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.     

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.     

Cell-free Translation of Potyviral RNA Including Extracts from Disease Resistant Soybean

Cell-free translation of soybean mosaic virus (SMV), strain G2 or G7, RNAs in the presence of fractionated leaf extracts from the cultivars Davis or Williams‘82 revealed no differences that were associated with disease resistance of the cultivars to the virus strains. Extracts of leaf tissue from Davis (resistant to strain G2) and susceptible Williams‘82 were fractionated into three broad peaks. Apparent proteinase activity was detected in peak II of both soybean cultivars in the cell-free translation of SMV-G2 or -G7 RNAs. Inhibition of translation occurred in the presence of peak extracts from either of the soybean cultivars at levels of 500 μ/ml and/or 1000 μ/ml of protein. No inhibition of proteolytic processing was observed by extracts from either cultivar.     

Resistance Genes in a 'Williams 82' x 'Hartwig' Soybean Cross to an Inbred Line of Heterodera glycines

The number of resistance genes in soybean to soybean cyst nematode (SCN) Heterodera glycines was estimated using progeny from a cross of ''Williams 82'' x ''Hartwig'' (derived from ''Forrest''³ x PI 437.654) screened with a fourth-generation inbred nematode line derived from a race 3 field population of SCN. Numbers of females developing on roots of inoculated seedlings were assigned to phenotype cells (resistant, susceptible, or segregating) using Ward''s minimum variance cluster analysis. The ratio obtained from screening 220 F₃ soybean families was not significantly different from a 1:8:7 (resistant:segregating:susceptible) ratio, suggesting a two-gene system for resistance. The ratio obtained from screening 183 F₂ plants was not significantly different from a 3:13 (resistant:susceptible) ratio, indicating both a dominant (Rhg) and a recessive (rhg) resistance gene.     

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 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.     

Peroxidase Activity in Soybeans Following Inoculation with Phytophthora sojae

The effects of race-specific resistance as conditioned by Rps genes (rps, Rps1-k, Rps2, Rps3, Rps6) in two genetic backgrounds (Williams & Harosoy) on accumulation of soluble peroxidases were determined by a soybean peroxidase capture assay (SPCA) after inoculation with P. sojae races 2, 7, or 25. Peroxidase activity increased in all isolines during the 72 h after inoculation, but reactions varied depending on time after inoculation, genetic background, Rps gene and P. sojae race. Peroxidase activity was higher in race-specific resistant than in susceptible reactions at 72 h. after inoculation, except for plants with the Rps2 gene which confers a unique form of root resistance in addition to the whole plant race-specific resistance. Williams isolines had larger increases in peroxidase activity than Harosoy isolines when data were averaged across Rps genes, and was most evident when plants were inoculated with race 2. When soybeans were inoculated with race 7 Rps1-k resistant plants had the highest increase in peroxidase activity, but Rps2 susceptible plants had a significantly higher peroxidase activity than plants with rps, Rps3, and Rps6 that were also susceptible. Results from inoculations with race 25 were somewhat different, Rps2 resistant plants had the highest increase in peroxidase activity; however, plants with the Rps3 or Rps6 gene that were also resistant did not have a significantly higher peroxidase activity than susceptible plants with the rps or Rps1-k 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.     

Identification and Candidate Gene Analysis of a Novel Phytophthora Resistance Gene Rps10 in a Chinese Soybean Cultivar

Resistance to Phytophthora sojae isolate PsMC1 was evaluated in 102 F_2∶3 families derived from a cross between the resistant soybean cultivar Wandou 15 and the susceptible cultivar Williams and genotyped using simple sequence repeat (SSR) markers. The segregation ratio of resistant, segregating, and susceptible phenotypes in the population suggested that the resistance in Wandou 15 was dominant and monogenic. Twenty-six polymorphic SSR markers were identified on soybean chromosome 17 (Molecular linkage group D2; MLG D2), which were linked to the resistance gene based on bulked segregation analysis (BSA). Markers Sattwd15-24/25 and Sattwd15-47 flanked the resistance gene at a distance of 0.5 cM and 0.8 cM, respectively. Two cosegregating markers, Sattwd15-28 and Sattwd15-32, were also screened in this region. This is the first Rps resistance gene mapped on chromosome 17, which is designated as Rps10. Eight putative genes were found in the mapped region between markers Sattwd15-24/25 and Sattwd15-47. Among them, two candidate genes encoding serine/threonine (Ser/Thr) protein kinases in Wandou 15 and Williams were identified and sequenced. And the differences in genomic sequence and the putative amino acid sequence, respectively, were identified within each candidate gene between Wandou 15 and Williams. This novel gene Rps10 and the linked markers should be useful in developing soybean cultivars with durable resistance to P. sojae.     

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.     

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.