A SNP molecular marker for detecting rice bacterial blight resistance gene Xa23 and application thereof

Abstract

The application discloses a SNP molecular marker for detecting rice bacterial blight resistance genes and application of the SNP molecular marker Xa23 . The SNP molecular marker is one of K_110532, K_110534, K_110536, K_110543 and K_110547. The SNP molecular marker screened by the application has good stability, is not affected by other gene effects and environmental factors, can be selected in an early generation, shortens a breeding period, improves breeding efficiency, has important significance for rice bacterial blight resistance variety improvement, and is suitable for Xa23 assisted selection breeding of the gene. The application is suitable for popularization and application.

Description

[0001] This case is a divisional application of the patent application with application number 2022115200132, application date November 30, 2022, entitled "An SNP molecular marker for detecting the rice bacterial blight resistance gene Xa23 and its application". Technical Field

[0002] This invention relates to the field of rice breeding, specifically to a method for detecting resistance genes to rice bacterial blight. Xa23 SNP molecular markers and their applications. Background Technology

[0003] Rice bacterial blight pathogen exhibits physiological race specialization, and the resistance of varieties is primarily controlled by major resistance genes in the nuclear genome. Since Japanese scientists analyzed the resistance responses of hybrids of *Prunus cerevisiae* and *Rhizopus lantaimas* to *Prunus chinensis*, identifying and naming dominant resistance genes, research on the identification and discovery of rice bacterial blight resistance genes has been ongoing. According to data released by the National Rice Data Center, 38 bacterial blight resistance genes have been confirmed and reported, numbered up to Xa38. Of these, 26 are dominant genes, and the rest are recessive genes; 26 have been mapped, and 8 of these genes have been cloned. Xa1 , Xa5 , Xa27 , Xa13 , Xa3 / Xa26 , Xa4 , Xa2 1 and Xa23 .

[0004] Currently, breeders have utilized molecular marker-assisted selection (MAS) to... Xa4 , Xa21 and Xa23 Many disease-resistant restorer lines have been bred, such as IR26, IR28, IR30, IR32, IR36, IR50, and IR54. Most hybrid rice varieties widely planted in China contain these disease-resistant restorer lines. Xa4 However, the long-term, large-scale use of a single resistance source has led to the evolution of new pathogenic strains, compromising the durability of disease resistance in hybrid rice. Therefore, how to utilize new disease-resistant genes to quickly improve the disease resistance of hybrid rice is an urgent problem to be solved in breeding.

[0005] Xa23 This is a broad-spectrum resistance gene for bacterial blight found in common wild rice, which can resist the new pathogenic race Xa21 discovered in Guangdong Province, my country. Xa23It is rapidly becoming a dominant resistance source for improving bacterial blight resistance in hybrid rice parents in my country. This is being achieved through marker-assisted selection to improve or breed varieties carrying molecular markers. Xa23 The restorer lines or strains include: CR6201, CR6271, CR6351, Minghui 86, C418, HB1471, HB1473, K10, H705, H706, and ZR21-sk1. The agronomic traits of combinations of K10 with Funong S and ZR21-sk1 with the sterile line II-32A are excellent. Improved or developed sterile lines include Jin 23A and Zhongjia A. In addition, the use of transgenic technology to improve the disease resistance of varieties has also shown initial success. Xa23 Gene transfer was performed into the disease-susceptible cultivar Jin Gang 30, resulting in the near-isogenic line CBB23.

[0006] In traditional breeding, phenotypic identification is used to infer the presence of resistance genes, while resistance spectrum analysis of bacterial blight pathogens is used for identification. Xa23 The gene exists, but this method requires a specific physiological race of the bacterial blight pathogen, and the inoculation conditions, including the rice's growth stage, climate, and temperature, are quite strict, making it unsuitable for large-scale identification and application. Because... Xa23 The gene has been cloned, and currently... Xa23 Research on gene introduction into rice mainly utilizes marker-assisted selection. The markers used in marker-assisted selection are generally RAPD, SSR, AFLP, RFLP, CAP, or dCAP, or based on PCR, enzyme digestion, or a combination of both. These methods require cumbersome and contamination-risk electrophoresis detection. The inventors developed a method that... Xa23 Tightly linked SNP molecular markers, combined with KASP detection technology, eliminate the need for gel electrophoresis and are not limited to costly restriction endonucleases, enabling high-throughput, rapid, and accurate identification in genetic breeding. Xa23 Genes, greatly improving the efficiency of gene transfer. Summary of the Invention

[0007] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a gene for resistance to rice bacterial blight. Xa23 Linked SNP molecular markers.

[0008] The present invention also proposes a primer set for detecting the above-mentioned SNP molecular markers.

[0009] The present invention also proposes a reagent kit.

[0010] This invention also proposes a gene chip.

[0011] This invention also proposes the application of the above-mentioned SNP molecular markers, primer sets, kits and / or gene chips.

[0012] The present invention also proposes a method for detecting the above-mentioned SNP molecular markers.

[0013] In a first aspect of the invention, a method for detecting resistance genes to rice bacterial blight is proposed. Xa23 The SNP molecular markers are K_110532, K_110534, K_110536, K_110543, and K_110547. The polymorphic site of K_110532 is located at position 22167870 on chromosome 11 of rice in the MSU7.0 genome version, with a polymorphism of A / T. The polymorphic site of K_110534 is located at position 22169591 on chromosome 11 of rice in the MSU7.0 genome version, with a polymorphism of [missing information]. The polymorphic site K_110536 is located at position 22174338 of chromosome 11 of rice in the MSU7.0 genome version, and its polymorphism is T / C; the polymorphic site K_110543 is located at position 22232666 of chromosome 11 of rice in the MSU7.0 genome version, and its polymorphism is A / G; the polymorphic site K_110547 is located at position 22249375 of chromosome 11 of rice in the MSU7.0 genome version, and its polymorphism is G / C.

[0014] In a second aspect of the invention, a primer set for amplifying the above-mentioned SNP molecular marker is proposed, the primer set comprising specific primers and universal primers, wherein the specific primer sequences include Primer X and Primer Y.

[0015] In some embodiments of the present invention, when the SNP molecular marker is K_110532, the specific primer sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2; when the SNP molecular marker is K_110534, the specific primer sequences are as shown in SEQ ID NO.4 and SEQ ID NO.5; when the SNP molecular marker is K_110536, the specific primer sequences are as shown in SEQ ID NO.7 and SEQ ID NO.8; when the SNP molecular marker is K_110543, the specific primer sequences are as shown in SEQ ID NO.10 and SEQ ID NO.11; and when the SNP molecular marker is K_110547, the specific primer sequences are as shown in SEQ ID NO.13 and SEQ ID NO.14.

[0016] In some embodiments of the present invention, when the SNP molecular marker is K_110532, the universal primer sequence is as shown in SEQ ID NO.3; when the SNP molecular marker is K_110534, the universal primer sequence is as shown in SEQ ID NO.6; when the SNP molecular marker is K_110536, the universal primer sequence is as shown in SEQ ID NO.9; when the SNP molecular marker is K_110543, the universal primer sequence is as shown in SEQ ID NO.12; and when the SNP molecular marker is K_110547, the universal primer sequence is as shown in SEQ ID NO.15.

[0017] In some embodiments of the present invention, the specific primers are respectively linked to FAM and HEX fluorescent adapter sequences.

[0018] In a third aspect of the invention, a kit is provided comprising the aforementioned primer set.

[0019] In a fourth aspect of the invention, a gene chip is provided, the gene chip comprising the primer set described above.

[0020] In a fifth aspect of the invention, the application of the above-mentioned SNP molecular markers, primer sets, kits, and / or gene chips is proposed, wherein the application is as follows: (1) In rice bacterial blight resistance genes Xa23 Applications in genotyping; (2) In the detection of bacterial blight resistance genes Xa23 Applications in; (3) Application in the identification and screening of rice varieties resistant to bacterial blight; (4) Application in molecular marker-assisted breeding of rice.

[0021] (5) Application in rice breeding; (6) Application in the preparation of rice breeding products.

[0022] According to the sixth aspect of the present invention, the detection of rice bacterial blight resistance genes using the above-described SNP molecular markers is described. Xa23 The method includes the following steps: S1. Extracting genomic DNA from rice; S2. Perform polymorphism detection on the SNP molecular markers in the genomic DNA extracted in step S1, and determine whether the rice material contains a bacterial blight resistance gene based on the detection results. Xa23 .

[0023] According to some embodiments of the present invention, in step S1, genomic DNA extraction is performed using a simplified CTAB method (hexadecyltrimethylammonium bromide method).

[0024] According to some embodiments of the present invention, in step S2, the SNP sites are detected using KASP (competitive allele-specific PCR) technology.

[0025] A rice breeding method includes the following steps: using the above-mentioned detection method, selecting rice varieties containing genes for resistance to bacterial blight. Xa23 The rice will be used for subsequent breeding.

[0026] According to embodiments of the present invention, a method for detecting rice bacterial blight resistance genes. Xa23 The SNP molecular markers have at least the following beneficial effects: This invention, through sequence alignment using a database of 3000 rice resequencing samples, identified SNP markers within gene linkage regions that are related to... Xa23 Co-separated specific SNP molecular markers, combined with KASP detection technology, eliminate the need for gel electrophoresis and are not limited to costly restriction endonucleases. Xa23 High-throughput, rapid, and accurate identification is possible in genetic breeding. Xa23 Genes, greatly improving the efficiency of gene transfer, and promoting Xa23 The application of genes in commercial breeding is of great significance. This invention has the advantages of simple operation, low cost, and short cycle. Furthermore, the markers exhibit good stability, are unaffected by other gene effects and environmental factors, allow for early-generation selection, shorten breeding cycles, and improve breeding efficiency. It is of great significance for improving rice varieties resistant to bacterial leaf blight and is suitable for… Xa23 Gene-assisted selection breeding. Suitable for widespread application.

[0027] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a flowchart of the molecular marker development process in Embodiment 1 of the present invention; Figure 2 This is a typing diagram of the K_110532 molecular marker in Example 1 of the present invention; Figure 3 This is a typing diagram of the K_110534 molecular marker in Example 2 of the present invention; Figure 4 This is a typing diagram of the K_110535 molecular marker in Example 3 of the present invention; Figure 5This is a typing diagram of the K_110543 molecular marker in Example 4 of the present invention; Figure 6 This is a typing diagram of the K_110547 molecular marker in Example 5 of the present invention; Figure 7 This is a diagram showing the genetic location verification results of the test example of the present invention. Detailed Implementation

[0029] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0030] Example 1: A method for detecting resistance genes to rice bacterial blight Xa23 SNP molecular marker K_110532 The design process of this molecular marker, such as Figure 1 As shown, based on the cloned gene Xa23 The sequence was located within the molecular marker region 22203734-22204676 on rice chromosome 11. SNPs were then extracted from this region by extending 50 kb to both sides of the region on chromosome 11, using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed for SNP mining. SNP selection was based on PIC values ​​and the presence of other SNPs within a 50 bp radius of the selected SNP. Primers were designed for the selected SNPs using BatchPrimer3. Primers containing these SNPs were then applied to these SNP sites. Xa23 Four gene donor materials from the rice varieties Huahui 7620, CBB23, R178, and 74, and other gene-free rice varieties, were used. Xa23 KASP reaction validation was performed on 19 rice varieties, and the KASP marker K_110532, which showed good association with the resistance donor material and good amplification effect, was selected. The marker was then screened and tested, as follows: 1 Primer Design Primers were synthesized by Invitrogen. Each set of primers contained three primers: two specific primers linked to the FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to the FAM and HEX fluorescent adapter sequences, respectively, as shown in Table 1. If the sample detected a fluorescent signal corresponding to the sequence linked to primer X, the sample was identified as genotype A at the detection site, and the rice sample was determined to be homozygous without resistance to bacterial blight. xa23 Genotype; if only the fluorescent signal corresponding to the fluorescent sequence linked to primer Y is detected, then the sample has the T genotype at the detection site, and the rice sample is determined to be homozygous with resistance to bacterial blight. Xa23 Genotype; if two fluorescences are detected simultaneously, the rice sample is determined to be a heterozygous variety with resistance to bacterial blight. Xa23 genotype.

[0031] Table 1. Tagging Information

[0032] 2. Sample Testing DNA extraction: Genomic DNA was extracted from rice using a simplified CTAB method, including the following steps: 1) Take a sample and place it in a 2.0 ml tube. Add two steel balls and 750 μL of CTAB solution beforehand, and shake to homogenize the sample for 1.5 min. 2) Heating at 65℃ with vibration for 0.5-1 hour; 3) Cool to room temperature, then add 750 ml of chloroform:isoamyl alcohol (24:1) solution to a fume hood and mix well; 4) Centrifuge at 12000 rpm for 10 min, and transfer about 500 ml of the supernatant to a new 1.5 ml centrifuge tube; 5) Add an equal volume of isopropanol solution, shake gently to mix, precipitate at -20℃ for more than 1 hour, centrifuge at 12000 rpm for 10 minutes, and discard the supernatant; 6) Add 1000ml of 70% ethanol, gently tap the precipitate, centrifuge at 1000rpm for 3min, and discard the supernatant; 7) Add 300 μL of H2O and dissolve overnight for later use.

[0033] KASP Reaction Assay: The KASP reaction assay was performed on the LGC SNPline genotyping platform. 20 ng of DNA sample was added to a microplate, dried, and then KASP reaction mixture was added. The reaction system is shown in Table 2. PCR amplification was performed in a water bath thermal cycler. The Touchdown PCR reaction conditions were: 94℃ pre-denaturation for 15 min; first amplification reaction: 94℃ denaturation for 20 s, annealing and extension at 65℃–57℃ for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 0.8℃ per cycle; second amplification reaction: 94℃ denaturation for 20 s, annealing and extension at 57℃ for 60 s, 26 cycles. After the reaction, the KASP reaction products were scanned using a Pherastar scanner to read the fluorescence data. The fluorescence scan results were automatically converted into images. The LGC SNPline genotyping platform and its accompanying reagents and consumables were purchased from LGC Ltd., UK.

[0034] Table 2 Reaction system for KASP detection

[0035] 3-labeled classification data Based on the above detection method, the molecular marker K_110532 was used to detect the presence of... Xa23 Twenty-three rice varieties, including gene donor materials, other gene donors, and susceptible materials, underwent KASP primary screening validation. The results are shown in Table 3. Xa23 Four rice varieties (Huahui 7620, CBB23, R178, and 74) showed T bases at the K_110532 testing site. Excluding three samples that showed no amplification, the remaining 12 samples did not contain [a specific gene]. Xa23 Both other gene donors for resistance to bacterial blight and susceptible materials in rice varieties showed the presence of base A at the test sites, consistent with the sequencing results.

[0036] Table 3 Initial Screening Data

[0037] 4. Specific detection To determine the specificity of the marker in this invention, the SNP molecular marker K_110532 was validated in a natural population using 188 samples according to the above-described detection method. These 188 samples included samples known to contain homozygous markers. Xa23 Varieties containing genes, those containing other bacterial blight resistance donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials, etc. Molecular markers in natural population genotyping results, such as... Figure 2 As shown, 5 known samples contain Xa23 Genetic testing showed the variety to be homozygous for resistance to bacterial leaf blight. Xa23 Genotype: All four F1 individual plants were found to be heterozygous. Xa23Genotype: Among 14 F2 individual plants, 3 homozygous individuals with resistance to bacterial blight were detected. Xa23 Genotype, 3 homozygous copies without resistance to bacterial blight xa23 Genotype and 8 heterozygotes with resistance to white leaf blight Xa23 Genotypes of 158 materials, including those containing other bacterial blight resistance gene donors, universally susceptible materials, common hybrid rice, and core rice breeding materials, were all tested and found to be homozygous for bacterial blight resistance. xa23 Genotyping results were consistent with the sequencing results of these 188 materials, indicating that the molecular marker K_110532 genotyping has high accuracy and can be conveniently and efficiently used to identify whether rice varieties contain [a specific genotype / propagation]. Xa23 Gene.

[0038] Example 2: A method for detecting resistance genes to rice bacterial blight. Xa23 SNP molecular marker K_110534 The design process of this molecular marker, such as Figure 1 As shown, based on the cloned gene Xa23 The sequence was located within the molecular marker region 22203734-22204676 on rice chromosome 11. SNPs were then extracted from this region by extending 50 kb to both sides of the region on chromosome 11, using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed for SNP mining. SNP selection was based on PIC values ​​and the presence of other SNPs within a 50 bp radius of the selected SNP. Primers were designed for the selected SNPs using BatchPrimer3. Primers containing these SNPs were then applied to these SNP sites. Xa23 Four gene donor materials from the rice varieties Huahui 7620, CBB23, R178, and 74, and other gene-free rice varieties, were used. Xa23 KASP reaction verification was performed on 19 rice varieties, and the KASP marker K_110534, which showed good association with the resistance donor material and good amplification effect, was selected. The marker was then screened and tested, as follows: 1 Primer Design Primers were synthesized by Invitrogen. Each set of primers contained three primers: two specific primers linked to the FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to the FAM and HEX fluorescent adapter sequences, respectively, as shown in Table 4. If the sample detected a fluorescent signal corresponding to the sequence linked to primer X, the sample was classified as having the T genotype at the detection site, indicating that the rice sample was homozygous and lacked resistance to bacterial blight. xa23 Genotype; if only the fluorescent signal corresponding to the fluorescent sequence linked to primer Y is detected, then the sample has genotype C at the detection site, and the rice sample is determined to be homozygous with resistance to bacterial blight.Xa23 Genotype; if two fluorescences are detected simultaneously, the rice sample is determined to be a heterozygous variety with resistance to bacterial blight. Xa23 genotype.

[0039] Table 4 Marking Information

[0040] 2. Sample Testing DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, the specific method being the same as in Example 1.

[0041] KASP reaction test: The specific method for the KASP reaction test is the same as in Example 1.

[0042] 3-labeled classification data Based on the above detection method, the molecular marker K_110534 was used to detect the presence of... Xa23 Twenty-three rice varieties, including gene donor materials, other gene donors, and susceptible materials, underwent KASP primary screening validation. The results are shown in Table 5. Xa23 Four rice varieties (Huahui 7620, CBB23, R178, and 74) showed C amplification at the K_110532 testing site. Excluding the three samples that showed no amplification, the remaining 12 samples did not contain [amplification gene]. Xa23 Both other gene donors for resistance to bacterial blight and susceptible materials in rice varieties showed the presence of the base T at the test site, consistent with the sequencing results.

[0043] Table 5 Initial Screening Data

[0044] 4. Specific detection To determine the specificity of the marker in this invention, the SNP molecular marker K_110534 was validated in a natural population using 188 samples according to the above-described detection method. These 188 samples included samples known to contain homozygous markers. Xa23 Varieties containing genes, those containing other bacterial blight resistance donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials, etc. Molecular markers in natural population genotyping results, such as... Figure 3 As shown, 5 known samples contain Xa23 Genetic testing showed the variety to be homozygous for resistance to bacterial leaf blight. Xa23 Genotype: All four F1 individual plants were found to be heterozygous. Xa23 Genotype: Among 14 F2 individual plants, 3 homozygous individuals with resistance to bacterial blight were detected. Xa23 Genotype, 3 homozygous copies without resistance to bacterial blight xa23 Genotype and 8 heterozygotes with resistance to white leaf blight Xa23Genotypes of 158 materials, including those containing other bacterial blight resistance gene donors, universally susceptible materials, common hybrid rice, and core rice breeding materials, were all tested and found to be homozygous for bacterial blight resistance. xa23 Genotyping results were consistent with the sequencing results of these 188 materials, indicating that the molecular marker K_110534 genotyping has high accuracy and can be conveniently and efficiently used to identify whether rice varieties contain [a specific genotype / gene]. Xa23 Gene.

[0045] Example 3: A method for detecting resistance genes to rice bacterial blight. Xa23 SNP molecular marker K_110536 The design process of this molecular marker, such as Figure 1 As shown, based on the cloned gene Xa23 The sequence was located in the molecular marker region 22203734-22204676 on chromosome 11 of the rice MSU7.0 genome version. SNPs were then extracted from this region by extending 50 kb to both sides of the region on chromosome 11, using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed for SNP mining. Selection was based on PIC values ​​and the presence of other SNPs within 50 bp of the selected SNP. Primers were designed for the selected SNPs using BatchPrimer3. For these SNPs, primers containing... Xa23 Four gene donor materials from the rice varieties Huahui 7620, CBB23, R178, and 74, and other gene-free rice varieties, were used. Xa23 KASP reaction verification was performed on 19 rice varieties, and the KASP marker K_110536, which showed good association with the resistance donor material and good amplification effect, was selected. The marker was then screened and tested, as follows: 1 Primer Design Primers were synthesized by Invitrogen. Each set of primers consisted of three primers: two specific primers linked to the FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to the FAM and HEX fluorescent adapter sequences, respectively, as shown in Table 6. If the sample detected a fluorescent signal corresponding to the sequence linked to primer X, the sample was classified as having the T genotype at the detection site, indicating that the rice sample was homozygous and lacked resistance to bacterial blight. xa23 Genotype; if only the fluorescent signal corresponding to the fluorescent sequence linked to primer Y is detected, then the sample has genotype C at the detection site, and the rice sample is determined to be homozygous with resistance to bacterial blight. Xa23 Genotype; if two fluorescences are detected simultaneously, the rice sample is determined to be a heterozygous variety with resistance to bacterial blight. Xa23 genotype.

[0046] Table 6 Marking Information

[0047] 2. Sample Testing DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, the specific method being the same as in Example 1.

[0048] KASP reaction test: The specific method for the KASP reaction test is the same as in Example 1.

[0049] 3-labeled classification data Based on the above detection method, the molecular marker K_110536 was used to detect the presence of... Xa23 Twenty-three rice varieties, including gene donor materials, other gene donors, and susceptible materials, underwent KASP primary screening validation. The results are shown in Table 7. Xa23 Four rice varieties (Huahui 7620, CBB23, R178, and 74) showed C amplification at the K_110536 testing site. Excluding the three samples that showed no amplification, the remaining 12 samples did not contain [amplification gene]. Xa23 Both other gene donors for resistance to bacterial blight and susceptible materials in rice varieties showed the presence of the base T at the test site, consistent with the sequencing results.

[0050] Table 7 Initial Screening Data

[0051] 4. Specific detection To test the specificity of the marker in this invention, the SNP molecular marker K_110536 was validated in a natural population using 188 samples according to the above detection method. The 188 samples included those known to contain homozygous markers. Xa23 Varieties containing genes, those containing other bacterial blight resistance donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials, etc. Molecular markers in natural population genotyping results, such as... Figure 4 As shown, 5 known samples contain Xa23 Genetic testing showed the variety to be homozygous for resistance to bacterial leaf blight. Xa23 Genotype: All four F1 individual plants were found to be heterozygous. Xa23 Genotype: Among 14 F2 individual plants, 3 homozygous individuals with resistance to bacterial blight were detected. Xa23 Genotype, 3 homozygous copies without resistance to bacterial blight xa23 Genotype and 8 heterozygotes with resistance to white leaf blight Xa23 Genotypes of 158 materials, including those containing other bacterial blight resistance gene donors, universally susceptible materials, common hybrid rice, and core rice breeding materials, were all tested and found to be homozygous for bacterial blight resistance. xa23Genotyping results were consistent with the sequencing results of these 188 materials, indicating that the molecular marker K_110536 genotyping has high accuracy and can be conveniently and efficiently used to identify whether rice varieties contain [a specific genotype / propagation]. Xa23 Gene.

[0052] Example 4: A method for detecting resistance genes to rice bacterial leaf blight Xa23 SNP molecular marker K_110543 The design process of this molecular marker, such as Figure 1 As shown, based on the cloned gene Xa23 The sequence was located within the molecular marker region 22203734-22204676 on chromosome 11 of the rice MSU7.0 genome version. SNPs were then extracted from this region by extending 50 kb to both sides of the region on chromosome 11, using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed for SNP site mining. SNP selection was based on PIC values ​​and the presence of other SNPs within 50 bp of the selected SNP site. Primers were designed for the selected SNP sites using BatchPrimer3. For these SNP molecular markers, primers containing... Xa23 Four gene donor materials from the rice varieties Huahui 7620, CBB23, R178, and 74, and other gene-free rice varieties, were used. Xa23 KASP reaction verification was performed on 19 rice varieties, and the KASP marker K_110543, which showed good association with the resistance donor material and good amplification effect, was selected. The marker was then screened and tested, as follows: 1 Primer Design Primers were synthesized by Invitrogen. Each set of primers contained three primers: two specific primers linked to the FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to the FAM and HEX fluorescent adapter sequences, respectively, as shown in Table 8. If the sample detected a fluorescent signal corresponding to the sequence linked to primer X, the sample was identified as genotype A at the detection site, and the rice sample was determined to be homozygous for bacterial blight resistance. Xa23 Genotype; if only the fluorescent signal corresponding to the fluorescent sequence linked to primer Y is detected, the sample is of genotype G at the detection site, and the rice sample is determined to be homozygous without resistance to bacterial blight. xa23 Genotype; if two fluorescences are detected simultaneously, the rice sample is determined to be a heterozygous variety with resistance to bacterial blight. Xa23 genotype.

[0053] Table 8 Marking Information

[0054] 2. Sample Testing DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, the specific method being the same as in Example 1.

[0055] KASP reaction test: The specific method for the KASP reaction test is the same as in Example 1.

[0056] 3-labeled classification data Based on the above detection method, the molecular marker K_110543 was used to detect the presence of... Xa23 Twenty-three rice varieties, including gene donor materials, other gene donors, and susceptible materials, underwent KASP primary screening validation. The results are shown in Table 9. Xa23 Four rice varieties (Huahui 7620, CBB23, R178, and 74) showed a result of base A at the K_110543 testing site. Excluding three samples that showed no amplification, the remaining 12 samples did not contain [the gene]. Xa23 Both other gene donors for resistance to bacterial blight and susceptible materials in rice varieties showed the presence of base G at the test sites, consistent with the sequencing results.

[0057] Table 9 Initial Screening Data

[0058] 4. Specific detection To determine the specificity of the marker in this invention, the SNP molecular marker K_110543 was validated in a natural population using 188 samples according to the above-described detection method. The 188 samples included those known to contain homozygous markers. Xa23 Varieties containing genes, those containing other bacterial blight resistance donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials, etc. Molecular markers in natural population genotyping results, such as... Figure 5 As shown, 5 known samples contain Xa23 Genetic testing showed the variety to be homozygous for resistance to bacterial leaf blight. Xa23 Genotype: All four F1 individual plants were found to be heterozygous. Xa23 Genotype: Among 14 F2 individual plants, 3 homozygous individuals with resistance to bacterial blight were detected. Xa23 Genotype, 3 homozygous copies without resistance to bacterial blight xa23 Genotype and 8 heterozygotes with resistance to white leaf blight Xa23 Genotypes of 158 materials, including those containing other bacterial blight resistance gene donors, universally susceptible materials, common hybrid rice, and core rice breeding materials, were all tested and found to be homozygous for bacterial blight resistance. xa23 Genotyping results were consistent with the sequencing results of these 188 materials, indicating that the molecular marker K_110543 genotyping has high accuracy and can be conveniently and efficiently used to identify whether rice varieties contain [a specific genotype / propagation]. Xa23 Gene.

[0059] Example 5: A method for detecting resistance genes to rice bacterial leaf blight. Xa23 SNP molecular marker K_110547 The design process of this molecular marker, such as Figure 1 As shown, based on the cloned gene Xa23 The sequence was located within the molecular marker region 22203734-22204676 on chromosome 11 of the rice MSU7.0 genome version. SNPs were then extracted from this region by extending 50 kb to both sides of the region on chromosome 11, using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed for SNP site mining. SNP selection was based on PIC values ​​and the presence of other SNPs within 50 bp of the selected SNP site. Primers were designed for the selected SNP sites using BatchPrimer3. For these SNP molecular markers, primers containing... Xa23 Four gene donor materials from the rice varieties Huahui 7620, CBB23, R178, and 74, and other gene-free rice varieties, were used. Xa23 KASP reaction verification was performed on 19 rice varieties, and the KASP marker K_110547, which showed good association with the resistance donor material and good amplification effect, was selected. The marker was then screened and tested, as follows: 1 Primer Design Primers were synthesized by Invitrogen. Each set of primers consisted of three primers: two specific primers linked to the FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to the FAM and HEX fluorescent adapter sequences, respectively, as shown in Table 10. If the sample detected a fluorescent signal corresponding to the sequence linked to primer X, the sample was classified as having the G genotype at the detection site, indicating that the rice sample was homozygous and lacked resistance to bacterial blight. xa23 Genotype; if only the fluorescent signal corresponding to the fluorescent sequence linked to primer Y is detected, then the sample has genotype C at the detection site, and the rice sample is determined to be homozygous with resistance to bacterial blight. Xa23 Genotype; if two fluorescences are detected simultaneously, the rice sample is determined to be a heterozygous variety with resistance to bacterial blight. Xa23 genotype.

[0060] Table 10 Marking Information

[0061] 2. Sample Testing DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, the specific method being the same as in Example 1.

[0062] KASP reaction test: The specific method for the KASP reaction test is the same as in Example 1.

[0063] 3-labeled classification data Based on the above detection method, the molecular marker K_110547 was used to detect the presence of... Xa23 Twenty-three rice varieties, including gene donor materials, other gene donors, and susceptible materials, underwent KASP primary screening validation. The results are shown in Table 11. Xa23 Four rice varieties (Huahui 7620, CBB23, R178, and 74) showed C amplification at the K_110547 testing site. Excluding three samples that showed no amplification, the remaining 12 samples did not contain [amplification gene]. Xa23 Both other gene donors for resistance to bacterial blight and susceptible materials in rice varieties showed the presence of base G at the test sites, consistent with the sequencing results.

[0064] Table 11 Initial Screening Data

[0065] 4. Specific detection To detect the specificity of the marker K_110547 in this invention, natural population validation of the SNP molecular marker was performed using 188 samples according to the above detection method. The 188 samples included those known to contain homozygous SNPs. Xa23 Varieties containing genes, those containing other bacterial blight resistance donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials, etc. Molecular markers in natural population genotyping results, such as... Figure 6 As shown, 5 known samples contain Xa23 Genetic testing showed the variety to be homozygous for resistance to bacterial leaf blight. Xa23 Genotype: All four F1 individual plants were found to be heterozygous. Xa23 Genotype: Among 14 F2 individual plants, 3 homozygous individuals with resistance to bacterial blight were detected. Xa23 Genotype, 3 homozygous copies without resistance to bacterial blight xa23 Genotype and 8 heterozygotes with resistance to white leaf blight Xa23 Genotypes of 158 materials, including those containing other bacterial blight resistance gene donors, universally susceptible materials, common hybrid rice, and core rice breeding materials, were all tested and found to be homozygous for bacterial blight resistance. xa23 Genotyping results were consistent with the sequencing results of these 188 materials, indicating that the molecular marker K_110547 genotyping has high accuracy and can be conveniently and efficiently used to identify whether rice varieties contain [a specific genotype / propagation]. Xa23 Gene.

[0066] Test case 1. Verification of the genetic location of SNP molecular markers Using 88 individual plants from the 1005S / R608 F2 segregating population, 16 SNP molecular markers that are polymorphic in the parent lines were tested. Xa23 Linked markers were used to verify their genetic locations. Valid data were used to construct a genetic map using JoinMap software. All five markers, K_110532, K_110534, K_110536, K_110543, and K_110547, were located at position 33.9 cM on chromosome 11, without segregation. Figure 7 As shown.

[0067] 2. Marked phenotypic validation Using an F2 hybrid population of 103 donor parents Hua 1015S and recurrent parents R608, the results were compared with... Xa23 The linkage markers K_110532, K_110534, K_110536, K_110543, and K_110547 were used for genetic phenotypic verification. During the rice booting stage, 103 individual plants of the F2 population and two parental varieties were inoculated with the bacterial blight strain PXO61. Disease severity was assessed 21 days later, and the phenotypic data showed 95% concordance between the phenotypic and genotype data. This further validated the feasibility and accuracy of the five SNP molecular markers provided in this application, and their potential applications. Xa23 Gene identification and assisted breeding.

[0068] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for detecting resistance genes to rice bacterial leaf blight Xa23 SNP molecular markers, characterized in that, The SNP molecular marker is K_110534; the polymorphic site of K_110534 is located at the 22169591st base on chromosome 11 of rice in the MSU7.0 genome version, and the polymorphism is T / C.

2. Used to amplify the rice bacterial blight resistance gene as described in claim 1. Xa23 Primer set of linked SNP molecular markers.

3. The primer set as described in claim 2, characterized in that, The primer set contains specific primers, the sequences of which are shown in SEQ ID NO.4 and SEQ ID NO.

5.

4. The primer set as described in claim 3, characterized in that, The specific primers are respectively linked to FAM and HEX fluorescent adapter sequences.

5. The primer set as described in claim 2, characterized in that, The primer set also includes universal primers, the sequences of which are shown in SEQ ID NO.

6.

6. A reagent kit, characterized in that, The kit includes the primer set as described in any one of claims 2-5.

7. A gene chip, characterized in that, The gene chip includes the primer set as described in any one of claims 2-5.

8. Any of the following applications of the SNP molecular marker as described in claim 1, the primer set as described in any one of claims 2-5, the kit as described in claim 6, or the gene chip as described in claim 7: (1) In rice bacterial blight resistance genes Xa23 Applications in genotyping; (2) In the detection of bacterial blight resistance genes Xa23 Applications in; (3) Application in the identification and screening of rice varieties resistant to bacterial blight; (4) Application in molecular marker-assisted breeding of rice; (5) Application in rice breeding; (6) Application in the preparation of rice breeding products.

9. Detection of rice bacterial blight resistance genes using the SNP molecular markers as described in claim 1. Xa23 The method is characterized by, The method includes the following steps: S1. Extracting genomic DNA from rice; S2. Perform polymorphism detection on the SNP molecular markers in the genomic DNA extracted in step S1, and determine whether the rice material contains a bacterial blight resistance gene based on the detection results. Xa23 .

10. A method for rice breeding, characterized in that, The method includes the following steps: selecting samples containing a bacterial blight resistance gene according to the method described in claim 9. Xa23 The rice will be used for subsequent breeding.

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