Screening For Molecular Genetic Markers Of Aggressive Behavior In Weaned Pigs After Mixing

It is a biological characteristic of pigs that a stable social hierarchy was maintained.Regrouping piglets after weaning is a common procedure in commercial pig farming to create homogenous groups and equalize competition.After mixing,aggressive behavior of pigs increased notably.To reduce aggressive behavior in pigs,a number of practical interventions have been explored,but the aggression after mixing is still unavoidable.The aggressive behavior of pigs after mixing,as an animal welfare concern,causes injury in the form of skin lesions,which affect growth performance,carcass and meat quality of pigs.Previous studies have shown that aggressive behavior is a heritable trait in pigs.Therefore,identification of molecular genetic markers of aggressive behavior is an important approach to reduce pig aggression.Candidate genes of aggressive behavior can be used in molecular breeding of pigs.A number of genes related to the serotonin neurotransmitter pathway,such as Monoamine oxidases A and B(MAOA and MAOB),Serotonin transporter(SLC6A4),Tryptophan hydroxylases 1 and 2(TPH1 and TPH2),Catechol-O-methyl transferase(COMT)and Dopamine-beta-hydroxylase(DBH),are implicated to anxiety and stress response of pigs.Identification of molecular genetic markers is important for reduction of aggression in pigs.Chinese indigenous pigs are more docile than European pigs,the difference on aggression between pig breeds indicates that the clear visible expression of aggression is heritable,and could be improved by genetic selection.Suhuai pigs have a mixed lineage containing 25%Chinese Huai pig breed and 75%Yorkshire pig breed.Exclusive single nucleotide polymorphisms(SNPs)in either Chinese domestic pigs or European pigs related to aggressive behavior could be identified on Suhuai pigs.The objective of this study is to identify the functional SNPs in porcine MAOA,5-HT1B and COMT gene related to aggression by association analyses between polymorphisms of genes and the scores of aggressive behavior parameters in weaned pigs after mixing.In the present study,a total of 500 weaned pigs were selected and blocked by sex and body weight.Nine or ten weaned pigs from different original litters were mixed in each pen.For the individually behavioral observation,pigs in each pen were marked numbers using a paint spraying on the backs and both sides of the body.Aggressive behavior score of each pig was assessed by observing the video and recording the frequency and time of fighting behaviors of each pig within 36 h after mixing in each pen.The most aggressive and the most docile pigs were selected in each pen by assessing their compositive aggressive scores(CAS).The ear tissue was sampled to extract the genomic DNA for genetic polymorphism analysis.Bioinformatics analysis was performed to predict the promoter,transcriptional binding sites and methylation sites of porcine candidate gene.Potential SNPs were identified between aggressive and submissive pig groups.The association analysis was performed between potential SNPs in porcine candidate gene and pig aggressive behaviors after mixing.The promoter activity of candidate gene MAOA were identified at the molecular level by double fluorescence activity test.1 Bioinformatics analysis of candidate genes related to pigs’ aggressive behaviorTo study the potential SNPs in the candidate genes of pig aggression,the promoter region,transcription factor binding site and CpG island of porcine MAOA,5-HT1B and COMT gene were predicted by the bioinformatics websites.A total of four core promoter regions and two transcription initiation sites were predicted in the promoter region of MAOA gene,ten SNPs were studied in this region previously.These SNP sites would lead to changes of transcription factor binding sites.Three core promoter regions and two transcription initiation sites were obtained in porcine 5-HT1B gene.Five SNPs were located in the predicted promoter region and lead to changes of transcription factor binding sites.Only one core promoter was predicted in the COMT gene promoter region.These results would provide important information for the studies of association analysis between gene polymorphism and aggressive behavior in pigs.2 Association analysis between gene polymorphism and aggressive behavior in pigsTo screen SNPs in porcine MAOA,5-HT1B and COMT gene associated with aggression,a total of 500 weaned Suhuai pigs from 76 litters multiparous sows were selected for mixing in this study.Behavior of pigs was recorded for 72 h after mixing using a digital video recording system.Ear tissues were collected for each pig.Pig aggressive behavior was assessed by observing the video and recording the frequency and time of fighting behavior for each pig in a pen in 36h after mixing.The most two aggressive pigs and the most two submissive pigs in each pen were selected.A total of 102 aggressive pigs and 102 submissive pigs were selected for the association analyses.To detect the porcine MAOA,5-HT1B and COMT gene polymorphisms in 5’-UTR,exons and 3’-UTR,targeted fragments were amplified by PCR and sequenced.Eight significant SNPs(P<0.05)were identified in gene MAOA.The results of linkage disequilibrium analyses indicated that four SNPs(rs321936011,rs331624976,rs346245147 and rs346324437)showed linkage inheritance,which results in five haplotypes(GCAA,ACGA,GCGA,ATGG and ACGG/GTAA).Pigs with wild genotypes were more aggressive than pigs with the mutant genotypes in four linked SNPs(P<0.05).The pigs with haplotype GCAA had greater aggressive indexes compared to pigs with haplotype GCGA and ATGG(P<0.05).Four potential SNPs were identified in porcine 5-HT1B and they may asscociated with aggressive behavior.Two SNPs in porcine COMT were identified and no association were found between these polymorphisms and aggressive behavior in pigs.3 Promoter identification and expression of different genotypes in promoter region of porcine MAOA gene in vitroTo study the promoter region and c.-30 G>A of porcine MAOA gene,the promoter region of porcine MAOA gene was cloned according to the results of bioinformatics analysis.Constructs containing variable length of truncated pig MAOA promoter(P1,P2,P3,P4,P5,and P6)and specific regions containing SNP c.-30 G>A(GG,AA)were amplified by pGL3-basic dual luciferase report gene system.PGL3-control was set as positive control.Transfections in 293T cells were assayed for luciferase activity using the Promega Dual Luciferase Assay system.For the MAOA promoter fragments,the luciferase activities of constructs P6,P5,P4 and P3 were greater than those of constructs P2 and P1(P<0.05).These results have shown that the core promoter region of the MAOA gene is located between-679 bp to-400 bp.To detected the in vitro expression of different genotypes,the significant SNP c.-30 G>A,one of the four linked SNPs,were selected.The luciferase reporter constructs of GG genotype had lower(P=0.031)luciferase activities compared to those of AA genotype.The luciferase activities of constructs with GG and AA genotypes were greater than that of the negative control pGL3-basic group(P<0.01)but less than that of the pGL3-control group(P<0.01).The c.-30 G>A may probably affect the expression of gene MAOA and the mutant individuals would be more submissive.It could be a reference for the association between porcine MAOA gene and aggressive behavior in pigs.