甘蓝型油菜polCMS育性恢复位点的全基因组关联分析

doi:10.3864/j.issn.0578-1752.2017.05.003

摘要/Abstract

摘要： 【目的】甘蓝型油菜波里马细胞质雄性不育（pol CMS）在中国已被广泛应用于杂交种育种，其育性恢复程度表现出受1对主效基因的控制，并受微效修饰基因的影响。通过全基因组关联分析方法挖掘育性恢复位点，并对候选基因进行比较分析。【方法】通过芸薹属60K SNP芯片对308份甘蓝型油菜自然群体进行基因型分型，并用pol CMS系301A作母本，与上述材料分别进行杂交得到308份F₁，每份F₁分别于2013年和2014年进行种植，每年2次重复，于始花期根据花粉育性和花蕊发育情况调查F₁植株的育性等级，同时对测交父本自然群体进行群体结构分析和亲缘关系评估，并结合测交父本的基因型分型结果和F₁的育性等级进行全基因组关联分析（GWAS）。从GWAS分析中显著的SNP左右100 kb区间或与显著SNP处于同一单体型块（R²＞0.5）的区间内预测候选基因，并对候选基因进行QTL比较分析和单体型或等位基因的效应分析。【结果】方差分析结果显示，两年F₁的育性等级存在显著差异（P＜0.01），但相关分析发现，两年的育性等级存在显著的正相关（r = 0.52，P＜0.001）。群体结构分析显示，所有测交父本被分为3个亚群（冬性、春性和半冬性），亲缘关系分析发现，任何2个材料之间平均亲缘关系值为0.072，73%的任意材料间亲缘关系值小于0.1，其中，约53%的材料亲缘关系值为0。GWAS分析共检测到13个与育性恢复程度显著关联的SNP，构成了6个候选区间，分别位于A01、A09、C03、C06和C08 5条染色体上，单个SNP解释的表型变异介于2.53%—9.96%。从中共预测到6个与育性恢复位点相关的候选基因，其中4个编码的蛋白含有恢复基因特有的PPR保守基序。共线性分析发现，4个候选基因中的2个（BnaA09g46700D和BnaC08g40710D）位于A09和C08染色体部分同源区间，且与已克隆的pol CMS育性恢复位点ORF2同源。另外2个新鉴定到的候选基因（BnaC03g45840D和BnaC06g13000D）连锁的SNP等位基因或单体型变化都与育性等级显著相关（P＜0.001）。【结论】通过GWAS分析鉴定到多个与油菜育性恢复有关的候选基因，开发基于与这些基因连锁位点或SNP的功能标记将有助于对该不育系统进行恢复系和保持系的筛选。

关键词: 甘蓝型油菜, 波里马细胞质雄性不育, 育性恢复基因, 全基因组关联分析, SNP

Abstract: 【Objective】 Polima system of cytoplasmic male sterility (pol CMS) in Brassica napus (rapeseed), controlled by a major gene as well as polygenes, has been widely used in China for hybrid rapeseed breeding. Genome-wide association study (GWAS) was performed in a rapeseed population to identify genetic loci and candidate genes for fertility restorer of pol CMS. 【Method】 301A, a rapeseed pol CMS line, was chosen as the female parent to cross with a panel of 308 accessions in natural population of rapeseed which has been genotyped previously using the 60 K Brassica SNP array. The F₁ hybrids were grown for fertility evaluation in 2013 and 2014, respectively, with two replications each year. The fertility of F₁ was classified according to pollen fertility and performance of pistil and stamen, and population structure and relative kinship of 308 male accessions were analyzed. GWAS was conducted by associating the fertility of F₁ with the single nucleotide polymorphisms (SNPs) of males. Candidate genes was identified from the region of 100 kb each side of the peak SNP or trait-associated SNPs at LD (R²＞0.5). Comparative analysis of QTL and haplotype effect evaluation for candidate genes were performed. 【Result】 A significant difference (P＜0.01) was found in the fertility of F₁ between two years, but a high correlation was detected in it between two years (r = 0.52, P＜0.001). The population structure analysis classified the 308 male accessions into three genetic groups (winter, spring and semi-winter). The relative kinships analysis found that 73% of the kinship coefficients between lines were ＜0.1 and 53% were equal to 0. A total of 13 SNPs were detected to be with significant association with the fertility of F₁, formed six genetic intervals on chromosomes A01, A09, C03, C06 and C08. Six genes related to fertility were predicted from the six intervals, and four of these could encode the PPR type proteins which is a conserve structure encoded by fertility restorer genes. Collinearity analysis revealed two PPR type candidate genes (BnaA09g46700D and BnaC08g40710D) detected in homoeologous regions between chromosome A09 and C08 were homology with ORF2, an open reading frame functioning as the reported rapeseed nuclear restorer gene of pol CMS. The other two PPR type candidates (BnaC03g45840D and BnaC06g13000D) were novel candidate restorer genes for rapeseed pol CMS, of which the linked alleles or haplotypes of SNPs were found to significantly associated with the fertility level of the F₁ (P＜0.001).【Conclusion】The present study identified several fertility restorer genes for pol CMS in rapeseed from both A and C subgenomes. Developing functional markers from the alleles or SNPs linked with the candidate genes will benefit the screening of restorer and maintainer lines in the pol CMS system.

Key words: Brassica napus, polima CMS, fertility restorer gene, GWAS, SNP

魏大勇，谭传东，崔艺馨，吴道明，李加纳，梅家琴，钱伟. 甘蓝型油菜polCMS育性恢复位点的全基因组关联分析[J]. 中国农业科学, 2017, 50(5): 802-810.

WEI DaYong, TAN ChuanDong, CUI YiXin, WU DaoMing, LI JiaNa, MEI JiaQin, QIANWei. Genome-Wide Association Study of the Fertility Restorer Loci for pol CMS in Rapeseed (Brassica napus L.)[J]. Scientia Agricultura Sinica, 2017, 50(5): 802-810.

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