SIXTY NEW HORSE MICROSATELLITES

James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 USA

A panel of 60 microsatellites have been developed for use in the horse. A small insert genomic library was constructed in the pUC18 plasmid vector using white blood cells from one Thoroughbred stallion as the source for DNA. The donor horse DNA was cut with restriction enzyme Sau3A and size selected by purification on an agarose gel to yield fragments of approximately 500bp. The size selected DNA was phosphatased to prevent insert concatemerization and then ligated into the vector. The vector was then used to transform electrocompetent DH10B E. coli cells. The library was screened using a radiolabeled mixture of four dinucleotide repeats [GC₁₅, GA₁₅, GT₁₅, AT₁₅] as a probe. Positive bacterial colonies were re-screened and isolated. Plasmid insert DNA was sequenced using automated cycle sequencing at the Cornell Biotechnology Sequencing Facility. PCR primers in the regions flanking the microsatellite repeats were designed with aid of the PRIMER3 program, synthesized, and optimized using a Robocycler (Stratagene). All reaction mixtures contained 50 mM KCl, 10 mM Tris HCl pH 8.3, 1.5 mM MgCl2, 0.25 mM dATP, dCTP, dTTP, dGTP, 0.5 uM of each primer, 50 ng DNA and 1 U Ampli Taq Polymerase (Perkin-Elmer). The forward primers were labeled on the 5' end with Elmer fluorescent dyes (FAM, NED HEX). Reactions were amplified in a Stratagene Robocycler using the following conditions: 95°C (5 min) for 1 cycle, 95°C (1 min), 58°C (1 min), 72°C (30 sec) for 33 cycles, 72°C (20 min) for 1 cycle. PCR products were electrophoresed on an Applied Biosystems 310 Genetic Analyzer. Alleles were sized using Genescan and Genotyper software. Heterozygosity and PIC (Polymorphism Information Content) values were determined using DNA from 30-34 horses of various breeds, including 1 Appaloosa, 1 Arabians, 1 Belgian, 2 Clydesdales, 1 Dale pony, 1 Hackney pony, 1 Haflinger, 1 Hanoverian, 1 Icelandic pony, 2 Miniature Shetland ponies, 1 Mississippi Fox Trotter, 2 Morgans, 2 Quarter horses, 1 American Saddlebred, 2 American Standardbreds, 2 Thoroughbreds, 1 Welsh Cob, 2 Welsh Mountain ponies, and 9 horses and ponies of mixed breeds. Results of heterozygosity testing and assignment of these new markers to horse chromosomes will be presented.