Sambucus williamsii seedling identification method based on DNA bar code

Abstract

The invention relates to the field of biological identification, and particularly discloses a method for identifying sambucus williamsii seedlings based on DNA barcodes, the DNA barcodes comprise ITS2 barcodes, universal primer sequences are SF2: 5 '-ATGCGATACTTGGTGTGAAT-3' and S3R: 5 '-GACGCTTCTCAGACTACAAT-3', the ITS2 barcodes are used for specifically amplifying ITS2 fragments, and the universal primer sequences are used for specifically amplifying the ITS2 fragments. The identification method comprises the following steps: extracting DNA of a sample, carrying out PCR amplification by using the primer, sequencing the product, comparing the obtained sequence with a database, and carrying out phylogenetic analysis, so as to determine the species. The method has the advantages of rapidness, accuracy and good repeatability, solves the problem that the flower-free and fruit-free period of elderberry is difficult to identify, and can be widely applied to the fields of elderberry seedling authenticity detection, Chinese herbal medicine source tracing, variety right protection, germplasm resource management and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological identification, and in particular to a DNA barcode for identifying elder seedlings, an identification method and application. BACKGROUND

[0002] Elder plants (Sambucus) are widely distributed medicinal and ornamental plant resources worldwide, and different species (such as Sambucus nigra and Sambucus williamsii) have significant differences in medicinal ingredients, activity and adaptability. Therefore, accurate and rapid species identification is crucial in seedling breeding, traditional Chinese medicine raw material procurement and variety right protection.

[0003] Traditional morphological identification methods are heavily dependent on the complete characteristics of reproductive organs such as flowers and fruits, and are extremely difficult to identify seedlings, vegetative plants or damaged specimens, and are easily influenced by subjective experience, making it difficult to ensure accuracy. With the development of molecular biology, DNA barcoding technology provides a revolutionary solution for species identification. This technology uses a standard, easily amplified and sequenced short DNA fragment in the genome to identify species.

[0004] In plants, commonly used DNA barcodes include chloroplast gene fragments (such as matK, rbcL) and ribosomal internal transcribed spacer (ITS). Among them, the ITS2 fragment is proven to be an effective barcode for distinguishing closely related species due to its faster evolution rate, strong primer universality, easy amplification and sequencing, and sufficient variation sites within and between species.

[0005] However, there is currently a lack of specific optimized and standardized DNA barcoding identification system for elder plants. Existing universal primers may have low amplification efficiency and poor specificity in specific species of elder plants, which cannot meet the requirements of accuracy, stability and high efficiency for commercial seedling identification. Therefore, developing a DNA barcoding identification method based on specific primers and standardized processes for elder plants has important practical significance and application value for ensuring the quality of traditional Chinese medicine, regulating the seedling market and protecting the rights and interests of breeders. SUMMARY

[0006] The purpose of the present application is to solve the problems existing in the prior art and to provide a rapid identification method for identifying elder seedlings based on DNA barcoding technology and its application.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: A DNA barcode-based sambucus seedling identification method, comprising: an ITS2 barcode, wherein a sequence of a disclosed primer pair of the ITS2 barcode primer comprises: an upstream primer S2F: 5'-ATGCGATACTTGGTGTGAAT-3' (SEQ ID NO: 1); a downstream primer S3R: 5'-GACGCTTCTCCAGACTACAAT-3' (SEQ ID NO: 2).

[0008] As a further technical solution of the present application, the ITS2 barcode primer is used for amplifying an ITS2 gene fragment of a sambucus plant, and the length of the amplification product is about 500 bp.

[0009] As a further technical solution of the present application, the DNA barcode-based sambucus seedling identification method comprises the following steps: S1: sample collection and processing: transplanting the sambucus seedling sample material (cutting) to be detected into a greenhouse, randomly collecting tender leaves from different parts of the plant as the sample to be detected after the leaves grow, about 0.5 g, and storing in color-changing silica gel at room temperature for subsequent identification; S2: DNA extraction: taking about 0.05-0.1 g of plant leaves, grinding them into powder in liquid nitrogen, transferring them to a 1.5 ml centrifuge tube, and extracting total DNA using a plant genomic DNA extraction kit according to the specified steps, and storing at -20℃ for standby; S3: DNA barcode amplification: using the DNA extracted in step S2 as a template, and performing PCR amplification reaction using the ITS2 barcode primer pair; S4: DNA barcode sequence determination: cutting and recovering the target PCR band, and entrusting a professional agency (Shanghai Genechem Biotech Co., Ltd.) to determine the DNA sequence, using the PCR amplification product obtained in step S3 as a sequencing primer, and sequencing the upstream and downstream primers; S5: DNA barcode sequence analysis: after the sequencing results obtained in step S4 are quality controlled and corrected, the barcode sequences of each sample are obtained, and then submitted to the NCBI nucleic acid database for Blast retrieval, analysis of homology with other plants, and determination of the species classification of the sample to be detected according to the sequence similarity and phylogenetic relationship.

[0010] As a further technical solution of the present application, in S3, the program of the PCR amplification reaction is: 95℃ pre-denaturation for 5 minutes; then 30 cycles of amplification, i.e. 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, and 72℃ extension for 30 seconds; and finally 72℃ terminal extension for 10 minutes.

[0011] As a further technical solution of the present application, in the S3, 5 μL of the PCR amplified product is separated by 1% agarose gel electrophoresis, and the electrophoresis result is detected.

[0012] As a further technical solution of the present application, the electrophoresis parameters are: 150 V, 100 mA, and the electrophoresis is observed and photographed under ultraviolet transmission light after 15 min.

[0013] As a further technical solution of the present application, the S5 specifically comprises: converting the barcode sequence of each sample into FASTA format, calculating the genetic distance between different elderberry samples based on the Kimura 2-parameter model using MEGA software, constructing a phylogenetic tree by the maximum likelihood method, evaluating the branch support rate by Bootstrap test (1000 times of repetition), and determining the evolutionary status and species identity of the to-be-tested sample in the elderberry genus according to the genetic distance and clustering analysis result.

[0014] As a further technical solution of the present application, the elderberry species identification kit comprises an ITS2 barcode primer in the DNA barcode for identifying the elderberry seedling.

[0015] The present application has the following beneficial effects: 1. High specificity and accuracy: the provided disclosure primer pair (S2F / S3R) is designed for the ITS2 region of the elderberry genus, has a high success rate of amplification, and the product has strong specificity, which can effectively distinguish common related species such as Sambucus nigra and Sambucus williamsii, and the identification result is accurate and reliable.

[0016] 2. High efficiency and rapidness: the process is standardized, and the DNA extraction to the sequencing result can be completed within 24 hours, which greatly shortens the identification period compared with the flower period observation identification lasting for several months, and is particularly suitable for batch and rapid detection of commercial seedlings.

[0017] 3. Good repeatability and stability: little affected by environmental conditions and plant development stage, stable results can be obtained for different parts of materials such as leaves and tender branches, and the experiment has good repeatability.

[0018] 4. Wide application range: not only can be used for identification of complete plants, but also can be used for identification of cuttings, seedlings and even dried medicinal material samples without roots and flowers, and application scenarios cover seedling production, market supervision, traditional Chinese medicine GAP base construction, customs quarantine, academic research and other fields.

[0019] 5. Provide systematic evolution information: not limited to simple sequence alignment, but also can reveal the genetic relationship and evolutionary status between different samples by constructing a phylogenetic tree and analyzing genetic distance, and provide more abundant genetic information. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 DNA extraction detection chart for Example 1 of the present application; Figure 2 Sample PCR amplification electrophoresis detection chart after Example 1 of the present application; Figure 3 Sample sequencing peak chart (partial results) in Example 1 of the present application; Figure 4 Sambucus nigra ITS2 sequence sequencing result alignment chart (part) in Example 1 of the present application; Figure 5 Sambucus nigra bar code ITS2 phylogenetic tree schematic diagram in Example 1 of the present application; Figure 6 Flow chart of a Sambucus nigra seedling identification method based on DNA barcoding proposed by the present application. DETAILED DESCRIPTION

[0021] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the present application will be further described below in conjunction with specific embodiments.

[0022] A Sambucus nigra seedling identification method based on DNA barcoding, characterized in that it comprises: an ITS2 barcoding primer, the primer pair sequence of the ITS2 barcoding primer comprising: an upstream primer S2F: 5'-ATGCGATACTTGGTGTGAAT-3' (SEQ ID NO: 1); a downstream primer S3R: 5'-GACGCTTCTCCAGACTACAAT-3' (SEQ ID NO: 2), and the ITS2 barcoding primer is shown in Table 1.

[0023] Table 1 DNA barcoding primer In a preferred embodiment, the ITS2 barcoding primer is used to amplify the ITS2 gene fragment of Sambucus plants, and the length of the amplification product is about 500 bp.

[0024] Please refer to the attached Figure 6 A Sambucus nigra seedling identification method based on DNA barcoding, comprising the following steps: S1: sample collection and processing: transplant the Sambucus nigra seedling sample material (cutting) to be detected into a greenhouse, collect tender leaves from different parts of the plant as test samples after the leaves grow, about 0.5 g, and store them in color-changing silica gel at room temperature for subsequent identification. S2: DNA extraction: about 0.05-0.1 g of plant leaves were taken, ground into powder in liquid nitrogen, transferred to a 1.5 ml centrifuge tube, and total DNA was extracted according to the specified steps using a plant genomic DNA extraction kit, and stored at -20°C for standby; S3: DNA barcode amplification: using the DNA extracted in step S2 as a template, PCR amplification reaction was performed using ITS2 barcode primer pairs, and the reaction system is shown in Table 2; Table 2 PCR reaction system S4: DNA barcode sequence determination: the target PCR band was cut and recovered, and a professional agency (Shanghai Genechem Biotech Co., Ltd.) was commissioned to determine the DNA sequence. The PCR amplification product obtained in step S3 was used as a sequencing primer, and the upstream and downstream primers were sequenced. S5: DNA barcode sequence analysis: after quality control and correction of the sequencing results obtained in step S4, the barcode sequences of each sample were obtained, and then submitted to the NCBI nucleic acid database for Blast search, analysis of homology with other plants, and determination of the species classification of the test sample according to the sequence similarity and phylogenetic relationship.

[0025] In a preferred embodiment, in S3, the PCR amplification reaction program is: 95°C pre-denaturation for 5 minutes; then 30 cycles of amplification, i.e. 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, and 72°C extension for 30 seconds; finally 72°C terminal extension for 10 minutes.

[0026] In a preferred embodiment, in S3, 5 μL of the PCR amplification product is separated by 1% agarose gel electrophoresis, and the electrophoresis result is detected.

[0027] In a preferred embodiment, the electrophoresis parameters are: 150V, 100 mA, and observation and photography under ultraviolet transmission light after electrophoresis for 15 min.

[0028] In a preferred embodiment, S5 specifically includes: converting each sample barcode sequence into FASTA format, calculating the genetic distance between different Sambucus samples based on the Kimura 2-parameter model using MEGA software, constructing a phylogenetic tree using the maximum likelihood method (Maximum Likelihood), evaluating the branch support rate through Bootstrap test (repeated 1000 times), and determining the evolutionary status and species identity of the test sample in the Sambucus genus based on the genetic distance and clustering analysis results.

[0029] A DNA barcode-based elder seedling identification method, the elder species identification kit comprises an ITS2 barcode primer for identifying the DNA barcode of the elder seedling.

[0030] Example 1 The DNA barcode-based elder seedling identification method according to the present application is used for identification experiments of elder seedlings.

[0031] (1) DNA extraction and barcode amplification The DNA band has no obvious tailing, indicating that the extracted genomic DNA is relatively complete and can meet the subsequent sequencing requirements Figure 1 ). The total DNA of the three samples was used as a template, and two elder samples were used as positive controls for PCR reaction with ITS2 barcode primers. The agarose gel electrophoresis results of the amplification products are shown in Figure 2 . Bright bands appear in all samples, and the amplification products are more and the effect is better, indicating that the selected barcode primer is suitable for the sample to be tested. The PCR amplification results show that the target band (about 500 bp) is consistent with the expected length of the amplified fragment.

[0032] (2) DNA barcode analysis At present, the DNA barcode of the nuclear gene used for plant classification and identification mainly focuses on the internal transcribed spacer (ITS) of ribosome. Based on the previous research and literature, this experiment selects ITS2 barcode as the detection object. According to the primer annealing temperature screening results, the sample PCR product is gel recovered and sequenced, and there is no abnormal situation such as high baseline, peak wrapping and miscellaneous peaks in the sequencing map Figure 3 . After comparing the determination results with the Blast tool in NCBI, it is found that the gene sequences of the three samples to be detected are 98%, 97% and 97% similar to the ITS region (MT796540) of Sambucus nigra, and the sequence alignment results and clustering analysis results are shown in Figure 4 and Figure 5 , which can confirm that the detection sample is Sambucus nigra.

[0033] (3) Conclusion 1) Sample number: No. 1, No. 2, No. 3.

[0034] 2) The electropherogram of the sample plant DNA after amplification is clear and the result is good.

[0035] 3) The PCR result of the sample DNA shows that only one band appears and no cross contamination is found.

[0036] 4) The name of the sample plant species is Sambucus nigra (scientific name Sambucus nigra).

[0037] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects: high specificity and accuracy: the specific primer pair (S2F / S3R) provided is optimized for the Sambucus ITS2 region, has high amplification success rate, strong product specificity, and can effectively distinguish common related species such as S. nigra and S. williamsii, and the identification result is accurate and reliable.

[0038] High efficiency and rapidness: the process is standardized, and from DNA extraction to obtaining sequencing results can be completed within 24 hours, which greatly shortens the identification period compared with the observation and identification of flowering period for several months, and is particularly suitable for batch and rapid detection of commercial seedlings.

[0039] Good repeatability and stability: little affected by environmental conditions and plant development stages, stable results can be obtained for different parts of materials such as leaves and twigs, and the experiment has good repeatability.

[0040] Wide application range: not only can be used for identification of complete plants, but also can be used for identification of cuttings, seedlings and even dried medicinal material samples without roots and flowers, and application scenarios cover seedling production, market supervision, traditional Chinese medicine GAP base construction, customs quarantine, academic research and other fields.

[0041] Providing phylogenetic information: not limited to simple sequence alignment, but also can reveal the genetic relationship and evolutionary status between different samples by constructing phylogenetic tree and analyzing genetic distance, and provide more abundant genetic information.

[0042] Those skilled in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to suggest that the scope of the present application is limited to these examples; under the idea of the present application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other changes of different aspects of the present application as described above. In order to be brief, they are not provided in details.

[0043] The present application is intended to cover all such alternatives, modifications and variations falling within the broad scope of the specification. Therefore, any omission, modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for identifying elderberry seedlings based on DNA barcoding, characterized in that, Comprising: ITS2 barcode, the sequence of the universal primer pair of the ITS2 barcode comprises: Upstream primer S2F: 5'-ATGCGATACTTGGTGTGAAT-3'; Downstream primer S3R: 5'-GACGCTTCTCCAGACTACAAT-3'.

2. The DNA barcoding method for identifying elder seedlings according to claim 1, wherein, The ITS2 barcode primer is used for amplifying the ITS2 gene fragment of the plant of Sambucus, and the length of the amplification product is about 500bp.

3. The DNA barcoding method for identifying elder seedlings according to claim 1, characterized in that, Comprising the following steps: S1: sample collection and processing: transplant the sample material of the Sambucus seedling to be tested to the greenhouse, and after the leaves grow, randomly collect tender leaves from different parts of the plant as the sample to be tested, and store them in the color-changing silica gel at room temperature; S2: DNA extraction: take 0.05-0.1g of plant leaves, grind them into powder in liquid nitrogen, transfer them to a 1.5ml centrifuge tube, and use a plant genomic DNA extraction kit to extract total DNA, which is stored at-20℃ for standby; S3: DNA barcode amplification: using the DNA extracted in step S2 as a template, the ITS2 barcode primer pair is used for PCR amplification reaction; S4: DNA barcode sequence determination: cut and recover the target PCR band, and entrust a professional agency to determine the DNA sequence, using the PCR amplification product obtained in step S3 as the sequencing primer, and sequencing the upstream and downstream primers; S5: DNA barcode sequence analysis: after the sequencing results obtained in step S4 are quality controlled and corrected, the barcode sequences of each sample are obtained, and then submitted to the NCBI nucleic acid database for Blast search, analysis of homology with other plants, and determination of the species classification of the sample to be tested according to the sequence similarity and phylogenetic relationship.

4. The DNA barcoding method for identifying elder seedlings according to claim 3, characterized in that, In the S3, the program of PCR amplification reaction is: 95℃ pre-denaturation for 5 minutes; then 30 cycles of amplification, i.e. 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, and 72℃ extension for 30 seconds; finally 72℃ terminal extension for 10 minutes.

5. The DNA barcoding method for identifying elder seedlings according to claim 4, wherein, In the S3, 5μL of the product after PCR amplification is separated by 1% agarose gel electrophoresis, and the electrophoresis result is detected.

6. The DNA barcoding method for identifying elder seedlings according to claim 5, wherein, The electrophoresis parameters are: 150V, 100mA, electrophoresis for 15min, observation under ultraviolet transmission light and photography.

7. The DNA barcoding method for identifying elder seedlings according to claim 3, wherein, The S5 specifically comprises: converting the barcode sequences of each sample into FASTA format, using MEGA software, calculating the genetic distance between different Sambucus samples based on Kimura 2-parameter model, and constructing a phylogenetic tree by maximum likelihood method, evaluating the branch support rate by Bootstrap test, and determining the evolutionary status and species identity of the sample to be tested in Sambucus based on the genetic distance and clustering analysis results.

8. The DNA barcoding method for identifying elder seedlings according to claim 3, characterized in that, The Sambucus species identification kit comprises the ITS2 barcode primer in the DNA barcode for identifying the Sambucus seedling according to any one of claims 1-2.

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