A method for local livestock and poultry traceability based on SNP markers

Abstract

The application provides a local livestock and poultry traceability method based on SNP markers, relates to the field of breed traceability, and comprises traceability gene library construction, kinship determination, traceability identification based on genomic data, and traceability identification process and result determination; wherein, the traceability gene library construction comprises source gene library sample collection, sample collection and information recording, gene analysis and data generation, and traceability gene library establishment; the kinship determination comprises repeated individual identification and parent-offspring identification; the traceability identification based on genomic data comprises repeated individual identification based on genomic information and parent-offspring relationship identification based on genomic information; the traceability identification process and result determination comprises sample receiving and quality control, data alignment analysis, and hierarchical result determination. The method realizes accurate traceability of livestock and poultry meat products by obtaining SNP marker information through genomics technology and realizing accurate traceability of livestock and poultry meat products by using kinship analysis, and effectively prevents false and inferior phenomena.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of breed tracing, in particular to a local livestock and poultry tracing method based on SNP markers. BACKGROUND

[0002] With the continuous improvement of the national economy and consumption capacity, consumers have higher requirements for the quality, flavor and traceability of livestock and poultry meat; local livestock and poultry breeds, as China's valuable genetic resources, are favored by the market due to their delicious meat and unique flavor, and have great market development potential and industry development prospects. However, after livestock and poultry are slaughtered and processed, their breed-specific phenotypic characteristics (such as coat color, body shape, etc.) will disappear, making it impossible to directly identify the breed origin of the terminal sold meat products by appearance; this makes it difficult for consumers to effectively identify whether the purchased meat products are the claimed high-quality local breeds, seriously hindering the healthy development of the market for high-quality and high-end local breed meat products. Therefore, establishing a technology that can accurately identify the identity of local breed meat products and ensuring that consumers can purchase authentic and reliable high-quality meat has become a key problem that needs to be solved in the local livestock and poultry industry.

[0003] At present, the mainstream tracing method in the industry relies on information identification systems such as two-dimensional codes, which record and associate information at breeding, slaughtering, circulation and other links to give products an electronic identity card. However, this method highly depends on manual recording and the integrity of the data chain, and has limitations such as many human intervention factors, missing or tampered data, etc., making it difficult to completely eliminate counterfeit and inferior products, and its reliability and credibility are often challenged.

[0004] In recent years, the development of genomics technology has provided a new way to solve the above problems. Based on genome resequencing or gene chip technology for genotyping, SNP (Single Nucleotide Polymorphism) marker information on individual DNA can be obtained, and by comparing the genotype data of marketed livestock and poultry meat products with the source local livestock and poultry breeding farms or breeding farms, and using genotype consistency rate and IBD (Identical By Descent) analysis, the product identity can be accurately identified through the genetic relationship between the marketed meat product individual and the source breeding farm individual; if two samples are determined to be the same independent individual or have a direct parent-child relationship, it can provide strong scientific evidence for the authenticity of high-quality local livestock and poultry breed meat products.

[0005] However, there is currently a lack of an effective SNP marker-based tracing system, making it difficult to scientifically, objectively and accurately trace local livestock and poultry breeds. SUMMARY

[0006] In view of the problems in the prior art, the present application aims to provide a local livestock and poultry traceability method based on SNP markers, which realizes accurate traceability of livestock and poultry meat products by obtaining SNP marker information through genomics technology, calculating genotype consistency rate and distribution probability of IBD fragments between individuals to determine the genetic relationship, thereby constructing a full-chain DNA identity authentication system from the source farm to the terminal market, effectively preventing fake and inferior phenomena, and providing technical support for the integrity and standardized management of local livestock and poultry industry.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] The present application provides a local livestock and poultry traceability method based on SNP markers, comprising the steps of: traceability gene library construction, first-level traceability determination based on genotype consistency rate, second-level traceability determination based on IBD, and final traceability result output;

[0009] The traceability method is automatically determined according to the following hierarchical rules:

[0010] First-level traceability determination: the system first calculates the genotype consistency rate G of the sample to be tested and the individual in the traceability gene library at the effective SNP site of the whole genome concordance , if G concordance > G th , it is determined that the sample to be tested and the individual in the library are repeated individuals, and a repeated individual matching traceability report is generated, and the subsequent second-level traceability determination is not performed; wherein the genotype consistency rate G concordance is the ratio of the number of SNP sites with completely consistent genotypes between the sample to be tested and the individual in the traceability gene library to the total number of common SNP sites in the whole genome detected in both samples, and the G th is a pre-set genotype consistency rate threshold;

[0011] Second-level traceability determination: if the first-level traceability determination is not passed, the system further calculates whether there is an IBD2 proportion of allele sites > IBD th in the library, if there is, it is immediately determined that the sample to be tested is the meat product of the corresponding individual in the library, and a repeated individual matching traceability report is also generated; wherein the IBD2 means that both alleles of the allele site of the sample to be tested and the individual in the traceability gene library are derived from the same source, and the IBD th is a pre-set IBD2 proportion threshold;

[0012] If the IBD2 determination is not passed, the system further scans to determine whether the sample to be tested simultaneously has an IBD1 proportion of allele sites greater than IBD shIBD0 ratio is not higher than 0.1, if any, it is determined that the sample to be tested and the pair of male and female livestock in the database are parent-offspring relationship, and a parent-offspring relationship matching traceability report is generated; wherein the IBD1 means that the sample to be tested and two alleles existing in the individual in the traceability gene database have only one allele with the same source, the IBD0 means that the sample to be tested and all individuals in the traceability gene database have no allele with the same source, and the IBD sh is a pre-set IBD1 ratio threshold value;

[0013] No association determination: if the sample to be tested cannot be associated with the gene database by any of the above rules, it is determined to be a non-product of this variety or system, and a traceability report is generated.

[0014] Preferably, in the secondary traceability determination process, the calculation method of IBD1 and IBD2 ratio is: based on method moment or site mixed information, the observed information of each site with the same state is summarized as the whole genome estimation; the cumulative information of multiplication or logarithmic addition is obtained by independent approximation of all sites, and the estimated value of IBD0, IBD1 and IBD2 is obtained by method moment or likelihood approximation, and then the relationship is judged according to the IBD ratio distribution; the state homology means that two individuals have the same allele.

[0015] More preferably, the specific calculation method of IBD ratio includes:

[0016] Collecting whole genome SNP data of the sample to be tested and individuals in the traceability database to obtain the genotype of each site and calculate the allele frequency of each site; for each site, the joint probability of the genotype of the sample to be tested and the individual in the traceability database at a certain SNP site j is calculated, and the average value of all sites is obtained to obtain the theoretical moment ; then, the theoretical moments , and under different IBD states IBD0, IBD1 and IBD2 are calculated respectively, and the following function equation group is established:

[0017] ;

[0018] In the formula, k0, k1 and k2 respectively represent the proportion of IBD0, IBD1 and IBD2 of two individuals at the genome level, which satisfies the constraint k0, k1, k2≥0 and k0+k1+k2=1; wherein represents the average theoretical moment of all sites, , and represent the theoretical moments under different IBD states IBD0, IBD1 and IBD2, respectively;

[0019] Solve k0, k1, k2 by the matrix equation of different genotypes, and then get the estimated value of IBD0, IBD1, IBD2 proportion.

[0020] Further preferably, the likelihood approximation is performed by EM iteration, and the specific method of obtaining k0, k1, k2 estimation value is as follows:

[0021] For each site j, calculate the corresponding IBD0, IBD1, IBD2 genotype joint probability under the site ; Where s represents the IBD state value, and P 0j , P 1j and P 2j represent the genotype joint probability under the corresponding IBD0, IBD1, IBD2 three IBD states at j site, respectively.

[0022] First, the E step is performed, and the initial value of k0, k1, k2 is assumed; calculate the posterior probability PE sj of the site j belonging to a certain IBD state:

[0023] ;

[0024] Wherein the formula ; s represents the IBD state value, and 0, 1 and 2 represent IBD0, IBD1, IBD2 three IBD states, respectively; k s ; s respectively takes 0, 1 and 2 to get k0, k1, k2 representing IBD0, IBD1, IBD2 three IBD states; ; s respectively takes 0, 1 and 2 to get genotype joint probability P 0j , P 1j and P 2j representing IBD0, IBD1, IBD2 three IBD states; according to the above formula, s respectively takes 0, 1 and 2 to get the corresponding posterior probability PE 0j , PE 1j and PE 2j representing IBD0, IBD1, IBD2 three IBD states;

[0025] Then, the M step is performed, and k0, k1, k2 are updated to the average value of the posterior probability PE sj of all sites, that is

[0026] ;

[0027] Wherein is the average value of the posterior probability PE sj of all sites; N represents the number of sites in the whole genome;

[0028] By With the foregoing mapping relationship of the (k0, k1, k2) vector group, the following is obtained The corresponding k0, k1, k2 values (herein represented as ) are used to replace the original k0, k1, k2 to calculate the E step, and the E step and M step iterations are repeated until the change in k0, k1, k2 is less than a preset threshold, at which point the k0, k1, k2 are the estimated values of the IBD0, IBD1, IBD2 ratios.

[0029] Preferably, the quality control standards adopted in the traceability gene library construction step include: removing low-quality site data with a detection rate < 95%, filtering low-frequency allele data with a low-frequency allele frequency < 0.05, and filtering strong linkage disequilibrium site data with r² > 0.8.

[0030] More preferably, the primary traceability determination based on genotype consistency and the secondary traceability determination based on IBD steps are specifically as follows:

[0031] Repeated individual identification based on genomic information: first, DNA extraction is performed on the to-be-tested sample, and genotyping is performed using the same gene chip platform as when the traceability gene library is constructed to obtain its whole genome SNP data; then, the genotype data of the to-be-tested sample is compared with the genotype data of all registered individuals in the traceability gene library one by one, first, primary traceability determination is performed: the genotype consistency between the to-be-tested sample and each individual in the gene library is calculated: if the genotype of the to-be-tested sample and a certain reference individual in the gene library satisfies G concordance >G th , it is determined that the to-be-tested sample is the meat product corresponding to the individual in the gene library; if G concordance <G th , secondary traceability determination is performed: the IBD proportion between the to-be-tested sample and each individual in the gene library is calculated: if the genotype of the to-be-tested sample and a certain reference individual in the gene library satisfies the proportion of IBD2 is greater than IBD th , it is determined that the to-be-tested sample is the meat product corresponding to the individual in the gene library, realizing direct tracing from the product to the source;

[0032] Parent-offspring relationship identification based on genomic information: first, the genotype data of the to-be-tested sample is obtained, and then the SNP data of the to-be-tested sample and the gene library livestock data are subjected to kinship analysis; if the to-be-tested sample and a pair of male and female livestock individuals in the gene library both exhibit high allele sharing, and the IBD1 proportion with the sire and the dam is greater than IBD sh , it can be determined that the to-be-tested sample is the offspring of the pair of parents.

[0033] More preferably, the traceability identification process and result determination step are specifically as follows:

[0034] Sample receiving and quality control: receiving market samples to be tested for livestock and poultry meat, performing DNA extraction and genotyping, using the same platform as the construction of the traceability gene library for genotyping, and adopting the same quality control standards for the generated genotype data;

[0035] Data comparison and analysis: comparing the SNP data of the quality-controlled test sample with all individual data in the traceability gene library for whole genome comparison, calculating the G concordance , IBD1 and IBD2 proportion of each individual in the library;

[0036] Hierarchical result determination: performing traceability identification and result determination according to the hierarchical rules.

[0037] Most preferably, the preset threshold is selected from one or more of the following criteria, the G th is not less than 95%, the IBD th is not less than 90%, the IBD sh is not less than 80%.

[0038] The following are the technical effects of the present application:

[0039] The present application uses SNP markers for traceability, and realizes identification based on genomic genetic information, avoiding the risk of manual intervention and data tampering, and the results are scientific and objective, difficult to forge, significantly improving the credibility and reliability of traceability; the traceability gene library focuses on parent generation breeding livestock and poultry, without the need for sequencing of commodity generation, significantly reducing the construction and operation cost of the traceability system, and having industrial application feasibility; through the two-level determination rules of repeated individuals and parent-child relationship, the combination of direct traceability and indirect traceability is realized, covering the traceability needs in different scenarios, with high identification accuracy, the present application is more accurate through the traceability method of repeated individuals with genotype consistency rate and IBD2 proportion, and more cost-saving through the indirect traceability method of parent-child relationship.

[0040] The present application constructs a full-chain DNA identity authentication system from the source farm to the terminal market, providing consumers with breed authenticity protection, helping the development of local livestock and poultry industry in a honest and standardized manner, and enhancing market consumer confidence. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The flowchart of local livestock and poultry traceability in the embodiment of the present application.

[0042] Figure 2 The genotype consistency rate distribution graph of repeated individual identification in the embodiment of the present application.

[0043] Figure 3 The IBD2 proportion distribution graph of repeated individual identification in the embodiment of the present application.

[0044] Figure 4 IBD1 proportion distribution map for parentage identification in the embodiments of the present application. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments.

[0046] Embodiment 1:

[0047] A local livestock and poultry tracing method based on SNP markers, comprising a tracing gene library construction, a kinship determination, a tracing identification based on genomic data, and a tracing identification process and result determination;

[0048] The tracing gene library construction specifically comprises (taking pigs as an example):

[0049] Source gene library sample collection: the sampling objects are parent generation breeding livestock and poultry of a local pig source core breeding population (i.e. parent generation breeding livestock and poultry for producing commodity generation meat livestock and poultry); for example: 1000 parent generation breeding pigs of a core breeding population of a certain local pig breed are selected as sampling objects;

[0050] Sample collection and information recording: biological tissue samples (such as ear tissue samples) of each breeding livestock and poultry are systematically collected, and key identity information such as individual number, gender, breed, etc. is accurately recorded synchronously to ensure that the sample and the individual information are one-to-one corresponding and unique; for example: ear tissue samples of each breeding livestock and poultry are collected by using an ear tissue sampler, numbered as S1-S1000, and the gender (300 boars, 700 sows), breed name and breeding batch information of each breeding pig are recorded synchronously;

[0051] Gene analysis and data generation: high-quality whole genome DNA is extracted from the collected samples, and whole genome resequencing or pig-specific gene chip is used for genotyping; strict quality control is performed on the original data (i.e. sequencing data or chip data), low-quality sites are removed, low-frequency alleles (MAF) and strong linkage disequilibrium (LD) or abnormal sites are filtered according to the set threshold, and high-quality SNP genotype data covering the whole genome are obtained; for example: whole genome DNA is extracted from the ear tissue samples by using the conventional phenol-chloroform method, and local pig 66K breeding gene chip is used for genotyping; the original data is quality controlled: low-quality sites with detection rate <95% are removed, low-frequency alleles with MAF <0.05 are filtered, strong LD sites (r²>0.8) are filtered by using PLINK software, and high-quality SNPs obtained by quality control are included in subsequent analysis;

[0052] Establishment of traceability gene library: integrate all genotype data of pig breeders passing quality control and corresponding identity information, construct a local pig breed-specific traceability gene library as an authoritative reference database for subsequent product traceability comparison; for example, enter the SNP genotype data of 1000 pig breeders and the corresponding identity information (number, gender, breed, breeding batch) into the database management system to construct a local pig breed-specific traceability gene library.

[0053] The application provides a local livestock and poultry traceability method based on SNP markers, which comprises the following steps: traceability gene library construction, first-level traceability determination based on genotype consistency rate, second-level traceability determination based on IBD, and final traceability result output.

[0054] The application adopts a layer-by-layer determination strategy of first-level traceability determination and second-level traceability determination. First-level traceability determination: the genotype consistency rate (G concordance ) of the sample to be tested and the individual in the traceability gene library at the effective SNP site of the whole genome is calculated. concordance If G th exceeds the preset identity threshold G th , it is determined that the sample to be tested and the individual in the library are repeated individuals (i.e. derived from the same livestock and poultry), and subsequent IBD calculation is not performed. Second-level traceability determination (IBD review and parentage identification): if the first-level traceability determination is not passed, the IBD coefficient (k0, k1, k2) of the sample to be tested and the individual in the library is calculated using the genomic homologous ancestor (IBD) algorithm. If the IBD2 proportion (k2) > IBD th , it is still determined as a repeated individual (this case is suitable for the case that the sample is degraded, resulting in a slightly lower consistency rate than G sh , but still has a high genetic identity). If the IBD1 proportion (k1) of the sample to be tested and a pair of male and female livestock and poultry in the library is both > IBD th , and the IBD0 proportion is not higher than 0.1, it is determined that the individual and the two individuals in the gene library are in a parent-child relationship, and the traceability of livestock and poultry is indirectly realized through the parent-child relationship.

[0055] The identification standard of the second-level traceability determination is that the IBD2 proportion of the sample to be tested and the individual in the traceability gene library is > IBD th .

[0056] The second-level traceability determination also includes traceability determination based on parentage. This case is suitable for the case that commodity livestock and poultry do not retain genotype data in the livestock and poultry gene library, and need to be traced through the identification of the parentage between their parents. This can save costs to a certain extent and provide more flexible options for the traceability of livestock and poultry. The identification standard is that the IBD1 proportion of the sample to be tested and a pair of parent individuals in the traceability gene library is both > IBD sh .

[0057] In the present application, the genotype consistency rate refers to the ratio of the number of SNP sites with complete genotype consistency between the sample to be tested and the individuals in the traceability gene library to the total number of common SNP sites in the whole genome effective genotype detected in both samples; the G th refers to the pre-set genotype consistency rate threshold. The IBD2 refers to that both alleles of the allele site in the sample to be tested and the individuals in the traceability gene library are derived from the same source; the IBD th refers to the pre-set IBD2 proportion threshold. The IBD1 refers to that there is only one allele derived from the same source in the two alleles existing in the sample to be tested and the individuals in the traceability gene library, and the IBD0 refers to that there is no allele derived from the same source in the sample to be tested and all individuals in the traceability gene library; the IBD sh refers to the pre-set IBD1 proportion threshold.

[0058] In the primary traceability determination, the genotype consistency rate calculation formula is:

[0059]

[0060] In the formula, N match is the number of sites with complete genotype consistency (such as AA vs. AA, AB vs. AB, and BB vs. BB) between the two samples; N common is the total number of common sites (i.e., intersection site number) of the effective genotype detected in both samples. Threshold setting: preferably, the genotype consistency rate threshold G th is set to ≥ 95.0%. This threshold is set based on the repeatability error rate of livestock and poultry gene chip technology, allowing a small amount of typing error or somatic mutation.

[0061] The kinship determination utilizes the proportion of genomic homologous common ancestor fragments (IBD) to accurately identify the kinship between the sample to be tested and the individuals in the traceability gene library, specifically including:

[0062] Duplicate individual identification: a duplicate individual refers to two samples derived from the same individual, and their genomes are almost completely identical; in a diploid organism, all SNP sites of the same organism are completely consistent in the alleles of the two chromosomes, which is reflected as a very high IBD2 proportion at the genomic level; when the IBD2 proportion of the sample to be tested and a reference individual in the gene library is > IBD th (e.g., IBD th is 90%), it is determined that the two are the same individual; by threshold IBD th , the misjudgment caused by genotyping error or somatic mutation can be effectively avoided.

[0063] Parentage test: the parentage relationship is vertical genetic relationship, offspring inherit half of the genome from each parent, so the offspring and the father (or mother) have the same allele at any genetic locus, which is reflected as a high proportion of IBD1 sharing at the genome level; when the IBD1 proportion of the test sample and the parent pair in the gene library is greater than IBD sh (For example, IBD sh is 80%) when the test sample is determined to be the offspring of the parent pair.

[0064] In the process of determining the relationship, the calculation method of IBD proportion is: based on the method moment or site mixed information (sample allele frequency and genotype combination frequency of each pair of individuals at each site), the IBS (identity-by-state) observation information of each site is summarized to the whole genome estimation; when the cumulative information (multiplication or logarithmic addition) of all sites is independently approximated, the estimated values of IBD0, IBD1 and IBD2 are obtained by method moment or likelihood approximation, so as to determine the relationship according to the IBD proportion distribution;

[0065] Among them, the method moment (Method of Moments, MoM) is used to obtain the estimated values of IBD0, IBD1 and IBD2, which are as follows:

[0066] First, the whole genome SNP data of two individuals are quality controlled (low-quality sites are filtered), the genotype (g1, g2) of each site is obtained, and the allele frequency of each site is calculated; for each site, the observed genotype joint probability of two individuals at a SNP site is calculated, and the average value of all sites is taken to obtain the average theoretical moment of all sites ; then, the theoretical moments of different IBD states IBD0, IBD1 and IBD2 are calculated , and respectively, and the following function equation group is established:

[0067] ;

[0068] In the formula, k0, k1 and k2 represent the proportion of IBD states IBD0, IBD1 and IBD2 of two individuals at the genome level, respectively, which satisfy the constraints k0, k1 and k2≥0 and k0+k1+k2=1; wherein represents the average theoretical moment of all sites, , and represent the theoretical moments of different IBD states IBD0, IBD1 and IBD2 respectively;

[0069] Solve k0, k1, k2 by matrix equation of different genotypes, and then get the estimated value of IBD0, IBD1, IBD2 proportion.

[0070] Further preferably, the likelihood approximation is performed by EM iteration, and the specific method of obtaining k0, k1, k2 estimation value is as follows:

[0071] For each site j, calculate the corresponding IBD0, IBD1, IBD2 genotype joint probability under the site ; Where s represents the IBD state value, and P 0j , P 1j and P 2j represent the genotype joint probability under the corresponding IBD0, IBD1, IBD2 three IBD states at j site.

[0072] First, the E step is performed, and the initial value of k0, k1, k2 is assumed; Calculate the posterior probability PE sj of the site j belonging to a certain IBD state:

[0073] ;

[0074] Wherein the formula ; s represents the IBD state value, respectively 0, 1 and 2 represent IBD0, IBD1, IBD2 three IBD states; k s , k1 and k2 are respectively taken as 0, 1 and 2 to get k0, k1 and k2 representing IBD0, IBD1, IBD2 three IBD states; , k1 and k2 are respectively taken as 0, 1 and 2 to get k0, k1 and k2 representing IBD0, IBD1, IBD2 three IBD states; 0j , P 1j and P 2j ; According to the above formula, P 0j , P 1j and P 2j representing the corresponding posterior probability PE sj of IBD0, IBD1, IBD2 three IBD states are obtained after s is respectively taken as 0, 1 and 2;

[0075] Then, the M step is performed, and k0, k1, k2 are updated to the average value of the posterior probability PE sj of all sites:

[0076] ;

[0077] Wherein is the average value of the posterior probability PE sj of all sites; N represents the number of sites in the whole genome;

[0078] By The mapping relationship with the (k0, k1, k2) vector group is obtained The corresponding k0, k1, k2 values are respectively represented as , instead of the original k0, k1, k2, calculate the E step, and repeat the E step and M step iterations until the change of k0, k1, k2 is less than the preset threshold, and the k0, k1, k2 at this time is the estimated value of the IBD0, IBD1, IBD2 ratio.

[0079] The traceability identification based on genomic data specifically includes:

[0080] Repeat individual identification based on genomic information: first, DNA extraction is performed on the sample to be tested, and the same gene chip platform as when constructing the traceability gene library is used for genotyping to obtain its whole genome SNP data; then, the genotype data of the sample to be tested is compared with the genotype data of all registered individuals in the traceability gene library one by one, first-level traceability determination is performed: the genotype consistency rate between the sample to be tested and each individual in the gene library is calculated: if the genotype of the sample to be tested and a certain reference individual in the gene library satisfies G concordance >G th , it is determined that the sample to be tested is the meat product corresponding to the individual in the gene library; if G concordance <G th , secondary traceability determination is performed: the IBD ratio between the sample to be tested and each individual in the gene library is calculated: if the genotype of the sample to be tested and a certain reference individual in the gene library satisfies IBD2 The proportion is greater than IBD th , it is also determined that the sample to be tested is the meat product corresponding to the individual in the gene library, realizing direct tracing from the product to the source;

[0081] Parent-offspring relationship identification based on genomic information: first, the genotype data of the sample to be tested is obtained, and then the SNP data of the sample to be tested and the gene library livestock data are analyzed for genetic relationship; if the sample to be tested and a pair of male and female livestock individuals in the gene library both show high allele sharing, and the IBD1 proportion with the sire and the dam is greater than IBD sh , and the IBD0 proportion is not higher than 0.1, it is determined that the sample to be tested is the offspring of the pair of parents.

[0082] The traceability identification process and result determination are specifically:

[0083] Sample receiving and quality control: receiving market samples of livestock and poultry meat to be tested, performing DNA extraction and genotyping, using the same platform gene chip or resequencing for genotyping as when constructing the traceability gene library; and the same quality control standard is adopted for the generated genotype data;

[0084] Data comparison analysis: After quality control, the SNP data of the test sample is compared with all individual data in the traceability gene library for whole genome comparison, and the genotype consistency rate, IBD1 and IBD2 proportion of each individual in the library are calculated;

[0085] Hierarchical result determination: According to the hierarchical rules, the traceability identification and result determination are carried out:

[0086] Primary traceability determination: the system first calculates the genotype consistency rate of the test sample and the individual in the traceability gene library at the effective SNP site of the whole genome, and if G concordance >G th , it is determined that the test sample and the individual in the library are duplicate individuals, and a traceability report of duplicate individual matching is generated, and no subsequent secondary traceability determination is performed;

[0087] Secondary traceability determination: if the primary traceability determination is not passed, the system calculates whether there is an IBD2 proportion>IBD th of the individual in the library, if there is, it is immediately determined that the test sample is the meat product corresponding to the individual in the library, and a traceability report of duplicate individual matching is also generated;

[0088] If the IBD2 determination is not passed, the system further scans to determine whether the test sample is simultaneously with the IBD1 proportion of the allele site of a pair of male and female livestock and poultry in the library is greater than IBD sh , and the IBD0 proportion is not higher than 0.1, if there is, it is determined that the test sample and the pair of male and female livestock and poultry in the library are parent-child relationship, and a traceability report of parent-child relationship matching is generated;

[0089] Irrelevant determination: if the test sample cannot be associated with the gene library by any of the above rules, it is determined to be a non-product of this variety or system, and a traceability report is generated.

[0090] Finally, the system automatically generates an electronic traceability report containing the following core contents, including:

[0091] a. Test sample number and detection date, determination result (duplicate individual / parent-child relationship / irrelevant);

[0092] b. Matching source field pig individual number;

[0093] c. Key genetic evidence (such as genotype consistency rate, IBD proportion value, etc.).

[0094] The report provides intuitive and authoritative scientific basis for market supervision, enterprise self-evidence and consumer inquiry, thereby completing the whole chain DNA identity authentication from market products to source population.

Claims

1. A method for local livestock and poultry traceability based on SNP markers, characterized in that The method comprises the steps of: constructing a gene library, determining the first level of traceability based on genotype consistency, determining the second level of traceability based on IBD, and outputting the final traceability result; the traceability method automatically determines according to the following hierarchical rules: first level of traceability determination: the system first calculates the genotype consistency G of the sample to be tested and the individual in the traceability gene library at the effective SNP site of the whole genome concordance , if G concordance > G th , it is determined that the sample to be tested and the individual in the library are repeated individuals, and a repeated individual matching traceability report is generated, and no subsequent second level of traceability determination is performed; wherein the genotype consistency G concordance refers to the ratio of the number of SNP sites with completely consistent genotypes between the sample to be tested and the individual in the traceability gene library to the total number of common SNP sites in the whole genome genotype detected in both samples; the G th refers to a pre-set genotype consistency threshold; second level of traceability determination: if the first level of traceability determination is not passed, the system calculates whether there is an IBD2 proportion of allele sites > IBD th of the individual in the library, if there is, it is immediately determined that the sample to be tested is the meat product corresponding to the individual in the library, and a repeated individual matching traceability report is also generated; wherein the IBD2 refers to that the two alleles of the allele site of the sample to be tested and the individual in the traceability gene library are from the same source, and the IBD th refers to a pre-set IBD2 proportion threshold; if the IBD2 determination is not passed, the system further scans to determine whether the sample to be tested is simultaneously with a pair of male and female livestock and poultry in the library IBD1 proportion of allele sites is greater than IBD sh , and the IBD0 proportion is not higher than 0.1, if there is, it is determined that the sample to be tested and the pair of male and female livestock and poultry in the library are parent-child relationship, and a parent-child relationship matching traceability report is generated; wherein the IBD1 refers to that the two alleles of the sample to be tested and the individual in the traceability gene library have and only have one allele from the same source, the IBD0 refers to that the sample to be tested has no allele from the same source with all individuals in the traceability gene library, and the IBD sh refers to a pre-set IBD1 proportion threshold; unrelated determination: if the sample to be tested cannot be associated with the gene library by any of the above rules, it is determined to be a non-product of the breed or the system, and a traceability report is generated.

2. The SNP marker based method for local livestock and poultry traceability according to claim 1, characterized in that: In the secondary traceability determination process, the calculation method of the IBD1 and IBD2 proportion is as follows: based on the method moment or locus mixed information, the observation information of the state homology of each locus is summarized as a whole genome estimation; the cumulative information of the multiplication or logarithmic addition of all loci independent approximation is multiplied or logarithmically added, the estimation value of IBD0, IBD1 and IBD2 is obtained through the method moment or likelihood approximation, and then traceability is performed according to the IBD proportion distribution; the state homology refers to two individuals having the same allele.

3. The SNP marker based method for local livestock and poultry traceability according to claim 2, characterized in that: The specific calculation method of the IBD estimation value comprises: collecting whole genome SNP data of the individual in the traceable database and the sample to be tested, obtaining the genotype of each site, and calculating the allele frequency of each site; for each site, the joint probability of the genotype of the individual in the traceable database and the sample to be tested at a certain SNP site is calculated, and the average value of all sites is taken to obtain the theoretical moment of all sites ; then, the theoretical moments , and under different IBD states IBD0, IBD1 and IBD2 are calculated respectively, and the following function equation group is established: ; in the formula, k0, k1 and k2 respectively represent the proportion of the IBD states IBD0, IBD1 and IBD2 of the two individuals at the genome level, and satisfy the constraints k0, k1 and k2≥0 and k0+k1+k2=1; wherein represents the average theoretical moment of all sites, , and represent the theoretical moments under different IBD states IBD0, IBD1 and IBD2 respectively; the estimation values of the proportions of IBD0, IBD1 and IBD2 are obtained by solving k0, k1 and k2 through the moment equation of different genotype combinations.

4. The SNP marker based method for local livestock and poultry traceability according to claim 3, characterized in that: The likelihood approximation is obtained by EM iteration, and the specific method for obtaining the k0, k1, k2 estimation value is as follows: for each site j, the corresponding IBD0, IBD1, IBD2 genotype joint probability of the site is calculated ; wherein s represents the IBD state value, and P 0j , P 1j and P 2j obtained after taking 0, 1 and 2 respectively represent the genotype joint probability under the corresponding IBD0, IBD1, IBD2 three IBD states at the j site; first, the E step is performed, and the initial value is assumed for k0, k1, k2; Calculate the posterior probability PE of site j belonging to a certain IBD state. sj : In the formula ;s represents the IBD state value, with 0, 1, and 2 representing IBD0, IBD1, and IBD2 respectively;k s By taking 0, 1 and 2 respectively, we can obtain k0, k1 and k2 representing the three IBD states of IBD0, IBD1 and IBD2; Taking values ​​of 0, 1, and 2 for s respectively yields the combined genotype probability P representing the three IBD states: IBD0, IBD1, and IBD2. 0j P 1j and P 2j The formula above calculates s, where s takes the values ​​0, 1, and 2 to represent the posterior probabilities PE corresponding to the three IBD states: IBD0, IBD1, and IBD2. 0j PE 1j and PE 2j Then, perform M steps to update k0, k1, and k2 to the posterior probabilities PE of all loci. sj Average value: ;in For the posterior probability PE of all sites sj The average value is calculated; N represents the number of loci in the entire genome; through The mapping relationship with the vector group (k0, k1, k2) is obtained. The corresponding k0, k1, and k2 values ​​are represented as follows: The original k0, k1, and k2 are replaced to calculate the E-step. The E-step and M-step iterations are repeated until the changes in k0, k1, and k2 are less than the preset threshold. At this point, k0, k1, and k2 are the estimated values ​​of the proportions of IBD0, IBD1, and IBD2.

5. The SNP marker based method for local livestock and poultry traceability according to any one of claims 1-4, characterized in that: The quality control standards adopted in the construction step of the traceability gene library include: removing low-quality locus data with a detection rate of less than 95%, filtering low-frequency allele data with a low-frequency allele frequency of less than 0.05, and filtering strong linkage disequilibrium locus data with r2 greater than 0.

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6. The SNP marker based method for local livestock and poultry traceability according to claim 5, characterized in that: The first-level traceability determination based on genotype consistency rate and the second-level traceability determination based on IBD are specifically as follows: repeated individual identification based on genomic information: first, DNA extraction is performed on the sample to be tested, and the same gene chip platform as that used in the construction of the traceability gene library is used for genotyping to obtain the whole genome SNP data; then, the genotype data of the sample to be tested is compared with the genotype data of all registered individuals in the traceability gene library one by one, first-level traceability determination is performed: the genotype consistency rate between the sample to be tested and each individual in the gene library is calculated: if the genotype of the sample to be tested and a reference individual in the gene library meets G concordance > th , the sample to be tested is determined to be the meat product corresponding to the individual in the gene library; if G concordance th , second-level traceability determination is performed: the IBD proportion between the sample to be tested and each individual in the gene library is calculated: if the genotype of the sample to be tested and a reference individual in the gene library meets the proportion of IBD2 greater than IBD th , the sample to be tested is also determined to be the meat product corresponding to the individual in the gene library, and direct traceability from the product to the source is realized; parent-offspring relationship identification based on genomic information: first, the genotype data of the sample to be tested is obtained, and then the SNP data of the sample to be tested and the data of the breeding stock in the gene library are analyzed for kinship; if the sample to be tested and a pair of male and female livestock individuals in the gene library both show high allele sharing, and the IBD1 proportion with the sire and the dam is greater than IBD sh , and the IBD0 proportion is not higher than 0.1, it is determined that the sample to be tested is the offspring of the pair of parents.​ 7. The SNP marker based method for local livestock and poultry traceability according to claim 6, characterized in that: The traceability identification process and the result determination step are as follows: sample receiving and quality control: receiving market samples to be tested for livestock and poultry meat, performing DNA extraction and genotyping, and using the same platform as the traceability gene library to perform genotyping; and the same quality control standards are adopted for the generated genotype data; data comparison and analysis: comparing and analyzing the SNP data of the quality-controlled sample to be tested with all individual data in the traceability gene library for whole genome comparison, calculating the genotype consistency rate, IBD1 and IBD2 proportion with each individual in the library; hierarchical result determination: performing traceability identification and result determination according to the hierarchical rules.

8. The SNP marker based method for local livestock and poultry traceability according to claim 7, characterized in that: The pre-set threshold is selected from one or more of the following criteria, the G th not less than 95%, the IBD th not less than 90%, the IBD sh not less than 80%.

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