CN102758022B - Internal transcribed spacer (ITS2) molecular marker identifying method for aquaculture of catfish hybrids - Google Patents

Internal transcribed spacer (ITS2) molecular marker identifying method for aquaculture of catfish hybrids Download PDF

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Publication number
CN102758022B
CN102758022B CN 201210278377 CN201210278377A CN102758022B CN 102758022 B CN102758022 B CN 102758022B CN 201210278377 CN201210278377 CN 201210278377 CN 201210278377 A CN201210278377 A CN 201210278377A CN 102758022 B CN102758022 B CN 102758022B
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catfish
its2
silurus meridionalis
gene fragment
meridionalis chen
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CN102758022A (en
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刘匆
郭睿
宋昭彬
盛晓洒
应汝华
陈齐勇
孙家贤
龚丽
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Tongwei Co Ltd
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Tongwei Co Ltd
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Abstract

The invention relates to an ITS2 molecular marker identifying method for aquaculture of catfish hybrids. Southern catfishes, catfishes and hybrids of the southern catfishes and the catfishes serve as objects of study, ITS2 gene sections in catfishes are selected to serve as molecular markers, the length difference the IST2 gene sections of the southern catfishes and the catfishes, and the southern catfishes, the catfishes and hybrids of the southern catfishes and the catfishes are identified successfully. The method has the advantages of being fast in experiment, convenient to operate, visual in identification and free from sequencing.

Description

The ITS2 molecular marker identification method of aquaculture catfish hybrid
Technical field
The present invention relates to the molecular marker identification method of fish hybrid, be specifically related to the ITS2 molecular marker identification method of aquaculture catfish hybrid.
Background technology
The SILURIFORMES fish belong to carnivorous demersal fish, are one of Teleosteis the most flourishing during fish are evolved.Silurus meridionalis Chen (crying again large mouthful catfish) ( Silurus meridionalisChen) and catfish ( Silurus asotusLinnaeus) all belonging to SILURIFORMES, catfish section, catfish and belong to, both delicious flavours, be of high nutritive value, is the high added value economic fish that China various places mainly cultivate.Silurus meridionalis Chen is distributed in the water systems such as China's Zhujiang River, the Min River, Xiang River, the Changjiang river, is a kind of predacious fish of large-scale ferociousness, has the characteristics such as growth is fast, individual greatly, disease resistance is strong, happiness inhabits opens wide water body, and gulf a small bay in a river, gravel section, river bed, fish brood amount is large, and the spawning time is long.Catfish is distributed in each water system of the whole nation except Qinghai-Tibet Platean and Xinjiang, and body colour is different and change to some extent with habitat, mainly inhabits the middle lower floor of rivers master stream or larger water bodys such as tributary, large-size lake and reservoir, its strong adaptability, inhabit hydrostatic or unhurried current waters, individuality is less, and growth is fast.Silurus meridionalis Chen and catfish body profile similarity, the Silurus meridionalis Chen of little individuality and catfish adult confuse lives together, and large individual the isolation with catfish lives.It is reported that male ratio is very low in the Silurus meridionalis Chen colony of artificial propagation.Investigation shows, when male Silurus meridionalis Chen lacks, may carry out the hybridization that female Silurus meridionalis Chen and other male catfish belong to kind, thereby the cross-breeding between species occurred in catfish produces.
In order to satisfy the needs of social production, obtain to have concurrently the seed of above 2 kinds of fish good characters, the bright grade of imperial court (cross experiment of large mouthful catfish and catfish, fresh water fishery, 2004,34(6): 41 ~ 43; The RAPD of three kinds of catfish genetic diversities analyzes, fresh water fishery, and 2005,35(4): 14 ~ 17) carried out the cross-breeding test of Silurus meridionalis Chen and catfish, and done certain hybrid identification work, filtered out 22 random primers and be used for Silurus meridionalis Chen, catfish and hybridization F thereof 1Generation three population diversity analyses found that hybridization F 1In generation, inherited 52 characteristic strips of large mouthful catfish and 32 characteristic strips of catfish.Yet the RAPD mark that its evaluation work is adopted is subjected to the restriction of method itself, and detection efficiency is low and repeated not high, has had a strong impact on promoting the use of of this method.In addition, only have Longhua etc. (catfish, large mouthful catfish and hybridization catfish blood physiology biochemical indicator thereof relatively reach the Transferrins,iron complexes alleles analysis, Changjiang University's journal (from section's version), 2006,3(2): 161 ~ 164; The blood type analysis of catfish, large mouthful of catfish and hybridization catfish thereof, water conservancy related fisheries, 2007,27(3): 19 ~ 20) to the research of the aspects such as blood physiology, blood type analysis and isozyme of Silurus meridionalis Chen, catfish and hybridization catfish thereof.As seen, be necessary to carry out molecule marker and the authenticate technology research of Silurus meridionalis Chen, catfish and hybrid generation thereof, for the hybrid identification in Silurus meridionalis Chen cultivation and the breeding practice provides reliable method.
Summary of the invention
Purpose of the present invention is for identifying the hybrid of Silurus meridionalis Chen and catfish, the hybrid and purebred resolution that form are not easily distinguishable on the molecular biology aspect.The present invention with Silurus meridionalis Chen, catfish and hybrid generation thereof as research object, catfish belong in the fish screening design ITS2 gene fragment as molecule marker, use expand the difference in length of ITS2 gene fragment in Silurus meridionalis Chen and catfish, successfully identified Silurus meridionalis Chen, catfish and hybrid generation thereof.
The ITS gene fragment is the part of ribosome-RNA(rRNA) transcribed spacer, has in the species to guard the large characteristics of variation between species.Adopt the method to be used for the evaluation of Silurus meridionalis Chen, catfish and hybrid generation thereof, have experiment fast, easy and simple to handle, evaluation is directly perceived, the advantage that need not to check order.
For achieving the above object, the present invention adopts following technical scheme:
The ITS2 molecular marker identification method of aquaculture catfish hybrid, it is characterized in that: there are difference in length in the ITS2 gene fragment of the Silurus meridionalis Chen behind the application pcr amplification and the ITS2 gene fragment of catfish, adopt agarose gel electrophoresis to identify Silurus meridionalis Chen, catfish and hybrid generation thereof.
The nucleotide sequence of the ITS2 gene fragment of described Silurus meridionalis Chen (SEQID NO:1) is:
CTCGTGCGTC GATGAAGAAC GCAGCCAGCT GCGAGAACTA ATGTGAATTG CAGGACACAT TGATCATCGA CACTTCGAAC GCACATTGCG GCCCCGGGTC CCTCCCGGGG CTACGCCTGT CTGAGGGTCG CTATTTCCAT CGATCGGGAA AAAAACGAAC CATACGCACG CGACTGGAAG CTCGCAGGCC GTCATCGGCC TTCGTCCTCC TAAAAGCAGA CGCTCTCTTT TTTCGTGTCG;
The nucleotide sequence of the ITS2 gene fragment of described catfish (SEQID NO:2) is:
CTCGTGCGTC GATGAAGAAC GCAGCCAGCT GCGAGAACTA ATGTGAATTG CAGGACACAT TGATCATCGA CACTTCGAAC GCACATTGCG GCCCCGGGTC CCTCCCGGGG CTACGCCTGT CTGAGGGTCG CTATTTCCAT CGATCGGGAA AAAAACGAAC GCGACTGGAA GCTCGCAGGC CGTCATCGGC CTTCGTCCTC CTAAAAGCAG ACGCTCTCTT TTTTCGTGTC G。
The ITS2 molecular marker identification method of described aquaculture catfish hybrid may further comprise the steps:
A gets respectively Silurus meridionalis Chen, catfish and hybridization sample thereof, extracts genomic dna;
B take total DNA of Silurus meridionalis Chen, catfish and hybridization sample thereof as template, carries out pcr amplification respectively under the guiding of ITS2 gene fragment specific primer;
The C agarose gel electrophoresis detects pcr amplification product, through electrophoresis detection, two gene bands of Silurus meridionalis Chen and catfish occur simultaneously on a swimming lane, then is the cross-fertilize seed of Silurus meridionalis Chen and catfish.
The base sequence of described ITS2 gene fragment specific primer is:
Forward primer (SEQID NO:3) 5 '-AACTCTTAGCGGTGGATCACTCGG-3 ';
Reverse primer (SEQID NO:4) 5 '-CGTGGAAAGGCGGCGGAGAT-3 '.
Beneficial effect of the present invention is: the present invention is owing to adopt dna sequence dna as molecule marker, therefore can be quickly and accurately with the catfish hybrid that is not easily distinguishable on the form with catfish is purebred differentiates, and identify the cross-fertilize seed of Silurus meridionalis Chen and catfish.Adopt the difference in length of ITS2 gene fragment in Silurus meridionalis Chen and catfish to identify the catfish hybrid, have experiment fast, easy and simple to handle, identify intuitively the advantage that need not to check order.
Description of drawings
Fig. 1 is that the length difference opposite sex of ITS2 gene detects electrophorogram.
Be labeled as among the figure: the molecular weight of right side numeral dna fragmentation, D1~D4 are the Silurus meridionalis Chen sample, and A1~A4 is the catfish sample; Z1~Z4 is the quadrature filial generation, and F1~F4 is the reciprocal cross filial generation, and M is Marker, and the present invention is used to be DL2000 DNA Marker (Tiangen company).
Embodiment
Below in conjunction with embodiment essentiality content of the present invention is described in further detail.
The ITS2 molecular marker identification method of aquaculture catfish hybrid is with Silurus meridionalis Chen, catfish and hybridization F thereof 1In generation, may further comprise the steps as research object:
The first step is got respectively Silurus meridionalis Chen, catfish and hybridization sample thereof, extracts genomic dna.
Because hybridization fry individuality is very little, extract the fry sample genomic dna so extract test kit (Tiangen company) with the ocean tissue DNA.
Extract the genomic dna of parent's Silurus meridionalis Chen and catfish fin ray sample with traditional phenol-chloroform extraction process:
A draws materials: get soaked in absolute ethyl alcohol the fin ray sample a little, the scissors that disinfects in alcohol shreds, and puts into 1.5 ml EP pipes.
B digestion: add 500 μ L lysis buffer (10 mmol/L Tris-Cl; 0.1 mol/L EDTA; 0.5 % SDS; PH 8.0), 5 μ L Proteinase Ks (20 mg/ml) mix, and put into 55 ℃ shaking table, and 60-70 rpm digestion is spent the night.
C extracting 1: take out the EP pipe, treat that solution is cooled to room temperature, add the saturated phenol of isopyknic Tris (about 500 μ L), slow mixing 10 min, centrifugal 10 min of 12000 rpm slowly move to supernatant liquor in another EP pipe with 1 ml rifle head.
D extracting 2: repeat the step of extracting 1, with saturated phenol again extracting once supernatant liquor is changed in another EP pipe.
E extracting 3: add the phenol-chloroform (1:1) of 400 μ L, slow mixing 5 min, centrifugal 10 min of 12000 rpm change supernatant liquor in another EP pipe gently.
F extracting 4: add the chloroform of 300 μ L, slow mixing 5 min, centrifugal 10 min of 12000 rpm, supernatant liquor is transferred in another EP pipe.
G precipitation: add the freezing dehydrated alcohol of pipe internal volume two volumes, in-20 ℃ of precipitation 20 min.Centrifugal 1 min of 8000 rpm.
H rinsing 1: add the freezing alcohol of 150 μ L, 75 %, after the rinsing, centrifugal 1 min of 8000 rpm.
I rinsing 2: with rinsing 1 step.
J rinsing 3: with the freezing dehydrated alcohol post rinse of 150 μ L once, behind centrifugal 1 min of 8000 rpm, discard supernatant, natural air drying.
K dissolving: add 50 μ L MilliQ ultrapure waters, the DNA of dissolution precipitation.
Second step filters out the ITS2 gene segment with difference in length.
(1) with reference to catfish 18S ribosomal rna gene sequence GQ465245 among the GenBank, use primer-design software Primer5.0 design primer I TS2-F(SEQID NO:3): 5 '-AACTCTTAGCGGTGGATCACTCGG-3 ' and TR(SEQID NO:5): 5 '-TAAATTCAGCGGGTCGTCTCGTC-3 ', and synthetic by Invitrogen company.
ITS2-F and TR are the primer of amplification ITS2 purpose fragment pair, and F wherein represent forward primer (or upstream primer), and TR during sequence length difference, represents reverse primer (or downstream primer) between the screening kind.
(2) pcr amplification
Each component is mixed (reaction system is 50 μ L) in the following order in the PCR of 0.2 mL pipe:
1. ultrapure water 36.6 μ L
2. Taq enzyme 10 * buffer 5 μ L
③MgCl 2(1.5 mM) 4 μL
④dNTPs(2.5 mM) 1 μL
5. primer I TS2-F(25 pM) 1 μ L
6. primer TR(25 pM) 1 μ L
7. template DNA (about 100 ng) 1 μ L
8. Taq archaeal dna polymerase (2.5 U μ L -1) 0.4 μ L
Amplification step is:
1. 94 ° of C denaturation 4 min
2. 94 ° of C sex change 30 s
3. 59 ° of C, 35 s that anneal
4. 72 ° of C extend 40 s
5. repeating step is 2.-4. 31 times
6. 72 ° of C fully extend 7 min
(3) the PCR product reclaims
Agarose gel electrophoresis with 1 % separates PCR product fragment, and puts into the EP pipe of 1.5 mL under ultraviolet lamp behind the cutting purpose band, reclaims and adopts DNA Agarose Extraction kit (Omega company) to reclaim test kit.The recovery product that electrophoresis detection is qualified send biotech firm's order-checking.
(4) data analysis and optimized expansion fragment
The sequence information that order-checking company is returned, carry out online BLAST(http after the artificial check and correction: //www.ncbi.nlm.nih.gov/BLAST) checking, the ITS2 sequence information of verifying errorless rear use 6.0 pairs of Silurus meridionalis Chens of DNAMAN Version and catfish is compared and is aided with artificial adjustment, choose conserved sequence SEQID NO:1 and SEQID NO:2 with Stable Length difference, use reverse primer ITS2-R(SEQID NO:4 in the middle of primer-design software Primer 5.0 designs) (5 '-CGTGGAAAGGCGGCGGAGAT-3 ') highlighting the segment of difference in length, and in parent and hybrid generation colony, increase.
ITS2-F and ITS2-R are the primer of amplification ITS2 purpose fragment pair, and F wherein represents forward primer (or upstream primer), and R represents reverse primer (or downstream primer).
In the 3rd step, take total DNA of Silurus meridionalis Chen, catfish and hybridization sample thereof as template, under the guiding of ITS2 molecule marker primer special, carry out pcr amplification respectively.
Use primer to ITS2-F(SEQID NO:3) (5 '-AACTCTTAGCGGTGGATCACTCGG-3 ') and ITS2-R(SEQID NO:4) (5 '-CGTGGAAAGGCGGCGGAGAT-3 '), pcr amplification ITS2 stable gene fragment in parent and hybrid generation colony.Each component is mixed (reaction system is 50 μ L) in the following order in the PCR of 0.2 ml pipe:
1. ultrapure water 35.6 μ L
2. Taq enzyme 10 * buffer 5 μ L
③MgCl 2(1.5 mM) 5 μL
④dNTPs(2.5 mM) 1 μL
5. primer I TS2-F(25 pM) 1 μ L
6. primer I TS2-R(25 pM) 1 μ L
7. template DNA (about 100 ng) 1 μ L
8. Taq archaeal dna polymerase (2.5 U μ L -1) 0.4 μ L
Amplification step is:
1. 94 ° of C denaturation 5 min
2. 94 ° of C sex change 30 s
3. 59 ° of C, 35 s that anneal
4. 72 ° of C extend 30 s
5. repeating step is 2.-4. 34 times
6. 72 ° of C fully extend 7 min
In the 4th step, agarose gel electrophoresis detects pcr amplification product.
The sepharose for preparing 2 %, and with loading electrophoresis behind 5 μ L PCR products adding point sample damping fluid and the mixing.Constant voltage 100 V electrophoresis 40 min use the UV gel imaging system to manifest the purpose band, difference in length analysis and hybrid identification between planting according to running gel figure behind the electrophoresis.
Agar gel preparation method of the present invention is as follows:
The preparation method of the sepharose of 1 %
Weigh in the balance and get 0.3 g agarose, pour in 1 * TAE damping fluid of 30 ml, shake up to be placed on to be heated in the microwave oven fully and dissolve, take out until slightly cool rear (preventing that high temperature from making the EB volatilization), adding EB 2 μ L.Glue plate (Bio-Rad company) is positioned over level attitude, then inserts the sample comb.Pour the sepharose of before preparation into glue plate inside groove.Half an hour is solidified rear taking-up comb until gel in cooling, gel is put into electrophoresis chamber (Bio-Rad company) and added 1 * TAE electrophoretic buffer make its submergence gel.The point sample damping fluid (tetrabromophenol sulfonphthalein) of 1/6 volume on point template is selected.
The preparation method of the sepharose of 2 %
Weigh in the balance and get 0.6 g agarose, pour in 1 * TAE damping fluid of 30 ml, shake up to be placed on to be heated in the microwave oven fully and dissolve, take out until slightly cool rear (preventing that high temperature from making the EB volatilization), adding EB 2 μ L.Glue plate (Bio-Rad company) is positioned over level attitude, then inserts the sample comb.Pour the sepharose of before preparation into glue plate inside groove.Half an hour is solidified rear taking-up comb until gel in cooling, gel is put into electrophoresis chamber (Bio-Rad company) and added 1 * TAE electrophoretic buffer make its submergence gel.The point sample damping fluid (tetrabromophenol sulfonphthalein) of 1/6 volume on point template is selected.
The key instrument that the present invention uses is as follows:
PCR instrument: Bio-Rad iCycler and MJ100
Whizzer: Eppendorf Centrifuge 5415D
Voltage stabilization and current stabilization electrophoresis apparatus: BIO-RAD power PAC 300
Gel imaging system: Alphalmage Multimage Light Cabinet
Electronic balance, incubator, steam sterilizer, shaking table and the experimental installation that some are commonly used.
Above-described embodiment result shows: the ITS2 gene fragment of Silurus meridionalis Chen has no report in GenBank, shown in the gene histogram of Fig. 1, two gene bands of Silurus meridionalis Chen and catfish appear in hybrid generation catfish simultaneously, and are consistent with 2 kinds of parental arrays respectively through sequence verification.
The above only is preferred embodiment of the present invention, and all equalizations of doing according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Sequence table
<110〉Tongwei Co., Ltd.
<120〉the ITS2 molecular marker identification method of aquaculture catfish hybrid
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 240
<212> DNA
<213〉Silurus meridionalis Chen (Silurus meridionalis)
<400> 1
ctcgtgcgtc gatgaagaac gcagccagct gcgagaacta atgtgaattg caggacacat 60
tgatcatcga cacttcgaac gcacattgcg gccccgggtc cctcccgggg ctacgcctgt 120
ctgagggtcg ctatttccat cgatcgggaa aaaaacgaac catacgcacg cgactggaag 180
ctcgcaggcc gtcatcggcc ttcgtcctcc taaaagcaga cgctctcttt tttcgtgtcg 240
<210> 2
<211> 231
<212> DNA
<213〉catfish (Silurus asotus)
<400> 2
ctcgtgcgtc gatgaagaac gcagccagct gcgagaacta atgtgaattg caggacacat 60
tgatcatcga cacttcgaac gcacattgcg gccccgggtc cctcccgggg ctacgcctgt 120
ctgagggtcg ctatttccat cgatcgggaa aaaaacgaac gcgactggaa gctcgcaggc 180
cgtcatcggc cttcgtcctc ctaaaagcag acgctctctt ttttcgtgtc g 231
<210> 3
<211> 24
<212> DNA
<213〉artificial sequence
<400> 3
aactcttagc ggtggatcac tcgg 24
<210> 4
<211> 20
<212> DNA
<213〉artificial sequence
<400> 4
cgtggaaagg cggcggagat 20
<210> 5
<211> 23
<212> DNA
<213〉artificial sequence
<400> 5
taaattcagc gggtcgtctc gtc 23

Claims (2)

1. the ITS2 molecular marker identification method of aquaculture catfish hybrid is characterized in that: the difference in length of the ITS2 gene fragment of the Silurus meridionalis Chen behind the application pcr amplification and the ITS2 gene fragment of catfish, evaluation Silurus meridionalis Chen, catfish and hybrid generation thereof;
The nucleotides sequence of the ITS2 gene fragment of described Silurus meridionalis Chen is classified as:
CTCGTGCGTC GATGAAGAAC GCAGCCAGCT GCGAGAACTA ATGTGAATTG CAGGACACAT TGATCATCGA CACTTCGAAC GCACATTGCG GCCCCGGGTC CCTCCCGGGG CTACGCCTGT CTGAGGGTCG CTATTTCCAT CGATCGGGAA AAAAACGAAC CATACGCACG CGACTGGAAG CTCGCAGGCC GTCATCGGCC TTCGTCCTCC TAAAAGCAGA CGCTCTCTTT TTTCGTGTCG;
The nucleotides sequence of the ITS2 gene fragment of described catfish is classified as:
CTCGTGCGTC GATGAAGAAC GCAGCCAGCT GCGAGAACTA ATGTGAATTG CAGGACACAT TGATCATCGA CACTTCGAAC GCACATTGCG GCCCCGGGTC CCTCCCGGGG CTACGCCTGT CTGAGGGTCG CTATTTCCAT CGATCGGGAA AAAAACGAAC GCGACTGGAA GCTCGCAGGC CGTCATCGGC CTTCGTCCTC CTAAAAGCAG ACGCTCTCTT TTTTCGTGTC G;
The base sequence of described ITS2 gene fragment specific primer is:
Forward primer 5 '-AACTCTTAGCGGTGGATCACTCGG-3 ';
Reverse primer 5 '-CGTGGAAAGGCGGCGGAGAT-3 '.
2. the ITS2 molecular marker identification method of aquaculture catfish hybrid according to claim 1 is characterized in that: may further comprise the steps:
A gets respectively Silurus meridionalis Chen, catfish and hybridization sample thereof, extracts genomic dna;
B take total DNA of Silurus meridionalis Chen, catfish and hybridization sample thereof as template, carries out pcr amplification respectively under the guiding of ITS2 gene fragment specific primer;
C electrophoresis detection pcr amplification product through electrophoresis detection, two gene bands of Silurus meridionalis Chen and catfish occur simultaneously on a swimming lane, then be the cross-fertilize seed of Silurus meridionalis Chen and catfish.
CN 201210278377 2012-08-07 2012-08-07 Internal transcribed spacer (ITS2) molecular marker identifying method for aquaculture of catfish hybrids Expired - Fee Related CN102758022B (en)

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CN106868112B (en) * 2017-01-20 2019-12-06 浙江省淡水水产研究所 molecular identification method for culter alburnus, triangular bream and filial generation thereof
CN116240276B (en) * 2022-09-28 2024-01-09 中国科学院水生生物研究所 Catfish male-specific molecular marker, primer, kit, application and male or pseudo male identification and production method of total female fish

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