PCR-RFLP genotyping protocol for chicken Mx gene G/A polymorphism associated with the S631N mutation.

The Mx (myxovirus resistance) gene codes for a protein with antiviral activity. Non-synonymous G/A polymorphism at position 2032 of chicken Mx cDNA results in a change at amino acid 631 of the Mx protein. This mutation has been shown to affect the antiviral activity of the Mx molecule, although recent studies have not confirmed this effect in response to some influenza strains. Nevertheless, the G/A polymorphism could be important for the chicken's response to other viruses. A robust PCR-RFLP protocol for genotyping chicken Mx gene polymorphism associated with the S631N mutation was developed. The F primer anneals to the last intron of the Mx gene, and the R primer anneals to the last exon of the gene, with an expected PCR product of 299 bp. PCR products were digested with Hpy8I. This enzyme cuts the sequence 5'-GTN|NAC-3', 2 bp downstream of the Mx polymorphism for the G allele, whereas the fragment containing the A allele is not cleaved. One hundred and twenty-seven chickens (commercial broilers, White Leghorn and New Hampshire) were genotyped using this protocol, and genotyping data were validated by sequencing. Full identity of results between the two genotyping methods was observed for all 127 samples, proving the reliability and robustness of this PCR-RFLP protocol.

Avian TAP genes: detection of nucleotide polymorphisms and comparative analysis across species.

TAP1 and TAP2 genes code for the two subunits of the transporter associated with antigen processing (TAP), and in chicken they are located between the two MHC class I genes. Using primers based on chicken sequences, the genomic regions corresponding to chicken TAP1 exons 6 to 7 and TAP2 exons 4 to 6 (which encode portions of the chicken TAP1 and TAP2 molecules corresponding to the human peptide-binding regions) were amplified and sequenced from chicken (70 birds), turkey (24), pheasant (6), and guinea fowl (7). A total of 80 within-species single nucleotide polymorphisms (SNPs) were identified. None of the chicken SNPs detected here was present in public databases. The SNP frequencies in chicken were 9.57 SNP/kb in TAP1 and 19.16 SNP/kb in TAP2, while turkey showed similar SNP frequencies in the two genes. Putative amino acid sequences were inferred to identify non-synonymous substitutions. The alignment of the consensus polypeptide sequences showed that most of the amino acid variations were conserved or semi-conserved substitutions. In conclusion, a high variability in the level of nucleotide polymorphism was observed within the two genes, with chicken showing the highest polymorphism rate in both genes. Most of the SNPs identified were within introns, and a general conservation of both amino acid numbers and characteristics of residues among and within the species was found. These data underline the functional importance of these molecules, but also suggest their capacity to bind different antigenic peptides.

Single nucleotide polymorphism discovery in the avian Tapasin gene.

Tapasin is a transmembrane glycoprotein located in the endoplasmic reticulum. Its function is to assist the assembly of major histocompatibility complex class I molecules. The chicken Tapasin gene includes 8 exons and is localized inside the major histocompatibility complex between the 2 class IIbeta genes. The aim of the current study was the estimation of single nucleotide polymorphism frequency within the avian Tapasin gene. The Tapasin gene sequence from exon 5 to exon 6 was amplified for the chicken, turkey, and pheasant, and sequences of different lengths were obtained. The sequence analysis based on PolyBayes identified 25 putative single nucleotide polymorphism sites when the 3 species were compared. The coding sequences were further translated and analyzed to identify amino acid substitutions. The results indicated that polymorphisms within this region of the gene was mainly observed in the heterozygous state. The level of conservation of the Tapasin gene sequence among species is likely to be related to the functional importance of the gene.

Effect of consanguinity on Argentinean Angus beef DNA traceability.

Since the 1990s several authors have envisaged the use of DNA to certify meat origin. Two major parameters must be assessed before a DNA based traceability protocol can be implemented in the food chain: (i) the information content of a DNA marker set in a specific livestock breed or group of breeds; (ii) the minimum number of DNA markers needed to obtain a statistically acceptable match probability. The objective of the present work was to establish the effect of different levels of inbreeding in the matching efficiency, and the minimum number of microsatellite markers needed, in a DNA based meat traceability program, starting from an 11-microsatellite marker panel. Samples were obtained from beef production farms in South America, where animals are typically bred under pasture-based extensive conditions. Three groups of animals with different consanguinity rates were sampled. Exclusion power (Q) was higher than 0.999998 and match probability lower than 3.01E-08, for the whole set of markers within each group. Both values were affected by consanguinity. To reach a two mismatch criteria exclusion power (Q(2)) of 99.99, six markers were needed in unrelated animals whereas seven markers were needed in related animals. To reach Q(2)=99.9999, 8 and 10 microsatellite markers, respectively, were needed. In general, one or two more microsatellite markers were needed to identify consanguineous animals. This study proved the DNA marker set used to be suitable for the identification of the meat from all slaughtered animals in Argentina, per week, month, and year.