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 gene Xa23 and application thereof, and 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 scheme has good stability, is not influenced 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, is suitable for assisted selection breeding of the Xa23 gene, and is suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of rice breeding, specifically to an SNP molecular marker for detecting the rice bacterial blight resistance gene Xa23 and its application. Background Technology

[0002] 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 Japanese bacterial strains, 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 genes have been mapped, and 8 of them have been cloned: Xa1, Xa5, Xa27, Xa13, Xa3 / Xa26, Xa4, Xa21, and Xa23.

[0003] Currently, breeders have used molecular marker-assisted selection (MAS) to develop many disease-resistant restorer lines, such as IR26, IR28, IR30, IR32, IR36, IR50, and IR54, using Xa4, Xa21, and Xa23. Most hybrid rice varieties widely planted in China contain the Xa4 gene; 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 rapidly improve the disease resistance of hybrid rice is an urgent problem to be solved in breeding.

[0004] Xa23 is a broad-spectrum resistance gene for bacterial blight discovered in common wild rice. It can resist a new pathogenic race of Xa21 found in Guangdong Province, my country. Therefore, Xa23 is rapidly becoming a leading source of resistance for improving bacterial blight resistance in hybrid rice parents in my country. Restorer lines or strains carrying Xa23, improved or bred using molecular marker-assisted selection, 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 male-sterile line II-32A are excellent. Improved or bred male-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. The Xa23 gene has been transferred into the disease-susceptible cultivar Jin Gang 30, resulting in the near-isogenic line CBB23.

[0005] Traditional breeding methods rely on phenotypic identification to infer the presence of resistance genes, using resistance spectrum analysis of bacterial blight pathogens to identify the Xa23 gene. However, this method requires specific physiological races of bacterial blight pathogens, and inoculation conditions, including the rice's growth stage, climate, and temperature, are quite strict, making it unsuitable for large-scale identification and application. Since the Xa23 gene has been cloned, current research on introducing it into rice mainly utilizes marker-assisted selection. Markers used in marker-assisted selection typically include RAPD, SSR, AFLP, RFLP, CAP, or dCAP, or require PCR, enzyme digestion, or a combination of both. These methods involve cumbersome and contamination-risk electrophoresis detection. The inventors have developed a SNP molecular marker closely linked to Xa23, combined with KASP detection technology. This eliminates the need for gel electrophoresis and is not limited to costly restriction enzymes, enabling high-throughput, rapid, and accurate identification of the Xa23 gene in genetic breeding, significantly improving the efficiency of gene transfer. Summary of the Invention

[0006] 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 an SNP molecular marker linked to the rice bacterial blight resistance gene Xa23.

[0007] The present invention also proposes a primer set for detecting the above-mentioned SNP molecular markers.

[0008] The present invention also proposes a reagent kit.

[0009] This invention also proposes a gene chip.

[0010] This invention also proposes the application of the above-mentioned SNP molecular markers, primer sets, kits and / or gene chips.

[0011] The present invention also proposes a method for detecting the above-mentioned SNP molecular markers.

[0012] In a first aspect of the invention, a SNP molecular marker for detecting the rice bacterial blight resistance gene Xa23 is provided, wherein the SNP molecular marker is at least one of K_110532, K_110534, K_110536, K_110543, and K_110547, wherein the polymorphic site of K_110532 is located at position 22167870 on chromosome 11 of rice in the MSU7.0 genome version, and the polymorphism is A / T; the polymorphic site of K_110534 is located at position 22167870 on chromosome 11 of rice in the MSU7.0 genome version. The polymorphism at position 2169591 is T / C; the polymorphic site K_110536 is located at position 22174338 on chromosome 11 of rice in the MSU7.0 genome version, and the polymorphism is T / C; the polymorphic site K_110543 is located at position 22232666 on chromosome 11 of rice in the MSU7.0 genome version, and the polymorphism is A / G; the polymorphic site K_110547 is located at position 22249375 on chromosome 11 of rice in the MSU7.0 genome version, and the polymorphism is G / C.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In some embodiments of the present invention, the specific primers are respectively linked to FAM and HEX fluorescent adapter sequences.

[0017] In a third aspect of the invention, a kit is provided comprising the aforementioned primer set.

[0018] In a fourth aspect of the invention, a gene chip is provided, the gene chip comprising the primer set described above.

[0019] 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:

[0020] (1) Application in the genotyping of the rice bacterial blight resistance gene Xa23;

[0021] (2) Application in detecting the bacterial blight resistance gene Xa23;

[0022] (3) Application in the identification and screening of rice with resistance to bacterial blight;

[0023] (4) Application in molecular marker-assisted breeding of rice.

[0024] (5) Application in rice breeding;

[0025] (6) Application in the preparation of rice breeding products.

[0026] According to a sixth aspect of the present invention, a method for detecting the rice bacterial blight resistance gene Xa23 using the above-described SNP molecular markers, the method comprising the following steps:

[0027] S1. Extracting genomic DNA from rice;

[0028] S2. Perform polymorphism detection on the SNP molecular markers in the genomic DNA extracted in step S1, and determine whether the rice material contains the bacterial blight resistance gene Xa23 based on the detection results.

[0029] According to some embodiments of the present invention, in step S1, genomic DNA extraction is performed using a simplified CTAB method (hexadecyltrimethylammonium bromide method).

[0030] According to some embodiments of the present invention, in step S2, the SNP sites are detected using KASP (competitive allele-specific PCR) technology.

[0031] A rice breeding method includes the following steps: using the above-mentioned detection method, selecting rice varieties containing the bacterial blight resistance gene Xa23 for subsequent breeding.

[0032] The SNP molecular markers for detecting the rice bacterial blight resistance gene Xa23 according to embodiments of the present invention have at least the following beneficial effects: The present invention, through sequence alignment using a database of 3000 rice resequencing samples, identifies specific SNP molecular markers co-segregated with Xa23 within gene linkage regions. Combined with KASP detection technology, it eliminates the need for gel electrophoresis and is not limited to costly restriction endonucleases. In Xa23 genetic breeding, it enables high-throughput, rapid, and accurate identification of the Xa23 gene, significantly improving gene transfer efficiency and playing a crucial role in promoting the application of the Xa23 gene in commercial breeding. The present invention has the advantages of simple operation, low cost, and short cycle. Furthermore, the markers exhibit good stability, unaffected by other gene effects and environmental factors, allowing for early-generation selection, shortening breeding cycles, and improving breeding efficiency. It is of great significance for improving rice varieties resistant to bacterial blight and is suitable for Xa23 gene-assisted selection breeding. It is suitable for widespread application.

[0033] 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

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0035] Figure 1 This is a flowchart of the molecular marker development process in Embodiment 1 of the present invention;

[0036] Figure 2 This is a typing diagram of the K_110532 molecular marker in Example 1 of the present invention;

[0037] Figure 3 This is a typing diagram of the K_110534 molecular marker in Example 2 of the present invention;

[0038] Figure 4 This is a typing diagram of the K_110535 molecular marker in Example 3 of the present invention;

[0039] Figure 5 This is a typing diagram of the K_110543 molecular marker in Example 4 of the present invention;

[0040] Figure 6 This is a typing diagram of the K_110547 molecular marker in Example 5 of the present invention;

[0041] Figure 7 This is a diagram showing the genetic location verification results of the test example of the present invention. Detailed Implementation

[0042] 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.

[0043] Example 1: A SNP molecular marker K_110532 for detecting the rice bacterial blight resistance gene Xa23.

[0044] The design process of this molecular marker, such as Figure 1 As shown, the Xa23 gene sequence was located in the molecular marker region 22203734-22204676 on chromosome 11 of rice. The region was then expanded 50kb to both sides of this region on chromosome 11, and SNP sites within this expanded region were extracted using a database of 3000 rice resequencing accesses. Simultaneously, donor and recipient resequencing data were analyzed to mine SNP sites. Selection was based on PIC values ​​and the presence of other SNP sites within 50bp of the selected SNP site. Primers were designed for the selected SNP sites using BatchPrimer3. KASP reaction validation was performed on four donor rice varieties containing the Xa23 gene (Huahui 7620, CBB23, R178, and 74) and 19 other rice varieties without Xa23. The KASP marker K_110532, which showed good association with the resistance donor material and strong amplification, was selected. The marker was then screened and tested, as detailed below:

[0045] 1 Primer Design

[0046] Primers were synthesized by Invitrogen. Each set of primers consisted of three primers: two specific primers linked to FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to FAM and HEX fluorescent adapter sequences, as shown in Table 1. If the sample detected fluorescence 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 a homozygous xa23 genotype without bacterial blight resistance. If only fluorescence corresponding to the sequence linked to primer Y was detected, the sample was identified as genotype T at the detection site, and the rice sample was determined to be a homozygous Xa23 genotype with bacterial blight resistance. If both fluorescence sequences were detected simultaneously, the rice sample was determined to be a heterozygous Xa23 genotype with bacterial blight resistance.

[0047] Table 1 Marking Information

[0048]

[0049] 2 Sample Testing

[0050] DNA extraction: Genomic DNA was extracted from rice using a simplified CTAB method, including the following steps:

[0051] 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.

[0052] 2) Heating at 65℃ with vibration for 0.5-1 hour;

[0053] 3) Cool to room temperature, then add 750 ml of chloroform:isoamyl alcohol (24:1) solution to a fume hood and mix well;

[0054] 4) Centrifuge at 12000 rpm for 10 min, and transfer about 500 ml of the supernatant to a new 1.5 ml centrifuge tube;

[0055] 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;

[0056] 6) Add 1000ml of 70% ethanol, gently tap the precipitate, centrifuge at 1000rpm for 3min, and discard the supernatant;

[0057] 7) Add 300 μL of H2O and dissolve overnight for later use.

[0058] 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.

[0059] Table 2 Reaction system for KASP detection

[0060] Final concentration Volume (μL) 100μM Primer C 0.42μM 0.0125 100μM Primer X 0.17μM 0.0050 100μM Primer Y 0.17μM 0.0050 2×KASP Master Mix 1× 1.4792 Ultrapure water 1.4983 DNA (dried) 3 Total volume 0.0125

[0061] 3-labeled classification data

[0062] Based on the above detection method, KASP screening was performed on 23 rice varieties containing the Xa23 gene donor, other gene donors, and susceptible materials using the molecular marker K_110532. The results are shown in Table 3. Four rice varieties containing the Xa23 gene—Huahui 7620, CBB23, R178, and 74—showed a T base at the K_110532 test site. Except for three materials that showed no amplification, the 12 rice varieties without Xa23, regardless of whether they were using other bacterial blight resistance gene donors or susceptible materials, all showed an A base at the test site, consistent with the sequencing results.

[0063] Table 3 Initial Screening Data

[0064] Material Name Material Specifications K_110532 IRBB7 Xa7 donor A DZ78 Xa7 donor A DV85 Xa7 donor A Hua Hui 1437 Xa7 and Xa21 donors A Hua Hui 1337 Xa7 and Xa21 donors A IRBB21 Xa21 donor A Zhonghui 8015 Xa21 donor A 66 Xa21 and Xa4 donors A 104 Xa21 and Xa4 donors A 108 Xa21 and Xa4 donors A Huahui7620 Pi9, Xa23 donors A CBB23 Xa23 donor T R178 Xa23 donor T 74 Xa23 donor T R900 Disease materials T Guangzhan 63-4S Disease materials A IR24 Disease materials A C815S Disease materials A Tianfeng A Disease materials A R207 Disease materials A Shu Hui 527 Disease materials A Minghui 86 Disease materials A

[0065] 4. Specific detection

[0066] To detect the specificity of the marker in this invention, the SNP molecular marker K_110532 was validated in natural populations using 188 materials according to the above detection method. The 188 materials included varieties known to contain homozygous Xa23 genes, materials containing other bacterial blight-resistant donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials. The genotyping results of the molecular marker in natural populations are as follows: Figure 2As shown, five varieties known to contain the Xa23 gene were identified as homozygous Xa23 genotypes with bacterial blight resistance. All four F1 plants were identified as heterozygous Xa23 genotypes. Among 14 F2 plants, three were homozygous Xa23 genotypes with bacterial blight resistance, three were homozygous Xa23 genotypes without bacterial blight resistance, and eight were heterozygous Xa23 genotypes with bacterial blight resistance. A total of 158 materials, including those containing other bacterial blight resistance gene donors, general-sensitive materials, common hybrid rice, and core rice breeding materials, were identified as homozygous Xa23 genotypes without bacterial blight resistance. The 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 the Xa23 gene.

[0067] Example 2: A SNP molecular marker K_110534 for detecting the rice bacterial blight resistance gene Xa23.

[0068] The design process of this molecular marker, such as Figure 1 As shown, the Xa23 gene sequence was located in the molecular marker region 22203734-22204676 on chromosome 11 of rice. The region was then expanded 50kb to both sides of this region on chromosome 11, and SNP sites within this expanded region were extracted using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed to mine SNP sites. Selection was based on PIC values ​​and the presence of other SNP sites within 50bp of the selected SNP site. Primers were designed for the selected SNP sites using BatchPrimer3. KASP reaction validation was performed on four donor materials (Huahui 7620, CBB23, R178, and 74) containing the Xa23 gene, and 19 other rice varieties without Xa23. The KASP marker K_110534, which showed good association with the resistant donor material and excellent amplification effect, was selected. The marker was then screened and tested, as detailed below:

[0069] 1 Primer Design

[0070] Primers were synthesized by Invitrogen. Each set of labeled primers consisted of three primers: two specific primers linked to FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to FAM and HEX fluorescent adapter sequences, as shown in Table 4. If the sample detected fluorescence 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 a homozygous xa23 genotype without bacterial blight resistance. If only fluorescence corresponding to the sequence linked to primer Y was detected, the sample was classified as having the C genotype at the detection site, indicating that the rice sample was a homozygous Xa23 genotype with bacterial blight resistance. If both fluorescence sequences were detected simultaneously, the rice sample was classified as a heterozygous Xa23 genotype with bacterial blight resistance.

[0071] Table 4 Marking Information

[0072]

[0073]

[0074] 2 Sample Testing

[0075] DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, the specific method being the same as in Example 1.

[0076] KASP reaction test: The specific method for the KASP reaction test is the same as in Example 1.

[0077] 3-labeled classification data

[0078] Based on the above detection method, KASP screening was performed on 23 rice varieties containing the Xa23 gene donor, other gene donors, and susceptible materials using the molecular marker K_110534. The results are shown in Table 5. Four rice varieties containing the Xa23 gene—Huahui 7620, CBB23, R178, and 74—showed a C base at the K_110532 test site. Except for three materials that showed no amplification, the 12 rice varieties without Xa23, regardless of whether they were using other bacterial blight resistance gene donors or susceptible materials, all showed a T base at the test site, consistent with the sequencing results.

[0079] Table 5 Initial Screening Data

[0080]

[0081]

[0082] 4. Specific detection

[0083] To detect the specificity of the marker in this invention, the SNP molecular marker K_110534 was validated in natural populations using 188 materials according to the above detection method. The 188 materials included varieties known to contain homozygous Xa23 genes, materials containing other bacterial blight-resistant donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials. The genotyping results of the molecular marker in natural populations are as follows: Figure 3 As shown, five varieties known to contain the Xa23 gene were identified as homozygous Xa23 genotypes with bacterial blight resistance. All four F1 plants were identified as heterozygous Xa23 genotypes. Among 14 F2 plants, three were homozygous Xa23 genotypes with bacterial blight resistance, three were homozygous Xa23 genotypes without bacterial blight resistance, and eight were heterozygous Xa23 genotypes with bacterial blight resistance. A total of 158 materials, including those containing other bacterial blight resistance gene donors, general-sensitive materials, common hybrid rice, and core rice breeding materials, were identified as homozygous Xa23 genotypes without bacterial blight resistance. The 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 the Xa23 gene.

[0084] Example 3: A SNP molecular marker K_110536 for detecting the rice bacterial blight resistance gene Xa23.

[0085] The design process of this molecular marker, such as Figure 1 As shown, the Xa23 gene sequence was located in the molecular marker region 22203734-22204676 on chromosome 11 of the rice MSU7.0 genome version. The region was then expanded 50kb to both sides of this region on chromosome 11, and SNP sites within this expanded region were extracted using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed to mine SNP sites. Selection was based on PIC values ​​and the presence of other SNP sites within 50bp of the selected SNP site. Primers were designed for the selected SNP sites using BatchPrimer3. KASP reaction validation was performed on four donor materials (Huahui 7620, CBB23, R178, and 74) containing the Xa23 gene, and 19 other rice varieties without Xa23. The KASP marker K_110536, which showed good association with the resistant donor material and excellent amplification effect, was selected from these SNP sites. The tags were then filtered and tested, as follows:

[0086] 1 Primer Design

[0087] Primers were synthesized by Invitrogen. Each set of labeled primers consisted of three primers: two specific primers linked to FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to FAM and HEX fluorescent adapter sequences, as shown in Table 6. If the sample detected fluorescence 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 a homozygous xa23 genotype without bacterial blight resistance. If only fluorescence corresponding to the sequence linked to primer Y was detected, the sample was classified as having the C genotype at the detection site, indicating that the rice sample was a homozygous Xa23 genotype with bacterial blight resistance. If both fluorescence sequences were detected simultaneously, the rice sample was classified as a heterozygous Xa23 genotype with bacterial blight resistance.

[0088] Table 6 Marking Information

[0089]

[0090] 2 Sample Testing

[0091] DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, the specific method being the same as in Example 1.

[0092] KASP reaction test: The specific method for the KASP reaction test is the same as in Example 1.

[0093] 3-labeled classification data

[0094] Based on the above detection method, KASP screening was performed on 23 rice varieties containing the Xa23 gene donor, other gene donors, and susceptible materials using the molecular marker K_110536. The results are shown in Table 7. Four rice varieties containing the Xa23 gene—Huahui 7620, CBB23, R178, and 74—showed base C at the K_110536 testing site. Except for three materials that showed no amplification, the 12 rice varieties without Xa23, regardless of whether they were using other bacterial blight resistance gene donors or susceptible materials, all showed base T at the testing site, consistent with the sequencing results.

[0095] Table 7 Initial Screening Data

[0096] Material Name Material Specifications K_110536 IRBB7 Xa7 donor T DZ78 Xa7 donor T DV85 Xa7 donor T Hua Hui 1437 Xa7 and Xa21 donors T Hua Hui 1337 Xa7 and Xa21 donors T IRBB21 Xa21 donor T Zhonghui 8015 Xa21 donor T 66 Xa21 and Xa4 donors T 104 Xa21 and Xa4 donors T 108 Xa21 and Xa4 donors T Huahui7620 Pi9, Xa23 donors T CBB23 Xa23 donor C R178 Xa23 donor C 74 Xa23 donor C R900 Disease materials C Guangzhan 63-4S Disease materials T IR24 Disease materials T C815S Disease materials T Tianfeng A Disease materials T R207 Disease materials T Shu Hui 527 Disease materials T Minghui 86 Disease materials T

[0097] 4. Specific detection

[0098] To detect the specificity of the marker in this invention, the SNP molecular marker K_110536 was validated in natural populations using 188 materials according to the above detection method. The 188 materials included varieties known to contain homozygous Xa23 genes, materials containing other bacterial blight-resistant donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials. The genotyping results of the molecular marker in natural populations are as follows: Figure 4 As shown, five varieties known to contain the Xa23 gene were identified as homozygous Xa23 genotypes with bacterial blight resistance. All four F1 plants were identified as heterozygous Xa23 genotypes. Among 14 F2 plants, three were homozygous Xa23 genotypes with bacterial blight resistance, three were homozygous Xa23 genotypes without bacterial blight resistance, and eight were heterozygous Xa23 genotypes with bacterial blight resistance. A total of 158 materials, including those containing other bacterial blight resistance gene donors, general-sensitive materials, common hybrid rice, and core rice breeding materials, were identified as homozygous Xa23 genotypes without bacterial blight resistance. The genotyping 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 the Xa23 gene.

[0099] Example 4: A SNP molecular marker K_110543 for detecting the rice bacterial blight resistance gene Xa23

[0100] The design process of this molecular marker, such as Figure 1 As shown, the Xa23 gene sequence was located in the molecular marker region 22203734-22204676 on chromosome 11 of the rice MSU7.0 genome version. The region was then expanded 50kb to both sides of this region on chromosome 11, and SNP sites within this expanded region were extracted using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed to mine SNP sites. Selection was based on PIC values ​​and the presence of other SNP sites within 50bp of the selected SNP site. Primers were designed for the selected SNP sites using BatchPrimer3. KASP reaction validation was performed on four donor materials (Huahui 7620, CBB23, R178, and 74) containing the Xa23 gene, and 19 other rice varieties without Xa23. The KASP marker K_110543, which showed good association with the resistant donor material and excellent amplification effect, was selected based on this invention. The tags were then filtered and tested, as follows:

[0101] 1 Primer Design

[0102] Primers were synthesized by Invitrogen. Each set of labeled primers consisted of three primers: two specific primers linked to FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to FAM and HEX fluorescent adapter sequences, respectively, as shown in Table 8. If the sample detected fluorescence 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 a homozygous Xa23 genotype with bacterial blight resistance. If only fluorescence corresponding to the sequence linked to primer Y was detected, the sample was identified as genotype G at the detection site, and the rice sample was determined to be a homozygous xa23 genotype without bacterial blight resistance. If both fluorescence sequences were detected simultaneously, the rice sample was determined to be a heterozygous Xa23 genotype with bacterial blight resistance.

[0103] Table 8 Marking Information

[0104]

[0105]

[0106] 2 Sample Testing

[0107] DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, the specific method being the same as in Example 1.

[0108] KASP reaction test: The specific method for the KASP reaction test is the same as in Example 1.

[0109] 3-labeled classification data

[0110] Based on the above detection method, KASP screening was performed on 23 rice varieties containing the Xa23 gene donor, other gene donors, and susceptible materials using the molecular marker K_110543. The results are shown in Table 9. Four rice varieties containing the Xa23 gene—Huahui 7620, CBB23, R178, and 74—showed base A at the K_110543 test site. Except for three materials that showed no amplification, the 12 rice varieties without Xa23, regardless of whether they were using other bacterial blight resistance gene donors or susceptible materials, all showed base G at the test site, consistent with the sequencing results.

[0111] Table 9 Initial Screening Data

[0112]

[0113]

[0114] 4. Specific detection

[0115] To detect the specificity of the marker in this invention, the SNP molecular marker K_110543 was validated in natural populations using 188 materials according to the above detection method. The 188 materials included varieties known to contain homozygous Xa23 genes, materials containing other bacterial blight-resistant donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials. The genotyping results of the molecular marker in natural populations are as follows: Figure 5 As shown, five varieties known to contain the Xa23 gene were identified as homozygous Xa23 genotypes with bacterial blight resistance. All four F1 plants were identified as heterozygous Xa23 genotypes. Among 14 F2 plants, three were homozygous Xa23 genotypes with bacterial blight resistance, three were homozygous Xa23 genotypes without bacterial blight resistance, and eight were heterozygous Xa23 genotypes with bacterial blight resistance. A total of 158 materials, including those containing other bacterial blight resistance gene donors, general-sensitive materials, common hybrid rice, and core rice breeding materials, were identified as homozygous Xa23 genotypes without bacterial blight resistance. The 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 the Xa23 gene.

[0116] Example 5: A SNP molecular marker K_110547 for detecting the rice bacterial blight resistance gene Xa23.

[0117] The design process of this molecular marker, such as Figure 1 As shown, the Xa23 gene sequence was located in the molecular marker region 22203734-22204676 on chromosome 11 of the rice MSU7.0 genome version. The region was then expanded 50kb to both sides of this region on chromosome 11, and SNP sites within this expanded region were extracted using a database of 3000 rice resequencing samples. Simultaneously, donor and recipient resequencing data were analyzed to mine SNP sites. Selection was based on PIC values ​​and the presence of other SNP sites within 50bp of the selected SNP site. Primers were designed for the selected SNP sites using BatchPrimer3. KASP reaction validation was performed on four donor materials (Huahui 7620, CBB23, R178, and 74) containing the Xa23 gene, and 19 other rice varieties without Xa23. The KASP marker K_110547, which showed good association with the resistant donor material and excellent amplification effect, was selected based on this invention. The tags were then filtered and tested, as follows:

[0118] 1 Primer Design

[0119] Primers were synthesized by Invitrogen. Each set of labeled primers consisted of three primers: two specific primers linked to FAM and HEX fluorescent sequences, respectively, and one universal primer. The 5' ends of the two specific primers were linked to FAM and HEX fluorescent adapter sequences, as shown in Table 10. If the sample detected fluorescence corresponding to the sequence linked to primer X, the sample was classified as genotype G at the detection site, indicating that the rice sample was a homozygous xa23 genotype without bacterial blight resistance. If only fluorescence corresponding to the sequence linked to primer Y was detected, the sample was classified as genotype C at the detection site, indicating that the rice sample was a homozygous Xa23 genotype with bacterial blight resistance. If both fluorescence sequences were detected simultaneously, the rice sample was classified as a heterozygous Xa23 genotype with bacterial blight resistance.

[0120] Table 10 Marking Information

[0121]

[0122] 2 Sample Testing

[0123] DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, the specific method being the same as in Example 1.

[0124] KASP reaction test: The specific method for the KASP reaction test is the same as in Example 1.

[0125] 3-labeled classification data

[0126] Based on the above detection method, KASP screening was performed on 23 rice varieties containing the Xa23 gene donor, other gene donors, and susceptible materials using the molecular marker K_110547. The results are shown in Table 11. Four rice varieties containing the Xa23 gene—Huahui 7620, CBB23, R178, and 74—showed base C at the K_110547 test site. Except for three materials that showed no amplification, the 12 rice varieties without Xa23, regardless of whether they were using other bacterial blight resistance gene donors or susceptible materials, all showed base G at the test site, consistent with the sequencing results.

[0127] Table 11 Initial Screening Data

[0128] Material Name Material Specifications K_110547 IRBB7 Xa7 donor G DZ78 Xa7 donor G DV85 Xa7 donor G Hua Hui 1437 Xa7 and Xa21 donors G Hua Hui 1337 Xa7 and Xa21 donors G IRBB21 Xa21 donor G Zhonghui 8015 Xa21 donor G 66 Xa21 and Xa4 donors G 104 Xa21 and Xa4 donors G 108 Xa21 and Xa4 donors G Huahui7620 Pi9, Xa23 donors G CBB23 Xa23 donor C R178 Xa23 donor C 74 Xa23 donor C R900 Disease materials C Guangzhan 63-4S Disease materials G IR24 Disease materials G C815S Disease materials G Tianfeng A Disease materials G R207 Disease materials G Shu Hui 527 Disease materials G Minghui 86 Disease materials G

[0129] 4. Specific detection

[0130] To detect the specificity of marker K_110547 in this invention, SNP molecular markers were validated in natural populations using 188 materials according to the above detection method. The 188 materials included varieties known to contain homozygous Xa23 genes, materials containing other bacterial blight-resistant donors, universally susceptible materials, common hybrid rice, core rice breeding materials, and segregating population materials. The molecular marker genotyping results in natural populations are as follows: Figure 6 As shown, five varieties known to contain the Xa23 gene were identified as homozygous Xa23 genotypes with bacterial blight resistance. All four F1 plants were identified as heterozygous Xa23 genotypes. Among 14 F2 plants, three were homozygous Xa23 genotypes with bacterial blight resistance, three were homozygous Xa23 genotypes without bacterial blight resistance, and eight were heterozygous Xa23 genotypes with bacterial blight resistance. A total of 158 materials, including those containing other bacterial blight resistance gene donors, general-sensitive materials, common hybrid rice, and core rice breeding materials, were identified as homozygous Xa23 genotypes without bacterial blight resistance. The 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 the Xa23 gene.

[0131] Test case

[0132] 1. Verification of the genetic location of SNP molecular markers

[0133] Using 88 individual plants from the 1005S / R608 F2 segregating population, 16 SNP molecular markers polymorphic in the parents and the Xa23 linkage marker were tested for genetic locus verification. Valid data were used to construct a genetic map using JoinMap software. Five markers, K_110532, K_110534, K_110536, K_110543, and K_110547, were all located at position 33.9 cM on chromosome 11, without segregation. Figure 7 As shown.

[0134] 2. Marked phenotypic validation

[0135] Genetic phenotypic verification of the Xa23-linked markers K_110532, K_110534, K_110536, K_110543, and K_110547 was performed using an F2 hybrid population of 103 donor parents (Hua1015S) and recurrent parents (R608). During the rice booting stage, 103 individual plants of the F2 population and two parent varieties were inoculated with bacterial blight strain PXO61. Disease severity was assessed 21 days later, and the phenotypic data showed 95% concordance between the phenotypic and genotypic data. This further validates the feasibility and accuracy of the five SNP molecular markers provided in this application, indicating their potential use for Xa23 gene identification and assisted breeding.

[0136] 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 marker products in rice bacterial blight resistance genes Xa23 Its application in genotyping is characterized by, The SNP molecular marker is K_110532. The polymorphic site of K_110532 is located at the base position 22167870 on chromosome 11 of rice in the MSU7.0 genome version, and the polymorphism is A / T.

2. A primer set for amplifying the SNP molecular marker as described in claim 1; the primer set comprising specific primers, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The primer set as described in claim 2, characterized in that, The specific primers are respectively linked to FAM and HEX fluorescent adapter sequences.

4. 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.

3.

5. A reagent kit, characterized in that, The kit includes the primer set as described in any one of claims 2-4.

6. A gene chip, characterized in that, The gene chip includes the primer set as described in any one of claims 2-4.

7. Any of the following applications of the primer set according to any one of claims 2-4, the kit according to claim 5, or the gene chip according to claim 6: (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 with resistance to bacterial blight.

8. Detection of rice bacterial blight resistance genes using the SNP molecular markers described in claim 1. Xa23 The method is characterized by, The method includes the following steps: S1. Extracting genomic DNA from rice materials; 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 ; If only the SNP molecular marker genotype A is detected, the rice material is determined to be a homozygous xa23 genotype without resistance to bacterial leaf blight. If only the SNP molecular marker genotype T is detected, the rice material is determined to be a homozygous Xa23 genotype with resistance to bacterial blight. If the genotype of the SNP molecular marker is detected as AT, the rice material is determined to be a heterozygous Xa23 genotype with resistance to bacterial blight.

9. 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 8. Xa23 The rice will be used for subsequent breeding.

Images