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

马铃薯晚疫病真菌致病疫霉基因组的分子细胞遗传学分析

基本信息

批准号：
18580043
负责人：
TAGA Masatoki
金额：
$ 2.37万
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：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

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.

疫霉是马铃薯最具破坏性的疫病——晚疫病的致病病原体。虽然该真菌的基因组序列计划正在进行中，但细胞遗传学的基本基因组信息非常缺乏。在本研究中，我们进行了分子细胞遗传学分析，以确定假单胞菌的核型和倍性。采用胚管爆裂法和秋水仙碱、秋水仙碱、环己胺化学处理相结合的方法制备有丝分裂染色体标本。用该方法制备的染色体用DAPI、Giemsa和硝酸银染色进行核型分析。标准二倍体菌株的染色体数多数为2n=10-14，包括一条或两条NOR染色体。姐妹染色单体呈盘绕状，异染色质在未完全凝聚的染色体中可见。克隆荧光原位杂交（FISH）探针，进行特异性染色体鉴定和细胞学倍性分析。以rDNA为探针，NOR位于染色体末端附近。用单拷贝基因NiaA和Piexol作为探针，每个细胞核的信号数量显示出倍性水平。建立了用流式细胞术测定病原菌核DNA含量的方法。以拟南芥为内参，估计病原菌基因组大小为250 ~ 300mb。对全世界收集的约60株菌株进行调查，发现菌株可分为单倍体、二倍体、三倍体、非整倍体和异核体。以前报道的四倍体在我们的收集中没有检测到。