山核桃属叶绿体基因组微卫星特征分析及分子标记开发

Characterization of Microsatellite in Chloroplast Genome of the Genus Carya and Marker Development

本研究利用山核桃属6个不同物种的叶绿体基因组(chloroplast genome DNA, cpDNA)序列信息,分析其简单重复序列(chloroplast simple sequence repeat, cpSSR)特征及开发分子标记。结果表明,山核桃属各物种叶绿体平均长度为163 048 bp,每个基因组平均有224个微卫星,cpSSR分布频率平均约为1.37个/kb。山核桃属cpSSR以A/T类型的单核苷酸重复基序为主,占微卫星总数的85.79%,各物种碱基重复基序类型较为一致;cpSSR在叶绿体基因组上的分布主要集中在大单拷贝区(large single copy, LSC)和小单拷贝区(small single copy, SSC),在两个反向重复序列(inverted repeat sequence, IRa和IRb)分布最少;山核桃属各物种cpSSR主要分布于cpDNA的非编码区。以薄壳山核桃的cpSSR为基准,其他物种与其进行BLAST比对,结果发现共有62种cpSSR (包括34种完全型,28种复合型cpSSR)具有种间差异,对具有种间差异的cpSSR进行引物设计,共成功设计出61对引物。以山核桃属5个物种共9个样品的DNA为模板,对这61对引物进行PCR扩增。选用的61对引物中有49对引物扩增出的产物有目的条带,其中6对引物扩增良好,在种间具有明显的多态性。本研究为山核桃属植物的遗传多样性、系统发育、种质资源鉴定等研究提供理论基础。

In this study, the chloroplast genome DNA(cpDNA) sequence of 6 species in Carya were used to analyze the characteristics of their chloroplast simple sequence repeat(cpSSR) and develop markers. The results showed that the average length of chloroplast genome in Carya was 163 048 bp, with an average of 224 microsatellites, and the average distribution frequency of cpSSR was about 1.37/kb. In Carya, the mononucleotide repeat motif of A/T was the predominant repeat type, accounting for 85.79% in total, and the type of motifs in different species were relatively consistent;the cpSSRs in cpDNA were mainly distributed in large single copy(LSC) and small single copy(SSC), with the lowest in two inverted repeat sequence(IRa and IRb);cpSSRs of Carya species were mainly distributed in the non-coding regions of cpDNA. Based on cpSSR of Carya illinoinensis, a BLAST comparison with other species revealed a total of 62 cpSSRs(including 34 perfect and 28 compound cpSSRs) exhibiting interspecific differences. Primers were designed for these cpSSRs with interspecies variation, and 61primers were successfully designed. Using 9 DNA samples from 5 different species in Carya as templates, these 61primers were amplified by PCR. Among 61 pairs of primers, 49 pairs of primers were amplified with target bands,and 6 pairs of primers were well amplified and showed obvious polymorphism between species. This study provides a theoretical basis for the study of genetic diversity, phylogeny and germplasm resource identification of Carya.