CN109988863A - EST-SSR for distinguishing different ecological type cloves is marked and the primer - Google Patents
EST-SSR for distinguishing different ecological type cloves is marked and the primer Download PDFInfo
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Abstract
The invention discloses the EST-SSR labels and the primer for distinguishing different ecological type cloves.EST-SSR label disclosed by the invention for distinguishing environmental cloves, is obtained by carrying out PCR amplification using the primer sets that 50 single stranded DNAs form shown in sequence 1-50 in sequence table.Primer sets of the invention identify identical or relevant plant to can be used for identifying polymorphic allele, distinguish plant and study the genetic diversity in population.Primer sets of the invention and EST-SSR label have preferable application in Genus Syringa, can be used for distinguishing the cloves of different ecological type in Genus Syringa, provide convenience to carry out the molecular mark of cloves and its sibling species.
Description
Technical field
The invention belongs to field of biotechnology, and more particularly, the present invention relate to distinguish different ecological type cloves
EST-SSR label and the primer.
Background technique
Cloves (Syringa) is the general designation of Oleaceae (Oleaceae) Genus Syringa (Syringa) perennial woody plant,
Existing more than 1000 years cultivation histories of China are the rare flowers of China.Whole world genus syringa is there are about 27 kinds, China cloves
About 22 kinds of platymiscium, wherein 18 kinds are endemic species.Cloves China distribution be concentrated mainly on southwest, northwest, North China and
The areas such as northeast, wherein the ground such as Sichuan, Yunnan and Tibet have 13 kinds of NATURAL DISTRIBUTIONs cloves and China cloves endemic species most
More places.Cloves is well-known with the strong fragrance of a flower, plant it is elegant it is beautiful, spend that numerous color is beautiful, inflorescence cluster is at coniform, habit is strong
Strong, cultivation is simple, deep to be liked by common people, is the excellent garden plants in China, is widely applied in afforestation.With
The depth excavation of the fast development of cloves industry, cloves germ plasm resource has been to be concerned by more and more people with innovation, right in the market
The demand of its new varieties is also very urgent.Previously, domestic and foreign scholars were concentrated mainly on its chemical component, medicine for the research of cloves
Reason activity, Lycopersicum esculentum technology and conventional hybridization breeding etc., and its genetic diversity is studied then relatively from molecular level
It is few.
DNA (Deoxyribonucleic acid) molecular labeling is that one kind based on explanations of genomic DNA polymorphism is new
Phenotypic marker can not only stablize heredity, and can directly reflect bion hereditary variation structure.In recent years, it successively produces
20 different kinds of molecules label is given birth to, its appearance greatly accelerates the development of science of heredity and functional genomics, is expected to become acceleration
The powerful tool of plant breeding research.Simple repeated sequence (Simple sequence repeat, SSR) is also known as microsatellite
(Microsatellite) it marks, DNA base sequence made of the motif tandem sequence repeats be made of 1-6 oligonucleotides.?
In numerous molecular labelings, SSR marker is due to having many advantages, such as that codominance, variation are abundant and easily detection is considered as ideal molecule
One of type is widely used in physianthropy, animals and plants and microorganism field.Currently, related Genus Syringa SSR marker exploitation
Also it is reported, original series are developed from limited expressed sequence or genomic data in public database
The SSR marker quantity arrived is considerably less, at present only 16 pairs of Genus Syringa SSR markers, is far from satisfying Genus Syringa germ plasm resource something lost
Pass the demand of Diversity, genetic linkage maps building, functional gene positioning and association analysis research etc..Therefore, fourth is developed
Perfume, which belongs to SSR molecular marker, becomes critical issue urgently to be resolved.
SSR marker basis source difference can be divided into two classes: genome SSR (Genomics SSR, gSSR) and expression sequence
Column label SSR (Expressed Sequence Tag SSR, EST-SSR).GSSR is located at code area and the non-coding of genome
Area, the exploitation of label is based on construction of gene library method (including SSR enriched library), and experimentation is many and diverse, time-consuming and laborious, effect
Rate is lower.In recent years, the EST resource of sharp increase was simple and quick with its, at low cost in database, from the genome of expression
Region, the diversity that can directly reflect related gene, and there is good versatility etc. between not of the same race, the category even species of section
Advantage has become the important sources of SSR marker.High-throughput transcript profile sequencing technologies can be with lower cost, in the shorter time
The interior molecular information for obtaining magnanimity provides a kind of simple and effective method for large-scale development SSR marker, is particularly suitable for
Lack the non-mode plant of genomic information.
Summary of the invention
The purpose of the present invention is a kind of ecotypic method of differentiation cloves and the primers.
Present invention firstly provides the methods of identification cloves genotype, which comprises using primer sets to fourth to be measured
Fragrant genomic DNA carries out PCR amplification, obtains PCR product, detects the size of each primer pair PCR product, determines the fourth to be measured
Fragrant genotype；
The primer sets include SO026, SO029, SO035, SO040, SO046, SO100, SO176, SO178, SO194,
SO197, SO222, SO244, SO403, SO424, SO432, SO448, SO464, SO509, SO518, SO532, SO872 and
At least one of SO922；
The SO026 is the primer pair that two single stranded DNAs form shown in sequence 1 and 2 in sequence table；
The SO029 is the primer pair that two single stranded DNAs form shown in sequence 3 and 4 in sequence table；
The SO035 is the primer pair that two single stranded DNAs form shown in sequence 7 and 8 in sequence table；
The SO040 is the primer pair that two single stranded DNAs form shown in sequence 9 and 10 in sequence table；
The SO046 is the primer pair that two single stranded DNAs form shown in sequence 11 and 12 in sequence table；
The SO100 is the primer pair that two single stranded DNAs form shown in sequence 13 and 14 in sequence table；
The SO176 is the primer pair that two single stranded DNAs form shown in sequence 17 and 18 in sequence table；
The SO178 is the primer pair that two single stranded DNAs form shown in sequence 19 and 20 in sequence table；
The SO194 is the primer pair that two single stranded DNAs form shown in sequence 21 and 22 in sequence table；
The SO197 is the primer pair that two single stranded DNAs form shown in sequence 23 and 24 in sequence table；
The SO222 is the primer pair that two single stranded DNAs form shown in sequence 25 and 26 in sequence table；
The SO244 is the primer pair that two single stranded DNAs form shown in sequence 27 and 28 in sequence table；
The SO403 is the primer pair that two single stranded DNAs form shown in sequence 31 and 32 in sequence table；
The SO424 is the primer pair that two single stranded DNAs form shown in sequence 33 and 34 in sequence table；
The SO432 is the primer pair that two single stranded DNAs form shown in sequence 35 and 36 in sequence table；
The SO448 is the primer pair that two single stranded DNAs form shown in sequence 37 and 38 in sequence table；
The SO464 is the primer pair that two single stranded DNAs form shown in sequence 39 and 40 in sequence table；
The SO509 is the primer pair that two single stranded DNAs form shown in sequence 41 and 42 in sequence table；
The SO518 is the primer pair that two single stranded DNAs form shown in sequence 43 and 44 in sequence table；
The SO532 is the primer pair that two single stranded DNAs form shown in sequence 45 and 46 in sequence table；
The SO872 is the primer pair that two single stranded DNAs form shown in sequence 47 and 48 in sequence table；
The SO922 is the primer pair that two single stranded DNAs form shown in sequence 49 and 50 in sequence table.
The primer sets may also include at least one of SO033, SO101 and SO329；
The SO033 is the primer pair that two single stranded DNAs form shown in sequence 5 and 6 in sequence table；
The SO101 is the primer pair that two single stranded DNAs form shown in sequence 15 and 16 in sequence table；
The SO329 is the primer pair that two single stranded DNAs form shown in sequence 29 and 30 in sequence table.
The primer sets can be by the SO026, SO029, the SO035, the SO040, the SO046, described
SO100, the SO176, the SO178, the SO194, the SO197, the SO222, the SO244, the SO403,
It is the SO424, the SO432, the SO448, the SO464, the SO509, the SO518, the SO532, described
Whole compositions in SO872 and the SO922.
The primer sets can also be by the SO026, the SO029, the SO035, the SO040, the SO046, institute
State the SO100, SO176, the SO178, the SO194, the SO197, the SO222, the SO244, described
SO403, the SO424, the SO432, the SO448, the SO464, the SO509, the SO518, the SO532,
Whole compositions in the SO872, the SO922, the SO033, the SO101 and the SO329.
It is the SO026, the SO029, the SO035, the SO040, the SO046, the SO100, described
SO176, the SO178, the SO194, the SO197, the SO222, the SO244, the SO403, the SO424,
It is the SO432, the SO448, the SO464, the SO509, the SO518, the SO532, the SO872, described
SO922, the SO033, the SO101 and the SO329 can be marked with fluorescent material.Fluorescent material can be marked in each primer
To the end 5' of forward primer.The fluorescent material that each primer pair is marked can be FAM.Serial No. odd number is single-stranded in sequence table
DNA is forward primer, and the single stranded DNA of Serial No. even number is reverse primer.
When carrying out PCR amplification, the annealing temperature of the SO026 can be 48 DEG C；The annealing temperature of the SO029 can be 48
℃；The annealing temperature of the SO035 can be 50 DEG C；The annealing temperature of the SO040 can be 50 DEG C；The annealing temperature of the SO046
Degree is 50 DEG C；The annealing temperature of the SO100 can be 50 DEG C；The annealing temperature of the SO176 can be 52 DEG C；The SO178's
Annealing temperature can be 52 DEG C；The annealing temperature of the SO194 can be 52 DEG C；The annealing temperature of the SO197 can be 52 DEG C；It is described
The annealing temperature of SO222 can be 52 DEG C；The annealing temperature of the SO244 can be 52 DEG C；The annealing temperature of the SO403 can be 52
℃；The annealing temperature of the SO424 can be 54 DEG C；The annealing temperature of the SO432 can be 54 DEG C；The annealing temperature of the SO448
Degree can be 54 DEG C；The annealing temperature of the SO464 can be 54 DEG C；The annealing temperature of the SO509 can be 54 DEG C；The SO518
Annealing temperature can be 54 DEG C；The annealing temperature of the SO532 can be 54 DEG C；The annealing temperature of the SO872 can be 56 DEG C；Institute
The annealing temperature for stating SO922 can be 60 DEG C；The annealing temperature of the SO033 can be 48 DEG C；The annealing temperature of the SO101 can be
50℃；The annealing temperature of the SO329 can be 52 DEG C.
When carrying out PCR amplification, each pair of primer is individually carried out.Concentration of each single stranded DNA in pcr amplification reaction system
It can be 10 μM.10 μ L reaction systems of each primer pair are as follows: 1 μ L, 2 × Power Taq PCR of template (25ng)
MasterMix (Beijing Ai Delai Biotechnology Co., Ltd, article No.: PC09) 5 μ L, forward and reverse primer each 0.5 μ L, ddH2O 3μ
L。
Carry out the reaction condition of PCR amplification are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, respective annealing temperature
30s, 72 DEG C of extension 1min, 30-35 circulation；72 DEG C of extension 10min.
The genotype may be embodied in above-mentioned each primer pair and carry out the EST-SSR that PCR amplification obtains with cloves genomic DNA
On label.
The method of the identification cloves genotype is in identification cloves polymorphic allele or cloves variant or cloves ecology
Application in type, also belongs to protection scope of the present invention.
Application of the method for the identification cloves genotype in identification cloves population in identical or associated genotype, also belongs to
In protection scope of the present invention.
Application of the method for the identification cloves genotype in research cloves population genetic diversity, also belongs to the present invention
Protection scope.
Application of the method for the identification cloves genotype in the evaluation of cloves Genetic Diversity of Germplasm, also belongs to this
The protection scope of invention.
Application of the method for the identification cloves genotype in the classification and/or identification of cloves germ plasm resource, also belongs to
Protection scope of the present invention.
Application of the method for the identification cloves genotype in cloves hybrid early stage identification, also belongs to protection of the invention
Range.
Application of the method for the identification cloves genotype in the building of cloves genetic linkage maps, also belongs to of the invention
Protection scope.
Application of the method for the identification cloves genotype in cloves molecular mark, also belongs to of the invention
Protection scope.
The present invention also provides the primer sets, the primer sets can be used for identifying cloves genotype.
The EST-SSR label that PCR amplification obtains is carried out with cloves genomic DNA by the primer sets, also belongs to the present invention
Protection scope.
The primer sets or EST-SSR label are raw in identification cloves polymorphic allele or cloves variant or cloves
Application in state type, also belongs to protection scope of the present invention.
The application of the primer sets or EST-SSR label in identification cloves population in identical or associated genotype,
Also belong to protection scope of the present invention.
The application of the primer sets or EST-SSR label in research cloves population genetic diversity, also belongs to this
The protection scope of invention.
The application of the primer sets or EST-SSR label in the evaluation of cloves Genetic Diversity of Germplasm, also belongs to
In protection scope of the present invention.
The primer sets or the EST-SSR mark the application in the classification and/or identification of cloves germ plasm resource, also belong to
In protection scope of the present invention.
The application of the primer sets or EST-SSR label in cloves hybrid early stage identification, also belongs to of the invention
Protection scope.
The application of the primer sets or EST-SSR label in the building of cloves genetic linkage maps, also belongs to this hair
Bright protection scope.
The primer sets or the EST-SSR mark the application in cloves molecular mark, also belong to this hair
Bright protection scope.
It whether is same ecotypic method the present invention also provides two kinds of cloves to be measured are distinguished, which comprises benefit
PCR amplification is carried out with genomic DNA of the primer sets to two kinds of cloves to be measured, PCR product is obtained, compares two kinds of cloves to be measured
The size of each primer pair PCR product, the size of every a pair of of primer PCR amplified production is all the same in the primer sets two kinds are to be measured
Cloves is or candidate is the same ecotype, at least of different sizes two kind of pair of primers pcr amplification product in the primer sets
Cloves to be measured is or candidate is different ecological type.
The cloves can be genus syringa.The cloves concretely ' Jin Yuan ' cloves, white lilac, syringa reticulata var mandshurica, small
Leaf cloves, Chinese lilac, pinnateleaf lilac root or branchlet, Ou Dingxiang, Persian lilac, Chinese lilac and/or velvety lilac flower.
It is demonstrated experimentally that carrying out PCR amplification, gained to different ecological type cloves genomic DNA using primer sets of the invention
PCR product is all different, and has polymorphism, can be used for identifying the cloves base determined with the EST-SSR that gained PCR product indicates
Because of type, it is yet further useful for distinguishing the different cloves ecotypes.Primer sets of the invention and EST-SSR label have in Genus Syringa
There is preferable application, can be used for distinguishing the cloves of different ecological type in Genus Syringa, for the molecule for carrying out cloves and its sibling species
Marker-assisted breeding is provided convenience.
Specific embodiment
Method in following embodiments is unless otherwise instructed conventional method.
Embodiment 1, the EST-SSR for distinguishing different ecological type cloves are marked
Present embodiments providing one group can be used for distinguishing the EST-SSR label of different ecological type cloves, and can expand
One group of EST-SSR primer of this group of EST-SSR label, primer information are as shown in table 1.
1,25 pair of polymorphism EST-SSR primer information of table
The primer of Serial No. odd number is forward primer in table 1, and the primer of Serial No. even number is reverse primer, Mei Duiyin
10 μ L reaction systems of object progress the PCR amplification are as follows: (north 1 μ L, 2 × Power Taq PCR MasterMix of template (25ng/ μ L)
Jing Aidelai Biotechnology Co., Ltd, article No.: PC09) 5 μ L, (forward and reverse primer is in reaction system by forward and reverse each 0.5 μ L of primer
In concentration be 10 μM), ddH2O 3μL.Template used is with the genomic DNA of cloves blade position.
Pcr amplification reaction condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, respective annealing temperature 30s, 72 DEG C of extensions
1min, 30-35 circulations；72 DEG C of extension 10min.
The stability of primer:
According to above-mentioned reaction system and reaction condition, using the primer in table 1 respectively with the blade part of 3 environmental cloves
The genomic DNA of position is that template carries out PCR amplification, and PCR product is detected with 2% agarose gel electrophoresis, followed by
FluorchemTM5500 (Alfa Innotech Corp., USA) gel imagers are observed and are recorded to electrophoresis result, knot
Fruit shows that PCR product band is clear, single, and the primer amplification in table 1 is stablized.
3 used environmental cloves are as follows: (Beijing holy well moistens clear afforestation science and technology to be had the lilac of 3 different genotypes
Limit company).
The polymorphism of primer:
Commission Beijing Bioisystech Co., Ltd, farsighted Boxing section utilizes the end 5' of the forward primer of each primer pair of table 1 later
FAM is marked.Then according to above-mentioned reaction system and reaction condition, with the genomic DNA at 8 environmental cloves blade positions
PCR amplification is carried out for template, in carrying out Capillary Electrophoresis on ABI3730DNA analyzer after PCR amplification, and data is collected, verifies
Polymorphism.Data are analyzed and read using GeneMarker version 2.2.0 software.Wherein, standard internal reference is LIZ600
(Applied Biosystems).8 used environmental cloves moisten the limited public affairs of clear afforestation science and technology both from Beijing holy well
Department.
The results show that 25 betweens of primers in table 1 different ecological type cloves have polymorphism, i.e., 25 pairs of each cloves
The pcr amplification product of primer is not exactly the same.
The versatility of primer:
Using the primer after the primer progress fluorescent marker by table 1 respectively with the base at the blade position of 10 environmental cloves
Because group DNA is template according to above-mentioned reaction system and reaction condition progress PCR amplification, obtained PCR product is subjected to capillary
Electrophoresis detection is analyzed and is read using GeneMarker version 2.2.0 software data, obtains the finger print data of each kind.
Wherein, standard internal reference is LIZ600 (Applied Biosystems).
10 used environmental cloves are as follows: ' Jin Yuan ' cloves, white lilac, syringa reticulata var mandshurica, daphne lilac, Chinese lilac, plumage
Leaf cloves, Ou Dingxiang, Persian lilac, Chinese lilac and velvety lilac flower.
' Jin Yuan ' cloves is documented in document " Guo Ling, Sun Yi, Cui Jiru Peking lilac new varieties ' Jin Yuan ' [J] forestry section
It learns, in 2008,44 (1) ".
White lilac, syringa reticulata var mandshurica, pinnateleaf lilac root or branchlet, Ou Dingxiang, Persian lilac, Chinese lilac are documented in document " Chen Xicang, Pei Juan
The fragrant Gansu genus syringa resource and development and utilization [J] special type Economic animals and plants, in 2017 (5) ".
Daphne lilac, Chinese lilac, velvety lilac flower are documented in document, and " Beijing area Meng Xin Genus Syringa germ plasm resource is collected
And landscape application [J] Landscape Architecture in Beijing, 2011 (2): in 35-42. ".
The results show that all primers are in ' Jin Yuan ' cloves, white lilac, syringa reticulata var mandshurica, daphne lilac, Chinese lilac, leatherleaf
Cloves, Ou Dingxiang have 100% versatility, i.e., amplifiable to arrive PCR product, and have polymorphism between product；SO101 and
SO329 primer fails in Persian lilac Successful amplification to PCR product；SO033 fails the Successful amplification in Chinese lilac and produces to PCR
Object.10 kinds of amplification rate range is in 92.0%-100.0%, averagely amplification rate 98.8%.Therefore EST-SSR of the invention draws
Object and EST-SSR label have preferable application in Genus Syringa, can be used for distinguishing the cloves of different ecological type in Genus Syringa,
It provides convenience to carry out the molecular mark of cloves and its sibling species.
Distinguish different ecological type cloves method include:
PCR amplification is carried out to the genomic DNA of two kinds of cloves to be measured respectively using 25 pairs of primers in table 1, obtain respectively to
Survey the pcr amplification product of cloves, more each cloves 25 to be measured is to the size of a pair of primer PCR amplified production every in primer, and 25 pairs
The size of every a pair of primer PCR amplified production two kinds of cloves to be measured all the same are identical environmental cloves in primer, and 25 pairs are drawn
At least the of different sizes two kind cloves to be measured of pair of primers pcr amplification product is different ecological type cloves in object.
The present invention can also judge whether cloves to be measured is together throughout one's life with following standard cloves using 25 pairs of primers in table 1
State type: ' Jin Yuan ' cloves, white lilac, syringa reticulata var mandshurica, daphne lilac, Chinese lilac, pinnateleaf lilac root or branchlet, Ou Dingxiang, Persian lilac, China
Cloves, velvety lilac flower, this method comprises:
PCR amplification is carried out using 25 pairs of primer pairs cloves to be measured in table 1, obtains the pcr amplification product of cloves to be measured, than
Size of each cloves 25 to a pair of primer PCR amplified production every in primer, such as cloves to be measured in cloves more to be measured and standard cloves
It is all the same with the size of the pcr amplification product of 25 pairs of primers of cloves a certain in standard cloves, then the cloves to be measured and the cloves
For the identical ecotype, such as cloves to be measured and the size of 25 pairs of primer PCR amplified productions of any cloves in standard cloves are endless
Exactly the same, then cloves to be measured is not any ecotype in standard cloves.
Sequence table
<110>Beijing Agricultural College
<120>for distinguishing the EST-SSR label and the primer of different ecological type cloves
<130> WHOI190028
<160> 50
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
tagttaagca acaatctaaa gc 22
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
ataagactgg accacgaa 18
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
cccatactct ccaactgt 18
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
ccaacttctt cccatcat 18
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 5
ggttcttcct acggtcat 18
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
aataaagcct tgtcagct 18
<210> 7
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 7
gcgagtggaa atgagtga 18
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 8
tacggtgatg atgatagttg 20
<210> 9
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 9
tgctgtcttc ctccttgt 18
<210> 10
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 10
gactcgcttg agattgga 18
<210> 11
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 11
gtgttccaac caccatct 18
<210> 12
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 12
ctgtttgcct gttccttc 18
<210> 13
<211> 22
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 13
gtaatcactc attttcgctt tt 22
<210> 14
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 14
gcattgcatg gctagggt 18
<210> 15
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 15
aaatcaacgg atcaacct 18
<210> 16
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 16
aacctggaaa caagaaca 18
<210> 17
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 17
ataccgtggt gccagaag 18
<210> 18
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 18
ggacccaatc aatgaagc 18
<210> 19
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 19
gctgggatat ttggtatg 18
<210> 20
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 20
gttgatgagg tccttgtt 18
<210> 21
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 21
catccgcatc gccacata 18
<210> 22
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 22
tcgccactca agaccacatt 20
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 23
tatgtaagca gaacgaggaa 20
<210> 24
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 24
gttgaaactt gggagacg 18
<210> 25
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 25
acagacggac ccagatag 18
<210> 26
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 26
accaacactc cacgaact 18
<210> 27
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 27
aggctgatgt ctgaggtt 18
<210> 28
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 28
gggaaagtat gagcgata 18
<210> 29
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 29
ttttcggttc ctcctgtc 18
<210> 30
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 30
atttgatgct gccctctt 18
<210> 31
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 31
tgccttgcct agtcacat 18
<210> 32
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 32
gacacggatt cggataaa 18
<210> 33
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 33
ttgacgggtg gataagga 18
<210> 34
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 34
gctcgcaagg atctggta 18
<210> 35
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 35
caggtcgatg gttgggtc 18
<210> 36
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 36
gggctggtag gatgttgc 18
<210> 37
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 37
cggcctctga ggtcctcctt 20
<210> 38
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 38
tctcccggta tggcacaa 18
<210> 39
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 39
aaatggaatg gtgggtgt 18
<210> 40
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 40
tgggtcctga aagtctcc 18
<210> 41
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 41
aaaagatagg cacagcaa 18
<210> 42
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 42
aactcgtcat cacggtag 18
<210> 43
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 43
tctcccaacc aaggtatt 18
<210> 44
<211> 19
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 44
taggcaacaa ggaactcaa 19
<210> 45
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 45
acttcctggc atgtcttc 18
<210> 46
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 46
ggatctaggg cttgttga 18
<210> 47
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 47
tgtggtggtg atgtctga 18
<210> 48
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 48
ggaatgaagg atttacgg 18
<210> 49
<211> 20
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 49
gacagagtgg cgatgggatt 20
<210> 50
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 50
tcgggtggaa ggagttgc 18
Claims (10)
1. the method for identifying cloves genotype, comprising: carry out PCR amplification using genomic DNA of the primer sets to cloves to be measured, obtain
To PCR product, the size of each primer pair PCR product is detected, determines the genotype of the cloves to be measured；
The primer sets include S0026, S0029, S0035, S0040, S0046, S0100, S0176, S0178, S0194,
S0197, S0222, S0244, S0403, S0424, S0432, S0448, S0464, S0509, S0518, S0532, S0872 and
At least one of S0922；
The S0026 is the primer pair that two single stranded DNAs form shown in sequence 1 and 2 in sequence table；
The S0029 is the primer pair that two single stranded DNAs form shown in sequence 3 and 4 in sequence table；
The S0035 is the primer pair that two single stranded DNAs form shown in sequence 7 and 8 in sequence table；
The S0040 is the primer pair that two single stranded DNAs form shown in sequence 9 and 10 in sequence table；
The S0046 is the primer pair that two single stranded DNAs form shown in sequence 11 and 12 in sequence table；
The S0100 is the primer pair that two single stranded DNAs form shown in sequence 13 and 14 in sequence table；
The S0176 is the primer pair that two single stranded DNAs form shown in sequence 17 and 18 in sequence table；
The S0178 is the primer pair that two single stranded DNAs form shown in sequence 19 and 20 in sequence table；
The S0194 is the primer pair that two single stranded DNAs form shown in sequence 21 and 22 in sequence table；
The S0197 is the primer pair that two single stranded DNAs form shown in sequence 23 and 24 in sequence table；
The S0222 is the primer pair that two single stranded DNAs form shown in sequence 25 and 26 in sequence table；
The S0244 is the primer pair that two single stranded DNAs form shown in sequence 27 and 28 in sequence table；
The S0403 is the primer pair that two single stranded DNAs form shown in sequence 31 and 32 in sequence table；
The S0424 is the primer pair that two single stranded DNAs form shown in sequence 33 and 34 in sequence table；
The S0432 is the primer pair that two single stranded DNAs form shown in sequence 35 and 36 in sequence table；
The S0448 is the primer pair that two single stranded DNAs form shown in sequence 37 and 38 in sequence table；
The S0464 is the primer pair that two single stranded DNAs form shown in sequence 39 and 40 in sequence table；
The S0509 is the primer pair that two single stranded DNAs form shown in sequence 41 and 42 in sequence table；
The S0518 is the primer pair that two single stranded DNAs form shown in sequence 43 and 44 in sequence table；
The S0532 is the primer pair that two single stranded DNAs form shown in sequence 45 and 46 in sequence table；
The S0872 is the primer pair that two single stranded DNAs form shown in sequence 47 and 48 in sequence table；
The S0922 is the primer pair that two single stranded DNAs form shown in sequence 49 and 50 in sequence table.
2. according to the method described in claim 1, it is characterized by: the primer sets further include in S0033, S0101 and S0329
At least one；
The S0033 is the primer pair that two single stranded DNAs form shown in sequence 5 and 6 in sequence table；
The S0101 is the primer pair that two single stranded DNAs form shown in sequence 15 and 16 in sequence table；
The S0329 is the primer pair that two single stranded DNAs form shown in sequence 29 and 30 in sequence table.
3. method according to claim 1 or 2, it is characterised in that: when carrying out PCR amplification, the annealing temperature of the S0026
It is 48 DEG C；The annealing temperature of the S0029 is 48 DEG C；The annealing temperature of the S0035 is 50 DEG C；The annealing temperature of the S0040
Degree is 50 DEG C；The annealing temperature of the S0046 is 50 DEG C；The annealing temperature of the S0100 is 50 DEG C；The annealing of the S0176
Temperature is 52 DEG C；The annealing temperature of the S0178 is 52 DEG C；The annealing temperature of the S0194 is 52 DEG C；The S0197's moves back
Fiery temperature is 52 DEG C；The annealing temperature of the S0222 is 52 DEG C；The annealing temperature of the S0244 is 52 DEG C；The S0403's
Annealing temperature is 52 DEG C；The annealing temperature of the S0424 is 54 DEG C；The annealing temperature of the S0432 is 54 DEG C；The S0448
Annealing temperature be 54 DEG C；The annealing temperature of the S0464 is 54 DEG C；The annealing temperature of the S0509 is 54 DEG C；It is described
The annealing temperature of S0518 is 54 DEG C；The annealing temperature of the S0532 is 54 DEG C；The annealing temperature of the S0872 is 56 DEG C；Institute
The annealing temperature for stating S0922 is 60 DEG C；The annealing temperature of the S0033 is 48 DEG C；The annealing temperature of the S0101 is 50 DEG C；
The annealing temperature of the S0329 is 52 DEG C.
4. method as claimed in any one of claims 1-3 is in identification cloves polymorphic allele or cloves variant or cloves ecology
Application in type.
5. application of the method as claimed in any one of claims 1-3 in identification cloves population in identical or associated genotype.
6. application of the method as claimed in any one of claims 1-3 in research cloves population genetic diversity,
Or, application of the method as claimed in any one of claims 1-3 in the evaluation of cloves Genetic Diversity of Germplasm,
Or, application of the method as claimed in any one of claims 1-3 in the classification and/or identification of cloves germ plasm resource,
Or, application of the method as claimed in any one of claims 1-3 in cloves hybrid early stage identification,
Or, application of the method as claimed in any one of claims 1-3 in the building of cloves genetic linkage maps,
Or, application of the method as claimed in any one of claims 1-3 in cloves molecular mark.
7. being primer sets described in claims 1 or 2 for identifying the primer sets of cloves genotype.
It is marked 8. the primer sets as described in claims 1 or 2 carry out the EST-SSR that PCR amplification obtains with cloves genomic DNA.
9. the label of EST-SSR described in primer sets described in claims 1 or 2 or claim 8 is more in identification cloves allele
Application in state property or cloves variant or the cloves ecotype；
Or, the label of EST-SSR described in primer sets described in claims 1 or 2 or claim 8 is identical in identification cloves population
Or the application in associated genotype,
Or, the label of EST-SSR described in primer sets described in claims 1 or 2 or claim 8 is more in research cloves population genetic
Application in sample,
Or, the label of EST-SSR described in primer sets described in claims 1 or 2 or claim 8 is more in cloves germ plasm resource heredity
Application in sample evaluation,
Or, classification of the label of EST-SSR described in primer sets described in claims 1 or 2 or claim 8 in cloves germ plasm resource
And/or the application in identification,
Or, the label of EST-SSR described in primer sets described in claims 1 or 2 or claim 8 is in cloves hybrid early stage identification
Application,
Or, the label of EST-SSR described in primer sets described in claims 1 or 2 or claim 8 is in cloves genetic linkage maps structure
Application in building,
Or, the label of EST-SSR described in primer sets described in claims 1 or 2 or claim 8 is educated in cloves molecular labeling auxiliary
Application in kind.
10. distinguishing whether two kinds of cloves to be measured are same ecotypic method, comprising: utilize primer described in claims 1 or 22
Group carries out PCR amplification to the genomic DNA of two kinds of cloves to be measured, obtains PCR product, compares two kinds of each primer pairs of cloves to be measured
The size of PCR product, in the primer sets size of every a pair of of primer PCR amplified production two kinds of cloves to be measured all the same be or
Candidate is the same ecotype, at least of different sizes two kind cloves to be measured of pair of primers pcr amplification product in the primer sets
For or candidate be different ecological type.
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| CN115453027A (en) * | 2022-10-11 | 2022-12-09 | 云南中烟工业有限责任公司 | Method for distinguishing clove and clove mother based on HS-SPME-GC-MS |
| CN116179738A (en) * | 2022-08-31 | 2023-05-30 | 中国医学科学院药用植物研究所海南分所 | Core primer group for identifying SSR molecular markers of agilawood varieties and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116179738A (en) * | 2022-08-31 | 2023-05-30 | 中国医学科学院药用植物研究所海南分所 | Core primer group for identifying SSR molecular markers of agilawood varieties and application |
| CN116179738B (en) * | 2022-08-31 | 2023-09-22 | 中国医学科学院药用植物研究所海南分所 | Core primer group for identifying SSR molecular markers of agilawood varieties and application |
| CN115453027A (en) * | 2022-10-11 | 2022-12-09 | 云南中烟工业有限责任公司 | Method for distinguishing clove and clove mother based on HS-SPME-GC-MS |
| CN115453027B (en) * | 2022-10-11 | 2023-09-15 | 云南中烟工业有限责任公司 | Method for distinguishing flos caryophylli and mother flos caryophylli based on HS-SPME-GC-MS |
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