Development of a universal test for sex determination in species of the bovidae family based on the analysis of amelogenin gene polymorphism

One of the important aspects of the population and evolutionary genetics study of animals is their sex determination using DNA markers. As a candidate locus for sex determination in both wild and domestic animal species is the amelogenin gene, since the variants of this gene located on the X and Y chromosomes are polymorphic along the sequence. The aim of our research was the development of the universal test system for sex determination in species of the Bovidae family based was the development of the universal test system for sex determination in species of the Bovidae family based on the analysis of amelogenin gene polymorphism. The studied sample set consisted of 139 individuals of domestic and wild animal species belonging to the 8 different genus of the family Bovidae including Bos (n=4), Bison (n=16), Ovis (n=38), Capra (n=65), Rupicapra (n=10), Tragelaphus (n=2), Pseudois (n=2) and Hemitragus (n=2). Samples of animal tissue obtained from farms, the mountain hunters club, reserves and zoos were used as biological material. The literature sources, recommendations of the International Society of Animal Genetics (ISAG) on markers for sex determination as well as amelogenin gene sequences deposited in the gene bank of the NCBI served as the basis for the test system development. The primers SE47 (forward: 5’-CAGCCAAACCTCCCTCTGC-3’) and SE48 (reverse: 5’-CCCGCTTGGCTTGTCTGTTGC-3 ’) were selected to amplify the amelogenin gene fragment, flanking the site with the Y-chromosome-specific deletion. To develop the test system, the possibilities of visual detection of amplicons by gel electrophoresis in agarose gel, as well as laser detection by capillary electrophoresis on an ABI3130xl DNA analyzer (Applied Biosystems, USA) were taken into account. As a result, differences in fragment lengths for the X and Y chromosomes in males and females of all studied species were identified, visually determined by gel electrophoresis. The lengths of the fragments specific for the X chromosome, depending on the species of animals, were 264 or 280 bp, for the Y chromosome - 202 or 217 bp. Using the genetic analyzer ABI3130xl allowed the implementation of the automatic laser detection of the obtained amplicons, which makes it possible to integrate the developed test system into the multiplex microsatellite analysis systems.

genus and species of the family Bovidae, sex determination, amelogenin gene

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