结合连锁分析和BSA-seq挖掘莲子产量和品质的关键候选基因

Lotus seed is rich in nutritional and pharmacological active ingredients. The seed yield of seed lotus cultivars is normally low, with the mean product of dried seed about 125-160 kg/667 m2. The nutritional and hygienical components of lotus seed, considered only as optional requests currently, are not set as the breeding goal. Therefore, understanding the genetic mechanisms controlling yield and quality of lotus seed and identifying their key candidate genes would accelerate the breeding of new seed lotus cultivars, and achieve the goal of high yield and good quality. In this project, SNP markers will be characterized by population whole-genome sequencing, and a genetic linkage map of lotus will be constructed based on the F2 population derived from two lotus cultivars that show significant difference in seed yield and quality. Phenotype data will be recorded for three years both for the yield (number of seedpod per unit area, number of carpel, seed setting rate, and single seed weight) and quality (the content of soluble sugar, protein, starch, and amylose etc.) of the fresh- and mature-seed, and QTLs controlling those traits will be identified. Lotus DNA of the F2 lines with extreme phenotypes are then pooled and subjected to BSA-seq, and candidate markers or regions closely linked with the target traits will be obtained. Combining the results of QTL mapping and BSA-seq, key candidate genes linked to the target traits will be finally identified. These candidate genes will be transformed into Arabidopsis thaliana for functional validation on seed development. The project will provide theoretical and technical support for the genetic improvement of lotus seed with high yield and good quality, and marker-assisted breeding of new seed lotus cultivars.

莲子具有丰富的营养保健和药理活性成分，但其产量较低。栽培子莲品种平均亩产带壳干莲子仅125-160 kg，且莲子品质（营养保健成分只作为辅助数据）未纳入子莲育种的目标范围，因此解析莲子产量和品质的遗传规律，定位与挖掘关键候选基因，可为高产优质子莲新品种的选育提供有效分子标记。本项目拟以已构建的亲本间莲子产量和品质存在显著差异的F2分离群体为材料，利用全基因组测序检测群体的SNP标记，构建莲遗传连锁图谱；检测鲜莲子和成熟莲子的产量（单位面积莲蓬数、心皮数、结实率、单粒莲子重等）和品质（可溶性糖、蛋白质、淀粉含量等）的QTL位点；选取F2群体中表型差异极端的单株构建混合DNA池并进行BSA-seq，获得与目标性状紧密关联的分子标记或候选区域；结合QTL定位和BSA-seq结果，获得目标性状的候选基因，利用转化拟南芥完成对候选基因的功能验证，可加快莲子产量与品质的遗传改良及分子辅助育种进程。

莲是我国种植面积最大、总产量和总产值均居于首位的水生蔬菜。其果实莲子具有丰富的营养保健和药理活性成分，特别是富含强抗氧化能力的黄酮化合物和抗消化功能的直链淀粉。解析莲子产量和品质性状的遗传机制，发掘其关键候选基因，对高产优质莲子遗传改良具有重要意义。通过本项目实施，取得的重要研究结果如下：（1）利用全基因组重测序技术检测了F2分离群体的全基因组SNP标记，构建了一张包括2201个分子标记、8个连锁群的莲遗传连锁图谱，图谱总长度为1029.96 cM。（2）连续3年调查F2分离群体的莲子产量和品质性状。各性状表现出连续性变异，呈明显的单峰分布，符合数量性状遗传特点。结实数与成熟莲子单粒重遗传力相对较高。（3）完成了三年的莲子产量和品质性状的QTL定位，一共检测到154个QTL区间，产量性状指标共检测到56个QTL区间，品质性状共检测到98个QTL区间，单个QTL对表型变异的解释率为1.98%-48.17%。多效性QTL有31个，三年间重复的QTL有5个。（4）开展了莲子产量和品质性状的BSA分析。在BSA与QTL位点重叠区间，挖掘到与莲子淀粉合成相关的候选基因Nn7g37382和Nn7g37394，产量性状候选基因Nn6g32047.76，Nn8g40052.4，Nn5g27097. 1和Nn5g26947.2。（5）完成了莲子发育和淀粉合成相关关键候选基因的时空表达模式和亚细胞定位分析，并进行了功能验证。通过瞬时表达证明了NnAGPL2a和NnGBSSI对莲子淀粉和直链淀粉合成和累积的作用；证明了NnLEAs基因在莲子芯发育及种子干燥脱水过程中的重要作用；初步确认了NnNF-YB1和NnNF-YB4对莲子发育的功能作用，将通过转化拟南芥突变体和病毒诱导基因沉默技术侵染莲蓬证明NnNF-YB1和NnNF-YB4调控莲子发育的功能。本项目为进一步解析莲子产量和品质性状的遗传机制奠定了良好基础，为快速高效选育高产、优质的特色子莲新品种提供分子选择依据。