CN105624307A - Microsatellite primers for identifying crassostrea hongkongensis, crassostrea ariakensis and crassostrea gigas and hybrid thereof and identification method - Google Patents
Microsatellite primers for identifying crassostrea hongkongensis, crassostrea ariakensis and crassostrea gigas and hybrid thereof and identification method Download PDFInfo
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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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- C12Q1/6876—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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses microsatellite primers for identifying crassostrea hongkongensis, crassostrea ariakensis and crassostrea gigas and a hybrid thereof and an identification method.The microsatellite primers are F:5'-CGACTGGTGGGAGTTTCTGAC-3' and R:5'-GCCGCTTCTATCTCCTTTGC-3'.Due to the fact that the external morphology of oysters changes greatly along with different living environments, identification is usually difficult only according to morphological characteristics; the oysters in the larval stage are more difficult to distinguish according to the morphological characteristics.By means of the microsatellite primers and the identification method, crassostrea hongkongensis, crassostrea ariakensis and crassostrea gigas individuals can be identified rapidly and accurately, and identification of the hybrid individual of the crassostrea hongkongensis, crassostrea ariakensis and crassostrea gigas individuals has the potential application value.Meanwhile, the microsatellite primers for identifying the crassostrea hongkongensis, the crassostrea ariakensis and the crassostrea gigas and the hybrid thereof and the identification method have the advantages of being simple in method, visual, accurate and effective in result, free of influences of environment and growth stage, low in cost and the like.
Description
Technical field:
The invention belongs to the shellfish molecular marker in aquatic wholesale market and species identification field, be specifically related to identify the huge Concha Ostreae in Hong Kong, the micro-satellite primers having bright Concha Ostreae, Pacific oyster and cenospecies thereof and authentication method.
Background technology:
Microsatellite DNA, repeat (STRs), simple repeated sequence (SSRs), SSLP (SSLP) also referred to as short series winding, refer to the nucleotide sequence being composed in series in units of 1��6 nucleotide in genome. In recent years, microsatellite marker is widely used in species identification with its advantage such as simply efficient and cenospecies is analyzed.
Concha Ostreae belongs to Mollusca, Bivalvia, Margarita order, Ostreidae, for global distribution monoid; Carnis ostreae delicious, rich in multiple essential amino acid, be a kind of important living marine resources, one of most important sea-farming grass economic category in the world especially, its cultivation total output and yield per unit area occupy first place at all of shellfish culture kind apoplexy due to endogenous wind. Concha Ostreae is also the economic shellfish that China's yield is maximum, wherein has the huge Concha Ostreae in Hong Kong of higher economic worth, has bright Concha Ostreae, Pacific oyster to carry out artificial cultivation in China. Hong Kong huge Concha Ostreae (Crassostreahongkongensis) like living in nearly river mouth or near have fresh water to inject place, be mainly distributed on Zhejiang, Fujian, Guangdong, Guangxi, Hainan, annual production is at more than 100 ten thousand tons. Its cedductor is loose, meat is delicate, delicious flavour, and the market price exceeds well over other Concha Ostreae kind. It is the high Concha Ostreae monoid of an economic worth of warm warm nature offshore growth, is the Main Economic kind of south China cultivation. Have that bright Concha Ostreae (Crassostreaariakensis) is coastal in China north and south all distribution, be concentrated mainly on Guangdong, Guangxi, be distributed in China's north and south less salt sea. Its meat flavour is delicious, nutritious, is the important sea-farming economic shellfish of all provinces of south China, the cultivation history of existing nearly two a century. Pacific oyster (Crassostreagigas) is distributed in China's North of Yangtze River, for worldwide breed variety, is concentrated mainly on the ground such as China Liaoning, Shandong, nearly 800,000 tons of annual production. Due to fast growth, environmental suitability is strong, meat flavour is delicious, is deeply favored by consumers in general. Often there is great change in the formalness of Concha Ostreae, simple dependence morphological characteristic is often difficult to differentiate with the difference of living environment. And the anatomical structure of Concha Ostreae soft body, chromosome number and difference of karyotype are only small, it is possible to provide classification evidence also little. In recent years, the molecular biology based on the mitochondrial genes such as CO I, 16SrDNA and ITS sequence difference is used to three's qualification, but between these gene orders, comparison in difference is little, it is difficult to make a distinction by straightforward procedures such as electrophoresis; Although HRM method is simple, but relevant large-scale instrument is needed to support; And the test period of checking order is longer, and cost is higher. Be badly in need of a kind of simple efficient method to the huge Concha Ostreae in Hong Kong, have bright Concha Ostreae, Pacific oyster individuality to carry out extensive species identification.
Additionally along with oyster culture industry being continuously increased improved Varieties demand, intervarietal hybridization research constantly carries out between Concha Ostreae, scholars have carried out the research of the huge Concha Ostreae �� Pacific oyster in Hong Kong, the bright Concha Ostreae of the huge Concha Ostreae in Hong Kong �� have, the bright Concha Ostreae cross combination of Pacific oyster �� have successively, and prove that the smart ovum of Some Species intermolecular hybrid Concha Ostreae can be in conjunction with, and normal development. The filial generation obtained is carried out Genetic identification is very necessary, because the phenomenons such as gynogenesis, androgenesis, monoploid often occurs in the interspecific hybrid Concha Ostreae. Be badly in need of a kind of straightforward procedure to the huge Concha Ostreae in Hong Kong, have bright Concha Ostreae, the hybrid individual of Pacific oyster carries out Molecular Identification.
Summary of the invention:
It is an object of the invention to provide a kind of simple, efficient and the huge Concha Ostreae in accurate qualification Hong Kong, the micro-satellite primers having bright Concha Ostreae, Pacific oyster and cenospecies thereof and authentication method.
1 couple micro-satellite (SSR) primer that the present invention filters out from 150 pairs of Hong Kong huge Concha Ostreae micro-satellite primers, with it to the huge Concha Ostreae in Hong Kong, each 90 the individual genomic DNAs of bright Concha Ostreae, Pacific oyster are had to carry out pcr amplification, species identification can be carried out simply efficiently by AFLP system, additionally triangular cenospecies Individual identification also there is is potential using value, it is achieved thereby that the purpose of the present invention.
The present invention identifies Hong Kong huge Concha Ostreae, has the micro-satellite primers of bright Concha Ostreae, Pacific oyster and cenospecies thereof, it is characterised in that described micro-satellite primers (SSR primer Ch409) is:
F:5 '-CGACTGGTGGGAGTTTCTGAC-3 ' (as shown in SEQIDNO.1);
R:5 '-GCCGCTTCTATCTCCTTTGC-3 ' (as shown in SEQIDNO.2).
The huge Concha Ostreae in Hong Kong of the present invention, there is the authentication method of bright Concha Ostreae, Pacific oyster and cenospecies thereof, it is characterised in that comprise the following steps:
Extracting the genomic DNA of Concha Ostreae sample, using this genomic DNA as template, carry out pcr amplification with above-mentioned micro-satellite primers, PCR primer carries out gel electrophoresis, and then typing judges;
Owing to the SSR primer Ch409 huge Concha Ostreae specific band scope in Hong Kong produced is between 308bp��347bp, there is bright Concha Ostreae specific band scope between 362bp��422bp, Pacific oyster specific band scope is between 206bp��242bp, allele overlay region is not had, it is possible to substantially distinguish the individuality of three seed oysters between three. If PCR primer, if occurring 1��2 band in 308bp��347bp interval, then this Concha Ostreae sample is the huge Concha Ostreae in Hong Kong, occurs that 1 band illustrates that this individuality is homozygote, occurs that 2 band then illustrate that this individuality is heterozygote; If occurring 1��2 band in 362bp��422bp interval, then this Concha Ostreae sample is for there being bright Concha Ostreae, occurs that 1 band illustrates that this individuality is homozygote, occurs that 2 band then illustrate that this individuality is heterozygote; If occurring 1��2 band in 206bp��242bp interval, then this Concha Ostreae sample is Pacific oyster, occurs that 1 band illustrates that this individuality is homozygote, occurs that 2 band then illustrate that this individuality is heterozygote; If PCR primer is simultaneously interval at 308bp��347bp and each appearance 1 band in 362bp��422bp interval, namely the individuality simultaneously having 1 specific band of parent is that the huge Concha Ostreae in Hong Kong hybridizes first filial generation with there being bright Concha Ostreae, lacks any one band therein and is pseudostationary; If PCR primer is simultaneously interval at 308bp��347bp and each appearance 1 band in 206bp��242bp interval, namely the individual of 1 specific band simultaneously having parent just hybridizes first filial generation for the huge Concha Ostreae in Hong Kong with Pacific oyster, and lacking any one band therein is pseudostationary; If PCR primer is simultaneously interval at 362bp��422bp and each appearance 1 band in 206bp��242bp interval, namely have the individuality of 1 specific band of parent for having bright Concha Ostreae and Pacific oyster hybridization first filial generation simultaneously, lack any one band therein and be pseudostationary.
Described using this genomic DNA as template, carrying out the preferred PCR reaction system of pcr amplification with above-mentioned micro-satellite primers is: DNA profiling 50ng, Buffer10mM, micro-satellite primers F and micro-satellite primers R each 0.2mM, dNTP0.2mM, Mg2+2.0Mm, Taq DNA polymerase 1U, supply 25 �� L with aquesterilisa; Response procedures is: 94 DEG C of denaturation 5min; 94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 10min.
The present invention filter out from 150 pairs of Hong Kong huge Concha Ostreae micro-satellite primers a pair can the huge Concha Ostreae in Hong Kong, have general in bright Concha Ostreae, Pacific oyster and can according to amplified band size discrimination three seed oyster, the micro-satellite primers that banding pattern is clear, reproducible, highly reliable simultaneously: Ch409, and also have potential using value at triangular cenospecies Individual identification.
Owing to great change often occurs the formalness of Concha Ostreae with the difference of living environment, simple dependence morphological characteristic is often difficult to differentiate; The Concha Ostreae of juvenile stage is difficult to differentiate between more according to morphological characteristic. And utilize the micro-satellite primers of the present invention, adopt the authentication method of the present invention, the huge Concha Ostreae in Hong Kong can be identified quickly and accurately, have bright Concha Ostreae, Pacific oyster individual, and also there is potential using value in triangular cenospecies Individual identification, simultaneously the present invention have that method is simple, visual result, accurate and effective, do not affected by environment and developmental stage, the advantage such as with low cost.
Accompanying drawing illustrates:
Fig. 1 is micro-satellite primers Ch409 at Pacific oyster, has the electrophoresis dying contrast collection of illustrative plates between the huge Concha Ostreae some individuals of bright Concha Ostreae, Hong Kong; 1��9 is that Pacific oyster is individual, wherein 2,3,4,5,6,7,8 is heterozygote, other are homozygote, 10��19 is have bright Concha Ostreae individual, wherein 11,12,14,16,17,19 is heterozygote, and other are homozygote, and 20��29 is that the huge Concha Ostreae in Hong Kong is individual, wherein 20,21,25,26,27,29 is heterozygote, and other are homozygote.
Detailed description of the invention:
Following example are further illustrating the present invention, rather than limitation of the present invention.
Embodiment 1:
Adopting phenol chloroform extraction method to extract the huge Concha Ostreae in Hong Kong, have each 90 the individual genomic DNAs of bright Concha Ostreae, Pacific oyster, concrete grammar is as follows:
Closed shell flesh is fully shredded, put in 1.5mL centrifuge tube after absorbing moisture with clean filter paper, add 400 �� l lysates and 10 �� l E.C. 3.4.21.64 (10mg/ml), mix on the oscillator, 55 DEG C of water-bath digestion 3��5h, until lysate clarification. Add the saturated phenol of equal-volume (200 �� L), chloroform/isoamyl alcohol (24:1) (200 �� L) mixed liquor extracting three times. Precipitating DNA with the dehydrated alcohol of 1mL, through 70% ethanol wash, room temperature is dissolved in after drying in the ultra-pure water of 100 �� L. UV spectrophotometer measuring DNA concentration and purity, and the integrity of DNA is detected with the agarose gel electrophoresis of 1%. Thus respectively obtain the huge Concha Ostreae in Hong Kong, have the genomic DNA of bright Concha Ostreae, Pacific oyster.
With the genomic DNA of three seed oysters of extraction for template, carry out pcr amplification reaction:
The cumulative volume of PCR reaction system is 25 �� L, and wherein various compositions and final concentration are respectively as follows: DNA profiling 50ng, Buffer10mM, primer (micro-satellite primers Ch409 sequence F and R) each 0.2mM, dNTP0.2mM, Mg2+2.0Mm, Taq DNA polymerase 1U, supply 25 �� L with aquesterilisa.
Wherein micro-satellite primers Ch409 sequence is:
F:5 '-CGACTGGTGGGAGTTTCTGAC-3 ' (as shown in SEQIDNO.1);
R:5 '-GCCGCTTCTATCTCCTTTGC-3 ' (as shown in SEQIDNO.2).
Response procedures is: 94 DEG C of denaturation 5min; 94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 1min, and degeneration repeats 30 times to extending three steps; 72 DEG C extend 10min.
PCR reaction takes 0.5 �� L amplified production at the 8% huge acrylamide gel electrophoresis of non denatured after terminating, 22V voltage stabilizing electrophoresis 4 hours, and gel is put in pallet from taking off glass plate after terminating by electrophoresis, cleans twice with deionized water, is subsequently adding 0.1%AgNO3Solution (AgNO31g, 37% formaldehyde 1.5mL, dH2O1000mL) dyeing 10min. Add deionized water after dyeing again to clean once, add nitrite ion (2%NaOH, 0.4% formaldehyde, 0.2%NaS2O3��dH2O1000mL) colour developing, till band is clear, scans under UMAX scanner, then carries out typing. Concrete outcome is as it is shown in figure 1, it will be seen from figure 1 that 1 band occurs in 308bp��347bp interval in the homozygote of the huge Concha Ostreae in Hong Kong, 2 band occurs in 308bp��347bp interval in the heterozygote of the huge Concha Ostreae in Hong Kong; There is 1 band in 362bp��422bp interval in the homozygote having bright Concha Ostreae, has the heterozygote of bright Concha Ostreae to occur 2 band in 362bp��422bp interval; There is 1 band in 206bp��242bp interval in the homozygote of Pacific oyster, and 2 band occurs in 206bp��242bp interval in the heterozygote of Pacific oyster.
Therefore, if Concha Ostreae sample to be measured, its DNA is extracted according to the method described above, with it for template, carrying out pcr amplification with micro-satellite primers Ch409, the huge acrylamide gel electrophoresis of PCR primer non denatured, then after dyeing, carry out typing, contrasting according to electrophoresis result and standard diagram, if occurring 1��2 band in 308bp��347bp interval, then this DNA sample is the huge Concha Ostreae in Hong Kong, occur that 1 band illustrates that this individuality is homozygote, occur that 2 band then illustrate that this individuality is heterozygote; If occurring 1��2 band in 362bp��422bp interval, then this DNA sample is for there being bright Concha Ostreae, occurs that 1 band illustrates that this individuality is homozygote, occurs that 2 band then illustrate that this individuality is heterozygote; If occurring 1��2 band in 206bp��242bp interval, then this DNA sample is Pacific oyster, occurs that 1 band illustrates that this individuality is homozygote, occurs that 2 band then illustrate that this individuality is heterozygote. This is marked at the huge Concha Ostreae in Hong Kong, have bright Concha Ostreae, Pacific oyster intermolecular hybrid kind to identify in potential application process be: sample is only simultaneously interval at 308bp��347bp and each in 362bp��422bp interval 1 band occurs, namely the individual of 1 specific band simultaneously having parent just hybridizes first filial generation for the real huge Concha Ostreae in Hong Kong with there being bright Concha Ostreae, and lacking any one band therein is pseudostationary; Sample is only simultaneously each in 308bp��347bp interval and 206bp��242bp interval there is 1 band, namely the individual of 1 specific band simultaneously having parent just hybridizes first filial generation for the real huge Concha Ostreae in Hong Kong with Pacific oyster, and lacking any one band therein is pseudostationary; Sample is only simultaneously each in 362bp��422bp interval and 206bp��242bp interval there is 1 band, namely the individual of 1 specific band simultaneously having parent just has bright Concha Ostreae and Pacific oyster hybridization first filial generation for real, lacks any one band therein and is pseudostationary.
Claims (3)
1. identify the huge Concha Ostreae in Hong Kong, have the micro-satellite primers of bright Concha Ostreae, Pacific oyster and cenospecies thereof for one kind, it is characterised in that described micro-satellite primers is:
F:5 '-CGACTGGTGGGAGTTTCTGAC-3 ';
R:5��-GCCGCTTCTATCTCCTTTGC-3����
2. the huge Concha Ostreae in Hong Kong, have the authentication method of bright Concha Ostreae, Pacific oyster and cenospecies thereof, it is characterised in that comprise the following steps:
Extracting the genomic DNA of Concha Ostreae sample, using this genomic DNA as template, carry out pcr amplification with the micro-satellite primers described in claim 1, PCR primer carries out gel electrophoresis, and then typing judges;
If PCR primer, if occurring 1��2 band in 308bp��347bp interval, then this Concha Ostreae sample is the huge Concha Ostreae in Hong Kong, occurs that 1 band illustrates that this individuality is homozygote, occurs that 2 band then illustrate that this individuality is heterozygote; If occurring 1��2 band in 362bp��422bp interval, then this Concha Ostreae sample is for there being bright Concha Ostreae, occurs that 1 band illustrates that this individuality is homozygote, occurs that 2 band then illustrate that this individuality is heterozygote; If occurring 1��2 band in 206bp��242bp interval, then this Concha Ostreae sample is Pacific oyster, occurs that 1 band illustrates that this individuality is homozygote, occurs that 2 band then illustrate that this individuality is heterozygote; 1 band occur if each in 308bp��347bp interval and 362bp��422bp interval while of PCR primer, the individuality namely simultaneously having 1 specific band of parent is that the huge Concha Ostreae in Hong Kong hybridizes first filial generation with there being bright Concha Ostreae; If PCR primer is simultaneously interval at 308bp��347bp and each appearance 1 band in 206bp��242bp interval, the individuality namely simultaneously having 1 specific band of parent is the huge Concha Ostreae in Hong Kong and Pacific oyster hybridization first filial generation; If PCR primer is simultaneously interval at 362bp��422bp and each appearance 1 band in 206bp��242bp interval, namely have the individuality of 1 specific band of parent for having bright Concha Ostreae and Pacific oyster hybridization first filial generation simultaneously.
3. authentication method according to claim 2, it is characterized in that, described using this genomic DNA as template, pcr amplification is carried out with the micro-satellite primers described in claim 1, its PCR reaction system is: each 0.2mM of DNA profiling 50ng, Buffer10mM, micro-satellite primers F and micro-satellite primers R, dNTP0.2mM, Mg2+2.0Mm, Taq DNA polymerase 1U, supply 25 �� L with aquesterilisa; Response procedures is: 94 DEG C of denaturation 5min; 94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 10min.
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Cited By (4)
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| CN108103211A (en) * | 2018-01-31 | 2018-06-01 | 中国科学院南海海洋研究所 | A kind of identification Tridacna (chamestrachea) maxima (R ding), the micro-satellite primers and identification method of Nova giant clam |
| CN108950016A (en) * | 2018-08-08 | 2018-12-07 | 中国科学院南海海洋研究所 | Identification Hong Kong oyster has bright oyster and its hybridizes micro-satellite primers, kit and identification method and the application of first filial generation |
| CN110592230A (en) * | 2019-09-03 | 2019-12-20 | 中国科学院南海海洋研究所 | Microsatellite primer, kit and identification method for identifying crassostrea hongkongensis and crassostrea viticola and first filial generation thereof |
| CN110760595A (en) * | 2019-11-06 | 2020-02-07 | 中国科学院南海海洋研究所 | Identification primer and method suitable for tridacna species common in trioyster and south China sea and hybridized species of tridacna species |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103211A (en) * | 2018-01-31 | 2018-06-01 | 中国科学院南海海洋研究所 | A kind of identification Tridacna (chamestrachea) maxima (R ding), the micro-satellite primers and identification method of Nova giant clam |
| CN108950016A (en) * | 2018-08-08 | 2018-12-07 | 中国科学院南海海洋研究所 | Identification Hong Kong oyster has bright oyster and its hybridizes micro-satellite primers, kit and identification method and the application of first filial generation |
| CN108950016B (en) * | 2018-08-08 | 2021-07-16 | 中国科学院南海海洋研究所 | Microsatellite primer, kit and identification method for identifying crassostrea hongkongensis, crassostrea gigas and hybridized first filial generation thereof and application |
| CN110592230A (en) * | 2019-09-03 | 2019-12-20 | 中国科学院南海海洋研究所 | Microsatellite primer, kit and identification method for identifying crassostrea hongkongensis and crassostrea viticola and first filial generation thereof |
| CN110592230B (en) * | 2019-09-03 | 2021-09-21 | 中国科学院南海海洋研究所 | Microsatellite primer, kit and identification method for identifying crassostrea hongkongensis and crassostrea viticola and first filial generation thereof |
| CN110760595A (en) * | 2019-11-06 | 2020-02-07 | 中国科学院南海海洋研究所 | Identification primer and method suitable for tridacna species common in trioyster and south China sea and hybridized species of tridacna species |
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| CN105624307B (en) | 2020-02-14 |
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