CN105803102A - KASP labeled primer for detecting Wx-D1 gene in waxy wheat K107Wx1 and application of KASP labeled primer - Google Patents
KASP labeled primer for detecting Wx-D1 gene in waxy wheat K107Wx1 and application of KASP labeled primer Download PDFInfo
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Abstract
The invention discloses a KASP labeled primer for detecting a Wx-D1 gene in waxy wheat K107Wx1 and application of the KASP labeled primer, and belongs to the technical field of molecular genetic breeding; and three groups of KASP primers provided by the invention, when used for detecting the Wx-D1 genotype in the waxy wheat K107Wx1, are low in human error and high in analysis throughput, and are suitable for detecting a great amount of samples. A codominant molecular marker provided by the invention, when applied to transfer breeding of the Wx-D1 gene in the waxy wheat K107Wx1, has an important practical significance for accelerating genetic improvement by virtue of the Wx-D1 gene in the waxy wheat K107Wx1 and f improving a breeding efficiency.
Description
Technical field
The invention belongs to molecular genetic breeding technical field, particularly to Waxy wheat Wx-D1 gene high flux molecular marker
Exploitation and application.
Background technology
Starch be wheat endosperm mainly comprise composition, play a crucial role in food and non-food industries.Starch contains 20%
～the amylose of 30% and the amylopectin of 70%～80%, wherein granule bound starch synzyme (Granule-bound
Starch synthase, GBSS I) be responsible for amylose synthesis, also known as Wx albumen.It is by being positioned at 7AS, 4AL and 7DS
Tri-gene code synthesis of Wx-A1, Wx-B1, Wx-D1.Any one Wx protein protomer disappearance or inactivation all can cause straight chain to form sediment
Powder content declines, and different content amylose directly affects the final utilization quality (Liu Yingchun, 2005) of Semen Tritici aestivi different product.
All there is the natural deletions such as Japan wheat breed Northeast 107 (Kanto107) disappearance Wx-Al in three Wx protein protomers
With Wx-Bl protein protomer, place of china kind Bai Huomai disappearance Wx-Dl protein protomer, its corresponding gene is respectively designated as Wx-
Alb, Wx-Blb and Wx-Dlb.There is not the Waxy wheat kind that three Wx protein protomers all lack in nature.Have at present and answer
Waxy wheat kind mostly cultivate on the basis of Kanto107 and Bai Huomai and obtain, and Wx-D1 gene class in Waxy wheat
Type is essentially Wx-Dlb.And Waxy wheat kind K107Wx1 that the present invention uses is that (1998) uses such as Yasui are through the examination of EMS chemistry
Agent induction Kanto107 sudden change is formed, and Wx-D1 gene type Wx-Dld may be different from Wx-Dlb gene (Yasui, 1998).
Therefore K107Wx1 is as a kind of new Waxy wheat germ plasm resource, at the Semen Tritici aestivi cultivated with study different Wx protein protomer deletion type
Kind and its quality characteristic aspect are significant.
Wx missing gene is all recessive mutation gene, selects year limit for length in breeding.When only 1-2 mutant gene exists
Time, even if Mutants homozygous also is difficult to directly select it according to phenotype.Therefore marker assisted selection is to cultivate difference directly
The most effectual way of chain content of starch wheat breed.The existing codominance mark of Wx-A1b and Wx-B1b gene in Waxy wheat K107Wx1
Note report and utilization, and Wx-D1 gene codominant marker have not been reported.
Single nucleotide polymorphism (SNP) labelling technique, belongs to a new generation's labelling technique.It is that between individuality, modal heredity becomes
Abnormity formula, the single nucleotide polymorphism often occurred has the replacement of base, the insertion of base and disappearance, is the heredity of plant complex character
The preferable molecular marker of research.Along with the development of biotechnology level, KASP(competitiveness ApoE gene) technology work
One of detection SNP classifying method for a kind of high flux, low cost and low fault rate, has weight in crop assistant breeding is applied
Act on.
Summary of the invention
An object of the present invention is to propose to divide as the detection SNP of a kind of high flux, low cost and low fault rate
Type method, for detecting the KASP labeled primer of Wx-D1 gene in Waxy wheat K107Wx1.
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'。
The four pairs of special primers (table 1) announced by Hu Yaping (2008) amplify K107Wx1, Kanto107 and comparison
China spring Wx-D1 gene order total length, send Hua Da genome company to check order, and utilizes DNAMAN software comparison correlated series to find to draw
In the Product Sequence of thing D3 amplification, K107Wx1 is that the product of product and the Kanto107 of template and China spring is in sequence 1484bp
There is a point mutation in place, bases G is mutated into A, produces termination codon, causes polypeptide chain synthesis to terminate in advance, it is impossible to synthesis
Wx-D1 protein protomer.Four pairs of primer sequence information are as shown in table 1:
Table 1 expands 4 pairs of specific primer sequences information needed for Semen Tritici aestivi Wx-D1 gene order total length.
According to SNP mutation information feature, design special complete KASP primer, be made up of primer 1, primer 2 and primer 3.Draw
Thing 1 is followed successively by the 22-45 position single stranded DNA of sequence 1 in sequence label A and sequence table from 5' end to 3' end.
Primer 2 is followed successively by the 22-45 position single stranded DNA of sequence 2 in sequence label B and sequence table from 5' end to 3' end.
Primer 3 is nucleotide sequence single stranded DNA as shown in sequence 3 in sequence table.
Further, described sequence label A nucleotides sequence is classified as the 1-21 position of sequence 1 in sequence table, sequence label B institute
The nucleotides sequence stated is classified as the 1-21 position 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
Row 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
。
Provided by the present invention comprise described complete for detecting the test kit of Wx-D1 gene in Waxy wheat K107Wx1
KASP primer, possibly together with fluorescent probe A, fluorescent probe B, quenching probes A, quenching probes A.
In the present invention, described fluorescent reporter group A is FAM, and fluorescent reporter group B is HEX, described fluorescent quenching base
Group is BHQ.
The most described fluorescent probe A, described fluorescent probe B, described quenching probes A and quenching probes B are to exist
It is Britain's LGC Products in KASP 2 × Master Mix, wherein said KASP 2 × Master Mix, catalog number
The orifice plate of 96/384 it is applicable to) for KBS-1016-002(.
It is a further object of the present invention to provide the application of above KASP labeled primer, i.e. be used for detecting Waxy wheat K107Wx1
Middle Wx-D1 gene.
The method of the genotype that detection or auxiliary detect Semen Tritici aestivi Wx-D1 gene to be measured is conventional method: with described to be measured little
Wheat genomic DNA is template, uses described test kit (described fluorophor A is FAM, and fluorophor B is HEX) to carry out PCR expansion
Increase, the product expanded is carried out fluorescence signal scanning, use Kluster Caller software that scan data is analyzed, root
According to analysis result according to Wx-D1 genotype in Waxy wheat K107Wx1 identified below: if the fluorescence letter of the product of Semen Tritici aestivi to be measured amplification
Number presents blueness through Kluster Caller software analysis, then in Waxy wheat K107Wx1, Wx-D1 genotype is GG；If treating
The fluorescent signal data of the product surveying Semen Tritici aestivi amplification presents redness through Kluster Caller software analysis, then Waxy wheat
In K107Wx1, Wx-D1 genotype is AA；If the fluorescent signal data of the product of Semen Tritici aestivi to be measured amplification is soft through Kluster Caller
Part analysis presents green, then in Waxy wheat K107Wx1, Wx-D1 genotype is AG.
Above KASP labeled primer can also be used for detecting the Wx-D1 gene of the filial generation of Waxy wheat K107Wx1.
Semen Tritici aestivi Wx-D1 genotype is GG or AG in the application, and Semen Tritici aestivi Wx-D1 protein protomer the most to be measured exists；If it is little
Wheat Wx-D1 genotype is AA, and Semen Tritici aestivi Wx-D1 protein protomer the most to be measured lacks.
The present invention is based on KASP technology, designs primer according to target gene key mutational site, utilizes prime end alkali
The specially coupling of base carries out typing to corresponding SNP.This technology can substantially increase detection with the multiple sample of high throughput testing
Efficiency, reduces time and cost of labor, is very beneficial for the deletion condition of field Large-scale Screening Wx-D1 protein protomer, shortens
The transformation process of the Wx-D1 gene of Waxy wheat K107Wx1, improves breeding efficiency.
Accompanying drawing explanation
Fig. 1 is that the codominant marker based on Wx-D1b gene delection type using the exploitations such as Shariflou expands respectively
Semen Tritici aestivi K107Wx1, Kanto107, the result of peaceful wheat 14 and Caiwx.
Fig. 2 be K107Wx1, Kanto107 and comparison China spring through special primer D3 expand after, its Product Sequence comparison
After near mutational site sequence information.
Fig. 3 is KASP molecular marker amplification parents K107Wx1, peaceful wheat 14 and its BC8F1 progeny population and comparison
The result of Kanto107.
Fig. 4 is KASP molecular marker amplification parents K107Wx1, peaceful wheat 14 and its BC8F2 progeny population and comparison
The result of Kanto107.
Detailed description of the invention
Principle and feature to the present invention are described below, and example is served only for explaining the present invention, is not intended to limit
Determine the scope of the present invention.
Material used in following embodiment, reagent etc., if without specified otherwise, the most commercially obtain.
The primer all synthesizes in Shanghai Jierui Biology Engineering Co., Ltd.
Wheat breed used is provided by country branch center, Yangzhou, wheat flour quality center, and the public can be from country's wheat flour quality
Branch center, heart Yangzhou obtains.
Wx-D1 gene high flux KASP indicia designs and its primer special sequence in embodiment 1, Waxy wheat K107Wx1
Exploitation.
The codominant marker based on Bai Huomai Wx-D1b gene using (2001) such as Shariflou to develop expands little respectively
Wheat K107Wx1, Kanto107, peaceful wheat 14 and Caiwx(Wx-D1b) time, in K107wx1, Wx-D1d gene is peaceful with common wheat
14 amplified band zero differences, are 840bp, and Waxy wheat Caiwx amplified band are 260bp, have substantially with other 3 Semen Tritici aestivi product
Difference, as shown in Figure 1.Result illustrate this codominant marker cannot be used for K107Wx1 Wx-D1 gene molecule marker auxiliary educate
Kind, and in K107Wx1, Wx-D1 gene delection type is different from Bai Huomai Wx-D1b, need to research and develop further and be applicable to
The SNP codominant marker of K107Wx1.
Wx-D1 gene-specific primer D1, D2, D3, D4(table 1 according to used by Hu Yaping (2008)), amplify pass
The total length of Wx-D1 gene order in east 107 and K107Wx1, cuts glue and reclaims order-checking, these two sections of sequences have been carried out comparison, result
Show as in figure 2 it is shown, in the Product Sequence of primer D3 amplification, K107Wx1 exists a point mutation (G → A) at 1484bp,
Produce termination codon, cause polypeptide chain synthesis to terminate in advance, it is impossible to synthesis Wx-D1 protein protomer.
According to SNP mutation information feature, design special complete KASP primer, be made up of primer 1, primer 2 and primer 3.Two
Bar forward primer (primer 1 and primer 2) 3' end is allelic variation bases G/A, and the sequence selection of downstream primer 3 to ensure amplification
Fragment is at 60-120bp.Forward primer 5' end connects fluorescence labels sequence, and wherein the 5' end of primer 1 is connected as FAM fluorescence mark
Signing sequence 5'-GAAGGTGACCAAGTTCATGCT-3', the 5' end of primer 2 is connected as HEX fluorescence labels sequence 5'-
GAAGGTCGGAGTCAACGG-3'.The present invention is based on the KASP labelling and specially of Wx-D1 Data mining in Waxy wheat K107Wx1
With primer sequence, its sequence is made up of sequence 1, sequence 2 and sequence 3, and in sequence 1, underlined sequences is FAM joint sequence, sequence
In 2, underlined sequences is HEX joint sequence.
Sequence 1 Primer-F1:
5'-GAAGGTGACCAAGTTCATGCTGTGGAGGGGCGCAAGATCAACTGG-3' 。
Sequence 2 Primer-F2:
5'-GAAGGTCGGAGTCAACGGATTGTGGAGGGGCGCAAGATCAACTGA-3' 。
Sequence 3 Primer-R:
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 K107Wx1, the method for Wx-D1 gene is set up.
1, genomic DNA is extracted:
Take the leaf tissue of wheat breed to be measured, use CTAB method to extract complete genome DNA.
2, with step one extract DNA as template, use embodiment 1 exploitation be used for detect Wx-in Waxy wheat K107Wx1
The primer special amplification PCR of the KASP labelling of D1 gene, obtains amplified production.
The preparation of KASP labeled primer working solution:
Respectively take forward primer 12 μ l(100 M), downstream primer 30 μ l(100 M), it is supplemented to 100 μ l with aseptic ultra-pure water, as
The primer working solution of KASP labelling uses.
Pcr amplification reaction system: include DNA profiling 2 μ l (20-30ng/ μ l), primer working solution 0.08 μ l, KASP 2
× Master Mix 2.5 μ l(LGC company, KBS-1016-002), it is supplemented to 5 μ l with aseptic ultra-pure water.Wherein KASP 2 ×
Master Mix is by fluorescent probe A, fluorescent probe B, quenching probes A and quenching probes B, and the Taq enzyme of high-fidelity, dNTP etc.
Composition.The sequence of fluorescent probe A is that 5'-GAAGGTGACCAAGTTCATGCT-3', 5' end connects a FAM fluorophor；Glimmering
The sequence of light probe B is that 5'-GAAGGTCGGAGTCAACGG-3', 5' end connects a HEX fluorophor；The sequence of quenching probes A
It is classified as 5'-AGCATGAACTTGGTCACCTTC-3', 3' end and connects a quenching group BHQ；The sequence of quenching probes B is
5'-AATCCGTTGACTCCGACCTTC-3', 3' end connects a quenching group BHQ.
Response procedures is as follows: 94 DEG C of denaturations 15min of the first step；Second step 94 DEG C of degeneration 20s, 61-55 DEG C (each are followed
Ring declines 0.6 DEG C) 60s, totally 10 circulations；3rd step 94 DEG C degeneration 20s, 55 DEG C of renaturation 60s, 28 circulations；10 DEG C of preservations.
Experiment arranges the blank (NTC) in reaction system without template DNA simultaneously, and each plate arranges 1 or many
Individual blank.
3, the fluorescent scanning of pcr amplification product:
Using Synergy H1MF microplate reader to be scanned pcr amplification product, FAM excitation wavelength is 585nm, launches wavelength
520nm；HEX excitation wavelength is 528nm, launches wavelength 560nm, system reference fluorescent ROX excitation wavelength 575nm, launches wavelength
610nm。
4, allelic gene typing:
(concrete grammar is with reference to Kluster Caller software to use Kluster Caller software to analyze microplate reader scan data
Description, the public can directly buy in LGC company), according to analysis result according to Semen Tritici aestivi Wx-D1 gene to be measured identified below
Concrete genotype, the genotype of the sample being aggregated in the display blueness close to X-axis is to connect the equipotential base of FAM fluorescence labels sequence
Because of type；The genotype of the sample being aggregated in the display redness close to Y-axis is the allelotype connecting HEX fluorescence labels sequence；
The genotype of middle display green sample be two kinds allelic；The sample of display pink colour is likely to be due to DNA poor quality, expands
Volume increase thing is not by clear and definite typing, and the sample of lower left corner display black is blank.
Specifically, it is as follows: if the fluorescent signal data of the amplified production of described Semen Tritici aestivi to be measured is through Kluster Caller
Software analysis is blueness, and the genotype of the most described Semen Tritici aestivi Wx-D1 gene is GG；If fluorescent signal data is through Kluster Caller
Software analysis takes on a red color, then the genotype of Semen Tritici aestivi Wx-D1 gene is AA；If fluorescent signal data is through Kluster Caller software
Analyze in green, then the genotype of Semen Tritici aestivi Wx-D1 gene is GA.
Embodiment 3, use KASP molecular marker expand checking between the peaceful wheat of K107Wx1, Kanto107 and common wheat 14
And utilize in breeding:
1, material to be tested:
Material to be tested includes: Waxy wheat K107Wx1(AA genotype) it is donor parents, non-Waxy wheat peaceful wheat 14(GG genotype) be
Recurrent parent, carries out the transformation of Wx-D1 protein protomer disappearance.K107Wx1 and Ning Mai 14 hybridization obtains corresponding F1 generation, F1 and wheel
Returning parent Ning Mai 14 8 generations that backcrossed obtains BC8F1 colony, and selfing obtains BC8F2 colony.Select Wx-D1 gene normal
Kanto107(GG genotype) it is the comparison of molecular marker assisting sifting.
2, KASP Markers for Detection:
Utilizing above-mentioned high flux molecular marker system anlysis parents, the genotype of BC8F1 colony, with known type
Kanto107 is comparison, and (in Fig. 3, sample 1 is blue to sample segment testing result, and sample 2 is red, and sample 3 is as shown in Figure 3
Black, remaining sample is green), blue sample is peaceful wheat 14 and comparison Kanto107, and genotype is GG, and red sample is
Donor parents K107Wx1, its genotype is AA, and all heterozygosis BC8F1 individual plant is green, genotype GA, and black is without mould
Plate comparison NTC, therefore this KSAP labelling can correctly distinguish aobvious, the recessive and heterozygosis individual plant that isozygotys.
It addition, for the accuracy verifying the genotype detection to Wx-D1 gene provided by the present invention further, invention
BC8F2 population segment individual plant is also detected by people, and (in Fig. 4, sample 1 is blue to result such as Fig. 4, and sample 2 is red, sample 3
For black, remaining sample is green), BC8F2 part individual plant has the homozygous recessive individual plant (AA) of redness, green heterozygous
Individual plant (GA) and blue dominant individual plant (GG), further demonstrate this KASP labelling to mutational site SNP by this step
The accuracy of typing.
In sum, three primers containing KASP molecular marker and the test kit of present invention design can be applicable to Wx-D1
Genotype screening, and the selection and use of the Wx-D1 gene containing K107Wx1, and this labelling is accurately and reliably, convenient and swift, carry
High speed, accelerates breeding process.
<110>Jiangsu Province's lane housing Institute of agricultural sciences
<120>a kind of KASP labeled primer detecting Wx-D1 gene in Waxy wheat K107Wx1 and application thereof
<160>3
<210>1
<211>45
<212>DNA
<213>artificial sequence
<220>
<223>design according to Wx-D1 gene order in Waxy wheat K107Wx1
<400>1
gaaggtgacc aagttcatgc tgtggagggg cgcaagatca actgg
<210>2
<211>45
<212>DNA
<213>artificial sequence
<220>
<223>design according to Wx-D1 gene order in Waxy wheat K107Wx1
<400>2
gaaggtcgga gtcaacggat tgtggagggg cgcaagatca actga
<210>3
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>design according to Wx-D1 gene order in Waxy wheat K107Wx1
<400>3
cgcgtagtag gggctcaccg tca
Claims (3)
1. the KASP labeled primer detecting Wx-D1 gene in Waxy wheat K107Wx1, it is characterised in that including:
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. detect the application of the KASP labeled primer of Wx-D1 gene in Waxy wheat K107Wx1 as claimed in claim 1, be used for examining
Survey Wx-D1 gene in Waxy wheat K107Wx1.
3. detect the application of the KASP labeled primer of Wx-D1 gene in Waxy wheat K107Wx1 as claimed in claim 1, be used for examining
Survey the Wx-D1 gene of the filial generation of Waxy wheat K107Wx1.
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| CN106636406A (en) * | 2016-12-26 | 2017-05-10 | 四川农业大学 | Molecular marker R207 coseparated with wheat few-tillering gene Ltn3 and application of molecular marker R207 |
| 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 |
| CN112662805A (en) * | 2021-01-26 | 2021-04-16 | 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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636406A (en) * | 2016-12-26 | 2017-05-10 | 四川农业大学 | Molecular marker R207 coseparated with wheat few-tillering gene Ltn3 and application of molecular marker R207 |
| 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 |
| CN112662805A (en) * | 2021-01-26 | 2021-04-16 | 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 |
| 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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| CN105803102B (en) | 2019-06-14 |
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