CN107868830B

CN107868830B - SNP locus for canine strain identification

Info

Publication number: CN107868830B
Application number: CN201711109552.6A
Authority: CN
Inventors: 刘毓文; 杨承; 耿灿
Original assignee: Shenzhen Bioinformagics Technology Co ltd
Current assignee: Shenzhen Bioinformagics Technology Co ltd
Priority date: 2017-11-11
Filing date: 2017-11-11
Publication date: 2020-12-11
Anticipated expiration: 2037-11-11
Also published as: CN107868830A

Abstract

The invention discloses a set of SNP loci for canine strain identification, which comprise the SNP loci shown in Table 2. The invention also discloses a chip for identifying the canine strains, which is used for measuring the alleles of the SNP loci given in the table 2.

Description

SNP locus for canine strain identification

Technical Field

The invention belongs to the field of gene detection, and particularly relates to a set of SNP loci for canine strain identification.

Background

Methods for canine genetic line identification are currently mainly divided into three major categories. The first type: the strain of the dog is judged according to the family information of the dog recorded on the record. The second type: and (5) judging the strain through the appearance characters of the dogs. In the third category: the strain is judged by DNA microsatellite loci (STR).

The technical disadvantages of the three types of products are as follows: the first type: 1) the dogs are required to keep systematic pedigree files all the time, and if the files are lost or tampered (a means is often used by a bad pet vendor to cheat consumers), the judgment of the pet line will be wrong. 2) Most of the mixed pet dogs which circulate in the market or are bred by pet owners have no systematic pedigree files, so that the strain composition cannot be accurately judged at all. The second type: 1) it is difficult to distinguish the breed of dogs with close breeds by the pet appearance. 2) The judgment is very subjective and cannot provide objective evidence support. 3) It is impossible to quantitatively determine how high the purity of the strain is. 4) If the variety is a blood-mixed variety, the composition of the variety cannot be accurately judged from the appearance. In the third category: 1) because of the small number of STR marker loci, accurate determinations rely on a vast number of canine databases. 2) The experimental process is difficult to be conveniently compatible with a second-generation sequencing platform, and cannot be effectively combined with other disease detection and character prediction based on second-generation sequencing DNA into an integrated experimental method. 3) Because the number of STR marker sites is small and the information content is limited, the method has acceptable resolution ratio for pure pedigrees, but can not accurately judge multi-breed mixed dogs.

Disclosure of Invention

We developed a computer algorithm to select the most representative 500 sites from 23 ten thousand DNA SNP markers of the canine, and the DNA data of the 500 sites is used to judge the strain composition of the canine.

Thus, in a first aspect, the present invention provides a set of SNP sites for use in canine strain identification, including the SNP sites set forth in table 2.

In one embodiment, the SNP sites include the SNP sites set forth in table 1.

In one embodiment, the canine lines include mini-cedirei, chrysis, penbroglie welshish and frizzled dogs.

Compared with the prior scheme I and the scheme II, DNA information in saliva or blood is used for strain judgment. Compared with the third scheme, the SNP (single-site nucleotide polymorphism) which can be conveniently detected by using a second-generation sequencing platform is used. We used fewer sites than scheme four.

Drawings

The invention is illustrated by the following figures

FIG. 1 shows the identification results of mini-SNPs, with the left column for analysis using only 500 SNP sites and the right column for analysis using 23 ten thousand sites in the whole genome.

FIG. 2 shows the results of the identification of the Cochly, the left column shows the analysis using only 500 SNP sites, and the right column shows the analysis using 23 ten thousand sites in the whole genome.

FIG. 3 shows the identification results of Chekyo penbutox Verlag, the left column is an analysis using only 500 SNP sites, and the right column is an analysis using 23 ten thousand sites in the whole genome.

FIG. 4 shows the results of identification of frizzled male dogs, with the analysis performed only at 500 SNP sites on the left column and the analysis performed at 23 ten thousand sites on the whole genome on the right column.

Detailed Description

Compared with the prior scheme I, the strain judgment is carried out by 1) DNA information in saliva or blood, the DNA of the dogs can be extracted from the dogs at any time, and the risk that the strain judgment cannot be carried out due to the loss of pedigree literature is avoided. 2) The DNA reading information is scientific and objective, and can be operated by a dog purchaser through a third-party platform, so that the possibility that a dog vendor falsify pedigree information to deceive a consumer is prevented. 3) The strain composition of the mixed-blood strain dogs without family tree information can also be judged. Compared with the second scheme, the strain identification based on the DNA data is scientific, accurate and objective. Although the appearance of similar canines is close, the difference in DNA level can be accurately judged by our algorithm. The algorithm can also accurately judge the components of each strain of the mixed-blood dogs. Compared with the third scheme in the prior art, the method uses SNP (single-site nucleotide polymorphism) which can be conveniently detected by using a second-generation sequencing platform, and simultaneously, the number of sites analyzed by the algorithm is far higher than STR (short tandem repeat), so that the accurate strain identification can be only carried out on the mixed-blood dogs. Compared with the prior scheme four, the method has the advantages that the number of used sites is small, and meanwhile, accurate judgment can be performed on the strain, so that the strain can be identified with low cost.

We developed statistical algorithms to select 500 canine SNP sites. The lines of the pets were identified using our analytical software system.

In the present invention, the canine genome was taken from CanFam3.1. chromosome numbers and SNP sites are given (see Table 1).

TABLE 1

1: ID, this ID is the code number of the canine SNP locus; 2: a chromosome; 3: position, the number representing the position on the chromosome; 4: a chrysosporium extract; 5: mini-sinarel; 6: bear; 7: a chryso group; two characters in 4-7 represent the genotype at each row site, and a "0" indicates that the site information does not meet the quality control criteria and is not used in the analysis. The chip platform has some sites which do not reach the standard, but the accurate judgment result is not greatly influenced as long as the proportion is not large.

The inventor screens 23 ten thousand DNA SNP marker information of the dogs to obtain the 500 SNP loci, and judges the strain composition of the dogs according to the canine DNA data of the 500 SNP loci.

Traditional methods of selection for ancestral informative-locus markers are based on the genetic differentiation coefficient Fst. This method is based on the Harvard Winberg equilibrium model, which requires computing statistics between each pair of lines for each locus, and then using the results of each pair of lines together. This model assumption cannot be established for a large number of non-naturally occurring lines (more than 200 artificial lines). However, the pure line analysis is considered as a classification problem in machine learning, and the feasibility and the representativeness of the known classification problem feature selection method are examined and measured by using a non-model method of the machine learning. Because a feature selection scheme under various parameters needs to be considered, the SVM and the CNN with higher operation speed relative to a grade mixed model are used as judgment standards of the feature selection method.

Table 2 (SNP sites distinguishing Mini Xunaire, Keli, Penburopex Verticillatus Kekiky and Pierce canis)

The SNP locus of the invention can be detected by a chip-based method, namely, a chip for identifying canine strains is designed, and the chip is used for measuring the allele of the SNP locus given in Table 2. Optionally, the chip is used to measure the alleles of the SNP sites given in Table 1. Also preferably, the canine lines include mini-cedirary, chrysis, penbroglie welshiuki and majestic male dogs. Still more preferably, the chip is an Illumina chip.

Examples of the experiments

1. Alleles of 23 ten thousand SNP sites were measured using a chip (microarray) at the 230K locus of Illumina canines.

1) Extracting a DNA sample from blood of each of mini-Xuenariy, Chery, Penburoe Welsh Checky dog and Cetrad;

2) the specific steps of the genotyping assay were as follows:

a. preparation of denatured single-stranded DNA: denaturing a DNA sample into a single strand by using sodium hydroxide, neutralizing a denaturant, and adding an enzyme amplification reaction solution;

b. whole genome amplification: putting the sample in the previous step into a 37-degree incubator for whole genome amplification, and reacting for 20-24 hours at 37 degrees;

c. amplified genome fragmentation: the amplified product is cut into segments with the size of hundreds of basic groups by enzyme;

d. and (3) precipitating DNA: adding isopropanol into the product after enzyme digestion, centrifuging for 20 minutes at 3000g to precipitate DNA, and drying for one hour at room temperature;

e. DNA dissolution: adding hybridization solution, carrying out vortex oscillation at 48 ℃ for 1 hour to fully dissolve the DNA in the hybridization solution;

f. spotting chips, DNA hybridization with chips: denaturing the DNA at 95 ℃ for 20 minutes, cooling to room temperature, starting the chip, and putting the spotted chip in a 48-DEG hybridization furnace for 16-24 hours, wherein the time is not more than 24 hours, taking care to avoid cross contamination among different samples;

g. cleaning the chip: washing off DNA which is not hybridized or incompletely hybridized on the chip, and only DNA which is completely matched with the chip can be remained on the chip;

h. single base extension and staining: using DNA hybridized to the chip as a template to carry out single base extension, wherein the extended base is pre-modified and can be combined with a dye, and different bases can be determined by corresponding dye colors;

i. cleaning a chip, coating and fixing: washing off redundant dye on the chip, and adding a fixing solution to fix a signal;

j. scanning the chip: placing the fixed chip into a chip groove of a HiScan scanner for scanning to obtain signals, wherein the scanning result can be further analyzed in software provided by Illumina corporation;

3) and (5) analyzing the genotyping result. Results of scans of the HiScan typing system were analyzed using Illumina GenomeStudio software. Clustering was performed based on the results of dye color upon single base extension, and the genotypes of the materials were classified into 3 classes (AA, BB, AB) based on the results of clustering.

2. We used only the 500 site information in Table 1 above to accurately perform line identification, and repeat the procedure in Table 1.

The experimental results are as follows:

the following results demonstrate that we used 500 SNPs to identify canine breeds very accurately, consistent with the results using all 23 ten thousand SNPs. In fig. 1-4 below, we project the canine information onto a two-dimensional plane, with the shaded portion representing the information for all of the pure dogs in the database of pure dogs. The star represents the position of the sample to be measured on the plane. And if the star is in the middle of the shadow, the corresponding information indicates that the sample to be detected is pure. The left column is the analysis only using 500 SNP loci, and the right column is the analysis made by 23 ten thousand loci in the whole genome, and the results are completely consistent and are consistent with the known information of the known dog to be detected.

The inventors found that the SNP sites in Table 2 were sufficient to distinguish mini-Xuerairy, Cheley, Penburopex Verlag and Cetraria frigida.

While the invention has been described in connection with preferred embodiments, it should be understood that the scope of the invention is not limited to the embodiments described herein. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

The application of SNP in strain identification of dogs, wherein the SNP consists of 500 SNP sites given in Table 1, and the dogs are mini-Xuenirah dogs, collies, Penbubrone Welsh Chekyo dogs and Taber-Februt dogs.