DNA bar code accD-psaI primer suitable for moss plants and application of DNA bar code accD-psaI primer
By designing DNA barcode accD-psaI primers suitable for mosses and combining them with the improved CTAB method and PCR amplification technology, the problems of versatility and resolution in moss identification and classification were solved, and efficient moss species identification and phylogenetic research were achieved.
Patent Information
- Application Number
- CN202510767974.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-10
- Publication Date
- 2025-09-19
AI Technical Summary
Existing DNA barcoding technology has poor versatility and low resolution in mosses, making it difficult to effectively identify and classify them.
A DNA barcode accD-psaI primer suitable for mosses was designed, with the nucleotide sequences "accD-psaI-DF, 5'-AGCCATTGTAATTGCTGGA-3'" and "accD-psaI-DR, 5'-ACCGGTGGAGTTACTGCT-3'". DNA was extracted using a modified CTAB method and identified by combining PCR amplification and sequencing technology.
High-resolution identification and classification of mosses were achieved, with amplification and sequencing success rates reaching 100%, and high resolution, suitable for the construction of phylogenetic trees.
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Figure CN120666070A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of molecular biology, and in particular relates to a DNA barcode accD-psaI primer suitable for mosses and an application thereof. Background Art
[0002] Mosses have important ecological value, ornamental value and medicinal value, and have broad market prospects. Due to its wide variety, only experts can accurately identify it by morphological characteristics. At present, DNA barcode technology has been widely used in the identification work of species (kinds) such as animals and plants. Researchers have proposed some chloroplast DNA barcodes that can be used for mosses, but these molecular markers are mostly from the research of angiosperms, are not the most suitable barcodes for mosses, and more or less have the problems such as poor versatility or low resolution. This makes the use of DNA barcode can not be well carried out in mosses. The application is based on the chloroplast full genome data of mosses, in order to screen out the high-resolution DNA barcode that is universal for mosses. Summary of the Invention
[0003] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a DNA barcode accD-psaI primer suitable for mosses and its application, which is a high-resolution DNA barcode universal for mosses, so that DNA barcode technology can be applied in the research of mosses.
[0004] The specific technical solution adopted in the present invention is:
[0005] The invention discloses a DNA barcode accD-psaI primer suitable for mosses. The nucleotide sequences of the primers are "accD-psaI-DF, 5'-AGCCATTGTAATTGCTGGA-3'" and "accD-psaI-DR, 5'-ACCGGTGGAGTTACTGCT-3'".
[0006] The primers are used in identifying mosses.
[0007] The primers are used in constructing a phylogenetic tree of mosses.
[0008] The application method comprises the following steps:
[0009] S1. Extract DNA from the moss species to be tested using a modified CTAB method;
[0010] S2. Design a PCR amplification program and perform PCR amplification using primers;
[0011] S3. Sequencing the PCR product;
[0012] S4. If the sequence of the PCR product matches a known species sequence in an existing database, it is identified as that known species; if no known species matches it, it is a new species or a species not included in the database.
[0013] The PCR amplification program is as follows: pre-denaturation, 80°C, 5 min; denaturation, 95°C, 1 min; annealing, 50°C, 1 min; extension, 65°C, 4 min; number of cycles, 35; and final extension, 65°C, 5 min.
[0014] The beneficial effects of the present invention are:
[0015] The accD-psaI gene fragment provided by this invention has not been previously reported as a DNA barcode. However, this study found that it meets the requirements for versatility and resolution of DNA barcodes and is therefore recommended as a potential DNA barcode for mosses. The forward and backward primers for amplification are "accD-psaI-DF, 5'-AGCCATTGTAATTGCTGGA-3'" and "accD-psaI-DR, 5'-ACCGGTGGAGTTACTGCT-3'," respectively.
[0016] The DNA barcode provided by the present invention can be used in the field of plant classification for moss species identification, molecular phylogeny and other related research; in the field of ecology and biodiversity research, it can be used for moss protection; in the field of horticulture research, it can be used for moss variety (species) identification, introduction and cultivation; in addition, it can also be used in forestry, traditional Chinese medicine research, agriculture and other related moss identification and other application work. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 is the sequence polymorphism site variation map of accD-psaI;
[0018] Figure 2 is the accD-psaI genetic distance;
[0019] Figure 3 To construct a strict consensus tree based on the accD-psaI fragment; DETAILED DESCRIPTION
[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:
[0021] The invention provides a DNA barcode accD-psaI primer suitable for mosses. The nucleotide sequences of the primers are "accD-psaI-DF, 5'-AGCCATTGTAATTGCTGGA-3'" and "accD-psaI-DR, 5'-ACCGGTGGAGTTACTGCT-3'".
[0022] The specific research process of the present invention is:
[0023] 1. DNA barcode screening and primer design:
[0024] Because chloroplast genomes are highly conserved in terms of gene content and structure, exhibiting single-copy sequences and a limited number of variable sites, this study, based on whole-genome data from mosses, aimed to identify a universal, high-resolution DNA barcode. To design primers suitable for amplification in mosses, we used 56 moss chloroplast genomes (covering 56 species from 26 orders, 33 families, 40 genera), obtained from GenBank and the Dryad database, as well as in-house production. Universal primers were then designed at both ends of the newly identified hypervariable regions of the DNA barcodes.
[0025] Table 1 Chloroplast genome
[0026]
[0027]
[0028] After screening, accD-psaI was found to be a highly variable region and was recommended as a candidate DNA barcode for mosses. Based on the primer design principles and DNA barcode screening criteria, the sequence polymorphism variation map calculated by DnaSP (such as Figure 1 ① The blue origin is the region where primers are designed for each gene; ② For primer design of non-coding region genes, since the design region is in the conserved coding region, the map of non-coding region genes includes part of the coding gene). Universal primers were designed on both sides of the appropriate hypervariable region for accD-psaI (Table 2).
[0029] Table 2 List of newly designed primers
[0030]
[0031] 2. Verification of the universality of new primers:
[0032] This study used 38 moss specimens, covering 31 species from 14 orders, 22 families, to verify the universality of the new primers. The method is as follows:
[0033] 1) First, the DNA of the experimental materials was extracted using the improved CTAB method.
[0034] 2) Design a PCR amplification program and use new primers for PCR amplification.
[0035] 3) The PCR products were sent to Beijing Sangon Biotechnology Co., Ltd. for sequencing.
[0036] 4) The sequence returned by sequencing is compared with the moss chloroplast genome containing annotation information to verify whether it is the target fragment.
[0037] The following PCR reaction program was used for amplification: initial denaturation, 80°C, 5 min; denaturation, 95°C, 1 min; annealing, 50°C, 1 min; extension, 65°C, 4 min; number of cycles, 35; and final extension, 65°C, 5 min.
[0038] Amplification results showed that the accD-psaI primers achieved 100% PCR amplification and sequencing success rates for the tested materials, demonstrating the versatility of the new primers, accD-psaI-DF and accD-psaI-DR, in mosses.
[0039] Table 3 Universal verification materials
[0040]
[0041]
[0042] 3. Verify the resolution of new primers:
[0043] In principle, DNA barcode resolution verification should be performed on closely related species. This study, based on a molecular phylogeny of the order Bryales, constructed by our research group based on chloroplast genes, selected several closely related species of the genus Pohlia Hedw., located at the bottom of the phylogeny, as well as species from other branches. This experiment involved 20 samples from 14 species across two families, eight genera, and the moss Philonotis fontana of the order Bryales as an outgroup.
[0044] 1) Perform DNA extraction, PCR amplification, and sequencing on the test materials using the same methods as those for universal verification 1)-3).
[0045] 2) Integrate by gene name, delete the base sites with inaccurate sequencing before and after the original sequence in Sequencher 5.3, and synthesize contigs of gene fragments with the same name and export them.
[0046] 3) Automatic alignment was performed using the MAFFT program in Phylosuite 1.1.15, and manual adjustment was performed using BioEdit 7.0.9.0.
[0047] 4) Statistically calculate the basic information and genetic distance of the successfully amplified target fragments.
[0048] 5) Use the maximum likelihood method (ML), Bayesian analysis method (BI), and neighbor joining method (NJ) to construct the phylogenetic tree. Figure 3 . This further confirmed the resolution of the new primers.
[0049] Table 4 Resolution verification materials
[0050]
[0051]
[0052] Primer resolution verification of accD-psaI:
[0053] 1) Amplification success rate: The new accD-psaI primers, "accD-psaI-DF" and "accD-psaI-DR," achieved 100% amplification and sequencing success rates for sample 21 (two families, eight genera, 14 species, and one species of Bryales). The sequence obtained was used for subsequent analysis.
[0054] 2) Basic information of amplified fragments
[0055] Table 5 Basic information statistics of amplified fragments
[0056]
[0057] 3) From the genetic distance between species counted by MEGA, it can be seen that the intraspecific distance of most species is smaller than the interspecific distance, which meets the screening criteria of DNA barcodes ( Figure 2 ).
[0058] 4) From the topological structure of the phylogenetic tree constructed based on the accD-psaI fragment, it can be seen that (e.g. Figure 3 ), the narrow-leaved loofah moss Pohlia timmioides samples with DNA numbers P35 and P43 were clustered into a branch (ML=100, BI=1, NJ=99). Combining the results of the ML tree, BI tree, and NJ tree, the branch relationship of this species cluster was stable and the support rate was close to 100%; Combining the results of the ML tree, BI tree, and NJ tree, the branch relationship of this species cluster was stable and the support rate was close to 100%; DNA numbers P20, K08, and P11 Pohlia cruda were clustered into a branch (ML=39, BI=0, NJ=63), which was clustered into one branch in both the ML and NJ trees; DNA numbers S09, P1, and P3 Pohlia lutescens were clustered into a branch (ML=87, BI=0.98, NJ=78). In summary, it was believed that the resolution of the accD-psaI fragment was high.
[0059] Figure 3 The figure represents the strict consensus tree constructed based on the accD-psaI fragment (with the Bayesian tree as the skeleton), and the numerical values represent the posterior probability of the ML tree, the bootstrap support rate of the BI tree, and the bootstrap support rate of the NJ tree, respectively.
[0060] In summary, the accD-psaI gene fragment has never been reported as a DNA barcode. This study found that it meets the requirements of DNA barcode versatility and resolution, and is therefore recommended as an alternative DNA barcode for mosses.
Claims
1. A DNA barcode accD-psaI primer suitable for mosses, characterized by: The nucleotide sequences of the primers are "accD-psaI-DF, 5'-AGCCATTGTAATTGCTGGA-3'" and "accD-psaI-DR, 5'-ACCGGTGGAGTTACTGCT-3'".
2. Use of the primer according to claim 1 in identifying mosses.
3. Use of the primers according to claim 1 in constructing a phylogenetic tree of mosses.
4. The application according to claim 2, characterized in that: The application method comprises the following steps: S1. Extract DNA from the moss species to be tested using a modified CTAB method; S2. Design a PCR amplification program and perform PCR amplification using primers; S3. Sequencing the PCR product; S4. If the sequence of the PCR product matches a known species sequence in an existing database, it is identified as that known species; if no known species matches it, it is a new species or a species not included in the database.
5. The application according to claim 4, characterized in that: The PCR amplification program is as follows: pre-denaturation, 80°C, 5 min; denaturation, 95°C, 1 min; annealing, 50°C, 1 min; extension, 65°C, 4 min; number of cycles, 35; and final extension, 65°C, 5 min.