INVESTIGATION
Iden ifica ion o Rep oduc ion-Rela ed Gene
Polymo phisms Using Whole T ansc ip ome
Sequencing in he La ge Whi e Pig Popula ion
Daniel Fische ,* As a Laiho,
†
A ila Gyenesei,
‡
and Anu Si onen*
,
1
*Na u al Resou ces Ins i u e Finland (Luke), G een Technology, Animal and Plan Genomics and B eeding, FI-31600
Jokioinen, Finland, †The Finnish Mic oa ay and Sequencing Cen e, Tu ku Cen e o Bio echnology, Uni e si y o Tu ku
and Åbo Akademi Uni e si y, Tykis öka u 6, FI-20520 Tu ku, Finland, and ‡Campus Science Suppo Facili ies, Vienna
Biocen e , A-1030 Vienna, Aus ia
ORCID ID: 0000-0003-2064-6960 (D.F.)
ABSTRACT Recen de elopmen s in high- h oughpu sequencing echniques ha e enabled la ge-scale
analysis o gene ic a ia ions and gene exp ession in di e en issues and species, bu gene exp ession
pa e ns and gene ic a ia ions in li es ock a e no well-cha ac e ized. In his s udy, we ha e used high-
h oughpu ansc ip omic sequencing o he Finnish La ge Whi e o iden i y gene exp ession pa e ns and
coding polymo phisms wi hin he b eed in he es is and o iduc . The main objec i e o his s udy was o
iden i y polymo phisms wi hin genes ha a e highly and specifically exp essed in male and/o emale
ep oduc i e o gans. The di e en ial exp ession (DE) analysis unde lined 1234 genes highly exp essed in
he es is and 1501 in he o iduc . Fu he mo e, we used a no el in-house R-package hoa deR o he
iden ifica ion o no el genes and hei o hologs, which unde lined 55 addi ional DE genes based on
o hologs in he human, cow, and sheep. Iden ifica ion o polymo phisms in he da ase esul ed in a o al o
29,973 a ian s, o which 10,704 we e known coding a ian s. Fi y-se en nonsynonymous SNPs we e
p esen among genes wi h high exp ession in he es is and 67 we e p esen in he o iduc , unde lining
possible influen ial genes o ep oduc ion ai s. Se en genes (PGR,FRAS1,TCF4,ADAT1,SPAG6,PIWIL2,
and DNAH8) wi h polymo phisms we e highligh ed as ep oduc ion- ela ed based on hei biological unc ion.
The exp ession and SNPs o hese genes we e confi med using RT-PCR and Sange sequencing. The iden ified
nonsynonymous mu a ions wi hin genes highly exp essed in he es is o o iduc p o ide a lis o candida e genes
o ep oduc ion ai s wi hin he pig popula ion and enable iden ifica ion o bioma ke s o sow and boa e ili y.
KEYWORDS
o iduc
es is
gene exp ession
polymo phism
SNP
pig
ansc ip ome
RNAseq
ep oduc ion
Du ing ecen yea s, gene ic s udies ha e e ealed an inc easing num-
be o associa ed ma ke s and causa i e genes ela ed o p oduc ion
and ep oduc ion ai s in li es ock [Online Mendelian Inhe i ance in
Animals (OMIA) (Len e e al. 2006) and The Animal Quan i a i e
T ai Loci (QTL) Da abase (Animal QTLdb) (Hu e al. 2013)]. Gene ic
ma ke s ha e p o ed especially impo an o ai s whose measu e-
men is di ficul , expensi e, only possible la e in li e in he s udy
subjec s, sex-limi ed, o no possible on selec ion candida es (Da is
and Denise 1998). Ad ances in molecula gene ics ha e led o he
iden ifica ion o se e al gene polymo phisms ha ha e an economic
impac on animal p oduc ion (Clop e al. 2006; G isa e al. 2002;
Milan e al. 2000; Mu phy e al. 2006; Si onen e al. 2006, 2011; an
Lae e e al. 2003). Recen de elopmen s in high- h oughpu sequenc-
ing echniques such whole ansc ip ome sequencing (RNAseq) ha e
enabled la ge-scale analysis o gene ic a ia ions and gene exp ession
in di e en issues and species. Se e al s udies o he ansc ip ome in
humans and model species ha e inc eased ou knowledge o issue-
specific gene exp ession. In addi ion, p e ious s udies ha e p o ided
Copy igh © 2015 Fische e al.
doi: 10.1534/g3.115.018382
Manusc ip ecei ed Janua y 21, 2015; accep ed o publica ion Ap il 23, 2015;
published Ea ly Online Ap il 27, 2015.
This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons
A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/),
which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium,
p o ided he o iginal wo k is p ope ly ci ed.
Suppo ing in o ma ion is a ailable online a www.g3jou nal.o g/lookup/suppl/
doi:10.1534/g3.115.018382/-/DC1
The da ase suppo ing he esul s o his a icle is a ailable in he GEO
eposi o y wi h he accession numbe GSE59149.
1
Co esponding au ho : Na u al Resou ces Ins i u e Finland (Luke), G een
Technology, Animal and Plan Genomics and B eeding, Mylly ie 1, FI-31600
Jokioinen, Finland. E-mail: anu.si onen@luke.fi
Volume 5 | July 2015 | 1351
some ounda ion o he ansc ip ional landscape o he pig based on
mic oa ay echnology (F eeman e al. 2012). T ansc ip omic analysis
o pig ep oduc i e o gans ( he placen a and es is) (Du e al. 2014;
Es e e-Codina e al. 2011) using RNAseq ha e p o ided u he in-
sigh s in he gene exp ession o hese issues. Howe e , he gene
exp ession pa e ns and a ia ions in li es ock species emain poo ly
unde s ood. Al hough he e e ence genome is a ailable o he pig,
enabling he iden ifica ion o candida e genes o a ious ai s, de-
ailed anno a ion and iden ifica ion o issue specificexp essionpa -
e ns s ill equi e addi ional s udies. T ansc ip ome sequencing also
enables comp ehensi e analysis o gene iso o ms and no el genes.
In his s udy we ha e used high- h oughpu ansc ip omic sequenc-
ing o he Finnish La ge Whi e b eed o iden i y gene polymo phisms
and exp ession pa e ns ela ed o ep oduc ion. The da ase con ains
sequences om samples collec ed om immo ile sho - ail spe m de ec
(ISTS)-a ec ed indi iduals and con ol animals. ISTS-a ec ed boa s a e
in e ile due o immo ile and sho spe m ails (Ande sson e al. 2000;
Suku a e al. 2002). The ISTS pheno ype is caused by an al e ed splicing
pa e n o exon 30 o he SPEF2 gene, which esul s in p ema u e ans-
la ion s op codons (Si onen e al. 2006). The cause o he al e ed splicing
pa e n was shown o be a ull-leng h L1 inse ion wi hin in on 30
(Si onen e al. 2006, 2007). We ha e in es iga ed and iden ified an
associa ion be ween he L1 inse ion and li e size in he Finnish La ge
Whi e pig popula ion (Si onen e al. 2012), which may be caused by
asignifican dec ease in PRLR exp ession in he ISTS-a ec ed and ca ie
sows (Si onen e al. 2014). Because he mechanisms unde lying he
causa i e mu a ion a e known, we assume ha he ISTS mu a ion does
no induce changes in he exp ession o o he genes be ween he ISTS-
a ec ed and con ol animals. The p esen in es iga ion es ed he hy-
po hesis ha RNAseq da a om wo es is and wo o iduc samples can
be used, fi s o he iden ifica ion o highly exp essed genes in hese
issues, second o disce ning genes specifically a ec ing male o emale
ep oduc ion, and hi d o cha ac e izing gene polymo phisms wi hin
iden ified genes. These gene ic polymo phisms se e as candida e a -
ian s in associa ion analysis and consequen ly po en ial bioma ke s o
pig e ili y. Fu he mo e, we ha e used he da a o he disco e y o 55
p e iously unanno a ed genes using a no el in-house analysis pipeline.
MATERIALS AND METHODS
Animal ma e ial
Tissue samples o he es is and o iduc om Finnish La ge Whi e
pigs we e collec ed a slaugh e . The da ase con ains samples o
ISTS-a ec ed and con ol animals. P e iously, we ha e s udied he
e ec o ISTS on gene exp ession wi hin he ISTS-associa ed egion
(Si onen e al. 2006, 2007, 2014), and we ha e shown ha only he
exp ession o SPEF2 and PRLR a e a ec ed. A slaugh e , sows we e
app oxima ely 4.5 mon hs old and had no been b ed. Boa s we e
ma u e and had been used o b eeding. The o iduc and es is issue
samples we e collec ed in RNAla e bu e (Qiagen) and s o ed a 280°.
Fo RNAseq analysis, ou samples we e used: he o iduc om an ISTS
homozygous sow and a con ol sow, and he es is om an ISTS
homozygous boa and a con ol boa . Fo RT-PCR, addi ional samples
o wo ISTS and con ol sows and boa s we e collec ed.
RNA ex ac ion and lib a y p epa a ion
To al RNA was ex ac ed using RNeasy Midi ki (Qiagen) ollowing
he manu ac u e ’s ins uc ions. The quali y and concen a ions o he
RNA we e checked using he Agilen 2100 Bioanalyze (Agilen ) and
Nanod op ND-2000 spec opho ome e (The mo Scien ific). Ribo-
somal RNA was emo ed wi h Ribominus
TM
Euka yo e Ki o RNA-
seq (In i ogen), and ibosomal RNA-deple ed o al RNA was
agmen ed using RNaseIII, o con e he whole ansc ip ome
sample o RNA o a size app op ia e o SOLiD Sys em sequencing.
A e clean-up using he Pu elink
TM
RNA Mic o ki , agmen ed
RNA samples wi h su ficien yield and an app op ia e size dis ibu-
ion we e eady o p epa a ion o amplified cDNA lib a ies. Quali y
o he agmen a ion was checked wi h he Bioanalyze . The ag-
men ed RNA sample was hyb idized and liga ed wi h he Adap o
Mix. RNA popula ion wi h liga ed adap o s was e e se- ansc ibed
o gene a e single-s anded cDNA copies o he agmen ed RNA
molecules. A e a clean-up s ep using he MinElu e PCR Pu ifica-
ion Ki , he sample was subjec ed o dena u ing gel elec opho esis,
and gel slices con aining cDNA o he desi ed size ange we e ex-
cised. The size-selec ed cDNA was amplified using 15 cycles o PCR
ha akes place in he gel slices. This s ep appends equi ed e minal
sequences o each molecule and gene a es a su ficien empla e o
SOLiD sequencing. A e PCR, he amplified cDNA was cleaned
using he Pu eLink
TM
PCR pu ifica ion ki . Lib a ies we e quan i-
a ed wi h wo di e en me hods: Qubi fluo ome e (In i ogen)
and quan i a i e PCR o ensu e accu acy. The SOLiD Lib a y Taq-
Man Quan i a ion Ki was used o de e mining he mola concen-
a ion o amplified empla e in a SOLiD lib a y. In qPCR, he
s anda d and unknown lib a y empla e a e amplified using wo
sequence-specific p ime s wi h a TaqMan fluo ogenic p obe labeled
wi h FAM dye and a dye quenche . Uni o mi y o agmen size o
lib a ies was confi med wi h he Bioanalyze . Templa ed bead p ep-
a a ion was pe o med by emulsion PCR (ePCR). SOLiD EZ Bead
ins umen a ion was used o empla ed bead p epa a ion.
T ansc ip omic da a analysis
The colo space eads ob ained om he SOLiD sequence we e
aligned agains he pig e e ence genome (Sus sc o a build 10.2) using
he s anda d whole ansc ip ome pipeline and he colo space align-
men ool p o ided by Applied Biosys ems and supplied wi h he
ins umen (Li eScope 2.1). Reads associa ed wi h ibosomal RNA,
ans e RNA, epea s, and o he unin o ma i e eads we e fil e ed
ou du ing he p ocess, as we e eads wi h mo e han 10 po en ial
alignmen s. Fo mapped eads, wo misma ches pe spli we e allowed,
wi h wo alid adjacen misma ches, which a e likely o be SNPs,
and we e coun ed only as a single misma ch. A e alignmen o
he e e ence genome, low-mapping-quali y eads we e disca ded
[mapQV (,10)] and unique eads we e associa ed wi h known genes
based on UCSC anno a ions, and he numbe o eads aligned wi hin
each gene was coun ed. FPKM alues we e calcula ed ollowing no -
maliza ion o he da a o emo e a ia ion be ween samples caused by
nonbiological easons, including lib a y size and gene leng h using he
Cu flinks so wa e ( 2.0.2) (T apnell e al. 2012). Thus, he alues
gene a ed a e independen o he o al numbe o eads in each sample
and make he da a compa able ac oss he sample se . Fo di e en ial
exp ession analysis wi h he Cu flinks so wa e, an FDR o no mo e
han 0.01 and a minimum log old-change o wo was se as a h esh-
old o a gene o be conside ed di e en ially exp essed (DE) be ween
g oups.
Gene classifica ion was pe o med wi h he Pan he (P o ein
ANalysis TH ough E olu iona y Rela ionships) classifica ion sys em
(Mi e al. 2005; Thomas e al. 2003). GO en ichmen analyses o he
Cu flinks esul s we e pe o med using Ag iGO (Du e al. 2010) and
GO illa (Eden e al. 2009). Ag iGO accep ed only p o ein IDs, which
limi ed he powe o he analysis due o a low numbe o iden ified
genes wi h he co ec ID. Thus, he knowledge o human gene unc-
ion was exploi ed o he iden ifica ion o en iched biological
1352 | D. Fische e al.
p ocesses and classifica ion analysis. Fo human genes, he lis o all
Ensembl anno a ed genes was used as he e e ence lis . Associa ed
gene names we e ob ained o pig genes by Bioma .
Gene a ian de ec ion
Fo iden ifica ion o polymo phisms in he Finnish La ge Whi e
ansc ip ome, we used he Genome Analysis Toolki (GATK)
(Dep is o e al. 2011; McKenna e al. 2010; an de Auwe a e al.
2013) p og am and SNP and Va ia ion Sui e 7 (Golden Helix, Inc.,
Bozeman, MT; www.goldenhelix.com) o fil e ing and anno a ion o
he iden ified a ian s. The da a we e fil e ed based on he ead dep h
(.5), geno ype quali y (.20), and quali y sco e (.80). The possible
e ec o amino acid subs i u ions on p o ein unc ion was analyzed
using SIFT (h p://www.ensembl.o g/Tools/VEP), and he e ec on
he seconda y s uc u e o he p o ein was analyzed using he CFSSP
(h p://www.biogem.o g/ ool/chou- asman/) p edic ion ool.
Gene o holog de ec ion
No el ansc ip s de ec ed by he Cu flinks sui e we e used o iden i y
gene ically ac i e, bu so a unanno a ed, egions in he Sus Sc o a
genome. De ec ed sequences ha we e longe han 130 bp and ha
had a FPKM alue o a leas 5 o a leas one o he samples we e
blas ed agains ull genomes in he NCBI da abase “ch omosomes”
using he in-house R-package hoa deR (h p://c an. -p ojec .o g/web/
packages/hoa deR/index.h ml). Hi s ha had an iden i y a io la ge
han 0.9 we e p ocessed u he . Due o he la ge amoun o hi s,
subsequen analysis ocused on hi s in he op h ee species (bos
au us, homo sapiens, and o is a ies). The genome assemblies o he
op species used we e as ollows: Homo sapiens, CRCh38; Bos Tau us,
UMD3.1; and O is A ies, OAR3.1. Hi s we e hen c oss-checked
agains he gene anno a ions o he species so ha he hi s could be
g ouped in o in e genic, in onic, and exonic hi s. The R-package
edgeR (Robinson, McCa hy and Smy h 2010) unc ion exac Tes
was hen applied o di e en ial exp ession es ing be ween he o i-
duc and es is samples and in onic/exonic hi s. Values o FDR less
han 0.01 we e conside ed o be di e en ially exp essed.
Genomic egions wi h mul iple hi s wi hin single genes we e
u he in es iga ed using a pai wise compa ison app oach based on
sliding windows. Se ing he window size o he smalles hi leng h, he
genomic sequences o Sus Sc o a and he hi o ganism we e di ided
in o chunks o his window size, ollowed by a pai wise sequence
simila i y compa ison be ween all chunks. The pai s wi h he la ges
simila i ies we e epo ed and, o isualiza ion o he egion,
indica ed as a colo ed ba using a colo spec um be ween ed and
g een o he deg ee o simila i y. Pai s wi h low simila i y (,0.3)
we e omi ed om he co esponding isualiza ions.
RT-PCR and sequencing
Fo analysis o gene exp ession wi h RT-PCR, RNA o he es is and
o iduc samples om wo con ols and ISTS homozygous animals
we e ex ac ed (RNeasy Midi ki ; Qiagen). To al RNA was e e se-
ansc ibed wi h andom p ime s and an RT-PCR ki (ImP om-II
Re e se T ansc ip ion Sys em; P omega) acco ding o he manu ac-
u e ’s ins uc ions. Syn hesized cDNA was amplified using gene-
specific p ime s (Suppo ing In o ma ion,Table S1). The housekeeping
gene ibosomal S18 (RIBS18) was used as a e e ence gene o calcu-
la e he ela i e exp ession. cDNA samples we e dilu ed o 20 ng/ml
p io o use. The qPCR was pe o med wi h a ViiA 7 Real-Time PCR
Sys em in 96-well mic o i e pla es using Absolu e qPCR SYBR
G een ROX Mix (VWR). Amplifica ion by qPCR con ained 12.5 ml
o Absolu e qPCR SYBR G een Mix, 100 ng o cDNA, and 70 nM o
each p ime in a final olume o 25 ml. Amplifica ions we e ini ia ed
wi h 15 min o enzyme ac i a ion a 95°, ollowed by 40 cycles o
dena u a ion a 95° o 15 sec, p ime annealing a 60° o 30 sec, and
ex ension a 72° o 30 sec. All samples we e amplified in iplica e,
and he mean alue was used in u he calcula ions. Raw da a we e
analyzed wi h he sequence de ec ion so wa e (Applied Biosys ems)
and ela i e quan i a ion was pe o med wi h GenEx so wa e (Mul iD).
Ra ios be ween he a ge and e e ence gene we e calcula ed using he
mean o hese measu emen s. A s anda d cu e o each p ime pai
was p oduced by se ially dilu ing a con ol cDNA and used o co ec
o di e ences in amplifica ion. A mel ing cu e analysis was pe -
o med allowing single p oduc -specific mel ing empe a u es o be
de e mined. No p ime –dime o ma ions we e gene a ed du ing he
applica ion o 40 eal- ime PCR amplifica ion cycles.
Fo sequencing, he RT-PCR amplicons we e pu ified using
ExoSAP-IT (Ame sham Biosciences). PCR agmen s we e sequenced
in bo h di ec ions wi h he same p ime s used o amplifica ion. Se-
quencing was pe o med on a MegaBace 500 capilla y DNA sequence
(Ame sham Biosciences) using DYEnamic ET Te mina o Ki s wi h
The mo Sequenase II DNA Polyme ase (Ame sham Biosciences).
RESULTS AND DISCUSSION
Da a e alua ion
The Li e Technologies’SOLiD 4 sequencing pla o m was used o
ansc ip omic sequencing o he po cine es is and o iduc samples.
App oxima ely 70 o 80 million eads we e ob ained o each sample
(Table 1). Be ween 30% and 65% o he eads could be mapped agains
he pig genome build 10.2. Pea son’s co ela ion be ween he es is
samples was 0.94, and was 0.76 be ween he mo e he e ogeneous
o iduc samples. Hie a chical clus e ing using Euclidean me ics and
a e age linkage me hod g ouped he pa allel issue samples clea ly
oge he as expec ed. A e fil e ing low-mapping-quali y eads [mapQV
(,10)], mos o he eads we e mapped on exons (27–41%), and many
we e mapped on in e genic egions (25–33%) (Table 1), indica ing
po en ially unanno a ed exp essed genes in he pig genome.
Iden ifica ion o candida e genes o ep oduc ion in
he pig es is and o iduc
Fo calcula ion o he no malized gene exp ession alues, we used
he Cu flinks so wa e. In o al, 16,595 (FPKM .0.2) genes we e
exp essed in he es is and 15,846 genes we e exp essed in he o iduc .
Genes wi h he highes exp ession ( op 80; Table S2) in he es is we e
associa ed wi h spe ma ogenesis as expec ed (Figu e S1A). In he
o iduc , he op 80 genes highligh ed ep oduc ion, egula ion o sys-
em p ocess, emb yonic de elopmen , egula ion o ac in polyme iza-
ion, and ansla ional elonga ion (Figu e S1C). A o al o 27 genes
had high exp ession in bo h he es is and o iduc (Figu e S1B). These
genes we e no en iched significan ly owa d any GO e ms, bu
many o hem (n = 15) we e associa ed wi h me abolic p ocesses
(Figu e S1D).
Di e en ially exp essed genes in he es is and o iduc
The analysis o gene exp ession di e ences be ween he es is and
o iduc highligh ed 1234 up egula ed genes in he es is and 1501 in
he o iduc . Significan ly en iched GO e ms (P,10
25
) in he es is
included ac osome eac ion, spe m–egg ecogni ion, spe m mo ili y,
spe ma id de elopmen , cell-cycle p ocess, and spe ma ogenesis (Fig-
u e 1A). In he o iduc , mo e di e se p ocesses we e significan , in-
cluding signaling, egula ion, and de elopmen al p ocesses (Figu e
1B). The op 30 DE genes based on old change (FC) a e lis ed in
Volume 5 July 2015 | Rep oduc ion-Rela ed Po cine T ansc ip ome | 1353
Table S3. Genes wi h a high FC be ween he es is and o iduc esul ed
in en ichmen in spe m mo ili y (SMCP,TNP1), spe ma ogenesis
(INSL3,PRM1,ADAM29,SPEM), and his one exchange and nucleo-
some disassembly (HIST1H2BA,TNP1) in he es is and in muscle
con ac ion (DES,TPM2,TNNC1,ACTG2), ex acellula nega i e
egula ion o signal ansduc ion (AGTR2, LTBP1), and mesenchymal-
epi helial cell signaling (TNC,HOXA5)in heo iduc .Se e al
pseudogenes (LOC100510878,LOC100516119,LOC100519930,and
LOC100626054) and ncRNA (LOC100523888,Sp y2d1-AS1)we e
also specifically exp essed among he op 30 exp essed genes in he
es is. In o al, 47 genes wi h a known ole in ep oduc ion we e
up egula ed in he o iduc and 52 genes we e up egula ed in he
es is (Table S4). These ep oduc ion- ela ed genes appea o be
sex-specific, unde lining possible candida e genes o ep oduc i e
pe o mance in he pig. In o al, 440 genes wi h a biological ole in
ep oduc ion we e iden ified in he da ase based on human gene
names. Gene names we e iden ified and ecognized by Pan he ool
o 10,320 exp essed genes.
No el ansc ip s and gene o hologs
Cu flinks p edic ed a la ge numbe o no el single-exon ansc ip s
(56,719). Du ing he mapping o sequence eads using Li e Technol-
ogies Li eScope so wa e, spliced eads we e only included o he
anno a ed genes, which p e en ed he cons uc ion o no el mul i-
exon ansc ip s. A e fil e ing he no el ansc ip s acco ding o
a minimum leng h (.130 bp) and FPKM alue (.5), a o al o 13,660
no el candida e ansc ip s we e iden ified (Figu e 2). Fu he mo e,
Cu flinks p edic ed 38,418 new iso o ms, 1595 ansc ip s wi h gene ic
o e lap wi h e e ence genes, and 1311 ansc ip s wi h exonic o e lap
wi h e e ence on he opposi e s and (Figu e 2). Fo 448 o hese
opposi e s and ansc ip s in he o iduc and o 787 in he es is,
he FPKM was .1. Func ional analysis o hese genes did no indica e
any en iched GO e ms, bu hey do ep esen possible egula o y
sequences o exp essed genes in he es is and o iduc .
Fo he iden ifica ion o gene o hologs in he da ase , we an
a blas sea ch agains he genomic sequences o all species a ailable
in he NCBI “ch omosome”da abase. Mos hi s (simila i y .90%)
we e iden ified in he Bos Tau us assembly UMD3.1 (n = 888). The
alignmen o genomic sequences be ween he pig and h ee o he
mammalian species (human, cow, and sheep) wi h he highes
amoun o sequence hi s showed high cohe ency be ween co e-
sponding ch omosomes (Figu e S2,A–C). The amoun o no el
and common hi s be ween he op h ee species is shown in Figu e
S2D. No el gene o hologs wi h in onic o exonic hi s in hese
genomes (n = 869) we e selec ed o di e en ial exp ession analysis
be ween he es is and o iduc , which esul ed in 152 DE hi s (FDR
#0.01). These hi s we e selec ed based on he c i e ion ha he hi
sequence was comple ely included in he exon o in on o an
anno a ed gene. The numbe o exonic hi s inc eased conside ably
when pa ial exon hi s we e included (Figu e S2E).In o al,DEhi s
we e iden ified in 55 unique genes. Mos hi s we e assigned o
nucleopo in 210kDa-like (NUP210L)gene(Table S5 and Figu e
3). NUP210L appea s o be es is-specific and p obably has a ole
in spe ma id de elopmen . Anno a ion o he NUP210L gene is
incomple e in he pig genome (10.2.73), whe e only 2 ou o 40
exons in he human (Ensembl da abase) a e anno a ed. Based
on ou exp ession da a, mos anno a ed exons in he human,
cow, and sheep a e also exp essed in he pig es is (Figu e 3, A–
C). Thi y-fi e genes in he es is and 20 in he o iduc showed
high exp ession (Table S5). These included some ecen anno a-
ions, which unde line possible no el ep oduc ion- ela ed genes.
To elucida e he possible ole o hese genes, we in es iga ed
he exp ession pa e n du ing he fi s wa e o mouse spe ma o-
genesis based on ou p e ious da a (Laiho e al. 2013) in which
Figu e 1 En iched GO e ms
among up egula ed genes (P,
10
25
). (A) En iched biological p o-
cesses among up egula ed genes
in he es is. (B) En iched biologi-
cal p ocesses among up egula ed
genes in he o iduc .
nTable 1 Mapping s a is ics
To al
Reads
Mapped
Reads Mapping % Coun ed on
Exons
Coun ed on
In ons
Coun ed on
In e genic Regions Exon % In on % In e genic %
ISTS es is 81,425,334 53,794,961 66 20,498,025 2,187,936 16,451,710 38.1 4.1 30.6
WT es is 68,655,352 20,507,264 30 5,995,191 1,177,538 5,882,955 29.3 5.7 28.7
ISTS o iduc 76,914,157 45,794,708 60 11,817,207 1,999,766 18,434,196 25.8 4.4 40.3
WT o iduc 72,020,521 44,993,220 63 10,457,619 2,467,954 17,987,423 23.1 5.5 40.0
Uncoun ed eads ailed he mapping quali y fil e .
1354 | D. Fische e al.
a mouse o holog was iden ified. Two genes wi h mouse o holog
4930538K18Rik (ENSBTAG00000017387/ENSOARG00000020461)
and 4930522H14Rik (C3H1o 185/C1o 185) showed inc eased ex-
p ession du ing spe ma ogenesis, wi h he highes mRNA le el a
pos na al day (PND) 28 (Figu e 4). Each ime poin co esponds o
he appea ance o specific cell ypes in he collec ed issue sample:
spe ma ogonia a PND 7; ea ly pachy ene spe ma ocy es a PND 14;
la e pachy ene spe ma ocy es a PND 17; ound spe ma ids a PND
21; and elonga ing spe ma ids a PND 28 (Laiho e al. 2013).
4930522H14Rik appea ed o be es is-specific in he mouse, and
4930538K18Rik showed high exp ession in he o iduc and es is
(h p://www.ncbi.nlm.nih.go /UniGene). This exp ession pa e n
indica es a ole o hese genes in la e s eps o spe ma id elonga ion.
The exp ession o NUP210L also inc eased du ing he fi s wa e o
spe ma ogenesis (Figu e 4).
Gene ic a ia ions in he Finnish La ge Whi e
A e he fil e ing s eps, a o al o 29,973 a ian s we e iden ified in he
da ase , o which 10,704 we e known coding a ian s (Table 2). These
included 1672 nonsynonymous a ian s, 8 addi ional s op codons,
a loss o 7 s op codons, 3436 unknown a ian s, and 1194 splicing
a ian s. Howe e , some o he splicing a ian s a ise om mapping
e o s and he e o e equi e confi ma ion p io o use in u he
expe imen s. Two o he s op loss a ia ions ha e been p e iously
epo ed, bu 13 appea o be no el (Table 3). A e de ailed analysis,
he a ia ion wi hin MCL1 appea s o be an anno a ion and splicing
e o . Based on he compa ison o he p edic ed p o ein sequence
ansla ed om ou exp essed sequences (Figu e S3A), he pig MCL1
p o ein sequence (ENSSSCP00000007094), and human MCL1
p o ein (ENSP00000358022), he anno a ion o he pig MCL1 is
incomple e and he p o ein p edic ion is inco ec (Figu e S3B).
Fu he mo e, mapping o he egion a ound he SNRPD3 a ia ion
con ained a high numbe o misma ches and unknown bases, and
he ac ha SNRPD3 is a splicesomal gene diminishes he eliabili y
o his a ia ion. The unc ional anno a ion o he es o he genes
con aining a s opgain a ia ion e ealed se e al ep oduc ion- ela ed
genes. DAZAP1 is exp essed mos abundan ly in he es is, bu i
does appea necessa y o no mal g ow h and de elopmen in mice
(Hsu e al. 2008). Deple ion o DAZAP1 causes male and emale
s e ili y, unde lining i s impo ance in ep oduc ion. Recen s udies
Figu e 2 Flowcha o he analysis pipeline o no el
ansc ip s. Se e al fil e ing s eps we e included o he
iden ifica ion o p e iously unanno a ed exons using
a blas sea ch agains he a ailable genomes in he
NCBI ch omosome da abase. The numbe o hi s o
each species is shown in he ba cha . Species wi h he
highes numbe o hi s (cow, human and sheep) we e
selec ed o iden ifica ion o no el DE genes be ween
he es is and o iduc in he pig.
Volume 5 July 2015 | Rep oduc ion-Rela ed Po cine T ansc ip ome | 1355

Figu e 3 Conse ed genomic sequences o NUP210L be ween pig and cow, human, and sheep. (A) Compa ison o he NUP210L genomic egion
be ween he pig and cow. (B) Compa ison o he NUP210L genomic egion be ween he pig and human. (C) Compa ison o he NUP210L
genomic egion be ween he pig and sheep. Log-exp ession le els (FPKM) a e shown as ed peaks in he es is and as blue in he o iduc , and he
simila i y o he hi is indica ed wi h he deepening shades o g een. Anno a ion o he genes in he pig is p esen ed abo e he alignmen , and
hose o he human, cow, o sheep a e shown below he alignmen .
1356 | D. Fische e al.
in mice ha e also shown he impo ance o PPRC1 o ea ly em-
b yonic de elopmen (He e al. 2012). Homozygous deficien
PPRC1 mice ail o o m egg cylinde s and die be o e emb yonic
day 6.5 (He e al. 2012). CETN2 is a calcium-binding p o ein and
a s uc u al componen o he cen osome. In human cells, Cen in 2
deple ion esul s in educ ion in ciliogenesis (G ase e al. 2007),
which implica es an e ec on spe m ail o ma ion and emale e-
p oduc ion h ough o iduc cilia. O he s opgain a ia ions we e
ound in UNC45, which has a ole in HSP90-media ed myosin mo-
o domain olding (Liu, S ikakulam and Winkelmann 2008), and
he e o e may ha e an impac on mea quali y in pigs. P2RX4 has
a ole in he esponse o endo helial cells o changes in blood flow
(Yamamo o e al. 2006), and ERCC8 is equi ed o DNA epai
(Henning e al. 1995). Mu a ions wi hin ERCC8 ha e been shown
ocauseCockaynesynd ome(Be olae al. 2006; Cao e al. 2004;
Henning e al. 1995; Ridley e al. 2005), which is cha ac e ized by
g ow h ailu e, impai ed de elopmen o he ne ous sys em, pho-
osensi i i y, and p ema u e aging (Knoch e al. 2012). Howe e ,
unde s anding he influence o hese polymo phisms on pheno ype
equi es u he in es iga ion. None o he s opgain a ia ions had
he homozygous geno ype o he mu a ed allele, bu he s oploss
a ia ions allowed homozygosi y (Table S6). Thus, he pheno ypic
e ec o hese a ia ions is clea ly less d ama ic.
When compa ed o he DE gene lis s, 57 nonsynonymous SNPs
we e p esen in up egula ed genes in he es is and 67 we e p esen
in he o iduc (Table S7). In he o iduc , he 67 genes we e in ol ed
in 16 di e en biological p ocesses, including, e.g., ep oduc ion, cel-
lula componen mo phogenesis, and cell cycle. The polymo phisms
in ep oduc ion- ela ed genes we e confi med by isualiza ion
wi h he In eg a i e genomics iewe (IGV) (Robinson e al. 2011;
Tho aldsdo i , Robinson and Mesi o 2013). Th ee o iduc genes
(FRAS1,TCF4, and PGR) we e associa ed wi h ep oduc ion, and
QTL egions o sow ep oduc i e ai s ha e been p e iously iden-
ified wi hin hese egions (Table S8). FRAS1 has been localized in
emb yonic epi helial basemen memb anes in he mouse (Chio aki
e al. 2007), and mu a ions wi hin he gene ha e been shown o
cause F ase synd ome in humans (Hoe ele e al. 2013; Ogu e al.
2011; Vogel e al. 2012). PGR, a p oges e one ecep o , is highly
exp essed in he o a y and in he o iduc , which makes i a good
candida e gene o sow ep oduc ion. P oges e one is c i ical o
success ul o ula ion and o he mul i- ace ed unc ioning o he
o iduc in mammalian ep oduc ion (Akison and Robke 2012).
Se e al nonsynonymous SNPs we e iden ified wi hin PGR (Table
S8). TCF4 is a ansc ip ion ac o and is widely exp essed. In he
es is, fi e up egula ed genes (ADAT1,SPAG6,PIWIL2,PKDREJ,
and DNAH8) wi h nonsynonymous a ia ions had a classified ole
in ep oduc ion. A QTL o male ep oduc ion has only been
iden ified o he genomic egion a ound DNAH8 (ch omosome
7: 39,286,129-39,569,290). This egion was associa ed wi h epidid-
ymis weigh . In addi ion, a high numbe o addi ional QTL ha e been
associa ed wi h his egion (n = 232; h p://www.animalgenome.o g/).
DNAH8 has a ole in spe m mo ili y and has been shown o be c ucial
o male e ili y (Fossella e al. 2000; Olds-Cla ke and Johnson 1993;
Saman e al. 2002). The iden ified QTL o epididymis weigh wi hin
heDNAH8 egionmaybeinfluenced by a lowe epididymal spe m
coun due o mal o med spe m. Fu he mo e, PKDREJ appea ed o
con ain se e al nonsynonymous SNPs, including wo dele e ious based
on SIFT analysis (Table S8). PKDREJ is a male ge m cell–specificpoly-
cys in, which is equi ed o ac osome eac ion du ing spe m–egg u-
sion (Bu scheid e al. 2006; Su on e al. 2006). PKDREJ has also been
iden ified on he su ace o ejacula ed boa spe ma ozoa (Zigo e al.
2013). Thus, hese a ian s may be conside ed s ong candida e poly-
mo phisms o boa e ili y, al hough iden ifica ion o a pheno ypic
e ec equi es u he s udy.
Valida ion o he ep oduc ion- ela ed SNPs and
exp ession di e ences be ween he es is and o iduc
Di e en ial exp essions iden ified in he RNAseq da a we e alida ed
by RT-PCR o selec ed genes in he es is and o iduc issue samples.
Fo he alida ion, we selec ed DE genes associa ed wi h ep oduc ion
ha con ained nonsynonymous SNPs, because hese genes ep esen
po en ial candida e genes o an e ec in ep oduc ion ai s. We
analyzed he exp ession di e ences be ween he es is and o iduc
samples o ou genes (FRAS1,TCF4,ADAT1,andSPAG6)byqPCR
and o wo addi ional genes (PIWIL2 and DNAH8)byRT-PCRand
an aga ose gel. All genes showed a simila exp ession pa e n in he
RT-PCR analysis, as de ec ed by RNAseq (Figu e 5). Tes is-specific
genes ADAT1,SPAG6,PIWIL2,andDNAH8 exhibi ed none o ex-
emely low exp ession in he o iduc . The genes wi h highe exp es-
sion in he o iduc compa ed o he es is in he RNAseq da a
appea ed o be p esen in he es is samples, bu a a much lowe le el
(Figu e 5B). Fu he mo e, he polymo phisms wi hin genes FRAS1,
ADAT1,SPAG6,DNAH8,andPGR we e confi med by Sange se-
quencing. Thus, hese polymo phisms ep esen po en ial candida es
o gene-assis ed selec ion.
A possible e ec o he alida ed SNPs was analyzed using he
SIFT and CFSSP p edic ion ools. All nonsynonymous SNPs we e
ole a ed, bu he SIFT sco e indica ed a possible e ec on p o ein
unc ion due o he le el o conse a ion o he amino acid sequence
a he SNP loca ion o FRAS1,ADAT1,SPAG6, and PIWIL2 (SIFT
sco e ,0.35) (Table S8). The e ec on p o ein seconda y s uc u e
nTable 2 Polymo phisms iden ified in he Finnish La ge Whi e
Coding 9510
Coding, splicing 7
Downs eam 1333
Downs eam, ups eam 25
In e genic 8667
In onic 4343
Splicing 1187
UTR3 3855
UTR3, UTR5 4
UTR5 539
Ups eam 503
To al 29,973
Figu e 4 Exp ession o NUP210L and 4930522H14Rik (C3H1o 185/
C1o 185) du ing he fi s wa e o spe ma ogenesis in he mouse.
Volume 5 July 2015 | Rep oduc ion-Rela ed Po cine T ansc ip ome | 1357
was also explo ed by CFSSP, which indica ed a shi in he helix
s uc u e a he SNP posi ion o SPAG6 and FRAS1 and he emo al
o a helix in PIWIL2. Al hough he e ec o he iden ified SNPs on
p o ein unc ion and pheno ypic di e ences is ye o be in es iga ed,
ou da a sugges possible causa i e mu a ions o di e ences in e-
p oduc i e pe o mance in he La ge Whi e pig popula ion.
CONCLUSIONS
The RNAseq echnology applied in he p esen s udy p o ides new
in o ma ion ega ding he ex en o a ia ion wi hin ep oduc ion-
ela ed genes in he es is and o iduc . In addi ion o p e iously an-
no a ed genes, we de ec ed 55 p e iously unanno a ed gene o hologs in
he pig based on blas analysis agains he human, cow, and sheep
genomes. The iden ified nonsynonymous mu a ions wi hin he highly
exp essed genes in he es is and o iduc unde line he possible iden i y
o genes a ec ing he e ili y in he Finnish La ge Whi e. Se e al
s opgain a ia ions we e also de ec ed highligh ing hepo en ialhigh-
impac gene polymo phisms o ep oduc i e and o he gene ic
diso de s. Fu he mo e, we alida ed se en nonsynonymous mu a ions
in es is-specific o o iduc -specific genes, which unde line he po en ial
o hese a ian sascandida es o selec ion in ep oduc ion ai s.
ACKNOWLEDGMENTS
The assis ance o Ta ja Ho i uo i in qPCR and Anneli Vi a in Sange
sequencing is g ea ly app ecia ed. This s udy was unded by he
Academy o Finland (h p://www.aka.fi/). The unde s had no ole in
s udy design, da a collec ion and analysis, decision o publish, o
p epa a ion o he manusc ip . The au ho s decla e ha hey ha e
no compe ing in e es s.
D.F. pa icipa ed in he bioin o ma ic analysis o he da a, de eloped he
in-house R-package Hoa deR, analyzed he da a using Hoa deR, and
helped d a he manusc ip . A.L. pe o med he Solid 4 sequencing un
and pa icipa ed in he mapping and DE analysis o he da a. A.G.
pa icipa ed in he design and execu ion o he sequencing p o ocol. A.S.
designed and coo dina ed he s udy, pe o med he qPCR and Sange
sequencing and pa o he bioin o ma ic analysis, and d a ed he
manusc ip . All au ho s ead and app o ed he final manusc ip .
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
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
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
?
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
111Leuex
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
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
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
442A gex

10
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
326Glyex

11
X 141373463 ? T/G 94 18 2304 S oploss CETN2 6 c.477A .Cp.

159Cysex

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DP, ead dep h; QD, quali y by dep h.
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