Forensic Identification of Sixteen Species of Chesapeake Bay Sportfishes Using Mitochondrial DNA Restriction Fragment-Length Polymorphism (RFLP) Analysis

Abstract

A method for the genetic identification of sixteen species of Chesapeake Bay sportfishes was developed using restriction fragment-length polymorphism (RFLP) analysis of two mitochondrial (mt) DNA gene regions amplified by the polymerase chain reaction (PCR). To facilitate molecular-based identifications we screened for genetic markers that revealed minimal intraspecific variation but consistently discriminated among the different species. Two mitochondrial markers were developed to provide redundancy for identifications, and 40 individuals of each species were screened with both markers. The primary marker was a region of mtDNA approximately 1,495 base-pairs (bp) in length that included part of the 12S ribosomal RNA (rRNA) gene and crossed into the adjacent 16S rRNA gene. When digested with the restriction enzymeRsa I, this region provided unique restriction patterms for all sixteen species. The second marker was a region of the mitochondrial NADH dehydrogenase 4 (ND4) gene approximately 1,700 bp in length and digested with the restriction enzymeBstO I. This marker also discriminated among all sixte en species, but revealed higher levels of intraspecific variation than the rRNA genes. An additional set of restriction profiles was obtained for this region using the enzymeAva II to provide identification in the event a novel restriction pattern might be encountered in future studies. Because a minimal amount of tissue is required for the PCR/RFLP analysis, these molecular markers should prove useful in identifying fishes both as adults and in their early life history stages.