CN114350845A - Rice storage-resistant gene excavation and molecular marker development - Google Patents
Rice storage-resistant gene excavation and molecular marker development Download PDFInfo
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Abstract
The invention discloses a rice storage-resistant gene excavation and molecular marker development method, which comprises the following steps: s1, rice germplasm resource collection: selecting 56 parts of main rice variety materials at the middle and lower reaches of Yangtze river, wherein the main rice variety materials comprise 25 indica rice and 31 japonica rice; s2, aging: the sample which is not aged is taken as a reference and is marked as CK; samples aged at a constant temperature and humidity (37 ℃ C., 85% RH) for 5 weeks and 7 weeks were designated as CD5 and CD7, respectively, and the germination percentage and other quantitative properties of the samples were measured for different treatments. By adopting the detection method designed by the invention, KASP molecular markers can be designed according to the identified SNP, so that a detection kit for identifying the storable varieties can be developed conveniently in the future, the storable rice varieties with better quality can be planted by screening the genotype of the storable rice and cultivating the new genotype of the storable quality, and therefore, the accurate regulation and control of grain storage are realized, and the detection method has important significance for reducing the loss of stored grain and ensuring the quality of the stored grain.
Description
Technical Field
The invention relates to the technical field of agriculture, in particular to paddy rice storage-resistant gene excavation and molecular marker development.
Background
At present, the storage technology for slowing down the deterioration of the paddy by controlling the storage environment conditions in the market is mature, the application of the genotype of the paddy in the grain storage safety is still in the initial exploration stage, the molecular marker related to the storage-resistant lipid of the paddy is developed, the genotype of the paddy with storage resistance can be screened, the new genotype with storage-resistant quality can be cultivated, the paddy variety which is suitable for storage and has better quality can be planted, so that the accurate regulation and control of the grain storage can be realized, the accurate regulation and control of the grain storage loss can be realized, the important significance is realized for reducing the grain storage loss and ensuring the grain storage quality, and the influence mechanism research on the storage resistance of the paddy is not clarified because the genetic mechanism of the storage resistance among the paddy varieties is complex, more genes determining the storage resistance are mainly caused by the regulation and control of micro-effect polygene.
Disclosure of Invention
The invention aims to provide the rice storage-resistant gene mining and molecular marker development, has the advantage of being convenient for developing and identifying a detection kit of a storage-resistant variety in the future, and solves the problems provided by the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: the method for mining the storage-resistant gene of the rice and developing the molecular marker comprises the following steps:
s1, rice germplasm resource collection:
selecting 56 parts of main rice variety materials at the middle and lower reaches of Yangtze river, wherein the main rice variety materials comprise 25 indica rice and 31 japonica rice;
s2, aging:
the sample which is not aged is taken as a reference and is marked as CK; accelerating and aging in a constant temperature and humidity box (37 ℃ and 85% RH) for 5 weeks and 7 weeks, respectively marking as CD5 and CD7, and simultaneously measuring the germination percentage, fatty acid value, malonaldehyde content, conductivity and other quantitative properties of the samples under different treatments;
s3, storage-tolerant phenotype identification:
extracting DNA of a rice sample, constructing a library after the DNA sample is detected to be qualified, carrying out machine sequencing after the library is detected to be qualified, adopting Illumina HiSeq/MiSeqe sequencing, carrying out sequencing data quality control analysis on original sequence data obtained by sequencing through FASTQ, comparing the data after sequencing filtration with a reference genome japonica rice variety Nipponbin through Hisat2 software, completing SNP detection development on samples in a group by utilizing GATK software, adopting Plink software, eliminating gene marker SNP with the minimum allele frequency of less than 5 percent and the deletion rate of more than 20 percent, and screening 353196 high-quality SNP markers for next phenotype association analysis;
s4, genome wide association analysis (GWAS) and transcriptome analysis:
(1) selecting a univariate mixed linear model by adopting Gemma software, and screening the obviously associated SNP sites by combining the genetic relationship analysis results among samples;
(2) performing correlation analysis on the phenotype data under the three environments respectively to detect storage-resistant sites which stably appear at different points;
(3) the threshold P-value of the significant associated SNP sites is 10 < -4 >, the total number of SNPs used for association analysis is 254815 (353196 SNPs are developed in the experiment), the functions of SNPs are annotated according to GFF3 annotation files of a Japanese sunny genome, and the Halpoview software is adopted to perform haplotype analysis on target candidate genes in combination with phenotypic data;
(4) significantly associated with the SNP site: through correlation analysis, the rice genotype germination rate is commonly correlated to 2071 SNP sites in the whole genome range, wherein strong signals are arranged on the correlation regions of No. 6 and No. 8 chromosomes, a plurality of SNP sites in the 1.2MB-1.5MB region on the No. 8 chromosome exceed a threshold value, the rice genotype fatty acid character is commonly correlated to 510 SNP sites in the whole genome range, wherein the correlation regions have strong signals on the No. 1, No. 7 and No. 9 chromosomes, the No. 1 chromosome and the No. 9 chromosome signals display strong correlation signals, the rice genotype malondialdehyde character is commonly correlated to 4034 SNP sites in the whole genome range, wherein the strong correlation signals are arranged on the No. 1, 3, 4, 5 and No. 8 chromosomes, the rice genotype conductivity character is commonly correlated to 1510 SNP sites in the whole genome range, and the strong correlation signals appear on the No. 1 chromosome 2;
(5) selecting 4 rice genotypes with extreme storage tolerance according to phenotype data to perform transcriptome sequencing;
(6) storage-tolerant genotype: xiushui 134 and Zhejiang 96; the non-storage-tolerant gene types of Nanjing 9108 and Suxiu 867 adopt a Trizol method to extract total RNA in the rice powder before and after aging, and strictly control the quality of an RNA sample: analyzing the RNA integrity of the sample and the existence of DNA pollution by agarose gel electrophoresis; detecting the purity of RNA by using a NanoPhotometer; then, the RNA concentration and the integrity are accurately quantified by using the Qubit2.0 and Agilent 2100, the sequence is carried out on the machine after the RNA quality is qualified, raw data 300G is obtained in total, a Nipponbare genome is used as a reference genome, Hisat2 software is selected for comparison, R package Deseq2 is used for counting differential expression genes, a rice genome annotation project database is compared, and 11039 differential genes are annotated in total;
(7) combining with transcriptome results, the test analyzes the expression patterns of 14 storage-tolerant candidate genes obtained by GWAS screening, and finds that three of the genes LOC _ Os06g09450.8, LOC _ Os01g45060.1 and LOC _ Os01g47350.1 respectively encode sucrose synthase, polygalacturonase and enoyl-CoA hydratase isomerase family proteins, the expression quantity difference is obvious in storage-tolerant and non-storage-tolerant genotypes, and the storage-tolerant genotypes can be up-regulated to express LOC _ Os06g09450.8 and LOC _ Os01g45060.1 to provide energy for stress reaction;
(8) significantly different genes: the gene is normally expressed in the non-storable genotype, 7 sites (chr1_25581589, chr1_270544634, chr6_4796306, chr6_4800231, chr6_4802754, chr6_4802754 and chr6_4802768) which are related to the storable shape are found in the LOC _ Os01g45060, LOC _ Os01g47350 and LOC _ Os06g09450 genes, and one related site (chr1_25579324) is dug in the upstream 1000bp range of the LOC _ Os01g 45060;
s5, screening of storage-resistant candidate genes:
the method comprises the steps of finding 8 strongly-associated SNP sites near 3 genes associated with the storage-tolerant traits, developing 8 corresponding pairs of fluorescent molecular markers based on the 8 sites, and further verifying and typing SNPs associated with the DNA samples of rice genomes of different varieties by combining a competitive Allele Specific PCR (KASP) technology;
s6, designing a universal fluorescent primer:
KASP requires preparation:
(1) the Primer Mix consists of two allele forward primers with different terminal bases and a reverse Primer, and the 5' ends of the two forward primers are respectively connected with detection Primer sequences with different sequences;
(2) two detection primers Master Mix with different fluorescence; 3) a DNA template;
the KASP reaction was carried out on a LightCycler480(LC480), and the PCR amplification reaction system and reaction program were determined by the method of Weiyu et al [25 ]:
adding 50-100 ng. mu.L-1 of the diluted DNA template into a 96-well plate respectively, and preparing the following reaction system for PCR amplification reaction: 5 μ L of template DNA, 2 XKASP Master mix5 μ L of KASP marker primer, 0.14 μ L of KASP marker primer, and the total reaction volume of each well is 10.14 μ L;
the PCR reaction program on LightCycler480(LC480) was:
(1) KASP thermal cycling: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 s; annealing at 61-55 deg.C for 60s, 10 cycles (0.6 deg.C reduction per cycle); denaturation at 94 ℃ for 20 s; annealing and extending at 55 ℃ for 60s for 26 cycles; reading at 37 ℃ for 1 min;
(2) and (3) KASP recovery: denaturation at 94 ℃ for 20 s; extending for 60s at 57 ℃, and repeating the steps (1) - (2) twice (totally 3 times); reading at 37 ℃ for 1 min;
(3) after the reaction is finished, judging the sample typing condition according to the detected 2 fluorescent signals, wherein different genotypes are obtained by different fluorescent signals;
s7, KASP molecular marker typing:
the experimental result proves that the 8 SNP markers exist in the storage-resistant material and are available molecular markers of the rice storage-resistant trait QTL, and the molecular markers can provide basis for obtaining individual plants of storage-resistant varieties later and lay a foundation for the subsequent breeding of the storage-resistant varieties.
Preferably, in step S3, the DNA of the rice sample is extracted by using a DNA extraction kit.
Compared with the prior art, the invention has the following beneficial effects:
by adopting the detection method designed by the invention, KASP molecular markers can be designed according to the identified SNP, so that a detection kit for identifying the storable varieties can be developed conveniently in the future, the storable rice varieties with better quality can be planted by screening the genotype of the storable rice and cultivating the new genotype of the storable quality, and therefore, the accurate regulation and control of grain storage are realized, and the detection method has important significance for reducing the loss of stored grain and ensuring the quality of the stored grain.
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FIG. 1 is a schematic flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The components of the present invention are all standard components or components known to those skilled in the art, and the structure and principle thereof can be known to those skilled in the art through technical manuals or through routine experimentation.
Referring to fig. 1, the mining of rice storage-resistant genes and the development of molecular markers includes the following steps:
s1, rice germplasm resource collection:
selecting 56 parts of main rice variety materials at the middle and lower reaches of Yangtze river, wherein the main rice variety materials comprise 25 indica rice and 31 japonica rice;
s2, aging:
the sample which is not aged is taken as a reference and is marked as CK; accelerating and aging in a constant temperature and humidity box (37 ℃ and 85% RH) for 5 weeks and 7 weeks, respectively marking as CD5 and CD7, and simultaneously measuring the germination percentage, fatty acid value, malonaldehyde content, conductivity and other quantitative properties of the samples under different treatments;
s3, storage-tolerant phenotype identification:
extracting DNA of a rice sample, constructing a library after the DNA sample is detected to be qualified, performing machine sequencing after the library is detected to be qualified, performing Illumina HiSeq/MiSeqe sequencing, performing sequencing data quality control analysis on original sequence data obtained by sequencing through FASTQ, comparing the data after sequencing filtration with a reference genome japonica rice variety Nipponbin through Hisat2 software, completing SNP detection development on samples in a group by utilizing GATK software, removing gene marker SNP (single nucleotide polymorphism) with the minimum allele frequency of less than 5% and the deletion rate of more than 20% by utilizing Plink software, and screening 353196 high-quality SNP markers for next phenotype association analysis in total, wherein a DNA extraction kit is adopted to extract the DNA of the rice sample in the step S3;
s4, genome wide association analysis (GWAS) and transcriptome analysis:
(1) selecting a univariate mixed linear model by adopting Gemma software, and screening the obviously associated SNP sites by combining the genetic relationship analysis results among samples;
(2) performing correlation analysis on the phenotype data under the three environments respectively to detect storage-resistant sites which stably appear at different points;
(3) the threshold P-value of the significant associated SNP sites is 10 < -4 >, the total number of SNPs used for association analysis is 254815 (353196 SNPs are developed in the experiment), the functions of SNPs are annotated according to GFF3 annotation files of a Japanese sunny genome, and the Halpoview software is adopted to perform haplotype analysis on target candidate genes in combination with phenotypic data;
(4) significantly associated with the SNP site: through correlation analysis, the rice genotype germination rate is commonly correlated to 2071 SNP sites in the whole genome range, wherein strong signals are arranged on the correlation regions of No. 6 and No. 8 chromosomes, a plurality of SNP sites in the 1.2MB-1.5MB region on the No. 8 chromosome exceed a threshold value, the rice genotype fatty acid character is commonly correlated to 510 SNP sites in the whole genome range, wherein the correlation regions have strong signals on the No. 1, No. 7 and No. 9 chromosomes, the No. 1 chromosome and the No. 9 chromosome signals display strong correlation signals, the rice genotype malondialdehyde character is commonly correlated to 4034 SNP sites in the whole genome range, wherein the strong correlation signals are arranged on the No. 1, 3, 4, 5 and No. 8 chromosomes, the rice genotype conductivity character is commonly correlated to 1510 SNP sites in the whole genome range, and the strong correlation signals appear on the No. 1 chromosome 2;
(5) selecting 4 rice genotypes with extreme storage tolerance according to phenotype data to perform transcriptome sequencing;
(6) storage-tolerant genotype: xiushui 134 and Zhejiang 96; the non-storage-tolerant gene types of Nanjing 9108 and Suxiu 867 adopt a Trizol method to extract total RNA in the rice powder before and after aging, and strictly control the quality of an RNA sample: analyzing the RNA integrity of the sample and the existence of DNA pollution by agarose gel electrophoresis; detecting the purity of RNA by using a NanoPhotometer; then, the RNA concentration and the integrity are accurately quantified by using the Qubit2.0 and Agilent 2100, the sequence is carried out on the machine after the RNA quality is qualified, raw data 300G is obtained in total, a Nipponbare genome is used as a reference genome, Hisat2 software is selected for comparison, R package Deseq2 is used for counting differential expression genes, a rice genome annotation project database is compared, and 11039 differential genes are annotated in total;
(7) combining with transcriptome results, the test analyzes the expression patterns of 14 storage-tolerant candidate genes obtained by GWAS screening, and finds that three of the genes LOC _ Os06g09450.8, LOC _ Os01g45060.1 and LOC _ Os01g47350.1 respectively encode sucrose synthase, polygalacturonase and enoyl-CoA hydratase isomerase family proteins, the expression quantity difference is obvious in storage-tolerant and non-storage-tolerant genotypes, and the storage-tolerant genotypes can be up-regulated to express LOC _ Os06g09450.8 and LOC _ Os01g45060.1 to provide energy for stress reaction;
(8) significantly different genes: the gene is normally expressed in the non-storable genotype, 7 sites (chr1_25581589, chr1_270544634, chr6_4796306, chr6_4800231, chr6_4802754, chr6_4802754 and chr6_4802768) which are related to the storable shape are found in the LOC _ Os01g45060, LOC _ Os01g47350 and LOC _ Os06g09450 genes, and one related site (chr1_25579324) is dug in the upstream 1000bp range of the LOC _ Os01g 45060;
s5, screening of storage-resistant candidate genes:
the method comprises the steps of finding 8 strongly-associated SNP sites near 3 genes associated with the storage-tolerant traits, developing 8 corresponding pairs of fluorescent molecular markers based on the 8 sites, and further verifying and typing SNPs associated with the DNA samples of rice genomes of different varieties by combining a competitive Allele Specific PCR (KASP) technology;
s6, designing a universal fluorescent primer:
KASP requires preparation:
(1) the Primer Mix consists of two allele forward primers with different terminal bases and a reverse Primer, and the 5' ends of the two forward primers are respectively connected with detection Primer sequences with different sequences;
(2) two detection primers Master Mix with different fluorescence; 3) a DNA template;
the KASP reaction was carried out on a LightCycler480(LC480), and the PCR amplification reaction system and reaction program were determined by the method of Weiyu et al [25 ]:
adding 50-100 ng. mu.L-1 of the diluted DNA template into a 96-well plate respectively, and preparing the following reaction system for PCR amplification reaction: 5 μ L of template DNA, 2 XKASP Master mix5 μ L of KASP marker primer, 0.14 μ L of KASP marker primer, and the total reaction volume of each well is 10.14 μ L;
the PCR reaction program on LightCycler480(LC480) was:
(1) KASP thermal cycling: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 s; annealing at 61-55 deg.C for 60s, 10 cycles (0.6 deg.C reduction per cycle); denaturation at 94 ℃ for 20 s; annealing and extending at 55 ℃ for 60s for 26 cycles; reading at 37 ℃ for 1 min;
(2) and (3) KASP recovery: denaturation at 94 ℃ for 20 s; extending for 60s at 57 ℃, and repeating the steps (1) - (2) twice (totally 3 times); reading at 37 ℃ for 1 min;
(3) after the reaction is finished, judging the sample typing condition according to the detected 2 fluorescent signals, wherein different genotypes are obtained by different fluorescent signals;
s7, KASP molecular marker typing:
the experimental result proves that the 8 SNP markers exist in the storage-resistant material and are available molecular markers of the rice storage-resistant property QTL, the molecular markers can be used for obtaining single plants of storage-resistant varieties later, a basis is provided for the subsequent breeding of the storage-resistant varieties, and a foundation is laid.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (2)
1. The method for mining the rice storage-resistant gene and developing the molecular marker is characterized in that: the method comprises the following steps:
s1, rice germplasm resource collection:
selecting 56 parts of main rice variety materials at the middle and lower reaches of Yangtze river, wherein the main rice variety materials comprise 25 indica rice and 31 japonica rice;
s2, aging:
the sample which is not aged is taken as a reference and is marked as CK; accelerating and aging in a constant temperature and humidity box (37 ℃ and 85% RH) for 5 weeks and 7 weeks, respectively marking as CD5 and CD7, and simultaneously measuring the germination percentage, fatty acid value, malonaldehyde content, conductivity and other quantitative properties of the samples under different treatments;
s3, storage-tolerant phenotype identification:
extracting DNA of a rice sample, constructing a library after the DNA sample is detected to be qualified, carrying out machine sequencing after the library is detected to be qualified, adopting Illumina HiSeq/MiSeqe sequencing, carrying out sequencing data quality control analysis on original sequence data obtained by sequencing through FASTQ, comparing the data after sequencing filtration with a reference genome japonica rice variety Nipponbin through Hisat2 software, completing SNP detection development on samples in a group by utilizing GATK software, adopting Plink software, eliminating gene marker SNP with the minimum allele frequency of less than 5 percent and the deletion rate of more than 20 percent, and screening 353196 high-quality SNP markers for next phenotype association analysis;
s4, genome wide association analysis (GWAS) and transcriptome analysis:
(1) selecting a univariate mixed linear model by adopting Gemma software, and screening the obviously associated SNP sites by combining the genetic relationship analysis results among samples;
(2) performing correlation analysis on the phenotype data under the three environments respectively to detect storage-resistant sites which stably appear at different points;
(3) the threshold P-value of the significant associated SNP sites is 10 < -4 >, the total number of SNPs used for association analysis is 254815 (353196 SNPs are developed in the experiment), the functions of SNPs are annotated according to GFF3 annotation files of a Japanese sunny genome, and the Halpoview software is adopted to perform haplotype analysis on target candidate genes in combination with phenotypic data;
(4) significantly associated with the SNP site: through correlation analysis, the rice genotype germination rate is commonly correlated to 2071 SNP sites in the whole genome range, wherein strong signals are arranged on the correlation regions of No. 6 and No. 8 chromosomes, a plurality of SNP sites in the 1.2MB-1.5MB region on the No. 8 chromosome exceed a threshold value, the rice genotype fatty acid character is commonly correlated to 510 SNP sites in the whole genome range, wherein the correlation regions have strong signals on the No. 1, No. 7 and No. 9 chromosomes, the No. 1 chromosome and the No. 9 chromosome signals display strong correlation signals, the rice genotype malondialdehyde character is commonly correlated to 4034 SNP sites in the whole genome range, wherein the strong correlation signals are arranged on the No. 1, 3, 4, 5 and No. 8 chromosomes, the rice genotype conductivity character is commonly correlated to 1510 SNP sites in the whole genome range, and the strong correlation signals appear on the No. 1 chromosome 2;
(5) selecting 4 rice genotypes with extreme storage tolerance according to phenotype data to perform transcriptome sequencing;
(6) storage-tolerant genotype: xiushui 134 and Zhejiang 96; the non-storage-tolerant gene types of Nanjing 9108 and Suxiu 867 adopt a Trizol method to extract total RNA in the rice powder before and after aging, and strictly control the quality of an RNA sample: analyzing the RNA integrity of the sample and the existence of DNA pollution by agarose gel electrophoresis; detecting the purity of RNA by using a NanoPhotometer; then, the RNA concentration and the integrity are accurately quantified by using the Qubit2.0 and Agilent 2100, the sequence is carried out on the machine after the RNA quality is qualified, raw data 300G is obtained in total, a Nipponbare genome is used as a reference genome, Hisat2 software is selected for comparison, R package Deseq2 is used for counting differential expression genes, a rice genome annotation project database is compared, and 11039 differential genes are annotated in total;
(7) combining with transcriptome results, the test analyzes the expression patterns of 14 storage-tolerant candidate genes obtained by GWAS screening, and finds that three of the genes LOC _ Os06g09450.8, LOC _ Os01g45060.1 and LOC _ Os01g47350.1 respectively encode sucrose synthase, polygalacturonase and enoyl-CoA hydratase isomerase family proteins, the expression quantity difference is obvious in storage-tolerant and non-storage-tolerant genotypes, and the storage-tolerant genotypes can be up-regulated to express LOC _ Os06g09450.8 and LOC _ Os01g45060.1 to provide energy for stress reaction;
(8) significantly different genes: the gene is normally expressed in the non-storable genotype, 7 sites (chr1_25581596, chr1_27054464, chr6_4796306, chr6_4800231, chr6_4802745, chr6_4802754 and chr6_4802768) which are related to the storable shape are found in the LOC _ Os01g45060, LOC _ Os01g47350 and LOC _ Os06g09450 genes, and one related site (chr1_25579324) is dug in the upstream 1000bp range of the LOC _ Os01g 45060;
s5, screening of storage-resistant candidate genes:
the method comprises the steps of finding 8 strongly-associated SNP sites near 3 genes associated with the storage-tolerant traits, developing 8 corresponding pairs of fluorescent molecular markers based on the 8 sites, and further verifying and typing SNPs associated with the DNA samples of rice genomes of different varieties by combining a competitive Allele Specific PCR (KASP) technology;
s6, designing a universal fluorescent primer:
KASP requires preparation:
(1) the Primer Mix consists of two allele forward primers with different terminal bases and a reverse Primer, and the 5' ends of the two forward primers are respectively connected with detection Primer sequences with different sequences;
(2) two detection primers Master Mix with different fluorescence; 3) a DNA template;
the KASP reaction was carried out on a LightCycler480(LC480), and the PCR amplification reaction system and reaction program were determined by the method of Weiyu et al [25 ]:
adding 50-100 ng. mu.L-1 of the diluted DNA template into a 96-well plate respectively, and preparing the following reaction system for PCR amplification reaction: 5 μ L of template DNA, 2 XKASP Master mix5 μ L of KASP marker primer, 0.14 μ L of KASP marker primer, and the total reaction volume of each well is 10.14 μ L;
the PCR reaction program on LightCycler480(LC480) was:
(1) KASP thermal cycling: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20 s; annealing at 61-55 deg.C for 60s, 10 cycles (0.6 deg.C reduction per cycle); denaturation at 94 ℃ for 20 s; annealing and extending at 55 ℃ for 60s for 26 cycles; reading at 37 ℃ for 1 min;
(2) and (3) KASP recovery: denaturation at 94 ℃ for 20 s; extending for 60s at 57 ℃, and repeating the steps (1) - (2) twice (totally 3 times); reading at 37 ℃ for 1 min;
(3) after the reaction is finished, judging the sample typing condition according to the detected 2 fluorescent signals, wherein different genotypes are obtained by different fluorescent signals;
(4) development of molecular marker sequences:
s7, KASP molecular marker typing:
the experimental result proves that the 8 SNP markers exist in the storage-resistant material and are available molecular markers of the rice storage-resistant trait QTL, and the molecular markers can provide basis for obtaining individual plants of storage-resistant varieties later and lay a foundation for the subsequent breeding of the storage-resistant varieties.
2. The rice storage-tolerant gene mining and molecular marker development according to claim 1, wherein: in the step S3, DNA of the rice sample is extracted by using a DNA extraction kit.
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