[논문]Investigation of PCR-RFLPs within Major Histocompatibility Complex B-G Genes Using Two Restriction Enzymes in Eight Breeds of Chinese Indigenous Chickens

Xu, R.F.; Li, K.; Chen, G.H.; Qiang, B.Y.Z.; Mo, D.L.; Fan, B.; Li, C.C.; Yu, M.; Zhu, M.J.; Xiong, T.A.; Liu, Bang

doi:10.5713/ajas.2005.942

Investigation of PCR-RFLPs within Major Histocompatibility Complex B-G Genes Using Two Restriction Enzymes in Eight Breeds of Chinese Indigenous Chickens 원문보기

Asian-Australasian journal of animal sciences, v.18 no.7, 2005년, pp.942 - 948

Xu, R.F. (Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology Huazhong Agricultural University) , Li, K. (Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology Huazhong Agricultural University) , Chen, G.H. (Department of Animal science and Technology, Yangzhou University) , Qiang, B.Y.Z. (Department of Animal Husbandry and Veterinary, College of Agriculture and Animal Husbandry, Tibetan University) , Mo, D.L. (Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology Huazhong Agricultural University) , Fan, B. (Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology Huazhong Agricultural University) , Li, C.C. (Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology Huazhong Agricultural University) , Yu, M. (Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology Huazhong Agricultural University) , Zhu, M.J. (Laboratory of Molecular Biology and Animal Bre) , Xiong, T.A. , Liu, Bang

Abstract ▼ AI-Helper

New polymorphism of major histocompatibility complex B-G genes was investigated by amplification and digestion of a 401bp fragment including intron 1 and exon 2 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique with two restriction enzymes of Msp I and Tas I in eight breeds of Chinese indigenous chickens and one exotic breed. In the fragment region of the gene, three novel single nucleotide polymorphisms (SNPs) were detected at the two restriction sites. We 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 a single mutation of A294G occurring at the site, also caused an identical substitution of amino acid, asparagine 54-to-serine, to that we described previously. And the transversion of G319C at Msp I site led to a non-synonymous substitution, glutamine 62-to-histidine. The new alleles and allele frequencies identified by the PCR-RFLP method with the two enzymes were characterized, of which the allele A and B frequencies at Msp I and Tas I loci were given disequilibrium distribution either in the eight Chinese local breeds or in the exotic breed. By comparison, allele A at Msp I locus tended to be dominant, while, the allele B at Tas I locus tended to be dominant in all of the breeds analyzed. In Tibetan chickens, the preliminary association analysis revealed that no significant difference was observed between the different genotypes identified at the Msp I and Tas I loci and the laying performance traits, respectively.

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문제 정의

, 2000). Their goals were to search for proper way to utilize the good characteristics of local chickens. However, the genetic polymorphism of B-G genes in Chinese indigenous breeds was rarely reported

가설 설정

(1) The number of animals in each population we detected is not enough to demonstrate the true event.

제안 방법

In this preliminary study, in order to find a potential valuable molecular marker of selection for higher laying intensity and egg weight in marker-assisted selection (MAS) program, we tentatively tried to search for the association of the novel polymorphisms by two restriction enzymes of Msp I and Tas I with the laying performance traits detected.
In this study, the genetic polymorphism in B-G gene was investigated by amplification of the 401 bp fragment spanning over parts of intron 1 and exon 2 encoding the Ig- V-like domains using PCR, followed by cloning, sequencing and restriction fragment length polymorphism analysis with two restriction enzymes of Msp I and Tas I in eight breeds of Chinese indigenous chickens and one commercial broiler breed.
The following statistical model was used to analyze the relationship between the genotypes at Msp I and Tas I loci of the B-G gene and laying performance traits using GLM procedure of SPSS11.0.
Then comparisons of the predicted sequences with the corresponding sequence in GenBank (AF388372) were done using the BLAST2 software provided by the NCBI server (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html) to assure that the expected sequences of chicken MHC B-G gene were isolated.

대상 데이터

A total of 381 indigenous Chinese chicken from eight different native breeds (Tibetan chicken, Langshan chicken, Silkies, Xianju chicken, Gushi chicken, White earlobes (Buff Baier) chicken, Beijing Fatty chicken and Chinese Game chicken) and commercial broiler chickens were used.
In this research, there are three novel SNPs within the MHC B-G gene were detected, which occurred correspondingly at the two restriction sites of Msp I (CaCGG) and Tas I (aAATT), respectively.

성능/효과

For all birds, no more than two allelic sequences were observed, suggesting that the primer pair specifically amplified a single locus, and no breed-specific sequence was found in different individuals tested from the nine chicken breeds, showing the universality of the primers used to amplify the fragment of MHC B-G gene.
For each bird, the sequence analysis either using direct PCR product or the clone of PCR product of genomic DNA from the same animal gave no more than two alleles, furthermore, by comparison the pattern types of PCR-RFLP observed in same bird, no variation appeared in all the detected chicken breeds in the condition.
However, only two genotypes of AA and AB were found in all the chicken breeds analyzed but in the Langshan chicken, single genotype (AA) was observed.
Taken together, we concluded that the designed primer pairs and the PCR conditions used in the study could be used to specifically, and sensitively amplify the alone, expected sequence of B-G gene.

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