CN108018290B - Plant anthocyanin synthesis control gene and application thereof - Google Patents
Plant anthocyanin synthesis control gene and application thereof Download PDFInfo
- Publication number
- CN108018290B CN108018290B CN201711269495.8A CN201711269495A CN108018290B CN 108018290 B CN108018290 B CN 108018290B CN 201711269495 A CN201711269495 A CN 201711269495A CN 108018290 B CN108018290 B CN 108018290B
- Authority
- CN
- China
- Prior art keywords
- chinese cabbage
- gene
- purple
- molecular marker
- marker2
- 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.)
- Active
Links
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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
-
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a plant anthocyanin synthesis control gene and application thereof. The disclosed gene sequence is shown in SEQ ID No. 1. The gene has an insertion mutation of a large fragment with the size of 3772bp in a first intron of white heading Chinese cabbage, and the gDNA sequence of the gene is shown as SEQ ID No. 5. Three pairs of specific PCR primers, namely Marker1, Marker2 and Marker8, are designed according to mutation in an intron in a gDNA nucleotide sequence of the gene, wherein the Marker1 and the Marker2 are codominant markers, and the Marker8 is a maternal Marker. Through PCR amplification and agarose gel electrophoresis identification of DNA of the target Chinese cabbage, the Marker1 and the Marker2 can quickly identify purple heart heterozygous strains and homozygous strains in the separated population, and the Marker8 can also distinguish the purple heart heterozygous strains and the homozygous strains of the separated population by combining the leaf ball characteristics.
Description
Technical Field
The invention belongs to the field of vegetable breeding and molecular genetics, and particularly relates to a gene and a molecular marker for controlling purple character of purple-heart Chinese cabbage and application thereof.
Background
Chinese cabbage (Brassica rapa L.) belongs to Brassica Brassica subspecies of Brassicaceae, is one of main vegetable crops originated in China, is also one of the vegetable crops with the largest planting area in China, and is closely related to the health of daily life of people. With the improvement of living standard of people, in recent years, the requirements of people on the quality of Chinese cabbages are increasingly improved, and the breeding of orange and purple Chinese cabbages is more and more valued by domestic and foreign scholars and breeders.
The purple Chinese cabbage is rich in anthocyanin, so that the stress resistance of the Chinese cabbage crops can be improved, the nutritional value of the health of human beings is extremely high, and the purple Chinese cabbage has the effects of resisting cancer, resisting oxidation and cardiovascular diseases and is widely concerned at home and abroad. However, the leaf bulb color of the purple-heart Chinese cabbage can be shown only after the leaf bulb is mature, and the leaf bulb is cut in the field and observed plant by plant, so that the operation has great destructiveness on germplasm materials, wastes time and labor, and greatly delays the breeding process. Therefore, cloning and controlling the gene of purple leaf ball of Chinese cabbage and researching the simple and accurate detection method are very important for innovating germplasm resources and accelerating breeding pace.
The applicant screens and clones the gene controlling the purple leaf ball of the Chinese cabbage by adopting a map-based cloning and candidate gene differential expression analysis method and combining with a sequencing result of the Chinese cabbage, develops a molecular marker and a dominant marker coseparated with the purple gene of the leaf ball of the Chinese cabbage according to the sequence difference of the gene, and lays a foundation for forming a molecular mechanism and molecular assisted breeding of the purple leaf ball of the Chinese cabbage and accelerating a breeding process.
Disclosure of Invention
The invention provides a plant anthocyanin synthesis control gene.
The sequence of the plant anthocyanin synthesis control gene provided by the invention is as follows:
(1) 1, SEQ ID NO; or
(2) 1, nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein; or
(3) A nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence of (1) or (2) and expresses the same functional protein.
The invention also provides an expression cassette, an expression vector, a cloning vector or an engineering bacterium comprising a nucleic acid comprising the invention.
The application of the gene of the invention in synthesizing anthocyanin in plants, wherein the plants comprise: chinese cabbage, non-heading Chinese cabbage, Arabidopsis thaliana, etc.
The invention also provides a construction method of the transgenic plant, which utilizes a gene engineering means to over-express the gene in a target plant and screen a positive transgenic plant.
The invention also provides a molecular marker related to the Chinese cabbage anthocyanin synthesis gene, which is characterized in that the molecular marker comprises an insertion mutation of a 3772bp fragment, and the gDNA sequence of the molecular marker is shown as SEQ ID No. 5.
The invention also provides a PCR detection kit for identifying the purple-heart Chinese cabbage material, which is characterized by comprising an upstream primer and a downstream primer for amplifying the molecular marker in the claim 5:
an upstream primer: 5'-ATGGAGGGTTCGTCCCAAG-3'
A downstream primer: 5'-TCAAGTTCCAGTTTCTCCATCC-3' are provided.
The invention also provides a PCR detection kit for identifying the purple-heart Chinese cabbage material, which is characterized by comprising an upstream primer and a downstream primer for amplifying the molecular marker in the claim 5:
Marker1-F：5’-TGGTGTACTTTGATCCTTCGTG-3’
Marker1-R：5’-ACTGCATTCGCGTCTCCTAC-3’
Marker2-F：5’-CTTTTCTGCACGAACCCG-3’
Marker2-R：5’-ATTCGCGTCTCCTACTCCATAT-3’
Marker8-F：5’-AAAAGGTGCATGGACTGCT-3’
Marker8-R：5’-TGTTTTGGTGTCGATGAAGAAG-3’。
the molecular marker disclosed by the invention is applied to molecular marker assisted breeding of purple Chinese cabbage.
The molecular marker disclosed by the invention is applied to identification of purple Chinese cabbage materials.
In the research of the invention, the specific PCR primer and the detection method designed by the inventor can not only carry out PCR amplification on DNA extracted from the seedling stage of the Chinese cabbage, but also can be used for screening a large amount of Chinese cabbage germplasm resources. The PCR Primer is designed based on the sequence difference between BrMYB2 gene and its allele of purple-heart Chinese cabbage by using Primer design software Primer 5.0. The Chinese cabbage variety and germplasm resource containing the gene can be quickly detected according to the existence, the length and the size of the amplified band, and the detection accuracy is high, the operation is simple and the cost is low. If a Chinese cabbage variety or germplasm resource contains the gene, the primer can be used for amplifying a specific segment with the length of 5442bp or 1665bp, wherein the large band is a plant with a white phyllosphere genetic background, and the small band is a plant with a purple phyllosphere genetic background.
The invention obtains a Chinese cabbage purple heart leaf ball character (/ related to anthocyanin synthesis) gene sequence and a detection method for rapidly detecting whether the gene and allele thereof are contained. Two pairs of codominant markers capable of carrying out heterozygous plant identification at the seedling stage and one pair of maternity bias markers capable of identifying the heterozygous plants by combining with the leaf ball character at the ball stage are developed, and the method can be used for quickly detecting the Chinese cabbage plants containing the gene, and has the advantages of high detection accuracy, simple operation and low cost. The method makes it possible to screen and identify the genotype in large quantity in purple Chinese cabbage germplasm resources, and can also be used as a marker character to be applied to purity identification of purple Chinese cabbage hybrids. The alleles (BrMYB2 and Brmyb2) for controlling purple character of the purple Chinese cabbage obtained by the invention can be used for mechanism research of a molecular mechanism for synthesizing anthocyanin of the purple Chinese cabbage.
Drawings
FIG. 1 shows the result of the amplification of the gene-specific PCR primers for BrMYB2/Brmyb2 of the present invention in cDNA. W represents the PCR amplification product of white cabbage containing Brmyb2 gene, P represents the amplification product of purple-heart cabbage containing BrMYB2 gene, and M represents the amplification product of the parent of purple cabbage containing BrMYB2 gene; m1 is DNA ladder.
FIG. 2 shows the result of amplification of BrMYB2/Brmyb2 gene in gDNA, where a 1665bp band is amplified from Chinese cabbage containing BrMYB2 gene, and a 5442bp band is amplified from common Chinese cabbage containing Brmyb2 gene. W represents the PCR amplification product of white cabbage containing Brmyb2 gene, P represents the amplification product of purple-heart cabbage containing BrMYB2 gene, and M represents the amplification product of the parent of purple cabbage containing BrMYB2 gene; m1 is DNA ladder; m2 is DNA ladder.
FIG. 3 shows the co-dominant Marker amplified by Marker1 and Marker2, the cabbage containing BrMYB2 gene has a band of about 300bp (306 bp for Marker1 and 348bp for Marker2), and the cabbage containing BrMYB2 gene has a band of about 4000bp (4081 bp for Marker1 and 4123bp for Marker 2). And the heterozygous strain can amplify the bands with two sizes. W represents the PCR amplification product of white cabbage containing Brmyb2 gene, P represents the amplification product of purple-heart cabbage containing BrMYB2 gene, and W + P represents the amplification product of heterozygous purple-heart cabbage.
FIG. 4 shows the dominant Marker amplified by Marker8, a 972bp band is amplified from Chinese cabbage containing Brmyb2 gene, while the Chinese cabbage containing BrMYB2 gene has no specific amplification product. If the strip can be amplified from the Chinese cabbage with the purple leaf ball character, the plant is a heterozygous plant; w represents the PCR amplification product of white cabbage containing Brmyb2 gene, P represents the amplification product of purple-heart cabbage containing BrMYB2 gene, and W + P represents the amplification product of heterozygous purple-heart cabbage.
Detailed Description
Materials used in the following examples:
the purple-heart Chinese cabbage strain 11S96 (Yasuan, Zhangha, Haoqiong, Zhangkeke, Shijiang super, anthocyanin accumulation characteristics and related gene expression analysis [ J ] gardening bulletin, 2012Vol.39(11): 2159-. The inventor researches an F1 group generated by hybridizing the strain with orange Chinese cabbage '14S 162' (14S162 is selected by selfing orange Chinese cabbage 'S941-6' multi-generation single plant, 'S941-6' is disclosed in ZL 200610104743.9) as a female parent, and then obtains a corresponding F2 group by single plant selfing. In the F2 colony, 10 purple and purple-free strains are selected to construct DNA pools, a BSA method is adopted to screen closely linked molecular markers in the whole genome, and as a result, a target gene is positioned on an A7 chromosome and is further cloned to obtain a gene sequence of the purple-heart Chinese cabbage, which is shown as SEQ ID No. 1. The allele sequence of white heading Chinese cabbage is shown as SEQ ID No. 5.
Example 1: extraction of Chinese cabbage genome total RNA
1) Grinding the material preserved at ultralow temperature by using liquid nitrogen, adding about 50mg of ground powder into 1mL of Bizol solution, violently mixing uniformly, and standing on ice for 15 min; centrifuging at 12000 Xg for 15min at 4 deg.C, collecting supernatant, adding 200 μ L chloroform, covering tube cover, shaking vigorously, and standing on ice for 15 min; centrifuging at 12000 Xg for 15min at 4 deg.C, carefully sucking supernatant into another centrifuge tube, adding equal volume precooled (-20 deg.C) isopropanol, turning upside down for several times, mixing, and precipitating at-20 deg.C for more than 2 h;
2) centrifugation was carried out at 10000 Xg for 10min at 4 ℃. The supernatant was discarded, and 1mL of 75% ethanol (killed enzyme-free ddH) was added2O preparation, namely containing 0.1 percent of DEPC, and sterilizing for 40 min); discarding supernatant, air drying RNA on ultra-clean bench for about 10min, adding 50-100 μ L enzyme-free ddH2Dissolving O; RQDNase I (TaKaRa, Dalian) is used for removing genomic DNA in RNA, and the specific method is as follows: the following reaction components were added to the centrifuge tube containing the crude RNA: 10 × Reaction Buffer10 μ L, RNA Inhibitor (40U/μ L)1 μ L, DNase I (5U/μ L)6 μ L, adding enzyme-free ddH2O to total volume of 100 μ L, mixing, centrifuging for a short time, and reacting at 37 deg.C for 30 min;
3) adding 300 μ L chloroform/isoamyl alcohol (24/1), vigorously mixing and shaking, adding 300 μ L enzyme-free ddH2O, mixing well, standing on ice for 15min, shaking once every few minutes; centrifuging at 12000rpm for 15min at 4 deg.C, carefully sucking supernatant, transferring into another centrifuge tube, adding equal volume of precooled (-20 deg.C) isopropanol, and precipitating at-20 deg.C for more than 2 h; centrifuging at 4 deg.C and 10000rpm for 10min, recovering precipitate, removing supernatant, and washing precipitate with 75% ethanol (standing at 4 deg.C) twice; the synthesis of the clean bench cDNA was carried out using TaKaRa's one-strand synthesis kit (D6110A), and the following operations were carried out:
4) the following reaction components were added to the PCR tube: about 5. mu.g total RNA, 1. mu.L oligo (dT)18 (50. mu. mol/L), 1. mu.L dNTP (10mmol/L), enzyme-free ddH2O to make up 10. mu.L; denaturation at 65 deg.C for 5min, and placing on ice for 2 min. The following ingredients were continuously added to the PCR tube: 5 × RT Buffer 4 μ L, RNase Inhibitor (40U/. mu.L) 0.5 μ L, PrimeScript Rtase (200U/uL)1 μ L or M-M μ LV Reverse Transcriptase (5U/. mu.L) 1 μ L, dd H2O 4.5 μ L, total volume 20 μ L, blending and centrifuging, 42 deg.C bath for 1H, 70 deg.C 15min to inactivate enzyme, then cooling at 4 deg.C, and standing for use.
Example 2: extraction of Chinese cabbage genome total DNA
1) Taking 0.2g of fresh tender leaf of Chinese cabbage with main veins removed, stuffing into 2mL centrifuge tube filled with steel balls (the steel balls need to be cleaned by 75% alcohol by mass), placing into liquid nitrogen for quick freezing, and grinding into powder by using a tissue grinder;
2) to the centrifuge tube was added 700 μ l of CTAB extract preheated at 65 ℃ (CTAB: 2%, Tris-HCl (pH 8.0): 100mmol/L, EDTA: 20mmol/L, NaCl: 1.4mol/L), then adding 10 mu L of beta-mercaptoethanol, and quickly mixing uniformly;
3) then putting the centrifuge tube into a 65 ℃ oven, shaking once every 5-10min in the middle, and carrying out warm bath for 45 min;
4) the centrifuge tube was removed and an equal volume of phenol was added: chloroform: shaking the mixture of isoamyl alcohol (25:24:1) for 15min, and centrifuging at 10000r/min for 10min at normal temperature;
5) the upper liquid phase (about 700. mu.l) was transferred to another centrifuge tube, and an equal volume of a mixed solution of chloroform and isoamyl alcohol (chloroform: isoamyl alcohol 24:1) gently shaking for 10min, and centrifuging at room temperature at 10000r/min for 10 min;
6) taking supernatant (about 500 mu l), adding 2 times volume of precooled absolute ethyl alcohol, gently mixing to make DNA agglomerate, precipitating at-20 ℃ for 30min, and centrifuging at 8000r/min at 4 ℃ for 5 min;
7) discarding the supernatant, adding 500 μ l of 75% ethanol by mass, washing the precipitate for 2 times, and air-drying the precipitate at room temperature;
8) adding 500 μ l sterile distilled water to dissolve DNA, adding 0.29 μ l RNaseA (10 μ g/μ l), mixing, centrifuging, and keeping the temperature at 37 deg.C for 30 min;
9) adding 50 mu L of NaAc solution with the concentration of 3mol/L and 2 times of precooled absolute ethyl alcohol, and gently mixing uniformly to enable DNA to agglomerate and precipitate for 30min at the temperature of minus 20 ℃;
10) centrifuging at 8000r/min for 5min at 4 ℃, discarding the supernatant, adding 75% ethanol by mass, cleaning the precipitate for 1-2 times, air drying the precipitate at room temperature, adding 400-500 μ l of enzyme-free ddH2And dissolving O for use.
Example 3: PCR amplification and gene detection of purple gene of Chinese cabbage
The total DNA in the example 2 is used as a template to respectively obtain purple genes of the purple Chinese cabbage, purple character genes in parents of the purple Chinese cabbage, alleles of the purple genes in the white leaf ball Chinese cabbage and detection of the genes.
1)20 μ l PCR reaction system: 50 ng/. mu.l of template DNA was 2. mu.l, 2 XTAQA Master Mix 10. mu.l, 10. mu.M of upstream and downstream primers 1. mu.l each, and the reaction system was supplemented with sterile deionized water to 20. mu.l.
The primer sequence is as follows:
an upstream primer: 5'-ATGGAGGGTTCGTCCCAAG-3'
A downstream primer: 5'-TCAAGTTCCAGTTTCTCCATCC-3'
2) The PCR amplification reaction is carried out on a PCR instrument
The temperature and reaction conditions were: pre-denaturation at 94 ℃ for 3 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 35 cycles; finally, the extension is carried out for 8min at 72 ℃ and the product is stored at 4 ℃. Wherein, the extension at 72 ℃ for 4min is used for cloning and detecting the gene in gDNA, and the extension at 72 ℃ for 1min is used for cloning and detecting the gene in cDNA.
The length of the PCR amplification product in the gene cDNA is 744bp in three materials (figure 1), and the sequencing result is shown in SEQ ID No. 1; the length of the PCR amplification product of the gene in gDNA is 1665bp and 5442bp in purple and non-purple materials respectively, and the gene sequencing result is shown as SEQ ID No. 5.
The ORF sequence of the gene was obtained using DNAMAN software and translated into an amino acid sequence as shown in SEQ ID No. 3.
When the gene is used for detecting the gene, an obtained amplification product is loaded on 1.5% agarose gel and electrophoresed, and is imaged on a gel imaging instrument after being dyed by ethidium bromide, and the result is shown in figure 2, a 1665bp strip is amplified from the Chinese cabbage containing the BrMYB2 gene, and a 5442bp strip is amplified from the Chinese cabbage containing the Brmyb2 gene.
Example 4: detection application of purple character codominant marker and maternal bias marker of Chinese cabbage
According to the obtained large fragment insertion in the gDNA sequence of the gene, a pair of codominant markers 2 (Marker1 and Marker2) capable of detecting hybrid strains is designed on conserved sequences at two ends of the insertion, and a pair of partial maternal markers (Marker8) capable of combining with the characters of purple Chinese cabbage segregation population is designed on the conserved sequence in the middle of the insertion. And (3) detecting the purple character codominant marker and the partial maternal marker of the Chinese cabbage by using the total DNA extracted in the example 2 as a template.
1)20 μ l PCR reaction system: 50 ng/. mu.l of template DNA was 2. mu.l, 2 XTAQA Master Mix 10. mu.l, 10. mu.M of upstream and downstream primers 1. mu.l each, and the reaction system was supplemented with sterile deionized water to 20. mu.l.
The primer sequence is as follows:
Marker1-F：5’-TGGTGTACTTTGATCCTTCGTG-3’
Marker1-R：5’-ACTGCATTCGCGTCTCCTAC-3’
Marker2-F：5’-CTTTTCTGCACGAACCCG-3’
Marker2-R：5’-ATTCGCGTCTCCTACTCCATAT-3’
Marker8-F：5’-AAAAGGTGCATGGACTGCT-3’
Marker8-R：5’-TGTTTTGGTGTCGATGAAGAAG-3’。
2) the PCR amplification reaction is carried out on a PCR instrument
The PCR amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 35 cycles; finally, the extension is carried out for 8min at 72 ℃ and the product is stored at 4 ℃. Wherein the extension at 72 ℃ for 4min is used for detecting Marker1 and Marker2 in gDNA, and the extension at 72 ℃ for 1min is used for detecting Marker8 in gDNA.
For detection of co-dominant labelled amplification products, the samples were loaded on a 1.5% agarose gel and electrophoresed, stained with ethidium bromide and imaged on a gel imager. The result of co-dominant labeling is shown in FIG. 3, the cabbage containing the BrMYB2 gene amplified a band of about 300bp (Marker1 is 306bp, Marker2 is 348bp), while the cabbage containing the Brmyb2 gene amplified a band of about 4000bp (Marker1 is 4081bp, Marker2 is 4123 bp). And the heterozygous strain can amplify the bands with two sizes.
For detection of the maternally-biased labeled amplification products, the samples were loaded on a 1.5% agarose gel and electrophoresed, stained with ethidium bromide and imaged on a gel imager. As shown in FIG. 4, the cabbage containing the Brmyb2 gene amplified a 972bp band, while the cabbage containing the BrMYB2 gene did not have a specific amplification product. If the Chinese cabbage with the purple leaf ball character can also amplify the strip, the plant is a heterozygous plant.
Example 5: application of purple character codominant marker and maternity bias marker of Chinese cabbage in auxiliary selection
The method is characterized in that an F2 segregation population hybridized by purple-heart Chinese cabbages and common Chinese cabbages is taken as a target, and auxiliary selection of the purple-heart Chinese cabbages is carried out by utilizing a codominant Marker2 pair (Marker1 and Marker2) and a pair (Marker8) of maternity biased markers.
1)20 μ l PCR reaction system: 50 ng/. mu.l of template DNA was 2. mu.l, 2 XTAQA Master Mix 10. mu.l, 10. mu.M of upstream and downstream primers 1. mu.l each, and the reaction system was supplemented with sterile deionized water to 20. mu.l.
The primer sequence is as follows:
Marker1-F：5’-TGGTGTACTTTGATCCTTCGTG-3’
Marker1-R：5’-ACTGCATTCGCGTCTCCTAC-3’
Marker2-F：5’-CTTTTCTGCACGAACCCG-3’
Marker2-R：5’-ATTCGCGTCTCCTACTCCATAT-3’
Marker8-F：5’-AAAAGGTGCATGGACTGCT-3’
Marker8-R：5’-TGTTTTGGTGTCGATGAAGAAG-3’。
2) the PCR amplification reaction is carried out on a PCR instrument
The PCR amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; then denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 35 cycles; finally, the extension is carried out for 8min at 72 ℃ and the product is stored at 4 ℃. Wherein the extension at 72 ℃ for 4min is used for detecting Marker1 and Marker2 in gDNA, and the extension at 72 ℃ for 1min is used for detecting Marker8 in gDNA.
The amplification products were loaded on a 1.5% agarose gel and electrophoresed, stained with ethidium bromide and imaged on a gel imager.
3) PCR amplification results
The marking results of the detection of 400F 2 segregating group single plants are completely consistent with the ball color, which indicates that the auxiliary selection by utilizing the purple character codominant mark and the partial maternal mark of the Chinese cabbage is feasible, and particularly, the homozygous and heterozygous purple-heart single plants can be simply and quickly distinguished by combining the partial maternal mark Marker8 with the phenotype.
Nucleotide sequence list electronic file
<110> northwest agriculture and forestry science and technology university
<120> plant anthocyanin synthesis control gene and application thereof
<141>
<160>
<210>1
<211>744
<212>DNA
<213> BrMYB2 gene sequence
<220>
<223>
<400>1
ATGGAGGGTTCGTCCCAAGGGTTGAAAAAAGGTGCATGGACTGCTGAAGAAGATAATCTCTTGAGGCAATGCATTGATAAGTATGGAGAAGGGAAATGGCACCAAGTTCCTTTAAGAGCTGGTCTAAATCGGTGCAGGAAGAGTTGTAGACTAAGATGGTTGAACTATTTGAAGCCAAGTATCAAGAGAGGAAAACTCAACTCCGATGAAGTTGATCTTCTTATTCGCCTTCATAAGCTTTTAGGAAACAGGTGGTCTTTAATTGCTGGTAGATTACCCGGTCGGACCGCCAATGACGTCAAAAATTACTGGAACACCCATTTGAGTAAGAAACATGAACCGGGTTGTAAGACCCAGATGAAAAAGAGAAACATTCCTTGCTCTTATACCACACCAGCCCAAAAAATCGACGTTTTCAAACCTCGACCTCGATCCTTCACCGTTAACAGCGGCTGCAGCCATAATAATGGCATGCCAGAAGCTGGCATTGTTCCTCTATGCCTTGGACACAACGATACTAATAATGTTTCTGAAAATATAATCACATGTAACAAAGATGATGATAAATCTGAGCTTGTTAGTCATTTAATGGATGGTCAGAATAGGTGGTGGGAAAGTTTGCTAGATGAGAGCCAAGATCCAGCTGCGCTCTTTCCAGAAACTACAGCAATAAAAAAGGGCGCAACCTCCGCGTTTGACGTTGAGCAACTTTGGAGCCTGTTGGATGGAGAAACTGGAACTTGA
<210>2
<211>744
<212>DNA
<213> Brmyb2 Gene sequence
<220>
<223>
<400>2
ATGGAGGGTTCGTCCCAAGGGTTGAAAAAAGGTGCATGGACTGCTGAAGAAGATAATCTCTTGAGGCAATGCATTGATAAGTATGGAGAAGGGAAATGGCACCAAGTTCCTTTAAGAGCTGGTCTAAATCGGTGCAGGAAGAGTTGTAGACTAAGATGGTTGAACTATTTGAAGCCAAGTATCAAGAGAGGAAAACTCAACTCCGATGAAGTTGATCTTCTTATGCGCCTTCATAAGCTTTTAGGAAACAGGTGGTCTTTAATTGCTGGTAGATTACCCGGTCGGACCGCCAATGACGTCAAAAATTACTGGAACACCCATTTGAGTAAGAAACATGAACCGGGTTGTAAGACCCAGATGAAAAAGAGAAACATTCCTTGCTCTTATACCACACCAGCCCAAAAAATCGACGTTTTCAAACCTCGACCTCGATCCTTCACCGTTAACAGCGGCTGCAGCCATAATAATGGCATGCCAGAAGCTGACATTGTTCCTCTATGCCTTGGACACAACGATACTAATAATGTTTCTGAAAATATAATCACATGTAACAAAGATGATGATAAATCTGAGCTTGTTAGTCATTTAATGGATGGTCAGAATAGGTGGTGGGAAAGTTTGCTAGATGAGAGCCAAGATCCAGCTGCGCTCTTTCCAGAAACTACAGCAATAAAAAAGGGCGCAACCTCCGCGTTTGACGTTGAGCAACTTTGGAGCCTGTTGGATGGAGAAACTGGAACTTGA
<210>3
<211>247
<212> amino acid
<213> Brmyb2 amino acid sequence
<220>
<223>
<400>3
MEGSSQGLKKGAWTAEEDNLLRQCIDKYGEGKWHQVPLRAGLNRCRKSCRLRWLNYLKPSIKRGKLNSDEVDLLIRLHKLLGNRWSLIAGRLPGRTANDVKNYWNTHLSKKHEPGCKTQMKKRNIPCSYTTPAQKIDVFKPRPRSFTVNSGCSHNNGMPEAGIVPLCLGHNDTNNVSENIITCNKDDDKSELVSHLMDGQNRWWESLLDESQDPAALFPETTAIKKGATSAFDVEQLWSLLDGETGT
<210>4
<211>247
<212>DNA
<213> nucleotide sequence of upstream primer CVA10-F2
<220>
<223>
<400>4
MEGSSQGLKKGAWTAEEDNLLRQCIDKYGEGKWHQVPLRAGLNRCRKSCRLRWLNYLKPSIKRGKLNSDEVDLLMRLHKLLGNRWSLIAGRLPGRTANDVKNYWNTHLSKKHEPGCKTQMKKRNIPCSYTTPAQKIDVFKPRPRSFTVNSGCSHNNGMPEADIVPLCLGHNDTNNVSENIITCNKDDDKSELVSHLMDGQNRWWESLLDESQDPAALFPETTAIKKGATSAFDVEQLWSLLDGETGT
<210>5
<211>1665
<212>DNA
<213> BrMYB2 molecular marker
<220>
<223>
<400>5
ATGGAGGGTTCGTCCCAAGGGTTGAAAAAAGGTGCATGGACTGCTGAAGAAGATAATCTCTTGAGGCAATGCATTGATAAGTATGGAGAAGGGAAATGGCACCAAGTTCCTTTAAGAGCTGGTATGTCTTTTTTTTTGATAACATAAGAGCTGGTATGCTACTTTTATTAATTTTCACACACACACACACACACATATAACTAATAAGTACGTATATTCTTTTTATTTTTCAGTACATTTATTCTCTTTCTCTCTGTCTAGTATTAGGAAATTAATTAACACCGGGGTACACAATCATTGTTTTTCTTTTCGTTTTAATGAAGGAATCATAGATTCATATGTTCTAATGTTTTTCATGAAAAAAAAAACATTTGCGTTCTTCATGTTTAATTACAAAGCGAGAAAATGTCAACTCTCTTTATTGATTCGTCGTTTTTCTTTTTTTTTTTGAGAAAGAGCTTTTTTGATTAGTGAACTTTTCTGCACGAACCCGTGTGTTTGTGTGGAATATGTTGTTTATTCTGGTGTACTTTGATCCTTCATGATAAAATTTTACTTCCTTTGTTATTAAATATAAGATATTTTGGTAGAAGCAAACATATTAAGAAAACTATTTTTTGTCTAGAAAATATCATTAAAACTATAAATTAATGGTGTTCAACCAATTACAAAATAGACTATTAAAATATGATTGGGTTCACAGTTTTTAATAAAGTAAAAGTTACCTAGAAAATTGAAAACATTTTATATATTGGATAATTAAAACATCAAAATTCAATAAAACATCCTATTTTTAGGAACATATGGAGTAGGAGACGCGAATGCAGTTTTTGCTCGTTCTTTTAATAATATTAAATGTCAATTATTGGTTTTGTAGGTCTAAATCGGTGCAGGAAGAGTTGTAGACTAAGATGGTTGAACTATTTGAAGCCAAGTATCAAGAGAGGAAAACTCAACTCCGATGAAGTTGATCTTCTTATTCGCCTTCATAAGCTTTTAGGAAACAGGTTTACATTCCAGACACAAATTCAACTGTATTTCGTATCCTCATTCGGTCTAATCTAATCATTTGATTTGTTTTTTTTTTTGATAAAAATACTTAAATTTATTTCATATGTAAATGATCCATTACTAAGTCAAATATATCCCTAATTTTTCAAATGCATGCTTAGGTGGTCTTTAATTGCTGGTAGATTACCCGGTCGGACCGCCAATGACGTCAAAAATTACTGGAACACCCATTTGAGTAAGAAACATGAACCGGGTTGTAAGACCCAGATGAAAAAGAGAAACATTCCTTGCTCTTATACCACACCAGCCCAAAAAATCGACGTTTTCAAACCTCGACCTCGATCCTTCACCGTTAACAGCGGCTGCAGCCATAATAATGGCATGCCAGAAGCTGGCATTGTTCCTCTATGCCTTGGACACAACGATACTAATAATGTTTCTGAAAATATAATCACATGTAACAAAGATGATGATAAATCTGAGCTTGTTAGTCATTTAATGGATGGTCAGAATAGGTGGTGGGAAAGTTTGCTAGATGAGAGCCAAGATCCAGCTGCGCTCTTTCCAGAAACTACAGCAATAAAAAAGGGCGCAACCTCCGCGTTTGACGTTGAGCAACTTTGGAGCCTGTTGGATGGAGAAACTGGAACTTGA
<210>6
<211>5442
<212>DNA
<213> molecular Label of Brmyb2
<220>
<223>
<400>6
ATGGAGGGTTCGTCCCAAGGGTTGAAAAAAGGTGCATGGACTGCTGAAGAAGATAATCTCTTGAGGCAATGCATTGATAAGTATGGAGAAGGGAAATGGCACCAAGTTCCTTTAAGAGCTGGTATGTCTTTTTTTTTTGATAACATAAGAGCTGGTATGCTACTTTTATTAATTTTCACACACACACACACACACACATATAACTAATAAGTACGTATATTCTTTTTATTTTTCAGTACATTTATTCTCTTTCTCTCTGTCTAGTATTAGGAAATTAATTAACACCGGGGTACACAATCATTGTTTTTCTTTTCGTTTTAATGAAGGAATCATAGATTCATATGTTCTAATGTTTTTCATGAAAAAAAACATTTGCGTTCTTCATGTTTAATTACAAAGCGAGAAAATGTCAACTCTCTTTATTGATTCGTCGTTTTTCTTTTTTTTTTTGAGAAAGAGCTTTTTTGATTAGTGAACTTTTCTGCACGAACCCGTGTGTTTGTGTGGAATATGTTGTTTATTCTGGTGTACTTTGATCCTTCGTGATAAAATTTTACTTCCTTTGTTCTTAAATATAAGATATTTTGGTAGAAGCATACATATTAAGAAAACTATTTTTTGTTTAGAAAATATCATTAAAACTATAAATTAATGGTGTTCAACCAATTACAAAATAGACTATTAAAATATGATTGGGTTCACAGTTTTTAATAAAGTAAAAGTTACCTAGAAAATTGAAAACATTTTATATCTAATACTATAAAGAAGACTATTCTCTTCTTCTCAGCTTGCCACGTCACTGAATCGAATTCTAACAATGCGACACGTGTTCGTGCTCATGTTATACTCTGCGTTTCATTAAAGCTTGATGTGGGCTGGTGTTTTCAATTACGGTTTTGATACATATTGGATCTCAATAATAATAGGCCCATTTCAAAATCATGAGAGATGCAAGCCGATTAGGGTTTCTGCTCTTCTTCTTCATCGACACCAAAACAGTTCCCAAACAAAAATGATGGGAGTTTCGAGGTATCTCTTTGTTGATCTTCGATGGCGTCGTTCTGGTTACTCTCCTAATTAGATGAAACGCTCCAAGTTACAGATCCATAGCCTTTATGACCTTCTTAACTCCAAAGACCGAAATGAGGCACGATCTGACGTTGAATGCGAGTTTACTCTCTGTGAGGCGGTGTAATCTACACAATATAAAGGTTAGCATCCTTCCCTTGAACATCATCCTCGCAAATTCTAATAAAACTTCAAGGTAGTAGCTTTTGTTTGATGTTTATCAAACTCTAGGTTTTGTTTGGTTCTTTGTCTTTCTATTTCCTCTCCCCAAGATCGTTAGATAAACATTTCGGATCGCTTTTGATAATTTTTTTTGTCTTATCTACAGTTGGCTCAACACAAGCCACATGGTGATGGAAGATCTTTGCAACAGTCGTTCACTGTGAAGCGGGGATTTTCGGAAGGCTATGGCAAAGATTCTGTGTGAGGAGATGAGTTGAGATGAGGATGAGAGTTCTTACTTTTCTTTGAAGAAATTGATTGACGGTTCAAGACAATTACTCCATTAGCGCCCGCGAACCTGTGGGAAGAGCTTTCGTAGTGTTTGGTTTACTCGCCGGTGGCGTTGCGGAAGTTCAATTCGTCGGCACCGTTTAAATGGCAACACCGACTCACTTCATCTTCATCTCTTTGGGACGACATCTGCGCCGTGGTAACATGCTTTCTTATTGTCGGTGTGGTGACGTCAGCCTCTCCACACATTCAAACGCATGTTATTGACAATCAAATTATTATGCAGGCTTTCTCTCAACATACAGAGCTTCCATAGTTATTGTTAAATCTTAATCATGTCTGAGGATACCATCACCGTCCCTCTCAGCTGCTGGAATGTACTCAGCTTGGTTGGATGGAGCGTCTGCTTGGCTGGAATTCAGAAAGTTCTGCTCCAAAGGAACCTGTCTGAACGTCAATGATGCCTTTGGCCAAGAATTATAACATGGTGGCTGATCCAAGAAATACCTGAACATTGCAATCTTCTTAAGAGCACCGAATGTAACATGTGACGAGGTGTCAAAAGCTCTTCTAGATTGTAAGATTGGTCTCCCATTTGGTTTGTTACTTAGGGGTAAAGTTCTATTTGCTTTGATCACTTCAACTTGCAAAATAGTGTTAACGGAACACATTCTATGCACTAATCTTATTTCCATCATTTTCTATTCACTCCACTAATCAAGTTCAGTCCAACGACTATTTATCATCCAGGAATCAAGTTCTATTTGTTAAATATATTTGTGAATCACACACAGCCAAGCTATCCACAAATTTATTGAACAATCTCTTCAAAGACAAACTTATAATACACTGCCACTTTCAAAAAATGTTTTGTGTACACTTCTTTGGACAGACGAAAACTATGACCGTCTTATTAATGGTAAGTCTGGCTCAAACCAAGCATCAATGCCATCACGTTCCTGCTCTTATTATTCTGAAAGGAGCAAGAGGAAAGCAGGTTGTAGCACAGTCATGTTCCAGCTCCATATTTCACAGAAGCCATCATTGCCGAGTTGAGTTGCAGTATTGTGTAAAAGTTTCTAACCACGAGGAGTTTAGTGTTGGAAAATATATTGGAGGTGCAGTGTTTATACCGAAAAGTTCTCAGGCAGAGATATTGTGTAGGTCTAAATGTCATAAAAGGACAGAAAATATAGTAGATGGAATTCTGTTTTTGTGTCCAGTTATATTCTCCGACTGTAGCTTGAAGAGCAAACTATCTAAGAGAGGTGAGATTGGTTTTAATCTGCCATTTCTGGAGAGTTGTGAAGATGTTGAAGGAGAGCAAGGTGAAGAATGTGATTGCTCACGCTGGGTTGTAAGAGACAGTCTTAGCTATTGGTTTCTGATTCTCAGATTTTTCAGCTGCAAATCTCAGACCGGTTTCTAAACACCAAAACAGCCTTTGGAAGACTTTCTTTTGTTGTGTGTTCTCCTGGCGGTGATGGGCCTACGGATCGCTGTGGTGACAGTGCAAAGGGGAGTGTTGTCGGGTGGTTATCAATTCAATACTATACAATTCTTTGCTTACAAAGCTGTGTAAGAGTATATTGTTATTATATAATGGTGAAAGAGTCGAAGTTGATGCTCACTGGAAGGAAATGAAGTAATGGAAAAAAAAACTCCACTAGATGCAAGAAACAATAGTTTTCTAAGTAGCGTTGAAATATTATCAATAAAAATAAAAGTATATAACAAACAATGTTTATTTGATAAGATTTCTAAACAATAAAGAGTGTTAAAGATTCGGCAAATAAATTATTTAGAATGTACATGATACAAAATATTGCAAAGTTTTTTTTTTTTTTTTGCTAAAAGTAAATGTGTTGTGTTGATTTAAAATATTGCAAAGTTGTTATGAAAAATGTAATATCTGAAGATACCATCGAAAACATTAAAACATTAAATTTTAACACATTTCCAACAGTTAAAAATATCAATTATCTTATATTATAAATGAATATGCTACGCAAAAAATGTACAATAATTCTTTTTATCAGCTACAGAAAACAGTTATGAAATTTTGTAACAAACATCAAATGTATTATTAACCGAAAGTCAACATTTTATCTATTAAAAAGTATTATACATGCTTATCAACGTCCCTACCACAAATTATAACTAATTAAATCAAGTTGAAGCCAATATCTAATACTCAATATTCTTTGTTCCAAAATCTTAATAATAAAAATGAAAAAATATTAGCAAAACATTGTTTACTTTTAATTTATTTTTGAAAATTATTTAGTATTAGTTAAGTTTATTAAATATTTAATTTGATATTGTAAATTTGAATATATACAATCAAATACACATTTTTAAAAAAATAGATGAAAGTATTTTTAAAACACGTATGTGTACCTAATAAATATGCAAAAAATCATACATGATAACTTTTTAAAAATAAAAAGCAATTAAATGTTATATGTAGATTTATTATTTATTATTTATGTCATTCAGATAAAAATATAGCAAAGGTGTAATAATATTCATAATATATACTAGAAAATTATATGCATCATTAAATTATATATTACTAAAAACTAATTTAAATTTTATAAACAATAAAATATAATTATGAAAGTAGTTTATATAATGAATAAAATATACAAAATTATAGTTAATTTAAATTAATAATGATATGAAAATAAAAATATGTCTTTACATAATCGAACAAAGAAAAAAATATAAAATAGTCTGACAACAAACAAAGATAGAAGAAATGAAATTAGTTGATTTATACGAAACAAATAAGTTAAAATATATATACTAACATAATAAAAATATAATACAATATTATTAAAAATCACAATAATTGTATTAGAAACCTACTAAATAGTAACAAAACTGACTTGAAAGAAAACAATTTTAAAAATAATGACCTGGATAAAATAATCATTTTAAAACAAAAATTTTAAAAATTATTTAATTTTCCGCCCGCCCGTAGGGCGGGTTTACTCTAGTATTGGATAATTGAAACATCAAAATTTTCAGTAAAACATCCTATTTTTAGGAACATATGGAGTAGGAGACGCGAATGCAGTTTTTGCTCGTTCTTTTAATAATATTAAATGTCAATTATTGGTTTTGTAGGTCTAAATCGGTGCAGGAAGAGTTGTAGACTAAGATGGTTGAACTATTTGAAGCCAAGTATCAAGAGAGGAAAACTCAACTCCGATGAAGTTGATCTTCTTATGCGCCTTCATAAGCTTTTAGGAAACAGGTTTACATTCAAGACACAAATTCAACTGTATTTCGTATCCTCATTCGGTCTAATCTAATCATTTGATTTGTTTTTTTTTTTGATAAAAAGTACTTAAATTTATTTCATATGTAAATGATCCATTACTAAGTCAAATATATCCCTAATTTTTCAAATGCATGCTTAGGTGGTCTTTAATTGCTGGTAGATTACCCGGTCGGACCGCCAATGACGTCAAAAATTACTGGAACACCCATTTGAGTAAGAAACATGAACCGGGTTGTAAGACCCAGATGAAAAAGAGAAACATTCCTTGCTCTTATACCACACCAGCCCAAAAAATCGACGTTTTCAAACCTCGACCTCGATCCTTCACCGTTAACAGCGGCTGCAGCCATAATAATGGCATGCCAGAAGCTGACATTGTTCCTCTATGCCTTGGACACAACGATACTAATAATGTTTCTGAAAATATAATCACATGTAACAAAGATGATGATAAATCTGAGCTTGTTAGTCATTTAATGGATGGTCAGAATAGGTGGTGGGAAAGTTTGCTAGATGAGAGCCAAGATCCAGCTGCGCTCTTTCCAGAAACTACAGCAATAAAAAAGGGCGCAACCTCCGCGTTTGACGTTGAGCAACTTTGGAGCCTGTTGGATGGAGAAACTGGAACTTGA
<210>7
<211>19
<212> Artificial sequence
<213> upstream primer
<220>
<223>
<400>7
5’- ATGGAGGGTTCGTCCCAAG-3’
<210>8
<211>22
<212> Artificial sequence
<213> downstream primer
<220>
<223>
<400>8
5’-TGGTGTACTTTGATCCTTCGTG-3’
<210>9
<211>22
<212> Artificial sequence
<213>Marker1-F
<220>
<223>
<400>9
5’-TGGTGTACTTTGATCCTTCGTG-3’
<210>10
<211>19
<212> Artificial sequence
<213>Marker1-R
<220>
<223>
<400>10
5’-ACTGCATTCGCGTCTCCTAC-3’
<210>11
<211>18
<212> Artificial sequence
<213>Marker2-F
<220>
<223>
<400>11
5’-CTTTTCTGCACGAACCCG-3’
<210>12
<211>22
<212> Artificial sequence
<213>Marker2-R
<220>
<223>
<400>12
5’-ATTCGCGTCTCCTACTCCATAT-3’
<210>13
<211>19
<212> Artificial sequence
<213>Marker8-F
<220>
<223>
<400>13
5’-AAAAGGTGCATGGACTGCT-3’
<210>14
<211>22
<212> Artificial sequence
<213>Marker8-R
<220>
<223>
<400>14
5’-TGTTTTGGTGTCGATGAAGAAG-3’
Claims (6)
1. The molecular marker related to the Chinese cabbage anthocyanin synthesis gene is characterized by comprising a purple Chinese cabbage molecular marker and a non-purple Chinese cabbage molecular marker, wherein the gDNA sequence of the purple Chinese cabbage molecular marker is shown as SEQ ID NO.5, and the gDNA sequence of the non-purple Chinese cabbage molecular marker is shown as SEQ ID NO. 6.
2. A PCR detection kit for identifying purple-heart Chinese cabbage materials, which is characterized by comprising an upstream primer and a downstream primer for amplifying the molecular marker of claim 1:
an upstream primer: 5'-ATGGAGGGTTCGTCCCAAG-3'
A downstream primer: 5'-TCAAGTTCCAGTTTCTCCATCC-3' are provided.
3. A PCR detection kit for identifying purple-heart Chinese cabbage materials, which is characterized by comprising an upstream primer and a downstream primer for amplifying the molecular marker of claim 1:
Marker1-F：5’-TGGTGTACTTTGATCCTTCGTG-3’
Marker1-R：5’-ACTGCATTCGCGTCTCCTAC-3’
Marker2-F：5’-CTTTTCTGCACGAACCCG-3’
Marker2-R：5’-ATTCGCGTCTCCTACTCCATAT-3’
Marker8-F：5’-AAAAGGTGCATGGACTGCT-3’
Marker8-R：5’-TGTTTTGGTGTCGATGAAGAAG-3’。
4. the application of the molecular marker of claim 1 in molecular marker assisted breeding of purple Chinese cabbage.
5. Use of the molecular marker of claim 1 for identifying purple-heart chinese cabbage material.
6. The use according to claim 5, comprising the steps of:
(1) extracting DNA of the Chinese cabbage to be detected;
(2) taking DNA as a template, and carrying out PCR amplification reaction by using an upstream primer and a downstream primer; the upstream primer is as follows: 5'-ATGGAGGGTTCGTCCCAAG-3', the downstream primer: 5'-TCAAGTTCCAGTTTCTCCATCC-3', respectively;
(3) and detecting the PCR amplification product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711269495.8A CN108018290B (en) | 2017-12-05 | 2017-12-05 | Plant anthocyanin synthesis control gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711269495.8A CN108018290B (en) | 2017-12-05 | 2017-12-05 | Plant anthocyanin synthesis control gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108018290A CN108018290A (en) | 2018-05-11 |
| CN108018290B true CN108018290B (en) | 2020-11-10 |
Family
ID=62078187
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711269495.8A Active CN108018290B (en) | 2017-12-05 | 2017-12-05 | Plant anthocyanin synthesis control gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108018290B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021189221A1 (en) * | 2020-03-24 | 2021-09-30 | 西北农林科技大学 | Selective breeding method for new germplasm of purple-orange chinese cabbage |
| CN113439657B (en) * | 2020-03-24 | 2022-12-02 | 西北农林科技大学 | Breeding method of purple-orange Chinese cabbage germplasm |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003100068A1 (en) * | 2002-05-29 | 2003-12-04 | Aresa Biodetection Aps | Reporter system for plants |
| CN102120762A (en) * | 2010-05-14 | 2011-07-13 | 昆明理工大学 | Arabidopsis anthocyanin pap1 purified protein and preparation method and applications thereof |
| CN104531754A (en) * | 2014-12-19 | 2015-04-22 | 西南大学 | Application of gene for interfering expression of LCYB and LCYE and simultaneously over-expressing PAP in preparation of brassica plant having red petals |
-
2017
- 2017-12-05 CN CN201711269495.8A patent/CN108018290B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003100068A1 (en) * | 2002-05-29 | 2003-12-04 | Aresa Biodetection Aps | Reporter system for plants |
| CN102120762A (en) * | 2010-05-14 | 2011-07-13 | 昆明理工大学 | Arabidopsis anthocyanin pap1 purified protein and preparation method and applications thereof |
| CN104531754A (en) * | 2014-12-19 | 2015-04-22 | 西南大学 | Application of gene for interfering expression of LCYB and LCYE and simultaneously over-expressing PAP in preparation of brassica plant having red petals |
Non-Patent Citations (3)
| Title |
|---|
| Anthocyanin biosynthetic genes in Brassica rapa;Ning Guo等;《BMC Genomics》;20141231;第15卷;第1-11页 * |
| PREDICTED: Brassica rapa transcription factor MYB114 (PAP1), transcript variant X1, mRNA, Accsession ID:XM_009106954.2;匿名;《GenBank数据库》;20161013;DEFINITION,CDS,ORIGIN * |
| 紫色芸薹属蔬菜花青素合成调控研究进展;李益等;《山东农业科学》;20141231;第46卷(第11期);第137-142页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108018290A (en) | 2018-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nelson et al. | The first gene-based map of Lupinus angustifolius L.-location of domestication genes and conserved synteny with Medicago truncatula | |
| Jarvis et al. | Simple sequence repeat marker development and genetic mapping in quinoa (Chenopodium quinoa Willd.) | |
| Shirkot et al. | Molecular identification of sex in Actinidia deliciosa var. deliciosa by RAPD markers | |
| CN107385055A (en) | The SNP marker of the genes of muskmelon unisexuality floral formation correlation ACS 7 and application | |
| CN106754954A (en) | A kind of corn MS8 gene mutation bodies and its method for identifying molecules and application | |
| CN108018290B (en) | Plant anthocyanin synthesis control gene and application thereof | |
| KR101047322B1 (en) | DNA marker for the identification of Anguilla eel species | |
| CN110512025A (en) | A kind of molecular labeling and its application with powdery mildew resistance gene in wheat PmJM23 close linkage | |
| CN107385053A (en) | A kind of four primer molecule labeling methods identified rice and produce als gene mutation | |
| Ramtekey et al. | Molecular tagging of photoperiod responsive flowering in Indian bean [Lablab purpureus (L.) Sweet] | |
| CN109825604B (en) | Molecular marker closely linked with peach aphid resistance character, primer, kit and method for detecting peach aphid resistance character and application of primer, kit and method | |
| CN108239675B (en) | Molecular marker TJcM02 for identifying melon unisexual flower and application thereof | |
| Daryono et al. | Development of random amplified polymorphism DNA markers linked to Powdery Mildew resistance gene in Melon | |
| CN111172317B (en) | Molecular marker HSRC3911 closely linked with major QTL site in flowering phase of sesame and application thereof | |
| KR101219546B1 (en) | Development of grain weevils resistive molecular markers in mung beans and uses thereof | |
| JP4581101B2 (en) | Papaya sex identification method using DNA marker | |
| CN110106270B (en) | Molecular marker coseparated from melon yellow seed coat and application thereof | |
| Jamil et al. | Estimation of genetic diversity in rice (Oryza sativa L.) genotypes using Simple Sequence Repeats | |
| CN107338323B (en) | Molecular marker closely linked with powdery mildew resistance of flue-cured tobacco variety TT7 and application thereof | |
| CN107130019B (en) | Molecular marker primer of rice local variety thin rice panicle blast resistance gene and application thereof | |
| CN113584199B (en) | Molecular marker related to cold non-retrogradation character of rice and application thereof | |
| CN115725772B (en) | InDel molecular marker for identifying seed coat color of cabbage type rape and application thereof | |
| CN114410826B (en) | InDel molecular marker co-separated from cucumber peel brightness gene and application thereof | |
| CN113584200B (en) | Molecular marker related to cold non-retrogradation character of rice and application thereof | |
| CN114369674B (en) | SNP marker linked with Indian pumpkin short vine gene CmDw-1, primer, kit and application thereof |
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 |