利用RAPD及rDNA ITS分子標誌分析馬祖原生百合遺傳歧異度

Abstract

為瞭解馬祖地區原生百合之分類地位及遺傳歧異度，本研究以逢機增幅多型性DNA（Random amplified polymorphic DNA, RAPD）及核糖體DNA（rDNA）內轉錄間隔區（Interal transcribed spacer, ITS）序列分子標誌進行遺傳歧異度分析。RAPD試驗包含馬祖、金門及中國廣西之原生百合25個樣本以及15個外源百合進行分析。從60個引子篩選出10個可產生多型性之引子，並選出35條清晰，亮度最亮之多型性片段。主座標分析（PCoA）及非加權平均法（UPGMA）集群分析得到之樹狀圖，可將馬祖、金門及中國廣西之原生百合百合與外緣百合群分開。馬祖南竿、東莒、西莒三個島及金門地區之分子變方分析（AMOVA）中，族群間的變異為13％，族群內個體間變異有87％，為主要的變異。 將馬祖原生百合及台灣與鐵炮百合之ITS片段，分別進行PCR產物直接定序及克隆定序，定序結果其 ITS序列長為626 ~627 bp，包含ITS1、5.8S基因與ITS2等三區，其長度分別為229、164及233~234 bp，GC含量為55.49%∼66.09%，變異位點主要發生在ITS1及ITS2。計算其遺傳距離以鄰近法（NJ）及非加權平均法分析之聚類樹狀圖，主要有(1)大部分的馬祖及金門地區百合和(2)台灣與鐵炮百合兩個分枝。ITS序列分析，直接定序結果以非加權平均法集群分析之結果較合理。而克隆定序以鄰近法之分析結果能得到較好的解釋。 綜合以上結果，雖然野百合對照樣本嫌少，但以RAPD及ITS序列分析均可將馬祖地區原生百合與外緣百合分開，且與野百合樣本分在同一群，故馬祖地區原生百合應為野百合（Lilium brownii F. E. Brown ex Miellez）。分子標誌分析結果，雖然馬祖原生百合與其他來源之野百合確有差異，但仍不足確定馬祖原生百合之獨特性，此部分尚需進一步之研究。

In order to establish genetic relationship of native lily in Matsu, Random Amplified Polymorphic DNA（RAPD）markers and rDNA Internal Transcribed Spacer（ITS）were analyzed. In RAPD analysis, 10 of 60 primers screened to provide polymorphic and reproducible bands. A total of 35 strong polymorphic bands were scored for 25 individuals of native lily collected in Matsu (21), Kinmen (3) and Guangxi China (1), and 15 individuals of outgroup. Principle coordinate analysis and unweighted pair group method with arithmetic mean（UPGMA）cluster analysis based on these RAPD profiles were performed. A clear distinction was found between the native lily in Matsu and outgroups. The results of analysis of molecular variance（AMOVA）of native lily in Matsu and Kinmen population indicated that 13％ of the total variation was attributable to the differences among population while 87％ was due to variation among individuals within population. The results of direct sequencing of PCR products and sequencing by cloning PCR products of the ITS and the 5.8S coding regions of nuclear ribosomal DNA in native lily in Matsu, L. longiflorum and L. formosanum were compared and phylogenetically analyzed. The length of ITS region was 626 to 627 bp in native lily in Matsu, including 229 bp of ITS1, 164 bp of 5.8S rRNA gene, and 233 to 234 bp of ITS2. The GC content was about 55.49%~66.09%. Variable sites mainly occurred in the ITS1 and ITS2. A dendrogram generated by neighbor-joining（NJ）and UPGMA cluster analysis. According to the dendrogram, two main clusters were generated: (1) native lily in Matsu and Kinmen, (2) L. longiflorum and L. formosanum. In ITS sequence analysis, direct sequencing data analyzed by UPGMA method would be more reasonable than NJ method, while the NJ method could get a better interpretation with cloning sequencing data. The results showed an explicit line of demarcation between native lily in Matsu and outgroup. Because the native lily in Matsu and other Lilium brownii were classified in the same group, we concluded that native lily in Matsu should be L. brownii. Owing to the sample number of L. brownii were too few, it needs further study to confirm that the native lily in Matsu is really unique.