CN106755561A - A kind of QTL related to soybean root dry weight, SNP marker and application - Google Patents
A kind of QTL related to soybean root dry weight, SNP marker and application Download PDFInfo
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Abstract
The present invention provides a kind of QTL related to soybean root dry weight, SNP marker and application, is related to soybean molecular breeding field, excavates with soybean root dry weight about new QTL, and use it for the seed selection of soybean root dry weight proterties.A kind of QTL related to soybean root dry weight, it is interval positioned at No. 9 M45250 M45247 of chromosome, positioned by SNP marker M45250, M45247.Molecular labeling with the QTL close linkages is M45250, M45247, and as shown in SEQ ID NO.1, the nucleotide sequence of M45636 is as shown in SEQ ID NO.2 for the nucleotide sequence of M45646.The invention also discloses a kind of application of the QTL related to soybean root dry weight, the QTL and molecular labeling can be used for molecular marker assisted selection breeding, the efficiency of selection of the larger material of soybean root dry weight is significantly improved, for further abundant soybean root dry weight regulated and control network provides a kind of cost-effective molecular breeding new way.
Description
Technical field
The present invention relates to soybean molecular breeding field, more particularly to a kind of QTL, SNP molecule related to soybean root dry weight
Mark and application.
Background technology
The root of the soybean vitals related to fertilizer utilization and soil environment stress.Root system is to soybean yields, quality
And resistance has a major impact, powerful root system can improve yield, improving quality and the enhancing resistance of soybean.Soybean Root
Dry weight is one of overall target of Analysing Root Characters.The root dry weight difference of different soybean varieties is obvious, the bigger capacity for the resistance to lodging of root dry weight
Stronger, yielding ability is better.The root dry weight of soybean is difficult to directly observation selection, and its heredity can be improved using molecular marker assisted selection
Improvement process.
At present, quantitative character gene locus therefor is carried out to soybean root dry weight related gene by linkage analysis
(Quantitative trait locus, QTL) Position Research it is less, the soybean root dry weight correlation QTL for having reported is distributed in 3
Number, No. 8, No. 10, No. 11, No. 13, No. 17, on No. 18 chromosomes, the related mechanism of action and application study is not yet fully entered
OK.Therefore, it is necessary to finding one can more effectively react soybean root dry weight QTL and relative SNP (Single
Nucleotide polymorphism, the polymorphism of mononucleotide) molecular labeling, and then in soybean root dry weight genetic improvement
Using.
The content of the invention
It is an object of the invention to provide a kind of QTL related to soybean root dry weight, SNP marker and application, to solve
Certainly above-mentioned technical problem.The present invention is excavated to 1 QTL related to root dry weight, and marker interval is only 0.125cM, screens SNP
Mark and objective trait close linkage, can be used for molecular marker assisted selection breeding, significantly improve the choosing of the larger material of root dry weight
Select efficiency.
The technical problems to be solved by the invention are realized using following technical scheme:
A kind of QTL related to soybean root dry weight, it is characterised in that:The QTL relevant with soybean root dry weight is located at No. 9 dyeing
The M45250-M45247 of body is interval, is positioned by SNP marker M45250, M45247.
A kind of SNP marker with QTL close linkages, it is characterised in that the SNP marker is M45250,
As shown in SEQ ID NO.1, the amplimer of M45250 is the nucleotide sequence of M45250:
M50F:5 '-TCATTCCAGAGAGTGGTTCA-3 ', as shown in SEQ ID NO.3;
M50R:5 '-GTTGTTTACTATTACGGTGCCC-3 ', as shown in SEQ ID NO.4.
A kind of SNP marker with QTL close linkages, it is characterised in that the molecular labeling is M45247, M45247
Nucleotide sequence as shown in SEQ ID NO.2, the amplimer of M45247 is:
M47F:5 '-GCCTCCTTTGTCATTCTTCT-3 ', as shown in SEQ ID NO.5;
M47R:5 '-TCTCTTTGGCAACCTGGA-3 ', as shown in SEQ ID NO.6.
Applications of the QTL related to soybean root dry weight in soybean root dry weight proterties seed selection.
Described SNP marker, the application in soybean root dry weight trait molecular marker assisted Selection seed selection, be according to
What following technical scheme was carried out:
1) material genomic DNA to be identified is extracted using CTAB methods
2) be utilized respectively M45250, M45247 primer enter performing PCR amplification, respectively obtain M45250 amplified productions and
M452476 amplified productions;
3) M45250 amplified productions and M45247 amplified productions are sequenced respectively;
4) it is big G root dry weights that M45250 amplified productions hold the 81st from 5 ', and M45247 amplified productions the 65th from 5 ' end are A
Root dry weight is big.
The present invention, further technical scheme:
A kind of QTL related to soybean root dry weight, it is characterised in that:The QTL relevant with soybean root dry weight, described QTL
Interval positioned at No. 9 chromosome mappings is M45250-M45247, and it is carried out according to following technical scheme:
First, by the wild beans hybridization of soybean varieties 1,000 and Chang Ling, to F1The seed that generation obtains carries out selfing, obtains generation high
For recombinant inbred lines (RIL);
2nd, using SLAF-seq (Specific-Locus Amplified Fragment Sequencing) technologies and
HighMap softwares develop High Density Molecular label to RIL segregating populations, carry out genetic map construction;
3rd, qtl analysis are carried out using the CIM methods of software rQTL, with LOD >=3 as standard, soybean root dry weight is lost
Pass mapping;
Hybridized using the wild beans of soybean varieties 1,000 and Chang Ling, select seed to continue to plant in 200 offsprings of generation,
Inbreeding of more generation obtains one by 200 F5:8The recombinant inbred lines of strain composition;Constructed using this recombinant inbred lines
20 genetic maps of chromosome of soybean, step includes:
(1) Parent and 200 progeny genome DNAs are extracted using CTAB methods;
(2) digestion, Select gene group are carried out respectively using RsaI and HaeIII to detecting qualified each sample genomic DNA
Segment ranges 364-414bp SLAF fragments, the SLAF fragments to obtaining carry out 3 ' end plus A treatment, connect Dual-index
Sequence measuring joints, PCR amplifications, purifying, sample mixing, cut glue choose purpose fragment, use IlluminaHiSeqTM after library quality inspection is qualified
2500 are sequenced;
(3) positioning result according to sequencing Reads in reference gene group, GATK carries out local anharmonic ratio to (Local
Realignment), GATK variations detection, samtools variation detections, takes the common factor that two methods of GATK and samtools are obtained
The steps such as variant sites, to ensure to detect the accuracy of the SNP for obtaining, finally screen available SNP labels for 111399;
(4) the SNP label filtering rules partially separate less than 70% and seriously according to integrity degree, filter out 4902 SNP marks
Sign, by SNP point be 20 linkage groups by the positioning with reference gene group, by calculating MLOD values between label two-by-two, it is common on
64 SNP labels of Figure 45, in units of linkage group, the linear rows of Marker in linkage group are obtained using HighMap software analysis
Row, and the genetic distance between adjacent Marker is estimated, total figure is finally given away from the genetic map for being 3,403.90cM.
The present invention, with LOD >=3 as standard, is carried out using many Interval Mappings of rQTL mapping softwares to soybean root dry weight
Qtl analysis.QTL interval M45250-M45247 are found on No. 2 chromosomes, as shown in table 1.
The soybean root dry weight QTL of table 1 is interval
A kind of application of the QTL related to soybean root dry weight, it is characterised in that:The QTL related to soybean root dry weight is tight
Chain SNP marker is M45250, M45247, and it is, using the genomic DNA of material to be identified as template, to be entered with primer
Fragment obtained by performing PCR amplification;
Wherein, the amplimer of M45250 is:
M50F:5 '-TCATTCCAGAGAGTGGTTCA-3 ', as shown in SEQ ID NO.3;
M50R:5 '-GTTGTTTACTATTACGGTGCCC-3 ', as shown in SEQ ID NO.4.
The amplimer of M45247 is:
M47F:5 '-GCCTCCTTTGTCATTCTTCT-3 ', as shown in SEQ ID NO.5;
M47R:5 '-TCTCTTTGGCAACCTGGA-3 ', as shown in SEQ ID NO.6.
Using comprising the following steps that for above-mentioned SNP marker auxiliary judgment root dry weight:
1. material genomic DNA to be identified is extracted using CTAB methods
1) take the fresh blade of soybean and add liquid nitrogen grinding powdering, take and be put into 1.5mL centrifuge tubes in right amount;
2) add 0.6mL preheating CTAB extract solutions, reverse mixing several times, water-bath one hour at a temperature of 65 DEG C, often
15min is mixed once, 12000rpm centrifugations 15min;
3) 0.6mL 24 is added:The chloroform of 1 volume ratio:Isoamyl alcohol, overturns and mixes 5-10 times, 10000rpm centrifugations
15min;
4) take supernatant solution to be transferred in another empty centrifuge tube, with 24:The chloroform of 1 (V/V):Isoamyl alcohol weight
New extracting once, then adds 50 μ L RNase (10mg/mL) to place 30min at room temperature;
5) isometric -20 DEG C of isopropanols of precooling, the 30min in -20 DEG C of refrigerators, 5000rpm centrifugation 10min are added to go
Clearly;
6) cleaned twice with 70% ethanol.With sterilizing water dissolves after drying, genomic templates DNA is obtained, by genome mould
It is standby that plate DNA is put into 4 DEG C of Refrigerator stores;
7) with 0.8% agar sugar detection DNA concentration, be diluted to working concentration for PCR expand;
2. be utilized respectively M45250, M45247 primer enter performing PCR amplification, respectively obtain M45250 amplified productions and
M452476 amplified productions;
1) PCR amplification system:Cumulative volume is 20 μ L, including 10ng genomic templates DNA 2 μ L, 2 × Es Taq
Each 2 μ L and ddH of primer of MasterMix 10 μ L, 10mM2O 4μL;
2) PCR amplification conditions:94 DEG C of predegeneration 3min;94 DEG C of denaturation 30s, 57 DEG C of annealing 45s, 72 DEG C of extension 45s;Circulation
35 times;72 DEG C extend 10min eventually;
3. root dry weight is judged according to sequence alignment result
M45250 amplified productions and M45247 amplified productions are carried out into sequencing analysis respectively, M45250 amplified productions are held from 5 '
81st in maternal 1,000 to be A in G, the wild beans in male parent Chang Ling;M45247 amplified productions are from 5 ' the 65th, ends in female parent
To be C in A, the wild beans in male parent Chang Ling in 1000;When these sites of filial generation SNP with female parent it is consistent when, root dry weight greatly, when with
Root dry weight is small when male parent is consistent, and the judging nicety rate of M45250 is 84.6% for the judging nicety rate of 89.7%, M45247, therefore
Above-mentioned SNP marker M45250, M45247 can be used as the codominant marker of soybean root dry weight.
The beneficial effects of the invention are as follows:
The interval of M45250-M45247 of the present invention is all ideal mark interval (being shown in Table 1) of soybean root dry weight, wherein
M45250 is LOD peaks, and its contribution rate is 14.12%.The additive effect of the QTL closely coupled with this mark is negative value, i.e.,
The 1000 QTL allele increases root dry weight.Therefore, either from the positioning of QTL, or contributions of the QTL to phenotype
From the point of view of rate, the interval of M45250-M45247 is all the ideal mark interval of soybean root dry weight.Using present invention offer such as table 1
It is further abundant Soybean Root that the significance of shown soybean root dry weight chromosome mapping interval M45250-M45247 is
Dry weight regulated and control network provides a kind of most economical effective molecular breeding new way.
Brief description of the drawings
Fig. 1 is to be located at genetic map and QTL mappings interval M45250-M45247 interval on No. 9 chromosomes.
Specific embodiment
In order that technological means, creation characteristic, reached purpose and effect that the present invention is realized are easy to understand, tie below
Specific embodiment is closed, the present invention, but following embodiments only the preferred embodiments of the present invention are expanded on further, and it is not all.
Based on the embodiment in implementation method, those skilled in the art obtain other realities on the premise of creative work is not made
Example is applied, protection scope of the present invention is belonged to.Experimental technique in following embodiments, unless otherwise specified, is conventional method,
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
First, the structure of genetic group
It is female parent with the Soybean Native Varieties 1,000 that root dry weight is big, the wild beans in the small Chang Ling of root dry weight are male parent, are carried out
Sexual hybridization, F1Generation selection is grown fine, and solid many individual plants are harvested, and then using single seed descent, breeding adds generation to F5, then
Individual plant is harvested, and next year kind in 3 generations of continuous plantation, obtains F into plant5:8Family, therefrom randomly selecting 200 familys carries out genetic map
Spectrum builds;
2nd, the structure of genetic map
1. parent and 200 filial generation F are extracted with CTAB methods5:8The DNA of family, is detected with Thermo nanodrop 2000
DNA concentration simultaneously detects DNA purity and integrality with 1% agarose electrophoresis;
2. using the SLAF-seq technologies and HighMap softwares pair of Beijing Biomarker Technologies Co., Ltd.'s independent research
2 parents and 200 filial generation exploitation High Density Molecular labels, carry out genetic map construction, comprise the following steps that:
(1) digestion scheme:Digestion prediction is carried out to having announced soybean reference gene group using digestion forecasting software, in selection
Enzyme cutting RsaI and HaeIII carries out digestion to detecting qualified each sample genome, and Select gene pack segment limit is in 364-
The SLAF fragments of 414bp;
(2) flow is sequenced:To SLAF fragment Klenow Fragment (3 ' → 5 ' exo -) (NEB) and dATP that obtain
3 ' ends plus A treatment, connection Dual-index sequence measuring joints, PCR amplification (pcr amplification primer things are carried out at 37 DEG C:F
AATGATACGGCGACCACCGA RCAAGCAGAAGACGGCATACG), purifying (Agencourt AMPure XP beads
(Beckman Coulter, High Wycombe, UK)), sample mixing, cut glue choose purpose fragment, library quality inspection it is qualified after use
IlluminaHiSeqTM2500 is sequenced;The accuracy of storehouse experiment is built for assessment, from paddy rice (Oryza sativa) conduct
Control (Control) carries out identical treatment participation and builds storehouse and sequencing;
(3) SNP labels and Genotyping:(3) positioning result according to sequencing Reads in reference gene group, GATK enters
Row part anharmonic ratio to (Local Realignment), GATK variation detection, samtools variation detection, take GATK and
The steps such as the common factor variant sites that two methods of samtools are obtained, to ensure to detect the accuracy of the SNP for obtaining, final screening
Available SNP labels are 111399;
(4) upper figure label filtration:In order to avoid the SNP clustering phenomenas on chromosome are to the shadow during genetic map construction
Ring, before being analyzed, part labels have been carried out with the treatment of removal redundancy, to each contig/supercontig, one
In the window (this project uses 120kb) of sizing, taking only one depth average highest Marker and representing the section is used for
Subsequent analysis, the 4902 SNP labels for filtering out altogether;
(5) linkage analysis:The 4902 SNP labels that will be filtered out, it is 20 to be divided SNP by the positioning with reference gene group
Individual linkage group, by calculating MLOD values between label two-by-two, figure 4 above 564, orientates icon note (Marker) as altogether;With even
Lock group is unit, and the linear array of Marker in linkage group is obtained using HighMap software analysis, and between estimating adjacent Marker
Genetic distance, finally give total figure away from the genetic map for 3403.90cM;
3rd, artificial infection idenfication parent and 200 root dry weights of filial generation
Test has carried out two batches in January, 2015 in December, 2016 in the Jilin Academy of Agricultural Science general intelligent greenhouse of peace
It is secondary.
1. soybean plant strain culture:Using river sand as cultivation medium, bucket 30cm high, diameter 25cm, every part of material 3 are cultivated
Bucket, 4 plants every barrel.The same period is sowed, and after emerging, 1L nutrient solutions (conventional formulation) is poured every 10 days.
2. root dry weight identification:The R7 phases are reached in material, the following root system of cotyledonary node is taken, after drying, is using accuracy
The balance of 0.001g obtains root dry weight data.
4th, the qtl analysis of soybean root dry weight
Using the average value of two batch root dry weights and the total figure of structure away from the genetic map for 3403.90cM, using rQTL
Many Interval Mappings of mapping software, with LOD >=3 as standard, qtl analysis are carried out to soybean root dry weight.Sent out on No. 9 chromosomes
Existing QTL intervals M45250-M45247, as shown in table 1.
The soybean root dry weight QTL of table 1 is interval
5th, the application of root dry weight QTL interval marks
The SNP marker of the QTL close linkage related to soybean root dry weight is M45250, M45247, and it is to wait to reflect
The genomic DNA of material is determined as template, and the fragment obtained by performing PCR amplification is entered with primer;The wherein nucleotide sequence of M45250
As shown in SEQ ID NO.1, the nucleotide sequence of M45247 is as shown in SEQ ID NO.2.
Wherein, the amplimer of M45250 is:
M50F:5 '-TCATTCCAGAGAGTGGTTCA-3 ', as shown in SEQ ID NO.3;
M50R:5 '-GTTGTTTACTATTACGGTGCCC-3 ', as shown in SEQ ID NO.4.
The amplimer of M45247 is:
M47F:5 '-GCCTCCTTTGTCATTCTTCT-3 ', as shown in SEQ ID NO.5;
M47R:5 '-TCTCTTTGGCAACCTGGA-3 ', as shown in SEQ ID NO.6.
Using comprising the following steps that for above-mentioned SNP marker auxiliary judgment root dry weight:
1. material genomic DNA to be identified is extracted using CTAB methods
1) take the fresh blade of soybean and add liquid nitrogen grinding powdering, take and be put into 1.5mL centrifuge tubes in right amount.
2) add 0.6mL preheating CTAB extract solutions, reverse mixing several times, water-bath one hour at a temperature of 65 DEG C, often
15min is mixed once, 12000rpm centrifugations 15min.
3) 0.6mL 24 is added:The chloroform of 1 (V/V):Isoamyl alcohol, overturns and mixes 5-10 times, 10000rpm centrifugations
15min。
4) take supernatant solution to be transferred in another empty centrifuge tube, with 24:The chloroform of 1 (V/V):Isoamyl alcohol weight
New extracting once, then adds 50 μ L RNase (10mg/mL) to place 30min at room temperature.
5) isometric -20 DEG C of isopropanols of precooling, the 30min in -20 DEG C of refrigerators, 5000rpm centrifugation 10min are added to go
Clearly.
6) cleaned twice with 70% ethanol.With sterilizing water dissolves after drying, genomic templates DNA is obtained, by genome mould
It is standby that plate DNA is put into 4 DEG C of Refrigerator stores.
7) with 0.8% agar sugar detection DNA concentration, be diluted to working concentration for PCR expand.
2. be utilized respectively M45250, M45247 primer enter performing PCR amplification, respectively obtain M45250 amplified productions and
M452476 amplified productions.
1) PCR amplification system:Cumulative volume is 20 μ L, including 10ng genomic templates DNA 2 μ L, 2 × Es Taq
Each 2 μ L and ddH of primer of MasterMix 10 μ L, 10mM2O 4μL。
2) PCR amplification conditions:94 DEG C of predegeneration 3min;94 DEG C of denaturation 30s, 57 DEG C of annealing 45s, 72 DEG C of extension 45s;Circulation
35 times;72 DEG C extend 10min eventually.
3. root dry weight is judged according to sequence alignment result
M45250 amplified productions and M45247 amplified productions are carried out into sequencing analysis respectively, M45250 amplified productions are held from 5 '
81st in maternal 1,000 to be A in G, the wild beans in male parent Chang Ling;M45247 amplified productions are from 5 ' the 65th, ends in female parent
To be C in A, the wild beans in male parent Chang Ling in 1000.When these sites of filial generation SNP with female parent it is consistent when, root dry weight greatly, when with
Root dry weight is small when male parent is consistent, and the judging nicety rate of M45250 is 84.6% for the judging nicety rate of 89.7%, M45247, therefore
Above-mentioned SNP marker M45250, M45247 can be used as the codominant marker of soybean root dry weight.
The QTL intervals of M45250-M45247 of the present invention are all ideal marks interval (being shown in Table 1) of soybean root dry weight,
Wherein M45250 is LOD peaks, and its contribution rate is 14.12%.The additive effect interval with the closely coupled QTL of this mark be
Negative value, i.e., 1,000 are the donor of the QTL.Therefore, either from the positioning that the QTL is interval, or QTL intervals are to phenotype
From the point of view of contribution rate, the interval with M45250-M45247 is all that the ideal mark of soybean root dry weight is interval.
Fig. 1 is to be located at genetic map and QTL mappings interval M45250-M45247 interval on No. 9 chromosomes, such as Fig. 1 institutes
Show, soybean root dry weight chromosome (09) the mapping interval M45250-M45247 as shown in table 1 provided using the present invention is only
0.125cM, apart from very little, and molecular labeling M45250, the M45247 for filtering out and soybean root dry weight close linkage, notable phase
Close, can be used for molecular marker assisted selection breeding, significantly improve the big material efficiency of selection of root dry weight, be further abundant soybean
Root dry weight regulated and control network provides a kind of most economical effective molecular breeding new way.
, but those skilled in the art once know basic creation although preferred embodiments of the present invention have been described
Property concept, then can make other change and modification to these embodiments.So, appended claims are intended to be construed to include excellent
Select embodiment and fall into having altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out various changes and modification without deviating from essence of the invention to the present invention
God and scope.So, if these modifications of the invention and modification belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising these changes and modification.
Sequence table
Sequence table
<110>Jilin Academy of Agricultural Science
<120>A kind of QTL related to soybean root dry weight, SNP marker and application
<160> 6
<170> PatentIn version 3.3
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<211> 231
<212> DNA
<213>Artificial sequence
<400> 1
tcattccaga gagtggttca acttctgctc cagctagtac tgcatcagtt tctgctggtc 60
aacagccggt caacatttct gttggcaagg aagtggttgt caatggcgag aatggatata 120
aggaaggcaa tggcaagcag agctctagtc atgacgattg tgatttgcaa gaatggtata 180
aaacttgttc tcatttagaa tagctaaaag ggcaccgtaa tagtaaacaa c 231
<210> 2
<211> 285
<212> DNA
<213>Artificial sequence
<400> 2
gcctcctttg tcattcttct acagtattcc acaccattta tttttgtatg aagttaaaat 60
atcaatgaac tcactgtcta gatatttaag catatacatg aatacaaagg attaaccatt 120
tgaagtttta gggtgagaag aggattcata tactaggcaa caataattat atcattttat 180
gggcatactt gatatgtttt ctaatcaatt gattttaata ttttacgtag cattgtttgt 240
taagtattac tgttaacctc catgttctcc aggttgccaa agaga 285
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
tcattccaga gagtggttca 20
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<400> 4
gttgtttact attacggtgc cc 22
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
gcctcctttg tcattcttct 20
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence
<400> 6
tctctttggc aacctgga 18
Claims (5)
1. a kind of QTL related to soybean root dry weight, it is characterised in that:The QTL relevant with soybean root dry weight is located at No. 9 chromosomes
M45250-M45247 it is interval, positioned by SNP marker M45250, M45247.
2. a kind of SNP marker of the QTL close linkages described in and claim 1, it is characterised in that the SNP molecules mark
M45250 is designated as, as shown in SEQ ID NO.1, the amplimer of M45250 is the nucleotide sequence of M45250:
M50F:5 '-TCATTCCAGAGAGTGGTTCA-3 ', as shown in SEQ ID NO.3;
M50R:5 '-GTTGTTTACTATTACGGTGCCC-3 ', as shown in SEQ ID NO.4.
3. the SNP marker of a kind of QTL close linkages described in claim 1, it is characterised in that the molecular labeling is
As shown in SEQ ID NO.2, the amplimer of M45247 is the nucleotide sequence of M45247, M45247:
M47F:5 '-GCCTCCTTTGTCATTCTTCT-3 ', as shown in SEQ ID NO.5;
M47R:5 '-TCTCTTTGGCAACCTGGA-3 ', as shown in SEQ ID NO.6.
4. applications of the QTL according to claim 1 in soybean root dry weight proterties seed selection.
5. the SNP marker according to claim any one of 2-3, in soybean root dry weight trait molecular marker assisted Selection
Application in seed selection, is carried out according to following technical scheme:
1) material genomic DNA to be identified is extracted using CTAB methods
2) primer for being utilized respectively M45250, M45247 enters performing PCR amplification, respectively obtains M45250 amplified productions and M452476
Amplified production;
3) M45250 amplified productions and M45247 amplified productions are sequenced respectively;
4) it is big G root dry weights that M45250 amplified productions hold the 81st from 5 ', and M45247 amplified productions hold the 65th to be done for A roots from 5 '
It is great.
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