CN110106256A - A kind of molecular labeling primer and its application identifying egg-shaped pompano and Bu Shi silvery pomfret Scad - Google Patents
A kind of molecular labeling primer and its application identifying egg-shaped pompano and Bu Shi silvery pomfret Scad Download PDFInfo
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- CN110106256A CN110106256A CN201910314393.6A CN201910314393A CN110106256A CN 110106256 A CN110106256 A CN 110106256A CN 201910314393 A CN201910314393 A CN 201910314393A CN 110106256 A CN110106256 A CN 110106256A
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Abstract
The purpose of the present invention is to provide a kind of molecular labeling primers and its application for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad; help to solve the problems such as ocean type mixes; for silvery pomfret Scad cultivation, silvery pomfret Scad protection of resources, rationally utilization and biodiversity research provide technical support.It is a kind of identify egg-shaped pompano and Bu Shi silvery pomfret Scad molecular labeling primer, including primer CSF and primer CSR, particular sequence it is as follows: CSF:GTCATACGCTCCCGAAGAT；CSR:TAGGGCTAAGCATAGTGGG.Molecular labeling primer is for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad;It will include the kit of the molecular labeling primer and PCR reaction system reagent applied to identification egg-shaped pompano and Bu Shi silvery pomfret Scad；Further include the molecular labeling for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad, further includes identifying its seed.There are the differences of 2 bases for two kinds of fish from the results of view, show as 1 conversion, 1 transversion, result is stablized, and repeatability is strong.
Description
Technical field
The invention belongs to field of molecular biotechnology, and in particular to a kind of identification egg-shaped pompano seed and Bu Shi silvery pomfret Scad seed
Molecular labeling primer and its application.
Background technique
Egg-shaped pompano (Trachinotus ovatus) and Bu Shi silvery pomfret Scad (Trachinotus blochii) are under the jurisdiction of perch
Shape mesh (Perciformes), Scad section (Carangidae), silvery pomfret Scad belong to (Trachinotus), are distributed widely in the Indian Ocean, peace
The temperate zone in ocean and the Atlantic Ocean, Tropical Ocean Area.Egg-shaped pompano and Bu Shi silvery pomfret Scad belong to the higher seafood fish of economic value, China sea
South, Guangdong and Fujian coast have just carried out the cage culture of both silvery pomfret Scad from the nineties in last century.Outside due to two kinds of silvery pomfret Scad
Shape is very much like, and coastal area of china various regions are referred to as yellow cured silvery pomfret, seawater orbfish or golden silvery pomfret.Although two kinds of silvery pomfret Scad are very much like, cloth
The thermophily of family name silvery pomfret Scad is poor compared with egg-shaped pompano, is only capable of smoothly overwintering in Hainan, is difficult in colder Guangdong and Fujian coast
High-volume death will occur whenever meeting with strong cold wave, cause serious economic loss for overwintering cultivation.
In actual production, due to that can not distinguish two kinds of silvery pomfret Scad seeds from the appearance, many Bu Shi silvery pomfret Scad seeds can be worked as
It sells and cultivates at egg-shaped pompano seed, undoubtedly will cause economic loss.Currently used for egg-shaped pompano and Bu Shi silvery pomfret Scad seed
Intuitive method yet there are no formal report.Therefore the reliable molecular labeling for finding egg-shaped pompano and Bu Shi silvery pomfret Scad, from the kind mixed
Identify in matter and distinguishes two kinds of silvery pomfret Scad as a urgent problem to be solved.
Summary of the invention
In view of the above problems, the purpose of the present invention is to provide a kind of molecular labelings for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad
Primer and its application help to solve ocean type the problems such as mixing, cultivate for silvery pomfret Scad, silvery pomfret Scad protection of resources, rationally utilize with
And biodiversity research provides technical support.
Technical scheme is as follows:
A kind of molecular labeling primer identifying egg-shaped pompano and Bu Shi silvery pomfret Scad, including primer CSF and primer CSR, primer CSF
Sequence as shown in SEQ ID NO:1, for the sequence of primer CSR as shown in SEQ ID NO:2, particular sequence is as follows:
CSF:GTCATACGCTCCCGAAGAT；
CSR:TAGGGCTAAGCATAGTGGG.
A kind of application for the molecular labeling primer identifying egg-shaped pompano and Bu Shi silvery pomfret Scad, which is characterized in that molecular labeling draws
Object further includes identifying its seed for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad.
Mirror method for distinguishing includes the following steps:
1) egg-shaped pompano and Bu Shi silvery pomfret Scad species composition sample populations are chosen, extract the DNA of two sample populations respectively；
2) DNA extracted respectively using step 1) is expanded primer CSF and primer CSR by PCR reaction system as template
Increase；
3) the resulting amplified production of step 2) purified, be sequenced, respectively obtain egg-shaped pompano and point of Bu Shi silvery pomfret Scad
The sequence of son label；
4) sequencing result is compared, its type is identified；
Wherein, the sequence of the molecular labeling of egg-shaped pompano described in step 3) is as shown in SEQ ID NO:3, Bu Shi silvery pomfret Scad's
The sequence of molecular labeling is as shown in SEQ ID NO:4；
PCR reaction system described in step 2) reagent composition include: ultrapure water 17.5ul, 10 × buffer2.5ul,
DNTPs 2ul, rTaq 0.15ul, each 1ul of DNA profiling 1ul, Primer；
The program of the amplification includes: 94 DEG C of 2~5min of initial denaturation, then 94 DEG C of denaturation 35sec, 50 DEG C~53 DEG C annealing
30~35sec, 72 DEG C of extension 35sec, 30~38 circulations, 3~10min of last 72 DEG C of extensions, 4 DEG C of preservations are carried out.
Further, the application for identifying egg-shaped pompano and the molecular labeling primer of Bu Shi silvery pomfret Scad will include described
Molecular labeling primer and the kit of PCR reaction system reagent be applied to identify egg-shaped pompano and Bu Shi silvery pomfret Scad；Further include
The molecular labeling is for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad.
Beneficial effects of the present invention are as follows:
The present invention carries out PCR amplification using the designed primer for identifying egg-shaped pompano and the molecular labeling of Bu Shi silvery pomfret Scad,
The sequence of molecular labeling is obtained, there are the differences of 2 bases for two kinds of fish from the results of view, and it shows as 1 and converts, 1 transversion,
Its result is stablized, and repeatability is strong；
The present invention provided from the level of genome, using the technology of molecular labeling one kind be advantageously applied to identify it is oval
The method of silvery pomfret Scad and Bu Shi silvery pomfret two different plant species of Scad, effectively prevents being difficult to differentiate between because of mode of appearance etc. and may cause economic damage
Lose, ecological environment destruction the problems such as, facilitate solve ocean germplasm mixed questions, for silvery pomfret Scad cultivate, silvery pomfret Scad protection of resources, rationally
Using and biodiversity research provide technical support；
Easy to operate on discrimination method of the invention, primer specificity is strong, using the high-efficient of PCR amplification, is easy to slap
It holds, identification result Stability and veracity with higher.
Specific embodiment
Below by way of specific case study on implementation, the present invention is described in further detail, it should be understood that these embodiments are only used
In illustrating the present invention rather than limit the scope of the invention, after the present invention has been read, those skilled in the art couple
It is as defined in the appended claims that the modification of various equivalent forms of the invention falls within the application.
If all raw materials and reagents of the invention are the raw material of conventional market, reagent without specified otherwise.
Embodiment 1
A kind of molecular labeling primer and its discrimination method identifying egg-shaped pompano and Bu Shi silvery pomfret Scad:
1) acquisition is chosen from the egg-shaped pompano and each 5 individuals of Bu Shi silvery pomfret Scad species in Sanya, Hainan littoral sea, composition
Sample populations extract the DNA of two sample populations respectively；
2) DNA extracted using step 1) is passed through PCR reaction system with primer CSF and primer CSR respectively and is expanded as template
Increase；
The composition of the PCR reaction system are as follows:
It is sequentially added in 0.2ml centrifuge tube:
PCR amplification program is 94 DEG C of initial denaturation 2min, and (94 DEG C of denaturation 15sec, 50 DEG C of annealing 15sec, 72 DEG C extend
15sec) × 38 circulation, 72 DEG C of extension 3min, 4 DEG C of preservations can long-term preservation.
3) PCR product is sequenced after purification, and the sequencing result of 2 groups is compared, identify its type.
Embodiment 2
A kind of molecular labeling primer and its discrimination method identifying egg-shaped pompano and Bu Shi silvery pomfret Scad:
1) acquisition is chosen from the egg-shaped pompano and each 5 individuals of Bu Shi silvery pomfret Scad seed in Sanya, Hainan littoral sea, composition
Sample populations extract the DNA of two sample populations respectively；
2) DNA extracted using step 1) is passed through PCR reaction system with primer CSF and primer CSR respectively and is expanded as template
Increase；
The composition of the PCR reaction system are as follows:
It is sequentially added in 0.2ml centrifuge tube:
PCR amplification program is 94 DEG C of initial denaturation 4min, and (94 DEG C of denaturation 35sec, 53 DEG C of annealing 30sec, 72 DEG C extend
15sec) × 30 circulation, 72 DEG C of extension 10min, 4 DEG C of preservations can long-term preservation.
3) PCR product is sequenced after purification, and the sequencing result of 2 groups is compared, identify its type.
Embodiment 3
A kind of molecular labeling primer and its discrimination method identifying egg-shaped pompano and Bu Shi silvery pomfret Scad:
1) acquisition is chosen from the egg-shaped pompano and each 5 individuals of Bu Shi silvery pomfret Scad seed in Sanya, Hainan littoral sea, composition
Sample populations extract the DNA of two sample populations respectively；
2) DNA extracted using step 1) is passed through PCR reaction system with primer CSF and primer CSR respectively and is expanded as template
Increase；
The composition of the PCR reaction system are as follows:
It is sequentially added in 0.2ml centrifuge tube:
PCR amplification program is 94 DEG C of initial denaturation 5min, and (94 DEG C of denaturation 35sec, 50 DEG C of annealing 35sec, 72 DEG C extend
35sec) × 38 circulation, 72 DEG C of extension 10min, 4 DEG C of preservations can long-term preservation.
3) PCR product is sequenced after purification, and the sequencing result of 2 groups is compared, identify its type.
It can be seen that by egg-shaped pompano seed and Bu Shi silvery pomfret Scad seed mtDNA CS gene 75bp sequence permutation result oval
There are the differences of 2 bases for silvery pomfret Scad seed and Bu Shi silvery pomfret Scad seed, show as 1 conversion, 1 transversion.
The mtDNA CS gene 75bp sequence alignment result of egg-shaped pompano seed and Bu Shi silvery pomfret Scad seed are as follows:
Egg-shaped pompano seed: ATGAAGCCCA ACCACGAAAG TGACTTTACA TAACCTGAAC
Bu Shi silvery pomfret Scad seed: * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * CT********
Ruler:.........10.........20.........30.........40
Egg-shaped pompano seed: CCACGAAAGC TAAGAAACAA ACTGGGATTA GATAC
Bu Shi silvery pomfret Scad seed: * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
Ruler:.........50.........60.........70....75
(wherein " * " indicates identical base sequence site)
Sequencing result shows that 5 tail egg-shaped pompano seed sample amplification results are consistent, while 5 tail Bu Shi silvery pomfret Scad seed samples
Amplification is always, as a result stable and repeatable strong.
Sequence table
<110>Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst
<120>a kind of molecular labeling primer and its application for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad
<141> 2019-04-18
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<400> 1
gtcatacgct cccgaagat 19
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
tagggctaag catagtggg 19
<210> 3
<211> 75
<212> DNA
<213> Artificial sequence
<400> 3
atgaagccca accacgaaag tgactttaca taacctgaac ccacgaaagc taagaaacaa 60
actgggatta gatac 75
<210> 4
<211> 75
<212> DNA
<213> Artificial sequence
<400> 4
atgaagccca accacgaaag tgactttaca ctacctgaac ccacgaaagc taagaaacaa 60
actgggatta gatac 75
Claims (8)
1. a kind of molecular labeling primer for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad, which is characterized in that including primer CSF and primer
The sequence of CSR, primer CSF are as shown in SEQ ID NO:1, and the sequence of primer CSR is as shown in SEQ ID NO:2.
2. the application of the molecular labeling primer described in claim 1 for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad, which is characterized in that point
Sub- labeled primer further includes identifying its seed for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad.
3. the application of the molecular labeling primer by identification egg-shaped pompano as claimed in claim 2 and Bu Shi silvery pomfret Scad, which is characterized in that
Mirror method for distinguishing includes the following steps:
1) egg-shaped pompano and Bu Shi silvery pomfret Scad species composition sample populations are chosen, extract the DNA of two sample populations respectively；
2) DNA extracted respectively using step 1) is expanded primer described in claim 1 by PCR reaction system as template
Increase；
3) the resulting amplified production of step 2 purified, be sequenced, respectively obtain the molecule mark of egg-shaped pompano and Bu Shi silvery pomfret Scad
The sequence of note；
4) sequencing result is compared, its type is identified.
4. the application of the molecular labeling primer by identification egg-shaped pompano described in claim 2 or 3 and Bu Shi silvery pomfret Scad, feature exist
In, the sequence of the molecular labeling of egg-shaped pompano described in step 3) as shown in SEQ ID NO:3, the molecular labeling of Bu Shi silvery pomfret Scad
Sequence is as shown in SEQ ID NO:4.
5. the application of the molecular labeling primer by identification egg-shaped pompano described in claim 2 or 3 and Bu Shi silvery pomfret Scad, feature exist
In the reagent composition of PCR reaction system described in step 2 includes: ultrapure water 17.5ul, 10 × buffer 2.5ul, dNTPs
2ul, rTaq 0.15ul, each 1ul of DNA profiling 1ul, Primer.
6. the application of the molecular labeling primer of identification egg-shaped pompano and Bu Shi silvery pomfret Scad as described in claim 5, which is characterized in that
The program of amplification described in step 2 includes: 94 DEG C of 2 ~ 5min of initial denaturation, then 94 DEG C of denaturation 35sec, 50 DEG C ~ 53 DEG C annealing 30
~ 35sec, 72 DEG C of extension 35sec, 30 ~ 38 circulations, 3 ~ 10min of last 72 DEG C of extensions, 4 DEG C of preservations are carried out.
7. the application of the molecular labeling primer by identification egg-shaped pompano described in claim 2 or 3 and Bu Shi silvery pomfret Scad, feature exist
In will include that the kit of the molecular labeling primer and PCR reaction system reagent is applied to identify egg-shaped pompano and cloth
Family name silvery pomfret Scad.
8. the application of the molecular labeling primer by identification egg-shaped pompano as claimed in claim 4 and Bu Shi silvery pomfret Scad, which is characterized in that
The molecular labeling is for identifying egg-shaped pompano and Bu Shi silvery pomfret Scad.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910314393.6A CN110106256B (en) | 2019-04-18 | 2019-04-18 | Molecular marker primer for identifying trachinotus ovatus and trachinotus branchii and application thereof |
| AU2020100468A AU2020100468A4 (en) | 2019-04-18 | 2020-03-27 | Molecular marker primer for identifying trachitotus ovatus and trachinotus blochii and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910314393.6A CN110106256B (en) | 2019-04-18 | 2019-04-18 | Molecular marker primer for identifying trachinotus ovatus and trachinotus branchii and application thereof |
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| CN110106256A true CN110106256A (en) | 2019-08-09 |
| CN110106256B CN110106256B (en) | 2020-12-01 |
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| CN201910314393.6A Expired - Fee Related CN110106256B (en) | 2019-04-18 | 2019-04-18 | Molecular marker primer for identifying trachinotus ovatus and trachinotus branchii and application thereof |
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| AU (1) | AU2020100468A4 (en) |
Cited By (6)
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| CN110521637A (en) * | 2019-08-26 | 2019-12-03 | 中国水产科学研究院南海水产研究所 | A kind of egg-shaped pompano family full-sibs construction method |
| CN110551732A (en) * | 2019-08-30 | 2019-12-10 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus antimicrobial peptide LEAP-2 gene and application thereof |
| CN111088370A (en) * | 2020-01-20 | 2020-05-01 | 中国水产科学研究院南海水产研究所 | Sex-specific molecular marker primer for Trachinotus ovatus, identification method and application of sex-specific molecular marker primer |
| CN112662785A (en) * | 2021-01-28 | 2021-04-16 | 中国水产科学研究院南海水产研究所 | Molecular marker primer pair, kit and identification method for identifying Benincasa quinqueradiata and Benincasa quinqueradiata |
| CN113667764A (en) * | 2021-04-25 | 2021-11-19 | 中国水产科学研究院南海水产研究所 | SNP molecular marker with cryptocaryon irritans disease resistance-related traits for trachinotus ovatus and application of SNP molecular marker |
| CN113684285A (en) * | 2021-04-25 | 2021-11-23 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus cryptocaryon irritans disease associated SNP molecular marker, primer and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110521637A (en) * | 2019-08-26 | 2019-12-03 | 中国水产科学研究院南海水产研究所 | A kind of egg-shaped pompano family full-sibs construction method |
| CN110551732A (en) * | 2019-08-30 | 2019-12-10 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus antimicrobial peptide LEAP-2 gene and application thereof |
| CN111088370A (en) * | 2020-01-20 | 2020-05-01 | 中国水产科学研究院南海水产研究所 | Sex-specific molecular marker primer for Trachinotus ovatus, identification method and application of sex-specific molecular marker primer |
| CN111088370B (en) * | 2020-01-20 | 2022-06-21 | 中国水产科学研究院南海水产研究所 | Sex-specific molecular marker primer for Trachinotus ovatus, identification method and application of sex-specific molecular marker primer |
| CN112662785A (en) * | 2021-01-28 | 2021-04-16 | 中国水产科学研究院南海水产研究所 | Molecular marker primer pair, kit and identification method for identifying Benincasa quinqueradiata and Benincasa quinqueradiata |
| CN112662785B (en) * | 2021-01-28 | 2023-03-14 | 中国水产科学研究院南海水产研究所 | Molecular marker primer pair, kit and identification method for identifying Benincasa quinqueradiata and Benincasa quinqueradiata |
| CN113667764A (en) * | 2021-04-25 | 2021-11-19 | 中国水产科学研究院南海水产研究所 | SNP molecular marker with cryptocaryon irritans disease resistance-related traits for trachinotus ovatus and application of SNP molecular marker |
| CN113684285A (en) * | 2021-04-25 | 2021-11-23 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus cryptocaryon irritans disease associated SNP molecular marker, primer and application thereof |
| CN113684285B (en) * | 2021-04-25 | 2022-05-10 | 中国水产科学研究院南海水产研究所 | Trachinotus ovatus cryptocaryon irritans disease associated SNP molecular marker, primer and application thereof |
| CN115011701A (en) * | 2021-04-25 | 2022-09-06 | 中国水产科学研究院南海水产研究所 | SNP molecular marker with cryptocaryon irritans disease resistance-related traits for trachinotus ovatus and application of SNP molecular marker |
| CN115011701B (en) * | 2021-04-25 | 2024-03-19 | 中国水产科学研究院南海水产研究所 | SNP molecular marker of character related to anti-cryptocaryon irritans of trachinotus ovatus and application thereof |
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| AU2020100468A4 (en) | 2020-04-30 |
| CN110106256B (en) | 2020-12-01 |
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