Microsatellite primer for identifying siniperca chuatsi and siniperca chuatsi germplasm and identification method

Abstract

The invention discloses a microsatellite primer for identifying germplasm of siniperca chuatsi and siniperca chuatsi and an identification method, and aims to effectively distinguish the siniperca chuatsi from the siniperca chuatsi The microsatellite primer comprises nucleotide sequences as shown in SEQ ID NO. 1-20, a nucleotide sequence as shown in SEQ ID NO. The identification method comprises the following steps: carrying out PCR amplification on extracted DNA of a sample to be identified, a siniperca chuatsi reference sample and a siniperca chuatsi reference sample by using the microsatellite primer, carrying out electrophoretic separation on the obtained amplification product, carrying out clustering analysis on the electrophoretic separation result, and judging that the sample to be identified is siniperca chuatsi if the sample to be identified is clustered to the siniperca chuatsi reference sample. The sample to be identified is clustered to the siniperca chuatsi reference sample, and the siniperca chuatsi reference sample is judged to be The method can accurately, quickly and effectively identify the germplasm of the siniperca chuatsi and the siniperca chuatsi, and provides technical support for field germplasm resource investigation of the siniperca chuatsi and the siniperca

Description

Technical Field

[0001] The invention relates to the field of animal molecular genetics, and in particular to a microsatellite primer and an identification method for identifying the germplasm of mandarin fish and big-eyed mandarin fish. Background Art

[0002] Mandarin fish (Siniperca chuatsi) belongs to the order Perciformes, subfamily Sinipercinae, genus Siniperca, and is a freshwater benthic carnivorous fish. It is large in size, fast in growth, and delicious in taste, and is an important valuable economic fish and aquaculture target in my country. However, in recent years, due to overfishing of rivers, deterioration of the ecological environment in waters, and inbreeding in aquaculture production, the natural germplasm resources have declined, the traits of aquaculture germplasm have degenerated, and fish diseases have continued to break out, which has affected the continuation of Mandarin fish germplasm and the normal development of its aquaculture industry.

[0003] The big-eyed mandarin fish also belongs to the genus Siniperca chuatsi, commonly known as sweet-scented osmanthus fish, white osmanthus, etc. It is widely distributed in the Yangtze River and the water system to its south in China, and is a species endemic to southern China. In recent years, due to human influence, the wild resources of big-eyed mandarin fish and other mandarin fish have declined sharply. The big-eyed mandarin fish was described and classified as a species in the early 20th century. Since then, scientific researchers have conducted relevant research on its morphological characteristics and classification, biology and breeding.

[0004] The big-eyed mandarin fish and the mandarin fish are very similar in appearance, and traditional taxonomy believes that they are different species. However, recently, some scholars have studied their systematic relationship from the aspects of morphology, cytology and molecular biology and found that the two mandarin fish have not yet reached species differentiation. In terms of the morphological differences between the big-eyed mandarin fish and the mandarin fish, researchers used multivariate analysis to find that although there is a certain degree of differentiation between the two, there is also a large amount of overlap, indicating that they are closely related. In the study of the chromosomes of the genus Mandarin fish, it was found that the chromosome karyotypes of the two mandarin fish were very similar, which also proved that the two mandarin fish were closely related. From the perspective of molecular biology research, the mtDNA structure of the two fish is very similar; the structure of their mitochondrial control region is extremely similar, and there is no obvious geographical structure and pedigree structure; the phylogenetic relationship of 9 mandarin fish species was analyzed using the complete sequence of the cytochrome b gene, and it was found that the genetic distance between the big-eyed mandarin fish and the mandarin fish was only 0.015-0.020, which is much smaller than the genetic distance between other species. The above studies all show that the two are very closely related and have not reached species differentiation. Therefore, it is urgent to develop a material and method for identifying mandarin fish and big-eyed mandarin fish to effectively distinguish mandarin fish and big-eyed mandarin fish. Summary of the invention

[0005] Studies on the morphology, cytology and molecular biology of mandarin fish and big-eyed mandarin fish have found that these two species of mandarin fish have not yet reached species differentiation, so it is necessary to propose a substance and method for identifying the germplasm of mandarin fish and big-eyed mandarin fish to effectively distinguish between mandarin fish and big-eyed mandarin fish.

[0006] The technical solution used to achieve the first invention objective of the present invention is: a microsatellite primer for identifying the germplasm of mandarin fish and big-eyed mandarin fish, comprising: a nucleotide sequence shown in SEQ ID NO.1-20.

[0007] The technical solution used to achieve the second invention objective of the present invention is: a method for identifying the germplasm of mandarin fish and big-eyed mandarin fish, comprising the following steps: S1. Extracting DNA from samples to be identified, reference samples of mandarin fish and reference samples of big-eyed mandarin fish; S2, using a microsatellite primer for identifying the germplasm of mandarin fish and big-eyed mandarin fish as described in the above technical solution to perform PCR amplification on the three sample DNAs obtained in step S1 to obtain amplified products; S3, performing electrophoresis separation on a 10% polyacrylamide gel on the PCR amplification product obtained in step S2 to obtain an electrophoresis separation result; S4. According to the electrophoresis separation result of step S3, the genotypes of the PCR amplification products of the sample to be identified, the mandarin fish reference sample and the big-eyed mandarin fish reference sample are counted; S5. Perform cluster analysis on the genotype results obtained in step S4 using genetic analysis software: if the clustering result of the sample to be identified is clustered to the mandarin fish reference sample, the sample to be identified is mandarin fish; if the clustering result of the sample to be identified is clustered to the big-eye mandarin fish reference sample, the sample to be identified is big-eye mandarin fish.

[0008] Compared with the prior art, the present invention has the following advantages: (1) effectively distinguishing between Siniperca chuatsi and Siniperca chuatsi by molecular means; (2) The PCR amplification system of the present invention saves half the cost compared with the traditional PCR system; (3) The method for identifying mandarin fish and big-eyed mandarin fish of the present invention is more accurate than the traditional method.

[0009] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Attached Figure 1 This is a cluster analysis diagram of Example 1 of the present invention.

[0011] Attached Figure 2 This is a cluster analysis diagram of Example 2 of the present invention. DETAILED DESCRIPTION

[0012] The present invention aims to effectively distinguish between Siniperca chuatsi and Siniperca chuatsi by molecular means. To achieve this goal, microsatellite primers for identifying Siniperca chuatsi and Siniperca chuatsi germplasm are first designed. The design method is as follows: The genome data of Siniperca scherzeri were downloaded from NCBI database. The genome data were analyzed by SSRHunter software. The fragments with microsatellite sequences were screened. 200 pairs of microsatellite sequences with repeat sequences of more than 3 bases were randomly selected. Primer 5.0 software was used to design primers for the above 200 pairs of microsatellites.

[0013] The principles of primer design are as follows: a. The primer sequence should be neither too close nor too far from the core sequence, and should be approximately 50-80 bases away; b. The target fragment (PCR product) is of appropriate size, about 100-400 bp; c. The annealing temperature of the primer sequence is between 50℃-60℃; d. The primer sequence must contain G and C, and the content should be between 40% and 60%.

[0014] During the primer design process, the conditions can be appropriately widened to screen suitable primers.

[0015] Fin rays of six mandarin fish and big-eye mandarin fish were taken, and PCR amplification and electrophoresis were performed on the six mandarin fish and big-eye mandarin fish respectively using the above 200 pairs of microsatellite primers. The PCR amplification reaction system was 25ul: 10×PCR Buffer 3ul, 2.5mmol / L dNTP 1ul, MgCl2 3ul, 1ul of upstream and downstream primers of the primer, 1ul of Taq enzyme, 2ul of DNA template, and 14ul of ultrapure water; the PCR amplification reaction program was: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 45 seconds, for a total of 35 cycles; finally extension at 72℃ for 10 minutes and storage at 4℃.

[0016] Through the above operations, microsatellite primers that can amplify PCR products in both mandarin fish and big-eyed mandarin fish were found, and the microsatellite primers were combined for double PCR test. The test method is: the screened microsatellite primers were combined in pairs, and then the combined primer sets were used to perform PCR amplification and electrophoresis on 6 mandarin fish and big-eyed mandarin fish respectively. The PCR amplification reaction system is 25ul: 10×PCR Buffer 3ul, 2.5mmol / L dNTP 1ul, MgCl23ul, 1ul of upstream and downstream primers of two pairs of primers, 1ul of Taq enzyme, 2ul of DNA template, and 12ul of ultrapure water; the PCR amplification reaction program is: pre-denaturation at 94℃ for 3 minutes; then denaturation at 94℃ for 30 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 45 seconds, for a total of 35 cycles; finally extension at 72℃ for 10 minutes and storage at 4℃.

[0017] Finally, 5 suitable dual PCR primer sets that can simultaneously amplify in mandarin fish and big-eyed mandarin fish were found, which are the 5 dual PCR systems of the present invention, and their nucleotide sequences are specifically: the nucleotide sequences shown in SEQ ID NO.1-20.

[0018] The detailed information of the five sets of dual PCR systems is shown in Table 1: Table 1. Sequence information of 5 sets of duplex PCR systems .

[0019] In the 5 sets of double PCR systems shown in Table 1, in set 1: GHDYG1F corresponds to the nucleotide sequence shown in SEQ ID NO.1; GHDYG1R corresponds to the nucleotide sequence shown in SEQ ID NO.2; GHDYG2F corresponds to the nucleotide sequence shown in SEQ ID NO.3; GHDYG2R corresponds to the nucleotide sequence shown in SEQ ID NO.4; Group 2: GHDYG3F corresponds to the nucleotide sequence shown in SEQ ID NO.5; GHDYG3R corresponds to the nucleotide sequence shown in SEQ ID NO.6; GHDYG4F corresponds to the nucleotide sequence shown in SEQ ID NO.7; GHDYG4R corresponds to the nucleotide sequence shown in SEQ ID NO.8; Group 3: GHDYG5F corresponds to the nucleotide sequence shown in SEQ ID NO.9; GHDYG5R corresponds to the nucleotide sequence shown in SEQ ID NO.10; GHDYG6F corresponds to the nucleotide sequence shown in SEQ ID NO.11; GHDYG6R corresponds to the nucleotide sequence shown in SEQ ID NO.12; Group 4: GHDYG7F corresponds to the nucleotide sequence shown in SEQ ID NO.13; GHDYG7R corresponds to the nucleotide sequence shown in SEQ ID NO.14; GHDYG8F corresponds to the nucleotide sequence shown in SEQ ID NO.15; GHDYG8R corresponds to the nucleotide sequence shown in SEQ ID NO.16; Group 5: GHDYG9F corresponds to the nucleotide sequence shown in SEQ ID NO.17; GHDYG9R corresponds to the nucleotide sequence shown in SEQ ID NO.18; GHDYG10F corresponds to the nucleotide sequence shown in SEQ ID NO.19; GHDYG10R corresponds to the nucleotide sequence shown in SEQ ID NO.20.

[0020] The above microsatellite primers are used to identify the germplasm of Siniperca chuatsi and Siniperca chuatsi, including steps S1-S5, and the specific operations and conditions are as follows.

[0021] S1. Extract DNA from samples to be identified, Siniperca chuatsi reference samples and Siniperca chuatsi reference samples.

[0022] The genomic DNA of Siniperca chuatsi and Siniperca chuatsi was extracted using the tissue genomic DNA extraction kit of Tiangen Biochemical Technology Co., Ltd. The specific steps are as follows: (1) Take an appropriate amount of fin rays and place them in a sterile centrifuge tube containing 1 mL of 95% alcohol. Cut them into pieces to form a cell suspension. Centrifuge at 10,000 r / min for 1 min. Pour off the supernatant, add 200 μL of buffer GA, and vortex until completely suspended. (2) Add 20 ul of Proteinase K solution, mix well, and place at 56°C overnight to allow complete digestion. Centrifuge briefly to remove water droplets on the inner wall of the tube cap before proceeding to the next step; (3) Add 200 μl of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 min. The solution should become clear. Centrifuge briefly to remove water droplets on the inner wall of the tube cap. (4) Add 200 μl of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap. (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000 r / min for 30 sec, discard the waste liquid, and place the adsorption column CB3 back into the collection tube; (6) Add 500 μL of buffer GD to the adsorption column CB3 (check whether anhydrous ethanol has been added before use), centrifuge at 12,000 r / min for 30 sec, discard the waste liquid, and place the adsorption column CB3 in a collection tube; (7) Add 600 μl of rinse solution PW to the adsorption column CB3 (check whether anhydrous ethanol has been added before use), centrifuge at 12,000 r / min for 30 sec, discard the waste liquid, and place the adsorption column CB3 in a collection tube; (8) Repeat step 7; (9) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 r / min for 2 min, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material; (10) Transfer the adsorption column CB3 to a clean centrifuge tube, add 50-200ul of elution buffer TE to the middle part of the adsorption membrane, leave it at room temperature for 2-5 minutes, centrifuge it at 12000 r / min for 2 minutes, and collect the solution in a centrifuge tube. To increase the yield of genomic DNA, add the solution obtained by centrifugation to the adsorption column CB3, leave it at room temperature for 2 minutes, and centrifuge it at 12000 r / min for 2 minutes; (11) Detect DNA purity and concentration by UV spectrophotometer and agarose gel electrophoresis and store at -20°C until use.

[0023] S2. Using the five sets of double PCR systems of the present invention, PCR amplification is performed on the DNA of the sample to be identified, the mandarin fish reference sample and the big-eyed mandarin fish reference sample to obtain amplified products.

[0024] The PCR amplification reaction system was 25ul: 3ul of 10×PCR Buffer, 1ul of 2.5mmol / L dNTP, 3ul of MgCl2, 1ul of each of the upstream and downstream primers of the two pairs of primers, 1ul of Taq enzyme, 2ul of DNA template, and 12ul of ultrapure water; the PCR amplification reaction program was: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 45 seconds, for a total of 35 cycles; finally extension at 72°C for 10 minutes and storage at 4°C.

[0025] S3. Perform electrophoresis separation on a 10% polyacrylamide gel on the PCR amplification product obtained in step S2 to obtain an electrophoresis separation result.

[0026] S4. According to the electrophoresis separation results of step S3, the genotypes of the PCR amplification products of the sample to be identified, the mandarin fish reference sample and the big-eyed mandarin fish reference sample obtained in 5 sets of double PCR systems are counted.

[0027] S5. Perform cluster analysis on the genotype results obtained in step S4 by using the genetic analysis software MEGA to obtain the clustering results of the samples to be identified. When the clustering results of the samples to be identified are clustered to the mandarin fish reference sample, the samples to be identified are mandarin fish; if the clustering results of the samples to be identified are clustered to the big-eyed mandarin fish reference sample, the samples to be identified are big-eyed mandarin fish.

[0028] The feasibility and advantages of the microsatellite primers and identification method of the present invention are demonstrated by the following examples.

[0029] Example 1 10 samples were taken from each of the mandarin fish and bigeye mandarin fish farms, wherein the 10 mandarin fish samples were marked as No. 1-10, and the 10 bigeye mandarin fish samples were marked as No. 11-20. According to the germplasm identification method of the present invention, DNA of the 20 samples was first extracted, and then the 5 sets of double PCR systems of the present invention were used to perform PCR amplification on the extracted DNA of the 20 samples, and the amplified products were separated by electrophoresis on a 10% polyacrylamide gel. After completion, the genotypes of the 20 samples at 10 pairs of microsatellite loci were counted, and a cluster analysis diagram of the 20 samples was drawn, which is Figure 1 .

[0030] analyze Figure 1 The results showed that samples 1-10 (Siniperca chuatsi) converged into one branch, and samples 11-20 (Bigeye Siniperca chuatsi) converged into one branch.

[0031] The above results prove that the microsatellite primers and identification method for the germplasm identification of Siniperca chuatsi and Siniperca chuatsi of the present invention are effective and accurate.

[0032] Example 2 10 samples were taken from each of the mandarin fish farm and the big-eye mandarin fish farm, wherein the 10 mandarin fish samples were listed as 1-10, and the 10 big-eye mandarin fish samples were listed as 11-20; then 10 juvenile fish individuals suspected to be mandarin fish or big-eye mandarin fish were taken as samples to be identified, and the samples to be identified were listed as 21-30, and the microsatellite primers and identification method of the present application were used to perform germplasm identification on the 10 samples to be identified.

[0033] The identification results are as follows Figure 2 The cluster analysis diagram shown in the figure shows the Figure 2 , we get: samples 1-10, 25, 27, 29, 30, 22, 24 converge into one branch, and samples 11-20, 23, 26, 21, 28 converge into one branch. This result shows that samples 25, 27, 29, 30, 22, 24 and sample 1-10 are the same species, Siniperca chuatsi; samples 11-20, 23, 26, 21, 28 are the same species, Siniperca chuatsi.

[0034] The above results prove that the microsatellite primers and identification method for the germplasm identification of mandarin fish and big-eyed mandarin fish of the present invention can identify the germplasm of juvenile individuals of mandarin fish or big-eyed mandarin fish; and further prove that the present invention can effectively identify the germplasm of mandarin fish and big-eyed mandarin fish.

Claims

1. A microsatellite primer for identifying germplasm of Siniperca chuatsi and Siniperca chuatsi, characterized in that: The microsatellite primers include: nucleotide sequences shown in SEQ ID NO.1-20.

2. A microsatellite primer for identifying germplasm of Siniperca chuatsi and Siniperca chuatsi according to claim 1, characterized in that: The nucleotide sequences shown in SEQ ID NO.1-20 constitute 10 pairs of microsatellite primer sets; The 10 pairs of microsatellite primers constitute 5 sets of duplex PCR systems; The information of the 5 sets of dual PCR systems is: 。 3. A method for identifying the germplasm of Siniperca chuatsi and Siniperca chuatsi, characterized in that: The identification method comprises the following steps: S1. Extracting DNA from samples to be identified, reference samples of mandarin fish and reference samples of big-eyed mandarin fish; S2. Using the microsatellite primers for identifying the germplasm of mandarin fish and big-eyed mandarin fish as described in claim 1 or 2, PCR amplification is performed on the three sample DNAs obtained in step S1 to obtain amplified products; S3, performing electrophoresis separation on a 10% polyacrylamide gel on the PCR amplification product obtained in step S2 to obtain an electrophoresis separation result; S4. According to the electrophoresis separation result of step S3, the genotypes of the PCR amplification products of the sample to be identified, the mandarin fish reference sample and the big-eyed mandarin fish reference sample are counted; S5. Perform cluster analysis on the genotype results obtained in step S4 using genetic analysis software to obtain cluster results of the sample to be identified: if the cluster result of the sample to be identified is clustered to the mandarin fish reference sample, the sample to be identified is mandarin fish; if the cluster result of the sample to be identified is clustered to the big-eye mandarin fish reference sample, the sample to be identified is big-eye mandarin fish.

4. A method for identifying the germplasm of mandarin fish and bigeye mandarin fish according to claim 3, characterized in that: The reaction system of PCR amplification in step S2 is 25ul: 10×PCR Buffer 3ul, 2.5mmol / L dNTP 1ul, MgCl2 3ul, 1ul each of the upstream and downstream primers of two pairs of primers, 1ul Taq enzyme, 2ul DNA template, and 12ul ultrapure water.

5. A method for identifying the germplasm of Siniperca chuatsi and Siniperca chuatsi according to claim 3, characterized in that: In step S2, the reaction procedure of PCR amplification is: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 35 cycles; and finally extension at 72°C for 10 minutes and storage at 4°C.

6. A method for identifying germplasm of mandarin fish and bigeye mandarin fish according to claim 3, characterized in that: In step S5, the genetic analysis software used is MEGA software.

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