Przewalski's gazelle SSR molecular marker combination, primer combination, kit and application

Abstract

The present invention provides a Przewalski's gazelle SSR molecular marker combination, a primer combination, a kit and an application, and relates to the technical field of molecular biology. Based on the whole genome sequencing data of Przewalski's gazelle, the present invention performs SSR primer design and SSR site screening, and the obtained 26 pairs of SSR primers can stably amplify the target product and are highly polymorphic, and can be used for the detection of genetic diversity of Przewalski's gazelle population, the analysis of population genetic structure, and the study of evolution and kinship. The polymorphic SSR molecular marker combination of Przewalski's gazelle obtained by screening can be used for individual identification of Przewalski's gazelle with high accuracy. The experimental results show that the SSR molecular marker combination of Przewalski's gazelle of the present invention can meet the needs of individual identification, and 33 fecal samples of Przewalski's gazelle subadults are individually identified, which come from 24 different individuals, which is consistent with the number of Przewalski's gazelle subadults in the Jiangxigou Przewalski's gazelle rescue center.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a Przewalski's gazelle SSR molecular marker combination, a primer combination, a kit and an application. Background Art

[0002] Przewalski's gazelle (Procapraprzewalskii), belonging to the order Artiodactyla, family Bovidae, genus Procaprzewalskii, is a unique animal on the Qinghai-Tibet Plateau. It is now only distributed in the area around Qinghai Lake. It is the flagship species of the Qinghai Lake Basin and a first-class key protected wild animal in my country. With the implementation of various protection measures and the strengthening of protection efforts, the population of Przewalski's gazelle has recovered rapidly. In May 2022, the data released by the Qinghai Provincial Forestry and Grassland Bureau showed that there were more than 2,800 adult individuals of Przewalski's gazelle in the wild.

[0003] Simple sequence repeats (SSR), also known as microsatellite sequences, are repeating units of 1 to 6 nucleotides. Multiple repetitions can produce tandem repeat sequences, which exist almost throughout the genome. The differences in repeating units and numbers of repetitions between different alleles make them polymorphic.

[0004] At present, many scholars have conducted extensive research on the population distribution and population size, habitat selection, feeding strategies and diet, and stress factors of Przewalski's gazelle. Some scholars have also used mitochondrial molecular markers to study the genetic diversity of Przewalski's gazelle populations. However, there are no reports on the use of SSR markers to identify Przewalski's gazelle individuals in the prior art. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a four-base SSR molecular marker combination, a primer combination, a kit and an application of Przewalski's gazelle. The present invention develops polymorphic SSR markers based on the whole genome sequencing data of Przewalski's gazelle, meeting the needs of individual identification of Przewalski's gazelle.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a Przewalski's gazelle SSR molecular marker combination, which includes one or more of PR-6, PR-7, PR-8, PR-10, PR-12, PR-14, PR-16, PR-22, PR-25, PR-26, PR-28, PR-30, PR-40, PR-42, PR-46, PR-53, PR-58, PR-63, PR-64, PR-65, PR-69, PR-71, PR-72, PR-85, PR-86, and PR-97; the SSR markers are amplified in sequence by the following primer pairs, and the sequences of the primer pairs are as shown in SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52.

[0008] Preferably, the SSR molecular marker combinations are PR-6, PR-8, PR-12, PR-22, PR-25, PR-30, PR-40, PR-58, PR-63, PR-69, PR-71, PR-72, PR-85, PR-86 and PR-97.

[0009] The present invention also provides a primer combination for SSR molecular markers of Przewalski's gazelle, comprising any one or more of the following primer pairs, wherein the sequences of the primer pairs are as follows: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-45 Shown in NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52.

[0010] The present invention also provides a kit for identifying Przewalski's gazelle individuals, comprising the above-mentioned SSR molecular marker primer combination.

[0011] Preferably, the kit further comprises a genome extraction reagent and a PCR reaction reagent.

[0012] Preferably, the kit further comprises reagents for capillary electrophoresis.

[0013] The present invention also provides the use of the above-mentioned SSR molecular marker primer combination or the above-mentioned kit in the individual identification analysis of Przewalski's gazelle.

[0014] The present invention also provides the use of the above-mentioned SSR molecular marker primer combination or the above-mentioned kit in the detection of genetic diversity of Przewalski's gazelle population.

[0015] The present invention also provides a method for identifying Przewalski's gazelle individuals, comprising: extracting DNA from a Przewalski's gazelle sample, performing PCR amplification on the genomic DNA using the above-mentioned primers, and performing capillary electrophoresis to determine the genotype of the above-mentioned SSR molecular marker sites for individual identification; when the genotypes of the SSR molecular marker sites are all the same or only one genotype at one site is different, they are determined to be the same individual.

[0016] Preferably, the sample comprises stool or tissue.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention designs SSR primers and screens SSR sites based on the whole genome sequencing data of Przewalski's gazelle. The obtained 26 pairs of SSR primers can stably amplify the target product and are highly polymorphic, which can be used for genetic diversity detection, population genetic structure analysis, evolution and kinship research of Przewalski's gazelle population. The screened polymorphic SSR molecular marker combination of Przewalski's gazelle can be used for individual identification of Przewalski's gazelle with high accuracy.

[0019] The experimental results show that the SSR molecular marker combination of Przewalski's gazelle of the present invention can meet the needs of individual identification. 33 fecal samples of sub-adult Przewalski's gazelle were individually identified, which came from 24 different individuals, which is consistent with the number of sub-adult Przewalski's gazelles in the Jiangxigou Przewalski's gazelle rescue center. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 33 fecal samples of sub-adult Przewalski's gazelle;

[0021] Figure 2 The agarose gel electrophoresis of the total genomic DNA of some fecal samples of Przewalski's gazelle; the leftmost is the 100bp DNA Ladder (Dye Plus) (TaKaRa), and 1-15 are the DNA samples of Przewalski's gazelle;

[0022] Figure 3 The allele frequencies of 26 SSR loci in 15 Przewalski's gazelle samples;

[0023] Figure 4 To screen the consistency probability of 15 SSR loci for individual identification;

[0024] Figure 5 Capillary electrophoresis of SSR loci of Przewalski's gazelle individual identification samples 2501 (A), 3201 (B), 3203 (C), and 3306 (D). DETAILED DESCRIPTION

[0025] The present invention provides a Przewalski's gazelle SSR molecular marker combination, which includes one or more of PR-6, PR-7, PR-8, PR-10, PR-12, PR-14, PR-16, PR-22, PR-25, PR-26, PR-28, PR-30, PR-40, PR-42, PR-46, PR-53, PR-58, PR-63, PR-64, PR-65, PR-69, PR-71, PR-72, PR-85, PR-86, and PR-97; the SSR markers are amplified in sequence by the following primer pairs, and the sequences of the primer pairs are as shown in SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52.

[0026] In the present invention, the SSR molecular marker combination is preferably PR-6, PR-8, PR-12, PR-22, PR-25, PR-30, PR-40, PR-58, PR-63, PR-69, PR-71, PR-72, PR-85, PR-86 and PR-97. The consistency probability test results show that the above 15 SSR molecular marker combinations can meet the individual identification needs.

[0027] The present invention also provides a primer combination for SSR molecular markers of Przewalski's gazelle, comprising any one or more of the following primer pairs, wherein the sequences of the primer pairs are as follows: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-45 Shown in NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52.

[0028] The more alleles there are at each locus, the richer the population polymorphism. A total of 143 alleles were detected in the amplification products of the SSR molecular marker primer combination of the Przewalski's gazelle of the present invention, and the number of alleles ranged from 2 (PR-97) to 10 (PR-14). It can be seen that the above-mentioned 26 SSR molecular marker primer combinations of Przewalski's gazelle of the present invention can stably amplify the target product and are highly polymorphic, and can be used for genetic diversity detection of Przewalski's gazelle populations, population genetic structure analysis, evolution and kinship research, etc.

[0029] The present invention also provides a kit for identifying Przewalski's gazelle individuals, comprising the above-mentioned SSR molecular marker primer combination.

[0030] The kit of the present invention also preferably includes a genome extraction reagent and a PCR reaction reagent.

[0031] The kit of the present invention preferably also includes reagents for capillary electrophoresis.

[0032] The present invention also provides the use of the above-mentioned SSR molecular marker primer combination or the above-mentioned kit in the individual identification analysis of Przewalski's gazelle.

[0033] The present invention uses 15 loci in the SSR molecular marker combination of Przewalski's gazelle to identify individuals of 33 sub-adult fecal samples of Przewalski's gazelle. The experimental results show that the 33 sub-adult fecal samples of Przewalski's gazelle come from 24 different individuals, which is consistent with the number of sub-adult Przewalski's gazelles in the Jiangxigou Przewalski's gazelle rescue center.

[0034] The present invention also provides the use of the above-mentioned SSR molecular marker primer combination or the above-mentioned kit in the detection of genetic diversity of Przewalski's gazelle population.

[0035] The above 26 Przewalski's gazelle SSR molecular marker primer combinations of the present invention can stably amplify the target product and are highly polymorphic, and can be used for genetic diversity detection, population genetic structure analysis, evolution and kinship research of Przewalski's gazelle populations, etc.

[0036] The present invention also provides a method for identifying Przewalski's gazelle individuals, comprising: extracting DNA from a Przewalski's gazelle sample, performing PCR amplification on the genomic DNA using the above-mentioned primers, and performing capillary electrophoresis to determine the genotype of the above-mentioned SSR molecular marker sites for individual identification; when the genotypes of the SSR molecular marker sites are all the same or only one genotype at one site is different, they are determined to be the same individual.

[0037] The sample of the present invention preferably includes feces or tissue, more preferably feces; the tissue more preferably includes fur, muscle, and blood.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] The experimental samples, experimental reagents, experimental instruments and equipment used in the embodiments of the present invention are as follows:

[0040] 1. Experimental samples

[0041] The 15 fecal samples of Przewalski's gazelle used for polymorphic SSR site screening were collected from Haiyan County, Gonghe County and Gangcha County of Qinghai Province. After being collected in the field, the fresh fecal samples were immediately stored in liquid nitrogen. After returning to the laboratory, they were transferred to a -80℃ refrigerator for storage until the total genomic DNA was extracted.

[0042] Individual identification samples were collected from the Jiangxigou Przewalski's gazelle rescue center. In April 2022, fecal samples of sub-adult Przewalski's gazelles were collected at the rescue center for 4 consecutive days, twice a day, to ensure that fecal samples of all sub-adult Przewalski's gazelles were collected. A total of 33 sub-adult fecal samples (such as Figure 1 shown).

[0043] 2. Experimental reagents

[0044] QIAamp Fast DNA Stool Mini Kit, TaKaRa Ex Taq Hot Start Version, anhydrous ethanol, 100 bp DNA Ladder, 6× loading buffer, 50× TAE, agarose, ethidium bromide (EB).

[0045] 3. Experimental instruments and equipment

[0046] ABI 3730XL genetic analyzer, ABI Veriti temperature gradient PCR instrument, Nanodrop 2000C spectrophotometer, Bio-rad electrophoresis system, Bio-rad gel imaging system, Millipore pure water system, high-speed refrigerated centrifuge, high-pressure sterilizer, 4℃ refrigerator, -20℃ refrigerator, -80℃ refrigerator, vortex oscillator, ice maker, constant temperature water bath, electronic balance, eppendorf pipette, microwave oven.

[0047] Example 1

[0048] Extraction and detection of total DNA from feces of Przewalski's gazelle

[0049] The kit method was adopted, and the kit used was QIAamp Fast DNA Stool (51604).

[0050] Preparation stage Ensure that Buffer AW1 and Buffer AW2 have been prepared according to the instructions on the kit label. Mix the buffers well before use. If a precipitate forms in Buffer ASL or Buffer AL, heat in a 70°C water bath until dissolved.

[0051] (1) DNA extraction First, weigh 180-220 mg of feces into a 2 mL centrifuge tube and place it on ice for pretreatment.

[0052] (2) Add 1 mL of Inhibit EX Buffer to the sample and vortex for 1 to 2 min until the sample is completely homogenized.

[0053] (3) Centrifuge at full speed of 14,000 rpm for 1.5 min to allow the feces to settle to the bottom of the centrifuge tube.

[0054] (4) Take 25 μL of proteinase K into a new 2 mL microcentrifuge tube.

[0055] (5) Pipette 600 μL of supernatant from the centrifuge tube in step 3 into a 2 mL centrifuge tube containing proteinase K.

[0056] (6) Add 600 μL of Buffer AL and vortex for 15 seconds to thoroughly mix the solution.

[0057] (7) Incubate at 70°C for 10 min, inverting 1-2 times to mix well and reduce droplets on the centrifuge tube cap. Place on ice to cool.

[0058] (8) Add 600 μL of pre-cooled anhydrous ethanol solution to the lysate and vortex to mix. Reduce the droplets on the centrifugal collection tube cap.

[0059] (9) Take 600 μL of the solution obtained in the previous step and add it to an adsorption column (the adsorption column is placed in a 2 mL collection tube). Centrifuge at full speed of 14,000 rpm for 1.5 min, pour out the waste liquid, and place the adsorption column in a new collection tube.

[0060] (10) Open the column cap, add another 600 μL of lysis buffer, centrifuge at 14,000 rpm for 1.5 min, discard the waste liquid, and place the adsorption column in a new collection tube.

[0061] (11) Repeat the previous step and load the third 600 μL lysate onto the column. Centrifuge at 14,000 rpm for 1.5 min, discard the waste liquid, and place the adsorption column into a new collection tube.

[0062] (12) Add 500 μL of Buffer AW1 to the column. Close the centrifuge tube cap tightly and centrifuge at full speed of 14000 rpm for 1.5 min. Remove the centrifuge tube and place it in a new 2 mL collection tube. Discard the old collection tube and the liquid in it.

[0063] (13) Add 500 μL of Buffer AW2 to the column. Close the centrifuge tube cap tightly and centrifuge at full speed of 14,000 rpm for 3 min. Remove the centrifuge tube and place it in a new 2 mL collection tube. Discard the old collection tube and the liquid in it.

[0064] (14) Take out the centrifuge tube, place it in a new 2 mL collection tube, and centrifuge at 14,000 rpm for 3 min. Transfer the column to a new 1.5 mL centrifuge tube, carefully add 200 μL Buffer ATE, let stand at room temperature for 2 min, and centrifuge at 14,000 rpm for 2 min to elute the DNA. Quickly place the DNA sample in the 1.5 mL centrifuge tube in a -20°C refrigerator.

[0065] After DNA extraction, the DNA concentration was measured by Nanodrop 2000C spectrophotometer, and the DNA quality was tested by 1% agarose gel electrophoresis. The specific results are shown in Figure 2 and Table 1.

[0066] Table 1 Results of total genomic DNA concentration test

[0067]

[0068] Depend on Figure 2 It can be seen that the extracted genomic total DNA has a bright main band, indicating that the DNA quality is relatively complete. As shown in Table 1, the total DNA concentration of the Przewalski's gazelle genome is between 2.6 (sample 3309) and 57.7 (sample 3302) ng / μL, which can be used for subsequent PCR experiments.

[0069] Example 2

[0070] Initial screening of SSR primers and detection of polymorphism

[0071] 1. SSR selection and primer design

[0072] The MISA microsatellite screening software was used to scan the entire genome of Przewalski's gazelle, and SSR loci with a repeat sequence of 4-6bp and a repeat number greater than 8 were identified, and a total of 1632 SSR loci that met the conditions were obtained. From the SSR loci with a repeat unit of 4bp and a repeat number between 10 and 70, 5 to 6 loci were randomly selected from each chromosome, totaling 150 loci, and primers were designed using the upstream and downstream sequences of the SSR loci using the Primer3 software.

[0073] Primer design follows the following principles: primer length should be 18-23bp; primer Tm value should be 55-63℃, with the optimum temperature around 59℃; the Tm difference between forward and reverse primers should be ≤5℃.

[0074] Among the 137 sites for which primers were successfully designed, 3 to 5 sites were randomly selected on each chromosome, totaling 100 pairs of primers. Sangon Biotechnology (Shanghai) Co., Ltd. was commissioned to synthesize the primers for subsequent PCR amplification.

[0075] 2. PCR amplification and specificity detection

[0076] The PCR reaction system (20 μL) is shown in Table 2. PCR amplification was performed on an ABI Veriti temperature gradient PCR instrument. The conditions for the first round of PCR amplification are shown in Table 3.

[0077] Table 2 PCR reaction system (20 μL)

[0078]

[0079] Table 3 PCR reaction program

[0080]

[0081] Take 3 μL of the PCR amplification product and perform 1.5% agarose gel electrophoresis at 220 V for 15 minutes to screen the SSR sites that can be specifically amplified.

[0082] The reaction conditions were optimized for unsatisfactory amplification results. Optimize the PCR amplification conditions for the cases where the electrophoresis results showed shallow bands or tailing bands. When the bands were shallow, the number of cycles was increased from 35 cycles to 37 cycles; when tailing bands appeared in the electrophoresis, the annealing temperature was increased from 60°C to 63°C. If multiple bands appeared, it proved that the microsatellite locus was not specific; when no bands appeared, the annealing temperature was lowered from 60°C to 57°C. 8 loci were optimized for PCR reaction conditions (Table 4).

[0083] Table 4 PCR reaction program optimization status table

[0084]

[0085] 3. Capillary electrophoresis polymorphism detection

[0086] Among the 15 individuals, three DNA templates were selected for PCR amplification of 100 SSR loci. The PCR products were subjected to agarose gel electrophoresis, and 60 specifically amplified SSR loci were screened out.

[0087] PCR amplification was performed using fluorescent adapter primers and forward primers, and the amplification system and conditions were the same as before. The amplified product was subjected to capillary electrophoresis on a 3037XL genetic analyzer to detect polymorphisms. The specific steps are as follows:

[0088] (1) Mix HiDi and 500 internal standards at a ratio of 130:1 to prepare a mix.

[0089] (2) Use a 96-well reaction plate to dispense the mix, adding 10 μL of the mix to each well.

[0090] (3) Add 0.5 μL of PCR product to a 96-well plate and start centrifugation until the speed reaches 4000 rpm.

[0091] (4) Heat the mixing plate at 95°C for 5 minutes using a metal bath heater to pre-denature the mixing plate. Immediately place the plate at -20°C after removing it from the heat.

[0092] (5) After cooling, take out, centrifuge at 4000 rpm, thaw and mix.

[0093] (6) Capillary electrophoresis was performed using a 3037XL genetic analyzer.

[0094] (7) Obtain and analyze the offline results.

[0095] Capillary electrophoresis detected 26 pairs of primers with high polymorphism, and the primer characteristics are shown in Table 5.

[0096] Table 526 Primer Characteristics of Highly Polymorphic SSR Sites

[0097]

[0098]

[0099] The polymorphism of 26 polymorphic SSR loci was further analyzed.

[0100] 4. SSR site polymorphism analysis

[0101] POPGENE1.31 was used to calculate the number of alleles N for 26 primer pairs and 15 samples. a , effective number of alleles N e , observed heterozygosity H e and the expected heterozygosity H o The polymorphism information content (PIC) of 26 SSR loci was evaluated using the analysis software PowerMarker3.25. The specific results are shown in Table 6 and Figure 3 .

[0102] Table 6 Polymorphic characteristics of 26 pairs of primers successfully amplified

[0103]

[0104]

[0105] The more alleles there are at each locus, the richer the population polymorphism is. A total of 143 alleles were detected in the amplification products of 26 pairs of primers of the present invention, and the number of alleles ranged from 2 (PR-97) to 10 (PR-14).

[0106] The polymorphic information content (PIC) is calculated by allele frequency and can reflect the diversity of SSR loci. When PIC>0.5, it indicates that the locus has high diversity and is a highly polymorphic locus; when PIC<0.25, it indicates that the locus has low diversity and is a low-polymorphic locus; and when the PIC value is between 0.25 and 0.5, it indicates that the locus has a medium diversity and is a moderately polymorphic locus. Among the 26 pairs of SSR primers of the present invention, there are 22 highly polymorphic loci, and the overall PIC value is between 0.3566 (PR-97) and 0.8122 (PR-14), with an average value of 0.7431, indicating that the SSR loci of the present invention have high polymorphism.

[0107] The expected heterozygosity of SSR loci (H e ) is higher, indicating that the genetic consistency of the population is lower, that is, the genetic diversity of the population is higher. eThe observed heterozygosity H o The number of alleles amplified by 26 pairs of primers in the present invention is 5.5±1.8815, and the number of effective alleles is 3.4926±1.0190. This result shows that the Przewalski's gazelle population has a high genetic diversity.

[0108] Depend on Figure 3 It can be seen that the highest allele frequency of the 26 SSR loci is 0.7 and the lowest is 0.03333.

[0109] It can be seen that the primers involved in the present invention can stably amplify the target product and are highly polymorphic, and can be used for the detection of genetic diversity of Przewalski's gazelle populations, population genetic structure analysis, evolution and kinship research, etc.

[0110] Example 3

[0111] Analysis of the individual identification ability of SSR loci

[0112] 1. Determination of SSR site consistency probability value

[0113] The consistency probability value refers to the probability that two individuals randomly selected from a population have the same genotype. This method is used to determine whether the number of SSR loci used can achieve individual identification. The appearance of the consistency probability value PIsibs can obtain a conservative lower limit on the number of loci required to complete individual identification. Based on the results of SSR polymorphic loci detection in 15 samples, this study used GenAlExV6.502 to analyze the genotypes of 26 loci in 15 individuals and calculated the consistency probability values ​​(PI and PIsibs). The specific results are shown in Figure 4 .

[0114] Depend on Figure 4 It can be seen that the PI value of these 15 SSR sites is 8.9020×10 -11 , indicating that when using this combination for individual identification, the probability of randomly selecting two Przewalski's gazelle individuals having the same haplotype is 8.9020×10 -11 ;PIsibs value is 4.1834×10 -5, indicating that the probability of any two Przewalski's gazelle individuals having the same haplotype at this SSR locus combination is 4.1834×10 -5 The combination can achieve an identification rate of one in ten thousand for individual identification. The current population of Przewalski's gazelle is only a few thousand, and the identification rate of one in ten thousand can meet the needs of individual identification of Przewalski's gazelle. It can be seen that this SSR locus combination can meet the needs of individual identification of Przewalski's gazelle.

[0115] 2. Identification of sub-adult Przewalski's gazelles at Jiangxigou Przewalski's gazelle rescue center

[0116] Using the 15 SSR loci obtained, 33 sub-adult Przewalski's gazelle samples collected from the Jiangxigou Przewalski's gazelle rescue center were individually identified by PCR amplification and capillary electrophoresis. Some electrophoresis results are shown in Figure 5 The Microsatellite Toolkit program was used to search for matching genotypes in the data and compared with the actual number of subadult animals in the rescue center to detect the recognition ability and accuracy of the developed SSR loci. The specific results are shown in Table 7.

[0117] The following principle is followed when identifying individuals: the genotypes at all microsatellite loci are the same or the genotypes at only one locus are different and are considered to be the same individual.

[0118] Table 7 Comparison results of 15 SSR loci genotypes in 33 fecal samples of Przewalski's gazelle sub-adults

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] As can be seen from Table 7, the genotypes of sample 218 and sample 403, sample 301 and sample 429, sample 304 and sample 446, sample 308 and sample 319, and sample 408 and sample 448 are completely consistent, and each of these five groups of stool samples comes from the same individual; sample 230 and sample 323 have the same genotype at each site, and sample 406 has only one genotype difference from sample 230 and sample 323, respectively, and it is judged that these three stool samples are from the same individual; sample 215 and sample 409, sample 216 and sample 410 have only one genotype difference, and according to the judgment criteria, it is judged that these two groups of samples are from the same individual.

[0125] In summary, there are 1 group (230-323-406) of 3 fecal samples from the same individual, and 7 groups (218-403, 301-429, 304-446, 308-319, 408-448, 215-409, 216-410) of 2 fecal samples from the same individual. The above results show that the 33 fecal samples of sub-adult Przewalski's gazelles detected are from 24 Przewalski's gazelles. According to the records of the rescue center, the number of newborn surviving individuals of Przewalski's gazelles in 2020 is 9, and the number of newborn surviving individuals in 2021 is 15, that is, there are 24 sub-adults by April 2022, indicating that the molecular recognition results are consistent with the actual situation. It can be seen that the individual identification results of Przewalski's gazelles carried out by the 15 SSR loci of the present invention are reliable.

[0126] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Primer combination of SSR molecular markers of Przewalski's gazelle, It is characterized in that It is composed of the following primer pairs, whose sequences are shown in SEQ ID NO.1-2, SEQ ID NO.5-6, SEQ ID NO.9-10, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.33-34, SEQ ID NO.35-36, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, and SEQ ID NO.51-52.

2. A kit for identifying Przewalski's gazelle individuals, It is characterized in that It includes the SSR molecular marker primer combination described in claim 1.

3. The kit according to claim 2, It is characterized in that The kit also includes a genome extraction reagent and a PCR reaction reagent.

4. The kit according to claim 2, It is characterized in that The kit also includes reagents for capillary electrophoresis.

5. Use of the SSR molecular marker primer combination according to claim 1 or the kit according to any one of claims 2 to 4 in individual identification analysis of Przewalski's gazelle.

6. Use of the SSR molecular marker primer combination according to claim 1 or the kit according to any one of claims 2 to 4 in detecting genetic diversity of a Przewalski's gazelle population.

7. A method for identifying individual Przewalski's gazelles, It is characterized in that include: Extracting total genomic DNA from a Przewalski's gazelle sample, performing PCR amplification on the total genomic DNA using the primer combination described in claim 1, and performing capillary electrophoresis to determine the genotype of the SSR molecular marker site for individual identification; When the genotypes at the SSR molecular marker sites are all the same or only one genotype at one site is different, they are determined to be the same individual; the sample is feces.

Images