Screening method of pig STR marker and application of pig STR marker in pig genotyping

Abstract

The invention provides a screening method of a pig STR marker and application of the pig STR marker in pig genotyping, and relates to the technical field of biology. The screening method comprises the following steps: (A) obtaining sequencing data of a plurality of pig genomes, and taking one of the sequencing data as a reference genome; (B) performing STR identification on the reference genome through MISA, and filtering preliminary screening data to obtain STR candidate sites; and (C) taking a fragment consisting of the filtered STR candidate sites and upstream and downstream flanking sequences as a reference sequence for subsequent STR typing. According to the screening method, the polymorphic pig STR marker for individual recognition and paternity test can be found in a pig genome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for screening pig STR markers and application of pig STR markers in pig gene typing. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Short Tandem Repeats (STRs) are a type of repetitive sequence widely found in DNA. The repeating units consisting of 2 to 6 nucleotides are repeated in tandem in the genome. STR loci are highly polymorphic in individuals, that is, different individuals may have different numbers of repeats at the same locus. STRs have important application value in individual identification due to their high polymorphism and genetic stability.

[0004] In the field of forensics, STR analysis is a common technique used in forensic medicine for personal identification and paternity testing. By detecting the allele size of a specific STR locus, an individual's genetic fingerprint can be generated. Using STR, the biological relationship between parents and children can be determined. Since each child inherits a set of STR alleles from both parents, parent-child relationships can be verified by comparison. In missing persons cases, STR analysis can be used to compare unidentified remains with reference samples provided by family members to help confirm identity. In medical research, STR can also be used to identify susceptibility genes for genetic diseases or genetic variants associated with specific diseases.

[0005] In agriculture and animal husbandry, STR is used to identify the breed and purity of plants and animals and to protect genetic resources. STR polymorphism can be used to study the genetic structure, migration patterns, and population history of a population. In large biobanks, STR is used to track and manage samples to ensure the uniqueness and traceability of samples. STR analysis can help construct an individual's genetic pedigree and understand family inheritance patterns and the transmission paths of genetic diseases.

[0006] The study of STR molecular markers in pigs has some applications in multiple fields. Through STR analysis, researchers evaluate the genetic diversity of different pig breeds, reveal the genetic differences between breeds and the genetic structure within the population. For example, the study may focus on the genetic differences between local breeds and commercial breeds to provide data support for the protection of genetic resources. STR markers are used to distinguish different pig breeds and determine their classification and origin. For example, STR analysis is used to distinguish European and Asian pig breeds and study their genetic relationships and evolutionary history. In pig breeding management, STR is used for parentage testing to ensure the accuracy of breeding plans. By analyzing multiple STR loci, pedigrees are constructed to evaluate genetic diversity and inbreeding. STR markers are an important tool for constructing genetic linkage maps. For example, researchers may use hundreds of STR markers to construct high-resolution genetic maps, providing a basis for subsequent gene positioning and QTL analysis. STR markers are used to study pig resistance to specific diseases, such as swine fever, PRRS, etc. Research may focus on identifying genetic markers associated with disease resistance to provide information for breeding.

[0007] At present, there are few studies on pig STR in terms of individual identification and paternity testing. In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide a porcine STR marker that can be used for individual identification and parentage testing.

[0009] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0010] In a first aspect, a method for screening porcine STR markers is provided, comprising the following steps:

[0011] (A) obtaining sequencing data of several pig genomes and using one of them as a reference genome;

[0012] (B) STR identification of the reference genome was performed using MISA, and the preliminary screening data was filtered to obtain STR candidate loci;

[0013] (C) The fragment consisting of the filtered STR candidate loci and upstream and downstream flanking sequences is used as the reference sequence for subsequent STR typing.

[0014] In a second aspect, a use of a porcine STR marker in porcine genotyping or the preparation of a porcine genotyping product is provided, wherein the porcine STR marker is obtained by the screening method described in the first aspect.

[0015] In a third aspect, the invention also provides the use of a detection substance in pig genotyping or in the preparation of a pig genotyping product, wherein the detection substance is used to detect the pig STR marker described in the second aspect.

[0016] In a fourth aspect, a porcine genotyping kit is also provided, which comprises one or more of primers, probes, chips and sequencing reagents for detecting the porcine STR markers described in the second aspect.

[0017] In a fifth aspect, a porcine genotyping detection device is also provided, the porcine genotyping detection device comprising: a data acquisition module and a data analysis module; the data acquisition module is used to obtain the site information of the porcine STR markers described in the second aspect of the individual to be detected;

[0018] The data analysis module records all allele information of the pig STR markers described in the second aspect. When the data analysis module is running, the locus information of the pig STR markers of the individual to be detected is compared with all allele information recorded by the data analysis module, and the genotyping of the individual to be detected at the STR locus is output.

[0019] In a sixth aspect, the present invention further provides the use of the method for screening pig STR markers described in the first aspect, the pig genotyping kit described in the fourth aspect, or the pig genotyping detection device described in the fifth aspect in any one of (I) to (IV):

[0020] (I) Pig genotyping;

[0021] (II) Individual identification or products prepared for use in individual identification;

[0022] (III) kinship identification or preparation of products for kinship identification;

[0023] (IV) Screening of germplasm resources or preparation of products for screening of germplasm resources.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention uses the whole genome resequencing analysis method to perform a whole genome analysis on the polymorphism of pig STR, and for the first time finds polymorphic pig STR markers in the pig genome for individual identification and paternity testing. The detection of the pig STR markers can distinguish the genotypes of individual pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1The minimum set statistics for simulating individual identification of the STR loci selected in Example 1. DETAILED DESCRIPTION

[0028] 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.

[0029] In a first aspect, a method for screening porcine STR markers is provided, comprising the following steps:

[0030] (A) Sequencing data of several pig genomes are obtained and one of them is used as a reference genome.

[0031] (B) STR identification was performed on the reference genome by MISA, and the preliminary screening data were filtered to obtain STR candidate loci.

[0032] (C) The fragment consisting of the filtered STR candidate loci and upstream and downstream flanking sequences is used as the reference sequence for subsequent STR typing.

[0033] In an optional embodiment, sequencing data of at least 100 to 1000 individuals are obtained in step (A), for example, may be but is not limited to at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000.

[0034] In an optional embodiment, the sequencing data of the pig genome includes pig resequencing data.

[0035] In an optional embodiment, Sscrofa11.1 is used as a reference genome.

[0036] In an optional embodiment, the upstream flanking sequence in step (C) is 50 to 150 bp, for example, but not limited to 50, 80, 100, 120 or 150 bp, preferably 100 bp.

[0037] In an optional embodiment, the downstream flanking sequence in step (C) is 50 to 150 bp, for example, but not limited to 50, 80, 100, 120 or 150 bp, preferably 100 bp.

[0038] In an optional embodiment, in step (C), the filtered STR candidate sites are extended by 100 bp upstream and downstream respectively as reference sequences for subsequent resequencing STR typing.

[0039] In an optional embodiment, the filtering in step (B) includes extracting the top 1000 sites in terms of STR / SNP PIC values.

[0040] In a second aspect, a use of a porcine STR marker in porcine genotyping or preparation of a porcine genotyping product is provided, wherein the porcine STR marker is obtained by the screening method described in the first aspect.

[0041] In an optional embodiment, the porcine STR markers include one or more of the sites shown in the following table.

[0042] Table 1 Information on pig STR marker loci

[0043]

[0044]

[0045] The reference genome is: Sscrofa11.1. The position range in the reference sequence represents the possible position range of the STR site in the genome.

[0046] In an optional embodiment, the porcine STR markers include at least 10 STR loci as shown in Table 1, for example, but not limited to, at least 10, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80 or all loci.

[0047] In an optional embodiment, the alleles of each STR locus in Table 1 include the range defined by the starting position to the ending position in the corresponding chromosome of the reference genome Sscrofa11.1, and also include the following alleles:

[0048] The allele of the ps011072 site also includes a sequence as shown in any one of SEQ ID NOs. 1 to 13; and / or, the allele of the ps011073 site also includes a sequence as shown in any one of SEQ ID NOs. 14 to 25; and / or, the allele of the ps011074 site also includes a sequence as shown in any one of SEQ ID NOs. 26 to 42; and / or, the allele of the ps011075 site also includes a sequence as shown in any one of SEQ ID NOs. 43 to 53; and / or, the allele of the ps011076 site also includes a sequence as shown in any one of SEQ ID NOs. 54 to 71; and / or, the allele of the ps011077 site also includes a sequence as shown in any one of SEQ ID NOs. 72 to 94; and / or, the allele of the ps011078 site also includes a sequence as shown in any one of SEQ ID NOs. 85 to 112; and / or, the allele of the ps011079 site also includes a sequence as shown in any one of SEQ ID NOs. NO.113-123; and / or, the allele at the ps011080 site also includes a sequence as shown in any one of SEQ ID NOs.124-135; and / or, the allele at the ps011081 site also includes a sequence as shown in any one of SEQ ID NOs.136-149; and / or, the allele at the ps011082 site also includes a sequence as shown in any one of SEQ ID NOs.150-165; and / or, the allele at the ps022245 site also includes a sequence as shown in any one of SEQ ID NOs.166-174; and / or, the allele at the ps022246 site also includes a sequence as shown in any one of SEQ ID NOs.175-188; and / or, the allele at the ps022247 site also includes a sequence as shown in any one of SEQ ID NOs.189-196; and / or, the allele at the ps022248 site also includes a sequence as shown in any one of SEQ ID NOs. NO.197-205; and / or, the allele at the ps022249 site also includes a sequence as shown in any one of SEQ ID NOs.206-220; and / or, the allele at the ps022250 site also includes a sequence as shown in any one of SEQ ID NOs.221-236; and / or, the allele at the ps022251 site also includes a sequence as shown in any one of SEQ ID NOs.237-249; and / or, the allele at the ps022252 site also includes a sequence as shown in any one of SEQ ID NOs.250-257; and / or, the allele at the ps022253 site also includes a sequence as shown in any one of SEQ ID NOs.258-265; and / or, the allele at the ps022254 site also includes a sequence as shown in any one of SEQ ID NOs.266-278; and / or, the allele at the ps022255 site also includes a sequence as shown in SEQ ID NO.279-287; and / or, the allele at the ps024677 site also includes a sequence as shown in any one of SEQ ID NOs.288-306; and / or, the allele at the ps024678 site also includes a sequence as shown in any one of SEQ ID NOs.307-317; and / or, the allele at the ps024679 site also includes a sequence as shown in any one of SEQ ID NOs.318-326; and / or, the allele at the ps024680 site also includes a sequence as shown in any one of SEQ ID NOs.327-338; and / or, the allele at the ps024681 site also includes a sequence as shown in any one of SEQ ID NOs.339-348; and / or, the allele at the ps024682 site also includes a sequence as shown in any one of SEQ ID NOs.349-362; and / or, the allele at the ps024683 site also includes a sequence as shown in SEQ ID NO.363-371; and / or, the allele at the ps024684 site also includes a sequence as shown in any one of SEQ ID NOs.372-387; and / or, the allele at the ps024685 site also includes a sequence as shown in any one of SEQ ID NOs.388-401; and / or, the allele at the ps024686 site also includes a sequence as shown in any one of SEQ ID NOs.402-416; and / or, the allele at the ps024687 site also includes a sequence as shown in any one of SEQ ID NOs.417-427; and / or, the allele at the ps035695 site also includes a sequence as shown in any one of SEQ ID NOs.428-446; and / or, the allele at the ps035696 site also includes a sequence as shown in any one of SEQ ID NOs.447-459; and / or, the allele at the ps035697 site also includes a sequence as shown in any one of SEQ ID NOs. NO.460-475; and / or, the allele at the ps035698 site also includes a sequence as shown in any one of SEQ ID NOs.476-488; and / or, the allele at the ps035699 site also includes a sequence as shown in any one of SEQ ID NOs.489-499; and / or, the allele at the ps035700 site also includes a sequence as shown in any one of SEQ ID NOs.500-511; and / or, the allele at the ps035701 site also includes a sequence as shown in any one of SEQ ID NOs.512-518; and / or, the allele at the ps035702 site also includes a sequence as shown in any one of SEQ ID NOs.519-532; and / or, the allele at the ps035703 site also includes a sequence as shown in any one of SEQ ID NOs.533-542; and / or, the allele at the ps035704 site also includes a sequence as shown in SEQ ID NO.543-558; and / or, the allele at the ps035705 site also includes a sequence as shown in any one of SEQ ID NOs.559-568; and / or, the allele at the ps040072 site also includes a sequence as shown in any one of SEQ ID NOs.569-579; and / or, the allele at the ps040073 site also includes a sequence as shown in any one of SEQ ID NOs.580-590; and / or, the allele at the ps040074 site also includes a sequence as shown in any one of SEQ ID NOs.591-600; and / or, the allele at the ps040075 site also includes a sequence as shown in any one of SEQ ID NOs.601-610; and / or, the allele at the ps040076 site also includes a sequence as shown in any one of SEQ ID NOs.611-618; and / or, the allele at the ps040077 site also includes a sequence as shown in SEQ ID NO.619-636; and / or, the allele at the ps040078 site also includes a sequence as shown in any one of SEQ ID NOs.637-644; and / or, the allele at the ps040079 site also includes a sequence as shown in any one of SEQ ID NOs.645-660; and / or, the allele at the ps040080 site also includes a sequence as shown in any one of SEQ ID NOs.661-671; and / or, the allele at the ps040081 site also includes a sequence as shown in any one of SEQ ID NOs.672-681; and / or, the allele at the ps040082 site also includes a sequence as shown in any one of SEQ ID NOs.682-688; and / or, the allele at the ps040867 site also includes a sequence as shown in any one of SEQ ID NOs.689-696; and / or, the allele at the ps040868 site also includes a sequence as shown in SEQ ID NOs. NO.697-711; and / or, the allele at the ps040869 site also includes a sequence as shown in any one of SEQ ID NOs.712-725; and / or, the allele at the ps040870 site also includes a sequence as shown in any one of SEQ ID NOs.726-739; and / or, the allele at the ps040871 site also includes a sequence as shown in any one of SEQ ID NOs.740-757; and / or, the allele at the ps040872 site also includes a sequence as shown in any one of SEQ ID NOs.758-771; and / or, the allele at the ps040873 site also includes a sequence as shown in any one of SEQ ID NOs.772-777; and / or, the allele at the ps040874 site also includes a sequence as shown in any one of SEQ ID NOs.778-791; and / or, the allele at the ps040875 site also includes a sequence as shown in SEQ ID NO.792-803; and / or, the allele at the ps040876 site also includes a sequence as shown in any one of SEQ ID NOs.804-817; and / or, the allele at the ps040877 site also includes a sequence as shown in any one of SEQ ID NOs.818-830; and / or, the allele at the ps060959 site also includes a sequence as shown in any one of SEQ ID NOs.831-838; and / or, the allele at the ps060960 site also includes a sequence as shown in any one of SEQ ID NOs.839-847; and / or, the allele at the ps060961 site also includes a sequence as shown in any one of SEQ ID NOs.848-855; and / or, the allele at the ps060962 site also includes a sequence as shown in any one of SEQ ID NOs.856-864; and / or, the allele at the ps060963 site also includes a sequence as shown in SEQ ID NO.865-870; and / or, the allele at the ps060964 site also includes a sequence as shown in any one of SEQ ID NOs.871-883; and / or, the allele at the ps060965 site also includes a sequence as shown in any one of SEQ ID NOs.884-892; and / or, the allele at the ps060966 site also includes a sequence as shown in any one of SEQ ID NOs.893-904; and / or, the allele at the ps060967 site also includes a sequence as shown in any one of SEQ ID NOs.905-910; and / or, the allele at the ps060968 site also includes a sequence as shown in any one of SEQ ID NOs.911-920; and / or, the allele at the ps060969 site also includes a sequence as shown in any one of SEQ ID NOs.921-928; and / or, the allele at the ps063255 site also includes a sequence as shown in SEQ ID NOs. NO.929-938; and / or, the allele at the ps063256 site also includes a sequence as shown in any one of SEQ ID NOs.939-949; and / or, the allele at the ps063257 site also includes a sequence as shown in any one of SEQ ID NOs.950-963; and / or, the allele at the ps063258 site also includes a sequence as shown in any one of SEQ ID NOs.964-970; and / or, the allele at the ps063259 site also includes a sequence as shown in any one of SEQ ID NOs.971-979; and / or, the allele at the ps063260 site also includes a sequence as shown in any one of SEQ ID NOs.980-987; and / or, the allele at the ps063261 site also includes a sequence as shown in any one of SEQ ID NOs.988-999; and / or, the allele at the ps063262 site also includes a sequence as shown in SEQ ID NO.1000-1008; and / or, the allele at the ps063263 site also includes a sequence as shown in any one of SEQ ID NOs. 1009-1017; and / or, the allele at the ps063264 site also includes a sequence as shown in any one of SEQ ID NOs. 1018-1031; and / or, the allele at the ps063265 site also includes a sequence as shown in any one of SEQ ID NOs. 1032-1045. .

[0049] In a third aspect, the invention also provides the use of a detection substance in pig genotyping or in the preparation of a pig genotyping product, wherein the detection substance is used to detect the pig STR marker described in the second aspect.

[0050] In an optional embodiment, the detection substance includes one or more of a primer, a probe, a chip, a sequencing reagent and a detection device.

[0051] In an optional embodiment, the primers are used to amplify the STR sites in Table 1, and the detection material may include multiple primer pairs, each primer pair amplifies any site in Table 1, and at least one primer pair amplifies a different STR site from the other primer pairs.

[0052] In an optional embodiment, the primers include several primer pairs capable of amplifying at least 10 sites in Table 1.

[0053] 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.

[0054] In an optional embodiment, the target fragment amplified by the primers covers the fragment defined by the position range of the STR loci in the reference sequence in Table 1.

[0055] In an optional embodiment, the probe binds to the gene fragment where the STR locus is located, or the probe binds to the target fragment amplified by the primer.

[0056] In an optional embodiment, the chip contains several probes that bind to the STR sites.

[0057] In an optional embodiment, the sequencing reagent is used to sequence the amplified fragment of the primer to obtain the STR site information in the amplified fragment.

[0058] It is understood that the detection material 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.

[0059] 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.

[0060] In a fourth aspect, a pig genotyping kit is also provided, which comprises one or more of primers, probes, chips and sequencing reagents for detecting the pig STR markers described in the second aspect.

[0061] In an optional embodiment, the primers are used to amplify the STR sites in Table 1, and the detection material may include multiple primer pairs, each primer pair amplifies any site in Table 1, and at least one primer pair amplifies a different STR site from the other primer pairs.

[0062] In an optional embodiment, the primers include several primer pairs capable of amplifying at least 10 sites in Table 1.

[0063] 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.

[0064] In an optional embodiment, the target fragment amplified by the primers covers the fragment defined by the position range of the STR loci in the reference sequence in Table 1.

[0065] In an optional embodiment, the probe binds to the gene fragment where the STR locus is located, or the probe binds to the target fragment amplified by the primer.

[0066] In an optional embodiment, the chip contains several probes that bind to the STR sites.

[0067] In an optional embodiment, the sequencing reagent is used to sequence the amplified fragment of the primer to obtain the STR site information in the amplified fragment.

[0068] It is understood that the porcine genotyping 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.

[0069] In a fifth aspect, a porcine genotyping detection device is also provided, the porcine genotyping detection device comprising: a data acquisition module and a data analysis module;

[0070] The data acquisition module is used to obtain the site information of the pig STR markers described in the second aspect of the individual to be detected.

[0071] In an optional embodiment, the locus information of the pig STR marker includes but is not limited to one or more of the nucleotide sequence of the STR locus, the core sequence and the number of core sequence repeats of the STR locus, the length of the fragment after amplification of the STR locus, and the nucleotide sequence of the fragment after amplification of the STR locus.

[0072] The data analysis module records all allele information of the pig STR markers described in the second aspect. When the data analysis module is running, the locus information of the pig STR markers of the individual to be detected is compared with all allele information recorded by the data analysis module, and the genotyping of the individual to be detected at the STR locus is output.

[0073] In an optional embodiment, all allele information of pig STR markers recorded by the data analysis module includes, but is not limited to, the nucleotide sequence of each allele at the STR site; the core sequence and the number of core sequence repeats of each allele at the STR site; the fragment length of each allele after amplification of the STR site using the same primers as those of the individual to be detected; and one or more of the nucleotide sequence of each allele fragment after amplification of the STR site using the same primers as those of the individual to be detected.

[0074] In an optional embodiment, the data analysis module records the site information in Table 1 and all the allele sequences of SEQ ID NOs. 1 to 1045.

[0075] In an optional embodiment, the pig genotyping detection device further includes a DNA extraction module, and the DNA extraction module is used to extract a DNA sample of the individual to be detected.

[0076] In an optional embodiment, the porcine genotyping detection device further comprises a nucleic acid amplification module, and the nucleic acid amplification module is used to amplify porcine STR markers in the DNA template.

[0077] In a sixth aspect, the present invention further provides the use of the method for screening pig STR markers described in the first aspect, the pig genotyping kit described in the fourth aspect, or the pig genotyping detection device described in the fifth aspect in any one of (I) to (IV):

[0078] (I) Pig genotyping;

[0079] (II) Individual identification or products prepared for use in individual identification;

[0080] (III) kinship identification or preparation of products for kinship identification;

[0081] (IV) Screening of germplasm resources or preparation of products for screening of germplasm resources.

[0082] In an optional embodiment, the pig genotyping includes obtaining the locus information of the pig STR markers described in the second aspect of the individual to be tested, and performing genotyping according to the locus information.

[0083] In an optional embodiment, the locus information of the pig STR marker of the individual to be detected is obtained, including but not limited to one or more of the nucleotide sequence of the STR locus, the core sequence and the number of core sequence repeats of the STR locus, the length of the fragment after amplification of the STR locus, and the nucleotide sequence of the fragment after amplification of the STR locus.

[0084] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0085] Example 1

[0086] 1. Data preparation:

[0087] The porcine genome sequence of version Sscrofa11.1 was downloaded from NCBI as our reference genome.

[0088] 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.

[0089] 2. Data acquisition, quality control and other pre-processing

[0090] Sscrofa11.1 was obtained from NCBI as our reference genome. STR identification was performed on the Sscrofa11.1 reference genome 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 100 bp each as the reference sequence for subsequent resequencing STR typing. Pig resequencing data information was collected from published literature. Resequencing data were downloaded from the EBI database to the Linux server using Aspera. Data quality control of resequencing data was performed using fastp.

[0091] 3. STR detection and typing

[0092] The processed resequencing data were aligned with the STR candidate reference sequence and the reference genome by BWA. The resequencing data aligned to the STR candidate reference sequence were tested and typed by HipSTR. Based on the typing results, the PIC, AF and number of genotypes of each locus were calculated.

[0093] 4. Results Analysis

[0094] We obtained resequencing data of 111 wild boars from 20 countries and resequencing data of 1082 resequencing data of 63 Chinese local and foreign domestic pig breeds from the EBI database, totaling 1193 individuals. Among the 1193 resequencing data, we selected 18 wild boar breeds and 42 domestic pig breeds, totaling 1084 samples as the data set. We detected 68,862 STR loci with a repeat unit of 2bp, of which AT had the highest proportion; 5122 STR loci with a repeat unit of 3bp, of which AAT had the highest proportion; 369 STR loci with a repeat unit of 4bp, of which CCCA had the highest proportion. The top 1000 loci with STR / SNP PIC values ​​were extracted 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, such as Figure 1 shown. Figure 1 To extract the top 1000 sites with the STR / SNP pic values, start with 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, repeat the calculation of 3, 4, 5... until the results of 1000 sites are reached.

[0095] In summary, we explored the STR characteristics in the pig genome, identified STR loci with differences between and within breeds, and provided new insights into the diversity and potential functions of STR variation in the pig genome. As second-generation and third-generation molecular markers, STR markers and SNP markers have significant advantages in individual identification and pedigree identification. In order to improve the accuracy of STR in individual identification, we recommend selecting 10 STR loci as a combination for individual identification, and for this purpose we recommend 88 loci as a candidate collection library (Table 2).

[0096] Table 2 Information on pig STR marker loci

[0097]

[0098]

[0099]

[0100] Table 3 Allele information of each STR locus

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] 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. A method for screening porcine STR markers, characterized in that: The steps include: (A) obtaining sequencing data of several pig genomes and using one of them as a reference genome; (B) STR identification of the reference genome was performed using MISA, and the preliminary screening data was filtered to obtain STR candidate loci; (C) The fragment consisting of the filtered STR candidate loci and upstream and downstream flanking sequences is used as the reference sequence for subsequent STR typing.

2. The screening method according to claim 1, characterized in that In step (A), sequencing data of at least 100 to 1000 individuals are obtained; Optionally, the sequencing data of the pig genome includes pig resequencing data; Optionally, Sscrofa11.1 is used as a reference genome.

3. The screening method according to claim 1, characterized in that The upstream and downstream flanking sequences in step (C) are independently 50 to 150 bp.

4. The screening method according to claim 1, characterized in that The filtering in step (B) includes extracting the top 1000 sites in terms of STR / SNPPIC values.

5. Use of porcine STR markers in porcine genotyping or in the preparation of porcine genotyping products, wherein the porcine STR markers are obtained by the screening method according to any one of claims 1 to 4.

6. The use according to claim 5, characterized in that: The porcine STR markers include one or more of the loci shown in the following table: The reference genome is: Sscrofa11.

1.

7. Use of a detection substance in pig genotyping or in the preparation of a pig genotyping product, wherein the detection substance is used to detect the pig STR marker according to any one of claims 5 or 6.

8. A pig genotyping kit, characterized in that: The method comprises one or more of primers, probes, chips and sequencing reagents for detecting the porcine STR markers according to any one of claims 5 or 6.

9. A pig genotyping detection device, characterized in that: Including data acquisition module and data analysis module; The data acquisition module is used to obtain the site information of the pig STR marker of any one of claims 5 or 6 of the individual to be detected; The data analysis module records all allele information of the pig STR markers described in any one of claims 5 or 6. When the data analysis module is running, the locus information of the pig STR markers of the individual to be detected is compared with all the allele information recorded by the data analysis module, and the genotyping of the individual to be detected at the STR locus is output.

10. Use of the method for screening porcine STR markers according to any one of claims 1 to 4, the porcine genotyping kit according to claim 8, or the porcine genotyping detection device according to claim 9 in any one of (I) to (IV): (I) Pig genotyping; (II) Individual identification or products prepared for use in individual identification; (III) kinship identification or preparation of products for kinship identification; (IV) Screening of germplasm resources or preparation of products for screening of germplasm resources.

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