RESEARCH ARTICLE Open Access
A combina ion o wo a ian s in PRKAG3 is
needed o a posi i e e ec on mea quali y in
pigs
Pekka Uima i
1,2*
and Anu Si onen
1
Abs ac
Backg ound: Colo and pH o mea measu ed 24 h pos mo em a e common selec ion objec i es in pig b eeding
p og ams. Se e al amino acid subs i u ions in PRKAG3 ha e been associa ed wi h a ious mea quali y ai s. In ou
p e ious s udy ASGA0070625, a SNP nex o PRKAG3, had he mos signi ican associa ion wi h mea quali y ai s in
he Finnish Yo kshi e. Howe e , he known amino acid subs i u ions, including I199V, did no show any associa ion.
The aims o his s udy we e o cha ac e ize u he a ia ion in PRKAG3 and i s p omo e egion, and o es he
associa ion be ween hese a ian s and he pH and colo o po k mea .
Resul s: The da a comp ised o 220 Finnish Land ace and 230 Finnish Yo kshi e a i icial insemina ion boa s wi h
p ogeny in o ma ion. We sequenced he coding and p omo e egion o PRKAG3 in hese and in h ee addi ional
wild boa s. Geno ypes om ou p e ious genome-wide scans we e also included in he da a. Associa ion be ween
SNPs o haplo ypes and mea quali y ai s (de eg essed es ima es o b eeding alues om Finnish na ional
b eeding alue es ima ion o pH, colo ligh ness and edness measu ed om loin o ham) was es ed using a linea
eg ession model. Sequencing e ealed se e al no el amino acid subs i u ions in PRKAG3, including K24E, I41V,
K131R, and P134L. Linkage disequilib ium was s ong among he no el a ian s, SNPs in he p omo e egion and
ASGA0070625, especially o he Yo kshi e. The s onges associa ions we e obse ed be ween ASGA0070625 and
he SNPs in he p omo e egion and pH measu ed om loin in he Yo kshi e and be ween I199V and pH measu ed
om ham in he Land ace. In con as , ASGA0070625 was no signi ican ly associa ed wi h mea quali y ai s in he
Land ace and I199V no in he Yo kshi e. Haplo ype analysis showed a signi ican associa ion be ween a haplo ype
consis ing o 199I and 24E alleles (o g.-157C o g.-58A alleles in he p omo e egion) and pH measu ed om loin
and ham in bo h b eeds (P- alues a ied om 1.72 × 10
−4
o 1.80 × 10
−8
).
Conclusions: We conclude ha haplo ype g.-157C - g.-58A - 24E - 199I in PRKAG3 has a posi i e e ec on mea
quali y in pigs. Ou esul s a e eadily applicable o ma ke -assis ed selec ion in pigs.
Keywo ds: Associa ion, Haplo ype, Mea quali y, Pig, SNP
Backg ound
Mea quali y cha ac e is ics such as wa e -holding cap-
aci y, ende ness, in amuscula a , and as e a e im-
po an o he mea indus y as well as o consume s
[1-3]. Since di ec measu emen o some o hese ai s
is conside ed labo ious in p ac ice, many b eeding p o-
g ams only use co ela ed ai s like pH and colo o
mea o selec ion. In Finland, pH and colo o loin ha e
been among he pig b eeding objec i es since 1983 and
mea quali y o ham since 2000 [1].
Se e al impo an genes a e known o ha e a majo e ec
on mea quali y and ca cass composi ion ai s in pigs,
including RYR1 ( yanodine ecep o 1) on ch omosome 6
[4]; PRKAG3 (p o ein kinase, AMP-ac i a ed, gamma 3
non-ca aly ic subuni ) on ch omosome 15 [5,6]; IGF2 (insu-
lin-like g ow h ac o 2) on ch omosome 2 [7,8]; CAST
(calpas a in) on ch omosome 2 [9]; and MC4R (melanoco -
in 4 ecep o ) on ch omosome 1 [10]. A p e ious genome-
wide associa ion s udy in he Finnish Yo kshi e popula ion
* Co espondence: [email p o ec ed]
1
MTT Ag i ood Resea ch Finland, Bio echnology and Food Resea ch, FI-31600
Jokioinen, Finland
2
Depa men o Ag icul u al Sciences, Animal B eeding, Uni e si y o Helsinki,
FI-00014 Helsinki, Finland
© 2014 Uima i and Si onen; licensee BioMed Cen al L d. 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/2.0), 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.
Uima i and Si onen BMC Gene ics 2014, 15:29
h p://www.biomedcen al.com/1471-2156/15/29
e ealed a s ong associa ion be ween pH measu ed om
loin and a ch omosomal egion a ound PRKAG3 [11].
Howe e , none o he epo ed amino acid subs i u ions
T30N, G52S, L53P, I199V, o R200Q we e as s ongly asso-
cia ed wi h pH as SNP ASGA0070625 (RS80816788, pos-
i ion 133,677,385 Sus Sc o a build 10.2), which is loca ed
nea he PRKAG3 gene. A u he s udy wi h mo e ma ke s
on he PRKAG3 egion was he e o e needed. Addi ionally,
he Finnish Land ace b eed couldbeusedasa alida ion
popula ion o he ini ial indings.
This a icle epo s no el a ia ions wi hin he
PRKAG3 gene in Finnish Yo kshi e and Land ace pigs,
shows he linkage disequilib ium (LD) s uc u e a ound
PRKAG3, and p esen s he esul s om ou associa ion
analysis be ween SNPs o haplo ypes and mea quali y
ai s. We show ha o achie e a posi i e e ec on mea
quali y ai s, pa icula ly in pH measu ed pos mo em
om loin and ham, he animal has o ca y bo h he
199I allele and he combina ion g.-157C - g.-58A - 24E.
This haplo ype was signi ican o mea quali y in bo h
b eeds.
Resul s
SNP disco e y
Sequencing o he exons and p omo e egion o
PRKAG3 e ealed ou no el amino acid subs i u ions:
K24E, I41V, K131R, and P134L (Table 1). The ou SNPs
ups eam o he PRKAG3 ansc ip ion ini ia ion si e e-
po ed by Ryan e al. [12] we e also de ec ed in he s ud-
ied Finnish Yo kshi e and Land ace popula ions. Two
addi ional synonymous subs i u ions we e obse ed a
amino acids 193 and 194, bu hese we e e y a e in
bo h popula ions ( equency less han 3%) and we e hus
excluded om la e analysis. Mino allele equencies o
he no el amino acid subs i u ions a ied om 0.17 o
0.20 and om 0.15 o 0.20 in he Yo kshi e and
Land ace, espec i ely. Alleles 53P and 200R we e ixed
in bo h popula ions. Allele 30T was also e y a e in he
Yo kshi e, wi h a equency o only 1% compa ed o 13%
in he Land ace, whe eas alleles 52S and 199I we e mo e
common in he Yo kshi e han in he Land ace.
Func ional analysis o he amino acid subs i u ions using
SIFT [13] (So ing In ole an F om Tole an , h p://si .bii.
a-s a .edu.sg/) unde lined h ee subs i u ions, K24E, I41V
and L53P, as damaging (Table 2). These e ec s appea ed o
be ansc ip speci ic indica ing di e ences be ween e ec s
o subs i u ions on p o ein iso o ms. Fo subs i u ions I41V
and L53P he SIFT sco es we e simila o all p o ein iso-
o ms, bu a clea di e ence o K24E was iden i ied
(Table 2).
Linkage disequilib ium
Figu e 1 shows he linkage disequilib ium (LD) wi hin
and a ound PRKAG3. O e all, LD was s onge and ex-
ended o e a longe ange in he Finnish Yo kshi e han
Land ace. In he Yo kshi e, g.-311A > G, g.-221G > A, g.-
157C > G, g.-58A > G, K24E, I41V, K131R, and P134L
we e in comple e LD wi h ASGA0070625, he SNP ha
showed he s onges associa ion wi h pH measu ed
om loin in ou p e ious whole-genome analysis [11].
In he Finnish Land ace, ASGA0070625 was in comple e
LD wi h g.-157C > G, g.-58A > G, and K24E. In e es -
ingly, he known amino acid subs i u ions T30N, G53S,
and I199V we e in e y weak LD wi h ASGA0070625
Table 1 Posi ions, alleles and mino allele equencies o he iden i ied SNPs in PRKAG3
Exon Va ia ion
a
SS numbe RS numbe Posi ion on ch omosome 15, bp
b
Alleles
c
Yo kshi e Land ace
ASGA0070625 80816788 133677385 A/G 0.21 0.28
g.-311A > G 196959880 133800456 G/A 0.19 0.13
g.-221G > A 196952335 133800546 A/G 0.19 0.13
g.-157C > G 196956394 133800610 G/C 0.20 0.26
g.-58A > G 196959698 133800709 G/A 0.20 0.26
2K24E 947848631 133801207 A/G 0.17 0.20
3 T30N 328566929 133802071 A/C 0.01 0.13
3I41V 947848632 133802103 A/G 0.20 0.16
3 G52S 343733804 133802136 A/G 0.43 0.12
4 L53P 337462352 133802499 T/C 0 0
4K131R 947848633 133802733 G/A 0.19 0.15
4P134L 947848634 133802742 T/C 0.19 0.16
5 I199V 133803828 A/G 0.45 0.30
5 R200Q 133803829 A/G 0 0
a
The no el amino acid subs i u ions a e gi en in i alics wi h co esponding dbSNP submission numbe s (h p://www.ncbi.nlm.nih.go /SNP/).
b
Based on Sus Sc o a build 10.2.
c
The mino allele is gi en i s .
Uima i and Si onen BMC Gene ics 2014, 15:29 Page 2 o 9
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and, hence, also wi h he no el amino acid subs i u ions
and SNPs in he p omo e egion o PRKAG3 in bo h
b eeds.
Haplo ypes
In o al, 12 di e en haplo ypes we e obse ed in ei-
he o he b eeds (Table 3). One o hese, HAP3, is
ac ually he same as he wild haplo ype iden i ied in
he sequences om he Eu opean wild boa s used in
his s udy (based on h ee wild boa samples). HAP3
was he mos common haplo ype in he Finnish
Land ace b eed ( equency 0.34), bu e y a e in he
Finnish Yo kshi e ( equency 0.01). HAP1, a haplo ype
simila o he wild haplo ype excep o a poin mu a-
ion o G o A a G52S, was he mos equen in he
Yo kshi e wi h a equency o 0.42. Again he e was a
no able di e ence in equencies be ween he wo
b eeds ( equency only 0.12 in he Land ace). HAP2,
ano he haplo ype simila o he wild haplo ype ex-
cep o a poin mu a ion o G o A a I199V, was
common in bo h b eeds, wi h equencies o 0.36 and
0.25 o he Yo kshi e and Land ace, espec i ely. The
o he haplo ypes (HAP4 o HAP12) join ly accoun ed
o 22% o 29% o he haplo ypes iden i ied in he
Yo kshi e and Land ace, espec i ely. Some o he
a e haplo ypes may be spu ious due o geno yping o
haplo yping e o s.
SNP associa ion
Based on he geno ypes om ou p e ious whole-
genome scans [11,14], he mos signi ican SNPs
a ec ing mea quali y ai s we e in he egion om
120 Mb o 140 Mb on ch omosome 15 in he
p oximi y o PRKAG3 (Figu e 2). The smalles
P- alue was obse ed o associa ion be ween six
SNPs (ASGA0070634, ASGA0070625, MARC0083357,
DBUN0002708, MARC0039273, and DIAS0002965)
and pH measu ed om loin in he Finnish Yo kshi e.
These six SNPs we e in comple e LD in he egion
om 133.64 Mb o 134.01 Mb. Thei es ima ed allele
subs i u ion e ec o he Yo kshi e was −0.059 ±
0.008 (P- alue = 7.28 × 10
−13
), co esponding o 1.3 SD
o he es ima ed polygenic e ec . Mino alleles A, A,
C, A, A,andGo ASGA0070634, ASGA0070625,
MARC0083357, DBUN0002708, MARC0039273, and
DIAS0002965, espec i ely, dec eased pH measu ed
Table 2 E ec o he iden i ied SNPs in PRKAG3 on p o ein sequence
Re e ence Posi ion on ch omosome 15, bp Amino acid co-o dina e T ansc ip SIFT
a
K24E 133801207 47 ENSSSCT00000017641 0.16
K24E 133801207 24 ENSSSCT00000033825 0.01
T30N 133802071 80 ENSSSCT00000017641 0.44
T30N 133802071 57 ENSSSCT00000033825 0.20
T30N 133802071 30 ENSSSCT00000036402 0.32
I41V 133802103 91 ENSSSCT00000017641 0.05
I41V 133802103 68 ENSSSCT00000033825 0.07
I41V 133802103 41 ENSSSCT00000036402 0.03
G52S 133802136 102 ENSSSCT00000017641 0.11
G52S 133802136 79 ENSSSCT00000033825 0.13
G52S 133802136 52 ENSSSCT00000036402 0.13
L53P 133802499 103 ENSSSCT00000017641 0.04
L53P 133802499 80 ENSSSCT00000033825 0.05
L53P 133802499 53 ENSSSCT00000036402 0.07
K131R 133802733 181 ENSSSCT00000017641 1.00
K131R 133802733 131 ENSSSCT00000036402 1.00
K131R 133802733 158 ENSSSCT00000033825 1.00
P134L 133802742 184 ENSSSCT00000017641 0.58
P134L 133802742 134 ENSSSCT00000036402 0.58
P134L 133802742 161 ENSSSCT00000033825 0.58
I199V 133803828 249 ENSSSCT00000017641 1.00
I199V 133803828 199 ENSSSCT00000036402 1.00
I199V 133803828 226 ENSSSCT00000033825 1.00
a
SIFT sco e <0.05 is conside ed as damaging.
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om loin. None o hese six SNPs showed s a is ically
signi ican associa ion wi h any o he measu ed ai s
in he Finnish Land ace (see Figu e 2 o pH
measu ed om loin, as an example). S a is ical
signi icance was claimed i he P- alue was below
2.0 × 10
−6
. This s ingen limi was se because he
obse a ions used in his s udy we e he same as
hose in ou p e ious genome-wide scans [11,14].
Because o he s ong LD wi h ASGA0070625, he
no el amino acid subs i u ions iden i ied in his s udy
and SNPs in he p omo e egion o PRKAG3 ga e
e y small P- alues o pH measu ed om loin o
he Finnish Yo kshi e (Table 4). The di e ences in P-
alues be ween ASGA0070625 and he SNPs which
we e in comple e LD wi h ASGA0070625 we e due o
sligh di e ences in he numbe o geno ypes a ailable
o each SNP. No signi ican associa ion was de ec ed
be ween I199V and he measu ed mea quali y ai s
o he Yo kshi e. Fo he Land ace, in con as , he
s onges associa ion was obse ed be ween I199V
and pH measu ed om ham (P- alue = 6.44 × 10
−7
wi h an allele subs i u ion e ec o 0.030 ± 0.006). The
associa ion be ween I199V and pH measu ed om
loin was also s ong in he Land ace (Table 4).
Figu e 1 Linkage disequilib ium (LD) exp essed as
2
alues (black colo indica ed comple e LD) wi hin and a ound PRKAG3 o he
Finnish Yo kshi e (A) and Land ace (B).
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Haplo ype associa ion
Fou haplo ypes we e iden i ied ha ing allele Go
ASGA0070625 (Table 3). When each o hese was es ed
agains all o he haplo ypes in he Yo kshi e da a, he
only s a is ically signi ican associa ion (P- alue = 6.35 ×
10
−8
, Table 5) was obse ed be ween HAP2 (wi h 199I
o allele A) and pH measu ed om loin. The subs i u-
ion e ec o his haplo ype was 0.039 ± 0.007. HAP2
was also he only haplo ype ha showed a signi ican as-
socia ion in he Finnish Land ace wi h bo h pH mea-
su emen s (Table 5). The haplo ype subs i u ion e ec s
o pH measu ed om loin and ham we e 0.031 ± 0.007
and 0.039 ± 0.007, espec i ely. Haplo ypes ca ying al-
lele Ao ASGA0070625 (HAP5-HAP12) had no signi i-
can associa ion wi h any o he es ed mea quali y
ai s. Haplo ypes wi h only one o wo obse a ions
(HAP6, HAP11, and HAP12) we e excluded om he
analysis.
Discussion
Mea colo and pH a e ai s which a e commonly in-
cluded in pig b eeding p og ams o imp o e he echno-
logical p ope ies o po k and o inc ease consume
g a i ica ion. Se e al s udies indica e ha PRKAG3 is
one o he key genes causing a ia ion in po k mea pH,
L* (ligh ness o colo ), and d ip loss be ween animals
[5,6,15-18]. The p o ein encoded by PRKAG3 is he skel-
e al muscle cell-speci ic egula o y subuni gamma 3 o
AMP-ac i a ed p o ein kinase (AMPK). AMPK is an en-
e gy senso which, when ac i a ed in esponse o cellula
me abolic s esses, di ec ly phospho yla es and inac i-
a es he key enzymes in ol ed in egula ing de no o
biosyn hesis o a y acid and choles e ol. The bes
known mu a ion in PRKAG3 is 200Q, which is ound
only in he Hampshi e pig b eed. The allele 200Q causes
a high con en o s o ed glycogen in whi e skele al mus-
cles, leading o low muscle pH 24 h pos mo em, poo
wa e -holding capaci y, and low p ocessing yield [5].
Fu he mo e, I199V and T30N a e epo ed o a ec pH
[6,15,17,18], and a ia ions in he p omo e egion o
PRKAG3 ha e been associa ed wi h gene exp ession and
mea quali y [12].
In his s udy, we cha ac e ized se e al no el amino acid
subs i u ions wi hin exons 2, 3, and 4. The SNPs cha ac e -
ized in he PRKAG3 p omo e egion co espond o hose
Table 3 Haplo ypes in PRKAG3 and hei equencies
123456789101112Wild boa
ASGA0070625 GGGGAAAAAAAA
g.-311A > G AAAAAAAGGGGGA
g.-221G > A GGGGGGGAAAAAG
g.-157C > G CCCCGGGGGGGGC
g.-58A > G AAAAGGGGGGGGA
K24E GGGGAGGAAAAAG
T30N CCCCAAACCCCAC
I41V GGGGGGGAAAGGG
G52S A G G A GGGGGGGGG
K131R AAAAAAAGGGAAA
P134L CCCCCCCTTTCCC
I199V G A G A G A G A GGGGG
Yo kshi e 0.418 0.363 0.007 0.007 0.002 0.002 0.007 0.080 0.087 0.027 0 0
Land ace 0.122 0.249 0.341 0 0.124 0 0 0.041 0.116 0 0.002 0.004
Figu e 2 P- alues (−log10) o he SNPs o pH measu ed om
loin. P- alues o he Finnish Yo kshi e a e ma ked wi h black
diamonds and o he Finnish Land ace wi h g een diamonds.
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epo ed by Ryan e al. [12]. Based on he genomic se-
quence he SNP g.-158C > G epo ed by Ryan e . al. co e-
sponds o ou g.-157C > G. The genomic loca ion o hese
SNPs is he same. All no el amino acid subs i u ions
(K24E, I41V, K131R, and P134L) and cha ac e ized SNPs in
he p omo e egion we e in comple e LD in he Finnish
Yo kshi e, based on Haplo iew analysis. The Finnish Land-
ace b eed showed mo e di e si y, wi h only g.-157C > G,
g.-58A > G, and K24E in comple e LD wi h each o he and
wi h ASGA0070625. S ong bu no comple e LD was also
epo ed by Ryan e al. [12] o SNPs in he p omo e e-
gion. No signi ican LD was ound be ween he p omo e
egion SNPs o no el amino acid subs i u ions and I199V.
This inding is simila o he obse a ion made by Ryan
e al. [12] ega ding LD be ween p omo e egion SNPs
and I199V.
A sligh ly di e en pic u e o LD can be d awn om
he haplo ype es ima es ob ained by he Fas PHASE p o-
g am (Table 3). Haplo ypes HAP5, HAP6, and HAP7
di ide he LD pa e n in o wo g oups. The i s g oup
comp ises he SNPs in comple e LD, namely
ASGA0070625, g.-157C > G, and g.-58A > G in he
Finnish Yo kshi e, and addi ionally K24E in Finnish
Land ace. Fo Land ace, his is exac ly he same esul
as gi en by Haplo iew. The second g oup includes SNPs
g.-311A > G, g.-221G > A, I41V, K131R, and P134L in
he Yo kshi e, bu no in he Land ace.
The e a e se e al explana ions o he di e en LD
ou comes om Haplo iew and Fas PHASE analyses,
such as di e en algo i hms and ways o ea ing missing
geno ypes be ween he wo p og ams. Geno yping o
phasing e o s may also cause spu ious haplo ypes. The
Table 4 P- alues o he associa ion be ween SNPs in PRKAG3 and mea quali y ai s
Yo kshi e Land ace
Loin Ham Loin Ham
Va ia ion pH L* a* pH L* a* pH L* a* pH L* a*
ASGA0070625 7.27 × 10
−13
5.52 × 10
−6
4.38 × 10
−5
3.47 × 10
−6
0.73 8.85 × 10
−3
0.02 0.03 0.03 0.02 0.34 0.79
g.-311A > G 6.83 × 10
−10
4.22 × 10
−5
6.28 × 10
−4
1.44 × 10
−4
0.74 0.05 0.28 0.36 0.36 0.60 0.93 0.79
g.-221G > A 6.83 × 10
−10
4.22 × 10
−5
6.28 × 10
−4
1.44 × 10
−4
0.74 0.05 0.33 0.62 0.36 0.64 0.98 0.77
g.-157C > G 7.94 × 10
−11
2.79 × 10
−5
4.29 × 10
−4
2.68 × 10
−5
0.56 0.03 0.02 0.06 0.37 0.05 0.38 0.83
g.-58A > G 7.94 × 10
−11
2.79 × 10
−5
4.29 × 10
−4
2.68 × 10
−5
0.56 0.03 0.03 0.09 0.36 0.06 0.41 0.92
K24E 5.80 × 10
−10
3.83 × 10
−5
3.16 × 10
−3
7.83 × 10
−5
0.38 0.03 0.23 0.27 0.16 0.15 0.57 0.54
T30N 0.13 0.39 0.46 0.12 0.61 0.36 0.02 6.51 × 10
−3
0.32 0.01 0.16 0.50
I41V 1.41 × 10
−11
1.43 × 10
−5
9.20 × 10
−5
1.96 × 10
−5
0.82 0.02 0.45 0.92 0.04 0.68 0.53 0.59
G52S 0.36 0.58 0.21 0.77 0.91 0.81 0.41 0.79 0.84 0.78 0.53 0.64
K131R 1.41 × 10
−11
1.43 × 10
−5
9.20 × 10
−5
1.96 × 10
−5
0.82 0.02 0.41 0.93 0.08 0.54 0.60 0.62
P134L 1.41 × 10
−11
1.43 × 10
−5
9.20 × 10
−5
1.96 × 10
−5
0.82 0.02 0.43 0.85 0.04 0.70 0.55 0.67
I199V 0.01 0.19 0.12 0.09 0.93 0.10 1.62 × 10
−5
0.09 0.04 6.44 × 10
−7
0.17 0.67
DBUN0002708 3.21 × 10
−10
4.15 × 10
−5
3.81 × 10
−4
3.36 × 10
−4
0.87 0.01 5.37 × 10
−3
0.02 0.05 0.07 0.37 0.75
Table 5 P- alues o he associa ions be ween haplo ypes in PRKAG3 and mea quali y ai s
Yo kshi e Land ace
Loin Ham Loin Ham
Haplo ype
a
pH L* a* pH L* a* pH L* a* pH L* a*
HAP1 0.47 0.90 0.14 0.76 0.69 0.66 0.41 0.89 0.92 0.65 0.61 0.57
HAP2 6.35 × 10
−8
6.00 × 10
−4
0.01 1.72 × 10
−4
0.97 0.01 8.69 × 10
−6
0.15 3.99 × 10
−3
1.80 × 10
−8
0.35 0.41
HAP3 0.15 0.51 0.17 0.40 0.03 5.98 × 10
−3
0.04 0.41 0.17 0.02 0.74 0.13
HAP4 0.42 0.06 0.24 0.88 0.23 0.38
HAP5 0.02 0.01 0.27 0.02 0.17 0.60
HAP7 0.73 0.44 0.78 0.16 0.80 0.32
HAP8 2.25 × 10
−4
3.24 × 10
−3
0.11 3.03 × 10
−3
0.82 0.35 0.12 0.05 0.83 0.12 0.08 0.48
HAP9 2.29 × 10
−4
0.05 4.98 × 10
−4
0.13 0.48 0.08 0.07 0.23 0.12 0.16 0.63 0.94
HAP10 0.04 0.17 0.50 0.11 0.38 0.42
a
Because o low numbe o obse a ions, haplo ypes HAP6, HAP11, and HAP12 we e excluded om he analysis.
Uima i and Si onen BMC Gene ics 2014, 15:29 Page 6 o 9
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la e iew is suppo ed by he ac ha haplo ypes
HAP6 and HAP7 a e ex emely a e in he Yo kshi e
and absen in he Land ace, while haplo ypes HAP11
and HAP12 a e comple ely absen in he Yo kshi e and
a e ca ied only by one o wo animals in Land ace.
Addi ionally, some haplo ypes may ha e been in oduced
in o one b eed om he o he h ough occasional in ol-
un a y c ossing o b eeds a he a m le el.
Single-SNP analysis yielded con o e sial esul s when
he wo b eeds we e compa ed. The SNP which was e-
po ed as highly signi ican o pH measu ed om loin
in he Finnish Yo kshi e in ou p e ious s udy [11] was
signi ican also in his analysis, gi en he ac ha mos
o he animals (Yo kshi e boa s) we e he same in bo h
analyses. Howe e , had he Finnish Land ace been used
as he alida ion popula ion o he p e ious s udy, he
signi icance o ASGA0070625 would no ha e been e-
pea ed and his SNP would ha e been claimed o be a
popula ion-speci ic ma ke o mea pH. Simila ly,
I199V was no epea ed in he Yo kshi e, aising a doub
ha I199V is b eed- o popula ion-speci ic. Bu when
haplo ypes ins ead o single SNPs we e used in he asso-
cia ion analysis, he esul s we e cohe en : he same
haplo ype (HAP2) was signi ican ly associa ed wi h pH
in bo h b eeds.
The haplo ype wi h bo h 199I and 24E alleles (o g.-157C
o g.-58A) was ound a o able o pH measu ed om loin
and ham in bo h b eeds. This p o ides s ong suppo o
he hypo hesis ha allele 199I alone does no c ea e a posi-
i e e ec on he pH le el in muscle pos mo em, bu he
animal has o ca y an addi ional a ia ion ei he in he p o-
mo e egion o PRKAG3 (g.-157C o g.-58A o bo h) o
glu ama e a amino acid posi ion 24 (o 47 depending on
he PRKAG3 iso o m used o naming). Analysis o he
SNP e ec on he p o ein unc ion sugges ed ha he e
maybesomedi e encesbe ween ansc ip s.Basedona
SIFT analysis [13], he K24E mu a ion showed a signi ican
(SIFT sco e < 0.05) e ec on he p o ein unc ion in he
ENSSSCT00000033825 ansc ip , bu no in ENSSSCT
00000017641 (Table 2). Thus he p omo e SNPs may
a ec he exp ession o a speci ic ansc ip , and oge he
wi h amino acid changes, may in luence he unc ion o
PRKAG3. In e es ingly, he haplo ype wi h he lowes
P- alue and a posi i e associa ion wi h mea quali y in bo h
o he s udied b eeds is simila o a wild boa haplo ype,
wi h he excep ion ha he wild boa s’199V is eplaced
by 199I.
Conclusions
A single mu a ion in ASGA00070625, in he p omo e
egion, in he amino acid a posi ions 24 o a 199 o
PRKAG3 is no alone su icien o c ea e a a o able e -
ec on mea quali y. Ins ead, a combina ion o a ia ions
o a haplo ype wi h he p omo e egion a ian s g.-
157C and g.-58A and amino acid subs i u ions 24E, and
199I o PRKAG3 is needed o achie e a posi i e impac
on mea quali y ai s, a leas in he Finnish Yo kshi e
and Land ace popula ions. The esul s p esen ed he e
can be di ec ly applied in ma ke -assis ed selec ion o
imp o e he quali y o po k mea .
Me hods
Animal ma e ial o his s udy included p e iously col-
lec ed semen and hai samples o he boa s hus no e h-
ical app o al was equi ed. All pheno ypic da a we e
kindly supplied by he Figen L d (h p://www. igen. i).
Animals and mea quali y measu emen s
The s udy included 220 Finnish Yo kshi e and 230
Finnish Land ace AI (a i icial insemina ion) boa s. Add-
i ionally, h ee Eu opean wild boa s we e sequenced, bu
no pheno ypic obse a ions we e a ailable o hese
boa s. B eeding alues o he s udied boa s we e es i-
ma ed using he ull na ional pig egis y da a including
mea quali y measu emen s om se e al housand ani-
mals. We used a single- ai BLUP p ocedu e o es ima e
a b eeding alue o mea pH, colo L* (ligh ness o
mea ) and a* ( edness o mea ). The s a is ical model in-
cluded slaugh e ba ch and sex as ixed, and li e and
animal as andom e ec s. The model was he same as
used in na ional b eeding alue es ima ion in Finland,
excep ha in he na ional e alua ion all mea quali y
ai s a e analyzed simul aneously by a mul i ai model,
whe eas in his s udy each ai was analyzed sepa a ely.
We selec ed he single- ai app oach o ensu e ha gen-
e ic co ela ion be ween ai s did no a ec he associ-
a ion esul s. The es ima ed b eeding alue (EBV)
e lec s he ela i e gene ic me i o an animal. EBVs a e
gene ally mo e eliable han he animal’s own pheno ype,
because hey a e based on all a ailable eco ds on ela-
i es and a e simul aneously co ec ed o speci ic sys-
ema ic and non-sys ema ic e ec s speci ied in he
es ima ion model. Mos o he mea quali y da a o a
speci ic AI boa is ob ained om i s p ogeny and i s ull-
and hal -sibs.
EBVs o mea quali y ai s a e based on measu e-
men s aken om animals aised in a es s a ion. Young
pigle s (on a e age 30 kg weigh ) a e aised up o 100 kg
li e weigh in a s anda dized es s a ion en i onmen .
A e he es pe iod, all bu he bes young boa s a e
sen o a slaugh e house whe e pH and colo L* and a*
o mea a e measu ed 24 h a e slaugh e . Fo his
s udy, colo L* and a* we e measu ed on a eshly cu
muscle su ace wi h a Minol a CR 300 colo ime e and a
CIELAB colo scale s anda d [19,20], and pH was de e -
mined using a Knick 752 pH me e and an Ingold 406
elec ode. Measu emen s we e aken om loin (longissi-
mus) and ham (semimemb anosus) muscles. Fo mo e
Uima i and Si onen BMC Gene ics 2014, 15:29 Page 7 o 9
h p://www.biomedcen al.com/1471-2156/15/29
in o ma ion on he measu emen p ocedu es, see Se ón-
Aimonen e al. [1].
The s udied Finnish Yo kshi e and Land ace boa s we e
bo n be ween 1992 and 2009, and included se e al ela i e
pai s such as si e-son, ull-sibs, g andsi e-g andson, e c.
A e age ela edness be ween boa s was 0.16 and 0.14 o
he Yo kshi e and Land ace, espec i ely.
Geno yping and sequencing
Pa o he SNP da a p esen ed in his s udy o igina e om
ou p e ious whole-genome analyses [11,14] using he Po -
cineSNP60 BeadChip (Illumina L d, San Diego, USA).
Geno yping was pe o med a FIMM (Ins i u e o Molecu-
la Medicine Finland, Helsinki, Finland) o a GeneSeek
(Lincoln, USA). DNA was ex ac ed ei he om hai olli-
cles o semen, wi h a a ge DNA concen a ion o 300 ng.
SNPs we e mapped o he pig genome build Ssc o a10.2.
We es ic ed ou s a is ical analysis o co e only SNPs lo-
ca ed in a 20-Mb egion su ounding PRKAG3 ( om
120 Mb o 140 Mb on ch omosome 15), because ou p e i-
ous analyses had shown ha he mos signi ican SNPs o
mea quali y we e in his egion.
A e designing p ime pai s o genomic sequence ana-
lysis, we ampli ied he DNA agmen s wi h gene-speci ic
p ime s. PCR amplicons we e pu i ied using ExoSAP-IT™
(GE Heal hca e, Pisca away, USA), and sequenced in bo h
di ec ions wi h he same p ime s as in he ampli ica ion
p ocedu es. Sequencing was pe o med on a 3500 × L Gen-
e ic Analyze (Applied Biosys ems, Ca lsbad, USA) using a
BigDye Te mina o 3.1 ki (Applied Biosys ems, Ca lsbad,
USA) and E OH p ecipi a ion.
S a is ical me hod
P io o associa ion analysis, he EBVs we e de eg essed
and hei weigh s we e calcula ed by he me hod p o-
posed by Ga ick e al. [21]. The me hod emo es pa en
a e age e ec s on EBV, so ha he de eg essed EBV
mo e closely e lec s he animal’s own pe o mance and
he pe o mance o i s o sp ing. The de eg ession p o-
cedu e also p e en s eg ession owa ds he popula ion
mean, which is ypical o EBVs which a e based on a
limi ed amoun o in o ma ion. Gene ally, he mo e eli-
able he de eg essed EBV is, he mo e weigh i ecei es
in he associa ion analysis.
Associa ion analysis was pe o med ei he o indi id-
ual SNPs o a combina ion o SNPs (haplo ype). Each
SNP/haplo ype was analyzed sepa a ely o associa ion
wi h mea quali y ai s using he ollowing mixed linea
model:
yi¼μþbxiþaiþei;
whe e y
i
is he de eg essed EBV o he mea quali y ai ;
x
i
is he numbe o mino alleles (0, 1, o 2) o he es ed
SNP o he numbe o copies o he es ed haplo ype (0:
an animal ca ies no copies; 1: an animal ca ies one
copy; 2: an animal ca ies wo copies); bis he co e-
sponding eg ession coe icien ; a
i
is a andom polygenic
e ec wi h a no mal dis ibu ion wi h mean 0 and a
a iance-co a iance s uc u e o Aσ
2a
, whe e Ais he
addi i e ela ionship ma ix and σ
2a
is he polygenic a i-
ance; and e
i
is a andom esidual e ec wi h a no mal
dis ibu ion wi h mean 0 and a a iance-co a iance
s uc u e o Iσ
2e
/w
i
, whe e Iis an iden i y ma ix, σ
2e
is
he esidual a iance, and w
i
is he weigh . Associa ion
analyses we e pe o med using he AI-REML me hod in
he DMU p og am package [22]. Haplo ypes we e es i-
ma ed wi h Fas PHASE [23], and linkage disequilib ium
plo s we e p oduced wi h Haplo iew [24].
Abb e ia ions
AI: A i icial insemina ion; AMPK: AMP-ac i a ed p o ein kinase; EBV: Es ima ed
b eeding alue; LD: Linkage disequilib ium.
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Au ho s’con ibu ions
PU ca ied ou he da a analysis and d a ed he manusc ip . AS pe o med
he sequencing and SNP calling, and helped o d a he manusc ip . Bo h
au ho s ead and app o ed he inal manusc ip .
Au ho s’in o ma ion
PU: cu en add ess: Depa men o Ag icul u al Sciences, Animal B eeding,
FI-00014 Uni e si y o Helsinki, Finland; AS: cu en add ess: MTT Ag i ood
Resea ch Finland, Bio echnology and Food Resea ch, FI-31600 Jokioinen,
Finland.
Acknowledgemen s
The au ho s wish o hank Ma ja-Liisa Se ón-Aimonen, who p o ided he
o iginal es ima ed b eeding alues o he boa s, and Ta ja Ho i uo i and
Anneli Vi a, who ca ied ou he echnical wo k on DNA ex ac ion and
sequencing. The esea ch was unded by he Minis y o Ag icul u e and
Fo es y o Finland.
Recei ed: 27 No embe 2013 Accep ed: 7 Feb ua y 2014
Published: 28 Feb ua y 2014
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doi:10.1186/1471-2156-15-29
Ci e his a icle as: Uima i and Si onen: A combina ion o wo a ian s in
PRKAG3 is needed o a posi i e e ec on mea quali y in pigs. BMC
Gene ics 2014 15:29.
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