JP6856928B2 - Method of selecting dogs using genotype and determining behavioral characteristics - Google Patents

Description

The present invention uses genetic polymorphisms, and behavioral characteristics such as obedience are important. Blind dogs, disaster relief dogs, assistance dogs, drug detection dogs, hearing guidance dogs, cancer detection dogs, police dogs, animal therapy dogs. , Animal-mediated education Social activity animals such as dogs (including assisting dogs for the physically handicapped), selection of domestic dogs, and disposition (personality) inspection.

Regarding behavioral characteristics and genotypes, we used GWAS analysis for guide dogs and non-guide dogs to find one gene and its genotype (SNP: single nucleotide polymorphism) SORCS1 from a large number of dog genes, and this is the selection of guide dogs. It was clarified that it is a useful marker related to behavioral traits.

Guide dogs are dogs stipulated in Article 14 of the Road Traffic Act that support safe and comfortable walking for the visually impaired. As of December 1, 2015, 984 dogs are registered as guide dogs nationwide. However, it is said that there are about 4,800 guide dog applicants, and it is estimated that there are about 7,800 people including potential applicants, and the supply of guide dogs to the visually impaired is not sufficient. In addition, the passing rate of training dogs is as low as 24%, and the breeding cost per dog is as high as about 5.3 million yen, so the penetration rate is from 1/2 compared to developed countries in Europe and the United States. It is said that it remains at 1/3.

Another issue is that guide dogs, both male and female, start training after being contraceptive and castrated, so even excellent guide dogs do not have the technology to pass on their genes to the next generation by breeding.

Artificial breeding technology was also developed for the efficient breeding of guide dogs, and in 2008, several puppies were obtained from fertilized eggs that were frozen and stored by Professor Suzuki of Obihiro University of Agriculture and Veterinary Medicine. This requires enormous research costs and time.

In addition, a technique that leads to estimation of guide dog suitability by examining and diagnosing a gene that is said to be related to the character of a dog at the puppy stage is also disclosed (see Patent Documents 1 to 3).

Japanese Patent No. 4839439 Japanese Patent No. 5023327 Japanese Patent No. 5023331

The present invention uses genetic polymorphisms, and behavioral characteristics such as obedience are important. Blind dogs, disaster relief dogs, assistance dogs, drug detection dogs, hearing guidance dogs, cancer detection dogs, police dogs, animal therapy dogs. The purpose is to select social activity animals such as dogs and animal-mediated education dogs (including assisting dogs for the physically handicapped) and domestic dogs, and to provide a method for examining disposition (personality) characteristics (behavioral characteristics).

The present inventors discriminated behavioral characteristics suitable for guide dogs and the like using genotype as an index, and diligently investigated whether dogs suitable for guide dogs and the like could be selected. Regarding behavioral characteristics and genotypes, the present inventors conducted a dog whole genome association analysis (GWAS) on blind and non-blind dogs, and among a large number of dog genes, one gene SORCS1 on chromosome 28 and its gene. We have found that the genotype (SNP: single nucleotide polymorphism) is a useful marker related to the selection and behavioral characteristics of blind dogs, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for determining the behavioral characteristics of a dog, which comprises analyzing a single nucleotide polymorphism of the Sortilin-related receptor CNS expressed (SORCS) 1 gene on chromosome 28 in a test dog.
[2] The method of [1], wherein the single nucleotide polymorphism of the SORCS1 gene on chromosome 28 is at least one single nucleotide polymorphism of the following (i) to (viii):
(i) A single nucleotide polymorphism of C or T at the 18801488th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 1 (NC_006610). .3: g.18801488C> T polymorphism);
(ii) A single nucleotide polymorphism of G or T at the 18801039th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 2 (NC_006610). .3: g.18801539G> T polymorphism);
(iii) A single nucleotide polymorphism of G or A at the 18809171th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 3 (NC_006610). .3: g.18809171G> A polymorphism);
(iv) A single nucleotide polymorphism of C or A at the 18820912th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 4 (NC_006610). .3: g.18820912C> A polymorphism);
(v) Single nucleotide polymorphism of T or A at base 18821030 of the entire base sequence of chromosome 28 of dogs, and single nucleotide polymorphism at base 26 of the base sequence shown by SEQ ID NO: 5 (NC_006610) .3: g.18821030T> A polymorphism);
(vi) A single nucleotide polymorphism of C or T at the .18821044th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 6 ( NC_006610.3: g.18821044C> T polymorphism);
(vii) A single nucleotide polymorphism of A or G at the 18823291th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 7 (NC_006610). .3: g.18823291A> G polymorphism); and
(viii) A single nucleotide polymorphism of T or C at the 18835904th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 8 (NC_006610). .3: g.18835904T> C polymorphism).
[3] The method of [1] or [2], wherein the test dog is determined to have obedience as a behavioral characteristic in any of the following cases (i) to (viii).
(i) A single nucleotide polymorphism of C or T at the 18801488th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 1 (NC_006610). .3: g.18801488C> T polymorphism) When the genotype of the site is C / C or C / T;
(ii) A single nucleotide polymorphism of G or T at the 18801039th base of the entire base sequence of chromosome 28 of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 2 (NC_006610). .3: g.18801539G> T polymorphism) When the genotype of the site is G / G or G / T;
(iii) A single nucleotide polymorphism of G or A at the 18809171th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 3 (NC_006610). .3: g.18809171 G> A polymorphism) When the genotype of the site is A / A or G / A;
(iv) A single nucleotide polymorphism of C or A at the 18820912th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 4 (NC_006610). .3: g.18820912C> A polymorphism) When the genotype of the site is A / A or C / A;
(v) Single nucleotide polymorphism of T or A at base 18821030 of the entire base sequence of chromosome 28 of dogs, and single nucleotide polymorphism at base 26 of the base sequence shown by SEQ ID NO: 5 (NC_006610) .3: g.18821030T> A polymorphism) When the genotype of the site is A / A or T / A;
(vi) A single nucleotide polymorphism in C or T at base 18821044 of the entire base sequence of chromosome 28 of a dog, and a single nucleotide polymorphism at base 26 of the base sequence shown in SEQ ID NO: 6 (NC_006610). .3: g.18821044C> T polymorphism) When the genotype of the site is T / T or C / T;
(vii) A single nucleotide polymorphism of A or G at the 18823291th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 7 (NC_006610). .3: g.18823291A> G polymorphism) When the genotype of the site is G / G or A / G; or
(viii) A single nucleotide polymorphism of T or C at the 18835904th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 8 (NC_006610). .3: g.18835904T> C polymorphism) When the genotype of the site is C / C or T / C.
[4] Any of [1] to [3], in which genomic DNA containing a single nucleotide polymorphism of intron 3 is extracted from a test dog and the base of the single nucleotide polymorphism site of an allele contained in the extracted DNA is identified. That way.
[5] Two types of probes having a pair of PCR primers that bind to the outside of the base sequence containing the single nucleotide polymorphism and a fluorescently labeled allelic gene-specific sequence for real-time PCR machine detection are prepared, and a PCR reaction is performed. The method of [4], which identifies the base of a single nucleotide polymorphism site of an allelic gene by real-time PCR performed by performing.
[6] The test dogs judged to have obedience as behavioral characteristics are blind guide dogs, disaster relief dogs, assistance dogs, drug detection dogs, hearing guide dogs, cancer detection dogs, police dogs, animal therapy dogs, etc. The method according to any one of [1] to [5], which is selected as a dog selected from the group consisting of an animal-mediated educational dog and a domestic dog.
[7] One of the following primer pairs (p1) to (p8) used for amplifying a DNA fragment containing a single nucleotide polymorphism site of the SORCS1 gene on chromosome 28 of a dog, [1] to Primer pair used in any of the methods [6]:
(p1) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801488C> T) in the base sequence of SEQ ID NO: 1;
(p2) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801539G> T) in the base sequence of SEQ ID NO: 2;
(p3) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18809171G> A) in the base sequence of SEQ ID NO: 3;
(p4) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18820912C> A) in the base sequence of SEQ ID NO: 4;
(p5) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821030T> A) in the base sequence of SEQ ID NO: 5;
(p6) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821044C> T) in the base sequence of SEQ ID NO: 6;
(p7) Primer capable of amplifying the region containing the 26th base (base of polymorphism site of NC_006610.3: g.18823291A> G) in the base sequence of SEQ ID NO: 7;
(p8) A primer capable of amplifying a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18835904T> C) in the base sequence of SEQ ID NO: 8.
[8] A primer pair according to any one of the following (a) to (e) used for amplifying a DNA fragment containing a single nucleotide polymorphism site of the SORCS1 gene on chromosome 28 of a dog, [1] to Primer pair used in any of the methods [6]:
(a) Forward: 5'-ATCCCTGCTGTCATACTT-3' (SEQ ID NO: 5'-ATCCCTGCTGTCATACTT-3' (SEQ ID NO:) to detect at least one polymorph of NC_006610.3: g.18801488C> T polymorphism and NC_006610.3: g.18801539G> T polymorphism. 14) and reverse: Primer pair of 5'-GATGAATGATTGCTTAGGGTT-3'(SEQ ID NO: 15);
(b) NC_006610.3: g.18809171G> A for detecting polymorphism: forward: 5'-TCTTTATTATAGGGAGCCTATAT-3'(SEQ ID NO: 16) and reverse: 5'-TGTTGGTACAAGGTTACCTAAA-3' (SEQ ID NO: 17) Primer pair;
(c) To detect at least one polymorph of NC_006610.3: g.18820912C> A polymorphism, NC_006610.3: g.18821030T> A polymorphism and NC_006610.3: g.18821044C> T polymorphism, Forward: 5'-CAAGATTGGGGCACTGAACT-3'(SEQ ID NO: 18) and reverse: 5'-AACGAGGCCTAAAGAGCTGA-3' (SEQ ID NO: 19) primer pair;
(d) NC_006610.3: g.18823291A> G Forward: 5'-TGCATATGGGCATCTCTACCC-3'(SEQ ID NO: 20) and Reverse: 5'-GCCATCACTTACTGAACGTC-3' (SEQ ID NO: 21) Primer pair; or
(e) Primer pair of forward: 5'-TTTAGGGCAGAATGACCCTG-3'(SEQ ID NO: 22) and reverse: 5'-AGGATGCCTTCTTCCTAGTGT-3' (SEQ ID NO: 23) for detecting NC_006610.3: g.18835904T> C ..
[9] A probe according to any one of the following (q1) to (q8) capable of hybridizing to a region containing a single nucleotide polymorphism site of the SORCS gene on chromosome 28 of a dog [1]. Probe used for any of the methods [6]:
(q1) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801488C> T) in the base sequence of SEQ ID NO: 1;
(q2) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801539G> T) in the base sequence of SEQ ID NO: 2;
(q3) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18809171G> A) in the base sequence of SEQ ID NO: 3;
(q4) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18820912C> A) in the base sequence of SEQ ID NO: 4;
(q5) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821030T> A) in the base sequence of SEQ ID NO: 5;
(q6) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821044C> T) in the base sequence of SEQ ID NO: 6;
(q7) A probe capable of hybridizing to a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18823291A> G) in the base sequence of SEQ ID NO: 7;
(q8) A probe capable of hybridizing to a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18835904T> C) in the base sequence of SEQ ID NO: 8.
[10] At least one pair of primer pairs (p1) to (p8) in [7], at least one pair of primer pairs (a) to (e) in [8], or (q1) to (q1) to [9]. A kit for determining behavioral characteristics of dogs, including at least one of the probes (q8).
[11] The test dogs judged to have obedience as behavioral characteristics are blind guide dogs, disaster rescue dogs, assistance dogs, drug detection dogs, hearing guide dogs, cancer detection dogs, police dogs, animal therapy dogs, etc. The kit of [10] selected as a dog selected from the group consisting of animal-mediated educational dogs and domestic dogs.

The SORCS1 gene is a gene that has been reported to be expressed in the central nervous system, and canine dog chromosome 28 (Canis lupus familiaris breed boxer chromosome 28, CanFam3.1, whole genome shotgun sequence: NCBI Reference Sequence: NC_006610. Eight single nucleotide polymorphisms present in 3) and present in Intron 3 are related to the behavioral characteristics of dogs, and by using the single nucleotide polymorphisms as markers, the behavioral characteristics of the test dog Furthermore, it is possible to select a dog suitable for a blind dog or the like.

It is a figure which shows the result of the genome-wide association study (GWAS) of the dog chromosome 28. The area surrounded by the ellipse is the area where a strong correlation is observed. It is a figure which shows the genotype of NC_006610.3: g.18801488C> T polymorphism, the allele frequency, and the pass rate in guide dogs and non-guide dogs. It is a figure which shows the genotype of NC_006610.3: g.18801539G> T polymorphism, the allele frequency, and the pass rate in guide dogs and non-guide dogs. It is a figure which shows the genotype of NC_006610.3: g.18809171G> A polymorphism, the allele frequency, and the pass rate in guide dogs and non-guide dogs. It is a figure which shows the genotype of NC_006610.3: g.18820912C> A polymorphism, the allele frequency, and the pass rate in guide dogs and non-guide dogs. It is a figure which shows the genotype of NC_006610.3: g.18821030T> A polymorphism, the allele frequency, and the pass rate in guide dogs and non-guide dogs. It is a figure which shows the genotype of NC_006610.3: g.18821044C> T polymorphism, the allele frequency, and the pass rate in guide dogs and non-guide dogs. It is a figure which shows the genotype of NC_006610.3: g.18823291A> G polymorphism, the allele frequency, and the pass rate in guide dogs and non-guide dogs. It is a figure which shows the genotype of NC_006610.3: g.18835904T> C polymorphism, the allele frequency, and the pass rate in guide dogs and non-guide dogs. It is a figure which shows the base sequence which contains each polymorphism in the SORCS1 gene intron on the 28th chromosome of a dog. It is a figure which shows 13 known single nucleotide polymorphisms used for guide dog selection. It is a figure which shows the comparison of the pass rate of the guide dog selected by using the single nucleotide polymorphism of this invention, and the known single nucleotide polymorphism (No. 1). It is a figure which shows the comparison of the pass rate of the guide dog selected by using the single nucleotide polymorphism of this invention, and the known single nucleotide polymorphism (No. 2). It is a figure which shows the comparison of the pass rate of the guide dog selected by using the single nucleotide polymorphism of this invention, and the known single nucleotide polymorphism (No. 3).

Hereinafter, the present invention will be described in detail.
The present invention analyzes a single nucleotide polymorphism (SNP) of a dog's Sortilin-related receptor CNS (central nervous system) expressed (SORCS) 1 gene and determines a method for selecting a dog or a dog's temperament (character) characteristics. How to do it.

The SORCS1 gene is a gene that has been reported to be expressed in the central nervous system, and canine chromosome 28 (Canis lupus familiaris breed boxer chromosome 28, CanFam3.1, whole genome shotgun sequence: NCBI Reference Sequence: NC_006610. It exists at positions 18835904 to 18801488 of the entire base sequence of 3).

In the present invention, the selected dogs are blind guide dogs, disaster rescue dogs, assistance dogs, drug detection dogs, hearing guide dogs, cancer detection dogs, police dogs, and animal therapy dogs, for which behavioral characteristics such as obedience are important. , Social activity animals such as animal-mediated education dogs (including assisting dogs for the physically handicapped), and domestic dogs. In the present invention, these dogs may be referred to as guide dogs and the like. Among these dogs, guide dogs are preferable. Behavioral characteristics such as obedience are also called temperament (personality) characteristics.

The determination of behavioral characteristics is a determination of whether or not the test dog has behavioral characteristics such as obedience suitable for the above-mentioned dog.

The present inventors have found that the single nucleotide polymorphism of the SORCS1 gene is associated with the behavioral characteristics of dogs, and completed the present invention, where the single nucleotide polymorphism is associated with the behavioral characteristics of dogs. "Yes" means that the single nucleotide polymorphism allele of the test dog is statistically related to the behavioral characteristics such as obedience of the test dog.

In the present invention, at least one single nucleotide polymorphism of the following eight single nucleotide polymorphisms (i) to (viii) existing on the intron 3 of the SORCS1 gene, preferably two, three, four, Analyze 5, 6, 7 or 8 single nucleotide polymorphisms. The behavioral characteristics of dogs can be determined based on the single nucleotide polymorphism present in the gene, and dogs suitable for guide dogs and the like can be selected. In the following (i) to (viii), the position of the base in the entire base sequence of chromosome 28 of the dog is shown.

(i) NC_006610.3: g.18801488C> T polymorphism A polymorphism of C or T in the 18801488th base of the entire base sequence of chromosome 28 of a dog, and the 26th base sequence shown in SEQ ID NO: 1. Polymorphism in the base of
(ii) NC_006610.3: g.18801539G> T polymorphism This is a G or T polymorphism in the 18801039th base of the entire base sequence of chromosome 28 in dogs, and is the 26th base sequence shown in SEQ ID NO: 2. Polymorphism in the base of
(iii) NC_006610.3: g.18809171G> A polymorphism A polymorphism of G or A in the 18809171th base of the entire base sequence of chromosome 28 of a dog, and the 26th base sequence shown in SEQ ID NO: 3. Polymorphism in the base of
(iv) NC_006610.3: g.18820912C> A polymorphism A polymorphism of C or A in the base 18820912 of the entire base sequence of chromosome 28 of a dog, and the 26th base sequence shown in SEQ ID NO: 4. Polymorphism in the base of
(v) NC_006610.3: g.18821030T> A polymorphism A polymorphism of T or A in the base 18821030 of the entire base sequence of chromosome 28 of a dog, and the 26th base sequence shown in SEQ ID NO: 5. Polymorphism in the base of
(vi) NC_006610.3: g.18821044C> T polymorphism A polymorphism of C or T in the base 18821044 of the entire base sequence of chromosome 28 of a dog, and the 26th base sequence shown in SEQ ID NO: 6. Polymorphism in the base of
(vii) NC_006610.3: g.18823291A> G polymorphism A or G polymorphism in the 18823291th base of the entire base sequence of chromosome 28 in dogs, and the 26th base sequence shown in SEQ ID NO: 7. Polymorphism in the base of
(viii) NC_006610.3: g.18835904T> C polymorphism A polymorphism of T or C in the base 18835904 of the entire base sequence of chromosome 28 of a dog, and the 26th base sequence shown in SEQ ID NO: 8. Polymorphism in the base of.

Canis lupus familiaris breed boxer chromosome 28, CanFam3.1, whole genome shotgun sequence of dogs containing NC_006610.3: g.18801488C> T polymorphism and NC_006610.3: g.18801539G> T polymorphism above ) The nucleotide sequence above is shown in SEQ ID NO: 9 and FIG. 10A, and the nucleotide sequence on chromosome 28 of the dog containing the NC_006610.3: g.18809171G> A polymorphism is shown in SEQ ID NO: 10 and FIG. 10B, NC_006610.3: The nucleotide sequence on chromosome 28 of dogs containing g.18820912C> A polymorphism, NC_006610.3: g.18821030T> A polymorphism and NC_006610.3: g.18821044C> T polymorphism is shown in SEQ ID NO: 11 and FIG. 10C. The nucleotide sequence on chromosome 28 of the dog containing the NC_006610.3: g.18823291A> G polymorphism is shown in SEQ ID NO: 12 and FIG. 10D, and the nucleotide sequence of the dog containing the NC_006610.3: g.18835904T> C polymorphism is shown in FIG. The nucleotide sequence on chromosome 28 is shown in SEQ ID NO: 13 and FIG. 10E.

Analysis of single nucleotide polymorphism refers to determining the type of base of a single nucleotide polymorphism site, that is, an allele, and may detect one chromosome on a pair of chromosomes or both chromosomes. The inclusion and detection of both chromosomes also includes the detection of homozygous or heterozygous at the single nucleotide polymorphism site. Each allele that opposes a particular allele contained in a sample isolated from a subject can be detected and genotyped. When only a certain allele is detected, it is homozygous having the allele homozygous, and when two alleles are detected, it is heterozygous having the two alleles heterozygously.

The above-mentioned single nucleotide polymorphism can be said to be a genetic marker for determining the behavioral characteristics of dogs and selecting dogs such as the above-mentioned guide dogs.

(i) For NC_006610.3: g.18801488C> T polymorphism, the test dog carrying the C allele is more homozygous for C / C than the test dog carrying the T allele. It can be determined that the C / T heterozygous test dog has behavioral characteristics more suitable for blind dogs such as obedience than the T / T homozygous test dog, and further, C / C It can be determined that the homozygous test dogs have behavioral characteristics more suitable for blind dogs such as obedience than the C / T heterozygous test dogs. Can be selected as a candidate for. That is, when the genotype of the single nucleotide polymorphism site at position 18801488 of the entire base sequence of chromosome 28 of the test dog is C / C or C / T, especially when it is C / C, the test dog is used. It can be determined that the dog has behavioral characteristics suitable for a guide dog or the like such as obedience, and such a test dog can be selected as a candidate for a guide dog or the like.

(ii) For NC_006610.3: g.18801539G> T polymorphism, the test dog carrying the G allele is more homozygous for G / G than the test dog carrying the T allele. It can be determined that the G / T heterozygous test dog has behavioral characteristics more suitable for blind dogs such as obedience than the T / T homozygous test dog, and further, G / G It can be determined that the homozygous test dogs have behavioral characteristics more suitable for blind dogs such as obedience than the G / T heterozygous test dogs. Can be selected as a candidate for. That is, when the genotype of the single nucleotide polymorphism site at position 18801539 of the entire base sequence of chromosome 28 of the test dog is G / G or G / T, especially when it is G / G, the test dog is submitted. It can be determined that the dog has behavioral characteristics suitable for guide dogs such as sex, and such test dogs can be selected as candidates for guide dogs and the like.

(iii) For NC_006610.3: g.18809171G> A polymorphism, the test dog carrying the A allele is more homozygous for A / A than the test dog carrying the G allele. It can be determined that the G / A heterozygous test dog has behavioral characteristics more suitable for blind dogs such as obedience than the G / G homozygous test dog, and further, A / A It can be determined that the homozygous test dogs have behavioral characteristics more suitable for blind dogs such as obedience than the G / A heterozygous test dogs. Can be selected as a candidate for. That is, when the genotype of the single nucleotide polymorphism site at position 18809171 of the entire base sequence of chromosome 28 of the test dog is A / A or G / A, especially when it is A / A, the test dog is used. It can be determined that the dog has behavioral characteristics suitable for a guide dog or the like such as obedience, and such a test dog can be selected as a candidate for a guide dog or the like.

(iv) For NC_006610.3: g.18820912C> A polymorphism, the test dog carrying the A allele is more homozygous for A / A than the test dog carrying the C allele. It can be determined that the C / A heterozygous test dog has behavioral characteristics more suitable for blind dogs such as obedience than the C / C homozygous test dog, and further, A / A It can be determined that the homozygous test dogs have behavioral characteristics more suitable for blind dogs such as obedience than the C / A heterozygous test dogs. Can be selected as a candidate for. That is, when the genotype of the single nucleotide polymorphism site at position 18820912 in the entire base sequence of chromosome 28 of the test dog is A / A or C / A, especially when it is A / A, the test dog is used. It can be determined that the dog has behavioral characteristics suitable for a guide dog or the like such as obedience, and such a test dog can be selected as a candidate for a guide dog or the like.

(v) For NC_006610.3: g.18821030T> A polymorphism, the test dog carrying the A allele is more homozygous for A / A than the test dog carrying the T allele. It can be determined that the T / A heterozygous test dog has behavioral characteristics more suitable for blind dogs such as obedience than the T / T homozygous test dog, and further, A / A It can be determined that the homozygous test dogs have behavioral characteristics more suitable for blind dogs such as obedience than the T / A heterozygous test dogs. Can be selected as a candidate for. That is, when the genotype of the single nucleotide polymorphism site at position 18821030 of the entire base sequence of chromosome 28 of the test dog is A / A or T / A, especially when it is A / A, the test dog is used. It can be determined that the dog has behavioral characteristics suitable for a guide dog or the like such as obedience, and such a test dog can be selected as a candidate for a guide dog or the like.

(vi) For NC_006610.3: g.18821044C> T polymorphism, the test dog carrying the T allele is more homozygous for T / T than the test dog carrying the C allele. It can be determined that the C / T heterozygous test dog has behavioral characteristics more suitable for blind dogs such as obedience than the C / C homozygous test dog, and further, the T / T It can be determined that the homozygous test dogs have behavioral characteristics more suitable for blind dogs such as obedience than the C / T heterozygous test dogs. Can be selected as a candidate for. That is, when the genotype of the single nucleotide polymorphism site at position 18821044 of the entire base sequence of chromosome 28 of the test dog is T / T or C / T, especially when it is T / T, the test dog is used. It can be determined that the dog has behavioral characteristics suitable for a guide dog or the like such as obedience, and such a test dog can be selected as a guide dog or the like or as a candidate for a guide dog or the like.

(vii) NC_006610.3: g.18823291A> For G polymorphisms, test dogs carrying the G allele are more homozygous for G / G than test dogs carrying the A allele. It can be determined that the A / G heterozygous test dog has behavioral characteristics more suitable for blind dogs such as obedience than the A / A homozygous test dog, and further, G / G It can be determined that the homozygous test dogs have behavioral characteristics more suitable for blind dogs such as obedience than the A / G heterozygous test dogs. Can be selected as a candidate for. That is, when the genotype of the single nucleotide polymorphism site at position 18823291 of the entire base sequence of chromosome 28 of the test dog is G / G or A / G, especially when it is G / G, the test dog is used. It can be determined that the dog has behavioral characteristics suitable for a guide dog or the like such as obedience, and such a test dog can be selected as a candidate for a guide dog or the like.

(viii) For NC_006610.3: g.18835904T> C polymorphism, test dogs carrying the C allele are more homozygous for C / C than test dogs carrying the T allele. It can be determined that the T / C heterozygous test dog has behavioral characteristics more suitable for blind dogs such as obedience than the T / T homozygous test dog, and further, C / C It can be determined that the homozygous test dogs have behavioral characteristics more suitable for blind dogs such as obedience than the T / C heterozygous test dogs. Can be selected as a candidate for. That is, when the genotype of the single nucleotide polymorphism site at position 18835904 of the entire base sequence of chromosome 28 of the test dog is C / C or T / C, especially when it is C / C, the test dog is used. It can be determined that the dog has behavioral characteristics suitable for a guide dog or the like such as obedience, and such a test dog can be selected as a candidate for a guide dog or the like.

Dogs selected as guide dogs, etc. or candidates for guide dogs, etc. are trained to become guide dogs, etc., and if they finally pass the test, they are used as guide dogs, etc.

In the method of the present invention, a sample may be collected from a test dog and a single nucleotide polymorphism may be analyzed for the DNA or RNA of the sample. That is, genomic DNA containing a single nucleotide polymorphism of intron 3 may be extracted from the test dog, and the base of the single nucleotide polymorphism site of the allele contained in the extracted DNA may be identified.

As the sample used for the analysis, any sample containing chromosomal DNA can be used, and examples thereof include blood, skin, oral mucosa, hair, urine, nails, and cells. Nucleic acids such as chromosomes, DNA, and RNA may be isolated and analyzed from these samples according to a conventional method.

Analysis of single nucleotide polymorphisms can be performed by ordinary gene polymorphism analysis methods. For example, an analysis method by sequence analysis in which direct sequencing is performed by a known method such as the dideoxy method or the Maxam-Gilbert method, or hybridization using a probe specific for a gene polymorphism or a microarray (DNA chip) on which the probe is immobilized. Methods, various methods using primers specific for gene polymorphism, etc. are mentioned, and more specifically, primer extension method (TaqMan® method), PCR-SSCP method, single-strand conformation polymorphism analysis. (SSCP; single strand conformation polymorphism), Invader method, single nucleotide primer method, PCR method, NASBA method, LCR method, SDA method, LAMP method, method using restricted fragment length polymorphism (RFLP), modified gradient gel electrophoresis Examples include the method (DGGE), the method using chemical cleavage of mismatch sites (CCM), the SNaPshot method, the MassArray method, the Pyrosequncing method, the SNP-IT method, the BeadArray method, the Scorpion method, and the MADI-TOF / MS method.

The analysis method by sequence analysis can be performed by a usual method. Specifically, a sequence reaction can be performed using a primer set at a position of several tens of bases on the 5'side of the single nucleotide polymorphism site, and the base of the single nucleotide polymorphism site can be determined from the analysis result. it can.

When a primer is used, a nucleic acid sample isolated from a test dog is amplified by using a primer corresponding to a part of the SORCS1 gene sequence containing a single nucleotide polymorphism site. That is, for example, a nucleic acid sample isolated from a test dog using a primer that is completely or almost completely complementary to one allele and a primer that is completely or almost completely complementary to only the other allele. Alleles with single nucleotide polymorphisms can be identified by whether or not is amplified. Also, only the primer corresponding to one of the alleles may be used.

When a probe is used, it consists of an oligonucleotide consisting of an oligonucleotide corresponding to a part of the SORCS1 gene sequence containing a single nucleotide polymorphism site or a complementary sequence thereof, or an oligonucleotide consisting of a sequence capable of hybridizing to those sequences under stringent conditions. Can be analyzed by hybridization with a nucleic acid sample isolated from a test dog or a nucleic acid sample amplified by a known method such as PCR.

Probes and primers for detecting single nucleotide polymorphisms of the SORCS1 gene can be appropriately designed based on the sequence information of single nucleotide polymorphisms.

The primer is a primer capable of identifying an allele of a single nucleotide polymorphism site of the SORCS1 gene, and is, for example, a sequence capable of amplifying a region having a sequence containing a single nucleotide polymorphism site of the SORCS1 gene. These primers may contain a sequence containing a single nucleotide polymorphism site, and the 3'and 5'sides of a region containing a single nucleotide polymorphism site (preferably a region having a length of 40 to 100,000 bases). It may be a primer pair of a forward primer and a reverse primer set at both ends of. The primer is a nucleotide sequence consisting of a base sequence containing a single nucleotide polymorphism site of the SORCS1 gene, a base sequence complementary to the base sequence, or a sequence capable of hybridizing to those sequences under stringent conditions (preferably a DNA fragment). ), And the number of bases is 5 to 50, preferably 10 to 30, and more preferably 15 to 25.

The probe is a nucleotide sequence (preferably a DNA fragment) consisting of a base sequence containing a single nucleotide polymorphism site of the SORCS1 gene, a base sequence complementary to the base sequence, or a sequence capable of hybridizing to those sequences under stringent conditions. ), And the number of bases is 5 to 50, preferably 10 to 30, and more preferably 10 to 25.

In addition, the number of primers or probes is several, preferably 1 to 5, more preferably 1 or 2, particularly preferably 1 in a continuous base sequence containing a single nucleotide polymorphism site of the base sequence of the SORCS1 gene. It may have a mismatch.

For example, a single nucleotide polymorphism present in an allelic gene has a pair of PCR primers that bind to the outside of the base sequence containing the single nucleotide polymorphism and a fluorescently labeled allelic gene-specific sequence for real-time PCR detection2. It can be investigated by real-time PCR performed by preparing different types of probes and performing a PCR reaction.

Examples of the primer include the following primers.
(p1) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801488C> T) in the base sequence of SEQ ID NO: 1;
(p2) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801539G> T) in the base sequence of SEQ ID NO: 2;
(p3) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18809171G> A) in the base sequence of SEQ ID NO: 3;
(p4) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18820912C> A) in the base sequence of SEQ ID NO: 4;
(p5) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821030T> A) in the base sequence of SEQ ID NO: 5;
(p6) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821044C> T) in the base sequence of SEQ ID NO: 6;
(p7) Primer capable of amplifying the region containing the 26th base (base of polymorphism site of NC_006610.3: g.18823291A> G) in the base sequence of SEQ ID NO: 7;
(p8) A primer capable of amplifying a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18835904T> C) in the base sequence of SEQ ID NO: 8.

Specifically, for example, the following primer pairs can be used.
(a) As PCR primers for detecting NC_006610.3: g.18801488C> T polymorphism and NC_006610.3: g.18801539G> T polymorphism, forward: 5'-ATCCCTGCTGTCATACTT-3'(SEQ ID NO: 14) And reverse: 5'-GATGAATGATTGCTTAGGGTT-3' (SEQ ID NO: 15) can be used.
(b) NC_006610.3: g.18809171G> A As polymorphic PCR primers, forward: 5'-TCTTTATTATAGGGAGCCTATAT-3'(SEQ ID NO: 16) and reverse: 5'-TGTTGGTACAAGGTTACCTAAA-3' (SEQ ID NO: 17). Can be used.
(c) NC_006610.3: g.18820912C> A polymorphism, NC_006610.3: g.18821030T> A polymorphism and NC_006610.3: g.18821044C> T polymorphism as PCR primers, forward: 5'-CAAGATTGGGGCACTGAACT -3'(SEQ ID NO: 18) and reverse: 5'-AACGAGGCCTAAAGAGCTGA-3' (SEQ ID NO: 19) can be used.
(d) NC_006610.3: g.18823291A> G As PCR primers for polymorphism, forward: 5'-TGCATATGGGCATCTCTACCC-3'(SEQ ID NO: 20) and reverse: 5'-GCCATCACTTACTGAACGTC-3' (SEQ ID NO: 21). Can be used.
(e) NC_006610.3: g.18835904T> C Polymorphism PCR primers include forward: 5'-TTTAGGGCAGAATGACCCTG-3'(SEQ ID NO: 22) and reverse: 5'-AGGATGCCTTCTTCCTAGTGT-3' (SEQ ID NO: 23). Can be used.

In addition, the following probes can be mentioned as probes.
(q1) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801488C> T) in the base sequence of SEQ ID NO: 1;
(q2) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801539G> T) in the base sequence of SEQ ID NO: 2;
(q3) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18809171G> A) in the base sequence of SEQ ID NO: 3;
(q4) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18820912C> A) in the base sequence of SEQ ID NO: 4;
(q5) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821030T> A) in the base sequence of SEQ ID NO: 5;
(q6) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821044C> T) in the base sequence of SEQ ID NO: 6;
(q7) A probe capable of hybridizing to a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18823291A> G) in the base sequence of SEQ ID NO: 7;
(q8) A probe capable of hybridizing to a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18835904T> C) in the base sequence of SEQ ID NO: 8.

The present invention is a probe used to analyze a single nucleotide polymorphism of the SORCS1 gene and a primer used to amplify a DNA fragment containing a single nucleotide polymorphism site to analyze a single nucleotide polymorphism (preferably at least a pair of primers). Includes set). It also includes a DNA chip on which a probe is immobilized. It also includes a kit for analyzing single nucleotide polymorphism sites of the SORCS1 gene, including probes, DNA chips, and primers.

The kit may also include restriction enzymes, polymerases, nucleoside triphosphates, nucleic acid-labeled molecules, buffers, instruction manuals and the like used for analysis of single nucleotide polymorphisms.

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Dog whole-genome-related analysis and search for SNPs 3 active blind dogs, 4 retired blind dogs, 7 passed dogs after training, and failed dogs (failed dogs) after training 14 Using DNA extracted from the whole blood of a total of 28 individuals using a commercially available kit (QIAGEN), genotypes of approximately 170,000 SNPs (single nucleotide polymorphisms) were determined by CanineHD BeadChip CanineHD BeadChip (illumina). Then, using SNP analysis software (SNP & Variation Suite7, Golden Helix), the differences in genotype frequencies of each SNP were statistically analyzed for the following A and B (GWAS analysis).
A Between two groups of 14 guide dogs (active guide dogs (3), retired guide dogs (4), train pass dogs (7)) and 14 guide dog fail groups B 7 post-training pass dog groups and post-training fail dogs Between 2 groups of 14 patients

Results (1) As a result of dog whole genome association analysis (GWAS) of guide dog group and non-guide dog group, a region showing a strong correlation with dog chromosome 28 was identified. FIG. 1 shows the results of GWAS analysis of dog chromosome 28. The region surrounded by the ellipse in FIG. 1 is the region where a strong correlation is observed. Next, as a result of searching the gene group in that region, it was clarified that eight SNPs having a correlation of p <0.05 or more are accumulated in SORCS1 which is said to be expressed in the central nervous system. The eight SNPs are shown in Table 1.
FIGS. 2 to 9 show the polymorphism genotypes, allele frequencies, and pass rates of Table 1 to 8 in guide dogs and non-guide dogs, respectively.

(2) A correlation was also confirmed between the two groups of dogs that passed the SORCS1 gene after training (7) and dogs that failed after training (14). These results revealed that the SORC1 gene and its genotype are related to the selection and behavioral characteristics of guide dogs.

FIG. 10 shows the nucleotide sequence containing each polymorphism in the SORCS1 gene intron. The underlined part of each sequence is the primer sequence described later, and the parentheses indicate the SNP site. FIG. 10A shows a base sequence (SEQ ID NO: 9) containing NC_006610.3: g.18801488C> T polymorphism and NC_006610.3: g.18801539G> T polymorphism, and FIG. 10B shows NC_006610.3: g.18809171G> A polymorphism. The nucleotide sequence containing the type (SEQ ID NO: 10) is shown in FIG. 10C as NC_006610.3: g.18820912C> A polymorphism, NC_006610.3: g.18821030T> A polymorphism and NC_006610.3: g.18821044C> T polymorphism. FIG. 10D shows a base sequence containing NC_006610.3: g.18823291A> G polymorphism (SEQ ID NO: 12), and FIG. 10E shows a base sequence containing NC_006610.3: g.18835904T> C polymorphism. The base sequence (SEQ ID NO: 13) containing the above is shown.

Detection of SNP in the SORC1 gene can be performed by determining the base sequence of the single nucleotide polymorphism moiety. For determination of the base sequence of the single nucleotide polymorphism part, for example, there are a direct sequencing method, an SNP typing method by the TaqMan method, and the like. Primers that can be used in these methods were designed as follows (Fig. 10).

As PCR primers for detecting NC_006610.3: g.18801488C> T polymorphism and NC_006610.3: g.18801539G> T polymorphism, forward: 5'-ATCCCTGCTGTCATACTT-3' (SEQ ID NO: 14) and reverse: 5'-GATGAATGATTGCTTAGGGTT-3'(SEQ ID NO: 15) can be used.
NC_006610.3: g.18809171G> A Polymorphism PCR primers can be forward: 5'-TCTTTATTAAGGGAGCCTATAT-3'(SEQ ID NO: 16) and reverse: 5'-TGTTGGTACAAGGTTACCTAAA-3' (SEQ ID NO: 17). it can.
NC_006610.3: g.18820912C> A polymorphism, NC_006610.3: g.18821030T> A polymorphism and NC_006610.3: g.18821044C> T polymorphism PCR primers include forward: 5'-CAAGATTGGGGCACTGAACT-3' (SEQ ID NO: 18) and reverse: 5'-AACGAGGCCTAAAGAGCTGA-3'(SEQ ID NO: 19) can be used.
NC_006610.3: g.18823291A> G As the PCR primer for the polymorphism, forward: 5'-TGCATATGGGCATCTCTACCC -3'(SEQ ID NO: 20) and reverse: 5'-GCCATCACTTACTGAACGTC-3' (SEQ ID NO: 21) can be used. it can.
NC_006610.3: g.18835904T> C polymorphism PCR primers can be forward: 5'-TTTAGGGCAGAATGACCCTG-3'(SEQ ID NO: 22) and reverse: 5'-AGGATGCCTTCTTCCTAGTGT-3' (SEQ ID NO: 23). it can.

Example 2 Comparison of effects with known single nucleotide polymorphisms used for guide dog selection In Japanese Patent No. 4839439, Japanese Patent No. 5023327, and Japanese Patent No. 5023331, as single nucleotide polymorphisms suitable for selection of guide dogs, Thirteen single nucleotide polymorphisms are listed.

FIG. 11 shows a known single nucleotide polymorphism. The underline in the table indicates p <0.05. As shown in FIG. 11, among the 13 known single nucleotide polymorphisms, only 5 had a significant difference P <0.05.

Among the known single nucleotide polymorphisms, DRD4 exon III, which had the least significant difference, and No. 3 to 5, No. 7, and 8 (Table 1), which are the most effective of the present invention, were about 8,700 times, the most significant difference. It has about 9 times more selection ability than the GLT-1 T471C.

Figures 12-1, 12-2, and 12-3 show a comparison of the pass rates of guide dogs selected using the single nucleotide polymorphism of the present invention and the known single nucleotide polymorphism. In FIGS. 12-1 and 12-2, the single nucleotide polymorphisms of the present invention are described from No. 8. When the single nucleotide polymorphism of the present invention is used, the pass rate may be 100%, and the pass rate of the blind guide dog selected using the single nucleotide polymorphism of the present invention was selected using a known single nucleotide polymorphism. It was found that the selection method using the single nucleotide polymorphism of the present invention, which is higher than the pass rate of blind dogs, is a highly sensitive selection method.

In Figures 12-1, 12-2 and 12-3, "blind" indicates the number of dogs that passed after training (active or retired), and "unblind" indicates the number of dogs that failed after training. Shown.

For the selection of guide dogs, dogs that are particularly friendly to humans and dogs that are calm are considered to be suitable for guide dogs. In addition, the dogs that failed this study had a high degree of excitement, superiority, low motivation, distraction, and separation anxiety, which were the dispositions of dogs that were unsuitable for guide dogs. Therefore, the present invention found in the GWAS analysis of blind dogs and dogs that have failed training is not only for selection of blind dogs, but also for disaster relief dogs, assistance dogs, drug detection dogs, cancer detection dogs, police dogs, and animal therapy dogs. , Animal-mediated education Social activities such as dogs It is effective in selecting dogs that are important for improving human welfare such as breeding domestic dogs.

SEQ ID NOs: 14-23 Primers

Claims (10)

A method for determining a dog's behavioral characteristics, including analysis of a single nucleotide polymorphism of the Sortilin-related receptor CNS expressed (SORCS) 1 gene on chromosome 28 in a test dog, wherein the behavioral characteristics are obedience. And
Method in which the single nucleotide polymorphism of the Sortilin-related receptor CNS expressed ( SORCS1) gene on chromosome 28 is at least one single nucleotide polymorphism of the following (i) to (viii):
(i) A single nucleotide polymorphism of C or T at the 18801488th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 1 (NC_006610). .3: g.18801488C> T polymorphism);
(ii) A single nucleotide polymorphism of G or T at the 18801039th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 2 (NC_006610). .3: g.18801539G> T polymorphism);
(iii) A single nucleotide polymorphism of G or A at the 18809171th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 3 (NC_006610). .3: g.18809171G> A polymorphism);
(iv) A single nucleotide polymorphism of C or A at the 18820912th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 4 (NC_006610). .3: g.18820912C> A polymorphism);
(v) Single nucleotide polymorphism of T or A at base 18821030 of the entire base sequence of chromosome 28 of dogs, and single nucleotide polymorphism at base 26 of the base sequence shown by SEQ ID NO: 5 (NC_006610) .3: g.18821030T> A polymorphism);
(vi) A single nucleotide polymorphism of C or T at the .18821044th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 6 ( NC_006610.3: g.18821044C> T polymorphism);
(vii) A single nucleotide polymorphism of A or G at the 18823291th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 7 (NC_006610). .3: g.18823291A> G polymorphism); and
(viii) A single nucleotide polymorphism of T or C at the 18835904th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 8 (NC_006610). .3: g.18835904T> C polymorphism). If: the test dogs either (i) ~ (viii), said test dogs judged to have a compliancy as behavioral characteristics, claim 1 Symbol placement methods.
(i) A single nucleotide polymorphism of C or T at the 18801488th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 1 (NC_006610). .3: g.18801488C> T polymorphism) When the genotype of the site is C / C or C / T;
(ii) A single nucleotide polymorphism of G or T at the 18801039th base of the entire base sequence of chromosome 28 of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 2 (NC_006610). .3: g.18801539G> T polymorphism) When the genotype of the site is G / G or G / T;
(iii) A single nucleotide polymorphism of G or A at the 18809171th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 3 (NC_006610). .3: g.18809171 G> A polymorphism) When the genotype of the site is A / A or G / A;
(iv) A single nucleotide polymorphism of C or A at the 18820912th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 4 (NC_006610). .3: g.18820912C> A polymorphism) When the genotype of the site is A / A or C / A;
(v) Single nucleotide polymorphism of T or A at base 18821030 of the entire base sequence of chromosome 28 of dogs, and single nucleotide polymorphism at base 26 of the base sequence shown by SEQ ID NO: 5 (NC_006610) .3: g.18821030T> A polymorphism) When the genotype of the site is A / A or T / A;
(vi) A single nucleotide polymorphism in C or T at base 18821044 of the entire base sequence of chromosome 28 of a dog, and a single nucleotide polymorphism at base 26 of the base sequence shown in SEQ ID NO: 6 (NC_006610). .3: g.18821044C> T polymorphism) When the genotype of the site is T / T or C / T;
(vii) A single nucleotide polymorphism of A or G at the 18823291th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 7 (NC_006610). .3: g.18823291A> G polymorphism) When the genotype of the site is G / G or A / G; or
(viii) A single nucleotide polymorphism of T or C at the 18835904th base of the entire base sequence of the 28th chromosome of a dog, and a single nucleotide polymorphism at the 26th base of the base sequence shown by SEQ ID NO: 8 (NC_006610). .3: g.18835904T> C polymorphism) When the genotype of the site is C / C or T / C. The method according to claim 1 or 2 , wherein a genomic DNA containing a single nucleotide polymorphism of intron 3 is extracted from a test dog, and a base of a single nucleotide polymorphism site of an allele contained in the extracted DNA is identified. Prepare two types of probes having a pair of PCR primers that bind to the outside of the base sequence containing the single nucleotide polymorphism and a fluorescently labeled allelic gene-specific sequence for real-time PCR machine detection, and perform the PCR reaction. The method according to claim 3 , wherein the base of the single nucleotide polymorphism site of the allelic gene is identified by real-time PCR carried out in. Test dogs judged to have obedience as behavioral characteristics are blind guide dogs, disaster relief dogs, assistance dogs, drug detection dogs, hearing dogs, cancer detection dogs, police dogs, animal therapy dogs, animal-mediated education. The method according to any one of claims 1 to 4 , wherein the dog is selected as a dog selected from the group consisting of a dog and a domestic dog. Any of the following primer pairs (p1) to (p8) used for amplifying a DNA fragment containing a single nucleotide polymorphism site of the SORCS1 gene on chromosome 28 of a dog, according to any one of claims 1 to 5 . Primer pair used in the method according to item 1.
(p1) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801488C> T) in the base sequence of SEQ ID NO: 1;
(p2) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801539G> T) in the base sequence of SEQ ID NO: 2;
(p3) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18809171G> A) in the base sequence of SEQ ID NO: 3;
(p4) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18820912C> A) in the base sequence of SEQ ID NO: 4;
(p5) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821030T> A) in the base sequence of SEQ ID NO: 5;
(p6) A primer capable of amplifying the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821044C> T) in the base sequence of SEQ ID NO: 6;
(p7) Primer capable of amplifying the region containing the 26th base (base of polymorphism site of NC_006610.3: g.18823291A> G) in the base sequence of SEQ ID NO: 7;
(p8) A primer capable of amplifying a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18835904T> C) in the base sequence of SEQ ID NO: 8. Any of the following primer pairs (a) to (e) used for amplifying a DNA fragment containing a single nucleotide polymorphism site of the SORCS1 gene on chromosome 28 of a dog, according to any one of claims 1 to 5 . Primer pair used in the method according to item 1.
(a) Forward: 5'-ATCCCTGCTGTCATACTT-3' (SEQ ID NO: 5'-ATCCCTGCTGTCATACTT-3' (SEQ ID NO:) to detect at least one polymorph of NC_006610.3: g.18801488C> T polymorphism and NC_006610.3: g.18801539G> T polymorphism. 14) and reverse: Primer pair of 5'-GATGAATGATTGCTTAGGGTT-3'(SEQ ID NO: 15);
(b) NC_006610.3: g.18809171G> A for detecting polymorphism: forward: 5'-TCTTTATTATAGGGAGCCTATAT-3'(SEQ ID NO: 16) and reverse: 5'-TGTTGGTACAAGGTTACCTAAA-3' (SEQ ID NO: 17) Primer pair;
(c) To detect at least one polymorph of NC_006610.3: g.18820912C> A polymorphism, NC_006610.3: g.18821030T> A polymorphism and NC_006610.3: g.18821044C> T polymorphism, Forward: 5'-CAAGATTGGGGCACTGAACT-3'(SEQ ID NO: 18) and reverse: 5'-AACG AGGCCTAAAGAGCTGA-3' (SEQ ID NO: 19) primer pair;
(d) NC_006610.3: g.18823291A> G Forward: 5'-TGCATATGGGCATCTCTACCC-3'(SEQ ID NO: 20) and Reverse: 5'-GCCATCACTTACTGAACGTC -3'(SEQ ID NO: 21) Primer pair; or
(e) Primer pair of forward: 5'-TTTAGGGCAGAATGACCCTG-3'(SEQ ID NO: 22) and reverse: 5'-AGGATGCCTTCTTCCTAGTGT-3' (SEQ ID NO: 23) for detecting NC_006610.3: g.18835904T> C .. A probe according to any one of the following (q1) to (q8) capable of hybridizing to a region containing a single nucleotide polymorphism site of the SORCS gene on chromosome 28 of a dog, according to claims 1 to 5 . Probe used in any one of the methods:
(q1) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801488C> T) in the base sequence of SEQ ID NO: 1;
(q2) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18801539G> T) in the base sequence of SEQ ID NO: 2;
(q3) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18809171G> A) in the base sequence of SEQ ID NO: 3;
(q4) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18820912C> A) in the base sequence of SEQ ID NO: 4;
(q5) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821030T> A) in the base sequence of SEQ ID NO: 5;
(q6) A probe capable of hybridizing to the region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18821044C> T) in the base sequence of SEQ ID NO: 6;
(q7) A probe capable of hybridizing to a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18823291A> G) in the base sequence of SEQ ID NO: 7;
(q8) A probe capable of hybridizing to a region containing the 26th base (base of the polymorphism site of NC_006610.3: g.18835904T> C) in the base sequence of SEQ ID NO: 8. At least one pair of primer pairs of claim 6 (p1) ~ (p8) , according to claim 7 (a) ~ at least one pair of primer pair (e), or according to claim 8 ( A kit for determining the behavioral characteristics of a dog, which comprises at least one of the probes q1) to (q8). Test dogs judged to have obedience as behavioral characteristics are blind guide dogs, disaster relief dogs, assistance dogs, drug detection dogs, hearing dogs, cancer detection dogs, police dogs, animal therapy dogs, animal-mediated education. The kit according to claim 9 , which is selected as a dog selected from the group consisting of dogs and domestic dogs.

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