白粉病（powdery mildew）是为害黄瓜的世界性病害之一，常对生产造成严重损失。国内外对于黄瓜白粉病的研究都比较重视，但是由于所使用研究材料的遗传背景、病原菌生理小种以及抗病性鉴定方法等的差异，不同学者的研究结果存在很大差异，已报道的抗病基因定位信息和与抗性的连锁标记尚不能满足分子育种的需要。本研究以抗白粉病的黄瓜自交系‘PI200815’和感病材料‘新泰密刺’选系‘931’为亲本构建 F2和 F2：3群体（189 个株系），结合人工接种抗病鉴定技术和 SSR 分子标记技术，对抗白粉病基因进行了遗传分析和 QTL 定位。同时，利用已完成全基因组重测序的黄瓜核心种质材料，进行了全基因组关联分析，在整个基因组范围内挖掘白粉病抗性基因。具体研究结果如下：
（1）通过对 P1、P2、F1、F2群体的抗病性鉴定，发现 F2群体中病情指数分布呈连续近似正太分布，推断其白粉病抗性是由多个基因控制的数量性状。
（2）利用基于黄瓜全基因组测序开发的 2112 对 SSR 引物，对亲本进行筛选，得到 129 对具有多态性；利用 F2分离群体构建了一张包含 129 对 SSR 标记、8 个连锁群的黄瓜遗传图谱。该图谱覆盖基因组长度 857.9 cM，平均图距 6.65cM，每个连锁群均可对应到黄瓜 7 条染色体。
（3）利用构建的遗传图谱，对F2和F2：3家系群体进行了三个季节的抗病鉴定和QTL定位分析，共检测到2个QTL位点，分别位于黄瓜1号和6号染色体，命名为pmQTL1.1和pmQTL6.1。其中，pmQTL1.1在三个季节中均被检测到，是一个主效的、稳定的QTL位点，位于标记SSR03860和SSR14445之间，LOD最高达到15.33，表型解释率最高达到37.9%。pmQTL6.1只在一个季节检测到，LOD值仅为3.31，表型解释率为7%，为微效的不稳定的QTL位点。
（4）利用生物信息学，结合基因组序列信息，对 pmQTL1.1 定位区域进行了基因预测，在标记 SSR03860-SSR14445 之间的物理距离约为 689kb，预测到 95 个候选基因，包含 2 个 NBS 类抗病蛋白，1 个控制细胞程序性死亡蛋白，2 个逆境调控蛋白。
（5）利用重测序的核心种质，进行了黄瓜白粉病抗性基因的全基因组关联分析，检测到 6个可靠的信号位点:pmG1.1、pmG2.1、pmG4.1、pmG5.1、pmG5.2、pmG5.3，其中 pmG2.1、pmG5.2和 pmG5.3 被重复检测到，pmG1.1、pmG5.2、pmG5.3 与前人获得的 QTL 定位结果相吻合。
关键词：黄瓜，白粉病，抗性基因，QTL，GWAS
AbstractPowdery mildew is one of the most important diseases for cucumber worldwide. It can cause seriouslosses for cucumber production. Studies on cucumber powdery mildew got more and more attention athome and abroad. But because of the difference between the genetic background of experimentmaterials, physiological strains and the methods used for the identification of the resistence, researchershad drawn various conclusions. The information about gene mapping of the resistance and themolecular markers linked to the resistance were not good enough to satisfy the marker assisted selection(MAS) breeding. In this study, F2and F2:3population, derived from cucumber lines PI200815 (highresistance to powdery mildew) and 931(a selected inbred line from the cultived variety “xintaimici”
with high susceptible to powdery mildew), were used as plant materials to analyze the inheritance anddetect quantitative trait loci (QTL) for the resistance. The artificial inoculation identification methodand SSR molecular marker technology were employed in the present study. At the same time, taking theadvantage of the whole-genome re-sequenced core germplasm, we conducted Genome-WideAssociation Study (GWAS) to detect the powdery mildew resistence gene in the whole genome. Themain results were summarized as following:
(1)We conducted the resistance identification for P1、P2、F1、F2and F2:3in three seasons. Theresults showed that the disease index (DI) in F2population had continuroius characteristics and fit thenomal distribution. It was concluded the powdery mildew resistence in PI200815 is a quantitative traitcontrolled by several genes.
(2)2112 pairs of SSR primers, developed by using cucumber whole genome sequence, were used toscreen the polymorphism between the two parental lines. There were 129 primers generatingpolymorphism amplicons. Using F2segregation population, a genetic map with 129 markers wasconstructed. It consisted of 8 linkage groups corresponding to 7 chromosomes of cucumber. The totallength of the map is 857.9cM with an average interval of 6.65cM.
(3) QTL analysis of the resistance to powdery mildew was conducted using the genetic map andthe identification results in three seasons. Two QTLs, named pmQTL1.1 and pmQTL6.1, were detectedand located on chromosome 1 (Chr.1) and Chr.6 of cucumber, respectively. The pmQTL1.1 was detectedin the all three seasons and considered as a steady major QTL with the flanking markers of SSR03860and SSR14445. It accounted for phenotypic variances of 37.9% with logarithm of odds (LOD) score of15.33. The pmQTL6.1 was detected only in one season with the LOD of 3.31 and phenotypic variancesof 7%. It was considered as an unstable minor QTL.
(4)According to the genome sequence information, the physical distance of the flanking markers ofpmQTL1.1 was 689kb containing 95 candidate genes. Among the candidate genes, there were twoNBS-like resistant proteins, one protein related to programmed cell death and two stress regulatedproteins.
(5)We conducted GWAS for the resistance gene using the re-sequenced core germplasm. Six steady singals were detected: pmG1.1, pmG2.1, pmG4.1, pmG5.1, pmG5.2 and pmG5.3. Three loci,pmG2.1, pmG5.2 and pmG5.3, were detected repeatedly. And pmG1.1, pmG5.2,pmG5.3 were consistentwith the previous QTL mapping results.
Key word: cucumber, powdery mildew, resistance gene, QTL, GWAS
目 录
第一章 绪 论
1.1 黄瓜白粉病及其抗性基因定位研究概况
1.1.1 黄瓜白粉菌简介
1.1.2 黄瓜白粉病抗病性鉴定方法
1.1.3 黄瓜白粉病发病规律及防治方法
1.1.4 抗性遗传规律研究
1.1.5 抗性分子标记与遗传定位的研究
1.2 黄瓜白粉病抗病基因挖掘的研究
1.2.1 植物病原互作模式的研究进展
1.2.2 植物抗病基因的类型
1.2.3 黄瓜抗白粉病基因挖掘的研究
1.3 黄瓜白粉病抗性基因全基因组关联分析（GWAS）
1.3.1 关联分析的优势
1.3.2 连锁不平衡与遗传连锁的关系
1.3.3 植物中影响 LD 的主要因素
1.3.4 关联分析的一般步骤
1.3.5 关联分析在黄瓜研究中的应用
1.4 本研究的目的意义与技术路线
1.4.1 研究目的与意义
1.4.2 技术路线
第二章 黄瓜种质 PI200815 抗白粉病基因定位研究
2.1 材料与方法
2.1.1 实验材料
2.1.2 白粉病抗病性鉴定
2.1.3 构建 SSR 连锁图谱
2.1.4 黄瓜白粉病抗病基因的 QTL 分析
2.1.5 主要试剂、仪器设备
2.2 PI200815 抗病基因定位结果与分析
2.2.1 遗传群体抗病性鉴定结果
2.2.2 SSR 多态性分析
2.2.3 遗传图谱的构建
2.2.4 黄瓜抗白粉病 QTL 定位分析
2.2.5 主效 QTL 区域基因预测
2.3 讨论
第三章 黄瓜白粉病抗病基因的全基因组关联分析
3.1 GWAS 材料方与法
3.1.1 实验材料
3.1.2 核心种质抗病性鉴定
3.1.3 全基因组关联分析
3.2 GWAS 结果与分析
3.2.1 抗病性鉴定结果
3.2.2 全基因组关联分析
3.2.3 关联分析结果与 QTL 结果比较
3.3 讨论
第四章 全文结论
参考文献
附 录
致 谢
