CN105349542A - Indel molecular marker related to wheat thousand seed weights and application - Google Patents
Indel molecular marker related to wheat thousand seed weights and application Download PDFInfo
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Abstract
The invention discloses an Indel molecular marker related to wheat thousand seed weights. On the basis of research of predecessors, the wheat TaGSK1 gene sequence disclosed by NCBI is used as a template design polymer, according to the sequence difference between wheat varieties yamai 8679 and shangmai with the different thousand seed weights, the Indel molecular marker related to the wheat thousand seed weights is developed, the different equipotential variations of the marker can be used for explaining 7.9-11.8% of the thousand seed weight phenotypic variation, strips with the size of amplification products being 624 bp have the synergistic effect on increase of the thousand seed weights, and strips with the size of amplification products being 609 bp have the synergistic effect on reduction of the thousand seed weights. The development of the molecular marker provides a feasible path for high-yield wheat molecule assistant breeding.
Description
Technical field
The present invention relates to genetically engineered and biology field, specifically, relate to the Indel molecule marker relevant to thousand grain weight of wheat and application.
Background technology
Wheat is one of most important food crop in the world, and along with the growth of population, the minimizing of cultivated area and improving constantly of grain-production cost, high yield, Breeding For Super High-yielding become the urgent problem solved in China's wheat breeding.Spike number, grain number per spike and thousand seed weight are the three elements that wheat yield is formed, and grain is heavily the quantitative character that key-gene and minor gene regulate and control jointly, thus understand fully that the incubation of the gene pairs High-Yield Wheat Cultivar participating in the heavy various process of regulation and control grain is significant.
Glycogen synthase kinase (GSKs) is a kind of serine/threonine protein kitase of high conservative, GSKs family plays important role in plant, existing evidence shows, abiotic stress response, the injury of plant GSKs possibility involved in plant are replied and brassinolide signal transduction, regulate a series of vital movement processes such as Floral development.Wheat glycogen synthetase kinase TaGSK1 is proceeded to coli strain and carries out Salt Tolerance Analysis, result shows, the salt resistance ability of transformant is higher than Host Strains.In addition, with the wheat mature embryo callus of quick salt for material, the TaGSK1 binary vector pBI12-gskl that successfully constructs is used to adopt agriculture bacillus mediated and biolistic bombardment two kinds of methods to be successfully made the Efficiency of Wheat Transformation of TaGSK1 gene respectively.As can be seen from the selection result, contrast callus most brownization in 0.5%NaCl is even dead, and transgenic calli then grows vigorous (Xu Tao etc., 2003, the separation andpreconcentration of wheat salt tolerant mutant TaGSK1 gene, Hebei Normal College master's Diplomarbeit).Therefore, this gene family member may have affects thousand grain weight of wheat, improves the potential value of wheat yield.At present, the research about this effect gene thousand grain weight of wheat is not yet had to report.
Along with developing rapidly of modern molecular biology technique, molecular marker assisted selection (MAS) is widely used in molecular breeding, due to the impact that it is not subject to environment and breeding generation, generation morning can be carried out and select and prediction, substantially reduce the wheat breeding time limit.Functional label is the mark that a class is developed based on gene particular sequence, with target gene be divided into from, substantially increase the accuracy of selection.Therefore the wheat molecule aid mark breeding that is developed as of functional label provides foundation.
Summary of the invention
The object of this invention is to provide the Indel molecule marker relevant to thousand grain weight of wheat and application.
In order to realize the object of the invention, the Indel molecule marker relevant to thousand grain weight of wheat provided by the invention, described Indel molecule marker and wheat TaGSK1 gene be divided into from, the primer pair for the described Indel molecule marker that increases is:
Upstream primer F:5 '-CGCAAATCAAAGCTCACC-3 ' (SeqIDNo.1)
Downstream primer R:5 '-CCATCCTACAAAGGCACA-3 ' (SeqIDNo.2)
The Indel molecular marker characteristic band relevant to the higher thousand seed weight of wheat of described primer pair amplifies is 624bp, and its nucleotide sequence is as shown in SeqIDNo.3; The Indel molecular marker characteristic band relevant to the lower thousand seed weight of wheat of described primer pair amplifies is 609bp, and its nucleotide sequence is as shown in SeqIDNo.4.
According to the sequence (Genbank:AF525086) of the wheat TaGSK1 gene that NCBI announces, utilize PrimerPremier5.0 software design between Henan wheat 8679 and Buddhist monk wheat two kinds, have the Indel primer of polymorphism for a pair, and screen between the wheat breed of thousand seed weight significant difference, in recombinant inbred lines (RILs) and natural population, analyze its effect to phenotype further, obtain the primer having specificity difference for a pair.
The present invention also provides the application of described Indel molecule marker in the Wheat Germplasm Resources of the higher thousand seed weight of screening, comprises the steps:
1) genomic dna of plant to be measured is extracted;
2) with the genomic dna of plant to be measured for template, utilize described primers F and R, carry out pcr amplification reaction;
3) detect pcr amplification product, if can amplify the characteristic bands of nucleotide sequence as shown in SeqIDNo.3, then plant to be measured is the wheat resource with higher thousand seed weight.
PCR amplification system is counted with 10 μ l: template DNA 100ng, and 10 μMs of primers F and R each 0.2 μ l, 2.5mMdNTP0.8 μ l, 5U/ μ lTaqDNA polysaccharase 0.12 μ l, containing 25mMMg
2+10 × PCR reaction buffer 1.0 μ l, ddH
2o complements to 10 μ l.
Pcr amplification program is: 94 DEG C 5 minutes; 94 DEG C 50 seconds, 54 DEG C 50 seconds, 72 DEG C 3 minutes, 36 circulations; 72 DEG C 10 minutes.
Preferably, step 3) middle employing 6% denaturing polyacrylamide gel electrophoresis detection pcr amplification product, then silver dye develops the color.
The present invention is also provided for the PCR detection kit identifying higher thousand seed weight Wheat Germplasm Resources, and described detection kit comprises primers F for the described Indel molecule marker relevant to thousand grain weight of wheat that increase and R.
The present invention further provides the Indel molecule marker application in wheat molecular marker assistant breeding relevant to thousand grain weight of wheat.
The present invention is on the basis that forefathers study, with the wheat TaGSK1 gene order that NCBI announces for template design primer, the Indel molecule marker relevant to thousand grain weight of wheat is developed according to the sequence difference between different thousand seed weight wheat breed Henan wheat 8679 and Buddhist monk wheat, the different allelic variation of this mark can explain the 7.9%-11.8% of thousand seed weight phenotypic variation, wherein, amplified production size is that the increase of band to thousand seed weight of 624bp has synergism, and amplified production size is that the reduction of band to thousand seed weight of 609bp has synergism.The High-yield Wheat marker assisted selection that is developed as of this molecule marker provides a feasible approach.
Accompanying drawing explanation
Fig. 1 is the equipotential type analysis of Indel molecule marker in Wheat Cultivars in the embodiment of the present invention 2; Wherein, swimming lane 1-23 be respectively that excellent 9415, horizontal excellent 18, the precious wheat 8 of Buddhist monk wheat, Bai Huomai, little jade flower, ligusticumic, Zheng wheat 366, western agriculture 979, Henan wheat 8679, many wheats No. 1, new 19, Shanxi wheat 41, capital winter 10, imperial state, river agriculture 638, Bai Manghong, Zheng wheat 98, M013, M016, white skin 224, stone are new 618, the white wheat in Wan County, Handan 5030.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
Check order in following examples and to be completed by raw work biotechnology Shanghai (share) company limited.
The exploitation of embodiment 1 wheat TaGSK1 gene function mark
According to the sequence (Genbank:AF525086) of the wheat TaGSK1 gene that NCBI announces, utilize PrimerPremier5.0 software design between Henan wheat 8679 and Buddhist monk wheat two kinds, have the Indel primer of polymorphism for a pair, and screen between the wheat breed of thousand seed weight significant difference, its effect to phenotype is analyzed further in recombinant inbred lines (RILs) and natural population, obtain the primer having specificity difference for a pair, primer sequence is as follows:
Upstream primer F:5 '-CGCAAATCAAAGCTCACC-3 ' (SeqIDNo.1)
Downstream primer R:5 '-CCATCCTACAAAGGCACA-3 ' (SeqIDNo.2)
The genotype that embodiment 2 wheat TaGSK1 gene is different and the dependency that grain weighs
1, the mensuration of wheat grain thousand seed weight
Random selecting 1000 complete wheat grains, twice repetition, weighs with millesimal balance, and twice mean value is designated as final thousand seed weight value.
2, SDS-Tris saturated phenol method extracting wheat leaves genomic DNA
(1) get the fresh young leaflet tablet of 2g, liquid nitrogen grinding becomes fine powder to be placed in 2ml sterile centrifugation tube.
(2) 1.2mlDNA Extraction buffer (200mMTris-Cl, pH8.0,250mMNaCl, 25mMEDTA, pH8.0,0.5%SDS, 2% β-ME mixes, and β-ME is fresh before use to add) is added.
(3) 55 DEG C of water-bath 45min, period, interrupted oscillation was fully to extract.
(4) the centrifugal 10min of 12000rpm under room temperature.
(5) shift in supernatant liquor to new 2ml sterile centrifugation tube, the saturated phenol/chloroform of equal-volume Tris adding precooling is different/amylalcohol (volume ratio is 25:24:1), in putting upside down mixing 15min on ice, period interrupted oscillation.
(6) the centrifugal 10min of 12000rpm under room temperature.
(7) supernatant liquor is shifted in new 2ml sterile centrifugation tube, repeating step (5), (6), fully to remove Deproteinization.
(8) shift supernatant liquor in new 1.5ml sterile centrifugation tube, the pH of the Virahol (300 μ l) and 0.1 times of volume that add 0.6 times of volume is the 5MNaAC solution 50 μ l of 5.2, fully leaves standstill 17min on ice after mixing mixing.
(9) occur that white flock DNA precipitates, 4 DEG C, the centrifugal 10min of 10000rpm.
(10) abandon supernatant, add 70% ethanol rinse 2 times of precooling, then use dehydrated alcohol rinsing 1 time, room temperature is dried, and adds 100 μ l and to spend the night dissolving containing 1 × TE damping fluid (or distilled water) of 2 μ l10mg/mlRNase enzymes, 10 times of diluted for use.
(11) by quality and the concentration of the Wheat volatiles DNA of 1% agarose Detection and Extraction.
3, the amplification of target product
PCR amplification system is counted with 10 μ l: template DNA 100ng, and 10 μMs of primers F and R each 0.2 μ l, 2.5mMdNTP0.8 μ l, 5U/ μ lTaqDNA polysaccharase 0.12 μ l, containing 25mMMg
2+10 × PCR reaction buffer 1.0 μ l, ddH
2o complements to 10 μ l.
Pcr amplification program is: 94 DEG C 5 minutes; 94 DEG C 50 seconds, 54 DEG C 50 seconds, 72 DEG C 3 minutes, 36 circulations; 72 DEG C 10 minutes.4 DEG C of preservations.
After PCR completes, add sex change Dual-indicator (DNA sample-loading buffer) 95 DEG C of sex change 10min in PCR instrument, be then placed in immediately on ice, after being down to room temperature, electrophoresis on 6% denaturing polyacrylamide gel, the then colour developing of silver dye.The wheat breed with higher thousand seed weight amplifies the target stripe of 624bp (SeqIDNo.3), and the kind with lower thousand seed weight amplifies the target stripe (Fig. 1) of 609bp (SeqIDNo.4).
4, the gene type assay in different thousand seed weight kind and the dependency with thousand seed weight
At Henan wheat 8679/ Buddhist monk wheat recombinant inbred lines (Henan wheat 8679 thousand seed weight 57.6g; Buddhist monk wheat thousand seed weight 19.6g, totally 146 familys) and the natural population that forms of 125 parts of Wheat Germplasm Resources (table 1) in respectively the allelic variation of variance analysis and TaGSK1 gene and the correlation analysis of thousand seed weight are carried out to two kinds of genotypic thousand seed weight, the results are shown in Table 2.
The genotype of table 1125 part Wheat Germplasm Resources and thousand seed weight are analyzed
Note: A genotype is 642bp banding pattern, 1 B gene type is 609bp banding pattern.
Table 2 result shows, the thousand seed weight of A genotype (624bp) is significantly higher than the thousand seed weight of 1 B gene type (609bp).Different genotype all reaches pole conspicuous level with the dependency of thousand seed weight in Liang Ge colony.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. the Indel molecule marker relevant to thousand grain weight of wheat, is characterized in that, described Indel molecule marker and wheat TaGSK1 gene be divided into from, the primer pair for the described Indel molecule marker that increases is:
Upstream primer F:5 '-CGCAAATCAAAGCTCACC-3 '
Downstream primer R:5 '-CCATCCTACAAAGGCACA-3 '
The Indel molecular marker characteristic band relevant to the higher thousand seed weight of wheat of described primer pair amplifies is 624bp, and its nucleotide sequence is as shown in SeqIDNo.3; The Indel molecular marker characteristic band relevant to the lower thousand seed weight of wheat of described primer pair amplifies is 609bp, and its nucleotide sequence is as shown in SeqIDNo.4.
2. the application of Indel molecule marker described in claim 1 in the Wheat Germplasm Resources of the higher thousand seed weight of screening, is characterized in that, comprise the steps:
1) genomic dna of plant to be measured is extracted;
2) with the genomic dna of plant to be measured for template, utilize primers F described in claim 1 and R, carry out pcr amplification reaction;
3) detect pcr amplification product, if can amplify the characteristic bands of nucleotide sequence as shown in SeqIDNo.3, then plant to be measured is the wheat resource with higher thousand seed weight.
3. application according to claim 2, is characterized in that, step 2) in PCR reaction use amplification system count with 10 μ l: template DNA 100ng, 10 μMs of primers F and each 0.2 μ l of R, 2.5mMdNTP0.8 μ l, 5U/ μ lTaqDNA polysaccharase 0.12 μ l, containing 25mMMg
2+10 × PCR reaction buffer 1.0 μ l, ddH
2o complements to 10 μ l.
4. application according to claim 2, is characterized in that, step 2) in PCR reaction use amplification program be: 94 DEG C 5 minutes; 94 DEG C 50 seconds, 54 DEG C 50 seconds, 72 DEG C 3 minutes, 36 circulations; 72 DEG C 10 minutes.
5. application according to claim 2, is characterized in that, step 3) middle employing 6% denaturing polyacrylamide gel electrophoresis detection pcr amplification product, then silver dye develops the color.
6. for the identification of the PCR detection kit of higher thousand seed weight Wheat Germplasm Resources, it is characterized in that, described detection kit comprises primers F for the Indel molecule marker described in claim 1 that increases and R, and the definition of primers F and R is with described in claim 1.
7. the application of Indel molecule marker in wheat molecular marker assistant breeding described in claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546776A (en) * | 2018-07-16 | 2018-09-18 | 四川农业大学 | The InDel labels and identification method of a kind of Triticum polonicum L of short stem and application |
| CN112280893A (en) * | 2020-12-16 | 2021-01-29 | 鲁东大学 | Molecular marker of wheat yield related gene TaD11-2A and application thereof |
| CN115772522A (en) * | 2022-11-30 | 2023-03-10 | 山东大学 | Application of knocking out TaSK2A by gene editing in increasing wheat grain length |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546776A (en) * | 2018-07-16 | 2018-09-18 | 四川农业大学 | The InDel labels and identification method of a kind of Triticum polonicum L of short stem and application |
| CN112280893A (en) * | 2020-12-16 | 2021-01-29 | 鲁东大学 | Molecular marker of wheat yield related gene TaD11-2A and application thereof |
| CN112280893B (en) * | 2020-12-16 | 2021-04-23 | 鲁东大学 | Molecular marker of wheat yield related gene TaD11-2A and application thereof |
| CN115772522A (en) * | 2022-11-30 | 2023-03-10 | 山东大学 | Application of knocking out TaSK2A by gene editing in increasing wheat grain length |
| 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 |
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