title: Detecting novel SNPs and breed-specific haplotypes at calpastatin gene in Iranian fat- and thin-tailed sheep breeds and their effects on protein structure.
abstract: Calpastatin has been introduced as a potential candidate gene for growth and meat quality traits. In this study, genetic variability was investigated in the exon 6 and its intron boundaries of ovine CAST gene by PCR-SSCP analysis and DNA sequencing. Also a protein sequence and structural analysis were performed to predict the possible impact of amino acid substitutions on physicochemical properties and structure of the CAST protein. A total of 487 animals belonging to four ancient Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Chall (fat-tailed), Zel-Atabay cross-bred (medium fat-tailed) and Zel (thin-tailed), were analyzed. Eight unique SSCP patterns, representing eight different sequences or haplotypes, CAST-1, CAST-2 and CAST-6 to CAST-11, were identified. Haplotypes CAST-1 and CAST-2 were most common with frequency of 0.365 and 0.295. The novel haplotype CAST-8 had considerable frequency in Iranian sheep breeds (0.129). All the consensus sequences showed 98-99%, 94-98%, 92-93% and 82-83% similarity to the published ovine, caprine, bovine and porcine CAST locus sequences, respectively. Sequence analysis revealed four SNPs in intron 5 (C24T, G62A, G65T and T69-) and three SNPs in exon 6 (c.197A>T, c.282G>T and c.296C>G). All three SNPs in exon 6 were missense mutations which would result in p.Gln 66 Leu, p.Glu 94 Asp and p.Pro 99 Arg substitutions, respectively, in CAST protein. All three amino acid substitutions affected the physicochemical properties of ovine CAST protein including hydrophobicity, amphiphilicity and net charge and subsequently might influence its structure and effect on the activity of Ca2+ channels; hence, they might regulate calpain activity and afterwards meat tenderness and growth rate. The Lori-Bakhtiari population showed the highest heterozygosity in the ovine CAST locus (0.802). Frequency difference of haplotypes CAST-10 and CAST-8 between Lori-Bakhtiari (fat-tailed) and Zel (thin-tailed) breeds was highly significant (P<0.001), indicating that these two haplotypes might be breed-specific haplotypes that distinguish between fat-tailed and thin-tailed sheep breeds.
authors.affiliation: Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. Electronic address: Mohsen.Ali@ut.ac.ir.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Aali
authors.affiliation: Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran; Excellent Center for Improving Sheep Carcass Quality and Quantity, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Moradi-Shahrbabak
authors.affiliation: Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
authors.forename: Hosein
authors.initials: H
authors.lastname: Moradi-Shahrbabak
authors.affiliation: Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
authors.forename: Mostafa
authors.initials: M
authors.lastname: Sadeghi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D005091:Exons; D005260:Female; D005787:Gene Frequency; D005828:Genetics, Population; D006239:Haplotypes / Q000235:genetics*; D007438:Introns; D007492:Iran; D008297:Male; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D020641:Polymorphism, Single Nucleotide; D018807:Polymorphism, Single-Stranded Conformational; D017386:Sequence Homology, Amino Acid; D034561:Sheep, Domestic / Q000235:genetics*; D013623:Tail / Q000033:anatomy & histology
chemical_list: D002135:Calcium-Binding Proteins; C033668:calpastatin
keywords: Arg; Asp; BLAST; BMS; Basic Local Alignment Search Tool; Breed-specific haplotype; CAST gene; Gln; Glu; HapMap; IMF; LD; Leu; NCBI; National Center for Biotechnology Information; Ovine CAST gene; PCR-SSCP; Pro; QTL; RCAN1 gene; SNP; Single nucleotide polymorphism (SNP); Single-strand conformational polymorphism (SSCP); arginine acid; aspartic acid; beef marbling score; calpastatin gene; glutamic acid; glutamine acid; haplotype map; intramuscular fat; leucine acid; linkage disequilibrium; polymerase chain reaction single-stranded conformation polymorphism; proline acid; quantitative trait loci; regulator of calcineurin 1 gene; single nucleotide polymorphism