IDENTIFICATION, MAPPING, AND SCREENING OF HARMFUL DNA MUTATIONS IN SAHIWAL CATTLE
| dc.contributor.advisor | S.K. NIRANJAN | |
| dc.contributor.author | GAURAV PATEL | |
| dc.date.accessioned | 2023-11-21T12:32:03Z | |
| dc.date.available | 2023-11-21T12:32:03Z | |
| dc.date.issued | 2022 | |
| dc.description.abstract | For the identification of deleterious mutations in cattle, the use of high throughput sequencing technology and related computational algorithms has become more pertinent, specifically in absence of genetic disease records or in vivo trials. The present study was carried out to identify the harmful mutations and to screen selected harmful mutations through PCR-based tests in Sahiwal cattle. Genomic ddRAD sequence data of twelve Sahiwal cattle were analyzed for the identification of harmful mutations, in silico. After initial quality control and discarding poor quality reads or raw sequence data, a total of 91.10 million reads were obtained. These reads were further aligned with the cattle (Bos taurus) reference genome ARS-UCD1.2 for variant calling and SNP identification in the Sahiwal genome with an average alignment rate of 97.5 percent. In the downstream analysis, a total of 1,62,377 SNPs and 14056 Indels were identified at read depth (RD10) in the Sahiwal cattle genome. The SNPs obtained at RD10 were further annotated with Ensemble - Variant Effect Predictor (VEP) and a total of 430 missense variants were identified. A total of 45 missense variants were found to be deleterious based on SIFT score obtained from the VEP. For further validation of the deleteriousness, all 45 deleterious nsSNPs, predicted with high confidence by SIFT, were analysed by PANTHER, PROVEAN, and PolyPhen-2. Total 18, 21, and 32 were found to be damaging through PANTHER, PROVEAN (Score ≤ -2.5), and PolyPhen-2.0, respectively. Further, using the consensus classifier of PredictSNP, the predictions of PredictSNP, MAPP, PhD-SNP, and SNAP were also obtained to identify deleterious nsSNPs, showing 29, 21, 20, and 19 nsSNPs as deleterious, respectively by these tools. Based on the results from all tools, four nsSNPs- C40Y, A242D, N234K, and D416Y, mapped on WDR4, ZNF280B, CCT6A, and CYP2C87 genes, were filtered out as common. Through I-Mutant2.0 and MUpro tools, change in protein stability was assessed, which revealed decrease in protein stability in almost all deleterious mutations. Using Project Hope, the effect of missense mutation on the protein was assessed, which revealed steric interference in ligand binding in C40Y of WDR4, repulsion of ligands, or other residues in A242D (ZNF280B), N234K (CCT6A) and D416 (CYP2C87), possibly. Differences in size and hydrophobicity of amino acids were found between wild and mutant structures for all four deleterious nsSNPs. Further, five non-sense mutations, causing termination of amino acid chain and one start lost mutation were identified in Sahiwal. With help of the Mapgene2chrom tool, these identified variants were represented chromosome-wise and compared with Bos taurus variants documented on Online Mendelian Inheritance in Animal (OMIA). In Bos taurus, maximum missense and nonsense deleterious mutations were found to be located at BTA2, followed by BTA19. Based on the associated function and effect of mutations of that gene in humans, two mutations, C40Y in the WDR4 gene and A242D in the ZNF280B gene were selected for genotyping. For A242D (ZNF280B) and for C40Y (WDR4) variants, PCR-RFLP (AluI enzyme) and allele-specific PCR assays were developed. Using PCR based assays, screening of 120 Sahiwal cattle was carried out for these two nsSNPs. After screening of A240D (ZNF280B), 104 animals were homozygous for the wild allele, 14 heterozygous, and 2 homozygous for the altered allele. At C40Y (WDR4) locus, 113 animals were homozygous for the wild allele, 7 heterozygous, and zero homozygous for the altered allele. However, no significant difference was observed among observed and expected genotype frequencies for both genotyped mutations, using Chi-square based statistics, indicating the absence of any natural selection against these mutants in the Sahiwal cattle. The study predicts four deleterious SNPs in Sahiwal (Bos indicus) cattle, which may be further studied in vitro and in vivo to validate their deleterious effects. Further, a larger population of Sahiwal cattle may be genotyped for these mutants to assess their purifying selection and genetic load in the population, if any. | |
| dc.identifier.uri | https://krishikosh.egranth.ac.in/handle/1/5810201632 | |
| dc.language.iso | English | |
| dc.pages | 77 p. | |
| dc.publisher | ICAR-NDRI, KARNAL | |
| dc.sub | Animal Genetics and Breeding | |
| dc.theme | ANIMAL GENETICS & BREEDING | |
| dc.these.type | M.V.Sc. | |
| dc.title | IDENTIFICATION, MAPPING, AND SCREENING OF HARMFUL DNA MUTATIONS IN SAHIWAL CATTLE | |
| dc.type | Thesis |