CN105349542B - The Indel molecular labeling related to thousand grain weight of wheat and application - Google Patents
The Indel molecular labeling related to thousand grain weight of wheat and application Download PDFInfo
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- CN105349542B CN105349542B CN201510907009.5A CN201510907009A CN105349542B CN 105349542 B CN105349542 B CN 105349542B CN 201510907009 A CN201510907009 A CN 201510907009A CN 105349542 B CN105349542 B CN 105349542B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of Indel molecular labeling related to thousand grain weight of wheat.The present invention is on the basis of forefathers study, the wheat TaGSK1 gene orders announced using NCBI is template design primers, the Indel molecular labeling related to thousand grain weight of wheat is developed according to the sequence difference between different mass of 1000 kernel wheat breed Henan wheats 8679 and Buddhist monk wheat, the different allelic variation of the mark can explain 7.9% the 11.8% of mass of 1000 kernel phenotypic variation, wherein, amplified production size is that increase of the 624bp band to mass of 1000 kernel has synergistic effect, and amplified production size is that reduction of the 609bp band to mass of 1000 kernel has synergistic effect.The exploitation of the molecular labeling provides a feasible approach for High-yield Wheat marker assisted selection.
Description
Technical field
The present invention relates to genetic engineering and biology field, specifically, is related to related to thousand grain weight of wheat
Indel molecular labelings and application.
Background technology
Wheat is one of most important cereal crops in the world, with the growth of population, the reduction of cultivated area and grain
The continuous improvement of production cost is eaten, high yield, Breeding For Super High-yielding turn into urgently to be solved the problems, such as in China's wheat breeding.Spike number, fringe
Grain number and mass of 1000 kernel are the three elements that wheat yield is formed, and grain is the quantitative character that key-gene and minor gene regulate and control jointly again,
Thus understand fully that the incubation for the gene pairs High-Yield Wheat Cultivar for participating in regulation and control grain weight various processes is significant.
Glycogen synthase kinase (GSKs) is a kind of highly conserved serine/threonine protein kitase, and GSKs families exist
Important role is play in plant, existing evidence shows, plant GSKs may participate in the abiotic stress response of plant, injury
A series of vital movement processes such as response and brassinosteroid signal transduction, regulation Floral development.By wheat glycogen synthetase
Kinases TaGSK1 is transferred to coli strain and carries out Salt Tolerance Analysis, the results showed that, the salt resistance ability of transformant is higher than Host Strains.
In addition, using the wheat mature embryo callus of quick salt as material, the TaGSK1 binary vectors pBI12-gskl successfully constructed is used
The Efficiency of Wheat Transformation that two methods of agriculture bacillus mediated and biolistic bombardment are successfully made TaGSK1 genes is respectively adopted.From sieve
Result is selected to can be seen that the most browning even death in 0.5%NaCl of control callus, and transgenic calli is then given birth to
Length is vigorous, and (Xu Tao etc., 2003, the separation and identification of wheat salt tolerant mutant TaGSK1 genes, Hebei Normal College master graduate
Paper).Therefore, gene family member, which may have, influences thousand grain weight of wheat, improves the potential value of wheat yield.At present,
There has been no the report of the research on the effect gene thousand grain weight of wheat.
With developing rapidly for modern molecular biology technique, molecular marker assisted selection (MAS) is widely used in point
In sub- breeding, because it is not influenceed by environment and breeding generation, early generation selection and prediction can be carried out, substantially reduces wheat
The breeding time limit.Functional label is a kind of mark based on the exploitation of gene particular sequence, isolates, substantially increases with target gene
The accuracy of selection.Therefore the exploitation of functional label provides foundation for wheat molecule aid mark breeding.
The content of the invention
It is an object of the invention to provide the Indel molecular labeling related to thousand grain weight of wheat and application.
In order to realize the object of the invention, the Indel molecular labeling related to thousand grain weight of wheat provided by the invention is described
Indel molecular labelings isolate with wheat TaGSK1 genes, and the primer pair for expanding the Indel molecular labelings is：
Sense primer F：5’-CGCAAATCAAAGCTCACC-3’(Seq ID No.1)
Anti-sense primer R：5’-CCATCCTACAAAGGCACA-3’(Seq ID No.2)
The Indel molecular marker characteristic band related to the higher mass of 1000 kernel of wheat of the primer pair amplifies is 624bp, its
Nucleotide sequence is as shown in Seq ID No.3；The Indel molecule mark related to the relatively low mass of 1000 kernel of wheat of the primer pair amplifies
Note characteristic bands are 609bp, and its nucleotide sequence is as shown in Seq ID No.4.
According to the sequence (Genbank for the wheat TaGSK1 genes announced on NCBI：AF525086), Primer is utilized
A pair of Indel primers between two kinds of Henan wheat 8679 and Buddhist monk wheat with polymorphism of the Software for Design of Premier 5.0, and
Screened between the wheat breed of mass of 1000 kernel significant difference, further in recombinant inbred lines (RILs) and natural population
Its effect to phenotype is analyzed, obtains a pair of primers for having specific difference.
The present invention also provides application of the Indel molecular labelings in the Wheat Germplasm Resources for screening higher mass of 1000 kernel,
Comprise the following steps：
1) genomic DNA of plant to be measured is extracted；
2) using the genomic DNA of plant to be measured as template, using the primers F and R, pcr amplification reaction is carried out；
3) pcr amplification product is detected, if it is possible to amplify the feature bar of the nucleotide sequence as shown in Seq ID No.3
Band, then plant to be measured is the wheat resource with higher mass of 1000 kernel.
PCR amplification system is calculated as with 10 μ l：Template DNA 100ng, 10 μM of primers Fs and each 0.2 μ l, 2.5mM dNTP of R
0.8 μ l, 5U/ μ l Taq archaeal dna polymerases 0.12 μ l, Mg containing 25mM2+10 × PCR reaction buffers 1.0 μ l, ddH2O is supplied
To 10 μ l.
PCR amplification programs are：94 DEG C 5 minutes；94 DEG C 50 seconds, 54 DEG C 50 seconds, 72 DEG C 3 minutes, 36 circulation；72 DEG C 10 points
Clock.
Preferably, pcr amplification product is detected using 6% denaturing polyacrylamide gel electrophoresis in step 3), then silver staining
Colour developing.
The present invention also provides the PCR detection kit for identifying higher mass of 1000 kernel Wheat Germplasm Resources, the detection examination
Agent box includes the primers F and R for being used for expanding the Indel molecular labeling related to thousand grain weight of wheat.
The present invention further provides the Indel molecular labeling related to thousand grain weight of wheat in wheat molecular marker assistant breeding
In application.
The present invention is drawn on the basis of forefathers study using the wheat TaGSK1 gene orders announced on NCBI as stencil design
Thing, developed according to the sequence difference between different mass of 1000 kernel wheat breed Henan wheats 8679 and Buddhist monk wheat related to thousand grain weight of wheat
Indel molecular labelings, the different allelic variation of the mark can explain the 7.9%-11.8% of mass of 1000 kernel phenotypic variation, its
In, amplified production size is that increase of the 624bp band to mass of 1000 kernel has synergistic effect, and amplified production size is 609bp's
Reduction of the band to mass of 1000 kernel has synergistic effect.The exploitation of the molecular labeling provides one for High-yield Wheat marker assisted selection
The feasible approach of bar.
Brief description of the drawings
Fig. 1 is equipotential type analysis of the Indel molecular labelings in Wheat Cultivars in the embodiment of the present invention 2；Wherein,
Swimming lane 1-23 is respectively Buddhist monk wheat, Bai Huomai, small beautiful flower, ligusticumic excellent 9415, horizontal excellent 18, precious wheat 8, Zheng wheat 366, western agriculture 979, Henan wheat
8679th, many wheats No. 1, new 19, Shanxi wheat 41, the capital winter 10, imperial state, river agriculture 638, Bai Manghong, Zheng wheat 98, M013, M016, white skin 224,
Stone is new 618, the white wheat in Wan County, Handan 5030.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular cloning:A laboratory manual, 2001), or the condition according to manufacturer's specification suggestion.
Sequencing is completed by raw work bioengineering Shanghai (share) Co., Ltd in following examples.
The exploitation of the wheat TaGSK1 gene functions of embodiment 1 mark
According to the sequence (Genbank for the wheat TaGSK1 genes announced on NCBI：AF525086), Primer is utilized
A pair of Indel primers between two kinds of Henan wheat 8679 and Buddhist monk wheat with polymorphism of the Software for Design of Premier 5.0, and
Screened between the wheat breed of mass of 1000 kernel significant difference, further in recombinant inbred lines (RILs) and natural population
Its effect to phenotype is analyzed, obtains a pair of primers for having specific difference, primer sequence is as follows：
Sense primer F：5’-CGCAAATCAAAGCTCACC-3’(Seq ID No.1)
Anti-sense primer R：5’-CCATCCTACAAAGGCACA-3’(Seq ID No.2)
The different genotype of the wheat TaGSK1 genes of embodiment 2 and the correlation of grain weight
1st, the measure of wheat seed mass of 1000 kernel
1000 complete wheat seeds are randomly selected, are repeated twice, are weighed with millesimal balance, average value is remembered twice
For final mass of 1000 kernel value.
2nd, SDS-Tris saturated phenols method extracting wheat leaves genomic DNA
(1) the fresh young leaflet tablets of 2g are taken, liquid nitrogen grinding is into being placed in 2ml sterile centrifugation tubes after fine powder.
(2) add 1.2ml DNA Extraction buffers (200mM Tris-Cl, pH8.0,250mMNaCl, 25mM EDTA,
PH8.0,0.5%SDS, 2% β-ME are well mixed, β-ME fresh additions before use).
(3) 55 DEG C of water-bath 45min, during which intermittent oscillation is fully to extract.
(4) 12000rpm centrifuges 10min at room temperature.
(5) supernatant is shifted into new 2ml sterile centrifugation tubes, the isometric Tris saturated phenols/chloroform for adding precooling is different/
Amylalcohol (volume ratio 25:24:1) 15min, is mixed in reverse on ice, during which intermittent oscillation.
(6) 12000rpm centrifuges 10min at room temperature.
(7) supernatant is shifted into new 2ml sterile centrifugation tubes, repeat step (5), (6), fully to remove deproteinized.
(8) shift supernatant into new 1.5ml sterile centrifugation tubes, add 0.6 times of volume isopropanol (300 μ l) and
The pH of 0.1 times of volume is the 5.2 μ l of 5M NaAC solution 50, and 17min is stood on ice after being sufficiently mixed mixing.
(9) there are white flock DNA precipitations, 4 DEG C, 10000rpm centrifuges 10min.
(10) supernatant is abandoned, 70% ethanol for adding precooling is rinsed 2 times, is then rinsed 1 time with absolute ethyl alcohol, and room temperature is dried,
Add 1 × TE buffer solutions (or distilled water) of the 100 μ l containing 2 μ l 10mg/ml RNase enzymes to dissolve overnight, 10 times of dilutions are standby.
(11) with the Wheat volatiles DNA of 1% agarose Detection and Extraction quality and concentration.
3rd, the amplification of target product
PCR amplification system is calculated as with 10 μ l：Template DNA 100ng, 10 μM of primers Fs and each 0.2 μ l, 2.5mM dNTP of R
0.8 μ l, 5U/ μ l Taq archaeal dna polymerases 0.12 μ l, Mg containing 25mM2+10 × PCR reaction buffers 1.0 μ l, ddH2O is supplied
To 10 μ l.
PCR amplification programs are：94 DEG C 5 minutes；94 DEG C 50 seconds, 54 DEG C 50 seconds, 72 DEG C 3 minutes, 36 circulation；72 DEG C 10 points
Clock.4 DEG C of preservations.
After the completion of PCR, add denaturation Dual-indicator (DNA sample-loading buffers) 95 DEG C of denaturation 10min, Ran Houli in PCR instrument
It is placed on ice, after room temperature is down to, the electrophoresis on 6% denaturing polyacrylamide gel, then silver staining colour developing.With compared with Gao Qian
The wheat breed of grain weight amplifies 624bp (Seq ID No.3) target stripe, and the kind with relatively low mass of 1000 kernel amplifies
609bp (Seq ID No.4) target stripe (Fig. 1).
4th, the genotyping in different mass of 1000 kernel kinds and the correlation with mass of 1000 kernel
The Buddhist monk wheat recombinant inbred lines of wheat 8679/ (the mass of 1000 kernel 57.6g of Henan wheat 8679 in Henan；Buddhist monk wheat mass of 1000 kernel
19.6g, totally 146 familys) and the natural population of 125 parts of Wheat Germplasm Resources (table 1) compositions in respectively to two kinds of genotype
Mass of 1000 kernel carries out variance analysis and the allelic variation of TaGSK1 genes and the correlation analysis of mass of 1000 kernel, the results are shown in Table 2.
The genotype of 1 125 parts of Wheat Germplasm Resources of table and mass of 1000 kernel analysis
Note：A genotype is 642bp banding patterns, and 1 B gene type is 609bp banding patterns.
The result of table 2 shows that the mass of 1000 kernel of A genotype (624bp) is significantly higher than 1 B gene type (609bp) mass of 1000 kernel.It is different
Genotype has reached the pole level of signifiance in Liang Ge colonies with the correlation of mass of 1000 kernel.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
1. the Indel molecular labeling related to thousand grain weight of wheat, it is characterised in that the Indel molecular labelings and wheat
TaGSK1 genes isolate, and the primer pair for expanding the Indel molecular labelings is：
Sense primer F：5’-CGCAAATCAAAGCTCACC-3’
Anti-sense primer R：5’-CCATCCTACAAAGGCACA-3’
The Indel molecular marker characteristic band related to the higher mass of 1000 kernel of wheat of the primer pair amplifies is 624bp, its nucleosides
Acid sequence is as shown in Seq ID No.3；The Indel molecular labeling related to the relatively low mass of 1000 kernel of wheat of the primer pair amplifies is special
Sign band is 609bp, and its nucleotide sequence is as shown in Seq ID No.4.
2. application of the Indel molecular labelings described in claim 1 in the Wheat Germplasm Resources for screening higher mass of 1000 kernel, its feature
It is, comprises the following steps：
1) genomic DNA of plant to be measured is extracted；
2) using the genomic DNA of plant to be measured as template, using primers F described in claim 1 and R, pcr amplification reaction is carried out；
3) pcr amplification product is detected, if it is possible to amplify the characteristic bands of the nucleotide sequence as shown in Seq ID No.3, then
Plant to be measured is the wheat resource with higher mass of 1000 kernel.
3. application according to claim 2, it is characterised in that the amplification system that PCR reactions use in step 2) is with 10 μ l
It is calculated as：Template DNA 100ng, 10 μM of primers Fs and each μ l, 5U/ μ l Taq archaeal dna polymerases of 0.2 μ l, 2.5mM dNTP 0.8 of R
0.12 μ l, Mg containing 25mM2+10 × PCR reaction buffers 1.0 μ l, ddH2O complements to 10 μ l.
4. application according to claim 2, it is characterised in that the amplification program that uses of PCR reactions is in step 2)：94℃
5 minutes；94 DEG C 50 seconds, 54 DEG C 50 seconds, 72 DEG C 3 minutes, 36 circulation；72 DEG C 10 minutes.
5. application according to claim 2, it is characterised in that using 6% denaturing polyacrylamide gel electricity in step 3)
Swimming detection pcr amplification product, then silver staining colour developing.
6. the PCR detection kit for identifying higher mass of 1000 kernel Wheat Germplasm Resources, it is characterised in that the detection kit
Comprising the primers F and R for expanding Indel molecular labelings described in claim 1, primers F and R definition are the same as claim 1 institute
State.
7. application of the Indel molecular labelings in wheat molecular marker assistant breeding described in claim 1.
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| CN108546776B (en) * | 2018-07-16 | 2020-03-31 | 四川农业大学 | InDel marker of poland dwarf wheat, identification method and application |
| CN112280893B (en) * | 2020-12-16 | 2021-04-23 | 鲁东大学 | Molecular marker of wheat yield related gene TaD11-2A and application thereof |
| CN115772522B (en) * | 2022-11-30 | 2023-11-07 | 山东大学 | Application of TaSK2A knocked out by gene editing in increasing wheat grain length |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1523110A (en) * | 2003-09-09 | 2004-08-25 | 河北师范大学 | Wheat glycogen synthetase kinase gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1523110A (en) * | 2003-09-09 | 2004-08-25 | 河北师范大学 | Wheat glycogen synthetase kinase gene |
Non-Patent Citations (3)
| Title |
|---|
| Isolation and characterization of TaGSK1 involved in wheat salt tolerance;Gui Ping Chen等;《Plant Science》;20031231;第165卷(第6期);第1369-1375页 * |
| 小麦糖原合成酶激酶（TaGSK1）的亚细胞定位及其功能鉴定;吴立柱;《中国优秀硕士学位论文全文数据库（农业科技辑）》;20041215(第04期);D047-143 * |
| 小麦糖原合成酶激酶基因(TaGSK1)的染色体定位;李亚青等;《华北农学报》;20061204;第21卷(第5期);第39-41页 * |
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