CN109055370A - Stalk WSC content gene label and application based on middle wheat 895 - Google Patents
Stalk WSC content gene label and application based on middle wheat 895 Download PDFInfo
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Abstract
The invention discloses a kind of, and the stalk WSC content gene based on middle wheat 895 is marked and is applied.The present invention provides special primer group, primer C shown in primer B shown in primer A, sequence 2 shown in sequence 1 and sequence 3 is formed.A kind of method that the present invention also protects stalk WSC content character for identifying wheat to be measured.Detect genotype of the wheat to be measured based on special SNP site.Using the genomic DNA of wheat to be measured as template, KASP is carried out using special primer group, fluorescent scanning is carried out, determines genotype of the wheat to be measured based on special SNP site.The stalk WSC content of TT genotype wheat is higher than CC genotype wheat.Special SNP site are as follows: the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.The present invention provides good tool for breeding utilization stalk WSC content main effect QTL site QWsc.caas-1RS.
Description
Technical field
The present invention relates to field of biotechnology more particularly to it is a kind of based on middle wheat 895 stalk WSC content gene label and
Using.
Background technique
Pustulation period is the critical period for determining wheat yield.China wheat main producing region is faced with sternly in the kernel grouting middle and later periods
The hot dry wind harm of weight, the underproduction is up to 10% or more when serious.The carbon source of Wheat in Grain Filling Stage is mainly derived from the light of boot leaf manufacture
Close the water soluble carbohydrates (Water soluble carbohydrate, WSC) stored in product and stalk and leaf sheath.
Under normal moisture condition, stalk WSC is 10%-20% to the contribution of yield, is up under drought stress to the contribution of yield
30%-50%.In wheat breed Evolution, leaf photosynthesis efficiency and stalk WSC store up and have measured significant improvement, right
It improves Ear weight and yield and there is significant contribution to degeneration-resistant, increase yield stability of improving the breed.However current wheat breed exists
Grouting middle and later periods stalk dry matter still has more delay, and there are larger genetic improvement spaces.Currently, the U.S., Australia and
It is relevant to transhipment that the countries such as international corn wheat improvement center (CIMMYT) and tissue pay much attention to Wheat in Grain Filling Stage WSC accumulation
Physiology and genetic research, using high WSC content as the important goal that future, wheat breeding was improved, to cope with global warming pair
The influence of Wheat Production.The wheat cereal crops important as China reinforce the research of stalk carbohydrate correlated inheritance to me
State's improving yield of wheat stable yields breeding is of great significance.
Studies have shown that stalk WSC content is in steady accumulated state from heading to Post flowering 20 days or so, then there is generation
It thanks to decaying, has been in reduced levels within 30 days or so after spending.The kind WSC early period amount of storing up of the resistance to heat stress of high yield is higher, generation in later period
The amount of thanking is higher, and it is less to be detained WSC amount in stalk.Analysis spends rear 10-30 days wheat culms WSC content to be of great significance.
Single nucleotide polymorphism (SNP) is the most common genetic variant form between plant individual, the mononucleotide often occurred
Polymorphism includes the replacement and insertion and missing of base, is the ideal molecular labeling of plant complex character genetic research.With
The development of biotechnology level, competitive ApoE gene (Kompetitive Allele Specific PCR,
KASP) one of method of the technology as a kind of detection SNP parting of high-throughput, low cost low fault rate, educates in crop auxiliary
Play a significant role in kind application.
According to the feature of molecular labeling, molecular labeling can be divided into dominant marker and codominant marker.KASP is marked,
Its dominant marker's type refers to that two homozygous genotype materials carry out F1 (hybridization first filial generation, the base of hybridization acquisition as parent
Because type be heterozygous genotypes) fluorescent scanning signal after SNP parting software automation parting, one of genotyping result and parent
The genotyping result of (homozygous genotype) is consistent, be often as the corresponding gene order of one of heterozygous genotypes base without
Method is caused by effectively expanding；In addition, for dominant marker, the corresponding gene order of one of two kinds of polymorphic bases base because
Can not effectively to be expanded, fluorescence signal is generally gathered in parting seat after Kluster Caller software automation parting
Near the origin for marking system, it is shown as pink colour.And codominant marker refers to the fluorescent scanning signal of F1 through parting software automatic parting direction
Afterwards, it is gathered in cornerwise middle position in parting coordinate system, it is shown in green, this is because in heterozygous genotypes more than two kinds
The corresponding gene order of state property base can by equal efficiency expand.
DH group (doubled haploid) is widely used material in crop genetic and breeding research, passes through monoploid material
Material chromosome doubling is formed, and genotype is homozygous.There are many ways to creating DH group, currently, in wheat genetic and breeding
The creation that wheat and corn hybrid method carries out DH group is generallyd use in research.
Summary of the invention
The object of the present invention is to provide a kind of, and the stalk WSC content gene based on middle wheat 895 is marked and is applied.
The present invention protects a species-specific primers group first, is made of primer A, primer B and primer C；
Primer A is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the replacement and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of identical function；
Primer B is following (b1) or (b2):
(b1) single strand dna shown in the sequence 2 of sequence table；
(b2) sequence 2 is had by the replacement and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of identical function；
Primer C is following (c1) or (c2):
(c1) single strand dna shown in the sequence 3 of sequence table；
(c2) sequence 3 is had by the replacement and/or deletion and/or addition of one or several nucleotide and with sequence 3
The DNA molecular of identical function.
The present invention also protects the application of the primer sets, for following (d1) or (d2) or (d3) or (d4) or (d5) or (d6)
Or (d7) or (d8):
(d1) genotype of the wheat based on special SNP site is identified；
(d2) the stalk WSC content character of auxiliary identification wheat；
(d3) wheat single plant or strain or strain or kind with high stalk WSC content of assisting sifting or breeding；
(d4) wheat single plant or strain or strain or kind with low stalk WSC content of assisting sifting or breeding；
(d5) product for identifying genotype of the wheat based on special SNP site is prepared；
(d6) product of stalk WSC content character of the preparation for assisting identification wheat；
(d7) preparation is for screening or the product of the high stalk WSC content wheat single plant of breeding or strain or strain or kind；
(d8) preparation is for screening or the product of the low stalk WSC content wheat single plant of breeding or strain or strain or kind；
The special SNP site is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
The present invention also protects a kind of kit, including the special primer group.
The kit further includes specific probe group.The specific probe group is visited by fluorescence probe A, quenching probes A, fluorescence
Needle B and quenching probes B composition；For fluorescence probe A as shown in the sequence 8 of sequence table, 5 ' ends connect fluorophor；Fluorescence probe B
As shown in the sequence 9 of sequence table, 5 ' ends connect fluorophor；Fluorophor in fluorescence probe A and fluorescence probe B is different；
For quenching probes A as shown in the sequence 10 of sequence table, 3 ' ends connect quenching group；11 institute of sequence of quenching probes B such as sequence table
Show, 3 ' ends connect quenching group.Fluorescence probe A specifically connects FAM fluorophor.Fluorescence probe B specifically connects HEX fluorescent base
Group.Quenching probes A specifically connects quenching group BHQ.Quenching probes B specifically connects quenching group BHQ.
The kit further includes 2 × Master of KASP Mix.
The present invention also protects the application of the kit, for as follows (d1) or (d2) or (d3) or (d4):
(d1) genotype of the wheat based on special SNP site is identified；
(d2) the stalk WSC content character of auxiliary identification wheat；
(d3) wheat single plant or strain or strain or kind with high stalk WSC content of assisting sifting or breeding；
(d4) wheat single plant or strain or strain or kind with low stalk WSC content of assisting sifting or breeding；
The special SNP site is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
A kind of method that the present invention also protects stalk WSC content character for identifying wheat to be measured, includes the following steps: to detect
Genotype of the wheat to be measured based on special SNP site；The stalk WSC content of TT genotype wheat is higher than CC genotype wheat.
The present invention also protects a kind of method for breeding wheat, includes the following steps: that detecting wheat to be measured is based on special SNP site
Genotype；TT genotype wheat is selected to carry out breeding.The purpose of the wheat breeding is the small of the high stalk WSC content of breeding
Wheat.
A kind of method that the present invention also protects stalk WSC content character for identifying wheat to be measured, includes the following steps:
(1) using the genomic DNA of wheat to be measured as template, KASP is carried out using the special primer group；
(2) after completing step (1), fluorescent scanning is carried out, determines genotype of the wheat to be measured based on special SNP site；
(3) judged according to genotype results: the stalk WSC content of TT genotype wheat is higher than CC genotype wheat.
The present invention also protects a kind of method for breeding wheat, includes the following steps:
(1) using the genomic DNA of wheat to be measured as template, KASP is carried out using the special primer group；
(2) after completing step (1), fluorescent scanning is carried out, determines genotype of the wheat to be measured based on special SNP site；
(3) selection TT genotype wheat carries out breeding.
The purpose of the wheat breeding is the wheat of the high stalk WSC content of breeding.
The present invention also protects a kind of specific DNA molecular, as shown in the sequence 4 of sequence table or the sequence 6 of sequence table.
The present invention also protects application of the specific DNA molecular in identification wheat culm WSC content character.The application
In, the specific DNA molecular is as detection target.
The special SNP site of any description above is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
In any description above method, the method that determines genotype of the wheat to be measured based on special SNP site are as follows: using more
Function microplate reader is scanned, and then carries out automated analysis to microplate reader scan data using Kluster Caller software,
It is described to be measured if being shown as pink colour (being gathered in genotyping result fluorescence signal coordinate system near coordinate axis origin)
Genotype of the wheat based on special SNP is TT.
In any description above method, the method that determines genotype of the wheat to be measured based on special SNP site are as follows: using more
Function microplate reader is scanned, and then carries out automated analysis to microplate reader scan data using Kluster Caller software；
It is described to be measured if being shown as pink colour (being gathered in genotyping result fluorescence signal coordinate system near coordinate axis origin)
Genotype of the wheat based on special SNP is TT；If being shown as blue (to be gathered in and lean in genotyping result fluorescence signal coordinate system
Near on the right side of nearly Y-axis), then genotype of the wheat to be measured based on special SNP is CC or CT.
Further, for being shown as blue sample, corresponding wheat lines can be selfed, detects and is selfed according to above-mentioned steps
Offspring's single plant of acquisition is based on the genotype of special SNP, if the genotype of each single plant of offspring of detection is non-TT homozygous,
Then wheat lines to be measured are that CC is homozygous；If each single plant part of offspring of detection is that TT is homozygous, partially homozygous for non-TT,
Then wheat lines to be measured are CT heterozygous.
In any description above method, in the reaction system of KASP, the concentration of primer A, primer B and primer C are followed successively by
0.192 μM, 0.192 μM and 0.48 μM.
In any description above method, the response procedures of KASP:
Step 1: 95 DEG C of initial denaturation 15min；
Step 2: 95 DEG C of 20s, 65 DEG C of 60s, 9 circulations；
Step 3: 95 DEG C of denaturation 20s, 57 DEG C of renaturation and extension 60s, 32 circulations.
Any description above wheat culm WSC content is the stalk WSC content that wheat plant spends latter 20 days.
The present inventor navigates to a main effect QTL site QWsc.caas-1RS, synergy on 1B the short arm of a chromosome
From middle wheat 895, (middle wheat 895 is 1BL/1RS translocation line to allele, and the synergy allele source of QWsc.caas-1RS is
1RS), explain that phenotypic variation is up to 7.4%-24.3%.On this plinth, the present invention is developed closely to be connected with QWsc.caas-1RS
The KASP primer sets of the SNP marker AX-111189530 of lock are breeding utilization stalk WSC content main effect QTL site
QWsc.caas-1RS provides good tool.The present invention can be used for wheat culm WSC content main effect QTL QWsc.caas-1RS
Molecular marker assisted selection breeding.Human error of the invention is low, and analysis throughput is high, right suitable for the detection of great amount of samples
Genetic improvement is carried out using the excellent allele of the QTL site in quickening, breeding efficiency is improved and is of great significance.
Detailed description of the invention
Fig. 1 is the genetic linkage map near 1B the short arm of a chromosome QTL QWsc.caas-1RS.
Fig. 2 is the result of embodiment 3.
Fig. 3 is the result of embodiment 4.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Test in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
The compact linkage molecule label AX- of embodiment 1, wheat culm WSC content QTL site QWsc.caas-1RS
The acquisition of 111189530 and its KASP primer sets
One, the acquisition of DH group
Middle wheat 895 and Yang Mai 16 are China Yellow River-Huai River region respectively and one of kind is promoted mainly in middle and lower reach of Yangtze River Winter Wheat Area, comprehensive
Economical character is excellent.Zhong Mai895Shi Institute of Crop Science, Chinese Academy of Agricultural Science and the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute
Cooperate Semi-winter wheat kind made of breeding, is authorized by the Yellow River and Huai He River south piece country within 2012.Middle 895 variable rate technology of wheat grouting speed
Rate is fast, and later period stalk WSC is detained few, yield height.
To raise wheat 16 as female parent, middle wheat 895 is male parent, constructs DH group, contains 174 familys.
Two, the acquisition of SNP marker AX-111189530
1, field trial and stalk WSC assay
DH group plants in 2016-2017 year in Henan Xinxiang and Shangqiu experiment station.Using RANDOMIZED BLOCK DESIGN, setting
It repeats three times, cell seeding method is taken to plant, each six row of cell, row long 3m, line-spacing 25cm.Field management is with reference to local examination
Station administration is tested, in addition emphasis carries out the prevention and control of plant diseases, pest control such as powdery mildew, stripe rust, aphid.
The florescence for recording each test material removes blade in spending latter 20 days from each cell 20 stems of random clip
And fringe, retain stalk, in 105 DEG C of water-removing 30min, then 80 DEG C of drying.Near infrared light is used referring to what Wang etc. (2014) constructed
Spectrometry measures stalk WSC content.Each sample carries out 3 technologies and repeats, and is averaged for statisticalling analyze.
2, genetic map construction
Before constructing genetic map, quality control first is carried out to material genotype data obtained, removal is without polymorphism, mark
Remember that miss rate is greater than 10% and label of the distortion separation greater than 30%.The BIN- of IciMapping V4.0 software is used in next step
Mapping function optimizes processing to remaining polymorphism mark, to be used for genetic map construction.Utilize JoinMap V4.0
The building of genetic map is carried out with MSTmap Online.
3, the discovery of QTL positioning and linked marker AX-111189530
Using QTL IciMapping V4.0 software, using complete composite interval mapping method (ICIM) to DH group 174
WSC content carries out QTL positioning under family varying environment.LOD value chooses 3.0 and is used as threshold value.
Utilize the heredity near the 1B the short arm of a chromosome QTL QWsc.caas-1RS of wheat 660K SNP chip label building
Linkage map is shown in Fig. 1.Positioning result shows, there are the main effect QTL site that one controls WSC content on 1B the short arm of a chromosome,
Left and right label is respectively AX-111189530 and AX-109507240, which explains that phenotypic variation reaches 7.4%-24.3%,
Synergy allele comes from middle wheat 895.
A large amount of sequence analyses, comparison and preliminary experiment are carried out, finds the flanking sequence of SNP marker AX-111189530 in 1B
There is specificity on chromosome.
Three, SNP marker AX-111189530 is converted into the primer sets that KASP is marked and is designed for the detection label
KASP label is converted by SNP marker AX-111189530, to be used for molecular marker assisted selection breeding.
Sequence 4 is the flanking sequence of SNP marker AX-111189530 in sequence table, and wherein Y indicates two kinds of the SNP marker
Polymorphism mononucleotide C or T, i.e., the nucleotide of the position is C or T in practical kind.
Alliance (IWGSC) IWGSC (https: //wheat- is sequenced in international Wheat volatiles with sequence 4 in sequence table
Urgi.versailles.inra.fr/ it) is retrieved, obtains 3 homologous sequences with 4 consistency of sequence 90% or more, point
Not as shown in sequence 5, sequence 6 (sequence 6 includes sequence 4, Y C) and sequence 7 in sequence table, it is respectively positioned in wheat 1A, 1B
With 1D chromosome.
Special SNP is located at the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles.Accordingly
, special SNP is located at the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
Design the primer sets based on KASP technology, abbreviation KASP primer sets.KASP primer sets are by two upstream primer (primers
A and primer B) and downstream primer (primer C) composition.
The nucleotide sequence of primer A is as shown in the sequence 1 of sequence table.
Sequence 1:
The nucleotide sequence of primer B is as shown in the sequence 2 of sequence table.
Sequence 2:
The nucleotide sequence of primer C is as shown in the sequence 3 of sequence table.
Sequence 3:5 '-TGGGTACTACAGCTCGGTGCTA-3 '.
The foundation of embodiment 2, method
1, the genomic DNA for extracting the blade of wheat to be measured obtains template solution through dilution.DNA in template solution is dense
Degree is about 30ng/ μ L.
2, KASP is carried out.
Primer working solution: taking primer A, primer B and primer C, is mended with sterile ultrapure water to 100 μ L, is mixed well.Primer work
Make in liquid, the concentration of primer A, primer B and primer C are followed successively by 12 μM, 12 μM and 30 μM.
2 × Master of KASP Mix is LGC Products (article No. KBS-1016-002).KASP 2×Master Mix
In contain fluorescence probe A, fluorescence probe B, quenching probes A, quenching probes B, high-fidelity Taq enzyme, dNTP, Mg2+Deng.Fluorescence probe
The sequence of A is 5 '-GAAGGTGACCAAGTTCATGCT-3 ', and 5 ' ends connect FAM fluorophor.The sequence of fluorescence probe B is
5 '-GAAGGTCGGAGTCAACGGATT-3 ', 5 ' ends connect HEX fluorophor.The sequence of quenching probes A is 5 '-
AGCATGAACTTGGTCACCTTC-3 ', 3 ' ends connect quenching group BHQ.The sequence of quenching probes B is 5 '-
AATCCGTTGACTCCGACCTTC-3 ', 3 ' ends connect quenching group BHQ.
Reaction system: 2 μ L of template solution, the 0.08 μ L of primer working solution, 2 KASP × Master of step 1 preparation
Mix2.5 μ L is mended with sterile ultrapure water to 5 μ L.In reaction system, the concentration of primer A, primer B and primer C are followed successively by 0.192 μ
M, 0.192 μM and 0.48 μM.In reaction system, the content of template DNA is about 60ng.
Response procedures:
Step 1: 95 DEG C of initial denaturation 15min；
Step 2: 95 DEG C of 20s, 65 DEG C of 60s, 9 circulations；
Step 3: 95 DEG C of denaturation 20s, 57 DEG C of renaturation and extension 60s, 32 circulations；10 DEG C of preservations.
3, fluorescent scanning is carried out.
After completing step 2, it is scanned using multi-function microplate reader.FAM excitation wavelength is 485nm, and launch wavelength is
520nm.HEX excitation wavelength is 535nm, launch wavelength 556nm.System reference fluorescent ROX excitation wavelength is 575nm, transmitting
Wavelength is 610nm.
4, allelic gene typing is carried out.
After completing step 3, it is (specific that automated analysis is carried out to microplate reader scan data using Kluster Caller software
Method can be obtained referring to Kluster Caller software document, the public from LGC company), it determines based on the analysis results to be measured small
McGee is in the genotype of special SNP.
It should be labeled as dominant marker, if the fluorescent signal data of the amplified production of the wheat to be measured is through Kluster
Caller software automation for pink colour analysis shows that (be gathered in genotyping result fluorescence signal coordinate system former close to reference axis
Near point), then genotype of the wheat to be measured based on special SNP is TT；
If the fluorescent signal data of the amplified production of the wheat to be measured is through Kluster Caller software analysis shows that being
Blue (near being gathered in genotyping result fluorescence signal coordinate system on the right side of Y-axis), then the wheat to be measured is based on spy
The genotype of different SNP may be CC or CT；
Further, for the above-mentioned sample for being shown as blue, corresponding wheat lines can be selfed, is detected according to above-mentioned steps
It is selfed genotype of offspring's single plant based on special SNP obtained, if the genotype of each single plant of offspring of detection is non-TT pure
Mould assembly, then wheat lines to be measured are that CC is homozygous；If each single plant part of offspring of detection, which is that TT is homozygous, (accounts for about detection sample
The ratio of quantity 25%), partially homozygous for non-TT, then wheat lines to be measured are CT heterozygous.
Embodiment 3 raises wheat 16, middle wheat 895 and part DH group family based on special SNP's using the detection of KASP primer sets
Genotype
Wheat to be measured are as follows: the part DH group family that the step of raising wheat 16, middle wheat 895, embodiment 1 one obtains.Raise wheat 16
The sample that the genotype based on special SNP for sequence verification is CC will raise the genomic DNA of wheat 16 as CC genotype
Check sample.Middle wheat 895 is the sample that the genotype based on special SNP of sequence verification is TT, by the gene of middle wheat 895
Check sample of the group DNA as TT genotype.The genomic DNA mixed in equal amounts of the genomic DNA and middle wheat 895 of wheat 16 will be raised,
Obtain the check sample of heterozygous genotypes.Using sterile ultrapure water as blank control sample.
Genotype of the wheat to be measured based on special SNP is detected using the method that embodiment 2 is established.
As a result see Fig. 2.The fluorescent signal data of wheat 16 is raised through Kluster Caller software analysis shows that being blue, gene
Type is CC.The fluorescent signal data of middle wheat 895 is through Kluster Caller software analysis shows that being pink colour, genotype TT.DH
Heterozygous genotypes are not present in group's family, and in DH family to be measured, fluorescent signal data is analyzed aobvious through Kluster Caller software
The sample genotype for being shown as blue is CC, and fluorescent signal data is through Kluster Caller software analysis shows that being the sample of pink colour
Genotype is TT.Near the genotyping result of the check sample of heterozygous genotypes is aggregated on the right side of X-axis, this is because working as template
In special SNP when being T, PCR amplification low efficiency, the result for causing HEX fluorescence signal very weak.To sum up, the KASP is labeled as aobvious
Property label, " TT " in site where KASP label and/or the primer sets can distinguish SNP marker AX-111189530 and
" C_ " genotype, wherein " C_ " is " CC " or " CT ".
Embodiment 4 assists identification wheat culm WSC content height using KASP primer sets
Wheat to be measured is 205 parts of existing wheat breeds (being shown in Table 1, national wheat flour quality center).
1, stalk WSC content detection
Wheat to be measured plants Yushan Hill East Germany state in 2016-2017 year.Using randomized complete-block design, setting weighs three times
Multiple, duplicate rows area, the long 1.0m of row, line width 20cm, field management is according to locality test station administration progress.Each cell is recorded to bloom
Phase, after spending 20 days from each cell 20 stems of random clip, remove blade and fringe, retain stalk, in 105 DEG C of water-removing 30min,
Then 80 DEG C of drying.Stalk WSC content is measured near infrared spectroscopy referring to the building such as Wang (2014).Each sample carries out
3 times technology repeats, and is averaged for statisticalling analyze.
2, genotype of the wheat to be measured based on special SNP is detected using the method that embodiment 2 is established.
The genomic DNA of wheat 16 will be raised as CC genotype check sample.Using the genomic DNA of middle wheat 895 as TT base
Because of the check sample of type.Using sterile ultrapure water as blank control sample.
As a result see Fig. 3.Two blank control samples are gathered in the lower left corner of fluorescence signal coordinate system, are shown as black.In
Wheat 895 and 107 parts of wheat breeds to be measured are gathered near blank control position, are shown as pink colour, genotype TT.It raises
Wheat 16 and other 98 parts of wheat breeds to be measured are gathered in X-axis near right positions, are shown as blue, it is considered that are bred as
Kind is homozygous genotype, therefore 98 parts of variety and genetypes are CC.
Genotype and stalk WSC content of 205 parts of wheat breeds to be measured based on special SNP the results are shown in Table 1.
Table 1
It is different to 205 parts of test group wheat breeds using the PROC TTEST model in international SAS9.2 statistical software
The WSC content of Genotype carries out t inspection, the results are shown in Table 2.The wheat breed that genotype is TT is than the wheat that genotype is CC
Kind stalk WSC content average value is high by 7.0%, and in the level of p < 0.01, above there were significant differences.The result shows that KASP primer sets can be with
For to improve molecule assisted selection of the wheat culm WSC content as target.
Table 2
SEQUENCE LISTING
<110>Institute of Crop Science, Chinese Academy of Agricultural Science
<120>the stalk WSC content gene label based on middle wheat 895 and application
<130> GNCYX181752
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 48
<212> DNA
<213> Artificial sequence
<400> 1
gaaggtgacc aagttcatgc tgagtcccaa gtgcataata ccacttac 48
<210> 2
<211> 48
<212> DNA
<213> Artificial sequence
<400> 2
gaaggtcgga gtcaacggat tgagtcccaa gtgcataata ccacttat 48
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
tgggtactac agctcggtgc ta 22
<210> 4
<211> 71
<212> DNA
<213> Triticum aestivum
<220>
<221> misc_feature
<222> (36)
<223> y =t or c
<400> 4
catgtttacg agtcccaagt gcataatacc acttaygtta acccactatg gacagcctaa 60
ttagcaccga g 71
<210> 5
<211> 433
<212> DNA
<213> Triticum aestivum
<400> 5
gctggttgta gtagggagta taatatacta gtatcatgca tatgatacta catccatagt 60
gcatactatt ataggcggtc tcatttattg ccatgcatga cacatagtgg cataacattt 120
attatgatat tgtatctacc tgtgttacta taaccctctc tctcttcttt aattgtctcc 180
catataaaca tgtttacgag tcccaagtgc atgataccgc ttatgttacc ccactatggc 240
cagcctaaca taatcagtat catgcgcttg ggactcgcaa acatgcttat gcggcagaca 300
attaaagaag agagaaggcg ttatagtaac ataggtagat accgtatcat aataaatgtt 360
atgctactat gtgtcatgca tggcaataaa tgagaccacc tatgatacta ctaaggcctc 420
ttttggttca tag 433
<210> 6
<211> 432
<212> DNA
<213> Triticum aestivum
<220>
<221> misc_feature
<222> (223)
<223> y =t or c
<400> 6
gagtatcata cactagtatc atgctacgtc tataatgcat agtatcatag atagattagt 60
atcatatatc gtatcattta ttgacataca agacacatag tagcataaaa ttaattatga 120
tatggtatct acctatatat gttatactat aaccctctct ctcttcttta attgtttgcc 180
atataagcat gtttacgagt cccaagtgca taataccact taygttaacc cactatggac 240
agcctaatta gcaccgagct gtagtaccca gttggggcgt cacttcatga gctgcacaag 300
gtcctgctaa ttaggctggt cataatggaa gtatcataag cggtattatg catgccaact 360
agattttttt gatgatgtag cacacaatta aatgaggaaa gagggtgtgt tgtaacatat 420
catgataccg ta 432
<210> 7
<211> 432
<212> DNA
<213> Triticum aestivum
<400> 7
acttagacta gtaacatgca tatgttacta gtctatgtta ctacctctat agtgcatagt 60
atcatagatt agtatcatag gtggtctcat ttattgccat gcatgacaca tagtagcatc 120
acatttatta tgttacggta tctacctatg ttactataac catctctctc ttctttaatt 180
gcctaccaca ttaccatgtt tgcgagtccc aagtgcatga tactacttat gttaccccca 240
ctatggccag cctaatagct aaccttgttg tgatgacaag ataggacatg gcacgcacct 300
ggacggagga actagtctgc attgtgcatt taagttttgt ctgaagtcaa tgtacctcta 360
ctttcaccag acttatagaa aaatgtgtca acattcacaa tgtcaaatca atatttttac 420
actcattata aa 432
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence
<400> 8
gaaggtgacc aagttcatgc t 21
<210> 9
<211> 21
<212> DNA
<213> Artificial sequence
<400> 9
gaaggtcgga gtcaacggat t 21
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence
<400> 10
agcatgaact tggtcacctt c 21
<210> 11
<211> 21
<212> DNA
<213> Artificial sequence
<400> 11
aatccgttga ctccgacctt c 21
Claims (10)
1. special primer group is made of primer A, primer B and primer C；
Primer A is following (a1) or (a2):
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the replacement and/or deletion and/or addition of one or several nucleotide and with sequence 1 identical
The DNA molecular of function；
Primer B is following (b1) or (b2):
(b1) single strand dna shown in the sequence 2 of sequence table；
(b2) have by sequence 2 by the replacement and/or deletion and/or addition of one or several nucleotide and with sequence 2 identical
The DNA molecular of function；
Primer C is following (c1) or (c2):
(c1) single strand dna shown in the sequence 3 of sequence table；
(c2) have by sequence 3 by the replacement and/or deletion and/or addition of one or several nucleotide and with sequence 3 identical
The DNA molecular of function.
2. the application of special primer group described in claim 1, for following (d1) or (d2) or (d3) or (d4) or (d5) or (d6)
Or (d7) or (d8):
(d1) genotype of the wheat based on special SNP site is identified；
(d2) the stalk WSC content character of auxiliary identification wheat；
(d3) wheat single plant or strain or strain or kind with high stalk WSC content of assisting sifting or breeding；
(d4) wheat single plant or strain or strain or kind with low stalk WSC content of assisting sifting or breeding；
(d5) product for identifying genotype of the wheat based on special SNP site is prepared；
(d6) product of stalk WSC content character of the preparation for assisting identification wheat；
(d7) preparation is for screening or the product of the high stalk WSC content wheat single plant of breeding or strain or strain or kind；
(d8) preparation is for screening or the product of the low stalk WSC content wheat single plant of breeding or strain or strain or kind；
The special SNP site is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
3. a kind of kit, including special primer group described in claim 1.
4. the application of kit described in claim 3, for as follows (d1) or (d2) or (d3) or (d4):
(d1) genotype of the wheat based on special SNP site is identified；
(d2) the stalk WSC content character of auxiliary identification wheat；
(d3) wheat single plant or strain or strain or kind with high stalk WSC content of assisting sifting or breeding；
(d4) wheat single plant or strain or strain or kind with low stalk WSC content of assisting sifting or breeding；
The special SNP site is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
5. a kind of method for the stalk WSC content character for identifying wheat to be measured includes the following steps: that detecting wheat to be measured is based on spy
The genotype of different SNP site；The stalk WSC content of TT genotype wheat is higher than CC genotype wheat；
The special SNP site is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
6. a kind of method of wheat breeding includes the following steps: to detect genotype of the wheat to be measured based on special SNP site；Choosing
It selects TT genotype wheat and carries out breeding；
The special SNP site is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
7. a kind of method for the stalk WSC content character for identifying wheat to be measured, includes the following steps:
(1) using the genomic DNA of wheat to be measured as template, KASP is carried out using special primer group described in claim 1；
(2) after completing step (1), fluorescent scanning is carried out, determines genotype of the wheat to be measured based on special SNP site；
(3) judged according to genotype results: the stalk WSC content of TT genotype wheat is higher than CC genotype wheat；
The special SNP site is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
8. a kind of method of wheat breeding, includes the following steps:
(1) using the genomic DNA of wheat to be measured as template, KASP is carried out using special primer group described in claim 1；
(2) after completing step (1), fluorescent scanning is carried out, determines genotype of the wheat to be measured based on special SNP site；
(3) selection TT genotype wheat carries out breeding；
The special SNP site is following (e1) or (e2):
(e1) the 36th nucleotide of DNA molecular shown in the sequence 4 of the sequence table in Wheat volatiles；
(e2) the 223rd nucleotide of DNA molecular shown in the sequence 6 of the sequence table in Wheat volatiles.
9. specific DNA molecular, as shown in the sequence 4 of sequence table or the sequence 6 of sequence table.
10. application of the specific DNA molecular described in claim 9 in identification wheat culm WSC content character.
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| CN116904636A (en) * | 2023-05-04 | 2023-10-20 | 扬州大学 | Molecular marker for detecting wheat stem WSC content QTL QWSC.caas-7DS and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060262A (en) * | 2018-02-08 | 2018-05-22 | 中国农业科学院作物科学研究所 | KASP marks relevant with wheat root character and its application |
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| CN108060262A (en) * | 2018-02-08 | 2018-05-22 | 中国农业科学院作物科学研究所 | KASP marks relevant with wheat root character and its application |
Non-Patent Citations (1)
| Title |
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| AWAIS RASHEED, 等: "Development and validation of KASP assays for genes underpinning key economic traits in bread wheat", 《THEOR APPL GENET》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116904636A (en) * | 2023-05-04 | 2023-10-20 | 扬州大学 | Molecular marker for detecting wheat stem WSC content QTL QWSC.caas-7DS and application |
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