Study of haplotype blocks structure and haplotype based genome scan in autosomal chromosomes to body weight gain trait in Zandi sheep breed

Average daily gain (ADG) is the most economically important trait in sheep industry.
In genome wide association study and genomic selection, determining of extent and level of linkage
disequilibrium (LD) and haplotype block structure are critical in sample size and marker density. Haplotype blocks are defined as long stretches of SNPs along a chromosome that have low recombination rates, which characterized by relatively few haplotypes. Understanding haplotype structure in genome can greatly facilitate LD analysis. Haplotype-based association analysis can offer a powerful approach for mapping functional genes (Gabriel et al., 2002). Therefore, the objective of this research were to study LD pattern, determine haplotype block structure and genome wide haplotype association study in Zandi sheep for identifying the genomic region associated with pre-weaning (AGW) and post-weaning daily gain (PWG). A total of 96 Iranian Zandi sheep were used in the study. The following two traits were analyzed: pre-weaning daily gain and post-weaning daily gain. Animals were genotyped using 50 K SNPChip panel. Quality control of the genotype data consisted in removing SNPs with a call rate less than 95 %, SNPs with a minor allele frequency (MAF) less than 5 %, individual with more than 10 % missing genotypes, and SNPs that deviated strongly from Hardy–Weinberg equilibrium (P < 10−6). LD between all pairs were calculated with r2 by PLINK v1.07. Haplotype blocks were identified on base algorithm Gabriel et al. (2002) for all autosomes, using Haploview software (Barrett et al. 2005), based on estimates of D' for all pairwise combinations of SNPs within each chromosome. Following Gabriel et al. (2002), a pair of SNPs is defined to be in “strong LD” if the upper 95% confidence bound of D' is > 0.98 (consistent with no historical recombination) and the lower bound is > 0.7. Using the Haploview default values for blocks (Gabriel et al. 2002), a haplotype block is defined as a region over which 95 % of informative SNP pairs show “strong LD”. The PLINK was used to generate the matrix using the GLM algorithm. In this analysis, because of the previous selection history of the flock, it was important to identify and correct for population stratification. To evaluate whether estimates were overinflated, we used the genomic inflation factor λ using the PLINK software (Purcell et al., 2007). We also assessed their deviation from the expected distribution of no SNPs being associated with the trait of interest using a quantile-quantile (Q-Q) plot, which is commonly used to analyze population stratification in GWAS. We use SNPEVG tool to show the (Q-Q) plot. The Bonferroni method was used to adjust for multiple testing from the number of SNP loci detected. We declared a significant SNP at the genome-wide significance level if the raw P-value was, 0.05/N, here N is the number of SNP loci tested in the analyses. Therefore, for each trait, the threshold P-value for declaring genome-wide significance was (0.05)/40,879=1.2×10-6. The exact positions of the annotated genes were extracted from the latest sheep genome Oar_v4.0 assembly along with the NCBI annotation release 102 of the sheep genome. To investigate whether the
significant SNPs detected in this study were within the range of previously identified QTL for relevant traits, we searched for meat or production QTL in the Animal QTLdb within a 1-Mb region on both sides of each significant haplotype. After quality control, 2 individuals were excluded, leaving 94 sheep for the association analysis. Additionally, we removed 1070 SNPs with call rates less than 95% and 7717 SNPs with MAF less than 0.05. A total of 40,879 SNPs passed these quality-control filters and were retained in the dataset. These SNPs were distributed across 26 autosomes, with the number of SNPs per chromosome ranging from 747 to 5694, and with a mean distance between adjacent SNPs ranging from 50.4 to 68.7 kb. Also, in this study, the extent of LD was 40 kb with r2=0.2. Overally, 1472 blocks were observed in the 7.58% of all SNPs were classified into haplotype blocks, covering 1.45% of the total autosomal genome size. The results showed a reduction in LD level with the increase in distance between markers. The average pre-weaning daily gain was 0.197±0.04 kg with an individual sheep range of 0.08-0.34 kg. Average post-weaning daily gain was 0.126±0.07 kg with an individual sheep range of 0.03-0.41 kg. The result from genomic control showed weak population stratification for AGW and PGW in between population of Zandi sheep. The genomic inflation factors (λgc) for the two traits were equal to 1.039 and 1.073, for AGW and PGW, respectively. However, as the Q-Q plots clearly show, there is no evidence of any systematic bias (λgc˂1.1) due to population structure or analytical approach in our case. Considering the significant fixed effects in the genomic wide association analysis, four haplotypes on chromosomes 3, 5, 6 and 7 identified to affect significantly AGW and PWG traits. The results of this study could provide a suite of novel SNP markers and candidate genes associated with growth traits and hence, may play an important role for understanding the biology of average daily gain in sheep.