Inventory of MHC alleles in Taiwan local chickens

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Local chicken breeds in Taiwan

General history

Six local chicken breeds are maintained in a conservation program at NCHU since 1982 (Lee 2006; Fig. 10, Table 4 & 5). Three breeds are Taiwan local chickens, Hsin-Yi originates from an aboriginal tribe in the central mountain of Taiwan, Ju-Chi from a village in central-south of Taiwan and Hua-Tung from east of Taiwan. Quemoy comes from Quemoy Island near Fu-Jian Province of China, Shek-Ki is from the GuangDong province of China and Nagoya is from Japan.
Hsin-Yi is a medium size red feathered chicken with no special pattern, white skin, and blue shanks. Ju-Chi is a small size and Hua-Tung is a medium size, both breeds have black plumage without special pattern and black shanks. Quemoy is a small size black plumage with gold laced feather on the neck, white skin, and blue shanks. Shek-Ki has yellow skin, yellow feather and yellow shanks with medium size. Nagoya has yellow plumage with black tail, and blue shanks with medium size. The history of local chicken breed in Taiwan can be traced back to the aborigines in the island who domesticated some jungle fowls. About 400 years ago, the Chinese immigrants came to Taiwan and brought in chickens from the southeastern China. And nearly 300 years ago, the Dutch and the Spanish had come to Taiwan for decades, and some European chickens might have been brought into Taiwan in this period. Later, Japanese ruled Taiwan from 1895 to 1945 and Rhode Island Red and White Leghorn were the earliest exotic breeds introduced to Taiwan in 1918. Also some Japanese breeds, such as Nagoya and Mikawa, and American breeds, such as Barred Plymouth Rock, were also introduced by the Japanese before 1925.
Most of chickens raised in Taiwan were the descendants of these chickens, before modern white broilers were introduced. These breeds have inhabited Taiwan for several generations and have become adapted to the environment. They have been selected by the local people, who recognized them as the native chickens of Taiwan (Lee 2006).

Breeds conservation program in NCHU

Conservation of chicken breeds was set up to study an array of traits, such as disease resistance or meat quality (Lee 2006). Recently, they were included in a survey of molecular diversity using a set of microsatellite markers which revealed different heterozygosity levels among them (Berthouly et al. 2008).
Table 5 shows the number of chickens for each breed that were used to set up the conservation programme in NCHU. Pedigree was recorded and random mating was used to produce chickens of each generation. The average generation interval varied from 1 to 2 years.

Population sampling for the thesis (years, numbers, family structure)

Table 7 shows the number of animals included in this thesis. In 2001, the sampling involved only half of the total population. The experimental protocols used for different sampling stages are listed in Table 7: samples from 2001 were used for population diversity (microsatellites, mtDNA, MC1R and LEI0258) and phenotypic data analysis (growth traits and immune responses), Samples from generation 2007 and 2008 were used for investigating the distribution of LEI0258 locus under a non-selection situation. Samples in 2009 were used for H6N1 low-pathogenic avian influenza virus challenge experiment, and to observe the antibody response for challenge and subsequence vaccines.

Molecular markers

mtDNA

The hypervariable sequence 1 (HVS-I) of the D-loop region was amplified using the same primers (Table 8) following Liu et al. (2006) protocol. PCR target region is showed in Figure 11. PCR was done using 20 ng of genomic DNA, with 1 pmol of each primer, and units of HotStarTaq Master Mix Kit (Qiagen) in a final volume of 25 ul. Initial denaturation for 15 min at 95°C was followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 1 min, followed by a 10 min extension at 72°C. PCR products were sequenced by Eurofins MWG Operon, using their standard protocol for purified PCR products. The Bioedit version 7.0.9.0 (Thomas, 1999) was used to assemble sequences and identify polymorphisms. The median-joining networks (Bandelt 1999) were constructed using the program Network 4.516 (http://www.fluxus-engineering.com/sharenet_rn.htm).

Microsatellite markers

A set of 24 microsatellite markers derived from the marker set of the AvianDiv European research project was genotyped on the LABOGENA facilities at Jouy-en-Josas. The list of 24 analyzed markers is listed in Table 9.

MC1R

Plumage, skin and eye color variations of the domestic fowl are used as selection criteria since the first mutant phenotypes appeared in early domesticated stocks (Smyth 1990). Plumage colour selection is not only for fancy breeding, but also as a standard for commercial poultry industry like layer production system (red plumage for brown-egg lines) or to satisfy the preferences requirement in different geographic markets (Groen 2003). The character of the genetics of cutaneous and ocular pigmentation which combined phenotypes and utility became particularly interesting for research.
Several loci control plumage or coat color in birds and mammals, such as Extended black (E), Dominant white (I), Recessive white (c) loci (Table 10; Smyth 1990, Kerje et al. 2003, Kerje et al. 2004 & Chang et al. 2006). E locus affects the distribution of black (eumelanin) and red (phaeomelanin) pigment, and Takeuchi et al. (1996) indicated a strong correlation between MC1R polymorphism and E locus. Further molecular studies confirmed that the gene product of the Extended black locus controlling plumage color in chicken was MC1R, which is located on chicken chromosome 11. Mutations in the MC1R sequence caused amino acid substitutions which were correlated with plumage color variation (Kerje et al. 2003 & Ling et al. 2003; Table 10).
PCR target region is showed in Figure 12, PCR primers for PCR are listed in Table 8. PCR was done using 20 ng of genomic DNA, with 1 pmol of each primer, and units of HotStarTaq Master Mix Kit (Qiagen) in a final volume of 25 ul. Initial denaturation for 15 min at 95°C was followed by 2 c ycles of 94°C for 45 s, 66°C for 30 s, 72°C for 1 min, and 2 cycles of 94°C for 45 s , 63°C for 30 s, 72°C for 1 min, and 2 cycles of 94°C for 45 s, 60°C for 30 s, 72°C for 1 min, and 26 cycles of 94°C for 45 s, 57°C for 30 s, 72°C for 1 min, followed b y a 15 min extension at 72°C. PCR products were sequenced by Eurofins MWG Operon, using their standard protocol for purified PCR products. SNPs analysis using the Staden package (Staden 1998) and defined genotype and haplotype. Phylogam tree was used Populations 1.2.30 (Langella 1999) to compute population distances and phylogenetic tree.

LEI0258

LEI0258 marker is an atypical Variable Number of Tandem Repeat (VNTR) located within the chicken MHC region. Chicken MHC region located on chromosome 16, LEI0258 marker is a minisatellite which exhibits a good correlation with serological typing of the chicken MHC (Fulton et al. 2006). The structure of LEI0258 locus contain a 12 bp repeat, a 13bp repeat and other polymorphisms, and can be a criteria to determinant different types of alleles (Fig. 13 & Fig. 14). Fulton et al. (2006) identified LEI0258 alleles from chicken stocks (Table 8), and proposed to use marker LEI0258 as a standard procedure to identify MHC alleles in any population.
Primers for PCR are the same as those used by McConnell et al 1999 in Table 9. PCR was done using 20 ng of genomic DNA, with 1 pmol of each primer, and units of HotStarTaq Master Mix Kit (Qiagen) in a final volume of 25 ul. The amplification protocol included a first cycle of initial denaturation for 15 min at 95°C, followed by 35 cycles of 94°C for 45 s, 62°C for 90 s, 72°C for 60 s, with a last cycle of extension for 15 min at 72°C. Gel m igration was realized with a 4% agarose gel for a first determination of allele size. Due to the complex structure of the LEI0258 sequence which contained a 13-bp and 12-bp repeat sequences and several SNP sites, sequencing is necessary to provide an unambiguous identification of alleles. Direct sequencing of PCR products was performed for homozygous genotypes (following Fulton et al. 2006) or after 4% agarose gel cutting and purification with QIAquick PCR Purification Kit (Qiagen) for heterozygous genotypes. Sequencing was done by Eurofins MWG Operon, using their standard protocol for purified PCR products. The Bioedit version 7.0.9.0 (Thomas 1999) was used to assemble sequences and to check the length (varying according to the number of repeats) and polymorphic sites (either single nucleotide polymorphisms or deletions) for each allele. Phylogam tree was used Populations 1.2.30 (Langella 1999) to compute population distances and phylogenetic tree.
Due to previously results showed Taiwan local chicken breeds have better disease resistance (Cheng et al. 1987, Fan et al. 1988, Cheng et al. 1990, Chen et al. 1991 and Chao & Lee 1991), and it is lack for study about investigate the relationship between Taiwanese local chickens’ serotypes and immune response or more thorough experiments to carry out. Therefore, the aim of the thesis is also to search for private LEI0258 alleles in local breeds and test their functional effect by vaccine or pathogen challenge experiment.

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Performance traits

Growth

Body weight (BW) was measured at various frequencies depending on the experiment. For the global analysis of diversity, three measures were retained: initial body weight at hatch (BW0), body weight at the time of rapid growth (BW12) and mature body weight (BW47). For the challenge experiment, body weight was measured every week from hatch to 16 weeks of age.

Heat adaptation

Panting is generally observed when ambient temperature is above 29°C with 50% humidity (Weaver 2002). For the global analysis of diversity, panting was recorded individually on a daily basis between 24 and 27 weeks of age, in the afternoon of the hot season when the ambient temperature was higher than 30°C (highest for 36°C, lowest for 31°C and average for 33.5°C with 61% humidity). Individual panting rate was calculated as the ratio of number of days where a given chicken exhibited panting on the total number of observed days

Immune traits

Immune response was investigated for the global analysis of diversity by using two different antigens. Sheep Red Blood Cells (SRBC) are frequently used as a non-pathogenic antigen for evaluating antibody response in selection experiments, and antibody response to NDV vaccination can be used as a selection criterion for immune response (Lamont et al. 2003). Newcastle disease is a major poultry disease in Taiwan (Yang et al. 1999) and vaccination against Newcastle disease virus (NDV) is a standard procedure recommended by government sanitary authorities. All day-old chicks were vaccinated against NDV in this study. At 8 weeks of age, all chickens were vaccinated again with 0.5 mL NDV vaccine, and serum was collected 7 and 14 days later. Haemagglutination inhibition tests were used to determine antibody titers against NDV. At 11 and 13 weeks of age, chickens were injected with 0.1 mL 0.25% SRBC intravenously and serum was collected 7 days later. Antibody titers of chickens against SRBC were determined by haemagglutination tests. The antibody titer was expressed as the log2 of reciprocal of the highest dilution.
Kinetics of antibody titers was studied by calculating NDD, as the difference between anti-ND antibody titer at day 14 and anti-ND antibody titer at day 7 post-inoculation, and SRBCD, as the difference between anti-SRBC antibody titer at secondary response and anti-SRBC antibody titer at primary response
For the challenge experiment, immune response to Avian influenza and secondary immune response was measured for Infectious Bronchitis (IB), Infectious Bursal Disease (IBD) and Newcastle disease.
Anti-AI titer: Chickens received a drop with 107 EID50 of H6N1 avian influenza viruses (A/chicken/Taiwan/0825/2006, provided by Dr. Poa-Chun Chang, Graduate Institute of Microbiology and Public Health, National Chung-Hsing University, Taiwan, R.O.C.) into eye and nose. Blood samples were collected at 0, 7, 14 and 21 days post-challenge. Anti-AI titers were measured by enzyme-linked immunosorbent assay (ELISA) with commercial test kit (IDEXX Laboratories, Inc., Westbrook, ME; http://www.idexx.com/view/xhtml/en_us/livestock-poultry/poultry/avian-influenza.jsf).
Anti-IB titer: Chickens were vaccinated again with 0.5 mL Infectious Bronchitis (IB) vaccine, and serum was collected 0, 14 and 28 days later. Anti-IB titers were measured by enzyme-linked immune-sorbent assay (ELISA) with commercial test kit (IDEXX Laboratories, Inc., Westbrook, ME).
Anti-IBD titer: Chickens were vaccinated again with 0.5 mL Infectious Bursal Disease (IBD) vaccine, and serum was collected 0, 14 and 28 days later. Anti-IBD titers were measured by enzyme-linked immunosorbent assay (ELISA) with commercial test kit (IDEXX Laboratories, Inc., Westbrook, ME).
The antibody responses to ND were measured by hemagglutination inhibition test (HIT), the antibody titer was expressed as the log2 of the reciprocal of the highest dilution.

Methods

Sequencing and sequence analysis

PCR products of mtDNA, MC1R and LEI0258, were sequenced and sequence alignment, editing and processing were performed with software BioEdit 7.0.9.0 (Hall 1999) and Staden package (Staden et al. 1998).
Mitochondrial DNA and LEI0258 sequence alignment and polymorphisms identification were done by using BioEdit. Haplotypes were scored manually. The median-joining networks (Bandelt et al. 1999) were constructed for mitochondrial DNA data using the program Network 4.516 (http://www.fluxus-engineering.com/sharenet_rn.htm).
The length variants (number of repeats) and polymorphic sites (either single nucleotide polymorphisms or deletions) of LEI0258 have been checked for each allele. MC1R SNP analysis was done with Staden package (Staden et al. 1998) to define genotypes and haplotypes.

Population genetics

Allele frequencies, number of alleles, expected and observed heterozygosity were calculated by using GENETIX 4.04 (Belkhir et al. 2004). Genetic distances (Nei 1983) were calculated and neighbor-joining (NJ) trees were established with Populations 1.2.30 (Langella 1999).
All marker data, including microsatellites, MC1R and LEI0258, were merged and treated with a Multiple CO-inertia Analysis (MCOA) by using R (R Core Development Team 2006) with ADE-4 package (Chessel et al. 2004; Dray & Dufour 2007). This method makes possible the extraction of common information from separate analyses, by setting up a reference typology, and comparing each typology separately. The efficiency of a marker is assessed by its typological value (Tv), the contribution of the marker to the construction of the reference typology, which is equal to the product of the variance (Var) multiply by the congruence with the consensus Cos² (i.e. the correlation between the scores of individual locus tables and the synthetic variable of the same rank) (Laloë et al. 2007).

Table of contents :

INTRODUCTION
BIBLIOGRAPHY
I. FAO’s recommendation and information system
II. Characterization
A. Sampling
B. Molecular markers
C. Phenotypes
III. Methods of population genetics
A. Within population parameters
B. Between population parameters
C. Bayesian methods to infer population structure
IV. Summary of results obtained on local chicken breeds in Asia
MATERIALS AND METHODS
I. Local chicken breeds in Taiwan
A. General history
B. Breeds conservation program in NCHU
II. Population sampling for the thesis (years, numbers, family structure)
III. Molecular markers
A. mtDNA
B. Microsatellite markers
C. MC1R
D. LEI0258
IV. Performance traits
V. Methods
A. Sequencing and sequence analysis
B. Population genetics
C. Analysis of performance data
D. Combining molecular and phenotypic data
E. Bayesian􀀃approach􀀃
F. Experimental challenge for H6N1 low pathogenic avian influenza virus
G. Experimental chickens
H. Viruses
I. MHC genotyping
J. Mating for specific MHC genotypes of experimental chickens and management
K. Challenge experiment and antibody responses measurement
L. Statistical analysis
RESULTS
I. Polymorphisms of LEI0258
A. Inventory of MHC alleles in Taiwan local chickens
B. Trends in frequencies (Tours 2010, annex 1)
II. Combining molecular data and performance traits
A. Paper accepted in Animal Genetics
B. Supplementary tables for paper 1
C. Complementary data and analyses
III. Immune response
A. AI challenge and MHC effects
B. AI challenge and vaccine response (AGAH congress 2, annex 2)
FINAL DISCUSSION and PERSPECTIVES
I. Characterizing genetic diversity of a subset of local breeds
II. Recommendations for the conservation program of local chicken breeds in Taiwan
III. Prospects for further studies
REFERENCES

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