CN1966723A - Determination primer and method for yellow croaker in different population - Google Patents
Determination primer and method for yellow croaker in different population Download PDFInfo
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Abstract
Identifying primers and method of different groups of large yellow croaker, relates to a kind of primer and large yellow croaker. The invention provides an identifying primer (5'-TGCCGCACTT-3') for identifying 3 geographical species of Daiqu, west Min-yue, and Naozhou, and cultivation species. The identifying method comprises the steps of: extracting large yellow croaker DNA with phenol/chloroform, constructing a PCR system (25uL) containing Buffer 10mM, dNTP 0.2mM, Mg2+ 1.5mM, primer 0.2mM, Taq 1U,large yellow croaker DNA 50ng, and water as balance, detecting the product with agarose gel electrophoresis, dyeing with ethidium bromide, observing with UV, taking a picture, and recording. Different species are identified through comparing the result of electrophoresis and the provided standard graph.
Description
Technical field
The present invention relates to a kind of primer and large yellow croaker, especially relate to diagnostic primers and the discrimination method of different population large yellow croaker.
Background technology
Large yellow croaker (Pseudosciaena crocea (Richardson)) has another name called yellow croaker, cucumber, yellow croaker, be under the jurisdiction of Perciformes (Perciformes), Anabantoidei (Percoidei), Sciaenidae (Scieanidae), yellow croaker belongs to (Pseudosciaena), once be one of China's famous " four Large Marine Ecosystem economic fishs ", its output is only second to hairtail and pelagic fishes.Distribution is all arranged in Shandong Peninsula of China's Huanghai Sea to the east of with the Lezhou Peninsula that reaches the South Sea in the south.China scientific worker had detailed research in 50～sixties of 20th century to the division of the geographical population (local family) of large yellow croaker, measure feature and population structure features such as life-span, age composition and age at sexual maturity according to forms such as gill raker number, fish glue number of branch and caudal peduncle height, and in conjunction with the characteristics of geographic range, comparative analysis 7 reproduction shoals of fish and 3 non-reproductions contain the data of the phase shoals of fish, propose the coastal large yellow croaker of China and can roughly be divided into tai-chu race, 3 geographical population of east Guangdong and island in Guangdong Province family.Tai-chu race comprises 4 large yellow croaker shoals of fish such as Jiangsu Dai Quyang, Lv Siyang, cat head ocean and ocean, Dongtou, is distributed in southern Yellow Sea to middle part, the East Sea; East Guangdong comprises 4 shoals of fish such as Fujian Guan Jingyang, off-lying sea, Xiamen, Nan'ao, Guangdong and Shanwei, mainly is distributed in southern East China Sea and the north, the South Sea (the mouth of the Zhujiang River); Island in Guangdong Province family is mainly the large yellow croaker shoal of fish in coastal waters, island in Guangdong Province, Guangdong, its main range of distribution be to the west of the mouth of the Zhujiang River to the east of the Qiongzhou Strait.Since the sixties in 20th century,, once be the condition that the large yellow croaker of one of four big fishing seasons has faced extinction because the artificial excessive and suitable large yellow croaker of fishing intensity lays eggs and the reproductive growth environment of forage is seriously damaged.Since 1985, cultured practice through in a few years, the work of large yellow croaker artificial breeding obtains breakthrough, and large-area subsequently breed obtains flourish.3 wild stockss of large yellow croaker also have fragmentary the distribution in each sea area at present, but because quantity is few and the market value height, and its market value is 20～30 times of cultured population price, often has illegal retailer to serve as wild individuality with the individuality of culturing and therefrom seek exorbitant profit.
Be usually used at present differentiating that the main method of large yellow croaker wild population and cultured population has following several: (1) ordinary people is usually according to the body colour of large yellow croaker bright orange or price whether; (2) shape and the quantity of experienced fisherman by seeing fish scale and gill raker; (3) method that is provided according to patent ZL2004 1 0060149.5 is carried out pcr amplification and order-checking by specific primer to plastosome CytB gene, according to the variation of 57,66,223,291 bases on the sequence.Above several method all can only be distinguished wild population and cultured population, 3 geographical population of tai-chu race, east Guangdong, island in Guangdong Province family and the judging criterion wherein that can't distinguish in the wild population have more subjective factor, are unfavorable for judging the source of large yellow croaker objective and accurately.The present invention utilizes a primer that the DNA of large yellow croaker is carried out pcr amplification, can distinguish 3 geographical population and 1 cultured population by the amplification collection of illustrative plates, has accurately quick, just objective, economical and practical characteristics.
Summary of the invention
The objective of the invention is to provides a kind of diagnostic primers that can differentiate 4 population large yellow croakers such as tai-chu race, 3 geographical population of east Guangdong and island in Guangdong Province family and cultured population simultaneously at the deficiency in the existing detection large yellow croaker population method.
Another object of the present invention is to utilize above-mentioned diagnostic primers to carry out the method that the different population large yellow croaker is differentiated.
The primer sequence that can differentiate tai-chu race, 3 geographical population of east Guangdong and island in Guangdong Province family and cultured population simultaneously of the present invention: 5 ' TGCCGCACTT-3 '.
Its step of the discrimination method of different population large yellow croaker of the present invention is as follows:
1) adopt the phenol chloroform extraction method to extract large yellow croaker DNA;
2) pcr amplification reaction: the cumulative volume of pcr amplification reaction system is 25uL, and its various compositions and final concentration are respectively: Buffer10mM, dNTP 0.2mM, Mg
2+1.5mM, primer 0.2mM, Taq 1U, large yellow croaker DNA 50ng uses the distilled water polishing to 25uL; Single primer sequence is: 5 ' TGCCGCACTT-3 ', and the PCR response procedures is:
95℃?5min→(94℃?40sec→42.5℃?40sec→72℃?60sec)×35cycles→72℃?5min→4℃∞；
3) after electrophoresis dying contrast collection of illustrative plates: PCR reaction finishes, getting product detects with agarose gel electrophoresis, ethidium bromide staining, and with the observation of ultraviolet transmission reflective analysis instrument, photograph, record, according to electrophoresis result and the standard diagram contrast that provides, if occur the band of a 1875bp in the collection of illustrative plates, then this DNA sample is from the large yellow croaker cultured population; If the band of 1 1250bp is only arranged in 1000～1500bp interval, then this DNA sample is from the wild large yellow croaker of tai-chu race; If the band of 1 1085bp is only arranged in 1000～1500bp interval, then this DNA sample is from the wild large yellow croaker of east Guangdong; If promptly there is the band of 1 1085bp that the band of 1 1250bp is arranged again in 1000～1500bp interval, then this DNA sample is from the wild large yellow croaker of island in Guangdong Province family.
Described PCR response procedures is to make part to revise on the basis of the response procedures of classics.Being used of this response procedures and this primer can obtain preferable experimental result.
When electrophoresis dying contrast collection of illustrative plates, after the PCR reaction finishes, get product 10uL, the agarose gel electrophoresis with 2.0% detects, ethidium bromide (EB, 5mg/L) dyeing.
Compare with existing detection large yellow croaker population method, the present invention is owing to utilize the primer sequence 5 ' TGCCGCACTT-3 ' can differentiate tai-chu race, 3 geographical population of east Guangdong and island in Guangdong Province family and cultured population simultaneously, therefore can differentiate the different geographical population of cultured large yellow croaker and wild large yellow croaker quickly.
Description of drawings
Fig. 1 is electrophoresis dying contrast collection of illustrative plates.In Fig. 1, M is DNA marker (Diamond
TM250bp DNA Marker), the 1st, tai-chu race large yellow croaker, the 2nd, east Guangdong large yellow croaker, the 3rd, island in Guangdong Province family large yellow croaker, the 4th, cultured large yellow croaker.
Embodiment
Embodiment 1
Select island in Guangdong Province family large yellow croaker and cultured large yellow croaker for use, adopt the phenol chloroform extraction method (to translate " molecular cloning experiment guide " (third edition) according to a conventional method referring to Huang Peitang etc., Science Press, in September, 2002) extract large yellow croaker DNA, design synthetic primer 5 ' TGCCGCACTT-3 ', its genome is increased, and amplification program is: 95 ℃ of 5min → (94 ℃ of 40sec → 42.5 ℃ 40sec → 72 ℃ of 60sec) * 35cycles → 72 ℃ 5min → 4 ℃ of ∞; Amplification through electrophoresis, dyeing and with the collection of illustrative plates contrast, the band of a 1875bp appears in the collection of illustrative plates, then this DNA sample is from the large yellow croaker cultured population; If promptly there is the band of 1 1085bp that the band of 1 1250bp is arranged again in the 1000-1500bp interval, then this DNA sample is from the wild large yellow croaker of island in Guangdong Province family.The cumulative volume of pcr amplification reaction system is 25uL, and its various compositions and final concentration are respectively: Buffer 10mM, dNTP 0.2mM, Mg
2+1.5mM, primer 0.2mM, Taq 1U, large yellow croaker DNA 50ng uses the distilled water polishing to 25uL.
When electrophoresis dying contrast collection of illustrative plates, after the PCR reaction finishes, get product 10uL, agarose gel electrophoresis with 2.0% detects, ethidium bromide (EB, 5mg/L) dyeing, and with the observation of ultraviolet transmission reflective analysis instrument, photograph, record, according to electrophoresis result and the standard diagram contrast that provides.
Similar to embodiment 1, its difference is to select for use tai-chu race and east Guangdong large yellow croaker, if the band of 1 1250bp is only arranged in 1000～1500bp interval, then this DNA sample is from the wild large yellow croaker of tai-chu race during the contrast collection of illustrative plates; If the band of 1 1085bp is only arranged in 1000～1500bp interval, then this DNA sample is from the wild large yellow croaker of east Guangdong.
Claims (4)
1. the diagnostic primers of different population large yellow croaker is characterized in that differentiating simultaneously that the primer of tai-chu race, 3 geographical population of east Guangdong and island in Guangdong Province family and cultured population is 5 ' TGCCGCACTT-3 '.
2. the discrimination method of different population large yellow croaker as claimed in claim 1 is characterized in that its step is as follows:
1) adopt the phenol chloroform extraction method to extract large yellow croaker DNA;
2) pcr amplification reaction: the cumulative volume of pcr amplification reaction system is 25uL, and its various compositions and final concentration are respectively: Buffer10mM, dNTP 0.2mM, Mg
2+1.5mM, primer 0.2mM, Taq 1U, large yellow croaker DNA 50ng uses the distilled water polishing to 25uL; Single primer sequence is: 5 ' TGCCGCACTT-3 ', and the PCR response procedures is:
95℃?5min→(94℃?40sec→42.5℃?40sec→72℃?60sec)×35cycles→72℃?5min→4℃∞；
3) after electrophoresis dying contrast collection of illustrative plates: PCR reaction finishes, getting product detects with agarose gel electrophoresis, ethidium bromide staining, and with the observation of ultraviolet transmission reflective analysis instrument, photograph, record, according to electrophoresis result and the standard diagram contrast that provides, if occur the band of a 1875bp in the collection of illustrative plates, then this DNA sample is from the large yellow croaker cultured population; If the band of 1 1250bp is only arranged in 1000～1500bp interval, then this DNA sample is from the wild large yellow croaker of tai-chu race; If the band of 1 1085bp is only arranged in 1000～1500bp interval, then this DNA sample is from the wild large yellow croaker of east Guangdong; If promptly there is the band of 1 1085bp that the band of 1 1250bp is arranged again in 1000～1500bp interval, then this DNA sample is from the wild large yellow croaker of island in Guangdong Province family.
3. the discrimination method of different population large yellow croaker as claimed in claim 2 is characterized in that after the PCR reaction finishes, getting product 10uL when electrophoresis dying contrast collection of illustrative plates, and the agarose gel electrophoresis with 2.0% detects, ethidium bromide staining.
4. the discrimination method of different population large yellow croaker as claimed in claim 3, the concentration that it is characterized in that ethidium bromide is 5mg/L.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974517A (en) * | 2010-10-22 | 2011-02-16 | 厦门大学 | Amplification primers for large yellow croaker IFNG (Interferon-Gamma Gene) and design method thereof |
| CN102758024A (en) * | 2012-08-08 | 2012-10-31 | 中山大学 | Selective molecular marker which for detecting little yellow croaker group structure and primer of marker and detection method of marker |
| CN102776284A (en) * | 2012-07-12 | 2012-11-14 | 宁波大学 | Primer and method for identifying pseudosciaena crocea of daiqu species and min-yuedong species |
| CN105525020A (en) * | 2016-02-01 | 2016-04-27 | 辽宁省海洋水产科学研究院 | Kit for rapidly identifying flathead fish and callionymus koreanus and identification method |
| CN106086167A (en) * | 2016-06-07 | 2016-11-09 | 江苏省海洋水产研究所 | A kind of Rapid identification Carnis Pseudosciaenae, Carnis Pseudosciaenae and the primer sequence of Collichthys lucidus and method |
| CN107449838A (en) * | 2017-04-20 | 2017-12-08 | 宁波大学 | It is a kind of that tai-chu race and the method for east Guangdong large yellow croaker are differentiated based on aliphatic acid composition otherness |
| CN108048576A (en) * | 2017-12-15 | 2018-05-18 | 浙江海洋大学 | Differentiate method of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea and application thereof |
| CN113897440B (en) * | 2021-10-29 | 2023-07-04 | 浙江省农业科学院 | Molecular marker for rapidly identifying sex of large yellow croaker in daiqu and application |
Families Citing this family (1)
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| CN103131765A (en) * | 2011-12-02 | 2013-06-05 | 汕头大学医学院 | Molecular biological method of identifying silver pomfret |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1266285C (en) * | 2004-06-28 | 2006-07-26 | 厦门大学 | Large yellow croaker mitochondrial molecule mark and its use in identification for cultivated population and wild population |
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2006
- 2006-11-28 CN CNB2006101352592A patent/CN100396794C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974517A (en) * | 2010-10-22 | 2011-02-16 | 厦门大学 | Amplification primers for large yellow croaker IFNG (Interferon-Gamma Gene) and design method thereof |
| CN101974517B (en) * | 2010-10-22 | 2013-01-02 | 厦门大学 | Amplification primers for large yellow croaker IFNG (Interferon-Gamma Gene) and design method thereof |
| CN102776284A (en) * | 2012-07-12 | 2012-11-14 | 宁波大学 | Primer and method for identifying pseudosciaena crocea of daiqu species and min-yuedong species |
| CN102776284B (en) * | 2012-07-12 | 2015-11-18 | 宁波大学 | The diagnostic primers of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race large yellow croaker and discrimination method thereof |
| CN102758024A (en) * | 2012-08-08 | 2012-10-31 | 中山大学 | Selective molecular marker which for detecting little yellow croaker group structure and primer of marker and detection method of marker |
| CN105525020A (en) * | 2016-02-01 | 2016-04-27 | 辽宁省海洋水产科学研究院 | Kit for rapidly identifying flathead fish and callionymus koreanus and identification method |
| CN106086167A (en) * | 2016-06-07 | 2016-11-09 | 江苏省海洋水产研究所 | A kind of Rapid identification Carnis Pseudosciaenae, Carnis Pseudosciaenae and the primer sequence of Collichthys lucidus and method |
| CN106086167B (en) * | 2016-06-07 | 2019-06-28 | 江苏省海洋水产研究所 | The primer sequence and method of a kind of Rapid identification Larimichthys crocea, little yellow croaker and Collichthys lucidus |
| CN107449838A (en) * | 2017-04-20 | 2017-12-08 | 宁波大学 | It is a kind of that tai-chu race and the method for east Guangdong large yellow croaker are differentiated based on aliphatic acid composition otherness |
| CN108048576A (en) * | 2017-12-15 | 2018-05-18 | 浙江海洋大学 | Differentiate method of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea and application thereof |
| CN113897440B (en) * | 2021-10-29 | 2023-07-04 | 浙江省农业科学院 | Molecular marker for rapidly identifying sex of large yellow croaker in daiqu and application |
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