CN103642912B - Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing - Google Patents
Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing Download PDFInfo
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Abstract
The invention provides a method for developing a mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing. The method comprises the following steps: obtaining a set of mung bean genome-wide transcription, and forming a sequence database; splicing sequencing sequences into a transcriptome by Trinity; taking the longest transcript in each gene as Unigene; carrying out bioinformatics analysis of a Unigene sequence; carrying out SSR detection on the Unigene by adopting MISA1.0; carrying out SSR primer design by using a Primer 3, and carrying out SSR primer polymorphism identification. 13134 pairs of SSR primers are successfully designed by application of the method; 50 pairs of primers are randomly selected to verify 8 parts of mung bean deoxyribonucleic acids (DNAs) from different countries, wherein 32 pairs of polymorphic primers are formed in all; the mung bean materials with different geographical origins can be distinguished by using the 32 pairs of SSR primers. The method disclosed by the invention is convenient, fast and accurate, and low in cost, and a new thought is provided for development of the mung bean SSR primer.
Description
Technical field
The present invention relates to molecular biology and information biology, specifically, relate to a kind of method based on transcript profile order-checking exploitation mung bean SSR primer.
Background technology
Mung bean (Vigna radiata) is a kind of pulse family, Papillionoideae Vigna plant, originates in India, Burma area.Present East Asia various countries generally plant, and also there are a small amount of plantation in Africa, Europe, the U.S., and China is one of source region of mung bean [Vigna radiata (L.) Wilczek], has the mung bean variety resource of wide variety.The states such as China, Burma are main mung bean export States.Because its breeding time is short, wide adaptability, and there is good nitrogen fixing capacity, so be the Important Economic crop of the plant husbandry rational distribution of resources, rotation of crops crop rotation, intercropping, the indispensable food crop of the mitigation disaster relief and poverty-stricken area farmer richness; Mung bean is rich in albumen, middle starch and lower fat simultaneously, is desirable nutritive health-care food.Seed and stem are extensively eaten, and have abundant nutritive value.Mung bean also can produce into numerous food as eaten the food such as bean sprouts, green bean vermicelli, green bean starch sheet, green gram wine, Semen phaseoli radiati cake raw, enjoys favor in the international market.In recent years, the turnout of world market to the demand of mung bean and whole world mung bean increases all to some extent, and the mung bean annual export volume of China is 20-30 ten thousand tons now, the general 400-500 dollar of export price.The social economic value of mung bean can not be ignored.But with staple crop as compared with corn, paddy rice, also quite delayed to the research of mung bean both at home and abroad, per unit area yield is still in lower level, the more aobvious weakness of research of molecular level.
Molecule marker is the genetic marker by between individuality based on genetic material inner nucleotide sequence variations, can reflect the difference on plant genetic basis on DNA level, is the direct reflection of DNA level genetic polymorphism.Simple repeated sequence (SSR) is distributed widely in the different positions of all kinds of eukaryotic gene group, and the multiplicity due to SSR is different different with repetition degree, makes it present the polymorphism of height.Compared with other molecular marking technique, SSR marker has that polymorphism information content is high, codominant inheritance, technology are simple, reproducible, high specificity, operation are convenient and the advantage such as discrete distribution has become one of molecule marker the most popular to people in genome, is considered to one of the highest molecule marker type of reliability.Widespread use in a lot of fields.But the main drawback of SSR marker is the sequence information that first will obtain tumor-necrosis factor glycoproteins both sides from these species, and designs primer, then just can be utilized.
SSR marker can be divided into genome SSR (gSSR) and expressed sequence tag SSR (EST-SSR), EST-SSR mark comes from the transcriptional domain of gene, compared with marking with gSSR, its polymorphism may be directly related with gene function, therefore, than gSSR mark, there is more high universalizable, more economical, more high-level efficiency.Utilize s-generation sequencing technologies can carry out large-scale high-flux sequence to the transcript within the scope of full-length genome, and the transcript profile data of the more magnanimity of checking order than EST can be produced, this is that the exploitation of functional genome's SSR marker provides abundanter and extremely valuable available stock.
The quantity of transcript profile sequence grows with each passing day, and making to obtain SSR by database search method becomes possibility.But the data produced from s-generation sequencing technologies are often extremely huge, and carry out format analysis processing to a large amount of est sequences, eliminate redundancy sequence etc. is still a no small workload.Perl is a kind of free and powerful programming language.It is used as Web programming, database processing, XML process and system management etc.Along with the development of information biology, in the operation that Perl has more been applied to biological data and retrieval, make to be treated as possibility to data in enormous quantities are unified.Carry out EST-SSR primer development on this basis and more can improve separation efficiency, save time and fund.
Current mung bean there is no whole genome sequence information, mung bean SSR primer comparatively small amt.For without with reference to genomic transcriptome analysis, first the sequence assembly of order-checking gained can be become transcript, take transcript as reference sequences, carry out subsequent analysis.S-generation high throughput sequencing technologies is utilized to obtain the transcript profile sequence information of a certain material in mung bean, the technology maturation of batch SSR primers development, will play important pushing effect to the location of mung bean important character gene, clone and molecular marker assisted selection breeding and comparative genomics research etc.
Summary of the invention
The object of this invention is to provide a kind of method based on transcript profile order-checking exploitation mung bean SSR primer.
In order to realize the object of the invention, a kind of method based on transcript profile order-checking exploitation mung bean SSR primer of the present invention, said method comprising the steps of:
1) transcript profile library is built: extract Catalase total serum IgE, isolate mRNA, reverse transcription synthetic double chain cDNA, purifying cDNA, add adenosine at cDNA end and connect sequence measuring joints, then reclaim 200-700bp fragment by agarose gel electrophoresis, pcr amplification is carried out to recovery fragment, namely build and obtain transcript profile library;
2) checked order in above-mentioned transcript profile library, utilize software Trinity sequencing sequence to be spliced into a complete transcript profile, get transcript the longest in every bar gene as Unigene, and bioinformatic analysis is carried out to Unigene sequence;
3) software MISA1.0 is adopted to carry out SSR detection to above-mentioned Unigene;
4) adopt software Primer3 to carry out SSR design of primers, and identify the polymorphism of SSR primer.
Wherein, sequence measuring joints described in step 1) is:
5′RNA Adapter：5′-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-3′
3′RNA Adapter：5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′。
Step 2) described in the version of software Trinity be v2012-10-05; Optimum configurations: min_kmer_cov is 2, and other parameter is default parameters.
Step 2) described in bioinformatic analysis include but not limited to gene annotation, CDS prediction and differential gene expression screening etc.Described gene annotation comprises gene expression amount annotation and/or annotation of gene function.Described differential gene expression screening comprises the enrichment of GO function significance and analyzes and/or Pathway significance enrichment analysis.
The parameter of carrying out the use of SSR design of primers in step 4) is: primer length 18-22bp, Tm55-65 DEG C, product size 100-300bp.
No. 1, Chinese medium green, No. 5, medium green is selected from for the identification of the mung bean of SSR primer polymorphism in step 4); Thailand VC2778A, TC1966; Russia 1810,1865; At least one in Australia ACC814, ACC41 etc.
The present invention also provides the mung bean SSR developed according to aforesaid method primer, and the sequence of described SSR primer is as shown in SEQ ID No.1-64.
The present invention further provides the application of mung bean SSR primer in mung bean molecular mark according to aforesaid method exploitation.
Particularly, a kind of method based on transcript profile sequence exploitation mung bean SSR primer provided by the invention, comprises the steps:
1) acquisition of transcript profile data
Extract Catalase total serum IgE, isolate mRNA, reverse transcription synthetic double chain cDNA, purifying cDNA, carries out end reparation, add A and connect sequence measuring joints, then carry out clip size selection with agarose gel electrophoresis, finally carry out pcr amplification, build transcript profile library, the sequencing library Illumina HiSeqTM2000 built up utilizes the method for two end sequencing (Paired-End) to check order, and obtains mung bean transcript profile sequencing data.The sequencing data amount of each sample individuality is 5GbClean Data.
2) identification of SSR sequence
First Perl language is installed, downloads est_trimmer.pl from http://pgrc.1pk-gatersleben.de/misa website, remove too short sequence and long sequence in transcript profile sequence; From http://www.bioinformatics, in org/cd-hit/, download CD_HIT software, remove redundant sequence.
Download from http://pgrc.1pk-gatersleben.de/misa website and use MISA software to identify and SSR positioning sequence, optimum configurations is as follows: the multiplicity of mononucleotide, dinucleotides, trinucleotide, tetranucleotide, pentanucleotide and Hexanucleotide is at least 10,6,5,3,3,3.
3) design of SSR primer
Use Primer3 Batch Design SSR primer, network address: http://sourceforge.net/projects/primer3/files/primer3/l.1.4/pri mer3-1.1.4-WINXP.zip/download, design of primers parameter is primer length 18-22bp, Tm55-65 DEG C, wherein, primer Tm differs 4 DEG C, and product size is 100-300bp.
4) SSR primer pair derives from the identification of polymorphisms of 8 parts of mung bean DNA of 4 country variants
From develop random selecting 50 pairs of primers 13134 pairs of SSR primers and carry out pcr amplification, adopt 8% native polyacrylamide gel electrophoresis to detect.
The invention provides a kind of without the side of genome with reference to transcript profile order-checking exploitation mung bean SSR primer, comprise the steps: the set obtaining mung bean full-length genome transcript, formation sequence database; With Trinity, sequencing sequence is spliced into a transcript profile, in this, as the reference sequences of subsequent analysis, gets transcript the longest in every bar gene as Unigene; Unigene sequence bioinformatic analysis; MISA1.0 is adopted to carry out SSR detection to Unigene; Carry out SSR design of primers with Primer3, and carry out SSR primer identification of polymorphisms.Present invention also offers and obtain the transcript profile information of mung bean and the method for functional gene.Apply present method successful design 13134 pairs of SSR primers, therefrom random selecting 50 pairs of primer pairs derive from country variant totally 8 parts of mung bean DNA verify, 46 pairs of SSR primers are wherein had clear band to be detected at 100-300bp, show that design of primers success ratio is higher, it is 32 right that wherein polymorphic primer has, and utilizes these 32 pairs of SSR primers can distinguish the mung bean material of different geographic origin.The inventive method is convenient, fast, accurate and with low cost, for mung bean SSR primer development provides new approaches.
Accompanying drawing explanation
Fig. 1 builds storehouse order-checking schematic flow sheet in the embodiment of the present invention 1.
Fig. 2 is RNA-seq data analysis schematic flow sheet in the embodiment of the present invention 1.
Fig. 3 is without with reference to genomic transcript profile analysis of biological information schematic flow sheet in the embodiment of the present invention 1.
Fig. 4 splices the Unigene staple diagram obtained in the embodiment of the present invention 1.
Fig. 5 is SSR density profile in the embodiment of the present invention 2.
Fig. 6 is part SSR repeating group element type and quantity in the embodiment of the present invention 2.
Fig. 7 be utilize in the embodiment of the present invention 3 part SSR primer pair derive from 4 countries (China, Thailand, Australia, each 2 parts of Russia) totally 8 parts of mung bean DNA carry out the result of polymorphism checking.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001) condition of, or according to manufacturer's specification sheets advising.
Test materials used in following examples, if no special instructions, all buys from routine biochemistry reagent shop and obtains.Trizol, RNase H and Superscript IIreversetranscriptase test kit is all purchased from Invitrogen company.DNA polymerase i is purchased from NEB company.In cDNA fragment, the joint sequence of grappling is purchased from by Illumina sequencing kit.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
The design of embodiment 1RNA-seq analysis and SSR primer
One, the acquisition of transcript profile data
Trizol reagent is utilized to extract mung bean whole strain seedling total serum IgE, with Oligo(dT) enrichment with magnetic bead mRNA.Add fragmentation buffer and mRNA is broken into short-movie section, take mRNA as template, with hexabasic base random primer synthesis Article 1 cDNA chain, then add damping fluid, dNTPs, RNase H and DNA polymerase I synthesizes Article 2 cDNA chain, end reparation is being done after adding EB buffer solution elution through QiaQuick PCR kit purifying, add A and connect sequence measuring joints, then clip size selection is carried out with agarose gel electrophoresis, finally carry out pcr amplification, the sequencing library IlluminaHiseq2000 built checks order.
Reverse transcription synthetic double chain cDNA, purifying cDNA, carries out end reparation, adds A and connects sequence measuring joints, then carries out clip size selection with agarose gel electrophoresis, finally carries out pcr amplification.The storehouse order-checking flow process of building of sample is shown in Fig. 1.Concrete grammar is as follows:
1. the extraction of mung bean Total RNA
Conventional Trizol method is adopted to extract, purifying, DNA enzymatic process, obtain Total RNA sample that concentration >=50ng/ μ l, total amount >=3 μ g, OD260/280 are 1.8-2.2 (electrophoresis detection and NanoDrop initial survey, more preferably select sample carry out Qubit quantitatively and Agilent2100 detection).
The separation of 2.mRNA and interrupting at random
Go out the mRNA with polyA with the Beads enrichment with oligo-dT, then utilize ultrasonic wave to interrupt at random, reclaim the fragment of 200-700bp.
The synthesis of 3.cDNA first chain and the second chain
The synthesis of cDNA first chain carries out with random 6 polymers and Superscript II reversetranscriptase test kit.CDNA second chain completes with RNase H and DNA polymerase i.
4. the joint sequence of grappling in cDNA fragment:
5′RNA Adapter(SEQ ID NO:1)：
5′-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-3′；
3′RNA Adapter(SEQ ID NO:2)：
5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′。
The pcr amplification of 15 circulations is carried out in 5.PCR amplification with the primer in above-mentioned joint sequence.
6. library construction and detection utilize the sequence obtained in above-mentioned steps, carry out library construction and detection according to Illumina company sample prep kit.
The order-checking of 7.RNA-seq
The library of building up is added to the concentration of 5-7pM in the respective channel of Illumina sequenator (GenomeAnalyzer II), runs 36 circulations.
8. data analysis
RNA-seq data analysis flow process is shown in Fig. 2.Reject impurity data, the result after RNA-seq assembling is integrated.What step before obtained is raw data, wherein containing the joint sequence added in 4 in steps, is called Clean reads after being removed, just can carry out splicing and assemble.Concrete grammar utilizes the Cleanreads that will obtain, and adopts the Trinity(version for transcript profile splicing: v2012-10-05; Optimum configurations: min_kmer_cov is 2, and other parameter is default parameters) software splices.Sequencing sequence is spliced into a transcript profile, in this, as the reference sequences of subsequent analysis with Trinity.Get transcript the longest in every bar gene as Unigene.
9. bioinformatic analysis
Without seeing Fig. 3 with reference to genomic transcript profile analysis of biological information flow process.Unigene sequence obtained above and albumen database nr, Swiss-Prot, KEGG and KOG are carried out blastx comparison (evalue < 0.00001), get the sequence direction that the best albumen of comparison result determines Unigene.If the comparison result between different sink is contradictory, the sequence direction of Unigene is then determined by the priority of nr, Swiss-Prot, KEGG and KOG, catch up with state 4 storehouses all less than Unigene, predict its coding region with software ESTScan and determine the direction of sequence.For the Unigene that can determine sequence direction, provide the sequence in its from 5 ' to 3 ' direction; For the Unigene that cannot determine sequence direction, provide the sequence that composite software obtains.Functional annotation is carried out to these genes, has comprised KOG classification and GO annotation.Partial analysis situation as shown in Figure 4.
Two, the identification of SSR primer
Perl language is installed, download est_trimmer.pl from http://pgrc.1pk-gatersleben.de/misa/ and run, remove in transcript profile sequence and be less than the too short sequence of 100bp and be greater than the long sequence of 2000bp, action command is: C: perl bin>perlest_trimmer, piA.fasta-amb=2,50-tr5=T, 5,50-tr3=A, 5,50-cut=100,2000.Export two file A.fasta.log and A.fasta.results (A is short title).CD_HIT software is downloaded from http://www.bioinformatics.org/cd-hit, it is utilized to remove redundant sequence: A.fasta.results to be copied in cd_hit folder and RNTO B.fasta, run cd_hit.exe, under Perl environment, action command is: C: perl bin cd_hit>cd_hit.exe-1B.fasta-oC.fasta-cl.00-n5-M2000, export three files, wherein C.fsata file is used for next step process (A, B and C are short title).Misa.pi program is downloaded with the SSR identified and positioning sequence from http://pgrc.1pk-gatersleben.de/misa/; Optimum configurations is as follows: the multiplicity of mononucleotide, dinucleotides, trinucleotide, tetranucleotide, pentanucleotide and Hexanucleotide is at least 10,6,5,3,3,3.By C.fsata file copy to C dish perl under bin catalogue, action command under Perl environment: C: perl bin>perlmisa.plC.fasta, produce C.fasta.misa and C.fasta.statistics two files after running, wherein C.fasta.misa is used for follow-up design of primers.
Three, the design of SSR primer
Primer3 module Batch Design SSR primer under use Perl environment: design of primers parameter is Tm55-65 DEG C, and primer length is 18-22bp.Under running p3_out.pi, Perl environment, action command is: C: perl bin>perlp3_in.plC.fasta.misa, create the input file of the primer3 of a C.fasta.p3in by name; Copy again C.fasta.p3in file to C dish perl bin primer3 under bin root directory, run the design of primers that primer3_core.exe realizes batch, under Perl environment, action command is: C: perl bin primer3 bin>primer3_core.exe<C.fasta. p3in>C.fasta.p3out, produce the file of a C.fasta.p3out by name; Finally by C.fasta.p3out file copy to C dish perl under bin catalogue, run p3_out.pi, its order is: C: perl bin>perl p3_out.pl C.fasta.p3outC.fasta.misa, obtain C.fasta.results file after operation, this is the primer designed.
The excavation in embodiment 2 mung bean high-throughput SSR site
Application aforesaid method uses Catalase to carry out high-flux sequence as material, utilizes Perl language to carry out the excavation in high-throughput SSR site to mung bean transcript profile sequence, obtains 83542 transcript profile sequences and 48693 unigenes(tables 1).What the SSR density distribution frequency of occurrences was the highest is the micro-satellite of single base, and that proportion is the highest is A/T, is secondly tetranucleotide (table 2, Fig. 5, Fig. 6).
Table 1 splices length frequency distribution situation
Table 2 repeats primitive situation
Primer3.0 Batch Design software is used to design acquisition SSR primer 13134 altogether right.Therefrom random selection 50 pairs of primers (see nucleotides sequence list), utilize Catalase DNA to carry out the detection of design of primers success ratio, result shows, has 46 pairs of SSR primers and clear band detected at 100-300bp, show that design of primers success ratio is higher.
8 parts of mung bean DNA that embodiment 3 utilizes SSR primer pair to derive from country variant carry out identification of polymorphisms
Extract 8 parts of mung bean DNA, detect its quality with 0.8% agarose gel electrophoresis method, DNA concentration dilution is placed on-20 DEG C to 50ng/ μ L and saves backup.Utilize the DNA of primer development material therefor to carry out design of primers success ratio PCR to identify.PCR reaction system adopts the reaction system of 10 μ L, comprising 40ng genomic dna, and 1 × Taq enzyme damping fluid (10mmolL
-1tris-HCl, pH8.8; 10mmol L
-1kCl; 10mmol L
-1(NH
4)
2sO
4; 1.5mmol L
-1mgCl
2; 0.1%Triton X-100), 1mmol L
-1dNTPs, upstream and downstream primer 0.25 μm of ol L
-1with 1U Taq archaeal dna polymerase.SSR response procedures is: 95 DEG C of denaturation 5min, and 95 DEG C of distortion 30s, 51-60 DEG C of annealing 45s, 72 DEG C extend 45s, carry out 32-35 circulation, and last 72 DEG C extend 5min.After reaction terminates, product adds 2 μ L sample loading buffers, with 100bp DNA ladder for DNA molecular amount standard, the non-denaturing polyacrylamide gel of 8% is adopted to carry out electrophoresis, electrophoretic buffer is 0.5 × TBE, 200V voltage stabilizing electrophoresis 2-2.5h, when moving on to bottom gel to sample loading buffer, electrophoresis terminates.After electrophoresis terminates, adopt argentation dyeing, finally gel is placed on gel imaging system and takes pictures.All Data duplications twice.
Choose 50 pairs of primer pairs, 8 parts of mung bean material DNA to verify, PAGE electrophorogram as shown in Figure 7.Result shows, 46 pairs of primers all detect polymorphic clear band in all material, wherein there is polymorphic primer to have 32 right, show that 32 couple (sequence is respectively as shown in SEQ ID No.1-64) in 46 pairs of primers can be used for distinguishing the mung bean material of different geographic origin.Show the method utilizing this mung bean transcript profile SSR primers development, be applicable to the exploitation of mung bean SSR primer.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Sequence illustrates:
SEQ ID No.1-64 is 32 pairs of mung bean SSR polymorphism primers, and wherein, SEQ ID No.1 and 2 is a pair SSR primer, and SEQ ID No.3 and 4 is a pair SSR primer, the like, annealing temperature and the amplified production size of 32 pairs of mung bean SSR primers are as shown in table 3.
SEQ ID No.65 and 66 is the joint sequence of grappling in cDNA fragment.
The title of table 3 32 pairs of mung bean SSR primers, annealing temperature and amplified production size
Reference
1. Wang Li is chivalrous, Cheng Xuzhen, Wang Suhua. mungbean germplasm resources, breeding and Advance in Genetic Studies. and Scientia Agricultura Sinica, 2009,42 (5): 1519-1527
2. Chen Xin, Yuan Xingxing, Chen Huatao, turns round and look at peace, Zhang Hongmei, Cui Xiaoyan, Chen Yu. mung bean research latest developments and future thrust. and Jinling School of Science and Technology journal, 2010,26 (2): 59-68
3. journey must be precious, Wang Suhua. Chinese mung bean variety the Study on Resources. and Crop Germplasm Resources, 1998, (4): 9-11
4. journey must be precious, Wang Suhua, Wang Lixia. mungbean germplasm resources Description standard and data standard [M]. and Beijing: Scientia Agricultura Sinica technology press, 2006:1-2
5. the long friend of Liu, Cheng Xuzhen, Wang Suhua, Wang Lixia, Sun Lei, Mei Li, Xu Nin. Mungbean Germplasm in China genetic diversity Journal of Sex Research. plant genetic resources journal, 2006,7 (4): 459-463
6. Zhao Dan, Cheng Xuzhen, Wang Lixia, Wang Suhua. plant genetic resources journal, 2010,11 (5): 583-588
7. Huanghai Sea swallow, Du Hongyan, black clouds tower Na, Liu Panfeng. based on the exploitation of the SSR molecular marker of bark of eucommia transcript profile sequence. forest-science, 2013,49 (5): 176-181
8. Lee little Bai, Xiang Lin, Jie Luo, marks woods, Tian Shengping, Xie Ming, Sun Chongbo recklessly. transcript profile order-checking (RNA-seq) strategy and the application of data on molecular markers development thereof. and Chinese cytobiology journal, 2013,35 (5): 1-8
9. grandson is clear and bright, Marvin's court, Ma Shuaipeng, Zhao Junsheng, Bai Lijun, Chen Jiezhen, Cai Changhe, Xiang Xu, Ou Liangxi. the SSR information analysis of lichee EST resource and EST-SSR marker development. and Scientia Agricultura Sinica, 2011,44 (19): 4037-4049
10. seedling snow rosy clouds, Li Haichao, Wang Yubing, Zhang Hao, Huang Yongping. from the method .CN102277351A obtaining gene information and functional gene without genome reference sequences species
11.CN201010197328
12.CN201010197347
Claims (3)
1., based on a method for transcript profile order-checking exploitation mung bean SSR primer, it is characterized in that, comprise the following steps:
One, the acquisition of transcript profile data
Trizol reagent is utilized to extract mung bean whole strain seedling total serum IgE, with Oligo (dT) enrichment with magnetic bead mRNA; Add fragmentation buffer and mRNA is broken into short-movie section, take mRNA as template, with hexabasic base random primer synthesis Article 1 cDNA chain, then add damping fluid, dNTPs, RNase H and DNA polymerase I synthesizes Article 2 cDNA chain, end reparation is being done after adding EB buffer solution elution through QiaQuick PCR kit purifying, add A and connect sequence measuring joints, then clip size selection is carried out with agarose gel electrophoresis, finally carry out pcr amplification, the sequencing library IlluminaHiseq 2000 built checks order;
Reverse transcription synthetic double chain cDNA, purifying cDNA, carries out end reparation, adds A and connects sequence measuring joints, then carries out clip size selection with agarose gel electrophoresis, finally carries out pcr amplification; Concrete grammar is as follows:
(1) extraction of mung bean Total RNA
Adopt conventional Trizol method to extract, purifying, DNA enzymatic process, acquisition concentration >=50ng/ μ l, total amount >=3 μ g, OD260/280 are the Total RNA sample of 1.8-2.2;
(2) mRNA separation and interrupt at random
Go out the mRNA with polyA with the Beads enrichment with oligo-dT, then utilize ultrasonic wave to interrupt at random, reclaim the fragment of 200-700bp;
(3) synthesis of cDNA first chain and the second chain
The synthesis of cDNA first chain carries out with random 6 polymers and Superscript II reversetranscriptase test kit; CDNA second chain completes with RNase H and DNA polymerase i;
(4) joint sequence of grappling in cDNA fragment:
5′RNA Adapter：
5′-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-3′；
3′RNA Adapter：
5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′；
(5) pcr amplification carries out the pcr amplification of 15 circulations with the primer in above-mentioned joint sequence;
(6) library construction and detection utilize the sequence obtained in above-mentioned steps, carry out library construction and detection according to Illumina company sample prep kit;
(7) order-checking of RNA-seq
The library of building up is added to the concentration of 5-7pM in the respective channel of Illumina sequenator, runs 36 circulations;
(8) data analysis
Reject impurity data, the result after RNA-seq assembling is integrated; What step before obtained is raw data, wherein containing the joint sequence added in 4 in steps, is called Clean reads after being removed, just can carry out splicing and assemble; Concrete grammar utilizes the Cleanreads that will obtain, and adopts the Trinity software for transcript profile splicing to splice; Trinity software version: v2012-10-05; Optimum configurations: min_kmer_cov is 2, and other parameter is that sequencing sequence is spliced into a transcript profile, in this, as the reference sequences of subsequent analysis by default parameters Trinity; Get transcript the longest in every bar gene as Unigene;
(9) bioinformatic analysis
Unigene sequence obtained above and albumen database nr, Swiss-Prot, KEGG and KOG are carried out blastx comparison, evalue < 0.00001, get the sequence direction that the best albumen of comparison result determines Unigene; If the comparison result between different sink is contradictory, the sequence direction of Unigene is then determined by the priority of nr, Swiss-Prot, KEGG and KOG, catch up with state 4 storehouses all less than Unigene, predict its coding region with software ESTScan and determine the direction of sequence; For the Unigene that can determine sequence direction, provide the sequence in its from 5 ' to 3 ' direction; For the Unigene that cannot determine sequence direction, provide the sequence that composite software obtains; Functional annotation is carried out to these genes, has comprised KOG classification and GO annotation;
Two, the identification of SSR primer
Perl language is installed, download est_trimmer.pl from http://pgrc.1pk-gatersleben.de/misa/ and run, remove in transcript profile sequence and be less than the too short sequence of 100bp and be greater than the long sequence of 2000bp, action command is: C: perl bin>perlest_trimmer, piA.fasta-amb=2,50-tr5=T, 5,50-tr3=A, 5,50-cut=100,2000; Exporting two file A.fasta.log and A.fasta.results, A is short title; CD_HIT software is downloaded from http://www.bioinformatics.org/cd-hit, it is utilized to remove redundant sequence: A.fasta.results to be copied in cd_hit folder and RNTO B.fasta, run cd_hit.exe, under Perl environment, action command is: C: perl bin cd_hit>cd_hit.exe-1B.fasta-oC.fasta-cl.00-n5-M2000, export three files, wherein C.fsata file is used for next step process, and A, B and C are short title; Misa.pi program is downloaded with the SSR identified and positioning sequence from http://pgrc.1pk-gatersleben.de/misa/; Optimum configurations is as follows: the multiplicity of mononucleotide, dinucleotides, trinucleotide, tetranucleotide, pentanucleotide and Hexanucleotide is at least 10,6,5,3,3,3; By C.fsata file copy to C dish perl under bin catalogue, action command under Perl environment: C: perl bin>perlmisa.plC.fasta, produce C.fasta.misa and C.fasta.statistics two files after running, wherein C.fasta.misa is used for follow-up design of primers;
Three, the design of SSR primer
Primer3 module Batch Design SSR primer under use Perl environment: design of primers parameter is Tm55-65 DEG C, and primer length is 18-22bp; Under running p3_out.pi, Perl environment, action command is: C: perl bin>perlp3_in.plC.fasta.misa, create the input file of the primer3 of a C.fasta.p3in by name; Copy again C.fasta.p3in file to C dish perl bin primer3 under bin root directory, run the design of primers that primer3_core.exe realizes batch, under Perl environment, action command is: C: perl bin primer3 bin>primer3_core.exe<C.fasta. p3in>C.fasta.p3out, produce the file of a C.fasta.p3out by name; Finally by C.fasta.p3out file copy to C dish perl under bin catalogue, run p3_out.pi, its order is: C: perl bin>perl p3_out.pl C.fasta.p3outC.fasta.misa, obtain C.fasta.results file after operation, this is the primer designed;
Then identify the polymorphism of SSR primer, the mung bean for the identification of SSR primer polymorphism is selected from No. 1, Chinese medium green, No. 5, medium green; Thailand VC2778A, TC1966; Russia 1810,1865; At least one in Australia ACC814, ACC41.
2. the mung bean SSR primer of method exploitation according to claim 1, it is characterized in that, the sequence of described SSR primer is as shown in SEQ ID No.1-64.
3. the application of mung bean SSR primer in mung bean molecular mark described in claim 2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277416A (en) * | 2010-06-10 | 2011-12-14 | 中国科学院上海生命科学研究院 | Transcriptome and functional gene for Ostrinia furnacalis Guenee |
| CN103233075A (en) * | 2013-05-09 | 2013-08-07 | 南京农业大学 | Method for developing dendranthema SSR (Simple Sequence Repeat) primer based on transcriptome sequencing |
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| CN103233075A (en) * | 2013-05-09 | 2013-08-07 | 南京农业大学 | Method for developing dendranthema SSR (Simple Sequence Repeat) primer based on transcriptome sequencing |
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