基于叶绿体psbA-trnH和核糖体5S rRNA基因间隔区序列的石斛种间和种内鉴别

Interspecific and intraspecific identification of Dendrobium based on the psbAtrnH intergenic region sequences and the 5S rRNA gene spacer sequences

采用直接测序法对12种石斛叶绿体psb A-trn H基因间隔区序列和核糖体5S r RNA基因间隔区序列进行测序,用克隆测序法对叠鞘石斛dq-2品种和dq-5品系(各20份样品)psb A-trn H基因间隔区序列进行测序,所得序列采用Squencher4.14拼接后,采用Bioedit 7.0软件对获得的psb A-trn H基因间隔区序列和5S r RNA基因间隔区序列进行序列比对和排序,用Dnasp5.0软件分析序列核苷酸多态性,用MEGA 5.2计算种内和种间kimura2-parameter(K2P)遗传距离,并构建系统发育树(NJ树).结果显示,石斛叶绿体psb A-trn H基因间隔区序列平均长度为742.3 bp,有72个变异位点,其中信息位点33个;核糖体5S r RNA基因间隔区序列平均长度为336.4 bp,存在213个变异位点,其中信息位点139个;以上两个基因片段的组合序列,能对叠鞘石斛、铁皮石斛、球花石斛、细叶石斛、细茎石斛、齿瓣石斛、鼓槌石斛、尖刀唇石斛和金钗石斛等9种石斛进行有效鉴别,可揭示铁皮石斛不同居群间的差异,而且可根据该组合序列中存在的一个单核苷酸多态性位点(Single Nucleotide Polymorphism,SNP)对叠鞘石斛dq-2品种和dq-5品系进行区分.本研究表明,psb A-trn H基因间隔区序列和5S r RNA基因间隔区序列组合后显示出多序列组合策略在石斛种间和种内的分子鉴别中的优势,可为石斛属植物及药材鉴别提供分子依据.

This research aimed to investigate the interspecific and intraspecific identification of Dendrobium by using the multi-locus method so as to provide a molecular basis for Dendrobium identification through the combination of chloroplast psbA-trnH intergenic region sequences and ribosome 5S rRNA gene spacer sequences.PCR direct sequencing was applied to detect the chloroplast psbA-trnH intergenic region sequences as well as the ribosome 5S rRNA gene spacer sequences of12 Dendrobium species,while the psbA-trnH intergenic region sequences of Dendrobium denneanum dq-2 variety and dq-51 ine were cloned and sequenced for single nucleotide polymorphism（SNP） analyzing.The sequences were analyzed by the software Sequencher4.14,Bioedit7.0,MEGA5.2 and Dansp5.0;the interspecific and intraspecific Kimara-2-Parameter（K2P）distances were also calculated.The phylogenetic tree（using Neighbor joining method） was constructed with Bulbophyllum odoratissimum and Bletilla striata as outgroup.The results showed an average length of chloroplast psbA-trnH gene sequences in Dendrobium as 742.3 bp,with 72 variable sites,including 33 information sites;the average length of the ribosome 5S rRNA gene spacer sequences in Dendrobium was 336.4 bp,with 213 variable sites including 139 information sites.Using psbAtrnH intergenic region sequences in combination with ribosome 5S rRNA gene spacer sequences can not only identify D.denneanum,D.hancockil,D.thysiflorum,D.devonianum,D.moniliforme,D.chrysotoxum,D.officinale,D.heterocarpum and D.nobile,but also differentiate D.officinale from different geographical populations,and distinguish the dq-2 variety and dq-51 ine with SNP in the multi locus of D.denneanum.