CN105238869A - FLC1 specific SNP molecular marker based on competitive allele specificity PCR technology and application - Google Patents
FLC1 specific SNP molecular marker based on competitive allele specificity PCR technology and application Download PDFInfo
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Abstract
The invention discloses an FLC1 specific SNP molecular marker based on the competitive allele specificity PCR technology and an application, and provides an application of polymorphism or genotype matter for detecting FLC1-13859895G/ASNP locuses in a Chinese cabbage genome in authenticating or assisting in authenticating the bolting state of Chinese cabbages. The experiment proves that a developed SNP marker specific primer can well classify bolting characters, high application value is achieved, and preselection and assistant breeding of bolting resisting genetic materials can be achieved.
Description
Technical field
The invention belongs to technical field of biotechnology, relate to the special SNP marker of FLC1 based on competitive ApoE gene technology and application.
Background technology
Chinese cabbage group is the maximum vegetable crop of China's cultivated area, and the effect in China's vegetable basket project holds the balance.In recent years, along with the raising of people's living standard, Chinese cabbage produces and supplied by guarantee quantity is main turn to improve quality, meet whole year production supply and high-end demand is master.Eating quality characteristic Chinese cabbage cultivar that is excellent, that can meet multiple demand more and more sells well in the market as spring chinese cabbage, baby cabbage and fast dish etc., and the spring and summer Chinese cabbage cultivated area of Spring cabbage and extremely frigid zones is expanded year by year.Bolting is the key constraints that restriction China's early Chinese cabbage and high altitude localities spring and summer Chinese cabbage produce in advance, and it produces most important impact to Chinese cabbage is suppress blade Middle nutrition substance accumulation, causes yield and quality to decline.Screen anti-bolting genetic resources, cultivating anti-bolting new variety is solve the Chinese cabbage bolting fundamental way of blooming in advance, is also one of most important breeding objective of current Chinese cabbage.
It is the quantitative character regulated and controled by complicated idiotype network that bolting is bloomed, and a lot of genes in this network are all subject to the regulation and control in epigenetic level.FLC is the key regulator controlling flowering of plant.Chinese cabbage BrFLC family has BrFLC1-3 and BrFLC5 tetra-genes, and in Arabidopis thaliana and Chinese cabbage, these BrFLC of overexpression all can cause delay of blooming, and this illustrates function and Arabidopis thaliana FLC similar (Schranzetal., 2002 of BrFLC; Kimetal., 2007; Huang is fine, and 2007).Recent studies have shown that being positioned at the chromosomal BrFLC2 of A02 is important gene (Zhaoetal., 2010 that regulation and control Chinese cabbage blooms; Xiaoetal., 2013); Also research display is had, variable sheer and flowering time closely related (former beautiful perfume etc., 2008 of BrFLC1; Yuanetal., 2009).
In the process that breeding practice and related biological are studied, molecule marker plays an important role.It screens at target resource, and the gene of the specific economical character of location regulation and control, gene pyramiding, the application of the aspects such as cultivar identification is more and more extensive.SNP belongs to molecule marker of new generation, have abundance high, detect easily realize the features such as automatization.The sequencing of different plant species full-length genome and comparing shows, the distribution of SNP on genome is extremely abundant, wants much extensive compared to SSR marker conventional in current breeding.SNP mutation rate is low, and the SNP being especially in coding region is high stability, and its genetic stability is more much higher than genetic markers such as SSR, and circulation ratio when genetic analysis or gene diagnosis, accuracy are all better than SSR.
SNPline genotype tests based on KASP (competitive ApoE gene) is the high-throughput SNP typing method of Britain LGC (LaboratoryoftheGovernmentChemist) company limited exploitation, it has accurately, flexible, low cost, high-throughout feature, has become one of main stream approach of snp analysis in the world at present.The core of the program is KASP technology, i.e. CompetitiveAllele-SpecificPCR.This technology comes SNP somatotype based on the special coupling of prime end base and detect InDels (InsertionsandDeletions inserts and disappearance).
Molecule marker plays a part more and more important in the resistance breeding work based on molecular marker assisted selection (MAS) technology.But in application process, be likely subject to the restriction of genetic background with the closely linked molecule marker of goal gene, need to detect the polymorphism of parent in different colonies; In addition, in reduction division process, the molecule marker of the type still also exists because karyomit(e) occurs to exchange and loses the possibility with goal gene close linkage relation, and this makes the situation (Hayashietal., 2006 that in goal gene qualification process, there will be wrong choosing or leak choosing; Ingvardsenetal., 2008).Therefore the molecule marker of development function gene seems particularly important.
Summary of the invention
An object of the present invention is to provide and detect the polymorphism in FLC1-13859895G/ASNP site or the purposes of genotypic material in Chinese cabbage genome.
The invention provides the application in the bolting state of qualification or assistant identification Chinese cabbage of the polymorphism that detects FLC1-13859895G/ASNP site in Chinese cabbage genome or genotypic material;
Or the invention provides the application in the bolting phase product of characterization or assistant identification Chinese cabbage of the polymorphism that detects FLC1-13859895G/ASNP site in Chinese cabbage genome or genotypic material.
Second object of the present invention is to provide and detects the polymorphism in FLC1-13859895G/ASNP site or the purposes of genotypic material in Chinese cabbage genome.
The invention provides the polymorphism that detects FLC1-13859895G/ASNP site in Chinese cabbage genome or genotypic material identify or assistant identification Chinese cabbage be early bolting Chinese cabbage or late bolting Chinese cabbage in application;
Or the invention provides the application in characterization or assistant identification Chinese cabbage are early bolting Chinese cabbage or late bolting Chinese cabbage product of the polymorphism that detects FLC1-13859895G/ASNP site in Chinese cabbage genome or genotypic material.
In above-mentioned application, in described detection Chinese cabbage genome, the polymorphism in FLC1-13859895G/ASNP site or genotypic material comprise the primer sets be made up of the single strand dna shown in the single strand dna shown in sequence 1, sequence 2 and the single strand dna shown in sequence 3.
Above-mentioned substance can be primer sets or the reagent containing primer sets or test kit etc.
The present invention's the 3rd object is to provide a kind of to identify or assistant identification Chinese cabbage to be measured is the method for early bolting Chinese cabbage or late bolting breeds of Chinese cabbage.
Method provided by the invention, comprise the steps: that detecting Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is AA, GG or GA, if described Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is AA, then described Chinese cabbage to be measured is or candidate is early bolting Chinese cabbage; If described Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is GG or GA, then described Chinese cabbage to be measured is or candidate is late bolting Chinese cabbage.
Bolting index 0-33.3 is late bolting material, and 77.7-100 is early bolting material.
In aforesaid method, the method for described detection Chinese cabbage to be measured genome FLC1-13859895G/ASNP loci gene type is as follows:
1) direct Sequencing;
2) ApoE gene is carried out with primer pair Chinese cabbage to be measured;
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 1, sequence 2 and the single strand dna shown in sequence 3.
In aforesaid method, described FLC1-13859895G/ASNP site is the 182nd Nucleotide in sequence 4 in sequence table.
The present invention's the 4th object is to provide the primer of qualification or assistant identification Chinese cabbage bolting to be measured.
Primer provided by the invention, is made up of the single strand dna shown in the single strand dna shown in sequence 1, sequence 2 and the single strand dna shown in sequence 3.
The present invention's the 5th object is to provide the PCR reagent of qualification or assistant identification Chinese cabbage bolting to be measured.
PCR reagent provided by the invention is the PCR reagent containing above-mentioned primer; The final concentration of every bar primer in described PCR reagent in described primer is 10 μMs;
The present invention's the 6th object is to provide the test kit of qualification or assistant identification Chinese cabbage bolting to be measured.
Test kit provided by the invention, for containing above-mentioned primer or above-mentioned test kit.
The present invention's the 7th object is to provide a kind of to identify or assistant identification Chinese cabbage to be measured is the method for early bolting Chinese cabbage or late bolting breeds of Chinese cabbage.
Method provided by the invention, comprise the steps: to carry out ApoE gene with above-mentioned primer pair primer to be measured, if described Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is AA, then Chinese cabbage to be measured is or candidate is early bolting Chinese cabbage; If described Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is GG or G/A, then Chinese cabbage to be measured is or candidate is late bolting Chinese cabbage.
In aforesaid method, the template of described ApoE gene is the genomic dna of Chinese cabbage to be measured.
The present invention has following advantage and effect relative to prior art:
1. molecule marker provided by the invention is functional type gene molecule marker.Putting into practice in breeding work, in application process, be likely subject to the restriction of genetic background with the closely linked molecule marker of goal gene.SNP marker involved in the present invention causes based on the sequence variations of functional gene FLC.Research shows that FLC is the key factor that control Chinese cabbage bolting is bloomed, and therefore this mark can avoid the impact of difference on detected result of genetic background to a great extent.
2. molecule marker provided by the invention can carry out special differentiation and detection to G or the A base of this SNP site.
3. molecule marker provided by the invention in actual applications, low cost, high-throughput.At present, the method detecting SNP has sequencing, fluorescence detection, DNA chip, mass spectroscopic assays etc., and these method major part costs are high, and speed is slow.Molecule marker provided by the invention only needs PCR and fluoroscopic examination two steps, and cost is low, flux is high, add specificity high (namely accuracy is high), is specially adapted in breeding practice.
4. molecule marker accuracy provided by the invention is high.Utilize natural population's test of 105 parts of different genetic stockss compositions to show, the SNP marker special primer of exploitation to the good somatotype of Bolting trait, can have good using value, can realize resisting bolting genetic stocks selection in advance and assistant breeding.
Accompanying drawing explanation
Fig. 1 is for being the gene type qualification figure of FLC1-13859895G/A locus specificity molecule marker (FLC1SNP) in 105 parts high generation selfing shaped materials.
Fig. 2 is be the qualification figure of FLC1-13859895G/A site contrast primer in 105 parts high generation selfing shaped materials.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The acquisition of embodiment 1, molecule marker
Anti-bolting resource is obtained in order to screen faster and better, promote the carrying out of molecular marker assisted selection breeding practice, by finding the order sequenced data analysis of resurveying of 5 anti-bolting Chinese cabbage materials and 5 easy bolting Chinese cabbage materials, the SNP site of a G/A sudden change is had in the base of FLC1 gene internal the 3763rd, the physical location in this site is positioned at A10 karyomit(e) the 13859895th bit base, this SNP site called after FLC1-13859895G/A site.
According to FLC1-13859895G/A site, design can be used for the special primer of the FLC1 of KASP technology as follows as molecule marker: upstream primer FLC1-FF, upstream primer FLC1-FV and downstream primer FLC1-R.
Above-mentioned FLC1-FF, FLC1-FV and FLC1-R primer entrusts Britain LGC (LaboratoryoftheGovernmentChemist government chemist laboratory) company limited to obtain.
FLC1-FF, FLC1-FV and FLC1-R primer sequence feature is as follows:
Upstream primer FLC1-FF:GAAGGTGACCAAGTTCATGCTGAACCATGTTTTGGCTAGCCAGa (sequence 1);
Upstream primer FLC1-FV:GAAGGTCGGAGTCAACGGATTGAACCATGTTTTGGCTAGCCAGg (sequence 2);
Downstream primer FLC1-R:AATCGGATCGAAACTTAAACCGTAAC (sequence 3).
The SNP site of the 3763rd of FLC1 gene on last bit base of above-mentioned upstream primer g/a corresponding A 10 karyomit(e).
The application of molecule marker in the early bolting and late bolting material of qualification in embodiment 2, FLC1-13859895G/A site
1, molecule marker is at the early bolting identified and late bolting material
1) DNA extraction
Conventional CTAB method extracts the genomic dna of 105 kinds of Chinese cabbage materials to be measured in table 1 respectively.
Detect the quality of extracted DNA with agarose electrophoresis and Nanodrop2100 respectively, find that the genomic dna extracted all reaches relevant specification of quality, namely agarose electrophoresis display DNA band is single, does not have obvious disperse; Nanodrop2100 detects A260/280 (DNA sample does not have protein contamination) between 1.8-2.0; A260/230 is (DNA sample salt ionic concentration is low) between 1.8-2.0; 270nm does not have obvious photoabsorption (DNA sample does not have phenol to pollute); DNA consumption for competitive ApoE gene technology for detection is the every sample of 4 ~ 10ng/.It is for subsequent use that dilution DNA concentration becomes 10ng/ μ l, obtains DNA to be measured.
2) based on competitive ApoE gene
According to the standard test flow process that Britain LGC (LaboratoryoftheGovernmentChemist government chemist laboratory) company limited provides, namely the experiment flow based on competitive ApoE gene technology is tested, the matched reagent that following reagent provides for being LGC company except specified otherwise, reagent dosage, usage and whole experimental procedure are according to the operational guidance GenetypingAssay of LGC company, and ManualPart#15004070Rev.B carries out.KASPar reaction is carried out in 384 microwell plates or 1536 microwell plates (Cat.No.04729749001, Roche), and reaction system is 3ul or 1ul.
Concrete steps are: first utilize K-pette separatory workstation in microwell plate, to add DNA profiling to be measured (4ng/ μ l) 1.5ul, 60 DEG C of oven dry.Then under Kraken operating system, utilize Meridian application of sample workstation to add 1 × Mastermix (KBS-1016-002 or Cat.No.KBS-1016-011 in each reacting hole, LaboratoryoftheGovernmentChemist) (two upstream primers of embodiment 1 and downstream primer mix according to molar concentration rate 6:6:15 with primer premixed liquid, each primer final concentration is 10 μMs), the complete Kube that is successively placed on by microwell plate immediately of Mix packing seals instrument and Fusion laser sealer instrument upper sealing film.PCR reaction is carried out in high-throughput water-bath system Hydrocycler, and specific procedure is 94 DEG C of denaturations, 15 minutes; 94 DEG C, DEG C-55 DEG C, 20 seconds (sex change)-61,1 minute (renaturation & extends: increase 10 with touchdown program and circulate, often circulation reduction by 0.6 DEG C); 94 DEG C, 20 seconds (sex change)-55 DEG C, continue amplification 26 circulation in 60 seconds.After amplification terminates, BMGPHERAstar instrument is utilized to detect fluorescent signal and check somatotype situation.If somatotype is insufficient, then continue amplification, every 3 circulations check somatotype situation, until somatotype is complete.
As shown in Figure 1, result display somatotype is respond well for result, and this mark can this site of special differentiation be the material of GG or the AA or heterozygosis G/A that isozygotys of isozygotying;
If the Nucleotide in Chinese cabbage FLC1-13859895G/A site to be measured is that AA isozygotys, then Chinese cabbage to be measured is early bolting;
Isozygoty or G/A heterozygosis if the Nucleotide in Chinese cabbage FLC1-13859895G/A site to be measured is GG, then Chinese cabbage to be measured is late bolting.
2, bolting detects
By part Chinese cabbage genetic germplasm sowing of 105 in table 1, detect bolting index, detection method can refer to as recorded in Publication about Document: Yu Yangjun etc., the Chinese cabbage authentication method of resistance to bolting in indoor seedling stage, China's Vegetable, 2002.
Bolting grade scale: 0 grade, without flower bud, shortening stem has no elongation; 1 grade, there is flower bud, the long < 1cm of shortening stem; 3 grades, have flower bud, shortening stem obviously extends, the long 1-2cm of shortening stem; 5 grades, bolting, the long 2-5cm of a kind of sedge; 7 grades, bolting, the long > 5cm of a kind of sedge; 9 grades, bloom, the long > 5cm of a kind of sedge.
Specific formula for calculation is as follows:
Bolting index 0-33.3 is late bolting material, and 77.7-100 is early bolting material.
Result is as shown in table 1, be detected as in late bolting material at 47 parts of boltings, molecular markers for identification FLC1-13859895G/A site is that the material that GG isozygotys has 35 parts, molecular markers for identification FLC1-13859895G/A site is the material 12 parts that AA isozygotys, through bolting conventional authentication, 47 kinds is late bolting, the inventive method identify that accuracy is 74.5%;
Be detected as in early bolting material at 58 parts of boltings, molecular markers for identification FLC1-13859895G/A site is that the material that AA isozygotys has 4 parts, molecular markers for identification FLC1-13859895G/A site is the material 3 parts that GG isozygotys, heterozygosis G/A site have 1 part, through bolting conventional authentication, 58 kinds is early bolting, the inventive method identify that accuracy is 93.2%.
Table 1 is the gene type of 105 parts of materials in FLC1-13859895G/A site and bolting index cartogram
Table note: all material is selfing six generation above homozygous inbred lines.
Therefore, can find out, method of the present invention and molecule marker can be used for detecting the bolting state of Chinese cabbage to be measured.
Comparative example 1,
One, the design and synthesis of primer
Devise other two groups according to FLC1-13859895G/A site and detect primer.
Primer sets 1:
Upstream primer FLC1-FF1:GAAGGTGACCAAGTTCATGCTCATGTTTTGGCTAGCCAGa;
Upstream primer FLC1-FV1:GAAGGTCGGAGTCAACGGATTCATGTTTTGGCTAGCCAGg;
Downstream primer FLC1-R1:AATCGGATCGAAACTTAAACCGTA (sequence 3).
Primer sets 2:
Upstream primer FLC1-FF2:GAAGGTGACCAAGTTCATGCTACCATGTTTTGGCTAGCCAGa;
Upstream primer FLC1-FV2:GAAGGTCGGAGTCAACGGATTACCATGTTTTGGCTAGCCAGg;
Downstream primer FLC1-R2:CGGATCGAAACTTAAACCGTAAC (sequence 3).
Two, apply
Adopt the method for 1 of embodiment 2, unlike the primer sets 1 replaced with respectively by primer in comparative example and primer sets 2.
The detected result of the primer sets 1 in comparative example is as following table 2 and the left figure of Fig. 2.
The detected result of the primer sets 2 in comparative example is as following table 2 and the right figure of Fig. 2.
Table 2 is for utilizing primer sets 1 and primer sets 2 to the gene type of 105 parts of materials and bolting index cartogram
Table note: "-", fails somatotype.
Can find out, these two groups of primers can not carry out gene type.
Claims (10)
1. detect the application in the bolting state of qualification or assistant identification Chinese cabbage of the polymorphism in FLC1-13859895G/ASNP site in Chinese cabbage genome or genotypic material;
Or detect the application in the bolting phase product of characterization or assistant identification Chinese cabbage of the polymorphism in FLC1-13859895G/ASNP site in Chinese cabbage genome or genotypic material.
2. detect the polymorphism in FLC1-13859895G/ASNP site in Chinese cabbage genome or genotypic material identify or assistant identification Chinese cabbage be early bolting Chinese cabbage or late bolting Chinese cabbage in application;
Or detect the application in characterization or assistant identification Chinese cabbage are early bolting Chinese cabbage or late bolting Chinese cabbage product of the polymorphism in FLC1-13859895G/ASNP site in Chinese cabbage genome or genotypic material.
3. application according to claim 1 and 2, is characterized in that: in described detection Chinese cabbage genome, the polymorphism in FLC1-13859895G/ASNP site or genotypic material comprise the primer sets be made up of the single strand dna shown in the single strand dna shown in sequence 1, sequence 2 and the single strand dna shown in sequence 3.
4. to identify or assistant identification Chinese cabbage to be measured is the method for early bolting Chinese cabbage or late bolting breeds of Chinese cabbage for one kind, comprise the steps: that detecting Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is AA, GG or GA, if described Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is AA, then described Chinese cabbage to be measured is or candidate is early bolting Chinese cabbage; If described Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is GG or GA, then described Chinese cabbage to be measured is or candidate is late bolting Chinese cabbage.
5. method according to claim 4, is characterized in that: the method for described detection Chinese cabbage to be measured genome FLC1-13859895G/ASNP loci gene type is as follows:
1) direct Sequencing;
2) ApoE gene is carried out with primer pair Chinese cabbage to be measured;
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 1, sequence 2 and the single strand dna shown in sequence 3.
6. according to described application arbitrary in claim 1-5, it is characterized in that: described FLC1-13859895G/ASNP site is the 182nd Nucleotide in sequence 4 in sequence table.
7. the primer of qualification or assistant identification Chinese cabbage bolting to be measured, is made up of the single strand dna shown in the single strand dna shown in sequence 1, sequence 2 and the single strand dna shown in sequence 3.
8. the PCR reagent of qualification or assistant identification Chinese cabbage bolting to be measured, is the PCR reagent containing primer according to claim 7; The final concentration of every bar primer in described PCR reagent in described primer is 10 μMs.
9. qualification or the test kit of assistant identification Chinese cabbage bolting to be measured, for containing primer according to claim 7 or test kit according to claim 8.
10. to identify or assistant identification Chinese cabbage to be measured is the method for early bolting Chinese cabbage or late bolting breeds of Chinese cabbage for one kind, comprise the steps: to carry out ApoE gene with primer pair according to claim 7 primer to be measured, if described Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is AA, then Chinese cabbage to be measured is or candidate is early bolting Chinese cabbage; If described Chinese cabbage genome FLC1-13859895G/ASNP loci gene type to be measured is GG or G/A, then Chinese cabbage to be measured is or candidate is late bolting Chinese cabbage;
The template of described ApoE gene is specially the genomic dna of Chinese cabbage to be measured.
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| CN105506163A (en) * | 2016-02-02 | 2016-04-20 | 北京市农林科学院 | SNP molecular marker for identifying and controlling purple character formation of Chinese cabbages and application thereof |
| CN105603088B (en) * | 2016-02-02 | 2020-01-03 | 北京市农林科学院 | SNP molecular site for identifying downy mildew resistance QTL on Chinese cabbage A08 chromosome and application thereof |
| CN105506163B (en) * | 2016-02-02 | 2019-01-22 | 北京市农林科学院 | A kind of SNP marker and its application that identification control purple properties of Chinese cabbages is formed |
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| CN105525024A (en) * | 2016-02-16 | 2016-04-27 | 北京市农林科学院 | Specific SNP molecular marker for identifying cabbage clubroot 4# physiological race resistance and application |
| CN106755437A (en) * | 2016-12-29 | 2017-05-31 | 中国农业科学院蔬菜花卉研究所 | A kind of SNP marker of identification of being bloomed for Chinese cabbage group bolting |
| CN109306379A (en) * | 2017-10-13 | 2019-02-05 | 广州健天基因技术有限公司 | For detecting primer, detection method and the kit of human EGFR gene T790M mutation |
| CN108034753A (en) * | 2018-01-15 | 2018-05-15 | 南京农业大学 | A kind of and the SNP marker of Chinese cabbage flowering time close linkage and its application |
| CN108517374A (en) * | 2018-06-14 | 2018-09-11 | 中国农业科学院蔬菜花卉研究所 | A kind of SNP marker and its application |
| 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 |
| 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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