CN113684296A - Sealwort EST-SSR primer group development and application thereof in genetic diversity - Google Patents
Sealwort EST-SSR primer group development and application thereof in genetic diversity Download PDFInfo
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Abstract
The invention discloses the development of a polygonatum rhizome EST-SSR primer group and application thereof in aspects of genetic diversity and the like, wherein 10 polygonatum rhizome germplasm resources are clustered into 3 groups according to sequence sources, EST-SSR site search, EST-SSR primer design, SSR primer screening, non-denaturing polyacrylamide gel electrophoresis, PCR amplification and data processing, and EST-SSR result analysis, wherein the group I comprises polygonatum cyrtonema from Anhui Chizhou, Guangxi Hejiang province, Zhejiang Xiju and Hunan Lou bottom, the group II comprises Jiangxi Xinfeng, Helingbao, Sichuan Yaan and Shaanxi Ningqiang, and the group III comprises polygonatum cyrtonema from Henan Lushan and Guizhou Dejiang. The EST-SSR marker can provide a valuable candidate marker for genetic diversity analysis and genetic map construction of polygonatum sibiricum, and also provides a technical means for molecular identification of interspecific species of polygonatum sibiricum and molecular assisted breeding of excellent varieties.
Description
Technical Field
The invention relates to the development of a sealwort EST-SSR primer group and application thereof in aspects of genetic diversity and the like, belonging to the technical field of molecular biology.
Background
Polygonatum sibiricum (Liliaceae) Polygonatum of Liliaceae is perennial herbaceous plant, and the genus plant is widely distributed in the world, and 31 species of China exist, wherein Polygonatum kingianum, Polygonatum sibiricum and Polygonatum cyrtonema are recorded in the Chinese pharmacopoeia (2020 edition). Modern pharmacological studies show that the sealwort has the medicinal values of resisting aging, regulating immunity, regulating blood fat, improving memory, resisting tumor, resisting bacteria and the like. The polygonatum plant resources in China are very rich, and in recent years, due to vigorous market demands, the polygonatum plant resources are excessively mined, so that the resources in a production area are rapidly reduced, even are nearly exhausted, and meanwhile, the living environment of the polygonatum is seriously damaged. Therefore, resource collection and protection are needed, artificial domestication and cultivation and genetic diversity research are carried out, a new way is provided for solving the problem of the deficiency of polygonatum plant resources, and effective references are provided for improved variety breeding, new germplasm innovation, interspecies identification and protection strategies.
The genetic diversity of the germplasm resources is the basis for researching the origin evolution of species, discovering new genes and improving the existing varieties. With the rapid development of high-throughput sequencing technology and the continuous reduction of sequencing cost, EST sequences obtained by transcriptome sequencing (RNA-Sep) are utilized, and valuable simple repetitive sequence sites are further explored for developing molecular markers. At present, the technology is applied to medicinal plants such as ginseng, liquorice, petunia and the like. The simple repeat sequence (SSR) is a DNA sequence formed by repeating elements consisting of 1-6 bases in series, and polymorphism of each site is caused due to different repetition times. SSR markers are distributed throughout the whole genome, and have the characteristics of codominance, good repeatability and high polymorphism, and SSR loci have conservation between genera and within-genus species. SSR markers can be classified into genome SSRs and expressed sequence tags SSRs (EST-SSRs) according to sources. For plants with incomplete genome sequencing, the EST-SSR marker can be rapidly and efficiently obtained by a transcriptome sequencing technology. Although the polymorphism is lower than that of a genome SSR marker, the sequence conservation is higher, the universality is better, and the polymorphism can be directly related to the gene function. The sealwort SSR molecular markers are slow to develop, only 225 pairs of currently published SSR molecular markers are available, and only 43 pairs of EST-SSR molecular markers are available, so that the requirements of genetic diversity analysis, genetic map construction, variety assisted breeding and the like of sealwort plants are far not met, and further development of the molecular markers is necessary.
The research carries out system analysis on the polygonatum rhizome transcriptome data obtained by utilizing a high-throughput sequencing technology, develops EST-SSR labeled primers, and carries out genetic analysis on 10 polygonatum rhizome germplasms through an EST-SSR technology so as to further understand the genetic relationship of polygonatum rhizome germplasm resources and lay a foundation for polygonatum rhizome genetic diversity analysis and germplasm resource identification.
Disclosure of Invention
The invention aims to provide the development of a sealwort EST-SSR primer group and the application thereof in aspects of genetic diversity and the like so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: the development of the polygonatum rhizome EST-SSR primer group and the application thereof in the aspects of genetic diversity and the like screen 154 pairs of polygonatum rhizome EST-SSR primers with high polymorphism and clear bands.
The method for developing the rhizoma polygonati EST-SSR primer group and the operation method thereof applied to the aspects of genetic diversity and the like comprises the following steps:
the method comprises the following steps: extracting genome DNA from 10 polygonatum germplasms from different producing areas by a CTAB method. Wherein the CTAB method comprises the following steps: (1) 25mg of the leaf blade is put into a sterilized mortar, and a small amount of liquid nitrogen of PVP is added for full grinding. The mixture was transferred to a 1.5mL centrifuge tube, centrifuged at 12000rpm for 5min, and the supernatant was discarded. (2) The washed tissue precipitate was mixed with 1mL CTAB extract, and washed with 65 deg.C water bath for 1h, during which time it was taken out every 15min, inverted and mixed. (3) Cooling to room temperature after water bath, transferring the cooled liquid to a 2mL centrifuge tube, adding 1mL24:1 chloroform isoamylol, reversing and uniformly mixing until the liquid is milky, centrifuging at 12000rpm for 5min, and taking the supernatant to the 2mL centrifuge tube; adding 800 μ L24:1 chloroform isoamyl alcohol, repeating the above steps; taking 1.5mL of the supernatant, adding isopropanol with the volume of 0.7 time,freezing in a refrigerator at-20 deg.C for more than 1 hr. (4) Taking the frozen supernatant, centrifuging at 2000rpm for 10min, removing the supernatant, adding 1mL of 75% ethanol for cleaning, and repeating the steps once; finally, the mixture is washed once by absolute ethyl alcohol and dried. Add 50. mu.LddH2And O. (5) The DNA solution was checked for quality using 1.5% agarose gel electrophoresis, while the DNA concentration and purity were checked using Nanodrop 2000. DNA samples were diluted to 50 ng/. mu.L and stored at-20 ℃ until use.
Step two: searching repeated sequence sites in the polygonatum sibiricum transcriptome by using MISA software, wherein the search setting standard is as follows: nucleotides 1, 2, 3, 4, 5 and 6bp, the repetition times are not less than 10, 6, 5 and 5 times in sequence, and the composite SSR (the distance between 2 SSRs) is less than 100 bp.
Step three: and (3) extracting a 300bp sequence containing the SSR repeated site flanking by using Primer3.0 software as a template sequence for marking and developing. The design standard of the primer is that the GC content is 30-70%, the size of the product is 100-300 bp, the Tm value is 55-65 ℃, the difference between the Tm values of the upstream primer and the downstream primer is not more than 5 ℃, and the length of the primer is 18-27 bp.
Step four: randomly selecting 500 pairs of primers for validity verification;
step five: the optimal renaturation temperature of the primer is screened on a common gradient PCR instrument; the PCR reaction system is 10 μ L, which comprises: mu.L (50 ng/. mu.L) of template DNA, 0.3. mu.L each of the upstream and downstream primers (10. mu.M), 2 XMaster Mix 5. mu.L, reaction volume 10. mu.L, using ddH2O make up volume; the reaction program is pre-denaturation at 94 ℃ for 5min, and then 35 cycles are carried out, wherein each cycle comprises denaturation at 94 ℃ for 30s, renaturation (48-64 ℃) for 30s, extension at 72 ℃ for 30s, final extension at 72 ℃ for 10min, and storage at 4 ℃; randomly selecting 3 polygonatum germplasms for screening primers and the optimal annealing temperature, wherein the optimal annealing temperature of each primer pair is determined by a gradient PCR test; screening out a primer with a target band and an optimal annealing temperature;
step six: performing non-denaturing polyacrylamide gel electrophoresis;
performing PCR amplification on primers with target bands for 10 polygonatum germplasms in different production places at the optimal annealing temperature, performing 8% non-denaturing polyacrylamide gel electrophoresis (200V and 90min) on products, dyeing electrophoresis gels with Cell Red Nucleic acid dye solution, and photographing and storing by using a gel imaging system;
step seven: processing data;
according to the statistical data of the existence of PCR product bands. Recording the amplification zone as 1 and the non-amplification zone as 0, and recording the data into Excel to obtain binary data for subsequent experimental analysis.
Compared with the prior art, the invention has the beneficial effects that:
through EST-SSR result analysis of the research of the invention, 10 polygonatum germplasm resources are clustered into three groups, wherein the group I comprises polygonatum floridumum from Anhui Chizhou, Guangxi Hezhou, Zhejiang Xiju and Hunan Rou bottom, the group II comprises Jiangxi Xinfeng, Henan Lingbao, Sichuan Yaan and Shaanxi Ningqiang polygonatum, and the group III comprises Yunnan polygonatum from Henan Lushan and Gui De Jiang. The primers screened by the method have the advantages of high accuracy, strong stability, simple and convenient operation, low cost and easy popularization and application in a large range.
Drawings
FIG. 1 is an electrophoresis diagram of a partial primer PCR product;
FIG. 2 is UPGMA cluster analysis of 10 sealwort germplasms based on EST-SSR markers.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
The method comprises the following steps: extracting genome DNA from 10 polygonatum germplasms from different producing areas by a CTAB method. Wherein the CTAB method comprises the following steps: (1) 25mg of the leaf blade is put into a sterilized mortar, and a small amount of liquid nitrogen of PVP is added for full grinding. The mixture was transferred to a 1.5mL centrifuge tube, centrifuged at 12000rpm for 5min, and the supernatant was discarded. (2) Adding 1mL CTAB extract into the cleaned tissue precipitate, and mixingHomogenizing, and taking out every 15min for 1h in a water bath at 65 ℃ and mixing reversely. (3) Cooling to room temperature after water bath, transferring the cooled liquid to a 2mL centrifuge tube, adding 1mL24:1 chloroform isoamylol, reversing and uniformly mixing until the liquid is milky, centrifuging at 12000rpm for 5min, and taking the supernatant to the 2mL centrifuge tube; adding 800 μ L24:1 chloroform isoamyl alcohol, repeating the above steps; taking the supernatant to a 1.5mL centrifuge tube, adding isopropanol with the volume of 0.7 times, and putting the centrifuge tube into a refrigerator with the temperature of 20 ℃ below zero for freezing for more than 1 h. (4) Taking the frozen supernatant, centrifuging at 2000rpm for 10min, removing the supernatant, adding 1mL of 75% ethanol for cleaning, and repeating the steps once; finally, the mixture is washed once by absolute ethyl alcohol and dried. Add 50. mu.LddH2O2. (5) The DNA solution was checked for quality using 1.5% agarose gel electrophoresis, while DNA concentration and purity were checked using Nanodrop2000, and the DNA sample was diluted to 50 ng/. mu.L and stored at-20 ℃ until use.
TABLE 1 Polygonatum sibiricum information table for different producing areas
Step two: searching repeated sequence sites in the polygonatum sibiricum transcriptome by using MISA software, wherein the search setting standard is as follows: nucleotides 1, 2, 3, 4, 5 and 6bp, the repetition times are not less than 10, 6, 5 and 5 times in sequence, and the composite SSR (the distance between 2 SSRs) is less than 100 bp.
Step three: and (3) extracting a 300bp sequence containing the SSR repeated site flanking by using Primer3.0 software as a template sequence for marking and developing. The design standard of the primer is that the GC content is 30-70%, the product size is 100-300 bp, the Tm value is 55-65 ℃, the difference between the Tm values of the upstream primer and the downstream primer is not more than 5 ℃, the length of the primer is 18-27 bp, and the GC content of the primer is 30-70%.
Step four: randomly selecting 500 pairs of primers for validity verification;
step five: the optimal renaturation temperature of the primer is screened on a common gradient PCR instrument; the PCR reaction system is 10 μ L, which comprises: mu.L (50 ng/. mu.L) of template DNA, 0.3. mu.L each of the upstream and downstream primers (10. mu.M), 2 XMaster Mix 5. mu.L, reaction volume 10. mu.L, using ddH2O make up volume; the reaction program is pre-denaturation at 94 ℃ for 5min, and then 35 cycles are carried out, wherein each cycle comprises denaturation at 94 ℃ for 30s, renaturation (48-64 ℃) for 30s, extension at 72 ℃ for 30s, final extension at 72 ℃ for 10min, and storage at 4 ℃; randomly selecting 3 polygonatum germplasms for screening primers and the optimal annealing temperature, wherein the optimal annealing temperature of each primer pair is determined by a gradient PCR test; screening out a primer with clear target band and optimal annealing temperature;
step six: native polyacrylamide gel electrophoresis:
performing PCR amplification on primers with target bands for 10 polygonatum germplasms in different production places at the optimal annealing temperature, performing 8% non-denaturing polyacrylamide gel electrophoresis (200V and 90min) on products, dyeing electrophoresis gels with Cell Red Nucleic acid dye solution, and photographing and storing by using a gel imaging system;
step seven: data processing:
according to the statistical data of the existence of PCR product bands. Recording the amplification zone as 1 and the non-amplification zone as 0, and recording the data into Excel to obtain binary data for subsequent experimental analysis.
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<400> 29
gctcctctcc cccaaaacaa 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tgcacaagct caacctttga 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
acaggactca cttccatgcc 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
actctctctc cccgactacg 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
cgtcgctccc cttgatttct 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
acgtgctgct caaatgcaag 20
<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gcacctcatt catcatcgtc g 21
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ggactacagc cattgtgcct 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gattcgagag ggcacaacca 20
<210> 38
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gggagtagaa gatgcaagcg t 21
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tgttgttgtg gagggtttgg 20
<210> 40
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
aggaaacacc ctctcgcac 19
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
aattgttcgt cggcggagat 20
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cttcgtcttg ggccatgact 20
<210> 43
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tcggggttta aatcgtttta tttgga 26
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
agatggcatt tcctcacgca 20
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ataatggtgg aggcaagccc 20
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
tgctctaaat tcgaggcgca 20
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
aacagagccc agctgattcc 20
<210> 48
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tgcaaacaaa gcagcaacat ct 22
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ttcatgggca gagactgtcg 20
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
caatgggtcc tgtatggcac t 21
<210> 51
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gccattgtgc agattctttg c 21
<210> 52
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tgccatgatg ctcaaccatc t 21
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tccctgacca ccaagtctca 20
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tgttgctgtt cccgatctcc 20
<210> 55
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
agaaaagtga ggcgaggagg 20
<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
tcccaagatc ccttcccctt 20
<210> 57
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
accacctgct gatgatgaat gt 22
<210> 58
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
taacccatgg ctgtcctgga 20
<210> 59
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
ccgatgacag gatcgacgtt 20
<210> 60
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
cccctcttct tctctgctcc 20
<210> 61
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
aaaccacgca ttgccatcag 20
<210> 62
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
aaggggtgaa ggagggatga 20
<210> 63
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gactcccaat tgcccttcca 20
<210> 64
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ccatgcatgc tcctctcgat 20
<210> 65
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cgccgagaac ataagcaagc 20
<210> 66
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ttcaaagcct tgggtgacgt 20
<210> 67
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
gggagggaat gtgggtgaag 20
<210> 68
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
tgattcctct ttgttgcccg a 21
<210> 69
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
ggaggttttt ccccgtgaca 20
<210> 70
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
ggttgtagtt tgttatcatg ccga 24
<210> 71
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ccaagtctct gcatccacga 20
<210> 72
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
cagcaagcaa caagaacccc 20
<210> 73
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
ctgcggagga taagtaggcg 20
<210> 74
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
accactctct gactccctct c 21
<210> 75
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ttcatggcgt cttcagaccc 20
<210> 76
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gccttgcaaa tgccgagaaa 20
<210> 77
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
tgtttgcagt agcccaccaa 20
<210> 78
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
ccatgtggca cgtaggactt 20
<210> 79
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ttgagacttc ggactcgctg 20
<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
caggatcagc ttcgcattgc 20
<210> 81
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
atcgaacgga gatcagcgtc 20
<210> 82
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
gagaggagtt ggggtttggg 20
<210> 83
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
agggccgtgc tcatagattg 20
<210> 84
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gtgccgtcct tgaagtacca 20
<210> 85
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
cagatcgcga agaatccgga 20
<210> 86
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gacttcgagc tacggggaag 20
<210> 87
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
agcaattaac ttacacttac ttgcct 26
<210> 88
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
tggcgatcct ccgatacaga 20
<210> 89
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
agctcgaatc acgttcacat ct 22
<210> 90
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
cttgggagca ttctagggtc a 21
<210> 91
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
atttcagaag cccccacctt 20
<210> 92
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gattcgctag cgcaaaacgt 20
<210> 93
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
gcccagcaga aagagaggag 20
<210> 94
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
tgttgtaacg gatccgcgaa 20
<210> 95
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
accctagtct cgatcgcaca 20
<210> 96
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
gtagatcagt gcttgccgct 20
<210> 97
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
ttcctcgtcc tcatcctcgt 20
<210> 99
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
acacttgggc cttctttggt 20
<210> 100
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
aagttacgat gatagttaac aagtgat 27
<210> 101
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
ccggacagtt ctgtgtgtca 20
<210> 102
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
aacctcttga ttcagccttg t 21
<210> 103
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
tcgcctttgc aagccttctt 20
<210> 104
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ctacaggcgg gtgctgaatt 20
<210> 105
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
gaaacgggtg ttaatggctg t 21
<210> 106
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
cccccttcgc tttctctgtt 20
<210> 107
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
atgttgccgt acacgtgtga 20
<210> 108
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ccggggtaca ctgcatctac 20
<210> 109
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
agcttctttg attcatagaa cagct 25
<210> 110
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
attcgtgtct gaagtggcgt 20
<210> 111
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
cgtcgatacc aactccgagg 20
<210> 112
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
tgtgattcag cagctggtgt 20
<210> 113
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
ctaggggcag ggatttcagc 20
<210> 114
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
tggtttgtca ccctgcaaga 20
<210> 115
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
acagtttgcg atattagagg gga 23
<210> 116
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
gtagggcaca gggaggaaag 20
<210> 117
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
gcttcattcg ccagctcaac 20
<210> 118
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
atgaatggtg tgggtgggtg 20
<210> 119
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cctagtccta gggttgccgt 20
<210> 120
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
gtgctgctca ctcccttctt 20
<210> 121
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gacggacatg acgagggatc 20
<210> 122
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
agcatgcaga gagaggagga 20
<210> 123
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
gatgcctgaa cacctccaca 20
<210> 124
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
acccctgatg aaccatgcag 20
<210> 125
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
cagagtgaag ctaaggtgcc a 21
<210> 126
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
tcctaggagg gtgggttacg 20
<210> 127
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
gcagaaccac caagactcca 20
<210> 128
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
aatgacgacg gcctcttcag 20
<210> 129
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
acccatactc gtgtctgctc 20
<210> 130
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
gaaaagactc gagccctccc 20
<210> 131
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
tcgatgcaga tgcggaatca 20
<210> 132
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
acgagaccat cttccctagc a 21
<210> 133
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
tcgacaagta tccgagtttc ca 22
<210> 134
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
atgtccttcg actcctcgga 20
<210> 135
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
catcgcatca tcagcagctg 20
<210> 136
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
caaacttgcc caaaccctcg 20
<210> 137
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
cagtgcgtgc attgtgtgaa 20
<210> 138
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
gtgaaggtga gcgtctcctc 20
<210> 139
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
gggaggtaga ggagcttcga 20
<210> 140
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
gcaattactg ccgctagtga 20
<210> 141
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
aagtcagttt aggcggtggc 20
<210> 142
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gagatccttc ctccgcttcg 20
<210> 143
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
tgccattagt gaacgcgtca 20
<210> 144
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
gacggcatcg gcaacttttt 20
<210> 145
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
ctcctcctcc tctcccttcc 20
<210> 146
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
gttcacgaag taggggtgca 20
<210> 147
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
cagttgcagc tctccacaga 20
<210> 148
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
ccaaacgtct ttcggcaaca 20
<210> 149
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
ggacagggct caatctaggc 20
<210> 150
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
ggttgtcgag gggtcgaaat 20
<210> 151
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
acctccctca gtggacagtc 20
<210> 152
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
acaaccaccc tcgtttccaa 20
<210> 153
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
ccaccaccca ccagaagaat 20
<210> 154
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
tcctggtgga gtaggcttca 20
<210> 155
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
attcccaggg cacatgtgaa 20
Claims (2)
1. The method for developing the rhizoma polygonati EST-SSR primer group and the operation method thereof applied to the aspects of genetic diversity and the like is characterized by comprising the following steps:
1) extracting genome DNA from 10 polygonatum germplasms from different producing areas by a CTAB method;
2) searching repeated sequence sites in the polygonatum sibiricum transcriptome by using MISA software, wherein the search setting standard is as follows: nucleotides 1, 2, 3, 4, 5 and 6bp, and the repetition times are not less than 10, 6, 5 and 5 times in sequence; the composite SSR (the distance between 2 SSRs) is less than 100 bp;
3) and (3) extracting a 300bp sequence containing the SSR repeated site flanking by using Primer3.0 software as a template sequence for marking and developing. The design standard of the primer is that the GC content is 30-70%, the size of the product is 100-300 bp, the Tm value is 55-65 ℃, the difference between the Tm values of the upstream primer and the downstream primer is not more than 5 ℃, and the length of the primer is 18-27 bp;
4) randomly selecting 500 pairs of primers for validity verification;
5) the optimal renaturation temperature of the primer is screened on a common gradient PCR instrument; the PCR reaction system is 10 μ L, which comprises: mu.L (50 ng/. mu.L) of template DNA, 0.3. mu.L each of the upstream and downstream primers (10. mu.M), 2 XMaster Mix 5. mu.L, reaction volume 10. mu.L, using ddH2O make up volume; the reaction program is pre-denaturation at 94 ℃ for 5min, and then 35 cycles are carried out, wherein each cycle comprises denaturation at 94 ℃ for 30s, renaturation (48-64 ℃) for 30s, extension at 72 ℃ for 30s, final extension at 72 ℃ for 10min, and storage at 4 ℃; selecting 3 rhizoma Polygonati germplasms (Polygonatum cyrtonema, Henan Lushan Polygonatum sibiricum, Henan Lingbao Polygonatum sibiricum) for screening primers and optimal annealing temperature, wherein the optimal annealing temperature of each primer pair is determined by gradient PCR experiment; screening out a primer with a target band and an optimal annealing temperature;
6) native polyacrylamide gel electrophoresis: at the optimal annealing temperature, primers with target bands are used for PCR amplification of 10 polygonatum germplasms in different production places, products are subjected to 8% non-denaturing polyacrylamide gel electrophoresis (200V and 90min), electrophoresis gel is stained by Cell Red Nucleic acid dye solution, and photographing and storage are performed by using a gel imaging system;
7) data processing: according to the statistical data of the existence of PCR product bands. Recording the amplification zone as 1 and the non-amplification zone as 0, and recording the data into Excel to obtain binary data for subsequent experimental analysis.
2. The method for developing the rhizoma polygonati EST-SSR primer group and the application and operation method thereof in aspects of genetic diversity and the like according to claim 1, wherein the CTAB method comprises the following steps: the method comprises the following steps: 25mg of the leaf blade is put into a sterilized mortar, and a small amount of liquid nitrogen of PVP is added for full grinding. The mixture was transferred to a 1.5mL centrifuge tube, centrifuged at 12000rpm for 5min, and the supernatant was discarded. Step (ii) ofII, secondly: the washed tissue precipitate was mixed with 1mL CTAB extract, and washed with 65 deg.C water bath for 1h, during which time it was taken out every 15min, inverted and mixed. Step three: cooling to room temperature after water bath, transferring the cooled liquid to a 2mL centrifuge tube, adding 1mL24:1 chloroform isoamylol, reversing and uniformly mixing until the liquid is milky, centrifuging at 12000rpm for 5min, and taking the supernatant to the 2mL centrifuge tube; adding 800 μ L24:1 chloroform isoamyl alcohol, repeating the above steps; taking the supernatant to a 1.5mL centrifuge tube, adding isopropanol with the volume of 0.7 times, and putting the centrifuge tube into a refrigerator with the temperature of 20 ℃ below zero for freezing for more than 1 h. Step four: taking the frozen supernatant, centrifuging at 2000rpm for 10min, removing the supernatant, adding 1mL of 75% ethanol for cleaning, and repeating the steps once; finally, the mixture is washed once by absolute ethyl alcohol and dried. Add 50. mu.LddH2And O. Step five: the DNA solution was checked for quality by electrophoresis on a 1.5% agarose gel, DNA concentration and purity by Nanodrop2000, and the DNA sample was diluted to 50 ng/. mu.L and stored at-20 ℃ until use.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114774571A (en) * | 2022-03-31 | 2022-07-22 | 海南医学院 | Traditional Chinese medicine intelligence-benefiting EST-SSR (expressed sequence tag-simple sequence repeat) labeled primer and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754886A (en) * | 2017-01-19 | 2017-05-31 | 北京林业大学 | Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers |
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