Use of VNTR Probes For Analysis of the Sheep Genome

Abstract

DNA fingerprints were prepared from sheep DNA. Optimal DNA fingerprinting conditions were established for the VNTR probes 33.15, M13 protein III, and alpha-globin 3'HVR, and the inheritance and variability of the detected fragment bands were assessed in a sample of Romney individuals. As with other eukaryotic species, the VNTR loci detected were inherited in a Mendelian fashion and exhibited considerable polymorphism. The extent of DNA fingerprint variability in the Romney sample was established by measuring the mean proportion of bands shared, the average band frequency, and the probability of exclusion (Pe). The DNA fingerprint variability in the Romney sample was then compared to previously described DNA fingerprint variability in human Caucasian populations with the same probes. The sheep DNA fingerprints were less variable than the human fingerprints. The Pe values for sheep were 3.6 x 10-3 for -33.15, 4.7 x 10-3 for M13 protein III, and 7.2 x 10-2 for alpha-globin 3'HVR, whereas in humans the Pe values were considerably lower, with 2.0 x 10-8 for -33.15, 1.3 x l0-la for M13 protein III, and 1.5 x 10-6 for alpha-globin 3'HVR. A set of standard DNA fingerprinting conditions were developed and then used to test further VNTR probes on sheep DNA. The probes included known multi-locus and single-locus VNTR probes of human origin, and cloned synthetic tandem repetitive sequences. All but two of the 18 VNTR probes hybridised to sheep DNA under the standard conditions, suggesting a wide variety of VNTR loci in the sheep genome. In addition, VNTR loci were cloned from sheep DNA by two processes, 'probe walking', and adaptor mediated PCR and cloning. The probe walking procedure resulted in the development of two novel multi-locus VNTR probes that detected highly variable DNA fingerprints in sheep. The adaptor mediated PCR and cloning procedure provided four multi-locus VNTR probes and ten single-locus VNTR probes. The variability of DNA fingerprints provided by each of the multi-locus VNTR probes that successfully hybridised to sheep DNA was assessed in a sample of l0 Romneys. Considerable differences in DNA fingerprint variability were observed, with Pe values ranging from 2.2 x l0-1 to 2.1 x 10-9. DNA fingerprints were prepared from sheep from four different flocks representing four different breeds; Merino, Romney, Dorset and Wiltshire. The variability of DNA fingerprints prepared with the VNTR probes 33.15, M13 protein III, and alpha-globin 3'HVR in combination with two enzymes, HinfI and HaeIII, was assessed in each flock. DNA fingerprint variability was consistently highest in the Merino flock, and lowest in the Wiltshire flock. Analysis of the Merino and Wiltshire flocks with with eight single-locus VNTR probes indicated the same trend in variability. These results, taken together with the known flock histories, suggested that sheep DNA fingerprint variability was related to genetic diversity. Considerably more genetic diversity was observed between each of the flocks than was observed within any individual flock. This trend was interpreted as a consequence of breed formation and flock foundation, during which genetic diversity was lost. Overall, however, the process of domestication has resulted in breeds that are genetically divergent, thus maintaining variability within the species. The use of DNA fingerprints for the detection of major genes contributing to quantitative traits was investigated. DNA fingerprint analysis of Romney flocks that had been selected for, or against, resistance to facial eczema (FE) indicated that some DNA fingerprint bands were more common within resistant flocks than within susceptible and control flocks. A possible explanation for this flock-specificity was a physical association between some VNTR loci and FE resistance loci. This hypothesis was tested by genetic linkage in a pedigree in which resistance to FE was segregating. The pedigree was initiated by crossing the resistant and susceptible flocks to provide F1 animals that were putatively heterozygous for resistance loci. Two F1 sires were then outcrossed with 150 ewes each to provide pedigrees segregating for resistance. Genetic linkage analysis was carried out in one of the pedigrees by comparing the inheritance of DNA fingerprint bands derived from the sire with the inheritance of resistance in the progeny. Resistance of the offspring was established by challenge with sporidesmin, the fungal toxin responsible for the symptoms of FE. Two criteria of resistance were measured; elevation of the enzyme gamma-glutamyl transferase (GGT) in the blood post-challenge, and elevation of glutamate dehydrogenase (GDH) in the blood post-challenge. A total of 455 paternal DNA fingerprint bands were assessed. One of these bands showed a pattern of inheritance that was similar to the inheritance of resistance. Analysis of offspring at each of the phenotypic extremes of resistance indicated a statistically significant association between this band and FE resistance measured by GGT elevation in the blood (P = 0.006), or by GDH elevation in the blood (P = 0.002).