MITOCHONDRIAL-DNA CONTROL REGION POLYMORPHISMS - GENETIC-MARKERS FOR ECOLOGICAL-STUDIES OF MARINE TURTLES

We describe a rapid and sensitive method for the detection of populati on-specific genetic markers in mitochondrial DNA (mtDNA) and the use o f such markers to analyse population structure of marine turtles. A se ries of oligonucleotide primers specific for the amplification of the mtDNA control region in Cheloniid turtles were designed from prelimina ry sequence data. Using two of these primers, a 384-385-bp sequence wa s amplified from the 5' portion of the mtDNA control region of 15 gree n turtles Chelonia mynas from 12 different Indo-Pacific rookeries. Fou rteen of the 15 individuals, including some with identical whole-genom e restriction fragment patterns, had sequences that differed by one or more base substitutions. Analysis of sequence variation among individ uals identified a total of 41 nucleotide substitutions and a l-bp inse rtion/deletion. Comparison with evidence from whole-genome restriction enzyme analysis of the same individuals indicated that this portion o f the control region is evolving approximately eight times faster than the average rate and that the sequence analysis detected approximatel y one fifth of the total variation present in the genome, Restriction enzyme analysis of amplified products from an additional 256 individua ls revealed significant geographic structuring in the distribution of mtDNA genotypes among five of the 10 rookeries surveyed extensively. A dditional geographic structuring of genotypes was identified through d enaturing gradient gel electrophoresis (DGGE) of amplified products. O nly two of the 10 rookeries surveyed could not be differentiated, indi cating that the Indo-Pacific C. mydas include a number of genetically differentiated populations, with minimal female-mediated gene flow amo ng them. Important applications for genetic markers in the conservatio n and management of marine turtles include the identification of appro priate demographic units for research and management (i.e. genetically discrete populations) and assessment of the composition of feeding an d harvested populations.