CN110241125A - Soybean 100-grain weight synergy gene and its molecular labeling and application - Google Patents
Soybean 100-grain weight synergy gene and its molecular labeling and application Download PDFInfo
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- CN110241125A CN110241125A CN201910702026.3A CN201910702026A CN110241125A CN 110241125 A CN110241125 A CN 110241125A CN 201910702026 A CN201910702026 A CN 201910702026A CN 110241125 A CN110241125 A CN 110241125A
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Abstract
The invention discloses a soybean 100-grain weight synergy gene and its molecular labeling and application, for the gene base sequence as shown in SEQ ID No.2, which has synergistic effect;The present invention identifies the gene GmSLK for being located at regulating and controlling soybean 100-grain weight on soybean Gm16 chromosome, and being overexpressed in arabidopsis for the gene can significantly improve its seed size.The label InDelSLK for identifying the gene is provided simultaneously.Important judgement value should be provided for soybean breeder selection labeled as identifying that its GmSLK is that synergy gene or leaky gene provide convenience in germ plasm resource and filial generation.
Description
Technical field
The invention belongs to breeding fields, and in particular to the excavation of the gene GmSLK of regulating and controlling soybean seed 100-grain weight and its answer
With.
Background technique
Soybean is the crop with Important Economic meaning, provides edible oil and protein etc. for human and animal.Now with
The continuous improvement of human life quality, it is also increasing to the demand of soybean, so improving the yield of soybean all the time all
It is the main target of domestic and international soybean breeder.However, important determinant of the 100-grain weight as soybean yields, is also arranged by breeder
For one of main breeding character.But soybean 100-grain weight character is the quantitative character by controlled by multiple genes, the shadow vulnerable to environment
It rings, this brings very big obstruction to researcher.
For soybean 100-grain weight character, many scholars at home and abroad are using different primary mapping populations and based on not isolabeling
Identification a large amount of QTL (Quantitative trait loci).But primary mapping population can make QTL position section
Excessive, lots of genes site separates simultaneously, causes Genetic Background Effects, increases the difficulty of main effect QTL finely positioning.Using secondary
Grade mapping population, such as answering for chromosome segment substitution line (chromosome segment substitution line, CSSL)
With having found desirable route to solve this problem.Ideally, chromosome segment substitution line genes of individuals group has donor
A small amount of segment of parental set of chromosome, genetic background height is consistent, undoubtedly carries out the ideal material of QTL positioning.Theoretically,
The character value of chromosome segment substitution line can be compared with the character value of recurrent parent, and Traits change is all because lead between two strains
The introgressed segments for entering strain have differences QTL and caused by, therefore, the chromosome segment substitution line for utilizing this homologous carries out
The accuracy of positioning can be improved in qtl analysis.
It, can be according to the positioning of chromosome segment substitution line in order to which QTL finely positioning and marker assisted selection is better achieved
As a result, the substitution line with important goal character detected is purposefully marked auxiliary backcrossing, secondary separation is constructed
These important sites are dispersed in different individuals by group, evaluate each candidate one by one under the consistent genetic background of height
The effect in site, it can be achieved that the positioning of candidate gene and marker assisted selection and gene functional research effective means.
Although located the largely QTL about soybean 100-grain weight at present, direct regulation and control soybean 100-grain weight is determined
Gene is really seldom, therefore excavates the functional study of the candidate gene and gene of regulating and controlling soybean 100-grain weight, can be soybean 100-grain weight point
Sub- assistant breeding provides useful information and important breeding material.
Summary of the invention
The technical problems to be solved by the invention are as follows: the crucial candidate gene GmSLK of a regulating and controlling soybean 100-grain weight is provided
And application, useful information and important breeding material are provided for soybean 100-grain weight marker assisted selection.
The technical solution of the present invention is as follows:
Soybean 100-grain weight synergy gene, base sequence is as shown in SEQ ID No.2.
The albumen of soybean 100-grain weight synergy gene coding, amino acid sequence is as shown in SEQ ID No.4.
Encode the polynucleotide passage of amino acid sequence shown in SEQ ID No.4.
Recombinant vector contains polynucleotide passage described above;Preferably, the sequence of the polynucleotide passage is such as
Shown in SEQ ID No.2.
The special primer of soybean 100-grain weight synergy gene described above is expanded, the upstream primer sequence of the special primer is
SEQ ID No.5, the downstream primer sequence of special primer are SEQ ID No.6.
The molecular labeling of soybean 100-grain weight synergy gene, the molecular labeling are located at No. 16 chromosome of soybean chromosome
The position of Chr16:35923082-35923257bp, the primer of the molecular labeling such as SEQ ID No.7 and SEQ ID No.8 institute
Show.
Whether identification soybean contains the method for soybean 100-grain weight synergy gene described above, with the gene of soybean to be identified
Group DNA is template, PCR amplification is carried out with primer shown in SEQ ID No.7 and SEQ ID No.8, if the expansion of 175bp can be obtained
Increase production object, then the soybean contains the soybean 100-grain weight synergy gene.
Soybean 100-grain weight synergy gene described above or albumen or recombinant vector or special primer or molecular labeling are big
Application in beans breeding.
Present invention finds a regulating and controlling soybean 100-grain weight gene GmSLK, the gene is located at soybean chromosome the 16th
Chromosome Chr16:35920962-35928190bp, full genome group leader 7229bp, the long 2640bp of complete encoding sequence analyze ratio
It compares compared with GmSLK gene order of the discovery from wild beans N24852 and cultivates the GmSLK gene order of beans NN1138-2 the
The more glutamine in the glutamic acid tandem repeat region of one exon.And the GmSLK gene pairs soybean from wild beans
100-grain weight has reduction effect, and the GmSLK gene pairs soybean 100-grain weight from cultivation beans has synergistic effect.By further fixed
Position one molecular labeling InDelSLK of discovery is located at GmSLK gene in the difference section of wild beans N24852 and cultivation beans NN1138-2
Between, therefore can be synergy gene or leaky gene the GmSLK gene that judge in different soybean varieties by this label,
There is important value in soybean breeder selection.
Compared with prior art, the invention has the following advantages:
The present invention identifies the gene GmSLK for being located at regulating and controlling soybean 100-grain weight on soybean Gm16 chromosome, the base
The overexpression of cause can significantly improve soybean 100-grain weight character.The label InDelSLK for identifying the gene is provided simultaneously.
It should be labeled as identifying that its GmSLK is synergy gene or the leaky gene side of providing in soya seeds resource and filial generation
Just, important judgement value is provided for soybean breeder selection.
Detailed description of the invention
Fig. 1: informative population schematic diagram;Using wild beans N24852 as male parent, cultivation beans NN1138-2 is recurrent parent, building
Chromosome segment substitution line (constructs) for Wang Wubin et al. 2013, and CSSL3068 is a family in substitution line.Secondary group
Using CSSL3068 as female parent, NN1138-2 is used as male parent, and hybridization, selfing obtain 294 offsprings, single-strain planting and harvest obtains
The F of 294 familys2Group, then each family plants a line, selfing, and harvests to obtain 294 F by plant2:3Family, similarly
Obtain F2:4And F2:5Family.
Fig. 2: parent and target group's 100-grain weight phenotype;(A) kind for being secondary group parent NN1138-2 and CSSL3068
The big logotype of son;(B) compare (P < 0.01 * *) for the 100-grain weight of secondary group parent NN1138-2 and CSSL3068;(C) fixed
Position group F2:3In plantation Nanjing (2015NJ) in 2015, F2:4Group in plantation in 2016 in Nanjing (2016NJ) and
F2:5Group planted Nanjing (2017NJ) and Anhui in 2017 when the number point for the 100-grain weight phenotype for applying (2017AH) respectively
Butut.
Fig. 3: finely positioning process;It (A) is the primary physical map of No. 16 chromosome;It (B) is the part of No. 16 chromosome
High density physical map;(C) high density of map spectrum analysis secondary group is combined using One marker analysis method (IciMapping2.0)
F2:3、F2:4And F2:5Phenotypic data in four environment;(D) using displacement graphing method analyze 11 recombination individual phenotypes and
Genotype positional candidate gene;(E) GmSLK gene structure display.
Fig. 4: GmSLK amplified band figure;GmSLK-N24852 represents GmSLK from wild beans N24852, GmSLK-
NN1138-2 represents GmSLK and represents GmSLK from substitution line from cultivation beans NN1138-2, GmSLK-CSSL3068
CSSL3068, Marker 5000bp, the band of red arrow instruction are 3000bp band.
Fig. 5: compare the seed size and fresh weight of parent's Grain Development different times.(A) compare parent NN1138-2 and
CSSL3068 is in Grain Development different times seed size;(B) compare parent NN1138-2 and CSSL3068 Grain Development not
Same time seed fresh weight.Each sample statistics analyze 3 biology and repeat to repeat (p < 0.01 * p < 0.05, * *) with 3 technologies.
Fig. 6: GmSLK parent's NN1138-2 and CSSL3068 Grain Development different times relative expression quantity.Internal reference base
Because of UBI3 gene, each sample statistics analyze 3 biology and repeat to repeat (p < 0.01 * p < 0.05, * *) with 3 technologies.
Fig. 7: transgenic arabidopsis analyzes GmSLK-NN1138-2 (left side) and GmSLK-CSSL3068 (the right) gene function
Energy.(A) relative expression quantity analysis of Transgenic wheat line, reference gene are Actin gene;(B) transgenic plant and wild
3 weeks plant forms sizes of type compare;(C) compare 1000 weights of transgenic plant and WT lines, each sample statistics
3 biology are analyzed to repeat to repeat (p < 0.001 * p < 0.05, * *) with 3 technologies.
Specific embodiment
The acquisition of 1. soybean 100-grain weight main effect site qSW16.1 of embodiment
(1), target group's building and plantation
Wang Wubin in 2013 et al. constructs a set of 151 family groups using wild beans N24852 and cultivation beans NN1138-2
At chromosome segment substitution line group, one of family CSSL3068 is had found according to the 100-grain weight phenotypic information of group's family
100-grain weight be 14.68g, the recurrent parent NN1138-2 100-grain weight (18.7g) that compares significantly becomes smaller, according to the label of group letter
Breath discovery CSSL3068 contains the wild site Sat_224 an of 100-grain weight.Utilize family CSSL3068 and circulation parent within 2014
This NN1138-2 hybridization building secondary group, obtains the F of 294 strains2Generation grade target group.2015 in Nanjing
(2015NJ) is by F2Secondary group plants 294 plants, and 3 repetitions, then every row individually harvests, and obtains the F of 294 familys2:3
Group measures F2:3The 100-grain weight phenotype of group's family.2016 in Nanjing (2016NJ) by F2:3294 strains are planted by group
Row, 3 repetitions, then every row individually harvests, and obtains the F of 294 familys2:4Group measures F2:4The 100-grain weight table of group's family
Type.2017 by F2:4294 plants of group are planted respectively in Nanjing (2017NJ) and Anhui when painting (2017AH), 3 weights
Multiple, then every row individually harvests, and obtains the F of 294 familys2:5Group measures F2:5The 100-grain weight phenotype (Fig. 1) of group's family.
(2), the measurement and analysis of parent and target group's 100-grain weight phenotype
Plant plantation, plant harvest, then selects development in 30 DEG C of constant temperature ovens to seed constant weight, each parent and family
Well, 100 full seeds weighing.
Seed size (Fig. 2A and B) first between two parents of identification and analysis, as a result, it has been found that parent NN1138-2
100-grain weight is significantly higher than the 100-grain weight (such as table 1) of parent CSSL3068 under four different environment.Also with SPSS
2.0 have statisticallyd analyze the frequency distribution (Fig. 2 C) of secondary group's 100-grain weight phenotype under four environment, as the result is shown all in company
Continuous distribution, and F2:5 group 100-grain weight average value is up to 16.94g in the environment of 2017AH, the group under 2015NJ environment
The minimum 13.88g of group's 100-grain weight average value (such as table 1), these results all show that soybean 100-grain weight phenotype is quantitative character, are
Controlled by multiple genes.In addition, target group all has lower phenotypic variation coefficient (CV) and higher something lost in different environments
Biography rate (h2), this, which illustrates that soybean 100-grain weight phenotype mainly makes a variation, is controlled by gene.
100-grain weight under 1 parent's NN1138-2 and CSSL3068 varying environment of table
CV, the coefficient of variation;h2, heritability.
(3), group's genotype identification
Secondary group F is extracted using CTAB method2294 strains soybean leaves genomic DNA, the primer is by English
The synthesis of Weihe River victory base (Shanghai) trading company.PCR reaction system 10ul, wherein containing 3ulDNA (15ul), upstream and downstream primer
(0.2mmol/L) each 1.5 μ L, 1.2 μ L Mgcl2(2.5mmol/L), 0.24 μ L dNTP (10mmol/L, N=A, C, G, T),
0.12 μ L Taq enzyme (5U/ul) and 1.4 μ L ddH2O.PCR response procedures: 94 DEG C of denaturation 5min;Then carry out 33 circulations
94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 40s;Thoroughly extend 10min using 72 DEG C;Last 4 DEG C of preservations.PCR expands
Increase production 8% polyacrylamide gel electrophoresis of object, the polymorphism of silver staining colour developing identification band.
(4), the building of genetic linkage maps
Two parents are analyzed using 49 SSR molecular markers on No. 16 chromosome, are had between statistical analysis parent more
The hereditary form with polymorphism mark between parent is identified, then to it in totally 7, the site of state property in secondary group's F2 strain
Linkage analysis is carried out, the genetic linkage maps (Fig. 3 A) of secondary group are constructed.
(5), molecular markers development
Existed using the soybean genome SSR marker information of the announcements such as Song and wild beans N24852 and cultivation beans NN1138-2
The InDel information of Gm16 chromosome devises new molecular labeling, statisticallys analyze the SSR between parent with polymorphism and marks
Note 3,16 InDel mark (table 2), the hereditary form with polymorphism mark between parent are identified in secondary group F2, so
Linkage analysis is carried out to it afterwards, constructs the high density map (Fig. 3 B) of secondary group.
The Molecular Marker Information of the present invention of table 2
(6), interpretation of result
The main effect of soybean 100-grain weight is shown using positioning result at the beginning of SAS9.4 combination secondary group phenotypic data and molecular data
Site qSW16.1 is located between No. 16 chromosome Sat_224 and Satt431.
In conjunction with high density map and secondary group F2:3、F2:4And F2:5Phenotypic data in four environment, utilizes single label
Analytic approach (IciMapping2.0) carries out finely positioning, is as a result positioned at soybean 100-grain weight main effect site qSW16.1
Between InDel16_01 and BARCSOYSSR_16_1215 two labels (Fig. 3 C).
The acquisition of 2 regulating and controlling soybean 100-grain weight gene GmSLK of embodiment
The determination of gene GmSLK
Soybean 100-grain weight main effect site qSW16.1 is positioned at Indel16_01 and BARCSOYSSR_ by finely positioning
Between 16_1215 two labels, find that there are two candidate bases altogether between the two labels with reference to gene order by comparing soybean
Cause, in order to further determine candidate gene, from F211 are picked in group in Gm16 chromosome 35889420bp-
There is the family of recombination in the region 35952811bp, passes through the base of this 11 recombinations familys and two parents of displacement graphing method comparative analysis
Because of type and 100-grain weight phenotypic data further progress finely positioning, InDel16_03 and BARCSOYSSR_16_1215 two are found
There is no recombination site between label, isolates (Fig. 3 D and E) with gene Glyma.16g198300.It is analyzed and is sent out by gene annotation
Existing gene Glyma.16g198300 belongs to LIM domain, and functional annotation is transcription regulatory factor (Transcriptional
Regulator, SLK2), therefore it is named as GmSLK.
Application of the 3 regulating and controlling soybean 100-grain weight gene GmSLK of embodiment in soybean breeder
The specific amplified upstream primer sequence of regulating and controlling soybean 100-grain weight character gene of the present invention is SEQ ID
No.5, specific amplified downstream primer sequence are SEQ ID No.6 (table 3).
The specific primer sequences of the amplification GmSLK gene of table 3
Using primer sequence SEQ ID No.5 and SEQ ID No.6, respectively with wild beans N24852 and two parents
The RNA of (NN1138-2 and CSSL3068) is code area (CDS) sequence of template amplification GmSLK.Amplification uses Takara
The high fidelity enzyme (Phanta Super-Fidelity DNA Poymerase, Vazyme) of company, PCR reaction system and program
Such as table 4 and table 5, amplified band size such as Fig. 4:
Table 4.PCR reaction system
Table 5.PCR response procedures
The CDS sequence for comparing the gene between two parents and wild beans N24852 by sequencing amplified production analysis is poor
It is different, it is found that sequence of the gene GmSLK in parent CSSL3068 and wild beans N24852 is consistent, nucleotide sequence such as SEQ ID
Shown in No.1, the amino acid sequence of coding is as shown in SEQ ID No.3.Illustrate the GmSLK of parent CSSL3068 from wild beans.
The GmSLK gene nucleotide series of beans NN1138-2 are cultivated as shown in SEQ ID No.2, the amino acid sequence of coding such as SEQ ID
Shown in No.4.The GmSLK gene of wild beans is in the glutamic acid tandem repeat region of first exon than cultivation beans NN1138-2
GmSLK gene it is more a glutamine (Fig. 3 E).And the GmSLK gene pairs soybean 100-grain weight from wild beans has reduction
Effect, the GmSLK gene pairs soybean 100-grain weight from cultivation beans (NN1138-2) have synergistic effect.
Find that InDel16_03 is located at GmSLK gene in wild beans N24852 and cultivation beans NN1138-2 by sequence alignment
Difference section, therefore Indel16_03 is named as InDelSLK.Therefore different soybean varieties can be judged by this label
In GmSLK gene be synergy gene or leaky gene, there is important value in soybean breeder selection.
Whether identification soybean contains the method for soybean 100-grain weight synergy gene described above, with the gene of soybean to be identified
Group DNA is template, PCR amplification is carried out with primer shown in SEQ ID No.7 and SEQ ID No.8, if the expansion of 175bp can be obtained
Increase production object, then the soybean contains the soybean 100-grain weight synergy gene.
The verifying of 4 GmSLK gene function of embodiment
In order to the influence of Rapid identification GmSLK gene pairs seed grain weight, this experimental selection mode crop arabidopsis
(Arabidopsis thaliana) Columbia-0 wild type as transgenic acceptor, is overexpressed parent respectively
The GmSLK (GmSLK-CSSL3068) of CSSL3068 and GmSLK (GmSLK-NN1138-2) gene for cultivating beans NN1138-2, point
Analysis is relatively more single to copy homozygotic 1000 weights, to verify the influence of GmSLK gene pairs seed grain weight.
(1) GmSLK gene expression dose is analyzed
In order to better understand the influence of GmSLK gene pairs soybean kernel developmental stage, GmSLK base is compared in this experimental analysis
Because of the expression of the Grain Development different times in parent (NN1138-2 and CSSL3068).8 single plant NN1138- are taken respectively
2 and CSSL3068 Post flowering 7_DAF, 14_DAF, 21_DAF, 28_DAF, 35_DAF and 42_DAF (DAF:day after
Flower seed), 3 biology of each sample repeat, and measure phenotype (Fig. 5) respectively and extract RNA and are (Real-time RT-
PCR) RT-PCR analyzes (Fig. 6).
Observation compares parent NN1138-2 and CSSL3068 in the seed size of Grain Development different times, discovery
The seed of NN1138-2 is all larger (Fig. 5 A) in the different developmental stage seeds CSSL3068 seed that compares, and passes through measurement seed
Fresh weight, the results showed that it is aobvious that the seed of NN1138-2 in different developmental stage seed fresh weights is significantly higher than CSSL3068 seed
It writes (Fig. 5 B).
Analysis compares GmSLK gene in the relative expression levels of the Grain Development different times of parent, this experiment is chosen big
UBI3 (ribosomal-ubiquitin fusion protein) gene of beans is as internal reference.Interpretation of result shows GmSLK base
Increase because of development of the expressed in abundance degree in parents with seed, and reaches maximum in 42_DAF period expression quantity.However,
GmSLK gene is much like in parents' Grain Development early period and mid-term (14_DAF-35_DAF) expression quantity level, but in 42_DAF
Period, GmSLK gene are significantly higher than the relative expression in parent CSSL3068 in the relative expression quantity level of parent NN1138-2
Amount is horizontal.
(2) GmSLK gene transgenic arabidopsis is verified
It is 3301 carrier of pCAMBIA selected by this research transformation of Arabidopsis thaliana, restriction enzyme site is (Bst EII and NcoI),
GmSLK gene magnification upstream and downstream primer is SEQ ID No.3 and SEQ ID No.4 (table 6) respectively.Use clone's recombination kit
(ClonExperss II) carries out recombining reaction.Then the over-express vector pCAMBIA 3301-GmSLK- that will be built
NN1138-2 and pCAMBIA 3301-GmSLK-CSSL30068 be transformed into respectively in DH5a competence carry out plasmid expand it is numerous, then
By clone identification, it is transferred in Agrobacterium EHA105 after extracting plasmid.By pollen soaking method, the overexpression that will be built respectively
Carrier is transferred in arabidopsis Columbia-0 wild type.
The gene constructed vector primer design of table 6GmSLK
Over-express vector pCAMBIA 3301 has glufosinate-resistant, the glufosinate that positive seedling passes through 1/2MSA+10mg/l
Culture medium is screened.The T1 of the Arabidopsis plant infected is harvested for seed, it is positive to screen T2 generation on resistance culture base again first
Seedling, then plantation harvests T2 seed, and survival/death rate is planted and statisticallyd analyze to T2 seed on resistance culture base, passes through card
Side detects and selects survival/death and meets 3:1, illustrates that the transgenic plant belongs to single copy plant, then plants and harvest T3 generation
Seed, then plant in the homozygous plants of resistance culture base selection survival rate 100%, plant and harvest T4 seed and count 1000
Weight phenotype.Plant is overexpressed for GmSLK-NN1138-2 and GmSLK-CSSL30068 and selects 3 single copy homozygous strains respectively
System, each strain plant 3 respectively, then take blade to carry out RT-PCR analysis, select the Actin gene of arabidopsis as internal reference
Gene detects the relative expression quantity (Fig. 7 A) of the foreign gene of transgenic plant.This experiment also observation analysis transgenic plant 3
The plant size of Zhou great little finds that GmSLK-NN1138-2 with the GmSLK-CSSL30068 plant being overexpressed compares wild type
Plant size does not have difference (Fig. 7 B).It harvests T5 seed and statisticallys analyze 1000 weights phenotype (Fig. 7 C), interpretation of result shows to compare
Compared with wild type, the GmSLK-NN1138-2 of overexpression can significantly increase grain weight, however it is significant to be overexpressed GmSLK-CSSL30068
Reduce grain weight.Therefore it can prove that the GmSLK genotype from wild beans or parent CSSL3068 belongs to leaky gene, it can be with
Seed grain weight is reduced, and the GmSLK genotype from cultivation beans NN1138-2 belongs to synergy gene, can increase seed grain weight,
This provides strong evidence for the Yield Breeding of soybean.
Sequence table
<110>Agricultural University Of Nanjing
<120>soybean 100-grain weight synergy gene and its molecular labeling and application
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2640
<212> DNA
<213>soybean (Glycine max)
<400> 1
atgacacctt tgcgagtggc aggtggatta acccaatcat catcaaattc tggaattttc 60
tatcaaggag atgggcagtc acagaatgta gttaactctc acttaagctc atcttttgtt 120
aactcgtcta gcacggtttc tggagcttct cgttcaaacc tgggtccggt ttctggggat 180
atgaataatg cagttttgaa cagtgtggca aactcagcac caagtgtagg agccagctct 240
ttagttacag atgcaaattc tgcactctct ggtggcccac atttgcagag aagtgccagc 300
gttaacacag actcatactt acgattacct gcctcaccta tgtcatttac atcaaataat 360
atcagtattt ctggatcatc agtgatggat gtttcctctg tagtacaaca gagctctcat 420
caagatcaga atgttcaaca attgcagcag aatcagcagc agccacaggg tgcttcaagt 480
gctatgtctt tgtctgcatc tcaaactggc ccttctatgc tccaaatggg tgcacaaatc 540
ccaggatctt tcattcaaga tccaaataat atgtctcatc tgtcaaagaa acctagaatg 600
gatatcaaac aggaagatat gatgcagcag caggttatac aacagattct tcagagacaa 660
gattccatgc aattccaggg tcgtaatccc cagttacagg ctttccttca gcagcagcag 720
cagagactga gacaacaaca aatgtttcag caaatgccac aattacaccg agcacacttg 780
cagcagcagc aacaacaaca acaaatgcaa ttgaggcagc agcagcagca gcagcaacaa 840
caacaacaac aacaacaaca acaacaacaa caacaacaac aacaacaaca acaacaacag 900
caacagcaag tgatgcagcc ctcttctgtg gtcaagcgtc catatgacag tagtgttagt 960
ggggtatgtg cccgtcgatt gatgcagtat ctctatcatc aaaggcaacg accaaatgat 1020
aatagtattg cctattggag aaaatttgtg gctgaatatt actctcctcg tgcaaagaaa 1080
cggtggtgct tgtcattata tagtaatgtt gggcatcacg cacttggtgt ttttccccaa 1140
gcatctatgg atgcatggca ctgtgacata tgtggttcta aatctggaag gggatttgag 1200
gcaacttatg aagtattacc tagacttaat gaaatcaaat ttggcagtgg agtaattgat 1260
gaactattgt ttctggacat gccacgtgaa atgagattcg cttctggtgc gatgatgtta 1320
gaatatggaa aagcagttca agagagtgta tatgagcagc ttcgtgttgt tcgtgaaggt 1380
caacttcgta tcatattcac tcaagacttg aagatattat cttgggagtt ctgtgcaagg 1440
tgccatgagg aacttcttcc tcgaaggttg gttgcaccac aggtcaacca gttagttcag 1500
gtagctaaaa aatgtcaaag tacaattgct gaaagtggtt ctgatggggt ttctcaacaa 1560
gacatccaaa caaacagcaa catgttgttg acagctgggg gtcagcttgc gaagattttg 1620
gagatgcaat cgctaaatga gttgggcttt tctaaaagat acgtgagatg tttgcaaatt 1680
tcggaggttg tcaatagcat gaaagaccta atagatatct gtgcagatca caaaattggt 1740
gccattgaga gtttgaaaaa ttttcctcgt ctagcaacag cctcaaaggt ccagatgcag 1800
aagatgcagg aaatggaaca gctagcaaat gttcaaggtc tgccaactga tcgaaacaca 1860
ctcaataagc taatggcact gaatcctgga ttgaacaacc atataaacaa ccctcataat 1920
atggttaatc gtggtgcttt gagtgggtca gcccaagctg ctttagcact gaacaactac 1980
caaaatcttc tcatgaggca aaattcaatg aactctagcc ctggctcact tcagcgcgaa 2040
gggtcctctt tcaataattc aaaccagagt ccatcttcag ctttgcaagg agctggtcct 2100
gctttaattc caggcccaat gcagaattca tctgttagtg gtttcccaag cccccgtcta 2160
cccccacagc agcagcaaca ccacctacaa cagccctcat taagtgcaaa tgctttactg 2220
caacaaaatc attcacaggg ttcccaagga aatcaagctc tgcagcagca gatgatccat 2280
caactactgc aggagatgtc aaataacaac gggggagtgc aaccacagtc tcttggtgga 2340
cccagtgcaa atatggcaaa gaatgcactg gggtttgggg gccattatcc atccttaagt 2400
ggaggttctg ccaatgttac aggaaacaat ggacctatgt caaggaataa tagcttcaaa 2460
acaactgcaa atagtgattc ttctgctgct ggtggcaaca atggattaaa ccagagaaca 2520
tctgagatgc cacaaaatct acatttgcaa gatgtggttc aggatattgg caatgaattc 2580
acggataatc ccttccttaa cagtgatctt gatgataaca tgggttttgg ctggaaggca 2640
<210> 2
<211> 2637
<212> DNA
<213>soybean (Glycine max)
<400> 2
atgacacctt tgcgagtggc aggtggatta acccaatcat catcaaattc tggaattttc 60
tatcaaggag atgggcagtc acagaatgta gttaactctc acttaagctc atcttttgtt 120
aactcgtcta gcacggtttc tggagcttct cgttcaaacc tgggtccggt ttctggggat 180
atgaataatg cagttttgaa cagtgtggca aactcagcac caagtgtagg agccagctct 240
ttagttacag atgcaaattc tgcactctct ggtggcccac atttgcagag aagtgccagc 300
gttaacacag actcatactt acgattacct gcctcaccta tgtcatttac atcaaataat 360
atcagtattt ctggatcatc agtgatggat gtttcctctg tagtacaaca gagctctcat 420
caagatcaga atgttcaaca attgcagcag aatcagcagc agccacaggg tgcttcaagt 480
gctatgtctt tgtctgcatc tcaaactggc ccttctatgc tccaaatggg tgcacaaatc 540
ccaggatctt tcattcaaga tccaaataat atgtctcatc tgtcaaagaa acctagaatg 600
gatatcaaac aggaagatat gatgcagcag caggttatac aacagattct tcagagacaa 660
gattccatgc aattccaggg tcgtaatccc cagttacagg ctttccttca gcagcagcag 720
cagagactga gacaacaaca aatgtttcag caaatgccac aattacaccg agcacacttg 780
cagcagcagc aacaacaaca acaaatgcaa ttgaggcagc agcagcagca gcagcaacaa 840
caacaacaac aacaacaaca acaacaacaa caacaacaac aacaacaaca acaacagcaa 900
cagcaagtga tgcagccctc ttctgtggtc aagcgtccat atgacagtag tgttagtggg 960
gtatgtgccc gtcgattgat gcagtatctc tatcatcaaa ggcaacgacc aaatgataat 1020
agtattgcct attggagaaa atttgtggct gaatattact ctcctcgtgc aaagaaacgg 1080
tggtgcttgt cattatatag taatgttggg catcacgcac ttggtgtttt tccccaagca 1140
tctatggatg catggcactg tgacatatgt ggttctaaat ctggaagggg atttgaggca 1200
acttatgaag tattacctag acttaatgaa atcaaatttg gcagtggagt aattgatgaa 1260
ctattgtttc tggacatgcc acgtgaaatg agattcgctt ctggtgcgat gatgttagaa 1320
tatggaaaag cagttcaaga gagtgtatat gagcagcttc gtgttgttcg tgaaggtcaa 1380
cttcgtatca tattcactca agacttgaag atattatctt gggagttctg tgcaaggtgc 1440
catgaggaac ttcttcctcg aaggttggtt gcaccacagg tcaaccagtt agttcaggta 1500
gctaaaaaat gtcaaagtac aattgctgaa agtggttctg atggggtttc tcaacaagac 1560
atccaaacaa acagcaacat gttgttgaca gctgggggtc agcttgcgaa gattttggag 1620
atgcaatcgc taaatgagtt gggcttttct aaaagatacg tgagatgttt gcaaatttcg 1680
gaggttgtca atagcatgaa agacctaata gatatctgtg cagatcacaa aattggtgcc 1740
attgagagtt tgaaaaattt tcctcgtcta gcaacagcct caaaggtcca gatgcagaag 1800
atgcaggaaa tggaacagct agcaaatgtt caaggtctgc caactgatcg aaacacactc 1860
aataagctaa tggcactgaa tcctggattg aacaaccata taaacaaccc tcataatatg 1920
gttaatcgtg gtgctttgag tgggtcagcc caagctgctt tagcactgaa caactaccaa 1980
aatcttctca tgaggcaaaa ttcaatgaac tctagccctg gctcacttca gcgcgaaggg 2040
tcctctttca ataattcaaa ccagagtcca tcttcagctt tgcaaggagc tggtcctgct 2100
ttaattccag gcccaatgca gaattcatct gttagtggtt tcccaagccc ccgtctaccc 2160
ccacagcagc agcaacacca cctacaacag ccctcattaa gtgcaaatgc tttactgcaa 2220
caaaatcatt cacagggttc ccaaggaaat caagctctgc agcagcagat gatccatcaa 2280
ctactgcagg agatgtcaaa taacaacggg ggagtgcaac cacagtctct tggtggaccc 2340
agtgcaaata tggcaaagaa tgcactgggg tttgggggcc attatccatc cttaagtgga 2400
ggttctgcca atgttacagg aaacaatgga cctatgtcaa ggaataatag cttcaaaaca 2460
actgcaaata gtgattcttc tgctgctggt ggcaacaatg gattaaacca gagaacatct 2520
gagatgccac aaaatctaca tttgcaagat gtggttcagg atattggcaa tgaattcacg 2580
gataatccct tccttaacag tgatcttgat gataacatgg gttttggctg gaaggca 2637
<210> 3
<211> 880
<212> PRT
<213>soybean (Glycine max)
<400> 3
Met Thr Pro Leu Arg Val Ala Gly Gly Leu Thr Gln Ser Ser Ser Asn
1 5 10 15
Ser Gly Ile Phe Tyr Gln Gly Asp Gly Gln Ser Gln Asn Val Val Asn
20 25 30
Ser His Leu Ser Ser Ser Phe Val Asn Ser Ser Ser Thr Val Ser Gly
35 40 45
Ala Ser Arg Ser Asn Leu Gly Pro Val Ser Gly Asp Met Asn Asn Ala
50 55 60
Val Leu Asn Ser Val Ala Asn Ser Ala Pro Ser Val Gly Ala Ser Ser
65 70 75 80
Leu Val Thr Asp Ala Asn Ser Ala Leu Ser Gly Gly Pro His Leu Gln
85 90 95
Arg Ser Ala Ser Val Asn Thr Asp Ser Tyr Leu Arg Leu Pro Ala Ser
100 105 110
Pro Met Ser Phe Thr Ser Asn Asn Ile Ser Ile Ser Gly Ser Ser Val
115 120 125
Met Asp Val Ser Ser Val Val Gln Gln Ser Ser His Gln Asp Gln Asn
130 135 140
Val Gln Gln Leu Gln Gln Asn Gln Gln Gln Pro Gln Gly Ala Ser Ser
145 150 155 160
Ala Met Ser Leu Ser Ala Ser Gln Thr Gly Pro Ser Met Leu Gln Met
165 170 175
Gly Ala Gln Ile Pro Gly Ser Phe Ile Gln Asp Pro Asn Asn Met Ser
180 185 190
His Leu Ser Lys Lys Pro Arg Met Asp Ile Lys Gln Glu Asp Met Met
195 200 205
Gln Gln Gln Val Ile Gln Gln Ile Leu Gln Arg Gln Asp Ser Met Gln
210 215 220
Phe Gln Gly Arg Asn Pro Gln Leu Gln Ala Phe Leu Gln Gln Gln Gln
225 230 235 240
Gln Arg Leu Arg Gln Gln Gln Met Phe Gln Gln Met Pro Gln Leu His
245 250 255
Arg Ala His Leu Gln Gln Gln Gln Gln Gln Gln Gln Met Gln Leu Arg
260 265 270
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
275 280 285
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Val
290 295 300
Met Gln Pro Ser Ser Val Val Lys Arg Pro Tyr Asp Ser Ser Val Ser
305 310 315 320
Gly Val Cys Ala Arg Arg Leu Met Gln Tyr Leu Tyr His Gln Arg Gln
325 330 335
Arg Pro Asn Asp Asn Ser Ile Ala Tyr Trp Arg Lys Phe Val Ala Glu
340 345 350
Tyr Tyr Ser Pro Arg Ala Lys Lys Arg Trp Cys Leu Ser Leu Tyr Ser
355 360 365
Asn Val Gly His His Ala Leu Gly Val Phe Pro Gln Ala Ser Met Asp
370 375 380
Ala Trp His Cys Asp Ile Cys Gly Ser Lys Ser Gly Arg Gly Phe Glu
385 390 395 400
Ala Thr Tyr Glu Val Leu Pro Arg Leu Asn Glu Ile Lys Phe Gly Ser
405 410 415
Gly Val Ile Asp Glu Leu Leu Phe Leu Asp Met Pro Arg Glu Met Arg
420 425 430
Phe Ala Ser Gly Ala Met Met Leu Glu Tyr Gly Lys Ala Val Gln Glu
435 440 445
Ser Val Tyr Glu Gln Leu Arg Val Val Arg Glu Gly Gln Leu Arg Ile
450 455 460
Ile Phe Thr Gln Asp Leu Lys Ile Leu Ser Trp Glu Phe Cys Ala Arg
465 470 475 480
Cys His Glu Glu Leu Leu Pro Arg Arg Leu Val Ala Pro Gln Val Asn
485 490 495
Gln Leu Val Gln Val Ala Lys Lys Cys Gln Ser Thr Ile Ala Glu Ser
500 505 510
Gly Ser Asp Gly Val Ser Gln Gln Asp Ile Gln Thr Asn Ser Asn Met
515 520 525
Leu Leu Thr Ala Gly Gly Gln Leu Ala Lys Ile Leu Glu Met Gln Ser
530 535 540
Leu Asn Glu Leu Gly Phe Ser Lys Arg Tyr Val Arg Cys Leu Gln Ile
545 550 555 560
Ser Glu Val Val Asn Ser Met Lys Asp Leu Ile Asp Ile Cys Ala Asp
565 570 575
His Lys Ile Gly Ala Ile Glu Ser Leu Lys Asn Phe Pro Arg Leu Ala
580 585 590
Thr Ala Ser Lys Val Gln Met Gln Lys Met Gln Glu Met Glu Gln Leu
595 600 605
Ala Asn Val Gln Gly Leu Pro Thr Asp Arg Asn Thr Leu Asn Lys Leu
610 615 620
Met Ala Leu Asn Pro Gly Leu Asn Asn His Ile Asn Asn Pro His Asn
625 630 635 640
Met Val Asn Arg Gly Ala Leu Ser Gly Ser Ala Gln Ala Ala Leu Ala
645 650 655
Leu Asn Asn Tyr Gln Asn Leu Leu Met Arg Gln Asn Ser Met Asn Ser
660 665 670
Ser Pro Gly Ser Leu Gln Arg Glu Gly Ser Ser Phe Asn Asn Ser Asn
675 680 685
Gln Ser Pro Ser Ser Ala Leu Gln Gly Ala Gly Pro Ala Leu Ile Pro
690 695 700
Gly Pro Met Gln Asn Ser Ser Val Ser Gly Phe Pro Ser Pro Arg Leu
705 710 715 720
Pro Pro Gln Gln Gln Gln His His Leu Gln Gln Pro Ser Leu Ser Ala
725 730 735
Asn Ala Leu Leu Gln Gln Asn His Ser Gln Gly Ser Gln Gly Asn Gln
740 745 750
Ala Leu Gln Gln Gln Met Ile His Gln Leu Leu Gln Glu Met Ser Asn
755 760 765
Asn Asn Gly Gly Val Gln Pro Gln Ser Leu Gly Gly Pro Ser Ala Asn
770 775 780
Met Ala Lys Asn Ala Leu Gly Phe Gly Gly His Tyr Pro Ser Leu Ser
785 790 795 800
Gly Gly Ser Ala Asn Val Thr Gly Asn Asn Gly Pro Met Ser Arg Asn
805 810 815
Asn Ser Phe Lys Thr Thr Ala Asn Ser Asp Ser Ser Ala Ala Gly Gly
820 825 830
Asn Asn Gly Leu Asn Gln Arg Thr Ser Glu Met Pro Gln Asn Leu His
835 840 845
Leu Gln Asp Val Val Gln Asp Ile Gly Asn Glu Phe Thr Asp Asn Pro
850 855 860
Phe Leu Asn Ser Asp Leu Asp Asp Asn Met Gly Phe Gly Trp Lys Ala
865 870 875 880
<210> 4
<211> 879
<212> PRT
<213>soybean (Glycine max)
<400> 4
Met Thr Pro Leu Arg Val Ala Gly Gly Leu Thr Gln Ser Ser Ser Asn
1 5 10 15
Ser Gly Ile Phe Tyr Gln Gly Asp Gly Gln Ser Gln Asn Val Val Asn
20 25 30
Ser His Leu Ser Ser Ser Phe Val Asn Ser Ser Ser Thr Val Ser Gly
35 40 45
Ala Ser Arg Ser Asn Leu Gly Pro Val Ser Gly Asp Met Asn Asn Ala
50 55 60
Val Leu Asn Ser Val Ala Asn Ser Ala Pro Ser Val Gly Ala Ser Ser
65 70 75 80
Leu Val Thr Asp Ala Asn Ser Ala Leu Ser Gly Gly Pro His Leu Gln
85 90 95
Arg Ser Ala Ser Val Asn Thr Asp Ser Tyr Leu Arg Leu Pro Ala Ser
100 105 110
Pro Met Ser Phe Thr Ser Asn Asn Ile Ser Ile Ser Gly Ser Ser Val
115 120 125
Met Asp Val Ser Ser Val Val Gln Gln Ser Ser His Gln Asp Gln Asn
130 135 140
Val Gln Gln Leu Gln Gln Asn Gln Gln Gln Pro Gln Gly Ala Ser Ser
145 150 155 160
Ala Met Ser Leu Ser Ala Ser Gln Thr Gly Pro Ser Met Leu Gln Met
165 170 175
Gly Ala Gln Ile Pro Gly Ser Phe Ile Gln Asp Pro Asn Asn Met Ser
180 185 190
His Leu Ser Lys Lys Pro Arg Met Asp Ile Lys Gln Glu Asp Met Met
195 200 205
Gln Gln Gln Val Ile Gln Gln Ile Leu Gln Arg Gln Asp Ser Met Gln
210 215 220
Phe Gln Gly Arg Asn Pro Gln Leu Gln Ala Phe Leu Gln Gln Gln Gln
225 230 235 240
Gln Arg Leu Arg Gln Gln Gln Met Phe Gln Gln Met Pro Gln Leu His
245 250 255
Arg Ala His Leu Gln Gln Gln Gln Gln Gln Gln Gln Met Gln Leu Arg
260 265 270
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
275 280 285
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Val Met
290 295 300
Gln Pro Ser Ser Val Val Lys Arg Pro Tyr Asp Ser Ser Val Ser Gly
305 310 315 320
Val Cys Ala Arg Arg Leu Met Gln Tyr Leu Tyr His Gln Arg Gln Arg
325 330 335
Pro Asn Asp Asn Ser Ile Ala Tyr Trp Arg Lys Phe Val Ala Glu Tyr
340 345 350
Tyr Ser Pro Arg Ala Lys Lys Arg Trp Cys Leu Ser Leu Tyr Ser Asn
355 360 365
Val Gly His His Ala Leu Gly Val Phe Pro Gln Ala Ser Met Asp Ala
370 375 380
Trp His Cys Asp Ile Cys Gly Ser Lys Ser Gly Arg Gly Phe Glu Ala
385 390 395 400
Thr Tyr Glu Val Leu Pro Arg Leu Asn Glu Ile Lys Phe Gly Ser Gly
405 410 415
Val Ile Asp Glu Leu Leu Phe Leu Asp Met Pro Arg Glu Met Arg Phe
420 425 430
Ala Ser Gly Ala Met Met Leu Glu Tyr Gly Lys Ala Val Gln Glu Ser
435 440 445
Val Tyr Glu Gln Leu Arg Val Val Arg Glu Gly Gln Leu Arg Ile Ile
450 455 460
Phe Thr Gln Asp Leu Lys Ile Leu Ser Trp Glu Phe Cys Ala Arg Cys
465 470 475 480
His Glu Glu Leu Leu Pro Arg Arg Leu Val Ala Pro Gln Val Asn Gln
485 490 495
Leu Val Gln Val Ala Lys Lys Cys Gln Ser Thr Ile Ala Glu Ser Gly
500 505 510
Ser Asp Gly Val Ser Gln Gln Asp Ile Gln Thr Asn Ser Asn Met Leu
515 520 525
Leu Thr Ala Gly Gly Gln Leu Ala Lys Ile Leu Glu Met Gln Ser Leu
530 535 540
Asn Glu Leu Gly Phe Ser Lys Arg Tyr Val Arg Cys Leu Gln Ile Ser
545 550 555 560
Glu Val Val Asn Ser Met Lys Asp Leu Ile Asp Ile Cys Ala Asp His
565 570 575
Lys Ile Gly Ala Ile Glu Ser Leu Lys Asn Phe Pro Arg Leu Ala Thr
580 585 590
Ala Ser Lys Val Gln Met Gln Lys Met Gln Glu Met Glu Gln Leu Ala
595 600 605
Asn Val Gln Gly Leu Pro Thr Asp Arg Asn Thr Leu Asn Lys Leu Met
610 615 620
Ala Leu Asn Pro Gly Leu Asn Asn His Ile Asn Asn Pro His Asn Met
625 630 635 640
Val Asn Arg Gly Ala Leu Ser Gly Ser Ala Gln Ala Ala Leu Ala Leu
645 650 655
Asn Asn Tyr Gln Asn Leu Leu Met Arg Gln Asn Ser Met Asn Ser Ser
660 665 670
Pro Gly Ser Leu Gln Arg Glu Gly Ser Ser Phe Asn Asn Ser Asn Gln
675 680 685
Ser Pro Ser Ser Ala Leu Gln Gly Ala Gly Pro Ala Leu Ile Pro Gly
690 695 700
Pro Met Gln Asn Ser Ser Val Ser Gly Phe Pro Ser Pro Arg Leu Pro
705 710 715 720
Pro Gln Gln Gln Gln His His Leu Gln Gln Pro Ser Leu Ser Ala Asn
725 730 735
Ala Leu Leu Gln Gln Asn His Ser Gln Gly Ser Gln Gly Asn Gln Ala
740 745 750
Leu Gln Gln Gln Met Ile His Gln Leu Leu Gln Glu Met Ser Asn Asn
755 760 765
Asn Gly Gly Val Gln Pro Gln Ser Leu Gly Gly Pro Ser Ala Asn Met
770 775 780
Ala Lys Asn Ala Leu Gly Phe Gly Gly His Tyr Pro Ser Leu Ser Gly
785 790 795 800
Gly Ser Ala Asn Val Thr Gly Asn Asn Gly Pro Met Ser Arg Asn Asn
805 810 815
Ser Phe Lys Thr Thr Ala Asn Ser Asp Ser Ser Ala Ala Gly Gly Asn
820 825 830
Asn Gly Leu Asn Gln Arg Thr Ser Glu Met Pro Gln Asn Leu His Leu
835 840 845
Gln Asp Val Val Gln Asp Ile Gly Asn Glu Phe Thr Asp Asn Pro Phe
850 855 860
Leu Asn Ser Asp Leu Asp Asp Asn Met Gly Phe Gly Trp Lys Ala
865 870 875
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atgacacctt tgcgagtggc 20
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tcatgccttc cagccaaaac cc 22
<210> 7
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
acaattacac cgagcaca 18
<210> 8
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cttgaccaca gaagaggg 18
Claims (9)
1. soybean 100-grain weight synergy gene, base sequence is as shown in SEQ ID No.2.
2. the albumen of soybean 100-grain weight synergy gene coding described in claim 1, amino acid sequence such as SEQ ID No.4 institute
Show.
3. encoding the polynucleotide passage of amino acid sequence shown in SEQ ID No.4.
4. recombinant vector contains polynucleotide passage as claimed in claim 3.
5. recombinant vector according to claim 4, which is characterized in that the sequence of the polynucleotide passage such as SEQ ID
Shown in No.2.
6. expanding the special primer of soybean 100-grain weight synergy gene described in claim 1, the upstream primer sequence of the special primer
It is classified as SEQ ID No.5, the downstream primer sequence of special primer is SEQ ID No.6.
7. the molecular labeling of soybean 100-grain weight synergy gene described in claim 1, which is located at soybean chromosome
The position of No. 16 chromosome Chr16:35923082-35923257bp, the primer of the molecular labeling such as SEQ ID No.7 and SEQ
Shown in ID No.8.
8. whether identification soybean contains the method for soybean 100-grain weight synergy gene described in claim 1, which is characterized in that with to
The genomic DNA for identifying soybean is template, carries out PCR amplification with primer shown in SEQ ID No.7 and SEQ ID No.8, if
The amplified production of 175bp can be obtained, then the soybean contains the soybean 100-grain weight synergy gene.
9. weight described in soybean 100-grain weight synergy gene or albumen as claimed in claim 2 or claim 5 described in claim 1
The application of group carrier or special primer as claimed in claim 6 or molecular labeling as claimed in claim 7 in soybean breeder.
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