CN105524993B - The molecular labeling HRM1 of barley grain length gene Lkl1 a kind of and its application - Google Patents
The molecular labeling HRM1 of barley grain length gene Lkl1 a kind of and its application Download PDFInfo
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Abstract
The present invention relates to barley field of molecular breeding, molecular labeling HRM1 and its application of a kind of barley grain length gene Lkl1 are specifically disclosed.Molecular labeling HRM1 provided by the invention with barley grain length gene Lkl1 close linkages, nucleotide sequence is as shown in SEQ ID No.1.Detection and analysis show that the molecular labeling can accurately track the long grain length gene of the barley, predict the grain length characteristic of barley, and then facilitate carry out Molecular design breeding.The invention also discloses a kind of methods of the molecular labeling of identification barley grain length gene Lkl1, it can reinforce the accuracy of grain length prediction using method provided by the present invention, it is used for breeding quickly to filter out the barley variety with the QTL for increasing grain length or strain, the selection and breeding process of barley high yield kind can be greatly speeded up.
Description
Technical field
The present invention relates to barley field of molecular breeding, specifically, being related to a kind of molecule mark of barley grain length gene Lkl1
Remember HRM1 and its application.
Background technology
Barley is most ancient in the world one of cereal crops, is served many purposes:Collect grain, feed, beer raw material with
And medicine, health food are.For its yield, barley is the fifth-largest crops in the whole world;It is the whole world by its cultivated area
It plants and shows that there are about 60,000,000 hm for the long-term sown area of barley at present with the related data of the fourth-largest cereal crops of production2, year is total
Yield is about 1.5 hundred million tons, is only second to corn, rice, wheat.Since nearly half a century, the mankind are in the production of barley and demand
The trend of sustainable growth.Statistical data in 2008 shows that barley year aggregate demand is 0.274 hundred million tons, this is in cereal crops
Demand be only second to corn and occupy second, therefore the selection and breeding of high yield barley variety are always the emphasis of breeder's concern.Meanwhile
Model plant one of of the barley as genetics research, is constantly subjected to the great attention of geneticist and molecular biologist.Point
Son label plays important role in the hereditary basis for understanding barley important economical trait, and the application of Molecular Markers Linkage Map is
Plant Genome structural analysis and compares and provide powerful.
Barley production character is the quantitative character of complexity, by multiple quantitative character gene locus therefor (Quantitative
Trait locus, QTL) control, the characteristic big, selection difficulty is high low, affected by environment with genetic force, so in selection and breeding
Cheng Zhong, the problem that traditional breeding method existence time is long, consumption is big, of high cost, achievement is small.Molecular mark is one
Kind carries out the effective ways of breeding using molecular labeling associated with specific trait as supplementary means, has not by environment item
Part limitation, the entire growth period advantages such as detectable, efficiency of selection height in development of plants.
Barley grain size is the important determinant of grain weight, and grain length directly affects barley grain size, and grain weight is
One of 3 constituent elements of barley production, therefore the long size of barley corn is an important factor for influencing yield.Therefore, barley is illustrated
The hereditary basis of the size of seed length and molecular mechanism are conducive to improve the yield and quality of barley simultaneously.
Barley grain size genetic research macro-progress is relatively slow, works mainly around seed gene outside Current Domestic
The Molecular mapping and its hereditary effect of QTL is carried out.Earliest Kjaer, G.Backes etc. are in nineteen ninety-five by barley grain length gene
It has been located in different chromosomes, has there is in recent years and successively research to do Position Research, such as Cassandra to barley grain length site
K has been located in the site by barley control grain length in 2013 on 6 chromosomes other than 4H.So far, it has reported
Grain length related gene QTL be located on 7 chromosomes, and have not been reported and more in-depth study done to complete essence to related locus
Fine positioning works.
Wild Barley AWCS276 has grain length big relative to barley variety Baudin, and grain is wide small, has good disease-resistant degeneration-resistant
Property etc. characteristics (Chen Guang steps on the heredity of barleys anti-reaping hook bacterial type base rot disease and its relationship [D] the Sichuan agricultures between Main Agronomic Characters
Sparetime university is learned, and 2013.).Meanwhile the grain length of barley variety Fleet is again smaller than Wild Barley AWCS276.Therefore, Wild Barley is utilized
AWCS276 and Fleet structure genetic researches group, further verifies the grain length characteristic of Wild Barley AWCS276, location control grain
Long gene finds the molecular labeling of close linkage, promotes the map based cloning of barley grain length gene, while being barley corn long material
Initiative and high yield breeding provide new gene resource, further utilize molecular marker assisted selection, will enhance the standard of grain length prediction
True property improves SOYBEAN IN HIGH-YIELD BREEDING efficiency, speeds up to the target for increasing barley per unit area yield.
Molecular marker assisted selection is selected independent of phenotype, i.e., not by environmental condition, Interaction among genes, genotype
With the influence of many factors such as environment interaction, but directly genotype is selected, thus breeding efficiency can be greatly improved.It is high
A kind of SNP and mutation that melting curve (High Resolution Melting, the HRM) technology of resolution is risen in the world in recent years
Research tool.This detection method has easy to operate, specificity is good, high-throughput, quick, testing cost is low, result is accurate etc.
Advantage, and realize real stopped pipe operation and receive universal concern.Therefore, it filters out and grain length gene close linkage
, and suitable for the molecular labeling of quantitative fluorescent PCR platform HRM technologies, barley grain length gene can not only be selected, effectively
Regulate and control barley grain length, while improving choosing flux, speed and accuracy, solves the technology bottle of large-scale promotion application
Neck improves Barley Breeding colony quality to scale and yield is of great significance.
Invention content
The purpose of the present invention is to provide the molecular labelings with barley grain length gene Lkl1 close linkages.
Another object of the present invention is to provide the fluorescence quantification PCR primers of the molecular labeling.
Third object of the present invention is to provide answering for the molecular labeling of above-mentioned barley grain length gene Lkl1 close linkages
With.
The purpose of the present invention is what is be achieved through the following technical solutions:
The new grain length gene Lkl1 of the present invention comes from Wild Barley AWCS276, which is located at barley 3H chromosomes.Lkl1
Barley grain length can be significantly improved, LOD value is more than 5.07, explains about 29.1% phenotypic variation.
The nucleotide sequence of molecular labeling HRM1 provided by the present invention is as shown in SEQ ID No.1.
Molecular labeling of the present invention and barley grain length gene Lkl1 common locations are in barley 3H chromosomes, and between Lkl1
Genetic distance be 0.1cM.
The present invention also provides a kind of methods of the molecular labeling of identification barley grain length gene Lkl1, with Plant samples to be measured
Genomic DNA as template, carry out fluorescent quantitative PCR with fluorescence quantification PCR primer, utilize amplification carry out gene
Type parting will be accredited as the plant containing barley grain length gene Lkl1 with AWCS276 points for same type of plant.
Optionally, it the described method comprises the following steps:
(1) using the genomic DNA of Plant samples to be measured as template, quantitative fluorescent PCR is carried out with fluorescence quantification PCR primer
Amplification:
(2) fluorescent quantitative PCR reaction system:Super 5 μ L of mixed liquor of SsoFast EvaGreen, sense primer and downstream
It is 10 μ L that each 300ng of primer, template DNA 100ng, Dnaes/RNase-free deionized water, which add to total amount,;
(3) quantitative fluorescent PCR program:95 DEG C of pre-degeneration 5min;94 DEG C of denaturation 15s, 53 DEG C of annealing 30s, totally 50 recycle;
Ranging from 65~95 DEG C of melting curve, number of every 0.2 DEG C of reading, time 10s;
(4) Genotyping is carried out using the result that step (3) obtains.
Wherein, the DNA of the plant to be identified is derived from the blade in barley sample tri-leaf period.
The present invention also provides the fluorescence quantification PCR primers of the molecular labeling, wherein upstream primer sequence such as SEQ ID
Shown in No.2, downstream primer sequence is as shown in SEQ ID No.3.
The present invention also provides applications of the molecular labeling HRM1 in Barley Breeding.
The present invention also provides applications of the molecular labeling HRM1 in prepare transgenosis plant, wherein the gene
For grain length gene Lkl1.
The present invention also provides application of the well-behaved invention the method in Barley Breeding improvement.
We additionally provide application of the fluorescence quantification PCR primer in molecular labeling assists barley SOYBEAN IN HIGH-YIELD BREEDING.
In the present invention, barley grain length gene Lkl1 and molecular labeling HRM1 are prepared by the following:
(1) using long grain length Wild Barley AWCS276 as female parent, using barley Baudin as paternal hybrid, hybrid is obtained
F1, F1 generation individual plant selfing obtain F2, obtain the F8 containing 130 strains for RIL groups using single seed descent, constitute genetic mapping
Group.
(2) DNA for using each strain of the CTAB methods extraction genetic mapping group, with DArT chip technologies, with parent
The DNA of AWCS276 and Baudin is masterplate, carries out Genotyping, obtains the genotype data of the RIL groups.Parent
The banding pattern of AWCS276 is denoted as A, and the banding pattern of parent Baudin is denoted as B.F8 groups strain banding pattern is denoted as A from AWCS276's,
It is denoted as B from Baudin.
(3) in F8 groups room described in barley full ripe stage species test identification seed grain length.
(4) JoinMap4.0 mapping softwares is utilized to connect the RIL groups genotype data structure barley molecule of acquisition
Collection of illustrative plates is locked, optimal reference numerals and flag sequence are found, determines the linkage group subsequently used.Utilize the area of software MapQTL 6.0
Between make graph model (Interval Mapping), and combine F8 group's grain length phenotypic datas that grain length gene Lkl1 is positioned at 3H dyes
On colour solid in the section of a 0.4cM.
(5) the DArT probe sequences in the section are obtained, by the gene order-checking data in barley Morex, obtain mesh
The more sequence informations of section are marked, electronics extends the corresponding sequence in DArT probe sequences both ends, and carries out sequence analysis, preliminary to sieve
It is selected to the target area of follow-up fluorescence quantification PCR primer design.
(6) fluorescence quantification PCR primer is designed, for subsequently screening.It is glimmering using 7.0 Software for Design of Beacon Designer
Fluorescent Quantitative PCR primer 10 is to (being shown in Table 1).Fluorescence quantification PCR primer design standard:18~25bp of primer length, amplified production are long
65~100bp is spent, 60 DEG C of annealing temperature, for G/C content between 40%~60%, SNP difference locus products annealing temperature differences are big
In 0.2 DEG C.
1 10 pairs of HRM primer sequences of table and expanding fragment length
(7) high-resolution fusion curve (HRM) is analyzed
A) between parent polymorphic molecular marker screening:10 pairs of primers for choosing above-mentioned design, with parent AWCS276 and
The DNA of Baudin is masterplate, carries out PCR amplification, obtains 1 pair of quantitative fluorescent PCR molecular labeling primer to work well, name altogether
For HRM1-F/R (nucleotide sequence is as shown in SEQ ID No.2 and 3).Amplified production is with polymorphism molecular labeling
HRM1, nucleotide sequence is as shown in SEQ ID No.1
B) the HRM analyses of F8 groups:With above-mentioned steps obtain the molecular labeling HRM1 with polymorphism PCR primer,
The DNA of parent AWCS276, Baudin and F8 groups plant is expanded, genotype identification is carried out, obtains molecular marker data.Parent
The type of AWCS276 is denoted as A, the long 92bp of amplified fragments size, and single base difference site is C.The type of parent Baudin is denoted as
B, the long 92bp of amplified fragments, single base difference site are T.F8 groups strain type origin is denoted as A in AWCS276, derives from
Baudin's is denoted as B.
Advantageous effect:
Present invention firstly discloses the grain length gene Lkl1 from barley AWCS276, are located at barley 3H chromosomes, significantly increase
It is long to increase wheat.The gene has higher utility value in barley production (regulation and control grain weight) breeding.
Present invention firstly discloses accurately detect the new grain length gene Lkl1 of barley AWCS276 based on quantitative fluorescent PCR platform
Molecular labeling HRM1, and be codominant marker, detection precise and high efficiency, amplification facilitate stabilization.
Molecular labeling HRM1 disclosed by the invention and grain length gene Lkl1 are extremely significantly correlated, and presentation isolates marker characteristic,
Accuracy for molecular marker assisted selection is high, improves the selection identification effect for the long grain length kind of barley for adapting to varying environment
Rate, and success rate is high.
Description of the drawings
Fig. 1 be positions of the barley AWCS276 long grain length gene Lkl1 on 3H chromosomes and with molecular labeling of the present invention
Genetic linkage map between HRM1.
Fig. 2 is that the F7RIL groups offspring of AWCS276 × Baudin makes the knot of genotyping of fluorescence quantification PCR primer
Fruit.
Fig. 3 is that the F7RIL groups offspring of AWCS276 × Fleet makes the knot of genotyping of fluorescence quantification PCR primer
Fruit.
Specific implementation mode
It below will the present invention is described in detail by specific implementation mode.It will be appreciated that following embodiment is given
Go out merely to play the purpose of explanation, is not used to limit the scope of the present invention.Those skilled in the art exists
In the case of without departing substantially from spirit of the invention and spirit, the present invention can be carry out various modifications and be replaced.
Embodiment 1
The present embodiment is used to illustrate the preparation method of barley grain length gene Lkl1 and molecular labeling HRM1:
(1) using long grain length Wild Barley AWCS276 as female parent, using barley Baudin as paternal hybrid, hybrid is obtained
F1, F1 generation individual plant selfing obtain F2, and the F8 containing 130 strains is obtained for RIL Canopy structure genetic mappings using single seed descent
Group.
(2) DNA for using each strain of the CTAB methods extraction genetic mapping group, with DArT chip technologies, with parent
The DNA of AWCS276 and Baudin is masterplate, carries out Genotyping, obtains the genotype data of the RIL groups.AWCS276's
Banding pattern is denoted as A, and the banding pattern of parent Baudin is denoted as B.F8 groups strain banding pattern is denoted as A from AWCS276's, derives from
Baudin's is denoted as B, heterozygous H.
(3) indoor species test is carried out after barley complete ripeness measures seed grain length.
(4) JoinMap4.0 mapping softwares is utilized to connect the RIL groups genotype data structure barley molecule of acquisition
Collection of illustrative plates is locked, optimal reference numerals and flag sequence are found, determines the linkage group subsequently used.Utilize the area of software MapQTL 6.0
Between make graph model (Interval Mapping), and combine F8 group's grain length phenotypic datas that grain length gene Lkl1 is positioned at 3H dyes
On colour solid in the section of a 0.4cM.
(5) the DArT probe sequences in the section are obtained, by the gene order-checking data in barley Morex, obtain mesh
The more sequence informations of section are marked, electronics extends the corresponding sequence in DArT probe sequences both ends, and carries out sequence analysis, preliminary to sieve
It is selected to the target area of follow-up fluorescence quantification PCR primer design.
(6) fluorescence quantification PCR primer is designed, for subsequently screening.It is glimmering using 7.0 Software for Design of Beacon Designer
Fluorescent Quantitative PCR primer 10 is to (as shown in table 1).Fluorescence quantification PCR primer design standard:18~25bp of primer length, amplification production
Object 65~100bp of length, 60 DEG C of annealing temperature, for G/C content between 40%~60%, SNP difference locus products annealing temperatures are poor
It is different to be more than 0.2 DEG C.
(7) high-resolution fusion curve (HRM) is analyzed
A) between parent polymorphic molecular marker screening:10 pairs of primers for choosing above-mentioned design, with parent AWCS276 and
The DNA of Baudin is masterplate, carries out PCR amplification, obtains 1 pair of quantitative fluorescent PCR molecular labeling to work well altogether.
B) the HRM analyses of F8 groups:With the molecular labeling HRM1 with polymorphism that above-mentioned steps obtain, parent is expanded
The DNA of AWCS276, Baudin and F8 group plant carries out genotype identification, obtains molecular marker data, as a result such as Fig. 2.Parent
The type of this AWCS276 is denoted as A, the long 92bp of amplified fragments size, and single base difference site is ' C '.The type of parent Baudin
It is denoted as B, the long 92bp of amplified fragments, single base difference site is ' T '.F8 groups strain type origin is denoted as A in AWCS276,
It is denoted as B from Baudin.
C) the Interval mapping model (Interval Mapping) of software MapQTL 6.0 is utilized, and combines F8 groups grain length
Phenotypic data finds that molecular labeling HRM1 is located in the section of grain length gene Lkl10.4cM, and the results are shown in Figure 1, can by figure
Know, grain length gene Lkl1 is located in label 3258132 | F | 0-44:C>G-44:C>G and 3258132 | F | 0--44:C>Between G
In the section of 0.4cM, and molecular labeling HRM1 distance DArT labels 3258132 | F | 0-44:C>G-44:C>G has 0.2cM, Lkl1
Gene and molecular labeling HRM1 distance 0.1cM are in close linkage.
Embodiment 2
The extraction of 1.1DNA
Test material chooses AWCS276, Baudin, and wherein Baudin is short grain length kind, and AWCS276 is long grain length product
Kind.The leaf DNA in barley sample tri-leaf period is extracted using CTAB methods.
The screening of the primer of 1.2 detection barley grain length gene Lkl1
1.2.1 design of primers
(1) the DArT probe sequences in the section are obtained, by the gene order-checking data in barley Morex, obtain mesh
The more sequence informations of section are marked, electronics extends the corresponding sequence in DArT probe sequences both ends, and carries out sequence analysis, preliminary to sieve
It is selected to the target area of follow-up fluorescence quantification PCR primer design.
(2) fluorescence quantification PCR primer is designed, for subsequently screening.It is glimmering using 7.0 Software for Design of Beacon Designer
Fluorescent Quantitative PCR primer 10 is to (being shown in Table 1).Fluorescence quantification PCR primer design standard:18~25bp of primer length, amplified production are long
65~100bp is spent, 60 DEG C of annealing temperature, for G/C content between 40%~60%, SNP difference locus products annealing temperature differences are big
In 0.2 DEG C.
1.2.2 the otherness between quantitative fluorescent PCR platform test primer and its parent
(1) leaf DNA in AWCS276, Baudin tri-leaf period is extracted.
(2) using the DNA obtained by step (1) as template, HRM1-F (sequence shown in SEQ ID No.2) of the present invention
It is that primer carries out fluorescent quantitative PCR with HRM1-R (sequence shown in SEQ ID No.3).
(3) fluorescent quantitative PCR reaction system:Super 5 μ L of mixed liquor of SsoFast EvaGreen, upstream and downstream primer are each
It is 10 μ L that 300ng, template DNA 100ng, Dnaes/RNase-free deionized water, which add to total amount,.
(4) quantitative fluorescent PCR program:95 DEG C of pre-degeneration 5min;94 DEG C of denaturation 15s, 53 DEG C of annealing 30s, totally 50 recycle;
Ranging from 65~95 DEG C of melting curve, number of every 0.2 DEG C of reading, time 10s.
(5) with step (4) acquired results file, BioRadPrecisionMelt softwares is imported and carry out Genotyping, as a result
Such as Fig. 2.During drawing melting curve, according to base-pair A=T, the temperature of C ≡ G unwindings is different,
Sample is divided into two types by BioRadPrecisionMelt.AWCS276 types are denoted as A, and Baudin types are denoted as B.
The applicability of primer sequence HRM1-F/R during 1.3 crowd surveillances
(1) be female parent with AWCS276, Fleet is that paternal hybrid obtains F1, and F1 selfings obtain F2, by single seed descent plus
In generation, verifies group to F7 for RIL.Extract the leaf DNA in each strain tri-leaf period in group.
(2) using the DNA obtained by step (1) as template, quantitative fluorescent PCR expansion is carried out using primer provided by the present invention
Increase, fluorescent dye is SsoFast EvaGreen.
(3) fluorescent quantitative PCR reaction system:The super mixed liquors of 5 μ L SsoFast EvaGreen, upstream and downstream primer are each
It is 10 μ L that 300ng, 100ng template DNA, Dnaes/RNase-free deionized waters, which add to total amount,.
(4) quantitative fluorescent PCR program:95 DEG C of pre-degeneration 5min;94 DEG C of denaturation 15s, 53 DEG C of annealing 30s, totally 50 recycle;
Ranging from 65~95 DEG C of melting curve, number of every 0.2 DEG C of reading, time 10s.
(5) with step (4) acquired results file, BioRadPrecisionMelt softwares is imported and carry out Genotyping, as a result
Such as Fig. 3.83 plants of strains of sampling observation at random, 46 plants of segments that can be amplified with AWCS276 same types, to contain long grain length gene
The plant of Lkl1, prediction strain plant grain length after maturation are larger.37 plants can amplify Type B piece identical with Baudin, Fleet
Section, for the plant without containing long grain length gene Lkl1, predicts that these plant grain length after maturation is smaller.
(6) species test measures the grain length of seed in barley full ripe stage rear chamber, the results are shown in Table 2, the molecule of long grain length gene Lkl1
Mark the result of HRM1 prediction genetic group tillering abilities.Plant identical with the long grain length gene Lkl1 types grain length that is averaged is
9.05mm is significantly higher than and the plant grain length of Baudin types (average 8.79mm).Actual result is consistent with expected results, explanation
The grain length gene Lkl1 of the present invention significantly increases grain length really;Simultaneously the present invention molecular labeling HRM1 can with
Tracking identification grain length gene Lkl1.
Table 2
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 (10)
1. a kind of molecular labeling HRM1 of barley grain length gene Lkl1, which is characterized in that the nucleotide sequence of the molecular labeling
As shown in SEQ ID No.1.
2. molecular labeling according to claim 1, which is characterized in that the molecular labeling is total with barley grain length gene Lkl1
Barley 3H chromosomes are positioned at, and the genetic distance between Lkl1 is 0.1cM.
3. molecular labeling according to claim 1 or 2, which is characterized in that barley grain length gene Lkl1 dramatically increases barley
Seed length, LOD value are more than 5.07.
4. a kind of method of the molecular labeling HRM1 of identification barley grain length gene Lkl1 described in claim 1, which is characterized in that
Using the genomic DNA of Plant samples to be measured as template, fluorescent quantitative PCR is carried out with fluorescence quantification PCR primer, utilizes expansion
Increase result and carry out genotyping, will divide with Wild Barley Awcs276 and be accredited as containing barley grain length for same type of plant
The plant of gene Lkl1;
The fluorescence quantification PCR primer includes:
The sense primer as shown in SEQ ID No.2, and the downstream primer as shown in SEQ ID No.3.
5. according to the method described in claim 4, it is characterized by comprising the following steps:
(1) using the genomic DNA of Plant samples to be measured as template, quantitative fluorescent PCR expansion is carried out with fluorescence quantification PCR primer
Increase;
(2) fluorescent quantitative PCR reaction system:Super 5 μ L of mixed liquor of SsoFast EvaGreen, sense primer and downstream primer
It is 10 μ L that each 300ng, template DNA 100ng, Dnaes/RNase-free deionized water, which add to total amount,;
(3) quantitative fluorescent PCR program:95 DEG C of pre-degeneration 5min;94 DEG C of denaturation 15s, 53 DEG C of annealing 30s, totally 50 recycle;It melts
Curve ranges are 65~95 DEG C, number of every 0.2 DEG C of reading, time 10s;
(4) Genotyping is carried out using the result that step (3) obtains.
6. the fluorescence quantification PCR primer of any one of the claim 1-3 molecular labelings, which is characterized in that sense primer
Sequence is as shown in SEQ ID No.2, and downstream primer sequence is as shown in SEQ ID No.3.
7. applications of the molecular labeling HRM1 described in claim 1 in Barley Breeding.
8. applications of the molecular labeling HRM1 described in claim 1 in prepare transgenosis plant, wherein the gene is grain length
Gene Lkl1.
9. application of the method in Barley Breeding improvement described in claim 4 or 5.
10. application of the fluorescence quantification PCR primer in molecular labeling assists barley SOYBEAN IN HIGH-YIELD BREEDING described in claim 6.
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CN104278051A (en) * | 2013-07-09 | 2015-01-14 | 中国科学院上海生命科学研究院 | Regulation and control gene of awn, grain length and number of grain per ear and application thereof |
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WO2001049104A2 (en) * | 1999-12-30 | 2001-07-12 | Pioneer Hi-Bred International, Inc. | Mqm mapping using haplotyped putative qtl-alleles: a simple approach for mapping qtl's in plant breeding populations |
CN104278051A (en) * | 2013-07-09 | 2015-01-14 | 中国科学院上海生命科学研究院 | Regulation and control gene of awn, grain length and number of grain per ear and application thereof |
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