Molecular mapping of soybean rust resistance in soybean accession PI 561356 and SNP haplotype analysis of the Rpp1 region in diverse germplasm

Soybean rust (SBR), caused by Phakopsora pachyrhizi Sydow, is one of the most economically important and destructive diseases of soybean [Glycine max (L.) Merr.] and the discovery of novel SBR resistance genes is needed because of virulence diversity in the pathogen. The objectives of this research were to map SBR resistance in plant introduction (PI) 561356 and to identify single nucleotide polymorphism (SNP) haplotypes within the region on soybean chromosome 18 where the SBR resistance gene Rpp1 maps. One-hundred F(2:3) lines derived from a cross between PI 561356 and the susceptible experimental line LD02-4485 were genotyped with genetic markers and phenotyped for resistance to P. pachyrhizi isolate ZM01-1. The segregation ratio of reddish brown versus tan lesion type in the population supported that resistance was controlled by a single dominant gene. The gene was mapped to a 1-cM region on soybean chromosome 18 corresponding to the same interval as Rpp1. A haplotype analysis of diverse germplasm across a 213-kb interval that included Rpp1 revealed 21 distinct haplotypes of which 4 were present among 5 SBR resistance sources that have a resistance gene in the Rpp1 region. Four major North American soybean ancestors belong to the same SNP haplotype as PI 561356 and seven belong to the same haplotype as PI 594538A, the Rpp1-b source. There were no North American soybean ancestors belonging to the SNP haplotypes found in PI 200492, the source of Rpp1, or PI 587886 and PI 587880A, additional sources with SBR resistance mapping to the Rpp1 region.     

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.     

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.     

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.     

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.     

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.     

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.     

Pooled analysis of data from multiple quantitative trait locus mapping populations

Quantitative trait locus (QTL) analysis on pooled data from multiple populations (pooled analysis) provides a means for evaluating, as a whole, evidence for existence of a QTL from different studies and examining differences in gene effect of a QTL among different populations. Objectives of this study were to: (1) develop a method for pooled analysis and (2) conduct pooled analysis on data from two soybean mapping populations. Least square interval mapping was extended for pooled analysis by inclusion of populations and cofactor markers as indicator variables and covariate variables separately in the multiple linear models. The general linear test approach was applied for detecting a QTL. Single population-based and pooled analyses were conducted on data from two F_2:3 mapping populations, Hamilton (susceptible) × PI 90763 (resistant) and Magellan (susceptible) × PI 404198A (resistant), for resistance to soybean cyst nematode (SCN) in soybean. It was demonstrated that where a QTL was shared among populations, pooled analysis showed increased LOD values on the QTL candidate region over single population analyses. Where a QTL was not shared among populations, however, the pooled analysis showed decreased LOD values on the QTL candidate region over single population analyses. Pooled analysis on data from genetically similar populations may have higher power of QTL detection than single population-based analyses. QTLs were identified by pooled analysis on linkage groups (LGs) G, B1 and J for resistance to SCN race 2 whereas QTLs on LGs G, B1 and E for resistance to SCN race 5 in soybean PI 90763 and PI 404198A. QTLs on LG G and B1 were identified in both PI 90763 and PI 404198A whereas QTLs on LG E and J were identified in PI 90763 only. QTLs on LGs G and B1 for resistance to race 2 may be the same or closely linked with QTLs on LG G and B1 for resistance to race 5, respectively. It was further demonstrated that QTLs on G and B1 carried by PI 90763 were not significantly different in gene effect from QTLs on LGs G and B1 in PI 404198A, respectively.     

Generation-means analysis and quantitative trait locus mapping of anthracnose stalk rot genes in maize

A generation-means analysis was performed on two maize populations, each segregating for genes conferring resistance to anthracnose stalk rot (ASR). The populations were derived from a cross of DE811ASR x DE811 and of DE811ASR x LH132. The resistant parent, DE811ASR, was obtained through introgression with MP305 as the donor and DE811 as the recurrent parent. The analysis revealed significant additive effects in both populations and a significant additive x dominant effect in the DES11ASR x DES11 population. Quantitative trait locus (QTL) mapping, using restriction fragment length polymorphism (RFLP)-based molecular markers, indicated a significant QTL on linkage group 4 in both populations. The QTL analysis confirmed additive inheritance in both populations. This work demonstrates a close correspondence between generation-means analysis and discrete observations using molecular markers. Linkage of a genetic marker to genes conferring resistance to ASR will be useful for the introgression of resistance into elite germplasm.This research was part of a thesis submitted by the first author in partial fulfillment of the requirements for a MS degree. Published as Miscellaneous Paper number 1491 of the Delaware Agricultural Experiment Station     

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.     

Mapping tightly linked genes controlling potyvirus infection at the Rsv1 and Rpv1 region in soybean.

Soybean mosaic virus (SMV) and peanut mottle virus (PMV) are two potyviruses that cause yield losses and reduce seed quality in infested soybean (Glycine max (L.) Merr.) fields throughout the world. Rsv1 and Rpv1 are genes that provide soybean with resistance to SMV and PMV, respectively. Isolating and characterizing Rsv1 and Rpv1 are instrumental in providing insight into the molecular mechanism of potyvirus recognition in soybean. A population of 1056 F2 individuals from a cross between SMV- and PMV-resistant line PI 96983 (Rsv1 and Rpv1) and the susceptible cultivar 'Lee 68' (rsv1 and rpv1) was used in this study. Disease reaction and molecular-marker data were collected to determine the linkage relationship between Rsv1, Rpv1, and markers that target candidate disease-resistance genes. F2 lines showing a recombination between two of three Rsv1-flanking microsatellite markers were selected for fine mapping. Over 20 RFLP, RAPD, and microsatellite markers were used to map 38 loci at high-resolution to a 6.8-cM region around Rsv1 and Rpv1. This study demonstrates that Rsv1 and Rpv1 are tightly linked at a distance of 1.1 cM. In addition, resistance-gene candidate sequences were mapped to positions flanking and cosegregating with these resistance loci. Based on comparisons of genetic markers and disease reactions, it appears likely that several tightly linked genes are conditioning a resistance response to SMV. We discuss the specifics of these findings and investigate the utility of two disease resistance related probes for the screening of SMV or PMV resistance in soybean.     

Genetic Analysis of Parasitism in the Soybean Cyst Nematode Heterodera Glycines

A genetic analysis of parasitic ability in the soybean cyst nematode Heterodera glycines was performed. To identify and characterize genes involved in parasitism, we developed three highly inbred H. glycines lines, OP20, OP25 and OP50, for use as parents for controlled crosses. Through these crosses, we have identified genes in the inbred parents that control reproduction of the nematode on hosts that carry resistance genes. These genes, designated as ror-* for reproduction on a resistant host, segregate in a normal Mendelian fashion as independent loci. Host range tests of F(1) generation progeny indicated that at least one parasitism gene in both the OP20 and OP50 lines for host PI 88788 was dominant. Parasitism genes in OP50 for hosts ``Peking' and PI 90763 are recessive. Two types of single female descent populations, a single backcrossed BC(1)F(2)-derived and a double backcrossed BC(2)F(1)-derived, were established on the susceptible soybean cultivar ``Lee 68.' Host range tests for parasitism in these lines demonstrated the presence of two independent genes in OP50, one for host PI 88788 designated ror-1 and one for host PI 90763 designated ror-2. OP20 carries two independent genes for parasitism on PI 88788, designated as alleles kr3 and kr4.     

Identification of quantitative trait loci associated with powdery mildew resistance in mungbean using ISSR and ISSR-RGA markers

To identify the powdery mildew (PM) resistance gene in mungbean, inter-simple sequence repeat (ISSR) markers and newly developed ISSR-anchored resistance gene analog (ISSR-RGA) markers were evaluated. When F_2:7 and F_2:8 recombinant inbred line populations derived from a cross between CN72 (susceptible cultivar in Thailand) and V4718 (resistant line from Asian Vegetable Research and Development Center) were evaluated for PM resistance under field conditions, the PM resistance gene from V4718 was found to be inherited as a single major gene. Fifteen out of 75 ISSR primers produced 27 DNA bands putatively associated with PM resistance in bulk segregant analysis (BSA). Ten ISSR primers were combined with four RGA primers homologous to the nucleotide-binding site and kinase domains of resistance (R) genes to generate 40 ISSR-RGA primer combinations. When these 40 ISSR-RGA primer combinations and 10 corresponding ISSR primers were used in BSA, 873 ISSR and 756 ISSR-RGA loci were amplified. Fifty-two of 756 ISSR-RGA loci were new, and 11 of these 23 ISSR-RGA loci were putatively associated with the PM resistance. Simple linear regression confirmed that 5 of the 27 ISSR markers and 3 of the 11 ISSR-RGA markers were significantly associated with the PM resistance gene. When these eight ISSR and ISSR-RGA markers were used for quantitative trait loci (QTL) analysis, multiple interval mapping identified a major QTL, qPMC72V18-1, explaining up to 92.4% of the phenotypic variation, flanked by I42PL229 and I85420 markers at the distance of 4 and 9 cM, respectively. These results suggest that ISSR and ISSR-RGA markers are highly efficient tools for mapping PM resistance gene in mungbean. The markers closely linked to the PM resistance gene will be useful for future marker-assisted selection to develop mungbean varieties resistant to PM.     

Molecular characterization of resistance to Heterodera glycines in soybean PI 438489B

Soybean (Glycine max L. Merr.) plant introduction (PI) 438489B is a newly found germplasm source that has resistance to multiple soybean cyst nematode (Heterodera glycines Ichinohe, SCN) races. We studied the inheritance of resistance to SCN races 1, 2, 3, 5 and 14 in PI 438489B using F₂ and F_2:3 families, which were generated by crossing to the susceptible cultivar ’Hamilton.’ The objectives of this study were to investigate the inheritance for resistance to SCN races in PI 438489B, to find molecular markers associated with resistances, and to study the allelic relationships among resistance loci for different SCN races. The results showed that the responses to SCN races were approximately normally distributed with large environmental effects, and were also highly correlated, which implied that genes giving resistance to different races were similar. The narrow-sense heritabilities of resistance to all five SCN races ranged from 0.55 to 0.88. Fifty one restriction fragment length polymorphism (RFLP) markers and 64 simple sequence repeat (SSR) markers were found to be polymorphic in the F₂ population. Quantitative trait loci (QTLs) associated with resistance to SCN races were anchored on soybean linkage groups (LGs) A1, A2, B1, B2, C1, C2, D1a, E and G. These QTLs explained 47.3%, 45.8%, 51.5%, 34.5% and 37.2% of the total phenotypic variances, respectively, for each race we investigated. Some QTLs for different races encompassed the same region of flanking markers; therefore, QTLs for multiple races may be linked or pleiotropic effects may be involved. Some loci provided resistance in a race-specific manner. Resistance to SCN race 14 had a different pattern compared to other races. Our results indicated that resistance to race 14 did not include loci on LGs A2 and G. These flanking markers associated with QTLs could be used to select for resistance to multiple SCN races in soybean breeding programs. Received: 25 March 2000 / Accepted: 4 August 2000     

Preliminary investigation of QTLs associated with seedling resistance to ascochyta blight from <Emphasis Type="Italic">Cicer echinospermum</Emphasis>, a wild relative of chickpea

Accessions from Cicer echinospermum, a wild relative of chickpea (Cicer arietinum L.), contain resistance to the fungal disease ascochyta blight, a devastating disease of chickpea. A linkage map was constructed based on an interspecific F(2) population, derived from a cross between a susceptible chickpea cultivar (Lasseter) and a resistant C. echinospermum accession (PI 527930). The linkage map incorporated 83 molecular markers, that included RAPD, ISSR, STMS and RGA markers; eight markers remained unlinked. The map comprised eight linkage groups and covered a map distance of 570 cM. Six out of the eight linkage groups were correlated to linkage groups from the integrated Cicer map using STMS markers. Quantitative trait loci (QTLs) associated with ascochyta blight resistance were detected using interval mapping and single-point analysis. The F(2) population was evaluated for seedling and stem resistance in glasshouse trials. At least two QTLs were identified for seedling resistance, both of which were located within linkage group 4. Five markers were associated with stem resistance, four of which were also associated with seedling resistance. QTLs from previous studies also mapped to LG 4, suggesting that this linkage group is an important region of the Cicer genome for resistance to ascochyta blight.