黄瓜幼苗下胚轴长度GWAS分析及候选基因挖掘

GWAS Analysis of Hypocotyl Length and Candidate Gene Mining in Cucumber Seedlings

【目的】挖掘与黄瓜幼苗下胚轴长度显著相关的SNP位点及候选基因,为揭示下胚轴长度的遗传基础和分子机制提供理论依据,为短下胚轴分子标记辅助选择育种奠定基础。【方法】以95份黄瓜核心种质为试验材料,分别于2016年春季、2017年春季、2017年秋季和2018年春季在中国农业科学院南口试验基地塑料大棚进行种植,在两叶一心期调查黄瓜幼苗的下胚轴长度;利用Structure 2.3.4软件分析群体结构,Haploview软件分析连锁不平衡的衰减;基于最优模型对下胚轴长度进行全基因组关联分析(GWAS),依据关联SNP位点的LD区间序列,预测与下胚轴长度相关的重要关联候选基因,并利用荧光定量PCR对预测基因进行表达模式分析。【结果】共检测到8个显著关联的位点(Hl1.1、Hl1.2、Hl2.1、Hl3.1、Hl3.2、Hl4.1、Hl5.1、Hl6.1),分别位于1、2、3、4、5、6号染色体,其中,Hl2.1、Hl3.1、Hl3.2、Hl5.1、Hl6.1等5个位点被重复检测到两次以上。通过分析关联SNP位点的LD区间序列,获得Csa1G074930、Csa1G475980、Csa2G381650、Csa3G141820、Csa4G051570、Csa3G627150、Csa5G174640、Csa6G3629708个与黄瓜下胚轴长度有关的候选基因,其中既有光形态建成、泛素化、激素信号通路等调控基因,也有调控网络下游参与细胞生长发育,调节细胞大小,直接调控黄瓜下胚轴长度的基因。多基因在不同黄瓜材料中的有机分布,形成了具有不同下胚轴长度的黄瓜种质。基因表达分析显示Csa1G074930、Csa1G475980、Csa2G381650、Csa4G051570、Csa5G174640在短下胚轴材料中高表达。Csa3G141820、Csa3G627150在长下胚轴材料中高表达。【结论】检测到Hl1.1、Hl1.2、Hl2.1、Hl3.1、Hl3.2、Hl4.1、Hl5.1、Hl6.1等8个与黄瓜下胚轴长度密切关联的SNP位点,挖掘到Csa1G074930、Csa1G475980、Csa2G381650、Csa3G141820、Csa4G051570、Csa3G627150、Csa5G174640、Csa6G362970等8个调控下胚轴长度的候选基因。

【Objective】The aim of this study was to identify SNP loci and candidate genes significantly correlated with cucumber hypocotyl length trait,which could provide a theoretical basis for revealing the genetic basis and molecular mechanism of cucumber hypocotyl length trait,and lay a foundation for marker-assisted selection breeding of cucumber hypocotyl length trait.【Method】The natural population including 95 cucumber germplasm was employed in this study,and seedlings were grown in the plastic house in Nankou Experimental Field of Chinese Academy of Agricultural Sciences in spring 2016,spring 2017,autumn 2017 and spring 2018,respectively.The hypocotyl length was measured at the two true leaves stage.Structure 2.3.4 software was used to analyze the population structure,and Haploview software was used to analyze the attenuation of linkage imbalance.Then,the whole genome association analysis of hypocotyl length was carried out based on the optimal model.The important candidate genes related to hypocotyl length were predicted according to the LD interval sequence of the associated SNP loci,and the expression pattern of candidate genes were performed by fluorescence quantitative PCR.【Result】A total of 8 loci,including Hl1.1,Hl1.2,Hl2.1,Hl3.1,Hl3.2,Hl4.1,Hl5.1 and Hl6.1,were detected on Chr.1,2,3,4 and 5,respectively.Five of them,Hl2.1,Hl3.1,Hl3.2,Hl5.1 and Hl6.1,were detected repeatedly in two or more different environments.By analyzing the LD interval sequences of the associated SNP loci,eight candidate genes,Csa1 G074930,Csa1 G475980,Csa2 G381650,Csa3 G141820,Csa4 G051570,Csa3 G627150,Csa5 G174640 and Csa6 G362970,were predicted,which were related to cucumber hypocotyl length.Some of the candidate genes involved in regulating plant photomorphogenesis,ubiquitination,and hormone signaling pathway.And some of them were downstream genes regulating cell growth,development and cell size,thus they directly regulated hypocotyl length.Thus,the varied distribution of above genes in different cucumber materials resulted in the different hypocotyl length cucumber germplasm.The organic distribution of polygenes in different cucumber materials formed cucumber germline with different Hypocotyl length.Gene expression analysis showed that Csa1 G074930,Csa1 G475980,Csa2 G381650,Csa4 G051570 and Csa5 G174640 were highly expressed in short hypocotyl materials and Csa3 G141820 and Csa3 G627150 were highly expressed in long hypocotyl materials.【Conclusion】Eight SNP loci linked with hypocotyl length,Hl1.1,Hl1.2,Hl2.1,Hl3.1,Hl3.2,Hl4.1,Hl5.1 and Hl6.1,were detected in this study.Eight candidate genes regulating hypocotyl length were predicted,including Csa1 G074930,Csa1 G475980,Csa2 G381650,Csa3 G141820,Csa4 G051570,Csa3 G627150,Csa5 G174640 and Csa6 G362970.