CN105803102B - It is a kind of to detect the KASP labeled primer of Wx-D1 gene and its application in Waxy wheat K107Wx1 - Google Patents
It is a kind of to detect the KASP labeled primer of Wx-D1 gene and its application in Waxy wheat K107Wx1 Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
In a kind of detection Waxy wheat K107Wx1Wx‑D1The KASP labeled primer of gene and its application belong to molecular genetic breeding technical field, are carried out in Waxy wheat K107Wx1 using three groups of KASP primers provided by the inventionWx‑D1The detection of genotype, human error is low, and analysis throughput is high, is applicable in the detection of great amount of samples.Codominant marker provided by the invention is applied to K107Wx1'sWx‑D1The transformation of gene, for accelerating using in Waxy wheat K107Wx1Wx‑D1Gene carries out genetic improvement, and improving breeding efficiency has important practice significance.
Description
Technical field
The invention belongs to molecular genetic breeding technical fields, in particular to Waxy wheatWx-D1Gene high throughput molecular labeling
Exploitation and application.
Background technique
Starch is the main constituents of wheat endosperm, is played a crucial role in food and non-food industries.Starch contains
20%~30% amylose and 70%~80% amylopectin, wherein granule bound starch synzyme (Granule-
Bound starch synthase, GBSS I) it is responsible for the synthesis of amylose, also known as Wx albumen.It is by being located at 7AS, 4AL
With 7DS'sWx-A1、Wx-B1、Wx-D1 Three gene coding synthesis.Any one Wx protein protomer missing or inactivation can all be led
Cause amylose content decline, different content amylose directly affect wheat different product it is final using quality (Liu Yingchun,
2005).
Wx-Al is lacked and there is such as Japanese wheat breed Northeast 107 (Kanto107) of natural deletions in three Wx protein protomers
With Wx-Bl protein protomer, place of china kind Bai Huomai lacks Wx-Dl protein protomer, and corresponding gene is respectively designated asWx- Alb、Wx-BlbWithWx-Dlb.The Waxy wheat kind that three Wx protein protomers all lack is not present in nature.Have and answer at present
Waxy wheat kind is cultivation acquisition on the basis of Kanto107 and Bai Huomai mostly, and in Waxy wheatWx-D1Gene class
Type is essentiallyWx-Dlb.And the Waxy wheat kind K107Wx1 that the present invention uses is that Yasui etc. (1998) is used and tried through EMS chemistry
Agent induction Kanto107 is mutated to be formed,Wx-D1Gene typeWx-DldIt may be different fromWx-DlbGene (Yasui, 1998).
Therefore Waxy wheat germ plasm resource K107Wx1 new as one kind, in the wheat for cultivating and studying different Wx protein protomer deletion types
It is significant in terms of kind and its quality characteristic.
Wx missing gene is all recessive mutation gene, and year limit for length is selected in breeding.When only 1-2 mutated gene exists
When, it is difficult to directly select it according to phenotype Mutants homozygous.Therefore marker assisted selection is to cultivate difference directly
The most effectual way of chain content of starch wheat breed.In Waxy wheat K107Wx1 Wx-A1bWithWx-B1bGene has codominance mark
Note report and utilization, andWx-D1Gene codominant marker has not been reported.
Single nucleotide polymorphism (SNP) labelling technique, belongs to labelling technique of new generation.It is that the most common heredity becomes between individual
Special-shaped formula, the single nucleotide polymorphism often occurred have the replacement of base, the insertion of base and missing, are plant complex character heredity
The ideal molecular labeling of research.With the development of biotechnology level, KASP(competitiveness ApoE gene) technology work
For one of a kind of high-throughput, low cost and detection SNP classifying method of low fault rate, there is weight in the application of crop assistant breeding
It acts on.
Summary of the invention
It is an object of the present invention to propose to divide as the detection SNP of a kind of high-throughput, low cost and low fault rate
Type method, for detecting in Waxy wheat K107Wx1Wx-D1The KASP labeled primer of gene.
KASP labeled primer of the present invention has:
1) primer 1:
5'-GAAGGTGACCAAGTTCATGCTGTGGAGGGGCGCAAGATCAACTGG-3'；WhereinGAAGGTGACCAAGTTCATGCTFor sequence label A；
2) primer 2:
5'-GAAGGTCGGAGTCAACGGATTGTGGAGGGGCGCAAGATCAACTGA-3'；WhereinGAAGGTCGGAGTCAACGGATTFor sequence label B；
3) primer 3:
5'-CGCGTAGTAGGGGCTCACCGTCA-3'。
K107Wx1, Kanto107 and control are amplified by four pairs of special primers (table 1) that Hu Yaping (2008) has been announced
China springWx-D1Gene order overall length, send Huada gene company to be sequenced, and compares correlated series discovery using DNAMAN software and draws
In the Product Sequence of object D3 amplification, K107Wx1 is the product and Kanto107 of template and the product of China spring in sequence 1484bp
There are a point mutation, bases G is mutated into A at place, generates terminator codon, causes polypeptide chain synthesis to terminate in advance, can not synthesize
Wx-D1 protein protomer.Four pairs of primer sequence information are as shown in table 1:
4 pairs of specific primer sequences information needed for table 1 expands wheat Wx-D1 gene order overall length.
According to SNP mutation information feature, special complete KASP primer is designed, is made of primer 1, primer 2 and primer 3.Draw
Object 1 is followed successively by 22-45 single stranded DNAs of sequence 1 in sequence label A and sequence table from the end 5' to the end 3'.
Primer 2 is followed successively by 22-45 single stranded DNAs of sequence 2 in sequence label B and sequence table from the end 5' to the end 3'.
Primer 3 is nucleotide sequence single stranded DNA as shown in sequence 3 in sequence table.
Further, the sequence label A nucleotides sequence is classified as 1-21 of sequence 1 in sequence table, sequence label B institute
The nucleotides sequence stated is classified as 1-21 of sequence 2 in sequence table.
More specifically, primer 1 is nucleotide sequence single stranded DNA as shown in sequence 1 in sequence table；Primer 2 is nucleotides sequence
Arrange the single stranded DNA as shown in sequence 2 in sequence table；Primer 3 is nucleotide sequence single stranded DNA as shown in sequence 3 in sequence table.
It is provided by the present invention to be used to detect in Waxy wheat K107Wx1Wx-D1The kit of gene includes described complete
KASP primer also contains fluorescence probe A, fluorescence probe B, quenching probes A, quenching probes A.
In the present invention, the fluorescent reporter group A is FAM, and fluorescent reporter group B is HEX, the fluorescent quenching base
Group is BHQ.
The fluorescence probe A, the fluorescence probe B, the quenching probes A and quenching probes B are to exist in the present invention
In KASP 2 × Master Mix, wherein 2 × Master of KASP Mix is Britain's LGC Products, catalog number
It is suitable for 96/384 orifice plate for KBS-1016-002().
It is a further object of the present invention to provide the applications of the above KASP labeled primer, that is, for detecting Waxy wheat K107Wx1
InWx-D1Gene.
Detection or auxiliary detect wheat to be measuredWx-D1The method of the genotype of gene is conventional method: with described to be measured small
Wheat genomic DNA is template, and the kit (for the fluorophor A for FAM, fluorophor B is HEX) is used to carry out PCR expansion
Increase, the product expanded is subjected to fluorescence signal scanning, scan data is analyzed using Kluster Caller software, root
It is determined in Waxy wheat K107Wx1 according to analysis result according to followingWx-D1Genotype: if the fluorescence letter of the product of wheat to be measured amplification
Number is analyzed through Kluster Caller software and blue is presented, then in Waxy wheat K107Wx1Wx-D1Genotype is GG；If to
The fluorescent signal data for surveying the product of wheat amplification is analyzed through Kluster Caller software is presented red, then Waxy wheat
In K107Wx1Wx-D1Genotype is AA；If the fluorescent signal data of the product of wheat amplification to be measured is soft through Kluster Caller
Green is presented in part analysis, then in Waxy wheat K107Wx1Wx-D1Genotype is AG.
The above KASP labeled primer can also be used for the filial generation of detection Waxy wheat K107Wx1Wx-D1Gene.
Wheat in the applicationWx-D1Genotype is GG or AG, then wheat Wx-D1 protein protomer to be measured exists；If small
WheatWx-D1Genotype is AA, then wheat Wx-D1 protein protomer missing to be measured.
The present invention is to utilize prime end alkali according to target gene key mutational site design primer based on KASP technology
The specially matching of base to carry out parting to corresponding SNP.The technology high-throughput can detect multiple samples, substantially increase detection
Efficiency reduces time and cost of labor, is very beneficial for the deletion condition of field Large-scale Screening Wx-D1 protein protomer, shortens
Waxy wheat K107Wx1'sWx-D1The transformation process of gene, improves breeding efficiency.
Detailed description of the invention
Fig. 1 be using the exploitations such as Shariflou based onWx-D1bThe codominant marker of gene delection type expands respectively
The result of wheat K107Wx1, Kanto107, Ning Mai 14 and Caiwx.
Fig. 2 is K107Wx1, Kanto107 and control China spring after special primer D3 amplification, and Product Sequence compares
The sequence information near mutational site afterwards.
Fig. 3 is KASP molecular labeling amplification parents K107Wx1, Ning Mai 14 and its BC8F1 progeny population and control
The result of Kanto107.
Fig. 4 is KASP molecular labeling amplification parents K107Wx1, Ning Mai 14 and its BC8F2 progeny population and control
The result of Kanto107.
Specific embodiment
The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit
Determine the scope of the present invention.
The materials, reagents and the like used in the following examples, if commercially being obtained without specified otherwise.
The primer is synthesized in Shanghai Jierui Biology Engineering Co., Ltd.
Wheat breed used is provided by national wheat flour quality center Yangzhou branch center, and the public can be from national wheat flour quality
Heart Yangzhou branch center obtains.
In embodiment 1, Waxy wheat K107Wx1Wx-D1The design of gene high throughput KASP label and its primer special sequence
Exploitation.
Using Shariflou etc. (2001) exploitation based on Bai HuomaiWx-D1bThe codominant marker of gene expands small respectively
Wheat K107Wx1, Kanto107, Ning Mai 14 and Caiwx(Wx-D1b) when, in K107wx1Wx-D1dGene is peaceful with common wheat
14 amplified band indifferences, are 840bp, and Waxy wheat Caiwx amplified band is 260bp, is had obviously with other 3 wheat product
Difference, as shown in Figure 1.As a result illustrate that the codominant marker cannot be used for K107Wx1'sWx-D1Gene molecule marker auxiliary is educated
Kind, and in K107Wx1Wx-D1Gene delection type is different from Bai HuomaiWx-D1b, need to further research and develop and be suitable for
The SNP codominant marker of K107Wx1.
According to used in Hu Yaping (2008)Wx-D1Gene-specific primer D1, D2, D3, D4(table 1), amplify pass
In 107 and K107Wx1 of eastWx-D1The overall length of gene order, gel extraction sequencing, this two sections of sequences is compared, as a result
Display as shown in Fig. 2, in the Product Sequence of primer D3 amplification, K107Wx1 at 1484bp there are a point mutation (G → A),
Terminator codon is generated, causes polypeptide chain synthesis to terminate in advance, Wx-D1 protein protomer can not be synthesized.
According to SNP mutation information feature, special complete KASP primer is designed, is made of primer 1, primer 2 and primer 3.Two
Upstream primer (the primer 1 and primer 2) end 3' is allelic variation bases G/A, and the sequence selection of downstream primer 3 will guarantee to expand
Segment is in 60-120bp.The end upstream primer 5' is connected with fluorescence labels sequence, and wherein the end 5' of primer 1 is connected as FAM fluorescence mark
Sequence 5'-GAAGGTGACCAAGTTCATGCT-3' is signed, the end 5' of primer 2 is connected as HEX fluorescence labels sequence 5'-
GAAGGTCGGAGTCAACGG-3'.The present invention is based in Waxy wheat K107Wx1Wx-D1The KASP of Data mining is marked and its specially
With primer sequence, sequence is made of sequence 1, sequence 2 and sequence 3, and underlined sequences are FAM joint sequence, sequence in sequence 1
Underlined sequences are HEX joint sequence in 2.
1 Primer-F1 of sequence:
5'-GAAGGTGACCAAGTTCATGCTGTGGAGGGGCGCAAGATCAACTGG-3' 。
2 Primer-F2 of sequence:
5'-GAAGGTCGGAGTCAACGGATTGTGGAGGGGCGCAAGATCAACTGA-3' 。
3 Primer-R of sequence:
5'-CGCGTAGTAGGGGCTCACCGTCA-3'。
Above-mentioned primer sequence is Shanghai Jierui Biology Engineering Co., Ltd's synthesis.
In embodiment 2, KASP Markers for Detection Waxy wheat K107Wx1Wx-D1The method of gene is established.
1, genomic DNA is extracted:
The leaf tissue for taking wheat breed to be measured extracts complete genome DNA using CTAB method.
2, the DNA extracted using step 1 is used to detect in Waxy wheat K107Wx1 as template using what embodiment 1 was developedWx- D1The primer special of the KASP label of gene expands PCR, obtains amplified production.
The preparation of KASP labeled primer working solution:
Respectively take 12 μ l(100 μM of upstream primer), 30 μ l(100 μM of downstream primer), 100 μ l are supplemented to sterile ultrapure water,
Primer working solution as KASP label uses.
Pcr amplification reaction system: 2 μ l of DNA profiling (20-30ng/ μ l), 0.08 μ l of primer working solution, 2 KASP are included
2.5 μ l(LGC company of × Master Mix, KBS-1016-002), 5 μ l are supplemented to sterile ultrapure water.Wherein KASP 2 ×
Taq enzyme of the Master Mix by fluorescence probe A, fluorescence probe B, quenching probes A and quenching probes B and high-fidelity, dNTP etc.
Composition.The sequence of fluorescence probe A is 5'-GAAGGTGACCAAGTTCATGCT-3', and the end 5' connects a FAM fluorophor；It is glimmering
The sequence of light probe B is 5'-GAAGGTCGGAGTCAACGG-3', and the end 5' connects a HEX fluorophor；The sequence of quenching probes A
It is classified as 5'-AGCATGAACTTGGTCACCTTC-3', the end 3' connects a quenching group BHQ；The sequence of quenching probes B is
The end 5'-AATCCGTTGACTCCGACCTTC-3', 3' connects a quenching group BHQ.
Response procedures are as follows: 94 DEG C of initial denaturation 15min of the first step；94 DEG C of 20s, 61-55 DEG C of denaturation of second step (each follow
Ring declines 0.6 DEG C) 60s, totally 10 recycle；94 DEG C of denaturation 20s of third step, 55 DEG C of renaturation 60s, 28 circulations；10 DEG C of preservations.
The blank control (NTC) for not adding template DNA in reaction system is tested while being arranged, each plate is arranged 1 or more
A blank control.
3, the fluorescent scanning of pcr amplification product:
Pcr amplification product is scanned using Synergy H1MF microplate reader, FAM excitation wavelength is 585nm, transmitted wave
Long 520nm；HEX excitation wavelength is 528nm, launch wavelength 560nm, system reference fluorescent ROX excitation wavelength 575nm, transmitted wave
Long 610nm.
4, allelic gene typing:
Using Kluster Caller software, to the analysis of microplate reader scan data, (specific method is referring to Kluster Caller
Software document, the public can directly buy in LGC company), based on the analysis results according to determining wheat to be measured as followsWx-D1Base
The specific genotype of cause, be aggregated in the genotype close to the sample of X-axis being displayed in blue be connect FAM fluorescence labels sequence etc.
Position genotype；Being aggregated in the genotype close to the sample of Y-axis being displayed in red is the allele for connecting HEX fluorescence labels sequence
Type；The genotype of centre display green sample is two kinds of allele；Show that the sample of pink colour may be due to DNA mass not
Good, for amplified production not by clear parting, the lower left corner shows that the sample of black is blank control.
Specifically, as follows: if the fluorescent signal data of the amplified production of the wheat to be measured is through Kluster Caller
Software analysis is blue, then the wheatWx-D1The genotype of gene is GG；If fluorescent signal data is through Kluster Caller
Software analysis takes on a red color, then wheatWx-D1The genotype of gene is AA；If fluorescent signal data is through Kluster Caller software
Analysis is in green, then wheatWx-D1The genotype of gene is GA.
Embodiment 3 expands verifying using KASP molecular labeling between K107Wx1, Kanto107 and the peaceful wheat 14 of common wheat
And it is utilized in breeding:
1, material to be tested:
Material to be tested includes: Waxy wheat K107Wx1(AA genotype) it is donor parents, the non-peaceful wheat 14(GG gene of Waxy wheat
Type) it is recurrent parent, it carries outWx-D1The transformation of protein protomer missing.K107Wx1 and Ning Mai 14 hybridization obtains corresponding F1 generation, F1
BC8F1 group is obtained with recurrent parent peaceful 8 generations of backcrossing of wheat 14, selfing obtains BC8F2 group.SelectionWx-D1Gene is normal
Kanto107(GG genotype) be molecular labeling assisting sifting control.
2, KASP Markers for Detection:
Utilize above-mentioned high-throughput molecular labeling system anlysis parents, the genotype of BC8F1 group, with known type
Kanto107 is control, and (sample 1 is blue to sample segment testing result in Fig. 3, and sample 2 is red, and sample 3 is as shown in Figure 3
Black, remaining sample are green), blue sample is peaceful wheat 14 and compares Kanto107, genotype GG, and red sample is
Donor parents K107Wx1, genotype AA, and whole heterozygosis BC8F1 single plants are green, genotype GA, black is no mould
Plate compares NTC, therefore KSAP label can correctly distinguish homozygous aobvious, recessive and heterozygosis single plant.
In addition, provided by the present invention right in order to further verifyWx-D1The accuracy of the genotype detection of gene, invention
People also detects BC8F2 population segment single plant, as a result as (sample 1 is blue to Fig. 4 in Fig. 4, and sample 2 is red, sample 3
For black, remaining sample is green), there are red homozygous recessive single plant (AA), green heterozygous in the single plant of the part BC8F2
The dominant single plant (GG) of single plant (GA) and blue further demonstrates KASP label to mutational site SNP by this step
The accuracy of parting.
In conclusion three containing KASP molecular labeling primer and kit that the present invention designs can be applied toWx-D1
Genotype screening, and contain K107Wx1'sWx-D1The selection and use of gene, and the label is accurate and reliable, it is convenient and efficient, it mentions
High rate, accelerates breeding process.
<110>Jiangsu Province's lane housing Institute of agricultural sciences
<120>in a kind of detection Waxy wheat K107Wx1Wx-D1The KASP labeled primer of gene and its application
<160>3
<210>1
<211>45
<212>DNA
<213>artificial sequence
<220>
<223>according in Waxy wheat K107Wx1Wx-D1Gene order design
<400>1
gaaggtgacc aagttcatgc tgtggagggg cgcaagatca actgg
<210>2
<211>45
<212>DNA
<213>artificial sequence
<220>
<223>according in Waxy wheat K107Wx1Wx-D1Gene order design
<400>2
gaaggtcgga gtcaacggat tgtggagggg cgcaagatca actga
<210>3
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>according in Waxy wheat K107Wx1Wx-D1Gene order design
<400>3
cgcgtagtag gggctcaccg tca
Claims (3)
1. in a kind of detection Waxy wheat K107Wx1Wx-D1The KASP labeled primer of gene, characterized by comprising:
1) primer 1:
5'-GAAGGTGACCAAGTTCATGCTGTGGAGGGGCGCAAGATCAACTGG-3'；WhereinGAAGGTGACCAAGTTCATGCTFor sequence label A；
2) primer 2:
5'-GAAGGTCGGAGTCAACGGATTGTGGAGGGGCGCAAGATCAACTGA-3'；WhereinGAAGGTCGGAGTCAACGGATTFor sequence label B；
3) primer 3:
5'-CGCGTAGTAGGGGCTCACCGTCA-3'。
2. as described in claim 1 in detection Waxy wheat K107Wx1Wx-D1The application of the KASP labeled primer of gene, for examining
It surveys in Waxy wheat K107Wx1Wx-D1Gene.
3. as described in claim 1 in detection Waxy wheat K107Wx1Wx-D1The application of the KASP labeled primer of gene, for examining
Survey the filial generation of Waxy wheat K107Wx1Wx-D1Gene.
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| CN106636406B (en) * | 2016-12-26 | 2020-08-21 | 四川农业大学 | Molecular marker R207 coseparated with wheat few-tillering gene Ltn3 and application thereof |
| CN107604087A (en) * | 2017-08-09 | 2018-01-19 | 四川农业大学 | The method for detecting wheat Wx B1 variations |
| CN111183894A (en) * | 2020-02-15 | 2020-05-22 | 四川农业大学 | Creating method of HMW-GS full-deletion waxy wheat |
| CN112662805B (en) * | 2021-01-26 | 2022-11-22 | 山东省农业科学院作物研究所 | KASP marker primer group for detecting waxy genes of wheat and application thereof |
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| WO2005055704A2 (en) * | 2003-12-03 | 2005-06-23 | Arcadia Biosciences, Inc. | Wheat having reduced waxy protein due to non-transgenic alterations of a waxy gene |
| CN104131092A (en) * | 2014-07-22 | 2014-11-05 | 华南农业大学 | High resolution melting curve-based multi-SNP identification method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005055704A2 (en) * | 2003-12-03 | 2005-06-23 | Arcadia Biosciences, Inc. | Wheat having reduced waxy protein due to non-transgenic alterations of a waxy gene |
| CN104131092A (en) * | 2014-07-22 | 2014-11-05 | 华南农业大学 | High resolution melting curve-based multi-SNP identification method |
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