WO2014131911A1 - Method for the selection of suids with better meat quality based on an snp in the gene that encodes cytosolic pepck - Google Patents

Method for the selection of suids with better meat quality based on an snp in the gene that encodes cytosolic pepck Download PDF

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WO2014131911A1
WO2014131911A1 PCT/EP2014/054097 EP2014054097W WO2014131911A1 WO 2014131911 A1 WO2014131911 A1 WO 2014131911A1 EP 2014054097 W EP2014054097 W EP 2014054097W WO 2014131911 A1 WO2014131911 A1 WO 2014131911A1
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seq
reference value
suid
nucleotide sequence
phenotype
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PCT/EP2014/054097
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French (fr)
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Pascual LÓPEZ BUESA
Carmen BURGOS SERRANO
José Alberto CARRODEAGUAS VILLAR
Luís VARONA AGUADO
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Universidad De Zaragoza
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like

Definitions

  • the present invention relates to the selection of suids with higher intramuscular fat content, together with lower back fat content, and/or with at least 30% more myoglobin in the muscle, and/or with a meat that is at least 19% less exudative, by means of the detection of the A/C polymorphism at position 2456 of the gene that encodes the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. Therefore, the invention belongs to the technical field of genetic selection in animals; in particular, to the fields of livestock production and the food industry.
  • the process for selecting animals with higher or lower intramuscular fat content or for reducing back fat thickness is the classical genetic selection.
  • the positive genetic correlation between both traits makes the joint genetic selection thereof difficult.
  • the use of selection techniques based on genetic markers may help to resolve this problem, provided that markers indicative of this phenotype are found.
  • Burgos et al., 2012 (Meat Science. 90: 309-313) describe the effect of the selection of pigs with higher intramuscular fat content for the production of cured ham on the basis of a pig genotype characterised by the presence of an A/G polymorphism at position 3072 of intron 3 of the gene that encodes the insulin-like growth factor (IGF2).
  • IGF2 insulin-like growth factor
  • the authors of the present invention have identified a polymorphism that modulates intramuscular fat content in suids, increasing it by about 25% (in the loin), and reduces back fat thickness by about 11 %; said polymorphism may be used as a genetic marker to select suids with higher intramuscular fat content, together with lower back fat content, thereby reducing the conversion index and increasing the efficiency of the animal raising process. Moreover, it improves the quality of fresh meat by reducing the exudation thereof (by between 24% and 19%) and increases its iron content (by about 30%). Meat with these characteristics has better quality both for obtaining fresh products (due to the lower percentage of exudation) and for obtaining cured products (due to the higher intramuscular fat content).
  • the inventors sequenced the promoter and encoding regions of the PEPCK gene in Pietrain-breed and Iberian-breed pigs. These two breeds were chosen because they show extreme phenotypes for the traits of interest: the Pietrain breed shows very low intramuscular fat contents together with very small back fat thicknesses; The Iberian breed presents high intramuscular fat contents and very large back fat thicknesses.
  • Pietrain animals show an SNP which involves one amino acid change (from methionine to leucine).
  • Said SNP is located at position 2456 of the gene that encodes PEPCK (SEQ ID NO: 1 ).
  • a statistical test (Chi-square Test) showed that the allele frequency of this mutation was significantly different in both breeds.
  • the sequencing of the PEPCK gene in nine pigs obtained by crossing Duroc breed pigs with Landrace x Large White breed pigs showed that, in said pigs, there was variability for the polymorphism detected in the Pietrain breed.
  • the genotype for said polymorphism was determined in a population of 202 of these animals, and studies were performed on the effect of the SNP on intramuscular fat content and back fat content thickness; these showed that a homozygous AA animal has about 25% more intramuscular fat and, simultaneously, about 1 1 % less back fat than a homozygous CC animal. This study further showed that AA animals provide better-quality meat, characterised by lower percentages of exudation (between 24% and 19% less) and greater iron content (30% more).
  • a first aspect of the invention relates to a method for selecting a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a suid comprising a phenotype whose meat is at least 19% less exudative than a reference value, hereinafter "selection method of the invention", which comprises:
  • suid is understood to mean a member of the family Suidae, a family of artiodactyl mammals that includes domestic pigs, wild boars and their closest relatives, up to 16 species, all of them originally distributed throughout Eurasia and Africa. These are non-ruminant omnivorous ungulates with primitive characteristics. Examples of suids include, without being limited thereto, pigs and wild boars.
  • the suid is a domestic pig or a wild boar.
  • Domestic pig (Sus scrofa domestica) is understood to mean a species of artiodactyl mammal of the family Suidae. It is a domestic animal used for human nutrition in some cultures, especially Western cultures.
  • Wild boar is understood to mean an artiodactyl mammal of the family Suidae, with the scientific name Sus scrofa, that is present in Europe, although there are also subspecies in America, Africa and Asia. It is different from the domestic pig in that it has a more pointed head, a longer snout, always erect ears, very thick, strong, uniform grey-colour hair, and large canine teeth that protrude from the mouth.
  • the method of the invention is aimed at selecting a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or comprising a phenotype whose meat is at least 19% less exudative than a reference value.
  • intramuscular fat is understood to mean the fat pad located between the muscle fasciculi, the fat contained inside the muscle fibres and the fat that is a part of the cell membranes.
  • Back fat is understood to mean the adipose tissue deposit located immediately beneath the epidermis, the thickness whereof is determined, amongst other causes, by genetic causes. It is one of the most important parameters in estimating the carcass yield, and the carcass is understood to be the set of tissues and organs extracted from the sacrificed animal once the viscerae have been removed and which will be processed at the slaughterhouse (muscle, fat and bones).
  • myoglobin or “muscle haemoglobin” is defined to be the muscle haemoprotein, which is structurally and functionally very similar to haemoglobin, relatively small, composed of a polypeptide chain with 153 amino acid residues, containing a haem group with an iron atom, and the function whereof is to store and transport oxygen.
  • the highest concentrations of myoglobin are found in skeletal muscle, especially in the red fibres, whose energy metabolism is more dependent on the oxidation of fats than on glycolysis, and in the cardiac muscle, where large quantities of O2 are required in order to meet the energy demands of contractions.
  • meat is understood to be “exudative” when it loses, especially in the cut surfaces, watery fluid due to changes in the myofibrils, a phenomenon that is favoured by protein denaturation and damage in the cell membranes.
  • excessive exudation leads to weight loss, with the consequent direct economic loss, makes packaged meat look worse, with the consequent rejection by consumers, and is closely related to loss of juiciness and increased meat hardness, which overall results in low-quality meats.
  • reference value is understood to be the mean of the values obtained upon measuring the phenotypic characteristic that is being selected in the suid population.
  • a suid is understood to comprise a phenotype with higher intramuscular fat content and lower back fat content than a reference value when the intramuscular fat content and the back fat content of said suid are, respectively, higher and lower than the mean intramuscular fat content and back fat content of the rest of suids present in the population.
  • a suid is understood to comprise a phenotype with at least 30% more myoglobin in the muscle than a reference value when the muscle myoglobin content of said suid is 30% higher than the mean quantity of myoglobin in the muscle in the rest of suids present in the population.
  • a suid is understood to comprise a phenotype whose meat is at least 19% less exudative than a reference value when the meat of said suid is 19% less exudative than the mean exudation of the meat in the rest of suids that are a part of the population.
  • the selection method of the invention comprises detecting, in a biological sample from said suid, an A/C polymorphism in the gene that encodes the cytosolic PEPCK (PEPCK-C) protein.
  • PPCK-C cytosolic PEPCK
  • biological sample refers to any matter that contains DNA.
  • biological samples wherefrom DNA may be obtained include, without being limited thereto, biological fluids (blood, saliva, urine, sperm, etc.), the epidermis, hair, faeces, tissue samples, etc. Obtainment of the biological sample must be performed under the adequate conditions using adequate material, such as swabs, tweezers, etc., and the samples must be stored independently in duly-labelled separate sterile jars or plastic bags, without any type of preservatives, preventing contamination by foreign biological material.
  • biological fluids blood, saliva, urine, sperm, etc.
  • the epidermis hair, faeces, tissue samples, etc.
  • Obtainment of the biological sample must be performed under the adequate conditions using adequate material, such as swabs, tweezers, etc., and the samples must be stored independently in duly-labelled separate sterile jars or plastic bags, without any type of preservatives, preventing contamination by foreign biological material.
  • nucleic acid which may be DNA or RNA.
  • DNA extraction may be performed using any method known to persons skilled in the art (Sambrock et al., 2001. "Molecular cloning: A Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vols.
  • these methods include, without being limited thereto, density gradient centrifugations, two- phase extraction using aqueous phenol or chloroform with ethanol, column chromatography, methods based on the DNA binding capacity to glass and/or silica surfaces, such as diatomaceous earth or glass bed preparations, using commercial kits, such as, for example, the kits "Q- Biogene fast DNA kit” or “QIAamp(R) DNA Blood Mini Kit” (Qiagen, Hilden, Germany) or “G-Spin lip” (Intron Biotechnology, Korea) or "Fast Prep System Bio 101 " (Qbiogene, Madrid, Spain).
  • RNA nucleic acid that is to be extracted from the sample
  • RNA there are commercial kits exclusively designed for this purpose which contain the adequate components to extract RNA under perfect conditions: high concentrations of chaotropic salts in the lysis buffer, in order to deactivate the RNases; silica membranes that favour RNA adsorption; DNases that eliminate the DNA, in order to obtain a high-purity RNA isolate, etc.
  • a commercial kit that fulfils the aforementioned characteristics is, for example, without being limited thereto, NucleospinTM RNA.
  • the selection method of the invention comprises detecting the A/C polymorphism in the gene that encodes the PEPCK protein in order to select those animals that are homozygous for the A allele.
  • the "phosphoenolpyruvate carboxykinase” (PEPCK) protein is an enzyme of the family of lyases that participates in various metabolic pathways, including glyceroneogenesis, because it provides activated forms of glycerol for triglyceride synthesis. It catalyses the conversion reaction of oxaloacetate into phosphoenolpyruvate and carbon dioxide, and consumes GTP, which is transformed into GDP.
  • PEPCK1 or PEPCK-C cytosolic
  • PEPCK2 or PEPCK-M mitochondrial
  • the sequences of these isoforms have 63% similarity; the precise function of the mitochondrial isoenzyme is unkwown.
  • the cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) protein is encoded by the PEPCK gene, the nucleotide sequence thereof is shown in NCBI database accession number: FJ668384.1 Gl:238863889.
  • the nucleotide sequence of the cytosolic PEPCK comprising the A2456C mutation is shown in SEQ ID NO: 1.
  • the gene that encodes the cytosolic PEPCK protein comprises a nucleotide sequence with at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 .
  • the gene encoding the PEPCK-C comprises a nucleotide sequence that hybridizes under low, medium, medium-high, high, or very high stringency conditions with the nucleotide sequence SEQ ID NO: 1 .
  • hybridize or “specifically hybridize” as used herein refers to a process where two complementary nucleic acid strands anneal to each other under appropriately stringent conditions.
  • low stringency conditions means for nucleotides of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
  • medium stringency conditions means for nucleotides of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
  • medium-high stringency conditions means for nucleotides of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
  • high stringency conditions means for nucleotides of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
  • polymorphism refers to a "single- nucleotide polymorphism", or "SNP”, and is defined as the variation of a single nucleotide between the genomes of individuals from the same species.
  • the A/C polymorphism is located at a position equivalent to nucleotide 2456 of SEQ ID NO: 1 determined by the optimal alignment of the sequences.
  • the A C polymorphism causes a change of leucine to methionine in the PEPCK protein, the mutation being located in amino acid 139 of said protein (SEQ ID NO: 2).
  • the complementary DNA (cDNA) obtained from the PEPCK-C protein comprising the amino acid sequence SEQ ID NO: 2 has the polymorphism located at nucleotide 415 of SEQ ID NO: 3.
  • the detection of the polymorphism of the invention may be performed using any of the techniques described in the state of the art. Examples of these processes include, without being limited to, sequencing, pyrosequencing, allele-specific oligonucleotide (ASO) dot-blot hybridisation analysis, nucleotide primer extension, PCR-based single-strand conformation polymorphism (SSCP) analysis, PCR-based restriction fragment length polymorphism (RFLP) analysis, real-time quantitative PCR (Q-PCR) and mass matrix using a mass spectrometer.
  • ASO allele-specific oligonucleotide
  • SSCP single-strand conformation polymorphism
  • RFLP PCR-based restriction fragment length polymorphism
  • Q-PCR real-time quantitative PCR
  • Allele-specific oligonucleotide (ASO) dot-blot hybridisation analysis is understood to mean the process aimed at detecting polymorphisms in specific genes, which may be performed by hybridising the DNA fragment with allele-specific oligonucleotide probes of a PCR-amplified gene fragment, using forward primers and reverse primers designed to incorporate a target SNP, for dot-spot hybridisation analysis.
  • RNA primer extension is understood to mean the production of sufficient multiple copies thereof to allow for relatively easy manipulation of the segment. Manipulation refers to both physical and chemical manipulation, i.e. the capacity to displace mass quantities of the segment and drive chemical reactions with the segment that generate detectable products.
  • PCR-based single-strand conformation polymorphism (SSCP) analysis is understood to mean the mutation tracking used in molecular diagnosis, based on the electrophoretic migration of DNA.
  • PCR-based restriction fragment length polymorphism (RFLP) analysis, invasive process is understood to mean the analysis of specific nucleotide sequences in DNA that are recognised and cut by restriction enzymes (also called restriction endonucleases), which vary amongst individuals.
  • RFLPs are DNA genetic markers and may be found in regions that encode proteins or exons, in introns or in the DNA that separates one gene from another.
  • Real-time quantitative PCR (qPCR or Q-PCR) or “real-time PCR” (RT- qPCR or RT-Q-PCR) is understood to mean a variant of the polymerase chain reaction (PCR) used to amplify and simultaneously quantify the deoxyribonucleic acid (DNA) amplification product.
  • PCR polymerase chain reaction
  • Mass matrix using a mass spectrometer is understood to mean the result obtained from the experimental technique that makes it possible to measure ions derived from molecules.
  • the mass spectrometer allows for a very precise analysis of the composition of different chemical elements and atomic isotopes, by separating the atomic nuclei as a function of their mass-to- charge (m/z) ratio.
  • detection of the polymorphism is performed by means of a process selected from the group consisting of direct sequencing, allele-specific oligonucleotide (ASO) dot-blot hybridisation analysis, mononucleotide primer extension, PCR-based single-strand conformation polymorphism (SSCP) analysis, PCR-based restriction fragment length polymorphism (RFLP) analysis, real-time quantitative PCR and mass matrix using a mass spectrometer.
  • ASO allele-specific oligonucleotide
  • SSCP single-strand conformation polymorphism
  • RFLP restriction fragment length polymorphism
  • said region comprises the 30 nucleotides adjacent to each side of nucleotide 2456 of SEQ ID NO: 1 .
  • Said primers may be designed by means of any of the standard processes in the state of the art, which are widely known to persons skilled in the art.
  • the primers used in the selection method of the invention comprise a nucleotide sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 .
  • the primers comprise the nucleotide sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
  • the selection method of the invention encompasses using at least one nucleic acid probe that comprises a nucleotide sequence with, at least, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 .
  • identity is explained further below in the present description.
  • the probe comprises the nucleotide sequence SEQ ID NO: 8 or SEQ ID NO: 9.
  • the presence of the A C polymorphism may be determined by means of real-time PCR, by designing two different probes capable of discriminating between the two alleles.
  • Real-time PCR is a method capable of quantifying the DNA in each PCR cycle in the form of fluorescence. This fluorescence is due to the modification undergone by the probes in each amplification cycle. Allelic discrimination takes place when two probes labelled with a different fluorochrome that are specific for each allele are used. At the end of the process, the fluorescence curves for the two probes will indicate the genotype.
  • labelling of the probes is based on the use of quenchers (quencher pigment or non-fluorescent quencher -NFQ- that increases the detection efficacy and the signal because it does not emit fluorescence) and probes labelled with a wide range of fluorophores (reporter pigment) with different excitation and emission spectra.
  • quenchers quencher pigment or non-fluorescent quencher -NFQ- that increases the detection efficacy and the signal because it does not emit fluorescence
  • probes labelled with a wide range of fluorophores (reporter pigment) with different excitation and emission spectra are examples of fluorophores (reporter pigment) with different excitation and emission spectra.
  • fluorescent labels include: FAM, VIC, HEX, TET, CY3, CY5.5, JOE, 6-ROX, cascade Blue, fluorescein, Texas red, rhodamine, rhodamine green, rhodamine red, rhodamine 6G, 6-TAMRA, 5-TMRIA, Alexa 430, Alexa 488, Alexa 594, Bodipy R6G, etc.
  • quenchers are, without being limited thereto, Methyl Red, ElleQuencher, Dabcyl, Dabsyl, TAMRA, etc.
  • probes that carry this type of labelling are, for example, TaqMan probes, molecular beacons (Molecular-Beacon-type probes), Scorpions probes, Amplifluor probes, Eclipse probes, etc.
  • step b) of the selection method of the invention comprises selecting that suid which comprises homozygosis for the A allele.
  • Homozygous or “homozygosis” is understood to mean that the two alleles of a diploid cell or organism at a given site are identical, i.e. that they have the same nucleotide for nucleotide change at the same position in their sequences.
  • Heterozygous is understood to mean the case of a diploid individual with different alleles, i.e. that they have a different nucleotide at the same position in their sequences.
  • a suid which comprises a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a phenotype whose meat is at least 19% less exudative than a reference value. Therefore, the meat obtained from said suid has better quality than that of a suid which does not present said polymorphism.
  • the phenotype of the selected suid comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
  • the phenotype of the selected suid comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value.
  • the phenotype of the selected suid comprises a meat that is at least between 19% and 24% less exudative than a reference value.
  • the authors of the present invention have discovered that the presence of the A C polymorphism in the gene that encodes the cytosolic PEPCK protein in suids is associated with higher intramuscular fat content and lower back fat content (in particular, between 19% and 25% more intramuscular fat and between 9% and 15% less back fat), with 30% more myoglobin in the muscle and/or a meat that is less exudative (in particular, between 19% and 24% less exudative).
  • said polymorphism is located at position 2456 of the PEPCK gene (SEQ ID NO: 1 ).
  • nucleotide sequence of the invention that comprises (i) a nucleotide sequence with at least 70% identity with SEQ ID NO: 1 and (ii) the A/C polymorphism at a position equivalent to nucleotide 2456 of said sequence SEQ ID NO: 1 by optimal alignment of the sequences.
  • isolated means a substance in a form or environment that does not occur in nature.
  • Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, and (2) any substance including, but not limited to, variant, nucleic acid, protein or peptide, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.
  • identity or “sequence identity” is understood to mean the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a different degree of identity, expressed as a percentage, will be obtained.
  • the degree of identity between two amino acid sequences may be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as, for example, BLAST [Altschul S.F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3): 403-10].
  • the BLAST programmes for example, BLASTN, BLASTX, and T BLASTX, BLASTP and TBLASTN, are in the public domain at The National Center for Biotechonology Information (NCBI) website.
  • NCBI National Center for Biotechonology Information
  • the skilled person in the art understands that mutations in the nucleotide sequence of genes that lead to conservative amino acid substitutions at non- critical positions for the functionality of the protein are evolutionarily neutral mutations which do not affect its global structure or its functionality. Said variants fall within the scope of the present invention.
  • variant means a polypeptide having (1 ) PEPCK- C activity and (2) the amino acid sequence SEQ ID NO: 2 comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions, with the proviso that the amino acid equivalent to the amino acid at position 139 of the SEQ ID NO: 2 by the optimal alignment of the sequences is not changed.
  • cytosolic PEPCK protein SEQ ID NO: 2 [encoded by the cytosolic PEPCK gene (SEQ ID NO: 1 )] which comprise insertions, deletions or modifications of one or more amino acids with respect to said sequence SEQ ID NO: 2, and, moreover, conserve the same functions as said protein SEQ ID NO: 2.
  • the nucleotide sequence of the invention comprises a nucleotide sequence with at least 80%, 90%, 95%, 98%, 99% or 100% identity with sequence SEQ ID NO: 1 .
  • the gene encoding the PEPCK-C comprises a nucleotide sequence that hybridizes under low, medium, medium-high, high, or very high stringency conditions with the nucleotide sequence SEQ ID NO: 1.
  • the terms low, medium, medium-high, high, or very high stringency conditions have been previously defined.
  • the invention relates to an isolated polypeptide encoded by the nucleotide sequence of the invention, hereinafter, "polypeptide of the invention".
  • polypeptide of the invention comprises an amino acid sequence with at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with SEQ ID NO: 2.
  • the polypeptide of the invention comprises a methionine at a position equivalent to amino acid 139 of the SEQ ID NO: 2 by optimal alignment of the sequences
  • the implementation of the present invention requires detection of the A/C polymorphism in the gene that encodes the cytosolic PEPCK protein.
  • said detection may be performed by means of numerous techniques that are widely known to the skilled person in the art, some of which entail using oligonucleotides that act as primers in order to amplify the gene region where the polymorphism is located.
  • the inventors of the present invention have designed some oligonucleotides that act as useful primers to amplify the region of the gene that encodes cytosolic PEPCK wherein the A C polymorphism is located.
  • oligonucleotide of the invention that comprises the nucleotide sequence selected from the group consisting of the sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
  • oligonucleotide refers to a nucleotide sequence comprising between 10 and 300 nucleotides, preferably between 10 and 200 nucleotides, more preferably between 10 and 100 nucleotides, even more preferably between 10 and 50 nucleotides, most preferably between 10 and 30 nucleotides in lenght.
  • primers of the invention are specifically designed to amplify the region of SEQ ID NO: 1 that comprises the A/C polymorphism at position 2456 of said sequence, i.e. they are capable of amplifying sequence SEQ ID NO: 1 .
  • said region comprises the 50, preferably 30, nucleotides adjacent to each side of nucleotide 2456 of SEQ ID NO: 1 .
  • primer is understood to mean that oligonucleotide which is capable of contiguously binding to a target sequence and serving as the starting-point for DNA synthesis when it is placed under conditions that initiate the synthesis of a primer extension product which is complementary to a nucleic acid chain.
  • the primers comprise a nucleotide sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 .
  • the primers of the invention comprise a nucleotide sequence which hybridizes under medium, high, or very high stringency conditions with the nucleotide sequence SEQ ID NO: 1 .
  • Hybridizations are typically and preferably conducted with prime-length nucleic acid molecules, preferably 10-100 nucleotides in length, more preferably 10-50 nucleotides in length, most preferably 10-30 nucleotides in length.
  • Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al . , 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N. Y. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementary will stably hybridize, while those having lower complementary will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al.
  • At least one of the primers in the pair comprises nucleotide sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7; preferably, the pair of primers is made up of sequences SEQ ID NO: 4 and SEQ ID NO: 5, or sequences SEQ ID NO: 6 and SEQ ID NO: 7.
  • Another aspect of the invention relates to a nucleic acid probe, hereinafter "probe of the invention", that comprises a nucleotide sequence capable of hybridising with SEQ ID NO: 1 in the region that comprises the A C polymorphism at position 2456, i.e. which is complementary to sequence SEQ ID NO: 1 .
  • said region comprises the 50, preferably 30, nucleotides adjacent to each side of nucleotide 2456 of SEQ ID NO: 1 .
  • the probe comprises a nucleotide sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with sequence SEQ ID NO: 1 , which, in another, even more particular embodiment, comprises nucleotide sequence SEQ ID NO: 8 or SEQ ID NO: 9.
  • the probe of the invention comprises a nucleotide sequence which hybridizes under medium, high, or very high stringency conditions with the nucleotide sequence SEQ ID NO: 1 .
  • the terms high, or very high stringency conditions have been previously with regards to the primers of the invention.
  • Probe is understood to mean a small-size DNA fragment, usually between 15 and 100 bases, preferably between 15 and 50 bases, used as a tool to detect the presence of DNA with an identical or similar complementary sequence.
  • the probe binds to the single-stranded target sequence by means of a hybridisation mechanism that forms a double-stranded structure; one of the strands belongs to the probe and the other belongs to the target sequence.
  • the probe may be labelled at the ends such that it may be detected. Examples of labels and types of probes that may be used within the context of the present invention have been mentioned above.
  • the oligonucleotides, the primers and the probes described in the present invention may be used to detect the A C polymorphism in the gene that encodes the cytosolic PEPCK protein.
  • another aspect of the invention relates to the use of an oligonucleotide of the invention, the probe of the invention and/or the pair of primers of the invention to detect the A/C polymorphism in the gene that encodes cytosolic PEPCK.
  • the gene that encodes the cytosolic PEPCK protein comprises a nucleotide sequence with at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with sequence SEQ ID NO: 1.
  • the A/C polymorphism is located at a position equivalent to nucleotide 2456 of sequence SEQ ID NO: 1 determined by the optimal alignment of the sequences.
  • oligonucleotide, the primers and the probe of the invention may be used to detect the A C polymorphism at position 2456 of the gene that encodes cytosolic PEPCK in the genome of an individual, in particular, a suid.
  • examples of the techniques that may be used to detect said polymorphism have been previously described in the method of the invention, and their use is routine practice for persons skilled in the art.
  • One example of these techniques is real-time or quantitative polymerase chain reaction (RT-PCR).
  • the detection of said polymorphism makes it possible to select a suid whose phenotype comprises a meat with higher intramuscular fat content and less back fat content, and/or with at least 30% more myoglobin in the muscle and/or that is at least 19% less exudative than a reference value.
  • another aspect of the invention relates to the use of the nucleotide sequence or the polypeptide of the invention, the oligonucleotide, the probe and/or the pair of primers of the invention to select a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a suid comprising a phenotype whose meat is at least 19% less exudative (in particular, between 19% and 24%) than a reference value.
  • the phenotype of the suid comprises, at least, between 19% and 25% more muscular fat and, at least, between 9% and 15% less back fat than a reference value. In another, even more particular embodiment, the phenotype of the suid comprises, at least, 20% more intramuscular fat and, at least, 11 % less back fat than a reference value.
  • the term "reference value" has been previously defined in the method of the invention and is applicable to the present inventive aspect.
  • genetic material is present in all the cells of an individual, except for the mature red blood cells of mammals, and, therefore, it will also be present in the meat from said individual, in particular, a suid. Therefore, the present invention makes it possible not only to select suids with the phenotypic characteristics previously mentioned in the present description, but also to select meat products from said suids which, as a consequence, also contain the A/C polymorphism at position 2456 of the gene that encodes PEPCK in their genetic material.
  • a meat product comprising higher intramuscular fat content and lower back fat content, at least 30% more myoglobin in the muscle, and/or at least 19% less exudation than other meat products.
  • a meat product is selected with higher quality than other meat products that do not present said characteristics.
  • another aspect of the present invention relates to the use of the nucleotide sequence or the polypeptide of the invention, the oligonucleotide, the probe and/or the pair of primers of the invention to select a meat product comprising higher intramuscular fat content and lower back fat content than a reference value, 30% more myoglobin than a reference value and/or at least 19% less exudation than a reference value.
  • Meat product or “meat” is understood to mean a foodstuff of animal origin, primarily made up of muscle tissue, which is consumed for nutrition. From the nutritional standpoint, meat is a habitual source of proteins, fats and minerals in the human diet. Most meat consumption by human beings comes from mammals, especially from ungulate animals, which are domesticated to provide food.
  • reference value is understood to be the mean of the values obtained from measuring the phenotypic characteristic that is being selected in the population/set of meat products.
  • a meat product is understood to present higher intramuscular fat content and lower back fat content than a reference value when the intramuscular fat content and the back fat content of said meat product are, respectively, higher and lower than the mean intramuscular fat content and back fat content of the rest of meat products which are a part of the population.
  • a meat product is understood to present at least 30% more myoglobin in the muscle than a reference value when the myoglobin content in the muscle of said meat product is 30% higher than the mean quantity of myoglobin in the muscle in the rest of meat products which are a part of the population.
  • a meat product is understood to present at least 19% less exudation than a reference value when said meat product comprises 19% less exudation than the mean exudation of the rest of meat products which are a part of the population.
  • exudation has been previously defined.
  • the meat product comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
  • the meat product comprises, at least, 20% more intramuscular fat and, at least, 11 % less back fat than a reference value.
  • the meat product comprises, at least, between 19% and 24% less exudation than a reference value.
  • methods comprising the use of the isolated nucleotide sequence, the isolated polypeptide, the nucleic acid probes the oligonucleotide and the primers disclosed in the present invention are also comprised within the scope of the present invention.
  • further aspects of the present invention relate to methods for detecting the A C polymorphism in the gene encoding the PEPCK-C protein and methods for selecting a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value and/or a suid comprising a phenotype whose meat is at least 19% less exudative than a reference value.
  • kit of the invention that comprises the oligonucleotide, the probe and/or the pair of primers described in the preceding inventive aspects, together with the corresponding particular embodiments.
  • kits of the invention to detect the A C polymorphism in the gene that encodes the cytosolic PEPCK protein.
  • the gene that encodes the cytosolic PEPCK protein comprises a nucleotide sequence with at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 .
  • the A C polymorphism is located at a position equivalent to nucleotide 2456 of sequence SEQ ID NO: 1 determined by the optimal alignment of the sequences.
  • kits of the invention to select a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a suid comprising a phenotype whose meat comprises at least 19% less exudation than a reference value.
  • the phenotype of the suid comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
  • the phenotype of the suid comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value.
  • the phenotype of the suid comprises a meat with, at least, between 19% and 24% less exudation than a reference value.
  • Another aspect of the invention relates to the use of the kit to select a meat product comprising higher intramuscular fat content and lower back fat content than a reference value, at least 30% more myoglobin than a reference value and/or at least 19% less exudation than a reference value.
  • the meaning of the term "reference value" has been previously defined.
  • the meat product comprises, at least, between 19% and 25% more muscular fat and, at least, between 9% and 15% less back fat than a reference value. In another, even more particular embodiment, the meat product comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value. In a particular embodiment, the meat product comprises, at least, between 19% and 24% less exudation than a reference value.
  • Figure 1 shows the structure of the cytosolic PEPCK gene. Position of the primers used in the sequencing of its encoding region.
  • FIG. 2 shows the sequences and alignments of the 27 animals studied.
  • the A C polymorphism is marked with an arrow and shading.
  • Figure 3 shows the PCR amplification of an 896-base fragment that contains the A2456C mutation.
  • the crosses represent failed amplifications from genomic DNA; the triangles, diamonds and squares represent amplifications from previously PCR-amplified fragments, as shown in Figure 3: the triangles are amplifications of homozygous CC animals, the squares are amplifications of heterozygous animals and the diamonds are samples of homozygous AA animals.
  • the circles belong to control amplifications (adding water instead of DNA).
  • Figure 4 shows a PCR amplification of the gene region of porcine cytosolic PEPCK that contains nucleotide 2456. Lanes 1 and 5 contain molecular weight markers; lanes 2-4 and 6-8 contain samples from 6 different animals.
  • Figure 5 is a SDS-PAGE showing the purified His-tagged PEPCK-C isoenzymes. MW, molecular weight markers.
  • Figures 6A, 6B and 6C are Lineweaver-Burks plots for kinetic calculations.
  • Figure 6A PEP as variable substrate
  • Figure 6B GDP as variable substrate
  • PEPCK-C-p.139Leu squares ( ⁇ )
  • PEPCK-C-p.139Met diamons ( ⁇ ).
  • Figures 7A and 7B are Lineweaver-Burks plots for kinetic calculations.
  • Figure 7A OAA as variable substrate
  • Figure 7B GTP as variable substrate.
  • PEPCK-C-p.139Leu squares ( ⁇ ); PEPCK-C-p,139Met, diamons ( ⁇ ).
  • Figure 8 is a SDS-PAGE analysis of trypsine digestion of both PEPCK-C isoenzymes (PEPCK-C-p.139Leu and PEPCK-C-p,139Met) after different time points.
  • Figure 9 is a picture of a three dimensional structure of PEPCK-C showing the substrate PEP in the center of the figure and the substituted amino acid methionine 139 in the right side of the figure.
  • Figure 10 shows the aligment of PEPCK-C protein regions from man to C. elegans to show the conserved methionine (bold) corresponding to position 139 of the pig protein.
  • Asterisks indicate perfectly conserved amino acids among the 7 species.
  • GenBank accession numbers are the following: pig, NP_001 1 16630; human, NP_002582; mouse, NP_035174; bovine, NP_777162; chicken, NP_990802; Drosophila, NP_001097367; C. elegans, NP_001021589.
  • the genomic DNA was extracted from different sources: muscle in the case of Iberian pigs, semen in the case of Pietrain pigs and a portion of the end of the tail in the case of animals from the Duroc x Landrace-Large White cross.
  • the analysis of the sequence of bases in the encoding region was performed by sequencing 5 PCR-amplified fragments of the PEPCK gene: PEPCK1 F/2R, PEPCK2F/3R, PEPCK3F/4R, PEPCK4F/5R and PEPCK5F/8R.
  • the sequences of these primers are shown in Table 1 .
  • Each PCR reaction included 1 unit of AccuPrimeTM Taq DNA Polymerase High Fidelity, 5 ⁇ of 10x buffer II, 0.5 ⁇ of the corresponding forward primer (20 pmol/ ⁇ ), 0.5 ⁇ of the corresponding reverse primer (20 pmol/ ⁇ ), 3.8 ⁇ of DMSO (Sigma-Aldrich, Saint Louis, United States), 2 ⁇ of genomic DNA (100 ng/ ⁇ ) and the necessary quantity of Milli-Q water to adjust the total reaction volume to 50 ⁇ .
  • the temperature programme used for these amplifications includes a denaturation step at 94°C for 2 minutes, followed by 35 denaturation cycles at 94°C for 30 seconds, hybridisation for 30 seconds at 55°C in the case of the PEPCK1 F/2R, PEPCK2F/3R and PEPCK3F/4R fragments, 53°C in the case of the PEPCK4F/R5 fragment and 55°C in the case of the PEPCK5F/8R fragment, and extension at 68°C for 1 minute 30 seconds, 1 minute 15 seconds, 1 minute 15 seconds, 1 minute and 1 minute for the PEPCK1 F/2R, PEPCK2F/3R, PEPCK3F/4R, PEPCK4F/5R and PEPCK5F/8R fragments, respectively, and a final extension step at 68°C for 10 minutes.
  • the purified product was eluted with 25 ⁇ of the elution buffer in the kit and the DNA concentration in this solution was measured using a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific, Wilmington, United States).
  • Each of the 5 purified PCR fragments were sent to zucchinias Genomicos (Paterna, Valencia, Spain) to be sequenced with both the forward primer used for their amplification and with the reverse primer.
  • the cDNA of PEPCK of each of these samples was constructed by binding the sequences of all the exons in the appropriate order.
  • the cDNA sequence of pig cytosolic PEPCK (SEQ ID NO: 3) was used as a reference.
  • the A2456C mutation in the porcine cytosolic PEPCK gene [NCBI, accession number: FJ668384 (version FJ668384.1 Gl:238863889)] potentially causes phenotypic effects in porcine meat and the porcine carcass.
  • the nucleotide sequence of the cytosolic PEPCK comprising the A2456C mutation is shown in SEQ ID NO: 1 .
  • the group of animals characterised was made up of a total of 202 pigs of both sexes (the males were castrated) from a Duroc x Landrace-Large White cross. These animals were raised in an experimental farm, fed "ad libitum” with a standard diet and sacrificed at 1 14.5 +/- 10.98 kg.
  • the genomic DNA of each of these animals was extracted from a sample from the terminal end of the tail, taken a few days after birth, using the Real Pure kit for genomic DNA extraction (Durviz, Paterna, Valencia), according to the manufacturer's instructions.
  • the genotype of 2456 A>C cytosolic PEPCK was determined by sequencing a fragment which was PCR-amplified with the PEPCK2F/3R pair of primers and subsequently purified as indicated in Example 1 , and/or by means of real-time PCR.
  • PEPCK2F 5 ' GTGTTGTCCAAGGGGGATCAGGAGGA-3 ' (SEQ ID NO: 4) and PEPCK3R: 5 ' -CCCCCTGCCTCGCACATTTAGGAT-3 ' (SEQ ID NO: 5)].
  • Each PCR reaction included 1 unit of AccuPrimeTM Taq DNA Polymerase High Fidelity, 5 ⁇ of the 10x buffer II, 0.5 ⁇ of a 20 pmol/ ⁇ solution of PEPCKF2, 0.5 ⁇ of a 20 pmol/ ⁇ solution of PEPCKR3, 3.8 ⁇ of DMSO (Sigma-Aldrich, Saint Louis, United States), 2 ⁇ of genomic DNA (100 ng/ ⁇ ) and the necessary quantity of Milli-Q water to adjust the total reaction volume to 50 ⁇ .
  • the temperature programme used includes a denaturation step at 94°C for 2 minutes, followed by 35 denaturation cycles at 94°C for 30 seconds, hybridisation for 30 seconds at 55°C, extension at 68°C for 1 minute 15 seconds, and a final extension step at 68°C for 10 minutes.
  • the PCR product was purified using the NucleoSpin R Extract II kit (Macherey-Nagel, Duren, Germany), following the manufacturer's instructions.
  • the purified product was eluted with 25 ⁇ of the elution buffer in the kit and sent toquelas Genomicos (Paterna, Valencia, Spain) to be sequenced with the PEPCKF2 primer; subsequently, the nucleotide, or nucleotides, present at the position where the mutation is located were identified in the chromatogram obtained.
  • the probes and the primers used in this assay were designed by means of the Assay by Design method from Applied Biosystems.
  • the reactions were performed in an ABI-PRISM 7000 thermocycler (Amersham Biosciences, United States) and each assay included 12.5 ⁇ of Taqman Universal Master Mix, 0.6 ⁇ of 40x Assay Mix, 5 ⁇ of a 2 ng/ ⁇ solution of genomic DNA or a 10 ng/ ⁇ solution of the fragment that was PCR-amplified with the PEPCKF2/3R pair of primers and subsequently purified, and 6.875 ⁇ of Milli- Q water.
  • the primers used were the following: SSPEPCKE4-F (5' CGCTGCCCCCTGCT 3' (SEQ ID NO: 6)) and SSPEPCKE4-R (5' CCCATGCTGAACGGGATGA 3' (SEQ ID NO: 7)), and the probes were: SSPEPCKE4-V (5' AGGTCGCACGCTGTAT 3' (SEQ ID NO: 8)), labelled with the fluorochrome VIC, and SSPEPCKE4-M (5' AGGTCGCACGCTGTAT 3' (SEQ ID NO: 9)), labelled with the fluorochrome FAM (the specific nucleotide to detect each allele of the polymorphism is highlighted in bold italics).
  • the temperature programme used includes an initial denaturation step at 95°C for 10 minutes, followed by 50 cycles at 92°C for 15 seconds and 1 minute at 61°C.
  • the animals were sacrificed at a local slaughterhouse close to their breeding area, after being made unconscious with CO 2 .
  • the carcass was allowed to cool for 6-8 hours.
  • the back fat thickness was measured with a caliper at the level of the fifth lumbar vertebra.
  • the right half of the carcass was divided into 10 parts: ham, loin, sirloin, shoulder, blade, streaky bacon, lard, ribs, cheek and trimmings, which were individually weighed.
  • the hams were dissected into three other parts: bone, subcutaneous adipose tissue with skin and muscle mass (including the intermuscular fat deposits), which were also individually weighed.
  • the lipids in a 10-gram sample of the preceding homogenates were extracted using the Bligh and Dyer method (1959) modified by Hanson and Olley (1963). A 5-ml aliquot was taken from the chloroform phase and dried under a nitrogen stream. The fat content was calculated in a gravimetric manner.
  • the exudation was determined by calculating the weight loss undergone by a loin fillet, free from the subcutaneous fat layer and weighing about 70 g, after 2, 4 and 7 days. In order to take this measurement and the following one, 8 loin fillets were cut from the region located between the second lumbar vertebra and the twelfth thoracic vertebra. The four closest to L2 were used for this measurement and the next four were used to determine the cook losses. 1.3.3.4 Cook losses: The exudation was determined by calculating the weight loss undergone by a loin fillet, free from the subcutaneous fat layer and weighing about 70 g, which was cut after being subjected to a thermal treatment at 75°C for 20 minutes. 2. Results and discussion
  • a Bayesian analysis was performed to study the association between the different genetic configurations of A2465C and the phenotypic variability in the traits of interest.
  • the analysis model was:
  • y k is the phenotypic record of the trait of interest
  • ⁇ is the general mean
  • S is the sex effect (2 levels)
  • Lj is the batch effect (four levels)
  • G k is the number of A alleles in individual k (0, 1 or 2)
  • a k is the age of individual k at the time of sacrifice
  • U k is the polygenic genetic value for individual k
  • e k is the residue
  • b g and b a are the co-variables with the number of A alleles and the age, respectively.
  • Uniform a priori distributions were assumed for the systematic effects, and variance components and a multivariate normal distribution were assumed for the polygenic effects:
  • A is the numerator relationship matrix (Wright, 1922) and * 7 " 3 is the polygenic additive variance.
  • A is the numerator relationship matrix (Wright, 1922) and * 7 " 3 is the polygenic additive variance.
  • A is the numerator relationship matrix (Wright, 1922) and * 7 " 3 is the polygenic additive variance.
  • a genealogy of 243 individuals was used. The analysis was performed by means of a Gibbs sampler (Gelfand and Smith, 1990, J. Amer. Statist. Assoc. 85, 398-409), with a single chain of 125,000 interactions after discarding the first 25,000.
  • the traits in Table 2 are of three types: 1 ) related to the conformation, including the carcass yields (back fat thickness, % of noble parts, % of various pieces, conformation of the leg, loin area); 2) related to the quality of the meat (pH 45 , pH 24 , exudation, cook losses); and 3) related to the fat content (intramuscular fat at the L-dorsi (loin), Ps. Major (sirloin), B. femoris; fat content in the subcutaneous adipose tissue; total fat content in the leg once the subcutaneous adipose tissue has been removed).
  • the especially relevant traits are not only those that have posterior Bayesian probability values above zero, close to 0 or to 1 , but also those with a direct relationship between them.
  • Table 3 it may be observed that the three exudation traits have a posterior Bayesian probability very close to 0, which indicates that the effect found (which ranges between 1 .015 and 1.54 percentage points more exudation at 2 and at 7 days, respectively) is a real effect and not an artifact. Since the mean percentage of exudation in the population studied is 4.30% and 8.12% at 2 and at 7 days, respectively, a homozygous AA animal presents a meat that is between 19% and 24% less exudative than that of a homozygous CC animal.
  • the back fat thickness value also has a significant posterior Bayesian probability above zero (0.0224).
  • the effect attributable to each A allele on this trait would be 1 .078 mm, which means that a homozygous AA individual has 2.16 mm less back fat thickness than a homozygous CC individual. Since the mean value for this trait in the population is 19.90 mm, a homozygous AA individual has a back fat thickness that is 10.9% smaller than that of a homozygous CC individual.
  • a allele of PEPCK is simultaneously associated with lower back fat values and higher intramuscular fat content values. Moreover, it is associated with meats that have a lower percentage of exudation and higher pH 24 values. Therefore, the selection of homozygous AA individuals makes it possible to improve the quality of the meat, since it undergoes less exudation and has more intramuscular fat, and improves the carcass conformation, since it comprises lower back fat thicknesses.
  • a very important characteristic of meat and meat products is the colour.
  • the colour is the first meat characteristic perceived by consumers and, for this reason, it is a key to their decision to purchase it.
  • One of the most important mechanisms that modulate the colour of meat is the content of its main pigment, myoglobin.
  • Myoglobin is an intracellular pigment that contains a haem group with an iron atom.
  • A2456C in PEPCK was decided to analyse both traits in a population of 60 animals that were very homogeneous in terms of weight (90.7 +/- 4.9 kg).
  • the meat from AA animals has 30% more myoglobin and haem iron than that from CC animals. Moreover, the instrumental colour analysis reveals that the meat from AA animals is redder. Both characteristics are commonly perceived as favourable by consumers, which makes the selection of AA pigs very interesting
  • the percentage of improvement in a given trait in a population by the selection of carrier animals for the alleles that modulate said trait in a given sense is dependent, in addition to on the magnitude of the effect associated with a given allele, on the initial proportion of animals in said population that are already carriers of the favourable alleles. Therefore, the potential percentage of improvement of a population will be greater the lower the frequency of said allele prior to starting the improvement process of selecting carrier animals for a given allele.
  • a number of animals from different breeds and crosses were genotyped using the method described in Example 2.
  • the genomic DNA was extracted from muscle, hair, tail or semen using the Real Pure kit for genomic DNA extraction (Durviz, Paterna, Valencia), according to the manufacturer's instructions. 1.3 Determination of the PEPCK genotype by means of real-time PCR
  • the genotype of cytosolic PEPCK at position 2456 was also determined by means of real-time PCR.
  • the probes and the primers used in this assay were designed using the Assay by Design method from Applied Biosystems.
  • the reactions were performed in an ABI-PRISM 7000 thermocycler (Amersham Biosciences, United States) and each assay included 12.5 ⁇ of Taqman Universal Master Mix, 0.6 ⁇ of 40x Assay Mix, 5 ⁇ of a 10 ng/ ⁇ solution of the fragment that was PCR-amplified with the PEPCKF2/3R pair of primers and subsequently purified, and 6.875 ⁇ of Milli-Q water.
  • the primers used were the following: SSPEPCKE4-F (5' CGCTGCCCCCTGCT 3' (SEQ ID NO: 6)) and SSPEPCKE4-R (5' CCCATGCTGAACGGGATGA 3' (SEQ ID NO: 7)), and the probes were: SSPEPCKE4-V (5' AGGTCGCACG4TGTAT 3' (SEQ ID NO: 8)), labelled with fluorochrome VIC, and SSPEPCKE4-M (5' AGGTCGCACGCTGTAT 3' (SEQ ID NO: 9)), labelled with fluorochrome FAM (the specific nucleotide to detect each allele of the polymorphism is highlighted with bold italics).
  • the temperature programme used includes an initial denaturation step at 95°C for 10 minutes, followed by 50 cycles at 92°C for 15 seconds and 1 minute at 61°C.
  • the polymorphism A2456C in pig's cvtosolic PEPCK changes the kinetic properties thereof and modifies the fat distribution in the pig
  • Backfat thickness was measured at L5 (lumbar vertebra 5) level with a caliper shortly after slaughtering. pH 4 5 (pH 45 minutes postmortem) and pH 24 (pH 24 hours postmortem) were measured in L.dorsi at L2 level with a PC3000 Oakton pHmeter equipped with a Hamilton penetration electrode (Bonaduz, Switzerland). Drip loss was measured in quadruplicate in L.dorsi samples taken between L2 and T14 (thoracic vertebra 14) as described by Honikel (1998). Fat content was analyzed from 10 g samples taken form L.dorsi at T12 level. Lipids were extracted by the method of Bligh & Dyer (1959) as modified by Hanson & Olley (1963). Fat content was calculated gravimetrically from an aliquot of 5 ml of the chlorophormic phase.
  • the PEPCK-C coding region was amplified by PCR using the following primers:
  • PEPCK1 F/2R (SEQ ID NO: 10/SEQ ID NO: 1 1 ): PEPCK2F/3R (SEQ ID NO: 4/SEQ ID NO: 5), PEPCK3F/4R (SEQ ID NO: 12/SEQ ID NO: 13), PEPCK4F/5R SEQ ID NO: 14/SEQ ID NO: 15) and PEPCK5F/8R SEQ ID NO: 16/SEQ ID NO: 17).
  • Temperature steps consisted of an initial denaturation at 94°C for 2 minutes followed by 35 cycles: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds (primers PEPCK 1 F/2R, PEPCK2F/3R, PEPCK3F/4R, PEPCK5F/8R) or 53°C for 30 seconds (primer PEPCK4F/R5) and extension at 68°C for 90 seconds (PEPCK1 F/2R), for 75 seconds (PEPCK2F/3R and PEPCK3F/4R) and for 60 seconds (PEPCK 4F/5R and PEPCK5F/8R). A final elongation step was carried out at 68°C for 10 minutes.
  • Fragments were analyzed by 2 % agarose electrophoresis at 100 V for 30 minutes.
  • the PCR products were purified using NucleoSpin ® Extract II kit (Macherey-Nagel, Duren, Germany) following manufacturer's instructions. DNA concentration was determined using a NanoDrop® 1000 and samples were sequenced (Sistemas Genomicos, Spain). Fragment assembly was carried out using Vector NTI for Mac (Life Technologies Corporation, USA). Sequence alignment was performed using ClustalW. 3.3 PEPCK-C cloning
  • PEPCK-C cDNA was amplified by PCR using PEPCK1 F (SEQ ID NO: 10) and PEPCK1 R [5 ' - AACGTGGGCTGTGCTCATTGCGGTG -3 ' (SEQ ID NO: 20)] as primers.
  • Each reaction consisted of 1 unit of AccuPrimeTM Taq DNA Polymerase High Fidelity, 2.5 ⁇ buffer II 10x, 0.25 ⁇ forward primer (20 pmol/ ⁇ ), 0.25 ⁇ reverse primer (20 pmol/ ⁇ ), 6.25 ⁇ DMSO, 2 ⁇ cDNA (100 ng/ ⁇ ) and milliQ water up to 25 ⁇ .
  • PCR was carried out by an initial denaturation step at 94°C for 2 minutes followed by 35 cycles: denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds and extension at 68°C for 130 seconds. Final extension was performed at 68°C for 10 minutes.
  • 2% agarose electrophoresis was carried out at 100 V for 30 minutes to analyze the amplified PEPCK-C fragment. Afterwards, nested PCR was performed using the following forward and reverse primers, respectively:
  • Reaction mix was: 1 unit of AccuPrimeTM Taq DNA Polymerase High Fidelity, 2.5 ⁇ buffer II 10x, 0.25 ⁇ forward primer (20 pmol/ ⁇ ), 0.25 ⁇ reverse primer (20 pmol/ ⁇ ), 1 .25 ⁇ DMSO, 2 ⁇ amplified cDNA (100 ng/ ⁇ ) and milliQ water up to 25 ⁇ .
  • Nested PCR steps were carried out by an initial denaturation at 94°C for 2 minutes and 35 cycles: denaturation at 94°C for 30 seconds, annealing at 63°C for 30 seconds and extension at 68°C for 130 seconds. Final extension was performed at 68°C for 10 minutes. Fragments were also electrophoretically analyzed.
  • PEPCK-C fragments were inserted into a pET28a(+) plasmid by digestion with Ndel and Notl (New England Biolabs) and ligation with T4 DNA ligase (New England Biolabs).
  • E.coli DH5a was transformed by heat shock, colonies were selected by growth in LB-agar with 30 ⁇ g/ml kanamycin, DNA was isolated using NucleoSpin Plasmid Quickpure (Macherey-Nagel, Duren, Germany) and sequenced. 1.4 Genotyping
  • RT-PCR Real Time-PCR
  • DNA fragment previously amplified by standard PCR.
  • Direct use of RT-PCR on genomic DNA failed to detect the A2456C substitution (data not shown).
  • the experimental approach was designed using Assay by Design (Applied Biosystems, USA). Reactions were carried out using an ABI-PRISM 7000 thermocycler (Amersham Biosciences, USA). RT-PCR steps consisted on an initial denaturation at 95°C for 10 minutes followed by 50 cycles at 92°C for 15 seconds and 61 °C for 60 seconds.
  • Each assay included 12.5 ⁇ Taqman Universal Master Mix (Applied Biosystems, USA), 0.6 ⁇ 40x Assay Mix, 5 ⁇ DNA (previously amplified by PCR using primers PEPCKF2/3R and purified using NucleoSpin ® Extract II kit). Blanks included all the components but DNA.
  • SSPEPCKE4-F SEQ ID NO: 6
  • SSPEPCKE4-R SEQ ID NO: 7
  • Taqman probes were SSPEPCKE4-V (SEQ ID NO: 8), containing the VIC fluorochrome, and SSPEPCKE4-M (SEQ ID NO: 9), containing the FAM fluorochrome.
  • E. coli BL21 (DE3) were transformed with the appropriate vectors and two colonies for each of the constructs were selected for protein expression.
  • Expression of proteins of the expected size was first assessed in a small scale protein expression experiment, growing bacteria in LB medium supplemented with 30 ⁇ 9/ ⁇ kanamycin at 37°C to an OD 6 oo of 0.5 and then inducing protein expression with 1 mM IPTG at 37°C for 3 hours. Analyses of clarified lysates after sonication in the presence of lysozyme by SDS-PAGE indicated that part of the desired protein was present in the pellet, suggesting aggregation into inclusion bodies. Therefore, lower induction temperatures were used for large scale preparations.
  • Bacteria were harversted by centrifugation at 8000 g for 10 minutes, resuspended in 12 ml of lysis buffer (20 mM sodium phosphate, pH 7.2, 10 mM DTT, 1 mg lysozyme) and sonicated with a Vibra-Cell sonicator (Sonics & Materials, Newtown, CT, USA) using 10 cycles (10 seconds on, 45 seconds off) at 50% power, on ice. The lysate was clarified by centrifugation at 18,000 g for 20 minutes at 4°C and the pellet was discarded.
  • lysis buffer 20 mM sodium phosphate, pH 7.2, 10 mM DTT, 1 mg lysozyme
  • Vibra-Cell sonicator Sonics & Materials, Newtown, CT, USA
  • Protein purification was carried out by affinity chromatography on a 5 ml FF crude HisTrap Column (GE Healthcare Life Sciences, Piscataway, NJ, USA), using an UPC-900 (GE Healthcare Life Sciences, Piscataway, NJ, USA) HPLC apparatus with UNICORN Manager software.
  • the column was first equilibrated with buffer A (50 mM NaH 2 PO 4 , pH 7.2, 300 mM NaCI, 10 mM imidazole, 10 mM DTT). After sample loading the column was washed with buffer A until absorbance at 280 nm reached the baseline.
  • the protein was then eluted with a 60 ml linear gradient at 5 ml/min from 100% buffer A to 100 % buffer B (same as buffer A but with 500 mM instead of 10 mM imidazole) and 5 ml fractions were collected.
  • Fractions were analyzed by SDS-PAGE and those containing PEPCK-C were pooled and concentrated, using Amicon® Ultra Centrifugal Filters 30kDa (Millipore, Tullagreen, Ireland). After reducing the sample volume by centrifugation at 4000 g from 10 ml to 1 ml, 9 ml of 20 mM HEPES, pH 7.2, 10 mM DTT was added and centrifuged again. This step was repeated 6 times for buffer exchange and imidazole elimination.
  • PEPCK activity was measured with coupled spectrophotometric assays in both reaction directions, as described by Johnson and Holyoak, (2010), using an UNICAM 500 spectralphotometer (Cambridge, UK). Assays in the direction of oxaloacetic acid synthesis were performed in 1 ml of a 100 mM HEPES buffer, pH 7.2, containing 10 mM DTT, 0.2 mM MnCI 2 , 2 mM MgCI 2 , 2 mM GDP, 0.2 mM NADH, 2 mM PEP, 100 mM KHC0 3 and 2 units of malate dehydrogenase. Assays were initiated by PEPCK addition.
  • Rates were calculated by measuring, from the initial slope of the graph, the decrease in absorbance at 340 nm after substracting the rate of spontaneous NADH oxidation.
  • Assays in the direction of phosphoenolpyruvate synthesis were performed in 1 ml of a 100 mM HEPES buffer, pH 7.2, containing 10 mM DTT, 0.2 mM MnCI 2 , 2 mM MgCI 2 , 1 mM GTP, 1 mM ADP, 0.2 mM NADH, PEPCK, and 5 units of each pyruvate kinase and lactate dehydrogenase. Reactions were initiated by addition of oxaloacetate (OAA).
  • OAA oxaloacetate
  • Rates were calculated by measuring, from the initial slope of the graph, the decrease in absorbance at 340 nm after substracting the rate of the blank which contained all the components of the mix but PEPCK.
  • Apparent values of Km, kcat and Km/kcat were calculated in triplicate in independent sets of assays using a non linear regression software (Leatherbarrow, 1987. Enzfitter; Biosoft, Cambridge UK). In these assays, saturating substrate concentrations (2 mM PEP, 2 mM GDP, 100 mM KHC0 3 ,1 mM GTP or 400 ⁇ OAA) were used except, logically, for the variable substrate.
  • PEPCK-C samples were submitted to proteolytic degradation using trypsin (Sigma) or Proteinase K (Durviz, Paterna, Spain). Reactions were performed at 39°C; aliquots were taken after different timepoints, mixed with standard SDS sample buffer and immediately boiled for 5 minutes. These samples were analyzed using SDS-PAGE in 12% polyacrylamide gels. Band quantification was performed with a densitometer Imagemaster "D Platinum 7 and software Image Scanner III both from GE Healthcare Life Sciences (Piscataway, NJ, USA).
  • y is the vector of phenotypic records for the analyzed trait
  • b is the vector of systematic effects, including covariates with age and the substitution effect of the analyzed polymorphism, a batch effect with four levels and a sex effect with two levels
  • p is the vector of litter effects and u recomprising the polygenic effects.
  • Xj,Wj and z are the rows of the incidence matrices (X,W and Z) corresponding to the ith phenotypic record
  • MVN multivariate Gaussian distributions
  • P MVN-MVN(0, la p ) gnc
  • prior distributions for systematic effects and variance components were assumed uniform with appropriate bounds. The analysis was performed through a single long chain of 500,000 interations of a Gibbs Sampler (Gelfand and Smith, 1990) after discarding the first 25,000 with the TM program (Legarra et al., 2008). Later on, samples for the substitution effect were used to compute the posterior probabilities above zero.
  • a SNP (A2456C) in pig ' s cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) results in a substitution of a methionine by a leucine in a highly conserved beta-strand located far away from the active site.
  • the substitution has strong effects on the enzyme kinetic properties increasing its catalytic efficiency up to 20 fold.
  • the C allele which is present at high frequency in several modern breeds, is strongly associated to lower meat quality and lower intramuscular fat content, and to enhanced backfat thickness.
  • the A allele is clearly associated to higher (up to 20.4%) IMF content in two of the three analyzed muscles (Bayesian posterior probability above cero ranging from 0.9973 in L. dorsi to 0.9800 in Ps. major) and also to reduced (9.9% lower) BT showing also a similar, although not as extreme, pattern than the transgenic PEPCK mouse.
  • a strong association was found between higher water holding capacity at several time points postmortem and the A allele. This elevated (up to 24%) water holding capacity is probably related to the higher values of pH 2 4 also associated to A alleles (Table 5).
  • the mutation detected in the PEPCK-C the substitution of a metionin with a Leucine in the position 139 give raised to changes in its kinetic characteristics. These changes are provoked by a change in the global 3D structure of the enzyme, originating that both isoenzymes show a different susceptibility to the protein hydrolysis.
  • the substitution is a conservative substitution (both the metionine and leucine are polar aliphatic amino acids of similar size) and is found in a beta-sheet far away from the active centre/site of the enzyme, the fact that the amino acid region around the position 139 shows a very high grade of conservation points out that said region is relevant for the functionality of the enzyme.

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Abstract

The present invention relates to a method for selecting a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or with at least 30% more myoglobin in the muscle than a reference value, and/or a phenotype whose meat is at least 9% less exudative than a reference value, which comprises: (a) detecting, in a biological sample from said suid, an A/C polymorphism in the gene that encodes the cytosolic PEPCK protein, and (b) selecting that suid comprising homozygosis for the A allele. Furthermore, the invention also relates to primers, probes and a kit to perform said method.

Description

METHOD FOR THE SELECTION OF SUIDS WITH BETTER MEAT QUALITY BASED ON AN SNP IN THE GENE THAT ENCODES CYTOSOLIC PEPCK The present invention relates to the selection of suids with higher intramuscular fat content, together with lower back fat content, and/or with at least 30% more myoglobin in the muscle, and/or with a meat that is at least 19% less exudative, by means of the detection of the A/C polymorphism at position 2456 of the gene that encodes the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. Therefore, the invention belongs to the technical field of genetic selection in animals; in particular, to the fields of livestock production and the food industry.
BACKGROUND ART
In the past decades, genetic selection in swine has been aimed at reducing the carcass fat content and, especially, the fat cover deposition (subcutaneous adipose tissue). This has been so, in the first place, due to consumers' demand for meats lower in fat, for aesthetic reasons and, to a lesser extent, because of the relationship between high intakes of animal fats and certain diseases. The fact that lean genotypes convert the fodder consumed into weight gain more efficiently, i.e. have lower conversion indices, has also played a very significant role. The latter factor, the conversion index, is the key to economic success in swine production, because feeding of the animals represents about 75% of the costs of pig production. As a consequence of genetic selection, the decrease in back fat thickness has been quite noteworthy. However, the reduction in back fat thickness has gone hand in hand with a reduction in intramuscular fat content. This factor is very relevant for the quality of fresh meat and, especially, of cured products. In fact, a defect of a large proportion of the swine produced is that they have very low intramuscular fat contents. Thus far, it was believed that an increase in intramuscular fat content was always accompanied by an increase in back fat thickness, which, given the higher energy cost of adipose tissue deposition as compared to muscle tissue deposition, inevitably entailed an increase in the conversion index and, consequently, a substantial increase in production costs. It should be borne in mind that the porcine sector is a sector with a high turnover but a narrow profit margin, if at all, and, for this reason, it is very important to reduce production costs in this sector.
The process for selecting animals with higher or lower intramuscular fat content or for reducing back fat thickness is the classical genetic selection. The positive genetic correlation between both traits makes the joint genetic selection thereof difficult. The use of selection techniques based on genetic markers may help to resolve this problem, provided that markers indicative of this phenotype are found. Burgos et al., 2012 (Meat Science. 90: 309-313) describe the effect of the selection of pigs with higher intramuscular fat content for the production of cured ham on the basis of a pig genotype characterised by the presence of an A/G polymorphism at position 3072 of intron 3 of the gene that encodes the insulin-like growth factor (IGF2). This publication shows that the presence of the A or G allele in pigs modifies the relative content of subcutaneous, intermuscular and intramuscular fat in the carcass and the meat, the A allele being, in general, the most favourable, because it considerably reduces subcutaneous fat content and slightly increases intramuscular fat content. Other studies performed on this specific polymorphism reveal that the expression of the A allele stimulates muscle growth at the expense of back fat growth, thereby favouring lean pig carcasses and lower back fat content. All these characteristics make this genotype desirable for production.
On the other hand, Nechtelberger D. et al. (2001. J Anim Sci. 79: 2798-2804) describe the association between the genetic variants of the fatty-acid- binding protein (FABP), in the heart and the adipocytes (H-FABP and A- FABP, respectively), and intramuscular fat content. In said publication, studies were performed on three different Austrian pig populations (Pietrain, Large White and Landrace), and it was discovered that said genetic variants present a polymorphism at the Mspl locus. However, they concluded that these genetic markers are not recommendable for the selection of Austrian pig populations in terms of their growth and the commercial characteristics of their meat.
Consequently, there is a need in the state of the art to provide new, alternative markers to those already described, in order to select suids that present higher intramuscular fat content but lower back fat content, favouring lean pig carcasses, increasing the yield and reducing production costs.
DETAILED DESCRIPTION OF THE INVENTION The authors of the present invention have identified a polymorphism that modulates intramuscular fat content in suids, increasing it by about 25% (in the loin), and reduces back fat thickness by about 11 %; said polymorphism may be used as a genetic marker to select suids with higher intramuscular fat content, together with lower back fat content, thereby reducing the conversion index and increasing the efficiency of the animal raising process. Moreover, it improves the quality of fresh meat by reducing the exudation thereof (by between 24% and 19%) and increases its iron content (by about 30%). Meat with these characteristics has better quality both for obtaining fresh products (due to the lower percentage of exudation) and for obtaining cured products (due to the higher intramuscular fat content).
In order to identify polymorphisms associated with the desired phenotype (higher intramuscular fat levels and lower back fat thicknesses), the inventors sequenced the promoter and encoding regions of the PEPCK gene in Pietrain-breed and Iberian-breed pigs. These two breeds were chosen because they show extreme phenotypes for the traits of interest: the Pietrain breed shows very low intramuscular fat contents together with very small back fat thicknesses; The Iberian breed presents high intramuscular fat contents and very large back fat thicknesses.
In the sequencing work, it was detected that Pietrain animals show an SNP which involves one amino acid change (from methionine to leucine). Said SNP is located at position 2456 of the gene that encodes PEPCK (SEQ ID NO: 1 ). A statistical test (Chi-square Test) showed that the allele frequency of this mutation was significantly different in both breeds. The sequencing of the PEPCK gene in nine pigs obtained by crossing Duroc breed pigs with Landrace x Large White breed pigs showed that, in said pigs, there was variability for the polymorphism detected in the Pietrain breed. Subsequently, by means of sequencing and RT-PCR analysis, the genotype for said polymorphism was determined in a population of 202 of these animals, and studies were performed on the effect of the SNP on intramuscular fat content and back fat content thickness; these showed that a homozygous AA animal has about 25% more intramuscular fat and, simultaneously, about 1 1 % less back fat than a homozygous CC animal. This study further showed that AA animals provide better-quality meat, characterised by lower percentages of exudation (between 24% and 19% less) and greater iron content (30% more).
Consequently, on the basis of this discovery, a number of inventive aspects were developed, which will be explained in detail below. Method for selecting the suid of the invention
A first aspect of the invention relates to a method for selecting a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a suid comprising a phenotype whose meat is at least 19% less exudative than a reference value, hereinafter "selection method of the invention", which comprises:
a) detecting, in a biological sample from said suid, an A/C
polymorphism in the gene that encodes the cytosolic PEPCK protein, and
b) selecting that suid comprising homozygosis for the A allele.
In the present invention, "suid" is understood to mean a member of the family Suidae, a family of artiodactyl mammals that includes domestic pigs, wild boars and their closest relatives, up to 16 species, all of them originally distributed throughout Eurasia and Africa. These are non-ruminant omnivorous ungulates with primitive characteristics. Examples of suids include, without being limited thereto, pigs and wild boars.
Therefore, in a particular embodiment, the suid is a domestic pig or a wild boar.
"Domestic pig" (Sus scrofa domestica) is understood to mean a species of artiodactyl mammal of the family Suidae. It is a domestic animal used for human nutrition in some cultures, especially Western cultures.
"Wild boar" is understood to mean an artiodactyl mammal of the family Suidae, with the scientific name Sus scrofa, that is present in Europe, although there are also subspecies in America, Africa and Asia. It is different from the domestic pig in that it has a more pointed head, a longer snout, always erect ears, very thick, strong, uniform grey-colour hair, and large canine teeth that protrude from the mouth.
The method of the invention is aimed at selecting a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or comprising a phenotype whose meat is at least 19% less exudative than a reference value. In the present invention, "intramuscular fat" is understood to mean the fat pad located between the muscle fasciculi, the fat contained inside the muscle fibres and the fat that is a part of the cell membranes.
"Back fat", "dorsal fat" or "subcutaneous fat" is understood to mean the adipose tissue deposit located immediately beneath the epidermis, the thickness whereof is determined, amongst other causes, by genetic causes. It is one of the most important parameters in estimating the carcass yield, and the carcass is understood to be the set of tissues and organs extracted from the sacrificed animal once the viscerae have been removed and which will be processed at the slaughterhouse (muscle, fat and bones).
In the present invention, the term "myoglobin" or "muscle haemoglobin" is defined to be the muscle haemoprotein, which is structurally and functionally very similar to haemoglobin, relatively small, composed of a polypeptide chain with 153 amino acid residues, containing a haem group with an iron atom, and the function whereof is to store and transport oxygen. The highest concentrations of myoglobin are found in skeletal muscle, especially in the red fibres, whose energy metabolism is more dependent on the oxidation of fats than on glycolysis, and in the cardiac muscle, where large quantities of O2 are required in order to meet the energy demands of contractions.
In the present invention, meat is understood to be "exudative" when it loses, especially in the cut surfaces, watery fluid due to changes in the myofibrils, a phenomenon that is favoured by protein denaturation and damage in the cell membranes. As known to persons skilled in the art, excessive exudation leads to weight loss, with the consequent direct economic loss, makes packaged meat look worse, with the consequent rejection by consumers, and is closely related to loss of juiciness and increased meat hardness, which overall results in low-quality meats. In the present invention, "reference value" is understood to be the mean of the values obtained upon measuring the phenotypic characteristic that is being selected in the suid population. Thus, in the present invention, a suid is understood to comprise a phenotype with higher intramuscular fat content and lower back fat content than a reference value when the intramuscular fat content and the back fat content of said suid are, respectively, higher and lower than the mean intramuscular fat content and back fat content of the rest of suids present in the population. Similarly, in the present invention, a suid is understood to comprise a phenotype with at least 30% more myoglobin in the muscle than a reference value when the muscle myoglobin content of said suid is 30% higher than the mean quantity of myoglobin in the muscle in the rest of suids present in the population.
In the present invention, a suid is understood to comprise a phenotype whose meat is at least 19% less exudative than a reference value when the meat of said suid is 19% less exudative than the mean exudation of the meat in the rest of suids that are a part of the population.
The skilled person in the art is knowledgeable about the techniques used to measure these phenotypic characteristics in suids. The illustrative Examples of the invention describe some of these techniques. In a first step [step (a)], the selection method of the invention comprises detecting, in a biological sample from said suid, an A/C polymorphism in the gene that encodes the cytosolic PEPCK (PEPCK-C) protein. In order to perform the first step, it is necessary to obtain a biological sample; this may be done with any of the methods described in the state of the art, which are routine practice for persons skilled in the art. Within the context of the present invention, "biological sample" refers to any matter that contains DNA. Examples of biological samples wherefrom DNA may be obtained include, without being limited thereto, biological fluids (blood, saliva, urine, sperm, etc.), the epidermis, hair, faeces, tissue samples, etc. Obtainment of the biological sample must be performed under the adequate conditions using adequate material, such as swabs, tweezers, etc., and the samples must be stored independently in duly-labelled separate sterile jars or plastic bags, without any type of preservatives, preventing contamination by foreign biological material.
Once the biological sample from the suid has been obtained, it must be processed to obtain the nucleic acid, which may be DNA or RNA. DNA extraction may be performed using any method known to persons skilled in the art (Sambrock et al., 2001. "Molecular cloning: A Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vols. 1 -3); these methods include, without being limited thereto, density gradient centrifugations, two- phase extraction using aqueous phenol or chloroform with ethanol, column chromatography, methods based on the DNA binding capacity to glass and/or silica surfaces, such as diatomaceous earth or glass bed preparations, using commercial kits, such as, for example, the kits "Q- Biogene fast DNA kit" or "QIAamp(R) DNA Blood Mini Kit" (Qiagen, Hilden, Germany) or "G-Spin lip" (Intron Biotechnology, Korea) or "Fast Prep System Bio 101 " (Qbiogene, Madrid, Spain).
In the event that the nucleic acid that is to be extracted from the sample is RNA, there are commercial kits exclusively designed for this purpose which contain the adequate components to extract RNA under perfect conditions: high concentrations of chaotropic salts in the lysis buffer, in order to deactivate the RNases; silica membranes that favour RNA adsorption; DNases that eliminate the DNA, in order to obtain a high-purity RNA isolate, etc. A commercial kit that fulfils the aforementioned characteristics is, for example, without being limited thereto, Nucleospin™ RNA.
After isolating the nucleic acid (DNA or RNA) from the biological sample from the suid, the selection method of the invention comprises detecting the A/C polymorphism in the gene that encodes the PEPCK protein in order to select those animals that are homozygous for the A allele. The "phosphoenolpyruvate carboxykinase" (PEPCK) protein is an enzyme of the family of lyases that participates in various metabolic pathways, including glyceroneogenesis, because it provides activated forms of glycerol for triglyceride synthesis. It catalyses the conversion reaction of oxaloacetate into phosphoenolpyruvate and carbon dioxide, and consumes GTP, which is transformed into GDP. Phosphoenolpyruvate carboxykinase is found in two isoforms, PEPCK1 or PEPCK-C (cytosolic), and PEPCK2 or PEPCK-M (mitochondrial). The sequences of these isoforms have 63% similarity; the precise function of the mitochondrial isoenzyme is unkwown. The cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) protein is encoded by the PEPCK gene, the nucleotide sequence thereof is shown in NCBI database accession number: FJ668384.1 Gl:238863889. The nucleotide sequence of the cytosolic PEPCK comprising the A2456C mutation is shown in SEQ ID NO: 1.
However, in a particular embodiment, the gene that encodes the cytosolic PEPCK protein comprises a nucleotide sequence with at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 . In another particular embodiment, the gene encoding the PEPCK-C comprises a nucleotide sequence that hybridizes under low, medium, medium-high, high, or very high stringency conditions with the nucleotide sequence SEQ ID NO: 1 . The term "hybridize" or "specifically hybridize" as used herein refers to a process where two complementary nucleic acid strands anneal to each other under appropriately stringent conditions. The term "low stringency conditions" means for nucleotides of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
The term "medium stringency conditions" means for nucleotides of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
The term "medium-high stringency conditions" means for nucleotides of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
The term "high stringency conditions" means for nucleotides of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C. In the present invention, the term "polymorphism" refers to a "single- nucleotide polymorphism", or "SNP", and is defined as the variation of a single nucleotide between the genomes of individuals from the same species. In a particular embodiment, the A/C polymorphism is located at a position equivalent to nucleotide 2456 of SEQ ID NO: 1 determined by the optimal alignment of the sequences. The A C polymorphism causes a change of leucine to methionine in the PEPCK protein, the mutation being located in amino acid 139 of said protein (SEQ ID NO: 2). The complementary DNA (cDNA) obtained from the PEPCK-C protein comprising the amino acid sequence SEQ ID NO: 2 has the polymorphism located at nucleotide 415 of SEQ ID NO: 3.
The detection of the polymorphism of the invention may be performed using any of the techniques described in the state of the art. Examples of these processes include, without being limited to, sequencing, pyrosequencing, allele-specific oligonucleotide (ASO) dot-blot hybridisation analysis, nucleotide primer extension, PCR-based single-strand conformation polymorphism (SSCP) analysis, PCR-based restriction fragment length polymorphism (RFLP) analysis, real-time quantitative PCR (Q-PCR) and mass matrix using a mass spectrometer. These processes will be considered in more detail below.
"Allele-specific oligonucleotide (ASO) dot-blot hybridisation analysis" is understood to mean the process aimed at detecting polymorphisms in specific genes, which may be performed by hybridising the DNA fragment with allele-specific oligonucleotide probes of a PCR-amplified gene fragment, using forward primers and reverse primers designed to incorporate a target SNP, for dot-spot hybridisation analysis.
"Mononucleotide primer extension" is understood to mean the production of sufficient multiple copies thereof to allow for relatively easy manipulation of the segment. Manipulation refers to both physical and chemical manipulation, i.e. the capacity to displace mass quantities of the segment and drive chemical reactions with the segment that generate detectable products. "PCR-based single-strand conformation polymorphism (SSCP) analysis" is understood to mean the mutation tracking used in molecular diagnosis, based on the electrophoretic migration of DNA.
"PCR-based restriction fragment length polymorphism (RFLP) analysis, invasive process" is understood to mean the analysis of specific nucleotide sequences in DNA that are recognised and cut by restriction enzymes (also called restriction endonucleases), which vary amongst individuals. RFLPs are DNA genetic markers and may be found in regions that encode proteins or exons, in introns or in the DNA that separates one gene from another.
"Real-time quantitative PCR" (qPCR or Q-PCR) or "real-time PCR" (RT- qPCR or RT-Q-PCR) is understood to mean a variant of the polymerase chain reaction (PCR) used to amplify and simultaneously quantify the deoxyribonucleic acid (DNA) amplification product.
"Mass matrix using a mass spectrometer" is understood to mean the result obtained from the experimental technique that makes it possible to measure ions derived from molecules. The mass spectrometer allows for a very precise analysis of the composition of different chemical elements and atomic isotopes, by separating the atomic nuclei as a function of their mass-to- charge (m/z) ratio. Therefore, in a particular embodiment, detection of the polymorphism is performed by means of a process selected from the group consisting of direct sequencing, allele-specific oligonucleotide (ASO) dot-blot hybridisation analysis, mononucleotide primer extension, PCR-based single-strand conformation polymorphism (SSCP) analysis, PCR-based restriction fragment length polymorphism (RFLP) analysis, real-time quantitative PCR and mass matrix using a mass spectrometer. In the event that the technique selected to detect the polymorphism requires DNA amplification, it will be necessary to use primers that are capable of amplifying the region of nucleotide sequence SEQ ID NO: 1 wherein the A C polymorphism is located. In a preferred embodiment, said region comprises the 30 nucleotides adjacent to each side of nucleotide 2456 of SEQ ID NO: 1 . Said primers may be designed by means of any of the standard processes in the state of the art, which are widely known to persons skilled in the art. In a particular embodiment, the primers used in the selection method of the invention comprise a nucleotide sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 . In another, even more particular embodiment, the primers comprise the nucleotide sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
The use of probes to detect polymorphisms is also routine practice for the skilled person in the art. In a particular embodiment, the selection method of the invention encompasses using at least one nucleic acid probe that comprises a nucleotide sequence with, at least, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 . The meaning of the term "identity" is explained further below in the present description. In another, even more particular embodiment, the probe comprises the nucleotide sequence SEQ ID NO: 8 or SEQ ID NO: 9. As known to the skilled person in the art, the presence of the A C polymorphism may be determined by means of real-time PCR, by designing two different probes capable of discriminating between the two alleles. Real-time PCR is a method capable of quantifying the DNA in each PCR cycle in the form of fluorescence. This fluorescence is due to the modification undergone by the probes in each amplification cycle. Allelic discrimination takes place when two probes labelled with a different fluorochrome that are specific for each allele are used. At the end of the process, the fluorescence curves for the two probes will indicate the genotype.
In general, labelling of the probes is based on the use of quenchers (quencher pigment or non-fluorescent quencher -NFQ- that increases the detection efficacy and the signal because it does not emit fluorescence) and probes labelled with a wide range of fluorophores (reporter pigment) with different excitation and emission spectra. Illustrative, non-limiting examples of fluorescent labels that may be used within the context of the present invention include: FAM, VIC, HEX, TET, CY3, CY5.5, JOE, 6-ROX, cascade Blue, fluorescein, Texas red, rhodamine, rhodamine green, rhodamine red, rhodamine 6G, 6-TAMRA, 5-TMRIA, Alexa 430, Alexa 488, Alexa 594, Bodipy R6G, etc. In the present invention, "quencher" is understood to mean a molecule that accepts energy from a fluorophore and dissipates it in the form of heat or fluorescence. Examples of quenchers are, without being limited thereto, Methyl Red, ElleQuencher, Dabcyl, Dabsyl, TAMRA, etc.
Examples of probes that carry this type of labelling are, for example, TaqMan probes, molecular beacons (Molecular-Beacon-type probes), Scorpions probes, Amplifluor probes, Eclipse probes, etc.
The study of the effect of this polymorphism on the intramuscular fat content and the back fat thickness, on the myoglobin in the muscle and/or the degree of exudation of the meat shows that homozygous animals for the A allele comprise higher intramuscular fat content and lower back fat content, at least 30% more myoglobin in the muscle and/or a meat that is at least 19% less exudative as compared to a homozygous animal for the C allele or a heterozygous animal (AC).
Therefore, once the A/C polymorphism has been detected as explained above, step b) of the selection method of the invention comprises selecting that suid which comprises homozygosis for the A allele. "Homozygous" or "homozygosis" is understood to mean that the two alleles of a diploid cell or organism at a given site are identical, i.e. that they have the same nucleotide for nucleotide change at the same position in their sequences. "Heterozygous" is understood to mean the case of a diploid individual with different alleles, i.e. that they have a different nucleotide at the same position in their sequences. Thus, by selecting a suid with homozygosis for the A allele, a suid is obtained which comprises a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a phenotype whose meat is at least 19% less exudative than a reference value. Therefore, the meat obtained from said suid has better quality than that of a suid which does not present said polymorphism.
In a particular embodiment, the phenotype of the selected suid comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
In another, more particular embodiment, the phenotype of the selected suid comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value.
In another particular embodiment, the phenotype of the selected suid comprises a meat that is at least between 19% and 24% less exudative than a reference value. Sequence of the invention, primers, probes and uses thereof
The authors of the present invention have discovered that the presence of the A C polymorphism in the gene that encodes the cytosolic PEPCK protein in suids is associated with higher intramuscular fat content and lower back fat content (in particular, between 19% and 25% more intramuscular fat and between 9% and 15% less back fat), with 30% more myoglobin in the muscle and/or a meat that is less exudative (in particular, between 19% and 24% less exudative). Preferably, said polymorphism is located at position 2456 of the PEPCK gene (SEQ ID NO: 1 ).
Therefore, another aspect of the present invention relates to an isolated nucleotide sequence, hereinafter "nucleotide sequence of the invention", that comprises (i) a nucleotide sequence with at least 70% identity with SEQ ID NO: 1 and (ii) the A/C polymorphism at a position equivalent to nucleotide 2456 of said sequence SEQ ID NO: 1 by optimal alignment of the sequences. The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, and (2) any substance including, but not limited to, variant, nucleic acid, protein or peptide, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.
In the present invention, "identity" or "sequence identity" is understood to mean the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a different degree of identity, expressed as a percentage, will be obtained. The degree of identity between two amino acid sequences may be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as, for example, BLAST [Altschul S.F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3): 403-10]. The BLAST programmes, for example, BLASTN, BLASTX, and T BLASTX, BLASTP and TBLASTN, are in the public domain at The National Center for Biotechonology Information (NCBI) website. The skilled person in the art understands that mutations in the nucleotide sequence of genes that lead to conservative amino acid substitutions at non- critical positions for the functionality of the protein are evolutionarily neutral mutations which do not affect its global structure or its functionality. Said variants fall within the scope of the present invention. In the context of the present invention, the term "variant" means a polypeptide having (1 ) PEPCK- C activity and (2) the amino acid sequence SEQ ID NO: 2 comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions, with the proviso that the amino acid equivalent to the amino acid at position 139 of the SEQ ID NO: 2 by the optimal alignment of the sequences is not changed. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.Thus, also comprised within the scope of the invention are those variants of the cytosolic PEPCK protein (SEQ ID NO: 2) [encoded by the cytosolic PEPCK gene (SEQ ID NO: 1 )] which comprise insertions, deletions or modifications of one or more amino acids with respect to said sequence SEQ ID NO: 2, and, moreover, conserve the same functions as said protein SEQ ID NO: 2.
In another particular embodiment, the nucleotide sequence of the invention comprises a nucleotide sequence with at least 80%, 90%, 95%, 98%, 99% or 100% identity with sequence SEQ ID NO: 1 .
In another particular embodiment, the gene encoding the PEPCK-C comprises a nucleotide sequence that hybridizes under low, medium, medium-high, high, or very high stringency conditions with the nucleotide sequence SEQ ID NO: 1. The terms low, medium, medium-high, high, or very high stringency conditions have been previously defined.
In another aspect, the invention relates to an isolated polypeptide encoded by the nucleotide sequence of the invention, hereinafter, "polypeptide of the invention". In a particular embodiment, the polypeptide of the invention comprises an amino acid sequence with at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with SEQ ID NO: 2. In another particular embodiment, the polypeptide of the invention comprises a methionine at a position equivalent to amino acid 139 of the SEQ ID NO: 2 by optimal alignment of the sequences
The implementation of the present invention requires detection of the A/C polymorphism in the gene that encodes the cytosolic PEPCK protein. As explained above, said detection may be performed by means of numerous techniques that are widely known to the skilled person in the art, some of which entail using oligonucleotides that act as primers in order to amplify the gene region where the polymorphism is located. To this end, the inventors of the present invention have designed some oligonucleotides that act as useful primers to amplify the region of the gene that encodes cytosolic PEPCK wherein the A C polymorphism is located.
Thus, another aspect of the invention relates to an oligonucleotide, hereinafter "oligonucleotide of the invention", that comprises the nucleotide sequence selected from the group consisting of the sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. The term "oligonucleotide" refers to a nucleotide sequence comprising between 10 and 300 nucleotides, preferably between 10 and 200 nucleotides, more preferably between 10 and 100 nucleotides, even more preferably between 10 and 50 nucleotides, most preferably between 10 and 30 nucleotides in lenght. Another aspect of the invention is a pair of primers, hereinafter called "primers of the invention", which are specifically designed to amplify the region of SEQ ID NO: 1 that comprises the A/C polymorphism at position 2456 of said sequence, i.e. they are capable of amplifying sequence SEQ ID NO: 1 . Preferably, said region comprises the 50, preferably 30, nucleotides adjacent to each side of nucleotide 2456 of SEQ ID NO: 1 .
In the present invention, "primer" is understood to mean that oligonucleotide which is capable of contiguously binding to a target sequence and serving as the starting-point for DNA synthesis when it is placed under conditions that initiate the synthesis of a primer extension product which is complementary to a nucleic acid chain.
In a particular embodiment of the preceding inventive aspect, the primers comprise a nucleotide sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 . In another particular embodiment, the primers of the invention comprise a nucleotide sequence which hybridizes under medium, high, or very high stringency conditions with the nucleotide sequence SEQ ID NO: 1 .
Hybridizations are typically and preferably conducted with prime-length nucleic acid molecules, preferably 10-100 nucleotides in length, more preferably 10-50 nucleotides in length, most preferably 10-30 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al . , 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N. Y. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementary will stably hybridize, while those having lower complementary will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al. , 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, N.J.
In another, more particular embodiment, at least one of the primers in the pair comprises nucleotide sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7; preferably, the pair of primers is made up of sequences SEQ ID NO: 4 and SEQ ID NO: 5, or sequences SEQ ID NO: 6 and SEQ ID NO: 7. Another aspect of the invention relates to a nucleic acid probe, hereinafter "probe of the invention", that comprises a nucleotide sequence capable of hybridising with SEQ ID NO: 1 in the region that comprises the A C polymorphism at position 2456, i.e. which is complementary to sequence SEQ ID NO: 1 . Preferably, said region comprises the 50, preferably 30, nucleotides adjacent to each side of nucleotide 2456 of SEQ ID NO: 1 .
In a particular embodiment, the probe comprises a nucleotide sequence with at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with sequence SEQ ID NO: 1 , which, in another, even more particular embodiment, comprises nucleotide sequence SEQ ID NO: 8 or SEQ ID NO: 9. In another particular embodiment, the probe of the invention comprises a nucleotide sequence which hybridizes under medium, high, or very high stringency conditions with the nucleotide sequence SEQ ID NO: 1 . The terms high, or very high stringency conditions have been previously with regards to the primers of the invention.
"Probe" is understood to mean a small-size DNA fragment, usually between 15 and 100 bases, preferably between 15 and 50 bases, used as a tool to detect the presence of DNA with an identical or similar complementary sequence. The probe binds to the single-stranded target sequence by means of a hybridisation mechanism that forms a double-stranded structure; one of the strands belongs to the probe and the other belongs to the target sequence. As previously explained in the preceding inventive aspect, the probe may be labelled at the ends such that it may be detected. Examples of labels and types of probes that may be used within the context of the present invention have been mentioned above. The skilled person in the art understands that the oligonucleotides, the primers and the probes described in the present invention may be used to detect the A C polymorphism in the gene that encodes the cytosolic PEPCK protein. Thus, another aspect of the invention relates to the use of an oligonucleotide of the invention, the probe of the invention and/or the pair of primers of the invention to detect the A/C polymorphism in the gene that encodes cytosolic PEPCK. In a particular embodiment, the gene that encodes the cytosolic PEPCK protein comprises a nucleotide sequence with at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with sequence SEQ ID NO: 1. In another particular embodiment, the A/C polymorphism is located at a position equivalent to nucleotide 2456 of sequence SEQ ID NO: 1 determined by the optimal alignment of the sequences.
The skilled person in the art is knowledgeable about how the oligonucleotide, the primers and the probe of the invention may be used to detect the A C polymorphism at position 2456 of the gene that encodes cytosolic PEPCK in the genome of an individual, in particular, a suid. Examples of the techniques that may be used to detect said polymorphism have been previously described in the method of the invention, and their use is routine practice for persons skilled in the art. One example of these techniques is real-time or quantitative polymerase chain reaction (RT-PCR).
Moreover, the detection of said polymorphism makes it possible to select a suid whose phenotype comprises a meat with higher intramuscular fat content and less back fat content, and/or with at least 30% more myoglobin in the muscle and/or that is at least 19% less exudative than a reference value. Therefore, another aspect of the invention relates to the use of the nucleotide sequence or the polypeptide of the invention, the oligonucleotide, the probe and/or the pair of primers of the invention to select a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a suid comprising a phenotype whose meat is at least 19% less exudative (in particular, between 19% and 24%) than a reference value. In another particular embodiment, the phenotype of the suid comprises, at least, between 19% and 25% more muscular fat and, at least, between 9% and 15% less back fat than a reference value. In another, even more particular embodiment, the phenotype of the suid comprises, at least, 20% more intramuscular fat and, at least, 11 % less back fat than a reference value.
The term "reference value" has been previously defined in the method of the invention and is applicable to the present inventive aspect. On the other hand, it is well-known that genetic material is present in all the cells of an individual, except for the mature red blood cells of mammals, and, therefore, it will also be present in the meat from said individual, in particular, a suid. Therefore, the present invention makes it possible not only to select suids with the phenotypic characteristics previously mentioned in the present description, but also to select meat products from said suids which, as a consequence, also contain the A/C polymorphism at position 2456 of the gene that encodes PEPCK in their genetic material. Thus, by analysing the DNA of a meat product, it is possible to select the meat product comprising higher intramuscular fat content and lower back fat content, at least 30% more myoglobin in the muscle, and/or at least 19% less exudation than other meat products. Thus, a meat product is selected with higher quality than other meat products that do not present said characteristics.
Therefore, another aspect of the present invention relates to the use of the nucleotide sequence or the polypeptide of the invention, the oligonucleotide, the probe and/or the pair of primers of the invention to select a meat product comprising higher intramuscular fat content and lower back fat content than a reference value, 30% more myoglobin than a reference value and/or at least 19% less exudation than a reference value.
"Meat product" or "meat" is understood to mean a foodstuff of animal origin, primarily made up of muscle tissue, which is consumed for nutrition. From the nutritional standpoint, meat is a habitual source of proteins, fats and minerals in the human diet. Most meat consumption by human beings comes from mammals, especially from ungulate animals, which are domesticated to provide food.
Within the context of the present inventive aspect, "reference value" is understood to be the mean of the values obtained from measuring the phenotypic characteristic that is being selected in the population/set of meat products.
Thus, in the present invention, a meat product is understood to present higher intramuscular fat content and lower back fat content than a reference value when the intramuscular fat content and the back fat content of said meat product are, respectively, higher and lower than the mean intramuscular fat content and back fat content of the rest of meat products which are a part of the population.
Likewise, in the present invention, a meat product is understood to present at least 30% more myoglobin in the muscle than a reference value when the myoglobin content in the muscle of said meat product is 30% higher than the mean quantity of myoglobin in the muscle in the rest of meat products which are a part of the population.
Similarly, a meat product is understood to present at least 19% less exudation than a reference value when said meat product comprises 19% less exudation than the mean exudation of the rest of meat products which are a part of the population. The meaning of the term "exudation" has been previously defined. In a particular embodiment, the meat product comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value. In another, even more particular embodiment, the meat product comprises, at least, 20% more intramuscular fat and, at least, 11 % less back fat than a reference value. In another particular embodiment, the meat product comprises, at least, between 19% and 24% less exudation than a reference value.
As the skilled person in the art understands, methods comprising the use of the isolated nucleotide sequence, the isolated polypeptide, the nucleic acid probes the oligonucleotide and the primers disclosed in the present invention are also comprised within the scope of the present invention.
Thus, further aspects of the present invention relate to methods for detecting the A C polymorphism in the gene encoding the PEPCK-C protein and methods for selecting a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value and/or a suid comprising a phenotype whose meat is at least 19% less exudative than a reference value.
Kit of the invention and uses thereof
The implementation of the present invention requires having the adequate materials and reagents to detect the A/C polymorphism in the gene that encodes cytosolic PEPCK, in order to select a suid with higher muscular fat content and lower back fat content, and/or higher iron content in the muscle and/or less exudation of the meat than a reference value, as explained throughout the present description. All the necessary materials to do so may be found as a part of a kit, although they may also be found individually in the state of the art. Therefore, another aspect of the invention relates to a kit, hereinafter "kit of the invention", that comprises the oligonucleotide, the probe and/or the pair of primers described in the preceding inventive aspects, together with the corresponding particular embodiments.
Another aspect of the invention relates to the use of the kit of the invention to detect the A C polymorphism in the gene that encodes the cytosolic PEPCK protein. In a particular embodiment, the gene that encodes the cytosolic PEPCK protein comprises a nucleotide sequence with at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 . In another, even more particular embodiment, the A C polymorphism is located at a position equivalent to nucleotide 2456 of sequence SEQ ID NO: 1 determined by the optimal alignment of the sequences.
Another aspect of the invention relates to the use of the kit of the invention to select a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a suid comprising a phenotype whose meat comprises at least 19% less exudation than a reference value.
The terms used in the present inventive aspect have been previously defined and explained in the preceding inventive aspects.
In a particular embodiment, the phenotype of the suid comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
In another, even more particular embodiment, the phenotype of the suid comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value. In another particular embodiment, the phenotype of the suid comprises a meat with, at least, between 19% and 24% less exudation than a reference value. Another aspect of the invention relates to the use of the kit to select a meat product comprising higher intramuscular fat content and lower back fat content than a reference value, at least 30% more myoglobin than a reference value and/or at least 19% less exudation than a reference value. The meaning of the term "reference value" has been previously defined.
In a particular embodiment, the meat product comprises, at least, between 19% and 25% more muscular fat and, at least, between 9% and 15% less back fat than a reference value. In another, even more particular embodiment, the meat product comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value. In a particular embodiment, the meat product comprises, at least, between 19% and 24% less exudation than a reference value.
The details of these inventive aspects have been previously explained in the preceding inventive aspects and are applicable to the kit of the invention and the uses thereof. Throughout the description and the claims, the words "comprises", "comprising" and variants thereof are not intended to exclude other technical characteristics, additives, components or steps. For persons skilled in the art, other objects, advantages and characteristics of the invention will arise partly from the description and partly from the practice of the invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following examples, drawings and sequence listing are provided by way of illustration and are not intended to be limiting of the present invention. DESCRIPTION OF THE DRAWINGS
Figure 1 shows the structure of the cytosolic PEPCK gene. Position of the primers used in the sequencing of its encoding region.
Figure 2 shows the sequences and alignments of the 27 animals studied. The A C polymorphism is marked with an arrow and shading. "Ref. Seq.": cDNA of PEPCK (SEQ ID NO: 3). A = adenine; C = cytosine; M = heterozygous animal that has an A allele and a C allele.
Figure 3 shows the PCR amplification of an 896-base fragment that contains the A2456C mutation. The crosses represent failed amplifications from genomic DNA; the triangles, diamonds and squares represent amplifications from previously PCR-amplified fragments, as shown in Figure 3: the triangles are amplifications of homozygous CC animals, the squares are amplifications of heterozygous animals and the diamonds are samples of homozygous AA animals. The circles belong to control amplifications (adding water instead of DNA). Figure 4 shows a PCR amplification of the gene region of porcine cytosolic PEPCK that contains nucleotide 2456. Lanes 1 and 5 contain molecular weight markers; lanes 2-4 and 6-8 contain samples from 6 different animals.
Figure 5 is a SDS-PAGE showing the purified His-tagged PEPCK-C isoenzymes. MW, molecular weight markers.
Figures 6A, 6B and 6C are Lineweaver-Burks plots for kinetic calculations. Figure 6A, PEP as variable substrate; Figure 6B, GDP as variable substrate; PEPCK-C-p.139Leu, squares (■); PEPCK-C-p.139Met, diamons (♦).
Figures 7A and 7B are Lineweaver-Burks plots for kinetic calculations. Figure 7A, OAA as variable substrate; Figure 7B, GTP as variable substrate. PEPCK-C-p.139Leu, squares (■); PEPCK-C-p,139Met, diamons (♦).
Figure 8 is a SDS-PAGE analysis of trypsine digestion of both PEPCK-C isoenzymes (PEPCK-C-p.139Leu and PEPCK-C-p,139Met) after different time points.
Figure 9 is a picture of a three dimensional structure of PEPCK-C showing the substrate PEP in the center of the figure and the substituted amino acid methionine 139 in the right side of the figure.
Figure 10 shows the aligment of PEPCK-C protein regions from man to C. elegans to show the conserved methionine (bold) corresponding to position 139 of the pig protein. Asterisks indicate perfectly conserved amino acids among the 7 species. GenBank accession numbers are the following: pig, NP_001 1 16630; human, NP_002582; mouse, NP_035174; bovine, NP_777162; chicken, NP_990802; Drosophila, NP_001097367; C. elegans, NP_001021589.
EXAMPLES
Example 1
Search for candidate genes for fatty infiltration traits
1 . Material and methods 1.1 Animals and samples
The genomic DNA was extracted from different sources: muscle in the case of Iberian pigs, semen in the case of Pietrain pigs and a portion of the end of the tail in the case of animals from the Duroc x Landrace-Large White cross.
1.2 Extraction and quantification of the genomic DNA The DNA from these samples was extracted using the Real Pure kit for genomic DNA extraction (Durviz, Paterna, Valencia), following the manufacturer's instructions. The DNA concentration of these extracts was determined using a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific, Wilmington, United States).
1.3 Sequencing of the encoding region of PEPCK
The analysis of the sequence of bases in the encoding region was performed by sequencing 5 PCR-amplified fragments of the PEPCK gene: PEPCK1 F/2R, PEPCK2F/3R, PEPCK3F/4R, PEPCK4F/5R and PEPCK5F/8R. The sequences of these primers are shown in Table 1 .
Size
Primer (SEQ ID NO ) Sequence
(bp)
PEPCK1 F 5'- GCGACCTTGGCATCCACACCCCTTA -3'
(SEQ ID NO: 10)
1026
PEPCK2R 5'- TTTAAAGCTCGCTGAGGCCACGGG -3'
(SEQ ID NO: 1 1 )
PEPCK2F 5'- GTGTTGTCCAAGGGGGATCAGGAGGA
(SEQ ID NO: 4) -3'
896
PEPCK3R 5'- CCCCCTGCCTCGCACATTTAGGAT -3'
(SEQ ID NO: 5)
PEPCK3F 5'- GCACCCTCCTAGGCCAAAGCATTG -3'
(SEQ ID NO: 12)
961
PEPCK4R 5'- ACAGCCAGAGAGAAGACGCCCTGA -3'
(SEQ ID NO: 13)
PEPCK4F 5'- GGGCATCAAGCTCACTTCCTGGAA -3'
(SEQ ID NO: 14)
790
PEPCK5R 5'- AC AC AC AG AG AAG AAAG G GC AG CG -3'
(SEQ ID NO: 15)
PEPCK5F 5'- TCCCCGCATCCCTCTGTACGTGTA -3'
789
(SEQ ID NO: 16) PEPCK8R 5'- CCAGAGACCCTGTGCCTGCGTGTC -3'
(SEQ ID NO: 17)
Table 1. Primers used for the amplification of the 5 fragments of the encoding region of the porcine cytosolic PEPCK gene. The size indicated is that of the amplified product. (F: Forward or direct primer; R: Reverse primer) The amplifications were performed in a Biometra UNO-Thermoblock thermocycler (Gottingen, Germany), using AccuPrime™ Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, United States) and the pairs of primers indicated in Table 1. Figure 1 shows a graphic representation of the positions whereat these primers hybridise in the gene in relation to the exon positions.
Each PCR reaction included 1 unit of AccuPrime™ Taq DNA Polymerase High Fidelity, 5 μΙ of 10x buffer II, 0.5 μΙ of the corresponding forward primer (20 pmol/μΙ), 0.5 μΙ of the corresponding reverse primer (20 pmol/μΙ), 3.8 μΙ of DMSO (Sigma-Aldrich, Saint Louis, United States), 2 μΙ of genomic DNA (100 ng/μΙ) and the necessary quantity of Milli-Q water to adjust the total reaction volume to 50 μΙ. The temperature programme used for these amplifications includes a denaturation step at 94°C for 2 minutes, followed by 35 denaturation cycles at 94°C for 30 seconds, hybridisation for 30 seconds at 55°C in the case of the PEPCK1 F/2R, PEPCK2F/3R and PEPCK3F/4R fragments, 53°C in the case of the PEPCK4F/R5 fragment and 55°C in the case of the PEPCK5F/8R fragment, and extension at 68°C for 1 minute 30 seconds, 1 minute 15 seconds, 1 minute 15 seconds, 1 minute and 1 minute for the PEPCK1 F/2R, PEPCK2F/3R, PEPCK3F/4R, PEPCK4F/5R and PEPCK5F/8R fragments, respectively, and a final extension step at 68°C for 10 minutes. In order to verify that the size of the PCR-amplified fragment was the correct one, 5 μΙ of the PCR product were loaded in 2% agarose gel (Biotools, Madrid, Spain) and electrophoresis was performed at 100 volts for 35 minutes. The molecular weight pattern used in these electrophoreses was the 100-bp ladder pattern (Biotools, Madrid, Spain). Subsequently, the product of each of these 5 PCRs was purified using the NucleoSpinR Extract II kit (Macherey-Nagel, Duren, Germany), according to the manufacturer's instructions. The purified product was eluted with 25 μΙ of the elution buffer in the kit and the DNA concentration in this solution was measured using a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific, Wilmington, United States). Each of the 5 purified PCR fragments were sent to Sistemas Genomicos (Paterna, Valencia, Spain) to be sequenced with both the forward primer used for their amplification and with the reverse primer.
In order to obtain the complete sequence of bases of each of the 5 PCR- amplified fragments of the PEPCK gene, the sequences obtained for each of them were assembled with their respective forward and reverse primers using the Vector NTI computer package.
Finally, the cDNA of PEPCK of each of these samples was constructed by binding the sequences of all the exons in the appropriate order. In order to locate the beginning and the end of each of said exons, the cDNA sequence of pig cytosolic PEPCK (SEQ ID NO: 3) was used as a reference.
1.4 Alignment of cDNA sequences
Alignment of the cDNA of PEPCK in the 9 pure Iberian pigs, the 9 Pietrain pigs and the 9 animals from the Duroc x Landrace-Large White cross were performed using the ClustalW programme.
2. Results and discussion The alignment of the cDNA sequences of PEPCK in the 27 animals analysed is shown in Figure 2. Of all the SNPs detected, only A2456C meets the two conditions of causing an amino acid change and segregating the Pietrain and Iberian breeds differently. Through a Chi-square test, it was detected that the A2456C mutation appeared in a significantly greater proportion (p = 0.0033) in Pietrain animals than in animals from the Iberian breed. In the latter, the 2456A allele seems to be fixed. The A2456C mutation leads to one amino acid change in the protein (one methionine is substituted by one leucine) at position 139 of the polypeptide chain.
The existence of variability for A2456C in the Duroc x Landrace/Large White animals (see Example 4), jointly with the fact that no additional mutations in the PEPCK gene associated with A2456C appear in these animals, makes it possible to use the Duroc x Landrace/Large White population to attempt to find associations between A2456C and the phenotypic traits of interest and/or related to the fat content.
3. Conclusion
The A2456C mutation in the porcine cytosolic PEPCK gene [NCBI, accession number: FJ668384 (version FJ668384.1 Gl:238863889)] potentially causes phenotypic effects in porcine meat and the porcine carcass. The nucleotide sequence of the cytosolic PEPCK comprising the A2456C mutation is shown in SEQ ID NO: 1 .
Example 2
Association study between A2456C in PEPCK and the phenotypic traits of interest (fatty infiltration and others)
Introduction In order to study whether the A2456C mutation is associated with changes in the traits of interest, particularly fatty infiltration and quality of the meat, a group of 202 animals from the Duroc x Landrace/Large White cross were analysed; their phenotypic characteristics are shown in Table 2 (Table 2 is shown further below).
In order to genotype these animals, a system was implemented based on the amplification of an 896-nucleotide fragment containing the A2456C mutation and the RT-PCR quantification, from this fragment, of the relative content of the two possible alleles. This strategy was chosen because the SNP does not cause the appearance of any new restriction site that would allow for analysis using the PCR-RFLP technique.
1 . Material and methods
1.1 Animals
The group of animals characterised was made up of a total of 202 pigs of both sexes (the males were castrated) from a Duroc x Landrace-Large White cross. These animals were raised in an experimental farm, fed "ad libitum" with a standard diet and sacrificed at 1 14.5 +/- 10.98 kg.
1.2 Determination of the PEPCK genotype The genomic DNA of each of these animals was extracted from a sample from the terminal end of the tail, taken a few days after birth, using the Real Pure kit for genomic DNA extraction (Durviz, Paterna, Valencia), according to the manufacturer's instructions. The genotype of 2456 A>C cytosolic PEPCK was determined by sequencing a fragment which was PCR-amplified with the PEPCK2F/3R pair of primers and subsequently purified as indicated in Example 1 , and/or by means of real-time PCR.
1.2.1 Determination of the PEPCK genotype by sequencing The amplification was performed in a Biometra UNO-Thermoblock (Gottingen, Germany) thermocycler, using AccuPrime™ Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, United States) and the PEPCK2F and PEPCK3R primers
[PEPCK2F:5'GTGTTGTCCAAGGGGGATCAGGAGGA-3' (SEQ ID NO: 4) and PEPCK3R: 5'-CCCCCTGCCTCGCACATTTAGGAT-3' (SEQ ID NO: 5)]. Each PCR reaction included 1 unit of AccuPrime™ Taq DNA Polymerase High Fidelity, 5 μΙ of the 10x buffer II, 0.5 μΙ of a 20 pmol/μΙ solution of PEPCKF2, 0.5 μΙ of a 20 pmol/μΙ solution of PEPCKR3, 3.8 μΙ of DMSO (Sigma-Aldrich, Saint Louis, United States), 2 μΙ of genomic DNA (100 ng/μΙ) and the necessary quantity of Milli-Q water to adjust the total reaction volume to 50 μΙ. The temperature programme used includes a denaturation step at 94°C for 2 minutes, followed by 35 denaturation cycles at 94°C for 30 seconds, hybridisation for 30 seconds at 55°C, extension at 68°C for 1 minute 15 seconds, and a final extension step at 68°C for 10 minutes.
Subsequently, the PCR product was purified using the NucleoSpinR Extract II kit (Macherey-Nagel, Duren, Germany), following the manufacturer's instructions. The purified product was eluted with 25 μΙ of the elution buffer in the kit and sent to Sistemas Genomicos (Paterna, Valencia, Spain) to be sequenced with the PEPCKF2 primer; subsequently, the nucleotide, or nucleotides, present at the position where the mutation is located were identified in the chromatogram obtained.
12.2 Determination of the PEPCK genotype by means of real-time PCR
The probes and the primers used in this assay were designed by means of the Assay by Design method from Applied Biosystems. The reactions were performed in an ABI-PRISM 7000 thermocycler (Amersham Biosciences, United States) and each assay included 12.5 μΙ of Taqman Universal Master Mix, 0.6 μΙ of 40x Assay Mix, 5 μΙ of a 2 ng/μΙ solution of genomic DNA or a 10 ng/μΙ solution of the fragment that was PCR-amplified with the PEPCKF2/3R pair of primers and subsequently purified, and 6.875 μΙ of Milli- Q water.
The primers used were the following: SSPEPCKE4-F (5' CGCTGCCCCCTGCT 3' (SEQ ID NO: 6)) and SSPEPCKE4-R (5' CCCATGCTGAACGGGATGA 3' (SEQ ID NO: 7)), and the probes were: SSPEPCKE4-V (5' AGGTCGCACGCTGTAT 3' (SEQ ID NO: 8)), labelled with the fluorochrome VIC, and SSPEPCKE4-M (5' AGGTCGCACGCTGTAT 3' (SEQ ID NO: 9)), labelled with the fluorochrome FAM (the specific nucleotide to detect each allele of the polymorphism is highlighted in bold italics). The temperature programme used includes an initial denaturation step at 95°C for 10 minutes, followed by 50 cycles at 92°C for 15 seconds and 1 minute at 61°C.
In each series of real-time PCR assays, two blanks, without DNA but with the rest of the components in the mixture, and three controls for each of the three possible genotypes were introduced.
1.3. Phenotypic characterisation 1.3.1 Carcass processing and conformation
The animals were sacrificed at a local slaughterhouse close to their breeding area, after being made unconscious with CO2. The carcass was allowed to cool for 6-8 hours. The back fat thickness was measured with a caliper at the level of the fifth lumbar vertebra. The right half of the carcass was divided into 10 parts: ham, loin, sirloin, shoulder, blade, streaky bacon, lard, ribs, cheek and trimmings, which were individually weighed. In turn, the hams were dissected into three other parts: bone, subcutaneous adipose tissue with skin and muscle mass (including the intermuscular fat deposits), which were also individually weighed. 1.3.2 Determination of the fat content
1.3.2.1 Preparation of the samples
In order to determine the intramuscular fat content in the Biceps femoris (B. femoris), Psoas major (Ps. major) and Longissimus dorsi (L-dorsi), a sample, weighing between 30 and 60 grams, was taken from the corresponding muscle, completely free from adipose tissue, and homogenised with a household meat mincer. In order to determine the mean fat content in the ham, the boneless ham, free from skin or adipose tissue, was homogenised with a mincer or "cutter" (Mainca CM-41 , Barcelona, Spain).
1.3.2.2 Analysis of the fat content
The lipids in a 10-gram sample of the preceding homogenates were extracted using the Bligh and Dyer method (1959) modified by Hanson and Olley (1963). A 5-ml aliquot was taken from the chloroform phase and dried under a nitrogen stream. The fat content was calculated in a gravimetric manner.
The analysis of the fat content in the subcutaneous fat samples (from the ham, in the region where the "V" cut is made) was performed in an analogous manner using a 0.5-g sample.
13.3 Quality of the meat Four measurements were taken of the quality of the meat:
1.3.3.1 H45. pH of the Longissimus dorsi at the level of the second lumbar vertebra at 45 minutes post-mortem. This measurement, as well as the following one, was made with an Oakton PC300 portable pH meter (Vernon Hills, Illinois, EEUU) equipped with a Foodtrode puncture electrode (Hamilton, Bonaduz, Switzerland).
13.3.2 H24. pH of the Longissimus dorsi at the level of the second lumbar vertebra at 24 hours post-mortem.
13.3.3 Exudation: The exudation was determined by calculating the weight loss undergone by a loin fillet, free from the subcutaneous fat layer and weighing about 70 g, after 2, 4 and 7 days. In order to take this measurement and the following one, 8 loin fillets were cut from the region located between the second lumbar vertebra and the twelfth thoracic vertebra. The four closest to L2 were used for this measurement and the next four were used to determine the cook losses. 1.3.3.4 Cook losses: The exudation was determined by calculating the weight loss undergone by a loin fillet, free from the subcutaneous fat layer and weighing about 70 g, which was cut after being subjected to a thermal treatment at 75°C for 20 minutes. 2. Results and discussion
The result of the implementation of the RT-PCR method:
1 ) RT-PCR did not work with genomic DNA (Figure 3 samples marked with an "x"). There is no increase in fluorescence in the genomic DNA samples, most likely because the fluorescent probes hybridise with other areas of the genome. 2) A PCR performed using genomic DNA made it possible to amplify an 896-nucleotide fragment containing A2456C (Figure 4).
3) The RT-PCR that failed using genomic DNA did work with the fragment amplified in point 2 (see the samples marked with diamonds, squares or triangles in Figure 3).
The results of the genotyping obtained by sequencing agreed in 100% of the cases with those obtained by means of RT-PCR. The allele distribution found in the 202 animals is shown in Table 4 of Example 4, where it may be observed that the three possible genotypes are represented in the population and that the frequency of both alleles makes it possible to perform an association study with the phenotypic traits of interest. Association study between A2465C and the traits of interest
A Bayesian analysis was performed to study the association between the different genetic configurations of A2465C and the phenotypic variability in the traits of interest.
The analysis model was:
y = + si + Lj + bgGk + + uk + e
where y k is the phenotypic record of the trait of interest, μ is the general mean, S, is the sex effect (2 levels), Lj is the batch effect (four levels), Gk is the number of A alleles in individual k (0, 1 or 2), Ak is the age of individual k at the time of sacrifice, Uk is the polygenic genetic value for individual k, e k is the residue, and bg and ba are the co-variables with the number of A alleles and the age, respectively. Uniform a priori distributions were assumed for the systematic effects, and variance components and a multivariate normal distribution were assumed for the polygenic effects:
u ~ N(0,Aaa 2) Where A is the numerator relationship matrix (Wright, 1922) and *7"3 is the polygenic additive variance. In order to construct the numerator relationship matrix, a genealogy of 243 individuals was used. The analysis was performed by means of a Gibbs sampler (Gelfand and Smith, 1990, J. Amer. Statist. Assoc. 85, 398-409), with a single chain of 125,000 interactions after discarding the first 25,000.
The inference regarding the association between the genetic configuration for A2465C and the traits of interest is focused on the posterior distribution for the bg parameter, which represents the substitution effect. The posterior mean and posterior standard deviation results, and the posterior probability above zero, are shown in Table 2.
Table 2. Mean values of the parameters for carcass conformation, composition and quality of the meat, and effects of the A2465C substitution thereon.
Substitution effect
Mean value (standard
Parameter (posterior standard 1P deviation)
deviation)
Back fat thickness
19.90 (5.73) 1.078 (0.5475) 0.0244 (mm)
Carcass yield (%) 78.05 (3.22) 0.587 (0.3560) 0.0496
Yield after dripping
76.35 (3.14) 0.813 (0.3590) 0.01 17 (%)
Yield of the noble
43.35 (4.98) -0.2490 (0.2020) 0.8912 parts (%)
% Loin 5.73 (0.67) -0.048 (0.077) 0.7334
% Sirloin 0.77 (0.10) 0.0124 (0.0108) 0.1254
% Ham 25.86 (1.26) -0.1484 (0.1281 ) 0.8767
% Shoulder 15.19 (0.80) -0.1228 (0.0844) 0.9271 % Streaky bacon 6.62 (1.22) -0.0065 (0.1223) 0.5210
% lard 12.23 (1.85) 0.2591 (0.2029) 0.1008
% lard in the leg 19.22 (3.58) 0.3959 (0.3806) 0.1491
% Muscle in the leg 64.93 (3.48) -0.5000 (0.3616) 0.0833
% Bone in the leg 15.85 (1.97) 0.0638 (0.2176) 0.3846 pH45 6.35 (0.03) 0.0061 (0.0293) 0.4175 pH24 5.64 (0.02) -0.0242 (0.0186) 0.9034
% Exudation at 2
4.30 (1.89) 0.5077 (0.2127) 0.0085 days
% Exudation at 4
6.51 (2.37) 0.7809 (0.2832) 0.0029 days
% Exudation at 7
8.12 (2.47) 0.7692 (0.3130) 0.0070 days
% Cook losses 33.90 (2.85) -0.0344 (0.021 1 ) 0.9485
% 2IMF loin 2.66 (0.95) -0.2620 (0.0949) 0.9971
% 2IMF sirloin 1.96 (0.44) -0.1187 (0.0524) 0.9883
% 2IMF B. femoris 2.95 (0.86) -0.0191 (0.0986) 0.5768
% total fat in the
77.80 (5.90) 0.1943 (0.5800) 0.3688 adipose tissue
% total fat in the
8.35 (1.54) -0.0674 (0.1465) 0.6773 leg
P = Posterior Bayesian probability above zero
2IMF = Intramuscular fat
The traits in Table 2 are of three types: 1 ) related to the conformation, including the carcass yields (back fat thickness, % of noble parts, % of various pieces, conformation of the leg, loin area); 2) related to the quality of the meat (pH45, pH24, exudation, cook losses); and 3) related to the fat content (intramuscular fat at the L-dorsi (loin), Ps. Major (sirloin), B. femoris; fat content in the subcutaneous adipose tissue; total fat content in the leg once the subcutaneous adipose tissue has been removed). In a Bayesian analysis, the especially relevant traits are not only those that have posterior Bayesian probability values above zero, close to 0 or to 1 , but also those with a direct relationship between them. For example, in Table 3, it may be observed that the three exudation traits have a posterior Bayesian probability very close to 0, which indicates that the effect found (which ranges between 1 .015 and 1.54 percentage points more exudation at 2 and at 7 days, respectively) is a real effect and not an artifact. Since the mean percentage of exudation in the population studied is 4.30% and 8.12% at 2 and at 7 days, respectively, a homozygous AA animal presents a meat that is between 19% and 24% less exudative than that of a homozygous CC animal. This phenomenon may well be related to the pH24 values of the meat, for which a quite high posterior Bayesian probability was also found (0.9034). It is well-known that high pH24 values lead to lower exudation values. This connection between the results for the exudation values and the pH24 values add even more coherence to the set of results in Table 3.
The back fat thickness value also has a significant posterior Bayesian probability above zero (0.0224). The effect attributable to each A allele on this trait would be 1 .078 mm, which means that a homozygous AA individual has 2.16 mm less back fat thickness than a homozygous CC individual. Since the mean value for this trait in the population is 19.90 mm, a homozygous AA individual has a back fat thickness that is 10.9% smaller than that of a homozygous CC individual. On the other hand, there are two fat content values, the intramuscular fat content in the L-dorsi and in the B. femoris, which show posterior Bayesian probability values very close to 1 . This is especially so in the case of the intramuscular fat value in the L-dorsi (P = 0.9971 ). The different sign of the estimated substitution effect as compared to the case of back fat thickness indicates that individuals with A alleles have higher intramuscular fat content than individuals with C alleles. The calculation of the estimated difference between a homozygous AA individual and a homozygous CC individual would be: 0.262 x 2 = 0.524 percentage points, which, taking into consideration that the mean value for this trait in the population studied is 2.66%, means 19.6% more intramuscular fat in the L-dorsi in a homozygous AA individual.
3. Conclusions The A allele of PEPCK is simultaneously associated with lower back fat values and higher intramuscular fat content values. Moreover, it is associated with meats that have a lower percentage of exudation and higher pH24 values. Therefore, the selection of homozygous AA individuals makes it possible to improve the quality of the meat, since it undergoes less exudation and has more intramuscular fat, and improves the carcass conformation, since it comprises lower back fat thicknesses.
Example 3
Myoglobin content and colour Introduction
A very important characteristic of meat and meat products is the colour. The colour is the first meat characteristic perceived by consumers and, for this reason, it is a key to their decision to purchase it. One of the most important mechanisms that modulate the colour of meat is the content of its main pigment, myoglobin. Myoglobin is an intracellular pigment that contains a haem group with an iron atom. In order to evaluate the influence of A2456C in PEPCK on the myoglobin content and the colour, it was decided to analyse both traits in a population of 60 animals that were very homogeneous in terms of weight (90.7 +/- 4.9 kg).
1 . Materials and methods The myoglobin and haem iron content in the Biceps femoris muscle was quantified using Hornsey's method (1956, Journal of the Sciences of Food and Agriculture 7, 534-541 ) modified by Gorospe et al. (1986, Alimentaria, 25-32). The instrumental measurement of colour was performed using a Minolta Chromameter CR-200 tri-stimulus colorimeter with illuminant D65. The measurements, in triplicate, were taken in the CIELAB colour space on the Biceps femoris muscle. The colour is expressed by means of the L* chromaticity coordinates, or lightness, a*, the red-green index, and b*, the yellow-blue index.
2. Results and discussion
Table 3. Mean values of the colour-related parameters and the effects of the A2465C substitution on said parameters.
Figure imgf000045_0001
1 P: Posterior Bayesian probability above zero.
As shown in Table 3, it is found very high posterior Bayesian probabilities for two traits that are very closely related, the "a" colour coordinate, or red-green index, and the myoglobin content. The magnitude of the effect of an allele on the latter parameter would be 0.2723 mg/g, which would cause a homozygous AA animal to have 0.54 mg/g more myoglobin than a CC animal, and, since the mean myoglobin content in this group of animals is 1 .84 mg/g, this means that homozygous AA animals have 30% more myoglobin than homozygous CC animals. This difference in the myoglobin content is at least partially responsible for the difference in colour detected in the "a" value.
3. Conclusions
The meat from AA animals has 30% more myoglobin and haem iron than that from CC animals. Moreover, the instrumental colour analysis reveals that the meat from AA animals is redder. Both characteristics are commonly perceived as favourable by consumers, which makes the selection of AA pigs very interesting
Example 4
Allele frequencies of the A and C alleles of PEPCK
Introduction
The percentage of improvement in a given trait in a population by the selection of carrier animals for the alleles that modulate said trait in a given sense is dependent, in addition to on the magnitude of the effect associated with a given allele, on the initial proportion of animals in said population that are already carriers of the favourable alleles. Therefore, the potential percentage of improvement of a population will be greater the lower the frequency of said allele prior to starting the improvement process of selecting carrier animals for a given allele. In order to investigate the current allele frequency of the 2465A and 2465C alleles of PEPCK, and thus be able to estimate the improvement potential, a number of animals from different breeds and crosses were genotyped using the method described in Example 2.
1 . Material and methods 1.1 Animals
The porcine crosses and breeds that were genotyped are shown in Table 4. A total of 13 wild boars were also genotyped.
Table 4. PEPCK genotypes of the animals analysed; allele frequencies (p) of the A allele, and allele frequencies (p) of C allele.
AA AC CC p (A allele) P (C allele)
Wild boar 10 3 0 0.88 0.12
Iberian 18 8 0 0.85 0.15
Duroc line 1 18 8 1 0.81 0.19
Duroc line 2 17 42 16 0.51 0.41
Duroc x Landrace-Large White 73 1 14 15 0.64 0.36
Pietrain x Large White 12 68 53 0.35 0.65
Pietrain 2 26 21 0.31 0.69
1.2 DNA extraction
The genomic DNA was extracted from muscle, hair, tail or semen using the Real Pure kit for genomic DNA extraction (Durviz, Paterna, Valencia), according to the manufacturer's instructions. 1.3 Determination of the PEPCK genotype by means of real-time PCR
The genotype of cytosolic PEPCK at position 2456 was also determined by means of real-time PCR. The probes and the primers used in this assay were designed using the Assay by Design method from Applied Biosystems. The reactions were performed in an ABI-PRISM 7000 thermocycler (Amersham Biosciences, United States) and each assay included 12.5 μΙ of Taqman Universal Master Mix, 0.6 μΙ of 40x Assay Mix, 5 μΙ of a 10 ng/μΙ solution of the fragment that was PCR-amplified with the PEPCKF2/3R pair of primers and subsequently purified, and 6.875 μΙ of Milli-Q water. The primers used were the following: SSPEPCKE4-F (5' CGCTGCCCCCTGCT 3' (SEQ ID NO: 6)) and SSPEPCKE4-R (5' CCCATGCTGAACGGGATGA 3' (SEQ ID NO: 7)), and the probes were: SSPEPCKE4-V (5' AGGTCGCACG4TGTAT 3' (SEQ ID NO: 8)), labelled with fluorochrome VIC, and SSPEPCKE4-M (5' AGGTCGCACGCTGTAT 3' (SEQ ID NO: 9)), labelled with fluorochrome FAM (the specific nucleotide to detect each allele of the polymorphism is highlighted with bold italics). The temperature programme used includes an initial denaturation step at 95°C for 10 minutes, followed by 50 cycles at 92°C for 15 seconds and 1 minute at 61°C.
In each series of real-time PCR assays, two blanks were introduced, without DNA but with the rest of the components in the mixture.
2. Results and discussion
The genotype of the animals from the different breeds and crosses, and the frequency of the A allele in these animals are shown in Table 4.
The results in Table 4 show that, particularly in modern breeds or crosses, there is considerable potential for improvement, since the percentage of favourable alleles (the A allele) ranges between 81 %, in a Duroc-breed line, and 31 % in the Pietrain breed. Even in the Iberian breed it was found that the frequency of the A allele is not 100%, but somewhat lower (85%), which means that there is certain room for selection in this breed.
3. Conclusions The A2456C mutation may be a very useful instrument for improvement in very diverse porcine populations. Example 5
The polymorphism A2456C in pig's cvtosolic PEPCK (PEPCK-C) changes the kinetic properties thereof and modifies the fat distribution in the pig
1 . Material and Methods
1.1 Animal material
202 pigs of both sexes (the males were castrated) from 9 sires and 32 dams of a Duroc x Landrace/Large White cross were raised in an experimental farm, fed ad libitum a standard diet and slaughtered after stunning with CO2 at an average weight of 1 14.5 +/-10.98 kg.
1.2 Phenotypic recording
Backfat thickness was measured at L5 (lumbar vertebra 5) level with a caliper shortly after slaughtering. pH45 (pH 45 minutes postmortem) and pH24 (pH 24 hours postmortem) were measured in L.dorsi at L2 level with a PC3000 Oakton pHmeter equipped with a Hamilton penetration electrode (Bonaduz, Switzerland). Drip loss was measured in quadruplicate in L.dorsi samples taken between L2 and T14 (thoracic vertebra 14) as described by Honikel (1998). Fat content was analyzed from 10 g samples taken form L.dorsi at T12 level. Lipids were extracted by the method of Bligh & Dyer (1959) as modified by Hanson & Olley (1963). Fat content was calculated gravimetrically from an aliquot of 5 ml of the chlorophormic phase.
1.3 Cloning and sequencing of pig PEPCK-C. 1.3.1 DNA extraction and quantification
DNA was isolated from 27 pig hair follicle samples using Real Pure genomic DNA extraction kit (Durviz, Paterna, Spain) following manufacturer's instructions. Proteinase K (20 mg/ml) was used at 55°C for 5 hours. DNA concentration was determined spectrophotometncally using NanoDrop® 1000 (Thermo Fischer Scientific, USA). 3.2 DNA Sequencing.
The PEPCK-C coding region was amplified by PCR using the following primers:
PEPCK1 F/2R (SEQ ID NO: 10/SEQ ID NO: 1 1 ): PEPCK2F/3R (SEQ ID NO: 4/SEQ ID NO: 5), PEPCK3F/4R (SEQ ID NO: 12/SEQ ID NO: 13), PEPCK4F/5R SEQ ID NO: 14/SEQ ID NO: 15) and PEPCK5F/8R SEQ ID NO: 16/SEQ ID NO: 17).
Each reaction was performed in Biometra UNO-Thermoblock thermocycler (Gottingen, Germany) using 1 unit of AccuPrime™ Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, USA), 5 μΙ buffer II 10x, 0.5 μΙ forward primer (20 pmol/μΙ), 0.5 μΙ reverse primer (20 pmol/μΙ), 3.8 μΙ DMSO (Sigma- Aldrich, San Louis, USA), 2 μΙ genomic DNA (100 ng/μΙ) and milliQ water up to 50 μΙ. Temperature steps consisted of an initial denaturation at 94°C for 2 minutes followed by 35 cycles: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds (primers PEPCK 1 F/2R, PEPCK2F/3R, PEPCK3F/4R, PEPCK5F/8R) or 53°C for 30 seconds (primer PEPCK4F/R5) and extension at 68°C for 90 seconds (PEPCK1 F/2R), for 75 seconds (PEPCK2F/3R and PEPCK3F/4R) and for 60 seconds (PEPCK 4F/5R and PEPCK5F/8R). A final elongation step was carried out at 68°C for 10 minutes. Fragments were analyzed by 2 % agarose electrophoresis at 100 V for 30 minutes. The PCR products were purified using NucleoSpin® Extract II kit (Macherey-Nagel, Duren, Germany) following manufacturer's instructions. DNA concentration was determined using a NanoDrop® 1000 and samples were sequenced (Sistemas Genomicos, Spain). Fragment assembly was carried out using Vector NTI for Mac (Life Technologies Corporation, USA). Sequence alignment was performed using ClustalW. 3.3 PEPCK-C cloning
Total cDNA was obtained from mRNA following the instructions from the Cells to cDNA kit (Ambion, USA). PEPCK-C cDNA was amplified by PCR using PEPCK1 F (SEQ ID NO: 10) and PEPCK1 R [5'- AACGTGGGCTGTGCTCATTGCGGTG -3' (SEQ ID NO: 20)] as primers. Each reaction consisted of 1 unit of AccuPrime™ Taq DNA Polymerase High Fidelity, 2.5 μΙ buffer II 10x, 0.25 μΙ forward primer (20 pmol/μΙ), 0.25 μΙ reverse primer (20 pmol/μΙ), 6.25 μΙ DMSO, 2 μΙ cDNA (100 ng/μΙ) and milliQ water up to 25 μΙ. PCR was carried out by an initial denaturation step at 94°C for 2 minutes followed by 35 cycles: denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds and extension at 68°C for 130 seconds. Final extension was performed at 68°C for 10 minutes. 2% agarose electrophoresis was carried out at 100 V for 30 minutes to analyze the amplified PEPCK-C fragment. Afterwards, nested PCR was performed using the following forward and reverse primers, respectively:
PEPCKsusFNde:
5'-CTAGGATCCATATGCCTCCTCAGCTCTCAAACGGCC -3' (SEQ ID NO: 18).
PEPCKsusRNot:
5'-CTAGGATCGCGGCCGCTCACATCTGGCTGATTCTCTGCTTC-3' (SEQ ID NO: 19).
Reaction mix was: 1 unit of AccuPrime™ Taq DNA Polymerase High Fidelity, 2.5 μΙ buffer II 10x, 0.25 μΙ forward primer (20 pmol/μΙ), 0.25 μΙ reverse primer (20 pmol/μΙ), 1 .25 μΙ DMSO, 2 μΙ amplified cDNA (100 ng/μΙ) and milliQ water up to 25 μΙ. Nested PCR steps were carried out by an initial denaturation at 94°C for 2 minutes and 35 cycles: denaturation at 94°C for 30 seconds, annealing at 63°C for 30 seconds and extension at 68°C for 130 seconds. Final extension was performed at 68°C for 10 minutes. Fragments were also electrophoretically analyzed. PEPCK-C fragments were inserted into a pET28a(+) plasmid by digestion with Ndel and Notl (New England Biolabs) and ligation with T4 DNA ligase (New England Biolabs). E.coli DH5a was transformed by heat shock, colonies were selected by growth in LB-agar with 30 μg/ml kanamycin, DNA was isolated using NucleoSpin Plasmid Quickpure (Macherey-Nagel, Duren, Germany) and sequenced. 1.4 Genotyping
Detection of polymorphism at position 2456 of the PEPCK-C gene was performed by Real Time (RT)-PCR using a DNA fragment previously amplified by standard PCR. Direct use of RT-PCR on genomic DNA failed to detect the A2456C substitution (data not shown). The experimental approach was designed using Assay by Design (Applied Biosystems, USA). Reactions were carried out using an ABI-PRISM 7000 thermocycler (Amersham Biosciences, USA). RT-PCR steps consisted on an initial denaturation at 95°C for 10 minutes followed by 50 cycles at 92°C for 15 seconds and 61 °C for 60 seconds. Each assay included 12.5 μΙ Taqman Universal Master Mix (Applied Biosystems, USA), 0.6 μΙ 40x Assay Mix, 5 μΙ DNA (previously amplified by PCR using primers PEPCKF2/3R and purified using NucleoSpin® Extract II kit). Blanks included all the components but DNA. SSPEPCKE4-F (SEQ ID NO: 6) and SSPEPCKE4-R (SEQ ID NO: 7) were used as primers.
Taqman probes were SSPEPCKE4-V (SEQ ID NO: 8), containing the VIC fluorochrome, and SSPEPCKE4-M (SEQ ID NO: 9), containing the FAM fluorochrome.
1.5 Protein expression and purification
E. coli BL21 (DE3) were transformed with the appropriate vectors and two colonies for each of the constructs were selected for protein expression. Expression of proteins of the expected size was first assessed in a small scale protein expression experiment, growing bacteria in LB medium supplemented with 30 μ9/ιτιΙ kanamycin at 37°C to an OD6oo of 0.5 and then inducing protein expression with 1 mM IPTG at 37°C for 3 hours. Analyses of clarified lysates after sonication in the presence of lysozyme by SDS-PAGE indicated that part of the desired protein was present in the pellet, suggesting aggregation into inclusion bodies. Therefore, lower induction temperatures were used for large scale preparations.
Large scale expression was carried out by growing a 20 ml preculture overnight which was then used to inoculate 2 L of LB medium supplemented with kanamycin. Bacteria were grown to an OD6oo of 0.5, the culture was cooled to 22°C, IPTG was added to 1 mM final concentration and incubation continued at 22°C for 24 hours.
Bacteria were harversted by centrifugation at 8000 g for 10 minutes, resuspended in 12 ml of lysis buffer (20 mM sodium phosphate, pH 7.2, 10 mM DTT, 1 mg lysozyme) and sonicated with a Vibra-Cell sonicator (Sonics & Materials, Newtown, CT, USA) using 10 cycles (10 seconds on, 45 seconds off) at 50% power, on ice. The lysate was clarified by centrifugation at 18,000 g for 20 minutes at 4°C and the pellet was discarded.
Protein purification was carried out by affinity chromatography on a 5 ml FF crude HisTrap Column (GE Healthcare Life Sciences, Piscataway, NJ, USA), using an UPC-900 (GE Healthcare Life Sciences, Piscataway, NJ, USA) HPLC apparatus with UNICORN Manager software. The column was first equilibrated with buffer A (50 mM NaH2PO4, pH 7.2, 300 mM NaCI, 10 mM imidazole, 10 mM DTT). After sample loading the column was washed with buffer A until absorbance at 280 nm reached the baseline. The protein was then eluted with a 60 ml linear gradient at 5 ml/min from 100% buffer A to 100 % buffer B (same as buffer A but with 500 mM instead of 10 mM imidazole) and 5 ml fractions were collected.
Fractions were analyzed by SDS-PAGE and those containing PEPCK-C were pooled and concentrated, using Amicon® Ultra Centrifugal Filters 30kDa (Millipore, Tullagreen, Ireland). After reducing the sample volume by centrifugation at 4000 g from 10 ml to 1 ml, 9 ml of 20 mM HEPES, pH 7.2, 10 mM DTT was added and centrifuged again. This step was repeated 6 times for buffer exchange and imidazole elimination.
Protein concentration was calculated by absorbance determination at 280 nm and by Bradford assays. 1.6 Enzyme assays and kinetic calculations
PEPCK activity was measured with coupled spectrophotometric assays in both reaction directions, as described by Johnson and Holyoak, (2010), using an UNICAM 500 spectralphotometer (Cambridge, UK). Assays in the direction of oxaloacetic acid synthesis were performed in 1 ml of a 100 mM HEPES buffer, pH 7.2, containing 10 mM DTT, 0.2 mM MnCI2, 2 mM MgCI2, 2 mM GDP, 0.2 mM NADH, 2 mM PEP, 100 mM KHC03 and 2 units of malate dehydrogenase. Assays were initiated by PEPCK addition. Rates were calculated by measuring, from the initial slope of the graph, the decrease in absorbance at 340 nm after substracting the rate of spontaneous NADH oxidation. Assays in the direction of phosphoenolpyruvate synthesis were performed in 1 ml of a 100 mM HEPES buffer, pH 7.2, containing 10 mM DTT, 0.2 mM MnCI2, 2 mM MgCI2, 1 mM GTP, 1 mM ADP, 0.2 mM NADH, PEPCK, and 5 units of each pyruvate kinase and lactate dehydrogenase. Reactions were initiated by addition of oxaloacetate (OAA). Rates were calculated by measuring, from the initial slope of the graph, the decrease in absorbance at 340 nm after substracting the rate of the blank which contained all the components of the mix but PEPCK. Apparent values of Km, kcat and Km/kcat were calculated in triplicate in independent sets of assays using a non linear regression software (Leatherbarrow, 1987. Enzfitter; Biosoft, Cambridge UK). In these assays, saturating substrate concentrations (2 mM PEP, 2 mM GDP, 100 mM KHC03,1 mM GTP or 400 μΜ OAA) were used except, logically, for the variable substrate.
1.7 Sensitivity to proteolytic cleavage Purified PEPCK-C samples were submitted to proteolytic degradation using trypsin (Sigma) or Proteinase K (Durviz, Paterna, Spain). Reactions were performed at 39°C; aliquots were taken after different timepoints, mixed with standard SDS sample buffer and immediately boiled for 5 minutes. These samples were analyzed using SDS-PAGE in 12% polyacrylamide gels. Band quantification was performed with a densitometer Imagemaster "D Platinum 7 and software Image Scanner III both from GE Healthcare Life Sciences (Piscataway, NJ, USA).
1.8 Statistical analysis
Data were analyzed for each trait separately by using a Bayesian approach. The assumed Bayesian likelihood was:
Figure imgf000055_0001
Where y is the vector of phenotypic records for the analyzed trait, b is the vector of systematic effects, including covariates with age and the substitution effect of the analyzed polymorphism, a batch effect with four levels and a sex effect with two levels, p is the vector of litter effects and u recomprising the polygenic effects. Further, Xj,Wj and z, are the rows of the incidence matrices (X,W and Z) corresponding to the ith phenotypic record
2
and e is the residual variance.
Prior distributions for litter and polygenic effects were the following
Figure imgf000055_0002
multivariate Gaussian distributions (MVN): )~ MVN(0, lap ) gnc| P )- MVN(0, Ασα ) ^ σρ σα ^gjpg †ne y^er anc| polygenic additive variance, respectively. In addition, prior distributions for systematic effects and variance components were assumed uniform with appropriate bounds. The analysis was performed through a single long chain of 500,000 interations of a Gibbs Sampler (Gelfand and Smith, 1990) after discarding the first 25,000 with the TM program (Legarra et al., 2008). Later on, samples for the substitution effect were used to compute the posterior probabilities above zero.
2. Results
As shown in Examples 1 to 4, a SNP (A2456C) in pig's cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) results in a substitution of a methionine by a leucine in a highly conserved beta-strand located far away from the active site. The substitution has strong effects on the enzyme kinetic properties increasing its catalytic efficiency up to 20 fold. The C allele, which is present at high frequency in several modern breeds, is strongly associated to lower meat quality and lower intramuscular fat content, and to enhanced backfat thickness.
The analysis of the PEPCK-C gene in 202 animals from the same Du x LD/LW cross to evaluate its effects on fat distribution and meat quality traits was carried out. The genotypic frequencies in this cross were 0.361 AA, 0.565 AC and 0.074 CC. Further, a Bayesian analysis of association between the two alleles and the phenotypic traits that were changed in PEPCK-C transgenic mouse (BF and IMF) and also with some meat quality traits were perform. The results are shown in Table 5. Parameter Mean (SD) Substitution effect CC vs. AA >P
(PSD) difference (%)
Backfat thickness (mm) 19.88 (5.74) 0.9796 (0.5631) 9.86 0.0409 pH45 6.34 (0.23) 0.0028 (0.0292) 0.09 0.4618 pH24 5.64 (0.15) -0.0215 (0.0192) -0.68 0.8686
% Drip loss after 2 days 4.25 (1.89) 0.3821 (0.2189) 17.98 0.0404
% Drip loss after 4 days 6.54 (2.37) 0.6065 (0.2874) 18.54 0.0174
% Drip loss after 7 days 8.08 (2.47) 0.5819 (0.3130) 14.40 0.0315
% 2IMF L. dorsi 2.67 (0.95) -0.2729 (0.0980) -20.44 0.9973
% 2IMF Ps. major 1.96 (0.44) -0.1085 (0.0528) -11.07 0.9800
% 2IMF B. femoralis 2.95 (0.87) 0.0040 (0.0992) 0.27 0.4839
% Fat content in adipose 77.78 (5.90) 0.1631 (0.5973) 0.42 0.3924 tissue
'P = Bayesian posterior probability above zero.
2IMF = Intramuscular fat.
Table 5
The A allele is clearly associated to higher (up to 20.4%) IMF content in two of the three analyzed muscles (Bayesian posterior probability above cero ranging from 0.9973 in L. dorsi to 0.9800 in Ps. major) and also to reduced (9.9% lower) BT showing also a similar, although not as extreme, pattern than the transgenic PEPCK mouse. In addition, a strong association was found between higher water holding capacity at several time points postmortem and the A allele. This elevated (up to 24%) water holding capacity is probably related to the higher values of pH24 also associated to A alleles (Table 5).
In order to demonstrate if the detected polymorphism in PEPCK-C was causative and not merely a consequence of linkage disequilibrium with the potential causative mutation(s), both isoenzymes as His-tagged proteins (Figure 5) were purified and characterized. His-tagged PEPCK has been shown to behave almost identically to the non His-tagged enzyme. Apparent values of Km, kcat and kcat/Km were calculated for the five PEPCK-C substrates (Table 6 and Figures 6A-6C and 7A-7B). Km (μΜ)
Substrate p.139Leu p.139Met Difference1 ,2
PEP 1091 ±12 193.3±43 5.65*
c
0
KHCO3 1 1450±1 7406±1246 1.54*
294
GDP 19.3±1.6 68.1 ±6.9 0.28*
c
0
OAA 5.4±0.6 8.6±1.1 0.62
GTP 21 .6±3.9 38.4±5.9 0.56
Figure imgf000058_0001
Substrate p.139Leu p.139Met Difference1 ,2
PEP 8.21 ±0.35 1 .06±0.05 7.74***
KHCO3 8.93±0.32 1 .16±0.04 7 7Q***
GDP 4.97±0.10 0.84±0.04 5.92**
OAA 6.87±0.20 2.46±0.09 2
GTP 5.91 ±0.37 2.66±0.19 2 22**
Figure imgf000058_0002
Substrate p.139Leu p.139Met Difference1 ,2
PEP 7.5 x 103 5.5 x 103 1 .36
KHCO3 7.8 x 102 1 .6 x 102 4.88**
GDP 2.6 x 105 1 .2 x 104 21 .66**
OAA 1.3x 106 2.9x105 4.48**
GTP 2.7 x 105 6.9 x 104 3.91 * 1Fold difference between PEPCK-C-p.139Leu and PEPCK-C-p.139Met.
2Welch's two-sample Mest; * p<0.05, ** p<0.01 , *** p< 0.001.
Table 6. Apparent values of Michaelis kinetic constants of pig PEPCK-C isoenzymes
Both isoenzymes showed significant differences in almost all the calculated kinetic constants but the difference was especially strong in the catalytic efficiency for GDP (over 20 fold larger in PEPCK-C p.139Leu) a consequence of a higher / cat and a lower Km value. Besides these differences, it is noteworthy that PEPCK-C-p.139Met has between 2.3 and 3 fold larger / cat values in the direction of PEP synthesis than in the direction of OAA synthesis whereas in PEPCK-C p.139l_eu they are similar in both reaction directions.
To search for structural differences, the sensitivity to proteolytic degradation of both isoenzymes using, separately, trypsin or proteinase K were tested. Whereas no differences were found in hydrolysis degree using proteinase K digestion after several digestion times (data not shown), PEPCK-C p.139Met was hydrolyzed by trypsin much faster yielding a different band pattern than PEPCK-C p.139l_eu (Figure 8). These differences in susceptibility to trypsin hydrolysis suggest that both isoenzymes have a notably different tertiary structure. Since the tridimensional structure of the PEPCK-C from different species is well known in the state of the art, the localization of the mutation Met139l_eu was analyzed in said tridimensional structure. The substituted amino acid is located in a beta-strand far away from the active site of the enzyme (Figure 9) but in a region of about 20 amino acids that is highly conserved from man to C. elegans (Figure 10).
4. Discussion
The mutation detected in the PEPCK-C, the substitution of a metionin with a Leucine in the position 139 give raised to changes in its kinetic characteristics. These changes are provoked by a change in the global 3D structure of the enzyme, originating that both isoenzymes show a different susceptibility to the protein hydrolysis. Although the substitution is a conservative substitution (both the metionine and leucine are polar aliphatic amino acids of similar size) and is found in a beta-sheet far away from the active centre/site of the enzyme, the fact that the amino acid region around the position 139 shows a very high grade of conservation points out that said region is relevant for the functionality of the enzyme. Although the Kcats of the isoenzyme PEPCK-139l_eu are higher than the Kcats of the PEPCK-139Met for all the substrates, the latter shows, in opposite to the first, an improved capacity of catalysed the reaction toward the synthesis of PEP in comparison to its capacity of synthesizing Oxaloacetate (OAA) [three time higher). Since the glyceroneogenic capacity of the PEPCK depend exclusively on its capacity of providing PEP to be used as precursor of triglycerides synthesis, these kinetic results show that, under particular relative concentration of substrate, the PEPCK139Met has more glyceroneogenic capacity. This fact explains that the homozygote PEPCK139Met animals show higher content of intramuscular fat than the homozygote PEPCK139l_eu animals.

Claims

1 . A method for selecting a suid that comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a suid comprising a phenotype whose meat is at least 19% less exudative than a reference value, which comprises:
a) Detecting in a biological sample from said suid, an A C
polymorphism in the gene encoding the cytosolic PEPCK protein, and
b) Selecting that suid comprising homozygosis for the A allele.
2. Method according to claim 1 , wherein the gene encoding the cytosolic PEPCK protein comprises a nucleotide sequence which has at least a 70, 80,
90, 95, 98, 99 or 100% identity with SEQ ID NO: 1 .
3. Method according to claim 1 or 2, wherein the A/C polymorphism is located at a position equivalent to nucleotide 2456 of the SEQ ID NO: 1 determined by the optimal alignment of the sequences.
4. Method according to anyone of claims 1 to 3, wherein detection of the polymorphism is performed by means of a process selected from the group consisting of direct sequencing, allele-specific oligonucleotide (ASO) dot-blot hybridisation analysis, mononucleotide primer extension, PCR-based single- strand conformation polymorphism (SSCP) analysis, PCR-based restriction fragment length polymorphism (RFLP) analysis, real-time quantitative PCR and mass matrix using a mass spectrometer.
5. Method according to claim 4, wherein detection of the polymorphism is performed by amplification of the PEPCK gene using primers comprising a nucleotide sequence which has at least 60, 70, 90, 90, 95, 98, 99 or 100% identity to SEQ ID NO: 1 .
6. Method according to claim 5, wherein the primers comprise the nucleotide sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
7. Method according to anyone of claims 1 to 6, wherein the detection of the polymorphism is performed by means of a nucleic acid probe comprising a nucleotide sequence which has at least 60, 70, 90, 90, 95, 98, 99 or 100% identity to SEQ ID NO: 1 .
8. Method according to claim 7, wherein the nucleic acid probe comprises the nucleotide sequence SEQ ID NO: 8 or SEQ ID NO: 9.
9. Method according to anyone of claims 1 to 8, wherein the phenotype of the suid comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
10. Method according to claim 9, wherein the phenotype of the suid comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value.
1 1 . Method according to anyone of claims 1 to 10, wherein the phenotype of the suid comprises meat that is, at least, between 19% and 24% less exudative than a reference value.
12. Method according to anyone of claims 1 to 1 1 , wherein the suid is a domestic pig or a wild boar.
13. An isolated nucleotide sequence that encodes the cytosolic PEPCK protein, which comprises (i) a nucleotide sequence with at least 70% identity with SEQ ID NO: 1 , and (ii) the A C polymorphism at a position equivalent to nucleotide 2456 of said sequence SEQ ID NO: 1 by optimal alignment of the sequences.
14. Nucleotide sequence according to claim 13, which comprises a nucleotide sequence which has at least 80%, 90%, 95%, 98% or 99% or 100% identity with sequence SEQ ID NO: 1 .
15. An isolated polypeptide encoded by a nucleotide sequence according to claim 13 or 14.
16. Polypeptide according to claim 15, comprising an amino acid sequence with at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with SEQ ID NO: 2.
17. Polypeptide according to claim 15 or 16, comprising a methionine at a position equivalent to amino acid 139 of the SEQ ID NO: 2 by optimal aligment of the sequences.
18. A nucleic acid probe comprising a nucleotide sequence capable of hybridizing with SEQ ID NO: 1 in the region comprising the polymorphism A/C in the position 2456.
19. Probe according to claim 18, wherein the probe comprises a nucleotide sequence with at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with the nucleotide sequence SEQ ID NO: 1 .
20. Probe according to claim 19, wherein the probe comprises the nucleotide sequence SEQ ID NO: 8 or SEQ ID NO: 9.
21 . An oligonucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
22. A pair of specific primers designed to amplify the region of SEQ ID NO: 1 comprising the A/C polymorphism at position 2456 of said sequence SEQ ID NO: 1 .
23. Pair of primers according to claim 22, wherein the primers comprise a nucleotide sequence which has at least 60, 70, 80, 90, 95, 98, 99 or 100% identity with SEQ ID NO: 1 .
24. Pair of primers according to claim 23 wherein at least one of the primers comprises the nucleotide sequence SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, preferably, the pair of primers comprises the sequences SEQ ID NO: 4 and SEQ ID NO: 5, or the sequences SEQ ID NO: 6 and SEQ ID NO: 7.
25. The use of a probe according to anyone of claims 18 to 20, of a oligonucleotide according to claim 21 , and/or of a pair of primers according to anyone of claims 22 to 24, to detect the A/C polymorphism in the gene encoding the cytosolic PEPCK protein.
26. Use according to claim 25, wherein the gene encoding the cytosolic PEPCK protein comprises a nucleotide sequence which has at least 70, 80,
90, 95, 98, 99 or 100% identity with the nucleotide sequence SEQ ID NO: 1.
27. Use according to claim 25 or 26, wherein the A C polymorphism is located at a position equivalent to nucleotide 2456 of the SEQ ID NO: 1 determined by the optimal alignment of the sequences.
28. The use of a nucleotide sequence according to claim 13 or 14, of a polypeptide according to anyone of claims 15 to 17, of a probe according to anyone of claims 18 to 20, of an oligonucleotide according to claim 21 , and/or of a pair of primers according to anyone of claims 22 to 24, to select a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value and/or a suid comprising a phenotype whose meat is at least 19% less exudative than a reference value.
29. Use according to claim 28, wherein the phenotype of the suid comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
30. Use according to claim 29, wherein the phenotype of the suid comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value.
31 . Use according to any of claims 28 to 30, wherein the phenotype of the suid comprises a meat with, at least, between 19% and 24% less exudation than a reference value.
32. The use of a nucleotide sequence according to claim 16 or 17, of a polypeptide according to anyone of claims 15 to 17, of a probe according to anyone of claims 18 to 20, of an oligonucleotide according to claim 21 , and/or of a pair of primers according to anyone of claims 22 to 24, to select a meat product comprising higher intramuscular fat content and lower back fat content than a reference value, at least 30% more myoglobin than a reference value and/or at least 19% less exudation than a reference value.
33. Use according to claim 32, wherein the meat product comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
34. Use according to claim 33, wherein the meat product comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value.
35. Use according to anyone of claims 32 to 34, wherein the meat product comprises, at least, between 19% and 24% less exudation than a reference value.
36. A kit comprising a probe according to anyone of claims 18 to 20, an oligonucleotide according to claim 21 , and/or a pair of primers according to anyone of claims 22 to 24.
37. Use of a kit according to claim 36, to detect the A C polymorphism in the gene encoding the cytosolic PEPCK protein.
38. Use according to claim 37, wherein the gene encoding the cytosolic PEPCK protein comprises a nucleotide sequence which has at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identity with SEQ ID NO: 1 .
39. Use according to claim 37 or 38, wherein the A C polymorphism is located at a position equivalent to nucleotide 2456 of the SEQ ID NO: 1 determined by the optimal alignment of the sequences.
40. The use of a kit according to claim 36, to select a suid comprising a phenotype with higher intramuscular fat content and lower back fat content than a reference value, and/or a suid comprising a phenotype with at least 30% more myoglobin in the muscle than a reference value, and/or a suid comprising a phenotype whose meat is at least 19% less exudative than a reference value.
41 . Use according to claim 40, wherein the phenotype of the suid comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
42. Use according to claim 41 , wherein the phenotype of the suid comprising, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value.
43. Use according to anyone of claims 40 to 42, wherein the phenotype of the suid comprises a meat with, at least, between 19% and 24% less exudation than a reference value.
44. Use of a kit according to claim 36, to select a meat product comprising higher intramuscular fat content and lower back fat content than a reference value, at least 30% more myoglobin than a reference value, and/or at least 19% less exudation than a reference value.
45. Use according to claim 44, wherein the meat product comprises, at least, between 19% and 25% more intramuscular fat and, at least, between 9% and 15% less back fat than a reference value.
46. Use according to claim 45, wherein the meat product comprises, at least, 20% more intramuscular fat and, at least, 1 1 % less back fat than a reference value.
47. Use according to anyone of claims 44 to 46, wherein the meat product comprises, at least, between 19% and 24% less exudation than a reference value.
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