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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 analyze the ITS sequences of 14 species of Dioscoreae Rhizoma germplasm, and to provide a basis for the molecular identification and evolutionary relationship of yam germplasm resources. Methods The ITS sequences were amplified by PCR and sequenced. The sequence alignment was performed by Clustal X (v1.83) software. Mega (V4.1) was used to calculate the sequence nucleotide number and genetic distance. Neighbor-joining, NJ) and Maximum parsimony (MP). Results The ITS sequences of 14 species of yam germplasms were 558-594 bp in length, of which ITS1 was 141-165 bp in length and ITS2 was 146-158 bp in length. ITS sequences had a large number of transversion, transversion and transversion / transversion ratios of 5.347 , ITS1 and ITS2 sequences showed 102 variation loci; K2-P genetic distances of 14 species of yam germplasm ranged from 0 to 0.517 2; Dioscorea opposita, Dioscorea persimilis, Dioscore japonica were closely related to form a single branch ; Dioscorea alata is closely related to Dioscorea fordii, aggregates into another branch and is located at the base of the developmental tree. CONCLUSIONS: The ITS sequence phylogenetic tree laid the foundation for the clarification of the evolutionary relationship of yam resources. The abundant variation sites in the sequence provided a scientific basis for the identification of yam.