Molecular cytogenetic analysis of the genome of the potato late blight fungus Phytophthora infestans

致病疫霉是马铃薯最具破坏性的病害晚疫病的病原体。虽然该真菌的基因组测序计划正在进行中，但细胞遗传学的基本基因组信息非常缺乏。本研究采用分子细胞遗传学方法对马铃薯晚疫病菌的核型和倍性进行了研究。染色体核型分析我们将芽管破裂法与秋水仙素、秋水仙胺和环己酰胺的化学处理相结合，建立了一种制备有丝分裂染色体标本的方法。用该方法制备的染色体用DAPI、Giemsa和银硝酸盐染色用于核型分析。二倍体标准株的染色体数目为2n=10-14，多数标本含有1条或2条NOR染色体。在不完全凝聚的染色体中，可观察到卷曲的姐妹染色单体和异染色质.克隆荧光原位杂交（FISH）探针，并进行FISH鉴定特异染色体和倍性细胞学分析。以rDNA为探针，NOR定位于染色体末端附近。以单拷贝基因NiaA和Piexol为探针，每个细胞核的信号数目可以反映倍性水平.流式细胞术（FCM）建立了用流式细胞术测定马铃薯晚疫病菌细胞核DNA含量的方法。以拟南芥（Arabidopsis thaliana）为内参，估计致病疫霉的基因组大小为250- 300 Mb。世界范围内的计算机科学研究概况。60个菌株可分为单倍体、二倍体、三倍体、非整倍体和异核体。以前报道的四倍体不能在我们的收集检测。

Phytophthora infestans is the causal pathogen of the most devastating potato disease called late blight. Although genome sequence project of this fungus is under way, basic genome information from cytogenetics is very scarce. In this study, we conducted molecular cytogenetic analyses to determine the karyotype and ploidy of P. infestans.1. Karyotype analysisWe developed a method to prepare mitotic chromosome specimens by combining the germ tube burst method and chemical treatment with colchicine, colcemid, and cyclohexamide. Chromosomes prepared with this method were stained with DAPI, Giemsa, and silver nitrate for karyotyping. Chromosome number of a standard diploid strain was 2n=10-14 in most specimens including one or two NOR chromosomes. Sister chromatids in a coiled shape and heterochromatines could be observed in not-fully condensed chromosomes.2. Fluorescence in situ hybridization (FISH)Probes were cloned and FISH was performed to identify specific chromosomes and analyze ploidy cytologically. With rDNA as a probe, NOR was located near the end of chromosome. Using single copy genes, NiaA and Piexol, as probes, the number of signals per nucleus was shown to indicate ploidy level.3. Flow cytometry (FCM)The method to measure nuclear DNA contents of P. infestans by FCM was established. Using Arabidopsis thaliana as the internal reference, genome size of P. infestans was estimated to be 250-300Mb. The survey of world-wide collection of ca. 60 strains showed that strains can be grouped into haploids, dipoids, triploids, aneuploids, and heterkaryons. Formerly reported tetraploids could not be detected in our collection.