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Theses (Ph.D.)
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ThesisItem Open Access EXOME AND PROTEOME PROFILING OF DEONI COWS WITH HIGH AND LOW REPRODUCTIVE PERFORMANCE(ICAR-SRS-NDRI, BENGALURU, 2022) JOEL DEVADASAN M; K.P. RAMESHAReproduction is an important factor for sustainable production in dairy cattle farming. Poor reproductive performance brings a detrimental effect on productivity and longevity in dairy cattle, thus improving the reproductive performance by all means is of paramount importance. Although all the reproduction processes were well defined and conserved, the genetic basis of controlling reproduction process is not fully understood, due to its complex nature. In the present study, we performed whole exome sequencing (WES) and comparative proteomics profiling in Deoni cows with differing reproductive performance to identify genes and protein related to reproductive performance. The Deoni cows of 6 to 9 years age were selected based on the days open and inter-calving period. In high reproductive performance (HRP) group cows mean days open and inter-calving period were 113±23.98 days and 398±24.85 days respectively. Likewise, in low reproductive performance (LRP) group cows mean days open and inter-calving period were 292±60.82 days and 576±58.90 days respectively. Using exome sequencing, we identified 312 and 301 SNPs in HRP and LRP cow groups respectively. A total of 221 genes from 312 SNPs in HRP cows group and 244 genes from 302 SNPs in LRP cows group were identified in the gene annotation. Gene Ontology revealed binding and catalytic activity in molecular functions while cellular, metabolic and biological processes in biological function were found to be enriched with genes identified in both groups. 12 different pathways enriched with genes involved in reproductive process were identified from the pathway analysis. In HRP cattle group ADAMTS19, TRPV4, HDGF, RANGAP1, ROR2, CNOT1, CELSR3, PTPRS, NFAM1, CTRC1, PLA2G4F, SHANK2, and SLIT3 while in LRP cattle group THBS4, SLC37A2, BRWD1, RABEP2, DGKI, ANKH, SCARF1, EPAS1, EIF4G3, and RABEP2 genes were found to be associated with reproduction related traits like calving ease, days open, implantation, calving interval and length of productivity. Using data independent acquisition (DIA) based LC-MS/MS, a total of 430 plasma proteins were identified in all biological replicate samples. In cyclical Deoni cows, a total of 20 proteins were differentially expressed (P<0.05) between high and low reproductive performance group; 15 upregulated while 5 downregulated. In pregnant Deoni cows, total of 35 proteins were differentially expressed (P<0.05) between high and low reproductive performance group; 23 upregulated while 12 downregulated. All these differentially expressed proteins were involved in oxidative stress control, autophagy, cell differentiation, cell migration, immune response, which are required for successful establishment of pregnancy. It is concluded that there is significant difference between Deoni cattle with differing reproductive performance at genome and proteome level. Further, our findings in this study contributes for understanding genetic basis of reproduction traits and aid in identifying probable candidate genes and proteins for enhancing the reproductive performance in dairy cattle.ThesisItem Open Access IDENTIFICATION OF SELECTION SIGNATURES AND IMPUTATION OF GENOTYPES USING HIGH DENSITY SNP DATA IN SAHIWAL CATTLE(ICAR-NDRI, KARNAL, 2022) SATISH KUMAR ILLA; ANUPAMA MUKHERJEEThe present study is aimed to assess the genomic diversity parameters, selection signatures, imputation of genotypes and genomic prediction using High Density SNP data in Sahiwal cattle. The average observed and expected heterozygosity values estimated as 0.33±0.19 and 0.33±0.18, respectively. The proportion of SNP observed in the bins (0.00-0.05, 0.05-0.10, 0.10-0.20, 0.20-0.30 and 0.30-0.40) viz., were 20.38, 4.79, 10.34, 14.76 and 22.54 percent, respectively. The linkage disequilibrium at 0-10 and 10-20 Kb distance were observed to be 0.36±0.39 and 0.26±0.32, respectively. The effective population size at 12th generation (60 years) was observed to be 97. The PCA analysis revealed no distinct structure within Sahiwal breed. However, three distinct clusters were observed when PCA was performed on the populations that include Sahiwal, Gir, Tharparkar, Ongole and Hariana breeds. Admixture analysis on these breeds revealed 4 as optimal cross entropy. Runs of Homozygosity (ROH) analysis was also performed and the optimum thresholds for ROH identification were found as Maximum Gap, Density, homozyg-window –snp, homozyg-window–threshold, homozyg-KB and homozyg- SNP as 250, 120, 10, 0.05, 30 and 20, respectively. The population consists greater number of longer and medium ROH segments. The trend line of Inbreeding due to pedigree (FPED) and ROH (FROH) was similar, however the magnitude is low. The correlations of FPED with various FROH ranged from medium to high except with 0-2 Mb segments. Significant ROH islands were located on BTA 1, 5, 7, 12, 14, 18, 19, 27. Hub genes related with host immunity and metabolism were also observed. The highest number of LD blocks were observed on BTA 6 and 21 as 124 and 21, respectively. However, the maximum block size was found on BTA5 and 21, respectively. Selection signature analysis revealed 14 genomic regions that harbors genes related with various economic traits. Gene prioritization analysis revealed four key genes such as KIT, MAP3K1, KDR and LEF1. High imputation accuracies (CORRSNP, PERCG) were observed when the reference population size is big and the SNP density is high. Beagle 5.0 is slightly advantageous than Minimac3 in terms of imputation of genotypes. Bayes A method is found to be effective for all the traits (production and reproduction) in Sahiwal.ThesisItem Open Access HIGH-THROUGHPUT PROFILING OF MILK PROTEOME ASSOCIATED WITH HEAT TOLERANCE IN DEONI AND HOLSTEIN FRIESIAN CROSSBRED CATTLE(ICAR-SRS-NDRI, BENGALURU, 2021) EKTA RANA; K.P. RAMESHAMilk production and its constituents are affected by many factors like breed, species, age, parity, stage of lactation, season, etc. Under field conditions, seasons significantly influence the milk production in dairy animals. Indigenous (Bos indicus) cattle survive and perform better under heat stress conditions when compared to exotic (Bos taurus) breeds or their crossbreds. However, the mechanisms at a molecular level associated with heat tolerance among these cattle are ill-understood till date. In the present study, high-throughput proteomic profiling of milk whey was performed to identify the subtle changes occurring at protein level during extreme seasons in Deoni and Holstein Friesian crossbred cows. A total of 412 proteins were identified in the milk whey samples by LC-MS/MS technique coupled with extensive bioinformatics analysis. Among which majority of the milk whey proteins were found to be associated with the defense mechanisms of the dairy cows. Complement and coagulation cascades pathway, Lysosome pathway and Phagosome pathway were found to be highly enriched biological pathways in global milk whey proteomics. Differential milk whey proteomic analysis compared between winter (average THI = 66.6) and summer (average THI = 82.2) seasons revealed that 27 and 54 proteins were up-regulated (fold > 1.5), whereas, 10 and 8 proteins were down-regulated (fold < 0.6) during summer season as compared to winter season in Deoni and Holstein Friesian crossbred cows, respectively. Gene ontology analysis of differentially expressed milk whey proteins compared between winter and summer seasons revealed that 38.09 and 48.15 percent of the enriched biological processes were related to the defense mechanism in Deoni and Holstein Friesian crossbred cows, respectively. On comparing Holstein Friesian crossbred cows with Deoni cows during summer season, 61.54 percent of the enriched biological processes was found to be associated with the defense mechanism of the animals. KEGG pathway analysis indicated that the complement and coagulation cascades pathway was highly enriched in Holstein Friesian crossbred cows as compared to Deoni cows during summer season. On narrowing down the milk whey proteins based on different criteria, three potential proteins having biological significance in relation to heat tolerance were selected for validation study by ELISA method. The statistical analysis revealed that the expression of haptoglobin protein during summer season was highly significant in both the breeds, whereas, the expression of peptidoglycan recognition protein 1 during summer season was highly significant in Deoni cows, but was non-significant in Holstein Friesian crossbred cows. It was concluded that a definite difference exists in the milk whey protein profile when compared between different seasons in dairy cows. Furthermore, the expression of haptoglobin protein during summer season was consistently higher (>1.5 folds) in both Deoni and Holstein Friesian crossbred cows by both LC-MS/MS and ELISA methods. Thus, it could be considered as one of the putative biomarkers to detect the level of heat tolerance in Deoni and Holstein Friesian crossbred cowsThesisItem Open Access WHOLE GENOME RE-SEQUENCING OF ZEBU CATTLE TO DELINEATE COPY NUMBER VARIATIONS (CNVs)(ICAR-NDRI, KARNAL, 2022) NARENDRA PRATAP SINGH; INDRAJIT GANGULYIndia has vast cattle genetic resources that contribute significantly to the dairy sector and to the country's overall agricultural economy. The total livestock population in India is estimated to be 535.78 million. There are 50.42 million exotic cattle and 142.11 million indigenous cattle in the overall cattle population of 193.46 million (Livestock Census, 2020). Till date, ICAR-National Bureau of Animal Genetic Resources has registered 50 breeds of cattle in India. Recent advances in Whole-genome sequencing technologies including high-throughput SNP genotyping assay are revolutionizing the field of livestock genomics. In addition, the availability of whole-genome sequences for individual species is redefining the future landscape of animal genomic research. Copy Number Variation (CNVs) constitutes a major component of genetic diversity. CNVs are the genomic structural variation that ranges in size from 50 base pairs (bp) to mega-base pairs (Mbp). Understanding the genetic diversity is crucial for breed improvement and conservation programmes. A substantial amount of overall genetic diversity in a species/breed is represented by its copy number variations (CNVs). CNVs are large insertions, deletions, and duplications in the genome that vary between individuals in a species. They are now extensively being used as markers for evaluation of genomic traits in domestic animals due to their major role in modulating complex traits by influencing gene expression, phenotypic variation, adaptation and disease susceptibility. To characterize CNVs/copy number variations regions (CNVRs), related diversity, and functional enrichment, 21 cattle whole genome re-sequencing data (10 ×) were obtained from Sahiwal, Kangayam, and Ladakhi breeds. Using the recent ARS-UCD1.2 reference assembly, genome-wide CNVs/CNVRs were identified using the CNVnator and CNVcaller software. PLINK (for PCA) and ADMIXTURE tools were used for the diversity analysis. ANNOVAR software was used for gene annotation, while PANTHER software was used for CNVRrelated gene enrichment analysis. The present study reported a total of 12,405 CNVs and 8,166 copy number variations regions (CNVRs). CNVRs were found to overlap a total of 2,922 genes. The functional significance of these genes was investigated further. CNVR function was determined by enrichment analysis, which comprised cellular component (GO: 0005575), binding (GO: 0005488), and molecular function (GO: 0003674), as well as the Gonadotropin-releasing hormone receptor pathway (GO: P06664) and metabolic process regulation (GO: 0019222). Principal component analysis (PCA) showed a separate clustering of Ladakhi cattle from the other two breeds. The admixture analysis at K = 2 revealed distinct genetic structuring of the studied populations. The clustering pattern of the breeds reveals importance of CNVRs, in view of hot versus cold adaptation, in particular. A total of 4,333 QTL were identified, which are significantly enriched for 373 traits. Systematic analysis revealed that the proportions of Milk, Production (other than milk), Reproduction, Health, Meat & carcass, and Exterior QTLs were 13.52 percent, 14.95 percent, 7.43 percent, 14.79 percent, and 6.9 percent, respectively.ThesisItem Open Access GENOME WIDE IDENTIFICATION OF SELECTION SWEEPS IN HIGH ALTITUDE BOVINES FOR MILK PRODUCTION AND ADAPTABILITY TRAITS(ICAR-NDRI, KARNAL, 2022) MOHAN, M.; S.K. NIRANJANLadakh (UT), a unique habitat with an altitude of more than 3000 m above mean sea level is the home of unique farm bovine species, well adapted to hypoxic conditions, subnormal temperature, high UV exposure, and sparse feed resources. The unique genetic architect of these native bovines is an interesting venture to study the adaptation; it also needs to explore the genomic region of milk production for their further genetic improvement. Genome wide comparison of high producing Zebu and exotic cattle with high altitude bovines might help to explore variety of hypothesis that may be utilized for genetic improvement for milk of native bovines without changing the genetic architect for adaptation. With hypothesizing the uniqueness of high altitude bovines and their differences with dairy cattle breeds, a technical program has been proposed with study group comprises native cattle (12), yak (12) and their hybrids (6) of Ladakh, and high producing cattle Sahiwal (12), Holstein Friesian (12) and Jersey (12). The samples were sequenced using ddRAD technology, data analysis done by creating a bioinformatic pipeline from open source tools. A total of 610058, 357981 and 311100 SNPs with novel proportion of 59.89, 86.93 and 81.26 % were detected in Ladakhi cattle, yak, and its hybrids respectively. Genome wide SNP density analysis showed a SNP occurs at every 4336, 7448 and 8563 bp intervals in Ladakhi cattle, yak, and its hybrids respectively. Total 59 high SNP dense regions were found common in all three-group influencing vital traits related to production, reproduction, and adaptation traits. Gene and Phenotype ontology of missense variants bearing genes accounted for the structural and physiological changes in high altitude bovines. Selection sweep scanning revealed 131, 78 and 67 highly significant genes were selected in individually Ladakhi cattle, yak, and its hybrids. These genes are involved in adaptive interactive gene networks influencing oxygen carrying, cardiovascular, cutaneous and energy metabolism systems. Selection sweep comparison between high altitude bovines with high producing cattle, showed former has similar selected regions related to milk production with high producing cattle and later has similar selected regions related to reproduction and adaptation with high altitude cattle. Among the High producing cattle based on greater proportion of selected regions, ranked correspondingly as Sahiwal, followed by Holstein Friesian and Jersey. This study implies that genetic variants occurred in high altitude bovines could be a retroaction to local environment. Adding value to above identified selection sweep also gives an idea on the gene level adaptation aided by nature’s selection process undergone in Himalayan bovines to cope up with harsh conditions and which would help for marker-based selection in future. Comparative selection sweep analysis of highaltitude bovines with high producing cattle throws light on the wide possibilities that could be used for selection and breeding strategies for breed improvement which in turn might develop socio-economic status of locals.ThesisItem Open Access GENOME WIDE ASSOCIATION AND BREEDING VALUE ESTIMATION FOR ECONOMIC TRAITS IN MURRAH BUFFALO(ICAR-NDRI, KARNAL, 2022) LINDA GEORGE; VERMA, ARCHANAThe present study was aimed to identify genome wide SNPs and their association with growth, production and reproduction traits and also to estimate and compare the breeding values for economic traits using ssGBLUP and BLUP in Murrah buffalos. Records of Murrah buffalo from 1965 to 2021 were used for the study, out of which 120 animals were genotyped using double digest Restriction site Associated DNA (ddRAD) sequencing approach. The phenotypic data were collected from National Dairy Research Institute, Karnal on Growth traits viz. 6M (month), 12M, 18M and 24M body weight, production traits like 305D (day) milk yield, lactation length (LL) and dry period (DP), Total milk yield (TMY) and reproduction traits like Age at first calving (AFC), Calving interval (CI) and First service period (FSP), Age at first service (AFS), period from calving to first AI and NRR56 days. The biallelic genotypic data consisted of 49459 markers post quality check. In GWAS study identified 25 significant SNPs (P < 1x10-6 ) associated with Age at first calving (AFC), Age at first service (AFS), period from calving to 1st AI, Service period (SP) and 6 month body weight (6M). 15 genetic variants overlapped with different QTL regions of already reported studies. In reproduction traits, significant SNPs present on MAK, AQP1, Ubiquitin-conjugating enzyme F2R1, KCNS3, CPNE4, PAH and VOPP1 genes and in growth traits, significant SNPs present on MTUS2 and SLC14A2 gene. Weighted ssGWAS able to identified genomic regions with more than 1% additive genetic variance in different traits. GWAS and WssGWAS able to identify major SNPs and its important QTL associated with each traits. Genetic parameter estimation was done using AI-REML and Gibbs sampling Bayesian approach using pedigree and genomic information data. Pedigree based and genomic information based heritability estimates have no difference. Bayesian approach provide better estimates of genetic parameters than AIREML approach based on accuracy of prediction. The estimate of heritability were moderate to high for growth traits (0.31±0.05 for 6M, 0.20±0.04 for 12M, 0.30±0.04 for 18M and 0.26±0.048 for 24M BW), low to moderate for production traits (0.083±0.04 for LL, 0.19±0.04 for TMY, 0.21±0.05 for 305D MY and 0.22±0.06 for DP) and low for reproduction traits (0.11±0.045 for AFC, 0.044±0.05 for CI and 0.091±0.05 for FSP). The breeding values of Murrah sires with 5 or more progenies for different economic traits were estimated using three sire evaluation methods viz., PBLUP (pedigree BLUP), ssGBLUP (single step Genomic BLUP) and ssBR (single step Bayesian Regression). In comparison of accuracy of prediction of breeding value of sires using single trait model, ssBR gave 10-67% more accuracy and in multitrait model ssGBLUP gave better accuracy of prediction than single trait model. So Single step genomic evaluation methodologies can be adopted for estimation of breeding value in Murrah Buffaloes.ThesisItem Open Access GENETIC EVALUATION OF MURRAH BUFFALOES FOR GROWTH, PRODUCTION AND REPRODUCTION TRAITS(ICAR-NDRI, KARNAL, 2022) POOJA JOSHI; VERMA, ARCHANAThe present study was undertaken for genetic evaluation of Murrah buffaloes for different economic traits. Records of body weight, production and reproduction data of Murrah buffalo maintained at ICAR-NDRI Karnal, over the period (1968-2019) were used in study. Animal model using AIREML approach was used for estimation of genetic parameters for growth, production and reproduction traits using WOMBAT software. (Co)variance components and genetic parameters were estimated using most appropriate model (based on LRT test) that account for direct and maternal effects fitted to each trait. The heritability estimate of BW, 3BW, 6BW, 9BW, 12BW, 18BW, 24BW, 30BW and 36BW were 0.19, 0.14, 0.37, 0.43, 0.10, 0.30, 0.30, 0.45 and 0.27 respectively, using traditional animal model and 0.25, 0.12, 0.20, 0.26, 0.26, 0.35, 0.35, 0.38 and 0.42 respectively, using Random regression model. RRM fitted to growth data, with sixth order Legendre polynomial for all the random effects was found to be best. Spearman rank correlation estimates between sire’s rank at different ages were higher using RRM. Lower error variance and higher additive variance was found for growth traits using RRMheterogenous model as compared to traditional animal model implying RRM was relatively more efficient. The correlation estimates obtained between different growth traits were positive and moderate to high. The heritability estimates for the production traits: TLMY, 305day milk yield and Lactation length were 0.20, 0.21 and 0.17 respectively and for the reproduction traits: AFS, AFC, SP and CI were 0.12, 0.25, 0.08 and 0.08 respectively. Significant increase of 182.98 kg in TLMY, 205.78 kg in 305day milk yield, 24 days for Lactation length and significant decline of 72 days in AFC was found in heifers having >650 gm of average daily gain during 9 to 18-month of growth phase. Association study based on body weight classes revealed heifers with more than 220 kg twelve-month body weight produced 7.1% more 305day milk yield and 11% more TLMY as compared to lighter heifers. It is recommended to use random regression model for genetic evaluation of growth traits.ThesisItem Open Access Sire evaluation using test day random refression models in murrah buffaloes(ICAR-NDRI, KARNAL, 2022) Ranjan, Ashish; ARCHANA VERMAThesisItem Open Access QUANTITATIVE TRAIT NUCLEOTIDE VARIATION IN SELECT CANDIDATE GENES ASSOCIATED WITH BEHAVIOURAL, PRODUCTIVE AND REPRODUCTIVE TRAITS IN DEONI CATTLE(ICAR-SRS-NDRI, BENGALURU, 2022) MANJUNATHA PRABHU B.H.; D.N. DASThe present work was carried out in Deoni cattle maintained at LRC, SRS, ICAR-NDRI, Bengaluru for identification of single nucleotide variation in Tyrosine Hydroxylase (TH) and Dopamine-β Hydroxylase (DBH) genes in Deoni cows and to study the association of genetic variants in Deoni cows for behavioural, productive and reproductive traits. The PCR-SSCP analysis of TH exons exhibited polymorphism banding patterns in exon1, exon2, exon 4, exon 11 and exon13. TH exons exhibited SNPs G49384059T, G49385677C C49387048A, C49389099 and T49390458A leading to transversion with amino acid change Arginine to Isoleucine, Arginine to Proline; Alanine to Glutamic acid and Leucine to Histidine in the translated sequence. The variation of TH exon1, exon2 and exon11 were found to have a significant association with the behavioural, productive and reproductive parameters in the Deoni cow. The PCR-RFLP analysis of the DBH exon exhibited polymorphism with a genotypic banding pattern in exon1, exon2, exon4, exon5, exon6 and exon10. DBH exons revealed SNPs G104499604A, C104501947T, A104503834G, G104504957A, C104505597A, A104509237C and A104514081G leading to transition and transversion leading to amino acid change Arginine to Lysine, Proline to Leucine, Glutamic acid to Glycine, Arginine to Histidine, Proline to Histidine, Glutamine to Proline, Histidine to Arginine in the translated sequence. The variations of DBH were found to have a significant association with most of the behavioural, productive and reproductive parameters in Deoni cows. The BLAST analysis between Deoni cows and other livestock species sequence revealed per cent homology between 78-100 per cent similarities. Finally, it concluded that the candidate genes (TH and DBH) were polymorphic in Deoni cattle indicating the genetic variability with identified SNPs or genotype patterns which were associated with behaviour, productive and reproductive traits which could be used as genetic markers in Marker Assisted Selection (MAS) after validating the same using a large number of Deoni cows.
