CN112592997A - Auxiliary breeding molecular marker of brown planthopper resistant gene Bph9 and application thereof - Google Patents
Auxiliary breeding molecular marker of brown planthopper resistant gene Bph9 and application thereof Download PDFInfo
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Abstract
The invention provides an auxiliary breeding molecular marker of a brown planthopper resistant gene Bph9 and application thereof. The invention discloses an SNP marker K120504 co-separated from a rice brown planthopper resistant gene Bph9 and having good amplification effect, the marker detects 22886030 site base (MSU7.0) of a rice 12 th chromosome, the polymorphism is T/G, and the primer sequence of the marker K120504 developed based on the KASP technology is shown as SEQ ID No: 1-3. The SNP molecular marker can be used for detecting that the locus of the Bph9 gene is a high-specificity locus, and can be conveniently and efficiently used for identifying whether the rice variety contains the Bph9 gene. The application method of the SNP molecular marker provided by the invention is accurate and reliable, is simple and convenient to operate, and is suitable for identification and auxiliary selective breeding of the Bph9 gene.
Description
Technical Field
The invention relates to the technical field of molecular biology and crop breeding, in particular to an auxiliary breeding molecular marker of a brown planthopper resistant gene Bph9 and application thereof.
Background
Rice is an important grain crop in China. Brown planthopper is a monophagic pest of rice, and rice plants are damaged by sucking phloem juice through a stylet, so that the rice grows slowly, tillering is delayed, and empty grains are increased. The brown planthopper is also a transmission medium of some rice viruses such as grass-like bushy stunt virus, odontoblast stunt virus and the like, and the production and the safety of rice are seriously influenced. At present, the control of brown planthopper mainly depends on chemical control, which not only increases the production cost and the drug resistance of pests, but also pollutes the environment, so the cultivation of brown planthopper resistant varieties by using host resistance is considered to be an effective way for controlling the harm of brown planthopper.
To date, at least 34 anti-brown planthopper gene loci have been identified and reported, 19 dominant genes, 15 recessive genes, 28 dominant anti-brown planthopper genes that have been mapped, and 4 resistance genes cloned, Bph3(Liu et al 2014), Bph14(Du et al 2009), Bph9(Zhao et al 2016), and Bph26(Ji et al 2016), respectively. The Sri Lanka rice variety Kahara na shows insect resistance to brown planthopper populations in east Asia and southeast Asia, Su and the like utilize SSR markers to position an insect-resistant gene Bph9 in Kahara a between markers RM463 and RM5341 on a 12 th chromosome, Zhao clones and analyzes Bph9, a BPH9 locus is a gene cluster on a 12 chromosome long arm, 8 alleles exist at the locus, and the most widely used Bph1, Bph2, Bph7, Bph18 and the like are alleles of BPH 9.
Due to the complicated and complicated insect-resistant phenotype identification process, the breeding efficiency of brown planthopper resistant varieties of rice is limited, and different insect-resistant genes are difficult to effectively polymerize by using conventional breeding means. Developing a molecular Marker closely linked or co-separated with the insect-resistant gene, and utilizing a Marker-assisted selection (MAS) technology to polymerize one or more target genes or QTLs, thereby breeding durable resistant varieties, delaying the degradation period of the insect-resistant varieties and preventing the occurrence of new biotypes of brown planthopper
The traditional rice insect-resistant breeding method is characterized in that phenotype selection is carried out on plants through resistance identification, the consumed time is long, the limitation of environmental conditions is easy, errors are easily caused by identification results, and the selection efficiency is low. The molecular marker-assisted selective breeding is simple and effective, the breeding cost can be reduced, the breeding period can be shortened, purposeful polygene polymerization can be carried out, the breeding efficiency is improved, and great social and economic benefits are brought. The markers mainly utilized in the literature reports are SSR and InDel markers, and have the defects of low polymorphism rate and small difference in breeding. EB or polyacrylamide used in the detection process is easy to cause pollution to the environment and harm to human bodies. The development of specific molecular markers coseparated with the brown planthopper resistant gene Bph9 and the establishment of an efficient and environment-friendly related detection system are of great significance in promoting the application of the Bph9 gene in commercial breeding.
Disclosure of Invention
The invention aims to develop a high-resistance, broad-spectrum and durable molecular marker of a brown planthopper-resistant gene Bph9, which can be used for identification of a Bph9 gene and auxiliary selective breeding.
The development process of the brown planthopper resistant gene Bph9 assisted breeding molecular marker is shown in figure 1. Earlier studies showed that the brown planthopper resistant gene Bph9 is derived from indica rice variety Kaharamana and is located between two SSR markers RM463 and RM5341 on the 12 th chromosome of rice (national rice data center).
The physical position of the corresponding reference genome Nipponbare (MSU7.0) is determined by using a Bph9 gene sequence published in a previous literature, and SNP sites on two sides of the gene interval and the vicinity of the gene interval are mined. And (3) extracting flanking sequences from the selected SNP sites, and performing primer design on the flanking sequences by using an online primer design website BatchPrimer 3.
Aiming at the candidate SNP markers, the donor material Pokkali containing the Bph9 gene, 20 parts of other insect-resistant gene donor materials, 2 parts of insect-susceptible control materials and 8 parts of common rice materials are subjected to KASP primary screening reaction verification, and the SNP marker K120504 which has good coseparation and amplification effects with the Bph9 donor material is selected.
And then natural population verification is carried out on the selected SNP markers linked with the resistance genes by using about 162 parts of materials, and the Bph9 gene locus detected by the invention is proved to be a high-specificity resistance locus and can be used for screening and detecting Bph 9.
In order to realize the purpose of the invention, the invention provides an auxiliary breeding molecular marker of a brown planthopper resistant gene Bph9, wherein the molecular marker is an SNP marker K120504 which is co-separated with a rice brown planthopper resistant gene Bph9, and the SNP marker detects 22886030 th basic group of a No. 12 chromosome of rice.
The invention also provides primers developed based on KASP technology and used for identifying the brown planthopper resistant gene Bph9 of rice, which comprise specific primers X, specific primers Y and universal primers C, wherein the primer sequences are respectively shown as SEQ ID NO: 1-3.
The invention also provides a detection reagent or a kit containing the primer.
The invention also provides application of the molecular marker, the primer, the detection reagent or the kit in identification of the brown planthopper resistant gene Bph9 of rice.
The invention also provides application of the molecular marker, the primer, the detection reagent or the kit in auxiliary breeding of the brown planthopper resistant gene Bph 9.
The invention also provides application of the molecular marker, the primer, the detection reagent or the kit in breeding of rice resources with brown planthopper resistance.
The application comprises the following steps:
1) extracting DNA of a rice sample to be detected;
2) 20ng of dried template DNA, 100UM specific primer X0.005. mu.L, 100UM specific primer Y0.005. mu.L, 100UM universal primer C0.0125. mu.L, 2 XKASP Master Mix 1.4792. mu.L, H2O1.4983 mu L, and carrying out PCR amplification;
3) and analyzing the genotype of the PCR amplification product by using a fluorescence detector.
Further, the PCR reaction conditions in step 2) are as follows: 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 reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 26 cycles.
Further, the step 3) is specifically as follows: typing the PCR amplification product by using biological software, if the PCR product of the sample only detects a fluorescent signal corresponding to the specific primer X, determining that the detection site is a base G, and judging that the tested rice sample is a homozygous non-pest resistant bph9 genotype; if only the fluorescent signal corresponding to the specific primer Y is detected, the detection site is a base T, and the rice sample to be tested is judged to be homozygous insect-resistant Bph9 genotype; if two fluorescence signals are detected simultaneously, the detection site is G: and T, judging that the rice to be detected is heterozygous anti-Bph 9 genotype. The allelic locus K120504-T is a rice plant with excellent resistance allelic type of brown planthopper.
By using the SNP marker K120504 provided by the invention, the allele type of the Bph9 gene in the rice variety to be detected is finally confirmed by detecting the 'Bph 9 gene locus' of a certain rice variety.
The invention has the advantages of simple operation, low cost and short period, and the marker has good stability, is not influenced by other gene effects and environmental factors, can be selected in the early generation, shortens the breeding period, improves the breeding efficiency, and is suitable for popularization and application. The invention has important significance for improving the brown planthopper resistant rice variety and is suitable for the auxiliary selective breeding of the Bph9 gene.
The KASP-based genotyping method is to monitor mutation sites by recording and analyzing fluorescent signals generated in the PCR process by a computer. The consistency between the detection result and the phenotype is high; electrophoresis is not needed in the detection process, so that aerosol pollution of a PCR product, environmental pollution of EB (Epstein-Barr) and harm of formaldehyde to a human body are completely avoided.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
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 described below, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive exercise.
FIG. 1 is a development flow chart of the molecular marker for auxiliary breeding of the brown planthopper resistance gene Bph 9.
FIG. 2 is a diagram showing the typing of a natural population by the SNP marker K120504 in example 2 of the present invention.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 acquisition of molecular marker for assisting breeding of brown planthopper-resistant gene Bph9 of rice
This example is used to illustrate the application of the molecular marker provided by the present invention in detecting brown planthopper resistant gene Bph9, and the specific steps are as follows:
1. primer design
The position of the brown planthopper resistant gene Bph9 is determined in a 12 th chromosome 22872945-2288634 interval of Nipponbare (MSU7.0) according to related documents, the SNP site and the flanking sequence specific to the Bph9 gene in the interval are extracted, and the online primer design website BatchPrimer3 (http:// probes. pw. usda. gov/BatchPrimer3/) is used for primer design. 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 Invitrogen corporation.
Table 1: molecular markers and primer information
The marker designed based on the KASP reaction principle and the single base difference of the anti-sensitive material can carry out Bph9 resistance gene detection on the rice material at high flux, and if only FAM fluorescence is detected in a sample, the base of the sample is Allele X; if only HEX fluorescence is detected, the base of the sample is Allele Y; if two kinds of fluorescence are detected simultaneously, the base of the site is in a heterozygous state.
2. Extracting genome DNA from rice leaf by simplified CTAB method
Firstly, sampling and putting the sample into 2.0mL Tube, adding two steel balls and 750 mu L CTAB solution in advance, and oscillating and homogenizing the sample for 1.5 min;
② vibrating and heating for 0.5-1h at 65 ℃;
cooling to room temperature, adding 750mL of chloroform/isoamyl alcohol (24: 1) solution in a fume hood, and mixing uniformly;
fourthly, 12000rmp is centrifuged for 10min, and 500mL of supernatant is taken and transferred into a new 1.5mL centrifuge tube;
adding an isovolumetric isopropanol solution, shaking gently, mixing uniformly, precipitating at-20 ℃ for more than 1 hour, centrifuging at 12000rmp for 10min, and removing supernatant;
sixthly, adding 1000mL of 70% ethanol, slightly flicking and precipitating, centrifuging for 3min at 1000rmp, and removing supernatant;
seventhly, 300 mu L H is added2O is dissolved overnight for use.
3. KASP reaction test
The KASP reaction assay was performed on the LGC SNPline genotyping platform. 20ng of DNA sample was added to the microplate, dried and added to the KASP reaction mixture, and the reaction system is shown in Table 2.
Table 2: reaction system for KASP detection
| Final concentration | Volume (μ L) | |
| 100UM Primer C | 0.42μM | 0.0125 |
| 100UM Primer X | 0.17μM | 0.0050 |
| 100UM Primer Y | 0.17μM | 0.0050 |
| 2x KASP Master Mix | 1x | 1.4792 |
| Ultrapure water | 1.4983 | |
| Total volume | 3 |
PCR amplification is completed in a water bath thermal cycler, and the Touchdown PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 15 min; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20 seconds, annealing at 65-57 ℃ and extending for 60 seconds for 10 cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.8 ℃; the second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 26 cycles. After the reaction is finished, a scanner Pherastar is used for reading fluorescence data of the KASP reaction product, and the result of fluorescence scanning can be automatically converted into a graph.
The LGC SNpline genotyping platform used in the invention and the consumable materials of the reagents matched with the platform are purchased from LGC company in the United kingdom.
4. Tagging typed data
The KASP preliminary screening reaction verification was performed using the marker K120504 for the donor material Pokkali of the insect-resistant gene Bph9, 20 parts of the other insect-resistant gene donors, 2 parts of the susceptible insect control and 8 parts of the common rice material, and the results are shown in Table 3. The donor variety of the Bph9 gene detected a genotype T resistant to insects at the K120504 test site, and all of the other 30 materials were genotype G not resistant to insects.
Table 3: mark K _120504 Primary sift data
Example 2 application of SNP marker K120504 of brown planthopper resistant gene Bph9 of rice
To test the specificity and utility of the markers of the invention, a natural population validation of SNP marker K120504 was performed using 162 materials. 162 parts of materials comprise insect-sensitive control materials, common hybrid rice and core rice breeding materials. The results of the markers in the natural population typing are shown in FIG. 2, except that Pokkali, CR 157-392-284 and Pokkalli 13 detected the homozygous insect-resistant Bph9 genotype, the other insect-susceptible control materials, the common hybrid rice and the core rice breeding material were all detected as homozygous Bph9 genotype free of brown planthopper resistance. Therefore, the SNP marker K120504 detected the Bph9 gene locus as a high-specificity resistant locus, and can be conveniently and efficiently used for identifying whether the rice variety contains the insect-resistant Bph9 gene.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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Claims (9)
1. An auxiliary breeding molecular marker of a brown planthopper resistant gene Bph9, which is characterized in that the molecular marker is an SNP marker K120504 which is co-separated from a rice brown planthopper resistant gene Bph9, and the SNP marker detects 22886030 th basic group of a No. 12 chromosome of rice.
2. The primers for identifying the brown planthopper resistant gene Bph9 of the rice are characterized by comprising specific primers X, specific primers Y and a universal primer C, wherein the primer sequences are respectively shown as SEQ ID NO: 1-3.
3. A detection reagent or kit comprising the primer of claim 2.
4. Use of the molecular marker for assisted breeding according to claim 1, the primer according to claim 2 or the detection reagent or kit according to claim 3 for identifying brown planthopper resistance gene Bph9 in rice.
5. Use of the molecular marker for assisted breeding according to claim 1, the primer according to claim 2 or the detection reagent or kit according to claim 3 in assisted breeding of brown planthopper-resistant gene Bph9 of rice.
6. Use of the molecular marker for assisted breeding according to claim 1, the primer according to claim 2 or the detection reagent or kit according to claim 3 for breeding rice resources with brown planthopper resistance.
7. Use according to claim 6, characterized in that it comprises the following steps:
1) extracting DNA of a rice sample to be detected;
2) 20ng of dried template DNA, 100UM specific primer X0.005. mu.L, 100UM specific primer Y0.005. mu.L, 100UM universal primer C0.0125. mu.L, 2 XKASP Master Mix 1.4792. mu.L, H2O1.4983 mu L, and carrying out PCR amplification;
3) and analyzing the genotype of the PCR amplification product by using a fluorescence detector.
8. The use of claim 7, wherein the PCR reaction conditions of step 2) are as follows: 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 reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 26 cycles.
9. Use according to claim 7 or 8, wherein step 3) is in particular: and (3) typing the PCR amplification product by using biological software, wherein the allelic locus K120504-T is a rice plant with excellent resistance and allelic type of brown planthopper.
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| CN114438242A (en) * | 2022-01-06 | 2022-05-06 | 武汉大学深圳研究院 | SNP marker for identifying brown planthopper resistant gene Bph43 of rice and application thereof |
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| CN114438242A (en) * | 2022-01-06 | 2022-05-06 | 武汉大学深圳研究院 | SNP marker for identifying brown planthopper resistant gene Bph43 of rice and application thereof |
| CN114438242B (en) * | 2022-01-06 | 2024-04-19 | 武汉大学深圳研究院 | SNP marker for identifying brown planthopper resistant gene Bph43 of rice and application thereof |
| CN117587158A (en) * | 2023-11-07 | 2024-02-23 | 四川省农业科学院植物保护研究所 | Tightly-linked SSR molecular marker of rice false smut resistance QTL and application thereof |
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