CN110452992A - A kind of labeling method of litopenaeus vannamei EST microsatellite locus primer - Google Patents
A kind of labeling method of litopenaeus vannamei EST microsatellite locus primer Download PDFInfo
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- 108091092878 Microsatellite Proteins 0.000 title claims abstract description 79
- 241000238553 Litopenaeus vannamei Species 0.000 title claims abstract description 29
- 238000002372 labelling Methods 0.000 title claims abstract description 7
- 238000013461 design Methods 0.000 claims abstract description 16
- 230000002068 genetic effect Effects 0.000 claims abstract description 13
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- 230000003321 amplification Effects 0.000 claims abstract description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 8
- 230000000694 effects Effects 0.000 claims abstract description 5
- 239000003550 marker Substances 0.000 claims abstract description 5
- 238000011895 specific detection Methods 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 14
- 238000000137 annealing Methods 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 241000238557 Decapoda Species 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 108700028369 Alleles Proteins 0.000 claims description 6
- 238000004925 denaturation Methods 0.000 claims description 6
- 230000036425 denaturation Effects 0.000 claims description 6
- 238000004043 dyeing Methods 0.000 claims description 4
- 230000003252 repetitive effect Effects 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 3
- 108091092584 GDNA Proteins 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 3
- 238000005251 capillar electrophoresis Methods 0.000 claims description 3
- 238000001962 electrophoresis Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 229920002401 polyacrylamide Polymers 0.000 claims description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims description 3
- 238000004153 renaturation Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims 1
- 238000005516 engineering process Methods 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 238000004321 preservation Methods 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 4
- 238000009399 inbreeding Methods 0.000 abstract description 4
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- 239000002253 acid Substances 0.000 description 3
- 230000005611 electricity Effects 0.000 description 3
- 241000238550 Penaeidae Species 0.000 description 2
- 241000927735 Penaeus Species 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 241000995704 Fenneropenaeus chinensis Species 0.000 description 1
- 241000530452 Litopenaeus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- 108091001629 crustin Proteins 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000009394 selective breeding Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
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Abstract
The invention discloses a kind of labeling methods of litopenaeus vannamei EST microsatellite locus primer, it the steps include: the sequence lookup containing microsatellite locus, micro-satellite primers design, microsatellite locus primer amplification effect and specific detection, microsatellite locus polymorphism detected, disclose the special primer of 6 pairs of EST microsatellite markers, method is simple, it is easy to operate, it is reproducible, it is reliable and stable.It is sequenced by transcript profile and obtains est sequence, excavate SSR site information, design primer constructs litopenaeus vannamei specificity SSR marker system, is applied to litopenaeus vannamei Parentage determination, Genetic Diversity of Germplasm analysis, auxiliary litopenaeus vannamei molecular genetic breeding or cultivation.The label that different familys can effectively be identified can be avoided the loss for causing inbreeding and genetic diversity, ensure the sustainable development of litopenaeus vannamei fine-variety breeding.
Description
Technical field
The present invention relates to a kind of biology techniques fields, and more specifically it is related to a kind of litopenaeus vannamei EST microsatellite
The labeling method of site primer.
Background technique
Litopenaeus vannamei (Litopenaeus vannamei) is also known as Penaeus Vannmei, is subordinate to Decapoda (Deeapoda),
Penaeidae (Penaeidae), shore Penaeus (Litopenaeus) originate in Central and South America Peru to the Pacific Ocean edge of Mexico
Bank is the Variety comprehensive of world's shrimp culture industry, is used as Crustin (Fenneropenaeus chinensis) within 1988
Substitute species introduce China, due to the litopenaeus vannamei advantages that have that the speed of growth is fast, the culture-cycle is short, cultivation is adaptable etc.,
Cultured area expands rapidly after 2000, has been one of the pillar industry of Chinese Fishery cultivation at present.It in recent years, is solution
Certainly germplasm status under one's control, China increase litopenaeus vannamei kind industry tackling key problem dynamics, are dedicated to litopenaeus vannamei germ plasm resource
It is researched and developed with fine-variety breeding etc..Unexpected mass incident and family selective breeding are the conventional means of current litopenaeus vannamei fine-variety breeding, find one
Kind can effectively identify the label of different familys, can be avoided the loss for causing inbreeding and genetic diversity, ensure all receives
Shore prawn fine-variety breeding sustainable development.
Microsatellite DNA, also known as short tandem repeat or simple sequence repeats.It is with the short core of 1~6 base
Thuja acid joins end to end for basic unit and forms tandem repetitive sequence, since number of repetition is different and repeats the incomplete of degree
Cause the length polymorphism in each site.Since the sequence at each microsatellite both ends is mostly relatively conservative single-copy sequence,
A pair of of specific primer can be designed according to its both ends, the microsatellite sequence of corresponding site is expanded by round pcr, then through electricity
Swimming analysis, can show the polymorphism of different genotype individual microsatellite.For microsatellite due to widely distributed, density is big, polymorphism
It is abundant, it then follows Mendel's law of segregation, codominant inheritance are easy to PCR amplification, and required DNA quantity is few, to DNA quality requirement
It is not high, it is as a result reproducible the advantages that, be widely used in genetic diversity, Genetic relationship, linkage map structure at present
It builds, functional gene positioning, the fields such as molecular mark.
Summary of the invention
In view of the deficienciess of the prior art, the invention reside in provide a kind of litopenaeus vannamei EST microsatellite locus primer
Labeling method, method is simple, easy to operate, reproducible, reliable and stable, can effectively identify the label of different familys,
It can be avoided the loss for causing inbreeding and genetic diversity, ensure the sustainable development of litopenaeus vannamei fine-variety breeding.
The invention is realized by the following technical scheme:
(1) sequence containing microsatellite locus is searched: being taken southern litopenaeus vannamei, is obtained by transcript profile sequencing analysis method
Microsatellite locus is obtained, the above repetitive unit of 3 bases is screened and microsatellite locus of the number of repetition greater than 5 times, picks out 52 altogether
A microsatellite locus；
(2) micro-satellite primers design: the gene order containing microsatellite obtained according to step (1), and selection meets primer
The sequence of design utilizes 5.0 design primer of Primer Premier；Major parameter setting are as follows: 18-23 nucleosides of primer length
Acid, mostly 20bp or so, annealing temperature are 59-60.5 DEG C, product 150-280bp；
(3) microsatellite locus primer amplification effect and specific detection: 1. extracting genome DNA: Beijing Ai Delai is used
The marine animal tissue gene group DNA rapidly extracting kit of Biotechnology Co., Ltd extracts group, Ecuador prawn flesh
The genomic DNA of meat tissue；2. the amplification of microsatellite PCR: using 52 pairs of micro-satellite primers of design, expanding litopenaeus vannamei
Genomic DNA；Reaction system is 25 μ l: primer each 1 μ l, 2.5 μ l 10 × buffer, 2 μ l dNTPs, 0.125 μ l TaKaRa
Ex Hot Start Vers ion (article No.: RR006A), 1 μ l gDNA, adds water to 25 μ l；3. PCR is reacted in ABI
It is carried out in 9700PCR instrument, amplification program is as follows: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, it is (each under each site annealing temperature
Site annealing temperature is as shown in full edition 2) renaturation 20s, 72 DEG C of extension 45s, 40 recycle；Final 72 DEG C of extensions 7min, last 4
DEG C save；4. PCR product is dyed through 1.2% agarose electrophoresis DuGreen and detected, 40 are filtered out without miscellaneous band, purpose band spy
Anisotropic high microsatellite locus；5. being sequenced to the PCR product of the single microsatellite locus of band using sanger and carrying out primer specific
Property verifying；
(4) microsatellite locus polymorphism is detected: 1. 8% denaturing polyacrylamide gel electrophoresis detection microsatellite position
Point polymorphism: the PCR product for 40 microsatellite locus that step (3) screens 8% denaturing polyacrylamide gel electricity
Swimming-DuGreen dyeing carries out Preliminary detection, and preliminary screening goes out 12 preferable microsatellite markers of polymorphism.2. sequenator detects
Amplified production: carrying out fluorescent marker to the primer of 12 microsatellite locus, and amplified production is kept in dark place, and surveys in ABI 3730XL
Sequence instrument carries out Capillary Electrophoresis and STR analysis accurately to determine number of alleles of 12 microsatellite locus in Different Individual
Mesh；3. analysis of genetic diversity: determining genotype according to the allele size of each microsatellite amplified production, use
PopGen 32 calculates genetic diversity parameter, to filter out 6 microsatellite locus with polymorphism (PIC > 0.25) (in detail
It is shown in Table 1, table 2).
Table 1 is the relevant informations such as annealing temperature, fragment length, the polymorphism of 6 microsatellite locus
Table 2 is 6 litopenaeus vannamei microsatellite locus and corresponding primer information
Compared with prior art, the present invention having the following advantages that and effect: method is simple, easy to operate, reproducible,
It is reliable and stable.It is sequenced by transcript profile and obtains est sequence, excavate SSR site information, design primer, building litopenaeus vannamei spy
Anisotropic SSR marker system is applied to litopenaeus vannamei Parentage determination, Genetic Diversity of Germplasm analysis, auxiliary vannamei boone pair
Shrimp molecular genetic breeding or cultivation.The label that different familys can effectively be identified, can be avoided and cause inbreeding and genetic diversity
Property loss, ensure the sustainable development of litopenaeus vannamei fine-variety breeding.
Specific embodiment
Present embodiment discloses a kind of labeling methods of litopenaeus vannamei EST microsatellite locus primer, mainly include following
Step:
(1) sequence containing microsatellite locus is searched: being taken southern litopenaeus vannamei, is obtained by transcript profile sequencing analysis method
Microsatellite locus is obtained, the above repetitive unit of 3 bases is screened and microsatellite locus of the number of repetition greater than 5 times, picks out 52 altogether
A microsatellite locus；
(2) micro-satellite primers design: the gene order containing microsatellite obtained according to step (1), and selection meets primer
The sequence of design utilizes 5.0 design primer of Primer Premier；Major parameter setting are as follows: 18-23 nucleosides of primer length
Acid, mostly 20bp or so, annealing temperature are 59-60.5 DEG C, product 150-280bp；
(3) microsatellite locus primer amplification effect and specific detection: 1. extracting genome DNA: Beijing Ai Delai is used
The marine animal tissue gene group DNA rapidly extracting kit of Biotechnology Co., Ltd extracts group, Ecuador prawn flesh
The genomic DNA of meat tissue；2. the amplification of microsatellite PCR: using 52 pairs of micro-satellite primers of design, expanding litopenaeus vannamei
Genomic DNA；Reaction system is 25 μ l: primer each 1 μ l, 2.5 μ l 10 × buffer, 2 μ l dNTPs, 0.125 μ l TaKaRa
Ex Hot Start Version (article No.: RR006A), 1 μ l gDNA, adds water to 25 μ l；3. PCR is reacted in ABI
It is carried out in 9700PCR instrument, amplification program is as follows: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, it is (each under each site annealing temperature
Site annealing temperature is as shown in full edition 2) renaturation 20s, 72 DEG C of extension 45s, 40 recycle；Final 72 DEG C of extensions 7min, last 4
DEG C save；4. PCR product is dyed through 1.2% agarose electrophoresis DuGreen and detected, 40 are filtered out without miscellaneous band, purpose band spy
Anisotropic high microsatellite locus；5. being sequenced to the PCR product of the single microsatellite locus of band using sanger and carrying out primer specific
Property verifying；
(4) microsatellite locus polymorphism is detected: 1. 8% denaturing polyacrylamide gel electrophoresis detection microsatellite position
Point polymorphism: the PCR product for 40 microsatellite locus that step (3) screens 8% denaturing polyacrylamide gel electricity
Swimming-DuGreen dyeing carries out Preliminary detection, and preliminary screening goes out 12 preferable microsatellite markers of polymorphism.2. sequenator detects
Amplified production: carrying out fluorescent marker to the primer of 12 microsatellite locus, and amplified production is kept in dark place, and surveys in ABI 3730XL
Sequence instrument carries out Capillary Electrophoresis and STR analysis accurately to determine number of alleles of 12 microsatellite locus in Different Individual
Mesh；3. analysis of genetic diversity: determining genotype according to the allele size of each microsatellite amplified production, use
PopGen 32 calculates genetic diversity parameter, to filter out 6 microsatellite locus with polymorphism (PIC > 0.25) (in detail
It is shown in Table 1, table 2).
Table 1 is the relevant informations such as annealing temperature, fragment length, the polymorphism of 6 microsatellite locus
Table 2 is 6 litopenaeus vannamei microsatellite locus and corresponding primer information
The foregoing is merely presently preferred embodiments of the present invention, is not intended to restrict the invention, all in design structure of the invention
Within think of, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (1)
1. a kind of labeling method of litopenaeus vannamei EST microsatellite locus primer, its step are as follows:
(1) sequence containing microsatellite locus is searched: being taken litopenaeus vannamei, is obtained microsatellite by transcript profile sequencing analysis method
Site screens the above repetitive unit of 3 bases and microsatellite locus of the number of repetition greater than 5 times, picks out 52 microsatellites altogether
Site；
(2) micro-satellite primers design: the gene order containing microsatellite obtained according to step (1), and selection meets design of primers
Sequence, utilize 5.0 design primer of Primer Premier；Major parameter setting are as follows: 18-23 nucleotide of primer length is more
For 20bp or so, annealing temperature is 59-60.5 DEG C, product 150-280bp；
(3) microsatellite locus primer amplification effect and specific detection: 1. extracting genome DNA: using Beijing Ai Delai biology
The marine animal tissue gene group DNA rapidly extracting kit of Science and Technology Ltd. extracts group, Ecuador prawn musculature
Genomic DNA；2. the amplification of microsatellite PCR: using 52 pairs of micro-satellite primers of design, expanding litopenaeus vannamei genome
DNA；Reaction system is 25 μ l: primer each 1 μ l, 2.5 μ l 10 × buffer, 2 μ l dNTPs, 0.125 μ l TaKaRa ExHot Start Version (article No.: RR006A), 1 μ l gDNA, adds water to 25 μ l；3. PCR is reacted in ABI 9700
It is carried out in PCR instrument, amplification program is as follows: 94 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, renaturation 20s under each site annealing temperature,
72 DEG C of extension 45s, 40 circulations；Final 72 DEG C of extensions 7min, last 4 DEG C of preservations；4. PCR product is through 1.2% agarose electrophoresis
DuGreen dyeing detection filters out 40 microsatellite locus high without miscellaneous band, purpose band specificity；5. single to band micro-
The PCR product in satellite site is sequenced using sanger and carries out primer specificity verifying；
(4) detect to microsatellite locus polymorphism: 1. 8% denaturing polyacrylamide gel electrophoresis detection microsatellite locus is more
State property: PCR product 8% denaturing polyacrylamide gel electrophoresis-for 40 microsatellite locus that step (3) screens
DuGreen dyeing carries out Preliminary detection, and preliminary screening goes out 12 preferable microsatellite markers of polymorphism；2. sequenator detection amplification
Product: carrying out fluorescent marker to the primer of 12 microsatellite locus, and amplified production is kept in dark place, ABI 3730XL sequenator into
Row Capillary Electrophoresis and STR analysis are accurately to determine allele number of 12 microsatellite locus in Different Individual；3. losing
It passes diversity analysis: genotype is determined according to the allele size of each microsatellite amplified production, calculated using PopGen 32
Genetic diversity parameter, so that 6 microsatellite locus with polymorphism (PIC > 0.25) are filtered out, it is specific as follows:
LvSSR16
F:TCAAGCAAAAAGAGAGTGCG
R:TCCCTGCCTTGAGCATTACT
LvSSR20
F:TGCGAGGTGATGTTCTAACG
R:CGCAAATCTATCGCTTCTCC
LvSSR25
F:GAGAGTGTGCGTTCCTGCTT
R:GCCTCCCGACTGTTTGAATA
LvSSR43
F:TCACCAGGTCAAGAAGGAGG
R:TCTTTCCTTCGTCCAGACTCA
LvSSR49
F:GCACAAAAGGAATCCTGGAA
R:TCATACACCTTCGACCCTCC
LvSSR51
F:TGGAGATTTCGGAACCTTTG
R:CGCATTAGTTGGTGCTGAGA。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113005204A (en) * | 2021-04-15 | 2021-06-22 | 中国科学院南海海洋研究所 | Vibrio-resistant related EST-STR marker of litopenaeus vannamei, specific primer and detection method thereof |
| CN113151503A (en) * | 2021-05-18 | 2021-07-23 | 中国水产科学研究院黄海水产研究所 | Litopenaeus vannamei microsatellite marked primer combination and application |
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| CN101942437A (en) * | 2010-08-23 | 2011-01-12 | 中山大学 | Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof |
| CN106007036A (en) * | 2016-07-12 | 2016-10-12 | 浙江省海洋水产养殖研究所 | Device for treating pollutants in penaeus vannamei aquaculture pond |
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| CN101942437A (en) * | 2010-08-23 | 2011-01-12 | 中山大学 | Specific primers for microsatellite markers of EST sequences of Litopenaeus vannamei and application thereof |
| CN106007036A (en) * | 2016-07-12 | 2016-10-12 | 浙江省海洋水产养殖研究所 | Device for treating pollutants in penaeus vannamei aquaculture pond |
| CN108611429A (en) * | 2018-05-10 | 2018-10-02 | 中山大学 | Litopenaeus vannamei disease resistance correlation EST-SSR molecular labelings and its application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113005204A (en) * | 2021-04-15 | 2021-06-22 | 中国科学院南海海洋研究所 | Vibrio-resistant related EST-STR marker of litopenaeus vannamei, specific primer and detection method thereof |
| CN113151503A (en) * | 2021-05-18 | 2021-07-23 | 中国水产科学研究院黄海水产研究所 | Litopenaeus vannamei microsatellite marked primer combination and application |
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Application publication date: 20191115 |