CN113981128A - EST-SSR marker developed based on dendrobium officinale transcriptome sequence and application - Google Patents
EST-SSR marker developed based on dendrobium officinale transcriptome sequence and application Download PDFInfo
- Publication number
- CN113981128A CN113981128A CN202111383204.4A CN202111383204A CN113981128A CN 113981128 A CN113981128 A CN 113981128A CN 202111383204 A CN202111383204 A CN 202111383204A CN 113981128 A CN113981128 A CN 113981128A
- Authority
- CN
- China
- Prior art keywords
- dendrobium
- est
- ssr
- primers
- marker
- 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.)
- Granted
Links
Images
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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of development and application of molecular marker technology, and particularly relates to an EST-SSR marker developed based on a dendrobium autumn transcriptome sequence and application thereof, wherein the microsatellite marker is numbered as follows: den2, Den7, Den12, Den17, Den18, Den19, Den24, Den27, Den38, Den46, Den47, Den51, Den57, Den60, Den63, Den72, Den75, Den82, Den94, and Den 95; the nucleotide sequences are respectively shown in SEQ ID NO. 1-SEQ ID NO. 20. The invention mainly utilizes the dendrobium nobile transcriptome data to carry out SSR marker development to obtain SSR loci with higher frequency, determines 20 pairs of primers which have the advantages of stable amplification, clear electrophoretic bands, rich polymorphism and the like, and can be effectively used in the research fields of the genetic diversity analysis of dendrobium nobile and dendrobium germplasm resources, the construction of high-density maps, the identification of variety purity and authenticity, molecular marker-assisted breeding and the like.
Description
Technical Field
The invention belongs to the field of molecular markers, and particularly relates to an EST-SSR marker developed by a dendrobium autumn transcriptome sequence, a primer and application thereof.
Background
The dendrobium nobile (Dendrobium hybrid) is a kind of excellent dendrobium nobile variety which is bred by hybridization with evergreen dendrobium nobile originally produced in tropical regions as parent, is one of the most ornamental populations in dendrobium, is popular with people due to beautiful flower appearance, rich and gorgeous color and longer flowering phase and vase period, and is an important cut flower and pot flower in ornamental orchid. In recent years, the method is deeply loved by consumers, becomes a new orchid variety with development potential emerging in the orchid market, and has wide market prospect. At present, the research on dendrobium officinale mainly focuses on the aspects of tissue culture technology, cultivation technology, flowering phase regulation and the like, and the research on the aspects of dendrobium officinale genetic diversity, genetic map construction, gene localization and the like is still few. The molecular markers used for the above research in dendrobium officinale are only universal primers such as RAPD, SRAP and the like, which greatly limits the molecular breeding process of dendrobium officinale.
An expressed sequence tag microsatellite (EST-SSR) is a novel molecular marker which is designed based on the simple sequence repetition of an expressed sequence tag, has the advantages of high polymorphism, codominant inheritance, good repeatability, low development cost and the like, is derived from a coding region of a genome, is closely linked with a functional gene, and is easier to obtain the information of gene expression. With the continuous enrichment of EST databases and the development of high-throughput sequencing technology, EST-SSR is developed and applied in plants in large quantities, but the application research of the EST-SSR in dendrobium autumn is not reported yet.
Therefore, the establishment of a method for screening EST-SSR primers based on the inflorescence transcriptome of dendrobium nobile, screening hybrid orchid EST-SSR primers, and analyzing the genetic diversity of germplasm resources of dendrobium nobile plants, constructing genetic maps and performing marker-assisted breeding is a problem to be solved by technical personnel in the field.
Disclosure of Invention
The invention aims to provide an EST-SSR marker developed based on a dendrobium autumn transcriptome sequence, wherein 20 pairs of EST-SSR marker primers come from the dendrobium autumn flower bud transcriptome sequence, the primers are rich in polymorphism, stable in amplification and easy to identify amplified bands, and are new markers which exist stably, and the blank of the dendrobium autumn for developing the SSR primers based on the transcriptome is filled.
In order to achieve the purpose, the technical scheme of the invention is as follows: provides an EST-SSR marker developed based on a dendrobium autumn transcriptome sequence, and the microsatellite marker is numbered as follows: den2, Den7, Den12, Den17, Den18, Den19, Den24, Den27, Den38, Den46, Den47, Den51, Den57, Den60, Den63, Den72, Den75, Den82, Den94, and Den 95; the nucleotide sequences are respectively shown in SEQ ID NO. 1-SEQ ID NO. 20.
In the early stage, the dendrobium autumn flower bud transcriptome sequencing is carried out, and a large amount of dendrobium autumn flower development regulating genes are obtained. In the obtained transcriptome sequence, adopting Microsatelite (MISA) software to identify and position SSR sites, designing SSR primers by Primer Premier 5.0 software, wherein the repeating units of the Primer design selection sites are dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, the minimum repeating times are 9 times, 7 times, 6 times, 5 times and 4 times respectively, the sequences of the sites which are more than 50bp away from the two ends of the sequence utilize the designed primers, the length of the primers is controlled to be 17-24bp, the length of the expected products is 100-300bp, and finally 100 pairs of primers are screened out. Taking DNA of 8 varieties of dendrobium officinale as a template, carrying out PCR amplification by using the 100 pairs of SSR primers, wherein the PCR reaction system is 20 mu L, and the PCR reaction system comprises: 40ng of DNA template, 1 XTaq buffer, 0.4. mu.L of dNTPs (10mM), 0.15mM/L Mg2+1.0. mu.L of upstream and downstream primers, 1.0. mu.L of Taq DNA polymerase (1U, Fermentas), reaction program 95 ℃ pre-denaturation for 3min, followed by 36 cycles, each cycle comprising: denaturation at 94 ℃ for 45s, renaturation at 60 ℃ for 30s, extension at 72 ℃ for 90s and final extension for 10 min. The PCR product was subjected to 2.5% agarose gel electrophoresis initial screening, and then verified by a full-automatic capillary electrophoresis apparatus QIAxcel Advanced (QIAgen, Germany), and finally 20 pairs of SSR labeled primers with clear bands, high polymorphism and good repeatability were obtained, and the primers are shown in Table 1.
TABLE 1 sequence Listing of EST-SSR primers for Dendrobium officinale
The invention also provides application of the dendrobium autumn EST-SSR primer based on the transcriptome in the fields of dendrobium autumn variety identification, genetic pedigree analysis, genetic map construction, germplasm resource protection and assisted breeding: the genetic relationship analysis is carried out on 42 dendrobium nobile varieties by using 20 pairs of SSR primers, the method has the characteristics of good repeatability, clear spectral bands, high polymorphism and the like, and can be widely applied to dendrobium nobile variety identification, high-density genetic linkage map construction and diversity analysis.
The invention also provides application of the dendrobium autumn EST-SSR primer based on the transcriptome in the fields of dendrobium plant species identification, genetic pedigree analysis, germplasm resource protection and assisted breeding: the 20 pairs of SSR primers are utilized to carry out genetic relationship analysis on 40 species of dendrobe, the 20 pairs of primers have good migration capacity in 40 original dendrobe species, and the marker provided by the invention can be applied to the researches such as genetic diversity analysis research, system evolution research, genetic linkage map structure, target character key gene positioning, molecular marker assisted breeding and the like of dendrobe plants.
Drawings
FIG. 1 is a graph of clustering analysis of 42 varieties of Dendrobium loddigesii Rolfe by using 20 pairs of primers.
FIG. 2 is a graph of cluster analysis of 40 dendrobe protospecies resources using 20 primer pairs.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 development of EST-SSR primers for Dendrobium loddigesii Rolfe with high polymorphism
The invention provides a transcriptome-based high-throughput sequencing method, and a bioinformatics method is combined to carry out SSR sequence search and SSR marker primer design and verification, and the specific implementation mode is as follows:
screening Unigene sequences rich in SSR sites: selecting dendrobium nobile flower buds to extract total RNA, constructing a transcriptome sequencing library, and performing high-throughput sequencing by adopting an Illumina four-generation sequencer. And (3) strictly filtering sequencing data, and then assembling to obtain Unigene, wherein the Unigene is used as background data for subsequent SSR primer development. Adopting MISA software to search the Unigene SSR locus, wherein the search criteria are as follows: the minimum repetition times of the dinucleotides, the trinucleotides, the tetranucleotides, the pentanucleotides and the hexanucleotides are respectively 9, 7, 6, 5 and 4. 5174 SSR sites were found in 63101 Unigene sequences of the Dendrobium loddigesii transcriptome, and were distributed among 4486 Unigenes with an occurrence frequency of 7.11%. Among them, the Unigene sequence containing 2 and more than 2 SSR sites has 552, and 1 SSR site appears in the average of 11.01 kb. The types of SSR sites detected in the transcriptome include types of one, two, three, four, five and six nucleotide repeat units and mixed SSRs, wherein the sites of the two and three nucleotide repeat units are more, 2960 and 1910 respectively, and the occurrence frequency is 57.21% and 36.92% respectively.
Designing SSR primers by using Primer Premier 5.0 software, wherein the repeating units of sites selected by Primer design are dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, the minimum repeating times are respectively 9 times, 7 times, 6 times, 5 times and 4 times, sequences of the sites which are more than 50bp from two ends of the sequences are utilized to design the primers, the length of the primers is controlled to be 17-24bp, the length of expected products is 100 bp and 300bp, 5289 pairs of primers are finally designed, and 100 pairs of primers are randomly selected for amplification.
Collecting young leaves of 8 varieties of Dendrobium (Dendrobium Tongchai Gold, Dendrobium Burana Charming, Dendrobium Enobi pure 'Splash', Dendrobium Sunny Red, Dendrobium aridian Blue, Dendrobium Coerulea Blue, Dendrobium Bangkok Green, Dendrobium Candy Stripe) by extracting a sample to be detected by using a Tiangen polysaccharide polyphenol plant DNA extraction kit, diluting the obtained DNA by 50 times according to the kit instruction, detecting the concentration by using a spectrophotometer method, detecting the mass by using an electrophoresis method, adjusting the concentration to be 40ng/L as a working solution for standby, and preserving the rest of mother solution at-80 ℃.
Taking DNA of 8 varieties of dendrobium officinale as a template, carrying out PCR amplification by using the 100 pairs of SSR primers, wherein the PCR reaction system is 20 mu L, and the PCR reaction system comprises: 40ng of DNA template, 1 XTaq buffer, 0.4. mu.L of dNTPs (10mM), 0.15mM/L Mg2+1.0. mu.L of upstream and downstream primers, 1.0. mu.L of Taq DNA polymerase (1U, Fermentas), reaction program 95 ℃ pre-denaturation for 3min, followed by 36 cycles, each cycle comprising: denaturation at 94 ℃ for 45s, renaturation at 60 ℃ for 30s, extension at 72 ℃ for 90s and final extension for 10 min. The PCR product is primarily screened by 2.5% agarose gel electrophoresis, and then verified by a full-automatic capillary electrophoresis apparatus QIAxcel Advanced (QIAgen, Germany), and finally 20 pairs of SSR marker primers with clear bands, high polymorphism and good repeatability are obtained.
Example 2. 20 markers were applied to analysis of genetic diversity of 42 species of Dendrobium loddigesii Rolfe
(1) Extracting the genome DNA of the dendrobium officinale variety in the table 2, and adjusting the concentration to 40 ng/L.
(2) The above DNA was used as a template, and the primers shown in Table 1 were used for amplification, and the PCR reaction system was 20. mu.L, which included: 40ng of DNA template, 1 XTaq buffer, 0.4. mu.L of dNTPs (10mM), 0.15mM/L Mg2+1.0. mu.L of upstream and downstream primers, 1.0. mu.L of Taq DNA polymerase (1U, Fermentas), reaction program 95 ℃ pre-denaturation for 3min, followed by 36 cycles, each cycle comprising: denaturation at 94 ℃ for 45s, renaturation at 60 ℃ for 30s, extension at 72 ℃ for 90s and final extension for 10 min. PCR products were analyzed by ABI 3730xl DNA analyzer, and amplified bands were analyzed by GeneMarker2.2 software in GeneScanTM500
And (6) carrying out statistics.
(3) And constructing a clustering graph of the obtained strip data through MEGA5.0 to obtain clustering analysis graphs of 42 varieties of dendrobium officinale.
(4) The clustering analysis is shown in FIG. 1, and the results show (Table 2) that 42 varieties can be classified into 5 classes at 0.150. Wherein, the I type contains 33 varieties, the plants are all high, the 33 varieties can be divided into 3 categories, the IA type contains 21 varieties, the 21 varieties have the common characters of high plants, round and flat petals and white flower bottom; the IB group comprises 7 varieties, and the common characters are plant height, slender and flat petals and purplish red flower color; the IC class comprises 5 varieties, and has the common characters of plant height, slender petals and pink flower color. Class II contains 2 varieties, medium plants, petals twisted. Class III contains 5 varieties, the plants are shorter, the petals are slender, and the flower color is pink. The IV group contains 1 variety, has small plant type, more flowers, small flowers and fragrance. The V type contains 1 variety, the plant type is medium, the number of flowers is large, and petals are twisted. The divided 5 major classes all have the same characters and have different characters with other classes, which shows that the 20 pairs of primers can be used for the research on the genetic diversity analysis and genetic relationship of dendrobium officinale variety resources.
TABLE 2 clustering results of the tested varieties and their common traits
[ example 3 ] application of 20 markers to genetic diversity analysis of 40 Dendrobium original species resources
(1) Genomic DNA of 40 dendrobium protoplasts in Table 3 was extracted and the concentration was adjusted to 40 ng/L.
(2) The above DNA was used as a template, and the primers shown in Table 1 were used for amplification, and the PCR reaction system was 20. mu.L, which included: 40ng of DNA template, 1 XTaq buffer, 0.4. mu.L of dNTPs (10mM), 0.15mM/L Mg2+1.0. mu.L of upstream and downstream primers, 1.0. mu.L of Taq DNA polymerase (1U, Fermentas), reaction program 95 ℃ pre-denaturation for 3min, followed by 36 cycles, each cycle comprising: denaturation at 94 ℃ for 45s, renaturation at 60 ℃ for 30s, extension at 72 ℃ for 90s, and finallyAnd then extending for 10 min. PCR products were analyzed by ABI 3730xl DNA analyzer, and amplified bands were analyzed by GeneMarker2.2 software in GeneScanTM500
And (6) carrying out statistics.
(3) And performing clustering graph construction on the obtained strip data through MEGA5.0 to obtain a clustering analysis graph of 40 dendrobium original strains.
(4) The cluster analysis is shown in fig. 2, and the results show (table 3) that 40 original species are classified into 8 major groups according to the source, the I group contains 29 species which are all from tropical and subtropical regions, and the IA group contains 13 species which are all from Yunnan and are all pattern groups; class IB contains 4 species, including 2 lantern groups, 1 model group and one scaly leaf group, all collected from india; IC groups included 8 species, 3 of which were bird foot groups, 2 were model groups, 1 was bird foot group, 1 was sword leaf group, and 1 was cylindrical scale leaf group, all collected from vietnam; the ID classes contain 4 varieties, all of which are pattern groups, collected from Hainan. Class II contains 4 species, including 3 bird foot groups and 1 australian group, all collected from australia. Class III 2 species, all model groups, were collected from guangxi. There were only 1 of IV, V, VI, VII and VIII, collected from Guizhou, Thailand, Malaysia and Indonesia, respectively. The clustering analysis can classify the original species with the same source into one class, which shows that the 20 pairs of primers can be used for dendrobe resource genetic diversity analysis and genetic relationship research.
TABLE 3 clustering results and source of original species
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.
Sequence listing
<110> research institute for tropical crop variety resources of Chinese tropical agricultural academy of sciences
<120> EST-SSR marker developed based on dendrobium officinale transcriptome sequence and application
<160>20
<170> SIPOSequenceListing 1.0
<210>1
<211>135
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>1
AGAGGAGAGGAGTTCGAAGAGACGGGATTACGATGAGAGCGAGAGGAGGAGGAGGAGGAG 60
GAGGAGGCTAATATAGAAGTGCGAGGGTTGGAACTGCTCGTGAGGGATAAGAGGCAGTAG 120
TAACCGTTGAGAACA 135
<210>2
<211>156
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>2
GTAACGCTGGTATTTGCTCTCAGATCAAACTAGAAGAAGAAGAAGAAGAAGAAGAGGTCG 60
CATGGCTGAAACCAAAGCAGAGACTCCATCACTGATATCCTCATCAAGAAAGCTTCCTGA 120
CTTCAAAAAATCAGTGAAGCTCAAATACGTCAAGCT 156
<210>3
<211>157
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>3
GTACCCGCCTTATAATGGTCTCTTCGAACCGAATTAGGCCAAAATATCAGCTCTGTTGGA 60
ATCACCAGACCATCGTAATCATCATCATCATCATCATCAAGATCTTGAATACAGATCCGT 120
CCAGAACTATATCTTTGGGTACTTAGCATCCTCACTC 157
<210>4
<211>148
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>4
CGGCCAGTGGAACAGTTTATATTATGGGCCTCAAATGTACGGCGGCGGCGGCGGCGGCGG 60
CTACGGGTTTTCCATTCCACCTCATGATCCAATGATGTATGCCGCAGCTTATGGCGCTTA 120
TCCCATGTATGGAAATCAACAGCAAGTG 148
<210>5
<211>147
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>5
GTTGATTACTCGCCAAATAGCTCGGCCGTTGCCTCGGAGGAGGAGGAGGAGGAGGACTTC 60
AGACAAGTGCCGTTTGAAAACAATGAGGTGGATGACGAGGCTGAAGAGTTCATTAGGAGG 120
TTTTATGAACAGCTACGAAGACAAAGC 147
<210>6
<211>158
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>6
TTTTTATTTTGGGGAATGACAACTAATTGTAATTCAAAATTTTAATAAGCGGCAATTGTC 60
TCAATCTTAGGAGTTTTTCACATCAAAATCTTATGTTTTCAACTTTTGATATATATATAT 120
ATATATATATAGTGTGCTCGCGCAATAGAAAGCTAACT 158
<210>7
<211>157
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>7
TTGATAATAGACCTAGCCAAGCAGAAGAAGAAGAAGAAGAAGAAAGTAGTGTTTACGTGA 60
ACGAGAAGACCGGAGAAATCAGTGGTCCAAGGGGGCCTGAACCAACACGTTACGGGGATT 120
GGGAGCGGAACGGTCGGTGCTACGACTTTTGATTAAT 157
<210>8
<211>151
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>8
AGGGAATAGAGACGTGAGCTTCTGGAGGAGCATCGGATTCAGGACTGTGAGGAGGCTGCT 60
GCTGCTGCTGCTGCTGCTGCTGCTGCTGCGTTGTGAATATTCGGTGTGGGGTGTTTGTTT 120
CCTGATCTTCTTCATTGATCTGCGAATCTTC 151
<210>9
<211>145
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>9
GGAATTTTTGTCATGCAGCTTATGTTTTATAACCCTAACCTATATATATATATATATATA 60
TTACAATCAAGATCTTTTATGAAACATAATGGCAAATAATCCTTGTGTATTAACATGACC 120
AAGAGTTATGCTCCTCGTCTTCAAA 145
<210>10
<211>154
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>10
AGCTAAAAGTGCACAGAAGACCATGGGAGGAGGAGGAGGAGGAGGAGAGAACGATAGCCG 60
ATGGCCGCCATGGCTACGGCCTCTGCTAGCGACGAGCTTCTTCGTTCAATGCAAGCACCA 120
CGCTGACGCTCACAAGAGCGAGTGCAACATGTAT 154
<210>11
<211>125
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>11
AGTGGAAAACTTTCCTTCTCCCTCTCCCTCTCCCTCTTCTCTCTCTCTCTCTCTCTCTAT 60
TATCTATAGCTTGCGTCGCAACGGTTTGGTGCTCCGACGACGGATGAGAGAAGATGGGAA 120
GATCC 125
<210>12
<211>128
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>12
CCAACGGCATTGTTGTGATGGAGCTGCCTCGACAGAGGAGGCAGCGGACGGAGCTCAGGC 60
GAGCCCTCGGCTGAGCCGCCGTTGGAGGAGGAGGAGGAGGAGGAGGTAGAAGAGCGATAG 120
TTGGGAGG 128
<210>13
<211>157
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>13
TTGTAGGAGGTATTGGACTGAGGGTGGATCCTTAAGAAATGTGCCAGTTGGTGGCGGTTC 60
AAGGAAGAATAAGAGATCTTCTTCTTCTTCTTCTTCTTTGGCTAATAACTCTTCTTCAAA 120
CACTACTACAACCATAGCATCATTGGTTTCTTCGTCT 157
<210>14
<211>132
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>14
CTTAAAACCGGCATCGCTATATTCTCCAGTAACGTCTCTATTAACGGGATCTCTAAGATT 60
GCCTCCACCTTCCTAACGCTTCGGAAGGAGGAGGAGGAGGAGGAGGAGCCGGAGCAAATA 120
GATGAGAAAGAG 132
<210>15
<211>131
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>15
TCTTTCTCTTTCATCGCTAGTGCTTCTTCTTCTTCTTCTTCTTCCTCTTCGACGAGCCTT 60
TCCACCGCCGGATTCGACGAAACCGCAGTGGATGCTCTTGGGAATGTCCAGGTTCTTACG 120
TCCTCTACTGG 131
<210>16
<211>150
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>16
GCTCATGTTGTTCCATTCCTGCATCATCATCATCATCATCATCAAGCATAGCAGGAGCAG 60
GTGCTGCAAGAGCTGGCGCAATGCCAGTCCGCTCTGATCGTGCCTTTTGAGCAAGAGCCC 120
GAAGCTCCTGCTCTTTCCTCTCTTTTTCCT 150
<210>17
<211>144
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>17
GGGAAACTCTGTGGATTGGAAGCGGCGGCGGCGGCGGCGGCGGCGTGCGGCAGCAACGGA 60
TAGCGGCGGCTGCGGCGGCGGCCGTACGATTGTTCCATATTTTGGTTTTTTTAATGCTTT 120
TGTCCTTGCGATTAACAGACTT 144
<210>18
<211>126
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>18
TTCCCATCATCAAAATGTCTAGCATATACAAGAATATATGTATATATATATATATATATA 60
TAATCATAAAAAAGCCTTTCTCTTTAACTTTCTCATTCAATCACATCTTCTCAAGCATCA 120
TTCTCC 126
<210>19
<211>145
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>19
TCCTTCACACGCAAGGATAAGTACATTATGCTTGCCGGCAATGATGGCCGCGTCACCTCC 60
TCCTCCTCCTCCTCCTTCAATAAGCCTGCTGCAGGTGACATGGTCGGCGAAGGCTCCGAC 120
GTCGTCCACAGTTAACTCAGTCATG 145
<210>20
<211>144
<212>DNA
<213> Dendrobium loddigesii (Dendrobium hybrid)
<400>20
AATTCGTAAAGCACCAAGTGAAAAATCCTTCATCCTTGAAAATAATATCGAGAGAGAGAG 60
AGAGAGAGAGAGAGATCCTAATTTAAAATGACAAAATAATTCAGCACTACTTAGTCTCTT 120
GATCTAGGATTTGCATGAAGGT 144
Claims (3)
1. EST-SSR marker developed based on dendrobium officinale transcriptome sequence is characterized in that: the microsatellite marker numbers are: den2, Den7, Den12, Den17, Den18, Den19, Den24, Den27, Den38, Den46, Den47, Den51, Den57, Den60, Den63, Den72, Den75, Den82, Den94, and Den 95; the nucleotide sequences are respectively shown in SEQ ID NO. 1-SEQ ID NO. 20.
2. The EST-SSR marker developed based on the dendrobium autumn transcriptome sequence according to claim 1, is characterized in that: the primer set comprises 20 pairs of primer sets, namely DenSSR2, DenSSR7, DenSSR12, DenSSR17, DenSSR18, DenSSR19, DenSSR24, DenSSR27, DenSSR38, DenSSR46, DenSSR47, DenSSR51, DenSSR57, DenSSR60, DenSSR63, DenSSR72, DenSSR75, DenSSR82, DenSSR94 and DenSSR95, and the primer sequences are respectively:
DenSSR2 F：AGAGGAGAGGAGTTCGAAGAGAC
DenSSR2 R：TGTTCTCAACGGTTACTACTGCC
DenSSR7 F：GTAACGCTGGTATTTGCTCTCAG
DenSSR7 R：AGCTTGACGTATTTGAGCTTCAC
DenSSR12F：GTACCCGCCTTATAATGGTCTCT
DenSSR12R：GAGTGAGGATGCTAAGTACCCAA
DenSSR17 F：CGGCCAGTGGAACAGTTTATATT
DenSSR17R：CACTTGCTGTTGATTTCCATACA
DenSSR18 F：GTTGATTACTCGCCAAATAGCTC
DenSSR18R：GCTTTGTCTTCGTAGCTGTTCAT
DenSSR19 F：TTTTTATTTTGGGGAATGACAAC
DenSSR19R：AGTTAGCTTTCTATTGCGCGAG
DenSSR24 F：TTGATAATAGACCTAGCCAAGCA
DenSSR24R：ATTAATCAAAAGTCGTAGCACCG
DenSSR27F：AGGGAATAGAGACGTGAGCTTCT
DenSSR27R：GAAGATTCGCAGATCAATGAAGA
DenSSR38 F：GGAATTTTTGTCATGCAGCTTAT
DenSSR38R：TTTGAAGACGAGGAGCATAACTC
DenSSR46 F：AGCTAAAAGTGCACAGAAGACCA
DenSSR46R：ATACATGTTGCACTCGCTCTTGT
DenSSR47 F：AGTGGAAAACTTTCCTTCTCCCT
DenSSR47R：GGATCTTCCCATCTTCTCTCATC
DenSSR51F：CCAACGGCATTGTTGTGAT
DenSSR51R：CCTCCCAACTATCGCTCTTCTAC
DenSSR57F：TTGTAGGAGGTATTGGACTGAGG
DenSSR57R：AGACGAAGAAACCAATGATGCTA
DenSSR60F：CTTAAAACCGGCATCGCTATATT
DenSSR60R：CTCTTTCTCATCTATTTGCTCCG
DenSSR63F：TCTTTCTCTTTCATCGCTAGTGC
DenSSR63R：CCAGTAGAGGACGTAAGAACCTG
DenSSR72F：GCTCATGTTGTTCCATTCCTG
DenSSR72R：AGGAAAAAGAGAGGAAAGAGCAG
DenSSR75F：GGGAAACTCTGTGGATTGGAAG
DenSSR75R：AAGTCTGTTAATCGCAAGGACAA
DenSSR82F：TTCCCATCATCAAAATGTCTAGC
DenSSR82R：GGAGAATGATGCTTGAGAAGATG
DenSSR94F：TCCTTCACACGCAAGGATAAGTA
DenSSR94R：CATGACTGAGTTAACTGTGGACG
DenSSR95F：AATTCGTAAAGCACCAAGTGAAA
DenSSR95R：ACCTTCATGCAAATCCTAGATCA。
3. the use of the primers in EST-SSR marker developed based on a dendrobium devoninum pauciflorum transcriptome sequence according to claim 2 in construction of a dendrobium devoninum pauciflorum genetic map, identification of dendrobium devoninum pauciflorum germplasm, breeding of dendrobium devoninum pauciflorum, analysis of dendrobium genetic diversity, analysis of dendrobium genetic relationship, molecular marker assisted breeding of dendrobium devoninum, analysis of dendrobium genetic diversity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111383204.4A CN113981128B (en) | 2021-11-22 | 2021-11-22 | EST-SSR marker developed based on autumn dendrobium transcriptome sequence and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111383204.4A CN113981128B (en) | 2021-11-22 | 2021-11-22 | EST-SSR marker developed based on autumn dendrobium transcriptome sequence and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113981128A true CN113981128A (en) | 2022-01-28 |
| CN113981128B CN113981128B (en) | 2023-10-20 |
Family
ID=79749613
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111383204.4A Active CN113981128B (en) | 2021-11-22 | 2021-11-22 | EST-SSR marker developed based on autumn dendrobium transcriptome sequence and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113981128B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102649955A (en) * | 2012-04-06 | 2012-08-29 | 安徽师范大学 | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker |
| AU2020103706A4 (en) * | 2020-01-14 | 2021-02-04 | Sichuan Agricultural University | Ssr molecular marker primer related to walnut black spot disease and application thereof |
| US20210040552A1 (en) * | 2019-07-12 | 2021-02-11 | Institute of Horticulture Research, Anhui Academy of Agricultural Science | Development of simple sequence repeat (ssr) core primer group based on whole genome sequence of pomegranate and application thereof |
| CN112725521A (en) * | 2021-03-02 | 2021-04-30 | 广东省农业科学院环境园艺研究所 | Dendrobium chrysotoxum SSR molecular marker primer composition and application thereof |
-
2021
- 2021-11-22 CN CN202111383204.4A patent/CN113981128B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102649955A (en) * | 2012-04-06 | 2012-08-29 | 安徽师范大学 | Dendrobium huoshanense microsatellite deoxyribonucleic acid (DNA) molecular marker |
| US20210040552A1 (en) * | 2019-07-12 | 2021-02-11 | Institute of Horticulture Research, Anhui Academy of Agricultural Science | Development of simple sequence repeat (ssr) core primer group based on whole genome sequence of pomegranate and application thereof |
| AU2020103706A4 (en) * | 2020-01-14 | 2021-02-04 | Sichuan Agricultural University | Ssr molecular marker primer related to walnut black spot disease and application thereof |
| CN112725521A (en) * | 2021-03-02 | 2021-04-30 | 广东省农业科学院环境园艺研究所 | Dendrobium chrysotoxum SSR molecular marker primer composition and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113981128B (en) | 2023-10-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108192990B (en) | SNP molecular marker related to watermelon peel background color and application thereof | |
| CN113430300B (en) | SSR molecular marker of mulberry variety Yuehen 123, core primer group and kit thereof, and application of SSR molecular marker | |
| CN113637794B (en) | SSR molecular marker of new variety of mulberry, namely Guangdong mulberry 201, and core primer group, kit and application thereof | |
| CN102703586B (en) | Method for constructing Prunus mume Sieb.et Zucc SSR (simple sequence repeat) genetic map | |
| CN113637789A (en) | Wheat stripe rust resistant gene YRTD121 linked KASP molecular marker, primer, kit and application | |
| CN113151532A (en) | Molecular marker Indel-LNT-36 closely linked with cucumber fruit multi-ventricular trait | |
| CN114182032B (en) | SNP molecular marker for detecting muskmelon seed coat color and application thereof | |
| CN113151553B (en) | Molecular marker coseparated with watermelon plant few lateral branch gene Clbl and application | |
| CN110093346A (en) | With the molecular marker and application thereof of purple tsai-tai a kind of sedge color gene linkage | |
| CN109266778B (en) | EST-SSR labeled primer developed based on hybrid blue transcriptome and application | |
| CN108642207A (en) | A kind of detection method for quick and precisely identifying cowberry platymiscium | |
| CN107034293B (en) | Sorbus alnifloria tree EST-SSR marks, its primer pair and application | |
| CN113278723B (en) | Composition for analyzing genetic diversity of Chinese cabbage genome segment or genetic diversity introduced in synthetic mustard and application | |
| CN113981128B (en) | EST-SSR marker developed based on autumn dendrobium transcriptome sequence and application thereof | |
| CN113151543B (en) | Primer group, method and kit for rapidly identifying calla seedling types by using SSR markers and application of primer group, method and kit | |
| CN109988863A (en) | EST-SSR for distinguishing different ecological type cloves is marked and the primer | |
| CN113462811B (en) | Fructus amomi SSR molecular marker primer group and application thereof | |
| CN108531636A (en) | A kind of molecular marked compound TJcM01 and its application for identifying muskmelon unisexual flower | |
| CN108588261A (en) | A kind of identification is located at InDel primers and its application of the late bolting QTL on radish R02 chromosomes | |
| CN108239675A (en) | A kind of molecular marked compound TJcM02 and its application for being used to identify muskmelon unisexual flower | |
| CN110643728B (en) | Method for improving breeding efficiency of poplar crossbreeding | |
| CN109628636B (en) | SSR molecular marker for identifying hybrid of Xinjiang grape and Kyoho grape and application thereof | |
| CN111926099A (en) | SSR molecular markers based on camellia transcriptome and application of SSR molecular markers in camellia plants | |
| CN108330164B (en) | Characteristic sequence, primer and identification method of apocarya variety Moore | |
| CN114634991B (en) | InDel marker for identifying high-variety coconuts and application 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 |