The PRKAG3 gene encodes for the regulatory gamma 3 subunit of adenosine monophosfate-activated protein kinase (AMPK) protein that acts as a regulator of carbohydrate and fat metabolism. Several single nucleotide polymorphisms (SNPs) have been identified in the porcine PRKAG3 gene. Two of them (c.595A>G or p.I199V and c.599G>A or p.R200Q) have been associated with variability of meat quality and carcass traits. In this study, we investigated the frequency of the c.595A>G and c.599G>A SNPs in 372 animals of five Italian autochthonous pig breeds (Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola and Nero Siciliano). Genomic DNA were extracted from hair roots or blood and SNPs genotyping was performed by PCR-RFLP protocols. The c.599A (p.200Q) allele was carried by only 3 Nero Siciliano pigs. All five breeds were polymorphic at the c.595A>G site. The c.595A (p.I199) allele was the less frequent in the analysed breeds with minor allele frequency ranging from 0.144 (Nero Siciliano) to 0.464 (Casertana). Based on identified allele frequencies, the c.595A>G SNP can be useful in association studies with meat, carcass and ham quality traits in the Italian local pig breeds

D\u2019Alessandro, E.

Dall'Olio, S.

Fontanesi, L.

Nanni Costa, L.

Russo, V.

Scotti, E.

English

DALL'OLIO, STEFANIA

SCOTTI, EMILIO

NANNI COSTA, LEONARDO

D’Alessandro, E.

RUSSO, VINCENZO

FONTANESI, LUCA

Archivio istituzionale della ricerca - Alma Mater Studiorum Università di Bologna

8th International Symposium on the Mediterranean Pig, Slovenia, Ljubljana, October 10th−12th, 2013.Acta argiculturae Slovenica, Supplement 4, 29–32, Ljubljana 2013COBISS: 1.08Agris category code: L10 AnALySIS Of PRKAG3 gene POLyMOrPhISMS In ItALIAn AUtOChthOnOUS PIg BreedSStefania dALL’OLIO 1, 2, emilio SCOttI 1, Leonardo nAnnI COStA 1, enrico d’ALeSSAndrO 3, Vincenzo rUSSO 1, Luca fOntAneSI 1 1 dipartimento di Scienze e tecnologie Agro-alimentari, Università di Bologna, Bologna, Italy2 Corresponding author: stefania.dallolio@unibo.it3 dipartimento di Scienze Veterinarie, Università di Messina, Messina, ItalyABSTRACTThe PRKAG3 gene encodes for the regulatory gamma 3 subunit of adenosine monophosfate-activated protein kinase (AMPK) protein that acts as a regulator of carbohydrate and fat metabolism. Several single nucleotide polymor-phisms (SnPs) have been identified in the porcine PRKAG3 gene. two of them (c.595A>g or p.I199V and c.599g>A or p.r200Q) have been associated with variability of meat quality and carcass traits. In this study, we investigated the fre-quency of the c.595A>g and c.599g>A SnPs in 372 animals of five Italian autochthonous pig breeds (Apulo-Calabrese, Casertana, Cinta Senese, Mora romagnola and nero Siciliano). genomic dnA was extracted from hair roots or blood and SnPs genotyping was performed by PCr-rfLP protocols. The c.599A (p.200Q) allele was carried by only 3 nero Siciliano pigs. All five breeds were polymorphic at the c.595A>g site. The c.595A (p.I199) allele was less frequent in the analysed breeds with minor allele frequency ranging from 0.144 (nero Siciliano) to 0.464 (Casertana). Based on identi-fied allele frequencies, the c.595A>g SnP can be useful in association studies with meat, carcass and ham quality traits in the Italian local pig breeds.Key words: PRKAG3 gene / pigs / breeds / allele / genotype frequencies1 INTRODUCTIONThe PRKAG3 gene encodes for the regulatory gamma 3 subunit of adenosine monophosfate-activated protein kinase (AMPK) that plays key roles as regula-tor of carbohydrate and fat metabolism in mammalian skeletal muscle cells (Barnes and Zierath, 2005). Sev-eral polymorphisms have been identified in the porcine PRKAG3 gene (Milan et al., 2000; Ciobanu et al., 2001, ryan et al., 2012). Of these mutations, the c.595A>g and c.599g>A single nucleotide polymorphisms (SnPs) (causing the p.I199V and p.r200Q aminoacid substitu-tions, respectively) were the most studied in different pig breeds and populations. The c.595A>g SnP has been associated with variation of different traits includ-ing water holding capacity, ph, colour, carcass traits and composition, and several quality traits of dry-cured ham (Ciobanu et al., 2001, gou et al., 2012; Santé-Lhoutellier et al., 2012; Škrlep et al., 2012). The pigs that carry the c.599A (p.200Q) allele have higher skeletal muscle glyco-gen content and lower rendement napole technological yield (this allele was previously referred to be the RN- al-lele) compared to animals that do not carry this allele. The c.599A allele is responsible of the so-called acid meat defect, characterised by very low ultimate ph, and/or hampshire type meat, due to the common presence of the p.200Q allele in hampshire and hampshire derived lines (Monin and Sellier, 1985; Le roy et al., 1990; Milan et al., 2000; Ciobanu et al., 2001; Lundström et al., 2006; Cherel et al., 2010; Scheffler et al., 2011). The aim of the current study was to investigated the allele and genotype frequencies of the c.595A>g and c.599g>A PRKAG3 SnPs in five Italian local pig breeds.Acta agriculturae Slovenica, Supplement 4 – 201330S. dALL’OLIO et al.2 MATERIALS AND METHODS 2.1 AnIMALS, genOMIC dnA extrACtIOn And genOtyPIngSamples were collected from Apulo-Calabrese (n = 70), Casertana (n = 28), Cinta Senese (n = 75), Mora romagnola (n = 91) and nero Siciliano (n = 108) pigs. Sampled animals were from different herds and registered to the Anagraphic Book of the national Pig Breeders Association (AnAS; http//www.anas.it). ge-nomic dnA was extracted from hair roots or blood and SnP genotyping was performed by PCr-rfLP analyses (Ciobanu et al., 2001; fontanesi et al., 2003). Briefly, the PCr product of 259 bp (Ciobanu et al., 2001) was digested with MbiI (c.599g>A) and BsahI (c.595A>g). PCr-rfLP products were resolved on 2.5% agarose gels visualized with 1xgelred nucleic Acid gel Stain.2.2 StAtIStICAL AnALySISAllele and genotype frequencies of the two analysed polymorphisms were determined. hardy-Weinberg equilibrium was evaluated using the hWe software pro-gram (Linkage Utility Programs, rockefeller University, new york, ny, USA). 3 RESULTS AND DISCUSSIONThe region of origin and demographic data (year 2011) of the analysed five breeds is reported in table 1 (AnAS, 2012). These local breeds belong to the Mediter-ranean type (southern european origin), characterized by a coloured coat and a high tendency for increased in-tramuscular fat, or are intermediate between european and Indochinese pigs (franci and Pugliese, 2007; Megens et al., 2008). Among the investigated breeds, the nero Siciliano population may have been crossbred heav-ily in recent times with commercial western european breeds (Megens et al., 2008; fontanesi et al., 2010). All investigated local breeds are adapted to local climate and marginal conditions and they are less efficient for pro-ductive (growth rate and feed conversion) and reproduc-tive performances (number of piglets born alive and teat numbers) and carcass traits (tendency to fatness) that other improved cosmopolitan breeds. however, the lo-cal breeds have in general better meat quality traits such as more red colour and lower cooking loss than the im-proved ones (franci and Pugliese, 2007). We analysed the two SnPs of the PRKAG3 gene lo-cated in a highly conserved region of the encoded protein which acts as a sensor of cellular energy status (Milan et al., 2000). Pigs from Apulo-Calabrese, Casertana, Cinta Senese and Mora romagnola were homozygous for the wild type c.599g (p.200r) allele. The c.599A (p.200Q) allele was carried, in heterozygous state (c.599Ag geno-type) only by 3 nero Siciliano pigs. This data suggested that hampshire or hampshire-derived lines could have contributed to shaping nero Siciliano genetic pool, as in part supported by other studies on coat colour (fontane-si et al., 2010). due to the low frequency of the c.599A allele in the nero Siciliano (equal to 0.014), this polymor-phic site might be of limited importance in determining the variability of meat and carcass quality traits in this breed. however, this breed should be further monitored to avoid excessive diffusion of this negative allele in the population.All breeds were polymorphic at the c.595A>g (p.I199V) polymorphism (table 2). This SnP does not deviate from hardy-Weinberg equilibrium in the geno-typed populations (P > 0.05). The c.595A (p.I199) allele was less frequent in all analysed breeds, in agreement with earlier studies performed in other commercial breeds and populations reared in Italy (fontanesi et al., 2003; 2008) and in the majority of modern breeds and crosses. In the Italian local breeds the frequency of the minor allele (c.595A), that has been shown to be favour-able for some meat quality traits such as ph and water holding capacity (Ciobanu et al., 2001; fontanesi et al., 2008), ranged from 0.144 (nero Siciliano) to 0.464 (Ca-Local pig breedsItalian region of  originCoat colour of  registered pigsnumber of boarsnumber of sowsnumber of  young candidatesApulo-Calabrese Calabria Black 93 527 3,523Casertana Campania dark gray 33 80 323Cinta Senese tuscany Black with white belt 170 1,035 1,552Mora romagnola romagna Black 67 235 683nero Siciliano Sicily Black 45 523 2,460Table 1: Details of Italian local breeds registered to the Anagraphic Book of the National Pig Breeders Association. The population data were referred to December 31, 2011 (ANAS, 2012)Acta agriculturae Slovenica, Supplement 4 – 2013 31AnALySIS Of PRKAG3 gene POLyMOrPhISMS In ItALIAn AUtOChthOnOUS PIg BreedSsertana). Combining the two investigated polymorphic sites, we identified 3 haplotypes, namely [199V:200r], [199I:200r] and, in nero Siciliano, [199V:200Q].4 CONCLUSIONSThe results indicated the absence of the negative c.599A allele in Italian local pig breeds, except for nero Siciliano. Based on identified allele frequencies, the c.595A>g SnP can be useful in association studies with production, meat, carcass and ham quality traits in the Italian local pig breeds. 5 ACKNOWLEDGMENTS We thank breeders and AnAS that collaborated during the sampling. This work was funded by University of Bologna rfO funds and Italian MiPAAf (Innovagen project).6 REFERENCESAnAS. 2012. relazione del comitato direttivo alla assemblea generale dei soci. roma, 26 giugno 2012. http://www.anas.it/circolari/201301712.Pdf (10. jun. 2013)Barnes B.r., Zierath J.r. 2005. role of AMP-activated protein kinase in the control of glucose homeostasis. 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Pork of low technological quality with Local pig breeds number of pigsc.595A>g (p.I199V) SnP genotype frequencyMAf (allele) c.595AA c.595Ag c.595ggApulo-Calabrese 70 0.350 (c.595A) 0.171 0.357 0.472Casertana 28 0.464 (c.595A) 0.286 0.357 0.357Cinta Senese 75 0.350 (c.595A) 0.083 0.533 0.383Mora romagnola 91 0.154 (c.595A) 0.011 0.286 0.703nero Siciliano 108 0.144 (c.595A) 0.028 0.231 0.741total 372Table 2: Minor allele (MAF) and genotype frequencies of the c.595A>G SNP in five Italian local pig breedsActa agriculturae Slovenica, Supplement 4 – 201332S. dALL’OLIO et al.a normal rate of muscle ph fall in the immediate post-mor-tem period: the case of the hampshire breed. Meat Science, 13: 49–63ryan M.t., hamill r.M., O’halloran A.M., davey g.C., Mc-Bryan J., Mullen A.M., Mcgee C., gispert M., Southwood O.I., Sweeney t. 2012. SnP variation in the promoter of the PrKAg3 gene and association with meat quality traits in pig. 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Analysis of PRKAG3 gene polymorphisms in Italian autochthonous pig breeds.

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Analysis of PRKAG3 gene polymorphisms in Italian autochthonous pig breeds.

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