Progress 11/01/01 to 10/31/04
Outputs
We hypothesized that a mutation in the turkey skeletal muscle ryanodine receptor isoforms alpha-RYR (aRYR) or beta-RYR (bRYR) underlies pale, soft, exudative (PSE) meat quality problems. In searching for evidence of mutations, we completed cloning and sequencing cDNAs of both RYR isoforms. There are 5050 and 4873 amino acid residues in the aRYR and in the bRYR, respectively. The estimated molecular weights are 555 kDa for the aRYR and 536 kDa for the bRYR. The two RYR isoforms share 66% amino acid sequence identity. Three highly variable regions in the primary sequence of the two isoforms were identified. We identified seven alternatively spliced transcript variants of aRYR. Two of them comprised deletions of 81 bp and 193 bp, respectively. Both deletions start at exon 13. The deletion of 193 bp (AS-193) corresponds to the entire exon 13, while the deletion of 81 bp (AS-81) is the product of the removal of the first 81 bp of exon 13. The 81 bp deletion results in a
27-amino-acid deletion. Four transcript variants were identified which correspond to the locations of one of the divergent regions mentioned previously (AS-1043, AS-401, AS-454, and AS-268 which carry deletions of 1043 bp, 401 bp, 454 bp and 268 bp, respectively). The deletions are clustered between exon 24 and exon 29. Each of these deletions and the 193-bp deletion introduce a premature stop codon in the cDNA sequence. Thus, these transcript variants would not encode a functional channel protein. The last transcript variant (AS-816) carrying a deletion of 816 bp corresponding to exon 91 was found in another divergent region. This deletion results in a 272-amino-acid deletion in the protein sequence. Upon analysis of the genomic aRYR DNA sequence in the region corresponding to AS-81 and AS-193, we identified two DNA alleles (aRYR-I and aRYR-II). Each allele was analyzed by digestion and hybridization. Results showed that within the region spanning exon 12 to exon 14, the major
difference between the two alleles resides in the size of introns. The coding sequences in exons 12-14 are identical in the two alleles. Based on the two aRYR alleles, turkeys could be grouped into three different genotypes: birds homozygous for aRYR-I, birds homozygous for aRYR-II, and heterozygous birds carrying aRYR-I and aRYR-II alleles. Based on the genotypes of turkey and the corresponding expression of the mRNA transcript variants, we concluded that birds expressing AS-81 or AS-193 could derive from both aRYR alleles. Each aRYR genotype was correlated with meat quality traits. The 15-min postmortem pH of muscle from the homozygous aRYR-II birds was significantly higher than that from the homozygous aRYR-I birds. The percentage of drip loss of muscle from birds homozygous for aRYR-II was also significantly lower than the homozygous aRYR-I birds. The differences of pH-15 and drip loss were not significant between the heterozygous population and either homozygous population. Our
results suggest that turkeys homozygous for aRYR-I are more likely to develop PSE meat under standard growth and slaughter processes. A patent application has been filed.
Impacts
As much as 40% of turkey meat suffers from quality defects with the onset of a period of hot, humid weather. We have identified genetic differences among turkeys which, when subjected to heat stress, result in either superior or inferior meat quality. The results from this project have been used to develop a simple genetic test that enables breeders to screen breeding stock for the desired genes for superior meat quality and thus reduce incidence of poor quality meat and consequent monetary losses by the processing industry.
Publications
- Chiang, W., Allison, C., Linz, J., Strasburg, G.M. 2004. Identification of two alpha-RYR alleles and characterization of alpha-RYR transcript variants in turkey skeletal muscle. Gene 330:177-184.
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Progress 01/01/03 to 12/31/03
Outputs
The objectives of this project are: 1) To correlate the presence or absence of the 27-amino-acid deletion in alpha-ryanodine receptor (RyR) with the incidence of pale, soft, exudative (PSE) turkey meat; 2) To complete cloning of the turkey beta-RyR cDNA, identifying sequence differences in turkeys with different ryanodine-binding activities. RT-PCR analysis of turkey alpha-RYR mRNA covering amino acids 376 to 615 (numbered according to the human sequence) revealed at least three transcript variants. One transcript was homologous to the mammalian skeletal muscle RyR1 sequence in this region. The second transcript variant (AS-81) was characterized by the absence of 81 bases located at the beginning of exon 13, while the third transcript variant (AS-193) carried a deletion of 193 bases, corresponding to the entire exon 13. Two alpha-RyR genomic DNA alleles (alpha-RyR-I and alpha-RyR-II) carrying the region of deletions in the turkey cDNA sequences were identified.
Nucleotide sequence analysis demonstrated that the two alleles are identical in exon sequences but different in intron sequences. Comparison of genomic and cDNA sequences indicated that both AS-81 and AS-193 transcript variants probably arose via alternative splicing. Consistent with this mechanism, the last eight nucleotides of the 81 bases form a consensus sequence for a splice acceptor site. Both alleles could give rise to the AS-81 and AS-193 transcript variants via alternative splicing. The results are being correlated with meat quality traits. An invention disclosure has been filed: MSU ID 03-059F Genetic Test for PSE-susceptible Turkeys.
Impacts
Genetic selection for faster growing, larger turkeys has resulted in an increased incidence of meat quality problems including pale, soft, exudative (PSE) meat. During periods of severe heat stress, the incidence of PSE meat may reach as high as 40% of the turkeys being slaughtered. We have evidence that there is a specific genetic marker which can be correlated with a predisposition of turkeys to develop PSE meat. If confirmed, we will be able to use a simple genetic test to identify turkeys and screen out those birds which are more likely to develop PSE meat. This would reduce the incidence of PSE meat and increase profitability of turkey processors and producers.
Publications
- Strasburg, G.M., Chiang, W. 2003. Genetic basis for pale, soft, exudative turkey meat. Proceedings of the 56th Reciprocal Meat Conference. pp. 17-22.
- Chiang, W., Strasburg, G.M. 2003. Recent advances in turkey ryanodine receptors. Proceedings of the XVIth European Symposium on the Quality of Poultry Meat and Xth European Symposium on the Quality of Eggs and Egg Products. Vol. 2. Quality of poultry meat. pp. 324-334.
- Chiang, W., Linz, J., Strasburg, G. 2003. Two alpha RyR genomic alleles in turkey skeletal muscle. Biophysical Journal 84:108A Part 2 Suppl. S.
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Progress 01/01/02 to 12/31/02
Outputs
The objectives of this project were to: 1) Identify a breed of turkeys that segregates for the susceptibility to the development of pale, soft, exudative (PSE) turkey meat; 2) Determine the association between calcium channel activity and the development of PSE meat in PSE-resistant and PSE-susceptible groups of turkeys within a breed; 3) subclone and sequence cDNA fragments from PSE-resistant and PSE-susceptible turkeys that are homologous to the region of the calcium channel gene containing the mutation in swine and humans. Most of the work this year has concentrated on objective 3 which is based on the fact that over 20 mutations in the primary structure of mammalian RYR1 have been associated with Malignant Hyperthermia (MH) and Central Core Disease (CCD) in human. At least 9 of these mutations cluster in the region of amino acid residues 35 to 614. One mutation in (R615C) is associated with porcine MH, and results in economic losses due to animal death or
abnormalities in muscle food quality resulting from rapid postmortem glycolysis. We hypothesized that one or more mutations exist in the region of the turkey alpha-RYR (homologous to RYR1) which give rise to this problem. RNA was purified from genetically unimproved turkeys and from a commercial line intensively selected for muscle growth. alpha-RYR cDNA was amplified through RT-PCR and was cloned into a plasmid vector. Analysis of the alpha-RYR cDNA covering amino acids 376 to 647 (human sequence) reveals two groups of sequence diversity in both turkey populations. One group is homologous to mammalian RYR sequences. The other group shows a 27-amino-acid-residue deletion (residue numbers in human sequence: 417-443). Analysis of the genomic DNA sequence indicates that there are two alleles which likely give rise to the differences in turkey alpha-RYR. Genotyping of turkeys is in progress in preparation for a study to determine whether one of the alleles is correlated with higher
frequency of PSE meat.
Impacts
Results from this study are essential for development of a genetic test which will enable turkey breeders to identify turkeys which are carriers of the undesirable channel protein gene. This will result in an overall improvement in turkey meat quality and smaller economic losses to processors and producers from inferior meat quality.
Publications
- Chiang, W., Linz, J., Maile, M., Strasburg, G. 2002. Mutation in turkey alpha-RyR genomic DNA. J. Anim. Sci. Vol. 80 (Supp. 1):129.
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