Application of pig SNP (Single Nucleotide Polymorphism) molecular marker combination in pig variety identification, kit and identification method

Abstract

The invention provides application of a pig SNP (Single Nucleotide Polymorphism) molecular marker combination in pig variety identification, a kit and an identification method, relates to the technical field of biology, and develops an SNP molecular marker combination for accurately identifying a plurality of pig varieties by applying a whole genome association analysis method for 18 wild boar varieties and 42 domestic pig varieties. By detecting the combination, a plurality of pig varieties can be accurately and efficiently identified, and the accuracy reaches 99.9%. The method is expected to provide a new variety identification technical means for pig variety right identification, germplasm resource protection and utilization, genetic evolution research and other demands.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of a pig SNP molecular marker combination in pig breed identification, a kit and an identification method. Background Art

[0002] Breeding chips are highly specialized genetic analysis tools that are based on microarray or super-multiplex PCR technology for rapid and efficient genotyping of a large number of genetic markers. These chips are a high-throughput genotyping tool designed for animal and plant breeding research and practice to help researchers and breeders understand and improve genetic traits.

[0003] As a high-throughput genotyping analysis tool, breeding chips have significant advantages in the field of animal and plant breeding. Compared with the traditional site-by-site analysis method, breeding chips can detect thousands or even hundreds of thousands of variant sites at a time, greatly improving the speed and efficiency of data collection, significantly reducing the time and labor required, and accelerating the breeding process. At the same time, breeding chip technology has high repeatability and low error rate, ensuring the accuracy and reliability of genotype data. For the analysis of specific genetic variation sites in a large number of samples, breeding chips are usually more cost-effective than high-throughput sequencing. The operation process of breeding chips is more standardized and simple, and the analysis speed is faster.

[0004] These advantages of breeding chips make them an indispensable tool in modern breeding programs, helping to improve breeding efficiency, shorten breeding cycles, and ultimately achieve genetic improvement goals. With the continuous advancement of genomics technology, the application of breeding chips will become more extensive and play a greater role in agricultural production.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to solve some limitations in traditional breeding methods, and to use the progress of molecular genetics to improve the efficiency and accuracy of animal and plant breeding, to use the method of whole genome association analysis to find molecular markers and haplotypes related to pig breed identification, to provide a molecular marker combination related to pig breed identification and its application, so as to at least solve one of the technical problems existing in the prior art.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: The present invention provides an application of a pig SNP molecular marker combination in pig breed identification or in the preparation of a pig breed identification product; the SNP molecular marker combination includes sites in the sites shown in Table 2, The reference genome is: Sscrofa11.1.

[0008] Furthermore, the pig breeds include Landrace pigs, Large White pigs, Duroc pigs, wild boars and Chinese local pigs.

[0009] The present invention also provides the use of the detection substance in pig breed identification or in the preparation of pig breed identification products, wherein the detection substance is used to detect the above pig SNP molecular marker combination.

[0010] Furthermore, the detection substance includes primers, probes, chips or detection equipment.

[0011] The present invention also provides a pig breed identification kit, comprising one or more of primers, probes, chips and sequencing reagents for detecting the above pig SNP molecular marker combination.

[0012] The present invention also provides a method for identifying pig breeds, comprising obtaining the site information of the pig SNP molecular marker combination of the individual to be detected, and performing breed identification according to the site information.

[0013] In addition, the present invention also provides the use of the above-mentioned pig SNP molecular marker combination, pig breed identification kit or pig breed identification method in pig breed identification; The pig breeds include Landrace, Large White, Duroc, Wild Boar and Chinese Native Pig.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention applies the whole genome association analysis method to 18 wild boar breeds and 42 domestic pig breeds, and develops a SNP molecular marker combination for accurate identification of multiple pig breeds. The detection of this combination can accurately and efficiently identify multiple pig breeds with an accuracy of 99.9%. The present invention is expected to provide new breed identification technology means for the needs of pig breed rights identification, germplasm resource protection and utilization, genetic evolution research, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 A diagram showing the minimum set statistics results of simulated individual identification provided by an embodiment of the present invention; Figure 2 A result diagram showing the relationship between the number of SSR loci and accuracy for variety identification provided in an embodiment of the present invention; Figure 3This is a graph showing the relationship between the number of SNP sites and accuracy for variety identification provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0017] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0018] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0019] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] According to one aspect of the present invention, there is provided a use of a pig SNP molecular marker combination in pig breed identification or in the preparation of a pig breed identification product; the SNP molecular marker combination includes the sites shown in Table 2. The reference genome is: Sscrofa11.1.

[0021] The present invention applies the whole genome association analysis method to 18 wild boar breeds and 42 domestic pig breeds, and develops a SNP molecular marker combination for accurate identification of multiple pig breeds. The detection of this combination can accurately and efficiently identify multiple pig breeds. When the number of selected sites is at least 82, the accuracy can reach 99.9%. The present invention is expected to provide new breed identification technology means for the needs of pig breed rights identification, germplasm resource protection and utilization, genetic evolution research, etc.

[0022] In an optional embodiment, the pig breeds include Landrace pigs, Large White pigs, Duroc pigs, wild boars and Chinese local pigs.

[0023] The present invention also provides the use of the detection substance in pig breed identification or in the preparation of pig breed identification products, wherein the detection substance is used to detect the above pig SNP molecular marker combination.

[0024] In an optional embodiment, the detection substance includes a primer, a probe, a chip or a detection device.

[0025] The primers are used to amplify the SNP sites in Table 2. The detection material may include multiple primer pairs, each primer pair amplifies any site in Table 2, and at least one primer pair amplifies a different SNP site from the other primer pairs.

[0026] In an optional embodiment, the primers include several primer pairs capable of amplifying at least 82 sites in Table 2.

[0027] In an optional embodiment, the primers include primers for nucleic acid amplification methods based on any principle acceptable in the art, such as but not limited to primers for PCR amplification, primers for fluorescent quantitative PCR amplification, or primers for isothermal nucleic acid amplification.

[0028] In an optional embodiment, the target fragment amplified by the primer covers the fragment defined by the position range of the SNP site in Table 2 in the reference sequence.

[0029] In an optional embodiment, the probe binds to the gene fragment where the SNP site is located, or the probe binds to the target fragment amplified by the primer.

[0030] In an optional embodiment, the chip contains several probes that bind to the SNP sites.

[0031] The sequencing reagent is used to sequence the amplified fragment of the primer to obtain the SNP site information in the amplified fragment.

[0032] It is understood that the detection substance also includes optional reagents for supporting detection, including but not limited to reagents for nucleic acid amplification reaction, DNA extraction reagents, target fragment detection reagents, sequencing library construction reagents, sequencing reagents and reference substances, etc. More specific examples include but are not limited to one or more of enzymes (polymerase, reverse transcriptase or recombinase), buffer components, metal ions, salts, fluorescent dyes, surfactants, dNTPs, color developers, gels, adapter sequences, magnetic beads, positive controls, negative controls and blank controls.

[0033] In an optional embodiment, the detection device includes, but is not limited to, a device for DNA extraction, a device for nucleic acid amplification, a device for amplification product analysis, or a device for sequencing.

[0034] The present invention also provides a pig breed identification kit, comprising one or more of primers, probes, chips and sequencing reagents for detecting the above pig SNP molecular marker combination.

[0035] The sequencing reagent is used to sequence the amplified fragment of the primer to obtain the SNP site information in the amplified fragment.

[0036] It is understood that the kit also includes optional reagents for supporting detection, including but not limited to reagents for nucleic acid amplification reaction, DNA extraction reagents, target fragment detection reagents, sequencing library construction reagents, sequencing reagents and reference substances, etc. More specific examples include but are not limited to one or more of enzymes (polymerase, reverse transcriptase or recombinase), buffer components, metal ions, salts, fluorescent dyes, surfactants, dNTPs, color developers, gels, adapter sequences, magnetic beads, positive controls, negative controls and blank controls.

[0037] The present invention also provides a method for identifying pig breeds, comprising obtaining the site information of the pig SNP molecular marker combination of the individual to be detected, and performing breed identification according to the site information.

[0038] In addition, the present invention also provides the use of the above-mentioned pig SNP molecular marker combination, pig breed identification kit or pig breed identification method in pig breed identification; The pig breeds include Landrace, Large White, Duroc, Wild Boar and Chinese Native Pig.

[0039] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market. Example 1

[0040] 1. Data preparation: The pig genome sequence of Sscrofa11.1 version was downloaded from NCBI as the reference genome of the present invention.

[0041] Resequencing data of 111 wild boars from 20 countries and resequencing data of 1082 pigs from 63 Chinese and foreign domestic pig breeds were obtained from the EBI database, totaling 1193 individuals.

[0042] 2. Data acquisition, quality control and other pre-processing The STR identification of Sscrofa11.1 reference genome was performed by MISA, and the preliminary screening data was filtered to obtain STR candidate loci. The upstream and downstream of the filtered STR candidate loci were extended by 100bp as the reference sequence for subsequent resequencing STR typing. Pig resequencing data information was collected from published literature. Aspera was used to download the resequencing data from the EBI database to the Linux server. Fastp was used to perform data quality control on the resequencing data.

[0043] 3. SNP and STR detection, typing and filtering The processed resequencing data are sequenced with the STR candidate reference sequence and the reference genome respectively through BWA. The resequencing data aligned to the STR candidate reference sequence are detected and typed by HipSTR. Based on the typing results, the polymorphic information content (PIC), allele frequency (AF) and number of genotypes of each locus are calculated. The resequencing data aligned to the reference genome are detected and typed by GATK. The STRs and SNPs of the initially screened resequencing data are filtered and screened to obtain the resequencing candidate STR and SNP sites. The specific filtering conditions are as follows: STR: The screened STR loci were first filtered for missing rate, and the loci with missing rate greater than 30% were filtered out. After filtering, they were arranged from large to small according to PIC, and the first 128 STR loci were selected as partial loci for chip design.

[0044] SNPs: (1) The selected SNP sites are filtered as follows: the missing rate cannot be higher than 0, the minimum allele frequency cannot be lower than 5%, the PIC cannot be lower than 0.4, the Havenberg equilibrium test cannot be lower than 1e-5, and the distance between two SNP sites cannot be lower than 100K (if it exists, one site will be discarded); (2) After filtering the remaining sites, the random forest model of machine learning was used to perform variety identification training. Based on the scoring of the training results output by the model, the top 966 SNP sites with higher scores were selected as the remaining sites for chip design.

[0045] 4. Result analysis: Table 1:

[0046] Table 2:

[0047] The resequencing data of 111 wild boars from 20 countries and 1082 resequencing data of 63 Chinese local and foreign domestic pig breeds were obtained from the EBI database, totaling 1193 individuals. Among the 1193 resequencing data, the present invention selected 18 wild boar breeds and 42 domestic pig breeds, totaling 1084 samples as the data set. The present invention detected a total of 74,353 STR loci and 184,050,820 SNP loci, and then the filtering steps listed above were performed.

[0048] For the molecular markers used for individual identification, the present invention extracts the top 1000 sites with STR / SNP PIC values ​​for minimum set comparison. When the minimum set number of STR markers is 6, individual distinction can be made, which is much smaller than SNP markers. STR is more suitable for individual identification.

[0049] Specifically, extract the top 1000 sites with STR / SNP pic values, start from the 2-site set (X-axis), simulate randomly selecting 2 sites from the screened STR / SNP data set, randomly select 10,000 times, and count the number of sites that can distinguish all sample sets in 10,000 times (Y-axis). After calculating the 2 sites, repeatedly calculate 3, 4, 5... until the results of 1000 sites are obtained, as shown in the figure. Figure 1 shown.

[0050] For the molecular markers used for variety identification, the present invention has compared the accuracy of machine learning training of STR / SNP sites. First, the present invention found that when the number of variant sites is not reduced, the variety identification accuracy of STR (90%) is lower than the accuracy of SNP (99.9%). The present invention also found that when the number of SNP sites is reduced to 82 sites, the accuracy is still 99.9%, while the accuracy begins to drop significantly when the number of STR sites is reduced to 390 sites. The number of STR sites used for variety identification is significantly higher than the number of SNP sites. The results are as follows: Figure 2 and Figure 3 As shown, Figure 2 and Figure 3The left main axis is the accuracy of the machine learning training model, corresponding to the green line, the right secondary axis is the number of variant sites used in training, corresponding to the yellow curve, and the horizontal axis is the number of cycles. Therefore, SNP is more suitable for variety identification. Example 2 Blind test to verify the accuracy of the SNP molecular marker combination of the present invention in pig breed identification

[0051] The SNP molecular marker combination of the present invention was used to test 30 pigs to obtain the genotyping information of the SNP marker combination of each individual. The identification results were compared with the sample breed information to calculate the identification accuracy.

[0052] Experimental results: 30 samples obtained genotyping, and a total of 10 Duchangda pigs, 3 Large White pigs, 8 Bama Xiang pigs, and 9 Tibetan pigs were identified. The accuracy rate was 93.33% when compared with the breed information registered with the samples.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of a pig SNP molecular marker combination in pig breed identification or preparation of pig breed identification products; the SNP molecular marker combination includes the sites shown in the following table: ， The reference genome is: Sscrofa11.

1.

2. The use according to claim 1, characterized in that: The pig breeds include Landrace, Large White, Duroc, Wild Boar and Chinese Native Pig.

3. Use of a detection substance in pig breed identification or in the preparation of pig breed identification products, wherein the detection substance is used to detect the pig SNP molecular marker combination according to claim 1 or 2.

4. The use according to claim 3, characterized in that: The detection substance includes primers, probes, chips or detection equipment.

5. A pig breed identification kit, characterized in that: It comprises one or more of primers, probes, chips and sequencing reagents for detecting the pig SNP molecular marker combination according to any one of claims 1 to 3.

6. A method for identifying pig breeds, characterized in that: It includes obtaining the site information of the pig SNP molecular marker combination of the individual to be tested, and performing breed identification based on the site information; The pig SNP molecular marker combination is the pig SNP molecular marker combination according to claim 1 or 2.

7. Use of the pig SNP molecular marker combination according to claim 1 or 2, the pig breed identification kit according to claim 5 or the pig breed identification method according to claim 6 in pig breed identification; The pig breeds include Landrace, Large White, Duroc, Wild Boar and Chinese Native Pig.

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