Soybean phytophthora resistance gene Rps8 maps closely to the Rps3 region

Root and stem rot is one of the major diseases of soybean. It is caused by the oomycete pathogen Phytophthora sojae. A series of resistance genes (Rps) have been providing soybean with reasonable protection against this pathogen. Among these genes, Rps8, which confers resistance to most P. sojae isolates, recently has been mapped. However, the most closely linked molecular marker was mapped at about 10 cM from Rps8. In this investigation, we attempted to develop a high-density genetic map of the Rps8 region and identify closely linked SSR markers for marker-assisted selection of this invaluable gene. Bulk segregant analysis was conducted for the identification of SSR markers that are tightly linked to Rps8. Polymorphic SSR markers selected from the Rps8 region failed to show cosegregation with Phytophthora resistance. Subsequently, bulk segregant analysis of the whole soybean genome and mapping experiments revealed that the Rps8 gene maps closely to the disease resistance gene-rich Rps3 region.     

The Avr1b locus of Phytophthora sojae encodes an elicitor and a regulator required for avirulence on soybean plants carrying resistance gene Rps1b

We have used map-based approaches to clone a locus containing two genes, Avr1b-1 and Avr1b-2, required for avirulence of the oomycete pathogen Phytophthora sojae (Kaufmann & Gerdemann) on soybean plants carrying resistance gene Rps1b. Avr1b-1 was localized to a single 60-kb bacterial artificial chromosome (BAC) clone by fine-structure genetic mapping. Avr1b-1 was localized within the 60-kb region by identification of an mRNA that is expressed in a race-specific and infection-specific manner and that encodes a small secreted protein. When the Avr1b-1 protein was synthesized in the yeast Pichia pastoris and the secreted protein infiltrated into soybean leaves, it triggered a hypersensitive response specifically in host plants carrying the Rps1b resistance gene. This response eventually spread to the entire inoculated plant. In some isolates of P. sojae virulent on Rps1b-containing cultivars, such as P7081 (race 25) and P7076 (race 19), the Avr1b-1 gene had numerous substitution mutations indicative of strong divergent selection. In other isolates, such as P6497 (race 2) and P9073 (race 25), there were no substitutions in Avr1b-1, but Avr1b-1 mRNA did not accumulate. Genetic complementation experiments with P6497 revealed the presence of a second gene, Avr1b-2, required for the accumulation of Avr1b-1 mRNA. Avr1b-2 was genetically mapped to the same BAC contig as Avr1b-1, using a cross between P7064 (race 7) and P6497. The Avr1k gene, required for avirulence on soybean cultivars containing Rps1k, was mapped to the same interval as Avr1b-1.     

Genetic characterization and fine mapping of the novel Phytophthora resistance gene in a Chinese soybean cultivar

Phytophthora root rot (PRR), caused by Phytophthora sojae Kaufmann & Gerdemann, is one of the most destructive diseases of soybean [Glycine max (L.) Merr.]. Deployment of resistance genes is the most economical and effective way of controlling the disease. The soybean cultivar ‘Yudou 29’ is resistant to many P. sojae isolates in China. The genetic basis of the resistance in ‘Yudou 29’ was elucidated through an inheritance study and molecular mapping. In response to 25 P. sojae isolates, ‘Yudou 29’ displayed a new resistance reaction pattern distinct from those of differentials carrying known Rps genes. A population of 214 F_2:3 families from a cross between ‘Jikedou 2’ (PRR susceptible) and ‘Yudou 29’ was used for Rps gene mapping. The segregation fit a ratio of 1:2:1 for resistance:segregation:susceptibility within this population, indicating that resistance in ‘Yudou 29’ is controlled by a single dominant gene. This gene was temporarily named RpsYD29 and mapped on soybean chromosome 03 (molecular linkage group N; MLG N) flanked by SSR markers SattWM82-50 and Satt1k4b at a genetic distance of 0.5 and 0.2 cM, respectively. Two nucleotide binding site-leucine rich repeat (NBS-LRR) type genes were detected in the 204.8 kb region between SattWM82-50 and Satt1k4b. These two genes showed high similarity to Rps1k in amino acid sequence and could be candidate genes for PRR resistance. Based on the phenotype reactions and the physical position on soybean chromosome 03, RpsYD29 might be a novel allele at, or a novel gene tightly linked to, the Rps1 locus.     

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.     

Molecular mapping of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI 567139B

Phytophthora root and stem rot (PRR), caused by the soil-borne oomycete pathogen Phytophthora sojae, is one of the most destructive diseases of soybean. PRR can be effectively controlled by race-specific genes conferring resistance to P. sojae (Rps). However, the Rps genes are usually non-durable, as populations of P. sojae are highly diverse and quick to adapt, and can be overcome 8–15 years after deployment. Thus, it is important to identify novel Rps genes for development of resistant soybean cultivars. PI 567139B is a soybean landrace carrying excellent resistance to nearly all predominant P. sojae races in Indiana. A mapping population consisting of 245 F₂ individuals and 403 F_2:3 families was developed from a cross between PI 567139B and the susceptible cultivar ‘Williams’, and used to dissect the resistance carried by PI 567139B. We found that the resistance in PI 567139B was conferred by two independent Rps genes, designated RpsUN1 and RpsUN2. The former was mapped to a 6.5 cM region between SSR markers Satt159 and BARCSOYSSR_03_0250 that spans the Rps1 locus on chromosome 3, while the latter was mapped to a 3.0 cM region between BARCSOYSSR_16_1275 and Sat_144, approximately 3.0–3.4 cM upstream of Rps2 on chromosome 16. According to the ‘Williams 82’ reference genome sequence, both regions are highly enriched with NBS-LRR genes. Marker assisted resistance spectrum analyses of these genes with 16 isolates of P. sojae, in combination with the mapping results, suggested that RpsUN1 was likely to be a novel allele at the Rps1 locus, while RpsUN2 was more likely to be a novel Rps gene.     

Targeted resistance gene mapping in soybean using modified AFLPs

The soybean [Glycine max (Merr.) L.] linkage group F contains a vital region of clustered genes for resistance to numerous pathogens including the soybean mosaic virus resistance gene, Rsv1. In order to develop new genetic markers that map to this gene cluster, we employed a targeted approach that utilizes the speed and high-throughput of AFLP, but modified it to incorporate sequence information from the highly conserved nucleotide binding site (NBS) region of cloned disease resistance genes. By using a labeled degenerate primer corresponding to the p-loop portion of the NBS region of resistance genes, such as N, L6, and Rps2, we were able to quickly amplify numerous polymorphic bands between parents of a population segregating for resistance to Rsv1. Of these polymorphic bands, bulk segregant analysis revealed four markers that were closely linked to Rsv1. These markers were cloned and used as probes for RFLP analysis. The four clones mapped to within a 6-cM region surrounding Rsv1, the closest being 0.4 cM away from the gene. Sequence analysis showed that all four clones contain the p-loop sequence corresponding to the degenerate primer and that one of the four clones contains an open reading frame sequence which when translated is related to the NBS region of other cloned disease resistance genes. The rapid identification of four markers closely linked to Rsv1 in soybean demonstrates the utility of this method for generating markers tightly linked to important plant disease resistance genes. Received: 25 September 1999 / Accepted: 3 November 1999     

Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene

 A soybean bacterial artificial chromosome (BAC) library, comprising approximately 45 000 clones, was constructed from high-molecular-weight nuclear DNA of cultivar Williams 82, which carries the Rps1-k gene for resistance against Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random sequences including four markers linked to the Rps1 locus and 16 cDNAs. We identified pools containing BACs for all sequences except for one cDNA. Additionally, when screened for possible contaminating BAC clones carrying chloroplast genes, no sequences homologous to two barley chloroplast genes were found. The estimated average insert size of the BAC clones was about 105 kb. The library comprises about four genome equivalents of soybean DNA. Therefore, this gives a probability of 0.98 of finding a specific sequence from this library. This library should be a useful resource for the positional cloning of Rps1-k, and other soybean genes. We have also evaluated the feasibility of an RFLP-based screening procedure for the isolation of BAC clones specific for markers that are members of repetitive sequence families, and are linked to the Rps1-k gene. We show that BAC clones isolated for two genetically linked marker loci, Tgmr and TC1-2, are physically linked. Application of this method in expediting the map-based cloning of a gene, especially from an organism, such as soybean, maize and wheat, with a complex genome is discussed. Received: 12 May 1998/Accepted: 24 August 1998     

Identification of a large cluster of coiled coil-nucleotide binding site–leucine rich repeat-type genes from the Rps1 region containing Phytophthora resistance genes in soybean

Fifteen Rps genes confer resistance against the oomycete pathogen Phytophthora sojae, which causes root and stem rot disease in soybean. We have isolated a disease resistance gene-like sequence from the genomic region containing Rps1-k. Four classes of cDNA of the sequence were isolated from etiolated hypocotyl tissues that express the Rps1-k-encoded Phytophthora resistance. Sequence analyses of a cDNA clone showed that the sequence is a member of the coiled coil-nucleotide binding site–leucine rich repeat (CC-NBS–LRR)-type of disease resistance genes. It showed 36% identity to the recently cloned soybean resistance gene Rpg1-b, which confers resistance against Pseudomonas syringae pv. glycinea, and 56% and 38% sequence identity to putative resistance gene sequences from lotus and Medicago truncatula, respectively. The soybean genome contains about 38 copies of the sequence. Most of these copies are clustered in approximately 600 kb of contiguous DNA of the Rps1-k region. We have identified a recombinant that carries both rps1-k- and Rps1-k-haplotype-specific allelomorphs of two Rps1-k-linked molecular markers. An unequal crossover event presumably led to duplication of alleles for these two physically linked molecular markers. We hypothesize that the unequal crossing over was one of the mechanisms involved in tandem duplication of CC-NBS–LRR sequences in the Rps1-k region.N.N. Narayanan, H. Gao, D.K. Santra, and S.S. Salimath contributed equally to this work.     

Isolation of ethylene-insensitive soybean mutants that are altered in pathogen susceptibility and gene-for-gene disease resistance

Plants commonly respond to pathogen infection by increasing ethylene production, but it is not clear if this ethylene does more to promote disease susceptibility or disease resistance. Ethylene production and/or responsiveness can be altered by genetic manipulation. The present study used mutagenesis to identify soybean (Glycine max L. Merr.) lines with reduced sensitivity to ethylene. Two new genetic loci were identified, Etr1 and Etr2. Mutants were compared with isogenic wild-type parents for their response to different soybean pathogens. Plant lines with reduced ethylene sensitivity developed similar or less-severe disease symptoms in response to virulent Pseudomonas syringae pv glycinea and Phytophthora sojae, but some of the mutants developed similar or more-severe symptoms in response to Septoria glycines and Rhizoctonia solani. Gene-for-gene resistance against P. syringae expressing avrRpt2 remained effective, but Rps1-k-mediated resistance against P. sojae races 4 and 7 was disrupted in the strong ethylene-insensitive etr1-1 mutant. Rps1-k-mediated resistance against P. sojae race 1 remained effective, suggesting that the Rps1-k locus may encode more than one gene for disease resistance. Overall, our results suggest that reduced ethylene sensitivity can be beneficial against some pathogens but deleterious to resistance against other pathogens.     

The soybean-Phytophthora resistance locus <Emphasis Type="Italic">Rps1</Emphasis>-k encompasses coiled coil-nucleotide binding-leucine rich repeat-like genes and repetitive sequences

Background  

A series of Rps (resistance to P ytophthora s ojae) genes have been protecting soybean from the root and stem rot disease caused by the Oomycete pathogen, Phytophthora sojae. Five Rps genes were mapped to the Rps1 locus located near the 28 cM map position on molecular linkage group N of the composite genetic soybean map. Among these five genes, Rps1-k was introgressed from the cultivar, Kingwa. Rps1-k has been providing stable and broad-spectrum Phytophthora resistance in the major soybean-producing regions of the United States. Rps1-k has been mapped and isolated. More than one functional Rps1-k gene was identified from the Rps1-k locus. The clustering feature at the Rps1-k locus might have facilitated the expansion of Rps1-k gene numbers and the generation of new recognition specificities. The Rps1-k region was sequenced to understand the possible evolutionary steps that shaped the generation of Phytophthora resistance genes in soybean.     

Mapping genes conferring resistance to Phytophthora root rot of soybean, Rps1a and Rps7.

A linkage map was constructed for two Phytophthora sojae Kauf. +Gerd. root rot resistance genes, Rps1a and Rps7, in soybean (Glycine max (L.) Merr.) using microsatellite or simple sequence repeat (SSR) markers. An F2 population consisting of 81 individuals derived from a cross between OX281, which carries Rps7, and Mukden, which carries Rps1a, was used as the mapping population. A linkage map consisting of 10 SSR markers was first constructed using the computer software MapMaker/EXP 3.0. Rps1a and Rps7 were then placed at two different loci in the same linkage group with LOD scores of 2.88 and 9.16, respectively. Rps1a and Rps7 were linked at a distance of 13.8 cM. Rps1a was flanked by Satt159 (0.7 cM) and Satt009 (3.2 cM). Rps7 was flanked by Satt009 (10.6 cM) and Satt125 (29.1 cM).     

Genetic analysis and fine mapping of RpsJS,a novel resistance gene to Phytophthora sojae in soybean [Glycine max (L.) Merr.]

Key message

We finely map a novel resistance gene ( RpsJS ) to Phytophthora sojae in soybean. RpsJS was mapped in 138.9 kb region, including three NBS-LRR type predicted genes, on chromosome 18.

Abstract

Phytophthora root rot (PRR) caused by Phytophthora sojae (P. sojae) has been reported in most soybean-growing regions throughout the world. Development of PRR resistance varieties is the most economical and environmentally safe method for controlling this disease. Chinese soybean line Nannong 10-1 is resistant to many P. sojae isolates, and shows different reaction types to P. sojae isolates as compared with those with known Rps (Resistance to P. sojae) genes, which suggests that the line may carry novel Rps genes or alleles. A mapping population of 231 F₂ individuals from the cross of Nannong 10-1 (Resistant, R) and 06-070583 (Susceptible, S) was used to map the Rps gene. The segregation fits a ratio of 3R:1S within F₂ plants, indicating that resistance in Nannong 10-1 is controlled by a single dominant gene (designated as RpsJS). The results showed that RpsJS was mapped on soybean chromosome 18 (molecular linkage group G, MLG G) flanked by SSR (simple repeat sequences) markers BARCSOYSSR_18_1859 and SSRG60752K at a distance of 0.9 and 0.4 cm, respectively. Among the 14 genes annotated in this 138.9 kb region between the two markers, three genes (Glyma18g51930, Glyma18g51950 and Glyma18g51960) are the nucleotide-binding site and a leucine-rich repeat (NBS-LRR) type gene, which may be involved in recognizing the presence of pathogens and ultimately conferring resistance. Based on marker-assisted resistance spectrum analyses of RpsJS and the mapping results, we inferred that RpsJS was a novel gene or a new allele at the Rps4, Rps5 or Rps6 loci.     

Genetic mapping and development of co-segregating markers of <Emphasis Type="Italic">RpsQ</Emphasis>, which provides resistance to <Emphasis Type="Italic">Phytophthora sojae</Emphasis> in soybean

Key message

The RpsQ Phytophthora resistance locus was finely mapped to a 118-kb region on soybean chromosome 3. A best candidate gene was predicted and three co-segregating gene markers were developed.

Abstract

Phytophthora root rot (PRR), caused by Phytophthora sojae, is a major threat to sustainable soybean production. The use of genetically resistant cultivars is considered the most effective way to control this disease. The Chinese soybean cultivar Qichadou 1 exhibited a broad spectrum resistance, with a distinct resistance phenotype, following inoculation with 36 Chinese P. sojae isolates. Genetic analyses indicated that the disease resistance in Qichadou 1 is controlled by a single dominant gene. This gene locus was designated as RpsQ and mapped to a 118-kb region between BARCSOYSSR_03_0165 and InDel281 on soybean chromosome 3, and co-segregated with Insert11, Insert144 and SNP276. Within this region, there was only one gene Glyma.03g27200 encoding a protein with a typical serine/threonine protein kinase structure, and the expression pattern analysis showed that this gene induced by P. sojae infection, which was suggested as a best candidate gene of RpsQ. Candidate gene specific marker Insert144 was used to distinguish RpsQ from the other known Rps genes on chromosome 3. Identical polymerase chain reaction amplification products were produced for cultivars Qichadou 1 (RpsQ) and Ludou 4 (Rps9). All other cultivars carrying Rps genes on chromosome 3 produced different PCR products, which all lacked a 144-bp fragment present in Qichadou 1 and Ludou 4. The phenotypes of the analyzed cultivars combined with the physical position of the PRR resistance locus, candidate gene analyses, and the candidate gene marker test revealed RpsQ and Rps9 are likely the same gene, and confer resistance to P. sojae.

     

Organization,expression and evolution of a disease resistance gene cluster in soybean

PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar "Williams 82" [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca(2+)-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.