山药种质资源核糖体rDNA-ITS区序列分析

Sequence analysis on rDNA-ITS region of germplasm resources from Dioscoreae Rhizoma

目的分析14种山药Dioscoreae Rhizoma种质ITS序列,为山药种质资源分子鉴别和进化关系研究提供依据。方法 PCR克隆扩增ITS序列并进行双向测序,Clustal X（v1.83）软件进行序列比对,Mega（V4.1）计算序列核苷酸比例及遗传距离,并构建邻接树（Neighbor-joining,NJ）和最大简约树（Maximum parsimony,MP）。结果 14种山药种质ITS序列全长为558~594 bp,其中ITS1长度为141~165 bp,ITS2长度为146~158 bp;ITS序列存在大量的转换、颠换,转化/颠换比率为5.347,ITS1和ITS2序列均显示102个变异位点;14种山药种质K2-P遗传距离为0~0.517 2;薯蓣Dioscorea opposita、褐苞薯蓣Dioscorea persimilis、日本薯蓣Dioscore japonica关系亲密,组成单一支系;参薯Dioscorea alata与山薯Dioscorea fordii关系亲密,聚为另一分支,并位于发育树基部。结论 ITS序列系统树为澄清山药类资源进化关系奠定了基础,序列中丰富的变异位点为多基原山药鉴别提供了科学依据。

Objective To provide the scientific evidence for molecular identification and phylogenetic evolution by analyzing internal transcribed spacer （ITS） sequences in 14 different germplasms from Dioscoreae Rhizoma. Methods The ITS regions were cloned by PCR amplification and sequenced bi-directionally. The ITS sequences were aligned using Clustal X software （version 1.83）, the genetic distances were calculated using Mega software （version 4.1）, and the phylogenetic trees were constructed through the Neighbor-joining （N J） and maximum parsimony （MP） methods. Results The length of ITS sequences obtained ranged from 558 to 594 bp, of which ITS1 was from 141 to 165 bp and ITS2 was from 146 to 158 bp. There were a lot of transitions and transversions in ITS sequences, and the transition/transversion ratio among 14 different germplasms was 5.347. Both in ITS1 and ITS2, 102 variable sites were further observed. The kimura 2-parameter （K2-P）genetic distance among 14 different germplasms ranged form 0 to 0.517 2, The phylogenetic trees suggested that there were close genetic relationship among Dioscorea opposita, D. persimilis, and D. japonica, they grouped closely into Clade I; Furthermore, D. alata and D. fordii also shared a closer genetic relationship comprised another clade （Clade II）. Conclusion The phylogenetic analyses based on ITS sequences can present a foundation for clearing the evolution of germplasm resources in Dioscoreae Rhizoma; The variable bases can also provide reliable molecular evidences for identifying different genotypic germolasms ofDioscoreae Rhizoma.