DNA barcoding sequences, primers and application and identification method for ulmus elongata identification

Abstract

The application belongs to the technical field of plant species identification, and particularly relates to a DNA barcode sequence for Ulmus prunifolia identification, primers and an application and identification method thereof. The DNA barcode sequence is composed of rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS three fragments in turn. The DNA barcode sequence has good primer universality and rich site variation, is easy to operate, can stably and accurately identify Ulmus prunifolia, and can obviously distinguish Ulmus prunifolia from its closely related species distributed in the same region. In addition, the application has important significance in the researches of Ulmus plant germplasm resource classification and evolution, protection and utilization by using the DNA barcode molecular marker technology.

Description

Technical Field

[0001] This invention belongs to the field of plant species identification technology, specifically relating to DNA barcode sequences, primers, their applications, and identification methods for identifying *Ulmus longiflora*. Background Technology

[0002] Elm with long-lasting leaves ( Ulmus elongata It belongs to the genus Ulmus in the family Ulmaceae. Ulmus The plant is a deciduous tree, reaching up to 30 meters in height; the bark is grayish-white, peeling off in irregular flakes; the leaves are elliptic or lanceolate-elliptic, 7-19 cm long and 3-8 cm wide, with a slightly oblique or nearly symmetrical base, and large, deep, double serrations along the margins; the petioles are 3-11 mm long; the stipules are mostly lanceolate to narrowly lanceolate, 7-18 mm long, and often fall off early. Flowers bloom in spring in racemose cymes, with a distinctly elongated, drooping inflorescence axis, and pedicels several times the length of the perianth. The inflorescence axis is 4-8 cm long and sparsely hairy; the samara is narrow and long, tapering to a point at both ends, resembling a spindle shape, 2-2.5 cm long and about 3 mm wide, sparsely hairy on both sides, with densely white, long cilia along the margins; the nut is located slightly upwards in the middle of the samara.

[0003] *Ulmus longiflorus* is a Tertiary relict plant endemic to my country, with fewer than 1,500 mature trees remaining in the wild. It has been listed as a Class II National Key Protected Plant and a wild plant with extremely small populations in China. Due to overlapping geographical distributions with closely related species such as *Ulmus spp.*, *Ulmus xingshanensis*, and *Ulmus parvifolia*, and because their morphological characteristics are easily influenced by environmental factors, traditional morphological identification methods struggle to accurately distinguish it from its sympatric relatives during non-flowering or non-fruiting periods. Furthermore, traditional methods often rely on the characteristics of flowers and fruits, which present significant difficulties in identifying incomplete samples such as wood, seeds, seedlings, or plant fragments, resulting in low identification efficiency and a high rate of misidentification. These limitations in identification techniques lead to frequent accidental felling, accelerating the rapid decline of the wild population of *Ulmus longiflorus*. In recent years, continuous human disturbance, habitat destruction, and global climate change have placed *Ulmus longiflorus* at constant risk of extinction, severely restricting its protection and management. Effective rescue and protection efforts for *Ulmus longiflorus* are urgently needed.

[0004] DNA barcoding technology, a method for species identification based on standardized short DNA fragments with high variability and easy amplification, has been widely used in the field of plant species identification. However, when using internationally accepted DNA barcoding, the DNA sequences of *Ulmus longiflora* are highly similar to those of its closely related species *Ulmus xingshanensis*, *Ulmus parvifolia*, and *Ulmus parvifolia*, which are distributed in the same region, and the interspecific genetic differences are small, making it difficult to distinguish them. Therefore, developing a DNA barcode that can accurately identify *Ulmus longiflora* is of significant scientific importance. Summary of the Invention

[0005] The purpose of this invention is to provide a novel DNA barcode capable of accurately identifying *Ulmus longiflorus*, specifically relating to a DNA barcode sequence, primers, and their application and identification method for the identification of *Ulmus longiflorus*. To achieve the above objective, this invention adopts the following technical solution.

[0006] The first objective of this invention is to provide a DNA barcode sequence for the identification of long-sequence elm. The DNA barcode sequence is composed of three fragments: rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS, sequentially tandemly spliced ​​together.

[0007] This invention is the first to develop a specific DNA barcode sequence applicable to *Ulmus longiflora* and its closely 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 longiflora* was constructed. This sequence can effectively determine whether a sample is *Ulmus longiflora*, solving the problem of difficulty in distinguishing *Ulmus longiflora* from its closely related species such as *Ulmus xingshanensis*, *Ulmus parvifolia*, and *Ulmus parvifolia*, which share a similar DNA sequence and exhibit small interspecific genetic differences, using internationally accepted DNA barcodes.

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

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

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

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

[0012] A third object of the present invention is to provide a reagent for identifying *Ulmus longiflorus*. The reagent comprises the primers described above.

[0013] A fourth objective of this invention is to provide the application of the DNA barcode sequence, primers, or reagents in the identification of long-sequence elm.

[0014] The fifth objective of this invention is to provide a method for identifying long-articulated elm using the aforementioned primers, specifically implemented according to the following steps: (1) Total DNA was extracted from the elm plant samples to be tested using the modified CTAB method.

[0015] (2) Using the total DNA of the elm plant sample to be tested as a template, PCR amplification was performed using the primers to obtain the amplification products of the three fragments 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 amplification products with a single band for bidirectional sequencing.

[0017] (4) The bidirectional sequencing results of the three fragments rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS of the elm plant sample to be tested were imported into the software and spliced ​​into a single sequence to obtain the complete three sequences of the elm plant sample rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS.

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

[0019] (6) Import the alignment results of each fragment obtained in step (5) into the 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 the software, calculate the K2P genetic distance, and construct a phylogenetic tree based on the maximum likelihood method. Determine whether the elm plant sample to be tested is a long-ordered elm based on the genetic distance results and the branch position in the phylogenetic tree.

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

[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; ddH2O added to 40 μL; the PCR amplification program is as follows: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 50 ℃~56 ℃ annealing for 15 s, 72 ℃ amplification for 15 s, for a total of 35 cycles; 72 ℃ final amplification for 5 min, and storage at 4 ℃.

[0023] Preferably, the elm plant sample 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. This invention is the first to develop a specific DNA barcode sequence applicable to *Ulmus longiflora* and its closely related species. By combining the rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS fragments, a precise and rapid DNA barcode identification technology system for *Ulmus longiflora* is constructed. This sequence can effectively determine whether a sample to be tested is *Ulmus longiflora*, solving the problem that internationally used DNA barcodes are difficult to distinguish between *Ulmus longiflora* and its closely related species *Ulmus xingshanensis*, *Ulmus parvifolia*, and *Ulmus parvifolia* due to the high similarity of their DNA sequences and the small interspecific genetic differences.

[0025] 2. This invention also provides primers for amplifying the aforementioned DNA barcode sequences and their applications. Experimental verification shows that the primers have the characteristics of clear amplified fragments, high resolution, and excellent sensitivity, enabling efficient, low-cost, and convenient identification of long sequences.

[0026] 3. This invention establishes a standard gene database covering rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS fragments by collecting multiple samples of identified species such as *Ulmus longiflora*, *Ulmus sibiricum*, *Ulmus xingshanensis*, and *Ulmus parvifolia*, providing a reliable reference for subsequent species identification. Attached Figure Description

[0027] Figure 1This is a gel electrophoresis image of total genomic DNA from eight samples in this 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 a gel electrophoresis image of primer amplification of ITS sequences from eight samples in this 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 a gel electrophoresis image of primer amplification of the rps16-trnQ-UUG sequence of 8 samples in this 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 a gel electrophoresis image of primer amplification of the trnS-GCU-trnG-GCC sequences of 8 samples in this 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 in this invention based on the combined fragments of rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Any modifications made based on the present invention are within the scope of protection 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 and reagents used are commercially available.

[0033] Example 1: Development of DNA barcode sequences specific to *Ulmus longiflora* and its closely related species The complete chloroplast genome sequences of *Ulmus longiflorus*, *Ulmus xingshanensis*, *Ulmus pumila*, and *Ulmus parvifolia* were obtained from GenBank (GenBank accession numbers: *Ulmus longiflorus*: MT165929.1, *Ulmus xingshanensis*: MT165921.1, *Ulmus pumila*: MT165942.1, *Ulmus parvifolia*: MT165940.1). Global alignment was performed using MAFFT v7.308, and nucleotide polymorphism (Pi) was analyzed using DnaSP v6.12.03. Based on the alignment variation sites and Pi values, hypervariable regions of the chloroplast genome were screened. Combined with chloroplast genome annotation information, two chloroplast DNA fragments with significant interspecific differences between *Ulmus longiflorus* and closely related species were finally selected: rps16-trnQ-UUG and trnS-GCU-trnG-GCC. Simultaneously, the ribosomal DNA transcription spacer region (ITS) was selected, as this region has good primer universality, a moderate number of sequences, and high variability, providing abundant specific sites.

[0034] Example 2: Primer design for specific DNA barcode sequences of *Ulmus longiflora* and its closely related species Based on the rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS fragment sequence information screened in Example 1, specific PCR primers for identifying long-ordered elm were designed. The PCR primer sequences are as follows: The primer pair used for amplifying rps16-trnQ-UUG is as follows: The nucleotide sequence of the forward primer rps16-trnQ-UUG-F, used to amplify rps16-trnQ-UUG, is shown in SEQ ID No. 1: 5′-TCCTTTTATCTTTGCCTTGA-3′.

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

[0036] The primer pair used for amplifying trnS-GCU-trnG-GCC is as follows: The nucleotide sequence of the forward primer trnS-GCU-trnG-GCC-F, used to amplify 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, used to amplify trnS-GCU-trnG-GCC, is shown in SEQ ID No. 4: 5′-TCGTTGAAACAAAAGAGGCCC-3′.

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

[0039] The nucleotide sequence of the reverse primer ITS-R, used to amplify ITS, is shown in SEQ ID No. 6: 5′-TCCTCCGCTTATTGATATGC-3′.

[0040] Example 3: Evaluation of the validity and feasibility of DNA barcode sequences 1. Collection of samples of *Ulmus longiflora* and its closely related species Plant samples of *Ulmus longiflora*, *Ulmus sibiricum*, *Ulmus xingshanensis*, and *Ulmus parvifolia* were collected from the wild and taken from plant leaves. Sample information is shown in Table 1.

[0041] Table 1 Sample Information 2. Sample preprocessing Weigh out appropriate amounts of each sample, wipe the surface with 75% ethanol to remove mud and impurities, grind into fine powder using a sample rapid grinder, and take 40 mg of the fine powder for later use.

[0042] 3. Extraction of genomic DNA from the sample Total genomic DNA was extracted from each sample using a modified CTAB method and analyzed by 1% agarose gel electrophoresis (e.g., Figure 1 (As shown) and the Nanodrop micro-volume spectrophotometer were used to detect DNA quality and concentration.

[0043] 4. Amplification, purification, and sequencing of the target fragment Using the extracted DNA as a template, the target fragment was amplified by PCR using the primers shown in SEQ ID No. 1 to 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, and 2 μL of DNA template; 20 μL of 2× Rapid Taq Master Mix PCR premix; and dd H2O to bring the total to 40 μL.

[0045] The 2 × Rapid Taq Master Mix PCR premix contains Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancers and optimizers, and stabilizers, at a concentration of 2 ×.

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

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

[0048] 5. Sequencing results assembly, alignment, and tandem assembly The sequencing results peak quality was checked using BioEdit v7.7.1, low-quality sequences at the beginning and end were removed, bases were manually proofread, and sequence assembly was completed. The corrected rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS sequences for each sample were finally obtained, as shown in SEQ ID No. 7~SEQ ID No. 30 of the sequence listing. Multiple sequence alignment of the rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS sequences was performed using MAFFT v7.308. The aligned sequences were then concatenated and assembled in the order of rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS using PhyloSuite v1.2.3 to construct a joint dataset.

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

[0050] The nucleotide sequence of YJF_CXY is 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 as SEQ ID No. 10: GAATCATTGGGTTTWGACMTTAMTTYGGTGATTTTTAATCGTTTCAAAATGGCAGCAACATACCATTTTTGTGATTTCTTTCCATCAAATAATCATATAAATGGTTGATTCTTGTTTAATACACTTTTAATTTGATCAAAAGAGTTTTACCAATTCAAAAAAAAAAAACTTTGGATTTGAAACTTGCTTGAATTGGATCTTTTCGATTTATATATCGAAAATAGACTTACAAAGTTGTCCCAATTTATTGATTGATACTAACCCTAGATTCTTGCCCCCGAGAAATGAATCAATACTTTCTGCTCGAGCTCCATCGTGTACAGTAAATTTATATCAAACCACAATACCCCCTCAAAAAAATGAGAGGTCTAGTGGAAAAGAACAAATGATGTCGAGTCAAGAGCACTTTCATTCCTATATAATTACTATATTATATAGTATATAATGGTGGATGTAAGACTCCACAACGGATCGTGTCCTTCAAGTCGCACGTTGCTTTCTACCACATCGTTTTAAACGAAGTTTTACCATAACATTCCTTTAGTTTGTAACCCGTATCTAATTGATTCCATCATGGAATTATGAATAGTCATTGGTTCGGTTGGTACTGGTACATAGTAATCTATACTTTATCTATACTTTTTTATCTATACTTTATTCATTTTATCTATACTTTATTCATTCAATATAAAAATAAAAAAAAAAT.

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

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

[0056] >The nucleotide sequence of NZX_LY is shown as 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~SEQ ID No.22: >The nucleotide sequence of YJF_CXY is shown in SEQ ID No.15: CGGAAAGAGRGGGATTCGAACCCCCGGTACAAAAAACTCGTACAACGGATTAGCAATCCGACGCTTTAGTCCMCTCMGCCMTCTCTCCCCCGATTGAAAAAAGAAAAKGACTATGTTACATTAGTAAACAAACATAAAACTTGAAAAAGCTCTTTTCCCTTCTTAATTTTTATTTTATTCMTCTTTCTTTTCTATTTTAAATTTTAATAGAATTTTTATAATTAACTATAATATAAATTATTAACATATAAATGTTAAATATAGTTTATAGTTTTTTATAATATAAAAGKGAAATAAACAATAAAAATAAAATAATATTAAATTTAAATTCTTTTTATTATTTATTAAATTCTTTTTAATTATTTATTTAATTATAATTATATATATATATATATATTTAATATTATATATAAATATTATATATATTTAATTAATTTTGAATTTAATTATATTAAATTTATATTTCATTTTTAATTTGAATATTATTTTAATATAATATTCAAAATATATATAAATAAAAATGAAAAATGAAATBTTTTTTTTTTTTATTTCATCCAAAGAAACCTTTTATT。

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

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

[0061] The nucleotide sequence of BZY_YS is shown as 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 as SEQ ID No. 20: CGGAAAGAGAGGGATTCGAACCCTCGGTACAAAAAACTCGTACAACGGATTAGCAATCCGACGCTTTAGTCCMCTCAGCCHTCTCTCCCCCGATTGAAAAAAGAAAAKGACTATGTTACATTAGTAAACAAACATAAAACTTGAAAAAAGCTCTTTTCCCTTCTTAATTTTGATTTTATTCATCTTTCTTTTCTATTTTTAAWAGAATTTTTTTTATAATTAAAWATAATTAAATTTATATAATTAAATATAAWATAAATTATTAACATATAAATGTTAAATATAGTTTTTTAWAAWATAAAAGKGAAATAAAAAATAAACAATAAAAAATAAACAATAAAATAATATTAAATTGAAATTCTTTTTATTAAATATTAAATTCTTTTTAATTATTTATTTATTATTTAATTTGAATTAATTTATTTATTATTTCATTTGAATTATATATAATTATAATTATAWATAATTCAATTTTGAATTATATATATTATATATATTTAAAWATATTTAAATTGAATATAATATTCAAAATAWATATAAATAAAAATATAAATAAAATCAAAATCTTTTTTTTTTTTTTATTTCATCCAAAGAAACCTTTTATT。

[0064] >The nucleotide sequence of NZX_LY is shown as 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 as SEQ ID No. 24: ACGACCCGCGAACACGTTGTTAAACCGGGGGGCGAGGGACCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACCGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGGAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGAATTGCARAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCCAAACCCCGTCGGGGCMWAAGGGTGGGGCGGATGCTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTACGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAACGCTCCCGGCGTGCCGTCTCGGAACGACCCCTGCGCGCGGCCTCGCGCCGCGCTTCCAACGCGACCCC。

[0068] >The nucleotide sequence of BZY_CXY is shown as 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 NZX_LY is shown as SEQ ID No. 29: ACGACCCGCGAACACGTTGTTAAATCGGGGGGCGAGGGRCCTCCGGGCCCCGACCCTCCCCCGGCGCTGGCCGCGGCACTGCCGTGCGCCAGCGTCAAACGAACCCCGGCGCTATCTGCGCCAAGGAAACCAAACGAACGAGCGCGCGACGGTCGGCCCGGAAACGGTGCCGTCGGAGCTGCGTCGTCTTCGATATGTCAAAACGACTCTCGGCAACGGATATCTCGGCTCTCGCATCGATGAARAACGTAGCGAAATGCGATACTTGGTGTGAATTGCARAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATCCGGCCGAGGGCACGTCTGCCTGGGCGTCACACACCGTTGCCCCCCCCAAACCCCGTCGGGGGAGAAGGGGGGCGGATGCTGGCCTCCCGTGAGCCTCGCCTCGCGGCTGGCCCAAATGCGAGATCTCTGCTGCGAGCGTCGCGGCGATGGTGGTTGTCGAATAACTCGGTGCCCCGTCGCGAGCGCCCCCAGCGTGCCGTCTCGGAACGACCCCTGCGCGCGGCCTCAAAACCGCGCTTCCAACGCGACCCC。

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

[0074] 6. Genetic distance analysis K2P genetic distances were calculated using MEGA v11.0.13, and the results (Table 2) show that the intraspecific genetic distances of *Ulmus longiflorus* samples ranged from 0.0006 to 0.0102. The minimum interspecific genetic distances between *Ulmus longiflorus* and *Ulmus santalinus*, *Ulmus xingshanensis*, and *Ulmus parvifolia* were 0.0119, 0.0144, and 0.0189, respectively. The intraspecific genetic distances of all *Ulmus longiflorus* samples were less than their minimum interspecific distances with other species. These results indicate that the selected sequences meet the DNA barcoding screening criteria and can provide a reliable basis for the identification of *Ulmus longiflorus*.

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

[0076] 8. Identification of unknown samples When it is necessary to identify whether an unknown sample is *Ulmus longiflorus*, the following steps can be followed: Extract total DNA from the unknown sample, amplify and sequence it using the *Ulmus longiflorus*-specific primers provided in this invention, obtain its rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS sequence fragments, and align them with the reference sequences 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 assembling the aligned results in the order 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 longiflorus* is less than its genetic distance from other species, and it clusters with *Ulmus longiflorus* individuals in the phylogenetic tree, then the unknown sample is determined to be *Ulmus longiflorus*; otherwise, it is not.

[0077] In summary, this invention addresses the limitations of traditional species identification methods by constructing an automated and standardized identification system using DNA barcoding molecular marker technology. This significantly reduces reliance on subjective experience and enables rapid and accurate identification of fresh leaves, dried leaves, and specimens of *Ulmus longiflora*. Furthermore, this invention can stably and accurately distinguish *Ulmus longiflora* from its closely related species at higher resolution, providing more refined species identification capabilities. Finally, the use of DNA barcoding molecular marker technology in this invention has significant implications for the classification, conservation, and utilization of *Ulmus* germplasm resources.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Those skilled in the art can make appropriate adjustments to the implementation scheme according to specific needs. Any modifications, substitutions, or improvements within the scope of the technical solution of the present invention are within the protection scope of the present invention. Therefore, the protection scope of the present invention should include all reasonable changes.

Claims

1. Primers for amplifying DNA barcode sequences for the identification of long-sequence elm, characterized in that, The DNA barcode sequence is composed of three fragments, rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS, which are sequentially tandemly spliced ​​together. The primers include primer pairs for amplifying rps16-trnQ-UUG, primer pairs for amplifying trnS-GCU-trnG-GCC, and primer pairs 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 to amplify trnS-GCU-trnG-GCC is shown in SEQ ID No. 3, and the nucleotide sequence of the reverse primer of the primer pair used to amplify trnS-GCU-trnG-GCC is shown in SEQ ID No.

4. The nucleotide sequence of the forward primer of the primer pair used to amplify ITS is shown in SEQ ID No. 5, and the nucleotide sequence of the reverse primer of the primer pair used to amplify ITS is shown in SEQ ID No.

6.

2. A reagent for identifying *Ulmus longiflorus*, characterized in that, The reagent includes the primers as described in claim 1.

3. The application of the primers of claim 1 or the reagents of claim 2 in the identification of long-ordered elm.

4. A method for identifying *Ulmus longiflora* using the primers described in claim 1, characterized in that, Includes the following steps: Total DNA was extracted from the samples of the *Ulmus* species to be tested. Using the total DNA of the elm plant sample to be tested as a template, PCR amplification was performed using the primers to obtain the amplification products of three fragments: rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS from the elm plant sample to be tested. The amplification products were analyzed by gel electrophoresis, and the amplification products showing a single band were selected for bidirectional sequencing. The bidirectional sequencing results of the amplified products were imported into software and assembled into a single sequence to obtain the sequences of three fragments: rps16-trnQ-UUG, trnS-GCU-trnG-GCC, and ITS of the complete sample of the elm species to be tested. The sequences of the three fragments rps16-trnQ-UUG, trnS-GCU-trnG-GCC and ITS from the elm plant samples to be tested were compared and cut with the nucleotide sequences of the corresponding target fragments of known species in the sequence listing. After concatenating the alignment results in the order rps16-trnQ-UUG+trnS-GCU-trnG-GCC+ITS, the genetic distance was calculated and a phylogenetic tree was constructed. Based on the genetic distance results and the branch position of the elm plant sample to be tested in the phylogenetic tree, it is determined whether the elm plant sample to be tested is *Ulmus longiflorus*.

5. The method according to claim 4, characterized in that, If the genetic distance between the elm plant sample to be tested and all individuals of *Ulmus longiflorus* is less than the genetic distance between it and other species, and it clusters with *Ulmus longiflorus* individuals in the phylogenetic tree, then the elm plant sample to be tested is *Ulmus longiflorus*.

6. The method according to claim 4, characterized in that, The PCR amplification procedure is as follows: Pre-denaturation at 95 ℃ for 3 min; The process involved denaturation at 95 °C for 15 s, annealing at 50 °C–56 °C for 15 s, amplification at 72 °C for 15 s, and repeating for 35 cycles. Final amplification was performed at 72 °C for 5 min, followed by storage at 4 °C.

7. The method according to claim 4, characterized in that, The elm plant samples to be tested include any one of fresh leaves, dried leaves, and specimens.

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