Integration ATAC-Seq and RNA-Seq Analysis of Mammary Placodes in Erhualian and Bamaxiang Pigs Identified Candidate Genes Influencing Pig Teat Number Variation

Abstract

Teat number is an important economic trait in pigs, affecting piglet health and survival. While numerous GWAS have identified candidate genes for teat number in Duroc, Landrace, and Large White pigs, the causal genes remain unclear, largely due to a lack of transcriptional and epigenetic studies on mammary placodes in 26-day-old pig embryos, a critical stage for teat formation. Erhualian and Bamaxiang pigs, derived from Chinese wild boars, serve as ideal models for studying genetic variation in teat number, with Erhualian averaging nearly 20 teats and Bamaxiang around 10. This study collected mammary placodes from these breeds at embryonic day 26 and performed ATAC-seq and RNA-seq to explore chromatin accessibility and gene expression. Results indicate widespread chromatin accessibility across mammary placodes. Of the 30,806 open chromatin regions (OCRs) identified, only 30 showed breed-specific differences, suggesting conserved accessibility patterns across breeds. OCRs are enriched in intergenic and promoter regions, and significantly overlap with QTL intervals for teat number. RNA-seq revealed 4432 differentially expressed genes between the two breeds, including WTN10B and WNT6, indicating breed-specific gene expression patterns. Combining ATAC-seq and RNA-seq results identified three protein-coding genes (ENSSSCG00000031037, ENSSSCG00000032042, and ENSSSCG00000039180) near 48.80 Mb on SSC14 that are associated with teat number according to pheWAS and GWAS data. FISH analysis confirmed that ENSSSCG00000031037 is specifically expressed in epithelial cells of mammary placodes, and this region is under stronger selection in Erhualian pigs, suggesting its role in the breed's higher teat number. In conclusion, this study integrates ATAC-seq and RNA-seq to construct a chromatin accessibility and gene expression map of pig mammary placodes. It identifies ENSSSCG00000031037, ENSSSCG00000032042, and ENSSSCG00000039180 as key candidate genes driving teat number differences, providing insights for understanding QTL intervals and identifying causal genes linked to teat number in pigs.