Identification of polymorphism in fatty acid binding protein 3 (FABP3) gene and its association with milk fat traits in riverine buffalo (Bubalus bubalis).

The fatty acid binding protein 3 (FABP3) gene, known to be associated with fat percentage of milk and meat in bovines, was screened among swamp and riverine buffaloes for polymorphism detection and further association with milk fat contents. An SNP g.307C > T was identified in the intron 2 (+53 exon 2) region of FABP3 gene of Indian buffaloes. The SNP identified was genotyped in 692 animals belonging to 15 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, by PCR-RFLP. A marked contrast was observed between the C and T allele frequencies in three types of buffaloes. The frequency of C allele ranged from 0.67 to 0.96 in pure swamp buffalo populations, with the highest in Mizoram (0.96). Whereas the frequency of T allele was high across all the Indian riverine buffalo breeds, ranging from 0.57 to 0.96. None of the genotypes at FABP3 g.307C > T locus was found to have significant association with milk fat and other production traits in Mehsana dairy buffalo breed. Our study revealed marked differences in the allele frequencies between riverine and swamp buffaloes at FABP3 g.307C > T locus, without any significant association with different milk traits in riverine buffaloes.

Sequence and topological characterization of Toll-like receptor 8 gene of Indian riverine buffalo (Bubalus bubalis).

In this study, buffalo (Bubalus bubalis) Toll-like receptor 8 (TLR8) gene has been characterized by sequence analysis and detecting polymorphism. Complete ORF of buffalo TLR8 gene was amplified using the RNA isolated from spleen tissue, which was found to be 3,102 nucleotides long encoding a 1,033 amino acid protein. Buffalo TLR8 had 10 nucleotide changes as compared to other livestock species resulting in six unique amino acid changes, four of them lying within leucine-rich repeat (LRR) domains. As compared to cattle (Bos indicus and Bos taurus), out of fifteen cysteine residues, fourteen were conserved and Cys at position 521 was replaced by Arg. Nine of the LRR domains had no amino acid change as compared to cattle, whereas LRR-C-terminus had maximum, five amino acid changes. Sequence characterization of 12 riverine and swamp buffaloes revealed presence of four polymorphic nucleotides, two of them were non-synonymous, one synonymous and one site in 3'UTR. PCR-RFLP genotyping of non-synonymous SNP 2758A>G (ILeu920Val) in Toll-interleukin-1 receptor domain of 463 swamp and riverine buffaloes showed a higher frequency of allele A in swamp (95 %) as compared to riverine (9.84 %) buffaloes.

Detection of occurrence of a recent genetic bottleneck event in Indian hill cattle breed Bargur using microsatellite markers.

The effective number of breedable individuals is a crucial determinant for maintaining genetic variability within a population. The population of Bargur, the hill cattle of South India, has gone down drastically by more than 93 % in the past three decades, and only a few thousand animals are available at present. The present study was undertaken to evaluate Bargur cattle for mutation drift equilibrium and to detect the occurrence of recent genetic bottleneck event, if any, in this population. About 50 unrelated animals, true to the type, were sampled and genotyped at 25 microsatellite loci. The mean observed heterozygosity (0.808 ± 0.023) was higher than the mean expected heterozygosity (0.762 ± 0.008) with 15 out of 25 microsatellite loci exhibiting heterozygosity excess when assumed under Hardy-Weinberg equilibrium. To evaluate Bargur cattle for mutation drift equilibrium, three tests were performed under three different mutation models, viz., infinite allele model (IAM), stepwise mutation model (SMM) and two-phase model (TPM). The observed gene diversity (H (e)) and expected equilibrium gene diversity (H(eq)) were estimated under different models of microsatellite evolution. All the 25 loci were found to exhibit gene diversity excess under IAM and TPM, while 22 loci were having gene diversity excess under SMM. All the three statistical tests, viz., sign test, standardized differences test, and Wilcoxon sign rank test, revealed significant (P < 0.01) deviation of Bargur cattle population from mutation-drift equilibrium under all the three models of mutation. Furthermore, the qualitative test of allele frequency distribution in Bargur cattle population revealed a strong mode shift from the normal L-shaped form suggesting that the population had experienced genetic bottleneck in the recent past. The occurrence of genetic bottleneck might have led to the loss of several rare alleles in the population, which point towards the need for efforts to conserve this important cattle germplasm. The present study is the first report in demonstrating the genetic basis of demographic bottleneck in an Indian cattle population.

PCR-SSCP analysis of leptin gene and its association with milk production traits in river buffalo (Bubalus bubalis).

Leptin gene has been found to be associated with various economic traits including milk production and fat quality in dairy animals. In the present study, we investigated genetic variations in intron 1 region of leptin gene in riverine buffaloes (Bubalus bubalis) using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing methods and associated them with milk traits. The study revealed three SSCP variants A, B and C among a total of 301 buffaloes from nine breeds. The frequency of variant C was found invariably high among all the breeds except in Marathwada buffalo. Variant A was found to be absent in Chilika, Nili-Ravi, Nagpuri and Pandharpuri breeds and also had the lowest frequencies in Mehsana, Jaffarabadi, Murrah and Toda breeds. Sequencing of SSCP variants revealed a total of five polymorphic sites, with three haplotypes. Statistical analysis revealed significantly high fat percentage at 150 days in SSCP variant B in Mehsana buffaloes. However, the associations of SSCP variants of leptin gene with total milk yield, 305 days milk yield and total fat yield were found to be non-significant. The present study is the first report on association analysis of leptin gene polymorphisms with milk production and milk quality traits in river buffalo.

Microsatellite-based genetic monitoring to detect cryptic demographic bottleneck in Indian riverine buffaloes (Bubalus bubalis).

The present study was undertaken to evaluate different Indian riverine buffalo breeds (Bubalus bubalis) for mutation drift equilibrium and occurrence of any recent genetic bottleneck. A total of 330 animals from seven different breeds were analyzed with a set of 24 heterologous microsatellite markers. Three different tests revealed significant heterozygosity excess in all the seven buffalo breeds studied when assumed under infinite alleles model of microsatellite evolution, while it was the reverse with no significant heterozygosity excess when assumed under conservative stepwise mutation model. Under the two-phase model, all the buffalo breeds except Mehsana were found to be in mutation drift equilibrium when evaluated by all the three statistical methods. Standardized differences test and Wilcoxon signed-rank test revealed significant heterozygosity excess suggesting possible cryptic demographic bottleneck in Mehsana buffaloes of Western India.

Seven novel single nucleotide polymorphisms identified within river buffalo (Bubalus bubalis) lactoferrin gene.

The present study aimed at identifying single-nucleotide polymorphic (SNP) sites in different coding and non-coding regions of lactoferrin gene in Indian riverine buffaloes. A total of 102 animals from six different river buffalo breeds were screened at six bubaline lactoferrin gene loci. Single-strand conformation polymorphism (SSCP) analysis revealed monomorphic patterns at three loci LtfE2, LtfE11, and LtfE14 while a total of eight distinct patterns were observed in the other three loci viz. LtfE5, LtfE10, and LtfE16 which correspond to respective exons and their flanking regions. Sequence analysis of different SSCP variants revealed the presence of two SNP sites within the coding (exon 16) region and five SNP sites in flanking non-coding regions (intron 4 and intron 9). Both SNPs within exon 16 were found to be synonymous. The SNPs and haplotypes identified in the present study could serve as potential markers for association with susceptibility/resistance to mastitis in buffaloes.