Table_7_Fine mapping of a QTL and identification of candidate genes associated with cold tolerance during germination in peanut (Arachis hypogaea L.) on chromosome B09 using whole genome re-sequencing.xlsx

Low temperatures significantly affect the growth and yield of peanuts. Temperatures lower than 12 °C are generally detrimental for the germination of peanuts. To date, there has been no report on precise information on the quantitative trait loci (QTL) for cold tolerance during the germination in peanuts. In this study, we developed a recombinant inbred line (RIL) population comprising 807 RILs by tolerant and sensitive parents. Phenotypic frequencies of germination rate low-temperature conditions among RIL population showed normally distributed in five environments. Then, we constructed a high density SNP-based genetic linkage map through whole genome re-sequencing (WGRS) technique and identified a major quantitative trait locus (QTL), qRGRB09, on chromosome B09. The cold tolerance-related QTLs were repeatedly detected in all five environments, and the genetic distance was 6.01 cM (46.74 cM - 61.75 cM) after taking a union set. To further confirm that qRGRB09 was located on chromosome B09, we developed Kompetitive Allele Specific PCR (KASP) markers for the corresponding QTL regions. A regional QTL mapping analysis, which was conducted after taking the intersection of QTL intervals of all environments into account, confirmed that qRGRB09 was between the KASP markers, G22096 and G220967 (chrB09:155637831–155854093), and this region was 216.26 kb in size, wherein a total of 15 annotated genes were detected. This study illustrates the relevance of WGRS-based genetic maps for QTL mapping and KASP genotyping that facilitated QTL fine mapping of peanuts. The results of our study also provided useful information on the genetic architecture underlying cold tolerance during germination in peanuts, which in turn may be useful for those engaged in molecular studies as well as crop improvement in the cold-stressed environment.

低温会显著影响花生的生长与产量。通常而言，低于12℃的温度会对花生萌发产生不利影响。截至目前，尚无针对花生萌发期耐冷性数量性状位点（quantitative trait loci, QTL）的精准相关报道。本研究以耐冷与冷敏感亲本构建了包含807个株系的重组自交系（recombinant inbred line, RIL）群体。该RIL群体在5个环境下的低温萌发率表型频率均呈正态分布。随后，本研究通过全基因组重测序（whole genome re-sequencing, WGRS）技术构建了高密度单核苷酸多态性（single nucleotide polymorphism, SNP）遗传连锁图谱，并在B09染色体上定位到一个主效数量性状位点qRGRB09。与耐冷性相关的QTL在全部5个环境中均被重复检测到，取并集后其遗传距离为6.01 cM（区间为46.74 cM至61.75 cM）。为进一步验证qRGRB09定位于B09染色体，本研究针对该QTL区间开发了竞争性等位基因特异性PCR（Kompetitive Allele Specific PCR, KASP）标记。通过整合所有环境的QTL区间取交集后开展的区域QTL定位分析证实，qRGRB09位于KASP标记G22096与G220967之间（chrB09:155637831–155854093），该区间大小为216.26 kb，共注释到15个基因。本研究证实了基于WGRS的遗传图谱在QTL定位与KASP基因分型中的应用价值，可为花生QTL精细定位提供有力支撑。本研究结果还解析了花生萌发期耐冷性的遗传基础，可为从事分子生物学研究以及低温胁迫环境下作物遗传改良的科研人员提供参考依据。