Whole blood ansc ip ome analysis e eals oo p in s o ca le
adap a ion o sub-a c ic condi ions
K. Pokha el* , M. Weldenegodguad*
†
, R. Popo
‡
, M. Honka ukia*
§
, H. Huuki*, H. Lindebe g
¶
,
J. Peippo*, T. Reilas*, S. Za o nyae ** and J. Kan anen*
*P oduc ion Sys ems, Na u al Resou ces Ins i u e Finland (Luke), Mylly ie 1, Jokioinen FI-31600, Finland.
†
Depa men o En i onmen al
and Biological Sciences, Uni e si y o Eas e n Finland, P.O. Box 1627, Kuopio FI-70311, Finland.
‡
Yaku ian Resea ch Ins i u e o Ag icul u e
(FGBNU Yaku skij NIISH), ul. Bes yzhe o-Ma linskogo 23/1, Yaku sk 67001, The Sakha Republic (Yaku ia), Russia.
§
The No dic Gene ic
Resou ces Cen e (No dgen), P.O. Box 115,

As NO-1431, No way.
¶
P oduc ion Sys ems, Na u al Resou ces Ins i u e Finland (Luke),
Halolan ie 31A, Maaninka FI-71750, Finland. **GBU Saha Ag oplem, ul. O dzhonkidze 20/204, Yaku sk 67700, The Sakha Republic
(Yaku ia), Russia.
Summa y Indigenous ca le b eeds in no he n Eu asia ha e adap ed o ha sh clima e condi ions. The
local b eeds a e impo an gene ic esou ces wi h cul u al and his o ical he i ages, and
he e o e, hei p ese a ion and gene ic cha ac e iza ion a e impo an . In his s udy, we
p o iled he whole-blood ansc ip ome o wo na i e b eeds (No he n Finnca le and
Yaku ian ca le) and one comme cial b eed (Hols ein) using high- h oughpu RNA
sequencing. Mo e han 15 000 genes we e iden i ied, o which wo, 89 and 162 genes
we e signi ican ly up egula ed exclusi ely in No he n Finnca le, Yaku ian ca le and
Hols ein ca le espec i ely. The unc ional classi ica ion o hese signi ican ly di e en ially
exp essed genes iden i ied se e al biological p ocesses and pa hways ela ed o signalling
mechanisms, cell di e en ia ion and hos –pa hogen in e ac ions ha , in gene al, poin
owa ds immuni y and disease esis ance mechanisms. The gene exp ession pa e n
obse ed in No he n Finnca le was mo e simila o ha o Yaku ian ca le, despi e sha ing
simila li ing condi ions wi h he Hols ein ca le included in ou s udy. In conclusion, ou
s udy iden i ied unique biological p ocesses in hese b eeds ha may ha e helped hem o
adap and su i e in no he n and sub-a c ic en i onmen s.
Keywo ds gene exp ession, Hols ein ca le, No he n Finnca le, RNA-seq, Yaku ian ca le
In oduc ion
As a esul o na u al and human-led selec ion, domes ic
animals ha e been able o adap , su i e, be p oduc i e and
ep oduce in challenging en i onmen s (Mi kena e al.
2010). Animals in he no he n hemisphe e (Lapland,
no he n Russia and Sibe ia) a e used o ood p oduc ion
and o he socio-cul u al needs and, hus, ha e been he
basis o human li e in he No h. These animals may ha e
di e en biological capaci ies o adap o ex emes in
empe a u e, dayligh and eed a ailabili y. Howe e , he e
has been g owing in e es owa ds imp o ing local b eeds
by in oducing gene ic ma e ial om mo e p oduc i e
b eeds, wi h a p e e ence owa ds comme cial b eeds.
In ensi e b eeding and he eplacemen o na i e b eeds
wi h comme cial b eeds may appea o be ad an ageous
a i s , bu hese p ac ices will ha e long-las ing conse-
quences. In addi ion o i e e sibly losing he unique
gene ic esou ces o na i e b eeds, we will also be losing
he cul u al and his o ical he i age associa ed wi h hose
local b eeds. The signa u es o adap a ion, as well as he
his o y o o ma ion, ha a e encoded in he na i e
b eeds may also ade away (Gaoua e al. 2015). The e-
o e, he local b eeds mus be gene ically cha ac e ized
and p ese ed.
He e, we s udied wo na i e ca le b eeds (No he n
Finnca le and Yaku ian ca le) and one in e na ional b eed
(Hols ein ca le). All hese b eeds a e used o milk and mea
p oduc ion. P e ious s udies using gene ic ma ke s ha e
indica ed he gene ic dis inc i eness among hese b eeds (Li
e al. 2007; Li & Kan anen 2010). Among he h ee b eeds,
Hols ein ca le ha e high economic impo ance and ha e
he sho es adap a ion his o y. Hols ein ca le a e he mos
Add ess o co espondence
J. Kan anen, P oduc ion Sys ems, Na u al Resou ces Ins i u e Finland
(Luke), Mylly ie 1, Jokioinen FI-31600, Finland.
E-mail: [email p o ec ed]
Accep ed o publica ion 04 Feb ua y 2019
doi: 10.1111/age.12783
1
©2019 S ich ing In e na ional Founda ion o Animal Gene ics
popula dai y b eed globally, wi h an in ensi e selec ion
p og amme and high milk p oduc ion; al hough hey
o igina ed in a empe a e clima e, hey ha e adap ed o
di e en pa s o he wo ld and can su i e in a ying
clima ic condi ions. No he n Finnca le and Yaku ian ca le
ha e economic, social and cul u al alues in p oducing milk
and mea in hese ma ginal egions, ha ing oles in local
ood cul u e and gas onomy and being cul u al symbols
(Kan anen e al. 2015). The No he n Finnca le b eed is
na i e o No he n Finland and Finnish Lapland. The b eed
nea ly became ex inc du ing he 1970s bu is cu en ly
main ained (cu en census is 850 cows) h ough ac i e
in i o and in i o conse a ion ac i i y. Yaku ian ca le a e
cha ac e ized by being pu eb ed abo iginal na i e ca le.
Adul Yaku ian cows (cu en popula ion is app oxima ely
1000 animals) ypically weigh 350–400 kg, and hei heigh
is 111 cm, on a e age. The animals a e well adap ed o
ha sh Sibe ian condi ions, whe e he empe a u e alls below
50 °C in long win e s (Kan anen e al. 2009).
High- h oughpu RNA sequencing (RNA-seq) has been
p o en o be an e icien me hod o s udying gene
exp ession (Wang e al. 2009). In ecen yea s, RNA-seq
has been applied o domes ic animals, wi h a majo ocus on
p oduc i i y ai s (Bai e al. 2016; Li e al. 2016; Sil a-
Vigna o e al. 2017; Pokha el e al. 2018). A numbe o
ansc ip ome s udies ha e been conduc ed in Hols ein (Cui
e al. 2014; Sand i e al. 2015; Bai e al. 2016; Li e al.
2016; Seo e al. 2016) wi h a p ima y ocus on milk ai s,
bu o da e, he e a e no epo s on gene exp ession s udies
in ei he Yaku ian ca le o No he n Finnca le. In his
s udy, we applied RNA-seq echnology and p o iled he
whole blood ansc ip ome o he h ee a o emen ioned
b eeds o cha ac e ize hei gene ic di e ences.
Ma e ials and me hods
Sample collec ion
Animal handling p ocedu es and sample collec ion we e
conduc ed in acco dance wi h legal egula ions app o ed by
he Russian au ho iza ion boa d (FS/UVN-03/163733/
07.04.2016) and he Animal Expe imen Boa d in Finland
(ESAVI/7034/04.10.07.2015). A 2.5-ml sample o blood
om each o h ee Yaku ian ca le, h ee No he n
Finnca le and h ee Hols ein ca le cows was collec ed in o
a PAXgene
â
Blood RNA IVD ube (Re . P eAnaly iX
â
)
du ing he win e pe iod o 2016 and 2017 and s o ed a
18 °C. Blood samples we e collec ed by jugula enipunc-
u e. All animals included in his s udy we e 4- o 8-yea -
old emales, excep o one 14-yea -old Hols ein cow (HC3).
RNA ex ac ion
The RNA was ex ac ed using he PAXgene
â
Blood RNA ki
(Re . 762174, Re . P eAnaly iX
â
), acco ding o he ki
manual, wi h mino adjus men s o he p o ocol: he
samples we e hawed a oom empe a u e o e nigh , he
ini ial cen i uga ion ime a 5000 gwas inc eased o
15 min, he ini ial pelle was esuspended in double he
amoun o BR1 bu e and di ided in o wo sepa a e
eac ions, he p o einase K incuba ion ime was ex ended
o 1 h and he columns we e incuba ed a oom empe a-
u e in elu ion bu e o 10 min p io o elu ion. The
concen a ion and quali y o he RNA was measu ed wi h a
spec opho ome e (NanoD op ND-1000), and he in eg i y
o he RNA was measu ed wi h an Agilen Bioanalyze
2100 using he Agilen 6000 RNA Nano ki (Re . 5067-
1511). All samples selec ed o sequencing had an RNA
in eg i y numbe alue o a leas 7.
Lib a y p epa a ion and sequencing
Lib a y p epa a ion and sequencing asks we e ou sou ced
o he Finnish Func ional Genomics Cen e in Tu ku,
Finland. The lib a y p epa a ion was pe o med acco ding
o Illumina’s T useq
â
mRNA sample p epa a ion guide
p o ocol. The high quali y o he lib a ies was con i med
wi h an Ad anced Analy ical F agmen Analyze , and he
concen a ions o he lib a ies we e quan i ied using Qubi
â
Fluo ome ic Quan i a ion (Li e Technologies). The a e age
RNA-seq lib a y agmen s we e in he ange o 250–
350 bp. Only he good lib a ies wi h RNA quali y numbe
alue g ea e han 7 we e sequenced wi h a pai ed-end
s a egy o gene a e 75-bp eads. Illumina’s s anda d
BCL2FASTQ2 so wa e was used o base calling and adap e
imming.
Compu a ional me hods
Raw sequence eads we e p e-p ocessed using FASTQC
0.11.8 (And ews n.d.) o de e mine he quali y o he
da a and o ob ain an o e iew o he sequencing da a. The
ou pu s om FASTQC we e summa ized using MULTIQC 1.5
(Ewels e al. 2016). As he aw da a did no ha e any
adap e s and he Ph ed quali y sco e o he eads om all
samples was g ea e han 30, we did no pe o m u he
imming. P ocessed pai ed-end eads we e mapped agains
he la es e sions o he ca le e e ence genome UMD3.1
and ansc ip ome (Ensembl elease 93) using STAR 2.6
(Dobin e al. 2013). Fi s , he genome indexes we e p e-
pa ed, and mapping was pe o med wi h de aul pa ame e s
using STAR 2.6 (Dobin e al. 2013). Mo eo e , a gene-le el
coun s ile o each sample was gene a ed as pa o he
STAR-alignmen pipeline by speci ying he ‘—quan Mode
GeneCoun s’ op ion. S a is ical analysis o ead coun s we e
ca ied ou using he DESEQ2 1.20.0 (Lo e e al. 2014)
Bioconduc o package (Appendix S1). A e pe o ming
s anda d di e en ial exp ession analysis s eps using DESEQ
unc ions, genes ha had ewe han i e ansc ip coun s
we e disca ded. Di e en ial gene exp ession be ween b eed
©2019 S ich ing In e na ional Founda ion o Animal Gene ics, doi: 10.1111/age.12783
Pokha el e al.2
g oups we e es ed as pai wise compa isons (Yaku ian
ca le s. No he n Finnca le, Yaku ian ca le s. Hols ein
and Hols ein s. No he n Finnca le). To iden i y genes
signi ican ly di e en ially exp essed be ween b eed g oups,
we se an adjus ed P- alue o 0.05 (Benjamini & Hochbe g
co ec ion). Fo he lis o signi ican ly di e en ially
exp essed genes, we e ie ed addi ional in o ma ion, such
as gene desc ip ion and ch omosomal loca ion, using he
BIOMART Bioconduc o package 2.36.1 (Du inck e al. 2009;
Smedley e al. 2009). Addi ional gene in o ma ion e ie ed
o di e en ially exp essed genes we e based on Ensembl
gene anno a ions o Cow (UMD3.1, Ensembl elease 92).
Finally, he signi ican ly di e en ially exp essed genes we e
assessed o hei unc ional oles. Gene On ology (GO)
e ms and Kyo o Encyclopaedia o Genes and Genomes
(KEGG) pa hway analyses we e pe o med using he CLUEGO
2.5.3 (Bindea e al. 2009) plugin in CYTOSCAPE 3.6.1
(Shannon e al. 2003). In addi ion o he de aul pa ame e s
in CLUEGO, we chose o obse e GO e ms in he ange o le el
3 and 5. Simila ly, o he GO e m o KEGG pa hway o be
displayed, a leas h ee genes and a minimum o 4% o he
o al genes needed o be p esen on ou lis . We used all
15 960 genes exp essed in ou da a as he backg ound lis
o compa ison. Mo eo e , he e ms o pa hways sha ed by
50% o he genes om ou lis we e g ouped oge he using
Kappa s a is ics (Kappa sco e h eshold o 0.4).
Resul s and discussion
F om nine samples, we ob ained 32.2 Gb o RNA-seq da a.
Mo e han 87% o he eads om each sample mapped o
he ca le e e ence genome, wi h Hols ein samples ha ing
he highes (91.6%) mapping a e ollowed by Yaku ian
ca le (88%) and Finnca le (87.9%). The sligh ly highe
mapping a e o Hols ein samples could be because ha
b eed is close o he He e o d b eed, which is he sou ce o
he e e ence assembly (Zimin e al. 2009). Wi h sequencing
cos s becoming cheape , ha ing b eed-speci ic e e ence
genomes would be use ul in he u u e.
The 15 960 o al numbe o genes exp essed in ou da a
(Table S1) co e 64.8% o known (n=24 616) ca le genes
epo ed in he anno a ion ile. A he indi idual le el, we
obse ed ha bo h he highes (FC8) and he lowes (FC7)
numbe o genes we e exp essed in No he n Finnca le
samples (Table 1). We belie e ha a compa a i ely lowe
numbe o eads in he sample FC7 led o a lowe numbe o
exp essed genes. We did no obse e any globin genes
among he op mos exp essed genes. Genes such as MHC
class I hea y chain (BOLA), uncoupling p o ein 2 (UCP2),
euka yo ic ansla ion elonga ion ac o 2 (EEF2) and imen in
(VIM) we e among he op mos exp essed genes. These
esul s con i m ha RNA ex ac ion using PAXgene
â
wo ked well.
We assessed he co-exp ession s a us o he op 500 mos
exp essed genes (i.e. he genes wi h highes base mean
alues) in each o he h ee b eed g oups. As seen in he
Venn diag am (Fig. 1), 386 o he op exp essed genes we e
p esen in all h ee b eeds. Hols ein samples had he highes
numbe (n=65) o unique genes, ollowed by Yaku ian
ca le (n=56) and No he n Finnca le (n=17). Mo eo e ,
he esul s showed ha No he n Finnca le had mo e
simila gene exp ession p o iles wi h Yaku ian ca le (53
uniquely sha ed genes) han wi h Hols ein ca le (44
uniquely sha ed genes). In con as , Yaku ian ca le and
Hols ein ca le sha ed he leas numbe o unique genes.
We hen looked mo e closely in o he genes ha we e
di e en ially exp essed be ween any wo o he h ee b eed
g oups and assessed wi h wha ypes o biological p ocesses
and/o biological pa hways hose genes we e associa ed.
Di e en ial gene exp ession be ween Yaku ian ca le
and No he n Finnca le
A o al o 189 ansc ip s we e signi ican ly di e en ially
exp essed be ween Yaku ian ca le and No he n Finnca le
(Table S2), o which 108 ansc ip s we e up egula ed in
Yaku ian ca le. The lis o di e en ially exp essed genes
included a numbe o cases o which he Ensembl IDs lack
gene desc ip ions. Ha ing a be e anno a ion would
ce ainly help in cha ac e izing he unc ions o hese
genes. The GO e ms associa ed wi h he up egula ed genes
in Yaku ian ca le included ‘na u al kille cell chemo axis’,
‘nega i e egula ion o i al p ocess’, ‘nega i e egula ion o
cy okine p oduc ion’ and ‘g anzyme-media ed apop o ic
Table 1 Sample summa y.
Sample ID B eed ENA accession no. No. o eads (million) Uniquely mapped eads % No. o genes exp essed
FC7 No he n Finnca le ERS2639473 49.4 87.2 12 106
FC8 No he n Finnca le ERS2639474 107.6 87.1 15 038
FC9 No he n Finnca le ERS2639475 89.4 89.7 14 811
HC1 Hols ein ERS2639476 104.4 91.7 14 768
HC2 Hols ein ERS2639477 62.8 91.0 14 140
HC3 Hols ein ERS2639478 62.4 92.4 13 831
YC6 Yaku ian ERS2639479 69.2 87.3 14 047
YC10 Yaku ian ERS2639480 69.8 88.2 13 736
YC11 Yaku ian ERS2639481 65.8 88.5 14 371
©2019 S ich ing In e na ional Founda ion o Animal Gene ics, doi: 10.1111/age.12783
Blood ansc ip omes o h ee ca le b eeds 3
signalling pa hway’ (Fig. 2a & Table S3). The KEGG
pa hways associa ed wi h up egula ed genes included
‘an igen p ocessing and p esen a ion’, ‘na u al kille cell
media ed cy o oxici y’ and ‘g a - e sus-hos disease’
(Table S4). Simila ly, he GO e ms associa ed wi h up eg-
ula ed genes in No he n Finnca le included ‘ egula ion o
lipid anspo ’, ‘e y h ocy e di e en ia ion’, ‘ egula ion o
cy okine-media ed signalling pa hway’ and ‘ egula ion o
p o ein ma u a ion’ (Fig. 2b, Table S5). ‘Mala ia’ (associ-
a ed genes HBA,HBB and THBS1) and ‘TCM- ecep o
in e ac ion’ (associa ed genes FN1,GP6 and THBS1) KEGG
pa hways we e ela ed o genes up egula ed in No he n
Finnca le. The biological p ocesses and pa hways ela ed o
diseases, immuni y and i al p ocesses could be associa ed
wi h adap a ion (Lib ado e al. 2015; Iso-Tou u e al. 2016).
Di e en ial gene exp ession be ween Yaku ian ca le
and Hols ein ca le
The highes numbe o signi ican ly di e en ially exp essed
genes was iden i ied be ween Yaku ian ca le and Hols ein
ca le (Table S6). Gi en he pheno ypic di e ences be ween
he b eeds, i was no su p ising o obse e a la ge numbe
o di e en ially exp essed genes. Yaku ian ca le ha e
pheno ypic cha ac e is ics, such as a small and compac
body size and a hick hai coa , ha help hem su i e in a
cold en i onmen . In con as , Hols ein ca le ha e a bigge
body size and a hin hai coa and a e hus no sui able o a
colde clima e. In addi ion, Hols ein ca le do no ace a
sho age o odde and a e main ained inside a ba n,
whe eas Yaku ian ca le may su e om ood sho ages and
a e pa ly ou side du ing he win e . The signi ican
di e ences be ween Yaku ian ca le and Hols ein ca le a e
in line wi h ea lie epo s (Decke e al. 2016; Zino ie a
e al. 2016; Yu chenko e al. 2018). Ou o 1418 genes ha
we e signi ican ly di e en ially exp essed be ween he wo
b eeds, 594 genes we e up egula ed in Yaku ian ca le.
Because o he high numbe o di e en ially exp essed
genes, we conside ed only hose genes wi h a log2 old
change g ea e han o less han 1.5 o GO and KEGG
pa hway analyses. Genes ha we e up egula ed in Yaku-
ian ca le we e associa ed wi h 108 GO e ms (Fig. 3a &
Table S7) and 18 KEGG pa hways (Fig. 3b & Table S8).
Simila ly, he genes ha we e up egula ed in Hols ein ca le
we e associa ed wi h 97 GO e ms (Fig. 3c & Table S9) and
16 KEGG pa hways (Fig. 3d & Table S10).
Di e en ial exp ession be ween No he n Finnca le and
Hols ein ca le
Be ween Finnca le and Hols ein ca le, we obse ed 250
signi ican ly di e en ially exp essed ansc ip s (Table S11),
wi h he majo i y o he genes (n=180) being up egula ed
in Hols ein ca le. Only one GO e m, ‘eicosanoid me abolic
p ocess’ (associa ed genes ALOX15,ALOX5 and HPGD), and
no KEGG pa hways we e iden i ied o genes ha we e
up egula ed in No he n Finnca le. In con as , 62 GO
e ms (Fig. 4 & Table S12) and 32 KEGG pa hways we e
associa ed wi h up egula ed genes in Hols ein ca le
(Table S13).
Uniquely di e en ially exp essed genes
We no iced ha he e was o e lap among many genes in
mo e han one compa ison (Fig. S1). The e o e, we com-
piled a lis o genes ha we e uniquely di e en ially
exp essed in each b eed. The numbe o uniquely di e en-
ially exp essed genes (Table 2) in each b eed ollowed a
simila pa e n o ha obse ed by he op 500 mos
exp essed genes (Fig. 1).
Yaku ian ca le had 139 uniquely di e en ially exp essed
genes, o which 89 we e epo ed o be up egula ed. We
no iced a numbe o cases in which mo e han one gene
om he same amily was up egula ed. Such cases could
p o ide highe con idence ela ed o gene unc ions. The
genes o ecep o s wi h mo e han one membe a e as
ollows: chemokines (CCL4 and CCL5), ca bohyd a e sul o-
ans e ases (CHST1 and CHST12), chemokine ecep o s
(CX3CR1 and CXCR6), g ow h a es s (GAS6 and GAS7),
g anzymes [GZMB ( wo pa alogs), GZMM and GZMH)],
insulin-like g ow h ac o binding p o eins (IGFBP4 and
IGFBP7) and na u al cy o oxici y igge ing ecep o s
(NCR1 and NCR3). Among hose on he lis we e BHLHE40
and PRKCG, which a e linked o ci cadian hy hm. The
impo ance o he ci cadian clock is pa icula ly applicable
o Yaku ian ca le, which mus be me abolically p epa ed
o ood sca ci y du ing long win e s (Ebling & Ba e
2008).
Fou g anzyme ansc ip s and pe o in we e up egula ed
in Yaku ian ca le. G anzymes a e se ine p o eases ha a e
used by cy o oxic lymphocy es o des oy malignan and
i us-in ec ed cells. G anzymes a e anspo ed in o he
cy oplasm o he a ge cell by pe o in 1 (PRF1), a e
Figu e 1 Co-exp ession o he op 500 genes among he h ee b eeds.
©2019 S ich ing In e na ional Founda ion o Animal Gene ics, doi: 10.1111/age.12783
Pokha el e al.4
which hey clea e speci ic p o eins and igge apop osis
(MacDonald e al. 1999; Russell & Ley 2002; Johnson e al.
2003). I has been sugges ed ha he e olu ion o
g anzymes is ela ed o species-speci ic immune challenges
(humans ha e i e g anzyme genes and mice ha e 10), and
hey a e main ained by gene duplica ions and al e a ions in
subs a e speci ici y (Kaise man e al. 2006). Th ee ou o
he six g anzymes known in ca le (Yang e al. 2018), wi h
wo nea by pa alogs o g anzyme B (Fig. S2) and PRF1, a e
all up egula ed in Yaku ian ca le. These indings sugges
ha Yaku ian ca le ha e a e y solid g anzyme-media ed
immune sys em.
The numbe o up egula ed genes was su icien o
iden i y signi ican GO e ms. The GO e ms associa ed wi h
he up egula ed genes included ‘nega i e egula ion o
cy okine p oduc ion’, ‘ egula ion o lymphocy e-media ed
immuni y’, ‘posi i e egula ion o leukocy e chemo axis’
and ‘cellula esponse o in e e on-gamma’ (Fig. 5a &
Table S14). Simila ly, pa hways such as ‘na u al kille cell-
media ed cy o oxici y’, ‘in lamma o y bowel disease (IBD)’,
‘A ican ypanosomiasis’ and ‘cy osolic DNA-sensing
pa hway’ we e ela ed o uniquely up egula ed genes in
Yaku ian ca le (Fig. 5b & Table S15). Two GO e ms,
‘ egula ion o lipid anspo ’ (associa ed genes ABCA1,
ABCG1,IRS2 and THBS1) and ‘ egula ion o endo helial
cell p oli e a ion’ (associa ed genes CCL24,ECM1 and
THBS1), and no KEGG pa hways we e ound o he
down egula ed genes.
The majo i y (162/200) o he uniquely di e en ially
exp essed genes in Hols ein ca le we e ound o be
up egula ed. The lis also includes a numbe o cases in
which mo e han one membe o he gene amily was
p esen . Genes encoding he solu e ca ie p o ein amily
(SLC16A12,SLC16A6,SLC25A37,SLC28A3,SLC2A9,
SLC45A4,SLC6A6 and SLC23A1), ing inge p o eins
(RNF149 and RNF24), no ch p o eins (NOTCH1,NOTCH2
and NOTCH3), lysine deme hylases (KDM4B and KDM6B),
in e e on ecep o s (IFNAR1,IFNAR2 and IFNGR1), CD
p o eins (CD101,CD164,CD300a and CD55) and o he s
we e up egula ed in Hols ein ca le samples. The GO e ms
associa ed wi h he up egula ed genes included ‘cellula
esponse o lipopolysaccha ide’, ‘ esponse o molecule o
(a)
(b)
Figu e 2 Func ional anno a ion o he signi ican ly di e en ially exp essed genes in Yaku ian ca le and No he n Finnca le; GO e ms associa ed
wi h up egula ed genes in (a) Yaku ian ca le and (b) No he n Finnca le. Simila GO e ms we e g ouped oge he , and hus, he pe cen age shown
a he end o each ep esen a i e GO e m indica es he o al pe cen ages o linked GO e ms. The signi ican GO e ms a he P<0.01 s a is ical
le el a e indica ed by double (**) as e isks and a he P<0.05 le el by a single (*) as e isk.
©2019 S ich ing In e na ional Founda ion o Animal Gene ics, doi: 10.1111/age.12783
Blood ansc ip omes o h ee ca le b eeds 5

(a)
(b)
(c)
(d)
Figu e 3 Func ional anno a ion o genes signi ican ly di e en ially exp essed be ween Yaku ian ca le and Hols ein ca le. (a) GO e ms and (b)
KEGG pa hways associa ed wi h up egula ed genes in Yaku ian ca le. (c) GO e ms and (d) KEGG pa hways associa ed wi h up egula ed genes in
Hols ein ca le. Simila GO e ms and KEGG pa hways we e g ouped oge he , and hus, he pe cen age shown a he end o each e m indica es he
o al pe cen ages o linked GO e ms and KEGG pa hways. The signi ican GO e ms and KEGG pa hways a he P<0.05 and P<0.01 s a is ical
le els a e indica ed by single (*) and double (**) as e isks espec i ely.
©2019 S ich ing In e na ional Founda ion o Animal Gene ics, doi: 10.1111/age.12783
Pokha el e al.6
bac e ial o igin’, ‘nega i e egula ion o leukocy e di e en-
ia ion’, ‘ egula ion o ansc ip ion om RNA polyme ase
II p omo e in esponse o s ess and phospha idylinosi ol
binding’ (Fig. 5c & Table S16). Simila ly, KEGG pa hways
such as ‘os eoclas di e en ia ion’, ‘Th1 and Th2 cell
di e en ia ion’ and ‘complemen and coagula ion cas-
cades’ we e ela ed o he up egula ed genes (Fig. 5d &
Table S17). We did no ind any GO e ms o KEGG
pa hways associa ed wi h he down egula ed genes.
Among o he unc ions, memb ane-bound solu e ca ie s
a e key o main aining physiological p ocesses, including
nu ien up ake, was e emo al and ion anspo (Hedige
e al. 2004). The up egula ed exp ession o eigh solu e
ca ie p o eins in Hols ein ca le samples sugges s he
highly impo an unc ion o hese genes. A ecen s udy
showed ha , du ing sloughing, amphibians inc ease he
a e o ion up ake o main ain in e nal homeos asis (Wu
e al. 2017). We specula e ha , wi h a compa a i ely bigge
body size and a hin hai coa , Hols ein ca le may ha e
de eloped a simila sys em o main aining in e nal home-
os asis in esponse o lowe empe a u es. The up egula ion
o h ee no ch p o eins (NOTCH1, NOTCH2 and NOTCH3)
and he biological p ocesses associa ed wi h hypoxia
(‘cellula esponse o oxygen le els’ and ‘cellula esponse
o hypoxia’) is in line wi h ea lie indings showing ha he
‘no ch signalling pa hway’ was ac i a ed in esponse o
hypoxia (Hiyama e al. 2011). This inding also sugges s
ha Hols ein ca le may no ha e adap ed well o win e
condi ions, as hypoxia is linked o low empe a u es (Rocha
& B anco 1998). ‘No ch signalling pa hway’ is also known
o ha e impo an oles in milk lac ose me abolism,
mamma y gland de elopmen and lac a ion, which a e all
associa ed wi h he high milk p oduc ion ai s o Hols ein
ca le (Poli i e al. 2004; Yalcin-Ozuysal e al. 2010). Mo e-
o e , NOTCH1 and NOTCH2 a e pa o he ‘Ras p o ein
signal ansduc ion’ and ‘ egula ion o ERK1 and ERK2
cascade’ p ocesses.
Ke a in 72 (KRT72, ENSBTAG00000007904) and ans-
memb ane p o ein 8 A (TMEM8A, ENSBTAG00000016588)
we e he only genes ha we e up egula ed exclusi ely in
No he n Finnca le. KRT72 is in ol ed in hai o ma ion,
and he unc ion o TMEM8A is unknown. Ou o se en
down egula ed genes, ou IDs (ENSBTAG00000038233,
ENSBTAG00000039691, ENSBTAG00000046611 and
ENSBTAG00000000930) did no ha e gene desc ip ions
a ailable a he ime o his w i ing. The emaining h ee
genes we e ace ylcholines e ase (ACHE), chemokine (C-C mo i )
ligand 3 (CCL3) and Bos au us egula o o G-p o ein signalling
2, 24 kDa (RGS2). The RGS amily o p o eins is in ol ed in
ine- uning he signalling ac i i ies o G-p o ein-coupled
ecep o s (Be man e al. 1996; Blume 2004). An inc eased
le el o RGS2 exp ession is linked o he egula ion o
nume ous biological ac i i ies, such as immune esponses,
bone o ma ion, ca dio ascula unc ion and anxie y (Zhang
& Mende 2014). CCL3,CCL4 and CCL5 encode membe s o
he CC chemokine amily o p o eins, which exhibi p oin-
lamma o y ac i i ies. Mo eo e , CCL3 inhibi s he p oli e -
a ion o haema opoie ic s em/p ogeni o cells (Cook 1996).
ACHE has nume ous unc ions, and i s ole in muscle
de elopmen , neu i ogenesis, cell adhesion, he ac i a ion o
dopamine neu ons and amyloid ib e assembly has been
e iewed ea lie (So eq & Seidman 2001).
Figu e 4 Func ional anno a ion o genes up egula ed in Hols ein ca le compa ed o No he n Finnca le. Simila GO e ms we e g ouped oge he ,
and hus, he pe cen age a he end o each e m indica es he o al pe cen ages o linked GO e ms. The signi ican GO e ms a he P<0.01
s a is ical le el a e indica ed by double (**) as e isks and a he P<0.05 le el by a single (*) as e isk..
Table 2 Numbe s o uniquely di e en ially exp essed genes in di e en
b eeds.
B eed
No. o uniquely
di e en ially
exp essed genes
Up egula ed
genes
Down egula ed
genes
Yaku ian ca le 139 89 52
No he n
Finnca le
927
Hols ein ca le 200 162 48
©2019 S ich ing In e na ional Founda ion o Animal Gene ics, doi: 10.1111/age.12783
Blood ansc ip omes o h ee ca le b eeds 7
Limi a ions o he s udy
Finally, we would like o highligh some o he limi a ions
o his s udy ha we a e awa e o . Fi s , ha ing mo e
samples could ha e gi en highe con idence o he esul s.
Compa isons in ol ing samples om empe a e clima es
would be equally in e es ing. Due o he quali y o he
gene anno a ions, many genes could no be unc ionally
in e p e ed. Mo eo e , due o he la ge numbe o
di e en ially exp essed genes, we did no look in de ail
a all he genes, and he e o e, many in e es ing genes
may no ha e been highligh ed in his s udy. Mo eo e ,
he di e ences in gene exp ession le el we obse ed
be ween he b eeds could be a ec ed by o he ac o s such
as he physiological s a us o he animal, managemen
and en i onmen al di e ences. Al hough we ied o
in e p e ou esul s based on model species, p ima ily
human s udies, some o he in e p e a ion may no be
accu a e. Pa icula ly, mos o he s udies we e based on
model species wi h mo e con olled se ings, whe eas he
animals in ou s udy, especially Yaku ian animals, li e in
ha sh en i onmen al condi ions.
Conclusions
In conclusion, using blood as a s a ing ma e ial, we we e
able o cap u e mo e han 60% o ca le genes o assess he
di e en ial gene exp ession in h ee b eeds. Gene exp ession
p o iles o wo impo an indigenous ca le b eeds ha a e
adap ed o he no he n and e en sub-a c ic clima e ha e
been epo ed o he i s ime. The gene exp ession p o iles
o No he n Finnca le appea ed o be mo e simila o
Yaku ian ca le han o Hols ein ca le, despi e sha ing
simila li ing condi ions wi h he Hols ein cows ha we e
analysed in his s udy. Mo eo e , we iden i ied b eed-
speci ic di e ences in main aining immuni y and disease
esis ance mechanisms among he b eeds included in ou
s udy. Some o ou esul s, based on Hols ein ca le, explain
how al e na i e biological p ocesses can help impo ed
animals cope wi h challenging en i onmen al condi ions.
S udies such as his one could iden i y gene ic ma ke s ha
may assis in animal b eeding and he sus ainable u iliza-
ion and conse a ion p ac ices o animal gene ic esou ces
in changing no he n Eu asian en i onmen s. One o he
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Figu e 5 Ne wo k ep esen a ion o GO and KEGG pa hways associa ed wi h uniquely di e en ially exp essed genes. Nodes ep esen GO e ms o
KEGG pa hways, wi h node size co esponding o he signi icance o e m en ichmen , and unc ionally ela ed g oups pa ially o e lap. Ne wo k o
(a) GO e ms and (b) KEGG pa hways associa ed wi h he genes up egula ed in Yaku ian ca le. Ne wo k o (c) GO e ms and (d) KEGG pa hways
associa ed wi h up egula ed genes in Hols ein ca le. The ep esen a i e GO e ms and KEGG pa hways a e highligh ed in bold.
©2019 S ich ing In e na ional Founda ion o Animal Gene ics, doi: 10.1111/age.12783
Pokha el e al.8
main limi a ions o ou s udy is ha we we e able o see
di e en ially exp essed genes based en i ely on only one
issue. This s udy migh ha e missed many impo an
genes, and he genes ha we e ound o be di e en ially
exp essed may ha e been associa ed wi h some ex e nal
ac o s (such as li ing condi ions, seasonal di e ence and
eeding). The e o e, u u e s udies based on addi ional
issues wi h a mo e con olled expe imen al design will
ce ainly p o ide a be e pic u e o gene ic basis o
adap a ion in no he n Eu asian condi ions.
Acknowledgemen s
We hank ou colleague Tuula Ma ja a Hamama om he
Na u al Resou ces Ins i u e Finland (Luke) o labo a o y
assis ance and Innoken y Ammoso o aluable assis ance
in sampling in Yaku ia. This s udy is pa o he A c ic-A k
p ojec , unded by he Academy o Finland (decision no.
286040). The au ho s wish o acknowledge he CSC –IT
Cen e o Science, Finland, o compu a ional esou ces.
This s udy was suppo ed by he Finnish Func ional
Genomics Cen e, Uni e si y o Tu ku and

Abo Akademi
and Biocen e Finland. Special hanks o he owne s o he
expe imen al animals o le ing us collec he blood
samples.
Au ho con ibu ions
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and S.Z. pa icipa ed in sample collec ion. H.H. ex ac ed
mRNA om blood samples. K.P. and M.W. pe o med RNA-
seq da a analyses. K.P. w o e he manusc ip . All au ho s
ead, e ised and app o ed he inal manusc ip .
Con lic o in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
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The aw sequence eads (FASTQ iles) ha e been deposi ed
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Blood ansc ip omes o h ee ca le b eeds 9