A Novel Phytophthora sojae Resistance Rps12 Gene Mapped to a Genomic Region That Contains Several Rps Genes

Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.


Introduction
Phytophthora root and stem rot (PRR), caused by Phytophthora sojae Kaufmann and Gerdemann, is one of the most devastating diseases in soybean [Glycine max (L.) Merr.] [1]. The disease was first reported in Indiana in 1948, in Ohio in 1951, and subsequently spread to all soybean-growing regions of the United States (US) [2]. It is most prevalent in the North a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 novel Rps gene on the lower arm of Chromosome 18. The present study was undertaken to map this potential novel Rps gene. We observed that a single dominant Phytophthora resistance gene, named Rps12, is tightly linked to the proximal side of the Rps4/6 locus in a 5.4 cM region between the SSR marker BARCSOYSSR_18_1840 and the NBSRps4/6-130/533 sequence.

Plant genetic material
The AX20925 recombinant inbred line (RIL) population was developed by crossing PI399036 (USDA-ARS National Soybean Germplasm Collection) with the germplasm line AR2, released by Iowa State University (S.R. Cianzio, D.R. Charlson, G. Gebhart, N. Rivera, P. Lundeen, and R. Shoemaker, unpublished). The cross was made at the Iowa State University research site at the University of Puerto Rico's Isabela Substation (ISU-PR) [60].
The individual F 2 plants were advanced to the F 6 generation by applying single-seed descent breeding method. One hundred seeds of each individual F 6 plant were planted and harvested in bulk to obtain F 7 seeds [recombinant inbred line (RILs)] used in this study [60]. In this study, 290 F 7 families [recombinant inbred lines (RILs)] were phenotyped for responses to a mixture of three P. sojae isolates [60] that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous for Phytophthora resistance and 148 were recessive homozygous and susceptible. In this molecular mapping study, 120 RILs of the 290 RILs were investigated. Eleven plants each from selected 120 RILs were scored again for responses to the P. sojae isolates in each of the three independent experiments. Among these 120 RILs, 59 were homozygous resistant and 61 were susceptible to the pathogen.

Phytophthora sojae isolates
Phytophthora sojae R17 (vir 1b, 1d, 3a, 3b, 3c, 5, 6), 1b,1c,1d,1k,2,4,7), and P7074 (vir 1b, 1d, 2, 3a, 3b, 3c, 4, 5, 6, 7, 8) isolates were used in this study (Table 1). Phytophthora sojae isolate R17 was obtained from Dr. Anne Dorrance (Ohio State University, Evaluation of genetic materials for phytophthora resistance The 120 RILs, the parents PI399036 and AR2 along with 14 differential lines and the susceptible cultivar 'Sloan' [12,61] with no known Rps genes were planted in vermiculite filled 237 mL Styrofoam cups (11 seeds per cup) and watered once a day. The differential lines include lines that carry Rps1a, Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3a, Rps3b, Rps3c, Rps4, Rps5, Rps6, Rps7, and Rps8 genes [19,62]. Seedlings were grown in the greenhouse for a week. Hypocotyls of seven-day old seedlings were inoculated using the wounded-hypocotyl inoculation technique [18][19][20][59][60][61][62][63]. An approximately 1 cm long slit was made with the needle tip in each hypocotyl, 1 cm below the cotyledonary node, and 0.2 to 0.4 mL of the culture slurry was placed into the slit using the syringe. Plants were kept in a dew chamber at 25˚C for 24 h in the dark after inoculations and then moved to a growth chamber at 25˚C with a 12 h photoperiod with light intensity 580 ± 75 μ mol PAR m -2 s -1 . The experiment was repeated two more times. Plants were rated seven days after inoculation as either R (resistant, <30% seedling death) or S (susceptible, !70% seedling death). Inocula were prepared using a modified version of the protocol described by Dorrance et al. [12]. Isolates were grown on soft V8 juice agar (12 g agar/liter) at 22˚C under dark conditions until the mycelia covered the entire plate. The colonized agar was cut in strips, placed in a 10-mL syringe and forced out through the syringe to prepare inoculum pulp. The macerated culture was placed in a syringe for a second time and a #18 needle was used to further macerate the culture. Macerated R17 and Val 12-11 cultures were mixed in a 1:1 ratio to prepare the mixed inoculum [63], which is virulent to soybean cultivars carrying Rps genes mapped to any of the Rps1 to 7 loci (Fig 1, Table 1). P. sojae strain P7074 [22,[64][65] was also used as a separate source of inoculum as it is virulent to soybean lines carrying Rps4, 5 and 6 ( Fig 1).

DNA preparation and bulked segregant analysis (BSA)
Prior to inoculation, one unifoliate leaf from each of 11 random plants per RIL was harvested, bulked and frozen in liquid nitrogen, and stored at -80˚C. The genomic DNA was extracted from the bulked leaf samples using the CTAB (cetyl trimethyl-ammonium bromide) method [66]. To identify microsatellite and molecular markers, we conducted bulked segregant analysis (BSA) [67] using pooled DNA samples of 10 homozygous resistant (Resistant Bulk) or 10 susceptible (Susceptible Bulk) RILs. One μg DNA from each selected RIL was used for pooling. Each DNA bulk was diluted to a final concentration of 50 ng DNA/μL.

Molecular marker analyses
Microsatellite (simple sequence repeats, SSR) and molecular markers were used to construct a linkage map of the genomic region carrying the putative novel Rps gene locus. Molecular markers based on previously reported NBSRps4/6 sequence [50] were developed for mapping the novel Rps gene (S2 Table). SSR primers were synthesized using the sequence data available at SoyBase (http://soybase.org/) (S2 Table). Primer sequences for SSR markers linked to RpsJS were obtained from a published report [54] (S2 Table). For SSR analysis, 50 ng DNA extracted from leaf samples of each resistant or susceptible RIL was used as the template in a 25 μL reaction containing 1X PCR reaction buffer ( Table), 1 min extension at 72˚C; followed by 10 min at 72˚C. The amplification products were separated on a 4% NuSieve TM 3:1 agarose (Lonza, USA) gel, stained with EtBr and then visualized under UV light using FOTO/ Analyst Express Systems (FOTODYNE Incorporated, USA). Thirty-four SSR markers covering the novel Rps gene region on Chromosome 18 (S2 Table) were evaluated for possible polymorphisms between the AR2 (susceptible), and PI399036 (resistant) parents, and resistant and susceptible bulks of BSA.

Linkage map construction and statistical analysis
The Chi square (χ2) analysis was performed to check the phenotypic data for goodness-of-fit to a Mendelian segregation ratio using Graphpad (http://www.graphpad.com/quickcalcs). To determine genetic distances, Mapmaker version 3.0 [68] and the Kosambi mapping function [69] were used. Marker order was determined using the log-likelihood (LOD) method with threshold 3.0. The linkage map of molecular markers and the Rps12 locus was constructed using MapChart 2.3 [70].

Results
Identification of a putative novel Rps gene PI399036 has been suggested to carry multiple Rps genes including known and unknown Rps genes [59,60,71]. Our previous study of two independent segregating populations suggested that there is a major Phytophthora resistance gene in the Rps4/6 region of this accession [60].
Here we determine the inheritance of the putative novel gene by evaluating F 2 and RILs for segregation of Phytophthora resistance against an inoculum mixture of Val 12-11 and R17 isolates, which together are virulent on soybean lines carrying all Phytophthora resistance genes mapped to the Rps1 to 7 loci. We also used the P7074 isolate in screening the RILs because this isolate can overcome the resistance encoded by Rps4, 5, and 6 mapped tightly to the Rps4/6 region (Fig 1; Table 1).

Putative mapping of the novel Rps gene by BSA
To putatively map the novel Rps gene, we evaluated 34 SSR markers from the Rps4/6 region for polymorphisms among the parents of the population, PI399036 and AR2 (S2 Table). The selected SSR markers encompass the genomic region that includes the RpsJS, Rps4, and Rps6 genes [27,[56][57]. Of the 34 SSR markers evaluated, 14 were polymorphic between PI399036 and AR2. These SSR markers were then used to putatively determine map location of the Rps gene by conducting BSA [67]. The BSA analysis revealed that the novel Rps gene was located in the Rps4/6 region. Of the 14 SSR markers, 11 showing close association to the novel Rps gene were further considered for mapping the 120 RILs (Fig 2).
In addition to the 14 polymorphic SSR markers, we determined if the NBSRps4/6 sequence previously reported to be the candidate for the Rps4 gene is polymorphic between the two parents, PI399036 and AR2 [50]. We designed NBSRps4/6 sequence-specific primers and amplified two PCR products of 130 and 533 bp in length, from both PI399036 and the resistant bulked DNA sample, but not from either AR2 or the susceptible bulked DNA sample (Fig 3). BSA analysis suggested that the amplified NBSRps4/6-like sequences co-segregate with the genomic region containing a putative novel Rps gene (Fig 3). The 130 bp and 533 bp PCR fragments showed 93% and 99% nucleic acid sequence identity, respectively, to the NBSRps4/6 sequence reported earlier [50]. The 130 and 533 bp NBSRps4/6-type fragments were named as NBSRps4/6-130 and NBSRps4/6-533, respectively.

Genetic mapping of the novel Rps gene
Two dominant markers, NBSRps4/6-130 and NBSRps4/6-533, and 11 co-dominant SSR markers (Figs 2 and 3 and S2 Table) from a genomic region of~3 Mb containing the novel Rps gene were used to construct a linkage map. We genotyped all 120 RILs (59 R and 61 S) for the 13 molecular markers (S3 Table). With the genotypic and phenotypic data of the mapping population, a genetic map consisting of the 11 SSR markers, the two dominant markers, NBSRps4/6-130 and NBSRps4/6-533, and the novel Rps gene locus was constructed. The new gene was mapped between the SSR markers, BARCSOYSSR_18_1840 and Sat_064 (BARCSOYSSR_18_1858) (Fig  4). Both the NBSRps4/6-130 and NBSRps4/6-533 markers were mapped 2.2 cM distal to the novel Rps locus, suggesting that the new Rps gene is unlikely to be allelic to Rps4. Based on the map positions of the molecular markers linked to previously reported Rps genes, it appears that Rps12 is mapped to a new locus which is distinct from the previously mapped Rps loci of the lower arm of Chromosome 18 (Fig 4A and 4B; S1 Fig).
The RpsJS gene has also been mapped to the Rps4/6 region between the molecular markers, BARCSOYSSR_18_1859 and SSRG60752K [54] (S1 Fig). Both of these markers mapped distal to Sat_064, which co-segregated with the Rps4/6 locus carrying Rps4 and Rps6 genes (S1 Fig). Our mapping data suggest that the novel Rps gene is located in the genomic region proximal to the Rps4, 6 and JS genes and distal to Rps5. We conclude that the gene is novel and named the Phytophthora resistance gene as Rps12 (Fig 4).

Discussion
It has been suggested that PI399036 contains multiple Rps genes including known and unknown Rps genes [59,60,71]. Several major and minor QTL for partial resistance to P. sojae have also been identified from this accession [60]. Our previous study indicated the presence of a novel Rps gene in the Rps4/6 region. Responses of the segregating RILs and parents to a P. sojae isolate and a mixture of two isolates established that the gene is distinct from Rps4, 5, and 6. In addition to these three Rps genes, RpsJS was mapped to the lower arm of Chromosome 18 [54]. To determine if the putative novel gene is distinct from Rps4, 5, 6 and JS, we investigated the molecular markers  Table). (B) The NBSRps4/6 specific molecular markers linked to the novel Rps gene. L, 100 bp DNA Ladder (New England Biolabs, USA); SP, susceptible parent AR2; RP, resistant parent PI399036; RB, bulk of 10 resistant homozygous RILs; SB, bulk of 10 susceptible RILs. NBSRps4/6-533, NBSRps4/6 specific NBSRps4/6-533 marker; NBSRps4/6-130, NBSRps4/6 specific NBSRps4/6-130 marker. that were shown to be linked to these Phytophthora resistance genes. The Rps4 and Rps6 genes were shown to co-segregate and Rps4 was tightly linked to Sat_064 [50]. Rps5 was shown to cosegregate with the RFLP marker T005_2, which is proximal to both the Satt191 and Satt472 SSR markers (Fig 4 and S1 Fig) [53,72]. Therefore, we conclude that the novel Phytophtora resistance gene identified in this investigation mapped to the novel locus, Rps12.
Rps12 is located in between two SSR markers, BARCSOYSSR_18_1840 and Sat_064, which span a region of 372 kb DNA. The genetic distance between these two loci is 5.8 cM (Fig 4). These results suggest that the Rps12 region is highly recombinogenic, with only 64 kb DNA/ cM. Thus, introgression of the gene using the BARCSOYSSR_18_1840 and Sat_064 to elite soybean lines would require molecular analyses of a relatively small segregating population (Fig 4). The Rps12 region contains 45 predicted genes, with on the average one gene in every 8 kb DNA. This means that the highly recombinogenic Rps12 region is gene-rich (S4 Table). It will therefore be feasible to map the candidate Rps12 genes to a small genetic interval through use of molecular markers and a large recombinant inbred line population.
Considering the fact that most identified disease resistance genes encode nucleotide binding site-leucine rich repeat (NBS_LRR) containing proteins, we investigated if there are any NBS-LRR-type genes in the Rps12 region [73]. We identified four clusters of eight NBS-LRRtype genes from this genomic region of the cultivar Williams 82, which has been sequenced (S4 Table) [74]. We observed that although NBSRps4/6 is closer to Rps12 as compared to Sat_064 in the genetic map (Fig 4B), in the Williams 82 genome its physical distance to Rps12 is larger than the distance between Rps12 and Sat_064. This could be due to a micro inversion in the Sat_064 region. Alternatively, this could be just an artifact resulting from misassembling of sequences in the Sat_064 region. The highly recombinogenic nature of the Rps12 region suggests that positional cloning of the gene could be facilitated through high density mapping of the Rps12 region using a large segregating population. It is expected that a few of the homozygous RILs for Rps12 contain QTL conditioned by minor genes for partial Phytophthora resistance reported earlier [60] and could be an invaluable germplasm for breeding soybeans.
In this study, we have demonstrated that the previously identified Rps4/6 locus is 2.2 cM distal to the Rps12 locus. To date, Rps1-k has been cloned and a strong candidate gene for Rps4 has been identified. Both encode NBS-LRR genes [29,50]. Several Rps loci have been shown to harbor NBS-LRR sequences, although their functional relevance to Rps genes is yet to be established [75][76]. Our data suggest that Rps12 could be an NBS-LRR-type sequence (Fig 5).
We have evaluated RILs carrying Rps12 against only three important P. sojae isolates, which can overcome resistance encoded by most known Rps genes ( Table 1). The study of RILs for their responses to three isolates indicate that the Rps12 gene is expected to have agronomical importance in conferring resistance to most P. sojae isolates that can defeat the Phytophthora resistance encoded by currently available Rps genes.