Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31145
標題: 以隨機增幅核酸多型性技術探討番茄黑黴病菌及龍葵葉斑病菌之遺傳差異 性
Analysis of Genetic Variation of Pseudocercospora fuligena and P. atromarginalis by Random Amplified polymorphic DNA (RAPD) technique
作者: 李慧真
Lee, Huey Jen
關鍵字: Pseudocercospora fuligena
番茄黑黴病菌
Pseudocercospora atromarginalis
RAPD
genetic variation
龍葵葉斑病菌
隨機增幅核酸多型性
遺傳差異性
出版社: 植物病理學系
摘要: 本研究供試菌株包含 33 個番茄黑黴病菌、33 個龍葵葉斑病菌及 20 個 危害茄科、 豆科和其他植物的尾子菌及假尾子菌之菌株, 抽取各供試菌 株的 total DNA 進行 RAPD 的增幅反應。於 RAPD 的先前試驗中,找到 適合番茄黑黴病菌及龍葵葉斑病菌兩菌進行 RAPD 的增幅反應之最佳各反 應物的濃度, 分別為 50 ng 的 DNA、含 1.5 mM 的 Mg+2 之反應緩衝液 、0.5 μ M 的引子、0.2 mM 的 dNTPs、 0.6 U / μ l 的 DynaZyme I DNA 聚合酵素及 0.5mg / ml 的小牛血清, 且其最佳增幅條件為 94 ℃ 60 秒, 42 ℃ 7 秒,72 ℃ 70 秒,2個循環周期;94 ℃ 3 秒,42 ℃ 7 秒,72 ℃ 70 秒,35 個循環周期;72 ℃ 4 分鐘。以Operon Technologies 公司所製造的 100 個隨機之 10 個鹽基的引子進行引子的 篩選, 選出 30 個能產生 1 - 13 條增幅產物且具再現性的引子,並應 用於探討番茄黑黴病菌、龍葵葉斑病菌及危害茄科、豆科和其他植物的尾 子菌及假尾子菌各菌株間的親源關係,且利用其RAPD 的條帶型式 ( banding patterns ) 計算出各供試菌株基因體的相似值, 並依 SAS (Statistic Analysis System Inc. ) 之階層分群法 ( hierarchial clustering method )中之均連法 ( average linkage method ) 畫出 各供試菌株的基因體樹狀圖 ( genomiccluster )。發現番茄黑黴病菌各 菌株間的相似值極高 ( 0.85 ∼ 0.99 ),雖從其樹狀圖 (其平均距離為 0.945 ) 中可將番茄黑黴病菌各菌株分為兩群, 但兩群各菌株間的平均 相似值為 0.89,可知番茄黑黴病菌各菌株屬於遺傳同源性的 (homogeneous); 同樣地,也發現龍葵葉斑病菌各菌株間的相似值極高 ( 0.91 ∼ 1.00 ), 從其樹狀圖 ( 其平均距離為1.04 ) 可知,除 pa-15、pa-25、pa-29 及 pa-34 等四菌株外, 可將龍葵葉斑病菌各菌株 區母為兩群, 兩群各菌株間的平均相似值為 0.95,可知龍葵葉斑病菌各 菌株亦屬於遺傳同源性的 (homogeneous);亦發現分離自不同地區的番茄 黑黴病菌及龍葵葉斑病菌各菌株間的相似值也很高,表示地源並無造成遺 傳差異性。有三菌株與番茄黑黴病菌及龍葵葉斑病菌具有親源關係, 其 親源關係最近為 Pseudocercospora solani-melogenicola、 其次為 P.piricola 及最遠為 P. eriobotryae。於其他植物的尾子菌及假尾子菌 各菌株間的相似值極低 ( 0.05 ∼ 0.23 ),顯示尾子菌及假尾子菌各菌 株間具有相當大的遺傳差異性存在, 亦可能尾子菌及假尾子菌仍具有寄 主專一性。於番茄黑黴病菌及龍葵葉斑病菌各菌株中,發現兩菌所產生的 RAPD 圖譜、所增幅的條帶數、多型性條帶數及其條帶範圍極為相似;亦 可得到共同的增幅條帶;且兩菌各菌株彼此間的相似值為 0.84 ∼ 0.97;並從其樹狀圖 ( 平均距離為 1.033 ) 中可將番茄黑黴病菌及龍葵 葉斑病菌各菌株分為兩群, A 群菌株全部為番茄黑黴病菌, 而 B 群菌 株則包含全部的 15 個龍葵葉班病菌株及 10 個番茄黑黴病菌菌株, 兩 群各菌株間的平均相似值為 0.87,且亦曾將此兩群各菌株間的相似值以 T - test 分析之,結果顯示此兩群並無顯著的差異,由以上種種証據顯 示,可確定番茄黑黴病菌及龍葵葉斑病菌是關係相當密切的兩個族群,應 屬同物異名 ( synonym )。本研究證實 RAPD 在供試尾子菌及假尾子菌之 屬間及種間的鑑別及探討其親源關係或遺傳差異上極具可行性,並提供一 更客觀及更簡便的遺傳標誌於供試尾子菌及假尾子中,且所建立的鑑定模 式亦可作為其他不易以傳統分類方法區分出之真菌分類時的參考模式。
The fungi tested in this study included each 33 isolates of Pseudocercosporafuligena (on Lycopersicon esculentum ) and P. atromarginalis (on Solanumnigrum), and 20 other isolates of Cercospora spp. and Pseudocercospora spp. wereused to perform analysis of their genetic variation by RAPD. Based on thepreliminary tests of searching suitable RAPD conditions, the results revealedthat optimal reaction mixtures contained DNA ( 50 ng ), 1 x buffer ( 1.5 mM Mg+2), primer ( 0.5 μ M), dNTPs ( 0.2 mM each ), DynaZyme I DNA polymerase ( 0.6 U/ μ l), and BSA( 0.5 mg / ml ) and the suitable amplification reaction is 2cycles at 94 ℃ for 60 s, 42 ℃ for 7 s, and 72 ℃ for 70 s, then 35 cycles at94 ℃ for 3 s, 42 ℃ for 7 s, and 72 ℃ for 70 s, and one cycle at 72 ℃ for 4min. Preliminary 100 different 10 mer primers (Operon Technologies Inc.,Alameda, CA, U. S. A. ) were tested for each 3 isolates of P. fuligena ( pf-10,pf-12, and pf-18 ) and P. atromarginalis ( pa-22, pa-24, and pa-29 ) among them,13 primers gave no banding pattern or poor amplification, the other 87 primersconsistantly produced common banding patterns. Therefore, 30 reproducibleprimers were selected for assessing genetic variation among isolates of P.fuligena, P. atromarginalis and other Cercospora spp. and Pseudocercospora spp.The banding patterns of RAPDs obtained from application of these primers wereused to calculate the genetic similarity of those fungi, and genetic cluster ofthose fungi were analyzed with the average linkage method of hierarchialclustering method in SAS (Statistic Analysis System Inc. ). The results of RAPDpatterns, genetic similarity and dendrogram indicated that isolates of P.fuligena formed a homogenous group, so did P. atromarginalis, except the 4isolates of P. atromarginalis ( pa-15, pa-30, pa-35 and pa-39 ); intraspecificand interspecific variation were not detected between isolates of P. fuligenaand P. atromarginalis from different areas; the 3 isolates (P. solani-melogenicola, P. piricola and P. eriobotryae ) showed the similar RAPD patternsas P. fuligena and P. atromarginalis did; isolates of other Cercospora spp. andPseudocercospora spp. were the host specific fungi. In conclusion, the twospecies, P. fuligena and P. atromarginalis were homogeneous based on RAPDanalysis could be a synonym. The RAPD is a very potential technique in using theunique RAPD bands as genetic markers for identification of interspecies andintergenus and assessing the genetic diversity and relationships among isolatesof Cercospora spp. and Pseudocercospora spp. This model should be asupplementary tool when the traditional taxonomy method can not solve theproblems caused by host specificity or morphology as the criteria in the fungusclassification.
URI: http://hdl.handle.net/11455/31145
Appears in Collections:植物病理學系

文件中的檔案:

取得全文請前往華藝線上圖書館

Show full item record
 
TAIR Related Article
 
Citations:


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.