利用分子標記RAPD、ISSR和DAMD來界定Astragalus rhizanthus複合群的物種

Delimitation of Species of the Astragalus rhizanthus Complex (Fabaceae) Using Molecular Markers RAPD, ISSR and DAMD

從印度喜馬拉雅不同地區收集了四十個隸屬於Astragalus rhizanthus複合群中的四個相關物種（A. rhizanthus、A. candolleanus、A. malacophyllus和A. pindreensis）；利用逢機擴增多型性DNA（Random Amplified Polymorphic DNA, RAPD）、簡單序列重複間序列（Inter Simple Sequence Repeats, ISSR）和直接擴增小衛星DNA（Directed Amplification of Minisatellite DNA, DAMD）來分析其種內及種間的關係。十八條RAPD、十五條ISSR和六條DAMD引子用來研究物種間的遺傳歧異度，總共獲得449條RAPD條帶、326條ISSR條帶以及179條DAMD條帶，其中將近98.44%的RAPD、99.38%的ISSR與98.32%的DAMD條帶為多型性的。並利用Jaccard的相似度公式（Jaccard’s similarity coefficient）計算出這些累計數據的相似性係數為0.23-0.82，並得到一個鄰近連接（Neighbor-joining）樹狀圖。由Jaccard相似係數估算出的遺傳相似性結果與形態特徵及先前的分類處理相符合。其中A. rhizanthus的種內遺傳相似性最高，接著為A. malacophyllus、A. candolleanus最後為A. pindreensis。本研究證實了這些分子標記能夠區分出關係密切的相近物種，並能夠分析其種內及種間的遺傳歧異度。

Forty accessions belonging to four closely related species of the Astragalus rhizanthus complex (i. e., A. rhizanthus, A. candolleanus, A. malacophyllus and A. pindreensis) were collected from different parts of the Indian Himalaya and analyzed for intra- and inter-specific relationship using Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats (ISSR) and Directed Amplification of Minisatellite DNA (DAMD). Eighteen RAPD, 15 ISSR and 6 DAMD primers were used to study the genetic diversity between the species. A total of 449 bands for RAPD, 326 bands from ISSR and 179 bands from DAMD were obtained. Nearly 98.44% of RAPD, 99.38% of ISSR and 98.32% of DAMD bands were polymorphic. Pair-wise genetic similarity for a cumulative data was determined using Jaccard's similarity coefficient which varied from 0.23 to 0.82 and a Neighbor-joining (NJ) dendrogram was generated. The genetic similarity from the data matrix estimated by Jaccard's coefficient shows that all four species are distinct in accordance with our morphological findings as well as previous taxonomic treatment in which these species have been treated distinctly. The highest degree of genetic similarity was observed within A. rhizanthus followed by A. malacophyllus, A. candolleanus and A. pindreensis. This study has proved that these markers have the ability to distinguish the closely allied species as well as analyze the genetic diversity within and between the species.