A DNA barcode clpP for mosses and its application

Abstract

The present invention relates to the field of molecular biology and discloses a DNA barcode (clpP) for mosses and its applications. The DNA barcode (clpP) comprises a clpP gene fragment. The primer sequences for obtaining the DNA barcode (clpP) are shown in SEQ ID NOs. 1 and 2. The DNA barcode (clpP) is used to identify mosses and construct a phylogenetic tree of mosses. This technical solution overcomes the problems of poor versatility and low resolution of existing DNA barcodes for mosses.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a DNA barcode for mosses. clpP and its applications. Background Art

[0002] Mosses are a group of higher plants in the plant kingdom that transitions from aquatic to terrestrial habitats. They possess significant ecological, ornamental, and medicinal value. Due to their diverse diversity, with approximately 23,000 species worldwide, accurate identification of mosses based on morphological characteristics is difficult.

[0003] With the rapid development and application of molecular technologies, DNA barcoding has greatly improved the accuracy of species demarcation and is widely used in moss phylogeny and evolutionary studies. However, relatively little research has been conducted on DNA barcoding for mosses. Furthermore, the DNA barcodes proposed for mosses suffer from poor versatility or low resolution, making them unsuitable for use as specialized DNA barcodes for mosses. Therefore, there is an urgent need to develop high-resolution DNA barcodes that are universal for mosses. Summary of the Invention

[0004] The present invention proposes a DNA barcode for mosses clpP The invention and its application solve the problems of poor universality or low resolution of DNA barcodes used for mosses in the prior art.

[0005] The technical solutions of the present invention are as follows:

[0006] The present invention proposes a DNA barcode for mosses clpP , the DNA barcode clpP Depend on clpP Gene fragment composition.

[0007] The present invention also proposes a method for obtaining the DNA barcode clpP The nucleotide sequences of the primers are shown in SEQ ID NO 1 and SEQ ID NO 2.

[0008] The present invention also proposes the DNA barcode clpP Application in identification of mosses.

[0009] The present invention also proposes DNA barcode clpP Application in constructing phylogenetic trees of mosses.

[0010] The present invention also provides a method for identifying mosses, comprising the following steps:

[0011] S1. Extract genomic DNA from the moss to be identified;

[0012] S2, using the genomic DNA of the moss to be identified as a template, performing PCR amplification with primers having nucleotide sequences as shown in SEQ ID NO 1 and SEQ ID NO 2 to obtain a PCR amplification product;

[0013] S3, sequencing the PCR amplification product obtained in S2;

[0014] S4. Construct a phylogenetic tree and identify mosses.

[0015] As a further technical solution, in S2, the reaction procedure of PCR amplification is: pre-denaturation at 80°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 48°C for 1 minute; extension at 65°C for 4 minutes, a total of 35 cycles, and final extension at 65°C for 5 minutes.

[0016] As a further technical solution, in S4, the method for constructing the phylogenetic tree is the maximum likelihood method, the Bayesian analysis method and the neighbor-joining method.

[0017] The working principle and beneficial effects of the present invention are:

[0018] 1. In the present invention, the first use clpP The gene fragment serves as the DNA barcode of the moss. When the DNA barcode is used to identify the moss, it has good versatility and high resolution, thereby achieving rapid and accurate identification of the moss.

[0019] 2. In this invention, for the first time, DNA barcode clpP Universal primers with the primer sequences shown as SEQ ID NO 1 and SEQ ID NO 2 were designed for the gene fragments. When the universal primers were used to perform PCR amplification and sequencing on the DNA of mosses, the PCR amplification success rate and the sequencing success rate were both as high as 100%.

[0020] 3. In the present invention, the use clpP Gene fragments can be used as DNA barcodes of mosses to identify mosses, construct phylogenetic trees of mosses, and further study the phylogeny of moss species and between species. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Figure 1 For the present invention clpP sequence polymorphism variation map;

[0023] In the figure: · is the region where primers are designed for each gene;

[0024] Figure 2 For the present invention clpP Genetic distance map of gene segments;

[0025] Figure 3 The present invention is based on clpP Phylogenetic tree constructed from gene fragments. DETAILED DESCRIPTION

[0026] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 are within the scope of protection of the present invention.

[0027] Example 1

[0028] 1. DNA barcode screening

[0029] Based on the chloroplast genome data of mosses, i.e., from the GenBank database, the Dryad database and self-produced sequences, a total of 56 moss chloroplast genomes (covering 26 orders, 33 families, 40 genera and 56 species of mosses) were screened for sequence hypervariable regions. clpP , as candidate DNA barcode gene fragments.

[0030] 2. DNA barcoding clpP Primer design

[0031] Comprehensive primer design principles and DNA barcode screening criteria, refer to Figure 1 The sequence polymorphism variation diagram calculated by DnaSP is shown. clpP Universal primers were designed on both sides of the hypervariable region, as shown in the following table:

[0032] Table 1 Primer list

[0033]

[0034] 3. DNA barcoding clpP Verification of universality

[0035] The universality of the new primers was verified using 38 moss specimens, involving 31 species from 14 orders, 22 families, and the specimen information is as follows:

[0036] Table 2 List of universal verification materials

[0037]

[0038] The experimental method is as follows:

[0039] (1) Extracting the genomic DNA of the test material using the modified CTAB method to obtain the genomic DNA of the test material;

[0040] (2) Using the genomic DNA of the test material as a template, PCR amplification was performed with primers to obtain PCR amplification primers; wherein the nucleotide sequences of the primers are shown in SEQ ID NO 1 and SEQ ID NO 2, and the PCR amplification reaction procedure was as follows: pre-denaturation at 80°C for 5 min, denaturation at 95°C for 1 min, annealing at 48°C for 1 min, extension at 65°C for 4 min, for a total of 35 cycles, and final extension at 65°C for 5 min;

[0041] (3) Send the PCR amplification products to Beijing Sangon Biotechnology Co., Ltd. for sequencing;

[0042] (4) Compare the sequence returned by sequencing with the moss chloroplast genome containing annotation information to verify whether it is the target fragment.

[0043] The experimental results are as follows: Using DNA barcodes clpP When the primers were used to perform PCR amplification and sequencing on the test materials, the PCR amplification success rate and sequencing success rate were both 100%, indicating that the candidate DNA barcode gene fragment clpP It has good versatility when used as a DNA barcode for mosses.

[0044] 4. DNA barcoding clpP Resolution verification

[0045] Reference has been made to the molecular phylogenetic tree of the order Bryales constructed based on chloroplast genes ( Pohlia (Bryophyta, Bryaceae) using chloroplastand nuclear ribosomal DNA.Phytotaxa.351(2): 141-153, public URL: https: / / doi.org / 10.11646 / phytotaxa.351.2.2), and selected the genus Luffa ( Pohlia Hedw.) and species in other branches. The experiment involved 20 samples of 14 species from 8 genera and 2 families in the order True Mosses, and selected the moss of the order Philonotis fontana As an outgroup, the sample numbers and information are as follows:

[0046] Table 3 Resolution verification material list

[0047]

[0048] The experimental method is as follows:

[0049] (1) The genomic DNA of the test materials was extracted using the modified CTAB method. The extracted genomic DNA was used as a template and PCR amplification was performed with primers to obtain PCR amplification primers. The nucleotide sequences of the primers are shown in SEQ ID NO 1 and SEQ ID NO 2. The PCR amplification reaction procedure was as follows: pre-denaturation at 80°C for 5 min, denaturation at 95°C for 1 min, annealing at 48°C for 1 min, extension at 65°C for 4 min, for a total of 35 cycles, and final extension at 65°C for 5 min. The PCR amplification products were sent to Beijing Sangon Biotechnology Co., Ltd. for sequencing.

[0050] (2) Integrate by gene name, delete the base sites before and after the original sequence that are not sequenced accurately in Sequencher 5.3, and synthesize the gene fragments with the same name into contigs for export;

[0051] (3) Automatic alignment was performed using the MAFFT program in Phylosuite 1.1.15, and manual adjustments were performed using BioEdit 7.0.9.0;

[0052] (4) Use MEGA evolutionary tree software to calculate the basic information and genetic distance of the successfully amplified target fragments;

[0053] (5) The phylogenetic tree was constructed using the Maximum Likelihood (ML) method, the Bayesian Inference (BI) method, and the Neighbor Joining (NJ) method. The Bayesian tree was used as the skeleton during the construction.

[0054] The experimental results are as follows:

[0055] (1) Use of DNA barcodes clpP When the primers were used to perform PCR amplification and sequencing on sample 21 from 2 families, 8 genera, 14 species and 1 species of Bryales, the PCR amplification success rate and sequencing success rate were both 100%;

[0056] (2) Basic information of the amplified fragment is shown in the following table:

[0057] Table 4 Basic information of amplified fragments

[0058] Clip Name Length / bp C / % V / % Pi S / % 524 79.39 20.61 0.0935 7.63

[0059] (3) The genetic distance between species in MEGA evolutionary tree statistics is as follows Figure 2 As shown in the figure, it can be seen that the intraspecific distance of most species is smaller than the interspecific distance, which meets the screening criteria of DNA barcoding;

[0060] (4) The constructed phylogenetic tree is as follows Figure 3 As shown in the figure, the 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. From the topological structure of the figure, it can be seen that species of the same family and genus are clustered on one branch, and the branch support rate is high. Pohlia timmioides The samples of the DNA number X1 and X2 were clustered into one branch (ML=90, BI=0.99, NJ=96), and the samples of the DNA number X1 and X2 were Pohlia hyaloperistoma Clustered into one branch (ML=95, BI=1, NJ=86), DNA number P20, K08 and P11 Pan-Luffa Pohlia cruda Clustered into one branch (ML=92, BI=0.99, NJ=81), DNA number S09, P1 and P3 Pohlia lutescens Clustered into one branch (ML=94, BI=1, NJ=94), the results of ML tree, BI tree, and NJ tree show that the branch relationship of the above four species is stable and the support rate is higher than 80%. The above results show that the DNA barcode of the present invention clpP Gene fragments have high resolution when used as DNA barcodes in mosses.

[0061] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A DNA barcode for mosses clpP The primer pair is characterized in that The DNA barcode clpP Depend on clpP Gene fragment composition, used to obtain the DNA barcode clpP The nucleotide sequences of the primer pair are shown as SEQ ID NO 1 and SEQ ID NO 2.

2. The DNA barcode according to claim 1 clpP Application of primer pairs in identifying mosses.

3. The DNA barcode according to claim 1 clpP Application of primer pairs in constructing phylogenetic trees of mosses.

4. A method for identifying mosses, characterized in that: The following steps are involved: S1. Extract genomic DNA from the moss to be identified; S2. Using the genomic DNA of the moss to be identified as a template, performing PCR amplification with the primer pair according to claim 1 to obtain a PCR amplification product; S3, sequencing the PCR amplification product obtained in S2; S4. Construct a phylogenetic tree and identify mosses.

5. The method for identifying mosses according to claim 4, wherein In S2, the reaction procedure of PCR amplification is: pre-denaturation at 80°C for 5 min, denaturation at 95°C for 1 min, annealing at 48°C for 1 min, extension at 65°C for 4 min, for a total of 35 cycles, and final extension at 65°C for 5 min.

6. The method for identifying mosses according to claim 4, wherein: In S4, the methods for constructing the phylogenetic tree are maximum likelihood method, Bayesian analysis method and neighbor-joining method.

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