Microsatellite markers of pseudogobio vaillanti and their application in genetic relationship identification

Abstract

The application discloses a pseudoacrossocheilus nanjiangensis microsatellite marker and application thereof in kinship identification, and belongs to the field of genetic engineering. The pseudoacrossocheilus nanjiangensis microsatellite marker comprises 10 pairs of microsatellite primers, the 10 pairs of microsatellite primers constitute 5 groups of double-PCR, and the sequence of the microsatellite primers is shown in the sequence table 1. The fin sample of the pseudoacrossocheilus nanjiangensis is taken, the pseudoacrossocheilus nanjiangensis sample DNA is extracted from the pseudoacrossocheilus nanjiangensis fin sample, the pseudoacrossocheilus nanjiangensis sample is subjected to PCR amplification by using the above microsatellite primers, the PCR amplification product is subjected to electrophoresis separation on a 10% polyacrylamide gel, the genotype of the PCR amplification product is counted according to the separation result, the cluster analysis diagram is drawn by using genetic analysis software MEGA, and the kinship of the pseudoacrossocheilus nanjiangensis is identified according to the cluster analysis result. The application fills the blank of the pseudoacrossocheilus nanjiangensis kinship research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of animal molecular genetics, and relates to a microsatellite marker primer of Culter oxycephaloides, and also relates to application of the microsatellite marker of the Culter oxycephaloides in kinship identification. BACKGROUND

[0002] The Culter oxycephaloides belongs to the Cypriniformes, Cyprinidae, Cultrinae and Culter, and is commonly known as duck-billed red fin and sharp-headed red fin. The Culter oxycephaloides is distributed in the upper and middle reaches of the Yangtze River and its affiliated water bodies. The Culter oxycephaloides is a medium-sized economic fish, which is widely distributed in the Yangtze River Basin, grows fast, and has a maximum weight of more than 3 kg. The Culter oxycephaloides has tender and delicious meat, and is rarely found in fishing catches. As a native fish in the Hanjiang River, the Culter oxycephaloides grows fast, has tender and delicious meat, and is favored by consumers. The market price of the Culter oxycephaloides is much higher than that of the four major fishes. However, the Culter oxycephaloides is prohibited from being caught in the key water areas of the Yangtze River Basin. Therefore, the artificial breeding and cultivation of the Culter oxycephaloides have a broad market prospect. According to the data query, the artificial breeding of the Culter oxycephaloides is still blank in China. SUMMARY

[0003] The present application aims to provide a microsatellite marker primer of Culter oxycephaloides, which can fill the blank of the research on the kinship of the Culter oxycephaloides.

[0004] The technical solution adopted by the present application is that the microsatellite marker of the Culter oxycephaloides includes 10 pairs of microsatellite primers, the 10 pairs of microsatellite primers form 5 groups of double-PCR, and the sequence of the microsatellite primer is shown in the sequence table 1.

[0005] Another object of the present application is to provide application of the microsatellite marker of the Culter oxycephaloides in kinship identification.

[0006] Another technical solution adopted by the present application is that the microsatellite marker of the Culter oxycephaloides is applied in kinship identification.

[0007] The method comprises the following steps:

[0008] Step 1, a fin sample of the Culter oxycephaloides is taken, and sample DNA of the Culter oxycephaloides is extracted from the fin sample of the Culter oxycephaloides;

[0009] Step 2, the sample of the Culter oxycephaloides is subjected to PCR amplification by using the above microsatellite primer;

[0010] Step 3, the PCR amplification product is subjected to electrophoresis separation on a 10% polyacrylamide gel, the genotype of the PCR amplification product is counted according to the separation result, a cluster analysis diagram is drawn by using genetic analysis software MEGA, and the kinship of the Culter oxycephaloides is identified according to the cluster analysis result.

[0011] PCR reaction system in PCR amplification process is 25ul: 10x PCR Buffer 3ul, 2.5mmol / L dNTP 2ul, MgCl2 3ul, 1ul of each of the upper and lower primers of two pairs of primers, 0.5ul of Taq enzyme, 2ul of DNA template, 10.5ul of ultrapure water.

[0012] PCR reaction program is: 94 DEG C pre-denaturation 3min; 94 DEG C denaturation 30s, annealing temperature 30s, 72 DEG C extension 30s, 30 cycles; 72 DEG C extension 10min; 4 DEG C preservation.

[0013] The beneficial effects of the present application are: the present application fills the blank of the research on the genetic relationship of Pseudohemiculter tenuis; the application of the Pseudohemiculter tenuis microsatellite marker in genetic relationship identification can avoid the phenomenon of inbreeding of Pseudohemiculter tenuis in artificial breeding. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the cluster analysis result graph of the application of the Pseudohemiculter tenuis microsatellite marker in genetic relationship identification. DETAILED DESCRIPTION

[0015] The present application will be described in detail below in combination with the drawings and specific embodiments.

[0016] The Pseudohemiculter tenuis microsatellite marker of the present application comprises 10 pairs of microsatellite primers, and the 10 pairs of microsatellite primers constitute 5 groups of double PCR.

[0017]

[0018] Example 1

[0019] DNA extraction

[0020] 1) Take the tail fin tissue of Pseudohemiculter tenuis less than 0.5g, place it in a 1.5mL Eppendorf tube, mark the number, and then sequentially add 450ul of STE DNA extraction buffer (10mmol / L Tris-HCl, pH8.0; 1mmol / L EDTA, pH8.0), 35ul of SDS (10%) and 15ul of protease K (0.2%) into each tube and invert to mix;

[0021] 2) Put the product mixed in 1) into a 55 DEG C water bath, invert and mix every 30min until the solution is clear and transparent, which needs about 2h;

[0022] 3) Take out, add 1ul of RnaseA into each tube, and place it in a water bath at 37 DEG C for 1h.

[0023] 4) Take out the Eppendorf tube, add an equal volume (about 700ul) of Tris-saturated phenol, and mix well on the DNA mixer for 30min, note that it cannot be shaken left and right.

[0024] 5) Place the Eppendorf tube in the centrifuge, set 4°C, 12000r / min for 10min, after the end, use the pipette to transfer the supernatant into another clean Eppendorf tube, mark the number.

[0025] 6) Add an equal volume of phenol-isoamyl alcohol mixture (phenol, chloroform, isoamyl alcohol ratio is 25:24:1) to the supernatant, and mix well on the DNA mixer for 15min. Repeat step 5.

[0026] 7) Add an equal volume of chloroform about 500ul to the supernatant, mix well on the DNA mixer for 15min, and repeat step 5.

[0027] 8) Add 1mL of anhydrous ethanol pre-cooled at -20°C to precipitate DNA in the supernatant, centrifuge at 12000r for 5min, and remove the supernatant.

[0028] 9) First, wash twice with 70% ethanol, then wash once with anhydrous ethanol, dry, then add 200ul of TE, dissolve thoroughly. Use spectrophotometer to detect the concentration, and dilute each DNA sample to 50ng / ul working solution.

[0029] Example 2

[0030] DNA quality detection

[0031] The integrity and purity of DNA were detected by 1% agarose gel electrophoresis. The operation steps of agarose gel electrophoresis detection of DNA are as follows:

[0032] Prepare 0.8% agarose gel with 1xTAE electrophoresis buffer, microwave heating for 1min to dissolve, brush the outer bottle wall with tap water to cool, when the gel cools to about 50℃, add 2ul EB dyeing liquid in a conical flask, mix quickly, pour into a clean gel tank to make gel, gently pull out the comb after the gel solidifies, get agarose gel;

[0033] Add 1xTAE electrophoresis buffer to the horizontal electrophoresis apparatus in advance, place the agarose gel in the horizontal electrophoresis apparatus, make sure that the electrophoresis buffer completely covers the agarose gel.

[0034] Take 5ul of prepared DNA sample and mix with 1ul of 6xloading buffer, then accurately add it to the sample well, avoid moving the gel position during the process.

[0035] Set the electrophoresis apparatus voltage to 100V, and the electrophoresis time to about 20min.

[0036] After the electrophoresis is completed, the power of the electrophoresis instrument is turned off, the agarose gel is taken out, and the electrophoresis result is observed in a gel imaging system.

[0037] Example 3

[0038] Genetic relationship identification of Pseudohemiculter tenuis

[0039] Ten fishing points are selected in the Yangtze River to carry out fish catching, two Pseudohemiculter tenuis are selected from each point to carry out sampling, and the genetic relationship of the 20 Pseudohemiculter tenuis is identified by using the method provided in the application. The DNA of the 20 Pseudohemiculter tenuis is extracted by using the method in Example 1. The DNA of all Pseudohemiculter tenuis is subjected to PCR amplification by using the 5 sets of double-PCR marker primers provided in the application. The PCR amplification products are subjected to polyacrylamide gel electrophoresis. The electrophoresis result is subjected to silver staining, the silver staining result is subjected to data separation, the genotypes of the PCR amplification products are counted according to the separation result, the cluster analysis graph is drawn by using the genetic analysis software MEGA software, and the genetic relationship of Pseudohemiculter tenuis is identified according to the cluster analysis result. As shown in the cluster analysis result, Figure 1 the 20 Pseudohemiculter tenuis individuals are divided into 10 branches, 1 and 2 are a branch, 3 and 4 are a branch, 5 and 6 are a branch, 7 and 8 are a branch, 9 and 10 are a branch, 11 and 12 are a branch, 13 and 14 are a branch, 15 and 16 are a branch, 17 and 18 are a branch, and 19 and 20 are a branch. The result is consistent with the sampling result, and it is indicated that the method can be used for genetic relationship identification of Pseudohemiculter tenuis.

Claims

1. Microsatellite-labeled primers for *Pseudo-sharp-headed tuna*, characterized in that, There are 10 pairs of microsatellite marker primers for *Pseudo-sharp-headed tanukii*, and the sequences of the microsatellite primers are shown in the table below: The 10 pairs of microsatellite primers were used to form 5 sets of duplex PCR.

2. The microsatellite marker primers for *Pseudo-sharp-headed tuna* as described in claim 1, characterized in that, The microsatellite marker primers for *Culter alburnus* were used to identify the phylogenetic relationships of *Culter alburnus*.

3. A method for identifying the phylogenetic relationship of *Pseudo-sharphead*, characterized in that, The application of the microsatellite marker primers for *Pseudo-sharp-headed tuna* as described in claim 2 specifically includes the following steps: Step 1: Take a sample of the fin rays of *Pseudo-sharphead shad* and extract DNA from the sample. Step 2: Perform PCR amplification on the *Pseudo-sharphead* sample using the microsatellite primers described in claim 1; Step 3: Separate the PCR amplification products by electrophoresis on a 10% polyacrylamide gel, and count the genotypes of the PCR amplification products based on the separation results; use the genetic analysis software MEGA to draw a cluster analysis diagram, and identify the phylogenetic relationships of the false sharphead trevally based on the cluster analysis results. The PCR reaction system in the PCR amplification process is 25ul: 10×PCR Buffer 3ul, 2.5mmol / L dNTP 2ul, MgCl2 3ul, each of the upstream and downstream primers of the two pairs of primers 1ul, Taq enzyme 0.5ul, DNA template 2ul, and ultrapure water 10.5ul. The PCR reaction program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, annealing at 30 s, extension at 72℃ for 30 s, 30 cycles; 72℃ extension for 10 min; storage at 4℃.

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