Molecular markers, primers and their applications for parentage testing and individual identification of sika deer

Abstract

The present invention provides molecular markers, primers and applications for sika deer paternity testing and individual identification, aiming to solve the practical application problems existing in the existing sika deer paternity testing. The present invention provides 16 highly polymorphic sites, including 15 four-base repeat sites and 1 six-base repeat site. The polymorphic information content of the 16 highly polymorphic sites is between 0.645 and 0.892, with an average of 0.7428. When the 16 sites are used for sika deer paternity testing, the three combined exclusion probabilities are respectively; 0.999824, 0.999999, and greater than 0.999999, all of which are greater than the universal value of 0.9973 for international paternity testing. When 8 of the sites are used for individual identification, the cumulative individual identification rate is greater than 99.99%, and the cumulative full sibling identification rate is greater than 99.97%. The present invention overcomes the problem of inability to accurately identify existing methods, is accurate and efficient for sika deer paternity testing and individual identification, has practical application value, and can be used in multiple sika deer varieties, with great application prospects.

Description

Technical Field

[0001] The invention belongs to the field of animal paternity testing and individual identification, and specifically relates to a molecular marker, a primer and an application thereof for paternity testing and individual identification of sika deer. Background Art

[0002] Sika deer (Cervus Nippon) belongs to the class Mammalia, order Artiodactyla, family Cervidae, genus Cervus. There are currently six subspecies of sika deer in China, including the Taiwan subspecies, the Northeast subspecies, the North China subspecies, the Shanxi subspecies, the Sichuan subspecies, and the South China subspecies. my country has a long history of domesticating sika deer. The rich resources of sika deer provided conditions for early capture and domestication, which can be traced back to the Shang Dynasty before the Common Era. Artificial breeding also has a history of more than 300 years. The sika deer raised in China mainly come from the Northeast Asia. According to relevant data, the total number of sika deer raised in my country is about 1.1 million, with the largest number of sika deer raised in Jilin Province, accounting for about 50% of the country's total. After years of artificial breeding, there are 7 varieties and one strain of sika deer that have passed the identification of the National Animal Husbandry and Poultry Committee, namely Aodong sika deer, Siping sika deer, Dongfeng sika deer, Xingkai Lake sika deer, Shuangyang sika deer, Dongda sika deer, Xifeng sika deer and Changbai Mountain sika deer breeding strain.

[0003] Sika deer breeding is carried out in some grassroots units, and there are problems such as lack of professional guidance, lack of breeding data records and unclear pedigree records. In order to further improve the production capacity of deer, breeding companies blindly introduce blood sources of other breeds, resulting in disordered hybridization between breeds, which has threatened the safety of germplasm resources and subsequent sika deer breeding work.

[0004] The development and application of sika deer paternity testing and individual identification technology can correct pedigree errors and assist in the establishment of complete pedigree archives, which is of great significance to the scientific selection and conservation of sika deer, breed improvement, and the formulation of breeding plans. Currently, microsatellite molecular marker technology is the most widely used in paternity testing and individual identification research of humans and other livestock.

[0005] Microsatellites, also known as short tandem repeats or simple sequence repeats (SSRs), are composed of a core sequence of 1 to 6 nucleotides as the basic repeating unit and the conserved flanking sequences on both sides, and are evenly distributed throughout the genome. The polymorphism of microsatellite markers lies in the core sequence. The more times the core sequence is repeated, the stronger the variability of the corresponding microsatellite locus and the greater the number of alleles. The conserved sequences on both sides of the repeat sequence are the embodiment of microsatellite specificity and are often used to design primers. Microsatellites are ideal molecular markers with the following characteristics: (1) High abundance. Microsatellites are widely distributed and abundant in the genome, with an average of 1 microsatellite per 5 to 10 kb. There are 1 million microsatellite loci in the human genome, accounting for about 3% of the total genome; (2) It is codominantly inherited, in accordance with Mendel's law of inheritance, and can easily distinguish between homozygous and heterozygous genes; (3) The degree of polymorphism is high. The more times a microsatellite is repeated, the easier it is to mutate, and the higher the polymorphism it has. In addition, the mutation rate of four-base repeat microsatellites is higher than that of two-base microsatellites; (4) The amount of DNA required is small, and the requirement for DNA quality is low. Microsatellites can use DNA samples from different sources, such as feces, fur, hair, tissues, and other samples that are easy to degrade or have been degraded. Microsatellites can be used for analysis; (5) Because the flanking sequences of microsatellites are relatively conservative, they have a certain degree of versatility and can be used for the amplification of closely related species. In addition, microsatellite markers also have the characteristics of simple typing, convenient operation, and relatively low application cost. Therefore, microsatellites are widely used in the study of population genetic diversity, genetic map construction, kinship identification, and marker-assisted breeding.

[0006] At present, the existing research methods for parentage identification of deer in China are based on 10 highly polymorphic microsatellite loci screened from the original data of simplified genome sequencing. Among the 10 loci, 6 are di-base repeats and 4 are tetra-base repeats. When the amplified repeat unit is di-base or tri-base repeat, in addition to the main band of the PCR product, a shadow band smaller than the main band will appear. Its existence complicates the correct reading of the alleles of the microsatellite loci and easily reads non-existent alleles. Therefore, the existing sika deer parentage identification method is not conducive to allele typing. When the genotypes of both parents are unknown, the cumulative exclusion probability is only 95.45%, which is far lower than the international paternity test universal value of 0.9973, and cannot meet practical applications. Summary of the invention

[0007] In view of the above problems in the paternity test of sika deer, the present invention has conducted a more in-depth study on the molecular markers of sika deer and developed a group of better high polymorphic sites. The 16 high polymorphic sites of the present invention include 15 four-base repeat sites and 1 six-base repeat site. The present invention can completely solve the problem of the inability to accurately identify the paternity test of sika deer in the current method, and fully meet the needs of actual detection.

[0008] The present invention first provides a group of SSR markers for sika deer parentage identification and individual identification, and the SSR marker locus information is as follows:

[0009] Table 1 Information of 16 microsatellite loci

[0010]

[0011]

[0012] The present invention also provides a primer set for sika deer parentage identification and individual identification, comprising 16 pairs of primer pairs, the sequences of the primer pairs are shown in SEQ ID NO.1-32 respectively; the primer set is used to amplify the SSR marker of claim 1. The 5' end of the primer carries a fluorescent marker (FAM, HEX, TAMRAD and ROX), and the specific information of the primer is shown in Table 2.

[0013] Table 2 Information of 16 microsatellite primers

[0014]

[0015]

[0016] The present invention also provides a method for paternity testing or individual identification of sika deer, using part or all of Table 2 as primers, and using sika deer genomic DNA as a template to perform fluorescent primer PCR reactions to obtain fluorescent PCR products, and then performing capillary electrophoresis and genotyping to determine whether the samples are in a parent-child relationship or whether they are derived from the same individual.

[0017] Furthermore, the present invention also provides a kit for paternity testing and / or individual identification of sika deer, the kit comprising the primer set in Table 2.

[0018] Furthermore, the present invention also provides a method for using the above-mentioned kit for sika deer parentage identification and / or individual identification. When the genotypes of the two parents to be identified are known, 5-16 pairs of primers can be used for identification; the 5 pairs of primers are primers for amplifying MHL06, MHL25, MHL44, MHL30, and MHL58 sites; when the genotypes of the two parents to be identified are known, CPE-P>99.99%.

[0019] When the genotype of one of the two parents to be identified is known, 9-16 pairs of primers can be used for identification: the 9 pairs of primers are primers for amplifying MHL06, MHL25, MHL44, MHL30, MHL58, MHL02, MHL03, MHL84, and MHL34 sites; in the method, when the genotype of one of the two parents to be identified is known, CPE-2>99.99%.

[0020] When the genotypes of both parents were unknown, all 16 primer pairs were used for parentage testing, and CPE-1 was >99.98%.

[0021] When used for sika deer individual identification, 5-8 pairs of primers can be used; the 5 pairs of primers are primers for amplifying the 5 loci MHL06, MHL25, MHL44, MHL30 and MHL58; when 5 pairs of primers are used, PID<0.000001, PID(sib) is 0.004446, and the minimum number of loci required for sika deer individual identification can be achieved;

[0022] The 8 pairs of primers are primer pairs for amplifying the 8 loci MHL02, MHL03, MHL06, MHL25, MHL30, MHL44, MHL58 and MHL84. When the 8 pairs of primers are used, the cumulative individual recognition rate is greater than 99.99%, and the cumulative full-sibling recognition rate is greater than 99.97%.

[0023] Beneficial effects:

[0024] 1. The present invention obtains highly polymorphic microsatellite loci and microsatellite primers based on whole genome screening, which are used for sika deer parentage identification and individual identification. The present invention includes 15 four-base repeat sites and 1 six-base repeat site. Studies have shown that with the increase in the number of repeating unit bases, the shadow bands outside the main band of the PCR product will gradually decrease, the distinction of polymorphism will become more obvious, and the genotyping results will be more accurate. The polymorphic information content of the 16 highly polymorphic loci of the present invention is between 0.645 and 0.892, with an average of 0.7428. When the number of loci used is 16, the three combined exclusion probabilities are; 0.999824, 0.999999, and greater than 0.999999, which are all greater than the universal value of 0.9973 for international paternity testing.

[0025] 2. The present invention has a wider practical application scope. In order to improve the accuracy and practicality of microsatellite primers, the present invention collects blood samples of multiple sika deer species for verification when verifying the primers, which fully proves the universal applicability of the primers of the present invention. It can be used in multiple sika deer species, expanding its application scope.

[0026] 3. When the present invention is applied to the identification of sika deer individuals, only 5-8 pairs of primers are needed, which greatly improves the detection efficiency and saves costs.

[0027] 4. The present invention has practical application value. The microsatellite markers selected by the present invention can provide rich genetic information, and the site combination has a high binding exclusion rate. It can be applied to parentage testing and individual identification to obtain accurate results. It is helpful to build pedigrees and clarify families in actual production, and provide a basis for the selection and breeding of sika deer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the distribution map of the 16 SSR marker loci of the present invention on chromosomes. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited thereto. The methods involved in the present invention are all commonly used methods in the art unless otherwise specified, and the reagents involved can be obtained from commercial sources unless otherwise specified.

[0030] Example 1: Acquisition of SSR markers for sika deer genome

[0031] Based on 136 sika deer whole genome sequences (the genome resequencing data was measured by our laboratory in the early stage, and the sika deer genome data has been uploaded to the GSA database), the screening steps for polymorphic microsatellite loci are as follows:

[0032] (1) 354,766 microsatellite loci were identified using MISA software;

[0033] (2) A total of 419,115 microsatellite loci were identified using the TRF software;

[0034] (3) The intersection of the microsatellite loci identified by the two software (1) and (2) was taken, and a total of 61,621 microsatellite loci were obtained;

[0035] (4) Screening sites with repeating unit base numbers of 4, 5, and 6 and a repeat number of 4 or more, a total of 11,258 microsatellite sites were obtained;

[0036] (5) After removing the chromosome double-terminal loci, 8915 microsatellite loci remained;

[0037] (6) Hip-STR software was used to perform STR detection on the 136 sika deer genome resequencing data obtained in the laboratory in the early stage and calculate the polymorphic information content. Sites with polymorphic information content greater than 0.7 were retained and then screened according to the principle of uniform chromosome distribution, resulting in a total of 198 sites;

[0038] (7) sites with repetitive sequences containing three or more consecutive base repeats (e.g., TTTA) were removed, leaving 143 microsatellite sites;

[0039] (8) Combining the 143 loci obtained above and the 31 microsatellite loci in the STR molecular markers and their applications developed by our research group based on the whole genome of sika deer in CN201710599880.2, 88 microsatellite loci evenly distributed on different chromosomes were selected from the above-mentioned loci, and primers were designed using the online version of primer3 (http: / / primer3.ut.ee).

[0040] (9) Among the 88 primer pairs, 42 pairs of primers with clear amplification bands were screened out, and the size of the amplified fragment was consistent with the target fragment.

[0041] (10) 240 sika deer samples were amplified and genotyped by capillary electrophoresis using 42 pairs of primers with good amplification effects. The genotyping data were sorted using Excel, manually corrected, and imported into the software Cervus3.0.7 using the AlleleFrequencyAnalysis program to calculate the polymorphic information content, expected heterozygosity, observed heterozygosity, and number of alleles for each marker. The results showed that 21 loci were highly polymorphic (PIC>0.5) and could provide rich genetic information.

[0042] (11) After arranging the obtained 21 polymorphic sites in descending order according to PIC, three kinds of combination exclusion probabilities are calculated in turn, as shown in Table 3. When the number of sites is 5, the combination exclusion probability 3 (CPE-P) is greater than 99.99%, that is, when the genotypes of both parents are known, these 5 sites can be used for paternity testing; when the number of sites is 9, the combination exclusion probability 2 (CPE-2) is greater than 99.99%, that is, when the genotype of one of the two parents is known, these 9 sites can be used for paternity testing; when the number of sites is 16, the combination exclusion probability 1 (CPE-1) reaches more than 99.98%, and when the genotypes of both parents are unknown, 16 microsatellite sites can be used for paternity testing analysis. Finally, the present invention selected the 16 microsatellite sites shown in Table 1. The above Table 1 lists the specific location information of the 16 microsatellite sites on the chromosome, including the number of repeated bases and the specific bases repeated. Figure 1 This is the distribution map of 16 microsatellite loci on chromosomes.

[0043] Table 3 Results of the combined exclusion probability analysis of 16 microsatellite loci

[0044]

[0045]

[0046] Example 2: Verification of STR markers for paternity testing of sika deer

[0047] 1 Primer synthesis

[0048] 16 pairs of microsatellite primers were synthesized for sika deer parentage identification. The 5' ends of the primers were labeled with different fluorescent markers (FAM, HEX, TAMRAD and ROX). The specific information of the primers is shown in Table 2 above.

[0049] 2. Genomic DNA Extraction

[0050] Blood samples (10 ml / sika deer) were collected from 103 pairs of parent-offspring with artificial pedigree records. Genomic DNA was extracted from the blood using a high-throughput magnetic bead nucleic acid extractor. The concentration and purity were measured using an ultra-micro spectrophotometer. After passing the test, the samples were stored in a -20°C refrigerator.

[0051] 3PCR amplification

[0052] The 16 primer pairs were used to perform PCR amplification using the sika deer genome as a template to establish a sika deer parentage identification method. The PCR amplification conditions are shown in Table 4.

[0053] Table 4 PCR amplification conditions

[0054]

[0055] The PCR reaction system is shown in Table 5.

[0056] Table 5 PCR system

[0057]

[0058] 4 Capillary electrophoresis genotyping

[0059] The Simulation of Parentage Analysis function in Cervus3.0.7 software was used to simulate the identification of the father. The number of simulated offspring was set to 10,000. In the case of only the genotype of the father, the matching numbers under the loose (80%) and strict (95%) confidence levels were both 9,966, and the matching ratio reached 99.66%. When the genotype of the mother was known and the matching of the father was performed, the matching numbers under the loose (80%) and strict (95%) confidence levels were both 9,998, and the matching ratio reached 99.98%. From this result, it can be seen that these 16 microsatellite loci can be used for subsequent parentage identification, and the identification results are reliable. Then the Parentage Analysis function was used to identify the father of the Zuojia sika deer breeding farm. The results are shown in Table 6. All 103 fawns in the Zuojia sika deer breeding farm were matched to the best father, the number of mismatched sites was ≤1, and the matching rate reached 100%. This shows that the 16 pairs of microsatellite primers of the present invention can be used for parentage identification of sika deer.

[0060] Table 6 Identification results of progeny father

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Note: In the confidence level of parent-child relationship assignment, * indicates strict confidence level

[0067] Example 3: Verification of SSR markers for identifying sika deer individuals

[0068] 1. Genomic DNA Extraction

[0069] A total of 50 sika deer were collected for the test, including 26 sika deer blood samples and fresh antler samples, with the blood samples and antler samples corresponding to the numbers; another 24 sika deer blood and hair follicle samples were collected, and the 24 hair follicle samples were randomly numbered ZJ-1 to ZJ-24. All samples came from the National Jilin Sika Deer Germplasm Resources Conservation Farm of the Specialty Products Research Institute of the Chinese Academy of Agricultural Sciences. The genomic DNA of sika deer hair follicles and antlers was extracted using the trace sample genomic DNA extraction kit (DP316) of Tiangen Biotechnology Co., Ltd. The operation steps were carried out according to the instructions of the kit, and the concentration and purity were determined by an ultra-micro spectrophotometer. After passing the test, it was stored in a -20℃ refrigerator.

[0070] 2 Primer synthesis

[0071] Eight pairs of microsatellite primers for sika deer parentage identification were synthesized, with different fluorescent labels (FAM, HEX, TAMRAD and ROX) at the 5' end of the primers. The eight pairs of primers are primers for amplifying MHL02, MHL03, MHL06, MHL25, MHL30, MHL44, MHL58 and MHL84 loci, respectively. The specific information of the primers is shown in Table 2.

[0072] 3PCR amplification

[0073] The 8 pairs of primers were used for PCR amplification using the sika deer genome as a template to establish a sika deer individual identification method. The PCR amplification conditions are shown in Table 7. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. in the dark and at low temperature. Capillary electrophoresis genotyping was performed using an ABI3730XL sequencer. The data were sorted in Excel and analyzed for individual identification using Cervus3.0.7 software.

[0074] Table 7 PCR amplification conditions

[0075]

[0076] The PCR reaction system is shown in Table 8.

[0077] Table 8 PCR system

[0078]

[0079] 4 Capillary electrophoresis genotyping

[0080] Using the above microsatellite locus genotyping data, individual identification was performed through the IdentityAnalysis function in Cervus3.0.7 software. The results of using 5 loci to identify 26 sika deer and their corresponding velvet samples (Table 9) showed that all individual blood samples and velvet samples were completely and accurately matched, with a matching accuracy of 100%. When 8 loci were used to identify 24 sika deer and 24 random hair follicle samples from the same source (Table 10), the results showed that all individuals were matched to a unique hair follicle sample, and the number of mismatched sites was 0. In summary, when the five loci MHL06, MHL25, MHL44, MHL30 and MHL58 were combined, PID < 0.000001, PID (sib) was 0.004446, reaching the minimum number of loci required for sika deer individual identification. When 8 loci are used in combination, the cumulative individual recognition rate is greater than 99.99%, and the cumulative full-sibling recognition rate is greater than 99.97%. Even if there are full-sibling individuals in the group, the probability of failure to recognize is only 0.000243. If more loci are added, the improvement in recognition rate is also minimal. Through verification, 5 loci can be applied to the actual individual recognition of sika deer. In actual applications, loci can be added according to the situation of the sample to be identified.

[0081] Table 9 Identification results of 26 sika deer blood samples and velvet skin

[0082]

[0083]

[0084] Table 10 Identification results of 24 sika deer blood and hair follicle samples

[0085]

[0086] Sequence Listing <110> Specialty Products Research Institute, Chinese Academy of Agricultural Sciences <120> Molecular markers, primers and their applications for parentage testing and individual identification of sika deer <141> 2022-06-20 <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial sequence <400> 1 accccaagca gcttttgac 19 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 tatggcaaaa agccatcaca 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 gtgtggccaa caggctaagt 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 caaacccaca cttcctccat 20 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <400> 5 tcccatctca actatctacc t 21 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <400> 6 ctactttaca cccatttcct a 21 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 ggtagcgggt agacagtgga 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <400> 8 ccaaggtggc catagtcagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <400> 9 attgaacctg ggtctcatgc 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 ccagatccat ctgagccact 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <400> 11 gatggagcct gtgggagata 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <400> 12 tctctggtga aggccatctt 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <400> 13 tcatttccat gtgcttgaca 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <400> 14 aggtgggcaa gcaaaatatg 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <400> 15 gctgcataat ttggggctta 20 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <400> 16 aatccctccc tgaaccattc 20 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <400> 17 ccaaaagcat tcagggagaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <400> 18 tcgcaaagag ttggacaaga 20 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <400> 19 ggaaatggtg aaggacagga 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <400> 20 aggggcaaga gattggagat 20 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <400> twenty one atcatgcaga ccacaagcag 20 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <400> twenty two tccattgcta ccagttgctg 20 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <400> twenty three atgttgctgc aaatggcata 20 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <400> twenty four gccaagacat gggaagtgat 20 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <400> 25 cagggagctg ctgtgtgata 20 <210> 26 <211> 20 <212> DNA <213> Artificial sequence <400> 26 agtggtgaag aacccgactg 20 <210> 27 <211> 20 <212> DNA <213> Artificial sequence <400> 27 gtccatggga ctttccagac 20 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <400> 28 tggcaggcag attctttctt 20 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <400> 29 accccattcc tcctttggta 20 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <400> 30 ctaagccacc cagcaatctc 20 <210> 31 <211> 20 <212> DNA <213> Artificial sequence <400> 31 atcagcacat ctgcatttgg 20 <210> 32 <211> 20 <212> DNA <213> Artificial sequence <400> 32 cgggtttgga ttagaatgga 20

Claims

1. SSR markers used for parentage testing and individual identification of sika deer, characterized by: The SSR marker locus information is as follows: The sequences of the SSR marker sites are amplified by 16 pairs of primers whose sequences are shown as SEQ ID NO.1-32.

2. A primer set for paternity testing and individual identification of sika deer, characterized in that: It comprises 16 pairs of primers, and the sequences of the primer pairs are respectively shown as SEQ ID NO.1-32; the primer set is used to amplify the SSR marker described in claim 1.

3. The primer set for parentage identification and individual identification of sika deer as claimed in claim 2, characterized in that: The 5' end of the primer is fluorescently labeled.

4. A method for identifying paternity of sika deer, characterized in that: Using the primer set described in claim 2 or 3, fluorescent primer PCR reactions are performed with sika deer genomic DNA as a template to obtain fluorescent PCR products, and then capillary electrophoresis and genotyping are performed to determine whether the samples are parent-child relationships.

5. A method for identifying sika deer individuals, characterized in that: By using the primer set described in claim 2 or 3, fluorescent primer PCR reactions are performed with sika deer genomic DNA as a template to obtain fluorescent PCR products, and then capillary electrophoresis and genotyping are performed to determine whether the samples are derived from the same individual.

6. A kit for paternity testing and / or individual identification of sika deer, characterized in that: The kit comprises the primer set according to claim 2 or 3.

7. The method for using the kit according to claim 6, characterized in that: When the genotypes of the two parents to be identified are known, 5 pairs of primers are used for identification; the 5 pairs of primers are primers for amplifying MHL06, MHL25, MHL44, MHL30, and MHL58 sites; the sequences of the 5 pairs of primers are shown in SEQ ID NO.7-8, SEQ ID NO.11-12, SEQ ID NO.21-22, SEQID NO.15-16, and SEQ ID NO.23-24, respectively; according to the method, when the genotypes of the two parents to be identified are known, CPE-P>99.99%.

8. The method for using the kit according to claim 6, characterized in that: When the genotype of one of the two parents to be identified is known, 9 pairs of primers are used for identification: the 9 pairs of primers are primers for amplifying MHL06, MHL25, MHL44, MHL30, MHL58, MHL02, MHL03, MHL84, and MHL34 sites; the sequences of the 9 pairs of primers are respectively shown in SEQ ID NO.7-8, SEQ ID NO.11-12, SEQ ID NO.21-22, SEQ ID NO.15-16, SEQ ID NO.23-24, SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.31-32, and SEQ ID NO.17-18; in the method, when the genotype of one of the two parents to be identified is known, CPE-2>99.99%.

9. The method for using the kit according to claim 6, characterized in that: When the genotypes of both parents are unknown, all 16 pairs of primers are used for parentage testing, and CPE-1>99.98%; the sequences of the 16 pairs of primers are shown in SEQ ID NOs. 1-32 respectively.

10. The method for using the kit according to claim 6, characterized in that: When used for sika deer individual identification: 5 pairs of primers are used, and the 5 pairs of primers are primers for amplifying the 5 loci MHL06, MHL25, MHL44, MHL30 and MHL58; when 5 pairs of primers are used, PID<0.000001, PID(sib) is 0.004446, which can achieve the minimum number of loci required for sika deer individual identification; The sequences of the five pairs of primers are shown as SEQ ID NO.7-8, SEQ ID NO.11-12, SEQ ID NO.21-22, SEQ ID NO.15-16, and SEQ ID NO.23-24, respectively; 8 pairs of primers are used, and the 8 pairs of primers are primer pairs for amplifying 8 sites, namely, MHL02, MHL03, MHL06, MHL25, MHL30, MHL44, MHL58, and MHL84; when 8 pairs of primers are used, the cumulative individual recognition rate is greater than 99.99%, and the cumulative full-sibling recognition rate is greater than 99.97%; the sequences of the 8 pairs of primers are respectively shown in SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.7-8, SEQ ID NO.11-12, SEQ ID NO.15-16, SEQ ID NO.21-22, SEQ ID NO.23-24, and SEQ ID NO.31-32.

Images