臺灣博碩士論文加值系統

番茄在高溫逆境下許多生理及生化的機制會受阻礙而使得產量降低。一般而言，番茄在高溫下，花粉粒的數目與活性、花粉管的發芽率、開花數、果實大小、果實軟化程度等許多與生長發育有關之生 理現象都會受到不同程度的影響；番茄在高溫下是否能正常結果及與產量相關之性狀多由數量遺傳因子所控制。台灣夏季環境高溫多雨，為限制番茄栽種產量的主要因素之一，因此透過分子遺傳輔助標誌選拔法（marker-assisted selection, MAS），配合傳統育種技術，理論上是可培育出高品質、高產量之番茄栽培品系。
本試驗所採用的親本為耐熱純系栽培品系番茄 CL5915 (母系)及不耐熱的野生種純系番茄 L4422 (父系)，mapping population為 CL5915及 L4422雜交產生之F1子代自交而得之158株F2分離族群，於民國93年夏季5~9月間種植於中國文化大學園藝學系之簡易網室中，調查單株總開花數、單株總果實數、平均單果重、開花率、結果率、種子數、甜度及產量等農藝性狀，分別計算各性狀之平均值、標準差及性狀間之相關係數。利用ISSR ( Inter simple sequence repeats )、AFLP ( Amplification fragment length polymorphism ) 及RAPD (Random amplified polymorphic DNA ) 的技術來分析性狀並找出在高溫逆境下與番茄品質及產量有關之數量性狀基因（QTL）連鎖的一些分子標記，再將所找到之QTL依相對遺傳距離繪製番茄遺傳圖譜。
試驗結果顯示，在調查的7種性狀中，有11組性狀間呈現顯著正相關。而在分子標誌分析上，找到了6個連鎖群，並且定位出23個QTL位置。其中跟產量有關的QTL有6個，分佈在group 2、3和4上；跟果實數有關的QTL有3個，分佈在group 2、3和4上；跟平均果重有關的QTL有3個，分佈在group 2和3上；跟甜度有關的QTL有2個，位在group 2和4上；跟開花數有關的QTL有4個，分佈在group 2、3和4上；跟結果率有關的QTL有5個，分佈在group 2、3和4上；而在目前研究的7種性狀中尚未發現到與種子數有關之QTL，若未來能增加符合孟德爾遺傳分離比3：1之marker數，將有機會找到。這些與番茄產量及品質數量性狀基因連鎖之分子標誌，可作為日後番茄耐熱育種的篩選標誌（selection markers），以期能縮短育種年限。

關鍵詞：番茄、耐熱性、逢機擴增多型DNA (RAPD)、簡單序列重複 (ISSR)、數量性狀基因座、標誌輔助選拔。

Heat stress may inhibit the physiological and biochemical mechanisms of tomato, and result in declining yields of the tomatoes. The activities and numbers of pollen, germination of pollen tube, flower number, fruit number, size and ripening of tomato are highly related to high temperature. During summer season in Taiwan, the production of tomato is decreased due to high temperature and high rainfall combination. Most tomato varieties are adaptive to warm and dry weather, and don’t show heat tolerance. Heat tolerance of tomato is a quantitatively inherited trait and easily affected by environmental conditions. Therefore, marker-assisted selection (MAS) may improve the traditional field selection
A total of 158 F2 plants derived from a heat tolerant CL 5915 crossed with a heat sensitive L4422 (L. Pimpinellifolium) were grown in the screen house at PCCU from May to September in 2004. Seven heat tolerance-related traits were scored: flower number, fruit number, fruit set, fruit weight, fruit yield, brix and seed number. The phenotypic data and correlation coefficient between traits were calculated. The objectives of this study were to identify and map the QTL linked to the heat tolerance traits in tomato, and to develop a database that enables use of RAPD and ISSR markers as selection tools for further improving heat tolerance in tomato.
The results show that 11 significantly positive correlation coefficients were found between the traits. Polymorphic bands were generated by 3 RAPD primers and 100 ISSR primers using PCR with optimal annealing temperatures. Informative bands were used for χ2 goodness-of-fit 3:1 test. Combination of the mentioned 7 traits and the informative markers, four linkage groups were constructed, and 23 QTLs of traits linked to the markers were identified using Mapmaker/Exp 3.0b and Mapmaker/QTL 1.1b softwares. Among these QTLs, 6 of them linked to yield on the groups of 2, 3 and 4, 3 linked to fruit weight on groups of 2 and 3, etc. Nevertheless, all these 23 markers didn’t show any linkage to the seed numbers. In general, different genes controlled different traits, and the same trait was controlled by different genes appeared to be specific to high temperature environment.
This work will expedite access to genetic variation necessary for ongoing crop improvement, and it will accommodate expansion of the production area in response to economic or climate demands, thereby reducing the environmental vulnerability of worldwide agriculture. MAS will not be fully accessible to plant breeders until the establishment of a procedure which is simple, rapid, accurate, efficient, cost effective and complementary to existing breeding protocols

Keyword： tomato、heat tolerance、RAPD、ISSR、QTL, Marker-assisted selection

目 錄
圖目錄………………………………………………………………......III
表目錄…………………………………………………………………..IV
中文摘要………………………………………………………………...V
英文摘要……………………………………………………………….VII
壹、前言……..……………………………………………………………1
貳、前人研究…….….……………………………………………………3
一、番茄……….………………………………..………….......……3
二、Mapping population ………………………...………………..…6
三、分子標誌發展與應用……………………...………………..….6
四、分子標誌技術的應用……………………………....…………11
五、連鎖群及QTL分析…………………………….…….…….....13
參、材料與方法…………………………………………….….….….…16
一、材料……………………………………...……….…….….…16
(1)試驗材料及生長情形....………………..………………...16
(2)性狀調查………………………………………………….17
二、方法……………………………………...…………….……..17
(1)DNA的萃取與定量………………………………………17
(2)基因型分析……………………………………………….18
(3)計算分離比……………………………………………….23
(4)基因型與外表型數據分析……………………………….24
肆、結果………………………………………………………………..25
一、番茄之外表型性狀調查與分析………………………………25
二、F2外表性狀之相關係數分析…………………………………31
三、分子標誌分析…………………………………………………33
四、連鎖群分析……………………………………………………37
五、QTL分析………………………………………………………41
伍、討論…………………………………………………………………49
一、耐熱環境與外表型分析………………………………………49
二、各性狀之相關係數分析………………………………………52
三、分子標誌分析…………………………………………………53
四、連鎖群與QTL分析…………………………………………..55
陸、結論…………………………………………………………………62
柒、參考文獻……………………………………………………………63
附錄……………………………………………………………………..68
圖 目 錄
圖一、F2世代耐熱相關性狀之分佈圖…………………………………27
圖二、利用Mapmark軟體所找出之基因連鎖圖譜………………….39圖三、QTL連鎖圖譜…………………………………………………..45
附圖一、民國93年交通部中央氣象局台北站之氣象資料圖……….68
附圖二、ISSR827之部分黏和溫度測試圖……..……………………..70
附圖三、ISSR838~849之部分親代測試圖……………………………70
附圖四、ISSR823之部分F2子代電泳圖……………………………..70
附圖五、AFLP ( E-ACG and M-CAG)之部分電泳圖…………………71
附圖六、RAPD D08之部分黏和溫度測試圖…………………………72
附圖七、RAPD P06之部分F2子代電泳圖……………………………72
附圖八、各性狀間之相關係性分佈圖…………………………………73
附圖九、ISSR-PCR之分析原理圖…………………………………….80
附圖十、AFLP-PCR之分析原理圖……………………………………81
附圖十一、RAPD-PCR之分析原理圖………………………………..82
表 目 錄
表一、F2分離族群、回交族群 mapping population之分離率…………15
表二、分子標誌技術比較表……………………………………………15
表三、各性狀之間之相關係數表………………………………………32
表四、ISSR條帶分析表………………………………………………..35
表五、RAPD條帶分析表……………………………………………….36
表六、以Mapmaker軟體分析番茄7種農藝性狀之QTL結果表……..47附表一、F2世代之性狀調查紀錄表……………………………………83
附表二、部分ISSR、AFLP及RAPD之F2子代1/0值表……………88

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