Acta agriculturae Slovenica, 88(november 2006)1, 11-18. http://aas.bf.uni-lj.si Agris category codes: L10 COBISS Code 1.01 EFFECT OF PPARGC-1 GENE ON BACKFAT THICKNESS IN PIGS * Tina FLISAR a), Tanja KUNEJ b), Milena KOVAČ c) and Peter DOVČ d) a) Univ. of Ljubljana, Biotechnical Fac, Zootechnical Dept, Groblje 3, SI-1230 Domžale, Slovenia, e-mail: tina.flisar@bfro.uni-lj.si. b) Same address as a), Asist., Ph.D., e-mail: tanja.kunej@bfro.uni-lj.si. c) Same address as a), Assoc.Prof, Ph.D., e-mail: milena@mrcina.bfro.uni-lj.si. d) Same address as a), Prof, Ph.D., e-mail: peter.dovc@bfro.uni-lj. si. Received July 27, 2006, accepted November 20, 2006. Delo je prispelo 27. julija 2006, sprejeto 20. novembra 2006. ABSTRACT PPARGC-1 gene is assumed to be a candidate gene with a major effect on fatness and meat quality. In this study, frequency of T/A substitution at position 1378 in PPARGC-1 gene in pigs was examined in boars of 4 different breeds. Frequency of genotypes and alleles were compared between animals with the thinnest and with the thickest backfat. Differences in genotype frequency between groups were significant in dam line of Slovenian Landrace (SL11) and sire line of Large White breed (LW66). Allele A was predominantly present in animals with thick backfat in SL11 (71.05%), LW66 (58%) and in sire line of Slovenian Landrace (67.65%). Differences in allele frequency were significant in SL11. We found a significant effect on phenotypic and breeding values for backfat in population of Large White breeds, where homozygotes TT had the thickest backfat, and heterozygotes the thinnest. In population of Slovenian Landrace breed the thickest backfat had homozygotes AA and the thinnest homozygotes TT. Similar results were obtained by analysis of gene effect on breeding values. Inconsistency could be explained by different background of allele, epistasis and intensity of selection. Key words: pigs / backfat / genetics / PPARGC-1 gene / polymorphism POVEZAVA GENA PPARGC-1 Z DEBELINO HRBTNE SLANINE † IZVLEČEK Gen za PPARGC-1 je kandidatni gen z velikim učinkom na zamaščenost in kvaliteto mesa. V prispevku smo preučevali frekvence točkovnih mutacij A -> T na mestu 1378 v 8. eksonu gena za PPARGC-1 pri merjascih štirih različnih linij dveh pasem. Frekvence genotipov in alel smo primerjali med merjasci s tanjšo (skupina A) ter merjasci z debelejšo hrbtno slanino (DHS), (skupina B). Razlike v frekvencah genotipov so bile značilne med skupinama merjascev pri maternalni liniji pasme slovenska landrace (SL11) ter pri terminalni liniji pasme large white (LW66). Alela A je bila pogostejša pri živalih z debelejšo DHS pri SL11 (71,05 %), LW66 (58 %) ter pri terminalni liniji pasme slovenska landrace (67,65 %). Razlike v frekvencah alel so bile značilne v populaciji SL11. Genotip je značilno vplival na DHS v populacijah LW11 in LW66, kjer so imeli debelejšo slanino homozigoti TT in tanjšo heterozigoti. V populacijah SL11 in SL55 so imeli najdebelejšo slanino genotipi AA in najtanjšo slanino homozigoti TT. * This article is part of a graduation thesis ´Effect of polymorphism T1378A in PPARGC-1 gene on fattening and growth rate traits in pigs´, issued by Tina Flisar, supervisor prof. Peter Dovč, Ph.D. † Prispevek je del diplomskega dela Tine Flisar z naslovom ´Vpliv polimorfizma T1378A v genu za PPARGC-1 na pitovne lastnosti pri prašičih´, mentor prof. dr. Peter Dovč. 12 Acta agriculturae Slovenica, 88(november 2006) 1. Nasprotujoči rezultati so lahko posledica genetskega ozadja alel, epistaze in intenzivnosti selekcije. Ključne besede: prašiči / debelina hrbtne slanine / genetika / gen PPARGC-1 / polimorfizem INTRODUCTION Although most genetic progress in pigs has been made by selection on predictor of breeding values, information, acquired from molecular genetics, can clarify some genetic factors with effect on production traits. Due to quantitative nature of production traits, the majority of genetics is oriented to identification and mapping quantitative trait locus (QTL) with effect on economically important traits. One of the most important traits is also meatiness, because in Slovenia the carcasses are still paid regarding the lean meat content. Growth of adipose tissue is a quantitative trait regulated by many transcription factors, included in the process of adaptive thermogenesis and biosynthesis of mitochondria (Rosen et al., 2000). One of them is protein PPARGC-1 (peroxisome proliferator-activated receptor-gamma coactivator-1), which is a transcriptional coactivator of the nuclear receptor PPAR? (peroxisome proliferator-activated receptor-gamma). It is an important factor in the process of adipocyte differentiation and muscle fibre type determination (Lin et al., 2002; Rosen et al., 2000). PPARGC-1 stimulates mitochondrial biogenesis and respiration in muscle cells. It induces activity of the genes included in adaptation of organism on temperature and nutritional changes (Knutti and Kralli, 2001; Puigserver et al., 1998). The mechanism of adipogenesis regulation and the role of PPAR? and PPARGC-1 were also described by Milosevic Berlic et al. (2004). The porcine PPARGC-1 gene (GenBank acc. no. AY346131) is located on chromosome 8 (Milosevic Berlic, 2002; Jacobs et al., 2006). The sequencing, polymorphisms analysis and mapping of porcine PPARGC-1 gene were reported by Jacobs et al. (2006). Substitution T/A at nucleotide position 1378 causes amino acid substitution (Cys›Ser) at position 430. According to Jacobs et al. (2006) Cys/Ser substitution could have major effects because of influences on disulfide bridges that might be present in the protein. Frequency distribution of mutation A1378T in PPARGC-1 was studied in Chinese and Western pig breeds because of remarkable differences in fatness (Kunej et al., 2005). Allele T was associated with fat type Chinese pig breeds, whereas the A allele was more frequent in lean type Western pigs (Kunej et al., 2005). Jacobs et al. (2006) studied the PPARGC-1 gene effect on performance in Meishan x White Composite resource population and the study did not reveal the effect of A1378T on performance. They found a significant correlation between SNP in exon 9 (A1747C) and leaf fat weight, backfat, and weight of the belly. Several QTLs with effect on backfat, located on chromosome 8 were reported (Bidanel and Rothschild, 2002; Rohrer, 2000; Bidanel et al., 2001). Some of these regions correspond to the location of PPARGC-1. PPARGC-1 is assumed to be a candidate gene with a major effect on backfat and other characteristic associated with fatness. This gene can be also considered as a candidate gene for meat quality (Knutti and Kralli, 2001). The purpose of this study was to find the frequency of mutation of PPARGC-1 gene in boars of different phenotype for backfat. The effect of PPARGC-1 gene on phenotypic and breeding values was analyzed using a linear model. Flisar, T. et al. Effect of PPARGC-1 gene on backfat thickness in pigs. 13 MATERIAL AND METHODS Data Preparation In this study, boars of dam and sire line of Slovenian Landrace (SL11 and SL55) and Large White (LW22 and LW66) breed were included. Boars were selected at intermediate selection stage in the year 2003 in the performance test and finished the test at 100 kg. Animals were divided in two groups based on backfat thickness (Table 1). Animals of each breed with the thinnest backfat were assigned to group A and those with the thickest backfat to group B. The total number of genotyped animals was 166. The differences between groups are evident (Table 1). Table 1. Statistics for backfat in group A and B Preglednica 1. Osnovna statistika za debelino hrbtne slanine v skupinah A in B A B Breed Pasma n x min max LW22 25 7.97 6.67 8.67 LW66 19 7.51 6.33 8.67 SL11 22 7.55 6.00 8.00 SL55 18 8.63 7.67 9.33 n x min max 21 12.48 11.33 15.00 25 10.29 9.33 12.67 19 13.26 12.33 16.67 17 15.86 14.67 17.67 n – number / število; x – mean / povprečje; min – minimum / najmanj; max – maximum / največ; PCR-RFLP analysis Genomic DNA was isolated from skin tissue samples by standard phenol-chloroform-isoamyl alcohol (25:24:1) extraction (Ausubel et al., 2000). The 200-bp fragment of PPARGC-1 gene was amplified with PCR reaction, which was carried out in a volume of 10 µl: 2 µl of template DNA, 1 x PCR buffer (Fermentas, Vilnius, Lithuania), 1mM MgCl2, 200 µM dNTP, 0.5 U Taq DNA polymerase (Fermentas, Vilnius, Lithuania) and 5 pmol of each primer. The primers used for amplification of target sequences were: PPARGC-1 – SSCP.F (5'- TAAAGATGCCGCCTCTGACT - 3') PPARGC-1 – SSCP.R (5'- CTGCTTCGTCGTCAAAAACA - 3'). The following amplification parameters were applied: 95 °C for 5 min followed by 31 cycles: 95 °C for 1 min, 56 °C for 1 min and 72 °C for 1 min. The reaction was completed by the final synthesis at 72 °C for 7 min. The PCR products were digested with 2U of AluI (Fermentas, Vilnius, Lithuania) at 37 °C overnight and analyzed on 2% agarose gels. Statistical Analysis Frequency distribution of genotypes and alleles were calculated. The statistical package SAS was used (SAS Inst. Inc., 2001). Genotype and allele frequencies between groups were tested using the multinomial model in proc logistic (SAS Inst. Inc., 2001). The effect of PPARGC-1 genotype on phenotypic values was analyzed with the following statistical model: 14 Acta agriculturae Slovenica, 88(november 2006) 1. yi]k =µ + G,+ S j + b(xiJk -x) + e iß [1] For analysis the effect of PPARGC-1 genotype on breeding values the following statistical model was used: y« µ + G,.+e, [2] where yijk is the phenotypic respectively breeding value for backfat, µ is intercept, Gi is PPARGC-1 genotype (i=1, 2, 3), S j is the season of selection at intermediate selection stage in performance test (j=1, 2, 3, …, 11, 12), b is the regression coefficient for body weight at the end of performance test, and eijk is a residual. RESULTS AND DISCUSSION Following digestion of PCR product with AluI, the T allele was cut into fragments of 121, 27, 31 and 21 bp (Figure 1). Allele A had one restriction site more at position 1379 and 121 bp band was cleaved into 61 and 60 bp band, consecutive the allele A had: 61, 60, 27, 31 and 21 bp bands. On agarose gels were defined: genotype AA had ?60 and ?30 bp, genotype TT 121 and ?30 bp and heterozygote AT had 3 bands: 121, ?60 and ?30 bp. 121 bp 61 bp 60 bp 31 bp 27 bp 21 bp 1 2 3 4 5 6 500 bp 200 bp 100 bp 80 bp TT AT AA AT TT AA Figure 1. Restriction patterns of samples. Slika 1. Restrikcijska analiza vzorcev. Three genotypes were identified in the studied populations: AA, TT and AT (Table 2). The distribution of PPARGC-1 genotypes frequencies demonstrated significant differences between group A and group B in dam line of Slovenian Landrace (SL11) and sire line of Large White breed (LW66). In the populations of SL11 the frequency of heterozygote was 54.5% in group A and 57.9% in group B. The frequency of TT was 22% in group A, whereas no genotype TT was found in group B. In populations of LW66, the difference of frequency of AT between groups was larger (78.9% in group A and 44% in group B). In group B genotype TT was present in 20%, whereas in group A in 5.3%. Group A and B of dam line of Large White (LW22) had a similar distribution of genotypes (Table 2). In both groups genotype AA was the most frequent (80 and 81%) and the frequency of heterozygote was similar (20% and 18%). In populations of SL55 the most frequent was genotype AT (50% and 64.7%). In group A the frequency of genotype TT was 11%, whereas in group B was not present. Flisar, T. et al. Effect of PPARGC-1 gene on backfat thickness in pigs. 15 Table 2. Frequency of genotypes of PPARGC-1 gene and test of differences between groups Preglednica 2. Frekvence genotipov gena PPARGC-1 in test razlik med skupinama Total AA AT TT Test Breed Group Pasma Skupina n n frequency % n frequency % n frequency % P-value LW22 A 25 20 80.0 5 20.0 0 0.0 1.0000 B 21 17 81.0 3 18.0 1 1.0 LW66 A 19 3 15.8 15 78.9 1 5.3 0.0772 B 25 9 36.0 11 44.0 5 20.0 SL11 A 22 5 22.7 12 54.5 5 22.7 0.0472 B 19 8 42.1 11 57.9 0 0.0 SL55 A 18 7 38.9 9 50.0 2 11.0 0.8330 B 17 6 35.3 11 64.7 0 0.0 Table 3 Frequency of alleles of PPARGC-1 gene and test of differences between groups Preglednica 3 Frekvenca alelov gena PPARGC-1 in test razlik med skupinama Group Total Allele A Allele T Test Breed Pasma Skupina n n frequency % n frequency % P-value LW22 A 50 45 90.00 5 10.00 0.7703 B 42 37 88.10 5 11.90 LW66 A 38 21 55.26 17 44.74 0.7974 B 50 29 58.00 21 42.00 SL11 A 44 22 50.00 22 50.00 0.0550 B 38 27 71.05 11 28.95 SL55 A 36 23 63.89 13 36.11 0.7407 B 34 23 67.65 11 32.35 Allele A was predominantly present in animals with thick backfat (group B) in SL11 (71.05%), LW66 (58%) and in 67.65% was present in SL55 (Table 3). In LW22 allele A was more frequent in animals with thin backfat. Differences in frequency of alleles were significant in SL11. The analysis revealed significant differences in frequency distribution of genotypes and alleles of PPARGC-1 gene between animals with different phenotype for backfat in populations 16 Acta agriculturae Slovenica, 88(november 2006) 1. of SL11 and LW66. These results support the assumption that T/A substitution at 1378 nucleotide position in PPARGC-1 gene influences the fatness characteristics (Kunej et al., 2005). Table 4 Least square means (LSM) and standard errors (SE) for phenotypic values for backfat for different breeds Preglednica 4 Ocenjene srednje vrednosti (LSM) in standardne napake (SE) za debelino hrbtne slanine (v mm) po pasmah in genotipih Breed Trait Analysis Pasma Lastnost Analiza AA AT TT P-value LW22 BF-2 1 10.34 ± 0.39 8.94 ± 0.82 10.95 ± 2.54 0.3006 2 10.30 ± 0.38 8.89 ± 0.82 0.1339 BF-3 1 10.15 ± 0.36 8.62 ± 0.76 10.17 ± 2.35 0.2104 2 10.11 ± 0.35 8.58 ± 0.76 0.0809 LW66 BF-2 1 10.20 ± 0.59 8.67 ± 0.34 10.40 ± 0.70 0.0248 2 10.00 ± 0.67 8.46 ± 0.34 0.0735 BF-3 1 9.92 ± 0.54 8.46 ± 0.32 10.13 ± 0.65 0.0184 2 9.73 ± 0.61 8.46 ± 0.34 0.0615 SL11 BF-2 1 11.16 ± 0.83 9.92 ± 0.66 8.38 ± 1.30 0.1624 2 11.13 ± 0.86 9.93 ± 0.68 0.2666 BF-3 1 11.24 ± 0.84 9.91 ± 0.66 8.44 ± 1.32 0.1603 2 11.22 ± 0.88 9.92 ± 0.69 0.2370 SL55 BF-2 1 11.30 ± 1.34 11.73 ± 1.13 7.30 ± 2.67 0.2928 2 11.38 ± 1.41 11.70 ± 1.16 0.8754 BF-3 1 11.56 ± 1.39 12.17 ± 1.17 7.60 ± 2.77 0.3010 2 11.67 ± 1.46 12.12 ± 1.20 0.8281 LW22 – dam line of Large White breed; LW66 – sire line of Large White; SL11 – dam line of Slovenian Landrace; SL55 – sire line of Slovenian Landrace; BF–2 – average of two measurements of backfat; BF-3 – average of 3 measurements of backfat; Analysis 1 – least square means for different genotypes AA, AT, and TT; Analysis 2 – comparison between homozygotes AA and heterozygotes AT; LW22 – ženska linija pasme Large White; LW66 – moška linija pasme Large White; SL11 – ženska linija slovenske landrace; SL55 – moška linija slovenske landrace; BF–2 – povprečje dveh meritev hrbtne slanine; BF-3 – povprečje treh meritev hrbtne slanine; Analiza 1 – povprečja najmanjših kvadratov za različne genotipe AA, AT in TT; Analiza 2 – primerjava homozigotov AA in heterozigotov AT; Results of analysis of the effect of PPARGC-1 genotype are shown as the least square means (Table 4). The analysis 1 was done to compare all three genotypes (AA, AT, and TT), whereas in the analysis 2 homozygotes TT were excluded because of the low number of observations and the estimates of mean values were less reliable. In population of Large White boars the thinnest backfat had heterozygotes, what could be the consequence of non-additive effects. The thickest backfat had homozygotes TT, what was in agreement with results obtained by Kunej et al. Flisar, T. et al. Effect of PPARGC-1 gene on backfat thickness in pigs. 17 (2005). In population of Slovenian Landrace boars the homozygotes had the thinnest backfat, whereas the homozygotes AA had the thickest backfat. A significant effect of PPARGC-1 genotype was found in population of Large White boars. The estimation of PPARGC-1 gene effect on breeding values revealed similar results (Table 5). The heterozygotes had the smallest predicted breeding values in Large White populations but in population of Slovenian Landrace boars the best predicted breeding values had homozygotes TT. We found a significant effect of PPARGC-1 gene on breeding values for backfat in population of sire line of Large White breed. Table 5 Preglednica 5 Least square means (LSM) and standard errors (SE) for breeding values for backfat for different breeds and genotypes Ocenjene srednje vrednosti (LSM) in standardne napake (SE) za plemenske vrednosti za debelino hrbtne slanine po pasmah in genotipih Breed Pasma Analysis Analiza AA AT TT P-value LW22 LW66 SL11 1 -1.0987 ± 0.1586 -1.6916 ± 0.3411 0.0580 ± 0.9648 0.1374 2 -1.0987 ± 0.1586 -1.6916 ± 0.3411 0.1223 1 0.4397 ± 0.1846 -0.1128 ± 0.1254 0.2308 ± 0.2611 0.0491 2 0.4398 ± 0.1864 -0.1128 ± 0.1266 0.0192 1 -1.9898 ± 0.4490 -2.7588 ± 0.3376 -3.8344 ± 0.7241 0.0980 2 -1.9898 ± 0.4742 -2.7589 ± 0.3565 0.2036 SL55 2 -0.5046 ± 0.5650 -0.5046 ± 0.5739 -0.2923 ± 0.4555 -0.2923 ± 0.4627 -3.0280 ± 1.4403 0.2097 0.7752 LW22 – dam line of Large White breed; LW66 – sire line of Large White; SL11 – dam line of Slovenian Landrace; SL55 –sire line of Slovenian Landrace; Analysis 1 – least square means for different genotypes AA, AT, and TT; Analysis 2 – comparison between homozygotes AA and heterozygotes AT; LW22 – ženska linija pasme Large White; LW66 – moška linija pasme Large White; SL11 – ženska linija slovenske landrace; SL55 – moška linija slovenske landrace; Analiza 1 – povprečja najmanjših kvadratov za različne genotipe AA, AT in TT; Analiza 2 – primerjava homozigotov AA in heterozigotov AT; CONCLUSIONS The comparison of the genotype frequency between groups of the thinnest (group A) and the thickest (group B) revealed differences in population of sire line Large White breed and dam line of Slovenian Landrace breed, but the distribution of allele differed between group only in population of dam line of Slovenian Landrace breed. The estimation of PPARGC-1 gene effect showed a significant effect of PPARGC-1 gene on phenotypic values in population of Large White boars, but the analysis of gene effect on breeding values revealed a significant effect only in sire line of Large White breed. Such conflicting results may be the consequences of different background of allele, epistasis, intensity of selection, and deficiency of experiment design. Although these results did not confirmed the effect of allele A to be involved in inhibited fat deposition in both populations this type of analysis (especially as data size increases) with porcine candidate genes provides useful information for better understanding of the process of fat deposition. Further evaluations and researches of effect of T1378A substitution in porcine 1 18 Acta agriculturae Slovenica, 88(november 2006) 1. PPARGC-1 gene should be studied on performance traits in different commercial pig populations. 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