CN116875736A - SNP molecular marker of rice blast resistance gene pizz, primer set and application - Google Patents
SNP molecular marker of rice blast resistance gene pizz, primer set and application Download PDFInfo
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- CN116875736A CN116875736A CN202311066111.8A CN202311066111A CN116875736A CN 116875736 A CN116875736 A CN 116875736A CN 202311066111 A CN202311066111 A CN 202311066111A CN 116875736 A CN116875736 A CN 116875736A
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 65
- 235000009566 rice Nutrition 0.000 title claims abstract description 65
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 45
- 239000003147 molecular marker Substances 0.000 title claims abstract description 22
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 64
- 238000000034 method Methods 0.000 claims abstract description 11
- 201000010099 disease Diseases 0.000 claims description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 210000000349 chromosome Anatomy 0.000 claims description 5
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 3
- 230000001717 pathogenic effect Effects 0.000 claims description 2
- 238000009395 breeding Methods 0.000 abstract description 17
- 230000001488 breeding effect Effects 0.000 abstract description 17
- 238000001514 detection method Methods 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 8
- 238000001962 electrophoresis Methods 0.000 abstract description 4
- 240000008467 Oryza sativa Japonica Group Species 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 240000002582 Oryza sativa Indica Group Species 0.000 abstract description 2
- 239000000443 aerosol Substances 0.000 abstract description 2
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- 238000006243 chemical reaction Methods 0.000 description 11
- 239000003550 marker Substances 0.000 description 6
- 238000013461 design Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 108700028369 Alleles Proteins 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 208000035240 Disease Resistance Diseases 0.000 description 2
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- 235000013339 cereals Nutrition 0.000 description 2
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- 241000894006 Bacteria Species 0.000 description 1
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- 241000196324 Embryophyta Species 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
- A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a SNP molecular marker of a rice blast resistance gene Pizt, a primer group and application thereof, and the SNP molecular marker of the rice blast resistance gene Pizt is applied to the PARMS technology to detect the Pizt gene of a rice material, so that the rice blast resistance gene Pizt can be rapidly and accurately detected in different germplasm resources such as indica rice, japonica rice and the like. The invention does not need complex procedures such as enzyme digestion, electrophoresis, sequencing and the like in the detection process, reduces pollution of PCR product aerosol and use of toxic substances such as EB and the like, can simultaneously carry out prospect selection in the early stage of molecular marker assisted breeding, reduces the scale of breeding population, accelerates the breeding process, and is beneficial to being applied to commercial molecular breeding of paddy rice by the high-efficiency and environment-friendly Pict gene.
Description
Technical Field
The invention relates to the field of rice breeding, in particular to SNP molecular markers of a rice blast resistance gene pizz, a primer set and application thereof.
Background
Rice is an important grain crop, rice blast can occur in the whole growth process of the rice, particles are not harvested when the rice blast is serious, grain safety is threatened, and the rice blast prevention and control method by utilizing the disease resistance gene of the rice is the most economical and effective mode and is environment-friendly. The rice blast resistance gene pizz shows broad-spectrum resistance to rice blast bacteria in different rice areas in China, and has higher utilization value in rice breeding.
The rice disease-resistant breeding is to perform phenotype selection on plants through resistance identification, so that the rice disease-resistant breeding consumes long time, is easily limited by environmental conditions, is easy to cause errors in identification results, and has low selection efficiency. The molecular marker is utilized to assist in selecting and breeding, so that the breeding cost can be reduced, the breeding period can be shortened, the purposeful polygene polymerization can be carried out, the breeding efficiency can be improved, and huge social and economic benefits can be brought.
Disclosure of Invention
In order to solve the technical problems, the invention provides an SNP molecular marker of a rice blast resistance gene pizz, a primer set and application thereof.
In order to achieve the above purpose, the invention is implemented according to the following technical scheme:
the invention provides an SNP molecular marker of a rice blast resistance gene pizz, wherein the SNP molecular marker is pizz-M1, the polymorphic site of the pizz-M1 is positioned at 10385731-10385732 base of the pizz gene of a rice chromosome 6, and the 10385731-10385732 base is TG or CT.
The second object of the invention is to provide a Primer set for detecting SNP molecular markers of rice blast resistance genes Pizt, wherein the Primer set comprises a specific Primer and a universal Primer, and the specific Primer comprises a Primer SeqPizt-M1Fg with a nucleotide sequence shown as SEQ ID NO.1 and a Primer SEQ Pizt-M1Ft with a nucleotide sequence shown as SEQ ID NO. 2; the universal Primer is Primer pizz-M1R with a nucleotide sequence shown as SEQ ID NO. 3.
Preferably, the 5 'end of the Primer Seq Pict-M1 Fg is connected with a FAM or HEX fluorescent sequence, and the 5' end of Primer SeqAlleleY is connected with a HEX or FAM fluorescent sequence.
The third purpose of the invention is to provide an application of SNP molecular markers of a rice blast resistance gene pizz in identification of rice pizz genotypes.
The fourth object of the invention is to provide an application of a primer group for detecting SNP molecular markers of rice blast resistance gene pizz in identifying rice pizz genotype, comprising the following steps:
s1, extracting genome DNA from rice leaves;
s2, detecting the genomic DNA extracted in the step S1 as a template, and detecting the Pizt-M1 molecular marker by using a PARMS technology by using a primer group for detecting the SNP molecular marker of the rice blast resistance gene Pizt; if the base of the SNP locus of the pizz-M1 is TG, judging that the tested rice sample is homozygous disease-resistant pizz genotype; if the base of the SNP locus of the Pizt-M1 is CT, judging that the tested rice sample is homozygous for the pathogenic Pizt genotype; if the SNP locus of the pizz-M1 detects TG and CT at the same time, judging that the rice to be detected is heterozygous disease-resistant pizz genotype.
Preferably, the genomic DNA is extracted from the rice leaf in the step S1 by a conventional simplified CTAB method.
Compared with the prior art, the invention has the following beneficial effects:
the rice blast resistance gene Pizt can be rapidly and accurately detected in different germplasm resources such as indica rice, japonica rice and the like by carrying out SNP molecular markers on the rice blast resistance gene Pizt and carrying out Pizt gene detection on rice materials by applying PARMS technical reaction. Compared with the most commonly used SSR technology based on PCR and electrophoresis, the PARMS SNP detection technology avoids the electrophoresis technology which is time-consuming and labor-consuming, directly reads genotype signals of alleles by adopting a fluorescent scanning mode, and can rapidly complete genotype analysis work by matching with data analysis software, thereby improving the working efficiency by orders of magnitude. The PARMS technology is very suitable for detection automation, and can achieve ultra-high throughput pizz gene identification of hundreds of thousands of data points per day and rice blast resistance resource screening by matching with a GeneMatrix high throughput genotyping system. PARMS is perfectly compatible with LGC KASP SNP detection hardware system and primer, so that seamless conversion can be realized. The invention does not need complex procedures such as enzyme digestion, electrophoresis, sequencing and the like in the detection process, reduces pollution of PCR product aerosol and use of toxic substances such as EB and the like, can simultaneously carry out prospect selection in the early stage of molecular marker assisted breeding, reduces the scale of breeding population, accelerates the breeding process, and is beneficial to being applied to commercial molecular breeding of paddy rice by the high-efficiency and environment-friendly Pict gene.
Drawings
FIG. 1 shows the positions of molecular markers according to an embodiment of the present invention.
FIG. 2 is a flow chart of PARMS SNP detection technique.
FIG. 3 is a typing chart of different varieties of molecular markers pizz-M1 in the embodiment of the invention.
FIG. 4 is a typing chart of the screening progeny molecular marker pizz-M1 in the embodiment of the invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
The embodiment discloses an SNP molecular marker of a rice blast resistance gene Pizt, the SNP molecular marker is Pizt-M1, the polymorphic site of Pizt-M1 is located at 10385731-10385732 base of the Pizt gene of a rice chromosome 6, the 10385731-10385732 base is TG or CT, and the position of the SNP marker Pizt-M1 on the rice chromosome is shown in figure 1. The specific design process is shown in figure 2, the physical position is determined through cloned target gene pizz, SNP locus and flanking sequence are extracted, and the marker is screened and tested through designing and synthesizing primer sequence of the marker, specifically as follows:
1) Primer design
According to the locating mark information of the literature, determining the No. 6 chromosome 10387793-10390465 interval of which the physical position of the pizz (GenBank: DQ 352040.1) is Japanese (MSU 7.0), extracting SNP locus and flanking sequences of the interval, and carrying out primer design by using an on-line primer design website http:// www.snpway.com. The design of the primers of the pizz-M1 is shown in Table 1, each group is marked with three primers, and the 5' ends of two specific primers are respectively connected with FAM and HEX fluorescent sequences. The primers were synthesized by martial arts and sciences.
TABLE 1
2) Sample detection
DNA extraction: genomic DNA was extracted from rice leaves using a simplified CTAB method.
PARMS reaction test: 10ul of PCR reaction system. 20ng of DNA sample was added to the PCR reaction plate, and after drying, PARMS Master mix reaction mixture (manufactured by Wohan market peptide biosciences Co., ltd.) was added to the reaction plate, and the reaction system was shown in Table 2.
TABLE 2
PCR amplification was completed in an ABI 7500 fluorescent quantitative PCR system, and the TouchDown PCR reaction conditions were: pre-denaturation at 94 ℃ for 15 min; the first step of amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 65-57 ℃ and extension for 60 seconds, 10 cycles, wherein the annealing and extension temperature of each cycle is reduced by 0.8 ℃; the second amplification step was performed by denaturation at 94℃for 20 seconds, annealing at 57℃and extension for 60 seconds, 32 cycles. After the PCR reaction is completed, fluorescence data is read, and the result of the fluorescence data is converted into a pattern.
3) Natural population verification
According to the detection method, the marker pizz-M1 is used for carrying out PARMS primary screening reaction verification on 94 rice varieties containing pizz genes, the results are shown in table 3, and the parting diagram is shown in figure 3.
TABLE 3 Table 3
As can be seen from Table 3, the variety carrying the pizz gene detects that the base TG is disease-resistant pizz type at the pizz-M1 test site, such as Longjing 31 and Liao Kai 79; the control which does not carry the pizz gene is all base CT or negative control which does not resist the disease pizz, the pizz gene is not widely applied to the detected japonica rice variety, the pizz gene can be introduced subsequently to directionally improve the rice blast resistance, and the pizz-M1 can be used for the high-efficiency detection of the rice pizz gene.
4) Offspring screening
According to the detection method, 55 offspring materials generated by hybridization of the dragon round-grained non-glutinous 31 and the Liaoxing 21 are detected by using the mark pizz-M1, and the detection result is shown in figure 4. The offspring material carrying the pure and disease-resistant genes pizz can be detected in the offspring material, the genotype is TG, the heterozygous offspring material carrying the disease-resistant genes pizz and other alleles detects the base TG/CT, and the genotype of the offspring material carrying the pure and other alleles is CT. The result shows that the SNP marker pizz-M1 is used for accurately detecting the pizz gene, and can detect whether the carried gene is homozygous. The rice strain carrying the homozygous disease resistance gene pizz can be effectively and rapidly screened when the SNP marker pizz-M1 is used for molecular marker assisted breeding.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.
Claims (7)
1. A SNP molecular marker of a rice blast resistance gene pizz, which is characterized in that: the SNP molecular marker is pizz-M1, the polymorphism site of the pizz-M1 is positioned at 10385731-10385732 base of the pizz gene of the chromosome 6 of the rice, and the 10385731-10385732 base is TG or CT.
2. A primer set for detecting SNP molecular markers of the rice blast resistance gene pizz of claim 1, characterized in that: the Primer group comprises a specific Primer and a universal Primer, wherein the specific Primer comprises a Primer SEQ Pict-M1 Fg with a nucleotide sequence shown as SEQ ID NO.1 and a Primer SEQ Pict-M1 Ft with a nucleotide sequence shown as SEQ ID NO. 2; the universal primer is PrimerPizt-M1R with a nucleotide sequence shown as SEQ ID NO. 3.
3. The primer set for detecting SNP molecular markers of rice blast resistance gene pizz according to claim 2, wherein: the 5 'end of the Primer Seq Pict-M1 Fg is connected with a FAM or HEX fluorescent sequence, and the 5' end of Primer SeqAlleleY is connected with a HEX or FAM fluorescent sequence.
4. Use of the SNP molecular marker of the rice blast resistance gene pizz according to claim 1 for identifying the genotype of rice pizz.
5. Use of the primer set for detecting the SNP molecular marker of rice blast resistance gene pizz according to claim 2 or 3 for identifying the genotype of rice pizz.
6. The use of a primer set for detecting SNP molecular markers of a rice blast resistance gene pizz according to claim 4, wherein the primer set comprises the following steps:
s1, extracting genome DNA from rice leaves;
s2, detecting the genomic DNA extracted in the step S1 as a template, and detecting the Pizt-M1 molecular marker by using a PARMS technology by using a primer group for detecting the SNP molecular marker of the rice blast resistance gene Pizt; if the base of the SNP locus of the pizz-M1 is TG, judging that the tested rice sample is homozygous disease-resistant pizz genotype; if the base of the SNP locus of the Pizt-M1 is CT, judging that the tested rice sample is homozygous for the pathogenic Pizt genotype; if the SNP locus of the pizz-M1 detects TG and CT at the same time, judging that the rice to be detected is heterozygous disease-resistant pizz genotype.
7. The application of the primer group for detecting SNP molecular markers of rice blast resistance genes pizz in the identification of rice pizz genotypes, according to claim 6, wherein the primer group is characterized in that: the genome DNA extracted from the rice leaves in the step S1 adopts a conventional simplified CTAB method.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117402994A (en) * | 2023-10-18 | 2024-01-16 | 辽宁省水稻研究所 | SNP molecular marker of rice blast resistance gene Pi50, primer set and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117402994A (en) * | 2023-10-18 | 2024-01-16 | 辽宁省水稻研究所 | SNP molecular marker of rice blast resistance gene Pi50, primer set and application |
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