CN113667763B - Biomarker, kit and method for identifying dogs with pickup behaviors - Google Patents

Abstract

The invention relates to a biomarker, a kit and a method for identifying dogs with a picking behavior. The biomarker included 20 SNP sites with 3 genes. The invention discovers 20 sites related to canine extraction behaviors, which comprise 3 genes related to behavioral or neurological diseases, and is used for distinguishing domestic dogs in high-grade groups from domestic dogs in low-grade groups. The experimental result shows that the chi-square value of different SNPs of each gene is the same, which suggests that the linkage disequilibrium phenomenon may exist. The SNP obtained by screening is probably the most strongly related to the canine recognition behavior, and can be suitable for identifying the canine with the recognition behavior.

Description

Biomarker, kit and method for identifying dogs with pickup behaviors

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a biomarker, a method and a kit for identifying a dog with a pickup behavior.

Background

Domestic dogs belong to the genus Canidae of the order Carnivora of the class Carnivorae of the phylum vertebrata and are one of the species with the most abundant genetic diversity in nature. The domestic dog is the domestic animal domesticated for the earliest time, and bears the most extensive work and hunting in human history. In the recent century of civilization evolution, with aesthetic requirements and the development of human society requirements, more than 400 varieties with different phenotypes and different sexual conditions were cultivated, and according to different functions of guard, compliance, hunting ability and the like of various dogs, different tasks such as hunting, grazing, guarding, transportation, rescue and the like were born, and even according to different hunting modes and commands of human, special behaviors such as tracking, pursuing, encirclement, fright flying, recovery and the like were shown (li jing, yue ruin, cheng super, dun dao, schintang plum, li, cao, sword li, etc.. the genetic research progress of the genome and phenotypic characteristics of domestic dogs [ J ]. chinese stockman heterosis, 2020,56(01): 58-65).

The police dog is one of the special bred dogs with police behaviors. Malinu amurensis is the leading police dog species in China, and plays a significant role in various fields of the first line with high excitability, sensitive smell, strong work desire and lasting endurance, especially in the criminal tracing search field (Liujiayi, Wu Rezai. many Aberd's heat seals, Dian \32751, the main behavior of Malinu amurensis has an influence on the tracing training [ J ] animal husbandry and veterinary science and technology information, 2019(04): 27-29). The police dog has the outstanding police performances of good compliance, long excitation, high alertness, sensitive smell, large and violent gallbladder, strong attacking force, high acquisition desire, good bouncing force, strong adaptability and the like, and is also one of the main police dogs for police and army in all countries in the world. The police dog species and Kunming dog species in China are the only police dog species cultured by Kunming police dog base, are the only military and police working dogs with independent intellectual property rights in China, are widely popularized and applied in China and south-east Asia countries, obtain important military, social and economic benefits, and fill the blank that China cannot culture qualified dogs by itself (Li light, Qiang Jing Ning, Song xing, Tang Tree Sheng, Peng Jian, Li Jie, Wan Jiu Sheng. research and review of Kunming dog [ J ]. China working dog 2007 (01): 13-15). Kunming dogs have three color strains in common: the Chinese pennisetum, the grass yellow and the black back, and the Kunming dog has stronger adaptability to plateau climate, severe cold environment and high temperature, and is widely applied to the aspects of border defense patrol, reconnaissance and case solving, drug and explosive detection and the like (Pengsheng country. Kunming dog reviews, China animal husbandry Candidymis Canine society, Nanchang police dog base, 17 th China national Candidae science and technology research institute argument, China animal husbandry Candidymis, Nanchang police dog base, China animal husbandry medical society, 2017: 38-50). They are all good police dogs in world reputation, which provides good materials for studying the molecular genetic mechanism of police dog police behaviors.

In the past decades, a large number of behavior test researches are widely applied to domestic dogs, and the behavior test researches are used for researching the behavior development rule of the dogs and predicting the behavior of adult dogs, essentially by applying certain external stimuli to the dogs, observing the reaction of the dogs to different stimuli, then carrying out statistics on proposed observation items, and finally comparing the difference of each dog.

The acquisition desire is an important factor influencing the training success rate of the dog, and a breeder hopes to clarify the genetic basis of the acquisition behavior for the basis of all training works. Slabebert reported that the inclusion test of 8-week-old puppies showed a high correlation with their likelihood of becoming a police dog after adulthood [ J.M, Slabebert, and, et al, Early prediction of adult polarity dog efficacy-a long dietary study [ J ]. Applied Animal taste Science,1999 ]. Joanna et al, using GWAS, revealed significant Genetic variation of most behavioral traits studied in the Lambda retriever population, identifying behavioral traits significantly associated with chromosome 4 and chromosome 22 sites [ Ilska, Joanna, Haskell, Marie J, Blott, Sarah C, et al.

The gene polymorphism of MAOB (T199C) at the site has influence on canine hunting reflex intensity, and the T allele has obvious influence on hunting reflex and hunting reflex of canine individuals and is a favorable allele for behavior traits of working dogs; and the microsatellite locus polymorphism analysis is also applied, and data show that the gene polymorphism of HTR2C (A840G) may have influence on the appetite reflex and hunting reflex of dogs [ Lidegui. research on the main behavior traits of dogs and related genes [ D ] of Huazhong university of agriculture, 2007 ].

Disclosure of Invention

Based on the above, one of the objects of the present invention is to provide a biomarker for identifying dogs having an act of taking.

The technical scheme for achieving the aim comprises the following steps.

A biomarker for identifying dogs with matched behaviors, comprising SNP locus rs852494264 of HOXC12 gene; and/or SNP site rs850750634 of TLE3 gene; and/or at least one of SNP sites rs8940078, rs24017227, rs9218315, rs9218316, rs24017222, rs9218313, rs24017212, rs851143465, rs851247740, rs24017208, rs24017206, rs24017205, rs24017204, rs8977717, rs8977718, rs24017177, rs24017176 and rs24017173 of LMX1A gene.

In one embodiment, the biomarker for identifying dogs with a picking behavior comprises at least 2, at least 3, at least 4, at least 5, at least 6, etc. or all of the SNP sites rs8940078, rs24017227, rs9218315, rs9218316, rs24017222, rs9218313, rs24017212, rs851143465, rs851247740, rs24017208, rs24017206, rs24017205, rs24017204, rs8977717, rs8977718, rs24017177, rs24017176, rs24017173 of the LMX1A gene.

In one embodiment, the identifying biomarkers with canine behavioral traits comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or 10 of the foregoing rs850750634, rs8940078, rs9218315, rs9218316, rs24017205, rs24017204, rs8977717, rs8977718, rs24017177, rs 24017173.

Also includes at least 1, at least 2, at least 3, or 4 of rs24017222, rs9218313, rs851143465, and rs 24017176.

And/or further comprises at least one, at least 2, at least 3, at least 4, at least 5, or 6 of rs852494264, rs24017227, rs851247740, rs24017206, rs8977717 and rs 24017212.

In one embodiment, the identifying biomarkers with pickup behavior dogs comprises identifying SNP site rs852494264 of HOXC12 gene; and SNP site rs850750634 of TLE3 gene. The invention also aims to provide application of the reagent or the method for detecting the biomarker in preparing a kit for identifying the dog with the act of taking.

It is another object of the present invention to provide a kit for identifying dogs having a picking behavior.

A kit for identifying dogs with a picking behavior comprises a reagent for detecting the biomarker.

In some of these embodiments, the kit is a biochip.

In some embodiments, the method for detecting the above biomarkers includes PCR, fluorescent quantitative PCR, RT-PCR, sequencing method, and liquid chip method.

It is another object of the present invention to provide a method for identifying dogs with a pickup behavior.

A method for identifying the dog with the match behavior includes such steps as detecting the biomarker in specimen, and judging if there is SNP mutation.

In some preferred embodiments, the dog is a police dog.

The invention researches the genetic mechanism of behavior acquisition of domestic dogs by behavior test and whole genome sequencing of two independent dogs. Through the analysis of GEMMA and FST and the experience of the inventor, 20 sites related to the extraction behavior are creatively found, and 3 genes related to the behavior or the neurological disease are contained, so as to distinguish a high-score population from a low-score population. The result shows that the chi-square value of different SNPs of each gene is the same, and suggests that the linkage disequilibrium phenomenon can exist. Some SNPs obtained through screening (such as rs850750634, rs8940078, rs9218315, rs9218316, rs24017205, rs24017204, rs8977717, rs8977718, rs24017177 and rs24017173) have the strongest relation with the identification behaviors, and are suitable for identifying dogs with the identification behaviors, in particular for police dogs.

Drawings

FIG. 1 is a GEMMA Manhattan plot of the acquisition behavior in example 1.

FIG. 2 is a graph of the retrieve behavior GEMMA QQ in example 1.

FIG. 3 is a FST Manhattan plot of the acquisition behavior in example 1.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present invention is further illustrated by the following specific examples, which are not intended to limit the scope of the invention.

Example 1

In the present study, the samples that we tested for the match were 20 Kunming dogs and 20 Malinula horses. The 20 Kunming dogs and 20 Bernoulli dogs were scored according to the police behavioral scoring criteria, which was done in our Kunming police base and their scoring criteria were as follows.

TABLE 1-1 basic information for Kunming dogs and Ardisia magna dogs

Table 1-1Basic Information of Kunming Dog and Belgian Malinois Dog

Tables 1-2 dog ranking behavior test item description and scoring criteria

Table 1-1Description of sub-tests and scoring standard about canine Fetch behavior

We performed sample sequencing on 20 Kunming dogs and 20 Bernoulli canines tested above, with the sampling done at Kunming police dogs.

1.2 extraction of DNA

The sample is whole blood in this research, and in order to guarantee the stability of DNA at the sample collection in-process, the family dog whole blood of gathering adds the heparin sodium blood anticoagulant to place the heparin tube in the liquid nitrogen container and preserve. After sampling is finished and the sample returns to the laboratory, the DNA preservative solution: the ratio of whole blood (1:3) was transferred to a specific sample storage tube for long-term storage at-80 ℃. In the research, a phenol-chloroform-isoamyl alcohol extraction method is adopted to extract the DNA of the whole blood, and the extracted DNA is quantitatively diluted to a proper concentration for a PCR fragment amplification experiment. The specific extraction method is as follows:

firstly, 150ml of whole blood or a small amount of tissue samples (fully sheared or ground), adding 450ul of STE buffer solution (30mM Tris-HCL, 200mM EDTA, 50mM NaCl, pH 8.0) and 75ul of SDS with the final concentration of 10%, mixing, adding 200mg/ml of protease K and 25ul, fully mixing, putting the mixture in a 56 ℃ water bath kettle for digestion for 8-12 hours until clarification, properly increasing the digestion time to ensure that the digestion effect is better, and shaking the mixture for several times.

Adding equal volume of 600-700ul of water saturated phenol or Tris saturated phenol (the pH value of phenol must be close to 8.0 to prevent the DNA from being retained on the interface of the organic phase and the aqueous phase), manually mixing the two phases slowly for 30 minutes or extracting the two phases by rotating the DNA mixer slowly for 24 hours, and carefully mixing the two phases. Centrifuge at 9000rpm/min at room temperature for 10 minutes and carefully transfer the supernatant to another clean Eppendorf tube.

Adding 300ul of phenol, chloroform: isoamyl alcohol (24:1)300ul, slowly mixed and extracted for 30 minutes or slowly rotated on a DNA mixer for 24 hours, centrifuged at 9000rpm/min for 10 minutes, and the supernatant was carefully transferred to another clean Eppendorf tube.

And then 600 + 700ul of chloroform: isopentanol (24:1, chloroform which makes proteins variable and helps to separate the liquid phase from the organic phase; isoamyl alcohol which helps to eliminate foam during extraction) was extracted for 10 minutes or slowly rotated on a DNA mixer for 24 hours, each time, 9000 revolutions/10 minutes of separation centrifugation, and the supernatant was transferred to another clean Eppendorf tube, and the process was repeated twice.

Fifthly, adding isopropanol (600ul) with the same volume for precooling for more than half an hour to precipitate DNA, standing at-20 ℃ for more than 2 hours to precipitate DNA (more overnight), and after centrifugation at 12000 r/min for 10 minutes, carefully removing supernatant to prevent the DNA precipitate at the bottom from being sucked out. 1000ml of 70% ethanol was added and carefully washed with shaking to remove some salts or other components detrimental to DNA dissolution, and after centrifugation at 13000rpm for 10 minutes, the supernatant was discarded and repeated twice.

Sixthly, completely removing 70% of ethanol as far as possible, and placing the DNA precipitate in an open Eppendorf tube at room temperature until visible trace ethanol is completely volatilized (taking care not to completely dry the DNA precipitate, otherwise, the DNA is difficult to dissolve).

Dissolving with appropriate amount of TE (10mM Tris-HCl,1mM EDTA, pH 8.0) buffer solution, dissolving at 37 deg.C, heating at 68 deg.C for inactivation, inactivating DNA degrading enzyme in water bath at 68 deg.C for 10 min after DNA is completely dissolved, and storing at 4 deg.C in refrigerator or long term at-20 deg.C for use.

1.3DNA quantification and dilution

And (3) detecting whether the DNA is degraded or not by using 1% agarose gel electrophoresis of the DNA sample stock solution. 1ul of the DNA stock solution was used for DNA concentration detection by an ultraviolet spectrophotometer, and the DNA sample was diluted to 50-100ng/ul with TE (10mM Tris-HCl,1mM EDTA, pH 8.0) as a working solution for PCR reaction. The stock solution is stored in a refrigerator at-20 deg.C for a long time, and the working solution is stored in a refrigerator at 4 deg.C for use.

1.4 Whole genome sequencing

Whole genome sequencing is carried out on each extracted DNA sample on an Illumina HiSeq4000 platform, and data above 50GB are obtained.

1.5 data processing

First, we align our sequenced original sequences to the reference genome of domestic dogs (version: canfam3.1) using BWA software, using version 0.7.10-r789, which results in binary BAM files, and then operate on the obtained BAM files with PICARD software set (version: 1.87) to remove redundant sequences. We then used the GATK software (version: 2.5-2-gf57256b) to perform local realignment and base quality correction of the sequences, resulting in the final BAM file we need.

Finally, detecting and filtering the whole genome Single Nucleotide Polymorphic Sites (SNPs), processing a BAM file through a UnifiedGenotypeCaller module in a GATK tool set to obtain an original VCF file, combining a domestic dog genome SNPs list published and verified in an Ensembl Database as a reference to perform quality correction on each SNP site in the obtained original VCF file to obtain a preliminarily filtered VCF file, and further filtering the obtained mutant sites. Deletion of insertions/deletions (INDELs) from the obtained VCF file and deletion of those SNPs with data deletion, trialles or near INDELs (not more than 5bp) from the VCF file to obtain a high-quality SNPs dataset.

1.6GWAS analysis

The linear mixture model is a powerful and efficient tool for interpreting population stratification, correlations and other confounding factors in genetic association tests, and we used GEMMA for the univariate linear mixture model GWAS analysis in this study. And respectively selecting a high group and a low group according to the scores of the matching behaviors, and accordingly selecting a corresponding data set from the VCF file, and combining the SNPs information of the Ardisia marnuensis group and the Kunming dog group. The VCF file is converted to BED file by using Plink and Vcftools, the input file for GEMMA is prepared, and Principal Component Analysis (PCA) is performed for controlling the population structure, with the PCA result as covariate.

In the first stage, according to the obtained data set, a genetic matrix is firstly generated by using GEMMA, and the relationship among samples is firstly subjected to sample structure analysis to obtain an output file of the genetic matrix.

In the second phase, the previous data set is also used, and the genetic matrix files obtained in the first step are taken together as input files, subjected to correlation tests and fitted into a univariate linear mixture model. A common test called the Wald test was chosen to test for association of SNPs, as it also takes into account relationships between individuals in the dataset. The top 100 selected sites by GEMMA were picked out as candidate regions.

Using the same data set, FST analysis was performed on each SNPs using Vcftools to examine the difference between high and low packets, and an excessively high FST value may be the cause of the behavioral difference.

And (3) picking out the overlapping part of the first 100 sites selected by GEMMA and the sites of FST, carrying out function annotation on each polymorphic site by ANNOVAR software, finding out genes contained in the sequence, and then further screening out genes related to behavioral or neurological diseases as candidate genes, wherein the picked sites are the candidate sites.

2.1 test of acquisition behavior

For canine police performance in two canine breeds (Kunming dog and Ardisia marnui dog), we focused on the identities described in the methods. In the test of the matching behaviors, scoring is carried out according to the scoring standard of the matching behaviors, high-grade grouping is selected as high-grade grouping, low-grade grouping is selected as low-grade grouping, and the number of the high-grade grouping and the low-grade grouping is equal to the number of the high-grade grouping and the low-grade grouping as much as possible.

TABLE 2-1 Canine behavior Low packet

Table 2-1Dogs'Fetch Behavior Low Group

TABLE 2-2 Canine engagement behavior high grouping

Table 2-1Dogs'Fetch Behavior High Group

2.2 Whole genome sequencing

The sequencing results for these 40 individuals were 17.65 x minimum depth and 27.38 x maximum depth. After final alignment, polymorphic site detection and filtration, we determined 5,776,352 autosomal Single Nucleotide Polymorphisms (SNPs) for further analysis.

2.3.1 chi-square test and FST analysis

We combined the genotypic data for the magnus and Kunming dog populations and artificially divided into two populations of high and low scoring individuals, and we performed GEMMA and FST validation analyses on these two populations in order to reflect the genetic differences between the two populations to the maximum. In the GEMMA results, it was found by annotation that we obtained 9 genes, and the results of the whole genome selection analysis are shown in FIGS. 1 and 2. Among the results of the FST analysis, the results of the whole genome selection analysis are shown in fig. 3.

We validated the first 100 sites in the GEMMA results in the first 5000 sites in the FST results, and we obtained 27 overlapping sites, which contained 4 genes. The inventor further screens the result to obtain genes and loci related to behavior or neurological disease, obtains 20 loci, and has high FST values, wherein 3 genes are related to nerve or behavior, and the gene list is shown in tables 2-3.

As can be seen from the table, p _ wald is less than 10 ^-11 The SNPs are extremely remarkable, which indicates that the strong association with traits at the positions is very likely to cause the acquisition behavior of domestic dogs, and the FST results are used for auxiliary verification, so that the FST values of the SNPs are all above 0.89, which indicates that the SNPs really have strong association with the acquisition behavior.

TABLE 2-3 family Canis engagement behavior Gene List

Example 2 site validation analysis

Then, 96 dogs including labrador, cadion deer dogs, hastelli, horse dogs, golden hair and other dog species are collected from dog houses all over the country; among them 53 labrador, 2 golden hair, 15 cadiperus cervidae, 5 dememu, 8 Fadou, 5 fagoid, 3 standard poonbine, 4 Siberia Hushiqi, 1 Penburoex Checky dog. The registration of character, pedigree, image was detailed, while its identity was scored. The scoring is shown in Table 3-1.

TABLE 3-1 dog breed and match score table

Collecting oral swabs of dogs, performing genotyping by using a 50K chip (manufactured by Tagmeifei company, 23 magic cube company for detection. the method comprises the steps of extracting DNA by using a Jimsanmag bead method oral swab DNA extraction kit, performing amplification reaction, fragmentation, enzyme digestion and other reactions to form a high-concentration fragment library with the fragment size of 25bp-125bp, performing specific binding with a probe on the chip after denaturation, washing out the non-specifically bound DNA, dyeing two different colors on an AT/GC channel, amplifying a site signal of the DNA so as to facilitate site information capture, scanning the dyed FDNA by a high-pixel camera, converting the FDNA into an optical signal, performing data processing according to the default parameters of an afymetrix official APT process), and performing data processing according to actual detection conditions, and further scoring the sample of the SNP marker by adopting the matching behavior phenotype, calculating the correlation and the correlation coefficient between the genotype and the phenotype, verifying the result found by the whole genome research, and obtaining 20 SNP genotypes of each sample by adopting the detection rate of 20 corresponding SNP sites to be detected which is greater than 86% according to the SNP typing experimental result shown in the following table 4-3.

TABLE 4-3 statistical table of SNP site detection rates

The 96 SNP typing experimental results and corresponding identification behaviors are subjected to correlation analysis, a Pearson correlation coefficient (Pearson correlation coefficient) describes the degree of linear correlation strength between two variables, and the correlation coefficient is represented by r. r describes the degree of linear correlation between two variables, 0.8-1.0: very strongly correlated, 0.6-0.8: strongly correlated, 0.4-0.6: moderate correlation, 0.2-0.4: weak correlation, 0.0-0.2: very weak or no correlation, with larger absolute values indicating stronger correlation.

The degree of correlation of the 20 site-specific accessibility behaviors was further verified by using the correlation coefficient, respectively. I count the correlation coefficient of the genotype frequency and the scoring average number of the individuals with 20 loci, and find that the correlation coefficient of rs850750634, rs8940078, rs9218315, rs9218316, rs24017205, rs24017204, rs8977717, rs8977718, rs24017177 and rs24017173 is more than 0.4, and is moderately correlated. rs24017222, rs9218313, rs851143465, rs24017176, rs852494264, rs24017227, rs851247740, rs24017206, rs24017208, rs24017212 are above 0.2, and are weakly correlated.

Therefore, 20 SNP loci in the 3 genes are verified to have correlation with the canine extraction behavior through the Pearson correlation test, and the SNP loci or the combination thereof can be effectively used for identifying the canine with the extraction behavior.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

Claims (12)

1. Use of a reagent or method for detecting biomarkers comprising rs850750634, rs8940078, rs9218315, rs9218316, rs24017205, rs24017204, rs8977717, rs8977718, rs24017177, and rs24017173 in the preparation of a kit for identifying canines with pickup behavior.

2. The use of claim 1, further comprising at least one of rs24017222, rs9218313, rs851143465, rs 24017176.

3. The use of claim 1 or 2, further comprising at least one of rs852494264, rs24017227, rs851247740, rs24017206, rs8977717, rs 24017212.

4. A kit for identifying a dog having a picking behavior, comprising reagents for detecting biomarkers comprising rs850750634, rs8940078, rs9218315, rs9218316, rs24017205, rs24017204, rs8977717, rs8977718, and rs24017177, rs 24017173.

5. The kit for identifying a dog having a picking behavior according to claim 4, further comprising at least one of rs24017222, rs9218313, rs851143465 and rs 24017176.

6. The kit for identifying a dog having a picking behavior according to claim 4 or 5, further comprising at least one of rs852494264, rs24017227, rs851247740, rs24017206, rs8977717, and rs 24017212.

7. The kit for identifying a dog with a picking behavior according to claim 4, wherein the method for detecting the biomarker comprises common PCR, fluorescent quantitative PCR, RT-PCR, sequencing method and liquid chip method.

8. The kit for identifying a dog having a picking behavior according to claim 4, wherein the dog is a police dog.

9. The kit for identifying a dog having an act of taking according to claim 4, wherein said kit is a biochip.

10. A method for identifying a dog with a picking behavior is characterized in that a sample to be tested is subjected to biomarker detection to detect whether SNP mutation exists, wherein the biomarker comprises rs850750634, rs8940078, rs9218315, rs9218316, rs24017205, rs24017204, rs8977717, rs8977718, rs24017177 and rs 24017173.

11. The method of claim 10, further comprising at least one of rs24017222, rs9218313, rs851143465, rs 24017176.

12. The method of claim 10 or 11, further comprising at least one of rs852494264, rs24017227, rs851247740, rs24017206, rs8977717, rs 24017212.

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