CN107217098A - The KASP molecular labeling related to wheat anti growing out character and its application - Google Patents
The KASP molecular labeling related to wheat anti growing out character and its application Download PDFInfo
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Abstract
The invention discloses the Wheat Molecular Breeding field KASP molecular labeling related to wheat anti growing out character and its application.(AX 111578083, AX 111624595 and AX 110772653) is marked based on 3 that SNP marker and its flanking sequence the are developed KASP for being located at the homologous group of wheat the 3rd, parting can be carried out to anti growing out candidate locus exactly, the ear germinating resistance of wheat is predicted, so that more easily anti growing out material is identified and screened in laboratory conditions.The influence that environmental factor and human error are brought to phenotypic evaluation can be prevented effectively from using molecular marker assisted selection breeding technique, can be used in combination with phenotypic evaluation.3 KASP molecular labelings of the present invention, can provide strong selection site, improve the accuracy of anti growing out breeding material selection, realize the target of wheat anti growing out molecular marker assisted selection breeding.
Description
Technical field
The present invention relates to molecular biology and technical field of crop propagation, specifically, it is related to and wheat Pre-harvest Sprouting Resistance
The related KASP molecular labelings of shape and its application.
Background technology
Wheat is third place in the world generalized grain crop, is only second to paddy rice and corn, worldwide plantation extensively.Wheatear
Germination (Pre-harvest Sprouting, PHS) refer to that wheat runs into before harvest it is unbroken overcast and rainy when, not and harvest just exists
A kind of phenomenon of head sprouting.Spike sprouting is a kind of global disaster, it was reported that, Major Wheat cultivation state in the world's takes as added
There is serious Spike sprouting to endanger in, Japan, Australia, China.There are 10 barley and wheat main producing regions in China, mainly with central China,
The southeast area of wheat such as middle and lower reach of Yangtze River area of wheat, southwestern area of wheat Spike sprouting harm are even more serious, as phenomenon is moved northward in rainfall in recent years,
Also often there is Spike sprouting phenomenon in the north Spike sprouting area of wheat such as Northern Winter area of China.
Spike sprouting of the wheat seed before harvest can not only bring the grain yield of large area to reduce, and also result in wheat seed
The related enzymatic activity such as hydrolysis is raised rapidly in grain, reserve substance content in reduction seed, unit weight (testing weight), is gone out
Powder rate and flour drop value (Falling number) decline, and cause the various processed food deteriorations of wheat, and can influence small
The storage of wheat seed.Therefore, the wheat resource that seed selection has ear germinating resistance has great practical significance to wheat breeding.
Molecular marker assisted selection breeding technique, is to utilize the molecular labeling chain with purpose character candidate gene, passes through
Molecular Biological Detection candidate gene parting, reaches the purpose to purpose character determination；Its advantage is simple and quick, not by environment
And the influence of environment interaction, can be quickly and efficiently select target resource material.Mononucleotide polymorphism (SNP,
Single Nucleotide Polymorphisms), refer to the DNA sequences caused by the variation of single nucleotide acid on genome
Row polymorphism, including conversion (transition), transversion (transversion), missing (deletion) and insertion
(insertion).SNP turn into third generation genetic marker, many phenotypic differences of human body, to neurological susceptibility of medicine or disease etc. all
May be relevant with SNP.Mainly there are following characteristics：1st, quantity is more, widely distributed；2nd, suitable for quick, scale examination；3rd, equipotential base
It is readily estimated because of frequency；4th, it is easy to Genotyping.Utilize the SNP association analysis methods and the SNP of candidate gene of full-length genome
The method of variance analysis, can find the SNP variation related to phenotype.
KASP (competitive ApoE gene, i.e. Kompetitive Allele-Specific PCR) technology is
The SNPline Genotyping detection schemes of LGC Genomics companies.The core of the program is KASP technologies, and this technology is base
Come in the special matching of prime end base to SNP partings and detect that (Insertions and Deletions are inserted InDels
Enter and lack).This technological merit is：As long as first, synthesis two universal fluorescent probes, two general quenching probes, then add
Synthesize multiple SNP PCR primers for specific site, it is possible to survey many sites；Second, because of fluorescence probe and spy is quenched
Pin is all expensive, and KASP methods can be visited compared to Taqman methods by universal fluorescent probe instead of the fluorescence for site
Pin, greatlys save cost.
Therefore, the SNP related to anti growing out is filtered out, and is developed as the KASP of its SNP site of energy precise Identification
Mark, is selected anti growing out material, in seed selection wheat anti growing out material, is reduced influence of the Spike sprouting to wheat, is improved small
There is important meaning in terms of wheat yield and quality.
Wheat in China distributional region is vast, because various regions natural conditions, planting system, variety type and the level of production are present
Different degrees of difference, obvious planting area is on the one hand formd, place of china kind is on the other hand greatly enriched
Diversity.Using extensive local varieties from various parts of the country, its fringe in 2012 to 2015 varying environments is sent out
Bud phenotypic evaluation, and the association analysis of full-length genome is carried out, 3 are identified positioned at the homologous group of wheat the 3rd and Spike sprouting phenotype
Related SNP marker.For this 3 further researchs of molecular labeling expansion, and it is to be aided in available for molecular labeling by this exploitation
The KASP marks of selection and use, can effectively improve wheat breeding efficiency and accuracy.
The content of the invention
It is an object of the invention to provide the KASP molecular labeling related to wheat anti growing out character and its application.
In order to realize the object of the invention, the present invention provides the KASP molecular labeling related to wheat anti growing out character, altogether
Comprising 3 molecular labelings, 1. the genetic distance between molecular labeling AX-111578083 and anti growing out gene myb10-A1 is
0.35Mb；2. the genetic distance between molecular labeling AX-111624595 and anti growing out section R-loci is 3.55Mb；3. divide
Genetic distance between son mark AX-110772653 and anti growing out section R-loci is 3.73Mb.Molecular labeling AX-
111578083rd, AX-111624595 and AX-110772653 are all from place of china kind wheat, respectively positioned at China spring
SURVEY sequences (IWGSC2.28) 3A chromosome 173.81Mb, 3D chromosome 112.63Mb and 112.80Mb, their nucleotides
Sequence is respectively such as SEQ ID NO:Shown in 1-3.
The present invention also provides the primer for being used for based on KASP technological development expanding the molecular labeling, including forward primer
AL1, forward primer AL2 and reverse primer C.
Wherein, amplifier molecule mark AX-111578083 primer sequence is respectively such as SEQ ID NO:Shown in 4-6, amplification point
Son mark AX-111624595 primer sequence is respectively such as SEQ ID NO:Shown in 7-9, amplifier molecule mark AX-110772653
Primer sequence respectively such as SEQ ID NO:Shown in 10-12.
The present invention is also provided is used for the identification KASP molecule mark related to wheat anti growing out character containing the primer
The detection kit of note.
The present invention also provides the application of the molecular labeling, primer or kit in the initiative of wheat anti growing out material.
The present invention also provides the molecular labeling, primer or kit and educated in molecular labeling auxiliary wheat anti growing out selection
Application in kind.
The present invention further provides the wheat money that the molecular labeling, primer or kit have ear germinating resistance in seed selection
Application in source.Comprise the following steps：
1) DNA of wheat samples to be measured is extracted；
2) the μ l of template DNA 1.00, primer mixed liquor 0.14 μ l, KASP Master that concentration is 50-100ng/ μ l are taken
Mix 5.00 μ l, 50mM MgCl2Solution 0.08 μ l, H2The μ l of O 3.78, enter performing PCR amplification；Forward primer in primer mixed liquor
AL1 and forward primer AL2 final concentration are 10 μM, final concentration of 10 μM of reverse primer C；
3) pcr amplification product genotype is analyzed using fluorescence detector.
Step 2) PCR reaction conditions are：95 DEG C of pre-degenerations 15 minutes；First step amplified reaction：95 DEG C are denatured 20 seconds, 68 DEG C
Gradient annealing simultaneously extends 60 seconds, and 10 circulations, the temperature of each cycle annealing and extension reduces by 1 DEG C；Second step amplified reaction, 94
DEG C denaturation 20 seconds, anneals for 57 DEG C and simultaneously extends 60 seconds, 30 circulations；12 DEG C of preservations.
Step 3) be specially：Parting is carried out to pcr amplification product using biological software：Loci AX-
111578083-G is carries the excellent equipotential type plant of anti growing out, and loci AX-111624595-C sends out to carry anti-fringe
The excellent equipotential type plant of bud, loci AX-110772653-T is to carry the excellent equipotential type plant of anti growing out.
Identify the primer and the strong and weak method of detection wheat anti growing out ability of wheat anti growing out in following (a)-(e)
In application fall within protection scope of the present invention：
(a) the strong wheat breed of screening anti growing out ability.
(b) the weak wheat breed of screening anti growing out ability.
(c) new variety of wheat of anti growing out is cultivated.
(d) application of the 3 KASP molecular labelings in the special anti growing out material initiative of wheat.
(e) application of the 3 KASP molecular labeling primers in the special anti growing out material initiative of wheat.
Wheat anti growing out candidate locus of the present invention and its molecular labeling are prepared by the following：
(1) natural population is constituted by collecting 272 portions of local varieties wheats from Chinese 10 Barley Regional.
(2) natural population's Spike sprouting multiple years are identified
In -2015 years 2012 under Wenjiang, Yaan, Chongzhou City totally 6 different environment, wheat seed is harvested in dough stage
Grain, is hung at shady and cool ventilation after natural air drying 7d, carries out artificial threshing.In having lid plastic culture dish 3- is used on pad
The filter paper of 5ml distilled water wetting carries out germination test, and three repetitions of experimental design each repeat 50 seeds.Every other day choose
Go out to have showed money or valuables one carries unintentionally and seed in a mouldy condition, and count respectively, percentage of seedgermination accounted for total seed number percentage with the 7th day chitting piece number
Represent, calculate germination percentage.
Germination percentage (%)=chitting piece number/(seed sum-mouldy number) × 100%
(3) genotype is scanned
A) extracting genome DNA:272 colony of natural population plant DNA are extracted using CTAB methods, its concentration is detected, and it is dilute
Release to 50-100ng/ul.
B) Wheat660SNP is scanned
1. all material delivers to Bo Ao companies Affymetrix platforms, and carries out Genomic DNA quality testings, uses
Wheat660SNP array carry out genome-wide screening.
2. Wheat660SNP array primary data analysis：Obtain 630517 SNP probes altogether with above-mentioned steps, pass through
The wheat reference gene group collection of illustrative plates IWGSC2.28 delivered at present, anchors to totally 312,831 SNP markers of single site, wherein
A genomic markers 122,338,1 B gene group echo 137,494, D genomic markers 52,999.
3. natural population's mark quality is controlled：It is less than by screening the variant, miss rate in 272 parts of place of china kinds
20% and minimum gene frequency (MAF) is more than 5% mark totally 178,803.
4. natural population's layered method：Selected 178,803 high-quality SNP markers utilize bioinformatics software
Structure2.34 assesses landrace population layering, selects optimal colony's result K=5, obtains Q values.
5. natural population's affiliation is assessed：Selected 178,803 high-quality SNP markers utilize bioinformatics
Software Kinship assesses local varieties group's affiliation, obtains K values.
6. Spike sprouting whole-genome association：Selected 178,803 high-quality SNP markers, and be previously obtained
Q and K values, add the Spike sprouting phenotype under 6 varying environments, utilize bioinformatics software Tassel 5.0 carry out full genome
Group association analysis (GWAS), identify 3 stabilizations and Spike sprouting phenotype select by force related molecular labeling AX-111578083,
AX-111624595 and AX-110772653.
(4) exploitation of KASP marks
3 SNP markers AX-111578083, AX-111624595 are carried out to wheat anti growing out candidate material for convenience
Identification and assisted Selection with AX-110772653, while in order to reduce in breeding cost and workload, enhancing breeding work
Operability, it is necessary to will with target molecule label probe sequence exploitation be based on conventional molecular biological means can be used for identification and
The KASP marks of screening, the following is the key step during related KASP marker developments：
1. probe sequence：
To develop the molecular labeling that round pcr can be used based on laboratory, molecular mark and application are conveniently used for,
The present invention has carried out is converted into common molecular mark by AX-111578083, AX-111624595 and AX-110772653 probe sequence
The experiment of note, it is known that SNP marker AX-111578083, AX-111624595 and AX-110772653 probe sequence respectively such as
Under：
AX-111578083:
CGTCACATGGAGCATCAGGATCGGTAGCTTCCCCC[A/G]
TGAGGATTGGACCAGGGATCACCACATAAGATCCA(SEQ ID NO:1)
AX-111624595:
TCCACCGTAGTGGGAGGCCTATGAACCAACGAATT[C/T]
GCGGAGGAGCCAACAAAAGGAGACCGCACGACAGATCTGCCCGAAGTGACACATGTCTCCACGACTGGCGTGTA
(SEQ ID NO:2)
AX-110772653:
GTGGTGTGGAACTGCCTCAACCGCGCCTCTTGGAG[A/T]
TGTGCCTCGCTGGACCCGAGTTCTTCCCCTCATCT(SEQ ID NO:3)
2. design of primers:
Designed according to known SNP marker AX-111578083, AX-111624595 and AX-110772653 probe sequence
Following primer：
AX-111578083:
Forward primer AL1:5’–3’GAAGGTGACCAAGTTCATGCTGATCGGTAGCTTCCCCCa(SEQ ID NO:4)
Forward primer AL2:5’–3’GAAGGTCGGAGTCAACGGATTGATCGGTAGCTTCCCCCg(SEQ ID NO:5)
Reverse primer C:5’–3’GTGATCCCTGGTCCAATC(SEQ ID NO:6)
AX-111624595:
Forward primer AL1:5’–3’GAAGGTGACCAAGTTCATGCTGGCCTATGAACCAACGAATTc(SEQ ID
NO:7)
Forward primer AL2:5’–3’GAAGGTCGGAGTCAACGGATTGGCCTATGAACCAACGAATTt(SEQ ID
NO:8)
Reverse primer C:5’–3’CAGTCGTGGAGACATGTGTCAC(SEQ ID NO:9)
AX-110772653:
Forward primer AL1:5’–3’GAAGGTGACCAAGTTCATGCTCAACCGCGCCTCTTGGAGa(SEQ ID NO:
10)
Forward primer AL2:5’–3’GAAGGTCGGAGTCAACGGATTCAACC GCGCCTCTTGGAGt(SEQ ID NO:
11)
Reverse primer C:5’–3’CGAGTTCTTCCCCTCATCT(SEQ ID NO:12)
3. primer detection:
Using 22 parts of place of china kind Wheat volatiles DNA as template, it is utilized respectively above primer and enters performing PCR amplification,
(Bio-Rad iQ5 are real-time by BIO-RAD iCycler Thermal Cycler w/iQ5Optical Module for RT-PCR
Quantitative real time PCR Instrument) phosphor collection is carried out at 37 DEG C, using bioinformatics software BioRadCFXManager to PCR primer
Carry out parting.
The present invention provides molecular labeling (primer) and the side that 3, Wheat Molecular Breeding field is used to screen anti growing out gene
Method and application.Analysis shows, are marked based on 3 that SNP marker and its flanking sequence the are developed KASP for being located at the homologous group of wheat the 3rd
Remember (competitive ApoE gene mark), parting can be carried out to anti growing out candidate locus exactly, prediction wheat
Ear germinating resistance, so that more easily anti growing out material is identified and screened in laboratory conditions.Utilize molecule
Marker assisted selection breeding technique can be prevented effectively from the influence that environmental factor and human error are brought to phenotypic evaluation.The present invention's
3 KASP molecular labelings, can provide strong selection site, improve the accuracy of anti growing out breeding material selection, realize that wheat resists
The target of Spike sprouting molecular marker assisted selection breeding.
Brief description of the drawings
Fig. 1 (is associated point for phenotype and the genotype association analysis result of place of china kind wheat multiple years of the present invention
Analyse notable site AX-111578083, AX-111624595 and AX-110772653), profile information comes from China spring SURVEY sequences
Arrange IWGSC2.28.
The KASP molecular labelings that Fig. 2 is SNP site AX-111578083 of the present invention are on 22 parts of China ground of known type
Qualification result in square kind, wherein loci AX-111578083-G are to carry the excellent equipotential type plant of anti growing out.
The KASP molecular labelings that Fig. 3 is SNP site AX-111624595 of the present invention are on 22 parts of China ground of known type
Qualification result in square kind, wherein loci AX-111624595-C are to carry the excellent equipotential type plant of anti growing out.
The KASP molecular labelings that Fig. 4 is SNP site AX-110772653 of the present invention are on 22 parts of China ground of known type
Qualification result in square kind, wherein loci AX-110772653-T are to carry the excellent equipotential type plant of anti growing out.
Fig. 5 marks (AX-111578083, AX-111624595 and AX- for the KASP of 3 different SNP sites of the present invention
110772653) type and corresponding Spike sprouting phenotype comparing result in 138 parts of China promote mainly kind.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular Cloning:A Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The positioning of the wheat place of china kind anti growing out candidate SNP locus of embodiment 1 and the acquisition of molecular labeling
1st, the local varieties wheat from Chinese ten Barley Regional is collected, planted refined in Sichuan Agricultural University respectively at 2012
Pacify farm, plant within 2013 in Sichuan Agricultural University's Wenjiang Experimental Base and Chongzhou City modernization industrial base, plant within 2014 in
Sichuan Agricultural University's Wenjiang Experimental Base and plant within 2015 in Chongzhou City of Sichuan Agricultural University modernize industrial base.
2nd, Spike sprouting is identified
Wheat dough stage seed is harvested, is hung at shady and cool ventilation after natural air drying 7d, artificial threshing is carried out.Having
The filter paper soaked on lid plastic culture dish middle berth with 3-5ml distilled water carries out germination test, three repetitions of experimental design, Mei Gechong
Multiple 50 seeds.Every other day choose and showed money or valuables one carries unintentionally and seed in a mouldy condition, and count respectively, percentage of seedgermination was germinateed with the 7th day
Seed number accounts for total seed number percentage and represented, calculates germination percentage.
Germination percentage (%)=chitting piece number/seed sum-mouldy number × 100%
3rd, genotyping
A) extracting genome DNA：Place of china kind genome DNA is extracted using CTAB methods.Its concentration is detected, and it is dilute
Release to 50-100ng/ul.
B) Wheat660SNP is scanned
1. all material delivers to Bo Ao companies Affymetrix platforms, and carries out Genomic DNA quality testings, uses
Wheat660SNP array carry out genome-wide screening.
2. Wheat660SNP array primary data analysis：Obtain 630517 SNP probes altogether with above-mentioned steps, pass through
The wheat reference gene group collection of illustrative plates IWGSC2.28 delivered at present, anchors to totally 312,831 SNP markers of single site, wherein
A genomic markers 122,338,1 B gene group echo 137,494, D genomic markers 52,999.
3. natural population's mark quality is controlled：It is less than by screening the variant, miss rate in 272 parts of place of china kinds
20% and minimum gene frequency (MAF) is more than 5% mark totally 178,803.
4. natural population's layered method：Selected 178,803 high-quality SNP markers utilize bioinformatics software
Structure2.34 assesses landrace population layering, selects optimal colony's result K=5, obtains Q values.
5. natural population's affiliation is assessed：Selected 178,803 high-quality SNP markers utilize bioinformatics
Software Kinship assesses local varieties group's affiliation, obtains K values.
6. Spike sprouting whole-genome association：Selected 178,803 high-quality SNP markers, and be previously obtained
Q and K values, add the Spike sprouting phenotype under 6 varying environments, utilize bioinformatics software Tassel5.0 carry out full genome
Group association analysis (GWAS), identify 3 stabilizations and Spike sprouting phenotype select by force related molecular labeling AX-111578083,
AX-111624595 and AX-110772653 (Fig. 1).The germination percentage corresponding to the different type of three SNP markers is carried out respectively
Variance analysis, it is anti growing out to identify loci AX-111578083-G, AX-111624595-C, AX-110772653-T
Excellent loci.
4th, the exploitation of KASP marks
3 SNP markers AX-111578083, AX-111624595 are carried out to wheat anti growing out candidate material for convenience
Identification and assisted Selection with AX-110772653, while in order to reduce in breeding cost and workload, enhancing breeding work
Operability, it is necessary to will with target molecule label probe sequence exploitation be based on conventional molecular biological means can be used for identification and
The KASP marks of screening.We download or extended before and after each SNP marker at least 35bp base sequences respectively, design two forward directions
Primer (AL1, AL2) and a shared reverse primer C.
3. primer detection:
Using 22 parts of place of china kind Wheat volatiles DNA as template, SEQ ID NO are utilized:Primer pair shown in 4-12
Template enters performing PCR amplification.
A.PCR amplification systems are as follows:
Forward primer AL1 and forward primer AL2 final concentration are 10 μM in primer mixed liquor, and reverse primer C end is dense
Spend for 10 μM.
B.PCR response procedures are as follows:
C.PCR product detections use BIO-RAD iCycler Thermal Cycler w/iQ5Optical Module
For RT-PCR (Bio-Rad iQ5 real-time fluorescence quantitative PCRs instrument) carry out phosphor collection at 37 DEG C, utilize bioinformatics software
BioRadCFXManager carries out parting to PCR primer, and is compareed with known type.
Application of the molecular labeling of the present invention of embodiment 2 in selection anti growing out candidate locus
1st, collect and promote mainly totally 138 parts of kind from Chinese each area of wheat, plant high in Sichuan Agricultural University in 2015-2016
State agricultural modernization base, and carry out germination percentage identification in dough stage sowing.
2nd, the kind of promoting mainly to harvest carries out KASP mark detections, and specific method is：Blade genome is extracted in tri-leaf period
STb gene；Using 138 parts of genomic DNAs for promoting mainly kind and 16 parts of known type local varieties (control) as substrate, with exploitation
KASP labeled primers enter performing PCR amplification respectively, and the primer is：
AX-111578083:
Forward primer AL1:5’–3’GAAGGTGACCAAGTTCATGCTGATCGGTAGCTTCCCCCa(SEQ ID NO:4)
Forward primer AL2:5’–3’GAAGGTCGGAGTCAACGGATTGATCGGTAGCTTCCCCCg(SEQ ID NO:5)
Reverse primer C:5’–3’GTGATCCCTGGTCCAATC(SEQ ID NO:6)
AX-111624595:
Forward primer AL1:5’–3’GAAGGTGACCAAGTTCATGCTGGCCTATGAACCAACGAATTc(SEQ ID
NO:7)
Forward primer AL2:5’–3’GAAGGTCGGAGTCAACGGATTGGCCTATGAACCAACGAATTt(SEQ ID
NO:8)
Reverse primer C:5’–3’CAGTCGTGGAGACATGTGTCAC(SEQ ID NO:9)
AX-110772653:
Forward primer AL1:5’–3’GAAGGTGACCAAGTTCATGCTCAACCGCGCCTCTTGGAGa(SEQ ID NO:
10)
Forward primer AL2:5’–3’GAAGGTCGGAGTCAACGGATTCAACCGCGCCTCTTGGAGt(SEQ ID NO:
11)
Reverse primer C:5’–3’CGAGTTCTTCCCCTCATCT(SEQ ID NO:12)
3rd, PCR primer detection uses BIO-RAD iCycler Thermal Cycler w/iQ5Optical Module
For RT-PCR (Bio-Rad iQ5 real-time fluorescence quantitative PCRs instrument) carry out phosphor collection at 37 DEG C, utilize bioinformatics software
BioRadCFXManager carries out parting to PCR primer, and is compareed with known type.
4th, genotypic results
When 1. being expanded with AX-111578083 primers, there are 129 parts to promote mainly kind and amplified loci AL1
(Allele A), only 7 parts materials (5.15%) have amplified excellent loci AL2 (Allele G), 2 parts of material deficiencies.
By variance analysis contrast two locis the germination percentage for promoting mainly kind in 0.05 level significant difference (P values=
0.0135), the corresponding kind average germination percentage of promoting mainly of excellent equipotential AL2 (Allele G) is 0.5350, loci AL1
(Allele A) corresponding kind average germination percentage of promoting mainly is 0.7848 (Fig. 2).
When 2. being expanded with AX-111624595 primers, there are 127 parts to promote mainly kind and amplified loci AL2
(Allele T), only 9 parts materials (6.62%) have amplified excellent loci AL1 (Allele C), 2 parts of material deficiencies.
By variance analysis contrast two locis the germination percentage for promoting mainly kind in 0.001 level difference extremely significantly (P values=
1.1E-09), the corresponding kind average germination percentage of promoting mainly of excellent equipotential AL1 (Allele C) is 0.2986, loci AL2
(Allele T) corresponding kind average germination percentage of promoting mainly is 0.8023 (Fig. 3).
When 3. being expanded with AX-110772653 primers, there are 121 parts to promote mainly kind and amplified loci AL1
(Allele A), only 15 parts materials (11.03%) have amplified excellent loci AL2 (Allele T), and 2 parts of materials lack
Lose.By variance analysis contrast two locis the germination percentage for promoting mainly kind in 0.001 level difference extremely significantly (P values
=5.3E-08), the corresponding kind average germination percentage of promoting mainly of excellent equipotential AL2 (Allele T) is 0.4518, loci AL1
(Allele A) corresponding kind average germination percentage of promoting mainly is 0.8108 (Fig. 4).
5th, (AX-111578083, AX-111624595 and AX-110772653) is marked in 138 parts of China using 3 KASP
Promote mainly in kind, parting is carried out to anti growing out candidate locus, Fig. 5 is as a result seen.As a result show：
1. the excellent loci of anti growing out identified in Chinese local varieties, promotes mainly in China and is equally fitted in kind
With experimental result is with being expected unanimously.Illustrate the anti growing out candidates of the present invention has the effect of anti growing out really.
2. the excellent loci of anti growing out identified in Chinese local varieties, the frequency in kind is promoted mainly in China
Very low (5.15%-11.03%).Illustrate that the anti growing out candidate locus mark of the present invention adapts to promote mainly cultivar for China
In.
3. 3 KASP molecular labelings of the invention, clearly, chromosome position is clear for sequence, primer, efficiently, accurate, through case
Example is implemented, and can be directly used in molecular marker assisted selection breeding.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
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Claims (9)
1. the KASP molecular labeling related to wheat anti growing out character, it is characterised in that altogether comprising 3 molecular labelings, 1. divide
Genetic distance between son mark AX-111578083 and anti growing out gene myb10-A1 is 0.35Mb；2. molecular labeling AX-
Genetic distance between 111624595 and anti growing out section R-loci is 3.55Mb；3. molecular labeling AX-110772653 with
Genetic distance between anti growing out section R-loci is 3.73Mb；The nucleotide sequence of 3 molecular labelings is respectively such as SEQ ID
NO:Shown in 1-3.
2. being used for based on KASP technological development expands the primer of molecular labeling described in claim 1, it is characterised in that including just
To primer AL1, forward primer AL2 and reverse primer C；
Wherein, amplifier molecule mark AX-111578083 primer sequence is respectively such as SEQ ID NO:Shown in 4-6, amplifier molecule mark
Remember AX-111624595 primer sequence respectively such as SEQ ID NO:Shown in 7-9, amplifier molecule mark AX-110772653's draws
Thing sequence is respectively such as SEQ ID NO:Shown in 10-12.
3. being used for containing primer described in claim 2 identifies the inspection of the KASP molecular labeling related to wheat anti growing out character
Test agent box.
4. molecular labeling described in claim 1, primer described in claim 2 or kit described in claim 3 are in the anti-fringe of wheat
Application in germination material initiative.
5. molecular labeling described in claim 1, primer described in claim 2 or kit described in claim 3 are in molecular labeling
Aid in the application in wheat anti growing out selection and use.
6. molecular labeling described in claim 1, primer described in claim 2 or kit described in claim 3 have in seed selection
Application in the wheat resource of ear germinating resistance.
7. application according to claim 6, it is characterised in that comprise the following steps：
1) DNA of wheat samples to be measured is extracted；
2) the μ l of template DNA 1.00, μ l, the KASP Master Mix of primer mixed liquor 0.14 that concentration is 50-100ng/ μ l are taken
5.00 μ l, 50mM MgCl2Solution 0.08 μ l, H2The μ l of O 3.78, enter performing PCR amplification；In primer mixed liquor forward primer AL1 and
Forward primer AL2 final concentration is 10 μM, final concentration of 10 μM of reverse primer C；
3) pcr amplification product genotype is analyzed using fluorescence detector.
8. application according to claim 7, it is characterised in that step 2) PCR reaction conditions are：95 DEG C of 15 points of pre-degenerations
Clock；First step amplified reaction：95 DEG C be denatured 20 seconds, 68 DEG C of Gradient annealings simultaneously extend 60 seconds, 10 circulation, each cycle annealing and
The temperature of extension reduces by 1 DEG C；Second step amplified reaction, 94 DEG C are denatured 20 seconds, and 57 DEG C are annealed and extended 60 seconds, 30 circulations；12
DEG C preserve.
9. the application according to claim 7 or 8, it is characterised in that step 3) be specially：Using biological software to PCR
Amplified production carries out parting：Loci AX-111578083-G is to carry the excellent equipotential type plant of anti growing out, equipotential position
Point AX-111624595-C is carries the excellent equipotential type plant of anti growing out, and loci AX-110772653-T is anti-to carry
The excellent equipotential type plant of Spike sprouting.
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| CN108642064A (en) * | 2018-05-21 | 2018-10-12 | 安徽农业大学 | Wheat seed suspend mode duration gene TaCNGC-2A and its functional label |
| CN109161609A (en) * | 2018-10-19 | 2019-01-08 | 河南省农业科学院小麦研究所 | SNP marker, detection method and the application of wheat leaf rust resistance gene Lr42 |
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| CN112322617A (en) * | 2020-11-27 | 2021-02-05 | 中国科学院成都生物研究所 | KASP molecular marker capable of identifying waxy property of barley grains and application thereof |
| CN113528700A (en) * | 2021-07-22 | 2021-10-22 | 湖北省农业科学院粮食作物研究所 | KASP molecular marker kit for detecting wheat ear germination resistance, detection method and application |
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