CN106754886A - Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers - Google Patents
Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers Download PDFInfo
- Publication number
- CN106754886A CN106754886A CN201710038608.7A CN201710038608A CN106754886A CN 106754886 A CN106754886 A CN 106754886A CN 201710038608 A CN201710038608 A CN 201710038608A CN 106754886 A CN106754886 A CN 106754886A
- Authority
- CN
- China
- Prior art keywords
- fructus lycii
- ssr
- primer
- black fruit
- sense primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000012163 sequencing technique Methods 0.000 title claims abstract description 29
- 238000013518 transcription Methods 0.000 title claims abstract description 23
- 230000035897 transcription Effects 0.000 title claims abstract description 23
- 238000012216 screening Methods 0.000 claims abstract description 12
- 238000013461 design Methods 0.000 claims abstract description 7
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 57
- 230000000692 anti-sense effect Effects 0.000 claims description 37
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 108091092878 Microsatellite Proteins 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 239000003292 glue Substances 0.000 claims description 8
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 108020004999 messenger RNA Proteins 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002299 complementary DNA Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000011833 salt mixture Substances 0.000 claims description 5
- 238000007605 air drying Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 210000002257 embryonic structure Anatomy 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000004570 mortar (masonry) Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 229930024421 Adenine Natural products 0.000 claims description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 3
- 239000007832 Na2SO4 Substances 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 229960000643 adenine Drugs 0.000 claims description 3
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000005251 capillar electrophoresis Methods 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000012154 double-distilled water Substances 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 229920002401 polyacrylamide Polymers 0.000 claims description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 238000007689 inspection Methods 0.000 claims 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical class OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 8
- 230000002068 genetic effect Effects 0.000 abstract description 7
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 239000003147 molecular marker Substances 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000002372 labelling Methods 0.000 abstract description 4
- 230000000052 comparative effect Effects 0.000 abstract description 2
- 238000012937 correction Methods 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 5
- SVPKNMBRVBMTLB-UHFFFAOYSA-N 2,3-dichloronaphthalene-1,4-dione Chemical compound C1=CC=C2C(=O)C(Cl)=C(Cl)C(=O)C2=C1 SVPKNMBRVBMTLB-UHFFFAOYSA-N 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000241838 Lycium barbarum Species 0.000 description 2
- 235000015459 Lycium barbarum Nutrition 0.000 description 2
- 241000169546 Lycium ruthenicum Species 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241001106041 Lycium Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 241000256247 Spodoptera exigua Species 0.000 description 1
- 102100023706 Steroid receptor RNA activator 1 Human genes 0.000 description 1
- 101710187693 Steroid receptor RNA activator 1 Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of method for obtaining black fruit fructus lycii SSR primers based on transcription sequencing, belong to biological molecule genetic molecule labelling technique technical field, its step is as follows：(1) transcript profile sequencing；(2) screening EST SSR sites；(3) design of primers；(4) DNA is extracted；(5) primer screening；(6) polymorphism primer screening is carried out using 8% native polyacrylamide gel electrophoresis.The method for obtaining black fruit fructus lycii SSR primers based on transcription sequencing of the invention, can have value higher as genome linkage map between correction distant relative's species and the method for comparative map in these two aspects.The biggest advantage is that it is low to develop simple, quick, expense.Using the general nature of the clear and definite black fruit fructus lycii SSR of SSR molecular marker technology, exploitation black fruit fructus lycii SSR primers, it is to carry out black fruit fructus lycii Genetic Diversity of Germplasm, linkage map structure and affiliation research using SSR molecular marker to lay the foundation, so as to preferably apply in molecular breeding.
Description
Technical field
It is more particularly to a kind of that black fruit fructus lycii is obtained based on transcription sequencing the present invention relates to a kind of method of acquisition SSR primers
The method of SSR primers, belongs to biological molecule genetic molecule labelling technique technical field.
Background technology
Simple repeated sequence (simple sequence Repeats, SSR), also referred to as microsatellite DNA
(Microsatellite DNA) refers to a class by the DNA of the primitive tandem sequence repeats of several (mostly 1~6) base compositions
Sequence, such as (AT) n, (GCA) n, (AATC) n are repeated, and it is distributed evenly in eukaryotic gene group extensively.Due to many
State property is high, technology is reproducible, it is easy to the advantages of operating, and since the early 1990s in last century, SSR marker is always as a kind of
Excellent molecular labeling and be widely used in the genetic analysis of species.People are to microsatellite DNA in the mankind, animal and plant
In generation and distribution done substantial amounts of research.The tradition exploitation of SSR marker is related to a series of complicated operating process, not only imitates
Rate is low, and due to the limitation of cost factor, cause for a long time SSR marker only model plant or economic worth compared with
Development and application is obtained on crop high.
The sequence at SSR two ends is relatively conservative single-copy sequence mostly, and people's sequences Design usually according to its two ends is special
Different primer is expanded, the change according to amplified production length and show different genotype individuality on each SSR site
Polymorphism.SSR originates according to sequence, is generally divided into genome gSSR (genomic SSR) and two kinds of EST-SSR (eSSR).
Black fruit fructus lycii (Lycium ruthenicum Murr.) is the perennial shrub plant of Solanaceae Lycium, is China west
A kind of northern distinctive wild plant in Desert Area, is distributed mainly on the ground such as Qinghai, Xinjiang, Inner Mongol, Ningxia, Gansu.This kind of salt tolerant,
Drought resisting, is distributed in saline-alkali soil wasteland, pond ground or roadside more, there is very strong adaptability to salt-affected soil.But with Ningxia haw matrimony vine
Compare, black fruit fructus lycii has obvious gap at the aspect such as breeding practice and cultivar identification, rarely seen in terms of molecule marking research
To the report for being related to SRAP, the report on its genomic information or transcription group information is had no.
With the increasingly increase to black fruit fructus lycii cultivar demand, the problem on black fruit fructus lycii molecular labeling auxiliary
In the urgent need to studying and solving.
At present, black fruit fructus lycii there is no genomic information, SSR primer negligible amounts.Therefore, how to be obtained based on transcription sequencing
Black fruit fructus lycii SSR primers, so that the affiliation preferably between research Different Provenances, for black fruit fructus lycii molecular breeding, is
Obtain a large amount of outstanding germ plasm resources to lay the foundation, just turn into the technical barrier that the technical field is badly in need of solving.
The content of the invention
An object of the present invention is to provide a kind of method for obtaining black fruit fructus lycii SSR primers based on transcription sequencing, so that
Affiliation preferably between research Different Provenances, for black fruit fructus lycii molecular breeding, to obtain a large amount of outstanding germ plasm resources
Lay the foundation.
To achieve the above object, the present invention takes following technical scheme：
A kind of method for obtaining black fruit fructus lycii SSR primers based on transcription sequencing, its step is as follows：
(1) 3 black fruit fructus lyciis of provenance are taken as sample, aerial part is done into transcript profile sequencing；
(2) using online microsatellite locus scanning tools SSRIT (or MISA softwares) detection EST-SSR sites, SSR >=
20bp, search is set to that trinucleotide is minimum to be repeated 6 times, and tetranucleotide is minimum to be repeated 5 times, and pentanucleotide is minimum to be repeated 4 times, and six
Nucleotides is minimum to be repeated 3 times；
(3) SSR design of primers is carried out using software primer3.0, primer parameter is set to length 18-25bp, Tm55-65
DEG C, primer size is 100-300bp；
(4) 10 DNA of provenance black fruit fructus lycii are extracted using CTAB methods, each provenance takes 25 samples as weight
It is multiple；
(5) primer in (3) and their corresponding SSR sites are carried out further using PRIMER PREMIER 5.0
Random screening is checked, and the primer sequence for being filtered out is sent to company and is synthesized, and synthesizes laggard performing PCR amplification；
(6) polymorphism primer screening is carried out using 8% native polyacrylamide gel electrophoresis：Show according to cementing fruit is run
Show band, primer of the same race can amplify differential band on Different Provenances ground, it is believed that with polymorphism.
Preferably, in the sequencing of transcript profile described in the step (1), treatment is divided into control group, salt treatment group and salt-mixture
Treatment group, wherein salt treatment group using 100mL, pH=7,200mmol/L NaCl solution, salt-mixture treatment group using 100mL,
PH=8.54, Na2CO3、NaCl、Na2SO4、NaHCO3According to 1:9:9:The mixed solution of the final concentration of 200mmol/L of 1 mixing.
Preferably, what transcript profile described in the step (1) was sequenced comprises the following steps that：It is total that CTAB methods extract black fruit fructus lycii
RNA, separates mRNA, is merged into double-strand cDNA, plus adenine and sequence measuring joints, and the fragment to size in 200bp-700bp is carried out
Amplification, is detected with the library after 2000 pairs of amplifications of Illumina HiSeq, and sequencing data is spliced using trinity softwares,
Transcript numbering most long in every gene is set to be 1 unigene.
Preferably, the PCR instrument that the amplifications of PCR described in the step (5) are used is BoiRad T100 models.
Preferably, comprising the following steps that for 10 DNA of provenance black fruit fructus lycii is extracted described in the step (4)：
1. the blade sample of Excised Embryos is taken, mortar adds (the blade amount of powder of addition in 2.0ml centrifuge tubes after grinding
The tip of the bottom of 2.0ml centrifuge tubes is not crossed；
2. (2%CTAB uses preceding addition 0.5-1% β-sulfydryl second to be rapidly added 65 DEG C of preheated μ l of CTAB 500
Alcohol), 65 DEG C of water-bath half an hour are put into after being well mixed, shake in every ten minutes once, makes CTAB fully be mixed with blade sample；
3. after taking out centrifuge tube placement 2 minutes, isometric chloroform/isoamyl alcohol (24 is added:1) mixed liquor, by mixed liquor with
After CTAB is mixed, vortex mixed makes CTAB be fully contacted with chloroform；
4. 12000rpm is centrifuged 10 minutes, in Aspirate supernatant to 1.5ml centrifuge tubes；
5. dliploid product absolute ethanol washing DNA precipitations are added, 12000rpm centrifugation 10min abandon supernatant；
6. the washed DNA of ethanol is air-dried (superclean bench blows or be concentrated in vacuo instrument)；
7. with 50 μ l ddH after air-drying2O dissolves, and obtains DNA concentration for 30ng/ μ l, and -20 DEG C of preservations are stand-by.
Preferably, what further random screening described in the step (5) was checked concretely comprises the following steps：SSR >=20bp, search
It is set to that trinucleotide is minimum to be repeated 6 times, tetranucleotide is minimum to be repeated 5 times, and pentanucleotide is minimum to be repeated 4 times, and Hexanucleotide is most
It is low to be repeated 3 times；Primer parameter is set to length 18-25bp, Tm 58-63 DEG C, and primer size is 100-300bp；77 pairs are chosen to draw
Thing.
Preferably, the amplifications of PCR described in the step (5) reaction system used is the 1 μ l of μ l, DNA of Mix 10, upstream
Primer and anti-sense primer each 1 μ l, ddH2O7μl；Course of reaction：95 DEG C of predegeneration, 5min, denaturation：95 DEG C, continue 30s, annealing：
60 DEG C, continue 30s, extend：72 DEG C, continue 30s, 34 circulations obtain amplified production.
Preferably, the detailed process of the step (6) is as follows：
1. 8% non-denaturing polyacrylamide gel is prepared：ddH2O 30mL, 10 × TBE 4mL, propylene
Acid amides 6mL, TEMED40 μ l, finally add the μ l of 20% ammonium persulfate 200, wait gel time
40min；
2. voltage 130v, electric current 250mA, power 30w, take 2.5 μ l amplified productions, run glue 1 hour 15 minutes；
3. the glue that will have been run is put into container, adds 200mL distilled water, is put to shaking table, opens shaking table 100r or so, then
Plus 2mL silver nitrate solutiones, shading reaction 10min；
4. liquid is outwelled, distilled water flushing 3 times adds the sodium hydroxide solutions of 200mL 1.5%, puts to shaking table, opens
Shaking table, then add 1mL formalins, shading reaction 10min.
It is a further object of the present invention to provide a kind of primer that can identify black fruit fructus lycii kind and seedling purity.
Above-mentioned purpose of the invention reaches by the following technical programs：
Identify the primer sets of black fruit fructus lycii kind, it is characterised in that：Including 11 pairs of SSR polymorphism primers：
(1)U20
Sense primer：CCCACTTCTCAAAAATGGTACAC, anti-sense primer：ATAGTTGCCAACAAACCCTTCTT；
(2)U21
Sense primer：GGATGAAGAAGAAGAGGATGACA, anti-sense primer：CTTCTCAAAAATGGTACACTGCC；
(3)U23
Sense primer：CTACTTCCATTTGTGGAAAGCTG, anti-sense primer：TAGCCAGTCTAATCTTCGGTTTG；
(4)U25
Sense primer：CAGGAAGGAGAAGAGTCTGATGA, anti-sense primer：TTATCATTAACGGCTTCCATTTG；
(5)U26
Sense primer：AATGGGGAAAGGTAAAGGAAGTT, anti-sense primer：CCTTGTGGAATTTTACTTTCCAAT；
(6)U27
Sense primer：CCACCCAGATAGTGGTGGTAATA, anti-sense primer：GCTGATGTTTTCACATTTGTCAC；
(7)U31
Sense primer：TAGGGTTTGAGGGTTTGAAGAAT, anti-sense primer：ATTATTATGGCTTCTTCACCTGG；
(8)U36
Sense primer：CTACCACTCCAACGTGTACCAAT, anti-sense primer：TTCTTGCTCTAATTCTGAAACCG；
(9)U42
Sense primer：GTCTCCATTTTACCCCTACCAAG, anti-sense primer：TTTGCAAATAAAATGCGATTATTG；
(10)U46
Sense primer：ATGAAGGCAATATTTAGGGCAGT, anti-sense primer：CAATTTCATATTTGTGCTCTGCAT；
(11)D1
Sense primer：TTCCAAGAACATTAGCACAAACA, anti-sense primer：TGGCACTTGTCCTAGTCCTAAAC.
Third object of the present invention is to provide a kind of method for identifying black fruit fructus lycii.
Above-mentioned purpose of the invention reaches by the following technical programs：
A kind of method for identifying black fruit fructus lycii, comprises the following steps：
Testing sample genomic DNA is extracted, with the genomic DNA as masterplate, is carried out with above-mentioned 11 pairs of SSR polymorphism primers
Pcr amplification reaction, described pcr amplification reaction includes as follows：
1st, with genomic DNA as masterplate, with U20, U21, U23, U25, U26, U27, U31, U36, U42, U46, D1 as upper
Anti-sense primer distinguishes amplified production, obtains the different amplified production of length；
2nd, glue is run using polyacrylamide gel electrophoresis or Capillary Electrophoresis, the product according to different length is distinguished not of the same race
Source ground kind.
The advantage of the invention is that：
By the present invention in that on the basis of carrying out transcript profile sequencing with the platforms of Illumina 2000, obtaining substantial amounts of SSR letters
Breath, detects, the part SSR to choosing is verified and polymorphism analysis, so as to obtain with universal polymorphism by PCR
Primer.It is contemplated that using the general nature of the clear and definite black fruit fructus lycii SSR of SSR molecular marker technology, exploitation black fruit fructus lycii SSR draws
Thing, is to carry out black fruit fructus lycii Genetic Diversity of Germplasm, linkage map using SSR molecular marker to build and affiliation research
Lay the foundation.
Below by the drawings and specific embodiments, the present invention will be further described, but is not meant to protect the present invention
Protect the limitation of scope.
Brief description of the drawings
Fig. 1 is the flow chart of the method that the present invention obtains black fruit fructus lycii SSR primers based on transcription sequencing.
Fig. 2 is 10 affiliation figures of provenance black fruit fructus lycii in the embodiment of the present invention 2.
Specific embodiment
Vegetable material used is as follows in example below：
1st, it is respectively as the black fruit fructus lycii of 3 provenances of sample：(1) Aksu, (2) Golmud, (3) quintar；
2nd, extracting the black fruit fructus lycii of 10 provenances of DNA samples is respectively：(1) Aksu (AKS), (2) quintar (J-
T), (3) Shaya (SY), (4) Bachu (BC), (5) Yongjing (YJ), (6) Guazhou County (GZ), (7) Alxa (ALS), (8) Golmud
(GEM), (9) big lattice strangle (DGL), (10) Kuerle (KEL).
As shown in figure 1, the flow chart of the method for black fruit fructus lycii SSR primers is obtained based on transcription sequencing for the present invention；Left side
The experimental procedure of transcript profile sequencing is described:(1) extract sample total serum IgE and using DNase I digestion DNA after, with carrying
The enrichment with magnetic bead eukaryote mRNA of Oligo (dT) (poly thymidine, T repeat oligonucleotides) (if prokaryotes, is then used
Enter next step after kit removal rRNA)；(2) add and interrupt reagent, the thermophilic in constant temperature blending instrument (Thermomixer),
MRNA is broken into short-movie section；(3) mRNA after interrupting is the chain cDNA of templated synthesis one；And then to prepare the synthesis of two chains anti-(4)
System is answered, synthesizes two chain cDNA, and reclaim using kits.
Right side describes SSR exploitations and design of primers：(5) the non repetitive sequence gene (Unigene) to assemble
As reference sequences, all of SSR is found out using SSR softwares MicroSAtellite (MISA)；(6) all SSR are repeated
Unit length of context on non repetitive sequence gene (Unigene) is screened, and is only retained context and is not less than
The SSR of 150bp, primer is designed with it.
Embodiment 1
A kind of method for obtaining black fruit fructus lycii SSR primers based on transcription sequencing, its step is as follows：
1) 3 black fruit fructus lyciis of provenance are taken as sample, aerial part is done into transcript profile sequencing, treatment is divided into control
Group, salt treatment group and salt-mixture treatment group, wherein salt treatment group are mixed using the NaCl solution of 100mL, pH=7,200mmol/L
Close salt treatment group and use 100mL, pH=8.54, Na2CO3、NaCl、Na2SO4、NaHCO3According to 1:9:9:1 mixing it is final concentration of
The mixed solution of 200mmol/L, takes blade and freezes in liquid nitrogen after treatment 6h, carries out transcript profile sequencing；
2) CTAB methods extract black fruit fructus lycii total serum IgE, separate mRNA, are merged into double-strand cDNA, plus adenine and sequence measuring joints,
Fragment to size in 200bp-700bp is expanded, and is examined with the library after 2000 pairs of amplifications of Illumina HiSeq
Survey, using trinity softwares (Trinity, be bythe Broad InstituteThe transcript profile de novo assemblings of exploitation are soft
Part, is made up of three independent software modules：Inchworm, Chrysalis and Butterfly.Three software is processed successively
The reads data of large-scale RNA-seq.) splicing sequencing data, make transcript numbering most long in every gene be 1
unigene；
3) using online microsatellite locus scanning tools SSRIT (or MISA softwares) screening EST-SSR sites, SSR >=
20bp, search is set to that trinucleotide is minimum to be repeated 6 times, and tetranucleotide is minimum to be repeated 5 times, and pentanucleotide is minimum to be repeated 4 times, and six
Nucleotides is minimum to be repeated 3 times；
4) SSR design of primers is carried out using software primer3.0, primer parameter is set to length 18-25bp, Tm 55-65
DEG C, primer size is 100-300bp；
5) 10 DNA of provenance black fruit fructus lycii are extracted using CTAB methods, each provenance takes 25 samples as repetition,
CTAB methods are cetyl trimethylammonium bromide, and DNA of plants extraction method is comprised the following steps that：
1. the blade sample of Excised Embryos is taken, mortar adds (the blade amount of powder of addition in 2.0ml centrifuge tubes after grinding
The tip of the bottom of 2.0ml centrifuge tubes is not crossed；
2. (2%CTAB uses preceding addition 0.5-1% β-sulfydryl second to be rapidly added 65 DEG C of preheated μ l of CTAB 500
Alcohol), 65 DEG C of water-bath half an hour are put into after being well mixed, shake in every ten minutes once, makes CTAB fully be mixed with blade sample；
3. after taking out centrifuge tube placement 2 minutes, isometric chloroform/isoamyl alcohol (24 is added:1) mixed liquor, by mixed liquor with
After CTAB is mixed, vortex mixed makes CTAB be fully contacted with chloroform；
4. 12000rpm is centrifuged 10 minutes, in Aspirate supernatant to 1.5ml centrifuge tubes；
5. dliploid product absolute ethanol washing DNA precipitations are added, 12000rpm centrifugation 10min abandon supernatant；
6. the washed DNA of ethanol is air-dried (superclean bench blows or be concentrated in vacuo instrument)；
7. with 50 μ l ddH after air-drying2O dissolves, and obtains DNA concentration for 30ng/ μ l, and -20 DEG C of preservations are stand-by；
6) primer screening is carried out using PRIMER PREMIER 5.0, primer sequence is sent to company and is synthesized, SSR >=
20bp, search is set to that trinucleotide is minimum to be repeated 6 times, and tetranucleotide is minimum to be repeated 5 times, and pentanucleotide is minimum to be repeated 4 times, and six
Nucleotides is minimum to be repeated 3 times；Primer parameter is set to length 18-25bp, Tm 58-63 DEG C, and primer size is 100-300bp；Choosing
77 pairs of primers are taken, synthesizes laggard performing PCR amplification, the PCR instrument that amplification is used is BioRad T100 models；Reaction system is Mix
10 1 μ l of μ l, DNA, sense primer and anti-sense primer each 1 μ l, ddH2O7μl；Course of reaction：95 DEG C of predegeneration, 5min, denaturation：
95 DEG C, continue 30s, annealing：60 DEG C, continue 30s, extend：72 DEG C, continue 30s, 34 circulations obtain amplified production；
7) polymorphism primer screening is carried out using 8% native polyacrylamide gel electrophoresis, process is as follows：
1. 8% non-denaturing polyacrylamide gel is prepared：ddH2O 30mL, 10 × TBE 4mL, acrylamide 6mL,
The μ l of TEMED 40, finally add the μ l of 20% ammonium persulfate 200, wait gel time 40min；
2. voltage 130v, electric current 250mA, power 30w, take 2.5 μ l amplified productions, run glue 1 hour 15 minutes；
3. the glue that will have been run is put into container, adds 200mL distilled water, is put to shaking table, opens shaking table 100r or so, then
Plus 2mL silver nitrate solutiones, shading reaction 10min；
4. liquid is outwelled, distilled water flushing 3 times adds the sodium hydroxide solutions of 200mL 1.5%, puts to shaking table, opens
Shaking table, then add 1mL formalins, shading reaction 10min；
5. according to the band run out of, same primer amplifies the otherness band of each provenance DNA, then it is assumed that the primer
With polymorphism, 77 to primer in select and draw as the 11 pairs of polymorphisms are high in table 1 below, can stablize amplification in all colonies
Thing.
Table 1
Embodiment 2 is using SSR primers identification black fruit fructus lycii
1st, DNA is extracted
10 DNA of provenance black fruit fructus lycii are extracted using CTAB methods, each provenance takes 25 samples as repeating, has
Body step is as follows：
1. the blade sample of Excised Embryos is taken, mortar adds (the blade amount of powder of addition in 2.0ml centrifuge tubes after grinding
The tip of the bottom of 2.0ml centrifuge tubes is not crossed；
2. (2%CTAB uses preceding addition 0.5-1% β-sulfydryl second to be rapidly added 65 DEG C of preheated μ l of CTAB 500
Alcohol), 65 DEG C of water-bath half an hour are put into after being well mixed, shake in every ten minutes once, makes CTAB fully be mixed with blade sample；
3. after taking out centrifuge tube placement 2 minutes, isometric chloroform/isoamyl alcohol (24 is added:1) mixed liquor, by mixed liquor with
After CTAB is mixed, vortex mixed makes CTAB be fully contacted with chloroform；
4. 12000rpm is centrifuged 10 minutes, in Aspirate supernatant to 1.5ml centrifuge tubes；
5. dliploid product absolute ethanol washing DNA precipitations are added, 12000rpm centrifugation 10min abandon supernatant；
6. the washed DNA of ethanol is air-dried (superclean bench blows or be concentrated in vacuo instrument)；
7. with 50 μ l ddH after air-drying2O dissolves, and obtains DNA concentration for 30ng/ μ l, and -20 DEG C of preservations are stand-by；
2nd, PCR amplifications
It is masterplate with the DNA that step 1 is extracted, the 11 pairs of SSR polymorphism primers developed with embodiment 1 enter performing PCR and expand, and expand
Increasing method with embodiment 1, specially：With genomic DNA as masterplate, with U20, U21, U23, U25, U26, U27, U31, U36,
U42, U46, D1 are that upstream and downstream primer distinguishes amplified production, obtain the different amplified production of length；
3rd, test and analyze
The amplified production of gained different length in step 2 is delivered into sequencing company and uses Capillary Electrophoresis order-checking, sequencing knot
Fruit detected using the hereditary variation that POPGENE 32 (Yeh et al., 2000) software calculates each colony of colony, Jin Erfen
The genetic diversity of Xi Ge colonies.Using unweighted mean method, based on Nei (1978) genetic distance, entered using NTSYSpc 2.1
Row cluster analysis.Result is as shown in Fig. 2 be 10 affiliation figures of provenance black fruit fructus lycii in the embodiment of the present invention 2.
Figure it is seen that Aksu is close with Kuerle, Xinjiang region kind is belonged to together；Bachu is close with Shaya, belongs to together
Xinjiang region kind；Golmud is close with big lattice Le, belongs to Qinghai Area kind together；Guazhou County is most close with Yongjing, and all with quintar
Close, these three belong to Gansu province kind.
Each technical characteristic of embodiment described above to make description succinct, can not implemented to the above in any combination
The all possible combination of each technical characteristic of example is described, as long as however, the combination of these technical characteristics not contradiction, all should
It is considered as the scope of this specification record.
The method for obtaining black fruit fructus lycii SSR primers based on transcription sequencing of the invention, can be used as base between correction distant relative's species
Because of group linkage map and the method for comparative map, there is value higher in these two aspects.The biggest advantage is that exploitation letter
Single, quick, expense is low.Using the general nature of the clear and definite black fruit fructus lycii SSR of SSR molecular marker technology, exploitation black fruit fructus lycii SSR draws
Thing, is to carry out black fruit fructus lycii Genetic Diversity of Germplasm, linkage map using SSR molecular marker to build and affiliation research
Lay the foundation, so as to preferably apply in molecular breeding.
The method for obtaining black fruit fructus lycii SSR primers based on transcription sequencing of the invention, obtains black in high-flux sequence means
On the basis of fruit matrimony vine transcript profile sequence, SSR sites are screened using online microsatellite locus scanning tools to gained sequence.With Ah
10 lycium ruthenicums of introduces a collection such as gram Soviet Union, Bachu, Shaya are examination material, and design primer carries out the checking of SSR sites, and sets up and optimize
SSR-PCR reaction systems, respectively with agarose and Polyacrylamide Gel Electrophoresis product.The mark succeeded in developing can be answered
For black fruit fructus lycii molecular breeding, so that the affiliation preferably between research Different Provenances, to obtain a large amount of outstanding germplasm
Resource lays the foundation.
SEQUENCE LISTING
<110>Beijing Forestry University
<120>Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers
<130>
<160> 22
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>U20 sense primers
<400> 1
cccacttctc aaaaatggta cac 23
<210> 2
<211> 23
<212> DNA
<213>U20 anti-sense primers
<400> 2
atagttgcca acaaaccctt ctt 23
<210> 3
<211> 23
<212> DNA
<213>U21 sense primers
<400> 3
ggatgaagaa gaagaggatg aca 23
<210> 4
<211> 23
<212> DNA
<213>U21 anti-sense primers
<400> 4
cttctcaaaa atggtacact gcc 23
<210> 5
<211> 23
<212> DNA
<213>U23 sense primers
<400> 5
ctacttccat ttgtggaaag ctg 23
<210> 6
<211> 23
<212> DNA
<213>U23 anti-sense primers
<400> 6
tagccagtct aatcttcggt ttg 23
<210> 7
<211> 23
<212> DNA
<213>U25 sense primers
<400> 7
caggaaggag aagagtctga tga 23
<210> 8
<211> 23
<212> DNA
<213>U25 anti-sense primers
<400> 8
ttatcattaa cggcttccat ttg 23
<210> 9
<211> 23
<212> DNA
<213>U26 sense primers
<400> 9
aatggggaaa ggtaaaggaa gtt 23
<210> 10
<211> 24
<212> DNA
<213>U26 anti-sense primers
<400> 10
ccttgtggaa ttttactttc caat 24
<210> 11
<211> 23
<212> DNA
<213>U27 sense primers
<400> 11
ccacccagat agtggtggta ata 23
<210> 12
<211> 23
<212> DNA
<213>U27 anti-sense primers
<400> 12
gctgatgttt tcacatttgt cac 23
<210> 13
<211> 23
<212> DNA
<213>U31 sense primers
<400> 13
tagggtttga gggtttgaag aat 23
<210> 14
<211> 23
<212> DNA
<213>U31 anti-sense primers
<400> 14
attattatgg cttcttcacc tgg 23
<210> 15
<211> 23
<212> DNA
<213>U36 sense primers
<400> 15
ctaccactcc aacgtgtacc aat 23
<210> 16
<211> 23
<212> DNA
<213>U36 anti-sense primers
<400> 16
ttcttgctct aattctgaaa ccg 23
<210> 17
<211> 23
<212> DNA
<213>U42 sense primers
<400> 17
gtctccattt tacccctacc aag 23
<210> 18
<211> 24
<212> DNA
<213>U42 anti-sense primers
<400> 18
tttgcaaata aaatgcgatt attg 24
<210> 19
<211> 23
<212> DNA
<213>U46 sense primers
<400> 19
atgaaggcaa tatttagggc agt 23
<210> 20
<211> 24
<212> DNA
<213>U46 anti-sense primers
<400> 20
caatttcata tttgtgctct gcat 24
<210> 21
<211> 23
<212> DNA
<213>D1 sense primers
<400> 21
ttccaagaac attagcacaa aca 23
<210> 22
<211> 23
<212> DNA
<213>D1 anti-sense primers
<400> 22
tggcacttgt cctagtccta aac 23
Claims (10)
1. a kind of that the method for obtaining black fruit fructus lycii SSR primers is sequenced based on transcription, its step is as follows：
(1) 3 black fruit fructus lyciis of provenance are taken as sample, aerial part is done into transcript profile sequencing；
(2) online microsatellite locus scanning tools SSRIT or MISA software detection EST-SSR sites are used, SSR >=20bp is searched
Rope is set to that trinucleotide is minimum to be repeated 6 times, and tetranucleotide is minimum to be repeated 5 times, and pentanucleotide is minimum to be repeated 4 times, Hexanucleotide
It is minimum to be repeated 3 times；
(3) SSR design of primers is carried out using software primer3.0, primer parameter is set to length 18-25bp, Tm 55-65 DEG C,
Primer size is 100-300bp；
(4) 10 DNA of provenance black fruit fructus lycii are extracted using CTAB methods, each provenance takes 25 samples as repetition；
(5) primer in (3) and their corresponding SSR sites are carried out using PRIMER PREMIER 5.0 further random
Screening test, the primer sequence for being filtered out is sent to company and is synthesized, and synthesizes laggard performing PCR amplification；
(6) polymorphism primer screening is carried out using 8% native polyacrylamide gel electrophoresis：The bar according to running cementing fruit
Band, primer of the same race can amplify differential band on Different Provenances ground, it is believed that with polymorphism.
It is 2. according to claim 1 that the method for obtaining black fruit fructus lycii SSR primers is sequenced based on transcription, it is characterised in that：Institute
State transcript profile described in step (1) sequencing in：Treatment is divided into control group, salt treatment group and salt-mixture treatment group, wherein salt treatment
Group uses the NaCl solution of 100mL, pH=7,200mmol/L, salt-mixture treatment group to use 100mL, pH=8.54, Na2CO3、
NaCl、Na2SO4、NaHCO3According to 1:9:9:The mixed solution of the final concentration of 200mmol/L of 1 mixing.
It is 3. according to claim 2 that the method for obtaining black fruit fructus lycii SSR primers is sequenced based on transcription, it is characterised in that：Institute
State comprising the following steps that for the sequencing of transcript profile described in step (1)：CTAB methods extract black fruit fructus lycii total serum IgE, separate mRNA, merge
Into double-strand cDNA, plus adenine and sequence measuring joints, the fragment to size in 200bp-700bp is expanded, and uses Illumina
Library after the amplification of HiSeq 2000 couples detected, sequencing data is spliced using trinity softwares, is made most long in every gene
Transcript numbering be 1 unigene.
It is 4. according to claim 3 that the method for obtaining black fruit fructus lycii SSR primers is sequenced based on transcription, it is characterised in that：Institute
State concretely comprising the following steps for further random screening inspection described in step (5)：SSR >=20bp, search is set to trinucleotide most
Low to be repeated 6 times, tetranucleotide is minimum to be repeated 5 times, and pentanucleotide is minimum to be repeated 4 times, and Hexanucleotide is minimum to be repeated 3 times；Primer is joined
Number is set to length 18-25bp, Tm 58-63 DEG C, and primer size is 100-300bp；Choose 77 pairs of primers.
It is 5. according to claim 4 that the method for obtaining black fruit fructus lycii SSR primers is sequenced based on transcription, it is characterised in that：Institute
It is BioRad T100 to state PCR described in step (5) and expand the PCR instrument for using.
It is 6. according to claim 5 that the method for obtaining black fruit fructus lycii SSR primers is sequenced based on transcription, it is characterised in that：Institute
State comprising the following steps that for 10 DNA of provenance black fruit fructus lycii of extraction described in step (4)：
1. the blade sample of Excised Embryos is taken, mortar is added in 2.0ml centrifuge tubes after grinding, and the blade amount of powder of addition did not had
The tip of the bottom of 2.0ml centrifuge tubes；
2. 65 DEG C of preheated CTAB 2%CTAB are rapidly added, preceding addition 0.5-1% beta -mercaptoethanols, 500 μ l, mixing is used
65 DEG C of water-bath half an hour are put into after uniform, shake in every ten minutes once, makes CTAB fully be mixed with blade sample；
3. after taking out centrifuge tube placement 2 minutes, isometric 24 are added:1 chloroform/isoamyl alcohol mixed liquor, by mixed liquor with
After CTAB is mixed, vortex mixed makes CTAB be fully contacted with chloroform；
4. 12000rpm is centrifuged 10 minutes, in Aspirate supernatant to 1.5ml centrifuge tubes；
5. dliploid product absolute ethanol washing DNA precipitations are added, 12000rpm centrifugation 10min abandon supernatant；
6. the washed DNA of ethanol is air-dried；
7. with 50 μ l ddH after air-drying2O dissolves, and obtains DNA concentration for 30ng/ μ l, and -20 DEG C of preservations are stand-by.
It is 7. according to claim 6 that the method for obtaining black fruit fructus lycii SSR primers is sequenced based on transcription, it is characterised in that：Institute
It is the 1 μ l of μ l, DNA of Mix 10, sense primer and each 1 μ of anti-sense primer to state the amplifications of PCR described in step (5) reaction system used
L, ddH2O7μl；Course of reaction：95 DEG C of predegeneration, 5min, denaturation：95 DEG C, continue 30s, annealing：60 DEG C, continue 30s, extend：
72 DEG C, continue 30s, 34 circulations obtain amplified production.
It is 8. according to claim 7 that the method for obtaining black fruit fructus lycii SSR primers is sequenced based on transcription, it is characterised in that：Institute
The detailed process for stating step (6) is as follows：
1. 8% non-denaturing polyacrylamide gel is prepared：ddH2O 30mL, 10 × TBE 4mL, acrylamide 6mL, TEMED 40 μ
L, finally adds the μ l of 20% ammonium persulfate 200, waits gel time 40min；
2. voltage 130v, electric current 250mA, power 30w, take 2.5 μ l amplified productions, run glue 1 hour 15 minutes；
3. the glue that will have been run is put into container, adds 200mL distilled water, is put to shaking table, opens shaking table 100r or so, then add
2mL silver nitrate solutiones, shading reaction 10min；
4. liquid is outwelled, distilled water flushing 3 times adds the sodium hydroxide solutions of 200mL 1.5%, puts to shaking table, opens shaking table,
Again plus 1mL formalins, shading reaction 10min.
9. the primer sets of black fruit fructus lycii kind are identified, it is characterised in that：Including 11 pairs of SSR primers：
(1)U20
Sense primer：CCCACTTCTCAAAAATGGTACAC, anti-sense primer：ATAGTTGCCAACAAACCCTTCTT；
(2)U21
Sense primer：GGATGAAGAAGAAGAGGATGACA, anti-sense primer：CTTCTCAAAAATGGTACACTGCC；
(3)U23
Sense primer：CTACTTCCATTTGTGGAAAGCTG, anti-sense primer：TAGCCAGTCTAATCTTCGGTTTG；
(4)U25
Sense primer：CAGGAAGGAGAAGAGTCTGATGA, anti-sense primer：TTATCATTAACGGCTTCCATTTG；
(5)U26
Sense primer：AATGGGGAAAGGTAAAGGAAGTT, anti-sense primer：CCTTGTGGAATTTTACTTTCCAAT；
(6)U27
Sense primer：CCACCCAGATAGTGGTGGTAATA, anti-sense primer：GCTGATGTTTTCACATTTGTCAC；
(7)U31
Sense primer：TAGGGTTTGAGGGTTTGAAGAAT, anti-sense primer：ATTATTATGGCTTCTTCACCTGG；
(8)U36
Sense primer：CTACCACTCCAACGTGTACCAAT, anti-sense primer：TTCTTGCTCTAATTCTGAAACCG；
(9)U42
Sense primer：GTCTCCATTTTACCCCTACCAAG, anti-sense primer：TTTGCAAATAAAATGCGATTATTG；
(10)U46
Sense primer：ATGAAGGCAATATTTAGGGCAGT, anti-sense primer：CAATTTCATATTTGTGCTCTGCAT；
(11)D1
Sense primer：TTCCAAGAACATTAGCACAAACA, anti-sense primer：TGGCACTTGTCCTAGTCCTAAAC.
10. a kind of method for identifying black fruit fructus lycii, comprises the following steps：
Testing sample genomic DNA is extracted, with the genomic DNA as masterplate, is carried out with 11 pairs of primers described in claim 9
Pcr amplification reaction, described pcr amplification reaction includes as follows：
(1) it is upper and lower with U20, U21, U23, U25, U26, U27, U31, U36, U42, U46, D1, with genomic DNA as masterplate
Trip primer difference amplified production, obtains the different amplified production of length；
(2) glue, is run using polyacrylamide gel electrophoresis or Capillary Electrophoresis, the product according to different length distinguishes Different Provenances
Ground kind.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710038608.7A CN106754886B (en) | 2017-01-19 | 2017-01-19 | The method for obtaining black fruit fructus lycii SSR primer is sequenced based on transcription |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710038608.7A CN106754886B (en) | 2017-01-19 | 2017-01-19 | The method for obtaining black fruit fructus lycii SSR primer is sequenced based on transcription |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106754886A true CN106754886A (en) | 2017-05-31 |
| CN106754886B CN106754886B (en) | 2019-12-03 |
Family
ID=58943678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710038608.7A Expired - Fee Related CN106754886B (en) | 2017-01-19 | 2017-01-19 | The method for obtaining black fruit fructus lycii SSR primer is sequenced based on transcription |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754886B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107201408A (en) * | 2017-07-15 | 2017-09-26 | 中国热带农业科学院南亚热带作物研究所 | A kind of method that exploitation sisal hemp SSR primers are sequenced based on transcript profile |
| CN107365874A (en) * | 2017-09-21 | 2017-11-21 | 中国农业科学院蔬菜花卉研究所 | Henry pocket orchid EST SSR label primers, its development approach and its application |
| CN108424975A (en) * | 2018-05-04 | 2018-08-21 | 广西壮族自治区农业科学院经济作物研究所 | Pueraria lobata SSR label primer pair based on the exploitation of transcript profile sequence and its application |
| CN108676907A (en) * | 2018-06-04 | 2018-10-19 | 贵州师范大学 | A method of it is sequenced based on transcript profile and obtains green hedge bavin SSR primers |
| CN109207560A (en) * | 2018-09-19 | 2019-01-15 | 广西壮族自治区林业科学研究院 | A method of exploitation bougainvillea SSR primer is sequenced based on transcript profile |
| CN109652587A (en) * | 2019-02-19 | 2019-04-19 | 江苏省中国科学院植物研究所 | A method of Chinese ilex germplasm is identified using the SSR molecular marker of transcript profile sequencing |
| CN112349347A (en) * | 2020-09-25 | 2021-02-09 | 江苏农林职业技术学院 | Development method of strawberry functional gene linked SSR marker |
| CN113684296A (en) * | 2021-07-20 | 2021-11-23 | 浙江理工大学 | Sealwort EST-SSR primer group development and application thereof in genetic diversity |
| CN114107538A (en) * | 2021-09-23 | 2022-03-01 | 北京林业大学 | Core primer group based on Chinese wolfberry variety SSR (simple sequence repeat) markers and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642912A (en) * | 2013-11-29 | 2014-03-19 | 中国农业科学院作物科学研究所 | Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing |
| CN105274198A (en) * | 2015-05-26 | 2016-01-27 | 江苏省农业科学院 | Method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome |
| CN106011228A (en) * | 2016-04-20 | 2016-10-12 | 中国科学院华南植物园 | EST-SSR core primer group for identifying variety of Chinese wolfberry and identification method and application thereof |
-
2017
- 2017-01-19 CN CN201710038608.7A patent/CN106754886B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642912A (en) * | 2013-11-29 | 2014-03-19 | 中国农业科学院作物科学研究所 | Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing |
| CN105274198A (en) * | 2015-05-26 | 2016-01-27 | 江苏省农业科学院 | Method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome |
| CN106011228A (en) * | 2016-04-20 | 2016-10-12 | 中国科学院华南植物园 | EST-SSR core primer group for identifying variety of Chinese wolfberry and identification method and application thereof |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107201408A (en) * | 2017-07-15 | 2017-09-26 | 中国热带农业科学院南亚热带作物研究所 | A kind of method that exploitation sisal hemp SSR primers are sequenced based on transcript profile |
| CN107365874B (en) * | 2017-09-21 | 2020-04-07 | 中国农业科学院蔬菜花卉研究所 | Huntington harlequin EST-SSR (expressed sequence tag-simple sequence repeat) labeled primer as well as development method and application thereof |
| CN107365874A (en) * | 2017-09-21 | 2017-11-21 | 中国农业科学院蔬菜花卉研究所 | Henry pocket orchid EST SSR label primers, its development approach and its application |
| CN108424975A (en) * | 2018-05-04 | 2018-08-21 | 广西壮族自治区农业科学院经济作物研究所 | Pueraria lobata SSR label primer pair based on the exploitation of transcript profile sequence and its application |
| CN108424975B (en) * | 2018-05-04 | 2023-04-18 | 广西壮族自治区农业科学院经济作物研究所 | Pueraria SSR marker primer pair developed based on transcriptome sequence and application thereof |
| CN108676907A (en) * | 2018-06-04 | 2018-10-19 | 贵州师范大学 | A method of it is sequenced based on transcript profile and obtains green hedge bavin SSR primers |
| CN109207560A (en) * | 2018-09-19 | 2019-01-15 | 广西壮族自治区林业科学研究院 | A method of exploitation bougainvillea SSR primer is sequenced based on transcript profile |
| CN109652587A (en) * | 2019-02-19 | 2019-04-19 | 江苏省中国科学院植物研究所 | A method of Chinese ilex germplasm is identified using the SSR molecular marker of transcript profile sequencing |
| CN109652587B (en) * | 2019-02-19 | 2020-10-23 | 江苏省中国科学院植物研究所 | Method for identifying holly germplasm by using SSR molecular marker of transcriptome sequencing |
| CN112349347A (en) * | 2020-09-25 | 2021-02-09 | 江苏农林职业技术学院 | Development method of strawberry functional gene linked SSR marker |
| CN112349347B (en) * | 2020-09-25 | 2023-11-10 | 江苏农林职业技术学院 | Strawberry functional gene linkage SSR marker development method |
| CN113684296A (en) * | 2021-07-20 | 2021-11-23 | 浙江理工大学 | Sealwort EST-SSR primer group development and application thereof in genetic diversity |
| CN114107538A (en) * | 2021-09-23 | 2022-03-01 | 北京林业大学 | Core primer group based on Chinese wolfberry variety SSR (simple sequence repeat) markers and application thereof |
| CN114107538B (en) * | 2021-09-23 | 2023-11-07 | 北京林业大学 | Core primer group based on wolfberry variety SSR (simple sequence repeat) markers and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106754886B (en) | 2019-12-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754886B (en) | The method for obtaining black fruit fructus lycii SSR primer is sequenced based on transcription | |
| CN105950747A (en) | Rice blast resistance gene Pi1 functional specificity molecular marker and application thereof | |
| CN107164459B (en) | Functional marker for identifying and screening tea trees with high catechin indexes and application of functional marker | |
| CN110951911B (en) | Tilia EST-SSR primer based on transcriptome as well as screening method and application thereof | |
| CN109536632B (en) | Rhododendron dauricum SSR primer pair developed based on transcriptome sequencing, screening method and application | |
| CN108754018B (en) | Screening method and application of acanthopanax senticosus target gene SSR molecular marker | |
| CN107475418B (en) | Molecular marker closely linked with tillering character of millet, primer and application | |
| CN107828858A (en) | A kind of method that exploitation beggar-ticks plant SSR primers are sequenced based on transcript profile | |
| CN112301142B (en) | Leymus speetanus anti-powdery mildew gene Pm12 molecular marker and application thereof | |
| Sogbesan et al. | DNA barcoding of tilapia species (Pisces: Cichlidae) from North-Eastern Nigeria | |
| CN116179738B (en) | Core primer group for identifying SSR molecular markers of agilawood varieties and application | |
| CN112349347B (en) | Strawberry functional gene linkage SSR marker development method | |
| CN114107545B (en) | CAPS molecular marker of wax gourd fruit and flour wax powder gene and application thereof | |
| CN116144819A (en) | SNP molecular marker closely linked with main effect QTL of pumpkin pulp carotenoid and application of SNP molecular marker | |
| CN108517373A (en) | It one InDel labeled primer pair for distinguishing five pepper cultivation kinds and its applies | |
| CN113493851B (en) | Application of 32 soybean InDel markers in detection of soybean genetic diversity | |
| CN107365873A (en) | Molecular labeling and its application with the millet leaf sheath color linkage of characters | |
| CN103540674B (en) | Method for authenticating geo-authentic crude drug radix ophiopogonis in different producing areas and kit for method | |
| CN106929587A (en) | The PCR primer method for designing of the genetic fragment containing SSR sites in cold orchid genome | |
| CN111363844A (en) | Water chestnut SSR primer group and application thereof | |
| CN108342503A (en) | The merely construction method of red Chinese scholartree SSR marker finger-print and application | |
| CN113136449B (en) | Molecular marker and primer pair for penis microsatellite, and preparation method and application thereof | |
| CN107557486A (en) | EST SSR primer sets and its acquisition methods based on the exploitation of root of bidentate achyranthes transcript profile | |
| CN113604593B (en) | DNA fragment related to linoleic acid content in oil tea seed oil and application thereof | |
| CN113584203B (en) | DNA fragment related to single fruit quality of camellia oleifera, SNP molecular marker closely linked with DNA fragment and application of DNA fragment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191203 |