CN110205329B - Saccharum cutting secret specific sequence and identification method thereof - Google Patents
Saccharum cutting secret specific sequence and identification method thereof Download PDFInfo
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- 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
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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.
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<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.
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