Identification of a new soybean rust resistance gene in PI 567102B

Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. and P. Syd. is one of the most economically important diseases of soybean (Glycine max (L.) Merr.). Durable resistance to P. pachyrhizi is the most effective long-term strategy to control SBR. The objective of this study was to investigate the genetics of resistance to P. pachyrhizi in soybean accession PI 567102B. This accession was previously identified as resistant to SBR in Paraguay and to P. pachyrhizi isolates from seven states in the USA (Alabama, Florida, Georgia, Louisiana, Mississippi, South Carolina, and Texas). Analysis of two independent populations, one in which F(2) phenotypes were inferred from F(2)-derived F(3) (F(2:3)) families and the other in which F(2) plants had phenotypes measured directly, showed that the resistance in PI 567102B was controlled by a single dominant gene. Two different isolates (MS06-1 and LA04-1) at different locations (Stoneville, MS and Ft. Detrick, MD) were used to independently assay the two populations. Linkage analysis of both populations indicated that the resistance locus was located on chromosome 18 (formerly linkage group G), but at a different location than either Rpp1 or Rpp4, which were previously mapped to this linkage group. Therefore, the SBR resistance in PI 567102B appeared to be conditioned by a previously unreported locus, with an underlying single dominant gene inferred. We propose this gene to be designated Rpp6. Incorporating Rpp6 into improved soybean cultivars may have wide benefits as PI 567102B has been shown to provide resistance to P. pachyrhizi isolates from Paraguay and the US.     

A new soybean rust resistance allele from PI 423972 at the <Emphasis Type="Italic">Rpp4</Emphasis> locus

Phakopsora pachyrhizi is a fungal pathogen and the cause of Asian soybean rust. P. pachyrhizi was first detected in the continental USA in 2004 and has since been a threat to the soybean industry. There are six described loci that harbor resistance to P. pachyrhizi (Rpp) genes. The resistance of PI 423972 was previously shown to be within 5 cM of the Rpp4 locus of PI 459025B, yet had differential reactions when challenged with P. pachyrhizi isolates India 1973 and Taiwan 1972. In this study, the resistance of PI 423972 was mapped to a 187.5 kb interval between the SNP markers GSM0543 and GSM0387 on chromosome 18 (51,397,064 to 51,584,617 bp, Glyma.Wm82.a2) that overlaps the interval for Rpp4 and is designated as Rpp4-b. A unique haplotype is described for PI 423972 that separates it from PI 459025B, 32 North American soybean ancestors, and all described sources of Rpp gene resistance.     

Molecular mapping of soybean rust (Phakopsora pachyrhizi) resistance genes: discovery of a novel locus and alleles

Soybean production in South and North America has recently been threatened by the widespread dissemination of soybean rust (SBR) caused by the fungus Phakopsora pachyrhizi. Currently, chemical spray containing fungicides is the only effective method to control the disease. This strategy increases production costs and exposes the environment to higher levels of fungicides. As a first step towards the development of SBR resistant cultivars, we studied the genetic basis of SBR resistance in five F(2) populations derived from crossing the Brazilian-adapted susceptible cultivar CD 208 to each of five different plant introductions (PI 200487, PI 200526, PI 230970, PI 459025, PI 471904) carrying SBR-resistant genes (Rpp). Molecular mapping of SBR-resistance genes was performed in three of these PIs (PI 459025, PI 200526, PI 471904), and also in two other PIs (PI 200456 and 224270). The strategy mapped two genes present in PI 230970 and PI 459025, the original sources of Rpp2 and Rpp4, to linkage groups (LG) J and G, respectively. A new SBR resistance locus, rpp5 was mapped in the LG-N. Together, the genetic and molecular analysis suggested multiple alleles or closely linked genes that govern SBR resistance in soybean.     

SNP assay to detect the ‘Hyuuga’ red-brown lesion resistance gene for Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi Syd., has the potential to become a serious threat to soybean, Glycine max L. Merr., production in the USA. A novel rust resistance gene, Rpp?(Hyuuga), from the Japanese soybean cultivar Hyuuga has been identified and mapped to soybean chromosome 6 (Gm06). Our objectives were to fine-map the Rpp?(Hyuuga) gene and develop a high-throughput single nucleotide polymorphism (SNP) assay to detect this ASR resistance gene. The integration of recombination events from two different soybean populations and the ASR reaction data indicates that the Rpp?(Hyuuga) locus is located in a region of approximately 371 kb between STS70887 and STS70923 on chromosome Gm06. A set of 32 ancestral genotypes which is predicted to contain 95% of the alleles present in current elite North American breeding populations and the sources of the previously reported ASR resistance genes (Rpp1, Rpp2, Rpp3, Rpp4, Rpp5, and rpp5) were genotyped with five SNP markers. We developed a SimpleProbe assay based on melting curve analysis for SNP06-44058 which is tighly linked to the Rpp?(Hyuuga) gene. This SNP assay can differentiate plants/lines that are homozygous/homogeneous or heterozygous/heterogeneous for the resistant and susceptible alleles at the Rpp?(Hyuuga) locus.     

Molecular mapping of two loci that confer resistance to Asian rust in soybean

Asian soybean rust (ASR) is caused by the fungal pathogen Phakopsora pachyrhizi Sydow & Sydow. It was first identified in Brazil in 2001 and quickly infected soybean areas in several countries in South America. Primary efforts to combat this disease must involve the development of resistant cultivars. Four distinct genes that confer resistance against ASR have been reported: Rpp1, Rpp2, Rpp3, and Rpp4. However, no cultivar carrying any of those resistance loci has been released. The main objective of this study was to genetically map Rpp2 and Rpp4 resistance genes. Two F(2:3) populations, derived from the crosses between the resistant lines PI 230970 (Rpp2), PI 459025 (Rpp4) and the susceptible cultivar BRS 184, were used in this study. The mapping populations and parental lines were inoculated with a field isolate of P. pachyrhizi and evaluated for lesion type as resistant (RB lesions) or susceptible (TAN lesions). The mapping populations were screened with SSR markers, using the bulk segregant analysis (BSA) to expedite the identification of linked markers. Both resistance genes showed an expected segregation ratio for a dominant trait. This study allowed mapping Rpp2 and Rpp4 loci on the linkage groups J and G, respectively. The associated markers will be of great value on marker assisted selection for this trait.     

Biphasic gene expression changes elicited by Phakopsora pachyrhizi in soybean correlate with fungal penetration and haustoria formation

Inoculation of soybean (Glycine max) plants with Phakopsora pachyrhizi, the causal organism of Asian soybean rust, elicits a biphasic response characterized by a burst of differential gene expression in the first 12 h. A quiescent period occurs from 24 to 48 h after inoculation, in which P. pachyrhizi continues to develop but does not elicit strong host responses, followed by a second phase of intense gene expression. To correlate soybean responses with P. pachyrhizi growth and development, we inoculated the soybean cultivar Ankur (accession PI462312), which carries the Rpp3 resistance gene, with avirulent and virulent isolates of P. pachyrhizi. The avirulent isolate Hawaii 94-1 elicits hypersensitive cell death that limits fungal growth on Ankur and results in an incompatible response, while the virulent isolate Taiwan 80-2 grows extensively, sporulates profusely, and produces a compatible reaction. Inoculated leaves were collected over a 288-h time course for microarray analysis of soybean gene expression and microscopic analysis of P. pachyrhizi growth and development. The first burst in gene expression correlated with appressorium formation and penetration of epidermal cells, while the second burst of gene expression changes followed the onset of haustoria formation in both compatible and incompatible interactions. The proliferation of haustoria coincided with the inhibition of P. pachyrhizi growth in the incompatible interaction or the beginning of accelerated growth in the compatible interaction. The temporal relationships between P. pachyrhizi growth and host responses provide an important context in which to view interacting gene networks that mediate the outcomes of their interactions.     

Distinct biphasic mRNA changes in response to Asian soybean rust infection

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is now established in all major soybean-producing countries. Currently, there is little information about the molecular basis of ASR-soybean interactions, which will be needed to assist future efforts to develop effective resistance. Toward this end, abundance changes of soybean mRNAs were measured over a 7-day ASR infection time course in mock-inoculated and infected leaves of a soybean accession (PI230970) carrying the Rpp2 resistance gene and a susceptible genotype (Embrapa-48). The expression profiles of differentially expressed genes (ASR-infected compared with the mock-inoculated control) revealed a biphasic response to ASR in each genotype. Within the first 12 h after inoculation (hai), which corresponds to fungal germination and penetration of the epidermal cells, differential gene expression changes were evident in both genotypes. mRNA expression of these genes mostly returned to levels found in mock-inoculated plants by 24 hai. In the susceptible genotype, gene expression remained unaffected by rust infection until 96 hai, a time period when rapid fungal growth began. In contrast, gene expression in the resistant genotype diverged from the mock-inoculated control earlier, at 72 h, demonstrating that Rpp2-mediated defenses were initiated prior to this time. These data suggest that ASR initially induces a nonspecific response that is transient or is suppressed when early steps in colonization are completed in both soybean genotypes. The race-specific resistance phenotype of Rpp2 is manifested in massive gene expression changes after the initial response prior to the onset of rapid fungal growth that occurs in the susceptible genotype.     

Genetic divergence between North American ancestral soybean lines and introductions with resistance to soybean cyst nematode revealed by chloroplast haplotype

Domesticated soybean [Glycine max (L.) Merr.] is a major crop with an established ancestral relationship to wild soybean (Glycine soja Sieb. & Zucc.) native to Asia. Soybean genetic diversity can be assessed at different levels by identification of polymorphic alleles at genetic loci, in either the plastid or nuclear genomes. The objective of this study was to evaluate genetic diversity based on chloroplast haplotypes for soybean genotypes present in the USDA germplasm resource collection. Shared chloroplast haplotypes represent broad groups of genetic relatedness. Previous work categorized three-quarters of the cultivated soybeans from Asia into a single haplotype group. Our results confirmed the close relationship of North American soybean ancestors and G. max plant introductions previously identified as representing potential sources of soybean genetic variation with the finding that these genotypes belonged to a single chloroplast haplotype group. Genetic diversity was identified in soybean genotypes determined to have a high density of single nucleotide polymorphisms and in a screen of accessions with resistance to soybean cyst nematode. Characterization of soybean plant introduction lines into chloroplast haplotype group may be an important initial step in evaluating the appropriate use of particular soybean genotypes.     

Fine mapping of the soybean aphid-resistance gene Rag2 in soybean PI 200538

The discovery of biotype diversity of soybean aphid (SA: Aphis glycines Matsumura) in North America emphasizes the necessity to identify new aphid-resistance genes. The soybean [Glycine max (L.) Merr.] plant introduction (PI) 200538 is a promising source of SA resistance because it shows a high level of resistance to a SA biotype that can overcome the SA-resistance gene Rag1 from ‘Dowling’. The SA-resistance gene Rag2 was previously mapped from PI 200538 to a 10-cM marker interval on soybean chromosome 13 [formerly linkage group (LG) F]. The objective of this study was to fine map Rag2. This fine mapping was carried out using lines derived from 5,783 F₂ plants at different levels of backcrossing that were screened with flanking genetic markers for the presence of recombination in the Rag2 interval. Fifteen single nucleotide polymorphism (SNP) markers and two dominant polymerase chain reaction-based markers near Rag2 were developed by re-sequencing target intervals and sequence-tagged sites. These efforts resulted in the mapping of Rag2 to a 54-kb interval on the Williams 82 8× assembly (Glyma1). This Williams 82 interval contains seven predicted genes, which includes one nucleotide-binding site-leucine-rich repeat gene. SNP marker and candidate gene information identified in this study will be an important resource in marker-assisted selection for aphid resistance and for cloning the gene.     

Genetics and mapping of adult plant rust resistance in soybean PI 587886 and PI 587880A

Two soybean accessions, PI 587886 and PI 587880A, previously identified as having resistance to Phakospora pachyrhizi Syd. (soybean rust, SBR) were used to create two populations (POP-1 and POP-2) segregating for SBR resistance. F₂-derived F₃ (F_2:3) families from each population were grown in a naturally SBR-infected field in Paraguay to determine inheritance and map resistance genes. Over 6,000 plants from 178 families in POP-1 and over 5,000 plants from 160 families in POP-2 were evaluated at R5 for lesion type: immune reaction (IR), reddish-brown (RB), or tan (TAN) colored lesions. Based on the lesion type present, each F_2:3 family was rated as resistant, segregating or susceptible and this classification was used to infer the F₂-phenotype and genotype. For both populations, the F₂ segregation ratios fit a 1:2:1 (resistant:segregating:susceptible) ratio expected for a single gene (P > 0.05). The RB lesions occurred almost exclusively in the heterozygous class, indicating incomplete dominance under the conditions of this study. Molecular markers flanking the locations of the known resistance genes were used to map the resistance gene in both populations to the Rpp1 locus. However, evaluation of PI 587886 and PI 587880A against eight P. pachyrhizi isolates indicated that the resistance allele in these two accessions was different from Rpp1. This test also demonstrated that these accessions were resistant to at least one P. pachyrhizi isolate collected in the southern US. This is the first report of using an adult plant field-screen with natural rust pressure to map SBR resistance.     

Genetic mapping revealed two loci for soybean aphid resistance in PI 567301B

The soybean aphid (Aphis glycines Matsumura) is the most damaging insect pest of soybean [Glycine max (L.) Merr.] in North America. New soybean aphid biotypes have been evolving quickly and at least three confirmed biotypes have been reported in USA. These biotypes are capable of defeating most known aphid resistant soybean genes indicating the need for identification of new genes. Plant Introduction (PI) 567301B was earlier identified to have antixenosis resistance against biotype 1 and 2 of the soybean aphid. Two hundred and three F_7:9 recombinant inbred lines (RILs) developed from a cross of soybean aphid susceptible cultivar Wyandot and resistant PI 567301B were used for mapping aphid resistance genes using the quantitative trait loci (QTL) mapping approach. A subset of 94 RILs and 516 polymorphic SNP makers were used to construct a genome-wide molecular linkage map. Two candidate QTL regions for aphid resistance were identified on this linkage map. Fine mapping of the QTL regions was conducted with SSR markers using all 203 RILs. A major gene on chromosome 13 was mapped near the previously identified Rag2 gene. However, an earlier study revealed that the detached leaves of PI 567301B had no resistance against the soybean aphids while the detached leaves of PI 243540 (source of Rag2) maintained aphid resistance. These results and the earlier finding that PI 243540 showed antibiosis resistance and PI 567301B showed antixenosis type resistance, indicating that the aphid resistances in the two PIs are not controlled by the same gene. Thus, we have mapped a new gene near the Rag2 locus for soybean aphid resistance that should be useful in breeding for new aphid-resistant soybean cultivars. Molecular markers closely linked to this gene are available for marker-assisted breeding. Also, the minor locus found on chromosome 8 represents the first reported soybean aphid-resistant locus on this chromosome.     

Genetic linkage mapping of the soybean aphid resistance gene in PI 243540

The soybean aphid (Aphis glycines Matsumura) is a pest of soybean [Glycine max (L.) Merr.] in many soybean growing countries of the world, mainly in Asia and North America. A single dominant gene in PI 243540 confers resistance to the soybean aphid. The objectives of this study were to identify simple sequence repeat (SSR) markers closely linked to the gene in PI 243540 and to position the gene on the consensus soybean genetic map. One hundred eighty-four F(2) plants and their F(2:3) families from a cross between the susceptible cultivar Wyandot and PI 243540, and the two parental lines were screened with the Ohio biotype of soybean aphid using greenhouse choice tests. A SSR marker from each 10-cM section of the consensus soybean map was selected for bulked segregant analysis (BSA) to identify the tentative genomic location of the gene. The BSA technique was useful to localize the gene to a genomic region in soybean linkage group (LG) F. The entire F(2) population was then screened with polymorphic SSR markers from this genomic region and a linkage map with nine SSR markers flanking the gene was constructed. The aphid resistance gene was positioned in the interval between SSR markers Satt334 and Sct_033 on LG F. These SSR markers will be useful for marker assisted selection of this gene. The aphid resistance gene from PI 243540 mapped to a different linkage group than the only named soybean aphid resistance gene, Rag1, from 'Dowling'. Also, the responses of the two known biotypes of the soybean aphid to the gene from PI 243540 and Rag1 were different. Thus, the aphid resistance gene from PI 243540 was determined to be a new and independent gene that has been named Rag2.     

Identification and molecular mapping of two soybean aphid resistance genes in soybean PI 587732

Soybean [Glycine max (L.) Merr.] continues to be plagued by the soybean aphid (Aphis glycines Matsumura: SA) in North America. New soybean resistance sources are needed to combat the four identified SA biotypes. The objectives of this study were to determine the inheritance of SA resistance in PI 587732 and to map resistance gene(s). For this study, 323 F₂ and 214 F₃ plants developed from crossing PI 587732 to two susceptible genotypes were challenged with three SA biotypes and evaluated with genetic markers. Choice tests showed that resistance to SA Biotype 1 in the first F₂ population was controlled by a gene in the Rag1 region on chromosome 7, while resistance to SA Biotype 2 in the second population was controlled by a gene in the Rag2 region on chromosome 13. When 134 F₃ plants segregating in both the Rag1 and Rag2 regions were tested with a 1:1 mixture of SA Biotypes 1 and 2, the Rag2 region and an interaction between the Rag1 and Rag2 regions were significantly associated with the resistance. Based on the results of the non-choice tests, the resistance gene in the Rag1 region in PI 587732 may be a different allele or gene from Rag1 from Dowling because the PI 587732 gene showed antibiosis type resistance to SA Biotype 2 while Rag1 from Dowling did not. The two SA resistance loci and genetic marker information from this study will be useful in increasing diversity of SA resistance sources and marker-assisted selection for soybean breeding programs.     

A novel locus for soybean aphid resistance

The soybean aphid (Aphis glycines Matsumura) is an important pest on soybean [Glycine max (L.) Merr.] in North America. Aphid resistance has recently been found on plant introduction (PI) 567543C, but little is known about its genetic control. The objectives of this study were to identify the resistance genes in PI 567543C with molecular markers and validate them in a different genetic background. A mapping population of 249 F₄ derived lines from a cross between PI 567543C and a susceptible parent was investigated for aphid resistance in both the greenhouse and the field. The broad sense heritability of aphid resistance in the field trial was over 0.95. The segregation of aphid resistance in this population suggests a major gene controlling the resistance. Bulked segregant analysis with molecular markers revealed a potential genomic region. After saturating this putative region with more markers, a genetic locus was mapped in an interval between Sat_339 and Satt414 on chromosome 16 (linkage group J) using the composite interval mapping method. This locus explained the majority of the phenotypic variation ranging from 84.7% in the field trial to 90.4% in the greenhouse trial. Therefore, the aphid resistance in PI 567543C could be mainly controlled by this gene. This aphid resistance gene was mapped on a different chromosome than the other resistance genes reported previously from other resistant germplasms. This gene appears to be additive based on the aphid resistance of the heterozygous lines at this locus. Thus, a new symbol Rag3 is used to designate this gene. Moreover, Rag3 was confirmed in a validation population. This new aphid-resistance gene could be valuable in breeding aphid resistant cultivars.     

Mapping and validation of a gene for soybean aphid resistance in PI 567537

The soybean aphid (Aphis glycines Matsumura) is a major pest on soybean [Glycine max (L.) Merr.] in North America. Aphid resistance has been found on plant introduction (PI) 567537, but its genetic characterization is unknown. The objectives of this study were to identify the resistance genes in PI 567537 using molecular markers and validate them in a different genetic background. A mapping population of 86 F4 lines from a cross between PI 567537 and a susceptible parent E00003 was investigated for aphid resistance in both greenhouse and field trials. A genomic region associated with the aphid resistance in PI 567537 was revealed on chromosome 16 (linkage group J) with molecular markers. This locus was coincidently located in the same region as Rag3 and explained most of the phenotypic variation, ranging from 87.4 % in the greenhouse trial to 78.9 % in the field trial. This resistance gene was further confirmed in an F2 population derived from a cross of PI 567537 × Skylla. The segregation of the F2 population indicated that the aphid resistance in PI 567537 was most likely controlled by a single dominant gene, which was the one we mapped in the F4-derived population. This gene was designated Rag3b since it is located in the same region as Rag3. The mapping of the aphid resistance gene in PI 567537 could be useful in marker-assisted selection when employing PI 567537 as an aphid resistance source.     

Genetic mapping of the powdery mildew resistance gene in soybean PI 567301B

Powdery mildew (PMD) of soybean [Glycine max (L.) Merr.] is caused by the fungus Microsphaera diffusa. Severe infection of PMD on susceptible varieties often causes premature defoliation and chlorosis of the leaves, which can result in considerable yield losses under favorable environmental conditions for disease development in the field. A total of 334 F(7)-derived recombinant inbred lines (RILs) from a cross of a PMD susceptible soybean cultivar Wyandot and PMD-resistant PI 567301B were used for genetic mapping of PMD resistance in PI 567301B and for development of molecular markers tightly linked to the gene. The result of the PMD screening for each line in the field was in agreement with that in the greenhouse test. The genetic map containing the PMD resistance gene was constructed in a 3.3?cM interval flanked by two simple sequence repeat (SSR) markers on chromosome 16. The PMD resistance gene was mapped at the same location with SSR marker BARCSOYSSR_16_1291, indicating that there was no recombination between the 334 RILs and this marker. In addition, a single nucleotide polymorphism (SNP) marker developed by high-resolution melting curve analysis and a cleaved amplified polymorphic sequence (CAPS) marker with Rsa1 recognition site were used for the genetic mapping. These two markers were also mapped to the same genomic location with the PMD resistance gene. We validated three tightly linked markers to the PMD resistance gene using 38 BC(6)F(2) lines and corresponding BC(6)F(2:3) families. The three marker genotypes of the backcross lines predicted the observed PMD phenotypes of the lines with complete accuracy. We have mapped a putatively novel single dominant PMD resistance gene in PI 567301B and developed three new molecular markers closely linked to the gene. Molecular markers developed from this study may be used for high-throughput marker-assisted breeding for PMD resistance with the gene from PI 567301B.     

Fine Mapping and Characterization of Candidate Genes that Control Resistance to Cercospora sojina K. Hara in Two Soybean Germplasm Accessions

Frogeye leaf spot (FLS), caused by the fungus Cercospora sojina K. Hara, may cause a significant yield loss to soybean growers in regions with a warm and humid climate. Two soybean accessions, PI 594891 and PI 594774, were identified to carry a high level of resistance similar to that conditioned by the Rcs3 gene in ''Davis''. Previously, we reported that the resistance to FLS in these two plant introductions (PIs) was controlled by a novel gene (s) on chromosome 13 that is different from Rcs3. To fine-map the novel FLS resistance gene(s) in these two PIs, F_{2: 3} seeds from the crosses between PI 594891 and PI 594774, and the FLS susceptible genotype ''Blackhawk'' were genotyped with SNP markers that were designed based on the SoySNP50k iSelect BeadChip data to identify recombinant events and locate candidate genes. Analysis of lines possessing key recombination events helped narrow down the FLS-resistance genomic region in PI 594891 from 3.3 Mb to a 72.6 kb region with five annotated genes. The resistance gene in PI 594774 was fine-mapped into a 540 kb region that encompasses the 72.6 kb region found in PI 594891. Sequencing five candidate genes in PI 594891 identified three genes that have several mutations in the promoter, intron, 5'', and 3'' UTR regions. qPCR analysis showed a difference in expression levels of these genes in both lines compared to Blackhawk in the presence of C. sojina. Based on phenotype, genotype and haplotype analysis results, these two soybean accessions might carry different resistance alleles of the same gene or two different gene(s). The identified SNPs were used to develop Kompetitive Allele Specific PCR (KASP) assays to detect the resistance alleles on chromosome 13 from the two PIs for marker-assisted selection.     

Detection and fine-mapping of SC7 resistance genes via linkage and association analysis in soybean

Soybean mosaic virus (SMV) disease is one of the most serious and broadly distributed soybean (Glycine max (L.) Merr.) diseases. Here, we combine the advantages of association and linkage analysis to i...