Molecular marker primer for tailed amphibian and application of molecular marker primer
By providing the molecular marker primer ISSRSEQ6 for tailed amphibians, and combining high-throughput sequencing and bioinformatics analysis, the problem of obtaining SNPs in amphibian population genetics research was solved, achieving non-destructive sampling and the acquisition of a large number of SNPs.
Patent Information
- Application Number
- CN202511221619.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-12-09
AI Technical Summary
Existing technologies are insufficient to reliably obtain a large number of SNPs in amphibian population genetics studies. In particular, the ISSR-SEQ method is not widely used in amphibians, and wild animal sampling is insufficient to meet the requirements for DNA library preparation.
A molecular marker primer ISSRSEQ6 for tailed amphibians is provided for PCR amplification, and combined with high-throughput sequencing and bioinformatics analysis, it enables PCR amplification of genomic DNA and acquisition of SNPs from tailed amphibians.
It enables non-invasive sampling of tailed amphibians, obtaining a large number of cloned products with specific sequences, and acquiring a large number of SNPs through high-throughput sequencing, making it suitable for genomic DNA analysis of trace and degraded samples.
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Figure CN121087186A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, and in particular to molecular marker primers for tailed amphibians and their applications. Background Technology
[0002] Environmental factors, climate, and disease can threaten the survival of endangered species with the constant threat of extinction. Therefore, prioritizing the protection of endangered species and preventing their extinction is a crucial and primary aspect of conservation biology. Amphibians are a large, mysterious, and seriously threatened group of vertebrates. The International Union for Conservation of Nature (IUCN) has conducted threat level assessments on 8,009 amphibian species, of which 3,364 are classified as Near Threatened or higher, representing 41% of this group, far exceeding other vertebrate groups (IUCN, 2025). Among amphibians, the proportion of those classified as Near Threatened or higher is even higher, reaching 65%. Therefore, strengthening the protection of biodiversity and genetic diversity of amphibians, especially tailed amphibians, is of great significance.
[0003] With the widespread adoption of high-throughput technologies, molecular markers based on single nucleotide polymorphism (SNP) big data are widely used in population genetics research. Compared to traditional molecular markers, such as microsatellite markers and Sanger sequencing, SNP data, due to its rich data volume, can reveal more deeply the genetic status of a target species population. Currently, there are many methods for obtaining SNPs, commonly including whole-genome sequencing and simplified genome sequencing. However, all these methods require high-quality and high-content genomic DNA from the target individual for DNA library preparation before high-throughput sequencing. Non-invasive sampling from wild animals often fails to meet the requirements for DNA library preparation.
[0004] In recent years, scientists have proposed the ISSR-SEQ method, which uses multiple tandem repeat sequences as primers to amplify the genomic DNA of an individual of a target species via PCR, thereby obtaining a large number of target sequences. This method can clone target DNA sequences from trace amounts of genomic DNA and slightly degraded DNA, and combine high-throughput sequencing technology and bioinformatics analysis to obtain a certain number of SNPs. However, this method is currently mainly used in plant population genetics research, and the number of SNPs successfully obtained is often small. In amphibian population genetics research, ISSR-SEQ lacks stable ISSR primers that can obtain a large number of target fragments.
[0005] In summary, in order to solve the problem of applying ISSR-SEQ to amphibian population genetics research, it is an urgent problem to develop a primer that can stably amplify the target sequence in large quantities. Summary of the Invention
[0006] In view of this, the present invention discloses molecular marker primers for tailed amphibians and their applications, so as to enable the application of ISSR-SEQ to amphibian population genetics research.
[0007] The technical solution provided by this invention, on the one hand, provides molecular marker primers for tailed amphibians, whose nucleotide sequence is: ISSRSEQ6; sequence: 5'-AATCAAATCAATCAAATACTC-3'.
[0008] Secondly, the present invention also provides the application of the molecular marker primers for tailed amphibians, which are used to perform PCR amplification of the genomic DNA of tailed amphibians to obtain SNPs.
[0009] Preferably, it includes:
[0010] 1) Perform PCR amplification on the target sample using the primers described in claim 1;
[0011] 2) Library construction and sequencing of the amplified products for high-throughput sequencing;
[0012] 3) Perform bioinformatics analysis on the sequencing results.
[0013] 4. The application according to claim 3, characterized in that, using the primers of claim 1 to perform PCR amplification of the target sample includes:
[0014] Step 1: Non-destructive sampling of the target species was carried out in the field. A 1-2 mm long sample of the tip of the forelimb toe was taken and preserved by immersing it in anhydrous ethanol to obtain toe tissue samples.
[0015] Step 2: Genomic DNA was extracted from the toe tissue sample using a blood / cell / tissue genomic DNA extraction kit;
[0016] Step 3: Use the primers and LA Taq enzyme to perform PCR amplification on the extracted genomic DNA.
[0017] Preferably, the amplification conditions in step 3 are as follows: Step 1: 94℃ for 4 min; Step 2 (30 cycles): 95℃ for 1 min — 52℃ for 1 min — 72℃ for 10 min; Step 3: 72℃ for 20 min.
[0018] Preferably, the library preparation and sequencing for high-throughput sequencing of the amplified products include:
[0019] Step 1: Use 2% agarose gel electrophoresis to check the PCR amplification results;
[0020] Step 2: Purify the amplification products using a universal DNA purification and recovery kit, and determine the DNA content using Qubit 4;
[0021] Step 3: Use The dsDNAFragmentase enzyme fragments the purified amplification product.
[0022] Step 4: Perform blunt-end and 5' phosphorylation on each fragmented sample, and purify the blunt-end product after the reaction is complete;
[0023] Step 5: Add “A” to the 3' end of the compound using Premix Ex Taq (Takara) at 72℃ for 20 min. After the reaction is complete, perform purification.
[0024] Step 6: Connect the individual-specific connector to the sample individual using an individual-specific connector with a 3' end suspended in a "T" shape;
[0025] Step 7: Mix the final products of each sample, which are each ligated with a different specific DNA adapter, and purify the DNA. Sequencing is performed using the Illumina Hiseq2500 platform to obtain pair-end 150bp sequencing results.
[0026] Preferably, the bioinformatics analysis of the sequencing results includes:
[0027] Step 1: Obtain individual sequences and use the fsplit script to classify individual data according to different individual-specific adapters;
[0028] Step 2: Use the cutadapt script to remove individual-specific adapters and 5' primer sequences;
[0029] Step 3: Randomly select the paired-end sequence of a sample, assemble it using SPAdes, and retain the sequence with a length greater than 200bp and a sequencing depth greater than 50.
[0030] Step 4: Use cd-hit-est to remove redundancy from the assembled sequence, and use the resulting sequence as a reference genome sequence for subsequent SNP extraction and analysis;
[0031] Step 5: Use GATK's SNP acquisition strategy to obtain SNPs from different individuals and integrate them to form the final VCF file.
[0032] This invention proposes molecular marker primers for tailed amphibians and their applications. These ISSR primers can be used to perform PCR amplification on amphibians to obtain a large number of cloned products with specific sequences. At the same time, tailed amphibians can be sampled non-invasively, and PCR amplification can be performed on samples from museum collections. Subsequent high-throughput sequencing yielded a large number of SNPs.
[0033] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit the disclosure of the present invention. Attached Figure Description
[0034] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.
[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0036] Figure 1 The image shows the effect of PCR amplification of 24 Liaoning clawed salamanders using ISSRSEQ6 primers, as provided in the embodiments of the present invention. The marker (1kb Plus DNA Ladder) has base lengths of 100, 200, 300, 400, 500, 700, 1000, 1600, 2000, 5000, 8000, and 10000 from bottom to top.
[0037] Figure 2 The image shows the effect of PCR amplification of 24 species of Chinese giant salamander using ISSRSEQ6 primers, as provided in the embodiments of this invention. The marker (1kb Plus DNA Ladder) has base lengths of 100, 200, 300, 400, 500, 700, 1000, 1600, 2000, 5000, 8000, and 10000 from bottom to top.
[0038] Figure 3 This image shows the effect of PCR amplification of 24 Far Eastern salamanders using ISSRSEQ6 primers, as provided in the embodiments of this invention. The marker (250-10000bp) has base lengths from bottom to top of 250, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 8000, and 10000. Detailed Implementation
[0039] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of systems consistent with some aspects of the invention as detailed in the appended claims.
[0040] Ideal ISSR primers can stably amplify a large number of specific bands in individuals of a target population, providing fundamental information for SNP acquisition. However, developing ideal ISSR primers for non-model species is challenging. Therefore, this implementation plan provides an ISSR primer that can be used for PCR amplification of amphibians to obtain a large number of cloned products with specific sequences. This primer allows for non-invasive sampling of tailed amphibians; PCR amplification of archival samples followed by high-throughput sequencing can yield a large number of SNPs.
[0041] Specifically, it includes the following:
[0042] The molecular marker primers for tailed amphibians have the following nucleotide sequences: ISSRSEQ6; sequence: 5'-AATCAAATCAATCAAATACTC-3'.
[0043] The aforementioned molecular marker primers for tailed amphibians were used to amplify the genomic DNA of tailed amphibians by PCR to obtain SNPs.
[0044] include:
[0045] 1. PCR amplification of the target sample using the primers described in claim 1;
[0046] 1) Field collection: Non-destructive sampling of the target species, taking the tips of the forelimb toes (~1-2 mm in length) and preserving them by immersing them in anhydrous ethanol;
[0047] 2) Genomic DNA extraction: Genomic DNA was extracted from toe tissue samples using the (Blood / Cell / Tissue Genomic DNA Extraction Kit) from Tiangen Biotech (Beijing) Co., Ltd.
[0048] 2. Library preparation and sequencing of the amplified products for high-throughput sequencing;
[0049] 1) Using the primers of this invention (name: ISSRSEQ6; sequence: 5'-AATCAAATCAATCAAATACTC-3'), PCR amplification was performed on three tailed salamanders (Liaoning clawed salamander, Northeast salamander, and Far Eastern Arctic salamander) collected by Takara brand LATaq enzyme. Table 1 below shows the nucleotide sequences of Liaoning clawed salamander, Northeast salamander, and Far Eastern Arctic salamander.
[0050] The amplification conditions were as follows: Step 1: 94℃ for 4 min; Step 2 (30 cycles, each cycle the target DNA is cloned exponentially once, which is an important process of PCR amplification): 95℃ for 1 min — 45℃ for 1 min — 72℃ for 6 min; Step 3: 72℃ for 20 min.
[0051] 2) Use agarose gel electrophoresis to view the PCR amplification results (agarose concentration: 2%; voltage: 100V; duration: 30min);
[0052] 3) DNA purification and content determination: DNA purification was performed using the (General DNA Purification and Recovery Kit) from Tiangen Biotech (Beijing) Co., Ltd. DNA content was determined using a Qubit 4 analyzer.
[0053] 4) Fragmentation: 2ug is used for each sample. The DNA was fragmented using dsDNAFragmentase for 20 minutes, followed by DNA purification.
[0054] 5) Each fragmented sample underwent blunt-end and 5' phosphorylation treatment. The 50 μL reaction system consisted of: 1 X Tango buffer (Fermentas), 0.1 mM of dNTPs, 3 U T4 DNA polymerase (Fermentas), 1 mM ATP, and 15 U T4 DNA polynucleotide kinase (Takara). The reaction conditions were 12℃ for 5 min, 37℃ for 5 min, and 80℃ for 15 min. After the reaction, the blunt-end product was purified.
[0055] 6) Add "A" to the 3' end of the compound using Premix Ex Taq (Takara) at 72℃ for 20 min. Purify the compound after the reaction.
[0056] 7) Individual-specific adapters were ligated into the samples. The individual-specific adapter DNA sequences with a "T" dangling at the 3' end were purchased from Sangon Biotech (Shanghai) Co., Ltd. The 50 μL reaction mixture consisted of 1X ligation buffer, 5 U T4 DNA ligase (Fermentas), and 5 μL adapter DNA (40 μM). The reaction was carried out overnight at 16°C.
[0057] 8) Mix the final products from each sample (each sample ligated with a different specific DNA adapter), purify the DNA, and send them to Sangon Biotech (Shanghai) Co., Ltd. for sequencing using the Illumina Hiseq2500 platform to obtain pair-end 150bp sequencing results. Adjust the sequencing volume according to the mixed sample volume to ensure that the sequencing volume of each sample is greater than 100Mb.
[0058] 3. Perform bioinformatics analysis on the sequencing results. The main analysis process is as follows:
[0059] 1) Individual sequence acquisition: The fsplit script is used to classify individual data according to different individual-specific adapters;
[0060] 2) The individual-specific adapters and 5' primer sequences were removed using the cutadapt script; the individual sequences obtained in this study are shown in Table 1;
[0061] Table 1. Species name, sample number, and NCBI SRA sequence number used for sequencing.
[0062]
[0063]
[0064]
[0065] 3) Randomly select the paired-end sequence of a sample, assemble it using SPAdes, and retain the sequence with a length greater than 300bp and a sequencing depth greater than 50.
[0066] 4) Use cd-hit-est to remove redundancy from the assembled sequence, and use the resulting sequence as a reference genome sequence for subsequent SNP extraction and analysis;
[0067] 5) Use GATK's SNP acquisition strategy to obtain SNPs from different individuals and integrate them to form the final VCF file.
[0068] The ISSRSEQ6 primers provided by this invention can be widely used in tailed amphibians to amplify a large number of species-specific target DNA fragments and obtain a large number of SNPs. The method, which uses toe-tip tissue sampling, does not harm endangered species and can be applied to tailed amphibians for non-invasive sampling to extract trace amounts of genomic DNA.
[0069] The ISSR-SEQ method based on ISSRSEQ6 primers can also be applied to genomic DNA from degraded samples of tailed amphibians, allowing for the reuse of mildly degraded samples and long-term preserved collections, and the cost of obtaining SNPs is relatively low.
[0070] To gain a more thorough understanding of the present invention, the present invention will be further described in detail below with reference to specific embodiments.
[0071] Example 1
[0072] The following species were selected as experimental subjects: Hynobiidae, Onychodactylus zhaoermii, and Salamandrella tridactyla. Twenty-four samples were collected from each species for non-destructive sampling to verify the feasibility of using ISSRSEQ6 primers for ISSR-SEQ. Figure 1-3 Electrophoresis images of PCR products from three species. Figure 1-3 The ISSRSEQ6 primers can stably amplify a large number of specific bands. Due to the small difference in band length and the large number of bands, the electrophoresis results may appear as sheets.
[0073] Ultimately, this embodiment successfully used ISSRSEQ6 primers to perform PCR amplification on three species of salamanders (Liaoning clawed salamander, Northeast salamander, and Far Eastern Arctic salamander). High-throughput sequencing yielded over 3600 SNPs in the Liaoning clawed salamander population, over 7000 SNPs in the Northeast salamander population, and over 2300 SNPs in the Far Eastern Arctic salamander population.
[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these changes and modifications should also be considered within the scope of protection of the present invention.
Claims
1. Molecular marker primers for caudate amphibians, characterized in that, The nucleotide sequence is: ISSR SEQ6; sequence: 5'-AATCAAATCAATCAAATACTC-3'.
2. Use of the molecular marker primer of the caudate amphibian according to claim 1, characterized in that, The genomic DNA of the caudate amphibian is subjected to PCR amplification to obtain SNPs.
3. The use according to claim 2, characterized in that, It comprises: 1) PCR amplification of the target sample using the primer according to claim 1; 2) library construction and sequencing of the amplified product for high-throughput sequencing; 3) bioinformatics analysis of the sequencing results.
4. Use according to claim 3, characterized in that, The PCR amplification of the target sample using the primer according to claim 1 comprises: Step 1: non-invasive sampling of the target species, field collection, taking 1-2 mm long of the toe tip of the forelimb, and immersing in anhydrous ethanol for preservation to obtain a toe tissue sample; Step 2: genomic DNA extraction of the toe tissue sample using a blood / cell / tissue genomic DNA extraction kit; Step 3: PCR amplification of the extracted genomic DNA using the primer according to claim 1 and LA Taq enzyme.
5. Use according to claim 4, characterized in that, The amplification conditions in step 3 are: first step: 94℃ for 4 min; second step: 30 cycles, 95℃ for 1 min, 45℃ for 1 min, and 72℃ for 6 min; third step: 72℃ for 20 min.
6. The use according to claim 3, characterized in that, The library construction and sequencing of the amplified product for high-throughput sequencing comprises: Step 1: 2% agarose gel electrophoresis is used to check the PCR amplification results; Step 2: a universal DNA purification and recovery kit is used to purify the amplified product, and Qubit 4 is used to determine the DNA content; Step 3: Use of The purified amplification products were fragmented using the dsDNA Fragmentase enzyme; Step 4: after fragmentation, each sample is subjected to end-blunting and 5' phosphorylation treatment, and the end-blunted product is purified after the reaction is completed; Step 5: 3' end "A" is added, and Premix Ex Taq (Takara) is used for the reaction, and the reaction conditions are 72℃ for 20 min, and purification is performed after the reaction is completed; Step 6: individual-specific adapters are connected to the sample using 3' end hanging "T" individual-specific adapters; Step 7: the final products of the samples each connected with different specific DNA adapters are mixed, and DNA purification is performed, sequencing is performed based on the Illumina Hiseq2500 platform, and pair-end 150bp sequencing results are obtained.
7. The use according to claim 3, characterized in that, The bioinformatics analysis of the sequencing results comprises: Step 1: individual sequences are obtained, and fsplit scripts are used to classify individual data according to different individual-specific adapters; Step 2: cutadapt scripts are used to remove individual-specific adapters and 5' end primer sequences; Step 3: double-end sequences of a randomly selected sample are assembled using SPAdes, and sequences with a length greater than 200 bp and a sequencing depth greater than 50 are retained; Step 4: cd-hit-est is used to remove redundancy from the assembled sequences, and the resulting sequences are used as reference genome sequences for subsequent SNPs extraction analysis; Step 5: GATK's SNPs acquisition strategy is used to obtain SNPs of different individuals and integrate to form a final VCF file.