利用rDNA ITS、RAPD及ISSR分子標誌評估安石榴之遺傳歧異度

Phylogenetic Relationships of Pomegranate (Punica granatum L.) Accessions by Using rDNA ITS, RAPD and ISSR Molecular Markers

為瞭解台灣現有收集自國外安石榴品系之遺傳歧異度，本研究以rDNA ITS（Interal Tramscribed Spacer）、RAPD（Randam Amplified Polymorphic DNA）及ISSR（Inter Simple Sequence Repeat）分子標誌進行遺傳相似性分析。利用聚合酶連鎖反應（PCR）增幅安石榴8個參試株系的ITS1和ITS2核醣體核酸（rDNA）基因與內轉錄間隔區，PCR產物經解序後，發現各株系間序列相似性100%。30個安石榴株系之核酸逢機增殖多型性（RAPD）分析，可由6個引子得到22個多型性片段，佔所有片段的29.72%，且發現由新加坡引進之品系皆擁有一個特有片段。將RAPD多型性片段進行未加權算數平均法（UPGMA）群叢分析，當相似性係數介於71%時分為5群，但群叢分析結果與果皮顏色並無相關性。簡單序列重複區間（ISSR）之DNA片段分析發現以dinucleotides motif:（GA）n序列作為引子所得到的多型性最高，由19個ISSR引子增幅得到72條多型性片段，佔所有片段的27.69%。分析ISSR產物發現有7個多型性片段可用於鑑別來自3個不同栽培株系的後裔。將ISSR多型性片段進行UPGMA群叢分析，相似性介於92%時區分為4群，而群叢分析結果則與引種地區來源一致。顯示RAPD和ISSR分子標誌技術，均有利於安石榴品種（系）之鑑別。 In order to establish identify genetic relationship among pomegranates available in Taiwan, seedling and cuttings of pomegranate introduced from China, Singapore and United States, were analyzed with ITS sequences of rDNA, ISSR and RAPD markers. Sequences of 5.8S rRNA and internal transcribed spacer (ITS) in eight samples of five lines showed no differences among theses lines. RAPD analysis revealed twenty-two polymorphic bands produced by six primers with a polymorphism rate of 29.72%. One specific band was found only in the Singapore line. According to UPGMA clustering in RAPD polymorphic bands, 30 lines of the pomegranate could be divided into five groups with their similarities of 71%. Dinucleotides motif: (GA)n sequence presented major part of pomegranate genome through ISSR analysis. Based on UPGMA clustering of ISSR polymorphic bands, 30 lines of the pomegranate in this study could be divided into four groups with their similarities of 92%. The result of ISSR clustering analysis coincided with the origins of pomegranate lines. We suggest that ISSR and RAPD markers could be used on identifying part of the pomegranate lines.