KR102269653B1 - A SNP marker composition and a method for diagnosis or prediction of osteochondrodyplasia in cats - Google Patents

Abstract

The present invention relates to a single nucleotide polymorphims (SNP) marker composition and method for predicting or diagnosing osteochondrosis in cats, and more particularly, TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4 ) It relates to a SNP marker composition and method for predicting or diagnosing osteochondrosis in cats, including an agent capable of amplifying or detecting a single nucleotide polymorphism (SNP) of a gene.

Description

A single nucleotide polymorphic marker composition for predicting or diagnosing osteochondrodysplasia in cats and a prediction or diagnosis method using the same {A SNP marker composition and a method for diagnosis or prediction of osteochondrodyplasia in cats}

The present invention relates to a single nucleotide polymorphims (SNP) marker composition and method for predicting or diagnosing osteochondrosis in cats, and more particularly, TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4) It relates to a SNP marker composition and method for predicting or diagnosing osteochondrosis in cats, including an agent capable of amplifying or detecting a single nucleotide polymorphism (SNP) of a gene.

Osteochondrodysplasia in cats is a representative incomplete dominant genetic disease caused by indiscriminate mating. Scottish folds, which are popular among cat lovers for their cute appearance features such as short legs and tail, and folded ears, Breeds such as American Curls and Munchkins are the most prevalent of the disease. Osteochondrodysplasia in cats enters the growth phase from 4 months of age, causing abnormalities in bone or cartilage production, causing problems in walking, and in severe cases, arthritis and bone tumors. If already onset, it can be corrected through periodic drug treatment and surgery. It is one of the diseases that require early diagnosis technology as it is difficult to diagnose and treat the disease at an early stage before the onset of the disease.

Currently, osteochondrodysplasia is diagnosed by X-ray or ultrasound examination from 5 to 6 months of age. First, it is difficult to diagnose early before 5 months of age. Second, general anesthesia is required for the examination. Thirdly, there is a disadvantage that the determination criteria and accuracy may vary depending on the growth stage and variety, so the development of a new diagnostic method is required in the field of veterinary medicine to compensate for this. In particular, in the case of a physical examination, when a hip dislocation is suspected, it is a method of measuring whether the pain in the hind leg and abduction of the leg are normal, and there is a disadvantage in that the accuracy is lowered because the criteria may vary depending on the growth stage. In addition, X-ray imaging is a method of evaluating osteochondrosis based on X-ray photos taken three times and a database, and it is necessary to calm the animal through anesthesia before the examination, and a specialist with specific qualifications Since it is diagnosed by the Inspector, there are problems such as the need to pay attention to the final reading because the judgment criteria are different for each inspection institution. Therefore, in order to diagnose or prevent osteochondrodysplasia in cats at an early stage and to minimize damage to pre-sale, it is necessary to identify the disease early and reduce the disease incidence by selective breeding and breeding. To this end, it is necessary to identify the genetic predisposition to the disease at an early stage. There is an urgent need for the development of a DNA diagnostic method.

(Non-Patent Document 001) Zhou Z, Sheng X, Zhang Z, Zhao K, Zhu L, et al. Differential Genetic Regulation of Canine Hip Dysplasia and Osteoarthritis. 2010, PLoS ONE 5(10).

(Non-Patent Document 002) Fels L, Distl O. Identification and Validation of Quantitative Trait Loci (QTL) for Canine Hip Dysplasia (CHD) in German Shepherd Dogs. 2014, PLoSONE 9(5).

(Non-Patent Document 003) Enrique Sachez-Molano., John A Woolliams., Ricardo Pong-Wong., et al. Quantitative trait loci mapping for canine hip dysplasia and its related traits in UK Labrador Retrievers. 2014, BMC Genomics 2014, 15:833

The present inventors discovered single nucleotide polymorphims (SNPs) highly correlated with osteochondrosis of cats for predicting or diagnosing osteochondrosis in cats, specifically amplifying them, and a composition capable of rapidly and accurately diagnosing them and to invent a method.

Accordingly, it is an object of the present inventors to provide a composition and method for predicting or diagnosing osteochondrosis in cats including novel SNP markers.

In order to achieve the above object, the present invention TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4 ) single nucleotide polymorphism (single nucleotide polymorphism, SNP) of the gene amplifying or detecting a cat bone comprising an agent Provided is a SNP marker composition for predicting or diagnosing chondrodysplasia.

In addition, the present invention includes a primer set or probe set capable of amplifying or detecting a polynucleotide comprising a single nucleotide polymorphism (SNP) of TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4) gene It provides a SNP marker composition for predicting or diagnosing osteochondrodysplasia in cats.

In addition, the present invention comprises the steps of 1) collecting a DNA sample from a cat;

2) performing PCR using the primer set of SEQ ID NO: 2 and SEQ ID NO: 3, or the probe set of SEQ ID NO: 4 and SEQ ID NO: 5 using the DNA collected in step 1) as a template;

3) examining the fluorescence value of the product amplified by PCR in step 2);

It provides a method for predicting or diagnosing osteochondrosis in cats, including.

In addition, the present invention provides a kit for predicting or diagnosing osteochondrosis in cats comprising a single nucleotide polymorphism (SNP) marker composition of the TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4) gene.

The marker composition and method comprising SNP for predicting or diagnosing osteochondrodysplasia in cats according to the present invention are first, in an objective and scientific way compared to physical examination or X-ray imaging, conventional imaging tests and DNA genotyping tests. In parallel, it is possible to minimize errors that may appear in the test results and improve accuracy. Second, even without clinical symptoms, early diagnosis is possible immediately after birth, which can contribute to reducing the incidence of diseases by avoiding genetic risk factors and breeding. Third, there is no need for anesthesia before the examination, and the examination can be easily performed using oral epithelial cells or hair roots. Fourth, it is possible to minimize the risk, cost, and time loss that may occur when an X-ray imaging test is requested overseas. It can be usefully used for early predictive diagnosis of feline osteochondrodysplasia.

The cat osteochondrosis diagnosis composition and method of the present invention is a method of analyzing the genotype, not the phenotype, and has the advantage of being able to discriminate osteochondrosis and quickly and easily diagnose without expensive equipment. A revolutionary alternative to

1 shows six cSNPs identified in TRPV4 candidate genes.
2 is the NCBI sequence of the TRPV4 gene.
3 is a specific amplification of the allele of G1127T, which determines cat osteochondrosis, using primers and probe sets corresponding to SEQ ID NOs: 2 to 5, (A) is a graph of allelic differentiation (Allelic); discrimination plot), and (B) is a graph showing amplification curves of each GG, GT, TT genotype and Negative Control labeled with a fluorescent substance.
4 shows the results of analyzing the genes of three allelic types (1127GG, 1127GT, 1127TT) analyzed using the real-time polymerase chain reaction method through DNA sequencing.

Hereinafter, the present invention will be described in detail.

The present invention is a TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4 ) SNP for predicting or diagnosing osteochondrosis in cats comprising an agent capable of amplifying or detecting single nucleotide polymorphism (SNP) of the gene A marker composition is provided. preferably TRPV4 The gene is SEQ ID NO: 1, TRPV4 A single nucleotide polymorphism (SNP) of a gene is one in which the 1127th base is G or T.

In addition, the present invention includes a primer set or probe set capable of amplifying or detecting a polynucleotide comprising a single nucleotide polymorphism (SNP) of TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4) gene It provides a SNP marker composition for predicting or diagnosing osteochondrodysplasia in cats. Preferably, TRPV4 The gene is SEQ ID NO: 1, TRPV4 A single nucleotide polymorphism (SNP) of a gene is one in which the 1127th base is G or T. More preferably, the composition is a primer set of SEQ ID NO: 2 and SEQ ID NO: 3 or a probe set of SEQ ID NO: 4 and SEQ ID NO: 5, even more preferably at the 5' end of SEQ ID NO: 4 and SEQ ID NO: 5 It is labeled with a fluorescent substance, VIC or FAM, and NFQ (Non Fluorescent quencher)-MGB (Minor Groove Binder) is bound to the 3' end of SEQ ID NO: 4 and SEQ ID NO: 5. In addition, preferably, the detection is a SNP marker composition for predicting or diagnosing osteochondrosis in cats, which is detected by a realtime PCR analysis using a Taqman probe.

The term "primer" of the present invention refers to an oligonucleotide, and conditions in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of nucleotides and a polymerization agent such as a DNA polymerase, and a suitable temperature and temperature. It can serve as a starting point for synthesis under the conditions of pH. Preferably, the primer is a deoxyribonucleotide and is single-stranded. The primer used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides. In addition, the primer may also include ribonucleotides.

"Probe" means a linear oligomer having a linkage or a natural or modified monomer comprising deoxyribonucleotides and ribonucleotides capable of hybridizing to a specific nucleotide sequence. Preferably, the probe is single-stranded for maximum efficiency in hybridization. The probe is preferably a deoxyribonucleotide.

As the probe used in the present invention, a sequence that is completely complementary to the sequence including the SNP may be used, but a sequence that is substantially complementary within a range that does not interfere with specific hybridization may be used. may be Preferably, the probe used in the present invention comprises a sequence capable of hybridizing to a sequence comprising 10-30 consecutive nucleotide residues comprising the SNP of the present invention. More preferably, the 3' end or 5' end of the probe has a base complementary to the SNP base. In general, since the stability of the duplex formed by hybridization tends to be determined by the matching of the sequences at the ends, in probes having a base complementary to the SNP base at the 3' or 5' end, the terminal portion is If not hybridized, these duplexes can dissociate under stringent conditions.

In particular, the primer set of SEQ ID NO: 2 and SEQ ID NO: 3 or the probe set of SEQ ID NO: 4 and SEQ ID NO: 5 specifically detects 1127 single nucleotide mutations of TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4) gene it can be done

For the primers, two pairs of primers including a forward primer and a reverse primer were prepared, respectively, and VIC or FAM was attached as a reporter to the 5' end of the fluorescent probe, and NFQ-MGB was attached as a quencher to the 3' direction.

More preferably, in the quantitative measurement by real-time PCR analysis, in the case of G1127T, the primer set consisting of the nucleotide sequences of SEQ ID NOs: 2 and 3 and the nucleotide sequences of SEQ ID NOs: 4 and 5 in order to quantitatively calculate the relative amplification amount It is preferable to use a gene expression analysis set consisting of probes labeled with VIC or FAM at the 5' end. And it is preferable that a quencher is bound to the 3' end of all probes for quenching, specifically, all of TaqMan NFQ-MGB are attached.

Even more preferably, the 5' end of SEQ ID NO: 4 is labeled with a fluorescent substance FAM, and the 5' end of SEQ ID NO: 5 is labeled with a fluorescent substance VIC, and the probe set of SEQ ID NO: 4 and SEQ ID NO: 5 A non-fluorescent quencher (NFQ)-minor groove binder (MGB) is bonded to the 3' end, respectively. The amplification of the present invention is carried out according to PCR (polymerase chain reaction), and according to a preferred embodiment of the present invention, the primers of the present invention are used in gene amplification reactions. More preferably, the amplification and detection of the present invention is more preferably detected by realtime PCR analysis.

The present invention is based on TaqMan SNP Genotyping technology using real-time polymerase chain reaction (Realtime PCR), and invents a diagnostic technology that can accurately diagnose osteochondrosis by efficiently analyzing the SNP of the TRPV4 gene, which determines osteochondrosis in cats. is doing TaqMan SNP genotyping is a method that applies the principle of 5'nuclease assays. When the probe and the corresponding SNP match, the fluorescent probe becomes active. When the probe mismatches with the corresponding SNP, the probe falls off. As a method using the principle that the above reaction does not proceed, two alleles can be analyzed simultaneously with only one PCR, so expensive equipment is not required compared to the existing DNA sequencing method, and time and cost can be saved.

In addition, the present invention comprises the steps of 1) collecting a DNA sample from a cat;

2) performing PCR using the primer set of SEQ ID NO: 2 and SEQ ID NO: 3, or the probe set of SEQ ID NO: 4 and SEQ ID NO: 5 using the DNA collected in step 1) as a template;

3) examining the fluorescence value of the product amplified by PCR in step 2);

It provides a method for predicting or diagnosing osteochondrosis in cats, including.

Preferably, a gene expression analysis set consisting of a primer set consisting of the nucleotide sequences of SEQ ID NOs: 2 and 3 and a probe labeled with VIC or FAM at the 5' end of the nucleotide sequences of SEQ ID NOs: 4 and 5 is used. And it is preferable that a quencher is bound to the 3' end of all probes for quenching, specifically, all of TaqMan NFQ-MGB are attached. In addition, the PCR is performed according to a realtime PCR analysis method using a Taqman probe.

In addition, the present invention provides a kit for predicting or diagnosing osteochondrosis in cats comprising a single nucleotide polymorphism (SNP) marker composition of the TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4) gene.

Preferably, the kit is a cat comprising a composition for detecting osteochondrosis in cats comprising a primer set represented by SEQ ID NO: 2 and SEQ ID NO: 3, or a probe set labeled with a fluorescent material of SEQ ID NO: 4 and SEQ ID NO: 5 Provided is a kit for detecting osteochondrosis.

The kit of the present invention optionally includes reagents necessary for PCR amplification, such as a buffer, a DNA polymerase (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)) thermostable DNA polymerase obtained from ), DNA polymerase cofactors and dNTPs. The kit of the present invention may be manufactured in a number of separate packaging or compartments containing the reagent components described above. After all, the present invention can rapidly and accurately amplify only the desired DNA amplified from the above-mentioned DNA fragment by a real-time PCR reaction among DNA collected from cats using the primer and probe set, and quantitatively measure the amount of amplification. have.

Hereinafter, the action and effect of the invention will be described in more detail through specific examples of the invention. However, these embodiments are merely presented as an example of the invention, and the scope of the invention is not defined thereby.

1. Sample Collection and DNA Extraction

(1) sample collection

A total of 216 oral epithelial cells, including Scottish, American Curl, and Munchkin, which are breeds with common cartilage or bone growth abnormalities, were obtained with cooperation from Haemaru Animal Hospital. to show the breed. In addition, for the reproducibility of the results, about 0.5 to 1 mL of blood was collected from the cat's jugular or radial cortical vein. Among the collected samples, in the Case group, Scottish and Munchkin varieties accounted for 81.3% and 18.7%, respectively, and in the Control group, Persian 44.6%, American Short Hair 27%, Munchkin 16.1%, and Scottish 12.5% accounted for the proportions. Overall, 75 Persian heads (34.7%) were the most common, followed by Scottish 60 heads (27.8%) and Munchkin 36 heads (16.7%).

Breeds Case Control Total N % N % N % Scottish 39 81.3 21 12.5 60 27.8 Munchkin 9 18.7 27 16.1 36 16.7 American Curl 0 0 3 1.8 3 1.4 American Short Hair 0 0 27 16.1 27 12.4 British Short Hair 0 0 15 8.9 15 7.0 Persian 0 0 75 44.6 75 34.7 48 100 168 100 216 100

(2) DNA extraction

For DNA isolation of samples collected from a total of 216 cat blood and oral epithelial cells, DNA was extracted using a Nucleogen kit (Nucleogen, KOR) according to the manufacturer's manual. The extracted DNA was measured for DNA concentration and purity using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA), and stored frozen at -20°C until analysis. The average concentration of DNA extracted from the sample was 129.78 ng/μl, and the values of 260/280 and 260/230 were 1.74 and 1.82, respectively, which were confirmed to be relatively good.

2. Polymorphism Analysis of Genes Associated with Feline Osteochondrosis

(1) Candidate gene regions related to osteochondrosis

Candidate genes related to osteochondrosis were selected as shown in Table 2 below.

No. Gene Name Coding exon Protein function No. Gene Name Coding
exon Protein functions One COL1A1 51 Type 1 collagen 17 PLOD2 19 ER procollagen processing 2 COL1A2 52 Type 1 collagen 18 TCIRG1 19 Osteoclast function 3 BMP1 20 C-propeptide cleavage 19 CLCN7 24 Osteoclast function 4 CRTAP 7 Collagen hydroxylation 20 OSTM1 6 Osteoclast homeostasis 5 FKBP10 9 Collagen processing 21 PLEKHM3 7 Osteoclast function 6 IFITM5 2 Mineralization 22 CA2 7 Osteoclast function 7 P3H1 15 Collagen hydroxylation 23 SNX10 7 Osteoclast homeostasis 8 PLS3 18 Actin binding 24 TRPV4 15 Ion channel transport 9 PPIB 5 Collagen hydroxy 25 TNFRSF11A 9 Osteoclast activation (RANK) 10 SEC24D 22 ER procollagen processing 26 TNFSF11 4 Osteoclast activation (RANKL) 11 SERPINF1 7 Collagen chaperoning 27 CTSK 7 Osteoclast function 12 SERPINH1 4 Mineralization 28 SOST 5 Osteoblast activation/Wnt signaling (antagonist 13 SP7 One Osteoblast regulation 29 LRP5 22 Osteoblast activation/Wnt signaling (receptor) 14 TMEM38B 6 Cation channel 30 NOTCH2 35 Notch signaling 15 WNT1 4 Osteoblast activation/Wnt signaling (ligand) 31 WNT3A 4 Wnt signaling (ligand) 16 P4HB 12 ER procollagen processing 32 MTHFR 12 Homocysteine metabolism

(2) Primer design for exon region amplification of genes related to osteochondrosis

In order to search for SNPs related to osteochondrosis in cats, the Primer3 program (http://primer3.ut. ee/), and the Primer was synthesized at the request of Bionics and MBiotech. Table 3 shows the primer nucleotide sequence of the TRPV4 gene and the size of the amplification product, and the target candidate gene was designed and synthesized in the same way.

Gene Primer no. Exon No. Primer F Primer R Size TRPV4 One Exon01 ATTACAACGGTGGCTTTGAA AGACGGGTGAACAGACAAGT 660 2 Exon02 GGTCTGGTTCCACGCTAGG GGGAAGACGCTAAGAGGAAA 391 3 Exon03 GATAGGGAGAGGCTCAGAGG TGGAAGGGGTAGTTATCGTTC 455 4 Exon04 TGGGTGGAGACGGTCAAG GAGGCTCAGGGAGGTGAG 390 5 Exon05 GACGTGGACACGGACTGT AGCAAGTGGGTGTGCATTT 564 6 Exon06 GCCCAGGACATGCTAAGTAA AGCTGGGACTCAGAGAGGTT 490 7 Exon07 ATCCTCGAGTGTCGCTCA TGTTCCTCAGCTCTCTCCTG 386 8 Exon08 AGGGACAGGAGAGAGCTGAG GGAGGAAAAAGCAGAGAAGG 403 9 Exon09 CCCTCCTTCTCTGCTTTTTC GTCCTGATGTTGGGCTACAG 353 10 Exon10 CCAGTGCATCCACTTTGTTT ACCTCCACCCTATCCCTTC 445 11 Exon11 GATCTGGGATGGAAAAGACC ACTCAGCACACGCTGGTT 250 12 Exon12 CTTTCCCCGCTGTCAAAT GGTCTCTCCGTTCTGGAAG 483 13 Exon13 ACCTTGAGGGCAGTAGGC ATTGCAATGGGTAGAAAGCA 272 14 Exon14 AAAGGGATCAAAAGGCAAAC AGAGGACAGCTCAGAGCAAA 380 15 Exon15 ATAAGCATACCTGCCCCTTC GGGTGGAGTAGAAACAGGTG 349

(3) PCR amplification of candidate genes

Using the sample prepared above, the gene exon region shown in Table 3 was amplified by PCR. The PCR reaction is pre-denatured at 95 °C for 15 minutes, then denatured at 95 °C for 30 seconds, annealed at 58 °C for 30 seconds, and at 72 °C for 30 seconds to 1 minute (if the product size is more than 1 kb, extension for 1 minute) After a total of 35 consecutive reactions, the final extension was performed at 72 ° C. for 10 minutes, and the PCR amplification product was electrophoresed on 2% agarose gel. As a result of confirming the PCR amplification product, a total of 32 genes and 296 kinds of primer sets Each PCR product amplified is shown in Table 4, and the same was performed for candidate genes of other targets.

TRPV4

Lane 1~15 : exon 1~15

Lane 1~15 : exon 1~15

(4) SNP search through DNA sequencing of candidate genes

After purifying the exon region amplification product of the osteochondrosis-related gene amplified for each cat breed using the LaboPass PCR purification system (Cosmogenetech, Korea), the nucleotide sequence was analyzed using BigDye v3.1 (Applied Biosystens, USA). , nucleotide sequences for other target candidate genes were also performed in the same manner.

The reaction solution was purified with ZR DNA Sequencing Clean-up Kit (Zymo Research, USA), and then POP-4 polymer (Applied Biosystems, USA), 36 cm X 16 channel capillary (Applied Biosystems, USA), 3130xl Genetic Analyzer (Applied) Biosystems, USA) was used for electrophoresis. Sequencing analysis was performed using Sequencing Analysis v5.2 and SeqMan Version 7.0.0.0 (DNASTAR Inc, USA) software. Table 5 below shows the nucleotide sequence results and the presence or absence of mutations of exon of the TRPV4 gene, and the same was performed for other target candidate genes.

Exon No. Exon region sequence (5'→ 3') Mutation confirmation in Exon One ATGGCGGATCCCAGCGGAGGCCCCYACRCAGGGCCYGGGGAGGTGGCTGAGACCCCCGGGGACGAGAGCGGGACCCCCGGGGCTGAGGCCTTCCCCCTCTCTTCTCTGGCCAACCTGTTTGAAGGGGAGGATGGCTCCCCCTCCCCCTCCCCGGCTGACACCAGCCGCYCCGCTGGGCCAGGTGATGGGCGACCAAATCTGCGCATGAAGTTCCAGGGTGCCTTCCGCAAGGGGGTGCCCAACCCCATTGACCTGCTGGAGTCCACCCTGTATGAGTCCTCGGTGGTGCCTGGACCCAAGAAGGCACCCATGGACTCGCTCTTTGACTATGGCACCTATCGTCATCACCCCAGCGACAACAGGCGGTGGAGGAAGAAGGTCATCGA TAC→CAC (Tyrosine→Histidine / Missense)
GCA→ACA (Alanine→Threonine / Missense)
GGA→GGG (Glycine / Synonymous)
CCT→CCC (Proline / Synonymous)
GAT→GAC (Aspartic acid / Synonymous)
CCC→TCC (Proline → Serine / Missense) 2
GAAGCAGCCGCAGAGCCCCAAAGCTCCTGCACCCCAGCCGCCCCCCATCCTTAAAGTCTTCAACCGGCCTATCCTCTTTGACATCGTGTCCCGGGGCTCTACTACTGACCTGGATGGGCTGCTTCCCTTCTTGCTGACCCACAAGAAGCGCCTGACTGACGAGGAGTTCCGGG
- 3
AGCCGTCCACAGGGAAGACCTGTCTGCCCAAGGCCCTGCTGAATCTGAGCAGTGGCCGTAATGACACCATCCCTGTGCTCCTGGACATCGCCGAGCGGACAGGCAACATGCGGGAGTTCATCAACTCGCCTTTCCGGGACATCTACTACCGAG
- 4
GCCAGACTGCCCTGCACATCGCCATCGAGCGCCGCTGCAAACACTACGTGGAGCTCCTGGTGGCCCAAGGAGCTGACGTCCATGCCCAGGCCCGGGGGCGCTTCTTCCAGCCCAAGGACGAGGGAGGCTATTTCTACTTTG
- 5
GCGAGCTGCCCCTGTCGCTGGCCGCCTGCACCAACCAGCCCCACATCGTCAACTACCTGACAGAGAACCCGCACAAGAAGGCGGACATGCGGCGGCAGGACTCCCGCGGMAACACGGTGCTGCACGCGCTGGTGGCCATCGCCGACAACACCCGTGAGGGAACTTACCAAGTTCKTCACCAAGAGATGCTCCAGTCAGGCCAACCGTGCAGTCAGTCAGCAGGCTGGCAGCTGGCTGCAGTCCAGCTGGA
GGA→GGC (Glycine / Synonymous)
GTC→TTC (Valine→Phenylalanine/Missense)
AAT→AAC (Asparagine / Synonymous) 6
GTCTTTCAGCACATCATTCGCCGGGAGGTGACAGACGAGGATACGAGGCACCTGTCCCGCAAGTTCAAGGACTGGGCCTACGGGCCGGTGTATTCCTCGCTGTATGACCTCTCCTCCCTGGACACGTGGGGAAGAGGCCTCTGTGCTGGAGATCCTGGTATAACAGCAAGATCGAG
- 7
AACCGCCACGAGATGCTGGCCGTGGAGCCCATCAATGAACTGCTGAGGGACAAGTGGCGCAAGTTCGGGGCTGTCTCCTTCTACATCAACGTGGTCTCCTATCTGTGCGCCATGGTCATCTTCACCCTCACCGCCTACTACCAGCCACTGGAGGGCACT
- 8
CCTCCGTACCCTTACCGCACCACCGTGGACTACCTGAGGCTGGCGGGCGAGATCATCACGCTCTTGACCGGCATCCTGTTCTTCTTTACCAAC - 9
ATCAAAGACTTGTTCATGAAGAAATGCCCGGGAGTGAATTCTCTCTTCATCGATGGCTCCTTCCAGCTACTCTA - 10
CTTCATCTACTCTGTGCTAGTGATTGTCTCAGCGGCCCTCTACCTGGCRGGAATTGAAGCCTACCTGGCTGTCATGGTCTTTGCCTTGGTCCTGGGCTGGATGAACGCCCTTTACTTTACCCGCGGGCTGAAGCTGACGGGAACCTATAGCATCATGATCCAGAAG
GCG→GCA (Alanine / Synonymous) 11
ATCCTCTTCAAAGACCTTTTCCGCTTCCTGCTGGTCTACTTGCTCTTCATGATTGGCTTATGCTTCAG - 12
CCCTGGTGTCCCTCCTGAACCCGTGCGCCAACATGA
AGGTGTGCGGCGAGGACCACACCAACTGCACGGTGCCCACGTACCCGTCCTGCCGCGACAGCGAGACCTTCAGCACCTTCCTCCTGGACCTCTTCAAGCTGACCATCGGCATGRGTGACCTGGAGATGCTGAGCAGCACCAAGTACCCCGTGGTCTTCATCATCCTGCTCGTCACTTACATCATCATCCGGCTGACTGACTGACATGGAGCTGGCAGCATGCATGAGCA - 13
TGGGCCACCACCATTCTGGACATCGAGCGTTCCTTCCCCGTGTTCCTGAGGAAGGCCTTCCGCTCTGGCGAGATGGTGACCGTGGGCAAGAGCTCAGACGGCACCCCAGACCGCAGGTGGTGCTTCAG - 14
GGTGGACGAGGTGAACTGGTCTCACTGGAACCAGAACTTGGGCATCATCAATGAGGACCCGGGCAAGAGCGAGAACTACCAGTACTATGGTTTCTCGCACACCGTGGGCCGGCTCCGGAGGG AGT→AAT (Serine→Asparagine / Missense) 15
ATCGGTGGTCCTCGGTGGTGCCGCGCGTGGTGGAGCTGAACAAGAACTCAAACCCAGACGACGTGGTGGTGCCTCTGGACAACATGGGGACCCCCAGCTGCGACGGCCACCAGCAGAGTTACCCCCCCAAGTGGAGGACAGATGCCGCTCCCCTCTAG
-

(5) TRPV4 Allele Frequency and Association Analysis of Gene SNPs

Among the total 32 candidate genes, 47 SNP mutations were identified in 10 genes, 31 synonymous cSNPs without amino acid change and 16 missense cSNPs inducing amino acid substitution were identified, respectively. Table 6 below shows the SNPs identified in the exon region of the osteochondrosis candidate gene.

Gene Exon No. Nucleotide change amino acid change Mutation type COL1A1 17 T/C Arginine synonymous 18 C/T Asparagine synonymous 19 T/A Alanine synonymous 19 C/T Glycine synonymous 25 C/T Glycine synonymous COL1A2 14 G/T Arginine synonymous 19 T/C Glycine synonymous 34 A/C Alanine synonymous 49 T/C Glycine synonymous FKBP10 06 T/C Serine → Proline missense P3H1 01 A/G Threonine synonymous 06 C/T Phenylalanine synonymous 08 A/G Glutamic acid synonymous 15 T/C Asparagine synonymous 15 G/C Alanine → Proline missense 15 G/A Valine → Isoleucine missense 15 G/A Arginine → Histidine missense P4HB 07 G/A Arginine → Histidine missense TRPV4 01 T/C Tyrosine → Histidine missense 01 G/A Alanine → Threonine missense 01 A/G Glycine synonymous 01 T/C Proline synonymous 01 T/C Aspartic acid synonymous 01 C/T Proline → Serine missense 05 A/C Glycine synonymous 05 G/T Valine → Phenylalanine missense 05 T/C Asparagine synonymous 10 G/A Alanine synonymous 12 G/A Glycine → Serine missense 14 G/A Serine → Asparagine missense TNFRSF11A 06 G/C Leucine synonymous 08 G/A Glutamic acid → Lysine missense 08 G/A Alanine → Threonine missense 08 C/T Threonine → Methionine missense 08 T/C Cysteine synonymous 08 T/C Methionine → Threonine missense 08 A/G Arginine synonymous LRP5 07 T/C Proline synonymous 08 A/G Alanine synonymous 08 G/T Threonine synonymous 08 G/A Alanine → Threonine missense 18 T/C Cysteine synonymous 18 T/C Cysteine synonymous NOTCH2 07 C/T Alanine synonymous 33 T/C Aspartic acid synonymous MTHFR 08 G/A Glutamic acid synonymous 09 C/T Aspartic acid synonymous

To investigate whether there is a correlation with osteochondrosis in cats, logistic regression analysis was performed by comparing the difference in allele frequency between the disease group and the control group and dividing them into dominant, recessive and additive, and the relative risk (OR: odd ratio) and 95% confidence interval and significance level (p>0.05) were calculated, respectively.

As a result of logistic regression analysis on the SNPs identified for a total of 32 candidate genes, no statistical significance was confirmed for the rest of the genes except TRPV4 (Transient Receptor Potential Vanilloid Family Member 4). TRPV4 is known as a gene involved in bone formation by causing calcium signal or depolarization in the cell membrane of the body.

Among the 6 cSNPs identified in the TRPV4 gene, all 4 cSNPs except c.169 C>T and c.2041 G>A were found to have high statistical significance (P<0.0001), but especially the c.1127 G>T SNP. In the T allele of the disease group, the genotype frequency was significantly (p<0.0001) higher than that of the control group. In addition, all 168 disease groups had GG type, whereas the control group did not observe any GG type, suggesting cat osteochondrosis. It was confirmed that it can be used for kit development by determining it as an early diagnosis DNA marker for 1 shows six cSNPs identified in TRPV4 candidate genes.

1. Preparation of primers and probes for real-time polymerase chain reaction of TRPV4 gene

TRPV4 as a gene for SNP detection derived through Example 1 as described above To verify the gene, the same sampling and DNA isolation and purification processes were repeated, and primers and probes were prepared for real-time polymerase chain reaction. Based on the genome sequence of the TRPV4 gene registered in the NCBI described in FIG. 2, a PCR primer set represented by SEQ ID NO: 2 and SEQ ID NO: 3 was designed so that the G1127T region was included and amplified. SEQ ID NOs: 4 and 5 are probe sets capable of detecting the GG type of No. 1127, SEQ ID NO: 4 is FAM labeled with a fluorescent material, SEQ ID NO: 5 is a probe set that detects the TT type of No. 1127, and VIC is labeled with a fluorescent material did. In addition, when 1127 is heterozygous such as GT type, it was designed to detect both FAM and VIC fluorescence values, and NFQ (Non Fluorescent quencher)-MGB (Minor Groove Binder) was quenched at the 3' end of all probes. . Table 7 shows the sequences of primers and probes.

SNPs Kinds SEQ ID NO: Primer/probe sequence (5'→3') G1127T TRPV4-F primer SEQ ID NO: 2 CGACAACACCCGTGAGAACA TRPV4-R primer SEQ ID NO: 3 GAGGCGGGCACACTTGAG TRPV4-G probe SEQ ID NO: 4 AAGTTCGTCACCAAGAT TRPV4-T probe SEQ ID NO: 5 CAAGTTCTTCACCAAGAT

2. Realtime PCR using primer and probe sets

For real-time polymerase chain reaction, 2 μl of template DNA (10 ng/μl), 3 μl of primer and probe set mixture, and 10 μl of TOPreal qPCR 2X PreMIX (TaqMan Probe, UDG plus / Enzynomics, Korea) were added, and then sterile distilled water was added. Thus, a reaction solution was prepared so that the final volume was 20 μl. The PCR reaction was performed at 60 °C for 30 seconds using a real-time gene amplification device QuantStudio 3 (ABI, USA) to remove contamination of the PCR product, and after performing a pre-read step of fluorescence values at 50 °C for 4 minutes, real-time PCR amplification was performed. For the amplification reaction, pre-denaturation was performed at 95°C for 15 minutes, denaturation was performed at 95°C for 15 seconds, and 40 cycles of annealing and extension were performed at 65°C for 1 minute, and finally The post-read step was performed at 60 °C for 30 seconds. As a template, genomic DNA extracted from oral epithelial cells, blood, and hair roots was used as described above, and cross-contamination was confirmed using distilled water as a negative control. Allelic discrimination plot and PCR amplication plot were confirmed using the real-time polymerase chain reactor Quantstudio 3 software program (QuantStudio™ Design & Analysis Software V.1.4.1).

3. Allelic analysis

The nucleotide mutations of G1127T, which determine osteochondrosis in cats, were analyzed using the real-time polymerase chain reactor QuantStudio 3 (Appplied Biosystems, USA) software program QuantStudio ^{™ Design & Analysis Software V.1.4.1 (Appplied Biosystems, USA), respectively.} did. Three types of genotypes were identified on the allelic discrimination plot of base 1127. Figure 3 is a specific amplification of the allele of G1127T for determining cat osteochondrosis using primers and probe sets corresponding to SEQ ID NO: 2 to SEQ ID NO: 5, (A) is a graph of allelic discrimination (Allelic); discrimination plot), and (B) is a graph showing amplification curves of each GG, GT, TT genotype and Negative Control labeled with a fluorescent substance. The TT genotype was shown in blue in the upper left corner, the GT genotype was shown in green in the center of the graph, and the TT genotype was shown in red in the lower right corner (Fig. 3). 4 shows the results of analysis through DNA sequencing in order to verify the analysis results using the real-time polymerase chain reaction method. As a result of amplifying the gene of each sample and analyzing it through DNA sequencing to verify whether it was amplified accurately and specifically, it can be confirmed that the nucleotide sequence of each corresponding allele was accurately amplified. It was confirmed that all base mutations of G1127T were exactly the same.

<110> (Korea Gene Information Institute Co., Ltd.) <120> A SNP marker composition and a method for diagnosis or prediction of osteochondrodyplasia in cats <130> INP19-143 <160> 50 <170> KoPatentIn 3.0 <210> 1 <211> 3290 <212> RNA <213> Felis catus <400> 1 aggagagtcg cgcggcgcgg gcgggcgggc gccggacggc cgggattcag gaagcgcctc 60 gcggtcccgg aggccgagca gcgcagacgg gtcccgggtc agcatggcgg atcccagcgg 120 aggccccccac gcaggacccg gggaggtggc tgagaccccc ggggatgaga gtgggacccc 180 cggggctgag gccttccccc tctcttctct ggccaacctg tttgaagggg aggatggctc 240 cccctccccc tccccggctg acaccagccg ccccgctggg ccaggtgatg ggcgaccaaa 300 tctgcgcatg aagttccagg gtgccttccg caagggggtg cccaacccca ttgacctgct 360 ggagtccacc ctgtatgagt cctcggtggt gcctggaccc aagaaggcac ccatggactc 420 gctctttgac tatggcacct atcgtcatca ccccagcgac aacaggcggt ggaggaagaa 480 ggtcatcgag aagcagccgc agagccccaa agctcctgca ccccagccgc cccccatcct 540 taaagtcttc aaccggccta tcctctttga catcgtgtcc cggggctcta ctactgacct 600 ggatgggctg cttcccttct tgctgaccca caagaagcgc ctgactgacg aggagttccg 660 ggagccgtcc acagggaaga cctgtctgcc caaggccctg ctgaatctga gcagtggccg 720 taatgacacc atccctgtgc tcctggacat cgccgagcgg acaggcaaca tgcgggagtt 780 catcaactcg cctttccggg acatctacta ccgaggccag actgccctgc acatcgccat 840 cgagcgccgc tgcaaacact acgtggagct cctggtggcc caaggagctg acgtccatgc 900 ccaggcccgg gggcgcttct tccagcccaa ggacgaggga ggctatttct actttggcga 960 gctgcccctg tcgctggccg cctgcaccaa ccagccccac atcgtcaact acctgacaga 1020 gaacccgcac aagaaggcgg acatgcggcg gcaggactcc cgcggaaaca cggtgctgca 1080 cgcgctggtg gccatcgccg acaacacccg tgagaacacc aagttcgtca ccaagatgta 1140 cgacctgctg ctgctcaagt gtgcccgcct cttccccgac agcaacctgg aggccgtgct 1200 caacaacgat gggctctcgc ccctcatgat ggccgccaag accggcaaga ttggggtctt 1260 tcagcacatc attcgccggg aggtgacaga cgaggatacg aggcacctgt cccgcaagtt 1320 caaggactgg gcctacgggc cggtgtattc ctcgctgtat gacctctcct ccctggacac 1380 gtgtggggaa gaggcctctg tgctggagat cctggtgtat aacagcaaga tcgagaaccg 1440 ccacgagatg ctggccgtgg agcccatcaa tgaactgctg agggacaagt ggcgcaagtt 1500 cggggctgtc tccttctaca tcaacgtggt ctcctatctg tgcgccatgg tcatcttcac 1560 cctcaccgcc tactaccagc cactggaggg cactcctccg tacccttacc gcaccaccgt 1620 ggactacctg aggctggcgg gcgagatcat cacgctcttg accggcatcc tgttcttctt 1680 taccaacatc aaagacttgt tcatgaagaa atgcccggga gtgaattctc tcttcatcga 1740 tggctccttc cagctactct acttcatcta ctctgtgcta gtgattgtct cagcggccct 1800 ctacctggca ggaattgaag cctacctggc tgtcatggtc tttgccttgg tcctgggctg 1860 gatgaacgcc ctttacttta cccgcgggct gaagctgacg ggaacctata gcatcatgat 1920 ccagaagatc ctcttcaaag accttttccg cttcctgctg gtctacttgc tcttcatgat 1980 tggctatgct tcagccctgg tgtccctcct gaacccgtgc gccaacatga aggtgtgcgg 2040 cgaggaccac accaactgca cggtgcccac gtacccgtcc tgccgcgaca gcgagacctt 2100 cagcaccttc ctcctggacc tcttcaagct gaccatcggc atgggtgacc tggagatgct 2160 gagcagcacc aagtaccccg tggtcttcat catcctgctc gtcacttaca tcatcctcac 2220 cttcgtgcta cttctcaaca tgctcatcgc cctcatgggg gagacggtgg gccaggtgtc 2280 caaggagagc aagcacatct ggaagctgca gtgggccacc accattctgg acatcgagcg 2340 ttccttcccc gtgttcctga ggaaggcctt ccgctctggc gagatggtga ccgtgggcaa 2400 gagctcagac ggcaccccag accgcaggtg gtgcttcagg gtggacgagg tgaactggtc 2460 tcactggaac cagaacttgg gcatcatcag tgaggacccg ggcaagagcg agaactacca 2520 gtactatggt ttctcgcaca ccgtgggccg gctccggagg gatcggtggt cctcggtggt 2580 gccgcgcgtg gtggagctga acaagaactc aaacccagac gacgtggtgg tgcctctgga 2640 caacatgggg acccccagct gcgacggcca ccagcagagt taccccccca agtggaggac 2700 agatgccgct cccctctagg ggctgtgggc ggggcagggt ctctggcctg ccgtccagcc 2760 ccgtcccccc acccacctgt ttctactcca cccgcatttc agtggtgcct cccagggcat 2820 cccccacgtg ctcccttgac ccccagaggc gagggccggg cggagatgaa ggggaggctc 2880 caggaccctc tggtccccag gctgcccccc cagctctgcc tccccacctg ggctcctggc 2940 tgcctgtctt actcccatgg agtcacataa gccaatgcca gggcctctct gtcccggggg 3000 gccccgacac ctgcctctcc attatttatt tgctctgcac tcaggaaagt ggtgtgatcc 3060 ctgcccccaa atggaacctg ggccgaggcc tcaggacctc attccaggtc actgccggcc 3120 agcccccagc cccagccggc gccctgggct gcgcacagca ccctgcgcag cagcggggtg 3180 gctttgctgt ggggccgggg ccctagggcg tggggccgcg cctgcggtgt gttctgtaaa 3240 gggtctggga tttgtcggtg ctcaataaat gcgcattcac tgatggtgaa 3290 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4-F primer <400> 2 cgacaacacc cgtgagaaca 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4-R primer <400> 3 gaggcgggca cacttgag 18 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> TRPV4-G probe <400> 4 aagttcgtca ccaagat 17 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4-T probe <400> 5 caagttcttc accaagat 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 6 attacaacgg tggctttgaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 7 agacgggtga acagacaagt 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 8 ggtctggttc cacgctagg 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 9 gggaagacgc taagaggaaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 10 gatagggaga ggctcagagg 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 11 tggaaggggt agttatcgtt c 21 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 12 tgggtggaga cggtcaag 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 13 gaggctcagg gaggtgag 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 14 gacgtggaca cggactgt 18 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 15 agcaagtggg tgtgcatatt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 16 gcccaggaca tgctaagtaa 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 17 agctgggact cagagaggtt 20 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 18 atcctcgagt gtcgctca 18 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 19 tgttcctcag ctctctcctg 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 20 agggacagga gagagctgag 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 21 ggaggaaaaa gcagagaagg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 22 ccctccttct ctgctttttc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 23 gtcctgatgt tgggctacag 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 24 ccagtgcatc cactttgttt 20 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 25 acctccaccc tatcccttc 19 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 26 gatctgggat ggaaaagacc 20 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 27 actcagcaca cgctggtt 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 28 ctttccccgc tgtcaaat 18 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 29 ggtctctccg ttctggaag 19 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 30 accttgaggg cagtaggc 18 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 31 attgcaatgg gtagaaagca 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 32 aaagggatca aaaggcaaac 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 33 agaggacagc tcagagcaaa 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer F <400> 34 ataagcatac ctgccccttc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TRPV4 primer R <400> 35 gggtggagta gaaacaggtg 20 <210> 36 <211> 386 <212> DNA <213> felis catus <400> 36 atggcggatc ccagcggagg ccccyacrca gggccygggg aggtggctga gacccccggg 60 gacgagagcg ggacccccgg ggctgaggcc ttccccctct cttctctggc caacctgttt 120 gaaggggagg atggctcccc ctccccctcc ccggctgaca ccagccgcyc cgctgggcca 180 ggtgatgggc gaccaaatct gcgcatgaag ttccagggtg ccttccgcaa gggggtgccc 240 aaccccattg acctgctgga gtccaccctg tatgagtcct cggtggtgcc tggacccaag 300 aaggcaccca tggactcgct ctttgactat ggcacctatc gtcatcaccc cagcgacaac 360 aggcggtgga ggaagaaggt catcga 386 <210> 37 <211> 173 <212> DNA <213> felis catus <400> 37 gaagcagccg cagagcccca aagctcctgc accccagccg ccccccatcc ttaaagtctt 60 caaccggcct atcctctttg acatcgtgtc ccggggctct actactgacc tggatgggct 120 gcttcccttc ttgctgaccc acaagaagcg cctgactgac gaggagttcc ggg 173 <210> 38 <211> 153 <212> DNA <213> felis catus <400> 38 agccgtccac agggaagacc tgtctgccca aggccctgct gaatctgagc agtggccgta 60 atgacaccat ccctgtgctc ctggacatcg ccgagcggac aggcaacatg cgggagttca 120 tcaactcgcc tttccgggac atctactacc gag 153 <210> 39 <211> 141 <212> DNA <213> felis catus <400> 39 gccagactgc cctgcacatc gccatcgagc gccgctgcaa acactacgtg gagctcctgg 60 tggcccaagg agctgacgtc catgcccagg cccgggggcg cttcttccag cccaaggacg 120 agggaggcta tttctacttt g 141 <210> 40 <211> 299 <212> DNA <213> felis catus <400> 40 gcgagctgcc cctgtcgctg gccgcctgca ccaaccagcc ccacatcgtc aactacctga 60 cagagaaccc gcacaagaag gcggacatgc ggcggcagga ctcccgcggm aacacggtgc 120 tgcacgcgct ggtggccatc gccgacaaca cccgtgagaa caccaagttc ktcaccaaga 180 tgtacgacct gctgctgctc aagtgtgccc gcctcttccc cgacagcaac ctggaggccg 240 tgctcaacaa ygatgggctc tcgcccctca tgatggccgc caagaccggc aagattggg 299 <210> 41 <211> 180 <212> DNA <213> felis catus <400> 41 gtctttcagc acatcattcg ccgggaggtg acagacgagg atacgaggca cctgtcccgc 60 aagttcaagg actgggccta cgggccggtg tattcctcgc tgtatgacct ctcctccctg 120 gacacgtgtg gggaagaggc ctctgtgctg gagatcctgg tgtataacag caagatcgag 180 180 <210> 42 <211> 159 <212> DNA <213> felis catus <400> 42 aaccgccacg agatgctggc cgtggagccc atcaatgaac tgctgaggga caagtggcgc 60 aagttcgggg ctgtctcctt ctacatcaac gtggtctcct atctgtgcgc catggtcatc 120 ttcaccctca ccgcctacta ccagccactg gagggcact 159 <210> 43 <211> 93 <212> DNA <213> felis catus <400> 43 cctccgtacc cttaccgcac caccgtggac tacctgaggc tggcgggcga gatcatcacg 60 ctcttgaccg gcatcctgtt cttctttacc aac 93 <210> 44 <211> 74 <212> DNA <213> felis catus <400> 44 atcaaagact tgttcatgaa gaaatgcccg ggagtgaatt ctctcttcat cgatggctcc 60 ttccagctac tcta 74 <210> 45 <211> 166 <212> DNA <213> felis catus <400> 45 cttcatctac tctgtgctag tgattgtctc agcggccctc tacctggcrg gaattgaagc 60 ctacctggct gtcatggtct ttgccttggt cctgggctgg atgaacgccc tttactttac 120 ccgcgggctg aagctgacgg gaacctatag catcatgatc cagaag 166 <210> 46 <211> 67 <212> DNA <213> felis catus <400> 46 atcctcttca aagacctttt ccgcttcctg ctggtctact tgctcttcat gattggctat 60 gcttcag 67 <210> 47 <211> 317 <212> DNA <213> felis catus <400> 47 ccctggtgtc cctcctgaac ccgtgcgcca acatgaaggt gtgcggcgag gaccacacca 60 actgcacggt gcccacgtac ccgtcctgcc gcgacagcga gaccttcagc accttcctcc 120 tggacctctt caagctgacc atcggcatgr gtgacctgga gatgctgagc agcaccaagt 180 accccgtggt cttcatcatc ctgctcgtca cttacatcat cctcaccttc gtgctacttc 240 tcaacatgct catcgccctc atgggggaga cggtgggcca ggtgtccaag gagagcaagc 300 acatctggaa gctgcag 317 <210> 48 <211> 128 <212> DNA <213> felis catus <400> 48 tgggccacca ccattctgga catcgagcgt tccttccccg tgttcctgag gaaggccttc 60 cgctctggcg agatggtgac cgtgggcaag agctcagacg gcaccccaga ccgcaggtgg 120 tgcttcag 128 <210> 49 <211> 122 <212> DNA <213> felis catus <400> 49 ggtggacgag gtgaactggt ctcactggaa ccagaacttg ggcatcatca atgaggaccc 60 gggcaagagc gagaactacc agtactatgg tttctcgcac accgtgggcc ggctccggag 120 gg 122 <210> 50 <211> 158 <212> DNA <213> felis catus <400> 50 atcggtggtc ctcggtggtg ccgcgcgtgg tggagctgaa caagaactca aacccagacg 60 acgtggtggt gcctctggac aacatgggga cccccagctg cgacggccac cagcagagtt 120 acccccccaa gtggaggaca gatgccgctc ccctctag 158

Claims (12)

delete delete delete SEQ ID NO: 1 TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4 ) The 1127th base of the gene is G or T Single nucleotide polymorphism (single nucleotide polymorphism, SNP) containing a polynucleotide amplifying or detecting A SNP marker composition for predicting or diagnosing osteochondrosis in cats, comprising a primer set of SEQ ID NO: 2 and SEQ ID NO: 3, and a probe set of SEQ ID NO: 4 and SEQ ID NO: 5. delete delete delete 5. The method of claim 4,
The 5' end of SEQ ID NO: 4 and SEQ ID NO: 5 is labeled with a fluorescent substance VIC or FAM, and NFQ (Non Fluorescent quencher)-MGB (Minor Groove Binder) is coupled to the 3' end of SEQ ID NO: 4 and SEQ ID NO: 5 The SNP marker composition for predicting or diagnosing osteochondrosis in cats. 5. The method of claim 4,
The detection is a SNP marker composition for predicting or diagnosing osteochondrodysplasia in cats, which is detected by a realtime PCR analysis method using a Taqman probe. 1) collecting a DNA sample from the cat;
2) Using the DNA collected in step 1) as a template, the 1127th base of the TRPV4 (Transient Receptor Potential cation channel, subfamily V, member 4 ) gene of SEQ ID NO: 1 is G or T single nucleotide polymorphism (single nucleotide) performing PCR using a primer set of SEQ ID NO: 2 and SEQ ID NO: 3 and a probe set of SEQ ID NO: 4 and SEQ ID NO: 5 capable of amplifying or detecting polymorphism (SNP);
3) examining the fluorescence value of the product amplified by PCR in step 2);
A method for predicting or diagnosing osteochondrosis in cats, comprising a. The method of claim 10, wherein the PCR is performed according to a realtime PCR analysis method using a Taqman probe. A kit for predicting or diagnosing osteochondrosis in cats comprising the composition of claim 4 .

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