盐胁迫下甘蓝型油菜发芽期下胚轴和根长的全基因组关联分析

doi:10.3864/j.issn.0578-1752.2017.01.002

摘要/Abstract

摘要： 【目的】解析甘蓝型油菜发芽期根和下胚轴发育及耐盐性的调控位点，筛选油菜耐盐性相关的候选基因，可为油菜耐盐性改良提供依据。【方法】以317份具有代表性的甘蓝型油菜自交系为材料，在正常生长和盐胁迫条件下进行沙培鉴定，利用芸薹属60K SNP芯片和全基因组关联分析鉴定正常生长与盐胁迫下甘蓝型油菜发芽期根和下胚轴长度显著关联的SNP，并确定其连锁不平衡区间。通过区间内基因的功能注释及盐胁迫下油菜幼苗根和叶片转录组差异表达基因筛选连锁不平衡区间内的重要候选基因，并以实时荧光定量PCR分析候选基因的组织特异性和盐胁迫诱导表达模式，提高候选基因筛选的准确性。【结果】正常生长和盐胁迫下甘蓝型油菜发芽期下胚轴和根长在不同材料间变异较大，频次分布表明目标性状均为数量性状，受多基因调控。全基因组关联分析模型比较表明，MLM+P+K模型为最优模型。以此模型对目标性状进行全基因组关联分析，检测到45个显著关联SNP，其中40个与下胚轴长度显著关联，5个与根长显著关联，单个SNP解释的表型变异分别为9.12%—14.46%和7.67%—8.93%。重复检测的显著相关SNP中，值得注意的是C04染色体的rs8970，同时与4个性状显著关联，表型贡献率为7.67%—12.35%，是唯一在下胚轴长和根长间重复检测到的显著关联SNP。11个重要关联SNP中有6个位于10—442 kb的连锁不平衡区块中。转录组分析表明，11个连锁不平衡区间共包含447个基因，其中15个受盐胁迫诱导表达。转录组和基因功能注释综合分析表明，BnaSRO1、BnaPAGR2、BnaNPH3、BnaMYB124、BnaSAM-Mtase、BnaBIN2、BnaUMAMIT11、BnaEXPA7、BnaRPT3、BnaEF-hand和BnaF3H很可能为各自区间的候选基因。实时荧光定量PCR结果证实除BnaNPH3外，其他基因均在根或下胚轴中受盐胁迫诱导上调表达。组织特异性分析还发现BnaUMAMIT11、BnaPAGR2和BnaEXPA7主要在萌发的根和下胚轴中特异表达，BnaRPT3、BnaBIN2和BnaMYB124虽然呈组成型表达，但在萌发阶段的下胚轴中表达量最高，证实这些基因很可能参与油菜发芽期根和下胚轴生长发育及耐盐性的调节。【结论】全基因组关联分析共鉴定出45个控制油菜发芽期根和下胚轴发育及耐盐性的显著关联SNP。连锁不平衡、转录组和基因功能注释综合分析初步鉴定出11个重要候选基因。

关键词: 甘蓝型油菜, 萌发, 耐盐性, 全基因组关联分析, 转录组

Abstract: 【Objective】 Identification of the regulatory loci and candidate genes governing salt stress tolerance in Brassica napus at germination stage could lay the foundation for improvement of B. napus salt resistance. 【Method】 In this study, 317 representative B. napus inbred lines were genotyped under normal and salt-stressed conditions in a sand culture system. Significant SNPs associated with root length and hypocotyl length in B. napus under normal and saline stress conditions and their linkage disequilibrium (LD) were determined by genome-wide association studies (GWAS), based on the Brassica 60 K SNP array. Candidate genes were selected based on the combination analyses results of functional annotation of genes within the LD blocks and transcriptome analyses of seedling roots and leaves in B. napus under saline stress treatments. Accuracy of candidate gene selection was improved by real-time quantitative reverse transcriptase PCR (qRT-PCR). 【Result】 Hypocotyl length and root length of B. napus showed large variation among accessions at germination stage under normal and salt-stressed conditions, and frequency distribution revealed that all the target traits were quantitative traits and controlled by polygenic genes. Comparison of different models showed that MLM+P+K model was the optimal model. Based on this model, GWAS identified 45 loci significantly associated with target traits, including 40 and 5 SNPs associated with hypocotyl length and root length, and each of SNP explained 9.12%-14.46% and 7.67%-8.93% of phenotypic variation, respectively. Among the significantly associated SNPs, rs8970 on chromosome C04 was the most notable, since it was the only SNP, which could be repeatedly detected between root length and hypocotyl length, and associated with four traits simultaneously, explaining 7.67%-12.35% of observed phenotypic variation. Of the 11 important significantly associated SNPs, 6 SNPs were distributed in 10 to 442 kb of linkage disequilibrium (LD) blocks. By combining differentially expressed genes detected by transcriptome analysis with LD block identification, 447 genes were identified within the 11 important LD intervals, of which 15 were activated by salt stress. BnaSRO1, BnaPAGR2, BnaNPH3, BnaMYB124, BnaSAM-Mtase, BnaBIN2, BnaUMAMIT11, BnaEXPA7, BnaRPT3, BnaEF-hand and BnaF3H were most likely the candidate genes within their LD blocks. Results of qRT-PCR detection showed that 10 candidate genes were induced by salt stress treatment in root or hypocotyl at germination stage, except for BnaNPH3. In addition, tissue-specificity detection of candidate genes also showed that BnaUMAMIT11, BnaPAGR2 and BnaEXPA7mainly expressed in the root and hypocotyl at germination stage, and BnaRPT3, BnaBIN2 and BnaMYB124 possessed the highest expression in hypocotyl, confirmed that these genes might be involved in development of root and hypocotyl and salt resistance of B. napus at germination stage. 【Conclusion】 A total of 45 significantly associated SNPs controlling development and salt resistance in root and hypocotyl of B. napus at germination stage were identified by GWAS. By combined LD block identification, transcriptome analyses and functional annotation, 11 important candidate genes were screened within different LD blocks.

Key words: Brassica napus, germination, salt tolerance, genome-wide association study, transcriptome

张蕊，邓文亚，杨柳，王亚萍，肖芳枝，禾健，卢坤. 盐胁迫下甘蓝型油菜发芽期下胚轴和根长的全基因组关联分析[J]. 中国农业科学, 2017, 50(1): 15-27.

ZHANG Rui, DENG WenYa, YANG Liu, WANG YaPing, XIAO FangZhi, HE Jian, LU Kun. Genome-Wide Association Study of Root Length and Hypocotyl Length at Germination Stage Under Saline Conditions in Brassica napus[J]. Scientia Agricultura Sinica, 2017, 50(1): 15-27.

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