A caps molecular marker of a rice blast-resistant crkbr gene promoter region and application thereof

Abstract

The application discloses a rice blast-resistant gene promoter region CAPS molecular marker and application thereof, and belongs to the technical field of plant molecular breeding. CRKBR The molecular marker comprises two polymorphic sites in a rice reference genome IRGSP-1.0 chromosome 7 CRKBR The two polymorphic sites are both C / T. By using a primer pair composed of SEQ ID NO. 1 and SEQ ID NO. 2 to perform PCR amplification, and using endonuclease Alw44I to perform enzyme cutting on the amplification product, if two bands of 266 bp and 347 bp are presented, it is the resistant type; if a single band of 613 bp is presented, it is the susceptible type. The CAPS molecular marker and the detection method thereof have the advantages of simple operation, low cost, intuitive and accurate results and the like, are suitable for rice blast-resistant germplasm resource identification and molecular marker assisted breeding, and have important significance for accelerating the rice disease-resistant breeding process.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to the development and application of molecular markers based on single nucleotide polymorphisms (SNPs) in the promoter region of rice blast resistance-related genes, and particularly to a rice blast resistance gene. CRKBR The CAPS (Cycleavage-amplified polymorphic sequence) molecular marker in the promoter region, its specific primer set, detection methods containing the marker, and its application in the identification of rice varieties resistant to rice blast. Background Technology

[0002] Rice is one of the world's most important food crops, and its safe production is directly related to global food security. Rice blast is caused by the rice blast fungus (…). Magnaporthe oryzae Rice blast is a devastating fungal disease caused by rice blast, often referred to as "rice cancer." It is characterized by its wide spread, severe damage, and difficulty in control, causing enormous losses to rice production year after year. Breeding and planting resistant varieties is the most economical, effective, and environmentally friendly strategy for controlling rice blast.

[0003] Traditional rice disease resistance breeding mainly relies on field phenotypic identification. This method is easily affected by environmental conditions, pathogen physiological races, and inoculation consistency, resulting in a long cycle, heavy workload, and low efficiency. The emergence of molecular marker-assisted selection (MAS) technology provides a powerful tool for breeding, enabling early, rapid, and accurate selection of resistant genes, thereby significantly shortening the breeding cycle and improving selection efficiency.

[0004] Through long-term artificial domestication and selection, cultivated rice has increasingly narrowed its genetic base, especially in terms of disease resistance genes. Wild rice, as the ancestor of cultivated rice, possesses abundant genetic variation and superior genes, making it a key germplasm resource for broadening the genetic base of cultivated rice and introducing new resistance sources. However, the key to discovering and utilizing disease resistance genes from wild rice lies in developing molecular markers that are closely linked to or co-segregated with their functional sequences.

[0005] Currently, although some molecular markers related to rice blast resistance have been developed, there is still a lack of specific molecular markers targeting key gene promoter regions from wild rice that directly affect gene expression or function. In particular, CAPS markers, which are developed based on SNPs, are simple to operate, low in cost, and provide intuitive results, are of great significance for large-scale and rapid screening of target genotypes in breeding practice. Therefore, developing a rice blast resistance gene derived from wild rice is crucial. CRKBR Highly efficient and reliable CAPS molecular markers that are directly associated with functional alleles are of great value for accelerating the breeding process of rice blast resistance. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for improving rice resistance to rice blast. CRKBR CAPS molecular markers directly associated with gene functional alleles, their specific primer sets, and detection methods. These markers enable rapid, accurate, and low-cost differentiation of rice materials carrying resistance and susceptibility alleles, providing an effective tool for marker-assisted breeding.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for screening rice for resistance to rice blast. CRKBR CAPS molecular markers in the gene promoter region, comprising two markers located on chromosome 7 of the rice reference genome IRGSP-1.0. CRKBR Polymorphic sites in the gene promoter region; wherein, the polymorphic sites are respectively located in CRKBR The gene promoter region contains polymorphic sites at -1704 bp and -1706 bp, both of which are C / T. When both polymorphic sites are C, the DNA sequence generates a recognition site for the restriction endonuclease Alw44I. CRKBR The nucleotide sequence of the gene promoter region is shown in SEQ ID NO.3.

[0008] Secondly, the present invention provides a set of specific primers for detecting the above-mentioned CAPS molecular markers. The primer set includes an upstream primer Pro-1F with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer Pro-1R with a nucleotide sequence as shown in SEQ ID NO.2. After PCR amplification using rice genomic DNA as a template and digestion of the amplified product with the restriction enzyme Alw44I, the number of bands in the digested product can be used to determine whether the rice is resistant to rice blast. Specifically: if only a single band of 613 bp appears in the digested product, it is a susceptible type; if two bands of 266 bp and 347 bp appear simultaneously in the digested product, it is a resistant type.

[0009] Thirdly, this invention provides a method for detecting rice using the aforementioned CAPS molecular markers and primer sets. CRKBR The method for determining the presence of rice blast resistance alleles in the gene promoter region includes the following steps: (1) Extract genomic DNA from the rice sample to be tested; (2) Using the genomic DNA obtained in step (1) as a template, perform PCR amplification with the above-mentioned specific primer set to obtain the amplification product; (3) The amplification product obtained in step (2) was digested with the restriction endonuclease Alw44I; (4) Perform electrophoretic analysis on the enzyme digestion products obtained in step (3); (5) Genotyping based on electrophoretic patterns (e.g.) Figure 4 As shown): If the electrophoresis result shows a 613 bp main band, the material is determined not to contain the rice blast resistance allele (susceptible type); if the electrophoresis result shows two main bands of 266 bp and 347 bp, the material is determined to contain the rice blast resistance allele (resistant type).

[0010] Further, the PCR amplification reaction program in step (2) is as follows: 94℃ pre-denaturation for 2 min; 98℃ pre-denaturation for 10 s, 63℃ annealing for 30 s, 68℃ extension for 1 min, cycled 35 times; and finally 68℃ extension for 10 min.

[0011] Fourthly, this invention provides the application of the above-mentioned CAPS molecular markers, specific primer sets, or detection methods in screening rice varieties resistant to rice blast.

[0012] The beneficial effects of this invention are as follows: (1) Highly targeted and accurate: This invention is based on rice with strong resistance to rice blast. CRKBR The gene (LOC_Os07g35680) has SNP sites (C / T) directly related to resistance function developed as molecular markers in its promoter region. By converting these to CAPS markers, resistance (C) and susceptibility (T) alleles can be accurately distinguished, and the detection results are highly consistent with genotype and field resistance phenotype (e.g., ...). Figure 4 , Figure 5 (As shown).

[0013] (2) Simple operation and low cost: The detection method provided by this invention is based on conventional PCR amplification, enzyme digestion and agarose gel electrophoresis technology. It does not require expensive sequencing or fluorescence detection equipment. The process is standardized and easy to operate, making it suitable for promotion and application in various breeding units, seed companies and laboratories.

[0014] (3) The results are intuitive and easy to interpret: the enzyme digestion and typing results can be presented by ordinary agarose gel electrophoresis, and the band patterns of resistant materials and susceptible materials are significantly different (e.g. Figure 4 As shown in the figure, it can be directly observed and judged by the naked eye, which greatly reduces the technical threshold and the risk of misjudgment.

[0015] (4) Wide range of applications and high practical value: This molecular marker can not only be used to discover resistance sources from wild rice or local varieties, but also directly used for resistance identification of cultivated rice varieties (lines), early directional selection of hybrid offspring, and purity and authenticity detection of bred varieties, which can effectively accelerate the breeding process of new varieties resistant to rice blast. Attached Figure Description

[0016] Figure 1This is a schematic diagram of the recognition site sequence of the restriction endonuclease Alw44I involved in this invention.

[0017] Figure 2 This is a schematic diagram showing the polymorphic sites (C / T) of the CAPS molecular marker described in this invention and their location in the promoter region of the rice CRKBR gene.

[0018] Figure 3 This is a comparison diagram of the results of using sequencing methods to verify the C / T polymorphism site in the promoter region of the CRKBR gene in this invention. The upper part (T-type) is the sequencing map of the susceptible variety Nip, and the lower part (C-type) is the sequencing map of the near-isogenic line (NIL) carrying the disease-resistant allele.

[0019] Figure 4 This is a graph showing the results of enzyme digestion electrophoresis typing of the CAPS molecular marker described in this invention. Lane M is the DNA molecular weight standard (Marker), lanes 1 and 2 are the undigested PCR products (approximately 613 bp) of resistant and susceptible materials, respectively, lane 3 is the product of susceptible material (T allele) digested with Alw44I (approximately 613 bp single band), and lane 4 is the product of resistant material (C allele) digested with Alw44I (approximately 266 bp and approximately 347 bp two characteristic bands).

[0020] Figure 5 This is a field comparison diagram of the blast resistance phenotypes of the near-isogenic line (NIL) of wild rice with the blast resistance gene CRKBR and its background parent Nipponbare (Nip), which intuitively shows the differences between the disease-resistant (NIL) and disease-susceptible (Nip) phenotypes corresponding to the molecular markers described in this invention. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of this invention, the following will provide a more detailed description of this application in conjunction with embodiments. The rice material used in this invention is Nipponbare (Nip), and the carrier... CRKBR The near-isogenic lines (NILs) of the disease resistance gene were all obtained from the 12-layer shelf of the mid-term collection of the National Crop Germplasm Bank in Beijing, China.

[0022] Example 1: Extraction of rice genomic DNA 1. Place approximately 100 mg of fresh leaves into a 2 mL centrifuge tube containing small steel balls. Place the centrifuge tube into a foam box containing liquid nitrogen and freeze for about 30 seconds. Then, remove it and place it in a grinder (SPEX Sample PrepGeno / Grinder) to grind the leaves into powder.

[0023] 2. Transfer the powder to a 2 mL centrifuge tube, add 800 μL of CTAB extraction buffer (preheated to 65°C, 100 mM Tris-HCl, pH 8.0; 20 mM EDTA, pH 8.0; 1.4 M NaCl; 2% CTAB; 1% PVP-40), and gently invert to mix. 3. Incubate in a 65℃ water bath for 60 minutes, gently inverting and mixing once every 15 minutes during this period.

[0024] 4. After cooling to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently, and place in a -20℃ refrigerator for 30 min. Then centrifuge at 12000 rpm / min for 10 min to precipitate the DNA and discard the supernatant.

[0025] 5. Add 1 mL of 95% ethanol to the centrifuge tube containing the DNA precipitate, soak and wash the DNA for 5 min, then centrifuge at 12000 rpm / min for 10 min, and discard the supernatant.

[0026] 6. Centrifuge at 4℃ and 12000 rpm for 15 minutes, and discard the supernatant. Wash the precipitate twice with 70% ethanol and air dry at room temperature.

[0027] 7. After the DNA precipitate has air-dried, add 100 μL of ddH2O and 1 μL of RNase. Store the dissolved sample at -20 °C for subsequent experiments.

[0028] Example 2: PCR amplification based on CAPS molecular markers 1. Primer preparation and target sequence localization Based on the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, specific primers Pro-1F and Pro-1R were synthesized. Figure 2 As shown, the 613 bp fragment amplified by the primer pair is located in the promoter region of the rice CRKBR gene (LOC_Os07g35680), which contains two key polymorphic sites (C / T); the nucleotide sequence of the promoter region of the rice CRKBR gene (LOC_Os07g35680) is shown in SEQ ID NO.3.

[0029] 2. Preparation of PCR reaction system (total volume 50 μL) 2× PCR Buffer for KOD FX Neo: 25 μL; 2 mM dNTPs: 10 μL; 10 μM Pro-1F primers: 1.5 μL; 10 μM Pro-1R primers: 1.5 μL; KOD FX Neo polymerase: 1 μL; Template DNA (approx. 50 ng / μL): 2 μL (approx. 100 ng); Sterile deionized water: bring to 50 μL.

[0030] Mix the above ingredients on ice and centrifuge briefly to collect the liquid at the bottom of the tube.

[0031] 3. PCR reaction procedure Pre-denaturation at 94℃ for 2 min; pre-denaturation at 98℃ for 10 s, annealing at 63℃ for 30 s, extension at 68℃ for 1 min, repeated 35 times; final extension at 68℃ for 10 min.

[0032] 4. PCR Product Verification: Take 35 μL of PCR product and perform electrophoresis on a 2% agarose gel. If the amplification is successful, a single, bright 613 bp band should be visible. Figure 4 (Undigested bands in lanes 1 and 2 of the middle swimming pool).

[0033] Example 3: Restriction endonuclease digestion analysis and genotyping 1. Enzyme digestion principle: This invention utilizes the restriction endonuclease Alw44I for typing. The recognition sequence of this enzyme is as follows: Figure 1 As shown. Figure 3 The sequencing results confirmed that the disease resistance (carrier) CRKBR The near-isogenic line (NIL) of the disease resistance gene (type C) introduces the Alw44I recognition site at this polymorphic site, while the disease-susceptible allele (type T) of the susceptible rice variety Nipponbare (Nip) does not have this recognition site.

[0034] 2. Preparation of the enzyme digestion system (total volume 50 μL): PCR product obtained in Example 2: 20 μL (approximately 1 μg); FastDigest Alw44I: 3 μL; 10×FastDigest Green Buffer: 5 μL; Sterile deionized water: to bring the total volume to 50 μL.

[0035] Gently whisk to mix, then briefly centrifuge.

[0036] 3. Enzymatic digestion reaction: Incubate the reaction system at 37°C for 10 hours, then treat at 80°C for 5 minutes to inactivate the enzyme.

[0037] 4. Electrophoretic analysis and genotyping of enzyme digestion products: All enzyme digestion products were subjected to electrophoresis on a 2.0% agarose gel. The results are as follows: Figure 4 As shown.

[0038] Susceptible type (Nip, T allele): The Alw44I restriction site is absent, the PCR product is not cleaved, and the electrophoresis pattern shows only a 613 bp major band (corresponding to...). Figure 4 (Lane 3 mode).

[0039] Disease-resistant type (NIL, C allele): The Alw44I restriction site is present; the 613 bp fragment is cleaved into two fragments, and the electrophoresis pattern shows two characteristic main bands at 266 bp and 347 bp (corresponding to...). Figure 4 (The pattern of lane 4). Based on this clear band pattern, genotyping can be performed intuitively and accurately.

[0040] like Figure 5 As shown, the molecular typing results are completely consistent with the field identification results of rice blast resistance phenotypes: the near-isogenic line (NIL) exhibits significant resistance, while Nipponbare (Nip) is severely susceptible. This fully demonstrates that the genotype-phenotype association of the molecular markers described in this invention is highly consistent, and the results are accurate and reliable.

Claims

1. A method for detecting rice CRKBR The application of the method of using alleles for rice blast resistance in the promoter region of rice blast in the screening of rice blast-resistant varieties is characterized by, The method includes the following steps: (1) Extract genomic DNA from the rice sample to be tested; wherein the rice to be tested is Nipponbare Nip or a near-isogenic line NIL carrying the CRKBR resistance gene; the near-isogenic line NIL carrying the CRKBR resistance gene is a near-isogenic line NIL of wild rice blast resistance gene CRKBR, its background parent is Nipponbare Nip, and the blast resistance gene CRKBR is LOC_Os07g35680; the nucleotide sequence of the promoter region of the blast resistance gene CRKBR is shown in SEQ ID NO.3; (2) Using the genomic DNA obtained in step (1) as a template, PCR amplification is performed using a specific primer set to obtain the amplification product; the primer set contains the upstream primer Pro-1F with the nucleotide sequence shown in SEQ ID NO.1 and the downstream primer Pro-1R with the nucleotide sequence shown in SEQ ID NO.2; (3) The amplification product obtained in step (2) was digested with the restriction endonuclease Alw44I; (4) Perform electrophoretic analysis on the enzyme digestion products obtained in step (3); (5) Genotyping based on electrophoresis patterns: If the electrophoresis results show a 613 bp main band, the rice sample to be tested is the susceptible variety Nipponbare Nip; if the electrophoresis results show two main bands of 266 bp and 347 bp, the rice sample to be tested is the near-isogenic line NIL carrying the CRKBR resistance gene, and the near-isogenic line NIL carrying the CRKBR resistance gene is a disease-resistant variety.

2. The method according to claim 1, characterized in that, The PCR amplification reaction program in step (2) is as follows: 94℃ pre-denaturation for 2 min; 98℃ pre-denaturation for 10 s, 63℃ annealing for 30 s, 68℃ extension for 1 min, cycled 35 times; and finally 68℃ extension for 10 min.

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