CN102251048A

CN102251048A - Method for identifying grass carps by using microsatellite primers

Info

Publication number: CN102251048A
Application number: CN2011102234378A
Authority: CN
Inventors: 李新辉; 杨计平; 李捷; 谭细畅; 王超; 李跃飞; 赖子尼; 吴瑞全; 吴茜
Original assignee: Pearl River Fisheries Research Institute CAFS
Current assignee: Pearl River Fisheries Research Institute CAFS
Priority date: 2011-08-05
Filing date: 2011-08-05
Publication date: 2011-11-23

Abstract

The invention discloses a method for identifying grass carps by using microsatellite primers. The sequences of the microsatellite primers used for identifying the grass carps are represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. In the invention, two pairs of specific microsatellite markers with clear banding pattern, high repeatability and high reliability for identifying the grass carps are screened out from 84 pairs of microsatellite primers for cyprinid fish as specific primers for identifying grass carps. The method can quickly and accurately identify grass carps without being influenced by environmental conditions and growth period and has the advantage of simple and convenient counting and the like.

Description

A kind of method of utilizing micro-satellite primers to identify grass carp

Technical field

The present invention relates to the dna molecular marker detection range, be specifically related to a kind of method of utilizing micro-satellite primers to identify grass carp.

Background technology

Grass carp (Ctenopharyngodon idellus) has another name called grass carp, careless grass carp, ctenopharyngodon idellus etc., is one of main cultured freshwater fish of China, is distributed in each big water system of the whole nation.In recent years because of overfishing, extensive construction hydro project, influences such as water pollution, the grass carp stock number is fallen sharply, in order to grasp group structure and germ plasm resource situation thereof as early as possible, thereby formulate and implement more scientific and reasonable fishery management policy, be necessary grass carp is set up the molecular recognition technology of a kind, microsatellite marker then becomes one of first-selected mark, studies show that, in eukaryote, approximately just there is 1 little satellite every 10-50kb, as in the fish genome, little satellite is considered to will occur once (Wright, 1993) every about 10kb, be distributed widely in the coding region, in intron and the non-coding region; The flanking sequence of microsatellite DNA is comparatively conservative, thereby the high efficiency and the stability of pcr amplification have been guaranteed, because the number of repeat unit purpose changes, therefore show length polymorphism (being SSR), this marking method especially shows less demanding to the DNA sample, even sample DNA has degraded to a certain degree, utilize little satellite analysis also can obtain result preferably, be very beneficial for analyzing particular sample (as the fossil sample, ethanol fixed fry etc.), because it has above-mentioned advantage, SSR is widely used in construction of genetic atlas, QTL, fields such as cultivar identification.

At present, discrimination method to grass carp mainly contains 3 kinds: (1) form method: identify by the formalness feature to grass carp, as build, muscle segment number etc., this method is easy, economical, intuitively, can both obtain qualification result preferably to the adult grass carp, but can not be applied to the identification research of young postlarva, because the young postlarva of early development can dewater after ethanol is fixing, distortion, some pigment can disappear to some extent, and can be with certain subjective colo(u)r, seriously restricted the feasibility .(2 of identification of morphology) the isozyme method: mainly be to be undertaken by polyacrylamide gel or starch gel electrophoresis, as the 10 kind isozyme electrophoresis analysis revealeds of Li Sifa (1990) to the Changjiang river four large Chinese carp original seeds, its isozymogram can be used as four large Chinese carp Idioplasm identification standards, but it is relevant period with tissue development that isozyme is expressed, limited its application in young postlarva is identified, (3) molecular labeling method: aspect molecule marker, RAPD is usually used in the species evaluation, as Xue Guoxiong etc. to the Changjiang river, the Zhujiang River, the RAPD analysis revealed of the grass carp of Heilungkiang 3 big water systems, the grass carp characteristic gene mapping of each water system can be used as the foundation that population is identified, but RAPD mark repeatability and less stable, Given this, microsatellite marker then is one of best molecule marker, at present, also there are a lot of scholars to be applied to the cultivar identification field, as publication number is the patent application of CN1370834A: the screening of " being suitable for the microsatellite DNA mark of pig variety classification " and scallop microsatellite marker and the application in species are identified thereof etc., the research of many microsatellite markers is also arranged about grass carp, but being used to analyze the genetic diversity of grass carp rather than being used for species, its purpose identifies, so, utilize the micro-satellite primers resource sharing, design and probe into a kind of little satellite technology and identify that the method for grass carp seems particularly important.

Summary of the invention

The objective of the invention is to according to the deficiencies in the prior art, a kind of microsatellite marker method of identifying grass carp accurately is provided, can be used for the early stage resource of grass carp and identify.

Above-mentioned purpose of the present invention is achieved by the following technical programs:

The present invention therefrom filters out one group (2 pairs of primers) and can identify the specificity micro-satellite primers of grass carp with reference to 79 pairs of cyprinid fish micro-satellite primers in the document of having delivered.By the identification of two kinds of little satellites, can be with other 28 kinds of fish and grass carp difference (seeing appendix).Grass carp microsatellite DNA mark of the present invention is mainly used in the early stage resource evaluation of fish and distinguishes grass carp, early stage resource fish COMMUNITY STRUCTURE, rivers ecological evaluation etc., and good reproducibility is a kind of reliable and effective molecule marker.

Concrete steps of the present invention are as follows:

1) traditional phenol-chloroform method extracts grass carp and other fish genomic dna (table 1);

2) design of primers and optimization, the ESTs sequence of search grass carp from NCBI dbEST, carry out searching of SSR with software SSRHutter1.3, carry out design of primers with Primer Premier5.0 then, obtain 84 pairs of grass carp EST-SSR primers, and these primer use temperature grads PCR instrument are carried out the optimization of annealing temperature;

3) pcr amplification

Is that template is carried out pcr amplification reaction with above-mentioned primer with the grass carp genomic dna, and the PCR reaction system is the primer of 20ul:0.5umol/L, the dNTP of 0.2 mmol/L, the Mg of 2 mmol/L ²⁺, 1 * PCR reaction buffer, the Taq archaeal dna polymerase of 1U; The PCR program parameter that is provided with during with these primers is: 94 ℃ of pre-sex change 3min, and 30 circulations: 94 ℃ of sex change 30s, Ta are annealing 30s down, and 72 ℃ are extended 45s, and last 72 ℃ are extended 7min;

4) the PCR product detects

Pcr amplification product is through 10% native polyacrylamide gel electrophoresis, and 220V voltage stabilizing electrophoresis 3 hours is used 0.1% AgNO then ₃Dyeing 1min, silver dye the back with 2% NaOH, 0.2% anhydrous Na ₂CO ₃, 0.4% formaldehyde colour developing 2min, till band is clear;

5) screening of grass carp monomorphism primer screens 59 pairs of grass carp monomorphism primers altogether in 84 pairs of micro-satellite primers, account for 70% of the primer.

6) screening of grass carp Auele Specific Primer, 59 pairs of monomorphism primers that obtain with step 5 carry out pcr amplification, electrophoresis to grass carp and other 28 kinds of fish by above-mentioned condition respectively, specificity microsatellite locus in order to the screening grass carp, result's demonstration: these primers are in these fish or amplify the fragment identical or close with the grass carp size, spawn can not increase, be chosen in 2 microsatellite locus that all do not have any amplified production in these 28 kinds of fish at last and use the site as the evaluation of grass carp, grass carp separates mutually with other fish the most at last.The primer sequence that obtains is shown in SEQ ID NO:1 ~ 4.

Compared with prior art, the present invention has following beneficial effect:

The present invention filters out 2 pairs of specificity microsatellite markers that are used to identify grass carp from 84 pairs of micro-satellite primers, banding pattern is clear, good reproducibility, good reliability, can be used as the characteristic primer of grass carp.

In view of grass carp was difficult for discerning by morphological specificity in the seedling etap, by application of the present invention, it can be differentiated out quickly and accurately that simultaneously, the present invention has the influence of envrionment conditions of not being subjected to and developmental stage, add up advantage such as easy.

Description of drawings

Fig. 1 is that primer C3210 is the intraindividual amplification collection of illustrative plates of grass carp different groups (1-10 is the Guiping sample, and 11-20 is the riverhead sample, and 21-30 is western ox sample);

Fig. 2 is that primer C41926 is the intraindividual amplification collection of illustrative plates of grass carp different groups (1-10 is the Guiping sample, and 11-20 is the riverhead sample, and 21-30 is western ox sample);

Fig. 3 is the amplification collection of illustrative plates (sequence number corresponding kind see appendix) of primer Q3210 in other 28 kinds of fish genomic dnas.

Embodiment

Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.

Embodiment

(1) extract grass carp and other fish genomic dna:

Clip blots the dehydrated alcohol on surface through the about 20mg of ethanol fixed fish fin ray with filter paper, adds 500ml STE lysate (100mM NaCL; 1mM EDTA (PH=8.0); 10 mM Tris-CL (PH=8.0)), shred, add 60ml SDS (10%) again, and the Proteinase K of 10ul (20mg/ml), 56 ℃ of water-baths are till the solution clarification; Add equal-volume phenol: chloroform: twice of primary isoamyl alcohol (25:24:1) extracting; The centrifugal 10min of 12000r gets supernatant; Add isopyknic chloroform: primary isoamyl alcohol (24:1) extracting once, the centrifugal 10min of 12000r gets supernatant; Add the ice-cold dehydrated alcohol of equal-volume, the centrifugal 10min of 12000r abandons supernatant; Add the washing with alcohol precipitation of 600ml 70%, the centrifugal 10min of 8000r outwells ethanol, treats that ethanol volatilizees to add the 80ul sterile distilled water when clean, and 4 ℃ of preservations are dissolved fully until DNA, survey its OD value and be diluted to 40ng/ml standby then with ultraviolet spectrophotometer.

(2) design of primers and optimization

Choose multiplicity in the double alkali yl tumor-necrosis factor glycoproteins more than 5 times and multiplicity at three bases more than 4 times or four base repetitive sequences, use primer-design software Primer Premier5.0 to carry out design of primers, obtain 84 pairs of EST-SSR primers, genomic dna mixture with indivedual grass carps in the different groups is a template then, carry out the optimization of primer, initial all primers all are 50 ℃ with the Tm value and increase, and carry out suitable increase and decrease according to amplification then, and scope is 45 ℃-65 ℃.

(3) pcr amplification

Primer with above-mentioned optimization carries out pcr amplification to genomic dna individual in the grass carp different groups, and reaction system is a 20ul:40ng grass carp genomic dna, the primer of 0.2mmol/L, the dNTP of 200mmol/L, the Mg2+ of 200mmol/L, 10 * PCR reaction buffer, the Taq archaeal dna polymerase of 1U; The PCR program parameter that is provided with during with these primers is: 94 ℃ of pre-sex change 3min, and 30 circulations: 94 ℃ of sex change 30s, Ta are annealing 30s down, and 72 ℃ are extended 45s, and last 72 ℃ are extended 7min;

(4) detection of amplified production

Pcr amplification product is through 10% native polyacrylamide gel electrophoresis, 220V voltage stabilizing electrophoresis 3 hours, then with 0.1% AgNO3 dyeing 1min, silver dyes the back and develops the color with 2% NaOH, 0.2% anhydrous Na2CO3,0.4% formaldehyde mixed solution, till when band is clear, and the preservation of taking pictures.

(5) screening of monomorphism primer

To in the grass carp different groups totally 96 individual genomic dnas carry out pcr amplification, electrophoresis, in 84 pairs of primers, screen 59 pairs of grass carp monomorphism primers altogether, account for 70% of the primer

(6) screening of grass carp Auele Specific Primer

59 pairs of monomorphism primers that obtain with above-mentioned (5) carry out pcr amplification, electrophoresis in order to screen the specificity microsatellite locus of grass carp to grass carp and other 28 kinds of fish by above-mentioned condition respectively, result's demonstration: these primers are in these fish or amplify the fragment identical or close with the grass carp size, spawn can not increase, be chosen in the microsatellite locus that does not all have any amplified production in these 28 kinds of fish at last and use the site as the evaluation of grass carp, grass carp separates mutually with other fish the most at last.

In sum, the present invention has following characteristics:

The present invention filters out the 2 pairs of specificity microsatellite markers that can discern grass carp from 84 pairs of grass carp EST-SSR primers, banding pattern is clear, good reproducibility, good reliability, can be used as the characteristic primer of grass carp, in view of grass carp was difficult for discerning by morphological specificity in the seedling etap, by application of the present invention, can differentiate out quickly and accurately, thereby be applied in the evaluation of young postlarva, in the hope of analyzing Zhujiang River middle and lower reaches group structure and grass carp change in resources situation.

Table 1

2 Bambusa Elopichthys bambusa	16 Guangdong triangular bream Megalobrama hoffmanni
		3 meal bar Hmiculter leucisxulus	17 catfish Silurus asotus
4 wiredrawn edge fish Ptychidio jordani	18 crucian carp Carassius auratus
		5 silver medal silver xenocypris Xenocypris argentea	19 Si Minnow Pseudogobio vaillanti
6 carp Cyprinus carpio	20 spot mandarin fish Siniperca scherzer
		7 She Minnow Saurogobio dabryi	21 big mandarin fish Siniperca kneri
8 spot Of-digestive-tract Mystus guttatus	The few squama mandarin fish Coreoperca whiteheadi of 22 China
		9 leiocassis crassilabris Leiocassis crassilabris	23 yellow tail silver xenocypris
The yellow forehead Pseudobagrus of 10 Wa Shi vachelli	24 Squaliobarbus curriculus Squaliobarbus curriculus
		11 lipstick hemibarbus labeo	25 Megalobrama amblycephalas
12 dace Cirrhina moitorella	26 Hainan red Culter Erydtroculter reclurviceps
		13 yellow forehead Pelteobagrus fulvidraco	27 flathead Aristichthys nobili
14 bream Parabramis pekinensis	28 black carp Mylopharyngodon piceus
		15 silver medal Minnow Squalidus argentatus	29 silver carp Hypophthalmichthys molitrix

SEQUENCE?LISTING

＜110〉China's Pearl River Fishery Research Institute of Aquatic Science Research Institute

＜120〉a kind of method of utilizing micro-satellite primers to identify grass carp

<130>

<160> 4

<170> PatentIn?version?3.2

<210> 1

<211> 20

<212> DNA

<213> C3210?F

<400> 1

gtacccatac?gaaatgaacc 20

<210> 2

<211> 20

<212> DNA

<213> C3210?R

<400> 2

gactttgagc?aatgtccacc 20

<210> 3

<211> 18

<212> DNA

<213> C41926?F

<400> 3

gcaagcaatg?gctgaggt 18

<210> 4

<211> 21

<212> DNA

<213> C41926?R

<400> 4

gtaaccctga?aatcctgacc?c 21

Claims

1. a method of utilizing little satellite technology to identify grass carp is characterized in that described method comprises the steps:

(1) extracts fish DNA (comprising grass carp and other 28 kinds of fish fin ray genomic dnas), measure its OD value, store for future use in-20 ℃; The ESTs sequence of grass carp among the search NCBI dbEST, carry out searching of SSR with software SSRHutter1.3, carry out design of primers with Primer Premier5.0 then and it is optimized, in 29 kinds of fish DNA samples, screen, screen 2 pairs of specificity micro-satellite primers of identification grass carp; Verify in the grass carp dna sample of 96 different sourcess that with these primers little satellite band spectrum is stable;

(2) respectively grass carp and the dilution of being carried of other fish genomic dna is 40ng/ml;

(3) optimize the annealing temperature of primer, and with these primers the genomic dna of individuality in the grass carp different groups is carried out pcr amplification, the PCR reaction system is the primer of 20ul:0.5umol/L, the dNTP of 0.2 mmol/L, the Mg of 2 mmol/L ²⁺, 1 * PCR reaction buffer, the Taq archaeal dna polymerase of 1U; The PCR program parameter that is provided with when using these primers is: 94 ℃ of pre-sex change 3min, and 30 circulations: 94 ℃ of sex change 30s, Ta are annealing 30s down, and 72 ℃ are extended 45s, and last 72 ℃ are extended 7min;

(4) pcr amplification product is through 10% native polyacrylamide gel electrophoresis, 220V voltage stabilizing electrophoresis 3 hours, then with 0.1% AgNO3 dyeing 1min, silver dyes the back with 2% NaOH, 0.2% anhydrous Na2CO3, the 0.4% formaldehyde 2min that develops the color, till band is clear, and the preservation of taking pictures;

(5) in 84 pairs of micro-satellite primers, screen 59 pairs of grass carp monomorphism primers, account for 70% of the primer;

(6) respectively grass carp and other 28 kinds of fish are carried out pcr amplification, electrophoresis by above-mentioned condition with above-mentioned monomorphism primer, filter out in these fish without any the primer of amplified production, grass carp separates mutually with other fish the most at last;

Described 2 pairs of specific primer sequences are shown in SEQ ID NO:1 ~ 4.