SHORT COMMUNICATION Open Access
Ancien DNA sheds ligh on he ances y o
p e-hispanic Cana ian pigs
Iñigo Olalde
1
, Juan Capo e
2
, Ma ía C Del-A co
3
, Pablo A oche
4
, Te esa Delgado
5
, Ra ael González-An on
6
,
Jo ge Pais
7
, Ma cel Amills
8
, Ca les Lalueza-Fox
1
and Osca Ramí ez
1*
Abs ac
Backg ound: Cana ian Black (CB) pigs belong o an au och honous and endange ed b eed, which is sp ead
h oughou he Cana ian a chipelago. I is commonly accep ed ha hey ep esen a elic o he pig popula ions
ha we e b ed by he Be be s in No h A ica o e millennia. I is impo an o no e ha he geog aphic isola ion o
he Cana y Islands has p ese ed his gene ic legacy in ac om o eign in og essions un il he Spanish conques
o he a chipelago in he 15
h
cen u y. Ten yea s ago, i was demons a ed ha , in CB pigs, he equency o he
Asian A2 cy och ome-b haplog oup eached 73%. The cu en wo k aimed a in es iga ing whe he his obse a ion
is explained by ei he a ecen o an ancien in og ession o CB pigs wi h Fa Eas e n pigs.
Resul s: Gene ic analyses o 23 ancien samples om p e-hispanic Cana ian pigs (420 o 2500 yea s be o e p esen )
showed ha Nea Eas e n and Fa Eas e n gene ic signa u es we e o ally absen in he p imi i e Cana ian
p e-hispanic pigs. Indeed, he haplo ypes de ec ed in hese pigs we e closely ela ed o hose o No h A ican and
Eu opean wild boa s.
Conclusions: Ou esul s demons a e ha he high equency o he Fa Eas e n mi ochond ial cy och ome B A2
haplo ype in mode n Cana ian Black pigs p obably co esponds o a ela i ely ecen in og ession wi h B i ish
b eeds.
Findings
The only li ing ep esen a i e o he domes ic swine ha
was in oduced in o he Cana y Islands 3000 YBP (yea s
be o e p esen ) is he Cana ian Black (CB) pig, which in
he 1980’s was e y nea ex inc ion. Howe e , an ac i e
conse a ion p og am has allowed he popula ion o
each a census o a ew hund ed indi iduals [1,2]. Ana-
lysis o mi ochond ial genome a ia ion in mode n CB
pigs e ealed ha he Fa Eas e n A2 cy och ome-b
(MT-CYB) haplog oup eached a high equency in his
b eed (up o 73%) [3]. Fa Eas e n haplo ypes a e absen
in local Eu opean b eeds, such as he Ibe ian, Mangali za,
Majo can Black o Basque pigs [4]. Clop e al. [3] p o-
posed wo al e na i e explana ions o he p esence o Fa
Eas e n haplo ypes in he mi ochond ial genome o CB
pigs i.e. ei he in og ession o CB pigs wi h imp o ed
B i ish b eeds ha had been ex ensi ely hyb idized wi h
Chinese sows in he 18
h
and 19
h
cen u ies o selec o
pigs wi h ea lie ep oduc i e ma u i y and inc eased a -
ness [5], o con e sely, a mo e ancien in oduc ion as a
consequence o he se lemen o Be be ibes in he
Cana y Islands. Al hough his la e in e p e a ion is less
likely, Fa Eas e n haplo ypes a e e y common in Eas
A ican b eeds such as he Muko a b eed [4] and i is pos-
sible ha hese haplo ypes may ha e di used wes wa ds,
as is he case o indicine alleles in ca le [6].
Ob iously, hese wo al e na i e scena ios canno be
asce ained wi h mi ochond ial da a om mode n CB
samples. Thus, we decided o su ey he mi ochond ial
a ia ion o 23 ancien pig samples ha co e a la ge
pe iod o ime ( om ≈420 o 2500 YBP) and ep esen
11 p e-Hispanic a cheological si es ac oss ou o he
se en Cana y Islands (Figu e 1) and [see Addi ional
ile 1; Addi ional ile 2: Table S1]. To al DNA was ex-
ac ed in labo a o ies ha a e dedica ed o he analysis o
* Co espondence: [email p o ec ed]
1
Ins i u de Biologia E olu i a (CSIC - Uni e si a Pompeu Fab a), Ba celona,
Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Gene ics
Selec ion
E olu ion
© 2015 Olalde e al.; licensee BioMed Cen al. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion 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 c edi ed. The C ea i e Commons Public Domain
Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle,
unless o he wise s a ed.
Olalde e al. Gene ics Selec ion E olu ion 
DOI 10.1186/s12711-015-0115-7
Tex o
ancien DNA a he Ins i u e o E olu iona y Biology and
Uni e si y o Pompeu Fab a in Ba celona by applying
p o einase-K diges ion ollowed by phenol-chlo o o m
p ecipi a ion and a column concen a ion (Amicon), as
desc ibed elsewhe e [7]. Ex ac s om skin samples we e
subsequen ly pu i ied wi h a gene clean silica me hod
using a DNA ex ac ion Ki (Fe men as, USA). To he bes
o ou knowledge, no p e ious wo k on mode n pigs had
e e been conduc ed a his labo a o y and s anda d p e-
cau ions o expe imen s in ol ing ancien DNA samples
we e ollowed (see Appendix).
Pig speci ic p ime s [see Addi ional ile 2: Table S2])
we e designed o ampli y wo non-o e lapping se-
quences o 89 (PCR1) and 77 bp (PCR2) co esponding
o he MT-CYB gene. Sequence PCR1 con ains he diag-
nos ic single nucleo ide polymo phisms (SNPs) ha a e
loca ed a posi ions 15036, 15038, 15041, 15044 and
15045 and di e en ia e Eu opean (E1, E2, E3 and E4)
om Asian (A1, A2, A3, A4) haplog oups [3,8] and se-
quence PCR2 con ains wo SNPs ha disc imina e be-
ween Nea Eas e n s Wes e n (Eu ope and No h
A ica) haplo ypes [4]. Each agmen was ampli ied
using a wo-s ep PCR p o ocol [9]. Ampli ied p oduc s
we e pu i ied wi h a gene clean silica me hod using he
DNA Ex ac ion Ki (Fe men as, USA) and cloned using
he Topo TA cloning ki (In i ogen, The Ne he lands).
Whi e colonies we e subjec ed o 30 cycles o PCR wi h
M13 uni e sal p ime s and subsequen ly sequenced wi h
an Applied BioSys ems 3100 DNA sequence , a he Se -
ei de Seqüenciació o he Uni e si a Pompeu Fab a
(Ba celona).
Amplicon sequences o PCR1 and PCR2 we e ob ained
om 21 o 23 and 8 o 10 specimens o p e-Hispanic
Cana y pigs, espec i ely. PCR1 was analyzed in all 23
ancien pig samples collec ed. Fo PCR2, we excluded
he samples o which PCR1 was no ampli ied and also
mos o he samples o mummi ied pig skin because,
a e pu i ica ion and ampli ica ions, he amoun o
DNA ex ac ed was no su icien [see Addi ional ile 2:
Table S1]. Al hough he high empe a u es o he cli-
ma ic condi ions o he Cana y Islands do no a o
DNA p ese a ion, he success a es o ampli ica ion
(91.3% and 80%, espec i ely) we e e y high [10,11]. Fo
iden ical empe a u e and en i onmen al se ings, p ese -
a ion o ancien DNA is highly co ela ed wi h sample age
[12]. Two o he samples o which no success ul ampli ica-
ion was achie ed (Buena is a 3 and Lanza o e 12, bo h
om Lanza o e) we e among he ou oldes ma e ials in
he assemblage. The hi d sample o which ampli ica ion
ailed (Guadayaque11/3) o igina ed om a lea he skin ha
was used as sh oud on he mummies. Possibly, he p es-
ence o inhibi o subs ances used du ing he embalming
p ocess may ha e p e en ed DNA ampli ica ion.
All PCR1 DNA sequences ob ained om he 21 an-
cien pig samples om he Cana y Islands belonged o a
single haplog oup, i.e. MT-CYB haplog oup E1 (Figu e 2)
Buena is a
El Bebede o
Cue a del
Tendal
Hoya
B unco
El Re ama
Cue a de
las Palomas
Cue a de
los Guanches
Cue a de
los Cabezazos
Guadayeque
A guineguín
Acusa
Figu e 1 Geog aphic loca ions o he a chaeological assemblies om whe e ancien samples we e collec ed. The size o he ci cles is
p opo ional o he numbe o samples analysed.
Olalde e al. Gene ics Selec ion E olu ion  Page 2 o 5
and [see Addi ional ile 2: Table S1, Addi ional ile 3:
Figu e S1]. Gi en ha haplog oup E1 is ep esen ed by
dis inc haplo ypes ha seg ega e in wild boa s om he
Nea Eas , Eu ope and No h A ica, i does no allow us
o conclude on he geog aphical o igin o he p imi i e
Cana ian p e-hispanic pigs. In e es ingly, he eigh an-
cien pig samples ha p o ided PCR2 amplicons ha -
bou ed Wes e n haplo ypes [see Addi ional ile 3: Figu e
S2]. I is impo an o no e ha Wes e n MT-CYB a i-
an s ha e negligible equencies in Nea Eas e n wild
boa s and hey p obably e lec in og ession wi h Wes -
e n pigs o e aliza ion o domes ic pigs [4,13]. This e-
sul ag ees well wi h da a on he au osomal nuclea
genome: based on 60K SNP geno ypes o Nea Eas e n
and Eu opean pigs and wild boa s, Manunza e al. [14]
demons a ed ha he e was no Nea Eas e n oo p in
in ex an Cana ian pigs. The absence o such signa u es
in he eigh p e-hispanic Cana ian samples ha a ied in
age om ≈960 o 2500 yea s sugges s ha , a he begin-
ning o he i s millennium BC, he Nea Eas signa u e
was absen , o a low equency, in domes ic pig popula-
ions om wes e n No h A ica.
Ou da a no only p o ide a i s glimpse on he mi o-
chond ial gene pool o pigs ha p obably ha e a Be be
ances y, bu also help o sol e a puzzling inding e-
po ed by Clop e al. [3] 10 yea s ago i.e. he high
n=2
n=7
n=3
n=33
n=11
n=9
n=11
n=11
n=8
n=27
n=12
n=4 n=4
E1
A1
A2
E2
n=21
Ancien Mode n
Wild boa
Figu e 2 Compa ison o equencies o mi ochond ial MT-CYB haplog oups in he ancien Cana ian pigs (desc ibed in his s udy) and
mode n pigs om A ica and he Cana y Islands and wild boa om Nea Eas and Eu ope desc ibed p e iously by Rami ez e al. [4].
Olalde e al. Gene ics Selec ion E olu ion  Page 3 o 5
equency o he Fa Eas e n A2 MT-CYB haplo ype in
CB pigs. Ou esul s on ancien DNA s ongly suppo
he o me hypo hesis i.e. he comple e absence o Fa
Eas e n haplo ypes in he da ase o ancien pig samples
( om 11 loca ions ac oss ou o he se en Cana y Islands)
sugges s ha na i e CB pigs did no ca y Fa Eas e n al-
leles. Acco ding o Ga cía-Ma ín and Capo e [2], pigmen-
a ion and ea mo phology and size o Cana ian pigs
esemble hose obse ed in he Be kshi e pig b eed. In e -
es ingly, he A2 haplo ype seg ega es a high equencies
(a ound 35%) in his B i ish local b eed [8], which p o ides
e idence o a ecen Be kshi e in oduc ion. F om an his-
o ical poin o iew, c ossb eeding o CB pigs wi h B i ish
and Ibe ian b eeds has been widely documen ed [15,16].
Ce ain insigh s abou he o igin o mode n domes ic
b eeds can only be ob ained h ough he analysis o an-
cien DNA [13,17–19]. In his s udy, pa ial sequencing
o he MT-CYB sequence in pig samples om Cana ian
a chaeological assemblages has allowed us o demon-
s a e ha he p esence o Asian MT-CYB alleles in CB
pigs was he esul o a ecen in og ession e en (p ob-
ably wi h B i ish b eeds). Howe e , asce ainmen bias is
a p oblem o p ope in e p e a ion o esul s ha a e
exclusi ely based on ma e nal da a. Analyses o ancien
samples based on whole-genome sequencing may con-
ibu e o be e unde s and he p ocess o domes ica-
ion and b eed o ma ion.
Addi ional iles
Addi ional ile 1: Sample in o ma ion. Desc ip ion o he pig ancien
samples ha we e used in his s udy and collec ed a di e en si es:
Tene i e [24–29], G an Cana ia [30] and Lanza o e [31–36].
Addi ional ile 2: Table S1. A chaeological si es and age o Cana ian
pig samples used. This Table p o ides he ID, lab codes, a cheological si e
and age o he Cana ian pig ancien samples om which cy och ome B
haplog oups (F agmen 1) and haplo ypes (F agmen 2) we e e ie ed.
Lis o p ime s used o ampli ica ion o he wo 89 and 77 bp sequences
o he MT-CYB gene. Table S2. p o ides he names and sequences o he
p ime s used o ampli y he wo 89 and 77 bp sequences o he MT-CYB
gene. P ime s we e designed wi h he P ime 3 so wa e (h p://
bioin o.u .ee/p ime 3-0.4.0/).
Addi ional ile 3: Figu e S1. Alignmen o he 48-bp sequences (PCR1)
o he MT-CYB gene agmen 1 ob ained om DNA om 21 Cana ian pig
ancien samples. Desc ip ion: The diagnos ic SNPs ha a e loca ed a
posi ions 15036, 15038, 15041, 15044 and 15045 (indica ed in ed)
di e en ia e Eu opean: E1 (GenBankID: KJ746666), E2 (EU531827) and E4
(GU211924) haplog oups and, also, Fa Asian: A1 (KP257599), A2 (KM215171),
A3 (AB015072), A4 (KJ746664) and A5 (GU135825) haplog oups. Figu e S2.
Alignmen o he 37-bp sequences (PCR2) o he MT-CYB gene agmen 1
ob ained om DNA om 8 Cana ian pig ancien samples. Desc ip ion: The
wo diagnos ic SNPs (indica ed in ed) di e en ia e haplo ypes o haplog oup
E1 p esen in wild boa indi iduals om he Nea Eas : H12 (GenBank ID:
EU531818), H31 (EU531827), H52 (EU531832), H53 (EU531833), and H54
(EU531834); and om Eu ope: H1 (AY237496), H4 (AY237512), H15
(AB015072), H16 (EU531821), H17 (EF061501), H18 (AY237516), H24
(AF136542), H27 (EU531824), H32 (EU531828), H34 (EU531830), H65
(AM492593) and H66 (AM492620).
Appendix
Sequencing o m DNA
DNA was ex ac ed ollowing he me hod desc ibed in
de ail in [20]. Ex ac ion p ocedu es we e pe o med in
an isola ed p e-PCR a ea, by adop ing he s anda d p e-
cau ions o ancien DNA s udies [21,22]. Mul iple ex-
ac ion and ampli ica ion nega i e con ols o moni o
o con amina ion in he eagen s we e added o each
PCR eac ion. Du ing he whole s udy, no ampli ica ion
p oduc s we e ob ained in hese blank PCR con ols.
Ampli ica ion was ca ied ou acco ding o a wo-s ep
PCR p o ocol epo ed by [9]. Bo h PCR s eps we e ca -
ied ou wi h 2 uni s AmpliTaq Gold (ABI, USA), 1X
AmpliTaq Gold bu e (ABI, USA), 2.5 mM MgCl
2
(ABI,
USA), and 500 μM o each dNTP. In he i s mul iplex
s ep, 150 nM o each p ime pai and 5 μl o DNA ex-
ac we e added o a inal eac ion olume o 20 μl. The
i s ampli ica ion s ep consis ed in a 12 min ac i a ion
s ep a 94°C, ollowed by 27 cycles a 94°C o 20 s, 50°C
o 20 s, and 72°C o 20 s. Fi e μl o a 1 o 10 dilu ion
o he p ima y ampli ica ion p oduc we e used as em-
pla e o he second PCR s ep. Condi ions we e he same
as o he i s s ep, excep ha he s anda d p ime con-
cen a ion was inc eased o 1.5 μM and ha 33 cycles
we e pe o med, ollowed by a inal s ep o 12 min a 72°C.
Blank and mock PCR con ols we e included in each
ampli ica ion s ep o moni o agains con amina ion.
PCR p oduc s we e isualized unde UV ligh and he
app op ia e bands we e excised om a low-mel ing poin
aga ose gel and pu i ied wi h a silica-based me hod. Sub-
sequen ly, he ampli ica ion p oduc s we e cloned in bac-
e ia (TOPO-TA cloning ki , In i ogen). Resul ing whi e
colonies we e picked, ampli ied wi h M13 uni e sal
p ime s and sequenced wi h an ABI3730 capilla y sequen-
ce (Applied Biosys ems).
We ollowed he c i e ia o au hen ici y desc ibed by
Gilbe e al. [23]: (i) ampli ica ions o bo h m DNA se-
quences o he Buena is a1 sample we e eplica ed in an
independen labo a o y (a he Uni e si a Pompeu
Fab a); (ii) o all he samples, mock ex ac ions and
PCR blank con ols we e ca ied ou and he e was no
e idence o con amina ion; and (iii) abou 30% o he
agmen s we e eplica ed wice [see Addi ional ile 3:
Figu es S1 and S2].
To assign he geno ype o he diagnos ic SNPs, we
used he majo i y ules consensus. I , o one o a ew
clones, addi ional subs i u ions a e ound p esen in he
sequence o one PCR bu no in he sequence o ano he
PCR om he same sample, i is easonable o a ibu e
hem o DNA damage, and hus hey we e no consid-
e ed in he analyses.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Olalde e al. Gene ics Selec ion E olu ion  Page 4 o 5
Au ho s’con ibu ions
JC, MCD-A, PA, TD, FJP, RG-A, MA, CL-F and OR con ibu ed o he design o
his esea ch. OR, and IO pe o med he expe imen al analyses. IO and OR
pe o med he da a analysis. IO, MA, CL-F and OR w o e he manusc ip . All
au ho s ead and app o ed he inal manusc ip .
Acknowledgemen s
IO has a p e-doc o al ellowship om he Basque Go e nmen (DEUI). OR is a
pos -doc o al esea che om he JAEDOC p og am co- unded by The Eu opean
Social Fund (ESF). This wo k has been ounded by he g an BFU2012-34157 o
C.L-F om he MINECO, Spain.
Au ho de ails
1
Ins i u de Biologia E olu i a (CSIC - Uni e si a Pompeu Fab a), Ba celona,
Spain.
2
Ins i u o Cana io de In es igaciones Ag a ias, La Laguna, Tene i e,
Spain.
3
Depa amen o de Geog a ía e His o ia, Uni e sidad de La Laguna, La
Laguna, Tene i e, Spain.
4
Depa amen o de Ciencias His ó icas, Uni e sidad
de Las Palmas de G an Cana ia, Las Palmas de G an Cana ia, Spain.
5
Museo
Cana io, Las Palmas de G an Cana ia, Spain.
6
Museo de la Na u aleza y el
Homb e-M A queológico, Tene i e, Spain.
7
Museo A queológico Benaho i a,
La Palma, Spain.
8
Cen e o Resea ch in Ag icul u al Genomics
(CSIC-IRTA-UAB-UB), Campus UAB, 08193 Bella e a, Spain.
Recei ed: 28 Oc obe 2014 Accep ed: 23 Ma ch 2015
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