Yu Fengqun, Zhang Xingguo, Huang Zhen, Chu Mingguang, Song Tao, Falk Kevin C, Deora Abhinandan, Chen Qilin, Zhang Yan, McGregor Linda, Gossen Bruce D, McDonald Mary Ruth, Peng Gary
Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada.
Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada.
PLoS One. 2016 Apr 14;11(4):e0153218. doi: 10.1371/journal.pone.0153218. eCollection 2016.
Clubroot, caused by Plasmodiophora brassicae, is an important disease on Brassica species worldwide. A clubroot resistance gene, Rcr1, with efficacy against pathotype 3 of P. brassicae, was previously mapped to chromosome A03 of B. rapa in pak choy cultivar "Flower Nabana". In the current study, resistance to pathotypes 2, 5 and 6 was shown to be associated with Rcr1 region on chromosome A03. Bulked segregant RNA sequencing was performed and short read sequences were assembled into 10 chromosomes of the B. rapa reference genome v1.5. For the resistant (R) bulks, a total of 351.8 million (M) sequences, 30,836.5 million bases (Mb) in length, produced 120-fold coverage of the reference genome. For the susceptible (S) bulks, 322.9 M sequences, 28,216.6 Mb in length, produced 109-fold coverage. In total, 776.2 K single nucleotide polymorphisms (SNPs) and 122.2 K insertion / deletion (InDels) in R bulks and 762.8 K SNPs and 118.7 K InDels in S bulks were identified; each chromosome had about 87% SNPs and 13% InDels, with 78% monomorphic and 22% polymorphic variants between the R and S bulks. Polymorphic variants on each chromosome were usually below 23%, but made up 34% of the variants on chromosome A03. There were 35 genes annotated in the Rcr1 target region and variants were identified in 21 genes. The numbers of poly variants differed significantly among the genes. Four out of them encode Toll-Interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat proteins; Bra019409 and Bra019410 harbored the higher numbers of polymorphic variants, which indicates that they are more likely candidates of Rcr1. Fourteen SNP markers in the target region were genotyped using the Kompetitive Allele Specific PCR method and were confirmed to associate with Rcr1. Selected SNP markers were analyzed with 26 recombinants obtained from a segregating population consisting of 1587 plants, indicating that they were completely linked to Rcr1. Nine SNP markers were used for marker-assisted introgression of Rcr1 into B. napus canola from B. rapa, with 100% accuracy in this study.
根肿病由芸薹根肿菌引起，是全球范围内十字花科作物的一种重要病害。一个对芸薹根肿菌3号生理小种有效的根肿病抗性基因Rcr1，先前已在小白菜品种“Flower Nabana”中定位到白菜A03染色体上。在本研究中，对2号、5号和6号生理小种的抗性被证明与A03染色体上的Rcr1区域有关。进行了混合分离群体RNA测序，并将短读序列组装成白菜参考基因组v1.5的10条染色体。对于抗性（R）混合群体，共产生3.518亿（M）条序列，长度为308.365亿碱基（Mb），对参考基因组的覆盖度为120倍。对于感病（S）混合群体，产生3.229亿条序列，长度为282.166亿碱基，覆盖度为109倍。总共在R混合群体中鉴定出77.62万个单核苷酸多态性（SNP）和12.22万个插入/缺失（InDel），在S混合群体中鉴定出76.28万个SNP和11.87万个InDel；每条染色体上约87%为SNP，13%为InDel，R和S混合群体之间78%为单态性变异，22%为多态性变异。每条染色体上的多态性变异通常低于23%，但在A03染色体上占变异总数的34%。在Rcr1目标区域注释了35个基因，在21个基因中鉴定到了变异。各基因的多变异数量差异显著。其中4个编码Toll样白细胞介素-1受体/核苷酸结合位点/富含亮氨酸重复序列蛋白；Bra019409和Bra019410含有较多的多态性变异，这表明它们更有可能是Rcr1的候选基因。利用竞争性等位基因特异性PCR方法对目标区域的14个SNP标记进行基因分型，并证实它们与Rcr1相关。用从1587株植物组成的分离群体中获得的26个重组体对选定的SNP标记进行分析，表明它们与Rcr1完全连锁。9个SNP标记用于将Rcr1从白菜标记辅助导入甘蓝型油菜，在本研究中准确率为100%。
