CN102732624A - Screening method of SRAP labels related to apostichopus japonicus fast-growing characteristics - Google Patents
Screening method of SRAP labels related to apostichopus japonicus fast-growing characteristics Download PDFInfo
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- CN102732624A CN102732624A CN2012102044344A CN201210204434A CN102732624A CN 102732624 A CN102732624 A CN 102732624A CN 2012102044344 A CN2012102044344 A CN 2012102044344A CN 201210204434 A CN201210204434 A CN 201210204434A CN 102732624 A CN102732624 A CN 102732624A
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Abstract
The invention disclosed a screening method of SRAP (sequence related amplified polymorphism) labels related to apostichopus japonicus fast-growing characteristics. According to the invention, an F (fast) group of 30 selected fast-growing apostichopus japonicus which has fast growing characteristics and an S (slow) group of 30 common wild apostichopus japonicus are adopted as materials; bulked segregant analysis (BSA) and an SRAP technology are combined; amplifications are respectively carried out upon mixed gene pools of the fast-growing F group and the common S group by using 340 pairs of primers; the PCR amplification products are subjected to denatured polyacrylamide gel electrophoresis, such that primer combinations of F06-R10, F08-R03, and F11-R11 with differential amplification bands are obtained; the occurrence frequencies of the differential bands 06-10-A, 08-03-B, and 11-11-C in the fast-growing F group are substantially higher than that in the common S group, such that the differential bands are the SRAP labels related to fast-growing characteristics. The screening method provided by the invention assists in providing a method for the development of apostichopus japonicus advantage-characteristic-related molecular labels, and in providing molecular label resources for apostichopus japonicus amount characteristic positioning and molecular-label-assisted apostichopus japonicus breeding.
Description
Technical field
The invention belongs to stichopus japonicus dna molecular genetic marker technology, be specifically related to a kind of screening and detection method of stichopus japonicus speed natural disposition shape related SR AP mark.
Background technology
Stichopus japonicus (Apostichopus japonicus) is subordinate to Echinodermata, Holothuroidea 、 Parapet hand order, Stichopodidae, is the important sea farming economic species of China.Screening cultivate have the optimum shape of dominant growth, resistance against diseases is strong, the improved seeds of good stress resistance, is to solve problems such as the degeneration of present stichopus japonicus germplasm, poor growth, disease pilosity, keeps pressing for of apostichopus japonicus culture industry sustainable and healthy development.The develop rapidly of modern molecular marking technique is for traditional stichopus japonicus genetic breeding provides effectively auxiliary and approach.
Sequence is correlated with, and (Sequence Related Amplifted Polymorphism is a kind of molecule marker system of PCR-based SRAP) to amplification polymorphism, is the molecule marker of new generation that is proposed by Li and doctor Quiros calendar year 2001.Abundant and characteristics that AT content enriches in promotor and the intron design primer and increase to GC content in the gene extron for it, produce polymorphum because of intron, promotor and the transcribed spacer of Different Individual is uneven in length.The SRAP mark has easy, moderate-yield, high codominance, repeatability, is easy to separate advantages such as band and order-checking, is fit to biological studies such as the assignment of genes gene mapping, gene clone, genetic map construction.
Genetic improvement or breed of variety are the power of apostichopus japonicus culture steady progression, and be along with seed selection work and the work of stichopus japonicus molecular biology research to the stichopus japonicus fast-growth strain deepen continuously, also more and more urgent to the demand of stichopus japonicus advantage proterties related molecular marker.Exploitation screening and the relevant mark of stichopus japonicus dominant growth proterties; Can in the stichopus japonicus breeding work, the heredity to relevant proterties dynamically follow the tracks of; Thereby strengthen the genetic manipulation ability of people greatly to stichopus japonicus dominant growth proterties; The final molecular marker assisted selection that realizes is accelerated the character inheritance progress, cultivates the stichopus japonicus new variety with quick growth traits.
Summary of the invention
The objective of the invention is for a kind of screening method of stichopus japonicus speed natural disposition shape related SR AP mark is provided, for stichopus japonicus quantitative character location provides molecule marker and method.
The present invention is material through 30 of the living F of speed (fast) group stichopus japonicus that have quick growth traits through seed selection are organized 30 of stichopus japonicus with common wild S (slow); Extract DNA; Adopt segregating population hybrid analysis BSA method and SRAP technology to combine, utilize 17 upstream primers, 20 downstream primers (table 1); Totally 340 pairs of combination of primers increase to 10 speed living group stichopus japonicus genome and 10 common wild group of stichopus japonicus genome blended gene pools with quick growth traits respectively; Pcr amplification product is used 8% denaturing polyacrylamide gel electrophoresis, and silver dyes, and obtains having combination of primers F06-R10, F08-R03, the F11-R11 (Fig. 1) of difference amplified band.Difference band combination of primers increases in 60 stichopus japonicus respectively with the same terms; The difference band is given birth to the F group frequency of occurrences in speed and is organized apparently higher than common S, and gained difference band 06-10-A, 08-03-B, 11-11-C are stichopus japonicus speed natural disposition shape related SR AP mark.
Description of drawings
Fig. 1: have difference band combination of primers polyacrylamide gel electrophoresis figure, A:06-10-A; B:08-03-B; C:11-11-C.
Embodiment
Explain further details in the face of the present invention down.
1, the extraction of stichopus japonicus genomic dna and detection
Extract each 30 of stichopus japonicus F group and S groups, cut open and get acquisition longitudinal muscle 100mg, liquid nitrogen grinding; Adding 700ml CTAB extraction damping fluid (100mmol/L Tris-HCl, pH 8.0,20mmol/L EDTA-Na2; 1.4mol/L NaCl, 2%CTAB, 0.1% beta-mercaptoethanol) and final concentration be 100 μ g/ml Proteinase Ks; 55 ℃ of digestion 3h or 37 ℃ spend the night, and equal-volume phenol chloroform (phenol: chloroform=1:1), chloroform extracting, diploid amasss ethanol sedimentation; The TE dissolving, ultraviolet spectrophotometer is quantitative, and-20 ℃ of preservations are subsequent use.
2, BSA (Bulked Segregant Analysis) divides the pcr amplification of pond and gene pool
Get 10 genes of individuals groups each 5 μ l dna solutions mixing formation corresponding F pond and S pond in F group and the S group.Utilize 17 upstream primers, 20 downstream primers (table 1, SEQ ID NO.1-37), totally 340 pairs of combination of primers are carried out pcr amplification to two pond DNA respectively, seek the difference band.The PCR reaction system is the template that the reaction system of 25 μ L comprises 100ng, 0.2mM dNTP, 0.4 μ M primer (forward and reverse difference), 1U Taq enzyme, 2.5 μ L, 10 * buffer.Reaction conditions is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 1min, 35 ℃ of renaturation 1min, 72 ℃ are extended 1min, carry out 5 circulations; 94 ℃ of sex change 1min, 50 ℃ of renaturation 1min, 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 5min, 4 ℃ of insulations.
Table 1 SRAP primer sequence table
Tab.1 The?sequences?of?SRAP?primers
3, SRAP amplification PCR product electrophoresis detection
(1) electrophoresis
Preparation 50ml denaturing polyacrylamide gel pours into the electrophoresis sheet glass, polymerization 2-3 hour.Electrophoresis chamber is installed, is injected 1 * TBE electrophoretic buffer, take out comb in the glue,, blow out wherein bubble and urea with suction pipe piping and druming point sample hole.50 ℃ of prerunning to temperature (establishing voltage 350 v, power 60 w).Get 12.5 μ L amplified productions+5 μ L sequencing gel sample-loading buffers, 94 ℃ of sex change 5 min immediately as on ice, get 6 μ L point samples, about electrophoresis 3.5 h.
(2) dyeing
Take off offset plate, dial down the glue on the offset plate gently, put into clear water immediately and wash, put into 500 mL, 1% salpeter solution then, vibrate gently about 5 min, fix.With the quick rinsing gel of distilled water 2 times, gel is put into 500 mL, 5min gently vibrates in 1% the cma staining liquid.From staining fluid, take out gel,, stir slightly, take out immediately in the yellow soda ash colour developing liquid that immerses 500 mL 60% and develop the color with the quick rinsing gel of distilled water 2 times.Bands of a spectrum appear after shaking 5 ~ 8 min gently behind the gel immersion colour developing liquid (1000 mL colour developing liquid is poured at twice).After isogel developed the color fully, fixedly stop buffer (front is solutions employed fixedly) was poured in the colour developing liquid, the color development stopping reaction.Taking out gel and immerse rinsing in the distilled water, takes a picture and record analysis with gel imaging system in the back.
4, difference band screening and not banding pattern statistics in the individuality on the same group
Show according to SRAP combination of primers amplified production electrophoresis result; Screening obtains having in the F group and occurs and at S group do not increase combination of primers F06-R10, F08-R03, the F11-R11 (Fig. 1) of difference band, the difference band is labeled as 06-10-A, 08-03-B, 11-11-C.The difference combination of primers with identical PCR condition to two colonies totally 60 stichopus japonicus carry out pcr amplification respectively, the statistical discrepancy band is organized the stichopus japonicus frequency of occurrences (table 2) in F group with S.06-10-A, 08-03-B, 11-11-C are respectively 83.3%, 90% and 86.7% in the F group frequency of occurrences, apparently higher than in the S group frequency of occurrences 23.3%, 30% and 13.3%, are stichopus japonicus speed natural disposition shape related SR AP mark.
The frequency of occurrences statistics of table 2 difference allelotrope fragment in individuality amplification banding pattern
Tab.2 Statistic?of?differential?bands?of?individual’s?banding?patterns
SEQUENCE?LISTING
< 110>Shandong Prov. Marine Aquatic Product Inst
< 120>SRAP primer
<130> ref
<160> 37
<170> PatentIn?version?3.3
<210> 1
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 1
tgagtccaaa?ccggata 17
<210> 2
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 2
tgagtccaaa?ccggagc 17
<210> 3
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 3
tgagtccaaa?ccggaat 17
<210> 4
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 4
tgagtccaaa?ccggacc 17
<210> 5
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 5
tgagtccaaa?ccggaag 17
<210> 6
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 6
tgagtccaaa?ccggaaa 17
<210> 7
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 7
tgagtccaaa?ccggacc 17
<210> 8
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 8
tgagtccaaa?ccggtgc 17
<210> 9
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 9
tgagtccaaa?ccggtca 17
<210> 10
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 10
tgagtccttt?ccggtaa 17
<210> 11
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 11
tgagtccttt?ccggtcc 17
<210> 12
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 12
tgagtccttt?ccggtgc 17
<210> 13
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 13
tgagtcgtat?ccggtct 17
<210> 14
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 14
tgagtcgtat?ccggagt 17
<210> 15
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 15
tgagtcgtat?ccggtag 17
<210> 16
<211> 19
<212> DNA
< 213>SRAP forward primer sequence
<400> 16
taaacaatgg?ctactcaag 19
<210> 17
<211> 17
<212> DNA
< 213>SRAP forward primer sequence
<400> 17
ctggtgaatg?ccgctct 17
210> 18
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 18
gactgcgtac?gaattaat 18
<210> 19
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 19
gactgcgtac?gaatttgc 18
<210> 20
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 20
gactgcgtac?gaattgac 18
<210> 21
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 21
gactgcgtac?gaatttga 18
<210> 22
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 22
gactgcgtac?gaattaac 18
<210> 23
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 23
gactgcgtac?gaattgca 18
<210> 24
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 24
gactgcgtac?gaattcaa 18
<210> 25
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 25
gactgcgtac?gaattctg 18
<210> 26
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 26
gactgcgtac?gaattcga 18
<210> 27
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 27
gactgcgtac?gaattcag 18
<210> 28
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 28
gactgcgtac?gaattcca 18
<210> 29
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 29
gactgcgtac?gaattgtc 18
<210> 30
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 30
gactgcgtac?gaattacg 18
<210> 31
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 31
gactgcgtac?gaattcgg 18
<210> 32
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 32
gactgcgtac?gaatttag 18
<210> 33
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 33
gactgcgtac?gaatttcg 18
<210> 34
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 34
gactgcgtac?gaattggt 18
<210> 35
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 35
gactgcgtac?gaatttcc 18
<210> 36
<211> 19
<212> DNA
< 213>SRAP reverse primer sequence
<400> 36
ccaaaaccta?aaaccagga 19
<210> 37
<211> 18
<212> DNA
< 213>SRAP reverse primer sequence
<400> 37
gcgaggatgc?tactggtt 18
Claims (4)
1. the screening method of stichopus japonicus speed natural disposition shape related SR AP mark; It is characterized in that; Operation steps is: giving birth to 30 of F (fast) group stichopus japonicus with the speed with quick growth traits is material with 30 of stichopus japonicus of common wild S (slow) group; Extract the stichopus japonicus genomic dna, adopt segregating population hybrid analysis BSA method and SRAP technology to combine, totally 340 pairs of combination of primers are carried out pcr amplification to two pond DNA respectively; Polyacrylamide gel electrophoresis, screening obtain in F group individuality the frequency of occurrences apparently higher than the difference band of the frequency of occurrences in S group individuality.
2. screening method as claimed in claim 1 is characterized in that, the combination of primers of said difference band is F06-R10, F08-R03, F11-R11, and the difference band is 06-10-A, 08-03-B, 11-11-C.
3. screening method as claimed in claim 1; It is characterized in that; Said segregating population hybrid analysis method is that the BSA method is specially from F group stichopus japonicus genome and S group stichopus japonicus genome these 10 of each samplings, and each is got 5 μ LDNA solution and mixes corresponding F mixing gene pool of formation and S mixing gene pool.
4. screening method as claimed in claim 1 is characterized in that, the reaction system of said pcr amplification is 25 μ L, comprises the template of 100ng, 0.2mM dNTP, and positive anti-primer is 0.4 μ M respectively, 1U Taq enzyme, 2.5 μ L, 10 * buffer; Reaction conditions is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 1min, 35 ℃ of renaturation 1min, 72 ℃ are extended 1min, carry out 5 circulations; 94 ℃ of sex change 1min, 50 ℃ of renaturation 1min, 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 5min, 4 ℃ of insulations.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994499A (en) * | 2013-01-05 | 2013-03-27 | 四川农业大学 | Ecotypic SRAP (Sequence Related Amplified Polymorphism) specificity molecular marker primer for determining switchgrass and ecotypic detection method of switchgrass |
| CN107937570A (en) * | 2018-01-16 | 2018-04-20 | 中国水产科学研究院黄海水产研究所 | A kind of genetic molecule for fast growing bechedemer breeding marks and application |
| CN107937569A (en) * | 2018-01-16 | 2018-04-20 | 中国水产科学研究院黄海水产研究所 | A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection |
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| CN102234644A (en) * | 2011-06-17 | 2011-11-09 | 中国水产科学研究院淡水渔业研究中心 | Method for establishing and optimizing exopalaemon modestus SRAP(sequence-related amplified polymorphism)-PCR(polymerase chain reaction) system |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994499A (en) * | 2013-01-05 | 2013-03-27 | 四川农业大学 | Ecotypic SRAP (Sequence Related Amplified Polymorphism) specificity molecular marker primer for determining switchgrass and ecotypic detection method of switchgrass |
| CN107937570A (en) * | 2018-01-16 | 2018-04-20 | 中国水产科学研究院黄海水产研究所 | A kind of genetic molecule for fast growing bechedemer breeding marks and application |
| CN107937569A (en) * | 2018-01-16 | 2018-04-20 | 中国水产科学研究院黄海水产研究所 | A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection |
| CN107937569B (en) * | 2018-01-16 | 2018-10-30 | 中国水产科学研究院黄海水产研究所 | A kind of molecular labeling and its application for stichopus japonicus growth traits assisted selection |
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Application publication date: 20121017 |