鲜食葡萄果实单萜合成关键基因的eQTL分析

eQTL Analysis of Key Monoterpene Biosynthesis Genes in Table Grape

【目的】通过对鲜食葡萄果实单萜合成关键基因进行eQTL定位及候选基因挖掘,深入了解单萜合成调控机制,为优良玫瑰香味葡萄新品种培育及种质改良奠定基础。【方法】以‘摩尔多瓦’ב瑞都香玉’F1代群体及亲本为供试材料,分别在转色期和成熟期采集葡萄果实样品;利用实时荧光定量qPCR技术对7个单萜合成途径基因(VvDXS1、VvDXS3、VvDXR、VvHDR、VvLiner、VvTerp和VvGermD)的表达量进行检测获得表达性状表型数据;基于区间作图法,采用MapQTL6.0软件,对单萜基因表达性状进行eQTL定位分析;将eQTL连锁标记定位到基因组区域,通过Ensembl Plants和NCBI数据库进行基因注释;利用葡萄全基因组芯片技术检测不同发育时期亲本果实样品中候选基因的表达谱。【结果】7个单萜合成基因表达量在F1代群体中呈现连续分布数量遗传特征;各个单萜基因表达之间具有显著的相关性;在转色期,7个表达性状一共定位到13个eQTL,主要位于1号、6号、14号、16号、17号、10号和12号等染色体上,表型解释率介于12.2%-23.5%。其中位于14号染色体的eQTL(qDXS1-v14、qHDR-v14-1和qTerp-v14)覆盖相同的遗传区间57.582-76.979 cM,qLiner-v10、qTerp-v10和qGermD-v10共定位到10号染色体相同的遗传区间;在成熟期,共检测到16个eQTL,主要位于1号、6号、12号、8号、13号和19号等染色体。qDXS1-m6-2、qDXR-m6-2、qLiner-m6和qGermD-m6共定位到6号染色体139.212-143.161 cM遗传区间;针对成熟期与转色期各个基因的表达量比值变化进行定位分析,共检测到18个eQTL,分别位于1号、3号、7号、10号、12号、15号和19号等染色体。定位于12号染色体的qDXS1-r12-1、qDXR-r12-1、qHDR-r12、qLiner-r12和qGermD-r12覆盖相同的遗传区间6.330-6.967 cM。对多个基因表达性状共定位的eQTL区域进行基因注释,共筛选到90个转录因子基因,表达谱及相关性分析最终确定11候选基因。其中4个候选基因(VIT_06s0009g01380、VIT_14s0006g02290、VIT_12s0028g01170和VIT_15s0046g00290)与激素信号通路调控相关,一个候选基因(VIT_12s0028g01110)编码光敏色素作用因子与光响应相关,还有一些编码Myb类、WRKY类转录因子或者未知功能蛋白基因。【结论】在两个不同的生长发育期共检测到37个与单萜合成基因表达性状连锁的eQTL,主要定位于6号、10号、12号和14号染色体。基于基因注释和表达谱分析结果,确定了包含VIT_06s0009g01380和VIT_14s0006g02290在内的11个可能的候选基因,这些候选基因与多个单萜基因表达高度相关。

【Objective】The eQTL mapping for monoterpene biosynthesis related gene expression traits were performed and the candidate genes were mined to deeply understand the regulation mechanism of monoterpene synthesis,so as to lay a foundation for the cultivation of new Muscat grape varieties and germplasm improvement.【Method】The F1 population generated by crossing Moldova and Ruiduxiangyu were used as materials in this study,and the grape berry samples were collected at verasion and ripening stage respectively.The phenotypic data of expression traits were obtained by detecting the expression levels of seven monoterpene synthesis pathway genes(VvDXS1,VvDXS3,VvDXR,VvHDR,VvLiner,VvTerp,and VvGermD)by using real-time quantitative qPCR technique.eQTL mapping of monoterpene gene expression traits were performed with the mapQTL6.0 software based on the interval mapping method.The associated markers of eQTL were mapped to the genomic region,and the genes within eQTLs were annotated and analyzed via the databases of Ensembl Plants and NCBI.The expression profiles of candidate genes in the samples of parents at different developmental stages were detected by grape whole genome microarray.【Result】The expression levels of seven monoterpene biosynthesis related genes in F1 population showed a continuous quantitative genetic distribution.A significant correlation between the expression of monoterpene genes was observed.At verasion,13 eQTLs for the seven expression traits were mapped on chromosome 1,6,14,16,17,10 and 12,respectively,and the phenotypic explanation value ranged from 12.2%to 23.5%.Among them,eQTLs(qDXS1-v14,qHDR-v14-1 and qTerp-v14)on chromosome 14 covered the same genetic interval of 57.582-76.979 cM,and qLiner-v10,qTerp-v10 and qGermD-v10 were co-located on chromosome 10.At the mature stage,16 eQTLs were detected,mainly located on chromosome 1,6,12,8,13 and 19.qDXS1-m6-2,qDXR-m6-2,qLiner-m6 and qGermD-m6 were co-located in the genetic interval 139.212-143.161 cM of chromosome 6.In addition,a total of 18 eQTLs on chromosomes 1,3,7,10,12,15 and 19 were detected for the change ratio of each gene expression between maturity and verasion,respectively.qDXS1-r12-1,qDXR-r12-1,qHDR-r12,qLiner-r12 and qGermD-r12 covered the same genetic interval of 6.330-6.967cM on chromosome 12.The eQTL region for multiple expression traits co-located were further annotated,90 transcription factor genes were screened,and 11 candidate genes were finally identified by expression profile and correlation analysis.Among them,four candidate genes of VIT_06s0009g01380,VIT_14s0006g02290,VIT_12s0028g01170 and VIT_15s0046g00290 were predicted to participate in the regulation of hormone signaling pathway,one candidate gene VIT_12s0028g01110 encodes a phytochrome interacting factor related to light response,and some other genes encode Myb,WRKY transcription factors or unknown functional proteins.【Conclusion】A total of 37 eQTLs linked to monoterpene synthesis gene expression traits were detected at two different development stages,which mainly located on chromosome 6,12 and 14.Based on the results of gene annotation and expression profile analysis,11 candidate genes including VIT_14s0006g02290 and VIT_06s0009g01380 were identified,and these candidate genes were highly correlated with the expression of multiple monoterpene genes.