Barcode analysis using mini-amplicons strategy for museum samples of neotropical primates Callithrix spp.

Abstract

The identification of species and the assignment of unidentified samples to a voucher reference database are among the most requested forensic analyses involving crimes against wildlife. Genomic DNA from forensic or museum samples, however, are frequently degraded, hampering the analysis of DNA fragments longer than 500bp. Among studies targeting non-human species identification, one of the most frequently used fragments is the mitochondrial Cytochrome C Oxidase subunit I (COI), in particular a ‘barcode' region containing approximately 650 base pairs (bp). The main objective of this work was to develop a set of primers to amplify five shorter overlapping COI fragments for the neotropical marmosets (Callithrix spp.) to be used in samples from museum specimens. Taxidermized skins, bones, and hair tissues were sampled out of 9 museum specimens with a range archival from 81 to 2 years; PCR were performed with five mini-amplicons primers pairs and with the primer pair for the whole barcode COI fragment, followed by Sanger sequencing. As results, no amplification was observed using the primer pair for the longer COI sequence; however, using the new set of mini-amplicon primers, all types of samples were amplified. We established optimal PCR conditions for the employed primers and conclude that the mini-amplicon strategy for COI region typing is best suited for hardly degraded samples in forensic research, and in particular for museum samples.