ARTICLE
OPEN
Received27Jan2014|Accepted12Jun2014|Published15Jul2014
Genomic analysis reveals lection for Asian genes in European pigs following human-mediated introgression
Mirte Bos1,Hendrik-Jan Megens1,Laurent A.F.Frantz1,Ole Madn1,Greger Larson2,Yogesh Paudel1, Naomi Duijvesteijn1,3,Barbara Harlizius3,Yanick Hagemeijer1,Richard P.M.A.Crooijmans1
&Martien A.M.Groenen1
The independent domestication of local wild boar populations in Asia and Europe about
10,000years ago led to distinct European and Asian pig breeds,each with very different
phenotypic characteristics.During the Industrial Revolution,Chine breeds were imported to
Europe to improve commercial traits in European breeds.Here we demonstrate the prence
of introgresd Asian haplotypes in European domestic pigs and lection signatures on some
loci in the regions,using whole genome quence data.The introgression signatures are
widespread and the Asian haplotypes are rarelyfixed.The Asian introgresd haplotypes are
associated with regions harbouring genes involved in meat quality,development and fertility.
We identify Asian-derived non-synonymous mutations in the AHR gene that associate with
incread litter size in multiple European commercial lines.Thefindings demonstrate that
incread fertility was an important breeding goal for early nineteenth century pig farmers,
and that Asian variants of genes related to this trait were preferentially lected during the
development of modern European pig breeds.
经销授权书
1Animal Breeding and Genomics Centre,Wageningen University,Wageningen6708WD,The Netherlands.2Durham Evolution and Ancient DNA, Department of Archaeology,Durham University,DH13LE Durham,UK.3TOPIGS Rearch Center IPG,6640AA Beuningen,The Netherland
s. Correspondence and requests for materials should be addresd to M.B.(email:mirte.bos@wur.nl).
T housands of years of lection on numerous traits in domesticated species such as dogs1,2,cows3and chickens4
has led to a wide range of distinct phenotypes that are not (or only rarely)obrved in the wild.Improvement of breeding practices,which involves making cross between different breeds and even(sub)species,has greatly improved productivity in intensive farming systems.Examples include the white ed colour in rice,shown to originate from a single mutation that swept through different subspecies following hybridization5,and the yellow skin allele that isfixed in the majority of modern western chicken breeds originating from admixture with the Grey junglefowl6.Human-mediated introgression of alleles is likely to have played a major role in the genomic architecture of many modern domestic species,including pigs.
The wild boar(Sus scrofa)originated B4million years ago in Southeast Asia and has since expanded its range over Eurasia,leading to the emergence of numerous geographically and genetically divergent populations7,8.The independent domestication of two of the populations in East Asia an
d western Eurasia led to distinct domesticated populations9–11. Hybridization and introgression between domestic pigs that originated from highly divergent wild populations has resulted in modern genomes that posss a mosaic of different haplotypes12,13.Although some geneflow may have taken place before the nineteenth century,it was certainly extremely rare given the geographic distance between Asia and Europe,and the lack of any historical records describing the importation of Asian breeds into Europe before the nineteenth century(or vice-versa). Mitochondrial studies have suggested that the introgression was mostly female driven12and the introduction of Asian pigs into Europe at the ont of the Industrial Revolution in the late eighteenth and early nineteenth centuries has been particularly well documented14,15.In parallel with increasing intensification of farming at that time,British pig breeders sought to improve productivity of the local breeds,and did so,in part,by importing Chine pigs.Chine pigs were renowned for having great mothering characteristics,superior meat quality,strong resistance to dias,better adaptation to living in sties and producing larger litters(415live born young).The lection for specific traits in European pig breeds has resulted in multiple lective sweeps in the genome of domesticates16.As European pig breeders deliberately introgresd Asian haplotypes into European local breeds,it is expected that the origin of haplotypes for which evidence of lection exists often stems from Asian introgression.Known examples include the EDNRB, IGF2and KITLG regions17–19,all of which the identified variants
EUWB (european wild boar)
ASDom
(asian domesticated)
Introgression
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2. LW-ASDom:
haplotypes shared
between ASDom
and large white LW
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1. LW-EUWB:
haplotypes shared
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and large white
Figure1|Experimental tup for the introgression detection.Arrows indicate the comparisons between groups that are ud for the IBD detection. Individuals from the LW breed are ud for all pairwi comparisons with individuals from two geographical and functional groups:EUWB and ASDom. The blue arrow indicates the human-mediated introgression from ASDom in to LW.
have considerable effect on the phenotypes.Interestingly,the lection criteria shifted through time.When Asian lines were first introgresd into European pigs,fatness was lected for, although now leanness is preferentially lected.The Large White (LW)breed is one of most widely ud breeds in commercial pig production and originated in the United Kingdom in the late nineteenth century.The breed is renowned for high growth rate, desirable carcass lean meat percentage and a desirable feed to body weight conversion ratio,but also for large litters,heavy milk production and gre
at mothering characteristics.As LW is thought to have a hybrid origin,some of the traits are potentially the result of lected Chine haplotypes.Becau of deliberate introduction of Asian germplasm into European pigs and subquent intensive artificial lection,Asian haplotypes in LW genomes are expected to be non-randomly distributed,but rather to be overreprented in regions that contain genes or regulatory elements that are linked to traits relevant for production.
Here we test this hypothesis and identify specific gene variants bred into European pigs involved in key production traits.More specifically,we interrogated the genomes of LW pigs to reveal patterns of introgresd and lected haplotypes,to unravel the genomic conquences of human-mediated hybridization and artificial lection.
Results
Evidence of introgression.We identified haplotypes in the LW pigs that were identical by decent(IBD)with individuals from both the original source of domestication,the European wild boars (EUWB),and the source of introgression,the Asian domestic group(ASDom,Fig.1).Individuals from different locations in Europe were ud to reprent the source of domestication, whereas individuals from three different Asian breeds were ud to reprent the pool of putative introgresd
haplotypes(e Supplementary Table1and Supplementary Methods for details). Average genetic differentiation(Fst,as defined by Weir and Cockerham20)between the LW and ASDom was0.33(s.d.0.
0.0008),while the average Fst value between LW and EUWB was 0.16(s.d.0.0.0006).The results show that the genomes of the LW pigs still share greater similarity with their EUWB ancestors than with ASDom.We ud an other independent method to further verify the existence of geneflow between ASDom and LW after lineage divergence between Asian and European Sus scrofa(D-statistics21,e Methods).We computed this statistic for each possible trio between(LW,ASDom and EUWB),and(LW,EUWB,ASDom)so that a significantly negative D(Z oÀ4)imply admixture between ASDom and LW.Our results demonstrate that all LW individuals posss roughly an equal degree of admixture with Asian pigs over their entire genomes,reflecting the human-mediated hybridization with Asian domestics in the late eighteenth and early nineteenth century (D¼À0.083±0.015,Z¼À20).
Introgression mapping.To infer whether a region was intro-gresd in multiple individuals,we calculated the frequency of all LW haplotypes that were of Asian or European origin for each bin of10,000bas in the genome.The relative fraction of Asian versus European haplotypes in the LW group is expresd as relative IBD(rIBD).Asian haplotype frequency in the LW population,for a given
locus,ranged from0.7(where1indicates all haplotypes are ASDom and none are EUWB)toÀ1(all haplotypes are IBD with EUWB,Fig.2a).The majority of the genome displays more similarity with the EUWB than with the Asian domesticated pigs.Despite this,every chromosome con-tained regions in which the signal for Asian ancestry was stronger than the European signature.A cutoff of two s.d.from the mean in the Z-transformed rIBD distribution allowed us to identify regions,which spans B1.3%of the genome of LW pigs,that were likely to be of Asian origin(Fig.2c,d and Supplementary Table2). This introgression pattern is probably the result of a combination of drift and lection.In contrast to dogs,where lection ems to have acted on a relatively small number of loci with large effects1,2,the introgression signal in the pigs is found at many loci,and the putatively lected Asian haplotype is rarely fixed.This pattern suggests that lection on Asian haplotypes,if prent,mostly involved complex multi-genic traits or genes influencing traits lected in opposing directions.A high rIBD signal in our analysis refers to a region that contains predominantly introgresd haplotypes,but this does not imply that the introgresd haplotypes are identical or similar.Regions that contain more Asian haplotypes just by chance,but have not been under lection,could result in a high rIBD signal.We ud an extended haplotype homozygosity test22to check for extended haplotypes in the LW population and compared the integrated haplotype score(iHS)signal with our rIBD values.This way,we can distinguish between regions that contain multiple Asian haplotypes and regions that cont
ain one or few particular elongated Asian haplotypes.We inferred a significant correlation between rIBD and integrated haplotype score(iHS) in bins of500kb over the full genome(Supplementary Fig.1a). To check whether the extended haplotype homozygosity in the LW pool was specific for the breed or obrved in more European breeds,we contrasted the LW signal with a reference pool of other European commercial pigs(Supplementary Fig.1b,c).
Genome-wide patterns of introgression.On a genome scale, many of the genes located within the regions where the LW pigs share more haplotypes with ASDom than with EUWB (s ZrIBD Z2),are associated with commercial traits such as meat quality(DNMT3A,SAL1,ME1,IGF2BP1),fertility(PGRMC2, KIF18A CDK20,AHR)and development(NRG1,AHR, Supplementary Table2),although no significant enriched Gene Ontology(GO)term was found.Gene-den regions on chro-mosome1and2display a high rate of alternating ASDom and EUWB haplotypes.For instance,the regions containing the CDK20and SAL1genes,which both have been associated with reproduction traits23,24,are only10–20kb long.The short tracts of shared haplotypes either indicate a high recombination frequency(corroborated by the recombination map for pig25),a more temporally distant hybridization episode and/or favourable European haplotypes surrounding the genes that could lead to positive
lection on recombinant haplotypes.The recombination landscape in Sus scrofa is known to be highly heterogeneous,and this probably results in an unequal distribution of haplotype length26.Longer Asian haplotypes will be found in regions of low recombination and therefore the introgression signal is easier to identify in regions with a low recombination rate.
Longest regions of introgression.Chromosomes8and9contain the largest concutive regions of inferred introgression in the LW genomes(defined as regions where rIBD40).To check whether the extended haplotype homozygosity in the LW pool was specific for the breed or obrved in more European breeds,we contrasted the LW signal with a reference pool of other European commercial pigs with the Rsb statistic27 (Supplementary Fig.1b,c).This analysis demonstrates that the region of introgression on chromosome8contains a stronger extended haplotype homozygosity(EHH)signal in the reference panel,and that the region on chromosome9contains a particularly strong signal in the LW population.We ud two independent methods,D-statistics and Fst,to support the
detected introgression in the regions in the LW (Fig.3a–e).To show that divergence between LW and ASDom was reduced in the introgresd regions,we calculated Fst for the regions parately.The Fst between ASDom and the LW was lower in both introgresd regions than between EUWB and LW (Fig.3c-e),thereby supporting the signal of Asian introgression (high rIBD).T
he D -statisctics for the regions on chromosome 9was lower than the genome-wide average,which corroborated our rIBD analysis (Fig.3b).The region on chromosome 8shows a wide distribution,indicating that some LW haplotypes contain the Asian signature,while others do not.Inconsistent clustering of European haplotypes within an Asian clade at this locus supports this hypothesis (Supplementary Fig.2).Curiously,the B 4Mb quence shows a clear signal of introgression,although a large part of the region is devoid of annotated genes.As this part of the genome has a relatively low recombination frequency 25,the region may extend considerably beyond the position of the actual favourable allele that has been lected for,due to genetic hitch-hiking and the short time since introgression.Alternatively,drift could have resulted in the prence of Asian haplotypes in this region.The PGRMC2gene,coding for the progesterone receptor,lies within the highest peak of Asian haplotypes in that region.Progesterone is an important hormone involved in female reproduction and maternal behaviour 28,traits that Asian pigs have been lected for extensively.Therefore,the Asian haplotype containing the PGRMC2gene could be associated with higher reproductive success in LW pigs and may have been subjected to lection pressure as a result.The Rsb signal suggest that in other European breeds,the proportion of Asian haplotypes is even higher for this locus (Supplementary Fig.1b,c).We ud genotype data from the Illumina Porcine 60K iSelect beadchip 29for an additional 5,143pigs from three European commercial lines to
screen allele frequencies in this region.Two genetic lines have been lected for reproductive traits since the establishment of the
lines (A and B),and one line for finishing traits (C).The SNP alleles in this 4-Mb region show a clear difference between the two reproduction-associated lines and the growth-associated line (Supplementary Fig.3).The findings could indicate that the Asian haplotypes in this region are associated with fertility,but further analys are needed to support this hypothesis.Introgression at the AHR locus .The 6.8-Mb region on chromosome 9has a large proportion of LW haplotypes that are nearly identical to two haplotypes found in the Asian Jianquhai breed (Supplementary Fig.2).Among the genes in this region of putative Asian introgression are multiple members of the TWIST gene family,transcription factors known to be involved in a variety of process,including embryonic development 30.The highest introgression peak in this region contains the AHR gene that has previously been associated with female reproductivity 31.Originally,the AHR gene has been identified as mediator of xenobiotic-induced toxicity 32,33.AHR has been shown not only to be involved in the respon to toxicity but also to be associated with fertility in mammals 34,35.The AHR gene ems to play an important role in the female reproductive system at multiple levels 31.In pigs,expression of AHR during the oestrous cycle and putative involvement of AHR in the regulation
of reproduction has been obrved 36,37.Furthermore,polymorphisms in human are known to occur predominantly in exon 10,which contains an important transactivation domain 38.We screened this gene in pigs for non-synonymous mutations and identified four non-synonymous mutations in exon 11of the AHR gene,which corresponds to exon 10of the human AHR gene.The variants of Asian origin were all in strong linkage diquilibrium.As the AHR is a strong candidate gene for which Asian variants have been lected since introgression during the Industrial Revolution,we examined this gene further (e
Methods).
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Figure 2|Distribution of regions in the genome where the LW contain introgresd haplotypes from ASDom.(a )The x axis shows the full length of all chromosomes,and the y axis reprents the relative frequency of LW haplotypes IBD with ASDom or EUWB,ranging from 1(all haplotypes are IBD with ASDom,and none with EUWB)to À1.The two longest regions of concutive introgression are indicated with arrows.(b )Distribution
of the relative proportion of IBD haplotypes in LW and the EUWB (green,IBDEUWB,0to À1)or ASDom (blue,IBDASDom,0to 1)in bins of 10Kbp.(c )Distribution of the rIBD scores for the LW haplotypes (rIBD ¼IBDEUWB–IBDASDom).(d )Z -transformed distribution of rIBD.
The iHS signal within the LW population is strongest for the AHR locus.The fact that the Rsb signal is also strong in the LW compared with other European breeds,indicates that the Asian haplotypes at the AHR locus were neverfixed in the ancestral population that led to current commercial breeds,bec
au the frequency and iHS signal differs between breeds(Supplementary Fig.1b,c).We ud an additional method to screen for lection, nSL,that has recently become available and should be robust to variation in recombination rate39.We averaged the rIBD,the nSL statistic and the corrected iHS P-value over bins of500kb for chromosome9(e Supplementary Fig.4a–c).All three statistics contain a high signal at the previously identified region on chromosome9that also contains the AHR gene.We show that the haplotype containing the ancestral allele at the AHR locus is more persistent than haplotypes containing the derived allele at this locus(Supplementary Fig.4d–f).A survey of wild boar populations and four other Sus species for the loci revealed that the ancestral haplotype is homozygous in all cloly related Sus species,and at high frequency in Asian domesticated pigs and European breeds.However,only derived haplotypes were found in European wild boar and the ancestral type was prent only at a low frequency in Asian wild boars.This suggests a history of lection for the ancestral state in domestics,after the derived state reached high frequency in the wild populations.
Effect of Asian haplotypes at the AHR locus.To examine the effect of the amino acid changes in the AHR protein on repro-ductive success,we ud genotype data from the Illumina Porcine 60K iSelect beadchip29for the same5,143pigs from three European commercial purebred lines for which estimat
ed breeding values(EBV)for the total number of piglets born (TNB)were available.We extracted genotypes for tho markers that fell within the region of high introgression(rIBD42).The AHR gene was the only annotated gene in this lected part of the genome.Either Asian or European heritage was assigned to the 60K haplotypes for the5,143commercial pigs at the AHR locus, bad on our re-quenced individuals and confirmation in the lab(e Methods).Haplotypes containing the Asian AHR variants had a significant effect on the EBV for total number born over all three lines(EBV–TNB0.0.04,P o0.0001,Table1). Although the EBV–TNB can be rather different between
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Figure3|Levels of differentiation between LW and ASDom or EUWB in regions of introgression.(a)Relative introgression fraction(rIBD)over
the full length of chromosome8and9.The longest regions of introgression are indicated with purple and blue.(b)Boxplot of D-statistics for the full genome(red)and the two longest regions of introgression as indicated in a on chr8(purple)and chr9(blue).The minimum,first quartile,median, third quartile and maximum are indicated with the box and whiskers with outliers41.5*IQR.D-statistics are computed for each possible trio with
LW¼P1,ASDom¼P2and EUWB¼P3,with the Sumatran S.scrofa as outgroup(O)resulting in378trios.(c–e)Distribution of Fst between
LW-ASDom(blue)and LW-EUWB(red)in bins of10Kbp.The left histogram shows the Fst distributions bad on the full genome(c),and the
other two show the Fst distribution for the regions of introgression on chr8(d)and chr9(e).
