A microsatellite marker of enteromorpha linza, a primer set and a method for identifying a family of enteromorpha linza

Abstract

The outbreak of Enteromorpha linza has a great influence, and the growth condition of Enteromorpha linza in a target area needs to be judged, so the present application provides a microsatellite marker of Enteromorpha linza, a primer set and a method for identifying the family of Enteromorpha linza. The microsatellite marker of Enteromorpha linza is YGHT1, YGHT2, YGHT3, YGHT4, YGHT5, YGHT6, YGHT7, YGHT8, YGHT9 and YGHT10; the primer set for amplifying the above microsatellite marker of Enteromorpha linza has a nucleotide sequence of SEQ ID NO. 11~30. The method for identifying the family of Enteromorpha linza is: sampling and extracting DNA; applying the above primer set to perform PCR amplification on the DNA; drawing a cluster analysis graph and analyzing the family result of Enteromorpha linza in a to-be-monitored area after electrophoresis and silver staining. The present application can quickly identify the genetic relationship of Enteromorpha linza, judge the growth condition of Enteromorpha linza, and provide a theoretical basis in the judgment of Enteromorpha linza disaster.

Description

Technical Field This invention relates to a microsatellite marker, primer set, and method for identifying pedigrees of *Ulva rimosa*, belonging to the field of molecular marker technology. Background Technology *Ulva procumbens*, also known as sea lettuce, sea spinach, and sea green seaweed, has a yellowish-green to dark green thallus. It is unbranched, lanceolate, linear, or obovate, with highly variable shapes, often spirally twisted, with wavy edges, gradually tapering at the base to form a distinct stalk. It grows on rocks or in marshes in the intertidal zone of bays, on rocks or gravel near the high tide line, and sometimes epiphytically on other large thallus species. Its growth is most vigorous from late spring to summer.

[0001] Although *Ulva spp.* is non-toxic, like red tides, its massive proliferation can block sunlight and hinder the growth of other seabed algae. Outbreaks of *Ulva spp.* can also severely impact the landscape and disrupt tourism and water sports—precisely the most significant negative impacts that people are trying to eliminate. The growth of *Ulva spp.* must be kept within a controllable range; otherwise, outbreaks will cause enormous damage. Therefore, it is necessary to establish a pedigree determination method for *Ulva spp.* to assess its growth status in a test area. Based on the assessment results, it can be determined in advance whether the growth of *Ulva spp.* in that area needs to be controlled. Summary of the Invention Outbreaks of *Ulva marimoides* affect the growth of marine algae and the landscape, and interfere with tourism and water sports. It is necessary to assess the growth status of *Ulva marimoides* in monitored areas to provide a basis for evaluating *Ulva marimoides* disasters. To address this problem, this invention provides a microsatellite marker, primer set, and method for identifying *Ulva marimoides* families. This method helps to quickly and accurately identify different families of *Ulva marimoides*, thereby assessing their growth status and providing a theoretical basis for evaluating *Ulva marimoides* disasters.

[0002] The technical solution adopted by the present invention to achieve the first objective is: a microsatellite marker for *Ulva rimosa*, including: YGHT1, YGHT2, YGHT3, YGHT4, YGHT5, YGHT6, YGHT7, YGHT8, YGHT9 and YGHT10.

[0003] The nucleotide sequences of YGHT1 to YGHT10 are shown in SEQ ID NO.1 to 10, respectively.

[0004] The technical solution adopted by this invention to achieve the second objective is as follows: a microsatellite marker primer set for *Ulva prolifera*, used to amplify the microsatellite markers of *Ulva prolifera* described in the above technical solution, wherein the primer set includes: Primer pairs for amplifying YGHT1 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.11 and SEQ ID NO.12; Primer pairs for amplifying YGHT2 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.13 and SEQ ID NO.14; Primer pairs for amplifying YGHT3 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.15 and SEQ ID NO.16; Primer pairs for amplifying YGHT4 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.17 and SEQ ID NO.18; Primer pairs for amplifying YGHT5 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.19 and SEQ ID NO.20; Primer pairs for amplifying YGHT6 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.21 and SEQ ID NO.22; Primer pairs for amplifying YGHT7 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.23 and SEQ ID NO.24; Primer pairs for amplifying YGHT8 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.25 and SEQ ID NO.26; Primer pairs for amplifying YGHT9 include: upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.27 and SEQ ID NO.28; Primer pairs for amplifying YGHT10 include upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO.29 and SEQ ID NO.30.

[0005] The technical solution adopted by this invention to achieve the third objective is as follows: a method for identifying the pedigree of *Ulva rimosa*, comprising the following steps: S1. Collect leaf samples of *Ulva rimosum* from the area to be monitored and extract DNA; S2. Using the primer set in the above technical solution, perform double PCR amplification on the DNA of the *Ulva prolifera* sample extracted in step S1 to obtain the PCR amplification product. S3. Perform electrophoresis and silver staining on the PCR amplification products obtained in step S2 to obtain the silver staining results. S4. Analyze the silver staining results obtained in step S3 using genetic analysis software to obtain the genetic distance between the samples to be tested; S5. Draw a UPGMA cluster analysis diagram based on the genetic distance obtained in step S4. Based on the cluster analysis diagram, obtain the pedigree results of the *Ulva rimosa* in the region to be identified.

[0006] The beneficial effects of the present invention are: (1) By applying the microsatellite markers, primer sets and methods of the present invention for identifying the family lineage of *Ulva rimosa*, the kinship between *Ulva rimosa* samples in the area to be monitored can be obtained, and the genetic diversity of *Ulva rimosa* in the area to be identified can be determined; (2) Furthermore, the phylogenetic results can identify the phylogenetic lineages of *Ulva fasciata* with good reproductive capacity, thereby inferring the reproductive capacity of *Ulva fasciata* in the region, providing a theoretical basis for assessing *Ulva fasciata* disasters, and further determining whether it is necessary to control the reproduction of *Ulva fasciata* in the region based on the inference results. (3) The method for identifying the lineage of *Ulva rimosa* of the present invention can quickly identify the lineage of *Ulva rimosa* samples in the area to be monitored. The lineage results are consistent with the sampling records and have high accuracy. Attached Figure Description

[0007] Figure 1 This is a UPGMA clustering analysis diagram of the genetic distance of *Ulva rimosa* samples in an embodiment of the present invention. Detailed Implementation Development of microsatellite markers for *Ulva rimosa*: (1) The simplified genome data of *Ulva rimosa* from this experiment were used for analysis to screen microsatellite fragments on a large scale. Primers were designed using the software RIMER PREMIER 5.0 with the following design conditions: primer length 17-24 bp, GC content 40-60%, and target fragment length 100-700 bp. A total of 200 primer pairs were designed.

[0008] (2) PCR amplification and screening of the above primers were performed using DNA from one edge tube of *Ulva prolifera*. The PCR amplification reaction system was 25 μl: 3 μl of 10×PCR Buffer, 1 μl of 2.5 mmol / L dNTP, 3 μl of MgCl2, 1 μl each of the forward and reverse primers, 0.5 μl of 5 U / μl Taq enzyme, 2 μl of DNA template, and 13.5 μl of ultrapure water. 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℃. The PCR products were separated by electrophoresis on a 12% non-denaturing polyacrylamide gel, and photographed after silver staining; microsatellite markers with clear bands were screened.

[0009] (3) For the selected microsatellite markers, use 6 pairs of *Ulva prolifera* DNA to continue screening for microsatellite markers with clear bands in accordance with the above method.

[0010] (4) Finally, the selected microsatellite primers were combined in pairs, and the primer pairs with good microsatellite reactions, no primer dimers, and clear bands were further screened using 6 edge tube Ulva DNA samples. The specific PCR reaction screening conditions are as follows: The PCR amplification reaction system is 25 μl: 10×PCR Buffer 3 μl, 2.5 mmol / L dNTP 1 μl, MgCl2 3 μl, each of the upstream and downstream primers of the two primer pairs 1 μl, 5 U / μl Taq enzyme 0.5 μl, DNA template 2 μl, and ultrapure water 11.5 μl. The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 3 minutes; then 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 45 seconds, for a total of 35 cycles; finally, 72℃ extension for 10 minutes, and storage at 4℃. The PCR products were separated by electrophoresis on a 12% non-denaturing polyacrylamide gel, and photographed after silver staining.

[0011] Ten microsatellite markers for *Ulva rimosa* were obtained: YGHT1, YGHT2, YGHT3, YGHT4, YGHT5, YGHT6, YGHT7, YGHT8, YGHT9 and YGHT10, with nucleotide sequences shown in SEQ ID NO.1~10.

[0012] Ten pairs of primers were obtained to amplify the microsatellite markers of the above-mentioned *Ulva rimosa*, with nucleotide sequences of SEQ ID NO. 11~30. These primers were used to form five sets of double PCR reaction systems for *Ulva rimosa*. The specific primer set information is shown in Table 1.

[0013] Table 1. Basic information on 10 pairs of microsatellite primers for *Ulva rimosa*. .

[0014] The application of the microsatellite markers and primer sets of this invention to identify *Ulva mariflora* families within the area to be monitored includes the following steps: S1. Collect leaf samples of *Ulva rimosa* from the area to be identified as a family and extract DNA.

[0015] S1-1. Sampling of *Ulva rimosa*: Five monitoring points were selected in the area where *Ulva rimosa* needs to be monitored for sampling. Three samples were selected from each monitoring point, for a total of 15 samples, namely 1-3, 4-6, 7-9, 10-12, and 13-15.

[0016] DNA extraction from S1-2 edge tube Ulva samples: (A) Collect 5g of leaf samples from each of the 15 *Ulva rimosa* specimens. (B) Preparation of extraction buffer I: Mix 5 mL of 2×CTAB buffer and 80 μL of β-mercaptoethanol in a centrifuge tube and preheat in a water bath at 65°C before use; (C) Place 15 samples of *Ulva rimosa* leaflets in liquid nitrogen and grind them into powder. Transfer the powder to centrifuge tubes containing extraction buffer I, mix well, and incubate in a water bath at 60°C for 60 minutes, shaking occasionally. (D) Remove the centrifuge tube and cool it to room temperature. Centrifuge at 10,000 rpm for 10 minutes at room temperature. (E) Transfer the supernatant to another centrifuge tube, add an equal volume of a mixture of chloroform and isoamyl alcohol (24:1 volume ratio), mix for 10 minutes, then centrifuge at 10,000 rpm at room temperature for 10 minutes; repeat this step once. (F) Add 2 / 3 volume of isopropanol to the centrifuge tube, let stand at -20°C for at least 30 minutes, and centrifuge at 10000 rpm at 4°C for 10 minutes. (G) After discarding the supernatant, add 3 mL of 70% pre-cooled alcohol and slowly invert to wash the precipitate. After standing for 15 minutes, centrifuge at 10000 rpm and 4°C for 10 minutes. (H) Discard the supernatant and absorb the alcohol, dissolve it in 100 μL of distilled water, let it stand for more than 10 minutes, and then transfer it into a 1.5 mL EP tube; (I) Add an equal volume of a mixture of chloroform and isoamyl alcohol, with a volume ratio of chloroform to isoamyl alcohol of 24:1, extract for 10-15 minutes, and then centrifuge at 10000 rpm at room temperature for 10 minutes. (J) Take the supernatant, record the volume, add 2 / 3 volume of isopropanol and 1 / 10 volume of 3mol / L sodium acetate. Both isopropanol and sodium acetate are pre-cooled, mixed well, and allowed to stand at -20℃ for 30 minutes. (K) Centrifuge at 10,000 rpm for 10 minutes at 4°C to remove the precipitate. Wash the DNA slowly by inverting with 1 mL of 75% pre-cooled alcohol. Centrifuge again at 10,000 rpm for 10 minutes and discard the alcohol. (L) Repeat step J once; (M) Allow the DNA to air dry naturally until no alcohol adheres to the walls, then dissolve it in 100 μL of sterile distilled water and store it in a 4°C refrigerator to complete the extraction of genomic DNA from *Ulva prolifera*.

[0017] S2. The DNA of the *Ulva prolifera* sample extracted in step S1 was amplified by dual PCR using the five dual PCR systems of the present invention to obtain PCR amplification products. 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 MgCl2, 1 μl each of the upstream and downstream primers of the two pairs of primers, 0.5 μl of 5 U / μl Taq enzyme, 2 μl of DNA template, and 11.5 μl of ultrapure water. 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℃.

[0018] S3. Separate the PCR amplification products obtained in step S2 by electrophoresis with 12% non-denaturing polyacrylamide gel, and take pictures after silver staining.

[0019] S4. The clear microsatellite electrophoresis images of the 5 groups of *Ulva rimosa* double PCR reaction systems obtained in step S3 are digitally processed to obtain the genetic distance between the samples to be tested.

[0020] S5. Using MEGA software, we analyzed and calculated and constructed a UPGMA cluster analysis diagram to analyze the family structure of *Ulva prolifera* in the monitored area.

[0021] like Figure 1 As shown, the 15 samples of *Ulva rimosa* clustered into 5 large branches: samples 4, 5, and 6 clustered into one branch; samples 10, 11, and 12 clustered into one branch; samples 7, 8, and 9 clustered into one branch; samples 1, 2, and 3 clustered into one branch; and samples 13, 14, and 15 clustered into one branch.

[0022] Based on the above results, it can be preliminarily determined that these 15 samples of *Ulva rimosa* came from 5 different families: samples 4, 5, and 6 belonged to one family; samples 10, 11, and 12 belonged to another family; samples 7, 8, and 9 belonged to another family; samples 1, 2, and 3 belonged to another family; and samples 13, 14, and 15 belonged to yet another family. This result is consistent with the sampling results, indicating that individuals from one family converged on the same branch, proving the accuracy of this technique. This demonstrates that the *Ulva rimosa* microsatellite marker, primer set, and family identification method of this invention can be used for family identification of *Ulva rimosa* with high accuracy.

[0023] By applying the method of identifying the pedigree of *Ulva spp.* of this invention, after identifying the pedigree of *Ulva spp.* in a selected area (an area with abundant and well-grown *Ulva spp.*), *Ulva spp.* samples with good reproductive capacity (those with a large number of individuals clustered on one branch) can be screened out, or a certain *Ulva spp.* sample is known to have good reproductive capacity. Then, the above *Ulva spp.* samples with good reproductive capacity are used as reference samples, and the *Ulva spp.* samples in the area to be monitored are used as monitoring samples. The method of this invention is used to identify the kinship between the reference samples and the monitoring samples. If there are many *Ulva spp.* samples in the monitoring samples that belong to the same pedigree as the reference samples, it can be preliminarily inferred that most of the *Ulva spp.* in the area to be monitored have good reproductive capacity, and thus an opinion can be given on whether the reproduction of *Ulva spp.* in the area to be monitored needs to be controlled.

Claims

1. A microsatellite-labeled primer set for *Ulva rimosa*, characterized in that, The primer set information is as follows:

2. A method for identifying the pedigree of *Ulva marginalis*, characterized in that, The method for identifying the pedigree of *Ulva rimosa* includes the following steps: S1. Collect leaf samples of *Ulva rimosum* from the area to be monitored and extract DNA; S2. The DNA of the *Ulva prolifera* sample extracted in step S1 is amplified by double PCR using the primer set described in claim 1 to obtain the PCR amplification product. S3. Perform electrophoresis and silver staining on the PCR amplification products obtained in step S2 to obtain the silver staining results. S4. Analyze the silver staining results obtained in step S3 using genetic analysis software to obtain the genetic distance between the samples to be tested; S5. Draw a UPGMA cluster analysis diagram based on the genetic distance obtained in step S4. Based on the cluster analysis diagram, obtain the pedigree results of *Ulva rimosa* in the area to be monitored.

3. The method for identifying the pedigree of *Ulva marginalis* according to claim 2, characterized in that, In step S2, the PCR amplification reaction system is 25 μl: 10×PCR Buffer 3 μl, 2.5 mmol / L dNTP 1 μl, MgCl2 3 μl, forward and reverse primers 1 μl each, 5 U / μl Taq enzyme 0.5 μl, DNA template 2 μl, and ultrapure water 11.5 μl.

4. The method for identifying the pedigree of *Ulva marginalis* according to claim 2, characterized in that, In step S2, the PCR amplification reaction program 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℃.

5. The method for identifying the pedigree of *Ulva marginalis* according to claim 2, characterized in that, In step S3, 12% polyacrylamide gel is used for electrophoresis and silver staining.

6. The method for identifying the pedigree of *Ulva marginalis* according to claim 2, characterized in that, In step S4, the genetic analysis software used is MEGA software.

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