CN109988863B - EST-SSR (expressed sequence tag-simple sequence repeat) marker for distinguishing different ecotypes of clove and primers used by EST-SSR marker - Google Patents
EST-SSR (expressed sequence tag-simple sequence repeat) marker for distinguishing different ecotypes of clove and primers used by EST-SSR marker Download PDFInfo
- Publication number
- CN109988863B CN109988863B CN201910345948.3A CN201910345948A CN109988863B CN 109988863 B CN109988863 B CN 109988863B CN 201910345948 A CN201910345948 A CN 201910345948A CN 109988863 B CN109988863 B CN 109988863B
- Authority
- CN
- China
- Prior art keywords
- clove
- sequences
- annealing temperature
- sequence table
- stranded dnas
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an EST-SSR marker for distinguishing different ecotypes of clove and a primer used by the EST-SSR marker. The EST-SSR marker for distinguishing the ecotype clove disclosed by the invention is obtained by carrying out PCR amplification on a primer group consisting of 50 single-stranded DNAs shown in sequences 1-50 in a sequence table. The primer sets of the invention can be used to identify allelic polymorphisms, identify identical or related plants, distinguish plants, and study genetic diversity in populations. The primer group and EST-SSR marker of the invention have better application in syringa, can be used for distinguishing different ecotypes of the syringa, and provide convenience for developing molecular marker assisted breeding of the syringa and related species.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an EST-SSR marker for distinguishing different ecotypes of clove and a primer used by the EST-SSR marker.
Background
Clove (Syringa) is a general name of perennial woody plants of Syringa (Syringa) of Oleaceae, has been cultivated in China for over 1000 years, and is a famous and precious flower in China. There are about 27 lilac plants all over the world, and there are about 22 lilac plants in China, 18 of which are special species. The distribution of the clove in China is mainly concentrated in regions such as southwest, northwest, northeast and northeast, wherein 13 naturally distributed cloves exist in the regions such as Sichuan, Yunnan and Tibet, and the clove is also the place with the most unique varieties of clove in China. The clove is famous for strong flower fragrance, the plant is elegant and beautiful, the flower is numerous and beautiful, the inflorescence clusters into a cone shape, the habit is strong, the cultivation is simple and easy, the clove is deeply loved by the world, is a good landscaping plant in China, and is widely applied to landscaping. With the rapid development of the clove industry, the deep excavation and innovation of clove germplasm resources are more and more concerned by people, and the demand on new species of the clove in the market is urgent. At present, researchers at home and abroad mainly concentrate on chemical components, pharmacological activity, cultivation and propagation technology, traditional crossbreeding and the like of the clove, and relatively little research on genetic diversity of the clove from a molecular level is carried out.
A DNA (deoxyriboneceic acid) molecular marker is a novel marker based on genome DNA polymorphism, can not only stabilize inheritance, but also directly reflect the individual genetic variation structure of organisms. In recent years, more than 20 molecular markers have been generated in sequence, and the emergence of the molecular markers greatly accelerates the development of genetics and functional genomics, and is expected to become a powerful tool for accelerating the research of plant breeding. Simple Sequence Repeat (SSR), also known as Microsatellite (Microsatelite), is a DNA base sequence consisting of a tandem repeat of motifs consisting of 1 to 6 oligonucleotides. Among the numerous molecular markers, SSR markers are considered to be one of ideal molecular marker types due to the advantages of co-dominance, abundant variation, easy detection and the like, and are widely applied to the fields of human medicine, animals, plants and microorganisms. At present, the development of syringa SSR markers is also reported, and the original sequences of the syringa SSR markers are derived from limited expression sequences or genome data in a public database, so that the quantity of the developed SSR markers is very small, only 16 pairs of syringa SSR markers exist at present, and the requirements of genetic diversity evaluation, genetic linkage map construction, functional gene positioning, association analysis research and the like of syringa germplasm resources are far from being met. Therefore, the development of syringa SSR molecular markers is a key problem to be solved urgently.
SSR markers can be divided into two categories depending on the source: genomic SSR (Genomics SSR, gSSR) and Expressed Sequence Tag SSR (Expressed Sequence Tag SSR, EST-SSR). gSSR is located in coding region and non-coding region of genome, and the development of marker is mainly based on gene library construction method (including SSR enrichment library), and its experimental process is complicated, time-consuming and labor-consuming, and its efficiency is low. In recent years, the EST resources which are rapidly increased in databases have become important sources of SSR markers due to the advantages of simplicity, rapidness, low cost, being derived from expressed genome regions, being capable of directly reflecting the diversity of related genes, having good universality among species of different species, genera and even families, and the like. The high-throughput transcriptome sequencing technology can obtain massive molecular information in a short time at a low cost, provides a simple and effective method for developing SSR markers on a large scale, and is particularly suitable for non-model plants lacking genome information.
Disclosure of Invention
The invention aims to provide a method for distinguishing the ecotype of clove and a primer used by the method.
The present invention first provides a method for identifying the genotype of clove, comprising: carrying out PCR amplification on genome DNA of the clove to be detected by utilizing the primer sets to obtain PCR products, detecting the size of the PCR products of each primer set, and determining the genotype of the clove to be detected;
the primer group comprises at least one of SO026, SO029, SO035, SO040, SO046, SO100, SO176, SO178, SO194, SO197, SO222, SO244, SO403, SO424, SO432, SO448, SO464, SO509, SO518, SO532, SO872 and SO 922;
the SO026 is a primer pair consisting of two single-stranded DNAs shown as sequences 1 and 2 in the sequence table;
the SO029 is a primer pair consisting of two single-stranded DNAs shown as sequences 3 and 4 in the sequence table;
the SO035 is a primer pair consisting of two single-stranded DNAs shown in sequences 7 and 8 in the sequence table;
the SO040 is a primer pair consisting of two single-stranded DNAs shown as sequences 9 and 10 in the sequence table;
the SO046 is a primer pair consisting of two single-stranded DNAs shown as sequences 11 and 12 in a sequence table;
the SO100 is a primer pair consisting of two single-stranded DNAs shown as sequences 13 and 14 in a sequence table;
the SO176 is a primer pair consisting of two single-stranded DNAs shown as sequences 17 and 18 in the sequence table;
the SO178 is a primer pair consisting of two single-stranded DNAs shown as sequences 19 and 20 in the sequence table;
the SO194 is a primer pair consisting of two single-stranded DNAs shown as sequences 21 and 22 in the sequence table;
the SO197 is a primer pair consisting of two single-stranded DNAs shown as sequences 23 and 24 in the sequence table;
the SO222 is a primer pair consisting of two single-stranded DNAs shown as sequences 25 and 26 in the sequence table;
the SO244 is a primer pair consisting of two single-stranded DNAs shown as sequences 27 and 28 in the sequence table;
the SO403 is a primer pair consisting of two single-stranded DNAs shown as sequences 31 and 32 in the sequence table;
the SO424 is a primer pair consisting of two single-stranded DNAs shown as sequences 33 and 34 in the sequence table;
the SO432 is a primer pair consisting of two single-stranded DNAs shown as sequences 35 and 36 in a sequence table;
the SO448 is a primer pair consisting of two single-stranded DNAs shown as sequences 37 and 38 in the sequence table;
the SO464 is a primer pair consisting of two single-stranded DNAs shown as sequences 39 and 40 in the sequence table;
the SO509 is a primer pair consisting of two single-stranded DNAs shown as sequences 41 and 42 in the sequence table;
the SO518 is a primer pair consisting of two single-stranded DNAs shown as sequences 43 and 44 in the sequence table;
the SO532 is a primer pair consisting of two single-stranded DNAs shown as sequences 45 and 46 in the sequence table;
the SO872 is a primer pair consisting of two single-stranded DNAs shown as sequences 47 and 48 in the sequence table;
and the SO922 is a primer pair consisting of two single-stranded DNAs shown as sequences 49 and 50 in the sequence table.
The primer set can further comprise at least one of SO033, SO101 and SO 329;
the SO033 is a primer pair consisting of two single-stranded DNAs shown as sequences 5 and 6 in the sequence table;
the SO101 is a primer pair consisting of two single-stranded DNAs shown as sequences 15 and 16 in a sequence table;
the SO329 is a primer pair consisting of two single-stranded DNAs shown as sequences 29 and 30 in the sequence table.
The primer set may be composed of all of the SO026, the SO029, the SO035, the SO040, the SO046, the SO100, the SO176, the SO178, the SO194, the SO197, the SO222, the SO244, the SO403, the SO424, the SO432, the SO448, the SO464, the SO509, the SO518, the SO532, the SO872 and the SO 922.
The primer set may further be composed of all of the SO026, the SO029, the SO035, the SO040, the SO046, the SO100, the SO176, the SO178, the SO194, the SO197, the SO222, the SO244, the SO403, the SO424, the SO432, the SO448, the SO464, the SO509, the SO518, the SO532, the SO872, the SO922, the SO033, the SO101, and the SO 329.
The SO026, the SO029, the SO035, the SO040, the SO046, the SO100, the SO176, the SO178, the SO194, the SO197, the SO222, the SO244, the SO403, the SO424, the SO432, the SO448, the SO464, the SO509, the SO518, the SO532, the SO872, the SO922, the SO033, the SO101, and the SO329 may be labeled with a fluorescent substance. Fluorescent substances may be labeled at the 5' end of each primer pair of the forward primers. The fluorescent substance labeled by each primer pair may be FAM. In the sequence table, the single-stranded DNA with the odd sequence number is used as a forward primer, and the single-stranded DNA with the even sequence number is used as a reverse primer.
When PCR amplification is carried out, the annealing temperature of the SO026 can be 48 ℃; the annealing temperature of the SO029 can be 48 ℃; the annealing temperature of the SO035 can be 50 ℃; the annealing temperature of the SO040 can be 50 ℃; the annealing temperature of the SO046 is 50 ℃; the SO100 annealing temperature may be 50 ℃; the SO176 annealing temperature may be 52 ℃; the annealing temperature of the SO178 may be 52 ℃; the annealing temperature of the SO194 may be 52 ℃; the annealing temperature of the SO197 can be 52 ℃; the SO222 annealing temperature may be 52 ℃; the SO244 annealing temperature may be 52 ℃; the annealing temperature of the SO403 may be 52 ℃; the annealing temperature of the SO424 can be 54 ℃; the annealing temperature of the SO432 may be 54 ℃; the SO448 may have an annealing temperature of 54 ℃; the SO464 annealing temperature may be 54 ℃; the annealing temperature of the SO509 may be 54 ℃; the annealing temperature of the SO518 may be 54 ℃; the annealing temperature of the SO532 can be 54 ℃; the annealing temperature of the SO872 may be 56 ℃; the annealing temperature of the SO922 may be 60 ℃; the annealing temperature of the SO033 may be 48 ℃; the annealing temperature of the SO101 may be 50 ℃; the SO329 anneal temperature may be 52 ℃.
When PCR amplification is carried out, each pair of primers is separately carried out. The concentration of each single-stranded DNA in the PCR amplification reaction system may be 10. mu.M. The 10. mu.L reaction system of each primer pair can be: 1. mu.L of template (25ng), 5. mu.L of 2 XPower Taq PCR MasterMix (Beijing Addela Biotech Co., Ltd., product number: PC09), 0.5. mu.L of forward and reverse primers, respectively, ddH2O 3μL。
The reaction conditions for PCR amplification can be as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 30s, extension at 72 ℃ for 1min, and 30-35 cycles; extension at 72 ℃ for 10 min.
The genotype can be expressed on the EST-SSR mark obtained by PCR amplification of clove genome DNA by the primer pairs.
The application of the method for identifying the clove genotype in identifying the clove allele polymorphism or clove variant or clove ecotype also belongs to the protection scope of the invention.
The application of the method for identifying the clove genotype in identifying the same or related genotypes in the clove population also belongs to the protection scope of the invention.
The application of the method for identifying the clove genotype in researching the genetic diversity of clove population also belongs to the protection scope of the invention.
The application of the method for identifying the clove genotype in evaluating the genetic diversity of clove germplasm resources also belongs to the protection scope of the invention.
The application of the method for identifying the clove genotype in the classification and/or identification of clove germplasm resources also belongs to the protection scope of the invention.
The application of the method for identifying the clove genotype in early clove hybrid identification also belongs to the protection scope of the invention.
The application of the method for identifying the clove genotype in constructing the clove genetic linkage map also belongs to the protection scope of the invention.
The application of the method for identifying the clove genotype in molecular marker assisted breeding of clove also belongs to the protection scope of the invention.
The invention also provides the primer group, and the primer group can be used for identifying the clove genotype.
EST-SSR marks obtained by PCR amplification of the primer group by using clove genome DNA also belong to the protection scope of the invention.
The application of the primer group or the EST-SSR marker in identifying the clove allele polymorphism or clove variant or clove ecotype also belongs to the protection scope of the invention.
The application of the primer group or the EST-SSR marker in identifying the same or related genotypes in the clove population also belongs to the protection scope of the invention.
The application of the primer group or the EST-SSR marker in researching the genetic diversity of the clove population also belongs to the protection scope of the invention.
The application of the primer group or the EST-SSR marker in the evaluation of genetic diversity of clove germplasm resources also belongs to the protection scope of the invention.
The application of the primer group or the EST-SSR marker in the classification and/or identification of clove germplasm resources also belongs to the protection scope of the invention.
The application of the primer group or the EST-SSR marker in early identification of clove hybrids also belongs to the protection scope of the invention.
The application of the primer group or the EST-SSR marker in the construction of the clove genetic linkage map also belongs to the protection scope of the invention.
The application of the primer group or the EST-SSR marker in clove molecular marker assisted breeding also belongs to the protection scope of the invention.
The invention also provides a method for distinguishing whether the two types of clove to be detected are in the same ecotype, which comprises the following steps: and performing PCR amplification on the genomic DNA of the two types of cloves to be detected by utilizing the primer group to obtain PCR products, comparing the sizes of the PCR products of the primer pairs of the two types of cloves to be detected, wherein the two types of cloves to be detected with the same size of the PCR amplification product of each pair of primers in the primer group are the same ecotype or candidates of the two types of cloves to be detected with the same size, and the two types of cloves to be detected with different sizes of the PCR amplification products of at least one pair of primers in the primer group are different ecotypes or candidates of the two types of cloves to be detected.
The flos Caryophylli can be plants of genus Eugenia. The flos Caryophylli can be 'JINYUAN' flos Caryophylli, cortex Syringae, Syringa amurensis Linne, lobular flos Caryophylli, mixed flos Caryophylli, Lupinus vulgaris, Eugenia caryophyllata, mosaic flos Caryophylli, Hua flos Caryophylli and/or Guandong flos Caryophylli.
Experiments prove that when the primer group disclosed by the invention is used for carrying out PCR amplification on genome DNA of different ecotypes of clove, the obtained PCR products are different and have polymorphism, so that the primer group can be used for identifying the genotype of the clove determined by EST-SSR represented by the obtained PCR products and further can be used for distinguishing different ecotypes of the clove. The primer group and EST-SSR marker of the invention have better application in syringa, can be used for distinguishing different ecotypes of the syringa, and provide convenience for developing molecular marker assisted breeding of the syringa and related species.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 EST-SSR marker for distinguishing different ecotypes of clove
The embodiment provides a group of EST-SSR markers which can be used for distinguishing different ecotypes of clove and a group of EST-SSR primers which can amplify the group of EST-SSR markers, and the primer information is shown in Table 1.
TABLE 1, 25 polymorphic EST-SSR primer information
In table 1, the primers with odd sequence numbers are forward primers, the primers with even sequence numbers are reverse primers, and the 10 μ L reaction system for performing PCR amplification on each pair of primers is as follows: mu.L of template (25 ng/. mu.L), 5. mu.L of 2 XPower Taq PCR MasterMix (Beijing Addela Biotech Co., Ltd., product number: PC09), 0.5. mu.L of forward and reverse primers, respectively (the concentration of the forward and reverse primers in the reaction system is 10. mu.M), ddH2O3. mu.L. The templates are genome DNA of clove leaf sites.
The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 30s, extension at 72 ℃ for 1min, and 30-35 cycles; extension at 72 ℃ for 10 min.
Stability of the primers:
according to the reaction system and the reaction conditions, the primers in the table 1 are used for respectively carrying out PCR amplification by taking the genome DNA of the leaf parts of 3 ecotype clove as templates, PCR products are detected by 2% agarose gel electrophoresis, and then Fluorchem is used for detectingTM5500(Alfa Inotech Corp., USA) gel imager observed and recorded the electrophoresis result, and the result shows that PCR product bands are all clear and single, and the amplification of the primers in Table 1 is stable.
The 3 ecotype lilac is: lilac of 3 different genotypes (Beijing Shengquan Qingyuan forest greening technology, Inc.).
Polymorphism of primer:
thereafter, Beijing Rui Boxing scientific Biotechnology Co., Ltd was requested to label the 5' -end of the forward primer of each primer pair in Table 1 with FAM. And then performing PCR amplification by using genome DNA of 8 ecotype clove leaf parts as templates according to the reaction system and the reaction conditions, performing capillary electrophoresis on the amplified PCR products on an ABI3730DNA analyzer, collecting data, and verifying polymorphism. Data were analyzed and read using GeneMarker version 2.2.0 software. The standard internal reference is LIZ600(Applied Biosystems). The 8 ecotype cloves are all from the afforestation science and technology company of Beijing Shengquan Qingyuan forest.
The results show that the 25 pairs of primers in table 1 have polymorphism between different ecotypes of syringae, i.e. the PCR amplification products of the 25 pairs of primers for each syringae are not completely the same.
The universality of the primers:
the primers obtained by carrying out fluorescence labeling on the primers in the table 1 are respectively used for carrying out PCR amplification by taking genome DNA of leaf parts of 10 ecotype cloves as templates according to the reaction system and the reaction conditions, the obtained PCR products are subjected to capillary electrophoresis detection, and data are analyzed and read by utilizing GeneMarker version 2.2.0 software to obtain fingerprint data of various varieties. The standard internal reference is LIZ600(Applied Biosystems).
The 10 ecotype lilac is: 'Jinyuan' clove, white clove, Syringa amurensis, cloves, assorted clove, Lupus lupulus, Eugenia caryophyllata, mosaic clove, Hua clove and Guandong clove.
The ' jinyuan ' clove is described in the document ' solieria, prosperity, discipline, new species of beijing clove ' jinyuan ' J.
Lilac, Syringa amurensis, Syringa oblata, Syringa reticulata, and Syringa huashanensis are described in the literature, "Chenxi storehouse, one of the fields of the family of Juan Fa & Gansu, plant resources and development and utilization of [ J ]. special economic animals and plants, 2017 (5)".
Cloves, assorted cloves, and guan dong cloves are described in "manxin. lilac genus germplasm resource collection and garden application in beijing garden [ J ], (2):35-42 ].
The results show that all primers have 100% of universality in 'gold garden' clove, white clove, Syringa amurensis, lobular clove, assorted clove, Lupus caryophyllus and syringa oblata, namely all primers can be amplified to PCR products, and polymorphism exists among the products; SO101 and SO329 primers failed to amplify successfully to PCR products in syringa japonica; SO033 did not amplify the PCR product successfully in syzygium aromaticum. The amplification rate of 10 species ranged from 92.0% to 100.0%, with an average amplification rate of 98.8%. Therefore, the EST-SSR primer and the EST-SSR marker have better application in the syringa, can be used for distinguishing the syringa with different ecotypes in the syringa, and provide convenience for developing molecular marker assisted breeding of the syringa and related species.
The method for distinguishing different ecotypes of clove comprises the following steps:
utilizing 25 pairs of primers in the table 1 to respectively perform PCR amplification on genome DNAs of two types of to-be-detected cloves to obtain PCR amplification products of the to-be-detected cloves, comparing the sizes of the PCR amplification products of each pair of primers in the 25 pairs of primers of the to-be-detected cloves, wherein the two types of to-be-detected cloves with the same size of the PCR amplification products of each pair of primers in the 25 pairs of primers are the same ecological clove, and the two types of to-be-detected cloves with different sizes of the PCR amplification products of at least one pair of primers in the 25 pairs of primers are different ecological clove.
The invention can also utilize 25 pairs of primers in the table 1 to judge whether the clove to be detected is in the same ecotype with the following standard clove: 'Jinyuan' clove, white clove, Syringa amurensis, lobular clove, assorted clove, Lupus lupulus, Eugenia caryophyllata, mosaic clove, Hua clove and Guandong clove, wherein the method comprises the following steps:
performing PCR amplification on the to-be-detected clove by using 25 pairs of primers in the table 1 to obtain a PCR amplification product of the to-be-detected clove, and comparing the sizes of the PCR amplification products of each pair of primers in the 25 pairs of primers of the to-be-detected clove and the standard clove, wherein if the sizes of the PCR amplification products of the 25 pairs of primers of the to-be-detected clove and one clove in the standard clove are the same, the to-be-detected clove and the standard clove are the same ecological type, and if the sizes of the PCR amplification products of the 25 pairs of primers of any clove in the to-be-detected clove and the standard clove are not completely the same, the to-be-detected clove is not any ecological type in the standard clove.
Sequence listing
<110> Beijing college of agriculture
<120> EST-SSR mark for distinguishing different ecotypes of clove and used primer
<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 (12)
1. The primer group for identifying the clove genotype consists of SO026, SO029, SO035, SO040, SO046, SO100, SO176, SO178, SO194, SO197, SO222, SO244, SO403, SO424, SO432, SO448, SO464, SO509, SO518, SO532, SO872, SO922, SO033, SO101 and SO 329;
the SO026 is a primer pair consisting of two single-stranded DNAs shown as sequences 1 and 2 in the sequence table;
the SO029 is a primer pair consisting of two single-stranded DNAs shown as sequences 3 and 4 in the sequence table;
the SO035 is a primer pair consisting of two single-stranded DNAs shown in sequences 7 and 8 in the sequence table;
the SO040 is a primer pair consisting of two single-stranded DNAs shown as sequences 9 and 10 in the sequence table;
the SO046 is a primer pair consisting of two single-stranded DNAs shown as sequences 11 and 12 in a sequence table;
the SO100 is a primer pair consisting of two single-stranded DNAs shown as sequences 13 and 14 in a sequence table;
the SO176 is a primer pair consisting of two single-stranded DNAs shown as sequences 17 and 18 in the sequence table;
the SO178 is a primer pair consisting of two single-stranded DNAs shown as sequences 19 and 20 in the sequence table;
the SO194 is a primer pair consisting of two single-stranded DNAs shown as sequences 21 and 22 in the sequence table;
the SO197 is a primer pair consisting of two single-stranded DNAs shown as sequences 23 and 24 in the sequence table;
the SO222 is a primer pair consisting of two single-stranded DNAs shown as sequences 25 and 26 in the sequence table;
the SO244 is a primer pair consisting of two single-stranded DNAs shown as sequences 27 and 28 in the sequence table;
the SO403 is a primer pair consisting of two single-stranded DNAs shown as sequences 31 and 32 in the sequence table;
the SO424 is a primer pair consisting of two single-stranded DNAs shown as sequences 33 and 34 in the sequence table;
the SO432 is a primer pair consisting of two single-stranded DNAs shown as sequences 35 and 36 in a sequence table;
the SO448 is a primer pair consisting of two single-stranded DNAs shown as sequences 37 and 38 in the sequence table;
the SO464 is a primer pair consisting of two single-stranded DNAs shown as sequences 39 and 40 in the sequence table;
the SO509 is a primer pair consisting of two single-stranded DNAs shown as sequences 41 and 42 in the sequence table;
the SO518 is a primer pair consisting of two single-stranded DNAs shown as sequences 43 and 44 in the sequence table;
the SO532 is a primer pair consisting of two single-stranded DNAs shown as sequences 45 and 46 in the sequence table;
the SO872 is a primer pair consisting of two single-stranded DNAs shown as sequences 47 and 48 in the sequence table;
the SO922 is a primer pair consisting of two single-stranded DNAs shown as sequences 49 and 50 in the sequence table;
the SO033 is a primer pair consisting of two single-stranded DNAs shown as sequences 5 and 6 in a sequence table;
the SO101 is a primer pair consisting of two single-stranded DNAs shown as sequences 15 and 16 in a sequence table;
the SO329 is a primer pair consisting of two single-stranded DNAs shown as sequences 29 and 30 in the sequence table.
2. A method of identifying the genotype of lilac comprising: carrying out PCR amplification on genome DNA of the clove to be detected by using the primer set as claimed in claim 1 to obtain PCR products, detecting the size of the PCR products of each primer pair, and determining the genotype of the clove to be detected.
3. The method of claim 2, wherein: when PCR amplification is carried out, the annealing temperature of the SO026 is 48 ℃; the annealing temperature of the SO029 is 48 ℃; the annealing temperature of the SO035 is 50 ℃; the annealing temperature of the SO040 is 50 ℃; the annealing temperature of the SO046 is 50 ℃; the annealing temperature of the SO100 is 50 ℃; the annealing temperature of the SO176 is 52 ℃; the annealing temperature of the SO178 is 52 ℃; the annealing temperature of the SO194 is 52 ℃; the annealing temperature of the SO197 is 52 ℃; the annealing temperature of the SO222 is 52 ℃; the annealing temperature of the SO244 is 52 ℃; the annealing temperature of the SO403 is 52 ℃; the annealing temperature of the SO424 is 54 ℃; the annealing temperature of the SO432 is 54 ℃; the annealing temperature of the SO448 is 54 ℃; the annealing temperature of the SO464 is 54 ℃; the annealing temperature of the SO509 is 54 ℃; the annealing temperature of the SO518 is 54 ℃; the annealing temperature of the SO532 is 54 ℃; the annealing temperature of the SO872 is 56 ℃; the annealing temperature of the SO922 is 60 ℃; the annealing temperature of the SO033 is 48 ℃; the annealing temperature of the SO101 is 50 ℃; the annealing temperature of the SO329 is 52 ℃.
4. Use of the primer set of claim 1, the method of claim 2 or 3 for identifying an allelic polymorphism or variant of clove or the ecotype of clove.
5. Use of the primer set of claim 1, the method of claim 2 or 3 for identifying the same or related genotype in a population of lilac.
6. The primer set of claim 1, the method of claim 2 or 3, and the use of the primer set of claim 1 or the method of claim 3 for studying genetic diversity of clove populations.
7. The primer group of claim 1 and the method of claim 2 or 3 are applied to the evaluation of genetic diversity of clove germplasm resources.
8. Use of the primer set of claim 1, the method of claim 2 or 3 for the classification and/or identification of lilac germplasm resources.
9. Use of the primer set of claim 1, the method of claim 2 or 3 for early stage identification of clove hybrids.
10. The primer group of claim 1 and the method of claim 2 or 3 are applied to the construction of the clove genetic linkage map.
11. The primer set of claim 1 and the method of claim 2 or 3 are applied to clove molecular marker-assisted breeding.
12. The method for distinguishing whether two types of clove to be detected are in the same ecotype comprises the following steps: the primer group of claim 1 is used for carrying out PCR amplification on genomic DNA of two types of clove to be detected to obtain PCR products, the sizes of the PCR products of the primer pairs of the two types of clove to be detected are compared, the two types of clove to be detected with the same size of the PCR amplification products of each pair of primers in the primer group are the same ecotype or candidates of the two types of clove to be detected with the same size, and the two types of clove to be detected with different sizes of the PCR amplification products of at least one pair of primers in the primer group are different ecotypes or candidates of the two types of clove to be detected with different sizes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910345948.3A CN109988863B (en) | 2019-04-26 | 2019-04-26 | EST-SSR (expressed sequence tag-simple sequence repeat) marker for distinguishing different ecotypes of clove and primers used by EST-SSR marker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910345948.3A CN109988863B (en) | 2019-04-26 | 2019-04-26 | EST-SSR (expressed sequence tag-simple sequence repeat) marker for distinguishing different ecotypes of clove and primers used by EST-SSR marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109988863A CN109988863A (en) | 2019-07-09 |
| CN109988863B true CN109988863B (en) | 2022-06-03 |
Family
ID=67135285
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910345948.3A Active CN109988863B (en) | 2019-04-26 | 2019-04-26 | EST-SSR (expressed sequence tag-simple sequence repeat) marker for distinguishing different ecotypes of clove and primers used by EST-SSR marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109988863B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652411A (en) * | 2018-12-28 | 2019-04-19 | 山东省林业科学研究院 | Fluorescent SSR primer combination and application thereof in construction of molecular fingerprint of new variety of white wax |
-
2019
- 2019-04-26 CN CN201910345948.3A patent/CN109988863B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652411A (en) * | 2018-12-28 | 2019-04-19 | 山东省林业科学研究院 | Fluorescent SSR primer combination and application thereof in construction of molecular fingerprint of new variety of white wax |
Non-Patent Citations (4)
| Title |
|---|
| characterization of Nuclear Microsatellite Markers for the Narrow Endemic Syringa josikaea Jacq. fil. ex Rchb;LENDVAY, B.等;《Notulae Botanicae Horti Agrobotanici Cluj-Napoca》;20130528;第41卷(第1期);第301-305页 * |
| Development of EST-SSR markers and association mapping with floral traits in Syringa oblata;Yunyao Yang等;《BMC Plant Biol》;20200921;第20卷(第1期);第1-13页 * |
| Microsatellite markers for common lilac (Syringa vulgaris L.);Inka Juntheikki-Palovaara等;《Plant Genetic Resources》;20131130;第11卷(第3期);摘要，第280页右栏第4段至第281页右栏第1段，表1，图1 * |
| 暴马丁香ISSR-PCR反应体系优化及引物筛选;杨晓霞等;《分子植物育种》;20140803;第12卷(第5期);第1018-1026页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109988863A (en) | 2019-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108779459B (en) | Cotton whole genome SNP chip and application thereof | |
| CN108715902B (en) | Plum blossom pendulous branch character SNP molecular marker and application thereof | |
| CN105219880B (en) | OncidiumLuridum belongs to EST-SSR labeled primers and its application | |
| CN108220402B (en) | Method for identifying pedigree relationship between Chinese cabbage germplasm and variety | |
| CN109988863B (en) | EST-SSR (expressed sequence tag-simple sequence repeat) marker for distinguishing different ecotypes of clove and primers used by EST-SSR marker | |
| CN113637789A (en) | Wheat stripe rust resistant gene YRTD121 linked KASP molecular marker, primer, kit and application | |
| CN110541041B (en) | SNP marker related to Chinese domestic horse dwarf trait and application thereof | |
| CN110029186B (en) | Peony SSR marker, primer pair thereof and construction of DNA molecular identity card | |
| CN109706231B (en) | High-throughput SNP (single nucleotide polymorphism) typing method for molecular breeding of litopenaeus vannamei | |
| CN110283929B (en) | SNP (single nucleotide polymorphism) marker 5-160 related to pepper phytophthora blight resistance gene as well as specific primer and application thereof | |
| WO2023207233A1 (en) | Neutral snapshot marker of peas and use thereof in population genetic diversity analysis | |
| CN108624710B (en) | SSR (simple sequence repeat) marker related to cucumber fruit length traits and application thereof | |
| CN115807122B (en) | SNP molecular marker for pineapple seed resource identification and application thereof | |
| CN113789407B (en) | SNP molecular marker combination for cyperus esculentus genotyping and application thereof | |
| CN113897457B (en) | KASP molecular marker linked with wheat stripe rust resistance QTL and application | |
| CN111793706B (en) | Cowpea InDel molecular marker detection primer group and kit | |
| CN108504771A (en) | A method of exploitation sugarcane SSR marker and identification Sugarcane Breeding affiliation | |
| CN116144816A (en) | KASP molecular marker for identifying orange peel color in seedling stage and application thereof | |
| CN108841983A (en) | A kind of SSR primer of sugarcane overall length transcript profile data large-scale development | |
| CN108517373A (en) | It one InDel labeled primer pair for distinguishing five pepper cultivation kinds and its applies | |
| CN104789650B (en) | Molecular detection method for brassica cultivar cytoplasm | |
| CN108165657B (en) | SSR (simple sequence repeat) marker related to cucumber fruit length traits and application thereof | |
| CN106676176A (en) | Method for performing SSR analysis on tetraploid alfalfa by utilizing multiple PCR | |
| KR20180055075A (en) | Single nucleotide polymorphism marker for selecting bacterial wilt-resistant or sensitive pepper cultivar and uses thereof | |
| KR101357497B1 (en) | EST-SSR primer derived from Ophiopogon japonicus and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |