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

ABSTRACT

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 (NJ) 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 0.5172. 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 germplasms of Dioscoreae Rhizoma.