CN113981128B - EST-SSR marker developed based on autumn dendrobium transcriptome sequence and application thereof - Google Patents
EST-SSR marker developed based on autumn dendrobium transcriptome sequence and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular marker technology development and application, and in particular relates to an EST-SSR marker developed based on an autumn dendrobium transcriptome sequence and application thereof, wherein the microsatellite marker number is as follows: den2, den7, den12, den17, den18, den19, den24, den27, den38, den46, den47, den51, den57, den60, den63, den72, den75, den82, den94 and Den95; the nucleotide sequences are respectively shown as SEQ ID NO. 1-SEQ ID NO. 20. The invention mainly utilizes the autumn dendrobium transcriptome data to carry out SSR marker development to obtain SSR sites with higher frequency, determines 20 pairs of primers, has the advantages of stable amplification, clear electrophoresis strip, rich polymorphism and the like, and can be effectively used in the research fields of genetic diversity analysis of autumn dendrobium and even dendrobium germplasm resources, construction of high-density maps, 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 an autumn dendrobium transcriptome sequence, a primer and application thereof.
Background
The dendrobium nobile (Dendrobium hybrid) is a great class of good dendrobium nobile varieties which are bred by hybridization by taking evergreen dendrobium nobile native to tropical areas as a parent, is one of the most ornamental populations in dendrobium nobile, is loved by people because of beautiful flower appearance, rich and gorgeous colors, long flowering period and bottle inserting period, and is an important cut flower and potted flower in ornamental orchids. In recent years, the orchid is deeply favored by consumers, and becomes a new orchid type with development potential in the orchid market, and has wide market prospect. At present, research on Guan Qiu dendrobium is mainly focused on aspects of tissue culture technology, cultivation technology, flowering phase regulation and the like, and little research on aspects of autumn dendrobium genetic diversity, genetic map construction, gene positioning and the like is performed. The molecular markers used for the research are only some general primers such as RAPD, SRAP and the like in the dendrobium candidum, which greatly limits the progress of dendrobium candidum molecular breeding.
The expressed sequence tag microsatellite (EST-SSR) is a novel molecular marker based on simple sequence repetitive design of the expressed sequence tag, has the advantages of high polymorphism, co-dominant inheritance, good repeatability, low development cost and the like, and is derived from a coding region of a genome, closely linked with a functional gene and easy to obtain gene expression information. Along with the continuous enrichment of EST databases and the development of high-throughput sequencing technology, EST-SSR is greatly developed and applied on plants, but the application research of EST-SSR in dendrobium nobile has not been reported yet.
Therefore, establishing a method for screening EST-SSR primers based on the autumn dendrobium inflorescence transcriptome, screening out the hybrid orchid EST-SSR primers, and analyzing genetic diversity of germplasm resources of the autumn dendrobium plant, constructing a genetic map and marking auxiliary breeding is a problem to be solved by the person skilled in the art.
Disclosure of Invention
The invention aims to provide an EST-SSR marker developed based on a autumn dendrobium transcriptome sequence, 20 pairs of EST-SSR marker primers are derived from the autumn dendrobium flower bud transcriptome sequence, the primers are rich in polymorphism, stable in amplification and easy to identify amplified bands, and the EST-SSR marker is a new marker which exists stably, so that the blank of developing the SSR primer based on the transcriptome of the autumn dendrobium is filled.
In order to achieve the above purpose, the technical scheme of the invention is as follows: the EST-SSR marker developed based on the autumn dendrobium transcriptome sequence is provided, and the microsatellite marker number is: den2, den7, den12, den17, den18, den19, den24, den27, den38, den46, den47, den51, den57, den60, den63, den72, den75, den82, den94 and Den95; the nucleotide sequences are respectively shown as SEQ ID NO. 1-SEQ ID NO. 20.
The transcriptome sequencing of the flower buds of the dendrobium nobile is carried out in the early stage, so that a large number of flower development regulation genes of the dendrobium nobile are obtained. In the obtained transcriptome sequence, microsatellite (MISA) software was used to identify and locate SSR sites,and designing SSR primers by using Primer Premier 5.0 software, wherein the repeat units of the Primer design selection sites are dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, the minimum repeat times are 9 times, 7 times, 6 times, 5 times and 4 times respectively, and the sequences with the site distances of more than 50bp at the two ends of the sequence are designed by using 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. 8 varieties of dendrobium candidum DNA are used as templates, the 100 pairs of SSR primers are used for PCR amplification, and a PCR reaction system is 20 mu L, wherein the PCR reaction system comprises: 40ng of DNA template, 1 XTaq buffer, 0.4. Mu.L of dNTP (10 mM), 0.15mM/L Mg 2+ 1.0. Mu.L of upstream and downstream primer, 1.0. Mu. L of Taq DNA polymerase (1U, fermentas), the reaction procedure was pre-denatured at 95℃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 finally extension for 10min. The PCR products were subjected to preliminary screening by 2.5% agarose gel electrophoresis, and then verified by a full-automatic capillary electrophoresis apparatus QIAxcel Advanced (QIAgen Biotechnology, germany), and 20 pairs of SSR labeled primers with clear bands, high polymorphism and good repeatability were finally obtained, and the primers are shown in Table 1.
TABLE 1 autumn dendrobium EST-SSR primer sequence table
The invention also provides application of the transcriptome-based autumn dendrobium EST-SSR primer in the fields of autumn dendrobium variety identification, genetic pedigree analysis, genetic map construction, germplasm resource protection and auxiliary breeding: the 20 pairs of SSR primers are used for carrying out genetic relationship analysis on 42 dendrobium nobile varieties, and the dendrobium nobile genetic linkage analysis method has the characteristics of good repeatability, clear band, 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 transcriptome-based autumn dendrobium EST-SSR primer in the fields of dendrobium plant species identification, genetic lineage analysis, germplasm resource protection and auxiliary breeding: the 20 pairs of SSR primers are used for carrying out genetic relationship analysis on 40 dendrobium species, and the 20 pairs of primers have good migration capability in 40 dendrobium primary species, so that the marker provided by the invention can be applied to researches such as genetic diversity analysis research, systematic evolution research, genetic linkage map structure, target character key gene positioning, molecular marker assisted breeding and the like of dendrobium plants.
Drawings
FIG. 1 is a graph of a cluster analysis of 42 varieties of dendrobium nobile resources using 20 pairs of primers.
FIG. 2 is a graph of a cluster analysis of 40 dendrobe primordial species resources using 20 pairs of primers.
Detailed Description
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification taken in conjunction with the drawings. The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way whatsoever. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 development of EST-SSR primers for Dendrobium candidum with high polymorphism
The invention provides a transcriptome-based high-throughput sequencing, which combines a bioinformatics method to search SSR sequences and design and verify SSR marker primers, and the specific implementation modes are as follows:
unigene sequence screening of SSR-rich sites: and (3) extracting total RNA from the flower buds of the dendrobium candidum, constructing a transcriptome sequencing library, and carrying out high-throughput sequencing by using an Illumina fourth-generation sequencer. The sequencing data are assembled after being strictly filtered, unigene is obtained, and the Unigene is used as background data for the subsequent development of SSR primers. SSR site searching is carried out on Unigene by adopting MISA software, and the searching standard is as follows: dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, hexanucleotides, with minimum number of repetitions of 9, 7, 6, 5, 4, respectively. 5174 SSR sites were found in the 63101 Unigene sequences of the autumn dendrobium transcriptome, distributed among 4486 Unigenes, and occurred at a frequency of 7.11%. Among them, there are 552 Unigene sequences containing 2 and more than 2 SSR sites, and 1 SSR site appears every 11.01kb on average. The types of SSR sites detected in the transcriptome comprise one, two, three, four, five and six nucleotide repeat unit types and mixed SSR, wherein the sites of the two and three nucleotide repeat units are more, 2960 and 1910 are respectively, and the occurrence frequencies are 57.21% and 36.92% respectively.
The SSR Primer is designed by Primer Premier 5.0 software, the repeat units of the Primer design selection sites are dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide, the minimum repeat times are 9 times, 7 times, 6 times, 5 times and 4 times respectively, the sequence with the site distance of more than 50bp at two ends of the sequence is designed by utilizing the Primer, the length of the Primer is controlled between 17 bp and 24bp, the length of the expected product is 100 bp to 300bp, the 5289 pairs of primers are finally designed, and 100 pairs of primers are randomly selected for amplification.
Collecting young leaves of 8 autumn dendrobium varieties (DendrobiumTongchai Gold, dendrobiumBurana Charming, dendrobiumEnobi Purple 'Splash', dendrobium Sunny Red, dendrobiumAridang Blue, dendrobiumCoerulea Blue, dendrobium Bangkok Green and Dendrobium Candy Stripe), extracting samples to be detected by using a plant DNA extraction kit of the plant polysaccharide and polyphenol, performing operation according to a kit instruction, diluting the obtained DNA by 50 times, detecting the concentration by a spectrophotometry, detecting the quality by an electrophoresis method, adjusting the concentration to 40ng/L as a working solution for standby, and storing the rest mother solution at-80 ℃.
8 varieties of dendrobium candidum DNA are used as templates, the 100 pairs of SSR primers are used for PCR amplification, and a PCR reaction system is 20 mu L, wherein the PCR reaction system comprises: 40ng of DNA template, 1 XTaq buffer, 0.4. Mu.L of dNTP (10 mM), 0.15mM/L Mg 2+ 1.0. Mu.L of upstream and downstream primer, 1.0. Mu. L of Taq DNA polymerase (1U, fermentas), the reaction procedure was pre-denatured at 95℃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 finally extension for 10min. The PCR product is subjected to preliminary screening by 2.5% agarose gel electrophoresis, and then full-automatic capillary electrophoresis is usedAnd (3) verifying by a QIAxcel Advanced instrument (QIAgen biological company, germany), and finally obtaining 20 pairs of SSR labeled primers with clear bands, high polymorphism and good repeatability.
Example 2 20 markers were applied to 42 genetic diversity analysis of dendrobium candidum variety material
(1) Genomic DNA of the autumn dendrobium variety in Table 2 was extracted and the concentration was adjusted to 40ng/L.
(2) The amplification was performed using the above DNA as a template and the primers shown in Table 1, with a PCR reaction system of 20. Mu.L, comprising: 40ng of DNA template, 1 XTaq buffer, 0.4. Mu.L of dNTP (10 mM), 0.15mM/L Mg 2+ 1.0. Mu.L of upstream and downstream primer, 1.0. Mu. L of Taq DNA polymerase (1U, fermentas), the reaction procedure was pre-denatured at 95℃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 finally extension for 10min. PCR products were analyzed by ABI 3730xl DNA analyzer and amplified bands were analyzed by GeneMarker2.2 software in GeneScan TM 500And (5) carrying out statistics.
(3) And constructing a cluster map of the obtained stripe data through MEGA5.0 to obtain a cluster analysis map of 42 autumn dendrobium varieties.
(4) The clustering analysis is shown in FIG. 1, and the results show (Table 2) that 42 varieties can be classified into 5 categories at 0.150. The I type contains 33 varieties, plants are higher, the 33 varieties can be divided into 3 subclasses, the IA type contains 21 varieties, the 21 varieties have common characters of high plants, round and flat petals and white flower ground color; the IB type contains 7 varieties, the common character is that the plants are high, the petals are slender and flat, and the flower color is purple red; the IC contains 5 varieties, the common characters are high plants, slender petals and pink flower colors. Class II contains 2 varieties, medium plants, petal twist. Class III contains 5 varieties, the plants are shorter, the petals are longer and longer, and the flower color is pink. Class IV contains 1 variety, has smaller plant type, more flowers, small flowers and fragrance. Class V contains 1 variety, is medium in plant type, has a large number of flowers and petals twisted. The 5 classes divided have the same characters and different characters from other classes, which indicates that the 20 pairs of primers can be used in the analysis of genetic diversity of autumn dendrobium variety resources and the research of genetic relationship.
TABLE 2 clustering results of test varieties and their consensus traits
Example 3 20 markers were applied to genetic diversity analysis of 40 dendrobe primordial species resources
(1) Genomic DNA of 40 dendrobe species in Table 3 was extracted and the concentration was adjusted to 40ng/L.
(2) The amplification was performed using the above DNA as a template and the primers shown in Table 1, with a PCR reaction system of 20. Mu.L, comprising: 40ng of DNA template, 1 XTaq buffer, 0.4. Mu.L of dNTP (10 mM), 0.15mM/L Mg 2+ 1.0. Mu.L of upstream and downstream primer, 1.0. Mu. L of Taq DNA polymerase (1U, fermentas), the reaction procedure was pre-denatured at 95℃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 finally extension for 10min. PCR products were analyzed by ABI 3730xl DNA analyzer and amplified bands were analyzed by GeneMarker2.2 software in GeneScan TM 500And (5) carrying out statistics.
(3) And constructing a cluster map of the obtained band data through MEGA5.0 to obtain a cluster analysis map of 40 dendrobium species native species.
(4) The clustering analysis is shown in fig. 2, and the results show (table 3) that 40 primary seeds are classified into 8 major categories according to different sources, the category I contains 29 seeds, namely from tropical subtropical regions, wherein the category IA contains 13 seeds, namely from Yunnan, and is a mode group; class IB contains 4 species, including 2 lantern groups, 1 pattern group and one leaf scale group, all collected from india; the IC class comprises 8 kinds, wherein 3 kinds of IC are bird foot groups, 2 kinds of IC are mode groups, 1 kind of IC are bird foot groups, 1 kind of IC are sword-leaf groups, 1 kind of IC are cylindrical scale-leaf groups, and the IC is collected from Vietnam; the ID class contains 4 kinds, all pattern groups, collected from hainan. Class II contains 4 species, including 3 bird feet and 1 australian group, all collected from australia. Class III 2, all pattern groups, were all collected from guangxi. Each of IV, V, VI, VII and VIII had only 1 species, collected from Guizhou, thailand, malaysia and Indonesia, respectively. Cluster analysis can classify the same source of the original species into one class, which indicates that the 20 pairs of primers can be used in dendrobe resource genetic diversity analysis and genetic relationship research.
TABLE 3 clustering results of test primordial species and their origins
The foregoing disclosure is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the claims herein, as equivalent variations of the claims herein will fall within the scope of the invention.
Sequence list
<110> national academy of Tropical agricultural sciences, tropical crop variety institute
<120> EST-SSR marker developed based on autumn dendrobium transcriptome sequence and application thereof
<160>20
<170> SIPOSequenceListing 1.0
<210>1
<211>135
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>1
AGAGGAGAGGAGTTCGAAGAGACGGGATTACGATGAGAGCGAGAGGAGGAGGAGGAGGAG 60
GAGGAGGCTAATATAGAAGTGCGAGGGTTGGAACTGCTCGTGAGGGATAAGAGGCAGTAG 120
TAACCGTTGAGAACA 135
<210>2
<211>156
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>2
GTAACGCTGGTATTTGCTCTCAGATCAAACTAGAAGAAGAAGAAGAAGAAGAAGAGGTCG 60
CATGGCTGAAACCAAAGCAGAGACTCCATCACTGATATCCTCATCAAGAAAGCTTCCTGA 120
CTTCAAAAAATCAGTGAAGCTCAAATACGTCAAGCT 156
<210>3
<211>157
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>3
GTACCCGCCTTATAATGGTCTCTTCGAACCGAATTAGGCCAAAATATCAGCTCTGTTGGA 60
ATCACCAGACCATCGTAATCATCATCATCATCATCATCAAGATCTTGAATACAGATCCGT 120
CCAGAACTATATCTTTGGGTACTTAGCATCCTCACTC 157
<210>4
<211>148
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>4
CGGCCAGTGGAACAGTTTATATTATGGGCCTCAAATGTACGGCGGCGGCGGCGGCGGCGG 60
CTACGGGTTTTCCATTCCACCTCATGATCCAATGATGTATGCCGCAGCTTATGGCGCTTA 120
TCCCATGTATGGAAATCAACAGCAAGTG 148
<210>5
<211>147
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>5
GTTGATTACTCGCCAAATAGCTCGGCCGTTGCCTCGGAGGAGGAGGAGGAGGAGGACTTC 60
AGACAAGTGCCGTTTGAAAACAATGAGGTGGATGACGAGGCTGAAGAGTTCATTAGGAGG 120
TTTTATGAACAGCTACGAAGACAAAGC 147
<210>6
<211>158
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>6
TTTTTATTTTGGGGAATGACAACTAATTGTAATTCAAAATTTTAATAAGCGGCAATTGTC 60
TCAATCTTAGGAGTTTTTCACATCAAAATCTTATGTTTTCAACTTTTGATATATATATAT 120
ATATATATATAGTGTGCTCGCGCAATAGAAAGCTAACT 158
<210>7
<211>157
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>7
TTGATAATAGACCTAGCCAAGCAGAAGAAGAAGAAGAAGAAGAAAGTAGTGTTTACGTGA 60
ACGAGAAGACCGGAGAAATCAGTGGTCCAAGGGGGCCTGAACCAACACGTTACGGGGATT 120
GGGAGCGGAACGGTCGGTGCTACGACTTTTGATTAAT 157
<210>8
<211>151
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>8
AGGGAATAGAGACGTGAGCTTCTGGAGGAGCATCGGATTCAGGACTGTGAGGAGGCTGCT 60
GCTGCTGCTGCTGCTGCTGCTGCTGCTGCGTTGTGAATATTCGGTGTGGGGTGTTTGTTT 120
CCTGATCTTCTTCATTGATCTGCGAATCTTC 151
<210>9
<211>145
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>9
GGAATTTTTGTCATGCAGCTTATGTTTTATAACCCTAACCTATATATATATATATATATA 60
TTACAATCAAGATCTTTTATGAAACATAATGGCAAATAATCCTTGTGTATTAACATGACC 120
AAGAGTTATGCTCCTCGTCTTCAAA 145
<210>10
<211>154
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>10
AGCTAAAAGTGCACAGAAGACCATGGGAGGAGGAGGAGGAGGAGGAGAGAACGATAGCCG 60
ATGGCCGCCATGGCTACGGCCTCTGCTAGCGACGAGCTTCTTCGTTCAATGCAAGCACCA 120
CGCTGACGCTCACAAGAGCGAGTGCAACATGTAT 154
<210>11
<211>125
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>11
AGTGGAAAACTTTCCTTCTCCCTCTCCCTCTCCCTCTTCTCTCTCTCTCTCTCTCTCTAT 60
TATCTATAGCTTGCGTCGCAACGGTTTGGTGCTCCGACGACGGATGAGAGAAGATGGGAA 120
GATCC 125
<210>12
<211>128
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>12
CCAACGGCATTGTTGTGATGGAGCTGCCTCGACAGAGGAGGCAGCGGACGGAGCTCAGGC 60
GAGCCCTCGGCTGAGCCGCCGTTGGAGGAGGAGGAGGAGGAGGAGGTAGAAGAGCGATAG 120
TTGGGAGG 128
<210>13
<211>157
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>13
TTGTAGGAGGTATTGGACTGAGGGTGGATCCTTAAGAAATGTGCCAGTTGGTGGCGGTTC 60
AAGGAAGAATAAGAGATCTTCTTCTTCTTCTTCTTCTTTGGCTAATAACTCTTCTTCAAA 120
CACTACTACAACCATAGCATCATTGGTTTCTTCGTCT 157
<210>14
<211>132
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>14
CTTAAAACCGGCATCGCTATATTCTCCAGTAACGTCTCTATTAACGGGATCTCTAAGATT 60
GCCTCCACCTTCCTAACGCTTCGGAAGGAGGAGGAGGAGGAGGAGGAGCCGGAGCAAATA 120
GATGAGAAAGAG 132
<210>15
<211>131
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>15
TCTTTCTCTTTCATCGCTAGTGCTTCTTCTTCTTCTTCTTCTTCCTCTTCGACGAGCCTT 60
TCCACCGCCGGATTCGACGAAACCGCAGTGGATGCTCTTGGGAATGTCCAGGTTCTTACG 120
TCCTCTACTGG 131
<210>16
<211>150
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>16
GCTCATGTTGTTCCATTCCTGCATCATCATCATCATCATCATCAAGCATAGCAGGAGCAG 60
GTGCTGCAAGAGCTGGCGCAATGCCAGTCCGCTCTGATCGTGCCTTTTGAGCAAGAGCCC 120
GAAGCTCCTGCTCTTTCCTCTCTTTTTCCT 150
<210>17
<211>144
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>17
GGGAAACTCTGTGGATTGGAAGCGGCGGCGGCGGCGGCGGCGGCGTGCGGCAGCAACGGA 60
TAGCGGCGGCTGCGGCGGCGGCCGTACGATTGTTCCATATTTTGGTTTTTTTAATGCTTT 120
TGTCCTTGCGATTAACAGACTT 144
<210>18
<211>126
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>18
TTCCCATCATCAAAATGTCTAGCATATACAAGAATATATGTATATATATATATATATATA 60
TAATCATAAAAAAGCCTTTCTCTTTAACTTTCTCATTCAATCACATCTTCTCAAGCATCA 120
TTCTCC 126
<210>19
<211>145
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>19
TCCTTCACACGCAAGGATAAGTACATTATGCTTGCCGGCAATGATGGCCGCGTCACCTCC 60
TCCTCCTCCTCCTCCTTCAATAAGCCTGCTGCAGGTGACATGGTCGGCGAAGGCTCCGAC 120
GTCGTCCACAGTTAACTCAGTCATG 145
<210>20
<211>144
<212>DNA
<213> autumn dendrobium (Dendrobium hybrid)
<400>20
AATTCGTAAAGCACCAAGTGAAAAATCCTTCATCCTTGAAAATAATATCGAGAGAGAGAG 60
AGAGAGAGAGAGAGATCCTAATTTAAAATGACAAAATAATTCAGCACTACTTAGTCTCTT 120
GATCTAGGATTTGCATGAAGGT 144
Claims (3)
1. EST-SSR markers developed based on autumn dendrobium transcriptome sequences are characterized in that: microsatellite markers are numbered: den2, den7, den12, den17, den18, den19, den24, den27, den38, den46, den47, den51, den57, den60, den63, den72, den75, den82, den94 and Den95; the nucleotide sequences are respectively shown as SEQ ID NO. 1-SEQ ID NO. 20.
2. An EST-SSR marker developed based on an autumn dendrobium transcriptome sequence according to claim 1, wherein: the primer group comprises 20 pairs of primer groups, 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 primers in EST-SSR markers developed based on autumn dendrobe transcriptome sequences according to claim 2 in construction of a autumn dendrobe genetic map, autumn dendrobe germplasm identification, autumn dendrobe breeding, autumn dendrobe genetic diversity analysis, autumn dendrobe genetic relationship analysis, autumn dendrobe molecular marker assisted breeding, dendrobe genetic relationship analysis and dendrobe genetic diversity analysis.
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| 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 |
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| 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 |
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