Physical mapping of chromosomes in plant-pathogenic fungi by fluorescence in situ hybridization (FISH)

通过荧光原位杂交 (FISH) 对植物病原真菌中的染色体进行物理定位

• 批准号: 13660053
• 负责人: TAGA Masatoki
• 金额: $ 1.79万
• 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660053/
• 关键词: chromosome; genetic map; fungi; FISH; fluorescence in situ hybridization; physical mapping; Nectria haematococca; Cochliobolus heterostrophus; マッピング; 植物病理学

Techniques of fluorescence in situ hybridization (FISH) including fiber-FISH were developed with two plant-pathogenic fungi, Nectria haemataococca and Cochliobolus heterostrophus, and physical mapping of their chromosomes was attempted with the established techniques. The summarized results obtained in this study are as follows.1. The method of preparing DNA fibers for fiber-FISH was established, in which protoplasts-containing agarose blocks for pulsed-field gel electrophoresis (PFGE) or PFGE-separated chromosomal DNA bands were used as the source of DNA fibers.2. Making contig map with cosmid clones for 1.6-Mb CD-chromosome of N. haematococca was shown to feasible by using multi-color fiber-FISH.3. Physical distance of RFLP linkage markers that locate near the breaking points of reciprocal translocation was determined with fiber-FISH in C. heterostrophus, and compared with the distance obtained in a genetic map. The results showed that crossing-over was not repressed near breaking points in meiosis and that the inserted alien fragment is unlikely to be large as expected previously.4. Physical mapping by FISH using interphase nuclei as targets was attempted with cosmid and plasmid probes in N. haematococca. Although the probe hybridization was successfully detected on the nuclei, mapping resolution was much lower than that of fiber-FISH. FISH on the interphase nuclei was thought to be useful to analyze the behavior and territory of chromosomes in the nucleus.5. Two-color FISH on the prometaphase or metaphase mitotic chromosomes of C. heterostrophus revealed that PKS1 and DEK1 which are involved in T-toxin production reside separately on the different chromosomes. It was also shown that chromosomes 6 and 12 are reciprocally translocated.

荧光原位杂交（FISH）技术，包括纤维FISH的开发与两种植物病原真菌，赤壳菌和异柄旋孢菌，并尝试与建立的技术，其染色体的物理作图。本研究的主要结果如下：1.建立了以原生质体中含有琼脂糖块的脉冲场凝胶电泳（PFGE）或PFGE分离的染色体DNA条带作为DNA纤维来源的DNA纤维的制备方法.用粘粒克隆制作了N.利用多色荧光原位杂交技术对红球菌进行鉴定是可行的。利用Fiber-FISH技术测定了位于相互易位断裂点附近的RFLP连锁标记的物理距离。heterostrophus，并与遗传图谱中获得的距离进行比较。结果表明，在减数分裂的断裂点附近，交换不受抑制，插入的外源片段不可能像以前预期的那样大.以间期核为靶，用粘粒和质粒探针，在N.红球菌属尽管探针杂交在细胞核上成功检测到，但映射分辨率远低于纤维-FISH。间期核上的FISH被认为有助于分析染色体在细胞核中的行为和区域.双色荧光原位杂交技术在有丝分裂前中期和中期染色体上的应用。Heterostrophus的研究表明，参与T-毒素产生的PKS 1和DEK 1分别位于不同的染色体上。结果还表明，6号和12号染色体是易位的。

项目成果

Tsuchiya et al.: "Physical mapping of plasmid and cosmid clones in filamentous fungi by fiber-FISH"Fungal Genetics and Biology. 37. 22-28 (2002)

Tsuchiya 等人：“通过纤维-FISH 对丝状真菌中的质粒和粘粒克隆进行物理定位”真菌遗传学和生物学。