CN112575117A - 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 PDF

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CN112575117A
CN112575117A CN202110018658.5A CN202110018658A CN112575117A CN 112575117 A CN112575117 A CN 112575117A CN 202110018658 A CN202110018658 A CN 202110018658A CN 112575117 A CN112575117 A CN 112575117A
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刘同坤
王新雅
何英子
李英
侯喜林
张昌伟
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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'-GAGCATCATCACCAAGCTCA-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

SSR molecular marker primers of non-heading Chinese cabbage and red-flowering Chinese cabbage and application thereof
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) The brassica chinensis and the brassica campestris belong to the variety of brassica napus subsp. 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 1, extracting genomic DNA of non-heading Chinese cabbage to be identified;
step 2, taking the DNA extracted in the step 1 as a template, and carrying out PCR amplification on the DNA by using the primer;
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 rapid 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, procedures, or conditions of the 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 are in accordance with 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, an SSR molecular marker primer BraSSR60 for a subsequent identification test is selected.
Figure DEST_PATH_IMAGE001
Through determination, in the amplified product,
the sequence of the rose flower with beautiful red (SEQ ID NO. 3) is as follows:
ATGTAACCGTTTCCTGGCTGCGCCTGAGCTTGTGTTGTTGTAGTTATCTGCTCCGAGCCTACTGGATTGTCATACCTGTTATCATCATCATCATCATCATCATCATATCACCAACGTAAATTATATGGTCCTCTTAATACCAAGAAACTAAAGAACCTCAAACAAAGACAAATCTTAGTAGCGAAGTAGACAAGCATGCATTTACCCTATCTGCAGTGTAGGATTGCATTCAAGAGGCTGGAATAGTCCTTGAGGCTGAGCTTGGTGATGATGCTC is 276bp in total,
the sequence of October red (SEQ ID NO. 4) is:
ATGTAACCGTTTCCTGGCTGCGCCTGAGCTTGTGTTGTTGCAGTTATCTGCTCCGAGCCTACTGGATTGTCATACCTGTTATCATCATCATCATATCACGTAAATTATATGGTCCTCTTAATACCAAGAAACTAAAGAACCTCAAACAAAGACAAATCTTAGTAGCGAAGTAGACAAGCATGCATTTACCCTATCTGCAGTGTAGGATTGCATTCAAGAGGCTGGAATAGTCCTTGAGGCTGAGCTTGGTGATGATGCTC total 260 bp.
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 ℃ (mercaptoethanol is added into the pre-heated GP1 before the experiment to enable the final concentration to be 0.1%), rapidly reversing and uniformly mixing, and then placing the centrifuge tube in a water bath at 65 ℃ for 20 min;
3) adding 700 mu L of chloroform, fully and uniformly mixing, and centrifuging at 12,000rpm for 5 min;
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 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 collection 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 discarded. 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 centrifugal tube, suspending and dripping 100 mu L of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 5min, centrifuging at 12,000rpm for 2min, and collecting the solution into the centrifugal tube;
11) the DNA was diluted to 50 ng/. mu.L by purity and concentration detection, and stored in a freezer at-20 ℃ for further 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 and reverse primers are BraSSR60, and the forward primer is BraSSR60-F: 5'-GAGCATCATCACCAAGCTCA-3'; reverse primer BraSSR-60-R: 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 electrophoresis is carried out for 1h 15min at 150v, silver staining is carried out for color development, electrophoresis images are photographed and collected, and the diversity is counted.
Specific gel solutions include: 7.5mL of polyacrylamide gel solution (specific solution preparation: 28.5g of acrylamide and 7.5g of methylene-bisacrylamide are weighed and dissolved by adding water to be 100 mL), 10 xTBE 3 mL, 20 mL of deionized sterile water, 190 muL of 10% ammonium persulfate solution and 40 muL of TEMED.
The specific glue dyeing process comprises the following steps:
1) after the glue is removed, 0.2 percent AgNO is added3Placing the solution on a shaking table and slowly shaking for 8-10 min;
2) pouring AgNO3Adding deionized water into the solution, and slowly shaking on a shaking table for 1min for 20 s;
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' are obviously different when the primer BraSSR60 is used for amplification, namely, the SSR molecular marker primer has specificity on the 'beautiful red rose' and the 'October red' of the red cabbage, 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. a molecular marking method for non-heading Chinese cabbage and red-flowering Chinese cabbage "Lianghong rose" and "October red", 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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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101260433A (en) * 2008-04-29 2008-09-10 上海交通大学 Method for marking non-heading cabbage molecule by utilizing SAMPL technique
CN109852717A (en) * 2018-12-19 2019-06-07 南京农业大学 It is a kind of for identifying the molecule labelling method of the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star
CN110894541A (en) * 2019-12-27 2020-03-20 山西农业大学 Functional molecular marker of anthocyanin synthesis regulation gene of brassica oleracea and application of functional molecular marker
WO2020168166A2 (en) * 2019-02-15 2020-08-20 BASF Agricultural Solutions Seed US LLC Clubroot resistant brassica plants

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101260433A (en) * 2008-04-29 2008-09-10 上海交通大学 Method for marking non-heading cabbage molecule by utilizing SAMPL technique
CN109852717A (en) * 2018-12-19 2019-06-07 南京农业大学 It is a kind of for identifying the molecule labelling method of the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star
WO2020168166A2 (en) * 2019-02-15 2020-08-20 BASF Agricultural Solutions Seed US LLC Clubroot resistant brassica plants
CN110894541A (en) * 2019-12-27 2020-03-20 山西农业大学 Functional molecular marker of anthocyanin synthesis regulation gene of brassica oleracea and application of functional molecular marker

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIHUANG等: "Construction of a New Molecular Barcode for Discriminating Plants and Animals with a Close Genetic Relationship", 《JOURNAL OF INTEGRATIVE AGRICULTURE》 *

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