利用RAPD與ISSR分子標誌技術鑑定台灣及菲律賓蝴蝶蘭物種

Phylogenetic Analysis of Five Phalaenopsis Species Native to Taiwan or the Philippines by Using RAPD and ISSR Molecular Markers

本研究以RAPD與ISSR分子標誌鑑定嘉義大學收集自國內之11份台灣蝴蝶蘭（Phalaenopsis aphrodite subsp. formosana E. A. Christ.，subsp. nov.）及17份菲律賓之Phal. amabilis，Phal. aphrodite，Phal. sanderiana，Phal. schilleriana及Phal. stuartiana等五個實生物種。本試驗由200個RAPD引子及100個ISSR引子選出12個RAPD引子及3個ISSR引子供使用，結果共獲得95個RAPD標誌片段及50個ISSR標誌片段。自UBC430 RAPD引子產生的450bp標誌片段，為台灣蝴蝶蘭之特有標誌片段。以RAPD標誌經Nei／Li’s法計算相似性和以UPGMA法建構之親緣樹狀圖顯示，台灣蝴蝶蘭與菲律賓之Phal. amabilis及Phal. aphrodite，相似性分別為73%及75%；以ISSR標誌法分析則分別為63%及71%；配合RAPD及ISSR標誌法分析結果均為71%。本研究結果顯示，台灣蝴蝶蘭有別於菲律賓之Phal. amabilis及Phal. aphrodite。合併RAPD和ISSR標誌之群集分析所獲得之親緣樹狀圖分群與Sweet研究之形態分群具一致性。 Eleven accessions of Taiwan Phalaenopsis (Phal. aphrodite subsp. formosana) and 17 accessions belonging to Phal. amabilis, Phal. aphrodite, Phal. sanderiana, Phal. schilleriana, or Phal. stuartiana, which were originated from Philippines and seed propagated in Taiwan, were collected and maintained by National Chiayi University. A total of 28 accessions were used for PCR amplification using RAPD and ISSR primers. Twelve 10-mer primers selected from 200 RAPD primers and three primers selected from 100 ISSR primers were used to access the genetic variation and interrelationships among the species in the genus Phalaenopsis. A total of 95 RAPD markers were scored using the 12 RAPD primers. A total of 50 ISSR markers were scored using the 3 ISSR primers. The 450 bp band in UBC430 RAPD primer was uniquely formed for Phal. aphrodite subsp. formosana. UPGMA analysis was performed and dendrograms were constructed using the data from RAPD. The similarity values between Phal. aphrodite subsp. formosana and Phal. amabilis, and between Phal. aphrodite subsp. formosana and Phal. aphrodite were 73% and 75% respectively. In contrast, the similarity values were 63% and 71% respectively using the data from ISSR. Cluster analysis combining RAPD and ISSR data separated the species and plants into 6 groups. The cluster of Phal. aphrodite subsp. formosana appeared different from Phal. amabilis and Phal. aphrodite. The results were consistent with the conventional taxonomy based on the morphological characteristics by Sweet.