Table_4_Construction of a High-Density Genetic Map and Analysis of Seed-Related Traits Using Specific Length Amplified Fragment Sequencing for Cucurbita maxima.docx

Seed traits are agronomically important for Cucurbita breeding, but the genes controlling seed size, seed weight and seed number have not been mapped in Cucurbita maxima (C. maxima). In this study, 100 F2 individual derived from two parental lines, “2013-12” and “9-6”, were applied to construct a 3,376.87-cM genetic map containing 20 linkage groups (LGs) with an average genetic distance of 0.47 cM using a total of 8,406 specific length amplified fragment (SLAF) markers in C. maxima. Ten quantitative trait loci (QTLs) of seed width (SW), seed length (SL) and hundred-seed weight (HSW) were identified using the composite interval mapping (CIM) method. The QTLs affecting SW, SL and HSW explained a maximum of 38.6%, 28.9% and 17.2% of the phenotypic variation and were detected in LG6, LG6 and LG17, respectively. To validate these results, an additional 150 F2 individuals were used for QTL mapping of SW and SL with cleaved amplified polymorphic sequence (CAPS) markers. We found that two major QTLs, SL6-1 and SW6-1, could be detected in both SLAF-seq and CAPS markers in an overlapped region. Based on gene annotation and non-synonymous single-nucleotide polymorphisms (SNPs) in the major SWand SL-associated regions, we found that two genes encoding a VQ motif and an E3 ubiquitin-protein ligase may be candidate genes influencing SL, while an F-box and leucinerich repeat (LRR) domain-containing protein is the potential regulator for SW in C. maxima. This study provides the first high-density linkage map of C. maxima using SNPs developed by SLAF-seq technology, which is a powerful tool for associated mapping of important agronomic traits, map-based gene cloning and marker-assisted selection (MAS)-based breeding in C. maxima.

种子性状在南瓜属（Cucurbita）育种中具有重要的农艺学价值，但目前尚未在笋瓜（Cucurbita maxima，简称C. maxima）中定位到控制种子大小、种子重量及种子数量的相关基因。本研究以两个亲本材料‘2013-12’和‘9-6’构建的100株F₂单株为试材，利用总计8406个特异性长度扩增片段（specific length amplified fragment，SLAF）标记，构建了包含20个连锁群（linkage groups，LGs）、总遗传距离为3376.87 cM、平均遗传距离为0.47 cM的笋瓜高密度遗传图谱。本研究采用复合区间作图法（composite interval mapping，CIM），共鉴定到控制种子宽度（seed width，SW）、种子长度（seed length，SL）及百粒重（hundred-seed weight，HSW）的10个数量性状位点（quantitative trait loci，QTLs）。上述分别调控SW、SL及HSW的QTLs可解释的表型变异率最高分别达38.6%、28.9%及17.2%，且分别定位在连锁群LG6、LG6及LG17上。为验证上述结果，本研究额外选取150株F₂单株，利用酶切扩增多态性序列（cleaved amplified polymorphic sequence，CAPS）标记对SW和SL进行QTL定位。研究发现，两个主效QTLs：SL6-1与SW6-1，可在SLAF-seq与CAPS标记的重叠区域中同时被检测到。基于主效SW及SL关联区域的基因注释信息与非同义单核苷酸多态性（non-synonymous single-nucleotide polymorphisms，SNPs）分析，本研究发现：在笋瓜中，两个分别编码VQ基序蛋白与E3泛素连接酶的基因可能为调控SL的候选基因，而包含F-box与富亮氨酸重复（leucine-rich repeat，LRR）结构域的蛋白则可能是调控SW的潜在调控因子。本研究首次利用SLAF-seq技术开发的单核苷酸多态性标记构建了笋瓜高密度遗传图谱，该图谱可作为笋瓜重要农艺性状关联定位、图位克隆及基于标记辅助选择（marker-assisted selection，MAS）育种的有力工具。