Practical strategy for identification of single nucleotide polymorphisms in fruiting mei (Prunus mume Sieb. et Zucc.) from amplified fragment length polymorphism fragments

Single nucleotide polymorphism (SNP) is the most abundant form of genetic variation among individuals within a species. SNPs can be used as markers for gene discovery and for assessment of diversity. We established a practical strategy for identification of SNPs in fruiting mei (Prunus mume Sieb. et Zucc.) from amplified fragment length polymorphism (AFLP) fragments. The main modification of this procedure was optimization of the reamplification of bands excised from an AFLP gel by using a single enzyme (EcoRI) in digestion reaction to generate larger AFLP fragments and to lower the number of bands on gels, using lower-concentration polyacrylamide gels (4%) and loading each sample into 4 continuous lanes, using a newly modified protocol for purification of AFLP bands from the gel, and using additional cycles for reamplification of AFLP bands. In this study, 15 groups of bands with identical migration distances from 10 fruiting mei cultivars were selected for purification. Eighty-one of the 150 chosen bands were successfully reamplified, and 67 of these reamplified polymerase chain reaction products yielded reliable sequences belonging to 13 groups. The alignment of 13 group sequences yielded 95 SNPs, for a total of 5252 bp. Among these SNPs, 73 were heterozygous in the loci of some individual cultivars. The SNP distribution was 58% transition, 40% transversion, and 2% indels. There was also 1 dinucleotide polymorphism and 1 tetranucleotide deletion.