Preimplantation genetic diagnosis has great potential in the horse, but information on evaluation of equine embryo biopsy samples is limited. Blastocysts were biopsied using a Piezo drill and methods for whole-genome amplification (WGA) investigated. Results for 33 genetic loci were then compared between biopsy samples from in vitro-produced (IVP) and in vivo-recovered (VIV) blastocysts. Under the experimental conditions described, WGA using the Qiagen Repli-g Midi kit was more accurate than that using the Illustra Genomiphi V2 kit (98.2% vs 25.8%, respectively). Using WGA with the Qiagen kit, three biopsy samples were evaluated from each of eight IVP and 19 VIV blastocysts, some produced using semen from stallions carrying the genetic mutations associated with the diseases hereditary equine regional dermal asthenia (HERDA), hyperkalemic periodic paralysis (HYPP) or polysaccharide storage myopathy 1 (PSSM1). Three of 81 biopsy samples (3.7%) returned <50% accuracy. In the remaining 78 samples, overall accuracy was 99.3% (2556/2574 loci interrogated). Accuracy did not differ significantly between samples from IVP and VIV blastocysts. Allele drop-out in heterozygous loci was 1.6% (17/1035). Accuracy for sex determination was 100%; accuracy for heterozygosity for disease-causing mutations was 97.7% (43/44). In conclusion, Piezo-driven embryo biopsy with WGA has >95% overall accuracy in IVP and VIV embryos, and this technique is suitable for use in a clinical setting.

There is growing interest in pre-implantation genetic diagnosis (PGD) for management of inherited genetic disease in the horse. In a previous study (Choi et al. 2010 Reproduction 140, 893–902), we demonstrated normal viability of equine blastocysts after biopsy. However, genome amplification was only moderately successful and only 1 of 2 analyses of heterozygous loci accurately detected both alleles. In the current study, we investigated different methods for amplification of DNA to improve the efficiency of PGD. To evaluate allele drop-out, multiple commonly-heterozygous gene loci were evaluated. In Experiment 1, using a piezo drill, 3 to 5 biopsy samples of 20 to 30 cells each were obtained from each of 4 in vitro-produced blastocysts. The samples and embryos were stored at –20°C, then shipped to the Veterinary Genetics Laboratory at the University of California, Davis. Whole genome amplification was done with an Illustra Genomiphi V2 kit (GE Healthcare, Waukesha, WI) before PCR for specific markers. Two disease-related (SCN4A and PPIB), one gender (AME) and 17 microsatellite identification markers were genotyped, for a total of 20 loci. Results for biopsy samples were compared with those for the corresponding embryo. A DNA signal was obtained from 14/15 biopsy samples, but for only 59.6% of the 280 total genotypes. Of 40 heterozygous loci, the signal from the corresponding biopsy sample showed only one allele (underwent allele dropout) in 60/80 instances (75%). In Experiment 2, 4 biopsies were obtained from each of 4 additional in vitro-produced blastocysts, then all samples were stored at –20°C. The Repli-G Mid kit (Qiagen, Valencia, CA) was used for whole genome amplification. Two disease-related (SCN4A and PPIB), 2 gender (AME and eSRY), 10 coat colour and 17 identification markers (total of 31 loci) were examined in each biopsy sample and were compared with results for the embryos. One biopsy sample was lost. Signal was obtained from 14/15 of the remaining biopsy samples and gave a 100% match at the 2 gender loci, 2 disease-related loci and 10 coat colour loci. One identification locus, LEX33, amplified in only 8 of 22 analyses. At the remaining 16 identification loci, 223/224 biopsy results matched those for the embryos. Overall, of 51 heterozygous loci among the 4 embryos, biopsy samples exhibited allele dropout in 1/180 instances (0.6%). In conclusion, results obtained using piezo-driven embryo biopsy and whole genome amplification using the Qiagen Repli-G kit have high accuracy and this technique may be suitable for use in a clinical setting. Further studies are needed with in vivo-derived embryos and to optimize accuracy of PCR of some identification markers. This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University and by Ms Kit Knotts.

Widespread genotyping of US dairy goat breeds for casein variants has not been reported, even though the genetic data could be of use in selective breeding programs. For instance, variability in the content of protein and solids in goat milk is attributed to allelic differences in the goat alpha(s1)-casein gene. Concentrations of alpha(s1)-casein in goat milk are positively correlated with milk components and coagulation properties. The alleles A and B are designated as strong alleles, resulting in the greatest amount of alpha(s1)-casein in goat milk, whereas the E allele produces intermediate amounts and the weak allele F produces the least concentrations of alpha(s1)-casein in goat milk. Here we report on one of the first surveys of the distribution of alpha(s1)-casein genotypes in US dairy goats. The population surveyed, consisting of a total of 257 American dairy goats representing 7 main dairy breeds, contained a greater predominance of the weaker alleles, E and F, than the strong alleles, A and B. Allele distribution was related to breed, with Toggenburg, Alpine, Saanen, and Oberhasli containing the most E and F alleles and LaMancha, Nubian, and Nigerian Dwarf the fewest. Quantification of alpha(s1)-casein production by 2-dimensional gel electrophoresis demonstrated that F/F animals had the least amount of alpha(s1)-casein protein in their milk compared with all other genotypes. The results indicate that genetic improvement of dairy goats in the United States could be achieved if an alpha(s1)-casein breeding scheme were adopted.

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex 'core' panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations.