CN109504793A - Azalea pontica SSR primer pair and screening technique and application based on transcript profile sequencing exploitation - Google Patents
Azalea pontica SSR primer pair and screening technique and application based on transcript profile sequencing exploitation Download PDFInfo
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Abstract
The invention discloses Azalea pontica SSR primer pairs and screening technique and application based on transcript profile sequencing exploitation.Belong to field of biotechnology.The primer is to analyze to obtain based on transcript profile sequencing and sequence, on the basis of transcript profile sequencing, mass data is screened, it obtains the unigene sequence rich in the site SSR and carries out the design of primers of SSR marker, overcome the obstacle that current Azalea pontica SSR molecular marker quantity is few, development efficiency is low.Using the validity of Azalea pontica group verifying SSR primer, lay a good foundation for genetics research such as Azalea pontica genetic diversity Journal of Sex Research, genetic linkage maps structure, QTL positioning, molecular marks.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to based on transcript profile sequencing exploitation Azalea pontica SSR primer pair and
Screening technique and application.
Background technique
Azalea pontica (Rhododendron molle G.Don) also known as Chinese azalea, Rhododendron molle, silently flower, Solanum lasiocarpum, eight
In fiber crops, tiger flower, belong to Ericaceae (Ericaceae) Rhododendron (Rhododendron) Azalea pontica subgenus (Subgenus
Pentanthera machaka).The seed of Azalea pontica is tiny, and germination and emergence is difficult, and artificial cuttage and division propagation are very
Difficulty survives, because artificially excavating caused by its medical value, so that wild Azalea pontica standing crop falls sharply, even in shape in imminent danger
State belongs to Threatened Plants of The First National List.Azalea pontica branch is graceful, corolla is very large, bright in colour, and Hua Weiwu splits funnel-form, great sight
Reward value is important the important cuckoo breeding resources of afforestation and ornamental plant and American-European countries.Azalea pontica complete stool device
Official has medical value, can relaxing tendons and activating collaterals, analgesia, wind-dispelling, dehumidifying, cough-relieving, scattered silt, be treat rheumatism valuable ingredient of traditional Chinese medicine,
The noisy sheep element contained is efficient medicinal insecticide, has high medical value, therefore has huge potentiality to be exploited and answer
Use prospect.Molecular marking technique is led in cultivar identification, genetic map construction, target gene positioning, molecular mark etc.
Domain is widely used, and is the important technical of flowers research and Breeding Application, but usable molecule in Azalea pontica breeding at present
Marker number is less, significantly limits it in Azalea pontica genetic diversity Journal of Sex Research, the assignment of genes gene mapping, genetic map construction, molecule
It is quickly and effectively applied in marker-assisted breeding.
Simple repeated sequence (Simple sequence repeats, SSR) is widely distributed in eukaryotic gene group,
The tandem repetitive sequence being made of 1-6 nucleotide has polymorphism information content height, codominant inheritance, reproducible, specific
It the features such as strong and technically simple, is ground in genetic diversity Journal of Sex Research, the assignment of genes gene mapping, genetic map construction, molecular mark
It is widely used in studying carefully.SSR marker can be divided into genome SSR (gSSR) and EST SSR (EST- according to existing position
SSR).Since the genomic information of Azalea pontica is not yet sequenced or sequencing data is not yet announced, and the SSR mark of allied species exploitation
Note, which there is no, determines whether across the species uses in Azalea pontica, so more using transcript profile data acquisition SSR molecular marker
Economic, quick, magnanimity.In addition, EST-SSR label derive from DNA transcript regions, compared to gSSR have higher versatility and
This category flag is often chain with functional gene, molecular mark and important goal trait associations analysis on have compared with
High application value.
The development of second generation high throughput sequencing technologies and maturation make that high-throughput acquisition EST-SSR mark is sequenced by transcript profile
Note becomes possibility.There has been no the relevant reports using Azalea pontica transcript profile data magnanimity exploitation polymorphism SSR marker at present.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides Azalea pontica SSR primer pairs and sieve based on transcript profile sequencing exploitation
Choosing method and application.The present invention carries out transcript profile sequencing to Azalea pontica petal material using RNA-seq technology, and data assembling is spelled
The sequence containing SSR marker is excavated after connecing, and the unigenes for meeting the requirement of SSR design of primers is carried out to the exploitation of SSR marker,
And random synthesis part SSR primer, carry out applicability verifying.The exploitation of the primer is the genetic diversity and heredity knot of Azalea pontica
The researchs such as structure research, genetic map construction, molecular mark, important character gene mapping and cloning lay the foundation.
To achieve the goals above, the invention adopts the following technical scheme:
In a first aspect, providing a kind of Azalea pontica EST-SSR primer pair based on transcript profile sequencing exploitation, in the detection process
Using 30 pairs of primers, the primer is respectively as follows:
Complete EST-SSR primer pair of the table 1.30 to Azalea pontica
。
Second aspect provides any purposes in the following A 1-A6 of above-mentioned Azalea pontica EST-SSR primer pair:
The application of A1, the EST-SSR primer pair in building Azalea pontica genetic map；
The application of A2, the EST-SSR primer pair in Azalea pontica Germplasm Identification；
The application of A3, the EST-SSR primer pair in Azalea pontica breeding；
The application of A4, the EST-SSR primer pair in Azalea pontica analysis of genetic diversity；
The application of A5, the EST-SSR primer pair in Azalea pontica Genetic relationship；
The application of A6, the EST-SSR primer pair in Azalea pontica molecular mark.
The third aspect provides a kind of kit for identifying Azalea pontica affiliation and hereditary capacity, including above-mentioned 30 pairs
EST-SSR primer.
Fourth aspect provides the method that exploitation Azalea pontica EST-SSR primer is sequenced based on transcript profile, including
(1) choose the fresh petal material of Azalea pontica full-bloom stage, after acquisition dry ice quick-frozen and be transferred quickly to -70 degree refrigerators protect
It deposits；Total serum IgE is extracted using the method for Trizol reagent with high salt and RNA column；
(2) mRNA enrichment with magnetic bead is first carried out to total serum IgE using chain specific transcriptional group library construction Kit；It will be enriched to
MRNA carry out double-strand cDNA synthesis, end repair connected with connector；Again with USER enzyme degradation the second chain of cDNA, retain true turn
The first chain information of mRNA of record；Agarose gel electrophoresis through PCR amplification and 2.5% recycles the segment of 300-500bp range,
Obtain sequencing library；Illumina Hiseq2500PE125 is recycled to carry out high throughput RNA-seq sequencing；
(3) filter data is crossed using FASTX and CUTADAPT software, and data assembling is carried out using Cufflinks software and is obtained
To Unigene, as the background data of subsequent SSR primer development；
(4) using MISA software to Unigene carry out the search of the site SSR, search criterion are as follows: dinucleotides, trinucleotide,
The minimum number of repetition of tetranucleotide, pentanucleotide, Hexanucleotide is respectively 10 times, 7 times, 5 times, 5 times, 5 times, filters out and meets
The sequence of SSR design of primers；
(5) using 3.0 software of primer to the Unigene sequence containing the site EST-SSR and flanking sequence greater than 50bp
Carry out design of primers, between 54-65 DEG C of primer annealing temperature, PCR product size 100-250bp, primer length 19-23nt it
Between, CG content 40%-60% avoids primer from generating dimeric structure and primer mispairing；
(6) the EST-SSR primer pair for selecting synthesis step (5) design carries out PCR amplification and 6% to Azalea pontica population
PAGE detected through gel electrophoresis verifies the validity and versatility of EST-SSR primer pair.
5th aspect, provides the method for obtaining Azalea pontica EST-SSR molecular labeling, comprising:
(1) using the genomic DNA of Azalea pontica as template, PCR expansion is carried out with the complete EST-SSR primer of above-mentioned Azalea pontica
Increase, obtains pcr amplification product；
(2) pcr amplification product obtained in (1) is subjected to electrophoresis, obtains Azalea pontica EST-SSR molecular labeling.
In the above method, the primer annealing condition that the PCR amplification uses can be 55-60 DEG C, 30s.
In the above method, the PCR temperature programming used in the PCR amplification can are as follows: 94 DEG C of initial denaturation 7min；Then
Recycle into 30: 94 DEG C of denaturation 30s, anneal at 55-60 DEG C 30s, 72 DEG C of extensions 30s, last 72 DEG C of extensions 5min.
6th aspect, provides the separation that the primer pair as described in table 1 is obtained using the genome of Azalea pontica as template amplification
EST-SSR label.
It is demonstrated experimentally that the present invention is based on 58 that the method that exploitation Azalea pontica EST-SSR primer is sequenced in transcript profile is spliced,
263 Unigene, the Unigene of the sequence containing SSR have 7,644, therefrom count 8,215 SSR, are shown in Table 2.Select synthesis
30 pairs of SSR primer pair Azalea pontica groups (30 individuals) carry out PCR amplification verify its validity, primer sequence is shown in Table 1.Using
The genetic diversity of 3.1 software statistics Dabie Mountain Azalea pontica population of Arlequin, every genetic diversity the results are shown in Table 3, big not
Mountain Azalea pontica population genetic diversity is in higher level.
The beneficial effects of the present invention are:
1, the present invention is by the background data of flower transcript profile data acquisition Azalea pontica EST-SSR marker development, method is quick,
Accurately, there is higher versatility and practicability than the gSSR that genomic data obtains.
2,30 EST-SSR of the invention label can perform well in the Study on Diversity of Azalea pontica population, can be by this
Invention provide EST-SSR apply the tag to the research of Azalea pontica Related species Genetic Diversity of Germplasm, genetic map construction,
The research such as the objective trait assignment of genes gene mapping, molecular mark.
Detailed description of the invention
Fig. 1 is the flow diagram based on transcript profile SSR primer development and application.
Fig. 2 is to implement RmE-1 primer diversity testing result in Azalea pontica population (30 individuals) in example.
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.In following embodiments
Experimental method be unless otherwise specified conventional method.The materials, reagents and the like used in the following examples, such as without special theory
It is bright, it is commercially available.
The exploitation of the EST-SSR primer of the high Azalea pontica of [embodiment 1] polymorphism
It is provided by the invention to be based on transcript profile high-flux sequence, and bioinformatics method is combined to carry out the lookup of SSR sequence
It designs and verifies with SSR label primer, specific embodiment is as follows:
(1) it is rich in the Unigene sequence screening in the site SSR: choosing Azalea pontica full-bloom stage petal material extraction total serum IgE, structure
Transcript profile sequencing library is built, high-flux sequence is carried out using Illumina bis- generations sequenator.Stringent filtering sequencing data, and to turning
Record group data are assembled, and Unigene is obtained, as the background data of SSR marker exploitation.Using MISA software pair
Unigene sequence carry out the site SSR search, search criterion are as follows: dinucleotides, trinucleotide, tetranucleotide, pentanucleotide,
Hexanucleotide number of repetition minimum 10,7,5,5 and 5 times.
Wherein, 58 spliced, 263 Unigene, the Unigene of the sequence containing SSR have 7,644, therefrom count
8,215 SSR, are shown in Table 2.
SSR repetitive sequence and number of repetition statistics in table 2Unigene
(2) site SSR and flanking sequence that are rich in (AG) n motif are greater than using 3.0 software of online software primer
The Unigene sequence of 50bp carries out design of primers, and between 53-65 DEG C of primer annealing temperature, PCR product size 100-250bp draws
Between object length 19-23bp, CG content 40%-60%, primer avoids generating dimeric structure, filters out and meets SSR primer and set
Count desired sequence.
(3) 8 Azalea pontica individuals are used, the SSR primer pair of 3% agarose gel electrophoresis screening polymorphism after PCR amplification.
SSR primer sequence is shown in Table 1.Primer synthesis is synthesized by Nanjing Genscript Biotechnology Co., Ltd. and is carried out by the way of RPC
Purifying.
Application of [embodiment 2] the 30 pairs of EST-SSR primers in Azalea pontica population genetic Study on Diversity
Detect EST-SSR label detection Azalea pontica genetic diversity the following steps are included:
(1) DNA is extracted: acquiring Azalea pontica population in In Dabie Mountainous Region of Hubei Province, totally 30 individuals (are locality distributed in east longitude
114.56 ° of -115.043 ° of E, north latitude 31.03 ° of -31.68 ° of N, height above sea level 350-1500m), number HDJ1-HDJ30.Using 3 ×
CTAB method extracts 30 Azalea pontica leaves genomic DNAs, and detects quality and uv-spectrophotometric through 1% agarose gel electrophoresis
Concentration is measured, guarantees that the purity OD260/OD280 of DNA is greater than 1.8.Vanes liquid nitrogen grinding, phenol/chloroform (volume ratio 1:1) are taken out
Mention, dehydrated alcohol precipitating, 75% ethanol washing, RNA enzyme digestion and etc. after, DNA is dissolved in 50 μ L TE solution, is diluted to
100ng/ μ l is placed on -20 DEG C of refrigerators and saves backup.
(2) PCR amplification: 10 μ l reaction systems include: ddH27.8 μ l, 10 × Buffer (Mg of O2+Ion) 1 μ l, dNTPs
(2.5mM each) 0.2 μ l, 10 μM of positive each 0.2 μ l of 0.15 μ l, Taq archaeal dna polymerase of anti-primer, DNA profiling (100ng/ μ l)
0.5μl.Response procedures are as follows: 94 DEG C of initial denaturation 7min；94 DEG C of denaturation 30sec are most suitable for annealing under annealing temperature (being shown in Table 1)
30sec, 72 DEG C of extension 30sec, coamplification 30 circulations；72 DEG C of additional extension 5min.Selected primer sequence is shown in Table 1.
(3) 6% PAGE glue detection: taking 3 μ l for pcr amplification product, add 1 μ l 6 × DNA loading buffer, into
Row 6%PAGE gel electrophoresis, PAGE glue develop the color through cma staining and sodium hydroxide solution.As a result it represents figure and sees Fig. 2.
(4) according to the presence or absence of primer amplification band and size, the SSR marker for counting exploitation is intraindividual in 30 Azalea ponticas
Genotype, using the genetic diversity of 3.1 software statistics Dabie Mountain Azalea pontica population of Arlequin, every genetic diversity knot
Fruit is shown in Table 3, and Dabie Mountain Azalea pontica population genetic diversity is in higher level.
The Azalea pontica population genetic diversity that table 3 is detected based on the SSR marker of transcript profile data mining
The EST-SSR primer detection of applicant's exploitation is the result shows that the present invention is a large amount of more using transcript profile data mining
State property EST-SSR primer, and population genetic diversity detection is carried out with these primers.Accordingly, the present invention is suitable for Azalea pontica
EST-SSR molecular labeling provided by the invention can be applied to more azalea plant researches by the exploitation of EST-SSR primer
In, be subsequent Azalea pontica and nearly edge species Genetic Diversity of Germplasm detection, genetic map construction, important character position,
The researchs such as molecular mark provide foundation.
Sequence table
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<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
gactcgtggt tgatttgaac 20
<210> 59
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
ctctttcctc cctagatcca 20
<210> 60
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
gaggagttgt cgagattgtc 20
Claims (6)
1. a kind of Azalea pontica EST-SSR primer pair based on transcript profile sequencing exploitation, which is characterized in that use in the detection process
30 pairs of primers, the primer are respectively as follows:
2. Azalea pontica EST-SSR primer pair according to claim 1, which is characterized in that the primer pair annealing temperature are as follows:
3. any purposes in the following A 1-A6 of Azalea pontica EST-SSR primer pair of any of claims 1 or 2:
The application of A1, the EST-SSR primer pair in building Azalea pontica genetic map；
The application of A2, the EST-SSR primer pair in Azalea pontica Germplasm Identification；
The application of A3, the EST-SSR primer pair in Azalea pontica breeding；
The application of A4, the EST-SSR primer pair in Azalea pontica analysis of genetic diversity；
The application of A5, the EST-SSR primer pair in Azalea pontica Genetic relationship；
The application of A6, the EST-SSR primer pair in Azalea pontica molecular mark.
4. a kind of kit for identifying Azalea pontica affiliation and hereditary capacity, which is characterized in that including claims 1 or 2 institute
The 30 pairs of EST-SSR primers stated.
5. a kind of method that exploitation Azalea pontica EST-SSR primer is sequenced based on transcript profile characterized by comprising
(1) choose the fresh petal material of Azalea pontica full-bloom stage, after acquisition dry ice quick-frozen and be transferred quickly to -70 degree refrigerators save；
Total serum IgE is extracted using the method for Trizol reagent with high salt and RNA column；
(2) mRNA enrichment with magnetic bead is first carried out to total serum IgE using chain specific transcriptional group library construction Kit；By what is be enriched to
MRNA carries out double-strand cDNA synthesis, end is repaired and connected with connector；Again with USER enzyme degradation the second chain of cDNA, retain true transcription
The first chain information of mRNA；Agarose gel electrophoresis through PCR amplification and 2.5% recycles the segment of 300-500bp range, i.e.,
Obtain sequencing library；Illumina Hiseq2500 PE125 is recycled to carry out high throughput RNA-seq sequencing；
(3) filter data is crossed using FASTX and CUTADAPT software, and data assembling is carried out using Cufflinks software and is obtained
Unigene, as the background data of subsequent SSR primer development；
(4) search of the site SSR, search criterion are as follows: dinucleotides, trinucleotide, four cores are carried out to Unigene using MISA software
The minimum number of repetition of thuja acid, pentanucleotide, Hexanucleotide is respectively 10 times, 7 times, 5 times, 5 times, 5 times, filters out and meets SSR and draw
The sequence of object design；
(5) the Unigene sequence containing the site EST-SSR and flanking sequence greater than 50bp is carried out using 3.0 software of primer
Design of primers, between 54-65 DEG C of primer annealing temperature, between PCR product size 100-250bp, primer length 19-23nt, CG
Content 40%-60% avoids primer from generating dimeric structure and primer mispairing；
(6) the EST-SSR primer pair for selecting synthesis step (5) design carries out PCR amplification to Azalea pontica population and 6%PAGE is solidifying
Gel electrophoresis detection, verifies the validity and versatility of EST-SSR primer pair.
6. primer pair of any of claims 1 or 2 is marked using the isolated EST-SSR that the genome of Azalea pontica is obtained as template amplification
Note.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112941151A (en) * | 2021-03-31 | 2021-06-11 | 西北农林科技大学 | Method for constructing EST-SSR (expressed sequence tag-simple sequence repeat) marker by using RNA-seq technology and application |
| CN113215220A (en) * | 2021-05-31 | 2021-08-06 | 福建农林大学 | Method for developing olive SSR molecular marker based on transcriptome sequencing |
| CN113584217A (en) * | 2021-09-06 | 2021-11-02 | 上海植物园 | Rhododendron hybrid variety identification method based on EST-SSR molecular marker |
| CN114317800A (en) * | 2021-12-27 | 2022-04-12 | 北京市农林科学院 | EST-SSR labeled primer developed based on arborvitae transcriptome sequence and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112941151A (en) * | 2021-03-31 | 2021-06-11 | 西北农林科技大学 | Method for constructing EST-SSR (expressed sequence tag-simple sequence repeat) marker by using RNA-seq technology and application |
| CN113215220A (en) * | 2021-05-31 | 2021-08-06 | 福建农林大学 | Method for developing olive SSR molecular marker based on transcriptome sequencing |
| CN113584217A (en) * | 2021-09-06 | 2021-11-02 | 上海植物园 | Rhododendron hybrid variety identification method based on EST-SSR molecular marker |
| CN113584217B (en) * | 2021-09-06 | 2023-11-14 | 上海植物园 | Method for identifying rhododendron hybrid varieties based on EST-SSR molecular markers |
| CN114317800A (en) * | 2021-12-27 | 2022-04-12 | 北京市农林科学院 | EST-SSR labeled primer developed based on arborvitae transcriptome sequence and application thereof |
| CN114317800B (en) * | 2021-12-27 | 2023-08-25 | 北京市农林科学院 | EST-SSR marker primer developed based on biota orientalis transcriptome sequence and application thereof |
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