CN102242222B - Method for classifying ctenopharyngodon idella based on expressed sequence tag-simple sequence repeats (EST-SSR) marker - Google Patents
Method for classifying ctenopharyngodon idella based on expressed sequence tag-simple sequence repeats (EST-SSR) marker Download PDFInfo
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Abstract
The invention discloses a method for classifying ctenopharyngodon idella based on an expressed sequence tag-simple sequence repeats (EST-SSR) marker, which comprises the following steps of: extracting the deoxyribonucleic acid (DNA) of the ctenopharyngodon idella, amplifying by using the EST-SSR marker, and analyzing the genetic structure of the ctenopharyngodon idella according to an amplification result. Compared with the conventional method, the method has the advantages of high purposiveness and direct action effect, and is easy to operate, high in detection speed, low in detection cost, and convenient to widely popularize and use.
Description
Technical field
The present invention relates to the sorting technique of a kind of fish, particularly a kind of grass carp sorting technique based on the EST-SSR mark.
Background technology
Grass carp
(Ctenopharyngodon idella)Having the advantages such as growth is fast, aquaculture cost is low, be the main cultivation object of pond, lake and reservoir, is one of most important breed variety in China's fresh water fishery.Grass carp is distributed in each great river River system in Asia, in China from Heilungkiang to the Xijiang River (except Xinjiang and the Qinghai-Tibet Platean without NATURAL DISTRIBUTION) have it to distribute, formed the genetic background of grass carp complexity and the germ plasm resource of enriching.In recent years, natural water environmental evolution and pollution make nature Grass Carp Population quantity obviously descend, and in production, endogamy causes germplasm to be degenerated, and has had influence on the normal development of grass carp aquaculture industry.For effectively protecting and utilize this freshwater fish resource, be necessary the development and utilization molecular genetic marker, the grass carp colony of different water systems is carried out the genetic construction analysis, get its genetic background clear, for the selection of grass carp seed selection population lays the foundation.At present, existing scholar adopts the detection techniques such as isozyme, mtDNA-RFLP, little satellite that the genetic construction of Grass Carp in Yangtze River System population is studied, but also few to the research work of grass carp population genetic structure between different water systems.
Expressed sequence tag (expressed sequence tag, EST) there are little satellite or the simple repeated sequence (EST-SSR) of some amount in, it not only has in genome the advantages such as evenly distribution, codominance and experimental repeatability be good, and cost of development is lower, has been successfully applied to the research fields such as population genetic structural analysis, QTL location and molecular marker assisted selection breeding.
Summary of the invention
The purpose of this invention is to provide a kind of grass carp sorting technique based on the EST-SSR mark.
The technical solution used in the present invention is:
The grass carp sorting technique of EST-SSR mark comprises the DNA that extracts grass carp, uses the EST-SSR mark to increase as primer; According to amplification, grass carp is classified, described EST-SSR mark is to being:
The reaction system of PCR is:
ddH
2O 14.9μL
10×PCR Buffer 2.0μL
MgCl
2（25 mmol/L） 0.8μL
4×dNTP（10mmol/L） 0.3μL
TaqEnzyme (5U/ μ L) 0.2 μ L
Upstream and downstream primer (10 μ mol/L) 0.4 μ L
Template DNA (40ng/ μ L) 1.0 μ L.
The reaction conditions of PCR is:
1) 94 ℃ of denaturation 4min,
2) 94 ℃ of sex change 30s,
3) annealing 30s,
4) 72 ℃ are extended 45s;
5) circulation step 2)～4) 30～40 times,
6) 72 ℃ are extended 7min.
The invention has the beneficial effects as follows:
Method of the present invention can identify genetic construction and the genetic background of different sources grass carp accurately and rapidly, for screening with set up grass carp moral education basic population reference is provided.
Description of drawings
Fig. 1 builds the UPGMA dendrogram of 5 colonies according to the genetic distance matrix between colony.
Embodiment
The applicant built the tissue cDNA such as grass carp brain, muscle, liver library in 2009, obtain 45 318 est sequences after going redundancy to process, average sequence length is 650bp, with trf(tandem repeats finder) software finds 5 556 little satellites from this sequence.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 Primer5.0 to carry out design of primers, obtain 118 pairs of EST-SSR primers.
The applicant utilizes 118 pairs of EST-SSR primer pair 50 tail grass carp samples of acquisition to carry out pcr amplification, wherein the 87 pairs of primer amplifications go out single band, 12 pairs of primers can amplify polymorphic bands without amplified production, 19 pairs of primers, and polymorphism primer accounts for 16.00% of all design primers.Wherein the double alkali yl tumor-necrosis factor glycoproteins is 15, accounts for 83.16%; 4 of four base repetitive sequences account for 16.84%.And double alkali yl repeat in take (AC) n/(GT) n is as main, accounts for 73.33%; (AT) n/(AT) n accounts for 13.33%, (CT) n/(AG) n accounts for 13.33%.
The sequence of 19 pairs of EST-SSR primers is as follows:
Concrete detection method is as follows:
The extraction of template DNA
1) after the isozyme 3mg that gets fish to be detected shreds, add lysate (the 10 mmol/L Tris-HCl of 0.5mL; 0.1 mol/L EDTA; 0.5% SDS; 30mg/L RNase; The 100mg/L Proteinase K, pH8.0), 55 ℃ digested 1 hour, and frequently shook gently therebetween;
2) add isopyknic phenol/chloroform/primary isoamyl alcohol (25:24:1), put upside down mixing, under room temperature after standing 5 minutes, 12000 rev/mins centrifugal 10 minutes, get supernatant liquor, then use the chloroform extracting once, under room temperature after standing 5 minutes, 12000 rev/mins centrifugal 10 minutes, get supernatant liquor;
3) add the dehydrated alcohol of 2 times of volumes, standing 10 minutes precipitation DNA of room temperature, 12000 rev/mins are centrifugal 10 minutes;
4) with 70% washing with alcohol 1 time, 12000 rev/mins centrifugal 2 minutes, suck supernatant, room temperature standing and drying 10 minutes adds 50 μ l TE(10mmol/L Tris-HCl; 1mmol/L EDTA, pH8.0) dissolving DNA, 4 ℃ of storages are standby.
The PCR reaction system:
ddH
2O 14.9μL
10×PCR Buffer 2.0μL
MgCl
2（25 mmol/L） 0.8μL
4×dNTP（10mmol/L） 0.3μL
TaqEnzyme (5U/ μ L) 0.2 μ L
Upstream and downstream primer (10 μ mol/L) 0.4 μ L
Template DNA (40ng/ μ L) 1.0 μ L.
The reaction conditions of PCR:
1) 94 ℃ of denaturation 4min,
2) 94 ℃ of sex change 30s,
3) annealing 30s,
4) 72 ℃ are extended 45s;
5) circulation step 2)～4) 32 times,
6) 72 ℃ are extended 7min.
Native polyacrylamide gel electrophoresis detects
(1) 8% non-denaturing polyacrylamide gel preparation (35ml)
Distilled water H
2O 22.259ml
30 ﹪ polyacrylamide stoste 9.33ml
10×TBE 3.27ml
20﹪AP 0.117 ml
TEMED 0.024ml。
Preparation steps:
1. the cleaning of sheet glass: water be stained with lotion the sheet glass scrub after, then rinse with ultrapure water, dry standby;
2. recording of gel: first the glass frame that fixes is tilted, gel is poured in the groove of sheet glass lentamente along comb slot edge, then plug immediately comb, allow gel overflow, can avoid producing like this bubble.Add a small amount of single water that steams at glue and comb contact surface, in order to avoid the atmospheric oxidation glue is surperficial.When waiting for the glue cohesion, examine to have or not and leak glue or Bubble formation.Generally 4-6min namely begins cohesion after encapsulating, and it is complete that 25 ℃ of left and right 20min of room temperature just can polymerization.When as low in room temperature, the corresponding prolongation of polymerization time.
(2) loading and gel electrophoresis
Amplified production and sample-loading buffer after the 3:1 mixing, are drawn 4 μ L mixture loadings with the capillary sample inlet device by volume, and electrophoresis on 8% non-denaturing polyacrylamide gel, electrophoretic buffer are 0.5 * TBE, voltage 230V, electrophoresis 2-3h.
Cma staining
(1) electrophoresis is complete takes out glue, is put in to fill in single plastic containers (Area Ratio glue is slightly large) that steam water rinsing 10s gently, removes distilled water;
(2) add 1% AgNO
3Solution, jog 3～4 min, outwell AgNO on decolorization swinging table
3Solution; With distilled water rinsing twice, be no more than 10s at every turn, outwell distilled water;
(3) add fast nitrite ion (NaOH 10g, Na
2CO
30.2g, HCHO 2ml, adding distil water is settled to 500ml) 50ml, go after vibration 30s;
(4) add the 500mL nitrite ion.In general 3min, banding pattern manifests;
(5) when banding pattern is clear, the glue taking-up is put in gel becomes phase system to take pictures, and carry out strip analysis.
Data statistics and analysis
(1) with POPGEN32 computed in software effective number of allele (effective numbers of allele,
Ne), the observation heterozygosity (observed heterozygosity,
Ho), the expectation heterozygosity (expected heterozygosity,
He), the Relative Hereditary distance (genetic distance,
D), genetic similarity (genetic similarity index,
S), the genetic variation and genetic differentiation index (genetic differentiation index,
Fst) and Hardy-Weinberg genetic divergence equilibrium index (
d).
In formula
P i Be
iIndividual allelic frequency,
nBe the allelotrope number.
The average observed heterozygosity (
Ho):
Ho=individual the sum of heterozygosis subnumber/observation observed;
In formula
P i Be
iIndividual allelic frequency.
Genetic similarity (
S):
Sij=
Nij/ [
Ni+
Nj+
Nij]
In formula
SijBe the genetic similarity between any two individualities,
NijFor
iIndividual and
jThe individual number of sites of sharing,
N i ,
N j Be respectively
iIndividual and
jThe site sum of individual amplification separately.
In formula
S ij Be the genetic similarity between two populations,
S i ,
S j Represent respectively population
iAnd population
jGenetic similarity.
This genetic variation and genetic differentiation index of group (
Fst): 1
-Fst=(1
-Fis) * (1
-Fit)
In formula
Fis,
Fst,
FitRepresent respectively coefficient of differentiation and total group inbreeding coefficient between inbreeding coefficient in colony, colony.
Hardy-Weinberg genetic divergence equilibrium index (
d):
D=Ho-He/He
(2) polymorphism information content (Polymorphic information content,
PIC),
In formula
P i , P j Be respectively
iWith
jIndividual allelic frequency,
nBe the allelotrope number.
(3) use MEGA 4 softwares according to the genetic similarity exponential sum genetic distance between colony, adopt non-weighting pairing arithmetic mean method (unweithted pair group method using arithmetic, UPGMA) build the genealogical tree of 5 grass carp colonies, to analyze the sibship between colony.
The applicant has carried out sample collecting in May, 2010-July to 5 different sources grass carp colonies, Jianli colony, Shishou colony and Changsha colony are respectively the wild-type grass carp sprays of river, Jianli, the Changjiang river section, the Changjiang river Shishou river section and Xiang River river, the Changjiang river section fishery harvesting, grow to sexual maturity; Qingyuan City colony and Zhaoqing colony be from the grass carp of Pearl River system Qingyuan City river section and river, Zhaoqing section, belonged to for four generations to breed the offspring, grows to sexual maturity, totally 181 tails.Sampling position and quantity see Table 1.Each sample clip fin ray is stored in 95% ethanol standby.
Use 19 pairs of micro-satellite primers to 5 grass carp colonies totally 181 tail grass carp samples carry out polymorphic detection, obtain altogether 93 allelotrope, the number of alleles that the every pair of primer detects is 2～8,4.89 of average out to, and the amplified production clip size is 87～339 bp; Its polymorphic information sees Table 2.
The applicant utilizes data statistics and analysis software to carry out genetic diversity and Hardy-Weinberg's equilibrium analysis to 181 tail grass carp samples, the average number of alleles that the statistic analysis result of population genetic diversity sees Table 3,5 grass carp colonies be (
Na) 3.526 3～4.315 8, the average effective number of alleles be (
Ne) 1.929 3～2.139 0, the average observed heterozygosity be (
Ho) 0.415 8～0.501 3, the average expectation heterozygosity be (
He) 0.450 6～0.502 8, average polymorphism information content be (
PIC) 0.4154～0.4604; The average polymorphism information content of Changsha colony and average expectation heterozygosity are up to 0.460 4 and 0.502 8; The average observation heterozygosity of Jianli colony and average expectation heterozygosity are minimum is 0.415 8 and 0.450 6.Genotypic by calculating
PValue check, departing from various degree all occured in 5 the grass carp Hardy-Weinberg of colony balance deviation constants, the results are shown in Table 4, and in Shishou colony, 16165 sites occur significantly to depart from, and 4703,17329 and 30977 sites occur extremely significantly to depart from; In the colony of Changsha, 23426 sites occur significantly to depart from, and 23460,5476,16165,4218,784,4996,17329 and 16158 sites occur extremely significantly to depart from; In the colony of Jianli, 23426 and 17329 sites occur significantly to depart from, and 4218 sites occur extremely significantly to depart from; In the colony of Zhaoqing, 4703,5476 and 23426 sites occur significantly to depart from, and 23460 and 13118 sites occur extremely significantly to depart from; In Qingyuan City colony, 23426 and 17329 sites occur significantly to depart from, and 4218 sites occur extremely significantly to depart from.
The applicant carries out genetic similarity and Genetic Distance Analysis between different grass carp colony, according to Nei(1978) calculate genetic distance and genetic similarity between colony, result shows: Jianli colony and Qingyuan City's population genetic distance be (0.0862) farthest, genetic similarity minimum (0.9174) illustrates that both sibships are far away; And Changsha colony and Shishou population genetic nearest (0.019 8), genetic similarity maximum (0.9804) illustrates that both sibships are nearest.Build the UPGMA dendrogram (Fig. 1) of 5 colonies according to the genetic distance matrix between colony.Can find out, 5 grass carp colonies have gathered into two, and Changsha colony, Jianli colony gather into one with Shishou colony; Zhaoqing colony gathers into one with Qingyuan City colony.
Annotate: diagonal lines is above is genetic similarity, and diagonal lines is following is genetic distance
19 of screening of utilization have and enrich polymorphic EST--SSR mark, grass carp DNA sample to different groups carries out the PCR detection, amplified production detects with 8% non-denaturing polyacrylamide, utilize biotechnology and data analysis software to carry out correlation analysis, the grass carp colony that utilizes that present method can be different with source in producing carries out its genetic construction and genetic cluster analysis, for the selected and grass carp fine-variety breeding of grass carp seed selection basic population lays the foundation.The method is compared with traditional method, has purpose strong, the direct advantage of action effect.And simple to operate, detect fast, testing cost is low, be convenient to extensively promote the use of.
＜110〉China's Pearl River Fishery Research Institute of Aquatic Science Research Institute
＜120〉based on the grass carp sorting technique of EST-SSR mark
<130>
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<170> PatentIn version 3.5
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tctaatactg tgcctgttct a 21
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cacttactta ctgcttgatt gt 22
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Claims (3)
1. based on the grass carp sorting technique of EST-SSR mark, comprise the DNA that extracts grass carp, use the EST-SSR mark to carrying out pcr amplification as primer; According to amplification, grass carp is classified, it is characterized in that: described EST-SSR mark is to being:
F:AAGCCCAAAGCCACAGAA SEQ ID NO：1
1 R:GCAGTAGAAGAGTGAAAAGCATC SEQ ID NO：2
F:GCTGCTGTCACTCCTAAACTC SEQ ID NO：3
2 R:TGAAAGGTCCAAAGAAAGAAC SEQ ID NO：4
3 F:AAGTGAGACTATGCTGATAAAACCG SEQ ID NO：5
R:ATTGAAACAGATGCCTGCTTG SEQ ID NO：6
F:CTGTGATGGGTAGATTTAGGG SEQ ID NO：7
4 R:GCCTGGTGAGTTGGTATGG SEQ ID NO：8
5 F：GCTAACACTCCTTTACTATCAT SEQ ID NO：9
R:TTGAAACGCTGATTGAGA SEQ ID NO：10
6 F:GGTCTCGTCTTCGCCATCCT SEQ ID NO：11
R:CCTGCGGTTTGCGTGGTAT SEQ ID NO：12
7 F:GAGCGACTCCAGTGAGATAA SEQ ID NO：13
R:CCAGGATGAACGAACAAATA SEQ ID NO：14
8 F:TCAGCCTTATGCCTTGGT SEQ ID NO：15
R:GCCACTGAACTGGTAACG SEQ ID NO：16
9 F:TCTAATACTGTGCCTGTTCTA SEQ ID NO：17
R:CACTTACTTACTGCTTGATTGT SEQ ID NO：18
10 F:TGTCGCTCAGTCATTCTCC SEQ ID NO：19
R:GCAAGTATTGTCAACCAAGTGT SEQ ID NO：20
F:TAAGTAACGCTCCTTGAAAA SEQ ID NO：21
11 R:GAGTGTCTGTTTGCCTGTG SEQ ID NO：22
F:AAGGAACAGCATAAACCGAAAT SEQ ID NO：23
12 R:GGAACCAAGCATCTGAAACTG SEQ ID NO：24
F:AGATAAACTGGGGCGGC SEQ ID NO：25
13 R:TGGAGACAGGTCGTCAACAC SEQ ID NO：26
F:CAGAGTTCGGAGACAAAGAG SEQ ID NO：27
14 R:GAATGGTGTTTCTATGTAAGTGT SEQ ID NO：28
15 F:TAAGGAAGTGATAGGGACATT SEQ ID NO：29
R:TAGTTGCTCGTTGTGATGTG SEQ ID NO：30
16 F:TGATTGCCAAGATTGTTTCG SEQ ID NO：31
R:ATACAGGGAGGTTATTACGACA SEQ ID NO：32
17 F:ATTTCAGTAGTAACCCATCAC SEQ ID NO：33
R:ACAGGACCAATAAGGAACA SEQ ID NO：34
18 F:GCAGGACTGAGACTGAAGGA SEQ ID NO：35
R:GAGGTCTTTGCGGTTGTAT SEQ ID NO：36
19 F:AAAATCCCAGTGAGACAATC SEQ ID NO：37
R:ATCCCATAATGCCTTGC SEQ ID NO：38
2. the grass carp sorting technique based on the EST-SSR mark according to claim 1, it is characterized in that: the reaction system of amplification is:
3. the grass carp sorting technique based on the EST-SSR mark according to claim 1 and 2, it is characterized in that: the reaction conditions of amplification is:
1) 94 ℃ of denaturation 4min,
2) 94 ℃ of sex change 30s,
3) annealing 30s,
4) 72 ℃ are extended 45s;
5) circulation step 2)～4) 30～40 times,
6) 72 ℃ are extended 7min.
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| CN103667272B (en) * | 2013-12-04 | 2015-08-12 | 天津市水生动物疫病预防控制中心 | The EST-SSR molecule marker relevant to mitten crab growth traits and application |
| CN103993081B (en) * | 2014-05-16 | 2015-11-04 | 中国水产科学研究院珠江水产研究所 | A kind of method differentiating gynogenetic grass carp and common grass carp |
| CN109468391B (en) * | 2018-12-29 | 2021-05-25 | 中国水产科学研究院长江水产研究所 | Double PCR method applied to black carp genetic relationship identification |
| CN109797226A (en) * | 2019-02-26 | 2019-05-24 | 中国水产科学研究院珠江水产研究所 | A kind of Macrobrachium rosenbergii classification method based on EST-SSR label |
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