Laboratory analysis:
A skin biopsy was received from the aborted fetus with hair samples from
the dam, the sire and the other foal for DNA genotyping and parentage
verification. Genomic DNA was extracted from the samples using an EZ-10
Spin Genomic DNA Minipreps purification kit following the manufacturer’s
protocol. A total of 12 microsatellite markers (AHT4, AHT5, ASB17,
ASB23, HMS6, HMS7, HTG4, VHL20, HMS3, ASB2, HTG10, HMS2) specific to
Equus caballus were used in this study. All markers are included in the
panel recommended by the International Society for Animal Genetics for
diversity studies and parentage verification. The 12 microsatellites are
amplified in one multiplex reaction using Stockmarks; horse genotyping
kit (Cat. No.: PN4336407 – Applied Biosystem - USA) according to the
method described by (Sargious et al., 2014). Fragment sizes of
microsatellite alleles were determined using Genetic analyzer 3500
(Applied Biosystem-USA) with the aid of Liz standard. The data obtained
is further analyzed using Gene Mapper V 4.1 software (Applied Biosystem,
USA). The Proposed nomenclature for the 12 equine short tandem repeat
loci investigated is based on the number of repeat units and is adopted
from the recommendation of International Society of Forensic Genetics
(ISFG) for the nomenclature of human STRs (Bozzini et al., 1996). Global
standardization of molecular marker profiles and their use within animal
parentage verification is currently governed by various institutes,
inclusive of ISAG (https://www.isag.us/)
Table (1):