A DNA barcode suitable for mosses

Abstract

This invention provides a DNA barcode suitable for bryophytes, wherein the gene of the DNA barcode is matK, and the primers for matK are matK-D-F and matK-D-R. The sequence of primer matK-D-F is (5'-3')TCGTCATCBGTTAAAGTAGTCC; the sequence of primer matK-D-R is (5'-3')CGTCRACGNATACAAGATGCT. When using the new matK primers for PCR amplification and sequencing of test materials, the success rate of both PCR amplification and sequencing reaches 100%, and different species can be distinguished. The new matK primers matK-D-F and matK-D-R have good versatility and high resolution for bryophytes.

Description

Technical Field

[0001] This invention belongs to the field of biological species identification technology, specifically relating to a DNA barcode applicable to bryophytes. matK Primer pairs. Background Technology

[0002] Bryophytes have significant ecological, ornamental, and medicinal value, and their market application prospects are broad. However, due to their tiny size, diverse species, and the reliance on specialized equipment such as microscopes for morphological research, accurate species identification based on morphological characteristics is typically only achievable by professional researchers.

[0003] Currently, DNA barcoding technology is widely used in the identification of species (varieties) of animals and plants. However, most DNA barcodes commonly used in bryophytes originate from angiosperm research and are not the most suitable barcodes for bryophytes, often suffering from poor universality or low resolution. This limits the effective application of DNA barcoding in bryophytes. matK While DNA barcodes, commonly used in angiosperms, show high identification rates when applied to bryophytes, their amplification success rate is low. To improve... matK To improve the success rate of barcode sequence amplification, this application redesigned the forward and reverse primers required for amplifying this DNA barcode. matK Its application effect in the identification of bryophytes. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides a DNA barcode for bryophytes that is versatile, high-resolution, and suitable for use in bryophytes. matK Primer pairs.

[0005] The technical solution adopted in this invention is: a DNA barcode suitable for bryophytes. matK The primer pair, the matK The primers used are matK-DF and matK-DR.

[0006] Furthermore, the primer matK-DF sequence is (5'-3') TCGTCATCBGTTAAAGTAGTCC; the primer matK-DR sequence is (5'-3') CGTCRACGNATACAAGATGCT.

[0007] Furthermore, the primers amplify bryophyte sequence bands of 550-750 bp.

[0008] Furthermore, the PCR amplification program for the primers matK-DF and matK-DR is as follows: denaturation, 95℃, 1 min; annealing, 50℃, 1 min; extension, 65℃, 1-4 min; cycle number, 30-45; final extension, 65℃, 5-10 min.

[0009] Furthermore, the Tm of the primer matK-DF is 53℃; the Tm of the primer matK-DR is 55℃.

[0010] Furthermore, the PCR amplification program for primers matK-DF and matK-DR also includes pre-denaturation at 80°C for 5 minutes.

[0011] The beneficial effects obtained by this invention are as follows: This invention uses matK When the new primers were used for PCR amplification and sequencing of the test materials, the success rates of both PCR amplification and sequencing reached 100%, and they were able to distinguish between bryophyte species. matK The new primers matK-DF and matK-DR have good versatility and high resolution for bryophytes.

[0012] In the field of plant classification, this invention can be used for the identification of bryophyte species and related research on molecular phylogeny; in the field of ecology and biodiversity research, it can be used for the protection of bryophytes; in the field of horticultural research, it can be used for the identification of bryophyte varieties (species) and their introduction and cultivation; in addition, it can also be used in forestry, traditional Chinese medicine research, agriculture and other applications related to the identification of bryophytes. Attached Figure Description

[0013] Figure 1 This is a sequence polymorphism variation diagram of the present invention;

[0014] Figure 2 For the present invention matK Genetic distance;

[0015] Figure 3 For based on matK The fragments are used to construct strictly consistent trees (with a Bayesian tree as the skeleton). The numerical values ​​represent the posterior probability of the ML tree, the expansion support rate of the BI tree, and the expansion support rate of the NJ tree, respectively. Detailed Implementation

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] A DNA barcode applicable to bryophytes matK The primer pair, the matK The primers used were matK-DF and matK-DR. The sequence of primer matK-DF was (5'-3') TCGTCATCBGTTAAAGTAGTCC; the sequence of primer matK-DR was (5'-3') CGTCRACGNATACAAGATGCT. The primer pair amplified bryophyte sequence bands ranging from 550 to 750 bp. The GC content of primer matK-DF was 41%; the GC content of primer matK-DR was 48%. The temperature range (Tm) of primer matK-DF was 53℃; the temperature range (Tm) of primer matK-DR was 55℃.

[0018] Experimental Data 1: DNA Barcoding Screening and Primer Design:

[0019] Because the gene content and structure of chloroplast genomes are highly conserved, and their sequences are single copies with certain variable sites, this study aims to screen for universal high-resolution DNA barcodes for bryophytes based on whole-genome data of chloroplasts. To design primers more suitable for bryophyte amplification, 56 chloroplast genome data from GenBank, Dryad, and self-produced bryophyte databases (involving 56 species from 40 genera and 33 families across 26 orders of bryophytes) were used in this study; universal primers were designed at both ends of the hypervariable regions of the newly screened DNA barcodes.

[0020] Experimental Data 2: Verification of the universality of the new primers:

[0021] This application utilizes specimen No. 38 of bryophytes, involving 31 species of bryophytes from 22 families and 14 orders, to... matK The universality of the new primers was verified. The method is as follows:

[0022] 1) First, DNA was extracted from the test materials using the modified CTAB method.

[0023] 2) Design a PCR amplification program and use new primers for PCR amplification.

[0024] 3) The PCR products were sent to Beijing Sangon Biotech Co., Ltd. for sequencing.

[0025] The sequence returned by sequencing is compared with the chloroplast genome of mosses containing annotation information to verify whether it is the target fragment.

[0026] Experimental data 3: Validating the resolution of the new primers:

[0027] DNA barcode resolution verification materials should, in principle, be closely related species. This application references an established molecular phylogenetic tree of the order Bryales constructed based on chloroplast genes, and selects the genus *Lysimachia* located at the base of this phylogenetic tree. Poland The experiment involved closely related species of Hedw. and species from other clades. The experiment included 20 samples from 14 species belonging to 8 genera and 2 families within the order Bryales, with *Hedw. hedw.* selected from the order Gloydioides. Philonotis fontana As an outgroup.

[0028] 1) DNA extraction, PCR amplification, and sequencing were performed on the test materials, using the same methods as the universality verification.

[0029] 2) Integrate by gene name, delete base sites that are not accurately sequenced before and after the original sequence in Sequencher 5.3, and synthesize contigs of gene fragments with the same name and export them.

[0030] 3) Use the MAFFT v7.130b program in Phylosuite 1.1.15 for automatic comparison, and use BioEdit 7.0.9.0 for manual adjustment.

[0031] 4) Statistically analyze the basic information and genetic distance of the successfully amplified target fragment.

[0032] 5) Three methods were used to construct a phylogenetic tree: Maximum Likelihood (ML), Bayesian Inference (BI), and Neighbor Joining (NJ). This was then used to evaluate the resolution of the new primers.

[0033] Analysis of experimental results:

[0034] 1. Primer design results

[0035] After screening, it was found that matK This region is a highly variable region and is recommended as a candidate barcode for bryophyte DNA. Based on primer design principles and DNA barcode screening criteria, and referring to the sequence polymorphism variation map calculated by DnaSP 5.10.01, as shown... Figure 1 ,right matK Universal primers were designed on both sides of the appropriate hypervariable region, as shown in Table 1.

[0036] Table 1 New Design matK Primer information

[0037]

[0038] 2. New primers matK Verification of universality:

[0039] The following PCR amplification program was used for amplification: pre-denaturation, 80℃, 5 min; denaturation, 95℃, 1 min; annealing, 50℃, 1 min; extension, 65℃, 4 min; cycle number, 35; final extension, 65℃, 5 min.

[0040] The results showed that using matK When the new primers were used for PCR amplification and sequencing of the test materials, both the PCR amplification success rate and the sequencing success rate reached 100%. This indicates that... matK The new primers matK-DF and matK-DR have good versatility for bryophytes.

[0041] 3. matK Primer resolution verification:

[0042] 1) Amplification success rate: matK The new primers matK-DF and matK-DR achieved a 100% amplification success rate for 20 samples from 14 species belonging to 8 genera and 2 families in the order Bryales.

[0043] 2) Basic information of the amplified fragment

[0044] Table 2 DNA Barcodes matK Resolution verification fragment information

[0045]

[0046] 3) Genetic distances between species as analyzed by MEGA show that intraspecific distances are smaller than interspecific distances in most species, meeting the screening criteria for DNA barcoding, such as... Figure 2 .

[0047] 4) Based on matK The topological structure of the phylogenetic tree constructed from the fragments can be seen (e.g.) Figure 3 Species of the same family and genus cluster on one branch, and the branching support is relatively high. Two narrow-leaved luffa species. Pohlia timmioides The samples aggregated into one molecule (ML=92, BI=1, NJ=99), with two moss spores. Pohlia hyaloperistoma The samples clustered into one molecule (ML=87, BI=0.96, NJ=68), consisting of 3 *Luffa sp.* species. Pohlia cruda The samples aggregated into one stream (ML=92, BI=1, NJ=89), containing 3 *Luffa beadica* species. Pohlia lutescens The samples clustered into one phylogenetic tree (ML=98, BI=1, NJ=99). Combining the results of ML, BI, and NJ tree analysis, the branching relationships among the four species were stable, with support rates mostly exceeding 80%. In conclusion, it is believed that... matK High segment resolution.

[0048] Conclusion: Previous matK While commonly used in bryophytes and offering high species resolution, primers have a low amplification success rate. The newly designed primers in this study significantly improve the amplification success rate in bryophytes.

[0049] Based on the results of primer universality verification and species resolution verification of the candidate DNA barcodes, matK As a candidate DNA barcode for bryophytes, the primers used for amplification were: matK-DF, (5'-3')TCGTCATCBGTTAAAGTAGTCC and matK-DR, (5'-3')CGTCRACGNATACAAGATGCT.

[0050] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A DNA barcode applicable to bryophytes matK The primer pair is characterized by: Used to obtain the matK The primers are matK-DF and matK-DR; The sequence of the primer matK-DF is (5'-3') TCGTCATCBGTTAAAGTAGTCC; the sequence of the primer matK-DR is (5'-3') CGTCRACGNATACAAGATGCT; The primers amplified bryophyte sequence bands ranging from 550 to 750 bp.

2. The DNA barcode for bryophytes according to claim 1 matK The primer pair is characterized by: The PCR amplification program for primers matK-DF and matK-DR was as follows: denaturation, 95℃, 1 min; annealing, 50℃, 1 min; extension, 65℃, 1-4 min; cycle number, 30-45; final extension, 65℃, 5-10 min.

3. The DNA barcode for bryophytes according to claim 1 matK The primer pair is characterized by: The Tm of the primer matK-DF is 53℃; the Tm of the primer matK-DR is 55℃.

4. A DNA barcode suitable for bryophytes according to claim 2 matK The primer pair is characterized by: The PCR amplification program for primers matK-DF and matK-DR also includes pre-denaturation at 80°C for 5 minutes.

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