CN105176989A - Primers and method for identifying takifugu obscurus fry and takifugu xanthopterus fry - Google Patents
Primers and method for identifying takifugu obscurus fry and takifugu xanthopterus fry Download PDFInfo
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- CN105176989A CN105176989A CN201510665512.4A CN201510665512A CN105176989A CN 105176989 A CN105176989 A CN 105176989A CN 201510665512 A CN201510665512 A CN 201510665512A CN 105176989 A CN105176989 A CN 105176989A
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- fry
- takifugu
- fugu
- xanthopterus
- obscurus
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- 241000980706 Takifugu obscurus Species 0.000 title claims abstract description 34
- 241000253385 Takifugu xanthopterus Species 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title abstract description 15
- 229920002684 Sepharose Polymers 0.000 claims abstract description 3
- 238000004043 dyeing Methods 0.000 claims abstract description 3
- 238000001962 electrophoresis Methods 0.000 claims abstract description 3
- 241001441723 Takifugu Species 0.000 claims description 20
- 238000012797 qualification Methods 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 2
- 238000012850 discrimination method Methods 0.000 claims description 2
- 238000012408 PCR amplification Methods 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000007400 DNA extraction Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract 1
- 241000251468 Actinopterygii Species 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 8
- 108091092878 Microsatellite Proteins 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000002068 genetic effect Effects 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
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
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- 238000013508 migration Methods 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 241000269799 Perca fluviatilis Species 0.000 description 1
- 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
- 244000144974 aquaculture Species 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
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- 235000013372 meat Nutrition 0.000 description 1
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- 239000003147 molecular marker Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
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- 230000009897 systematic effect Effects 0.000 description 1
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Abstract
The invention relates to primers and a method for identifying takifugu obscurus fry and takifugu xanthopterus fry. The pair of primers is named as F-FXF147, and the sequences of the primers are shown as SEQ ID No.1 and SEQ ID No.2. In the verification process, a common DNA extraction reagent kit is used for extracting the genomic DNA of the takifugu obscurus fry and the genomic DNA of the takifugu xanthopterus fry, a DNA sample of takifugu obscurus and a DNA sample of takifugu xanthopterus are subjected to PCR amplification through the primers, PCR products are detected through 2.2% sepharose gel (EB dyeing) electrophoresis, and the fry with strips around 300 bp are the takifugu obscurus, and the fry with strips around 600 bp are the takifugu xanthopterus. According to the primers and the method for identifying the takifugu obscurus fry and the takifugu xanthopterus fry, verification can be conducted on the basis that only a small number of tail fins are sheared instead of killing the fry, and the primers and the method have the advantages of being high in stability, good in repeatability, high in practicability, easy to operate, capable of quickly achieving verification and the like.
Description
Technical field
The invention belongs to aquaculture field, relate to the qualification of a kind of Protocols in Molecular Biology and belong to fry method together, more particularly, the present invention relates to and utilize microsatellite marker accurately to differentiate fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry.
Background technology
Fugu obscurus (Takifugufasciatus) is under the jurisdiction of Tetraodontiformes (Letrodontiforms), Molidae (Tetraodontidae), Fugu genus (Fugu), be commonly called as river Puffer, bubble fish, belong to migration fishes, be distributed in the East China Sea and the Yellow Sea, the Bohai Sea and the middle and lower reach of Yangtze River, the high fishery products of a kind of economic worth, ranking first of the Changjiang river " four your name fishes ", is the model in traditional cuisines.
Fugu Xanthopterus (Temminck et Schlegel) (Takifuguxanthopterus) is under the jurisdiction of Tetraodontiformes (Letrodontiforms), Molidae (Tetraodontidae), Fugu genus (Fugu), be commonly called as yellowfin Fugu, flower ship bar etc., also migration fishes are belonged to, itself and fugu obscurus to perch sea area close with mating period, mainly be distributed in the East Sea, the South Sea, the Huanghai Sea and the Bohai Sea, also enter rivers mouth.
Fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) differ from one another, and because of ecological habit difference, both meats are far different.Fugu Xanthopterus (Temminck et Schlegel) has a very wide distribution, and output is higher; Fugu obscurus build is comparatively large, and the speed of growth is very fast, and its taste flavor and nutritive value are far away higher than Fugu Xanthopterus (Temminck et Schlegel).Along with the market demand of two kinds of Fugus expands day by day, the demand of its seed also grows with each passing day, and therefore the source of fry is the key that two kinds of Fugus cultivate with accurate discriminating.But on morphology, the fry outward appearance build of fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) is quite similar, if gain knowledge without professional classification, only go to distinguish very easily to produce with naked eyes and obscure.Even therefore cultivate veteran fisherman can not go accurately to distinguish this two kinds of Fugu fries, once mixed breed, not only increase the workload of later stage adult fish classification process, also there will be species hybridization, and then cause kind of matter to mix, product qualitative change is bad.In addition; on market, the juvenile fish of two kinds of Fugus is usually also confused; due to fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) market price obvious difference; cause some businessman for seeking economic interests; Fugu Xanthopterus (Temminck et Schlegel) fry is served as fugu obscurus fry to sell by mistake; the interests of human consumer are not only invaded, also the extreme influence economic benefit of fisherman and river Puffer breeding enterprise.Therefore, a kind of accurate method differentiated and distinguish 2 kinds of Fugus of active demand on market.
Microsatellite DNA in genome is with Mendelian fashion heredity; express in codominance; and have that distribution is wide, genetic information content is high and be convenient to the advantages such as PCR detection simultaneously; in genetics-breeding in fish research, apply that microsatellite marker carries out species population genetic construction and diversity analysis, the structure of molecular genetic linkage map, species develop and the research such as sibship; the differentiation of accurate systematics can be carried out to different plant species; accurately determine evolutionary path and the taxonomy of species, and then have object to protect to germ plasm resource, gene pool.Research shows that micro-satellite flanking sequence comparatively closely or between kind is quite guarded in sibship, therefore, in view of current Fugu belongs to the uncertainty of fry Morphological Identification, the present invention uses microsatellite molecular marker effectively differentiate 2 kinds of Fugu fries and distinguish.
Summary of the invention
The object of this invention is to provide a kind of method quick and precisely differentiating fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry, to overcome, existing to there is subjectivity from morphology qualification strong and effectively can not identify the defect of two kinds of fries, and the present invention is not putting to death on the basis of fry, only need cut off a small amount of tail fin or other Fish tissue part can be differentiated, there is stability high, simple to operate reproducible, practical and the advantage such as quick and precisely can to differentiate.
Technical scheme of the present invention is
A diagnostic primers for fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) juvenile fish, called after F-FXF147, sequence is as follows:
F：5’-AACAAGTGCATGGCTGAGTG-3’(SEQIDNo.1)
R：5’-TTGACAGATGTGGAGCTGATG-3’(SEQIDNo.2)
The discrimination method of fugu obscurus of the present invention and Fugu Xanthopterus (Temminck et Schlegel) fry, comprises the following steps:
1) 2 kinds of Fugu qualification sample DNAs are extracted;
2) by above-mentioned primer pair step 1) sample that extracts carries out DNA cloning, obtains PCR primer;
3) detect PCR primer with 2.2% sepharose (EB dyeing) electrophoresis, the band of about 300bp size is fugu obscurus, and the band of about 600bp size is Fugu Xanthopterus (Temminck et Schlegel).
Pcr amplification reaction system of the present invention can adopt the 2 × TaqPlusMix provided by Jie Yi bio tech ltd, Shanghai, and the inside comprises the required composition of PCR reaction system, only needs disposable adding, simple to operate, minimizing experimental error.
The present invention sets up based on molecular biology method to carry out mirror method for distinguishing to fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry first, utilize the special micro-satellite primers F-FXF147 from fugu obscurus synthesis, screening, establish the novel method that a common PCR reaction just can realize discriminating 2 kinds of Fugu fries.Present method is without the need to relying on the taxonomy of fishes knowledge of specialty, the rich experiences with the discriminating of long campaigns fish are not needed yet, only need to get PCR primer carry out agarose gel electrophoresis detect stablized, the amplified band of clear, obvious difference, cost is low, simple to operate, practical, to Fugu breeding production, there is certain directive function.
Accompanying drawing explanation
The amplification of Fig. 1: F-FXF147 primer in fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) 2 colonies.
M-Marker; 1-12 is Fugu Xanthopterus (Temminck et Schlegel); 13-24 is fugu obscurus.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment
1. the extraction and quantitatively of fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry genomic dna
(fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) all come from Jiangsu Zhongyang Group Co., Ltd. to the genomic dna of employing Shanghai JaRa bio-engineering cells/tissue gene group DNA extraction kit rapid extraction 2 kinds of Fugu fry tail fins (10 ~ 30mg), through morphology comprehensive identification, be defined as fugu obscurus and each 12 tails of Fugu Xanthopterus (Temminck et Schlegel)), test kit extracts that than traditional method for extracting sample DNA, sample DNA has that extraction efficiency is high, speed is fast, extract DAN quality high.
2. the screening of microsatellite locus and the synthesis of primer
First Beijing hundred Tyke biotech firm RNA is utilized to extract the RNA of the fugu obscurus fry of identifying in test kit extraction 1, and give Shanghai Jing Neng biotech firm and carry out IllminaHiseq2000 transcript profile high-flux sequence, Tophat software is adopted to carry out assembling and the splicing of sequence, then with the microsatellite locus that may exist in SSRHunt software prediction sequence, utilize Primer5.0 software design primer, and synthesize its primer by Shanghai JaRa biotech firm.
3. the screening of fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry diagnostic primers
Utilize the DNA of two kinds of Fugu tail fin samples in 1, adopt the amplification situation of primer in fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry in PCR instrument inspection 2
1) PCR amplification system
Pcr amplification reaction system (20 μ L) is as follows: aqua sterilisa (7.4 μ L), 2 × TaqPlusMix (10 μ L) is provided by Jie Yi bio tech ltd, Shanghai, the positive anti-primer of 10uM (each 0.8 μ L) is provided by Shanghai Jierui Biology Engineering Co., Ltd, Fugu juvenile fish tail fin template DNA (1 μ L).
2) pcr amplification reaction
In 95 DEG C of denaturation 5min in PCR amplification instrument; 30 circulations: 94 DEG C of sex change 30sec, anneal at 52 DEG C 30sec, and 72 DEG C extend 30sec; Last 72 DEG C extend 5min, 4 DEG C of preservations.
3) agarose gel electrophoresis detects
Get pcr amplification reaction product 5 μ L and carry out agarose gel electrophoresis detection, EB dyes and uses ultraviolet gel imaging system to take pictures.
4) determination of diagnostic primers
Pick out can in fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry Successful amplification and product through 3) detect the pair of primers that can draw significant difference band, described primer sequence is:
F：5’-AACAAGTGCATGGCTGAGTG-3’(SEQIDNo.1)；
R：5’-TTGACAGATGTGGAGCTGATG-3’(SEQIDNo.2)。
This to the electrophorogram of primer products therefrom as shown in Figure 1, compares with standard Marker according to electrophoretic band: the band of about 300bp size is fugu obscurus, and the band of about 600bp size is Fugu Xanthopterus (Temminck et Schlegel).
Claims (2)
1. differentiate a primer for fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry, it is characterized in that described primer called after F-FXF147, its sequence is as follows:
F：5’-AACAAGTGCATGGCTGAGTG-3
，；
R：5’-TTGACAGATGTGGAGCTGATG-3
，。
2. a discrimination method for fugu obscurus and Fugu Xanthopterus (Temminck et Schlegel) fry, is characterized in that, comprises the following steps:
1) 2 kinds of Fugu fry DNA to be identified are extracted;
2) carry out DNA cloning with the 2 kinds of Fugu qualification samples of primer pair described in claim 1, obtain PCR primer;
3) detect PCR primer with 2.2% sepharose EB dyeing electrophoresis, the band of about 300bp size is fugu obscurus, and the band of about 600bp size is Fugu Xanthopterus (Temminck et Schlegel).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 | Publication Date |
|---|---|
| CN105176989A true CN105176989A (en) | 2015-12-23 |
| CN105176989B CN105176989B (en) | 2018-05-04 |
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| CN201510665512.4A Expired - Fee Related 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109852703A (en) * | 2019-04-02 | 2019-06-07 | 南京师范大学 | One kind SNP marker relevant to fugu obscurus coefficient of condition and its application |
| 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 |
| CN111979336A (en) * | 2020-08-05 | 2020-11-24 | 南京师范大学 | Specific primer and method for identifying fugu obscurus, fugu rubripes and hybrid fugu rubripes |
| CN112795661A (en) * | 2020-11-20 | 2021-05-14 | 南京师范大学 | Microsatellite marker, primer, method and application related to growth traits of fugu obscurus |
| CN117925863A (en) * | 2024-03-25 | 2024-04-26 | 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 |
-
2015
- 2015-10-13 CN CN201510665512.4A patent/CN105176989B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| MA ET AL: "Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of obscure puffer (Takifugu obscurus) and cross-species amplification", 《CONSERV GENET》 * |
| SONG ET AL: "Construction of a microsatellite-based genetic linkage map for half-smooth tongue sole Cynoglossus semilaevis", 《CURRENT ZOOLOGY》 * |
| 程长洪等: "暗纹东方鲀微卫星筛选和群体遗传多样性分析", 《江西农业大学学报》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109852703A (en) * | 2019-04-02 | 2019-06-07 | 南京师范大学 | One kind SNP marker relevant to fugu obscurus coefficient of condition and its application |
| 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 |
| CN111979336A (en) * | 2020-08-05 | 2020-11-24 | 南京师范大学 | Specific primer and method for identifying fugu obscurus, fugu rubripes and hybrid fugu rubripes |
| CN112795661A (en) * | 2020-11-20 | 2021-05-14 | 南京师范大学 | Microsatellite marker, primer, method and application related to growth traits of fugu obscurus |
| 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 |
| CN117925863A (en) * | 2024-03-25 | 2024-04-26 | 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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| CN105176989B (en) | 2018-05-04 |
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