Identification of sequence variations by PCR-RFLP and its application to the evaluation of cpDNA diversity in wild and cultivated soybeans

The diversity and maternal lineage in wild and cultivated soybeans have pre viously been assayed using restriction fragment length polymorphism (RFLP) and sequencing analyses of chloroplast DNA (cpDNA). Here we describe a meth od based on PCR-RFLP for the identification of nucleotides at four mutation sites in noncoding regions of cpDNA. Of the four sites, two were located i n restriction enzyme sites and two were not. For the latter two sites, new primers were designed to artificially create restriction sites that spanned them. The PCR-RFLP method enabled us to identify nucleotides at each of th e four mutation sites easily and reliably. Fifty-seven wild and sixty-seven cultivated soybeans of different origins and different cpDNA types (types I, II, and III) were assayed. All of the samples tested could be classified into four haplotypes. All of the type-I and -II accessions had the same nu cleotides at each of the four mutation sites, while all of the type-III acc essions, except for 3 wild ones, had nucleotides that were different from t hose of types I and II. A sequencing analysis revealed that the 3 wild acce ssions possessed other single-base variations in the non-coding regions of trnH-psbA and trnT-trnL. The results of this study suggest that the type-I and type-II chloroplast genomes form a group that is distinct from the type -In: chloroplast genome.