AU2020100468A4 - Molecular marker primer for identifying trachitotus ovatus and trachinotus blochii and application thereof - Google Patents
Molecular marker primer for identifying trachitotus ovatus and trachinotus blochii and application thereof Download PDFInfo
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Abstract
An objective of the present invention is to provide a molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii and application thereof, which can help solve the problems of mixed marine species and the like, and provide technical support for pompano breeding, pompano resource conservation, rational use and biodiversity research. The molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii includes a primer CSF and a primer CSR, and specific sequences are as follows: CSF: GTCATACGCTCCCGAAGAT; and CSR: TAGGGCTAAGCATAGTGGG. The molecular marker primer is used for identifying Trachitotus ovatus and Trachinotus blochii; and a kit including the molecular marker primer and a PCR reaction system reagent is applied to the identification of Trachitotus ovatus and Trachinotus blochii. The application also includes that molecular markers are used for identifying Trachitotus ovatus and Trachinotus blochii, and the application also includes identifying fries thereof According to results, the two fishes have a difference of 2 bases, which is shown as 1 transformation and 1 transversion. The results are stable and the reproducibility is high.
Description
MOLECULAR MARKER PRIMER FOR IDENTIFYING
TRACHITOTUS OVATUS AND TRACHINOTUS BLOCHIIAND
APPLICATION THEREOF
TECHNICAL FIELD
The present invention belongs to the field of molecular biotechnology, and particularly relates to a molecular marker primer for identifying Trachitotus ovatus fries and Trachinotus blochii fries and application thereof.
BACKGROUD
Both Trachitotus ovatus and Trachinotus blochii belong to Trachinotus, Carangidae, Perciformes, and are widely distributed in temperate zone and tropical sea areas of the Indian Ocean, the Pacific Ocean and the Atlantic Ocean. Both Trachitotus ovatus and Trachinotus blochii are marine fishes with high economic value. The cage culture of the two kinds of pompanos has been carried out in Hainan, Guangdong and Fujian coastal areas since the 1990s. Since the two kinds of pompanos are very similar in shape, they are both called golden pompano in coastal areas of China. Although the two kinds of pompanos are very similar, Trachinotus blochii has poor temperature resistance than Trachitotus ovatus, can only survive winter smoothly in Hainan and cannot survive winter in coasts of colder Guangdong and Fujian. Mass deaths occur whenever there is a strong cold wave, causing serious economic losses.
In actual production, since it is impossible to distinguish two kinds of pompano fries from the appearance, many Trachinotus blochii fries are regarded as Trachitotus ovatus fries for sales and culture, which will undoubtedly cause economic losses. A current method for visually identifying Trachitotus ovatus and Trachinotus blochii fries has not been officially reported. Therefore, how to search for reliable molecular markers of Trachitotus ovatus and Trachinotus blochii and identify the two kinds of pompanos from mixed species has become an urgent problem to be solved.
SUMMARY
In view of the foregoing problems, an objective of the present invention is to provide a molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii and application thereof, which can help solve the problems of mixed marine species and the like, and provide technical support for pompano breeding, pompano resource conservation, rational
2020100468 27 Mar 2020 use and biodiversity research.
The technical solution of the present invention is as follows:
a molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii, including a primer CSF and a primer CSR, where a sequence of the primer CSF is shown in SEQ ID NO: 1, a sequence of the primer CSR is shown in SEQ ID NO: 2, and specific sequences are as follows:
CSF: GTCATACGCTCCCGAAGAT;
CSR: TAGGGCTAAGCATAGTGGG.
Application of the molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii is provided, where the molecular marker primer is used for identifying Trachitotus ovatus and Trachinotus blochii, and the application also comprises identifying fries thereof.
An identification method includes the following steps:
1) : selecting Trachitotus ovatus and Trachinotus blochii to form sample populations, and extracting DNAs of the two sample populations respectively;
2) : respectively using the DNAs extracted in step 1) as templates to amplify the primer CSF and the primer CSR through a PCR reaction system;
3) : purifying and sequencing amplified products obtained in step 2) to obtain sequences of molecular markers of Trachitotus ovatus and Trachinotus blochii respectively; and
4) : comparing sequencing results to identify the species;
where the sequence of the molecular marker of Trachitotus ovatus in step 3) is shown in SEQ ID NO: 3, and the sequence of the molecular marker of Trachinotus blochii is shown in SEQ ID NO: 4;
a reagent of the PCR reaction system in step 2) includes 17.5 ul of ultra-pure water, 2.5 ul of 10 x buffer, 2 ul of dNTPs, 0.15 ul of rTaq, 1 ul of DNA template, and 1 ul of each primer; and a procedure for the amplification comprises pre-denaturation at 94°C for 2-5 min, then denaturation at 94°C for 35 s, annealing at 50-53°C for 30-35 s, extension at 72°C for 35 s, proceeding of 30-38 cycles, final extension at 72°C for 3-10 min, and storage at 4°C.
Further, according to the application of the molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii, a kit including the molecular marker primer and the reagent of the PCR reaction system applied to the identification of Trachitotus ovatus and Trachinotus blochii', and the application also includes that the molecular markers are used for identifying Trachitotus ovatus and Trachinotus blochii.
2020100468 27 Mar 2020
Beneficial effects of the present invention are as follows:
According to the present invention, the designed primers of the molecular markers for identifying Trachitotus ovatus and Trachinotus blochii are utilized to perform PCR amplification to obtain the sequences of the molecular markers. According to results, the two fishes have a difference of 2 bases, which is shown as 1 transformation and 1 transversion. The results are stable and the reproducibility is high.
The present invention provides a method favorable for identifying two different species, Trachitotus ovatus and Trachinotus blochii, from the genomic level by using a molecular marker technology, thereby effectively avoiding the problems of economic losses and destruction of the ecological environment and the like caused by difficulty in distinguishing appearances and the like, which can help solve the problems of mixed marine species, and provide technical support for pompano breeding, pompano resource conservation, rational use and biodiversity research.
The identification method of the present invention is simple to operate, has strong primer specificity and high efficiency of PCR amplification, and is easy to grasp. The identification results have high accuracy and stability.
DESCRIPTION OF THE EMBODIMENTS
The present invention is further described in detail below by way of specific embodiments. These embodiments are only intended to illustrate the present invention and are not intended to limit the protection scope of the present invention. Modifications of various equivalent forms of the present invention by those skilled in the art after the reading of the present invention are defined by the appended claims of the present application.
Unless otherwise stated, all the raw materials and reagents of the present invention are conventional raw materials and reagents on the market.
Embodiment 1
A molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii and an identification method thereof:
1) : Select 5 individuals of each of Trachitotus ovatus and Trachinotus blochii species acquired from coastal waters of Sanya of Hainan Province to form sample populations, and extract DNAs of the two sample populations respectively.
2) : Use the DNAs extracted in step 1) as templates to amplify the primer CSF and the primer CSR respectively through a PCR reaction system.
The composition of the PCR reaction system is:
2020100468 27 Mar 2020 the following are sequentially added into a 0.2 ml centrifuge tube:
ultra-pure water 17.5 ul x buffer 2.5 ul dNTPs 2 ul rTaq 0.15 ul template 1 ul primer 1 ul for each
A procedure for PCR amplification includes pre-denaturation at 94°C for 2 min, (denaturation at 94°C for 15 s, annealing at 50°C for 15 s, extension at 72°C for 15 s) χ 38 cycles, extension at 72°C for 3 min, and storage at 4°C, where PCR products can be stored for a long term.
3): Purify and then sequence the amplified products, and compare sequencing results of the two populations to identify the species.
Embodiment 2
A molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii and an identification method thereof:
1) : Select 5 individuals of each of Trachitotus ovatus and Trachinotus blochii fries acquired from coastal waters of Sanya of Hainan Province to form sample populations, and extract DNAs of the two sample populations respectively.
2) : Use the DNAs extracted in step 1) as templates to amplify the primer CSF and the primer CSR respectively through a PCR reaction system.
The composition of the PCR reaction system is:
the following are sequentially added into a 0.2 ml centrifuge tube:
ultra-pure water 17.5 ul x buffer 2.5 ul dNTPs 2 ul rTaq 0.15 ul template 1 ul primer 1 ul for each
A procedure for PCR amplification includes pre-denaturation at 94°C for 4 min, (denaturation at 94°C for 35 s, annealing at 53°C for 30 s, extension at 72°C for 15 s) x 30 cycles, extension at 72°C for 10 min, and storage at 4°C, where PCR products can be stored for a long term.
3) : Purify and then sequence the amplified products, and compare sequencing results of the
2020100468 27 Mar 2020 two populations to identify the species.
Embodiment 3
A molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii and an identification method thereof:
1) : Select 5 individuals of each of Trachitotus ovatus and Trachinotus blochii fries acquired from coastal waters of Sanya of Hainan Province to form sample populations, and extract DNAs of the two sample populations respectively.
2) : Use the DNAs extracted in step 1) as templates to amplify the primer CSF and the primer CSR respectively through a PCR reaction system.
The composition of the PCR reaction system is:
the following are sequentially added into a 0.2 ml centrifuge tube:
ultra-pure water 17.5 ul x buffer 2.5 ul dNTPs 2 ul rTaq 0.15 ul template 1 ul primer 1 ul for each
A procedure for PCR amplification includes pre-denaturation at 94°C for 5 min, (denaturation at 94°C for 35 s, annealing at 50°C for 35 s, extension at 72°C for 35 s) x 38 cycles, extension at 72°C for 10 min, and storage at 4°C, where PCR products can be stored for a long term.
3) : Purify and then sequence the amplified products, and compare sequencing results of the two populations to identify the species.
It can be seen from 75bp sequence sequencing results of mtDNA 12S rRNA genes of Trachitotus ovatus fries and Trachinotus blochii fries, the Trachitotus ovatus fries and the Trachinotus blochii fries have a difference of 2 bases, which is shown as 1 transformation and 1 transversion.
The 75bp sequence sequencing results of the mtDNA 12S rRNA genes of the Trachitotus ovatus fries and the Trachinotus blochii fries are:
Trachitotus ovatus fries: ATGAAGCCCA ACCACGAAAG TGACTTTACA
TAACCTGAAC
Tfdchifiotusblochii
Ruler:.........10.........20.........30.........40
Trachitotus ovatus fries: CCACGAAAGC TAAGAAACAAACTGGGATTAGATAC
Trachinotus blochii fries:
2020100468 27 Mar 2020
Ruler:.........50.........60.........70....75 (where * indicates the same base sequence site)
The sequencing results showed that the amplification results of the five Trachitotus ovatus fries were consistent, and the amplification results of the five Trachinotus blochii fries were consistent. The results are stable and the reproducibility is high.
Sequence table <110> South China Sea Fisheries Research Institute, CAFS <120> Molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii and application thereof <141> 2019-04-18 <160>4 <170> SIPO Sequence Listing 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
2020100468 27 Mar 2020 <212>DNA <213> Artificial sequence <400> 4 atgaagccca accacgaaag tgactttaca ctacctgaac ccacgaaagc taagaaacaa 60 actgggatta gatac 75
Claims (5)
1) : selecting Trachitotus ovatus and Trachinotus blochii to form sample populations, and extracting DNAs of the two sample populations respectively;
1. A molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii, comprising a primer CSF and a primer CSR, wherein a sequence of the primer CSF is shown in SEQ ID NO: 1, and a sequence of the primer CSR is shown in SEQ ID NO: 2.
2) : respectively using the DNAs extracted in step 1) as templates to amplify the primers according to claim 1 through a PCR reaction system;
2. Application of the molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii according to claim 1, wherein the molecular marker primer is used for identifying Trachitotus ovatus and Trachinotus blochii, and the application also comprises identifying fries thereof, preferably, wherein an identification method comprises the following steps:
3. The application of the molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii according to claim 2, wherein the sequence of the molecular marker of Trachitotus ovatus in step 3) is shown in SEQ ID NO: 3, and the sequence of the molecular marker of Trachinotus blochii is shown in SEQ ID NO: 4, preferably, wherein the molecular markers are used for identifying Trachitotus ovatus and Trachinotus blochii.
3) : purifying and sequencing amplified products obtained in step 2) to obtain sequences of molecular markers of Trachitotus ovatus and Trachinotus blochii respectively; and
4. The application of the molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii according to claim 2, wherein a reagent of the PCR reaction system in step 2) comprises 17.5 ul of ultra-pure water, 2.5 ul of 10 x buffer, 2 ul of dNTPs, 0.15 ul of rTaq, 1 ul of DNA template, and 1 ul of each primer, preferably, wherein a procedure for the amplification in step 2) comprises pre-denaturation at 94°C for 2-5 min, then denaturation at 94°C for 35 s, annealing at 50-53°C for 30-35 s, extension at 72°C for 35 s, proceeding of 30-38 cycles, final extension at 72°C for 3-10 min, and storage at 4°C.
4) : comparing sequencing results to identify the species.
5. The application of the molecular marker primer for identifying Trachitotus ovatus and Trachinotus blochii according to claim 2, wherein a kit comprising the molecular marker primer
2020100468 27 Mar 2020 and the reagent of the PCR reaction system is applied to the identification of Trachitotus ovatus and Trachinotus blochii.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910314393.6A CN110106256B (en) | 2019-04-18 | 2019-04-18 | Molecular marker primer for identifying trachinotus ovatus and trachinotus branchii and application thereof |
| CN201910314393.6 | 2019-04-18 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110521637B (en) * | 2019-08-26 | 2021-11-09 | 中国水产科学研究院南海水产研究所 | Method for constructing full-sibling family of egg-shaped pompano |
| 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 |
| 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 |
| 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 |
| CN113322328A (en) * | 2021-04-25 | 2021-08-31 | 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 |
| CN113186299A (en) * | 2021-04-25 | 2021-07-30 | 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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| CN103131759A (en) * | 2011-12-01 | 2013-06-05 | 汕头大学医学院 | Molecular biological method of identifying sparus latus |
| CN103131769A (en) * | 2011-12-02 | 2013-06-05 | 汕头大学医学院 | Molecular biological method of identifying trachinotus ovatus |
| CN103131765A (en) * | 2011-12-02 | 2013-06-05 | 汕头大学医学院 | Molecular biological method of identifying silver pomfret |
| CN102912012B (en) * | 2012-08-30 | 2013-12-25 | 浙江海洋学院 | Marine fish mitochondrion 12S rRNA (ribosomal ribonucleic acid) gene amplification primer and design and amplification method thereof |
| CN107841563A (en) * | 2017-11-22 | 2018-03-27 | 镇江华大检测有限公司 | Differentiate the molecular specificity labeled primers and method of catfish |
| CN109457035B (en) * | 2018-11-26 | 2020-09-18 | 中国水产科学研究院南海水产研究所 | SSR fluorescence labeling primer for parent-child identification of trachinotus ovatus and application thereof |
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