Local livestock and poultry tracing method based on SNP (Single Nucleotide Polymorphism) marker

Abstract

The invention provides a local livestock and poultry traceability method based on an SNP marker, and relates to the field of variety traceability, and the method comprises the steps of traceability gene bank construction, genetic relationship determination, traceability identification based on genome data, and traceability identification process and result determination. Wherein the traceability gene pool construction comprises source gene pool sample collection, sample collection and information recording, gene analysis and data generation, and traceability gene pool establishment; the genetic relationship judgment comprises repeated individual identification and paternity test; the traceability identification based on the genome data comprises repeated individual identification based on genome information and parent-child relationship identification based on the genome information; the traceability identification process and result judgment comprises sample receiving and quality control, data comparison and analysis and hierarchical result judgment. According to the method, SNP marker information is obtained through a genomics technology, accurate source tracing of livestock and poultry meat products is achieved through genetic relationship analysis, and the counterfeit and shoddy phenomena are effectively eradicated.

Description

Technical Field

[0001] This invention relates to the field of breed traceability technology, specifically to a local livestock and poultry traceability method based on SNP markers. Background Technology

[0002] With the continuous improvement of national economic level and consumption capacity, consumers have put forward higher requirements for the quality, flavor, and traceability of livestock and poultry meat. Local livestock and poultry breeds, as valuable genetic resources in my country, are highly favored by the market due to their delicious meat and unique flavor, possessing enormous market development potential and promising industrial development prospects. However, after slaughter and processing, the unique phenotypic characteristics of livestock and poultry breeds (such as coat color and body shape) disappear, making it impossible to directly identify the breed origin of meat products sold at the end through appearance. This makes it difficult for consumers to effectively distinguish whether the meat products they purchase are from the claimed high-quality local breeds, seriously restricting the healthy development of the market for high-quality, high-end local breed meat products. Therefore, establishing a technology that can accurately identify the identity of local breed meat products, ensuring that consumers purchase authentic and reliable high-quality meat, has become a key issue that the local livestock and poultry industry urgently needs to address.

[0003] Currently, most mainstream traceability methods in the industry rely on information identification systems such as QR codes. This method records and links information from breeding, slaughtering, and distribution stages to give products an electronic identity. However, such methods are highly dependent on manual recording and the integrity of the data chain, and have limitations such as many human intervention factors, data loss or tampering, etc., making it difficult to completely eliminate counterfeit and substandard products, and their reliability and credibility are often challenged.

[0004] In recent years, the development of genomics technology has provided new solutions to the above problems. Genotyping based on genome resequencing or gene chip technology can obtain SNP (Single Nucleotide Polymorphism) marker information on an individual's DNA. By comparing the genotype data of commercially available livestock and poultry meat products with those from local livestock and poultry breeding farms or farms, and using genotype consistency rate and homologous ancestor (IBD) analysis, the kinship between commercially available meat products and those from source farms can be accurately identified. If two samples are determined to be from the same independent individual or have a direct parent-child relationship, strong scientific evidence can be provided to confirm the authenticity of high-quality local livestock and poultry breed meat products.

[0005] However, there is currently a lack of an effective traceability system based on SNP markers, making it difficult to achieve scientific, objective, and accurate traceability of local livestock and poultry breeds. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to provide a local livestock and poultry traceability method based on SNP markers. This method obtains SNP marker information through genomics technology, calculates the genotypic consistency rate and IBD fragment distribution probability among individuals using SNP markers to determine kinship, thereby achieving accurate traceability of livestock and poultry meat products. This constructs a full-chain DNA identity authentication system from the source farm to the end market, effectively preventing counterfeit and substandard products, and providing technical support for the integrity and standardization management of the local livestock and poultry industry.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides a local livestock and poultry tracing method based on SNP markers, including the following steps: constructing a tracing gene bank, determining primary tracing based on genotype consistency rate, determining secondary tracing based on IBD, and outputting the final tracing results;

[0009] The source tracing method automatically determines the source according to the following hierarchical rules:

[0010] First-level source identification: The system first calculates the genotypic similarity (G) between the sample to be tested and individuals in the source gene bank at effective SNP loci across the entire genome. concordance If G concordance >G th If the sample to be tested is found to be a duplicate individual in the database, a source tracing report matching the duplicate individual is generated, and no further secondary source tracing is performed; wherein the genotype consistency rate G concordance G refers to the ratio of the number of SNP loci with completely identical genotypes in the tested sample and individuals in the source gene bank to the total number of shared SNP loci with valid genome-wide genotypes detected in both samples. th This refers to a pre-set threshold for genotype concordance rate;

[0011] Secondary source tracing determination: If the primary source tracing determination is not passed, the system will then calculate whether there is an allele locus with an IBD2 ratio greater than IBD. th If an individual exists in the database, the sample to be tested is immediately identified as the meat product of that individual in the database, and a traceability report for duplicate individual matching is generated; wherein, IBD2 refers to the fact that both alleles at the allele locus in the sample to be tested and the individuals in the traceability gene database have the same origin. th This refers to the pre-set IBD2 ratio threshold;

[0012] If the IBD2 determination is not passed, the system will further scan to determine whether the sample being tested simultaneously has an IBD1 ratio greater than IBD1 at a certain allele locus in a pair of male and female livestock in the database. shFurthermore, the proportion of IBD0 is not higher than 0.1. If it exists, the test sample is determined to be related to the male and female livestock in the database as offspring, and a parentage matching traceability report is generated. Here, IBD1 refers to the test sample sharing one and only one allele from the same source as individuals in the traceability gene database, and IBD0 refers to the test sample not sharing any alleles from the same source as any individual in the traceability gene database. sh This refers to the pre-set IBD1 ratio threshold;

[0013] No association determination: If the sample to be tested cannot establish an association with the gene bank through any of the above rules, it is determined to be a product of a different variety or a different system, and a traceability report is generated.

[0014] Preferably, in the secondary tracing process, the calculation method for the ratio of IBD1 and IBD2 is as follows: based on method moments or site mixture information, the observation information of state homology at each site is summarized into a whole-genome estimate; the cumulative information of multiplication or logarithmic summation when all sites are independently approximated is obtained by method moments or likelihood approximation to obtain the estimated values ​​of IBD0, IBD1, and IBD2, and then the kinship is determined according to the IBD ratio distribution; state homology refers to two individuals having the same alleles.

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

[0016] Whole-genome SNP data of the test sample and individuals in the source database were collected to obtain the genotype of each locus and calculate the allele frequency at each locus. For each locus, the joint probability of the observed genotypes of the test sample and individuals in the source database at a certain SNP locus j was calculated, and the average of all loci was taken to obtain the theoretical moments of all loci. Then, the theoretical moments were calculated for different IBD states IBD0, IBD1, and IBD2. , and And establish the following system of functional equations:

[0017] ;

[0018] In the formula, k0, k1, and k2 represent the proportions of IBD states IBD0, IBD1, and IBD2 at the genomic level for two individuals, respectively, satisfying the constraints k0, k1, and k2 ≥ 0 and k0 + k1 + k2 = 1; where The average theoretical moment representing all sites, , and These represent the theoretical moments under different IBD states, IBD0, IBD1, and IBD2, respectively.

[0019] By solving the moment equations for different genotype combinations, k0, k1, and k2 can be used to obtain the estimated proportions of IBD0, IBD1, and IBD2.

[0020] More preferably, the likelihood approximation is performed through EM iteration, and the specific method for obtaining the estimates of k0, k1, and k2 is as follows:

[0021] For each locus j, calculate the combined probability of the IBD0, IBD1, and IBD2 genotypes at that locus. Where s represents the IBD state value, P is obtained by taking 0, 1 and 2 respectively. 0j P 1j and P 2j These represent the combined probabilities of genotypes corresponding to the three IBD states, IBD0, IBD1, and IBD2, at locus j, respectively.

[0022] First, perform the E-step, assigning initial values ​​to k0, k1, and k2; then calculate the posterior probability PE of locus j belonging to a certain IBD state. sj :

[0023] ;

[0024] 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 ;

[0025] Then, perform M steps to update k0, k1, and k2 to the posterior probabilities PE of all loci. sj The average value, i.e.

[0026] ;

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

[0028] pass The aforementioned mapping relationship with the vector group (k0, k1, k2) yields the following: The corresponding k0, k1, and k2 values ​​(represented here as follows) Replace the original k0, k1, and k2 to calculate the E step, and repeat the E step and M step iterations until the change in k0, k1, and k2 is less than the preset threshold. At this time, k0, k1, and k2 are the estimated values ​​of the proportions of IBD0, IBD1, and IBD2.

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

[0030] More preferably, the steps for primary source tracing based on genotype consistency rate and secondary source tracing based on IBD are as follows:

[0031] Identification of duplicate individuals based on genomic information: First, DNA is extracted from the sample to be tested, and genotyping is performed using the same gene chip platform used to construct the source gene bank to obtain its whole-genome SNP data; then, the genotype data of the sample to be tested is compared one by one with the genotype data of all registered individuals in the source gene bank. First, a primary source determination is performed: the genotype similarity rate between the sample to be tested and each individual in the gene bank is calculated; if the genotype of the sample to be tested and a reference individual in the gene bank satisfy G… concordance >G th If G, then the sample to be tested is identified as the meat product corresponding to that individual in the gene bank; if G concordance <G th Then, a secondary source determination is performed: the IBD ratio between the test sample and each individual in the gene bank is calculated; if the genotype of the test sample and a reference individual in the gene bank satisfies the condition that the IBD2 ratio is greater than the IBD... th If the sample is identified as the meat product of the corresponding individual in the gene bank, then direct traceability from product to source is achieved.

[0032] Genomic-based paternity testing involves first obtaining the genotype data of the sample to be tested, then performing a kinship analysis between the SNP data of the sample and the livestock and poultry data in the gene bank; if the sample and a pair of male and female livestock and poultry individuals in the gene bank both show a high degree of allele sharing, and the proportion of IBD1 in the sample is greater than that in the paternal and maternal parents, then the paternal-female sample will be identified. sh If so, it can be determined that the sample to be tested is the offspring of these parents.

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

[0034] Sample Receiving and Quality Control: We receive livestock and poultry meat samples from the market for testing, perform DNA extraction and genotyping, and use gene chips or resequencing on the same platform as the one used to construct the traceability gene bank for genotyping; and we apply the same quality control standards to the generated genotype data.

[0035] Data comparison and analysis: The SNP data of the quality-controlled test samples are compared with the whole genome data of all individuals in the source gene bank, and the G values ​​are calculated with each individual in the bank. concordance The ratio of IBD1 to IBD2;

[0036] Hierarchical result determination: Source tracing and result determination are carried out according to the hierarchical rules.

[0037] Most preferably, the preset threshold is selected from one or more of the following criteria, wherein G th Not less than 95%, the IBD th Not less than 90%, the IBD sh No less than 80%.

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

[0039] This invention employs SNP markers for tracing and identifies individuals based on genomic genetic information, avoiding the risks of human intervention and data tampering. The results are scientifically objective, difficult to falsify, and significantly improve the credibility and reliability of tracing. The tracing gene bank focuses on parent breeding stock, eliminating the need for sequencing commercial offspring, greatly reducing the construction and operation costs of the tracing system and making it feasible for industrial application. Through two-level determination rules of duplicate individuals and parent-child relationships, it combines direct and indirect tracing, covering tracing needs in different scenarios and achieving high identification accuracy. The tracing method for duplicate individuals based on genotype consistency rate and IBD2 ratio is more accurate, while the indirect tracing method based on parent-child relationships is more cost-effective.

[0040] This invention establishes a full-chain DNA identity authentication system from the source farm to the end market, providing consumers with a guarantee of breed authenticity, helping the local livestock and poultry industry to develop with integrity and standardization, and enhancing market consumer confidence. Attached Figure Description

[0041] Figure 1 This is a flowchart illustrating the process of tracing the origins of local livestock and poultry in an embodiment of the present invention.

[0042] Figure 2 This is a distribution chart of genotype consistency rate for duplicate individual identification in an embodiment of the present invention.

[0043] Figure 3 This is a distribution map of the IBD2 proportion for duplicate individual identification in an embodiment of the present invention.

[0044] Figure 4 This is a distribution chart of IBD1 ratios for paternity testing in an embodiment of the present invention. Detailed Implementation

[0045] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0046] Example 1:

[0047] A local livestock and poultry traceability method based on SNP markers includes the construction of a traceability gene bank, determination of kinship, traceability identification based on genomic data, and traceability identification process and result determination;

[0048] The construction of the gene bank for tracing origins specifically includes (taking pigs as an example):

[0049] Source gene bank sample collection: The sampling objects are the parent breeding livestock and poultry of the core breeding group of local pigs (i.e., the parent breeding livestock and poultry used to produce commercial meat livestock and poultry); for example: select 1,000 parent breeding pigs of the core breeding group of a certain local pig breed as the sampling objects;

[0050] Sample collection and information recording: Systematically collect biological tissue samples (such as ear tissue samples) from each breeding livestock and poultry, and simultaneously and accurately record key identification information such as individual number, sex, and breed to ensure that the sample and individual information correspond one-to-one and are unique; for example: use an ear tissue sampler to collect ear tissue samples from each breeding livestock and poultry, numbered S1-S1000, and simultaneously record the sex (300 boars and 700 sows), breed name, and breeding batch information of each breeding pig.

[0051] Genetic analysis and data generation: High-quality whole-genome DNA was extracted from the collected samples, and genotyping was performed using whole-genome resequencing or pig-specific gene chips. Strict quality control was implemented on the raw data (i.e., sequencing data or chip data) to remove low-quality sites and filter low-frequency alleles (MAF) and strong linkage disequilibrium (LD) or anomalous sites according to set thresholds, obtaining high-quality SNP genotyping data covering the entire genome. For example, whole-genome DNA was extracted from ear tissue samples using the conventional phenol-chloroform method, and genotyping was performed using a local pig 66K breeding gene chip. Quality control was performed on the raw data: low-quality sites with a detection rate <95% were removed, low-frequency alleles with MAF <0.05 were filtered, and strong LD sites (r²>0.8) were filtered using PLINK software. The high-quality SNPs obtained from the quality control were included in subsequent analyses.

[0052] Establishment of a traceability gene bank: Integrate the genotype data and corresponding identity information of all breeding pigs that have passed quality control to build a traceability gene bank for local pig breeds, which will serve as an authoritative reference database for subsequent product traceability comparison; for example, enter the SNP genotype data and corresponding identity information (number, sex, breed, breeding batch) of 1,000 breeding pigs into the database management system to build a traceability gene bank for that local pig breed.

[0053] This invention provides a local livestock and poultry tracing method based on SNP markers, including the following steps: construction of a tracing gene bank, primary tracing determination based on genotype consistency rate, secondary tracing determination based on IBD, and final tracing result output.

[0054] This invention employs a layered determination strategy consisting of primary source tracing and secondary source tracing. Primary source tracing involves calculating the genotypic similarity (Gr) between the test sample and individuals in the source gene bank at effective SNP loci across the entire genome. concordance If G concordance Exceeding the preset identity threshold G th If the first-level source determination fails, the sample to be tested and an individual in the database are considered duplicates (i.e., originating from the same animal), and further IBD calculations are not performed. Secondary source determination (IBD verification and paternity testing): If the primary source determination fails, the IBD coefficients (k0, k1, k2) between the sample to be tested and individuals in the database are calculated using the Genomic Common Ancestor (IBD) algorithm. If the IBD2 ratio (k2) > IBD... th The individual was still identified as a duplicate (this applies when the consistency rate is slightly lower than G due to sample degradation). th However, cases where there is still a high degree of genetic identity are still present. If the IBD1 ratio (k1) of the sample to be tested and a certain pair of male and female livestock in the library is both greater than IBD1, then... sh If the IBD0 ratio is not higher than 0.1, then the individual is determined to be related to two individuals in the gene pool as parents, and the traceability of livestock and poultry can be indirectly achieved through the parent-child relationship.

[0055] The identification criterion for secondary source tracing is that the ratio of IBD2 in the sample to be tested to that in the source gene bank is greater than IBD2. th .

[0056] Secondary traceability determination also includes traceability based on parentage. This applies when commercial livestock and poultry do not retain genotype data in the livestock and poultry gene bank, and traceability is required. It can be achieved by identifying the parent-child relationship between the parents, which can save costs to some extent and provide a more flexible option for livestock and poultry traceability. The identification standard is that the IBD1 ratio of the sample to be tested and a certain pair of parents in the traceability gene bank are both greater than IBD1. sh .

[0057] In this invention, the genotype concordance rate refers to the ratio of the number of SNP loci with completely identical genotypes in the tested sample and individuals in the source gene bank to the total number of shared SNP loci with valid genome-wide genotypes detected in both samples; the G th This refers to a pre-set threshold for genotype concordance. IBD2 refers to the fact that both alleles at the allele locus in the tested sample and the individuals in the source gene bank originate from the same source; the IBD... th This refers to the pre-set IBD2 proportion threshold. IBD1 refers to a situation where the tested sample and individuals in the gene pool share exactly one allele of the same origin; IBD0 refers to a situation where the tested sample and all individuals in the gene pool do not share any alleles of the same origin. sh This refers to the pre-set IBD1 ratio threshold.

[0058] In the primary source tracing determination, the formula for calculating the genotype consistency rate is:

[0059]

[0060] In the formula, N match N represents the number of loci with completely identical genotypes in two samples (e.g., AA vs. AA, AB vs. AB, BB vs. BB); common This represents the total number of shared loci (i.e., intersection sites) where valid genotypes were detected in both samples. Threshold setting: Preferably, the genotype concordance rate threshold G... th The threshold is set to ≥95.0%. This threshold is based on the repeatability error rate of livestock and poultry gene chip technology, allowing for a very small number of typing errors or somatic mutations.

[0061] Phylogenetic relationship determination utilizes the proportion of genomic homologous ancestral fragments (IBDs) to accurately identify the kinship between a sample and an individual in a gene bank. Specifically, this includes:

[0062] Identification of duplicate individuals: Duplicate individuals are two samples originating from the same individual, with almost identical genomes. In diploid organisms, the alleles on both chromosomes at all SNP loci are completely identical, manifesting as an extremely high IBD2 ratio at the genomic level. When the IBD2 ratio of the sample to be tested and a reference individual in the gene pool is greater than the IBD2 ratio, the individual is considered a duplicate individual. th (e.g., IBD) th When the threshold IBD is 90%, the two are considered to be the same person; th It effectively avoids misjudgments caused by genotyping errors or somatic cell mutations.

[0063] Paternity testing: Parentage is a vertical inheritance relationship, where offspring inherit half of the genome from each parent. Therefore, offspring will share at least one allele with any gene locus of the father (or mother), which manifests as a high proportion of IBD1 sharing at the genomic level. When the IBD1 ratio of the sample being tested and that of a particular pair of parents in the gene pool is greater than IBD1, paternity testing is performed. sh (e.g., IBD) sh When the percentage is 80%, the sample to be tested is determined to be the offspring of the parents.

[0064] In the process of determining kinship, the IBD proportion is calculated as follows: based on method moments or locus mixture information (sample allele frequencies and genotype combination frequencies of each pair of individuals at each locus), the IBS (identity-by-state) observation information of each locus is summarized into a genome-wide estimate; when all loci are independently approximated, the accumulated information (multiplication or logarithmic summation) is used to obtain the estimated values ​​of IBD0, IBD1, and IBD2 through method moments or likelihood approximation, and thus the kinship is determined based on the IBD proportion distribution;

[0065] Specifically, the estimation values ​​of IBD0, IBD1, and IBD2 are obtained using the Method of Moments (MoM) as follows:

[0066] First, quality control was performed on the whole-genome SNP data of the two individuals (filtering out low-quality loci), obtaining the genotype (g1, g2) for each locus, and calculating the allele frequency for each locus. For each locus, the observed genotype co-occurrence probability of the two individuals at a given SNP locus was calculated, and the average of all loci was taken to obtain the theoretical moment for all loci. Then, the theoretical moments were calculated for different IBD states IBD0, IBD1, and IBD2. , and And establish the following system of functional equations:

[0067] ;

[0068] In the formula, k0, k1, and k2 represent the proportions of IBD states IBD0, IBD1, and IBD2 at the genomic level for two individuals, respectively, satisfying the constraints k0, k1, and k2 ≥ 0 and k0 + k1 + k2 = 1; where The average theoretical moment representing all sites, , and These represent the theoretical moments under different IBD states, IBD0, IBD1, and IBD2, respectively.

[0069] By solving the moment equations for different genotype combinations, k0, k1, and k2 can be used to obtain the estimated proportions of IBD0, IBD1, and IBD2.

[0070] More preferably, the likelihood approximation is performed through EM iteration, and the specific method for obtaining the estimates of k0, k1, and k2 is as follows:

[0071] For each locus j, calculate the combined probability of the IBD0, IBD1, and IBD2 genotypes at that locus. Where s represents the IBD state value, P is obtained by taking 0, 1 and 2 respectively. 0j P 1j and P 2j These represent the combined probabilities of genotypes corresponding to the three IBD states, IBD0, IBD1, and IBD2, at locus j, respectively.

[0072] First, perform the E-step, assigning initial values ​​to k0, k1, and k2; then calculate the posterior probability PE of locus j belonging to a certain IBD state. sj :

[0073] ;

[0074] 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 ;

[0075] Then, perform M steps to update k0, k1, and k2 to the posterior probabilities PE of all loci. sj Average value:

[0076] ;

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

[0078] pass 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.

[0079] Origin tracing and identification based on genomic data specifically includes:

[0080] Identification of duplicate individuals based on genomic information: First, DNA is extracted from the sample to be tested, and genotyping is performed using the same gene chip platform used to construct the source gene bank to obtain its whole-genome SNP data; then, the genotype data of the sample to be tested is compared one by one with the genotype data of all registered individuals in the source gene bank. First, a primary source determination is performed: the genotype similarity rate between the sample to be tested and each individual in the gene bank is calculated; if the genotype of the sample to be tested and a reference individual in the gene bank satisfy G… concordance >G th If G, then the sample to be tested is identified as the meat product corresponding to that individual in the gene bank; if G concordance <G th Then, a secondary source determination is performed: the IBD ratio between the test sample and each individual in the gene bank is calculated; if the genotype of the test sample and a reference individual in the gene bank satisfies the condition that the IBD2 ratio is greater than the IBD... th If so, the sample to be tested is identified as the meat product of the corresponding individual in the gene bank, thus achieving direct traceability from product to source;

[0081] Genomic-based paternity testing involves first obtaining the genotype data of the sample to be tested, then performing a kinship analysis between the SNP data of the sample and the livestock and poultry data in the gene bank; if the sample and a pair of male and female livestock and poultry individuals in the gene bank both show a high degree of allele sharing, and the proportion of IBD1 in the sample is greater than that in the paternal and maternal parents, then the paternal-female sample will be identified. sh If the IBD0 ratio is not higher than 0.1, then the sample to be tested is determined to be the offspring of the parents.

[0082] The specific procedures and results determination for tracing the source are as follows:

[0083] Sample Receiving and Quality Control: We receive livestock and poultry meat samples from the market for testing, perform DNA extraction and genotyping, and use gene chips or resequencing on the same platform as the one used to construct the traceability gene bank for genotyping; and we apply the same quality control standards to the generated genotype data.

[0084] Data comparison analysis: The SNP data of the test sample after quality control is compared with the data of all individuals in the source gene bank, and the genotypic similarity rate, IBD1 and IBD2 ratios are calculated.

[0085] Hierarchical result determination: Source tracing and result determination are performed according to the aforementioned hierarchical rules.

[0086] First-level source tracing determination: The system first calculates the genotypic similarity between the sample to be tested and individuals in the source gene bank at effective SNP loci across the entire genome. If G concordance >G th If the sample to be tested is determined to be a duplicate individual in the database, a source tracing report matching the duplicate individual will be generated, and no further secondary source tracing will be performed.

[0087] Secondary source tracing determination: If the primary source tracing determination is not passed, the system will then calculate whether there is an allele locus with an IBD2 ratio greater than IBD. th If an individual exists in the database, the sample to be tested is immediately determined to be the meat product of that individual in the database, and a traceability report matching duplicate individuals is also generated.

[0088] If the IBD2 determination is not passed, the system will further scan to determine whether the sample being tested simultaneously has an IBD1 ratio greater than IBD1 at a certain allele locus in a pair of male and female livestock in the database. sh If the IBD0 ratio is not higher than 0.1, the sample to be tested is determined to be related to the male and female livestock in the database, and a parent-child relationship traceability report is generated.

[0089] No association determination: If the sample to be tested cannot establish an association with the gene bank through any of the above rules, it is determined to be a product of a different variety or a different system, and a traceability report is generated.

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

[0091] a. Sample number and testing date, and determination result (duplicate individual / parent-child relationship / no connection);

[0092] b. The individual breeding pig ID from the matching source farm;

[0093] c. Key genetic evidence (such as genotype concordance rate, IBD ratio, etc.).

[0094] This report provides intuitive and authoritative scientific evidence for market supervision, corporate self-certification, and consumer inquiries, thus completing the entire chain of DNA identity verification from market products to the source population.

Claims

1. A method for tracing local livestock and poultry based on SNP markers, characterized in that... The process includes the following steps: construction of the source gene bank, primary source tracing determination based on genotype concordance rate, secondary source tracing determination based on IBD, and final source tracing result output; the source tracing method automatically determines the source according to the following hierarchical rules: Primary source tracing determination: The system first calculates the genotype concordance rate G between the sample to be tested and individuals in the source gene bank at effective SNP loci across the entire genome. concordance If G concordance >G th If the sample to be tested is found to be a duplicate individual in the database, a source tracing report matching the duplicate individual is generated, and no further secondary source tracing is performed; wherein the genotype consistency rate G concordance G refers to the ratio of the number of SNP loci with completely identical genotypes in the tested sample and individuals in the source gene bank to the total number of shared SNP loci with valid genome-wide genotypes detected in both samples. th This refers to a pre-set threshold for genotype concordance rate; Secondary source tracing judgment: If the primary source tracing judgment is not passed, the system will then calculate whether there is an IBD2 ratio greater than IBD at any allele locus. th If an individual exists in the database, the sample to be tested is immediately identified as the meat product of that individual in the database, and a traceability report for duplicate individual matching is generated; wherein, IBD2 refers to the fact that both alleles at the allele locus in the sample to be tested and the individuals in the traceability gene database have the same origin. th This refers to the pre-set IBD2 ratio threshold; if the IBD2 threshold is not met, the system will further scan to determine whether the sample being tested simultaneously has an IBD1 ratio greater than IBD1 at a certain allele locus in a pair of male and female livestock in the database. sh Furthermore, the proportion of IBD0 is not higher than 0.

1. If it exists, the test sample is determined to be related to the same male and female livestock in the database, and a parentage matching traceability report is generated. Here, IBD1 refers to the test sample and individuals in the traceability gene database having exactly one allele from the same source, while IBD0 refers to the test sample and all individuals in the traceability gene database having no alleles from the same source. sh This refers to the pre-set IBD1 ratio threshold; No association determination: If the sample to be tested cannot establish an association with the gene bank through any of the above rules, it is determined to be a product of a different variety or a different system, and a traceability report is generated.

2. The local livestock and poultry traceability method based on SNP markers according to claim 1, characterized in that: In the secondary source tracing process, the calculation method for the IBD1 and IBD2 ratio is as follows: based on method moments or site mixture information, the observation information of state homology at each site is summarized into a whole-genome estimate; the cumulative information of multiplication or logarithmic summation when all sites are independently approximated is obtained by method moments or likelihood approximation to obtain the estimated values ​​of IBD0, IBD1, and IBD2, and then source tracing is performed according to the IBD ratio distribution; state homology refers to two individuals having the same alleles.

3. The local livestock and poultry traceability method based on SNP markers according to claim 2, characterized in that: The specific calculation method for IBD estimates includes: collecting whole-genome SNP data of individuals in the test sample and the source database, obtaining the genotype of each locus, and calculating the allele frequency at each locus; for each locus, calculating the joint probability of the observed genotypes of individuals in the test sample and the source database at a certain SNP locus, and taking the average of all loci to obtain the theoretical moments for all loci. Then, the theoretical moments were calculated for different IBD states IBD0, IBD1, and IBD2. , and And establish the following system of functional equations: In the formula, k0, k1, and k2 represent the proportions of IBD states IBD0, IBD1, and IBD2 at the genomic level for two individuals, respectively, satisfying the constraints k0, k1, and k2 ≥ 0 and k0 + k1 + k2 = 1; where The average theoretical moment representing all sites, , and These represent the theoretical moments under different IBD states, IBD0, IBD1, and IBD2, respectively. By solving the moment equations for different genotype combinations, k0, k1, and k2 are obtained, thus yielding the estimated proportions of IBD0, IBD1, and IBD2.

4. The local livestock and poultry traceability method based on SNP markers according to claim 3, characterized in that: The likelihood approximation is performed through EM iteration. The specific method for obtaining the estimates of k0, k1, and k2 is as follows: For each locus j, calculate the joint probability of the IBD0, IBD1, and IBD2 genotypes at that locus. Where s represents the IBD state value, P is obtained by taking 0, 1 and 2 respectively. 0j P 1j and P 2j These represent the combined probabilities of genotypes at site j corresponding to the three IBD states: IBD0, IBD1, and IBD2. First, we perform the E-step, assigning initial hypothetical values ​​to k0, k1, and 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 local livestock and poultry traceability method based on SNP markers according to any one of claims 1-4, characterized in that: The quality control standards adopted in the construction steps of the traceability gene bank include: removing low-quality site data with a detection rate of <95%, filtering low-frequency allele data with a low frequency allele frequency of <0.05, and filtering strong linkage disequilibrium site data with r²>0.

8.

6. The local livestock and poultry traceability method based on SNP markers according to claim 5, characterized in that: The specific steps for primary source identification based on genotype concordance rate and secondary source identification based on IBD are as follows: Identification of duplicate individuals based on genomic information: First, DNA is extracted from the sample to be tested, and genotyping is performed using the same gene chip platform used when constructing the source gene bank to obtain its whole-genome SNP data; subsequently, the genotype data of the sample to be tested is compared one by one with the genotype data of all registered individuals in the source gene bank. Primary source identification is then performed: the genotype concordance rate between the sample to be tested and each individual in the gene bank is calculated: if the genotype of the sample to be tested and a reference individual in the gene bank satisfy G… concordance >G th If G, then the sample to be tested is identified as the meat product corresponding to that individual in the gene bank; if G concordance <G th Then, a secondary source determination is performed: the IBD ratio between the test sample and each individual in the gene bank is calculated; if the genotype of the test sample and a reference individual in the gene bank satisfies the condition that the IBD2 ratio is greater than the IBD... th If the sample is identified as the meat product of the corresponding individual in the gene bank, direct traceability from product to source is achieved. For paternity testing based on genomic information: first, the genotype data of the sample is obtained; then, the SNP data of the sample is compared with the breeding livestock data in the gene bank for kinship analysis. If the sample and a pair of male and female livestock individuals in the gene bank both show high allele sharing, and the proportion of IBD1 in the sample is greater than that in the paternal and maternal lines, then the test results are considered valid. sh If the IBD0 ratio is not higher than 0.1, then the sample to be tested is determined to be the offspring of the parents.

7. The local livestock and poultry traceability method based on SNP markers according to claim 6, characterized in that: The specific steps for traceability identification and result determination are as follows: Sample reception and quality control: Receive livestock and poultry meat samples from the market to be tested, perform DNA extraction and genotyping, and use gene chips or resequencing on the same platform as the traceability gene bank for genotyping; and apply the same quality control standards to the generated genotype data; Data comparison and analysis: Compare the SNP data of the quality-controlled sample to be tested with the whole genome data of all individuals in the traceability gene bank, and calculate the genotype consistency rate, IBD1 and IBD2 ratios with each individual in the bank; Hierarchical result determination: Conduct traceability identification and result determination according to the hierarchical rules.

8. The local livestock and poultry traceability method based on SNP markers according to claim 7, characterized in that: The preset threshold is selected from one or more of the following criteria, wherein G th Not less than 95%, the IBD th Not less than 90%, the IBD sh No less than 80%.

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