CN110724735B - SNP locus and primer for rapidly identifying individual sex of fugu obscurus and method thereof - Google Patents
SNP locus and primer for rapidly identifying individual sex of fugu obscurus and method thereof Download PDFInfo
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Abstract
The invention discloses a SNP locus and a primer for rapidly identifying individual sex of fugu obscurus and a method thereof, wherein the base sequence of the SNP locus is shown as SEQ ID NO.1, the invention obtains a specific SNP locus by carrying out genome analysis on male and female individuals of three independent genetic groups of the fugu obscurus, and designs two pairs of specific PCR primers for male and female of the locus for identifying the sex of the fugu obscurus individual; by improving the DNA extraction method, the extraction of the fin-shaped DNA can be completed within 10 minutes, the operation is simple and rapid, and through PCR amplification and agarose gel electrophoresis, male individuals with DNA bands are obtained, and female individuals are obtained on the contrary, so that the separation is easy. The method has the characteristics of high speed, high accuracy, simple and convenient operation, low cost and the like, and can finish the identification of the genetic sex of the takifugu obscurus within 2 hours.
Description
Technical Field
The invention belongs to the field of aquaculture, and particularly relates to SNP (single nucleotide polymorphism) loci and primers for quickly identifying individual sex of Takifugu obscurus and a method thereof, which are used for quickly identifying genetic sex of Takifugu obscurus.
Background
Puffer fish is one of the important cultured fishes in China. The puffer fish has white and tender meat, delicious taste and high economic value, and enjoys the reputation of the king of fish. With the gradual release of the culture, processing and sale of the takifugu rubripes and the takifugu obscurus in China, the takifugu rubripes culture industry is rapidly increased and developed, and the total culture yield reaches 30686 tons in 2017 (statistics yearbook in fishery of China, 2018). The takifugu obscurus is one of the famous three delicacies in Yangtze river, the white son (spermary) of a male individual is more called 'Xishi milk', the flavor is unique, and the takifugu obscurus is deeply loved by consumers, so that the male individual has higher value than the female individual, the culture benefit is higher, and if the full-male culture can be realized, the culture benefit of the takifugu obscurus can be obviously improved. Therefore, the research on the sex-controlled breeding of the puffer fish is developed, the market demand is met, the breeding benefit is improved, and the quality improvement and the efficiency improvement of the puffer fish breeding industry are promoted, so that the puffer fish breeding industry is developed to be green. The problem of stable and efficient identification of male and female individuals is mainly solved in the research of development breeding control.
Most fishes (including puffer fish) cannot be directly distinguished from sexes such as appearances, and the traditional identification method mainly comprises two types, namely, after individual sexual maturity is waited, the male and female fishes are identified by observing extruded semen or ovum in a breeding period, the method has the advantages of no fish body damage, simple operation, long waiting period and high breeding cost, and particularly the puffer fish which can be sexually mature for 2-3 years. The other type is dissecting gonad tissues of fish bodies, and identifying male and female by a method such as tissue slicing, and the like.
With the development of molecular biology technology, at present, various molecular marker technologies have been widely applied to fish sex control breeding research, such as Random Amplified Polymorphic DNA (RAPD), amplified Fragment Length Polymorphism (AFLP), microsatellite (SSR), single Nucleotide Polymorphism (SNP) marker technology, genome sequencing analysis, and the like, and a batch of site and DNA markers specific to sex chromosomes or linked to sex chromosomes are successfully screened, thereby laying a foundation for the identification of fish sex chromosomes and the production of all-male or all-female fish.
PCR identification technology based on sex specific sites has been widely applied to fish breeding research and breeding practice. At present, several methods for identifying the sex of fishes in the genus of fugu have been developed based on this principle, but some disadvantages such as complicated operation, long time consumption, high cost, etc. exist. When a small amount of samples are identified, a good identification effect can be obtained, but when large-scale sample identification is carried out, the tedious experimental process undoubtedly greatly increases the identification labor cost and the material cost, and the probability of misoperation (mainly mutual pollution among the samples) can be increased, so that the result misjudgment is caused, and the risk of identification failure is increased. This risk is particularly reflected in the DNA extraction stage, the conventional DNA extraction method involves many steps such as lysis, extraction, precipitation, washing, dissolution, etc., the operation is cumbersome, often takes several hours to complete DNA extraction, and there is a risk of cross-contamination in the extraction process (especially when the sample size is large), and the complicated operation also means that more reagents and consumables are consumed, and the cost is greatly increased. In addition, the conventional identification method usually requires multiple PCR reactions or multiple PCR reaction systems, or distinguishes DNA electrophoresis bands with different sizes, and has the problems of long time consumption, low efficiency, difficulty in learning and the like. Therefore, there is a need for a simple, rapid, low-cost and easily mastered method of sex determination.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides an SNP locus and a primer for rapidly identifying the individual sex of the fugu obscurus and a method thereof, and provides a new SNP locus and primer and a DNA extraction scheme for rapidly identifying the individual sex of the fugu obscurus, so that the method is a method for identifying the genetic sex of the fugu obscurus, which has the advantages of simple and rapid operation, low cost, reliable result and easy mastering.
The technical scheme is as follows: in order to achieve the purpose, the SNP locus for rapidly identifying the individual sex of the fugu obscurus is shown as SEQ ID NO. 1.
The SNP locus is a Takifugu obscurus male and female specific SNP locus obtained by analyzing the male and female individual genomes of three independent genetic groups of the Takifugu obscurus, and the locus has high specificity.
The primer for rapidly identifying the individual sex of the fugu obscurus is two pairs of male specific PCR primers, wherein two pairs of male specific PCR primers are designed according to the female and male specific SNP sites of the fugu obscurus, and the base sequences are respectively shown as SEQ ID NO. 2-5:
SEQ ID NO.2：MSP1-F ACCTGTGGCTCACGGCGACC
SEQ ID NO.3：MSP1-R CAGATACCATTTGTTGTGAAGATC
SEQ ID NO.4：MSP2-F GCTCCTGGAAGGCTCTGTCG
SEQ ID NO.5：MSP2-R GTGACAGCGCTTCCCAGTGT。
the method for rapidly identifying the individual sex of the takifugu obscurus comprises the following steps: the method comprises the steps of rapidly extracting DNA as a template by an improved alkaline lysis method, and carrying out PCR amplification reaction and agarose gel electrophoresis by combining a primer for rapidly identifying the individual sex of the fugu obscurus to realize the rapid identification of the genetic sex of the fugu obscurus; the improved alkaline cracking method for rapidly extracting DNA is characterized in that fin-shaped tissues are cracked in NaOH solution for 5-10 minutes, the DNA extraction can be completed, the cracking products do not need to be processed and are directly used for subsequent PCR amplification reaction, and the cracking solution can be directly used as a template for PCR amplification.
Preferably, the improved alkaline lysis method for rapidly extracting DNA comprises the following steps: clipping about 5mg of fin ray tissues at the tail end of the tail fin of the fish body to be detected, so that the fish body can be gradually recovered, and the physical damage to the fish body is avoided; the fin ray tissue is placed in 50mM NaOH solution for cracking for 10 minutes, then DNA extraction can be completed, the steps of extraction, precipitation, washing, dissolution and the like are omitted, and the product does not need to be processed and can be directly used for subsequent PCR reaction.
The primer for rapidly identifying the individual sex of the takifugu obscurus is one of two pairs of male specific PCR primers or two pairs of primers are used simultaneously to perform PCR amplification reaction.
Wherein the PCR amplification reaction system comprises 2.5 mu L of 10 XTaq buffer solution and 0.75 mu L of 50mM MgCl 2 0.5. Mu.L of 10mM dNTPs, 10. Mu.M of each of the upstream and downstream primers 0.5. Mu.L and 1. Mu.L of 1U/. Mu.L TaqDNA polymerase; mu.L of template DNA.
Wherein the PCR amplification reaction comprises the following steps: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 seconds, annealing at 63 ℃ for 30 seconds, and extension at 72 ℃ for 20 seconds, wherein the process is circulated for 35 times, and the PCR amplification product can be directly used for agarose gel electrophoresis.
If two pairs of primers are used simultaneously and the amplification results of the two pairs of primers can be verified mutually, accidental errors caused by misoperation during large-scale identification are further avoided, the reliability and the accuracy of the identification result are ensured, and the accuracy can reach 100%.
The invention obtains a specific male and female SNP locus by analyzing the genome of male and female individuals in a Fugu obscurus population, designs primers at the upstream and downstream of the SNP locus, and amplifies and sequences in the genome of three Fugu obscurus populations (respectively taken from Takipedia and Fugu zhenjiang farms, belonging to a genetic independent population) to identify that the SNP locus is male specific. Primers only recognizing male SNP are further designed, male and female can be identified through PCR and agarose gel electrophoresis, and only male individuals have amplified bands.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the invention provides the SNP locus for rapidly identifying the individual sex of the fugu obscurus, and the primer is designed to identify the individual sex of the fugu obscurus, so that the result is accurate, and the operation is simple and rapid.
The method for rapidly identifying the individual sex of the takifugu obscurus emphasizes that the DNA of the fish body to be detected is extracted by an improved and optimized alkaline lysis method, the DNA extraction can be completed within 10 minutes only by one-step operation (the conventional DNA extraction method usually needs several hours), and the time required by sex identification is greatly shortened; the consumption of a plurality of operation flows and reagents and consumables is reduced, and the labor cost and the material cost are obviously reduced; meanwhile, the risk of DNA template pollution during large-scale identification is obviously reduced, two groups of primers are used for assisting in simultaneous detection, and the reliability and accuracy of an identification result are obviously improved. In addition, the electrophoresis results are simple "with" or "without", that is, the individuals with the DNA amplified fragment are male individuals, and the individuals without the corresponding band are female individuals. The identification method is simple and easy to distinguish and master.
In view of the conservative property of the sex determination mode of the Takifugu fish, the invention can also be popularized and applied to Takifugu rubripes, takifugu leopardus, takifugu isoperianum and other Takifugu fish.
Drawings
FIG. 1 is a schematic view of the operation of the present invention, showing the time required for each step;
FIG. 2 shows the result of electrophoresis using the first set of primers according to the present invention. By comparing the identification effects of an alkaline lysis method and a conventional DNA extraction method (DNA extraction kit, manufactured by Promega corporation in the United states) and respectively verifying in three independent genetic groups (total 147), the reliability of the invention is verified;
FIG. 3 shows the result of electrophoresis using the second set of primers according to the present invention. The reliability of the invention is verified by comparing the identification effects of the alkaline lysis method and the DNA extraction kit and verifying in three independent genetic groups respectively.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the drawings.
Example 1
The operation flow is shown as 1.
1. Sample collection
The takifugu obscurus used in the present example was collected from three independent genetic groups of zhenju zhenjiang and nantong, respectively, containing 32, 43, 72 individuals, and named as groups 1, 2, 3 in sequence. Each individual was separately organized into gonads and fin rays, the gonads were fixed in a 4% PFA solution, and the fin rays were stored in absolute ethanol in one-to-one correspondence.
2. Gonadal tissue section and physiological sex identification
And embedding the fixed gonad tissue, then carrying out tissue sectioning, dyeing by adopting a hematoxylin and eosin dyeing method, observing the gonad tissue structure by using a microscope after dyeing, thereby identifying the physiological sex, wherein the identification result is that a population 1 comprises 16 males and 16 females, a population 2 comprises 24 males and 19 females, and a population 3 comprises 44 males and 28 females.
3. Genomic DNA extraction
The alkaline cracking method comprises the following steps: clipping about 5mg of fin ray tissue and placing the fin ray tissue in a 1.5mL centrifuge tube; 200 mul of 50mM NaOH solution is added, the mixture is cracked for 10 minutes at 95 ℃, and the lysate can be used as a DNA template for subsequent PCR reaction, namely 1 mul.
In addition, the genomic DNA of the takifugu obscurus individuals was extracted by using the DNA extraction kit of Promega corporation and the improved alkaline lysis method of the present invention as a comparison.
The DNA extraction kit comprises the following steps: clipping about 5mg of fin ray tissue and placing the fin ray tissue in a 1.5mL centrifuge tube; adding 500 mu L of nucleic lysine solution into the mixture, and carrying out water bath at 65 ℃ for 30 minutes; adding 10 μ L of 20mg/ml proteinase K, and water bathing at 55 deg.C until fin ray digestion is complete; centrifuging at 14000rpm for 4-8 minutes, and taking the supernatant to a new 1.5ml centrifuge tube; adding 3 μ L of RNase solution, mixing, standing at 37 deg.C for 15-30 min; cooling to room temperature, adding 200 μ L Protein precipitation Solution, rapidly reversing and mixing, and standing on ice for 10 min; centrifuging at 14000rpm for 4 minutes to precipitate the protein; aspirate approximately 600 μ Ι _ of supernatant and transfer to a new 1.5mL centrifuge tube; adding isopropanol with the same volume, slightly reversing and uniformly mixing until white linear DNA appears, and standing at 4 ℃ or-20 ℃ overnight to ensure that the DNA is fully separated out; centrifuging at 14000rpm for 1 min, precipitating DNA, and discarding the supernatant; adding 600 mu L of 70% ethanol, slightly reversing and mixing, washing DNA, and centrifuging at 14000rpm for 1 minute; then 600. Mu.L of 70% ethanol was added, the mixture was gently inverted and mixed to wash the DNA, and the mixture was centrifuged at 14000rpm for 1 minute; after discarding the supernatant, the supernatant was sufficiently dried for about 5 minutes, 50. Mu.L of TE buffer or sterilized water was added thereto, and the DNA was dissolved in a water bath at 65 ℃ for 1 hour or overnight at 4 ℃.
4. Takifugu obscurus sex specific site analysis
Designing primers (the primer sequences are as follows) according to the gene sequence (SEQ ID NO. 1) of the SNP locus, taking the DNA of three population 147 individuals extracted by the cleavage products in the step 3 or a kit method respectively as a template, amplifying the fragments and sequencing for SNP identification (the PCR process is the same as the step 5). Through primer amplification and amplification product sequencing, comparing with the slicing result in the step 2, identifying and confirming that all physiological male individuals of the three groups have G/C genotypes, and all physiological female individuals have C/C genotypes, which indicates that the SNP locus can be used for identifying the individual sex of the takifugu obscurus; then, male specific primers are designed according to the G/C difference.
SEQ ID NO.6：SNP-F ACCTGTGGCTCACGGCGACC
SEQ ID NO.7：SNP-R ATCAGAGCAGCACATCCAGATCTC
5. Genetic type identification of fugu obscurus
And (3) respectively carrying out PCR (polymerase chain reaction) reaction by taking the DNA of 147 individuals of the three groups respectively extracted by an alkaline lysis method and a kit method as templates, and detecting and analyzing reaction products by agarose gel electrophoresis.
The PCR reaction system is as follows: 2.5. Mu.L of 10 XTaq buffer, 0.75. Mu.L of 50mM MgCl 2 0.5. Mu.L of 10mMdNTPs, 0.5. Mu.L of each of 10. Mu.M upstream and downstream primers, and 1. Mu.L of 1U/. Mu.L Taq DNA polymerase; mu.L of template DNA.
The PCR reaction steps are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 sec, annealing at 63 ℃ for 30 sec, and extension at 72 ℃ for 20 sec, which were cycled 35 times.
The PCR primer sequences are respectively:
SEQ ID NO.2：MSP1-F ACCTGTGGCTCACGGCGACC
SEQ ID NO.3：MSP1-R CAGATACCATTTGTTGTGAAGATC
SEQ ID NO.4：MSP2-F GCTCCTGGAAGGCTCTGTCG
SEQ ID NO.5：MSP2-R GTGACAGCGCTTCCCAGTGT。
6. analysis of results
The amplification results of the first set of primers (MSP 1-F, MSP 1-R) are shown in FIG. 2, and the amplification results of the second set of primers (MSP 2-F, MSP 2-R) are shown in FIG. 3.
The left sides of FIGS. 2 and 3 are the amplification results using DNA extracted by the kit method as a template. M is a molecular weight marker, and the molecular weights shown in the figure are respectively 500bp and 250bp; lane is Lane marker. As shown, in all three populations, the male individuals amplified DNA bands of either 349bp (FIG. 2) or 336bp (FIG. 3), while the female individuals had no corresponding bands.
The right sides of fig. 2 and 3 are amplification results using DNA extracted by alkaline lysis as a template. As shown in the figure, the alkaline lysis method greatly reduces the time and cost required for identification, but can obtain the same identification effect as the kit method. In addition, the result of the step 6 shows that the identification purpose can be achieved by only one pair of primers, the two pairs of primers are respectively used simultaneously, and the amplification results of the two pairs of primers can be verified mutually, so that accidental errors caused by misoperation during large-scale identification are further avoided, the reliability and the accuracy of the identification result are ensured, and the accuracy can reach 100%.
Sequence listing
<110> university of river and sea
<120> SNP locus and primer for rapidly identifying individual sex of fugu obscurus and method thereof
<160> 7
<170> SIPOSequenceListing 1.0
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<211> 4711
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<213> Fugu obscurus SNP (SNP)
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cctgttctgt aatggtttgt cgtgtactca gttgtattag aattattatt tttgtacagc 60
agacagagtg tgttgttcat gcaggtgtgc atcagtggga gtgttgctgg aaatgtgggg 120
aaactcctct cacattccca ggactgataa ccattcactt gagatgggtc taaatttctg 180
ttcccagagt caccatgatc ctgcaacagt tgctgacttt agctgtgggt gagtacattc 240
actgttcaaa gatcgaatat cacatgattt ttgtaaataa tctgcatgct tgactttaaa 300
tgtaacagtg tgagtgttta tatttaggat tggggcatga ataatgcagt catcgtgttt 360
ttaacagtga cacttttgac tttaaggaat acttaggctt tagttaaaag gtcccaattt 420
aatcatgaaa cgatgtatca gataaatgct tttgtacatg gatgacaaca tttttgtgtg 480
ggacattaag acatcgctca accacaaggt caaaccctcc ttaaatgttt ttatcattaa 540
tgtcttgtgt gctgaaagat atatagttta ccccaaatta aaacatttcc attttctaac 600
tggagggttt ttgtgggtta tttttttttg aggggtttta tcagtgtggg tcttctctgt 660
tatttggaag gctacgcaga tttaaagaat gctcaagtgg gtctgactga aggactcttt 720
aacaaagtta ataatgtatc agtaataata ctgacccatt gaagcacaaa tcatgaatcc 780
ggtattcagt cctttgccag tggctgttcc tcctctgtct tcctcaacag aatgcatcct 840
aataagtgtc tccagtcaat cttctccaca gcagagacgc tgtgcgtacc atgtgactga 900
caagggggac gtgtacacaa ctgctggcaa tgtgagcggg tcggtgcagc tctgcaagaa 960
cacccagtgc tgtgttggct actacgtagt gatcgatggc cggcccaagg ctgacgttct 1020
cggtgaggag atacaaacaa ggacggctgg attacattca aaaggaatgt ctgtgcatcc 1080
tcgcgggtct gctgttgatt tcttctgttg cagcttgtga tacagtggag aagcgctgca 1140
cagacacaac ctgcaaggcc caggtgttcc ggaatgtccg caccttcaag tgcgtttgca 1200
acacggacct gtgcaacagc aacgtcacct ggagccaaaa cgcacgtgaa gagtctcaac 1260
acgccagctc ctattacaaa ggtgtcgcgc cacatctgcg ccacgtgctc tgcgctcctg 1320
agaaatcatt aggagccttt ctttgtggtt ctggcatgat gttcatggaa aaacctcaat 1380
aaataaataa aattccaaaa atcaatccag tctctcggaa tcatggcaca gctccccaaa 1440
tgggagtacg acgcctcccg gtggaaggct gcagtaaaag aatcagctcc tctttccata 1500
ttcttgataa cactcaaact ggaatgtact cctccagaaa ggaaagacta attctacatt 1560
aaattcattc caattttacc aaacaaatta ttttgtatta gaaatccccc ccaaatggaa 1620
agattaccgg tactttccaa gctaagacca tatgttttct actgtgcttt ttaaaacatt 1680
attaatcatt tgtcatttat ggtgctttta gatgaaacca tgacaaccgc tctcatttcg 1740
attggaatct tgctcgccct cggcctcctg attgttgcca tccagtccag aaacttcttc 1800
acggtgaaaa gtaagtagaa aagatacatt gttctttatc aaacagattt tgtatcacaa 1860
aaaacatccc ccgcattttg aagccaccat tcaccctttt tgtaaatgta gtttgacctt 1920
ggattcactc agctgaactg accttggcgt ttatctctgc tccgacactc ctttgtgctt 1980
ccatcctttc tgtgtgactc ttcaaacaca tttctcttcg ctcctcagtc agcctcttta 2040
atttaaagca ccctttaagt catatcattt caagtgaagc cttttcccgt ctgctctctc 2100
catttgagat aagaattcaa ggtctcttga cacttatgac ttctcgttgc tgcactccca 2160
ccgaaaaaag gagccttctg acattgacgc tacagacgtt gagctgcagc aggtgagaag 2220
gtgaacgaaa ttctggtgaa tgacatcacc actgctttca tctcagtgtt gtacatctgc 2280
aggttttgtg ccatgggaat tttgcaacgg tttggcttgg gacacaccag gaatccagag 2340
tagccgtgaa ggttttccct gcaagctgca aacgcaaatt tactgcagag aaggaaattt 2400
acgagctccc gctaatgagg cacggtggaa tcgtccattt cctgggaaca gcgaggaagc 2460
cttgtggaga cagttggctc attgtcctgc aacttgctga aaatgtaagt tgtgagccgt 2520
cattctgctg agtggtgaga agaaccgaga tacagggggg acggaaagtc ttcacaccct 2580
cagaaacctt gtgtcatggt gatgtctcgt gtcaaaatga aaaaaaaaaa atcatttccc 2640
acctcatcaa tccacattct acagcccaaa tccggaattt gtgaattttc ccacaaatta 2700
atgaaaaaaa gggaaaactg catcattaaa tgaacataag tattcacact cctgtgggtc 2760
agtcaggtgc tccctatttc ttttgatcag ctttgagatg gtcccacatc ttcatcagag 2820
tccagctgtg ttgaattaaa tggactgaag gtgactttta aagcacacct ctctgaataa 2880
ggccttagag tttgttctgc aagccagaga actgaagagc tcagagacag cagtagcaag 2940
acacagatgt ggacaagact agggggaaaa atctgcttca tttactgttt ctaagagctc 3000
agcggtcttc cttattccca tatggaacag gacttttctt actgctggtt ggcagcacca 3060
caacacacca aaaacagagc tgtgagtaat tattcagcct ttttcgtgtg tcttccaatg 3120
agtaaattca ggatttgtca ggtcccgatt gtagacgagg cttcaacaag aggggaaagg 3180
aaaagcagaa agaaggatgt tttctatcag tactctacat tattgaaagc aacagaagcg 3240
cgtacctgcc cctcttctgt aacgttagtt gttacccagt gttctgtatt tctctggtcc 3300
ggcatgttct cattgacata aacatctgcc tttatttctt gattccaggg ttctctccgc 3360
acattcttgc gtgaaaactc cgttgactgg acgtcatcac tgaagttatg cctctctttg 3420
tcccaggggc tagcctatct tcactctgac ctccatagac acggtgtgtg tgtgtgtgtg 3480
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtatt gtatctgttc tgaacttggc 3540
acggctgtaa ggatttacaa tacataagca aggaactctc agctcagttc tgatgctaac 3600
cacacaaggc aatcttgttt gataaacttt gctgccgtta aaccttcaaa tcatcctttg 3660
ctgcacgttt ttcttgcaga tgcacacaaa ccacctgtgg ctcacggaga cctgagcagc 3720
tccaacgtgc ttgtgagagc cgatggcgcc tgcgtcctgt gcgatttcgg atgttccgcc 3780
atcctgcatt ctttttcagg atgtcacaga cagagcaaca caactagccc gttggtgagc 3840
agagacgtcg cgcctaaaac acgcgagaaa accatcgctc gggcaattgt gtgcgttttt 3900
ctgctttggc cgtaaaacac aatgtgcaaa acgtgcagct cttgactcta actacagaac 3960
ctcattcagt ggggcacgtt gcgctacatg tcccctgagc tcctggaagg ctctgtccat 4020
cttcacaaca aatggtatct gatgcgcgcc gacgtctact ctctggcact gctgctgtgg 4080
gagatctgga tgtgctgctc tgatttcagt cacggtacgg tgagcagcaa tgtgcaggtg 4140
ctgtgcagca ggcaggacat ttcccccagt gtttgttttt ctgatcctgt ttacaggcgg 4200
tgatgctcct ccacggcatc agttgccata tgaatctgag ctgggagcca atgtctccat 4260
agagagcctc attttgcgag tgtgtcacat gggcatgaga ccttacatac cgcaacactg 4320
ggaagcgctg tcacaggtag tcctaaatat aatatccatg cagttcatgt tttgttactc 4380
aactcaactc ttttataact cgcaggggtc tgccttggag gagattctga cagattcttg 4440
ggactctgaa ccagatgctc gtctgtctgc tcagtgcgtt gcagacagac tggtctctct 4500
tcagtcttac cataatgtac ctgttaagca cctgaaagct tctatttgtg tggcaactgc 4560
ttagctgtat ccactagatc ctctgctaca ccttatttgt gtatttctta gagtatcttt 4620
tatgttctcg aagtataatt ttactttaac aagttttgtg cctggtttct atttttttta 4680
actggcatct attaccaagt ctgccaacat a 4711
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acctgtggct cacggcgacc 20
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cagataccat ttgttgtgaa gatc 24
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gctcctggaa ggctctgtcg 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtgacagcgc ttcccagtgt 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acctgtggct cacggcgacc 20
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atcagagcag cacatccaga tctc 24
Claims (5)
1. A method for rapidly identifying the individual sex of Fugu obscurus is characterized by comprising the following steps: the method comprises the steps of rapidly extracting DNA as a template by an improved alkaline lysis method, and carrying out PCR amplification reaction and agarose gel electrophoresis by combining a primer for rapidly identifying the individual sex of the fugu obscurus to realize the rapid identification of the genetic sex of the fugu obscurus; the improved alkaline cracking method for rapidly extracting DNA is characterized in that fin-shaped tissues are cracked in NaOH solution for 5-10 minutes, the DNA extraction is completed, and the cracked products are directly used for the subsequent PCR amplification reaction without being processed; the primers are two pairs of male specific PCR primers, and the base sequences are respectively shown in SEQ ID NO. 2-5:
SEQ ID NO.2：MSP1-F ACCTGTGGCTCACGGCGACC
SEQ ID NO.3：MSP1-R CAGATACCATTTGTTGTGAAGATC
SEQ ID NO.4：MSP2-F GCTCCTGGAAGGCTCTGTCG
SEQ ID NO.5：MSP2-R GTGACAGCGCTTCCCAGTGT。
2. the method of claim 1, wherein the primer for rapid sex determination of Fugu obscurus is any one of two pairs of male specific PCR primers or two pairs of primers used simultaneously for PCR amplification reaction.
3. The method for rapid sex identification of Fugu obscurus L according to claim 1, wherein the PCR amplification reaction system comprises 2.5 μ L of 10 XTaq buffer, 0.75 μ L of 50mM MgCl 2 0.5. Mu.L of 10mM dNTPs, 10. Mu.M of each of the upstream and downstream primers 0.5. Mu.L and 1. Mu.L of 1U/. Mu.LTaqA DNA polymerase; mu.L of template DNA.
4. The method for rapidly identifying the individual sex of takifugu obscurus according to claim 1, wherein the PCR amplification reaction comprises the following steps: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 seconds, annealing at 63 ℃ for 30 seconds, and extension at 72 ℃ for 20 seconds, wherein the process is circulated for 35 times, and PCR amplification products are directly used for agarose gel electrophoresis.
5. The method for rapidly identifying the sex of takifugu obscurus individuals according to claim 1, wherein the agarose gel electrophoresis is performed on male individuals and female individuals with the DNA amplification fragments.
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