Monika Sodhi

DGAT1 and ABCG2 polymorphism in Indian cattle (Bos indicus) and buffalo (Bubalus bubalis) breeds

BackgroundIndian cattle (Bos indicus) and riverine buffalo (Bubalus bubalis) give a poor yield of milk but it has a high fat and protein percentage compared to taurine cattle. The identification of QTLs (Quantitative Trait Loci) on BTA14 and BTA6 and its subsequent fine mapping has led to identification of two non conservative mutations affecting milk production and composition. Our objective was to estimate the frequency of K232A (DGAT1 – diacylglycerol – acyltransferase 1) and Y581S (ABCG2 – ATP binding cassette sub family G member 2) polymorphisms in diverse cattle and buffalo breeds of India having large variation in terms of milk production.ResultsWe screened the reported missense mutations in six cattle and five buffalo breeds. The DGAT1K and ABCG2Y alleles were found to be fixed in Indian cattle and buffalo breeds studied.ConclusionThis study provides an indirect evidence that all the Indian cattle and buffalo breeds have fixed alleles with respect to DGAT1 and ABCG2 genes reported to be responsible for higher milk fat yield, higher fat and protein percent.

Evaluation of Genetic Differentiation in Bos indicus Cattle Breeds from Marathwada Region of India Using Microsatellite Polymorphism

Elucidation of genetic variability and genetic relationship among breeds has direct relevance with the issues of sustainable use of domestic animal genetic resources. In the present study, genetic polymorphism was evaluated using 22 microsatellite loci in unrelated samples of Red Kandhari and Deoni cattle breeds inhabiting the same geographical area of Marathwada region in Maharashtra state (western India). This work was mainly aimed at assessing the current genetic diversity to understand whether the two zebu populations in question are genetically differentiated. A total of 164 alleles were detected with an average of 5.82 and 5.86 alleles per locus (MNA) in Red Kandhari and Deoni breeds, respectively. The estimated mean observed (Ho) and expected (He) heterozygosity were 0.47 and 0.64 in Red Kandhari vs. 0.57 and 0.69 in Deoni cattle, respectively, demonstrating considerable level of genetic variation in both the populations. Mean estimates of F statistics were: F (FIT) = 0.315±0.035, f(FIS) = 0.231±0.031, θ(FST) = 0.110±0.022, with both the breeds exhibiting significant deficit of heterozygotes (FIS = 0.179 in Deoni; 0.278 in Red Kandhari). The multilocus FST values implied that 11.0% of the total genetic variation corresponds to breed and were statistically greater than zero for the two populations, suggesting population division. The evaluation of exact test also indicated that allele frequencies across all the loci differed significantly (P < 0.001) between two zebu breeds, further supporting population differentiation. Different genetic distance measures showed considerable levels of distances between the two cattle breeds (0.318 = Neis standard DS; 0.250 = Neis DA; 0.416 = Cavalli-Sforza and Edwardss DC; 0.164 = Reynolds, and 2.64 = Delta mu square (dμ)2. Bayesian statistical approach to assign each individual to the population also supported considerable differentiation between the two cattle breeds, possibly reflecting the limited gene flow between the two Marthwada cattle populations. The existence of cohesive breeding structure of both the breeds was further substantiated by allele-sharing distance measures (DAS) among individual animals. The results of this study thus revealed that the two Bos indicus breeds sharing the common breeding tracts are genetically differentiated enough as separate breeds.

Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis)

Gene expression studies require appropriate normalization methods for proper evaluation of reference genes. To date, not many studies have been reported on the identification of suitable reference genes in buffaloes. The present study was undertaken to determine the panel of suitable reference genes in heat-stressed buffalo mammary epithelial cells (MECs). Briefly, MEC culture from buffalo mammary gland was exposed to 42 °C for one hour and subsequently allowed to recover at 37 °C for different time intervals (from 30 m to 48 h). Three different algorithms, geNorm, NormFinder, and BestKeeper softwares, were used to evaluate the stability of 16 potential reference genes from different functional classes. Our data identified RPL4, EEF1A1, and RPS23 genes to be the most appropriate reference genes that could be utilized for normalization of qPCR data in heat-stressed buffalo MECs.

Identification of suitable housekeeping genes for normalization of quantitative real‐time PCR data during different physiological stages of mammary gland in riverine buffaloes (Bubalus bubalis)

Gene expression analysis unravels the complex changes or relations at transcriptomic level. To nullify all type of errors that can be incorporated during any stage of RNA extraction into cDNA synthesis and for reliable results, the data obtained from qPCR have to be normalized using the appropriate/suitable housekeeping genes (HKGs). Unfortunately, till date, no such HKG has been reported for bubaline mammary gland. The objective of the present study was thus to identify and validate the potential HKGs for the gene expression studies in buffalo mammary gland. Mammary tissues from twelve buffaloes during different physiological stages: pre-pubertal (heifer), lactation and involution were obtained for the present study. A total of 16 potential HKGs (GAPDH, β-actin, UXT, β2M, A2M, RPl4, RPS9, RPS15A, RPS18, RPS23, HMBS, HPRT1, GTP, EEF1A1, UB1 and RPL22) from different functional classes were evaluated. The analysis revealed that the expression of EEF1A1, RPl4, β2M and RPS15A was most consistent across different physiological stages of buffalo mammary gland. On the other hand, β-actin, A2M, RPL22 and GAPDH were the least stable genes making them unsuitable as HKGs. Based on our analysis, we recommend the use of EEF1A1, RPl4, β2M and RPS15A genes as suitable HKGs for accurate normalization of gene expression data in bubaline mammary gland.

Identification of internal control genes in milk-derived mammary epithelial cells during lactation cycle of Indian zebu cow

The present study aims to evaluate the suitability of 10 candidate genes, namely GAPDH, ACTB, RPS15A, RPL4, RPS9, RPS23, HMBS, HPRT1, EEF1A1 and UBI as internal control genes (ICG) to normalize the transcriptional data of mammary epithelial cells (MEC) in Indian cows. A total of 52 MEC samples were isolated from milk of Sahiwal cows (major indigenous dairy breed of India) across different stages of lactation: Early (5-15 days), Peak (30-60 days), Mid (100-140 days) and Late (> 240 days). Three different statistical algorithms: geNorm, Normfinder and BestKeeper were used to assess the suitability of these genes. In geNorm analysis, all the genes exhibited expression stability (M) values below 0.5 with EEF1A1 and RPL4 showing the maximum expression stability. Similar to geNorm, Normfinder also identified EEF1A1 and RPL4 as two of the most stable genes. In Bestkeeper algorithm as well, all the 10 genes showed consistent expression levels. The analysis showed that four genes, that is, EEF1A1, RPL4, GAPDH and ACTB exhibited higher coefficient of correlation to the Bestkeeper index, lower coefficient of variance and standard deviation, indicating their superiority to be used as ICG. The present analysis has provided evidence that RPL4, EEF1A1, GAPDH and ACTB could probably act as most suitable genes for normalizing the transcriptional data of milk-derived mammary epithelial cells of Indian cows.

Variations in the Regulatory Region of Alpha S1-Casein Milk Protein Gene among Tropically Adapted Indian Native (Bos Indicus) Cattle.

Regulatory region of milk protein alpha S1-casein (αS1-CN) gene was sequenced, characterized, and analyzed to detect variations among 13 Indian cattle (Bos indicus) breeds. Comparative analysis of 1,587 bp region comprising promoter (1,418 bp), exon-I (53 bp), and partial intron-I (116 bp) revealed 35 nucleotide substitutions (32 within promoter region, 1 in exon-I, and 2 in partial intron-I region) and 4 Indels. Within promoter, 15 variations at positions −1399 (A > G), −1288 (G > A), −1259 (T > C), −1158 (T > C), −1016 (A > T), −941 (T > G), −778 (C > T), −610 (G > A), −536 (A > G), −521 (A > G), −330 (A > C), −214 (A > G), −205 (A > T), −206 (C > A), and −175 (A > G) were located within the potential transcription factor binding sites (TFBSs), namely, NF-κE1/c-Myc, GATA-1, GATA-1/NF-E, Oct-1/POU3F2, MEF-2/YY1, GATA-1, AP-1, POU1F1a/GR, TMF, GAL4, YY1/Oct-1, HNF-1, GRalpha/AR, GRalpha/AR, and AP-1, respectively. Seventy-four percent (26/35) of the observed SNPs were novel to Indian cattle and 11 of these novel SNPs were located within one or more TFBSs. Collectively, these might influence the binding affinity towards their respective nuclear TFs thus modulating the level of transcripts in milk and affecting overall protein composition. The study provides information on several distinct variations across indicine and taurine αS1-CN regulatory domains.

Milk proteins and human health: A1/A2 milk hypothesis.

Sir, Milk from dairy cows has been regarded as nature’s perfect food, providing an important source of nutrients including high quality proteins, carbohydrates and selected micronutrients. More than 95% of the cow milk proteins are constituted by caseins and whey proteins. Among the caseins, beta casein is the second most abundant protein and has excellent nutritional balance of amino acids. Different mutations in bovine beta casein gene have led to 12 genetic variants and out of these A1 and A2 are the most common. The A1 and A2 variants of beta casein differ at amino acid position 67 with histidine (CAT) in A1 and proline (CCT) in A2 milk as a result of single nucleotide difference. This polymorphism leads to a key conformational change in the secondary structure of expressed β-casein protein. Gastrointestinal proteolytic digestion of A1 variant of β-casein (raw/processed milk) leads to generation of bioactive peptide, beta casomorphin 7 (BCM7). [1] Infants may absorb BCM-7 due to an immature gastrointestinal tract whereas adults gather the biological activity locally on the intestinal brush boarder. In hydrolysed milk with variant A1 of beta-casein, BCM-7 level is 4-fold higher than in A2 milk. Initial studies on indigenous cow (Zebu type), buffalo and exotic cows (taurine type) have revealed that A1 allele is more frequent in exotic cattle while Indian native dairy cow and buffalo have only A2 allele, [2] and hence are a source for safe milk. Recently, a relationship between disease risk and consumption of a specific bovine β-casein fraction with either A1 or A2 genetic variants has been identified. BCM7 is suggested to be associated as a risk factor for human health hazards as it can potentially affect numerous opioid receptors in the nervous, endocrine and immune system. It is also known to be an oxidant of low dietary lipoproteins (LDL) and oxidation of LDL is believed to be important in formation of arterial plaque. Epidemiological evidences claim that consumption of beta-casein A1 milk is associated as a risk factor for type-1 diabetes, coronary heart disease, arteriosclerosis, sudden infant death syndrome, autism, schizophrenia etc. [3,4] A broad range of studies from American and European investigations has shown reduction in autistic and schizophrenic symptoms with decrease in A1 milk intake. [5] Further, animal trials have also supported the linking of type-1 diabetes to milk exposure in general and A1 beta-casein in particular. Populations, which consume milk containing high levels of β-casein A2 variant, have a lower incidence of cardiovascular disease and type-1 diabetes. The A1/A2 hypothesis is both intriguing and potentially very important for public health if it is proved correct. It should be taken seriously and deeper research is needed to verify the range and nature of BCM7 interactions with the human gastrointestinal tract and whole organism. This requires more of animal trials and generation of data on human subjects having the problems related to A1/A2 beta-casein milk consumption.

Genetic Relationship Among Indian Termites Based on DNA Sequence of Mitochondrial 12S Ribosomal RNA Gene

Partial 12S gene fragments were amplified by using specific primers in nine species of termites of the genus Odontotermes, Microtermes and Microcerotermes (Isoptera: Termitidae: Macrotermitinae), and the PCR products were subjected to sequence analysis. The sequences obtained were characterized to see the frequencies of each nucleotide bases, and high A+T content was observed. The divergence of the species was found be lower within the same family, and highest with species from the family Rhinotermitidae. Phylogenetic tree drawn on the basis of distance Neighbour-joining method revealed clustering of individuals according to their genera and families.

Analysis of genetic variations across regulatory and coding regions of kappa-casein gene of Indian native cattle (Bos indicus) and buffalo (Bubalus bubalis).

The promoter region of kappa-casein (κ-CN) gene in Indian native cattle and buffalo breeds was sequenced and analyzed for nucleotide variations. Sequence comparison across breeds of Indian cattle revealed a total of 7 variations in the promoter region, of which − 515 G/T, − 427 C/T, − 385 C/T, − 283 A/G and − 251 C/T were located within consensus binding sites for octamer-binding protein (OCT1)/pregnancy specific mammary nuclear factor (PMF), activator protein-2 (AP2), hepatocyte nuclear factor (HNF-1) and GAL4 transcription factors (TFs), respectively. These variations might be involved in gain or loss of potential transcription factor binding sites (TFBSs). Unlike the other 4 variants, the − 283 (A/G) variant located within HNF-1 TFBS was specific to Indian cattle as this change has not been observed in the Bos taurus sequence. Other TFBSs viz., MGF, TBP, NF-1, milk box and C/EBP were conserved across species. For the Indian native buffalo breeds, only 3 changes were identified in the promoter region; − 305 (A/C), − 160 (T/C) and − 141 (A/G) and most of the TFBSs were found to be conserved. However, deletion of two adjacent nucleotides located in and around binding site for C/EBP TF was identified in buffalo when compared with promoter sequence of bovine κ-CN. For κ-CN of Indian native cattle, a strong linkage disequilibrium (LD) was observed for variations 515 G/T, − 427 C/T and − 385 C/T in the promoter region; and for variations at codons 136 and 148 of exon-IV. Further, among intragenic haplotypes, variation − 427 C/T was found to be in LD with variations at codons 136 and 148. The information generated in the present work provides comprehensive characterization of κ-CN gene promoter and coding regions in Indian cattle and buffaloes and reported variations could become important candidates for carrying out further research in dairy traits.

Genetic Polymorphisms in the Bovine Toll-Like Receptor 4 (TLR4) and Monocyte Chemo Attractant Protein-1(CCL2) Genes: SNPs Distribution Analysis in Bos indicus Sahiwal Cattle Breed

Toll-like receptor 4 gene (TLR4) that recognizes the Gram negative bacterial ligand LPS was sequenced in the Bos indicus Sahiwal cattle breed. Ninety four single nucleotide polymorphisms (SNPs) were detected within 10.8 kb gene region. Seventeen of the SNPs were in the coding regions and the one at position 9589(A > G) in exon3 resulted in an amino acid change from Valine to Isoleucine. These SNPs led to generation of 27 TLR4 gene haplotypes. All the Sahiwal animals studied presently showed the occurrence of the genotype CC at gene position 9662, which codes for the amino acid threonine at position 674 of the TLR4 protein, and which had been reported to be associated with lower somatic cell score and, therefore, a lower susceptibility to mastitis, in Taurus cattle. This nucleotide configuration of the Toll-like receptor 4 gene of the Bos indicus Sahiwal cattle breed could possibly indicate toward a lower susceptibility to mastitis in the Sahiwal animals. Monocyte chemo-attractant protein-1 (CCL2) gene encoding for small inducible cytokine A2 that belongs to the CC chemokine family was also sequence characterized in these Sahiwal animals. The CCL2 gene was observed to have 12 polymorphic sites in 3.3 kb region of which one SNP at position 2500 (A > G) in exon 3 resulted in amino acid change from Valine to Isoleucine at position 46 of the mature CCL2 peptide. Seventeen haplotypes of the CCL2 gene were predicted corresponding to 12 genotypes detected.