CN108034696A - A kind of method based on transcript profile sequencing SSR primers development - Google Patents
A kind of method based on transcript profile sequencing SSR primers development Download PDFInfo
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Abstract
The invention discloses a kind of method based on transcript profile sequencing SSR primers development.This method is based on existing whole genome sequence data and its annotation, screens candidate SSR sites for its genomic exon sequences and designs SSR amplimers.The data for being then based on multiple individual transcript profile sequencings carry out ePCR verifications, finally filter out the good SSR sites of polymorphism and obtain its amplimer.Method provided by the invention greatly reduces construction cycle and manpower with the laboratory PCR used in the past with polyacrylamide gel electrophoresis, and compares and construction cycle and exploitation funds are greatly reduced compared with the screening technique of sequencing and typing using laboratory PCR.Method proposed by the present invention is applicable not only to SSR primer developments, applies also for all SSR primer developments with reference gene group species, is with a wide range of applications.
Description
Technical field
The invention belongs to SSR primer development technical fields, and in particular to a kind of new method of SSR primers development.
Background technology
Microsatellite marker (microsatellite), the short tandem repeat that is otherwise known as (short tandem
Repeats, STRs) or simple repeated sequence (simple sequence repeats), it is to be uniformly distributed in eukaryotic gene
Simple repeated sequence in group, is made of the tandem repeat of 2~6 nucleotide, since the number of repetition of recurring unit exists
Enriched between individual in variability and quantity, therefore the application of microsatellite marker is very extensive.Microsatellite marker codominance
The characteristics of enable it to be used to study allele, distinguish the homozygote or heterozygote of diploid (or polyploid), this be AFLP,
RAPD codominances mark can not be accomplished.In addition, the primer amplified of microsatellite has good repeatability and fidelity
Property, facilitate the exchange of each laboratory monitoring.At present, microsatellite marker be widely applied to gene linkage and genetic map construction,
Genetic diversity Journal of Sex Research, pedigree and development research, disease detection and cultivar identification, parent's analysis and individual, pure lines are examined.
As microsatellite marker is abundant on collection of illustrative plates, the advantage of bigger will become apparent from out.But primer in microsatellite analysis
Obtain need know nucleotide sequence in advance, its extensively using be limited to from specific species separate microsatellite locus difficulty with
Spend.The maximum difficulty of microsatellite marker separation is the acquisition of primer.The genetic markers such as same RAPD, AFLP are different, and microsatellite is ground
Study carefully firstly the need of the sequence information for knowing site, to design primer from the conserved sequence of repetitive sequence both ends flank.Thus
The exploitation of micro-satellite primers is the key using the technology.It is very limited to be currently known the species of microsatellite locus, this is because micro-
The acquisition in satellite site need to pass through clone, screening by hybridization, sequencing, therefore need to spend certain manpower, money, this is limited
A large amount of uses of microsatellite marker.Various microsatellite locus beneficiation technologies developed in recent years solve to a certain extent
Determine this problem.The existing most common method for obtaining microsatellite locus includes：1. micro- defend is searched from public database
Championship point；2. primer transfer amplification between the close species of genetic distance；3. microsatellite locus is screened from genomic DNA.Wherein, most
Convenient, most economical method is exactly that micro-satellite primers are obtained from the document delivered, but the method is only limited to using on object
The species that existing primer development goes out, and the quantity of primer will not increased, and can only stop on the original basis.For nearly edge
The application of kind primer, its scope of application has much actually；The microsatellite locus of original species in other thing Interspecific transfer amplifications,
How is its polymorphism.When these problems to share micro-satellite primers between different plant species, blindness is larger, can instruct operation
Theoretical foundation deficiency.Best approach is exactly that microsatellite locus is screened from genomic DNA and carries out the exploitation of primer.
Microsatellite locus usually passes through electrophoretic analysis or sequencing analysis and basis by PCR amplification, amplified production at present
Size separation allele is detected.In fact, the SSR sites developed from huge genomic data often count
It is bad to measure huge and polymorphism.The problem of this brings to us is exactly that still workload is huge after the identification of SSR sites.Grind
The person of studying carefully usually needs to design hundreds of pairs of primers easily, expands thousands of a samples, and carries out electrophoresis or sequence verification one by one to them,
The manpower, material resources and financial resources not only expended, which are appointed, can not so despise, and the effective SSR sites finally obtained are also (more than ten pairs to tens very limited
It is right).Therefore, develop a set of efficiently quick SSR primer developments method has very great show to the research for carrying out SSR sites
Sincere justice.
SSR marker development approach is in addition, there will be to be also divided into developing and based on EST based on genome SSR
SSR is developed.SSR marker based on expressed sequence exploitation conservative in Spherical scanning is more preferable, more efficient.And based on expression
The SSR marker of data is the expressed sequence based on a certain period, directly related with gene function and trait phenotypes, rare
Endangered animal and plant germ plasm resource and genetic diversity assessment etc. have important researching value.With producing shockingly for sequencing data
Hair, excavates from transcript profile data and develops new SSR marker as a kind of simple efficient mode.But it is currently based on transcript profile
The method that developing SSR mark is sequenced remains unchanged in the presence of the situation for needing a large amount of checking tests.
The content of the invention
The purpose of the present invention is in view of the deficienciess of the prior art, providing a kind of based on transcript profile sequencing developing SSR mark
Remember the new method of primer, this method searches for SSR sites from the exon sequence of genome, and designs corresponding PCR amplification primer,
First pass through extron and carry out ePCR verifications, finally carry out ePCR verifications without the transcript that ginseng assembling obtains using individual, it is final to obtain
To the good SSR marker of polymorphism and its primer.This method provides to study the identification of the genetic diversity and germ plasm resource of species
A kind of efficiently quick new way.
In order to realize above-mentioned technical purpose, a kind of method based on transcript profile sequencing SSR primers development, including following step
Suddenly：
(1) sample of 2 Different Individuals is at least gathered, total serum IgE is extracted respectively and carries out transcript profile sequencing library structure；
(2) transcript profile sequencing and data filtering；
(3) each sample individually carries out transcript without ginseng assembling, obtains each individual all transcripts；
(4) the SSR screenings based on exon sequence and design of primers：Reference gene group sequence, extraction are downloaded from database
All exon sequences, and using 2.0 softwares of GMATA identification SSR sites, and design primer；The primer GMATA that will be obtained
2.0 ePCR functions carry out ePCR to exon sequence, exclude the primer pair of specificity difference；
(5) the ePCR verifications of SSR primers：The ePCR functions for the SSR primers GMATA 2.0 that (4) step is screened are to
(3) step obtains each individual transcript and carries out ePCR respectively, and primer is screened from result.
Step (1) extract total serum IgE and carry out transcript profile sequencing library structure detailed process it is as follows：Total serum IgE uses
QIAGEN RNeasy Protect Animal Blood Kit (73224) are extracted, and are checked with agarose gel electrophoresis
The quality of RNA；Transcript profile sequencing library structure uses VAHTSTM mRNA-seq v2Library Prep Kit for(NR602-01) kit, i.e., each sample take 1 μ g total serum IgEs, are then purified with polyadenylic acid Beads enrichment
MRNA is obtained, 98 DEG C of processing in 8 minutes carry out fragmentation in VAHTS Frag/Prime Buffer with bivalent cation；Purifying
The first chains of cDNA and the synthesis of the second chain are carried out afterwards, then carry out end-filling, dATP adds end to be connected with connector；Ago-Gel
Electrophoresis selection 150-200bp fragments carry out magnetic beads for purifying, carry out RCR amplified libraries afterwards：98 DEG C of 10s denaturation, 60 DEG C of 30s knots
Close, 72 DEG C of 30s extensions, cyclic amplification 12 times；Finally, it is sequenced after confirming to Library Quality detection.
Step (2) transcript profile is sequenced and the detailed process of data filtering is as follows：MRNA libraries use Illumina
Hiseq2500 is sequenced；Sequencing data is N content filtering out low quality sequence and connector polluted sequence, low quality sequence
More than 30% or sequence of the low quality base contents more than 10%, quality testing is carried out with fastqc software defaults parameter afterwards,
Ensure that data are effective.
Detailed process of step (3) transcript without ginseng assembling is as follows：Using assembling streams of the Trinity without reference gene group
Journey, default parameters individually carry out each sample transcript assembling, and each individual obtains the transcript profile sequential file of oneself
Trinity.fasta。
The search criterion for the SSR that step (4) is identified is：Minimum Repeated m otif is dinucleotides, maximum Repeated m otif
For ten nucleotide；At least it is repeated 5 times in exon sequence.
Step (4) design primer condition be：Amplified production length is between 120bp~400bp；Product flanking sequence is grown
Spend for 400bp；Optimum annealing temperature Tm is 60 DEG C；Maximum template length is 2000bp.
Step (4) and step (5) the ePCR procedure parameters are：Word length is 12, consecutive word a length of 1, and maximum missing is 1,
Maximum mispairing is 0, and amplified production length is 100-1000bp.
The standard of screening primer described in step (5) is：The amplified production Fragment Differential at least in two different samples
For the integral multiple of SSR repetitive unit motif length.
Compared with the prior art, the advantageous effects that technical scheme is brought have：It is sequenced and is utilized based on transcript profile
EPCR carries out the polymorphic detection verification of SSR primers twice, compared with the conventional method, can greatly save manpower, material resources and financial resources,
It is a kind of new method of Efficient Development SSR marker.
Brief description of the drawings
Fig. 1 is the electrophoresis result for two pairs of primers that present invention screening obtains.
Embodiment
Present invention is further elaborated by the following examples, but model is protected not as to the claims in the present invention
The restriction enclosed.The operation of all kits carries out to specifications, at transcript profile Library development flow not specified (NS) according to normal process into
OK.
Embodiment 1
First, the extraction of total serum IgE and transcript profile library construction
4 Rhinopithecus roxellana samples of the present embodiment pick up from Shennongjia, Hubei Province national park.The method of cylinder is blown by gold with anesthesia
After silk monkey anesthesia, take hind leg vein blood dry ice to transport -80 DEG C of preservations and be used for subsequent extracted total serum IgE.Total serum IgE uses QIAGEN
RNeasy Protect Animal Blood Kit (73224) are extracted, and the matter of RNA is checked with agarose gel electrophoresis
Amount.Transcript profile sequencing library structure uses VAHTSTM mRNA-seq v2Library Prep Kit for
(NR602-01) kit.It is exactly that each sample takes 1 μ g total serum IgEs, is then purified with polyadenylic acid Beads enrichment in simple terms
MRNA is obtained, (98 DEG C 8 points of fragmentation is carried out in the processing of VAHTS Frag/Prime Buffer high temperatures with bivalent cation
Clock).The first chains of cDNA and the synthesis of the second chain are carried out after purifying, then carries out end-filling, dATP adds end to be connected with connector.Fine jade
Sepharose electrophoresis selection 150-200bp fragments carry out magnetic beads for purifying, carry out RCR amplified libraries afterwards：98 DEG C of 10s denaturation, 60
DEG C 30s is combined, 72 DEG C of 30s extensions, cyclic amplification 12 times.Finally, Library Quality Agilent2100Bioanalyzer
Detection is quantified with final before (Agilent Technologies) and qPCR carry out machine.
2nd, transcript profile sequencing and data filtering：
MRNA libraries are sequenced using Illumina Hiseq2500.N content is more than 30% or low-quality in initial data
Amount low quality sequence of the base contents more than 10% is filtered first.Connector pollution is gone using cutadapt1.9 versions
Remove.Sequencing data carries out quality testing, it is ensured that data after low quality data and connector pollution is filtered out with fastqc softwares
Effectively.Quality data using Trinity 2.2.0 default parameters assemble without reference gene group transcript, each sample list
Solely assembling, obtains respective Trinity.fasta files.Row statistics such as table 1 are put into transcriptome.
3rd, the search of SSR sites and amplimer design
RefSeq databases are downloaded under the genome reference sequences (Rox v1) of golden monkey and corresponding comment file
(http://www.ncbi.nlm.nih.gov/refseq).First write in perl script foundation comment file and include sub-information, from
All exon sequences are extracted in genome sequence file.Then SSR search is carried out using Windows editions 2.0 softwares of GMATA,
2~10 nucleotide repeating units are searched, and number of repetition is more than or equal to the SSR sites of 5 times.As a result search altogether outer aobvious
Sub-district dinucleotides repeat 5011, Trinucleotide repeats 2733, tetranucleotide repeat 217, pentanucleotide repeat 60,
Hexanucleotide repeats 42 and eight nucleotide repeat 2.The Primer 3 embedded using MGATA carries out design of primers, sets ginseng
Number is：Amplified production length is between 120bp~400bp；Product flanking sequence length is 400bp；Optimum annealing temperature Tm is
60℃；Maximum template length is 2000bp.As a result it is right to have successful design primer 5279 altogether, selects 5 pairs to be illustrated in table 2 at random.
4th, the screening of SSR marker and the verification of polymorphism
EPCR programs are embedded in first with GMATA, the genomic exon sequences of above onestep extraction are carried out as template
ePCR.EPCR procedure parameters are：Word length word size are 12, and continuous word length contiguous word are 1, maximum missing max
Indels is 1, and maximum mispairing max mismatch are 0, and amplified production length is 100-1000bp.Then there will be a plurality of amplification
The PCR primer of band filters out, and obtains 5253 pairs of SSR primers.This process purpose is, amplification in candidate's SSR primers is arrived more
The unqualified primer of a band and the shared SSR sites with pair of primers exclude, in order to avoid disturb follow-up SSR polymorphisms to verify.
Next by all SSR primers, ePCR amplifications are carried out respectively by template of the transcript sequence that each sample assembles.Four
The succeed SSR marker number of amplification of sample is respectively：Sample S1 totally 1520；Sample S2 totally 1502；Sample S3 totally 888
It is a；Sample S4 totally 755；The SSR sites of Successful amplification have 1305 at least in two samples.In all samples in statistical result
There is the SSR sites of polymorphism product in this.The standard of the good primer of screening polymorphism is：At least in two different samples
Amplified production fragment is variant.As a result totally 17 couples of SSR and amplimer (table 3, table 4) with polymorphism are obtained.Product fragment
The more SSR primers of length polymorphic bands in all samples are better.
5th, the verification of SSR primers polymorphism in 9 Rhinopithecus roxellanas
In addition 9 golden monkey sample transcript profile data are taken to be assembled into transcript respectively, it is polymorphic with 17 couple that the 4th step is screened
Property EST-SSR primers carry out ePCR amplifications, amplification such as table 5.17 EST-SSR sites of the results show are in these golden monkeys
Same phenotype goes out different degrees of polymorphism in body.Random 9 samples of wherein two pairs of primer pairs of selecting carry out PCR experiments, and poly- third
Acrylamide Gel electrophoresis results such as Fig. 1.Illustrate that the method provided by the invention based on transcript profile data screening SSR marker accurately has
Effect, primer can apply in the Diversity Detection and Relationship iden- tification of Rhinopithecus roxellana.
The result that 4 Rhinopithecus roxellana sample transcript profiles assemble respectively in 1 embodiment 1 of table
| Sample ID | S1 | S2 | S3 | S4 |
| Transcript sum | 182210 | 173808 | 81807 | 71073 |
| N50 length (bp) | 1504 | 1346 | 869 | 699 |
Part candidate's SSR marker and corresponding based on the identification of Rhinopithecus roxellana genomic exon sequences in 2 embodiment 1 of table
Amplimer
The partial results that screening verification is carried out to candidate's SSR marker are sequenced in 3 embodiment 1 of table based on Rhinopithecus roxellana transcript profile
17 couple screened in 4 embodiment 1 of table has the SSR and amplimer sequence of polymorphism
Table 5
Sequence table
<110>Central South University
<120>A kind of method based on transcript profile sequencing SSR primers development
<160> 44
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 1
atttgggaga agggcagagt 20
<210> 2
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 2
agcaactcac acacacacac a 21
<210> 3
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 3
atttgggaga agggcagagt 20
<210> 4
<211> 22
<212> DNA
<213>Unknown (Unknown)
<400> 4
ggcccacatc tgtacataac aa 22
<210> 5
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 5
caattcccct ctcctcttcc 20
<210> 6
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 6
caggggctgg agtttgatta 20
<210> 7
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 7
ccaaaagaaa accccatcaa 20
<210> 8
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 8
ctgggtgtga gcctgtaatg 20
<210> 9
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 9
cctggtagct caacctcctg 20
<210> 10
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 10
cacgcccatc tttaccattt 20
<210> 11
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 11
gagccccatg acttttctca 20
<210> 12
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 12
gaagccatga gaatggagga 20
<210> 13
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 13
cagcttatcc aaagctctcc a 21
<210> 14
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 14
gtccctccct tccactcttc 20
<210> 15
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 15
ggtaaccaca ccaggtcagc 20
<210> 16
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 16
cccagtgaga agacctttgc 20
<210> 17
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 17
gattcccctg aatccctacc 20
<210> 18
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 18
ggtagtcgaa gccgtagctg 20
<210> 19
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 19
ttttggggtg tctctgtgtg 20
<210> 20
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 20
tcttgggcct acctgaattg 20
<210> 21
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 21
caggcccttc ttcctctagc 20
<210> 22
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 22
gaagaaatgg gcacctttga 20
<210> 23
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 23
aaatatctgg ggagggaagg 20
<210> 24
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 24
ccatcccctt tgcttttaca 20
<210> 25
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 25
aggaccactt agcccaacct 20
<210> 26
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 26
aaatgccacg tctgctcttc 20
<210> 27
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 27
ggggctgtct gaaaactgtg 20
<210> 28
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 28
ttcctttggg gatatgatgc 20
<210> 29
<211> 22
<212> DNA
<213>Unknown (Unknown)
<400> 29
agctttgtgt gaaaaccagt ca 22
<210> 30
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 30
gggttttaga aaggcagcaa 20
<210> 31
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 31
gtatggtggg ccctaggaaa 20
<210> 32
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 32
ctctgggact cctgtgcttc 20
<210> 33
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 33
caagcctggt gaagaggaag 20
<210> 34
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 34
caggtgatct tggggagaga 20
<210> 35
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 35
ggttgggagt tcaagaccag 20
<210> 36
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 36
ccgaagacct taagcccaaa 20
<210> 37
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 37
gggagcatct tctgtgtcaa 20
<210> 38
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 38
tgtacagcat gtcggtctga a 21
<210> 39
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 39
ttagcggtca ctgccttagc 20
<210> 40
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 40
gactccccat gctcctctc 19
<210> 41
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 41
aagaggctga ggttgtggaa 20
<210> 42
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 42
tcagcacaag tccgtcagtc 20
<210> 43
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 43
catgactctc ctggtccaca 20
<210> 44
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 44
cccaactctc tgcattattc g 21
Claims (9)
- A kind of 1. method based on transcript profile sequencing SSR primers development, it is characterised in that：Comprise the following steps：(1) sample of 2 Different Individuals is at least gathered, total serum IgE is extracted respectively and carries out transcript profile sequencing library structure；(2) transcript profile sequencing and data filtering；(3) each sample individually carries out transcript without ginseng assembling, obtains each individual all transcripts；(4) the SSR screenings based on exon sequence and design of primers：Reference gene group sequence is downloaded from database, extraction is all Exon sequence, and using 2.0 softwares of GMATA identification SSR sites, and design primer；By obtained primer GMATA 2.0 EPCR functions to exon sequence carry out ePCR, exclude specificity difference primer pair；(5) the ePCR verifications of SSR primers：The ePCR functions for the SSR primers GMATA 2.0 that (4) step is screened are to (3) step Obtain each individual transcript and carry out ePCR respectively, primer is screened from result.
- 2. the method according to claim 1 based on transcript profile sequencing SSR primers development, it is characterised in that：Step (1) extract total serum IgE and carry out transcript profile sequencing library structure detailed process it is as follows：Total serum IgE uses QIAGEN RNeasy Protect Animal Blood Kit (73224) are extracted, and the matter of RNA is checked with agarose gel electrophoresis Amount；Transcript profile sequencing library structure uses VAHTSTM mRNA-seq v2 Library Prep Kit for (NR602-01) kit, i.e., each sample take 1 μ g total serum IgEs, then purify to obtain mRNA with polyadenylic acid Beads enrichment, With bivalent cation, 98 DEG C of processing in 8 minutes carry out fragmentation in VAHTS Frag/Prime Buffer；Carried out after purifying The first chains of cDNA and the synthesis of the second chain, then carry out end-filling, dATP adds end to be connected with connector；Agarose gel electrophoresis selects 150-200bp fragments carry out magnetic beads for purifying, carry out RCR amplified libraries afterwards：98 DEG C of 10s denaturation, 60 DEG C of 30s are combined, 72 DEG C of 30s Extension, cyclic amplification 12 times；Finally, it is sequenced after confirming to Library Quality detection.
- 3. the method according to claim 1 based on transcript profile sequencing SSR primers development, it is characterised in that：Step (2) transcript profile is sequenced and the detailed process of data filtering is as follows：MRNA libraries use Illumina Hiseq2500 is sequenced；Sequencing data is N content filtering out low quality sequence and connector polluted sequence, low quality sequence More than 30% or sequence of the low quality base contents more than 10%, quality testing is carried out with fastqc software defaults parameter afterwards, Ensure that data are effective.
- 4. the method according to claim 1 based on transcript profile sequencing SSR primers development, it is characterised in that：Detailed process of step (3) transcript without ginseng assembling is as follows：Using assembling flow paths of the Trinity without reference gene group, write from memory Recognize parameter and transcript assembling is individually carried out to each sample, each individual obtains the transcript profile sequential file of oneself Trinity.fasta。
- 5. the method according to claim 1 based on transcript profile sequencing SSR primers development, it is characterised in that：The search criterion for the SSR that step (4) is identified is：Minimum Repeated m otif is dinucleotides, and maximum Repeated m otif is ten Nucleotide；At least it is repeated 5 times in exon sequence.
- 6. the method according to claim 1 based on transcript profile sequencing SSR primers development, it is characterised in that step (4) is set Meter primer condition be：Amplified production length is between 120bp~400bp；Product flanking sequence length is 400bp；Most preferably move back Fiery temperature Tm is 60 DEG C；Maximum template length is 2000bp.
- 7. the method according to claim 1 based on transcript profile sequencing SSR primers development, it is characterised in that：Step (4) and Step (5) the ePCR procedure parameters are：Word length is 12, consecutive word a length of 1, and maximum missing is 1, and maximum mispairing is 0, amplification production Thing length is 100-1000bp.
- 8. the method according to claim 1 based on transcript profile sequencing SSR primers development, it is characterised in that：Step (5) institute The standard for the screening primer stated is：Amplified production Fragment Differential is SSR repetitive units motif at least in two different samples The integral multiple of length.
- 9. the method according to claim 1 based on transcript profile sequencing SSR primers development, it is characterised in that：Step (1) institute The sample stated is Rhinopithecus roxellana.
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| CN108754018A (en) * | 2018-07-27 | 2018-11-06 | 大连民族大学 | A kind of screening technique of wilsonii target gene SSR molecular marker and application |
| CN110491448A (en) * | 2019-07-15 | 2019-11-22 | 广州奇辉生物科技有限公司 | A kind of method, system, platform and storage medium handling PCR primer |
| CN110791573A (en) * | 2019-12-05 | 2020-02-14 | 云南大学 | Microsatellite locus and primer suitable for identifying golden monkey individual |
| CN113337578A (en) * | 2021-06-17 | 2021-09-03 | 集美大学 | Method for efficiently screening positive SNP of aquatic animals based on transcriptome data |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108754018A (en) * | 2018-07-27 | 2018-11-06 | 大连民族大学 | A kind of screening technique of wilsonii target gene SSR molecular marker and application |
| CN110491448A (en) * | 2019-07-15 | 2019-11-22 | 广州奇辉生物科技有限公司 | A kind of method, system, platform and storage medium handling PCR primer |
| CN110791573A (en) * | 2019-12-05 | 2020-02-14 | 云南大学 | Microsatellite locus and primer suitable for identifying golden monkey individual |
| CN110791573B (en) * | 2019-12-05 | 2022-06-03 | 云南大学 | Microsatellite locus and primer suitable for identifying golden monkey individual |
| CN113337578A (en) * | 2021-06-17 | 2021-09-03 | 集美大学 | Method for efficiently screening positive SNP of aquatic animals based on transcriptome data |
| CN113337578B (en) * | 2021-06-17 | 2022-11-08 | 集美大学 | Method for efficiently screening positive SNP (Single nucleotide polymorphism) of aquatic animal based on transcriptome data |
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