CN102776183A - Amplimer of large yellow croaker mitochondrion complete genome sequence and application thereof - Google Patents
Amplimer of large yellow croaker mitochondrion complete genome sequence and application thereof Download PDFInfo
- Publication number
- CN102776183A CN102776183A CN2012102414939A CN201210241493A CN102776183A CN 102776183 A CN102776183 A CN 102776183A CN 2012102414939 A CN2012102414939 A CN 2012102414939A CN 201210241493 A CN201210241493 A CN 201210241493A CN 102776183 A CN102776183 A CN 102776183A
- Authority
- CN
- China
- Prior art keywords
- large yellow
- yellow croaker
- amplimer
- genome sequence
- whole genome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses an amplimer of a large yellow croaker mitochondrion complete genome sequence and an application thereof. The 15 pairs of amplimer are applied, a gene order is respectively shown as SEQ ID No: 1-30, the amplimer is further used for obtaining a Tai Qu family large yellow croaker mitochondrion complete genome sequence and a Min-Yue East family large yellow croaker mitochondrion complete genome sequence which are respectively shown as SEQ ID No: 31 and SEQ ID No: 32, and an amplimer of large yellow croakers in a high variation region is obtained. The amplimer and the application have the advantages that the Tai Qu family large yellow croaker and Min-Yue East family large yellow croaker mitochondrion complete genome sequences can be quickly and efficiently obtained respectively through the 15 pairs of amplimers; and the amplimer of the large yellow croakers in the high variation region provides support for the research of species identification, geographical population identification, germ plasm resources and genetic diversity of the large yellow croakers, and further provide the basis for developing an identification technology of Tai Qu family large yellow croakers and Min-Yue East family large yellow croakers.
Description
Technical field
The present invention relates to biological technical field, especially relate to a kind of amplimer and application thereof of large yellow croaker plastosome whole genome sequence.
Background technology
Large yellow croaker (Pseudosciaena crocea); Have another name called yellow croaker, yellow croaker; Be under the jurisdiction of Perciformes (Perciformes), Sciaenidae (Sciaenidae), yellow croaker genus (Pseudosciaena); Being one of main economic fish of China's marine fishing, is the maximum fish species of Chinese seawater cage cultured output.The coastal large yellow croaker of China roughly is divided into tai-chu race, Fujian-East Guangdong family, three geographical population of island in Guangdong Province family.The tai-chu race large yellow croaker is golden yellow or brave yellow, and is glossy, and the gill filament is clear to be scarlet or red-purple; Eyeball is full, brawniness, high resilience; Be the superfine product in the yellow croaker family, it mainly is distributed in Dai Quyang fishing ground, Zhe Bei Zhoushan, because excessive fishing for; Environmental degradation, present wild tai-chu race large yellow croaker is very rare.Because Fujian-East Guangdong family large yellow croaker is similar with tai-chu race large yellow croaker formalness; Be difficult to distinguish; Some trade companies even Fujian-East Guangdong family large yellow croaker and tai-chu race large yellow croaker obscured sale on the market, therefore the swindle human consumer carries out species with large yellow croaker similar on the profile and identifies and seem very necessary.
At present, differentiate that through sequence difference fish similar on the profile are a kind of very convenient methods accurately, wherein the research of plastosome sequence difference is wherein important a kind of method.Plastosome is intracellular important organelle; Belong to matrocliny and have self and overlap genome; It is semiautonomous organelle; And because characteristics such as it is simple in structure, recombinate hardly, rate of evolution is fast, the different zones rate of evolution there are differences, it is extensive to make it aspect the research of kind discriminating, phyletic evolution and population genetics, use ten minutes.Different biological Mitochondrial Genome Overview all demonstrate great variety at aspects such as structure, gene number and sequence in the gene modes, so the strongest evidence takes place the complete sequence of Mitochondrial Genome Overview research becoming animal molecular system.The Chinese patent name is called wise sieve salmon plastosome whole genome sequence and amplimer (application number CN200910073114.8) thereof; Heilongjiang Province boar mitochondrial genom sequence clone and evolutionary analysis (application number: CN200810172959.8) etc. respectively the full genome of plastosome is checked order, and the plastosome whole genome sequence of the large yellow croaker of different geographical population (like Fujian-East Guangdong family large yellow croaker and tai-chu race large yellow croaker) undetermined still up to the present.
Summary of the invention
Technical problem to be solved by this invention provides a kind of amplimer and application thereof of large yellow croaker plastosome whole genome sequence; Utilize this amplimer can obtain plastosome whole genome sequence and the large yellow croaker high variant area amplimer of different types of large yellow croaker, for the Molecular Identification of protection genetics, evolutionary genetics and the species thereof of large yellow croaker provides material base.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of amplimer of large yellow croaker plastosome whole genome sequence, and the amplimer of large yellow croaker plastosome whole genome sequence is 15 pairs, gene order is as follows respectively:
(1) upstream primer of the 1st pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AGTCAACGGCGTAAAGAGTGG-3 ', downstream primer is: 5 '-TACCCTTCTGGGAAAGTTGT-3 ';
(2) upstream primer of the 2nd pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CCTGGGAAATGAATAGAAGT-3 ', downstream primer is: 5 '-AAAATCATGGCATAGATAGA-3 ';
(3) upstream primer of the 3rd pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-TTACGACCTCGATGTTGGATCAG-3 ', downstream primer is: 5 '-GGAAGCACTAGGAGTTTTGAGT-3 '
(4) upstream primer of the 4th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AAGGGCCACTTTGATAGAGTG-3 ', downstream primer is: 5 '-GTGAGTGCGGGGGTTTTGGCTCA-3 ';
(5) upstream primer of the 5th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-TTGCCTACTCCTCAATTGCCCAC-3 ', downstream primer is: 5 '-CTTATGTTATTCATTCGGGGGAA-3 ';
(6) upstream primer of the 6th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CCTCTACCTAATTTTTGGTGC-3 ', downstream primer is: 5 '-ATGCGGTTGGCTTGAAATCAA-3 ';
(7) upstream primer of the 7th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AGCACTTGAACAAAAGCTCACTT-3 ', downstream primer is: 5 '-GTCGAAGAGGCTTACAGTCATGGTCA-3 ';
(8) upstream primer of the 8th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-GCAAGCGTTAGCCTTTTAAGC-3 ', downstream primer is: 5 '-CAGGGGCTGGGGTCTACTAT-3 ';
(9) upstream primer of the 9th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CCTACACTTCTTATCCCTATTCTAAT-3 ', downstream primer is: 5 '-GGAGAAAGGGAGGCGAGCGGT-3 ';
(10) upstream primer of the 10th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AGTATTAGTGACTTCCAATCA-3 ', downstream primer is: 5 '-TGTAGAATAGAAAATAAGTGCC-3 ';
(11) upstream primer of the 11st pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CCTCTTAACATCCCTGCAAATC-3 ', downstream primer is: 5 '-TTCCTAAGACCAACGGATGAGC-3 ';
(12) upstream primer of the 12nd pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AAAACATTAGATTGTGATTCTAA-3 ', downstream primer is: 5 '-GACTCGGAGGCTGTAAATAG-3 ';
(13) upstream primer of the 13rd pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-GCCTGGCCCTCACTGGTACC-3 ', downstream primer is: 5 '-AGCGGGTGGGTTTTGCGTAG-3 ';
(14) upstream primer of the 14th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CTCTAACCAGGACTAATGGCTTG-3 ', downstream primer is: 5 '-TGATTATCAATCAATGTCCC-3 ';
(15) upstream primer of the 15th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-TCAACATTAATTACCACGCT-3 ', downstream primer is: 5 '-GGGGTATCTAATCCCAGTTT-3 '.
A kind of application of amplimer of large yellow croaker plastosome whole genome sequence, the tai-chu race large yellow croaker plastosome whole genome sequence that utilizes described amplimer acquisition is shown in SEQ ID NO:31.
A kind of application of amplimer of large yellow croaker plastosome whole genome sequence, the Fujian-East Guangdong family large yellow croaker plastosome whole genome sequence that utilizes described amplimer acquisition is shown in SEQ ID NO:32.
A kind of application of amplimer of large yellow croaker plastosome whole genome sequence, the large yellow croaker high variant area amplimer that utilizes described amplimer to obtain, gene order is following:
B1 upstream primer 5 '-AGACCTTCTAGGCTTTGCAATC-3 ',
B1 downstream primer 5 '-GGAGCTTTCTAGGGCTCATCT-3 ';
B2 upstream primer 5 '-TTATTTTCTATTCTACACCCTGGC-3 ',
B2 downstream primer 5 '-AGAGGGAATACCCCGCTGT-3 '.
Compared with prior art; The invention has the advantages that: the present invention discloses 15 pairs of amplimers of large yellow croaker plastosome whole genome sequence first; 15 pairs of amplimers carry out pcr amplification to the large yellow croaker sample, obtain the specific amplification products of clip size between 900-1560bp respectively, need not high-quality dna profiling and long-chain round pcr; Clone's success ratio is high, and the product that every pair of primer amplification obtained does not need the direct order-checking of walking method can survey logical; The sequence of adjacent area amplified production has the above lap of 80bp; Make things convenient for the splicing of sequencing result; Can obtain tai-chu race large yellow croaker and Fujian-East Guangdong family large yellow croaker plastosome whole genome sequence fast and efficiently respectively; And different large yellow croakers are carried out homologous sequence compare; Large yellow croaker high variant area sequence analyzed obtain the high variation of large yellow croaker zone amplication primer, for kind evaluations, geographical population discriminating, germ plasm resource and the Genetic Diversity of large yellow croaker provides support, for the further authentication technique of exploitation tai-chu race large yellow croaker and Fujian-East Guangdong family large yellow croaker provides basic.
Description of drawings
Fig. 1 is the agarose gel electrophoresis synoptic diagram as a result of tai-chu race large yellow croaker pcr amplification product;
Fig. 2 is the agarose gel electrophoresis synoptic diagram as a result of Fujian-East Guangdong family large yellow croaker pcr amplification product.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
Specific embodiment one
The amplimer of large yellow croaker plastosome whole genome sequence of the present invention is 15 pairs, and gene order is as follows:
The amplified production of institute of the present invention designed primer should cover the full genome of whole plastosome, and the amplified production length of every pair of primer is at 900-1560bp, and the sequence that obtains of adjacent two pairs of primers overlapping be more than the 80bp.
Specific embodiment two
The method of design of the amplimer of large yellow croaker plastosome whole genome sequence
One, the screening of template
Land BCBI (http://www.ncbi.nlm.nih.gov/) website; Search and determinand kind belong to the species mitochondrial genome complete sequence of same section even same genus; If there are many sequences can supply to utilize; Compare the sequence of the species that preferential selection and species sibship to be measured are nearest through blast software.
Two, according to template sequence design primer
The sequence that screening is obtained is as template, is designed for the primer of pcr amplification with PCR primer-design software Primer Premier5.0, and concrete thinking is following:
1) length of pcr amplification will be below 1600bp; Because reading length, the generation sequenator ABI3730 of main flow order-checking now can arrive 800 to 1000bp; And the order-checking accuracy rate is high in the 800bp, when therefore designing primer, makes positive and negative two-way order-checking can once survey logical and need not go on foot and move;
2) sequence of adjacent area amplified production has the above lap of 80bp, and the accuracy of 40bp left and right sides sequence is low before the order-checking, has the above lap of 80bp to make things convenient for the splicing of sequencing result and the accuracy of sequence;
3) avoid the secondary structure district of primer, some primer amplification reason invalid or the order-checking failure is the influence of primer iteron secondary structure;
4) length in the complementary district of general primer is 19-25bp, and G+C content is preferably in 45%-55%, does not occur surpassing 3 continuous G or C at 3 ' end, otherwise causes non-specific amplification easily;
5) according to the result of these species or the research of close species Mitochondrial Genome Overview; Design of primers is in conserved regions; And comprise the height variation come in as much as possible; Primer can not only be used for amplification and obtains splicing large yellow croaker plastosome whole genome sequence like this, can also be used to analyzing the molecule polymorphum of different geographical population.
Specific embodiment three
The tai-chu race large yellow croaker plastosome whole genome sequence that the present invention utilizes 15 pairs of amplimers of large yellow croaker plastosome whole genome sequence to obtain is shown in SEQ ID NO:31.
Tai-chu race large yellow croaker plastosome complete genome sequence is 16467bp altogether, contains 22 tRNA, 2 rRNA and 13 protein coding genes and 1 D-loop district.
Specific embodiment four
Fujian-East Guangdong family large yellow croaker plastosome whole genome sequence that the present invention utilizes 15 pairs of amplimers of large yellow croaker plastosome whole genome sequence to obtain is shown in SEQ ID NO:32.
Tai-chu race large yellow croaker chondriogen is formed and is seen table 2 with the variance analysis of Fujian-East Guangdong family large yellow croaker.Tai-chu race large yellow croaker and Fujian-East Guangdong family large yellow croaker Mitochondrial Genome Overview total length is 16467bp, 64 in difference site, and both similaritys are 99.6%, 37 genes of encoding altogether comprise 13 protein coding genes.Remove outside the tRNA gene order of short sequence; Secondly D-loop regional differentiation the highest (2.01%) between tai-chu race and Fujian-East Guangdong family large yellow croaker chondriogen is ND3 (0.86%), CO II (0.72%), ND6 (0.57%) and ND 4 (0.43%) etc.
Specific embodiment five
Utilize 15 pairs of primer amplifications in the above-mentioned specific embodiment one to obtain tai-chu race large yellow croaker and Fujian-East Guangdong family large yellow croaker plastosome whole genome sequence, concrete steps are following:
1, the extraction of large yellow croaker Mitochondrial Genome Overview template DNA
1.1 gather tai-chu race, Fujian-individual each 1 tail of East Guangdong family large yellow croaker breed, take from Xiangshan, Zhejiang Huang in November, 2010 and keep away the Ao aquaculture net cage;
1.2 get the about 0.1g of fish body muscle of back (removing scale and skin), the phenol/total DNA of chloroform method extracting large yellow croaker is preserved subsequent use down for-20 ℃;
2, large yellow croaker plastosome full length sequence amplification
2.1 design of primers and pcr amplification
Template DNA: each 1 of tai-chu race and Fujian-East Guangdong family large yellow croaker; The amplimer of amplification large yellow croaker plastosome full length sequence is seen embodiment 1.The template DNA concentration of PCR reaction is about 100ng, and the reaction system TV is 25 μ L, 10 * Buffer, 2.5 μ L wherein, dNTPs 4 μ L (each 2.5mmol/L), each 1 μ L (10 μ mol/L) of primer, Taq enzyme 0.2 μ L (5U/ μ L).The PCR reaction conditions is: 95 ℃ of preparatory sex change 3min; 94 ℃ of sex change 45s, 54 ℃ of annealing 45s, 72 ℃ are extended 1min30s, 35 circulations; Last 72 ℃ are extended 10min.The PCR product adopts test kit to cut glue purification.
The PCR product agarose gel electrophoresis result who adopts tai-chu race large yellow croaker template DNA to obtain is as shown in Figure 1; 1-15 is for being template with the total DNA of tai-chu race large yellow croaker; The band that the amplimer of large yellow croaker plastosome whole genome sequence increases out to 1-15PCR, M:marker; Adopt designed primer pcr amplification tai-chu race large yellow croaker plastosome whole genome sequence efficient height of the present invention and band special.
Adopt the PCR product agarose gel electrophoresis result that Fujian-East Guangdong family large yellow croaker template DNA obtains as shown in Figure 2; 1-15 is for being template with Fujian-total DNA of large yellow croaker of East Guangdong family; The band that the amplimer of large yellow croaker plastosome whole genome sequence increases out to 1-15PCR, M:marker.Adopt designed primer pcr amplification of the present invention Fujian-East Guangdong family large yellow croaker plastosome whole genome sequence efficient height and band special.
2.2 clone and order-checking
With 15 PCR products difference purifying of tai-chu race large yellow croaker and Fujian-East Guangdong family large yellow croaker, spending the night for 4 ℃ with the pMD18-T carrier respectively then is connected transformed competence colibacillus cell E.coli DH5 α.With machine testing, each clone all sees 3 positive and negative two-way order-checkings of positive colony off, to guarantee the accuracy of sequence with primer M13F/R.
2.3 sequence assembly
Adopt softwares such as DAMBE, Vector NTI 7.0 to splice respectively the sequencing result of tai-chu race large yellow croaker and Fujian-East Guangdong family large yellow croaker, the chondriogen full length sequence of tai-chu race large yellow croaker and Fujian-East Guangdong family large yellow croaker is analyzed respectively then.
2.4 adopt sequence analysis software that the tai-chu race large yellow croaker of acquisition and the chondriogen full length sequence of Fujian-East Guangdong family large yellow croaker are carried out structure, functional analysis respectively
| Numbering | Amplification region | Product length | Upstream primer (5 '-3 ') | Downstream primer (5 '-3 ') |
| B1 | 15068-62 | 1462 | AGACCTTCTAGGCTTTGCAATC | GGAGCTTTCTAGGGCTCATCT |
| B2 | 10805-11589 | 785 | TTATTTTCTATTCTACACCCTGGC | AGAGGGAATACCCCGCTGT |
Utilize the DNAStar software package that sequence is carried out searching of ORFs, the acquisition of sequence complementary strand; Utilize the ORFfinder software of NCBI website to accomplish the translation of albumen coded sequence and the investigation of amino acid coding situation; Compare with the template sequence in online software of blast and the specific embodiment two and to confirm each chondriogen and the particular location of other non-coding sequence in sequence, analytical results is as shown in table 2.
Specific embodiment six
The design of sequential analysis of large yellow croaker high variant area and amplimer
With the tai-chu race that obtains and each chondriogen full length sequence of 1 of Fujian-East Guangdong family large yellow croaker; Compare with Vector NTI7.0 software in conjunction with the large yellow croaker mtDNA complete sequence (sequence number: FJ595214 and NC011710) on the NCBI and to find out high variant area; To B1 and B2, its gene order is as shown in table 3 at the conservative region district at high region of variability two ends design primer:
The principle of invention: with 2 pairs of primers; B1 upstream primer 5 '-AGACCTTCTAGGCTTTGCAATC-3 ', B1 downstream primer 5 '-GGAGCTTTCTAGGGCTCATCT-3 '; B2 upstream primer 5 '-TTATTTTCTATTCTACACCCTGGC-3 '; B2 downstream primer 5 '-AGAGGGAATACCCCGCTGT-3 ' carries out pcr amplification to tai-chu race large yellow croaker and Fujian-East Guangdong family high region of variability of large yellow croaker; The laggard line data analysis of cloning and sequencing, two geographical population sample numbers have 25 respectively.Adopt the mitochondrial D-loop of B1 amplification large yellow croaker zone; To 2 partial sequence fragment binding analysis in this sequence; Detect 2 OTU altogether; Be respectively OTU-D1:GGCATTTAGGCACAAGGTC and CTTCTGAGTTGTGCCCCC, OTU-D2:GGCATTTAGGCACAAGGTT and TTTCTGAGTTGTGCCCCC; 25 samples of tai-chu race are the D1 type, in Fujian-25 samples of East Guangdong family, have 9 to be the D1 type, have 16 to be the D2 type.On the basis of above-mentioned experiment; Use primer to the mitochondrial ND4 of B2 amplification large yellow croaker zone; To 2 partial sequence fragments in this sequence also binding analysis; Detect 2 OTU, be respectively N1:CAGGCCCCACCATACTAAT and GACAGGAACCGGCACCC, N2:CAGGCCCCACCATACTAAC and AACAGGAACCGGCACCC; 25 samples of tai-chu race are the N1 type, in Fujian-East Guangdong family 9 D1 types sample, have 4 to be the N1 type, have 5 to be the N2 type.
OTU somatotype to above-mentioned D-loop and ND4 zone carries out binding analysis, can be divided into 3 type: D1-N1, D1-N2 and D2.The tai-chu race large yellow croaker is the D1-N1 type; And can be divided into 2 types in Fujian-East Guangdong family large yellow croaker: D1-N2 type and D2 type,
Certainly, above-mentioned explanation is not a limitation of the present invention, and the present invention also is not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement also should belong to protection scope of the present invention.
Claims (4)
1. the amplimer of a large yellow croaker plastosome whole genome sequence, the amplimer that it is characterized in that large yellow croaker plastosome whole genome sequence is 15 pairs, gene order is as follows respectively:
(1) upstream primer of the 1st pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AGTCAACGGCGTAAAGAGTGG-3 ', downstream primer is: 5 '-TACCCTTCTGGGAAAGTTGT-3 ';
(2) upstream primer of the 2nd pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CCTGGGAAATGAATAGAAGT-3 ', downstream primer is: 5 '-AAAATCATGGCATAGATAGA-3 ';
(3) upstream primer of the 3rd pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-TTACGACCTCGATGTTGGATCAG-3 ', downstream primer is: 5 '-GGAAGCACTAGGAGTTTTGAGT-3 '
(4) upstream primer of the 4th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AAGGGCCACTTTGATAGAGTG-3 ', downstream primer is: 5 '-GTGAGTGCGGGGGTTTTGGCTCA-3 ';
(5) upstream primer of the 5th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-TTGCCTACTCCTCAATTGCCCAC-3 ', downstream primer is: 5 '-CTTATGTTATTCATTCGGGGGAA-3 ';
(6) upstream primer of the 6th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CCTCTACCTAATTTTTGGTGC-3 ', downstream primer is: 5 '-ATGCGGTTGGCTTGAAATCAA-3 ';
(7) upstream primer of the 7th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AGCACTTGAACAAAAGCTCACTT-3 ', downstream primer is: 5 ' GTCGAAGAGGCTTACAGTCATGGTCA-3 ';
(8) upstream primer of the 8th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-GCAAGCGTTAGCCTTTTAAGC-3 ', downstream primer is: 5 '-CAGGGGCTGGGGTCTACTAT-3 ';
(9) upstream primer of the 9th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CCTACACTTCTTATCCCTATTCTAAT-3 ', downstream primer is: 5 ' GGAGAAAGGGAGGCGAGCGGT-3 ';
(10) upstream primer of the 10th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AGTATTAGTGACTTCCAATCA-3 ', downstream primer is: 5 '-TGTAGAATAGAAAATAAGTGCC-3 ';
(11) upstream primer of the 11st pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CCTCTTAACATCCCTGCAAATC-3 ', downstream primer is: 5 '-TTCCTAAGACCAACGGATGAGC-3 ';
(12) upstream primer of the 12nd pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-AAAACATTAGATTGTGATTCTAA-3 ', downstream primer is: 5 '-GACTCGGAGGCTGTAAATAG-3 ';
(13) upstream primer of the 13rd pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-GCCTGGCCCTCACTGGTACC-3 ', downstream primer is: 5 '-AGCGGGTGGGTTTTGCGTAG-3 ';
(14) upstream primer of the 14th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-CTCTAACCAGGACTAATGGCTTG-3 ', downstream primer is: 5 '-TGATTATCAATCAATGTCCC-3 ';
(15) upstream primer of the 15th pair of large yellow croaker plastosome whole genome sequence amplimer is: 5 '-TCAACATTAATTACCACGCT-3 ', downstream primer is: 5 '-GGGGTATCTAATCCCAGTTT-3 '.
2. the application of the amplimer of the described large yellow croaker plastosome of claim 1 whole genome sequence is characterized in that: the tai-chu race large yellow croaker plastosome whole genome sequence that utilizes described amplimer acquisition is shown in SEQ ID NO:31.
3. the application of the amplimer of the described large yellow croaker plastosome of claim 1 whole genome sequence is characterized in that: the Fujian-East Guangdong family large yellow croaker plastosome whole genome sequence that utilizes described amplimer acquisition is shown in SEQ ID NO:32.
4. the application of the amplimer of the described large yellow croaker plastosome of claim 1 whole genome sequence is characterized in that: the large yellow croaker high variant area amplimer that utilizes described amplimer to obtain, and gene order is following:
B1 upstream primer 5 '-AGACCTTCTAGGCTTTGCAATC-3 ',
B1 downstream primer 5 '-GGAGCTTTCTAGGGCTCATCT-3 ';
B2 upstream primer 5 '-TTATTTTCTATTCTACACCCTGGC-3 ',
B2 downstream primer 5 '-AGAGGGAATACCCCGCTGT-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210241493.9A CN102776183B (en) | 2012-07-12 | 2012-07-12 | A kind of amplimer of large yellow croaker plastosome whole genome sequence and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210241493.9A CN102776183B (en) | 2012-07-12 | 2012-07-12 | A kind of amplimer of large yellow croaker plastosome whole genome sequence and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102776183A true CN102776183A (en) | 2012-11-14 |
| CN102776183B CN102776183B (en) | 2015-08-12 |
Family
ID=47121291
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210241493.9A Active CN102776183B (en) | 2012-07-12 | 2012-07-12 | A kind of amplimer of large yellow croaker plastosome whole genome sequence and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102776183B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074335A (en) * | 2012-11-22 | 2013-05-01 | 浙江海洋学院 | Goby mitochondrion COIII and ND3 gene amplimer, design and amplification method |
| CN104911182A (en) * | 2015-06-01 | 2015-09-16 | 中国水产科学研究院 | Fish mitochondria genome-wide DNA amplification |
| CN107449853A (en) * | 2017-04-20 | 2017-12-08 | 宁波大学 | It is a kind of to differentiate tai-chu race and the method for east Guangdong large yellow croaker |
| CN107557478A (en) * | 2017-09-15 | 2018-01-09 | 中国长江三峡集团公司中华鲟研究所 | A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer |
| CN109868272A (en) * | 2019-03-18 | 2019-06-11 | 湖南农业大学 | The amplimer and amplification method of dark fund reddish tone mouse mitochondria whole genome sequence |
| CN110684850A (en) * | 2019-09-04 | 2020-01-14 | 浙江海洋大学 | Primer sequence and method for rapidly identifying large and small yellow croakers |
| CN110846381A (en) * | 2019-11-08 | 2020-02-28 | 河南师范大学 | Primer combination for amplification of carp mitochondrial DNA fragment and application thereof |
| CN112725466A (en) * | 2021-02-20 | 2021-04-30 | 安徽农业大学 | Amplification primer and amplification method of chicken mitochondrial whole genome sequence |
| CN113046445A (en) * | 2021-03-30 | 2021-06-29 | 拱北海关技术中心 | DNA barcodes, primers, kit and method for identifying vertebrates |
| CN114686600A (en) * | 2022-02-24 | 2022-07-01 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI643866B (en) * | 2017-08-17 | 2018-12-11 | 陳喆洺 | Primer pairs for lates calcarifer, mitochondrial hypervariable region and method for detecting perch dna by using the primer pairs |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698842A (en) * | 2009-10-22 | 2010-04-28 | 厦门大学 | Amplification primer for grouper catostomidae mitochondrial genome complete sequence and design method thereof |
| CN101724630A (en) * | 2008-10-24 | 2010-06-09 | 刘娣 | Complete sequence cloning and evolutionary analysis on boar mitochondrial genome in Heilongjiang Province |
-
2012
- 2012-07-12 CN CN201210241493.9A patent/CN102776183B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724630A (en) * | 2008-10-24 | 2010-06-09 | 刘娣 | Complete sequence cloning and evolutionary analysis on boar mitochondrial genome in Heilongjiang Province |
| CN101698842A (en) * | 2009-10-22 | 2010-04-28 | 厦门大学 | Amplification primer for grouper catostomidae mitochondrial genome complete sequence and design method thereof |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074335A (en) * | 2012-11-22 | 2013-05-01 | 浙江海洋学院 | Goby mitochondrion COIII and ND3 gene amplimer, design and amplification method |
| CN103074335B (en) * | 2012-11-22 | 2014-07-09 | 浙江海洋学院 | Goby mitochondrion COIII and ND3 gene amplimer, design and amplification method |
| CN104911182A (en) * | 2015-06-01 | 2015-09-16 | 中国水产科学研究院 | Fish mitochondria genome-wide DNA amplification |
| CN104911182B (en) * | 2015-06-01 | 2018-05-22 | 中国水产科学研究院 | Fish mitochondria complete genome DNA expands |
| CN107449853A (en) * | 2017-04-20 | 2017-12-08 | 宁波大学 | It is a kind of to differentiate tai-chu race and the method for east Guangdong large yellow croaker |
| CN107557478A (en) * | 2017-09-15 | 2018-01-09 | 中国长江三峡集团公司中华鲟研究所 | A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer |
| CN109868272A (en) * | 2019-03-18 | 2019-06-11 | 湖南农业大学 | The amplimer and amplification method of dark fund reddish tone mouse mitochondria whole genome sequence |
| CN109868272B (en) * | 2019-03-18 | 2020-07-24 | 湖南农业大学 | Amplification primer and amplification method of blackhead red-headed mouse mitochondrion whole genome sequence |
| CN110684850A (en) * | 2019-09-04 | 2020-01-14 | 浙江海洋大学 | Primer sequence and method for rapidly identifying large and small yellow croakers |
| CN110846381A (en) * | 2019-11-08 | 2020-02-28 | 河南师范大学 | Primer combination for amplification of carp mitochondrial DNA fragment and application thereof |
| CN112725466A (en) * | 2021-02-20 | 2021-04-30 | 安徽农业大学 | Amplification primer and amplification method of chicken mitochondrial whole genome sequence |
| CN113046445A (en) * | 2021-03-30 | 2021-06-29 | 拱北海关技术中心 | DNA barcodes, primers, kit and method for identifying vertebrates |
| CN113046445B (en) * | 2021-03-30 | 2022-02-08 | 拱北海关技术中心 | DNA barcodes, primers, kit and method for identifying vertebrates |
| CN114686600A (en) * | 2022-02-24 | 2022-07-01 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
| CN114686600B (en) * | 2022-02-24 | 2023-12-12 | 宁波大学 | Primer group and method for meat detection based on seven-fold PCR technology |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102776183B (en) | 2015-08-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102776183B (en) | A kind of amplimer of large yellow croaker plastosome whole genome sequence and application thereof | |
| Moeseneder et al. | Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes | |
| CN100532573C (en) | Method of screening pacific oyster EST micro-satellite mark | |
| CN103789301B (en) | The Auele Specific Primer of Portunus trituberculatus Miers microsatellite marker and screening method | |
| CN103571963B (en) | Primer, kit and detection method for detecting recessive white feather locus genotype of chicken | |
| CN107326077A (en) | A kind of molecular labeling for differentiating spotted maigre genetic sex and its application | |
| CN105349671B (en) | Microsatellite sequence and screening technique suitable for clean brook crow soft-shelled turtle population analysis | |
| CN104946790B (en) | A kind of PCR method for 8 kinds of animal derived materials of identification of tracing to the source | |
| CN103667275A (en) | Oil-tea SSR molecular marker | |
| WO2016064264A1 (en) | Multiplex pcr kit for the detection of pig, dog, cat, rat and monkey derived materials for halal authentication | |
| CN103937802A (en) | DNA barcoding standard gene sequence of Rizhao Blepharipoda liberata and species identification method based thereon | |
| CN102140522A (en) | Detection method for Apostichopus japonicas AjE101 micro-satellite DNA label | |
| CN102146460A (en) | Detecting method for Litopenaeus vannamei Boone LvE165 microsatellite DNA marker | |
| CN105969862A (en) | Method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers | |
| CN108950017B (en) | Universal amplification primer for mitochondria 16S rRNA gene of tridacnidae species in southern China and screening method thereof | |
| CN105349543A (en) | DNA (deoxyribonucleic acid) sequence used for identifying genetic sex of fenneropenaeus chinensis as well as obtaining method and application thereof | |
| KR101796306B1 (en) | Mircrosatellite marker for detecting Haliotis gigantean and method of detecting Haliotis gigantean using the same | |
| CN105624289B (en) | Primer sets and its application utilize the method for primer sets progress germplasm resource for cotton analysis of genetic diversity | |
| CN104988148B (en) | A kind of swamp type water buffalo SSR primers and its application | |
| CN102876777B (en) | The special primer of brown croaker EST microsatellite marker and screening method | |
| CN104975012A (en) | Microsatellite molecular marker sites of platichthys stellatus and primers | |
| Massana et al. | Metagenomic retrieval of a ribosomal DNA repeat array from an uncultured marine alveolate | |
| CN107849566A (en) | For detecting the method and kit of powdery mildew | |
| CN106222271A (en) | A kind of method of microsatellite genetic marker monitoring kind matter | |
| KR101630814B1 (en) | SNP molecular markers associated with distinction of grape understock variety and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |