Study on Genetic Variation in Milk Genes and Their Association with Milk Performance Traits in Indigenous Cattle Breeds
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Date
2017
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Rajasthan University of Veterinary and Animal Sciences, Bikaner
Abstract
The present study was undertaken to explore the genetic variability in exon-3 and exon-4 of prolactin (PRL); and DGAT1 gene and their association with milk performance and milk composition traits in indigenous cattle breed of Rathi, Sahiwal and Kankrej. 225 apparently healthy and lactating cows from breeds of Rathi (n=75), Sahiwal (n=75) and Kankrej (n=75) were selected randomly for the collection of blood and milk samples. The phenotypic information on various lactation traits was recorded from official records maintained in the farm. 100 ml milk from selected animals was collected for the analyses of different milk constituents. About 2ml jugular vein blood was collected in sterile vacutainer from all the selected animals. Genomic DNA from whole blood was extracted through spin column method. The purity (OD ratio 260/280) and concentration (ng/µl) of extracted genomic DNA was determined by Nano-drop spectrophotometer and 0.8% agarose electrophoresis. Species specific primers were used to amplify the 156 bp, 294 bp and 411 bp region of PRL exon-3, PRL exon-4 and DGAT1 gene, respectively. Two approaches such as restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP) was carried out to detect the genetic variation in selected gene regions. Bioinformatics tools such as Chromas, Bioedit and Clustal W were used for single nucleotide polymorphisms (SNPs) detection and analysis after gene sequencing. Generated sequences were submitted to international database, NCBI GENBANK for assignment of accession number. Neighbourhood Joining (NJ) phylogenetic tree was constructed through bootstrap test method in MEGA7 bioinformatics software. The genetic structure of the population and genetic variation was analysed through POPGENE program. Univariate analysis of variance was carried out through general linear model (GLM) procedure of SPSS software to detect the association of different lactation performance and milk composition traits with detected genetic variation in selected genomic regions. The RFLP analysis of PRL exon-3 through Rsa I enzyme detected two genotypic patterns in all the studied breeds in the genotypic frequency of 0.28 and 0.72 for GG and AG genotype, respectively and gene frequency of 0.64 (G allele) and 0.36 (A allele) on overall basis. SSCP analysis detected three different genotype in Rathi cattle whereas two genotype each in Sahiwal and Kankrej cattle. AG genotype of PRL exon-3 gene in Rathi cattle demonstrated two different SSCP genotypic patterns, ‘P2’ and ‘P3’. The RFLP frequency of AG genotype (0.72) in Rathi was redistributed for P2 (0.57) and P3 (0.15) SSCP pattern. Sufficient genetic variation in the form of heterozygosity, PIC, fixation index and Shannon information index was detected for PRL exon-3 locus however all the populations were significantly deviated from Hardy-Weinberg equilibrium. The genetic distance between Rathi and Sahiwal was observed lower than between distance of Sahiwal and Kankrej. Three transition and one transversion type of SNP were detected in PRL exon-3 locus of studied breeds. Five different haplotypes were constructed on the basis of detected SNP’s in 156-bp amplified fragment of exon-3 of PRL-3 gene. The evolutionary divergence among the different alleles of studied breeds was observed lower than mean
distance. Four conserved regions were identified among studied breeds. Both RFLP analysis of PRL exon-4 locus with Rsa I enzyme and SSCP analysis revealed three different genotypic patterns in an overall frequency of 0.21, 0.56 and 0.23 for GG, AG and AA genotype with gene frequency of 0.49 and 0.51 for G and A allele, respectively, in Hardy-Weinberg equilibrium except in Sahiwal cattle. Observed (HO) and expected heterozygosity (HE), PIC, fixation index and Shannon information index revealed sufficient genetic variation in PRL exon-4 locus however Rathi revealed positive FIS value. The genetic between Sahiwal and Kankrej was less than distance between Rathi and Sahiwal. SNPs detection revealed seven transitions and three transversion SNPs in PRL exon-4 locus with the presence of six difference haplotypes. The distance between alleles of studied breeds ranged below the mean value of 0.0117±0.0051. The A allele of PRL exon-4 locus in all the three breeds shared the clade directly or indirectly with Bos taurus sequence. Six conserved regions were identified in between studied breeds. RFLP analysis of DGAT1 gene through Eae I restriction enzyme revealed monomorphic pattern KK in all the samples whereas SSCP analysis revealed five different conformational banding patterns for lysine (KK) variants in studied animals with an overall genotypic frequency of 0.32, 0.16, 0.19, 0.16 and 0.17 for K1K1, K1K2, K1K3, K2K2 and K2K4, respectively. The gene frequency of K1, K2, K3 and K4 lysine variant was observed 0.49, 0.33, 0.10 and 0.08, respectively in Hardy-Weinberg disequilibrium. Observed (HO) and expected heterozygosity (HE), PIC, fixation index and Shannon information index revealed sufficient genetic variation in lysine variant of DGAT1 gene. The genetic distance between Rathi and Sahiwal was observed less than Rathi and Kankrej cattle or lysine variants of DGAT1 gene. SNP analysis reflected three transition and seven transversion mutation which revealed four different haplotype sequences. The evolutionary distance between any two lysine variant in indigenous cattle observed below mean distance and ranged between 0.000 to 0.01746. Different lysine variant of indigenous cattle shared different branches with other species or breed on phylogenetic tree. Six different conserved regions were identified among different identified alleles of DGAT1 gene in studied breeds. Association analysis revealed significantly higher total milk yield (TMY) and milk protein content for AG genotype of PRL exon-3 and AA genotype of PRL exon-4 locus in all studied breeds whereas breed specific variable effect was observed for 305-day milk yield (305-day MY), daily milk yield (DMY) and lactation length (LL) in different parity. No significant effect of lysine variants of DGAT1 gene was observed on different lactation traits however significant effect was observed on milk fat, protein and SNF. Thus the present study concluded the significant effect of prolactin gene on milk yield and protein percentage and DGAT1 gene on milk fat, protein and SNF.
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