DNA bar code ycf2 primer suitable for moss plants and application of DNA bar code ycf2 primer

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a DNA (deoxyribonucleic acid) bar code ycf2 primer applicable to moss plants and application of the DNA bar code ycf2 primer applicable to the moss plants, and the nucleotide sequence of the primer is' ycf2-D-F, 5 '-GGATCAGATTCCACATTTGAGT-3' 'and' ycf2-D-R, 5 '-TAGACCTTTCGTAGAGAACG-3' '. The DNA bar code provided by the invention can be applied to related researches such as species identification and molecular system development of moss plants in the field of plant classification; in the fields of ecology and biodiversity research, the method can be used for protecting moss plants; in the field of gardening research, the method can be used for identifying, introducing and cultivating moss plant varieties (species); in addition, the method can also be used for application work related to moss plant identification and the like in forestry, traditional Chinese medicine research, agriculture and the like.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular relates to a DNA barcode ycf2 primer suitable for mosses and its application. 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 ycf2 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 ycf2 primer suitable for mosses. The nucleotide sequences of the primers are "ycf2-DF, 5'-GGATCAGATTCCACATTTTGAGT-3'" and "ycf2-DR, 5'-TAGACCTTTCTCGTAGAGAACG-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 of the primer comprises the following steps:

[0009] S1. Extract DNA from the moss to be tested;

[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, 30; and final extension, 65°C, 5 min.

[0014] The beneficial effects of the present invention are:

[0015] The ycf2 gene fragment provided by this invention has not been previously reported as a DNA barcode. 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 "ycf2-DF, 5'-GGATCAGATTCCACATTTTGAGT-3'" and "ycf2-DR, 5'-TAGACCTTTCTCGTAGAGAACG-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 This is the polymorphic site variation map of the ycf2 sequence;

[0018] Figure 2 is the genetic distance of ycf2;

[0019] Figure 3 The topology of the phylogenetic tree constructed based on the ycf2 fragment; DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0021] The present invention provides a DNA barcode ycf2 primer suitable for mosses. The nucleotide sequences of the primers are shown in SEQ ID NO 1 and SEQ ID NO 2, specifically: "ycf2-DF, 5'-GGATCAGATTCCACATTTTGAGT-3'" and "ycf2-DR, 5'-TAGACCTTTCTCGTAGAGAACG-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, their sequences are single-copy and contain a certain number of variable sites, this application, based on complete chloroplast genome data from mosses, aims to screen for a universal, high-resolution DNA barcode for mosses. To design primers more suitable for moss amplification, we used 56 moss chloroplast genomes (covering 26 orders, 33 families, 40 genera, and 56 species) from the GenBank and Dryad databases, as well as self-generated data (see Table 1 below). Universal primers were then designed at both ends of the newly screened DNA barcode hypervariable regions.

[0025] Table 1 Chloroplast genome

[0026]

[0027]

[0028] After screening, ycf2 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 , where 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 ycf2 (Table 2).

[0029] Table 2 List of newly designed primers

[0030]

[0031] 2. Verification of the universality of new primers:

[0032] This application uses 38 moss specimens (see the universal verification material table), involving 14 orders, 22 families, and 31 species of specimens 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] 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, 30; and final extension, 65°C, 5 min.

[0038] Amplification results showed that the success rates of PCR amplification and sequencing using ycf2 reached 97.30%, demonstrating the versatility of the new primers "ycf2-DF" and "ycf2-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 materials should be selected from closely related species. This study, based on a molecular phylogenetic tree of the order Bryales constructed based on chloroplast genes, selected several closely related species of the genus Pohlia Hedw., located at the bottom of the phylogenetic tree, as well as species from other branches. This experiment involved 20 samples from 2 families, 8 genera, 14 species, and 8 species within the order Bryales (see the Resolution Verification Materials table). Philonotis fontana of the order Bryales was selected 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) Three methods, namely Maximum Likelihood (ML), Bayesian Inference (BI), and Neighbor Joining (NJ), were used to construct phylogenetic trees to verify the resolution of the new primers.

[0049] Table 4 Resolution verification materials

[0050]

[0051]

[0052] Primer resolution verification of ycf2:

[0053] 1) Amplification success rate: The new ycf2 primers "ycf2-DF" and "ycf2-DR" achieved a 100% amplification and sequencing success rate for 20 samples from 2 families, 8 genera, 14 species, and the order Bryales. The sequences obtained were used for subsequent analysis.

[0054] 2) Basic information of the amplified fragment is shown in Table 5 below

[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 ycf2 fragment, it can be seen that ( Figure 3A strict consensus tree was constructed based on the ycf2 fragment (with the Bayesian tree as the skeleton, and 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, respectively). Species of the same family and genus are clustered on one branch, and the branch support rate is high. Two samples of Pohlia timmioides clustered into one branch (ML=99, BI=1, NJ=99). The combined results of ML, BI and NJ trees showed that the branch relationship of this species clustered was stable and the support rate was close to 100%. Two samples of Pohlia hyaloperistoma clustered into one branch (ML=96, BI=0.94, NJ=66). The combined results of ML, BI and NJ trees showed that the branch relationship of this species clustered was stable and the support rate was high. Three samples of Pohlialutescens clustered into one branch (ML=91, BI=0.98, NJ=97). The combined results of ML, BI and NJ trees showed that the branch relationship of this species clustered was stable and the support rate was close to 100%. Cruda samples clustered into a single branch (ML=54, BI=0, NJ=80). This species did not form a single branch in the BI tree, and its support in the ML and NJ trees was not very high. This study suggests that the stability of the branch formed by this species is moderate. In summary, the resolution of the ycf2 fragment is good.

Claims

1. A DNA barcode ycf2 primer suitable for mosses, characterized by: The nucleotide sequences of the primers are "ycf2-DF, 5'-GGATCAGATTCCACATTTTGAGT-3'" and "ycf2-DR, 5'-TAGACCTTTCTCGTAGAGAACG-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 to be tested; 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, 30; and final extension, 65°C, 5 min.

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