渝产大叶黄精及其近缘种的psbA-trnH序列分析

Analysis of PsbA-trnH Sequences of Polygonatum kingianum var.grandifolium Plant and Its Related Species in Chongqing

目的基于中药材DNA条形码分子鉴定法,对渝产大叶黄精及其近缘种进行psbA-trnH序列分析。方法采用试剂盒法提取DNA,扩增psbA-trnH序列并双向测序,CodonCode Aligner软件拼接,利用MEGA软件进行比对、计算、分析以及构建系统发育树。结果共分析出9个物种77条psbA-trnH序列(自测获取序列大叶黄精40条、近缘种24条,另从GenBank下载相关序列13条),长度为601~615 bp,GC含量为35.16%~35.49%,其中,大叶黄精psbA-trnH主导单倍型序列长度602 bp、GC含量35.22%;比对出单倍型12个,变异位点19个;计算出不同品种之间K2-P遗传距离为0.0017~0.0249,总体平均值为0.0121;UPGMA发育树能对大叶黄精与研究中其它品种进行有效聚类区分。结论psbA-trnH序列可作为大叶黄精及其易混淆近缘品种区分的有效辅助手段,为重庆市黄精药材种源保障提供参考。

Objective To analyze the psbA-trnH sequences of Polygonatum kingianum var.grandifolium plant and its related species in Chongqing based on DNA barcode molecular identification method of traditional Chinese medicine.Methods The DNA were extracted using the reagent kit method,and bidirectional sequencing was performed after amplification of their psbA-trnH sequences.And then the sequences were assembled by CodonCode Aligner,the phylogenetic trees were constructed after sequences alignment and computation in MEGA.Results A total of 77 psbA-trnH sequences of 9 species were achieved(40 sequences of Polygonatum kingianum var.grandifolium plant and 24 sequences of its related species were obtained by self-testing,and the other 13 related sequences were downloaded from GenBank),with the length of 601 to 615 bp and the GC content of 35.16%to 35.49%.Among the above sequences,the psbA-trnH sequence of master haplotype of P kingianum var.grandifolium was 602 bp in length and its GC content was 35.22%.The 12 haplotypes of the species were identified with the 19 variable sites.Based on Kimura 2-Parameter(K2-P)model,the genetic distance of the nine species was range from 0.0017 to 0.0249,with the overall average value of 0.0121.The constructed UPGMA phylogenetic tree was an effective method in clustering and distinguishing between P kingianum var.grandifolium and the other species.Conclusion The psbA-trnH sequences of P kingianum var.grandifolium can use as an effective auxiliary tool for discriminating it from easily-confused species,providing a reference for ensuring the source of Polygonati Rhizoma in Chongqing.