SITE-DIRECTED MUTAGENESIS WITH THE POLYMERASE CHAIN-REACTION FOR IDENTIFICATION OF SIBLING SPECIES OF MYTILUS

The large sample sizes needed for population and biogeographic studies make sequencing impractical as a means of quantifying genotype freque ncies. In a previous study, we identified sequence variation in the mi tochondrial 16S ribosomal gene from mussels in a sibling species group (Mytilus spp.) that discriminated mussels introduced to the west coas t of North America (Mytilus galloprovincialis) from native mussels (My tilus trossulus). In this study, we used site-directed mutagenesis by the polymerase chain reaction (PCR) to transform a single diagnostic n ucleotide substitution into a restriction site. By using a large (51 b p) oligomer as a primer, a size shift in cut and uncut PCR products wa s visible on an agarose gel. Thus, PCR followed by a restriction diges tion allowed identification of species-specific haplotypes in hours ra ther than days. This method is applicable to any study in which rapid detection of genotypes is desired. We show the presence of M. trossulu s haplotypes in Monterey Bay, California, extending its southern range . We also present evidence for heteroplasmic individuals that contain mitochondrial haplotypes indicative of both species.