毛花猕猴桃雄株花期及花粉性状与SNP标记全基因组关联分析

Actinidia eriantha is one of important and unique variety in Actinidiaceae in China, belong to functional dioecious plants. Flowering time and pollen characters is an important target trait of male plant breeding A. eriantha. Male plants with early flowering and high activity pollen have a great deal of sense for interspecific cross breeding and manual pollination. Matching of excellent male plants have great significance for the commercial production of kiwifruit. In this study, the 200 male core collections of the wild A.eriantha, which had obvious difference in flowering period and prominent characters about pollen, were selected as the experimental materials. The population structure of the tested materials was analyzed by the SNP makers developed through the method of whole genome re-sequencing (WGR) and the STRUCTURE software. The genome-wide association studies (GWAS) were performed using TASSEL MLM (mixed linear model) program through the flowering and pollen related phenotypic traits of the male populations. The association loci of SNP and excellent alleles were explored and mined which had a significant effect on the early stage of flowering period, pollen quantity, pollen yield and pollen viability. The candidate genes related to early flowering and high activity pollen of male plants in A. eriantha were studied by gene hunting and functional annotation in the association areas, and their functions were verified. The results could provide a theoretical basis for the molecular marker assisted breeding and revealing the regulation mechanism of flowering of male plants in A. eriantha.

毛花猕猴桃(Actinidia eriantha )是我国独有的猕猴桃属中的一个重要种类，属功能性雌雄异株植物，花期和花粉性状是毛花猕猴桃雄株选育的重要目标性状，早花型和高活性花粉型雄株对于猕猴桃种间杂交育种和人工授粉具有重要意义。本研究拟以前期筛选收集的200份花期及花粉活性变异广泛且差异明显的野生毛花猕猴桃雄株核心种质群体为试材，利用全基因组重测序（WGR）技术开发的SNP标记结合STRUCTURE软件对供试材料进行群体结构解析，通过居于该雄株种质群体花期及花粉活性表型性状采用TASSEL软件中的MLM模型对其进行全基因组关联分（GWAS），寻找对其早花及高活性花粉相关性状有显著影响的SNP关联位点及优异等位基因；进一步通过关联区域基因搜寻与功能注释，挖掘其早花及高活性花粉性状相关候选基因，并进行功能验证，可为毛花猕猴桃雄株分子辅助育种、揭示调控其雄株开花的分子机制提供理论基础。

本研究以野生毛花猕猴桃雄性种质为材料，对其花和叶片相关的11个数量性状进行综合分析与评价；利用RAD-seq技术对其群体结构和连锁不平衡进行分析；通过对11个数量性状与SNP标记的关联分析，鉴定出与目标性状显著关联的SNP位点，并对其候选基因进行挖掘，为在基因水平上解析雄株相关重要性状的调控机制提供理论依据。主要研究结果如下：.野生毛花猕猴桃雄株花和叶片相关数量性状变异丰富，单花花粉量变异最大（50.71%），单花花瓣数变异最小（7.23%）。数量性状聚类分析表明，在欧式距离为1.24处可将种质划分为Ⅳ大类群，其中第Ⅱ类群花粉活力较高，第Ⅲ类群单花雄蕊数、花冠直径、单花药花粉量和单花花粉量较高；第Ⅳ类群的花瓣花青苷含量及叶片总酚含量较高。.RAD测序共获得164.84 Gb的数据量，与参考基因组的平均比对率高达95.70 %。经过滤共获得946 337个高一致性的SNP标记，遍布于整个基因组。LD平均衰减距离为0.1 ~ 0.3 Kb，大约每0.73 kb DNA序列长度上有1个SNP。系统发育树和群体结构分析表明，143份种质材料可被分成两大类群，进一步通过主成分分析可划分为4个亚群，对应所选种质材料的4个不同地理来源。.在MLM（QK）模型下，基于0.01显著水平（-log10(P) > 6.67），检测到3个SNP与花粉量（APF、APA）相关，共找到候选基因14个，均位于5号染色体，且APF与APA其中1个显著关联的SNP位置一致；与单花花瓣数相关的2个SNP共找到15个候选基因，位于19号和22号染色体；与花冠直径相关的1个SNP共找到10个候选基因，位于22号染色体；与花瓣花青苷含量相关的2个SNP共找到10个候选基因，位于1号和5号染色体；与叶片AsA含量相关的2个SNP共找到14个候选基因，位于5号和24号染色体；与叶片总酚含量相关的1个SNP共找到2个候选基因，位于3号染色体。.进一步选择雄株叶片中与AsA相关的2个SNP多态性位点进行验证。Sanger测序的验证结果与重测序筛选结果的匹配率为98.95%。重测序结果显示，24号染色体上有3个SNP位点为杂合突变，而Sanger测序结果显示，这些位点与参考基因组一致，不是杂合突变。