Radhika G.MARYKUTTY THOMAS2023-02-172023-02-172020-12-16https://krishikosh.egranth.ac.in/handle/1/5810193981Submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Animal Genetics and BreedingPopulation 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.EnglishThesis