CN111979336B - Specific primer and method for identifying fugu obscurus, fugu rubripes and hybrid fugu rubripes - Google Patents
Specific primer and method for identifying fugu obscurus, fugu rubripes and hybrid fugu rubripes Download PDFInfo
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Abstract
The invention discloses a specific primer and a method for identifying fugu obscurus, fugu rubripes and hybrid fugu rubripes, wherein the specific primer comprises a forward primer and a reverse primer, and the nucleotide sequences of the primers are respectively shown in SEQ ID NO. 1-2. Firstly, collecting a puffer fish sample to be identified, and extracting genome DNA; then, three globefish genome DNAs are used as templates, and PCR amplification is carried out on the three globefish genome DNAs by using primers; and finally, carrying out electrophoresis on the amplification products and identifying according to band difference. The method for identifying the takifugu obscurus, the takifugu rubripes and the takifugu hybridus by using the specific primers does not need complex steps such as microsatellite on-board sequencing and the like, can quickly and accurately distinguish the three fishes according to the number and positions of bands, and has the advantages of good stability, strong practicability, simplicity in operation, high accuracy and the like.
Description
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to a specific primer and a method for identifying fugu obscurus, fugu rubripes and hybrid fugu rubripes.
Background
Fugu obscurus (D.Don) ((R))Takifugu fasciatus) And Fugu rubripes (Takifugu rubripes) Commonly known as puffer fish, belonging to Osteichthyes, subfamily of Amygdales (Actinopterygii), Fugu (Letrodontiforms), Fugu (Tetraodontidae), Takifugu (Takifugu genus) (Fugu flavidus)Takifugu) And is mainly distributed in yellow sea, Bohai sea, offshore sea areas, rivers and lakes in China. The food has the advantages of delicious meat and high nutritive value, is deeply loved by consumers and is an aquatic product with higher economic value.
In crossbreeding, the genotypes of the parents are recombined, so that the offspring can have the excellent characters of the parents at the same time, even some shape characteristics which the parents do not have can be generated, and certain heterosis can be realized. The hybrid takifugu produced by the hybridization of female takifugu obscurus and male takifugu rubripes has the rich flavor of takifugu obscurus and the characteristic of fast growth of takifugu rubripes. Although the appearance and body type differences of the three kinds of fugu obscurus are obvious in the adult fish period and can be distinguished by naked eyes, the appearance and body types of the three kinds of fugu obscurus are very similar in the juvenile fish period and cannot be distinguished by naked eyes. Further, it is difficult to distinguish a part of puffer fish tissues, processed meat products, and the like from the appearance. With the outstanding problems of germplasm mixing, serious quality degradation, disordered hybridization and the like faced by the cultivation of the fugu obscurus in recent years, a species identification method for distinguishing the fugu obscurus, the fugu rubripes and the fugu hybridum is urgently needed.
Species identification has been a crucial research step in taxonomy, or even in almost all biological fields. Therefore, accurate identification and classification of species is particularly important. As a second generation molecular marker, the microsatellite has the characteristics of polymorphism, high heterozygosity rate, codominant inheritance and the like. In recent years, the development of microsatellite markers in puffer fish species is mainly applied to the analysis of genetic diversity among different geographical populations of puffer fish, germplasm evolution among closely related species, species identification and the like. However, so far, there are few reports on the intergeneric species identification of fugu, and no reports on the identification of fugu obscurus, fugu rubripes and fugu hybride have been found yet. The traditional method for identifying species by using a microsatellite marker technology is to add 18bp specific fluorescent fragments in the front of a primer pair, add a fluorescent reagent in a PCR amplification system, carry out capillary fluorescence electrophoresis detection on amplified PCR products by using a DNA sequencer ABI 3730xl, read the locus genotype of a microsatellite according to the peak value of allele, and detect the genotype difference among different species or the polymorphism of different primers. The operation is complicated, time-consuming and high in cost.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a specific primer for effectively distinguishing the identification between the species of the takifugu obscurus, the takifugu rubripes and the hybrid takifugu obscurus; the primer group can rapidly and accurately identify the three kinds of fugu obscurus.
The invention also provides the method for identifying the takifugu obscurus, the takifugu rubripes and the hybrid takifugu obscurus.
The technical scheme is as follows: in order to achieve the above object, the specific primers of the present invention for identifying fugu obscurus, fugu rubripes and fugu hybride [ fugu obscurus (female) × fugu rubripes (male) ] and fugu hybride, which is a hybrid produced by the hybridization of female fugu obscurus and male fugu rubripes ] comprise a forward primer and a reverse primer, and the nucleotide sequences thereof are respectively shown in SEQ ID nos. 1-2:
forward primer SEQ ID No. 1: 5'-AAAGGGGAGTTTTTCCTTGCCACT-3', respectively;
reverse primer SEQ ID NO. 2: 5'-CCCACATTCTCTCCATCTCCTCCT-3' are provided.
The kit for identifying the method of the takifugu obscurus, the takifugu rubripes and the hybrid takifugu obscurus comprises the specific primer.
The method for identifying the takifugu obscurus, the takifugu rubripes and the hybrid takifugu obscurus by using the specific primers comprises the following steps of:
(1) extracting the genomic DNA of the fugu rubripes to be identified;
(2) carrying out PCR amplification on 3 kinds of fugu rubripes DNA by using the specific primer;
(3) and detecting the PCR product by agarose gel electrophoresis.
Wherein, the PCR amplification reaction system in the step (2) is 7.4 mu L of sterilized water, 10 mu L of 2 xTaq Plus Mix and 0.8 mu L of DNA1 mu L of each 10uM forward and reverse specific primers.
Wherein, the PCR amplification reaction program in the step (2) is pre-denaturation at 95 ℃ for 5 min; 40 cycles of: denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, and extension at 72 ℃ for 30 sec; finally, extension is carried out for 7min at 72 ℃ and storage is carried out at 4 ℃.
Wherein, the PCR product in the step (3) is used for detecting that the Fugu obscurus has 3 bands, and the positions are respectively about 150bp, 800bp and 1500 bp; the takifugu rubripes has 3 bands, and the positions of the bands are respectively about 150bp, 1000bp and 1800 bp; the hybrid takifugu has 5 bands in the positions of 150bp, 800bp, 1000bp, 1500bp and 1800bp separately.
The invention optimizes the microsatellite marking technology, does not need to add fluorescent primers and fluorescent reagents, can identify the fugu obscurus, the fugu rubripes and hybrids thereof by the electrophoretic map only by optimizing PCR amplification conditions and electrophoresis conditions, and is simple and efficient.
The invention utilizes microsatellite screening software MISA to screen and analyze the takifugu obscurus whole genome microsatellite. And then carrying out batch development on primers according to a large number of microsatellite locus binding scripts and primer software screened from the whole genome, and selecting related primers for synthesis. DNA of the tail fins of the takifugu obscurus, the takifugu rubripes and the hybrid takifugu rubripes is extracted, the DNA is used as a template for amplification, and the positions of bands are observed by agarose gel electrophoresis to distinguish the 3 kinds of takifugu rubripes. In the experimental process, the inventor finds that the microsatellite primer has low specificity, and different amplification conditions (annealing temperature, extension time and cycle number) and agarose concentration have influence on the electrophoresis result. The invention finally determines the amplification condition as the optimal condition by changing the amplification condition and the agarose concentration.
The invention identifies and distinguishes different species according to the number and the position of bands through PCR amplification and agarose gel electrophoresis. The species identification of 3 kinds of puffer fish is realized simply and efficiently, and the method is convenient and low in cost.
Has the beneficial effects that: compared with the prior art, the invention has the following advantages:
(1) the invention provides a primer and a method for identifying fugu obscurus, fugu rubripes and hybrid fugu obscurus, which are not limited by the age, sex, population and the like of three fish individuals, and have strong primer stability and high accuracy.
(2) By using the primer designed by the invention, the method can obtain stable, clear and obviously different amplification strips only by PCR amplification and agarose gel electrophoresis detection, and has the advantages of low cost, simple operation and strong practicability.
Drawings
FIG. 1 shows the results of amplification electrophoresis of 3 groups of Fugu obscurus, Fugu rubripes and Fugu hybridus. M-Marker; 1-8 is Fugu obscurus; 9-16 is Fugu rubripes; 17-24 is hybrid takifugu obscurus;
FIG. 2 shows the results of partial amplification electrophoresis in three groups of Fugu obscurus, wild Fugu rubripes and cultivated Fugu hybridus in different regions. M-Marker; 1-8 is Fugu obscurus; 9-16 is Fugu rubripes; 17-24 is hybrid takifugu obscurus;
FIG. 3 shows the electrophoresis result of 3 groups of Fugu obscurus, Fugu rubripes and Fugu hybridus amplified by changing the PCR reaction conditions and using specific primers. M-Marker; 1-8 is Fugu obscurus; 9-16 is Fugu rubripes; 17-24 is hybrid takifugu obscurus;
FIG. 4 shows the electrophoresis results of 3 groups of Fugu obscurus, Fugu rubripes and Fugu hybridus amplified by microsatellite fluorescent primers. M-Marker; 1-8 is Fugu obscurus; 9-16 is Fugu rubripes; 17-24 is hybrid takifugu obscurus;
FIG. 5 shows the electrophoresis results of 3 groups of Fugu obscurus, Fugu rubripes and Fugu hybridus amplified by means of microsatellite fluorescent primer and microsatellite amplification system. M-Marker; 1-8 is Fugu obscurus; 9-16 is Fugu rubripes; 17-24 is Fugu hybridus.
Detailed Description
The invention is further illustrated by the following figures and examples.
The invention is based on SEQ ID NO: 1 and SEQ ID NO: 2, extracting genomic DNA of the takifugu obscurus, the takifugu rubripes and the hybrid takifugu rubripes, carrying out PCR amplification and agarose gel electrophoresis result analysis, and identifying the three kinds of takifugu rubripes according to electrophoresis difference band results. Can be applied to the identification of the fries of the takifugu obscurus, the takifugu rubripes and the hybrid takifugu obscurus.
Example 1
(1) Obtaining samples of Fugu obscurus, Fugu rubripes and Fugu hybridus
The material selection comprises 24 samples of 8 takifugu obscurus, takifugu rubripes and takifugu hybridus of determined types, wherein takifugu obscurus seedlings come from Jiangsu Zhongyang group Limited company (Haian base), are cultured in Korea experimental base of Nanjing City Water sciences research institute for 5 months and are selected; 5 months old Fugu rubripes and Fugu hybride were obtained from Jiangsu Zhongyang group GmbH (Haian base).
(2) Extraction of fugu obscurus, fugu rubripes and hybrid fugu obscurus genome DNA
10-30 mg of tail fin is taken from each fish, and a DNA extraction kit (purchased from Beijing Baitaeke biology company) is utilized to extract the genome DNA of 24 puffer fish.
(3) Screening of microsatellite loci and synthesis of primers
Firstly, a microsatellite screening software MISA is used for screening Takifugu obscurus whole genome microsatellites, primers are developed in batches by using Primer 5.0 software in combination with scripts, and 100 pairs of primers are selected and synthesized (the primers are synthesized by Shanghai Czeki organisms).
(4) Analysis of experimental results of Fugu obscurus, Fugu rubripes and Fugu hybridus
And (3) detecting the amplification condition of the primer synthesized in the step (3) in the puffer fish sample DNA extracted in the step (2) by adopting PCR amplification and agarose gel electrophoresis. The specific process is as follows:
1) PCR amplification system
The PCR amplification reaction (20. mu.L) was as follows: sterilized water (7.4. mu.L), 2 XTaq Plus Mix (10. mu.L) (purchased from Shanghai-Country Probiotics technologies, Ltd.), 10uM forward and reverse primers (0.8. mu.L each), and Fugu ocellatus tail fin DNA (1. mu.L).
2) PCR amplification reaction
Pre-denaturing for 5min at 95 ℃ on a PCR amplification instrument; 40 cycles: denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, and elongation at 72 ℃ for 30 sec; finally, extension is carried out for 7min at 72 ℃ and storage is carried out at 4 ℃.
3) Agarose gel electrophoresis detection
And 5 mu L of DNA Marker 20004 mu L, PCR amplification reaction product is taken for agarose gel electrophoresis detection, EB staining is carried out, and a picture is taken by an ultraviolet gel imaging system.
4) Determining specific primers according to electrophoresis results
100 pairs of microsatellite primers are synthesized in total, a pair of primers which can be successfully amplified in the Fugu obscurus, the Fugu rubripes and the hybrid Fugu rubripes and can obtain obvious difference bands in the three products through 3) detection are selected through PCR amplification and agarose electrophoresis experiments, and the primer sequences are as follows:
SEQ ID NO.1：F：5′-AAAGGGGAGTTTTTCCTTGCCACT-3′；
SEQ ID NO.2：R：5′-CCCACATTCTCTCCATCTCCTCCT-3′。
the electrophoretogram of the product obtained by the pair of primers is shown in FIG. 1, and according to the comparison of an electrophoresis band with a standard Marker: 3 bands appear, and the positions of the bands are respectively around 150bp, 800bp and 1500bp, and the bands are takifugu obscurus; 3 bands appear, and the red-fin eastern globefish is respectively positioned at about 150bp, 1000bp and 1800 bp; 5 bands appear, the positions of which are respectively around 150bp, 800bp, 1000bp, 1500bp and 1800bp are the takifugu hybridus. The Fugu obscurus, the Fugu rubripes and the hybrid Fugu obscurus can be distinguished by electrophoresis.
Example 2
(1) Obtaining different groups of Fugu obscurus, Fugu rubripes and Fugu hybridus:
the method comprises the following steps of taking 60 samples of 20 takifugu obscurus, takifugu rubripes and takifugu hybridus with determined varieties, wherein the 20 takifugu obscurus with 5 months ages are respectively from Jiangsu Zhongyang group Gmbycis company Limited (Haian, 8 tails), Zhenjiang Source fishery science and technology Limited (Yangzhong, 6 tails) and Jiangyin Shenhong Kong Sanxian breed Limited (Jiangyin, 6 tails); 20-tailed 3-year-old wild takifugu rubripes is taken from Shandong Taishi island; the 20-tailed 5-month-old cultivated hybrid takifugu was obtained from Jiangsu Zhongyang group GmbH (Haian base).
(2) Extraction of fugu obscurus, fugu rubripes and hybrid fugu obscurus genome DNA
Specific genomic DNA extraction procedure reference was made to procedure (1) of example 1.
(3) Analysis of experimental results of Fugu obscurus, Fugu rubripes and Fugu hybridus
The specific experimental procedure is as in example 1 (4). The primer of example 1 was used to perform PCR amplification and agarose gel electrophoresis test on 60 total Takifugu rubripes individuals, and the results were identical to those of example 1. The 20 Fugu obscurus from different families in different regions has identical electrophoresis bands of 8 Fugu obscurus from the same family in example 1, and has 3 bands in the band positions of 150bp, 800bp and 1500bp separately. The 20-tailed wild fugu rubripes has the same electrophoresis bands as those of the fugu rubripes cultivated by 8-tailed Zhongyang group GmbH in example 1, and 3 bands appear, and the band positions are respectively about 150bp, 1000bp and 1800 bp. The electrophoresis bands of the 20-Zhongwei Fugu hybridus bred by the Zhongyang group member Co., Ltd are the same as those of the 5-8-Jiangsu Fugu hybridus bred by different groups of the Zhongyang group member Co., Ltd in the example 1, and 5 bands appear, and the band positions are respectively about 150bp, 800bp, 1000bp, 1500bp and 1800 bp. The primer has the identification accuracy of 100% in all the individuals of Fugu obscurus, Fugu rubripes and Fugu hybride.
Comparative example 1
(1) Obtaining samples of Fugu obscurus, Fugu rubripes and Fugu hybridus
The acquisition of the specific three population samples was referred to in step (1) of example 1.
(2) Extraction of fugu obscurus, fugu rubripes and hybrid fugu obscurus genome DNA
Specific genomic DNA extraction procedure reference was made to procedure (2) of example 1.
(3) Screening of microsatellite primers and synthesis of primers
The specific primer pair of primer reference example 1 was used in this comparative example.
(4) Analysis of experimental results of Fugu obscurus, Fugu rubripes and Fugu hybridus
And (3) detecting the amplification condition of the primers in the three parts by using the puffer fish sample DNA extracted in the step (2) and adopting PCR amplification and agarose gel electrophoresis.
The specific process is as follows:
1) PCR amplification system
The PCR amplification system was referred to in step (4) of example 1.
2) PCR amplification reaction
Pre-denaturing for 5min at 95 ℃ on a PCR amplification instrument; 35 cycles: denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, and extension at 72 ℃ for 2 min; finally, extension is carried out for 7min at 72 ℃ and storage is carried out at 4 ℃.
3) Agarose gel electrophoresis detection
Agarose gel electrophoresis detected step (4) of reference example 1.
4) Determining specific primers according to electrophoresis results
The electrophoretogram of the product obtained under the amplification condition is shown in FIG. 3, and the amplified bands of the Fugu obscurus, the Fugu rubripes and the Fugu hybridus are darker and more disordered. And compared with the fig. 1 and 2, the 3 kinds of puffer fish individual bands in fig. 3 are not successfully amplified. Thus, in comparison with examples 1 and 2, the modification of the PCR amplification reaction did not accurately distinguish between Fugu obscurus, Fugu rubripes and Fugu hybride.
Comparative example 2
(1) Obtaining samples of Fugu obscurus, Fugu rubripes and Fugu hybridus
The acquisition of the specific three population samples was referred to in step (1) of example 1.
(2) Extraction of fugu obscurus, fugu rubripes and hybrid fugu obscurus genome DNA
Specific genomic DNA extraction procedure reference was made to procedure (2) of example 1.
(3) Screening of microsatellite primers and synthesis of primers
In this comparative example, the specific primer pair of primer reference example 1 was used, but an 18bp fluorescent fragment was added before the specific primer pair, the sequence of the fluorescent primer pair being:
SEQ ID NO.3：
F：5′-TGTAAAACGACGGCCAGTAAAGGGGAGTTTTTCCTTGCCACT-3′；
SEQ ID NO.4：R：5′-CCCACATTCTCTCCATCTCCTCCT-3′。
this primer pair was sent to the company for synthesis (primers were synthesized by Shanghai Czeri Bio Inc.).
(4) Analysis of experimental results of Fugu obscurus, Fugu rubripes and Fugu hybridus
And (3) detecting the amplification condition of the fluorescent primer synthesized in the step (3) in the puffer fish sample DNA extracted in the step (2) by PCR amplification and agarose gel electrophoresis. The specific PCR amplification system, PCR amplification reaction and agarose gel electrophoresis detection process refer to step (4) of example 1.
1) Determining specific primers according to electrophoresis results
The electrophoretogram of the products obtained by the pair of primers is shown in FIG. 4, and the amplified bands of the portions of the Fugu obscurus, the Fugu rubripes and the Fugu hybridus are darker and more disordered. And compared with the fig. 1 and 2, the individual bands of the 3 kinds of puffer fishes in fig. 4 are not successfully amplified. Therefore, the specific primers added with the 18bp fluorescent fragments can not accurately distinguish the fugu obscurus, the fugu rubripes and the hybrid fugu rubripes.
Comparative example 3
(1) Obtaining samples of Fugu obscurus, Fugu rubripes and Fugu hybridus
The acquisition of the specific three population samples was referred to in step (1) of example 1.
(2) Extraction of fugu obscurus, fugu rubripes and hybrid fugu obscurus genome DNA
Specific genomic DNA extraction procedure reference was made to procedure (2) of example 1.
(3) Screening of microsatellite primers and synthesis of primers
In this comparative example, the specific primer pair of primer reference example 1 was used, but an 18bp fluorescent fragment was added before the specific primer pair, the sequence of the fluorescent primer pair being:
SEQ ID NO.3：
F：5′-TGTAAAACGACGGCCAGTAAAGGGGAGTTTTTCCTTGCCACT-3′；
SEQ ID NO.4：R：5′-CCCACATTCTCTCCATCTCCTCCT-3′。
This primer pair was sent to the company for synthesis (primers were synthesized by Shanghai Czeri Bio Inc.).
(4) Analysis of experimental results of Fugu obscurus, Fugu rubripes and Fugu hybridus
And (3) detecting the amplification condition of the fluorescent specific primer synthesized in the step (3) by PCR amplification and agarose gel electrophoresis by using the tetrodotoxin sample DNA extracted in the step (2).
The specific process is as follows:
1) microsatellite fluorescent primer PCR amplification system
The microsatellite fluorescent primer PCR amplification system (10. mu.L) is as follows: sterilized water (3.64. mu.L), 2 XTaq Plus Mix (5. mu.L) (purchased from Shanghai-Probiotics technology Co., Ltd.), 10uM fluorescent forward primer (0.04. mu.L), reverse primer (0.16. mu.L), HEX fluorescent reagent (0.16. mu.L), and Fugu ocellatus tail fin DNA (1. mu.L).
2) PCR amplification reaction
The PCR amplification reaction was referred to in step (4) of example 1.
3) Agarose gel electrophoresis detection
Agarose gel electrophoresis detected step (4) of reference example 1.
4) Determining specific primers according to electrophoresis results
The electrophoretogram of the product obtained by changing the microsatellite primer and the PCR amplification system is shown in FIG. 5, and the amplification bands of the portions of the Fugu obscurus, the Fugu rubripes and the Fugu hybridus are darker and more disordered. Compared with fig. 1 and fig. 2, the individual bands of 3 kinds of puffer fishes in fig. 5 are not successfully amplified, and partial individual bands have tailing phenomenon. Therefore, the method cannot accurately distinguish between Fugu obscurus, Fugu rubripes and Fugu hybride.
The comparative examples 1, 2 and 3 respectively use the primers and the common PCR amplification system which change the amplification reaction conditions and add the 18bp specific fluorescent fragment and use the primers and the microsatellite fluorescent reagent amplification system which add the 18bp specific fluorescent fragment to amplify the 3 kinds of puffer fishes, the bands are darker and more messy, and the identification of the 3 kinds of puffer fishes cannot be carried out, which shows that the 3 kinds of puffer fishes can be effectively identified only by adopting the specific primers and the amplification reaction conditions of the invention.
In summary, the microsatellite primers for identifying the takifugu obscurus, the takifugu rubripes and the hybrid takifugu obscurus have universal applicability in three different groups and are not limited by age, gender and geographical position. The microsatellite molecular marking method or the identification method for identifying the takifugu obscurus, the takifugu rubripes and the hybrid takifugu obscurus overcomes the defects of complicated operation, time and labor waste of the traditional method, and has the advantages of simple operation, strong practicability, time and labor saving, low cost, good stability and high accuracy.
Sequence listing
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Claims (1)
1. A method for identifying Fugu obscurus, Fugu rubripes and hybrid Fugu obscurus by using specific primers is characterized by comprising the steps of extracting the genomic DNA of the Fugu obscurus to be identified, carrying out PCR amplification on the DNA of the Fugu obscurus by using the specific primers and detecting PCR products;
the PCR amplification reaction system comprises 7.4 mu L of sterilized water, 10 mu L of 2 xTaq Plus Mix, 0.8 mu L of each forward and reverse specific primer of 10uM and 1 mu L of DNA;
the PCR amplification reaction program is pre-denaturation at 95 ℃ for 5 min; 40 cycles: denaturation at 94 ℃ for 30sec, annealing at 58 ℃ for 30sec, and extension at 72 ℃ for 30 sec; finally, extending for 7min at 72 ℃, and storing at 4 ℃;
the PCR product detection is agarose gel electrophoresis detection amplification bands, wherein the Fugu obscurus has 3 bands, and the positions are respectively about 150bp, 800bp and 1500 bp; the takifugu rubripes has 3 bands, and the positions of the bands are respectively about 150bp, 1000bp and 1800 bp; the hybrid takifugu has 5 bands with positions of about 150bp, 800bp, 1000bp, 1500bp and 1800 bp;
the specific primers for identifying the takifugu obscurus, the takifugu rubripes and the hybrid takifugu rubripes comprise a forward primer and a reverse primer, and the nucleotide sequences of the primers are respectively shown in SEQ ID NO. 1-2:
Forward primer SEQ ID No. 1: 5'-AAAGGGGAGTTTTTCCTTGCCACT-3';
reverse primer SEQ ID NO. 2: 5'-CCCACATTCTCTCCATCTCCTCCT-3' are provided.
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