterilization. The pods are first rinsed with 70% EtOH for 2 to 3 minutes and then placed in 10% Clorox bleach for an additional 30 minutes on a platform shaker at approximately 130 RPM. Finally, the pods are rinsed multiple times with sterile water to remove any residual bleach. Embryo separation can be started immediately after the pods are sterilized, 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 embryos can be rescued. Individual pods are placed in a sterile Petri dish and opened using a scalpel and forceps. An incision is made along the length of the outbred 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 husk. Carefully remove the seed from the pod and place in a sterile Petri dish under a dissecting microscope. Very fine forceps are required to separate the embryo from the seed. Holding the forceps with one hand, gently move one side of the seed away from the embryo with the umbilical cord facing up. The other hand uses another pair of tweezers to remove the seed coat from one side of the seed containing the embryo. Peel off the membrane around the embryo, and push the embryo upward from the bottom. The embryo should be through the globular development stage, and preferably through the early heart (heart) development stage (mid-term to late heart stage, cotyledon stage and early maturity stage embryo are desired). The separated embryo is transferred to embryo rescue medium, such as soybean ER1-1. Now the embryo can be processed to induce chromosome doubling. (For detailed information about chromosome doubling, see below) the separated embryo is retained on embryo rescue medium at 24 ℃ for 21 to 30 days. The embryo can be kept in the dark during the entire incubation of ER1-1, can start incubation in the dark and complete under illumination, or can carry out the entire incubation under illumination. There is no callus induction stage in this scheme. Buds develop directly from embryos.

[0430] Chromosome doubling process: colchicine or trifluralin can be used for inducing chromosome doubling.Ideally, chemical treatment is carried out to late heart stage distant hybridization embryo (or larger) to separate and induce chromosome doubling immediately after separation at any time up to 1 week.Double agent can be mixed in solid or liquid culture medium and apply several hours or up to several days.Trifluralin is used with 10-40uM concentration in solid or liquid culture medium.In addition, colchicine is used with 0.4-1mg / ml concentration in solid or liquid culture medium.After chemical treatment, embryo is transferred in fresh embryo rescue culture medium.Colchicine or trifluralin can be handled immediately after embryo rescue.

[0431] Shoot regeneration: The developing embryo is transferred from the rescue medium to a germination medium (e.g., soybean ER GSMv2) at 24°C under light for about 3 to 5 weeks. Alternatively, the developing embryo can be transferred from the rescue medium to an elongation medium (e.g., soybean E1 0 No TCV) at 24°C under light for about 3 to 5 weeks. The developing buds can be transferred from the culture medium plate to a Phytocons containing a germination or elongation medium for further bud development. The established buds are moved to the soil. Initial plant care is essential for the survival of these buds.

[0432] Ploidy Analysis: Ploidy analysis was performed using flow cytometry. Leaf tissue for ploidy analysis was collected from shoots in culture or after establishment in soil. Tissue was 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 is sufficient. Samples were prepared according to the instructions in the Sysmex kit. Each sample group contained untreated F1 plants (not treated to induce chromosome doubling) as a control. The following is a description of the method: Distant hybridization - Our distant hybridization success rate is significantly higher than reported in the literature. Female flowers are not emasculated, which saves time and reduces the risk of damage to the stigma.

[0433] The above examples clearly illustrate the advantages of the present invention. Although the present invention has been described with reference to specific details of certain embodiments, such details are not intended to be considered as limitations on the scope of the present invention unless or to the extent they are included in the appended claims.

[0434] Throughout the application, various patents, patent publications and non-patent publications are cited. The disclosures of these patents, patent publications and non-patent publications are hereby incorporated into this application in their entirety by reference to more fully describe the current state of the art to which the invention belongs.

Claims

1. A method for producing a soybean plant having increased resistance to ASR, the method comprising the steps of: a. Providing a Penghu soybean plant line or progeny thereof comprising the chromosome interval shown in SEQ ID NO: 1; b. Implement embryo rescue; c. Collecting seeds produced by the method of b); d. Regenerating the seeds of c) into plants.

2. The method of claim 1, wherein the Penghu soybean plant line of a) is PI339656, PI591576 or a progeny thereof.

3. A method for identifying or selecting a soybean plant having an ASR resistance allele derived from Penghu soybean, the method comprising the following steps: a. isolating nucleic acids from soybean plants; b. detecting the presence of a molecular marker associated with increased ASR resistance in the nucleic acid, wherein the molecular marker is located within the interval corresponding to SEQ ID NO: 1, and wherein the molecular marker is a favorable marker as described in Table 1; and c. thereby identifying or selecting soybean plants having the ASR resistance allele derived from Penghu soybean. The method of claim 3 , wherein the marker is T at position 3,275,655 of SEQ ID NO:

1.

5. The method of claim 3, wherein the marker is T at position 3,274,672 of SEQ ID NO:

1. The method of claim 3 , wherein the marker is T at position 3, 291, 351 of SEQ ID NO:

1.

7. A method of producing a soybean plant having increased resistance to Asian soybean rust (ASR), the method comprising: a. introducing a chromosomal interval associated with ASR resistance comprising SEQ ID NO: 1 into the genome of a soybean plant; b. isolating nucleic acid from said soybean plant or its progeny plant; c. detecting the presence of a molecular marker associated with the chromosome interval in the nucleic acid, wherein the molecular marker is any favorable molecular marker as described in Table 1; and d. Thereby identifying soybean plants with increased ASR resistance.

8. The method of claim 7, wherein the marker is T at position 3,275,655 of SEQ ID NO:

1.

9. The method of claim 7, wherein the marker is T at position 3,274,672 of SEQ ID NO:

1.

10. The method of claim 7, wherein the marker is T at position 3, 291, 351 of SEQ ID NO: 1.

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