Chloroplast DNA variations of cultivated radish and its wild relatives

Chloroplast DNA sequences were investigated to further elucidate the evolution of cultivated radish. This is the first known attempt to analyze intrageneric DNA sequences of chloroplast DNA in Raphanus. The nucleotide sequence of the 5′-matK region (690 bp) was determined for 17 accessions including cultivated radish (Raphanus sativus), wild radish (R. raphanistrum), and East Asian wild radish (Raphanus sativus var. hortensis f. raphanistroides Makino). Three parsimony-informative sites showed that cultivated radish has two distinctly diverged chloroplast DNA sequences. When considering the evolutionary rate of the matK sequences, it was difficult to hypothesize that a new haplotype had occurred in cultivated radish and then rapidly spread among the cultivated radish varieties. Alternatively, it is far more likely that cultivated radish received two distinct haplotypes from its wild ancestor(s). For more detailed analyses, five different chloroplast regions were amplified through the use of five newly constructed primers. The total length of the amplified regions was 32.8 kb, which covered both the coding and non-coding DNA sequences. In total, 137 restriction fragments were detected. The results of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses were consistent with that of the cpDNA sequences. We found that the combined data of the matK sequences and the PCR-RFLP analysis indicated that Raphanus raphanistrum was not the maternal ancestor of cultivated radish, and that the geographical distribution pattern of the haplotypes suggests that the East Asian wild radish has contributed to the establishment of the East Asian cultivated radish. The present PCR-RFLP method using the newly developed primers works for other plant species and for other purposes.