Microsatellite polymorphism between and within broiler populations

Two independent broiler chicken populations were genotyped with microsatellite markers to determine genetic polymorphisms within and among broiler populations. Birds were genotyped with primers from the US Poultry Genome Mapping Kits 1 and 2. The 59 primer sets selected for this study provided wide genomic coverage. All 59 primer sets amplified a polymerase chain reaction product in Population L, whereas 57 primer sets produced a product in Population C. The average allele number per line per microsatellite was 2.8 and 2.9 for Populations L and C, respectively. Considering the 57 primer pairs generating product in both lines, 72.3% of the total alleles were unique to one or the other population. This study illustrates the high polymorphism level in broiler populations of microsatellites amplified from primers developed from Red Jungle Fowl or White Leghorn sequences

Differences in Major Histocompatibility Complex Frequencies after Multitrait, Divergent Selection for Immunocompetence

White Leghorn chickens from lines selected for four immune-response traits (IR lines) were serotyped for B system alloantigens characterizing the haplotypes and genotypes to examine the effect of divergent selection for multitrait immunocompetence on MHC haplotype and genotype frequencies. The selected lines were derived from the Ottawa Strain 7. The selection index included four immunocompetence traits: antibody production against Mycoplasma gallisepticum (MG) and Pasteurella multocida, inflammatory response to phytohemagglutinin, and reticuloendothelial carbon clearance. The four lines include two replicates of high and low multitrait-immunocompetence lines. After four cycles of selection, significant differences (P \u3c .05) in several B system haplotype frequencies were observed, both among IR lines and between the IR lines and the Ottawa Strain 7. The B2 haplotype frequency was greater in all IR lines than in the Ottawa Strain 7. The B21 frequency was less in both high lines than in the Ottawa Strain 7. In comparisons among lines, frequencies of B21 were greater in both replicates of the low lines and the B12 and B19 frequencies were significantly greater (P \u3c .05) in the high lines. A gene substitution model showed effects (P \u3c .10) of specific haplotypes on MG and on the index. The B2 haplotype had a positive effect associated with MG. Haplotype B21 was positively associated with the multitrait index. Haplotype B13 had a negative effect on both MG and the index. Significant differences (P \u3c .01) in genotype frequencies were also noted among the IR lines. Associations between specific MHC haplotypes or genotypes and immune-response traits may offer insight into MHC-mediated mechanisms of disease resistance

Performance comparison of dwarf laying hens segregating for the naked neck gene in temperate and subtropical environments

This study compares laying performances between two environments of dwarf laying hen lines segregating for the naked neck mutation (NA locus), a selected dwarf line of brown-egg layers and its control line. Layers with one of the three genotypes at the NA locus were produced from 11 sires from the control line and 12 sires from the selected line. Two hatches produced 216 adult hens in Taiwan and 297 hens in France. Genetic parameters for laying traits were estimated in each environment and the ranking of sire breeding values was compared between environments. Laying performance was lower, and mortality was higher in Taiwan than in France. The line by environment interaction was highly significant for body weight at 16 weeks, clutch length and egg number, with or without Box-Cox transformation. The selected line was more sensitive to environmental change but in Taiwan it could maintain a higher egg number than the control line. Estimated heritability values in the selected line were higher in France than in Taiwan, but not for all the traits in the control line. The rank correlations between sire breeding values were low within the selected line and slightly higher in the control line. A few sire families showed a good ranking in both environments, suggesting that some families may adapt better to environmental change

Editing the genome of chicken primordial germ cells to introduce alleles and study gene function

With continuing advances in genome sequencing technology, the chicken genome assembly is now better annotated with improved accuracy to the level of single nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck, turkey and zebra finch have now been sequenced. A great opportunity exists in avian biology to use genome editing technology to introduce small and defined sequence changes to create specific haplotypes in chicken to investigate gene regulatory function, and also perform rapid and seamless transfer of specific alleles between chicken breeds. The methods for performing such precise genome editing are well established for mammalian species but are not readily applicable in birds due to evolutionary differences in reproductive biology. A significant leap forward to address this challenge in avian biology was the development of long-term culture methods for chicken primordial germ cells (PGCs). PGCs present a cell line in which to perform targeted genetic manipulations that will be heritable. Chicken PGCs have been successfully targeted to generate genetically modified chickens. However, genome editing to introduce small and defined sequence changes has not been demonstrated in any avian species. To address this deficit, the application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for performing small and defined sequence changes was investigated in this thesis. Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9 (SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained showed that small sequences changes ranging from a single to a few nucleotides could be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT, SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate specific homozygous and heterozygous genotypes. This finding demonstrates the utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with high efficiency in PGCs. Since PGCs can be converted into pluripotent stem cells that can potentially differentiate into many cell types from the three germ layers, genome editing of PGCs can, therefore, be used to generate PGC-derived avian cell types with defined genetic alterations to investigate the host-pathogen interactions of infectious avian diseases. To investigate this possibility, the chicken ANP32A gene was investigated as a target for genetic resistance to avian influenza virus in PGC-derived chicken cell lines. Targeted modification of ANP32A was performed to generate clonal lines of genome-edited PGCs. Avian influenza minigenome replication assays were subsequently performed in the ANP32A-mutant PGC-derived cell lines. The results verified that ANP32A function is crucial for the function of both avian virus polymerase and human-adapted virus polymerase in chicken cells. Importantly, an asparagine to isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support avian influenza polymerase function. This genetic change can be introduced into chickens and validated in virological studies. Importantly, the results of my investigation demonstrate the potential to use genome editing of PGCs as an approach to generate many types of unique cell models for the study of avian biology. Genome editing of PGCs may also be applied to unravel the genes that control the development of the avian germ cell lineage. In the mouse, gene targeting has been extensively applied to generate loss-of-function mouse models to use the reverse genetics approach to identify key genes that regulate the migration of specified PGCs to the genital ridges. Avian PGCs express similar cytokine receptors as their mammalian counterparts. However, the factors guiding the migration of avian PGCs are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit genes which have been implicated in controlling mouse PGC migration. The results showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient PGCs colonise the developing gonads in reduced numbers and are significantly reduced or absent from older stages. This finding shows a conserved role for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other genes suspected to be involved in controlling the development of avian germ cells can be investigated using this approach to increase our understanding of avian reproductive biology. Finally, the methods developed in this thesis for editing of the chicken genome may be applied in other avian species once culture methods for the PGCs from these species are develope