Primers and method for identifying the genetic relationship of hydrilla verticillata by microsatellite markers

Abstract

In view of the expansion of the artificial cultivation of Hydrilla verticillata, which brings about the problems of the sharp degradation of genetic diversity, the reduction of excellent traits, the slow growth speed and the small individual, and further affects the crab cultivation, a primer and a method for identifying the genetic relationship of Hydrilla verticillata by using microsatellite markers are provided, the problems are solved by screening high-quality Hydrilla verticillata through the identification of the genetic relationship with high-quality Hydrilla verticillata populations; the primer is five groups of double-PCR microsatellite primers, including the nucleotides shown in SEQ ID NO:1-20; the DNA of the Hydrilla verticillata sample whose genetic relationship needs to be identified is amplified by PCR using the primer, then the amplification product is subjected to electrophoresis separation and the separation result is analyzed, and the genetic relationship between the Hydrilla verticillata samples is obtained; the five groups of double-PCR primers of the present application have clear microsatellite marker bands and high polymorphism, and can quickly identify the distance of the genetic relationship of Hydrilla verticillata, and further quickly screen high-quality Hydrilla verticillata populations.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a primer and identification method for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers. Background Technology

[0002] Hydrilla verticillata has many advantages: it has a wide range of habitats, strong adaptability and bioaccumulation ability, making it an ideal plant for purifying wastewater. It can form huge underwater forests in water bodies, playing an important role in the stability of the structure and function of aquatic ecosystems, and has a relatively fast adsorption effect on lead.

[0003] Cultivating *Hydrilla verticillata* can also bring good economic and social benefits to crab farmers. Compared with other aquatic plants, *Hydrilla verticillata* has the following advantages: (1) It has a wide temperature adaptability and can grow at temperatures above 5℃; (2) It does not pollute the water quality of crab ponds; (3) It has strong regeneration ability; (4) *Hydrilla verticillata* is not easy to break, grows vigorously, does not consume fertilizer, and even if it is broken by crabs, it is very easy to take root and survive, resulting in high yield; (5) It has good palatability, is liked by crabs, and can be used as high-quality green fodder for crabs.

[0004] However, with the expansion of artificial cultivation of *Hydrilla verticillata*, its genetic diversity has drastically degraded. Inbreeding has become rampant, leading to a decrease in desirable traits, slower growth, and smaller individual specimens. These problems have further impacted the income of farmers.

[0005] The above problems can be solved by screening high-quality *Hydrilla verticillata* populations. In terms of screening high-quality species, this can be achieved by identifying the phylogenetic relationship with high-quality species. Summary of the Invention

[0006] This invention addresses the problems arising from the expanded artificial cultivation of *Hydrilla verticillata*, including a sharp decline in genetic diversity, a reduction in desirable traits, slower growth rates, smaller individual sizes, and further impacts on crab farming. It provides primers and a method for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers. By identifying phylogenetic relationships with superior *Hydrilla verticillata* populations, high-quality *Hydrilla verticillata* can be screened, thus resolving the aforementioned issues.

[0007] The technical solution adopted to achieve the first objective of this invention is: a primer for identifying the phylogenetic relationship of *Hydrilla verticillata* using microsatellite markers, wherein the primers are 5 sets of dual PCR microsatellite primers; the primers include the nucleotides shown in SEQ ID NO: 1-20.

[0008] The technical solution adopted to achieve the second objective of this invention is: a method for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers, comprising the following steps: S1. Delineate the sampling area for *Hydrilla verticillata*, obtain *Hydrilla verticillata* samples from the sampling area, and extract DNA from the *Hydrilla verticillata* samples. S2. Using the five sets of dual PCR primers described in the above technical solution, the DNA of the *Hydrilla verticillata* sample obtained in step S1 is amplified by PCR to obtain PCR amplification products. S3. Separate the PCR amplification products obtained in step S2 by electrophoresis to obtain the electrophoretic separation results; S4. Analyze the electrophoretic separation results obtained in step S3 using analysis software to determine the phylogenetic relationship between the *Hydrilla verticillata* samples.

[0009] The beneficial effects of the present invention are: (1) The present invention discloses a five-group dual PCR system composed of 10 pairs of microsatellite markers. The microsatellite marker bands are clear and highly polymorphic, and are suitable as primers for identifying the kinship of *Hydrilla verticillata*. (2) Dual PCR can quickly identify the phylogenetic relationship between different *Hydrilla verticillata* populations, and thus quickly select high-quality *Hydrilla verticillata* populations. (3) The method of identifying the kinship of *Hydrilla verticillata* in this invention can bring more benefits to *Hydrilla verticillata* farmers by screening out high-quality *Hydrilla verticillata* populations, such as those with faster growth rates; and releasing the selected high-quality *Hydrilla verticillata* populations into the wild can help rivers to carry out ecological restoration more quickly.

[0010] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0011] Appendix Figure 1 This is a diagram showing the phylogenetic analysis results of 10 *Hydrilla verticillata* samples in Example 1 of the present invention.

[0012] In the attached figures, 1-10 are all samples of *Hydrilla verticillata*. Detailed Implementation

[0013] Before identifying the phylogenetic relationship of *Hydrilla verticillata*, it is necessary to extract the DNA of *Hydrilla verticillata*. The method is as follows: DNA is extracted from *Hydrilla verticillata* tissue samples using the Tiangen Plant DNA Extraction Kit. The specific method is to follow the instructions.

[0014] The screening and synthesis of microsatellite primers for identifying the phylogenetic relationships of *Hydrilla verticillata* are as follows: Primers containing microsatellite markers were designed from a transcriptome library of *Hydrilla verticillata*. Transcriptome sequencing was used to screen for unigenes containing *Hydrilla verticillata* microsatellite repeat sequences. Microsatellite-specific primers were then designed from sequences containing microsatellite loci detected in the *Hydrilla verticillata* transcriptome library, and their polymorphism was detected. After screening, 10 pairs of microsatellite markers were selected to form 5 duplex PCR systems. The selected microsatellite markers showed clear bands and high polymorphism, making them suitable as primers for identifying the phylogenetic relationships of *Hydrilla verticillata*. The primers were synthesized at Shanghai Sangon Biotech Co., Ltd.

[0015] The specific primer information is as follows: The microsatellite loci were used to perform PCR amplification on samples of *Hydrilla verticillata*, and the specific method is as follows: 1. Take a sample of *Hydrilla verticillata* to be tested and extract DNA from the sample; 2. PCR amplification was performed on *Hydrilla verticillata* samples using the microsatellite loci selected above; the PCR amplification products were separated by electrophoresis on a 10% polyacrylamide gel; the genotypes of the PCR amplification products were statistically analyzed based on the separation results. In step 2: (1) The PCR reaction system is 25ul: 10×PCR Buffer 3ul, 2.5mmol / L dNTP 2ul, MgCl2 3ul, upstream and downstream primers 1ul each, Taq enzyme 0.5ul, DNA template 2ul, ultrapure water 10.5ul; (2) The PCR reaction program is: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, annealing at 30s, extension at 72℃ for 30s, 35 cycles; 72℃ extension for 10min; store at 4℃.

[0016] The following example illustrates how the primers of this invention were used to identify the phylogenetic relationships among individuals of *Hydrilla verticillata*.

[0017] Example 1 Five areas of *Hydrilla verticillata* were randomly selected in Hongze Lake for sampling, with two samples collected from each area. Samples 1 and 2 were collected from the first area, samples 3 and 4 from the second area, samples 5 and 6 from the third area, samples 7 and 8 from the fourth area, and samples 9 and 10 from the fifth area.

[0018] DNA was extracted from 10 samples of *Hydrilla verticillata* using the methods described above.

[0019] The DNA of these 10 *Hydrilla verticillata* individuals was amplified using the PCR amplification method disclosed in the above technical solution and separated by electrophoresis. Based on the electrophoretic separation results, a clustering phylogenetic tree was constructed using mega5.0 software. The phylogenetic relationship between individuals was identified based on the cluster analysis diagram, and the closeness of the phylogenetic relationship among these 10 *Hydrilla verticillata* individuals was measured.

[0020] The phylogenetic results of these 10 individuals of *Hydrilla verticillata* are attached. Figure 1 As shown, the analysis appendix Figure 1 It can be seen that: samples 1 and 2 are closely related, samples 3 and 4 are relatively close, samples 5 and 6 are relatively close, samples 7 and 8 are relatively close, and samples 9 and 10 are relatively close.

[0021] Further analysis is as follows: (1) The individuals of *Hydrilla verticillata* in the second and third water areas converged together, indicating that the individuals of *Hydrilla verticillata* in the second and third water areas were closely related; (2) The individuals of *Hydrilla verticillata* in the fourth and fifth water areas converged together, and then converged with the individuals of *Hydrilla verticillata* in the first water area, indicating that the individuals of *Hydrilla verticillata* in the fourth and fifth water areas were closely related, and then converged with the individuals of *Hydrilla verticillata* in the first water area.

[0022] The above identification methods can be used to determine the phylogenetic relationships between samples of *Hydrilla verticillata*, allowing for the selection of different *Hydrilla verticillata* populations for cultivation.

[0023] When it is necessary to screen for fast-growing *Hydrilla verticillata*, the general steps are as follows: (1) Obtain fast-growing *Hydrilla verticillata* as a standard sample, extract its DNA, and perform PCR amplification using the microsatellite primers of this invention to obtain the amplification results of the standard sample; (2) Take samples of *Hydrilla verticillata* from the waters to be screened and extract its DNA, and perform PCR amplification using the microsatellite primers of this invention to obtain the amplification results of the sample to be screened; (3) Separate the amplification products of the standard sample and the amplification products of the sample to be screened by electrophoresis under the same conditions to obtain the electrophoretic separation results of the standard sample and the electrophoretic separation results of the sample to be screened; (4) Use mega5.0 software to perform a clustering development tree on the electrophoretic separation results of the sample to be screened and the electrophoretic separation results of the standard sample, and identify the kinship between the sample to be screened and the standard sample based on the cluster analysis diagram; (5) Based on the above results, screen *Hydrilla verticillata* populations that are closely related to the standard sample, and further screen waters with high-quality *Hydrilla verticillata* populations.

Claims

1. A primer for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers, characterized in that, The primers described are five sets of dual-PCR microsatellite primers. The information for these five sets of dual-PCR microsatellite primers is as follows:

2. A method for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers, characterized in that... The method for identifying the phylogenetic relationships of *Hydrilla verticillata* includes the following steps: S1. Delineate the sampling area for *Hydrilla verticillata*, obtain *Hydrilla verticillata* samples from the sampling area, and extract DNA from the *Hydrilla verticillata* samples. S2. Using the 5 sets of dual PCR primers described in claim 1, the DNA of the *Hydrilla verticillata* sample obtained in step S1 is amplified by PCR to obtain PCR amplification products. S3. Separate the PCR amplification products obtained in step S2 by electrophoresis to obtain the electrophoretic separation results; S4. Analyze the electrophoretic separation results obtained in step S3 using analysis software to determine the phylogenetic relationship between the *Hydrilla verticillata* samples.

3. The method for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers according to claim 2, characterized in that... In step S2, the PCR reaction system used for PCR amplification is 25 μL: 10×PCR Buffer 3 μL, 2.5 mmol / L dNTP 2 μL, MgCl2 3 μL, upstream and downstream primers 1 μL each, Taq enzyme 0.5 μL, DNA template 2 μL, and ultrapure water 10.5 μL.

4. The method for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers according to claim 2, characterized in that, In step S2, the PCR amplification procedure is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, annealing at 30 s, extension at 72℃ for 30 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃.

5. The method for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers according to claim 2, characterized in that, In step S3, the PCR amplification products are separated by electrophoresis on a 10% polyacrylamide gel.

6. The method for identifying the phylogenetic relationships of *Hydrilla verticillata* using microsatellite markers according to claim 2, characterized in that, In step S3, the analysis software used is mega5.0 software; the electrophoretic separation results obtained in step S4 are used to perform clustering development tree analysis on mega5.0 software, and the phylogenetic relationship between the *Hydrilla verticillata* samples is obtained based on the clustering analysis diagram.

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