Development of genetic markers to sex subadult loggerhead sea turtles (Caretta caretta) using AFLP technology
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Stegman, Claire Elaine
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Abstract
Genomics is transforming our ability to investigate the integrative biology of organisms. One technique for sampling genome-wide levels of polymorphism a priori is amplified fragment length polymorphism (AFLP), which can generate large datasets of potentially polymorphic loci using relatively few primer combinations and restriction endonucleases. The objectives of this study are to assess sex-linked patterns and develop sex-linked markers using AFLP to sex subadult loggerheads non-invasively. For this study, DNA isolated from whole blood samples was processed with an AFLP genotyping kit, and products were visualized using the Beckman Coulter CEQ 8000 Genetic Analysis System. Sixty-four primer pair combinations were tested on 32 samples with known sex (16 male; 16 female). Analyses of sex-specific amplification patterns based on signal strength, amplification frequencies, and fixed polymorphisms screened among thousands of loci revealed no strictly sex-specific fragments, although one primer pair, EAGG-MCTG, did show heavily male biased amplification frequency. Several primer pairs exhibited significant levels of genetic differentiation between sexes (AMOVA with minimum p value >0.05), which could indicate the presence of differential genes that may play a role in the sex differentiation or determination pathways of this species. The genetic diversity and evenness of each primer pair was also compared between sexes using the Shannon index to indicate where high levels of diversity are found. Higher levels of diversity could potentially stem from sex based genetic differences and therefore indicate which primer pairs may be the best candidates for expanded genomic investigation.