Refinement and validation of a microsatellite based identification and parentage testing panel in horses

Abstract

The power of microsatellite markers lies in their ability to identify. Whether it is the identification of genes and associating them with known phenotypes or identifying and discerning individuals from one another, the role they play in the genetic field has been immense. Parentage testing of horses today is done via molecular means as opposed to serology. Microsatellite marker panels are decided upon by bodies such as the International Society for Animal Genetics (ISAG) in order to uphold international genotyping standards. The current horse microsatellite marker panel is not fully characterized and many markers are amplified by primers originally designed for linkage studies and were never intended for multiplex PCR analysis. The aim of this study was to refine and validate the current marker panel used for horses through sequencing of the repeat elements and flanking regions as well as the design of new primers for the setup of a marker panel incorporating more microsatellites and better primers. Sequencing of microsatellite flanking regions revealed that much variation lies within the regions flanking a microsatellite repeat element. Sequencing of the repeat element showed that not all markers are simple repeats, as was previously thought. The primers used to amplify microsatellite markers for horses were re-designed in the course of this study, utilizing knowledge gained from flanking region variation and repeat element length. New primers and known allele sizes allowed for the implementation of a nomenclature system in horses based on repeat element length as opposed to alphabet letters. By incorporating more markers into the panel it was hoped that a greater discriminatory power would be achieved. Measures of genetic diversity such as Observed Heterozygosity and Polymorphism Information Content showed negligible differences between the two panels however genotyping data from the old ISAG panel of nine markers showed that the probability of excluding an individual in a parentage test was better when using more markers.