Hypervariable markers in the chicken genome.

This thesis describes the characterization of minisatellite sequences in the chicken genome. A DNA fingerprinting protocol was established using four types of minisatellite probe in chickens. Segregation analysis was carried out on three pedigrees. Linkage was higher than in several other avian species, with groups of up to four cosegregating bands being detected. Although some overlap among probes occurred, the probes detected mostly independent sets of loci. Two commercial egg-laying lines were analyzed in detail. The mean band sharing coefficient was 0.5 - 0.6 and band frequencies in the lines were bimodally distributed. In an Investigation of rare breed populations, within population coefficients were found to be around 0.9 and between population values were around 0.15. These populations were highly inbred, and require genetic management. The charomid cloning system was used to isolate chicken minisatellites. Library 1 (11 - 21 kb size fraction) yielded 35,000 clones and Library 2 (2-7 kb) yielded 115,000 clones. 1,710 and 4,275 clones for Libraries 1 and 2 respectively, were transferred into microtitre wells and replicated on to nylon membranes. For Library 1, the total number of positively hybridizing minisatellites was 28 (1.64% of clones). For Library 2, the total number of positives was 113 (2.64%). 55 cloned fragments were tested against four unrelated chickens: 30 revealed variable single locus patterns. The mean number of alleles detected with clones isolated from Library 1 was 4.8 + 1.8 (s.d.), mean number of heterozygotes out of 4 was 2.3 + 1.4. The mean number of alleles detected with clones isolated from Library 2 was 3.8 + 1.5, and the mean number of heterozygotes out of four was 1.9 + 1.2. Detailed characterization was made of fifteen probes. Probes were tested against four families of 13, 13, 10 and 17 offspring. Informative segregations were produced in 51% of parent-offspring comparisons. No allelic mutation was observed, therefore the mean mutation rate was less than 0.003 per gamete (95% confidence maximum). Three probes were linked on an autosome, and one was Z chromosome linked. Heterozygosity and allelic variability were measured in 67 individuals. Mean heterozygosity ranged between 50% and 84%. Heterozygosity values were slightly lower than those found using the same system in humans (Armour et al. 1990), but very similar to those found in peafowl (Hanotte et al. 1991). There was no significant difference in the level of variability revealed by probes from the two libraries, though the mean number of heterozygous individuals among the different sample sets and mean percentage heterozygosity were generally higher for Library 1 than Library 2. Genetic variability within, and genetic distance among commercial egg-layer and broiler lines were analysed using distance matrices and parsimony. Both egg-layers and broiler lines showed similar levels of within-line variability and extremely large allele frequency differences. Genetic distance among lines were very high, reflecting the small population sample size. Maximum parsimony techniques failed to cluster individuals according to their population of origin. The proportion of the probe sequence made up of DNA flanking the minisatellite, and size differences within an individual when digested with different restriction enzymes, was tested using a panel of unrelated individuals digested separately with AluI and HaeIII. DNA samples were also digested with ten restriction enzymes. Variability, allele size and pattern clarity were analyzed for each probe/enzyme combination. For nine of these (60%), MboI, the cloning enzyme, gave the most interpretable pattern with the smallest allele size. However, occasionally, such as with cGgaMS134, AluI or HaeIII produced more informative low molecular weight patterns.