CN107365874A - Henry pocket orchid EST SSR label primers, its development approach and its application - Google Patents
Henry pocket orchid EST SSR label primers, its development approach and its application Download PDFInfo
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Abstract
The invention discloses one group of EST SSR label primer group based on the exploitation of Henry pocket orchid transcript profiles sequence, including 34 pairs of primers, the nucleotide sequence of the primer is as shown in sequence table SEQ ID NO.1~SEQ ID NO.68.The invention also discloses the reagent for including Henry pockets orchid EST SSR label primers of the present invention.The present invention further discloses a kind of method of exploitation Henry pocket orchid EST SSR label primers.Henry pockets orchid EST SSR label primers provided by the invention have many advantages, such as rich polymorphism, favorable repeatability, amplification is stable and is easy to statistics etc., available for the molecular studies that Henry pockets are blue and the Genetic Diversity of Germplasm of Paphiopedilum especially other kinds of pocket orchid subgenus is analyzed, molecular mark and cypripedium are related.
Description
Technical field
The present invention relates to the EST- based on blue (Paphiopedilum henryanum) the transcript profile sequence exploitation of Henry pockets
SSR label primer, its development approach and its application, belong to biological technical field.
Background technology
Henry Paphiopedilums originate in China Guang Xixi in orchid family (Orchidaceae) Paphiopedilum (Paphiopedilum) plant
South, southeastern Yunnan and North Vietnam, are grown in the height above sea level 900-1300m hidden gap in In Limestone Area or evergreen broad-leaved
The ground of more stones or draining well in woods and spinney.Now all cypripediums are put into《Endangered species of wild fauna and flora kind is international
Trade pact》The register of annex one and be prohibited trade.Pocket is blue to be used as a kind of endangered species, and the research of its genetic diversity becomes ten
Point important, the blue child care work of pocket is more very urgent.
Simple repeated sequence (Simple sequence repeat, SSR) mark is reproducible because high with polymorphism,
High specificity, easy to detect, codominance, the features such as marking covering whole gene group and be uniformly distributed, it has also become genetic diversity
One of critically important a kind of genetic marker (Phuekvilai, Prattana＆Pongtongkam, Pradit＆ in analysis
Peyachoknagul, Surin " Development of Microsatellite Markers for Vanda Orchid, "
Kasetsart Journal-Natural Science, 2009,43 (3)：497-506；Kalia R K, Rai M K, Kalia
S etc., " Microsatellite markers：An overview of the recent progress in plants, "
Euphytica, 2011,177 (3)：309-334).Compared with genome SSR marker, EST-SSR derives from expressing gene group area
Domain, the mark of " absolute " can be provided for functional gene, development cost is low, thing cross-species amplification it is higher (Zhu Zhendong, Jia Jizeng, it is " small
The development and application of wheat SSR marker ",《Heredity》, 2003, (03)：355-360；Varshney, R.K. etc.,
“Interspecific transferability and comparative mapping of barley EST-SSR
Markers in wheat, rye and rice, " Plant Science, 2005.168 (1)：The 195-202 pages).In recent years
Sequencing technologies continue to develop, and transcript profile sequencing technologies (RNA-seq) are the new transcript of discovery and gene expression abundance and SSR sites
Excavation provide opportunity (Zhang H, Wei L, Miao H, etc. " Development and validation of
Genic-SSR markers in sesame by RNA-seq, " BMC Genomics, 2012,13 (1)：316-316；
Noakes A G, Best T, Staton M etc., " Cross amplification of 15EST-SSR markers in the
Genus Fraxinus, " Conservation Genetics Resources, 2014,6 (4)：969-970；Adal AM,
Demissie Z A, Mahmoud S S etc., " Identification, validation and cross-species
Transferability of novel Lavandula EST-SSRs, " Planta, 2015,241 (4)：987-1004), it is right
In the non-mode biology either biology without genome sequencing, marked using SSR of the transcript profile sequencing exploitation with gene information
Remember to analyze the genetic diversity of biology be also a kind of simple efficient research method, this method also multiple report in orchid
(Tsai CC, Shih HC, Wang HV, Lin YS, Chang CH etc., " RNA-Seq SSRs of Moth Orchid and
Screening for Molecular Markers across Genus Phalaenopsis (Orchidaceae), " PLOS
ONE, 2015,10 (11)：e0141761).But so far, there is not been reported for the research of cypripedium EST-SSR marks.
Therefore, EST-SSR primers are developed using Henry pocket orchid transcript profiles sequence information, it will and Paphiopedilum blue to Henry pockets
The especially Genetic Diversity of Germplasm of other kinds of pocket orchid subgenus, Genetic relationship, the important character assignment of genes gene mapping, molecule mark
Remember that molecular studies of assistant breeding and cypripedium correlation etc. play important impetus.
The content of the invention
The invention discloses one group of EST-SSR labeled primer group based on the exploitation of Henry pocket orchid transcript profiles sequence, including 34
To primer, the nucleotide sequence of the primer is as shown in sequence table SEQ ID NO.1~SEQ ID NO.68.Invention additionally discloses
Include the reagent of Henry pockets orchid EST-SSR labeled primers of the present invention.The present invention further discloses for developing henry
The method of sharp pocket orchid EST-SSR labeled primers, including：(a) obtained based on the sequencing splicing of Henry pocket orchid organs transcript profile
Unigenes sequence data design EST-SSR primers；(b) Henry pocket orchid plant sample genomic DNAs are extracted；(c) step is utilized
Suddenly the EST-SSR primers that (a) is obtained, performing PCR is entered as template using DNA obtained by step (b)；(d) by PCR primer obtained by step (c)
Electrophoresis is carried out, and electrophoresis result is analyzed, so as to filter out the Henry pocket orchid EST-SSR primers with desired characteristic.This
The Henry pocket orchid EST-SSR labeled primers that invention provides have many advantages, such as rich polymorphism, favorable repeatability, amplification
It is stable and be easy to statistics etc., available for Henry pockets are blue and the heredity of the germ plasm resource of its Paphiopedilum especially other kinds of pocket orchid subgenus
The related molecular studies of diversity analysis, molecular mark and cypripedium.
The first aspect of the present invention provides the EST-SSR as shown in table 1 below based on the exploitation of Henry pocket orchid transcript profiles sequence
Primer pair：
The Henry pocket orchid EST-SSR polymorphism primer information of table 1
The sequence number of above-mentioned primer is respectively SEQ ID NO.1~SEQ ID NO.68.
In one embodiment, any bar primer in Henry pockets orchid EST-SSR primer pairs of the present invention can be with
It is detectably labeled.Labeling method is well known in the art.The mark includes those commonly used in the art, for example, described
Mark includes but is not limited to fluorescence labeling, isotope marks etc..Preferably, the mark is fluorescence labeling.Further real
Apply in scheme, the forward primer in the primer pair is labeled.In a further embodiment, reversely drawing in the primer pair
Thing is labeled.
In one embodiment, the primer in Henry pockets orchid primer pair of the present invention can be due to one or more cores
Insertion, missing, replacement or the modification means well known by persons skilled in the art of thuja acid are modified and changed, so as to improve
The specificity of the primer, for belonging to together or the molecular biology research of other plant of the same race or obtaining other desired performances.
The second aspect of the present invention provides one group of EST-SSR primer based on the exploitation of Henry pocket orchid transcript profiles sequence, institute
Stating primer sets includes any combination of the above-mentioned primer pair of the present invention.
The third aspect of the present invention is provided by primer pair of the present invention or primer sets from Henry pocket orchid genomes
Isolated DNA fragmentation or gene outcome, i.e. EST-SSR mark.These EST-SSR marks can be used for Henry pockets blue and other
The related molecular studies of pocket orchid germ plasm resource analysis of genetic diversity, molecular mark and cypripedium.Especially
Ground is related to functional gene therefore auxiliary available for molecular labeling because these EST-SSR marks are from DNA transcript regions
Help correlative study such as genetic linkage map structure, the association analysis of important character mark of correlation, separation and identification new gene etc. of breeding.
The present invention also provides Henry pockets orchid, the other plants of Paphiopedilum using EST-SSR primers of the present invention structure
The even SSR finger-prints of orchid.
The fourth aspect of the present invention provides the reagent for separating Henry pocket orchids EST-SSR marks, and the reagent includes
Any pair of primer of the present invention or one group of primer.
In one embodiment, reagent of the present invention includes one group of primer as described above, wherein the primer
Every pair of primers in group is individually packed.
The fourth aspect of the present invention provides a kind of kit, and the kit includes present invention reagent as described above.
The fourth aspect of the present invention provides a kind of side based on Henry pocket orchid transcript profiles sequence exploitation EST-SSR primers
Method, methods described include：
(a) unigenes obtained based on the sequencing splicing of Henry pocket orchid organs transcript profile sequence data designs EST-
SSR primers；
(b) Henry pocket orchid plant sample genomic DNAs are extracted；
(c) the EST-SSR primers obtained using step (a), performing PCR is entered as template using DNA obtained by step (b)；
(d) PCR primer obtained by step (c) is subjected to electrophoresis, and electrophoresis result is analyzed, so as to filter out Henry pockets
Blue EST-SSR primers.
In one embodiment, the step (a) is carried out as follows：The sequencing of Henry pocket orchid organs transcript profile is spliced
To unigenes sequence data；It is each with MIcroSAtellite identification tool (MISA) software lookup
SSR sites that may be present in unigene (Thiel, T., Michalek, W., Varshney, R.K. and Graner, A.,
“Exploiting EST databases for the development and characterization of gene-
Derived SSR-markers in barley (Hordeum vulgare L.), " Theoretical and Applied
Genetics, 2003,106,411-422), the standard of the lookup in SSR sites is：2nd, three, four, five and Hexanucleotide repetition
The number of sequence motifs is not less than 7,5,5,5 and 5 times respectively；With sequences Design of the Primer3 softwares based on SSR sites both wings
SSR primers.
In one embodiment, the screening strategy of design of primers is：Length (most suitable 20bp) between 17~24bp, melt
Temperature (Tm) is solved between 55~62 DEG C, pcr amplification product size between 100~350bp, the GC ratios that contain 40%~
Between 60% (most suitable is 50%), other are default setting.Using primer sets cooperation the drawing as SSR sites closest to design requirement
Thing.
In one embodiment, the step (b) includes the CTAB method batch extracting sample DNAs with improvement, specific behaviour
Make as follows：
1. with liquid nitrogen by the blue blade grind into powder of Henry pockets, the CTAB (v/ containing beta -mercaptoethanol of 65 DEG C of preheatings are added
V1%)；
2. in 65 DEG C of water-bath 1h；
3. taking-up is cooled to room temperature (or being put into refrigerator), the chloroform of precooling is added：Isoamyl alcohol (24: 1), jog 5min；
4. centrifuging, supernatant is taken, the precooling absolute ethyl alcohol of 2 times of volumes, mixing of gently turning upside down are added after being repeated twice；
5. -20 DEG C of placement 30min, DNA unite precipitation, liquid are discarded；
6. adding 70% ethanol washing precipitation, turn upside down 6-8 times, supernatant is abandoned after centrifugation, then washed with 90% ethanol；
7. ventilation dries up 2h, transparent, alcohol-free taste is dried to DNA；
8. add sterilized water to redissolve.
It is with the advantage of the CTAB methods extraction DNA after improvement：Liquid is directly discarded after DNA unites, is dexterously avoided
Various contamination precipitations caused by centrifugal process, the DNA purity that finally obtains and concentration quality is set to increase substantially.
In one embodiment, the step (c) is carried out by touchdown PCR (Touch Down PCR), its reactant
It is as shown in table 2 below：
The PCR reaction systems of table 2
Using the mode of touchdown PCR, there is amplification efficiency height, the few and versatile advantage of non-specific amplification.
In one embodiment, electrophoresis is carried out to PCR primer using 8% polyacrylamide gel, contaminated using argentation
Color, finally gel is placed on gel imaging system and taken pictures, to identify the polymorphic bandses on electrophoresis pattern, so as to screen shaping
The Henry pocket orchid EST-SSR primers that band is clear, polymorphism is high, specificity is good.
In further embodiment, the EST- of Paphiopedilum other plant is developed using method of the present invention
SSR primers, or even the EST-SSR primers available for the other plant beyond exploitation orchid family other plant or orchid family.
Another aspect of the present invention additionally provide EST-SSR primers of the present invention between Henry pocket orchid different populations,
Application between different cultivars, between cypripedium and between orchid in progress analysis of genetic diversity.
In the present invention, " pair of primers " is used interchangeably with " primer pair ", is referred to by forward primer and reverse primer group
Into pair of primers；" one group of primer " is used interchangeably with " primer sets ", refers to include two pairs of primers or more to the one of primer
Group primer.
The advantageous effects that technical scheme is brought are provided in embodiment of the present invention is：Henry pockets are developed first
Blue EST-SSR primers, there is provided 34 pairs of rich polymorphisms, favorable repeatability, specificity are high, the Henry pockets of amplification stabilization are blue
EST-SSR primers, it is Henry pocket orchid germ plasm resources analysis of genetic diversity, molecular mark and cypripedium phase
The molecular studies of pass provide basis；It is related to functional gene because these EST-SSR marks derive from DNA transcript regions, and
Cross-species amplification is good, therefore available for the molecular biology research of congener, is used especially for molecular mark
Correlative study such as genetic linkage map structure, the association analysis of important character mark of correlation, separation and identification new gene etc.；Also, this
The method that invention provides is simply easily efficient, production cost is low, it can also be used to the exploitation of Paphiopedilum other plant EST-SSR primers.
Foregoing teachings are only schematical and to be never intended to be restricted.Except above-mentioned schematically aspect, implement
Mode and feature, by reference to following detailed description, further aspect, embodiment and feature will be more readily understood.
Brief description
By reference to following accompanying drawings, further aspect of the invention, feature will be more readily understood.People in the art
Member is it should be understood that these accompanying drawings only symbolically elaborate according to certain embodiments of the present invention, and should not be taken as
Limitation of the scope of the invention.
Fig. 1 shows the DNA Ago-Gels of 33 parts of different Henry pockets orchid materials from 2 Henry pocket orchid wild populations
Electrophoretogram.
Fig. 2-Figure 35 respectively illustrates polymorphism amplification of 34 pairs of primers on 33 parts of Henry pocket orchid samples listed by table 1
As a result electrophoretogram.
Figure 36 shows different prosperous using 33 parts of 34 pairs of primer pairs that table 1 is listed from 2 Henry pocket orchid wild populations
Sharp pocket orchid material carries out the UPGMA dendrograms after cluster analysis.
Embodiment
Hereinafter, some exemplary embodiments are simply just described.As one skilled in the art will recognize that
Like that, without departing from the spirit or scope of the present invention, described embodiment can be changed by various different modes.
Therefore, accompanying drawing and description are considered essentially illustrative rather than restrictive.
The exploitation of the Henry pocket orchid EST-SSR primers of embodiment 1.
1.EST-SSR design of primers
Experiment material：Henry pocket orchid organs are obtained from vegetable or flower institute of Chinese Academy of Agricultural Sciences greenhouse；The present invention is all
The various reagents or medium component used in embodiment are through commercially available.
1) transcript profile library is built：Henry pocket orchid organ total serum IgEs are extracted, separate mRNA, reverse transcription is synthesized and purified
CDNA, end is repaired plus adenosine connection sequence measuring joints, and it is 200~700bp to reclaim size by agarose gel electrophoresis
Fragment, will recovery fragment enter performing PCR amplification, i.e., structure obtain transcript profile library.
2) acquisition of transcript profile data：The transcript profile library that step 1) obtains is sequenced, obtains transcript profile sequencing number
According to sequencing data is spliced, obtains unigenes sequence data.
3) SSR sites are searched：It is each with MIcroSAtellite identification tool (MISA) software lookup
SSR sites that may be present in individual unigene.The standard of the lookup in SSR sites is：2nd, three, four, five and Hexanucleotide weight
The number of complex sequences motif is not less than 7,5,5,5 and 5 times respectively.
4) EST-SSR primers are designed：With sequences Design SSR primer of the Primer3 softwares based on SSR sites both wings.Draw
Thing design screening strategy be：Length (most suitable 20bp) between 17~24bp, melting temperature (Tm) between 55~62 DEG C,
Pcr amplification product size between 100~350bp, the GC ratios contained between 40%~60% (most suitable is 50%), other
For default setting.Primer using the primer sets cooperation closest to design requirement as SSR sites.For PCR amplification SSR primers by
Raw work bioengineering (Shanghai) limited company synthesis.
2. plant sample extracting genome DNA
Experiment material：The sun that Henry pocket orchid plant leaf blades are taken respectively from Wenshan Prefecture of Yunnan Province Malipo country rushes population
(TYC) 8 plants of plants and big 25 plants of plants for doing population (DN).
It is as follows with the CTAB method batch extracting sample DNAs of improvement, concrete operations：
(1) 2ml centrifuge tubes (sterilizing), blade, lid size two panels are filled；
(2) liquid nitrogen grinding adds 65 DEG C of μ L of CTAB 800 (v/v containing beta -mercaptoethanol 1%) preheated into powder；
(3) 65 DEG C of water-bath 1h, shake once for every ten minutes；
(4) take out and be cooled to room temperature (or being put into refrigerator), add the chloroform of precooling：The μ L of isoamyl alcohol (24: 1) 800, jog
5min (hand)；
(5) 13000rpm centrifuges 10min, takes the μ L of supernatant 450 into 1.5 μ L centrifuge tubes, then extract once (24: the μ of Isosorbide-5-Nitrae 50
L), clear 300 μ L are sucted, add the μ L of precooling absolute ethyl alcohol 600 of 2 times of volumes, mixing of gently turning upside down；
(6) -20 DEG C of placement 30min, DNA unite precipitation, hold DNA groups with pipette tips, liquid is discarded；
(7) ethanol of 1ml 70% washing precipitation is added, is turned upside down 6-8 times, 7500rpm centrifuges 5min, supernatant, 1ml
90% ethanol washing precipitation, 7500rpm centrifuge supernatant；
(8) ventilation drying 2h, transparent, alcohol-free taste is dried to DNA；
(9) plus 100 μ L sterilized waters redissolve.
Enter row agarose gel electrophoresis, as a result (Fig. 1) shows that extracted STb gene electrophoretic band is clear, illustrates extraction
DNA mass is good.
3. enter performing PCR amplification using EST-SSR primers
Using the genome DAN extracted above as template, enter performing PCR amplification using the EST-SSR primers designed above, so as to
Identify primer validity.PCR reaction systems are as shown in table 2.
The analysis of 4.PCR amplified productions
Electrophoresis is carried out to PCR primer using 8% polyacrylamide gel, dyed using argentation, band is complete after developing the color
After full presentation, gel is placed on gel imaging system and taken pictures.According to electrophoresis pattern, screening band is clear, polymorphism is high, special
The good Henry pocket orchid EST-SSR primers of property.
As a result 34 pairs of primer pairs that expanding effect is good and polymorphism is high are filtered out, the primer pair is as shown in table 1.As Fig. 2-
Shown in Figure 35, detect to obtain 94, allele altogether using 34 pairs of primers, the variation amplitude of each pair primer allele 2-5 it
Between, average number of alleles is 2.74, shows that the ESR-SSR primers that the present invention develops can be completely used for the blue heredity of Henry pockets
Diversity analysis.
Embodiment 2. carries out analysis of genetic diversity using Henry pocket orchid EST-SSR primers
Experiment material：Henry pocket orchids laboratory sample rushes population obtained from the sun of Wenshan Prefecture of Yunnan Province Malipo country respectively
(TYC) 8 plants of plants and big 25 plants of plants for doing population (DN).
It has been shown that, observation and expectation are analyzed and (see below table 3) to the amplification polymorphism information of this 34 pairs of EST-SSR primers
Heterozygosity scope is the distribution of 0.0303-1.000 and 0.0597-0.6793, Shannon information index respectively in 0.14-
Between 1.25, average value 0.61, show that the genetic variation and genetic differentiation of this 33 parts of Henry pocket orchid materials is relatively abundanter.
3 34 pairs of EST-SSR primer amplification polymorphism information analyses of table
In addition, also using 33 parts of this 34 pairs of primer pair above-mentioned two wild population (sun rushes population and big does population) not
Same Henry pocket orchids material carries out cluster analysis, as shown in figure 36.As a result show, 33 parts of samples are divided into 5 major classes, and heredity is similar
Material is divided into two groups by property coefficient at 0.71, and I, II, Group III are one group, and sample standard deviation comes arrogant lane population (DN), IV, V
For one group, sample standard deviation rushes population (TYC) from the sun, the genetic similarity system between this blue different wild population of explanation Henry pockets
Number is smaller, and hereditary basis is wider.Big to do in population, genetic similarity index is at 0.77, and material can be divided into 3 major classes.Too
In rushing population, genetic similarity index is at 0.70, you can material is divided into 2 major classes.It can be seen that Henry pockets are blue different wild
The material of population has otherness, and hereditary variation is relatively large, 34 pairs of EST-SSR primers that exploitation goes out, can be successful
Population is distinguished, the hereditary variation that the Henry pockets of same population are blue is relatively large, and hereditary basis is wider.
Embodiment 3. carries out General Use Analysis using Henry pocket orchid EST-SSR primers in cypripedium
General Use Analysis is carried out to other six kinds of cypripediums from Paphiopedilum using Henry pocket orchid microsatellites, as a result
(seeing below table 4) shows 34 pairs of EST-SSR primers in purple hair pocket blue (P.villosum), beautiful pocket blue (P.venustum), coloured silk
Cloud pocket orchid (P.wardii), long valve pocket blue (P.dianthum), homochromy pocket blue (P.concolor), Paphiopedilum micranthum
(P.micranthum) amplification success rate is respectively 0.85,0.82,0.74,0.68,0.56,0.56, illustrates this 34 couples of EST-
SSR primers have higher versatility, particularly the amplification rate in pocket orchid subgenus high in Paphiopedilum, it is seen that with Henry pockets
Blue genetic distance is nearer, and the amplification efficiency of SSR primers is higher.Prove 34 couple using the sequencing exploitation of Henry pocket orchids transcript profile
EST-SSR primers can be used for the research of the genetic diversity and genetic structure of nearly edge species.
Amplification situation of the 4 34 pairs of EST-SSR primers of table in other six kinds of cypripediums
The foregoing is only a specific embodiment of the invention, but protection scope of the present invention is not limited thereto, any
Those familiar with the art the invention discloses technical scope in, its various change or replacement can be readily occurred in,
These should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the guarantor of the claim
Shield scope is defined.
Sequence table
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<211> 20
<212> DNA
<213>Artificial sequence
<400> 13
gcaaatccat tcagccctgc 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<400> 14
cgacatggtc tgagaggagc 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
<400> 15
cactggggga tcttcgaagg 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
cagcacctct tcgcgtaaga 20
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence
<400> 17
cttgggactc tttcctcggc 20
<210> 18
<211> 20
<212> DNA
<213>Artificial sequence
<400> 18
ccaggaggct ctcagctttc 20
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence
<400> 19
ctcccaatct ccaccgacaa 20
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence
<400> 20
tggatcccag ggaaggtagg 20
<210> 21
<211> 20
<212> DNA
<213>Artificial sequence
<400> 21
tgcagatgag cccatgcatt 20
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<400> 22
tcacgcctgt atttctgcgt 20
<210> 23
<211> 20
<212> DNA
<213>Artificial sequence
<400> 23
ttgattggcg cagtgaaagc 20
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence
<400> 24
ccactgatcg cagaccatca 20
<210> 25
<211> 20
<212> DNA
<213>Artificial sequence
<400> 25
ggcacttaca gcaaggctct 20
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence
<400> 26
gagacctggg cccatcaaaa 20
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence
<400> 27
acgcggcaaa aatgatgagg 20
<210> 28
<211> 20
<212> DNA
<213>Artificial sequence
<400> 28
aggagggttc atgcagtagt 20
<210> 29
<211> 20
<212> DNA
<213>Artificial sequence
<400> 29
caggctccaa aacaaggcac 20
<210> 30
<211> 20
<212> DNA
<213>Artificial sequence
<400> 30
gggactgggg agtaaaaggc 20
<210> 31
<211> 20
<212> DNA
<213>Artificial sequence
<400> 31
caggttgaca gcaatgtcgc 20
<210> 32
<211> 20
<212> DNA
<213>Artificial sequence
<400> 32
gccgcagctt ttcggataag 20
<210> 33
<211> 22
<212> DNA
<213>Artificial sequence
<400> 33
agtagaggat ctggtcaacg ga 22
<210> 34
<211> 22
<212> DNA
<213>Artificial sequence
<400> 34
tgcaaggttc tagagtgcat ga 22
<210> 35
<211> 20
<212> DNA
<213>Artificial sequence
<400> 35
agcgtgtttg gactagagca 20
<210> 36
<211> 20
<212> DNA
<213>Artificial sequence
<400> 36
tcggggatgc acatggaaaa 20
<210> 37
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 37
gctccatagc tggcgatcat 20
<210> 38
<211> 20
<212> DNA
<213>Artificial sequence
<400> 38
tctttctgac ggcgcaagat 20
<210> 39
<211> 20
<212> DNA
<213>Artificial sequence
<400> 39
gggtggggag agtaggagtt 20
<210> 40
<211> 20
<212> DNA
<213>Artificial sequence
<400> 40
gccacaactt gttttcccgg 20
<210> 41
<211> 20
<212> DNA
<213>Artificial sequence
<400> 41
ttcgtcctca cttcttcgcc 20
<210> 42
<211> 20
<212> DNA
<213>Artificial sequence
<400> 42
gaagaggaga tggctcgtgg 20
<210> 43
<211> 20
<212> DNA
<213>Artificial sequence
<400> 43
ccacgtgtga cagaatccca 20
<210> 44
<211> 20
<212> DNA
<213>Artificial sequence
<400> 44
ggctcccgac gaggaattac 20
<210> 45
<211> 20
<212> DNA
<213>Artificial sequence
<400> 45
tcaacaagga cgggttggtc 20
<210> 46
<211> 20
<212> DNA
<213>Artificial sequence
<400> 46
ttgatgtggc acaagcacac 20
<210> 47
<211> 20
<212> DNA
<213>Artificial sequence
<400> 47
agagaaggag caccaatggc 20
<210> 48
<211> 20
<212> DNA
<213>Artificial sequence
<400> 48
cttcgatgga tcatgggcga 20
<210> 49
<211> 22
<212> DNA
<213>Artificial sequence
<400> 49
ccaccagctt tcatatcctc ca 22
<210> 50
<211> 20
<212> DNA
<213>Artificial sequence
<400> 50
gcccatgctg tgcaaaaaga 20
<210> 51
<211> 20
<212> DNA
<213>Artificial sequence
<400> 51
aagaagtggc ttccatggca 20
<210> 52
<211> 20
<212> DNA
<213>Artificial sequence
<400> 52
gcaaaaccaa ggtgtcgtcc 20
<210> 53
<211> 20
<212> DNA
<213>Artificial sequence
<400> 53
agccaacaca gccacgataa 20
<210> 54
<211> 20
<212> DNA
<213>Artificial sequence
<400> 54
aagaaccatc accaccaccc 20
<210> 55
<211> 20
<212> DNA
<213>Artificial sequence
<400> 55
caaacctcgc tcggaagact 20
<210> 56
<211> 20
<212> DNA
<213>Artificial sequence
<400> 56
agggtttcta tcgcttggcc 20
<210> 57
<211> 20
<212> DNA
<213>Artificial sequence
<400> 57
gctgccaatt cgaatggagg 20
<210> 58
<211> 20
<212> DNA
<213>Artificial sequence
<400> 58
gctgccgatt ctccttcctt 20
<210> 59
<211> 20
<212> DNA
<213>Artificial sequence
<400> 59
gaaaaccgat tggcgtcgag 20
<210> 60
<211> 20
<212> DNA
<213>Artificial sequence
<400> 60
ttcccttctc ccatttccgg 20
<210> 61
<211> 20
<212> DNA
<213>Artificial sequence
<400> 61
agctatgagg aactgcgctg 20
<210> 62
<211> 20
<212> DNA
<213>Artificial sequence
<400> 62
tgttgacatg atccgtggca 20
<210> 63
<211> 20
<212> DNA
<213>Artificial sequence
<400> 63
agcttgaagt acttgggggc 20
<210> 64
<211> 20
<212> DNA
<213>Artificial sequence
<400> 64
tccaccttct cctcctcact 20
<210> 65
<211> 20
<212> DNA
<213>Artificial sequence
<400> 65
ccttcctcat gatccgtccg 20
<210> 66
<211> 20
<212> DNA
<213>Artificial sequence
<400> 66
ttagctctct cccctccgag 20
<210> 67
<211> 20
<212> DNA
<213>Artificial sequence
<400> 67
ggcgattgac ctctgctgat 20
<210> 68
<211> 20
<212> DNA
<213>Artificial sequence
<400> 68
tcggctttgg ttgctgagat 20
Claims (10)
1. based on the EST-SSR primer pairs of Henry pocket orchid transcript profiles sequence exploitation, the primer pair is selected from following (1)-(34) item
Any one：
(1) primer pair PH_SSR011, the nucleotide sequence of its forward primer is as shown in SEQ ID NO.1, the sequence of its reverse primer
Row are as shown in SEQ ID NO.2；
(2) primer pair PH_SSR025, the nucleotide sequence of its forward primer is as shown in SEQ ID NO.3, the sequence of its reverse primer
Row are as shown in SEQ ID NO.4；
(3) primer pair PH_SSR026, the nucleotide sequence of its forward primer is as shown in SEQ ID NO.5, the sequence of its reverse primer
Row are as shown in SEQ ID NO.6；
(4) primer pair PH_SSR041, the nucleotide sequence of its forward primer is as shown in SEQ ID NO.7, the sequence of its reverse primer
Row are as shown in SEQ ID NO.8；
(5) primer pair PH_SSR060, the nucleotide sequence of its forward primer is as shown in SEQ ID NO.9, the sequence of its reverse primer
Row are as shown in SEQ ID NO.10；
(6) primer pair PH_SSR172, the nucleotide sequence of its forward primer as shown in SEQ ID NO.11, its reverse primer
Sequence is as shown in SEQ ID NO.12；
(7) primer pair PH_SSR272, the nucleotide sequence of its forward primer as shown in SEQ ID NO.13, its reverse primer
Sequence is as shown in SEQ ID NO.14；
(8) primer pair PH_SSR334, the nucleotide sequence of its forward primer as shown in SEQ ID NO.15, its reverse primer
Sequence is as shown in SEQ ID NO.16；
(9) primer pair PH_SSR343, the nucleotide sequence of its forward primer as shown in SEQ ID NO.17, its reverse primer
Sequence is as shown in SEQ ID NO.18；
(10) primer pair PH_SSR351, the nucleotide sequence of its forward primer as shown in SEQ ID NO.19, its reverse primer
Sequence is as shown in SEQ ID NO.20；
(11) primer pair PH_SSR356, the nucleotide sequence of its forward primer as shown in SEQ ID NO.21, its reverse primer
Sequence is as shown in SEQ ID NO.22；
(12) primer pair PH_SSR372, the nucleotide sequence of its forward primer as shown in SEQ ID NO.23, its reverse primer
Sequence is as shown in SEQ ID NO.24；
(13) primer pair PH_SSR376, the nucleotide sequence of its forward primer as shown in SEQ ID NO.25, its reverse primer
Sequence is as shown in SEQ ID NO.26；
(14) primer pair PH_SSR383, the nucleotide sequence of its forward primer as shown in SEQ ID NO.27, its reverse primer
Sequence is as shown in SEQ ID NO.28；
(15) primer pair PH_SSR413, the nucleotide sequence of its forward primer as shown in SEQ ID NO.29, its reverse primer
Sequence is as shown in SEQ ID NO.30；
(16) primer pair PH_SSR416, the nucleotide sequence of its forward primer as shown in SEQ ID NO.31, its reverse primer
Sequence is as shown in SEQ ID NO.32；
(17) primer pair PH_SSR425, the nucleotide sequence of its forward primer as shown in SEQ ID NO.33, its reverse primer
Sequence is as shown in SEQ ID NO.34；
(18) primer pair PH_SSR426, the nucleotide sequence of its forward primer as shown in SEQ ID NO.35, its reverse primer
Sequence is as shown in SEQ ID NO.36；
(19) primer pair PH_SSR430, the nucleotide sequence of its forward primer as shown in SEQ ID NO.37, its reverse primer
Sequence is as shown in SEQ ID NO.38；
(20) primer pair PH_SSR447, the nucleotide sequence of its forward primer as shown in SEQ ID NO.39, its reverse primer
Sequence is as shown in SEQ ID NO.40；
(21) primer pair PH_SSR535, the nucleotide sequence of its forward primer as shown in SEQ ID NO.41, its reverse primer
Sequence is as shown in SEQ ID NO.42；
(22) primer pair PH_SSR547, the nucleotide sequence of its forward primer as shown in SEQ ID NO.43, its reverse primer
Sequence is as shown in SEQ ID NO.44；
(23) primer pair PH_SSR548, the nucleotide sequence of its forward primer as shown in SEQ ID NO.45, its reverse primer
Sequence is as shown in SEQ ID NO.46；
(24) primer pair PH_SSR592, the nucleotide sequence of its forward primer as shown in SEQ ID NO.47, its reverse primer
Sequence is as shown in SEQ ID NO.48；
(25) primer pair PH_SSR645, the nucleotide sequence of its forward primer as shown in SEQ ID NO.49, its reverse primer
Sequence is as shown in SEQ ID NO.50；
(26) primer pair PH_SSR651, the nucleotide sequence of its forward primer as shown in SEQ ID NO.51, its reverse primer
Sequence is as shown in SEQ ID NO.52；
(27) primer pair PH_SSR658, the nucleotide sequence of its forward primer as shown in SEQ ID NO.53, its reverse primer
Sequence is as shown in SEQ ID NO.54；
(28) primer pair PH_SSR669, the nucleotide sequence of its forward primer as shown in SEQ ID NO.55, its reverse primer
Sequence is as shown in SEQ ID NO.56；
(29) primer pair PH_SSR687, the nucleotide sequence of its forward primer as shown in SEQ ID NO.57, its reverse primer
Sequence is as shown in SEQ ID NO.58；
(30) primer pair PH_SSR698, the nucleotide sequence of its forward primer as shown in SEQ ID NO.59, its reverse primer
Sequence is as shown in SEQ ID NO.60；
(31) primer pair PH_SSR716, the nucleotide sequence of its forward primer as shown in SEQ ID NO.61, its reverse primer
Sequence is as shown in SEQ ID NO.62；
(32) primer pair PH_SSR800, the nucleotide sequence of its forward primer as shown in SEQ ID NO.63, its reverse primer
Sequence is as shown in SEQ ID NO.64；
(33) primer pair PH_SSR809, the nucleotide sequence of its forward primer as shown in SEQ ID NO.65, its reverse primer
Sequence is as shown in SEQ ID NO.66；With
(34) primer pair PH_SSR877, the nucleotide sequence of its forward primer as shown in SEQ ID NO.67, its reverse primer
Sequence is as shown in SEQ ID NO.68；
Or more primer pair any combination.
2. Henry pockets orchid EST-SSR primer pairs as claimed in claim 1 a, wherein primer in the primer pair can be examined
Geodetic marks.
3. Henry pockets orchid EST-SSR primer pairs as claimed in claim 2, wherein the mark is fluorescence labeling.
4. based on the EST-SSR primer sets of Henry pocket orchid transcript profiles sequence exploitation, it is any that the primer sets include claim 1-3
Any combination of primer pair described in.
5. the reagent for separating Henry pocket orchids EST-SSR marks, the reagent are included as described in claim any one of 1-3
Primer pair or primer sets as claimed in claim 4.
6. reagent as claimed in claim 5, wherein the reagent includes primer sets as claimed in claim 4, wherein described
Every pair of primers in primer sets is individually packed.
7. a kind of kit, the kit includes the reagent as described in claim 5 or 6.
8. a kind of method based on Henry pocket orchid transcript profiles sequence exploitation EST-SSR primers, methods described include：
(a) unigenes obtained based on the sequencing splicing of Henry pocket orchid organs transcript profile sequence data design EST-SSR is drawn
Thing；
(b) Henry pocket orchid plant sample genomic DNAs are extracted；
(c) the EST-SSR primers obtained using step (a), performing PCR is entered as template using DNA obtained by step (b)；
(d) PCR primer obtained by step (c) is subjected to electrophoresis, and electrophoresis result is analyzed, so as to filter out Henry pockets orchid
EST-SSR primers；
Wherein described primer is as claimed in claim 1.
9. method as claimed in claim 8, wherein the step (b) includes the CTAB method batch extracting sample DNAs with improvement,
Concrete operations are as follows：
1. with liquid nitrogen by the blue blade grind into powder of Henry pockets, the CTAB of 65 DEG C of preheatings are added；
2. in 65 DEG C of water-bath 1h；
3. taking-up is cooled to room temperature (or being put into refrigerator), the chloroform of precooling is added：Isoamyl alcohol (24：1), jog 5min；
4. centrifuging, supernatant is taken, the precooling absolute ethyl alcohol of 2 times of volumes, mixing of gently turning upside down are added after being repeated twice；
5. -20 DEG C of placement 30min, DNA unite precipitation, liquid are discarded；
6. adding 70% ethanol washing precipitation, turn upside down 6-8 times, supernatant is abandoned after centrifugation, then washed with 90% ethanol；
7. ventilation dries up 2h, transparent, alcohol-free taste is dried to DNA；
8. add sterilized water to redissolve.
10. method as claimed in claim 8 or 9, wherein the step (c) is carried out by touchdown PCR, its response procedures is：
94 DEG C of denaturation 5min；94 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 40s, circulate 10 times；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s, circulation 15
It is secondary；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, circulate 10 times；Followed by 72 DEG C of extension 10min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815509A (en) * | 2017-12-12 | 2018-03-20 | 福建省农业科学院作物研究所 | SSR primer sets and its application based on the exploitation of okra transcript profile sequence |
| CN107974510A (en) * | 2017-12-06 | 2018-05-01 | 中国农业科学院蔬菜花卉研究所 | Paphiopedilum armeniacum EST-SSR labeled primers, its development approach and its application |
| CN109266778A (en) * | 2018-11-20 | 2019-01-25 | 福建省农业科学院作物研究所 | EST-SSR labeled primer and application based on the exploitation of hybrid orchid transcript profile |
| CN110564722A (en) * | 2019-10-18 | 2019-12-13 | 西南林业大学 | Method for extracting paphiopedilum genome DNA by using improved CTAB method |
| CN110699480A (en) * | 2019-11-18 | 2020-01-17 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Primer group for hybridizing orchidaceae EST-SSR (expressed sequence tag-simple sequence repeat) markers and screening method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754886A (en) * | 2017-01-19 | 2017-05-31 | 北京林业大学 | Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers |
| CN107142324A (en) * | 2017-06-23 | 2017-09-08 | 广东省农业科学院蚕业与农产品加工研究所 | Mulberry tree EST SSR molecular markers and its core primers group and application |
-
2017
- 2017-09-21 CN CN201710864768.7A patent/CN107365874B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754886A (en) * | 2017-01-19 | 2017-05-31 | 北京林业大学 | Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers |
| CN107142324A (en) * | 2017-06-23 | 2017-09-08 | 广东省农业科学院蚕业与农产品加工研究所 | Mulberry tree EST SSR molecular markers and its core primers group and application |
Non-Patent Citations (4)
| Title |
|---|
| DONG-MEI LI ET AL: "Floral Transcriptome Analyses of Four Paphiopedilum Orchids with Distinct Flowering Behaviors and Development of Simple Sequence Repeat Markers", 《PLANT MOL BIOL REP》 * |
| YUFENG XU ET AL: "Development and characterization of polymorphic EST-SSR markers for Paphiopedilum henryanum (Orchidaceae)", 《APPLICATIONS IN PLANT SCIENCES》 * |
| 陈业等: "23种中国兜兰属植物亲缘关系的ISSR分析", 《西南大学学报》 * |
| 陈业等: "兜兰属植物ISSR－PCR反应体系的优化", 《山地农业生物学报》 * |
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| CN107974510A (en) * | 2017-12-06 | 2018-05-01 | 中国农业科学院蔬菜花卉研究所 | Paphiopedilum armeniacum EST-SSR labeled primers, its development approach and its application |
| CN107974510B (en) * | 2017-12-06 | 2022-08-30 | 中国农业科学院蔬菜花卉研究所 | EST-SSR (expressed sequence tag-simple sequence repeat) marker primer of paphiopedilum armeniacum, development method and application thereof |
| CN107815509A (en) * | 2017-12-12 | 2018-03-20 | 福建省农业科学院作物研究所 | SSR primer sets and its application based on the exploitation of okra transcript profile sequence |
| CN107815509B (en) * | 2017-12-12 | 2021-04-06 | 福建省农业科学院作物研究所 | SSR primer group developed based on okra transcriptome sequence and application thereof |
| CN109266778A (en) * | 2018-11-20 | 2019-01-25 | 福建省农业科学院作物研究所 | EST-SSR labeled primer and application based on the exploitation of hybrid orchid transcript profile |
| CN109266778B (en) * | 2018-11-20 | 2021-07-27 | 福建省农业科学院作物研究所 | EST-SSR labeled primer developed based on hybrid blue transcriptome and application |
| CN110564722A (en) * | 2019-10-18 | 2019-12-13 | 西南林业大学 | Method for extracting paphiopedilum genome DNA by using improved CTAB method |
| CN110699480A (en) * | 2019-11-18 | 2020-01-17 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Primer group for hybridizing orchidaceae EST-SSR (expressed sequence tag-simple sequence repeat) markers and screening method |
| CN110699480B (en) * | 2019-11-18 | 2023-10-24 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Primer group for hybridization of EST-SSR (expressed sequence tag-simple sequence repeat) markers of cymbidium kanran and screening method |
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