InDel marker for detecting rice xanthomonas oryzae pv. oryzae resistance gene x21 genotype and application

Abstract

This invention provides a method for detecting genes that resist bacterial blight in rice. Xa21 The InDel marker for genotypes and its application belongs to the field of rice breeding technology. The upstream primer sequence for the InDel marker is Xa21-InDel-F: 5'-TCTGCTCTACACTGCAATATGAAATGA-3', and the downstream primer sequence is Xa21-InDel-R: 5'-TAGTACCAAGTATTTTTCATGGTCAATG-3'. The Xa21-InDel marker was used to detect IRBB21 and 37 hybrid japonica rice restorer lines. The results showed that the InDel marker designed in this invention can accurately identify whether different varieties contain genes for resistance to bacterial blight. Xa21 And heterozygous genotype. This detection method has high accuracy and specificity. Xa21 Molecular marker-assisted selection of genes has strong practical applications in breeding.

Description

Technical Field

[0001] This invention relates to a method for detecting genes that resist bacterial blight in rice. Xa21 The InDel genotype marker belongs to the field of rice breeding technology. Background Technology

[0002] Rice growth and development are affected by various diseases. Bacterial blight is one of the most serious bacterial diseases affecting rice, severely restricting yield. Bacterial blight has a wide range of occurrence and spreads rapidly, generally causing a 10%-30% yield reduction, and in severe cases, more than 50% reduction, or even total crop failure. Rice bacterial blight was first discovered in Kyushu, Japan in 1884, and subsequently occurred in many regions of Asia, northern Australia, Africa, and the United States. Its pathogen is *Xanthomonas oryzae* (rice xanthomonas). Xanthomonas oryzae pv. Oryzae , Xoo It was separated in 1911. Years of rice breeding and production practices both domestically and internationally have shown that selecting and planting rice varieties carrying genes resistant to bacterial blight is the most environmentally friendly, effective, and economical way to control rice bacterial blight.

[0003] Currently, a total of 38 resistance genes for rice bacterial blight have been reported, of which... Xa1 , Xa3 / Xa26 , xa5 , Xa10 , xa13 , Xa21 , Xa23 , xa25 and Xa27 Nine disease-resistant genes have been successfully cloned. These genes are derived from wild rice species (such as *Rhizophora longifolia*). Rice longistaminata L. ) white leaf blight resistance gene Xa21 This is the first bacterial blight resistance gene cloned in rice. In recent years, Chinese rice breeders have been using genes carrying this gene. Xa21 Using the near-isogenic line IRBB21 as a donor, a batch of genes carrying the gene were bred using molecular marker-assisted selection breeding technology. Xa21 New rice varieties with this gene all exhibit strong resistance to rice blast and have significant value for production applications. Currently, most literature utilizes the STS molecular marker PTA248 for... Xa21 Molecular marker-assisted selection breeding. BLAST analysis using the PTA248 primer sequence in the Nipponbare genome sequence (IRGSP-1.0) revealed that the PTA248 marker is located at positions 21,049,068-21,048,425 bp on chromosome 11. Xa21 Alleles in Japanese Sun Os11g0559200Located at positions 20,802,978-20,806,262 bp on chromosome 11, there is a physical distance of 242 kb between the marker and the target gene. In assisted selection breeding using the PTA248 marker, recombination between the marker and the target gene is possible, potentially leading to the loss of the target gene. Therefore, the development of… Xa21 Functional molecular markers within genes are important for improving... Xa21 The efficiency of gene utilization in breeding for resistance to bacterial blight is of great significance. Summary of the Invention

[0004] This invention aims to solve the above-mentioned problems, thereby providing a method for detecting genes that resist bacterial blight in rice. Xa21 InDel markers for genotypes. This invention is based on... Xa21 Based on sequence differences in introns, a pair of InDel markers were designed. These markers were used to analyze IRBB21, Nipponbare, its F1 hybrids, and 37 hybrid japonica rice restorer lines. Xa21 Genotyping confirmed that this marker can accurately identify whether different rice varieties contain genes that resist bacterial blight. Xa21 And heterozygous genotypes, which can be used Xa21 Molecular marker-assisted selection breeding.

[0005] The technical solution of the present invention to solve the above problems is as follows: Detection of rice bacterial blight resistance genes Xa21 The InDel marker for the genotype, wherein the sequence of the InDel marker is: Xa21-InDel-F: 5'-TCTGCTCTACACTGCAATATGAAATGA-3', Xa21-InDel-R: 5'-TAGTACCAAGTATTTTTCATGGTCAATG-3'.

[0006] Another object of the present invention is to provide the InDel marker for detecting rice bacterial blight resistance genes. Xa21 Applications of genotypes.

[0007] Another object of the present invention is to provide a method for detecting genes that resist bacterial blight in rice. Xa21 Genotyping method.

[0008] Detection of rice bacterial blight resistance genes Xa21 The genotyping method includes the following steps: (1) Using the genomic DNA of the sample to be tested as a template, the target sequence is amplified by PCR using InDel labeling; the InDel-labeled sequence is: Xa21-InDel-F: 5'-TCTGCTCTACACTGCAATATGAAATGA-3', Xa21-InDel-R: 5'-TAGTACCAAGTATTTTTCATGGTCAATG-3'; The PCR reaction program was as follows: 95℃, 5 min; 94℃, 30 s, 58℃, 30 s, 72℃, 30 s, 35 cycles; 72℃, 10 min. (2) Electrophoresis was performed using a 3% agarose gel. (3) Judge based on electrophoresis results Xa21 The genotype.

[0009] InDel (insertion-deletion) refers to the insertion or deletion of DNA fragments of different sizes at the same locus in the genome of closely related species or different individuals of the same species. It is a phenomenon caused by gaps in homologous sequence alignment. It is characterized by high accuracy and good reproducibility. InDel polymorphic molecular markers are markers used for PCR amplification based on specific primers designed for the sequences flanking the insertion / deletion site, and can be genotyped using a convenient electrophoresis platform. InDel markers have high accuracy and good stability, avoiding ambiguity in subsequent analyses due to specificity and complexity. Furthermore, InDel markers can amplify mixed DNA samples and highly degraded trace DNA samples, and perform effective genotyping. InDel markers have a high distribution density and have been widely used in genetic analysis and molecular-assisted breeding of rice.

[0010] In summary, the present invention has the following beneficial effects: This invention is achieved through Xa21 InDel molecular markers were designed based on differential intron sequences of genes. Genotyping of IRBB21, Nipponbare, and their F1 hybrids revealed that the Xa21-InDel marker could accurately identify the gene. Xa21 Homozygous and heterozygous varieties of the gene were identified. This demonstrates that the InDel marker designed in this invention can accurately identify whether different varieties contain the gene for resistance to bacterial blight. Xa21 And heterozygous genotypes, which can be used Xa21 Molecular marker-assisted selection breeding of genes. Attached Figure Description

[0011] Figure 1 For different rice varieties Xa21 Allele intron sequence alignment and Xa21-InDel marker design; Figure 2 Verification of the Xa21-InDel marker; Figure 3Results of testing Xa21-InDel markers on 37 hybrid japonica rice restorer lines. Detailed Implementation

[0012] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0013] Example 1 1. Rice materials carry Xa21 The gene donor material IRBB21 was obtained from the Guangdong Provincial Germplasm Bank of Biological Resources, while the remaining hybrid japonica rice restorer lines were developed by our research group. The tested materials were planted at the Zhuangxing Comprehensive Experimental Station of the Shanghai Academy of Agricultural Sciences.

[0014] 2. Rice DNA extraction Take 100 mg of rice leaves, cut them into small pieces with scissors, put them into a 2 mL centrifuge tube, add 650 μL of 1.5 × CTAB solution (1.5% CTAB, 75 mmol / L Tris-HCl, 15 mmol / L EDTA, 1.05 mol / L NaCl, pH 8.0) and two steel balls with a diameter of 6 mm. Grind the mixture with a vibratory grinder at a frequency of 70 Hz for 90 s, then incubate at 56℃ for 30 min. Add 450 μL of chloroform, shake vigorously, and centrifuge at the highest speed for 10 min. Transfer the supernatant to a new centrifuge tube, add twice the amount of anhydrous ethanol, mix well, freeze for 10 min, then centrifuge at 12,000 r / min for 10 min. Discard the supernatant, wash once with 75% ethanol, air dry, add 200 μL of double-distilled water, and store at -20℃ for later use.

[0015] 3. Design of the InDel tag Xa21 Derived from wild rice with long-stemmed leaves, this gene contains two exons and one intron, with a coding region of 3075 bp and an intron of 843 bp. In the rice variety Nipponbare... Xa21 Gene alleles LOC_ Os11g35500 The coding region and intron lengths are 3111 bp and 797 bp, respectively (https: / / rapdb.dna.affrc.go.jp / ). Xa21 Compared to introns of alleles in other cultivated rice, this gene intron has two larger insertion sites, InDel1 and InDel2 (…). Figure 1 ).

[0016] The designed InDel marker sequence is as follows: Xa21-InDel-F: 5'-TCTGCTCTACACTGCAATATGAAATGA-3', Xa21-InDel-R: 5'-TAGTACCAAGTATTTTTCATGGTCAATG-3'.

[0017] The Xa21-InDel mark is carried in Xa21 A 184 bp DNA fragment can be amplified in the rice variety IRBB21, while other varieties such as Nipponbare, which do not contain this gene, do not. Xa21 A 160 bp DNA fragment was amplified in the rice variety containing the gene.

[0018] 4. Detection methods for molecular markers The total volume of the PCR amplification system was 10 μL, including 2×TSINGKE Master Mix, 0.2 μL each of forward and reverse primers (10 μM), 1 μL of DNA template, and 3.6 μL of double-distilled water. The PCR reaction program was as follows: 95℃, 5 min; 94℃, 30 s, 58℃, 30 s, 72℃, 30 s, 35 cycles; 72℃, 10 min. The PCR products were detected by 3% agarose gel electrophoresis.

[0019] 5. Results and Analysis Genotyping of IRBB21, Nipponbare, and their F1 hybrids was performed using InDel markers. Electrophoresis results of the PCR products showed that the Xa21-InDel marker could accurately identify the genotypes. Xa21 Homozygous and heterozygous varieties of the gene. The Xa21-InDel marker in carriers... Xa21 The IRBB21 rice variety can amplify a 184 bp DNA fragment, while the Nipponbare variety amplifies a 160 bp DNA fragment. In the F1 generation, both 184 bp and 160 bp DNA fragments are amplified simultaneously. Figure 2 ).and Figure one The designed Xa21-InDel markers yielded the expected results.

[0020] Example 2 Xa21-InDel markers were used to detect IRBB21 and 37 hybrid japonica rice restorer lines. The detection method was the same as in Example 1. The results showed that Xa21-InDel markers were present in... Xa21 Varieties with and without genes Xa21 The varieties of genes have very good genotyping ( Figure 3 This indicates that the InDel marker designed in this invention can accurately identify whether different varieties contain genes that resist bacterial blight. Xa21 And heterozygous genotype, suitable for use Xa21 Molecular marker-assisted selection breeding.

Claims

1. Detection of the rice IRBB21 bacterial blight resistance gene Xa21 The primer pair for genotype is characterized by, The sequences of the primer pair are as follows: Xa21-InDel-F: 5'-TCTGCTCTACACTGCAATATGAAATGA-3', Xa21-InDel-R: 5'-TAGTACCAAGTATTTTTCATGGTCAATG-3'.

2. The primer pair described in claim 1 is used to detect the rice IRBB21 bacterial blight resistance gene. Xa21 Applications of genotypes.

3. Detection of the rice IRBB21 bacterial blight resistance gene Xa21 The genotyping method is characterized by, Includes the following steps: Using the genomic DNA of the rice IRBB21 sample as a template, the target sequence was amplified by PCR using primer pairs; the sequences of the primer pairs are as follows: Xa21-InDel-F: 5'-TCTGCTCTACACTGCAATATGAAATGA-3', Xa21-InDel-R: 5'-TAGTACCAAGTATTTTTCATGGTCAATG-3'; The PCR reaction program was as follows: 95℃, 5 min; 94℃, 30 s; 58℃, 30 s; 72℃, 30 s; 35 cycles; 72℃, 10 min. Electrophoresis was performed using a 3% agarose gel. Judging from the electrophoresis results Xa21 The genotype.

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