Melatonin receptor 1A gene is the prime receptor mediating the effect of melatonin at the neuroendocrine level for control of seasonal reproduction in sheep. The aims of this study were to examine the polymorphism pattern of coding sequence of MTNR1A gene in Chokla sheep, a breed of Indian arid tract and to identify new variations in relation to its aseasonal status. Genomic DNAs of 101 Chokla sheep were collected and an 824 bp coding sequence of Exon II was amplified. RFLP was performed with enzyme RsaI and MnlI to assess the presence of polymorphism at position C606T and G612A, respectively. Genotyping revealed significantly higher frequency of M and R alleles than m and r alleles. RR and MM were found to be dominantly present in the group of studied population. Cloning and sequencing of Exon II followed by mutation/polymorphism analysis revealed ten mutations of which three were non-synonymous mutations (G706A, C893A, G931C). G706A leads to substitution of valine by isoleucine Val125I (U14109) in the fifth transmembrane domain. C893A leads to substitution of alanine by aspartic acid in the third extracellular loop. G931C mutation brings about substitution of amino acid alanine by proline in the seventh transmembrane helix, can affect the conformational stability of the molecule. Polyphen-2 analysis revealed that the polymorphism at position 931 is potentially damaging while the mutations at positions 706 and 893 were benign. It is concluded that G931C mutation of MTNR 1A gene, may explain, in part, the importance of melatonin structure integrity in influencing seasonality in sheep.

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane-bound G-coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out-of-season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen-2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out-of-season breeding. GG and CC genotypes were having a higher prevalence in the studied population.

Seasonality of animals is an important adaptive trait for successful survival and production during limited food availability and extreme environmental conditions. Photoperiodic changes in day length are utilized by these seasonal animals as an important environmental cue for regulating their annual rhythms of reproduction cycles. Melatonin is an important hormone which is secreted by the pineal gland in proportion to darkness and its effect is mediated by melatonin receptor subtypes, principally MTNR1A. In the present study, polymorphism in the coding sequence at two important SNPs (C606T and G612A),known to be markers for out of season breeding in sheep were studied by PCR-RFLP in a panel of four breeds of sheep from subtemperate and subtropical arid conditions, respectively. The frequencies of 'G' and 'A' alleles with reference to G612A SNP did not differ considerably among all the breeds of sheep. Frequency of 'T' allele of the C606T SNP was found to be dom inantly higher in subtemperate sheep breeds in comparison to subtropical sheep breeds. Identified SNPs in the coding region were mostly synonymous and did not lead to any change in conformation of the MTNR1A receptor protein.

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