Many bands were detected on an electrophoretic profile of double-stranded (ds) RNA preparation from a single strain of Fusarium poae isolated from wheat. When the purified dsRNA sample was deep-sequenced by a next-generation sequencer, sixteen virus-like assembled contigs with predicted amino acid sequences showing homologies to respective viral RNA-dependent RNA polymerases (RdRps) were found by BLAST analysis. Fourteen out of sixteen sequences showed homologies to RdRps of known mycoviruses, that is, four mitoviruses, two narnaviruses, two partitiviruses, an alternavirus, a fusarivirus, a hypovirus, a victorivirus, and two unclassified mycoviruses, Sclerotinia sclerotiorum dsRNA mycovirus-L and Aspergillus foetidus slow virus 2, respectively. The other two putative viral RdRp sequences showed homologies to those of members of negative-stranded RNA viruses, the Ophiovirus and the Phlebovirus respectively, which mycoviruses had been not ever assigned to. Based on genome structure and phylogenetic analysis, both viruses were thought to be members of novel respective negative-stranded RNA virus groups. The presences of all sixteen viral RdRp sequences identified by BLAST analysis were confirmed by sequencing RT-PCR products generated from the starting dsRNA material using primers designed from the de novo assembled sequences of respective putative mycoviruses. Since the single strain of F. poae was considered to be multiply infected with mycoviruses from novel taxonomical groups in addition to many common mycoviruses, the RNA virome of the strain was found to be highly diverse.

Restriction landmark genome scanning (RLGS) is a 2-DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, NotI as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites directly. We employed isoschizomers, MspI and HpaII, that recognize the same sequence (CCGG) but have different methylation sensitivity. We carried out the RLGS analysis of Arabidopsis thaliana ecotype Columbia, and obtained a pair of spot patterns with MspI and HpaII. We detected 22 spots in both patterns. In comparison of them, 18% of the spots were polymorphic, which indicated the methylation of C(5m)CGG sites. Further analyses revealed an additional methylated site of NotI. Moreover, 52 and 54 restriction enzyme sites were also analyzed in two other ecotypes, Wassilewskija and Landsberg erecta, respectively. Consequently, 15% of the 52 common sites showed methylation polymorphism among the three ecotypes. The restriction sites analyzed in this study were located in or near genes, and contribute new data about the correlation between methylation status and gene expression. Therefore, this result strongly indicates that the improved RLGS method is readily applicable to practical analyses of methylation dynamics, and provides clues to the relationship between methylation and gene expression.

The National Institute of Agrobiological Sciences (NIAS) is implementing the NIAS Genebank Project for conservation and promotion of agrobiological genetic resources to contribute to the development and utilization of agriculture and agricultural products. The project’s databases (NIASGBdb; http://www.gene.affrc.go.jp/databases_en.php) consist of a genetic resource database and a plant diseases database, linked by a web retrieval database. The genetic resources database has plant and microorganism search systems to provide information on research materials, including passport and evaluation data for genetic resources with the desired properties. To facilitate genetic diversity research, several NIAS Core Collections have been developed. The NIAS Rice (Oryza sativa) Core Collection of Japanese Landraces contains information on simple sequence repeat (SSR) polymorphisms. SSR marker information for azuki bean (Vigna angularis) and black gram (V. mungo) and DNA sequence data from some selected Japanese strains of the genus Fusarium are also available. A database of plant diseases in Japan has been developed based on the listing of common names of plant diseases compiled by the Phytopathological Society of Japan. Relevant plant and microorganism genetic resources are associated with the plant disease names by the web retrieval database and can be obtained from the NIAS Genebank for research or educational purposes.

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