Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31759
標題: 利用RAPD與AFLP的技術篩選西瓜抗蔓割病之分子標記
Screening of DNA markers related to Fusarium wilt resistance in watermelon based on RAPD and AFLP techniques
作者: 林盈宏
Lin, Ying-Hong
關鍵字: Watermelon
西瓜蔓割病
Fusarium wilt
RAPD
AFLP
SCAR
西瓜
RAPD
SCAR
AFLP
出版社: 植物病理學系
摘要: 西瓜 [(Citrullus lanatus (Thunb.) Matsum. & Nakai)],是台灣的栽培面積最大宗的葫蘆科作物,也是台灣的重要蔬菜作物之一。在西瓜的真菌病害中,以西瓜蔓割病 (Fusarium wilt) 最為嚴重,是西瓜產業的限制因子之一。西瓜蔓割病主要由 Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hanson 引起。台灣的商業栽培品種對於本病害大多不具抗性,且尚無化學藥劑可有效防治本病,故培育抗病品種為防治本病害之最有效策略。近年來,行政院農委會農試所鳳試分所陳甘澍氏培育出一抗西瓜蔓割病的新品系,命名為 JSB 品系,此抗病品系選自於一感病 Sugar Baby (SB) 品系。兩者間除了抗西瓜蔓割病的性狀呈現顯著差異外,其他諸如果皮顏色、果肉顏色、果形以及果實大小等園藝性狀並無特殊的差異,故極適合作為研究西瓜抗蔓割病遺傳之材料。本研究的目的主要是利用RAPD 與 AFLP 的技術以篩選西瓜抗蔓割病之分子標記 (DNA markers),進而以該分子標記設計專一性引子對,祈建立西瓜快速篩選抗病品種之系統。本實驗係利用 OPG05 RAPD 引子成功增幅出一條僅於抗病材料中出現之專一性 RAPD 片段,命名為 OPG05899;同時根據此序列針對抗病或感病材料分別設計出不同的專一性引子對,分別命名為 aR2、gR3與G-SCAR。經最佳 PCR 反應條件測試後,aR2、gR3 與G-SCAR引子對最適合的黏合溫度 (annealing temperature) 分別是62℃、64℃與54℃;黏合時間皆為 30 秒,在 36 (aR2、gR3) 或35 (G-SCAR) 個反應 (cycle) 下有最佳的結果。測試 12 個台灣常用的商業感病品種與抗病品種(系)後,發現除了抗病品種 Crimson Sweet 外,gR3 引子對無法在其他感病或抗病品種 DNA 中增幅出與感病相關的核酸片段;此外,針對抗病特性所設計之專一性引子對 aR2 與G-SCAR,於 12 個商業感病品種與六個抗病品種(系)中,皆無此抗性相關之專一性核酸片段被增幅出。目前本篩選系統正積極測試中,祈有助於未來西瓜的抗病育種工作。
Watermelon, Citrullus lanatus (Thunb.) Matsum. & Nakai, is one of the most important vegetable crops. Its cultivation area is the largest among the Cucurbitaceae in Taiwan. Among the watermelon fungal diseases, Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hanson, is one of the major limiting factors for watermelon production. The commercial cultivars of watermelon used in Taiwan usually do not resist to Fusarium wilt. So far, no chemicals can control this disease economically and effectively. The best way to solve this vascular wilt problem is to breed the resistant varieties which may not only increase the quality and yield of watermelon but also reduce the use of synthetic chemicals. This practice will also reduce the production cost and decrease environmental pollution. In Taiwan, the genetics of watermelon plants resist to Fusarium wilt has not been studied yet. Recently, a resistant inbred line JSB, which was derived from a spontaneous mutant of the susceptible Sugar Baby (SB), was obtained in Fengshan Tropical Horticultural Experiment Station (FTHES). Several random amplified polymorphic DNA (RAPD) markers were found to be related to Fusarium wilt resistance. The goal of this research is to establish a better system for screening resistant watermelon varieties and to elucidate the mechanisms of Fusarium wilt. Based on the information obtained from Mr. K.-S. Chen of the FTHES, the OPG05 random primer, which could generate an 899-bp DNA fragment (OPG05899) only in the resistant JSB line but not in the susceptible SB variety by RAPD, was used to design a specific PCR (polymerase chain reaction) program. According to the sequence of OPG05899, different primer sets were designed to screen the resistant or susceptible watermelon lines/varieties. The aR2 primer pair was found to produce a 340-bp DNA fragment (aR2340) only in the resistant JSB line. The gR3 primer pair was found to produce a 532-bp DNA fragment (gR3532) only in the DNA of susceptible SB variety. DNA isolated from twelve commercial susceptible cultivars and six resistant were also tested by these primer sets in specific PCR programs. The developed PCR system could only differentiate resistant JSB line from susceptible SB varieties but not for other resistant cultivars/lines. In the future, we will use molecular biotechnology to understand the differences in resistant (JSB) and susceptible (SB) lines/variety in molecular level. We will screen for more molecular markers related to Fusarium wilt resistance and analyze the relationships between resistance-related genes and disease resistance based on the results of amplified fragment length polymorphism (AFLP). The progress of this research could be applied to the breeding program for resistance to Fusarium wilt in watermelon and other Cucurbitaceae.
URI: http://hdl.handle.net/11455/31759
Appears in Collections:植物病理學系

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