Characterization of Genetic Polymorphism of Novel MHC B-LBⅡ Alleles in Chinese Indigenous Chickens

Genetic polymorphism of the major histocompatibility complex （MHC） B-LBⅡ gene was studied by amplification of exon 2 using PCR, followed by cloning and DNA sequencing in eight indigenous Chinese chicken populations. To reveal the genetic variation of the B-LB Ⅱ gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LB Ⅱsequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identification of 31 novel MHC B-LBⅡ alleles from 30 unrelated individuals of Chinese chickens that were sampled. These are the first designators for the alleles of chicken MHC B-LBⅡ gene based on the rule of assignment for novel mammalian alleles. Sequence alignment of the 31 B-LB Ⅱ alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informative and 17 singleton variable sites were observed. Among the polymorphic sites, the nucleotide substitutions in the first and second positions of the codons accounted for 36.76% and 35.29%, respectively. The sequence similarities between the alleles were estimated to be 90.6%-99.5%. The relative frequencies of synonymous and nonsynonymous nucleotide substitutions within the region were 2.92%±0.94% and 14.64%±2.67%, respectively. These results indicated that the genetic variation within exon 2 appeared to have largely arisen by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of the β1 domain coded by exon 2 revealed 6 synonymous mutations and 27 nonsynonymous substitutions at the 33 disparate sites. In particular, the nonsynonymous substitutions at the putative peptide-binding sites are considered to be associated with immunological specificity of MHC B-LB Ⅱ molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.

Expression of B Locus Beta 2 (<i>BLB2</i>) Gene at Cytolytic and Latent Immune Response Stages of Immunocompetence in Nigerian Indigenous Chickens

An economically-important trait in poultry for which gene identification continues to be a challenge is immune response. The objective of the study was to quantitate the expression of major histocompatibility complex (MHC) class II BLB2 gene at cytolytic and latent immune response stages in Nigerian indigenous chickens. A total of 108 Nigerian indigenous chickens (NIC) were sourced across the South-western states in Nigeria. The birds were inoculated with sheep red blood cells (SRBC), after which blood samples were obtained (5 days post-inoculation) and antibody haemagglutination test was carried out to place the birds into groups of high and low antibody titre levels. The categorisation of the birds resulted in six groups of normal feather high, normal feather low, naked neck high, naked neck low, frizzle feather high and frizzle feather low antibody groups. A total of 48 chicks were selected from the progeny for gene expression studies. Surgical excision of thymus and spleen was carried out for the detection of cytolytic and latent responses of the birds. β-actin was used as the endogenous control and the critical threshold method (2–ΔΔCт) was carried out for the determination of fold change. The fold change of spleen tissue expression at cytolytic immune response of the birds was 30,362.44 compared to latent response 294.07;and the fold change of thymus expression at cytolytic immune response of the birds was 51.98 compared to latent response 5.24. At both cytolytic and latent stages of immune response to SRBC antigen, BLB2 expression in the spleen was comparatively higher than in the thymus and the height of transcriptional activity was associated with the cytolytic stage. The birds of high titre at both the cytolytic and the latent responses had higher mRNA expression. This study concluded that BLB2 gene expression in the Nigerian indigenous chicken was induced at the cytolytic stage and repressed at the latent stage. During avian infections, the category of high immune response birds would perform better than the low immune response counterpart;and the protective response that BLB2 gene offers will be repressed from one time point to the other.

Integrative analysis of hypothalamic transcriptome and genetic association study reveals key genes involved in the regulation of egg production in indigenous chickens

Indigenous chicken products are increasingly favored by consumers due to their unique meat and egg quality.However,the relatively poor egg-laying performance largely impacts the economic benefits and hinders sustainable development of the local chicken industry.Thus,excavating key genes and effective molecular markers associated with egg-laying performance is necessary to improve egg production via genetic selection in indigenous breeds.In the present study,comparative hypothalamic transcriptome between pre-laying(15 weeks old)and peak-laying(30 weeks old)Lushi blueshelled-egg(LBS)chicken was performed.A total of 518 differentially expressed genes(DEGs)were identified.Among the DEGs,64 genes were enriched in 10 Gene Ontology(GO)terms associated with reproductive regulation via GO analysis and considered as potential candidate genes regulating egg-laying performance.Of the 64 genes,16 showed high connectivity(degree≥12)by protein–protein interaction(PPI)network analysis and were considered as potential core candidate genes(PCCGs).To further look for key candidate genes from the PCCGs,firstly,the expression patterns of the 16 genes were examined in the hypothalamus of two indigenous breeds(LBS and Gushi(GS)chickens)between the pre-laying and peak-laying stages using quantitative real-time PCR(qRT-PCR).Eleven out of the 16 genes showed significantly differential expression(P<0.05)with the same changing trends in the two breeds.Then,correlations between the expression levels of the above 11 genes and egg numbers and reproductive hormone concentrations in serum were investigated in high-yielding and low-yielding GS chickens.Of the 11 genes,eight showed significant correlations(P<0.05)between their expression levels and egg numbers,and between expression levels and reproductive hormone concentration in serum.Furthermore,an association study on single nucleotide polymorphisms(SNPs)identified in these eight genes and egg production traits was carried out in 640 GS hens,and a significant association(P<0.05)between the SNPs and egg numbers was confirmed.In conclusion,the eight genes,including CNR1,AP2M1,NRXN1,ANXA5,PENK,SLC1A2,SNAP25 and TRH,were demonstrated as key genes regulating egg production in indigenous chickens,and the SNPs sites within the genes might be served as markers to provide a guide for indigenous chicken breeding.These findings provide a novel insight for further understanding the regulatory mechanisms of egglaying performance and developing molecular markers to improve egg production of indigenous breeds.

Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

To reveal genetic variation of MHC B-G gene at Chinese native chickens, two PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the B-G gene and used to amplify two DNA fragments in ten Chinese indigenous chicken breeds and one introduced breed. The fragments were cloned and sequenced to assure that the expected sequences of chicken B-G gene were isolated. Of which the 189 bp fragment encompassing the most variable region within exon 2 of B-G gene was employed for PCR-SSCP assay, this method provided evidence for the presence of at least 56 B-G genotypes in the Chinese chickens sampled. It revealed a high degree of diversity in B-G genes of Chinese local breeds; particularly, high variation of B-G gene was confirmed with the presence of 48 B-G genotypes within Tibetan chicken population. Not only can the B-G genotypes be used to preliminarily screen new B-G alleles, but also they would be utilized to investigate MHC haplotypes and matched unrelated donors for bone marrow transplantation in immune researches. Another fragment of 401 bp size spanning over partial intron 1 and exon 2 of B-G gene was employed for PCR-RFLP analysis with two restriction enzymes of Msp Ⅰ and Tas Ⅰ in the breeds sampled. In this part of the gene, three novel SNPs were detected at the two restriction sites. It was more generally found the transition of two nucleotides of A294G and T295C occurred at Tas Ⅰ restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that an alone mutation of A294G occurring at the site, which also caused the substitution of amino acid, asparagine 54-to-serine; and we haven＇t found only single mutation occurred at position 295 of the restriction site. At the Msp Ⅰ site, the transversion of G318C led to a non-synonymous substitution, glutamine 62-to-histidine. The variations at expression level caused by the genetic variability of B-G gene may bring about the changes in immune specificity of B-G antigen finally. Furthermore, two alleles, A and B, were identified at Msp Ⅰ and Tas Ⅰ loci of B-G gene, respectively. The allele frequencies were estimated, which gave a nonsymmetrical distribution either in the eight Chinese local breeds or in the introduced breed. By comparison, allele A at Msp Ⅰ locus was tended to be dominative, while, the allele B at Tas Ⅰ locus was tended to be prevalent in the breeds analyzed. It is concluded that the genetic variability of B-G gene revealed by the PCR-SSCP and RFLP assays in Chinese native chickens provide molecular data for further investigating the varied immune functions of B-G antigen; and the PCR-RFLPs at Msp Ⅰ and Tas Ⅰ loci of B-G gene might be used as genetic markers in selecting for the traits of disease resistance in chicken breeding.

Genetic Diversity and Population Structure of Three Strains of Indigenous Tswana Chickens and Commercial Broiler Using Single Nucleotide Polymormophic (SNP) Markers

The Tswana chicken is native to Botswana and comprises strains such as the naked neck, normal, dwarf, frizzled, and rumples. The origins of the different strains of Tswana chicken remain unknown and it is not yet clear if the different strains represent distinct breeds within the large Tswana chicken population. Genetic characterization of different strains of Tswana chickens using SNP arrays can elucidate their genetic relationships and ascertain if the strains represent distinct breeds of Tswana chicken population. The aim of this study was therefore to investigate population structure and diversity and to estimate genetic distances/identity between the naked neck, normal and dwarf strains of Tswana chickens. A total of 96 chickens (normal strain (n = 39), naked neck strain (n = 32), dwarf strain (n = 13) and commercial broiler (n = 12)) were used in the study. SNP genotyping was carried out using the Illumina chicken iSelect SNP 60 Bead chip using the Infinium assay compatible with the Illumina HiScan SQ genotyping platform. The observed heterozygosity (Ho) values were 0.610 ± 0.012, 0.611 ± 0.014, 0.613 ± 0.0006 for normal, naked neck and dwarf strains of Tswana chickens respectively and averaged 0.611 ± 0.016 across the three strains of Tswana chickens compared to Ho of 0.347 ± 0.023 in commercial broiler chicken. The expected heterozygosity (He) values were 0.613 ± 0.00012, 0.614 ± 0.00013, 0.608 ± 0.00021 for normal, naked neck and dwarf strains of Tswana chickens respectively and averaged 0.612 ± 0.00015 across the three strains of Tswana chickens compared to He of 0.577 ± 0.00022 in commercial broiler chicken. Principal component analysis (PCA) was used to get an insight into the population structure of indigenous Tswana chickens. The first two principal components revealed a set of three clusters. The normal strain of Tswana chicken and commercial broiler clustered together in one group. The dwarf strain clustered separately in one group and the naked neck and normal strains clustered together in the last group. The separate clustering of the dwarf strain from the rest of Tswana chicken strains suggests significant genetic uniqueness of the dwarf strain and very close genetic similarities between the normal and naked neck strains. The clustering pattern was confirmed by less genetic differentiation and less genetic distances between the naked neck and normal strains of Tswana chicken than between the two strains and the dwarf strain of Tswana chicken.

Epidemiological Investigation of Avian Leukemia in Some Indigenous Chicken Breeds of China

[ Objective] The paper was to understand the natural infection status of avian leukemia in some indigenous chicken breeds of China. [ Method ] Using ELISA assay and virus isolation method, epidemiological investigation of ALV-AB and ALV-J avian leukemia of 10 indigenous chicken breeds were conducted. ALV dynamics were monitored in F2 generation of four chicken lines. ALV pollution of attenuated live vaccines used in raising process was also inspected through sampling method. [ Result] The positive rate of ALV-P27 antigen was 0 -62.1% ; the positive rate of ALV-AB antibody was 0 -25.0% ; the positive rate of ALV-J antibody was 0 - 59.0% ; the positive rate of virus isolation was 0 - 22.0%. The positive rate of ALV-P＇27 antigen in 1 -day-old chick mecunium of four lines was 6.0% - 67.0%. The positive rate of virus isolation in 6-week-old chickens was 2.0% - 34.3%. Two kinds of vaccines from two hatches pro- duced by a manufacturer were polluted by ALV. [ Conclusion] Most indigenous chickens were infected by ALV. There were great differences among different breeds of indigenous chicken, which might be related to ALV genetic resistance of different indigenous chickens. The ALV positive rates of F： chicks were slightly enhanced in some lines, which might be related to vaccine pollution.

Genetic diversity and population structure of indigenous chicken breeds in South China

A total of 587 individuals from 12 indigenous chicken breeds from South China and two commercial breeds were genotyped for 26 microsatellites to investigate the genetic diversity and population structure.All microsatellites were found to be polymorphic.The number of alleles per locus ranged from 5 to 36,with an average of12.10±7.00(SE).All breeds,except White Recessive Rock,had high allelic polymorphism(>0.5).Higher genetic diversity was revealed in the indigenous chicken breeds rather than in the commercial breeds.Potential introgression from the commercial breeds into the indigenous chickens was also detected.The population structure of these indigenous chicken breeds could be explained by their geographical distribution,which suggested the presence of independent history of breed formation.Data generated in this study will provide valuable information to the conservation for indigenous chicken breeds in future.