開發最佳雙限制酶切位點標定法及其於甜瓜育種之應用

Abstract

分子標誌為遺傳研究與作物育種之重要工具。隨著次世代定序的發展，開發高通量分子標誌並進行基因型分型已成為作物遺傳育種研究的常用方法，但針對目標物種發展適合的最佳化流程仍為挑戰。為此，本研究奠基於次世代定序，優化雙限制酶切位點標定法 (double digest restricted associated DNA sequencing, ddRADseq) 之分子標誌開發與基因型分型，並以甜瓜抗白粉病之遺傳分析與育成抗病近同源系作為方法驗證。我們以電腦模擬限制酶於甜瓜基因體之切位並以小規模實驗評估限制酶組合。之後分別以PstI和XbaI作為稀有切位限制酶與常見切位限制酶TaqαI組合並加入SphI與MseI排除序列，應用於單一F2分離族群以建立高通量分子標誌基因型資料校正法及建立高密度連鎖圖譜之最佳化流程。基於此最佳化流程，以三個F2族群（A6、B2和C4）定位甜瓜抗白粉病的數量性狀位基因座 (quantitative trait loci, QTL)。分別在A6族群中偵測到位於第2條染色體的qPM2，在B2族群中偵測到位於第5條染色體的qPM5.B2與在C4族群中偵測到第5與第12條染色體上的qPM5.C4與qPM12。其後，針對第2、5和12條染色體上的QTL開發一系列TaqMan分子標誌用於分子標誌輔助回交育種之前景選拔與重組選拔，並結合ddRADseq評估輪迴親基因體恢復率進行背景選拔。選用A6與C4族群的抗病親作為供給親，並分別與帶有綠色及橙色果肉的優良親本作為輪迴親進行分子標誌輔助回交增進優良甜瓜親本的白粉病抗病性。經過二到三個回交世代後，成功將單一抗病QTL導入優良輪迴親的遺傳背景中，最終在綠色和橙色果肉的輪迴親遺傳背景中分別獲得6個帶有白粉病抗病QTL的近同源系。從分子標誌開發完成到抗病近同源系的育成僅花費三年半的時間，顯示基於次世代定序的高通量基因分型技術可以有效與作物育種結合，提升作物分子育種的效率。

Molecular markers are important tools for genetic studies and crop breeding. With the development of next-generation sequencing, high-throughput genotyping has become a common tool for crop breeding. However, optimizing the genotyping process for each target crop is still challenging. Therefore, this study aimed to optimize the double digest restriction-site associated DNA sequencing (ddRADseq) genotyping platform and implement the optimized procedure in melon as a proof-of-concept. The framework consists of the identification of the genetic architecture of powdery mildew resistance in melon and the development of the powdery mildew resistance near-isogenic lines. We applied in silico digestion and empirical tests to evaluate candidate enzyme combinations in melon. Two candidate enzyme combinations were further applied to an F2 population, which were PstI-TaqαI-SphI and XbaI-TaqαI-MseI, for optimization of a high-throughput genotyping data error correction and linkage map construction. Based on the optimized procedure, quantitative trait locus (QTL) mapping of melon powdery mildew resistance was applied to three F2 populations, A6, B2, and C4. QTL were identified on chromosomes 2, 5, and 12, which were qPM2 in A6, qPM5.B2 in B2 and qPM5.C4, and qPM12 in C4. A series of TaqMan assays targeting QTL were developed and validated for foreground and recombinant selection, complemented with the ddRADseq genotyping system to evaluate the recurrent parent genome recovery. Three marker-assisted backcrossing (MABC) programs using resistant donor parents from A6 and C4 crossed with elite susceptible recurrent parents with green and orange fruit flesh were implemented. After two to three cycles of MABC, individual QTL was successfully introgressed into elite genetic backgrounds, giving six powdery mildew resistance near-isogenic lines in each green- and orange-flesh background. In three and a half years, we have achieved from marker development to the production of isogenic lines. This study demonstrated the power of high-throughput genotyping and its efficient implementation in molecular breeding.