Specific primer for identifying sibirica and myriophylla and identification method

Abstract

This invention belongs to the field of ecological restoration and aquatic plant germplasm identification technology. It provides specific primers and identification methods for *Myriophyllum sieboldii* and *Myriophyllum sieboldii*, based on microsatellite marker (SSR) technology. Ten pairs of highly polymorphic microsatellite primers were screened and used to form five sets of dual PCR primer combinations for amplifying DNA fragments of the target species. Using confirmed *Myriophyllum sieboldii* and *Myriophyllum sieboldii* as basic reference samples, a genetic clustering model was constructed. DNA extraction, PCR amplification, electrophoresis analysis, and UPGMA clustering analysis were performed on the individuals to be identified, thereby achieving accurate species identification of suspected individuals. This invention enables rapid, accurate, and low-cost germplasm identification during the plant's vegetative growth stage and is applicable to wetland ecological restoration, germplasm resource protection, and aquatic vegetation surveys.

Description

Technical Field

[0001] This invention belongs to the field of ecological restoration, specifically involving a specific primer and identification method for identifying Siberian Myriophyllum and Myriophyllum verticillatum; used to accurately distinguish between Siberian Myriophyllum (M. sibiricum Kom.) and Myriophyllum verticillatum L. Background Technology

[0002] *Myriophyllum* L. is a genus of perennial aquatic herbs belonging to the family Haloragaaceae, with approximately 45 species worldwide, including 5 species and 1 variety recorded in my country. The morphology of vegetative organs in this genus is highly homogeneous, and common characteristics such as pinnately lobed leaves, whorled leaf arrangement, and spike-like inflorescences pose significant challenges to classical morphological identification at the species level. This is especially true in complexes (such as the *M. verticillatum*–*M. sibiricum* complex), where plants exhibit phenotypic plasticity with environmental gradients, further blurring interspecific boundaries. *M. sibiricum* Kom. and *M. verticillatum* L. are the two most common native *Myriophyllum* species used in freshwater ecological restoration, aquatic trade, and wetland monitoring in my country. During their vegetative growth stages, their macroscopic morphology is almost indistinguishable: quantitative traits such as internode length, leaf angle, and leaf color exhibit continuous variation and overlap. Currently, the only reliable diagnostic feature is the microscopic sculptural markings on the edge of mature fruit—M. sibiricum mericarps have 0.05–0.08 mm tubercles on the back, while M. verticillatum fruits are completely smooth. However, the flowering and fruiting periods of the *Myriophyllum* genus are short and not concentrated, and field surveys often only collect vegetative bodies without flowers or fruits. In addition, the fruits require scanning electron microscopy for observation, which is costly and time-consuming, seriously hindering aquatic vegetation surveys, germplasm resource protection, and purity testing of seedlings for ecological engineering.

[0003] With the popularization of DNA barcoding and population genetics technologies, species / population level identification using simple repetitive sequences (SSRs, also known as microsatellites) has become the international mainstream. SSRs have advantages such as codominance, high polymorphism, good reproducibility, and tolerance to trace amounts of DNA templates, and have been successfully applied to the identification and invasion monitoring of aquatic plants such as *Utricularia verticillatum*, *Utricularia sibiricum*, and *Utricularia sibiricum*. However, microsatellite resources in the genus *Myriophyllum* are extremely scarce: as of this application, no SSR markers specifically developed for *M. verticillatum* or *M. sibiricum* have been found in public databases (NCBI / EMBL); existing studies mostly rely on DNA barcoding such as ITS / trnL-F, but the genetic distance between species in the genus *Myriophyllum* is too small (<0.3%), and the barcode resolution is insufficient to reliably distinguish these two closely related species. Faced with the "blind spots" in morphological identification and the "resolution bottleneck" of DNA barcoding, there is an urgent need to develop a germplasm identification technology for *Myriophyllum* that is highly polymorphic, species-specific, and can achieve rapid, low-cost, and large-sample-volume detection at the vegetative stage. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention aims to provide specific primers for identifying *M. verticillatum* and *M. sibiricum*. For the first time, a set of microsatellite (SSR) core loci applicable to *M. verticillatum* and *M. sibiricum* has been isolated and validated. Through genetic distance clustering, accurate identification of the two *M. verticillatum* species can be achieved at the seedling stage or in any tissue fragment, providing reliable technical support for seed purity testing, invasive species monitoring, and new variety rights protection in aquatic ecological restoration.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A specific primer for identifying *Myriophyllum sibiricum* Kom. and *Myriophyllum verticillatum* L. was developed. This invention is the first to use primers in the related genus *Myriophyllum ursuri*. Myriophyllum propinquum A. Cunn Ten microsatellite molecular marker sites were screened from the genome, and their nucleotide sequences are shown in SEQ ID NO.21-SEQ ID NO.30. Based on these microsatellite molecular marker sites, corresponding specific primers were designed for the phylogenetic analysis of *Myriophyllum sibiricum* Kom. and *Myriophyllum verticillatum* L. Ten pairs of specific primers were used in five groups for duplex PCR. The primer information is shown in Table 1. Table 1. Primer pair information for microsatellite molecular marker sites Among them, primer pair ZZJD1 is used in combination with primer pair ZZJD2; primer pair ZZJD3 is used in combination with primer pair ZZJD4; primer pair ZZJD5 is used in combination with primer pair ZZJD6; primer pair ZZJD7 is used in combination with primer pair ZZJD8; and primer pair ZZJD9 is used in combination with primer pair ZZJD10.

[0006] Another technical objective of this invention is to provide a method for identifying *Myriophyllum sibiricum* Kom. and *Myriophyllum verticillatum* L., comprising the following steps: (1) Genomic DNA was extracted from *Myriophyllum sibiricum* Kom. and *Myriophyllum verticillatum* L. as the basic reference species; genomic DNA was also extracted from individual algae to be tested. (2) Using the three sets of genomic DNA obtained in step (1) as templates, perform double PCR amplification using the five sets of double PCR primers described in claim 2 to obtain three sets of amplification products; (3) The three groups of amplification products obtained in step (2) were subjected to electrophoresis and silver staining using polyacrylamide gel; (4) Analyze the silver staining results of step (3) using genetic analysis software, draw a UPGMA cluster analysis diagram, and identify the species of the individual to be identified based on the clustering results. If the individual to be identified is clustered into the Siberian Myriophyllum branch, then the individual to be identified is Siberian Myriophyllum; if the individual to be identified is clustered into the Myriophyllum branch, then the individual to be identified is Myriophyllum.

[0007] In step (2), the reaction system for duplex PCR amplification is 25 μL: 10×PCR Buffer 3 μL, 2.5 mmol / L dNTP 1 μL, 2 mmol / L MgCl2 3 μL, each of the upstream and downstream primers of the two pairs of primers 1 μL, 0.5 U / μL Taq enzyme 0.5 μL, DNA template 2 μL, and ultrapure water 11.5 μL.

[0008] The duplex PCR amplification reaction program in step (2) is as follows: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles; finally, extension at 72℃ for 10 minutes, and storage at 4℃.

[0009] In step (3), the PCR products were electrophoresed and silver-stained with 12% polyacrylamide gel.

[0010] The genetic analysis software mentioned in step (4) can be MEGA software.

[0011] Compared with the prior art, the advantages and beneficial effects of the present invention include: Breaking through the limitations of morphological identification: This invention overcomes the problem that Siberian Myriophyllum and Myriophyllum vegetans are highly similar in morphology during their vegetative growth period and are difficult to distinguish, and provides an objective and accurate identification method based on DNA molecular markers.

[0012] High efficiency, speed, and low cost: Compared with traditional identification methods that rely on the microscopic structure of mature fruits, this invention does not require waiting for the plant to flower and bear fruit, and can be used for identification at any growth stage, saving time and costs, and is suitable for large-scale sample screening.

[0013] High polymorphism and stability: The 10 microsatellite primer pairs selected are highly polymorphic, have stable amplification, and good repeatability. They can clearly distinguish between two closely related species, ensuring the reliability of the identification results.

[0014] Dual PCR system optimization: The dual PCR amplification system improves detection efficiency, reduces experimental steps and reagent consumption, and is suitable for high-throughput sample processing.

[0015] With broad application prospects, this invention is applicable to various aspects of wetland ecological restoration projects, such as seed purity testing, invasive species monitoring, germplasm resource management, and new variety rights protection. It has good promotional value and social and economic benefits. Attached Figure Description

[0016] Figure 1 This is the UPGMA cluster analysis diagram of Myriophyllum sp. in Example 1, where the numbers 1-20 correspond to samples 1-20 in Example 1. Figure 2 This is the UPGMA cluster analysis diagram of Myriophyllum spicatum in Example 2, where the numbers 1-26 correspond to samples 1-20 in Example 1 and samples 21-26 in Example 2, respectively. Figure 3 The image shows the UPGMA cluster analysis of Myriophyllum spicatum in Example 3. The numbers 1-20 correspond to samples 1-20 in Example 1, and 27-36 correspond to samples 27-36 in Example 3. Detailed Implementation

[0017] The applicant will now clearly and completely describe the technical solution of the present invention with reference to specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0018] The algae samples used in the following examples were all taken from the Shiyan Lake Wetland in Shenzhen; unless otherwise specified, the experimental methods used were conventional methods; and unless otherwise specified, the materials and reagents used were commercially available.

[0019] Example 1: Ten Siberian Myriophyllum strains identified as the base reference species (M. sibiricum Kom.) and ten myriophyllum sp. (M. verticillatum L.) Cluster analysis verification 1.1 Obtaining the genomic DNA of the basic reference species DNA was extracted from the leaves of ten identified *Myriophyllum sp.* strains (1-10) and ten strains (11-20) from Siberia. Genomic DNA was extracted using the Tiangen novel plant genomic DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing). The specific procedure is as follows: (1) Use tweezers to take about 100 mg of the dry weight of the 1-20 Myriophyllum sp. sample into a 2 mL centrifuge tube (to avoid cross-contamination, the tweezers used to take the plant tissue should be burned on an alcohol lamp each time). Add two grains of quartz sand and place the tube on a shaker to grind the tissue. Grind the tissue in both directions for 1 minute until it is powdery. After centrifuging for 30 seconds, add 500 μL of buffer LP1 and 6 uRNase A (10 mg / mL). Vortex for 1 minute (check if the tissue adheres to the wall; if it does, add another 100 μL of buffer LP1). Dry bath at 65°C for 10 minutes (300 rpm) and then place the tube in an ice box for 2 minutes.

[0020] (2) Add 150 μL of buffer LP2, vortex for 1 min to mix thoroughly, centrifuge at 12000 rpm for 5 min, and transfer the supernatant (150 μL each time, twice) to a new 2 mL centrifuge tube.

[0021] (3) Add 450 μL of buffer LP3 (1.5 times the volume of the supernatant) and vortex for 15 seconds.

[0022] (4) Place the adsorption column CB3 into the collection tube, and pour the contents of 2.3 into the adsorption column CB3. Centrifuge at 12000 rpm for 1 min in a centrifuge, and keep the adsorption column CB3 in the collection tube.

[0023] (5) Add 600 μL of washing buffer PW to the adsorption column CB3 retained in the collection tube of (4) to wash the DNA, centrifuge at 12000 rpm for 1 min, and retain the adsorption column CB3 in the collection tube.

[0024] (6) Repeat the rinsing process of (5) with rinsing solution PW (if the filter membrane of adsorption column CB3 is green, rinse once with 500 μL of anhydrous ethanol, and the rinsing process is the same as that of rinsing solution PW) until the filter membrane of adsorption column CB3 is colorless.

[0025] (7) After centrifuging the collection tube treated in (6) at 12000 rpm for 2 min, open the cap of the adsorption column CB3 and place it horizontally to air dry. It can be placed at room temperature for 30 min or in a 32℃ oven for 10 min to completely dry the residual rinsing solution in the adsorption column CB3. (The adsorption membrane should be slightly wrinkled and almost odorless when you get close to it is ideal.) (8) The genomic DNA of Myriophyllum sp. in the adsorption column CB3 was collected into centrifuge tubes using elution buffer (TE) (ensuring the pH value was in the range of 7.0-8.5) to obtain genomic DNA of 20 individuals.

[0026] Before amplification, the 20 Myriophyllum sp. genomic DNA samples obtained above need to be tested and preserved through the following steps: a. The success of extraction was determined by 1% agarose gel electrophoresis, and the concentration was determined by micro-spectrophotometer.

[0027] b. According to the experimental requirements, the genomic DNA of Myriophyllum spicatum was diluted 5 times as a working solution and stored in a refrigerator at 4°C for later use. The remaining mother solution was stored in a refrigerator at -20°C for long-term preservation.

[0028] 1.2 Obtaining microsatellite molecular marker sites and designing and synthesizing corresponding primers Microsatellite search software was used to explore the genomes of multiple species of the same genus and *Myriophyllum* published in NCBI. After preliminary screening, *Myriophyllum ursuri* (a species of the same genus) was selected. Myriophyllum propinquum A. Cunn The genome was used as a further screening target; subsequently, *Myriophyllum ursuri* (…) was selected as the target genome. Myriophyllum propinquum A.Cunn The genome (Genebank: GCA_044231635.1) was scanned to find microsatellite fragments, and 20 Myriophyllum ursulphureus genomic DNAs were obtained. For the fragments containing microsatellites, microsatellite molecular marker sequences with repeat units of more than 2 bases were selected as subsequent target fragments. The corresponding microsatellite primers were designed and sent to a primer company for synthesis, which were used for the amplification of microsatellite molecular marker sequences.

[0029] 1.3 Microsatellite molecular site primer screening Twenty *Myriophyllum ursuri* samples obtained from the above steps were selected. Myriophyllum propinquum A.Cunn Six genomic DNA sequences were randomly selected as templates to perform preliminary screening of the microsatellite primers synthesized in step 1.2, selecting microsatellite molecular marker sequences with good polymorphism, few deletions, and stable amplified bands.

[0030] The PCR amplification reaction system was 25 μL: 3 μL 10×PCR Buffer, 1 μL 2.5 mmol / L dNTP, 3 μL 2 mmol / L MgCl2, 1 μL each of forward and reverse primers, 0.5 μL 0.5 U / μL Taq enzyme, 2 μL DNA template, and 13.5 μL ultrapure water.

[0031] The PCR amplification reaction program was as follows: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles; finally, extension at 72℃ for 10 minutes, and storage at 4℃.

[0032] 1.4 Evaluation of microsatellite molecular markers and detection of the universality of primers for polymorphic sites Based on the selected microsatellite molecular markers with good polymorphism, few deletions, and stable amplified bands, 20 Myriophyllum genomic DNA samples were used for PCR amplification again, and the microsatellite molecular markers were evaluated.

[0033] The PCR amplification reaction system consisted of 25 μL: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL each of forward and reverse primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 13.5 μL of ultrapure water.

[0034] The PCR amplification reaction program was as follows: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles; finally, extension at 72℃ for 10 minutes, and storage at 4℃.

[0035] 1.5 Duplex PCR Amplification After amplifying the genomic DNA of 20 *Myriophyllum sibiricum* samples, microsatellite markers with good polymorphism, few deletions, and stability were still able to be amplified for further screening. The specific screening method was as follows: the screened microsatellite primers were randomly paired to form duplex PCR primer sets. The DNA of the 20 samples was amplified using a duplex PCR system. Ten samples were selected as duplex PCR systems that could stably amplify microsatellite markers with good polymorphism, few deletions, and stable amplified bands. Finally, 10 microsatellite markers were selected for identifying *Myriophyllum sibiricum* Kom. and *Myriophyllum verticillatum* L., as follows: The microsatellite molecular marker (SEQ ID NO.21) corresponding to primer pair ZZJD1 is: tggttgttcatcttaatacgaaatcattttagcttataatttcatcataagtttgaccactttgattttcttattatttaatatatatatatatatataactacttaaattcatgagaatatcctaaatcctaaacccttagttataTGTGTGT GTGTGTGttggcaatatttaattaaatgtacatatatagtgtttagattgaatttgaaataataaaaatacatatattcttagaattaatttgtaaattcacccttttaatttaaaaacttattactaatatagttgtaaatcaaattaataaagt The microsatellite molecular marker (SEQ ID NO.22) corresponding to primer pair ZZJD2 is: ctcacctatctagaggagatttatgacttaaatgaactaaatgtgaaacttctaacttaaatgtgcatgaatcatattttattttgttcattaaaaatcctgaatctcacatatctaagtgagactaaagcacataattatggccatcatTATATA TATATAcatatatacgtgtgtgtatataaagatacaatttccggcatgtaacatgttcaattctaactaaatccaatagtggggccactctcttagcggtagcgcctaaagctaaaattaaataacgtttatatcattatatggtttaggaaatga The microsatellite molecular marker (SEQ ID NO.23) corresponding to primer pair ZZJD3 is: aacgtaaaatgaaaaacgaaaacggaaaacgataatcgattaacggtaaaaattgtgtttggattgcttattcaaagcgaattataaaattaaaatgagttataattaaatttagctttataatatatttttttgggtaaaatgaaataATAATAATAATAATAA TAATAATAATAATAATAAtttttttactagaatttttttcgatctttcatataattaaacttattttgcgtacttatataaataattaacacaaattttaaaaggtattgataataaaatagtaatttaaattattttgtaaactcaatatgaagtttaagtattcaa The microsatellite molecular marker (SEQ ID NO.24) corresponding to primer pair ZZJD4 is: gatcttaatatatatatagagagagagagaagaagagaataaaatgtccattggtctcagtattctaaataggtgaaacttatggtctaaataaactaaatgtgatatgatttaaaggcacataacatgatcctcatataatgtaatataATATATA TATATATtctatcaaaattttctttttatttgttgataatatttgcaatttattcaatttcaagcaaaagtttgaaagtaaattttggtaaaagtgtggtctaaatttttgtacacccagttaggaaaaaagaaatagtggagatgaattgtgcac The microsatellite molecular marker (SEQ ID NO.25) corresponding to primer pair ZZJD5 is: atcgatgtattaaataaattatttttataatttattaatatttttaatatgtcaatataaatgtgtatcaattaagtcgcacgtagcgtatctggtgaaattataagaacagattattttgtattgattgtaaggtaaccaatgcactgaATATATAT ATATATATtcataactacaaatttactaacaattaacaacggatataaagaaaacatatacagcgtataaaagagattagtatggagcgcacaaaaaacgttaagttattgacttagaatcaatatatatcgcatcaggttgatgaaaagacattcat The microsatellite molecular marker (SEQ ID NO.26) corresponding to primer pair ZZJD6 is: acatcatatacacatgccgtattacactcgtatgtttcaataatacgttggccaatggtccagattaaagataatagataacttaaaaatgtcaaagtctaagatacaatgttaaaataaccaaagatagacctgaaaaaaagaaaaaaaAGAGAG AGAGAGttgattagtaatgctgcaactactaatatggaatgacaaatggcttttgaaatacgatgtgacaaatggcttctcaaaaatgttaaagtcttaaatgcaagcattgaatgtactaaacttatatttttatatttttaaacatcacttatt The microsatellite molecular marker (SEQ ID NO.27) corresponding to primer pair ZZJD7 is: cctttctaccttccttttgagctttatgtgaatacttattaccttcgatcctaaagtatgatcttatggagcatttattatacgatgatttggaaggattcaagacactaagtgtggggtggaagtgagaaggtatgaagagtgaatagtTATATATAT ATATATATAaaatcgtttatcaacaaactttgttgataaataagaaagaaataaaagattgctcttaagaatagccatcttgttgattgagaagtgagtgaaaactggcgaattaggaaagtggtgaatgaggtagcataaagagtaggatgaatgatg The microsatellite molecular marker (SEQ ID NO.28) corresponding to primer pair ZZJD8 is: ccattgttatattgaaattacagtttaaacatttagttttattaaattacagaacatatctcatactttggctaattacagatttgttccctaattttcagcatgattaattttgatcctaatcatattatattctgtattttagttttgggATATATATATATATATATATATATATATAT ATATATATATATATATATATATATATATATgcatgcattatgttatggtaattgtagaattacattaagattcacagtttatcatgctatttggatttatgcttgatattctatacttcaatgatgcatgcaaatatatttttatgcctaaagatatttttatgcctaaagatattgtctaagtgatctttatgattt The microsatellite molecular marker (SEQ ID NO.29) corresponding to primer pair ZZJD9 is: aaaaataaagacagatactgagcgaaaaaaagtgccaacaaatcttcacactgcagcatctgcattcaaacaattgtgggaaaaggagttgttgggaccgccgtgcgcagcgtcgtgtcccgcccgtacctatgcgtccctcatcgagctCGCGC GCGCGagcctgatgcaaaatttggacaagtttatacgacttgagtctacctcatctttacccaaaatcaattggcaatgggttgaggcaactcaagacatatgaacctaccaaatcttctctacctctccaatgtgggattttaactcatccact The microsatellite molecular marker (SEQ ID NO.30) corresponding to primer pair ZZJD10 is: attgaacaaaagagcatgttggggagctccctcaacctcgtgtttcaaggccttaacagccagtgcagaaataaggacaagtttatcttaatagtcaaaagacttatagaatcatcaatttctttgagcttcaaataagtcaaaagattacATATATA TATATATgtgaaagctctgatgataaagaaatgtgtgagtaaaaagtattaacgatatatggactgaaaatactaacggcgataaaaatcattaaattttttcttcatcttgtagaaaaatacaagcaaatgattctactgtgcactaagatgaaca The amplification primer pairs used for the above microsatellite molecular markers are shown in Table 1: Table 1. Primer pair information for microsatellite molecular marker sites The PCR amplification reaction system was 25 μL: 3 μL 10×PCR Buffer, 1 μL 2.5 mmol / L dNTP, 3 μL 2 mmol / L MgCl2, 1 μL each of the forward and reverse primers of the two primer pairs, 0.5 U / μL Taq enzyme, 2 μL DNA template, and 11.5 μL ultrapure water.

[0036] The PCR amplification reaction program was as follows: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles; finally, extension at 72℃ for 10 minutes, and storage at 4℃.

[0037] 1.6 Testing and Identification The PCR products of Myriophyllum sp. were separated by electrophoresis using a 12% non-denaturing polyacrylamide gel, and photographed after silver staining.

[0038] Ten optimal microsatellite marker primer pairs were selected. Primer pairs ZZJD1 and ZZJD2 were used together; ZZJD3 and ZZJD4 were used together; ZZJD5 and ZZJD6 were used together; ZZJD7 and ZZJD8 were used together; and ZZJD9 and ZZJD10 were used together. Primer information is shown in Table 1.

[0039] 1.7 Genetic Structure Analysis The silver staining results of the double PCR amplification were digitized (PCR products were read using Gene Marker software), and a UPGMA cluster analysis diagram was constructed using MEGA software. The results are shown below. Figure 1 As shown, these 20 *Myriophyllum sp.* strains divided into two branches: strains 1-10 clustered in one branch, and strains 11-20 clustered in another. The clustering analysis results are consistent with the sampling classification. This experiment confirms that the method described in this technique for identifying *Myriophyllum sp.* Siberianus and *Myriophyllum sp.* germplasm is accurate.

[0040] This invention demonstrates that the 10 pairs of microsatellite primers for Myriophyllum sp. provided by this invention can be used for germplasm identification of Myriophyllum sp. Siberianus and Myriophyllum sp., providing a new technical method and means for studying and identifying the relationship between Myriophyllum sp. Siberianus and Myriophyllum sp.

[0041] Example 2: Verifying the accuracy of identifying *Myriophyllum sieboldii* and *Myriophyllum sieboldii*. Three strains of Siberian Myriophyllum (21-23) and three strains of Myriophyllum (24-26) were taken, and the germplasm of these six plants were identified and verified using this technique.

[0042] 2.1 Genomic DNA extraction from samples to be identified The genomic DNA of these six plants was extracted using the Tiangen novel plant genomic DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing). The specific procedure is as follows: (1) Take about 100 mg of the dry weight of the 21-26 Myriophyllum sp. sample into a 2 mL centrifuge tube using tweezers (to avoid cross-contamination, the tweezers used to take the plant tissue should be burned on an alcohol lamp each time). Add two grains of quartz sand and place the tube on a shaker to grind the tissue. Grind the tissue in both directions for 1 minute until it is powdery. After centrifuging for 30 seconds, add 500 μL of buffer LP1 and 6 uRNase A (10 mg / mL). Vortex for 1 minute (check if the tissue adheres to the wall; if it does, add another 100 μL of buffer LP1). Dry bath at 65°C for 10 minutes (300 rpm) and then place the tube in an ice box for 2 minutes.

[0043] (2) Add 150 μL of buffer LP2, vortex for 1 min to mix thoroughly, centrifuge at 12000 rpm for 5 min, and transfer the supernatant (150 μL each time, twice) to a new 2 mL centrifuge tube.

[0044] (3) Add 450 μL of buffer LP3 (1.5 times the volume of the supernatant) and vortex for 15 seconds.

[0045] (4) Place the adsorption column CB3 into the collection tube, and pour the contents of (3) into the adsorption column CB3. Centrifuge at 12000 rpm for 1 min in a centrifuge, and keep the adsorption column CB3 in the collection tube.

[0046] (5) Add 600 μL of washing buffer PW to the adsorption column CB3 retained in the collection tube of (4) to wash the DNA, centrifuge at 12000 rpm for 1 min, and retain the adsorption column CB3 in the collection tube.

[0047] (6) Repeat the rinsing process of (5) with rinsing solution PW (if the filter membrane of adsorption column CB3 is green, rinse once with 500 μL of anhydrous ethanol, and the rinsing process is the same as that of rinsing solution PW) until the filter membrane of adsorption column CB3 is colorless.

[0048] (7) After centrifuging the collection tube treated in (6) at 12000 rpm for 2 min, open the cap of the adsorption column CB3 and place it horizontally to air dry. It can be placed at room temperature for 30 min or in a 32℃ oven for 10 min to completely dry the residual rinsing solution in the adsorption column CB3. (The adsorption membrane should be slightly wrinkled and almost odorless when you get close to it is ideal.) (8) The genomic DNA of Myriophyllum in the adsorption column CB3 was collected into centrifuge tubes using elution buffer (TE) (ensuring the pH value was in the range of 7.0-8.5) to obtain genomic DNA of 6 individuals.

[0049] Before amplification, the six Myriophyllum sp. genomic DNA samples obtained above need to be tested and preserved through the following steps: a. The success of extraction was determined by 1% agarose gel electrophoresis, and the concentration was determined by micro-spectrophotometer.

[0050] b. According to the experimental requirements, the genomic DNA of Myriophyllum spicatum was diluted 5 times as a working solution and stored in a refrigerator at 4°C for later use. The remaining mother solution was stored in a refrigerator at -20°C for long-term preservation.

[0051] 2.2 PCR product amplification reaction and electrophoresis detection The DNA of six suspected individuals and 10 reference *Myriophyllum siberianense* and 10 reference *Myriophyllum siberianense* strains were amplified using the five sets of dual PCR primers described in this patent. Gene analysis software was used to perform cluster analysis on the PCR amplification results and those of 20 basic reference species to determine whether the suspected individuals clustered into *Myriophyllum siberianense* or *Myriophyllum siberianense*. The species of the suspected individuals was determined based on the clustering results. If the individual to be identified clustered into the *Myriophyllum siberianense* branch, then the individual to be identified was *Myriophyllum siberianense*; if the individual to be identified clustered into the *Myriophyllum siberianense* branch, then the individual to be identified was *Myriophyllum siberianense*.

[0052] The amplification primer pairs used for the above microsatellite molecular markers are shown in Table 1.

[0053] The silver staining results were analyzed using genetic analysis software to generate UPGMA cluster analysis diagrams, and the species of the individuals to be identified were determined based on the clustering results.

[0054] The PCR amplification reaction system consisted of 25 μL: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL each of the forward and reverse primers of the two primer pairs, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 11.5 μL of ultrapure water.

[0055] The PCR amplification reaction program was as follows: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles; finally, extension at 72℃ for 10 minutes, and storage at 4℃.

[0056] The PCR products were subjected to electrophoresis and silver staining using 12% polyacrylamide gel.

[0057] The genetic analysis software mentioned can be MEGA software.

[0058] The results are as follows Figure 2As shown, these 26 *Myriophyllum sp.* strains were divided into two branches: strains 1-10 and 21-23 clustered into one branch; strains 11-20 and 24-26 clustered into another branch. The clustering analysis results are consistent with the sampling classification. This experiment confirms that the method described in this technique for identifying *Myriophyllum sp.* Siberiani and *Myriophyllum sp.* germplasm is accurate.

[0059] Example 3: Application of germplasm identification for Siberian myriocarpus and suspected individuals of myriocarpus. Ten individuals (27-36) of uncertain species, identified as either Siberian Myriophyllum or Myriophyllum, were selected for germplasm identification.

[0060] 3.1 Genomic DNA extraction from samples to be identified The genomic DNA of these 10 plants was extracted using the Tiangen novel plant genomic DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing). The specific procedure is as follows: (1) Take about 100 mg of the dry weight of the 27-36 Myriophyllum sp. sample into a 2 mL centrifuge tube using tweezers (to avoid cross-contamination, the tweezers used to take the plant tissue should be burned on an alcohol lamp each time). Add two grains of quartz sand and place the tube on a shaker to grind the tissue. Grind the tissue in both directions for 1 minute until it is powdery. After centrifuging for 30 seconds, add 500 μL of buffer LP1 and 6 uRNase A (10 mg / mL). Vortex for 1 minute (check if the tissue adheres to the wall; if it does, add another 100 μL of buffer LP1). Dry bath at 65°C for 10 minutes (300 rpm) and then place the tube in an ice box for 2 minutes.

[0061] (2) Add 150 μL of buffer LP2, vortex for 1 min to mix thoroughly, centrifuge at 12000 rpm for 5 min, and transfer the supernatant (150 μL each time, twice) to a new 2 mL centrifuge tube.

[0062] (3) Add 450 μL of buffer LP3 (1.5 times the volume of the supernatant) and vortex for 15 seconds.

[0063] (4) Place the adsorption column CB3 into the collection tube, and pour the contents of 2.3 into the adsorption column CB3. Centrifuge at 12000 rpm for 1 min in a centrifuge, and keep the adsorption column CB3 in the collection tube.

[0064] (5) Add 600 μL of washing buffer PW to the adsorption column CB3 retained in the collection tube of (4) to wash the DNA, centrifuge at 12000 rpm for 1 min, and retain the adsorption column CB3 in the collection tube.

[0065] (6) Repeat the rinsing process of (5) with rinsing solution PW (if the filter membrane of adsorption column CB3 is green, rinse once with 500 μL of anhydrous ethanol, and the rinsing process is the same as that of rinsing solution PW) until the filter membrane of adsorption column CB3 is colorless.

[0066] (7) After centrifuging the collection tube treated in (6) at 12000 rpm for 2 min, open the cap of the adsorption column CB3 and place it horizontally to air dry. It can be placed at room temperature for 30 min or in a 32℃ oven for 10 min to completely dry the residual rinsing solution in the adsorption column CB3. (The adsorption membrane should be slightly wrinkled and almost odorless when you get close to it is ideal.) (8) Collect the Myriophyllum genomic DNA from the adsorption column CB3 into centrifuge tubes using elution buffer (TE) (ensuring the pH value is within the range of 7.0-8.5) to obtain genomic DNA from 10 individuals.

[0067] Before amplification, the 10 Myriophyllum sp. genomic DNA samples obtained above need to be tested and preserved through the following steps: a. The success of extraction was determined by 1% agarose gel electrophoresis, and the concentration was determined by micro-spectrophotometer.

[0068] b. According to the experimental requirements, the genomic DNA of Myriophyllum spicatum was diluted 5 times as a working solution and stored in a refrigerator at 4°C for later use. The remaining mother solution was stored in a refrigerator at -20°C for long-term preservation.

[0069] 3.2 PCR product amplification reaction and electrophoresis detection The DNA of 10 suspected individuals and 10 reference *Myriophyllum siberianense* and 10 *Myriophyllum sp.* strains were amplified using the five sets of dual PCR primers described in this patent. Gene analysis software was used to perform cluster analysis on the PCR amplification results and the PCR amplification results of 20 basic reference species to determine whether the suspected individuals clustered into *Myriophyllum siberianense* or *Myriophyllum sp.* strains. The species of the suspected individuals was determined based on the clustering results. If the individual to be identified clustered into the *Myriophyllum siberianense* branch, then the individual to be identified was *Myriophyllum siberianense*; if the individual to be identified clustered into the *Myriophyllum sp.* branch, then the individual to be identified was *Myriophyllum sp.*

[0070] The amplification primer pairs used for the above microsatellite molecular markers are shown in Table 1.

[0071] The silver staining results were analyzed using genetic analysis software to generate UPGMA cluster analysis diagrams, and the species of the individuals to be identified were determined based on the clustering results.

[0072] The PCR amplification reaction system consisted of 25 μL: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL each of the forward and reverse primers of the two primer pairs, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 11.5 μL of ultrapure water.

[0073] The PCR amplification reaction program was as follows: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles; finally, extension at 72℃ for 10 minutes, and storage at 4℃.

[0074] The PCR products were subjected to electrophoresis and silver staining using 12% polyacrylamide gel.

[0075] The genetic analysis software mentioned can be MEGA software.

[0076] The results are as follows Figure 3 As shown, these 30 *Myriophyllum spicatum* plants are divided into two branches. Plants 1-10, 28-30, 32, 34, and 36 cluster in one branch, while plants 11-20, 27, 31, 33, and 35 cluster in another. The cluster analysis results can clearly identify the 10 plants that are uncertain to be identified as either *Myriophyllum siberianense* or *Myriophyllum spicatum*. Among them, plants 28-30, 32, 34, and 36 are *Myriophyllum siberianense*, while plants 27, 31, 33, and 35 are *Myriophyllum spicatum*.

[0077] The above experiments verified that the *Myriophyllum spicatum* population selected in this experiment from the Shiyan Lake wetland in Shenzhen indeed has a higher capacity and efficiency in improving wetland restoration than other selected populations. This experiment also showed that the genetic distance between the *Myriophyllum spicatum* population from the Shiyan Lake wetland and the baseline population is the closest. Therefore, using this technology to screen for *Myriophyllum spicatum* populations with longer individual body lengths, more vigorous growth, and faster growth can effectively improve the efficiency of wetland ecological restoration.

Claims

1. A specific primer for identifying *Myriophyllum sieboldii* and *Myriophyllum spicatum*, characterized in that, The specific primers consist of 10 pairs, namely primer pair ZZJD1-primer pair ZZJD10, and the nucleotide sequences of the primers are shown in SEQ ID NO.1-SEQ ID NO.

20.

2. A method for identifying *Myriophyllum sieboldii* and *Myriophyllum spicatum*, characterized in that, The method uses a combination of duplex PCR primers to amplify the DNA of the sample to be tested, followed by cluster analysis to determine whether the sample is *Myriophyllum sibiricum* Kom. or *Myriophyllum verticillatum* L. Five sets of duplex PCR primer pairs are used for the duplex PCR reaction; primer pairs ZZJD1 and ZZJD2 are used together; primer pairs ZZJD3 and ZZJD4 are used together; primer pairs ZZJD5 and ZZJD6 are used together; primer pairs ZZJD7 and ZZJD8 are used together; and primer pairs ZZJD9 and ZZJD10 are used together.

3. The method based on claim 1 or 2, characterized in that, The method is based on 10 Myriophyllum polymorphic microsatellite molecular markers, the nucleotide sequences of which are shown in SEQ ID NO.21-SEQ ID NO.

30.

4. The method based on claim 1 or 2, characterized in that, Includes the following steps: (1) Using Siberian myriocarpus and myriocarpus as basic reference species, genomic DNA was extracted from samples of the two basic reference species respectively; (2) Extract genomic DNA from the sample to be tested; (3) Using the two basic reference species samples and the genomic DNA of the sample to be tested obtained in step (2) as templates, PCR amplification was performed using the double PCR primer pair to obtain three sets of amplification products; (4) The three groups of amplification products obtained in step (3) were subjected to electrophoresis and silver staining using polyacrylamide gel; (5) Analyze the silver staining results of step (4) using genetic analysis software and draw a UPGMA cluster analysis diagram; if the sample to be tested is clustered into the Siberian Myriophyllum branch, then the sample to be tested is determined to be Siberian Myriophyllum; if the sample to be tested is clustered into the Myriophyllum branch, then the sample to be tested is determined to be Myriophyllum.

5. The method according to claim 4, characterized in that, The PCR amplification reaction system in step (2) is 25 μL: 10×PCR Buffer 3 μL, 2.5 mmol / L dNTP 1 μL, 2 mmol / L MgCl2 3 μL, each of the upstream and downstream primers of the two pairs of primers 1 μL, 0.5 U / μL Taq enzyme 0.5 μL, DNA template 2 μL, and ultrapure water 11.5 μL.

6. The method according to claim 4 or 5, characterized in that, The PCR amplification reaction program in step (2) is as follows: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 45 seconds, for a total of 35 cycles; finally, extension at 72℃ for 10 minutes, and storage at 4℃.

7. The method according to claim 4 or 5, characterized in that, In step (3), the PCR products were electrophoresed and silver-stained with 12% polyacrylamide gel.

8. The method according to claim 4 or 5, characterized in that, The genetic analysis software mentioned in step (4) is MEGA software.

9. A kit for analyzing the genetic diversity of Myriophyllum sp. populations, characterized in that, The kit includes at least the primers as described in claim 1 or 2.

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