ANALYSIS OF GONADOTROPIN RELEASING HORMONE RECEPTOR GENE POLYMORPHISMS AND THEIR ASSOCIATION WITH LITTER SIZE IN NATIVE GOAT BREEDS OF KERALA
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Date
2022-03-08
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY
Abstract
The present study was carried out with the objectives of molecular characterization of gonadotropin releasing hormone receptor (GNRHR) gene and its association analysis with litter size trait in native goat breeds of Kerala. The study goat population comprised of Attappady black (n=155) and Malabari (n=185) goats sampled from farmers herd in their respective breeding tracts and conservation units. The target regions of GNRHR characterization encompassed promoter, first, second and third exons, and 3’ untranslated regions (UTR) regions. Ten polymorphisms (c.-1129T>G, c.-1069A>G, c.-978A>C, c.-605A>G, c.-33A>G,c.-29T>G, c.48G>A, c.75G>A, c.209T>G and c.*212A>G) were identified in GNRHR of native goats by DNA pool sequencing assay and two of them in promoter region (c.-1129T>G and c.-33A>G) were novel ones. There was one non-synonymous single nucleotide polymorphism (SNP) in exon I (c.209T>G). Two loci (c.-978A>C and c.-33A>G) were polymorphic only in Malabari goats. Analysis of promoter sequence revealed the presence of multiple transcription start sites (TSS) with the major one being located at 806 nucleotides upstream of translation start site (c.-806). The polymorphisms in 5’UTR modified transcription factor binding sites and mi-RNA target sites which regulate the GNRHR gene expression. The SNPs in the promoter and exons also altered the primary, secondary and tertiary structure of mRNA and protein.The identified SNPs were screened in the whole study population by various genotyping methods. Three SNPs at loci c.-1129T>G, c.-978A>C and c.*212A>G were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and five SNPs at c.-33A>G, c.-29T>G, c.48G>A, c.75G>A and c.209T>G loci were genotyped by single strand conformation polymorphism (SSCP) sequencing. The one each SNP at c.-1069A>G locus and c.-605A>G locus were genotyped by Tetra primer amplification refractory mutation system (ARMS-PCR) and high resolution melt curve (HRM) assay respectively. There were presence two genotypes TT and TG at locus c.-1129T>G and three genotypes in c.-1069A>G, c.-978A>C, c.-605A>Gand c.*212A>G loci. The SSCP assay and sequencing showed six haplotypes and seven diplotypes encompassing the polymorphisms at c.-33A>G, c.-29T>G,
c.48G>A, c.75G>A and c.209T>G loci. Population genetic analysis indicated that all polymorphic loci except c.-605A>G were in Hardy-Weinberg equilibrium (HWE) in both breeds. The inbreeding coefficient (FIS) ranged between -0.12 to 0.62 in Attappady Black and
-0.11 to 0.55 in Malabari goats. There were six and four loci pairs in linkage disequilibrium (LD) at significant level (p<0.01) in Attappady Black and Malabari goats, respectively. The highly significant and strong LD was observed among the haplotypic loci c.-29T>G, c.48G>A and c.209T>G loci in both Atttappady Black and Malabari goats. The low values of FST (0.0081) and Nei’s unbiased measure genetic divergence (0.9970) indicated low genetic differentiation among the two breeds with respect to GNRHR loci.The overall (mean ± S.E) of litter size at birth was 1.9900 ± 0.0440 in Malabari goats. The association analysis of the GNRHR variants with litter size trait using ordinal logistic regression showed that the genotypes at c.-605A>Glocus had a significant (p<0.01) influence on litter size at birth in second and third with GG genotypes favouring multiple births in Malabari goats. The diplotypes of GNRHR promoter and exon 1 had a statistically significant (p<0.01) association with litter size at birth of all four parities. The heterozygotes viz; D1D3
diplotypes and D1D2 diplotypes had the highest odds of having multiple birth in first two parities and in the subsequent parities respectively. Thus results imply that GNRHR is a possible candidate gene for the selection and improvement of litter size in Malabari goats.