A primer and method for rapid detection of sex of grass carp fry

Abstract

The present invention provides a primer and method for quickly detecting the sex of grass carp fry, and relates to the field of biological detection technology. The method for quickly detecting the sex of grass carp fry specifically comprises the following steps: Step 1, sample acquisition: obtain several grass carps, determine the male and female individuals by gonad morphology, extract tissue samples of the male and female individuals respectively and store them at low temperature; Step 2, extract total DNA of the sample to be tested: extract muscle tissue genomic DNA of 3 female fish and 3 male fish using TIANamp Marine Animals DNA kit, measure the concentration and OD260 / OD280 ratio of DNA using NanoDrop2000 ultra-micro spectrophotometer, and detect the integrity of DNA by 1.0% agarose electrophoresis; Step 3, primer detection. The primer and detection method proposed by the present invention can avoid the difficulty of conventionally killing fish to obtain gonads, and the removed gonads also need to be paraffin sliced ​​to identify the male and female, which is a cumbersome process, and realize that the sex of grass carp fry can be identified by only one PCR amplification and agarose gel electrophoresis detection.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a primer and a method for quickly detecting the sex of grass carp fry. Background Art

[0002] Grass carp (Ctenopharyngodon idellus) is the first of my country's "four major carps". It has the characteristics of fast growth, good meat quality, easy breeding, strong adaptability, etc., and its bait source is wide, suitable for breeding in rural areas. It is the freshwater farmed fish with the highest output in my country. In 2023, the annual output of grass carp will reach 5.94 million tons, accounting for 21.4% of the country's freshwater farmed fish output.

[0003] Grass carp has a relatively long growth cycle, usually taking 3-4 years to reach sexual maturity. In mature grass carp, male and female individuals can be distinguished by observing features such as mouth shape, genital pores, and abdominal morphology; however, in the juvenile stage, especially in juveniles under 1 year old, the morphological differences between male and female individuals are not obvious, and the testes of male fish only develop to stage II, and the ovaries develop to stage I, and the gonads are in the shape of thin lines. It is difficult to kill the fish and remove the gonads, and the removed gonads also need to be paraffin-sectioned to identify the sex, which is a cumbersome process. Therefore, it is very necessary to develop a method that can quickly identify the genetic sex of grass carp juveniles. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the shortcomings of the prior art, the present invention provides a primer and method for quickly detecting the sex of grass carp fry, which solves the problem that it is difficult to kill the fish and take out the gonads, and the removed gonads also need to be made into paraffin sections to identify the sex, which is a cumbersome process.

[0006] (II) Technical solution

[0007] To achieve the above object, the present invention is implemented by the following technical scheme: a primer for rapid detection of the sex of grass carp fry: the primer is CL-2F / CL-2R;

[0008] Among them, CL-2F: 5′-AGATGTCCTCGTGAAGTCAA-3′;

[0009] CL-2R:5'-CATAACAGAGCCTAAGTGCC-3'.

[0010] Preferably, a method for quickly detecting the sex of grass carp fry comprises the following steps:

[0011] Step 1: Sample acquisition:

[0012] Several grass carps were obtained, and the sexes were determined by gonad morphology. Tissue samples were extracted from the male and female individuals and stored in a -20°C refrigerator.

[0013] Step 2: Extract the total DNA of the sample to be tested:

[0014] The TIANamp Marine Animals DNA kit was used to extract genomic DNA from muscle tissues of three female and three male fish. The DNA concentration and OD260 / OD280 ratio were measured by NanoDrop2000 ultra-micro spectrophotometer, and the integrity of the DNA was detected by 1.0% agarose gel electrophoresis.

[0015] Step 3: Primer detection:

[0016] The primers are used to perform PCR amplification on the sample to be tested, and the amplified product is visualized as an amplified band in the agarose gel electrophoresis diagram, thereby detecting and determining the gender of the sample to be tested;

[0017] The amplification reaction system is:

[0018] PCR reaction system 50 μL, including 2× Taq PCR MasterMix 25 μL, DNA 1 μL, primer F 1 μL, primer R 1 μL, ddH2O 22 μL;

[0019] The PCR product was detected and observed by 1% agarose gel electrophoresis. The gel was run at 120V for 30 minutes. The agarose gel was placed in a gel imaging system to observe the presence of bands, and then the gender of the sample to be tested was determined.

[0020] Preferably, the amplification reaction conditions in the step 3 are pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 1 min, and after 35 cycles, extension at 72°C for 5 min.

[0021] Preferably, the method for determining the sex of the sample to be tested in step 3 comprises detecting the PCR amplification product by agarose gel electrophoresis, and if the obtained agarose gel electrophoresis diagram contains an amplification band, the sample to be tested is judged to be male.

[0022] (III) Beneficial effects

[0023] The present invention provides a primer and method for quickly detecting the sex of grass carp fry. It has the following beneficial effects:

[0024] The invention provides a primer and a method for quickly detecting the sex of grass carp fry. DNA is extracted from muscle tissue of grass carp for PCR amplification and agarose gel electrophoresis detection. The PCR primers provided by the invention are used to identify the sex of grass carp according to whether amplified bands appear in agarose gel electrophoresis detection. This greatly avoids the difficulty of conventionally killing fish to obtain gonads, and the extracted gonads also need to be sliced ​​into paraffin to identify the sex, which is a cumbersome process. The sex of grass carp fry can be identified by only one PCR amplification and agarose gel electrophoresis detection. That is, the genetic sex of grass carp can be identified quickly, accurately and at low cost by using the marker, and the operation is simple and the actual use is more economical and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the PCR amplification results of the grass carp sex marker candidate primers of the present invention, wherein (a) is the amplification result of CL-1F / CL-1R, (b) is the amplification result of CL-2F / CL-2R, (c) is the amplification result of CL-1F / CL-3R, and (d) is the amplification result of CL-3F / CL-4R;

[0026] Figure 2 Schematic diagram of the PCR amplification verification results of the grass carp-specific sex marker primers of the present invention, wherein (e) is another example of the amplification result of CL-2F, and (f) is another example of the amplification result of CL-2R. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The embodiment of the present invention provides a primer for quickly detecting the sex of grass carp fry, the primer is CL-2F / CL-2R;

[0029] Among them, CL-2F: 5′-AGATGTCCTCGTGAAGTCAA-3′;

[0030] CL-2R:5'-CATAACAGAGCCTAAGTGCC-3'.

[0031] Based on the primer, a method for quickly detecting the sex of grass carp fry is proposed, which specifically includes the following steps:

[0032] Step 3: Primer detection:

[0033] Primers CL-2F: 5'-AGATGTCCTCGTGAAGTCAA-3'; CL-2R: 5'-CATAACAGAGCCTAAGTGCC-3' were used to perform PCR amplification on the sample to be tested, and the amplified product was visualized by the amplified band in the agarose gel electrophoresis diagram, and then the gender of the sample to be tested was determined;

[0034] The amplification reaction system is:

[0035] PCR reaction system 50 μL, including 2× Taq PCR MasterMix 25 μL, DNA 1 μL, primer F 1 μL, primer R 1 μL, ddH2O 22 μL;

[0036] The amplification reaction conditions were pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 1 min, and after 35 cycles, extension at 72°C for 5 min;

[0037] The PCR product was detected and observed by 1% agarose gel electrophoresis, that is, 1% agarose gel was prepared, 4 μl of PCR reaction solution and DL2000 marker were added to the agarose gel wells, and the gel was run at 120 V for 30 minutes. The agarose gel was placed in a gel imaging system to observe the presence of the band. If the amplified band was present, the sample to be tested was judged to be male.

[0038] Example:

[0039] The acquisition and design of the primers and the specific extension methods include:

[0040] 1. Sample collection:

[0041] Thirty two-year-old grass carps were purchased from a farm. After dissection and observation of gonadal morphology, it was determined that there were 13 females and 17 males and females, respectively. A small amount of muscle tissue was taken from each sample and stored in a -20°C refrigerator.

[0042] 2. DNA extraction:

[0043] The phenol-chloroform method was used to extract DNA from fish muscle tissue as follows:

[0044] 1) Use scissors to take a small amount of muscle tissue and put it into a 1.5ml centrifuge tube, chop it into small pieces, and add 500μl STE rinse solution.

[0045] 2) At room temperature, centrifuge at 10,000 rpm for 1 min, collect the precipitate, add 500 μl lysis buffer and 10 μl proteinase K, shake gently, and digest in a 55°C water bath for 4 h, shaking once every 2 h.

[0046] 3) Add an equal volume of DNA extraction buffer, slowly invert the tube back and forth for 10 minutes, and centrifuge at 10,000 rpm for 10 minutes at 4°C.

[0047] 4) Transfer the upper aqueous phase to a new 1.5 ml centrifuge tube, add an equal volume of DNA extraction solution: chloroform (1:1), invert for 10 minutes, and centrifuge at 10,000 rpm for 7 minutes at 4°C.

[0048] 5) Transfer the upper aqueous phase to a new 1.5 ml centrifuge tube, add an equal volume of chloroform (500 μl), invert for 10 min, centrifuge at 10,000 rpm for 5 min at 4°C, and take the supernatant solution.

[0049] 6) Add twice the volume of pre-cooled anhydrous ethanol, shake gently to precipitate DNA, centrifuge at 12000rpm for 4min at 4℃, rinse twice with 1ml70% ethanol, dry at room temperature for 30min, add 50μl of sterile water, and store at -20℃ for later use.

[0050] 7) Prepare 1% agarose gel, take 4 μl DNA and 1 μl 6× DNA Buffer and mix them evenly. Then add the mixture into the agarose gel wells and run the gel at 150 V for 12 minutes. Place the agarose gel into the gel imaging system to determine the integrity of the DNA bands.

[0051] 3. Primer design:

[0052] The DNA of 10 female fish (F1-F10) and 10 male fish (M1-M10) were randomly selected for whole genome resequencing. The resequencing data of F1-F10 and M1-M10 were split into a short sequence primer set of L=20 bp sequence, and each short sequence in the array was queried in the F1-F3 and M1-M3 sequencing data in turn. If the primer can be found in all male (M1-M10) resequencing data but not in female (F1-F10) resequencing data, the primer sequence is used as a candidate male-specific primer sequence. The candidate primer sequences are shown in Table 1.

[0053] Table 1 Candidate sex-specific primers for grass carp

[0054] Primers Primer (F) CL-1F 5'-GAGGCAGCCAATAAGGAGTC-3' CL-2F 5'-AGATGTCCTCGTGAAGTCAA-3' CL-3F 5'-CACAGCCCAGACTGAGATAG-3' CL-1R 5'-CTTGTTTCAGCGGCAGGTGT-3' CL-2R 5'-CATAACAGAGCCTAAGTGCC-3' CL-3R 5'-TGTTTCAGCGGCAGGTGTTT-3' CL-4R 5'-ACTCAAGAAGACAAACACCC-3'

[0055] 4. PCR detection:

[0056] PCR amplification was performed according to primers CL-1F / CL-1R, CL-2F / CL-2R, CL-1F / CL-3R, and CL-3F / CL-4R. The PCR reaction system was 50 μL, including 2×Taq PCR MasterMix 25 μL, DNA 1 μL, primer F 1 μL, primer R 1 μL, and ddH2O 22 μL. The PCR reaction conditions were 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 59°C annealing for 30 s, 72°C extension for 1 min, and 72°C extension for 5 min after 35 cycles. The PCR product was detected and observed by 1% agarose gel electrophoresis, that is, 1% agarose gel was prepared, 4 μl of PCR reaction solution and DL2000 marker were added to the agarose gel wells, and the gel was run at 120 V for 30 minutes. The agarose gel was placed in a gel imaging system to observe the presence of bands.

[0057] 5. PCR results:

[0058] As attached Figure 1 As shown, the gel electrophoresis results showed that primer CL-2F / CL-2R had an amplified band in male fish but no amplified band in female fish, while the other three pairs of primers CL-1F / CL-1R, CL1F / CL-3R, and CL-3F / CL-4R had amplified bands in both male and female fish; primer CL-2F / CL-2R can be used for genetic sex identification of grass carp.

[0059] Specifically, 60 2-year-old grass carps were purchased, and their gonads were dissected to determine their physiological sex. Then, 20 male and 20 female individuals were taken to extract muscle genomic DNA, and PCR amplification was performed using primers CL-2F / CL-2R. The PCR reaction system and reaction conditions were the same as above. Figure 2 As shown, the results show that this primer also has specific amplification bands only in male fish, and the accuracy of genetic sex identification is 100%. This marker can be used to identify the genetic sex of grass carp quickly, accurately and at low cost.

[0060] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for quickly detecting the sex of grass carp fry, characterized in that: The specific steps include: Step 1: Sample acquisition: Several grass carps were obtained, and the sexes were determined by gonad morphology. Tissue samples were extracted from the male and female individuals and stored in a -20°C refrigerator. Step 2: Extract the total DNA of the sample to be tested: The TIANamp Marine Animals DNA kit was used to extract genomic DNA from muscle tissues of three female and three male fish. The DNA concentration and OD260 / OD280 ratio were measured by NanoDrop2000 ultra-micro spectrophotometer, and the integrity of the DNA was detected by 1.0% agarose gel electrophoresis. Step 3: Primer detection: The primers CL-2F / CL-2R were used to perform PCR amplification on the sample to be tested, and the amplified product was visualized by the amplified band in the agarose gel electrophoresis diagram, and the gender of the sample to be tested was determined by detection; Among them, CL-2F: 5′-AGATGTCCTCGTGAAGTCAA-3′; CL-2R:5'-CATAACAGAGCCTAAGTGCC-3'; The amplification reaction system of the PCR amplification is: PCR reaction system 50 μL, including 2× Taq PCR MasterMix 25 μL, DNA 1 μL, primer F 1 μL, primer R 1 μL, ddH2O 22 μL; The PCR product was detected and observed by 1% agarose gel electrophoresis. The gel was run at 120V for 30 minutes. The agarose gel was placed in a gel imaging system to observe the bands and determine the gender of the sample to be tested. The method for determining the sex of the sample to be tested in step 3 includes detecting the PCR amplification product by agarose gel electrophoresis, and judging that the sample to be tested is male if the amplification band is contained in the obtained agarose gel electrophoresis diagram.

2. A method for rapidly detecting the sex of grass carp fry according to claim 1, characterized in that: The amplification reaction conditions in step 3 are as follows: pre-denaturation at 95° C. for 5 min, denaturation at 95° C. for 30 s, annealing at 59° C. for 30 s, extension at 72° C. for 1 min, and after 35 cycles, extension at 72° C. for 5 min.

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