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標題: 立枯絲核菌第四融合群(Rhizoctonia solani AG-4)致病性之核酸序列擴增區段(SCAR)分子標記篩選研究
The study of SCAR molecular marker for pathogenicity of Rhizoctonia solani AG-4
作者: 黃信琇
Huang, Hsin-Hsiu
關鍵字: Rhizoctonia solani;立枯絲核菌;RAPD;SCAR;逢機增幅多型性DNA;核酸序列擴增區段
出版社: 植物學系
立枯絲核菌(Rhizoctonia solani Kühn) 為一世界性分布之土壤病媒植物病原真菌,會造成寄主植物如水稻、花生等倒伏、莖腐及葉斑,此菌之形態,寄主專一性與致病性都會因地理位置不同而有明顯的差異。傳統的致病性測試耗日費時,而且環境因子的細微差異可能會影響測試的結果,因此我們希望能應用分子生物學技術,開發一快速且準確之病原性偵測方式,以供檢疫及學術研究上之應用。
核酸序列擴增區段(sequence characterized amplified region, SCAR),是對逢機增幅多型性聚合酶鏈鎖反應(random amplified polymorphic DNA, RAPD)所產生的多型性條帶進行DNA定序,再根據所得DNA序列,設計另一組核酸引子,可專一性地放大單一產物,提供作為DNA分子標記。
本實驗選取15株不同強弱致病性之立枯絲核菌第四融合群(R. solani AG-4)菌株之總體DNA,分別利用100條核酸引子進行RAPD-PCR,其中引子UBC-96增幅的片段可區分致病性與非致病性菌株,此專一性DNA片段長度為397 bps,經回收、選殖、定序後,藉由所得到的核酸序列設計出專一性核酸引子SCA96f與SCA96r。以此核酸引子對R. solani AG-4進行SCAR-PCR,可以在致病性菌株上得到一條363 bps之專一性片段,此即為致病性之SCAR分子標記。

Rhizoctonia solani Kühn is a world-wide soil-born pathogenic fungus; significantly causing damping-off, stem rot, root rot, and leaf spot in rice, peanuts etc.. There are difference in morphology, host specificity and pathogenicity from different geographic locations. It wastes lot of time by traditional pathogenic determination, and the results will been easily effected by slightly variation of enviroment factor. Therefore we would like to develop a fast and precise identification method, based on molecular technology for quarantine and research pathogen.
SCAR, sequence characterized amplified region, according the DNA sequence result of RAPD (random amplified polymorphic DNA) fragments which appeared on pathogenisity isolates to design a pair of primers specific to one of those products for DNA molecular marker.
RAPD was used to screen 100 primers of UBC with total DNA of pathogenic and non- pathogenic R.solani AG-4 isolates. The primer UBC-96 was selected, and the specific DNA was amplified of pathologenic isolates. The specific DNA fragment 397 bps was amplified, recoverd, cloned, and sequenced. Then a pair of specific primers SCA96f and SCA96r were by the nucleotide sequence of the 397 bps DNA fragment. A 363bps fagment was amplified by the specific primers SCA96f/SCA96r of pathogenic isolates, can be as SCAR marker of the R. solani AG-4.
Appears in Collections:生命科學系所

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