CN110184383A - Molecular marker panel and its application in Genes of Pre-harvest Sprouting molecular labeling - Google Patents
Molecular marker panel and its application in Genes of Pre-harvest Sprouting molecular labeling Download PDFInfo
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- CN110184383A CN110184383A CN201910574842.0A CN201910574842A CN110184383A CN 110184383 A CN110184383 A CN 110184383A CN 201910574842 A CN201910574842 A CN 201910574842A CN 110184383 A CN110184383 A CN 110184383A
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The application belongs to Wheat Molecular Breeding technical field, and in particular to molecular marker panel and its application in Genes of Pre-harvest Sprouting molecular labeling.Molecular labeling has 4, including a SSR marker and three KASP labels altogether, and wherein SSR marker is Xgwm383b label；Three KASP labels are as follows: A009711, A009180, A009716.In breed of variety, the approach that favorable genes are shifted into wheat in Triticum tauschii mainly formulates a series of wheat/Triticum tauschii substitution line, and Triticum tauschii chromosome is imported into Wheat Background by the means of distant hybridization.For this breeding method, the application is with actual F2For group, molecular marker panel is devised, to be used to detect, track the Triticum tauschii chromosome situation imported in Wheat Background, and is used to refer to lead wheat anti growing out rearing new variety in turn with preferable practical value.
Description
Technical field
The application belongs to Wheat Molecular Breeding technical field, and in particular to molecular marker panel and its in wheat ear germinating base
Because of the application in molecular labeling.
Background technique
Wheat ear germinating (Pre-harvest sprouting, PHS) be before a kind of harvesting wheat after physiological maturity because of yin
Head sprouting phenomenon caused by rain or wet environment.Spike sprouting phenomenon not only influences wheat yield, but also seriously affects processing
Quality, seed storage and the seeding quality of next year, in turn result in different degrees of economic loss.
Wheat ear germinating is a kind of worldwide climate damage, and there is Spike sprouting phenomenon hair in wheat belt all over the world
It is raw, caused by lose it is also more serious.China's Spike sprouting mainly appears on the Yangtze river basin, the southwestern area of wheat and Northeasten Spring Wheat Area of China, yellow
The Guanzhong, Shanxi in Huaihe River wheat area also once multiple occurrence of large-area Spike sprouting.Because it causes damages greatly, various countries breeding scholar is to wheatear
Germination all takes much count of, and has conducted extensive research to its identification method, resistance mechanism, Genetic Mechanisms and molecular labeling etc., but
There has been no breakthroughs for the breeding of wheat anti growing out.Therefore, Spike sprouting problem be still wheat breeding problem anxious to be resolved it
One.
In recent years, it achieves with the molecular labeling of wheat ear germinating and development compared with quantum jump.Such as: Anderson etc.
(RFLP Analysisi of genomic regions associated with resistance to pre-harvest
Sprouting in wheat. Crop Science.1993) 8 and the associated genome area of Spike sprouting are found, it orients
10 RFLP labels relevant to ear germinating resistance；In addition, can also be used for ear germinating resistance using the morphological feature that gene controls
Label, such as: found on the site 2B, 2D control epicuticle wax site it is i.e. related to ear germinating resistance (King,
Epicuticular waxes and regulation of ear wetting and pre-harvest sprouting in
Barley and wheat. Euphytica. 2000).In the prior art, for part main effect QTL and the anti growing out degree of association
More research has also been made.But generally, controlled due to ear germinating resistance by multiple sites, for going deep into for different loci
Research and relevant to Spike sprouting molecular labeling is developed for different loci, it is small for assisting sifting anti growing out
Wheat variety still has highly important technological value.
Summary of the invention
The application purpose is moieties label primer, so as to mark to part anti growing out related gene
Note, and it is used to refer to the cultivation of impedance Spike sprouting new variety of wheat in turn.
Details are as follows for the technical solution that the application is taken.
For marking the molecular marker panel of anti growing out related gene, 4 altogether, including a SSR marker and three
KASP label (SSR marker is pair of primers, and each KASP is labeled as a set of primer), wherein SSR marker is named as Xgwm383b
Label, three KASP labels are respectively designated as: A009711, A009180, A009716 label；Base sequence such as SEQ ID
Shown in NO.1 ~ 11, particular sequence are as follows:
Xgwm383b:
Xgwm383b-F:5'- ACGCCAGTTGATCCGTAAAC-3',
Xgwm383b-R:5'- GACATCAATAACCGTGGATGG-3'；
A009711:
A009711-1:5'-TGGTGATTAGCATCATCGGAATGG-3',
A009711-2:5'-TTGGTGATTAGCATCATCGGAATGT-3',
A009711-3:5'-ATCAAATCTATCGAGTTAAAGCTGCCCAA-3'；
A009180:
A009180-1:5'-TAAGTGAATTTTTAAAGTTCGCATACCCT-3',
A009180-2:5'-AGTGAATTTTTAAAGTTCGCATACCCC-3',
A009180-3:5'-CAACGGCGTACCCCGGATTTTAAAT-3'；
A009716:
A009716-1:5'-GTTGCTATGTAACGGAATAAGAACG-3',
A009716-2:5'-CGTTGCTATGTAACGGAATAAGAACT-3',
A009716-3:5'-CCAAATAGAAGTATCACTTGAACAATGCTT-3'.
Application of the molecular marker panel in anti growing out genetic marker cooperates existing Xcfd 223 to anti growing out
Gene carries out molecular labeling, when concrete application:
Xcfd 223 and A009711 cooperation is marked；
A009711 and A009180 cooperation is marked；
A009716 and Xgwm383b cooperation is marked；
The Xcfd 223, particular sequence are as follows:
Xcfd 223:
5'-AAGAGCTACAATGACCAGCAGA-3',
5' -GCAGTGTATGTCAGGAGAAGCA-3'。
Have statistics and show that white wheat than red grain wheat is easier to Spike sprouting under normal circumstances, think this is because
Red R gene is transcription regulaton factor, it influences seed by adjusting the expression for several genes that control flavonoids synthesizes
Dormant trait, to influence ear germinating resistance.But since white wheat has than red grain wheat, flour extraction is high, purchasing price is high etc.
Advantage, therefore excavating white wheat anti growing out gene and cultivating white grain anti growing out kind is still main research emphasis.
In existing research, the sibling species Triticum tauschii of wheat due to many merits, as resistance, disease resistance and its
His merit, therefore important gene source usually as wheat breed hereditary variation and supplied as common wheat D group chromosome
Body kind is used to carry out genetic improvement to wheat.In practical breed of variety, favorable genes are shifted into wheat in Triticum tauschii approach
A series of wheat/Triticum tauschii substitution line is mainly formulated, is imported into Triticum tauschii chromosome by the means of distant hybridization small
In wheat background.For this breeding method, the application is with actual F2For group, molecular marker panel is devised, thus with
It detects, track the Triticum tauschii chromosome situation imported in Wheat Background, and be used to refer to lead wheat anti growing out new varieties in turn
It cultivates；In conjunction with practical study result evidence, related molecular marker provided herein is for instructing wheat anti growing out new product
Kind, which is cultivated, has preferable practical value.
Detailed description of the invention
Fig. 1 is to 1884 plants of F27 days germination indexes of group measure statistical chart；
Fig. 2 is using SSR primer Xcfd223-3D to F2The amplification in population segment material and parent's 18 and T093 of week compares knot
Fruit, in figure: 1-36 indicates part F2Group；M indicates that Marker, T indicate Triticum tauschii T093；Z indicates all wheats 18；
Fig. 3 is wheat F2The positioning of 3D chromosome Spike sprouting QTL in group；It is 4 SSR of positioning on the right side of chromosome in left figure
Label and 10 KASP labels, chromosome left side are the genetic distance of each label in genetic map, and genetic distance unit is inner
It rubs；Red-label under color image is the QTL position of anti growing out；Right figure shows the corresponding LOD of the last label of chromosome
(logarithm of the odds) value, the corresponding LOD value of three QTL is respectively 119,70 and 20.
Specific embodiment
Explanation is further explained to the application below with reference to embodiment.Before introducing specific embodiment, with regard to following realities
The background for applying part experimental material in example, which is briefly introduced, to be described as follows.
Experimental material:
Circulation background parent week wheat 18(is easy Spike sprouting material), Triticum tauschii T093(because there is longer dormant period anti-fringe hair
Bud), it is that can disclose the common germ plasm resource obtained；
1884 parts of F involved in following embodiments2Material is formulated by diploid Triticum tauschii T093 and all hybridization of wheat 18,
It formulates process are as follows: hybridizes week 18 with T093, hybrid F1It doubles to obtain through IMMATURE EMBRYOS CULTURE and colchicine artificial octuple
Body synthetic wheat (AABBDDDD, 2n=8x=56)；Again with week 18 backcrossing, selfing, therefrom select anti growing out single plant continue with
Week 18 hybridizes, then is selfed and to form F2Group；Concrete operations refer to the prior art, repeat no more；
During experiment, parents' Derived Populations F2And parent week wheat 18, Triticum tauschii T093 are planted and are tested in the school in He'nan University
Field, when plantation: selecting 10 at random as unit of family each in group, uniline is uniformly sowed, and spacing in the rows is set as 10cm, row length
It is set as 1.0m, line-spacing is set as 25cm, Routine Management cultivation；
4 SSR primers involved in following embodiments win Radix Polygalae biotechnology Co., Ltd by Beijing three and synthesize offer；
Related KASP(Kompetitive Allele Specific PCR) it marks by the biological skill of middle radix curcumae label (Beijing)
The synthesis of art limited liability company provides.
Embodiment
It should be noted that the major technique thinking of the application are as follows: by being led to related gene segment in Triticum tauschii T093
Enter the phenotype for whether playing reduction effect to malting after wheat to determine, further combined with technologies such as SSR marker, qtl analysis,
Specific designs provide molecular marker panel, so as to lay the foundation for further wheat anti growing out breed of variety.Therefore,
The present embodiment is briefly introduced with regard to related experiment situation and is described as follows.
(1) anti growing out phenotypic evaluation
Each wheat plant carries out listing mark in florescence in April, enters dough stage within Post flowering 35 days or so, to wheat head whole chlorosis
Afterwards, every part of material takes air-dried 7 days outside the consistent stem fringe wheat head room of 5 maturations after, the wheat head of each material is subjected to seed hair
Bud experiment.Specific experiment method is described below.
Grain number of germinateing measurement is referring to (Characterization of QTL controlling genetic such as Imtiaz
variation for pre-harvest sprouting in synthetic backcross derived wheat
Lines. Genetics 2008) method counted, with specific reference to as follows:
The wheat head carries out artificial threshing, and each kind takes the seed of 50 full health at random, its ventral groove is neatly arranged downwards
In the culture dish for being lined with double-layer filter paper, it is added appropriate distilled water in culture dish, covers between postposition culture in 22 DEG C of tissue cultures
Between cultivated, keep filter paper wet during experiment；
It is showed money or valuables one carries unintentionally with the rupture of seed kind skin and is denoted as germination, the kernal number of statistics germination daily, and remove, until counting down to the 7th day, press
Following formula calculates 7 days germination indexes；
。
To 1884 plants of F2Germination index counted, as a result as shown in Figure 1.Analysis is it can be seen that in F2 In group
Germination index is distributed within the scope of 0-100%, without apparent proportionate relationship, therefore belongs to Inheritance of Quantitative Characters.
(2) SSR marker is analyzed
Using round pcr, further labeled analysis, used SSR marker (each SSR mark are carried out using 4 SSR markers
It is denoted as a primer pair) come specifically:
Xcfd152:
Xcfd152-F:5'-TGGAAGTCTGGAACCACTCC -3',
Xcfd152-R:5'-GCAACCAGACCACACTCTCA -3；
Xgwm383b:
Xgwm383b-F:5'- ACGCCAGTTGATCCGTAAAC- 3',
Xgwm383b-R:5'-GACATCAATAACCGTGGATGG- 3'；
Xcfd 223:
Xcfd 223-F:5'-AAGAGCTACAATGACCAGCAGA- 3',
Xcfd 223-R:5'- GCAGTGTATGTCAGGAGAAGCA- 3'；
Xgpw 5094:
Xgpw 5094-F:5'-GACGATCAACAGCGAGTCAA-3',
Xgpw 5094-R:5'-TTACAATCTCACCCTGGCAA-3'.
Before PCR amplification, first to March from field Triticum tauschii T093, Zhou Mai 18 collected and F2The children of group's strain
Leaflet tablet sample extraction genomic DNA, and in this, as PCR amplification template.It is extracted when extracting DNA using CTAB method
(Rogers S O. Extraction of DNA from plant tissues. Plant molecular biology
Manual .1989).
When PCR amplification, the design of 10 μ L reaction systems is as follows:
DNA sample, 2 μ l(50ng/ul)；
10 PCR buffer(Mg2+), 1 μ l；
D NTP (2.5mmol/L), 1 μ l；
Primer F (10umol/L), 0.5 μ l；
Primer R (10umol/L), 0.5 μ l；
TaqE (5U/ul), 0.1 μ l；
ddH2O, 4.9 ul.
After reaction system is mixed, PCR amplification is carried out by following procedure:
95 DEG C of 3 min of initial denaturation；
95 DEG C of denaturation 30 s, 58 DEG C of annealing 45 s, 72 DEG C of 1 min of extension, totally 35 recycle；
72 DEG C of 10 min of extension.
To the direct electrophoretic analysis of pcr amplification product (or 16 DEG C save backup).When carrying out electrophoresis to pcr amplification product,
Non denatured 6 × Loading the buffer of 2 μ L is added in PCR product, carries out 8% polyacrylamide gel electrophoresis detection after mixing.
It is taken a picture to electrophoresis result and records banding pattern, banding pattern identical with parent's Triticum tauschii T093 is denoted as " 2 ", with parent
The identical banding pattern of this week wheat 18 is denoted as " 0 ", and heterozygosis banding pattern is denoted as " 1 ", and missing or the banding pattern that not detected are denoted as " -1 ".
Part of test results is as shown in Figure 2.
(3) BSA (Bulked Segregant Analys) is analyzed
Using segregating population bulked segregant analysis (BSA), in F2Extremely anti-pond and pole each 60 plants of pond of sense are selected in group.With existing merchant
SNP detection is carried out to parent's week 18, Triticum tauschii T093, extremely anti-pond and pole sense pond after the wheat 660k chip for increasing hair.
In conjunction with the statistical result of step (2), comprehensive analysis thinks:
After being compared with wheat IWGSC_RefSeq_v1.0 version, for F24 SSR markers can be by 3D chromosomal target area
Section is located in the section 538M-565M, further combined with the SNP testing result of wheat 660k chip, in this section, anti-pond and sense
52 difference SNP are found altogether in pond.Further according to the flanking sequence of this 52 SNP site two sides, KASP primer has been designed and developed
Group, it is final successfully to obtain 10 KASP molecular labelings altogether, on this basis further to 18, T093 and 1884 plants of F of parent's week2
Parting is carried out.
(4) QTL positioning analysis
In conjunction with the genotyping result and 4 SSR primer electrophoresis results in step (3) based on KASP, with QTL IciMapping v
4.0 carry out qtl analysis.
Need to illustrate when, although designed KASP label include it is multiple, typically have 3, specifically:
A009711:
A009711-1:5'-TGGTGATTAGCATCATCGGAATGG-3',
A009711-2:5'-TTGGTGATTAGCATCATCGGAATGT-3',
A009711-3:5'-ATCAAATCTATCGAGTTAAAGCTGCCCAA-3'；
A009180:
A009180-1:5'-TAAGTGAATTTTTAAAGTTCGCATACCCT-3',
A009180-2:5'-AGTGAATTTTTAAAGTTCGCATACCCC-3',
A009180-3:5'-CAACGGCGTACCCCGGATTTTAAAT-3'；
A009716:
A009716-1:5'-GTTGCTATGTAACGGAATAAGAACG-3',
A009716-2:5'-CGTTGCTATGTAACGGAATAAGAACT-3',
A009716-3:5'-CCAAATAGAAGTATCACTTGAACAATGCTT-3'.
When concrete application, existing Xcfd 223 and aforementioned SSR marker Xgwm383b is cooperated to carry out anti growing out gene
Molecular labeling, specific combined application mode are as follows:
Xcfd 223 and A009711 cooperation is marked；
A009711 and A009180 cooperation is marked；
A009716 and Xgwm383b cooperation is marked；
The Xcfd 223, particular sequence are as follows:
Xcfd 223:
5'-AAGAGCTACAATGACCAGCAGA-3',
5' –GCAGTGTATGTCAGGAGAAGCA-3'。
It should be noted that Xcfd223 label belongs to discloses sequence in the prior art, and for details, reference can be made to: " An
Advanced Backcross Population through Synthetic Octaploid Wheat as a
" Bridge ": Development and QTL Detection for Seed Dormancy " (Dale Zhang et.al,
Frontiers in Plant Science, 2017).
It using IciMapping4.0 software, is detected by 1000 arrangements, anti growing out LOD critical value is 2.3604.With
The method of complete Interval mapping is to F2Group carries out qtl analysis such as following table, detects 3 QTL sites altogether.
Result is made a concrete analysis of as shown in following table and Fig. 3.
Table 1:F2Additivity QTL position, effect and the contribution rate of group's anti growing out character
。
To the qtl analysis of upper table, the results show that there are 3 QTLs relevant to Spike sprouting on 3D chromosome:
First QTL is between xcfd223 and A009711, physical distance 2.1M, 5.25 c M of genetic distance between two labels,
The LOD value of this QTL is 119, phenotype contribution rate 24.16%, additive effect -16.61；
Second QTL is between A009711 and A009180, and physical distance is 6.9M, 17.44 c of genetic distance between two labels
M.The LOD value of this QTL is 70, phenotype contribution rate 15.49%, additive effect -12.59；
Third QTL is between A009716 and xgwm383b, and genetic distance is 43.56 c M between two labels.This QTL's
LOD value is 22, phenotype contribution rate 4.74%, additive effect -6.03.
Additive effect is that negative value can illustrate that the introgressed segment from Triticum tauschii T093 plays the role of reduction to grain germination,
The germination percentage of Wheat Grain Germination is reduced, molecular mark is used for, research in terms of wheat anti growing out and is educated
Kind work.
It is to be understood that " the An Advanced Backcross Population through that published an article
Synthetic Octaploid Wheat as a “Bridge”: Development and QTL Detection for
Seed Dormancy " in by building 201 BC3F4Group has carried out Primary Location to Spike sprouting QTL, and telltale mark is
Xcfd223.Herein on the basis of Spike sprouting QTL Primary Location, Population is expanded, to 1884 plants of F2Group utilizes BSA
660k chip analysis and KASP marker development have been carried out, in conjunction with SSR marker, Spike sprouting QTL positioning section has been further reduced, fringe
The germination section QTL is 9M, wherein identifying 3 QTLs relevant to Spike sprouting, while also being tested the QTL of Primary Location
Card, to further determine that Spike sprouting gene provides reference.
SEQUENCE LISTING
<110>He'nan University
<120>molecular marker panel and its application in Genes of Pre-harvest Sprouting molecular labeling
<130> none
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>engineer
<400> 1
acgccagttg atccgtaaac 20
<210> 2
<211> 21
<212> DNA
<213>engineer
<400> 2
gacatcaata accgtggatg g 21
<210> 3
<211> 24
<212> DNA
<213>engineer
<400> 3
tggtgattag catcatcgga atgg 24
<210> 4
<211> 25
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<213>engineer
<400> 4
ttggtgatta gcatcatcgg aatgt 25
<210> 5
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<213>engineer
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atcaaatcta tcgagttaaa gctgcccaa 29
<210> 6
<211> 29
<212> DNA
<213>engineer
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taagtgaatt tttaaagttc gcataccct 29
<210> 7
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<400> 7
agtgaatttt taaagttcgc atacccc 27
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caacggcgta ccccggattt taaat 25
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gttgctatgt aacggaataa gaacg 25
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<210> 11
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ccaaatagaa gtatcacttg aacaatgctt 30
Claims (4)
1. being used to mark the molecular marker panel of anti growing out related gene, which is characterized in that molecular labeling has 4 altogether, including
One SSR marker and three KASP labels, wherein SSR marker is named as Xgwm383b label, the sequence such as institute of SEQ ID NO.1 ~ 2
Show；Three KASP labels are respectively designated as: A009711, A009180, A009716 label, the sequence such as institute of SEQ ID NO.3 ~ 11
Show, particular sequence are as follows:
Xgwm383b:
Xgwm383b-F:5'- ACGCCAGTTGATCCGTAAAC-3',
Xgwm383b-R:5'- GACATCAATAACCGTGGATGG-3'；
A009711:
A009711-1:5'-TGGTGATTAGCATCATCGGAATGG-3',
A009711-2:5'-TTGGTGATTAGCATCATCGGAATGT-3',
A009711-3:5'-ATCAAATCTATCGAGTTAAAGCTGCCCAA-3'；
A009180:
A009180-1:5'-TAAGTGAATTTTTAAAGTTCGCATACCCT-3',
A009180-2:5'-AGTGAATTTTTAAAGTTCGCATACCCC-3',
A009180-3:5'-CAACGGCGTACCCCGGATTTTAAAT-3'；
A009716:
A009716-1:5'-GTTGCTATGTAACGGAATAAGAACG-3',
A009716-2:5'-CGTTGCTATGTAACGGAATAAGAACT-3',
A009716-3:5'-CCAAATAGAAGTATCACTTGAACAATGCTT-3'.
2. application of the molecular marker panel described in claim 1 in anti growing out genetic marker, which is characterized in that for fighting
Spike sprouting gene carries out molecular labeling.
3. application of the molecular marker panel as claimed in claim 2 in anti growing out genetic marker, which is characterized in that for losing
Pass the label of the anti growing out gene shifted in Triticum tauschii genome into Wheat volatiles in breeding.
4. the molecule labelling method of the anti growing out gene using molecular marker panel described in claim 1, which is characterized in that match
It closes existing Xcfd 223 and molecular labeling is carried out to anti growing out gene, when concrete application:
Xcfd 223 and A009711 cooperation is marked；
A009711 and A009180 cooperation is marked；
A009716 and Xgwm383b cooperation is marked；
The Xcfd 223, particular sequence are as follows:
Xcfd 223:
5'-AAGAGCTACAATGACCAGCAGA-3',
5'-GCAGTGTATGTCAGGAGAAGCA-3'。
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