臺灣博碩士論文加值系統

在物種演化上，物種內的相互雜交往往造成遺傳特性上的差異，利用分析方便、穩定性高且易於推廣之ISSR分子標誌可進行物種的ISSR DNA指紋資料庫之建立及遺傳相似性分析。為了解種原的歧異性帶給生物遺傳演化上的影響，本實驗材料所選用的物種為芸香科(Rutaceae)中柑橘屬(Citrus)的甜橙（C. sinensis [L.] Osbeck），柑橘屬的果實內含有具抗癌、抗發炎效果的花青素及茄紅素，而其中的甜橙原產於印度東北部、中國西南部及緬甸地區，自十五世紀起便廣泛栽種於地中海地區，並經過變異選拔而衍生出不同性狀的後代栽培種，分成一般甜橙(Common Orange)、臍橙(Navel Orange)、血橙(Blood Orange)、無酸橙(Acidless Orange)等四類，由於外觀不易分辨，故選用來作為探討種原歧異的物種。
本試驗以20種甜橙的栽培種為材料，利用ISSR分子生物技術來探討物種的種原變異性，試驗結果顯示可產生多型性條帶訊號的核酸引子有8個，共可獲得51條條帶，平均每一引子可得條帶為6.4條。分析使用之8條核酸引子序列發現3’-anchored primer 之擴增效果較佳，以2個鹽基之簡單重複序列為主。所得之指紋分析資料經Jaccard’s遺傳相似係數運算，得各栽培種間的遺傳相似性介於17~72﹪之間，依據UPGMA進行群聚分析，繪製出樹狀分枝圖。若依遺傳距離0.40作為分群依據，可將所有樣品分成4個主要群組；品種差異性較大的cv. White Siletta及cv. Sanguinello個別成為獨立的第1,2群；第3群則為無酸橙類，但彼此間遺傳相似度都未超過0.45，具有較大的差異性；第4群包含樣品最多，酸度較高的一般甜橙及臍橙類的一群，臍橙類中以cv. Frost及cv. Washington這兩種臍橙的相似性最高，遺傳相似性達72%。
可呈現多型性條帶的8個甜橙ISS當中除了差異性較大的cv. Parson Brown, cv. Valencia, cv. Da Xue Gan之外，尚可再劃分出一般甜橙中含籽量較少及酸度較低的一群以及R引子全部為dinucleotides motif， 發現為(AC)n序列者佔6個。故(AC)n大量存在於甜橙基因內，值得進一步利用此microsatellite DNA序列，設計一系列的ISSR分析用引子，以進行更多的甜橙遺傳相關性之研究。藉由對種原之遺傳背景有深入的探討與了解，有助於我們在遺傳雜交組合的多樣性上的應用。

Concerning the evolution of species, the mutual hybridization among the biological species often causes the difference on the hereditary characteristic. Utilizing the convenient and stabile ISSR （inter simple sequence repeat）molecular maker can analyse hereditary similarity and make the setting-up of ISSR DNA fingerprint database among the biological species. In this study, we utilize Sweet Orange (C. sinensis [L.] Osbeck) for the experiment, the twenty cultivars of Sweet Orange can be divided into four groups, including Common Orange、Navel Orange、Blood Orange and Acidless Orange. Objectives were to assess genetic similarity among the cultivars. There were 51 polymorphic bands produced by 8 selected ISSR primers. The result of ISSR suggested that among the dinucleotides motifs, the poly(AC)n motif was more abundant and gave the largest number of polymorphism. Genetic similarity among the twenty cultivars was detected from 17% to 72% on ISSR marker basis. The cluster analysis of genetic similarity by UPGMA (Unweighted Pair-group Mean Arithmetic) revealed that four major groups could be recognized. The most interesting is the 4th group which includes most kinds can be observed that are mainly divied from two groups, one includes the Common Orange with fewer seeds and lower acidity and the other one includes the Navel Orange and Common Orange with high acidity. Among the Navel Orange, there is the highest similarity between cv. Frost and cv. Washington. Hereditary similarity value is up to 72% . Thus, it can be seen the quick and reliable molecular tool is useful in selecting the best parents and obtaining new combinations.

中 文 摘 要……………………………………………………..…...Ⅰ
英 文 摘 要…………………………………………………..……...Ⅲ
謝 誌………………………………………………………..………...Ⅴ
目錄……………………………………………………..……………Ⅵ
圖表目錄……………………………………………..……………….Ⅷ
第一章 緒論……………………………………………..………………1
第一節 研究緣起………………………………..…….………………...1
1-1 前言…………………………………………..……………………...1
1-2遺傳變異分析技術與研究…………………………………………..2
1-3 ISSR分子生物技術…………………………….…………………..4
第二節 研究題材與目的………………………………………………..6
2-1研究題材概述………………………………………………………..6
2-2研究目的……………………………………………………………10
第二章 材料與方法…………………………………..………………..12
一、 樣本收集……………………………….…………….……12
二、 材料準備………………….…………………………….…12
三、 葉片DNA萃取方法………………………………………12
四、 ISSR分子標誌分析……………………………………….14
五、 PCR擴增產物電泳分析…………………………………..15
六、 分子標誌統計分析……………………………………..…15
第三章 結果………………………………………………….……..….20
一、 增幅條帶核酸引子…………………..……………………20
二、 ISSR所得條帶統計分析……………………..…………...20
三、 所得樹狀分枝圖………………………………..…………21
四、 主成份分析…………………………...…………………...21
第四章 討論….………………………………………………………...34
一、 增幅條帶核酸引子………………………………………..34
二、 樹狀分枝圖……………………………………………..…34
三、 主成份分析……………………………………………..…36
第五章 結論……..………………………………………………….….38
第六章 參考文獻………………………………………..…...………...40

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