Next-generation sequencing to identify candidate genes and develop diagnostic markers for a novel Phytophthora resistance gene, RpsHC18, in soybean
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A novel Phytophthora sojae resistance gene RpsHC18 was identified and finely mapped on soybean chromosome 3. Two NBS–LRR candidate genes were identified and two diagnostic markers of RpsHC18 were developed.
Phytophthora root rot caused by Phytophthora sojae is a destructive disease of soybean. The most effective disease-control strategy is to deploy resistant cultivars carrying Phytophthora-resistant Rps genes. The soybean cultivar Huachun 18 has a broad and distinct resistance spectrum to 12 P. sojae isolates. Quantitative trait loci sequencing (QTL-seq), based on the whole-genome resequencing (WGRS) of two extreme resistant and susceptible phenotype bulks from an F2:3 population, was performed, and one 767-kb genomic region with ΔSNP-index ≥ 0.9 on chromosome 3 was identified as the RpsHC18 candidate region in Huachun 18. The candidate region was reduced to a 146-kb region by fine mapping. Nonsynonymous SNP and haplotype analyses were carried out in the 146-kb region among ten soybean genotypes using WGRS. Four specific nonsynonymous SNPs were identified in two nucleotide-binding sites–leucine-rich repeat (NBS–LRR) genes, RpsHC18-NBL1 and RpsHC18-NBL2, which were considered to be the candidate genes. Finally, one specific SNP marker in each candidate gene was successfully developed using a tetra-primer ARMS-PCR assay, and the two markers were verified to be specific for RpsHC18 and to effectively distinguish other known Rps genes. In this study, we applied an integrated genomic-based strategy combining WGRS with traditional genetic mapping to identify RpsHC18 candidate genes and develop diagnostic markers. These results suggest that next-generation sequencing is a precise, rapid and cost-effective way to identify candidate genes and develop diagnostic markers, and it can accelerate Rps gene cloning and marker-assisted selection for breeding of P. sojae-resistant soybean cultivars.
We thank Professors Lijuan Qiu and Tianfu Han in the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Professor Zhiping Huang in Anhui Academy of Agricultural Sciences, and Professor Ran Xu in Shandong Academy of Agricultural Sciences for supplying the soybean cultivars tested in this study. The work was supported by the Special Fund for Agroscientific Research in the Public Interest (201303018), the Program of Protection of Crop Germplasm Resources (2016NWB036-12) from the Ministry of Agriculture of the People’s Republic of China, the National Infrastructure for Crop Germplasm Resources (NICGR2017-008), and the Scientific Innovation Program of Chinese Academy of Agricultural Sciences.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
The experiments were performed in accordance with all relevant Chinese laws.
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