CN107779522B - Specific codominant molecular marker of brown planthopper resistant gene Bph15 of rice and application thereof - Google Patents
Specific codominant molecular marker of brown planthopper resistant gene Bph15 of rice and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of genetic engineering, and particularly discloses a specific codominant molecular marker of a brown planthopper resistant gene Bph15 of rice and application thereof. The invention develops and verifies the specific codominant molecular markers B15InD1 and B15InD2 of Bph15 by comparing the genome sequence of the Bph15 fine localization interval with the corresponding sequences of other rice varieties. The molecular marker can effectively detect whether the pest-resistant variety B5 and the derived variety (line) thereof contain the brown planthopper resistant gene Bph15 of rice, greatly improve the selection efficiency of brown planthopper resistant rice and obtain the brown planthopper resistant rice variety containing the Bph15 gene. The co-dominant molecular marker provided by the invention can be used for assisting in breeding by means of the molecular marker, and can be used for gathering a plurality of brown planthopper resistant gene loci through gene polymerization to obtain a high-quality broad-spectrum resistant breeding material, so that the degradation period of insect-resistant rice varieties is delayed, and the occurrence of new biotypes of brown planthoppers is prevented.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a specific codominant molecular marker of a brown planthopper resistant gene Bph15 of rice and application thereof.
Background
Rice is one of the main grain crops in China, and the production of the rice is directly related to the grain safety, the income increase of farmers and the rural stability in China. The rice planting process faces various plant diseases and insect pests, and serious threats are formed on the yield of the rice when the damage is serious. Wherein, the brown planthopper is a rice pest with strong explosive power and great harmfulness. Its imagoes and nymphs can directly prick the phloem juice of paddy rice, resulting in yellow leaves and even withering of plants. In addition, the brown planthopper can also transmit viruses and induce various rice diseases such as rice jagged leaf dwarf, grassy dwarf and the like in the feeding process, and the yield can be reduced even the rice is completely harvested when the rice diseases are seriously attacked. Outbreaks of brown planthopper groups mostly occur in the rice mature filling stage. At the initial stage of insect pest occurrence, the nymphs of the brown planthoppers are small in insect bodies, strong in concealment and difficult to find, so that the nymphs of the brown planthoppers cannot be prevented and controlled in time and can be propagated in large quantities, at the moment, rice plants grow vigorously, the operation of applying the insecticide to the bases of the rice plants is very difficult, the insect pests are popular, and harm and yield reduction are caused frequently. Brown planthopper only happened in local rice area of China before the last 60 years. Subsequently, with changes in climate, environment, planting structure, farming system, cultivation method, and the like, brown planthopper is spread from south to north in the area where it is harmful, the occurrence frequency increases, and the degree of harm becomes worse. According to the records of Chinese agricultural yearbook, the generation area of rice planthopper (mainly brown planthopper) in China is more than 2000 million hectares every year, and the direct yield loss caused by the damage of the rice planthopper every year is more than 100 million tons (Louyongguge root, Chengjia safety, 2011). Brown planthopper has serious threat to the rice production safety in China.
The control of brown planthopper has been relied on for the most part by the application of chemical insecticides. The pesticide is applied in a large amount, so that the damage of the brown planthopper is controlled to a certain extent and in a certain time period, and the natural enemy of the brown planthopper in the rice field is eliminated. In addition, due to the fact that the chemical insecticide is applied in large quantities in successive years, the drug resistance of the brown planthopper is increased in multiple times, and the prevention and control effect of the chemical insecticide is very limited. In addition, some pesticides such as triazophos, pyrethroid insecticides, bactericides, herbicides and the like have the effect of stimulating reproduction of brown planthopper, so that the increase of rice planthopper is more aggravated, and the aim of sustainable treatment of the rice planthopper cannot be realized by means of specific pesticides (Cheng Jia' an and the like, 2008). Meanwhile, the continuous and large-scale use of the chemical pesticide aggravates the pollution of ecological environment and grains, increases the production cost of farmers and brings challenges to the income increase of the farmers in a new period.
The internal reason that brown planthopper continuously erupts and becomes rampant in successive years is that the main rice varieties planted in large areas in China have generally poor brown planthopper resistance, hybrid rice varieties have high plant types, large field groups in middle and later periods, thick stems and leaves, high field canopy degree and proper nutrition, are beneficial to rapid propagation of brown planthopper, show super-sensitivity to brown planthopper and other insect pests, and easily form epidemic outbreak situation when the insect source base is large and the climate conditions are proper, so that serious harm is caused (Hanchuan, Liu photo Jie and other 2003). Practice proves that the cultivation of brown planthopper-resistant rice varieties by using brown planthopper-resistant genes is the most economic, effective, safe and ecological method in the comprehensive prevention and control of brown planthoppers.
To date, 30 major genes against brown planthopper have been identified and reported in cultivated and wild rice sources (Hu et al.2016 Recent progress on the genetics and molecular planning of brown plant resistance in rice.Rice,9: 30). In addition to bph5 and bph8, the other major genes against brown planthopper have been mapped to different chromosomes of rice using molecular markers. A research group of the Wuhan university Hoyle professor obtains a rice variety B5 which shows high resistance to the biological types 1, 2 and 3 of several common brown planthoppers by multi-generation hybridization breeding of medicinal wild rice (Oryza officinalis Wall ex Watt) and common cultivated rice Zhenshan 97B. From B5, 2 dominant brown planthopper-resistant genes Bph14 and Bph15 are identified and genetic analysis shows that the resistance contribution of Bph15 to B5 is stronger than that of Bph 14. Bph15 is finely localized to the fourth chromosomal short arm (Yang et al 2004; Lv et al 2014).
Due to the complexity of insect resistance identification of rice materials, a seedling stage group identification method is generally adopted in the backcross breeding of conventional transformation insect-resistant genes, and the identification method cannot identify a single plant and can only determine the insect resistance of the current generation through insect resistance identification of offspring. Therefore, transformation of an insect-resistant gene by a conventional breeding method is inefficient, and it is often difficult to efficiently introduce or aggregate different insect-resistant genes. The molecular marker technology for positioning the insect-resistant gene can accelerate the process of insect-resistant gene breeding, save the cost and improve the breeding efficiency. On the basis of obtaining the molecular Marker which is tightly linked with the insect-resistant gene, the invention can purposefully conduct the introduction and the polymerization of the insect-resistant gene by means of a Marker-assisted selection (MAS) technology, breed the durable resistant variety, delay the degradation period of the insect-resistant variety and prevent the occurrence of new biotypes of brown planthoppers. Thereby making important contributions to the development of environment-friendly and resource-saving agriculture by using less pesticides and reducing grain loss. In the breeding process, the molecular marker closely linked with the brown planthopper resistant gene is used for developing brown planthopper resistant molecular marker assisted breeding, so that the effective way for breeding brown planthopper resistant rice is formed.
Disclosure of Invention
The invention aims to provide a specific codominant molecular marker of a brown planthopper resistant gene Bph15 of rice and application thereof. By detecting the specific codominant molecular markers of the brown planthopper resistant genes Bph15, the resistance of rice plants to brown planthoppers can be predicted, and the selection progress of brown planthopper resistant rice varieties is accelerated.
The technical scheme of the invention is as follows:
in a first aspect, the invention provides a specific codominant molecular marker of a brown planthopper resistant gene Bph15 of rice, which is obtained by PCR amplification of one of the following primer pairs:
1) codominant marker primer B15InD 1:
forward primer sequence: TGCACCTGGATCAAATGTTTCT, respectively;
reverse primer sequence: GCCCTTCCCTCTCCATATCC, respectively;
2) codominant marker primer B15InD 2:
forward primer sequence: AGGAACAGTGACACGTAGCA, respectively;
reverse primer sequence: GGAGAGTTCAGTTTGCCATCC are provided.
The specific codominant molecular marker of the brown planthopper resistant gene Bph15 of the rice is a 460bp amplified fragment amplified by a codominant marker primer B15InD 1; or 537bp amplified fragment using codominant marker primer B15InD 2.
The invention provides application of the specific codominant molecular marker in detection of brown planthopper resistant gene Bph15 of rice.
The invention provides application of the specific codominant molecular marker in breeding of rice with brown planthopper resistance.
In a second aspect, the invention provides a primer pair for detecting a brown planthopper resistant gene Bph15 of rice, which comprises the following steps:
1) codominant marker primer B15InD 1:
forward primer sequence: TGCACCTGGATCAAATGTTTCT, respectively;
reverse primer sequence: GCCCTTCCCTCTCCATATCC, respectively; or
2) Codominant marker primer B15InD 2:
forward primer sequence: AGGAACAGTGACACGTAGCA, respectively;
reverse primer sequence: GGAGAGTTCAGTTTGCCATCC are provided.
The invention provides application of the primer pair in detection of brown planthopper resistant gene Bph15 of rice.
The invention provides application of the primer pair in breeding brown planthopper resistant rice.
In a third aspect, the invention provides a method for detecting a brown planthopper resistant gene Bph15 of rice, which comprises the steps of taking the genomic DNA of the rice to be detected as a template, carrying out PCR amplification by using the codominant marker or the primer pair provided by the invention, and detecting an amplification product: if the codominant marker primer B15InD1 is used, 460bp amplified fragments can be amplified; or
The co-dominant marker primer B15InD2 can be used for amplifying 537bp amplified fragment, and then the existence of the brown planthopper resistant gene Bph15 in the rice to be detected is marked.
When the method is used for detecting the amplification product, the detection and the resolution can be carried out only by using common agarose gel electrophoresis.
In a fourth aspect, the invention provides a method for screening brown planthopper-resistant rice, which comprises the steps of carrying out PCR amplification by using the co-dominant marker or the primer pair provided by the invention and taking the genomic DNA of the rice to be detected as a template, and detecting an amplification product: if the codominant marker primer B15InD1 is used, 460bp amplified fragments can be amplified; or
The codominant marker primer B15InD2 can be used for amplifying 537bp amplification fragments, so that the existence of the brown planthopper resistant gene Bph15 in the rice to be detected is marked, and the brown planthopper can be shown to be resistant to insects, thereby realizing the purpose of screening.
In a fifth aspect, the invention provides detection reagents and kits comprising the aforementioned primer pairs.
The invention also provides application of the molecular marker or the primer pair or the kit containing the molecular marker or the primer pair in breeding brown planthopper-resistant rice.
The invention also provides application of the molecular marker or the primer pair or the kit containing the molecular marker or the primer pair in the auxiliary breeding of the molecular marker for resisting brown planthopper of rice and the improvement of rice germplasm resources.
And the application of the method for detecting the brown planthopper resistant gene Bph15 of the rice or the method for screening brown planthopper resistant rice in breeding brown planthopper resistant rice.
Further, the invention also provides a screening process of the co-dominant molecular marker, which comprises the following steps:
1) development of Bph 15-specific co-dominant molecular marker:
the genome sequence of the fine localization segment of Bph15 was analyzed by Standard Nucleotide BLAST in the NCBI database (https:// BLAST. NCBI. nlm. nih. gov/BLAST. cgi.
2) Verification of Bph15 specific codominant molecular marker:
7 co-dominant molecular markers developed by a fine positioning segment sequence of Bph15 are used for detecting different anti-brown planthopper gene resistance sources, common breeding parents without anti-brown planthopper genes and population materials of Bph15, and finally the co-dominant molecular markers B15InD1 and B15InD2 are determined to be capable of specifically distinguishing Bph15 from other rice varieties and are Bph15 specific co-dominant molecular markers. Y15 is an insect-resistant material derived from B5 (containing Bph14 and Bph15), does not contain Bph14 and only contains Bph15 gene, and is prepared by pairing 9311// Y15/9311BC1F1The population of (A) carries out the genotype and the BC thereof1F2The phenotype identification of the offspring resisting brown planthopper determines that the codominant molecular markers are cosegregating with the phenotype of resisting brown planthopper, and indicates that the codominant molecular markers can be used for detecting the existence of Bph 15.
3) The application of the Bph15 specific codominant molecular marker in breeding:
hybridizing Y15 (from B5, containing no Bph14 and containing Bph15 and resisting brown planthopper) with excellent restorer line 9311 widely used in breeding, screening hybrid offspring containing Bph15 by using Bph15 specific codominant molecular marker, backcrossing by using 9311 as recurrent parent, and backcrossing to BC according to the process4F1Selfing to obtain BC4F2. Application of Bph15 specific codominant molecular marker to BC4F2Genotyping, and corresponding BC4F23And carrying out phenotype identification on the progeny against brown planthopper, determining the co-dominant molecular markers to be co-separated with the phenotype against brown planthopper again, and further showing that the Bph15 specific co-dominant molecular markers can be applied to molecular marker-assisted selection breeding practice of Bph15 to cultivate rice varieties against brown planthopper containing Bph 15.
Based on the technical scheme, the invention has the beneficial effects that:
the locus of the brown planthopper resistant gene positioned by the codominant molecular marker provided by the invention is definite, and the brown planthopper resistance of rice plants can be predicted by detecting the specific codominant molecular marker of the Bph15 gene. Furthermore, the method can be used for detecting the genotype of the rice variety or strain to judge whether the rice variety or strain to be detected has the resistance of the brown planthopper, and then quickly screening the insect-resistant variety or strain for rice breeding. Secondly, the Bph15 specific codominant marker provided by the invention is a codominant insertion and deletion marker developed on both sides of a specific insertion or deletion region of Bph15 after comparing a fine positioning interval sequence of Bph15 with other rice variety sequences in a database, has sequence difference with breeding parent materials widely applied in the current production, and can be directly applied to Bph15 brown planthopper resistant breeding practice. In addition, the molecular marker provided by the invention is easy to detect, can be detected and distinguished through conventional agarose gel electrophoresis, and is different from the traditional SSR marker which can be separated and distinguished only through complicated PAGE gel electrophoresis and silver staining.
The molecular marker provided by the invention can be used for molecular marker-assisted breeding, has a definite selection target and saves cost. In the traditional breeding method, parents with insect-resistant genes and cultivars are firstly collected to carry out a series of hybridization, and the rice cultivars are identified and selected for the brown planthopper resistance, so the operation is very complicated and is influenced by the environment. In addition, before insect-resistant identification, firstly, insect sources are obtained and bred to brown planthopper groups, and meanwhile, the insect sources are required to be inoculated and the seedling ages of rice seedlings are required to be synchronous, which brings troubles to breeding work, and if the relationship among the insect sources, the seedlings and the environment cannot be effectively processed, the reliability of the brown planthopper-resistant phenotype identification result is very low. Therefore, the insect-resistant breeding is time-consuming, difficult and high in cost. However, by detecting the locus of the gene resisting the brown planthopper, a single plant with high brown planthopper resistance can be identified in the seedling stage, other plants are eliminated, the production cost is saved, the selection efficiency of the brown planthopper-resistant rice is greatly improved, and the breeding period of rice varieties is greatly shortened.
The co-dominant molecular markers B15InD1 and B15InD2 provided by the invention can be used for assisting breeding by means of molecular markers, and can be used for gathering a plurality of brown planthopper resistant gene loci through gene polymerization to obtain a high-quality broad-spectrum resistant breeding material, so that the degradation period of insect-resistant rice varieties is prolonged, and the occurrence of new biotypes of brown planthoppers is prevented.
Drawings
FIG. 1 shows the verification of specific co-dominant molecular markers B15InD1 and B15InD2 of Bph15 in different anti-brown planthopper gene resistant sources and conventional rice varieties without anti-brown planthopper genes. M represents a molecular weight standard. Lanes numbered 1 to 17 correspond to rice varieties B5 (containing Bph14 and Bph15), mdgo (Bph1), ASD7(Bph2), Swarnalata (Bph6), T12(Bph7), Pokkali (Bph9), IR71033-121-15(Bph21), 9311, Jiangxiang 929, Yuzhenxiang, Xuanhui 1577, Meixiang, late Xiang 98, Huazhan, Huanghua viscos, Guanghuangsi, Guangzhan 63S, respectively.
FIG. 2 shows specific co-dominant molecular markers B15InD1 and B15InD2 of Bph15 at 9311// Y15/9311BC1F1Validation in the population. M represents a molecular weight standard. Y15 is derived from B5 (containing Bph14 and Bph15), does not contain Bph14 and only contains a insect-resistant parent of the Bph15 gene; 9311 is a perceptual parent. Lanes 1-22 are 22 randomly selected 9311// Y15/9311BC1F1And (4) rice strain. +/+ represents material containing homozygous Bph15 site after detection by Bph15 specific codominant molecular markers B15InD1 and B15InD2, +/-represents material containing heterozygous Bph15 site, -/-represents material not containing Bph15 site. The resistance value is the result of the phenotypic identification of the rice line for resisting brown planthopper, wherein each BC1F1Resistance value of (D) is from its corresponding BC1F2And identifying the brown planthopper resistance of the offspring.
FIG. 3 shows the validation of Bph 15-specific co-dominant molecular markers B15InD1 and B15InD2 in molecular marker-assisted selective breeding. M represents a molecular weight standard. Y15 is derived from B5 (containing Bph14 and Bph15), does not contain Bph14 and only contains a insect-resistant parent of the Bph15 gene; 9311 is a perceptual parent. 1-22 are 22 randomly selected 9311// Y15/9311BC4F2And (4) rice strain. +/+ represents material containing homozygous Bph15 site after detection by Bph15 specific codominant molecular markers B15InD1 and B15InD2, +/-represents material containing heterozygous Bph15 site, -/-represents material not containing Bph15 site. The resistance value is the result of the phenotypic identification of the rice line for resisting brown planthopper, wherein each BC4F2Resistance value of (D) is from its corresponding BC4F23And identifying the brown planthopper resistance of the offspring.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The rice varieties contemplated herein are available to those skilled in the art in a conventional manner or commercially available manner.
Example 1 development and verification of specific codominant molecular marker of brown planthopper resistant gene Bph15 of rice
1. Development of rice brown planthopper resistant gene Bph15 specific codominant molecular marker
The genomic sequence of the finely localized region in which Bph15 is located was subjected to Standard Nucleotide BLAST analysis in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM＝ blastn&PAGE_TYPE＝BlastSearch&LINK_LOC＝blasthome) Comparing the genome sequence of the fine positioning segment where the Bph15 is located with the genome sequence of the corresponding interval of other rice varieties, listing the specific insertion/deletion existing in the genome sequence of the Bph15 fine positioning segmentMissing fragments; further utilizing Primer3 online software design to develop 7 co-dominant molecular markers easy to detect: (http://primer3.ut.ee/)。
The genome DNA of different brown planthopper gene resistant sources and conventional rice varieties without brown planthopper resistant genes (rice variety B5 (containing Bph14 and Bph15), Mudgo (Bph1), ASD7(Bph2), Swarnalata (Bph6), T12(Bph7), Pokkali (Bph9), IR71033-121-15(Bph21), 9311, fragrance 929, Jatropha essence, Xuanhui 1577, Meixiang, late fragrance 98, Huazhan, Huanghua sticky, Guangxiang silk sprout, Guangdong silk sprout 63S) is extracted by a CTAB method (Murray MG & Thompson, 1980Rapid isolation of high-molecular-weight plant DNA. nucleic Acids Res 8: 4321-4325).
The 7 codominant molecular markers which are designed and developed by the sequence of the fine positioning segment where the Bph15 is located are used for carrying out PCR amplification on the DNA of the rice varieties, and the PCR amplification system is as follows: 20ng of rice genome DNA template, 1U of Taq DNA polymerase, 2.0 ul of 10 XPCR buffer, 0.2ul of 10mmol each dNTPs and 0.2 uM of primers, and finally ddH is used2O made up the reaction to 20. mu.l. The PCR reaction conditions are as follows: firstly, 94 ℃ for 3 min; followed by 35 cycles of 94 ℃ for 30sec, 55 ℃ for 30sec, and 72 ℃ for 30 sec; then 5min at 72 ℃. And (3) loading the PCR product on 1% agarose gel for electrophoresis to detect the banding pattern of the PCR product. The results show that the codominant molecular marker B15InD1 (forward primer sequence: TGCACCTGGATCAAATGTTTCT; reverse primer sequence: GCCCTTCCCTCTCCATATCC) and B15InD2 (forward primer sequence: AGGAACAGTGACACGTAGCA; reverse primer sequence: GGAGAGTTCAGTTTGCCATCC) can specifically distinguish the Bph15 parent material B5 from other rice varieties (FIG. 1): in the Bph15 parent material B5, the co-dominant molecular marker B15InD1 can amplify a PCR product with the size of 460bp, B15InD2 can amplify a PCR product with the size of 537bp, and the PCR product has a band size which is obviously different from that of the PCR products amplified in other rice varieties and has polymorphism.
2. Verification of Bph15 specific codominant molecular marker
In order to verify specific co-dominant molecular markers B15InD1 and B15InD2 of Bph15, the invention takes an excellent restorer line 9311 (brown planthopper) widely applied in breeding as a female parent and takes a Bph15 parent material Y15 (from B5, does not contain Bph14 and contains Bph1)5, brown planthopper resistance) as a male parent to obtain F1. Further using 9311 as male parent, pair 9311/Y15F1Backcrossing to obtain 9311// Y15/9311BC1F1. The invention randomly selects 60 BC1F1The genotype of the materials is firstly analyzed by using Bph15 specific codominant molecular markers B15InD1 and B15InD2 developed by the invention, and 60 BC are determined1F1The total of 31 parts of the material contained Bph15 (both contained Bph15 in a heterozygous state, and 2 bands were observed in the electrophoresis result in FIG. 2), and 29 parts of the material did not contain Bph15 (22 pieces of 9311// Y15/9311BC in FIG. 2)1F1Genotype of Rice line and its BC1F2Anti-brown planthopper phenotype). To determine these BC1F1Whether the genotype of the material is consistent with the phenotype of brown planthopper resistance or not is judged, and 60 BC are added1F1Selfing the material to obtain corresponding BC1F2The 60 parts of BC are further examined by adopting a seedling stage group method1F2Resistance manifestation of materials (in BC)1F2Pedigree resistance rating for BC1F1Brown planthopper resistant phenotype of individual plants).
To ensure parental and BC1F2The population growth is consistent, and all the tested materials (including insect-resistant parent Y15, insect-susceptible parent 9311 and insect-susceptible control variety TN1) are soaked in seeds for germination before sowing. 60 seeds of each family (variety) are sowed in a bread box which is 58cm long, 38cm wide and 9cm high and is filled with nutrient soil with the thickness of 7 cm. Each box was seeded with 3 replicates per material, with 3 replicates each of insect-resistant parent Y15, insect-susceptible parent 9311, and insect-susceptible control TN1 randomly seeded. Thinning seedlings after 7 days of sowing, and eliminating weak seedlings. When the seedlings grow to the period of two leaves and one heart, 2-3 instar brown planthopper nymphs are inoculated according to the proportion of 9 heads/seedling, and a nylon gauze is covered. When all of the insect-susceptible varieties TN1 (local No. 1 in Taiwan) died, resistance evaluations were performed on each individual plant at 0, 1, 3, 5, 7 or 9 levels (Table 1) by referring to the method of Huang et al (Huang Z et al, 2001Identification and mapping of two brown plant resistance genes in rice, Theor. appl. Gene.102, 929-934), and the resistance level of each family was calculated by weighted average for the parent material and the population.
TABLE 1 evaluation grading Standard for anti-Nilaparvata lugens
60 BC granules1F1The genotype of the material (reflected by the analysis of the Bph15 specific codominant molecular markers B15InD1 and B15InD2 developed by the invention) is compared with the anti-brown planthopper phenotype (identified by a seedling stage group method), and the genotype (or reflected by the analysis of the Bph15 specific codominant molecular markers B15InD1 and B15InD2 developed by the invention) is found to be completely matched with the anti-brown planthopper phenotype, and the consistency is 100%: 29 parts BC without Bph151F1The materials are all sensitive to brown planthopper, and the resistance value is 9; 31 parts BC containing Bph15 in heterozygous state1F1The materials were resistant to brown planthopper, the average resistance value was around 4.3, and the materials showed heterozygous resistance (FIG. 2, 22 randomly selected 9311// Y15/9311BC1F1Rice strain line is representative). The results show that the Bph15 specific codominant molecular markers B15InD1 and B15InD2 developed by the invention are cosegregated with the phenotype of brown planthopper resistance, and the linked molecular markers can be used for detecting the existence of Bph 15.
Example 2 application of specific codominant molecular marker of brown planthopper resistant gene Bph15 of rice in molecular marker-assisted selective breeding
In the traditional brown planthopper resistant breeding, brown planthopper resistant material parents are selected to be hybridized with cultivated varieties, the selection needs to be carried out by carrying out brown planthopper resistant character identification on filial generations, and in view of the special condition of brown planthopper resistant identification, the brown planthopper resistant phenotype of a single plant of the material of the last generation can be reflected only by the identification of progeny materials. Therefore, the traditional brown planthopper resistant breeding is time-consuming, difficult and high in cost. However, by applying the molecular marker linked with the brown planthopper resistant gene, a single brown planthopper resistant plant can be identified in the seedling stage, other plants are eliminated, the production cost is saved, the selection efficiency of brown planthopper resistant rice is greatly improved, and the breeding period of rice varieties is greatly shortened. In this embodiment, two Bph 15-linked molecular markers provided in embodiment 1 of the present invention are used for molecular marker-assisted selective breeding, which are specifically implemented as follows:
hybridizing an excellent restorer line 9311 (brown planthopper) widely applied in breeding with a Bph15 parent material Y15 (from B5, without Bph14, with Bph15 and resistant to brown planthopper) as a female parent to obtain F1(ii) a Using 9311 as male parent pair 9311/Y15F1Backcrossing to obtain 9311// Y15/9311BC1F1. The Bph15 specific codominant molecular markers B15InD1 and B15InD2 developed by the invention are used for screening to obtain BC containing Bph151F1Backcrossing with 9311 to obtain BC2F1(ii) a Application of Bph15 specific codominant molecular markers B15InD1 and B15InD2 to BC2F1Screening to obtain BC containing Bph152F1Backcrossing with 9311 to obtain BC3F1(ii) a Application of Bph15 specific codominant molecular markers B15InD1 and B15InD2 to BC3F1Screening to obtain BC containing Bph153F1Backcrossing with 9311 to obtain BC4F1。
In this example, the backcross material was not phenotypically identified against brown planthopper, and only the Bph 15-specific co-dominant molecular markers B15InD1 and B15InD2 were used for testing to determine the presence of Bph 15. Randomly selecting 48 BC4F1The material was genotyped (as demonstrated by the Bph15 specific co-dominant molecular marker assay developed in the present invention) and its corresponding BC4F2The comparison of the anti-brown planthopper phenotype (identified by a seedling stage group method) shows that the genotype (embodied by the developed Bph15 specific codominant molecular marker) is completely consistent with the anti-brown planthopper phenotype, and the consistency is 100%: 25 parts BC without Bph154F1The materials are all sensitive to brown planthopper, and the resistance value is 9; 23 parts BC containing Bph15 (heterozygous state)4F1The materials were resistant to brown planthopper with an average resistance value between 4.2 and 4.6 (showing heterozygous resistance). The results show that the specific codominant molecular markers B15InD1 and B15InD2 of the Bph15 developed by the invention are cosegregation with the phenotype of brown planthopper resistance, and the linked molecular markers can be used for detecting the existence of the Bph 15.
Applied to parent materials Y15 and 9311 molecular marker with polymorphism for 23 BC containing Bph15 in heterozygous state and resisting brown planthopper4F1Performing whole genome scan, selecting a BC with the vast majority of genetic background replaced by 9311 genetic background4F1From its BC4F248 of the materials are randomly selected, the genotype of the materials is analyzed by using the developed Bph15 specific codominant molecular marker, and 48 BC are determined4F2The materials contained 12 parts of the material containing pure Bph15, 23 parts of the material containing heterozygous Bph15 and 13 parts of the material not containing Bph 15. To further determine these BC4F2Whether the genotype of the material is consistent with the brown planthopper resistant phenotype or not, and determining the BC4F2Selfing the material to obtain corresponding BC4F23The 48 parts of BC are inspected by adopting a seedling stage group method4F23Resistance manifestation of materials (with these BC)4F23Pedigree resistance rating for BC4F2Brown planthopper resistant phenotype of individual plants).
The results show that these BC4F2The genotype of the material (embodied by the developed Bph15 specific codominant molecular marker) is completely consistent with the brown planthopper resistant phenotype, and the consistency is 100%: 13 parts BC without Bph154F2The materials are all sensitive to brown planthoppers, and the resistance value is about 9; 23 parts BC containing Bph15 in heterozygous state4F2The average resistance value of the material is between 4.2 and 4.6, and the material shows heterozygous resistance to brown planthopper; 12 BC containing homozygous Bph154F2The mean resistance value of the material was around 2.7, and it was resistant to brown planthopper (FIG. 3, with 22 BC randomly selected)4F2Rice strain line is representative). These BC containing homozygous Bph154F2Compared with the parent 9311, the genetic background except the Bph15 locus has been replaced by the 9311 genetic background, other excellent agronomic traits of the 9311 used as a male parent for breeding by a three-line method and a two-line method are retained, and the material is also resistant to brown planthopper because of containing the homozygous Bph15 gene, and can be used as an improved 9311 used as a male parent for breeding hybrid rice resistant to brown planthopper.
The results also further prove that the developed Bph15 specific codominant molecular marker is coseparated with the brown planthopper resistant phenotype, and the linked molecular markers can be applied to molecular marker assisted selective breeding practice of Bph15 to cultivate brown planthopper resistant rice varieties containing Bph 15. Meanwhile, the molecular marking method provided by the invention can accurately screen the rice material of the brown planthopper resistant gene Bph15, thereby greatly improving the breeding efficiency.
Sequence listing
<110> Wuhan university
<120> specific codominant molecular marker of brown planthopper resistant gene Bph15 of rice and application thereof
<130> KHP171116848.7
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<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ggagagttca gtttgccatc c 21
Claims (2)
1. An application of a primer pair in breeding rice with brown planthopper resistance is disclosed, wherein the primer pair is any one of the following:
1) codominant marker primer B15InD 1:
forward primer sequence: TGCACCTGGATCAAATGTTTCT, respectively;
reverse primer sequence: GCCCTTCCCTCTCCATATCC, respectively; or
2) Codominant marker primer B15InD 2:
forward primer sequence: AGGAACAGTGACACGTAGCA, respectively;
reverse primer sequence: GGAGAGTTCAGTTTGCCATCC, respectively;
if the codominant marker primer B15InD1 is used, a 460bp amplified fragment can be amplified; or
The codominant marker primer B15InD2 can be used for amplifying 537bp amplification fragments, and then the rice to be detected is marked as brown planthopper resistant rice.
2. A method for screening brown planthopper resistant rice is characterized in that rice genome DNA to be detected is used as a template, a primer pair is utilized for PCR amplification, and an amplification product is detected: if the codominant marker primer B15InD1 is used, 460bp amplified fragments can be amplified; or
The codominant marker primer B15InD2 can be used for amplifying 537bp amplification fragments, and then the rice to be detected is marked to show insect resistance to brown planthopper; the primer pair is any one of the following:
1) codominant marker primer B15InD 1:
forward primer sequence: TGCACCTGGATCAAATGTTTCT, respectively;
reverse primer sequence: GCCCTTCCCTCTCCATATCC, respectively; or
2) Codominant marker primer B15InD 2:
forward primer sequence: AGGAACAGTGACACGTAGCA, respectively;
reverse primer sequence: GGAGAGTTCAGTTTGCCATCC are provided.
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