利用AFLP比對系統分析文心蘭栽培種之遺傳圖譜

Abstract

本文即是以AFLP（amplified fragment length polymorphism）技術，針對文心蘭不同栽培種間的親源關係進行鑑定及分析。實驗中係以EcoRI及MseI限制酵素切割文心蘭幼葉組織之DNA，並在DNA片段兩端接上接合子（adaptors），利用接合子及鄰近的序列設計與其互補之引子（primers），且此引子之特點為3端分別延伸出三個不同核甘酸組合，再以不同引子進行放射性元素標定多型性片段放大反應，即可選擇性的放大某些DNA片段，經由電泳分析，可觀察到不同長度片段，此結果即為AFLP圖譜。 將圖譜以Nei&Li遺傳相似性公式分析文心蘭不同栽培種間的親源關係，得到的分析結果顯示，在形態上殊異的栽培品系，其AFLP圖譜分析亦呈現較低的相似係數和較高的親源距離值，表示AFLP技術分析所得結果與由外部型態分析結果相符。 在AFLP技術上則以實驗證明固定的限制酵素切割時間能得到更高的圖譜再現率；並在不同的引子組合中找出16個適於於文心蘭遺傳距離分析所使用的組合。 未來期望能建立完整的文心蘭栽培種AFLP圖譜庫，便於進行早期不適育種目標幼苗之淘汰和品系鑑定，確實做到理論研究與實際應用之結合。

Amplified restriction fragment polymorphism (AFLP) is a PCR-based DNA fingerprinting technique. The technique involves three steps (I) restriction of the DNA and ligation of oligonucleotide adapters, (II) selective amplification of sets of restriction fragments and (III) analysis of the amplified fragments on denaturing polyacrylamind gels. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. This method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. To investigate the genetic diverification of oncidium by use of AFLP markers, 56 primer combinations were applied to generate AFLP patterns with eight oncidium lines, e.g. Gold 1. Gold 2. Gower Ramsey. Golden Star. Hamana, Shonan, Volcano Queen and Yellow King respectively. With seven primer combinations, the generated AFLP fingerprintings are hard to detect the polymorphism from eight lines. With the remaining 47 primer combinations, on average about 138 amplification products were generated and the polymorphism rate between the eight lines was generally over 60%. At present study, AFLP markers were successfully employed to detect genetic differentiation among eight oncidium lines. Nei & Li coefficient of genetic similarity was used to partition the total genetic variation in between. From the genetic distance cluster map, we can find that the distance between eight lines is 18?36%. The lines can be differentiated into two groups: (I) Gold 1. Gold 2. Gower Ramsey. Hamana. Shonan and Volcano Queen (II) Gold Star and Yellow King. The results are coincident to phenotypic variation. In conclusion the AFLP technique is an adequate and powerful tool to evaluate genetic diversification.