SNP molecular markers associated with fear and / or aggression behavior towards strangers in dogs and uses thereof

Abstract

The application provides a SNP molecular marker related to the fear behavior and / or attack behavior of a dog to a stranger and an application thereof, and belongs to the technical field of molecular biology. The SNP molecular marker comprises a first molecular marker and / or a second molecular marker. The nucleotide sequence of the first molecular marker is shown in SEQ ID NO:1, the 170th position is a polymorphic site, and the polymorphism is T / G. The nucleotide sequence of the second molecular marker is shown in SEQ ID NO:2, the 240th position is a polymorphic site, and the polymorphism is A / G. The SNP molecular marker can be used to identify the degree of the fear behavior and / or attack behavior of a dog to a stranger.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to SNP molecular markers and their applications related to canine fear and / or aggression towards strangers. Background Technology

[0002] Domestic dogs, or simply dogs, are humankind's closest friends and have always played an important role in human life. For example, police dogs, guide dogs, companion dogs, and sheepdogs are loyal companions that play an important role in our lives and production. In more and more families, dogs are even regarded as important family members.

[0003] Fear is an emotional state caused by any stimulus that may pose a danger. Fear triggers a survival-oriented behavioral response in animals; however, excessive fear, while a mental illness in humans, is considered a behavioral problem in dogs, affecting their relationship with humans and potentially severely damaging their health. For example, dogs that exhibit fear of strangers have significantly shorter lifespans compared to domestic dogs that are not afraid. Furthermore, excessive fear of strangers can render police dogs and guide dogs unable to perform their duties, leading to their elimination and significantly increasing breeding costs.

[0004] In addition, dogs exhibit aggressive behavior. While moderate aggression is important for dogs to protect themselves and their families, excessive aggression can sometimes lead to disputes and even very serious social problems. It is estimated that more than 4 million dog bite incidents occur annually in the United States. Aggressive behavior places severe stress on the relationship between people and their dogs. Furthermore, harm to victims can lead to significant financial and legal burdens for owners.

[0005] Currently, there are many reports on canine behavior research. However, existing technologies for investigating the fear and aggression of breed dogs towards strangers mainly rely on behavioral tests or questionnaires to obtain scores on the dogs' fear and aggression towards strangers. This allows researchers to determine whether the dog exhibits fear and aggression towards strangers, or the degree and intensity of such behavior. Furthermore, there is a lack of research on the genetic mechanisms of canine fear and aggression towards strangers, making it impossible to effectively identify the degree of fear and aggression towards strangers at the molecular level. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide SNP molecular markers related to canine fear and / or aggression towards strangers and their applications. Based on the SNP molecular markers of the present invention, the degree of canine fear and / or aggression towards strangers can be effectively identified at the molecular level.

[0007] This invention provides SNP molecular markers associated with fear and / or aggression in dogs toward strangers, including a first molecular marker and / or a second molecular marker; the nucleotide sequence of the first molecular marker is shown in SEQ ID NO: 1, with a polymorphic site at position 170, exhibiting T / G polymorphism; the nucleotide sequence of the second molecular marker is shown in SEQ ID NO: 2, with a polymorphic site at position 240, exhibiting A / G polymorphism.

[0008] Preferably, the polymorphic site of the first SNP marker is located at position 20310833 on canine chromosome 18;

[0009] The polymorphic site of the second SNP marker is located at position 20331216 on canine chromosome 18;

[0010] The SNP molecular markers are based on the canine reference genome canfam3.1.

[0011] This invention provides a primer set for identifying the SNP molecular markers described in the above scheme, the primer set comprising a first molecular marker primer set and a second molecular marker primer set;

[0012] The first molecular marker primer set includes a first upstream primer and a first downstream primer; the nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 3; the nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 4;

[0013] The second molecular marker primer set includes a second upstream primer and a second downstream primer; the nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 5; the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 6.

[0014] The present invention also provides a kit for identifying the SNP molecular markers described in the above scheme, comprising the primer set described in the above scheme.

[0015] The present invention also provides the application of the SNP molecular markers described above as detection targets, or the primer sets or the kits described above, in identifying the degree of fear and / or aggression in dogs towards strangers, or in breeding dogs with low or high levels of fear and / or aggression towards strangers.

[0016] Preferably, dogs with the first SNP molecular marker polymorphic site gene G exhibit significantly higher levels of fear and / or aggression towards strangers than dogs with the gene T; and dogs with the second SNP molecular marker polymorphic site gene G exhibit significantly higher levels of fear and / or aggression towards strangers than dogs with the gene A.

[0017] Preferably, the dogs include large dogs, medium dogs, and small dogs.

[0018] Preferably, the dogs include Great Danes, Beagles, Shih Tzus, French Bulldogs, Pomeranians (Hadorian breed), 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.

[0019] The present invention also provides a method for identifying the degree of fear and / or aggression in dogs towards strangers, or for breeding dogs with low or high levels of fear and / or aggression towards strangers, comprising the following steps:

[0020] Detect the genes in the test dog whose SNP molecular markers are located at polymorphic sites as described in the above protocol;

[0021] When the gene at the polymorphic site of the first SNP molecular marker is G and / or the gene at the polymorphic site of the second SNP molecular marker is G, the dog is judged to have a high degree of fear and / or aggression towards strangers.

[0022] Alternatively, when the gene at the polymorphic site of the first SNP molecular marker is T and the gene at the polymorphic site of the second SNP molecular marker is A, the dog is judged to have a low degree of fear and / or aggression towards strangers.

[0023] This invention also provides the application of the SNP molecular markers described above as detection targets in the veterinary field or in canine breeding and training.

[0024] This invention provides SNP molecular markers associated with fear and / or aggression towards strangers in dogs, including a first molecular marker and / or a second molecular marker. The nucleotide sequence of the first molecular marker is shown in SEQ ID NO: 1, with a polymorphic site at position 170 exhibiting T / G polymorphism. The nucleotide sequence of the second molecular marker is shown in SEQ ID NO: 2, with a polymorphic site at position 240 exhibiting A / G polymorphism. Dogs with the G gene at the polymorphic site of the first SNP molecular marker show significantly higher levels of fear and / or aggression towards strangers than dogs with the T gene. Similarly, dogs with the G gene at the polymorphic site of the second SNP molecular marker show significantly higher levels of fear and / or aggression towards strangers than dogs with the A gene. Therefore, the SNP molecular markers of this invention can be used to identify the degree of fear and / or aggression towards strangers in dogs or to breed dogs with low or high levels of fear and / or aggression towards strangers, thus shortening the breeding time and reducing the cost of judging breed behavior. Attached Figure Description

[0025] Figure 1 This is a Manhattan plot of canine aggression towards strangers. 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 there is a more significant association between this point and aggression towards strangers.

[0026] Figure 2 This is a QQ graph of canine aggression towards strangers. 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.

[0027] Figure 3 This is a Manhattan plot of canine fear of strangers. 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 fear of strangers.

[0028] Figure 4 This is a QQ graph of a dog's fear of strangers. 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.

[0029] Figure 5 The image shows a PCR gel image of the locus, where M is the marker (8000bp), 1 is 18:20310833, and 2 is 18:20331216. Detailed Implementation

[0030] This invention provides SNP molecular markers associated with fear and / or aggression in dogs towards strangers, including a first molecular marker and / or a second molecular marker; the nucleotide sequence of the first molecular marker is shown in SEQ ID NO: 1, with a polymorphic site at position 170, exhibiting T / G polymorphism; the nucleotide sequence of the second molecular marker is shown in SEQ ID NO: 2, with a polymorphic site at position 240, exhibiting A / G polymorphism.

[0031] In this invention, the polymorphic site of the first SNP marker is located at position 20310833 on canine chromosome 18; the polymorphic site of the second SNP marker is located at position 20331216 on canine chromosome 18; the SNP molecular markers are based on the canine reference genome canfam3.1.

[0032] In this invention, individuals with the first SNP marker having a polymorphic site gene of G exhibit significantly higher levels of fear behavior and / or separation anxiety behavior than individuals with a gene of A.

[0033] Individuals with the second SNP marker having the T genotype showed significantly higher levels of fear behavior and / or separation anxiety than individuals with the C genotype.

[0034] This invention provides a primer set for identifying the SNP molecular markers described in the above scheme, the primer set comprising a first molecular marker primer set and a second molecular marker primer set;

[0035] The first molecular marker primer set includes a first upstream primer and a first downstream primer; the nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 3; the nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 4;

[0036] The second molecular marker primer set includes a second upstream primer and a second downstream primer; the nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 5; the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 6.

[0037] The present invention also provides a kit for identifying the SNP molecular markers described in the above scheme, comprising the primer set described in the above scheme.

[0038] The present invention also provides the application of the SNP molecular markers described above as detection targets, or the primer sets or the kits described above, in identifying the degree of fear and / or aggression in dogs towards strangers, or in breeding dogs with low or high levels of fear and / or aggression towards strangers.

[0039] In this invention, the dogs preferably include large, medium, and small breeds; more preferably, they include Great Danes, Beagles, Shih Tzus, French Bulldogs, Pomeranians (Hadori breed), Chihuahuas, Miniature Poodles, Yorkshire Terriers, Jack Russell Terriers, Dachshunds, Cocker Spaniels, Shar Peis, Norwegian Deerhounds, Rottweilers, English Bulldogs, Springer Spaniels, Skipjack 'n' Rolls-Royces, Pugs, Welsh Corgis, Havanese, Siberian Huskies, Border Collies, and others. Lottie Shepherd, Greyhound, Newfoundland, Cavalier King Charles Spaniel, Weimaraner, Poodle, Belgian Terrier, Eurasian Dog, English Setter, Golden Retriever, German Shorthaired Pointer, Finnish Spitz, Labrador Retriever, Boston Terrier, Border Terrier, Brittany Hound, New St. Shepherd Duckweed, German Shepherd, Boxer, Mastiff, Australian Shepherd, Doberman Pinscher, Gordon Setter, Bernese Mountain Dog, and Irish Wolfhound.

[0040] The present invention also provides a method for identifying the degree of fear and / or aggression in dogs towards strangers, or for breeding dogs with low or high levels of fear and / or aggression towards strangers, comprising the following steps:

[0041] Detect the genes in the test dog whose SNP molecular markers are located at polymorphic sites as described in the above protocol;

[0042] When the gene at the polymorphic site of the first SNP molecular marker is G and / or the gene at the polymorphic site of the second SNP molecular marker is G, the dog is judged to have a high degree of fear and / or aggression towards strangers.

[0043] Alternatively, when the gene at the polymorphic site of the first SNP molecular marker is T and the gene at the polymorphic site of the second SNP molecular marker is A, the dog is judged to have a low degree of fear and / or aggression towards strangers.

[0044] This invention also provides the application of the SNP molecular markers described above as detection targets in the veterinary field or in canine breeding and training.

[0045] The English abbreviations and key terms involved in this invention are defined as follows:

[0046] GWAS - Genome-wide association analysis;

[0047] SNP - Single Nucleotide Polymorphism;

[0048] P-value - the probability of obtaining the sample observation result or a more extreme result when the null hypothesis is true.

[0049] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0050] Example 1

[0051] This example uses behavioral data from C-BARQ (Canine Behavioral Assessment & Research Questionnaire) (https: / / vetapps.vet.upenn.edu / cbarq / ).

[0052] 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, because 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.

[0053] Here, we combine canine aggression towards strangers and canine fear of strangers into a single term: canine fear-induced aggression towards strangers, i.e., aggressive behavior caused by fear. C-BARQ defines canine aggression towards strangers as a threatening or hostile reaction from a dog when a stranger (such as a delivery person or food delivery worker) approaches or intrudes into the dog's or owner's personal space, territory, or home area. This reaction may include barking, growling, barking teeth, or even lunging and biting. If the dog does not bark or growl, it scores 0 points; if it does bark, growl, or bark teeth, it scores 0-2 points; and if it lunges, lunges, tears, or attempts to tear, it scores 3 points or more. This embodiment currently covers 47 common dog breeds. Table 1 shows the number of individuals for each breed with available genotype data and the C-BARQ scoring for breed-specific aggression towards strangers.

[0054] Table 1. Scoring of Aggressive Behavior Towards Strangers by Different Dog Breeds

[0055]

[0056]

[0057]

[0058] C-BARQ defines a dog's fear of strangers as exhibiting anxious or fearful behavior when a stranger (delivery person or courier) approaches or intrudes into the dog's personal space, territory, or home area. Typical characteristics of mild to moderate fear include: avoiding eye contact, hiding from the feared object, crouching or curling up, tail lowered or tucked between legs, whimpering and howling, freezing, and trembling. Extreme fear is characterized by exaggerated withdrawal, attempting to escape, retreat, or hide from the feared object, person, or situation. A score of 0 is given if the dog does not exhibit any of these behaviors; a score of 0-2 is given if some of these behaviors are present; and a score of 3 or higher is given if there is intense escape, retreat, or hiding. This embodiment currently covers 47 common dog breeds, and Table 2 shows the number of individuals for each breed with available genotype data and the dog's fear of strangers score.

[0059] Table 2. Scoring of Fear Behavior Towards Strangers by Different Dog Breeds

[0060]

[0061]

[0062]

[0063] 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 dogs have undergone intense artificial selection, the genetic differences between individuals of the same breed are small, and behavioral differences between individuals are also smaller than those between different breeds. Therefore, we used a genome-wide association analysis of breed average data with individual genotype data.

[0064] The workflow for genome-wide association analysis (GWAS) of 2737 individuals is as follows: First, 180,117 SNPs and genotype data for 4224 canine individuals were obtained from Illumina 173kCanine HDarray sequencing (a genetic chip specifically designed for dogs by Illumina, ensuring its scientific validity). After excluding breeds without phenotypic data, principal component analysis (PCA) was performed on individuals using PLINK software. Then, a generalized model was used to calculate the PCA results for each trait to be included in the covariates. Subsequently, sex and the PCA results were used as covariates, and GEMMA software was used to calculate the kinship matrix among all individuals. Finally, the covariate files, kinship matrix, and files containing individual information were used to perform association analysis using a mixed linear model in GEMMA software. After correction using multiple hypothesis testing, loci significantly associated with the trait of aggression towards strangers were obtained. The results of the association analysis were visualized using R. The GWAS results are shown below. Figure 1 As shown in Figures 2, 3, and 4.

[0065] The sites were then annotated after being corrected by multiple hypothesis testing (P<3.2254735E-7), and mutation prediction was performed using ensembl (https: / / asia.ensembl.org / index.html). Two SNP sites that were significantly associated with the degree of fear-aggressive behavior towards strangers were obtained. These sites are located between the genes GNAT3 and CD36. The site information is shown in Table 3.

[0066] Table 3. Results of Genome-wide Association Analysis

[0067] 18:20310833 T G P<3.2254735E-7 Intergenic region 18:20331216 A G P<3.2254735E-7 Intergenic region

[0068] Example 2

[0069] Two pairs of primers were designed near the two SNPs. The specific implementation process and the DNA sequences of the primers and PCR amplification products are shown below.

[0070] Sequences near two sites were obtained from the UCSC Genome Browser Home and the National Center for Biotechnology Information database; primers were designed and synthesized using PrimerPremier 5.0 software (Table 4).

[0071] Table 4 Primer-related information

[0072]

[0073]

[0074] DNA was extracted from the dog cells to be tested using a DNA extraction kit (catalog number: 69504) provided by QIAGEN Ltd.

[0075] PCR amplification was performed using a kit from Mona Biotechnology Co., Ltd. (catalog number: MP05501). The PCR reaction mixture (50 μL) consisted of: 1 μL DNA template, 2 μL upstream primer, 2 μL downstream primer, and 25 μL MonAmp. TM×Taq Mix Pro, add deionized water to a final volume of 50 μL. PCR reaction program: pre-denaturation 94℃, 3 min; denaturation 94℃, 15 s; annealing temperature determined according to primer design (58℃ for site 18:20310833T>G, 60℃ for site 18:20331216A>G), 15 s; extension 72℃, 10 s; final extension 72℃, 5 min, for 34 cycles. Amplified products were detected by 1% agarose gel electrophoresis (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 gel image showed that the bands amplified by the two primer pairs were unique and clear, consistent with expectations. Figure 5 The amplified product sequences and the locations of the target SNPs are shown in Table 5.

[0076] Table 5. Sequence correlation of amplified products

[0077]

[0078]

[0079] Note: The bolded bases in the table are the target SNP sites.

[0080] We then scored the behavior of some of the 57 breeds of dogs, including 5 Chihuahuas, 10 Golden Retrievers, 10 Rottweilers, 7 Siberian Huskies, 2 Newfoundlands, 15 Greyhounds, and 8 Dachshunds. The scores are shown in Tables 6-7. After obtaining the genotypes of the two SNPs mentioned above for all the dogs, we summarized them in Tables 8-9.

[0081] Table 6. Scoring Table for Dog Breeds and Aggressive Behavior Towards Strangers

[0082]

[0083]

[0084] Table 7. Scoring Table for Dog Breeds and Fear of Strangers

[0085]

[0086]

[0087] Table 8. A summary of genotypes and their ratings for aggressive behavior towards strangers in different dog breeds.

[0088]

[0089]

[0090] Table 9. A summary of genotypes and fear of strangers in different dog breeds.

[0091]

[0092]

[0093]

[0094] The above research shows that 18:20310833T>G and 18:20331216A>G can be used to assess fear-aggression behavior in dogs. If a dog has mutant bases (ALT) at both sites, its fear-aggression behavior is higher. If the bases are from the reference genome (REF), its fear-aggression behavior is lower.

[0095] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a primer set or kit for detecting SNP molecular markers associated with fear and / or aggression in dogs in the preparation of products for identifying the degree of fear and / or aggression in dogs or for breeding dogs with low or high levels of fear and / or aggression in dogs. The SNP molecular marker includes a first molecular marker and a second molecular marker; the nucleotide sequence of the first molecular marker is shown in SEQ ID NO: 1, with the 170th position being a polymorphic site and exhibiting T / G polymorphism; the nucleotide sequence of the second molecular marker is shown in SEQ ID NO: 2, with the 240th position being a polymorphic site and exhibiting A / G polymorphism. The polymorphic site of the first SNP marker is located at position 20310833 on canine chromosome 18; the polymorphic site of the second SNP marker is located at position 20331216 on canine chromosome 18; the SNP molecular markers are based on the canine reference genome canfam3.1; The primer set includes a first molecular marker primer set and a second molecular marker primer set; The first molecular marker primer set includes a first upstream primer and a first downstream primer; the nucleotide sequence of the first upstream primer is shown in SEQ ID NO: 3; the nucleotide sequence of the first downstream primer is shown in SEQ ID NO: 4; The second molecular marker primer set includes a second upstream primer and a second downstream primer; the nucleotide sequence of the second upstream primer is shown in SEQ ID NO: 5; the nucleotide sequence of the second downstream primer is shown in SEQ ID NO: 6; The kit includes the primer set described above; Dogs with both the first and second SNP molecular markers having a polymorphic site gene of G exhibited significantly higher levels of fear and / or aggression towards strangers than dogs with a T gene and a second SNP molecular marker having a polymorphic site gene of A.

2. Use according to claim 1, characterized in that, The dogs include large, medium, and small dogs.

3. Use according to claim 1 or 2, characterized in that, 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.

Images