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

建立了两种植物病原真菌赤霉病菌（Nectria haemataococca）和异strophus （Cochliobolus heterostrophus）的荧光原位杂交技术（FISH），包括纤维原位杂交技术（fiber-FISH），并尝试利用已建立的技术对其染色体进行物理定位。本研究总结的结果如下：1。建立了制备用于纤维- fish的DNA纤维的方法，其中含有琼脂糖块的原生质体用于脉冲场凝胶电泳（PFGE）或PFGE分离的染色体DNA条带作为DNA纤维的来源。2 .利用多色纤维- fish，证明了用cosmid克隆制作1.6 mb cd染色体contig图谱是可行的。利用fiber-FISH测定了位于互易位断裂点附近的RFLP连锁标记的物理距离，并与遗传图谱中获得的距离进行了比较。结果表明，在减数分裂断裂点附近，交叉不受抑制，插入的外来片段不可能像先前预期的那样大。以间期细胞核为靶点，尝试用球粒和质粒探针在红嗜血杆菌中进行FISH物理定位。虽然在细胞核上成功检测到探针杂交，但作图分辨率远低于纤维- fish。间期细胞核上的FISH被认为有助于分析细胞核内染色体的行为和范围。双色FISH检测显示，参与t毒素产生的PKS1和DEK1分别位于不同的染色体上。6号和12号染色体是相互易位的。

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.