CN103642912A - 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 transcribing group 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.Generally plant various countries, East Asia now, 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 that type is various.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 in the international market favor.In recent years, world market all increases to some extent to the turnout of the demand of mung bean and whole world mung bean, and the mung bean annual export volume of China is ten thousand tons of 20-30 now, the general 400-500 dollar of export price.The social economic value of mung bean can not be ignored.Yet, compare as corn, paddy rice with staple crop, both at home and abroad the research of mung bean is also quite lagged behind, per unit area yield is still in lower level, the more aobvious weakness of the research of molecular level.
Molecule marker is that to take genetic material inner nucleotide sequence variations between individuality be basic genetic marker, can on DNA level, reflect the difference on plant genetic basis, 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 groups, because the multiplicity of SSR is different different with repetition degree, makes it present the polymorphism of height.Compare with other molecular marking technique, SSR mark has that polymorphism information content is high, codominant inheritance, technology are simple, reproducible, high specificity, operation is convenient and in genome the advantage such as discrete distribution become one of molecule marker the most popular to people, be considered to one of molecule marker type that reliability is the highest.Widespread use in a lot of fields.But the main drawback of SSR mark is first will from these species, obtain the sequence information of tumor-necrosis factor glycoproteins both sides, and designs primer, then just can be utilized.
SSR mark can be divided into genome SSR (gSSR) and expressed sequence tag SSR (EST-SSR), EST-SSR mark comes from the transcriptional domain of gene, compare with gSSR mark, its polymorphism may be directly related with gene function, therefore, than gSSR mark, there is more high universalizable, more economical, high-level efficiency more.Utilize s-generation sequencing technologies to carry out large-scale high-flux sequence to the transcript in full genome range, and can produce than EST, check order magnanimity more transcribe group data, the exploitation of the SSR of Zhe Wei functional genome mark provides abundanter and extremely valuable available stock.
The quantity of transcribing group sequence grows with each passing day, and making to obtain SSR by database search method becomes possibility.But the data that produce from s-generation sequencing technologies are often extremely huge, and a large amount of est sequences is carried out to format analysis processing, and 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 processing and system management etc.Along with the development of information biology, Perl has more been applied in the operation and retrieval of biological data, makes data unification in enormous quantities be treated as possibility.Carry out on this basis EST-SSR primer development and more can improve separation efficiency, save time and fund.
Mung bean there is no whole genome sequence information, mung bean SSR primer comparatively small amt at present.For without with reference to the genomic group analysis of transcribing, can first the sequence assembly of order-checking gained be become to transcript, take transcript as reference sequences, carry out subsequent analysis.That utilizes that s-generation high throughput sequencing technologies obtains a certain material in mung bean transcribes group sequence information, the technology maturation of 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. in batches.
Summary of the invention
The object of this invention is to provide a kind of method based on transcribing group order-checking exploitation mung bean SSR primer.
In order to realize the object of the invention, a kind of method based on transcribing group order-checking exploitation mung bean SSR primer of the present invention, said method comprising the steps of:
1) build and transcribe group library: extract the total RNA of Catalase, isolate mRNA, reverse transcription synthetic double chain cDNA, purifying cDNA, at cDNA end, add adenosine and connect sequence measuring joints, then by agarose gel electrophoresis, reclaim 200-700bp fragment, to reclaiming fragment, carry out pcr amplification, build and obtain transcribing group library;
2) checked order in the above-mentioned group library of transcribing, utilize software Trinity that sequencing sequence is spliced into a complete group of transcribing, get transcript the longest in every gene as Unigene, and Unigene sequence is carried out to bioinformatic analysis;
3) adopt software MISA1.0 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) version of the Trinity of software described in is v2012-10-05; Parameter arranges: min_kmer_cov is 2, and other parameter is default parameters.
Step 2) bioinformatic analysis described in includes 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 that the enrichment of GO function significance is analyzed and/or the enrichment of Pathway significance is analyzed.
The parameter of carrying out the use of SSR design of primers in step 4) is: primer length 18-22bp, Tm55-65 ℃, product size 100-300bp.
Mung bean for the identification of SSR primer polymorphism in step 4) 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 etc.
The present invention also provides the mung bean SSR developing 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 in mung bean molecular mark according to the mung bean SSR primer of aforesaid method exploitation.
Particularly, a kind of method based on transcribing group sequence exploitation mung bean SSR primer provided by the invention, comprises the steps:
1) transcribe the acquisition of group data
Extract the total RNA of Catalase, isolate mRNA, reverse transcription synthetic double chain cDNA, purifying cDNA, carries out end reparation, add A and connect sequence measuring joints, then with agarose gel electrophoresis, carry out clip size selection, finally carry out pcr amplification, build and transcribe group library, the sequencing library of building up utilizes the method for two end sequencings (Paired-End) to check order with Illumina HiSeqTM2000, obtains mung bean and transcribes group sequencing data.The sequencing data amount of each sample individuality is 5GbClean Data.
2) identification of SSR sequence
First Perl language is installed, from http://pgrc.1pk-gatersleben.de/misa website, is downloaded est_trimmer.pl, remove and transcribe sequence too short in group sequence and long sequence; From http://www.bioinformatics, in org/cd-hit/, download CD_HIT software, remove redundant sequence.
From http://pgrc.1pk-gatersleben.de/misa website, download and use MISA software with SSR identification and positioning sequence, parameter arranges 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 ℃, wherein, primer Tm value differs 4 ℃, 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 and 13134 pairs of SSR primers, choose at random 50 pairs of primers and carry out pcr amplification, adopt 8% native polyacrylamide gel electrophoresis to detect.
The invention provides a kind of side that transcribes group order-checking exploitation mung bean SSR primer without genome reference, comprise the steps: to obtain the set of the full genome transcript of mung bean, formation sequence database; With Trinity, sequencing sequence is spliced into one and transcribes group, using this reference sequences as subsequent analysis, get transcript the longest in every gene as Unigene; Unigene sequence bioinformatic analysis; Adopt MISA1.0 to carry out SSR detection to Unigene; With Primer3, carry out SSR design of primers, and carry out SSR primer identification of polymorphisms.The present invention also provides the method for transcribing group information and functional gene that obtains mung bean.Application present method has successfully designed 13134 pairs of SSR primers, therefrom choose at random 50 pairs of primer pairs derive from country variant totally 8 parts of mung bean DNA verify, wherein there are 46 pairs of SSR primers clear band to be detected at 100-300bp, show that design of primers success ratio is higher, wherein polymorphic primer has 32 pairs, 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 organizing analysis of biological information schematic flow sheet with reference to genomic transcribing in the embodiment of the present invention 1.
Fig. 4 is the Unigene staple diagram that in the embodiment of the present invention 1, splicing obtains.
Fig. 5 is SSR density profile in the embodiment of the present invention 2.
Fig. 6 is part SSR repeating group unit's type and quantity in the embodiment of the present invention 2.
Fig. 7 utilizes part SSR primer pair to derive from 4 countries (respectively 2 parts of China, Thailand, Australia, Russia) totally 8 parts of results that mung bean DNA carries out polymorphism checking in the embodiment of the present invention 3.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is all according to normal experiment condition, as Sambrook equimolecular cloning experimentation handbook (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer specification sheets suggestion.
Test materials used in following examples, if no special instructions, all buys and obtains from routine biochemistry reagent shop.Trizol, RNase H and Superscript IIreversetranscriptase test kit are all purchased from Invitrogen company.DNA polymerase i is purchased from NEB company.In cDNA fragment, the joint sequence of grappling is purchased from the sequencing kit by Illumina.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
The design of embodiment 1RNA-seq analysis and SSR primer
One, transcribe the acquisition of group data
Utilize Trizol reagent to extract the total RNA of the whole strain seedling of mung bean, use with Oligo(dT) enrichment with magnetic bead mRNA.Add fragmentation buffer that mRNA is broken into short-movie section, take mRNA as template, with the synthetic article one cDNA chain of hexabasic base random primer, then add damping fluid, dNTPs, RNase H and DNA polymerase I to synthesize second cDNA chain, through QiaQuick PCR test kit purifying and after adding EB buffer solution elution, doing end reparation, add A and connect sequence measuring joints, then with agarose gel electrophoresis, carry out clip size selection, finally carry out pcr amplification, the sequencing library building checks order with IlluminaHiseq2000.
Reverse transcription synthetic double chain cDNA, purifying cDNA, carries out end reparation, adds A and connects sequence measuring joints, then with agarose gel electrophoresis, carries out clip size selection, 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
Adopt conventional Trizol method to extract, purifying, DNA enzyme is processed, the Total RNA sample that to obtain concentration >=50ng/ μ l, total amount >=3 μ g, OD260/280 be 1.8-2.2 (electrophoresis detection and NanoDrop initial survey, more preferably select that sample carries out Qubit quantitatively and Agilent2100 detection).
2.mRNA separated and interrupting at random
Use with the magnetic bead of oligo-dT and isolate the mRNA with polyA, then utilize ultrasonic wave to interrupt at random, reclaim the fragment of 200-700bp.
Synthesizing of 3.cDNA the first chain and the second chain
The synthetic of cDNA the first chain is to carry out with random 6 polymers and Superscript II reverse transcriptase test kit.CDNA the second chain is to complete 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 that 5.PCR amplification is carried out 15 circulations with the primer in above-mentioned joint sequence.
6. library construction and detection utilize the sequence obtaining in above-mentioned steps, according to the sample prep kit of Illumina company, carry out library construction and detection.
The order-checking of 7.RNA-seq
Concentration by the library of building up with 5-7pM is added in the respective channel of Illumina sequenator (Genome Analyzer II), moves 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 adding in 4 in steps, is called Clean reads after being removed, just can splice and assemble.Concrete grammar is to utilize the Cleanreads obtaining, and adopts for the Trinity(version of transcribing group splicing: v2012-10-05; Parameter setting: min_kmer_cov is 2, and other parameter is default parameters) software splices.With Trinity, sequencing sequence is spliced into one and transcribes group, using this reference sequences as subsequent analysis.Get transcript the longest in every gene as Unigene.
9. bioinformatic analysis
Without seeing Fig. 3 with reference to the genomic group analysis of biological information flow process of transcribing.Unigene sequence obtained above and albumen database nr, Swiss-Prot, KEGG and KOG are carried out to blastx and compare (evalue < 0.00001), get the sequence direction that the best albumen of comparison result is determined Unigene.If the comparison result between different sink is contradictory, by the priority of nr, Swiss-Prot, KEGG and KOG, determine the sequence direction of Unigene, catch up with state 4 storehouses all less than Unigene, with software ESTScan, predict its coding region and determine the direction of sequence.For the Unigene that can determine sequence direction, provide its from 5 ' to the sequence of 3 ' direction; For the Unigene that cannot determine sequence direction, provide the sequence that composite software obtains.These genes have been carried out to functional annotation, comprised KOG classification and GO annotation.Partial analysis situation as shown in Figure 4.
Two, the identification of SSR primer
Perl language is installed, from http://pgrc.1pk-gatersleben.de/misa/, download est_trimmer.pl operation, removal is transcribed in group sequence and is less than the too short sequence of 100bp and is greater than the long sequence of 2000bp, and 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).From http://www.bioinformatics.org/cd-hit, download CD_HIT software, utilize it to remove redundant sequence: A.fasta.results is copied in cd_hit folder and RNTO B.fasta, operation 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 processed (A, B and C are short title) for next step.From http://pgrc.1pk-gatersleben.de/misa/, download misa.pi program with the SSR identification and positioning sequence; Parameter arranges 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, after operation, produce C.fasta.misa and two files of C.fasta.statistics, wherein C.fasta.misa is for follow-up design of primers.
Three, the design of SSR primer
Use primer3 module Batch Design SSR primer under Perl environment: design of primers parameter is Tm55-65 ℃, and primer length is 18-22bp.Operation p3_out.pi, under Perl environment, action command is: C: perl bin>perlp3_in.plC.fasta.misa, produced 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, operation primer3_core.exe realizes design of primers in batches, 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, operation p3_out.pi, its order is: C: perl bin>perl p3_out.pl C.fasta.p3out C.fasta.misa, after operation, obtain C.fasta.results file, this is the primer designing.
The excavation in embodiment 2 mung bean high-throughput SSR sites
Application aforesaid method is used Catalase to carry out high-flux sequence as material, utilizes Perl language to transcribe group sequence to mung bean and carries out the excavation in high-throughput SSR site, obtains 83542 and transcribes group sequence 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 splicing length frequency distribution situation
Table 2 repeats primitive situation
Use primer3.0 Batch Design software to design altogether and obtain 13134 pairs of SSR primers.Therefrom 50 pairs of primers of random selection (seeing nucleotides sequence list), utilize Catalase DNA to carry out the detection of design of primers success ratio, and result shows, has 46 pairs of SSR primers and clear band detected at 100-300bp, shows 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, with 0.8% agarose gel electrophoresis method, detect its quality, DNA concentration dilution is placed on to-20 ℃ to 50ng/ μ L and saves backup.Utilize the DNA of primer development material therefor to carry out design of primers success ratio PCR evaluation.PCR reaction system adopts the reaction system of 10 μ L, comprising 40ng genomic dna, and 1 * Taq enzyme buffer liquid (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 μ mol L
-1with 1U Taq archaeal dna polymerase.SSR response procedures is: 95 ℃ of denaturation 5min, and 95 ℃ of distortion 30s, 51-60 ℃ of annealing 45s, 72 ℃ are extended 45s, carry out 32-35 circulation, and last 72 ℃ are extended 5min.After reaction finishes, product adds 2 μ L sample loading buffers, take 100bp DNA ladder as DNA molecular amount standard, the non-denaturing polyacrylamide gel of employing 8% carries out electrophoresis, electrophoretic buffer is 0.5 * TBE, 200V voltage stabilizing electrophoresis 2-2.5h, while moving on to gel bottom to sample loading buffer, electrophoresis finishes.After electrophoresis finishes, adopt argentation dyeing, finally gel is placed on gel imaging system and is taken pictures.Twice of all Data duplication.
Choose 8 parts of mung bean material DNA of 50 pairs of primer pairs and 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 pairs, show that 46 couples of 32 couples (sequence is respectively as shown in SEQ ID No.1-64) in primer can be used for distinguishing the mung bean material of different geographic origin.Show to utilize this mung bean to transcribe the method for group 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, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence explanation:
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 of SSR primer, and SEQ ID No.3 and 4 is a pair of 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 32 pairs of mung bean SSR primers of table 3, annealing temperature and amplified production size
Reference
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3. journey must be precious, Wang Suhua. Chinese mung bean variety the Study on Resources. and Crop Germplasm Resources, 1998, (4): 9-11
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Claims (10)
1. the method based on transcribing group order-checking exploitation mung bean SSR primer, is characterized in that, comprises the following steps:
1) build and transcribe group library: extract the total RNA of Catalase, isolate mRNA, reverse transcription synthetic double chain cDNA, purifying cDNA, at cDNA end, add adenosine and connect sequence measuring joints, then by agarose gel electrophoresis, reclaim 200-700bp fragment, to reclaiming fragment, carry out pcr amplification, build and obtain transcribing group library;
2) checked order in the above-mentioned group library of transcribing, utilize software Trinity that sequencing sequence is spliced into a complete group of transcribing, get transcript the longest in every gene as Unigene, and Unigene sequence is carried out to bioinformatic analysis;
3) adopt software MISA1.0 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.
2. method according to claim 1, is characterized in that, sequence measuring joints described in step 1) is:
5′RNA?Adapter：5′-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-3′
3′RNA?Adapter：5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′。
3. method according to claim 1, is characterized in that step 2) described in the version of software Trinity be v2012-10-05; Parameter arranges: min_kmer_cov is 2, and other parameter is default parameters.
4. method according to claim 1, is characterized in that step 2) described in bioinformatic analysis include but not limited to gene annotation, CDS prediction and differential gene expression screening.
5. method according to claim 4, is characterized in that, described gene annotation comprises gene expression amount annotation and/or annotation of gene function.
6. method according to claim 4, is characterized in that, described differential gene expression screening comprises that the enrichment of GO function significance is analyzed and/or the enrichment of Pathway significance is analyzed.
7. method according to claim 1, is characterized in that, the mung bean for the identification of SSR primer polymorphism in step 4) 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.
8. method according to claim 1, is characterized in that, the parameter of carrying out the use of SSR design of primers in step 4) is: primer length 18-22bp, Tm55-65 ℃, product size 100-300bp.
9. according to the mung bean SSR primer of method exploitation described in claim 1-8 any one, it is characterized in that, the sequence of described SSR primer is as shown in SEQ ID No.1-64.
10. the application of mung bean SSR primer in mung bean molecular mark described in claim 9.
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