CN110093346B

CN110093346B - Molecular marker linked with chromogene of brassica oleracea and application thereof

Info

Publication number: CN110093346B
Application number: CN201910423365.8A
Authority: CN
Inventors: 王晓武; 程锋; 武剑; 张鑫; 张亢
Original assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Current assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2019-12-27
Anticipated expiration: 2039-05-21
Also published as: CN110093346A

Abstract

The invention discloses a molecular marker linked with a chromogene of a brassica oleracea bolt and application thereof. The related gene of the chromogene of the purple flowering Chinese cabbage is positioned on the No. 7 chromosome. The gene Indel is positioned between 25181252-25181356 of chromosome 7 from the 5' end by taking the 3.0 version of Chinese cabbage as reference. The molecular marker can be used for identifying the bolting purple vegetables or the genotypes thereof, and further can be used for biotechnology-assisted breeding.

Description

Molecular marker linked with chromogene of brassica oleracea and application thereof

Technical Field

The invention relates to molecular assisted breeding, in particular to an InDel molecular marker linked with a chromogene of a brassica oleracea and application thereof.

Background

Chinese cabbage vegetables (Brassica rapa L.) belong to Brassica (Brassica) of Brassicaceae and comprise a plurality of important vegetables and oil crops, and have a cultivation history of more than two thousand years in China; the genetic resources are extremely rich, including a plurality of subspecies and varieties such as Chinese cabbage, turnip, and flowering cabbage; is one of the vegetable crops with the widest distribution and the largest planting area in China, and plays a significant role in agricultural production. The brassica oleracea and the flowering cabbage are two different cabbage crops, wherein veins and leaves of the brassica oleracea are purple, leaves of the flowering cabbage are green, and the veins and the leaves of the flowering cabbage are mainly caused by different anthocyanin contents in the two different cabbage crops.

Along with the gradual improvement of living standard, people pay more attention to the nutritional quality of vegetables. Anthocyanin and flavonol glycoside are used as important flavonoid plant secondary metabolites and widely exist in fruits and vegetables. Researches show that the two substances are not only vital to the growth and development of plants, but also have the health-beneficial effects of resisting oxidation and tumors, improving cardiovascular diseases and the like. Therefore, fruits and vegetables rich in the two active substances are more and more favored by consumers. In addition, the natural variation characteristics and the genetic mechanism of anthocyanin in the cabbage crops are researched, theoretical basis is provided for further cloning key genes, improving the nutritional quality of the cabbage vegetables and cultivating new varieties of cabbage rich in anthocyanin, and a foundation is laid for mining and utilizing important gene resources.

With the development of molecular biology technology, people make a great deal of research work on molecular markers, gene positioning and gene expression analysis related to the bolting gene. In addition, in brassica vegetables containing anthocyanin, because the genetic background is complex, anthocyanin synthesis regulation and control pathways are different, and regulation and control genes are different. Existing studies show that purple control genes are initially located in RBr varieties of Chinese cabbage, purple leaf Chinese cabbage, purple turnip, Chinese cabbage hybridized with red leaf mustard and the like, are respectively located on chromosomes A09, A03, A07, A02 and the like, and have large differences in physical positions (Burdzinski et al, 2007; Wang et al, 2014; Hayashi et al, 2010; Zhang et al, 2013). In the purple cauliflower mutant and collard localization studies, the purple gene is localized to MYB transcription factor BoMYB2 of R2R3 family on C06 chromosome (Chiu et al, 2010; Yan et al, 2018). Regarding the location of the bolting color gene in the brassica oleracea, guoning et al utilized the QTL method, using the genome of cabbage version 1.5 as the reference sequence, and finally located at 6068769-8028904 on chromosome a09 (Guo N et al, 2015). These studies suggest that the purple trait is not controlled by the same gene in different species. In addition, the genetic mechanism of the purple regulatory gene is different in different varieties, and some genes are found to be expressed as quality traits and controlled by a single gene, and some genes are expressed as quantitative traits and have main effective sites. These studies suggest that the synthesis and regulation of anthocyanin in brassica is highly complex.

Disclosure of Invention

The invention provides the re-sequencing data according to the brassica chinensis bolts and the flowering cabbage and the descendant F₂And determining a deletion or/and insertion mutation by using sequencing data of the population pool, wherein the deletion or/and insertion mutation is a nucleic acid sequence length polymorphism related to the color gene of the bolting purple. The present invention has been accomplished, at least in part, based on this. Specifically, the present invention includes the following.

In a first aspect of the invention, a synthetic oligonucleotide is provided that comprises a unique sequence of a bolting color gene of a purple bolting chromosome A07.

In certain embodiments, the oligonucleotide comprises the sequence set forth in SEQ ID NO. 3:

5 '-GGGAATCGATCCAACTTTGTTTC(A) n-3', wherein (A) n is poly A sequence and n is natural number between 25-110. Preferably, the synthetic oligonucleotide is 95-130bp in length.

In certain embodiments, the oligonucleotide further comprises other sequences in addition to the sequence set forth in SEQ ID NO. 5, examples of other sequences including a sequence upstream of position 25181252 of the chromogene of the Brassica oleracea, and a sequence downstream of position 25181356 of the chromogene of the Brassica oleracea. Preferably, examples of the other sequences are the sequence shown by SEQ ID NO. 4 and the sequence shown by SEQ ID NO. 5.

In a second aspect of the present invention, a method is provided for identifying whether a plant contains a brassica oleracea bolt, comprising the step of detecting the presence or absence of a unique sequence of a brassica chromogene located on chromosome a07 of the brassica oleracea bolt in the DNA of the plant to be identified.

In certain embodiments, the method comprises the steps of:

(1) a step of extracting sample DNA from a plant sample to be identified;

(2) a step of amplifying nucleic acids containing said unique sequences from said sample DNA by PCR using forward and reverse primers, wherein said forward primer is capable of specifically binding to a first target sequence region, said reverse primer is capable of specifically binding to a second target sequence region, said first target sequence region being any contiguous region from position 25181155 to position 25181272 of the chromogene of the brassica oleracea, said second target sequence region being any contiguous region from position 25181360 to position 25181690 of the chromogene of the brassica oleracea; and

(3) the plants were identified by electrophoretically detecting the length of the sample PCR product.

Preferably, further comprising (4) a step of amplifying a nucleic acid containing the unique sequence from a control DNA by PCR using the forward and reverse primers; and (5) comparing the length of the sample PCR product with the length of the control PCR product.

In certain embodiments, the control DNA is DNA from a brassica oleracea and/or DNA from a heart.

In a third aspect of the invention, a method for identifying the genotype of a bolting gene in a plant is provided, which comprises the step of detecting the content of a unique sequence of the bolting gene located on the purple flowering cabbage A07 chromosome in the plant DNA to be identified.

In certain embodiments, the method comprises the steps of:

(1') a step of extracting a sample DNA from a plant to be identified;

(2') a step of amplifying a nucleic acid containing the unique sequence from the sample DNA and a control DNA by PCR using forward and reverse primers under the same conditions, wherein the forward primer is capable of specifically binding to a first target sequence region, the reverse primer is capable of specifically binding to a second target sequence region, the first target sequence region is any continuous region from position 25181155 to position 25181272 of the chromogene of the brassica oleracea, the second target sequence region is any continuous region from position 25181360 to position 25181690 of the chromogene of the brassica oleracea, the control DNA is DNA from a homozygous brassica oleracea plant and/or DNA from a heterozygous brassica oleracea plant; and

(3') a step of comparing the content of the sample PCR product with the content of the control PCR product.

The fourth aspect of the invention provides application of a gene fragment as a molecular marker in breeding of a brassica oleracea, wherein the gene fragment comprises a sequence positioned between 25181252-25181356 th site of a brassica oleracea chromogene.

Drawings

FIG. 1 is the comparison result of the color genes of the flowering Chinese cabbage in 3.0 version of the genome.

FIG. 2 is a diagram showing the result of electrophoresis of PCR amplification products of DNA of a brassica oleracea and DNA of a flowering cabbage.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

Unless otherwise specified, the gene position in the present invention refers to a position relative to the 3.0 version of Chinese cabbage.

The invention relates to a Brassica oleracea' Brassica vegetable which is a Brassica vegetable (Brassica rapa L.) of Brassica of Cruciferae (Cruciferae). Examples of the brassica vegetables include chinese cabbage, pakchoi, turnip, and flowering cabbage, among many subspecies and varieties.

The "variety" of the present invention refers to a variety which is not classified (i.e., variety, subspecies, variants) at the time of being originally published in the academic journal, and later, as people know the variety more thoroughly and deeply, it is found that some individuals or groups of plants in the variety have variations which are different from the characteristics of the variety recognized at the time of originally publishing the variety, and the variations are obvious and stable, and it is worth to divide them separately for distinction, and the type having variations is named by a plant specialist and is published as a variety in the variety according to different situations. In contrast, a type which has the original characteristics and in which no significant variation has occurred is referred to as the original variant of the variety.

"nucleic acid" in the present invention includes deoxyribonucleic acid (i.e., DNA) and ribonucleic acid (i.e., RNA). In the present invention, DNA is preferred. Nucleic acids of the invention also include protein-encoding nucleic acids and non-protein-encoding nucleic acids. The present invention is preferably a nucleic acid encoding a protein.

In the present invention, "capable of specifically binding" means that the sequence of the forward primer binds to the first target sequence region and/or the sequence of the reverse primer binds to the second target sequence region under stringent conditions, without binding to other parts of the DNA. The first target sequence region refers to any continuous region from the 25181155 th site to the 25181272 th site of the chromogene of the brassica campestris. Preferably, the first target sequence region is any contiguous region within the sequence shown in SEQ ID NO. 4. The second target sequence region refers to any continuous region from the 25181360 th site to the 25181690 th site of the chromogene of the brassica oleracea. Preferably, the second target sequence region is any contiguous region within the sequence shown in SEQ ID NO. 5. More preferably, the second target sequence region refers to any continuous region from position 25181360 to position 25181450 of the chromogene of the Brassica oleracea.

In the present invention, "stringent conditions" refer to conditions under which a target sequence hybridizes to the target sequence to a greater extent than can be detected (for example, the average of background measurement values + the standard error of background measurement values × 2 or more). Stringent conditions are sequence-dependent and will vary depending on the environment in which the hybridization is performed. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified that are 100% complementary to the primers. In certain embodiments, stringent conditions refer to nucleic acid hybridization conditions at 5 XSSC, 50% formamide, and 42 ℃. In certain embodiments, stringent conditions refer to high stringency conditions, i.e., prehybridization and hybridization at 42 ℃ in 5 XSSC, 0.3% SDS, 200. mu.g/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard southern blotting procedures for sequences at least 100 nucleotides in length, for 12 to 24/hour. The material was finally washed three times each for 15 minutes using 0.2 x SSC, 0.2% SDS at 65 ℃.

[ synthetic oligonucleotides ]

In a first aspect of the invention, synthetic oligonucleotides are provided that comprise a unique sequence of a bolting color gene of chromosome a07 of the brassica oleracea.

The "unique sequence" of the present invention belongs to an "insertion-deletion marker", which refers to the difference in the whole genome between the two parents. One parent has a certain number of nucleotide insertions or deletions in its genome relative to the other parent (Jander et al, 2002).

The unique sequence in the present invention refers to a gene fragment that is present only in a bolting plant in all subspecies and varieties of, for example, brassica vegetables. Preferably, the unique sequence refers to a fragment of the gene that is present in the brassica oleracea but not in the core. More preferably, the unique sequence of the invention refers to a gene fragment which exists only in the chromosome A07 of the brassica oleracea and does not exist in the chromosome A07 of the flowering cabbage. Also preferably, the unique sequences of the present invention refer to sequences beginning at position 25181252 and about 95-130pb downstream of the chromogene of chromosome A07 of the Brassica oleracea.

In certain embodiments, the oligonucleotides of the invention comprise the sequence set forth in SEQ ID NO. 3:

5 '-GGGAATCGATCCAACTTTGTTTC(A) n-3'. Wherein (A) n is a poly A sequence, and n is a natural number between 25 and 110.

In certain embodiments, the synthetic oligonucleotides of the invention further comprise additional sequences in addition to the sequence shown in SEQ ID NO. 3. Other sequences may be sequences of the first and second cds regions of the bolting gene, as well as partial sequences within the third cds region. Examples include the sequence upstream of the 25181252 th site of the chromogene of a bolting gene, and the sequence downstream of the 25181356 th site of the chromogene of a bolting gene. Preferably, the sequence preceding the insertion position in the third cds region, i.e., the sequence shown in SEQ ID NO. 4, the sequence following the insertion position in the third cds region, i.e., the sequence shown in SEQ ID NO. 5, or the sequence preceding the insertion position in the third cds region in the chromogene cDNA of the Brassica oleracea, i.e., the sequence shown in SEQ ID NO. 7, are examples of other sequences.

[ method for identifying whether plant contains a purple flowering cabbage ]

In a second aspect of the invention, a method is provided for identifying whether a plant comprises a bolting. Preferably, the plant herein refers to a plant from the family brassicaceae (Cruciferae), more preferably, the plant herein is a plant from the genus Brassica (Brassica), and even more preferably, the plant herein is a plant from a Brassica vegetable (Brassica rapa L.). In certain embodiments, where a "plant" is a type of plant, the methods of the present invention refer to methods for identifying whether the plant is a bolting plant. In certain embodiments, where a "plant" is a mixture of multiple types of plants or plant constituents thereof, the methods of the present invention refer to methods for identifying whether a plant contains a bolting in that plant.

The method comprises the step of detecting whether a unique sequence of a bolting color gene located in a purple bolting A07 chromosome exists in DNA of a plant to be identified. Specific sequences have been described in the first aspect and will not be described in detail here.

In the present invention, the method of detecting the presence or absence of the unique sequence of the chromogene located in chromosome A07 of the Brassica oleracea can adopt any method known in the art, and examples thereof include a method of amplifying a nucleic acid containing the unique sequence, a method of directly sequencing the genome of a plant, and the like.

In certain embodiments, the methods of the present invention for identifying whether a plant comprises a brassica oleracea bolt comprise the step of amplifying a gene sequence containing a unique sequence from the DNA of a plant sample to be identified by PCR using forward and reverse primers. The forward primer can be specifically combined with a first target sequence region, and the first target sequence region is any continuous region from 25181155 th site to 25181272 th site of the chromogene of the brassica oleracea. Similarly, the reverse primer herein is capable of specifically binding to the second target sequence region. The second target sequence region is any continuous region from 25181360 th site to 25181690 th site of the chromogene of the brassica oleracea. That is, the region to be amplified covered by the forward primer and the reverse primer of the present invention contains at least the entire sequence of the unique sequence of the present invention.

In an exemplary embodiment, the method for identifying whether a plant is a brassica bolting comprises the steps of:

(1) extracting sample DNA from a plant sample to be identified;

(2) amplifying a gene sequence containing a unique sequence from a sample DNA by PCR using forward and reverse primers;

(3) and detecting the length of the PCR product of the sample by electrophoresis to identify whether the plant is the brassica oleracea.

In step (1), the "plant sample to be identified" means the whole plant or a part of the plant, for example, a plant leaf or a plant stem, etc.

In additional exemplary embodiments, the method for identifying whether a plant comprises a brassica oleracea bolting further comprises (4) a step of amplifying a gene sequence containing a unique sequence from a control DNA by PCR using the same forward and reverse primers as in step (2); and (5) comparing the length of the sample PCR product with the length of the control PCR product.

In exemplary embodiments, the control DNA may be DNA from a brassica oleracea as a positive control, or DNA from a brassica oleracea as a negative control. Alternatively, the method of the present invention may use both the positive control and the negative control described above.

In the present invention, the PCR conditions of step (2) and step (4) are preferably the same, and steps (2) and (4) may be performed simultaneously or separately in a time-spaced sequence.

[ method for identifying the genotype of the bolting gene in plants ]

In a third aspect of the invention, a method for identifying the genotype of a bolting gene in a plant is provided. The plant in the method is preferably a bolting plant. The genotype according to the invention is preferably homozygous or heterozygous.

The method comprises the step of detecting the content of the specific sequence of the bolting color gene of the purple flowering cabbage A07 chromosome in the DNA of the plant to be identified. The method for detecting the content of the unique sequence can adopt any method known in the art, and is not limited thereto. The content in the method can be absolute content or relative content.

In certain embodiments, the amounts of the invention refer to the relative amounts relative to a control or reference. The control or reference herein may be a content obtained from a control DNA under the same conditions, or may be control or reference data obtained by repeating the operation a plurality of times under prescribed conditions (standard conditions).

In exemplary embodiments, a method for identifying the genotype of a bolting gene in a plant may comprise the steps of:

(1') a step of extracting a sample DNA from a plant to be identified;

(2') a step of amplifying a gene sequence containing the unique sequence from the sample DNA and the control DNA by PCR using forward and reverse primers under the same conditions;

(3') comparing the content of the sample PCR product with the content of the control PCR product.

The forward and reverse primers in the method may be the same as those described in the second aspect, and are not described herein again.

The control DNA in the method can be DNA from homozygous brassica oleracea plants as a positive control, and can also be DNA from heterozygous brassica oleracea plants as a negative control. The control DNA of the present invention may be used in combination with the positive control and the negative control.

[ use of Gene fragment as an identification molecular marker in Breeding Carex chinensis ]

In a fourth aspect of the invention, there is provided the use of a gene fragment as a molecular marker in molecular breeding. Preferably, the gene fragment comprises at least a portion of the sequence located between positions 25181252-25181356 of the chromogene of the brassica oleracea.

The gene fragment provided by the invention is used as an InDel marker for identifying a molecular marker of the brassica oleracea L.var.latifolia.

Example 1

This example is the development of an InDel marker closely linked to the chromogene of a brassica oleracea.

Indel markers are derived from resequencing data of heart and bolting material. Namely, the genomic DNA of the flowering cabbage and the genomic DNA of the purple flowering cabbage are respectively extracted, the concentration range is 800-1300 ng/mu l, 10 mu g of each sample is sent to a company for sequencing, and the two samples are subjected to re-sequencing on an Illumina Genome Analyzer sequencing system platform. Wherein, about 32Gb double-ended (PE) reads were obtained for each of the two samples.

Then using the flowering cabbage and the purple flowering cabbage as F₁And selecting F after hybridization₂Medium green and purple each 25 individuals, constructing extreme pools and sequencing. Wherein, the two mixing pools respectively obtain about 11Gb double-ended (PE) reads. The genome of cabbage version 3.0 was selected as a reference genome, and SNP development was performed using bwa and samtools.

Firstly, double-end sequencing (PE) reads are aligned to a genome of a cabbage version 3.0, secondly, at least 3 pairs of reads are allowed to support the site, and then, sites with the quality value of less than 10, the DP value of less than 5 and 5bp near InDel are filtered out so as to obtain a reliable SNP site. Finding out the site information consistent with the parent homozygous site in the offspring pool, and finally calculating the snp _ index. Referring to anthocyanin synthesis pathway genes (41) in arabidopsis, anthocyanin synthesis related structural genes and regulation genes in Chinese cabbage are predicted, and total 73 related genes are obtained (Guo et al, 2014). Finally, the bolting color gene of the brassica oleracea is positioned between 25180918-25181774 from the 5' end of the A07 chromosome, and an insert exists between 25181252-25181356. The specific analysis results are shown in FIG. 1.

Next, a forward primer InDelzx _ A07-F with the sequence shown in SEQ ID NO. 1 and a reverse primer InDelzx _ A07-R with the sequence shown in SEQ ID NO. 2 were designed. And amplifying the DNA of the brassica oleracea and the DNA of the brassica oleracea by utilizing forward and reverse primers. Theoretically, the length of the fragment amplified in the DNA of the brassica oleracea is about 331 bp; the length of the amplified fragment in the cabbage heart DNA is 230 bp.

The accuracy of the InDel marker was verified as follows:

the reaction system of PCR amplification is as follows: 2ul DNA, 0.8ul each of the primers forward and reverse, 10ul of 2 × Rapid Taq Master Mix and 6.4ul double distilled water.

The procedure for PCR amplification was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 15 seconds, extension at 72 ℃ for 15 seconds, and 35 cycles; keeping the temperature at 72 ℃ for 5 minutes, and storing at 4 ℃.

3. Agarose gel plate preparation and electrophoresis:

preparation of 1% agarose gel: 0.3g agarose and 0.5% TAE 30 ml.

4. Mixing, heating for 3min, adding 3 μ l type I nucleic acid coloring agent, pouring gel as soon as possible, and immediately inserting comb with thickness of 1.0 mm.

It should be noted that the teeth of the comb are in a straight line, no air bubbles are left at the comb holes, and the sample can be spotted by pouring 0.5 Xelectroplate buffer solution after 30 min. Three replicates of each sample were made for this experiment.

5. Electrophoresis conditions were 150V for 10 min.

The PCR electrophoresis results are shown in FIG. 2. The results show that: wherein lane A is shown as 5k Marker; B. c, D Lane results show that the DNA fragment is a single piece with a size of 230bp, which is identical with the material of the cabbage heart; E. the results in lane F, G show: the DNA fragment is one, the size is about 330bp, and the DNA fragment is matched with the material of the brassica oleracea.

By comparing the fragment obtained by amplifying the DNA of the flowering Chinese cabbage with the fragment obtained by amplifying the DNA of the flowering Chinese cabbage, a sequence is inserted into the A07 chromosome of the flowering Chinese cabbage, and the sequence is shown as SEQ ID NO. 6.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

Sequence listing

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Claims

1. A synthetic oligonucleotide, comprising a sequence unique to the bolting gene of chromosome a07 of the brassica oleracea, and the sequence of said oligonucleotide is 5 '-GGGAATCGATCCAACTTTGTTTC(A) n-3', wherein n is a natural number between 25-80.

2. The synthetic oligonucleotide according to claim 1, wherein the oligonucleotide is 95-103bp in length.

3. A method for identifying whether a plant contains a bolting DNA, characterized by comprising the step of detecting whether a unique sequence exists in the DNA of the plant to be identified, wherein the unique sequence is shown as 5 '-GGGAATCGATCCAACTTTGTTTC(A) n-3', and n is a natural number between 25 and 80.

4. The method for identifying whether a plant contains a purple bolting according to claim 3, comprising the steps of:

(1) a step of extracting sample DNA from a plant sample to be identified;

(2) a step of amplifying a nucleic acid containing the unique sequence from the sample DNA by PCR using forward and reverse primers, wherein the forward primer is capable of specifically binding to a first target sequence region, the reverse primer is capable of specifically binding to a second target sequence region, the first target sequence region is any continuous region within the sequence shown by SEQ ID NO. 4, and the second target sequence region is any continuous region within the sequence shown by SEQ ID NO. 5; and

5. The method of claim 4, further comprising the steps of (4) amplifying nucleic acid from control DNA by PCR using the forward and reverse primers; and (5) comparing the length of the sample PCR product with the length of the control PCR product.

6. The method for identifying whether a plant comprises a bolting according to claim 5, characterized in that said control DNA is DNA from a bolting and/or DNA from a heart.

7. A method for identifying the homozygous genotype or the heterozygous genotype of the bolting chromogene in the purple flowering cabbage is characterized by comprising the step of detecting the content of a special sequence in DNA of the purple flowering cabbage, wherein the special sequence is shown as 5 '-GGGAATCGATCCAACTTTGTTTC(A) n-3', and n is a natural number between 25 and 80.

8. The method for identifying a bolting gene in a brassica bolting according to claim 7, as homozygous genotype or heterozygous genotype, comprising the steps of:

(1') extracting sample DNA from the brassica oleracea;

(2') a step of amplifying nucleic acids containing said unique sequence from said sample DNA and a control DNA by PCR under the same conditions using forward and reverse primers, wherein said forward primer is capable of specifically binding to a first target sequence region, said reverse primer is capable of specifically binding to a second target sequence region, said first target sequence region is any continuous region within the sequence shown by SEQ ID NO. 4, said second target sequence region is any continuous region within the sequence shown by SEQ ID NO. 5, said control DNA is DNA from a homozygous Brassica oleracea plant and/or DNA from a heterozygous Brassica oleracea plant; and

9. The gene fragment is used as a molecular marker in breeding of the brassica oleracea, wherein relative to the position of a 3.0 version genome of Chinese cabbage, the gene fragment is at least a part of a sequence positioned between 25181252-th 25181356-th sites of the brassica oleracea color gene.