Microsatellite genotyping method

Abstract

The invention provides a microsatellite genotyping method, and belongs to the technical field of genotyping. According to the microsatellite genotyping method, a bar code primer is introduced into a microsatellite primer, and the bar code primer comprises a bar code and a universal primer. The universal primer can be used for increasing PCR products obtained by the microsatellite primer so as to supplement a target product obtained by the microsatellite primer, the problem of amplification of a low-quality DNA template is solved, the library building success rate is remarkably increased to 90-100% from 60-70%, the library building and sequencing times are remarkably reduced, the sample number of each time of library building and sequencing is increased, and the library building and sequencing efficiency is improved. The method has the remarkable advantages of low cost and high accuracy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genotyping, and particularly relates to a microsatellite genotyping method. BACKGROUND

[0002] Microsatellite is also known as simple sequence repeats (SSR), short tandem repeats (STR) or simple sequence length polymorphism (SSLP). The number of base pairs in the repeated unit of microsatellite DNA varies, mainly including 2-4bp, 2-5bp, 2-6bp, 1-6bp, 1-9bp and 1-10bp. Current data shows that the microsatellite DNA with 2bp repeated unit is the most abundant, followed by 3bp and 4bp.

[0003] Microsatellite is randomly distributed on the genome. In eukaryotes, microsatellite covers the entire genome, and there is a microsatellite site every 10-50kb. Each microsatellite site is single copy on the genome, and the number of repeated units varies from several to hundreds, and the fragment length is between tens to 300bp. The polymorphism of microsatellite is that the number of repetitions is different at the same site in the population, each site has multiple alleles, and is co-dominant inheritance. The characteristics of the wide distribution of microsatellite sites on the genome, the extremely active mutation rate, multiple alleles and co-dominant inheritance make microsatellite markers become a powerful technical means, and are widely used in detection of genetic diversity, identification of species or strains, individual identification, analysis of pedigree relationship, breeding of plants and animals, genetic diseases and tumors, forensic medicine and many other fields.

[0004] At present, the most widely used in microsatellite markers is PCR technology. Because the flanking sequence of each microsatellite core sequence is quite different, these core sequences and their flanking sequences constitute many independent sites in genomic DNA. The microsatellite polymorphism information of a specific site can be obtained by designing primers with flanking sequences as reference for PCR amplification. The detection of microsatellite alleles is earliest by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then gradually replaced by capillary electrophoresis. Both methods use length variation of PCR products to distinguish different alleles, and cannot accurately analyze the point mutations and the number of microsatellite repeats in the products, which limits the accuracy of microsatellite genotyping.

[0005] In addition, endangered animal population analysis often needs to use low-quality non-invasive samples such as feces. The concentration of target DNA in such DNA is low, and the contamination rate is high. When PCR amplification is performed, the concentration of the target product is often low, and it is difficult to perform subsequent microsatellite genotyping analysis. SUMMARY

[0006] The application provides a microsatellite genotyping method, which introduces a barcode primer into a microsatellite primer of a to-be-tested species, the barcode primer comprises a barcode and a universal primer, a target product is amplified by using the universal primer, the concentration of the target PCR product is significantly increased, so that subsequent library construction and sequencing analysis are met, and the barcodes are used to distinguish samples and increase the number of samples for each library construction and sequencing.

[0007] The application provides a microsatellite genotype analysis method, which comprises the following steps: (1) taking genomic DNA of a to-be-tested species as a template, performing PCR amplification by using a microsatellite primer, and obtaining a plurality of microsatellite site PCR products by amplification; (2) mixing the plurality of microsatellite site PCR products obtained in step (1) as a template, performing PCR amplification by using a barcode primer, and obtaining a plurality of barcode PCR products by amplification; the barcode primer comprises a sample barcode sequence and a universal primer; (3) mixing the plurality of barcode PCR products in step (2) as a sequencing library for sequencing, and performing microsatellite genotyping on the to-be-tested species according to the sequencing result.

[0008] In a preferred mode of the application, the to-be-tested species in step (1) comprises a giant panda.

[0009] In a preferred mode of the application, the 19 bases at the 5' end of the forward primer and the 20 bases at the 5' end of the reverse primer of the microsatellite primer in step (1) are both from the universal primer.

[0010] In a preferred mode of the application, the microsatellite primer designed for the giant panda comprises at least one of the following pairs: Ame-u10, Ame-u13, Ame-u15, GPY-20, Ame-u26, GP-01, GP-08, GP-901 and GPL-31. The primer pair Ame-u10 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 3 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 4. The primer pair Ame-u13 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 5 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 6. The primer pair Ame-u15 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 7 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 8. The primer pair GPY-20 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 9 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 10. wherein the primer pair Ame-u26 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 11 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 12; wherein the primer pair GP-01 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 13 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 14; wherein the primer pair GP-08 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 15 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 16; wherein the primer pair GP-901 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 17 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 18; wherein the primer pair GPL-901 comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 19 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 20.

[0011] In a preferred mode of the present application, the PCR amplification procedure in step (1) comprises: 98℃ pre-denaturation for 2 min; 5℃ denaturation for 30 s, 55℃ to 45℃ cooling annealing for 30 s, 72℃ extension for 45 s, 35 cycles; 72℃ final extension for 15 min, 4℃ keeping.

[0012] In a preferred mode of the present application, the sample barcode sequence of the forward primer and the reverse primer of each pair of the barcode primers in step (2) is the same. The sample barcode sequence comprises at least one of ATGCAT, CAGAGT, CATGCA and TGACAG.

[0013] In a preferred mode of the present application, the universal primer of the giant panda in step (2) comprises a forward primer TongF and a reverse primer TongR, wherein the nucleotide sequence of the forward primer TongF is shown in SEQ ID No. 1, and the nucleotide sequence of the reverse primer TongR is shown in SEQ ID No. 2.

[0014] In a preferred mode of the present application, the PCR amplification procedure in step (2) comprises: 98℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, 25 cycles; 72℃ final extension for 15 min, 4℃ keeping.

[0015] In a preferred mode of the present application, the concentration of each primer in step (1) and step (2) is 20 pmol / μL.

[0016] In a preferred mode of the present application, the sequencing in step (3) comprises second-generation sequencing.

[0017] Beneficial effects: the present application provides a microsatellite genotype analysis method, by introducing barcodes and universal primers; the 19 bases at the 5' end of the forward primer of the microsatellite primer and the 20 bases at the 5' end of the reverse primer are both from the universal primer. The sample barcode of the present application can distinguish samples, the universal primer does not amplify any mammalian DNA sequence, and is used to increase the PCR product obtained by the microsatellite primer to supplement the target product obtained by the microsatellite primer. The present application combines the universal primer and the microsatellite primer together to amplify the microsatellite PCR product of high-quality DNA and low-quality DNA of multiple species such as giant pandas.

[0018] The universal primer in the barcode primer of the present application can increase the concentration of the target product that is difficult to amplify by conventional primers, thereby significantly increasing the library sequencing success rate of 60~70% low-quality DNA to 90~100%, and overcoming the shortcoming of multiple non-specific amplification bands and difficulty in distinguishing real microsatellite genotypes caused by capillary electrophoresis, and the microsatellite typing result is more reliable; and the present application uses barcode primers for microsatellite genotype analysis, according to the data output of 1G per sequencing library, 660~1000 PCR products can be mixed in each sequencing library, which significantly reduces the number of library sequencing and increases the number of samples per library sequencing, and has the significant advantages of low cost and high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a microsatellite primer PCR amplification product electropherogram of high-quality DNA of giant panda, in which 1: GPL-31, 2: GP-901, M: marker; Figure 2 It is a microsatellite length distribution frequency diagram assembled from high-quality DNA; Figure 3 It is a microsatellite primer PCR amplification product electropherogram of low-quality DNA of giant panda, in which 1: GPL-31, 2: GP-901, 3: GP-08, 4: GP-01, 5: Ame-u26, 6: GPY-20, 7: Ame-u15, 8: Ame-u13, 9: Ame-u10, M: marker; Figure 4 It is a microsatellite length distribution frequency diagram assembled from low-quality DNA; Figure 5 It is a microsatellite typing price comparison result diagram of each sample of each site by capillary electrophoresis and microsatellite sequencing; Figure 6 It is a capillary electrophoresis diagram of two sites GP08 (left) and Ame-u13 (right); Figure 7 It is a capillary electrophoresis diagram of site GPL-31; Figure 8 The application compares the amount of PCR product of low-quality DNA of the giant panda by using the universal primer and the conventional primer. DETAILED DESCRIPTION

[0020] The application provides a microsatellite genotype analysis method, comprising the following steps: (1) using genomic DNA of a to-be-tested species as a template, performing PCR amplification by using a microsatellite primer, and obtaining several microsatellite site PCR products by amplification; (2) mixing the several microsatellite site PCR products obtained in step (1) as a template, performing PCR amplification by using a barcode primer, and obtaining several barcode PCR products by amplification; the barcode primer comprises a sample barcode sequence and a universal primer; (3) mixing the several barcode PCR products in step (2) as a sequencing library for sequencing, and performing microsatellite genotyping on the to-be-tested species according to the sequencing result.

[0021] In the process of performing the microsatellite genotype analysis, the barcode primer of the to-be-tested species and the microsatellite primer designed based on the to-be-tested species are needed; the barcode primer comprises a sample barcode sequence and a universal primer; 19 bases at the 5' end of the forward primer of the microsatellite primer and 20 bases at the 5' end of the reverse primer are both from the universal primer.

[0022] The design method of the microsatellite sequencing primer set can be used for multiple species, and the giant panda is taken as an example in the application, but it cannot be identified as the entire protection range of the application.

[0023] In the primer set designed in the application, the sample barcode sequences of the forward primer and the reverse primer of each pair of the barcode primer are the same; the sample barcode sequence comprises at least one of ATGCAT, CAGAGT, CATGCA and TGACAG. The sample barcode in the application can be used to distinguish samples.

[0024] In an embodiment of the application, the primer set is designed for the giant panda, and the universal primer of the giant panda comprises a forward primer TongF and a reverse primer TongR, wherein the nucleotide sequence of the forward primer TongF is shown in SEQ ID No. 1, and the nucleotide sequence of the reverse primer TongR is shown in SEQ ID No. 2. The primer does not amplify any mammalian DNA sequence, and is used to increase the PCR product obtained by the microsatellite primer to supplement the target product obtained by the microsatellite primer.

[0025] TongF: 5'-GTGCCAGCCGCCGCGGTAC-3'; TongR: 5'-GGACTACTAGGGTGTCTAAC-3'.

[0026] The barcode sequence and the universal primer are combined to obtain the barcode primer described in Table 1 of the present application. The length of the target product of the giant panda microsatellite primer described in the present application is between 110 and 220 bp. In the examples, nine giant panda microsatellite sites are taken as examples to design the microsatellite primers shown in Table 2. The 19 bases at the 5' end of the forward primer and the 20 bases at the 5' end of the reverse primer of the giant panda microsatellite primer described in the present application are both from the universal primer. The universal primer and the microsatellite primer are combined to amplify the giant panda microsatellite PCR product. In the present application, the primers are synthesized by a biological company and then dissolved in sterile water to 20 pmol / μL for subsequent experiments.

[0027] Table 1 barcode primer

[0028] Table 2 microsatellite primer

[0029] The working concentration of each primer in the subsequent PCR of the present application is 20 pmol / μL.

[0030] When the microsatellite primer amplification of the present application is performed, each pair of primers is subjected to one PCR. In the examples of the present application, nine pairs of microsatellite primers are designed, so nine PCRs are required for each giant panda DNA using the nine microsatellite sites shown in Table 2. The system of the microsatellite PCR described in the present application is 20 μL, including: 1 μL of DNA template, 1 μL of microsatellite forward and reverse primers, 0.5 μL of DNA polymerase (2.5 units), 2 μL of 10 times enzyme buffer, 2 μL of 2.5 mM dNTPs, and the rest of sterile water. The program of the microsatellite PCR described in the present application includes: 98℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, annealing temperature from 55℃ to 45℃ in turn for 30 s (every 1℃ one gradient, 2 cycles for each temperature, and finally 45℃ for 15 cycles), 72℃ extension for 45 s, a total of 35 cycles; 72℃ extension for 15 min, and keeping at 4℃ for 30 min. Electrophoresis is performed after the microsatellite PCR described in the present application to detect whether the target fragment is amplified and the band brightness, and the concentration of the target fragment of each microsatellite site is calculated. The DNA template described in the present application can be a high-quality DNA template, such as DNA extracted from blood or muscle tissue; or a low-quality DNA template, such as DNA extracted from feces.

[0031] The application mixes the PCR products of different microsatellite sites of the same individual, and calculates the volume of the PCR product to be added for each microsatellite site according to the concentration of the target product of the 9 microsatellite sites of each giant panda DNA, so that each site has the same amount of PCR target fragment after mixing. The total amount of PCR product of each individual after mixing is 1 μg, and the concentration is 50 ng / μL. When performing the mixing, if the concentration of the PCR product of a certain site is not enough, the subsequent barcode primer or universal primer can be used to amplify the PCR product as a template.

[0032] The application uses the mixed DNA of the PCR products of different primers of the same sample as a template, and uses the barcode primers shown in Table 1 to amplify. The system of the amplification is 20 μL, including: 2 μL of DNA template, 1 μL of each of the barcode primers (one barcode primer is used for each sample), 0.5 μL of DNA polymerase (5 units), 2 μL of 10 times enzyme buffer, 2 μL of 2.5 mM dNTPs, and the rest of sterile water. The PCR program of the amplification includes: denaturation at 98℃ for 2 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 45 s, 25 cycles; extension at 72℃ for 15 min, and keeping at 4℃ for 30 min. The application uses the amplified product to perform electrophoresis to detect whether the target fragment is amplified and its concentration.

[0033] The application uses the amplified product to mix as a sequencing library, and calculates the volume of the PCR product to be added for each sample according to the total DNA amount of 2 μg and 4 samples, so that the amount of DNA of each sample is 0.5 μg for mixing to build a library for sequencing. If the number of samples in a single library is increased, the amount of DNA of each sample can be correspondingly reduced to ensure that the total DNA amount is at least 2 μg, at which time the barcode primer needs to be increased. Different barcode primers are needed for different samples in each library in order to determine the sequence of each sample.

[0034] This invention involves sequencing the obtained sequencing libraries after mixing. The sequencing includes next-generation sequencing (NGS). The PCR products of each library are purified again using magnetic beads after adding Illumina adapters to obtain the sequencing libraries, which are then sequenced using Illumina PE150 / PE250. The raw sequencing files are then base-called and converted to Bcl2Fastq to obtain raw sequencing sequences in FASTQ format. Trimmomatic reads are then removed, discarding reads shorter than 36 bytes and low-quality bases at the beginning and end, resulting in high-quality reads. These reads are then assembled using flash and converted to FASTA files. Sequences for each microsatellite locus in each sample are extracted, and the frequency of unique microsatellite sequences at each locus in each sample is calculated. Based on the frequency and the number of repetitions of microsatellite repeats, the genotype of each individual and each microsatellite locus is obtained, which can then be used for subsequent individual identification and population genetic structure analysis.

[0035] To further illustrate the present invention, the following detailed description of a microsatellite sequencing primer set and a giant panda microsatellite genotyping method provided by the present invention is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1: Effect analysis on high-quality DNA samples 1. Total DNA was extracted from the blood tissues of 12 giant pandas using the phenol-chloroform method.

[0037] 2. Using the DNA obtained in step 1 as a template, amplification was performed using the microsatellite primers shown in Table 2. The results are as follows: Figure 1 As shown, the PCR products obtained by amplifying high-quality DNA samples using microsatellite primers have high brightness and no extraneous bands.

[0038] 3. After mixing the products obtained in step (2), amplify them using the barcode primers shown in Table 1, mix them again, and then perform next-generation sequencing.

[0039] Microsatellite sequencing status as follows Figure 2 As shown, sequencing results of 12 high-quality panda DNA samples obtained in step 1 yielded a total of 9.42 G of data, derived from combinations of three sets of barcode primers (Table 1) (each set consisted of PCR products from four barcode primers mixed together). After pairing and assembly, this invention obtained a total of 9,720,300 microsatellite sequences, averaging approximately 90,000 sequences per locus. The lengths of these sequences ranged from 150 to 210 bp, with 160 bp being the most frequent.

[0040] According to the statistical analysis of 9 microsatellite loci of 12 samples, it is found that the repetition rate of the 108 genotypes obtained by the second generation sequencing data is between 75.3-90.5%, the number of unique sequences is between 1028-3291, two loci (GP08 and Ame-u13) fail to find microsatellite repeat sequences, and the number of sequences containing microsatellite core repeat components of the remaining 7 loci is between 58790-79531, the distribution frequency conforms to the rule that the longer the number of microsatellite repeats is, the lower the frequency is, and accordingly the microsatellite genotypes of 7 loci of 12 samples are obtained. After inspection, the accuracy of the 84 genotypes is 100%.

[0041] Example 2: Effect analysis of low-quality DNA samples 1. The Qiagen fecal DNA extraction kit (QIAamp DNA Stool Mini Kit) was used to extract total DNA from 12 giant panda feces.

[0042] 2. The fecal DNA obtained in step 1 was used as a template, and the microsatellite primers shown in Table 2 were used for amplification, and the results are shown in Figure 3 The brightness of the PCR products obtained by different microsatellite primers amplifying low-quality DNA samples is quite different, some primers have brighter target bands, and some have weaker bands, and there are many primer dimers. There are also many heterozygous bands in the amplification of low-quality DNA samples.

[0043] 3. The products obtained in step (2) were mixed and amplified using the barcode primers shown in Table 1, and then mixed again for second-generation sequencing.

[0044] The microsatellite sequencing is shown in Figure 4 According to the sequencing results of 12 panda fecal DNA samples (low quality, containing various contaminants) obtained in step 1, the present application obtained a total of 8.78G data, which came from 3 groups of barcode primers (Table 1) (each 4 barcode primers were mixed to obtain 1 group). After paired assembly, the present application obtained a total of 9.078 million microsatellite sequences, with an average of about 84,000 sequences per locus. The length distribution of these sequences is between 150-290bp, and the frequency of 160bp is the highest. Compared with high-quality DNA, low-quality DNA amplification produces a large number of heterozygous bands.

[0045] According to the statistical analysis of 9 microsatellite loci of 12 samples, the present application found that the repeat rate of the 108 genotypes of the second generation sequencing data was between 27.4-88.5%, the number of unique sequences was between 1571-25576, two loci (GP08 and Ame-u13) failed to find microsatellite repeat sequences, and the remaining 7 loci contained microsatellite core repeat components, and the number of sequences was between 110-9546. The distribution frequency of the microsatellite sequences of the 7 loci conforms to the rule that the longer the number of microsatellite repeats, the lower the frequency of occurrence, and accordingly the microsatellite genotypes of the 7 loci of the 12 samples are obtained. After inspection, the accuracy of the 84 genotypes is 100%.

[0046] Example 3: The number of PCR products that can be mixed in a single library of microsatellite sequencing According to the data obtained in Example 1 and Example 2, the data output of each sequencing library is 1G, 660-1000 PCR products can be mixed in each sequencing library, then 2-3.3 ng of each PCR product should be taken, and the optimal PCR product is 720, then the optimal amount of each PCR product is 2.8 ng, so that the number of microsatellite sequences that can be obtained for each sample at each locus is 900-2000, which can fully meet the requirements of microsatellite genotyping. Since the primers of each microsatellite locus are different, the number of barcodes should be designed according to the corresponding sample number when designing the number of PCRs to be added in each sequencing library. The barcodes of the samples mixed together must be different, so that each sample can be distinguished in the sequencing results.

[0047] Comparative Example 1: Cost comparison between microsatellite sequencing and capillary electrophoresis At present, the price of using capillary electrophoresis to analyze microsatellite genotypes is 9 yuan per sample per locus, and the price of 660,000 sequences obtained by microsatellite sequencing is 400 yuan (separate library, 1G data volume). This data volume can be used to analyze the microsatellite genotypes of 660-1000 PCR products. According to 660 PCRs, the price of each sample per locus is 0.61 yuan, which is only 6.8% of the price of capillary electrophoresis (9 yuan / locus) Figure 5 ), the price is reduced by about 14 times.

[0048] Comparative Example 2: Accuracy comparison between microsatellite sequencing and capillary electrophoresis (1) Microsatellite sequencing can show whether the microsatellite sequence is successfully amplified at the locus, while capillary electrophoresis cannot show whether the locus contains microsatellite repeat sequences Microsatellite sequencing data showed that two microsatellite loci (GP08 and Ame-u13) of the giant panda failed to find microsatellite repeats, in which GP08 amplified microsatellite sequences, but the most abundant sequence did not show microsatellite repeats, and all sequences of Ame-u13 did not have microsatellite repeats, so the present application excluded these two loci.

[0049] Capillary electrophoresis can only show the length and abundance information of the analyzed sequences Figure 6 According to the conventional capillary electrophoresis analysis, the capillary electrophoresis of these two loci (GP08 and Ame-u13) showed that the genotype of GP08 should be 173\173, and the genotype of Ame-u13 should be 153\155. However, microsatellite sequencing showed that the peak 173.4 of GP08 did not contain microsatellite repeats, and the two main peaks 152.6 and 154.6 of Ame-u13 did not contain microsatellite repeats, so the genotype obtained by capillary electrophoresis was wrong.

[0050] (2) Microsatellite sequencing can distinguish whether the sequences with similar lengths contain microsatellite sequences, while capillary electrophoresis cannot distinguish The capillary electrophoresis diagram of locus GPL-31 is shown in Figure 7 There are two main peaks at 172.6 and 195.6, which cannot confirm the true microsatellite peak; and the microsatellite sequencing of the present application shows that 195.6 here is the microsatellite genotype of the locus.

[0051] (3) Microsatellite sequencing can show point mutations in microsatellite sequences, while capillary electrophoresis cannot distinguish alleles with the same length but with point mutations Capillary electrophoresis for microsatellite typing can only distinguish alleles with different lengths, and cannot reveal alleles with the same length but with point mutations. Microsatellite sequencing directly obtains the sequence of microsatellite alleles, so it can be used to distinguish alleles with the same length but with point mutations, thereby ensuring the accuracy of the results of microsatellite alleles.

[0052] Comparative Example 3: Comparison of success rate of microsatellite target fragment amplification of low-quality DNA In order to compare the amplification efficiency of universal primers in low-quality DNA, the present application carried out the following two experiments, and the total volume of each PCR was 20 μL: Conventional primer amplification: directly use the microsatellite primers of the giant panda, i.e. delete the universal primer sequence in all primers in Table 2 to obtain the fragments as conventional primers. These conventional microsatellite primers are used to amplify 12 low-quality panda fecal samples. At the same time, in order to facilitate comparison, the present application also uses the same primers to perform re-amplification using the first PCR product as a template.

[0053] Universal primer amplification: the same 12 low-quality panda fecal samples were amplified by microsatellite primers (Table 2) first, and then amplified by bar code primers (Table 1) and the first PCR products as templates.

[0054] The amounts of target products obtained from the two experiments are shown in Table 2. Figure 8 Although the amounts of PCR products of the two primers are not significantly different (P>0.05), 32 times of PCR with conventional primers failed to amplify enough DNA (2 ng) for microsatellite sequencing, and the success rate of amplification was 70.4%, while 108 times of PCR with universal primers obtained enough DNA for microsatellite sequencing, and the success rate of amplification was 100%. Figure 8 The results also show that for samples with high DNA concentration, there is no significant difference in the amount of target product obtained by conventional primers or universal primers; but for samples with low DNA concentration, it is difficult for conventional primers to amplify the target fragment, while the probability of universal primers amplifying the target fragment is significantly increased (P<0.001).

[0055] After repeated experiments, the universal primer in the bar code primer can increase the concentration of the target product that is difficult to amplify by conventional primers, and the success rate of library sequencing of low-quality DNA can be increased from 60-70% to 90-100%.

[0056] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which belong to the protection scope of the present application.

Claims

1. A method for microsatellite genotyping, characterized by, The method comprises the following steps: (1) using genomic DNA of a species to be tested as a template, performing PCR amplification by using microsatellite primers to obtain several microsatellite locus PCR products; (2) mixing the several microsatellite locus PCR products obtained in step (1) as a template, performing PCR amplification by using barcode primers to obtain several barcode PCR products; The barcode primers comprise a sample barcode sequence and a universal primer; (3) mixing the several barcode PCR products in step (2) as a sequencing library for sequencing, and performing microsatellite genotyping on the species to be tested according to the sequencing results.

2. The microsatellite genotyping method according to claim 1, wherein, The species to be tested in step (1) comprises a giant panda.

3. The microsatellite genotyping method according to claim 1, wherein, The 19 bases at the 5' end of the forward primer and the 20 bases at the 5' end of the reverse primer of the microsatellite primers in step (1) are both from the universal primer.

4. The microsatellite genotyping method according to claim 2, wherein The microsatellite primers designed for the giant panda comprise at least one of the following pairs: Ame-u10, Ame-u13, Ame-u15, GPY-20, Ame-u26, GP-01, GP-08, GP-901 and GPL-31; The primer pair Ame-u10 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 3 and a reverse primer with the nucleotide sequence shown in SEQ ID No. 4; The primer pair Ame-u13 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 5 and a reverse primer with the nucleotide sequence shown in SEQ ID No. 6; The primer pair Ame-u15 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 7 and a reverse primer with the nucleotide sequence shown in SEQ ID No. 8; The primer pair GPY-20 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 9 and a reverse primer with the nucleotide sequence shown in SEQ ID No. 10; The primer pair Ame-u26 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 11 and a reverse primer with the nucleotide sequence shown in SEQ ID No. 12; The primer pair GP-01 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 13 and a reverse primer with the nucleotide sequence shown in SEQ ID No. 14; The primer pair GP-08 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 15 and a reverse primer with the nucleotide sequence shown in SEQ ID No. 16; The primer pair GP-901 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 17 and a reverse primer with the nucleotide sequence shown in SEQ ID No. 18; The primer pair GPL-901 comprises a forward primer with the nucleotide sequence shown in SEQ ID No. 19 and a reverse primer with the nucleotide sequence shown in SEQ ID No.

20.

5. The microsatellite genotyping method according to claim 1, wherein The program of the PCR amplification in step (1) comprises: 98℃ pre-denaturation for 2 min; 5℃ denaturation for 30 s, 55℃ to 45℃ cooling annealing for 30 s, 72℃ extension for 45 s, 35 cycles; 72℃ final extension for 15 min, 4℃ keeping.

6. The microsatellite genotyping method according to claim 1, wherein, The sample barcode sequences of the forward primer and the reverse primer of each pair of the barcode primers in step (2) are the same; The sample barcode sequence comprises at least one of ATGCAT, CAGAGT, CATGCA and TGACAG.

7. The microsatellite genotyping method according to claim 2, wherein The universal primer of the giant panda in step (2) comprises a forward primer TongF and a reverse primer TongR, wherein the nucleotide sequence of the forward primer TongF is shown as SEQ ID No. 1, and the nucleotide sequence of the reverse primer TongR is shown as SEQ ID No.

2.

8. The microsatellite genotyping method according to claim 7, wherein The program of the PCR amplification in step (2) comprises: 98℃ pre-denaturation for 2min; 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 45s, 25 cycles; 72℃ terminal extension for 15min, 4℃ keeping.

9. The microsatellite genotyping method according to claim 1, wherein, The concentration of each primer in steps (1) and (2) is 20pmol / μL.

10. The microsatellite genotyping method according to claim 1, wherein, The sequencing in step (3) comprises second-generation sequencing.

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