Two F_6:8 recombinant inbred line (RIL) populations were used for initial mapping: 103 RILs and 130 RILs were constructed from a cross between Meng8206 × Linhedafenqing and Meng8206 × Zhengyang148, respectively. An F_2:3 secondary population was used for fine mapping: 159 lines were constructed from a cross between Meng8206 × Linmeng6-46 (Supplementary Figure 1). The soybean lines Meng8206, Linhedafenqing, Zhengyang148 and Linmeng6-46 were obtained from National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing, China. Meng8206 was also obtained from the Chinese National Soybean GeneBank (CNSGB).

To clarify the response type of Meng8206 to P. sojae, 15 differentials were used, and each cultivar had an independent Rps gene. The 15 differential cultivars included Harlon (Rps1a), Harosoy13XY (Rps1b), Williams79 (Rps1c), PI103091 (Rps1d), Williams82 (Rps1k), L76-1988 (Rps2), Chapman (Rps3a), PRXI46-36 (Rps3b), PRXI 45-48 (Rps3c), L85-2352 (Rps4), L85-3059 (Rps5), Harosoy62XY (Rps6), Harosoy (Rps7), Yudou25 (RpsYu25), and LuDou4 (Rps9). In addition, Williams (no known Rps gene) was a susceptible variety used as an inoculation reference.

A total of eight P. sojae isolates (Supplementary Table 1) with different virulence capabilities were provided by Professor Yuanchao Wang of Nanjing Agricultural University and maintained on V8 juice agar medium (10% V8 vegetable juice, 0.02% CaCO₃ and 1.0% Bacto-agar). These isolates were used to evaluate the resistance identified among the parents and 15 differential cultivars. P. sojae HeN08-35 (virulence formula is 3a, 3c, 4, 5, 6 and 7) was used to evaluate two mapping populations.

A modified hypocotyl inoculation technique was utilized for disease evaluation in this experiment (Sun et al., 2011, 2014). All materials were planted in plastic pots containing vermiculite; the mycelia from 7-day-old seedlings were maintained on V8 juice agar and subsequently inoculated onto wounded hypocotyls. After inoculation, the seedlings were placed in a high humidity mist chamber for 48 h and subsequently transferred to a greenhouse at 25°C with a 14-h light/10-h dark photoperiod for 5 days. Two F_6:8 RILs and F_2:3 family reactions were evaluated at 5 days post-inoculation (DPI) and recorded as the percentage of dead seedlings. Each family had 30 plants scored. The standard criterion of each family is as follows: if the percentage of dead seedlings >80%, then this family was recorded as homozygous susceptible (S); if the percentage of dead seedlings <20%, then this family was recorded as homozygous resistant (R); and if the percentage of dead seedlings is between 21 and 79%, then this family was recorded as heterozygous resistant (Rs) (Gordon et al., 2006; Ping et al., 2015).

The genomic DNA was extracted from the young leaves of two RIL populations according to Zhang and Wang (2004) and used to construct the genomic DNA library after Taq I digestion according to Baird et al. (2008). The 400- to 700-bp DNA fragments were sequenced using the Illumina HiSeq 2000 standard protocol for MSG (multiplexed shotgun genotyping), and 90-mer paired-end reads were generated (Andolfatto et al., 2011). SOAP2 (Li et al., 2009) software was used for aligning the sequenced reads to the Williams 82 reference genome. SNP calling and genotyping were conducted using RealSFS software (Yi et al., 2010) based on the Bayesian estimation. Subsequently, using a three-standard filter, 50 < depth < 2500, a probability of site mutation ≥95%, and every SNP loci separated by at least 5 bp, we obtained high confidence SNPs.

Bin maps were constructed using a sliding window approach. The sliding window contained 15 SNPs. As the window slides, the genotypes are called and recombination breakpoints are determined. The same genotype across the entire RIL population was recognized as a single recombination bin (Huang et al., 2009).

Plant genomic DNA was extracted from young leaves using the CTAB method with minor modifications (Allen et al., 2006). Resistant and susceptible bulks for the bulk segregation analysis (BSA) were, respectively, formed using the plant genomic DNA of 10 resistant and 10 susceptible F₂ individuals (Michelmore et al., 1991), and the DNA concentration for the two bulks was greater than 50 ng/μl.

According to the initial mapping physical position, the sequence was downloaded from Phytozome Glyma1.0¹, and simple repeat sequences were assessed using SSR Hunter 1.3². New SSR markers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA). In addition, some markers from Soybase³ and the published paper (Zhang et al., 2013b) were used. PCR was conducted according to Sun et al. (2014).

A goodness-of-fit to the Mendelian segregation ratio was calculated using Chi-square (χ²) analysis to examine the segregation patterns of the phenotypes and selected SSR markers. The resistance locus for initial mapping was detected using composite interval mapping (CIM) in QTL Cartographer 2.5(threshold value 2.5) (Wang et al., 2012). The linkage map of RpsHN for fine mapping was constructed using Joinmap 4.0 linkage analysis software (van Ooijen, 2006). The linkage groups were analyzed with a log-likelihood (LOD) threshold of 3.0.

Meng8206 (R) and LinMeng6-46 (S) seedlings were cultivated for 7 days and subsequently inoculated with isolate HeN08-35. Approximately 1-cm samples of the treated hypocotyl tissues were collected at five time points. Total RNA was extracted from the plants using the RNA Simple Total RNA kit (TIANGEN, China). cDNA was synthesized using the Prime Script^TM RT Reagent Kit (TaKaRa, Japan) using a standard protocol. The experiment was repeated three times.

The CDS sequences for the candidate genes were obtained from Phytozome⁴. The primers for qRT-PCR were designed using Primer Premier 5.0. In addition, the housekeeping gene Actin was used as a control. These primers are shown in Supplementary Table 2, and qRT-PCR was conducted using a Light Cycler 480 instrument.

The 8 different isolates were applied to evaluate 4 parents and 15 differentials. The results showed that Meng8206 was resistant to the HeN08-35 isolate and was susceptible to the other 7 isolates (Supplementary Table 3). The phenotype reaction of Meng8206 was different for each of the 15 differentials, conferred through an independent Rps gene. The three parents Zhengyang148, Linhedafenqing and Linmeng6-46 were susceptible to all selected P. sojae isolates, and their phenotype reactions were the same as Williams. In addition, we also analyzed the genetic diversity and phenotypic relationships of the four parents and 15 differentials to 8 P. sojae using cluster tree analysis in the NTSYS program (Figure 1). When the coefficient was more than 0.8, Meng8206 existed independently as a subgroup, suggesting that Meng8206 may possess a novel Rps gene.

In initial mapping populations, among the 103 F_6:8 RILs derived from a cross of Meng8206 × Linhedafenqing, 48 RILs were homozygous resistant (R), 55 RILs were homozygous susceptible (S) to P. sojae isolate HeN08-35, and the actual segregation ratio was consistent with expected ratio 1:1 (χ² = 0.49 and p = 0.49) (Table 1). This result suggested that Meng8206 resistance was controlled by a single gene. Thus, this locus was temporarily designated RpsHN. Among the 130 F_6:8 RILs derived from a cross of Meng8206 × Zhengyang148, 44 RILs were homozygous resistant (R), and 86 RILs were homozygous susceptible (S) to P. sojae HeN08-35. The phenotype data showed no clearly inheritance mechanisms of quality traits. Thus, it was decided to perform QTL analysis for mapping of PRR resistance in this population.

In the fine mapping population, among the 159 F_2:3 individuals derived from a cross of Meng8206 × Linmeng6-46, 38 were homozygous resistant (R), 69 were segregating individuals Rs, 52 were homozygous susceptible (S) to P. sojae HeN 08-35, and the actual segregation ratio was consistent with the expected ratio 1:2:1 (χ² = 4.77, p = 0.09) (Table 2). This result suggested that Meng8206 resistance was controlled by a single dominant gene RpsHN.

To analyze the resistance locus of Meng8206 in the two RIL populations, a bins map was constructed using a sliding window approach. A total of 2600 bins were identified in the RIL population derived from a cross of Meng8206 × Linhedafenqing; the genetic distance was 2626.00 cM, and the average genetic distance between markers was 1.01 cM. A total of 2267 bins were identified in the RIL population derived from a cross of Meng8206 × Zhengyang148; the genetic distance was 2584.38 cM, and the mean genetic distance between markers was 1.14 cM.

For the Meng8206 × Linhedafenqing RIL population, a resistance locus was detected on Chr03 with an LOD score of 56.89 using CIM. This locus was located between marker bin249 and bin250, at nucleotide positions 3,515,595 and 4,237,477, respectively, determined through a BLAST search in Glyma1.0. The additive effect of this locus was 0.51 and resistant allelic effect came from Meng8206 (Supplementary Figures 2A,B and Table 3). Interestingly, only a significant resistance locus with an LOD score of 22.66 was also identified on Chr. 03 in the Meng8206 × Zhengyang148 RILs population, and this locus was located between marker bin282 and bin283, at nucleotide positions 3,564,629 and 4,734,455, respectively. The additive effect of this locus was 0.34 and resistant allelic effect came from Meng8206 (Supplementary Figures 2C,D and Table 3). Based on the physical location of the marker, an intersection was detected in the two RILs populations. Because Meng8206 was the same resistance parent in the two RIL populations, the two loci was the same locus.

We conducted fine mapping on the region between markers bin249 and bin283. The physical distance of bin249 and bin283 is approximately 1218.8 kb (Figures 2B,C). In this region, 33 and 6 SSR markers were selected according to Song et al. (2010) and Zhang et al. (2013b), respectively. The two markers satt009 and satt1k2a showed polymorphisms between Meng8206 and Linmeng6-46 using the BSA method.

In addition, a total of 183 repeat motifs (SSR loci) in this region were identified using SSR hunter and used for the fine mapping of RpsHN. Four SSR markers, SSRSOYN-14, SSRSOYN-15, SSRSOYN-25 and SSRSOYN-44, showed polymorphisms between Meng8206 and Linmeng6-46 using the BSA method (Supplementary Table 4). Together with satt009 and satt1k2a, six polymorphic marker segregation patterns were revealed by analyzing 159 F_2:3 families, consistent with the 1:2:1 ratio (Supplementary Table 5).

A genetic map, including six SSR markers and RpsHN was constructed, and RpsHN was closely linked to the SSR markers SSRSOYN-25 and SSRSOYN-44 at genetic distances of 1.6 and 1.0 cM, respectively (Figure 2A).

The genomic region of Williams 82 was delimited by the markers SSRSOYN-25 and SSRSOYN-44. A BLAST search showed that the physical distance of SSRSOYN-25 and SSRSOYN-44 are at nucleotide position 4,227,863 and 4,506,526 in Glyma1.0, is appropriately 278.7 kb (Figure 2B). A total of eight genes were annotated according to the Glyma 1.0 (Supplementary Table 6). Among these genes, Glyma.03g04260 and Glyma.03g04300 encoded NB-ARC domain-containing disease resistance protein. Glyma.03g04340 encodes serine/threonine protein kinase (STK), which is involved in plant disease resistance. These genes were predicted as possible candidate genes.

To confirm whether Glyma.03g04260, Glyma.03g04300 and Glyma.03g04340 were induced under the treatment of P. sojae, the expression patterns of three genes were examined using qRT-PCR analysis in Meng8206 and Linmeng6-46. As shown in Figure 3, compared with the control (0 h), the expression of Glyma.03g04260 was down-regulated at 12, 36, and 48 h after treatment in the resistant line Meng8206 and the susceptible line Linmeng6-46, and the expression of Glyma.03g04300 did not significantly change in Linmeng6-46 and was up-regulated in Meng8206 at 12, 36, and 48 h after treatment. Glyma.03g04340, compared with Glyma.03g04300, had opposite expression levels. These results showed that three genes were induced by P. sojae HeN08-35. Thus, Glyma.03g04260, Glyma.03g04300 and Glyma.03g04340 were considered as potential candidate genes.