CN110343781A

CN110343781A - A kind of identification method of rice Wx gene mutation and its application

Info

Publication number: CN110343781A
Application number: CN201910718870.5A
Authority: CN
Inventors: 郭涛; 陈淳; 周丹华; 黄翠红; 严贤诚; 孙凯; 王慧; 陈志强
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-08-05
Filing date: 2019-08-05
Publication date: 2019-10-18
Anticipated expiration: 2039-08-05
Also published as: CN110343781B

Abstract

The invention discloses a kind of identification methods of rice Wx gene mutation, comprising: extracts rice plant genome building DNA mixing pit to be measured, while extracting the genomic DNA of control rice plant；4 pairs of nest-type PRC primers are designed for Wx gene；Nest-type PRC is carried out respectively as template using the genomic DNA of the DNA mixing pit of rice plant genome to be measured building and control rice plant；HRM analysis is carried out to PCR product, determines doubtful mutation mixing pit；The corresponding single plant DNA of doubtful mutation mixing pit is extracted respectively, single plant DNA is mixed with adjoining tree DNA and carries out nest-type PRC, and amplified production carries out HRM analysis and obtains doubtful mutation single plant, is that template carries out PCR row agarose gel electrophoresis of going forward side by side and identifies using its DNA.TILLING technology is combined the screen mutation applied to rice Wx gene with HRM detection means by the present invention, provides new approaches for rice mutagenic progeny efficiently and directionally screening.

Description

A kind of identification method of rice Wx gene mutation and its application

Technical field

The invention belongs to the identification method of field of plant genetic project technology more particularly to a kind of rice Wx gene mutation and It is applied.

Background technique

Directional induction genome abrupt local technology (TILLING) is reverse-genetics approach, in gene functional research And screen mutation etc. plays an important role.The technology is at the end of the nineties in last century by U.S. Fred A kind of efficient detection that the research group of the Steven Henikoff leader of Hutchinson Cancer Research Center grows up The method of SNP, and mutant screening has been carried out using EMS mutagenesis group of the TILLING to arabidopsis for the first time.At present quasi- In the crops such as southern mustard, corn and soybean, tomato, rape, tobacco, peanut, cotton, radish, wheat, barley, rice, sorghum extensively Using.

Earlier T ILLING is to utilize denaturing high-performance liquid chromatography (Denaturing High Performance Liquid Chromatography, DHPLC) detection.But since its test period is long, detection efficiency is low, the limitation such as at high cost, Replaced quickly by other more effective detection methods.The most classical, the Two Colour Fluorescence detection system being most widely used gradually is sent out Transform into ripe, this method is based on the digestion to mismatch site and to the electrophoresis point of the digestion products with different colours fluorescence From come the presence that detects mutation.For efficiency, the accuracy for improving detection, cost, many new abrupt climatic change means are constantly reduced It is introduced sequentially into.Its middle high-resolution solubility curve (HRM) technology is by detecting the DNA double chain in conjunction with saturated fluorescence dyestuff The variation tendency of fluorescence intensity in dehybridization procedure and then the difference realization for being depicted as solubility curve, and passing through analysis curve Detection and classification to DNA double chain difference, the detection technique be not necessarily to digestion system, have it is easy to operate, at low cost, high-throughput, The advantages that high-resolution, high sensitivity and specificity.Using TILLING combination HRM detection technique in tomato, rape, great Bai It is applied in the crops such as dish, wheat.

Rice is as important cereal crops and unifacial leaf mode plant, general also fewer for the application of TILLING The screen mutation that TILLING combination HRM technology is applied to rice target gene has not been reported.The Wx gene of rice is control The key gene of rice grain amylose content, function variation directly result in the variation of amylose content and influence rice product Matter has important value in rice quality breeding.Currently, widely applied Wx gene has Wx in rice breeding^aAnd Wx^bTwo Kind, both genotype are long-grained nonglutinous rice, japonica rice differentiation as a result, wherein Wx^aIt is the genotype being widely present in long-grained nonglutinous rice, this gene The amylose content that type determines is middle high type；Wx^bIt is the genotype of low amylose content in control in japonica rice.With The rice varieties demand of the development of social economy, different amylose contents becomes more diverse, existing Wx^aAnd Wx^bTwo kinds of bases Because being difficult to meet demand, it is necessary to create more Wx allelic variant genes using mutagenesis means and serve breeding.

Summary of the invention

In order to solve the above technical problem, the present invention provides a kind of identification methods of rice Wx gene mutation, main to utilize TILLING-HRM system screens the mutation of rice mutagenic progeny, and it is prominent to realize the target gene based on " sample mixes pond " Become mass screening.

In order to achieve the above object, the present invention adopts the following technical scheme that:

A kind of identification method of rice Wx gene mutation, which comprises the steps of:

(1) using not mutagenized rice plant as check plant, with mutagenized rice plant for plant to be measured Strain extracts plant genome building DNA mixing pit to be measured, while extracting the genomic DNA of adjoining tree；

(2) according to the Wx gene order announced, in 4,9,10 exon regions and Wx^aAnd Wx^bDifference site is set Nest-type PRC outer primer and inner primer are counted, is with the genomic DNA of the DNA mixing pit of plant to be measured building and adjoining tree respectively The outer primer of the nest-type PRC primer is added in template, carries out the 1st wheel PCR amplification, ddH is added after amplification₂O dilution；

(3) nest-type PRC inner primer and fluorescent dye are added using product after PCR dilution in step (2) as template, progress the 2 wheel PCR amplifications after amplification, need to carry out 1 circulation again；

(4) the 2nd wheel amplified production obtained by step (3) is centrifuged respectively and is transferred in HRM detection plate, carry out HRM detection Analysis, using HRM curve corresponding to adjoining tree as benchmark line, HRM curve and adjoining tree corresponding to plant more to be measured The maximum fluorescence discrepancy delta F of corresponding HRM curve；If | Δ F | > 0.05, it is considered as the result of plant and adjoining tree to be measured not Together, there are the plant in mutational site in the preliminary judgement DNA mixing pit, are considered as doubtful mutation mixing pit；

(5) the corresponding single plant of doubtful mutation mixing pit that screening obtains in step (4) is extracted into DNA respectively and is diluted, it will Single plant DNA and adjoining tree DNA is mixed in a certain ratio carry out nested PCR amplification, and amplified production carries out HRM detection and analysis, The same step of method (2)-(4)；

(6) PCR amplification is carried out as template to screen obtained doubtful mutation single plant DNA, the primer is institute in step (2) State the outer primer of nido RCR primer；

(7) PCR product is detected through agarose gel electrophoresis, and band is obvious and single, is sequenced, and is determined specific prominent Displacement point；

Wherein,

The nest-type PRC outer primer and inner primer for the design of the 4th exon region of Wx gene order, sequence is such as Under:

EX4-O outer primer-upstream primer: 5 '-CAGATCATCACAAGATCGATTAGC -3 ' (SEQ ID NO:1),

EX4-O outer primer-downstream primer: 5 '-AACCTGAAATCACCAGTGGAAG -3 ' (SEQ ID NO:2),

EX4-I inner primer-upstream primer: 5 '-CCAAGTTTTTGTGGTGCAATTC -3 ' (SEQ ID NO:3),

EX4-I inner primer-downstream primer: 5 '-TAAGCTCAGTCCAACTGCTAAATG -3 ' (SEQ ID NO:4)；

The nest-type PRC outer primer and inner primer for the design of the 9th exon region of Wx gene order, sequence is such as Under:

EX9-O outer primer-upstream primer: 5 '-CGACGGGTATGAGTAAGATTCTAAG -3 ' (SEQ ID NO:5),

EX9-O outer primer-downstream primer: 5 '-CATTCGTTCTTACCGTGGTTG -3 ' (SEQ ID NO:6),

EX9-I inner primer-upstream primer: 5 '-AGATCCTTTTGAGCTGACAACC -3 ' (SEQ ID NO:7),

EX9-I inner primer-downstream primer: 5 '-GTACTTGGCGGTGATGTACTTG -3 ' (SEQ ID NO:8)；

The nest-type PRC outer primer and inner primer for the design of Wx gene order exon10 region, sequence is such as Under:

EX10-O outer primer-upstream primer: 5 '-AGAACGAATGCATTCTTCACAAGA -3 ' (SEQ ID NO:9),

EX10-O outer primer-downstream primer: 5 '-GCCTCACCCCTTCTAATTATTTGA -3 ' (SEQ ID NO:10),

EX10-I inner primer-upstream primer: 5 '-ACAAGGCAAGAATGAGTGACAA -3 ' (SEQ ID NO:11),

EX10-I inner primer-downstream primer: 5 '-GCATATCGTGCAAGTGTGTCTT -3 ' (SEQ ID NO:12)；

It is described to be directed to Wx gene order Wx^aAnd Wx^bNest-type PRC outer primer and inner primer are designed in difference site, and sequence is such as Under:

Wxab-O outer primer-upstream primer: 5 '-GAGGGAGAGGGGGAGAGAGAGATC -3 ' (SEQ ID NO:13),

Wxab-O outer primer-downstream primer: 5 '-TCCAGCCCAACACCTTACAGAAAT -3 ' (SEQ ID NO:14),

Wxab-I inner primer-upstream primer: 5 '-CTTCACTTCTCTGCTTGTGTTGTT -3 ' (SEQ ID NO:15),

Wxab-I inner primer-downstream primer: 5 '-CATGTGATCGATCTGAATAAGAGG -3 ' (SEQ ID NO:16).

Further, DNA mixing pit ratio is 1~9:1 in the step (2).

Further, the 2nd wheel PCR reaction system in the step (3) are as follows: 4.0 μ L 2 × Taq Plus Master Mix, each 0.3 μ L of inner primer (10 μm of ol/L), 1.0 μ L template DNAs, Evagreen saturated fluorescence dyestuff 0.3 μ L, ddH₂O mend to 10 μ L, and add 15 μ L mineral oil.

Further, in the step (3), the 2nd wheel PCR response procedures are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s, most suitable annealing temperature annealing 30s, 72 DEG C of extension 30s, 15 circulations；72 DEG C of extension 5min.

Further, in the step (3) after the 2nd wheel PCR amplification, then carrying out the program of 1 circulation is 95 DEG C of changes Property 30s, 25 DEG C of annealing 1min.

Further, nest-type PRC inner primer obtained by the 2nd wheel PCR expands target fragment length < 500bp.

Further, single plant DNA and adjoining tree DNA is mixed in the step (5) in 1:1 ratio and carries out nest-type PRC Amplification.

Further, the dissolution program of the HRM detection is determined according to target fragment G/C content, the purpose of low G/C content It is 66 DEG C of starting, 96 DEG C of end, 62 DEG C of holding, exposure value 180, the target fragment dissolution of high GC content that segment, which dissolves program, The initial temperature of program is set as 75 DEG C, and other parameters are constant.

Further, the doubtful mutation single plant that screening obtains in the step (6) carries out the reaction system and journey of PCR amplification Sequence are as follows: 25.0 μ L 2 × Taq Plus Master Mix, each 2 μ L of primer (10 μm of ol/L), 2.0 μ L template DNAs, ddH₂O is mended To 50 μ L.PCR program is 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, the most suitable annealing temperature annealing 30s of primer, 72 DEG C of extensions 30s, 35 circulations；72 DEG C of extension 5min.

Compared with prior art, the invention has the following beneficial effects:

(1) compared with traditional TILLING method, the TILLING-HRM system that the present invention uses is not necessarily to digestion system, Easy to operate, sensitivity is higher.

(2) detection method provided by the invention carries out screen mutation in a manner of DNA mixing pit, and filtering out has mutation base Single plant identification is carried out behind the pond of cause again, and traditional abrupt climatic change need to be sequenced respectively a single plants up to ten thousand and determine genotype.The two Compare, present invention significantly reduces mutant to detect workload；In addition, single of the present invention is 96 or 384 DNA detectable Mixing pit can complete detection for 10 minutes, greatly improve detection efficiency, reduce testing cost indirectly.

(3) the present invention provides the primer sequences for the Wx detection in Gene Mutation for being suitable for mixing pit strategy, and are applied successfully It is screened in Wx mutated gene, for instructing the screening of mutated gene in rice that there is more practical value.

Detailed description of the invention

Fig. 1 a is that XF2-9-3, XF2-9-5 different proportion mix pond HRM testing result in embodiment 1.

Fig. 1 b is that H481, H353 different proportion mix pond HRM testing result in embodiment 1.

Fig. 1 c is that B88, B92 different proportion mix pond HRM testing result in embodiment 1.

Fig. 2 a is Wxab-O, Wxab-I primer extension product HRM testing result in embodiment 1.

Fig. 2 b is EX2-O, EX2-I primer extension product HRM testing result in embodiment 1.

Fig. 2 c is EX4-O, EX4-I primer extension product HRM testing result in embodiment 1.

Fig. 2 d is EX9-O, EX9-I primer extension product HRM testing result in embodiment 1.

Fig. 2 e is EX10-O, EX10-I primer extension product HRM testing result in embodiment 1.

Fig. 2 f is EX14-O, EX14-I primer extension product HRM testing result in embodiment 1.

Fig. 3 a is Wxab-O, Wxab-I primer extension product HRM testing result in embodiment 2.

Fig. 3 b is EX4-O, EX4-I primer extension product HRM testing result in embodiment 2.

Fig. 3 c is EX9-O, EX9-I primer extension product HRM testing result in embodiment 2.

Fig. 3 d is EX10-O, EX10-I primer extension product HRM testing result in embodiment 2.

Fig. 4 is the single plant HRM difference curve that EX10-O and EX10-I primer extension product detects in embodiment 2.

Fig. 5 a is that comparison result is sequenced in mutation single plant in embodiment 3.

Fig. 5 b is that single plant amino acid alignment result is mutated in embodiment 3.

Fig. 5 c is that peak figure is sequenced in the Sanger of mutation single plant control in embodiment 3.

Fig. 5 d is the Sanger sequencing peak figure that single plant is mutated in embodiment 3.

Fig. 6 is implementation flow chart of the invention.

Specific embodiment

No. 31 rice dry seeds of China Airlines Limited pass through " practicing No. ten " recoverable scientific experiment Seeds of First Post-flight and respectively in Lanzhou The energetic ion radioactivity line that Contemporary Physics research institute, state utilizes baryon research device HIRFL to provide¹²C⁶⁺It carries out at irradiation Reason,¹²C⁶⁺Irradiation energy 80.55MeV/u, dosage 80Gy, dosage rate 60Gy/min, while retaining untreated control.Mutagenesis The seed of processing recycling is a mutagenesis generation, and the main fringe of single plant after plantation is mixed to harvest to obtain two generation of mutagenesis.

Embodiment 1

The determination of DNA mixing pit multiplying power is used for using 3 groups of rice with known base difference, material to be shown in Table 1.

1 base difference donor material information of table

According to GRAMENE (http://www.gramene.org/) announce Wx gene order, its 2nd, 4,9,10, Nest-type PRC primer is designed in 14 exon regions and Wxa and Wxb difference site.In addition each in Pi2 and Pik-H4 variant sites 1 pair of nest-type PRC primer is designed, all primers are as shown in table 2.Primer 5.0 software design of Primer Premier.By Shanghai The synthesis of Sheng Gong bioengineering Co., Ltd.Suitable annealing temperature is determined using grads PCR.

2 nest-type PRC primer sequence of table

Genomic DNA is extracted using paramagnetic particle method to 3 groups of rice in table 1, the DNA of extraction uses microplate spectrophotometer (EPOCH2T) it measures concentration and is adjusted to 50ng/ μ L.By this 3 groups genomic DNAs with known base difference uniformly press 1:1, The DNA mixing pit that the ratio mixing of 3:1,5:1,7:1,9:1,11:1 constitute 18 different multiplyings is tested and analyzed for HRM, knot Fruit such as Fig. 1 a- Fig. 1 c.Wherein XF2-9-3, XF2-9-5 different proportion mix pond detection, and detection gene is Pik-H4； H481,H353 Different proportion mixes pond detection, and detection gene is Pi2；B88, B92 different proportion mix pond detection, and detection gene is Wx, the primer For Wxab-O and Wxab-I.

In HRM difference curve, will | Δ F | > 0.05 regards significant difference as, shows that there are doubtful mutation.XF2-9-3, The DNA mixing pit of each multiplying power of two groups of materials of XF2-9-5 and H481, H353 can be detected difference (Fig. 1 a and Fig. 1 b). The DNA mixing pit that B88, B92 are constituted can not detect difference (Fig. 1 c) in the ratio of 11:1.

The testing result of this comprehensive each ratio DNA mixing pit of 3 groups of materials, mixing pit ratio difference at 9:1 (10 times of ponds) Curve is all 0.05 or more.In view of the case where there may be heterozygotes in actually detected, and the extraction of paddy DNA uses The method extracted again after blade is mixed in advance causes the amount of the DNA of each material to have differences, so to guarantee abrupt climatic change Sensitivity, multiplying power of the 4 times of ponds of final choice as crowd surveillance.

Two generation of mutagenesis group DNA mixing pit is constructed using 4 times of ponds, utilizes 6 pairs of nidos in each site design of Wx gene PCR primer carries out HRM detection to 8 DNA mixing pits selected at random, as a result as shown in Fig. 2 a- Fig. 2 f.

Wherein the amplified production dissolution peak of EX2-O, EX2-I primer is single, but temperature corresponding to its dissolution peak is excessively high Cause peak shape imperfect (Fig. 2 b)；EX14-O, EX14-I primer extension product respectively have in about 79 DEG C and 92 DEG C one it is obvious Dissolution peak, show that its amplified production is not single or product has multiple dissolution regions (Fig. 2 f).Wxab-O/Wxab-I,Ex4- O/Ex4-I, Ex9-O/Ex9-I, Ex10-O/Ex10-I tetra- can be used for the screening of Wx mutated gene to primer.

Embodiment 2

4736 parts of rice space-induced line SP2 single plants construct 1184 4 times of DNA mixing pits, 4848 parts of rice heavy ion mutagenesis M2 single plant constructs 1212 4 times of DNA mixing pits.It is expanded using the 4 pairs of nest-type PRC primers filtered out in embodiment 2, often Product after the PCR amplification of a PCR96 orifice plate 93 DNA mixing pits of placement and 3 comparison DNAs, and unified diluted concentration is extremely HRM detection and analysis are carried out after 50ng/ μ L.

It is 4 pairs of nest-type PRC primer amplifications of one of them 96 orifice plate DNA mixing pit of space mutagenesis shown in Fig. 3 a- Fig. 3 d The HRM testing result of product.As can be seen that expanding in Wxab-O and Wxab-I primer extension product, EX9-O and EX9-I primer There are 2,4,1 respectively in the detection of volume increase object and EX10-O and EX10-I primer extension product | Δ F | > 0.05 difference is bent Line is considered as doubtful mutation pond (Fig. 3 a, Fig. 3 c, Fig. 3 d).EX4-O, EX4-I primer extension product, which have no, detects difference curve (Fig. 3 b).

Secondary verifying is carried out to doubtful mutation pond.Final SP2 group DNA mixing pits of space mutagenesis screen 9 altogether and doubtful dash forward Become pond, wherein Wxa and 3, Wxb difference site primer region, 4 detection zone of exon 3,9 detection zone of exon 2, 10 detection zone of exon 1；Heavy ion mutagenesis M2 group DNA mixing pit screens 14 doubtful mutation ponds altogether, wherein Wxa with 5, Wxb difference site primer region, 4 detection zone of exon 3,9 detection zone of exon 2,10 detection zone 4 of exon It is a.

By the screening of the Wx gene of whole group DNA mixing pits, 23 doubtful mutation ponds have been obtained, these are doubtful It is mutated the corresponding single plant in pond and DNA is extracted in control respectively, and unified diluted concentration mixes building 2 to press 1:1 after 50ng/ μ L Times pond carries out amplification using the corresponding primer in the region of doubtful mutation and HRM is tested and analyzed.In 23 doubtful mutation ponds, There are 18 to obtain repeated authentication, illustrates the method for mixing pit accuracy with higher.Further select EX10-O and 1 difference curve that EX10-I is detected is verified (Fig. 4).By multiple authentication, the difference exists, and shows that the single plant exists There is mutation in exon10 region, which is heavy ion mutagenesis M2.

Embodiment 3

The doubtful mutation single plant heavy ion mutagenesis M2 filtered out in embodiment 2 is sequenced, sequence alignment result is shown The mutation single plant produces the conversion (Fig. 5 a) of C → T in the 115th site of exon10, and codon becomes UCU by CCU, For missense mutation.The mutation causes coding proline to become encoding serine (Fig. 5 b), thus it is speculated that it can produce the function of Wx gene It is raw to influence.Peak figure is sequenced in comparison control and the Sanger of the mutation single plant mutated site, it is found that the mutant is heterozygous mutant (figure 5c,5d)。

The present invention is dashed forward using the Wx gene of TILLING-HRM system and 4 pairs of nest-type PRC primer pair mutagenic progeny groups Become screening, can detect that the replacement of single base, finally obtained 1 heterozygous mutant single plant, it was demonstrated that the system is with higher High sensitivity, this method are the effective ways (Fig. 6) of the acquisition various new allelic variants of rice.

Embodiment described above is only that preferred embodiment of the invention is described, and is not carried out to the scope of the present invention It limits, without departing from the spirit of the design of the present invention, those of ordinary skill in the art make technical solution of the present invention Various changes and improvements, should all fall into claims of the present invention determine protection scope in.

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Claims

1. a kind of identification method of rice Wx gene mutation, which comprises the steps of:

(1) it using not mutagenized rice plant as check plant, with mutagenized rice plant for plant to be measured, mentions Plant genome building DNA mixing pit to be measured is taken, while extracting the genomic DNA of adjoining tree；

(2) according to the Wx gene order announced, in 4,9,10 exon regions and Wx^aAnd Wx^bDesign nido in difference site PCR outer primer and inner primer add respectively using the DNA mixing pit of plant to be measured building and the genomic DNA of adjoining tree as template Enter the outer primer of the nest-type PRC primer, carries out the 1st wheel PCR amplification, ddH is added after amplification₂O dilution；

(3) using product after PCR dilution in step (2) as template, nest-type PRC inner primer and fluorescent dye is added, carries out the 2nd wheel PCR amplification after amplification, need to carry out 1 circulation again；

(4) the 2nd wheel amplified production obtained by step (3) is centrifuged respectively and is transferred in HRM detection plate, carry out HRM detection and analysis, Using HRM curve corresponding to adjoining tree as benchmark line, corresponding to HRM curve corresponding to plant more to be measured and adjoining tree HRM curve maximum fluorescence discrepancy delta F；If | Δ F | > 0.05, it is different from the result of adjoining tree to be considered as plant to be measured, tentatively Determine that there are the plant in mutational site in the DNA mixing pit, are considered as doubtful mutation mixing pit；

(5) the corresponding single plant of doubtful mutation mixing pit that screening obtains in step (4) is extracted into DNA respectively and is diluted, by single plant DNA and adjoining tree DNA is mixed in a certain ratio carry out nested PCR amplification, and amplified production carries out HRM detection and analysis, method Same step (2)-(4)；

(6) PCR amplification is carried out as template to screen obtained doubtful mutation single plant DNA, the primer is nest described in step (2) The outer primer of formula RCR primer；

(7) PCR product is detected through agarose gel electrophoresis, and band is obvious and single, is sequenced, and determines specific mutation position Point；

Wherein,

The nest-type PRC outer primer and inner primer for the design of the 4th exon region of Wx gene order, sequence are as follows:

EX4-O outer primer-upstream primer: 5 '-CAGATCATCACAAGATCGATTAGC -3 ',

EX4-O outer primer-downstream primer: 5 '-AACCTGAAATCACCAGTGGAAG -3 ',

EX4-I inner primer-upstream primer: 5 '-CCAAGTTTTTGTGGTGCAATTC -3 ',

EX4-I inner primer-downstream primer: 5 '-TAAGCTCAGTCCAACTGCTAAATG -3 '；

The nest-type PRC outer primer and inner primer for the design of the 9th exon region of Wx gene order, sequence are as follows:

EX9-O outer primer-upstream primer: 5 '-CGACGGGTATGAGTAAGATTCTAAG -3 ',

EX9-O outer primer-downstream primer: 5 '-CATTCGTTCTTACCGTGGTTG -3 ',

EX9-I inner primer-upstream primer: 5 '-AGATCCTTTTGAGCTGACAACC -3 ',

EX9-I inner primer-downstream primer: 5 '-GTACTTGGCGGTGATGTACTTG -3 '；

The nest-type PRC outer primer and inner primer, sequence for the design of Wx gene order exon10 region is as follows:

EX10-O outer primer-upstream primer: 5 '-AGAACGAATGCATTCTTCACAAGA -3 ',

EX10-O outer primer-downstream primer: 5 '-GCCTCACCCCTTCTAATTATTTGA -3 ',

EX10-I inner primer-upstream primer: 5 '-ACAAGGCAAGAATGAGTGACAA -3 ',

EX10-I inner primer-downstream primer: 5 '-GCATATCGTGCAAGTGTGTCTT -3 '；

It is described to be directed to Wx gene order Wx^aAnd Wx^bNest-type PRC outer primer is designed in difference site and inner primer, sequence are as follows:

Wxab-O outer primer-upstream primer: 5 '-GAGGGAGAGGGGGAGAGAGAGATC -3 ',

Wxab-O outer primer-downstream primer: 5 '-TCCAGCCCAACACCTTACAGAAAT -3 ',

Wxab-I inner primer-upstream primer: 5 '-CTTCACTTCTCTGCTTGTGTTGTT -3 ',

Wxab-I inner primer-downstream primer: 5 '-CATGTGATCGATCTGAATAAGAGG -3 '.

2. the identification method of rice Wx gene mutation as described in claim 1, which is characterized in that DNA is mixed in the step (2) Conjunction pond ratio is 1~9:1.

3. the identification method of rice Wx gene mutation as described in claim 1, which is characterized in that the 2nd wheel in the step (3) PCR reaction system are as follows: 4.0 μ L 2 × Taq Plus Master Mix, each 0.3 μ L of inner primer (10 μm of ol/L), 1.0 μ L templates DNA, Evagreen saturated fluorescence dyestuff 0.3 μ L, ddH₂O is mended to 10 μ L, and adds 15 μ L mineral oil.

4. the identification method of rice Wx gene mutation as claimed in claim 2, which is characterized in that the 2nd wheel PCR reaction interval Sequence are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s, most suitable annealing temperature annealing 30s, 72 DEG C of extension 30s, 15 circulations；72℃ Extend 5min.

5. the identification method of rice Wx gene mutation as described in claim 1, which is characterized in that the 2nd wheel in the step (3) After PCR amplification, then carrying out the program of 1 circulation is 95 DEG C of denaturation 30s, 25 DEG C of annealing 1min.

6. the identification method of rice Wx gene mutation as described in claim 1, which is characterized in that nido obtained by the 2nd wheel PCR PCR inner primer expands target fragment length < 500bp.

7. the identification method of rice Wx gene mutation as described in claim 1, which is characterized in that will be single in the step (5) Strain DNA and adjoining tree DNA is mixed in 1:1 ratio and is carried out nested PCR amplification.

8. the identification method of rice Wx gene mutation as described in claim 1, which is characterized in that the dissolution of the HRM detection Program is determined according to target fragment G/C content, and the target fragment dissolution program of low G/C content is 66 DEG C of starting, is terminated 96 DEG C, is protected 62 DEG C are held, the initial temperature of exposure value 180, the target fragment dissolution program of high GC content is set as 75 DEG C, and other parameters are constant.

9. the identification method of rice Wx gene mutation as described in claim 1, which is characterized in that screening in the step (6) Obtained doubtful mutation single plant carries out the reaction system and program of PCR amplification are as follows: 25.0 μ L 2 × Taq Plus Master Mix, each 2 μ L of primer (10 μm of ol/L), 2.0 μ L template DNAs, ddH₂O is mended to 50 μ L.PCR program is 95 DEG C of initial denaturation 5min；95 DEG C denaturation 30s, primer most suitable annealing temperature are annealed 30s, and 72 DEG C of extensions 30s, 35 recycle；72 DEG C of extension 5min.

10. such as application of the described in any item identification methods of claim 1-9 in screening rice Wx gene mutation.