WO2004020663A2 - Marqueurs genetiques permettant de diagnostiquer la predisposition a l'heredite ou a l'expression du phenotype d'imperforation de l'anus chez des animaux de compagnie, domestiques et d'elevage - Google Patents

Marqueurs genetiques permettant de diagnostiquer la predisposition a l'heredite ou a l'expression du phenotype d'imperforation de l'anus chez des animaux de compagnie, domestiques et d'elevage Download PDF

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WO2004020663A2
WO2004020663A2 PCT/EP2003/008786 EP0308786W WO2004020663A2 WO 2004020663 A2 WO2004020663 A2 WO 2004020663A2 EP 0308786 W EP0308786 W EP 0308786W WO 2004020663 A2 WO2004020663 A2 WO 2004020663A2
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nucleic acid
animals
mammals
microsatellite
genome
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PCT/EP2003/008786
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German (de)
English (en)
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WO2004020663A3 (fr
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Hans-Rudolf Fries
Georg Thaller
Sabine Wiedemann
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Förderverein Biotechnologieforschung Der Deutschen Schweineproduktion E.V.
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Priority to AU2003260386A priority Critical patent/AU2003260386A1/en
Priority to EP03790882A priority patent/EP1529119A2/fr
Publication of WO2004020663A2 publication Critical patent/WO2004020663A2/fr
Publication of WO2004020663A3 publication Critical patent/WO2004020663A3/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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the use of a first nucleic acid for determining the predisposition to the expression or inheritance of the phenotype "afterlessness" 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 on chromosome 1 of the pig or in a homologous position in the genome of other mammals, specifically in the area of a microsatellite selected from the group consisting of SW2185, SW1621, SW1902, S0155, and S0320; or on chromosome 3 of the pig or in a homologous position in Genome of other mammals, in the area of microsatellite S0002; or on chromosome 9 of the pig or in a homologous position in the genome of other mammals, in the area of a microsatellite selected from the group consisting of SW2401 and S0081; or on chromosome 12 of the pig or in a homologous
  • the invention further relates to methods for determining the predisposition to the expression or inheritance of the characteristic "afterlessness" 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
  • the invention relates to a kit, at least containing a pair of primers for the amplification of one of the above 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 that binds to one of the above-mentioned second nucleic acids, or a specific antibody or an antibody fragment that binds to the second nucleic acid disclosed above.
  • hereditary defects in newborn piglets cause considerable financial losses, both through direct animal loss and the associated veterinary treatment costs. It also consists of Due to the Animal Welfare Act ⁇ 11 b paragraphs 1 and 2 there is a need to avoid the spread of such defects within animal breeding and to save pain, suffering and agony for affected animals.
  • the hereditary defect "Atresia ani” means the congenital lack of anus opening.
  • the clinical picture "Atresia recti" can also be found. In this form of the disease, the rectum is missing or ends blindly in the pelvic cavity.
  • 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). The fact that it is relatively easy to identify the molecular genetic markers and that they are available in large numbers are advantages over other marker systems such as biochemical or immunological markers.
  • 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 Around 65,000 to 100,000 microsatellites 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 March 5, 2001). A small number of ETL traits in pigs and other mammals can be predicted using genetic markers. To date, however, there is no possibility of demonstrating a predisposition to inheritance or the expression of the phenotype "afterlessness".
  • Genotyping or genome screening procedures determine whether the presence of certain polymorphic sections of DNA or specific alleles of a gene correlates with the inheritance or expression of a phenotype (association). It can be assumed that the polymorphic DNA associated with the trait is located in the vicinity of the gene responsible for the phenotype and is therefore, with a certain probability, inherited together with it. Become two or more High-frequency polymorphic markers are inherited together, so they define a quasi-stable genetic "haplotype". The association of a specific haplotype with a phenotype (eg that of "afterlessness”) can be used as a diagnostic or prognostic marker that enables statements to be made about the likelihood of occurrence or about hitting a phenotype.
  • the present invention is therefore based on the object of providing methods and methods by means of which animals can be identified which are predisposed to the expression of the phenotype "anuslessness" or inherit such a predisposition Characterized claims solved embodiments.
  • the invention thus relates to the use of a first nucleic acid for determining the predisposition to the expression or inheritance of the phenotype "afterlessness" 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 on chromosome 1 of the pig or in a homologous position in the genome of other mammals, specifically in the area of a microsatellite selected from the group consisting of SW2185, SW1621, SW1902, S0155, and S0320; or on chromosome 3 of the pig or in a homologous position in Genome of other mammals, in the area of microsatellite S0002; or on chromosome 9 of the pig or in a homologous position in the genome of other mammals, in the area of a microsatellite selected from the group consisting of SW2401 and S0081; or on chromosome 12 of the pig or in a homolog
  • 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. It is preferably DNA, but can also be RNA or a DNA or RNA derivative such as PNA.
  • 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 at least 21 nucleotides, most preferably at least 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. In these cases, 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 those nucleic acids that are used in a 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, hybridization probes and primers claimed in the present case which hybridize with the second nucleic acids, preferably under stringent conditions.
  • higher or higher stringency hybridization conditions are understood to mean, for example, 0.2-0.5 ⁇ 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 are dependent on the size or length of the nucleic acid and its nucleotide composition and are to be determined by a person skilled in the art by hand-testing.
  • the solution used for hybridization contains a detergent such as SDS in a concentration of 0.1% to 0.5% and a collection of non-specific nucleic acids to saturate non-specific binding sites.
  • the basic principles of hybridization and the requirements for a hybridization probe are well known to the person skilled in the art. For example, see Maniatis, et al. Molecular Cloning: A laboratory manual, Cold Spring Harbor Press, New York, 1982 or Harnes and Higgins, Nucleic acid hybridization: a practical approach, IRL Press, Oxford 1985.
  • 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.
  • atresia ani is understood by the person skilled in the art to mean the innate lack of anus opening. In addition to this simple manifestation of anuslessness the clinical picture "Atresia recti” can also be found. In this form of the disease the rectum is missing or ends blindly in the pelvic cavity. Both diseases are characterized by a short survival time of the male animals after birth, in female piglets is in some cases caused by a fistula Vagina allows the eradication and thus extends the lifespan.
  • microsatellite SW2185 is located at position SSC1 67.6 cM
  • SW1621 is located at position SSC1 79.6 cM
  • SW1902 is located at position SSC1 83.4 cM
  • S0155 is located at position SSC1 93.9 cM
  • S0320 is located at position SSC1 112.5 cM located
  • S0002 located at position SSC3 102.2 cM
  • S0081 located at position SSC9 77.0 cM is
  • microsatellite SW2185 on chromosome 1 of the pig denotes a position on chromosome 1 which is specific for a population and comprises DNA sections 5cM upstream and / or downstream of the indicated position, preferably up to 10cM upstream and / or downstream, more preferably up to 20cM upstream and / or downstream and most preferably up to 30cM upstream and / or downstream of the indicated position on the chromosome.
  • the microsatellites If the microsatellite is terminal, ie located at the end of the chromosome, in particular less than 30cM from End removed, the upstream or downstream area can also be shorter than 5 cm.
  • 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” denotes nucleic acid segments in the genome of other mammals that have a sequence identity with the second nucleic acid disclosed above, at least preferably 40%, over the entire sequence length or in specific genes located here or at one or more loci or parts thereof with a length of at least 100 nucleotides.
  • 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, Genetics Computer Group, University Research Park, 575 Madison, Wl 53711).
  • Bestfit 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 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 region of 8 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 first nucleic acid can be identical or essentially identical to the + strand or the - strand.
  • a first nucleic acid also includes the fact that more than one (first) nucleic acid can be used in the use according to the invention. These can be, for example, two, three or four nucleic acids.
  • second nucleic acid mean both the + strand and the - strand. If two first nucleic acids are identical or essentially identical to the second nucleic acid, the first nucleic acid can be identical or essentially identical to the + strand, while the other first nucleic acid can be identical or essentially identical to the - strand. In this case, it is preferred that the “alignment” of the first nucleic acid is in opposite directions, which enables 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 expression of the phenotype "afterlessness".
  • the invention disclosed here enables the time of the selection to be shifted significantly forward, so that it no longer depends on the phenotypic expression of the feature
  • the introduction of molecular biological markers can bring about a significant increase in the efficiency of the selection process, which includes the determination of the genotype of the test subject / mammal at one or more loci in a region of the above-mentioned second nucleic acids, preferably in two regions, more preferably three, even more preferably four, more preferably five, most preferably in six areas and the assessment of an individual as suitable for breeding or not suitable including information about the coupling phase e.g. between the genotyped marker locus and the genes responsible for the defect.
  • the genotypes of suitable and unsuitable mammals may differ Distinguish the number of copies of a repetitive nucleotide sequence within the microsatellite locus under consideration.
  • This different nature of the nucleic acid can be represented 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 mammals, on egg cells, or on samples of body fluids such as sperm, urine, blood, tear fluid and other secretions. These can be taken from the animal before diagnosis.
  • anal atresia also known as atresia ani
  • the deep form of anal atresia is one of them
  • the most common form of disease in pigs where the rectum ends blindly at the intact anal membrane, which forms a septum between the endodermal and ectodermal section of the anal canal.
  • the anus pit is usually completely created.
  • atresia recti is a thicker layer of connective tissue between the blind rectum and the surface of the body.
  • Rectal fistulas In severe cases, the rectum can also be completely absent and end blind in the pelvic cavity (Russe, 1991). Rectal fistulas often occur in connection with both anal atresias. These fistulas can affect the rectum with the vagina Bladder or the ureter ve bind (Lambrecht, 1987). In some cases there are also ano-cutaneous fistulas, that is, a connection of the rectum to the surface of the skin, which then lies in the place of the non-existing anus. In animals with the high form of anal atresia (atresia recti), a recto-urethral or recto-vaginal fistula is formed in many cases (Lambrecht, 1989).
  • the different forms and characteristics of the anal atresia in piglets correspond to the different forms in humans.
  • the prevalence of such diseases in humans is 0.048% (4.8 diseases per 10000 children born alive) (Stoll, 1997).
  • the spectrum of the different forms ranges from ectopic anus to atresia ani and recti with urogential fistula to complex deformations of the cloaca.
  • the classification and definition of the disease is handled very differently in the literature.
  • such diseases are not divided into special disease categories, so that the term anal atresia is a collective term for causally different malformations with very differentiated development in the embryonic phase. For example, sewers are sometimes regarded as a different cause.
  • Atresia ani and Atresia recti have been described in numerous other species. These include cattle (Dreyfuss, 1989) and American buffalo (Bison Bison) (Marler, 1977). There are also case studies in sheep (Dennis, 1972), cats and dogs (McAfee, 1976). It can therefore be assumed that anal atresia occurs in most domestic and farm animal species, albeit with very different frequencies.
  • anal atresia is often seen in connection with syndromes.
  • anal atresia often occurs together with Hirschsprung's disease (congenital enlargement of the large intestine) or in connection with VACTERL syndrome.
  • Menlister-Hall and Sacral Agenesis malformations of the urogenital tract and defects of the anorectal system are often found in newborn children.
  • This cloacal membrane migrates dorsally towards the rectum in the course of the normal embryonic development of the rectum, caused by the growth of the sexual bump. This shift is an important part of the division of the cloaca into the urogenital system and rectum by the urogenital septum. In animals with anal atresia, a displacement of the sewage membrane together with the adjacent mesenchymal components could not be observed. In animals with high forms of anal atresia (atresia recti), the cloaca membrane in the dorsal area is also greatly shortened.
  • the aim of statistical methods is to demonstrate the co-segregation of a marker allele with the characteristic in a family.
  • the methods used for this which are used to map disease-correlated genes, can be divided into two categories: (1) Parametric methods for coupling analysis, which are based on a genetic model. These methods presuppose the exact knowledge of the inheritance as well as the parameters that provide information about the occurrence of the disease in the population. This includes the frequency of the disease allele and the penetrance. (2) Nonparametric methods for coupling analysis. Their methods do not assume a specific inheritance model and are therefore often referred to as model-free procedures. Parametric coupling analysis is the classic method for coupling analysis.
  • this method is based on the observation and determination of recombinant and non-recombinant offspring within a family, to estimate the recombination rate ⁇ between marker and phenotype.
  • the recombination rate results from the quotient of the number of observed recombinants divided by the number of possible meiosis.
  • the statistical test then checks whether the recombination frequency is significantly less than 0.5 (Eiston, 1998, Ott, 1999).
  • 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 to the expression or inheritance of the phenotype "afterlessness". This preferred embodiment is particularly suitable, the reliability of the detection to increase.
  • the invention further encompasses preferred embodiments, the second nucleic acid disclosed above being a microsatellite or a sequence flanking it.
  • the microsatellite is SW2185, SW1621, SW1902, S0155, S0320, S0002, SW2401, S0081, SW957 and S0229.
  • the flanking sequence lies outside of the repetitive sequences typical of the microsatellite and preferably comprises a range of 1 kB upstream or downstream of the repetitive sequences.
  • primers or hybridization probes for determining the predisposition to the expression or inheritance of the phenotype “afterlessness.
  • the primers or hybridization probes come from the range of microsatellites SW2185, SW1621, SW1902, S0155, S0320, S0002, SW2401, S0081, SW957 or S0229 and are identified by the SEQ ID given below
  • the invention relates to the use of at least one primer or at least one
  • Hybridization probe which comprises or consists of a nucleotide sequence which is selected from the group consisting of SW2185 (SEQ ID NO: 9 and 10), SW1621 (SEQ ID NO: 5 and 6), SW1902 (SEQ ID NO: 7 and 8), S0155 (SEQ ID NO: 1 and 2), S0320 (SEQ ID NO: 3 and 4), S0002 (SEQ ID NO: 11 and 12), SW2401 (SEQ ID NO: 17 and 18), S0081 (SEQ ID NO: 13 and 14), SW957 (SEQ ID NO: 21 and 22) and S0229 (SEQ ID NO: 19 and 20).
  • a further preferred embodiment of the invention relates to the use of two primers, the primers being oriented in opposite directions with respect to the complementary DNA region and thus, for example, enabling PCR amplification.
  • the second nucleic acid is a specific gene or a part of a gene.
  • genes selected from the group consisting of SHH Sonic hedgehog, IHH Indian hedgehog, DHH Desert hedgehog, PTCH1 Patched homolog 1, PTCH2 Patched homolog 2, PRKAR 1 Protein kinase cAMP-dependent regulatory type I, HIP Hedgehog-interacting protein, GLI 1 GLI -Kruppel family member GLI 1, GLI 2 GLI- Kruppel family member GLI 2, GLI 3 GLI-Kruppel family member GLI 3, SMOH Smoothened, CKTSF1B1 Cysteine Knot Superfamily 1, FGF4 Fibroblast growth factor 4, FGF10 Fibroblast growth factor 10, FGF8 Fibroblast growth factor 8, RARA retinoid acid receptor alpha, SOX9 / SRY (Sex determining region Y) -box 9, BMP2 bone morpho genetic protein 2, BMP4 Bone morphogenetic protein 4, NOG Noggin, FMN Formin, ALDH1A1 Aldehyde dehydrogenase 1 family member A1, ALDH1
  • Genes or parts of genes in the sense of the invention can comprise the coding as well as the non-coding sections of the DNA, ie introns, exons and regulatory areas such as promoters or other control elements of gene expression.
  • 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 a first nucleic acid for determining the predisposition for the expression or inheritance of the phenotype "afterlessness" in a mammal, the first nucleic acid having a length of at least 8 nucleotides and being identical or essentially identical to one second nucleic acid, which occurs on chromosome 1 of the pig or in a homologous position in the genome of other mammals, namely in the range of microsatellites SW1621 and SW1902, the common presence on the same chromosome of an individual from allele 1 (146bp, according to Rohrer et al.
  • microsatellite SW1621 and allele 2 150 bp, according to Rohrer et al., 1996) of the microsatellite SW1902 defines a haplotype that correlates with a predisposition to the expression or inheritance of the phanotype "afterlessness".
  • the invention relates to the use of the disclosed first or second nucleic acids for the selection of domestic, breeding or farm animals with the missing feature "afterlessness".
  • the domestic, breeding or Farm animals cattle, dog, cat, rabbit, buffalo, camel, alpaca, mink, pig, goat, sheep, horse, donkey, rat or mouse.
  • a genomic screen is used on multiple mammals in a population.
  • the term "several mammals” includes at least two animals one Population, preferably at least 5 animals, more preferably at least 8 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.
  • 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 "afterlessness"
  • a common inheritance of nucleic acids with the phenotypic expression of the characteristic "afterlessness” implies a genetic coupling of the nucleic acid to the characteristic.
  • markers are the microsatellites SW2185, SW1621, SW1902, S0155, and S0320 on chromosome 1 of the pig or of microsatellites in a homologous manner Position in the genome of other mammals; or microsatellite S0002 on chromosome 3 of the pig or microsatellites in a homologous position in the genome of other mammals; or the microsatellites SW2401 and S0081 on chromosome 9 of the pig or of microsatellites in a homologous position in the genome of other mammals; or the Microsatellites SW957 and S0229 on chromosome 12 of the pig or of microsatellites in a homologous position in the genome of other mammals
  • other nucleic acid sequences can also be used as markers, provided that they are identical or essentially identical to that in the sense of the invention second nucleic acid disclosed in the invention or in one of the above-mentioned nucleic acid
  • the person skilled in the art can readily use or develop detection methods for determining the predisposition for the expression of the phenotype "afterlessness".
  • the invention also discloses methods for determining the predisposition for the expression of the phenotype "afterlessness" in domestic and breeding -, or farm animals, whereby the animals, their fertilized or unfertilized egg cells, their sperm, tissue samples or samples of body fluids are checked for the presence, Expression or nature of one of the above-mentioned second nucleic acids is tested.
  • test methods are preferably in vitro test methods. Different "forms or textures" of nucleic acids can be caused, for example, by insertions, duplications, deletions, substitutions or translocations.
  • Inserts or deletions 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 represented 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 be, for example, a closely related gene variant, which in extreme cases is only distinguished from the related gene sequence by a single nucleotide exchange.
  • Such different “forms or qualities” of the nucleic acid can optionally be represented with the aid of RFLP analyzes (restriction fragment length polymorphisms (RFLPs) or by nucleic acid sequencing.
  • RFLPs restriction fragment length polymorphisms
  • the domestic, breeding or farm animals are cattle, dog, cat, rabbit, buffalo, camel, alpaca, mink, pig, goat, sheep, horse, donkey, rat or mouse.
  • the methods according to the invention also relate to other mammals, in particular humans.
  • 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 hybridization is carried out, wherein a hybridization probe with a length of at least 8 nucleotides binds to the second nucleic acid, or sequencing of the second nucleic acid is carried out, or detection is carried out with a specific antibody or antibody fragment or antibody derivative or an aptamer, wherein 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 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.
  • a typical reaction mixture of 12.5 ⁇ l is composed, for example, as follows: 0.20 ⁇ M primer, 200 ⁇ M dNTPs, 0.50 U Taq polymerase, 1.25 ⁇ l 10 x buffer, 1.50 ⁇ l DNA (50ng / ⁇ l) and made up to 12.5 ⁇ l with H 2 O.
  • the reaction conditions listed in the method part of this application are preferably selected.
  • 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 80 nucleotides, preferably at least 70 nucleotides, more preferably at least 50 nucleotides, even more preferably at least 30 nucleotides and most preferably 20, 17, 15, 13, 12 or 8 nucleotides.
  • the nucleotide sequences of the primers can be put together as desired from the second nucleic acid sequences disclosed above, provided that they have at least 8 consecutive nucleotides exhibit.
  • primers with the target sequence within the second nucleic acid can also lead to base mismatches, provided that hybridization occurs 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 methods are described, for example, in Newton, PCR, BIOS Scientific Publishers Limited, 1994, and subsequent editions.
  • RT-PCR reverse polymerase chain reaction
  • other amplification methods have also been developed in recent years, which also represent preferred embodiments of the invention.
  • amplification methods are, for example, the “Ligase Chain Reaction” (LCR, EPA 320308), “Cydic 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).
  • 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 , still more preferably 200, 300, 400, 500, 600, 700, 800 or 1000 nucleotides and most preferably up to 5000 nucleotides attached to one of the second nucleic acids disclosed above 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 (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor and all subsequent editions).
  • Another preferred detection method for determining the predisposition to the expression of the phenotype "afterlessness" is the sequencing of one of the second nucleic acids disclosed above. Sequencing methods are known from the prior art and require no further explanation for the person skilled in the art. For example, here Sambrook et al.
  • the primers are understood according to the invention to be nucleic acids which are preferably at least 70 nucleotides, more preferably at least 50 Nucleotides, more preferably at least 30 nucleotides and most preferably 20, 17, 15, 13, 12 or 8 nucleotides
  • the properties shown for the PCR primers apply accordingly to sequencing primers.
  • primers or hybridization probes which are derived from the second nucleic acids mentioned above, 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.
  • nucleic acids, hybridization probes and primers can also be constructed from base derivatives. A number of modifications change the chemistry of the phosphodiester backbone of the DNA or RNA, the sugar or heterocyclic bases.
  • the useful modofications include phosphorothioates; Phosphorodithioates, in which both oxygen atoms not involved in hydrogen bonding by sulfur, Phosphoramides, alkyl phosphotriesters and / or boranophosphates are replaced.
  • Achiral phosphate derivatives include S'-O'- ⁇ '-S phosphorothioates, 3'-S-5'-0-phosphorothioates, 3'-CH2-5'-0-phosphonates and 3'-NH-5'- 0 phosphoroamidates.
  • the entire backbone of the phosphodiester can be replaced by peptide bonds.
  • Sugar modifications are used to change stability or affinity.
  • the A anomer of deoxyribose can be used with the base inverted with respect to the natural B anomer.
  • the 2'-OH group of the ribose can be changed to the corresponding 2'-0-methyl or 2'-0-allyl sugar, whereby a gain in stability is achieved without impairing the binding affinity.
  • Some other useful substitutions include deoxyuridine instead of deoxythymidine; 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine instead of deoxycytidine.
  • 5-Propyyl-2'-deoxyuridine and 5-propyyl-2'-deoxycytidine can replace deoxythymidine and deoxycytidine and thus increase affinity and biological activity.
  • the nucleic acids can have a label for detection.
  • radioactive labeling for example with 35 S, 32 P or 3 H fluorescent labeling, biotin labeling, digoxigenin labeling,
  • Suitable markers include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2 ', 7'-dimethoxy-4', 5'-dichloro-6-carboxyfluorescein ( JOE), 6-Carboxy-X-Rhodamine (ROX), 6-Carboxy-2 ', 4', 7 ', 4,7-Hexachlorofluorescein (HEX), 5-Carboxyfluorescein (5-FAM) or NNN'.N' -Tetramethyl-ö-carboxyrhodamine (TAMRA).
  • fluorochromes e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM),
  • the label can also be part of a multi-stage system, the nucleic acid being conjugated with biotin, or with a hapten or a similar substance that has a high-affinity binding partner, for example avidin, specific antibodies, etc., in which case the binding partner with a detectable compound is conjugated.
  • the label can be conjugated with a primer and / or the nucleotides in the pool of the amplification reaction can be provided with a suitable label so that the label is inserted into the new resulting amplification product is installed.
  • double strands that have arisen in a hybridization reaction can also be detected by DNA double strand specific antibodies. Said antibodies are characterized in that they only bind to double-stranded DNA, but not to single-stranded DNA.
  • 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. Fv, 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. be made visible by labeling the primary antibodies or is detected with the aid of antibody-binding second antibodies, which in turn are then labeled.
  • the antibodies can e.g. be modified with fluorescent substances, by radioactive labeling or an enzymatic labeling.
  • Immunological detection methods using specific antibodies, as well as the generation of antibodies and fragments or derivatives thereof are, as already mentioned, 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 mammals in a population.
  • the term "Several mammals" comprises at least two animals of a population, preferably up to 5 animals, more preferably 8 animals, more preferably 10 animals, more preferably 50 animals, still more preferably 250 animals, most preferably 1500 animals.
  • 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, still more preferably 1000 nucleotides, most preferably up to 5000 nucleotides or longer, is inherited together with the feature "afterlessness".
  • Preferred markers are the microsatellites SW2185, SW1621, SW1902, S0155 , and S0320 on pig chromosome 1 or the microsatellites in a homologous position in the genome of other mammals; or microsatellite S0002 on pig chromosome 3 or a microsatellite in a homologous position in the genome of other mammals; or the mic rosatellites SW2401 and S0081 on chromosome 9 of the pig or the microsatellites in a homologous position in the genome of other mammals; or the microsatellites SW957 and S0229 on chromosome 12 of the pig or the microsatellites in a homologous position in the genome of other mammals.
  • nucleic acid sequences can also be used as markers, provided that they are identical or essentially identical in the sense of the invention to the second nucleic acid disclosed in the invention or are in one of the above-mentioned nucleic acid regions.
  • 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 binding to the + strand and another primer 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 70 nucleotides, more preferably at least 50 nucleotides, even more preferably at least 30 nucleotides and most preferably 25, 22, 20, 17, 15, 13, 12 or 8 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, RIA, 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 others Detection methods. Immunological detection methods and methods for generating specific antibodies are known from the prior art.
  • the components of the kit can be packaged 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 Information content (info) of the nonparametric multipoint coupling analysis when increasing the marker density to SSC1.
  • Figure 2 Course of the NPL a ⁇ S pt statistics when increasing the marker density to SSC 1.
  • Figure 3 Information content of the nonparametric single point coupling analysis when increasing the marker density to SSC 1.
  • Figure 5 Information content of the multipoint NPL statistics on SSC1 in coupling analyzes with different numbers of families and different marker density.
  • Figure 7 Information content of the Singlepoint NPL statistics on SSC1 in coupling analyzes with different numbers of families and different marker density.
  • Figure 8 Frequency distribution of the haplotypes, derived from the markers SW 1621 and SW 1902 on SSC1.
  • the haplotypes of the piglets are marked in blue and the haplotypes of the boars in yellow.
  • the genealogical examination was carried out with ten microsatellite markers distributed over the genome, which also represented the first set of markers for genome-wide typing. At the time of typing, there was no information about the number of alleles and the allele frequencies existing in the populations for these markers. According to Garber (1983), the probability PE ⁇ for the detection of an incorrect parentage was determined for each marker. This calculation takes into account that only a typed parent, usually the boar or in some cases the mother animal, is available for the pedigree control in the animal material. The allele frequencies required for this were estimated from the genotypes of the typed boars and piglets, since no typing information was available from the dams.
  • the 20 microsatellite markers used are characterized in more detail in Table 1. The number of alleles, the chromosome (SSC), the heterozygosity, the polymorphism information content (PIC) and the probability PE ⁇ for the detection of an incorrect parentage are listed for each marker.
  • Table 1 Characterization of the 20 microsatellite markers for parentage control.
  • Table 2 Characteristics of anuslessness and the sex of the typed piglets.
  • Atresia ani and Atresia recti piglets were classified in one disease category and the two forms of anuslessness were considered the same defect.
  • the animal material without Atresia recti animals was evaluated. The number of families with 2 HG was reduced by 2, 4 families with 3 HG lost 1 HG, so that 17 families with 2 HG and 1 family with 3 HG were available for the evaluation. The other family material remained unchanged.
  • Table 3 shows the breeds of all 72 afterless piglets that were in the typing set. The most common is also with 51.4% the cross with DL as the mother breed and Pl as the father breed.
  • Table 3 Breed and crossbreeds of the afterless animals.
  • the supplementary family material consisted of 31 animals, including 23 free piglets, 7 boars and 1 mother sow.
  • Table 4 shows the sex and the nature of the defect in these piglets.
  • Table 5 also gives an overview of the breeds of boars and sows of the 23 afterless animals.
  • Table 4 Characteristics of anuslessness and sex of the piglets.
  • Table 5 Breed of boars and sows with ateless animals (supplementary family material).
  • Marker card The distribution of the microsatellite markers over the genome was aimed at a marker spacing of 20 cM. The criteria for compiling the primer set was the achievement of high quality PCR Amplificates and a minimum number of three amplifying alleles. The information came from a Pietrain x Mangalitza resource population. For the genome-wide typing for defect gene mapping, 130 microsatellites, including the markers from the parentage control, were selected from this set and assembled into multiplex groups of 10-12 markers. The distances on the genetic map of the pig genome and the order of the markers were taken from the database of the US Meat Animal Research Center (Rohrer et al., 1996).
  • the distances between the marker loci on this map were estimated on the basis of recombination rates averaged over both sexes. With a few exceptions, the distance between the markers used was 20-25 cM.
  • a total of eight markers were typed on the sex chromosome (SSC X). Within the pseudo-autosomal region of the sex chromosomes (SSC XY), two markers were used for typing, but PCR amplificates could only be obtained for the SW949 marker. As part of the parametric and nonparametric evaluation, only a coupling analysis between the microsatellite marker SW949 and the supposed disease locus could be carried out.
  • the allele frequencies from the entire typed family material were estimated. Since marker genotypes were only determined for one parent, mostly the father animal, it was not possible to estimate the allele frequencies directly in the parent generation.
  • the transferred maternal alleles were derived from the genotypes of the piglets and boars and the allele frequencies in the maternal population were calculated. Typing the microsatellite markers
  • the first step in the preparation was to wash the sperm cells with 1 x PBS to completely remove the seminal plasma.
  • 500 ⁇ l of native pork sperm were mixed with 1 ml of 1 ⁇ PBS (pH 7.4, 140 mM NaCl, 2.7 mM KCI, 6.5 mM Na 2 HP0 4 , 1.5 mM KH 2 PO 4 ) in a 2 ml Eppendorf tube. Centrifugation was carried out for 3 minutes and at a low speed (3,000 rpm) to prevent the sperm cells from sticking together. The supernatant was then poured off. This washing step was repeated at least twice until the supernatant was no longer viscous.
  • the sperm cell pellet was completely resuspended with 1 ml of 1 ⁇ PBS after centrifugation. After the washing step, the sperm pellet was suspended in 1 ml of lysis buffer (pH 7.4, 1% SDS, 20 mM Tris, 4 mM Na 2 EDTA, 100 mM NaCl) and 150 ⁇ l ProteinaseK (20 mg / ml in bidist. H 2 O) and 50 ⁇ l DTT (1, 4-dithiothreitol, pH 5.2, 1M in 0.01 M NaAcetat) added. Incubation took place overnight at 55 ° C. After the incubation, the solution should be clear and transparent.
  • lysis buffer pH 7.4, 1% SDS, 20 mM Tris, 4 mM Na 2 EDTA, 100 mM NaCl
  • ProteinaseK 20 mg / ml in bidist. H 2 O
  • DTT 1, 4-dithiothreitol, pH 5.2, 1
  • the mixture was incubated again with 150 ⁇ l ProteinaseK (20 mg / ml).
  • the solution was then transferred to a Vacutainer SST 9.5 ml tube (368510, Becton Dickinson). This was followed by extraction with 1000 ⁇ l phenol (pH 7.9; buffered with Tris) and a mixture of chloroform and isoamyl alcohol in a ratio of 24: 1.
  • the DNA precipitation was carried out in a 15 ml Sarstedt tube with 0.8 volume percent isopropanol.
  • the DNA was air-dried and taken up in 150 ⁇ l TE (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA). The DNA yield varied between 10-22 ⁇ g depending on the quality of the sperm.
  • the piglet's muscle tissue which contains a lot of RNA, was treated with 400 ⁇ g RNase (pH 7.4, 20 mg / ml in 10mM NaAcetat (pH 5.2)) for 30 min before the phenol / chloroform extraction. incubated at 37 ° C. The solution was then transferred together with 200 ⁇ l TE (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA) into a Vacutainer SST 9.5 ml tube (368510, Becton Dickinson). Following air drying, the DNA was taken up in 200 ⁇ l TE (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA). The DNA yield varied between 30-60 ⁇ g.
  • the DNeasy kit from Qiagen (cat. No. 29308) was used for DNA extraction from ear tissue.
  • the fluorometer Hoefer DyNA Quant 200, Amersham Pharmacia Biotech
  • 200 ng of Calf thymus DNA 100 ⁇ g / ml in bidist. H 2 O
  • 2 ⁇ l DNA in 2 ml TNE measuring solution pH 7.4, 10 mM Tris, 1 mM EDTA Na 2 • 2H 2 O, 0.2 mM NaCl, 0.1 ⁇ g / ml Hoechst H 33258 (bisbenzimide)
  • 2 ml DNA in 2 ml TNE measuring solution pH 7.4, 10 mM Tris, 1 mM EDTA Na 2 • 2H 2 O, 0.2 mM NaCl, 0.1 ⁇ g / ml Hoechst H 33258 (bisbenzimide)
  • Measurements were made at an excitation wavelength of 350 nm and an emission wavelength of 456 nm.
  • the measured DNA concentration for the TNE measuring solution described above was proportional to the bound Hoechst H 33258 amount in the DNA double strand.
  • the samples were adjusted to a concentration of 25 ng / ⁇ l with TE (pH 8.0) and applied to a 0.8% agarose gel (ethidium bromide) as a control.
  • PCR reactions For the PCR reactions, 200 ⁇ l DNA (25 ng / ⁇ l) were placed in 96-well microtiter plates and covered with 2 drops of mineral oil to protect against evaporation. Mixing the standard master mix (50 mM KCI, 10 mM Tris-HCl (pH 8.3), 200 ⁇ M per dNTP, 1.5 mM MgCI 2l 5 pmol per primer and 0.5 units Perkin- Bucket of AmpliTaq polymerase) for the PCR reactions with a total volume of 20 ⁇ l was carried out in two Eppendorf tubes (1.5 ml) for 52 batches each. A Biomek 2000 (Laboratory Automation Workstation, Beckmann) was used for pipetting the PCR.
  • standard master mix 50 mM KCI, 10 mM Tris-HCl (pH 8.3), 200 ⁇ M per dNTP, 1.5 mM MgCI 2l 5 pmol per primer and 0.5 units Perkin- Bucket of AmpliTaq
  • 6-FAM-labeled PCR products were generally diluted 1:30, TET-labeled PCR products 1:20 and HEX-labeled products 1:10.
  • the standard dilution factor was corrected from the intensity of the PCR bands on the agarose gel.
  • multiplex approaches with up to 12 different markers per animal were created and with bidest.
  • H 2 0 set to the desired concentration.
  • Polyacrylamide gel electrophoresis For the polyacrylamide gel electrophoresis, a 5% acrylamide gel mixture (21 g urea, 8.4 ml acrylamide solution 30%, 6.0 ml 10 x TBE, 10.0 ml bidist. H 2 O) was prepared, filtered and with the help of a membrane vacuum pump for 20 min. degassed. 20 ⁇ l of TEMED (tetramethylethylenediamine, Ameresco, 0761) and 300 ⁇ l of APS (ammonium persulfate, ameresco, 0486) were then mixed in, the gel mixture was poured between glass plates and polymerized in a horizontal position for 1 hour.
  • TEMED tetramethylethylenediamine
  • APS ammonium persulfate, ameresco, 0486
  • the runtime was between 2 and 3 hours, depending on the fragment length of the microsatellites.
  • 1 ⁇ TBE 90 mM trisborate (pH 8.3), 2 mM Na 2 EDTA
  • the fragment lengths were analyzed with.
  • the first evaluation of the typed genotypes showed 134 conflicts between offspring and parents at a total of 43 different markers.
  • the majority of the offspring-parent couples found inconsistencies in one or two marker loci.
  • the microsatellite markers were amplified again so that the mix-ups of the PCR samples could also be excluded.
  • Table 6 Estimated allele frequency r for a non-amplifying allele in the microsatellite markers with genotype conflicts between parents and offspring.
  • the first step of the statistical analysis consisted of the genome-wide nonparametric and parametric coupling analysis of the family material.
  • the marker density in interesting chromosome regions was increased in a second step of the evaluations and the supplementary family material was also genotyped.
  • the family material was examined at 130 markers and evaluated with the Allegro 1.0 program.
  • NPL statistics were calculated for the nonparametric evaluation of the data material. It was used for both test statistics a coupling analysis between the individual markers and the disease locus (single point coupling analysis) was carried out. These test statistics are called NPLpairs spt and NPLaii S p t . A coupling analysis between the markers and the disease locus (multipoint coupling analysis) was also carried out for both NPL statistics, which are referred to as NPL pa irs mpt and NPL a n mpt .
  • the result overviews show the NPL values, the error probabilities of the NPL value (p values) and the information content (info) of the family material calculated by Allegro 1.0 at the respective marker position.
  • p values the error probabilities of the NPL value
  • info the information content of the family material calculated by Allegro 1.0 at the respective marker position.
  • the informativity was 0.45 and 0.47, respectively.
  • the results of the coupling analysis between the individual markers and the disease locus are shown below.
  • the NPL a n S pt and the NPL a ⁇ S p t statistics were calculated for each marker of the genome-wide typing. The results of this evaluation are shown in Table 8. The highest values of the NPL statistics were found on the SSC1.
  • the genetic model of the disease ie the mode of inheritance, the disease parallel frequency and the penetrance of the disease in the population must be specified.
  • the parameters in question were therefore chosen as they are possible or likely to be assumed based on the available data.
  • a monogenic recessive inheritance with a very low penetrance of 1% was assumed.
  • the assumed parallel disease frequency was varied between 0.10 and 0.30 in the statistical evaluation.
  • LOD score calculations based on a monogenic recessive inheritance, with almost complete penetrance of 90%, with disease parallel frequencies of 0.10 and 0.30 are given.
  • LOD scores between several markers and the disease locus multipoint LOD scores
  • m p t the LOD score between several markers and the disease locus
  • coupling analyzes between individual markers and the disease locus were carried out, which are called the LOD score late .
  • Table 9 shows the results of the genome-wide parametric evaluation.
  • the chromosome (SSC), the marker or the position between two markers that has reached the highest LOD scorem Pt under the specified parameters (penetrance, parallel disease frequency) are indicated.
  • the chromosomes for which a positive LOD score mp t could be determined are highlighted in gray.
  • the significance limit of the parametric coupling analysis, which is at an LOD score mp t of 3 was not reached at any marker position of the genome with the exception of SSC 1.
  • an LOD score mpt of 2.38 with a frequency of the disease allele of 0.3 was calculated for SSC 1.
  • a higher LOD score mpt of 2.72 is achieved with a frequency of the disease allele of 0.1.
  • the LOD score mpt is 3.02 and 2.88.
  • Positive LOD-Score mp t values resulted on SSC 3 with incomplete penetrance (1%). They were 1.41 and 1.21.
  • a positive LOD score mpt of 0.41 was only achieved for the disease parallel frequency of 0.3.
  • the LOD score mpt with a value of -0.75 was in the negative range.
  • SSC 12 showed a LOD score of 0.3 with a disease parallel frequency of 0.3 and a penetrance of 1% 1.07 determined.
  • SSC 9 and SSC 13 chromosomes the LOD score sp t values for the same parameters were 0.55, 0.53 and 0.57
  • Table 9 The maximum LOD scores mp of the genome-wide coupling analysis.
  • Table 10 shows the results of the Singlepoint LOD score analysis.
  • the evaluation was carried out under the same comparative parameters, with regard to the parallel disease frequency and penetrance, as the multipoint coupling analysis.
  • the chromosomes that have reached a positive LOD score are highlighted in gray in the table.
  • the highest LOD score values were also on SSC 1.
  • a higher LOD score of 2.40 is achieved with the same penetrance and with a frequency of the disease allele of 0.1.
  • the maximum LOD score is between 2.00 and 1.28.
  • Table 10 The maximum LOD scores late values of the genome-wide coupling analysis.
  • results of the multipoint coupling analysis 8 additional microsatellite markers were genotyped on the SSC 1.
  • the course of the NPL a n t mp statistics on SSC 1 to 14 markers black graph
  • the course engineering of the original NPL a n m pt statistic is faced with 6 markers.
  • the maximum NPL value in the evaluation with 14 markers is somewhat lower than in the first analysis. In contrast, the information content at the NPL maximum was increased significantly from 0.47 to 0.74.
  • Information content (info) of the multipoint coupling analysis The additionally typed markers not only increased the information content at the position of the NPL maximum, but also in the proximal area of SSC 1 (see FIG. 1). Compared to the first genome-wide analysis, the information content in the interesting chromosome range (70-90 cM) is now between 0.74 and 0.88. The information content in the distal area of the chromosome is 0.40. The typing of further microsatellite markers in this region could thus lead to a decisive improvement in the information content.
  • the information content at the individual marker positions of the single point coupling analysis is shown in FIG. 3. It can be seen that increasing the marker density, especially in the proximal area of the SSC 1, significantly increased the information content.
  • the supplementary family material was also included in the typing.
  • Table H Comparison of the NPL a n mpt statistics on SSC1 in the coupling analysis with different family material and variable marker density.
  • Table 12 Comparison of the NPL a ⁇ late statistics on SSC1 in the coupling analysis with different family material and variable marker density.
  • a TDT was carried out to check for coupling and association between the individual marker alleles and the disease locus. Specifically, these were SSC 1, SSC 3, SSC 8, SSC 9, SSC 12, SSC 13 and SSC 15. The significance limit depends on the number of microsatellite markers on which a TDT was carried out. It is separate for each chromosome, along with the results. Results of the TDT on SSC1
  • a TDT test was carried out on 14 markers for chromosome SSC 1. The results are shown in Table 13.
  • the order of the markers in the table reflects the arrangement on the chromosome.
  • the marker SW2185 which had shown the NPL a ⁇ maximum of 2.05 in the confirmation study with 14 markers, only achieved a significant error probability of 0.015 for the T mH e t test statistics.
  • Table 13 Results of the TDT test statistics T m and T m Het on 14 microsatellite markers from SSC1 (significance limit for multiple tests ⁇ m
  • t 0.003).
  • a TDT test with the transmitted and non-transmitted coupling phases or haplotypes was also carried out on the markers SW1621 and SW1902.
  • Allegro 1.0 was used to derive the most likely coupling phases of the markers based on the available family structure and marker information. This possibility was used to determine the haplotypes of the affected HG and the typed Elteri for the markers on SSC 1.
  • the frequencies of the haplotypes for the markers SW1621 and SW1902 are shown in FIG. These two marker positions showed significant results both in the nonparametric coupling analysis and in the test for coupling and association with the TDT.
  • the first position of the haplotype indicates the marker allele on the marker SW1621, the second position shows the allele on the microsatellite marker SW1902.
  • the alleles are numbered consecutively from 1 to 3 on marker SW1621 and from 1 to 8 on marker SW1902.
  • a total of 14 different haplotypes could be derived from the affected piglets and 11 from the boars.
  • the result of the TDT showed with an error probability of p equal to 1 x 10 "4 (level of significance 0.05) that this haplotype was preferentially transmitted to the diseased animals.
  • the results of the TDT on the six markers of the SSC 12 are shown in Table 17.
  • the marker SW957 showed significant test values for the TDT with an error probability of 0.013 and 0.003.
  • Table 17 Results of the TDT test statistics T m and T m Het on 7 microsatellite markers from SSC12 (significance limit for multiple tests ⁇ m
  • t 0.007).
  • Example 6 Additional study data for the TDT on association with the occurrence of anuslessness for the markers SW1621 and SW1902
  • Table 20 shows the results of the TDT test statistics for association for the individual markers and for the corresponding haplotypes (allele combinations).
  • Example 7 Additional study data for the TDT for the markers SW1621 and SW1902 on healthy test animals. From a further work with another question, test material was available at the chair for animal breeding from healthy piglets, which came from boars, which also had afterless piglets. The basic consideration of the test carried out was that these boars pass on the allele associated with the lack of anus to healthy offspring less frequently. Due to the existing circumstances, the low incidence in the population and the low penetrance, it would be expected that no difference can be determined whether these boars transmit the associated or the alternative allele to the healthy offspring. The corresponding results are summarized in Table 21. The TDT test statistics in Table 21 are not significant and differ significantly from those in Table 20.
  • Table 21 Test statistics and error probabilities (p) for the association of marker alleles or haplotypes of markers SW1621 and SW1902 in healthy test animals
  • Example 8 Additional study data for allele frequency estimation for the markers SW1621 and SW1902 on afterless animals. As part of the extensive data and material collection in the field, a number of individual afterless piglets were recorded, of which the parentage was unknown and which was neither found in coupling studies by families nor in Association studies of parent-offspring pairs could be included. These animals can be considered as an independent sample and are suitable for a more precise estimate of the frequencies of associated marker alleles or haplotypes in atless piglets. Furthermore, the associations found so far can be checked from frequency differences between affected and healthy piglets. The estimates of the allele frequencies for both markers are shown in Table 22. It turns out that the associated allele is by far the most common in afterless animals.
  • Table 22 Estimates of allele frequencies for markers SW1621 and SW1902 for afterless piglets
  • Table 23 Estimates of the allele frequencies for markers SW1621 and SW1902 in pure breed animals of the breeds Pietrain and Manual Landrasse

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Abstract

Utilisation d'un premier acide nucléique pour déterminer la prédisposition à l'hérédité ou à l'expression du phénotype d'imperforation de l'anus chez un mammifère, ce premier acide nucléique ayant une longueur d'au moins 8 nucléotides et étant identique ou essentiellement identique à un second acide nucléique qui se trouve sur le chromosome 1 du porc ou dans une position homologue dans le génome d'autres mammifères, et plus précisément dans la région d'un microsatellite choisi dans le groupe constitué par SW2185, SW1621, SW1902, S0155 et S0320, ou sur le chromosome 3 du porc ou dans une position homologue dans le génome d'autres mammifères, et plus précisément dans la région du microsatellite S0002, ou sur le chromosome 9 du porc ou dans une position homologue dans le génome d'autres mammifères, et plus précisément dans la région d'un microsatellite choisi dans le groupe constitué par SW2401 et S0081, ou sur le chromosome 12 du porc ou dans une position homologue dans le génome d'autres mammifères, et plus précisément dans la région d'un microsatellite choisi dans le groupe constitué par SW957 et S0229. La présente invention concerne en outre des procédés permettant de déterminer la prédisposition à l'expression ou à l'hérédité de la caractéristique d'imperforation de l'anus chez des mammifères, de préférence chez les animaux de compagnie, domestiques et d'élevage, selon lesquels les mammifères, leurs ovules fécondés ou non, ou leur sperme sont analysés à la recherche de la présence, des caractéristiques ou de l'expression des deux acides nucléiques susmentionnés. La présente invention concerne en outre un kit, contenant au moins une paire d'amorces pour l'amplification de l'un des deux 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 ayant une longueur d'au moins 8 nucléotides qui se lie à l'un des deux acides nucléiques susmentionnés, ou un anticorps spécifique ou un fragment d'anticorps qui se lie au second acide nucléique mentionné ci-dessus.
PCT/EP2003/008786 2002-08-09 2003-08-07 Marqueurs genetiques permettant de diagnostiquer la predisposition a l'heredite ou a l'expression du phenotype d'imperforation de l'anus chez des animaux de compagnie, domestiques et d'elevage WO2004020663A2 (fr)

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AU2003260386A AU2003260386A1 (en) 2002-08-09 2003-08-07 Genetic markers for diagnosing predisposition to heredity or expression of the imperforate anus phenotype in domestic animals, breeding animals and working animals
EP03790882A EP1529119A2 (fr) 2002-08-09 2003-08-07 Marqueurs genetiques permettant de diagnostiquer la predisposition a l'heredite ou a l'expression du phenotype d'imperforation de l'anus chez des animaux de compagnie, domestiques et d'elevage

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EP02017954 2002-08-09
EP02017954.5 2002-08-09

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CN102719532A (zh) * 2012-05-16 2012-10-10 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 一种通过微卫星标记检测陶赛特羊早期生长发育的方法

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EP1529119A2 (fr) 2005-05-11
AU2003260386A1 (en) 2004-03-19

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