CN107974510A - Paphiopedilum armeniacum EST-SSR labeled primers, its development approach and its application - Google Patents
Paphiopedilum armeniacum EST-SSR labeled 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 paphiopedilum armeniacum transcript profile sequence, including 13 pairs of primers, the nucleotide sequence of the primer is as shown in sequence table SEQ ID NO.1~SEQID NO.26.The invention also discloses the reagent for including paphiopedilum armeniacum EST SSR label primers of the present invention.The present invention further discloses a kind of method for developing paphiopedilum armeniacum EST SSR label primers.Paphiopedilum armeniacum EST SSR label primers provided by the invention have many advantages, such as rich polymorphism, favorable repeatability, amplification are stablized and easy to count, Genetic Diversity of Germplasm analysis, molecular mark and the relevant molecular studies of cypripedium available for other kinds of paphiopedilum armeniacum and Paphiopedilum.
Description
Technical field
The present invention relates to the EST- developed based on paphiopedilum armeniacum (Paphiopedilum armeniacum) transcript profile sequence
SSR label primer, its development approach and its application, belong to biological technical field.
Background technology
Paphiopedilum armeniacum belongs to orchid family (Orchidaceae) Paphiopedilum (Paphiopedilum) plant, is the distinctive original in China
Non-hibernating eggs, there is the title of " orchid giant panda ", since its distributed areas is narrow, habitat loss, resource are excavated by predatoriness, causes apricot
Yellow pocket orchid population quantity rapidly reduce and it is endangered, be difficult in original producton location find its wild resource.Now all Paphiopedilums are planted
Thing is put into《Convention on International Trade in Endangered Species of Wild Fauna and Flora》One register of annex and be prohibited trade.Therefore paphiopedilum armeniacum
The research of genetic diversity becomes particularly significant, and the child care work of pocket orchid is more very urgent.
Simple repeated sequence (Simple sequence repeat, SSR) based on genome or transcript profile data mining
Mark is also known as microsatellite marker, there is high polymorphism, reproducible, high specificity, easy to detect, codominance, mark to cover whole
A genome and the features such as be uniformly distributed, it has also become one of critically important genetic marker in analysis of genetic diversity
(Phuekvilai, Prattana＆Pongtongkam, Pradit＆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).With
Genome SSR marker is compared, and EST-SSR derives from expressing gene group region, and the mark of " absolute " can be provided for functional gene, is opened
Send out cost it is low, thing cross-species amplification it is higher (Zhu Zhendong, Jia Jizeng, " 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).For the non-mode biology either biology without genome sequencing, profit
(RNA-Seq) SSR marker of exploitation with gene information is sequenced with transcript profile come to analyze the genetic diversity of biology be also a kind of
Simple efficient research method, this method in orchid also repeatedly report (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).It is blue in pocket
In category, Zeng Songjun etc. developed in 2010 10 pairs of homochromy pocket orchids genome SSR marker (Li, L., Zeng, S., Zheng,
F., Chen, Z., Wu, K., Zhang, J. and Duan, J., " Isolation and Characterization of
10Polymorphic Microsatellite Loci in Paphiopedilum concolor(Batem.)Pfitzer
(Orchidaceae) and Cross-species Amplification, " Hortscience, 2010,45 (8)：1286-
1287), this laboratory succeeded using the transcript profile data of paphiopedilum armeniacum have developed 13 pairs have polymorphism EST-SSR draw
Thing.
We utilize paphiopedilum armeniacum transcript profile sequence information exploitation EST-SSR primers for this, it will to paphiopedilum armeniacum and pocket
The Genetic Diversity of Germplasm that other kind of Cymbidium, Genetic relationship, the important character assignment of genes gene mapping, molecular mark
And relevant molecular studies of cypripedium 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 paphiopedilum armeniacum transcript profile sequence, including 13
To primer, the nucleotide sequence of the primer is as shown in sequence table SEQ ID NO.1~SEQ ID NO.26.Invention additionally discloses
Include the reagent of paphiopedilum armeniacum EST-SSR labeled primers of the present invention.The present invention further discloses for developing apricot
The method of yellow pocket orchid EST-SSR labeled primers, including：(a) obtained based on the sequencing splicing of paphiopedilum armeniacum floral organ transcript profile
The sequence data design EST-SSR primers of unigenes；(b) paphiopedilum armeniacum plant sample genomic DNA is extracted；(c) step is utilized
Suddenly the EST-SSR primers that (a) is obtained, PCR is carried out by template of DNA obtained by step (b)；(d) by PCR product obtained by step (c)
Electrophoresis is carried out, and electrophoresis result is analyzed, so as to filter out the paphiopedilum armeniacum EST-SSR primers with desired characteristic.This
The paphiopedilum armeniacum EST-SSR labeled primers that invention provides have many advantages, such as rich polymorphism, favorable repeatability, amplification
Stablize and easy to count, available for the analysis of the Genetic Diversity of Germplasm of paphiopedilum armeniacum and other kinds of its Paphiopedilum, divide
Sub- marker-assisted breeding and the relevant molecular studies of cypripedium.
The first aspect of the present invention provides the EST-SSR as shown in table 1 below based on the exploitation of paphiopedilum armeniacum transcript profile sequence
Primer pair：
1 paphiopedilum armeniacum EST-SSR polymorphism primer information of table
The sequence number of above-mentioned primer is respectively SEQ ID NO.1~SEQ ID NO.26.
In one embodiment, any bar primer in paphiopedilum armeniacum 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 not limited to fluorescent marker, isotope marks etc..Preferably, the mark is fluorescent marker.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 paphiopedilum armeniacum 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 paphiopedilum armeniacum transcript profile 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 paphiopedilum armeniacum genome
Isolated DNA fragmentation or gene outcome, i.e. EST-SSR mark.These EST-SSR marks are available for paphiopedilum armeniacum and other
Pocket orchid germ plasm resource analysis of genetic diversity, molecular mark and the relevant molecular studies of cypripedium.Especially
Ground is related to functional gene therefore auxiliary available for molecular labeling since these EST-SSR marks are from the transcript regions of DNA
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 the paphiopedilum armeniacum using EST-SSR primers of the present invention structure, the other plants of Paphiopedilum
The even SSR finger-prints of orchid.
The fourth aspect of the present invention provides the reagent for separating paphiopedilum armeniacum 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 fifth aspect of the present invention provides a kind of kit, and the kit includes present invention reagent as described above.
The sixth aspect of the present invention provides a kind of side based on paphiopedilum armeniacum transcript profile sequence exploitation EST-SSR primers
Method, the described method includes：
(a) sequence data of the unigenes obtained based on the sequencing splicing of paphiopedilum armeniacum floral organ transcript profile designs EST-
SSR primers；
(b) paphiopedilum armeniacum plant sample genomic DNA is extracted；
(c) the EST-SSR primers obtained using step (a), PCR is carried out by template of DNA obtained by step (b)；
(d) PCR product obtained by step (c) is subjected to electrophoresis, and electrophoresis result is analyzed, so as to filter out apricot yellow pocket
Blue EST-SSR primers.
In one embodiment, the step (a) is carried out as follows：The sequencing of paphiopedilum armeniacum floral organ transcript profile is spliced
To the sequence data of unigenes；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 sequence 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, melts
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 blade grind into powder of paphiopedilum armeniacum, the CTAB (v/v containing beta -mercaptoethanol of 65 DEG C of preheatings are added
1%)；；
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 sterile 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, make the DNA purity that finally obtains and concentration quality 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：
2 PCR reaction systems of table
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 product using fluorescent capillary electrophoresis tube method, to identify electrophoresis pattern
On polymorphic bands, so as to filter out, band is clear, polymorphism is high, the good paphiopedilum armeniacum EST-SSR primers of specificity.
In a further embodiment, using the EST- of method of the present invention exploitation Paphiopedilum other plant
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 paphiopedilum armeniacum 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 solution is brought are provided in embodiment of the present invention is：Apricot yellow pocket is developed first
Blue EST-SSR primers, there is provided 13 pairs of rich polymorphisms, favorable repeatability, specificity are high, the Henry pockets of amplification stabilization are blue
EST-SSR primers, for the analysis of paphiopedilum armeniacum Genetic Diversity of Germplasm, molecular mark and cypripedium phase
The molecular studies of pass provide basis；It is related to functional gene since these EST-SSR marks derive from the transcript regions of DNA, 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 attached drawings, further aspect of the invention, feature will be more readily understood.People in the art
Member is it should be understood that these attached 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 part sample in 170 parts of different paphiopedilum armeniacum material samples from 5 paphiopedilum armeniacum wild populations
The DNA agarose gel electrophoresis figures of product.
Fig. 2-Figure 131 shows polymorphism amplification knot of 13 pairs of primers on the paphiopedilum armeniacum sample of part listed by table 1
The fluorescence electrophoresis figure of fruit, wherein：
Fig. 2-Figure 11 show primer XH039 YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-14,
The fluorescence electrophoresis figure of polymorphism amplification on LJ-7, LJ-27 sample；
Figure 12-Figure 21 shows primer XH047 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 22-Figure 31 shows primer XH097 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 32-Figure 41 shows primer XH115 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 42-Figure 51 shows primer XH116 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 52-Figure 61 shows primer XH119 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 62-Figure 71 shows primer XH137 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 72-Figure 81 shows primer XH146 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 82-Figure 91 shows primer XH176 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 92-Figure 101 shows primer XH179 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 102-Figure 111 shows primer XH206 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 112-Figure 121 shows primer XH224 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；With
Figure 122-Figure 131 shows primer XH252 in YL-3, YL-11, PH-6, PH-13, LW-7, LW-33, WM-8, WM-
14th, the fluorescence electrophoresis figure of the polymorphism amplification on LJ-7, LJ-27 sample；
Figure 132 shows 170 part differences of the 13 pairs of primer pairs listed using table 1 from 5 paphiopedilum armeniacum wild populations
Paphiopedilum armeniacum 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, attached drawing and description are considered essentially illustrative rather than restrictive.
The exploitation of 1. paphiopedilum armeniacum EST-SSR primers of embodiment
1.EST-SSR design of primers
Experiment material：The various reagents or medium component used in all embodiments of the invention are through commercially available.
1) transcript profile library is built：Paphiopedilum armeniacum floral organ total serum IgE is extracted, separates mRNA, reverse transcription is synthesized and purified
CDNA, end is repaired plus adenosine connection sequence measuring joints, and it is 200~700bp to recycle size by agarose gel electrophoresis
Fragment, will recycling fragment carry out 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 the sequence data of unigenes.
3) SSR sites are searched：It is each with MIcroSAtellite identification tool (MISA) software lookup
SSR sites that may be present in a 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 sequence 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：Paphiopedilum armeniacum plant leaf blade be taken respectively from Nujiang Autonomous Prefecture of Yunnan Province Lan Pinglajing towns, Fugong County Pi He townshiies,
170 plant in Lushui County Lao Wo townshiies, Longyang District willow township and Longyang District watt Ma Cun.
It is as follows with the CTAB method batch extracting sample DNAs of improvement, concrete operations：
(1) 2ml centrifuge tubes (sterilizing), fills blade, tube cover size two panels；
(2) liquid nitrogen grinding adds 65 DEG C of 800 μ L of CTAB (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：800 μ L of isoamyl alcohol (24: 1), jog
5min (hand)；
(5) 13000rpm centrifuges 10min, takes 450 μ L of supernatant into 1.5 μ L centrifuge tubes, then extract once (24: 50 μ of Isosorbide-5-Nitrae
L), clear 300 μ L are sucted, add the 600 μ L of precooling absolute ethyl alcohol 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) 70% ethanol of 1ml 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 sterile waters redissolve.
Into row agarose gel electrophoresis, the STb gene electrophoretic band that the results show (Fig. 1) is extracted is clear, illustrates extraction
DNA mass is good.
3. carry out PCR amplification using EST-SSR primers
Using the genome DAN extracted above as template, PCR amplification is carried out using the EST-SSR primers designed above, so that
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 product using fluorescent capillary electrophoresis tube method.It is clear, polymorphic according to electrophoresis pattern, screening band
Property high, paphiopedilum armeniacum EST-SSR primers that specificity is good.
As a result 13 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 131, detect to obtain 96, allele altogether using 13 pairs of primers, the variation amplitude of each pair primer allele is in 2-21
Between, average number of alleles is 7.38, shows that the EST-SSR primers of the invention developed can be completely used for the something lost of paphiopedilum armeniacum
Pass diversity analysis.
Embodiment 2. carries out analysis of genetic diversity using paphiopedilum armeniacum EST-SSR primers
Experiment material：Paphiopedilum armeniacum laboratory sample is obtained from Nujiang Autonomous Prefecture of Yunnan Province Lan Pinglajing towns (LJ), Fugong County respectively
River township (PH), Lushui County Lao Wo townshiies (LW), 170 plant of Longyang District willow township (YL) and Longyang District watt Ma Cun (WM).
It has been shown that, observation and expectation are analyzed and (see below table 3) to the amplification polymorphism information of this 13 pairs of EST-SSR primers
Heterozygosity scope is that the distribution of 0.1934-0.9722 and 0.0278-0.8066, Shannon information index exists respectively
Between 0.1709-2.2988, average value 1.05, shows that the genetic variation and genetic differentiation of this 170 parts of paphiopedilum armeniacum materials is relatively abundanter.
Understood using the analysis of genetic diversity (seeing below table 4) of 13 pairs of primer pairs, 5 paphiopedilum armeniacum population, paphiopedilum armeniacum
The polymorphic site percentage (PPL) of 5 population changes between 83.33%-100%, average value 93.33%.Wherein Lu
The old nest of water (LW) and the Baoshan watt horse (WM) population polymorphic site percentage up to 100%, Baoshan willow (YL) population polymorphism
Site percentage minimum 83.33%.Nei ' s gene diversity indexes (h) luffing of 5 population between 0.3978-0.5003,
Average value is 0.4564；Shannon indexes (I) change between 0.7207-0.9938, average value 0.8803.5 paphiopedilum armeniacums
In population, the genetic diversity index h and I of Lushui old nest (LW) population show as highest, but LJ population is minimum.It is overall
From the point of view of, the genetic diversity very abundant of 5 population.
3 13 pairs of EST-SSR primer amplifications polymorphism information analyses of table
4 Yunnan Nujiang two sides paphiopedilum armeniacum population genetic diversity of table
The cluster analysis of genetic affinity between 3. population of embodiment
Experiment material：Paphiopedilum armeniacum laboratory sample respectively be obtained from Nujiang Autonomous Prefecture of Yunnan Province Lan Pinglajing towns, Fugong County Pi He townshiies,
170 plant in Lushui County Lao Wo townshiies, Longyang District willow township and Longyang District watt Ma Cun.
Genetic variation and genetic differentiation degree (the table between apricot yellow population is evaluated with Nei ' s genetic distances (D) and genetic identity (IN)
5).The genetic similarity of 5 population is very high, changes between 0.9188-0.9683；Correspondingly, its genetic distance change width
Degree is also little, and between keeping 0.0323-0.0847, genetic identity is higher between showing paphiopedilum armeniacum population, and genetic distance is less than normal.
Further UPMGA cluster analyses show, in 5 paphiopedilum armeniacum population of Yunnan Province Nujiang two sides, LY and LW's
Genetic distance is minimum, and affiliation is nearer；LJ and PH population genetic distance is maximum, and affiliation farther out, is shown in Figure 132.
The genetic distance (lower triangle) and Nei ' s genetic identities (upper three of 5 Yunnan Nujiang two sides paphiopedilum armeniacum population of table
Angle)
Embodiment 4. carries out General Use Analysis using paphiopedilum armeniacum EST-SSR primers in cypripedium
General Use Analysis is carried out to other four kinds of cypripediums from Paphiopedilum using paphiopedilum armeniacum microsatellite, as a result
(seeing below table 6) shows 13 pairs of EST-SSR primers in Malipo pocket blue (P.malipoerise), Paphiopedilum micranthum
(P.micranthum), De Shi pockets blue (P.delenatii), the amplification success rate of homochromy pocket blue (P.concolor) are respectively
0.62nd, 0.54,0.62,0.69, illustrate that this 13 pairs of EST-SSR primers have higher versatility in Paphiopedilum.Prove to utilize
13 pairs of EST-SSR primers of paphiopedilum armeniacum transcript profile sequencing exploitation can be used for the genetic diversity and genetic structure of nearly edge species
Research.
Amplification situation of the 6 13 pairs of EST-SSR primers of table in other four kinds of cypripediums
The above description is merely a specific embodiment, 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 be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the guarantor of the claim
Protect subject to scope.
Sequence table
<110>Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science
<120>Paphiopedilum armeniacum EST-SSR labeled primers, its development approach and its application
<141> 2017-11-26
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 1
gttcgcctca ttgaagcatt 20
<210> 2
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 2
gggatttata gagctcgccc 20
<210> 3
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 3
tccagccttc cacttttcac 20
<210> 4
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 4
ggaggaacgt acttggtgga 20
<210> 5
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 5
gggtcagcgt ctgctatctc 20
<210> 6
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 6
aagccctctt atacagcgca 20
<210> 7
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 7
gaacggtcct gatcccacta 20
<210> 8
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 8
aaggtttgga ggaagctggt 20
<210> 9
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 9
agtcgcacaa gggacagact 20
<210> 10
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 10
actgtctgtg gttcccaagg 20
<210> 11
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 11
gctgaaactg ggaaccttca 20
<210> 12
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 12
ggagtggtga tcctgctgat 20
<210> 13
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 13
gaagaggttc gtgttggctc 20
<210> 14
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 14
caagcgtagc aaggaatgaa 20
<210> 15
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 15
cccttctccg tttcctctct 20
<210> 16
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 16
cttcctctgc tttctcacgc 20
<210> 17
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 17
tttagcaaag accacagggg 20
<210> 18
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 18
atcagcaggt ccagcaattc 20
<210> 19
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 19
tgcttggaca gatgatgagc 20
<210> 20
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 20
tcttactgct cctgggttgg 20
<210> 21
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 21
ctagtcctaa cactcgcgcc 20
<210> 22
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 22
aagagacggg gtgaaatcct 20
<210> 23
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 23
cctgaatcag cattttgcct 20
<210> 24
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 24
ctcggttcct tttcccttct 20
<210> 25
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 25
gggaggagaa ggagttggtc 20
<210> 26
<211> 20
<212> DNA
<213>Paphiopedilum armeniacum (paphiopedilum armeniacum artificial sequence)
<400> 26
ggctgggaac acattcagtt 20
Claims (10)
1. based on the EST-SSR primer pairs of paphiopedilum armeniacum transcript profile sequence exploitation, the primer pair is selected from following (1)-(13) item
Any one：
(1) primer pair PA_SSR039, 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_SSR047, 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_SSR097, 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_SSR115, 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_SSR116, 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_SSR119, 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_SSR137, 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_SSR146, 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_SSR176, 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_SSR179, 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_SSR206, 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_SSR224, 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_SSR252, 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；
Or its any combination.
2. paphiopedilum armeniacum EST-SSR primer pairs as claimed in claim 1 a, wherein primer in the primer pair can be examined
Geodetic marks.
3. paphiopedilum armeniacum EST-SSR primer pairs as claimed in claim 2, wherein the mark is fluorescent marker.
4. based on the EST-SSR primer sets of paphiopedilum armeniacum transcript profile 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 paphiopedilum armeniacum EST-SSR marks, the reagent include such as claim 1-3 any one of them
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 that EST-SSR primers as claimed in claim 1 are developed based on paphiopedilum armeniacum transcript profile sequence, the side
Method includes：
(a) the sequence data design EST-SSR of the unigenes obtained based on the sequencing splicing of paphiopedilum armeniacum floral organ transcript profile is drawn
Thing；
(b) paphiopedilum armeniacum plant sample genomic DNA is extracted；
(c) the EST-SSR primers obtained using step (a), PCR is carried out by template of DNA obtained by step (b)；
(d) PCR product obtained by step (c) is subjected to electrophoresis, and electrophoresis result is analyzed, so as to filter out paphiopedilum armeniacum
EST-SSR primers.
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 blade grind into powder of Henry pockets orchid, 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: isoamyl alcohol (24: 1), jog 5min is added；
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 sterile 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, are circulated 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, are circulated 10 times；Followed by 72 DEG C of extension 10min.
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