CN110205329B - Saccharum cutting secret specific sequence and identification method thereof - Google Patents
Saccharum cutting secret specific sequence and identification method thereof Download PDFInfo
- Publication number
- CN110205329B CN110205329B CN201910533250.4A CN201910533250A CN110205329B CN 110205329 B CN110205329 B CN 110205329B CN 201910533250 A CN201910533250 A CN 201910533250A CN 110205329 B CN110205329 B CN 110205329B
- Authority
- CN
- China
- Prior art keywords
- sequence
- saccharum
- specific sequence
- primer
- dna
- 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.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 15
- 241000209051 Saccharum Species 0.000 title claims description 20
- 238000012408 PCR amplification Methods 0.000 claims abstract description 11
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 238000001962 electrophoresis Methods 0.000 claims description 8
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000012154 double-distilled water Substances 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 abstract description 34
- 240000000111 Saccharum officinarum Species 0.000 abstract description 17
- 235000007201 Saccharum officinarum Nutrition 0.000 abstract description 17
- 241000894007 species Species 0.000 abstract description 17
- 239000003147 molecular marker Substances 0.000 abstract description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 abstract description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 6
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 241000404041 Chamaemelum Species 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Chemical class 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- KEQXNNJHMWSZHK-UHFFFAOYSA-L 1,3,2,4$l^{2}-dioxathiaplumbetane 2,2-dioxide Chemical compound [Pb+2].[O-]S([O-])(=O)=O KEQXNNJHMWSZHK-UHFFFAOYSA-L 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010071602 Genetic polymorphism Diseases 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 241000746444 Saccharum sp. Species 0.000 description 1
- 229920001284 acidic polysaccharide Polymers 0.000 description 1
- 150000004805 acidic polysaccharides Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 210000004283 incisor Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a sugarcane cutting hand secret specific sequence and a molecular marker thereof, wherein software blast is used for comparing a sugarcane tropical species LA pure with a DNA sequence of a cutting hand secret AP85-441 to obtain 6 cutting hand secret specific DNA sequences, primers are respectively designed according to the 6 sequences, 2 tropical species and 15 cutting hand secret genomic DNAs are extracted by a CTAB method, and 6 pairs of primers are respectively used for carrying out PCR amplification on the genomic DNAs, so that the results show that the 6 pairs of primers can distinguish the tropical species from the cutting hand secret with clear and bright bands, and the invention is beneficial to the identification and research of sugarcane hybrid species.
Description
Technical Field
The invention relates to the fields of bioinformatics and molecular biology, in particular to a Saccharum sinensis Hance specific sequence and an identification method thereof.
Background
The dense handlings are wild species of sugarcane thin stems, are rich in variety, wide in distribution and strong in perennial root and stress resistance, and are one of the most contributed resources in sugarcane breeding. In the sugarcane breeding, the efficient utilization and scientific management of the genotypes and useful genes of a germplasm resource gene bank are very critical, and a cross breeding plan aiming at sugarcane parents with different genotype genetic backgrounds is drawn up while the genetic diversity of the sugarcane gene bank is improved, so that the method has great practical significance for widening the sugarcane genetic basis and expanding the heterosis. Therefore, the research on genetic diversity of the cleft hand density has important significance for the breeding of excellent sugarcane varieties.
Molecular markers are genetic markers based on nucleotide sequence variations in the genetic material between individuals, and are a direct reflection of genetic polymorphisms at the DNA level. The DNA molecular marker has the advantages that DNA of different tissues can be used for marker analysis at different stages of biological development, the marker is neutral, the expression of target characters is not influenced, no linkage is generated with bad characters, and the detection means is simple and rapid. With the development of molecular biology technology, there are dozens of DNA molecular marker technologies, and the DNA molecular marker technologies are widely applied to the aspects of genetic breeding, genome mapping, gene localization, species genetic relationship identification, gene library construction, gene cloning and the like.
At present, most sugarcane varieties are close in relativity and have close blood relationship, the excellent rate of offspring is extremely low, and the bred new variety is difficult to break through in the aspects of yield, sugar content and resistance. The excellent characters of strong stress resistance and barren resistance, wide adaptability and the like of the sugarcane relative genus are applied to the high-priced breeding by hybridizing the relative genus and the distant genus. Since there are many species of hybrids, the interspecific hybrids include tropical species, cleft hand, Chinese species, Indian species and wild species with large stem. However, at present, no method can rapidly identify whether the hybrid contains the blood margin of the cleft hand secret, so that a specific molecular marker for rapidly identifying the blood margin of the cleft hand secret is necessary to be developed, and development and utilization of the germplasm resources of the cleft hand secret are facilitated. According to the invention, the genome data of the cleft hand secret and the tropical seed is utilized to carry out comparative analysis to obtain 6 cleft hand secret specific sequences, and primers are designed according to the 6 sequences, and the primers can amplify bright and clear bands only in the cleft hand secret genome DNA through PCR amplification and agarose gel electrophoresis verification.
Disclosure of Invention
The invention aims to explore specific sequences of the Saccharum incisum density, identify whether hybrid seeds contain incisor density blood margins, and provide an economic and efficient identification method for the chromosome research of complex polyploid plants such as sugarcane.
In order to achieve the purpose, the invention adopts the following technical scheme:
the Saccharum sinensis Roxb specific sequence is obtained by analyzing genome comparison data of Saccharum sinensis Roxb and tropical seeds, and has the following names: the nucleotide sequences of the sequence 1C, the sequence 2C-1, the sequence 2C-2, the sequence 5D, the sequence 6D and the sequence 7C are respectively shown in SEQ ID NO. 1-7.
The primer sequence of the sequence 1C is as follows:
an upstream primer: 5 'GCGGATGGTTTCTCTTAGGTTC 3';
a downstream primer: 5 'TAATGGCGTTAGGGAGTGGTTG 3';
the primer sequence of the sequence 2C-1 is as follows:
an upstream primer: 5 'ATCACGAGAATCGGAAAGGAAT 3';
a downstream primer: 5 'AGGAAGAGTGGAAGATGAAAGC 3';
the primer sequence of the sequence 2C-2 is as follows:
an upstream primer: 5 'TCCAAGCCAAACCAGACAGAGC 3';
a downstream primer: 5 'TAAACGCCAGCCAACTAAAACA 3';
the primer sequence of the sequence 5D is as follows:
an upstream primer: 5 'AGTTGACCTCTCGGAATCCATCG 3';
a downstream primer: 5 'ACCTTGACCGTGAAAGTCTCGCC 3';
the primer sequence of the sequence 6D is as follows:
an upstream primer: 5 'CACGGGAGAAAACCAACAGGC 3';
a downstream primer: 5 'TATGATTACCCACGACAAGAA 3';
the primer sequence of the sequence 7C is as follows:
an upstream primer: 5 'GATTGGCCTCTTTTGTGTTTTT 3';
a downstream primer: 5 'CACGGTTCATTTCTTCTTCTCG 3'.
The method for identifying the Saccharum sp.cleft specific sequence comprises the following steps:
(1) comparing and analyzing genome data of Saccharum sinensis Roxb and tropical seeds to obtain 6 specific sequences of Saccharum sinensis Roxb;
(2) carrying out PCR amplification on the 6 cleft hand-tight specific sequences to obtain a PCR product;
(3) the PCR product was identified by agarose gel electrophoresis.
In the step (2), the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 1min, 35 cycles, and final extension at 72 ℃ for 10 min.
In the step (2), the PCR amplification reaction system is: the upstream and downstream primers, F/R, were each 0.25. mu.L, DNA template 2. mu.L, Mix 5. mu.L, and ddH2O was added to make up the total volume to 10. mu.L.
In the step (3), the agarose gel electrophoresis identification conditions are as follows: after loading, electrophoresis is carried out by using 125V voltage, and the result is checked after 15 min.
The result shows that the specific sequence of the cleft hand density can distinguish the tropical species from the cleft hand density and obtain clear and bright bands.
The invention has the advantages that: the invention utilizes the genome data of the Saccharum incarnatum champ and tropical seeds to compare and analyze to obtain the specific sequence of the chamaemelum incarnatum, designs the corresponding PCR primers according to different sequences, proves that the primers can only amplify DNA of the chamaemelum incarnatum, the specificity of the primers is good, the amplified band is single and bright, the PCR primers provide an important tool for rapidly identifying whether the Saccharum officinarum hybrid seeds contain the bloody margin of the chamaemelum incarnatum, and the invention is beneficial to the identification and research of the Saccharum officinarum hybrid varieties.
Drawings
FIG. 1 is an electrophoretogram of specific sequence 1C amplified DNA of different cleft and tropical species;
FIG. 2 is an electrophoresis chart of different cleft hand density and tropical seed DNA amplification with specific sequence 2C-1;
FIG. 3 is an electrophoresis chart of DNA amplification of different cleft hand densities and tropical species by specific sequence 2C-2;
FIG. 4 is an electrophoretogram of specific sequence 5D amplified for different cleft and tropical species of DNA;
FIG. 5 is an electrophoretogram of specific sequence 6D amplified for different cleft and tropical species of DNA;
FIG. 6 is an electrophoretogram of specific sequence 7C amplified DNA of different cleft and tropical species;
wherein Lane 1 is LA Purple, Lane 2 is Badila, Lane 3 is AP85-441, Lane 4 is SES208, Lane 5 is cliff 12, Lane 6 is Yunnan 84-268, Lane 7 is Guizhou 78-II-28, Lane 8 is Sichuan 88-16, Lane 9 is Guangdong 30, Lane 10 is NP-X, Lane 11 is Fujian 89-I-17, Lane 12 is Yunnan 82-106, Lane 13 is Yunnan 82-29, Lane 14 is Fujian 87-1-4, Lane 15 is Fujian 87-1-11, Lane 16 is Fujian 89-1-9, Lane 17 is Sichuan 78-2-11, and Lane 18 is DL5000 DNA Maker; LA pure and Badila are tropical seeds, and others are clenched.
Detailed Description
Example 1: primer design and PCR amplification
The Saccharum sinensis Roxb specific sequence is obtained by analyzing genome comparison data of Saccharum sinensis Roxb and tropical seeds, and has the following names: the nucleotide sequences of the sequence 1C, the sequence 2C-1, the sequence 2C-2, the sequence 5D, the sequence 6D and the sequence 7C are respectively shown in SEQ ID NO. 1-7.
The molecular marker primer sequences corresponding to the sequence 1C, the sequence 2C-1, the sequence 2C-2, the sequence 5D, the sequence 6D and the sequence 7C are shown in SEQ ID NO. 8-18.
Primer design was performed on the 6 Customidium specific sequences using Primer Premier 5.0, the Primer sequences are shown in Table 1, and the setting conditions were as follows: firstly, the primer and the template sequence are closely complementary; secondly, the length of the primer is 18 plus or minus 6 bp; thirdly, the GC content of the primer sequence is 40-60%; fourthly, stable dimer or hairpin structure is prevented from being formed between primers; fifthly, the primer can not generate mismatching at the non-target site of the template sequence.
TABLE 1 cleft hand secret specific sequence amplification primers
Carrying out PCR amplification on the 6 cleft hand-tight specific sequences, wherein the conditions of the PCR amplification are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 1min, 35 cycles, and final extension at 72 ℃ for 10min to finally obtain a PCR product.
Example 2: extraction of DNA by CTAB method
CTAB is a cationic detergent with the property of precipitating nucleic acids and acidic polysaccharides from solutions of low ionic strength. In a solution of high ionic strength, CTAB forms a complex with proteins and polysaccharides, but does not precipitate nucleic acids. Then extracting with organic solvent to remove protein, polysaccharide, phenols and other impurities, and precipitating with ethanol to separate out nucleic acid.
The technology for extracting the DNA of the saccharum plant by the CTAB method comprises the following steps:
(1) adding liquid nitrogen into fresh sugarcane leaves, and fully grinding;
(2) rapidly transferring the powder into a 2ml centrifuge tube, adding 800L of a 2 xCTAB extraction buffer solution (respectively adding 2% by volume of beta-mercaptoethanol and PVP) preheated at 65 ℃, uniformly mixing, and keeping the temperature at 65 ℃ for 30min while gently shaking for 2-3 times;
(3) taking out the centrifuge tube, cooling to room temperature, adding equal volume of chloroform/isoamyl alcohol (24: 1), reversing, mixing to milk white, standing at room temperature for 10min, and centrifuging at 10000rpm for 10 min;
(4) slowly sucking out the supernatant, transferring about 600L into another 2ml sterile centrifuge tube, and adding 2 times of volume of absolute ethyl alcohol; slowly and reversely mixing, standing at-20 deg.C for 30-60min, centrifuging at 12000rpm for 10min, and removing supernatant;
(5) the precipitate was washed twice with 1ml 70% ethanol, the residue was aspirated as far as possible with a 200. mu.L pipette, blown dry and 60. mu.L ddH2O was added.
Because a large number of homologous sequences exist in genomes of the cleft hand secret and the tropical seeds, the blood margin of the cleft hand secret in the sugarcane cultivar cannot be quickly identified only by using the traditional genome in situ hybridization technology.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Sequence listing
<110> Fujian agriculture and forestry university
<120> Saccharum cleft hand secret specific sequence and identification method thereof
<130> 2019
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 640
<212> DNA
<213> Artificial
<400> 1
gggtttcgct agagttcggt taattggctt tctaatctcc cggtcaattg aagtcaaggt 60
cttagtcccc ctcccccttc tacaggggcc catgccttcc cttatacacc ggagaggttg 120
tgggagtgca aagcggatgg tttctcttag gttctctcac ccttggcttc agacttcttc 180
tttgcaccgt cggtcatcct ttgtcctgta gttaggggcc gacgaaaccg caaccactcc 240
ctaacgccat tattgcagct gggtgctgac tgccaggtgc tgtctgtgca gcggtacctt 300
ccctaggtga gccttctcct ggcttcattg gcatgccagg ctccttctcc tgcccaaaaa 360
ggctgccaag tgggcccaga tcctctactc tgtgggtttg gaggagttcc ttgtctgaca 420
catcgtgccc tgagcgtgac cctatcgagg gagtggtgct tctatgccta tcaggctgtg 480
gccgcatggt agattccgga gcttgcttcc gcctttgccc agggcaatca ttcaccccga 540
gccaaggctg gcacaattgc gattgccatt attttgcctg ggctcccctg ggtcaagaat 600
gcctagcgct ttggttgagc cctcgagttc ccaagcagga 640
<210> 2
<211> 376
<212> DNA
<213> Artificial
<400> 2
gatgctagcg aatctcaggt ggttgtttat tatgagcatt cccagcatca cgagaatcgg 60
aaaggaattc tatggagagg aaggagcatg caaaaggttg agagttattc tattgtgggg 120
attggagaac ttggaggagt ggtggacaac acggtcaggt gaagaaggcg acgagttctt 180
aatccctaag ttgcacaagt tggatgtatg ggactgccca aagttgaagt tcctgccata 240
tccccccaaa agtatgaatt gggacttgac caacagcgat gaggtgctac caccagtgca 300
tgggtttggg aggctttcat cttccactct tccttttcaa gccagaatct caagcaagag 360
ttttactgct gacaag 376
<210> 3
<211> 793
<212> DNA
<213> Artificial
<400> 3
ggccaggtat gaaacgagcc attctccagt ttgtccagtc ttgtgcagtt tgcctccaag 60
ccaaaccaga cagagctaag tcactaggac tactgcagcc gcttccggtg cctagaactg 120
cttgggaaat tatttcgctg gactttgttg agggcctccc attgtctggc agttataatg 180
ctattctcat ggtggtggtt aagtattcta agtacaccca tttcttgccc ctgaggcatc 240
ctttcacagc agcttcagtg gcaagacttt tcattgacaa tatctaccgt ctacatggtt 300
taccccagtc aatcatctcc gatcgtgacc ggatttttac cagcagactc tggcaactcc 360
tgttttagtt ggctggcgtt tagttaagga tgagttcctc ttatcaccct cagaccgacg 420
gtcagaccga gcgcgtcaat caatgccttg agacgttctt gagatgtttt cttcatgctt 480
gtcctagccg ctggattcat tggcttgcct tagcagaatt ctagtacaat acgagctctc 540
attctgccct gggccattcc cccttcgaag tactgtatgg ctatcctcct cggcttctgg 600
gtgttgatat ctcggctgct gcacctgtgc tggacctgca acaatggtca gaggagcgcg 660
atctgatgca cagcttggtc cgtcttcatc tccagcgtgc tcaggaaagg atgcaccgtc 720
aggccaacaa gaccaggtct gaatgctcat ttcaagttgg cgatcaggtt tacctcaaaa 780
cttcaaccgt atg 793
<210> 4
<211> 324
<212> DNA
<213> Artificial
<400> 4
tttgtaaaga ctttattggg tactatcaag ctagaagaag ttgacctctc ggaatccatc 60
gagggtgtca agtatatgat ttttgaaaag aacggcattc ctccggcatc tcagagattg 120
gtattcaatg gagtgcgggc tgacaaggag tggtatacct tggaggaata cggcttccag 180
tatggaacag atatccatca ggtcctttat ctccgtggcg gtggcactga ccttaagatt 240
aatatatttg tcacgaagct cgacggcgag actttcacgg tcaaggtgcc gagcctcatg 300
agtcgcatct ctgacgtcag gtgc 324
<210> 5
<211> 300
<212> DNA
<213> Artificial
<400> 5
ggaacacttg tagcgtacgt acagaagtta cacgggagaa aaccaacagg cgcccagatg 60
cagtaatgga gattttggac gggatttaat ggcaatggca gctaaggaga gaaaaattaa 120
agtcaatggc atagaagcaa agccctattt ttcaatggca caaaacacac aagtttcaat 180
ggcatagaac aaattttctc ttatttctgt tgtggtatat atgaccatct gaaagcttgt 240
gtgacagtta gcttcttgtc gtgggtaatc ataggttttg taacaaagga agagaaccat 300
<210> 6
<211> 316
<212> DNA
<213> Artificial
<400> 6
tgtagatata gattcgaaaa cgtcctttct gagattggcc tcttttgtgt ttttttacaa 60
cgacatggat ttattagtta aggtcaatta agcagtgtta cacggatacc cttcaaaaaa 120
ggtcaagcac tcatacgtac ataggtgctc tccgagaaga agaaatgaac cgtgctgtcc 180
ataaaatcta agcatagaca atctgggtgt ttagcaatcc tgattaatac tgtgcaaaaa 240
gacttttgtg ttcttttgaa ttcttctttc tcaaaccacc ttccgttttc cttggtcaat 300
ggtcatagcc gttcgc 316
<210> 7
<211> 22
<212> DNA
<213> Artificial
<400> 7
gcggatggtt tctcttaggt tc 22
<210> 8
<211> 22
<212> DNA
<213> Artificial
<400> 8
taatggcgtt agggagtggt tg 22
<210> 9
<211> 22
<212> DNA
<213> Artificial
<400> 9
atcacgagaa tcggaaagga at 22
<210> 10
<211> 22
<212> DNA
<213> Artificial
<400> 10
aggaagagtg gaagatgaaa gc 22
<210> 11
<211> 22
<212> DNA
<213> Artificial
<400> 11
tccaagccaa accagacaga gc 22
<210> 12
<211> 22
<212> DNA
<213> Artificial
<400> 12
taaacgccag ccaactaaaa ca 22
<210> 13
<211> 23
<212> DNA
<213> Artificial
<400> 13
agttgacctc tcggaatcca tcg 23
<210> 14
<211> 23
<212> DNA
<213> Artificial
<400> 14
accttgaccg tgaaagtctc gcc 23
<210> 15
<211> 21
<212> DNA
<213> Artificial
<400> 15
cacgggagaa aaccaacagg c 21
<210> 16
<211> 21
<212> DNA
<213> Artificial
<400> 16
tatgattacc cacgacaaga a 21
<210> 17
<211> 22
<212> DNA
<213> Artificial
<400> 17
gattggcctc ttttgtgttt tt 22
<210> 18
<211> 22
<212> DNA
<213> Artificial
<400> 18
cacggttcat ttcttcttct cg 22
Claims (6)
1. The Saccharum sinensis Roxb specific sequence is characterized in that the sequence is obtained by comparing and analyzing the Saccharum sinensis Roxb specific sequence 1C according to genome data of a Saccharum sinensis Roxb and a tropical seed genome, and the nucleotide sequence is shown as SEQ ID NO. 1.
2. The Saccharum root cutter specific sequence of claim 1, wherein the primer sequence of sequence 1C is:
an upstream primer: 5 'GCGGATGGTTTCTCTTAGGTTC 3';
a downstream primer: 5 'TAATGGCGTTAGGGAGTGGTTG 3'.
3. The method of claim 1, comprising the steps of:
(1) comparing and analyzing genome data of the Saccharum sinensis Roxb and tropical seeds to obtain a specific sequence 1C of the Saccharum;
(2) carrying out PCR amplification on the cleft hand density specific sequence 1C to obtain a PCR product;
(3) the PCR product was identified by agarose gel electrophoresis.
4. The method of claim 3, wherein in step (2), the PCR amplification conditions are: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 1min, 35 cycles, and final extension at 72 ℃ for 10 min.
5. The method of claim 3, wherein in step (2), the PCR amplification reaction system comprises: the upstream and downstream primers, F/R (Forward primer/Reverse primer), were each 0.25. mu.L, DNA template 2. mu.L, Mix 5. mu.L, and ddH2O was added to make up the total volume to 10. mu.L.
6. The method of claim 3, wherein the conditions for identifying the Saccharum root-knot specific sequence in step (3) are as follows: after loading, electrophoresis is carried out by using 125V voltage, and the result is checked after 15 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910533250.4A CN110205329B (en) | 2019-06-19 | 2019-06-19 | Saccharum cutting secret specific sequence and identification method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910533250.4A CN110205329B (en) | 2019-06-19 | 2019-06-19 | Saccharum cutting secret specific sequence and identification method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110205329A CN110205329A (en) | 2019-09-06 |
| CN110205329B true CN110205329B (en) | 2021-04-06 |
Family
ID=67793604
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910533250.4A Expired - Fee Related CN110205329B (en) | 2019-06-19 | 2019-06-19 | Saccharum cutting secret specific sequence and identification method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110205329B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226446A (en) * | 2020-10-22 | 2021-01-15 | 云南省农业科学院甘蔗研究所 | Sugarcane SPSB gene isoform 1 and identification method and application thereof |
| CN113073134B (en) * | 2021-04-06 | 2022-03-01 | 南通大学 | Novel method for carrying out accurate chromosome counting by applying sugarcane centromere specific repeat sequence |
| CN113584184B (en) * | 2021-08-04 | 2023-07-28 | 南通大学 | Molecular marker system for identifying authenticity of sugarcane hybrid and development method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017859A (en) * | 2014-03-13 | 2014-09-03 | 南宁泰格瑞农业科技有限公司 | Method for identifying sugarcane germplasm resources based on SSR (Simple Sequence Repeats) and CE (capillary electrophoresis) technique |
| CN104561364B (en) * | 2015-02-05 | 2016-04-13 | 云南省农业科学院甘蔗研究所 | A kind of method of rapid detection saccharum SPSB gene pleiomorphism and application |
| CN105713988B (en) * | 2016-04-28 | 2019-03-22 | 福建农林大学 | The molecular labeling primer and application of the anti-leaf rust gene loci of Rapid identification sugarcane |
-
2019
- 2019-06-19 CN CN201910533250.4A patent/CN110205329B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN110205329A (en) | 2019-09-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103305510B (en) | Rice blast resistance gene Pi9 gene specificity molecular marker Pi9SNP as well as preparation and application thereof | |
| Liu et al. | Genome‐wide development of microRNA‐based SSR markers in Medicago truncatula with their transferability analysis and utilization in related legume species | |
| CN110205329B (en) | Saccharum cutting secret specific sequence and identification method thereof | |
| CN110894542A (en) | Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer | |
| CN107881256B (en) | Single nucleotide polymorphism marker site, primer pair, kit and application for identifying bitter kernel/sweet kernel characteristics of peach fruit | |
| CN105200052B (en) | Estimate molecular labeling, primer and the method for tobacco N introgressed segment left end length | |
| KR101151239B1 (en) | Marker for ms, a genic multiple-allele male sterile gene in chinese cabbage and uses thereof | |
| KR100842434B1 (en) | Ssr primer derived from ginseng and use thereof | |
| CN110331222B (en) | Molecular marker related to cotton fertility restoration and application thereof | |
| CN108531636B (en) | Molecular marker TJcM01 for identifying melon unisexual flower and application thereof | |
| CN108239675B (en) | Molecular marker TJcM02 for identifying melon unisexual flower and application thereof | |
| CN106676171B (en) | Molecular detection method for cotton polygene pyramiding breeding | |
| CN114410821A (en) | InDel molecular marker for identifying amaranth leaf color character and application thereof | |
| CN107460246A (en) | A kind of method of fast positioning peach target gene | |
| KR101432281B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| CN108330164B (en) | Characteristic sequence, primer and identification method of apocarya variety Moore | |
| CN111321237A (en) | SSR marker-based efficient breeding kit for 'Hui' self-fruitful progeny | |
| CN115725772B (en) | InDel molecular marker for identifying seed coat color of cabbage type rape and application thereof | |
| CN111235292B (en) | Rye 4RS chromosome arm specific KASP molecular marker and application thereof | |
| KR101432284B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| KR101432283B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| KR101432290B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| KR101432287B1 (en) | SSR markers and Genetic linkage map using Intraspecific population of Capsicum annuum | |
| CN113073134B (en) | Novel method for carrying out accurate chromosome counting by applying sugarcane centromere specific repeat sequence | |
| CN114438239B (en) | Molecular marker for identifying large 10 of mulberry variety Yuehong, identification primer group, kit and application |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210406 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |