CN110317897A - The relevant SNP marker 5-162 of capsicum epidemic disease resistant gene and its specific primer and application - Google Patents
The relevant SNP marker 5-162 of capsicum epidemic disease resistant gene and its specific primer and application Download PDFInfo
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Abstract
The invention belongs to agricultural biological technical fields, and in particular to capsicum epidemic disease resistant gene SNP marker 5-162 and its specific primer and application.Present invention determine that relevant to capsicum epidemic disease resistance shape SNP site in capsicum genome, using above-mentioned SNP site, it the DNA to pepper plant need to only detect in seedling stage, so that it may capsicum epidemic disease resistant gene be judged, can be widely applied in molecular mark.
Description
Technical field
The invention belongs to agricultural biological technical fields, and in particular to the relevant SNP marker 5-162 of capsicum epidemic disease resistant gene
And its specific primer and application.
Background technique
Capsicum epidemic disease is one of the Major Diseases for influencing capsicum production, by phytophthora blight of pepper (Phytophthora
Capsici Leonian) cause, capsicum may occur in which that root system is downright bad after being infected by phytophthora, the symptoms such as stem, leaf and fruit rot,
Cause heavy losses.Introducing epidemic disease resistant gene through hybridization is the main means for cultivating pepper disease resistance kind, the difficult point of this method
It is capsicum material to the genetics of resistance rule multiplicity of epidemic disease, mainly by 2 or 2 or more controlled by multiple genes, single dominant gene
Control and single dominant gene add modifier Controlling model, and resistant gene has small species specificity and hazard symptoms specificity
Feature.
Molecular mark is based on genotype screening germ plasm resource, avoids or reduces the error of Phenotypic Selection, can be big
It is big to shorten breeding process, it is the important means for improving transformation accuracy and efficiency.It is anti-that prior art discloses multiple capsicum epidemic diseases
Property molecular labeling, however, these molecular labelings there is also genotype and phenotype it is inconsistent the problems such as, influence its material screening and
Application in molecular mark.For different resistant materials exploitation with related gene it is chain it is closer, adaptability is higher,
Detecting more efficient molecular labeling facilitates selection and the breed breeding of capsicum epidemic disease resistant material.
Summary of the invention
The purpose of the present invention is to provide a kind of relevant SNP marker 5-162 of capsicum epidemic disease resistant gene.
A further object of the present invention is to provide the KASP specific primers of above-mentioned SNP marker.
A further object of the present invention is to provide the applications of above-mentioned SNP marker and primer.
A further object of the present invention is to provide the methods for identifying anti-epidemic disease capsicum.
A further object of the present invention is to provide the detection kits for identifying anti-epidemic disease capsicum.
The relevant SNP marker 5-162 of the capsicum epidemic disease resistant gene of specific embodiment according to the present invention, the SNP mark
The nucleotide sequence of note is as shown in SEQ ID No.1, and wherein the 22nd bit base of sequence shown in SEQ ID No.1 is indicated with R, R
Represent G or A.
GGCCATAAAGGTTTTGAATTTRGAAAATGAAGAAGTATGCAAGAGGTT
The KASP of the relevant SNP marker 5-162 of the capsicum epidemic disease resistant gene of specific embodiment is special according to the present invention
Property primer, 5-162 primer sequence are as follows:
Preceding primer A1:5 '-GGCCATAAAGGTTTTGAATTTG-3 ',
Preceding primer A2:5 '-GGCCATAAAGGTTTTGAATTTA-3 ',
Primer C:5 '-AACCTCTTGCATACTTCTTCATTTTC-3 ' afterwards.
The KASP specific primer of specific embodiment according to the present invention, the 5 ' ends of described preceding primer A1, A2 are added with fluorescence
Label, wherein the 5 ' ends of the preceding primer A1 are added with FAM fluorescence labels sequence, and the 5 ' ends of the preceding primer A2 are added with HEX fluorescence
Sequence label.
The KASP specific primer of specific embodiment according to the present invention, the FAM fluorescence labels sequence are 5 '-
GAAGGTGACCAAGTTCATGCT-3 ', the HEX fluorescence labels sequence are 5 '-GAAGGTCGGAGTCAACGGATT-3 '.
The present invention provides the application of capsicum epidemic disease resistant gene SNP marker, especially in capsicum annuum marker-assisted breeding
In application.
The method of the anti-epidemic disease capsicum of identification of specific embodiment according to the present invention comprising apply capsicum epidemic disease resistance base
The step of identifying the epidemic disease resistance of capsicum material to be measured because of relevant SNP marker 5-162
When the epidemic disease disease resistance phenotype of capsicum is " susceptible ", which is A:A genotype, i.e., shown in SEQ ID No.1
22nd A's of nucleotide sequence is homozygous；
When the epidemic disease disease resistance phenotype of capsicum is " disease-resistant ", which is G:G genotype, i.e., shown in SEQ ID No.1
The 22nd of nucleotide sequence is the homozygous of G；Alternatively, the SNP site is A:G genotype, i.e. nucleosides shown in SEQ ID No.1
The 2nd of acid sequence is the heterozygous of A and G.
The method of the anti-epidemic disease capsicum of identification of specific embodiment according to the present invention, including using the KASP specificity
Primer carries out the step of PCR detection.
The detection kit of the anti-epidemic disease capsicum of identification of specific embodiment according to the present invention, wherein containing the KASP
Specific primer.
Also contain fluorescence probe A, fluorescence probe B, quenching probes A and quenching probes B in the kit.
The nucleotides sequence of the fluorescence probe A is classified as the preceding primer A1 of above-mentioned 5-162 primer, and 5 ' ends connect fluorescent base
Group A；The nucleotides sequence of the quenching probes A is classified as the reverse complementary sequence of above-mentioned A1 sequence, and 3 ' ends connect quenching group；
The nucleotides sequence of the fluorescence probe B is classified as the preceding primer A2 of 5-162 primer, and 5 ' ends connect fluorophor B；Institute
The nucleotides sequence for stating quenching probes B is classified as the reverse complementary sequence of above-mentioned A2 sequence, and 3 ' ends connect quenching group.
In the present invention, the fluorophor A is specially FAM fluorescence labels sequence；The fluorophor B is specially HEX
Fluorescence labels sequence；The quenching group is specially Q.
Beneficial effects of the present invention:
SNP marker according to the present invention can not be by capsicum epidemic disease resistance artificial infection idenfication, only in seedling stage to peppery
The DNA of pepper plant is detected, so that it may be judged capsicum epidemic disease resistant gene, can be widely applied to molecular labeling auxiliary and educate
Kind.SNP marker of the invention can carry out mass detection with KASP genotyping technique, improve efficiency, when shortening identification
Between.
Detailed description of the invention
Fig. 1 is that KASP marks amplification of the 5-162 in cross combination；Wherein, blue dot is and capsicum epidemic disease resistance
The single plant (G:G genotype) of parent's phase homogenic type, green point are the susceptible material of capsicum epidemic disease (A:A genotype), and red point is
Heterozygous single plant (A:G genotype)；
Fig. 2 is that KASP marks amplification of the 5-162 in 44 capsicum materials；Wherein, blue dot is and capsicum epidemic disease
The single plant (G:G genotype) of resistance parent's phase homogenic type, green point is the susceptible material of capsicum epidemic disease (A:A genotype).
Specific embodiment
The acquisition of 1 capsicum epidemic disease resistant gene associated SNP positions of embodiment and the determination of corresponding KASP primer
1. test material
With susceptible material 77013, IVF2014032 and H3, disease-resistant material C M334, perennial, 0601M and RP4 and its
F1 is test material.
2. phenotypic evaluation
Above-mentioned each capsicum material and its Resistance Identification of F1 generation phenotype use artificial infection idenfication method, each material 3 times
Repeat, 10 plants of every repetition, according to People's Republic of China's agricultural industry criteria " pepper disease resistance identification technology regulation part 1:
Capsicum blight-resistant identification technology regulation " (NYT 2060.1-2011) progress.
The result shows that the disease index of 77013, IVF2014032 and H3 is respectively 80.00,80.48 and 75.38, it is greater than
70, belong to susceptible material；The disease index of CM334, perennial, 0601M and RP4 are respectively 18.09,48.11,39.26 and
3.33, less than 70, belong to disease-resistant material.
3. the acquisition of capsicum epidemic disease resistant gene associated SNP positions and the determination of corresponding KASP primer
(1) genome resurveys sequence and molecular labeling design
The genome of susceptible material 77013 and disease-resistant material 0601M is carried out resurveying sequence, is ginseng with disease-resistant material C M334
Genome is examined, determines the SNP site in sample, and designs KASP primer.
Each pair of KASP molecular labeling contains 3 primer sequences: A1, A2, C.
Wherein, A1, A2 are only that the SNP site of end has differences, at the 5 ' ends of A1, A2 respectively added with different fluorescence marks
Sign sequence.
FAM fluorescence labels sequence: 5 '-GAAGGTGACCAAGTTCATGCT-3 '；
5 '-GAAGGTCGGAGTCAACGGATT-3 ' of HEX fluorescence labels sequence.
FAM fluorescence labels sequence and HEX fluorescence labels sequence and KASP V4.0 2X Master mix 96/384, Low
Sequence in Rox with fluorescence is complementary, and fluorescence can be excited after pairing.
C is reverse complementary sequence.
The PCR amplification system of KASP label includes DNA profiling, primer mixture, fluorescence probe A, fluorescence probe B, is quenched
The Taq enzyme of probe A and quenching probes B and high-fidelity, dNTP etc..Primer mixture includes primer A1, A2 and C.Fluorescence probe
The sequence of A is 5 '-GAAGGTGACCAAGTTCATGCT-3 ', and 5 ' ends connect 1 fluorophor FAM；The sequence of fluorescence probe B
For 5 '-GAAGGTCGGAGTCAACGGATT-3 ', 5 ' ends connect 1 fluorophor HEX；The sequence of quenching probes A is 5 '-
AGCATGAACTTGGTCACCTTC-3 ', 3 ' ends connect quenching group Q；The sequence of quenching probes B is 5 '-
AATCCGTTGACTCCGACCTTC-3 ', 3 ' ends connect quenching group Q.
The nucleotide sequence information of 1 KASP of table label
(2) screening of polymorphism SNP marker
Using the susceptible material 77013 of above-mentioned KASP primer pair, IVF2014032, H3 and disease-resistant material C M334,
Perennial, 0601M and RP4 and its F1 generation carry out PCR amplification.
Reading need to use 480 fluorescent quantitation instrument of Roche after KASP pcr amplification reaction, carry out KASP Genotying, read
Fluorescence data is taken, based on the analysis results according to the following specific genotype for determining testing gene: being aggregated in the display indigo plant close to X-axis
The genotype of the sample of color is to connect the allelotype of FAM fluorescence labels sequence, is aggregated in close to the display green in Y-axis
The genotype of sample is to connect the allelotype of HEX fluorescence labels sequence, and the genotype for the sample that centre is displayed in red is two
The heterozygous of kind allele, the lower left corner show that the sample of black is with H2The blank control of O alternate template.
5-162, phenotype and genotype identification result such as table 2 are combined into the consistent KASP primer sets of capsicum epidemic disease resistant phenotype
With shown in Fig. 1, molecular labeling parting is consistent with parental phenotypes, it can be used for the SNP marker auxiliary of capsicum epidemic disease resistant material
Selection.
5-152,5-155 and 5-163 label cannot accurately distinguish anti-sense parent and its F1, i.e., do not have on this SNP between parents
There is polymorphism, not can be carried out the molecular labeling parting of group.
2 molecular labeling 5-162 of table is in capsicum parents and F1 generation parting situation
The polymorphism of SNP site is that 22 of sequence shown in SEQ ID NO.1 are A or G.
When the epidemic disease disease resistance phenotype of capsicum is " susceptible ", which is A:A genotype, i.e. SEQ ID No.1 institute
Show the 22nd of nucleotide sequence for the homozygous of A；
When the epidemic disease disease resistance phenotype of capsicum is " disease-resistant ",
(1) SNP site be G:G genotype, i.e., the 22nd of nucleotide sequence shown in SEQ ID No.1 be G homozygosis
Type；Or
(2) SNP site be A:C genotype, i.e. the 22nd of nucleotide sequence shown in SEQ ID No.1 be A and G it is miscellaneous
Mould assembly.
Embodiment 2 screens anti-epidemic disease capsicum material using molecular labeling 5-162 from natural population
44 parts of capsicum materials are selected, is marked using KASP and carries out PCR amplification.Experimental result is shown in Table 3, Fig. 2
Parting situation of the 3 molecular labeling 5-162 of table in capsicum natural population
The result shows that the corresponding SNP site of molecular labeling 5-161 is respectively when the phenotype for trying capsicum is " disease-resistant "
G:G genotype；When the phenotype for trying capsicum is " susceptible ", the corresponding SNP site of molecular labeling 5-161 is respectively A:A.Cause
This, screens anti-epidemic disease material using molecular labeling 5-161 of the present invention from capsicum natural population.
Sequence table
<110>Vegetable & Flower Inst., Chinese Academy of Agriculture Science
<120>the relevant SNP marker 5-162 of capsicum epidemic disease resistant gene and its specific primer and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 48
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<221> allele
<222> (22)..(22)
<223> R = G OR A
<400> 1
ggccataaag gttttgaatt trgaaaatga agaagtatgc aagaggtt 48
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggccataaag gttttgaatt tg 22
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ggccataaag gttttgaatt ta 22
<210> 4
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aacctcttgc atacttcttc attttc 26
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gaaggtgacc aagttcatgc t 21
<210> 6
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gaaggtcgga gtcaacggat t 21
Claims (10)
1. the relevant SNP marker 5-162 of capsicum epidemic disease resistant gene, which is characterized in that the nucleotide of the SNP marker 5-162
Sequence is as shown in SEQ ID No.1, and wherein the 22nd bit base of sequence shown in SEQ ID No.1 is A or C.
2. the KASP specific primer of the relevant SNP marker 5-162 of capsicum epidemic disease resistant gene described in claim 1, special
Sign is that the primer sequence is as follows:
Preceding primer A1:5 '-GGCCATAAAGGTTTTGAATTTG-3 ',
Preceding primer A2:5 '-GGCCATAAAGGTTTTGAATTTA-3 ',
Primer C:5 '-AACCTCTTGCATACTTCTTCATTTTC-3 ' afterwards.
3. KASP specific primer according to claim 2, which is characterized in that 5 ' the end difference of the preceding primer A1 and A2
Added with fluorescence labels sequence.
4. KASP specific primer according to claim 2, which is characterized in that the 5 ' ends of the preceding primer A1 are added with FAM
Fluorescence labels sequence, the 5 ' ends of the preceding primer A2 are added with HEX fluorescence labels sequence.
5. KASP specific primer according to claim 4, which is characterized in that the FAM fluorescence labels sequence is 5 '-
GAAGGTGACCAAGTTCATGCT-3 ', the HEX fluorescence labels sequence are 5 '-GAAGGTCGGAGTCAACGGATT-3 '.
6. the application of the relevant SNP marker 5-162 of capsicum epidemic disease resistant gene described in claim 1.
7. the relevant SNP marker 5-162 of capsicum epidemic disease resistant gene described in claim 1 is in capsicum annuum marker-assisted breeding
In application.
8. the method for identifying anti-epidemic disease capsicum, which is characterized in that the method includes applying capsicum epidemic disease described in claim 1
The step of relevant SNP marker 5-162 of resistant gene identifies the epidemic disease resistance of capsicum material to be measured.
9. the method for the anti-epidemic disease capsicum of identification according to shown in claim 8, which is characterized in that
When the corresponding SNP site of the relevant SNP marker 5-162 of capsicum epidemic disease resistant gene be A:A homozygous gene type when, then to
It is susceptible for surveying the epidemic disease disease resistance phenotype of capsicum material；
When the corresponding SNP site of the relevant SNP marker 5-162 of capsicum epidemic disease resistant gene is that G:G homozygosity genotype or A:G are miscellaneous
When mould assembly genotype, then the epidemic disease disease resistance phenotype of capsicum material to be measured is disease-resistant.
10. identifying the detection kit of anti-epidemic disease capsicum, which is characterized in that containing as claimed in claim 2 in the kit
KASP specific primer.
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Cited By (1)
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| CN111961753B (en) * | 2020-09-28 | 2022-07-01 | 中国农业科学院蔬菜花卉研究所 | SNP (Single nucleotide polymorphism) marker related to resistance gene of pepper and tomato leaf blight virus, and specific primer and application thereof |
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