花生出仁率主效QTL cqSPA09的精细定位和候选基因鉴定

Seed yield improvement is the most important objective of peanut breeding. The high shelling percentage breeding is an efficient way to increase seed yield. Recent research on shelling percentage is still limited to rough QTLs, the molecular mechanism underlying shelling percentage remains unclear. In our previous study, a major QTL cqSPA09（explained 10.47%-17.41% phenotypic variation）was detected repeatedly in a genetic interval of 1.5cM (physical interval of 3.5Mb) on A09 chromosome using RILs. In this project, the homozygous progenies of the heterozygotes at the target interval will be re-sequenced to develop new polymorphic molecular marker, and employed to construct the near isogenic lines (NILs). The NILs will be screened, and the recombined lines in the target interval will be identified. Finally cqSPA09 will be fine-mapped by combining genotype and phenotype of progenies of recombined lines. The differences in transcriptome profiles between pods of NILs will be analyzed. By integrating the functional prediction, different expression level and sequence variation of genes located in the target interval, the candidate genes of cqSPA09 will be isolated. The allelic variations of cqSPA09 will be detected by association analysis, and the molecular markers will be developed for high shelling percentage breeding. The identification and isolation of cqSPA09 will provide solid foundation for clarifying the molecular mechanism underlying shelling percentage, and carry molecular design breeding of peanut.

提高籽仁产量是花生育种的核心目标，培育高出仁率品种是提高籽仁产量的有效途径。目前花生出仁率的研究停留在QTL初定位水平，出仁率形成分子机理未知。项目前期利用RIL群体将一个出仁率主效QTL cqSPA09定位于A09连锁群上，四年贡献率为10.47%-17.41%，目标区间遗传距离1.5cM，物理距离3.5Mb。本项目拟利用目标区间残余杂合单株的纯合分离后代进行深度测序，开发目标区间分子标记，构建、分析近等基因系群体基因型和表型。鉴定目标区间重组单株，结合次级分离群体的表型及基因型鉴定结果，精细定位cqSPA09。同时对近等基因系的不同发育时期荚果进行转录组测序，基于定位区间的基因功能预测和结构、表达差异，鉴定候选基因，开展候选基因的关联分析，明确等位变异，开发高出仁率分子标记。cqSPA09基因的鉴定和分离对解析控制出仁率的分子机制，开展花生分子设计育种具有重要意义。

而籽仁产量是花生的经济产量，花生增产的核心是提高籽仁产量。花生出仁率是评价花生品种籽仁生产效率的重要指标。而花生荚果壳质量是决定出仁率的负向因子，荚果壳质量主要由荚果壳厚度决定，同时荚果壳厚度也是荚果壳的机械强度的影响因子之一，荚果壳厚度也是影响机械化脱壳的重要因素。.项目在前期花生出仁率主效QTL cqSPA09初定位的基础上，构建目标区段的姊妹近等基因系，构建了局部高密度遗传连锁图谱，通过重组单株基因型和表型检测, 精细定位cqSPA09，将QTL cqSPA09定位在了45Kbp区间内，这一区间有注释基因14条，设计引物对这14个基因进行了基因组序列分析和表达分析。花生荚果壳发育的细胞学观察揭示在荚果发育中期表皮细胞层开始出现发育差异，而对荚果壳发育过程的转录组学分析发现木质素代谢途径基因的差异表达可能是高低出仁率形成的分子基础。基于对定位区间内基因的功能预测、表达量和结构差异分析，确定*****K*为出仁率QTL cqSPA09候选基因，*****K*基因在荚果皮发育早中期特异表达，且在转录组分析中其转录本存在一个移码突变。本研究为高出仁率育种提供了关键的基因资源，并且进一步研究*****K*的分子功能对揭示木质素在荚果发育过程中的分子机理具有重要意义。.