8th World Congress on Genetics Applied to Livestock Production, August 13-18, 2006, Belo Horizonte, MG, Brasil SEX VERIFICATION OF ARAB HORSES USING PCR-RFLP WITHIN ZFX/ZFY GENE M.H. Banabazi 1 , A. Javanmard 2 , N. Asadzadeh 3 and H.R. Seyedabadi 1 1 Dept. of Biotechnology, Animal Science Research Institute of IRAN (ASRI), First Dehghan Villa, P.O.Box: 1483, Karaj, IRAN. 2 Dept. of Genomics, West and North-West Agriculture Biotechnology Research Institute (ABRII-T). 3 Dept. of Animal Production and Management , Animal Science Research Institute of IRAN (ASRI), First Dehghan Villa, P.O.Box: 1483, Karaj, IRAN. INTRODUCTION Molecular sexing of mammals is normally done by PCR amplification of Y chromosomal fragments such as SRY, or coamplification of homologous fragments from both sex chromosomes such as amelogenin, or ZFX/ZFY. During the sex determination using the SRY region, male samples yield a product, whereas female samples do not. Therefore, this system requires two primer sets, one for the SRY region and another as an internal positive control. On the other hand, the amelogenin and the zinc finger (ZFX/ZFY) regions require only one primer set to amplify a region of the X–Y homologous gene. In the ZFX/ZFY system, sex is determined by the appearance of sex-specific bands in restriction fragment length polymorphism (RFLP) assays after PCR. Aasen and Medrano (1990) assayed ZFX/ZFY for humans, cattle, sheep, and goats using universal primers. They amplified 447 or 445 bp fragments from male or female genomic DNA corresponding to the ZFY or ZFX genes. They could not find RFLPs in horse fragments with the group of enzymes chosen for the other species. However, Senese et al. (1999) reported a HaeIII PCR-RFLP in the ZFY/ZFX genes of horses. In order to estimation accuracy of sex determination, we determine the sex of arab horses using the ZFX/ZFY regions and then verify them by comparing with real sex. MATERIAL AND METHODS Blood sampling and DNA extraction. Whole blood samples were collected form 20 arabian horses (10 individuals from each sex) in Khuzestan province-IRAN. DNA was extracted using modified salting out procedure (Miller et al., 1988). PCR and genotyping. Reactions were carried out using following primers (Senese et al. 1999): 5'-ATAATCACTggAgAgCCACAAgCT-3' 5'-gCACTTCTTTggTATCTgAgAAAgT-3' Total volume of 25μl of each PCR reaction were contained 1X of PCR buffer, 2mM of MgCl2, 200μM of dNTPs, 10-20pM of each primer, 1U of Taq polymerase and 50-100ng of genomic DNA. Thermal program was included 29 cycles denaturation at 92°c for 2min, annealing at 58°c for 1min and extension at 72°c for 1min. PCR products were digested by HaeIII enzyme overnight in 37°c. The restricted fragments were then electrophoresed on 1.8% agarose gel which stained by ethidium boromide.