利用关联作图法挖掘薄壳山核桃种仁单宁含量关键等位变异

Pecan is an important nut tree in the world, which has been widely cultivated in China. However, the quality of the kernel is reduced by high tannin concentration and astringency. Therefore, obtaining of molecular markers which association with tannin content of the kernel, is crucial for the germplasm innovation in this plant. Phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) are rate-limiting enzymes in the tannin synthesis pathway. In the present study, two PAL genes and three CHS genes, which shown high expression level in kernels, will be cloned and genotyped in an association population including 200 germplasm resources of pecan, and single nucleotide polymorphism (SNP) markers will be mined from each gene using Sanger sequencing. The structure of the associated group will assess using 30 simple sequence repeats (SSR) markers. Then, a linkage disequilibrium (LD) mapping method will be employed to analyze the association of molecular markers with the content of total tannin, total flavonoid, condensed tannin, ellagic acid and gallic acid in the associated group. After that, the SNP which exhibited significant correlation with kernel tannin content will be evaluated its influence of changes in the amino acid sequence and protein structure. Finally, the key mutation site will be converted into functional marker. In this research, we focus on the establishment of candidate gene-based LD mapping method of pecan and obtaining significant related SNP or haplotype with tannin content in this plant. This study will provide scientific basis for the utilization of marker-assisted selection (MAS) in pecan using LD strategy.

薄壳山核桃是重要的干果树种，近年来我国广泛引种，但因其种仁单宁含量高，多数品种涩味明显，造成品质降低。利用种仁单宁含量有广泛差异的种质资源，挖掘与单宁含量相关分子标记，对加快薄壳山核桃种仁低单宁育种具有重要意义。本研究以200份薄壳山核桃种质为材料，检测种仁5种单宁组分含量；用简单重复序列标记分析群体结构；克隆单宁合成限速酶苯丙氨酸解氨酶和查尔酮合酶基因，通过测序在群体中挖掘各基因单核苷酸多态性(SNP)位点；使用连锁不平衡(LD)策略，并考虑群体结构，计算基因内LD衰退距离，分析SNP及单倍型与种仁单宁含量的相关性，筛选显著相关等位变异；预测其对氨基酸序列和蛋白质结构的影响；将关键等位变异转化为功能标记。通过研究，明确基因内LD衰退距离，构建薄壳山核桃候选基因关联作图技术体系，获得与种仁单宁含量显著相关的等位变异，为关联作图应用于薄壳山核桃种仁低单宁分子标记辅助育种的可行性提供科学依据。

薄壳山核桃是世界范围的重要干果树种，因种仁单宁含量高、多数品种涩味明显，显著降低坚果品质。开发与种仁单宁含量关联的分子标记，有望加快种仁低单宁品种选育，推动产业发展。本研究利用基于候选基因的连锁不平衡策略，开展薄壳山核桃单宁含量关联等位变异挖掘。克隆了2个苯丙氨酸解胺酶基因和3个查尔酮合酶基因作为连锁不平衡作图的候选基因，在关联群体中共检测到519个等位变异，包括137个Indel和382个SNP。利用SNP和SSR标记，明确了关联群体的群体结构；明确了总单宁、缩合单宁、类黄酮总量、鞣花酸和没食子酸的含量分布。利用等位基因频率相关性系数的平方评估SNP间的LD值，发现随着基因长度的增加连锁不平衡迅速消失，在700bp处衰退至0.2以下，表明基于候选基因的LD作图在薄壳山核桃中是可行的。使用MLM模型，结合群体结构Q矩阵和Kinship矩阵开展关联作图，挖掘到10个与总单宁、缩合单宁、类黄酮总量、鞣花酸含量显著关联的SNP，基因型对表型的贡献率介于2.83%~8.21%。其中有3个SNP属于非同义替换，可能通过影响编码蛋白质的空间构象影响酶的活性，进而导致不同基因型个体单宁含量的差异。本研究明确了基于候选基因的LD作图方法在薄壳山核桃中的可行性，挖掘到10个与单宁含量显著相关的等位变异，有望促进薄壳山核桃品质性状相关的分子标记辅助育种。