甘蓝型油菜种子和角果皮中硫苷含量的动态变化及转录组关联分析

中国农业科学 2018,51(4):635-651Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2018.04.004甘蓝型油菜种子和角果皮中硫苷含量的 动态变化及转录组关联分析田志涛1，赵永国1，Lenka Havlickova2，He Zhesi2，Andrea L Harper2，Ian Bancroft2，邹锡玲1，张学昆1，陆光远1（1中国农业科学院油料作物研究所/农业部油料作物生物学重点实验室，中国武汉430062；2CNAP，University of York，York YO10 5DD，UK）摘要：【目的】探索甘蓝型油菜不同发育时期种子和角果皮硫苷含量的动态变化规律，通过转录组关联分析解析控制硫苷含量变异的遗传机制。

【方法】在由113个油菜品种构成的自然群体中，利用高效液相色谱法检测不同发育时期油菜种子及角果皮中的硫苷含量。

提取油菜幼嫩叶片mRNA并进行mRNA-Seq测序，开发了355 536个单核苷酸多态性标记（SNP）和116 098个基因表达量标记（GEM）。

将开发的SNPs和GEMs分别导入到混合线性模型（MLM）和回归分析模型中进行关联分析，获得与油菜硫苷表型变异显著相关的遗传位点，进一步通过序列比对和功能预测筛选确定候选基因。

【结果】授粉后15 d（15 DAP），油菜种子和角果皮的硫苷含量变异范围分别是1.69—20.45和1.47—25.23 μmol·g-1。

25 DAP种子、25 DAP角果皮以及成熟种子的硫苷变异范围分别是2.17—147.21、0.73—130.77和8.87—111.83 μmol·g-1。

后两个时期（即25 DAP和成熟期）的硫苷含量表现出较大变异，适合进行转录组关联分析。

基于mRNA-Seq测序数据，经质量控制后获得256 397个高质量的SNP标记（MAF＞0.01）和53 889个GEM标记（平均基因表达量＞0.4）。

利用SNP标记对成熟种子、25 DAP种子和25 DAP角果皮硫苷含量进行关联分析，分别检测到167、158和3个显著关联位点（-log10P＞6.71）。

其中，与成熟种子显著关联的SNP标记形成5个明显的关联峰，分别位于A2、A9、C2、C7和C9染色体上。

同时，利用GEM标记进行的关联分析中，分别检测到127、16和24个与成熟种子、25 DAP种子、25 DAP角果皮硫苷含量显著关联的位点（-log10P＞6.03），这些位点主要分布在A2、A8、A9、C2和C9染色体上并形成明显的关联峰。

其中，成熟种子和25 DAP角果皮在A9、C2和C9染色体上形成共同的关联峰。

通过与公共数据库的基因组序列比对和功能注释，共筛选到295个功能基因，其中25个基因涉及硫苷代谢网路途径，直接参与硫苷的调控。

其余基因虽然没有直接参与硫苷代谢过程，但是它们大多属于转录因子基因、刺激响应因子基因，涉及细胞过程或是具有催化活性，因而推测在硫苷积累中同样起到重要作用。

【结论】油菜角果皮中的硫苷含量较高，而且与种子硫苷含量显著正相关，是硫苷合成或转运的重要器官。

共检测到328个SNP显著标记和144个GEM显著标记，筛选到25个与硫苷合成或者转运有关的功能基因以及73个功能未知的新基因。

关键词：甘蓝型油菜；硫代葡萄糖苷；动态变化；转录组关联分析；单核苷酸多态性标记；基因表达量标记Dynamic and Associative Transcriptomic Analysis of Glucosinolate Content in Seeds and Silique Walls of Brassica napus TIAN ZhiTao1, ZHAO YongGuo1, LENKA Havlickova2, HE Zhesi2, ANDREA L Harper2,IAN Bancroft2,ZOU XiLing1, ZHANG XueKun1, LU GuangYuan1(1Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062, China; 2CNAP, University of York, York YO10 5DD, UK)收稿日期：2017-08-21；接受日期：2017-11-07基金项目：国家自然科学基金（31371663）联系方式：田志涛，E-mail：tianzhi_tao@。

赵永国，E-mail：zhaoyongguo@。

田志涛和赵永国为同等贡献作者。

通信作者陆光远，E-mail：luwiz@636 中国农业科学51卷Abstract:【Objective】To assess the dynamic and underlying genetic control of glucosinolate (GS) content in seeds and silique walls at different developing stages. We conducted Associative Transcriptomics (AT) in a panel of European winter oilseed rape. 【Method】The glucosinolate content in developing and mature seeds and silique wall samples from the rapeseed panel (n=113) was determined by HPLC. Using mRNA-seq data derived from the young leaves of B. napus, functional genotypes were inferred for the panel, which included both sequence variation (i.e. Single Nucleotide Polymorphism, SNP) and transcript abundance (Gene Expression Markers, GEM). A total number of 355 536 SNPs and transcript abundance were scored across the rapeseed panel using a reference sequence of 116 098 ordered coding DNA sequence (CDS) gene models for resolving the genetic factors controlling GS variation. All significantly associated markers were further traced back to a CDS gene model, of which the function can be inferred by sequence blast and prediction using public Brassica and Arabidopsis databases. 【Result】The GS content in seeds and silique walls 15 days after pollination (DAP) was very low, with an average of 7.58 μmol·g-1 (1.69-20.45 μmol·g-1) and 4.81 μmol·g-1 (1.47-25.23 μmol·g-1), respectively. The GS content for mature seeds, seeds at 25 DAP and silique walls at 25 DAP varied from 8.87 to 111.83 μmol·g-1, 2.17 to 147.21 μmol·g-1 and 0.73 to 130.77 μmol·g-1, respectively. The observed extensive phenotypic variations for GS content at these two stages made them suitable for A T analysis. In total, 256,397 informative SNPs with minor allele frequency (MAF) ＞ 0.01 and 53,889 GEMs with mean expression level＞0.4 were plotted after AT analysis. With these SNPs markers, a total number of 167, 158 and 3 SNPs were shown to be significantly associated (-log10P＞6.71) with GS content of mature seeds, seeds at 25 DAP, and silique walls at 25 DAP, respectively. Among these, 5 association peaks distributing across chromosome A2, A9, C2, C7 and C9 were found for mature seeds. Meanwhile, 127, 16 and 24 GEMs were significantly (-log10P＞6.03) associated with GS content of mature seeds, seeds at 25 DAP, and silique walls at 25 DAP, respectively, which also unambiguously defined 5 association peaks in A2, A8, A9, C2, and C9. The association peaks in A9, C2 and C9 were common for both mature seeds and silique walls at 25 DAP. Based on the Arabidopsis homologous gene annotations, 25 genes derived from the significant SNPs or GEMs were inferred and predicted to be involved in GS metabolic pathways. The other genes derived from AT analysis, though not directly involved in GS metabolism, were classified as transcription factors, factors responding to stimulus or involved in cellular process, catalytic activity, and thus were also predicted to play an important role in GS accumulation in the different tissues of Brassica napus. 【Conclusion】The GS content in silique walls was found to be high and positively correlated with that of seeds, indicating that silique wall was an important organ for GS synthesis or translocation. A total number of 328 SNPs and 144 GEMs were found to be significantly associated with GS content for different tissues. From these, 25 genes were predicted to be directly involved in GS accumulation or translocation, and 73 genes with uncharacterized function were also inferred.Key words:Brassica napus; glucosinolates; dynamics; Associative Transcriptomics; SNP; GEM0 引言【研究意义】甘蓝型油菜是世界上仅次于大豆的第二大油料作物，同时也是中国最主要的冬季油料作物，其产油量占国产油料作物产油量的45%以上[1-2]。