CN112575117B - SSR molecular marker primers of non-heading Chinese cabbage and red-flowering Chinese cabbage and application thereof - Google Patents
SSR molecular marker primers of non-heading Chinese cabbage and red-flowering Chinese cabbage and application thereof Download PDFInfo
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- CN112575117B CN112575117B CN202110018658.5A CN202110018658A CN112575117B CN 112575117 B CN112575117 B CN 112575117B CN 202110018658 A CN202110018658 A CN 202110018658A CN 112575117 B CN112575117 B CN 112575117B
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses a group of SSR molecular marker primers for identifying 'beautiful red rose' and 'October red' of non-heading Chinese cabbage and red flowering Chinese cabbage and application thereof, belonging to the technical field of vegetable variety identification and improved variety. The SSR molecular marker primer has the sequence as follows: brcSSR60-F:5 'GAGCATCACCAAGCTCCA-3', brcSSR60-R:5 'ATGTAACCGTTTCCTGGCTG-3'. The SSR molecular marker primers and the detection method thereof disclosed by the invention can be used for effectively distinguishing the 'beautiful red rose' and 'October red' varieties of the non-heading Chinese cabbage and the red vegetable. The method is not influenced by the growth and development stages of plants and the environment, is simple to operate, is accurate and rapid to detect, and can effectively supervise and arbitrate when varieties are mixed up or disputed.
Description
Technical Field
The invention belongs to the technical field of vegetable variety identification and improved variety, and particularly relates to a group of SSR molecular marker primers and a molecular marker method for identifying 'beautiful red roses' and 'October red' of non-heading Chinese cabbage and red flowering Chinese cabbages.
Background
Chinese cabbage Young cabbage not heading (A) and Young cabbageBrassica campestris ssp. chinensis var. purpurea) Also called purple flowering cabbage and red rape, belongs to the brassica variety of brassicaceae and the variety of the subspecies of Chinese cabbage, is native to China, mainly distributed in Yangtze river regions such as Xiang, hue and Chuan, and is the main vegetable supplied all the year round in the south. The red flowering cabbage contains various vitamins required by human bodies, such as protein, vitamin C, anthocyanin and the like, has very rich nutritive value, has a health-care function, and is popular with consumers. The red flowering Chinese cabbage is gradually introduced and planted in various regions throughout the country due to the good nutritional value and economic value. With the increase of the breeding process of the red flowering cabbage, more and more excellent varieties are cultivated. When varieties are mixed up, the identification is easily influenced by external environment only through morphological character identification, and the identification result is often inaccurate.
Microsatellite repetition or Simple Sequence Repeat (SSR) is a DNA tandem repeat sequence which is composed of a plurality of nucleotides as repeat units, two sides of each SSR repeat sequence are relatively conservative single copy sequences and are widely distributed at different positions of a genome, each site is determined by a designed primer sequence, and the SSR marker has the advantages of high repeatability, codominant inheritance, high Polymorphic Information Content (PIC), multiallelic property and the like and is widely applied to genetic diversity analysis, variety purity identification, core germplasm fingerprint map construction and the like of different plant germplasm resources. SSR markers show that there are extensive polymorphisms in individual length when PCR analysis is performed on specific gene loci using different primers. The SSR marker is used for amplifying the microsatellite fragments through PCR reaction, because each core sequence is different in series repetition, PCR products with different lengths can be amplified through the PCR reaction, and the fragments are separated through polyacrylamide gel electrophoresis, so that the size of the fragments is separated, and the SSR marker has the advantages of specificity, accuracy and rapidness. The identification is carried out by the technology, and the method is not influenced by tissue types, growth and development stages and external environments, and has simple technology, thereby having very wide application prospect in the aspect of variety identification.
Disclosure of Invention
The non-heading Chinese cabbage and red flowering Chinese cabbage are two excellent varieties sold in the market, and if seeds are mixed up, the appearance is difficult to distinguish and the period is long. Aiming at the technical problem, the invention provides a group of SSR molecular marker primers for identifying 'beautiful red rose' and 'October red' of the non-heading Chinese cabbage and the red flowering Chinese cabbage, and the developed SSR molecular marker primers and the detection method thereof can be used for distinguishing the 'beautiful red rose' from the 'October red'. The method is not influenced by the growth and development stages of plants and the environment, is simple to operate, is accurate and rapid to detect, and can effectively supervise and arbitrate when varieties are mixed up or disputed.
In order to achieve the purpose, the invention adopts the following technical scheme:
a group of SSR molecular marker primers for identifying 'beautiful red rose' and 'October red' of non-heading Chinese cabbage and red flowering Chinese cabbage have the following sequences:
BrcSSR60-F: 5’- GAGCATCATCACCAAGCTCA-3’（SEQ ID NO.1），
BrcSSR60-R: 5’- ATGTAACCGTTTCCTGGCTG-3’（SEQ ID NO.2）。
a molecular marking method for non-heading Chinese cabbage and red flowering Chinese cabbage 'Lianghong rose' and 'October red' comprises the following steps:
step 3, carrying out electrophoretic separation on the PCR amplification product obtained in the step 2 to obtain a separation band pattern of each sample;
and 4, analyzing the obtained electrophoresis separation result so as to distinguish the 'beautiful red rose' and 'October red' of the non-heading Chinese cabbage and the red vegetable.
A kit for identifying the 'beautiful red rose' and 'October red' of the non-heading Chinese cabbage and the red cabbage comprises the SSR molecular marker primer.
The SSR molecular marker primer and the detection method thereof disclosed by the invention can effectively distinguish two varieties of 'beautiful red rose' and 'October red' of the non-heading Chinese cabbage and the red flowering Chinese cabbage. The method is not influenced by the growth and development stages of plants and the environment, is simple to operate, is accurate and quick to detect, and can effectively supervise and arbitrate when varieties are mixed up or disputed.
Drawings
FIG. 1 shows the electrophoresis results of "Lianghong rose" and "October red" of the non-heading Chinese cabbage and the red flowering Chinese cabbage. Wherein: marker is DL2000 Marker, lane 1 is Lianghong rose, lane 2 is October Red.
Detailed Description
The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples were carried out according to the conventional conditions in the art.
68 pairs of primers are purposefully screened from a non-heading Chinese cabbage database, an SSR molecular marker primer database and SSR primers reported in a reference document, and the 68 pairs of SSR primers are synthesized to carry out primer polymorphism screening.
The specific screening process is as follows: firstly extracting DNA of 'beautiful red rose' and 'October red' of the red cabbage, then respectively carrying out PCR amplification on 68 pairs of SSR primers by using genomic DNA of the 'beautiful red rose' and the 'October red' as templates, carrying out polyacrylamide gel electrophoresis after the amplification, carrying out gel staining after the electrophoresis is finished, finally judging each pair of amplified strips, if polymorphism occurs in the two different varieties in the amplified strips of a certain pair of primers, determining that the pair of primers can be used for subsequent accurate identification tests, and otherwise, indicating that the pair of primers is not suitable for identifying the two varieties of the 'beautiful red rose' and the 'October red'. Finally, through the selection process, the SSR molecular marker primer BraSSR60 for subsequent identification tests is selected.
Through determination, in the amplification product,
the sequence (SEQ ID NO. 3) of the rose flower with beautiful red is as follows:
<xnotran> ATGTAACCGTTTCCTGGCTGCGCCTGAGCTTGTGTTGTTGTAGTTATCTGCTCCGAGCCTACTGGATTGTCATACCTGTTATCATCATCATCATCATCATCATCATATCACCAACGTAAATTATATGGTCCTCTTAATACCAAGAAACTAAAGAACCTCAAACAAAGACAAATCTTAGTAGCGAAGTAGACAAGCATGCATTTACCCTATCTGCAGTGTAGGATTGCATTCAAGAGGCTGGAATAGTCCTTGAGGCTGAGCTTGGTGATGATGCTC 276bp, </xnotran>
The sequence of October red (SEQ ID NO. 4) is:
<xnotran> ATGTAACCGTTTCCTGGCTGCGCCTGAGCTTGTGTTGTTGCAGTTATCTGCTCCGAGCCTACTGGATTGTCATACCTGTTATCATCATCATCATATCACGTAAATTATATGGTCCTCTTAATACCAAGAAACTAAAGAACCTCAAACAAAGACAAATCTTAGTAGCGAAGTAGACAAGCATGCATTTACCCTATCTGCAGTGTAGGATTGCATTCAAGAGGCTGGAATAGTCCTTGAGGCTGAGCTTGGTGATGATGCTC 260bp. </xnotran>
Example 1
The molecular marking method for identifying the 'beautiful red rose' and 'october red' of the non-heading Chinese cabbage and red flowering Chinese cabbage provided by the embodiment comprises the following steps:
(1) Extraction of genome DNA of non-heading Chinese cabbage
DNA was extracted according to the instructions of the plant genome DNA rapid extraction kit (purchased from Beijing Tiangen Biochemical technology Co., ltd.), as follows:
1) Taking about 100mg of fresh plant tissues, adding liquid nitrogen, and fully grinding;
2) Rapidly transferring the ground powder to a centrifuge tube which is pre-filled with 700 mu L of pre-heated buffer solution GP1 at 65 ℃ (adding mercaptoethanol into the pre-heated GP1 before the experiment to enable the final concentration to be 0.1%), rapidly reversing and uniformly mixing, and placing the centrifuge tube in a water bath at 65 ℃ for 20min;
3) Adding 700 mu L of chloroform, fully and uniformly mixing, and centrifuging at 12,000rpm for 5min;
4) Carefully transferring the upper-layer water phase obtained in the previous step into a new centrifuge tube, adding 700 mu L of buffer solution GP2, and fully and uniformly mixing;
5) Transferring the uniformly mixed liquid into an adsorption column CB3, centrifuging at 12,000rpm for 30s, and discarding the waste liquid;
6) Adding 500 muL of a buffer solution GD (firstly checking whether absolute ethyl alcohol is added before use) into an adsorption column CB3, centrifuging at 12,000rpm for 30s, pouring out waste liquid, and putting the adsorption column CB3 into a collecting pipe;
7) Adding 600 muL of rinsing liquid PW (firstly checking whether absolute ethyl alcohol is added before use) into the adsorption column CB3, centrifuging at 12,000rpm for 30s, pouring out waste liquid, and putting the adsorption column CB3 into a collecting pipe;
8) Repeating the operation step 7;
9) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm for 2min, and the waste liquid was decanted. Placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
10 Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 100 mu L of elution buffer TE into the middle part of the adsorption film, standing for 5min at room temperature, centrifuging for 2min at 12,000rpm, and collecting the solution into the centrifuge tube;
11 DNA was tested for purity and concentration, diluted to 50 ng/. Mu.L, and stored in a freezer at-20 ℃ until use.
(2) SSR amplification of genome of non-heading Chinese cabbage
An amplification system with the total volume of 10 mu L of SSR-PCR is adopted, and the method specifically comprises the following steps: mu.L of 2 XTaq MasterMix, 1. Mu.L of each forward and reverse primer at a concentration of 10. Mu.M, 1. Mu.L of template DNA at a concentration of 50 ng/. Mu.L, and 10. Mu.L of template DNA supplemented with deionized sterile water.
Wherein the forward primer is BraSSR60, and the forward primer is BraSSR 60-F5; reverse primer BraSSR-60-R is 3 '-ATGTAACCGTTTCCTGGCTG-5'.
The PCR amplification procedure was: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 1min, 28 cycles, extension at 72 deg.C for 7min, and storage at 4 deg.C.
(3) Polyacrylamide gel electrophoresis detection
After the PCR amplification is finished, 8% non-denaturing polyacrylamide gel electrophoresis is adopted, the electrophoresis buffer solution is 1 xTBE, 1.8 mu L of sample is loaded, after 150v electrophoresis is carried out for 1h 15min, silver staining is carried out for color development, electrophoresis images are photographed and collected, and the diversity is counted.
Specific gel solutions include: polyacrylamide gel solution (concrete solution preparation: 28.5g acrylamide and 7.5g methylene-bisacrylamide are weighed and added with water to dissolve and fix the volume to 100 mL) 7.5mL, 10 XTBE 3 mL, deionized sterile water 20 mL,10% ammonium persulfate solution 190 μ L and TEMED 40 μ L.
The specific glue dyeing process comprises the following steps:
1) Removing glue, adding 0.2% AgNO 3 Placing the solution on a shaking table, and slowly shaking for 8-10min;
2) Pouring AgNO 3 Adding deionized water into the solution, and slowly shaking on a shaking table for 1min for 20s;
3) Adding a color developing solution containing 1.5% of sodium hydroxide and 1.0% of formaldehyde, and slowly shaking on a shaking table until the color development is successful;
4) Pouring off the color developing solution, washing for three times by using deionized water, wrapping by using a preservative film, airing and observing.
As shown in figure 1, electrophoresis lanes of 'beautiful red rose' and 'October red' amplified by the primer BraSSR60 have obvious difference, namely, the SSR molecular marker primer has specificity to the 'beautiful red rose' and the 'October red' of the non-heading Chinese cabbage red vegetable, and can be used for identifying two varieties of the 'beautiful red rose' and the 'October red'.
Sequence listing
<110> Nanjing university of agriculture
<120> SSR molecular marker primers of non-heading Chinese cabbage and brassica rapa brassicae and application thereof
<130> 20210107
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gagcatcatc accaagctca 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgtaaccgt ttcctggctg 20
<210> 3
<211> 276
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgtaaccgt ttcctggctg cgcctgagct tgtgttgttg tagttatctg ctccgagcct 60
actggattgt catacctgtt atcatcatca tcatcatcat catcatatca ccaacgtaaa 120
ttatatggtc ctcttaatac caagaaacta aagaacctca aacaaagaca aatcttagta 180
gcgaagtaga caagcatgca tttaccctat ctgcagtgta ggattgcatt caagaggctg 240
gaatagtcct tgaggctgag cttggtgatg atgctc 276
<210> 4
<211> 260
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgtaaccgt ttcctggctg cgcctgagct tgtgttgttg cagttatctg ctccgagcct 60
actggattgt catacctgtt atcatcatca tcatatcacg taaattatat ggtcctctta 120
ataccaagaa actaaagaac ctcaaacaaa gacaaatctt agtagcgaag tagacaagca 180
tgcatttacc ctatctgcag tgtaggattg cattcaagag gctggaatag tccttgaggc 240
tgagcttggt gatgatgctc 260
Claims (3)
1. A group of SSR molecular marker primers for identifying 'beautiful red rose' and 'October red' of non-heading Chinese cabbage and red flowering Chinese cabbage have the following sequences:
BrcSSR60-F: 5’- GAGCATCATCACCAAGCTCA-3’
BrcSSR60-R: 5’- ATGTAACCGTTTCCTGGCTG-3’。
2. an identification method of 'beautiful red rose' and 'october red' of non-heading Chinese cabbage and red vegetable, which is characterized in that: the method comprises the following steps:
step 1, extracting genomic DNA of non-heading Chinese cabbage to be identified;
step 2, using the DNA extracted in the step 1 as a template, and carrying out PCR amplification on the DNA by using the primer of claim 1;
step 3, carrying out electrophoretic separation on the PCR amplification product obtained in the step 2 to obtain a separation band pattern of each sample;
and 4, analyzing the obtained electrophoresis separation result so as to distinguish the 'beautiful red rose' and 'October red' of the non-heading Chinese cabbage and the red vegetable.
3. A kit for identifying the 'beautiful red rose' and 'october red' of the non-heading Chinese cabbage and red flowering Chinese cabbage is characterized in that: comprising the primer of claim 1.
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| CN101260433B (en) * | 2008-04-29 | 2010-11-17 | 上海交通大学 | Method for marking non-heading cabbage molecule by utilizing SAMPL technique |
| CN109852717B (en) * | 2018-12-19 | 2022-04-08 | 南京农业大学 | Molecular marking method for identifying non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star |
| CN113453540B (en) * | 2019-02-15 | 2024-03-05 | 巴斯夫农业种子解决方案美国有限责任公司 | Anti-clubroot brassica plants |
| CN110894541B (en) * | 2019-12-27 | 2022-05-17 | 山西农业大学 | Functional identification system of anthocyanin synthesis regulation gene based on molecular marker and application |
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