Development of the cytogenetic techniques to analyze chromosome rearrangements in plant-pathogenic filamentous fungi

开发细胞遗传学技术来分析植物病原丝状真菌中的染色体重排

• 批准号: 07660059
• 负责人: TAGA Masatoki
• 金额: $ 1.28万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660059/
• 关键词: plant-pathogenic fungi; filamentous fungi; chromosome; fluorescence in situ hybridization; pulsed field gel electrophoresis; cytogenetics

1.Background and objectivesOne of the major causes for genetic variation of plat-pathogenic filamentous fungi is the change of complement chromosomes of the genomes. Such chromosomal changes are thought to be constituted of aneuploidy and structural rearrangements of chromosomes such as translocation and inversion. Presently techniques that enable detailed cytogenetic analyzes are not available for filamentous fungi. The purpose of this study is to develope novel cytogenetic techniques to elucidate chromosomal changes, especially the rearrangements, in plant fungal pathogens. We set up three concrete goals, that is, (1) application of fluorescence in situ hybridization (FISH) to identify specific chromosomes, (2) detection of chromosomal rearrangements by FISH and pulsed field gel electrophoresis (PFGE), (3) search for the kinds and frequencies of chromosomal rearrangements in fungal populations.2.MaterialsThree plant pathogens, Fusarium solani f.sp.pisi, Botrytis cinerea and Alternari … More a alternata, were used.3.Results and conclusions(1)FISH and PFGE techniquesExperimental protocols for FISH and PFGE were successfully developed especially for F.solani. Using our PFGE prosocol the chromosomes in size range between ca.6-Mb and ca.500-kb could be clearly separated in a sigle run, which enabled us to analyze karyotypic polymorphisms of F.solani. Regarding FISH four types of probes, that is, plasmid cosmid, total nuclear DNA,PCR-amplified DNA of specific chromosomes were labeled with either biotin or DIG.With these probes specific chromosomes became detectable among the somatic metaphase complement chromosomes of the genome.(2)Identification and characterization of specific chromosomesNucleolar-organizing chromosomes was identified by FISH and PFGE using rDNA as a probe in F.solani, A.alternata and B.cinerea. The number of nucelolar-organizing chromosomes was only one in F.solani and B.cinerea, while two were detected in A.alternata. The morphological change of rDNA in the course of nuclear division was evident for F.solani.B chromosome in F.solani was detected by three types of FISH (ordinary FISH with B chromosome-specific cosmid clone, genomic in situ hybridization, and chromosome painting). This was the first success of cytological detection and visualization of B chromosome in fungi.Simultaneous identification of two chromosomes was accomplished by two-color FISH using probes of plasmid-cosmid clones or chromosome painting coupled with hybridization detection by FITC and rhodamine.In this study we have not succeeded in showing the chromosomal rearrangement yet mainly because of the shortage of time. Less

1.背景和目的平病丝状真菌遗传变异的主要原因之一是基因组互补染色体的改变。这种染色体变化被认为是由非整倍体和染色体的结构重排组成的，如易位和倒位。目前，丝状真菌还没有能够进行详细细胞遗传学分析的技术。本研究的目的是开发新的细胞遗传学技术来阐明植物真菌病原菌中的染色体变化，特别是重排。我们建立了三个具体的目标，即(1)应用荧光原位杂交(FISH)来鉴定特定的染色体，(2)用FISH和脉冲场凝胶电泳法(PFGE)检测染色体重排，(3)寻找真菌种群中染色体重排的种类和频率。2.材料3种植物病原菌，茄病镰刀菌，番茄灰霉病菌和链格孢霉…结果与结论(1)FISH和PFGE技术成功地建立了FISH和PFGE的实验方法。利用我们的PFGE原醇，可以一次分离出大小在约6-Mb到约500-kb之间的染色体，这使得我们能够分析F.solani的核型多态性。对于FISH的四种类型的探针，即质粒粘粒、总核DNA，用生物素或DIG标记特定染色体的PCR扩增DNA，通过这些探针可以在基因组的体细胞中期补充染色体中检测到特定的染色体。(2)特定染色体的鉴定和特征以rDNA为探针，采用FISH和PFGE方法对三种植物的核仁组成染色体进行了鉴定。水稻纹枯病菌和灰霉菌的核仁组织染色体只有1条，而互隔交链孢霉有2条。用3种FISH技术(普通FISH结合B染色体特异粘粒克隆、基因组原位杂交和染色体涂染)检测了水稻纹枯病菌的B染色体。这是真菌中B染色体的细胞学检测和可视化的第一次成功。两条染色体的同时鉴定是通过双色FISH结合FITC和罗丹明的杂交检测来完成的。较少

项目成果

赤松 創 ら: "パルスフィールドゲル電気泳動法(PFGE)および蛍光染色法によるAlternaria属、植物病原菌の染色体解析" 日本植物病理学会報. 63. 250-251 (1997)

So Akamatsu 等人：“使用脉冲场凝胶电泳 (PFGE) 和荧光染色对链格孢属植物病原菌进行染色体分析”日本植物病理学会通报 63. 250-251 (1997)。

Akamatsu, H.et al.: "Chromosome analyzes of Alternaria fungi by pulsed field gel electrophoresis and fluorescence staining. (in Japanese)" Ann.Phytopathol.Soc.Jpn.63. 250-251 (1997)

Akamatsu, H.et al.：“通过脉冲场凝胶电泳和荧光染色对链格孢属真菌进行染色体分析。（日语）”Ann.Phytopathol.Soc.Jpn.63。

多賀正節: "蛍光in situハイブリダイゼーションによるNectria haematococca体細胞染色体の分子細胞遺伝学的解析" 日本植物病理学会報. 62. 299 (1996)

Masanori Taga：“通过荧光原位杂交对 Nectria haematococca 体细胞染色体进行分子细胞遗传学分析”日本植物病理学会通报 62. 299 (1996)。

M.Taga et al: "Comparison of different karyotyping methods in filamenfous ascomycetes-a case study of Nectria haemafococca (anamorph:Fusarium solani)" Mycological Research. (印刷中). (1998)

M.Taga 等人：“丝状子囊菌不同核型分析方法的比较 - Nectria haemafococca（无性型：茄镰孢）的案例研究”真菌学研究（1998 年出版）。

多賀 正節 ら: "蛍光 in situ ハイブリダイゼーションによる Nectria haematococca 体細胞染色体の分子細胞遺伝学的解析" 日本植物病理学会報. 62. 299 (1996)

Masosetsu Taga 等：“通过荧光原位杂交对 Nectria haematococca 体细胞染色体进行分子细胞遗传学分析”日本植物病理学会通报 62. 299 (1996)。