Pig variety identification method based on liquid phase chip technology and application thereof

Abstract

The invention belongs to the technical field of gene detection, and relates to a detection method of nucleic acid, in particular to a pig breed identification method based on a liquid chip technology and application of the pig breed identification method. According to the invention, a specific SNP marker combination aiming at 73 pig varieties is established for the first time, the specific SNP marker combination is used for constructing a liquid phase chip for pig variety identification, and targeted capture and high-throughput sequencing are combined, so that the distinguishing accuracy of 95% or more among the 73 pig varieties is successfully realized. The method disclosed by the invention has the advantages of wide variety types, high cost performance, high accuracy and short detection period, and can be used for identifying the variety of landrace, large white pig, duroc, 28 wild boars and 42 domestic pigs.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and relates to a method for measuring nucleic acids, and more specifically to a method for identifying pig breeds based on liquid phase chip technology and its application. Background Technology

[0002] Pig breeds are the core carriers of pig genetic resources, and the development of breed identification technology is directly related to the effective utilization and protection of these resources. Early identification methods, which relied on morphological characteristics, became ineffective due to the increasing crossbreeding and blurring of morphological differences. The emergence of molecular marker technology has enabled breed identification to move from the phenotypic level to the genotypic level, but accurately capturing breed-specific genetic characteristics remains the core direction of technological research and development.

[0003] With the intensive development of the livestock industry, higher requirements have been placed on breed identification: it is necessary not only to distinguish common breeds, but also to cover rare and endangered breeds; it is necessary not only to be accurate for individual samples, but also to support efficient testing of batch samples. This has driven the development of breed identification technology towards higher precision and higher throughput.

[0004] Liquid-phase microarray / targeted capture sequencing is a low-cost, high-throughput locus detection technology that emerged after the maturation of next-generation sequencing. Its earliest and most widespread applications were in whole-exome capture sequencing and genetic disease locus detection in medical research. In recent years, with decreasing costs, targeted capture sequencing technology has gradually expanded from basic medicine and molecular diagnostics to agricultural breeding. In agricultural breeding, this technology was first applied to animal breeding (such as livestock, poultry, and aquaculture), where breeding costs are high, and later began to be widely used in crop breeding and precision identification.

[0005] Although pig breeding chips of different densities have been launched on the market, these chips are mainly intended for a comprehensive evaluation of the germplasm of a specific breed. They are not universally applicable, and their high SNP density leads to high development and testing costs, large data volumes, and long analysis cycles, making them unsuitable for rapid identification of pig breeds.

[0006] Therefore, developing a pig breed identification chip and system that is adaptable to a wide range of breed types, cost-effective, accurate, and has a short testing cycle would enable better identification of breed components in core breeding pig populations. Summary of the Invention

[0007] To address the aforementioned problems, in a first aspect, the present invention provides a specific SNP marker combination for pig breed identification, the combination consisting of 1094 SNP marker sites, the information of which is shown in Table 1.

[0008] Table 1: SNP marker sites

[0009] chr1:238762805-238762806 chr13:207616132-207616133 chr1:27521529-27521530 chr1:257033391-257033392 chr1:218186471-218186472 chr1:257535397-257535398 chr1:241195738-241195739 chr1:248857779-248857780 chr1:221862224-221862225 chr3:14732346-14732347 chr1:260789396-260789397 chr1:31062080-31062081 chr1:262854002-262854003 chr1:250641862-250641863 chr1:255984186-255984187 chr1:251745681-251745682 chr1:263744943-263744944 chr1:248900103-248900104 chr8:72602260-72602261 chr1:262422669-262422670 chr1:250070290-250070291 chr1:262278987-262278988 chr1:238808210-238808211 chr1:261930768-261930769 chr1:187690102-187690103 chr1:264366924-264366925 chr6:150775533-150775534 chr1:261620191-261620192 chr1:251757956-251757957 chr1:257941244-257941245 chr1:253286235-253286236 chr1:261947062-261947063 chr17:2860639-2860640 chr1:261705968-261705969 chr1:226699743-226699744 chr1:257603342-257603343 chr1:252311102-252311103 chr13:203520563-203520564 chr1:249019121-249019122 chr1:240172509-240172510 chr1:258776790-258776791 chr1:249191764-249191765 chr13:193427893-193427894 chr1:261933765-261933766 chr1:258388490-258388491 chr1:202362448-202362449 chr13:181235803-181235804 chr14:43574847-43574848 chr1:261568154-261568155 chr1:251201160-251201161 chr13:205302472-205302473 chr1:257006906-257006907 chr6:168743594-168743595 chr13:203504813-203504814 chr1:257614422-257614423 chr14:130783805-130783806 chr6:143354190-143354191 chr1:249223289-249223290 chr15:136645768-136645769 chr2:83106435-83106436 chr1:256751905-256751906 chr13:205740377-205740378 chr1:265045792-265045793 chr14:137127604-137127605 chr1:217486741-217486742 chr6:20987558-20987559 chr1:261717109-261717110 chr1:256978734-256978735 chr1:258441279-258441280 chr18:37212017-37212018 chr1:252985354-252985355 chr1:261359097-261359098 chr1:250797309-250797310 chr4:94971405-94971406 chr13:194870226-194870227 chr1:256764916-256764917 chr13:194096323-194096324 chr9:135402003-135402004 chr1:258557094-258557095 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chr1:263296095-263296096 chr1:250900549-250900550 chr1:265109819-265109820 chr1:253274027-253274028 chr13:177121878-177121879 chr1:261801479-261801480 chr1:260046106-260046107 chr10:36263802-36263803 chr1:265141489-265141490 chr1:262463184-262463185 chr1:239615323-239615324 chr1:226470926-226470927 chr5:99000709-99000710 chr8:123096266-123096267 chr1:251939727-251939728 chr1:257203165-257203166 chr13:206404343-206404344 chr1:199607670-199607671 chr1:250350494-250350495 chr1:254647106-254647107 chr1:254527600-254527601 chr6:150669742-150669743 chr1:248604053-248604054 chr13:194960767-194960768 chr8:136199360-136199361 chr1:261411058-261411059 chr1:253462053-253462054 chr1:260147913-260147914 chr1:258447105-258447106 chr3:33232388-33232389 chr1:265112068-265112069 chr1:238811514-238811515 chr1:207791466-207791467 chr14:108789459-108789460 chr13:189933331-189933332 chr1:264980814-264980815 chr1:259246423-259246424 chr1:256623369-256623370 chr1:260607458-260607459 chr1:259026535-259026536 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[0010] Furthermore, the SNP marker sites are used to distinguish at least 73 pig breeds, including Landrace, Large White, Duroc, 28 wild boar breeds and 42 domestic pig breeds.

[0011] Furthermore, the SNP marker sites can also be used to distinguish different strains within the same variety.

[0012] Furthermore, the SNP marker sites can be used to distinguish different strains within the Landrace pig breed.

[0013] Furthermore, the different strains within the Landrace pig breed include Danish, American, Canadian, and French strains.

[0014] Furthermore, the SNP marker sites can be used to distinguish different strains within the Large White pig breed.

[0015] Furthermore, the different strains within the Large White pig breed include Danish, American, Canadian, and French strains.

[0016] In a second aspect, the present invention provides a molecular probe array for pig breed identification, characterized in that the molecular probe array targets a specific SNP marker array as described herein.

[0017] In a third aspect, the present invention provides a liquid phase chip for pig breed identification, the chip comprising probes corresponding to each SNP site in the specific SNP marker combination described herein, the probes being capable of specifically hybridizing and enriching genomic DNA fragments containing the SNP sites.

[0018] In a fourth aspect, the present invention provides a method for identifying pig breeds, the method comprising the following steps:

[0019] (a) Extracting genomic DNA from the pig samples to be tested;

[0020] (b) Targeted capture of the genomic DNA obtained in step (a) using molecular probe combinations as described herein or liquid phase chips as described herein, enriching DNA fragments containing the 1094 SNP marker sites;

[0021] (c) Perform high-throughput sequencing on the enriched DNA fragments to obtain sequencing data;

[0022] (d) Analyze the sequencing data to determine the genotype of the sample to be tested at the 1094 SNP marker sites;

[0023] (e) The genotype obtained in step (d) is compared with the reference SNP genotype of the pig breed to identify the breed of the pig to be tested.

[0024] Furthermore, the pig samples to be tested are derived from blood, tissue, hair, oral swabs, or meat products.

[0025] Furthermore, the reference SNP genotypes for the pig breeds include the genotypes of pig samples of known breeds at the 1094 SNP marker sites.

[0026] Furthermore, the pig breeds include at least 73 pig breeds, including Landrace, Large White, Duroc, 28 wild boar breeds and 42 domestic pig breeds.

[0027] Furthermore, the method also includes identifying different strains within the same variety.

[0028] Furthermore, the reference SNP genotypes for the pig breeds also include the genotypes of pig samples with known breed lines at the 1094 SNP marker sites.

[0029] Furthermore, the method also includes determining whether the tested pig is purebred based on the comparison results with the reference SNP genotype of the pig breed. Specifically, the homozygosity of breed-specific SNP markers in the tested pig sample can be used to determine whether an individual is purebred.

[0030] Furthermore, when the pig to be tested is a hybrid, the method further includes determining the hybrid combination of the pig to be tested based on the comparison results with the reference SNP genotype of the pig breed. This hybrid combination can be determined by analyzing the proportion of specific SNP markers from different parent breeds in the hybrid individuals.

[0031] In a fifth aspect, the present invention provides a system for identifying pig breeds, the system comprising:

[0032] As described in this article, liquid phase chips;

[0033] High-throughput sequencer;

[0034] The data processing unit stores a reference SNP genotype database of pig breeds and is configured with an algorithm for comparing and analyzing the genotype of the sample to be tested with the reference database.

[0035] In a sixth aspect, the present invention provides the use of SNP marker combinations as described herein in the preparation of reagents or kits for pig breed identification.

[0036] The beneficial effects of this invention are:

[0037] This invention screened SNP markers related only to breed characteristics from whole-genome data of 73 breeds. Through further filtering, 1094 SNP marker loci were obtained for constructing a liquid-phase chip for pig breed identification. This SNP marker locus combination has specific markers for 28 wild boar breeds and 42 domestic pig breeds, achieving 100% identification coverage and broad applicability. By utilizing targeted capture sequencing technology, SNP target locus information can be detected, successfully achieving a differentiation accuracy of over 95% between pig breeds. The method of this invention is applicable to a wide range of breeds, cost-effective, accurate, and has a short detection cycle. It can be used for the identification of components in Landrace, Large White, Duroc, 28 wild boar breeds, and 42 domestic pig breeds. Detailed Implementation

[0038] This invention provides a specific SNP marker combination for pig breed identification, the combination consisting of 1094 SNP marker sites, the information of which is shown in Table 1.

[0039] In some embodiments, the pig breed-specific SNP marker combinations of the present invention are obtained by screening using the following methods:

[0040] 1) Population stratification analysis was performed on the genomic data of 73 varieties to clarify the genetic distances between varieties;

[0041] 2) Machine learning algorithms (such as random forest) are used to screen for markers that contribute the most to variety classification from genome-wide SNPs. The screening criteria are as follows:

[0042] 2.1) Filter out sites with a variance less than 0.2;

[0043] 2.2) Filter out sites that were detected in less than 60% of the samples;

[0044] 2.3) Filter out sites with a correlation coefficient greater than 0.5 or less than -0.5 (keep 1 and remove 1).

[0045] 2.4) Filter sites where the Heat-Wen balance is below 1e-10;

[0046] 2.5) Filter sites with a PIC value less than 0.4;

[0047] 2.6) Sites with a base distance of less than 2000 bp between filtering sites (keep 1 site and remove 1 site).

[0048] 3) Through cross-validation, markers that showed cross-reactivity among closely related varieties were eliminated, and 1094 core markers were finally identified.

[0049] The present invention also provides a liquid phase chip for pig breed identification, comprising probes corresponding to each SNP site in a specific SNP marker combination as described herein, the probes being capable of specifically hybridizing and enriching genomic DNA fragments containing the SNP sites.

[0050] In some embodiments, the design of the liquid phase chip of the present invention follows the following principles:

[0051] 1) The probe design not only matches the SNP site, but also covers the sequences 40-60 bp upstream and downstream of it, which enhances the specific binding with the DNA of the target variety and reduces cross hybridization with non-target varieties.

[0052] 2) The probe has a length of 110 bp, a GC content between 30% and 80%, and ≤5 homology regions. The selection priority is: gene region > gene promoter region (2Kb) > gene downstream adjacent region (2Kb) > gene intergenic region. Combined with the principle of uniform chromosome distribution, the probe is obtained.

[0053] 3) By optimizing probe concentration and hybridization conditions, we can ensure that even if the sample DNA is partially degraded, we can still accurately detect variety-specific markers.

[0054] This invention also provides a method for identifying pig breeds, the method comprising the following steps:

[0055] (a) Extracting genomic DNA from the pig samples to be tested;

[0056] (b) Using the molecular probe combination of claim 3 or the liquid phase chip of claim 4, the genomic DNA obtained in step (a) is targeted and captured to enrich DNA fragments containing the 1094 SNP marker sites;

[0057] (c) Perform high-throughput sequencing on the enriched DNA fragments to obtain sequencing data;

[0058] (d) Analyze the sequencing data to determine the genotype of the sample to be tested at the 1094 SNP marker sites;

[0059] (e) The genotype obtained in step (d) is compared with the reference SNP genotype of the pig breed to identify the breed of the pig to be tested.

[0060] In some implementations, the DNA library required in the targeted capture process can be a single library or a mixed library; when a mixed library is used, the detection efficiency is higher. A mixture of 24 DNA libraries is preferred, balancing efficiency and accuracy.

[0061] In some implementations, 30 representative individuals of each variety are tested to construct a reference SNP genotype map for that variety, which includes the allele frequency range for each marker.

[0062] In some implementations, the genotype of the pig sample to be tested is compared with the reference genotype map of each breed, the matching score is calculated, and the breed identification result is determined.

[0063] In some implementations, pig breeds are identified by calculating genetic distance, using machine learning algorithms such as support vector machines and random forests, principal component analysis (PCA), or model-based cluster analysis such as ADMIXTURE.

[0064] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0065] Example 1: Acquisition of target SNP sites

[0066] Genome resequencing data from 1256 pigs was used for whole-genome genotyping. 172 samples were self-tested sequencing data from participating units, including data from six Large White pig breeds. The remaining 1084 sequencing data were downloaded from shared databases, including data from 28 wild boar breeds and 42 domestic pig breeds, as well as from public databases. Loci were filtered based on the following criteria: a. Loci with variance less than 0.2; b. Loci detected in less than 60% of samples; c. Loci with correlation coefficients greater than 0.5 / less than -0.5 (one retained, one removed); d. Loci with Hardy-Wenz balance below 1e-10; e. Loci with a PIC less than 0.4; f. Loci with a base distance less than 2000 bp (one retained, one removed). Finally, 1094 loci (as shown in Table 1) were selected for the development of a pig breed identification chip.

[0067] Example 2: SNP marker combinations for pig breed identification

[0068] 1. Synthesis and evaluation of liquid phase chip probes

[0069] 1) Probe synthesis

[0070] Based on the principle of DNA complementarity, one or more probes covering the target 1K site are designed for each SNP site to be tested. These biotin-modified probes can hybridize with the target region in the denatured resequencing library to form double strands. Magnetic beads coated with streptavidin are used to adsorb the biotin-carrying molecules. After elution, amplification and sequencing, the genotype of the target site is finally obtained.

[0071] 2) Probe evaluation

[0072] Three pig DNA samples were collected, and high-throughput sequencing was performed after library construction and capture using liquid-phase chip reagents loaded with probes. The alignment rate, coverage, capture rate, and uniformity were statistically analyzed to evaluate the suitability of the probes.

[0073] The test results are shown in Table 2 below. All indicators are good and meet expectations.

[0074] Table 2: Evaluation Results of Liquid Chip Probes

[0075]

[0076] 2. Multi-library hybrid capture

[0077] Porcine DNA libraries with different adapters were mixed in quantities of 1, 6, 10, 15, and 24. The libraries with different mixed quantities were hybridized with synthetic liquid-phase chip probes. After elution, amplification, and sequencing, the alignment rate, coverage, and capture rate of each sample were analyzed. The results showed that there was no significant difference in alignment rate, coverage, and capture rate between the mixed capture of 24 libraries and the capture of a single sample.

[0078] Table 3: Comparison of alignment rate, coverage rate, and capture rate for mixed capture of different numbers of libraries.

[0079]

[0080] 3. Analyze the components of 10 pig DNA samples.

[0081] The above methods were used to detect and sequence 10 pig DNA samples. The genotyping results of 1094 SNP sites in these samples were obtained through mutation detection, compared with standard chromatograms, and calculated and analyzed to determine the breed identification results.

[0082] The results of breed identification of 10 pig DNA samples are shown in Table 3-5.

[0083] Table 3: Variety Identification Results and Purity Analysis

[0084] sample Duroc pig Changbai pig Big White Pig Variety identification results 20240011250 0.00% 0.31% 99.69% It is very likely a purebred Great White. 20240011251 0.00% 0.20% 99.80% It is very likely a purebred Great White. 20240011252 0.00% 0.01% 99.99% It is very likely a purebred Great White. 20240011253 0.00% 0.01% 99.99% It is very likely a purebred Great White. 20240011254 0.00% 0.24% 99.76% It is very likely a purebred Great White. 20240011255 0.00% 0.95% 99.05% It is very likely a purebred Great White. 20240011256 0.00% 99.97% 0.03% It is very likely a purebred Changbai. 20240011257 0.00% 99.70% 0.30% It is very likely a purebred Changbai. 20240011258 0.00% 99.75% 0.25% It is very likely a purebred Changbai. 20240011259 0.00% 65.23% 34.77% It is very likely a crossbred pig.

[0085] Table 4: Distribution Analysis of Large White Pig Strains

[0086] sample Dan series American Canadian French system Strain Reference Recommendations 20240011250 0.00% 55.20% 0.00% 44.80% It is very likely a hybrid of alchemy and elixirs. 20240011251 0.00% 95.30% 0.00% 4.70% It is very likely American. 20240011252 0.00% 98.53% 0.00% 1.47% It is very likely American. 20240011253 0.00% 99.61% 0.00% 0.39% It is very likely American. 20240011254 0.00% 99.29% 0.00% 0.71% It is very likely American. 20240011255 0.00% 98.87% 0.00% 1.13% It is very likely American.

[0087] Table 5: Distribution Analysis of Landrace Pig Strains

[0088] sample Dan series American Canadian French system Strain Reference Recommendations 20240011256 99.97% 00.00% 00.00% 0.03% It is very likely the Dan system. 20240011257 99.97% 00.00% 00.00% 0.30% It is very likely the Dan system. 20240011258 99.97% 00.00% 00.00% 0.25% It is very likely the Dan system.

[0089] As can be seen from the results in Tables 3-5, the specific SNP marker combination of the present invention can clearly distinguish between different purebred pig breeds, hybrid pig breeds and strains, with a distinction accuracy of over 95%.

[0090] It should be noted that while the preferred embodiments of the present invention are provided in this specification, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A specific SNP marker combination for pig breed identification, characterized in that, The combination consists of 1094 SNP marker sites, the information of which is shown as follows: 。 2. The SNP marker combination according to claim 1, wherein The SNP marker sites are used to distinguish at least 73 pig breeds, including Landrace, Large White, Duroc, 28 wild pig breeds and 42 domestic pig breeds.

3. The SNP marker combination according to claim 2, wherein The SNP marker sites are also used to distinguish different strains within the same breed.

4. A combination of molecular probes for the identification of pig breeds, characterized in that The molecular probe combination targets the specific SNP marker combination of any one of claims 1-3.

5. A liquid chip for pig breed identification, characterized by, The chip comprises probes corresponding to each SNP site in the specific SNP marker combination of any one of claims 1-3, which can specifically hybridize and enrich the genomic DNA fragments containing the SNP sites.

6. A method for identifying a pig breed, characterized by, The method comprises the following steps: (a) extracting genomic DNA of the pig sample to be tested; (b) using the molecular probe combination of claim 4 or the liquid chip of claim 5 to target capture the genomic DNA obtained in step (a) and enrich DNA fragments containing the 1094 SNP marker sites; (c) performing high-throughput sequencing on the enriched DNA fragments to obtain sequencing data; (d) analyzing the sequencing data to determine the genotype of the sample to be tested at the 1094 SNP marker sites; (e) comparing the genotype obtained in step (d) with the reference SNP genotype of the pig breed, thereby identifying the breed of the pig to be tested.

7. The method of claim 6, wherein, The pig sample to be tested is derived from blood, tissue, hair, buccal swab or meat product.

8. The method of claim 6, wherein, The reference SNP genotype of the pig breed includes the genotype of a pig sample known to the breed at the 1094 SNP marker sites.

9. The method of claim 6, wherein, The pig breed includes at least 73 pig breeds, including Landrace, Large White, Duroc, 28 wild pig breeds and 42 domestic pig breeds.

10. The method of claim 6, wherein, The method further comprises identifying different strains within the same breed.

11. The method of claim 6, wherein, The method further comprises determining whether the pig to be tested is a purebred according to the comparison result with the reference SNP genotype of the pig breed.

12. The method of claim 6, wherein, In the case that the pig to be tested is a hybrid, the method further comprises determining the hybrid combination of the pig to be tested according to the comparison result with the reference SNP genotype of the pig breed.

13. A system for pig breed identification, characterized by, The system comprises: The liquid chip of claim 5; A high-throughput sequencer; A data processing unit, which stores a reference SNP genotype database of pig breeds and is configured with an algorithm for comparing and analyzing the genotype of the sample to be tested with the reference database.

14. Use of the SNP marker combination of any one of claims 1-3 in the preparation of a reagent or kit for pig breed identification.

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