CN105624328B - The high-throughput molecular labeling and its labeling method of identification leaf muld of tomato resistance and application - Google Patents

The high-throughput molecular labeling and its labeling method of identification leaf muld of tomato resistance and application Download PDF

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CN105624328B
CN105624328B CN201610217298.0A CN201610217298A CN105624328B CN 105624328 B CN105624328 B CN 105624328B CN 201610217298 A CN201610217298 A CN 201610217298A CN 105624328 B CN105624328 B CN 105624328B
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CN105624328A (en
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程琳
张生
国家进
陈福东
张晓燕
董甜
李晓杰
刘岳
王如芳
董静
于彩玉
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Shandong Shouguang Vegetable Industry Group Co Ltd
Shandong Shouguang Vegetable Seed Industry Group Co Ltd
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Abstract

The invention discloses the high-throughput molecular labeling of identification leaf muld of tomato resistance and its labeling method and applications, molecular labeling is specifically to be matched using prime end base according to target gene critical specificity site design primer Cf-5-Al lele-F1, primer Cf-5-Al lele-F2 and primer Cf-5-R and carry out Genotyping to target gene；The present invention is high-throughput molecular marker systems using the SNPLine platform of PCR, so it is full-automatic to be able to achieve operating process, reduces human error；Analysis throughput is high, and daily achievable 500,000 SNP genotypings are suitble to a large amount of samples while detecting.The KASP molecular labeling of high-throughput detection based on leaf muld of tomato disease-resistant gene Cf-5 design, and it is applied to high flux screening and the identification of leaf muld of tomato disease-resistant plant, time and cost of labor can be greatlyd save, improve the breeding efficiency of molecular marker assisted selection, harm to the tomato excellent variety, effectively control leaf muld of tomato of cultivating leafmold resistance, it is of great significance and the innovation of breeding method that one important and technological innovation.

Description

The high-throughput molecular labeling and its labeling method of identification leaf muld of tomato resistance and application
Technical field
The invention belongs to agricultural biological technical fields, are related to a kind of for identifying the high-throughput molecule of leaf muld of tomato resistance Label and its labeling method and application.
Background technique
Tomato is important one of the vegetable crop in China, is played a very important role in vegetable at protected field production.Kind Eggplant leaf mold (Cladosporium fulvum Cooke) is one of the worldwide disease for threatening tomato in greenhouse production, seriously The quality for influencing tomato, leads to the tomato underproduction, brings massive losses to tomato in greenhouse production.Leaf muld of tomato in 1883 Britain is reported for the first time, fast because spreading speed, can break out and cause disaster in the short time, quickly just by the great attention of scientists. Leaf muld of tomato after TaiWan, China generation, occurs successively the 1930s in China mainland, with protection ground area Expand, harmfulness is more obvious.
In recent years, domestic and international scientist deeply widely study and explore to leaf muld of tomato.Studies have shown that Cultivating leaf mold disease-resistant variety is the prevention and treatment most effective approach of leaf muld of tomato.With progress of research, multiple leafs muld of tomato Disease-resistant gene has been accredited and has cloned.Wherein, Cf-5 gene is important one of leaf muld of tomato disease-resistant gene.At home and abroad In the numerous disease-resistant tomato varieties cultivated, Cf-5 plays irreplaceable role.Therefore, the exploitation efficient molecular labeling of Cf-5 is outstanding It is important.Wordregen etc. (1994) is developed in " WSL6 " tomato tight with Mi and Cf-2/Cf-5 using molecular labeling mapping Close chain SCAR molecular labeling REX-1.But there is recombination to a certain extent in Mi gene and Cf-5 gene, seriously affect The selection accuracy of molecular labeling REX-1.Yu Shuancang etc. (2005) develops codominance CAPS molecule according to Cf-5 gene order Label, this label need digestion, at high cost, experimental arrangement is cumbersome, detection efficiency is low.
In conclusion currently, tomato breeding still lacks the molecular labeling of accurate and efficient Cf-5 gene, seriously affect kind The breeding efficiency of eggplant leafmold resistance kind.Therefore, accurate, efficient and practical Cf-5 high throughput molecular labeling is developed, it is right Anti- leaf muld of tomato excellent variety is cultivated, the harm of leaf muld of tomato is effectively controlled, is of great significance.
Summary of the invention
Object of the present invention is to be directed to need digestion in the prior art, at high cost, experimental arrangement is cumbersome, and detection efficiency is low to be waited not Foot and defect, provide it is a kind of it is at low cost, quickly and efficiently, the high throughput of the high identification leaf muld of tomato resistant gene Cf-5 of accuracy rate Molecular labeling and its labeling method and application.
Technical solution of the present invention is as follows:
High-throughput molecular labeling provided by the present invention for detecting resistant gene Cf-5 includes primer Cf-5-Al lele- F1, primer Cf-5-Al lele-F2 and primer Cf-5-R.Primer is comprising following sequence of primer, and sequence is as follows:
Cf-5-Al lele-F1:
5’–GAAGGTGACCAAGTTCATGCTGCTGAAATCATGTTTCCTGATCTT-3’
(underscore part is FAM fluorescence labels sequence)
Cf-5-Al lele-F2:
5’-GAAGGTCGGAGTCAACGGATTGCTGAAATCATGTTTCCTGATCTC-3’
(underscore part is HEX fluorescence labels sequence)
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '
More specifically, design primer Cf-5-F1, primer Cf-5-F2, primer Cf-5-R, and in above-mentioned primer Cf-5-F1 Corresponding fluorescence labels sequence is added respectively with the 5 ' ends of primer Cf-5-F2.
Wherein, primer Cf-5-F1, primer Cf-5-F2, primer Cf-5-R are to include following sequence of primer:
Cf-5-F1:5 '-GCTGAAATCATGTTTCCTGATCTT-3’
Cf-5-F2:5 '-GCTGAAATCATGTTTCCTGATCTC-3’
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '
Preferably, fluorescence labels sequence are as follows:
Cf-5-F1adaptor:5 '-GAAGGTGACCAAGTTCATGCT-3 ' (FAM fluorescence labels sequence)
Cf-5-F2adaptor:5 '-GAAGGTCGGAGTCAACGGATT-3 ' (HEX fluorescence labels sequence)
Preferably, molecular labeling primer are as follows:
Cf-5-Al lele-F1:
5’–GAAGGTGACCAAGTTCATGCTGCTGAAATCATGTTTCCTGATCTT-3’
(underscore part is FAM fluorescence labels sequence)
Cf-5-Al lele-F2:
5’–GAAGGTCGGAGTCAACGGATTGCTGAAATCATGTTTCCTGATCTC-3’
(underscore part is HEX fluorescence labels sequence)
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '
The present invention also provides a kind of high-throughput molecule labelling method for identifying leaf muld of tomato resistance, key steps are as follows:
A extracts tomato complete genome DNA using CTAB method；
B is using primer Cf-5_Allele-F1, primer Cf-5_Allele-F2 and primer Cf-5-R with tomato full-length genome DNA is that template carries out PCR amplification；
C carries out fluorescent scanning to pcr amplification product；
D analyzes allelic gene typing result.
Preferably, PCR reaction system is KASP Genotyping PCR reaction system: 5ngDNA, 0.07ul in above-mentioned steps b 72 × assay of KASP mix, 2.5ul KASP V4.02 × Master Mix, adds ddH2O to 5ul.
Preferably, KASP72 × assay mix by concentration is 100uM in above-mentioned KASP Genotyping PCR reaction system Primer Cf-5_Allele-F1, primer Cf-5_Allele-F2 and primer Cf-5-R and ddH2O presses the volume ratio of 12 ︰, 12 ︰, 30 ︰ 46 It is mixed to get.
In KASP Genotyping PCR reaction system KASP V4.02 × Master Mix by fluorescence probe A, fluorescence probe B, Quenching probes A and quenching probes B, high fidelity enzyme and dNTP composition.Above-mentioned fluorescence probe A are as follows: 5 '- 5 ' the ends of GAAGGTGACCAAGTTCATGCT-3 ' connect 1 FAM fluorophor；Fluorescence probe B are as follows: 5 '- 5 ' the ends of GAAGGTCGGAGTCAACGGATT-3 ' connect 1 HEX fluorophor；Quenching probes A are as follows: 5 '- 3 ' the ends of AGCATGAACTTGGTCACCTTC-3 ' connect quenching group BHQ；Quenching probes B are as follows: 5 '- 3 ' the ends of AATCCGTTGACTCCGACCTTC-3 ' connect quenching group BHQ.
Amplified reaction program are as follows:
1:94 DEG C of initial denaturation 15min of stage；2:94 DEG C of 20s of stage, 65-55 DEG C of (1.0 DEG C of each cycle down) 1min, totally 10 A circulation；3:94 DEG C of 20s of stage, 55 DEG C of 1min, totally 26 recycle.
Preferably, to allelic gene typing and interpretation of result in step d: gained amplified production progress fluorescence signal is swept It retouches, tomato Cf-5 genotype is analyzed according to scanning result.If the tomato amplified production fluorescent signal data to be measured is through Kraken Blue is presented in software analysis in gained parting dendrogram, then the tomato Cf-5 genotype to be measured is T ︰ T, as tomato leaf mold Sick disease-resistant plant；If red is presented, genotype is C ︰ C, as disease plant；If green is presented, genotype is T ︰ C, i.e., For heterozygosis disease-resistant plant.
The present invention also provides the high-throughput molecular labelings for identifying leaf muld of tomato resistant gene Cf-5 in anti-tomato Application in leaf mold breed breeding.
By adopting the above-described technical solution, the beneficial effects of the present invention are: the present invention is based on KASP The SNPline gene of (Kompetitive Allele-Specific PCR, competitive ApoE gene) technology point Type detection technique platform utilizes the specially matching of prime end base according to target gene critical specificity site design primer SNP parting is carried out to target gene.And the SNPLine platform of based on PCR is high-throughput molecular marker systems, institute in this approach can Enough realize that operating process is full-automatic, reduces human error；Analysis throughput is high, compatible 96,384,1536 porous plates, achievable daily 20 to 500000 SNP genotypings are very suitable to a large amount of samples while detecting.It is set with leaf muld of tomato disease-resistant gene Cf-5 The high-throughput detection KASP molecular labeling of meter can in high flux screening and the identification for being applied to leaf muld of tomato disease-resistant plant To greatly save time and cost of labor, breeding efficiency is improved.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is that the present invention is illustrated using 1536 hole sample panel SNP genotyping result of tomato Cf-5 high throughput molecular marker analysis Figure；
In figure, A-T ︰ T genotype (disease-resistant), for blue；
B-T ︰ C genotype (heterozygosis is disease-resistant), for green；
C-C ︰ C genotype (susceptible), for red；
D- pink colour indicates that DNA poor quality or concentration are too low, and amplified production is not by clear parting.
Specific embodiment
With reference to the accompanying drawings and examples, the present invention is further explained.
Embodiment 1: the high-throughput molecular labeling design of identification leaf muld of tomato disease-resistant gene Cf-5
(1) the tomato material with Cf-5 resistance and the tomato material without Cf-5 resistance each 20 of separate sources are taken Remaining part extracts its complete genome DNA with CTAB method:
1) tomato leaf is freeze-dried, 0.6g sample is taken to be added to the 1.5mL centrifuge tube of the CTAB containing 1mL preheating after grinding In, during which 65 DEG C of water-bath 30min are overturned 3-5 times；
2) 400 μ L chloroforms are added: isoamyl alcohol mixes liquid (24 ︰ 1 of volume ratio), mixing of turning upside down, 12000rpm centrifugation 15min；
3) supernatant is taken, isometric isopropanol is added, is mixed by inversion, in -20 DEG C of placement 30min；
4) 12000rpm is centrifuged 10min, abandons supernatant, 70% ethanol wash 1 time；
5) it dries, 60 μ L ddH is added2O dissolution, -20 DEG C of storages are spare.
(2) the high-throughput molecular markers development of tomato Cf-5 gene
Ncbi database retrieve tomato Cf-5 gene C DS sequence, design primer using above-mentioned tomato sample DNA as template into Row PCR amplification carries out PCR amplification.
Primer sequence for the amplification of above-mentioned specific fragment are as follows:
1F:5 '-AGCAGATGAAATCCCTCGGTC-3 '
1R:5 '-CCTCGCTGCTTCTTTCTCCTT-3 '
PCR reaction system (20 μ L system) are as follows: 2 × Taq Mix, 10 0.5 0.5 μ L of μ L, 1R (10 μM) of μ L, 1F (10 μM), DNA profiling 20-100ng, ddH2O is supplied.
PCR amplification program: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 60s, 32 are followed Ring；72 DEG C of extension 10min.Wherein, PCR instrument is the S1000Thermal Cycler of BIO-RAD company.PCR product agarose Electrophoresis detection is tapped and recovered the purpose band that length is about 960bp, send Beijing Invitrogen company after TA connection, clone Sequencing.Sequencing result is compared and is analyzed, 7 single base mutation sites and 1 insertion mutation site are obtained.It is respectively as follows: 2750T/ C, T is inserted between 2770G/A, 2867A/C, 2928T/C, 2943G/A, 3159T/G, 3498A/G, 3254-3255.
There are T-C single base mutation sites at tomato Cf-5 gene C DS 2750 nucleotide of sequence, according to the SNP Point design is used for the KASP molecular labeling of high flux screening:
Cf-5-F1:5 '-GCTGAAATCATGTTTCCTGATCTT-3’
Cf-5-F2:5 '-GCTGAAATCATGTTTCCTGATCTC-3’
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '
Cf-5-F1 and Cf-5-F2 is added into corresponding fluorescence labels sequence at 5 ' ends, sequence is as follows:
Cf-5-F1adaptor:5 '-GAAGGTGACCAAGTTCATGCT-3 ' (FAM fluorescence labels sequence)
Cf-5-F2adaptor:5 '-GAAGGTCGGAGTCAACGGATT-3 ' (HEX fluorescence labels sequence)
Obtaining corresponding Cf-5 gene high throughput molecular labeling primer is comprising following sequence of primer:
Cf-5-Allele-F1:
5’–GAAGGTGACCAAGTTCATGCTGCTGAAATCATGTTTCCTGATCTT-3’
(underscore part is FAM fluorescence labels sequence)
Cf-5-Al lele-F2:
5’–GAAGGTCGGAGTCAACGGATTGCTGAAATCATGTTTCCTGATCTC-3’
(underscore part is HEX fluorescence labels sequence)
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '
Above-mentioned primer is synthesized by Shanghai Invitrogen biotech firm.
Embodiment 2: the foundation of high-throughput Markers for Detection tomato Cf-5 genetic method is utilized
One, genomic DNA is extracted
Referring to 1 genome DNA extracting method of embodiment.
Two, PCR amplification
Using different tomato dna group DNA as template, PCR amplification is carried out using molecular labeling of the present invention, obtains PCR expansion Increase production object.
Genotyping PCR reaction system:
96 orifice plates: 10ng genomic DNA, 5ul KASP V4.02 × Master Mix, 0.14ul KASP72 × assay Mix adds ddH2O to 10ul.
384 orifice plates: 5ngDNA, 2.5ul KASP V4.02 × Master Mix, 0.07ulKASP 72 × assay mix, Add ddH2O to 5ul.
1536 orifice plates: 72 × assay of 5ngDNA, 2.5ul KASP V4.02 × Master Mix, 0.07ulKASP Mix adds ddH2O to 5ul.
Wherein, KASP V4.02 × Master Mix be LGC Products, for 96/384 orifice plate KASP V4.02 × Master Mix catalogue number is KBS-1016-002；KASP V4.02 × Master Mix product for 1536 orifice plates Catalog number (Cat.No.) is KBS-1016-011.
In KASP Genotyping PCR reaction system KASP V4.02 × Master Mix by fluorescence probe A, fluorescence probe B, Quenching probes A and quenching probes B, high fidelity enzyme and dNTP composition.Above-mentioned fluorescence probe A are as follows: 5 '- 5 ' the ends of GAAGGTGACCAAGTTCATGCT-3 ' connect 1 FAM fluorophor；Fluorescence probe B are as follows: 5 '- 5 ' the ends of GAAGGTCGGAGTCAACGGATT-3 ' connect 1 HEX fluorophor；Quenching probes A are as follows: 5 '- 3 ' the ends of AGCATGAACTTGGTCACCTTC-3 ' connect quenching group BHQ；Quenching probes B are as follows: 5 '- 3 ' the ends of AATCCGTTGACTCCGACCTTC-3 ' connect quenching group BHQ.
Primer Cf-5-Allele-F1, the primer Cf-5-Allele- that 72 × assay of KASP mix is 100uM by concentration F2, primer Cf-5-R and ddH2O is mixed to get by the volume ratio of 12 ︰, 12 ︰, 30 ︰ 46.
KASP Genotyping pcr amplification reaction program are as follows:
1:94 DEG C of initial denaturation 15min of stage；2:94 DEG C of 20s of stage, 65-55 DEG C of (1.0 DEG C of each cycle down) 1min, is followed altogether Ring 10 times；3:94 DEG C of 20s of stage, 55 DEG C of 1min are recycled 26 times altogether.Wherein PCR water-bath thermal cycle is Hydrocycler 16-32 The high throughput thermally circulatory system is suitable for 96,384 and 1536 orifice plates.
The blank control for not adding template DNA in the single reaction system of experiment while setting, only sets in each PCR plate Set 2 blank controls.
Three, the fluorescent scanning of pcr amplification product
Pcr amplification product is scanned using two-way single excitation plate reader PHERA star, FAM excitation wavelength is 485nm, launch wavelength 520nm, HEX excitation wavelength are 528nm, launch wavelength 560nm, system reference fluorescent ROX excitation Wavelength is 575nm, launch wavelength 610nm.
At least three repetition is arranged in each pcr amplification product sample.
Four, allelic gene typing
Plate reader PHERA star scan data is analyzed using Kraken TM software, is determined based on the analysis results to be measured The specific genotype of tomato Cf-5 gene (referring to attached drawing 1).Being aggregated in the sample genotype being displayed in blue close to X-axis is connection The allelotype (referring to Figure 1A) of FAM fluorescence labels sequence, being aggregated in the sample genotype being displayed in red close to Y-axis is connection The allelotype (referring to Fig. 1 C) of HEX fluorescence labels sequence, the sample genotype of centre display green are heterozygous (referring to figure 1B), show that the sample of pink colour may be too low due to DNA poor quality or concentration, amplified production is not by clear parting (referring to figure 1D)。
Further interpretation of result is as follows: if the tomato amplified production fluorescent signal data to be measured is analyzed through Kraken software Blue is presented in gained parting dendrogram, then the tomato Cf-5 genotype to be measured is T ︰ T, the as disease-resistant plant of leaf muld of tomato Strain；If red is presented, genotype is C ︰ C, as disease plant；If green is presented, genotype is T ︰ C, and as heterozygosis is anti- Sick plant.
Embodiment 3: application of the high-throughput molecular labeling of tomato Cf-5 gene in tomato variety breeding
1) tomato breeding material is detected with the high-throughput molecular labeling of tomato Cf-5 gene, including F3 is for material 200 parts, F4 for 200 parts of breeding material, F5 for 200 parts of breeding material and F6 for 200 parts of breeding material, specific method is referring to implementing Example 2.
2) Fulvia fulva artificial infection idenfication is carried out to the tomato single plant that Markers for Detection is crossed.It is used in the 4-5 leaf phase Concentration is that bacteria suspension (under 200 × visual field) the sprinkling tomato blade face of 7-8 spore is inoculated with, and observes after 2 weeks, records tomato Plant incidence.
3) high-throughput molecular marker analysis result and artificial bacteria inoculation's result system are compared into analysis, discovery genotype and phenotype are kissed Conjunction rate is up to 100% (being shown in Table 1).
1 tomato Cf-5 gene high throughput molecular marker gene type analysis result of table and phenotype investigate the rate of coincideing

Claims (3)

1. the method for identifying leaf muld of tomato resistance, it is characterised in that: the method is by the primer pair using molecular labeling Tomato complete genome DNA carries out PCR amplification and identifies realization；
The primer of the molecular labeling are as follows:
Cf-5-Allele-F1:
5'-GAAGGTGACCAAGTTCATGCTGCTGAAATCATGTTTCCTGATCTT-3'；
Cf-5-Allele-F2:
5'-GAAGGTCGGAGTCAACGGATTGCTGAAATCATGTTTCCTGATCTC-3'；
Cf-5-R:5 '-CTTAGATTTCCAGTCGAGATCAAG-3 '；
It the described method comprises the following steps:
A extracts tomato complete genome DNA using CTAB method；
B is using the primer Cf-5-Allele-F1, primer Cf-5-Allele-F2 and primer Cf-5-R with tomato full genome Group DNA is that template carries out PCR amplification；The system of the PCR amplification is KASP Genotyping PCR reaction system, the KASP base Because of parting PCR reaction system are as follows: 5ngDNA, 0.07 μ l 72 × assay of KASP mix, 2.5 μ l KASP V4.02 × Master Above-mentioned three kinds of material mixings are added ddH by Mix2O to 5 μ l；
Wherein, primer Cf-5-Allele-F1, primer Cf-5-Allele- that KASP72 × assay mix is 100 μM by concentration F2 and primer Cf-5-R and ddH2O is mixed to get by the volume ratio of 12 ︰, 12 ︰, 30 ︰ 46；
KASP V4.02 × Master Mix is by fluorescence probe A, fluorescence probe B, quenching probes A and quenching probes B, high fidelity enzyme It is formed with dNTP；Wherein, fluorescence probe A are as follows: it is glimmering to connect 1 FAM in the 5 ' ends of 5 '-GAAGGTGACCAAGTTCATGCT-3 ' Light group；Fluorescence probe B are as follows: connect 1 HEX fluorophor in the 5 ' ends of 5 '-GAAGGTCGGAGTCAACGGATT-3 '；It quenches Go out probe A are as follows: connects quenching group BHQ in the 3 ' ends of 5 '-AGCATGAACTTGGTCACCTTC-3 '；Quenching probes B are as follows: 3 ' the ends of 5 '-AATCCGTTGACTCCGACCTTC-3 ' connect quenching group BHQ；
C carries out fluorescent scanning to pcr amplification product；
D analyzes allelic gene typing result.
2. the method for identification leaf muld of tomato resistance as described in claim 1, it is characterised in that: expanded in the step b anti- Answer program are as follows: 1:94 DEG C of initial denaturation 15min of stage；2:94 DEG C of 20s of stage, 65-55 DEG C of 1min, wherein each circulation decline 1.0 DEG C, totally 10 recycle；3:94 DEG C of 20s of stage, 55 DEG C of 1min, totally 26 recycle.
3. the method for identification leaf muld of tomato resistance as described in claim 1, it is characterised in that: fluorescence is swept in the step c Retouching is to be analyzed using Kraken software scan data, the genotype of tomato Cf-5 is determined based on the analysis results, if to be measured Tomato amplified production fluorescent signal data is analyzed through Kraken software is presented blue in gained parting dendrogram, then described to be measured Tomato Cf-5 genotype is T ︰ T, as leaf muld of tomato disease-resistant plant；If red is presented, genotype is C ︰ C, as susceptible Plant；If green is presented, genotype is T ︰ C, as heterozygosis disease-resistant plant.
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