Genomic and transcriptomic analyses reveal selection of genes for puberty in Bama Xiang pigs

2018·3 MB·

by  Yang, Yang

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
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HuZL,HumphraySJ,HuntT,HornshøjH,JeonJT,JernP,JonesM,Jurka
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datatoGSA.A.C.A.,H.B.X.andY.P.Z.revisedthemanuscript. Allauthors LunneyJK,MaJ,MadsenO,MannK,MatthewsL,McLarenS,MorozumiT,
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