Soybean is one of the most important crops in the world. There are a large number of soybean accessions in China, among which many PRR-resistant cultivars/lines were identified in a previous study [10, 13, 14, 52-56]. In the present study, the Guizao1 cultivar was PRR resistant to P. sojae PNJ4 and PNJ1, thus differing from the other soybean cultivars tested (Table 1). Genetic analyses indicated that resistance to P. sojae PNJ4 in Guizao1 was controlled by a single locus.
To more finely map the PRR resistance locus, RpsGZ was mapped in an RIL population based on genotyping through resequencing, resulting in the integration of 54,002 SNPs into 3748 recombination bin units. These markers were then employed to construct a high-density bin linkage map with an average distance of 0.81 cM between adjacent markers . The map exhibited well-distributed linkage distances and a higher resolution than the conventional map, and gene/QTL mapping was thus more accurate and reliable. The position of RpsGZ was refined through fine mapping to a 367,371 bp interval between 4,003,401 and 4,370,772 bp on chromosome 3, which was the region rich in Rps genes.
Previous studies have identified 17 known Rps genes (alleles) and mapped them to chromosome 3 before RpsGZ, including five alleles of Rps1 (Rps1a, 1b, 1c, 1d, 1k) [23, 24, 39, 57, 58], Rps7 , Rps9 , RpsYu25 , an Rps gene in Waseshiroge , RpsYD29 , an Rps gene in E00003 soybean within the Rps1k interval , RpsHC18 , RpsQ , RpsHN , RpsX , RpsWY , and RpsUN1 . Nevertheless, the positional relationships of these Rps genes had not been confirmed, and some of the mapping intervals for these Rps genes overlapped. Therefore, whether these genes were allelic or located at a new locus needed to be confirmed.
In the present study, RpsGZ was found to be a distinct gene from the Rps1 alleles because five varieties carrying Rps1 (1a, 1b, 1c, 1d and 1k) were PRR susceptible to P. sojae PNJ4, although the candidate region of RpsGZ partly overlapped with the region of Rps1. The Wayao cultivar (RpsWY) was susceptible to P. sojae PNJ4, Guizao1 was resistant to P. sojae PNJ4 , and these two mapping parents exhibited different resistance reactions, suggesting that RpsGZ may be different from RpsWY. Compared with the nucleotide positions of the Rps genes mapped to chromosome 3 (Table 4) according to the Glyma 2.0 soybean gene annotation database (http://soybase.org/), the positional information for RpsGZ suggested that RpsGZ was distinct from 9 known Rps genes, including Rps1a, Rps1b, Rps1c, Rps1d, Rps9, RpsQ, RpsX, RpsYu25 and RpsHC18.
In addition, Rps7 was mapped to a 14,483,755 bp genomic region (3,931,955–18,415,710 bp) flanked by the SSR markers Satt009 and Satt125 . RpsUN1 was localized to the region between 4,020,587 and 4,171,402 bp, flanked by two SSR markers, BARCSOYSSR_03_0233 and BARCSOYSSR_03_0246, based on the Glyma 2.0 soybean gene annotation database of the Williams 82 genome sequence. Among the regions of four other known Rps genes according to the Glyma1.0 annotations, the Waseshiroge Rps gene was located between Satt009 and T003044871 and may reside in the nucleotide region between 3,910,260 and 4,486,048 bp of the Williams 82 genome . The Rps gene in cv. E00003 was positioned within the interval of 4,475,877 to 4,563,799 bp . RpsHN was mapped to a 278.7 kb genomic region flanked by the SSR markers SSRSOYN-25 and SSRSOYN-44 and may reside at nucleotide position 4,227,863 and 4,506,526 bp . RpsYD29 was flanked by the markers SattWM82-50 and Satt1k4b, which were located at nucleotide positions 3,857,715 and 4,062,474 bp . RpsGZ was also located in a region between 4,022,530 and 4,483,231 bp in GlymaWm82.a1.v1. Therefore, RpsGZ and the Rps7, RpsHN, RpsUN1, RpsYD29, and Rps genes from Waseshiroge and E00003 may be tightly linked genes, different alleles of the same gene, or identical alleles of the same gene. However, further confirmation is needed. Moreover, if the sources of resistance mentioned above carry different resistance genes, a pyramiding effect of different resistance genes may increase the resistance of soybean cultivars to P. sojae.
The NBS-LRR genes are the extremely large family of plant disease resistance genes , and the local tandem duplication of NBS genes has created many homogenous clustered loci in each legume genome studied to date . Meziadi et al. suggested that the NBS-LRR proteins are encoded by one of the largest and most variable multigene families and are often organized into complex clusters of tightly linked genes in plants . In soybean, 319 putative NBS-LRR genes and 175 disease resistance QTLs have been found, among which 36 NBS-LRR genes are clustered on chromosome 3, and most of the NBS-LRR genes are located at the front end of chromosome 3 . The 17 identified Rps genes were all mapped to regions between 2,943,883 and 9,228,144 bp on chromosome 3. In addition, some genes or QTLs for resistance to abiotic or biotic stresses in soybean have been mapped near the region of RpsGZ on chromosome 3. For instance, the QTL Raso1 for major foxglove aphid resistance was mapped to a 63-kb interval containing an NBS-LRR-type R-like gene and two other genes in the Williams 82 sequence assembly . A minor foxglove aphid resistance QTL in PI 366121 , two soybean sudden death syndrome resistance QTLs, di1 [68,69] (also known as qRfs6 ) and SDS14-1 , and the major QTLs or dominant loci underlying salt tolerance in the soybean cultivars Tiefeng8 and Jidou12 [72,73] might be clustered in the region as Rps resistance genes. Among the 19 genes in the region close to RpsGZ detected in this study, five gene candidates were NB-ARC domain and leucine-rich repeat-containing (NBS-LRR) genes, which are a typical type of so-called R-genes. NBS-LRR-type genes have been implicated in the resistance of Rps1k . qRT-PCR analysis showed differential expression patterns of the NBS-LRR-type gene Glyma.03g05300 between Guizao1 and BRSMG68, and this gene may be involved in defence mechanisms against disease.