CN110423839B - SNP molecular marker of brown planthopper resistant major gene bph3 of rice as well as KASP detection method and application thereof - Google Patents
SNP molecular marker of brown planthopper resistant major gene bph3 of rice as well as KASP detection method and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker of a brown planthopper resistant major gene bph3 of rice, which is positioned in OsLecRK3 in a gene cluster forming the major gene bph 3; the SNP loci of the molecular markers comprise SNP locus 1 and SNP locus 2, which are respectively located at 6968474 site or 6968050 site of No.4 chromosome of rice, and the corresponding molecular marker polymorphisms are respectively C/T, C/T. Accordingly, in combination with the KASP technique, the inventors also designed a corresponding detection primer set and established a corresponding detection method. The invention can rapidly identify the bph3 gene in the rice variety by using the KASP detection technology, has convenient operation and accurate and reliable detection result, is suitable for identification and auxiliary breeding of the bph3 gene of the rice, and solves the problems of low efficiency and long breeding period of the traditional breeding.
Description
Technical Field
The invention belongs to the technical field of plant molecular markers, and particularly relates to an SNP molecular marker of a brown planthopper resistant major gene bph3 of rice, and a KASP detection method and application thereof.
Background
Rice is one of the most important grain crops in China. The brown planthopper harms the long triangular rice planting area in China in the 60 th century, then quickly becomes a main pest in the southwest rice planting area, and in recent years, brown planthopper populations erupt in successive years. Brown planthoppers inhabit the base of rice clusters, wither rice plants by piercing and absorbing the sap of the phloem of the rice stem leaves, and can reduce the yield of rice and even stop harvesting the rice in severe cases. The brown planthopper spreads rice dwarf disease when eating, and simultaneously promotes rice sheath blight and sclerotinia rot of rice. At present, chemical pesticides are the main means for preventing and treating brown planthopper of rice, however, excessive use of pesticides can pollute the environment, destroy ecological balance, influence rice quality and cause great hidden danger to grain safety. The most economical and effective method for preventing and controlling the harm of brown planthopper is to excavate resistance genes from brown planthopper resistant varieties and then transfer the resistance genes into cultivated rice varieties by means of traditional breeding, molecular genetic technology and the like, thereby improving the resistance level of the cultivated rice.
At present, 34 brown planthopper resistance genes are found and reported, wherein 19 dominant genes and 15 recessive genes exist, the number of the located major resistance genes reaches 28, and the major resistance genes such as bph3, bph9, bph14, bph18, bph26, bph29, bph32 and bphi008a are successfully cloned. bph3 is located between SSR markers RM 589-RM 588 on the short arm of the rice chromosome 4, the genetic distance is 0.9cM and 1.4cM (Jairin et al, 2007), the gene cluster is a resistance gene cluster formed by 3 genes (OsLecrRK 1, osLecrRK 2 and OsLecrRK 3) for encoding plasma membrane lectin receptor, the donor parent of the gene cluster is Rathu Heenati, and the gene cluster is a variety (Liu et al, 2015) with broad-spectrum and lasting resistance to brown planthopper.
Traditional backcross breeding of insect-resistant rice varieties depends on phenotype selection of rice, environmental conditions, genotypes and the like can reduce the efficiency of phenotype selection, and the breeding period is long. The molecular marker based on DNA polymorphism is widely applied to the aspects of crop genetic map construction, marker positioning of important agronomic trait genes and the like at present, in particular to a marker-assisted selection (MAS) technology, which utilizes the molecular marker closely linked with insect-resistant genes to carry out indirect selection, has the advantages of good stability, simple and convenient operation, no environmental influence and allele implicit relation interference, capability of polymerizing a plurality of insect-resistant genes and the like, and improves the breeding efficiency of insect-resistant rice.
The type of molecular marker commonly used today is SNP. KASP (competitive Allele Specific PCR) is one of the mainstream international SNP detection methods due to its economic and flexible characteristics, replaces the traditional high-throughput sequencing, and plays an important role in SNP typing research.
Disclosure of Invention
The invention aims to solve the technical problem of providing an SNP molecular marker of a brown planthopper resistant major gene bph3 of rice, a KASP detection method and application thereof.
In order to solve the technical problem, the invention adopts the following technical scheme:
the SNP molecular marker of the brown planthopper resistant major gene bph3 of the rice is positioned in OsLecRK3 in a gene cluster forming the major gene bph 3; the SNP loci of the molecular markers comprise SNP locus 1 and SNP locus 2, which are respectively located at 6968474 th site or 6968050 th site of rice chromosome 4, and the corresponding molecular marker polymorphism is C/T, C/T respectively.
The gene sequences of one of the gene members OsLecRK3 of the bph3 gene cluster in the brown planthopper-resistant rice and the brown planthopper-non-resistant rice are respectively cloned, and are obtained by analysis and comparison, wherein the specific base sequences of the SNP site 1 and the SNP site 2 respectively contain SEQ ID No.1 and SEQ ID No.2.
The molecular marker is used for assisting in identifying the brown planthopper resistant major gene bph3 of the rice.
The detection primer set of the molecular marker comprises a primer set of SNP site 1 and a primer set of SNP site 2, wherein the primer set of SNP site 1 comprises primers PrimerX, primerY and PrimerC which respectively have base sequences of SEQ.ID.NO.3, SEQ.ID.NO.4 and SEQ.ID.NO. 5; the primer set for SNP site 2 includes primers PrimerX ', primerY ', primerC ' having base sequences of seq.id.no.6, seq.id.no.7, seq.id.no.8, respectively.
The detection primer group is used for detecting the brown planthopper resistant major gene bph3 of rice.
A kit containing the detection primer group.
The kit is applied to rice germplasm resource improvement or variety identification.
The kit is applied to cultivation of rice with brown planthopper resistant major genes.
The KASP detection method of the molecular marker takes rice genome DNA as a template and utilizes a specific detection primer group to carry out KASP reaction detection; the detection primer set comprises a primer set of SNP site 1 and a primer set of SNP site 2, the primer set of SNP site 1 comprises primers PrimerX, primerY and PrimerC, and the primers have base sequences of SEQ.ID.NO.3, SEQ.ID.NO.4 and SEQ.ID.NO.5 respectively; the primer set for SNP site 2 includes primers PrimerX ', primerY ', primerC ' having base sequences of seq.id.no.6, seq.id.no.7, seq.id.no.8, respectively.
In the above method for detecting molecular marker KASP, the Touchdown PCR reaction conditions in the KASP reaction detection are as follows: 1min at 30 ℃; 15min at 94 ℃; the first step of amplification reaction, at 94 ℃ for 20sec and 61-55 ℃ for 1min, wherein the annealing and extension temperature is reduced by 0.6 ℃ in each cycle, and the total number of cycles is 10; a second step of amplification reaction, at 94 ℃ for 20sec and 55 ℃ for 1min, for 27 cycles; 1min at 30 ℃.
The SNP molecular marker of the brown planthopper resistant major gene bph3 of the rice is obtained by the inventor through high-throughput sequencing comparison of 7 rice varieties, and the molecular marker is positioned in a plasma membrane lectin receptor kinase gene OsLecRK3 which is one of three gene members in a major gene bph3 gene cluster; the SNP loci of the molecular markers comprise SNP locus 1 and SNP locus 2, which are respectively located at 6968474 site or 6968050 site of No.4 chromosome of rice, and the corresponding molecular marker polymorphisms are respectively C/T, C/T. Accordingly, in combination with the KASP technique, the inventors also designed a corresponding detection primer set and established a corresponding detection method. The invention can efficiently detect 2 SNP molecular markers of the major gene bph3 of the brown planthopper resistance rice, which are functional markers in the gene, and the specificity of the site for detecting the major gene bph3 of the brown planthopper resistance rice is high; the KASP detection technology can be used for rapidly identifying the bph3 gene in the rice variety, the operation is convenient, the detection result is accurate and reliable, the method is suitable for identification and auxiliary breeding of the bph3 gene of the rice, and the problems of low efficiency and long breeding period of the traditional breeding are solved.
Drawings
FIG. 1 shows the result of typing of SNP site 1 of a sample.
FIG. 2 shows the result of typing of SNP site 2 in a sample.
In the figure: the 1 red small circle represents the "CC" homozygous genotype, the 2 blue small circle represents the "TT" homozygous genotype, the 3 purple small circle represents the "CT" heterozygous genotype, and the 4 black small circle represents the "NTC" i.e.water control, as well as the undetected sample.
Detailed Description
To illustrate the invention in detail, it is further illustrated by the following examples. The experimental methods used in the examples are conventional methods unless otherwise specified, and the materials, reagents and the like used therein are commercially available.
Through carrying out whole genome high-throughput sequencing on 7 rice varieties (1205, RH, 91, 58, 1209, 1208 and 1298), comparing the result with a reference genome in a rice genome database to obtain SNP sites of the whole genome, and then screening out the SNP sites with better polymorphism by carrying out methods such as experiment reliability filtration and the like on all the SNP sites. The method comprises the following steps: the SNP molecular markers of the major gene bph3 of the brown planthopper resistance of the rice comprise SNP site 1 and SNP site 2 which are respectively positioned at 6968474 site or 6968050 site of No.4 chromosome of the rice, and the corresponding molecular marker polymorphisms are respectively C/T, C/T. And further confirming 2 SNP sites in the invention by cloning bph3 gene sequences of brown planthopper resistant rice and brown planthopper non-resistant rice and comparing the sequences. The specific base sequences of SNP site 1 and SNP site 2 comprise SEQ.ID.NO.1 and SEQ.ID.NO.2.
SNP site 1: TCTGATACCATCCTACCCACACAAGTGCTTTCACTGGGGACGGCACTCCACAGCCGTCTCCTCGCCACAGACTATTCCAATGGCCGATTTCAACTAAAAGY [ C/T ] TCAACGAGATGGTAATCTTGTTATGTATCCAGATGCTGTACCTTCTGGATACTTATACGATCCATATTGGGCTAGTAACACAGTGGACAATGGCTCACAGCTAGTGTTCAATGAAAC;
SNP site 2: TGGTGGGTAGTGGAGCATGCGGCTTTAACAGTTACTGCACCATTGATGGCACCAAGAACACGACAAGTTGTTTATGCCCACAGAAY [ C/T ] TATAAGTTCATCGATGATAAAAGGAAATACAAAGGCTGCAGGCCAGATTTTGAGCCACAAAACTGTGATCTGGATGAGACGACAG.
According to the design principle of the SNP marker primer of the KASP high-throughput technology platform of LGC, the primer LG-SNP is designed and is used for the molecular marker-assisted selection of the rice bph3 gene. Specific primer information is shown in the following table:
TABLE 1 specific primer information
The specific primer group is used for detecting the brown planthopper resistant major gene bph3:
for SNP site 1: if only the base C is detected, judging that the bph3 gene in the rice sample to be detected has the brown planthopper resistance function, and meanwhile judging that the rice sample to be detected has the brown planthopper resistance; if only the base T is detected, judging that the bph3 gene in the rice sample to be detected does not have the brown planthopper resistance function, and simultaneously judging that the rice sample to be detected does not have the brown planthopper resistance; if bases C and T are detected simultaneously, the rice sample to be detected is judged to contain heterozygous bph3 genes.
For SNP site 2: if only the base C is detected, judging that the bph3 gene in the rice sample to be detected has the brown planthopper resistance function, and meanwhile judging that the rice sample to be detected has the brown planthopper resistance; if only the base T is detected, judging that the bph3 gene in the rice sample to be detected does not have the brown planthopper resistance function, and simultaneously judging that the rice sample to be detected does not have the brown planthopper resistance; if bases C and T are detected at the same time, the rice sample to be detected is judged to contain heterozygous bph3 genes.
1. Extraction of genomic DNA of rice to be tested
94 parts of rice material were collected, and genomic DNA was extracted. The quality and concentration of the extracted rice genome DNA are detected by using 1% agarose gel electrophoresis and SMA4000, the completeness of a genome DNA band is ensured, no tailing is caused, and A260/280 is ensured to be between 1.8 and 2.0, and A260/230 is between 1.8 and 2.0. According to the KASP reaction principle and requirements of LGC, the genomic DNA sample was diluted to 5 ng/. Mu.L for use.
2. KASP-specific PCR reactions
Used in the invention
The real-time fluorescence quantitative PCR working platform and the reagents thereof are purchased from LGC company in England. According to the following tableAnd (3) configuring a PCR reaction system.
TABLE 2 PCR reaction System
Wherein 72 × KASP assay mix is obtained by mixing PrimerX/PrimerX ' (100 μ M), primerY/PrimerY ' (100 μ M), primerC/PrimerC ' (100 μ M) and ddH2O in a ratio of 12.
TABLE 3 PCR reaction conditions
3. Results of genotyping
The results of typing 94 rice samples and 2 pure water negative controls with SNP molecular marker 1 are shown in FIG. 1, the red small circle represents "CC" homozygous genotype, the blue small circle represents "TT" homozygous genotype, the purple small circle represents "CT" heterozygous genotype, the black small circle at the lower left corner represents "NTC", i.e., water control sample and undetected sample, and the genotypes are judged by detecting two fluorescence intensities in PCR products. Each small circle in the figure represents the genotype of each rice sample or a pure water control sample, so that 27 parts of disease-resistant homozygous CC genotype samples, 34 parts of disease-susceptible homozygous TT genotype samples, 21 parts of heterozygous genotype samples and 6 parts of black small circles in the batch of rice samples to be detected represent that 6 parts are not detected and no signal exists for 6 parts.
The typing results of 94 rice samples using SNP molecular marker 2 are shown in FIG. 2, the red small circle represents "CC" homozygous genotype, the blue small circle represents "TT" homozygous genotype, the purple small circle represents "CT" heterozygous genotype, the black small circle at the lower left corner represents "NTC", i.e., water control sample and undetected sample, and the genotypes are judged by detecting two fluorescence intensities in PCR products. Each small circle in the figure represents the genotype of each rice sample, so that 26 parts of disease-resistant homozygous genotype samples, 27 parts of disease-susceptible homozygous genotype samples, 24 parts of heterozygous genotype samples, 10 parts of undetected samples and 7 parts of no signal exist in the batch of rice samples to be detected.
Sequence listing
<110> Nanning VierKai Biotech Co., ltd
<120> SNP molecular marker of brown planthopper resistant major gene bph3 of rice, KASP detection method and application thereof
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ctcgccacag actattccaa tggccgattt caactaaaag ycttcaacga gatggtaatc 120
ttgttatgta tccagatgct gtaccttctg gatacttata cgatccatat tgggctagta 180
acacagtgga caatggctca cagctagtgt tcaatgaaac 220
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tggtgggtag tggagcatgc ggctttaaca gttactgcac cattgatggc accaagaaca 60
cgacaagttg tttatgccca cagaayctta taagttcatc gatgataaaa ggaaatacaa 120
aggctgcagg ccagattttg agccacaaaa ctgtgatctg gatgagacga cag 173
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atacataaca agattaccat ctcgttgaa 29
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acataacaag attaccatct cgttgag 27
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Claims (7)
1. The SNP molecular marker of the brown planthopper resistant major gene bph3 of the rice is used for assisting in identifying the brown planthopper resistant major gene bph3 of the rice, and is characterized in that: the SNP loci of the molecular marker comprise SNP locus 1 and SNP locus 2, wherein the SNP locus 1 is located at the 101 th base of the sequence shown in SEQ ID NO.1, and the molecular marker polymorphism is C/T; the SNP site 2 is located at the 86 th base of the sequence shown in SEQ ID NO.2, and the molecular marker polymorphism is C/T.
2. The detection primer group of the SNP molecular marker of the major gene bph3 of the brown planthopper resistance of the rice is characterized by comprising a primer group of an SNP site 1 and a primer group of an SNP site 2, wherein the primer group of the SNP site 1 comprises primers PrimerX, primerY and PrimerC which are respectively a base sequence shown in SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5; the primer set of SNP site 2 includes primers PrimerX ', primerY ', primerC ' which are the base sequences shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.
3. The detection primer set of claim 2 is used for detecting the brown planthopper resistant major gene bph3 of rice.
4. A kit comprising the detection primer set according to claim 2.
5. The use of the kit of claim 4 for breeding rice plants with brown planthopper resistant major genes.
6. A method for detecting KASP of SNP molecular marker of rice brown planthopper resistant major gene bph3 is characterized in that the DNA of rice genome is used as a template, and a specific detection primer group is used for KASP reaction detection; the detection primer group comprises a primer group of an SNP locus 1 and a primer group of an SNP locus 2, wherein the primer group of the SNP locus 1 comprises primers PrimerX, primerY and PrimerC which are respectively base sequences shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the primer set of SNP site 2 includes primers PrimerX ', primerY ', primerC ' which are the base sequences shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.
7. The method of claim 6, wherein the Touchdown PCR reaction conditions in the KASP reaction assay are: 1min at 30 ℃; 15min at 94 ℃; the first step of amplification reaction, at 94 ℃ for 20sec and 61-55 ℃ for 1min, wherein the annealing and extension temperature is reduced by 0.6 ℃ in each cycle, and the total number of cycles is 10; a second step of amplification reaction, at 94 ℃ for 20sec and 55 ℃ for 1min, for 27 cycles; 1min at 30 ℃.
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