Capture probe for detecting chloroplast protein coding region of Chinese herbaceous peony group and kit and application thereof
By designing capture probes used for the chloroplast protein coding region of the Paeonia group combined with second-generation sequencing technology, the time-consuming and insufficient resolution of the germplasm identification of the Paeonia group in the prior art was solved, and efficient and low-cost germplasm identification was achieved.
Patent Information
- Application Number
- CN202510668567.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-22
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art is difficult to quickly and effectively identify the medicinal plant germplasm of the peony group. The traditional methods take a long time and lack resolution. High-throughput sequencing has problems of data redundancy and high analysis complexity.
A set of capture probes for the chloroplast protein coding region of the peony group was designed. Combined with second-generation sequencing technology, hybrid capture and PCR amplification after capture, efficient detection of the chloroplast protein coding region was achieved.
The rapid germplasm identification of medicinal plants in the peony group was achieved, and a high-throughput and low-cost germplasm identification scheme was provided, which improved data utilization and identification accuracy.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular biology, and particularly relates to a group of capture probes for detecting chloroplast protein coding regions of peony group, a kit thereof, and applications thereof. Background Art
[0002] The Paeonia group (Sect. Paeoniae) is a perennial herbaceous plant family in the Paeoniaceae family. It is generally believed to comprise approximately 22 species, widely distributed across the temperate climates of Eurasia. Seven species and two subspecies of Paeoniae are native to China. White and red peony root varieties, both derived from the dried roots of medicinal plants in the Paeoniaceae group, are commonly used in clinical practice as traditional Chinese medicines. They promote blood circulation, dissipate blood stasis, clear the liver, and relieve pain. They are used for symptoms such as abdominal pain caused by blood stasis, amenorrhea, dysmenorrhea, and chest and flank pain. However, due to the high morphological similarity among species in the Paeoniaceae group, rapid identification of medicinal plants or herbs using traditional methods such as traits and microscopy is difficult.
[0003] At present, the molecular markers used for the identification and molecular systematics of peony species are mainly common gene sequences such as nuclear gene I TS sequences, chloroplast gene matK sequences, and rcbL. However, the variation sites provided by single gene fragments are limited, the resolution is insufficient, and they cannot achieve good identification of germplasm and molecular systematic analysis. In addition, traditional PCR amplification and Sanger sequencing technologies have low throughput and long time consumption, which cannot meet the needs of rapid identification of large-scale germplasm resources. The chloroplast genome has the characteristics of structural conservation (circular tetrad structure), maternal inheritance, and moderate mutation rate. It is independent of the nuclear genome and carries complete genetic information. Its protein coding region sequence is highly stable and can provide richer phylogenetic information. In recent years, it has become an ideal molecular marker for plant classification and species identification.
[0004] Despite the significant reduction in the cost of high-throughput sequencing technology, direct sequencing of the entire chloroplast genome still presents challenges such as data redundancy (interference from the nuclear genome) and high analytical complexity. Probe hybridization capture technology can enrich target regions (such as chloroplast protein-coding regions) and, combined with next-generation sequencing, significantly improve data utilization and reduce costs. However, the design and optimization of specific probes for the chloroplast protein-coding regions of the Paeonia lactiflora group are currently incomplete, resulting in a lack of an efficient, standardized identification system for this group of species. Summary of the Invention
[0005] In response to the above problems, in a first aspect, the present invention proposes a set of capture probes for detecting chloroplast protein coding regions of medicinal plants of the Paeonia lactiflora group, wherein the nucleotide sequences of the capture probes are shown in SEQ ID NO: 1 to SEQ ID NO: 1144.
[0006] Furthermore, the capture probe is a single-stranded DNA probe.
[0007] Furthermore, the capture probe is labeled with biotin.
[0008] In a second aspect, the present invention provides a kit for detecting the chloroplast protein coding region of Paeonia lactiflora, comprising the capture probe.
[0009] Furthermore, the kit also includes streptavidin magnetic beads and library hybridization reaction reagents.
[0010] Furthermore, the kit further comprises at least one of the following functional components:
[0011] Targeted sequencing library purification reagents;
[0012] PCR premix reaction reagents;
[0013] Post-capture PCR amplification reaction reagents.
[0014] In a third aspect, the present invention provides a method for detecting chloroplast protein coding sequences in the Paeonia lactiflora group, comprising the following steps:
[0015] hybridizing the genomic DNA library of the sample to be tested with the capture probe to obtain a hybridization reaction solution;
[0016] separating the DNA fragments hybridized with the capture probe from the hybridization reaction solution;
[0017] The DNA fragment is used as a template for post-capture PCR amplification, the obtained PCR product is subjected to second-generation sequencing, and the sequencing result is subjected to data analysis to obtain the chloroplast protein coding sequence of the medicinal plant of the peony group.
[0018] In a fourth aspect, the present invention proposes the use of the capture probe or the kit in capturing the chloroplast protein coding region of Paeonia lactiflora.
[0019] In a fifth aspect, the present invention proposes the use of the capture probe or the kit in the germplasm tracing of the Paeonia lactiflora group.
[0020] In a sixth aspect, the present invention proposes the use of the capture probe or the kit in species identification, genetic diversity analysis and / or chloroplast genome assembly of the Paeonia lactiflora group.
[0021] Beneficial effects of the present invention:
[0022] The capture probe provided by the present invention comprehensively covers 78 chloroplast protein coding genes of the peony group medicinal plants, has the characteristics of uniformity, specificity and high capture efficiency for the captured areas, and can realize rapid detection of the chloroplast protein coding regions of the peony group medicinal plants.
[0023] The method of probe hybridization capture combined with second-generation sequencing proposed in the present invention can realize the germplasm identification of medicinal plants in the peony group.
[0024] The capture probe provided by the present invention can provide reliable information for DNA barcode research of medicinal plants in the Paeonia lactiflora group.
[0025] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0027] Figure 1 Shown are germplasm identification diagrams of three peony group medicinal plants constructed from 78 peony group chloroplast protein encoding genes in the examples of the present invention. DETAILED DESCRIPTION
[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.
[0029] Example 1
[0030] The design of capture probes for the chloroplast protein coding regions of species in the Paeonia lactiflora group includes the following steps:
[0031] (1) Construction of a chloroplast protein coding sequence collection
[0032] The chloroplast genome of the peony group species P. lactiflora NC_040983 was downloaded from the NCBI database, and 78 non-repeated protein-coding genes (a total of 84 protein-coding gene sequences, excluding the inverted repeats of 6 genes - ndhB, rpl2, rps12, rps7, ycf15, and ycf2) were extracted using Geneious Prime software to obtain the chloroplast protein-coding sequence collection.
[0033] (2) Probe design
[0034] Based on the obtained chloroplast protein-coding sequence collection, a "shingled" probe design approach was employed based on a multi-factor algorithm to design multi-layered probes of 100 nucleotides in length for the target region. After comprehensive evaluation, 1,144 capture probes were designed and synthesized using a high-throughput electrochemical method (commissioned to Aigitaikang Biotechnology (Beijing) Co., Ltd.). These probes achieved 100% coverage of all sequences in the chloroplast protein-coding sequence collection and ensured uniform and effective capture depth across all regions of the chloroplast protein-coding genes. The nucleotide sequences of the designed capture probes are shown in SEQ ID NOs: 1 to 1144.
[0035] In the present invention, the capture probe is preferably a single-stranded DNA modified with a biotin group at the 5' end, so as to facilitate subsequent hybridization of the capture probe with the target region DNA to form a complex for separation.
[0036] Example 2
[0037] The capture probe designed in Example 1 is used to detect the chloroplast protein coding region of the medicinal plants of the Paeonia lactiflora group and identify the germplasm, including the following steps:
[0038] (1) Construction of genomic DNA library of the sample to be tested:
[0039] Dried leaves of three peony group medicinal plants were used as test samples. Genomic DNA was extracted using the QIAGEN DNeasy Plantmini Kit. The genomic DNA was fragmented and end-repaired and adapter-ligated to construct a genomic DNA library.
[0040] (2) Hybridization capture:
[0041] 1. Preliminary preparation for hybrid capture experiment. The specific preparation process is as follows:
[0042] 1.1 Take out the blocking reagent Hyb Human Block from the -20℃ refrigerator and place it on an ice box to melt. After melting, vortex briefly to mix and centrifuge it immediately. Place it on an ice box for storage.
[0043] 1.2 Insert the matching oligonucleotide fragments Take the Blocking Oligo out of the -20℃ freezer, place it on an ice box to melt, vortex briefly to mix, centrifuge briefly, and place it on an ice box for storage;
[0044] 1.3 Take out the genomic DNA library to be hybridized and captured from the -20℃ refrigerator, place it on an ice box to melt, vortex briefly to mix and centrifuge instantaneously, and place it on an ice box temporarily; vortex briefly to mix and centrifuge instantaneously. If there is precipitation, remove the TargetSeq Heat Hyb Buffer v2 in a 37°C water bath and use it only after the reagent is completely dissolved.
[0045] 2. Hybridization of the genomic DNA library with the probe. The specific process is as follows:
[0046] 2.1 Take 750ng of genomic DNA library and add it to a PCR tube and mark it;
[0047] 2.2 Place the PCR tube in a vacuum centrifuge, open the PCR tube cap, and concentrate to dryness;
[0048] 2.3 After the genomic DNA library is concentrated, prepare the hybridization reaction solution according to Table 1;
[0049] Table 1 Hybridization reaction system
[0050]
[0051] 2.4 Add the hybridization reaction solution to the dried genomic DNA library, vortex for 30 seconds to ensure that the genomic DNA library dried at the bottom of the tube is dissolved, and briefly centrifuge;
[0052] 2.5 Set the PCR instrument parameters as follows, place the hybridization reaction solution on the PCR instrument, run the program according to Table 2, and hybridize for 12-18 hours to obtain the hybridization product.
[0053] Table 2 Hybridization reaction procedures
[0054]
[0055] 3. Preparation before capture experiment (performed 30 minutes before the end of hybridization reaction program), the specific process is as follows:
[0056] 3.1 Take the streptavidin magnetic beads Cap Beads out of the 4°C refrigerator in advance, mix thoroughly and equilibrate at room temperature for 30 minutes;
[0057] 3.2 Prepare 80% ethanol by volume using anhydrous ethanol and nuclease-free water in advance and store it at room temperature;
[0058] 3.3 Take out the Wash Buffer 1. If there is precipitation, heat the Wash Buffer 1 in a 37℃ water bath and wait until the precipitation is completely dissolved before use. If there is no precipitation, it can be used directly.
[0059] 3.4 TargetSeq Take out Wash Buffer 2v2 and preheat it in a 60°C water bath;
[0060] 3.5 Add 50 μL of Cap Beads to a new PCR tube and place on a magnetic rack for 1 minute. Wait for the solution to clear and discard the supernatant.
[0061] 3.6 Remove the PCR tube from the magnetic stand and add 180 μL of Binding Buffer. Mix by pipetting or vortexing to resuspend the Cap Beads.
[0062] 3.7 After centrifugation, place the PCR tube on a magnetic rack for 1 minute. Wait for the solution to clear and discard the supernatant.
[0063] 3.8 Repeat steps 3.6 to 3.7 twice, washing the Cap Beads three times with Binding Buffer.
[0064] 3.9 Remove the PCR tube from the magnetic stand, add 180 μL Binding Buffer, pipette or vortex to mix, and obtain the CapBeads resuspension. Proceed immediately to step 4.
[0065] 4. Target region DNA capture, the specific process is as follows:
[0066] 4.1 Remove the hybridization product from step 2 from the PCR instrument, centrifuge briefly, and then put the PCR tube back into the PCR instrument. Add 180 μL of the Cap Beads resuspension prepared in step 3 to the hybridization product and mix thoroughly by pipetting.
[0067] 4.2 Close the tube cap, remove the PCR tube from the thermal cycler, and place it on a vertical rotary mixer at a speed not exceeding 10 rpm. Allow to combine at room temperature for 30 minutes.
[0068] 4.3 Remove the PCR tube, centrifuge briefly, and place on a magnetic rack for 2 minutes. After the solution is clear, discard the supernatant.
[0069] 4.4 Remove the PCR tube from the magnetic stand and add 150 μL of Wash Buffer 1 to the tube. Gently pipette to mix to resuspend the magnetic beads. Replace the tube cap and wash on a vertical rotary mixer at room temperature for 15 minutes at a speed not exceeding 10 rpm.
[0070] 4.5 Remove the PCR tube, centrifuge briefly, and place on a magnetic rack for 2 minutes. After the solution is clear, discard the supernatant.
[0071] 4.6 Remove the PCR tube from the magnetic stand and add 150 μL of TargetSeq preheated at 60°C. Wash Buffer 2v2, gently pipette to mix, centrifuge briefly, place on a constant temperature shaker or metal bath, and incubate at 60℃ for 10 min;
[0072] 4.7 Remove the PCR tube, centrifuge briefly, and place on a magnetic rack for 2 minutes. After the solution is clear, discard the supernatant.
[0073] 4.8 Repeat steps 4.6 to 4.7 twice. For panels with probe coverage less than 200 kb, this can further improve capture specificity and stability.
[0074] 4.9 Remove the PCR tube from the magnetic stand and add 150 μL of TargetSeq preheated at 60°C. Wash Buffer 2v2, gently pipette to mix, centrifuge briefly, place on a constant temperature shaker or metal bath, and incubate at 60℃ for 10 min;
[0075] 4.10 Remove the PCR tube, centrifuge briefly, gently pipette to mix, transfer all the liquid (including the magnetic beads) to a new PCR tube, place the new PCR tube on a magnetic stand for 2 minutes, wait for the solution to clear, and discard the supernatant;
[0076] 4.11 Keep the PCR tube on the magnetic rack and add 200 μL of 80% ethanol to the tube. Let it stand for 30 seconds and then completely discard the ethanol solution (you can centrifuge it briefly to centrifuge the liquid on the wall to the bottom of the tube, and then use a 10 μL pipette to discard the ethanol solution remaining at the bottom). Dry the magnetic beads at room temperature to completely evaporate the residual ethanol.
[0077] 4.12 Add 24 μL of Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic stand, vortex briefly to resuspend the magnetic beads, and proceed with the amplification reaction in step 5.
[0078] 5. Post-capture PCR amplification, the specific process is as follows:
[0079] 5.1 Take out the Post PCR Master Mix and Post PCR Primer from the -20℃ refrigerator in advance and place them on an ice box to melt. After melting, mix the Post PCR Master Mix by inverting it upside down. Vortex the Post PCR Primer briefly and then centrifuge it.
[0080] 5.2 Before PCR, check again whether the Post PCR Primer is consistent with the genomic DNA library platform.
[0081] 5.3 Prepare the PCR reaction solution according to Table 3 below:
[0082] Table 3 Post-capture PCR reaction system
[0083] Reagents volume The magnetic bead suspension obtained in step 4 24 μL Post PCR Primer 1 μL Post PCR Master Mix 25 μL Total volume 50μL
[0084] 5.4 After the PCR reaction solution is prepared, use a pipette to mix it evenly and quickly transfer it to the PCR instrument.
[0085] 5.5 Set up the PCR instrument as follows: Place the PCR reaction solution on the PCR instrument and run the PCR reaction program according to Table 4;
[0086] Table 4 PCR reaction program
[0087]
[0088]
[0089] 5.6 After the PCR reaction is completed, remove the Post PCR product, centrifuge briefly, and proceed to magnetic bead purification in step 6.
[0090] 6. Purification after amplification. The specific process is as follows:
[0091] 6.1 Remove the purified magnetic beads, mix well and equilibrate at room temperature for 30 minutes;
[0092] 6.2 Add 1.1 times the volume of magnetic beads (55 μL) to the PCR product from step 5, pipette or vortex to mix, and let stand at room temperature for 5 minutes;
[0093] 6.3 Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clear.
[0094] 6.4 Keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds;
[0095] 6.5 Keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL of 80% ethanol solution to the PCR tube again, let it stand for 30 seconds, and then completely discard the supernatant (you can centrifuge it briefly to centrifuge the liquid on the wall to the bottom of the tube, and then use a 10 μL pipette to discard the ethanol solution remaining at the bottom);
[0096] 6.6 Ensure that the PCR tube is on the magnetic rack and stand at room temperature for 3-5 minutes to dry the magnetic beads and completely evaporate the residual ethanol;
[0097] 6.7 Add 25 μL of Nuclease-Free Water, remove the PCR tube from the magnetic stand, pipette or vortex to mix, and let it stand at room temperature for 2 minutes;
[0098] 6.8 Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clear.
[0099] 6.9 Use a pipette to aspirate 23 μL of supernatant and transfer it to a new PCR tube. Store the captured library in a -20°C freezer for one month.
[0100] 6.10 Take 1 μL of captured library and measure the library concentration using the Qubit dsDNA HS Assay Kit on a Qubit 4.0 Fluorometer. Record the library concentration.
[0101] 6.11 Take 1 μL of the captured library and perform fragment quality check using a fragment analyzer. The fragment size should be basically consistent with the pre-library size.
[0102] 7. Sequencing: The captured library was sequenced using the NovaSeq 6000 (Illumina) sequencing platform to obtain raw sequencing data.
[0103] (III) Assembly of chloroplast protein coding regions and germplasm identification. The specific process is as follows:
[0104] High-throughput sequencing of the enriched capture library was performed on the Illumina platform. AdapterRemoval2 was used to quality control and remove adapters from the raw reads. The Concatenate Sequence module of PhyloSuite software was used to concatenate 78 non-duplicate protein-coding genes extracted from P. lactiflora NC_040983, which served as the reference protein-coding sequence. Clean reads were aligned to the reference protein-coding sequence using the Burrows-Wheeler alignment tool, and PCR duplicate reads were removed using Picard tools. Finally, sam2 consensus (https: / / github.com / edgardomortiz / sam2consensus) was used to obtain the chloroplast protein-coding sequences of the three samples to be identified, using a 75% threshold.
[0105] The chloroplast genomes of nine peony species were downloaded from the NCBI database and 78 non-repeated protein-coding genes were extracted. The MAFFT module of PhyloSuite software was used to perform multiple alignments of the 78 non-repeated chloroplast protein-coding genes, and the sequences were concatenated using the Concatenate Sequence module. The chloroplast protein-coding sequences of the three samples to be identified and the nine peony species were combined. The RAxML software was used to set the GTR+GAMMA substitution model, with Paeonia ostii as the outgroup, and a maximum likelihood (ML) tree was constructed using 1000 bootstrap values (e.g., Figure 1 As shown in the figure, the three samples to be identified, JCG, BBS and ZJBH, were clustered into one branch with Paeonia lactiflora, and the three samples to be identified were Paeonia lactiflora.
[0106] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A set of capture probes for detecting chloroplast protein coding regions of Paeonia lactiflora, characterized in that: The nucleotide sequences of the capture probes are shown in SEQ ID NO: 1 to SEQ ID NO: 1144.
2. The capture probe for detecting the chloroplast protein coding region of the Paeonia lactiflora group according to claim 1, characterized in that The capture probe is a single-stranded DNA probe.
3. The capture probe for detecting the chloroplast protein coding region of the Paeonia lactiflora group according to claim 1 or 2, characterized in that The capture probe is labeled with biotin.
4. A kit for detecting chloroplast protein coding regions in the peony group, characterized in that: The capture probe comprises the capture probe according to any one of claims 1 to 3.
5. The kit for detecting the chloroplast protein coding region of the peony group according to claim 4, characterized in that The kit also includes streptavidin magnetic beads and library hybridization reaction reagents.
6. The kit for detecting the chloroplast protein coding region of the peony group according to claim 4, characterized in that The kit further comprises at least one of the following functional components: Targeted sequencing library purification reagents; PCR premix reaction reagents; Post-capture PCR amplification reaction reagents.
7. A method for detecting chloroplast protein coding sequences in the Paeonia lactiflora group, characterized in that: The following steps are involved: hybridizing a genomic DNA library of a sample to be tested with the capture probe according to any one of claims 1 to 3 to obtain a hybridization reaction solution; separating the DNA fragments hybridized with the capture probe from the hybridization reaction solution; The DNA fragment is used as a template for post-capture PCR amplification, the obtained PCR product is subjected to second-generation sequencing, and the sequencing result is subjected to data analysis to obtain the chloroplast protein coding sequence of the medicinal plant of the peony group.
8. Use of the capture probe according to any one of claims 1 to 3 or the kit according to any one of claims 3 to 6 in capturing chloroplast protein coding regions of Paeonia lactiflora.
9. Use of the capture probe according to any one of claims 1 to 3 or the kit according to any one of claims 3 to 6 in tracing the germplasm of Paeonia lactiflora.
10. Use of the capture probe according to any one of claims 1 to 3 or the kit according to any one of claims 3 to 6 in species identification, genetic diversity analysis and / or chloroplast genome assembly of the Paeonia lactiflora group.