【第一章】黃瓜白粉病抗病基因探析緒論
【第二章】黃瓜種質PI200815抗白粉病基因定位研究
【第三章】黃瓜白粉病抗病基因的全基因組關聯分析
【結論/參考文獻】黃瓜白粉病抗性基因挖掘結論及參考文獻
摘 要
白粉?。╬owdery mildew）是為害黃瓜的世界性病害之一，常對生產造成嚴重損失。國內外對于黃瓜白粉病的研究都比較重視，但是由于所使用研究材料的遺傳背景、病原菌生理小種以及抗病性鑒定方法等的差異，不同學者的研究結果存在很大差異，已報道的抗病基因定位信息和與抗性的連鎖標記尚不能滿足分子育種的需要。本研究以抗白粉病的黃瓜自交系‘PI200815’和感病材料‘新泰密刺’選系‘931’為親本構建 F2和 F2：3群體（189 個株系），結合人工接種抗病鑒定技術和 SSR 分子標記技術，對抗白粉病基因進行了遺傳分析和 QTL 定位。同時，利用已完成全基因組重測序的黃瓜核心種質材料，進行了全基因組關聯分析，在整個基因組范圍內挖掘白粉病抗性基因。具體研究結果如下：
（1）通過對 P1、P2、F1、F2群體的抗病性鑒定，發現 F2群體中病情指數分布呈連續近似正太分布，推斷其白粉病抗性是由多個基因控制的數量性狀。
（2）利用基于黃瓜全基因組測序開發的 2112 對 SSR 引物，對親本進行篩選，得到 129 對具有多態性；利用 F2分離群體構建了一張包含 129 對 SSR 標記、8 個連鎖群的黃瓜遺傳圖譜。該圖譜覆蓋基因組長度 857.9 cM，平均圖距 6.65cM，每個連鎖群均可對應到黃瓜 7 條染色體。
（3）利用構建的遺傳圖譜，對F2和F2：3家系群體進行了三個季節的抗病鑒定和QTL定位分析，共檢測到2個QTL位點，分別位于黃瓜1號和6號染色體，命名為pmQTL1.1和pmQTL6.1。其中，pmQTL1.1在三個季節中均被檢測到，是一個主效的、穩定的QTL位點，位于標記SSR03860和SSR14445之間，LOD最高達到15.33，表型解釋率最高達到37.9%。pmQTL6.1只在一個季節檢測到，LOD值僅為3.31，表型解釋率為7%，為微效的不穩定的QTL位點。
（4）利用生物信息學，結合基因組序列信息，對 pmQTL1.1 定位區域進行了基因預測，在標記 SSR03860-SSR14445 之間的物理距離約為 689kb，預測到 95 個候選基因，包含 2 個 NBS 類抗病蛋白，1 個控制細胞程序性死亡蛋白，2 個逆境調控蛋白。
（5）利用重測序的核心種質，進行了黃瓜白粉病抗性基因的全基因組關聯分析，檢測到 6個可靠的信號位點:pmG1.1、pmG2.1、pmG4.1、pmG5.1、pmG5.2、pmG5.3，其中 pmG2.1、pmG5.2和 pmG5.3 被重復檢測到，pmG1.1、pmG5.2、pmG5.3 與前人獲得的 QTL 定位結果相吻合。
關鍵詞：黃瓜，白粉病，抗性基因，QTL，GWAS
Abstract
Powdery 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 錄
第一章 緒 論
1.1 黃瓜白粉病及其抗性基因定位研究概況
1.1.1 黃瓜白粉菌簡介
1.1.2 黃瓜白粉病抗病性鑒定方法
1.1.3 黃瓜白粉病發病規律及防治方法
1.1.4 抗性遺傳規律研究
1.1.5 抗性分子標記與遺傳定位的研究
1.2 黃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進展
1.2.2 植物抗病基因的類型
1.2.3 黃瓜抗白粉病基因挖掘的研究
1.3 黃瓜白粉病抗性基因全基因組關聯分析（GWAS）
1.3.1 關聯分析的優勢
1.3.2 連鎖不平衡與遺傳連鎖的關系
1.3.3 植物中影響 LD 的主要因素
1.3.4 關聯分析的一般步驟
1.3.5 關聯分析在黃瓜研究中的應用
1.4 本研究的目的意義與技術路線
1.4.1 研究目的與意義
1.4.2 技術路線
第二章 黃瓜種質 PI200815 抗白粉病基因定位研究
2.1 材料與方法
2.1.1 實驗材料
2.1.2 白粉病抗病性鑒定
2.1.3 構建 SSR 連鎖圖譜
2.1.4 黃瓜白粉病抗病基因的 QTL 分析
2.1.5 主要試劑、儀器設備
2.2 PI200815 抗病基因定位結果與分析
2.2.1 遺傳群體抗病性鑒定結果
2.2.2 SSR 多態性分析
2.2.3 遺傳圖譜的構建
2.2.4 黃瓜抗白粉病 QTL 定位分析
2.2.5 主效 QTL 區域基因預測
2.3 討論
第三章 黃瓜白粉病抗病基因的全基因組關聯分析
3.1 GWAS 材料方與法
3.1.1 實驗材料
3.1.2 核心種質抗病性鑒定
3.1.3 全基因組關聯分析
3.2 GWAS 結果與分析
3.2.1 抗病性鑒定結果
3.2.2 全基因組關聯分析
3.2.3 關聯分析結果與 QTL 結果比較
3.3 討論
第四章 全文結論
參考文獻
附 錄
致 謝
