A molecular marker related to canine anxiety aggression behavior, amplification primer and application thereof

Abstract

The application provides a molecular marker related to anxiety attack behavior of a dog, an amplification primer and application thereof, and belongs to the technical field of molecular biology. The molecular marker comprises the following SNP site combination: a first SNP marker exists G / A polymorphism at the 8013736th site on chromosome 10, a second SNP marker exists A / G polymorphism at the 8019503th site on chromosome 10, a third SNP marker exists C / T polymorphism at the 8030670th site on chromosome 10 and a fourth SNP marker exists A / C polymorphism at the 8059173th site on chromosome 10. The molecular marker can be used for identifying the dog with anxiety attack behavior and judging the degree of the anxiety attack behavior of the dog. The application discloses the genetic mechanism of whether the dog has anxiety attack behavior from the aspect of molecular biology, and provides a new idea for greatly shortening the dog breeding time and helping veterinarians or dog trainers to quickly judge the behavior of the dog.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a molecular marker, amplification primer and its application related to canine anxiety and aggression behavior. Background Technology

[0002] Domestic dogs, also known simply as canines, were the first animals domesticated by humans and are excellent companion animals. Their behavior is more similar to that of humans, and they can both be considered predators who cooperate with members of their pack. Compared to other domesticated animals, domestic dogs are unique in that they are products of human selection based on behavioral patterns. Domestic dogs have always been valued by humans, undertaking various tasks in human life, including guarding, hunting, carrying loads, herding sheep, guiding, and companionship. In just 200-300 years, humans have selectively bred domestic dogs with these abilities to meet different needs.

[0003] Almost all animals exhibit aggressive behavior, and dogs are no exception. While aggression is a normal behavior in all species, it becomes problematic when it develops at an abnormal intensity or in an abnormal environment, interfering with the relationship between humans and their dogs. Dogs exhibit a wide variety of aggressive behaviors, and some dogs often display separation anxiety after being separated from their owners, which can escalate into attacks on their owners; this behavior is considered anxious aggression.

[0004] Currently, there are numerous reports on canine behavior research. However, existing techniques for assessing anxiety and aggression in purebred dogs primarily rely on behavioral tests or questionnaires to obtain scores on the dogs' anxiety and aggression behaviors, thereby determining whether the dog exhibits such behaviors or the intensity of these behaviors. Research on the genetic mechanisms underlying anxiety and aggression is lacking. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a molecular marker, amplification primer and its application related to canine anxiety-aggressive behavior, so as to effectively identify dogs with anxiety-aggressive behavior and the intensity of anxiety-aggressive behavior at the molecular biology level.

[0006] This invention provides a molecular marker associated with canine anxiety-aggressive behavior, including a first SNP molecular marker, a second SNP molecular marker, a third SNP molecular marker, and a fourth SNP molecular marker;

[0007] The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO:1, and a G / A polymorphism exists at position 164;

[0008] The nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO:2, and an A / G polymorphism exists at position 37;

[0009] The nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO:3, and a C / T polymorphism exists at position 84;

[0010] The nucleotide sequence of the fourth SNP molecular marker is shown in SEQ ID NO:4, and an A / C polymorphism exists at position 38.

[0011] Preferably, the first SNP molecular marker is located at position 8013736 on chromosome 10;

[0012] The second SNP molecular marker is located at position 8019503 on chromosome 10;

[0013] The third SNP molecular marker is located at position 8030670 on chromosome 10;

[0014] The fourth SNP molecular marker is located at position 8059173 on chromosome 10;

[0015] The SNP molecular markers are based on the canine reference genome canfam3.1.

[0016] The present invention provides a primer set for amplifying the molecular marker, including a primer pair for amplifying a first SNP molecular marker, a primer pair for amplifying a second SNP molecular marker, a primer pair for amplifying a second SNP molecular marker, and a primer pair for amplifying a second SNP molecular marker.

[0017] The primer pair for amplifying the first SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:5 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:6;

[0018] The primer pair for amplifying the second SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:7 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:8;

[0019] The primer pair for amplifying the third SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:9 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:10;

[0020] The primer pair for amplifying the fourth SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:11 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:12.

[0021] This invention provides a kit for identifying dogs exhibiting canine anxiety and aggression, comprising the primer set and PCR amplification reagents.

[0022] This invention provides the application of the molecular marker, the primer set, or the kit in predicting and / or screening dogs with canine anxiety-aggressive behavior.

[0023] Preferably, the canine anxiety-aggressive behavior includes aggression towards the owner and separation anxiety behavior when the dog is separated from the owner.

[0024] Preferably, the dogs include large dogs, medium dogs, and small dogs;

[0025] The breeds mentioned include Great Danes, Beagles, Shih Tzus, French Bulldogs, Pomeranians (Hadori lineage), Chihuahuas, Miniature Poodles, Yorkshire Terriers, Jack Russell Terriers, Dachshunds, Cocker Spaniels, Shar Peis, Norwegian Deerhounds, Rottweilers, English Bulldogs, Springer Spaniels, Skipjack Pikes, Pugs, Welsh Corgis, Havanese, Siberian Huskies, Border Collies, Shetland Sheepdogs, Greyhounds, Newfoundlands, Cavalier King Charles Spaniels, Weimaraners, Poodles, Belgian Terriers, Eurasian Dogs, English Setters, Golden Retrievers, German Shorthaired Pointers, Finnish Spitz, Labrador Retrievers, Boston Terriers, Border Terriers, Brittany Spaniels, New Zealand Shepherds, German Shepherds, Boxers, Mastiffs, Australian Shepherds, Doberman Pinschers, Gordon Setters, Bernese Mountain Dogs, and Irish Wolfhounds.

[0026] Preferably, the application includes the following steps:

[0027] The sequences of the first, second, third, and fourth SNP markers in the gene of the dog to be tested are detected to determine the genotype of the dog at the marker sites. Based on the genotype of the dog to be tested, the tendency for anxious and aggressive behavior is determined.

[0028] If the dog being tested has the GG genotype in the first SNP marker, then the degree of aggression towards its owner and / or separation anxiety towards its owner is significantly higher than that of dogs with the AA genotype.

[0029] If the dog being tested has the AA genotype in the second SNP marker, then the degree of aggression towards its owner and / or separation anxiety towards its owner is significantly higher than that of dogs with the GG genotype.

[0030] If the dog being tested has the CC genotype in the third SNP marker, then the degree of aggression towards the owner and / or separation anxiety towards the owner is significantly higher than that of dogs with the TT genotype.

[0031] If the dog being tested has the AA genotype in the fourth SNP marker, then the degree of aggression towards its owner and / or separation anxiety towards its owner is significantly higher than that of dogs with the CC genotype.

[0032] Preferably, the Great Dane, Beagle, and Shih Tzu are more aggressive towards their owners, while the Australian Shepherd and Irish Wolfhound are less aggressive towards their owners.

[0033] Beagles and Miniature Poodles exhibit higher levels of separation anxiety towards their owners, while Norwegian Deerhounds and Eurasian Dogs exhibit lower levels of separation anxiety.

[0034] This invention provides the application of the molecular marker, the primer set, or the kit in the veterinary field or in canine breeding and training.

[0035] This invention provides molecular markers associated with canine anxiety and aggression behavior, including a first SNP molecular marker, a second SNP molecular marker, a third SNP molecular marker, and a fourth SNP molecular marker. The sequence of the first SNP molecular marker is shown in SEQ ID NO.1, with the base at position 164 being G or A, and this marker is located at position 8013736 of canine chromosome 10. The sequence of the second SNP molecular marker is shown in SEQ ID NO.2, with the base at position 37 being A or G, and this marker is located at position 8019503 of canine chromosome 10. The sequence of the third SNP molecular marker is shown in SEQ ID NO.3, with the base at position 84 being C or T, and this marker is located at position 8030670 of canine chromosome 10. The sequence of the fourth SNP molecular marker is shown in SEQ ID NO.4, with the base at position 38 being A or C, and this marker is located at position 8059173 of chromosome 10. All four SNP sites in the molecular markers were significantly associated with anxious and aggressive behavior in dogs. If all four SNP sites in a dog were reference bases, the dog exhibited lower levels of anxious and aggressive behavior; if the SNP sites were mutant bases, the behavior was higher. Therefore, these molecular markers can be used to differentiate the levels of anxious and aggressive behavior in dogs, reducing breeding time and costs for veterinarians or dog behavior trainers to quickly identify behavioral problems in different breeds. Attached Figure Description

[0036] Figure 1 The Manhattan plot represents the aggressive behavior of dogs towards their owners. The horizontal axis represents chromosomes, and the vertical axis represents the -log(P) value of the association between the locus and the trait calculated by the software. Each point represents a SNP locus. The smaller the -P value of the locus, the larger the -log(P) value, indicating a more significant association between this point and the aggressive behavior of dogs towards their owners.

[0037] Figure 2 This is a QQ graph showing the aggressive behavior of a domestic dog towards its owner. The horizontal axis represents the expected P-value, and the vertical axis represents the actual calculated P-value for each site. Sites located above the standard line indicate that the calculation results are highly reliable.

[0038] Figure 3 This is a Manhattan plot of separation anxiety behavior in domestic dogs. The horizontal axis represents chromosomes, and the vertical axis represents the -log(P) value of the association between the locus and the trait calculated by the software. Each point represents a SNP locus. If the -P value of the locus is smaller, then the -log(P) value is larger, indicating that the point is more significantly associated with the dog's aggressive behavior towards its owner.

[0039] Figure 4 This is a QQ graph of separation anxiety behavior in domestic dogs. The horizontal axis represents the expected P-value, and the vertical axis represents the actual calculated P-value for each site. Sites located above the standard line indicate that the calculation results are highly reliable.

[0040] Figure 5 The image shows the SNP site PCR amplification electrophoresis diagram provided by this invention. M: Marker (8000bp), 1: 10: 8013736, 2: 10: 8019503, 3: 10: 8030670, 4: 10: 8059173. Detailed Implementation

[0041] This invention provides a SNP molecular marker associated with canine anxiety-aggressive behavior, including a first SNP molecular marker, a second SNP molecular marker, a third SNP molecular marker, and a fourth SNP molecular marker;

[0042] The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO:1, and a G / A polymorphism exists at position 164;

[0043] The nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO:2, and an A / G polymorphism exists at position 37;

[0044] The nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO:3, and a C / T polymorphism exists at position 84;

[0045] The nucleotide sequence of the fourth SNP molecular marker is shown in SEQ ID NO:4, and an A / C polymorphism exists at position 38.

[0046] In this invention, the first SNP molecular marker is located at position 8013736 on chromosome 10; the second SNP molecular marker is located at position 8019503 on chromosome 10; the third SNP molecular marker is located at position 8030670 on chromosome 10; and the fourth SNP molecular marker is located at position 8059173 on chromosome 10. The SNP molecular markers are based on the canine reference genome canfam3.1.

[0047] The present invention provides a primer set for amplifying the SNP molecular marker, including a primer pair for amplifying a first SNP molecular marker, a primer pair for amplifying a second SNP molecular marker, a primer pair for amplifying a second SNP molecular marker, and a primer pair for amplifying a second SNP molecular marker.

[0048] The primer pair for amplifying the first SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:5 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:6;

[0049] The primer pair for amplifying the second SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:7 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:8;

[0050] The primer pair for amplifying the third SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:9 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:10;

[0051] The primer pair for amplifying the fourth SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:11 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:12.

[0052] The present invention provides a kit for identifying dogs exhibiting canine anxiety and aggression behaviors, characterized in that it comprises the primer set and PCR amplification reagents.

[0053] This invention provides the application of the SNP molecular marker, the primer set, or the kit in predicting and / or screening dogs with canine anxiety-aggressive behavior.

[0054] In this invention, the canine anxiety-aggressive behavior preferably includes aggressive behavior towards the owner and separation anxiety behavior when the dog is separated from the owner.

[0055] In this invention, the anxious aggressive behavior preferably includes aggressive behavior towards the owner and separation anxiety behavior that arises when the dog is separated from its owner. Aggressive behavior towards the owner is defined as a threatening or hostile reaction to the owner or other family members when the dog perceives itself as being challenged, manipulated, stared at, trampled on, or approached or threatened while eating. This reaction may include barking, growling, baring teeth, or lunging and biting. Separation anxiety behavior is defined as vocalizations or destructive behaviors that occur when the dog is separated from its owner, usually accompanied by or involving anxious behaviors, including restlessness, trembling, loss of appetite, excessive salivation, whimpering, barking, growling, or scratching at doors, floors, windows, curtains, etc.

[0056] The application described in this invention is suitable for various dog breeds, including large, medium, and small dogs. In embodiments of this invention, it is applied to Great Danes, Beagles, Shih Tzus, French Bulldogs, Pomeranians (Hadori lineage), Chihuahuas, Miniature Poodles, Yorkshire Terriers, Jack Russell Terriers, Dachshunds, Cocker Spaniels, Shar Peis, Norwegian Deerhounds, Rottweilers, English Bulldogs, Springer Spaniels, Schipperkes, Pugs, Welsh Corgis, Havanese, Siberian Huskies, Border Collies, Shetland Sheepdogs, Greyhounds, Newfoundlands, and Knightsbill Terriers. Behavioral evaluation and genotypic analysis were conducted on the following breeds: King Leigh, Weimaraner, Poodle, Belgian Terrier, Eurasian, English Setter, Golden Retriever, German Shorthaired Pointer, Finnish Spitz, Labrador Retriever, Boston Terrier, Border Terrier, Brittany Spaniel, New St. Shepherd's Duckweed, German Shepherd, Boxer, Mastiff, Australian Shepherd, Doberman Pinscher, Gordon Setter, Bernese Mountain Dog, and Irish Wolfhound. The results of this invention's embodiments indicate that Great Danes exhibit higher levels of aggression towards their owners, while Irish Wolfhounds exhibit lower levels of aggression. Beagles show higher levels of separation anxiety towards their owners, while Eurasians show lower levels.

[0057] In this invention, the application includes the following steps:

[0058] The sequences of the first, second, third, and fourth SNP markers in the gene of the dog to be tested are detected to determine the genotype of the dog at the marker sites. Based on the genotype of the dog to be tested, the tendency for anxious and aggressive behavior is determined.

[0059] If the dog being tested has the GG genotype in the first SNP marker, then the degree of aggression towards its owner and / or separation anxiety towards its owner is significantly higher than that of dogs with the AA genotype.

[0060] If the dog being tested has the AA genotype in the second SNP marker, then the degree of aggression towards its owner and / or separation anxiety towards its owner is significantly higher than that of dogs with the GG genotype.

[0061] If the dog being tested has the CC genotype in the third SNP marker, then the degree of aggression towards the owner and / or separation anxiety towards the owner is significantly higher than that of dogs with the TT genotype.

[0062] If the dog being tested has the AA genotype in the fourth SNP marker, then the degree of aggression towards its owner and / or separation anxiety towards its owner is significantly higher than that of dogs with the CC genotype.

[0063] In this invention, the results of the detection and analysis of the above-mentioned various dog breeds show that Great Danes, Beagles, and Shih Tzus are more aggressive towards their owners, while Australian Shepherds and Irish Wolfhounds are less aggressive towards their owners; Beagles and Miniature Poodles exhibit a higher degree of separation anxiety towards their owners, while Norwegian Deerhounds and Eurasian Shepherds exhibit a lower degree of separation anxiety towards their owners.

[0064] Given that the aforementioned molecular markers can accurately and quickly predict or screen whether dogs exhibit anxious and aggressive behavior, and the degree of such behavior, this invention provides applications of these molecular markers in veterinary medicine or in dog breeding and training. The detection results of these molecular markers can quickly and accurately help veterinarians or dog trainers assess the level of anxious and aggressive behavior in dogs, while also providing new insights for dog breeding.

[0065] In this invention, the molecular marker detection method preferably uses the sequence of the molecular marker as a template to design PCR amplification primers, amplifies the DNA of the dog to be identified using a conventional PCR amplification method, and the amplification product is sequenced to analyze the genotype of the molecular marker. When the base is a reference base, the dog exhibits higher anxiety and aggression behavior, and when the base is a mutated base, the dog exhibits lower anxiety and aggression behavior.

[0066] The following detailed description, in conjunction with embodiments, illustrates a molecular marker, amplification primer, and their application related to canine anxiety and aggression behavior provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] Screening methods for SNP loci associated with canine anxiety-aggressive behavior

[0069] This example uses behavioral data from C-BARQ (Canine Behavioral Assessment & Research Questionnaire) (https: / / vetapps.vet.upenn.edu / cbarq / ). C-BARQ, developed by researchers at the University of Pennsylvania, is a method for obtaining behavioral data from dogs through questionnaires. Instead of directly observing and measuring animal behavior, C-BARQ distributes questionnaires to dog owners or handlers, who then score the dogs based on indirect behavioral information. The advantage of this questionnaire is that, since owners or handlers are the humans with the closest relationship to the dog, the results are more representative of reality. For ease of analysis, C-BARQ calculates each dog's behavioral score as the average of the scores for questionnaire items related to that behavior. Breed averages are based on the sum of individual dog scores divided by the number of individuals of each breed.

[0070] Here, anxious aggression is defined as aggressive behavior in dogs toward their owners and anxious behavior that arises when dogs are separated from their owners.

[0071] 1) Scoring results for dog's aggressive behavior towards its owner

[0072] The questionnaire defines aggressive behavior towards the owner as a dog's reaction to being challenged, manipulated, stared at, trampled on, or approached or threatened while eating, or a threatening or hostile response to the owner or other family members, such as barking, growling, baring teeth, or even lunging and biting. If the dog does not exhibit the above behaviors, it is scored as 0 points; if it exhibits some of the above behaviors, it is scored as 0 to 2 points; if it frequently exhibits the above behaviors, it is scored as 3 points or more.

[0073] This experiment involved 47 common domestic dog breeds. The number of individuals for each breed with available genotype data and the C-BARQ scores for dog aggression towards owners are shown in Table 1.

[0074] Table 1. Scoring Table for Aggressive Behavior Towards Owners by Different Dog Breeds

[0075]

[0076]

[0077]

[0078] 2) Scoring results of separation anxiety behavior in dogs

[0079] Separation anxiety behavior is defined as vocalizations or destructive behaviors in dogs when separated from their owners. It is usually accompanied by or accompanied by anxious behaviors, including restlessness, trembling, loss of appetite, excessive drooling, whimpering, barking, growling, or scratching at doors, floors, windows, curtains, etc. Owners need to determine the frequency of separation-related behaviors exhibited by their dogs when they leave alone or are about to leave. If these behaviors have never occurred, score 0; if they occur occasionally or sometimes, score 0-2; if they occur frequently or always, score 3 or higher.

[0080] This experiment involved 47 common dog breeds. The number of individuals of each breed with available genotype data and the degree of separation anxiety behavior of the dogs when separated from their owners are shown in Table 2.

[0081] Table 2. Scoring Table for Separation Anxiety Behavior of Different Dog Breeds

[0082]

[0083]

[0084]

[0085] The C-BARQ surveyed over 50,000 dogs and then calculated breed averages, which were based on the sum of individual dog scores divided by the number of individuals for each breed. Because the dogs have undergone intense artificial selection, the genetic differences between individuals of the same breed are small, and behavioral differences between individuals are smaller than those between different breeds. Therefore, this study used genome-wide association analysis of breed average data with individual genotype data.

[0086] 3) Genome-wide association analysis and site annotation

[0087] The procedure for genome-wide association analysis of 2,737 canine individuals is as follows.

[0088] First, 180,117 SNPs from canine sequencing using the Illumina 173k CanineHD array (datadryad.org, doi:10.5061 / dryad.266k4) and genotype data from 4,224 individuals were downloaded from NCBI. [i] This chip is a genetic chip specifically designed for dogs by Illumina, ensuring its scientific validity. PLINK software was used to eliminate breeds lacking phenotypic data. Principal component analysis (PCA) was then performed on individuals using PLINK software to minimize the impact of population stratification on subsequent analyses. The glm() function in R was then used to progressively calculate the PCA results for each trait that needed to be included in the covariates using a generalized linear model. [ii] Subsequently, individual sex and PCA results were used as covariates to calculate kinship matrices among all individuals using GEMMA software. Finally, the covariate files, kinship matrices, and files containing individual information were used in a genome-wide association analysis (GWAS) using a mixed linear model within GEMMA software to calculate SNP loci associated with canine aggression towards owners and separation anxiety. After correction using multiple hypothesis testing, loci significantly associated with aggression towards owners and separation anxiety were obtained. The association analysis results were visualized using R, resulting in QQ plots and Manhattan plots.

[0089] The results of the genome-wide association analysis are as follows: Figures 1-4 As shown. From Figure 1 and Figure 3 The two Manhattan plots reveal a significant association between aggressive behavior towards the owner and separation anxiety behavior on chromosome 10. Figure 2 and Figure 4 The QQ plot shows that the site is located above the standard line, which means that the calculation results are highly reliable.

[0090] The result will then be corrected using multiple hypothesis testing (P < 3.2254735E). -7 The loci were annotated, and mutation prediction was performed using ensembl (https: / / asia.ensembl.org / index.html). Four SNP loci significantly associated with both aggression towards owners and separation anxiety were obtained. These loci are located on or near the MSRB3 gene, which is currently considered to play an important role in dog domestication. Locus information is shown in Table 3.

[0091] Table 3. Results of Genome-wide Association Analysis

[0092]

[0093] Example 2

[0094] Four pairs of primers were designed near the four SNP sites screened in Example 1. The specific implementation process and the DNA sequences of the primers and PCR amplification products are shown below.

[0095] Sequences near four sites were obtained from the UCSC Genome Browser Home and the National Center for Biotechnology Information database; primers were designed using Primer Premier 5.0 software (Table 4) and synthesized artificially.

[0096] Table 4 Primer sequence correlation

[0097]

[0098]

[0099] DNA was extracted from the dog cells to be tested using a DNA extraction kit (catalog number: 69504) provided by QIAGEN Ltd.

[0100] PCR amplification was performed using a kit (catalog number: MP05501) manufactured by Mona Biotechnology Co., Ltd. The PCR reaction system (50 μL) consisted of 1 μL DNA template, 2 μL upstream primer, 2 μL downstream primer, 25 μL MonAmp™×Taq Mix Pro, and deionized water to a final volume of 50 μL. The PCR reaction program was as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 15 s; 60℃ for 15 s; extension at 72℃ for 10 s, 34 cycles; final extension at 72℃ for 5 min. The amplified products were detected by electrophoresis on a 1% agarose gel (0.25 g agarose and 25 mL 1×TAE) and observed using a high-sensitivity chemiluminescence imaging system (Yunnan Chien Technology Co., Ltd.; model: Chemidoc XRS+). The electrophoresis results showed that the bands amplified by the four primer pairs were unique and clear, consistent with expectations. Figure 5 The amplified products were sequenced and aligned, and the locations of the target SNPs are shown in Table 5.

[0101] Table 5. Sequence Correlation of Amplified Products

[0102]

[0103]

[0104] Note: The bolded bases in the table are the target SNP sites.

[0105] Subsequently, behavioral scores were assigned to some of the 28 breeds of dogs, including 6 Great Danes, 7 Beagles, 5 Shih Tzus, 2 Australian Shepherds, 2 Irish Wolfhounds, 2 Miniature Poodles, 2 Eurasian Dogs, and 2 Norwegian Deerhounds. The scores are shown in Tables 6 and 7. The genotypes of the above four SNPs for all the dogs were obtained and summarized in Tables 8 and 9.

[0106] Table 6. Scoring Table for Dog Breeds and Aggressive Behavior Towards Owners

[0107]

[0108] Table 7 Scoring Table for Dog Breeds and Separation Anxiety Behavior

[0109]

[0110]

[0111] Table 8. A summary of genotypes and their ratings for aggressive behavior towards owners in different dog breeds.

[0112]

[0113] Table 9. Overview of Genotype and Separation Anxiety Behavior Scores for Different Dog Breeds

[0114]

[0115] Analysis of the above loci revealed that the base types of four SNPs—10:8013736G>A, 10:8019503A>G, 10:8030670C>T, and 10:8059173A>C—can be used to assess canine anxiety-aggressive behavior. When all four loci are homozygous for the reference genome bases, the canine exhibits higher levels of anxiety-aggressive behavior (aggression towards the owner and separation anxiety). Conversely, when all four loci are homozygous for mutant bases, the canine exhibits lower levels of anxiety-aggressive behavior, consistent with the phenotypic expression of canine anxiety-aggressive behavior.

[0116] References:

[0117] [i]HAYWARD JJ,CASTELHANO MG,OLIVEIRA KC,et al.Complex disease andphenotype mapping in the domestic dog[J].Nat Commun,2016,7:10460.

[0118] [ii]Wang S, Strandberg E, Arvelius P, Clements DN, Wiener P, FriedrichJ. Genome-wide association studies for canine hip dysplasia in single and multiple populations-implications and potential novel risk loci. BMCGenomics.2021 Sep 2;22(1):636.doi:10.1186 / s12864-021-07945-z.PMID:34474664; PMCID:PMC8414704.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecular marker associated with canine anxious-aggressive behavior, characterized in that, Including the first SNP molecular marker, the second SNP molecular marker, the third SNP molecular marker, and the fourth SNP molecular marker; The nucleotide sequence of the first SNP molecular marker is shown in SEQ ID NO:1, and a G / A polymorphism exists at position 164; The nucleotide sequence of the second SNP molecular marker is shown in SEQ ID NO:2, and an A / G polymorphism exists at position 37; The nucleotide sequence of the third SNP molecular marker is shown in SEQ ID NO:3, and a C / T polymorphism exists at position 84; The nucleotide sequence of the fourth SNP molecular marker is shown in SEQ ID NO:4, and an A / C polymorphism exists at position 38.

2. The molecular marker according to claim 1, characterized in that, The first SNP molecular marker is located at position 8013736 on chromosome 10; The second SNP molecular marker is located at position 8019503 on chromosome 10; The third SNP molecular marker is located at position 8030670 on chromosome 10; The fourth SNP molecular marker is located at position 8059173 on chromosome 10; The SNP molecular markers are based on the canine reference genome canfam3.

1.

3. A primer set for amplifying the molecular marker of claim 1 or 2, characterized in that, This includes primer pairs for amplifying the first SNP molecular marker, primer pairs for amplifying the second SNP molecular marker, primer pairs for amplifying the third SNP molecular marker, and primer pairs for amplifying the fourth SNP molecular marker. The primer pair for amplifying the first SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:5 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:6; The primer pair for amplifying the second SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:7 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:8; The primer pair for amplifying the third SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:9 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:10; The primer pair for amplifying the fourth SNP molecular marker includes a forward primer with a nucleotide sequence as shown in SEQ ID NO:11 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

12.

4. A kit for identifying dogs exhibiting canine anxiety and aggression, characterized in that, Includes the primer set and PCR amplification reagents as described in claim 3.

5. The use of the primer set of claim 3 or the kit of claim 4 in the preparation of products for predicting and / or screening dogs with canine anxiety-aggressive behavior, said canine anxiety-aggressive behavior including canine aggression towards its owner and / or separation anxiety behavior when separated from its owner.

6. The application according to claim 5, characterized in that, The breeds of dogs include Great Danes, Beagles, and Shih Tzus.

7. The application according to claim 5, characterized in that, Great Danes, Beagles, and Shih Tzus are more aggressive towards their owners, while Australian Shepherds and Irish Wolfhounds are less aggressive. Beagles and Miniature Poodles exhibit higher levels of separation anxiety towards their owners, while Norwegian Deerhounds and Eurasian Dogs exhibit lower levels of separation anxiety.

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