DNA bar code atpB-rbcL primer suitable for moss plants and application of DNA bar code atpB-rbcL primer

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a DNA bar code atpB-rbcL primer suitable for moss plants and application of the DNA bar code atpB-rbcL primer. The nucleotide sequences of the primers are as follows: 'atpB-rbcL-D-F, 5 '-AGGTCCCTCCCTACAATTC-3' ', and'atpB-rbcL-D-R, 5'-AGTAACATATACGGG (ACC)-3 ''. When the novel atpB-rbcL primer is used for carrying out PCR amplification and sequencing on a test material, the success rate can reach 100%. It is proved that the novel primer has good universality in moss plants. The DNA bar code identification method based on the atpB-rbcL can be widely applied to the research fields of moss plant species identification, molecular system development, ecology, biodiversity and the like.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a DNA barcode atpB-rbcL primer suitable for mosses and an application thereof. Background Art

[0002] Mosses have important ecological, ornamental, and medicinal value, and have broad market prospects. Due to their wide variety, only experts can accurately identify them based on their morphological characteristics. Currently, DNA barcoding technology has been widely used in the identification of species (varieties) such as animals and plants. Most of the DNA barcodes commonly used in mosses come from research on angiosperms, such as atpB-rbcL. Although the identification rate of atpB-rbcL is high when applied to mosses, the amplification success rate is low. In order to ensure the amplification success rate of atpB-rbcL, this application attempts to redesign the primers used during amplification in order to improve the amplification success rate of atpB-rbcL. 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 atpB-rbcL primer suitable for mosses, which can improve the success rate of atpB-rbcL amplification.

[0004] The specific technical solution adopted in the present invention is:

[0005] A DNA barcode atpB-rbcL primer suitable for mosses, wherein the nucleotide sequences of the primers are "atpB-rbcL-DF, 5'-AGGTCCCTCCCTACAACTC-3'" and "atpB-rbcL-DR, 5'-AGTAACATCTAATACGGG(ACC)-3'".

[0006] The primers are used in identifying mosses.

[0007] The application method comprises the following steps:

[0008] S1. Extract DNA from the moss to be tested;

[0009] S2. Design a PCR amplification program and perform PCR amplification using primers;

[0010] S3. Sequencing the PCR product;

[0011] 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.

[0012] The PCR amplification program was as follows: pre-denaturation, 95°C, 5 min; denaturation, 95°C, 1 min; annealing, 52°C, 1 min; extension, 65°C, 4 min; number of cycles, 35; and final extension, 65°C, 5 min.

[0013] The beneficial effects of the present invention are:

[0014] This application discloses new primers for atpB-rbcL, "atpB-rbcL-DF" and "atpB-rbcL-DR". The success rate of PCR amplification and sequencing of test materials using the new atpB-rbcL primers can reach 100% (see Figure 1 The new primers demonstrate excellent versatility across mosses, enabling the atpB-rbcL-based DNA barcoding method to be widely applied in moss species identification, molecular phylogeny, ecology, and biodiversity research. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Agarose gel electrophoresis diagram for universal verification

[0016] Figure 2 is the polymorphic site variation map of the sequence; DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0018] 1. Design of new primers for DNA barcode atpB-rbcL:

[0019] In order to design primers more suitable for moss amplification, we used 56 moss chloroplast genomes (covering 26 orders, 33 families, 40 genera and 56 species of mosses) from the GenBank database, the Dryad database and our own production for this study (Table 1).

[0020] Table 1 Chloroplast genome

[0021]

[0022]

[0023] This application integrates the primer design principles and DNA barcode screening standards, and refers to the sequence polymorphism variation map calculated by DnaSP ( Figure 2 ), universal primers were designed on both sides of the appropriate hypervariable regions of atpB-rbcL (Table 2). Figure 2 The blue origin is the region where primers are designed for each gene. For primer design of non-coding genes, since the design region is in the conserved coding region, the map of non-coding genes includes part of the coding gene.

[0024] Table 2 Primer list

[0025]

[0026] 2. Verification of the universality of new primers:

[0027] This study used 38 moss specimens, representing 31 species from 14 orders, 22 families, and 31 species, to verify the universality of the new atpB-rbcL primers. The method is as follows:

[0028] 1) First, DNA was extracted from the experimental materials using the modified CTAB method.

[0029] 2) Design a PCR amplification program and use new primers for PCR amplification.

[0030] 3) The PCR products were sent to Beijing Sangon Biotechnology Co., Ltd. for sequencing.

[0031] 4) The sequence returned by sequencing is compared with the moss chloroplast genome containing annotation information to verify whether it is the target fragment.

[0032] The following PCR amplification protocol was used for amplification: initial denaturation at 95°C for 5 minutes; denaturation at 95°C for 1 minute; annealing at 52°C for 1 minute; extension at 65°C for 4 minutes; number of cycles: 35; final extension at 65°C for 5 minutes. The basic information of the atpB-rbcL amplified fragment is shown in Table 3.

[0033] Table 3 Basic information statistics of atpB-rbcL

[0034]

[0035] The amplification results showed that the success rate of PCR amplification and sequencing of the test materials (see Table 3) by the new primers "atpB-rbcL-DF" and "atpB-rbcL-DR" for atpB-rbcL was 100%, proving that the new primers "atpB-rbcL-DF" and "atpB-rbcL-DR" for atpB-rbcL have good versatility in mosses, as shown in Table 3. Figure 1 As shown, Figure 1 The correspondence between the electrophoresis sequence number and the DNA number in Table 3 is as follows:

[0036] 1.DJ11; 2.DJ12; 3.DJ15; 4.DJ16; 6.DJ18; 7.DJ20; 8.DJ29; 10.DJ30; 11.DJ31; 12.DJ32; 1 3.DJ34; 14.DJ38; 15.DJ41; 16.DJ43; 17.DJ44; 18.DJ48; 20.DJ49; 21.DJ50; 22.DJ53; 23. DJ54; 24.DJ56; 25.DJ58; 26.DJ59; 27.DJ60; 28.DJ61; 29.DJ62; 30.DJ67; 33.DJ68; 34.DJ 71; 37.DJ72; 38.DJ73; 39.DJ77; 40.DJ78; 42.DJ80; 43.DJ81; 45.DJ21; 46.DJ74; 47.DJ82.

[0037] Table 3 Universal verification materials

[0038]

[0039]

Claims

1. A DNA barcode atpB-rbcL primer suitable for mosses, characterized by: The nucleotide sequences of the primers are "atpB-rbcL-DF, 5'-AGGTCCCTCCCTACAACTC-3'" and "atpB-rbcL-DR, 5'-AGTAACATCTAATACGGG(ACC)-3'".

2. Use of the primer according to claim 1 in identifying mosses.

3. 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.

4. The application according to claim 3, characterized in that: The PCR amplification program was as follows: pre-denaturation, 95°C, 5 min; denaturation, 95°C, 1 min; annealing, 52°C, 1 min; extension, 65°C, 4 min; number of cycles, 35; and final extension, 65°C, 5 min.

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