WO2003066891A2 - Marqueurs genetiques pour le diagnostic de l'expression des mamelles rentrees chez les animaux domestiques, d'elevage et utiles - Google Patents

Marqueurs genetiques pour le diagnostic de l'expression des mamelles rentrees chez les animaux domestiques, d'elevage et utiles Download PDF

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WO2003066891A2
WO2003066891A2 PCT/EP2003/001045 EP0301045W WO03066891A2 WO 2003066891 A2 WO2003066891 A2 WO 2003066891A2 EP 0301045 W EP0301045 W EP 0301045W WO 03066891 A2 WO03066891 A2 WO 03066891A2
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nucleic acid
chromosome
microsatellite
mammals
nucleotides
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PCT/EP2003/001045
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German (de)
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WO2003066891A3 (fr
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Torsten Hardge
Karl Schellander
Klaus Wimmers
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Förderverein Biotechnologieforschung Der Deutschen Schweineproduktion E.V.
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Priority to AU2003210201A priority Critical patent/AU2003210201A1/en
Priority to EP03737302A priority patent/EP1472378A2/fr
Publication of WO2003066891A2 publication Critical patent/WO2003066891A2/fr
Publication of WO2003066891A3 publication Critical patent/WO2003066891A3/fr

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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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the invention relates to the use of a first nucleic acid for determining the predisposition for the expression or inheritance of the quantitative feature of inverted teat in a mammal, the first nucleic acid having a length of at least 8 nucleotides and being identical or essentially identical to a second nucleic acid that occurs in the region of microsatellite S0220 on chromosome 6 of the pig or in a homologous position in the genome of other mammals; or from microsatellite SW2443 on chromosome 2 of the pig or in a homologous position in the genome of other mammals; or from microsatellite S0097 on chromosome 4 of the pig or in a homologous position in the genome of other mammals; or from microsatellite S0007 on chromosome 14 of the pig or in a homologous position in the genome of other mammals; or from microsatellite SW1301 on chromosome 1 of the pig or in a homologous position in the genome of
  • the invention further relates to methods for determining the predisposition for the expression or inheritance of the quantitative feature of teat in mammals, preferably in domestic, breeding or farm animals, whereby the mammals, their fertilized or unfertilized egg cells, or their sperm on the presence, nature or expression of the above-mentioned second nucleic acid.
  • the invention relates to a kit containing at least one pair of primers for the amplification of one of the above-mentioned second nucleic acids, one primer each binding to the + strand and another primer to the - strand of the nucleic acid, or a hybridization probe with a length of at least 8 nucleotides which binds to one of the second nucleic acids mentioned above, or a specific antibody or an antibody fragment which binds to the second nucleic acid disclosed above.
  • Genetic maps are created through coupling analyzes (Morton 1955; Xu 1997; Penalver 1999). Two prerequisites are required for this, namely an animal population with a known lineage (family structure) and numerous polymorphic loci (Archibald and Haley 1998). Molecular genetic markers can be used as polymorphic loci (Jarne and Lagoda 1996; Hui Liu 1998; Luikart and England 1999). A molecular genetic marker can be defined as a section of the genetic material that has or has a specific property. It is a marked locus that is inherited from generation to generation (Nagel 1996; O ⁇ rien et al. 1999).
  • RFLPs restriction fragment length polymorphisms
  • microsatellites Jarne and Lagoda 1996; Montaldo and Herrera-Meza 1998.
  • Most RFLPs are diallelic and, according to Hui Liu (1998), have low PIC (Polymorphism Information Content) values compared to microsatellites.
  • PIC Polymorphism Information Content
  • Microsatellites are numerous and have high PIC values. Around 65,000 to 100,000 microsatellite loci are evenly distributed in the pig genome (Ellegren 1993; Schlötterer 1997; Dounavi 2000). The identification of microsatellites is carried out by various laboratories, the current number of identified microsatellites is 1286 (as of 05.03.2001). A large number of quantitative performance characteristics of pigs and other mammals can already be achieved with the help of genetic Markers are predicted. To date, however, there is no possibility of demonstrating predisposing teat.
  • the present invention is therefore based on the object of providing genetic markers by means of which animals are identified which are predisposed to the expression of teats or pass on such a predisposition. This object is achieved according to the invention by providing the embodiments characterized in the patent claims.
  • the invention thus relates to the use of a first nucleic acid for determining the predisposition for the expression or inheritance of the quantitative feature of inverted teat in a mammal, the first nucleic acid having a length of at least 8 nucleotides and being identical or essentially identical to a second nucleic acid, which occurs in the region of microsatellite S0220 on chromosome 6 of the pig or in a homologous position in the genome of other mammals; or from microsatellite SW2443 on chromosome
  • the first nucleic acid is preferably DNA.
  • the second nucleic acid disclosed above is preferably genomic DNA or cDNA, but can also be an RNA transcript of this DNA. Genomic DNA and cDNA are mostly double-stranded, but the use according to the invention also includes single-stranded DNA molecules.
  • the first nucleic acid is preferably an oligonucleotide, but in certain embodiments can also be a polynucleotide.
  • the first nucleic acid mentioned usually has a length of at least 8 nucleotides, preferably at least 15 nucleotides, more preferably at least 18 nucleotides, even more preferably 21 nucleotides, most preferably 25 nucleotides.
  • the first nucleic acid can also be up to 50 nucleotides, more preferably up to 100 nucleotides, even more preferably up to 1000 nucleotides and most preferably up to 5000 nucleotides long or longer.
  • the first or second nucleic acid comprises whole genes or even groups of genes.
  • the first or second nucleic acid has a length of up to 1000 nucleotides, preferably up to 5000 nucleotides, for example up to 25000 nucleotides, such as up to 150,000 nucleotides.
  • Hybridization probes are nucleic acids that are used in hybridization and bind to homologous nucleic acids.
  • the hybridization probe is preferably a radioactively labeled nucleic acid or it contains modified nucleotides.
  • the invention also includes such modifications of the nucleic acids claimed here.
  • Hybridization probes and primers the second with the nucleic acids, preferably under stringent conditions' hybridize.
  • stringent hybridization conditions are understood to mean, for example, 0.2 ⁇ SSC (0.03 M NaCl, 0.003M sodium citrate, pH 7) at 65 ° C.
  • the hybridization temperature is below 65 ° C., for example above 55 ° C., preferably above 50 ° C.
  • Stringent hybridization temperatures depend on the size or length of the nucleic acid and its nucleotide compositions and are to be determined by a person skilled in the art by manual tests.
  • predisposition refers to the presence of a hereditary disposition, which can possibly result in an inheritance of the hereditary disposition and / or the expression of a characteristic.
  • the characteristic teat is a particularly broad and flat teat with a centrally located indentation of different thickness, where the teat tip can be invisible, instead of the teat tip a crater-shaped pit is then formed is mostly filled with black colored dirt particles.
  • teat teats can occur in all mammals, so the term “mammal” is to be viewed comprehensively and includes humans.
  • microsatellite S0220 is at position SSC6 98cM, microsatellite SW2443 at position SSC2 20cM, microsatellite SS40097 100cM, microsatellite S0007 at position SSC14 70cM, microsatellite SSW1301 at position SSC1 160cM and microsatellite S0164 at position SSC3 70cM. Since genetic mapping positions are dependent on the population, this position specification is subject to fluctuations.
  • microsatellite S0220 on chromosome 6 of the pig denotes a position on chromosome 6 which is specific for a population and comprises 5 cm upstream and / or downstream of the indicated position, preferably up to 10 cm upstream and / or downstream, more preferably 20 cm upstream and / or downstream and most preferably 30 cm upstream and / or downstream of the indicated position on the chromosome, the same applies to the other microsatellites mentioned.
  • microsatellite S0220 is defined as the region between microsatellites S0087 to S0003
  • microsatellite SW2443 is defined as the region between microsatellites SW2443 to SW240
  • microsatellite S0097 is defined as the region between microsatellites S0001 to S0097
  • microsatellite S0007 as the area between microsatellites S0007 to SWC27
  • microsatellite SW1301 defined as the area between microsatellites S0155 to SW1301
  • microsatellite S0164 defined as the area between microsatellites SW72 to SW2570.
  • the comparative genome maps between different species are based on the mapping of one or more loci in the genome of the species in question.
  • "Homologous position” refers to nucleic acid segments in the genome of other mammals that have a sequence identity with the second nucleic acid disclosed above, of at least 95% sequence identity, preferably at least 30%, stronger over the entire sequence length or in specific genes located here or at one or more loci preferably at least 40%, even more preferably at least 50% and particularly preferably 75% sequence identity
  • the sequence identity is preferably determined by the FASTA, BLAST (Basic Local Alignment Search Tool) or Bestfit algorithms of the GCG sequence analysis program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Ge ⁇ etics Computer Group, University Research Park, 575 Madison, Wl 53711).
  • the parameters are preferably set so that the percentage of identity is calculated over the entire length of the reference sequence and homology gaps ("gaps" ) of up to 5% of the G total number of nucleotides are allowed.
  • the so-called optional parameters are preferably left at their preset values.
  • the term “essentially identical” means that, for example, 7 nucleotides are identical in a range of 8 nucleotides. However, the invention also includes embodiments in which 4, 5 or 6 of the 8 nucleotides have the corresponding sequence of second nucleic acid are identical.
  • the first nucleic acid can be identical or essentially identical to the + strand as well as the - strand.
  • the term "a first nucleic acid” in the sense of the invention also includes that more than one (first) nucleic acid in the use according to the invention can be used. This can be, for example, two, three or four nucleic acids.
  • second nucleic acid can mean both the + strand and the - strand.
  • the first nucleic acid can be identical or essentially identical to the + strand during the other first nucleic acid may be identical or substantially identical to the strand, in which case it is preferred that the "Alignment" of the first nucleic acid is in opposite directions, which enables a PCR to be carried out.
  • nucleic acids are made available for the first time, which allow a targeted molecular-biological diagnosis of the predisposition to the teat. It must be taken into account in particular that the teat assessment is still carried out at a very early point in time, which mostly does not or only insufficiently permits detection of teats. With the invention disclosed here, the time of the selection can be shifted significantly forward, so that there is no longer any need to wait for the phenotypic expression of the feature. In addition, the introduction of molecular biological markers can significantly increase the efficiency of the selection process.
  • This selection process includes the determination of the genotype of the subject / mammal at one or more loci in a region of the above-mentioned second nucleic acids, preferably in two regions, more preferably three, more preferably four, more preferably five, most preferably in six regions and the assessment of an individual as suitable or unsuitable for breeding, including information about the coupling phase between the genotyped marker locus and the genes responsible for the defect expression.
  • the genotypes of suitable and unsuitable mammals can be distinguished by a different number of copies of a repetitive nucleotide sequence within the microsatellite locus under consideration.
  • nucleic acid can be shown in a PCR reaction using suitable primers and is reflected in different PCR product sizes and / or different restriction fragment lengths.
  • the tests are usually performed on tissue samples from the mammal, or on samples of sperm, egg, urine or blood.
  • a preferred embodiment of the invention relates to the use of combinations of at least two, three, four, five or six of the above-mentioned nucleic acids for determining the predisposition for the expression or inheritance of the quantitative feature of the teat. This preferred embodiment is particularly suitable for increasing the reliability of the detection.
  • the second nucleic acid disclosed above being a microsatellite or a sequence flanking it.
  • the microsatellite is S0164, SW1301, S0007, S0097, S2443 or S0220, or one of the neighboring microsatellites SW72 and S0002, SW1301, SW857 and SWC27, S0214, SW240, S0087, S0003, SW2570, S0155, S0001.
  • Microsatellite S0164 is preferred here, more preferably SW1301, even more preferably S0007, more preferably S0097, even more preferably S2443 and most preferably S0220.
  • the flanking sequence lies outside the repetitive sequences typical of the microsatellite and preferably comprises a region of 1 kB upstream or downstream of the repetitive sequences.
  • the second nucleic acid is a specific gene.
  • the genes ryrl, TGFB1, GPI, POU2F2, EAH, Tenascingen, HSPA5, orosomucoid gene, steroidogenic factor 1, relaxingen, GGTA1, TSHB, ATP1A1, NGFB, PKLR, OAT, MLB2, Npy4R, PLAU, parathyroid hormone-like are preferred (parathyroid-hormone-like hormones) and parathyroid hormone receptor 1, as well as all other genes located in the nucleic acids disclosed above but not listed here individually.
  • Genes in the sense of the invention can include the coding as well as the non-coding sections of the DNA, ie introns, exons and regulatory areas such as promoters.
  • such genes can also be surrounded by flanking sequences, which preferably comprise a region of 1 kB upstream or downstream of the genes.
  • the invention relates to the use of the disclosed second nucleic acids for the selection of domestic, breeding or farm animals with missing teats.
  • the domestic, breeding or farm animals are cattle, pigs, goats, sheep, horses, donkeys, rats or mice.
  • a genome screen is carried out on several related mammals in a population.
  • the term "several related mammals” includes at least two animals of a population, preferably at least 5 animals, more preferably at least 10 animals, even more preferably at least 10 animals, more preferably at least 50 animals, even more preferably at least 250 animals, most preferably 1500 animals
  • the animals are ultimately derived from a pair of parents
  • the genome screen is used to examine whether a nucleic acid of at least 8 nucleotides in length, preferably up to 50 nucleotides, more preferably 350 nucleotides, even more preferably 1000 nucleotides, most preferably up to 5000 nucleotides or is inherited for a longer time together with the characteristic teat, especially in this case it is clarified, for example with the aid of nucleic acid amplification methods or hybridization methods, which marker is inherited together with the characteristic teat.
  • a common inheritance of nucleic acids with the phenotypic expression of the teat implies genetic coupling of the nucleic acid to the trait.
  • Preferred markers are microsatellite S0220 on chromosome 6 of the pig or from microsatellites in a homologous position in the genome of other mammals; or from SW2443 microsatellite on pig chromosome 2 or from microsatellites in a homologous position in the genome of other mammals; or from microsatellite S0097 on chromosome 4 of the pig or from microsatellites in a homologous position in the genome of other mammals; or from microsatellite S0007 on pig chromosome 14 or from microsatellites in a homologous position in the genome of other mammals; or from SW1301 microsatellite on pig chromosome 1 or from microsatellites in a homologous position in the genome of other mammals; or from microsatellite S0164 on chromosome 3 of
  • the preferred marker here is microsatellite S0164, more preferably SW1301, even more preferred S0007, more preferably S0097, even more preferably S2443 and most preferably S0220.
  • other nucleic acid sequences can also be used as markers, provided they are identical or essentially identical in the sense of the invention to the second nucleic acid disclosed in the invention or lie in one of the above-mentioned nucleic acid regions.
  • nucleic acid segments disclosed in the invention can readily use or develop detection methods for determining the predisposition to teat.
  • the invention also discloses methods for determining the predisposition for the expression or inheritance of the quantitative feature of teat in domestic, breeding, or farm animals, the animals, their fertilized or unfertilized egg cells, or their sperm, depending on the presence, expression or nature of a of the above second nucleic acid tests.
  • Different "forms or textures" of nucleic acids can be caused, for example, by insertions, duplications, deletions, substitutions or translocations. Inserts or deletions, for example, result in a changed nucleic acid length. Duplications are a phenomenon usually observed in the generation of microsatellites.
  • This length polymorphism can, for example are shown in a PCR reaction and is reflected when using suitable flanking primers, for example in the case of insertion in a longer PCR product.
  • the different "expression or nature” can also, for example be a closely related gene variant, which in extreme cases differs from the related gene sequence only by a single nucleotide exchange.
  • Such different “forms or qualities” of the nucleic acid can optionally be carried out with the aid of RFLP analyzes (restriction fragment length polymorphisms (RFLPs) or through
  • the methods according to the invention also relate to other mammals, in particular humans.
  • the domestic, breeding or farm animals are cattle, pigs, goats, sheep, horses, donkeys, rats or mice.
  • a PCR amplification is carried out with complementary primers with a length of at least 8 nucleotides, one primer binding to the + strand and another primer in opposite orientation to the - strand of the second nucleic acid, or it will a hybridization is carried out, wherein a hybridization probe with a length of at least 8 nucleotides binds to the second nucleic acid, or a sequencing of the second nucleic acid is carried out, or a detection is carried out with a specific antibody or antibody fragment or antibody derivative or an aptamer, the Antibody or the antibody fragment or the antibody derivative or the aptamer is specifically directed against the second nucleic acid.
  • the reaction mixture also contains an excess of deoxynucleoside triphosphates and a DNA polymerase, for example Taq polymerase.
  • a DNA polymerase for example Taq polymerase.
  • the primers bind to the nucleic acid and the DNA polymerase extends the primers based on the nucleotide sequence specified in the nucleic acid.
  • the cycles and reaction conditions shown in Table 4 are preferably selected.
  • the annealing temperature of a primer is influenced by its adenine + tymine and cytosine + guanine content. 2 ° C are calculated for each adenine and tymin, while 4 ° C is calculated for each cytosine and guanine.
  • the quality of a PCR reaction is directly influenced by the primer concentration, the changing amount of dNTP in the PCR mix and the quality of the Taq DNA polymerase.
  • PCR reactions with a final volume of 12.5 ⁇ l.
  • the PCR reactions were set up at 25 ⁇ l.
  • the reaction mixture at 12.5 ⁇ l final volume was composed as follows: 0.20 ⁇ M primer, 200 ⁇ M dNTPs, 0.50 U Taq polymerase, 1.25 ⁇ l 10 ⁇ buffer, 1.50 ⁇ l DNA (50ng / ⁇ l) and made up to 12.5 ⁇ l with H 2 O.
  • Primers are those nucleic acids that are at least 8 nucleotides in length and bind to one of the second nucleic acids disclosed above.
  • Preferred primers have a length of at least 8 nucleotides, preferably at least 13, 15, 17 or 20 nucleotides, more preferably at least 30 nucleotides, even more preferably at least 50 nucleotides and most preferably at least 70 nucleotides.
  • the nucleotide sequences of the primers can be combined as desired from the second nucleic acid sequences disclosed above, provided that they have at least 8 consecutive nucleotides.
  • primers when the primers are hybridized with the target sequence within the second nucleic acid, base mismatches can also occur (provided that they are hybridized under the chosen reaction conditions, which can lead to an elongation reaction).
  • a primer should have 7 identical nucleotides within 8 neighboring nucleotides.
  • the invention also includes those embodiments in which 4, 5 or 6 of the 8 nucleotides are identical to the corresponding sequence of the second nucleic acid.
  • the basic principles of the PCR methodology must be observed, the process steps and reaction conditions of which are state of the art. In detail, however, the method steps may nevertheless require adjustment by a person skilled in the art.
  • PCR reverse polymerase chain reaction
  • RT-PCR reverse polymerase chain reaction
  • Amplification methods have been developed in recent years, further amplification methods, which are also preferred embodiments of the invention.
  • Other amplification methods are for example the “Ligase Chain Reaction” (LCR, EPA 320308), “Cyclic Probe Reaction” (CPR,), “Strand Displacement Amplification” (SDA, Walker et al., Nucleic Acids Res. 1992 (7): 1691-6. ) or “Transciption-based amplification systems” (TAS, Kwoh et al Proc. Nat. Acad Sei. USA 86: 1173 (1989), Gingeras et al., PCT Application WO 88/10315).
  • the hybridization probe is understood to mean a nucleic acid with a length of at least 8 nucleotides, preferably up to 50 nucleotides, more preferably up to 100 nucleotides, even more preferably up to 1000 nucleotides and most preferably up to 5000 nucleotides, which are attached to one of the above disclosed second nucleic acids binds.
  • the first nucleic acid is preferably provided with a detectable label, such as a radioactive or fluorescent label. Examples of hybridization methods are dot blot, northern blot, reverse northern blot, in situ hybridization or southern blot.
  • primers are understood to be those nucleic acids which comprise at least 8 nucleotides, preferably up to 13, 15, 17 or 20 nucleotides, more preferably up to 30 nucleotides, even more preferably up to 50 nucleotides and most preferably up to 70 nucleotides.
  • the properties shown for the PCR primers apply accordingly to sequencing primers.
  • primers or hybridization probes which are derived from the above-mentioned second nucleic acids, are suitable for special detection methods, for example PCR methods, sequencing or hybridization methods, and which are not, or less are suitable.
  • the primers or hybridization probes for use in the invention can also be present, for example, in larger DNA or RNA sequences, for example flanked by restriction sites, and moreover Nucleic acids, hybridization probes and primers can also be constructed from base derivatives.
  • the nucleic acids can have a label for detection. Examples of this are radioactive labeling, for example with 32 P or 3 H fluorescent labeling, biotin labeling, digoxigenin labeling,
  • Peroxidase labeling or labeling with an alkaline phosphatase Another preferred detection method is the detection of the first or second nucleic acid with a specific antibody or antibody fragment or antibody derivative or an aptamer. This method generates specific antibodies that recognize the first or second nucleic acids. Fragments of antibodies are e.g. Fab or F (ab) 2 fragments
  • Derivatives include scFvs.
  • Aptamers are nucleic acids that bind specifically to a target molecule due to their three-dimensional structure.
  • Methods for generating specific antibodies are known from the prior art.
  • the specificity of binding to the genomic nucleic acid can e.g. by competition experiments with radioactively labeled desired target nucleic acid and unwanted, e.g. randomly selected nucleic acid can be tested.
  • Common detection methods in which the antibodies are used are e.g. ELISA or RIPA but also immunofluorescence and other detection methods.
  • the antibodies specifically bind the first or second nucleic acids. Antibody binding can e.g.
  • the antibodies can e.g. be modified with fluorescent substances, by radioactive labeling or an enzymatic labeling.
  • immunological detection methods using specific antibodies are known from the prior art. Examples include Harlow et al., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press and all subsequent editions.
  • a genome screen is carried out on several related mammals in a population.
  • the term “several related mammals” includes at least two animals in a population, preferably up to 5 animals, more preferably 10 animals, even more preferably 10 animals, more preferably 50 animals, even more preferably 250 animals, on most preferred 1500 animals.
  • the genome screen examines whether a nucleic acid of at least 8 nucleotides in length, preferably up to 50 nucleotides, more preferably 350 nucleotides, even more preferably 1000 nucleotides, most preferably up to 5000 nucleotides or longer, is inherited together with the feature teat.
  • a common inheritance of nucleic acids with the phenotypic expression of the inverted tip implies a genetic coupling of the nucleic acid to the gene controlling the characteristic and also indicates which marker allele is located on the same homologous chromosome as the allele involved in the phenotypic expression of the inverted tip on the defect gene, ie in which coupling phase there is marker locus and defect locus.
  • Preferred markers are microsatellite S0220 on pig chromosome 6 or microsatellites in a homologous position in the genome of other mammals; or SW2443 microsatellite on pig chromosome 2 or microsatellites in a homologous position in the genome of other mammals; or microsatellite S0097 on pig chromosome 4 or microsatellites in a homologous position in the genome of other mammals; or microsatellite S0007 on pig chromosome 14 or microsatellites in a homologous position in the genome of other mammals; or SW1301 microsatellite on pig chromosome 1 or microsatellites in a homologous position in the genome of other mammals; or microsatellite S0164 on chromosome 3 of the pig or microsatellites in a homologous position in the genome of other mammals.
  • the preferred marker here is microsatellite S0164, more preferably SW1301, even more preferably S0007, more preferably S0097, even more preferably S2443 and most preferably S0220.
  • other nucleic acid sequences can also be used as markers, provided that they are identical or essentially identical to the second nucleic acid disclosed in the invention in the sense of the invention.
  • sequences flanking the microsatellites for example as target sequences for the PCR primers, can also be included in the analysis.
  • the invention further relates to a kit containing at least one pair of primers for the amplification of the second nucleic acid, one primer each on the + strand and another primer binds to the strand of this nucleic acid; or a hybridization probe with a length of at least 8 nucleotides that binds to the second nucleic acid; or an antibody or an antibody fragment or an antibody derivative or an aptamer that specifically binds the first or second nucleic acid.
  • primers are understood to be those nucleic acids which comprise at least 8 nucleotides, preferably up to 20 nucleotides, more preferably up to 30 nucleotides, even more preferably up to 50 nucleotides and most preferably up to 70 nucleotides.
  • the nucleotide sequences of the primers can be combined as desired from the second nucleic acid sequences disclosed above, provided that they have at least 8 consecutive nucleotides.
  • a primer should have at least 7 nucleotides identical to the target sequence within 8 neighboring nucleotides.
  • the invention also includes those embodiments in which 4, 5 or 6 of the 8 nucleotides are identical to the corresponding sequence of the second nucleic acid.
  • Kits based on hybridization methods contain a hybridization probe.
  • the hybridization probe can be up to 50 nucleotides long, more preferably up to 100 nucleotides, even more preferably up to 1000 nucleotides, and most preferably up to 5000 nucleotides or longer.
  • the hybridization probe is preferably a radioactively labeled nucleic acid or it contains modified nucleotides.
  • Kits for the detection of nucleic acids on ELISA, RIPA or similar basis contain a specific antibody or an antibody fragment or an antibody derivative or an aptamer. Antibodies or antibody fragments or antibody derivatives or aptamers are specifically directed against the first or second nucleic acid. Common detection methods in which the kits are used are, for example, ELISA or RIPA, but also immunofluorescence and other detection methods. Immunological detection methods and methods for generating specific antibodies are known from the prior art.
  • the components of the kit can be packed in containers such as vials, optionally also in buffers and / or solutions. Optionally, one or more of the components can be packaged in the same container. Additionally or alternatively, one or more components can be absorbed on a solid support, such as on nitrocellulose filters, nylon membranes, or on the well of a microtiter plate.
  • Figure 1 Coupling card on chromosome 1. The figure shows gender-neutral, female and male coupling cards of the markers SW1515, SW1851,
  • FIG. 1 QTL effects for inverted teats on chromosome 1. A significant effect in the range of microsatellite SW1301 was identified.
  • Figure 3 Coupling card on chromosome 4.
  • the figure shows gender-neutral, female and male coupling cards of markers S0227, S0001, S0214, S0097 on chromosome 4.
  • Figure 4 QTL effects for inverted teats on chromosome 4. From the microsatellite S0001, the graph rises to the location of the marker S0097, where it reaches its maximum value.
  • Figure 5 Coupling card on chromosome 6.
  • the figure shows gender-neutral, female and male coupling cards of markers S0035, S0087, S0220 and S0003 on chromosome 6.
  • Figure 6 QTL effects for teat on chromosome 6. The highest NPL value for the feature teat was identified on chromosome 6 in the area of marker S0220.
  • FIG 7 Coupling card on chromosome 14.
  • the figure shows gender-neutral, female and male coupling cards of markers SW857, S0007 and SWC27 on chromosome 14.
  • Figure 8 QTL effects for inverted teats on chromosome 14. The NPL value increases to 4.8 in the localization of microsatellite S0007.
  • Figure 9 Coupling card on chromosome 8.
  • the figure shows gender-neutral, female and male coupling cards of markers SW 2410, S0086, S0144, SW61 on chromosome 8.
  • Figure 11 Coupling card on chromosome 10. The figure shows gender-neutral, female and male coupling cards of markers SW830, S0070, SW2067 on chromosome 10.
  • Figure 12 QTL effects for teats on chromosome 10.
  • the course of the NPL graphic shows a maximum of less than 2.5, which is below the level of significance and otherwise remains below 1.5.
  • Figure 13 Coupling card on chromosome 10. The figure shows gender-neutral, female and male coupling cards of the markers SO143
  • Figure 14 QTL effects for teats on chromosome 12.
  • the NPL value shows a peak of two and is therefore clearly not significant.
  • Figure 15 Coupling card on chromosome 16.
  • the figure shows gender-neutral, female and male coupling cards of markers S0111, S0026 and S0061 on chromosome 16.
  • Figure 16 QTL effects for teats on chromosome 16.
  • the NPL value rises between the microsatellite markers S0111 and S0026 and reaches its maximum value of 4.2.
  • Figure 17 Coupling card on chromosome 18. The figure shows gender-neutral, female and male coupling cards of markers SW1023, SW787 and SWR414 on chromosome 18.
  • Figure 18 QTL effects for teats on chromosome 18.
  • the NPL value on chromosome 18 rises above 3.5 in the location of the marker SWR414.
  • Figure 19 a-c QTL regions for the feature teat on chromosomes 1 (Sscrl), 2 (Sscr2) and 6 (Sscr6) in animals of commercial origins of the German Landrace breed and their crossing with German noble pig.
  • microsatellites and other loci and microsatellite typing A total of 72 microsatellite loci, which are evenly distributed over the autosomes, were selected and typed.
  • the microsatellites used are summarized in Tables 1 and 2.
  • the typings of two microsatellites on chromosome 8 (S0086 and S0144) were provided by the Institute for Farm Animal Science, Humbold University Berlin.
  • the coupling card of the American group (USDA-MARC) was used.
  • the coupling card of the European group (PiGMaP) was used to select the four microsatellite loci, S0035, S0087, S0220 and S0003.
  • Table 3 Selected Type I markers and candidate genes, their function and effects on the mammary gland
  • Leptin receptor mediates growth and mediator effect
  • Estrogen receptor mediates effect of the growth of the duct system, Oka et al, 1991, Gravert, 1983, Nicholson et al, ESR1 Ostrogen's teat development 1988
  • Parathyroid hormone epithehal-mesenchymale mediates effect of receptor 1, interactions during Lanske et al, 1996 PTHLH PTHR1 development of the mammary gland
  • the polymerase chain reaction is a fast and effective method with which nucleotide sequences can be enzymatically amplified (amplified) in vitro.
  • the method mainly consists of repeating three steps. In the first step, the DNA is denatured (the two DNA strands are separated), in the second, the temperature of the primers attaches to their complementary sequences. In the last step, the Taq polymerase supplements the single strands to the double strands.
  • Table 4 shows the program used in the present work.
  • Table 4 PCR programs used. The annealing temperatures vary between 55 and 65 ° C.
  • the annealing temperature of a primer is influenced by its adenine + tymine and cytosine + guanine content. 2 ° C are calculated for each adenine and tymin, while 4 ° C is calculated for each cytosine and guanine.
  • the quality of a PCR reaction is directly influenced by the primer concentration, the changing amount of dNTP in the PCR mix and the quality of the Taq DNA polymerase.
  • PCR reactions with a final volume of 12.5 ⁇ l were set up.
  • the PCR reactions were set up at 25 ⁇ l. With a final volume of 12.5 ⁇ l, the reaction mixture was composed as follows:
  • a DNA sequencer (DNA Analyzer 4200, LI-COR) was used for the electrophoretic separation of the PCR products. PCR reactions can show different results, which result from different band intensities. This is particularly the case with multiplex PCR reactions.
  • the LI-COR sequencer system also enables weaker bands to be detected. With the "Intensity adjustment" function, the weaker bands can be made clearer and analyzed. An overview of the LI-COR system is provided by Mclndoe et al. (1996).
  • the glass plates were rinsed thoroughly with warm water and detergent to remove gel residues and dried thoroughly with a lint-free paper towel.
  • the rinsed glass plates were then washed again with double-distilled water and rubbed with alcohol (70% ethanol). After washing, binding silane was pipetted onto the upper glass plate in the area of the gel pocket comb and wiped off with the paper towel. This allows stable gel pockets to be created.
  • Two spacers, which determine the thickness of the gel, were inserted between the two glass plates. The glass plates lying on top of each other were then assembled with brackets.
  • the software program "ONE-DSCAN" was used for the fragment length determination of the alleles.
  • the program calculates the fragment length of the samples to be examined based on the previously entered fragment lengths of the standard samples. Fragments were used as standard, the fragment lengths of 75 bp, 100 bp, 105 bp, 120 bp, 145 bp, 175 bp, 200 bp, 204 bp, 230 bp, 255 bp, 300 bp, both at 700 nm and 800 nm, 1 ⁇ l of each of the fragments mentioned was mixed together and from this Mix 0.7 ⁇ l of mixture on the gel. linkage analysis
  • the program CRI-MAP was used for the coupling analysis, which estimates the genetic distances between loci according to the Kosambi mapping function and using the maximum likelihood method.
  • multi-loci gene maps can be created quickly and fully automatically and differences in recombination rates between the sexes can be taken into account.
  • Various options were used in the CRI-MAP program.
  • With the "Build” function a genetic gene map was calculated by successively incorporating the loci.
  • the "Twopoint” function two-point coupling analyzes were calculated for each pair of markers. The resulting arrangement of the loci was checked with the "Flipsn" function.
  • Female, male and gender-neutral coupling cards were created with the CRI-MAP program.
  • the main goal of QTL studies is to localize disease-causing or performance-determining chromosome regions in the genome of farm animals or humans.
  • the program packages "Genehunter” (Kruglyak et al., 1996) and “Allegro” (Gudbjartsson et al. 2000) were used for the QTL analysis in this work.
  • the two programs are statistical analysis programs that can perform both parametric and nonparametric analyzes with many genetic markers. This is particularly necessary if you want to map the genes that are responsible for a complex disease and family tree data are available, as is the case in the analyzed population.
  • the Allegro program is a new version of Genehunter and includes all of its functions, but can accommodate larger families and work faster.
  • the program packages perform nonparametric analyzes as follows.
  • the individuals of a pedigree are divided into " founder” and "Non founder” individuals.
  • the founders are individual parents (sows and boars) whose ancestor information is not known or is not taken into account, while the descendants of founder individuals represent the non-founder animals.
  • Each parent animal (founder) has two alleles, a paternal allele and a maternal allele.
  • inheritance vectors can be created which represent all possible states of the parental alleles in the offspring concerned.
  • inheritance vectors in which one progeny receives a maternal allele from the boar and the other a paternal allele are excluded.
  • the other inheritance vectors are considered to be equally probable and a "normalized score" Z (v) is calculated, which has a mean value of 0 and a variance of 1 under the null hypothesis Ho (no link between disease locus and marker allele).
  • v normalized score
  • NPL non parametric lodscore
  • Example 1 Creation of a resource population and isolation of the DNA A test population was created in the F 2 generation of which there are high incidence of teats.
  • the F 2 test population is based on the reciprocal crossing of the Berlin miniature pig with Duroc.
  • a Duroc boar with four sows of the breed Berlin miniature pig and a miniature pig boar with five Duroc sows were mated repeatedly using artificial insemination.
  • the animals were mated to one another by the F ⁇ generation.
  • the resource population the characteristics of the mammal quality, in particular the number of teats and their location, were recorded. In the population, 53.6% of the animals are affected by a mammal anomaly.
  • the proportion of animals that show at least one teat is 42.2%.
  • the phenotypic teat anomalies were recorded in 969 F2 animals of the resource population.
  • the evaluation of the suckles took place approximately between the 130th and 200th day of life. This period largely coincides with that of the youngster selection in breeding practice.
  • DNA was isolated from both blood and tissue (tail and ears). The isolation of high molecular DNA was carried out in the following steps by disrupting the cells and releasing the DANN using ProteineaseK digestion, phenol-chloroform extraction and ethanol precipitation.
  • the F 2 generation of the test population was initially genotyped using 69 microsatelite markers, which cover the genome with an average spacing of 35 cM. After evaluating the gel images and assigning the genotypes, the genetic maps were calculated and the statistical analysis was carried out to identify QTL regions. To narrow the QTL regions, additional microsatellites and type I markers were typed in two test areas; a total of 85 DNA markers were analyzed in this way. Nonparametric approaches were used for statistical analysis. In order to achieve a level of significance of 1% in a genome-wide study, an NPL value of approximately 4.3 is necessary for an F 2 population. The corresponding NPL value at a significance level of 5% is approximately 3 (Lander and Kruglyak 1995).
  • NPL> 4.3 A total of six QTL regions with highly significant NPL values (NPL> 4.3) were identified that contain one or more genes that have a significant effect on the occurrence of teats. The results are summarized in the table below.
  • the length of the entire chromosome 1 is 145.3 cM for the gender-neutral map in this study, while it is 110.4 cM for the female map and 143.6 cM for the male map.
  • the coupling map of chromosome 1 is shown in Figure 1.
  • the microsatellite markers used are highly polymorphic.
  • the microsatellites SW1515 and SW1851 each have four alleles, the microsatellite S0155 has three, SW1301 has seven alleles and is highly informative. A significant effect could be identified on chromosome 1 with the microsatellites used.
  • the NPL value in the localization of microsatellite SW1301 reached 4.7.
  • the QTL effects on chromosome 1 are shown in Figure 2.
  • the microsatellites S0227, S0001, S0214 and S0097 were genetically mapped on chromosome 4.
  • the length of the entire chromosome 4 in the gender-neutral map is 117.7 cM in this study, while it is 165.9 cM in the female map and 101.5 cM in the male map.
  • Coupling map of chromosome 4 is shown in FIG. 3.
  • FIG. 4 shows the result of the coupling analysis for teats on chromosome 4. There is no effect in the area between microsatellites S0227 and S0001. From the microsatellite S0001, the graph rises to the location of the marker S0097, where it reaches its maximum value and drops immediately. On chromosome 4, an NPL value of 5.0 was achieved for the feature teat.
  • the microsatellite loci S0035, S0087, S0220, S0003 were mapped on chromosome 6.
  • the length of the gender-neutral coupling card of chromosome 6 is 147.7 cM.
  • the length of the female coupling card is 193.4 and that of the male 184.4 cM. 5 shows the microsatellites that were used in this work.
  • the microsatellite markers S0035, S0087, S0220 and S0003 were used on chromosome 6. The distribution of the 4 microsatellites on chromosome 6 is even. The number of alleles is three for S0220 and S0035, four for S0003 and five for S0087.
  • the S0220 microsatellite delivers a high PIC value of 0.85.
  • the PIC of the other microsatellite markers is 0.80 for S0035, 0.67 for S0003 and 0.64 for S0087.
  • the highest NPL value for the feature teat was identified on chromosome 6 in the area of marker S0220.
  • the NPL value reaches a maximum value of 8.8. This value is highly significant.
  • the course of the NPL values on chromosome 6 is shown in FIG. 6.
  • the SW857, S0007 and SWC27 microsatellites were genetically mapped on chromosome 14.
  • the length of the entire chromosome 14 is 95.2 cM for the gender-neutral map in this investigation, while it is 90.2 cM in the female map and 100.7 cM in the male map.
  • Microsatellites on chromosome 14 are shown in FIG. 7.
  • Genotyping with the microsatellites SW857, S0007 and SWC27 resulted in over 2736 genotypes for QTL analysis on chromosome 14.
  • the microsatellite markers S0007 and SWC27 used were not so highly polymorphic with PIC values of 0.60 and 0.68.
  • the PIC value for microsatellite SW857 is 0.85.
  • the number of alleles identified is three for SWC27 and four for SW857 and S0007.
  • microsatellites SW2410, S0086, S0144 and SW61, were genetically mapped on chromosome 8, the length of the gender-neutral coupling card being 101.0 cM.
  • the lengths of the female and male coupling cards 116.4 and 87.7 cM were estimated on chromosome 8.
  • the gender-neutral, female and male coupling map of four microsatellites on chromosome 8 is shown in FIG. 9.
  • the microsatellite markers SW2410, S0086, S0144 and SW61 for chromosome 8 were selected and genotyped for the analysis of gene effects on teats.
  • KIT1 on chromosome 8 was also included in the analysis.
  • FIG. 10 shows the result of the coupling analysis for teats on chromosome 8.
  • Example 8 QTL and coupling analysis of chromosome 10
  • the SW830, S0070 and SW2067 microsatellites were genetically mapped on chromosome 10.
  • the length of the entire chromosome 10 is 135 cM in the gender-neutral map in this investigation, while it is 134.6 cM in the female map and 129.3 cM in the male map.
  • the coupling map of three microsatellites on chromosome 10 are shown in FIG. 11.
  • NPL value of 2.3 was identified on chromosome 10.
  • the PIC value of markers SW2067 and SW830 is low at 0.32 and 0.50 respectively, while marker S0070 has a larger PIC value of 0.85.
  • the graph of gene effects on teats on chromosome 10 runs below 1.5 with a maximum value above 2.5, which is far below the level of significance.
  • the course of the NPL on chromosome 10 is shown in FIG.
  • microsatellites S0143, SW874 and SW605, were genetically mapped on chromosome 12, with the length of the gender-neutral coupling card being 90.8 cM. On chromosome 12, the lengths of the female and male coupling cards were estimated to be 116.4 and 87.7 cM.
  • the gender-neutral, female and male coupling map of 4 microsatellites on chromosome 8 are shown in FIG. 13. An NPL value of 1.9 was identified on chromosome 12.
  • the microsatellites selected for QTL analysis on chromosome 12 are evenly distributed. Genotyping with the microsatellites S0143, SW874 and SW605 resulted in over 2736 genotypes for QTL analysis on chromosome 12.
  • microsatellite markers used were highly polymorphic with PIC values of 0.87 for SW605, 0.81 for SW874 and 0, 63 for S0143.
  • the number of alleles identified is six for SW874; four for SW605 and three for S0143.
  • the course of the NPL values on chromosome 12 is shown in FIG. 14. The maximum value is two.
  • the coupling between the markers S0143, SW874 and SW605 on chromosome 12 and the characteristic teat is clearly not significant.
  • Example 10 QTL and coupling analysis of chromosome 16
  • the microsatellite loci S0111, S0026 and S0061 were mapped on chromosome 16.
  • the length of the gender-specific coupling map of chromosome 16 is 94.7 cM.
  • the length of the female coupling card is 103.6 and the length of the male coupling card of chromosome 16 is 87.8 cM.
  • the gender-neutral, female and male coupling map of three microsatellites on chromosome 16 are shown in FIG. 15.
  • Three microsatellite loci, S0111, S0026 and S0061, were selected and genotyped for QTL analysis for the feature teat on chromosome 16. 2736 genotypes were available for performing the QTL analysis.
  • the allele number of microsatellite markers is three for S0061, four for S0026 and five for S0111.
  • the PIC value is 0.70 for marker S0061, 0.82 for marker S0111 and 0.87 for marker S0026.
  • the gene effect on chromosome 16 is particularly high with the microsatellite markers S0026 and S0061 with a value of 4.2.
  • the curve rises between the microsatellite markers S0111 and S0026 and reaches its maximum value on this chromosome of 4.2.
  • marker S0026 drops to 3.3 and rises again to form a second peak with a value of 4.1.
  • the course of the NPL on chromosome 16 was shown in FIG. 16.
  • the SW1023, SW787 and SWR414 microsatellites were genetically mapped on chromosome 18.
  • the length of the entire chromosome 18 is 68.8 cM for the gender-specific map in this investigation, while it is 78 cM in the female map and 61.3 cM in the male map.
  • Microsatellites on chromosome 18 are shown in FIG. 17.
  • FIG. 18 shows the result of the association analysis for teats on the chromosome
  • Microsatellite S2443 a value of 7.9.
  • microsatellites SW72 (51, 1 cM), S0164 (25.6 cM) SW2570 (32.6 cM) S0002 were genetically mapped on chromosome 3 with the specified distances. A significant effect could be identified on chromosome 3 with the microsatellites used become.
  • Ear notch samples from rated gilts as well as further ear notch or sperm samples from their parents were collected in breeding establishments from three breeding organizations. These animals were used for the coupling analysis and the investigation of the
  • the sample material from the commercial breeding populations comprises 201
  • Table 3 provides an overview of the animal material in the breeding population.
  • Table 6 Number of gilts and genetic structure of the sample material from the breeding populations
  • Breeding associations B C Total gilts rated (excluding parents) 105 30 66 201 affected 84 30 57 171 not affected 21 0 9 30 paternal half-sibling groups 19 8 10 37
  • the coupling analysis in animals from commercial herds shows a QTL with a maximum NPL of 2.6 for chromosome 6 when calculated over the entire animal material with 70 affected sibpairs from the breed Academic Landrasse, DL, and their crossing with Deutscher Edelschwein, DE This is in the position in which a QTL region with NPL 3.8 (SSC6 position OcM microsatellite S0035) was detected in animals of the DUMI resource population, but not the maximum NPL of 8.8 (SSC6 position 120 cM microsatellite) S0220) for this chromosome.
  • Including only the 45 affected sibpairs of the DL breed shows a maximum NPL of 1.9 in the position of the microsatellite S0220.
  • the development of the mammary gland is regulated by a number of hormones.
  • hormones In particular, the influence of sex hormones and metabolic hormones on the development of the mammary gland has been established and has been experimentally proven in various animal species (Oka et al., 1991; Günther, 1984). The effects of these hormones are mediated by various growth factors (Oka et al., 1991). It can be assumed that the development of lactation abnormalities is due to a dysregulation of the hormonal control. Therefore, genes coding for hormones, hormone receptors and growth factors are candidate genes for the analysis of teat abnormalities.
  • SNPs were identified by comparative sequencing of the project Transcripts of an adult animal of the breeds Duroc, Berlin miniature pig, Hampshire, Pietrain, German landrace, German noble pig as well as the F1 and F2 of the resource population.
  • the other candidate genes are genotyped using the polymorphisms described in the literature.
  • Chi-square tests and transmission disequilibrium tests were carried out for statistical analysis of the relationship between genotypic variation in the candidate gene locations and the trait.
  • the chi-square tests were carried out with the program package SAS (SAS, 2000), the TDTs with the program TDT / S-TDT program 1.1 (Shman et al., 1993).
  • genes TGFB1, LEPR on chromosome 6 and RLN on chromosome 1 map within the QTL regions identified here also represent positional candidate genes.
  • the test results are supported by the fact that the position of the leptin receptor and the TGFB1 on chromosome 6 is in the chromosome area, which also had a highly significant NPL score (8.8) in the QTL analysis.
  • the relaxin maps to chromosome 1 in the area of the QTL.

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Abstract

La présente invention concerne l'utilisation d'un premier acide nucléique pour déterminer la prédisposition à l'expression ou à la transmission héréditaire de la caractéristique quantitative des mamelles rentrées chez un mammifère, ce premier acide nucléique ayant une longueur d'au moins 8 nucléotides et étant identique ou essentiellement identique à un deuxième acide nucléique qui est présent dans le domaine du microsatellite S0220 sur le chromosome 6 du porc ou dans une position homologue dans le génome d'autres mammifères, ou du microsatellite SW2443 sur le chromosome 2 du porc ou dans une position homologue dans le génome d'autres mammifères, ou du microsatellite S0097 sur le chromosome 4 du porc ou dans une position homologue dans le génome d'autres mammifères, ou du microsatellite S0007 sur le chromosome 14 du porc ou dans une position homologue dans le génome d'autres mammifères, ou du microsatellite SW1301 sur le chromosome 1 du porc ou dans une position homologue dans le génome d'autres mammifères, ou du microsatellite S0164 sur le chromosome 3 du porc ou dans une position homologue dans le génome d'autres mammifères. La présente invention concerne en outre des procédés permettant de déterminer la prédisposition à l'expression ou à la transmission héréditaire de la caractéristique quantitative des mamelles rentrées chez des mammifères, de préférence des animaux domestiques, d'élevage ou utiles, qui consistent à analyser les mammifères, leurs cellules reproductrices fécondées ou non fécondées ou leur sperme à la recherche de la présence ou de l'expression du deuxième acide nucléique mentionné ci-dessus. La présente invention concerne enfin un kit contenant au moins une paire d'amorces pour l'amplification d'un des deuxièmes acides nucléiques susmentionnés, une amorce se liant au brin + et une autre amorce se liant au brin de l'acide nucléique, ou une sonde d'hybridation d'une longueur d'au moins 8 nucléotides qui se lie à un des deuxièmes acides nucléiques susmentionnés, ou un anticorps spécifique ou un fragment d'anticorps qui se lie au deuxième acide nucléique susmentionné.
PCT/EP2003/001045 2002-02-05 2003-02-03 Marqueurs genetiques pour le diagnostic de l'expression des mamelles rentrees chez les animaux domestiques, d'elevage et utiles WO2003066891A2 (fr)

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Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
CASSADY J P ET AL: "Identification of quantitative trait loci affecting reproduction in pigs." JOURNAL OF ANIMAL SCIENCE. UNITED STATES MAR 2001, Bd. 79, Nr. 3, März 2001 (2001-03), Seiten 623-633, XP001165627 ISSN: 0021-8812 *
CLAYTON G A ET AL: "INHERITANCE OF TEAT NUMBER AND TEAT INVERSION IN PIGS" ANIMAL PRODUCTION, Bd. 33, Nr. 3, 1981, Seiten 299-304, XP008022045 ISSN: 0003-3561 *
HIROOKA H ET AL: "A whole-genome scan for quantitative trait loci affecting teat number in pigs." JOURNAL OF ANIMAL SCIENCE, Bd. 79, Nr. 9, September 2001 (2001-09), Seiten 2320-2326, XP001165727 ISSN: 0021-8812 *
KRUGLYAK L ET AL: "Parametric and nonparametric linkage analysis: a unified multipoint approach." AMERICAN JOURNAL OF HUMAN GENETICS. UNITED STATES JUN 1996, Bd. 58, Nr. 6, Juni 1996 (1996-06), Seiten 1347-1363, XP002920344 ISSN: 0002-9297 *
KUENZI M J ET AL: "Relaxin acts directly on rat mammary nipples to stimulate their growth." ENDOCRINOLOGY. UNITED STATES JUL 1995, Bd. 136, Nr. 7, Juli 1995 (1995-07), Seiten 2943-2947, XP002254837 ISSN: 0013-7227 *
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ROHRER G A ET AL: "A comprehensive map of the porcine genome" PCR METHODS AND APPLICATIONS, COLD SPRING HARBOR, NY, US, Bd. 6, Nr. 5, Mai 1996 (1996-05), Seiten 371-391, XP002193166 ISSN: 1054-9803 *
ROHRER G A: "Identification of quantitative trait loci affecting birth characters and accumulation of backfat and weight in a Meishan-White Composite resource population." JOURNAL OF ANIMAL SCIENCE, Bd. 78, Nr. 10, Oktober 2000 (2000-10), Seiten 2547-2553, XP001165628 ISSN: 0021-8812 *
See also references of EP1472378A2 *
TRAKOOLJUL N ET AL: "A highly polymorphic repetitive polypyrimidine/polypurine (CCTTT)n sequence in the 5' untranslated sequence of the porcine androgen receptor gene." ANIMAL GENETICS. ENGLAND AUG 2000, Bd. 31, Nr. 4, August 2000 (2000-08), Seiten 288-289, XP002254835 ISSN: 0268-9146 *
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