CN107287288A - The EST SSR markers special primer and screening technique of a kind of Chinese torreya transcript profile sequence - Google Patents
The EST SSR markers special primer and screening technique of a kind of Chinese torreya transcript profile sequence Download PDFInfo
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Abstract
The invention belongs to molecular biology DNA marker technology and application field, and in particular to a kind of screening technique of the EST SSR marker special primers of Chinese torreya transcript profile sequence, including：Chinese torreya benevolence transcript profile data are justified to thin Chinese torreya kind Renhe first and use Trinity software combinations, transcript sequence is obtained；Then more than the 1kb obtained using MISA programs to screening Unigene does the search of SSR sites, be respectively successively 9 according to number of repetition for 26 base repeat unit numbers of repetition, the microsatellite fragment of 8,5,5,5 times carry out screening separation, so as to obtain the ESTs sequences repeated containing microsatellite；Then design of primers is carried out using Primer3 programs, finally the repeatability, stability and polymorphism to EST SSR markers carry out overall merit, obtain EST SSR markers.The present invention can be conveniently and quickly used for doing the analysis that Chinese torreya belongs to different type germ plasm resource and genetic diversity in inter-species and Chinese torreya kind.
Description
Technical field
The invention belongs to molecular biology DNA marker technology and application field, and in particular to Chinese torreya (Torreya
Grandis) EST (the Express Sequence Tag-Simple Sequence Repeat, expressed sequence of transcript profile sequence
Label)-SSR (simple sequence repeats) mark special primer and screening technique, belong to molecular marking technique field.
Background technology
Chinese torreya (Torreya grandis) is under the jurisdiction of Chinese torreya category (Torreya Arn), and whole world Chinese torreya platymiscium has 7 kind 2
Mutation, including Florida Chinese torreya (Torreya taxifolia), California nutmeg (Torreya californica), kaya
(Torreya nucifera), Chinese torreya (Torreya grandis), Bashan Mountain Chinese torreya (Torreya fargesii), Sichuan Chinese torreya
(Torreya parvifolia) Yunnan Chinese torreya (Torreya fargesii var.yunnanensis) and Jiulong shan Mountain Chinese torreya
(Torreya grandis Fort.ex Lind.var.jiulongshanensis), Chinese torreya is that China's distribution is most wide, cultivates profit
With history at most, one of economic value highest seeds.Character variation is complicated in Chinese torreya species kind, there is many natural variation classes
Type, also the Chinese torreya kind comprising artificial cultivation.Chinese torreya has character stable, best in quality (benevolence oil content up to 65% or so, no
Saturated fatty acid is up to more than 80%) the features such as.But it is prominent to there is single, variety deterioration of cultivar etc. in current Chinese torreya production
Problem, while Chinese torreya juvenile phase is long, carrying out fine-variety breeding using conventional cross-breeding means generally requires more than ten years or even decades
Time, this greatly hinder Chinese torreya fine-variety breeding, new varieties initiative.Therefore, inhomogeneity in Chinese torreya and its Chinese torreya kind is developed
The molecular genetic marker of type, detection genetic diversity, research genetic structure, and then implement marker assisted selection, educated for shortening
The process of kind, raising breeding efficiency, which all has, to be of great significance.
SSR is the genetic marker being widely present in eucaryon and prokaryotic gene group, with codominance, spreadability it is wide,
Contain much information, the advantage such as simple to operate.It is widely used in the fruit trees such as apple, cherry, citrus, grape.Traditional SSR primers
Mainly obtained from genomic library, its step is complicated, workload is big, and development cost is high.In recent years, high throughput sequencing technologies are fast
Exhibition is hailed, EST-SSR (microsatellite) is the accessory substance of ESTs sequencing plans, and development cost is relatively low, codominant inheritance, polymorphism
Height, repeatability and stability are good, are best suitable at present for one of labelling technique of Germplasm Identification analysis.In Chinese torreya platymiscium molecule
In terms of marker development, Jin Zexin etc. establishes Torreya jackii ISSR-PCR reaction system, and it is real that Liang Dan et al. establishes Chinese torreya AFLP
Test system and the identification of Chinese torreya male and female plant has been carried out using AFLP, Liu Haokai etc. utilizes the female Chinese torreya 4 of SRAP technical research
The genetic diversity of population, wears the positive sex for waiting and Chinese torreya seedling being identified with RAPD and SCAR mark, and Zhang Dangquan etc. utilizes RAPD
Several cultivars of Chinese torreya are classified.The blade transcript profile data based on 1 Chinese torreya tree such as Yi, from 4600
1713 SSR sites are obtained in Unigene, 108 pairs of primers are devised, wherein 37 pairs of primers have amplified production, 19 pairs of primers exist
Polymorphism is detected in test sample.Further, since EST is the expression fragment of functional gene, the microsatellite mark found wherein
Advantage of the note with direct mark function gene, and may be associated with some production traits, this to genetic map construction and
Marker assisted selection has very high application value.
The SSR primer marks carried out using ' thin Chinese torreya ' and circle Chinese torreya seed transcript profile data and exploitation, may be important Chinese torreya
Economic characters are associated, and reach the purpose of marker assisted selection, and can further carry out the research of Chinese torreya functional gene.At present
It yet there are no Chinese torreya and belong to the research report that different type EST-SSR is marked in inter-species and Chinese torreya kind.
The content of the invention
The present invention is intended to provide a kind of based on thin Chinese torreya seed transcript profile sequence and the sequence exploitation of circle Chinese torreya seed transcript profile
EST- SSR special primers, the EST-SSR based on thin Chinese torreya seed transcript profile sequence and the sequence exploitation of circle Chinese torreya seed transcript profile is special
Primer has the advantages that amplification is stable, electrophoretic band is clear, rich polymorphism, can be effectively used for Chinese torreya and belongs in inter-species and Chinese torreya kind not
The research fields such as same type analysis of genetic diversity, Variety fingerprinting structure and molecular marker assisted selection breeding.
To realize the goal of the invention of the present invention, inventor provides following technical scheme：
Inventor provide firstly the EST-SSR mark special primers of Chinese torreya transcript profile sequence, described special primer point
It is not：
ZAFU-1：
F:GGCTATGCTACACCCAAAGAA (SEQ ID NO.1),
R：GGGGCACCACCTATTGTATG(SEQ ID NO.2)；
ZAFU-2：
F：TCAAAGTGCAACCGGTACAA (SEQ ID NO.3),
R:CAACAGGCCAACATGGAGTA(SEQ ID NO.4)；
ZAFU-3：
F：GGGTTACCCCCTTGCTTTAT (SEQ ID NO.5),
R:CCCTACTTTATTTCCGTGCG(SEQ ID NO.6)；
ZAFU-4：
F：GAATTCCCATTCCCATTGTG (SEQ ID NO.7),
R:ACCCCCTTCTGCTCTGATTT(SEQ ID NO.8)；
ZAFU-5：
F：AATGAATGCGTGTTACGCTG (SEQ ID NO.9),
R:TTGGAGCGGAAGGAATAATG(SEQ ID NO.10)；
ZAFU-6：
F:CCAATTTGTGGAGCGTTTCT (SEQ ID NO.11),
R:TGTGGAAAGGTGGTGAACAA(SEQ ID NO.12)；
ZAFU-7：
F:TTTTCCAACTCCAACCCTTG (SEQ ID NO.13),
R:ATGTTTGGGGTTGACGTGTT(SEQ ID NO.14)；
ZAFU-8：
F:AATTGGCCCTTCATTCAACA (SEQ ID NO.15),
R:CTAGTGGGTGCATTTGAGCC(SEQ ID NO.16)；
ZAFU-9：
F:GGAGCGAGCGTAGCGTATAG (SEQ ID NO.17),
R:GTTGCTGCATCTTCCTCCTC(SEQ ID NO.18)；
ZAFU-10：
F:GCCTGGTCTGCTTCTTGTTC (SEQ ID NO.19),
R:AGGCAAAGGCTGCAATAGAA(SEQ ID NO.20)；
ZAFU-11：
F:ACATCTGCAAGGCAAGGTTC (SEQ ID NO.21),
R:TTGAATTTTCACCAGGCTCC(SEQ ID NO.22)；
ZAFU-12：
F:TGCTAATGTTTGCTTTGAATGG (SEQ ID NO.23),
R:AACAACCTAAACTTTTGCCGAA(SEQ ID NO.24)；
ZAFU-13：
F:GAAATTAAACGCGTTCAGGG (SEQ ID NO.25),
R:CATTGCTGTCCGTCTCGTAA(SEQ ID NO.26)；
ZAFU-14：
F:CACAATCCTCCATTCATCCA (SEQ ID NO.27),
R:GCCACTGGTGTGATTCTCAA(SEQ ID NO.28)；
ZAFU-15：
F:ATTTGATACGACGGAAACGG (SEQ ID NO.29),
R:GGTGGTCAATATCCCATGCT(SEQ ID NO.30)；
ZAFU-16：
F:AAGGTTGCCACCTCAGTCAC (SEQ ID NO.31),
R:ACAGAACGTCTCCAACCGAC(SEQ ID NO.32)。
The step of Chinese torreya EST-SSR is analyzed is from the thin Chinese torreya benevolence transcript profile data in inventor laboratory and the benevolence transcription of circle Chinese torreya
Group data use Trinity software combinations, obtain transcript sequence.Assembling be obtained 246862 Transcript and
142213 Unigene.Utilize MISA programs (MIcroSAtellite identification tool, http://
Pgrc.ipk-gatersleben.de/misa/misa.html) more than the 1kb obtained to screening Unigene does SSR sites
Search.Be respectively successively 9 according to number of repetition for 2-6 base repeat unit number of repetition, the microsatellite of 8,5,5,5 times
Fragment carries out screening separation, so as to obtain the ESTs sequences repeated containing microsatellite.
Primer is designed to Chinese torreya EST-SSR, the flanking sequence design using Primer3 programs at microsatellite two ends is specifically drawn
Thing.The main principle of design of primers：Primer length is controlled in 20bp；58 DEG C -62 DEG C of annealing temperature；The Tm value phases of upstream and downstream primer
Difference is not more than 2 DEG C；PCR primer length is between 100-300bp；G/C content is between 40%-60%；Each site produces 3 pairs
Candidate drugs.
Inventor additionally provides a kind of screening technique of the EST-SSR marks of Chinese torreya transcript profile sequence, including：First to thin
Chinese torreya kind Renhe circle Chinese torreya benevolence transcript profile data use Trinity software combinations, obtain transcript sequence, and assembling is obtained 246862
Bar Transcript and 142213 Unigene；Then MISA programs (MIcroSAtellite identification are utilized
tool,http://pgrc.ipk-gatersleben.de/misa/misa.html) to screening obtained more than 1kb's
Unigene does the search of SSR sites, be respectively successively 9 according to number of repetition for 2-6 base repeat unit number of repetition, 8,
5th, 5, the microsatellite fragment of 5 times carries out screening separation, so as to obtain the ESTs sequences repeated containing microsatellite；Then utilize
Primer3 programs carry out design of primers, the main principle of design of primers：Primer length control is in 58 DEG C -62 of 20bp, annealing temperature
DEG C, the Tm values of upstream and downstream primer be more or less the same in 2 DEG C, PCR primer length between 100-300bp, G/C content is in 40%-
Between 60%, each site produce 3 pairs of candidate drugs；Finally EST-SSR repeatability, stability and the polymorphisms marked is entered
Row overall merit, obtains EST-SSR marks, and the special primer of the described EST-SSR screened marks is respectively：
ZAFU-1：
F:GGCTATGCTACACCCAAAGAA (SEQ ID NO.1),
R：GGGGCACCACCTATTGTATG(SEQ ID NO.2)；
ZAFU-2：
F：TCAAAGTGCAACCGGTACAA (SEQ ID NO.3),
R:CAACAGGCCAACATGGAGTA(SEQ ID NO.4)；
ZAFU-3：
F：GGGTTACCCCCTTGCTTTAT (SEQ ID NO.5),
R:CCCTACTTTATTTCCGTGCG(SEQ ID NO.6)；
ZAFU-4：
F：GAATTCCCATTCCCATTGTG (SEQ ID NO.7),
R:ACCCCCTTCTGCTCTGATTT(SEQ ID NO.8)；
ZAFU-5：
F：AATGAATGCGTGTTACGCTG (SEQ ID NO.9),
R:TTGGAGCGGAAGGAATAATG(SEQ ID NO.10)；
ZAFU-6：
F:CCAATTTGTGGAGCGTTTCT (SEQ ID NO.11),
R:TGTGGAAAGGTGGTGAACAA(SEQ ID NO.12)；
ZAFU-7：
F:TTTTCCAACTCCAACCCTTG (SEQ ID NO.13),
R:ATGTTTGGGGTTGACGTGTT(SEQ ID NO.14)；
ZAFU-8：
F:AATTGGCCCTTCATTCAACA (SEQ ID NO.15),
R:CTAGTGGGTGCATTTGAGCC(SEQ ID NO.16)；
ZAFU-9：
F:GGAGCGAGCGTAGCGTATAG (SEQ ID NO.17),
R:GTTGCTGCATCTTCCTCCTC(SEQ ID NO.18)；
ZAFU-10：
F:GCCTGGTCTGCTTCTTGTTC (SEQ ID NO.19),
R:AGGCAAAGGCTGCAATAGAA(SEQ ID NO.20)；
ZAFU-11：
F:ACATCTGCAAGGCAAGGTTC (SEQ ID NO.21),
R:TTGAATTTTCACCAGGCTCC(SEQ ID NO.22)；
ZAFU-12：
F:TGCTAATGTTTGCTTTGAATGG (SEQ ID NO.23),
R:AACAACCTAAACTTTTGCCGAA(SEQ ID NO.24)；
ZAFU-13：
F:GAAATTAAACGCGTTCAGGG (SEQ ID NO.25),
R:CATTGCTGTCCGTCTCGTAA(SEQ ID NO.26)；
ZAFU-14：
F:CACAATCCTCCATTCATCCA (SEQ ID NO.27),
R:GCCACTGGTGTGATTCTCAA(SEQ ID NO.28)；
ZAFU-15：
F:ATTTGATACGACGGAAACGG (SEQ ID NO.29),
R:GGTGGTCAATATCCCATGCT(SEQ ID NO.30)；
ZAFU-16：
F:AAGGTTGCCACCTCAGTCAC (SEQ ID NO.31),
R:ACAGAACGTCTCCAACCGAC(SEQ ID NO.32)。
Preferably, in a kind of screening technique of the EST-SSR marks of Chinese torreya transcript profile sequence of the present invention, it is described
Special primer to thin Chinese torreya, Chinese torreya staminiferous plant, great circle Chinese torreya, Jiulong shan Mountain Chinese torreya female plant, Jiulong shan Mountain Chinese torreya staminiferous plant, Torreya jackii female plant, Torreya jackii
Staminiferous plant, Bashan Mountain Chinese torreya staminiferous plant and kaya female plant enter performing PCR amplification, wherein：Amplification system cumulative volume is 10 μ L, containing 10 × Buffer
(Mg2+) 1 μ L, the μ L of dNTP mix 0.8, each μ L of 0.5 μ L, rTaq enzyme 0.05 of upstream and downstream primer, template 0.5 μ L and dd H2O
6.65 μ L, are placed in PCR instrument after being well mixed and expand；Amplification program is：95 DEG C of pre-degeneration 5min, then carry out 34 circulations,
Each circulation includes 94 DEG C of denaturation 45s, 54- 58 DEG C of annealing 30s, 72 DEG C of extensions 1min, last 72 DEG C of extensions 3min, 4 DEG C of guarantors
Deposit, the optimum annealing temperature of each primer fluctuates in expected annealing temperature and optimized between 10 DEG C, until expected product
It is clear single.
Preferably, in a kind of screening technique of the EST-SSR marks of Chinese torreya transcript profile sequence of the present invention, it is described
Special primer to thin Chinese torreya, Chinese torreya staminiferous plant, great circle Chinese torreya, Jiulong shan Mountain Chinese torreya female plant, Jiulong shan Mountain Chinese torreya staminiferous plant, Torreya jackii female plant, Torreya jackii
Staminiferous plant, Bashan Mountain Chinese torreya staminiferous plant and kaya female plant enter performing PCR amplification, and the detection method of PCR primer is as follows：Obtained product is expanded to use
1.2% agarose gel electrophoresis, main technologic parameters are：Voltage 5- 10V/cm, 15-20 minute；Detect the spy of amplified production
After the opposite sex, then separated with 8% native polyacrylamide gel electrophoresis, main technologic parameters are：Electrophoretic buffer be 1 ×
TBE, voltage 180v, electrophoresis 90min；Then detected again with cma staining system, analysis determines polymorphism.
Preferably, in a kind of screening technique of the EST-SSR marks of Chinese torreya transcript profile sequence of the present invention, screening
Reproducible, stable polymorphic abundant microsatellite marker is obtained, its step is obtained using at least two transcriptome analysis
Polymorphic primer, continues with a large amount of its repeatability of individual detection and stability, while obtaining its polymorphic feature.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention, which can be conveniently and quickly used for cooking Chinese torreya, belongs to different type germ plasm resource and genetic diversity in inter-species and Chinese torreya kind
The analysis of property, molecule Population Genetics, the identification of germ plasm resource, the research of genetic map construction and functional gene, is further used
In the Molecular design breeding and resource investigation of Chinese torreya.
Brief description of the drawings
Fig. 1 is results of the ZAFU-1 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Fig. 2 is results of the ZAFU-2 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Fig. 3 is results of the ZAFU-3 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Fig. 4 is results of the ZAFU-4 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Fig. 5 is results of the ZAFU-5 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Fig. 6 is results of the ZAFU-6 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Fig. 7 is results of the ZAFU-7 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Fig. 8 is results of the ZAFU-8 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Fig. 9 is results of the ZAFU-9 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Figure 10 is results of the ZAFU-10 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Figure 11 is results of the ZAFU-11 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Figure 12 is results of the ZAFU-12 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Figure 13 is results of the ZAFU-13 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Figure 14 is results of the ZAFU-14 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Figure 15 is results of the ZAFU-15 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
Figure 16 is results of the ZAFU-16 in the EST-SSR native polyacrylamide gel electrophoresis detections marked,
In figure, the Torreya jackii female plant of 15 Jiulong shan Mountain Chinese torreya staminiferous plant of thin 2 Chinese torreya staminiferous plant of Chinese torreya 3 great circle Chinese torreya, 4 Jiulong shan Mountain Chinese torreya female plant 6
The kaya female plant of 7 Torreya jackii staminiferous plant, 8 Bashan Mountain Chinese torreya staminiferous plant 9, that used in Marker is 1000bp.
Embodiment
With reference to embodiment, present disclosure is further illustrated.It should be appreciated that the implementation of the present invention is not limited to
In the following examples, any formal accommodation and/or change made to the present invention fall within the scope of the present invention.
In the present invention, if not refering in particular to, all part, percentage are unit of weight, and all equipment and raw material etc. are
It is commercially available or the industry is conventional.If without specializing, the method that embodiment is used is technology generally in the art.
Main material, reagent and instrument and equipment explanation in embodiment：
Eye scissors, tweezers, 1.5ml centrifuge tubes, micropipettor, micropipettor pipette tips.Deoxyribonucleotide
DNTP, thermal polymerization Taq enzyme, compact centrifuge (QspinTM, BAYGENE), turbula shaker (QL-866 types,
QILINEBEIER), pH meter (Mettler Toledo 320PH Meter), high-pressure steam sterilizing pot (SANYO), electrophoresis apparatus
(DYY-6C types, Beijing 6 one), electrophoresis apparatus (DYY-12C types, Beijing 6 one), DNA sequence analysis electrophoresis apparatus (DYCZ-20C types,
Beijing 6 one), speed governing use oscillator (HY-2 types, Shanghai state China), water-bath (upper Nereid is grand), gel imaging system (Bio-
Rad GD2000), PCR instrument (ABI Veriti 96well Thermal Cycler).
The thin Chinese torreya of sample of the present invention, Chinese torreya staminiferous plant, great circle Chinese torreya, Jiulong shan Mountain Chinese torreya female plant, Jiulong shan Mountain Chinese torreya staminiferous plant, Torreya jackii are female
Strain, Torreya jackii staminiferous plant, Bashan Mountain Chinese torreya staminiferous plant and kaya female plant.In 9 Chinese torreya individual plants, Bashan Mountain Chinese torreya staminiferous plant picks up from Hubei Province's Baokang
County；Torreya jackii male and female plant, picks up from Zhejiang Province's Tonglu County；Jiulong shan Mountain Chinese torreya male and female plant, picks up from Zhejiang Province Suichang County；Kaya female plant,
Pick up from Nanjing；Thin Chinese torreya, great circle Chinese torreya and Chinese torreya staminiferous plant pick up from Fuyang area of Hangzhou, Zhejiang province city.
Conventional medication phenol chloroform extraction liquid (25 used in the embodiment of the present invention:24:1), formaldehyde, sodium hydroxide (analysis
It is pure), silver nitrate, sodium chloride (analysis is pure), urea, acrylamide, methene, Tris- alkali, boric acid, ethylenediamine tetra-acetic acid (EDTA),
Ammonium persulfate, lauryl sodium sulfate (SDS), absolute ethyl alcohol is purchased from Chinese medicines group agarose, ethidium bromide, deionization formyl
Amine, TEMED, Proteinase K etc. is purchased from Takara companies, and deoxyribonucleotide dNTP, thermal polymerization Taq enzyme is public purchased from TIANGEN
Department, plasmid library sequencing is completed by Sangon Biotech (Shanghai) Co., Ltd., and primer is by giving birth to work bioengineering (Shanghai)
Limited company synthesizes；
Key instrument includes used in the embodiment of the present invention：Compact centrifuge (QspinTM, BAYGENE), turbula shaker
(QL-866 types, QILINEBEIER), pH meter (Mettler Toledo 320pH Meter), high-pressure steam sterilizing pot
(SANYO), electrophoresis apparatus (DYY-6C types, Beijing 6 one), electrophoresis apparatus (DYY-12C types, Beijing 6 one), DNA sequence analysis electrophoresis
Instrument (DYCZ-20C types, Beijing 6 one), speed governing use oscillator (HY-2 types, Shanghai state China), it is water-bath (upper Nereid is grand), solidifying
Glue imaging system (Bio-Rad GD2000), PCR instrument (ABI Veriti 96well Thermal Cycler).
The experimental method of unreceipted actual conditions in embodiment, is the author such as Sambrook according to normal condition
Molecular Cloning: A Laboratory room handbook (New York：Cold Spring Harbor Laboratory Press, 1989) described in
's.
Embodiment 1
1st, the screening of the source in SSR sites and EST-SSR sequences
Inventor uses Trinity software groups from the thin Chinese torreya benevolence transcript profile data in laboratory and circle Chinese torreya benevolence transcript profile data
Dress, obtains transcript sequence.246862 Transcript and 142213 Unigene are obtained in assembling.Utilize MISA programs
(MIcroSAtellite identification tool,http://pgrc.ipk- gatersleben.de/misa/
Misa.html) more than the 1kb obtained to screening Unigene does the search of SSR sites.Repeated for 2-6 base repeat unit
Number of times is respectively successively 9 according to number of repetition, the microsatellite fragment of 8,5,5,5 times, so as to obtain what is repeated containing microsatellite
ESTs sequences.Flanking sequence using primer-design software Primer3.0 at microsatellite two ends designs special primer.
2nd, the design of EST-SSR labeled primers
Flanking sequence in microsatellite duplicate block utilizes software Primer3 programming primers, and primer is required to meet following
Condition：Primer length is controlled in 20bp；58 DEG C -62 DEG C of annealing temperature；The Tm values of upstream and downstream primer are more or less the same in 2 DEG C；PCR
Product length is between 100-300bp；G/C content is between 40%-60%；Each site produces 3 pairs of candidate drugs.
3rd, the optimization of primer
The optimum annealing temperature of each primer fluctuates in expected annealing temperature and optimized between 10 DEG C, until expected mesh
Product can be by steady and audible amplification.PCR amplification response procedures be：95 DEG C of pre-degeneration 5min；Then 34 circulations are carried out,
Each circulation includes 94 DEG C of denaturation 45s, 54-58 DEG C of annealing 30s, 72 DEG C of extension 1min；Last 72 DEG C of extensions 3min, 4 DEG C of preservations.
Reaction system is：The μ L of cumulative volume 10, wherein (including 1.5mMMg containing 10 × Buffer2+) 1 μ L, dNTP mix, 0.8 μ L, up and down
Swim each 0.5 μ L, rTaq enzyme 0.05 μ L, ddH of primer2The μ L of O 6.65, the μ L of template 0.5.Template is random 10 Chinese torreyas individual
DNA mixing pits.Obtained product is expanded to be detected with 1.2% agarose gel electrophoresis-EB coloring systems, selection it is single or
Miscellaneous band is few, specific product, and higher temperature is used as optimum annealing temperature.
4th, the determination in EST-SSR sites
The polymorphism that these EST-SSR marks are surveyed in 35 physical examinations is chosen according to the annealing temperature optimized in step 3. PCR
Response procedures are：95 DEG C of pre-degeneration 5min, then carry out 34 circulations, and each circulation includes 94 DEG C of denaturation 45s, and 54-58 DEG C is moved back
Fiery 30s (annealing temperature is different because of different primers), 72 DEG C of extension 1min；Last 72 DEG C of extensions 3min.Reaction system is：It is overall
10 μ L of product, wherein (including 1.5mMMg containing 10 × Buffer2+) 1 μ L, dNTP mix 0.8 μ L, each 0.5 μ L of upstream and downstream primer,
The μ L of rTaq enzymes 0.05, template 0.5 μ L, ddH2O 6.65μL.Pcr amplification product is electric with 8% non-denaturing polyacrylamide gel
Swimming separation, constant pressure 180v, electrophoresis 90min, cma staining system is detected, is dried post analysis and is determined polymorphism, finally sieves
Select 16 Chinese torreya EST-SSR marks, the specifying information such as table 1 of these marks.
16 Chinese torreya EST-SSR marks that the exploitation of table 1 is obtained
Embodiment 2
1st, the DNA of Chinese torreya is extracted
Comprise the following steps that：
CTAB is put into water-bath (30min) in 65 DEG C of water-baths in advance, fresh sample is taken with ice chest, is placed in after shredding
In 2ml centrifuge tube (sample size is about the 1/4 of pipe).An a diameter of 4mm or so steel is added in each centrifuge tube
Pearl, soaks 5-7 minutes in liquid nitrogen, after abundant precooling, is ground with homogeneous instrument.Plus 800 μ l shift to an earlier date the CTAB extract solutions of water-bath
In centrifuge tube (beta -mercaptoethanol for Jia 1% in CTAB extract solutions), centrifuge tube is placed in water-bath 40min in 65 DEG C of water-baths,
Period, which turns upside down, rocks once.12000rpm, centrifuges 10min by 4 DEG C, takes supernatant, plus isometric 24:1 chloroform-isoamyl
Alcohol (chloroform:Isoamyl alcohol=24:1), fully shake up, stand 5-8min.12000rpm, centrifuges 10min, takes supernatant by 4 DEG C, plus etc.
The 24 of volume:1, fully shake up, 12000rpm, 4 DEG C, centrifuge 10min, take supernatant, plus -20 DEG C of isometric precooled isopropyls
Alcohol, fully shakes up, it is seen that flocculent deposit.It is placed in -20 DEG C of refrigerator 40min or so.12000rpm, centrifuges 10min, outwells liquid by 4 DEG C
Body, plus 500 μ L75% ethanol are washed twice, are placed in fume hood 15-20min, are air-dried to translucent, plus the ddH after sterilizing2O
30-40μl.Detect quality.
2nd, PCR is expanded
As shown in table 1, PCR reaction systems are the optimum annealing temperature of each EST-SSR labeled primers：95 DEG C of pre-degenerations
5min；Then 34 circulations are carried out, each circulation includes 94 DEG C of denaturation 45s, 54-58 DEG C of annealing 30s, 72 DEG C of extension 1min；Most
72 DEG C extend 3min, 4 DEG C of preservations afterwards.Reaction system is：The μ L of cumulative volume 10, wherein containing 10 × Buffer (1.5mMMg2+) 1 μ L,
DNTP mix0.8 μ L, each 0.5 μ L, rTaq enzyme 0.05 μ L, ddH of upstream and downstream primer2The μ L of O 6.65, the μ L of template 0.5.Amplification is obtained
Product detected with 1.2% agarose gel electrophoresis-EB coloring systems.
3rd, electrophoresis detection
(voltage 5-10V/cm, 15-20 minute) is detected through agarose gel electrophoresis, the PCR primer of specific amplified uses 8%
Native polyacrylamide gel electrophoresis separation, electrophoretic buffer be 1 × TBE, constant pressure 180V, power 200W, 90 points of electrophoresis
Dyed and developed the color after clock, film is put into silver staining liquid first, 5min or so is shaken on earthquake instrument, silver staining liquid is poured out,
Use ddH2O is rinsed twice.Then film is put into nitrite ion and developed the color, concussion on earthquake instrument until have clearly bar take out of
Existing, film uses ddH after taking out2O is rinsed twice, and gel is placed in preservation of observing and take pictures on visible ray lamp box.Silver staining liquid is configured：
ddH2O 450ml, absolute ethyl alcohol 50ml and AgNO30.75g.Nitrite ion is configured：ddH2O 450ml, 20%NaOH 50ml and first
Aldehyde 2ml.
Dried polyacrylamide gel electrophoresis result is as shown in Fig. 1 to Figure 16.
SEQUENCE LISTING
<110>Zhejiang A ＆ F University
<120>The EST-SSR mark special primers and screening technique of a kind of Chinese torreya transcript profile sequence
<130> 20170306
<160> 32
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
ggctatgcta cacccaaaga a 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ggggcaccac ctattgtatg 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
tcaaagtgca accggtacaa 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
caacaggcca acatggagta 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
gggttacccc cttgctttat 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<400> 6
ccctacttta tttccgtgcg 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<400> 7
gaattcccat tcccattgtg 20
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence
<400> 8
acccccttct gctctgattt 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
aatgaatgcg tgttacgctg 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
ttggagcgga aggaataatg 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<400> 11
ccaatttgtg gagcgtttct 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
tgtggaaagg tggtgaacaa 20
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<400> 13
ttttccaact ccaacccttg 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<400> 14
atgtttgggg ttgacgtgtt 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
<400> 15
aattggccct tcattcaaca 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
ctagtgggtg catttgagcc 20
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence
<400> 17
ggagcgagcg tagcgtatag 20
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence
<400> 18
gttgctgcat cttcctcctc 20
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence
<400> 19
gcctggtctg cttcttgttc 20
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence
<400> 20
aggcaaaggc tgcaatagaa 20
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence
<400> 21
acatctgcaa ggcaaggttc 20
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<400> 22
ttgaattttc accaggctcc 20
<210> 23
<211> 22
<212> DNA
<213>Artificial sequence
<400> 23
tgctaatgtt tgctttgaat gg 22
<210> 24
<211> 22
<212> DNA
<213>Artificial sequence
<400> 24
aacaacctaa acttttgccg aa 22
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence
<400> 25
gaaattaaac gcgttcaggg 20
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence
<400> 26
cattgctgtc cgtctcgtaa 20
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence
<400> 27
cacaatcctc cattcatcca 20
<210> 28
<211> 20
<212> DNA
<213>Artificial sequence
<400> 28
gccactggtg tgattctcaa 20
<210> 29
<211> 20
<212> DNA
<213>Artificial sequence
<400> 29
atttgatacg acggaaacgg 20
<210> 30
<211> 20
<212> DNA
<213>Artificial sequence
<400> 30
ggtggtcaat atcccatgct 20
<210> 31
<211> 20
<212> DNA
<213>Artificial sequence
<400> 31
aaggttgcca cctcagtcac 20
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence
<400> 32
acagaacgtc tccaaccgac 20
Claims (5)
1. the EST-SSR mark special primers of Chinese torreya transcript profile sequence, it is characterised in that described special primer is respectively：
ZAFU-1：
F:GGCTATGCTACACCCAAAGAA (SEQ ID NO.1),
R：GGGGCACCACCTATTGTATG(SEQ ID NO.2)；
ZAFU-2：
F：TCAAAGTGCAACCGGTACAA (SEQ ID NO.3),
R:CAACAGGCCAACATGGAGTA(SEQ ID NO.4)；
ZAFU-3：
F：GGGTTACCCCCTTGCTTTAT (SEQ ID NO.5),
R:CCCTACTTTATTTCCGTGCG(SEQ ID NO.6)；
ZAFU-4：
F：GAATTCCCATTCCCATTGTG (SEQ ID NO.7),
R:ACCCCCTTCTGCTCTGATTT(SEQ ID NO.8)；
ZAFU-5：
F：AATGAATGCGTGTTACGCTG (SEQ ID NO.9),
R:TTGGAGCGGAAGGAATAATG(SEQ ID NO.10)；
ZAFU-6：
F:CCAATTTGTGGAGCGTTTCT (SEQ ID NO.11),
R:TGTGGAAAGGTGGTGAACAA(SEQ ID NO.12)；
ZAFU-7：
F:TTTTCCAACTCCAACCCTTG (SEQ ID NO.13),
R:ATGTTTGGGGTTGACGTGTT(SEQ ID NO.14)；
ZAFU-8：
F:AATTGGCCCTTCATTCAACA (SEQ ID NO.15),
R:CTAGTGGGTGCATTTGAGCC(SEQ ID NO.16)；
ZAFU-9：
F:GGAGCGAGCGTAGCGTATAG (SEQ ID NO.17),
R:GTTGCTGCATCTTCCTCCTC(SEQ ID NO.18)；
ZAFU-10：
F:GCCTGGTCTGCTTCTTGTTC (SEQ ID NO.19),
R:AGGCAAAGGCTGCAATAGAA(SEQ ID NO.20)；
ZAFU-11：
F:ACATCTGCAAGGCAAGGTTC (SEQ ID NO.21),
R:TTGAATTTTCACCAGGCTCC(SEQ ID NO.22)；
ZAFU-12：
F:TGCTAATGTTTGCTTTGAATGG (SEQ ID NO.23),
R:AACAACCTAAACTTTTGCCGAA(SEQ ID NO.24)；
ZAFU-13：
F:GAAATTAAACGCGTTCAGGG (SEQ ID NO.25),
R:CATTGCTGTCCGTCTCGTAA(SEQ ID NO.26)；
ZAFU-14：
F:CACAATCCTCCATTCATCCA (SEQ ID NO.27),
R:GCCACTGGTGTGATTCTCAA(SEQ ID NO.28)；
ZAFU-15：
F:ATTTGATACGACGGAAACGG (SEQ ID NO.29),
R:GGTGGTCAATATCCCATGCT(SEQ ID NO.30)；
ZAFU-16：
F:AAGGTTGCCACCTCAGTCAC (SEQ ID NO.31),
R:ACAGAACGTCTCCAACCGAC(SEQ ID NO.32)。
2. the screening technique of the EST-SSR marks of a kind of Chinese torreya transcript profile sequence, it is characterised in that justify first to thin Chinese torreya kind Renhe
Chinese torreya benevolence transcript profile data use Trinity software combinations, obtain transcript sequence, and assembling is obtained 246862
Transcript and 142213 Unigene；Then more than the 1kb obtained using MISA programs to screening Unigene is SSR
Site is searched for, and is respectively successively 9 according to number of repetition for 2-6 base repeat unit number of repetition, 8,5,5,5 times micro- defend
Star fragment carries out screening separation, so as to obtain the ESTs sequences repeated containing microsatellite；Then drawn using Primer3 programs
Thing is designed, the main principle of design of primers：Primer length control 20bp, 58 DEG C -62 DEG C of annealing temperature, upstream and downstream primer Tm
Value be more or less the same in 2 DEG C, PCR primer length between 100-300bp, G/C content between 40%-60%, each site produce
3 pairs of candidate drugs；Repeatability, stability and the polymorphism finally marked to EST-SSR carries out overall merit, obtains EST-SSR
Mark, the special primer of the described EST-SSR screened marks is respectively：
ZAFU-1：
F:GGCTATGCTACACCCAAAGAA (SEQ ID NO.1),
R：GGGGCACCACCTATTGTATG(SEQ ID NO.2)；
ZAFU-2：
F：TCAAAGTGCAACCGGTACAA (SEQ ID NO.3),
R:CAACAGGCCAACATGGAGTA(SEQ ID NO.4)；
ZAFU-3：
F：GGGTTACCCCCTTGCTTTAT (SEQ ID NO.5),
R:CCCTACTTTATTTCCGTGCG(SEQ ID NO.6)；
ZAFU-4：
F：GAATTCCCATTCCCATTGTG (SEQ ID NO.7),
R:ACCCCCTTCTGCTCTGATTT(SEQ ID NO.8)；
ZAFU-5：
F：AATGAATGCGTGTTACGCTG (SEQ ID NO.9),
R:TTGGAGCGGAAGGAATAATG(SEQ ID NO.10)；
ZAFU-6：
F:CCAATTTGTGGAGCGTTTCT (SEQ ID NO.11),
R:TGTGGAAAGGTGGTGAACAA(SEQ ID NO.12)；
ZAFU-7：
F:TTTTCCAACTCCAACCCTTG (SEQ ID NO.13),
R:ATGTTTGGGGTTGACGTGTT(SEQ ID NO.14)；
ZAFU-8：
F:AATTGGCCCTTCATTCAACA (SEQ ID NO.15),
R:CTAGTGGGTGCATTTGAGCC(SEQ ID NO.16)；
ZAFU-9：
F:GGAGCGAGCGTAGCGTATAG (SEQ ID NO.17),
R:GTTGCTGCATCTTCCTCCTC(SEQ ID NO.18)；
ZAFU-10：
F:GCCTGGTCTGCTTCTTGTTC (SEQ ID NO.19),
R:AGGCAAAGGCTGCAATAGAA(SEQ ID NO.20)；
ZAFU-11：
F:ACATCTGCAAGGCAAGGTTC (SEQ ID NO.21),
R:TTGAATTTTCACCAGGCTCC(SEQ ID NO.22)；
ZAFU-12：
F:TGCTAATGTTTGCTTTGAATGG (SEQ ID NO.23),
R:AACAACCTAAACTTTTGCCGAA(SEQ ID NO.24)；
ZAFU-13：
F:GAAATTAAACGCGTTCAGGG (SEQ ID NO.25),
R:CATTGCTGTCCGTCTCGTAA(SEQ ID NO.26)；
ZAFU-14：
F:CACAATCCTCCATTCATCCA (SEQ ID NO.27),
R:GCCACTGGTGTGATTCTCAA(SEQ ID NO.28)；
ZAFU-15：
F:ATTTGATACGACGGAAACGG (SEQ ID NO.29),
R:GGTGGTCAATATCCCATGCT(SEQ ID NO.30)；
ZAFU-16：
F:AAGGTTGCCACCTCAGTCAC (SEQ ID NO.31),
R:ACAGAACGTCTCCAACCGAC(SEQ ID NO.32)。
3. a kind of screening technique of the EST-SSR marks of Chinese torreya transcript profile sequence as claimed in claim 2, it is characterised in that
Described special primer is to thin Chinese torreya, Chinese torreya staminiferous plant, great circle Chinese torreya, Jiulong shan Mountain Chinese torreya female plant, Jiulong shan Mountain Chinese torreya staminiferous plant, Torreya jackii female plant, length
Leaf Chinese torreya staminiferous plant, Bashan Mountain Chinese torreya staminiferous plant and kaya female plant enter performing PCR amplification, wherein：Amplification system cumulative volume is 10 μ L, containing 10 ×
Buffer(Mg2+) 1 μ L, the μ L of dNTP mix 0.8, each μ L of 0.5 μ L, rTaq enzyme 0.05 of upstream and downstream primer, template 0.5 μ L and dd
H2The μ L of O 6.65, are placed in PCR instrument after being well mixed and expand；Amplification program is：95 DEG C of pre-degeneration 5min, then follow for 34 times
Ring, each circulation includes 94 DEG C of denaturation 45s, 54-58 DEG C of annealing 30s, 72 DEG C of extensions 1min, last 72 DEG C of extensions 3min, 4 DEG C of guarantors
Deposit, the optimum annealing temperature of each primer fluctuates in expected annealing temperature and optimized between 10 DEG C, until expected product
It is clear single.
4. a kind of screening technique of the EST-SSR marks of Chinese torreya transcript profile sequence as claimed in claim 2, it is characterised in that
Described special primer is to thin Chinese torreya, Chinese torreya staminiferous plant, great circle Chinese torreya, Jiulong shan Mountain Chinese torreya female plant, Jiulong shan Mountain Chinese torreya staminiferous plant, Torreya jackii female plant, length
Leaf Chinese torreya staminiferous plant, Bashan Mountain Chinese torreya staminiferous plant and kaya female plant enter performing PCR amplification, and the detection method of PCR primer is as follows：Expand obtained production
Thing is with 1.2% agarose gel electrophoresis, and main technologic parameters are：Voltage 5-10V/cm, 15-20 minute；Detect amplified production
Specificity after, then with 8% native polyacrylamide gel electrophoresis separation, main technologic parameters are：Electrophoretic buffer is 1
× TBE, voltage 180v, electrophoresis 90min；Then detected again with cma staining system, analysis determines polymorphism.
5. a kind of screening technique of the EST-SSR marks of Chinese torreya transcript profile sequence as claimed in claim 2, it is characterised in that
Screening obtains reproducible, stable polymorphic abundant microsatellite marker, and its step is obtained using at least two transcriptome analysis
The polymorphic primer arrived, continues with a large amount of its repeatability of individual detection and stability, while obtaining its polymorphic feature.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815509A (en) * | 2017-12-12 | 2018-03-20 | 福建省农业科学院作物研究所 | SSR primer sets and its application based on the exploitation of okra transcript profile sequence |
| CN108165652A (en) * | 2018-02-12 | 2018-06-15 | 杭州师范大学 | For the specific molecular marker TGMI001 of Chinese torreya seedling stage sex identification |
| CN108359738A (en) * | 2018-02-06 | 2018-08-03 | 浙江农林大学 | A kind of Chinese torreya EST-SSR primers and Variety fingerprinting construction method |
| CN113481313A (en) * | 2021-04-28 | 2021-10-08 | 浙江省林业科学研究院 | Multiple fluorescence SSR (simple sequence repeat) labeled primers and method for identifying three Chinese torreya varieties |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130134480A (en) * | 2012-05-31 | 2013-12-10 | (주)아모레퍼시픽 | Extract of torreya sp.treated enzyme |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130134480A (en) * | 2012-05-31 | 2013-12-10 | (주)아모레퍼시픽 | Extract of torreya sp.treated enzyme |
Non-Patent Citations (1)
| Title |
|---|
| GUANMEI YI等: "Mining and characterizing EST-SSR markers from ESTs of Torreya grandis", 《BIOCHEMICAL SYSTEMATICS AND ECOLOGY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815509A (en) * | 2017-12-12 | 2018-03-20 | 福建省农业科学院作物研究所 | SSR primer sets and its application based on the exploitation of okra transcript profile sequence |
| CN107815509B (en) * | 2017-12-12 | 2021-04-06 | 福建省农业科学院作物研究所 | SSR primer group developed based on okra transcriptome sequence and application thereof |
| CN108359738A (en) * | 2018-02-06 | 2018-08-03 | 浙江农林大学 | A kind of Chinese torreya EST-SSR primers and Variety fingerprinting construction method |
| CN108359738B (en) * | 2018-02-06 | 2021-08-27 | 浙江农林大学 | Torreya sinensis EST-SSR primer and variety fingerprint construction method |
| CN108165652A (en) * | 2018-02-12 | 2018-06-15 | 杭州师范大学 | For the specific molecular marker TGMI001 of Chinese torreya seedling stage sex identification |
| CN113481313A (en) * | 2021-04-28 | 2021-10-08 | 浙江省林业科学研究院 | Multiple fluorescence SSR (simple sequence repeat) labeled primers and method for identifying three Chinese torreya varieties |
| CN113481313B (en) * | 2021-04-28 | 2022-06-10 | 浙江省林业科学研究院 | Multiple fluorescence SSR (simple sequence repeat) labeled primers and method for identifying three Chinese torreya varieties |
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