CN110885896A

CN110885896A - For rice Wxop/hpKASP molecular marker for gene detection and application thereof

Info

Publication number: CN110885896A
Application number: CN201911122155.1A
Authority: CN
Inventors: 彭彦; 毛毕刚; 韶也; 胡远艺; 赵炳然
Original assignee: Hunan Hybrid Rice Research Center
Current assignee: Hunan Hybrid Rice Research Center
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-03-17

Abstract

The invention discloses a method for preparing rice Wx^op/hpThe KASP molecular marker for gene detection comprises a forward specific primer F1, a forward specific primer F2 and a reverse universal primer R1; the KASP molecular marker can rapidly and accurately develop Wx in a plurality of samples^op/hpHigh-throughput detection of genotype, accurate identification of Wx^op/hpWhether the gene exists or not and the type of the 77 th base of the 4 th exon of the Wx locus, the operation is simple and convenient, the accuracy is high, and the detection period is short. The invention also discloses the application of the KASP molecular marker in rice Wx^op/hpApplication in gene detection and breeding of Wx-containing^op/hpUse of new lines of allelic rice by combining Wx^op/hpThe new rice line with lower amylose content is successfully cultured after being introduced into a target variety, so that the breeding and improvement of the new rice variety with good taste quality are accelerated, and the method has very important significance.

Description

For rice Wxop/hpKASP molecular marker for gene detection and application thereof

Technical Field

The invention belongs to the technical field of molecular genetics, and particularly relates to a KASP (Kasp-assisted protein polymorphism) technology-based applicationIn rice Wx^op/hpMolecular marker for gene detection and application thereof.

Background

Rice is the first major food crop in China. In recent years, with the improvement of the quality of life of people, the dietary structure and the eating habits are greatly changed, and the requirements on the rice quality, particularly the taste quality, are higher and higher. Therefore, rice quality improvement has become one of the focuses of breeding research. Edible rice is mainly part of the endosperm of seeds, and the main component constituting the endosperm is starch, which generally accounts for 90% of the dry weight of the endosperm. Therefore, the composition and structure of starch are the most important factors in determining rice cooking and taste quality (Tian et al, PNAS).

There are two main types of rice starch, namely amylose (amylose) and amylopectin (amylopectin). Amylose content is generally considered to be a determining factor in rice cooking, processing and eating quality. The major gene for regulating amylose content is waxy gene Wx, and multiple alleles exist at the gene locus, and at least 8 published Wx alleles exist at present, including Wx and Wx^a、Wx^b、Wxⁱⁿ、Wx^mw、Wx^op/hp、Wx^mqAnd Wx^mpAnd the like. wx is present in glutinous rice and premature termination of wx transcription is caused by a deletion of 23bp in the second exon (Wanchana et al, Plant Science). In non-waxy varieties, SNP polymorphism of intron 1 + G-T at position 1 of the Wx locus forms Wx^aAnd Wx^bTwo genotypes (Wang et al, Plant Journal). Wx^aThe SNP polymorphism of A-C at 62 th base of 6 th exon of genotype forms WxⁱⁿGene, Wx^bSNP mutation at the same locus of genotype is named as Wx^mwA gene. Wx^mq、Wx^mpAnd Wx^op/hpThe rice containing 3 'dark endosperm genes' has lower amylose content, and the endosperm is cloudy, but has very good taste. Wx^mqIs a low amylose content mutant gene, Wx, obtained by chemical mutagenesis of the rice variety of Japan^mpIs at Wx^mqOn the basis of the presence of a new allele of the fifth exon by back-mutation (Yang et al, Plant Breeding). Wx^op/hpIs a new allele found in Yunnan, a rice variety "Haopi" with a low amylose content (9.7%). By sequence analysis, Wx^op/hpThe base at the 5' cleavage point of the 1 st intron is G, and Wx^aThe same, but with an A/G SNP mutation at exon 4 at base 77 (Mikami et al, the or Appl Genet). Mutation of this site mutated the 165 th aspartate encoded by exon 4 to glycine, eventually leading to reduced gene transcription, reduced GBSS enzyme activity, and finally a significant reduction in synthesized amylose (Liu et al, plantamol Biol). Therefore, a rapid and accurate identification of the rice dark endosperm mutant gene Wx is established^op/hpThe method has great significance for accelerating the application of the gene in rice quality improvement.

At present, in the aspect of rice quality improvement research, different researchers have developed corresponding molecular markers aiming at different Wx alleles. E.g. for Wx^a、Wx^b、Wxⁱⁿ、Wx^mqAllelic development of CAPS molecular markers for Wx^mq、Wx^mwAnd Wx^hpAllele develops the specific molecular marker of the four-primer amplification hindered mutant system PCR. However, the molecular marker is complicated to operate, and particularly, the CAPS marker often has the phenomenon that accurate identification of different genotypes is difficult due to incomplete enzyme digestion. Competitive allele specific PCR (KASP) is a fluorescence-based SNP detection technology and has the advantages of simplicity, rapidness, accuracy and the like. Currently in rice for Wx^a、Wx^b、Wxⁱⁿ、Wx^mqAllelic variations developed a molecular detection marker for KASP, whereas Wx^op/hpThe KASP molecular marker of the multiple alleles of the excellent rice taste quality is not reported. The invention aims to establish a set of KASP-based technology for rice Wx^op/hpThe molecular marker of the allele detection is applied to the molecular marker-assisted selection, so that the breeding and the improvement of the new rice variety with excellent taste quality are accelerated.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the disadvantages and drawbacks mentioned in the background art, and to establishApplication of KASP technology to rice Wx^op/hpMolecular marker for allele detection and rice Wx using the molecular marker^op/hpGene detection and an application method for assisting in breeding a new rice line with excellent taste quality.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

be used for rice Wx^op/hpThe KASP molecular marker for gene detection comprises a forward specific primer F1, a forward specific primer F2 and a reverse universal primer R1; the forward specific primer F1 comprises a last specific base 1 and a plurality of bases which are positioned at the upstream of the specific base 1 and can realize PCR reaction, wherein the last specific base 1 is A at the 77 th base of the 4 th exon of the rice Wx locus; the forward specific primer F2 comprises the last specific base 2 and a plurality of bases which are positioned at the upstream of the specific base 2 and can realize PCR reaction, wherein the last specific base 2 is G at the 77 th base of the 4 th exon of the rice Wx locus.

In the KASP molecular marker, the sequence of the forward specific primer F1 is preferably: 5'-GGAGTCGACCGTGTGTTCATCGA-3', respectively; the sequence of the forward specific primer F2 is as follows: 5'-GGAGTCGACCGTGTGTTCATCGG-3', respectively; the sequence of the reverse universal primer R1 is as follows: 5'-CAAACCTGAAATCACCAGTGGAAGC-3' are provided.

Preferably, the 5' end of the forward specific primer F1 further comprises a FAM fluorescent signal tag 5'-GAAGGTGACCAAGTTCATGCT-3'; the 5' end of the forward specific primer F2 further comprises a HEX fluorescent signal tag 5'-GAAGGTCGGAGTCAACGGATT-3'.

Based on a general technical concept, the invention also provides a KASP molecular marker in rice Wx^op/hpThe application of the gene detection comprises the following steps:

(1) extracting a sample DNA template of the rice leaf to be detected; preparing a specific KASP Assay mix containing the forward specific primer F1 and the forward specific primer F2, and a general KASPMaster mix containing the reverse general primer R1;

(2) mixing the sample DNA template extracted in the step (1), the specific KASP Assay mix, the general KASP Mastermix and water to prepare a reaction system, and adding the reaction system into a 96-hole PCR micropore reaction plate for PCR amplification;

(3) scanning the fluorescent signal of the PCR amplification product obtained in the step (2), analyzing the scanned fluorescent signal to obtain a genotype scatter diagram, and judging whether the rice to be detected has Wx according to the analysis result of the scatter diagram^op/hpAllele and judging the 77 th base type of the 4 th exon of the Wx locus.

In the above application, preferably, in the step (1), a DNA template of a rice leaf to be detected is extracted by a CTAB method; in the reaction system in the step (2), the volume concentration of the sample DNA template is 10%, the volume concentration of the specific KASP Assaymix is 1.4%, and the volume concentration of the general KASP Master mix is 50%; the concentration of DNA in the sample DNA template is 20-30 ng/ul.

Preferably, in the step (2), the running procedure of the PCR amplification is as follows:

firstly, pre-denaturation at 94 ℃ for 15 min;

the second step is that: denaturation at 94 ℃ for 20s, then annealing and extension at 61-55 ℃ for 60s for 10 cycles, each cycle decreasing by 0.6 ℃;

the third step: denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and performing 26 cycles to obtain a PCR amplification product.

Preferably, in the step (3), a Roche Light Cycler480 instrument is used for scanning the fluorescence signal, the ordinate in the scattergram represents the HEX fluorescence value, the abscissa represents the FAM fluorescence value, and the coordinate where the scattergram is located is the fluorescence value of the sample represented by the scattergram.

Preferably, in the step (3), it is determined whether there is Wx in the rice to be tested according to an analysis result of the scatter plot^op/hpAllele and judging the type of the 77 th base of the 4 th exon of the Wx locus, and the specific operation comprises the following steps: observing the scatter diagram, wherein the scatter displayed near the origin of the coordinate axis is a blank contrast, and the scatter displayed near the origin of the non-coordinate axis is indicated as detected fluorescence; wherein the scatter displayed near the abscissa axis is represented as detecting onlyUntil FAM fluorescence, namely the base 77 of the 4 th exon of the Wx locus in the sample represented by the scattered dot is A; the scatter plot shown near the ordinate axis indicates that only HEX fluorescence was detected, i.e., the scatter plot represents G at base 77 of exon 4 of the Wx locus in the sample; the scatter plot shown at a position away from both the abscissa and the ordinate indicates that FAM fluorescence and HEX fluorescence are detected simultaneously, i.e., the scatter plot represents a heterozygous type of A and G at the 77 th base of the 4 th exon of the Wx locus in the sample.

Based on a general technical concept, the invention also correspondingly provides a KASP molecular marker for breeding Wx-containing hybrid seeds^op/hpThe application of the allele rice new strain comprises the following steps:

A) to carry Wx^op/hpTaking the rice variety of the allele as a donor parent and taking the rice variety to be improved as an acceptor parent to carry out hybridization;

B) wx using said KASP molecular marker in the progeny of the cross obtained after said step A)^op/hpDetecting gene, selecting carried Wx according to the detection result^op/hpProgeny plants of the allele;

C) carrying Wx obtained after the step B)^op/hpContinuously backcrossing the plants of the allelic genes and the receptor parent until the plants are bred, which not only keep the excellent characters of the receptor parent, but also carry Wx^op/hpNew lines of rice with alleles.

If necessary, breeding the strain containing Wx^op/hpNew lines of rice with allele-specific base types, carrying Wx, selected in step B)^op/hpGrouping the progeny plants of the allele according to the 77 th base type of the 4 th exon of the Wx locus to obtain three groups of progeny plants of A: A homozygoty, G: G homozygoty and A: G heterozygote, and then selecting the plants containing specific base types from the progeny plants to carry out continuous backcross with the receptor parent until the Wx plants which not only retain the excellent properties of the receptor parent but also carry the specific base types are bred^op/hpNew lines of rice with alleles.

In the above application, preferably, the specific operation of step B) includes the following steps: mixing the sample DNA template of the filial generation obtained after the step A), the specific KASP Assay mix containing the forward specific primer F1 and the forward specific primer F2, the general KASP Master mix containing the reverse general primer R1 and water to prepare a reaction system, adding the reaction system into a 96-hole PCR micropore reaction plate for PCR amplification, scanning the obtained PCR amplification product with a fluorescence signal, analyzing the scanned fluorescence signal to obtain a gene typing scatter diagram and a fluorescence value, observing the scatter diagram, wherein the scatter diagram displayed near the origin of a coordinate axis is a blank control, and the scatter diagram displayed near the origin of a non-coordinate axis is indicated as detected fluorescence; wherein, the scatter dots displayed near the abscissa axis indicate that only FAM fluorescence is detected, namely, the scatter dots represent that the 77 th base of the 4 th exon of the Wx locus in the sample is A, and the samples are divided into A: A group; the scatter plot shown near the ordinate axis indicates that only HEX fluorescence is detected, i.e., the scatter plot represents a sample in which G is located at the 77 th base of the 4 th exon of the Wx locus, and the samples are divided into G: G groups; the scatter plot shown at a position away from the abscissa and the ordinate indicates that FAM fluorescence and HEX fluorescence are simultaneously detected, i.e., the scatter plot represents a heterozygous type of A and G at the 77 th base of the 4 th exon of the Wx locus in the sample, and is classified into A: G group.

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

the KASP molecular marker is designed according to the SNP polymorphism of A-G at the 77 th base of the 4 th exon of the Wx locus and is named as KASP-Wx^op/hp. The KASP molecular marker can rapidly and accurately develop Wx in a plurality of samples^op/hpHigh-throughput detection of genotype, accurate identification of Wx^op/hpWhether the gene exists or not and the type of the 77 th base of the 4 th exon of the Wx locus, the operation is simple and convenient, the accuracy is high, and the detection period is short. It is also possible to combine Wx with conventional hybridization and backcrossing methods^op/hpThe new rice line with lower amylose content is successfully cultured after being introduced into a target variety, so that the breeding and improvement of the new rice variety with good taste quality are accelerated, and the method has very important significance.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 shows Wx in example 1^op/hpGene detection results;

FIG. 2 shows Wx in example 2^op/hpGene detection results;

FIG. 3 is a graph of the amylose content of rice in the parent and new lines of rice in example 2.

Detailed Description

In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

Example 1:

the invention is used for the rice Wx^op/hpThe KASP molecular marker for gene detection comprises two forward specific primers and a reverse universal primer, and the sequence information of the primers is as follows:

the sequence of the forward specific primer F1 is: 5'-GGAGTCGACCGTGTGTTCATCGA-3' (shown as SEQ ID No: 1), and the 5' end also comprises a FAM fluorescent signal label 5'-GAAGGTGACCAAGTTCATGCT-3' (shown as SEQ ID No: 4);

the sequence of the forward specific primer F2 is: 5'-GGAGTCGACCGTGTGTTCATCGG-3' (shown as SEQ ID No: 2), and a HEX fluorescent signal tag 5'-GAAGGTCGGAGTCAACGGATT-3' (shown as SEQ ID No: 5) at the 5' end;

the sequence of the reverse universal primer R1 is as follows: 5'-CAAACCTGAAATCACCAGTGGAAGC-3' (shown in SEQ ID No: 3).

Wx gene allelic variation analysis is carried out on nearly 300 parts of global rice core germplasm resource resequencing data in earlier stage, and carried Wx is excavated^op/hp12 parts of genotype rice material, and Wx development on these materials by using the KASP molecular marker of this example^op/hpAnd detecting and verifying allele.

Materials: including carrying Wx^op/hpType and not carrying Wx^op/hpType 20 parts of rice material and using "Haopi" (carrying Wx)^op/hp) And (5) making a positive control material.

The method comprises the following specific steps: extracting DNA of rice leaves by adopting a CTAB method; the formulation comprises KASP-Wx^op/hpLabeling the primer specific KASP Assay mix; the genotyping experiment was run in a Roche Light Cycler480 (real-time fluorescent quantitative PCR system) and a 10u1 reaction system was added to a 96-well PCR microwell reaction plate, the reaction system included: DNA template 1u1(20-30ng/ul), KASP Assay mix (LGC, KBS-2100-100-0L1)0.14u1, KASP Master mix (LGC, KBS-1016-016)5u1, and water to supplement 10u1 final system for PCR amplification; the PCR running program comprises: pre-denaturation at 94 ℃ for 15 min; a second step of denaturation, renaturation and extension, wherein the temperature is 94 ℃ for 20s, and the temperature is reduced from 61 ℃ to 55 ℃ (0.6 ℃ per cycle) for 60s, and the number of cycles is 10; third, denaturation, renaturation and extension are carried out for 26 cycles at 94 ℃ for 20s and 55 ℃ for 60 s; fourthly, scanning fluorescence analysis data at 37 ℃ for 1min, scanning fluorescence signals of PCR amplification products by a Roche Light Cycler480 instrument, analyzing the scanned fluorescence signals, and converting the result obtained by analysis into a gene typing scatter diagram and a fluorescence value (shown in figure 1); a scatter plot and FAM/HEX fluorescence values were obtained for each PCR amplification product. Each point in the scatter plot represents the fluorescence of a corresponding sample, with the HEX fluorescence (533nm-580nm) on the ordinate and the FAM fluorescence (465nm-510nm) on the abscissa.

The criteria are as follows: gray scattered points displayed near the origin of the coordinate axis are blank contrast, and scattered points displayed near the origin of the non-coordinate axis are expressed as detected fluorescence; wherein a blue scatter plot shown near the abscissa axis indicates that only FAM fluorescence is detected, i.e., the scatter plot represents A at the 77 th base of the 4 th exon of the Wx locus in the sample; a green scatter plot shown near the ordinate axis indicates that only HEX fluorescence is detected, i.e., the scatter plot represents a G at base 77 of exon 4 of the Wx locus in the sample; the red dispersion spot shown at a position away from both the abscissa and the ordinate indicates that FAM fluorescence and HEX fluorescence are simultaneously detected, i.e., the dispersion spot represents a heterozygous type of A and G at the 77 th base of the 4 th exon of the Wx locus in the sample.

FIG. 1 shows Wx^op/hpAccording to the gene detection result, the blue scattered point is that only FAM fluorescence is detected in a 96-well plate, which indicates that the 77 th base of the 4 th exon of the Wx gene locus in the samples is A; green spots are samples where only HEX fluorescence was detected in 96-well plates, indicating that in these samples, G was at base 77 of exon 4 of the Wx locus; the grey scatter is the negative control in this experiment.

The result obtained by the detection method is completely consistent with the result of Wx gene allelic variation analysis performed in earlier stage, which shows that the KASP molecular marker of the invention can accurately identify Wx^op/hpThe presence or absence of a gene, and the type of base 77 of exon 4 of the Wx locus. The detection method is simple, convenient, rapid and accurate, and can accelerate Wx^op/hpThe application of the gene in the rice quality improvement is of great significance.

Example 2:

assisted selection of a molecular marker KASP containing Wx in example 1^op/hpA new line of rice with alleles, comprising the following steps:

the invention uses rice variety Haopi (carrying Wx)^op/hpGene) as donor parent, using rice variety Zhenshan 97 (carrying Wx) to be improved^aGene, carrying no Wx^op/hpGene) is a recurrent parent, and continuous backcross breeding is carried out.

Wx in the lower generation Using the KASP molecular marker of example 1^op/hpDetecting gene, selecting carried Wx according to the detection result^op/hpProgeny population of alleles and based on the type of base 77 of exon 4 of the Wx locusThe three groups of descendants A: A, G: G and A: G are obtained by grouping. As shown in FIG. 2, the genotype of the sample of the blue scatterbar is Zhenshan 97 genotype (A: A); the genotype of the sample at the green dispersion point is the "Haopi" genotype (G: G); the sample genotype of the red dispersion point is a heterozygous genotype of two alleles (A: G); the grey scatter is the negative control in this experiment.

The high generation population is further selected by combining the amylose content, and after a certain generation, the high generation population can breed the strain which not only retains the excellent properties of the original receptor parent, but also carries Wx^op/hpNew lines of rice with alleles. The new lines of rice were analyzed for amylose content (as shown in FIG. 3). Data display containing Wx^op/hpNew rice strain (NIL-Wx) of gene^op ^/hp) The apparent amylose content of the rice is reduced to about 10 percent, which shows that the amylose content of the rice of the new rice line is obviously reduced.

Sequence listing

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Claims

1. Be used for rice Wx^op/hpThe KASP molecular marker for gene detection is characterized by comprising a forward specific primer F1, a forward specific primer F2 and a reverse universal primer R1;

the forward specific primer F1 comprises a last specific base 1 and a plurality of bases which are positioned at the upstream of the specific base 1 and can realize PCR reaction, wherein the last specific base 1 is A at the 77 th base of the 4 th exon of the rice Wx locus;

the forward specific primer F2 comprises the last specific base 2 and a plurality of bases which are positioned at the upstream of the specific base 2 and can realize PCR reaction, wherein the last specific base 2 is G at the 77 th base of the 4 th exon of the rice Wx locus.

2. The KASP molecular marker according to claim 1, wherein the sequence of the forward specific primer F1 is: 5'-GGAGTCGACCGTGTGTTCATCGA-3', respectively; the sequence of the forward specific primer F2 is as follows: 5'-GGAGTCGACCGTGTGTTCATCGG-3', respectively; the sequence of the reverse universal primer R1 is as follows: 5'-CAAACCTGAAATCACCAGTGGAAGC-3' are provided.

3. The KASP molecular marker according to claim 1 or 2, wherein the 5' end of the forward specific primer F1 further comprises a FAM fluorescent signal tag 5'-GAAGGTGACCAAGTTCATGCT-3'; the 5' end of the forward specific primer F2 further comprises a HEX fluorescent signal tag 5'-GAAGGTCGGAGTCAACGGATT-3'.

4. A KASP molecular marker as claimed in any one of claims 1 to 3 in rice Wx^op/hpThe application in gene detection is characterized by comprising the following steps:

(1) extracting a sample DNA template of the rice leaf to be detected; preparing a specific KASP Assay mix containing the forward specific primer F1 and the forward specific primer F2, and a general KASP Mastermix containing the reverse general primer R1;

(2) mixing the sample DNA template extracted in the step (1), the specific KASP Assay mix, the general KASP Master mix and water to prepare a reaction system, and adding the reaction system into a 96-hole PCR micropore reaction plate for PCR amplification;

5. The use of claim 4, wherein in the step (1), the CTAB method is adopted to extract the DNA template of the rice leaf to be detected; in the reaction system in the step (2), the volume concentration of the sample DNA template is 10%, the volume concentration of the specific KASP Assay mix is 1.4%, and the volume concentration of the general KASP Master mix is 50%; the concentration of DNA in the sample DNA template is 20-30 ng/ul.

6. The use of claim 4, wherein in the step (2), the PCR amplification is performed as follows:

firstly, pre-denaturation at 94 ℃ for 15 min;

7. The use of claim 4, wherein in step (3), the fluorescence signal scan is performed by using a Roche LightCycler480 instrument, the ordinate in the scattergram represents HEX fluorescence value, the abscissa represents FAM fluorescence value, and the coordinate where the scattergram is located is the fluorescence value of the sample represented by the scattergram.

8. The use according to any one of claims 4 to 7, wherein in the step (3), it is determined whether the rice to be tested has Wx or not based on the analysis result of the scatter diagram^op/hpAllele and judging the type of the 77 th base of the 4 th exon of the Wx locus, and the specific operation comprises the following steps: observing the scatter diagram, wherein the scatter displayed near the origin of the coordinate axis is a blank contrast, and the scatter displayed near the origin of the non-coordinate axis is indicated as detected fluorescence; wherein the content of the first and second substances,

the scatter plot shown near the abscissa axis indicates that only FAM fluorescence is detected, i.e., the scatter plot represents a at base 77 of exon 4 of the Wx locus in the sample;

the scatter plot shown near the ordinate axis indicates that only HEX fluorescence was detected, i.e., the scatter plot represents G at base 77 of exon 4 of the Wx locus in the sample;

the scatter plot shown at a position away from both the abscissa and the ordinate indicates that FAM fluorescence and HEX fluorescence are detected simultaneously, i.e., the scatter plot represents a heterozygous type of A and G at the 77 th base of the 4 th exon of the Wx locus in the sample.

9. A method of selectively breeding a plant containing Wx comprising the KASP molecular marker of any one of claims 1 to 3^op/hpThe application of the allele rice new strain is characterized by comprising the following steps:

B) wx using said KASP molecular marker in the progeny of the cross obtained after said step A)^op/hpDetecting gene, selecting carried Wx according to the detection result^op/hp(ii) an allelic plant;

10. The application according to claim 9, wherein the specific operation of step B) comprises the following steps: mixing the sample DNA template of the filial generation obtained after the step A), the specific KASP Assay mix containing the forward specific primer F1 and the forward specific primer F2, the general KASP Master mix containing the reverse general primer R1 and water to prepare a reaction system, adding the reaction system into a 96-hole PCR micropore reaction plate for PCR amplification, scanning the obtained PCR amplification product with a fluorescence signal, analyzing the scanned fluorescence signal to obtain a gene typing scatter diagram and a fluorescence value, observing the scatter diagram, wherein the scatter diagram displayed near the origin of a coordinate axis is a blank control, and the scatter diagram displayed near the origin of a non-coordinate axis is indicated as detected fluorescence; wherein the content of the first and second substances,

the scatter plot shown near the abscissa axis indicates that only FAM fluorescence is detected, i.e., the scatter plot represents a group A in which the 77 th base of exon 4 of the Wx locus is A in the sample;

the scatter plot shown near the ordinate axis indicates that only HEX fluorescence is detected, i.e., the scatter plot represents a sample in which G is located at the 77 th base of the 4 th exon of the Wx locus, and the samples are divided into G: G groups;

the scatter plot shown at a position away from the abscissa and the ordinate indicates that FAM fluorescence and HEX fluorescence are simultaneously detected, i.e., the scatter plot represents a heterozygous type of A and G at the 77 th base of the 4 th exon of the Wx locus in the sample, and is classified into A: G group.