基于关联分析的野生毛花猕猴桃AsA富集相关基因发掘及功能解析

Recently, association analysis is a procedure for explore and identification excellent genes based on linkage disequilibrium (LD). Association analysis is an approach to identify the relationship of candidate genes with traits in a given population. This study intends to take the germplasm populations with rich variation of ascorbic acid (AsA) content in wild kiwifruit (Actinidia eriantha Benth) fruit in jiangxi province as experimental material. Association analysis between SSR loci and AsA content trait was performed using TASSEL MLM (mixed linear model) program. In the main effect QTLs segment screening and discover regulation AsA synthesis of key enzyme -GalDH、GalLDH、GalUR purpose gene through the analysis of candidate genes associated with wild kiwifruit (A. eriantha Benth) GalDH、GalLDH and GalUR gene sequence differences and their relationship with AsA synthesis ability. At the same time, the related genes cDNA will be systemetically analized by SYBR Green I real-time quantitative PCR during the expression level of different development periods.The cDNA of candidate genes will be obtained through RACE technology, and their function will be analyzed by gene transformation as well as the key genes related to rich AsA accumulation in wild kiwifrui could be found, which could receal the molecular character of AsA accumulation in wild kiwifruit from the gene level.

基于连锁不平衡的关联分析是目前发掘与鉴定优异基因的有效手段，可用于鉴定群体内目标性状与候选基因的关系。本研究以江西省境内抗坏血酸（AsA）含量具丰富变异的野生毛花猕猴桃(Actinidia eriantha Benth)种群为试验材料，利用覆盖猕猴桃全基因组的SSR标记，结合该野生群体AsA含量性状应用Tassel软件中的MLM进行全基因组关联分析，定位其AsA富集相关的主效QTLs。然后在主效QTLs区段内发掘调控AsA合成的关键酶GalDH、GalLDH和GalUR基因，通过基于候选基因的关联分析探究不同等位基因序列差异及其与AsA合成能力的关系，同时采用SYBR Green I实时荧光Q-PCR对其相关基因的表达水平进行分析，利用RACE技术获得其候选基因全长cDNA并进行遗传转化，解析候选基因的功能，探明其AsA合成相关的关键基因，从基因水平揭示野生毛花猕猴桃AsA富集的分子特征。

基于野生毛花猕猴桃资源的调查与收集，对其果实表型性状进行变异分析和SSR遗传多样性分析；并对随机采集的不同植株的野生毛花猕猴桃成熟叶片和幼嫩果实的AsA含量进行了测定与分析，旨在为野生毛花猕猴桃资源的合理开发利用及探讨AsA积累机制提供理论依据。主要研究结果如下：. 1、野生毛花猕猴桃果实表型性状分析及SSR遗传多样性分析.根据UPOV公布的猕猴桃属种测定标准对供试材料的果实表型性状进行观测。参照猕猴桃遗传连锁图谱，选用分布于猕猴桃基因组中的70对SSR引物对供试材料进行遗传多样性分析。结果表明，随机采集的70份野生毛花猕猴桃种质资源中，其果实表型性状和DNA分子水平均存在丰富的变异和较高的遗传多样性。21对多态性高的SSR引物共检测出127个等位变异位点，对供试的野生毛花猕猴桃种质的区分率为100%。. 2、野生毛花猕猴桃叶片和果实AsA含量变异分析.供试的野生毛花猕猴桃成熟叶片和幼嫩果实的AsA含量存在丰富的变异，成熟叶片AsA含量变异系数为41.30%，幼嫩果实AsA含量变异系数为21.88%；通过对成熟叶片和幼嫩果实 AsA含量分布的频率的分析，成熟叶片AsA含量主要集中在1.00~2.75mg · g-1 FW，占总数的77%。幼嫩果实AsA含量在10.00~17.00mg · g-1 FW 区间内集中分布，分布频率为80%。. 3、野生毛花猕猴桃叶片和果实AsA含量与SSR标记关联分析.供试的野生毛花猕猴桃成叶和幼果的AsA含量存在丰富的变异，通过群体结构分析可将70份野生毛花猕猴桃种质材料分为4个亚群；GLM模型分析结果显示，在P<0.01水平上，检测有7个SSR标记与叶片和果实AsA含量紧密相关，各个标记对其叶片和果实AsA含量变异的解释率在0.0825-0.1755；MLM模型分析结果显示，在P<0.01的水平上，共检测到有6个SSR标记分别与叶片和果实AsA含量紧密相关，各个标记的解释率在0.0736-0.1546之间。利用SSR标记分析了70份野生毛花猕猴桃种质的叶片和果实AsA含量自然变异及群体遗传结构，并通过2种关联分析模型，分别寻找到了7个与叶片和果实AsA含量相关联、6 个与叶片和果实AsA含量相关联的标记。