CN106755354B - One kind molecular labeling TaSnRK2.4A relevant to thousand grain weight of wheat and stalk soluble sugar content and its application - Google Patents
One kind molecular labeling TaSnRK2.4A relevant to thousand grain weight of wheat and stalk soluble sugar content and its application Download PDFInfo
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Abstract
The invention discloses a kind of molecular labeling relevant to thousand grain weight of wheat and stalk soluble sugar content and its applications.One mononucleotide polymorphism site corresponds to sequence 1 from 5 ' ends the 30th, is found in wheat polymorphism group.In natural population, this SNP is primarily present two kinds of haplotypes: haplotype first (G) and haplotype second (A).It is proved by association analysis, in the homozygous type of both haplotypes, the wheat of haplotype first homozygosis, mass of 1000 kernel and stalk soluble sugar content extremely significant (or significant) are lower than the wheat of haplotype second homozygosis.The present invention also provides the molecular labelings of the SNP.It is demonstrated experimentally that the SNP described by detection, can find mass of 1000 kernel and the higher wheat of stalk soluble sugar content.The present invention provides a new method for the molecular marker assisted selection breeding of wheat, is of great significance in cultivating High-Yield Wheat Cultivar or research.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to one kind are related to thousand grain weight of wheat and stalk soluble sugar content
Molecular labeling TaSnRK2.4A and its application.
Background technique
Wheat is that one of main cereal crops, production are closely related with mankind's grain security in the world.Arid is shadow
Ring the main abiotic limiting factor of Wheat Production.Protein kinase is the hinge of adverse circumstance signal transmitting network, in environment stress
Extremely important effect is played in responsing reaction.Sucrose non-fermented protein kinase (sucrose non-fermental
Protein kinase, SnRK) it is a kind of serine/threonine protein kitase, wherein SnRK2 participates in turning for a variety of adverse circumstance signals
It leads, plays a crucial role in terms of plant stress-resistance, root system development and crop yield.How SnRK2 to be used for degeneration-resistant
High yield crops rearing new variety, is not only molecular biologist and geneticist's focus of attention and breeder compels highly necessary
It solves the problems, such as.The excellent allele of SnRK2 is excavated from Germplasm Resources of Farm Crop, then passes through conventional hybridization and molecular labeling
Assisted Selection, which combines, to be used, undoubtedly most direct, most effective, and most easy received variety of crops improvement side
Method.Conventional breeding generally relies on Phenotypic Selection, although obtaining great success, takes time and effort, breeding process is slow；Compare and
Speech, molecular marker assisted selection breeding can targetedly be selected from generation to generation early, and operation is simple, and can be efficient
Using favorable genes, the process of breeding can be dramatically speeded up.
Wheat TaSnRK2.4 gene participates in the response to a variety of environment stresses, and overexpression can significantly improve plant pair
Resistance (Maoet al.TaSnRK2.4, a SNF1-typeserine/threonine protein kinase of a variety of adverse circumstances
of wheat(TriticumaestivumL.),confers enhanced multi-stress tolerances
InArabidopsis.J Exp Bot, 2010,61:683-96), therefore be the favorable genes resource of the degeneration-resistant molecular breeding of wheat,
However so far, the molecular labeling of TaSnRK2.4 gene is not yet developed, also have no way of finding out about it molecular labeling and economical character
Relationship.
Summary of the invention
It is an object of the present invention to provide a kind of methods that Traits of Wheat to be measured is identified in identification or auxiliary.
Method provided by the invention includes the following steps: the genotype for detecting the exit site of wheat population genome to be measured,
Determine that Traits of Wheat to be measured is as follows according to the genotype of exit site:
The wheat population mass of 1000 kernel that the genotype of exit site is GG is less than wheat population thousand that the genotype of exit site is AA
Weight；
And/or the wheat population stalk soluble sugar content that the genotype of exit site is GG is less than the genotype of exit site
The wheat population stalk soluble sugar content of AA；
The exit site is sequence 1 the 30th from 5 ' ends, is located at 3A chromosome, gene regions the 2478th.
In the above method, the method for the genotype of the exit site of the detection wheat population genome to be measured includes following step
It is rapid: PCR amplification to be carried out to any one section in the genomic DNA of the wheat to be measured DNA fragmentation including the exit site, and will
Pcr amplification product digestion identification, obtains the DNA fragmentation digestion products including exit site, true according to digestion products clip size
The genotype of the exit site of the fixed wheat population genome to be measured.
In the above method, any one section of DNA including the exit site in the genomic DNA to wheat to be measured
Single strand dna shown in the primer pair single strand dna shown in sequence 2 and sequence 3 of segment progress PCR amplification forms；
Enzyme used in the digestion of the DNA fragmentation including the exit site is Hpa II.
In the above method, the E that the wheat population genome to be measured is determined according to the digestion products X clip size
The genotype in site is as follows:
If the digestion products X is only the DNA fragmentation of 212bp, the wheat to be measured is AA in the genotype of exit site；
If the digestion products X is the DNA fragmentation of 28bp, 184bp, the wheat to be measured is in the genotype of exit site
GG。
Another object of the present invention is to provide a kind of side of the genotype of exit site for detecting wheat population genome to be measured
Method.
Method provided by the invention, the side of the genotype of the exit site including the middle detection wheat population genome to be measured
Method.
Third purpose of the present invention is to provide a kind of product for identifying or assisting to identify Traits of Wheat to be measured.
Product provided by the invention, for the substance of the genotype of the exit site of detection wheat population genome to be measured.
In the said goods, the substance includes II enzyme of primer pair 1 and Hpa；
Single strand dna shown in the single strand dna shown in sequence 2 of primer pair 1 and sequence 3 forms.
In the said goods, the product is kit.
The substance of the genotype of the exit site of above-mentioned detection wheat population genome to be measured is being identified or is assisting identification to be measured small
Application in wheat character is also the scope of protection of the invention；
Or, the substance of the genotype of the exit site of above-mentioned detection wheat population genome to be measured is in preparation identification or auxiliary mirror
Application in fixed Traits of Wheat product to be measured is also the scope of protection of the invention.
The above method is also the present invention cultivating the application in the wheat that mass of 1000 kernel is high and/or stalk soluble sugar content is high
The range of protection.
4th purpose of the invention is to provide a kind of screening or cultivation mass of 1000 kernel is high and/or stalk soluble sugar content is high
The method of wheat population.
Method provided by the invention includes the following steps: the wheat that the genotype for screening or cultivating above-mentioned exit site is AA
Group.
The target sequence of the PCR amplification is sequence 1 from 5 ' end 1-212bp.
It is confirmed as the wheat to be measured of genotype A, mass of 1000 kernel and stalk soluble sugar content extremely significant (or significant) are low
In the wheat to be measured for being confirmed as genotype B.
The present invention has found mononucleotide at 1 by the analysis of variance to TaSnRK2.4A gene in wheat polymorphism group
Polymorphic site is named as exit site.The present invention develops dCAPS label according to exit site, is named as AM.This SNP is in natural group
There are two kinds of haplotypes in body: haplotype first (G) and haplotype second (A).Association analysis shows the homozygous class of both haplotypes
In type, the mass of 1000 kernel of haplotype first and stalk soluble sugar content extremely significant (or significant) are lower than haplotype second.It is demonstrated experimentally that logical
The detection SNP is crossed, mass of 1000 kernel and the relatively high wheat of stalk soluble sugar content can be found.The present invention is wheat point
Sub- marker assisted selection breeding provides a new method, is of great significance in cultivating High-Yield Wheat Cultivar or research.
Detailed description of the invention
Fig. 1 is that SNP according to the present invention develops molecular labeling digestion products electrophoresis detection result.
Wherein, swimming lane M is 100bp DNA ladder.As shown in the figure is to be carried out with the PCR product of II pair of Hpa different wheats
The banding pattern of digestion.Swimming lane A contains the band of a 212bp；Swimming lane G contains two and is followed successively by 184bp's and 28bp from top to bottom
Band, since electrophoresis time is longer, the small fragment item of 28bp is more fuzzy.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Embodiment 1, SNP relevant to thousand grain weight of wheat and stalk soluble sugar content and its label polymorphic detection
1, the special primer of the genomic DNA fragment of the SNP site containing wheat is expanded
Inventor's 32 parts of Guard cell kinds (from national germplasm resource bank) from table 1 have found 1 change
Ectopic sites, selecting this SNP exploitation is molecular labeling, (is named as E from the 30th of 5 ' ends corresponding to sequence 1
Point, there are the polymorphisms of A and G)；There are two kinds of haplotypes in wheat natural variation group for this exit site:
Haplotype first: G；
Haplotype second: A.
1,32 part of Guard cell kind of table
According to the sequence difference of wheat different genes group, designs genome specificity primer pair PE amplification and exist comprising exit site
Interior DNA fragmentation, primer pair PE are made of the single stranded DNA of entitled pe1 and pe2, and pe1 is sequence 2 (5 '-
CAAACTCACCTTTCCATCATACTCACGCC-3 ') shown in single stranded DNA, pe2 be sequence 3 (5 '-
ATATGCGATTTCGGCTACTCCAAGGTTG-3 ') shown in single stranded DNA.
2, sequence polymorphism detection and the foundation of methods of genotyping
1) genomic DNA for extracting wheat to be measured, carries out PCR amplification with genome specific primer (F and R), obtains PCR expansion
Increase production object；
F:5 '-CATGAGTAAAACTTGCATACTATATTTCTC-3 ' (sequence 4)；
R:5 '-TCCGCGGCTCCGGC-3 ' (sequence 5).
2) 50 times are diluted for template with the pcr amplification product of step 1), PCR expansion is carried out using the primer pair PE in step 1
Increase, obtains pcr amplification product.
The system of PCR amplification are as follows: 2 μ L of template DNA (10pg-1 μ g), 2 × UtaqPCR MasterMix12.5 μ L, primer
Pe1 (10 μm of ol/L) and pe2 (10 μm of ol/L) each 1.0 μ L mends ddH2O to 25.0 μ L.
PCR amplification condition are as follows: 95 DEG C of 5min, 95 DEG C of 50s, 59 DEG C of 45s, 72 DEG C of 50s, 33 circulations；72 DEG C of 10min, 4 DEG C
It saves.
3) by II digestion of Hpa of the PCR product of step 2), digestion products are obtained；It is solidifying that digestion products carry out 4% agarose
Gel electrophoresis detects, each clip size in identification record digestion products, and judges according to the following method and record wheat to be measured in institute
The case where stating exit site:
If the digestion products are the DNA fragmentation of 212bp (the 1st -212bp of sequence 1), the wheat to be measured is in institute
State the wheat (the swimming lane A in Fig. 1) that exit site is A homozygous (being expressed as A/A)；
If the digestion products are 184bp (the 29th -212bp of sequence 1), 28bp (the 1st -28bp of sequence 1)
DNA fragmentation, then the wheat to be measured is in the wheat (the swimming lane G in Fig. 1) that the exit site is G homozygous (being expressed as G/G).
4) according to step 3) as a result, it is following I-II type that wheat, which was divided into the case where site E and F,
Type:
I: G/G (i.e. haplotype first is homozygous)；
II: A/A (i.e. haplotype second is homozygous).
It is the situation on a homologue before above-mentioned "/", is the feelings on another homologue after above-mentioned "/"
Condition.
3, using the molecular labeling to natural population carry out parting and with mass of 1000 kernel and stalk soluble sugar content character into
Row association analysis
Each wheat difference in the natural population formed with 262 parts of Guard cells (from national germplasm resource bank)
Parting is carried out according to the method for step 2 as wheat to be measured, sequence verification is carried out to the amplified production size of part wheat at random,
The results are shown in Table 2.
The case where site E and F, counts in table 2, wheat natural population
Note: "-" indicates no PCR product.
With General linear model (GLM) model in Tassel2.1 software to two kinds of haplotypes and mass of 1000 kernel,
Stalk soluble sugar content character is associated analysis.
As a result, it has been found that the mass of 1000 kernel of haplotype I and stalk soluble sugar content extremely significant (or significant) are lower than haplotype
Ⅱ.To natural population studies have shown that haplotype II is the favorable genes type for improving mass of 1000 kernel and stalk soluble sugar content.
Two kinds of genotype correlation shape statistical results in table 3, wheat natural population
Note:**It is extremely significant (P < 0.01) to represent Traits change between different genetic wheat varieties.
Sequence table
<110>Institute of Crop Science, Chinese Academy of Agricultural Science
<120>it a kind of molecular labeling TaSnRK2.4A relevant to thousand grain weight of wheat and stalk soluble sugar content and its answers
With
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 212
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
caaactcacc tttccatcat actcacgccr ggatagcacc tctggtgcaa tatatgctgg 60
cgtccccact gctgatttgg gccttgaatg caatactgat gactgatcca gaaatgggaa 120
gaacagtttc tgtgagaatt tacagacatc gataacttga aggttcaata tgtgggttca 180
gggacaacct tggagtagcc gaaatcgcat at 212
<210> 2
<211> 29
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 2
caaactcacc tttccatcat actcacgcc 29
<210> 3
<211> 28
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 3
atatgcgatt tcggctactc caaggttg 28
<210> 4
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 4
catgagtaaa acttgcatac tatatttctc 30
<210> 5
<211> 14
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 5
tccgcggctc cggc 14
Claims (10)
1. a kind of method that identification or auxiliary identify Traits of Wheat to be measured, includes the following steps: to detect wheat population gene to be measured
The genotype of the exit site of group, determines that Traits of Wheat to be measured is as follows according to the genotype of exit site:
The wheat population mass of 1000 kernel that the genotype of exit site is GG is less than the wheat population mass of 1000 kernel that the genotype of exit site is AA；
And/or the genotype of exit site be GG wheat population stalk soluble sugar content be less than exit site genotype be AA's
Wheat population stalk soluble sugar content；
The exit site is sequence 1 the 30th from 5 ' ends.
2. according to the method described in claim 1, it is characterized by: the exit site of detection wheat population genome to be measured
The method of genotype include the following steps: the DNA fragmentation in the genomic DNA to wheat to be measured including the exit site into
Row PCR amplification, and pcr amplification product digestion is identified, the DNA fragmentation digestion products including exit site are obtained, according to digestion
Product clip size determines the genotype of the exit site of the wheat population genome to be measured.
3. according to the method described in claim 2, it is characterized by: including the E in the genomic DNA to wheat to be measured
It is single-stranded shown in the primer pair single strand dna as shown in sequence 2 and sequence 3 of DNA fragmentation progress PCR amplification including site
DNA molecular composition；
Enzyme used in the digestion of the DNA fragmentation including the exit site isHpaⅡ。
4. according to the method in claim 2 or 3, it is characterised in that:
The genotype of the exit site that the wheat population genome to be measured is determined according to the digestion products X clip size is
It is as follows:
If the digestion products X is only the DNA fragmentation of 212bp, the wheat to be measured is AA in the genotype of exit site；
If the digestion products X is the DNA fragmentation of 28bp, 184bp, the wheat to be measured is GG in the genotype of exit site.
5. a kind of identification or auxiliary identify the product of Traits of Wheat to be measured, for the exit site for detecting wheat population genome to be measured
The substance of genotype；The substance includes 1 He of primer pairHpaII enzyme；
Single strand dna shown in the single strand dna shown in sequence 2 of primer pair 1 and sequence 3 forms.
6. product according to claim 5, it is characterised in that: the product is kit.
7. the substance of the genotype of the exit site of the detection wheat population genome to be measured in claim 5 or 6 identification or
Auxiliary identifies the application in Traits of Wheat to be measured.
8. the substance of the genotype of the exit site of the detection wheat population genome to be measured in claim 5 or 6 reflects in preparation
Fixed or auxiliary identifies the application in Traits of Wheat product to be measured.
9. any the method is in cultivating the wheat that mass of 1000 kernel is high and/or stalk soluble sugar content is high in claim 1-4
Application.
10. a kind of screening or the method for cultivating the wheat population that mass of 1000 kernel is high and/or stalk soluble sugar content is high, including it is as follows
It is the wheat population of AA that step:, which screening, or cultivates the genotype of any exit site in claim 1-4.
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| CN102220297A (en) * | 2011-05-31 | 2011-10-19 | 中国农业科学院作物科学研究所 | Stress resistance associated protein TaSnRK2.3 and coding gene and use thereof |
| CN102399760A (en) * | 2011-10-28 | 2012-04-04 | 中国农业科学院作物科学研究所 | Plant stress tolerance related protein TaSnRK2.10 as well as coding gene and application thereof |
| CN103820476A (en) * | 2014-01-24 | 2014-05-28 | 山东农业大学 | Gene relevant to wheat thousand seed weight, functional marker and application thereof |
| CN104342484A (en) * | 2013-07-23 | 2015-02-11 | 中国农业科学院作物科学研究所 | Molecular marker related with wheat thousand grain weight and applications thereof |
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| CN102220297A (en) * | 2011-05-31 | 2011-10-19 | 中国农业科学院作物科学研究所 | Stress resistance associated protein TaSnRK2.3 and coding gene and use thereof |
| CN102399760A (en) * | 2011-10-28 | 2012-04-04 | 中国农业科学院作物科学研究所 | Plant stress tolerance related protein TaSnRK2.10 as well as coding gene and application thereof |
| CN104342484A (en) * | 2013-07-23 | 2015-02-11 | 中国农业科学院作物科学研究所 | Molecular marker related with wheat thousand grain weight and applications thereof |
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| 植物和小麦SnRK2基因家族的研究进展;张洪映等;《安徽农业科学》;20141231;第42卷(第13期);3805-3807 * |
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