Combination of Psibalski gazelle SSR molecular markers, primer combination, kit and use

Abstract

The present invention provides a combination of Psibalsk gazelle SSR molecular markers, a primer combination, a kit and uses, and relates to the technical field of molecular biology. Based on the whole-genome sequencing data of Psibalsk gazelle, SSR primer design and SSR loci were screened. The obtained 26 pairs of SSR primers can stably amplify the target product and are highly polymorphic, and can be used for detecting the genetic diversity of the Psibalsk gazelle population, analyzing the genetic structure of the population, and studying evolution and blood relationship. The combination of the screened Psibalsk gazelle polymorphic SSR molecular markers can identify individuals of Psibalsk gazelle with high accuracy. The experimental results show that the combination of Psibalsk gazelle SSR molecular markers of the present invention can meet the needs of individual identification, and individual identification was performed on 33 fecal samples of Psibalsk gazelle subadults derived from 24 different individuals, which is consistent with the number of Psibalsk gazelle subadults in the Jiangxigou Psibalsk gazelle rescue center.

Description

Technical Field

[0001] This application claims the priority of a Chinese patent application filed with the China National Intellectual Property Administration on May 29, 2023, with the application number 202310615110.8 and the invention title "Combination of Procapra przewalskii SSR Molecular Markers, Primer Combinations, Kits and Uses", and all its contents are incorporated herein by reference.

[0002] The present invention belongs to the technical field of molecular biology, and particularly relates to combinations of Procapra przewalskii SSR molecular markers, primer combinations, kits and uses.

Background Art

[0003] Procapra przewalskii belongs to the order Artiodactyla, family Bovidae, and genus Procapra, and is an animal endemic to the Tibetan Plateau. It is currently only distributed in the surrounding areas of Qinghai Lake, is a symbolic species of the Qinghai Lake Basin, and is a first-class protected wild animal in China. With the implementation of various protection measures and the strengthening of protection efforts, the number of Procapra przewalskii populations has recovered rapidly. According to data released by the Qinghai Provincial Forestry and Grassland Bureau in May 2022, it is shown that the number of adult individuals of wild Procapra przewalskii exceeded 2,800.

[0004] Simple sequence repeats (SSRs), also known as microsatellite sequences, use 1 - 6 nucleotides as repeat units. By repeating multiple times, tandem repeat sequences can be generated. They are present almost throughout the genome and become polymorphic due to differences in repeat units and the number of repeats between different alleles.

[0005] At present, many scholars have conducted a lot of research on the population distribution and population number of Przewalski's gazelle, the selection of habitats, foraging strategies and feeding habits, and damage factors. Some scholars have also used mitochondrial molecular markers to study the genetic diversity of Przewalski's gazelle populations. However, in the prior art, there is no report on the individual identification of Przewalski's gazelle using SSR markers.

Summary of the Invention

Problems to be Solved by the Invention

[0006] In view of this, the object of the present invention is to provide a combination of 4-base SSR molecular markers, a primer combination, a kit and its use for Przewalski's gazelle. The present invention develops polymorphic SSR markers based on the whole genome sequencing data of Przewalski's gazelle to meet the needs of individual identification of Przewalski's gazelle.

Means for Solving the Problems

[0007] To achieve the above object of the invention, the present invention provides the following technical solutions.

[0008] The present invention provides a combination of Psathyrella skiergel SSR molecular markers, and the combination of the SSR molecular markers 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, PR-97. The SSR markers are sequentially amplified 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 ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52.

[0009] Preferably, the combination of the SSR molecular markers is 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.

[0010] The present invention further provides a combination of Psibalsk gazelle SSR molecular marker primers, including any one or more of 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 ID ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52.

[0011] The present invention further provides a kit for individual identification of Psibalsk gazelle, which includes the above combination of SSR molecular marker primers.

[0012] Preferably, the kit further includes a genomic extraction reagent and a PCR reaction reagent.

[0013] Preferably, the kit further includes reagents for capillary electrophoresis.

[0014] The present invention further provides the use of the above combination of SSR molecular marker primers, or the above kit, in the individual identification analysis of Psibalsk gazelle.

[0015] The present invention further provides the use of the above combination of SSR molecular marker primers, or the above kit, in the detection of genetic diversity of Psibalsk gazelle populations.

[0016] The present invention further provides a method for individual identification of Pseudois nayaur, which includes extracting the DNA of a Pseudois nayaur sample, performing PCR amplification on the genomic DNA using the above primers respectively, performing capillary electrophoresis, determining the genotypes of the SSR molecular marker loci, and performing individual identification. When all the genotypes of the SSR molecular marker loci are the same, or only one genotype of one locus is different, it is determined that they are of the same individual.

[0017] Preferably, the sample includes feces or tissue.

Advantages of the Invention

[0018] Compared with the prior art, the present invention has the following beneficial effects. Based on the whole-genome sequencing data of Pseudois nayaur, the present invention designs SSR primers and screens SSR loci. The obtained 26 pairs of SSR primers can stably amplify the target products and are highly polymorphic, and can be used for detecting the genetic diversity of the Pseudois nayaur population, analyzing the genetic structure of the population, and studying evolution and blood relationship. The combination of the screened polymorphic SSR molecular markers of Pseudois nayaur can perform individual identification on Pseudois nayaur with a high accuracy rate. The experimental results show that the combination of the Pseudois nayaur SSR molecular markers of the present invention can meet the needs of individual identification. Individual identification is performed on 33 fecal samples of sub-adult Pseudois nayaur derived from 24 different individuals, which is consistent with the number of sub-adult Pseudois nayaur in the Jiangxigou Pseudois nayaur Rescue Center.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0020] The present invention provides a combination of Psibalsk gazelle SSR molecular markers. The combination of the SSR molecular markers 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, PR-97. The SSR markers are sequentially amplified 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 ID ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52.

[0021] In the present invention, the combination of the SSR molecular markers 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 detection results of the identity probability indicate that the combination of the above 15 SSR molecular markers can meet the needs of individual identification.

[0022] The present invention provides a combination of Psibalskij gazelle SSR molecular marker primers, including any one or more of 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 ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, SEQ ID NO.49-50, SEQ ID NO.51-52.

[0023] The more alleles there are at each locus, the richer the polymorphism of the population. A total of 143 alleles were detected from the amplification products of the combination of Psibalskij gazelle SSR molecular marker primers of the present invention, and the number of alleles varied from 2 (PR-97) to 10 (PR-14). The combination of the above 26 Psibalskij gazelle SSR molecular marker primers of the present invention can stably amplify the target product and is highly polymorphic, and it has been found that it can be used for detecting the genetic diversity of the Psibalskij gazelle population, analyzing the genetic structure of the population, and studying evolution and blood relationship.

[0024] The present invention further provides a kit for individual identification of Psibalskij gazelle, including the above combination of SSR molecular marker primers.

[0025] The kit described in the present invention preferably further includes a genomic extraction reagent and a PCR reaction reagent.

[0026] The kit described in the present invention preferably further includes reagents for capillary electrophoresis.

[0027] The present invention further provides the use of the above SSR molecular marker primer combinations or the above kits in the individual identification analysis of Przewalski's gazelle.

[0028] The present invention uses 15 loci in the combination of Przewalski's gazelle SSR molecular markers to perform individual identification on 33 fecal samples of Przewalski's gazelle subadults. The experimental results show that the 33 fecal samples of Przewalski's gazelle subadults are derived from 24 different individuals, which is consistent with the number of Przewalski's gazelle subadults in the Jiangxigou Przewalski's Gazelle Rescue Center.

[0029] The present invention further provides the use of the above SSR molecular marker primer combinations or the above kits in the detection of genetic diversity of Przewalski's gazelle populations.

[0030] The combination of the above 26 Przewalski's gazelle SSR molecular marker primers of the present invention can stably amplify the target product and is highly polymorphic, and can be used for the detection of genetic diversity of Przewalski's gazelle populations, the analysis of population genetic structure, the study of evolution and blood relationship, etc.

[0031] The present invention provides a method for individual identification of Przewalski's gazelle, which includes extracting DNA from a Przewalski's gazelle sample, performing PCR amplification on the genomic DNA using the above primers respectively, performing capillary electrophoresis, determining the genotypes of the above SSR molecular marker loci, and performing individual identification, and determining that they are the same individual when all the genotypes of the above SSR molecular marker loci are the same or only one genotype of one locus is different.

[0032] The samples described in the present invention preferably contain feces or tissues, more preferably feces, and the tissues more preferably include fur, muscle, and blood.

[0033] Hereinafter, the technical solutions provided by the present invention will be described in detail with reference to examples, but it should not be understood that they limit the protection scope of the present invention.

[0034] The experimental samples, experimental reagents, experimental instruments and equipment used in the examples of the present invention are as follows.

[0035] 1. Experimental samples Fifteen fecal samples of Przewalski's gazelle used for screening polymorphic SSR loci were collected from Haiyan County, Gonghe County and Gangcha County in Qinghai Province. When fresh fecal samples were collected in the field, they were immediately stored in liquid nitrogen, and after returning to the laboratory, they were transferred to a -80°C refrigerator and stored until genomic DNA was extracted. Individual identification samples were collected from the Przewalski's gazelle rescue center in Jiangxigou. In April 2022, fecal samples of sub-adult Przewalski's gazelles were collected at the rescue center, collected twice a day for 4 consecutive days, and it was ensured that all fecal samples of sub-adult Przewalski's gazelles were collected. A total of 33 fecal samples of sub-adults were collected over 4 days (as shown in Figure 1).

[0036] 2. Experimental reagents QIAamp Fast DNA Stool Mini Kit, TaKaRa Ex Taq Hot Start Version, absolute ethanol, 100 bp DNA Ladder, 6×loading buffer, 50×TAE, agarose, Ethidium bromide (EB).

[0037] 3. Experimental instruments and equipment ABI 3730 XL gene analyzer, ABI Veriti thermal gradient PCR device, Nanodrop 2000C spectrophotometer, Bio-rad electrophoresis system, Bio-rad gel imaging system, Millipore pure water system, high-speed refrigerated centrifuge, autoclave, 4°C refrigerator, -20°C refrigerator, -80°C refrigerator, vortex oscillator, ice maker, constant temperature water bath, electronic balance, eppendorf pipette, microwave oven.

[0038] Example 1 Extraction and detection of total DNA from the fecal genome of Psibalskij gazelle Using the kit method, the kit used was QIAamp Fast DNA Stool (51604). In the preparation stage, it was ensured that Buffer AW1 and Buffer AW2 were prepared according to the instructions on the kit label. Before use, the buffer was mixed uniformly. If a precipitate formed in Buffer ASL or Buffer AL, it was heated in a 70°C water bath until dissolved. (1) For DNA extraction, first, 180 - 220 mg of feces was weighed into a 2 mL centrifuge tube and pretreated on ice. (2) 1 mL of Inhibit EX Buffer was added to the sample, and it was intermittently shaken and vortexed for 1 - 2 min until the sample became completely homogeneous. (3) Centrifuged at a full speed of 14000 rpm for 1.5 min to precipitate the feces at the bottom of the centrifuge tube. (4) 25 μL of proteinase K was placed in a new 2 mL microcentrifuge tube. (5) 600 μL of the supernatant was aspirated from the centrifuge tube in step 3 into the 2 mL centrifuge tube containing proteinase K. (6) 600 μL of Buffer AL was added and vortexed for 15 s to thoroughly mix the solution. (7) Incubated at 70°C for 10 min, inverted up and down 1 - 2 times during incubation to mix uniformly, and reduced the droplets on the lid of the centrifuge collection tube. Cooled on ice. (8) 600 μL of pre-cooled absolute ethanol solution was added to the lysate, and vortexed to mix uniformly. The droplets on the lid of the centrifugation collection tube were reduced. (9) 600 μL of the solution obtained in the previous step was added to the adsorption column (the adsorption column was placed in a 2 mL collection tube). Centrifuged at a full speed of 14,000 rpm for 1.5 min, the waste liquid was discarded, and the adsorption column was placed in a new collection tube. (10) The lid of the column was opened, and an additional 600 μL of lysate was added. Centrifuged at a full speed of 14,000 rpm for 1.5 min, the waste liquid was discarded, and the adsorption column was placed in a new collection tube. (11) The previous step was repeated, and 600 μL of the lysate for the third time was loaded onto the column. Centrifuged at a full speed of 14,000 rpm for 1.5 min, the waste liquid was discarded, and the adsorption column was placed in a new collection tube. (12) 500 μL of Buffer AW1 was added to the column. The lid of the centrifuge tube was covered tightly and centrifuged at a full speed of 14,000 rpm for 1.5 min. The centrifuge tube was taken out and placed in a new 2 mL collection tube. The old collection tube and the liquid in it were discarded. (13) 500 μL of Buffer AW2 was added to the column. The lid of the centrifuge tube was covered tightly and centrifuged at a full speed of 14,000 rpm for 3 min. The centrifuge tube was taken out and placed in a new 2 mL collection tube. The old collection tube and the liquid in it were discarded. (14) After taking out the centrifuge tube and placing it in a new 2 mL collection tube, it was centrifuged at a full speed of 14,000 rpm for 3 min. The column was transferred to a new 1.5 mL centrifuge tube, 200 μL of Buffer ATE was carefully added, left standing at room temperature for 2 min, and centrifuged at a full speed of 14,000 rpm for 2 min to elute the DNA. The DNA sample in the 1.5 mL centrifuge tube was quickly placed in a freezer at -20 °C. After the DNA extraction was completed, the DNA concentration was detected with a Nanodrop 2000C spectrophotometer, and the quality of the DNA was detected by 1% agarose gel electrophoresis. The specific results are shown in Figure 2 and Table 1.

[0039]

Table 1

[0040] As can be seen from Figure 2, there is one bright main band in the extracted total genomic DNA, indicating that the quality of the DNA is relatively complete. As can be seen from Table 1, the concentration of total genomic DNA of Przewalski's gazelle ranges from 2.6 (sample 3309) to 57.7 (sample 3302) ng / μL, which can be used for subsequent PCR experiments.

[0041] Example 2 Primary screening of SSR primers and polymorphism detection

[0042] 1. Selection of SSRs and primer design Using MISA microsatellite screening software, the entire genome of Przewalski's gazelle was scanned to identify SSR loci with repeat sequences of 4 - 6 bp and a repeat number greater than 8. A total of 1632 SSR loci meeting the conditions were obtained. From the SSR loci with a repeat unit of 4 bp and a repeat number of 10 - 70, 5 - 6 loci per chromosome, a total of 150 loci were randomly selected. Using the upstream and downstream sequences of the SSR loci, primer design was performed by Primer3 software. The primer design follows the following principles. The length of the primer is 18 - 23 bp, the Tm value of the primer is 55 - 63 °C, the optimal temperature is about 59 °C, and the difference in Tm between the forward primer and the reverse primer is ≤ 5 °C. For the 137 loci where primer design was successful, 3 - 5 loci per chromosome, a total of 100 pairs of primers were randomly selected, and the synthesis of the primers was entrusted to Sangon Biotech (Shanghai) Co., Ltd. for use in subsequent PCR amplification.

[0043] 2. PCR amplification and specificity detection The PCR reaction system (20 μL) is shown in Table 2, and PCR amplification was performed on an ABI Veriti temperature gradient PCR instrument. The conditions for the first - round PCR amplification are shown in Table 3.

[0044]

Table 2

[0045]

Table 3

[0046] For 3 μL of the PCR amplification product, 1.5% agarose gel electrophoresis was carried out at a voltage of 220 V for 15 minutes to screen for SSR loci that could be specifically amplified. For the non-ideal amplification results, the reaction conditions were optimized. For the cases where the bands in the electrophoresis results were faint or the bands were trailing, the PCR amplification conditions were optimized. When the bands were faint, the number of cycles was increased from 35 to 37. When the bands were trailing in the electrophoresis diagram, the annealing temperature was raised from 60 °C to 63 °C. When multiple bands appeared, it indicated that the microsatellite locus did not have specificity. When no bands appeared, the annealing temperature was lowered from 60 °C to 57 °C. The PCR reaction conditions were optimized for 8 loci (Table 4).

[0047]

Table 4

[0048] 3. Polymorphism detection by capillary electrophoresis From 15 individuals, 3 DNA templates were selected for PCR amplification of 100 SSR loci. Agarose gel electrophoresis was carried out on the PCR products, and 60 SSR loci that were specifically amplified were screened from them. PCR amplification was carried out using fluorescent linker primers and forward primers, and the amplification system and conditions were the same as before. Capillary electrophoresis was carried out on the amplification products using a 3037XL genetic analyzer to detect polymorphisms. The specific steps are as follows. (1) HiDi and GeneScan TM 500LIZ TMThe dyeSizeStandard was mixed at a ratio of 130:1 and prepared into the mix. (2) The mix was dispensed into a 96-well reaction plate, and 10 μL of the mix was added to each well. (3) 0.5 μL of the PCR product was added to the 96-well plate, centrifugation was started, and it was stopped at 4000 rpm. (4) Using a metal bath heater, it was heated at 95 °C for 5 min to initially denature the mixing plate, and immediately put into -20 °C as soon as it was taken out. (5) After cooling, it was taken out, centrifuged at 4000 rpm, thawed, and mixed uniformly. (6) Capillary electrophoresis was performed using a 3037XL genetic analyzer. (7) The results of capillary electrophoresis were obtained and analyzed. By capillary electrophoresis, 26 pairs of primers with high polymorphism were detected, and the characteristics of the primers are shown in Table 5.

[0049]

Table 5

[0050] Further polymorphism analysis was performed on 26 polymorphic SSR loci.

[0051] 4. Polymorphism Analysis of SSR Loci Using POPGENE1.31, the number of alleles N a of 15 samples of 26 pairs of primers, the effective number of alleles N e the observed heterozygosity H e and the expected heterozygosity H oEach was calculated, and using PowerMarker 3.25, its polymorphism information content PIC was calculated. Using the analysis software PowerMarker 3.25, the polymorphisms of 26 SSR loci were evaluated. The specific results are shown in Table 6 and Figure 3.

[0052]

Table 6

[0053] The more alleles at each locus, the richer the polymorphism of the population. A total of 143 alleles were detected in the amplification products of the 26 pairs of primers of the present invention, and the number of alleles varied from 2 (PR-97) to 10 (PR-14). The polymorphism information content PIC is calculated by allele frequency and can reflect the degree of diversity of the SSR locus. When PIC > 0.5, it indicates that the diversity of the locus is high and it is a highly polymorphic locus. When PIC < 0.25, it indicates that the diversity level of the locus is low and it is a lowly polymorphic locus. When the PIC value is between 0.25 and 0.5, it indicates that the diversity level of the locus is moderate and it is a moderately polymorphic locus. There are 22 highly polymorphic loci among the 26 pairs of SSR primers of the present invention. The overall PIC value ranges from 0.3566 (PR-97) to 0.8122 (PR-14), and the average value is 0.7431, indicating that the SSR loci of the present invention have high polymorphism. The expected heterozygosity (H e ) of the SSR locus is higher, indicating that the genetic identity of the population is lower, that is, the genetic diversity of the population is higher. The H e of the 26 SSR loci of the present invention ranges from 0.4805 (PR-97) to 0.8598 (PR-14), and the average value is 0.7117 ± 0.1008. The observed heterozygosity H oIt ranges from 0.2667 (PR-8) to 1.0000 (PR-30, PR-40), and the average value is 0.6436 ± 0.1968. The number of alleles obtained by comprehensively amplifying the 26 pairs of primers of the present invention is 5.5 ± 1.8815, and the effective number of alleles is 3.4926 ± 1.0190. This result indicates that the Przewalski's gazelle population has high genetic diversity. As can be seen from Figure 3, the allele frequencies of the 26 SSR loci range from a maximum of 0.7 to a minimum of 0.03333. The primers described in the present invention can stably amplify the target product and are highly polymorphic. It has been found that they can be used for detecting the genetic diversity of the Przewalski's gazelle population, analyzing the genetic structure of the population, and studying evolution and blood relationships.

[0054] Example 3 Analysis of the individual discrimination ability of SSR loci

[0055] 1. Measurement of the identity probability value of SSR loci The identity probability value refers to the probability that two randomly selected individuals in a population have the same genotype. This method is used to determine whether the number of SSR loci used can achieve individual discrimination. The appearance of the PIsibs of the identity probability value gives a conservative lower limit of the number of loci required to achieve individual discrimination. Based on the detection results of the SSR polymorphic loci of 15 samples in this study, GenAlEx V6.502 was used to analyze the genotypes of 26 loci of 15 individuals, and the identity probability values (PI and PIsibs) were calculated. The specific results are shown in Figure 4. As can be seen from Figure 4, the PI values of these 15 SSR loci, namely 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, PR-97, are 8.9020×10 -11 When using this combination for individual discrimination, if two Przewalski's gazelle individuals are randomly selected, the probability that their haplotypes are the same is 8.9020×10 -11It is shown to be. The PIsibs value is 4.1834×10 -5 and when considering the blood relationship, the probability that the haplotypes of the combinations of the SSR loci of any two Przewalski's gazelle individuals are the same is 4.1834×10 -5 It is shown to be. When using this combination for individual identification, a discrimination rate of 1 in 10,000 can be achieved. The current population of Przewalski's gazelles is only several thousand, and a discrimination rate of 1 in 10,000 can meet the needs of Przewalski's gazelle individual identification. It was found that the combination of the SSR loci can meet the needs of Przewalski's gazelle individual identification.

[0056] 2. Individual Identification of Sub - adult Przewalski's Gazelles in the Jiangxigou Przewalski's Gazelle Rescue Center Using the obtained 15 SSR loci, individual identification was performed on 33 sub - adult Przewalski's gazelles collected by the Jiangxigou Przewalski's Gazelle Rescue Center through PCR amplification and capillary electrophoresis. Some of the electrophoresis results are shown in Figure 5. The Microsatellite Tool kit program was used to search for matching genotypes in the data and compare them with the actual number of sub - adult animals in the rescue center to detect the discriminatory power and accuracy of the developed SSR loci. The specific results are shown in Table 7. Individuals were identified according to the following principle. If the genotypes of all microsatellite loci are the same, or only one genotype of one locus is different, they are regarded as the same individual.

[0057]

Table 7

[0058] 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 identical, and each pair of these 5 sets of fecal samples is from the same individual. Each locus of Sample 230 and Sample 323 has the same genotype, and Sample 406 differs from Sample 230 and Sample 323 by only one genotype, and it was determined that these 3 fecal samples are from the same individual. Sample 215 and Sample 409, and Sample 216 and Sample 410 differ by only one genotype, and according to the judgment criteria, these 2 sets of samples were determined to be from the same individual respectively. From the above, there is 1 set (230 - 323 - 406) where 3 fecal samples are from the same individual, and there are 7 sets (218 - 403, 301 - 429, 304 - 446, 308 - 319, 408 - 448, 215 - 409, 216 - 410) where 2 fecal samples are from the same individual. The above results indicate that the 33 detected fecal samples of Przewalski's gazelle subadults are from 24 Przewalski's gazelle individuals. According to the records of the rescue center, the number of newly born surviving Przewalski's gazelles in 2020 was 9, and the number of newly born surviving individuals in 2021 was 15, that is, by April 2022, there were 24 subadults, indicating that the molecular identification results are consistent with the actual situation. It was found that the individual identification results of Przewalski's gazelle using 15 SSR loci in the present invention are reliable.

[0059] The above are only preferred embodiments of the present invention. Those skilled in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be regarded as within the protection scope.

Claims

1. A combination of Psibalskij gazelle SSR molecular markers, wherein the combination of the SSR molecular markers 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, PR-97, the combination of the SSR molecular markers is sequentially amplified 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 ID NO. 43-44, SEQ ID NO. 45-46, SEQ ID NO. 47-48, SEQ ID NO. 49-50, SEQ ID NO. 51-52. A combination of Psibalskij gazelle SSR molecular markers characterized by this.

2. The combination of the SSR molecular markers is 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 combination of Psibalskij gazelle SSR molecular markers according to Claim 1, characterized by this.

3. A combination of Psibalskij gazelle SSR molecular marker primers, comprising any one or more of the following primer pairs, wherein 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 ID NO. 43-44, SEQ ID NO. 45-46, SEQ ID NO. 47-48, SEQ ID NO. 49-50, SEQ ID NO. 51-52. A combination of Psibalskij gazelle SSR molecular marker primers characterized by the above.

4. A kit for individual identification of Psibalskij gazelle, comprising the combination of SSR molecular marker primers according to claim 3. A kit for individual identification of Psibalskij gazelle characterized by the above.

5. The kit according to claim 4, further comprising a genomic extraction reagent and a PCR reaction reagent.

6. The kit according to claim 4, further comprising a reagent for capillary electrophoresis.

7. Use of the combination of SSR molecular marker primers according to claim 3 or the kit according to any one of claims 4 to 6 in the individual identification analysis of Psibalskij gazelle.

8. Use of the combination of SSR molecular marker primers according to claim 3 or the kit according to any one of claims 4 to 6 in the detection of genetic diversity of Psibalskij gazelle populations.

9. A method for identifying an individual of Przewalski's gazelle, comprising extracting DNA from a sample of Przewalski's gazelle, performing PCR amplification on the genomic DNA using the primers described in claim 3, performing capillary electrophoresis, determining the genotype of the SSR molecular marker locus described in claim 1, and performing individual identification. Determining that the individuals are the same when all of the genotypes of the SSR molecular marker loci are the same or only one genotype at one locus is different. A method for identifying an individual of Przewalski's gazelle, characterized by including this.

10. The method according to claim 9, wherein the sample includes feces or tissue.

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