Application of AFLP Technique That Uses Non-radioactive Fluorescent Primers to the Detection of Genetic Diversity in Japanese Rice Cultivars and Cloning of DNA Sequences Derived from an Indica Genome

Abstract

To perform rapid amplified fragment length polymorphism (AFLP) analysis and efficiently clone AFLP fragments with non-radioactive primers, we modified the current AFLP protocol. When the modified procedure was applied to the rice genome, 30 to 80 AFLP fragments (∼40 to 400 bp in length) could be detected in a single PCR reaction. To assess the suitability of the technique for the analysis of the rice genome, we first applied it to quantify genetic diversity in a sample of 55 predominantly Japanese modern and domestic cultivars. In this analysis, more than 75% of the Japanese rices were differentiated by the combination of six prinrer pairs. Then we used the technique to identify and clone AFLP fragments unique to the Japanese variety Kinuhikari compared to those found in the closely related cultivar Koshihikari. In this cloning, we obtained sequences derived from chromosomal segments of an Indica genome, which had introgressed into the Kinuhikari genome. These two studies demonstrated that the modified AFLP technique is suitable for the genome analysis in rice.