臺灣博碩士論文加值系統

本試驗目的在利用ISSR及SSR DNA分子標誌來探討：(1)豆類物種間簡單重複序列及其鄰近區域之DNA序列變異 (2) SSR及ISSR DNA分子標誌跨物種利用之可行性 (3)豆類物種間之分類或是演化關係；調查材料包括7個屬12個物種，即紅豆、綠豆、大豆、花生、菜豆、紅花菜豆、豇豆、豌豆、蠶豆、白鳳豆、紅鳳豆及樹豆等作物。利用SSR分子標誌轉移利用率評估引子設計之來源物種和其他物種間的相似程度，發現豌豆和大豆基因組間在引子黏合序列上保守性較高。結果證實SSR分子標誌在跨物種轉移利用是可行的，本試驗亦篩選得到兩組可在12個物種以及禾本科水稻共同利用之SSR引子。也發現在不同物種所增幅長度相近的專一性片段間，亦存在多量的DNA序列變異，而僅利用大豆AG81引子所增幅之DNA序列變異資訊，即可區分遠緣物種。由於豆類物種間的基因組結構差異甚大，因此本試驗篩選得到大量的物種特有之ISSR DNA分子條帶，可利用於物種間或是屬間物種的遺傳變異分析。而在遺傳相似度、群集分析或主成分分析結果均顯示豇豆屬內的紅豆、綠豆及豇豆三個物種之遺傳距離甚遠，和屬間物種的遺傳距離相近，故試驗結果傾向支持應將三個物種進一步區分之建議。SSR DNA序列分析以及ISSR分析的結果均顯示蠶豆與其他調查之豆類作物間有最大之遺傳距離，在蠶豆品種之AG81 DNA片段中，簡單重複序列結構幾乎已經不存在，亦可說明蠶豆較其他物種在簡單重複序列區域具有較大之DNA序列變異。本試驗結果不但證實ISSR分子標誌可有效利用於分析豆類物種間之遺傳變異，亦顯示SSR DNA分子標誌甚具有跨物種應用的潛力。

To study genetic variation among legumes, ISSR and SSR DNA markers were analyzed around simple sequence repeats and their flanking regions for (1) the variation of simple sequence repeated DNAs and their flanking regions among legume crops (2) the feasibility of applying SSR and ISSR DNA markers in different species of legumes (3) the phylogenetic analysis of legume crops. Twelve important legume crops (33 varieties), including soybean (Glycine max), peanut (Arachis hypogaea), small red bean (Vigna angularis), mungbean (V. radiata), cowpea (V. unguiculata), pea (Pisum sativum), broad bean (Vicia faba), jack bean (Canavalia ensiformis/ gladiata), common bean (Phaseolus vulgaris/ coccineus) and pigeon pea (Cajanus cajan), were investigated. The transferability of SSR DNA markers was also used to evaluate the genetic similarity between the core species (soybean), which the SSR primesrs were originally designed, and other species. The conservative in primer annealing regions was found in pea and soybean. The experimental results indicate that it is applicable of using SSR markers of a species for the other legumes. In this study, two primer pairs able to amplify SSR sequences for 12 legume crops and rice were discovered. Also, lots of DNA sequence variations were identified among the similar length amplicons of the legume crops. And the distant species could be by discriminated by merely using DNA sequences amplified by soybean AG81 primer pairs. Because of the divergence in size of legume genomes, a great deal of ISSR primers were identified for a specific crop. These primers are available for detecting genetic variation among or within legume crops at molecular level. Because the long genetic distance among small red bean, mungbean and cowpea was found in the genetic similarity, cluster analysis and principle component coordinate analysis, each of the three species was suggested to become an isolated genera. According to SSR DNA and ISSR analysis, the broad bean had the largest genetic distance to the other legume crops. The finding of the degenerated repeated sequence in the AG81 segments of the broad bean also indicated that lots of DNA sequence variation had occurred in the region. Results from this study not only have proved the usefulness of ISSR DNA markers in detecting genetic variation among legume crops, but also have demonstrated the potential of applying SSR DNA markers in the other species.

目 錄
中文摘要
英文摘要
一、前言 ••••••••••••••••••••••••••••••1
二、前人研究 ••••••••••••••••••••••••••••3
三、材料與方法•••••••••••••••••••••••••••13
四、結果
(一)跨物種SSR分子標誌之轉移利用
1.利用SSR分子標誌跨物種轉移效率評估物種相似度•••••••••25
2. SSR引子產生跨物種專一性片段之DNA序列變異分析 •••••••29
3.利用物種間之SSR及其相鄰區域之DNA序列變異探討物種間關係•••34
(二)利用ISSR分子標誌進行物種間關係探討
1.豆類物種間ISSR分子標誌多型性分析••••••••••••••36
2.豆類物種間之遺傳相似性分析及UPGMA群聚分析 •••••••••36
3.豆類物種間之主成分分析••••••••••••••••••••41
五、討論
(一)跨物種SSR分子標誌之轉移利用
1.利用SSR分子標誌跨物種轉移效率評估物種相似度•••••••••44
2. SSR引子產生跨物種專一性片段之DNA序列變異分析 •••••••47
3.利用物種間之SSR及其相鄰區域之DNA序列變異探討物種間關係 ••49
(二)利用ISSR分子標誌進行物種間關係探討
1.豆類物種間ISSR分子標誌多型性分析••••••••••••••50
2. 豆類物種間之遺傳相似性分析及UPGMA群聚分析•••••••••51
3.豆類物種間之主成分分析••••••••••••••••••••53
六、結論••••••••••••••••••••••••••••••55
參考文獻••••••••••••••••••••••••••••••57
附 表 •••••••••••••••••••••••••••••••61


圖 目 錄
圖1.大豆AG81 SSR引子在12種豆類作物所增幅之DNA序列••••••28
圖2.菜豆屬、豇豆屬與刀豆屬內作物間及大豆不同AG81片段之DNA 序列比對 •••••••••••••••••••••••••••31
圖3.大豆AG81 SSR引子經PCR增幅12種豆類作物之產物 •••••••33
圖4.以鄰近連接法分析AG81 DNA序列資訊所得到28個豆類種原之 分群圖 ••••••••••••••••••••••••••••35
圖5.以UPGMA法分析AG81 DNA序列資訊所得到28個豆類種原之 分群圖•••••••••••••••••••••••••••••35
圖6. ISSR 880引子經PCR增幅12種豆類作物之產物 ••••••••••37
圖7.以UPGMA法分析533個ISSR DNA分子標誌產生之多型性得 到之群聚分析圖•••••••••••••••••••••••••40
圖8.利用ISSR分子標誌第一及第二主成分所繪之33個豆類作物種原 關係分布圖•••••••••••••••••••••••••••43
圖9.利用ISSR分子標誌第一、第二及第三主成分所繪之33個豆類作物 種原關係分布圖•••••••••••••••••••••••••43

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