臺灣常見杜鵑花(Rhododendron spp.)栽培品種之葉部形態調查及遺傳歧異性分析

Studies on leaf morphological variation and genetic diversity of Rhododendron cultivars in Taiwan

湯智欽(Chih-Chin Tang)

指導教授 ： 張育森

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02465

摘要

摘要

本研究以41個杜鵑栽培品種(cultivar)為材料，進行形態調查及遺傳歧異性分析，期能建立一個精確快速的品種鑑別方法，並利用分子標誌探討41個栽培品種的親緣關係。
臺灣常見的杜鵑大致可分為平戶杜鵑、皋月杜鵑、久留米杜鵑及台灣原生杜鵑四大類。針對這幾類杜鵑品種間的分類關係，歷來之分類處理，大部分著重於形態之描述，缺乏有系統之調查與研究。本研究以平戶杜鵑、皋月杜鵑、久留米杜鵑及台灣原生杜鵑四大類為材料，進行葉部形態資料分析，並將其葉片與花朵照像記錄。在形態方面，依葉長、葉寬及葉柄長三個葉部形態特徵，能夠順利的將平戶杜鵑與皋月杜鵑和久留米杜鵑區別開來。皋月杜鵑及九留米杜鵑的葉長、葉寬及葉柄長三個葉部形態特徵相當接近，以葉寬為X軸，葉長葉寬比為Y軸做圖則可將皋月杜鵑及九留米杜鵑大致分為兩群。臺灣原生杜鵑與平戶杜鵑、皋月杜鵑、久留米杜鵑的葉片形態有明顯不同，可直接觀察葉片做出區別。臺灣原生杜鵑的葉長平均值明顯落在平戶杜鵑與皋月杜鵑和久留米杜鵑之間。本研究所觀察記錄之花朵照片可作為各品種區別之參考。
杜鵑栽培品種(系)名稱甚為混雜，利用葉片之性狀進行多變量分析，瞭解杜鵑收集品系之親緣關係。以11個葉片性狀做群集分析的結果可將41個栽培品種分群，經主成份向量分析，前二個主成份共可解釋78.93%的變異量，杜鵑收集品系在兩軸的排列結果與群集分析之分析結果相似，第一群(Ⅰ)平戶杜鵑；第二群(Ⅱ)烏來杜鵑；第三群(Ⅲ)皋月杜鵑；第四群(Ⅳ)久留米杜鵑；第五群(Ⅴ)金毛杜鵑；第六群(Ⅵ)毛杜鵑。
利用逢機擴增多型性DNA (random amplified polymorphic DNA，簡稱RAPD)分子標誌技術分析，藉以評估41個栽培品種(分別為平戶杜鵑11種、皋月杜鵑12種、久留米杜鵑5種以及台灣原生杜鵑13個收集品系)間之遺傳距離，經由18個引子中篩選出7個具多型性且再現性良好的引子，計獲得42個多型性DNA片段。而综合7個引子之多型性片段，經未加權平均法(unweighted pair-group method with arithmetic mean，UPGMA)集群分析得到之樹狀圖，明顯有同種聚集一起之趨勢。在所分析的品種中，遺傳相似度係數在0.36~1之間，編號7號、9號和11號3種平戶杜鵑的遺傳相似度係數為1，顯示其親緣關係最近，編號18號的皋月杜鵑與其餘試驗品種的遺傳相似度係數為0.36，與其他品種親緣關係最遠。综合結果，RAPD分析應可作為杜鵑栽培種分群的有效方法之一。
研究分析核糖體核酸( ribosomal DNA, rDNA )內轉錄間隔區( internal transcribed spacer, ITS )之序列， 以一組核苷酸引子( primer )，利用聚合酵素連鎖反應( polymerase chain reaction, PCR )，將所收集到的13個臺灣原生杜鵑的5.8S核糖體核酸( rDNA )基因與內轉錄間隔區( internal transcribed spacer, ITS )選殖並定序，以探討所收集到的13個臺灣原生杜鵑的親緣關係。將13個收集品系的臺灣原生杜鵑的ITS序列進行比較排列後發現，在ITS1區域有3個變異處，在ITS2區域有6個變異處。以Kimura 2-parameters換算杜鵑間兩兩的遺傳距離( genetic distance )，發現這13個收集品系的遺傳距離介於0~0.00609間。經未加權平均法( unweighted pair-group method with arithmetic mean，UPGMA )集群分析，完成親緣關係樹狀圖( phylogenetic tree )。根據樹狀圖可得到1個主要分群，以烏來杜鵑為主。

關鍵字

杜鵑 ； 葉部形態 ； 形態特徵 ； 遺傳歧異性 ； 主成份分析 ； 集群分析 ； 逢機增殖多型性DNA

並列摘要

Abstract
In this study, we study on morphological variation and genetic diversity of 41 Rhododendron cultivars in Taiwan. During the analysis, We hope to establishment a rapid and accurate detection method. We also hope to use the molecular markers to analyze the relationships among 41 Rhododendron cultivars in Taiwan.
We can usually see the 4 major groups of Rhododendron in Taiwan, they are Hirado azalea cultivars, Satsuki cultivars, Kurume azalea cultivars and Rhododendron native in Taiwan. In the past, most taxonomy work was based on morphological description, but lack in systematic investigation. In this study, we recorded the flower and leaf shapes by digital camera, together with leaf morphological analysis of the 4 major groups of Rhododendron in Taiwan. In view of morphological characteristics, leaf length, leaf mid width and leaf stalk ( petiole ) length can distinguish Hirado azalea cultivars, Satsuki cultivars and Kurume azalea cultivars into two groups, the one is Hirado azalea cultivars and the second group is Satsuki cultivars and Kurume azalea cultivars. Leaf morphological characteristics of Satsuki cultivars and Kurume azalea cultivars are very similar. We can distinguish between Satsuki cultivars and Kurume azalea cultivars and determined by the two axes on the basis of leaf mid width and leaf length/leaf mid width. We can usually distinguish between Hirado azalea cultivars, Satsuki cultivars, Kurume azalea cultivars and Rhododendron native in Taiwan by their leaves—the leaf of Rhododendron native in Taiwan are different from others. The leaf average length of Rhododendron native in Taiwan are between Satsuki cultivars and Kurume azalea cultivars. In this study, the pictures of flower records can be used as reference resources to distinguish the cultivars.
The varieties ( lines ) of Rhododendron were not clearly classified in Taiwan. The objective of this study was to identify the genetic relationship among the 41 Rhododendron cultivars through numerical analysis of the leaf characters. Using cluster analysis of the 11 leaf characters, 41 cultivars were divided into six main groups. The ordination pattern of the principal component analysis was similar to the grouping pattern of the cluster analysis, and the first two principal components derived the principal component analysis explained 78.93% of the observed variation. The first group(Ⅰ) included Hirado azalea cultivars. Cultivars in the second group (Ⅱ) were Rhododendron kanehirai. The third group (Ⅲ) were Satsuki cultivars. The fourth group (Ⅳ) were Kurume azalea cultivars. The fifth group (Ⅴ) were Rhododendron oldhamii. The sixth group (Ⅵ) hybrid strains.
A total of 41 cultivars of Rhododendron were evaluated by using random amplified polymorphic DNA ( RAPD ) marker. Objectives were to evaluate genetic distance within those 41 cultivars. Screening from 18 primers and 42 polymorphic DNA fragment were derived from 7 primers. A dendrogram generated by UPGMA ( unweighted pair-group method with arithmetic mean ) clusteranalysis. The results also show the similar varieties were cluster into the same subgroup. Similarity indexes among 41 cultivars were extremely from 0.36 to 1. The number 7, 9, 11 of Hirado azalea cultivars had 100% similarity indexes, indicating the closest relationship. Otherwise, the number 18 of Satsuki cultivars having 36% similarity indexes showed the farthest genetic relationship with other tested Rhododendron cultivars. In brief, that the RAPD markers is an effectively and reliably method for analysis of the genetic distance of Rhododendron cultivars.
The entire internal transcribed spacer ( ITS ) region between 18S and 26S ribosomal RNA ( rRNA ) genes among 13 Rhododendron accessions in Taiwan were amplified by polymerase chain reaction ( PCR ). The primers of PCR, IT1: 5’-TCGTAACAAGGTTTCCGTAGGT-3’ and IT2: 5’-GTAAGTTTCTTCTCCTCCGCT-3’ were designed for amplification. Genetic relationship of 13 Rhododendron accessions in Taiwan was derived from the sequence analysis of the internal transcribed spacer ( ITS ) region of ribosomal DNA. Sequences of complete ITS region, including ITS1, 5.8S rDNA, and ITS2, were obtained by direct sequencing of polymerase chain reaction ( PCR ) amplified fragments. Aligned sequences of ITS1 and ITS2 , we found the ITS1 region contained 3 variable sites and the ITS2 region contained 6 variable sites. A distance matrix of sequence divergence was caculated using Kimura’s two parameter model. Divergene values among the accessions were extremely low ranging from 0.00 to 0.609%. A dendrogram generated by UPGMA ( unweighted pair-group method with arithmetic mean ) cluster analysis. According to the dendrogram, 1 main cluster were generated among 13 Rhododendron accessions in Taiwan.

並列關鍵字

Rhododendron ； leaf pattern ； morphological character ； genetic diversity ； principal component analysis ( PCA ) ； cluster analysis ； RAPD

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