CN106755437A - A kind of SNP marker of identification of being bloomed for Chinese cabbage group bolting - Google Patents
A kind of SNP marker of identification of being bloomed for Chinese cabbage group bolting Download PDFInfo
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Abstract
The present invention relates to a kind of SNP marker of identification of being bloomed for Chinese cabbage group bolting.The SNP marker is the nucleotides of Pi3+1 that is located at the exon 3 of BrFLC5 genes on Chinese cabbage group A03 chromosomes, and it is A or G.The bolting flowering time of the GG genotype individuals of the SNP marker is later than the individuality of GA and AA genotype.SNP marker of the present invention can high throughput identification Chinese cabbage group control bolting bloom related gene B rFLC5 presence functional variants site, through the Detection accuracy of test-target loci gene type may be up to absolutely, with high efficiency and accuracy；It is simultaneously easy to operate, cheap, can effectively carry out marker assisted selection in Chinese cabbage group bolting resistant property molecular breeding.
Description
Technical field
The invention belongs to Chinese cabbage group molecular breeding technology field, and in particular to one kind is opened for Chinese cabbage group bolting
The SNP marker of flower identification.
Background technology
Chinese cabbage group polytype vegetable crop such as including Chinese cabbage, pakchoi, tender flower stalk, water dish, Wuta-tsai, turnip
And as the turnip type rape of oil crops, cultivate extensive in worldwide, it is with a long history.It is cabbage that bolting is bloomed
One of Main Agronomic Characters of crop, bolting opens the marketing quality for the time spent directly affecting Chinese cabbage group.In the spring of China
In the production cultivation of the autumn and winter Chinese cabbage group of season and extremely frigid zones, experience low temperature can frequently result in bolting in advance and bloom so that
It completely loses commodity, a great problem as puzzlement production.It is exactly all the time white to improve the Tolerance to immature bolting of kind
The important breeding objective of dish class vegetables.
FLC is inhibiting factor of blooming, and vernalization path in flowering of plant regulated and control network is take part in and from main path, in nutrition
Grow to reproductive growth change regulated and control network in play an important role.Low temperature can suppress the expression of FLC genes, so as to release
FLC is to the inhibitory action bloomed so that flowering of plant.The loss of function of FLC genes can cause requirement reduction of the plant to low temperature, no
Need to experience the process that low temperature suppresses FLC gene expressions, so as to show as prematurity.
The ancestors of Chinese cabbage experienced the process (Wang et al., 2011) of genome tripling after breaking up with arabidopsis.
There are 4 homologous genes of FLC, respectively BrFLC1, BrFLC2, BrFLC3 and BrFLC5 in Chinese cabbage genome, wherein
BrFLC1, BrFLC2, BrFLC3 are replicated from genome tripling, and BrFLC5 is then that arabidopsis experiences with the common ancestor of Chinese cabbage
Genome α replicates what is produced, and this copy has been lost in arabidopsis gene group, but is remained in Chinese cabbage group
(Yang et al.,2006).4 FLC homologous genes of Chinese cabbage group are located on different chromosome, wherein BrFLC1
In A10 chromosomes, BrFLC2 is located at A02 chromosomes, BrFLC3 and BrFLC5 be located at A03 chromosomes (Schranz et al.,
2002)。
Zhang Xueming etc. (2014) has found that the exon 3 of BrFLC5 has Pi3+1 (G-A) variations, and this variation causes
BrFLC5 genes are unable to normal transcription.The flowering time for carrying the Chinese cabbage group in A genotype site relatively carries G genotype site
Chinese cabbage group significantly do sth. in advance.The bolting of Chinese cabbage group can be sooner or later predicted using the variation in this site.
Competitive ApoE gene (KASP) technology is a kind of high flux, low cost, low false detection rate
SNP classifying methods, it has also become one of main stream approach of snp analysis in the world, extensively should in terms of crop molecular breeding
With.The SNP marker of BrFLC5 of the exploitation based on KASP technologies is possible to the earlier evaluations for Chinese cabbage bolting resistant property, effectively improves
The efficiency of the molecular breeding of resistance to bolting.
The content of the invention
The technical problems to be solved by the invention are directed to the deficiencies in the prior art offer one kind and are taken out for Chinese cabbage group
A kind of sedge is bloomed the SNP marker of identification, and the SNP marker can identify that Chinese cabbage group control bolting related gene B rFLC5 of blooming is deposited
Functional variants site, so as to effectively carrying out marker assisted selection in Chinese cabbage group bolting resistant property molecular breeding,
It is easy to operate.
Therefore, the invention provides a kind of SNP marker of identification of being bloomed for Chinese cabbage group bolting, the SNP marker
It is the nucleotides of Pi3+1 that is located at the exon 3 of BrFLC5 genes on Chinese cabbage group A03 chromosomes, it is A or G.
According to the present invention, the position of SNP is to be based on the Chinese cabbage genome sequence of 1.5 versions and determine.
In some embodiments of the invention, the bolting flowering time of the GG genotype individuals of the SNP marker is later than GA
With the individuality of AA genotype.
Present invention also offers a kind of primer that the SNP marker is expanded for PCR, its sequence information is：
Positive FAM fluorescent primers：5'-GAAGGTGACCAAGTTCATGCTTGAGCTACTAGAACTTGTGGAAAGG-3'；
Positive VIC fluorescent primers：5'-GAAGGTCGGAGTCAACGGATTCATGAGCTACTAGAACTTGTGGAAAGA-3'；
Reverse primer：5'-GGAGGAGAGCAAAAATAGTCTCAG-3'.
Invention further provides a kind of application of SNP marker in Chinese cabbage group bolting blooms identification.
According to the present invention, the bolting flowering time of the GG genotype individuals of the SNP marker is later than GA and AA genotype
It is individual.
In some embodiments of the invention, described application is comprised the following steps：
A, extracts the DNA of Chinese cabbage group to be measured, obtains DNA profiling；
B, special KASP Primer mix and general KASP is added in the DNA profiling to step A extractions
Mastermix, enters performing PCR amplification, obtains PCR primer；
C, fluorimetric analysis are carried out to PCR primer, and the fluorescence sent according to PCR primer is opened the bolting of Chinese cabbage group
Flower is identified；
The KASP Primer mix contain three species specific primers, respectively：
Positive FAM fluorescent primers：5'-GAAGGTGACCAAGTTCATGCTTGAGCTACTAGAACTTGTGGAAAGG-3'；
Positive VIC fluorescent primers：5'-GAAGGTCGGAGTCAACGGATTCATGAGCTACTAGAACTTGTGGAAAGA-3'；
Reverse primer；5'-GGAGGAGAGCAAAAATAGTCTCAG-3'；
The KASP Master mix include following each component：General TRET cassette fluorescent primers, ROX internal references
Dyestuff, KlearTaq archaeal dna polymerases and dNTP.
According to the present invention, if the fluorescence that PCR primer sends is FAM fluorescence, Chinese cabbage group to be measured is the GG of SNP marker
Genotype individuals；
If the fluorescence that PCR primer sends is VIC fluorescence, Chinese cabbage group to be measured is the AA genotype individuals of SNP marker；
If the fluorescence that PCR primer sends is the mixing fluorescence of FAM and VIC, Chinese cabbage group to be measured is the GA of SNP marker
Genotype individuals.
In some embodiments of the invention, the reaction system of the PCR amplifications is：
| Component | 96 orifice plates | 384 orifice plates | 1536 orifice plates |
| DNA profiling | 5μl | 2.5μl | 0.625μl |
| KASP Primer Mix | 5μl | 2.5μl | 0.625μl |
| KASP Master Mix | 0.14μl | 0.07μl | 0.0175μl |
| Reaction cumulative volume | 10.14μl | 5.07μl | 1.2675μl |
The step of PCR is expanded be：
In other embodiments of invention, the PCR amplifications are further comprising the steps of：
Beneficial effects of the present invention are：SNP marker of the present invention can high throughput identification Chinese cabbage group control take out
A kind of sedge is bloomed the functional variants site of related gene B rFLC5 presence, can be high through the Detection accuracy of test-target loci gene type
Up to absolutely, with high efficiency and accuracy；
Using detectable 1536 samples of PCR of the mark (when experiment is using 1536 microwell plate), or 384 samples
(when experiment is using 384 microwell plate) or 96 samples (when experiment is using 96 microwell plate), the testing cost of each sample is only 0.3
Unit/sample (1536 orifice plate), 0.8-1.0 units/sample (384 orifice plate), 1.8 yuan/sample (96 orifice plate), easy to operate, price is low
It is honest and clean, can effectively carry out marker assisted selection in Chinese cabbage group bolting resistant property molecular breeding.
Brief description of the drawings
Fig. 1 carries out the result schematic diagram of fluoroscopic examination for the pcr amplification product in embodiment 1 to 281 parts of Chinese cabbage materials.
Specific embodiment
To be readily appreciated that the present invention, the present invention is described more detail below.
The SNP marker of identification of being bloomed for Chinese cabbage group bolting involved in the present invention, is to be located at Chinese cabbage group A03
The nucleotides of Pi3+1 of the exon 3 of BrFLC5 genes on chromosome, it is A or G；Nucleotides sequence containing the SNP site
Row such as SEQ ID NO:Shown in 4, SNP site is that the 63rd nucleotides y, y of the sequence are A or G；The GG bases of the SNP marker
The individuality of GA and AA genotype is later than because of the individual bolting flowering time of type.
According to the present invention, the position of SNP is the Chinese cabbage genome sequence (Bra022771 (B.rapa based on 1.5 versions
Chromosome V1.5) 2013-05-17) and determine, network address：http://brassicadb.org。
According to SNP mutation information feature, the special set of competitive ApoE gene primer of design, including two
Bar forward primer (positive FAM fluorescent primers and forward direction VIC fluorescent primers), a reverse primer；Its sequence information is：
Positive FAM fluorescent primers：5'-GAAGGTGACCAAGTTCATGCTTGAGCTACTAGAACTTGTGGAAAGG-3'；
Positive VIC fluorescent primers：5'-GAAGGTCGGAGTCAACGGATTCATGAGCTACTAGAACTTGTGGAAAGA-3'；
Reverse primer：5'-GGAGGAGAGCAAAAATAGTCTCAG-3'.
The base of positive FAM fluorescent primers end is G, and the base of positive VIC fluorescent primers end is A, reverse primer sequence
The selection of row will ensure the fragment length of amplification in 60-120bp, to ensure the integrality of amplification.Two 5' ends of forward primer
Public fluorescence labels sequence is connected with, wherein the 5' ends of forward direction FAM fluorescent primers are connected as FAM fluorescence labeling sequences 5'-
GAAGGTGACCAAGTTCATGCT-3', the 5' ends of positive VIC fluorescent primers are connected as VIC fluorescence labeling sequences 5'-
GAAGGTCGGAGTCAACGGATT-3'.The sequence of " two forward primers " of the present invention is not limited to positive FAM fluorescence and draws
Thing sequence and forward direction VIC fluorescent primer sequences；Positive special primer 5'-ACTAGAACTTGTGGAAAGG-3' and 5'-
The 5' ends of CATGAGCTACTAGAACTTGTGGAAAGA-3' can connect other public fluorescence labeling sequences；For example, also may be used
VIC fluorescence labeling sequences are connected with the 5' ends of 5'-ACTAGAACTTGTGGAAAGG-3', in 5'-
The 5' ends connection FAM fluorescence labeling sequences of CATGAGCTACTAGAACTTGTGGAAAGA-3', form two new forward primers
Sequence, during subsequent detection, if the fluorescence that PCR primer sends is FAM fluorescence, Chinese cabbage group to be measured is the AA genes of SNP marker
Type is individual；If the fluorescence that PCR primer sends is VIC fluorescence, Chinese cabbage group to be measured is the GG genotype individuals of SNP marker；If
The fluorescence that PCR primer sends is the mixing fluorescence of FAM and VIC, then Chinese cabbage group to be measured is the GA genotype individuals of SNP marker.
SNP marker involved in the present invention can be applied to Chinese cabbage group bolting bloom identification in, the SNP marker
The bolting flowering time of GG genotype individuals is later than the individuality of GA and AA genotype.
In some embodiments of the invention, described application is comprised the following steps：
A, extracts the DNA of Chinese cabbage group to be measured, obtains DNA profiling；
B, 15ng/ μ l are diluted to by the DNA profiling that step A is obtained, and are subsequently adding special KASP Primer mix and are led to
KASP Mastermix, enter performing PCR amplification, obtain PCR primer；
The KASP Primer mix contain three species specific primers, respectively：
Positive FAM fluorescent primers：5'-GAAGGTGACCAAGTTCATGCTTGAGCTACTAGAACTTGTGGAAAGG-3'；
Positive VIC fluorescent primers：5'-GAAGGTCGGAGTCAACGGATTCATGAGCTACTAGAACTTGTGGAAAGA-3'；
Reverse primer；5'-GGAGGAGAGCAAAAATAGTCTCAG-3'；
The KASP Master mix include following each component：General TRET cassette fluorescent primers, ROX internal references
Dyestuff, KlearTaq archaeal dna polymerases and dNTP, purchased from LGC companies (Laboratory of the Government
Chemist British governments chemist laboratory, http://www.lgcgroup.com/) KASP Master mix kits；
The Mix containing 2 × Master in the kit, MgCl2And DMSO, wherein 2 × Master Mix are the present invention mentioning
KASPMaster mix；MgCl2It is due to the detection and genotyping signal that can not succeed caused by high or low G/C contents with DMSO
In the case of add what is used in KASP Master mix, the present invention does not use MgCl2And DMSO, user can be according to experiment
Actual conditions selection add and said components or be added without；
The reaction system of PCR amplification is：
The step of PCR is expanded be：
In the step of above-mentioned PCR is expanded 2, the 1st amplification of circulation is 94 DEG C of holdings 20s, 61 DEG C of holding 60s；2nd is followed
The amplification of ring is 94 DEG C of holdings 20s, 60.4 DEG C of holding 60s；……；10th amplification of circulation keeps 20s, 55.6 DEG C for 94 DEG C
Keep 60s；
If by above-mentioned steps enter performing PCR amplification after, fluorimetric analysis display genotyping result it is not ideal, can continue into
The PCR amplifications that row following steps are carried out,
C, fluorimetric analysis are carried out to PCR primer, and the fluorescence sent according to PCR primer is opened the bolting of Chinese cabbage group
Flower is identified；
Fluoroscopic examination can utilize (Laboratory of the Government Chemist British governments of LGC companies
Chemist laboratory, http://www.lgcgroup.com/) SNPline detected that other can also be used can detect
The instrument of FAM fluorescence and VIC fluorescence is detected；
If the fluorescence that PCR primer sends is FAM fluorescence, Chinese cabbage group to be measured is the GG genotype individuals of SNP marker；
If the fluorescence that PCR primer sends is VIC fluorescence, Chinese cabbage group to be measured is the AA genotype individuals of SNP marker；
If the fluorescence that PCR primer sends is the mixing fluorescence of FAM and VIC, Chinese cabbage group to be measured is the GA of SNP marker
Genotype individuals.
Embodiment
To make the present invention easier to understand, the present invention is further described below in conjunction with drawings and Examples,
These embodiments only serve illustrative, it is not limited to range of application of the invention.Raw material or group used in the present invention
If divide can be obtained by commercial sources or conventional method without specified otherwise.
Primer employed in following embodiments synthesizes acquisition by Invitrogen (Shanghai) Trading Co., Ltd.；Used
Breeds of Chinese cabbage by Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science provide.
Embodiment 1：The Pi3+1 existed using BrFLC5 in the SNP marker identification breeds of Chinese cabbage based on KASP technological development
(G-A) make a variation.
(1) extraction of breeds of Chinese cabbage complete genome DNA：Breeds of Chinese cabbage leaf tissue to be measured (281 portions of Chinese cabbage) is taken, is used
CTAB methods extract complete genome DNA, and the complete genome DNA of each breeds of Chinese cabbage is diluted into 15ng/ μ l；
(2) primer is diluted：The primer dilution information provided according to synthetic primer company is diluted, first by dry powder primer
Be centrifuged, dry powder primer when preventing from uncapping near the mouth of pipe is splashed, according to primer dilute information, by positive FAM fluorescent primers,
Positive VIC fluorescent primers and reverse primer (sequence information such as sequence table SEQ ID NO:1、SEQ ID NO:2 and SEQ ID NO:3
It is shown) concentration be diluted to 10 μM；
(3) KASP Primer mix are prepared：Take positive FAM fluorescent primers, each 12 μ of forward direction VIC fluorescent primers for having diluted
L, the μ l of reverse primer 30, add aseptic ultra-pure water (ddH2O) 46 μ l, obtain the KASP Primer mix of 100 μ l；
(4) PCR reaction systems are prepared：According to the addition of each component shown in table 1, PCR reaction systems are prepared；
Table 1：The addition of each component in PCR reaction systems
| Component | 96 orifice plates | 384 orifice plates | 1536 orifice plates |
| DNA profiling | 5μl | 2.5μl | 0.625μl |
| KASP Primer Mix | 5μl | 2.5μl | 0.625μl |
| KASP Master Mix | 0.14μl | 0.07μl | 0.0175μl |
| Reaction cumulative volume | 10.14μl | 5.07μl | 1.2675μl |
Blank (NTC) without DNA profiling is set simultaneously, and each reaction plate sets one or more blanks；
(5) PCR amplifications：After microwell plate is installed, pcr amplification reaction is followed the steps below；
If by above-mentioned steps enter performing PCR amplification after, fluorimetric analysis display genotyping result it is not ideal, can continue into
The PCR amplifications that row following steps are carried out,
(7) fluoroscopic examination：After PCR reactions terminate, the 7900HT Fast produced using Applied Biosystems companies
Real-Time PCR System machines are scanned to pcr amplification product, carry out fluorimetric analysis；And utilize software
Allelic Discrimination modules in SDS2.3 carry out parting, the excitation wave of two fluorescence (FAM fluorescence and VIC fluorescence)
Length is different with launch wavelength, is detected that testing result is as shown in Figure 1 using above-mentioned instrument；According to testing result, according to such as
Subscript accurately determines the genotype of Pi3+1 (G-A) variant sites of BrFLC5 presence in breeds of Chinese cabbage to be measured；
The standard is specially：The fluorescence signal of breeds of Chinese cabbage pcr amplification product to be measured shows by SDS2.3 software analysis
It is following 3 class to show that fluorescence signal gathers, including is aggregated in the blue round dot close to Y-axis, and representative sample has VIC fluorescence, then described
Breeds of Chinese cabbage BrFLC5 Pi3+1 (G-A) variant sites genotype be AA；The red spots close to X-axis are aggregated in, are represented
Sample has FAM fluorescence, then the genotype of the variant sites is GG；The green round dot near coordinate diagonal is aggregated in, is represented
Sample has two kinds of fluorescence values, then the genotype of the variant sites is heterozygous GA.If breeds of Chinese cabbage pcr amplification product to be measured
Fluorescence signal by SDS2.3 software analysis in black × number, irregular to be dispersed in coordinate arbitrary portion, the then change dystopy
The genotype of point fails to be identified；It is distributed near the origin in the coordinate lower left corner, legend is sky for the sample of solid black square frame
White control, blank should not both have FAM fluorescence values, without VIC fluorescence values yet.
(8) interpretation of result：Using above-mentioned testing result and the breeds of Chinese cabbage the bolting time (from be seeded into bolting when
Between) and flowering time (from the time spent is seeded into out), the correlation between analysis three；The present embodiment uses 281 parts altogether
Chinese cabbage carries out above-mentioned analysis, and the calculating of correlation is calculated by software I BM SPSS Statistics19.
There are 4 BrFLC genes in Chinese cabbage group, the wherein G-A functions of the Pi6+1 positions of the exon 6 of BrFLC1 become
Ectopic sites (hereinafter referred to as " BrFLC1 target sites ") are significantly correlated with flowering time.Therefore in control BrFLC1 target sites
G-A variant sites (hereinafter referred to as " the BrFLC5 mesh of the Pi3+1 positions of the exon 3 of BrFLC5 is carried out under conditions of genotype
Mark point ") genotype and bolting time (and the correlation analysis between flowering time.
281 parts of above-mentioned two target site genotype information, variety type, bolting time of breeds of Chinese cabbage in the present embodiment
The details of (from the time for being seeded into bolting) and flowering time (from the time spent is seeded into out) are as shown in table 2.In table
The genotype that " FLC1 " represents the G-A variant sites of the Pi6+1 positions of the exon 6 in gene BrFLC1 (is known gene
Type), the genotype that " FLC5 " represents the G-A variant sites of the Pi3+1 positions of the exon 3 in gene BrFLC5 (is by upper
State the genotype of case step detection).
Analysis result shows, under conditions of the genotype of BrFLC1 target sites is for AA, the base of BrFLC5 target sites
Because type is the bolting time of AA breeds of Chinese cabbage and flowering time and the bolting time and flowering time that genotype is GG breeds of Chinese cabbage
Significant difference is respectively provided with, SNP site is AA's for the bolting time of the breeds of Chinese cabbage of GG and flowering time are significantly later than SNP site
Breeds of Chinese cabbage (bolting time P=0.045, flowering time P=0.007)；When the genotype of BrFLC1 target sites is the bar of GG
Under part, the genotype of BrFLC5 target sites is the bolting time of AA breeds of Chinese cabbage and flowering time and the material that genotype is GG
The bolting time be also respectively provided with significant difference, SNP site is that the bolting time of the breeds of Chinese cabbage of GG and flowering time are significantly later than
SNP site is the breeds of Chinese cabbage (bolting time P=0.000, flowering time P=0.000) of AA.
By to randomly selected from 9 281 parts of Chinese cabbage groups of different cultivation populations (including Chinese cabbage, little Bai
Dish, purple tsai-tai, turnip, oil use Chinese cabbage, water dish etc.) carry out molecular marker screening, as a result show, this mark can detect entirely
281 parts of the portion genotype in material object site, and carry the storeroom flowering time of different BrFLC5 allele and take out
There is significant difference in a kind of sedge time.
It should be noted that embodiment described above is only used for explaining the present invention, do not constitute to of the invention any
Limitation.By referring to exemplary embodiments, invention has been described, it should be appreciated that word wherein used is descriptive
With explanatory vocabulary, rather than limited vocabulary.The present invention can be made within the scope of the claims by regulation
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it is related to
And specific method, material and embodiment, it is not intended that the present invention is limited to wherein disclosed particular case, conversely, this hair
It is bright to can be extended to other all methods and applications with identical function.
SEQUENCE LISTING
<110>Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science
<120>A kind of SNP marker of identification of being bloomed for Chinese cabbage group bolting
<130> 2016
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 46
<212> DNA
<213>Positive FAM fluorescent primers
<400> 1
gaaggtgacc aagttcatgc ttgagctact agaacttgtg gaaagg 46
<210> 2
<211> 48
<212> DNA
<213>Positive VIC fluorescent primers
<400> 2
gaaggtcgga gtcaacggat tcatgagcta ctagaacttg tggaaaga 48
<210> 3
<211> 24
<212> DNA
<213>Reverse primer
<400> 3
ggaggagagc aaaaatagtc tcag 24
<210> 4
<211> 147
<212> DNA
<213>Nucleotide sequence containing SNP site
<400> 4
gatcttcagt caaaatctct gaactatagt tcacaccatg agctactaga acttgtggaa 60
agyttagtac taactgagac tatttttgct ctcctccttt cattacaaat atattagggt 120
ttcctgtcaa tctgtgcata tatgcag 147
Claims (10)
1. a kind of SNP marker of identification of being bloomed for Chinese cabbage group bolting, the SNP marker is to be located at Chinese cabbage group A03
The nucleotides of Pi3+1 of the exon 3 of BrFLC5 genes on chromosome, it is A or G.
2. SNP marker according to claim 1, it is characterised in that the position of SNP is the Chinese cabbage gene based on 1.5 versions
Organize sequence and determine.
3. SNP marker according to claim 1 and 2, it is characterised in that the GG genotype individuals of the SNP marker are taken out
A kind of sedge flowering time is later than the individuality of GA and AA genotype.
4. a kind of primer for SNP marker described in PCR amplification claim any one of 1-3, its sequence information is：
Positive FAM fluorescent primers：5'-GAAGGTGACCAAGTTCATGCTTGAGCTACTAG AACTTGTGGAAAGG-3'；
Positive VIC fluorescent primers：5'-GAAGGTCGGAGTCAACGGATTCATGAGCTACTA GAACTTGTGGAAAGA-3'；
Reverse primer：5'-GGAGGAGAGCAAAAATAGTCTCAG-3'.
5. application of the SNP marker described in a kind of any one of claim 1-3 in Chinese cabbage group bolting blooms identification.
6. application according to claim 5, it is characterised in that the bolting of the GG genotype individuals of the SNP marker is bloomed
Time is later than the individuality of GA and AA genotype.
7. the application according to claim 5 or 6, it is characterised in that comprise the following steps：
A, extracts the DNA of Chinese cabbage group to be measured, obtains DNA profiling；
B, special KASP Primer mix and general KASP Mastermix is added in the DNA profiling to step A extractions,
Enter performing PCR amplification, obtain PCR primer；
C, fluorimetric analysis are carried out to PCR primer, the fluorescence sent according to PCR primer the bolting of Chinese cabbage group is bloomed into
Row identification；
The KASP Primer mix contain three species specific primers, respectively：
Positive FAM fluorescent primers：5'-GAAGGTGACCAAGTTCATGCTTGAGCTACTAGAACTTGTGGAAAGG-3'；
Positive VIC fluorescent primers：5'-GAAGGTCGGAGTCAACGGATTCATGAGCTACTAGAACTTGTGGAAAGA-3'；
Reverse primer；5'-GGAGGAGAGCAAAAATAGTCTCAG-3'；
The KASP Master mix include following each component：General TRET cassette fluorescent primers, ROX internal references dye
Material, KlearTaq archaeal dna polymerases and dNTP.
8. application according to claim 7, it is characterised in that to be measured if the fluorescence that PCR primer sends is FAM fluorescence
Chinese cabbage group is the GG genotype individuals of SNP marker；
If the fluorescence that PCR primer sends is VIC fluorescence, Chinese cabbage group to be measured is the AA genotype individuals of SNP marker；
If the fluorescence that PCR primer sends is the mixing fluorescence of FAM and VIC, Chinese cabbage group to be measured is the GA genes of SNP marker
Type is individual.
9. the application according to claim any one of 5-8, it is characterised in that the reaction system of PCR amplification is：
The step of PCR is expanded be：
10. application according to claim 9, it is characterised in that the PCR amplifications are further comprising the steps of：
Priority Applications (1)
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| CN107338309A (en) * | 2017-07-28 | 2017-11-10 | 华智水稻生物技术有限公司 | A kind of often SNP marker with cultivar scent gene badh2 and the application of rice |
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| CN109609671A (en) * | 2018-11-09 | 2019-04-12 | 北京市农林科学院 | A kind of detection Vegetable Crops of Brassica vegetable leaf marginal slit carves the SNP marker and its application of character |
| CN109609671B (en) * | 2018-11-09 | 2022-07-05 | 北京市农林科学院 | SNP molecular marker for detecting leaf edge cracking character of brassica species and vegetables and application thereof |
| CN109880927A (en) * | 2019-03-20 | 2019-06-14 | 江苏省农业科学院 | Detect SNP marker primer and its application of rape BnALS1R gene |
| CN110295248A (en) * | 2019-05-30 | 2019-10-01 | 河南省农业科学院园艺研究所 | For detecting KASP molecular labeling and its application of Chinese cabbage wax powder character |
| CN110295248B (en) * | 2019-05-30 | 2024-01-09 | 河南省农业科学院园艺研究所 | KASP molecular marker for detecting Chinese cabbage wax powder character and application thereof |
| CN110484645A (en) * | 2019-09-05 | 2019-11-22 | 北京市农林科学院 | Molecular labeling relevant to bolting character of Chinese cabbage and its application |
| CN110484645B (en) * | 2019-09-05 | 2020-07-28 | 北京市农林科学院 | Molecular marker related to bolting character of Chinese cabbage and application thereof |
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Application publication date: 20170531 |