Identification of functional single nucleotide polymorphisms in the porcine SLC6A4 gene associated with aggressive behavior in weaned pigs after mixing

Lay Summary The current study identified the functional single nucleotide polymorphisms for the porcine SLC6A4 (serotonin transporter) gene associated with aggressive behavior of pigs after mixing. We investigated the underlying molecular regulation mechanism of those functional variations. SNP rs345058216 (c.-1694C > G) affects binding of the transcription factor MAZ (MYC associated zinc finger protein) to the core promoter region of the SLC6A4 gene, whereas SNP rs332335871 (c.*1586G > A) affects the binding of miR-671-5p to the 3MODIFIER LETTER PRIME-UTR of SLC6A4 gene. These results suggest that rs345058216 and rs332335871 could be used as candidate molecular markers for aggressive behavior of pigs after mixing. miR-671-5p and transcription factor MAZ mediated porcine SLC6A4 expression level, providing the possible molecular mechanism of aggressive behavior regulation. Variation in genes of the serotonergic system influences aggressive behavior by affecting serotonin levels in the central and cortical nervous system. SLC6A4 (serotonin transporter) is a master regulator of 5-HT signaling and involved in the regulation of aggressive behavior in humans and rodents. To identify potential functional single nucleotide polymorphisms (SNPs) for the porcine SLC6A4 gene associated with aggressive behavior, a total of 500 pigs (268 barrows and 232 gilts) were selected and mixed in 51 pens. Their behavior was recorded and observed for 72 h after mixing. Based on a composite aggressive score (CAS), the most aggressive and the least aggressive pigs within each pen were selected separately (a total of 204 pigs). Ear tissue was sampled to extract genomic DNA. Eight SNPs in the 5MODIFIER LETTER PRIME-flanking region, coding region, and 3MODIFIER LETTER PRIME-untranslated region (3MODIFIER LETTER PRIME-UTR) of SLC6A4 were genotyped, of which 6 SNPs had significant differences (P < 0.05) in allele frequency between the most aggressive and least aggressive pigs. Luciferase activity was greater in plasmids of genotype GG than plasmids of genotype CC of rs345058216 (P < 0.01). Computational analysis nominated MAZ as putative transcription factor (TF) with higher probability to bind the SLC6A4 promoter at the SNP (rs345058216) site. Also, we demonstrated that MAZ overexpression modulates SLC6A4 promoter activity in allele-specific manner with an in vitro assay. In addition, we demonstrated that SLC6A4 was a direct target of miR-671-5p. The dual luciferase reporter gene assay and cell transfection were performed to examine the role of miR-671-5p in regulating SLC6A4 expression. The luciferase assays revealed that the SNP rs332335871 affects regulation of miR-671-5p in SLC6A4 expression. After overexpression of miR-671-5p in porcine primary neural cells, the SLC6A4 mRNA levels can be significantly reduced. In conclusion, we here found that miR-671-5p and MAZ mediated porcine SLC6A4 expression level, which provides the possible molecular mechanism of aggressive behavior.