Dot-blot Analysis for Identification of japonica Rice Cultivars and Genotyping of Recombinant Inbred Lines

Abstract

Dot-blot analysis of rice cultivars was performed using 14 cultivar-specific sequences identified previously by AFLP and PCR-RF-SSCP analyses as probes. DNA samples prepared from leaves by a simple method without the use of organic solvent were found to be usable for dot-blot analysis. Although more than a single grain of rice was needed as material for direct dot-blotting of genomic DNA, PCR amplification of DNA enabled detection of cultivar-specific signals in a DNA sample from a single grain by dot-blot analysis. All 31 rice cultivars examined could be distinguished from each other by analysis using ten cultivar-specific probes. Dot-blot analysis was also applied to genotyping of recombinant inbred lines (RILs), the results of which corresponded to those by electrophoretic analysis of PCR products amplified with specific primer sequences. The probe sequences deleted in ‘Nipponbare’ were mapped on the rice chromosome by BlastN search using all the Kasalath BAC-end sequences and by analysis of RILs and chromosome segment substitution lines (CSSLs). Dot-blot analysis using cultivar-specific sequences was found to be applicable for the identification of cultivars and genotyping of plants in rice.