Selection of a stable reference gene for relative copy number profiling of porcine chromosomal genes using SYBR green qPCR.

Reference gene with stable copy number is essential for normalization in qPCR based copy number assay. Present study aims to identify a suitable reference gene in pigs for qPCR based relative copy number profiling of chromosomal genes. A total of 30 crossbred pigs of both sexes were cyto-screened and gDNA was extracted from the pigs having numerically normal karyotypes. The copy number stability was studied for 7 genes (FSHB, IL4, IGF1R, TCF24, BRMS1L, ARMC1 and SRSF4) selected on the basis of the chromosomal location, reports of single copy and lack of involvement in structural chromosomal abnormalities. The copy number was estimated from Ct values in 3 technical replicates using 6 animals from either sex for each gene. The stability was evaluated from the variations in Ct values using different (Delta Ct, geNorm, BestKeeper and normFinder) algorithms. While the moderate variation was observed among relative copy number stabilities among the genes, comprehensive ranking revealed the most stable gene for normalization (IGF1R > FSHB > TCF24 > IL4 > ARMC1> SRSF4 > BRMS1L) across the samples. The selected reference gene was validated using DNA of cyto-screened pigs to find out ratio of X and Y chromosome fragments using qPCR based copy number analysis.

Distribution analysis of single nucleotide polymorphisms related to feet and legs and their association with lameness in Vrindavani cattle.

Lameness is one of the most serious economic problems in dairy animals. The present study was undertaken to identify the SNPs associated with lameness in Vrindavani, a synthetic crossbred cattle developed in India. A total of 187 cows were screened for lameness at Cattle and Buffalo Farm, Indian Veterinary Research Institute, Izatnagar, India and 16.04% prevalence was noticed. The effect of age, body weight, parity, and peak yield was not significant on lameness score. Heritability estimate of lameness score was low (0.07 ± 0.01). A total of 90 Vrindavani cows were genotyped by PCR-RFLP for 10 SNPs that were reported to have association with feet and legs in cattle. Five SNPs displayed significant departure from Hardy Weinberg equilibrium. Varying levels of heterozygosity, polymorphic information content, and allelic diversity were noticed at different SNPs. Odds ratio and least squares analysis revealed a significant association (P < 0.05) of three SNPs with lameness in the herd. Cows with AG, TT, and GG genotypes respectively at rs41632254, rs41603160, and rs41636945 loci were less prone to lameness. These genetic markers would certainly aid in development of an early detection system for lameness.