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

以两种植物病原真菌Nectria haematao occa和Cochliobolus Heterostrophus为材料，建立了包括纤维FISH在内的荧光原位杂交技术，并对它们的染色体进行了物理定位。本研究取得的主要成果如下：1.研究成果。目的：1.建立了一种制备DNA纤维的方法，该方法以含有原生质体的脉冲场凝胶电泳(PFGE)琼脂糖块或PFGE分离的染色体DNA条带作为DNA纤维的来源。用多色纤维FISH制作红球线虫1.6-Mb CD染色体粘粒克隆的重叠群图谱是可行的。用Fibre-FISH方法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，并与遗传图谱上的距离进行了比较。结果表明，在减数分裂断裂点附近的交叉没有被抑制，插入的外源片段不太可能像之前预期的那样大。利用粘粒和质粒探针，以间期核为靶点，尝试FISH对红球线虫的物理定位。虽然在细胞核上成功检测到了探针杂交，但作图分辨率远低于纤维FISH。间期细胞核上的FISH被认为有助于分析染色体在核中的行为和区域。双色FISH显示，参与T毒素产生的PKS1和DEK1分别位于不同的染色体上。研究还表明，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.