Identification and validation of a regulatory mutation upstream of the BMP2 gene associated with carcass length in pigs

Abstract

Background: Carcass length is very important for body size and meat production for swine, thus understanding the genetic mechanisms that underly this trait is of great significance in genetic improvement programs for pigs. Although many quantitative trait loci (QTL) have been detected in pigs, very few have been fine-mapped to the level of the causal mutations. The aim of this study was to identify potential causal single nucleotide polymorphisms (SNPs) for carcass length by integrating a genome-wide association study (GWAS) and functional assays.

Results: Here, we present a GWAS in a commercial Duroc × (Landrace × Yorkshire) (DLY) population that reveals a prominent association signal (P = 4.49E-07) on pig chromosome 17 for carcass length, which was further validated in two other DLY populations. Within the detected 1 Mb region, the BMP2 gene stood out as the most likely causal candidate because of its functions in bone growth and development. Whole-genome gene expression studies showed that the BMP2 gene was differentially expressed in the cartilage tissues of pigs with extreme carcass length. Then, we genotyped an additional 267 SNPs in 500 selected DLY pigs, followed by further whole-genome SNP imputation, combined with deep genome resequencing data on multiple pig breeds. Reassociation analyses using genotyped and imputed SNP data revealed that the rs320706814 SNP, located approximately 123 kb upstream of the BMP2 gene, was the strongest candidate causal mutation, with a large association with carcass length, with a ~ 4.2 cm difference in length across all three DLY populations (N = 1501; P = 3.66E-29). This SNP segregated in all parental lines of the DLY (Duroc, Large White and Landrace) and was also associated with a significant effect on body length in 299 pure Yorkshire pigs (P = 9.2E-4), which indicates that it has a major value for commercial breeding. Functional assays showed that this SNP is likely located within an enhancer and may affect the binding affinity of transcription factors, thereby regulating BMP2 gene expression.

Conclusions: Taken together, these results suggest that the rs320706814 SNP on pig chromosome 17 is a putative causal mutation for carcass length in the widely used DLY pigs and has great value in breeding for body size in pigs.

Fig. 1

Genome-wide association results for carcass length in 686 DLY pigs. a Manhattan plot, with the genome-wide significance threshold at − log₁₀P > 6 indicated by the dotted line. The top SNP rs80965549 is located in the intergenic region upstream of the BMP2 gene. b Quantile–quantile (QQ) plot. The red line represents the 95% confidence level for the null hypothesis of no association between SNPs and the trait. The black dots represent the P values of all SNPs. c Effect of the top SNP rs80965549 on carcass length.

Fig. 2

Fine-mapping of the SSC17 QTL for carcass length and detection of putative causal variants. a LocusZoom plot for the regional association analysis with an additional 196 SNPs that were genotyped and passed quality control. Different colors indicate different linkage disequilibrium values of the top SNP with other SNPs. b LocusZoom plot for the regional association analysis with imputed SNP genotypes. c LocusZoom plot for the association analysis conditioning on the lead SNP rs320706814, depicting the secondary association signal at SNP rs318639793. d Bayesian fine-mapping of the QTL. The 95% credible set of loci comprised only one variant (rs320706814), located at 15,626,425 bp, with the maximum posterior probability of causality

Fig. 3

Comparison of the effects of four haplotypes on carcass length. The haplotypes are composed of the top seven SNPs that were most significantly associated with carcass length, specifically (in order from 1 to 7), rs342071386, rs333646524, rs345818757, rs323371124, rs336843722, rs343796635, and rs320706814. The linkage disequilibrium (r²) of the second-most significant SNP, #1, rs342071386, with other SNPs (including the most significant SNP, #7, rs320706814) are presented. Two effect-size categories (with favorable, Q, or unfavorable, q, effects on carcass length), with haplotype frequencies in parentheses

Fig. 4

EMSA and transfection assays assessing the significance of the SNP rs320706814 (C > T substitution) on gene expression. a, b EMSA using nuclear extracts from MC3T3-E1 and MLO-Y4 cells. c, d Luciferase assays of the rs320706814 reporter constructs in MC3T3-E1 and MLO-Y4 cells. Three independent experiments were performed in two cell lines. Three replicates were performed for each experiment. The means ± s.d. and comparative significance of each group are shown