Discovery and Characterization of Tetranucleotide Short Tandem Repeats in North American Bears (Ursids)

Background: Accurate individual identification is essential to wildlife crime investigation or associating individuals to source populations in conservation genetics. Current methodology utilized dinucleotide short tandem repeats (STRs) that can be difficult to type accurately and have high mutation rates. Tetranucleotide STRs, like those used in human forensics and population genetics, are more stable and can have a more diverse allele composition due to inherent motif substructure, making them more robust and informative for finer grained individualization. STRs are currently typed by PCR amplification followed by electrophoretic sizing that cannot identify polymorphisms in the repeat motif structure often observed at tetranucleotide loci. Hypothesis: The main objective of this study was to perform a preliminary identification of a suite of new tetranucleotide repeat STR loci, for each of the three primary bear species in North America: the American black bear, the brown (grizzly) bear, and the polar bear. These loci would be more informative than existing panels of dinucleotide STR loci reported in the literature and used in wildlife genetics. Methods: In this study, barcoded, adaptor-ligated genome libraries were prepared for four individuals from each bear species from enzymatically fragmented ursine DNA to be interrogated by massively parallel sequencing. Preliminary libraries for the set of samples were size selected and quantified to ensure sufficient concentrations for downstream processing. These preliminary libraries were enriched for tetranucleotide STR sequence regions prior to sequencing using biotinylated RNA baits designed to capture twelve sequence motifs common to mammals. The enriched libraries were sequenced using the Ion Torrent™ Personal Genome Machine™ (PGM) and the data analyzed using the NextGENe® software using a Floton assembly method for de novo alignment to synthetically reference sequences based on the nucleotide structure of the targeted STR motifs as reference genomes for the bear species examined in this study do not exist. Loci were sorted by species and then selected within a species based on the polymorphism observed among the individual samples. Results and Conclusions: Several tetranucleotide STR loci were identified within each bear species. Loci were characterized based upon common repeat sequence and motif structure, and individualized based on unique sequence composition in regions flanking the repeat region. . Further characterization of individual alleles observed at the loci within and between the bear species is underway, and additional individuals will be typed at the optimized loci to establish allelic frequency distributions, genetic stability, and establish their value as individualizing markers. All unambiguous sequences identified in this study will be submitted to Genbank.