DNA bar code sequence and primer for identifying long-sequence elm, application of DNA bar code sequence and primer and identification method

Abstract

The invention belongs to the technical field of plant species identification, and particularly relates to a DNA bar code sequence for identifying long-sequence elm, a primer, application of the DNA bar code sequence and the primer and an identification method. The DNA bar code sequence is formed by sequentially connecting and splicing three fragments, namely rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS in series. The DNA bar code sequence provided by the invention has good primer universality and abundant site variation, is easy to operate, can be used for stably and accurately identifying the ulmus pumila, and can obviously distinguish the ulmus pumila from sibling species distributed in the same domain of the ulmus pumila. In addition, a DNA bar code molecular marker technology is utilized, and the method has important significance in research aspects of classification evolution, protection and utilization and the like of germplasm resources of the ulmus plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant species identification, and particularly relates to a DNA barcode sequence, a primer, and their applications and identification methods for the identification of Background Art

[0002] Ulmus elongata Ulmus elongata belongs to the genus Ulmus Ulmus of the family Ulmaceae. The plant is a deciduous tree, up to 30 meters tall; the bark is grayish-white, peeling off in irregular flakes; the leaves are elliptical or lanceolate-elliptical, 7 cm to 19 cm long and 3 cm to 8 cm wide, with the base slightly oblique or nearly symmetrical, the leaf margin has large and deep double serrations, and the petiole is 3 mm to 11 mm long; the stipules are mostly lanceolate to narrowly lanceolate, 7 mm to 18 mm long, and often fall off early. The flowers bloom in spring, with a racemose cyme, and the inflorescence axis is significantly elongated and drooping, and the pedicel is several times as long as the perianth. The fruiting axis is 4 - 8 cm long, with sparse hairs; the samara is narrow and long, gradually narrowing and pointed at both ends, spindle-shaped, 2 cm to 2.5 cm long, about 3 mm wide, with sparse hairs on both sides, and dense white long cilia on the edge, and the fruit nucleus is slightly above the middle of the samara.

[0003] Ulmus elongata is a tertiary relict plant endemic to China. There are less than 1500 mature trees in the wild, and it has been listed as a national second-class protected plant and a wild plant with an extremely small population in China. Since Ulmus elongata has a geographical distribution overlap with its related species Ulmus pumila, Ulmus bergmanniana, and Ulmus parvifolia, and its morphological characteristics are easily affected by the environment and tend to be the same, it is difficult to accurately distinguish it from its sympatric related species by traditional morphological identification methods during the non-flowering or non-fruiting period. In addition, traditional methods often rely on the characteristics of organs such as flowers and fruits, and there are great difficulties in identifying incomplete samples such as wood, seeds, seedlings, or plant fragments, with low identification efficiency and high misjudgment rate. Due to the problem of limited identification technology, the phenomenon of mis-cutting and mis-felling occurs frequently, accelerating the rapid decline of the wild population of Ulmus elongata. In recent years, continuous human interference, habitat destruction, and global climate change have led to the risk of extinction of Ulmus elongata at any time, seriously restricting its protection and management, and there is an urgent need to carry out effective rescue and protection work on Ulmus elongata.

[0004] DNA barcode technology is a method for species identification based on standardized short DNA fragments with high variability and easy amplification, and has been widely used in the field of plant species identification. However, when using internationally common DNA barcodes for identification, the DNA sequences of Ulmus elongata and its sympatric related species Ulmus pumila, Ulmus bergmanniana, and Ulmus parvifolia are highly similar and the interspecific genetic differences are small, making it difficult to distinguish them. Therefore, developing a DNA barcode that can accurately identify Ulmus elongata has important scientific significance. Summary of the Invention

[0005] The object of the present invention is to provide a novel DNA barcode capable of accurately identifying Ulmus elongata, and specifically relates to a DNA barcode sequence, primers, and their application and identification method for identifying Ulmus elongata. To achieve the above object, the present invention adopts the following technical solutions.

[0006] The first object of the present invention is to provide a DNA barcode sequence for identifying Ulmus elongata. The DNA barcode sequence is composed of three fragments, rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS, concatenated in sequence.

[0007] The present invention has developed for the first time a specific DNA barcode sequence applicable to Ulmus elongata and its related species. By combining three fragments, rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS, an accurate and rapid DNA barcode identification technology system for Ulmus elongata has been constructed. This sequence can effectively determine whether the sample to be tested is Ulmus elongata, solving the problem that it is difficult to distinguish the internationally common DNA barcode because the DNA sequences of Ulmus elongata and its sympatric related species, such as Ulmus pumila, Ulmus bergmanniana, and Ulmus parvifolia, are highly similar and the interspecific genetic differences are small.

[0008] The second object of the present invention is to provide primers for amplifying the aforementioned DNA barcode sequence. The primers include: a primer pair for amplifying rps16-trnQ-UUG, a primer pair for amplifying trnS-GCU-trnG-GCC, and a primer pair for amplifying ITS.

[0009] The nucleotide sequence of the forward primer of the primer pair for amplifying rps16-trnQ-UUG is as shown in SEQ ID No.1, and the nucleotide sequence of the reverse primer of the primer pair for amplifying rps16-trnQ-UUG is as shown in SEQ ID No.2.

[0010] The nucleotide sequence of the forward primer of the primer pair for amplifying trnS-GCU-trnG-GCC is as shown in SEQ ID No.3, and the nucleotide sequence of the reverse primer of the primer pair for amplifying trnS-GCU-trnG-GCC is as shown in SEQ ID No.4.

[0011] The nucleotide sequence of the forward primer of the primer pair for amplifying ITS is as shown in SEQ ID No.5, and the nucleotide sequence of the reverse primer of the primer pair for amplifying ITS is as shown in SEQ ID No.6.

[0012] The third object of the present invention is to provide a reagent for identifying Ulmus elongata. The reagent contains the above primers.

[0013] The fourth object of the present invention is to provide the application of the DNA barcode sequence, primer or reagent in the identification of Ulmus elongata

[0014] The fifth object of the present invention is to provide a method for identifying Ulmus elongata using the primer, which is specifically implemented according to the following steps: (1) Extract the total DNA of the Ulmus plant sample to be tested by the improved CTAB method.

[0015] (2) Using the total DNA of the Ulmus plant sample to be tested as a template, perform PCR amplification using the primer respectively to obtain amplification products of three fragments of rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS of the sample to be tested.

[0016] (3) Perform gel electrophoresis analysis on the PCR amplification products, and select the amplification products with single band display for bidirectional sequencing.

[0017] (4) Import the bidirectional sequencing results of the three fragments of rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS of the Ulmus plant sample to be tested into software and splice them into one sequence to obtain the complete rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS sequences of the Ulmus plant sample to be tested.

[0018] (5) Align and trim the rps16-trnQ-UUG sequence of the Ulmus plant sample to be tested with the rps16-trnQ-UUG reference sequences recorded as SEQ ID No.7 to SEQ ID No.14, align and trim the trnS-GCU-trnG-GCC sequence of the Ulmus plant sample to be tested with the trnS-GCU-trnG-GCC reference sequences recorded as SEQ ID No.15 to SEQ ID No.22, and align and trim the ITS sequence of the Ulmus plant sample to be tested with the ITS reference sequences recorded as SEQ ID No.23 to SEQ ID No.30.

[0019] (6) Import the results of multiple sequence alignment and trimming of each fragment obtained in step (5) into software in the order of rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS for splicing.

[0020] (7) Import the splicing results obtained in step (6) into software, calculate the K2P genetic distance, and construct a phylogenetic tree based on the maximum likelihood method. Judge whether it is Ulmus elongata according to the genetic distance results and the branching position of the Ulmus plant sample to be tested in the phylogenetic tree.

[0021] Preferably, if the genetic distance between the sample of the elm plant to be tested and all individuals of U. elongata is less than its genetic distance from other species, and it clusters with U. elongata individuals in the phylogenetic tree, then the sample of the elm plant to be tested is U. elongata.

[0022] Preferably, the PCR amplification reaction system is as follows: 40 μL of PCR reaction mixture, including 2 μL of forward primer, 2 μL of reverse primer, and 2 μL of DNA template; 20 μL of 2× Rapid Taq Master Mix PCR premix; supplemented with ddH2O to 40 μL; the PCR amplification program is: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 50 °C - 56 °C for 15 s, amplification at 72 °C for 15 s, for a total of 35 cycles; final amplification at 72 °C for 5 min, and stored at 4 °C.

[0023] Preferably, the sample of the elm plant to be tested includes any one of fresh leaves, dried leaves, and specimens.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention has developed for the first time a specific DNA barcode sequence applicable to U. elongata and its related species. By combining the rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS fragments, an accurate and rapid DNA barcode identification technology system for U. elongata is constructed. This sequence can effectively determine whether the sample to be tested is U. elongata, and solves the problem that it is difficult to distinguish the internationally common DNA barcode because the DNA sequences of U. elongata and its sympatric related species, U. pumila, U. bergmanniana, and U. parvifolia, are highly similar and the interspecific genetic differences are small.

[0025] 2. The present invention also provides primers for amplifying the aforementioned DNA barcode sequence and their applications. Experimental verification shows that the primers have characteristics such as clear amplified fragments, high resolution, and excellent sensitivity, and can achieve efficient, low-cost, and convenient identification of U. elongata.

[0026] 3. The present invention has established a standard gene database covering the rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS fragments by collecting multiple samples of U. elongata, U. pumila, U. bergmanniana, and U. parvifolia with determined species, providing a reliable reference for subsequent species identification. Description of the Drawings

[0027] Figure 1This is the gel electrophoresis diagram of the total genomic DNA of 8 samples in the present invention. From left to right: M: Marker; 1: YJF_CXY; 2: WYX_CXY; 3: BZY_CXY; 4: BZY_YS; 5: YDS_YS; 6: HJX_XSY; 7: NZX_LY; 8: BZY_LY.

[0028] Figure 2 This is the gel electrophoresis diagram of the primer amplification of ITS sequences of 8 samples in the present invention. From left to right: M: Marker; 1: YJF_CXY; 2: WYX_CXY; 3: BZY_CXY; 4: BZY_YS; 5: YDS_YS; 6: HJX_XSY; 7: NZX_LY; 8: BZY_LY.

[0029] Figure 3 This is the gel electrophoresis diagram of the primer amplification of rps16 - trnQ - UUG sequences of 8 samples in the present invention. From left to right: M: Marker; 1: YJF_CXY; 2: WYX_CXY; 3: BZY_CXY; 4: BZY_YS; 5: YDS_YS; 6: HJX_XSY; 7: NZX_LY; 8: BZY_LY.

[0030] Figure 4 This is the gel electrophoresis diagram of the primer amplification of trnS - GCU - trnG - GCC sequences of 8 samples in the present invention. From left to right: M: Marker; 1: YJF_CXY; 2: WYX_CXY; 3: BZY_CXY; 4: BZY_YS; 5: YDS_YS; 6: HJX_XSY; 7: NZX_LY; 8: BZY_LY.

[0031] Figure 5 This is the maximum - likelihood phylogenetic tree constructed based on the combined fragments of rps16 - trnQ - UUG, trnS - GCU - trnG - GCC and ITS in the present invention. Detailed implementation mode

[0032] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Any transformation based on the present invention falls within the protection scope of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional methods well - known to those skilled in the art, and the materials, reagents, etc. used can be obtained through commercial channels.

[0033] Example 1: Development of specific DNA barcode sequences for Ulmus elongata and its related species Retrieve the complete chloroplast genome sequences of *Ulmus elongata*, *Ulmus bergmanniana*, *Ulmus pumila* and *Ulmus parvifolia* from GenBank (GenBank accession numbers: *Ulmus elongata*: MT165929.1, *Ulmus bergmanniana*: MT165921.1, *Ulmus pumila*: MT165942.1, *Ulmus parvifolia*: MT165940.1). Perform global alignment using MAFFT v7.308 and analyze nucleotide polymorphism (Pi) through DnaSP v6.12.03. Screen the highly variable regions of the chloroplast genome based on the alignment variable sites and Pi values. Combining with the chloroplast genome annotation information, finally select two chloroplast DNA fragments with significant differences between *Ulmus elongata* and its related species: rps16-trnQ-UUG and trnS-GCU-trnG-GCC. At the same time, select the ribosomal DNA gene transcription spacer (ITS), which has good primer universality, moderate sequence quantity and high variability, and can provide rich specific sites.

[0034] Example 2: Primer Design for Specific DNA Barcode Sequences of *Ulmus elongata* and Its Related Species Based on the sequence information of rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS fragments screened in Example 1, design specific PCR primers for identifying *Ulmus elongata*. The sequences of the PCR primers are as follows: The primer pair for amplifying rps16-trnQ-UUG is as follows: The nucleotide sequence of the forward primer rps16-trnQ-UUG-F of the primer pair for amplifying rps16-trnQ-UUG is shown in SEQ ID No.1: 5′-TCCTTTATCTTTGCCTTGA-3′.

[0035] The nucleotide sequence of the reverse primer rps16-trnQ-UUG-R of the primer pair for amplifying rps16-trnQ-UUG is shown in SEQ ID No.2: 5′-CTTCAGAAACTACCCATT-3′.

[0036] The primer pair for amplifying trnS-GCU-trnG-GCC is as follows: The nucleotide sequence of the forward primer trnS-GCU-trnG-GCC-F of the primer pair for amplifying trnS-GCU-trnG-GCC is shown in SEQ ID No.3: 5′-TGATCCGGGTCGTAATCCTG-3′.

[0037] The nucleotide sequence of the reverse primer trnS-GCU-trnG-GCC-R of the primer pair for amplifying trnS-GCU-trnG-GCC is shown in SEQ ID No. 4: 5′-TCGTTGAAACAAAAGAGGCCC-3′.

[0038] The primer pairs for amplifying ITS are as follows: The nucleotide sequence of the forward primer ITS-F of the primer pair for amplifying ITS is shown in SEQ ID No. 5: 5′-TCCGTAGGTGAACCTGCGG-3′.

[0039] The nucleotide sequence of the reverse primer ITS-R of the primer pair for amplifying ITS is shown in SEQ ID No. 6: 5′-TCCTCCGCTTATTGATATGC-3′.

[0040] Example 3: Evaluation of the effectiveness and feasibility of DNA barcode sequences 1. Collection of samples of Ulmus elongata and its related species Plant samples of Ulmus elongata, Ulmus pumila, Ulmus bergmanniana, and Ulmus parvifolia were collected from the wild, taken from plant leaves, and the sample information is shown in Table 1.

[0041] Table 1 Sample information 2. Sample pretreatment Appropriate amounts of the samples were weighed respectively, the surfaces were wiped with 75% ethanol to remove sediment and impurities, and then ground into fine powder using a sample rapid grinder. 40 mg of the fine powder was taken for standby.

[0042] 3. Extraction of genomic DNA from samples The total genomic DNA of each sample was extracted using the modified CTAB method, and the DNA quality and concentration were detected by 1% agarose gel electrophoresis (as Figure 1 shown) and Nanodrop ultra-micro spectrophotometer.

[0043] 4. Amplification, purification, and sequencing of target fragments Using the extracted DNA as a template, the target fragments were PCR amplified using the primers shown in SEQ ID No. 1~SEQ ID No. 6.

[0044] The specific PCR amplification reaction system is as follows: 40 μL of PCR reaction mixture, including 2 μL of forward primer, 2 μL of reverse primer, 2 μL of DNA template; 20 μL of 2× Rapid Taq Master Mix PCR premix; supplemented with dd H2O to 40 μL.

[0045] Among them, the 2× Rapid Taq Master Mix PCR premix contains Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, enhancer and optimizer for PCR reaction, and stabilizer, with a concentration of 2×.

[0046] The procedure of PCR amplification is as follows: Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 53 °C for 15 s, amplification at 72 °C for 15 s, for a total of 35 cycles; final amplification at 72 °C for 5 min, stored at 4 °C.

[0047] After the PCR products were detected by 1% agarose gel electrophoresis (130 V, 30 min), the PCR amplification products with single and bright bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. The gel electrophoresis diagrams of primers for ITS, rps16-trnQ-UUG, and trnS-GCU-trnG-GCC sequences of each sample are as Figure 2 , Figure 3 and Figure 4 shown.

[0048] 5. Assembly, alignment, and concatenation of sequencing results Use BioEdit v7.7.1 to check the quality of the sequencing result peak diagram, remove the low-quality sequences at the beginning and end, manually proofread the bases and complete the sequence assembly, and finally obtain the corrected sequences of rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS of each sample, as shown in SEQ ID No.7~SEQ ID No.30 in the sequence listing. Perform multiple sequence alignment on rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS sequences respectively by MAFFT v7.308. Use PhyloSuite v1.2.3 to concatenate and splice the aligned sequences in the order of rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS to construct a combined dataset.

[0049] The rps16-trnQ-UUG nucleotide sequences of each sample are as shown in SEQ ID No.7~SEQ ID No.14.

[0050] >The nucleotide sequence of YJF_CXY is as shown in SEQ ID No.7: GAATCATTGGGTTTAGACATAACTTCGGTGATTTTTAATCGTTTCAAAATGGCAGCAACATACCATTTTTGTGATTTCTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCCCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCGCAATGAGAGGTCTAGTGGAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATTAAGACTCCACAATGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTATTCATTCAATATAAAATAACAATATAAAAT。

[0051] >The nucleotide sequence of WYX_CXY is shown in SEQ ID No.8: GAATCATTGGGTTTAGACATAACTTCGGTGATTTTTAATCGTTTCAAAATGGCAGCAACATACCATTTTTGTGATTTCTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCCCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCGCAATGAGAGGTCTAGTGGAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATTAAGACTCCACAATGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTATTCATTCAATATAAAATAACAATATAAAAT。

[0052] >The nucleotide sequence of BZY_CXY is shown in SEQ ID No.9: GAATCATTGGGTTTAGACATAACTTCGGTGATTTTTAATCGTTTCAAAATGGCAGCAACATACCATTTTTGTGATTTTTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCCCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCTCAAAAAAATGAGAGGTCTAGTGGAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATTAAGACTCCACAATGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTATTCATTCAATATAAAATAACAATATAAAAT。

[0053] >The nucleotide sequence of BZY_YS is shown in SEQ ID No. 10: GAATCATTGGGTTTWGACMTTAMTTYGGTGATTTTTAATCGTTTCAAAATGGCAGCAACATACCATTTTTGTGATTTCTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCCCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCTCAAAAAAATGAGAGGTCTAGTGGAAAAAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATGTAAGACTCCACAACGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTTTTATCTATACTTTATTCATTTTATCTATACTTTATTCATTCAATATAAAAATAAAAAAAAAAT。

[0054] >The nucleotide sequence of YDS_YS is shown in SEQ ID No. 11: GAATCATTGGGTTTAGACATTACTTCGGTGATTTTTAATCGTTTCAAAATGGCAGCAACATACCATTTTTGTGATTTCTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCCCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCTCAAAAAAATGAGAGGTCTAGTGGAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATGTAAGACTCCACAACGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTTTTATCTATACTTTATTCATTTTATCTATACTTTATTCATTCAATATAAAAATAAAAAAAAAT。

[0055] >The nucleotide sequence of HJX_XSY is shown in SEQ ID No. 12: GAATCMTTGGGKTTDGVCMTTAHTTYGGKGRTTTTTWATYGTTTYAAAAWGGCAGCAACATACCATTTTTGTGATTTCTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCCCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCTCAAAAAAATGAGAGGTCTAGTGGAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATGTAAGACTCCACAACGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTTTTATCTATACTTTATTCATTCAATATAAAAATAAAAAAAAAAAA。

[0056] >The nucleotide sequence of NZX_LY is shown in SEQ ID No.13: GAATCMTTGGGTTTWGRCMTTAHTTCGGTGATTTTGAATCGTTTCAAAATGGCAGCAACATACCATTTTTGTGATTTCTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCTCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCTCAAAAAAATGAGAGGTCTAGTGGAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATGTAAGACTCCACAATGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTATTCATATCTATACTTGATTCATTCAATATAAAAATAAAAAAAAAAAA。

[0057] >The nucleotide sequence of BZY_LY is shown in SEQ ID No. 14: GAATCMTTGGGKTTAGVCMTTWHTTBGGKGDTTTTTWATCGTTTCAAAATGGCAGCAACATACCATTTTTGTGATTTCTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCCCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCTCAAAAAAATGAGAGGTCTAGTGGAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATGTAAGACTCCACAACGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTTTTATCTATACTTTATTCATTCAATATAAAAATAAAAAAAAAAAA。

[0058] The trnS-GCU-trnG-GCC nucleotide sequences of each sample are shown in SEQ ID No.15 to SEQ ID No.22: >The nucleotide sequence of YJF_CXY is shown in SEQ ID No.15: CGGAAAGAGRGGGATTCGAACCCCCGGTACAAAAAACTCGTACAACGGATTAGCAATCCGACGCTTTAGTCCMCTCMGCCMTCTCTCCCCCGATTGAAAAAAGAAAAKGACTATGTTACATTAGTAAACAAACATAAAACTTGAAAAAGCTCTTTTCCCTTCTTAATTTTTATTTTATTCMTCTTTCTTTTCTATTTTAAATTTTAATAGAATTTTTATAATTAACTATAATATAAATTATTAACATATAAATGTTAAATATAGTTTATAGTTTTTTATAATATAAAAGKGAAATAAACAATAAAAATAAAATAATATTAAATTTAAATTCTTTTTATTATTTATTAAATTCTTTTTAATTATTTATTTAATTATAATTATATATATATATATATTTAATATTATATATAAATATTATATATATTTAATTAATTTTGAATTTAATTATATTAAATTTATATTTCATTTTTAATTTGAATATTATTTTAATATAATATTCAAAATATATATAAATAAAAATGAAAAATGAAATBTTTTTTTTTTTTATTTCATCCAAAGAAACCTTTTATT。

[0059] >The nucleotide sequence of WYX_CXY is shown in SEQ ID No. 16: CGGAAAGAGAGGGATTCGAACCCTCGGTACAAAAAACTCGTACAACGGATTAGCAATCCGACGCTTTAGTCCMCTCASCCATCTCTCCCCCGATTGAAAAAAGAAAATGACTATGTTACATTAGTAAACAAACATAAAACTTGAAAAAGCTCTTTTCCCTTCTTAATTTTTATTTTATTCATCTTTCTTTTCTATTTTAAATTTTAATAGAATTTTTATAATTAACTATAATATAAATTATTAACATATAAATGTTAAATATAGTTTATAGTTTTTTATAATATAAAAGTGAAATAAACAATAAAAATAATATTAAATTTAAATTCTTTTTATTATTTATTAAATTCTTTTTAATTATTTATTTAATTATAATTATATATATATTTAATATTATATATAAATATTATATATATTTAATTAATTTTGAATTTAATTATATTAAATTTATATTTCATTTTTAATTTGAATATTATTTTAATATAATATTCAAAATATATATAAATAAAAATGAAAAATGAAATCTTTTTTTTTTTTATTTCATCCAAAGAAACCTTTTATT。

[0060] >The nucleotide sequence of BZY_CXY is shown in SEQ ID No. 17: CGGAAAGAGAGGGATTCGAACCCTCGGTACAAAAAACTCGTACAACGGATTAGCAATCCGACGCTTTAGTCCACTCAGCCATCTCTCCCCCGATTGAAAAAAGAAAATGACTATGTTACATTAGTAAACAAACATAAAACTTGAAAAAAGCTCTTTTCCCTTCTTAATTTTTATTTTATTCATCTTTCTTTTCTATTTTAAATTTTAATAGAATTTTTATAATTAACTAGAATATAAATTATTAACATATAAATGTTAAATATAGTTTTTTATAATATAAAAATAATATAAAAGTGAAATAAACAATAAAAATAAAATAATATTCAATTTAAATTCTTTTTATTATTTATTAAATTCTTTTTAATTATTTATTTAATTATTTATTTAATTATAATAATTATTATAATAATTATATATATTTAATATTATAATATTATATATAAATATTATATATTATTATATATATTTAATTAATTAATTATATATATTTAATATATATAAATAAAAATTCAATTTTTTTTTTTTTTATTTCATCCAAAGAAACCTTTTATT。

[0061] >The nucleotide sequence of BZY_YS is shown in SEQ ID No. 18: CGGAAAGAGRGGGATTCGAACCCTCGGTACAAAAAACTCGTACAACGGATTAGCAATCCGACGCTTTAGTCCMCTCMGCCHTCTCTCCCCCGATTGAAAAAAGAAAAKGACTWTGTTHCHTTAGTAAACAAACHTAAAACTTGAAAAAAGCTCTTTTCCCTTCTTAATTTTGATTTTWTTCHTYTTTCTTTTCTWTTTTTAATAGAATTTTTTTTWTCHTTAAATATAATTAAATTTHCHTTAAAWATAATTAAATTTATATATATATAATTAAATTTWTWTAATTAAATATTTAAATATAATATTATATTATAATATATATAATATAAATTATTAACATATAAATGTTAAATATAGTTTTTTWTAATATAAAAATATAAAAGKGAAATAAAAAATAAACAATAAAATAATATTAAATTGAAATTCTTTTTATTAAATATTAAATTCTTTTTAATTATTTAATTTGAATTAATTTATTTATTATTTCATTTGAATTATATATATTATATATATATTGAAATATATTTAAATTGAATATAATATTCAAAATATATATAAATAAAAATATAAATAAAATCTTTTTTTTTTTTTTTTATTTCATCCAAAGAAACCTTTTATT。

[0062] >The nucleotide sequence of YDS_YS is shown in SEQ ID No. 19: MGGAAAGAGRGGGATTCGAACCCYCGGKACAAAAAAHTBGTHCAACGGATTAGCAATCCGACGCTTTAGTCCMCTCMGCCHTYTYTCCCCCGATTGAAAAAAGAAAAKGAHTWTGTTHCHTTAGTAAACAAACMTAAAACTTGAAAAAAGCTYTTTTCCCTTYTTAATTTTGATTTTATTCATCTTTYTTTTYTWTTTTTAAWAGAATTTTTTTTWTCHTTAAAWATAATTAAATTTWTWTWWWWWTAATTAAATTTWTWTAATTAAATWTTTAAATATAATWTTWTWTTATAATATATATAATATAAATTWTTAACMTWTAAADGTTAAAWWTWGTTTTTTWTAATATAAAAATATAAAAGKGAAATAAAAAATAAACAATAAAATAATATTAAATTGAAATTYTTTTTWTTAAATWTTAAATTYTTTTTAATTATTTAATTTGAATTAATTTWTTTWTTWTTTCMTTTGAATTATWTWTWTTATATATATATTGAAATATATTTAAATTGAATATAATATTCAAAATATATATAAATAAAAATATAAATAAAATCTTTTTTTTTTTTTTTTTATTTCATCCAAAGAAACCTTTTATT。

[0063] >The nucleotide sequence of HJX_XSY is shown in SEQ ID No.20: CGGAAAGAGAGGGATTCGAACCCTCGGTACAAAAAACTCGTACAACGGATTAGCAATCCGACGCTTTAGTCCMCTCAGCCHTCTCTCCCCCGATTGAAAAAAGAAAAKGACTATGTTACATTAGTAAACAAACATAAAACTTGAAAAAAGCTCTTTTCCCTTCTTAATTTTGATTTTATTCATCTTTCTTTTCTATTTTTAAWAGAATTTTTTTTATAATTAAAWATAATTAAATTTATATAATTAAATATAAWATAAATTATTAACATATAAATGTTAAATATAGTTTTTTAWAAWATAAAAGKGAAATAAAAAATAAACAATAAAAAATAAACAATAAAATAATATTAAATTGAAATTCTTTTTATTAAATATTAAATTCTTTTTAATTATTTATTTATTATTTAATTTGAATTAATTTATTTATTATTTCATTTGAATTATATATAATTATAATTATAWATAATTCAATTTTGAATTATATATATTATATATATTTAAAWATATTTAAATTGAATATAATATTCAAAATAWATATAAATAAAAATATAAATAAAATCAAAATCTTTTTTTTTTTTTTATTTCATCCAAAGAAACCTTTTATT。

[0064] >The nucleotide sequence of NZY_LY is shown in SEQ ID No. 21: CGGAAAGAGAGGGATTCGAACCCTCGGTACAAAAAAACTCTTTTCCCTTCTTAATTTTGATTTTATTCATCTTTCTTTTCTATTTTTAATAGAATTTTTTATATATAATTTTTATAATTAAATATAATATAAATTATTAACATATAAATTTTAAATATAGTTTTTTATAATATATAGTGAAATAAAAATAAACAATAAAATAATATTAAATTGAAATTCTTTTATTAAATATTAAATTCTTTTTAATTATTTATTTATTATTTAATTTTTTAATTATATAATTTAGTTATATTTATATATAATTAAAATTATATATATTTTGATATATATTTAAATTGAATATAATATTCAAAAAATATATAAATAAAATCAAAATCTTTTTATTTCCACGGCTTGGCTTGGTCAGTACCTAGCTG。

[0065] >The nucleotide sequence of BZY_LY is shown in SEQ ID No.22: CGGAAAGAGAGGGATTCGAACCCTCGGTACGATTAACTCGTACAACGGATTAGCAATCCGCCGCTTTAGTCCACTCAGCCATCTCTCCCAATTGAAAAAGATAATTACTATATGAGATAGCACATAAGATAAAGGAAAGAATCTTTCTTTCTCTCTTTTCTTCTTTCTATATTATATAGATATGTACAACTTTTATCATCAATTTCCTTTATTTCTTTATCTAAAGTAAAGGAAGGGCTCAGAAGAGCCAAGAATATCAAGAAAAATAAAGAAGACCTCTTTTCTTTGTCTTGATTTTGTTCGAAAGGACCCTCTTATTCTCATGGCCTGGTCTGGTCAGTACCCAGCCG。

[0066] The ITS nucleotide sequences of each sample are shown in SEQ ID No.23 to SEQ ID No.30: >The nucleotide sequence of YJF_CXY is shown in SEQ ID No.23: ACGACCCGCGAACACGTTGTTAAACCGGGGGGCGAGGGACCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACCGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGGAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCAAACCCCGTCGGGGCMRAAGGGTGGGGCGGATGCTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTACGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAACGCTCCCGGCGTGCCGTCTCGGAACGACCCCTGCGCGCGGCCTCGCGCCGCGCTTCCAACGCGACCCC。

[0067] >The nucleotide sequence of WYX_CXY is shown in SEQ ID No. 24: ACGACCCGCGAACACGTTGTTAAACCGGGGGGCGAGGGACCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACCGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGGAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCARAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCCAAACCCCGTCGGGGCMWAAGGGTGGGGCGGATGCTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTACGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAACGCTCCCGGCGTGCCGTCTCGGAACGACCCCTGCGCGCGGCCTCGCGCCGCGCTTCCAACGCGACCCC。

[0068] >The nucleotide sequence of BZY_CXY is shown in SEQ ID No. 25: ACGACCCGCGAACACGTTGTTAAACCGGGGGGCGAGGGACCTCCGGGCCCCGAGCCCTCCCCCGGSGCTGGTCGCGGCACCGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGAAACGGTGCCGTCCGAGCCGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAARAACGTAGCGAAATGCGATACTTGGTGTGAATTGCARAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACGCCGTTGCCCCCCCCAACCCCKTSGGGGGRGKGGGGGGGCGGATGCTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTCTGCTGCGGGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAGCGTCCCCGGCGTGCCGTCTCGGAACGACCCCTGCGCGCGGCCTCGCGCCGCGCTTCCAACGCGACCCC。

[0069] >The nucleotide sequence of BZY_YS is shown in SEQ ID No. 26: ACGACCCGCGAACACGTTGTTAAATTGGGGGGCGAGGGGTCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACTGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGAAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCAAACCCCGTCGGGGAAAAAGGGGGGCGGATGYTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTCTGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAACGCCCCCAGCGTGCTGTCTCGGAACGACCCCTGCGCGCGGCCTCGCGCCGCGCTTCCAACGCGACCCC。

[0070] >The nucleotide sequence of YDS_YS is shown in SEQ ID No. 27: ACGACCCGCGAACACGTTGTTAAATTGGGGGGCGAGGGGTCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACTGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGAAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCAAACCCCGTCGGGGAAAAAGGGGGGCGGATGYTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTCTGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAACGCCCCCAGCGTGCTGTCTCGGAACGACCCCTGCGCGCGGCCTCGCGCCGCGCTTCCAACGCGACCCC。

[0071] >The nucleotide sequence of HJX_XSY is shown in SEQ ID No. 28: ACGACCCGCGAACACGTTGTTAAATYGGGGGGCGAGGGGTCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACTGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGAAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCAAACCCCGTCGGGGAAGAAGGGGGGCGGATGCTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTCTGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAACGCCCCCAGCGTGCTGTCTCGGAACGACCCCTGCGCGCGGCCTCGCGCCGCGCTTCCAACGCGACCCC。

[0072] >The nucleotide sequence of NZY_LY is shown in SEQ ID No. 29: ACGACCCGCGAACACGTTGTTAAATCGGGGGGCGAGGGRCCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACTGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGAAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAARAACGTAGCGAAATGCGATACTTGGTGTGAATTGCARAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCCAAACCCCGTCGGGGGAGAAGGGGGGCGGATGCTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTCTGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAGCGCCCCCAGCGTGCCGTCTCGGAACGACCCCTGCGCGCGGCCTCAAAACCGCGCTTCCAACGCGACCCC。

[0073] >The nucleotide sequence of BZY_LY is shown in SEQ ID No. 30: ACGACCCGCGAACACGTTGTTAAATYGGGGGGCGAGGGGTCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACTGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGAAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCAAACCCCGTCGGGGAAGAAGGGGGGCGGATGCTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTCTGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAACGCCCCCAGCGTGCTGTCTCGGAACGACCCCTGCGCGCGGCCTCGCGCCGCGCTTCCAACGCGACCCC。

[0074] 6. Genetic distance analysis The K2P genetic distance was calculated based on MEGA v11.0.13. The results showed (Table 2) that the intraspecific genetic distance of Ulmus elongata samples was 0.0006 - 0.0102, and the minimum interspecific genetic distances between Ulmus elongata and Ulmus pumila, Ulmus bergmanniana, and Ulmus parvifolia were 0.0119, 0.0144, and 0.0189, respectively. The intraspecific genetic distance of all Ulmus elongata samples was less than the minimum interspecific distance from other species. The above results indicate that the selected sequences meet the DNA barcode screening criteria and can provide a reliable basis for the identification of Ulmus elongata.

[0075] Table 2 Intraspecific and interspecific K2P distances of specific DNA barcode sequences 7. Phylogenetic analysis The maximum likelihood (ML) phylogenetic tree was constructed based on IQ-TREE v 2.2.0.3, with the bootstrap repeat value set to 1000. Figure 5The results showed that the phylogenetic tree constructed using the combined fragment of rps16-trnQ-UUG+trnS-GCU-trnG-GCC+ITS could clearly separate Ulmus elongata from its related species, demonstrating that this sequence has significant discriminatory ability for Ulmus elongata.

[0076] 8. Identification of unknown samples When it is necessary to identify whether an unknown sample is Ulmus elongata, the following steps can be taken: extract the total DNA of the unknown sample, amplify and sequence it using the specific primers for Ulmus elongata provided by the present invention to obtain its rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS sequence fragments, and align and trim them with the reference sequences of SEQ ID No.7~SEQ ID No.14, SEQ ID No.15~SEQ ID No.22, and SEQ ID No.23~SEQ ID No.30 respectively. After trimming the results and splicing them in the order of rps16-trnQ-UUG+trnS-GCU-trnG-GCC+ITS, calculate the K2P genetic distance and construct a phylogenetic tree. If the genetic distance between the unknown sample and all individuals of Ulmus elongata is less than its genetic distance from other species and clusters with the individuals of Ulmus elongata in the phylogenetic tree, it is determined that the unknown sample is Ulmus elongata; otherwise, it is not.

[0077] In summary, in view of the limitations of traditional species identification methods, the present invention constructs an automated and standardized identification system through DNA barcoding molecular marker technology, significantly reducing the dependence on subjective experience judgment, and can quickly and accurately identify samples such as fresh leaves, dried leaves, and specimens of Ulmus elongata. In addition, the present invention can stably and accurately distinguish Ulmus elongata from its related species at a higher resolution, providing a more refined species identification ability. Finally, the present invention uses DNA barcoding molecular marker technology, which has important significance in the classification, protection, and utilization of germplasm resources of the genus Ulmus.

[0078] The above is only the preferred embodiment of the present invention and does not limit the present invention. Those skilled in the art can make appropriate adjustments to the implementation plan according to specific needs. Any modification, replacement, or improvement within the scope of the technical solution of the present invention belongs to the protection scope of the present invention. Therefore, the protection scope of the present invention should include all reasonable changes.

Claims

1. A DNA barcode sequence for identification of Ulmus pumila, characterized in that: The DNA barcode sequence is composed of three fragments, rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS, which are sequentially connected in series.

2. A primer for amplifying the DNA barcode sequence according to claim 1, characterized in that: The primers include a primer pair for amplifying rps16-trnQ-UUG, a primer pair for amplifying trnS-GCU-trnG-GCC, and a primer pair for amplifying ITS; The nucleotide sequence of the forward primer of the primer pair used to amplify rps16-trnQ-UUG is shown in SEQ ID No. 1, and the nucleotide sequence of the reverse primer of the primer pair used to amplify rps16-trnQ-UUG is shown in SEQ ID No. 2; The nucleotide sequence of the forward primer of the primer pair used for amplifying trnS-GCU-trnG-GCC is shown in SEQ ID No. 3, and the nucleotide sequence of the reverse primer of the primer pair used for amplifying trnS-GCU-trnG-GCC is shown in SEQ ID No. 4; The nucleotide sequence of the forward primer of the primer pair for amplifying ITS is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer of the primer pair for amplifying ITS is shown as SEQ ID No.

6.

3. A reagent for identifying Ulmus longituba, characterized in that: The reagents include the primers according to claim 2.

4. Use of the DNA barcode sequence according to claim 1, the primer according to claim 2 or the reagent according to claim 3 in the identification of Ulmus pumila.

5. A method for identifying Ulmus longissima using the primers described in claim 2, characterized in that: The steps include: Extracting total DNA from the Ulmus plant samples to be tested; Using the total DNA of the Ulmus genus plant sample to be tested as a template, PCR amplification is performed using the primers to obtain amplified products of three fragments of rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS of the Ulmus genus plant sample to be tested; Performing gel electrophoresis analysis on the amplified products, and selecting the amplified products showing a single band for bidirectional sequencing; The bidirectional sequencing results of the amplified products were imported into the software and spliced ​​into one sequence to obtain the sequences of the three fragments of rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS of the complete Ulmus plant sample to be tested; The sequences of the three fragments rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS of the Ulmus plant sample to be tested are aligned with the nucleotide sequences of the corresponding target fragments of the known species in the sequence list; The alignment results were spliced ​​in the order of rps16-trnQ-UUG+trnS-GCU-trnG-GCC+ITS, the genetic distance was calculated, and the phylogenetic tree was constructed; According to the genetic distance result and the branch position of the Ulmus genus plant sample to be tested in the phylogenetic tree, it is determined whether the Ulmus genus plant sample to be tested is Ulmus longicornis.

6. The method according to claim 5, characterized in that If the genetic distance between the Ulmus plant sample to be tested and all individuals of Ulmus longiflorum is smaller than the genetic distance between it and other species, and it is clustered into a branch with the individuals of Ulmus longiflorum in the phylogenetic tree, then the Ulmus plant sample to be tested is Ulmus longiflorum.

7. The method according to claim 5, characterized in that The amplification program of the PCR amplification is: Pre-denaturation at 95 °C for 3 min; Denaturation at 95 °C for 15 s, annealing at 50 °C-56 °C for 15 s, and amplification at 72 °C for 15 s were repeated for 35 cycles; The final amplification was performed at 72°C for 5 min and the amplified fragments were stored at 4°C.

8. The method according to claim 5, characterized in that The Ulmus plant sample to be tested includes any one of fresh leaves, dried leaves and specimens.

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