A gene gn1a associated with lodging resistance in rice R498 Linked SNP molecular markers and their applications

Abstract

This invention discloses a gene gn1a related to lodging resistance in rice. R498 Linked SNP molecular markers and their applications, wherein the SNP molecular marker is located at base 5271730 on chromosome 1 of Nipponbare rice, with a polymorphism of A / C. This invention utilizes the gn1a gene, which is associated with lodging resistance in rice. R498 Linked SNP molecular markers, combined with KASP detection technology, solve the technical problems in traditional molecular marker-assisted breeding. They eliminate the need for agarose gel electrophoresis, enabling rapid, accurate, efficient, and high-throughput identification of gn1a. R498 Genes can accelerate the breeding process of new lodging-resistant varieties.

Description

Technical Field

[0001] This invention belongs to the field of agricultural molecular biology, specifically relating to a gene gn1a related to lodging resistance in rice. R498 Linked SNP molecular markers and their applications. Background Technology

[0002] Lodging is a major factor limiting the widespread application of high-yield rice varieties. With the continuous decrease in rice planting area and the increasing labor costs, mechanized planting methods have been widely adopted. This, coupled with the large-scale use of chemical fertilizers, has led to increasingly serious lodging problems in rice. Lodging increases harvesting costs and causes losses in yield and quality. Therefore, improving lodging resistance in high-yield rice varieties is particularly important.

[0003] High-yielding, heavy-panicle rice is a major crop in southwestern my country, exhibiting excellent traits such as high yield and lodging resistance. Shuhui 498, as a representative of high-yielding, heavy-panicle rice, is frequently used in basic research. Related technologies indicate that Shuhui 498 contains nonfunctional gn1a... R498 This is a key factor in its resistance to lodging. Carrying gn1a R498 The phenotype plants showed significantly higher stem diameter and bending strength than the control group, gn1a R498 By increasing stem diameter and promoting adventitious root growth, the lodging resistance of rice is improved. Shuhui 498 has significant application value in the process of improving lodging resistance in high-yield rice varieties.

[0004] Currently, most molecular markers used for breeding lodging-resistant rice varieties require cumbersome gel electrophoresis detection, resulting in low automation and low throughput, which significantly limits the breeding process. Therefore, developing molecular markers similar to gn1a is crucial. R498 Linked SNP molecular markers can, on the one hand, promote the application of Shuhui 498 in the breeding of lodging-resistant new varieties; on the other hand, they can rapidly and efficiently screen lodging-resistant rice varieties, thus accelerating the breeding process of new varieties. Summary of the Invention

[0005] 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 gn1a related to lodging resistance in rice. R498 Linked SNP molecular markers.

[0006] The present invention also proposes a primer set for detecting the above-mentioned SNP molecular markers.

[0007] The present invention also proposes a reagent kit.

[0008] This invention also proposes a gene chip.

[0009] This invention also proposes the application of the above-mentioned SNP molecular markers, primer sets, kits and / or gene chips.

[0010] The present invention also proposes a method for detecting the above-mentioned SNP molecular markers.

[0011] According to a first aspect of the present invention, a gene gn1a related to lodging resistance in rice... R498 Linked SNP molecular markers, the SNP molecular markers being located at base position 5271730 on chromosome 1 of Nipponbare 1, with a polymorphism of A / C.

[0012] According to a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular marker includes specific primers and universal primers, wherein the specific primer sequences include Primer X and Primer Y.

[0013] In some embodiments of the present invention, the specific primers comprise nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0014] According to some embodiments of the present invention, the SNP molecular marker further includes a universal primer sequence with a nucleotide sequence as shown in SEQ ID NO.3.

[0015] In some embodiments of the present invention, the specific primers are respectively linked to FAM and HEX fluorescent adapter sequences.

[0016] In some embodiments of the present invention, the primer set is used in rice genotype detection.

[0017] According to a third aspect of the present invention, a kit is provided, the kit comprising the above-described primer set.

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

[0019] According to a fifth aspect of the present invention, any of the following applications of the above-described SNP molecular markers, primer sets, kits, or gene chips are:

[0020] (1) In rice lodging resistance-related gene gn1a R498 Applications in genotyping;

[0021] (2) In the detection of rice lodging resistance-related gene gn1a R498 Applications in;

[0022] (3) Application in the identification and screening of rice with lodging resistance traits;

[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 lodging resistance-related gene gn1a using the above-mentioned SNP molecular markers is proposed. R498 The method includes the following steps:

[0027] S1. Extracting genomic DNA from rice;

[0028] S2. Perform polymorphism detection on the SNP molecular markers on the genomic DNA extracted in step S1, and determine whether the rice material contains the rice lodging resistance-related gene gn1a based on the genotype. R498 .

[0029] In some embodiments of the present invention, only the base corresponding to primer X is detected, indicating that the tested rice material carries gn1a. R498 Type gene; if only the base corresponding to primer Y is detected, it is determined that the tested rice material does not carry gn1a. R498 Type gene; if fluorescence signals corresponding to Primer X and Primer Y are detected simultaneously, the test material is a heterozygous genotype.

[0030] In some embodiments of the present invention, preferably, in step S1, the genomic DNA is extracted from rice using the simplified CTAB method (hexadecyltrimethylammonium bromide method).

[0031] In some embodiments of the present invention, preferably, in step S2, the SNP molecular marker is detected using KASP (competitive allele-specific PCR) technology.

[0032] In some embodiments of the present invention, the composition of the KASP reaction mixture for detecting SNP molecular markers using KASP technology is as follows:

[0033]

[0034] In some embodiments of the present invention, the amplification program for detecting SNP molecular markers using KASP technology is as follows: 94℃ for 15 min; 94℃ for 20 s, 65℃-57℃ for 60 s, 10 cycles, with the temperature decreasing by 0.8℃ in each cycle; 94℃ for 20 s, 57℃ for 60 s, 30 cycles.

[0035] A rice breeding method includes the following steps: using the genotype detection method described above, selecting rice varieties containing the lodging resistance-related gene gn1a. R498 The rice will be used for subsequent breeding.

[0036] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention utilizes the gn1a gene, which is related to lodging resistance in rice. R498 Linked SNP molecular markers, combined with KASP detection technology, solve the technical problems in traditional molecular marker-assisted breeding. They eliminate the need for agarose gel electrophoresis, enabling rapid, accurate, efficient, and high-throughput identification of gn1a. R498 Genes can accelerate the breeding process of new lodging-resistant varieties.

[0037] 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

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0039] Figure 1 This is a flowchart of the molecular marker development process in Embodiment 1 of the present invention;

[0040] Figure 2 This is a diagram showing the typing results of OS900305_K02 in Shuhui 498 and Hedao 98 in Embodiment 1 of the present invention;

[0041] Figure 3 This is a diagram showing the typing results of the marker OS900305_K02 in 60 rice samples in Example 1 of this 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] Embodiment of the present invention: a gene gn1a related to lodging resistance in rice. R498 Linked SNP molecular markers

[0044] The design process of this molecular marker, such as Figure 1 As shown, based on the cloned target gene gn1aR498 Location, find gn1a R498 Linked SNP molecular markers were developed by performing whole-genome sequencing on Shuhui 498 and Hedao 98 to identify specific SNP sites linked to the target gene. These sites were then validated using KASP reactions on the core parental materials to select the SNP sites that best differentiated between different material types and achieved optimal amplification. SNP sites and flanking sequences were extracted, and primer sequences for the markers were designed and synthesized. The markers were then screened and tested, as detailed below:

[0045] 1 Primer Design

[0046] Related studies have shown that Gn1a is located in the region 5270835-5275522 on chromosome 1. Whole-genome sequencing of Shuhui 498 and Hedao 98, and comparison with BAM files, revealed a link to Gn1a. R498 Linked SNP molecular markers revealed an A / C variation at position 5271730 on chromosome 1 of the reference genome, Nipponbare. When the detected base was A, it indicated that this rice variety carried gn1a. R498 When the detected base is C, it indicates that the rice variety does not carry the gn1a gene. R498 Type gene. 100 bp flanking sequences before and after this locus were extracted from Nipponbare DNA, and primers were designed using the BatchPrimer3 primer design website. The marker OS900305_K02 consisted of three primers, as shown in Table 1. Two specific primers had their 5' ends linked to FAM and HEX fluorescent adapter sequences, respectively, and one universal primer. Primers were synthesized by Sangon Biotech.

[0047] Molecular markers designed based on the KASP reaction principle and material single-base differences can be used for high-throughput gn1a analysis of rice materials. R498 Type gene detection. If only the fluorescence signal corresponding to Primer X is detected in the PCR product, then the detection site base is A, and the test material carries gn1a. R498 Type gene; if only the fluorescence signal corresponding to Primer Y is detected, the detection site base is C, and the test material does not carry gn1a. R498 If fluorescent signals corresponding to Primer X and Primer Y are detected simultaneously, the base sequence at the detection site is A:C, and the test material is a heterozygous genotype.

[0048] Table 1 Marking Information

[0049]

[0050]

[0051] 2 Sample Testing

[0052] DNA extraction: Genomic DNA was extracted from rice leaves using a simplified CTAB method, including the following steps:

[0053] (1) Take about 30 mg of leaf material into a 1.3 mL 96-well plate and place it in a freeze dryer and vacuum for 12 hours or more;

[0054] (2) After vacuuming, use a bead separator to add two steel balls to each well, cover with a silicone film, grind in a high-throughput grinder for 1 minute, and then centrifuge in a deep well plate centrifuge to centrifuge the ground tissue to the bottom of the well.

[0055] (3) Add 700 μL of CTAB extraction solution to each well using a TECAN pipetting workstation, shake to mix, and then place in a 65℃ water bath for about 1-1.5 h. Every 20 min, take out 1.3 mL of the 96-well plate and shake it several times on a vortex mixer.

[0056] (4) After the warm bath, take out 1.3 mL of 96-well plate and place it in a deep well plate refrigerated centrifuge. Centrifuge at 4000 rpm for 10 min.

[0057] (5) Using a TECAN pipetting workstation, transfer 380 μL of supernatant from each well to a new 1.3 mL 96-well plate, add an equal volume of chloroform, invert and mix well, let stand for 2 min, place in a deep well plate refrigerated centrifuge, centrifuge at 4000 rpm for 10 min.

[0058] (6) After centrifugation, use a TECAN pipetting station to extract 250 μL of the supernatant into a 0.8 mL 96-well plate pre-filled with 250 μL of isopropanol, vortex to mix, and place it in a -20℃ refrigerator for precipitation for 1 hour or more.

[0059] (7) Take out 0.8 mL of 96-well plate and place it in a deep well plate refrigerated centrifuge. Centrifuge at 4000 rpm for 15 min.

[0060] (8) Discard the supernatant, add 250 μL of 70% ethanol to each well using a TECAN pipetting station, vortex several times, centrifuge at 5000 rpm for 15 min;

[0061] (9) Discard the supernatant and dry it in a 65℃ oven for 30 minutes;

[0062] (10) Add 200 μL of sterile ultrapure water to each well and let it dissolve overnight at room temperature for later use.

[0063] KASP reaction assay: The KASP reaction assay was performed on the Douglas Arraytape genotyping platform. The reaction system is shown in Table 2. As can be seen from the table, the amplification system used for the PCR amplification reaction, in 0.8 μL increments, consisted of: 20-50 ng of dried sample DNA, 0.0013 μL each of two 100 μM specific primers, 0.0033 μL of 100 μM universal primers, 0.3945 μL of 2×KASPMaster Mix, and 0.3996 μL of ultrapure water. 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 temperatures decreasing by 0.8℃ per cycle; second amplification reaction: 94℃ denaturation for 20 s, annealing and extension at 57℃ for 60 s, 30 cycles. After the reaction is complete, the fluorescence data of the KASP reaction products are read using an Arraytape scanning system, and the results of the fluorescence scan are automatically converted into images.

[0064] Table 2 Reaction system for KASP detection

[0065] 100μM Primer C 0.42μM 0.0033μL 100μM Primer X 0.17μM 0.0013μL 100μM Primer Y 0.17μM 0.0013μL 2×KASP Master Mix 1× 0.3945μL Ultrapure water 0.3996μL DNA (dried) 20ng-50ng Total volume 0.8μL

[0066] 3-labeled classification data

[0067] According to the above detection method, the KASP reaction of Shuhui 498 and Hedao 98 was verified by labeling with OS900305_K02, with 10 replicates for each material.

[0068] Test results as follows Figure 2 As shown, the detection result of Shuhui 498 at the test site is base A, carrying gn1a. R498 The genotype phenotype indicates lodging resistance; the Hedao 98 variety tested at the test site showed base C, indicating it did not carry gn1a. R498 Type 1 gene, poor or no lodging resistance.

[0069] 4. Specificity and Practicality Testing

[0070] To test the specificity and practicality of the marker OS900305_K02 in this invention, 60 rice materials with known genotypes were subjected to KASP reaction typing according to the above detection method, with 4 replicates for each material, including Shuhui 498, Hedao 98, core rice breeding materials, and conventional rice materials.

[0071] The results of the tagging and typing are as follows Figure 3 As shown, the detection result of Shuhui 498 at the test site is base A, carrying gn1a. R498The genotype phenotype indicates lodging resistance; Hedao 98 and 58 other rice materials showed base C at the test site, indicating they did not carry gn1a. R498 The gene type indicates poor or no lodging resistance. The results were consistent with expectations; the marker OS900305_K02 of this invention was effective in detecting gn1a. R498 The genotype test has high specificity and can quickly and accurately identify whether the lodging resistance gene gn1a is present in the test material. R498 .

[0072] The reagents and consumables used in this invention with the Douglas Arraytape genotyping platform were all purchased from LGC Ltd. in the UK.

[0073] 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 gene associated with lodging resistance in rice gn1a R498 The following applications of linked SNP molecular marker-based KASP primer sets or kits containing said KASP primer sets: (1) Application in the identification and screening of rice varieties with lodging resistance traits; (2) Application in marker-assisted breeding of lodging resistance in rice; (3) Application in the preparation of products for marker-assisted breeding of lodging resistance traits in rice; The KASP primer set is shown in SEQ ID NO. 1-3; If the test result is base A, then the rice sample carries... gn1a R498 The genotype indicates lodging resistance; if the test result is base C, then the tested rice does not carry the gene. gn1a R498 Genotype with poor or no lodging resistance.

2. A method for screening rice varieties with lodging resistance traits, characterized in that, The method includes the following steps: S1. Extract genomic DNA from the rice samples; S2. Using the KASP primer set in claim 1, perform the analysis on the genomic DNA extracted in step S1 to obtain the genes related to lodging resistance in rice. gn1a R498 The polymorphism detection of linked SNP molecular markers was used to determine whether the rice species tested had lodging resistance based on their genotype. If the test result is base A, then the rice sample carries... gn1a R498 The genotype indicates lodging resistance; if the test result is base C, then the tested rice does not carry the gene. gn1a R498 Genotype with poor or no lodging resistance.

3. A method for breeding lodging-resistant rice, characterized in that, The process includes the following steps: selecting rice lodging resistance-related genes according to the method described in claim 2. gn1a R498 The rice will be used for subsequent breeding.

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