Myriophyllum spicatum screening method for improving wetland ecological restoration efficiency, kit and application

Abstract

The embodiment of the invention discloses a myriophyllum verticillatum screening method for improving wetland ecological restoration efficiency, a kit and application, and can be used for screening myriophyllum verticillatum groups with high water purification efficiency. Specifically, five groups of primer pairs of a microsatellite marker technology can be provided to analyze the genetic relationship between a myriophyllum verticillatum group identified as high water purification efficiency and a to-be-identified myriophyllum verticillatum group, so as to screen the myriophyllum verticillatum group close to the genetic relationship with the myriophyllum verticillatum group with high water purification efficiency, and further rapidly evaluate the genetic relationship of the myriophyllum verticillatum. Meanwhile, a double PCR system is adopted for analyzing the genetic relationship of the myriophyllum elatinoides, technical support is provided for analyzing the relationship between the myriophyllum elatinoides in different regions and groups, the genetic relationship of different myriophyllum elatinoides groups can be rapidly and accurately identified, the growth condition of the myriophyllum elatinoides can be judged, and the application prospect is wide. The myriophyllum verticillatum population close to the genetic relationship with the known myriophyllum verticillatum population with good water purification effect can be quickly found, so that the wetland ecological restoration efficiency can be effectively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of ecological restoration, in particular to a Myriophyllum screening method for improving wetland ecological restoration efficiency, a kit and application thereof. BACKGROUND

[0002] Myriophyllum spp. is a perennial submerged plant of Haloragaceae, with rhizomes in the sediment, erect stems branching, and leaflets in a silken full split. It grows well in a slightly alkaline, warm water, and a sunny environment, and overwinters with rhizomes. Myriophyllum is known as a "denitrification wizard" that can absorb nitrogen and phosphorus in water and sediment through roots, stems and leaves. Studies have shown that its removal efficiency for ammonium nitrogen, nitrate nitrogen and total nitrogen can reach more than 90%, and the removal rate for total phosphorus can reach more than 45%, effectively preventing water eutrophication. After planting Myriophyllum, water transparency is significantly improved (e.g., from 0.5 meters to 2.3 meters in the Hangzhou Bay case), dissolved oxygen concentration is increased (average 9.3 mg / L), and pH value is increased, promoting ammonia volatilization and phosphorus precipitation. Myriophyllum competes with phytoplankton (especially blue-green algae such as Microcystis) to inhibit their growth, reducing algal density (algal density in the restoration area is only 1 / 10 of that in the non-restoration area), while increasing phytoplankton diversity index to prevent single algae outbreak. At the same time, the "green Myriophyllum wetland ecological treatment technology" is used in southern rural areas, combining straw substrate and Myriophyllum wetland to treat aquaculture wastewater, farmland drainage and domestic sewage at low cost, so that the effluent COD, ammonia nitrogen and other indicators meet the national first-level A standard.

[0003] Due to the strong nitrogen and phosphorus absorption capacity and ecological adaptability of Myriophyllum, it has become a "green engine" for water restoration, especially in the treatment of eutrophic wastewater, showing high efficiency and low cost. However, different groups of Myriophyllum have different water purification efficiencies, and the use of Myriophyllum groups with high water purification efficiency can better improve the efficiency of wetland ecological restoration. SUMMARY

[0004] The present application provides a Myriophyllum screening method for improving wetland ecological restoration efficiency, a kit and application thereof, which can quickly find a method for Myriophyllum groups with high water purification efficiency, and has great application value.

[0005] In a first aspect, the present application provides a Myriophyllum screening method for improving wetland ecological restoration efficiency, which screens Myriophyllum groups with high water purification efficiency, and the method comprises: The five groups of primers of the microsatellite marker technology are used for analyzing the genetic relationship of the populations of Myriophyllum verticillatum identified as high water purification efficiency and the populations of Myriophyllum verticillatum to be identified, so as to screen the populations of Myriophyllum verticillatum which are close to the populations of Myriophyllum verticillatum with high water purification efficiency. The ten pairs of primers of the microsatellite marker technology are divided into five groups for double PCR reaction, and the ten pairs of primers are respectively as follows: HWZ1-F, the sequence is shown as SEQ ID No. 1, and HWZ1-R, the sequence is shown as SEQ ID No. 2; HWZ2-F, the sequence is shown as SEQ ID No. 3, and HWZ2-R, the sequence is shown as SEQ ID No. 4; HWZ3-F, the sequence is shown as SEQ ID No. 5, and HWZ3-R, the sequence is shown as SEQ ID No. 6; HWZ4-F, the sequence is shown as SEQ ID No. 7, and HWZ4-R, the sequence is shown as SEQ ID No. 8; HWZ5-F, the sequence is shown as SEQ ID No. 9, and HWZ5-R, the sequence is shown as SEQ ID No. 10; HWZ6-F, the sequence is shown as SEQ ID No. 11, and HWZ6-R, the sequence is shown as SEQ ID No. 12; HWZ7-F, the sequence is shown as SEQ ID No. 13, and HWZ7-R, the sequence is shown as SEQ ID No. 14; HWZ8-F, the sequence is shown as SEQ ID No. 15, and HWZ8-R, the sequence is shown as SEQ ID No. 16; HWZ9-F, the sequence is shown as SEQ ID No. 17, and HWZ9-R, the sequence is shown as SEQ ID No. 18; HWZ10-F, the sequence is shown as SEQ ID No. 19, and HWZ10-R, the sequence is shown as SEQ ID No. 20; The annealing temperature of each pair of primers is 56 DEG C.

[0006] Further, in the method for screening Myriophyllum verticillatum for improving the efficiency of wetland ecological restoration provided in the application, HWZ1 and HWZ2 are used in cooperation; HWZ3 and HWZ4 are used in cooperation; HWZ5 and HWZ6 are used in cooperation; HWZ7 and HWZ8 are used in cooperation; and HWZ9 and HWZ10 are used in cooperation.

[0007] In a second aspect, the present application further provides a kit comprising the primers used in the screening method for Myriophyllum spicatum for improving the efficiency of wetland ecological restoration provided in the first aspect.

[0008] In a third aspect, the present application further provides an application of the primers of the microsatellite marker for identifying the genetic relationship of Myriophyllum spicatum population, wherein the primers used in the screening method for Myriophyllum spicatum for improving the efficiency of wetland ecological restoration provided in the first aspect are applied to identify the genetic relationship of Myriophyllum spicatum population.

[0009] In a fourth aspect, the present application further provides a method for identifying the genetic relationship of Myriophyllum spicatum population, comprising: S1, extracting the Myriophyllum spicatum genomic DNA of the Myriophyllum spicatum population identified as having high water purification efficiency and the Myriophyllum spicatum population to be identified, respectively; S2, using the Myriophyllum spicatum genomic DNA extracted in S1 as a DNA template, and performing PCR amplification using the primers of the five groups provided in the screening method for Myriophyllum spicatum for improving the efficiency of wetland ecological restoration to obtain an amplification product; S3, performing electrophoresis and silver staining of the amplification product using polyacrylamide gel to obtain a silver staining result; S4, based on the silver staining result, using genetic analysis software to draw a UPGMA clustering analysis diagram to obtain a clustering result, and identifying and distinguishing the genetic relationship of the Myriophyllum spicatum population according to the clustering result.

[0010] Further, in the method for identifying the genetic relationship of Myriophyllum spicatum population provided in the present application, the reaction system for PCR amplification has a volume of 25 μL, which comprises: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of each of the upper and lower primers of the two pairs of primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 13.5 μL of ultrapure water.

[0011] Further, in the method for identifying the genetic relationship of Myriophyllum spicatum population provided in the present application, the reaction procedure for PCR amplification is as follows: Pre-denaturation at 94°C for 3 minutes; Denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 45 seconds, in sequence, for a total of 35 cycles; Extension at 72°C for 10 minutes, and preservation at 4°C.

[0012] Further, in the method for identifying the genetic relationship of Myriophyllum spicatum population provided in the present application, the amplification product is subjected to electrophoresis and silver staining using 12% polyacrylamide gel.

[0013] Further, in the method for identifying the genetic relationship of Myriophyllum verticillatum populations provided in the application, the genetic analysis software comprises MEGA software.

[0014] The Myriophyllum verticillatum screening method, kit and application for improving the efficiency of wetland ecological restoration provided in the application can screen Myriophyllum verticillatum populations with high water purification efficiency, and can provide five groups of primers for analyzing the genetic relationship of Myriophyllum verticillatum populations identified as having high water purification efficiency and Myriophyllum verticillatum populations to be identified, so as to screen Myriophyllum verticillatum populations with close genetic relationship to Myriophyllum verticillatum populations with high water purification efficiency, and then the genetic relationship of Myriophyllum verticillatum can be quickly evaluated. The genetic relationship of Myriophyllum verticillatum is analyzed by using a duplex PCR system, which can greatly save drugs and time by about 50% compared with a traditional PCR system for analyzing the genetic relationship of species, and a Myriophyllum verticillatum genetic relationship evaluation method is provided, which provides technical support for analyzing the relationship between Myriophyllum verticillatum in different regions and populations, and helps to quickly and accurately identify the genetic relationship of different Myriophyllum verticillatum populations, so as to judge the growth status of Myriophyllum verticillatum, quickly find Myriophyllum verticillatum populations with close genetic relationship to Myriophyllum verticillatum populations with known high water purification effect, and then the efficiency of wetland ecological restoration can be effectively improved. The Myriophyllum verticillatum screening method, kit and application for improving the efficiency of wetland ecological restoration have high practicability and great popularization and utilization value. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative labor.

[0016] Figure 1 UPGMA cluster analysis diagram of Myriophyllum verticillatum provided in the embodiments of the present application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0018] It should be understood that when used in the specification and the appended claims, the terms "comprise" and "include" indicate the presence of described features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or collections thereof.

[0019] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0020] It is further to be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items, and that the term "at least one of' encompasses any and all possible combinations of one or more of the associated listed items.

[0021] The present application provides a screening method of Myriophyllum for improving the efficiency of wetland ecological restoration, which screens Myriophyllum population with high water purification efficiency, and the method comprises the following steps: The five groups of primers of the microsatellite marker technology are provided to analyze the genetic relationship of the Myriophyllum population identified as high water purification efficiency and the Myriophyllum population to be identified, so as to screen the Myriophyllum population with close genetic relationship with the Myriophyllum population with high water purification efficiency, thereby providing technical support for the relationship analysis between different regions and populations of Myriophyllum, which is helpful to quickly and accurately identify the genetic relationship of different Myriophyllum populations, and then the growth status of Myriophyllum can be judged, and the Myriophyllum population with close genetic relationship with the known Myriophyllum population with good water purification effect can be quickly found, thereby the efficiency of wetland ecological restoration can be effectively improved.

[0022] The ten pairs of primers of the microsatellite marker technology are divided into five groups for double PCR reaction, and the ten pairs of primers are respectively: HWZ1-F, the sequence is shown as SEQ ID No. 1, and HWZ1-R, the sequence is shown as SEQ ID No. 2; HWZ2-F, the sequence is shown as SEQ ID No. 3, and HWZ2-R, the sequence is shown as SEQ ID No. 4; HWZ3-F, the sequence is shown as SEQ ID No. 5, and HWZ3-R, the sequence is shown as SEQ ID No. 6; HWZ4-F, the sequence is shown as SEQ ID No. 7, and HWZ4-R, the sequence is shown as SEQ ID No. 8; HWZ5-F, the sequence is shown as SEQ ID No. 9, and HWZ5-R, the sequence is shown as SEQ ID No. 10; HWZ6-F, the sequence is shown as SEQ ID No. 11, and HWZ6-R, the sequence is shown as SEQ ID No. 12; HWZ7-F, the sequence is shown as SEQ ID No. 13, and HWZ7-R, the sequence is shown as SEQ ID No. 14; HWZ8-F, sequence as shown in SEQ ID No. 15, HWZ8-R, sequence as shown in SEQ ID No. 16; HWZ9-F, sequence as shown in SEQ ID No. 17, HWZ9-R, sequence as shown in SEQ ID No. 18; HWZ10-F, sequence as shown in SEQ ID No. 19, HWZ10-R, sequence as shown in SEQ ID No. 20; wherein the annealing temperature of each pair of primers is 56°C.

[0023] Specifically, the sequence of HWZ1-F is: TGGGATGAGTAAGATGAC, and the sequence of HWZ1-R is TATAACCGTGAAAGAAAT; The sequence of HWZ2-F is: CACTCGCTCTACGCCCTC, and the sequence of HWZ2-R is: TATCAACAACTTCAAAAGCAATG; The sequence of HWZ3-F is: AATCCACTGTCTCCCTCC, and the sequence of HWZ3-R is: TTTCAAACTGACACGCATAG; The sequence of HWZ4-F is: GCTTCGTTAGCACCCTAT, and the sequence of HWZ4-F is: TCATGCGCTTCGTATTCC; The sequence of HWZ5-F is: TGTAATGAAGGGCATGATAT, and the sequence of HWZ5-R is: TCGAGCTAGATGGAGAAAC; The sequence of HWZ6-F is: GACTGCTGATGGTGACCCTGAT, and the sequence of HWZ6-R is: CCGACGAAGTACCTGCCTCT; The sequence of HWZ7-F is: AGTCAGTGGGCTTTCAGTTT, and the sequence of HWZ7-R is: AGGGAAGTCGGTGGTAGG; The sequence of HWZ8-F is: ACCGAAGTCAGAATCAAGTCAA, and the sequence of HWZ8-R is: ATTAGGTGTTATCAACTTAGTTGGAA; The sequence of HWZ9-F is: TCGGGAGTGAGATTGTTG, and the sequence of HWZ9-R is: GCAGTGCTGGTGTTTGTT; The sequence of HWZ10-F is: CAGTAGCAACAACCGTGT, and the sequence of HWZ10-R is: TCTAATGGCCCAATAGATAG.

[0024] Further, in some embodiments, HWZ1 is used in cooperation with HWZ2; HWZ3 is used in cooperation with HWZ4; HWZ5 is used in cooperation with HWZ6; HWZ7 is used in cooperation with HWZ8; and HWZ9 is used in cooperation with HWZ10.

[0025] In some embodiments, the present application also provides a kit comprising the primers used in the screening method for improving the efficiency of wetland ecological restoration provided by the present application. Specifically, the kit can be used for analyzing the genetic diversity of a population of Myriophyllum.

[0026] In some embodiments, the present application also provides an application of the primers of the microsatellite marker for identifying the genetic relationship of a population of Myriophyllum, wherein the primers used in the screening method for improving the efficiency of wetland ecological restoration provided by the present application are applied to identify the genetic relationship of a population of Myriophyllum.

[0027] In some embodiments, the present application also provides a method for identifying the genetic relationship of a population of Myriophyllum, comprising: S1, extracting the genomic DNA of Myriophyllum from a population of Myriophyllum which has been identified as having high water purification efficiency and a population of Myriophyllum to be identified, respectively; S2, using the genomic DNA of the population of Myriophyllum extracted in S1 as a DNA template, and performing PCR amplification using the primers provided in the screening method for improving the efficiency of wetland ecological restoration provided by the present application, which are divided into five groups, to obtain an amplification product; S3, performing electrophoresis and silver staining of the amplification product using polyacrylamide gel to obtain a silver staining result; S4, based on the silver staining result, using genetic analysis software to draw a UPGMA clustering analysis diagram to obtain a clustering result, and identifying and distinguishing the genetic relationship of the population of Myriophyllum according to the clustering result.

[0028] In some embodiments, the reaction system for PCR amplification has a volume of 25 μL, which comprises: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of each of the upper and lower primers of the two pairs of primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 13.5 μL of ultrapure water.

[0029] In some embodiments, the reaction program for PCR amplification is as follows: Pre-denaturation at 94°C for 3 minutes; Denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 45 seconds, in turn, for a total of 35 cycles; Extension at 72℃ for 10 min and storage at 4℃.

[0030] In some embodiments, the amplified products are subjected to electrophoresis and silver staining using 12% polyacrylamide gel.

[0031] In some embodiments, the genetic analysis software includes MEGA (Molecular Evolutionary Genetics Analysis).

[0032] In some embodiments, the present application also provides a primer screening method for identifying the microsatellite marker of Myriophyllum verticillatum population, which is used to screen the primer for identifying the microsatellite marker of Myriophyllum verticillatum population provided by the present application, and the method comprises: Providing a microsatellite sequence with N repeating units of more than 2 bases to design N pairs of primers; wherein N is greater than or equal to 10; Based on the microsatellite marker technology, the M pairs of primers are screened using the Myriophyllum verticillatum population sample genomic DNA template to obtain ten pairs of primers.

[0033] In the present application, before screening the primers, the Myriophyllum verticillatum population sample needs to be selected and the Myriophyllum verticillatum population genomic DNA needs to be extracted.

[0034] Specifically, in the process of selecting the Myriophyllum verticillatum population sample, the Myriophyllum verticillatum population with high water purification efficiency is selected, and specifically, the Myriophyllum verticillatum population with longer mature individual body length, lush growth, and faster growth can be selected as the basic population (which is defaulted as the Myriophyllum verticillatum population with high water purification efficiency), the Myriophyllum verticillatum seedlings from different wetlands are selected as the Myriophyllum verticillatum population to be identified, and the direct genetic relationship between the Myriophyllum verticillatum population to be identified and the basic population is detected.

[0035] In the process of extracting the Myriophyllum verticillatum population genomic DNA, the Myriophyllum verticillatum population with longer mature individual body length, lush growth, and faster growth can be selected as the basic population, and 6 Myriophyllum verticillatum (sample marked as 1-6) are selected, 6 Myriophyllum verticillatum (sample marked as 7-12) are randomly selected in the water area of Tangxun Lake in Wuhan, 6 Myriophyllum verticillatum (sample marked as 13-18) are randomly selected in the water area of East Lake Wetland in Wuhan, 6 Myriophyllum verticillatum (sample marked as 19-24) are randomly selected in the water area of Panlong Lake Wetland in Wuhan, and 6 Myriophyllum verticillatum (sample marked as 25-30) are randomly selected in the water area of Jin Yin Lake Wetland in Wuhan.

[0036] The extraction of Myriophyllum verticillatum genomic DNA uses Tiangen new plant genomic DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing), and the specific operation process is as follows: 1. Take about 100 mg of the plant dry weight of the 1-30 Myriophyllum aquaticum sample obtained in step (1) with tweezers and place it in a 2 mL centrifuge tube. To avoid cross contamination, the tweezers used to take the plant tissue should be burned on an alcohol lamp. Then add two quartz sands and place them on a shaker for crushing treatment. The positive and negative sides should be shaken for 1 minute until the plant tissue becomes powder. After centrifugation for 30 seconds, add 500 μL of buffer LP1 and 6 uRNase A (10 mg / mL) and vortex for 1 minute (check if it is wall-hung, if it is wall-hung, add 100 uL of buffer LP1). After 10 minutes of dry bath shaking at 65°C (300 rpm), place it in an ice box for 2 minutes of ice bath; 2. Add 150 μL of buffer LP2 and vortex for 1 minute to mix thoroughly. Centrifuge at 12000 rpm for 5 minutes, and transfer the supernatant (take 150 μL each time, twice) to a new 2 mL centrifuge tube; 3. Add 450 μL of buffer LP3 (1.5 times the volume of the supernatant) and vortex for 15 seconds; 4. Place the adsorption column CB3 in the collection tube, and pour the mixture obtained in step 2.3 into the adsorption column CB3. Centrifuge at 12000 rpm for 1 minute, and keep the adsorption column CB3 in the collection tube; 5. Add 600 μL of rinse solution PW to the adsorption column CB3 kept in the collection tube in step 2.4 to rinse the DNA. Centrifuge at 12000 rpm for 1 minute, and keep the adsorption column CB3 in the collection tube; 6. Repeat the rinse process with rinse solution PW in step 2.5 (if the filter membrane of the adsorption column CB3 appears green, rinse it once with 500 μL of absolute ethanol, and the rinse process is the same as that with rinse solution PW). Repeat until the filter membrane of the adsorption column CB3 is colorless; 7. After centrifuging the collection tube treated in step 2.6 at 12000 rpm for 2 minutes, open the cover of the adsorption column CB3 and place it horizontally to dry. It can be directly placed at room temperature for 30 minutes or in a 32°C oven for 10 minutes to completely dry the residual rinse solution in the adsorption column CB3. The filter membrane should be slightly wrinkled, and there should be almost no alcohol smell when you approach it; 8. Collect the Myriophyllum aquaticum genomic DNA in the adsorption column CB3 into a centrifuge tube by elution buffer (TE) (ensure that the pH value is in the range of 7.0-8.5), and obtain 24 Myriophyllum aquaticum genomic DNAs.

[0037] In addition, before amplification, the 30 Myriophyllum aquaticum genomic DNAs obtained above need to be detected and preserved by the following steps: A. Detect whether the extraction is successful by 1% agarose gel electrophoresis, and determine the concentration by a micro spectrophotometer; B, according to the experimental requirements, the genome DNA of Myriophyllum is diluted 5 times as working solution and placed in the refrigerator at 4 DEG C for standby, and the remaining mother liquor is placed in the refrigerator at -20 DEG C for long-term preservation.

[0038] Further, in the process of screening the primer of the microsatellite marker for identifying the genetic relationship of the Myriophyllum population, the primer can be obtained, and the primer can be preliminarily screened, then the primer of the polymorphic site is detected, then the double-PCR amplification is carried out, and finally the detection and identification are carried out, and then 10 pairs of better primers can be finally screened, and the genetic structure analysis can also be carried out according to the result of the double-PCR amplification.

[0039] In the process of obtaining the primer, the microsatellite search software can be used to search for the microsatellite fragment in the Myriophyllum population genome and the transcriptome obtained by the previous sequencing, 500 microsatellite sequences with more than 2 base pairs of repeating units are selected from the fragments containing the microsatellite, the microsatellite primer is designed by using the Primer 6 software, 500 pairs of primers are designed, and the primer company is sent for synthesis.

[0040] In the process of preliminarily screening the primer, 6 Myriophyllum population sample genomic DNA templates extracted from the Myriophyllum population genomic DNA template are randomly selected to preliminarily screen the synthesized primer, and the microsatellite marker with good polymorphism, less deletion and stable amplified bands is selected.

[0041] In the process of preliminarily screening the primer, 6 Myriophyllum population sample genomic DNA templates extracted from the Myriophyllum population genomic DNA template are randomly selected to preliminarily screen the synthesized primer, and the microsatellite marker with good polymorphism, less deletion and stable amplified bands is selected.

[0042] In the process of preliminarily screening the primer, 6 Myriophyllum population sample genomic DNA templates extracted from the Myriophyllum population genomic DNA template are randomly selected to preliminarily screen the synthesized primer, and the microsatellite marker with good polymorphism, less deletion and stable amplified bands is selected.

[0043] PCR reaction system: the volume of PCR amplification reaction system is 25 μL, which respectively is: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of each of two pairs of primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, 13.5 μL of ultrapure water. The reaction program of PCR amplification is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 45 seconds, for 35 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0044] In the process of carrying out double-PCR amplification, after the amplification of the genomic DNA of the 30 Myriophyllum group samples, microsatellite markers with good polymorphism, less deletion and stability can still be screened out, and the double-PCR primer group is randomly combined into two groups, and the double-PCR amplification system is used to amplify the DNA of the above-mentioned 30 samples, and 18 samples can be screened out. Microsatellite molecular markers with good polymorphism, less deletion and stable bands amplified by double-PCR system can be stably amplified.

[0045] PCR reaction system: the volume of PCR amplification reaction system is 25 μL, which respectively is: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of each of two pairs of primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, 13.5 μL of ultrapure water. The reaction program of PCR amplification is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 45 seconds, for 35 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0046] Finally, in the process of detection and identification, the above Myriophyllum group PCR product can be separated by electrophoresis on 12% non-denaturing polyacrylamide gel, and photographed after silver staining, and then ten pairs of better primers can be screened out. The sequences of the ten pairs of primers are: HWZ1: F: TGGGATGAGTAAGATGAC R: TATAACCGTGAAAGAAAT HWZ2: F: CACTCGCTCTACGCCCTC R: TATCAACAACTTCAAAAGCAATG HWZ3: F: AATCCACTGTCTCCCTCC R:TTTCAAACTGACACGCATAG HWZ4:F:GCTTCGTTAGCACCCTAT R:TCATGCGCTTCGTATTCC HWZ5:F:TGTAATGAAGGGCATGATAT R:TCGAGCTAGATGGAGAAAC HWZ6:F:GACTGCTGATGGTGACCCTGAT R:CCGACGAAGTACCTGCCTCT HWZ7:F:AGTCAGTGGGCTTTCAGTTT R:AGGGAAGTCGGTGGTAGG HWZ8:F:ACCGAAGTCAGAATCAAGTCAA R:ATTAGGTGTTATCAACTTAGTTGGAA HWZ9:F:TCGGGAGTGAGATTGTTG R:GCAGTGCTGGTGTTTGTT HWZ10:F:CAGTAGCAACAACCGTGT R:TCTAATGGCCCAATAGATAG The annealing temperature for each primer pair is 56℃.

[0047] In this application, primer pair HWZ1 is used in combination with primer pair HWZ2, primer pair HWZ3 is used in combination with primer pair HWZ4, primer pair HWZ5 is used in combination with primer pair HWZ6, primer pair HWZ7 is used in combination with primer pair HWZ8, and primer pair HWZ9 is used in combination with primer pair HWZ10.

[0048] The microsatellite molecule M1 corresponding to primer pair HWZ1 is labeled as shown in SEQ ID No. 21, specifically: CattgttgattttgtttatagttcatgtttaacattaaatttataagaattgatatgttaatgttctgagaaaaatgaaaatatacctaaggtacacctaactaggtgtgtcataggcatttttatatgtatgttataaccgtgaaagaaATATATATATgaaattgataaattacgaaaccatcatcttgtgtctatgacgtacctatctaggtgtaccttcgccacattttcatacatccgtgatattttatatggttgcagaataatttttgaatagtcatcttactcatcccattaaaaattttcc The microsatellite molecular marker M2 corresponding to the primer pair HWZ2 is shown in SEQ ID No. 22, and specifically is: gtcgtcatcatcatcatattcatacagaaaattataaacattgttttcttaatatcaacaacttcaaaagcaatggattctgcattttcggattggagagaagagggagaaaaactagcctgtgaaaaattaaagtgaaattggagaaggTATATATATAgtattagtagtagagagtctcagaggaagaggaaacgagcgcgagaggttaacggttgggaggggaggatctgcagcagaaccacgcgagggcgtagagcgagtggctgaaattttttctgatgtttccgacttagggtttctctctcgc The microsatellite molecular marker M3 corresponding to the primer pair HWZ3 is shown in SEQ ID No. 23, and specifically is: aaaaaaaaacaaaaacaggagtgtaaacaaatgaagaaagatgtattatacatgtagcatgcagtaaatatttagggtttcaaactgacacgcatagctttagtaagaagaagaagaagaagaagaagaagaagaagaagaagaagaagaAGGAGGAGGAGGAGGAGGAGGAGGAGGttaagcccatacggccatgagggacatgcatccagcaccaagaaggagggagacagtggatttaagtcgaagtgcagcactcttttgcagttttgtgctcatgaaatcaacagccaagagatttacgagtgccatataaataagaattcc The microsatellite molecular marker M4 corresponding to the primer pair HWZ4 is shown as SEQ ID No. 24, in particular: tcatgcgcttcgtattcccctagagtttgctcaataaaaattgagtgtacctatatgttatgacaattttactagctttgtattatatatttttatgagatgtatgaaaatatgtataaaatatgtataaatattttttatctcatttttTATATATATAataaaagtttatattcgtcttttagatttaagtatattccatcataaatttataattgtaaagattcaaatttttattttcatatcgtgaatttctttttatcttctatattattattattatagggtgctaacgaagcctttcacaagg The microsatellite molecular marker M5 corresponding to the primer pair HWZ5 is shown as SEQ ID No. 25, in particular: cttgatctttatcaactgggtttgcttcgagctagatggagaaaccctgtttgacagcggtgcttcatgaagacaacaaccaagagctcacttccagctttactgcaaatcttccggtaacttttcttcttctctttccttttcttttttTCTCTCTCTCtttaactgaagatggaatttgtgtataaagttaggatctttatggctatatcatgcccttcattacatggaaaatttaggcgtatttatatgtacttacttcgtttgttatgacaacaaaatggatcgcaccagcaccagcaccagcatc The microsatellite molecular marker M6 corresponding to the primer pair HWZ6 is shown as SEQ ID No. 26, in particular: tgaattgctggaccatagctcggaaattcttggtgtcggtgttgagcaccggcgtgggagttcgcttggaggccctggaccggcggcgtgctggcttagccacgcggccttcagggttcatcaacaacgactgctgatggtgaccctgatGAGGAGGAGGAGGAGccgccgcttcgccttgaaaagagtacgacgacgacgacctacttggtagatgatttgagctagtagtagtcgtagtagagatttgatcggcggcgaacatggcagaggcaggtacttcgtcggaagatgatcggaggacgtcgttactga The microsatellite molecular marker M7 corresponding to the primer pair HWZ7 is shown as SEQ ID No. 27, in particular: acggagatcttcaagtatgaaacagcagcccttgaagccgacaacaagctgcttgccattttcgttctctctgtaacttggaagaaggaaggagggtgaaaatggcgccgagggagggaagtcggtggtagggactgatgattgaaaccg ACACACACAC caaaaccaaaccttaaaacctaaacagtagcagtaaatgaaactgaaagcccactgactaaagattccaagacacccccaccaccaccaaactctgtctctattagctatttatagtaaatcttcttcattttcgagacccttttttgga The microsatellite molecular marker M8 corresponding to the primer pair HWZ8 is shown as SEQ ID No. 28, in particular: acggagatcttcaagtatgaaacagcagcccttgaagccgacaacaagctgcttgccattttcgttctctctgtaacttggaagaaggaaggagggtgaaaatggcgccgagggagggaagtcggtggtagggactgatgattgaaaccg ACACACACAC caaaaccaaaccttaaaacctaaacagtagcagtaaatgaaactgaaagcccactgactaaagattccaagacacccccaccaccaccaaactctgtctctattagctatttatagtaaatcttcttcattttcgagacccttttttgga The microsatellite molecular marker M9 corresponding to the primer pair HWZ9 is shown as SEQ ID No. 29, in particular: ttgtcagtaactcattatttttctgatgattttttttttggtgttattcgttctggtacccgacgaagatgtgcagtgctggtgtttgtttcttcttttttttctgattaattaattaagtacattatatactttgctaagtaagaattcTATATATATATATATATATATAatattgttttttattaaaaatattacaatcccatcatcgttatgaatcaacaatctcactcccgatagatagatagatatatatatatatatatatattgtttttttattaaaaatattataatctcgtcttcattatgaatcgaccata The microsatellite molecular marker M10 corresponding to the primer pair HWZ10 is shown as SEQ ID No. 30, specifically: ttgtcagtaactcattatttttctgatgattttttttttggtgttattcgttctggtacccgacgaagatgtgcagtgctggtgtttgtttcttcttttttttctgattaattaattaagtacattatatactttgctaagtaagaattcTATATATATATATATATATATAatattgttttttattaaaaatattacaatcccatcatcgttatgaatcaacaatctcactcccgatagatagatagatatatatatatatatatatattgtttttttattaaaaatattataatctcgtcttcattatgaatcgaccata In addition, in the process of genetic structure analysis, the obtained double-PCR amplification results can be digitized, specifically, the PCR products can be read by using the Gene Marker software, and the UPGMA cluster analysis diagram can be constructed by using the MEGA software for analysis and calculation. Figure 1As shown, the numbers 1-30 in the figure correspond to samples 1-30 in the examples. The 30 Myriophyllum populations are divided into three branches, 1-6 and 13-18 are clustered into one branch, 7-12 are clustered into one branch, 19-24 and 25-30 are clustered into one branch. Therefore, it can be determined that the clustering analysis result is consistent with the sampling classification (1-6 are clustered into one branch, which indicates that the 6 Myriophyllum populations of mature individuals with longer body length, more vigorous growth and faster growth as the basic population are more closely related, 13-18 are clustered into one branch, which indicates that the 6 Myriophyllum populations in the water area of East Lake Wetland in Wuhan are more closely related, 1-6 and 13-18 are clustered into one branch, which indicates that the 6 Myriophyllum populations in the water area of East Lake Wetland in Wuhan and the 6 basic Myriophyllum populations are more closely related; 7-12 are clustered into one branch, which indicates that the 6 Myriophyllum populations randomly selected from the water area of Tangxun Lake in Wuhan are more closely related; 19-24 are clustered into one branch, which indicates that the 6 Myriophyllum populations randomly selected from the water area of Panlong Lake Wetland in Wuhan are more closely related; 25-30 are clustered into one branch, which indicates that the 6 Myriophyllum populations randomly selected from the water area of Jin Yin Lake Wetland in Wuhan are more closely related, which further indicates that the technology can accurately determine the genetic relationship of Myriophyllum, and the 6 Myriophyllum populations in the water area of East Lake Wetland in Wuhan and the 6 basic Myriophyllum populations are more closely related.

[0049] Therefore, it can be determined that the 10 pairs of Myriophyllum microsatellite primers provided in the present application can be used to evaluate the genetic relationship of Myriophyllum, and provide a new technical method and means for studying the genetic diversity and genetic relationship analysis of Myriophyllum.

[0050] In some embodiments, to verify the effect of Myriophyllum from different regions on the efficiency of wetland ecological restoration, 5 wetland areas with equal area can be selected, and 50% of Myriophyllum from each region can be planted. After three months of planting, the water quality data of each wetland area is as follows in Table 1: Table 1

[0051] As can be seen from Table 1, by planting different populations of Myriophyllum, it can be determined that the Myriophyllum populations in the water area of East Lake Wetland in Wuhan and the basic Myriophyllum populations have the best ecological restoration effect.

[0052] As shown above, the above experiments verify that the ability and efficiency of the Myriophyllum populations in the water area of East Lake Wetland in Wuhan selected in the present experiment to improve wetland restoration are indeed higher than those of the other selected populations, and the present experiment also shows that the genetic distance between the Myriophyllum populations in the water area of East Lake Wetland in Wuhan and the basic populations is the closest. Therefore, using the present technology to screen Myriophyllum populations with longer body length, more vigorous growth and faster growth can effectively improve the efficiency of wetland ecological restoration.

[0053] The above merely provides the specific implementation of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the present application, and these modifications or replacements should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for screening of cattail for improving the efficiency of wetland ecological restoration, characterized in that, The method comprises: Providing five groups of primers of microsatellite marker technology to analyze the genetic relationship of the Myriophyllum spicatum population identified as high water purification efficiency and the Myriophyllum spicatum population to be identified, so as to screen the Myriophyllum spicatum population with close genetic relationship with the Myriophyllum spicatum population with high water purification efficiency; The primers of the microsatellite marker technology are ten pairs, and the ten pairs of primers are divided into five groups for double PCR reaction. HWZ1-F, the sequence is shown as SEQ ID No. 1, HWZ1-R, the sequence is shown as SEQ ID No. 2; HWZ2-F, the sequence is shown as SEQ ID No. 3, HWZ2-R, the sequence is shown as SEQ ID No. 4; HWZ3-F, the sequence is shown as SEQ ID No. 5, HWZ3-R, the sequence is shown as SEQ ID No. 6; HWZ4-F, the sequence is shown as SEQ ID No. 7, HWZ4-R, the sequence is shown as SEQ ID No. 8; HWZ5-F, the sequence is shown as SEQ ID No. 9, HWZ5-R, the sequence is shown as SEQ ID No. 10; HWZ6-F, the sequence is shown as SEQ ID No. 11, HWZ6-R, the sequence is shown as SEQ ID No. 12; HWZ7-F, the sequence is shown as SEQ ID No. 13, HWZ7-R, the sequence is shown as SEQ ID No. 14; HWZ8-F, the sequence is shown as SEQ ID No. 15, HWZ8-R, the sequence is shown as SEQ ID No. 16; HWZ9-F, the sequence is shown as SEQ ID No. 17, HWZ9-R, the sequence is shown as SEQ ID No. 18; HWZ10-F, the sequence is shown as SEQ ID No. 19, HWZ10-R, the sequence is shown as SEQ ID No. 20; The annealing temperature of each pair of primers is 56°C.

2. The method for screening of Myriophyllum spicatum for improving the efficiency of wetland ecological restoration according to claim 1, characterized in that, HWZ1 is used in cooperation with HWZ2; HWZ3 is used in cooperation with HWZ4; HWZ5 is used in cooperation with HWZ6; HWZ7 is used in cooperation with HWZ8; and HWZ9 is used in cooperation with HWZ10.

3. A kit characterized in that, The primers used in the Myriophyllum spicatum screening method for improving the efficiency of wetland ecological restoration in claim 1 or 2.

4. Use of a primer of a microsatellite marker for identifying the genetic relationship of a Myriophyllum spicatum population, characterized in that, The primers used in the Myriophyllum spicatum screening method for improving the efficiency of wetland ecological restoration in claim 1 or 2 are applied to identify the genetic relationship of Myriophyllum spicatum populations.

5. A method of identifying the genetic relationship of a population of Myriophyllum spicatum, characterized by, It comprises: S1, extracting Myriophyllum spicatum genomic DNA from the Myriophyllum spicatum population identified as high water purification efficiency and the Myriophyllum spicatum population to be identified, respectively; S2, using the Myriophyllum spicatum genomic DNA extracted in S1 as a DNA template, and using the five groups of primers provided in the Myriophyllum spicatum screening method for improving the efficiency of wetland ecological restoration in claim 1 or 2 to perform PCR amplification, to obtain an amplification product; S3, using polyacrylamide gel for electrophoresis and silver staining of the amplification product, to obtain a silver staining result; S4, based on the silver staining results, using genetic analysis software to draw UPGMA cluster analysis chart to get clustering results, and identifying and distinguishing the genetic relationship of the group of fox tail reed according to the clustering results.

6. The method of identifying the genetic relationship of a population of Myriophyllum spicatum of claim 5, wherein, The reaction system of the PCR amplification has a capacity of 25 μL, which respectively includes: 3 μL of 10×PCR Buffer, 1 μL of 2.5 mmol / L dNTP, 3 μL of 2 mmol / L MgCl2, 1 μL of upper and lower primers of two pairs of primers, 0.5 μL of 0.5 U / μL Taq enzyme, 2 μL of DNA template, and 13.5 μL of ultrapure water.

7. The method of identifying the genetic relationship of a population of Myriophyllum spicatum of claim 5, wherein, The reaction procedure of the PCR amplification is as follows: Pre-denaturation at 94 ℃ for 3 minutes; Denaturation at 94 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, and extension at 72 ℃ for 45 seconds in turn, for 35 cycles; Extension at 72 ℃ for 10 minutes, and preservation at 4 ℃.

8. The method of identifying the genetic relationship of a population of Myriophyllum spicatum of claim 5, wherein, The amplification product is subjected to electrophoresis and silver staining by using 12% polyacrylamide gel.

9. The method of identifying the genetic relationship of a population of Myriophyllum spicatum of claim 5, wherein, The genetic analysis software includes MEGA software.

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