I. D. Gupta

Genotyping of β-Lactoglobulin gene by PCR-RFLP in Sahiwal and Tharparkar cattle breeds

BackgroundImprovement of efficiency and economic returns is an important goal in dairy farming, as in any agricultural enterprise. The primary goal of dairy industry has been to identify an efficient and economical way of increasing milk production and its constituents without increasing the size of the dairy herd. Selection of animals with desirable genotypes and mating them to produce the next generation has been the basis of livestock improvement and this would continue to remain the same in the coming years. The use of polymorphic genes as detectable molecular markers is a promising alternative to the current methods of trait selection once these genes are proven to be associated with traits of interest in animals. The point mutations in exon IV of bovine β-Lactoglobulin gene determine two allelic variants A and B. These variants were distinguished by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) analysis in two indigenous Bos indicus breeds viz. Sahiwal and Tharparkar cattle. DNA samples (228 in Sahiwal and 86 in Tharparkar) were analyzed for allelic variants of β-Lactoglobulin gene. Polymorphism was detected by digestion of PCR amplified products with Hae III enzyme, and separation on 12% non-denaturing gels and resolved by silver staining.ResultsThe allele B of β-Lactoglobulin occurred at a higher frequency than the allele A in both Sahiwal and Tharparkar breeds. The genotypic frequencies of AA, AB, and BB in Sahiwal and Tharparkar breeds were 0.031, 0.276, 0.693 and 0.023, 0.733, 0.244 respectively. Frequencies of A and B alleles were 0.17 and 0.83, and 0.39 and 0.61 in Sahiwal and Tharparkar breeds respectively. The Chi-square test results (at one degree of freedom at one per cent level) revealed that the Tharparkar population was not in Hardy-Weinberg equilibrium as there was a continuous migration of animals in the herd studied, where as, the results are not significant for the Sahiwal population.ConclusionGenotype frequencies of AA were the lowest compared to that of BB genotype in Sahiwal cattle while AB genotypes were more frequent in Tharparkar cattle. The frequency of A allele was found to be lower than that of B allele in both the breeds studied. These results further confirm that Bos indicus cattle are predominantly of β-Lactoglobulin B type than Bos taurus breeds.

Bovine kappa-casein gene polymorphism and its association with milk production traits

Point mutations in exon IV of the bovine κ-casein (CSN3) gene determine two allelic variants, A and B. These variants were distinguished by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis in the indigenous Sahiwal and Tharparkar cattle breeds. DNA samples (252 Sahiwal and 56 Tharparkar) were analyzed for allelic variants of the CSN3 gene. Polymorphism was detected by digestion of PCR-amplified products with HindIII, HhaI and HaeIII restriction enzymes, followed by separation on 3% agarose gels, and resolved by ethidium bromide staining. Allele A of the κ-casein gene occurred at a higher frequency than allele B, in both Sahiwal and Tharparkar breeds. The genotypic frequencies of AA, AB, and BB in the Sahiwal and Tharparkar breeds were 0.758, 0.230 and 0.012, and 0.0.732, 0.250 and 0.018, respectively. The frequencies of alleles A and B in the Sahiwal and Tharparkar breeds were 0.873 and 0.127, and 0.857 and 0.143, respectively. Genotype BB of the kappa-casein gene had more influence on the monthly milk yield, 305-days milk yield, monthly solids-not-fat (SNF) yield, and monthly protein yield, in the Sahiwal cattle.

Characterization of promoter region of CD14 gene and association with clinical mastitis in Karan Fries cattle.

Cluster of Differentiation (CD14) protein was identified as a differentiation marker on the surface of monocytes and macrophages and was characterized as a receptor for bacterial endotoxin (LPS). It is regarded as the first described pattern-recognition receptor (PRR) and one of the excellent candidates for mastitis resistance in cattle. Present study was carried out with the objective to characterize and identify genetic polymorphism of promoter region of CD14 gene and its association with clinical mastitis in lactating Karan Fries cattle maintained at NDRI Karnal. Twelve SNPs were found in the complete sequence of 553 base pairs of promoter region of CD14 gene of Karan Fries cows. Cows were also screened using PCR-RFLP with Hpy188I restriction enzyme which revealed three genotypes AA, AB and BB. All three genotypes differed significantly regarding mastitis incidence.

Molecular characterization and combined genotype association study of bovine cluster of differentiation 14 gene with clinical mastitis in crossbred dairy cattle.

Aim: The present study was undertaken with the objectives to characterize and to analyze combined genotypes of cluster of differentiation 14 (CD14) gene to explore its association with clinical mastitis in Karan Fries (KF) cows maintained in the National Dairy Research Institute herd, Karnal. Materials and Methods: Genomic DNA was extracted using blood of randomly selected 94 KF lactating cattle by phenol-chloroform method. After checking its quality and quantity, polymerase chain reaction (PCR) was carried out using six sets of reported gene-specific primers to amplify complete KF CD14 gene. The forward and reverse sequences for each PCR fragments were assembled to form complete sequence for the respective region of KF CD14 gene. The multiple sequence alignments of the edited sequence with the corresponding reference with reported Bos taurus sequence (EU148610.1) were performed with ClustalW software to identify single nucleotide polymorphisms (SNPs). Basic Local Alignment Search Tool analysis was performed to compare the sequence identity of KF CD14 gene with other species. The restriction fragment length polymorphism (RFLP) analysis was carried out in all KF cows using Helicobacter pylori 188I (Hpy188I) (contig 2) and Haemophilus influenzae I (HinfI) (contig 4) restriction enzyme (RE). Cows were assigned genotypes obtained by PCR-RFLP analysis, and association study was done using Chi-square (χ2) test. The genotypes of both contigs (loci) number 2 and 4 were combined with respect to each animal to construct combined genotype patterns. Results: Two types of sequences of KF were obtained: One with 2630 bp having one insertion at 616 nucleotide (nt) position and one deletion at 1117 nt position, and the another sequence was of 2629 bp having only one deletion at 615 nt position. ClustalW, multiple alignments of KF CD14 gene sequence with B. taurus cattle sequence (EU148610.1), revealed 24 nt changes (SNPs). Cows were also screened using PCR-RFLP with Hpy188I (contig 2) and HinfI (contig 4) RE, which revealed three genotypes each that differed significantly regarding mastitis incidence. The maximum possible combination of these two loci shown nine combined genotype patterns and it was observed only eight combined genotypes out of nine: AACC, AACD, AADD, ABCD, ABDD, BBCC, BBCD, and BBDD. The combined genotype ABCC was not observed in the studied population of KF cows. Out of 94 animals, AACD combined genotype animals (10.63%) were found to be not affected with mastitis, and ABDD combined genotyped animals was observed having the highest mastitis incidence of 15.96%. Conclusion: AACD typed cows were found to be least susceptible to mastitis incidence as compared to other combined genotypes.

Reversible magnetic resonance imaging changes in a case of neuroleptic malignant syndrome

Neuroleptic malignant syndrome (NMS) is a life-threatening neurologic emergency associated with the use of mainly typical antipsychotic drugs. It is characterized by fever, altered mental status, generalized rigidity, autonomic instability, myoclonus, raised creatine phosphokinase, rhabdomyolysis, and leukocytosis. Neuroimaging (brain computed tomography/magnetic resonance imaging [MRI]) is usually normal in most of the cases of NMS. Magnetic resonance imaging findings have not been well elucidated in NMS as yet. Very few cases have been reported worldwide. We herein, report a case of a 42-year-old patient of NMS, who presented to us with reversible changes in MRI brain. This case report highlights the possible MRI changes in NMS and their plausible mechanism.

Candidate SNP of CACNA2D1 Gene Associated with Clinical Mastitis and Production Traits in Sahiwal (Bos taurus indicus) and Karan Fries (Bos taurus taurus × Bos taurus indicus)

ABSTRACT The present study was conducted to identify polymorphisms in CACNA2D1 gene and their association with clinical mastitis and production traits. Exon 18 and its flanking regions were screened for the presence of SNPs. Statistical analysis was performed to identify association of period of birth, breed, and genotype with mastitis incidence on randomly selected 103 Sahiwal and 102 Karan Fries cattle. PCR-RFLP analysis revealed that g.38819398G > A mutation in exon 18 (269 bp amplicon) of CACNA2D1 gene resolved into AA, AG, and GG genotypes in Sahiwal and Karan Fries cattle. Wald chi-square analysis revealed that the period of birth, breed, and genotype were significantly associated with mastitis incidence. GG genotyped cattle were found to be less susceptible to mastitis. Least square analysis revealed that GG and AG genotype animals of G38819398A SNP of CACNA2D1 gene in Sahiwal as well as in Karan Fries cattle were associated with higher average milk yields during 1st, 2nd, and 3rd lactations (P < 0.01). These observations and their differential association with the incidence of mastitis and production traits can be utilized as an aid to selection for simultaneous improvement of both antagonistic traits; however, validation of results on large number of animals is warranted.

Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls

Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls. Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme. Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study. Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible.

Phylogenetic analysis and comparative study of Sahiwal cattle CD14 gene

Cluster of Differentiation-14 (CD14) gene is one of the strong candidate genes for mastitis resistance trait. Bovine CD14 gene spans over 2630 bp having 2 exons. Present investigation is based on phylogenetic analysis and comparative study of CD14 gene in Sahiwal cattle. A total of 100 lactating Sahiwal cattle maintained at NDRI, Karnal (Haryana) were included in the present study. Sequence analysis revealed that Sahiwal cattle CD14 gene sequence is 73 to 99% identical with several species. Phylogenetic analysis indicated that Sahiwal cattle (Bos indicus) and Bos taurus are genetically the most similar (99%) followed by Bubalus bubalis. The predicted peptide sequence revealed 373 amino acids precursor corresponding to coding sequence of CD14 gene of Sahiwal cattle. Amino acid length of CD14 protein was found different in reported species. BLAST analysis also revealed that Sahiwal cattle CD14 protein is 73 to 100% similar with other reported species. The sites for leucine-rich repeats, leucine rich nuclear export signal, putative N-linked glycosylation, O-linked glycosylation and disulphide bridges were predicted and comparative study was done.