Development of PCR-based genetic test for detection of TTF1 mutation causing abortion in Holstein Friesian cattle.

A stop-gain mutation (rs715966442; BTA11: 1,02,463,944 nucleotide position) in transcription termination factor, RNA polymerase I (TTF1) gene causes abortion in Holstein Friesian (HF) cattle. A PCR-restriction fragment length polymorphism (PCR-RFLP)-based genetic test has been developed and validated to screen the TTF1 mutation locus in HF cattle. The mutation locus was screened in 80 HF and HF crossbreds using the protocol, which revealed two animals as carriers of the mutant TTF1 allele. The test employed is cost-effective, rapid and precise and can be utilized as an effective tool for the screening of TTF1 mutation carriers in HF cattle population.

Genome-wide selection signatures address trait specific candidate genes in cattle indigenous to arid regions of India.

The peculiarity of Indian cattle lies in milk quality, resistance to diseases and stressors as well as adaptability. The investigation addressed selection signatures in Gir and Tharparkar cattle, belonging to arid ecotypes of India. Double digest restriction-site associated DNA sequencing (ddRAD-seq) yielded nearly 26 million high-quality reads from unrelated seven Gir and seven Tharparkar cows. In all, 19,127 high-quality SNPs were processed for selection signature analysis. An approach involving within-population composite likelihood ratio (CLR) statistics and between-population FST statistics was used to capture selection signatures within and between the breeds, respectively. A total of 191 selection signatures were addressed using CLR and FST approaches. Selection signatures overlapping 86 and 73 genes were detected as Gir- and Tharparkar-specific, respectively. Notably, genes related to production (CACNA1D, GHRHR), reproduction (ESR1, RBMS3), immunity (NOSTRIN, IL12B) and adaptation (ADAM22, ASL) were annotated to selection signatures. Gene pathway analysis revealed genes in insulin/IGF pathway for milk production, gonadotropin releasing hormone pathway for reproduction, Wnt signalling pathway and chemokine and cytokine signalling pathway for adaptation. This is the first study where selection signatures are identified using ddRAD-seq in indicine cattle breeds. The study shall help in conservation and leveraging genetic improvements in Gir and Tharparkar cattle.

Whole genome resequencing revealed genomic variants and functional pathways related to adaptation in Indian yak populations.

The present study aims to identify genomic variants through a whole genome sequencing (WGS) approach and uncover biological pathways associated with adaptation and fitness in Indian yak populations. A total of 30 samples (10 from each population) were included from Arunachali, Himachali and Ladakhi yak populations. WGS analysis revealed a total of 32171644, 27260825, and 32632460 SNPs and 4865254, 4429941, and 4847513 Indels in the Arunachali, Himachali, and Ladakhi yaks, respectively. Genes such as RYR2, SYNE2, BOLA, HF1, and the novel transcript ENSBGRG00000011079 were found to have the maximum number of high impact variants in all three yak populations, and might play a major role in local adaptation. Functional enrichment analysis of genes harboring high impact SNPs revealed overrepresented pathways related to response to stress, immune system regulation, and high-altitude adaptation. This study provides comprehensive information about genomic variants and their annotation in Indian yak populations, thus would serve as a data resource for researchers working on the yaks. Furthermore, it could be well exploited for better yak conservation strategies by estimating population genetics parameters viz., effective population size, inbreeding, and observed and expected heterozygosity.

Novel insights into genome-wide associations in Bos indicus reveal genetic linkages between fertility and growth.

Bos indicus breed Sahiwal, famous for its optimum performance, has so far been genetically improved for performance traits based on phenotypic records and the genomic knowhow regarding genes, regions and biological processes underlying the complex quantitative traits is lacking. In this context, a Genome-wide Association Study was performed for fertility and growth traits in Sahiwal cattle to shed light on its genomic profile. A total of 46 SNPs were found associated with the traits at genome-wide suggestive threshold of P ≤ 10-4. USP32, LRPPRC, PLA2G10, RRN3 and ASAP1 were identified as putative candidate genes for body weight at different ages. However, several genes mapped for growth traits like GREB1, PLA2G10, RAD51C, BIRC6, TEX14 and PEBP4 had significant physiological underpinnings in determining fertility of the animals. Moreover, Quantitative trait loci (QTL) identification revealed potential overlaps with the already reported QTLs for both fertility and growth for most of the traits. Further, candidate SNP enrichment analysis revealed an enriched biological process for birth weight with a significant reproductive role. Based on the findings, genetic linkages underlying fertility and growth could be discerned in Sahiwal population and may be utilized for improving fertility traits in future.

Genetic admixture and population structure analysis of Indian water buffaloes (Bubalus bubalis) using STR markers.

BACKGROUND: India has a vast riverine and swamp buffalo diversity adapted to various agro-ecological conditions. In the present study, genetic diversity data for 10 different buffalo populations of India, using 20 highly polymorphic microsatellite markers has been generated for the genetic diversity analysis. The buffalo populations of Eastern Odisha state, were the primary focus. METHODS AND RESULTS: The minimal spanning network based on Bruvo's distance, PCA (Principal Component Analysis) based on the Fst (Fixation Index) values, and genetic admixture analysis using both the STRUCTURE and 'snapclust' were performed. The analysis could identify the Manda population as distinct from other Odisha buffalo breeds as well as adjoining Chhattisgarhi buffalo breeds. The total observed number of alleles ranged between 143 (Manda) and 301 (Paralakhemundi) with an average of 204 alleles per breed. The Sambhalpuri buffalo population also clustered into two separate subpopulations, half of the unique sub-population located geographically south-wards, displayed no admixture with any of the adjacent buffalo populations. The Manda buffalo population has shown sufficient allelic richness and heterozygosity under random mating being practiced in the field conditions. CONCLUSIONS: The study has led to the identification of the Manda as a distinct buffalo population, and the germplasm has been registered as a new Indian buffalo breed. Whereas, the Sambhalpuri population requires elaborate analysis to confirm the existence of two distinct sub-populations.

Genomic Diversity Profiling and Breed-Specific Evolutionary Signatures of Selection in Arunachali Yak.

Arunachali yak, the only registered yak breed of India, is crucial for the economic sustainability of pastoralist Monpa community. This study intended to determine the genomic diversity and to identify signatures of selection in the breed. Previously available double digest restriction-site associated DNA (ddRAD) sequencing data of Arunachali yak animals was processed and 99,919 SNPs were considered for further analysis. The genomic diversity profiled based on nucleotide diversity, π (π = 0.041 in 200 bp windows), effective population size, Ne (Ne = 83) and Runs of homozygosity (ROH) (predominance of shorter length ROHs) was found to be optimum. Subsequently, 207 regions were identified to be under selective sweeps through de-correlated composite of multiple signals (DCMS) statistic which combined three individual test statistics viz. π, Tajima's D and |iHS| in non-overlapping 100 kb windows. Mapping of these regions revealed 611 protein-coding genes including KIT, KITLG, CDH12, FGG, FGA, FGB, PDGFRA, PEAR1, STXBP3, olfactory receptor genes (OR5K3, OR5H6 and OR1E1) and taste receptor genes (TAS2R1, TAS2R3 and TAS2R4). Functional annotation highlighted that biological processes like platelet aggregation and sensory perception were the most overrepresented and the associated regions could be considered as breed-specific signatures of selection in Arunachali yak. These findings point towards evolutionary role of natural selection in environmental adaptation of Arunachali yak population and provide useful insights for pursuing genome-wide association studies in future.

Current status and unique attributes of Indian Chilika buffalo for adaptation to brackish water ecology.

Chilika buffalo is native to the Eastern coast of India and well adapted to the largest coastal brackish water lagoon of Asia, Chilika Lake. We present here a report on the Chilika buffalo breed emphasizing the conservational urgency based on unique biochemical and molecular evidence related to liver and kidney functions while comparing it with tropically adapted other water buffalo breeds (Bubalus bubalis) of India. It is found that the Chilika buffalo breed has a better ability to withstand a long dehydration period as evident from its better glomerular filtration and higher expression of the ion transport channel. Mitochondrial D-loop sequencing results have shown these buffaloes being closer to swamp-type buffaloes of Bangladesh and northeast India and represent a unique "hybrid zone" on the eastern coast of India. Conservation of such uniquely adapted germplasm is crucial owing to the current global trend, where the introduction of exotic breeds has negatively impact "sui-generis" germplasm and they require higher managerial resource consumption for maintaining higher productivity. Further, the introduction of unconventional fisheries activities has proved detrimental to the lagoon ecosystem, potentially causing more threat to the buffalo's population.

Understanding the genomic architecture of clinical mastitis in Bos indicus.

This study elucidated potential genetic variants and QTLs associated with clinical mastitis incidence traits in Bos indicus breed, Sahiwal. Estimated breeding values for the traits (calculated using Bayesian inference) were used as pseudo-phenotypes for association with genome-wide SNPs and further QTL regions underlying the traits were identified. In all, 25 SNPs were found to be associated with the traits at the genome-wide suggestive threshold (p ≤ 5 × 10-4) and these SNPs were used to define QTL boundaries based on the linkage disequilibrium structure. A total of 16 QTLs were associated with the trait EBVs including seven each for clinical mastitis incidence (CMI) in first and second lactations and two for CMI in third lactation. Nine out of sixteen QTLs overlapped with the already reported QTLs for mastitis traits, whereas seven were adjudged as novel ones. Important candidates for clinical mastitis in the identified QTL regions included DNAJB9, ELMO1, ARHGAP26, NR3C1, CACNB2, RAB4A, GRB2, NUP85, SUMO2, RBPJ, and RAB33B genes. These findings shed light on the genetic architecture of the disease in Bos indicus, and present potential regions for fine mapping and downstream analysis in future.

Alternate PCR assays for screening of JH1 mutation associated with embryonic death in Jersey cattle.

Jersey haplotype (JH) 1, a stop-gain lethal mutation in the CWC15 gene, causes embryonic losses in Jersey cattle. Two PCR based assays using Amplification Refractory Mutation System (T-ARMS-PCR) and restriction fragment length polymorphism (PCR-RFLP) were developed for screening of the JH1 in cattle. During the screening, seven among 30 Indian Jersey bulls were identified as carriers of the mutant JH1 allele, the first time in the country. These PCR assays are economical, rapid and accurate; and can be used separately or in combination for screening and cross-validation of the JH1 carriers in Jersey cattle.

Diversity analysis at MHC class II DQA locus in buffalo (Bubalus bubalis) indicates extensive duplication and trans-species evolution.

Variation at MHC Class II-DQA locus in riverine and swamp buffaloes (Bubu) has been explored in this study. Through sequencing of buffalo DQA, 48 nucleotide variants identified from 17 individuals, reporting 42 novel alleles, including one pseudogene. Individual animal displayed two to seven variants, suggesting the presence of more than two Bubu-DQA loci, as an evidence of extensive duplication. dN values were found to be higher than dS values at peptide binding sites, separately for riverine and swamp buffaloes, indicating locus being under positive selection. Evolutionary analysis revealed numerous trans-species polymorphism with alleles from water buffalo assigned to at least three different loci (Bubu-DQA1, DQA2, DQA3). Alleles of both the sub-species intermixed within the cluster, showing convergent evolution of MHC alleles in bovines. The results thus suggest that both riverine and swamp buffaloes share con-current arrangement of DQA region, comparable to cattle in terms of copy number and population polymorphism.

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.

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.