WO2003066891A2 - Genetic markers for the diagnosis of the expression of inverted nipples in pets, breeding animals and domestic cattle - Google Patents

Abstract

The invention relates to the use of a first nucleic acid for determining the predisposition for the expression or heredity of the quantitative characteristic of inverted nipples in a mammal, whereby the first nucleic acid is present in the region of microsatellite S0220 or microsatellite SW2443 or microsatellite S0097 or microsatellite S0007 or microsatellite SW1301 or microsatellite S0164. The invention also relates to methods for determining the predisposition for the expression or heredity of the quantitative characteristic of inverted nipples in mammals, especially in pets, breeding animals and domestic cattle, wherein the mammals, and the fertilised or non-fertilised eggs thereof or the sperm thereof are tested in the presence of, constitution of or expression of the above-mentioned second nucleic acid. The invention further relates to a kit containing at least one primer pair for the amplification of the second nucleic acid mentioned above, whereby a primer respectively binds to the + strand and another primer binds to the - strand of the nucleic acid, or a hybridisation probe having a length of at least 8 nucleotides which binds to the above-mentioned second nucleic acid, or a specific anti-body or an anti-body fragment which binds to the second above-mentioned nucleic acid.

Description

 Genetic markers for the diagnosis of the form of teats

Domestic, breeding and farm animals

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 other mammals; or from microsatellite S0164 on chromosome 3 of the pig or in a homologous position in the genome of other mammals. 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. Finally, 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.

A number of documents are cited in the description. The disclosure content of these documents, including instructions for use from manufacturers, is hereby incorporated by reference. Training a Functional suckling is decisive for the number of raised piglets and thus also for the fertility in pigs. Only piglets with sufficient milk intake can survive the suckling period. Mammal deficiencies have a particularly negative effect on the rearing of large litters and can increase piglet mortality to 40% to 50% without litter compensation. The most common mammal anomaly is the "inverted teat", followed by inoperative teat and intermediate teat. In addition, the number of functional teats in the front and rear umbilical area of the mammal is important, since the number of teat defects varies in the mammal regions. Although the glandular tissue If the teats are not changed, the piglets are not able to suck up these complexes and therefore avoid them even with large litters, which leads to an increased disposition to mastitis and piglet mortality.

The described negative effects of mammal anomalies on the economic result in pig farming lead to a priority consideration of the mammalian quality in pig breeding. A key position is selling gilts. Mammals such as teats, pickling and defects in the teat distribution lead to the exclusion of a breeding sow, for which only the respective slaughter price can be achieved. The breeding associations incur additional losses due to the exclusion of boar piglets and insemination boars with offspring information due to mammal anomalies. However, it must be taken into account that the boar piglets' teat scoring takes place at a very early point in time, which usually does not permit the detection of teats.

The development of molecular genetic methods, especially the discovery of microsatellites as polymorphic markers, has made it possible to identify the molecular genetic basis of many economically important traits and hereditary defects. The number of genetically mapped loci in pigs is currently (Rothschild 2001) around 2000 markers and genes. This enables a genome-wide search for genome regions that influence the feature under consideration. These regions are called quantitative trait loci (QTL). Many QTL examinations have been and are being carried out with regard to characteristics such as Reproduction performance, fattening and slaughter performance, meat quality, disease resistance, immune response and other characteristics performed.

In the past ten years, gene mapping in pigs has developed rapidly. This was mainly the achievement of three research groups, namely the PiGMaP program in Europe (Archibald et al. 1991; Archibald et al. 1995; Yerle et al. 1997) and the Nordic consortium (Ellegren et al. 1994; Marklund et al. 1996) and in the USA the Meat Animal Research Center of the US Department of Agriculture (USDA) (Rohrer et al. 1994).

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; OΕrien 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. Genome analysis in domestic pigs is mainly dominated by two molecular marker types. These types of markers are restriction fragment length polymorphisms (RFLPs) and 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. In addition, the presentation of RFLPs is time-consuming and costly (Botstein et al. 1980; Winter et al, 1992; Yue 1999). Microsatellites, on the other hand, 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

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 homologous positions in the genome of other mammals, or from microsatellite S0164 on chromosome

3 of the pig or in a homologous position in the genome of other mammals. 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. As a primer in PCR or sequencing reactions, 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. When used as Hybridization probe, for example in a Southern blot, 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. In some cases, 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 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. For the purposes of this invention, 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. In the case of shorter fragments, for example nucleic acid molecules of up to 20 nucleotides, 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. 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.

The term "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. Basically, teat teats can occur in all mammals, so the term "mammal" is to be viewed comprehensively and includes humans. The teat teats and their different forms in humans and animals are well known to the person skilled in the art, however reference is made to the relevant literature, such as Günther et al., 1985; Brevern et al., 1994; Wendt and Haider, 1994; Steffens 1993.

Based on the gender-neutral PiGMaP coupling map (Brown and Archibald, 1995; Muladno et al., 1996; Korwin-Kossakowska et al., 1998), 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. The region "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.

If the microsatellite is remote, ie located at the end of the chromosome, in particular less than 30 cm from the end, the upstream or downstream region can also be shorter than 5 cm. In a preferred embodiment, the position of 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 and 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, Geπetics Computer Group, University Research Park, 575 Madison, Wl 53711). When using 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 G total number of nucleotides are allowed. When using Bestfit, the so-called optional parameters are preferably left at their preset values.

For the purposes of this invention, 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. Furthermore, the term "second nucleic acid" can 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 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.

Mammal anomalies have a clearly negative impact on the economic result in animal breeding, since they usually lead to the exclusion of a breeding sow. With the invention, 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. For example, 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. This different nature of the 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.

Of course, molecular biological characterization of the predisposition to teat can also be of particular interest to humans. It is conceivable that the identification of a predisposed teat predisposition leads to new therapeutic approaches against this defect. 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.

Preferred embodiments are furthermore encompassed by the invention, the second nucleic acid disclosed above being a microsatellite or a sequence flanking it. In particularly preferred embodiments, 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.

In a further preferred embodiment of the invention, 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. In addition, according to the invention, such genes can also be surrounded by flanking sequences, which preferably comprise a region of 1 kB upstream or downstream of the genes. In another preferred embodiment, the invention relates to the use of the disclosed second nucleic acids for the selection of domestic, breeding or farm animals with missing teats. In a particularly preferred embodiment of the use according to the invention, the domestic, breeding or farm animals are cattle, pigs, goats, sheep, horses, donkeys, rats or mice.

In another preferred embodiment of the use according to the invention, 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 pig or from microsatellites in a homologous position in the genome of other mammals. 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. In addition to the microsatellites mentioned, 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.

Starting from the nucleic acid segments disclosed in the invention, the person skilled in the art 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

Nucleic acid sequencing are shown.

In addition to the domestic, breeding or farm animals explicitly listed here, the methods according to the invention also relate to other mammals, in particular humans. In a preferred embodiment of the method according to the invention, the domestic, breeding or farm animals are cattle, pigs, goats, sheep, horses, donkeys, rats or mice.

In a further preferred embodiment of the method according to the invention, 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. In the PCR reaction, one primer binds to the + strand and another primer in the opposite orientation to the - strand of the second nucleic acid disclosed above. In addition to the above-mentioned nucleic acid and an excess of primers, the reaction mixture also contains an excess of deoxynucleoside triphosphates and a DNA polymerase, for example Taq polymerase. With suitable buffer and reaction conditions, the primers bind to the nucleic acid and the DNA polymerase extends the primers based on the nucleotide sequence specified in the nucleic acid. By raising and lowering the reaction temperature, the polymerization products detach from the nucleic acid, so that other nucleic acids, usually the excess primers, can bind to the nucleic acid. A cyclical repetition of these temperature conditions consequently results in an amplification of the nucleic acid sequence. 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. In the present work, except for the microsatellites on chromosomes 1, 4 (except S0097) and Chromosome 6, PCR reactions with a final volume of 12.5 μl. For the microsatellites on the chromosomes mentioned, the PCR reactions were set up at 25 μl. The reaction mixture at 12.5 μl final volume was composed as follows: 0.20 μM primer, 200 μM dNTPs, 0.50 U Taq polymerase, 1.25 μl 10 × buffer, 1.50 μl DNA (50ng / μl) and made up to 12.5 μl 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. As an alternative to this, 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. However, 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. Of course, 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. For example, reference is made here to laboratory books known to the person skilled in the art that describe this method, for example to Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor and all subsequent editions. PCR methods are described, for example, in Newton, PCR, BIOS Scientific Publishers Limited, 1994, and subsequent editions. In addition to PCR amplification, the reverse polymerase chain reaction (RT-PCR) is also a preferred detection method. In addition to the above

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).

Another preferred detection method for determining the predisposition to teat is hybridization with a hybridization probe. 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. Another preferred detection method 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, see Maniatis, et al. Molecular Cloning: A laboratory manual, Cold Spring Harbor Press, New York, 1982, which describes the Sanger and Maxam / Gilbert methods. According to the invention, 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.

Starting from the nucleic acids disclosed above, the person skilled in the art can determine, without undue effort, which 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. be made visible by labeling the primary antibodies or is detected with the aid of antibody-binding second antibodies, which are then labeled on your part. The antibodies can e.g. be modified with fluorescent substances, by radioactive labeling or an enzymatic labeling. As already mentioned, 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.

In a preferred embodiment of the method according to the invention, 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. In particular, it is clarified here, for example with the aid of nucleic acid amplification methods or hybridization methods, which marker allele is inherited together with the characteristic 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. In addition to the microsatellites mentioned, 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. As already mentioned above, the 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.

PCR-based kits contain a pair of primers for the amplification of one of the second nucleic acids mentioned above. According to the invention, 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. However, 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.

The figures show:

Figure 1: Coupling card on chromosome 1. The figure shows gender-neutral, female and male coupling cards of the markers SW1515, SW1851,

S0155 and SW1301 on chromosome 1.

Figure 2: 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.

Figure 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 10: QTL effects for teats on chromosome 8. The maximum NPL is 3.8.

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 | SW874 and SW605 on chromosome 12.

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.

The following examples illustrate the invention.

material and methods

Selection of 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.

Table: List of typed microsatellite markers

For the selection of the microsatellite loci, in addition to four microsatellite loci on chromosome 6, 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 2: Primer sequences of the examined microsatellite markers

When selecting the microsatellites, the high informativity of the microsatellites and an even distribution on chromosomes were taken into account. Furthermore, polymorphisms have been genotyped in some genes:

Table 3: Selected Type I markers and candidate genes, their function and effects on the mammary gland

Candidate function hormone effect on the reference mammary gland

Epithelial proliferation

TGF-beta, TGFB growth factor Soriano et al., 1996 glandular tissue

Hypophysenspez.

Transcription factor f. extraparenchymal proliferation

Transcription factor, Oka et al., 1991; Growth hormone of the glandular tissue

POU1 F1

Insulin-like

Cell differentiation during growth factor 1, growth factor Oka et al., 1991; Breast development IGF1

Follicular morphological development

Lactogenesis McNeilly, 1997 stimulating the mammary gland Hormone, FSHB

Prolactin, lobulo-alveolar development Oka et al, 1991, e ta et

Lactogenesis PRL of the mammary gland, 1991

Prolaktinrezeptor, mediated effect of the development and oka et al, 1991, Mehta et PRLR prolactin differentiation of mammary gland ^al <1991

Growth hormone, metabolism, extraparenchymal proliferation oka et al, 1991, Pump et GH1 differentiation of glandular tissue ^{a |} , ¹⁹⁹³

Growth hormone-regulates growth-extraparenchymal proliferation oka et al, ₁ 991, Purup et

Releasing hormone, hormone secretion of the glandular tissue ^al . ¹⁹⁹³

GHRHR

Leptin, mediator for growth and

Lactogenesis Moschos et al, 2002 LEP Differentiation of the mammary gland

Leptin receptor, mediates growth and mediator effect

Moschos et al, 2002 LEPR Leptins Differentiation of the mammary gland

Tate-Ostroff & Bn ges,

Androgen receptor, mediates effect of sex dimorphism, 1988, Imperato-McGinley AR Androgenic teat development et al, 1986

Relaxin, morphological / functional Kuenzi et al 1995, Zhao et

Birth preparation RLN1 Development of the mammary gland al 1999

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-endogenous al-mesenchymal epithe

Holick et al, 1994, similar hormone, antipro ferative interactions during Wysolmerski et al, 1998 PTHLH factor development of the mammary gland

Parathyroid hormone epithehal-mesenchymale mediates effect of receptor 1, interactions during Lanske et al, 1996 PTHLH PTHR1 development of the mammary gland

Polymerase chain reaction (PCR)

The polymerase chain reaction (PCR) 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. In Table 4 shows the program used in the present work.

Table 4: PCR programs used. The annealing temperatures vary between 55 and 65 ° C.

Temperature function (° C) Duration of cycles

Initial denaturation 94 3 minutes

Denaturation 94 1 minute

Annealing 55-65 1 minute 30-40 cycles

Extension 72 1 minute

Final extension 72 10 minutes

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. In the present work, with the exception of the microsatellites on chromosomes 1, 4 (except S0097) and chromosome 6, PCR reactions with a final volume of 12.5 μl were set up. For the microsatellites on the chromosomes mentioned, the PCR reactions were set up at 25 μl. With a final volume of 12.5 μl, the reaction mixture was composed as follows:

0.20 μM dNTPs 200.00 μM per primer

Taq polymerase 0.50 U

10 x buffer 1, 25 μl

DNA (50ng / μl) 1.50 μl

Supplemented with H ₂ O to 12.50 μl

Representation of the PCR product in the DNA sequencer

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).

Preparation of the polyacrylamide gel

To prepare the gel, 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. For a 41 cm glass plate, 15 ml Sequagel XR, 3.75 ml Sequagel buffer, 200 ml DMSO, 150 ml 10% APS were mixed together and poured between the glass plates. The gel was polymerized for 1 hour. After one hour, the gel was installed in the DNA sequencer and the sequencer for the pre-run of the electrophoresis was switched on.

Determination of the allele length

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 μl of each of the fragments mentioned was mixed together and from this Mix 0.7 μl 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. With the program, 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. With 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.

QTL analysis

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. With the help of an inheritance vector, it can be tracked whether the patemale or maternal allele is passed on to the affected offspring has been. For all possible allele combinations in the offspring, inheritance vectors can be created which represent all possible states of the parental alleles in the offspring concerned. Using the marker genotypes, 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). Ultimately, the standard normal distribution under the null hypothesis becomes Test statistics NPL (non parametric lodscore) calculated.

There are two different errors in the statistical analysis of the data, false positive (type I) and false negative (type II). The false positive results are the main problem in the QTL analysis. To reduce false positive results in the genome scan as much as possible, a level of significance is set. In order to achieve a significant level of 1% in a genome-wide investigation, 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).

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. For the generation of the F generation (parent animals), 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. For the generation of the F ₂ animals, the animals were mated to one another by the F ^ generation. In 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. For the investigation, 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.

Example 2: Microsatellite mapping and coupling analysis

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).

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.

Table 5: Number of markers typed per chromosome and observed maximum NPL values for different chromosomes

Example 3: QTL and coupling analysis of chromosome 1

The microsatellites SW1515, SW1851, S0155 and. SW1301 genetically mapped. 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. Four microsatellite markers, distributed as evenly as possible, were used on chromosome 1 for QTL analysis. 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.

Example 4: QTL and coupling analysis of chromosome 4

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. The

Coupling map of chromosome 4 is shown in FIG. 3.

The microsatellite markers were used for the analysis of gene effects on teats

S0227, S0001, S0214 and S0097 selected for chromosome 4. The markers are evenly distributed on the own coupling card as well as on the USDA-MARC card so that the whole chromosome can be analyzed well. The microsatellites used with their large number of alleles (S0097 = 3, S0227 = 4, S0214 = 5 and S0001 = 6) and high PIC values (S0227 = 0.74, S0214 = 0.77, S0001 and S0097 each 0.78 ) are suitable markers for QTL analysis. 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.

Example 5: QTL and coupling analysis of chromosome 6

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.

Example 6: QTL and coupling analysis of chromosome 14

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. The gender neutral, female and male coupling map of three

Microsatellites on chromosome 14 are shown in FIG. 7.

Using the microsatellite marker S0007, a significantly significant effect was identified on chromosome 14 (NPL value of 4.8). The

Coupling analysis from other microsatellite markers, SW857 and SWC27, is meaningless with a low NPL below 2.

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.

The course of the NPL value on chromosome 14 was shown in FIG. 8.

Example 7: QTL and coupling analysis of chromosome 8

Four 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. In addition to the 4 microsatellites, KIT1 on chromosome 8 was also included in the analysis.

The NPL value on chromosome 8 rises from 0 to 1.5 between microsatellites SW2410 and S0086 and drops immediately after the marker S0086 has been localized to the location of KIT1. After the localization of KIT1, the curve rises regularly to the localization of microsatellite SW61, where it reaches its maximum value. The maximum value is 3.8. 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.

An NPL value of 2.3 was identified on chromosome 10. Three microsatellites, namely SW830, S0070 and SW2067, were selected and genotyped for QTL analysis for the feature teat on chromosome 10. 2736 genotypes were available for performing the QTL analysis. The number of alleles is four for SW2067 and S0070 and five for SW830. 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.

Example 9: QTL and coupling analysis of chromosome 12

Three 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. The 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. Immediately after marker S0026, it 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.

Example 11: QTL and coupling analysis of chromosome 18

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. The gender neutral, female and male coupling card of three

Microsatellites on chromosome 18 are shown in FIG. 17.

Results of QTL analysis on chromosome 18

FIG. 18 shows the result of the association analysis for teats on the chromosome

18 reproduced. On chromosome 18, similar to chromosome 16, was identified a positive but insignificant NPL value. The NPL value rises particularly high for the SWR414 microsatellite marker with a value of 3.7. 2736 genotypes were available for performing the QTL analysis. The allele number of microsatellite markers is six for SW787, four for SW1023 and four for SWR414. The PIC value is 0.73 for the marker SW787 and 0.78 for the marker SW1023 and SWR414. The course of the NPL value on chromosome 18 is shown in FIG.

Example 12: QTL and coupling analysis of chromosome 2

The microsatellites SW2443, SW240, SW1564 and

S0226 and the TypeI markers FTH, STS2 and C3 genetically mapped. The genetic

Card looks like this: SW2443 - 42.8 cM - FTH - 20.2 cM - SW240 - 22.9 cM -

STS2 - 13.8 cM - C3 18.4 cM - SW1564 - 24.8 cM - S0226.

With the microsatellites used, a significant one could be found on chromosome 2

Effect can be identified. The NPL value reached in the localization of

Microsatellite S2443 a value of 7.9.

Example 13: QTL and coupling analysis of chromosome 3

The 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. The NPL value in the localization of microsatellite S0164 reached 4.0.

Example 14: QTL and coupling analysis in commercial herd animals

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

Effects of direct biological and positional candidate genes in genome areas that had an impact on the shape of the teat in the resource population.

The sample material from the commercial breeding populations comprises 201

Tissue and DNA samples from gilts with teats that come from families come in which at least one animal had teats, as well as their parents. In addition to 37 paternal half-sibling groups with up to 22 animals per group, the population contains 37 "classic affected sibpairs" (37 sibling groups of at least two animals that have the defect phenotypically). In the offspring of 5 pairs of parents, litters with more than two affected offspring were born observed, which increases the total number of affected sibpairs to 70. 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

"classic affected sibpairs" 15 14 8 37 affected sibpair with station wagons 70

)

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 Deutsche 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 evaluation of the data from the crossing animals alone (15 affected sibpairs) results in a maximum NPL of 6. 8 on the microsatellite S0035, which analyzes the data of the animals of commercial herds separately according to breeds (DL or DL ^* DE), as well as the two-peaked course of the NPL value curve via the chromo som 6 for the experimental population, suggesting that at least two QTLs segregate on this chromosome. The QTL on chromosome 2 could be confirmed in the examined animals of the commercial herds. This results in a maximum NPL of 1, 8 for all animals; only for the DL animals of 1, 9 and only for the cross animals of 2.4 in the position of the marker S2443 as for the animals of the resource population. The QTL identified in the experimental population on chromosome 1 could not be traced for the animals of commercial origin. (Table 7, Figure 19a-c).

Tab. 7: QTL regions for the characteristic "teat" in animals from commercial herds

Example 15: Candidate gene analysis

The development of the mammary gland is regulated by a number of 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.

For the gene transforming growth factor beta 1, TGFB1, androgen receptor, AR, parathyroid hormone-like hormone, PTHLH, and relaxin, RLN, 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 (TDT) 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 (Spielman et al., 1993).

A total of 15 genes were genotyped. These genes represent direct biological candidate genes for the characteristic teat. The genes TGFB1, LEPR on chromosome 6 and RLN on chromosome 1 map within the QTL regions identified here also represent positional candidate genes.

Two candidate genes (estrogen receptor and prolactin) were not polymorphic in the F ₂ resource families. For the androgen receptor gene and the gene for the growth factor TGFB1 and for PTHLH, the project was carried out with the cloning and sequencing and detection of polymorphisms, SNPs (single nucleotide polymorphisms) not previously described in the literature:

The results of the Chi-square test for association and the transmission disequilibrium test for association and coupling indicate a significant relationship between gene variants of the leptin receptor, LEPR, (TDT p = 0.003), the pituitary-specific transcription factor, POU1 F1, part of the growth hormone axis (TDT p = 0.01), the transforming growth factor beta 1, TGFB1 (TDT p = 0.02) and the relaxin, RLN, (TDT p = 0.0009) and the appearance of teats. 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.

Claims

claims

1. Use of a first nucleic acid to determine the predisposition for the expression or inheritance of the quantitative characteristic 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

(a) microsatellite S0220 on chromosome 6 of the pig or in a homologous position in the genome of other mammals; or

(b) SW2443 microsatellite on pig's chromosome 2 or in a homologous position in the genome of other mammals; or

(c) microsatellite S0097 on chromosome 4 of the pig or in a homologous position in the genome of other mammals; or

(d) S0007 microsatellite on pig chromosome 14 or in a homologous position in the genome of other mammals; or

(e) microsatellite SW1301 on chromosome 1 of the pig or in a homologous position in the genome of other mammals; or

(f) Microsatellite S0164 on chromosome 3 of the pig or in a homologous position in the genome of other mammals.

2. Use of a combination of at least two, three, four, five or six of the nucleic acids mentioned in claim 1 for determining the predisposition for the expression or inheritance of the quantitative characteristic of the Stülpzitze.

3. Use according to one of claims 1 to 2, wherein the second nucleic acid is a microsatellite or a sequence flanking the microsatellite.

4. Use according to one of claims 1 to 2, wherein the second nucleic acid is derived from a gene from the group consisting of ryrl, TGFB1, GPI, POU2F2, EAH or one of these genes.

5. Use according to one of claims 1 to 2, wherein the second nucleic acid is derived from a gene from the group consisting of tenascingen, HSPA5, orosomucoid gene, steroidogenic factor 1, relaxin gene, GGTA1, parathyroid hormone-like hormone, parathyroid hormone receptor 1 or is one of these genes.

6. Use according to one of claims 1 to 2, wherein the second nucleic acid is derived from a gene from the group consisting of TSHB, ATP1 A1, NGFB, PKLR or one of these genes.

7. Use according to one of claims 1 to 2, wherein the second nucleic acid is derived from a gene from the group consisting of OAT, MLB2, Npy4R, PLAU or one of these genes.

8. Use of the second nucleic acid according to one of claims 1 to 7 for the selection of domestic, breeding, or farm animals with missing teats.

9. Use according to claim 8, wherein the domestic, breeding or farm animals are from the group consisting of cattle, pigs, goats, sheep, horses, donkeys, rats and mice.

10. Use according to claim 8, wherein a genome screen is performed on several related mammals in a population.

11. A method for determining the predisposition for the expression or inheritance of the quantitative feature of inverted teats in mammals, preferably in domestic, breeding or farm animals, whereby the animals, their fertilized or unfertilized egg cells, or their sperm are dependent on the presence, nature or Expression of a second nucleic acid mentioned in one of claims 1 to 7 is tested.

12. The method according to claim 11, wherein the domestic, breeding or farm animals are from the group consisting of cattle, pork, goat, sheep, horse, donkey, rat and mouse.

13. The method according to claim 11 or 12, wherein one

(a) performing a PCR amplification with complementary primers with a length of at least 8 nucleotides, wherein one primer binds to the + strand and another primer binds the strand of the second nucleic acid from claims 1 to 7 in opposite orientation; or

(b) carries out a hybridization, a hybridization probe with a length of at least 8 nucleotides binding to the second nucleic acid from claims 1 to 7; or

(c) sequencing the second nucleic acid from claims 1 to 7

(d) performing a detection with a specific antibody or antibody fragment or antibody derivative or aptamer, the antibody or the antibody fragment or the antibody derivative or the aptamer being specifically directed against a first or second nucleic acid from claims 1 to 7.

14. The method of claim 13, wherein a genomic screen is performed on multiple related mammals in a population.

15. Kit at least

(a) a pair of primers for the amplification of the second nucleic acid mentioned in claims 1 to 7, one primer each binding to the + strand and a further primer to the - strand of this nucleic acid; or

(b) a hybridization probe with a length of at least 8 nucleotides which binds to the second nucleic acid from claims 1 to 7; or (c) a specific antibody or an antibody fragment or an antibody derivative or an aptamer which binds to the first or second nucleic acid from claims 1 to 7.