US20060037090A1 - Selecting animals for desired genotypic or potential phenotypic properties - Google Patents

Selecting animals for desired genotypic or potential phenotypic properties Download PDF

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US20060037090A1
US20060037090A1 US11/177,498 US17749805A US2006037090A1 US 20060037090 A1 US20060037090 A1 US 20060037090A1 US 17749805 A US17749805 A US 17749805A US 2006037090 A1 US2006037090 A1 US 2006037090A1
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igf2
seq
gene
qtl
binding
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Leif Andersson
Goran Andersson
Michel Georges
Nadine Buys
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LEIGE UNIVERSITY OF
Universite de Liege
Gentec BV
Melica
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Universite de Liege
Gentec BV
Melica
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Priority to US11/357,718 priority Critical patent/US7732137B2/en
Assigned to GENTEC B.V., UNIVERSITY OF LIEGE, MELICA HB reassignment GENTEC B.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GEORGES, MICHEL, ANDERSSON, GORAN, ANDERSSON, LEIF, BUYS, NADINE
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
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    • 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
    • C12Q2600/154—Methylation markers
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the invention relates to methods to select animals, such as mammals, in particular, domestic animals such as breeding animals or animals destined for slaughter for having desired genotypic or potential phenotypic properties, in particular, related to muscle mass and/or fat deposition or, in the case of mammals, to teat number.
  • a domestic animal is defined as an animal being purposely selected or having been derived from an animal having been purposely selected for having desired genotypic or potential phenotypic properties.
  • domestic animals provide a rich resource of genetic and phenotypic variation. Traditionally, domestication involves selecting an animal or its offspring for having desired genotypic or potential phenotypic properties. This selection process has in the past century been facilitated by growing understanding and utilization of the laws of Mendelian inheritance.
  • One of the major problems in breeding programs of domestic animals is the negative genetic correlation between reproductive capacity and production traits. This is, for example, the case in cattle (a high milk production generally results in slim cows and bulls), poultry (broiler lines have a low level of egg production and layers have generally very low muscle growth), pigs (very prolific sows are in general fat and have comparatively less meat), or sheep (high prolific breeds have low carcass quality and vice versa).
  • WO 00/36143 provides a method for selecting an animal for having desired genotypic or potential phenotypic properties comprising testing the animal for the presence of a parentally imprinted qualitative or quantitative trait locus (QTL).
  • QTL quantitative trait locus
  • a breeding program wherein knowledge of the parental imprinting character of a desired trait as demonstrated herein is utilized, increases the accuracy of the breeding value estimation and speeds up selection compared to conventional breeding programs. For example, selecting genes characterized by paternal imprinting is provided to help increase uniformity; a (terminal) parent homozygous for the “good or wanted” alleles will pass them to all offspring, regardless of the other parent's alleles, and the offspring will all express the desired parent's alleles. This results in more uniform offspring.
  • Alleles that are interesting or favorable from the maternal side are often the ones that have opposite effects to alleles from the paternal side.
  • meat animals such as pigs
  • alleles linked with meat or carcass quality traits such as—intramuscular fat or muscle mass
  • alleles linked with reduced back fat could be fixed in the sire lines.
  • Other desirable combinations are, for example, fertility, teat number and/or milk yield in the female line with increased growth rates, reduced back fat and/or increased muscle mass in the male lines.
  • the purpose of breeding programs in livestock is to enhance the performances of animals by improving their genetic composition.
  • QTL Quantitative Trait Loci
  • the invention provides a method for selecting an animal for having desired genotypic or potential phenotypic properties comprising testing the animal for the presence of a qualitative or quantitative trait locus (QTL).
  • QTL quantitative trait locus
  • IGF2-intron3-nt3072 is part of the evolutionary conserved CpG island with a regulatory function, located between Differentially Methylated Region 1 (DMR1) and a matrix attachment region previously defined in mice (11-13).
  • the 94 bp sequence around the mutation shows about 85% sequence identity to both human and mouse and the wild-type nucleotide at IGF2-intron3-nt3072 is conserved among the three species ( FIG. 4A ).
  • a qualitative trait nucleotide (QTN) occurs three bp downstream of an eight bp palindrome also conserved between the three species.
  • the methylation status of the 300 bp fragment centered on IGF2-intron3-nt3072 and containing 50 CpG dinucleotides was examined by bisulphite sequencing in four month old Q pat /q mat and q pat /Q mat animals.
  • electrophoretic mobility shift analyses were performed using 27 bp oligonucleotides spanning the QTN and corresponding to the wild-type (q) and mutant (O) sequences.
  • Nuclear extracts from murine C2C12 myoblast cells, human HEK293 cells, and human HepG2 cells were incubated with radioactively labeled q or Q oligonucleotides.
  • the data show that the CpG island contains both Enhancer and Silencer functions so that there may be several nuclear factors binding to this CpG island except for the one already shown here.
  • the results provide a method for isolating such nuclear factors and a stretch of oligonucleotides that can be used to fish out such proteins.
  • Pigs carrying the mutation have a three-fold increase in IGF2 mRNA expression in postnatal muscle.
  • the mutation abrogates in vitro interaction with a nuclear factor, most likely a repressor.
  • the mutation has experienced a selective sweep in several pig breeds.
  • All 19 Q-bearing chromosomes shared a haplotype in the 90 kilobase pairs (kb) interval between the microsatellites PULGE1 and SWC9 (IGF2 3′-UTR), which was not present among the q chromosomes and was, therefore, predicted to contain the QTL.
  • the nine q chromosomes exhibited six distinct marker haplotypes in the same interval. This region is part of the CDKN1C-H19 imprinted domain and contains INS and IGF2 as the only known paternally expressed genes.
  • the invention provides a method for selecting an animal for having desired genotypic or potential phenotypic properties comprising testing the animal, a parent of the animal or its progeny for the presence of a nucleic acid modification affecting the activity of an evolutionary conserved CpG island, located in intron 3 of an IGF2 gene and/or affecting binding of a nuclear factor to an IGF2 gene.
  • the invention provides a method for selecting an animal for having desired genotypic or potential phenotypic properties comprising testing a nucleic acid sample from the animal for the presence of a single nucleotide substitution.
  • a nucleic acid sample can, in general, be obtained from various parts of the animal's body by methods known in the art.
  • Traditional samples for the purpose of nucleic acid testing are blood samples or skin or mucosal surface samples, but samples from other tissues can be used as well, in particular, sperm samples, oocyte or embryo samples can be used.
  • the presence and/or sequence of a specific nucleic acid can be determined with methods known in the art, such as hybridization or nucleic acid amplification or sequencing techniques known in the art.
  • the invention also provides testing such a sample for the presence of nucleic acid, wherein the QTN or allele associated therewith is associated with the phenomenon of parental imprinting, for example, where it is determined whether a paternal or maternal allele comprising the QTN is capable of being predominantly expressed in the animal.
  • the invention provides a method wherein the nuclear factor is capable of binding to a stretch of nucleotides, which, in the wild-type pig, mouse or human IGF2 gene, is part of an evolutionary conserved CpG island, located in intron 3 of the IGF2 gene. Binding should preferably be located at a stretch of nucleotides spanning a QTN (qualitative trait nucleotide) that comprises a nucleotide (preferably a G to A) transition, which, in the pig, is located at IGF2-intron3-nt3072. It is preferred that the stretch is functionally equivalent to the sequence as shown in FIG.
  • QTN quantitative trait nucleotide
  • Functional equivalence also entails a sequence homology of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferred at least 90% of the stretch overlapping the QTN.
  • the stretch is preferably from at least 5 to about 94 nucleotides long, more preferably from about 10 to 50, most preferably from about 15 to 35 nucleotides, and it is preferred that it comprises a palindromic octamer sequence as identified in FIG. 4 .
  • the invention provides a method wherein the nucleic acid modification comprises a nucleotide substitution, whereby in the pig, the substitution comprises a G to A transition at IGF2-intron3-nt3072 (SEQ ID NO:6 and SEQ ID NO:5).
  • the substitution comprises a G to A transition at IGF2-intron3-nt3072 (SEQ ID NO:6 and SEQ ID NO:5).
  • the in vivo effect of the mutation on IGF2 expression was studied in a purpose-built Q/q ⁇ Q/q intercross counting 73 offspring.
  • a deletion encompassing DMR0, DMR1, and the associated CpG island derepresses the maternal IGF2 allele in mesodermal tissues in the mouse (12)
  • the effect of the intron3-nt3072 mutation on IGF2 imprinting in the pig was tested. This was achieved by monitoring transcription from the paternal and maternal IGF2 alleles in tissues of q/q, Q pat /q mat , and q pat /Q mat animals that were heterozygous for the SWC9 microsatellite located in the IGF2 3′UTR.
  • Imprinting could not be studied in Q/Q animals that were all homozygous for SWC9.
  • IGF2 was shown to be expressed exclusively from the paternal allele in skeletal muscle and kidney, irrespective of the QTL genotype of the fetuses.
  • weak expression from the maternal allele was observed in skeletal muscle, however, at comparable rates for all three QTL genotypes ( FIG. 6A ). Only the paternal allele could be detected in four-month-old kidney (data not shown). Consequently, the mutation does not seem to affect the imprinting status of IGF2.
  • the Q allele was expected to be associated with an increased IGF2 expression since IGF2 stimulates myogenesis (6).
  • the relative mRNA expression of IGF2 was monitored at different ages in the Q/q ⁇ Q/q intercross using both Northern blot analysis and real-time PCR ( FIGS. 6B and C).
  • the expression levels in fetal muscle and postnatal liver was about ten-fold higher than in postnatal muscle. No significant difference was observed in fetal samples or in postnatal liver samples, but a significant three-fold increase of postnatal IGF2 mRNA expression in skeletal muscle was observed in (Q/Q or Q pat /q mat ) versus (q pat /Q mat or q/q) progeny.
  • the invention provides a method for modulating mRNA transcription of an IGF2 gene in a cell or organism provided with the gene comprising modulating binding of a nuclear factor to an IGF2 gene, in particular, wherein the nuclear factor is capable of binding to a stretch of nucleotides (as identified above) that in the wild-type pig, mouse or human IGF2 gene is part of an evolutionary conserved CpG island, located in intron 3 of the IGF2 gene.
  • the significant difference in IGF2 expression revealed by real-time PCR was confirmed using two different internal controls, GAPDH ( FIG. 6C ) and HPRT (15). An increase of all detected transcripts originating from the three promoters (P2-P4) located downstream of the mutated site was found.
  • a method according to the invention is herein provided allowing testing for, and modulation of, desired genotypic or potential phenotypic properties comprising muscle mass, fat deposition or teat numbers (of mammals). Such testing is applicable in man and animals alike (animals herein defined as including humans).
  • a desirable breeding combination as provided herein comprises, for example, increased teat number in the female line with increased growth rates, reduced back fat and/or increased muscle mass in the male lines. It is herein also shown that the mutation influences teat number.
  • the Q allele that is favorable with respect to muscle mass and reduced back fat is the unfavorable allele for teat number. This strengthens the possibility of using the paternal imprinting character of this QTL in breeding programs.
  • paternal lines can be selected for the Q allele that will increase muscle mass and reduce back fat, characteristics that are of more importance in the paternal lines. Terminal sires that are homozygous QQ will pass the full effect of increased muscle mass and reduced back fat to the slaughter pigs, while selection of parental sows that express the q allele will allow for the selection of sows that have more teats and suckle more piglets without affecting slaughter quality.
  • the invention also provides a method for identifying a compound capable of modulating mRNA transcription of an IGF2 gene in a cell or organism provided with the gene comprising providing a first cell or organism having a nucleic acid modification affecting the activity of an evolutionary conserved CpG island, located in intron 3 of an IGF2 gene and/or affecting binding of a nuclear factor to an IGF2 gene and a second cell or organism not having the modification further comprising providing the first or second cell or organism with a test compound and determining IGF2 mRNA transcription in the first and second cell or organism and selecting a compound capable of modulating IGF2 mRNA transcription.
  • An example of such a compound as identifiable herewith comprises a stretch of oligonucleotides spanning a QTN (qualitative trait nucleotide) that comprises a nucleotide (preferably a G to A) transition, which in the pig, is located at IGF2-intron3-nt3072. It is preferred that the stretch is functionally equivalent to the sequence as shown in FIG.
  • functional equivalence preferably entails that the stretch is spanning the QTN and preferably overlaps with at least two or three nucleotides at or on both sides of the QTN, although the overlaps may be longer.
  • Functional equivalence also entails a sequence homology of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, most preferred at least 90%, of the stretch overlapping the QTN.
  • the stretch is preferably from at least 5 to at about 94 nucleotides long, more preferably from about 10 to 50, most preferably from about 15 to 35 nucleotides, and it is preferred that it comprises a palindromic octamer sequence as identified in FIG. 4 .
  • An alternative compound as provided herein comprises a functional analogue of the stretch, the alternative compound or oligonucleotide analogue functionally at least capable of modulating the activity of an evolutionary conserved CpG island, located in intron 3 of an IGF2 gene and/or modulating binding of a nuclear factor to an IGF2 gene, preferably effecting modulation at the site of the QTN.
  • EMSA electrophoretic mobility shift analyses
  • such compounds e.g., the identified nuclear factor in FIG. 2
  • compounds competing with the binding of the factor to the IGF2 gene can be further identified and selected.
  • a typical example of such an EMSA is given in the detailed description.
  • the nuclear factor is capable of binding to a stretch of nucleotides that in the wild-type pig, mouse or human IGF2 gene, is part of an evolutionary conserved CpG island, located in intron 3 of the IGF2 gene.
  • Oligonucleotide compounds or probes spanning the QTN are herein provided that have the desired effect.
  • Such compounds or probes are preferably functionally equivalent to the sequence 5′-GATCCTTCGCCTAGGCTC(A/G)CAGCGCGGGAGCGA-3′(SEQ ID NO: 1).
  • the invention also provides a method for identifying a compound capable of affecting the activity of an evolutionary conserved CpG island, located in intron 3 of an IGF2 gene and/or modulating binding of a nuclear factor to an IFG2 gene comprising providing a stretch of nucleotides that in the wild-type pig, mouse or human IGF2 gene, is part of an evolutionary conserved CpG island, located in intron 3 of the IGF2 gene.
  • testing may be done with single oligonucleotides or analogues thereof, or with a multitude of such oligonucleotides or analogues in an array fashion, and may further comprise providing a mixture of DNA-binding proteins derived from a nuclear extract of a cell and testing these with the array or analogue or oligonucleotide under study. Testing may be done as well with test compounds provided either singularly or in an array fashion and optionally further comprises providing a test compound and determining competition of binding of the mixture of DNA-binding proteins to the stretch of nucleotides in the presence or absence of test compound(s).
  • the invention thus provides a compound identifiable with a method as described herein.
  • a compound is, for example, derived from a stretch of oligonucleotides spanning a QTN (qualitative trait nucleotide) that comprises a nucleotide (preferably a G to A) (SEQ ID NO:6 and SEQ ID NO:5) transition, which in the pig, is located at IGF2-intron3-nt3072. It is preferred that the stretch is functionally equivalent to the sequence as shown in FIG.
  • oligonucleotide compound is preferably from at least 5 to at about 94 nucleotides long, more preferably from about 10 to 50, most preferably from about 10 to 35 nucleotides, and it is preferred that it comprises a palindromic octamer sequence as identified in FIG. 4 .
  • An alternative compound or functional analogue as provided herein comprises a functional analogue of the oligonucleotide compound, the alternative compound or oligonucleotide analogue functionally at least capable of modulating the activity of an evolutionary conserved CpG island, located in intron 3 of an IGF2 gene and/or modulating binding of a nuclear factor to an IGF2 gene, preferably effecting the modulation at the site of the QTN.
  • electrophoretic mobility shift analyses such compounds, e.g., the identified nuclear factor in FIG. 2 , or compounds competing with the binding of the factor to the IGF2 gene, can be further identified and selected.
  • a typical example of such an EMSA is given in the detailed description.
  • the invention also provides a pharmaceutical composition comprising a compound as provided herein, and use of such a compound, for the production of a pharmaceutical composition for the treatment of obesity or for the treatment of muscle deficiencies. Furthermore, the invention provides a method for modulating mRNA transcription of an IGF2 gene in a cell or organism provided with the gene comprising treating or providing the cell or organism with a compound as provided herein.
  • the results have important practical implications.
  • the IGF2*Q mutation increases the amount of meat produced, at the expense of fat, by 3-4 kg for an animal slaughtered at the usual weight of about 100 kg.
  • the high frequency of IGF2*Q among major pig breeds implies that this mutation affects the productivity of many millions of pigs in the Western world.
  • the development of a simple diagnostic DNA test now facilitates the introgression of this mutation to additional breeds. This could be an attractive way to improve productivity in local breeds as a measure to maintain biological diversity.
  • the diagnostic test will also make it possible to investigate if the IGF2*Q mutation is associated with any unfavorable effects on meat quality or any other trait.
  • Applications of these insights are manifold. Applications in animals typically include diagnostic tests of the specific causative mutation in the pig and diagnostic tests of these and possible other mutations in this CpG island in humans, pigs or other meat-producing animals.
  • transgenic animals with modified constitution of this CpG island or with modified expression of nuclear factors interacting with this sequence
  • the invention provides the use of pharmaceutical compounds (including oligonucleotides) or vaccination to modulate IGF2 expression by interfering with the interaction between nuclear factors and the CpG island provided herein.
  • pharmaceutical compounds including oligonucleotides
  • vaccination to modulate IGF2 expression by interfering with the interaction between nuclear factors and the CpG island provided herein.
  • one may treat the animals with a drug, if not for producing meat, then at least in experimental animals for studying the therapeutic effects of the compounds.
  • diagnostic tests of mutations predisposing to diabetes, obesity or muscle deficiency are particularly provided and pharmaceutical intervention to treat diabetes, obesity or muscle deficiency by modulating IGF2 expression based on interfering with the interaction between nuclear factors and the CpG island as provided herein is typically achievable with compounds, such as the above-identified nucleotide stretches or functional analogues thereof as provided herein.
  • FIG. 1 QTL genotyping by marker-assisted segregation analysis.
  • the graphs show, for 14 paternal half-sib pedigrees (P1, P2, . . . P14), the phenotypic mean ⁇ 2 standard errors of the offspring sorted into two groups according to the homologue inherited from the sire. The number of offspring in each group is given above and below the error bars, respectively.
  • the upper graph corresponds to the boars that were shown to be heterozygous “Qq” for the QTL, the lower graph to the boars that were shown to be homozygous at the QTL.
  • Pedigrees for which the percentage of lean meat was measured as “% lean cuts” N EZER et al.
  • FIG. 2 A. Schematic representation of the human 11p15 imprinted domain according to Onyango et al. (2000).
  • B BAC contig spanning the porcine orthologue of the 11p15 imprinted domain, assembled by STS content mapping. The length of the horizontal bars does not reflect the actual physical size of the corresponding BACs.
  • C Marker haplotypes of the five “Q” chromosomes (diamonds) and six “q” chromosomes (circles). Closely linked SNPs ( ⁇ 5 kb) were merged into poly-allelic multisite haplotypes (cfr. Table 2).
  • the chromosome segments highlighted in green correspond to the haplotype shared by all “Q” chromosomes and, therefore, assumed to contain the QTL.
  • the chromosome segment highlighted in red corresponds to a haplotype shared by chromosome “q 4 ” and chromosomes “Q 1 ⁇ 4 ” that, therefore, excludes the QTL out of this region. The resulting most likely position of the QTL is indicated by the arrow.
  • FIG. 3 Average probability for two chromosomes to be identical-by-descent at a given map position conditional on flanking marker data (p(IBD
  • FIG. 4 (A) DNA sequence polymorphisms identified in a 28.6 kb segment spanning the porcine TH (exon 14), INS and IGF2 genes. The average (C+G) content of a moving 100-bp window is shown on a gray scale (black 100%, white 0%). The positions of evolutionary conserved regions (7) including the DMR1 and associated CpG island in IGF2 intron 3 are marked by horizontal cylinders. The Viewgene program (25) was used to highlight the 258 differences between the reference Q P208 sequence, four Q, and ten q chromosomes. The position of the causative intron3-nt3072G ⁇ A mutation is marked by an asterisk.
  • FIG. 5 (A) Percentage methylation determined by bisulphite sequencing for the 300 bp fragment centered around intron3-nt3072, containing 50 CpG dinucleotides, in liver and skeletal muscle of four-month-old Q pat /q mat and q pat /Q mat individuals. The number of analyzed chromosomes as well as the standard errors of the estimated means are given. Pat and Mat refer to the paternal and maternal alleles, respectively, determined based on the intron3-nt3072G ⁇ A mutation.
  • the pig IGF2 fragments contained 578 bp from intron 3 (nucleotide 2868 to 3446) including the causative G to A transition at nucleotide 3072.
  • FIG. 6 Analysis of IGF2 mRNA expression.
  • A Imprinting analysis of IGF2 in skeletal muscle of qq, Q Pat /q Mat , and q Pat /Q Mat animals before birth and at four months of age. The QTL and SWC9 genotypes of the analyzed animals are given. In these, the first allele is paternal, the second maternal. The lanes corresponding to PCR product obtained from genomic DNA are marked by continuous lines, those corresponding to RT-PCR products by the dotted lines. The three SWC9 alleles segregating in the pedigree (1, 2, and 8) are marked by arrows. The RT-PCR controls without reverse transcriptase were negative (not shown).
  • Haplotype sharing refines the location of an imprinted QTL with major effect on muscle mass to a 90 Kb chromosome segment containing the porcine IGF2 gene.
  • LD linkage disequilibrium
  • the ability to deduce QTL genotype from phenotype can be improved by using “clones” (e.g., recombinant inbred lines) (e.g., D ARVASI 1998), by means of progeny-testing (e.g., G EORGES et al. 1995), or by marker-assisted segregation analysis (e.g., R IQUET et al. 1999).
  • clones e.g., recombinant inbred lines
  • progeny-testing e.g., G EORGES et al. 1995
  • marker-assisted segregation analysis e.g., R IQUET et al. 1999.
  • LIT-1 KVLQT1-AS
  • IGF2 IGF2-AS
  • R EIK and W ALTER 2001 Fifteen imprinted transcripts are known to map to the orthologous domain on distal mouse chromosome MMU7, of which four are paternally expressed: Lit-1 (Kvlqt1-as), Ins2, Igf2 and Igf2-as (e.g., http://www.mgu.har.mrc.ac.uk/imprinting/imprinting.html; O NYANGO et al. 2000). Because of its known function in myogenesis (F LORINI et al. 1996), IGF2 stood out as a prime positional candidate.
  • the pedigree material used for this work comprised a subset of previously described Piétrain ⁇ Large White F2 pedigrees (N EZER et al. 2000; H ANSET et al. 1995), as well as a series of paternal half-sib pedigrees sampled in commercial lines derived from the Pietrain and Large White breeds (Buys, personal communication). In the F2 animals, “% lean cuts” was measured as previously described (H ANSET et al.
  • the QTL genotype of each sire was determined from the Z-score, corresponding to the log10 of the likelihood ratio L H 1 /L H 0 , where L H1 corresponds to the likelihood of the pedigree data assuming that the boar is of “Qq” genotype, and L H 0 corresponds to the likelihood of the pedigree data assuming that the boar is of “QQ” or “qq” genotype.
  • n the number of informative offspring in the corresponding pedigree
  • y i the phenotype of offspring i
  • ⁇ overscore (y) ⁇ is the average phenotype of the corresponding pedigree computed over all (informative and non-informative) offspring
  • ⁇ is the residual standard deviation maximizing L
  • a is the Q to q allele substitution effect.
  • a was set at zero when computing L H 0 , and at +1% for “R” offspring and ⁇ 1% for “L” offspring when computing L H1 (N EZER et al. 1999).
  • Boars were considered to be “Qq” when Z>2, “QQ” or “qq” when Z ⁇ 2 and of undetermined genotype if 2>Z> ⁇ 2.
  • QTL genotyping by marker-assisted segregation analysis A series of paternal half-sib families was genotyped counting at least 20 offspring for two microsatellite markers located on the distal end of chromosome SSC2 and spanning the most likely position of the imprinted QTL: SWR2516 and SWC9 (N EZER et al. 1999; J EON et al. 1999). These families originated either from a previously described Piétrain ⁇ Large White F2 pedigree (N EZER et al. 2002) or from two composite pig lines derived from Large White and Piétrain founder animals (Nadine Buys, personal communication).
  • Offspring were slaughtered at a constant weight of approximately 105 Kgs, and a series of phenotypes collected on the carcasses including “% lean meat,” measured either as lean cuts” (experimental cross) or as “Piglog” (composite lines) (see Materials & Methods).
  • H 0 postulating that the corresponding boar was homozygous at the QTL
  • H 1 postulating that the boar was heterozygous at the QTL.
  • H 0 corresponds to QTL genotypes “QQ” or “qq,” and H 1 to genotype “Qq.”
  • Likelihoods were computed using “% lean meat” as phenotype (as the effect of the QTL was shown to be most pronounced on this trait in previous analyses) and assuming a Q to q allele substitution effect of 2.0% (N EZER et al. 1999).
  • the SWC9 marker was known from previous studies to correspond to a (CA) n microsatellite located in the 3′UTR of the porcine IGF2 gene (N EZER et al. 1999; J EON et al. 1999; A MARGER et al. 2002).
  • a BLAST search was performed with the sequence of the porcine SWR2516 marker (gi
  • Porcine sequence tagged sites were then developed across the orthologous region of the human 11p15 imprinted domain. Sixteen of these were developed in genes (TSSC5, CD81, KVLQT1 (3 ⁇ ), TH (2 ⁇ ), INS (3 ⁇ ), IGF2 (3 ⁇ ), H19 (3 ⁇ )), and five in intergenic regions (IG IGF2-H19 , IG H19-RL23mep (4 ⁇ )).
  • the corresponding primer sequences were derived from the porcine genomic sequence, when available (A MARGER et al. 2002), or from porcine-expressed sequence tags (EST) that were identified by BLAST searches using the human orthologues as query sequences (Table 1).
  • a porcine BAC library (F AHRENKRUG et al. 2001) was screened by filter hybridization using (i) human cDNA clones corresponding to genes known to map to 11p15, as well as (ii) some of the 21 previously described porcine STS. Seven of the identified BACs were shown by PCR to contain at least one of the porcine STS available in the region and were kept for further analysis, together with two BACs that were previously shown to span the TH-H19 region (A MARGER et al. 2002). Three additional STS were developed from BAC end sequences (389B2T7, 370C17T7, 370SP6). 370SP6 revealed a highly significant BLAST hit (expected value 10 ⁇ 7 ) downstream from the ASCL2 gene providing an additional anchor point between the human and porcine sequence.
  • the BAC contig shown in FIG. 2 was assembled. It confirms the overall conservation of gene order between human and pigs in this chromosome region and indicates that the gap remaining in the human sequence between the INS and ASCL2 genes may not be larger than 55 Kb.
  • the first boar that proved to be homozygous for the QTL by marker-assisted segregation analysis (P8) carried the “q 4 ” haplotype on one if its chromosomes. Its other haplotype, therefore, had to be of “q” genotype as well and was referred to as “q 6 .”
  • Boar P9 appeared to be heterozygous “Q 1 /Q 2 .”
  • Boars P10 and P11 carried the “Q 1 ” haplotype shared by six of the “Qq” boars.
  • the other chromosomes of boars P10 and P11 which were IBS as well, were placed in the “Q” pool and referred to as “Q 3 .”
  • Homozygous boar P12 carried haplotype “Q 2 .”
  • its homologue was referred to as “Q 4 .”
  • boars P13 and P14 were identified as being, respectively, “Q 3 Q 4 ” and “Q 2 Q 5 .”
  • the marker genotypes of the resulting five “Q” and five “q” chromosomes are shown in FIG. 2 .
  • closely linked ( ⁇ 5 Kb) SNPs were merged into a series of polyallelic multisite haplotypes.
  • the correspondence between SNP genotype and haplotype number is given in Table 2.
  • the average pair-wise IBD probability amongst the five “Q” chromosomes is superior to 0.4 over the entire KVLQT1(SSR)-IG (H19-RL23mrp) interval and exceeds 0.9 in the PULGE3-IGF2 interval.
  • the equivalent parameter averages 0.25 in the same region. It is worthwhile noting, however, that even amongst “q” chromosomes, the average pair-wise IBD probability peaks just above 0.4 between TH and INS, which is thought to reflect a “q”-specific haplotype signature.
  • chromosome “q 4 ” carries a KVLQT1(I12)-PULGE3 haplotype that is IBS with the ancestral “Q” haplotype in the KVLQTI(I12)-PULGE3 interval.
  • the probability that this IBS status reflects IBD was estimated at 0.50 using the coalescent model of M EUWISSEN and G ODDARD (2001). Assuming IBD, this would position the QTL in the PULGE3-SWC9 interval, measuring less than 90 Kb and containing TH, INS and IGF2 as the only known genes.
  • haplotype-sharing approaches in fine-mapping QTL in livestock also suggests that QTL may be mapped in these populations by virtue of the haplotype signature resulting from intense selection on “Q” alleles, i.e., haplotypes of unusual length given their population frequency.
  • the feasibility of this approach has recently been examined in human populations for loci involved in resistance to malaria (S ABETI et al. 2002). QTL could thus be identified in livestock in the absence of phenotypic data.
  • QTLs Quantitative Trait Loci
  • the imprinted IGF2-linked QTL is one of the major porcine QTLs for body composition. It was first identified in intercrosses between the European Wild Boar and Large White domestic pigs and between Pietrain and Large White pigs (1, 2). The data showed that alleles from the Large White and Pietrain breeds, respectively, were associated with increased muscle mass and reduced back-fat thickness, consistent with the existing breed differences in the two crosses. A paternally expressed IGF2-linked QTL was subsequently documented in intercrosses between Chinese Meishan and Large White/Landrace pigs (3) and between Berkshire and Large White pigs (4). In both cases the allele for high muscle mass was inherited from the lean Large White/Landrace breed.
  • one of the 19 Q-chromosomes (P208) and six q-chromosomes were re-sequenced for a 28.6 kb segment containing IGF2, INS, and the 3′ end of TH.
  • This chromosome collection was expanded by including Q- and q-chromosomes from (i) a Wild Boar/Large White intercross segregating for the QTL (2), (ii) a Swedish Landrace boar showing no evidence for QTL segregation in a previous study (8), (iii) F 1 sires from a Hampshire/Landrace cross showing no indication for QTL segregation (9), and (iv) an F 1 sire from a Meishan/Large White intercross.
  • a Japanese Wild Boar was included as a reference for the phylogenetic analysis; the QTL status of this animal is unknown, but it is assumed that it is homozygous wild-type (q/q).
  • the causative mutation would correspond to a DNA polymorphism for which the two alleles segregate perfectly between Q- and q-chromosomes.
  • the G to A transition at IGF2-intron3-nt3072 is the only polymorphism fulfilling this criterion, implying that it is the causative Quantitative Trait Nucleotide (QTN) (10). So far, 12 large sire families were tested where the sire is heterozygous A/G at this position and all have showed evidence for QTL segregation.
  • QTN Quantitative Trait Nucleotide
  • IGF2-intron3-nt3072 is part of an evolutionary conserved CpG island of unknown function (7), located between Differentially Methylated Region 1 (DMR1) and a matrix attachment region previously defined in mice (11-13).
  • DMR1 Differentially Methylated Region 1
  • the 94 bp sequence around the mutation shows about 85% sequence identity to both human and mouse, and the wild-type nucleotide at IGF2-intron3-nt3072 is conserved among the three species ( FIG. 4A ).
  • the QTN occurs three bp downstream of an eight bp palindrome also conserved between the three species.
  • the methylation status of the 300 bp fragment centered on IGF2-intron3-nt3072 and containing 50 CpG dinucleotides was examined by bisulphite sequencing in four-month-old Q pat /q mat and q pat /Q mat animals.
  • paternal and maternal chromosomes were shown to be essentially unmethylated (including the IGF2-intron3-nt3071 C residue), irrespective of the QTL genotype of the individual (3.4% of CpGs methylated on average; FIG. 5A ).
  • the CpG island was more heavily, and also differentially, methylated in liver. 33% of the CpGs were methylated on the maternal alleles versus 19% on the paternal allele.
  • this CpG island behaves as a previously unidentified DMR in liver, the repressed maternal allele being more heavily methylated than the paternal allele, which is the opposite of what is documented for the adjacent DMR1 in the mouse.
  • electrophoretic mobility shift analyses were performed using oligonucleotide probes spanning the QTN and corresponding to the wild-type (q) and mutant (O) sequences.
  • Nuclear extracts from murine C2C12 myoblast cells, human HEK293 cells, and human HepG2 cells were incubated with radioactively labeled q or Q oligonucleotides.
  • One specific band shift (complex C1 in FIG. 5B ) was obtained with the wild-type (q) but not the mutant (O) probe using extracts from C2C12 myoblasts; similar results were obtained using both methylated and unmethylated probes.
  • the in vivo effect of the mutation on IGF2 expression was studied in a purpose-built Q/q ⁇ Q/q intercross counting 73 offspring.
  • a deletion encompassing DMR0, DMR1, and the associated CpG island derepresses the maternal IGF2 allele in mesodermal tissues in the mouse (12)
  • the effect of the intron3-nt3072 mutation on IGF2 imprinting in the pig was tested. This was achieved by monitoring transcription from the paternal and maternal IGF2 alleles in tissues of q/q, Q pat /q mat , and q pat /Q mat animals that were heterozygous for the SWC9 microsatellite located in the IGF2 3′UTR.
  • Imprinting could not be studied in Q/Q animals that were all homozygous for SWC9.
  • IGF2 was shown to be expressed exclusively from the paternal allele in skeletal muscle and kidney, irrespective of the QTL genotype of the fetuses.
  • weak expression from the maternal allele was observed in skeletal muscle, however, at comparable rates for all three QTL genotypes ( FIG. 6A ). Only the paternal allele could be detected in four-month-old kidney (data not shown). Consequently, the mutation does not seem to affect the imprinting status of IGF2.
  • the Q allele was expected to be associated with an increased IGF2 expression since IGF2 stimulates myogenesis (6).
  • the relative mRNA expression of IGF2 was monitored at different ages in the Q/q ⁇ Q/q intercross, using both Northern blot analysis and real-time PCR ( FIGS. 6B and 6C ).
  • the expression levels in fetal muscle and postnatal liver was about ten-fold higher than in postnatal muscle. No significant difference was observed in fetal samples or in postnatal liver samples, but a significant three-fold increase of postnatal IGF2 mRNA expression in skeletal muscle was observed in (Q/Q or Q pat /q mat ) versus (q pat /Q mat or q/q) progeny.
  • IGF2 expression revealed by real-time PCR was confirmed using two different internal controls, GAPDH ( FIG. 6C ) and HPRT (15). An increase of all detected transcripts originating from the three promoters (P2-P4) located downstream of the mutated site was found. Combined, these results provide strong support for IGF2 being the causative gene.
  • the lack of significant differences in IGF2 mRNA expression in fetal muscle and postnatal liver are consistent with the previous QTL study, showing no effect of the IGF2 locus on birth weight and weight of liver (2).
  • the results have important practical implications.
  • the IGF2*Q mutation increases the amount of meat produced, at the expense of fat, by 3-4 kg for an animal slaughtered at the usual weight of about 100 kg.
  • the high frequency of IGF2*Q among major pig breeds implies that this mutation affects the productivity of many millions of pigs in the Western world.
  • the development of a simple diagnostic DNA test now facilitates the introgression of this mutation to additional breeds. This could be an attractive way to improve productivity in local breeds as a measure to maintain biological diversity.
  • the diagnostic test will also make it possible to investigate whether the IGF2*Q mutation is associated with any unfavorable effects on meat quality or any other trait.
  • the long template PCR products were subsequently purified using Geneclean (Polylab) and sequenced using the Big Dye Terminator Sequencing or dGTP Big Dye Terminator kits (Perkin Elmer).
  • the primers used for PCR amplification and sequencing are available as supplementary information.
  • the sequence traces were assembled and analyzed for DNA sequence polymorphism using the Polyphred/Phrap/Consed suite of programs (21).
  • the genotype was determined by pyrosequencing with a Luc 96 instrument (Pyrosequencing AB).
  • a 231 bp DNA fragment was PCR amplified using Hot Star Taq DNA polymerase and Q-Solution (QIAGEN) with the primers pyrol8274F (5′-Biotine-GGGCCGCGGCTTCGCCTAG-3′) (SEQ ID NO: 2) and pyro18274R (5′-CGCACGCTTCTCCTGCCACTG-3′ (SEQ ID NO:3)).
  • the sequencing primer pyro18274seq: 5′-CCCCACGCGCTCCCGCGCT-3′ SEQ ID NO:4) was designed on the reverse strand because of a palindrome located 5′ to the QTN.
  • DNA-binding proteins were extracted from C2C12, HEK293, and HepG2 cells as described (22).
  • Gel shift assays were performed with 40 fmole 32 P-labeled ds-oligonucleotide, 10 ⁇ g nuclear extract, and 2 ⁇ g poly dI-dC in binding buffer (15 mM Hepes pH 7.65, 30.1 mM KCl, 2 mM MgCl 2 , 2 mM spermidine, 0.1 mM EDTA, 0.63 mM DTT, 0.06% NP-40, 7.5% glycerol).
  • the membrane was hybridized with pig-specific IGF2 and GAPDH cDNA probes using ExpressHyb hybridization solution (Clontech).
  • the quantification of the transcripts was performed with a Phosphor Imager 425 (Molecular Dynamics).
  • Real-time PCR were performed with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems).
  • TaqMan probes and primers were designed with the Primer Express software (version 1.5); primer and probe sequences are available as supplementary material.
  • PCR reactions were performed in triplicate using the Universal PCR Master Mix (Applied Biosystems).
  • the mRNA was quantified as copy number using a standard curve. For each amplicon, a ten-point calibration curve was established by a dilution series of the cloned PCR product.
  • Bisulphite sequencing was performed according to Engemann et al. (23). Briefly, high molecular weight genomic DNA was isolated from tissue samples using standard procedures based on proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. The DNA was digested with EcoRI, denatured, and embedded in low melting point agarose beads. Non-methylated cytosine residues were converted to uracil using a standard bisulphite reaction.
  • the region of interest was amplified using a two-step PCR reaction with primers complementary to the bisulphite-converted DNA sequence (PCR1-UP: 5′-TTGAGTGGGGATTGTTGAAGTTTT-3′ (SEQ ID NO:7), PCR1-DN: 5′-ACCCACTTATAATCTAAAAAAATAATAAATATATCTAA-3′ (SEQ ID NO:8), PCR2-UP: 5′-GGGGATTGTTGAAGTTTT-3′ (SEQ ID NO:9), PCR2-DN: 5′-CTTCTCCTACCACTAAAAA-3′ (SEQ ID NO:10)).
  • the amplified strand was chosen in order to be able to differentiate the Q and q alleles.
  • Plasmid DNA was purified using the modified Plasmid Mini Kit (QIAGEN) and sequenced using the Big Dye Terminator Kit (Perkin Elmer) and an ABI3100 sequence analyzer.
  • C2C12 myoblast cells were plated in six-well plates and grown to ⁇ 80% confluence.
  • Cells were transiently co-transfected with a Firefly luciferase reporter construct (4 ⁇ g) and a Renilla luciferase control vector (phRG-TK, Promega; 80 ng) using 10 ⁇ g Lipofectamine 2000 (Invitrogen).
  • the cells were incubated for 24 hours before lysis in 100 ⁇ l Triton Lysis Solution. Luciferase activities were measured with a Mediators PhL luminometer (Diagnostic Systems) using the Dual-Luciferase reporter Assay System (Promega).
  • RT-PCR analysis of the highly polymorphic SWC9 microsatellite located in IGF2 3′UTR was used to determine the IGF2 imprinting status.
  • the analysis involved progeny groups from heterozygous sires.
  • Total RNA was extracted from the gluteus muscle using Trizol Reagent (Life Technology) and treated with RNase-free DNase I (Roche Diagnostics GmbH).
  • cDNA was synthesized using the 1 st Strand cDNA Synthesis Kit (Roche Diagnostics GmbH).
  • the SWC9 marker was amplified using the primers UP (5′-AAGCACCTGTACCCACACG-3′ (SEQ ID NO:11)) and DN (5′-GGCTCAGGGATCCCACAG-3′ (SEQ ID NO:12)).
  • the 32 P-labeled RT-PCR products were separated by denaturing PAGE and revealed by autoradiography.
  • the statistical analysis confirms that the mutation influences teat number.
  • the Q allele that is favorable with respect to muscle mass and reduced back fat is the unfavorable allele for teat number. This strengthens the possibility of using the paternal imprinting character of this QTL in breeding programs. Selecting maternal lines for the q allele will enhance teat number, a characteristic that is favorable for the maternal side.
  • paternal lines can be selected for the Q allele that will increase muscle mass and reduce back fat, characteristics that are of more importance in the paternal lines. Terminal sires that are homozygous QQ will pass the full effect of increased muscle mass and reduced back fat to the slaughter pigs, while selection of parental sows that express the q allele will have more teats without affecting slaughter quality.

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Cited By (9)

* Cited by examiner, † Cited by third party
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US20060288433A1 (en) * 1998-12-16 2006-12-21 University Of Liege Selecting animals for parentally imprinted traits
US20100281547A1 (en) * 2003-01-10 2010-11-04 Leif Andersson Selecting animals for desired genotypic or potential phenotypic properties
US20100304353A1 (en) * 2007-07-16 2010-12-02 Pfizer Inc Methods of improving a genomic marker index of dairy animals and products
US20100324356A1 (en) * 2007-12-17 2010-12-23 Pfizer, Inc. Methods for improving genetic profiles of dairy animals and products
US20110123983A1 (en) * 2007-09-12 2011-05-26 Pfizer Inc. Methods of Using Genetic Markers and Related Epistatic Interactions
KR20140118236A (ko) * 2013-03-28 2014-10-08 대한민국(농촌진흥청장) 돼지의 유두 수 판단용 ｓｎｐ 마커 및 이의 용도
EP3153030A1 (fr) 2007-11-29 2017-04-12 Monsanto Technology LLC Produits de viande ayant des niveaux accrus d'acides gras bénéfiques
WO2017096218A1 (fr) * 2015-12-03 2017-06-08 The Penn State Research Foundation Régions génomiques ayant une variation épigénétique qui contribuent aux différences phénotypiques du bétail
CN114807384A (zh) * 2022-04-14 2022-07-29 华南农业大学 一种与鸡屠体性状相关的snp分子标记及其应用

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BR0317329A (pt) 2002-12-31 2005-11-08 Mmi Genomics Inc Composições, métodos e sistemas para inferir traços bovinos
WO2007133075A2 (fr) * 2006-05-12 2007-11-22 Institute For Pig Genetics B.V. Effets d'empreinte pour caractéristiques de reproduction chez le porc sur le chromosome ssc2
EP2027771B1 (fr) * 2007-08-24 2012-06-13 Hermitage Pedigree Pigs Ltd. Séquençage de l'ADN mitochondrial par référence avec la fertilité comme moyen d'optimisation de lignées d'élevage de truies
DE102012109704A1 (de) * 2012-10-11 2014-04-17 Epcos Ag Keramisches Bauelement mit Schutzschicht und Verfahren zu dessen Herstellung
CN107779517B (zh) * 2017-10-26 2018-12-21 华南农业大学 一种影响杜洛克种猪瘦肉率性状的分子标记及其应用
CN110714082B (zh) * 2019-09-03 2021-10-15 中国农业大学 一种与猪乳头数相关的snp位点及其检测方法和应用

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DE69936539T2 (de) * 1998-12-16 2008-02-14 University of Liège Auswahl von tieren nach parental geprägten merkmalen
ATE440967T1 (de) * 2003-01-10 2009-09-15 Univ Liege Auswahl von tieren für gewünschte genotypische und potentielle phänotypische eigenschaften auf der basis eines einzelnen nukleotidpolymorphismus (snp) im intron 3 des igf2-gens

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060288433A1 (en) * 1998-12-16 2006-12-21 University Of Liege Selecting animals for parentally imprinted traits
US7892736B2 (en) 1998-12-16 2011-02-22 University Of Liege Selecting animals for parentally imprinted traits
US20100281547A1 (en) * 2003-01-10 2010-11-04 Leif Andersson Selecting animals for desired genotypic or potential phenotypic properties
US20100304353A1 (en) * 2007-07-16 2010-12-02 Pfizer Inc Methods of improving a genomic marker index of dairy animals and products
US20110123983A1 (en) * 2007-09-12 2011-05-26 Pfizer Inc. Methods of Using Genetic Markers and Related Epistatic Interactions
EP3153030A1 (fr) 2007-11-29 2017-04-12 Monsanto Technology LLC Produits de viande ayant des niveaux accrus d'acides gras bénéfiques
US20100324356A1 (en) * 2007-12-17 2010-12-23 Pfizer, Inc. Methods for improving genetic profiles of dairy animals and products
KR20140118236A (ko) * 2013-03-28 2014-10-08 대한민국(농촌진흥청장) 돼지의 유두 수 판단용 ｓｎｐ 마커 및 이의 용도
KR101595011B1 (ko) 2013-03-28 2016-02-18 대한민국 돼지의 유두 수 판단용 ｓｎｐ 마커 및 이의 용도
WO2017096218A1 (fr) * 2015-12-03 2017-06-08 The Penn State Research Foundation Régions génomiques ayant une variation épigénétique qui contribuent aux différences phénotypiques du bétail
US10697014B2 (en) 2015-12-03 2020-06-30 The Penn State Research Foundation Genomic regions with epigenetic variation that contribute to phenotypic differences in livestock
CN114807384A (zh) * 2022-04-14 2022-07-29 华南农业大学 一种与鸡屠体性状相关的snp分子标记及其应用

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