石斛属叶绿体DNA序列的系统发育及变异位点分析

目的对石斛属叶绿体IRa(trn V-GAC~trn R-ACG)序列进行系统发育和变异位点分析,深入了解石斛属10个物种此序列所含9个基因的异同,为石斛属植物的系统进化历程及DNA条形码的研究作重要探索。方法用改良试剂盒法提取石斛属10个样本总DNA,自行设计引物,采用聚合酶链式反应(polymerase chain reaction,PCR)及T载体对PCR产物进行克隆转化,扩增石斛属叶绿体IRa序列的DNA序列,并与NCBI数据库进行序列比对完成序列拼接;绘制此区域序列的基因结构图,对10条目的序列进行差异位点、遗传距离、系统进化关系等分析。结果目的序列长约7 800 bp,10条目的序列共存在55个差异位点,在基因trn V-GAC、rrn16、trn I-GAU、trn A-UGC、orf42、rrn23及基因间隔区上分布的个数分别是1、8、12、6、1、9和18个;系统发育分析将石斛属10个样本聚为4个类群。结论建立了测定石斛属叶绿体IRa序列的方法,对石斛属这一序列的系统发育和变异位点分析表明,此区域序列丰富,且较为集中分布的变异位点将为石斛属DNA条形码的进一步研究、石斛属资源的道地性评价及相关的种质资源鉴定提供较好的理论依据。

Phylogenetic and mutation sites analysis on chloroplast DNA sequences in Dendrobium Sw.

Objective To analyze the phylogenetic and mutation sites of IRa (trnV-GAC to trnR-ACG) sequence among 10 Dendrobium Sw. samples, to understand the similarities and differences of nine genes in this sequence, and to explore the system evolution and DNA barcode in species of Dendrobium Sw. Methods The improved kit method was used to extract total DNA and design primers, PCR method and T-vector cloning transformation were used to amplify the region of IRa and the sequences were obtained after the amplified fragment sequences were blasted in NCBI database and splicing. Genetic structures were drawn, and variable sites, genetic distance and phylogenetic relationship among 10 sequences were analyzed. Results The lengths of sequences were about 7 800 bp and 55 variable sites were found. The numbers of mutation points distributeed on trnV-GAC, rrn16, trnI-GAU, trnA-UGC, orf42, rrn23, and intergenic region were 1, 8, 12, 6, 1, 9, and 18, respectively. These 10 samples were clustered to four latitude-dependent groups based on IRa sequences. Conclusion A set of methods on sequence IRa is established. Phylogenetic and mutation point analysis indicate that there is a rich and relatively concentrated distribution variation loci among the research sequences. And it will provide a good theoretical basis on the further study of DNA barcodes, authentic assessment and identification of Dendrobium Sw. germplasm resources.