The candidate gene OLFML2A possibly contributed to the variation of total number of teats in Meishan and Erhualian pigs by influencing the formation of mammary placodes.

The teat number trait in pigs has significant economic value, with Meishan and Erhualian pigs having the highest counts globally. To uncover the genetic basis of teat number variation, this study employed genome re-sequencing for a total of 1 119 individuals. Genome-wide association study (GWAS), meta-analysis, and Bayesian fine mapping were conducted. The GWAS from two breeds revealed multiple significant quantitative trait locus (QTL) for total teat number (TNT) mainly on SSC1, SSC5, SSC10, and others. Bayesian fine mapping targeted the most significant QTL in both Meishan pig GWAS and meta-analysis, mapping it to SSC1: 264 609 542 - 266 079 236 bp. Within this QTL, eighteen protein-coding genes were annotated. Bayesian fine mapping was performed for the second most significant QTL in meta-analysis, which also was the most significant QTL in Erhualian pig GWAS, identifying its mapping at SSC5: 58 228 193 - 59 198 414 bp. One protein-coding gene was annotated within this QTL. After determining the anatomical location of the mammary placodes, RNA-seq results from mammary placodes of extremely high and low 26-day-old Erhualian pig embryos were integrated, and found that only OLFML2A showed significant differential expression among these nineteen candidate genes. OLFML2A was primarily enriched in the extracellular matrix and extracellular matrix organisation pathways, which may play a role in the interaction between epithelial and stromal cells in mammary placodes. In addition, the utilisation of PigGTEx data for transcriptome-wide association study revealed a significant association between the expression level of OLFML2A in the placenta and TNT in Meishan pig. The results of phenome-wide association study further validate the significant association of OLFML2A with teat number traits in lean-type commercial pigs. Molecular validation experiments confirmed that OLFML2A specifically expresses mRNA and protein in mammary placodes. Reverse transcription-quantitative PCR results further confirmed significant differences in gene expression in the mammary placodes between extremely high and low 26-day-old Erhualian pig embryos, with the high group showing significantly higher expression levels. Differential expression analysis of mammary placodes from different litters further confirmed that the differential expression of OLFML2A was not caused by the litter effect of the sows. After integrating multiple omics, we have tentatively identified the OLFML2A gene as a potential causal gene responsible for teat number variation in Meishan and Erhualian pigs. This gene could potentially influence the development of mammary placodes in Meishan and Erhualian pigs, consequently influencing the phenotype of teat number.