gene-09-00118 Ap il 9, 2018 Time: 17:46 # 1
ORIGINAL RESEARCH
published: 10 Ap il 2018
doi: 10.3389/ gene.2018.00118
Edi ed by:
Jo am Mwashigadi Mwacha o,
In e na ional Cen e o Ag icul u al
Resea ch in he D y A eas (ICARDA),
E hiopia
Re iewed by:
Shahin Eghbalsaied,
Islamic Azad Uni e si y, I an
Cla e A. Gill,
Texas A&M Uni e si y, Uni ed S a es
Da id W agg,
The Uni e si y o Edinbu gh,
Uni ed Kingdom
Mou ad Rekik,
In e na ional Cen e o Ag icul u al
Resea ch in he D y A eas (ICARDA),
Jo dan
*Co espondence:
Meng-Hua Li
[email p o ec ed]
†These au ho s ha e con ibu ed
equally o his wo k.
Special y sec ion:
This a icle was submi ed o
Li es ock Genomics,
a sec ion o he jou nal
F on ie s in Gene ics
Recei ed: 13 Decembe 2017
Accep ed: 23 Ma ch 2018
Published: 10 Ap il 2018
Ci a ion:
Xu S-S, Gao L, Xie X-L, Ren Y-L,
Shen Z-Q, Wang F, Shen M,
Eyþó sdó i E, Hallsson JH,
Kisele a T, Kan anen J and Li M-H
(2018) Genome-Wide Associa ion
Analyses Highligh he Po en ial
o Di e en Gene ic Mechanisms
o Li e Size Among Sheep B eeds.
F on . Gene . 9:118.
doi: 10.3389/ gene.2018.00118
Genome-Wide Associa ion Analyses
Highligh he Po en ial o Di e en
Gene ic Mechanisms o Li e Size
Among Sheep B eeds
Song-Song Xu1,2†, Lei Gao3,4†, Xing-Long Xie1,2, Yan-Ling Ren5, Zhi-Qiang Shen5,
Feng Wang6, Min Shen3,4, Emma Eyþó sdó i 7, Jón H. Hallsson7, Ta yana Kisele a8,
Juha Kan anen9and Meng-Hua Li1,2*
1CAS Key Labo a o y o Animal Ecology and Conse a ion Biology, Ins i u e o Zoology, Chinese Academy o
Sciences (CAS), Beijing, China, 2College o Li e Sciences, Uni e si y o Chinese Academy o Sciences, Beijing, China,
3Ins i u e o Animal Husband y and Ve e ina y Medicine, Xinjiang Academy o Ag icul u al and Reclama ion Science, Shihezi,
China, 4S a e Key Labo a o y o Sheep Gene ic Imp o emen and Heal hy B eeding, Xinjiang Academy o Ag icul u al and
Reclama ion Science, Shihezi, China, 5Shandong Binzhou Academy o Animal Science and Ve e ina y Medicine Academy,
Binzhou, China, 6Ins i u e o Sheep and Goa Science, Nanjing Ag icul u al Uni e si y, Nanjing, China, 7Facul y o Na u al
Resou ces and En i onmen al Sciences, Ag icul u al Uni e si y o Iceland, Bo ga nes, Iceland, 8All-Russian Resea ch
Ins i u e o Gene ics and Fa m Animal B eeding, Russian Academy o Sciences, Moscow, Russia, 9P oduc ion Sys ems,
Na u al Resou ces Ins i u e Finland, Jokioinen, Finland
Rep oduc ion is an impo an ai in sheep b eeding as well as in o he li es ock.
Howe e , despi e i s impo ance he gene ic mechanisms o li e size in domes ic sheep
(O is a ies) a e s ill poo ly unde s ood. To explo e gene ic mechanisms unde lying he
a ia ion in li e size, we conduc ed mul iple independen genome-wide associa ion
s udies in i e sheep b eeds o high p oli icacy (Wadi, Hu, Icelandic, Finnsheep, and
Romano ) and one low p oli icacy (Texel) using he O ine In inium HD BeadChip,
espec i ely. We iden i ied di e en se s o candida e genes associa ed wi h li e size in
di e en b eeds: BMPR1B,FBN1, and MMP2 in Wadi; GRIA2,SMAD1, and CTNNB1 in
Hu; NCOA1 in Icelandic; INHBB,NF1,FLT1,PTGS2, and PLCB3 in Finnsheep; ESR2 in
Romano and ESR1,GHR,ETS1,MMP15,FLI1, and SPP1 in Texel. Fu he anno a ion
o genes and bioin o ma ics analyses e ealed ha di e en biological pa hways could
be in ol ed in he a ia ion in li e size o emales: ho mone sec e ion (FSH and LH)
in Wadi and Hu, placen a and emb yonic le hali y in Icelandic, olliculogenesis and
LH signaling in Finnsheep, o ula ion and p eo ula o y ollicle ma u a ion in Romano ,
and es ogen and ollicula g ow h in Texel. Taken oge he , ou esul s p o ide new
insigh s in o he gene ic mechanisms unde lying he p oli icacy ai in sheep and o he
mammals, sugges ing a ge s o selec ion whe e he aim is o inc ease p oli icacy in
b eeding p ojec s.
Keywo ds: sheep, p oli icacy, genome-wide associa ion s udy, biological pa hways, egula ion
INTRODUCTION
Rep oduc ion is one o he mos impo an ai s in li es ock p oduc ion pa icula ly o emales.
Selec ion o highe p oli icacy in domes ic sheep (O is a ies) has led o a iable li e size (LS)
wi hin and among b eeds. Fo example, indi idual li e size o 1 o 8 has been eco ded in he Hu
sheep and Finnsheep (Yue, 1996;Da is e al., 2006a).
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P e ious s udies epo ed ha he excep ional p oli icacy o
he Boo oola Me ino was a ibu ed o a single majo gene, while
a numbe o mu a ions o a majo e ec on li e size ha e been
iden i ied in o he sheep b eeds (Table 1; see also Xu and Li,
2017). Vage e al. (2013) de ec ed a mu a ion FecGFin gene GDF9
s ongly associa ed wi h li e size in No wegian Whi e Sheep and
Finnish Land ace (Finnsheep) using a genome-wide associa ion
analysis. Dema s e al. (2013) epo ed he mu a ions FecXG in
G i e e sheep and FecXOin Olkuska sheep associa ed wi h he
highly p oli ic pheno ype by a genome-wide associa ion analysis.
Cao e al. (2016) ound ha nine candida e genes including he
well-known FecB mu a ion played impo an oles in he a iable
li e size in Hu and Small- ailed Han sheep h ough me hyla ed
DNA-immunop ecipi a ion sequencing da a. Miao e al. (2016)
iden i ied a se o di e en ially exp essed genes (e.g., FecB)
be ween low- and high-p oli icacy b eeds (Do se s. Small- ailed
Han sheep) h ough implemen ing in eg a ed analysis o miRNAs
and lncRNAs. Lassoued e al. (2017) ound he mu a ion FecXBa
associa ed wi h he p oli icacy in Tunisian Ba ba ine. Despi e i s
g ea impo ance he gene ic mechanisms o he high p oli icacy
ai in domes ic sheep a e s ill poo ly unde s ood, pa ly due
o sho age o s udies conduc ed ac oss mul iple p oli ic sheep
b eeds. To da e, nume ous ecundi y-associa ed mu a ions ha e
been iden i ied in di e en sheep b eeds, bu e y ew mu a ions
ha e been consis en ly de ec ed ac oss he b eeds. Despi e he
ep oduc ion o ewes can be a ec ed by he complex in e ac ions
o en i onmen al condi ions (i.e., clima e, densi y, and ood
abundance) (Wilson e al., 2009), p e ious s udies sugges ed ha
gene ic ac o could play impo an oles in he a iable li e size
o ewes.
In his s udy, we conduc ed mul iple independen genome-
wide associa ion s udies (GWAS) on li e size in he sheep
b eeds o high (Wadi, Hu, Icelandic, Finnsheep, and Romano )
and low (Texel) p oli icacy wi h a li e size anging om 1 o
6 om di e en geog aphic egions (Figu e 1A) and gene ic
o igins (Figu e 1B) o he wo ld, espec i ely. Wadi sheep is
a high-p oli icacy na i e b eed om he Shandong P o ince
o China (Peng e al., 2017). Hu sheep is amous o ea ly
sexual ma u i y and high ecundi y, and a e dis ibu ed in he
Taihu Lake a ea o Eas e n China (Yue, 1996). Icelandic and
TABLE 1 | Gene ics a ian s associa ed wi h he ecundi y in sheep.
Gene Mu a ion Name, allele symbol Founde b eeds Re e ence
BMP15 V299D In e dale, FecXI Romney, In e dale Galloway e al., 2000
Q291Te Hanna, FecXH Romney Galloway e al., 2000
S367I Belcla e, FecXB Belcla e Han ahan e al., 2004
Q239R Galway, FecXG Belcla e, Camb idge,
Small- ailed Han
Han ahan e al., 2004
C321Y Lacaune, FecXL Lacaune Bodin e al., 2007
1P154S159 Rasa A agonesa, FecXR Rasa A agonesa Ma inez-Royo e al.,
2008;Mon eagudo
e al., 2009
T317I G i e e, FecXG G i e e (F ance) Dema s e al., 2013
N337H Olkuska, FecXO Olkuska (Poland) Dema s e al., 2013
c.301G >T,
c.310insC,
c.302_304delCTA
Ba ba ine, FecXBa Tunisian Ba ba ine Lassoued e al., 2017
Unknown Woodlands, FecXW Woodlands Fea y e al., 2007
BMPR1B Q249R Boo oola, FecBB Boo oola Me ino,
Ga ole, Ja anese,
Small- ailed Han,
Wadi, Hu
Mulsan e al., 2001;
Souza e al., 2001;
Wilson e al., 2001;
Chu e al., 2011;Zhang
e al., 2011;Cao e al.,
2016
GDF9 S395F High Fe ili y, FecGH Belcla e, Camb idge Han ahan e al., 2004
S427R Thoka, FecGT Icelandic Nicol e al., 2009
F345C Emb apa, FecGE San a Ines Sil a e al., 2011
V371M FecGF No wegian Whi e
Sheep, Finnsheep
Land ace, Belcla e
Vage e al., 2013;
Mullen and Han ahan,
2014
R315C Vaca ia, FecGV B azilian sheep de Souza e al., 2012
R87H FecGI Baluchi Mo adband e al., 2011
B4GALNT2 Lacaune, FecLL Lacaune D ouilhe e al., 2013
Woodlands Wood-land, FecX2W Coopwo h Da is, 2005
OLKUSKA Olkuska Da is, 2004
BELLE-ILE Belle-Ile Da is, 2005
Unknown FecW Da is e al., 2006b
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Xu e al. Gene ic Basis o Sheep Rep oduc ion
FIGURE 1 | (A) Geog aphic loca ions o i e sheep b eeds o high (WAD, Wadi sheep; HUS, Hu sheep; ICE, Icelandic sheep; FIN, Finnsheep; and ROM, Romano
sheep) and one low (TEX, Texel sheep) p oli icacy. (B) Neighbo -joining ee o he six sheep b eeds wi h 1000 boo s ap eplica es.
Finnsheep (Finnish Land ace) sheep a e no he n Eu opean
high- ecundi y b eeds (Mullen and Han ahan, 2014;Ei iksson
and Sigu dsson, 2017). Romano sheep om he Volga Valley
shows ou s anding ep oduc ion quali ies: ea ly sexual ma u i y,
ou -o -season b eeding and ex ao dina y p oli icacy (Denisko a
e al., 2017). The Texel sheep is a ela i ely low-p oli icacy b eed
o iginally om he island o Texel in he Ne he lands and excels
in muscle g ow h and lean ca casses (Casas e al., 2004). Ou
esul s will be impo an o u he gene ic imp o emen o
he ai and o be e unde s anding he molecula basis o
ep oduc ion in sheep as well as o he mammals.
MATERIALS AND METHODS
Sample Collec ion and Pheno yping
A o al o 522 ewes om i e sheep b eeds o high (Wadi,
n= 160; Hu, n= 117; Icelandic, n= 54; Finnsheep, n= 54;
and Romano , n= 78) and one low (Texel, n= 59) p oli icacy
we e collec ed om a ms in China, Iceland, Finland, and Russia
(Figu e 1A). Animals included we e as un ela ed as possible
based on analysis o pedig ee eco ds and a me s’ knowledge.
Da a o he pheno ype o li e size and he o al numbe o
li e s collec ed om a m eco ds a e shown in Figu e 2. The
li e size anged om 1 o 6 based on pa i y om 1 o 11
in six sheep b eeds. Genomic DNA was ex ac ed om he ea
ma ginal issues ollowing a s anda d phenol/chlo o o m me hod
and was dilu ed o 50 ng/µl o he SNP BeadChip geno yping
(Köchl e al., 2005), excep o he Icelandic samples which we e
isola ed om whole-blood using Mas e Pu eTM Comple e DNA
Pu i ica ion Ki (Epicen e Bio ech) ollowing he manu ac u e s
p o ocol.
Geno yping and Quali y Con ol
All he samples we e geno yped using he O ine In inium HD
BeadChip acco ding o he manu ac u e ’s p o ocol. Geno ypes o
FIGURE 2 | Pheno ypic dis ibu ion o li e size in he six sheep b eeds (WAD,
Wadi sheep; HUS, Hu sheep; ICE, Icelandic sheep; FIN, Finnsheep; ROM,
Romano sheep; and TEX, Texel sheep).
a o al o 606,006 SNPs we e ob ained (geno ype and pheno ype
da ase s1). We implemen ed quali y con ol o hese SNPs using
PLINK 1.07 so wa e (Pu cell e al., 2007). The SNPs o
indi iduals we e excluded i hey me any o he c i e ia: (1) no
ch omosomal o physical loca ion, (2) call a e <0.95, (3) missing
geno ype equency >0.05, and/o (4) mino allele equency
(MAF) <0.05. SNPs we e excluded om he analysis i a p- alue
o Fishe ’s exac es o Ha dy–Weinbe g equilib ium less han
0.001.
Gene ic Rela ionships and Popula ion
S uc u e
To in es iga e he gene ic ela ionships and popula ion s uc u e
among he six domes ic sheep, we pe o med global FST,
1h ps://www.animalgenome.o g/ eposi o y/pub/CAAS2018.0302/
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Xu e al. Gene ic Basis o Sheep Rep oduc ion
neighbo -joining (NJ) ee and p inciple componen analysis
(PCA). The global FST alue was calcula ed using GENEPOP
4.2 (Raymond and Rousse , 1995). The gene ic dis ances
be ween popula ions we e calcula ed using an iden i y by s a e
(IBS) simila i y ma ix (Kang e al., 2010). Then, he dis ances
we e used o cons uc a NJ ee wi h 1000 boo s aps using
he package PHYLIP .3.695 (Felsens ein, 1989). In addi ion,
PCA was conduc ed using he Sma PCA p og am om he
EIGENSOFT package e sion 4.2 (Pa e son e al., 2006) based
on he geno ypes da a.
Genome-Wide Associa ion Analysis
To explo e gene ic s uc u e wi hin he b eeds, mul idimensional
scaling (MDS) analysis was pe o med based on he independen
SNPs using PLINK 1.07. Fi s ly, we implemen ed he op ion
o ‘indep-pai wise 50 5 0.05’ in PLINK 1.07, which calcula ed
pai wise linkage disequilib ium (LD) in a 50-SNP-window shi ed
a a pace o i e SNPs. I he LD es ima e was 2>0.05, one o he
pai s o SNPs was emo ed (Pu cell e al., 2007). The independen
SNPs e ained by he LD c i e ia we e hen used in he MDS
analysis, and he esul s we e plo ed using he GenABEL package
in R 3.2.2 (Aulchenko e al., 2007).
We pe o med genome-wide associa ion s udies wi hin i e
sheep b eeds o high p oli icacy (Wadi, Hu, Icelandic, Finnsheep,
and Romano ) and one low p oli icacy (Texel) using he
case/con ol design. We anked all indi iduals wi hin he b eeds
acco ding o hei li e size om he highes o lowes . Then, we
selec ed indi iduals om wo ails o each b eed as ‘case’ and
‘con ol,’ espec i ely. Based on he dis ibu ion o pheno ypes,
114 samples (LS ≥2) in Wadi, 66 samples (LS ≥2) in Hu,
20 samples (LS >2) in Icelandic, 37 samples (LS ≥2.5) in
Finnsheep, 40 samples (LS ≥2.5) in Romano and 28 samples
(LS ≥1.6) in Texel sheep we e selec ed as ‘cases,’ while 28 samples
(LS = 1) in Wadi, 15 samples (LS = 1) in Hu, 15 samples
(LS ≤1.75) in Icelandic, 9 samples (LS ≤2) in Finnsheep, 26
samples (LS ≤2) in Romano and 14 samples (LS ≤1.33) in
Texel sheep we e selec ed as ‘con ols.’ In he GWAS, we used
he unc ion o “q sco e” in he GenABEL package. Associa ed
SNPs we e iden i ied a bo h he genome-wide and ch omosome-
wise signi icance le els (p<0.05) a e he Bon e oni co ec ion
(Bon e oni, 1936). To accoun o sys ema ic biases caused by
wi hin-popula ion subs uc u e, he i s and second dimensions
om he MDS analyses we e used as he co a ia es (P ice e al.,
2006). The co ela ion analysis be ween li e size and pa i y
wi hin b eeds showed ha he e we e signi ican e ec s be ween
li e size and pa i y in ou b eeds (Wadi, Hu, Icelandic, and
Texel), and he e ec o pa i y 1 on li e size was less han
ha o pa i ies 2 h ough 10 (Supplemen a y Table S1 and
Supplemen a y Figu e S1). Howe e , he pa i y o indi iduals
wi hin b eeds was di e en , and we mainly ocused on he mean
o li e size o indi idual ( o al li e size/pa i y) in pe b eed.
The e o e, we excluded he e ec o pa i y om he model.
The Quan ile–Quan ile (Q–Q) plo s we e isualized by plo ing
he dis ibu ion o ob ained s. expec ed genome-wide p- alues.
Fo geno ype e ec o po en ial SNPs on li e size in each
b eed, di e ences be ween means we e analyzed by he S uden ’s
- es . The p<0.05 was conside ed s a is ically signi ican . All
he esul s we e p esen ed as mean ±s anda d e o (SE).
We implemen ed pai wise es s o linkage disequilib ium (LD)
be ween he mos signi ican SNPs and hei lanking SNPs wi hin
app oxima ely 1 Mb ups eam and downs eam using PLINK
1.07. Regional associa ion plo s we e gene a ed using he R
package 3.2.2.
Bioin o ma ics Analysis
We anno a ed he genes associa ed wi h li e size in each b eed
using he O. a ies assembly Oa _ .4.02. Fu he , we submi ed
he genes o he DAVID (da abase o anno a ion, isualiza ion
and in eg a ed disco e y) da abase3 o gene on ology (GO)
en ichmen and pa hways analyses (Huang e al., 2009a,b). The
p- alue o 0.1 and a leas wo genes om he inpu gene lis in he
en iched ca ego y we e conside ed o he en iched GO e ms.
Also, we in es iga ed he p o ein–p o ein in e ac ion ne wo k
o he candida e genes using he STRING da abase e sion 10.5
(Szkla czyk e al., 2017). In addi ion, di e en ial exp essions o
he candida e genes in a ious issues we e examined using he
EMBL-EBI Exp ession A las da abase4(Pe yszak e al., 2016).
RESULTS
Popula ion Rela ionship and
Di e en ia ion
Pai wise FST alue a ied om 0.023 o 0.104 among he
popula ions wi h he leas gene ic di e en ia ion obse ed
be ween Wadi and Hu sheep b eeds (Supplemen a y Table S2).
The NJ ee showed ha hese b eeds we e clus e ed in o wo
majo g oups acco ding o hei Chinese and Eu opean o igins
(Figu e 1B). A simila geog aphic pa e n was seen in he PCA
analyses wi h he g ouping o Wadi and Hu sheep sepa a ed om
he o he ou Eu opean b eeds (Supplemen a y Figu e S2).
Genome-Wide Associa ion Analysis
A e he quali y con ol, 508,444 SNPs and 114 indi iduals (91
cases s. 23 con ols) in Wadi, 506,031 SNPs and 80 indi iduals
(66 cases s. 14 con ols) in Hu, 443,125 SNPs and 23 indi iduals
(8 cases s. 15 con ols) in Icelandic, 492,165 SNPs and 37
indi iduals (28 cases s. 9 con ols) in Finnsheep, 465,794 SNPs
and 38 indi iduals (29 cases s. 9 con ols) in Romano , 475,955
SNPs and 39 indi iduals (28 cases s. 11 con ols) in Texel
sheep we e e ained in he wo king da ase o he GWAS. We
did ind se e al animals ou lying he clus e s o cases, which
migh cause biases in he associa ion analyses (Supplemen a y
Figu e S3). We ha e epea ed he associa ion analyses wi hou
hese animals, and ound he esul s a e e y simila . Thus, we
did no exclude hese animals in he associa ion analyses due
o he small sample size o he b eeds. The esul ing genomic
in la ion ac o s we e equal o 1.07 in Wadi, 1.14 in Hu, 1.12
in Icelandic, 1.14 in Finnsheep, 1.10 in Romano , and 1.05 in
2h p://www.ncbi.nlm.nih.go /genome? e m=o is%20a ies
3h ps://da id.nci c .go /
4h ps://www.ebi.ac.uk/gxa/home/
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FIGURE 3 | Manha an plo s o GWAS a e shown on (A) Wadi, (B) Hu, (C) Icelandic, (D) Finnsheep, (E) Romano and (F) Texel sheep. The 5% genome-wide
signi ican h eshold alue is indica ed by a do ed line. The signi ican SNPs su ounding he genes p e iously epo ed o be associa ed wi h ep oduc ion a e
anno a ed a he ch omosome-wise and genome-wide 5% signi icance a e he Bon e oni co ec ion.
Texel sheep, sugges ing well-con olled popula ion s a i ica ions
(Supplemen a y Figu e S4).
In Wadi sheep, we de ec ed 59 and 8 SNPs a he ch omosome-
wise and genome-wide (p<1.92 ×10−6) 5% signi icance
a e he Bon e oni co ec ion, espec i ely (Figu e 3A and
Supplemen a y Tables S3,S4). We obse ed a high le el o LD
be ween he op signi ican SNP s416717560 and s421635584
loca ed in gene BMPR1B (Figu e 4A). Fo he SNP s416717560,
a e age li e size o indi iduals wi h he G/G geno ype (n= 115,
LS = 2.05 ±0.06) was signi ican ly (p<0.01) highe han
ha o he ewes wi h he A/G (n= 15, LS = 1.47 ±0.16)
geno ype (Figu e 5A). Also, we ound h ee addi ional signi ican
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Xu e al. Gene ic Basis o Sheep Rep oduc ion
FIGURE 4 | Plo s o egional associa ion esul s o he op signi ican SNP ( ed squa e) and hei nea SNPs in (A) Wadi, (B) Hu, (C) Icelandic, (D) Finnsheep,
(E) Romano , and (F) Texel sheep. Di e en colo s ep esen he 2 alues o pai -wise LD es ima es.
SNPs ( s429416173, s402803857, and s160917020) neighbo ing
genes BMPR1B,FBN1, and MMP2 (Table 2 and Supplemen a y
Table S3).
In Hu sheep, we iden i ied 98 and 9 SNPs a he ch omosome-
wise and genome-wide (p<2.18 ×10−6) 5% signi icance
a e Bon e oni co ec ion (Figu e 3B and Supplemen a y
Tables S3,S4). The egional plo showed ha he op signi ican
SNPs s429755189 and s420460180 on ch omosome 17 we e in
an LD block ha con ained gene GRIA2 (Figu e 4B). Fo he
s429755189, a e age li e size o indi iduals wi h he geno ypes
G/G (n= 38, LS = 1.99 ±0.07) and A/G (n= 52, LS = 1.94 ±0.06)
we e signi ican ly (p<0.001) highe han ha o ewes wi h he
geno ype A/A (n= 20, LS = 1.40 ±0.09) in he p esen popula ion
(Figu e 5B). Among hese signi ican SNPs, 3 ( s406357666,
s427436644 and s412185353) a e loca ed wi hin he genes
SMAD1 and CTNNB1 (Table 2 and Supplemen a y Table S3).
In Icelandic sheep, we ound 22 SNPs a he ch omosome-
wise 5% signi icance a e he Bon e oni co ec ion (Figu e 3C
and Supplemen a y Tables S3,S4). The op signi ican SNP
s429836421 on ch omosome 3 was loca ed wi hin gene NCOA1
(Figu e 4C). Fo s429836421, a e age li e size o indi iduals
wi h he A/G geno ype (n= 19, LS = 2.03 ±0.05) is signi ican ly
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FIGURE 5 | Geno ypic dis ibu ions o he op signi ican SNPs o he li e size (LS) pheno ype in (A) Wadi, (B) Hu, (C) Icelandic, (D) Finnsheep, (E) Romano , and
(F) Texel sheep, espec i ely. The means LS we e calcula ed o a ious b eeds. Numbe o ewes pe g oup o geno ype is men ioned. Pai wise s a is ical
compa isons be ween means o geno ype’s clades we e pe o med using S uden ’s - es . ∗p<0.05, ∗∗p<0.01, ∗∗∗ p<0.001.
(p<0.05) highe han ha o he ewes wi h he geno ype A/A
(n= 33, LS = 1.81 ±0.04) (Figu e 5C).
In Finnsheep, we de ec ed 102 and 6 SNPs a he ch omosome-
wise and genome-wide (p<3.64 ×10−6) 5% signi icance
a e he Bon e oni co ec ion, espec i ely (Figu e 3D and
Supplemen a y Tables S3,S4). The egional plo e ealed
s ong LD be ween he op signi ican SNP s412280524 and
i s neighbo ing SNPs s401960737 and s407751830 ha bo ed
gene INHBB (Figu e 4D). Fo he SNP s412280524, li e size
o ewes wi h he geno ype A/A (n= 40, LS = 2.84 ±0.09)
is signi ican ly (p<0.001) highe han ha o he ewes wi h
he geno ype A/G (n= 13, LS = 2.08 ±0.16) (Figu e 5D).
Also, i e addi ional signi ican SNPs ( s160509574, s417444297,
s404890873, s401746929, and s402764237) we e ound o be
loca ed nea o genes FLT1,NF1,PTGS2, and PLCB3 (Table 2 and
Supplemen a y Table S3).
In Romano sheep, we iden i ied 77 and 2 SNPs a he
ch omosome-wise and genome-wide (p<4.56 ×10−6)
5% signi icance a e he Bon e oni co ec ion (Figu e 3E
and Supplemen a y Tables S3,S4). The op signi ican SNP
s423810437 on ch omosome 7 was in he gene ESR2 (Figu e 4E).
Fo s423810437, li e size o ewes wi h he geno ype A/A (n= 69,
LS = 2.50 ±0.06) is signi ican ly (p<0.001) highe han ha
o he ewes wi h he geno ype A/G (n= 8, LS = 1.79 ±0.18)
(Figu e 5E).
In Texel sheep, we obse ed 133 SNPs a he ch omosome-
wise 5% signi icance a e he Bon e oni co ec ion (Figu e 3F
and Supplemen a y Tables S3,S4). The egional plo showed
ha he op signi ican SNPs s161146164 and s413776054 on
ch omosome 16 we e in a s ong LD egion con aining one
unc ional gene GHR (Figu e 4F). Fo s161146164, li e size
o ewes wi h he geno ype A/A (n= 53, LS = 1.64 ±0.05)
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TABLE 2 | Genome-wide and ch omosome-wise signi ican SNPs and associa ed genes.
Popula ion SNP Ch Posi ion (bp) MAF p-unadjus ed p-adjus ed Genes Loca ion
Wadi s416717560∗6 29295803 0.07 3.65E-08 8.19E-09 BMPR1B130UTR
s421635584∗6 29361782 0.05 4.36E-06 9.78E-07 BMPR1B1In on
s429416173 6 29302788 0.2 7.55E-05 2.75E-05 BMPR1B1CDS
s402803857 7 58598895 0.1 4.96E-05 2.93E-05 FBN11In on
s160917020∗14 23133427 0.19 1.10E-06 3.71E-07 MMP2 Downs eam
Hu s429755189∗17 41621298 0.43 1.94E-06 3.21E-07 GRIA21In on
s420460180 17 41621269 0.29 8.50E-06 2.43E-06 GRIA21In on
s406357666 17 12487861 0.19 1.40E-05 2.66E-05 SMAD11In on
s427436644 19 13639996 0.32 7.69E-05 2.14E-05 CTNNB1 Downs eam
s412185353 19 13641870 0.33 1.51E-04 4.49E-05 CTNNB1 Downs eam
Icelandic s429836421 3 32030054 0.16 4.55E-05 3.63E-05 NCOA11In on
Finnsheep s412280524∗2 184578329 0.09 2.62E-05 5.32E-07 INHBB Downs eam
s401960737∗2 184579671 0.09 2.62E-05 5.32E-07 INHBB Downs eam
s160509574 10 31933001 0.27 1.50E-05 4.71E-05 FLT11In on
s417444297 11 18552961 0.11 4.20E-05 5.65E-05 NF1 Downs eam
s404890873 12 65662842 0.05 1.87E-04 1.59E-05 PTGS2 Ups eam
s401746929 21 41915064 0.08 1.85E-03 1.75E-04 PLCB3 Ups eam
s402764237 21 41919836 0.08 1.85E-03 1.75E-04 PLCB3 Ups eam
Romano s423810437∗7 73335157 0.07 1.65E-05 3.12E-06 ESR2150 lanking egion
Texel s409969387 8 75353388 0.08 1.11E-03 1.21E-04 ESR1 In on
s410595930 14 23645021 0.06 1.33E-04 1.46E-04 SPP11In on
s401207152 14 25147418 0.06 1.33E-04 1.46E-04 MMP15 Downs eam
s161146164 16 31834495 0.06 1.33E-04 9.11E-06 GHR1CDS
s413776054 16 31834942 0.06 1.33E-04 9.11E-06 GHR CDS
s426666828 16 31882869 0.18 1.88E-04 7.54E-05 GHR1In on
s413148060 21 30950537 0.15 1.02E-04 4.17E-05 ETS1 Ups eam
s405994606 21 31001548 0.15 1.02E-04 4.17E-05 ETS11In on
s161612044 21 31009743 0.14 5.41E-04 1.01E-04 ETS11In on
s412251543 21 31178275 0.1 4.01E-03 1.46E-04 ETS1/FLI1 Ups eam/Downs eam
Fo genes he bes SNP o which is loca ed ou side o ups eam/downs eam 150 kb egion. Ch ., ch omosome; MAF, Mino Allele F equency. The p-unadjus ed
co esponds o exac p o he Fishe ’s es . The p-adjus ed co esponds o he co ec ed signi icance o GWAS a e p inciple componen adjus men . The SNPs wi h
symbol (∗) deno e ha bon e oni-co ec ed genome-wide signi ican SNPs. The genes wi h symbol (1) deno e ha he SNPs a e in agenic, o he wise hey a e he nea es
genes ups eam and downs eam o he es ed SNPs.
is signi ican ly (p<0.01) highe han ha o he ewes wi h
he geno ype A/C (n= 6, LS = 1.15 ±0.14) (Figu e 5F).
The wo mu a ions ( s161146164, Asn >His; s413776054,
P o >Se ) cause he amino acid change in coding egion o he
GHR gene. In addi ion, we ound eigh addi ional signi ican
SNPs ( s426666828, s409969387, s410595930, s401207152,
s413148060, s405994606, s161612044, and s412251543)
su ounding genes ESR1,ETS1,FLI1,SPP1, and MMP15
(Table 2 and Supplemen a y Table S3).
In addi ion o he sou ce b eed whe e he a ge SNPs
ha e been de ec ed, we u he assessed geno ype e ec o
he mos signi ican SNPs on li e size in he o he i e
sheep b eeds. In gene al, geno ypes o he a ge SNPs did
no show signi ican associa ion wi h inc eased li e size
in he b eeds o he han he sou ce b eed (Supplemen a y
Table S7). Ne e heless, we obse ed some excep ions. Fo
example, he geno ype A/G o s429836421, which was iden i ied
in Icelandic sheep, showed signi ican associa ions wi h inc eased
li e size in bo h Icelandic and Hu sheep b eeds. Howe e ,
a lack o homozygo es o he SNPs such as he geno ype
G/G o s412280524 in Finnsheep, G/G o s423810437 in
Romano and C/C o s161146164 in Texel sheep could be
because o low equency o he mu a ions and small sample
size.
Bioin o ma ics Analysis
We ound signi ican ly (p<0.1) en iched GO e ms associa ed
wi h ep oduc ion o he candida e genes. The GO clus e s
we e p ima ily en iched in he ca ego ies o o a ian and oocy e
de elopmen (PTGS2,BMPR1B,INHBB,CTNNB1,MMP2,
MMP15,FBN1,GHR, and SPP1), phospholipase C ac i i y (FLT1
and ESR1), SMAD p o ein (INHBB and SMAD1) and BMP
signaling (SMAD1 and BMPR1B) and posi i e egula ion o
ansc ip ion (NCOA1,FLI1,ESR1,ESR2,CTNNB1,ETS1, and
BMPR1B), all o which a e in ol ed in he olliculogenesis,
ollicle g ow h and g anulosa cell p oli e a ion (Figu e 6
and Supplemen a y Table S5). Ano he ele an GO ca ego y
was hindb ain de elopmen (SMAD1 and CTNNB1), which
pa icipa ed in egula ing o ula ion (Bai d e al., 2006). In
addi ion, we de ec ed 11 genes (i.e., PLCB3,ESR1,ESR2,
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FIGURE 6 | Gene on ology (GO) en ichmen s based on he unc ional genes su ounding he signi ican SNPs a he ch omosome-wise 5% le el.
FIGURE 7 | P o ein–p o ein in e ac ion ne wo ks iden i ied by using STRING
da abase. Each line indica ed known signaling pa hways and p o ein
complexes.
MMP2,NCOA1,CTNNB1,INHBB,SMAD1,BMPR1B,PTGS2,
and GRIA2) in ol ed in es ogen, hy oid ho mone, TGF-be a,
e og ade endocannabinoid and hippo signaling pa hways, and
hese pa hways played impo an oles in egula ing ollicle
g ow h and o ula ion in li es ock (Supplemen a y Table S5).
Howe e , we obse ed di e en GO e ms o he candida e genes
in di e en sheep b eeds. Fo example, I-SMAD binding we e
en iched in Hu sheep, and ch oma in binding we e en iched
in Texel sheep (Supplemen a y Table S6). In he gene ne wo k
analysis, we obse ed ha 16 genes (i.e., BMPR1B,FBN1,MMP2,
SMAD1,CTNNB1,GRIA2,NCOA1,FLT1,NF1,PTGS2,PLCB3,
ESR2,ESR1,ETS1,SPP1, and GHR) showed p o ein–p o ein
in e ac ions in he ne wo k (Figu e 7). Exp ession da a u he
showed ha he genes BMPR1B,FBN1,MMP2,GRIA2,SMAD1,
CTNNB1,NCOA1,NF1,FLT1,PTGS2,PLCB3,ESR2,ESR1,GHR,
ETS1,MMP15,FLI1, and SPP1 we e ei he highly o mode a ely
exp essed in ep oduc ion- ela ed issues such as o a y, u e ine
ce ix, placen a, co pus lu eum, ce ebellum, pi ui a y gland o
u e us in sheep (Figu e 8). Also, gene INHBB showed a high
exp ession in o a y and u e us o Mus musculus5.
DISCUSSION
In his s udy, we conduc ed mul iple independen GWAS in
di e en sheep b eeds o in es iga e he gene ic mechanisms
unde lying he li e size in sheep. Coupled wi h popula ion
ela ionship and bioin o ma ics analyses, he GWAS iden i ied
di e en genes associa ed wi h he li e size in di e en b eeds
and e ealed hei di e en ially gene ic egula ion mechanisms
associa ed wi h ollicle g ow h and o ula ion in he ep oduc ion
o ewes.
The di e se biological pa hways iden i ied om he no el
genes anno a ion play an impo an ole in ollicle g ow h and
o ula ion o emales in di e en sheep b eeds (Figu e 9). The
h ee genes iden i ied in Wadi sheep, BMPR1B,FBN1, and
5h ps://www.ebi.ac.uk/gxa/home/
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