CN104894286B - A pair of functional labels for the high mass of 1000 kernel wheat breed of seed selection - Google Patents
A pair of functional labels for the high mass of 1000 kernel wheat breed of seed selection Download PDFInfo
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Abstract
The invention provides the functional label that a pair are used for the high mass of 1000 kernel wheat breed of seed selection, the mark is located on wheat 7B chromosomes, and its nucleotide sequence is as shown in the SEQ ID NO.1 and SEQ ID NO.2 in sequence table.The site is the site for the presence major gene resistance that forefathers had not reported, because the functional label is designed based on this specific gene order, isolated with the gene, relatively general SSR molecular marker, it is favorably improved the Objective and specific aim of breeding selection, the accuracy of selection is substantially increased, field of wheat breeding is can be applied to.The molecular labeling is used for into wheat breeding, and by environment etc., other extraneous factors are not influenceed, and can be screened wheat breed in mesoderm growing early stage, saved production cost, accelerate breeding process.
Description
Technical field
The present invention relates to field of molecular breeding, and in particular to a kind of function mark of wheat breed for the high mass of 1000 kernel of seed selection
Note and this be marked at application in molecular breeding.
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
Eat production cost continuous improvement, high yield, Breeding For Super High-yielding by be China's wheat breeding eternal theme.Spike number, grain number per spike and
Mass of 1000 kernel is the three elements that wheat yield is constituted, and the genetic force of wherein mass of 1000 kernel is of a relatively high, belongs to major gene resistance regulation and control plus micro-
The quantitative character of genetic modification is imitated, many functional genes reported finally all have different contributions, therefore thousand to mass of 1000 kernel
Grain weight regulated and control network it is complex, dissect grain weight Regulation Mechanism, research related gene and its between reciprocal effects, to height
The seed selection of yield wheat new varieties has important references value.
BHLH transcription factors are the second major class transcription factors in plant, and non-life is grown and respond in regulation and control plant
Played an important role in thing stress.Forefathers' research has shown that bHLH transcription factor families participate in the distribution of soluble sugar, GA and closed
Into with decompose and ABA signal pathways.During wheat grain filling, the distribution condition of soluble sugar influences whether that the grouting of seed is filled
Real degree, finally influences whether the seed size and grain weight of wheat.
PTF1 genes, which are one, has the transcription factor of bHLH domains.OsPTF1 in paddy rice most early in being cloned, and its is right
P deficiency shows tolerance, and in the transgenic paddy rice for being overexpressed OsPTF1, the efficiency that phosphorus is utilized is significantly improved.In phosphorus
Under the conditions of the water planting of shortage, the tillering ability of transfer-gen plant, root biomass, plant phosphorus content it is all higher than WT lines
30% or so.In water planting and field experiment, the tiller number of the transfer-gen plant under reduced phosphorus levels, spike weight and phosphorus content are also same
Sample is higher than wild type by about 20% or so.The research such as Zhang Juren is again showed that, ZmPTF1 is overexpressed in corn and is conducive to plant
Root development, the increase of biomass, the enhancing of the tillering ability of plant and seed become big.By biomass experiment and efficiently
The liquid-phase chromatographic analysis component of soluble sugar, it has also been found that the carbon metablism approach of corn is by ZmPTF1 expressions and phosphorus concentration
Influence.Fructose-1,6-diphosphonic acid salt enzyme and sucrose phosphate synthase during overexpression ZmPTF1 increase Sucrose synthesis is in leaf
Expression in piece.On the contrary, the expression of the gene that is related in sucrose decomposition metabolic process in root can be reduced by being overexpressed ZmPTF1.Cause
And, ZmPTF1 is by regulating and controlling fructose-1,6-diphosphate enzyme, sucrose phosphate synthase (SPS1), invertase (Ivr1 and Ivr2b)
And the expression in the case of hypophosphate such as sucrose synthase (SUSl, SUS2 and ShlB), match somebody with somebody so as to take part in sugar.Gao Jianrui
Corn inbred line A318 normally and in Spike sprouting seed embryo is carried out Deng using corn Affymetrix expression profiles of gene chip
Analysis of gene differential expression, as a result shows, ZmPTF1 genes lower expression in the maize of Spike sprouting.However, having in wheat
The correlative study for closing PTF1 genes has not been reported.
With developing rapidly for Protocols in Molecular Biology, molecular marker assisted selection (MAS) is widely used in molecule and educated
In kind, because it is not influenceed by environment and breeding generation, early generation selection can be carried out and predicted, wheat breeding is substantially reduced
The time limit.Functional label is the mark that a class is developed based on gene particular sequence, is isolated with target gene, substantially increases selection
Accuracy.Therefore the exploitation of functional label provides foundation for wheat molecule aid mark breeding.
Because wheat is allohexaploid, its genome comparison is complicated, and it is less that the functional gene relevant with grain weight is identified.
The functional gene cloned is predominantly located at 2A, and the former is wide and grain weight the major gene resistance of control grain of the homologous clone from paddy rice
TaGW2, the latter is the cell membrane invertase gene TaCWI related to Grain Weight in Common Wheat.Therefore, seek a kind of based on wheat berry heavy phase
The utility function mark of correlation gene sequence, will be to provide support using the Molecular tools new varieties that select and breed high yielding wheat.
The content of the invention
In order to overcome above-mentioned technological deficiency, it is used for the high mass of 1000 kernel wheat of seed selection it is an object of the present invention to provide a pair
The functional label of kind, to aid in improving yield of wheat molecular breeding.
To achieve the above object, the present invention is adopted the following technical scheme that：
The preferred aspect of the present invention, which provides a pair, is used for the functional label of the high mass of 1000 kernel wheat breed of seed selection, the mark
Note is located on wheat 7B chromosomes, and its nucleotide sequence is as shown in the SEQ ID NO.1 and SEQ ID NO.2 in sequence table.
Another preferred aspect of the present invention provides a kind of method for obtaining above-mentioned functions mark, comprises the following steps：
1) est sequence of wheat is searched on NCBI, electronic cloning includes a part of 5 ' UTR of UTR and 3 ' TaPTF1 gene orders；
2) using the sequence of the Electronic Assemblies as template, PCR amplifications are obtained including ORF in wheat cDNA
2012bp, performing PCR amplification is entered in Wheat volatiles DNA, the total length of TaPTF1-B genes is obtained；
3) arranged with this genome sequence obtained as template, a pair of molecular labelings with polymorphism of design.
Further, above-mentioned steps 2) in wheat cDNA PCR amplifications obtain the nucleic acid of 2012bp including ORF
Sequence is as shown in the SEQ ID NO.4 in sequence table.
Further, above-mentioned steps 2) in the nucleotide sequence of TaPTF1-B genes that obtains be such as the SEQ in sequence table
Shown in ID NO.3.
Present invention also offers application of the above-mentioned functional label in wheat breeding.
Whether a preferred aspect of the invention, above-mentioned application is to judge plant containing increase mass of 1000 kernel effect using it
Gene loci, concretely comprise the following steps：
The leaf DNA of target plant is expanded using it, electrophoresis detection amplified fragments are to detect whether as with higher thousand
The kind of weight；
Judgment criteria is：Kind with higher mass of 1000 kernel amplifies the 438bp as shown in SEQ ID NO.5 in sequence table
Target stripe, the kind with relatively low mass of 1000 kernel amplifies the target bar of the 422bp as shown in SEQ ID NO.6 in sequence table
Band.
The present invention is by having cloned a Partial Fragment for controlling grain weight major gene resistance：TaPTF1-B, and develop a pair
Functional label based on this specific gene order.The site is the site for the presence major gene resistance that forefathers had not reported.
Because the functional label is designed based on this specific gene order, isolated with the gene, relatively general SSR points
Son mark, the Objective and specific aim for being favorably improved breeding selection substantially increases the accuracy of selection, can be applied to wheat
Breeding field.Plus other extraneous factors are not influenceed by environment etc., wheat breed can be screened in mesoderm growing early stage, save production
Cost, accelerates breeding process.
Brief description of the drawings
Fig. 1 is techniqueflow chart of the invention；
The electrophoresis result figure of the wheat for 12 different cultivars that Fig. 2 expands for the functional label of the present invention；Wherein 1 is molecule
Label；2 be the amplification of capital 411；3 be the red amplification of awns spring 21；4 be the amplification of tobacco grower 15；5 be that new wheat 19 expands knot
Really；6 be No. 6 amplifications of space；7 be the peace amplification of agriculture 1116；8 be peace agriculture 0942-13 amplifications；9 be that new wheat 13 is expanded
Increase result；10 be the continuous amplification of wheat 37；11 be Bai Huomai amplifications；12 be the middle amplification of wheat 1139；13 be 02Y151
Amplification；And
Fig. 3 is to carry out chromosome mapping figure to target gene using the system of China spring-nullisomic four；Wherein, swimming lane 1 is molecule
Label；Swimming lane 2 is the amplification of capital 411；Swimming lane 3 is the red amplification of awns spring 21；Swimming lane 4 is that China spring (CS) expands knot
Really；Swimming lane 5-10 is respectively CS N7A-T7D, CS N7A-T7B, CS N7B-T7A, CS N7B-T7D, CS N7D-T7A, CS
N7D-T7B amplification.
Embodiment
Following examples are used to further illustrate the present invention, but should not be construed as limiting the invention.Without departing substantially from this
On the premise of spirit and essence, modification or replacement made for the present invention belong to scope of the invention.
Sequence (Genebank of the invention by using the TaPTF1 genes on NCBI：DQ979392.1), on NCBI
The est sequence of wheat is searched for, electronic cloning includes a part of 5 ' UTR of UTR and 3 ' TaPTF1 gene orders.With the electronics group
The sequence of dress is template, and PCR amplifications are obtained including 2012bp (the SEQ ID in sequence table including ORF in wheat cDNA
NO.4), performing PCR amplification is entered in Wheat volatiles DNA, the total length 3097bp (SEQ in sequence table of TaPTF1-B genes are obtained
ID NO.4).In addition, arranged with this genome sequence obtained as template, a pair using the Software for Design of Primer Premier 5.0
There is the Indel of polymorphism between in Beijing 411 and red awns spring 21, it is analyzed in recombinant inbred lines (RILs) and natural population
Effect to phenotype, is eventually developed the functional label being closely related with Grain Weight in Common Wheat for a pair, the deoxyribose of the functional label
Nucleotides sequence is classified as:
Upstream primer sequence：5 ' GAGTACACGGGCATTCTA 3 ' (SEQ ID NO.1 in such as sequence table)
Downstream primer sequence：5 ' TTCCCTGTTGGTTGTCATTC 3 ' (SEQ ID NO.2 in such as sequence table)
By the chromosome mapping of wheat aneuploid material, the gene is located on wheat 7B chromosomes.The present invention
Obtain above-mentioned functions mark particular technique flow such as Fig. 1.
Show the present invention with following specific embodiment：
Embodiment 1：The clone of wheat TaPTF1-B genes
What the method for being obtained by electronic cloning combination Standard PCR of TaPTF1-B genes was carried out.According to small on NCBI
Sequence (the Genebank of wheat TaPTF1 genes：DQ979392.1 wheat ESTs databases) are searched for, the est sequence that screening is obtained
(CJ904603, CJ916459.1, FF580499.1) carries out electronic splicing, obtains TaPTF1 gene orders.With the splicing
CDNA sequence is template, using Primer Premier5.0 software Design primers to R1, R2, R3, G1, for TaPTF1 genes
CDNA is obtained, and primer pair G1 is also used for the acquisition of genomic DNA simultaneously, and specific nucleotide sequence is shown in Table 1.PCR is reacted respectively with small
Wheat cDNA and genomic DNA are template, are carried out on BIO-RAD My Cycler 1.0PCR instrument, 10ul reaction systems, specifically
Reaction system and PCR amplification conditions it is as follows：
After PCR amplifications, then 1.5% agarose gel electrophoresis carries out gel extraction under uviol lamp, and last TA clones survey
Sequence, sequencing is given birth to work sequencing company by Shanghai and completed.
Table 1 is used for the primer sequence for expanding TaPTF1-B genes on wheat 7B chromosomes
Embodiment 2：The exploitation of wheat TaPTF1-B gene functions mark
With the wheat TaPTF1-B gene orders (SEQ ID NO.3 in sequence table) that are obtained by PCR method for template, profit
With the Software for Design of Primer Premier 5.0 with polymorphism between a pair in Beijing 411 and 21 two kinds of red awns spring
Indel primers, and screened between the wheat breed of mass of 1000 kernel significant difference, in the weight built by capital 411 and red awns spring 21
Its effect to phenotype is analyzed in the natural population of group inbred line population (RILs) and 125 parts of germ plasm resource compositions.The primer sequence
Row are as follows：
Upstream primer sequence：5 ' GAGTACACGGGCATTCTA 3 ' (SEQ ID NO.1 in such as sequence table)
Downstream primer sequence：5 ' TTCCCTGTTGGTTGTCATTC 3 ' (SEQ ID NO.2 in such as sequence table)
Embodiment 3：Wheat TaPTF1-B genes difference equipotential type and the correlation of grain weight
First, the measure of wheat seed mass of 1000 kernel
1000 complete wheat seeds are taken at random, repeats, is weighed with millesimal balance, average value is designated as twice twice
Final mass of 1000 kernel value.
2nd, SDS-Tris saturated phenols method extracting wheat leaves genomic DNA
(1) wheat (capital 411 of 12 different cultivars is taken respectively；The red awns spring 21；Tobacco grower 15；New wheat 19；Space 6；An Nong
1116；Pacify agriculture 0942-13；New wheat 13；Continuous wheat 37；Bai Huomai；Middle wheat 1139；02Y151；) the fresh young leaflet tablets of 2g, liquid nitrogen
Grind to form and be placed in after fine powder in 2ml sterile centrifugation tubes.
(2) add 1.2ml DNA Extraction buffers (200mM Tris-cl, pH8.0,250mM NaCl, 25mM EDTA,
PH8.0,0.5%SDS, 2% β-ME are well mixed, and β-ME are fresh before use to be added).
(3) 55 DEG C of water-bath 45min, during which intermittent oscillation is fully to extract.
(4) at room temperature, 12000rpm, 10min.
(5) transfer supernatant into new 2ml sterile centrifugation tubes, add precooling isometric Tris saturated phenols/chloroform it is different/
(volume ratio is 25 to amylalcohol：24：1) 15min, is mixed in reverse on ice, during which intermittent oscillation.
(6) at room temperature, 12000rpm, 10min.
(7) transfer supernatant is into new 2ml sterile centrifugation tubes, repeat step (5), (6), fully to remove deproteinized.
(8) transfer supernatant adds 0.6 times of isopropanol (300 μ l) and 1/10 times of body into new 1.5ml sterile centrifugation tubes
Product (50 μ l) NaAc (pH5.2), 17min is stood after being sufficiently mixed mixing on ice.
(9) there is white flock DNA precipitations, 4 DEG C, 10000rpm, 10min.
(10) supernatant is abandoned, 70% ethanol for adding precooling is rinsed 2 times, is then rinsed 1 time with absolute ethyl alcohol, room temperature is dried,
Add 100 μ l wherein 1 × TE buffer solutions (or distilled water) containing 2 μ l 10mg/ml RNase enzymes and stay overnight dissolving, 10 times of dilutions are standby
With.
(11) 12 kinds of Wheat volatiles DNA of Detection and Extraction quality and concentration is distinguished with 1% agarose.
3rd, the amplification of target product
PCR reaction systems：
PCR amplification conditions are referring to embodiment 1.
After the completion of PCR, plus denaturation Dual-indicator (DNA loading buffer) 95 DEG C of denaturation 10min in PCR instrument, so
After be immediately placed on ice, after room temperature is down to, the electrophoresis on 6% denaturing polyacrylamide gel, then silver staining develop the color, as a result
See Fig. 2.It can be seen in fig. 2 that the band of the wheat amplification of 12 kinds of different cultivars concentrates on two positions, with higher mass of 1000 kernel
Kind amplifies 438bp (SEQ IDNO.5 in such as sequence table) target stripe, and the kind with relatively low mass of 1000 kernel is amplified
422bp (SEQ IDNO.6 in sequence table) target stripe.
4th, the positioning of the body of TaPTF1-B genes China spring nullisomic four
Chromosome mapping is carried out to the gene using wheat nulli-tetrasomes material, scarce 7A is marked at this respectively with 7B and
The nullisomic that the Nullisomic wheat of 7D replacements, scarce 7B are substituted with 7A and 7B respectively with the 7A and 7D Nullisomic wheats substituted and scarce 7D respectively
Detected in wheat, find to can't detect the gene in the Nullisomic wheat that 7B is substituted with 7A and 7D respectively is lacked, as a result the base
Because being located on wheat 7B chromosomes, it is illustrated in fig. 3 shown below.
5th, in different mass of 1000 kernel kinds genotyping and the correlation with mass of 1000 kernel
Utilize the red recombinant inbred lines of awns spring 21 in capital 411/ (the mass of 1000 kernel 37.02g of capital 411；The red mass of 1000 kernel of awns spring 21
19.14g, totally 174 familys) and 125 parts of germ plasm resource compositions natural population, the mass of 1000 kernel of different genotype is carried out respectively
The correlation analysis of the allelic variation and mass of 1000 kernel of variance analysis and TaPTF1-B genes, the results are shown in Table 2：
Mass of 1000 kernel significance difference analysis in the different groups of table 2 between two kinds of genotype
Note：* is P<0.01, reach the pole level of signifiance；A genotype is 438bp banding patterns；1 B gene type is 422bp banding patterns
As seen from Table 2, the mass of 1000 kernel of A genotype (438bp) is significantly higher than 1 B gene type (422bp) mass of 1000 kernel.Can
See, this pair of functional label obtained by the present invention has reached extremely notable water in Liang Ge colonies with the correlation of mass of 1000 kernel
It is flat.
Claims (5)
1. a pair of functional labels being closely related with Grain Weight in Common Wheat, it is characterised in that the mark is located on wheat 7B chromosomes, with
The nucleotide sequence for the functional label that higher thousand grain weight properties are closely related is and relatively low as shown in the SEQ ID NO.5 in sequence table
The nucleotide sequence for the functional label that thousand grain weight properties are closely related is as shown in the SEQ ID NO.6 in sequence table.
2. the primer pair for expanding functional label described in claim 1, it is characterised in that its nucleotide sequence is as in sequence table
Shown in SEQ ID NO.1 and 2.
3. a kind of method for obtaining primer pair described in claim 2, it is characterised in that comprise the following steps：1) searched on NCBI
The est sequence of wheat, electronic cloning includes a part of 5 ' UTR of UTR and 3 ' TaPTF1 gene orders；
2) using the sequence of the Electronic Assemblies as template, PCR amplifications obtain the 2012bp including ORF in wheat cDNA,
Enter performing PCR amplification in Wheat volatiles DNA, obtain the total length of TaPTF1-B genes；
3) arranged with this genome sequence obtained as template, a pair of functional labels for expanding and Grain Weight in Common Wheat is closely related of design
Primer；
Step 2) in wheat cDNA PCR amplifications obtain the nucleotide sequence including the 2012bp including ORF in such as sequence table
SEQ ID NO.4 shown in；
Step 2) in obtain TaPTF1-B genes nucleotide sequence be as shown in the SEQ ID NO.3 in sequence table.
4. application of the functional label described in claim 1 in wheat breeding.
5. application according to claim 4, it is characterised in that judge plant whether containing increase mass of 1000 kernel effect using it
Gene loci, concretely comprise the following steps：
The leaf DNA of the primer pair amplifies target plant of above-mentioned functions mark, electrophoresis are expanded using being used for described in claim 2
Amplified fragments are detected to detect whether as the kind with higher mass of 1000 kernel；
Judgment criteria is：Kind with higher mass of 1000 kernel amplifies the mesh of the 438bp as shown in SEQ ID NO.5 in sequence table
Band is marked, the kind with relatively low mass of 1000 kernel amplifies the target stripe of the 422bp as shown in SEQ ID NO.6 in sequence table.
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| Title |
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| Developing Rice with High Yield under Phosphorus Deficiency: Pup1 Sequence to Application;Joong Hyoun Chin;《Plant Physiology》;20110731;第156卷;1202-1216 * |
| Development and application of gene-based markers for the major rice QTL Phosphorus uptake 1;Chin JH;《Theor Appl Genet》;20101231;第120卷;1073-1086 * |
| 农杆菌介导磷高效转录因子 OsPTF1 转化大豆研究;梁慧珍;《分子植物育种》;20131231;第11卷(第6期);688-693 * |
| 小麦磷高效基因TaPHR1的分子标记辅助选择与功能分析;张超;《中国优秀硕士学位论文全文数据库 农业科技辑》;20141215(第12期);D047-57 * |
| 玉米低磷响应基因ZmPAP18的表达特征与序列变异分析;苏顺宗;《分子植物育种》;20130621;第11卷;全文 * |
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