Bar code and identification method for identifying phoebe wood

Abstract

The invention belongs to the technical field of phoebe wood identification, and relates to a bar code for identifying phoebe wood and an identification method, the bar code comprises a rpl32-trnLUAG bar code, a ycf1 bar code and a rpl32-trnLUAG + ycf1 combined bar code, the sequence of the rpl32-trnLUAG + ycf1 combined bar code is shown as SEQ ID NO.5-9, the rpl32-trnLUAG + ycf1 combined bar code is used for identifying phoebe zhennan, phoebe tenuifolia, phoebe bournei, phoebe sheareri and phoebe chekiangensis in phoebe, and the primer sequence of the rpl32-trnLUAG + ycf1 combined bar code is shown as SEQ ID The method for identifying the phoebe wood adopts the rpl32-trnLUAG and ycf1 bar codes, and identifies phoebe zhennan, phoebe bournei, phoebe sheareri and phoebe zhejiangensis in the phoebe on the basis of SeqMan software and a phylogenetic evolutionary tree method. The bar code for identifying the phoebe wood, provided by the invention, has richer sequence information sites, so that the species identification capability is improved, and accurate identification of five types of phoebe wood with extremely close structural characteristics is realized.

Description

Technical Field

[0001] This invention belongs to the field of molecular biological detection for wood identification, specifically involving barcodes and identification methods for identifying Phoebe zhennan wood. Background Technology

[0002] According to the national standard "Names of Major Timbers in China" (GB / T18513-2001), Nanmu refers to timber from the genus Phoebe. Renowned worldwide for its precious material, fine texture, uniform color, strong luster, and good dimensional stability, it has been widely used in ancient architecture such as aristocratic palaces, gardens, and temples. In modern times, Nanmu has been used in various product forms, including beads, handicrafts, stationery, and furniture. With people's aspirations for a better life, the demand for Nanmu resources continues to increase, leading to illegal and over-exploitation of Nanmu timber.

[0003] Phoebe zhennan is a typical mature forest tree species, highly valued throughout history, and was even the "imperial wood" used by emperors during the Ming and Qing dynasties. Historically, human logging was severe, with trees used for the construction of royal palaces, gardens, altars, and mausoleums, leading many species to become endangered or vulnerable (IUCN Red List). Currently, Phoebe zhennan, Phoebe bournei, and Phoebe chekiangensis are also listed as Class II protected species in my country's "National Key Protected Wild Plants List." Because the wood structure characteristics of the Phoebe genus and its close relative, the Machilus sp., are extremely similar, the sale of high-value Phoebe zhennan wood products as inferior or counterfeit products is frequent in the timber trade. To more effectively protect my country's precious forest resources, accurate identification of Phoebe zhennan species is crucial. This not only provides technical support for the protection of important Phoebe zhennan forest resources and the effective implementation of related laws and regulations, but also provides important scientific basis for the supervision of the wood products and furniture industry chain.

[0004] General identification methods based on wood anatomy are generally insufficient for distinguishing between the genera *Phoebe* and *Machilus*, let alone accurately identifying them at the species level. However, DNA molecular marker methods provide an effective means for the accurate identification of *Phoebe*.

[0005] DNA barcodes are standardized, sufficiently variable, easily amplifiable, and relatively short DNA fragments. In recent years, DNA barcodes have become an important tool for species identification. However, single, internationally recognized DNA barcodes, such as rbcL and matK, have limited ability to identify species, especially those with complex evolutionary relationships. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a barcode and identification method for identifying Phoebe zhennan wood.

[0007] To achieve the objectives of this invention, the following technical solutions will be adopted.

[0008] A type of rpl32-trnL UAG Barcode, the rpl32-trnL UAG The DNA barcode sequences, as shown in PV940116, PV940101, PV940087, PV940108, and PV940097, are used to identify Phoebe zhennan, Phoebe bournei, Phoebe pekinensis, Phoebe zhennan var. chinensis, and Phoebe zhennan var. zeylanica within the genus Phoebe; the rpl32-trnL UAG The primer sequences for the barcode are shown in SEQ ID Nos. 1-2. Among them, PV940116, PV940101, PV940087, PV940108, and PV940097 can be viewed at the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / #! / landingpage).

[0009] A type of rpl32-trnL UAG The method for identifying Phoebe zhennan wood using barcodes includes the following steps:

[0010] S201. Select the heartwood or sapwood of the wood sample, grind it and filter it through a sieve to obtain a wood powder sample, and store it in a -80℃ low temperature freezer for later use.

[0011] S202. Extract DNA from the wood flour sample;

[0012] S203. Using the DNA sample as a template, amplify the rpl32-trnL sequence of the plasmid genome using PCR. UAG Barcode sequence to obtain amplification products;

[0013] S204. Sequencing the amplified product yields rpl32-trnL UAG Sequence, to obtain rpl32-trnL UAG Barcode;

[0014] S205, using the rpl32-trnL UAG Barcodes were used to identify Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan, and Phoebe zhennan in the genus Phoebe based on SeqMan software and phylogenetic tree method;

[0015] in:

[0016] The rpl32-trnL UAG The primer sequences for the barcode are shown in SEQ ID No. 1-2;

[0017] The PCR amplification reaction process is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, cycled 35 times; 72℃ final extension for 5 min to obtain the amplified DNA target fragment.

[0018] The PCR amplification reaction system includes: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA.

[0019] A YCF1 barcode, with sequences as shown in PV940150, PV940135, PV940121, PV940142, and PV940131, is used to identify Phoebe zhennan, Phoebe bournei, Phoebe zhennan, Phoebe zhennan var. chinensis, and Phoebe zhennan var. zealosa. The primer sequences for the YCF1 barcode are shown in SEQ ID Nos. 3-4. PV940150, PV940135, PV940121, PV940142, and PV940131 can be viewed on NCBI.

[0020] A method for identifying Phoebe zhennan wood using the YCF1 barcode includes the following steps:

[0021] S401. Select the heartwood or sapwood of the wood sample (in the case of no heartwood), grind and filter through a sieve to obtain a wood powder sample, and store it in a -80℃ low temperature freezer for later use.

[0022] S402. Extract DNA from the wood flour sample;

[0023] S403. Using the DNA sample as a template, amplify the ycf1 barcode sequence in the plasmid genome sequence using PCR to obtain the amplification product;

[0024] S404. Sequencing the amplified product to obtain the ycf1 sequence, and then generating the ycf1 barcode.

[0025] S405. Using the ycf1 barcode, and based on SeqMan software and phylogenetic tree method, identify Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan and Phoebe zhinan in the Phoebe genus;

[0026] in:

[0027] The primer sequences for the ycf1 barcode are shown in SEQ ID No. 3-4;

[0028] The PCR amplification reaction process is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, cycled 35 times; 72℃ final extension for 5 min to obtain the amplified DNA target fragment.

[0029] The PCR amplification reaction system includes: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA.

[0030] A type of rpl32-trnL UAG +ycf1 combined barcode, the rpl32-trnL UAG The +ycf1 combined barcode sequence is shown in SEQ ID No. 5-9 and is used to identify Phoebe zhennan, Phoebe bournei, Phoebe zhennan, Phoebe zhennan var. chinensis, and Phoebe zhennan var. zebrina within the Phoebe genus; the rpl32-trnL UAG The primer sequences for the +ycf1 combined barcode are shown in SEQ ID No. 1 to 4.

[0031] A type of rpl32-trnL UAG The method for identifying Phoebe zhennan wood using the +ycf1 combination barcode includes the following steps:

[0032] S601. Select the heartwood or sapwood of the wood sample (in the case of no heartwood), grind and filter through a sieve to obtain a wood powder sample, and store it in a -80℃ low temperature freezer for later use.

[0033] S602. Extract DNA from the wood flour sample;

[0034] S603. Using the DNA sample as a template, amplify the rpl32-trnL sequence of the plasmid genome using PCR. UAG The ycf1 barcode sequence was used to obtain the amplification product;

[0035] S604. Sequencing the amplified product yields rpl32-trnL UAG And ycf1 sequence, to obtain rpl32-trnL UAG and ycf1 barcode;

[0036] S605, using the rpl32-trnL UAG The barcode ycf1 was used to identify Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan, and Phoebe zhennan in the genus Phoebe based on SeqMan software and phylogenetic tree method;

[0037] in:

[0038] The rpl32-trnL UAGThe primer sequences for the +ycf1 combined barcode are shown in SEQ ID Nos. 1 to 4;

[0039] The PCR amplification reaction process is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, cycled 35 times; 72℃ final extension for 5 min to obtain the amplified DNA target fragment.

[0040] The PCR amplification reaction system includes: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA.

[0041] As a preferred embodiment of the present invention, the amount of the Phoebe zhennan wood to be identified is 300 grams.

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] The barcode for identifying Phoebe zhennan wood provided by this invention has richer sequence information sites, thereby improving species identification ability and enabling accurate identification of five Phoebe zhennan species with very similar structural features. This breaks through the limitation of traditional wood identification technology that cannot identify wood to the "species" level. Furthermore, it constructs an optimal method for high-resolution barcode identification suitable for identifying wood of closely related species. This method has high identification accuracy, requires less sampling, reduces wood consumption by 40% compared to existing technologies, and is not affected by factors such as sampling location and original sample state. Attached Figure Description

[0044] Figure 1 This is a flowchart of the method described in this invention;

[0045] Figure 2 For combined barcode rpl32-trnL UAG Phylogenetic trees of twenty species of *Machilus* were constructed using the Neighbor-Joining (NJ) method, as shown in the figure. The 55 sequences of the 20 *Machilus* species were downloaded from NCBI (see Table 2). Fragment sequences of the target barcodes were obtained through analysis using the Geneious software. The phylogenetic trees were then constructed using the "bionj" function in the "ape" R package, and cluster analysis was performed. The results showed that rpl32-trnL UAGThe +ycf1 barcode can effectively distinguish not only 10 species of Phoebe genus, including Phoebe zhennan, Phoebe hui, Phoebe bournei, Phoebe sheareri, and Phoebe chekiangensis, but also 10 species of Phoebe genus, including Phoebe yaiensis, Phoebe neurantha, Phoebe lanceolata, Phoebe calcarea, Phoebe hungmoensis, Phoebe macrocarpa, Phoebe glaucophylla, Phoebe minutiflora, Phoebe puwenensis, and Phoebe neuranthoides.

[0046] Figure 3 For combined barcode rpl32-trnL UAG Phylogenetic trees of five species of *Machilus* were constructed using the Neighbor-Joining (NJ) clustering method. Cluster analysis was performed on the amplified barcodes of the five *Machilus* species to verify the accuracy, specificity, and effectiveness of the amplified sequences. In the figure, each "leaf" represents a species of *Machilus*, and each "vein" represents a sample. Specifically: PZ (*Phoebe zhennan*) represents *Machilus* var. *hui* (blue), PB (*Phoebe bournei*, pink), PS (*Phoebe sheareri*, orange), and PC (*Phoebe chekiangensis*, green. The outgroup is *Machilus yunnanensis* (NCBI download, Accession number: NC_028073). Each species clusters independently into a single "leaf," and the same color indicates the same "leaf." The results show that rpl32-trnL UAG The +ycf1 combination barcode can effectively distinguish between Phoebe zhennan, P. hui, P. bournei, P. sheareri, and P. chekiangensis.

[0047] Figure 4 Specimens of unknown genus *Machilus* based on rpl32-trnL UAGThe NJ tree constructed with ycf1; each color in the figure represents a species of the genus Phoebe. Phoebe sp. (specimen) was subjected to cluster analysis by constructing an NJ phylogenetic tree with Phoebe zhennan, P. hui, P. bournei, P. sheareri, P. chekiangensis and the outgroup Machilus yunnanensis downloaded from the NCBI database. The results showed that the specimen and P. sheareri clustered together, that is, the sample is P. sheareri.

[0048] Figure 5 The NJ phylogenetic tree is constructed based on the barcode ycf1. Fifty-five sequences from 20 species of *Phoebe* in the figure were downloaded from NCBI (see Table 2). Fragment sequences of the target barcodes were obtained using the Geneious software, and the phylogenetic tree was constructed using the "bionj" function in the "ape" R package for cluster analysis. The results show that ycf1 can effectively distinguish not only *Phoebe chekiangensis*, *Phoebe hui*, and *Phoebe sheareri*, but also nine other *Phoebe* species: *Phoebe yaiensis*, *Phoebe neurantha*, *Phoebe lanceolata*, *Phoebe calcarea*, *Phoebe hungmoensis*, *Phoebe macrocarpa*, *Phoebe glaucophylla*, *Phoebe minutiflora*, and *Phoebe puwenensis*.

[0049] Figure 6 To construct the NJ phylogenetic tree based on barcode ycf1, cluster analysis was performed on the amplified barcodes of five species of *Machilus* wood collected from the NCBI database and their corresponding tree species to verify the accuracy, specificity, and effectiveness of the amplified sequence of barcode ycf1. PZ: *Phoebe zhennan*, PH: *P. hui* (small-leaved *Machilus*), PB: *P. bournei*, PS: *P. sheareri* (purple), and PC: *P. chekiangensis* (Zhejiang *Machilus*) were marked in blue. The outgroup for verification was *Machilus yunnanensis* (downloaded from NCBI, Accession number: NC_028073). The data in the figure show that the three colors effectively distinguish *P. chekiangensis*, *P. hui*, and *P. sheareri*.

[0050] Figure 7 For barcode-based rpl32-trnL UAGThe constructed NJ phylogenetic tree, consisting of 55 sequences from 20 species of the genus *Machilus*, was downloaded from NCBI (see Table 2). Fragment sequences of the target barcodes were obtained through analysis using the *geneious* software. The phylogenetic tree was then constructed using the *bionj* function in the *ape* R package, followed by cluster analysis. The results showed that rpl32-trnL UAG It can effectively distinguish between Phoebe chekiangensis, Phoebe bournei, and Phoebe sheareri, as well as seven species of the genus Phoebe: Phoebe calcarea, Phoebe yaiensis, Phoebe hungmoensis, Phoebe macrocarpa, Phoebe glaucophylla, Phoebe minutiflora, and Phoebe puwenensis.

[0051] Figure 8 For barcode-based rpl32-trnL UAG The constructed NJ phylogenetic tree was used to perform cluster analysis on the barcodes amplified from five species of *Machilus* wood collected and their corresponding species in the NCBI database, verifying the barcode rpl32-trnL. UAG The accuracy, specificity, and effectiveness of the amplified sequences were assessed. PZ: *Phoebe zhennan*, PH: *P. hui*, and PB: *P. bournei* were marked in green; PS: *P. sheareri* was marked in purple; and PC: *P. chekiangensis* was marked in pink. Five species of *Machilus* were validated, with the outgroup being *Machilus yunnanensis* (downloaded from NCBI, Accession number: NC_028073). The data in the figure show that the three colors effectively distinguish *P. chekiangensis*, *P. bournei*, and *P. sheareri*.

[0052] Figure 9 Primer rpl32-trnL was used for specimens of unknown *Machilus* species. UAGThe amplified sequences were aligned to the NCBI website using BLAST. The results show that the species with the highest sequence consistency is *Phoebe zhennan*, meaning the unknown *Phoebe* wood specimen is *Phoebe zhennan*. The parameters are as follows: Description: Sequence description: Detailed information about the sequence containing the high-scoring matching fragment; Scientific Name: Species name; Max Score: The higher the score, the higher the similarity between the two sequences; Total Score: The cumulative score when multiple fragments of a sequence match the query sequence; Query Cover: The coverage rate between the aligned sequence and the target sequence; higher coverage indicates higher similarity; E value: E-value or expected value: The closer the E-value is to zero, the greater the probability that the input sequence and the current sequence are the same; Per.ident: Consistency: Percentage of sequence alignment consistency; Acc.Len: Sequence length; Accession: Sequence accession number.

[0053] Figure 10 The image shows the results of Blast alignment of the amplified sequence of the unknown genus *Machilus* wood specimen using primer ycf1 on the NCBI website; the results indicate that the species with the highest sequence alignment consistency is *P. sheeareri*, meaning the unknown genus *Machilus* wood specimen is *P. sheeareri*.

[0054] Figure 11 Primer rpl32-trnL UAG Gel image after PCR amplification; where P: positive control; N: negative control; B: blank control; Mark: nucleic acid molecular marker DL3000, the brightest bands from bottom to top are 500bp and 1000bp, respectively, and those above 1000bp are 1200bp, 1500bp, 2000bp and 3000bp, respectively, primer rpl32-trnL UAG The amplified sequence is approximately 1200 bp in length;

[0055] Figure 12 This is a gel image of primer ycf1 after PCR amplification; where P: positive control; N: negative control; B: blank control; Mark: nucleic acid molecular marker DL3000. The brightest bands from bottom to top are 500bp and 1000bp, respectively, and those above 1000bp are 1200bp, 1500bp, 2000bp and 3000bp, respectively. The amplified sequence of primer ycf1 is about 1000bp long. Detailed Implementation

[0056] To facilitate understanding of the present invention, embodiments are provided below. Those skilled in the art should understand that these embodiments are merely illustrative and should not be considered as specific limitations of the invention. Unless otherwise specified, all raw materials used in this invention are commercially available or prepared according to conventional methods in the art. Unless otherwise defined or stated, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0057] Example 1 of the present invention: Design of primers specific to 20 species of the genus *Machilus*:

[0058] (1) Download 55 chloroplast genes from 20 species of the genus *Machilus* from GenBank, as shown in Table 1;

[0059] (2) Geneious software was used to compare sequences and identify and determine the differential sites among the five species of the genus Phoebe, especially Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan and Phoebe zhennan.

[0060] (3) Specific fragments were screened for 20 species of the genus *Machilus* using NCBI software, and two pairs of primers were set using Primer Premier5, which were then verified by Primer-BLAST. The specific primer sequences are shown in Table 2.

[0061] Table 1 shows the genome information of plastid DNA in the genus *Machilus*.

[0062]

[0063]

[0064]

[0065] Table 2 shows the primer sequences:

[0066]

[0067] Example 2 of the present invention: the identification ability of barcodes for 20 species of Phoebe genus specific primer combinations (1) Download Phoebe genus chloroplast genes from GenBank, see Table 1;

[0068] (2) Geneious software was used to compare sequences and identify and determine the differential sites among the five species of the genus Phoebe, especially Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan and Phoebe zhennan.

[0069] Table 3 shows rpl32-trnL UAGMultiple sequence alignment differential sites of +ycf1:

[0070]

[0071] (3) Specific fragments were screened for 20 species of the genus *Machilus* using NCBI software, and two pairs of primers were set using Primer Premier5, which were then verified by Primer-BLAST. The specific primer sequences are shown in Table 2.

[0072] (4) Based on the combined barcode rpl32-trnL UAG The NJ phylogenetic tree constructed by +ycf1 was used for cluster analysis of 55 sequences from 20 species of the genus *Phoebe* downloaded from GenBank. The results showed that this assemblage could successfully distinguish not only five species of *Phoebe* (*Phoebe zhennan*), *Phoebe hui*, *Phoebe bournei*, *Phoebe sheareri*, and *Phoebe chekiangensis*, but also 10 other species of the genus *Phoebe*, such as... Figure 2 As shown.

[0073] As an embodiment 3 of the present invention: an rpl32-trnL UAG The method of identifying Phoebe zhennan wood using the +ycf1 DNA barcoding combination includes the following steps:

[0074] (1) Standard sample collection: Five standard samples of the genus Phoebe were collected, namely Phoebe nanmu, Phoebe bournei, Phoebe minnanensis, Phoebe zebrina, and Phoebe zhennan, totaling 33 samples. They were collected from Sichuan, Hunan and Zhejiang respectively. Specific collection information is shown in Table 4.

[0075] (2) Sample preparation: The heartwood or sapwood (in the case of no heartwood) was selected as the sample. The outer surface of the wood sample was removed by a scalpel blade disinfected with 70% alcohol to avoid external contamination. The wood sample was cut into several wood chips and ground with a ball mill for 2 minutes at a frequency of 30 cps. After grinding, the wood powder was passed through an 80-mesh sieve and dispensed into several 1.5 mL centrifuge tubes, with 300 mg of wood powder in each tube. The tubes were then stored in a -80℃ freezer for later use.

[0076] (3) DNA extraction: DNA was extracted from five species of Phoebe wood in a sterilized clean working environment using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).

[0077] (4) PCR amplification and sequencing: The primer pairs were the four DNA barcode sequence primers from Example 1. PCR amplification was performed using DNA extract as a template. The reaction system was 20 μL: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA. All PCR reactions were performed on a PCR amplification instrument.

[0078] The reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, repeated 35 times; final extension at 72℃ for 5 min. This yielded efficiently amplified DNA fragments. The amplified products were then purified and subjected to bidirectional direct sequencing.

[0079] Sequencing quality was assessed using SeqMan software. Low-quality portions at both ends were removed, and the remaining portions were then evaluated. Only those meeting quality requirements were used for sequence assembly and proofreading. rpl32-trnL of five species of Phoebe wood were analyzed. UAG The barcode sequences ycf12 and ycf12 are shown in Table 2.

[0080] (5) Specific primer selection and determination: Sequences were assembled using SeqMan software, and sequence identity analysis was performed by the National Center for Biotechnology Information (NCBI). The results showed that rpl32-trnL UAG The two primer pairs, ycf1 and ycf1, can identify five species of Phoebe wood: Phoebe bournei, Phoebe simonii, Phoebe pekinensis, Phoebe zhennan, and Phoebe zhennan. Figure 3 As shown.

[0081] Table 4 shows the sample source information:

[0082]

[0083] As an example 4 of the present invention: DNA identification of an unknown Phoebe sp. wood specimen (1) A wood sample was taken from a forestry university. Through anatomical analysis, it was identified as Phoebe sp. wood, but the specific tree species could not be determined. Therefore, based on the rpl32-trnL described in Example 3 UAG The method of using +ycf1 combined DNA barcoding to identify Phoebe zhennan wood includes the following steps:

[0084] (2) Prepare wood flour and grind it to 80 mesh.

[0085] (3) Wood DNA extraction:

[0086] In a sterilized, ultra-clean working environment, DNA was extracted from the wood using the DNA extraction method described in Example 2. The DNA then underwent purification to a final concentration of 1–100 ng / μL.

[0087] (4) PCR amplification reaction and sequencing.

[0088] Wood DNA extract was used as a template for PCR amplification. Primers were listed as NO:1–NO:4. The reaction volume was 20 μL: 7.2 μL ddH2O, 10 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA. All PCR reactions were performed on a PCR instrument. The reaction program was: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, for 35 cycles; final extension at 72℃ for 5 min. This yielded efficiently amplified DNA fragments. The amplified products were purified and then subjected to bidirectional direct sequencing.

[0089] (5) Sequence alignment analysis and tree species identification.

[0090] Two pairs of specific primers, rpl32-trnL, were used for the unknown sample. UAG PCR amplification and sequencing were performed on ycf1. The PCR amplification products were bidirectionally sequenced, and an NJ tree was constructed for cluster analysis. The analysis results are as follows: Figure 4 As shown, an unknown Phoebe sp. wood specimen and P. sheareri clustered together, with the specimen being P. sheareri.

[0091] As an embodiment 5 of the present invention: rpl32-trnL UAG Amplification rates of DNA barcodes and ycf1 DNA barcodes

[0092] PCR amplification was performed on 33 wood samples, as shown in Table 5. Successful sequencing was indicated by "Yes" if the sequencing results matched the sample information, and failed sequencing was indicated by "NO" if no amplification was achieved. The results show that the sequencing was based on rpl32-trnL... UAG The amplification rate of the sample was 87.88%, and the amplification rate of the sample based on ycf1 was 93.94%. The amplification effect was good.

[0093] Table 5. Based on DNA barcode rpl32-trnL UAG Sample amplification rate of ycf1

[0094]

[0095]

[0096]

[0097] Y:ycf1,R:rpl32-trnL UAG

[0098] As an embodiment 6 of the present invention: based on rpl32-trnL UAG Barcode identification capabilities for 20 species of the genus *Machilus*:

[0099] (1) Download the chloroplast genes of the genus *Machilus* from GenBank, as shown in Table 1;

[0100] (2) Geneious software was used to compare sequences and find and determine the differential sites in the sequences of different species in the genus Phoebe, as shown in Table 6;

[0101] Table 6 shows rpl32-trnL UAG Multiple sequence alignment differential sites:

[0102]

[0103] (3) NCBI software was used to screen for specific fragments from 55 species of the genus *Machilus*, and Primer Premier 5 was used to set a pair of primers, which were then verified by Primer-BLAST. The specific primer sequences are shown in Table 2.

[0104] (4) Based on barcode rpl32-trnL UAG The constructed NJ phylogenetic tree was used for cluster analysis of 55 sequences from 20 species of the *Phoebe* genus downloaded from GenBank. The results showed that this assemblage could successfully distinguish not only three species of *Phoebe* (including *Phoebe zhennan*, *Phoebe bournei*, *Phoebe zhennan*, *Phoebe zei*, and *Phoebe zhennan*), but also 10 other species of the *Phoebe* genus, such as... Figure 5 As shown.

[0105] Example 7 of the present invention: Amount of wood powder used for DNA extraction from Phoebe zhennan wood in Zhejiang

[0106] (1) Sample preparation: The heartwood of Phoebe zhennan was selected. The outer surface of the wood sample was removed with a scalpel sterilized with 70% alcohol to avoid external contamination. The wood sample was cut into several wood chips and ground with a ball mill for 2 minutes at a frequency of 30 cps. After grinding, the wood powder was passed through 80-mesh, 100-mesh, 150-mesh and 200-mesh sieves respectively and dispensed into 1.5 mL centrifuge tubes with wood powder of 300 mg, 400 mg and 500 mg per tube respectively. The tubes were stored in a -80℃ low temperature freezer for later use.

[0107] (3) DNA extraction: DNA was extracted from Phoebe zhennan wood in a sterilized clean working environment using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).

[0108] (4) PCR amplification and sequencing: The primer pairs were the barcode sequences in Example 1 (Table 2). PCR amplification was performed using DNA extract as a template. The reaction system was 20 μL: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA. All PCR reactions were performed on a PCR amplification instrument.

[0109] The reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, repeated 35 times; final extension at 72℃ for 5 min. This yielded efficiently amplified DNA fragments. The amplified products were then purified and subjected to bidirectional direct sequencing.

[0110] The results showed that DNA could be successfully extracted from wood flour passing through 80-mesh, 100-mesh, 150-mesh, and 200-mesh sieves at amounts of 300 mg, 400 mg, and 500 mg, respectively. The DNA was then analyzed using rpl32-trnL. UAG The sequence amplified by the primers was from *Phoebe zhennan*. Subsequent experiments can use a sieve that passes through an 80-mesh sieve, and 300 mg of wood flour.

[0111] As an embodiment 8 of the present invention: an rpl32-trnL UAG The method of identifying Phoebe wood using DNA barcoding includes the following steps:

[0112] (1) Standard sample collection: Five standard samples of the genus Phoebe were collected, namely Phoebe zhennan, Phoebe bournei, Phoebe zhennan, Phoebe zhennan zebrina and Phoebe bournei, totaling 33 species. They were collected from Sichuan, Hunan and Zhejiang respectively. Specific collection information is shown in Table 4.

[0113] (2) Sample preparation: The heartwood or sapwood of the wood was selected as the sample. The outer surface of the wood sample was removed with a scalpel blade disinfected with 70% alcohol to avoid external contamination. The wood sample was cut into several wood chips and ground with a ball mill for 2 minutes at a frequency of 30 cps. After grinding, the chips were passed through an 80-mesh sieve and the fine wood powder was dispensed into several 1.5 mL centrifuge tubes, with 300 mg of wood powder in each tube. The tubes were then stored in a -80℃ low-temperature freezer for later use.

[0114] (3) DNA extraction: DNA was extracted from five species of Phoebe wood in a sterilized clean working environment using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).

[0115] (4) PCR amplification and sequencing: The primer pairs were the four DNA barcode sequences from Example 1. PCR amplification was performed using DNA extract as a template. The reaction system was 20 μL: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA. All PCR reactions were performed on a PCR amplification instrument.

[0116] The reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, repeated 35 times; final extension at 72℃ for 5 min. This yielded efficiently amplified DNA fragments. The amplified products were then purified and subjected to bidirectional direct sequencing.

[0117] Sequencing quality was assessed using SeqMan software. Low-quality portions at both ends were removed, and the remaining portions were then evaluated. Only those meeting quality requirements were used for sequence assembly and proofreading. rpl32-trnL of five species of Phoebe wood were analyzed. UAG The barcode sequences are shown in Table 1.

[0118] (5) Specific primer selection and determination: Sequences were assembled using SeqMan software, and sequence identity analysis was performed by the National Center for Biotechnology Information (NCBI). The results showed that rpl32-trnL UAG The primers can identify five species of Phoebe wood: Phoebe bournei, Phoebe zhennan, Phoebe pekinensis, Phoebe zhennan zeylan, and Phoebe zhennan. Figure 7 As shown.

[0119] Example 9 of the present invention: DNA identification of an unknown Phoebe sp. wood specimen

[0120] (1) A wood specimen was taken from a forestry university. Anatomical analysis identified it as a species of *Phoebe*, but the specific tree species could not be determined. Therefore, based on an rpl32-trnL... UAG The method of identifying Phoebe wood using DNA barcoding includes the following steps:

[0121] (2) Prepare wood flour and grind it through an 80-mesh sieve.

[0122] (3) DNA extraction from wood. DNA was extracted from the wood in a sterilized, ultra-clean working environment, following the DNA extraction method described in Example 2. The DNA was further purified to a final concentration of 1–100 ng / μL.

[0123] (4) PCR Amplification and Sequencing. Wood DNA extract was used as a template for PCR amplification. Primers were listed as NO:1–NO:4. The reaction mixture was 20 μL, containing 7.2 μL ddH2O, 10 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA. All PCR reactions were performed on a PCR instrument. The reaction program was: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, repeated 35 times; final extension at 72℃ for 5 min. This yielded a efficiently amplified DNA fragment. The amplified product was purified and then subjected to bidirectional direct sequencing.

[0124] (5) Sequence alignment analysis and tree species identification.

[0125] Primer rpl32-trnL was used for specimens of unknown *Machilus* species. UAG Perform PCR amplification and sequencing, and then gel the amplified sample, such as... Figure 11 As shown (P: positive control; N: negative control; B: blank control; Mark: nucleic acid molecular marker DL3000), rpl32-trnL UAG Bidirectional sequencing was performed on the PCR amplification products of specific primers, based on the specific primer rpl32-trnL. UAG The analytical results for identifying specimens of the unknown genus *Machilus*, such as... Figure 9 As shown, the unknown nanmu wood specimen is Phoebe sheareri.

[0126] As an embodiment 10 of the present invention: wood sample barcode rpl32-trnL UAG amplification rate

[0127] PCR amplification was performed on 33 wood samples (Table 10). Sequencing success was indicated by "Yes" if the sequencing results matched the sample information, and failure was indicated by "NO" if the amplification was unsuccessful. The results showed that the amplification rate of ycf1-based samples was 87.88% (see Table 5). The amplification effect was good.

[0128] Example 11 of the present invention: Design of primers specific to 20 species of the genus *Machilus*.

[0129] (1) Download the chloroplast genes of the genus *Machilus* from GenBank as shown in Table 1;

[0130] (2) Geneious software was used to compare sequences and identify and determine the differential sites among the five species of the genus Phoebe, especially Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan and Phoebe zhennan.

[0131] Table 8 shows the differentially aligned sites of ycf1 multiple sequence alignments:

[0132]

[0133] (3) Specific fragments were screened for 20 species of the genus *Machilus* using NCBI software, and two pairs of primers were set using Primer Premier5, which were then verified by Primer-BLAST. The specific primer sequences are shown in Table 2.

[0134] (4) Based on the NJ phylogenetic tree constructed using the barcode ycf1, cluster analysis was performed on 55 sequences of 20 species from the genus *Machilus* downloaded from GenBank. The results showed that this phylogenetic tree could successfully distinguish not only five species of *Machilus* (including *Machilus nanmu*, *Machilus zeylindrica*, *Machilus zeylindrica*, *Machilus stenoptera*, and *Machilus purpurea*), but also ten other species of *Machilus*. (See...) Figure 9 .

[0135] Example 12 of the present invention: Amount of wood powder used for DNA extraction from Phoebe zhennan wood

[0136] (1) Sample preparation: The heartwood of the Phoebe zhennan wood was selected, and the outer surface of the wood sample was removed with a scalpel sterilized with 70% alcohol to avoid external contamination. The wood sample was cut into several wood chips and ground with a ball mill for 2 minutes at a frequency of 30 cps. After grinding, the wood powder was passed through 80-mesh, 100-mesh, 150-mesh and 200-mesh sieves respectively and dispensed into 1.5 mL centrifuge tubes with 300 mg, 400 mg and 500 mg of wood powder per tube, respectively. The tubes were then stored in a -80℃ low temperature freezer for later use.

[0137] (3) DNA extraction: DNA was extracted from the Phoebe zhennan wood in a sterilized clean working environment using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).

[0138] (4) PCR amplification and sequencing: The primer pairs were the barcode sequences in Example 1 (Table 1). PCR amplification was performed using DNA extract as a template. The reaction system was 20 μL, consisting of 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA. All PCR reactions were performed on a PCR amplification instrument.

[0139] The reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, repeated 35 times; final extension at 72℃ for 5 min. This yielded efficiently amplified DNA fragments. The amplified products were then purified and subjected to bidirectional direct sequencing.

[0140] The results showed that DNA could be successfully extracted from wood flour passing through 80-mesh, 100-mesh, 150-mesh, and 200-mesh sieves at amounts of 300 mg, 400 mg, and 500 mg, respectively. The sequence amplified using the ycf1 primer was identified as *P. sheareri*. Subsequent experiments could use a 100-mesh sieve with 300 mg of wood flour.

[0141] As an embodiment 13 of the present invention: a method for identifying Phoebe zhennan wood using the YCF1 barcode, comprising the following steps

[0142] (1) Standard sample collection: Five standard samples of the genus Phoebe were collected, namely Phoebe zhennan, Phoebe bournei, Phoebe zhennan, Phoebe zhennan zebrina and Phoebe bournei, totaling 33 species. They were collected from Sichuan, Hunan and Zhejiang respectively. The specific collection information is shown in Table 4.

[0143] (2) Sample preparation: The heartwood or central part of the wood was selected as the sample. The outer surface of the wood sample was removed by a scalpel sterilized with 70% alcohol to avoid external contamination. The wood sample was cut into several wood chips and ground with a ball mill for 2 minutes at a frequency of 30 cps. After grinding, the wood chips were passed through an 80-mesh sieve and the fine wood powder was dispensed into several 1.5 mL centrifuge tubes, with 300 mg of wood powder in each tube. The tubes were then stored in a -80℃ low-temperature freezer for later use.

[0144] (3) DNA extraction: DNA was extracted from five species of Phoebe wood in a sterilized clean working environment using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).

[0145] (4) PCR amplification and sequencing: The primer pairs were the four DNA barcode sequence primers from Example 1 (Table 2). PCR amplification was performed using DNA extract as a template. The reaction system was 20 μL: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA. All PCR reactions were performed on a PCR amplification instrument.

[0146] The reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, repeated 35 times; final extension at 72℃ for 5 min. This yielded efficiently amplified DNA fragments. The amplified products were then purified and subjected to bidirectional direct sequencing.

[0147] SeqMan software was used to assess the sequencing quality of the sequencing results, removing low-quality portions at both ends and assessing the quality of the remaining portions. Only those portions that met the quality requirements could be used for sequence assembly and proofreading.

[0148] Specific primer selection and determination: Sequences were assembled using SeqMan software, and sequence identity analysis was performed by the National Center for Biotechnology Information (NCBI). Results showed that primer ycf1 could identify five species of Phoebe wood: *Phoebe bournei*, *Phoebe zhennan*, *Phoebe leucantha*, *Phoebe zhennan*, and *Phoebe zhennan*. Figure 10 As shown.

[0149] Example 14 of the present invention: DNA identification of wood specimens from an unknown genus of Phoebe sp.

[0150] (1) A wood specimen was taken from a forestry university. Anatomical analysis identified it as a species of *Phoebe*, but the specific tree species could not be determined. Therefore, a method for identifying *Phoebe* wood based on ycf1 DNA barcoding was proposed, comprising the following steps:

[0151] (2) Prepare wood flour and grind it through an 80-mesh sieve.

[0152] (3) DNA extraction from wood. DNA was extracted from the wood in a sterilized, ultra-clean working environment, following the DNA extraction method described in Example 2. The DNA was further purified to a final concentration of 1–100 ng / μL.

[0153] (4) PCR Amplification and Sequencing. Wood DNA extract was used as a template for PCR amplification. Primers were listed as NO:1–NO:4. The reaction volume was 20 μL: 7.2 μL ddH2O, 10 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA. All PCR reactions were performed on a PCR instrument. The reaction program was: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, repeated 35 times; final extension at 72℃ for 5 min. This yielded a efficiently amplified DNA fragment. The amplified product was purified and then subjected to bidirectional direct sequencing.

[0154] (5) Sequence alignment analysis and tree species identification.

[0155] Unknown samples were amplified and sequenced using specific primers ycf1. The amplified samples were then gelled, as shown in the image. Figure 12 As shown, the PCR amplification products of the ycf1-specific primers were subjected to bidirectional sequencing. The analysis results based on the specific primer ycf1 for identifying unknown *Phoebe* specimens are as follows. Figure 10 As shown, the unknown nanmu wood specimen is Phoebe sheareri.

[0156] Example 15 of the present invention: Amplification rate of DNA barcode ycf1 in wood samples

[0157] PCR amplification was performed on 33 wood samples (Table 10). Sequencing success was indicated by "Yes" if the sequencing results matched the sample information, and failure was indicated by "NO" if the amplification was unsuccessful. The results showed that the amplification rate of ycf1-based samples was 93.94% (see Table 5). The amplification effect was good.

[0158] SEQ ID No. 5:

[0159]

[0160] SEQ ID No.6:

[0161]

[0162] SEQ ID No.7:

[0163]

[0164] SEQ ID No.8:

[0165]

[0166] SEQ ID No.9:

[0167]

[0168] The preferred embodiments of the present application have been described above with reference to the accompanying drawings, but this does not limit the scope of the claims of the present application. Any modifications, equivalent substitutions, and improvements made by those skilled in the art without departing from the scope and substance of the embodiments of the present application shall be within the scope of the claims of the present application.

Claims

1. An rpl32-trnL UAG Barcode, characterized in that: The rpl32-trnL UAG The barcode sequence, as shown in PV940116, PV940101, PV940087, PV940108, and PV940097, is used to identify Phoebe zhennan, Phoebe bournei, Phoebe zhennan, Phoebe zhennan var. chinensis, and Phoebe zhennan var. zeylanica within the Phoebe genus; the rpl32-trnL UAG The primer sequences for the barcode are shown in SEQ ID No. 1 to 2.

2. An rpl32-trnL UAG The method for identifying Phoebe wood using barcodes is characterized by: Includes the following steps: S201. Select the heartwood or sapwood of the wood sample, grind it and filter it through a sieve to obtain a wood powder sample, and store it in a -80℃ low temperature freezer for later use. S202. Extract DNA from the wood flour sample; S203. Using the DNA sample as a template, amplify the rpl32-trnL sequence of the plasmid genome using PCR. UAG Barcode sequence to obtain amplification products; S204. Sequencing the amplified product yields rpl32-trnL UAG Sequence, to obtain rpl32-trnL UAG Barcode; S205, using the rpl32-trnL UAG Barcodes were used to identify Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan, and Phoebe zhennan in the genus Phoebe based on SeqMan software and phylogenetic tree method; in: The rpl32-trnL UAG The primer sequences for the barcode are shown in SEQ ID No. 1-2; The PCR amplification reaction process is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, cycled 35 times; 72℃ final extension for 5 min to obtain the amplified DNA target fragment. The PCR amplification reaction system includes: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA.

3. A YCF1 barcode, characterized in that: The sequence of the ycf1 barcode, as shown in PV940150, PV940135, PV940121, PV940142, and PV940131, is used to identify Phoebe zhennan, Phoebe bournei, Phoebe zhennan, Phoebe zhennan var. chinensis, and Phoebe zhennan var. zeylanica in the genus Phoebe zhennan; the primer sequences of the ycf1 barcode are shown in SEQ ID No. 3 to 4.

4. A method for identifying Phoebe zhennan wood using the YCF1 barcode, characterized in that: Includes the following steps: S401. Select the heartwood or sapwood of the wood sample (in the case of no heartwood), grind and filter through a sieve to obtain a wood powder sample, and store it in a -80℃ low temperature freezer for later use. S402. Extract DNA from the wood flour sample; S403. Using the DNA sample as a template, amplify the ycf1 barcode sequence in the plasmid genome sequence using PCR to obtain the amplification product; S404. Sequencing the amplified product to obtain the ycf1 sequence, and then generating the ycf1 barcode. S405. Using the ycf1 barcode, and based on SeqMan software and phylogenetic tree method, identify Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan and Phoebe zhinan in the Phoebe genus; in: The primer sequences for the ycf1 barcode are shown in SEQ ID No. 3-4; The PCR amplification reaction process is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, cycled 35 times; 72℃ final extension for 5 min to obtain the amplified DNA target fragment. The PCR amplification reaction system includes: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA.

5. An rpl32-trnL UAG +ycf1 combined barcode, characterized in that: The rpl32-trnL UAG The +ycf1 combined barcode sequence is shown in SEQ ID No. 5-9 and is used to identify Phoebe zhennan, Phoebe bournei, Phoebe zhennan, Phoebe zhennan var. chinensis, and Phoebe zhennan var. zebrina within the Phoebe genus; the rpl32-trnL UAG The primer sequences for the +ycf1 combined barcode are shown in SEQ ID No. 1 to 4.

6. An rpl32-trnL UAG The method for identifying Phoebe zhennan wood using the +ycf1 combination barcode is characterized by: Includes the following steps: S601. Select the heartwood or sapwood of the wood sample (in the case of no heartwood), grind and filter through a sieve to obtain a wood powder sample, and store it in a -80℃ low temperature freezer for later use. S602. Extract DNA from the wood flour sample; S603. Using the DNA sample as a template, amplify the rpl32-trnL sequence of the plasmid genome using PCR. UAG The ycf1 barcode sequence was used to obtain the amplification product; S604. Sequencing the amplified product yields rpl32-trnL UAG And ycf1 sequence, to obtain rpl32-trnL UAG and ycf1 barcode; S605, using the rpl32-trnL UAG The barcode ycf1 was used to identify Phoebe zhennan, Phoebe bournei, Phoebe simonii, Phoebe zhennan, and Phoebe zhennan in the genus Phoebe based on SeqMan software and phylogenetic tree method; in: The rpl32-trnL UAG The primer sequences for the +ycf1 combined barcode are shown in SEQ ID Nos. 1 to 4; The PCR amplification reaction process is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, cycled 35 times; 72℃ final extension for 5 min to obtain the amplified DNA target fragment. The PCR amplification reaction system includes: 19 μL ddH2O, 7.2 μL Mix, 0.4 μL forward primer, 0.4 μL reverse primer, and 2 μL template DNA.

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