CN105176989B - It is a kind of to differentiate fugu obscurus and the primer and method of Fugu Xanthopterus (Temminck et Schlegel) fry - Google Patents
It is a kind of to differentiate fugu obscurus and the primer and method of Fugu Xanthopterus (Temminck et Schlegel) fry Download PDFInfo
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- CN105176989B CN105176989B CN201510665512.4A CN201510665512A CN105176989B CN 105176989 B CN105176989 B CN 105176989B CN 201510665512 A CN201510665512 A CN 201510665512A CN 105176989 B CN105176989 B CN 105176989B
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- 241000980706 Takifugu obscurus Species 0.000 title claims abstract description 29
- 241000253385 Takifugu xanthopterus Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title abstract description 11
- 241001441723 Takifugu Species 0.000 claims abstract description 25
- 238000001962 electrophoresis Methods 0.000 claims abstract description 4
- 238000004043 dyeing Methods 0.000 claims abstract description 3
- 238000010367 cloning Methods 0.000 claims description 2
- 238000012850 discrimination method Methods 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 abstract description 10
- 238000012408 PCR amplification Methods 0.000 abstract description 7
- 238000007400 DNA extraction Methods 0.000 abstract description 2
- 241000251468 Actinopterygii Species 0.000 description 11
- 108091092878 Microsatellite Proteins 0.000 description 8
- 239000000047 product Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
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- 241000894007 species Species 0.000 description 4
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- 238000009395 breeding Methods 0.000 description 3
- 230000000366 juvenile effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 241001441728 Molidae Species 0.000 description 2
- 241001441724 Tetraodontidae Species 0.000 description 2
- 241001441726 Tetraodontiformes Species 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
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- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000872315 Takifugu fasciatus Species 0.000 description 1
- 241001222097 Xenocypris argentea Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
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- 230000004087 circulation Effects 0.000 description 1
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- 235000019634 flavors Nutrition 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000009403 interspecific hybridization Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
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Abstract
Differentiate fugu obscurus and the primer and method of Fugu Xanthopterus (Temminck et Schlegel) fry the present invention relates to a kind of, pair of primers of the present invention is named as F FXF147, its sequence is as shown in SEQ ID No.1 and 2.2 kinds of Fugu fry genomic DNAs are extracted with common DNA extraction kit and PCR amplification is carried out to 2 kinds of Fugu DNA samples of primer pair with this during identification, PCR product is detected with 2.2% Ago-Gel (EB dyeing) electrophoresis again, the band of 300bp or so sizes is that the band of fugu obscurus, 600bp or so size is Fugu Xanthopterus (Temminck et Schlegel).The present invention only needs a small amount of tail fin of clip to be differentiated there is that stability is high, reproducible, highly practical, easy to operate and can quick and precisely differentiate on the basis of fry without putting to death.
Description
Technical field
The invention belongs to aquaculture field, is related to a kind of Protocols in Molecular Biology identification and belongs to fry method together, more specifically
Ground is said, the present invention relates to the use of microsatellite marker and fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry are accurately differentiated.
Background technology
Fugu obscurus (Takifugu fasciatus) is under the jurisdiction of Tetraodontiformes (Letrodontiforms), Molidae
(Tetraodontidae), Fugu category (Fugu), is commonly called as river Puffer, bubble fish, belongs to migration fishes, be distributed in the East Sea, Huang
Sea, the Bohai Sea and the middle and lower reach of Yangtze River, are a kind of high aquatic products of economic value, rank first of the Changjiang river " four big fishes ", to pass
Model in system cuisines.
Fugu Xanthopterus (Temminck et Schlegel) (Takifugu xanthopterus) is under the jurisdiction of Tetraodontiformes (Letrodontiforms), Molidae
(Tetraodontidae), Fugu category (Fugu), is commonly called as yellowfin Fugu, flower ship bar etc., also falls within migration fishes, its with it is dark
Line Fugu inhabit sea area and mating period is close, the East Sea, the South Sea, the Huanghai Sea and the Bohai Sea are mainly distributed on, also into rivers mouth.
Fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) are with their own characteristics, because ecological habit difference, both meats are far different.Striped east
Square Puffer has a very wide distribution, and yield is higher；Fugu obscurus build is larger, and the speed of growth is very fast, its taste flavor and nutritive value
Significantly larger than Fugu Xanthopterus (Temminck et Schlegel).Expand day by day with the market demand of two kinds of Fugus, the demand of its seed is also all with day
Increase, therefore the source of fry and accurate discriminating are the keys of two kinds of Fugu cultivation.But on morphology, fugu obscurus and bar
The fry appearance build of line Fugu is quite similar, if gaining knowledge without professional classification, only with visually go distinguish easily produce obscure.
Even therefore cultivating veteran fisherman can not accurately go to distinguish both Fugu fries, once mixed breed,
The workload of later stage adult fish classification processing is not only increased, there is also interspecific hybridization, and then causes germplasm to mix, quality becomes
It is bad.In addition, the juvenile fish of two kinds of Fugus of in the market is also usually confused, due to fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) market
Selling price variance is obvious, causes some businessmans to seek economic interests, Fugu Xanthopterus (Temminck et Schlegel) fry is served as fugu obscurus fry by mistake
Sell, not only invaded the interests of consumer, also significantly impacted the economic benefit of fisherman and river Puffer breeding enterprise.Therefore, city
A kind of accurate method differentiated and distinguish 2 kinds of Fugus of active demand on field.
Microsatellite DNA in genome is expressed, and have be distributed wide, something lost at the same time with Mendelian fashion heredity in codominance
The advantages that passing information content height and being detected easy to PCR, species group is carried out in genetics-breeding in fish research using microsatellite marker
The research such as body genetic structure and diversity analysis, the structure of molecular genetic linkage map, species evolution and affiliation, can be to difference
Species carry out the differentiation of accurate systematics, the evolutionary path and taxonomy of accurate definite species, so to germ plasm resource,
Gene pool is purposefully protected.Research show microsatellite flanking sequence affiliation is relatively near or kind between be quite conservative
, therefore, in view of the uncertainty of Fugu category fry Morphological Identification at present, the present invention is using microsatellite molecular marker to 2
Kind Fugu fry effectively differentiate and distinguish.
The content of the invention
The object of the present invention is to provide a kind of method for quick and precisely differentiating fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry, with
The defects of overcoming the existing identification from morphology strong there are subjectivity and cannot effectively identifying two kinds of fries, and the present invention exists
On the basis of not putting to death fry, only need to cut off a small amount of tail fin or other Fish tissue parts can be differentiated there is stability
It is high, easy to operate reproducible, highly practical and the advantages that can quick and precisely differentiate.
The technical scheme is that
A kind of diagnostic primers of fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) juvenile fish, are named as F-FXF147, sequence is as follows：
F：5’-AACAAGTGCATGGCTGAGTG-3’(SEQ ID No.1)
R：5’-TTGACAGATGTGGAGCTGATG-3’(SEQ ID No.2)
The discrimination method of fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry of the present invention, comprises the following steps：
1) 2 kinds of Fugu identification sample DNAs are extracted；
2) sample extracted with above-mentioned primer pair step 1) carries out DNA cloning, obtains PCR product；
3) PCR product is detected with 2.2% Ago-Gel (EB dyeing) electrophoresis, the band of 300bp or so sizes
For fugu obscurus, the band of 600bp or so sizes is Fugu Xanthopterus (Temminck et Schlegel).
The pcr amplification reaction system of the present invention can use the 2 × Taq provided by Shanghai Jie Yi bio tech ltd
Plus Mix, the inside include the required component of PCR reaction systems, only need to disposably add, and easy to operate, reduction experiment misses
Difference.
The present invention is to be set up first based on molecular biology method to fugu obscurus and the progress of Fugu Xanthopterus (Temminck et Schlegel) fry
Reflect method for distinguishing, using the special micro-satellite primers F-FXF147 for synthesizing, screening from fugu obscurus, establishes one commonly
PCR reaction can realize differentiate 2 kinds of Fugu fries new method.This method is known without relying on the taxonomy of fishes of specialty
Know, it is not required that there are the rich experiences that long campaigns fish differentiate, it is only necessary to take PCR product to be detected into row agarose gel electrophoresis
Stablized, clear, the obvious amplified band of difference, cost is low, easy to operate, highly practical, has to Fugu breeding production
There is certain directive function.
Brief description of the drawings
Fig. 1：Amplification of the F-FXF147 primers in 2 colonies of fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel).
M-Marker；1-12 is Fugu Xanthopterus (Temminck et Schlegel)；13-24 is fugu obscurus.
Embodiment
Below in conjunction with attached drawing embodiment, the present invention is described in further detail.
Embodiment
1. the extraction of fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry genomic DNA and quantitative
Using Shanghai JaRa bio-engineering cells/tissue gene group DNA extraction kit 2 kinds of Fugu fries of rapid extraction
(fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) are limited both from Jiangsu Zhong Yang groups share for the genomic DNA of tail fin (10~30mg)
Company, through morphology comprehensive identification, is determined as fugu obscurus and each 12 tail of Fugu Xanthopterus (Temminck et Schlegel)), kit extract sample DNA compared with
Traditional method for extracting sample DNA have the characteristics that extraction efficiency is high, speed is fast, extraction DAN mass is high.
2. the screening of microsatellite locus and the synthesis of primer
First with the fugu obscurus fry identified in hundred Tyke biotech firms of Beijing RNA extracts kits extraction 1
RNA, and give Shanghai Jing Neng biotech firms carry out Illmina Hiseq2000 transcript profile high-flux sequences, using Tophat
Software carries out the assembling and splicing of sequence, then with microsatellite locus that may be present in SSRHunt software prediction sequences, utilizes
Primer5.0 software Design primers, and its primer is synthesized by Shanghai JaRa biotech firm.
3. the screening of fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry diagnostic primers
Using the DNA of two kinds of Fugu tail fin samples in 1, primer is in fugu obscurus and striped in examining 2 using PCR instrument
Amplification situation in Fugu fry
1) PCR amplification system
Pcr amplification reaction system (20 μ L) is as follows：Aqua sterilisa (7.4 μ L), 2 × Taq Plus Mix (10 μ L) by Shanghai all
Beneficial bio tech ltd provides, and the positive anti-primers of 10uM (each 0.8 μ L) are provided by Shanghai Jierui Biology Engineering Co., Ltd, east
Square Puffer juvenile fish tail fin template DNA (1 μ L).
2) pcr amplification reaction
In 95 DEG C of pre-degeneration 5min in PCR amplification instrument；30 circulations：94 DEG C are denatured 30sec, and anneal 30sec at 52 DEG C,
72 DEG C of extension 30sec；Last 72 DEG C of extensions 5min, 4 DEG C of preservations.
3) agarose gel electrophoresis detects
5 μ L of pcr amplification reaction product are taken to be detected into row agarose gel electrophoresis, EB is dyed and used ultraviolet gel imaging system
Take pictures.
4) diagnostic primers is definite
Pick out can in fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry Successful amplification and product through 3) detect can draw it is bright
The pair of primers of the other band of significant difference, the primer sequence are：
F：5’-AACAAGTGCATGGCTGAGTG-3’(SEQ ID No.1)；
R：5’-TTGACAGATGTGGAGCTGATG-3’(SEQ ID No.2).
This electrophoretogram to primer products therefrom is as shown in Figure 1, according to electrophoretic band compared with standard Marker：300bp
The band of left and right size is that the band of fugu obscurus, 600bp or so size is Fugu Xanthopterus (Temminck et Schlegel).
Claims (2)
1. a kind of differentiate fugu obscurus and the primer of Fugu Xanthopterus (Temminck et Schlegel) fry, it is characterised in that the primer is named as F-
FXF147, its sequence are as follows：
F：5’-AACAAGTGCATGGCTGAGTG-3,；
R：5’-TTGACAGATGTGGAGCTGATG-3,。
2. a kind of fugu obscurus and the discrimination method of Fugu Xanthopterus (Temminck et Schlegel) fry, it is characterised in that comprise the following steps：
1) 2 kinds of Fugu fry DNA to be identified are extracted；
2) 2 kinds of Fugu identification samples of primer pair carry out DNA cloning described in claim 1, obtain PCR product；
3) PCR product is detected with 2.2% Ago-Gel EB dyeing electrophoresis, the band of 300bp or so sizes is dark line
The band of Fugu, 600bp or so size is Fugu Xanthopterus (Temminck et Schlegel).
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| CN201510665512.4A CN105176989B (en) | 2015-10-13 | 2015-10-13 | It is a kind of to differentiate fugu obscurus and the primer and method of Fugu Xanthopterus (Temminck et Schlegel) fry |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106755473A (en) * | 2017-03-29 | 2017-05-31 | 中国水产科学研究院 | A kind of Fugu belongs to the molecular identification method of fish male and female sex |
| CN109852703B (en) * | 2019-04-02 | 2022-02-11 | 南京师范大学 | SNP molecular marker related to fugu obscurus fullness and application thereof |
| CN110241225A (en) * | 2019-04-29 | 2019-09-17 | 中国水产科学研究院 | The SNP marker of species identification for river Puffer combines |
| CN111979336B (en) * | 2020-08-05 | 2022-07-29 | 南京师范大学 | Specific primer and method for identifying fugu obscurus, fugu rubripes and hybrid fugu rubripes |
| CN112795661B (en) * | 2020-11-20 | 2023-01-31 | 南京师范大学 | Microsatellite marker, primer, method and application related to fugu obscurus growth traits |
| CN117925863B (en) * | 2024-03-25 | 2024-06-18 | 海南热带海洋学院崖州湾创新研究院 | Method for identifying puffer fish, takifugu megaly and takifugu hexakistrodon |
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Non-Patent Citations (3)
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| Construction of a microsatellite-based genetic linkage map for half-smooth tongue sole Cynoglossus semilaevis;SONG et al;《Current Zoology》;20131231;99–108 * |
| Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of obscure puffer (Takifugu obscurus) and cross-species amplification;Ma et al;《Conserv Genet》;20091231;摘要，第956页左栏第2段-第957页左栏第2段，表1-2 * |
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