CN110257488A - A kind of high-resolution solubility curve detection method of OsNramp5 gene specific locus mutation - Google Patents
A kind of high-resolution solubility curve detection method of OsNramp5 gene specific locus mutation Download PDFInfo
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Abstract
The invention discloses a kind of high-resolution solubility curve detection methods of OsNramp5 gene specific locus mutation, low cadmium rice strain can be quickly formulated by knocking out OsNramp5 gene, in the gene knockout that conventional CRISPR/Cas9 is mediated, the screening that the identification of transformed plant present age mutant and offspring remove label Mutants homozygous genotype is detected according to conventional sequencing approach, time and effort consuming, higher cost.In order to overcome disadvantage as described above, the present invention provides in a kind of work for knocking out OsNramp5 gene using CRISPR/Cas9, it quickly can detect whether to mutate in the transformed plant present age, and separate the high-resolution solubility curve detection method of offspring's Rapid identification homozygous mutation individual in mutant.Method provided by the invention has flux big, and speed is fast, advantage at low cost.
Description
Technical field
The invention belongs to Rice molecular breeding technical fields, are related to a kind of high score of OsNramp5 gene specific locus mutation
Resolution solubility curve detection method.
Background technique
Rice is a kind of crops for being easy to cadmium, and ploughing to be acidified or pollute causes active cadmium content in soil to increase
Add, and exacerbate Cadmium accumulation in rice, the exceeded event of rice cadmium is caused to take place frequently.Excess free enthalpy cadmium seriously endangers health,
Some areas in south China, rice cadmium is exceeded to have become the food-safety problem for causing social extensive concern.Selection kind
Planting low cadmium-accumulation rice varieties is the optimum method that current low cost quickly solves the problems, such as rice cadmium pollution.Studies have shown that rice
Middle OsNramp5 gene function Inactivating mutations may make that cadmium content is greatly reduced in seed [Ishikawa S, Ishimaru Y,
Igura M,et al.Ion-beam irradiation,gene identification,and marker-assisted
breeding in the development of low-cadmium rice[J].Proc Natl Acad Sci U S A,
2012,109 (47): 19166-19171], therefore OsNramp5 gene mutation body has significant application value.
The progress of gene editing technology can be used in the targeting mutation of gene, comprising being turned using zinc lipoprotein nuclease (ZFN), class
Record [Gaj, T., et al.ZFN, TALEN, the and CRISPR/ such as factorial effect protein nucleic acid enzyme (TALEN) and CRISPR system
Cas-based methods for genome engineering.Trends Biotechnol,2013,31(7):397-
405], using gene editing technology generate double-strand break induction generate mutation be usually the several bases of 1- missing or/and
It is inserted into (InDel) mutation, it is difficult to be detected by the method for regular-PCR combination general electrophoresis, it is therefore desirable to by DNA sequencing
Method carry out, especially gone in gene editing plant generations label homozygous mutation single plant identification work in, according to sequencing
Detect to mutation quite time-consuming laborious, and higher cost, thus needs more time saving and energy saving and cheap method.
Mononucleotide polypeptide (SNP) or InDel mutation can be detected by high-resolution solubility curve analytic approach.?
OsNramp5 gene carries out in the work of targeting knockout, by high-resolution solubility curve detection method to particular target series jump
Carrying out detection can be improved test efficiency, reduce cost.
Summary of the invention
The shortcomings that present invention is for target position point mutation detection time and effort consuming is carried out with sequencing approach after targeted gene disruption, mentions
It has supplied one kind when carrying out gene editing to the important control gene OsNramp5 of low cadmium character, specific gene editing sites has been carried out
The high-resolution solubility curve method of detection.
Unless otherwise specified, implementation of the invention by using the molecular biology of this field routine, biotechnology and
Agriculture field technology.Unless otherwise specified, term use herein is generally understood with those skilled in the art
Meaning.
The invention discloses a kind of high-resolution solubility curve detection method of OsNramp5 gene specific locus mutation, institutes
It states OsNramp5 gene specific site and refers to that rice genomic DNA sequence is identical as sequence shown in SEQ ID No.1 or exists
The site of 95% or more sequence similarity.
Preferably, the OsNramp5 gene specific locus mutation is completed by CRISPR/Cas9 system.
Any of the above-described scheme is preferably, and the CRISPR/Cas9 system uses sgRNA boot sequence (guide
It sequence is) sequence shown in SEQ ID No.2.
Any of the above-described scheme is preferably comprising the steps of:
(1) targeted gene disruption rice plant DNA is prepared；
(2) PCR reaction is carried out using primer pair；
(3) PCR product is transferred to the PCR reaction plate of high-resolution solubility curve analysis instrument adaptation, carries out high-resolution
The analysis of rate solubility curve；
(4) genotype is analyzed according to high-resolution solubility curve.
Any of the above-described scheme is preferably, and prepares targeted gene disruption rice plant with conventional method in the step (1)
DNA。
Any of the above-described scheme is preferably, and primer pair is SEQ ID No.3 and SEQ ID No.4 institute in the step (2)
Show the primer pair of sequence composition.
Any of the above-described scheme is preferably, and in the step (3), high-resolution solubility curve is carried out between 60-95 DEG C
Analysis.
Any of the above-described scheme is preferably, and PCR reactive component is as shown in table 1 in the step (2).
Any of the above-described scheme is preferably, when PCR reacts in the step (2), temperature program(me) are as follows: 94 DEG C of 2min；95℃
5S, 60 DEG C of 20S, 72 DEG C of 20S, 50 circulations；72℃1min；95℃3min；16℃2min.
Any of the above-described scheme is preferably, and addition high-resolution solubility curve analysis may be selected after the step (2)
One or both of general double chain oligonucleotide temperature internal standard, the double chain oligonucleotide temperature internal standard include in high temperature
Mark and 2 kinds of low temperature internal standard, the high temperature internal standard positive-sense strand be GCGGTCAGTCGGCCTAGCGGTAGCCAGCTGCGGCACTG
CGTGACGCTCAG, the low temperature internal standard positive-sense strand are ATCGTGATTTCTATAGTTATCTAAGTAGTTGGCATTAATAA
TTTCATTTT。
PCR reactive component in 1 HRM of table analysis
Any of the above-described scheme is preferably, and step (3) the middle high-resolution solubility curve analysis instrument is Idaho
96 system of lightscanner.
Any of the above-described scheme is preferably, in the step (4) solubility curve based on Genotyping should with to shining into
Row compares, and solubility curve is at least one of wild type peak, saltant type peak and heterozygosis peak.
Beneficial effect
High-resolution solubility curve (HRM) analysis is that the high throughput of Genotyping is carried out to SNP and short-movie section InDel variation
Cost effective method.High-resolution solubility curve method provided by the invention is suitable for but is not limited only to following scene:
(1) carry out OsNramp5 gene target knock out when, transformation seedlings the present age (be defined as T0 generation, offspring is followed successively by T1,
T2, T3 etc.) whether plant pair mutates is detected；
(2) when carrying out OsNramp5 gene target knockout, homozygous mutant genotypes are screened in T1 generation or later offspring；
(3) it is used for the molecular marker assisted selection breeding of OsNramp5 gene specific mutation type.
Method provided by the invention has the advantages that compared with common detecting methods:
(1) for wild type, miscellaneous when carrying out Genotyping to SNP or short-movie section InDel variation using general electrophoresis method
The resolution ratio of these three genotype of mould assembly and saltant type is poor, in addition completely can not parting, in contrast, side provided by the invention
Method can distinguish most heterozygous mutants of target site with wild type or homozygous mutant, and can be to specific mutation
Homozygous mutant, wild type and heterozygous carry out parting, thus the advantage incomparable with general electrophoresis method simultaneously.
(2) conventional PCR product sequencing needs to carry out PCR product purifying then further sequencing, time and effort consuming, cost compared with
Height, and method provided by the invention has detection time fast, flux is big, high-efficient, low in cost advantage.Therefore this hair is applied
The method of bright offer can improve efficiency and save the cost.
Detailed description of the invention
Fig. 1 is CRISPR/Cas9 carrier schematic diagram used in the present invention.LB, RB, the left and right boundary T-DNA；HPT, tide are mould
Plain resistance marker；2 × 35S, concatenated 35S promoter；Cmr- ccdB expression cassette, chloramphenicol resistance gene and expression of suicide gene
Box；SgRNA Gu Jia &Poly (T) 7, sgRNA skeleton and the terminator of 7 T base compositions；Corn Ubi gene promoter, driving
The promoter of Cas9 gene expression；The Cas9 of rice codon optimization encodes Cas9 gene；AarI, for boot sequence clone's
Restriction enzyme site；Pst I-Sna BI-Mlu I, three single restriction enzyme enzyme recognition sites；HPT and Cas9 are omitted in figure
Gene respectively expresses required 3 ˊ UTR and Nos polyA signal sequence of terminator CaMV；
Fig. 2 is the 5 kind OsNramp5 gene mutation body target position point mutation feelings formulated by CRISPR/Cas9 system
Condition.WT, wild type；#- number, mutation type number；The grey person of highlighting is and CRISPR/Cas9 system in wild-type sequence
The corresponding gene order site of system sgRNA boot sequence, wherein underlining base shows that the starting of U6 promoter turns in Cas9 system
Bases G is recorded, underscore base CGG indicates that spCas9 PAM site NGG, black inverted triangle indicate Cas9 nucleic acid cleavage sites,
All sequences direction is 5 ' -3 '；It lacks base in mutant sequence to be replaced with short-term, insertion base underlines display, remaining
For person identical as wild type；Figure digits right indicates missing (being indicated with "-") or the base number for being inserted into (being indicated with "+")；
Fig. 3 is the solubility curve map of different samples in the detection of high-resolution solubility curve.Figure A is shown as 3 kinds of double equipotentials
Genic mutation type (i.e. #01/#05, #03/#29, #06/#29) and wild type (WT/WT) can good discriminations；Figure B shows one pair
Three kinds of different genotypes of allelic mutation genotype (i.e. #05/#29) offspring Different Individual (i.e. homozygous #05/#05, #29/#
29 and heterozygous #05/#29) between each other can good discrimination)；Scheme C and shows wild type (WT/WT) and homozygous mutant #05/#05
It can good discrimination；Saltant type number mutant sequence is referring to fig. 2；Horizontal axis is temperature, and the longitudinal axis is the fluorescence of normalization and differentiation
It is worth curve.
Specific embodiment
Following embodiments are further explanations for the content of present invention using as the explaination to the technology of the present invention content, but
Substantive content of the invention is not limited in described in following embodiments, those skilled in the art can with and should know appoint
What simple change or replacement based on true spirit should belong to protection scope of the presently claimed invention.
Embodiment 1
The experimental method of the dated actual conditions in end in the following example, usually according to normal condition, such as books " molecule gram
Grand experiment guide (the 3rd edition) " (J. Pehanorm Brooker, D.W. Russell write；Huang Peitang etc. is translated；Science Press, 2008) institute in
The condition stated, or according to condition proposed by manufacture reagent or device manufacturer.
Embodiment 1, a kind of initiative of OsNramp5 gene specific locus mutation
Mutagenesis is carried out to OsNramp5 gene specific site by CRISPR/Cas9 system, is carried out as follows:
1.CRISPR vector construction
CRISPR/Cas9 vector construction.The CRISPR/Cas9 carrier that this research uses is voluntarily constructs, and carrier is with double base
Carrier pCUbi1390 (obtains) [Peng H, Zhang by adding corn Ubiquitin3 promoter on pCAMBIA1390 skeleton
Q,Li Y,et al.Aputative leucine-rich repeat receptor kinase,OsBRR1,is involved
In rice blast resistance.Planta, 2009,230 (2): 377-385] based on, at Kpn I and Bam HI
It is inserted into the Cas9 gene of rice codon optimization at point, continues the site the Hind III insertion side before Ubiquitin3 promoter
The rice U6 promoter-sgRNA frame member in the same direction to Cas9 gene [is inserted between promoter and sgRNA frame in component
One section of both ends is the Cm of Aar I siteR- ccdB expression cassette is to facilitate sgRNA boot sequence (guide sequence) segment
Clone；The transcription initiation site bases G of U6 promoter is included in], final carrier is named as pCUbi1390Cas9-U6 (such as
Shown in Fig. 1), carrier is in Escherichia coli ccdB SurvivalTM 2 T1RIt is saved in bacterial strain (Invitrogen).It is close containing rice
Numeral optimized Cas9 gene initial carrier and gives [Miao J, Guo D, Zhang J, et by the laboratory Qu Lijia
al.Targeted mutagenesis in rice using CRISPR-Cas system.Cell Res,2013,23(10):
1233-1236], rice U6 promoter is amplified from OryzasativaLcv.Nipponbare genomic DNA, the article delivered with reference to the laboratory Zhu Jiankang
[Feng Z,Zhang B,Ding W,et al.Efficient genome editing in plants using a
CRISPR/Cas system.Cell Res, 2013,23 (10): 1229-1232], sgRNA skeleton is tested from Feng Zhang
Room [Cong L, Ran F A, Cox D, et al.Multiplex genome engineering using CRISPR/Cas
Systems.Science, 2013,339 (6121): 819-823], CmR- ccdB expression cassette is purchased since Invitrogen
Gateway carrier pENTR1A.
Design CRISPR/Cas9 target site.Utilize online tool CRISPR-P (http://cbi.hzau.edu.cn/
Crispr/) sgRNA boot sequence (the guide designed for knockout OsNramp5 gene (RAP ID:Os07g0257200)
Sequence), using the boot sequence as shown in SEQ ID No.2, positive-sense strand of the corresponding target-gene sequence in exon10
Upper (SEQ ID No.3 sequence respectively corresponds OryzasativaLcv.Nipponbare with reference to genome ORF position: 3673-3692, with reference to the position mRNA CDS:
1006-1025)。
Synthesize go-ahead sequence DNA connector.When carrier construction, synthesis go-ahead sequence DNA connector is first required according to vector construction
Oligonucleotide.Positive-sense strand is cttgGGCAGAGCTCCACTATTAC (5 ' -3 '), antisense strand aaacGTAATAGTGGAGCTCTGCC
(5′-3′).It is connector after two oligonucleotides are annealed, connector both ends are 5 ' cantilevered out ends of 4 bases.Connector production method:
With 1 times of TE buffer solution oligonucleotide, concentration is 100pmol/ μ L, and complementary oligonucleotide is respectively taken mixed in equal amounts, 2% body is added
(annealing system is 10mM Tris-Cl to long-pending 5M NaCl in the method, and 1mM EDTA, 100mM NaCl, oligonucleotide is respectively
50pmol/ μ L), mix, 95 degree 2 minutes in PCR instrument, cooled to room temperature, connector is made, 50 times of connector dilution at
1pmol/ μ l is spare.
Vector linearization.With Aar I digestion pCUbi1390Cas9-U6 carrier, directly ethanol precipitation is simultaneously after the completion of digestion
Dry dissolution is spare after 70% ethanol wash desalination, and concentration is 50-100ng/ μ L.
Connection.With 50-100ng carrier, 1pmol connector, T4 ligase and buffer (Takara) each 1 μ L, 10 μ L are overall
Product, 16 degree of connection 2h.
Convert Escherichia coli.10 μ L whole connection products, all 100 μ LDH5 α Escherichia coli Competents of access are thin
Born of the same parents (Takara) are converted by the method that businessman provides, with the plate screening positive colony containing kanamycins.
Sequencing.Monoclonal is chosen, is sequenced with following primer pair monoclonal: OsU6-F
(TTGAGCGATTACAGGCGAAAGTG) (for detecting clone's correctness), 35S-F (TGACGCACAATCCCACTATCCTTC)
(riddled basins promoter primer, for detecting carrier integrality), Cas9-R-1 (TCGAGCCTGCGGGACTTAGAG；
Cas9 5 ' holds primer, is located at antisense strand, for detecting carrier integrality)；C126(TCGTGAAGAAGACCGAGGTT；Cas9
3 ' end primers, are located at positive-sense strand, this primer designs [Miao J, Guo D, Zhang J, et al.Targeted by Miao etc.
Mutagenesis in rice using CRISPR-Cas system.Cell Res, 2013,23 (10): 1233-1236],
For detecting carrier integrality), it chooses correct clone's expanding propagation and to extract plasmid spare.
2. genetic transformation
Kind Kasalath, China is selected to account for, five rich B, five mountains silk seedling and middle morning 35 are as transformation receptor.The Ti that will be built
Binary plasmid carrier is converted into EHA105 Agrobacterium, and Agrobacterium competent cell preparation method uses CaCl2 method, and conversion uses
Multigelation method.For kind Kasalath, genetic transformation carries out [27] using the method for Toki etc.；China accounts for, five rich B, five mountains
The conversion of silk seedling and middle morning 35 entrusts Wuhan Biorun Bio-Tech. Co., Ltd. to complete.
3. obtaining OsNramp5 gene target site mutant
The gene knockout target site in transgenic positive plant is tentatively examined by the method for PCR product direct Sequencing
It surveys, thinks there is mutation if having set peak in sequencing result peak figure, continue for segment to be cloned into plasmid vector and be sequenced, often
A sample at least five positive colony therefrom analyzes two respective genotype of allele, if sequencing result without set peak,
Sequence alignment is carried out with compareing, learns genotype.PCR primer: TTCGTGGCGCTGCTGATAAAC (forward direction) and
AGAGCGGAGAAATATGGACGAAAGT (reversed).PCR reaction system: 5 μ l 10 × PCR Buffer (Mg2+plus；
Takara), 4 μ l dNTP Mixture (each 2.5mM), 5 μ l primers (forward and reverse mixing, each 5pmol/ μ l), 0.5 μ l Takara
RTaq (5U/ μ l), 2 μ l DMSO (≤99.5%), 2 μ l DNA, 31.5 μ l H2O, 50 μ l of total volume.PCR temperature program(me): 95 DEG C
2min；94 DEG C of 30S, 58 DEG C of 30S, 72 DEG C of 60S, 40 circulations；72℃2min.PCR product direct Sequencing primer (amplified fragments
It is interior, it is located at antisense strand): GTGCACCCCTACAATTCGTCAGT.Sequencing commission Hangzhou Qing Ke Bioisystech Co., Ltd completes.
Sequencing result is analyzed, knows each strain catastrophe.All strain catastrophes are as shown in table 2, in mutating strain series
Target site catastrophe as shown in Fig. 2, different mutating strain series respectively genotype is as shown in table 3.
2 different cultivars positive transformants gene mutation situation of table statistics
The genotype of 3 five kind OsNramp5 gene editing strains of table
Embodiment 2 detects OsNramp5 gene specific locus mutation using high-resolution solubility curve detection method
High-resolution solubility curve method is provided using the present invention to plant the transformation seedlings T0 generation that the method using embodiment 1 obtains
Strain is analyzed, and is compared with sequencing result, and discovery the method provided by the present invention can identify nearly all heterozygosis or double
Allelic mutation genotype, typical testing result are shown in Fig. 3 (A).
To embodiment 1 obtain mutant T1 for plant, filtering out not hygromycin using leaf section hygromycin infusion method
After being free of the single plants of transgene components such as resistance marker, high-resolution solubility curve detection method pair of the present invention is utilized
It is mutated single plant and carries out homozygote identification.90% or more strain can preferably distinguish homozygous and heterozygous as the result is shown, and 40% or so
Homozygous and heterozygous can not only be distinguished, moreover it is possible to distinguish two kinds of homozygous genotypes, typical qualification result is shown in Fig. 3 (B).
In order to which embodiment 2 is obtained the molecular marker assisted selection breeding that part mutated gene is applied to future, this is utilized
The invention high-resolution solubility curve detection method analyzes the homozygous genotype of multiple mutant with wild type,
As a result, it has been found that number is that the genotype of #05, #06, #09 and #14 perfect can be distinguished as shown in Figure 2, typical qualification result is shown in Fig. 3
(C).Thus base is mutated to containing these in the molecular marker assisted selection breeding in future using method provided by the invention
Because the individual of type is identified.
The specific method is as follows for high-resolution solubility curve analysis detection:
96 system of high-resolution solubility curve analysis and utilization Idaho lightscanner carries out, and is expanded using small fragment
Method.PCR primer and amplicon: CTGGGCAAGTCGAGTGCGAT is (positive；That is SEQ ID No.3) and
ATTACCTGCATGATGTACTGTCC is (reversed；That is SEQ ID No.4), amplified production 104bp.PCR reaction system: 1 μ l 10
×PCR Buffer(Mg2+plus；Takara), 0.8 μ l dNTP Mixture (each 2.5mM), 1 μ l primer (forward and reverse mixing,
Each 5pmol/ μ l), 0.1 μ l Takara rTaq (5U/ μ l), 0.4 μ l DMSO (>=99.5%), 1 μ l DNA, 1 μ l LC-
Green fluorescent dye, 4.7 μ l H2O, 10 μ l of total volume, above plus 20 μ l mineral oil.PCR temperature program(me): 94 DEG C of 2min；95℃
5S, 60 DEG C of 20S, 72 DEG C of 20S, 50 circulations；72℃1min；95℃3min；16℃2min.By PCR system together with paraffin oil whole
It is transferred in dedicated PCR plate (Bio-rad, black shell/white well PCR plate, article No. hsp9665) and carries out HRM
Temperature range 65-95 degree is analyzed in analysis.Data Analysis Services carry software with system and carry out, and analysis mode selects small fragment method.
It may be selected to add high-resolution solubility curve and analyze to be marked with to improve in general temperature have the different samples of phase homogenic type molten
The concentration degree of solution curve, to improve the accuracy of analytical effect.It is designated as double-strand oligonucleotide in temperature, is divided into high temperature and low temperature internal standard
Two kinds, respective sense strand sequence be respectively GCGGTCAGTCGGCCTAGCGGTAGCCAGCTGCGGCACTGCGTGACGCTCAG and
High/low temperature internal standard may be selected when test and add by ATCGTGATTTCTATAGTTATCTAAGTAGTTGGCATTAATAATTTCATTTT
Add or only add one of high temperature and low temperature internal standard.
Sequence table
<110>Jiangxi Province's super rice research and development center (academy of agricultural sciences, Jiangxi Province, rice breeding center, Hainan)
<120>the high-resolution solubility curve detection method of a kind of OsNramp5 gene specific locus mutation
<130> 1110000000
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 104
<212> DNA
<213> Oryza sativa
<400> 1
ctgggcaagt cgagtgcgat cgtgtacggc gtggcactgt tggcatctgg gcagagctcc 60
actattaccg gcacatacgc tggacagtac atcatgcagg taat 104
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized sequence (Artificial Sequence)
<400> 2
gggcagagct ccactattac 20
<210> 3
<211> 20
<212> DNA
<213>artificial synthesized sequence (Artificial Sequence)
<400> 3
ctgggcaagt cgagtgcgat 20
<210> 4
<211> 23
<212> DNA
<213>artificial synthesized sequence (Artificial Sequence)
<400> 4
attacctgca tgatgtactg tcc 23
Claims (8)
1. a kind of high-resolution solubility curve detection method of OsNramp5 gene specific locus mutation, which is characterized in that described
OsNramp5 gene specific site refers to that rice genomic DNA sequence is identical as sequence shown in SEQ ID No.1 or there are 95%
The site of the above sequence similarity.
2. a kind of high-resolution solubility curve detection side of OsNramp5 gene specific locus mutation according to claim 1
Method, which is characterized in that the OsNramp5 gene specific locus mutation is completed by CRISPR/Cas9 system.
3. a kind of high-resolution solubility curve detection side of OsNramp5 gene specific locus mutation according to claim 2
Method, which is characterized in that the sgRNA boot sequence that the CRISPR/Cas9 system uses includes sequence shown in SEQ ID No.2
Column.
4. the high-resolution solubility curve of OsNramp5 gene specific locus mutation according to any one of claim 1-3
Detection method, which is characterized in that comprise the steps of:
(1) targeted gene disruption rice plant DNA is prepared；
(2) PCR reaction is carried out using primer pair；
(3) PCR product is transferred to the PCR reaction plate of high-resolution solubility curve analysis instrument adaptation, it is molten to carry out high-resolution
Solution curve analysis；
(4) genotype is analyzed according to high-resolution solubility curve.
5. a kind of high-resolution solubility curve detection side of OsNramp5 gene specific locus mutation according to claim 4
Method, which is characterized in that primer pair is the primer of the composition of sequence shown in SEQ ID No.3 and SEQ ID No.4 in the step (2)
It is right.
6. the high-resolution solubility curve detection method of OsNramp5 gene specific locus mutation according to claim 4
Preparation process, which is characterized in that addition high-resolution solubility curve may be selected after the step (2) and analyze general double-strand
Oligonucleotides temperature internal standard.
7. the high-resolution solubility curve detection method of OsNramp5 gene specific locus mutation according to claim 4
Preparation process, which is characterized in that in the step (3), the analysis of high-resolution solubility curve is carried out between 60-95 DEG C.
8. the high-resolution solubility curve detection method of OsNramp5 gene specific locus mutation according to claim 4
Preparation process, which is characterized in that solubility curve based on Genotyping should be compared with control in the step (4), molten
Solution curve is at least one of wild type peak, saltant type peak and heterozygosis peak.
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