CN106520939B - A kind of method and its application of madai Germplasm Identification - Google Patents
A kind of method and its application of madai Germplasm Identification Download PDFInfo
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Abstract
The invention discloses a kind of method and its application of madai Germplasm Identification, are successively expanded using two pairs of specific primers, and combine the reciprocal cross offspring of digestion method identification red porgy, black porgy and the two.The method of the invention can fast and accurately identify the germplasm of madai in molecular level；And can understand and identify red porgy, black porgy and the two reciprocal cross offspring, important evidence is provided for cultivar identification, plasm resource protection and the breeding etc. of madai.
Description
Technical field
The invention belongs to molecular biology fields, are related to fish molecular marker breeding method, and in particular to a kind of madai kind
The method and its application of matter identification.
Background technique
Red porgy Pagrosomus major belongs to Perciformes, porgy section, red porgy category, and black porgy Spraus macrocephalus belongs to perch
Shape mesh, porgy section, black porgy category, they are all the important sea-farming economic fish in China.Red porgy is big with individual, growth is fast, meat
The features such as matter is good, color is beautiful, disease resistance is strong, but it is sensitive to salinity, temperature change, and resistance is poor；Black porgy is to temperature, salinity
Adaptation range is wider, but grows slowly, and it is long to form the period.By red porgy and black porgy crossbreeding, can obtain excellent with parents
The hybrid generation of character pushes the development of madai aquaculture to improve madai economic benefit of aquaculture.After hybridization in generation, just
Offspring (red porgy ♀ × black porgy ♂) and reciprocal cross offspring (black porgy ♀ × red porgy ♂) is handed over to embody different production performance, wherein orthogonal
Offspring has preferable heterosis, hybrid vigor, and production performance is excellent.In production, cenospecies formalness is again much like with parent, just
It hands over offspring to be also easy to obscure with reciprocal cross offspring, therefore, in production easily causes madai germplasm to mix, production performance is caused to decline
It degenerates with germplasm.
Summary of the invention
The technical issues of solution: in order to overcome the drawbacks of the prior art, obtaining a kind of fast and accurately detection method, and
Important evidence is provided for the cultivar identification, plasm resource protection and breeding etc. of madai, the present invention provides a kind of madai germplasm mirror
Fixed method and its application.
A kind of technical solution: method of madai Germplasm Identification comprising the steps of:
(1) using madai genomic DNA as template, specific amplification is carried out using primer A, identifies red porgy, black porgy and the two
Filial generation；The sequence of the primer A is NO:1~2 SEQ ID；
(2) the filial generation genomic DNA obtained is identified as template using step (1), specific expansion is carried out using primer B
Increase；The sequence of the primer B is NO:3~4 SEQ ID；
(3) amplified production of digestion step (2), identifies orthogonal offspring and reciprocal cross offspring.
Primer sequence is as shown in table 1:
The primer sequence of 1 madai Germplasm Identification of table
Preferably, step (3) digestion process is using restriction endonuclease MspI.
Preferably, the system of step (3) digestion are as follows: 10 μ L of amplified production, sterilize distilled water 18 μ L, 10 × buffer
2 μ L of Tango, enzyme 10~20U, 37 DEG C of digestion 12h.
Preferably, the amplification system of step (1) and step (2) are as follows: 20 μ L of total volume, wherein 2 μ containing 10 × reaction buffer
L, Mg2+200 μm of ol/L of 2mmol/L, dNTP, upstream and downstream primer each 0.2 μm of ol/L, Taq enzyme 0.5U, DNA profiling 100ng~
200ng complements to 20 μ L with sterilizing distilled water.
Preferably, the condition of step (1) amplified reaction are as follows: 94 DEG C of 2min；94 DEG C of 30s, 57 DEG C of annealing 30s, 72 DEG C of 30s,
30 circulations；72 DEG C of extension 5min, 4 DEG C of preservations.
Preferably, the condition of step (2) amplified reaction are as follows: 94 DEG C of 2min；94 DEG C of 30s, 55 DEG C of annealing 30s, 72 DEG C of 30s,
30 circulations；72 DEG C of extension 5min, 4 DEG C of preservations.
Preferably, the amplified production of step (1) is separated by electrophoresis using 8.0% non-denaturing polyacrylamide gel.
Preferably, step (3) digestion products are separated by electrophoresis using 3% Ago-Gel.
Application of the method for the madai Germplasm Identification in madai breeding.
The utility model has the advantages that the method for (1) madai Germplasm Identification of the present invention can fast and accurately be identified in molecular level
The germplasm of madai；(2) the method, which can understand, identifies red porgy, black porgy and the two reciprocal cross offspring, be madai cultivar identification,
Plasm resource protection and breeding etc. provide important evidence.
Detailed description of the invention
Fig. 1 is red porgy, black porgy and reciprocal cross offspring's qualification result figure；
Wherein, M is molecular weight standard, and 1~10 is red porgy, and 11~19 be orthogonal offspring, and 20~29 be black porgy, and 30~35 are
Reciprocal cross offspring；
Fig. 2 is filial generation qualification result figure；
Wherein, M is molecular weight standard, and 1~12 is orthogonal offspring, and 13~23 be reciprocal cross offspring；
Fig. 3 is red porgy, black porgy and the two filial generation qualification result figure in embodiment 3；
Wherein, M is molecular weight standard, and 1~12,35~37 be filial generation, and 13~24 be red porgy, and 25~34 be black porgy；
Fig. 4 is 3 filial generation qualification result figure of embodiment；
Wherein, M is molecular weight standard, and 1~12 is orthogonal offspring, and 13~25 be reciprocal cross offspring.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modification made by the method for the present invention, step or condition and replaces, belong to the present invention
Range.Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
1 primer A feasibility analysis of embodiment
The determining red porgy of germplasm, each 10 tail of black porgy, 9 tails of orthogonal offspring (red porgy ♀ × black porgy ♂), reciprocal cross offspring (black porgy ♀
× red porgy ♂) 6 tails, totally 35 samples, are adopted from the base Jiangsu Prov. Inst. of Marine Aquatic Products Lv Si.
Every tail fish takes 30 μ L haemocytes to extract genomic DNA, respectively to extract obtained genomic DNA as template, carries out
PCR amplification.The sense primer sequence of primer A are as follows: 5 ' AACACTTATCGCACGGCTCAG3 ', antisense primer sequence are as follows: 5 '
CCCTCTGAGATCTTCACCTCCA 3′；20 μ L of PCR system total volume, wherein 2 μ L, Mg containing 10 × reaction buffer2+2mmol/
200 μm of ol/L of L, dNTP, upstream and downstream primer each 0.2 μm of ol/L, Taq enzyme 0.5U, DNA profiling 100ng~200ng, it is double with sterilizing
Distilled water supplies volume to 20 μ L.PCR reaction condition are as follows: 94 DEG C of 2min；94 DEG C of 30s, 57 DEG C of annealing 30s, 72 DEG C of 30s, 30
Circulation；72 DEG C of extension 5min, 4 DEG C of preservations.Each sample take 3 μ L of PCR reaction product use 8.0% non-denaturing polyacrylamide
Gel is separated by electrophoresis, and after argentation dyeing, photographs to record electrophoresis result.
As shown in Figure 1, red porgy has the band of 1 255bp, black porgy has the band of 1 271bp, the hybridization of red porgy and black porgy
Offspring's (orthogonal and reciprocal cross) has 2 band, respectively 255bp and 271bp.Therefore, primer A can be by red porgy, black porgy and its miscellaneous
Offspring is handed over to be distinguished.
2 primer B combination digestion feasibility analysis of embodiment
Orthogonal offspring (red porgy ♀ × black porgy ♂) 12 tails that type determines, reciprocal cross offspring (black porgy ♀ × red porgy ♂) 11 tails, altogether
23 samples are adopted from the base Jiangsu Prov. Inst. of Marine Aquatic Products Lv Si.
Every tail fish takes 30 μ L haemocytes to extract genomic DNA, respectively to extract obtained genomic DNA as template, carries out
PCR amplification.The sense primer sequence of primer B are as follows: 5 ' GCATAACACTGAAGCTGTTAAGATGG3 ', antisense primer sequence are as follows:
5′CAATGTTTATCACTGCTGAGTACCCT 3′；20 μ L of PCR system total volume, wherein 2 μ L, Mg containing 10 × reaction buffer2+
200 μm of ol/L of 2mmol/L, dNTP, upstream and downstream primer each 0.2 μm of ol/L, Taq enzyme 0.5U, DNA profiling 100ng~200ng are used
Sterilizing double distilled water supplies volume to 20 μ L.PCR reaction condition are as follows: 94 DEG C of 2min；94 DEG C of 30s, 55 DEG C of annealing 30s, 72 DEG C
30s, 30 circulations；72 DEG C of extension 5min, 4 DEG C of preservations.Each sample takes 7 μ L of PCR reaction product to be carried out with 1.5% agarose
As a result electrophoresis detection all expands the purpose band of 1 235bp or so.In remaining PCR reaction product, each sample preparation
Digestion system: 10 μ L of PCR reaction product, sterilizing 18 μ L, 10 × buffer Tango of double distilled water 2 μ L, MspI enzyme 15U, 37 DEG C
After digestion 12h, digestion products photograph to record electrophoresis result with 3% agarose electrophoresis.As shown in Fig. 2, orthogonal offspring has 2
Band, respectively 141bp and 93bp, reciprocal cross offspring have 1 band, 235bp.Therefore, primer B combination digestion method can incite somebody to action
Orthogonal offspring and reciprocal cross offspring are distinguished.
3 madai Germplasm Identification of embodiment
12 tail of red porgy to be checked, 10 tail of black porgy, red porgy and black porgy hybridize 15 tail of porgy, from Jiangsu Prov. Inst. of Marine Aquatic Products Lv Si
It adopts in base.
The every tail fishtail fin of clip simultaneously extracts genomic DNA, carries out PCR amplification, electrophoresis using method as described in Example 1
Detection.As shown in figure 3, red porgy has the band of 1 255bp, black porgy has the band of 1 271bp, and red porgy and black porgy hybridize porgy
Have 2 band, respectively 255bp and 271bp, 12 tail red porgy detected, 10 tail black porgies be all it is purebred, it is mixed without germplasm
It is miscellaneous.
15 tails hybridize porgy and carry out PCR amplification, detection, again digestion using method as described in Example 2 again, and digestion products are used
3% agarose electrophoresis photographs to record electrophoresis result.As shown in figure 4, thering are 12 tails to have 2 band in 15 tails hybridization porgy, respectively
141bp and 93bp is orthogonal madai (red porgy ♀ × black porgy ♂), has 3 tails there was only the band of 1 235bp, is reciprocal cross madai (black porgy
♀ × red porgy ♂).
SEQUENCE LISTING
<110>China Aquatic Science Research Academy Fresh Water Fishery Research Center
Jiangsu Prov. Inst. of Marine Aquatic Products
<120>a kind of method and its application of madai Germplasm Identification
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
aacacttatc gcacggctca g 21
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
ccctctgaga tcttcacctc ca 22
<210> 3
<211> 26
<212> DNA
<213>artificial sequence
<400> 3
gcataacact gaagctgtta agatgg 26
<210> 4
<211> 26
<212> DNA
<213>artificial sequence
<400> 4
caatgtttat cactgctgag taccct 26
Claims (2)
1. a kind of method of madai Germplasm Identification, which is characterized in that comprise the steps of:
(1) using madai genomic DNA as template, specific amplification is carried out using primer A, identifies the miscellaneous of red porgy, black porgy and the two
Hand over offspring；The sequence of the primer A is NO:1 ~ 2 SEQ ID；
(2) the filial generation genomic DNA obtained is identified as template using step (1), specific amplification is carried out using primer B；Institute
The sequence for stating primer B is NO:3~4 SEQ ID；
(3) amplified production of digestion step (2), identifies orthogonal offspring and reciprocal cross offspring；
Wherein, step (3) digestion process is using restriction endonuclease MspI, the system of step (3) digestion are as follows: amplified production
10 μ L, sterilize 18 μ L, 10 × buffer Tango of distilled water, 2 μ L, enzyme 10~20U, 37 DEG C of 12 h of digestion；
The amplification system of step (1) and step (2) are as follows: 20 μ L of total volume, wherein 2 μ L, Mg containing 10 × reaction buffer2+
200 μm of ol/L of 2mmol/L, dNTP, upstream and downstream primer each 0.2 μm of ol/L, Taq enzyme 0.5U, the DNA profiling ng of 100 ng~200,
20 μ L are complemented to sterilizing distilled water；
The condition of step (1) amplified reaction are as follows: 94 DEG C of 2min；94 DEG C of 30s, 57 DEG C of annealing 30s, 72 DEG C of 30s, 30 circulations；
72 DEG C of extension 5min, 4 DEG C of preservations；The condition of step (2) amplified reaction are as follows: 94 DEG C of 2min；94 DEG C of 30s, 55 DEG C of annealing 30s,
72 DEG C of 30s, 30 circulations；72 DEG C of extension 5min, 4 DEG C of preservations；
The amplified production of step (1) is separated by electrophoresis using 8.0% non-denaturing polyacrylamide gel, and step (3) digestion produces
Object is separated by electrophoresis using 3% Ago-Gel.
2. application of the method for madai Germplasm Identification in madai breeding described in claim 1.
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| CN106119389B (en) * | 2016-08-23 | 2019-07-05 | 江苏省海洋水产研究所 | A method of identifying black porgy and black porgy red porgy reciprocal cross filial generation |
| CN110338104A (en) * | 2018-04-03 | 2019-10-18 | 嘉丰海洋国际股份有限公司 | The method of Oreochromis aureus feminized is induced using the immersion treatment of ethinylestradiol |
| CN110643715B (en) * | 2019-10-11 | 2022-12-23 | 江苏省海洋水产研究所 | Method for rapidly identifying compound gobies and javanica fish |
| CN114350815B (en) * | 2021-12-30 | 2024-03-26 | 江苏省海洋水产研究所 | Molecular marking method for identifying red sea bream and red sea bream filial generation by ISSR primer |
| CN114480666B (en) * | 2021-12-30 | 2024-03-22 | 江苏省海洋水产研究所 | Method for distinguishing black sea bream and two filial generations thereof by using ISSR molecular markers |
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