CN106755384A - A kind of micro-satellite primers and standard diagram and method for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon - Google Patents
A kind of micro-satellite primers and standard diagram and method for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon Download PDFInfo
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- CN106755384A CN106755384A CN201611149602.9A CN201611149602A CN106755384A CN 106755384 A CN106755384 A CN 106755384A CN 201611149602 A CN201611149602 A CN 201611149602A CN 106755384 A CN106755384 A CN 106755384A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
Standard diagram and method the present invention relates to a kind of micro-satellite primers and for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon.The present invention filters out 1 pair of micro-satellite primers that can be general in the flat Rockfish of Korea and cabezon from 70 pairs of flat Rockfish micro-satellite primers of Xu Shi, and the difference according to this primer amplified band in three species carries out species identification.The genomic DNA of the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon is extracted using phenol chloroform method, and with its be template enter performing PCR amplification obtain amplified production, gel electrophoresis and dyeing are carried out to amplified production, and contrasted with the standard diagram for providing, so as to can fast and efficiently differentiate that the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon are individual.
Description
Technical field
The invention belongs to seawater fish molecular labeling and species identification technology in aquatic wholesale market, and in particular to a kind of
Come from the flat Rockfish of Xu Shi a pair of micro-satellite primers and its for differentiate the flat Rockfish of Xu Shi, the standard diagram of the flat Rockfish of Korea and cabezon and
Method.
Background technology
Rockfish sections fish include more than 300 a kind in worldwide, and from coastal waters intertidal zone to a km, deep deep-sea is all
It is distributed, possesses extremely abundant species and habitat, is highly important in world fisheries, particularly China's fishery
Economic fish.Flat Rockfish category and Chang Rockfish category are ovoviviparity fish, and the flat Rockfish of Xu Shi are commonly called as black Jun, are that China commonly sees cold warm nature bottom rock
Reef fingerling, in often inhabiting coastal waters cay area, clear water gravel region and weed-filled sea area, cave, the colonization property of battalion half
Life, does not carry out long range migration.Cabezon is warm warm nature coastal waters demersal fishes, in inhabiting cay and marine alga clump, movable model
Enclose small, also without remote migration.The correlative study of the flat Rockfish of Korea is also fewer, is also to perch in bottom waters.Three kinds of fish
Distribution is close, is distributed mainly on waters on the south coastal area of china and the Korea peninsula, Hokkaido, Japan.In recent years, due to mistake
Degree is fished for and the reason, the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon resource slump of disastrous proportions such as environmental change.The differentiation and identification of species
It is the basis of various research work, but the profile of the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon adult is more similar, directly passes through
Morphological feature carries out identifying relatively difficult that the discriminating in particularly its mesoderm growing early stage stage is more difficult.It is badly in need of a kind of simple efficient
Method Rockfish flat to Xu Shi, the flat Rockfish of Korea and cabezon individuality carry out extensive species identification.
The content of the invention
The present invention discloses a kind of micro-satellite primers, and it comes from the flat Rockfish of Xu Shi, the primer（HJ5-132）Sequence be：
F: CCCTCCTCTTCCATCTGT
R: ACCCTGAATGAGTTACCG。
The present invention filters out a pair from 70 pairs of flat Rockfish micro-satellite primers of Xu Shi can be in flat Rockfish of Xu Shi, the flat Rockfish of Korea, brown
In Chang Rockfish it is general and can according to three individualities of species of amplified band size discrimination, while banding pattern it is clear, reproducible, can
By the strong micro-satellite primers of property：HJ5-132.Its nucleotides sequence is classified as：
GATCTTTCTCAGAGACAGTGTAAAACTCCCACACGGCTGACATTTACTTTCACTTTTATTTGTAAACAAACCA
CACGTGCACACGGCGCCCGCTGTCATGCCAGAGTCGTGTAGTCTCCAGCCAGTGGCTTCTTCTCTGAATCAGCAGCT
GGTTACTGTTGTCAGATGTTCGGCCAATAAATCGGTGCATCTCTGTAACCTAAATCTAGTTCCAGATAGCATTAACA
ATTTCACCCCCCCTCCTCTTCCATCTGTAGAGCCCAGTATACAGTGTTGGTCCCTGCCCTGTGGAATACTGAGGTCC
AAGACTGGGAGCTTAAATGTATGAACAGTCTGGTGTTGGACGAGAGCCACCATGGACCCAACGCAGGTATCTCTCAC
ACACGCACACACGCACGCACGCACGGCACGCACGCACGCACACATACACACACACACACACACATTACACACACAAA
GCAGTTGTTCTTAAGCAAAGGCTAATAATGCAAAGGGACACAGGGGACCACTCGGCACACACAGTCGCTCAAACTCC
GGTAACTCATTCAGGGTACATCACTTAACCTCGGCACACATGC。
Invention additionally discloses a kind of standard diagram for for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon, pass through
Following steps are realized：
1）Selection can completely recognize the ripe flat Rockfish of Xu Shi, the flat Rockfish of Korea, each 60 of the cabezon of its species, and each is individual for clip
A small amount of fin is fully shredded respectively, moisture is absorbed with clean filter paper, it is respectively put into 1.5mL centrifuge tubes, add 400 μ l
Lysate and 10 μ l concentration are 10mg/ml Proteinase Ks, are mixed on the oscillator, and 55 DEG C of water-baths digest 3~5 h, until lysate
Untill clarification；
2）200 μ L saturated phenols, 200 μ L24 are added in centrifuge tube respectively:1 chloroform/isoamyl alcohol mixed liquor is extracted three times；
3）DNA is precipitated with the absolute ethyl alcohol of 600 μ L respectively, through 70% ethanol wash, room temperature is dissolved in the ultra-pure water of 50 μ L after drying
In；
4）UV spectrophotometer measuring DNA concentration and purity, and with 1% agarose gel electrophoresis detect DNA integrality,
If integrality meets the requirements；
5）It is template with the three species gene group DNA for extracting, carries out pcr amplification reaction：The cumulative volume of PCR reaction systems is 25
μ L, wherein various composition and final concentration are respectively：The mM of DNA profiling 50 ng, Buffer 10, primer each 0.2 mM, dNTP
0.2 mM, Mg2+2.0 mM, Taq archaeal dna polymerase 1U, 25 μ L are supplied with aqua sterilisa；Described primer is foregoing primer
（HJ5-132）Sequence；Response procedures are：94 DEG C of predegenerations 5min, 94 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extensions
1min, denaturation is repeated 30 times to three steps are extended, 72 DEG C of extension 10min；
6）After PCR reactions terminate, 0.2 μ L amplified productions are taken in 8% native polyacrylamide gel electrophoresis, 22 V voltage stabilizings electrophoresis 4
Hour, be put into pallet from being removed on glass plate gel after terminating by electrophoresis, is cleaned with deionized water twice, is subsequently adding 0.1%
AgNO3Solution dyes 10min；Add deionized water to clean again once after dyeing, nitrite ion colour developing is added, until band is clear
Untill, the flat Rockfish of Xu Shi, the flat Rockfish of Korea, the collection of illustrative plates of cabezon are respectively obtained, three kinds of collection of illustrative plates are merged to form mark as shown in drawings
Quasi- collection of illustrative plates.
Further, described AgNO3Solution is that ratio is 1g AgNO3In 1000mL dH2The solution of O；Described is aobvious
Color liquid is 2%NaOH, 0.4% formaldehyde, 0.2%NaS2O3、dH2The mixed solution of O 1000mL.
Invention additionally discloses a kind of method for for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon, comprising as follows
Step：
a）The fin of a little Rockfish to be reflected is cut, according to claim 2 step 1）-4）Phenol chloroform method extract Rockfish fishes to be identified
Individual genomic DNA；
b）Genomic DNA to extracting is carried out such as claim 2 step 5）PCR amplification, obtain amplified production；
c）Amplified production is carried out such as claim 2 step 6）Polyacrylate hydrogel electrophoresis and dyeing, obtain contrast collection of illustrative plates；
d）The standard diagram that is obtained with claim 2 of collection of illustrative plates will be contrasted to be contrasted, if
There is 1~2 band in 305bp~330bp is interval, then the DNA sample is the flat Rockfish of Xu Shi, 1 band explanation occurs
The individuality is homozygote, 2 band occurs and then illustrates that the individuality is heterozygote；
If occurring 1~2 band in 286bp~295bp is interval, the DNA sample is the flat Rockfish of Korea, 1 band occurs and says
The bright individuality is homozygote, 2 band occurs and then illustrates that the individuality is heterozygote；
If occurring 1~2 band in 252bp~270bp is interval, the DNA sample is cabezon, 1 band explanation occurs
The individuality is homozygote, 2 band occurs and then illustrates that the individuality is heterozygote.
The present invention have method simple, visual result, accurate and effective, do not influenceed by environment and developmental stage, it is with low cost
The advantages of.
Brief description of the drawings
Fig. 1 is the present invention for differentiating the standard diagram of the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon.
Specific embodiment
Implementation method 1, invention additionally discloses a kind of standard for for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon
Collection of illustrative plates, is achieved by the steps of：
1）Selection can completely recognize the ripe flat Rockfish of Xu Shi, the flat Rockfish of Korea, each 60 of the cabezon of its species, and each is individual for clip
A small amount of fin is fully shredded respectively, moisture is absorbed with clean filter paper, it is respectively put into 1.5mL centrifuge tubes, add 400 μ l
Lysate and 10 μ l concentration are 10mg/ml Proteinase Ks, are mixed on the oscillator, and 55 DEG C of water-baths digest 3~5 h, until lysate
Untill clarification；
2）200 μ L saturated phenols, 200 μ L24 are added in centrifuge tube respectively:1 chloroform/isoamyl alcohol mixed liquor is extracted three times；
3）DNA is precipitated with the absolute ethyl alcohol of 600 μ L respectively, through 70% ethanol wash, room temperature is dissolved in the ultra-pure water of 50 μ L after drying
In；
4）UV spectrophotometer measuring DNA concentration and purity, and with 1% agarose gel electrophoresis detect DNA integrality,
If integrality meets the requirements；
5）It is template with the three species gene group DNA for extracting, carries out pcr amplification reaction：The cumulative volume of PCR reaction systems is 25
μ L, wherein various composition and final concentration are respectively：The mM of DNA profiling 50 ng, Buffer 10, primer each 0.2 mM, dNTP
0.2 mM, Mg2+2.0 mM, Taq archaeal dna polymerase 1U, 25 μ L are supplied with aqua sterilisa；Described primer is foregoing primer
（HJ5-132）Sequence；Response procedures are：94 DEG C of predegenerations 5min, 94 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extensions
1min, denaturation is repeated 30 times to three steps are extended, 72 DEG C of extension 10min；
6）After PCR reactions terminate, 0.2 μ L amplified productions are taken in 8% native polyacrylamide gel electrophoresis, 22 V voltage stabilizings electrophoresis 4
Hour, be put into pallet from being removed on glass plate gel after terminating by electrophoresis, is cleaned with deionized water twice, is subsequently adding 0.1%
AgNO3Solution dyes 10min；Add deionized water to clean again once after dyeing, nitrite ion colour developing is added, until band is clear
Untill, the flat Rockfish of Xu Shi, the flat Rockfish of Korea, the collection of illustrative plates of cabezon are respectively obtained, three kinds of collection of illustrative plates are merged to form mark as shown in drawings
Quasi- collection of illustrative plates.
Claims (4)
1. a kind of micro-satellite primers, it is characterised in that：It comes from the flat Rockfish of Xu Shi, the primer（HJ5-132）Sequence be：
F: CCCTCCTCTTCCATCTGT
R: ACCCTGAATGAGTTACCG。
2. a kind of standard diagram for for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon, it is characterised in that by as follows
Step is realized：
1）Selection can completely recognize the ripe flat Rockfish of Xu Shi, the flat Rockfish of Korea, each 60 of the cabezon of its species, and each is individual for clip
A small amount of fin is fully shredded respectively, moisture is absorbed with clean filter paper, it is respectively put into 1.5mL centrifuge tubes, add 400 μ l
Lysate and 10 μ l concentration are 10mg/ml Proteinase Ks, are mixed on the oscillator, and 55 DEG C of water-baths digest 3~5 h, until lysate
Untill clarification；
2）200 μ L saturated phenols, 200 μ L24 are added in centrifuge tube respectively:1 chloroform/isoamyl alcohol mixed liquor is extracted three times；
3）DNA is precipitated with the absolute ethyl alcohol of 600 μ L respectively, through 70% ethanol wash, room temperature is dissolved in the ultra-pure water of 50 μ L after drying
In；
4）UV spectrophotometer measuring DNA concentration and purity, and with 1% agarose gel electrophoresis detect DNA integrality,
If integrality meets the requirements；
5）It is template with the three species gene group DNA for extracting, carries out pcr amplification reaction：The cumulative volume of PCR reaction systems is 25
μ L, wherein various composition and final concentration are respectively：The mM of DNA profiling 50 ng, Buffer 10, primer each 0.2 mM, dNTP
0.2 mM, Mg2+2.0 mM, Taq archaeal dna polymerase 1U, 25 μ L are supplied with aqua sterilisa；Described primer is claim 1 institute
The sequence stated；Response procedures are：94 DEG C of predegenerations 5min, 94 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 1min, denaturation
Repeated 30 times to three steps are extended, 72 DEG C of extension 10min；
6）After PCR reactions terminate, 0.2 μ L amplified productions are taken in 8% native polyacrylamide gel electrophoresis, 22 V voltage stabilizings electrophoresis 4
Hour, be put into pallet from being removed on glass plate gel after terminating by electrophoresis, is cleaned with deionized water twice, is subsequently adding 0.1%
AgNO3Solution dyes 10min；Add deionized water to clean again once after dyeing, nitrite ion colour developing is added, until band is clear
Untill, the flat Rockfish of Xu Shi, the flat Rockfish of Korea, the collection of illustrative plates of cabezon are respectively obtained, three kinds of collection of illustrative plates are merged to form mark as shown in drawings
Quasi- collection of illustrative plates.
3. a kind of standard diagram for for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon according to claim 2,
It is characterized in that：Described AgNO3Solution is that ratio is 1g AgNO3In 1000mL dH2The solution of O；Described nitrite ion is
2%NaOH, 0.4% formaldehyde, 0.2%NaS2O3、dH2The mixed solution of O 1000mL.
4. a kind of method for for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon, it is characterised in that comprise the following steps：
a）The fin of a little Rockfish to be reflected is cut, according to claim 2 step 1）-4）Phenol chloroform method extract Rockfish fishes to be identified
Individual genomic DNA；
b）Genomic DNA to extracting is carried out such as claim 2 step 5）PCR amplification, obtain amplified production；
c）Amplified production is carried out such as claim 2 step 6）Polyacrylate hydrogel electrophoresis and dyeing, obtain contrast collection of illustrative plates；
d）The standard diagram that is obtained with claim 2 of collection of illustrative plates will be contrasted to be contrasted, if
There is 1~2 band in 305bp~330bp is interval, then the DNA sample is the flat Rockfish of Xu Shi, 1 band explanation occurs
The individuality is homozygote, 2 band occurs and then illustrates that the individuality is heterozygote；
If occurring 1~2 band in 286bp~295bp is interval, the DNA sample is the flat Rockfish of Korea, 1 band occurs and says
The bright individuality is homozygote, 2 band occurs and then illustrates that the individuality is heterozygote；
If occurring 1~2 band in 252bp~270bp is interval, the DNA sample is cabezon, 1 band explanation occurs
The individuality is homozygote, 2 band occurs and then illustrates that the individuality is heterozygote.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611149602.9A CN106755384A (en) | 2017-02-15 | 2017-02-15 | A kind of micro-satellite primers and standard diagram and method for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon |
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|---|---|---|---|
| CN201611149602.9A CN106755384A (en) | 2017-02-15 | 2017-02-15 | A kind of micro-satellite primers and standard diagram and method for differentiating the flat Rockfish of Xu Shi, the flat Rockfish of Korea and cabezon |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998539A (en) * | 2018-08-15 | 2018-12-14 | 浙江海洋大学 | Cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies |
| CN109022558A (en) * | 2018-08-15 | 2018-12-18 | 浙江海洋大学 | The flat Rockfish genome SNP marker method of Xu Shi based on enzymes combinations Genotyping sequencing technologies |
| CN109452203A (en) * | 2018-11-28 | 2019-03-12 | 山东省海洋资源与环境研究院 | A kind of flat Rockfish deep water mesh cage large size seedling seed breeding method of Xu Shi |
| CN111118003A (en) * | 2020-01-17 | 2020-05-08 | 中国海洋大学 | Sebastes schlegeli hilgendorf muscle satellite cell in-situ hybridization probe primer, probe and positioning method |
| CN112410436A (en) * | 2020-11-26 | 2021-02-26 | 中国水产科学研究院黄海水产研究所 | Sebastes schlegeli low-oxygen-resistant individual screening molecular marker and screening method |
-
2017
- 2017-02-15 CN CN201611149602.9A patent/CN106755384A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998539A (en) * | 2018-08-15 | 2018-12-14 | 浙江海洋大学 | Cabezon genome SNP marker method based on enzymes combinations Genotyping sequencing technologies |
| CN109022558A (en) * | 2018-08-15 | 2018-12-18 | 浙江海洋大学 | The flat Rockfish genome SNP marker method of Xu Shi based on enzymes combinations Genotyping sequencing technologies |
| CN109452203A (en) * | 2018-11-28 | 2019-03-12 | 山东省海洋资源与环境研究院 | A kind of flat Rockfish deep water mesh cage large size seedling seed breeding method of Xu Shi |
| CN111118003A (en) * | 2020-01-17 | 2020-05-08 | 中国海洋大学 | Sebastes schlegeli hilgendorf muscle satellite cell in-situ hybridization probe primer, probe and positioning method |
| CN111118003B (en) * | 2020-01-17 | 2022-04-08 | 中国海洋大学 | Sebastes schlegeli hilgendorf muscle satellite cell in-situ hybridization probe primer, probe and positioning method |
| CN112410436A (en) * | 2020-11-26 | 2021-02-26 | 中国水产科学研究院黄海水产研究所 | Sebastes schlegeli low-oxygen-resistant individual screening molecular marker and screening method |
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