DE10155999A1 - Determining the condition of exo- and endo-skeleton in animals, for selecting animals for breeding, comprises a method based on genotype of the diastrophic dysplasia sulfate transporter gene - Google Patents

Abstract

Determining the condition and quality of: (i) a paw or hoof horn; and (ii) the skeletal system in animals, comprises analyzing the diastrophic dysplasia sulfate transporter gene (X) for the presence of mutations at positions 4117 and/or 4259 (of a 6524 base pair sequence (S1), given in the specification).

Description

The present invention relates to a method for determining of the nature and quality of the claw and hoof horn and the skeletal system in hoofed and hoofed animals - conventional referred to as foundation and exterior - by means of Genome analysis.

According to the state of the art, the quality of the foundation and the exterior of an animal to determine the contra Stability of its claws and hooves against animal and posture-specific environmental conditions with the help of biomet defined methods. Here, in particular Hoof or claw angle and heel height and the diagonal of the hoof or claw footprint (Huber et al., 1984). One assumes that one is under average and undesirably soft claw or an under average and undesirably soft hoof with one against angulation reduced above average, enlarged Diagonal and shortened heel height correlated. This to name is based on the observation that a soft claw is subject to stronger signs of abrasion and therefore the angulation of the claw tip is reduced. To be more precise say about the goodness of the foundation and the exterior of one To be able to meet certain animal breeds, a number of Animals of the breed in question are examined to be sure represent that genetically predisposed foundation qualities  not through extreme, accidental or systematic environmental influences have been falsified.

Basically, the dimensions of angulation, heel height and diagonal of a hoof or a claw and the by means of ma data from thematic relationships only indicative for a good or bad quality of foundation and extent deliver interior. Statements about the inheritance of claw quality on the offspring are difficult, Un studies in several generations of the descendants of one each subject animal and extensive statistics cal calculations are carried out.

In addition, the claw strength is often from practice reasons of stability simply based on the heel height estimates what leads to further inaccuracies made statements about the quality of the hoof or hoof creativity leads.

These problems described for hooves and claws meet in analogous to the determination / determination of the skeleton systems (exterior) based on pelvis width and height to.

At least to determine the strength of claws and hooves To standardize, some measuring methods have already been proposed provides with which, using special equipment Hardness, abrasion resistance and the necessary shear force analy can be used (Kamara and Gravert, 1971). The biometri measuring the claws or hooves and building a corresponding physical tests is very time consuming agile and live animal with an increased injury risk connected to humans and animals. In the practical animal For this reason, these methods have not yet been bred can put.  

The present invention is based on the object To provide procedures with which the quality of the Funda reproducible and the exterior of an individual animal can be voted.

One solution to this problem is to provide egg nes procedure of the type mentioned above that distinguishes that one selects the genome or the DNA of one subjects the animal to a genome analysis and that in the gene for the diastrophic dysplasia sulfate transporter (DTDST gene) by means of direct or indirect verification procedures the presence of nucleotide mutations in position 4117 and / or position 4259, each according to the sequence listing SEQ ID NO. 1, analyzed.

This is a new procedure for quality inspection of stealing enhorn, hoof horn and skeletal systems in hoofed and hoofed animals provided that this is different from the state of the art nik stands out that the parameters used for the evaluation are not geometric or biometric quantities as before, but the nucleotide positions 4117 and 4259 in bovine DTDST gene according to sequence listing SEQ ID NO. 1. With this The test or assessment procedure is the target or standard value the wild-type occupation of these nucleotide positions, and Abwei Indications indicate a positive or negative state. Which of the two is available ultimately becomes for each Rin the or animal breed determined separately, that in a representative group of animals of a certain Breed with the same genome analysis result (in the Nucleotide positions 4117 and 4259 of the bovine DTDST gene) actual condition of the claw horn, hoof horn and ske lettsystems compared to the wild type.

The position information 4117 and 4259 refer to the Nucleotide sequence according to sequence listing SEQ ID NO. 1. Should it turns out in the future that the gene is longer or  is shorter, the expert can by comparing the new Se sequence with the sequence according to SEQ ID NO. 1 clearly determine which positions in the new sequence the positions 4117 and 4259 according to the present patent application. The genome analysis can be carried out, for example, in that to examine double-stranded or single-stranded DNAs of the DTDST gene compared to the wild-type nucleotide sequence according to sequence listing SEQ ID NO. 1 or that to double-stranded or single-stranded DNA fragments of the investigating DTDST gene compared to corresponding Partial sequences of the wild-type nucleotide sequence according to sequence pro tokoll SEQ ID NO. 1 lets it wander in an electric field and an existing nucleotide polymorphism based on the dodging migration rate of DNA or DNA Fragments of the DTDST gene to be examined compared to Wild type sequence or the wild type sequence fragments (Detected).

This analysis can just as well be carried out using a wild-type Divide nucleotide sequence (according to sequence listing SEQ ID NO. 1) appropriate ligation behavior or hybridization behavior can be detected.

This procedure is based on the genetic analysis of the diastrophi Dysplasia sulfate transporter gene (DTDST gene). The Human DTDST gene is from Hästbacka et al. (1994) sequenced and have been characterized. Mutations in the DTDST gene lead to severe disorders of cellular sulfate transport and too serious changes in bone growth. (Homozy Goth mutants are not viable and die the embryo nalen early death).

In the course of the development of the present invention, it is ge were also able to sequence the DTDST gene from cattle. In further studies of this bovine DTDST gene  then surprisingly bovine DTDST gene mutants were ge find a nucleotide exchange at position 4117 and Po sition 4259 in the sequence listing SEQ ID NO. 1, näm C4117 to T4117 and T4259 to G4259.

In addition, it was also surprisingly found that these cattle mutants with the heterozygous genotypes C4117 / T4117 or T4117 / C4117 and T4259 / G4259 or G4259 / T4259 as well as the homozygous genotypes T4117 / T4117 and G4259 / G4259 do not show the serious physiological disorders, that are found in humans, but that they change you have changed sulfur metabolism, which changes with a different composition of the claw horn or hoof goes. This change is the cause of the differences cheek or hoof strength. Bone growth is also changed in these cattle mutants compared to the wild type, in particular, the size growth and the pool width are sig significantly lower.

With the method according to the invention, the poss the quality and quality of the fun daments, especially the claw and hoof horn at hoof and Hoofed animals, especially cattle, and / or the prospective size and pelvic width of the animal based on the poly Morphism C / C, C / T, T / C and T / T at position 4117 and the Po Lymorphism T / T, T / G, G / T, G / G at position 4259 in the sequence protocol SEQ ID NO. 1 to determine.

Evidence of the presence or absence of the mutations at positions 4117 and 4259 in the bovine DTDST gene it can either directly or indirectly consequences. In particular, the Sequencing of the (from the animal to be examined) isolated DNA is suggested, for example Ligation procedures and hybridization procedures.  

A particularly advantageous direct detection method is based on the surprising discovery that the two nucleotides exchanges from C4117 to T4117 and from T4259 to G4259 DTDST gene mutants result in comparison to the DTDST wild-type gene one PstI interface less (position 4117) and one Have additional DdeI interface (item 4259). A enzymatic cleavage of this mutant bovine DTDST gene with the restriction enzyme PstI therefore provides in comparison to DTDST wild-type gene fewer cleavage products, while one zymatic cleavage of this mutated bovine DTDST gene with the restriction enzyme DdeI compared to the DTDST wild-type gene provides an additional cleavage product. The division pro products can e.g. B. by means of a gel electrophoretic appearance can be proven without any problems.

The mutation at position 4259 also results in an out swap the amino acid isoleucine520 to serine520, and this Amino acid exchange can also be used for indirect detection of the mutations according to the invention are used. (The Mutation at position 4117 does not change the Ami acid sequence).

An easy in practice, i. H. with common and relative one fold means of realization variant of the he The method according to the invention is characterized in that first of all from a DNA-containing cell or body fluid samples of the animal, in particular from a blood or sperm or tissue sample that isolates and purifies the DNA that the isolated and purified DNA of a polymerase chain Subject to reaction (PCR) using primers which are suitable for the nucleotide sequence according to the sequence protocol SEQ ID NO. 1 at least in the area of position 4117 and to amplify in the area of position 4259 that one the amplificates obtained in this way (i.e. partial sequences nucleus otide sequence according to sequence listing SEQ ID NO. 1, the  Nucleotide positions 4117 and / or 4259 included) with the Restriction enzyme DdeI or isoschizomers of this enzyme or with the restriction enzyme PstI or isoschizomers of this ene zyms cleaves, and that the resulting cleavage pro products separated by gel electrophoresis, visible by staining (visualizable) makes and with regard to the existence or missing (depending on the choice of restrictions zyms) of additional cleavage pro compared to the wild type products evaluates. In this variant of the method, the comes for the evaluation used nucleotide polymorphism (C / C, C / T, T / C and T / T at position 4117 and T / T, T / G, G / T, G / G on Position 4259, each in the sequence listing SEQ ID NO. 1) indi rectifies in the restriction fragment length polymorphism Cleavage expressed with the restriction enzyme. The straight zygote and homozygote mutants show (depending on Choice of the restriction enzyme used) additional or missing interfaces for the restriction enzymes DdeI and PstI more or less cleavage products d. H. restriction fragments than the homozygous wild type.

Forward primers with a Nucleotide sequence, which is the nucleotide sequence of position 3858 to position 3877 according to sequence listing SEQ ID NO. 1 to at least includes, and reverse primer with one nucleotide sequence which the nucleotide sequence from position 4321 to Po sition 4339 according to sequence listing SEQ ID NO. 1 at least around summarizes, used.

With the method according to the invention it is possible to hoof and To detect and select hoofed animals that are passing through have a high, genetically determined claw hardness, and / or who qualify through their growth in size. The The process thus represents a new possibility for measurable and standardized determination of the foundation and exterior original quality in hoofed and hoofed animals. It is simple  chen and in the prior art methods of molecular Largenetic laboratory diagnostics feasible, delivers more precise Statements than the conventional biometric methods and is much cheaper than this. Because the procedure of random or systematic environmental factors of the proban dentieres is unaffected, the thus determined apply Measurement results and the resulting statements about Funda ment and exterior quality even if the animal in question is brought into another environment and allow as a result its an extensive standardization.

All types of DNA can be used as test material Tissues and body fluids of the test animal used become.

A particular advantage of the method according to the invention is that it enables prenatal diagnosis. This procedure can be used even in the prenatal state will give how the foundation and exterior quality will express. This is for practical breeding work by of special interest.

To determine the extent to which the quality of the foundation corresponds to the Descendants of the test person are inherited, are not expensive offspring exams necessary. In the previous system the assessment of the foundation and the claws over the general made my usual linear exterior description.

The invention is illustrated below with the aid of an embodiment explained in more detail.  

example 1

A subject animal gets blood, sperm or other DNA-containing Tissue removed and the DNA isolated and ge cleans. With the help of the polymerase chain reaction, the Ge Section of the bovine genome between positions 3858 and Posi tion 4339 according to sequence listing SEQ ID NO. 1 using of forward primers with the nucleotide sequence 5'-CATTGGGTTTGCTATCACTG-3 '(corresponds to positions 3858 to 3877 in the sequence listing SEQ ID NO. 1) and backward primers (rever se primer) with the nucleotide sequence 5'-ACTCAGAAGCCAAAGGCTT-3 ' (corresponds to positions 4321 to 4339 in the sequence listing SEQ ID NO. 1) amplified. The amplificate obtained is re restriction enzyme (DdeI) cleaved and the reaction products applied and visualized on an agarose gel. The ban denmuster provides information about the genotypes and thus about your own as well as the potentially inheritable foundation and The subject's claw quality.

Isolation of the DAN

The isolation of DNA from blood, sperm or tissue was done with carried out using common standard procedures.

Reaction approach for the PCR

The following components are pipetted into a reaction mixture of 25 µl:
10 mmol / l KCl, 10 mmol / l (NH ₄ ) ₂ SO ₄ , 20 mmol / l Tris-HCl pH 8.75, 2 mmol / l MgSO ₄ , 0.1% Triton ™ X-100, 100 µm / ml BSA, 0.2 mmol / l dNTP, 20 µmol of the primers given below 0.1 pmol of the purified DNA of the test subject, 1 U Taq-DNA Polymera se.
Forward primer: 5'-CATTGGGTTTGCTATCACTG-3 ',
Reverse primer: 5'-AGGCCTTTGGCTTCTGAGT-3 '.

The following temperature profile was used:
Initial denaturation of the starting DNA by heating to 94 ° C for 3 min. then 30 cycles with the following characteristics: 94 ° C for 3 min. 56 ° C for 1 min. 72 ° C for 1 min. This was followed by heating to 72 ° C. for 5 minutes. Finally, the reaction mixture was cooled down to 4 ° C.

restriction digestion

Reaction buffer 50 mmol / l Tris-HCl, 10 mmol / l MgCl ₂ , 100 mmol / l NaCl. The amplification products were cleaved with 5 U DdeI / µg DNA for 2 hours at 37 ° C.

Separation and visualization of the reaction products

Running buffer (10 × concentrate): 130 mmol / l Tris, 45 mmol / l boric acid, 2.5 mmol / l EDTA
Loading buffer: 20% sucrose, 0.05% orange G.

The reaction products are taken up in the loading buffer, applied to a 1.5% agarose gel in 1 × TBE buffer and visualized the DNA with 0.05 µg / ml dimidium bromide.

Depending on the genotype, the present primers result in ei ne different number of fragments. In the amplifi graced area are at the allele A, which the DTDST wild-type gene corresponds to three DdeI recognition sequences. Dar resulting in 4 fragments with the lengths 18 bp, 30 bp, 156 bp and 279 bp, and accordingly 4 bands. With the allele B, which contains the mutations, are identified by the additional  DdeI interface at position 4259 instead of the wild type question elements with the length 279 bp two fragments with the lengths 212 bp and 67 bp were generated and consequently 5 bands were obtained.

The band pattern can also be used to determine whether the subject animal hetero regarding the mutated DTDST gene zygot or homozygous is: The presence of gangs with frag Ment lengths of 18, 30, 67, 156, 212 and 279 bp indicate the heterozygous status (mutant allele / wild-type allele), the pre presence of bands with fragment lengths of 18, 30, 67, 156 and 212 bp indicates the homozygous status (mutant Al lel / mutant allele).

The drawings show:

Fig. 1 the detection according to the invention the Nukleotidvarian th at position 4259 (the ge with dimidium bromide stained gel shows the band patterns of the individual Ge genotypes after cleavage with the restriction enzyme DdeI. M = 1 kB ladder) and

Fig. 2 detection of the nucleotide variants according to the invention at position 4117 (the gel stained with dimidium bromide shows the band patterns of the individual gene types after cleavage with the restriction enzyme PstI. M = 1 kB ladder).

With proof of the point mutation at position 4259 or 4117 the genotype of an animal can be linked to it as described Clearly determine positions. The connection between the Genotype and the quality of the hoof or hoof horn consists of the present genotype.  

Animals with the genotype AA (see Fig. 1 and 2) have flat (low rear heel height) and rather soft claws or hooves (poor quality, not preferred).

Animals that have the genotype AB (see Fig. 1 and 2) show a medium high rear heel height and have a medium hardness (moderate quality, conditionally preferred).

Animals with the genotype BB (see Fig. 1 and 2), however, have a steep claw or hooves (high rear heel height) and the horn is hard (high quality, preferred).

Depending on the breeding goal, the geno can be determined types AA, AB or BB a statement about the expected Qua the horn and the heel height become.  

SEQUENCE LISTING

Claims (4)

1. A method for determining the nature and quality of the claw and hoof horn and the skeletal system in hoof and claw animals, characterized in that the genome or the DNA of a selected animal is subjected to a genome analysis and the gene for the diastatic dysplasia sulfate transporter (DTDST gene) for the presence of nucleotide mutations at position 4117 and / or position 4259 according to the sequence listing SEQ ID NO. 1 analyzed. 2. The method according to claim 1, characterized in that to position the gene for nucleotide exchange 4117 and / or position 4259 according to sequence listing SEQ ID NO. 1 analyzed. 3. The method according to claim 1 or 2, characterized in that you can make from a DNA-containing cell or body fluid test, especially from a blood, sperm or tissue sample, isolate the animal's DNA and purify that this isolated and purified DNA of a polymerase chain Reaction (PCR) subjects using primers to do so are suitable, the nucleotide sequence according to the sequence listing SEQ ID NO. 1 least in the area of heading 4117 and / or in the area of position 4259 to amplify that the amplicons obtained here (partial sequences) with the Re restriction enzyme DdeI or with the restriction enzyme PstI or with an isoschizomer of one of these two restrictions zyme splits, and that the resulting splitting pro separates products by gel electrophoresis, makes them visualizable  and in terms of additional or compared to the wild type missing (depending on the choice of the restriction enzyme used) Fission products are evaluated. 4. The method according to claim 3, characterized in that as a primer forward primer with a nucleotide sequence which the nucleotide sequence from position 3858 to position 3877 according to sequence listing SEQ ID NO. 1 at least includes, and re upward primer with a nucleotide sequence which is the nucleotide sequence from position 4321 to position 4339 according to sequence pro tokoll SEQ ID NO. 1 at least includes.