| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
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 analysis
We 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.
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