PHYLOGENETIC STUDIES OF SHEEP BREEDS OF KHYBER PAKHTUNKHWA THROUGH DNA MARKERS

Abstract

This dissertation communicate the results of our scientific endeavor on molecular characterisation and phylogenetic analysis of sheep breeds present in Khyber Paktunkhwa Province of Pakistan. The study is based upon 487 individuals of 10 local sheep breeds of Khyber Paktunkhwa, selected for blood sampling and genotyped with mitochondrial D-loop and microsatellite markers (STR’s). Analyses of the DNA data showed the presence of three haplogroups (HapA, HapB and HapC). The 124 sequences out of 159 were clustered with Haplogroup A (77.99%), 30 with haplogroup B (18.87%) and 5 with haplogroup C (3.14%). Moreover the mtDNA dloop sequenced data exhibited 106 haplotypes with 252 polymorphic sites. The nucleotide diversity and haplotype diversity was found 0.03250±0.00315 and 0.9854±0.0041, respectively. The Phylogenetic analysis showed that most of the breeds clustered with two wild sheep i-e: Ovis ammon and Ovis orientalis anatolica. The mean diversity for Shannon’s index (SHUA) was observed 0.073-0.212. The AMOVA analysis for mtDNA and microsatellite data revealed that genetic diversity was mainly observed within populations (97.84 % and 96.69%) while a small portion of the total diversity was noted among populations (2.16% and 3.31%), respectively. The Chi-square test for Hardy Weinberg Equilibrium (HWE) showed significant deviation in 21 of 80 (26.25 %) test loci and 2.63 breeds have deviation from HWE for each locus. Garza and Wlliamson statistics showed highest value (0.357±0.194) for Rambouillet and lowest (0.256±0.173) for Damani sheep and was subjected to bottleneck effect and hence reduction in population size. The clustering pattern on STR based PCoA plot exhibited close association with mitochondrial DNA based clustering pattern. Moreover, it was observed that most of sheep breeds were found scattered on the plot. The mean value of nucleotide variances (D) and the mean value of nucleotide replacements per spot between breeds (Dxy) showed the lowest distance between Gauder and Waziri (D: 11.429 and Dxy: 0.02037) while the highest distances were recorded between Damani and Kaghani (D: 34.535 and Dxy: 0.05552). Analysis of 185 animals STR data revealed 121 alleles for studied loci, with a mean 15.13±8.27 alleles per locus, ranging from 31 in INRA063 to 6 in D5S2. The number of alleles (NA) ranged from 5.00±2.27 in Rambouillet to 8.00±4.34 in Hashtnagri with a mean of 6.51±3.06 across all loci. It was observed that for all loci observed heterozygosity (HO) was higher than expected heterozygosity (HE). The (HE) and (HO) as measures of genetic diversity for all breed was 0.754 and 0.996 alleles, respectively. The mean coefficient of inbreeding ((FIS) was recorded -0.399 while the highest values (-0.559) was recored for Rambouillet and lowest (-0.304) for Afghani sheep. The Polymorphic information contant (PIC) values were noted from 0.933 (AF) to 0.955 (WZ). The overall Fu’s FS test value was -79.766. The values for different studied sheep breeds ranged from -2.756 in Waziri to 6.740 in Ramghani; most of the FST values were negative. The overall Tajima’s D value was recorded as -2.355. The mean values for coefficient of inbreeding among population (FIS), within populations (FIT) and average genetic differentiation (FST) were -0.191, -0.089 and 0.086, respectively for all loci. The values for Inbreeding coefficient within individuals (GIS) and coefficient of gene differentiation (GST) were noted as -0.154 and 0.050, respectively for all loci. The negative value of FIS, FIT and GIS indicated heterozygote excess or more out breeding compared with HWE expectations. On the basis of current findings it is concluded that sheep breeds of Khyber Pakhunkhwa are more diverse and the selected genetic stock can be utilized for obtaining valuable products. Successful completion of this research project has not only established a strong baseline of molecular taxonomy of sheep but have also elaborateed the population structure of sheep breeds of the region. The first hand information presented here has filled the scientific gape of the knowledge of molecular biology of the native breeds of sheep, on the other hand our findings can be utilized in breeding superior breeds and establishing sustainable ways for conservation and sustainable development of sheep in Pakistan.