ZOOLOGICAL RESEARCH Genomic and transcriptomic analyses reveal selection of genes for puberty in Bama Xiang pigs YangYang1,2,AdeniyiC.Adeola1,Hai-BingXie1,*,Ya-PingZhang1,2,* 1StateKeyLaboratoryofGeneticResourcesandEvolution,YunnanLaboratoryofMolecularBiologyofDomesticAnimals,KunmingInstitute ofZoology,ChineseAcademyofSciences,KunmingYunnan650204,China 2KunmingCollegeofLifeScience,UniversityofChineseAcademyofSciences,KunmingYunnan650204,China ABSTRACT fivemonthsobservedinmostotherEastAsiandomesticpigs TheBamaXiangpig(BMX)isafamousearly-maturing (China National Commission of Animal Genetic Resources, 2011). Thus, the BMX pig provides an important model to Chinese indigenous breed with a two-end black coat. study early maturity and pigmentation evolution during pig To uncover the genetic basis of the BMX phenotype, domestication. weconductedcomparativegenomicanalysesbetween Inthisstudy,weperformedgenomicscreeningofsignatures BMX and East Asian wild boars and Laiwu pigs, of positive selection in the BMX pig in comparison with those respectively. Genes under positive selection were oftheEastAsianwildboar(EAW)andLaiwu(LWU)pigs. The enrichedinpathwaysassociatedwithgonadalhormone LWU pig has a normal maturity process (at six months) and blackcoatcolor(ChinaNationalCommissionofAnimalGenetic andmelaninsynthesis,consistentwiththephenotypic Resources,2011),butsharesthesamedomesticationoriginas changesobservedduringdevelopmentinBMXpigs. We thatoftheBMXpigfromEastAsianwildboars(Wuetal.,2007). alsoperformeddifferentiallyexpressedgeneanalysis Incombinationwithtranscriptomicanalysis,weidentifiedgenetic basedonRNA-seqdatafrompituitarytissuesofBMX variations that were putatively associated with the maturity and and Large White pigs. The CTTNBP2NL, FRS2, pigment phenotype in the BMX pig. The primary goal of this KANK4, and KATNAL1 genes were under selection studywastoexplorethe(1)genomicevolutionarypatternshaped by development of the BMX pig, and (2) genetic variations and exhibited expressional changes in the pituitary associatedwithselectionforearlymaturityandcoatcolorinthe tissue, which may affect BMX pig puberty. Our study BMXpig. demonstrated the positive selection of early maturity MATERIALSANDMETHODS in the development of BMX pigs and advances our knowledgeontheroleofregulatoryelementsinpuberty SamplecollectionandresequencingofgenomicDNA evolutioninpigs. We downloaded the genome resequencing data of six BMX, six LWU, and 13 EAW pigs from the NCBI database Keywords: Puberty; Bama Xiang pig; Pituitary; (Supplementary TableS1),as reported from earlierstudies Differentiallyexpressedgenes INTRODUCTION Received: 30March2018;Accepted: 10April2018;Online: 19June Pigsareimportantproteinfoodsourceandgloballydistributed animaldomesticatedfromEurasianwildboars9000yearsago 2018 (Larson et al., 2005). During their long domestication history, Foundationitems:ThisworkwassupportedbygrantsfromtheNational pigshaveexhibitedabundantphenotypicvariationincoatcolor, NaturalScienceFoundationofChina(31472000),National973Program reproduction, and growth. This phenotypic diversity provides of China (2013CB835203), and Animal Branch of the Germplasm valuable animal models for genetic studies (Ciobanu et al., BankofWildSpecies,ChineseAcademyofSciences(LargeResearch 2001;Rubinetal.,2012). InfrastructureFunding) The Bama Xiang pig (BMX) from Guangxi Zhuang AutonomousRegionisawell-knownvarietynotedforitsearly *Corresponding authors, E-mail: [email protected]; zhangyp maturity and two-end black pigmentation. The female BMX @mail.kiz.ac.cn pig matures at about three months, in sharp contrast to the DOI:10.24272/j.issn.2095-8137.2018.068 424 SciencePress ZoologicalResearch39(6):424–430,2018 (Ai et al., 2015; Groenenet al., 2012; Li et al., 2013; Rubin six pituitaries (GSA number: CRA000876) were used for et al., 2012). Sequence reads were filtered by removing comparativeanalysis.TheRNA-seqcleanreadsweremapped adaptors and low-quality bases using Trimmomatic (Bolger et onto the Sus scrofa 10.2 genome using Tophat2 (Kim et al., al.,2014). GenomicreadswerealignedtotheSusscrofa10.2 2013).AnewmergedGTFannotationfilewasgeneratedusing genome (Groenenet al., 2012) with the BWA program (Li & CufflinksandCuffmerge(Trapnelletal.,2010). Durbin, 2010). Alignment was performed with a maximum of Differentially expressed genes (DEGs) were identified by sixmismatchesallowedforeach100-bpfragment. DESeq2 (Love et al., 2014). As DESeq2 required a raw read count table as input, we obtained the raw read count using Singlenucleotidepolymorphism(SNP)callingandannotation HTseq(Andersetal.,2014).AnFDRof<0.05wasusedasthe SNP calling was performed with the DNA sequencing data cutoffforDEGs. TheClueGOplugininCytoscape(Mlecniket of the 25 pig genomes using HaplotypeCaller in the GATK al.,2018)wasusedforgeneontology(GO)enrichmentanalysis. toolkit (McKenna et al., 2010). Only polymorphic SNPs were GOtermswithaP-valueof<0.05weredefinedasenriched. included in this analysis. VariantFiltration and SelectVariants wereusedtofilterSNPsbyqualityscore(QUAL<20)anddepth RESULTS ofcoverage(DP<10). SNPswereannotatedwiththeGTFfile SelectivesignaturesinBMXpiggenomes downloaded from the NCBI website (https://www.ncbi.nlm.nih. The whole genomes of six BMX, 13 EAW, and six LWU gov/). Forannotation, SNPswereclassifiedbytheirlocations pigs were used to identify genomic variants underlying the in protein-coding (synonymous or non-synonymous), intronic, developmentofBMXpigs.Genomicreadswerequalityfiltered intergenic,anduntranslatedregion(UTR)sequences. and mapped to the pig reference genome (Sus scrofa 10.2) Selectivesweepdetection (see Materials and Methods). A total of 43.5 million SNPs Populationfixationindex(F )(Weir&Cockerham,1984),cross were identified in the 25 individuals. To identify signatures of ST populationextendedhaplotypehomozygosity(XPEHH)(Sabeti selection in BMX pig genomes, we analyzed the FST (Weir et al., 2007), and nucleotide diversity (π) (Tajima, 1983) were & Cockerham, 1984), π (Tajima, 1983), and XPEHH (Sabeti usedtosearchforsignalsofselectionintheBMXpiggenome. et al., 2007) metrics along chromosomes in comparison to Weanalyzedselectivesweepsin10-kbnon-overlappingsliding those of EAW and LWU pigs (Figure 1). Selective signatures windows.Windowscontaining<5SNPswereremovedfromthe were screened in 10-kb non-overlapping sliding windows and analysis. adjacentselectivewindowswerecombined.Asrecentartificial The F values between BMX and other pigs (LWU and selectionproduceslongstretchesofhaplotypesinapopulation ST EAW)werecalculatedusingVCFtools(Daneceketal.,2011). (Amaral et al., 2008), a clustering of selective windows with Haplotypes were inferred using Beagle software (Browning intervalsoflessthanninewindowswaschosenasanindicator & Browning, 2007). Comparing the BMX pigs with LWU and of a putatively selective sweep. Sweep regions greater than EAWpigs,XPEHHwascalculatedtoexaminehaplotypesthat 30 kb in length were included in the following analyses. This showed low levels of linkage decay in the BMX populations procedure resulted in the identification of 257 genes within (Coopetal.,2009;Pickrelletal.,2009;Pritchardetal.,2010). 339 putative selective sweeps in the BMX pig genomes (SupplementaryTableS2). The nucleotide diversities of BMX (π ), EAW (π ), BMX EAW and LWU (π ) pigs were then calculated separately using Functional enrichment analysis of the gene set revealed LWU VCFtools (Danecek et al., 2011). To identify regions that significant overrepresentation of 59 terms (Table 1). showedlownucleotidediversityintheBMXpigscomparedto Interestingly, several biological processes related to sexual the LWU and EAW pigs, we calculated π divided by π maturitywereinvolved,including“pituitaryglanddevelopment” EAW BMX andπ dividedbyπ ,respectively. (BMP4,FGF10,andTBX19),“endocrinesystemdevelopment” LWU BMX (BMP4,CRH,FGF10,NEUROD1,andTBX19),and“negative GenomicwindowsshowinghighF ,lownucleotidediversity, ST regulation of reproductive process” (BMP4, CSN2, and ZP4). and high XPEHH values were identified as having signatures The pituitary is the most important endocrine gland involved of selection in BMX pigs. Candidate selective sweeps were ingrowth andreproduction regulation. Inaddition, fivegenes chosenfromgenomicwindowsidentifiedsimultaneouslybyall (FGF10, FGF2, MITF, PDGFC, and PDGFD) were involved threestatisticsashavingvaluesabovethetop10%thresholdin in “Melanoma”, which might be associated with the two-end theirempiricaldistributions. black pigmentation observed in BMX pigs, which differs from RNA sample collection, sequencing, and identification of most Chinese domestic pigs with black coats (China National differentiallyexpressedgenes CommissionofAnimalGeneticResources,2011). PituitarytissueswerecollectedfromthreeBMXpigsandthree Althoughmanymissensemutationsareassociatedwiththe LargeWhitepigsforRNAsequencing(RNA-seq). Thesepigs domestication of pigs (Fang et al., 2009; Li et al., 2010; Ren were females at 85 days after birth. The RNA-seq libraries et al., 2011; Rubin et al., 2012), whether regulatory variants with an inserted size of 250 bp were prepared using the play a role and their effects on the domestication processes Illumina standard RNA-seq library preparation pipeline and compared to coding variants remain unknown. To address sequenced on the Illumina Hiseq 2000 platform, with 150-bp this issue, we compared the evolutionary patterns for SNPs paired-end reads generated. In total, the transcriptomes of identifiedfromtheselectiveregionsintheBMXpiggenomes.A ZoologicalResearch39(6):424–430,2018 425 totalof830949SNPswereidentifiedfrom339sweeps.Among pigs, respectively. We identified a dataset consisting of 11 these, 3 668 SNPs were located in coding sequences and missense SNPs from 10 genes (Supplementary Table S3). 827 542 SNPs were located in noncoding sequences. We Alignmentofthe10proteinsequencesshowedthattheflanking first focused on protein coding region variations located in sequencesattheresiduesencodedbythe11SNPsdisplayed the339sweeps. Weexaminedhighlydifferentiatedmissense lowlevelcross-speciesconservation,implyingweakfunctional mutations and analyzed their presence in DNA sequences constraints on these protein-coding variants. This indicated encoding conserved protein sequences. We concentrated on that regulatory elements rather than missense variants may missense SNPs with F values up to the chromosomal top havegreatereffectsonBMXpigdevelopment. ST 0.1% threshold and compared BMX pigs to LWU and EAW Figure1WholegenomeselectivesignaturesofBMXpigs X-axisshowsthepositionsofthewindowsalongthe1–18autosomechromosomes. Differentchromosomesaredisplayedindifferentcolors. Y-axisshows theFST(A),XPEHH(B),πEAW/BMX(C),andπLWU/BMX(D)values. FSTandXPEHHvalueswerecalculatedbetweenBMXand(LWU+EAW)pigs. πEAW/BMXand πLWU/BMXwereobtainedfromπEAWandπLWUdividedbyπBMX,respectively. Comparativetranscriptomicanalysisofpituitarytissues al., 2005;Rubin et al., 2012), the DEGs between BMX and We conducted transcriptomic profiling to examine gene LargeWhitepigsprovidedvaluableinformationonexpressional expression changes due to selection in the BMX genomes as changesinBMXpigsiftheyalsoexhibitedselectivesignatures regulatory variants exhibited a profound role during BMX pig in the BMX genome. The DEGs were enriched in the development. We chose pituitary tissue as the pituitary gland following biological processes: “Estrogen signaling pathway” secretes reproduction-related hormones involved in maturity (ADCY6, ADCY8, CREB3L1, CREB5, FOS, GABBR2, GNAS, regulation. In our experimental design, we collected pituitary GRM1, KCNJ9, MMP9, PIK3CG, PIK3R5, and PRKACA), tissues from three BMX and three Large White pigs at 85 “Ovarian steroidogenesis” (ADCY6, ADCY8, ALOX5, GNAS, daysafterbirth. Atthisstage, BMXpigsareclosetomaturity, LDLR, LHCGR, and PRKACA), “Oocyte meiosis” ( ADCY6, whereas Large White pigs do not reach maturity until eight ADCY8,CAMK2B,CAMK2D,CCNE1,CDC20,CPEB2,ESPL1, monthsofage(ChinaNationalCommissionofAnimalGenetic MAD2L2, MOS, PRKACA, and YWHAZ), “Developmental Resources,2011).Thetranscriptomesfromthepituitarytissues maturation” ( C12H17orf47, CDC20, EDNRB, FAM101B, LTF, were sequenced, from which we obtained 35 million mapped PLP1, PRKACA, RBPJ, TAL1, WASH1, and WNT10B), and reads, on average, for each individual. We compared the “Progesterone-mediatedoocytematuration”(ADCY6,ADCY8, geneexpressionlevelsinthepituitaryandfound1600DEGs CPEB2, MAD2L2, MOS, PIK3CG, PIK3R5, and PRKACA) between BMX and Large White pigs. Although Large White (Supplementary Table S4). These pathways all participate in pigs were domesticated from European wild boars (Larson et sexualmaturity. 426 www.zoores.ac.cn Table1FunctionalenrichmenttermsofpositivelyselectedgenesinBMXpigs. GOID GOterm P-value* GO:0004080 Neuroactiveligand-receptorinteraction 24.0E-15 GO:0060449 Budelongationinvolvedinlungbranching 100.0E-6 GO:0032740 Positiveregulationofinterleukin-17production 140.0E-6 GO:0060393 Regulationofpathway-restrictedSMADproteinphosphorylation 230.0E-6 GO:0060389 Pathway-restrictedSMADproteinphosphorylation 250.0E-6 GO:0032946 Positiveregulationofmononuclearcellproliferation 310.0E-6 GO:0050671 Positiveregulationoflymphocyteproliferation 310.0E-6 GO:0070665 Positiveregulationofleukocyteproliferation 370.0E-6 GO:0048636 Positiveregulationofmuscleorgandevelopment 400.0E-6 GO:0045844 Positiveregulationofstriatedmuscletissuedevelopment 400.0E-6 GO:1901863 positiveregulationofmuscletissuedevelopment 450.0E-6 GO:0005412 Arrhythmogenicrightventricularcardiomyopathy(ARVC) 490.0E-6 GO:0005218 Melanoma 560.0E-6 GO:0045740 PositiveregulationofDNAreplication 600.0E-6 GO:0009798 Axisspecification 600.0E-6 GO:0090090 NegativeregulationofcanonicalWntsignalingpathway 600.0E-6 GO:0032660 Regulationofinterleukin-17production 660.0E-6 GO:0051155 Positiveregulationofstriatedmusclecelldifferentiation 660.0E-6 GO:0032620 Interleukin-17production 780.0E-6 GO:0042506 TyrosinephosphorylationofStat5protein 780.0E-6 GO:0051145 Smoothmusclecelldifferentiation 800.0E-6 GO:0061037 Negativeregulationofcartilagedevelopment 920.0E-6 GO:0048546 Digestivetractmorphogenesis 950.0E-6 GO:0005410 Hypertrophiccardiomyopathy(HCM) 990.0E-6 GO:0051149 Positiveregulationofmusclecelldifferentiation 1.2E-3 GO:0035270 Endocrinesystemdevelopment 1.3E-3 GO:0055025 Positiveregulationofcardiacmuscletissuedevelopment 1.3E-3 GO:0042310 Vasoconstriction 1.5E-3 GO:0060602 Branchelongationofanepithelium 1.5E-3 GO:0001709 Cellfatedetermination 2.0E-3 GO:0021536 Diencephalondevelopment 1.8E-3 GO:0021983 Pituitaryglanddevelopment 2.2E-3 GO:0042307 Positiveregulationofproteinimportintonucleus 2.3E-3 GO:0000561 Glycerolipidmetabolism 2.4E-3 GO:0004730 Long-termdepression 2.6E-3 GO:0003338 Metanephrosmorphogenesis 2.4E-3 GO:0030199 Collagenfibrilorganization 2.4E-3 GO:0060441 Epithelialtubebranchinginvolvedinlungmorphogenesis 3.0E-3 GO:0030219 Megakaryocytedifferentiation 3.3E-3 GO:0042383 Sarcolemma 3.3E-3 GO:0009799 Specificationofsymmetry 3.3E-3 GO:0051153 Regulationofstriatedmusclecelldifferentiation 3.3E-3 GO:0010092 Specificationoforganidentity 3.3E-3 GO:1900182 Positiveregulationofproteinlocalizationtonucleus 3.7E-3 GO:0005033 Nicotineaddiction 4.0E-3 GO:0004140 Regulationofautophagy 5.9E-3 GO:2000242 Negativeregulationofreproductiveprocess 5.9E-3 GO:0070664 Negativeregulationofleukocyteproliferation 5.1E-3 GO:0003401 Axiselongation 5.1E-3 GO:0009948 Anterior/posterioraxisspecification 5.5E-3 GO:0055024 Regulationofcardiacmuscletissuedevelopment 5.1E-3 GO:0004940 TypeIdiabetesmellitus 7.4E-3 GO:0048259 Regulationofreceptor-mediatedendocytosis 6.9E-3 GO:0007193 Adenylatecyclase-inhibitingG-proteincoupledreceptorsignalingpathway 11.0E-3 GO:0009880 Embryonicpatternspecification 11.0E-3 GO:0005160 Transforminggrowthfactorbetareceptorbinding 9.5E-3 GO:0031623 Receptorinternalization 11.0E-3 GO:0048286 Lungalveolusdevelopment 9.5E-3 GO:0010862 Positiveregulationofpathway-restrictedSMADproteinphosphorylation 10.0E-3 *:P-valueobtainedfromClueGOplugininCytoscapeusingdefaultparaments.OnlytermswithP<0.05arelisted. ZoologicalResearch39(6):424–430,2018 427 As both artificially selected genes and DEGs showed dockingproteinboundtoFGFreceptors,withaknockoutstudy functional enrichment in maturity regulation, it is possible in mice indicating its involvement in epididymal development that differential gene expression was favored by the andspermmaturation(Xuetal.,2013). Inthepituitarytissue, artificial selection of variants from some sweep loci. We we found that the expression level of FRS2 in BMX pigs was thereforeidentifiedartificiallyselectedgeneswithalteredgene abouttwicethatfoundinLargeWhitepigs.Inaddition,KANK4 expression. In our analysis, a total of 22 artificially selected wasfoundtobedifferentiallyexpressedinthepituitarytissues genes exhibited differential gene expression in the pituitary of BMX and Large White pigs. Although few studies have (P<0.05; FDR<0.05; Table 2). Interestingly, four genes reportedonthefunctionofKANK4,itislocatedinasignificant (CTTNBP2NL,FRS2, KANK4, andKATNAL1)wererelatedto pig quantitative trait locus (QTL) for age at puberty (Bidanel reproduction(Figure2). ComparedtothatintheLargeWhite et al., 2008). KATNAL1, which encodes Katanin Catalytic pigs,theexpressionoftheCTTNBP2NLgenewasincreasedin subunit A1 like 1, has been implicated in both meiosis and BMXpigs.CTTNBP2NLhasbeenreportedtoaffectconception spermiogenesis(Smithetal.,2012). rates in bovines (Sugimoto et al., 2013). FRS2 encodes a Figure 2 Normalized read counts of significantly differentially expressed genes CTTNBP2NL (A), FRS2 (B), KANK4 (C), and KATNAL1(D)inthepituitary Foreachsubgraph,redandgreenboxesrepresentgeneexpressioninthepituitariesofBMXandLargeWhitepigs,respectively. Upperwhiskerismaximum value,blackhorizontallineinboxismedian,andlowerwhiskerisminimumvalue. DISCUSSION presentstudyrevealedfourloci(CTTNBP2NL,FRS2,KANK4, and KATNAL1) showing strong signatures of selection and We conducted a comparative genomic analysis of BMX pigs functional association with puberty. In addition, the four withdomesticpigsandwildboarsfromEastAsia,andrevealed genesexhibiteddifferentialexpressionbetweenearlyandlate selective signatures potentially associated with selection for maturityinpigbreedsinthepituitary. Thisresultindicatesthat early maturity and the two-end black phenotype. During regulatoryelementsmayhaveconsiderableeffectonBMXpig domestication,selectionisbasedonthosefunctionsandtraits development. thatarefavoredinpigbreeds. Asdescribedearlier, selective Coat color is a trait that is usually under strong selection signaturesweredetectedforthespecificphenotypeoftheBMX in different breeds. In previous studies, several genes have pig. Thisstudyshedslightontheevolutionofpubertyinpigs beenshowntobeassociatedwithcoatcolorvariants,including andprovidesimportantinformationandcandidategenesforpig MC1R,KIT,andEDNRB(Kijasetal.,1998;Wiener&Wilkinson, breeding. 2011; Wilkinsonetal.,2013). However,wefoundnoselective Puberty is an important trait related to economic output, signatureofMC1R.Thereasonforthismaybethatselectionof with early maturity resulting in early slow growth. For MC1Roccurredduringearlydomestication,andmostChinese instance, Western commercial breeds are characterized with indigenous pig breeds share the same haplotype (Li et al., late maturity for enlarged body size (Rubin et al., 2012). The 2010). Earlier research has indicated that mutations in MITF, 428 www.zoores.ac.cn PAX3,EDNRB,andKIT togethermayexplainalargeproportion REFERENCES ofwhitespottingphenotypesinhorses(Hauswirthetal.,2012). In BMX pigs, the two-end black phenotype is a complex trait AiH,FangX,YangB,HuangZ,ChenH,MaoL,ZhangF,ZhangL,Cui resulting in the diversification of white areas on the body. We L, He W, Yang J, Yao X, Zhou L, Han L, Li J, Sun S, Xie X, Lai B, Su hypothesizethatacorrelationexistsbetweencoatcolorvariants Y, Lu Y, Yang H, Huang T, Deng W, Nielsen R, Ren J, Huang L. 2015. anddifferentcombinationsofcandidategenes. Adaptationandpossibleancientinterspeciesintrogressioninpigsidentified bywhole-genomesequencing.NatureGenetics,47(3):217–225. Table2Overlapbetweenpositivelyselectedgenesand AmaralAJ,MegensHJ,CrooijmansRPMA,HeuvenHCM,GroenenMAM. differentiallyexpressedgenesfrompituitarytissuein 2008.Linkagedisequilibriumdecayandhaplotypeblockstructureinthepig. BMXpigs. Genetics,179(1):569–579. Gene Normalizedcountfoldchange(BMX/LW) P-value* AndersS,PylPT,HuberW.2014.HTSeq–aPythonframeworktoworkwith MGAT5B 0.08 7.57E-05 high-throughputsequencingdata.Bioinformatics,31(2):166–169. BHLHE22 2.57 0.000538 BidanelJP,RosendoA,IannuccelliN,RiquetJ,GilbertH,CaritezJC,Billon FAM174B 0.43 0.000589 Y,AmiguesY,PrunierA,MilanD.2008.Detectionofquantitativetraitlocifor SFT2D2 0.28 0.001381 teatnumberandfemalereproductivetraitsinMeishanxLargeWhiteF2pigs. TRHDE 2.13 0.005172 Animal,2(6):813–820. COL23A1 0.12 0.006991 FRMD4B 2.45 0.007354 BolgerAM,LohseM,UsadelB.2014.Trimmomatic: aflexibletrimmerfor CDKN2B 0.14 0.007441 Illuminasequencedata.Bioinformatics,30(15):2114–2120. FRS2 2.09 0.008626 BrowningSR,BrowningBL.2007.Rapidandaccuratehaplotypephasing ATG4C 2.16 0.010881 andmissing-datainferenceforwhole-genomeassociationstudiesbyuseof PELI2 1.90 0.012313 localizedhaplotypeclustering.AmericanJournalofHumanGenetics,81(5): FEZ2 2.12 0.018321 1084–1097. LGALS12 0.13 0.020835 TYRP1 0.13 0.025025 China National Commission of Animal Genetic Resources. 2011. Animal KCND3 1.81 0.03048 Genetic Resources in China: Pigs. Beijing: China Agriculture Press. (in FNDC1 0.22 0.030808 Chinese) ATL3 0.46 0.033021 CiobanuDC,DayAE,NagyA,WalesR,RothschildMF,PlastoGS.2001. KANK4 0.34 0.036521 GeneticvariationintwoconservedlocalRomanianpigbreedsusingtype1 KATNAL1 1.99 0.037825 CTTNBP2NL 2.27 0.039048 DNAmarkers.GeneticsSelectionEvolution,33(4):417–432. ANO3 2.50 0.044986 CoopG,PickrellJK,NovembreJ,KudaravalliS,LiJ,AbsherD,MyersRM, DYNC1I2 1.67 0.04703 Cavalli-SforzaLL,FeldmanMW,PritchardJK.2009.Theroleofgeography *:P-valuesarecorrectedbyBenjamini-HochbergFDR<0.05. inhumanadaptation.PLoSGenetics,5(6):e1000500. Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA, OnelimitationofthisstudywastheidentificationoftheDEG Handsaker RE, Lunter G, Marth GT, Sherry ST, McVean G, Durbin R. directioninRNA-seqanalysis. AsbothBMXandLargeWhite 1000GenomesProjectAnalysisGroup.2011.Thevariantcallformatand pigsaredomesticated,itwasdifficulttoidentifythedirectionof VCFtools.Bioinformatics,27(15):2156–2158. geneexpressionwithouttheuseofanoutgroup. Weanalyzed FangM,LarsonG,RibeiroHS,LiN,AnderssonL.2009.Contrastingmode the overlap between DEGs (between BMX and Large White ofevolutionatacoatcolorlocusinwildanddomesticpigs.PLoSGenetic, pigs)andtheartificiallyselectedgenesinBMXpigs(compared 5(1):e1000341. to LWU and EAW pigs). The shared genes were more likely GroenenMA,ArchibaldAL,UenishiH,TuggleCK,TakeuchiY,RothschildMF, to be changed in BMX pigs. We did not focus on DEGs that Rogel-GaillardC,ParkC,MilanD,MegensHJ,LiS,LarkinDM,KimH,Frantz didnotexhibitselectivesignaturesintheBMXpiggenome. In LA,CaccamoM,AhnH,AkenBL,AnselmoA,AnthonC,AuvilL,BadaouiB, addition, future analysis of Asian and European pig genome BeattieCW,BendixenC,BermanD,BlechaF,BlombergJ,BolundL,Bosse datawillhelpclarifybreeddevelopmentinpigs. M,BottiS,BujieZ,BystromM,CapitanuB,Carvalho-SilvaD,ChardonP, COMPETINGINTERESTS ChenC,ChengR,ChoiSH,ChowW,ClarkRC,CleeC,CrooijmansRP, DawsonHD,DehaisP,DeSapioF,DibbitsB,DrouN,DuZQ,EversoleK, Theauthorsdeclarethattheyhavenocompetinginterests. FadistaJ,FairleyS,FarautT,FaulknerGJ,FowlerKE,FredholmM,FritzE, AUTHORS’CONTRIBUTIONS GilbertJG,GiuffraE,GorodkinJ,GriffinDK,HarrowJL,HaywardA,HoweK, HuZL,HumphraySJ,HuntT,HornshøjH,JeonJT,JernP,JonesM,Jurka Y.P.Z.,H.B.X.,andY.Y.designedthestudy. H.B.X.supervisedtheanalyses. Y.Y.performedgenomeassemblyandannotation. Y.Y.extractedRNAand J,KanamoriH,KapetanovicR,KimJ,KimJH,KimKW,KimTH,Larson wrotethemanuscriptwiththeotherauthors’input. Y.Y.submittedRNA-seq G,LeeK,LeeKT,LeggettR,LewinHA,LiY,LiuW,LovelandJE,LuY, datatoGSA.A.C.A.,H.B.X.andY.P.Z.revisedthemanuscript. Allauthors LunneyJK,MaJ,MadsenO,MannK,MatthewsL,McLarenS,MorozumiT, readandapprovedthefinalmanuscript. MurtaughMP,NarayanJ,NguyenDT,NiP,OhSJ,OnteruS,PanitzF,Park ZoologicalResearch39(6):424–430,2018 429 EW,ParkHS,PascalG,PaudelY,Perez-EncisoM,Ramirez-GonzalezR, hardsweeps,softsweeps,andpolygenicadaptation.CurrentBiology,20(4): ReecyJM,Rodriguez-ZasS,RohrerGA,RundL,SangY,Schachtschneider R208–R215. K,SchraiberJG,SchwartzJ,ScobieL,ScottC,SearleS,ServinB,Southey Ren J, Duan Y, Qiao R, Yao F, Zhang Z, Yang B, Guo Y, Xiao S, Wei BR,SperberG,StadlerP,SweedlerJV,TaferH,ThomsenB,WaliR,WangJ, R, Ouyang Z, Ding N, Ai H, Huang L. 2011. A missense mutation in WangJ,WhiteS,XuX,YerleM,ZhangG,ZhangJ,ZhangJ,ZhaoS,Rogers PPARDcausesamajorQTLeffectonearsizeinpigs.PLoSGenetics,7(5): J,ChurcherC,SchookLB.2012.Analysesofpiggenomesprovideinsight e1002043. intoporcinedemographyandevolution.Nature,491(7424):393–398. Rubin CJ, Megens HJ, Barrio AM, Maqbool K, Sayyab S, Schwochow D, Hauswirth R, Haase B, Blatter M, Brooks SA, Burger D, Drögemüller C, Wang C, Carlborg Ö, Jern P, Jørgensen CB, Archibald AL, Fredholm M, GerberV,HenkeD,JandaJ,JudeR,MagdesianKG,MatthewsJM,Poncet, Groenen MAM, Andersson L. 2012. Strong signatures of selection in the PA;SvanssonV,TozakiT,Wilkinson-WhiteL,PenedoMCT,RiederS,LeebT. domesticpiggenome.ProceedingsoftheNationalAcademyofSciencesof 2012.MutationsinMITFandPAX3cause“splashedwhite”andotherwhite theUnitedStatesofAmerica,109(48):19529–19536. spottingphenotypesinhorses.PLoSGenetics,8(4):e1002653. Sabeti PC, Varilly P, Fry B, Lohmueller J, Hostetter E, Cotsapas C, KijasJMH,WalesR,TörnstenA,ChardonP,MollerM,AnderssonL.1998. Xie X, Byrne EH, McCarroll SA, Gaudet R, Schaffner SF, Lander ES, Melanocortinreceptor1(MC1R)mutationsandcoatcolorinpigs.Genetics, The International HapMap Consortium. 2007. Genome-wide detection 150(3):1177–1185. and characterization of positive selection in human populations. Nature, Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. 2013. 449(7164):913–918. TopHat2:accuratealignmentoftranscriptomesinthepresenceofinsertions, SmithLB,MilneL,NelsonN,EddieS,BrownP,AtanassovaN,O’BryanMK, deletionsandgenefusions.GenomeBiology,14(4):R36. O’DonnellL,RhodesD,WellsS,NapperD,NolanP,LalanneZ,Cheeseman Larson G, Dobney K, Albarella U, Fang M, Matisoo-Smith E, Robins J, M,PetersJ.2012.KATNAL1regulationofsertolicellmicrotubuledynamics LowdenS,FinlaysonH,BrandT,WillerslevE,Rowley-ConwyP,Andersson is essential for spermiogenesis and male fertility. PLoS Genetics, 8(5): L,CooperA.2005.Worldwidephylogeographyofwildboarrevealsmultiple e1002697. centersofpigdomestication.Science,307(5715):1618–1621. Sugimoto M, Sasaki S, Gotoh Y, Nakamura Y, Aoyagi Y, Kawahara Li H, Durbin R. 2010. Fast and accurate long-read alignment with T, Sugimoto Y. 2013. Genetic variants related to gap junctions and Burrows-Wheelertransform.Bioinformatics,26(5):589–595. hormonesecretioninfluenceconceptionratesincows.Proceedingsofthe LiJ,YangH,LiJR,LiHP,NingT,PanXR,ShiP,ZhangYP.2010.Artificial National Academy of Sciences of the United States of America, 110(48): selection of the melanocortin receptor 1 gene in Chinese domestic pigs 19495–19500. duringdomestication.Heredity,105(3):274–281. Tajima F. 1983. Evolutionary relationship of DNA sequences in finite LiM,TianS,JinL,ZhouG,LiY,ZhangY,WangT,YeungCKL,ChenL,Ma populations.Genetics,105(2):437–460. J,ZhangJ,JiangA,LiJ,ZhouC,ZhangJ,LiuY,SunX,ZhaoH,NiuZ,Lou Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren P,XianL,ShenX,LiuS,ZhangS,ZhangM,ZhuL,ShuaiS,BaiL,Tang MJ, Salzberg SL, Wold BJ, Pachter L. 2010. Transcript assembly and G,LiuH,JiangY,MaiM,XiaoJ,WangX,ZhouQ,WangZ,StothardP,Xue quantification by RNA-Seq reveals unannotated transcripts and isoform M,GaoX,LuoZ,GuY,ZhuH,HuX,ZhaoY,PlastowGS,WangJ,Jiang switchingduringcelldifferentiation.NatureBiotechnology,28(5):511–515. Z,LiK,LiN,LiX,LiR.2013.Genomicanalysesidentifydistinctpatterns WeirBS,CockerhamCC.1984.EstimatingF-statisticsfortheanalysisof ofselectionindomesticatedpigsandTibetanwildboars.NatureGenetics, populationstructure.Evolution,38(6):1358–1370. 45(12):1431–1438. Wiener P, Wilkinson S. 2011. Deciphering the genetic basis of animal LoveMI,HuberW,AndersS.2014.Moderatedestimationoffoldchangeand domestication. Proceedings of the Royal Society B: Biological Sciences, dispersionforRNA-seqdatawithDESeq2.GenomeBiology,15(12):550. 278(1722):3161–3170. McKenna A, Hanna M, Banks E, Sivachenko A,CibulskisK,KernytskyA, Wilkinson S, Lu ZH, Megens HJ, Archibald AL, Haley C, Jackson IJ, GarimellaK,AltshulerD,GabrielS,DalyM,DePristoMA.2010.TheGenome GroenenMAM,CrooijmansRPMA,OgdenR,WienerP.2013.Signatures AnalysisToolkit:aMapReduceframeworkforanalyzingnext-generationDNA of diversifying selection in European pig breeds. PLoS Genetics, 9(4): sequencingdata.GenomeResearch,20(9):1297–1303. e1003453. MlecnikB,GalonJ,BindeaG.2018.Comprehensivefunctionalanalysisof Wu GS, Yao YG, Qu KX, Ding ZL, Li H, Palanichamy MG, Duan ZY, largelistsofgenesandproteins.JournalofProteomics, 171: 2–10.DOI: Li N, Chen YS, Zhang YP. 2007. Population phylogenomic analysis of doi.org/10.1016/j.jprot.2017.03.016. mitochondrial DNA in wild boars and domestic pigs revealed multiple PickrellJK,CoopG,NovembreJ,KudaravalliS,LiJZ,AbsherD,Srinivasan domesticationeventsinEastAsia.GenomeBiology,8(11):R245. BS, Barsh GS, Myers RM, Feldman MW, Pritchard JK. 2009. Signals of XuB,YangL,HintonBT.2013.Theroleoffibroblastgrowthfactorreceptor recent positive selection in a worldwide sample of human populations. substrate2(FRS2)intheregulationoftwoactivitylevelsofthecomponents GenomeResearch,19(5):826–837. of the extracellular signal-regulated kinase (ERK) pathway in the mouse PritchardJK,PickrellJK,CoopG.2010.Thegeneticsofhumanadaptation: epididymis.BiologyofReproduction,89(2):48. 430 www.zoores.ac.cn