CN110093346A - With the molecular marker and application thereof of purple tsai-tai a kind of sedge color gene linkage - Google Patents
With the molecular marker and application thereof of purple tsai-tai a kind of sedge color gene linkage Download PDFInfo
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Abstract
The present invention discloses the molecular marker and application thereof with purple tsai-tai a kind of sedge color gene linkage.Purple tsai-tai a kind of sedge color oncogene related gene of the invention is located on No. 7 chromosomes.It is reference, position of the gene Indel between the 25181252-25181356 from 5 ' ends of No. 7 chromosome with 3.0 version of Chinese cabbage.Molecular marker of the present invention can be used for identifying purple tsai-tai or its genotype, and then can be used for biotechnology assistant breeding.
Description
Technical field
The present invention relates to marker assisted selections, in particular to the InDel molecular marker with purple tsai-tai a kind of sedge color gene linkage
And application thereof.
Background technique
Chinese cabbage group (Brassica rapa L.) belongs to Cruciferae (Cruciferae) Brassica genus (Brassica),
Crop is used comprising many important vegetables and oil, has more than 2,000 years cultivation histories in China；Genetic resources is extremely abundant, packet
Include multiple subspecies such as Chinese cabbage, pakchoi, turnip, tender flower stalk and mutation；It is that China's distribution is most wide, the maximum vegetables of cultivated area make
One of object occupies very important status in agricultural production.Purple tsai-tai and cabbage heart are two different Chinese cabbage groups, wherein
The blade presentation green of purple and cabbage heart can be all presented in the vein and blade of purple tsai-tai, this is mainly in two class difference Chinese cabbage crops
Anthocyanidin content it is different caused by.
Along with stepping up for living standard, people pay attention to the nutritional quality of vegetables further.Anthocyanin and flavonols
Glycosides is widely present in fruits and vegetables as important flavonoids Secondary metabolites.Research shows that these two types of substances
Growth and development not only for plant is most important, and is good for one's health with anti-oxidant, antitumor and improvement angiocarpy etc.
The effect of.Therefore the fruits and vegetables rich in these two types of active materials are more and more popular with consumers.Furthermore Chinese cabbage group is studied
The natural variation feature and genetic mechanism of middle anthocyanin improve the nutriment of Chinese cabbage group for further clone's key gene
Matter and cultivation are rich in anthocyanin Chinese cabbage new varieties offer theoretical foundation, to excavate and being laid the foundation using important genetic resources.
With the development of molecular biology technology, people to a kind of sedge color base because of relevant molecular labeling, the assignment of genes gene mapping and
Gene expression analysis has done a large amount of research work.In addition, in vegetables of the Brassica genus containing anthocyanin, since genetic background is multiple
Miscellaneous, anthocyanin synthesis regulation access has differences, and controlling gene is not identical.Existing studies have shown that is in RBr kind Chinese cabbage, purple
It just located purple respectively in leaf pakchoi, purple turnip, Chinese cabbage for hybridize with red autumnal leaves leaf mustard etc. and control gene, these genes are distinguished
On the chromosomes such as A09, A03, A07 and A02, there are great differences for physical location (Burdzinski et al., 2007；
Wang et al.,2014；Hayashi et al.,2010；Zhang et al.,2013).And in purple cauliflower mutant
And in the Position Research of collard, purple gene has navigated to the myb transcription factor of R2R3 family on C06 chromosome
BoMYB2(Chiu et al.,2010；Yan et al.,2018).About a kind of sedge color base in purple tsai-tai because positioning, Guo Ning et al.
Using the method for QTL, using the genome of 1.5 version of Chinese cabbage as reference sequences, is eventually positioned on A09 chromosome
At 6068769-8028904 (Guo N et al., 2015).These researchs imply Purpled traits in different plant species not by phase
Same gene control.In addition, there is also differences for the genetic mechanism of purple controlling gene in different mutation, sent out in different researchs
Existing to show as qualitative character, by Dominant gene, some is then quantitative character, and there are main effect sites.These research explanations,
The synthesis of anthocyanin has the complexity of height with regulation in Brassica genus.
Summary of the invention
The present invention, which is provided, resurveys ordinal number accordingly and offspring F according to purple tsai-tai and cabbage heart2The sequencing data that pond is mixed by group is true
A kind of fixed missing or/insertion mutation, for purple tsai-tai a kind of sedge color base because of a kind of relevant nucleic acid sequence length polymorphism.At least
It is based in part on this and completes the present invention.Specifically, the present invention includes the following contents.
The first aspect of the present invention, provides a kind of oligonucleotides of synthesis, and the oligonucleotides includes purple tsai-tai A07 dye
A kind of sedge color base of colour solid because characteristic sequences.
In certain embodiments, the oligonucleotides includes sequence shown in following SEQ ID NO:3:
5 '-GGGAATCGATCCAACTTTGTTTC (A) n-3 ', wherein (A) n is poly A sequence, n is between 25-110
Natural number.Preferably, the length of the oligonucleotides of synthesis is 95-130bp.
In certain embodiments, the oligonucleotides further wraps other than comprising sequence shown in SEQ ID NO:5
Containing other sequences, the example of other sequences includes sequence and purple tsai-tai a kind of sedge of purple tsai-tai a kind of sedge color base because of the 25181252nd upstream
Sequence of the color base because of the 25181356th downstream.Preferably, the example of other sequences be SEQ ID NO:4 shown in sequence and
Sequence shown in SEQ ID NO:5.
The second aspect of the present invention, provide it is a kind of for plant identification whether include purple tsai-tai method comprising to
Detected whether in the DNA of plant identification exist positioned at purple tsai-tai A07 chromosome a kind of sedge color base because characteristic sequences the step of.
In certain embodiments, method includes the following steps:
(1) the step of sample DNA is extracted from plant sample to be identified；
(2) step of the nucleic acid containing the characteristic sequences is expanded from the sample DNA by PCR using forward and reverse primer
Suddenly, wherein the forward primer can be specifically bound to the first target sequence area, the reverse primer can be specifically bound to
Second target sequence area, the first target sequence Qu Weicong purple tsai-tai a kind of sedge color base rise to the 25181272nd extremely because of the 25181155th
Between any continuum, the second target sequence Qu Weicong purple tsai-tai a kind of sedge color base rises because of the 25181360th to the
25181690 any continuous regions between；With
(3) plant is identified by the length of electrophoresis detection sample P CR product.
It preferably, further comprise that (4) are expanded from comparison DNA by PCR containing described using forward and reverse primer
The step of nucleic acid of characteristic sequences；(5) length of the sample P CR product is carried out with the length for compareing PCR product
The step of comparing.
In certain embodiments, the comparison DNA is the DNA from the purple tsai-tai and/or DNA from cabbage heart.
The third aspect of the present invention, provide for a kind of sedge color base in plant identification because genotype method comprising detection
In DNA of plants to be identified positioned at purple tsai-tai A07 chromosome a kind of sedge color base because characteristic sequences content the step of.
In certain embodiments, method includes the following steps:
(1 ') extracts the step of sample DNA from plant to be identified；
(2 ') are expanded from the sample DNA and comparison DNA using forward and reverse primer by PCR under the same conditions to be contained
The step of nucleic acid of the characteristic sequences, wherein the forward primer can be specifically bound to the first target sequence area, it is described anti-
It can be specifically bound to the second target sequence area to primer, the first target sequence Qu Weicong purple tsai-tai a kind of sedge color base is because of
25181155 are risen to the 25181272nd any continuum between, the second target sequence Qu Weicong purple tsai-tai a kind of sedge color
Gene the 25181360th is risen to the 25181690th any continuous region between, and the comparison DNA is from homozygosis
The DNA of type purple tsai-tai plant and/or DNA from heterozygous purple tsai-tai plant；With
The step of content of the sample P CR product is compared by (3 ') with the content of the control PCR product.
The fourth aspect of the present invention provides purposes of the genetic fragment as molecular marked compound in purple tsai-tai breeding, wherein
The genetic fragment includes to be located at purple tsai-tai a kind of sedge color base because of the sequence between 25181252-25181356.
Detailed description of the invention
Fig. 1 is a kind of sedge color base of purple tsai-tai and cabbage heart because of the comparison result on 3.0 version genome of Chinese cabbage.
Fig. 2 is the pcr amplification product electrophoresis result figure of purple tsai-tai DNA and cabbage heart DNA.
Specific embodiment
The existing various exemplary embodiment that the present invention will be described in detail, the detailed description are not considered as to limit of the invention
System, and it is understood as the more detailed description to certain aspects of the invention, characteristic and embodiment.
It should be understood that it is to describe special embodiment that heretofore described term, which is only, it is not intended to limit this hair
It is bright.In addition, for the numberical range in the present invention, it is thus understood that specifically disclose the range upper and lower bound and they it
Between each median.Median and any other statement value in any statement value or stated ranges or in the range
Lesser range is also included in the present invention each of between interior median.These small range of upper and lower bounds can be independent
Ground includes or excludes in range.
Unless otherwise stated, all technical and scientific terms used herein has the routine in field of the present invention
The normally understood identical meanings of technical staff.Although the present invention only describes preferred method and material, of the invention
Implement or also can be used and similar or equivalent any method and material described herein in testing.The institute mentioned in this specification
There is document to be incorporated by reference into, to disclosure and description method relevant to the document and/or material.It is incorporated to any
When document conflicts, it is subject to the content of this specification.Unless otherwise stated, " % " is the percentage based on weight.
Unless otherwise noted, the gene location in the present invention refers to the position for 3.0 version of Chinese cabbage.
" purple tsai-tai " of the invention refers to a kind of cabbage of Cruciferae (Cruciferae) Brassica genus (Brassica)
Vegetables (Brassica rapa L.).The example of Chinese cabbage group includes multiple subspecies such as Chinese cabbage, pakchoi, turnip, tender flower stalk
And mutation.
" mutation " of the invention refers to one kind when being initially published on academic journal, is not plant lower grade (to become
Kind, subspecies, modification), later as understanding of the people to this kind is gradually comprehensive, gos deep into, discovery has in this kind
Individual plants or group have the variation different from the feature of this kind recognized when initially delivering this kind, and it is obvious to make a variation
And stablize, it is worth them individually to mark off come to show difference, botanist there will be the type of variation to name to this, and
According to different situations, delivered as the mutation in this kind.In contrast, there is original feature, there is no obviously making a variation
Type be known as the former mutation of this mutation.
" nucleic acid " of the invention includes DNA (i.e. DNA) and ribonucleic acid (i.e. RNA).In the present invention preferably
DNA.Nucleic acid of the invention further includes the nucleic acid of coding protein and the nucleic acid of non-coding albumen.The present invention preferably encodes albumen
The nucleic acid of matter.
In the present invention, " can specifically bind " refers to the sequence of forward primer under strict conditions and the first target sequence area
In conjunction with and/or reverse primer sequence in conjunction with the second target sequence area, without DNA other parts combine.Wherein, first
Target sequence area refers to from purple tsai-tai a kind of sedge color base is because of the 25181155th to the 25181272nd any continuum between.
Preferably, the first target sequence area is any continuous region in sequence shown in SEQ ID NO:4.Second target sequence area refers to
Any continuous region from purple tsai-tai a kind of sedge color base is because of the 25181360th to the 25181690th between.Preferably,
Two target sequence areas are any continuous region in sequence shown in SEQ ID NO:5.It is highly preferred that the second target sequence area refers to
Any continuous region from purple tsai-tai a kind of sedge color base is because of the 25181360th to the 25181450th between.
" stringent condition " refers to than being able to detect bigger degree (such as average+back of background measured value in the present invention
The measured value of standard error × 2 of scape measured value or more) condition that hybridizes with its target sequence.Stringent condition is sequence dependent
, it is different and different according to the environment hybridized.By control hybridization and/or the stringency of wash conditions, can identify
It is 100% complementary target sequence to primer.In certain embodiments, stringent condition refers to 5 × SSC, 50% formamide
With the nucleic acid hybridization conditions at 42 DEG C.In certain embodiments, stringent condition refers to high stringency conditions, i.e., is extremely for length
The sequence of few 100 nucleotide, it then follows standard DNA western blot procedure, in 5 × SSC, 0.3%SDS, 200 micrograms/ml at 42 DEG C
It shears and prehybridization and hybridization 12 to 24/ hours in the salmon sperm DNA and 50% formamide that are denaturalized.Material finally uses 0.2 ×
SSC, 0.2%SDS are washed three times at 65 DEG C, and 15 minutes every time.
[oligonucleotides of synthesis]
The first aspect of the present invention provides the oligonucleotides of synthesis, it includes a kind of sedge color base of purple tsai-tai A07 chromosome because
Characteristic sequences.
" characteristic sequences " of the invention belong to a kind of " InDel label " (insertion-deletion), refer to two kinds of parents
Difference in this in full-length genome.For another opposite parent, have in the genome of one of parent a certain number of
Nucleotides inserted or missing (Jander et al., 2002).
Characteristic sequences in the present invention, which refer to, for example exists only in seaweed in all subspecies of Chinese cabbage group and mutation
The genetic fragment of a kind of sedge plant.Preferably, characteristic sequences, which refer to, is present in purple tsai-tai, but is not present in the genetic fragment in cabbage heart.
It is highly preferred that characteristic sequences of the invention refer to that existing only in purple tsai-tai A07 chromosome in Chinese cabbage group may be not present
In the genetic fragment of cabbage heart A07 chromosome.It is further preferred that characteristic sequences of the invention refer to from purple tsai-tai A07 chromosome
A kind of sedge color base because the 25181252nd start to downstream about 95-130pb sequence.
In certain embodiments, oligonucleotides of the invention includes sequence shown in SEQ ID NO:3:
5'-GGGAATCGATCCAACTTTGTTTC(A)n-3'.Wherein, (A) n is poly A sequence, and n is between 25-110
Natural number.
In certain embodiments, shown in the SEQ ID NO:3 other than sequence, the oligonucleotides of synthesis of the invention into
One step includes other sequences.Other sequences can be a kind of sedge color base because of the sequence in first area cds and second area cds, Yi Ji
Partial sequence in three areas cds.The example includes sequence and purple tsai-tai of purple tsai-tai a kind of sedge color base because of the 25181252nd upstream
Sequence of a kind of sedge color base because of the 25181356th downstream.Preferably, before the example of other sequences is insertion position in the area third cds
The sequence in face, i.e. sequence shown in SEQ ID NO:4, the subsequent sequence in insertion position, i.e. SEQ ID NO in the area third cds:
Sequence in sequence shown in 5 or purple tsai-tai a kind of sedge color gene cDNA in the 3rd area cds before insertion position, i.e. SEQ ID
Sequence shown in NO:7.
[for plant identification whether include purple tsai-tai method]
The second aspect of the present invention, provide it is a kind of for plant identification whether include purple tsai-tai method.Preferably, herein
Plant refer to the plant from Cruciferae (Cruciferae), it is highly preferred that plant herein is from Brassica genus
(Brassica) plant, it is further preferred that plant herein is from Chinese cabbage group (Brassica rapa L.)
Plant.In certain embodiments, " plant " herein is a seed type plant, and method of the invention at this time refers to for identifying
The plant whether be purple tsai-tai method.In certain embodiments, " plant " herein is a plurality of types of plants or its plant
The mixture of object ingredient, method of the invention at this time refer to for identify in the plant whether include purple tsai-tai method.
The method of the present invention includes detect whether to exist in the DNA of plant to be identified to be located at purple tsai-tai A07 chromosome
A kind of sedge color base because characteristic sequences the step of.It has been illustrated in the first aspect about characteristic sequences, details are not described herein.
In the present invention, detect whether exist positioned at purple tsai-tai A07 chromosome a kind of sedge color base because characteristic sequences method
Any method known in the art can be used, it further includes direct that the example, which includes the method for nucleic acid of the amplification comprising characteristic sequences,
The method etc. that Plant Genome is sequenced.
In certain embodiments, whether of the invention includes the method for purple tsai-tai including the use of positive and negative for plant identification
The step of gene order containing characteristic sequences is expanded from the DNA of plant sample to be identified by PCR to primer.Herein just
The first target sequence area can be specifically bound to primer, first target sequence Qu Weicong purple tsai-tai a kind of sedge color base rises because of the 25181155th
To the 25181272nd any continuum between.Similarly, reverse primer herein can be specifically bound to second
Target sequence area.Second target sequence Qu Weicong purple tsai-tai a kind of sedge color base plays to the 25181690th appointing between because of the 25181360th
What continuous region.That is, the region to be amplified that forward primer and reverse primer of the invention is covered has included at least the present invention
Characteristic sequences full sequence.
In an exemplary embodiment, for plant identification whether be purple tsai-tai method the following steps are included:
(1) sample DNA is extracted from plant sample to be identified；
(2) the step of gene order containing characteristic sequences being expanded from sample DNA by PCR using forward and reverse primer；
(3) identify whether the plant is purple tsai-tai by the length of electrophoresis detection sample P CR product.
In step (1), " plant sample to be identified " refers to a part of plant entirety or plant, for example, leaves of plants or plant
Object stem etc..
It whether include that the method for purple tsai-tai further comprises for plant identification in other exemplary implementation scheme
(4) the gene sequence containing characteristic sequences is expanded from comparison DNA by PCR using with identical forward and reverse primer in step (2)
The step of column；(5) the step of length of sample P CR product being compared with the length of control PCR product.
In an exemplary embodiment, comparison DNA can be the DNA from purple tsai-tai as positive control, can also be with
It is the DNA from cabbage heart as negative control.Optionally, method of the invention can use above-mentioned positive control and yin simultaneously
Property control.
In the present invention, the PCR condition of step (2) and step (4) is preferably identical, and step (2) and (4) can carry out simultaneously
Or it is successively individually carried out with interval time.
[for a kind of sedge color base in plant identification because genotype method]
The third aspect of the present invention, provide it is a kind of for a kind of sedge color base in plant identification because genotype method.This method
In plant be preferably purple tsai-tai plant.Genotype of the invention preferably refers to homozygous or heterozygous.
The method of the present invention includes be located in the DNA for detecting plant to be identified a kind of sedge color base of purple tsai-tai A07 chromosome because
Characteristic sequences content the step of.Any method known in the art can be used in the method for detection characteristic sequences content, to this
It does not limit.Content in this method can be absolute content, be also possible to relative amount.
In certain embodiments, content of the invention refers to the relative amount relative to control or reference substance.Herein
Control or reference substance can be the content obtained under the same conditions by comparison DNA, be also possible in defined condition (standard bar
Part) under can be by repeating the operation several times the control or reference data of acquisition.
In an exemplary embodiment, for a kind of sedge color base in plant identification because the method for genotype may include following step
It is rapid:
(1 ') extracts the step of sample DNA from plant to be identified；
(2 ') are expanded from the sample DNA and comparison DNA using forward and reverse primer by PCR under the same conditions to be contained
The step of gene order of the characteristic sequences；
The step of content of sample P CR product is compared by (3 ') with the content of control PCR product.
Forward and reverse primer in this method can be identical as forward and reverse primer described in second aspect, and details are not described herein.
Comparison DNA in this method can be the DNA from homozygous purple tsai-tai plant as positive control, can also be with
It is the DNA from heterozygous purple tsai-tai plant as negative control.Comparison DNA of the invention can also use above-mentioned sun simultaneously
Property control and negative control.
[purposes of the genetic fragment as identification molecular marked compound in purple tsai-tai breeding]
The fourth aspect of the present invention provides purposes of the genetic fragment as molecular marked compound in molecular breeding.Preferably,
Genetic fragment includes to be located at purple tsai-tai a kind of sedge color base because of at least part sequence between 25181252-25181356.
Genetic fragment of the invention marks the molecular marked compound for identifying purple tsai-tai as InDel, further can be used as
The marker assisted selection tool of purple tsai-tai breeding, has very important significance.
Embodiment 1
The present embodiment is the exploitation marked with purple tsai-tai a kind of sedge color base by the InDel of close linkage.
Indel label derives from the heavy sequencing data of cabbage heart and purple tsai-tai material.Cabbage heart and seaweed are namely extracted respectively
A kind of sedge genomic DNA, concentration range send company's sequencing in Illumina Genome in 800-1300ng/ μ l, each 10 μ g of sample
Two parts of samples are carried out resurveying sequence on Analyzer sequencing system platform.Wherein, two kinds of samples respectively obtain about 32Gb both-end
(pair-ends,PE)reads。
It then is F with cabbage heart and purple tsai-tai1, F is picked out after hybridization2Green and each 25 single plant of purple, it is right to construct extreme pond
After be sequenced.Wherein, two mixed ponds respectively obtain about 11Gb both-end (pair-ends, PE) reads.Select the base of 3.0 version of Chinese cabbage
Because a group conduct refers to genome, bwa and samtools is utilized to carry out SNP exploitation.
Both-end sequencing (pair-ends, PE) reads is compared onto the genome of 3.0 version of Chinese cabbage first, is secondly being permitted
Perhaps 3 couples of reads are no less than and support the site, then filter out mass value less than 10, DP value less than 5 and near InDel
The site of 5bp is to obtain reliable SNP site.Found in filial generation pond with the consistent site information in parent's homozygosis site, most
After calculate snp_index.With reference to anthocyanin synthesis pathway gene (41) in arabidopsis, predict that the anthocyanin in Chinese cabbage synthesizes phase
Structural gene and controlling gene are closed, 73 related genes (Guo et al., 2014) is amounted to.Finally position purple tsai-tai a kind of sedge color base
Because A07 chromosome from 5 ' end 25180918-25181774 between, deposited between 25181252-25181356
In Insert Fragment.It is as shown in Figure 1 to make a concrete analysis of result.
Next, design forward primer InDelzx_A07-F, sequence is as shown in SEQ ID NO:1 and reverse primer
InDelzx_A07-R, sequence is as shown in SEQ ID NO:2.Using forward and reverse primer pair purple tsai-tai DNA and cabbage heart DNA into
Row amplification.Theoretically, the fragment length expanded in purple tsai-tai DNA is about 331bp；Amplification obtains in cabbage heart DNA
Fragment length is 230bp.
The accuracy of InDel marker is verified, the verification operation is as follows:
The reaction system of 1.PCR amplification are as follows: 2ulDNA, 2 × Rapid Taq of primer forward and reverse each 0.8ul, 10ul
Master Mix and distilled water 6.4ul.
2.PCR amplification program are as follows: 95 DEG C initial denaturation 3 minutes；95 DEG C are denaturalized 15 seconds, and 60 DEG C are annealed 15 seconds, 72 DEG C of extensions
15 seconds, 35 circulations；72 DEG C keep the temperature 5 minutes, 4 DEG C of preservations.
3. the preparation of Ago-Gel offset plate and electrophoresis:
The Ago-Gel of configuration 1%: 0.3g agarose and 0.5%TAE 30ml.
4. mixing then heating 3min, the I type nucleic acid staining agent of 3 μ l is added, encapsulating and plugs comb, comb at once as early as possible
With a thickness of 1.0mm.
It is worth noting that the tooth of comb will point-blank, and comb at hole can not there are bubble, after 30min on
0.5 × battery plate buffer can point sample.This is tested each sample and does three repetitions.
5. deposition condition is 150V, 10min.
PCR electrophoresis result is as shown in Figure 2.As the result is shown: wherein A swimming lane is shown as 5k Marker；B, C, D swimming lane result
It has been shown that, DNA fragmentation are one, and size 230bp matches with the material of cabbage heart；E, F, G swimming lane is as the result is shown: DNA fragmentation is
One, size is about 330bp, is matched with the material of purple tsai-tai.
By comparing the segment obtained in cabbage heart DNA cloning and the segment obtained in purple tsai-tai DNA cloning it can be found that
A Duan Xulie is inserted on the A07 chromosome of purple tsai-tai, sequence is as shown in SEQ ID NO:6.
Without departing substantially from the scope or spirit of the invention, the specific embodiment of description of the invention can be done more
Kind improvements and changes, this will be apparent to those skilled in the art.Other realities obtained by specification of the invention
Applying mode for technical personnel is apparent obtain.Present specification and embodiment are merely exemplary.
Sequence table
<110>Vegetable & Flower Inst., Chinese Academy of Agriculture Science
<120>with the molecular marker and application thereof of purple tsai-tai a kind of sedge color gene linkage
<130> BH1900065-1
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 1
agatggtcac tgatagcg 18
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 2
cacatgattc agataccgaa g 21
<210> 3
<211> 24
<212> DNA
<213> Brassica rapa L.
<220>
<221> polyA_site
<222> (24)..(24)
<400> 3
gggaatcgat ccaactttgt ttca 24
<210> 4
<211> 95
<212> DNA
<213> Brassica rapa L.
<400> 4
agatggtcac tgatagcggg aagattgcct ggacgaaccg acgacgaagt aaggatccat 60
tgggaaattt acttagagaa gaaactcatg aaaat 95
<210> 5
<211> 334
<212> DNA
<213> Brassica rapa L.
<400> 5
gggaatcgat ccaactaatc atcgtatcta ccatcacacc aactacactt ctagacgatt 60
cgtcaatgcc tcgtataaga aacatgaaac cgatattatt agtgatcaat cttcttcggt 120
atctgaatca tgtgatatga aactattacc cgtttcaagt accaatagct ctgaggctaa 180
tgctagttct ggaaacagcc ggttgcctga cctcaacatc ggtctcgtcc cgataaagac 240
cgtgacttct ttgccagatg gctcccttca agaacctagc ggatcctcta accatggttc 300
aacgagtcaa gaaacacttc ttctttttca gtga 334
<210> 6
<211> 103
<212> DNA
<213> Brassica rapa L.
<400> 6
gggaatcgat ccaactttgt ttcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60
gggaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 103
<210> 7
<211> 257
<212> DNA
<213> Brassica rapa L.
<400> 7
atgaacaaaa ttagccacgg cgctctctct cggccttccg gaatgctgca ccgtgccaag 60
aggtatagag ggagaaagta cgcaaagcca gaacttaaag aaagcaactt ctcaaaagac 120
gaggacgatc tcatcctcaa gcttcatgca cttcttggca atagatggtc actgatagcg 180
ggaagattgc ctggacgaac cgacgacgaa gtaaggatcc attgggaaat ttacttagag 240
aagaaactca tgaaaat 257
Claims (10)
1. a kind of oligonucleotides of synthesis, which is characterized in that the oligonucleotides includes a kind of sedge color base of purple tsai-tai A07 chromosome
The characteristic sequences of cause.
2. the oligonucleotides of synthesis according to claim 1, which is characterized in that the oligonucleotides includes following SEQ ID
Sequence shown in NO:3:
5 '-GGGAATCGATCCAACTTTGTTTC (A) n-3 ', wherein natural number of the n between 25-110.
3. the oligonucleotides of synthesis according to claim 2, which is characterized in that the length of the oligonucleotides is 95-
130bp。
4. it is a kind of for plant identification whether include purple tsai-tai method, which is characterized in that including in the DNA of plant to be identified
Detect whether exist positioned at purple tsai-tai A07 chromosome a kind of sedge color base because characteristic sequences the step of.
5. it is according to claim 4 for plant identification whether include purple tsai-tai method, which is characterized in that including following
Step:
(1) the step of sample DNA is extracted from plant sample to be identified；
(2) the step of nucleic acid containing the characteristic sequences being expanded from the sample DNA by PCR using forward and reverse primer,
Wherein the forward primer can be specifically bound to the first target sequence area, and the reverse primer can be specifically bound to second
Target sequence area, the first target sequence Qu Weicong purple tsai-tai a kind of sedge color base rise to the 25181272nd because of the 25181155th between
Any continuum, the second target sequence Qu Weicong purple tsai-tai a kind of sedge color base because the 25181360th rise to the 25181690th
Any continuous region between；With
(3) plant is identified by the length of electrophoresis detection sample P CR product.
6. it is according to claim 5 for plant identification whether include purple tsai-tai method, which is characterized in that further packet
Include (4) using described forward and reverse primer by PCR from comparison DNA the step of amplification of nucleic acid；(5) the sample P CR is produced
The step of length of object is compared with the length of the control PCR product.
7. it is according to claim 6 for plant identification whether include purple tsai-tai method, which is characterized in that the control
DNA is the DNA from the purple tsai-tai and/or DNA from cabbage heart.
8. it is a kind of for a kind of sedge color base in plant identification because genotype method, which is characterized in that including detecting plant to be identified
In DNA positioned at purple tsai-tai A07 chromosome a kind of sedge color base because characteristic sequences content the step of.
9. it is according to claim 8 for a kind of sedge color base in plant identification because genotype method, which is characterized in that including
Following steps:
(1 ') extracts the step of sample DNA from plant to be identified；
(2 ') are expanded from the sample DNA and comparison DNA by PCR containing described using forward and reverse primer under the same conditions
The step of nucleic acid of characteristic sequences, wherein the forward primer can be specifically bound to the first target sequence area, it is described reversely to draw
Object can be specifically bound to the second target sequence area, and the first target sequence Qu Weicong purple tsai-tai a kind of sedge color base is because of the 25181155th
It rises to the 25181272nd any continuum between, the second target sequence Qu Weicong purple tsai-tai a kind of sedge color base is because of the
25181360 are risen to the 25181690th any continuous region between, and the comparison DNA is from homozygous seaweed
The DNA of a kind of sedge plant and/or DNA from heterozygous purple tsai-tai plant；With
The step of content of the sample P CR product is compared by (3 ') with the content of the control PCR product.
10. purposes of the genetic fragment as molecular marked compound in purple tsai-tai breeding, wherein the genetic fragment includes to be located at purple
Tender flower stalk a kind of sedge color base is because of at least part sequence between 25181252-25181356.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110512022A (en) * | 2019-09-02 | 2019-11-29 | 中国农业科学院蔬菜花卉研究所 | Identify the molecular labeling and method of Vegetables in Brassica interspecific hybrid and progeny material A06 and C05 chromosome separation situation |
| 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 |
| CN116326473A (en) * | 2023-03-10 | 2023-06-27 | 贵州师范大学 | Breeding method of cabbage-cabbage type rape substitution line variety rape |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107581059A (en) * | 2017-10-27 | 2018-01-16 | 四川省农业科学院经济作物育种栽培研究所 | A kind of purple Chinese cabbage breeding method |
| CN109156337A (en) * | 2018-09-18 | 2019-01-08 | 贵州省油菜研究所 | A kind of ornamental rape flower selection |
-
2019
- 2019-05-21 CN CN201910423365.8A patent/CN110093346B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107581059A (en) * | 2017-10-27 | 2018-01-16 | 四川省农业科学院经济作物育种栽培研究所 | A kind of purple Chinese cabbage breeding method |
| CN109156337A (en) * | 2018-09-18 | 2019-01-08 | 贵州省油菜研究所 | A kind of ornamental rape flower selection |
Non-Patent Citations (1)
| Title |
|---|
| 陈路格: "紫菜薹紫色相关基因的QTL分析与pur07初定位", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110512022A (en) * | 2019-09-02 | 2019-11-29 | 中国农业科学院蔬菜花卉研究所 | Identify the molecular labeling and method of Vegetables in Brassica interspecific hybrid and progeny material A06 and C05 chromosome separation situation |
| 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 |
| CN110894541B (en) * | 2019-12-27 | 2022-05-17 | 山西农业大学 | Functional identification system of anthocyanin synthesis regulation gene based on molecular marker and application |
| CN116326473A (en) * | 2023-03-10 | 2023-06-27 | 贵州师范大学 | Breeding method of cabbage-cabbage type rape substitution line variety rape |
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