三七叶绿体分子标记的开发与应用研究

Development and application of chloroplast molecular markers in Panax notoginseng

该研究采用三七叶绿体全基因组开发分子标记(cpSSR,cpSNP,cpIndel),对来源于云南省9个主要三七产地,共计89份三七个体进行实验验证,以期为后续三七种质资源的评价与筛选提供理论支撑。该研究使用的4个三七叶绿体全基因组下载自NCBI数据库,GenBank编号分别为KJ566590,KP036468,KR021381,KT001509。通过序列比对分析,确定多态性位点(SNP与Indel)30个,其中cpSNP 16个,cpIndel 14个;并且cpSNP与cpIndel在基因间区的占比远超基因区。开发的cpSSR 87~89个,重复单元以三碱基为主,占比70%~71%,二碱基最少,占比7%。根据比对分析结果,开发cpDNA分子标记18个,其中cpSSR引物7个,cpIndel引物6个,cpSNP引物5个。该研究采用MatK与ycf1基因为对照。根据DNA凝胶电泳条带分析结果确认引物cpSSR-5,pgcpir019,pncp08可用于区分9个不同三七栽培居群。通过比较分析序列长度、群体π和平均核苷酸差异数等指标表明cpSSR-5与pgcpir019可用于区分人参属不同物种,但pncp08仅可用于区分三七不同栽培居群。此外,引物pncp-M(基于MatK基因)区分三七栽培居群效果弱于cpSSR-5,pgcpir019,pncp08。

The molecular markers(cpSSR, cpSNP and cpIndel) were developed based on the whole genome sequence of Panax notoginseng chloroplast genome, which provide a powerful tool for the evaluation and analysis of the future P. notoginseng germplasm resources. The 89 P. notoginseng samples from 9 groups were used for the experiment, and the data for the study were derived from NCBI and the GenBank numbers were: KJ566590, KP036468, KR021381 and KT001509. Through sequence alignment, 30 polymorphic sites(SNP and Indel) were identified, including 16 cpSNP and 14 cpIndel;cpSNP and cpIndel accounted for far more than the gene region in the intergenic region. The developed cpSSR reached 87-89, the repeat unit was mainly composed of trinucleotide, accounting for 70%-71%, and the dinucleotide was the least, accounting for 7%. Eighteen cpDNA molecular markers were developed, including 7 cpSSR primers, 6 cpIndel primers, and 5 cpSNP primers. The MatK gene and ycf1 primers were chosen as control. According to the results of DNA gel electrophoresis, cpSSR-5, pgcpir019 and pncp08 can be used to distinguish different cultivated populations of P. notoginseng. Among them, cpSSR-5 and pgcpir019 can also be used to distinguish the inter-species resources of ginseng by comprehensive sequence length, population π value and average nucleotide difference. However, pncp08 can only be used to distinguish different populations of P. notoginseng. In addition, the effect of distinguishing the groups of P. notoginseng, which the primer pncp-M(based on the MatK gene) is weaker than the cpSSR-5, pgcpir019 and pncp08.