A combination of two variants in PRKAG3 is needed for a positive effect on meat quality in pigs

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 h p://www.biomedcen al.com/1471-2156/15/29 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. Uima i and Si onen BMC Gene ics 2014, 15:29 Page 3 o 9 h p://www.biomedcen al.com/1471-2156/15/29 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). Uima i and Si onen BMC Gene ics 2014, 15:29 Page 4 o 9 h p://www.biomedcen al.com/1471-2156/15/29 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. Uima i and Si onen BMC Gene ics 2014, 15:29 Page 5 o 9 h p://www.biomedcen al.com/1471-2156/15/29 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 h p://www.biomedcen al.com/1471-2156/15/29 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 Re e ences 1. Se ón-Aimonen ML, Honka aa a M, Se enius T, Mäki-Tanila A, Puon i M: Gene ic a ia ion o loin and ham quali y in Finnish Land ace and La ge Whi e pigs. Ag ic Food Sci 2007, 16:89–102. 2. Moelle SJ, Mille RK, Edwa ds KK, Ze by HN, Logan KE, Ald edge TL, S ahl CA, Boggess M, Box-S e ensmeie JM: Consume pe cep ions o po k ea ing quali y as a ec ed by po k quali y a ibu es and end-poin cooked empe a u e. Mea Sci 2010, 84:14–22. 3. Sellie P: Gene ics o mea and ca cass ai s. In The Gene ics o he Pig. 2nd edi ion. Edi ed by Ro hschild MF, Ru insky A. Chippenham, UK: CAB In e na ional; 2011:355–386. 4. 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Submi you nex manusc ip o BioMed Cen al and ake ull ad an age o : • Con enien online submission • Tho ough pee e iew • No space cons ain s o colo figu e cha ges • Immedia e publica ion on accep ance • Inclusion in PubMed, CAS, Scopus and Google Schola • Resea ch which is eely a ailable o edis ibu ion Submi you manusc ip a www.biomedcen al.com/submi Uima i and Si onen BMC Gene ics 2014, 15:29 Page 9 o 9 h p://www.biomedcen al.com/1471-2156/15/29