Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Poster: Genome Sequencing & ESTs
          

ISOLATION OF DISEASE RESISTANCE GENE ANALOGS IN ITALIAN RYEGRASS

¹ Forage Crop Research Institute, Japan Grassland Farming & Forage Seed Association, 388-5 Higashiakada, Nishinasuno, Tochigi 329-2742, Japan

² Yamaguchi Agricultural Experiment Station, 1419 Oouchi-mihori, Yamaguchi, Yamaguchi 753-0214, Japan

The large-scale cloning of resistance gene analogs (RGAs) was attempted in Italian ryegrass. Total DNA was extracted from mature leaf of Italian ryegrass by CTAB method, and then used for PCR template DNA. Primer design and PCR amplification were carried out according to Collins et al.(1998). PCR products were fractionated by 1.5% agarose gel electrophoresis, and expected size of DNA fragments (300-500bp) were extracted from the gel. After TA cloning of the purified DNA fragments, PCR clones were picked up at random, and subjected to one pass sequencing for sequence similarity search with public database. To date we have sequenced 12,000 clones and succesfully obtained 79 unique sequences which have shown sequence similarity to known NBS-LRR type proteins in current data base. The additional sequencing will be done up to 24,000 clones with different primer sets reported by others. Our data and resource will facilitate genomics research of disease resistance in Italian ryegrass.