利用測序基因分型建立甜瓜高密度連鎖圖譜

Abstract

測序基因分型（GBS）技術雖然能以低成本有效率地產生大量分子標幟，但是使用上受限於其潛在的錯誤序列資訊以及高比例的缺值。我們在本研究中使用GBS技術獲得109個甜瓜(Cucumis melo L.)重組自交系之大量單一核苷酸多型性（SNP）資訊，並將這些SNP分子標幟依據其在參考基因體序列DHL92上的物理位置排列。藉由調查SNP分子標幟彼此間的連鎖關係，我們發現有多個物理圖譜的片段與連鎖關係之調查結果相抵觸。剔除有明顯錯誤的SNP位點後，我們挑選出4,110個高可信度的SNP位點作為比對基準，對其餘的SNP位點進行獨立性檢定，通過檢定的分子標幟再使用軟體TASSEL 5.2內建的Full-Sib Family Haplotype (FSFHap) 演算法填補缺值。最終獲得共22,933個SNP位點，其平均缺值率為0.281%。以此最終產出之基因型資料建立連鎖圖譜，連鎖圖譜共涵蓋12條染色體，總長共1,088.3 cM，而分子標幟間的最大間距為6.0 cM。利用此連鎖圖譜我們成功地定位到多個已知控制果實性狀的數量性狀基因座（QTLs），此結果也驗證了我們採用的資料處理流程之可行性。此外對於64個收集系的遺傳分析顯示存在著明顯的族群結構且收集系的遺傳分群與瓜果外觀型態存在著關聯性。連鎖失衡衰退的分析顯示在兩個收集系分群中有著不同的連鎖失衡衰退速度。本研究顯示GBS技術確實可以有效地應用於定位甜瓜的QTLs上，另外也有助於對甜瓜的基因體結構做更進一步的研究。

Although genotyping-by-sequencing (GBS) enables the efficient and low-cost generation of large numbers of markers, the utility of resultant genotypes are limited, because they are enormously error-prone and contain high proportions of missing data. In this study, we generated single nucleotide polymorphism (SNP) markers for 109 recombinant inbred lines (RILs) of melon (Cucumis melo L.) using the GBS approach and ordered them according to their physical position on the draft double haploid line DHL92 genome. Next, by investigating associations between these SNPs, we discovered that some segments on the physical map conflict with linkage relationships. Therefore, to filter out error-prone loci, 4,110 SNPs in which we have a high degree of confidence were selected as anchors to test independence with respect to unselected markers, and missing data in the resultant dataset was then filled in using the Full-sib Family Haplotype (FSFHap) algorithm in the software TASSEL 5.2. On the basis of this analysis, 22,933 loci that have an average rate of missing data of 0.281% were used to construct a genetic map, which spans 1,088.3 cM across 12 chromosomes and has a maximum spacing of 6.0 cM. Use of this high-quality linkage map enabled the identification of several quantitative trait loci (QTL) known to control traits in fruit and validated our approach. The genetic analysis of 64 other diverse accessions showed the strong population structure and also the association between genetic groups and the types of fruit appearance in melon collection. The results of linkage disequilibrium (LD) suggested that the different level of LD decay existed in two subgroups of accessions. This study highlights the utility of GBS markers for the identification of trait-associated QTLs in melon and facilitates further investigation of genome structure.