Loading...
Thesis
Browse
Search Results
ThesisItem Open Access EVALUATION OF CANDIDATE GENES ASSOCIATED WITH CANINE MAMMARY TUMOUR(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-03-08) ARYA GOPAL; Dr. Radhika G.Canine mammary tumour (CMT) is a common disease reported in female dogs especially from middle age to old age. In the current study, screening of mutation hotspots within the candidate genes namely Breast cancer 1 and 2(BRCA1 and BRCA2) and association of the detected mutations with CMT was performed. Further, characterisation and expression profiling of Tissue Inhibitor of Metalloproteinase 2 gene (TIMP2) was conducted. The polymorphism study in BRCA1 was conducted using High Resolution Melt Curve (HRM) analysis, in 50 CMT affected and 50 normal female dogs above five years of age. Representative samples showing different melt curve patterns on HRM of canine BRCA 1, were sequenced and the presence of the variant -1173C>T within the promoter region was confirmed. The sequence results depicted three different genotypes namely CC, CT and TT within the population under study. The association analysis revealed significant association of the variant -1173C>T with CMT. The TT genotype was obtained only in CMT affected dogs and seven out of 11 dogs with TT genotype had a history of mammary tumour recurrence. The variant analysis within canine BRCA2 was done using PCR-RFLP which detected the missense variant 4304A>G within exon 11 of BRCA2. The digested fragments, showing three different band patterns represented three different genotypes namely AA, AG and GG. On post hoc analysis, a significant association of the variant with CMT could not be obtained. The relative expression of TIMP2 in mammary tumour samples and normal glands was studied using GAPDH as housekeeping gene. The expression profile illustrated a tenfold reduced expression of TIMP2 in tumour samples when compared with normal glands. Characterisation of the coding region TIMP2 revealed 484 bp sequence, which showed 100 per cent similarity with Canis lupus familiaris (Sequence ID: NM_001003082.1). In the present study, the genetic association of two variants within BRCA1 and BRCA2 with CMT was studied, and the role of TIMP2 in incidence of CMT was analysed. The observations from current research implied the possibility of utilising the variant -1173C>T within BRCA1, either for early detection of CMT or for predicting its prognosis and taking appropriate precautions in dogs.ThesisItem Open Access ANALYSIS OF GONADOTROPIN RELEASING HORMONE RECEPTOR GENE POLYMORPHISMS AND THEIR ASSOCIATION WITH LITTER SIZE IN NATIVE GOAT BREEDS OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-03-08) SARANYA S.K.; Dr. Marykutty ThomasThe present study was carried out with the objectives of molecular characterization of gonadotropin releasing hormone receptor (GNRHR) gene and its association analysis with litter size trait in native goat breeds of Kerala. The study goat population comprised of Attappady black (n=155) and Malabari (n=185) goats sampled from farmers herd in their respective breeding tracts and conservation units. The target regions of GNRHR characterization encompassed promoter, first, second and third exons, and 3’ untranslated regions (UTR) regions. Ten polymorphisms (c.-1129T>G, c.-1069A>G, c.-978A>C, c.-605A>G, c.-33A>G,c.-29T>G, c.48G>A, c.75G>A, c.209T>G and c.*212A>G) were identified in GNRHR of native goats by DNA pool sequencing assay and two of them in promoter region (c.-1129T>G and c.-33A>G) were novel ones. There was one non-synonymous single nucleotide polymorphism (SNP) in exon I (c.209T>G). Two loci (c.-978A>C and c.-33A>G) were polymorphic only in Malabari goats. Analysis of promoter sequence revealed the presence of multiple transcription start sites (TSS) with the major one being located at 806 nucleotides upstream of translation start site (c.-806). The polymorphisms in 5’UTR modified transcription factor binding sites and mi-RNA target sites which regulate the GNRHR gene expression. The SNPs in the promoter and exons also altered the primary, secondary and tertiary structure of mRNA and protein.The identified SNPs were screened in the whole study population by various genotyping methods. Three SNPs at loci c.-1129T>G, c.-978A>C and c.*212A>G were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and five SNPs at c.-33A>G, c.-29T>G, c.48G>A, c.75G>A and c.209T>G loci were genotyped by single strand conformation polymorphism (SSCP) sequencing. The one each SNP at c.-1069A>G locus and c.-605A>G locus were genotyped by Tetra primer amplification refractory mutation system (ARMS-PCR) and high resolution melt curve (HRM) assay respectively. There were presence two genotypes TT and TG at locus c.-1129T>G and three genotypes in c.-1069A>G, c.-978A>C, c.-605A>Gand c.*212A>G loci. The SSCP assay and sequencing showed six haplotypes and seven diplotypes encompassing the polymorphisms at c.-33A>G, c.-29T>G, c.48G>A, c.75G>A and c.209T>G loci. Population genetic analysis indicated that all polymorphic loci except c.-605A>G were in Hardy-Weinberg equilibrium (HWE) in both breeds. The inbreeding coefficient (FIS) ranged between -0.12 to 0.62 in Attappady Black and -0.11 to 0.55 in Malabari goats. There were six and four loci pairs in linkage disequilibrium (LD) at significant level (p<0.01) in Attappady Black and Malabari goats, respectively. The highly significant and strong LD was observed among the haplotypic loci c.-29T>G, c.48G>A and c.209T>G loci in both Atttappady Black and Malabari goats. The low values of FST (0.0081) and Nei’s unbiased measure genetic divergence (0.9970) indicated low genetic differentiation among the two breeds with respect to GNRHR loci.The overall (mean ± S.E) of litter size at birth was 1.9900 ± 0.0440 in Malabari goats. The association analysis of the GNRHR variants with litter size trait using ordinal logistic regression showed that the genotypes at c.-605A>Glocus had a significant (p<0.01) influence on litter size at birth in second and third with GG genotypes favouring multiple births in Malabari goats. The diplotypes of GNRHR promoter and exon 1 had a statistically significant (p<0.01) association with litter size at birth of all four parities. The heterozygotes viz; D1D3 diplotypes and D1D2 diplotypes had the highest odds of having multiple birth in first two parities and in the subsequent parities respectively. Thus results imply that GNRHR is a possible candidate gene for the selection and improvement of litter size in Malabari goats.ThesisItem Open Access CHARACTERISATION OF BOVINE INTEGRIN BETA 6 GENE AND ANALYSIS OF ITS EXPRESSION WITH REFERENCE TO FOOT AND MOUTH DISEASE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-03-08) AKHILA M. R.; Dr. K. A BinduFoot and mouth disease (FMD) is a highly contagious viral disease of livestock. The disease causes significant economic setbacks in the dairy industry. The present study was carried out to identify single nucleotide variations in Integrin beta 6 (ITGβ6), to evaluate its association with FMDV infection and to compare the expression profile in infected and non-infected cattle. A total of 50 FMD infected crossbred, 100 non-infected crossbred and 50 Vechur cattle were screened for polymorphism using PCR-SSCP. Representative samples of each SSCP pattern were sequenced to ascertain the genotypes. The exons 1, 2 and 14 of ITGβ6 were analysed for polymorphism detection. Similar banding patterns (AA with two bands) were observed for exon 1 in all the animals studied. The PCR-SSCP analysis of exon 2 revealed two genotypes BB (two bands) and BC (three bands) and on further sequencing one synonymous SNP, c.29G>A was revealed. The genotype BC was significantly (p0.01) higher in non-infected cattle. Three banding patterns (two, three and four bands) were observed for exon 14 and further sequencing revealed a synonymous SNP, c.133C>T and a non-synonymous SNP, c.135G>A. The diplotypes II (CC, GG) and HI (CT, GG) were identified in crossbred cattle while an additional diplotype IJ (CC, GA) was observed in Vechur cattle. The diplotype HI was significantly (p0.01) higher in non-infected cattle. The relative expression of ITGβ6 was studied in the infected and non-infected cattle with normalization using the reference gene (GAPDH). The expression was downregulated by 0.49 fold in the infected group than the non-infected group. The results obtained from this study indicate the potential role of ITGβ6 on FMD infection and hence offers a chance to explore the possibility of breeding for FMD resistance in future.ThesisItem Open Access ASSESSMENT OF SIRE EVALUATION METHODS IN CROSSBRED CATTLE OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-02-10) M.PRIYADHARSHINI; Dr. K. AnilkumarThe objective of the present study was to assess the sire evaluation methods in crossbred cattle of Kerala by using age at first calving (AFC) and first lactation milk yield (FLMY). The records of 898 crossbred cows sired by 203 bulls spread over a period of 18 years (2002-2019) were collected from different field centres of ICAR-FPT, KVASU. The effect of genetic factors such as genetic group, sire batches and non-genetic factors including the period of birth and season of birth for AFC and period, season, age at first calving for FLMYand source of sires from for both traits were analyzed through Least squares analysis. The present study revealed that overall least square means (±standard error) for AFC was 1041.66± 6.73 days and for FLMY 2780.17 ± 15.72 kg. The sire batches had significant influence (P<0.01%) on AFC and FLMY. The period and season of birth was found to have non significant effect on AFC. Effect of calving period was significant (P<0.05%) on FLMY. However, season of calving and AFC was found to have non-significant influence on FLMY. The genetic group of sire and sources of sire showed non-significant influence on both traits under study. The genetic parameters for AFC and FLMY were estimated using Harvey and REML method. The breeding values of sires with four models were estimated and compared for both traits. BLUP-SM showed lowest error variance and highest coefficient of determination when compared to other models. Relative efficiency of both these traits was estimated with respect to error variance i.e. BLUP-SM. The rank and product moment correlation was statistically significant (P<0.01%) by all four models. The high correlation between BLUP-SM and BLUP-AM indicate higher degree of similarity between both models. So the BLUP-SM and BLUP-AM were equally effective for sire evaluation based on AFC and FLMY. The sires were ranked based on most efficient method for both the traits. Since low AFC is preferred, the sires with lowest AFC value ranked first and the sire with highest AFC value was ranked last in that order and vice versa for FLMY. In conclusion, BLUP-SM was better model when different models were evaluated based on accuracy measures R2, C.V, RMSE, MAE and MAPE for evaluation of bulls of crossbred cattle.ThesisItem Open Access EXPRESSION PROFILING OF ECTO-NOX DISULFIDE THIOL EXCHANGER 2 (ENOX2) GENE IN RESPONSE TO HEAT STRESS IN GOATS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022) AISHWARY VIBHUTIGoats are considered as a heat resilient species among livestock owing to their genetic, physiological, behavioural and morphological characteristics. The Ecto-NOX Disulfide-Thiol Exchanger 2 (ENOX2) gene belongs to ECTO NOX family of cell surface NADH oxidases coding ENOX2 protein. It regulates cell growth, apoptosis and cytoskeleton remodelling, host defence at cellular level during different stress and disease conditions. The present study is planned with an objective to find out the expression profile of ENOX2 gene in response to heat stress and identify single nucleotide polymorphisms (SNPs) in Malabari md Attappady black goats of Kerala. Based on physiological parameters both breeds are grouped as heat stress susceptible (HSS – RT >39 °C, RR >80 breaths/min. and HR >95 beats/min.) and heat stress tolerant (HST – RT <38.5°C, RR <40 breaths/min. and HR <80 beats/min.). The temperature humidity index (THI) was found to havesignificantly high correlation with rectal temperature, respiratory rate and heart rate in both breeds. Total RNA was isolated from whole blood from HSS and HST group of Attappady black (12) and Malabari (12) goats. The relative expression of ENOX2gene is higher in HSS compared to HST group. Higher expression of 5.27 fold was noticed in Malabari whereas in Attappady black it was 1.74 fold. The PCR-SSCP analysis revealed two novel SNPs at intron 1 (g.l42 A>T) and exon 2 (g.154 G>A).Two genotypes AA, BB were observed in the 245bp fragment (E1-I1) with a frequency of 0.21 and 0.79 in Attappady black. Only BB genotype was observed in Malabari goats. Two genotypes, AA and AB were observed in 245bp fragment (I1-E2-I2) with overall frequency of 0.18 and 0.82, respectively. The AB genotype was abundant in both the population. Association study of SNP in E1-I1 region revealed that the BB genotype of Attappady black goats had significantly (p≤0.01) higher respiratory rate and heart rate compared to AA genotype. The SNP at I1-E2-I2region revealed significant (p≤0.01) association of AB genotypes with respiratory rate and body length in both Attappady black and Malabari breeds. The present study concludes that ENOX2 gene can be considered as a promising candidate gene for selection of heat resilient goats.ThesisItem Open Access INSERTION/ DELETION (InDel) POLYMORPHISMS IN CANDIDATE GENES AND ITS ASSOCIATION WITH BODY MEASUREMENT TRAITS IN NATIVE GOAT BREEDS OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-02-02) MANJUTHA S.; Dr. E.M. MuhammedIndia has 34 well-defined goat breeds in their genetic database which is distributed among four major regions: the temperate Himalayas, the north-western regions, the southern peninsulas and the eastern regions. Goats are highly valued as multipurpose animals capable of boosting rural economies, as much as they are involved in food production. India has a high demand for goat meat, which is one of the choicest and most sought-after meats in the country. Kerala is home to two goat breeds- Attappady Black, which is known for its disease resistance and Malabari, which is known for its prolificacy. Livestock are increasingly selected for economic traits using genetic markers like single nucleotide polymorphism (SNP) and insertion/deletion polymorphisms (InDel). A total of 60 Malabari goats maintained in the University farm, Pookode, 67 Attappady Black and 50 Malabari goats maintained in the university farm, Mannuthy and 43 Attappady Black and 17 Malabari goats maintained in the Livestock research farm (LRS), Thiruvazhamkunnu were the experimental animals included in the study. Among those breeds, the animals were grouped into yearling(18-month-old), matured but not yet mated and adult (36-month-old), kidded at least once. From each goat, body measurements including body length (BL), heart girth (HG), body weight (BW), height, chest width (ChW), chest depth (ChD), cannon circumference (CC), hip width (HW) and height at the hip cross (HHC) were taken. From those measurements, calculated the body measurement indices such as the body trunk index (BTI), the body length index (BLI), the heart girth index (HGI), the cannon circumference index (CCI) and the chest width index (ChWI). For our polymorphic study, blood samples were collected from each goat, DNA extracted from blood, amplify the DNA and genotyping study was done.The study investigated five InDel polymorphisms in four candidate genes in Attappady Black and Malabari goats viz., two InDel polymorphisms in PR/SET domain family 6 (PRDM6), one InDel polymorphism each in myostatin (MSTN), insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) and growth hormone receptor (GHR) and found that 15bp insertion and unidentified insertion were detected within the intron2 region of goat IGF2BP1 gene, 9bp insertion was detected within the upstream region of goat PRDM6 gene, 5bp insertion was detected within 5’UTR of goat myostatin gene, 12bp deletion was detected within the intron 1 region of PRDM6P1 gene and 14bp deletion was not detected within the intron 1 region of goat GHR gene. Body measurement traits and their ratios were significantly different across breeds, ages and breed x age interactions (p <0.05). Especially in adult Attappady Black goats, body length, body weight and chest depth were found to be significantly different (p<0.05) and in yearling Attappady Black goats, cannon circumference index was found to be significantly different (p<0.05). In adult Malabari goat, body length, body weight and hip width were found to be significantly different (p<0.05) and yearling Malabari, body height were found to be significantly different (p<0.05). Body weight was found to be significantly different (p<0.05) in breed* age interaction. An association study was conducted between identified InDel and body measurement traits. It was revealed that no significant association between body measurement traits and detected InDel in IGF2BP1 gene, PRDM6 gene and MSTN gene. But in PRDM6P1 gene, body height, cannon circumference and cannon circumference index were found to be significantly associated with 12bp InDel polymorphism. A Chi square test was also used to determine whether Hardy-Weinberg equilibrium (HWE) existed in the screened population. In both Attappady Black and Malabari goat breeds, IGF2BP1gene, MSTN gene and PRDM6P1 gene were deviated from the state of HWE (p <0.05). In Attappady Black goats, PRDM6 gene was deviated from the state of HWE (p <0.05) but in Malabari PRDM6 gene was in the state of Hardy- Weinberg equilibrium (p >0.05). A number of other population genetic parameters were also calculated, including allele frequency, genotype frequency, observed heterozygosity (Ho), heterozygosity (He) and the polymorphic information content (PIC). It was found that the allele frequencies of IGF2BP1 gene, the frequencies of insertion “I” allele was greater than the deletion “D” allele in both breeds. In PRDM6 gene, the frequencies of deletion “D” allele was higher than insertion “I” allele in Malabari goats and vice-versa in Attappady Black goat breeds. In both MSTN gene and PRDM6P1 gene, the frequencies of insertion “I” allele was lower than the deletion “D” allele in both breeds. found that the expected heterozygosity (He) in the PRDM6 and PRDM6P1 genes in both breeds was higher than the observed heterozygosity (Ho).This may have been caused by large proportion of inbreeding. As a result of mixing two isolated populations, observed heterozygosity (Ho) for the IGF2BP1 gene and MSTN gene was greater than expected heterozygosity (He). The polymorphic information content was examined and found that, the polymorphic information content (PIC) was examined and found that, it was moderate level of genetic diversity in Attappady Black goats and high level of genetic diversity in Malabari goats in IGF2BP1 gene, while in PRDM6 and MSTN gene, it was found that high and low level of genetic diversity respectively. in case of PRDM6P1 gene, it was low level of genetic diversity in Attappady Black goats and moderate level of genetic diversity in Malabari goats.From these findings, it was concluded that InDel markers found in the present study may provide a theoretical basis for use of InDel as a marker for marker assisted selection of growth traits among Attappady Black and Malabari goats. Especially PRDM6P1 gene may be directly used as an InDel marker for the selection of goat based on body height, cannon circumference and cannon circumference for meat production as well as growth aspectsThesisItem Open Access ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE IMMUNE-RELATED GENES WITH MASTITIS IN CROSSBRED CATTLE(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-07-10) ARIFA ABDUL KHADER; Dr. C.N. DineshMastitis is the most prevalent and costly disease which affects the economics of the dairy farm. Even though several strategies like antibiotic therapy, improved herd sanitation, and vaccination have been tried to treat and reduce the incidence of mastitis, effective treatment and control of mastitis continue to be a major headache for the dairy farmer. Since differences in susceptibility to mastitis in cattle are known to have a strong genetic background, genetic selection for improved mastitis resistance in dairy cattle is considered to be an alternate strategy. Animal breeders often favour MAS based on the SCC or SCS to select mastitis-resistant animals. A total of 105 lactating crossbred cattle maintained in the ILFC, Pookode of the KVASU, and two private farms located in Wayanad and Kozhikode districts of Kerala state were included in the present study. Milk samples were collected from individual quarters separately from each animal and were graded using CMT and SCC. Based on the CMT reaction, 23.81 per cent, 42.86 per cent and 33.33 per cent of animals were found to have healthy udder, subclinical mastitis and clinical mastitis, respectively. Based on SCC, 46.67 per cent, 38.10 per cent and 15.24 per cent were found to have healthy udder, subclinical mastitis and clinical mastitis, respectively. The genotyping and polymorphism study in selected SNPs in five immune-related genes was done using the PCR-RFLP assay. The SNPs and genes included in this study were rs467234707 of gene EPS15L1, rs433539538of gene PTX3, rs45930378 of gene MAST3, rs480534837 of gene PDGFD and rs800533391 of gene STAB2. Moreover, their association with mastitis susceptibility or resistance in the population was also analyzed. For the SNP rs467234707 of EPS15L1gene, the PCR-RFLP revealed a single banding pattern of 467bp size. Further, all the individuals were of AA genotype. The SNP rs433539538 of the PTX3 gene also revealed a monomorphic pattern with all the animals showing TT genotypes (226bp and 100bp). The SNP rs480534837 of gene PDGFD and SNP rs800533391 of STAB2 gene also showed monomorphic patterns, with GG genotype (254bp and 154bp) for SNP rs480534837 and GG genotype (76bp and 241bp) for SNP rs800533391. However, in the PCR-RFLP assay, the SNP rs45930378 of the MAST3 gene exhibited two genotypes viz.TT and TA in the cattle population under study. The frequencies of TT genotype (108bp and 220bp) and TA genotype (108bp, 220bp and 328bp) were 0.924 and 0.076, respectively and the allele frequencies for T and A were 0.962 and 0.038, respectively. The Chi-square test showed that the locus was in HWE in the populations under study. Moreover, the association study revealed that there was no association between the genotypes and udder health status (subclinical mastitis and healthy udder).ThesisItem Open Access GENOME-WIDE SEARCH FOR GENETIC DIVERSITY, SELECTION SIGNATURES AND BREED SPECIFIC MARKERS IN NATIVE GOAT BREEDS OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2020-12-16) MARYKUTTY THOMAS; Radhika G.Population genomics study was carried out in Attappady Black and Malabari goats, the two native goat breeds of Kerala with objectives of (1) genome wide assessment of genetic diversity (2) genome wide scan for selection signatures (3) detection and validation of breed specific markers (4) identification, validation and association analysis of prolificacy linked candidate SNP markers. Whole genome SNP genotypic data obtained by high throughput genotyping of reference population (24 each of Attappady Black and Malabari goats) using goat SNP50 BeadChip were used for genome wide scans. After quality control, 47,100 polymorphic SNP markers were available for downstream analysis, demonstrating low influence of ascertainment bias. Analysis of autozygosity and demographic history using runs of homozygosity (ROH) revealed mean ± S. E. of ROH length (Mb) as 4.02±0.38 and 6.00±0.53 in Attappady Black and Malabari goats respectively. The higher proportion of short ROH segments (2 to 4 and 4 to 8 Mb) in both breeds denoted ancient inbreeding due to common ancestors between 6 to 25 generations ago. Genomic inbreeding coefficient based on ROH was extremely low and was 0.07 and 0.52 per cent in Attappady and Malabari goats respectively. Investigation of linkage disequilibrium (LD), considering 0.794 million SNP pairs showed low and comparable level of LD (r2=0.07) between adjacent SNP markers (marker interval, 0.17 Gb) in both breeds, indicating the high genetic diversity and low selection pressure in native goats. The LD estimates also denoted the requisite for a denser SNP array to capture useful genomic information for selection and association analysis. Historical effective population size estimated from LD decay was 518 and 371 in Malabari and Attappady Black goats respectively at 17 generations back. Other population genetic indices like minor allele frequency (0.30), observed heteroygosity (0.38), expected heterozygosity (0.39) and FIS (0.03) also supported genetically diverse nature of native goats. Though genetic differentiation among two breeds was low (FST, 0.018), on Principal Component Analysis, first two principal components which accounted for 0.09 proportion of variance, grouped the sampled animals in two distinct breed clusters. Model based clustering using STRUCTURE showed that relative proportions of Attappady Black and Malabari ancestral blocks at k=2 (based on least cross validation error and k =number of sub populations) were 0.93 and 0.07 respectively in the genome of Attappady Black and 0.12 and 0.88 respectively in the genome of Malabari goats. This implied the distinct ancestral influence of two breeds. Selection signature analysis that facilitates detection of genomic regions under natural and artificial selection was performed using outFLANK and hapFLK analysis. The outFLANK analysis that detects ancient selection signals, detected 104 numbers of highly differentiated SNP loci at genome wide levels of significance (p<0.001). Gene ontology analysis using DAVID identified important genes that are related to coat colour (MITF and WNT2) and mammary gland development (FGFR2 and GLI2) in ancient sweep regions. The hapFLK analysis that exposes recent selection signatures yielded seven significant (p<0.005) sweep regions including the genomic area that harbours casein cluster and quantitative trait loci for milk production on chromosome 6. Gene ontology enrichment analysis of 166 putative selective genes that was linked to recent sweep regions using Panther listed significantly (p≤0.05) over-represented 13 Panther pathways that included TGF-beta signalling pathway and GnRH receptor pathway. Development of breed genetic traceability for Attappady Black and Malabari goats could enhance breed profitability and sustainability of its production system. Breed genetic traceability for Attappady Black goats was developed by adopting deterministic approach using breed specific markers. High throughput genome wide SNP genotypic data of reference goat population was used to identify candidate breed specific markers. Nine putative Attappady breed specific markers were selected and were validated for breed specificity by converting them into PCR-RFLP markers and genotyped in large sample goat population. On validation, four markers retained their breed specificity with probability of identification (Pi) ranging from 0.24 to 0.66. Panel of four Attappady Black breed specific markers yielded Pi of 0.92. Similar protocol was adopted for genetic breed traceability of Malabari goats as well. Seven candidate Malabari breed specific markers were selected and further genotyped in large sample goat population by PCR-RFLP. Three candidate markers confirmed their breed specificity on validation, with Pi ranging from 0.21 to 0.56. Panel of three Malabari breed specific markers had Pi of 0.73. Genome wide screening of high throughput genotypic data of reference goat population for candidate SNP markers useful for litter size trait selection in native goats of Kerala identified 49 putative candidate SNP markers. Population genetic analysis of putative candidate markers based on genotypic data of reference goat population revealed that five candidate SNPs were either significantly (p≤0.05) deviated from Hardy Weinberg equilibrium (HWE) or with significant (p≤0.05) genotype frequency differences among two breeds. These five candidate markers that were on SETDB2, SOX6, AHCTF1, FAT4 and USP16 genes were genotyped in large population by PCR-RFLP method and analysed for their association with prolificacy trait. Population genetic analysis based on genotypic data in large population showed that SETDB2, SOX6 and USP16 were significantly (p≤0.05) deviated from HWE denoting potential selection. Association analysis illustrated that genotype of USP16 gene variant had highly significant (p≤0.01) influence on litter size trait of native goats of Kerala. The USP16 gene variant could be a useful SNP marker for litter size enhancement in native goats of Kerala.ThesisItem Open Access GENETIC VARIABILITY OF TOLL LIKE RECEPTOR 4 AND BETA DEFENSIN 4 GENES AND ITS ASSOCIATION WITH SOMATIC CELL SCORE IN CROSSBRED CATTLE OF KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-10-21) KIYEVI G. CHISHI; KIYEVI G. CHISHI; Jamuna Valsalan; Jamuna ValsalanThe main objectives of present study were to identify the single nucleotide variations in exon 3 of TLR4 and exon 1 and 2 of DEFB4 associated with somatic cell score (SCS) and 305 Days Milk Yield (305DMY) and to estimate breeding value in crossbred cattle of Kerala. To assess the genetic variability, PCR-SSCP analysis was done. Blood samples of 200 lactating crossbred cattle were collected from different farms of Kerala Veterinary and Animal Sciences University and field centres of ICAR- FPT scheme, KVASU. Representative samples of each SSCP patterns were sequenced to ascertain the genotypes. The 231 bp fragment of TLR4 (exon 3) was found to polymorphic and two genotypes viz. CC and CD were obtained with frequency 77% and 23% respectively. The frequency of C and D allele in crossbred were found to be 0.88 and 0.12. On sequencing, it revealed that C to T transition had led to one non-synonymous change at 2021th position of ORF. Cattle with CC genotype were more related (p≤0.05) to lower SCS and higher 305 DMY compared to CD genotypes. Monomorphic band patterns were obtained for 192 bp fragment of exon 1 of DEFB4. The 229 bp fragment of exon 2 of DEFB4 was found to be polymorphic and three genotypes viz. PP, QQ and PR were obtained with frequency 54%, 34% and 12%, respectively. The frequency of P, Q and R alleles were found to be 0.60, 0.34 and 0.06, respectively. On sequencing, four non-synonymous SNP (c.121T>A, c.131G>C, c.134T>G and c.188G>C) and one stop-lost SNP (c.191A>G) was obtained. Cattle with PP and QQ genotype were more related (p≤0.05) to lower SCS compared to PR genotypes. Exon 2 of DEFB4 was not significantly associated with 305 DMY. Overall least square mean of SCS and 305 DMY were 4.85 and 2409.35 kg, respectively. The heritability estimates for SCS and 305 DMY were 0.003±0.340 and 0.359±0.076.The average breeding value of 200 crossbred cattle sired by 78 bulls for SCS was 4.16 and for 305DMY was 2743.60 kg. TLR4 and DEFB4 can be used as potential candidate genes for mastitis resistance and SCS would be included in the selection criteria of crossbred cattle of Kerala.
