WO2004020663A2 - Genetic markers for diagnosing predisposition to heredity or expression of the imperforate anus phenotype in domestic animals, breeding animals and working animals - Google Patents

Abstract

The invention relates to the use of a first nucleic acid for determining predisposition to expression or heredity of the imperforate anus phenotype in a mammal, said first nucleic acid having a length of at least 8 nucleotides and being identical or essentially identical to a second nucleic acid on chromosome 1 of the pig or in a homologous position in the genome of other mammals, that is in the region of a microsatellite selected from the group consisting of SW2185, SW 1621, SW 1902, S0155, and S0320; or on chromosome 3 of the pig or in a homologous position in the genome of other mammals, that is in the region of the microsatellite S0002; or on chromosome 9 of the pig or in a homologous position in the genome of other mammals, that is in the region of a microsatellite selected from the group consisting of SW2401 and S0081; or on chromosome 12 of the pig or in a homologous position in the genome of other mammals, that is in the region of a microsatellite selected from the group consisting of SW957 and S0229. The invention also relates to methods for determining predisposition to expression or heredity of the imperforate anus characteristic in mammals, preferably in domestic animals, breeding animals and working animals, whereby the mammals, the fertilised ovules or non-fertilised ovules thereof, or the sperm thereof are tested for the presence, structure or expression of the second abovementioned nucleic acids. Disclosed is also a kit containing at least one pair of primers for amplifying one of the second abovementioned nucleic acids, respectively one primer binding to the + strand of the nucleic acid and another primer binding to the strand of the nucleic acid, or a hybridisation probe having a length of at least 8 nucleotides which binds to one of the second abovementioned nucleic acids, or a specific antibody or an antibody fragment which binds to the second abovementioned nucleic acid.

Description

Genetic markers for the diagnosis of the predisposition to inheritance or expression of the phenotype "afterlessness" in domestic, breeding and farm animals

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 position in the genome of other mammals, in the area of a microsatellite selected from the group consisting of SW957 and S0229. 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 Finally, 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.

In pig production, 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. In addition to this simple form 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 in some cases a fistula to the vagina enables the decoction and thus extends the lifespan. The heredity of the anuslessness has been proven several times, the exact inheritance of the defect is not yet known. Several studies have indicated that individual genes could play a role.

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) about 2000 markers and genes. This enables a genome-wide search for genome regions that influence the feature under consideration. These regions are called Economic Trait Loci (ETL). Many ETL studies have been and are being carried out with regard to characteristics such as reproductive performance, fattening and slaughter performance, meat quality, disease resistance, immune response and other characteristics.

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 by coupling analyzes (Morton 1955; Xu 1997; Penalver 1999). Two prerequisites are required for this, namely an animal population with a known one Pedigree (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 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. It should be borne in mind here that such prognostic or diagnostic detection methods are independent of the identification of the gene causing the phenotype. This is important because establishing the molecular basis of a phenotype is often very difficult and time-consuming, especially in connection with multifactorial phenotypes.

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 homologous position in the genome of other mammals r, in the area of a microsatellite selected from the group consisting of SW957 and S0229. 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. 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 at least 21 nucleotides, most preferably at least 25 nucleotides. When used as a 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 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. For the purposes of this invention, 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. 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 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. Usually, 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.

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 term "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.

Based on the coupling map of the US Meat and Animal Center (Rohrer et al., 1996 Genome Research 6: 371-391, a constantly updated version can also be found at http://www.genome.iastate.edu/maps/index.html ), which was created on the basis of gender-averaged recombination rates, 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, SW2401 located at position SSC9 57.1 cM, S0081 located at position SSC9 77.0 cM. SW957 located at position SSC12 33.4 cM and S0229 located at position SSC12 19.4 cM. Since genetic mapping positions are dependent on the population, this position specification is subject to fluctuations, which, however, is readily known to the person skilled in the art knowing the teaching according to the invention empower to implement them. The region "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 same applies to the other microsatellites mentioned. 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. , more preferably at least 50%, even more preferably at least 75% and particularly preferably at least 95% 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, Genetics 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 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 region of 8 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. The first nucleic acid can be identical or essentially identical to the + strand or the - strand.

For the purposes of the invention, the term “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. Furthermore, the term “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.

Depending on the population, anomalies of the anus can have a significantly 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 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 In addition, 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. For example, 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.

Of course, a molecular-biological characterization of the predisposition to inheritance or expression of the phenotype "afterlessness" can also be of particular interest to humans. It is conceivable that the identification of a predisposition to inheritance or expression of the phenotype "afterlessness" would lead to new therapeutic approaches against this defect leads.

Under the inaccurate term "anuslessness", various malformations of the intestinal tract are summarized in pigs, which correspond in shape and form to anal atresia in humans (van der Putte, 1986). The deep form of anal atresia, also known as atresia ani, 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. In the high form of anal atresia, atresia recti is a thicker layer of connective tissue between the blind rectum and the surface of the body. 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). Among the piglets with the deep shape of the anal atresia (atresia ani), an Evidence of vestibular and anocutaneous fistulas in the male animals with the exception of a few piglets. After the preparation of histological specimens, it can be seen that the inner fistula opening in the rectum blind sac bears the essential features of a normal anus in many cases (Lambrecht, 1989). Both an internal (sphincter intemus) and an external sphincter (sphincter externus) can be found and the ephithelium of the fistula mouth is often identical to that of an anal opening (Lambrecht, 1987). The data in the literature on the prevalence of anal atresia in pigs are very different and vary between 0.1-1% within the population, with clear differences between the individual breeds being observed. The affected piglets usually die within one to three days. In some cases, the animals can be between one and three weeks old. The most common cause of death is intoxication due to constipation. The strongly distended belly is characteristic. Sometimes, however, the strong scaling of the intestinal contents also leads to an intestinal rupture. In the further course, the released feed particles cause inflammation of the abdominal cavity (peritonitis) with lethal consequences. Female animals with recto-vaginal fistulas, on the other hand, may be able to survive for a long time, as they are able to vomit through the vagina.

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. It should be noted that the classification and definition of the disease is handled very differently in the literature. In many cases, 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. An exact differentiation of these diseases is also difficult because the embryological and pathological background of these diseases has so far only been is insufficiently known. In addition to the human field, 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.

In humans, anal atresia is often seen in connection with syndromes. For example, anal atresia often occurs together with Hirschsprung's disease (congenital enlargement of the large intestine) or in connection with VACTERL syndrome. In combination with Pallister-Hall and Sacral Agenesis, malformations of the urogenital tract and defects of the anorectal system are often found in newborn children.

The aetology of anorectal defects has not been fully clarified, the previous ideas about the occurrence of the defects are mostly based on morphological and histological studies on a small number of human and porcine embryos (Kluth, 1995, Kluth 1997, van der Putte, 1986, Nievelstein, 1998 ). A very important animal model for the development of anorectal defects are the studies of van der Putte's, 1986 on porcine embryos. He examined 41 embryos and fetuses between 24 and 120 days of gravity. In his opinion, the development of anal atresia is due to a defect in the dorsal cloaca membrane. 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 result of this too short a cloaca membrane is an anus misalignment, which is an "ectopic" Takes position and opens into the urogenital tract. According to Van der Putte's investigations, the different forms of the disease (Atresia ani and Atresia recti) can be regarded as the same cause. In his opinion, the different forms of anal atresia are caused by the degree of deformation of the cloaca membrane in the embryonic development.

The results of the segregation analysis carried out by Thaller (1992) for the trait of nocturnal gave clear indications of the influence of a single gene. The overall frequency of anuslessness in the pig population was estimated at 0.10%, with clear differences in frequency between the breeds of the animals concerned. This study also showed that abnormal littermates were very rare to find.

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. In its original form, this method is based on the observation and determination of recombinant and non-recombinant offspring within a family, to estimate the recombination rate θ 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). In addition to the difficulty of specifying the mode of inheritance of complex diseases, it has often been problematic in human genetics to collect DNA from large families with several living diseased individuals. An alternative to the classic parametric LODscore coupling analysis is therefore the ASP (affected sib-pairs) approach developed by (Suarez, 1978). This method is based on the assumption that sick siblings have inherited the same alleles from their common ancestors both at the disease locus and at the immediately adjacent markers. Since these alleles are identical in terms of parentage, they are referred to as "identical by descent" (IBD). The statistical test for coupling between the disease locus and the marker checks whether there are significant deviations from the expected IBD distribution. With a full-sibling pair, one would expect, for example, that they share a common allele with a probability of po = 1/4, an allele with pι = 1/2 and two alleles with p ₂ = 1/4. For a family with a half-sibling pair, p ₀ and p-ι = 1/2 and the probability p ₂ = 0. This means that under the null hypothesis: no coupling between disease locus and marker locus, the expected value for the sharing of alleles among siblings is 0.5 and when coupling is greater than 0.5. A χ ² test is used (Lander, 1994). The advantage of the ASP method is that it is not parametric and does not require any assumptions about the specific mode of inheritance of the disease. The ASP method is more robust than the classic LODscore coupling analysis. Accordingly, even in the presence of incomplete penetrance, phenocopies, genetic heterogeneity and complex inheritance, IBD alleles can still be detected between the affected siblings. On the other hand, if knowledge of the mode of inheritance of a disease can be assumed to be certain, the LODscore coupling analysis is significantly more meaningful than the nonparametric "allelesharing" method (Lander, 1994).

For the application of nonparametric and parametric methods for the

Several software programs have been developed to address problems in human genetics. This includes the Genehunter program (Kruglyak, Daly et al. 1996). For genome-wide coupling analysis on which this patent application is based Study, the Allegro 1.0 program (Gudbjartsson, Jonasson et al. 2000) was used, which is a successor to Genehunter 2.0. It offers the possibility of evaluating complex family tree structures, including the entire marker information, using both nonparametric and parametric methods. The nonparametric evaluation methodology includes various "non parametric linkage ^{, r} (NPL) statistics, which can be calculated between individual markers and the disease locus as well as with the inclusion of several markers. Part of the parametric evaluation is the calculation of a classic LODscore test statistic However, the precise knowledge of the mode of inheritance as well as the special parameters such as penetrance of the disease, the phenocopy rate and the frequency of the defect allele in the population is necessary. In the case of complex diseases, an exact characterization of these parameters is often not clearly determinable and therefore problematic.

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. In particularly preferred embodiments, 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.

Yet another preferred embodiment of the invention relates to the use of 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

Numbers defined. A particularly preferred embodiment of the

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.

One or more genes located in the vicinity of the microsatellites listed above are probably of crucial importance for the expression of the phenotype "afterlessness". Accordingly, in a further preferred embodiment of the invention the second nucleic acid is a specific gene or a part of a gene. Preferred here the 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, ALDH1A2 Aldehyde dehydrogenase 1 family member A2, ALDH1A3 Aldehyde dehydrogenase 1 family member A3, CYP19A1 Cytochrome P4y A, family 19, subfamily polypeptidel, PML Promyelocytic leukemia, HOXA11 Homeobox A11 (Homeobox A dark), HOXA13 Homeobox A13 (Homeobox A dark), HOXB1 Homeobox B1 (Homeobox dark B), HOXB8 Homeobox B8 (Homeobox dark B), HOXB9 Homeobox B9 (Homeobox dark B), HOXB5 Homeobox B5 (Homeobox cluster B), HOXD13 Homeobox D13 (Homeobox D cluster) and all other genes in the nucleic acids disclosed above but not listed here. 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. 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 a further preferred embodiment, 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. , 1996) of the 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".

In another preferred embodiment, 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". In a particularly preferred embodiment of the use according to the invention, the domestic, breeding or Farm animals cattle, dog, cat, rabbit, buffalo, camel, alpaca, mink, pig, goat, sheep, horse, donkey, rat or mouse.

In another preferred embodiment of the invention

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. In the genome screen, it is examined 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 "afterlessness" In particular, this clarifies, for example with the aid of nucleic acid amplification methods or hybridization methods, which marker allele is inherited together with the characteristic "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. Preferred 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 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 that in the sense of the invention second nucleic acid disclosed in the invention or 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 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. These 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, 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 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.

In a preferred embodiment of the method according to the invention, the domestic, breeding or farm animals are cattle, dog, cat, rabbit, buffalo, camel, alpaca, mink, pig, goat, sheep, horse, donkey, rat or mouse. 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 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 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.

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 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 12.5μl is composed, for example, as follows: 0.20μM primer, 200μM dNTPs, 0.50 U Taq polymerase, 1.25μl 10 x buffer, 1.50 μl DNA (50ng / μl) and made up to 12.5 μl 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. As an alternative to this, (if identical) hybridization of the 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. 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 methodology, 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 aforementioned amplification methods, other amplification methods have also been developed in recent years, which also represent preferred embodiments of the invention. Further 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).

Another preferred detection method for determining the predisposition to the expression of the phenotype "afterlessness" is hybridization with a hybridization probe. Here, 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 , 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. , 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor (and all subsequent editions referenced), in which the methods according to Sanger and Maxam / Gilbert are shown 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.

On the basis of 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 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. In addition, 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'-δ'-S phosphorothioates, 3'-S-5'-0-phosphorothioates, 3'-CH2-5'-0-phosphonates and 3'-NH-5'- 0 phosphoroamidates. In the case of peptide nucleic acids, 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. Examples of this are radioactive labeling, for example with ³⁵ S, ³² P or ³ H fluorescent labeling, biotin labeling, digoxigenin labeling,

Peroxidase labeling or labeling with an alkaline phosphatase. The nucleic acids used in the hybridization or amplification reaction can furthermore be provided with various suitable markers. 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). 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. In the case of the amplification reaction, 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. Alternatively, 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.

In a preferred embodiment of the method according to the invention, 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". 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 "afterlessness". A common inheritance of nucleic acids with the phenotypic expression of "afterlessness" implies a genetic coupling of the nucleic acid to the characteristic controlling gene and also indicates which marker allele is located on the same chromosome as the allele involved in the phenotypic expression of the "afterlessness" of the defect gene, ie in which coupling phase there are marker allele and defect allele. 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. In addition to the microsatellites mentioned, other 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. 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 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.

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

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.

The figures show:

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 ιι _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 4: Course of the NPL _mpt statistics on SSC1 in coupling analyzes with different numbers of families and different marker density.

Figure 5: Information content of the multipoint NPL statistics on SSC1 in coupling analyzes with different numbers of families and different marker density.

Figure 6: Course of the NPLgpj 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 following examples illustrate the invention.

material and methods

Descent Control:

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 _Π 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 _Π for the detection of an incorrect parentage are listed for each marker.

Table 1: Characterization of the 20 microsatellite markers for parentage control.

Structure of animal material for qenome-wide typing:

After the pedigree control, the animal material consisted of the following 27 shark and full-sibling families:

15 families with 2 half siblings (HG) 5 families with 3 HG

2 families with 2 full siblings (VG)

3 families with 2 VG and 1 additional HG 1 family with 2 VG and 2 additional HG

1 family with relatives of the boar (fathers of the afterless animals are themselves HG) There were a total of 106 animals available for genome-wide typing. These are divided into 72 afterless piglets, 31 boars and 3 sows. Table 2 shows the sex and the severity of the defect in the typed piglets.

Table 2: Characteristics of anuslessness and the sex of the typed piglets.

For the coupling analysis, Atresia ani and Atresia recti piglets were classified in one disease category and the two forms of anuslessness were considered the same defect. In a further step, 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.

DL = Deutsche Landrasse, DE = Deutsches Edelschwein, Pl = Pietrain, PIC = PIC Hybrideberber, CAM = Camborough 26 (PIC Hybridsau), BHZP = Federal Hybrid Breeding Program

Structure of the supplementary animal material:

In the course of the genome-wide typing, the sample material was supplemented by further HG and VG families with atlos piglets. These families were targeted in specific chromosome regions and used to confirm the results. This additional sample material consisted of 4 individual afterless piglets that complemented existing HG families and 8 further new families. These are made up as follows:

4 families with 2 HG 3 families with 3 HG 1 family with 2 VG

In total, 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).

Structure of family material for coupling analysis on the sex chromosome SSC X and in the pseudoautosomal region SSC XY

For the coupling analysis on the X chromosome, which is referred to as SSC X, diseased full siblings were typed together with the respective mothers. For this purpose, VG families from the animal material of the genome-wide typing as well as VG families from the supplementary family material were used. In total there were 9 families with two VG each. Among the 18 piglets there was a mixed VG pair, with one female and one male piglet, one female VG pair and 7 male VG pairs. The coupling analysis in the pseudoautosomal region (SSC XY), however, was based on the family material.

Selection of the typing markers

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.

Allelfrequenzschätzung:

For the calculation of the probability P _en (detection of an incorrect parentage) as well as for the calculation of the information content of the markers, the allele frequencies from the entire typed family material (parents and piglets) 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. For the parametric and nonparametric coupling analysis, 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

Isolation of genomic DNA from native pig sperm:

The first step in the preparation was to wash the sperm cells with 1 x PBS to completely remove the seminal plasma. For this purpose, 500 μl 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 μl ProteinaseK (20 mg / ml in bidist. H ₂ O) and 50 μl 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. If this was not the case, the mixture was incubated again with 150 μl 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 μl 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. After washing twice with 70% ETOH and then centrifuging for 10 min at 10,000 rpm, the DNA was air-dried and taken up in 150 μl TE (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA). The DNA yield varied between 10-22 μg depending on the quality of the sperm.

Isolation of genomic DNA from muscle tissue:

To prepare DNA from muscle tissue, 30 - 40 mg frozen tissue was cut into small pieces with a scalpel and in a 2 ml Eppendorf tube with 1 ml lysis buffer (pH 8.0, 1% SDS, 50 mM Tris, 100 mM EDTA, 100 mM NaCI) added. After the addition of 100 μl ProteinaseK (20 mg / ml in bidist. H ₂ O) the sample mixed on the vortex and incubated overnight at 55 ° C. The piglet's muscle tissue, which contains a lot of RNA, was treated with 400 μg 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 μl 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 μl TE (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA). The DNA yield varied between 30-60 μg.

Isolation of genomic DNA from ear tissue:

The DNeasy kit from Qiagen (cat. No. 29308) was used for DNA extraction from ear tissue. The "Animal Tissue" protocol was followed.

DNA quantification using fluorescence emission:

Before starting the measurement, the fluorometer (Hoefer DyNA Quant 200, Amersham Pharmacia Biotech) was calibrated. For this purpose, 200 ng of Calf thymus DNA (100 μg / ml in bidist. H ₂ O) were used as reference DNA. For quantification, 2 μl 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 μg / ml Hoechst H 33258 (bisbenzimide)) were mixed in a cuvette. Measurements were made at an excitation wavelength of 350 nm and an emission wavelength of 456 nm. In the concentration range of 10 - 500 ng / μl DNA, 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. After the DNA measurement, the samples were adjusted to a concentration of 25 ng / μl with TE (pH 8.0) and applied to a 0.8% agarose gel (ethidium bromide) as a control.

Polymerase chain reaction (PCR):

For the PCR reactions, 200 μl DNA (25 ng / μl) 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 μM 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 μl 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. From the robotic station, 18 μl of master mix were placed in 96-well microtiter plates and then mixed with 2 μl of DNA from the DNA plate. Two T gradients, a T1 thermal cycler, two UNO thermoblocks from Biometra Whatman and two PTC-100 machines from MJ Research were used to carry out the PCR cycles. The standard PCR conditions were: 4 min denaturation at 94 ° C, followed by 30 cycles with 30 sec at 94 ° C, 45 sec at primer-specific annealing temperature, 1 min 30 sec extension at 72 ° C and final cooling to 4 ° C , As a control, PCR amplificates were randomly applied to a 2% agarose gel. The fluorochrome marking resulted in the dilution factor for the genotyping: 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. Depending on the fragment length and fluorochrome marking, 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 μl of TEMED (tetramethylethylenediamine, Ameresco, 0761) and 300 μl 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. In each case 1.8 μl were taken from the multiplex PCR mixture and with 2.0 μl ABI-Dye (pH 8.0, 25 mM EDTA: formamide = 1: 5, mixed with a pipette tip Dextran Blue (Fluka, 31393)) and 0.3 μl internal length standard TAMRA 500 (ABI Prism, length standard) mixed in a microtiter plate. Before applying the gel in an ABI Prism ® 377 DNA sequencer (Perkin Elmer), the samples were denatured for 3 min in a PCR machine at 94 ° C. and then Chilled to 4 ° C. 2.0 μl of the denatured samples were applied to the acrylamide gel. At an operating temperature of 51 ° C and an applied voltage of 2500 V, 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) was used as the running buffer for the electrophoresis. The fragment lengths were analyzed with. the computer programs ABI Prism 377 Collection and the GeneScan 3.1 from Perkin Elmer. The definition and determination of the marker allele "allele calling" was carried out with the Genotyper 2.5 program.

Example 1: Results of checking for consistent genotypes

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. A family with three half-siblings, who showed eight, nine and eleven marker conflicts, was removed from the further typing and statistical evaluation, although the parentage check had not given cause for complaint. In all other conflicts, after checking the genotypes for transmission errors, the microsatellite markers were amplified again so that the mix-ups of the PCR samples could also be excluded.

After this repetition, the total number of parent-NK conflicts decreased to 52, which occurred at 12 different markers. No offspring-parent pairs with more than four marker conflicts could be observed. In most cases, it had been shown that alleles from heterozygous animals with different intensities of the PCR amplificates had led to incorrect detection of the alleles. A second repetition reduced the existing conflicts again. The resulting 28 discrepancies at six different markers could not be resolved by typing again. A simulation study showed that these inconsistencies are not due to incorrect parentage. You would in In the worst case, expect at least 8 conflicts per animal-parent pair if you randomly assign the genotypes of the offspring to the genotypes of any parent. It is therefore very unlikely that with 130 typed markers there will be conflicts at one, two or three markers due to incorrect parentage. The existence of non-amplifying alleles (zero alleles) was tested on the six microsatellite markers which appeared to have discrepancies despite repeated typing. Table 6 shows the allele frequencies r determined for the possible zero allele together with the standard error of r. For two markers, SW797 and SW1881, a t-test showed significant evidence for the existence of null alleles.

Table 6: Estimated allele frequency r for a non-amplifying allele in the microsatellite markers with genotype conflicts between parents and offspring.

Example 2: Genome-wide coupling analysis

The first step of the statistical analysis consisted of the genome-wide nonparametric and parametric coupling analysis of the family material. As an independent confirmation study, the marker density in interesting chromosome regions was increased in a second step of the evaluations and the supplementary family material was also genotyped. For the genome-wide coupling analysis, the family material was examined at 130 markers and evaluated with the Allegro 1.0 program.

Results of the genome-wide nonparametric coupling analysis:

The NPL statistics, NPL _pa j _rs and NPL _a ιι, 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. Within the pseudoautosomal region on the sex chromosomes (SSC XY), a coupling analysis with the supposed disease locus could only be carried out for a microsatellite marker SW949.

Nonparametric coupling analysis between multiple markers and the disease locus (multipoint coupling analysis):

The results of the genome-wide coupling analysis are shown in Table 7. The highest NPL _pa j _{rs mp} t and NPL _a n _mpt values for each chromosome (SSC) are listed, as well as the p-value and the information _content at the position of the maximum NPL value. The highest value was achieved by the NPL statistics on SSC1 on marker SW1621. There the NPUπ mpt was 2.57 (p = 0.006) and the NPL ^ _r s _mp t was 2.59 (p = 0.006). At 0.47 and 0.46, respectively, the informativity at the position of the NPL maximum was only in the middle area (FIG. 4). On the SSC 12 the marker S0229 also showed high NPL values. The NPL _a n _mp t and the NPL _pa irs mpt were 1.94 (p = 0.028) and 1.90 (p = 0.030), respectively. In addition, there was a high information content of 0.88 within the examined families at this marker position. At the position of marker S0002 on SSC 3, a value of 1.90 (p = 0.031) was found for the NPL _a ιι _m pt and 1.86 (p = 0.030) for the NPL _{pairs mpt} . The informativity was 0.45 and 0.47, respectively. NPL statistics with an error probability of less than 0.1 could be found on SSC 13 [NPL _a n _mpt 1.57 (p = 0.060); NPLp _ai rs m _P t 1 -66 (p = 0.050)] and on SSC 8 [NPL _a ι, m _pt 1.37 (p = 0.087); NPLpairs m _pt 1 -31 (p = 0.096)] can be found. With an NPL _a n _mpt of 1.13 (p = 0.130) and an NPL _pa i _rS m _{Pt of} 1.11, the SSC 9 was only slightly above the p value of O.1. Table 7: Significant NPL _a n and NPL _pairs values of the individual chromosomes in the context of the nonparametric coupling analysis (multipoint coupling analysis).

Nonparametric coupling analysis between the individual markers and the disease locus (singlepoint coupling analysis):

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 ιι _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 NPL _a ιι _S p _t on marker SW1621 was 2.14 (p = 0.019) and the NPL _pa irs mpt 2.13 (p = 0.018). The information content at this marker position was in the lower range at 0.38. On SSC 12 (S0229) an NPL _{aπ spt} of 1.61 (p = 0.056) and an NPLpairs spt of 1.57 (p = 0.060) were found. The information content there was 0.80 and 0.79, respectively. The NPL values on SSC 8 were 1.49 (p = 0.069) for the NPL _a ιι _sp t statistics and 1.54 for the NPLpairs (p = 0.063). On the SSC 3 the NPL _a ιι _S pt 1 -33 (p = 0.095) and the NPL _pai rs _S pt 1-36 (p = 0.089). The error probabilities of the NPL statistics were greater than 0.1 for all other chromosomes. On SSC 9, the NPLaii spt was 0.75 (p = 0.226) and the NPL _pa irs spt 0.75 (p = 0.226). And for the SSC13 the values of the NPL _a n _{spt were} 0.85 (p = 0.197) and for the NPL _pair s _sp t it was 0.90 (p = 0.184).

Table 8: The highest NPL _a n and NPL _paιrs values of the individual chromosomes in the

Framework of the nonparametric coupling analysis (single point coupling analysis).

'Maximum NPL _paιrs statistics on SSC 5 at marker SW1463

Parametric coupling analysis with a variable genetic model

For the parametric coupling analysis, 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. However, this is not exactly possible in the case of anal atresia because there is no reliable information about the mode of inheritance, the prevalence and the penetrance of this defect. For the parametric coupling analysis, 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. As a comparison, 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. As part of the parametric evaluation, LOD scores between several markers and the disease locus (multipoint LOD scores) were calculated, which are referred to as the LOD score _m p _t . In addition, coupling analyzes between individual markers and the disease locus (single point LOD scores) were carried out, which are called the LOD score _late .

Genome-wide multipoint LOD-Score coupling analysis

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. With incomplete penetrance (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. With a penetrance of 90%, 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. With a penetrance of 90%, a positive LOD score _mpt of 0.41 was only achieved for the disease parallel frequency of 0.3. At a frequency of 0.1, 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. For the SSC 8, 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.

LOD score 0.3; 0.90: parallel patient frequency 0.3 / penetrance 0.90 LOD score 0.1; 0.90: parallel disease frequency 0.1 / penetrance 0.90 LOD score 0.3; 0.01: Disease allelence 0.3 / penetrance 0.01 LOD score 0.1; 0.01: Disease allence 0.1 / penetrance 0.01

1 2 Maximum LOD score 0.1; 0.90 and maximum LOD score 0.1; 0.01 on SSC 5 between SW413 and SWR453

Maximum LOD score 0.1; 0.01 on SSC 8 between S0069 and SO 144

Maximum LOD score 0.1; 0.01 on SSC 13 between SW398 and S0289

Maximum LOD score 0.1; 0.01 on SSC 15 between SW964 and SWR1533

Maximum LOD score 0.3; 0.01 on SSC X on marker SW1943

Genome-wide single point LOD score 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. Here too, the chromosomes that have reached a positive LOD score are _highlighted in gray in the table. In this analysis, the highest LOD score values were also on SSC 1. In the case of incomplete penetrance of 1% and a frequency of the disease allele of 0.3 a LOD score 2.05 _late on microsatellite markers SW1621 calculated. A higher LOD score of 2.40 is achieved with the same penetrance and with a frequency of the disease _allele of 0.1. With a penetrance of 90%, the maximum LOD score is _between 2.00 and 1.28. On SSC 12 and SSC 3 values of 1.13 and 1.04 were achieved for a pentrance of 1% and a disease parallel frequency of 0.3 LOD-Score _sp t. With a frequency of the disease allele of 0.1, the LOD score _p t is 0.25 and 0.47. Positive LOD _{scores late} with a pentrance of 1% and a frequency of 0.3 are also evident on SSC 11 and SSC 13 with values of 0.91 and 0.71. As well as SSC 8, SSC 2 and SSC 10 with LOD scores _late of 0.57, 0.42 and 0.40.

Table 10: The maximum LOD scores _late values of the genome-wide coupling analysis.

Maximum LOD score 0.3; 0.01 to SSC 6 S0099

² maximum LOD score 0.1; 0.01 to SSC 8 S0144

³ maximum LOD score 0.1; 0.90 on SSC 10 SW920

⁴ maximum LOD score 0.1; 0.90 on SSC 14 SWC27 LOD score 0.3; 0.90: parallel disease frequency 0.3 / penetrance 0.90 LOD score 0.1; 0.90: parallel disease frequency 0.1 / penetrance 0.90 LOD score 0.3; 0.01: Parallel disease frequency 0.3 / penetrance 0.01 LOD score 0.1; 0.01: Parallel disease frequency 0.1 / penetrance 0.01 Example 3: Coupling analysis when increasing the marker density to SSC1

After the genome-wide coupling analysis, a clear allele sharing of IBD alleles between the affected siblings was found on SSC 1 at marker SW1621, which was statistically significant. The non-parametric evaluation in the distal area of the chromosome resulted in an NPL _a ιι _mpt of 2.57 (p = 0.006) and for the parametric coupling analysis LOD-Scorβmpt values between 2.38 and 2.72. In order to improve the information content (info) at the individual marker positions, the marker density was increased on SSC 1.

Nonparametric coupling analysis

Results of the multipoint coupling analysis: To confirm the results, 8 additional microsatellite markers were genotyped on the SSC 1. The nonparametric coupling analysis could thus be carried out on 14 markers. This resulted in an NPLaii m _P t of 2.39 (p = 0.009) on the marker SW1621, with an information content of 0.74. 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. Singlepoint coupling analysis results:

For the NPL _a ιι _S p _t statistics, the inclusion of 8 additional markers resulted in a maximum value of 1.95 (p = 0.028) on the marker SW1621. The information content at this point was 0.35. The NPL _a ιι _S pt is therefore lower after increasing the marker density on SSC1 than in the first evaluation [NPL _a ιι _sp t = 2.14 (p = 0.018)]. The course of the NPL _a ιι _Sp t statistics on all 14 genotyped markers on SSC 1 (black graph) is shown in FIG. 2 in comparison to the first coupling analysis with 6 markers (blue graph). In this case, a high NPL _a ιι _s t value of 1.83 (p = 0.036) shows in addition to the markers SW1621, even at the S0155 microsatellite marker.

Information content (info) of the Singlepoint coupling analysis:

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 highest information content was achieved on the microsatellite markers S0316 and S0155 and was 0.61. At the position of the marker SW1621, however, there was only a value of 0.35. At this point, the information content was on a similar level to that in the first coupling analysis on SSC1 with 6 markers (Info = 0.38).

Example 4: Coupling analysis on SSC1 when expanding the family material

Nonparametric coupling analysis

Multipoint linkage analysis:

To confirm the first genome-wide coupling analysis on SSC 1, the supplementary family material was also included in the typing. When these additional 8 families without the individual piglets supplementing the existing HG were evaluated separately, the NPL value _mpt was 2.61 (p = 0.007) on the marker SW2185. This is approx. 15 cM proximal to the marker SW1621. In the next Step by step, the 27 families, along with the 8 new families, were evaluated at all 14 markers. This resulted in two NPL Maximas, one due to marker SW2185 as in the separate evaluation of the 8 additional families with an NPL _a ιi mpt of 2.05 (p = 0.022). Another maximum was at marker SW1621 with an NPL _a ii _mp t of 2.00 (p = 0.025) (see Figure 1). The marker SWR982 also showed an NPL statistics with an NPL _a ιι _pt of 2.00 (p = 0.025) which was at a very high level. A summary of the different coupling analyzes on SSC 1 on different family material and with different marker density is given in Table 11.

Table H: Comparison of the NPL _a n _mpt statistics on SSC1 in the coupling analysis with different family material and variable marker density.

Informativity of multipoint coupling analysis:

When the 8 additional families were evaluated separately, a high information content of 0.91 at the maximum of the NPL value (SW2185) was achieved (FIG. 5).

Single point analysis:

The separate evaluation of the 8 complementary families on 14 markers resulted in a maximum NPL _a ιι _S pt of 1.68 (p = 0.044) for the single point coupling analysis on the marker SW2185. The informativity at this marker position was 0.62. On marker SW1621, the NPL _a ιι _S pt was -0.15 (p = 0.586) and the informativity was 0.45 in the middle range. When evaluating the family material together, an NPL _a ιι _sp t of 0.71 (p = 0.240) was achieved on the marker SW2185 and an NPL _a ιι _sp t of 1.40 (p = 0.083) on the SW1621 marker. The informativity at the two marker positions was 0.36 and 0.35 in the lower area. However, the maximum value of the NPL _a ιι _spt statistics was reached at marker S0155. It was 1.44 (p = 0.077) with an average informativity of 0.52. A comparative representation of the NPL statistics of these two coupling analyzes with the first coupling analysis (27 families / 6 markers) on SSC1 is shown in FIG. 6. For a better overview, the most important results of the various single point coupling analyzes on SSC1 are summarized in Table 12. The maximum values of the NPL statistics are highlighted in gray in the table.

Table 12: Comparison of the NPL _a π _late statistics on SSC1 in the coupling analysis with different family material and variable marker density.

Informativity of the Singlepoint coupling analysis:

The course of the informativity about the individual markers in the single point coupling analysis is shown in FIG. Compared to the initial coupling analysis on SSC1, which achieved an informativity of 0.38 on marker SW1621, the information _spt value was 0.45 when evaluating the 8 additional families and 0.35 when evaluating the entire material. The position of the marker SW2185 showed an informativity of 0.62 in the evaluation of the 8 supplementary families and a value of 0.36 in the overall evaluation.

Example 5: Results of the TDT (Transmission-Disequilibrium Test)

For the chromosomes which carry possible candidate genes or which had shown a significant result in the NPL statistics in the nonparametric coupling analysis, 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 marker, the χ ² test variables for the two test statistics T _m and T _m Het _>, ^the number of degrees of freedom and the probability of error are given. The order of the markers in the table reflects the arrangement on the chromosome. The marker SW2185, which had shown the NPL _a ιι 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. For the marker SW1621 and the 6.0 cM distal SW1902, however, highly significant χ ² values could be determined for the test statistics T _m and TmHet. The probability of error at this time was for T _m 2 x 10 ^"6 (SW1621) and 0.002 (SW1902) and T _m Het 7 x ^10" x 10 ^"4 (SW1902). At ⁷ (SW1621) and 2, the position of the marker SW1621 was the maximum of the NPL _a n statistics of 2.57 (p = 0.006) in the first coupling analysis with 6 markers, whereas in the confirmation study with 14 markers and enlarged family material the NPL _a ιι mp _t was only 2.00 (p = At the position of marker SW1902, the NPL _a ιι value in the confirmation study (14 markers) was 1.56 (p = 0.064). Markers S0155 and S0320 also showed significant error probabilities for both TDT test statistics. These were 0.016 and 0.016, respectively 0.028 for T _m and at 0.006 or 0.002 for TmHet.

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 α _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 haplotype 1-2 TDT, ie allele 1 (SW1621) = 146bp-allele 2 (SW1902) = 150bp was the most common haplotype (60.4%) both in the sick piglets and in the boars with 50.7% (see Figure 8). 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.

Results of the TDT on SSC3

The results of the TDT on SSC3 are shown in Table 14. At the location of the NPLaii mpt maximum of 1.90 (p = 0.031) on marker S0002, the error probability of the T _m test statistic was 0.349 and the p-value of T _m Het was 0.225. The TDT is therefore not significant on this marker. Somewhat lower error probabilities were achieved on the SW902 marker with 0.206 and 0.185, which, however, were also above the significance limit of 0.05. Table 14: Results of the TDT test statistics T _m and T _m Het on 7 microsatellite markers from SSC 3.

(Significance limit for multiple testing α _m | _t = 0.007)

Results of the TDT on SSC 8

The TDT showed on SSC 8 on marker S0069 with 0.043 and 0.024 a significant result of the T _m and T _m H _e t test statistics (see Table 15). Within the nonparametric evaluation, the NPL _a ιimpt at this position was 1.37 (p = 0.087) with an average informativity of 0.48. After correction for multiple testing, the alleles of marker S0069 were no longer significant. No significant result of the TDT could be determined for all other markers on SSC 8.

Table 15: Results of the TDT test statistics Tm and Tm Het on 7 microsatellite markers from SSC 8 (limit of significance for multiple tests _m ι _t = 0.007).

Results of the TDT on SSC9

The results of the TDT on SSC9 are shown in Table 16. A significant result was achieved with both test statistics on marker S0081. The Error probabilities were 0.023 for the Tm and 0.001 for the T _mH et test size. At the position of marker S0081, an NPL _a ιimp _t of 0.67 (p = 0.251) was achieved in the nonparametric evaluation. The maximum of the NPL statistics was at the position of marker SW2401, which with an NPLaiimpt of 1.13 (p = 0.130) is about 19.9 cM ahead of marker S0081.

Results of the TDT on SSC12

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. However, the maximum NPLaiimp _{t of} the non-pararmetric evaluation was not at marker SW957 but 14.1 cM proximal from this position at marker S0229 with a value of 1.94 (p = 0.028) and an informativity of 0.89. The NPLaiimpt on the marker SW957 was only 1.78 (p = 0.040) with an informativity of 0.68.

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 α _m | _t = 0.007).

Results of the TDT on SSC13

The results of the TDT on SSC13 are shown in Table 18. No significant result was found on the 6 microsatellite markers of the chromosome. The marker S0068 with the maximum NPL _a n _m pt of 1:57 (p = 0.061) could likewise not obtain a significant result.

Table 18: Results of the TDT test statistics T _m and T _m Het on 6 microsatellite markers

Result of the TDT on SSC 15

The results of the TDT for chromosome 15 are shown in Table 19. Although the results of the nonparametric coupling analysis in this study showed no evidence of a coupling of the markers with the disease locus (max.NPLaii mpt = 0.05 SWHI _Θ ), a TDT was carried out with regard to the coupling between the marker SW2072 and Atesia ani found by Hori, 2001 carried out. No significant test result (p = 0.933) for the TDT could be achieved on marker SW2072. However, marker S0148, which is approximately 22.1 cM distal to marker SW2072, showed a significant test result with an error probability of 0.011 and 0.025 for the T _m and T _m Het statistics. At marker S0355, which is 3.1 cM distal from SW2072, the TmHet statistics showed a result 9.21 with an error probability of p = 0.056. However, after the significance level was corrected for multiple testing 0.007, none of the markers showed a significant result Table 19: Results of the TDT test statistics T _m and T _m Het on 7 microsatellite markers from SSC15 (significance limit for multiple tests α _m ι _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).

The separate analysis of the newly added data material, consisting of 22 parent-offspring pairs, does not allow independent confirmation of the relationships found so far. The main reason for this is that only a few parent-offspring pairs proved to be informative. However, the informative pairs show the expected pattern of inheritance, ie the alleles and haplotypes associated with the occurrence of atrophy are passed on more often from the parent to the offspring in the partial material than would be expected if there were no relationship. (Table 20: Test statistics and error probabilities (p) for the association of! Marker alleles or haplotypes of markers SW1621 and SW1902 with the

| Occurrence of anuslessness

Data TestSW1621 SW1902 Haplotype procedure TDT P TDT P TDT p

Data from Tm 32.10 1.07x10 ^" '19.58 0.001 32.49 0.001 Table 13 Tmhet 34.27 2.68xl0 ^{" 8} 23.39 2.81xl0 ^"4 37.32 1.99xl0 ^{" 4} and 4 maternal HG families

Additional Tm 2.04 0.153 4.04 0.133 5.62 0.060 Data material Tmhet 1.80 0.180 3.47 0.177 3.75 0.154 (22 parents-NK pairs)

'Total Tm 33.24 6.06xl0 ^{~ s} 20.50 0.001 35.74 3.57xl0- ⁴ Tmhet 36.67 1.09xl0 ^-8 24.21 1.98xl0 ^"4 39.68 8.12xl0 ^{" 5}

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. The finding that the TDT values are close to the significance limit was analyzed further and was able to determine the unfavorable distribution of allele frequencies in the original breeds (see below ) to be led back. 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

Data TestSW1621 SW1902 process TDT p TDT p

Test animals Tm 5.35 0.069 10.49 0.063 Tmhet 4.80 0.091 10.67 0.058

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

Marker afterless piglets

All!

SW1621

146 ^* 0.87

148 0.06

150 0.07

SW1902

142 0

144 0.02

146 0.05

150 ^* 0.73

152 0.14

154 0.06 Alleles associated with anuslessness Example 9: Additional study data for allele frequency estimation for markers SW1621 and SW1902 on healthy animals of the Pietrain and Deutsche Landrasse breeds

In order to obtain an overview of the occurrence of the alleles associated with the markers SW 1621 and SW1902 in the most important Bavarian pig breeds, samples of 125 Pietrain boars and 123 boars of the German Landrace were examined. Table 23 shows the estimates for the frequencies of the corresponding alleles. The alleles associated with anuslessness are present with a relatively high frequency in the two races.

Table 23: Estimates of the allele frequencies for markers SW1621 and SW1902 in pure breed animals of the breeds Pietrain and Deutsche Landrasse

Marker breed

Allele Pietrain German Landrace

SW1621

146 ^* 0.61 0.78

148 0.35 0.03

150 0.04 0.19

SW1902

142 0 0

144 0 0.12

146 0.20 0.02

150 ^* 0.60 0.59

152 0.20 0.20

154 0 0.06 Alleles associated with afteriosity

Claims

claims

1. Use of a first nucleic acid to determine 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 substantially identical to a second nucleic acid which occurs,

(a) on chromosome 1 of the pig or in a homologous position in the genome of other mammals, in the region of a microsatellite selected from the group consisting of SW2185, SW1621, SW1902, S0155, and S0320;

(b) on chromosome 3 of the pig or in a homologous position in the genome of other mammals, in the region of the microsatellite S0002;

(c) on chromosome 9 of the pig or in a homologous position in the genome of other mammals, in the region of a microsatellite selected from the group consisting of SW2401 and S0081; or

(d) on chromosome 12 of the pig or in a homologous position in the genome of other mammals, in the region of a microsatellite selected from the group consisting of SW957 and S0229.

2. Use of a combination of at least two or more of the nucleic acids mentioned in claim 1 for determining the expression or inheritance of the phanotype "afterlessness".

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 3, wherein the microsatellites SW1621 and SW1902 are detected together and one

Characterize haplotypes that are associated with a predisposition to the expression or inheritance of the phenotype "afterlessness".

5. Use according to claim 4, wherein the haplotype by the common inheritance of:

(a) Allele 1 (146bp) of the SW1621 microsatellite; and

(b) Allele 2 (150 bp) of the microsatellite SW1902 is defined.

6. Use according to one of claims 1 to 5, wherein the second nucleic acid is derived from a gene 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 Y) -box 9, BMP2 bone morphogenetic protein 2, BMP4 bone morphogenetic protein 4, NOG

Noggin, FMN Formin, ALDH1A1 Aldehyde dehydrogenase 1 family member A1, ALDH1A2 Aldehyde dehydrogenase 1 family member A2, ALDH1A3 Aldehyde dehydrogenase 1 family member A3, CYP19A1 Cytochrome P450, family 19, subfamily A, polypeptidel, PML Homeyoboxox11 AoxobloxXA11 homeukobloxA11 A cluster), HOXA13 Homeobox A13 (Homeobox

A cluster), HOXB1 Homeobox B1 (Homeobox cluster B), HOXB8 Homeobox B8 (Homeobox cluster B), HOXB9 Homeobox B9 (Homeobox cluster B), HOXB5 Homeobox B5 (Homeobox cluster B), HOXD13 Homeobox D13 (Homeobox D cluster).

7. Use of a second nucleic acid mentioned in one of claims 1 to 6 for the selection of domestic, breeding or farm animals without the phanotype "afterlessness".

8. Use according to claim 7, wherein the domestic, breeding or farm animals are from the group consisting of cattle, dog, cat, rabbit, buffalo, camel, alpaca, mink, pig, goat, sheep, horse, donkey, Rat and mouse.

9. Use according to claim 8, wherein a genome screen is carried out on several mammals of a population.

10. In vitro methods for determining the predisposition to the expression or inheritance of the phanotype "afterlessness" in mammals, preferably in domestic, breeding or farm animals, the animals, their fertilized or unfertilized egg cells, or their sperm for the presence,

Tests the nature or expression of a second nucleic acid mentioned in one of claims 1 to 6.

11. The method according to claim 10, wherein the domestic, breeding or farm animals are from the group consisting of cattle, dog, cat, rabbit, buffalo,

Camels, alpaca, mink, pig, goat, sheep, horse, donkey, rat and mouse.

12. The method according to claim 10 or 11, wherein one

(a) performing a PCR amplification with complementary primers with a length of at least 8 nucleotides, with one primer attached to the +

Strand and another primer in opposite orientation the strand which binds the second nucleic acid mentioned in one of claims 1 to 6; or

(b) performing a hybridization, wherein a hybridization probe with a length of at least 8 nucleotides is attached to that in one of the

Claims 1 to 6 mentioned second nucleic acid binds; or

(c) sequencing the second nucleic acid mentioned in one of claims 1 to 4; or

(d) performs a detection with a specific antibody or antibody fragment or antibody derivative or aptamer, the antibody or antibody fragment or antibody derivative or aptamer specifically against one in one of the Claims 1 to 6 mentioned first or second nucleic acid is directed.

13. The method of claim 12, wherein a genomic screen is performed on multiple mammals in a population.

14. Kit at least

(a) a pair of primers for the amplification of the second nucleic acid mentioned in claims 1 to 6, wherein one primer binds to the + strand and another 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 mentioned in one of claims 1 to 6; or

(c) a specific antibody or an antibody fragment or an antibody derivative or an aptamer which is linked to that in one of the

Claims 1 to 6 said first or second nucleic acid binds; in one or more containers.