A SNP molecular marker linked to the maize stalk rot resistance gene ZmAuxRP1 and its application

Abstract

The present invention discloses a SNP molecular marker linked to a corn stalk rot resistance gene ZmAuxRP1 and an application thereof, wherein the SNP molecular marker is located at the base at position 267362668 of chromosome 1 of the reference genome Maize B73 genome sequences AGPv4, and the polymorphism is C / G. The present invention utilizes a specific SNP molecular marker linked to corn stalk rot resistance and combines it with KASP detection technology, without the need for agarose gel electrophoresis, to rapidly, accurately, efficiently, and with high throughput identify corn stalk rot resistance materials, thereby accelerating the breeding process of new corn stalk rot resistance varieties.

Description

Technical Field

[0001] The invention belongs to the field of agricultural molecular biology, and in particular relates to a SNP molecular marker linked to a corn stalk rot resistance gene ZmAuxRP1 and an application thereof. Background Art

[0002] Corn stalk rot is a type of disease caused by a variety of pathogens, which seriously affects corn production. There are up to 20 fungi or bacteria in the world that can infect corn and induce stalk rot, which are divided into 5 types, including Pythium, Gibberella, Fusarium, Colletotrichum and bacteria. Corn stalk rot is mainly caused by Pythium and Fusarium, among which Fusarium is the main pathogen of stalk rot, and among Fusarium, Fusarium graminearum, Fusarium solani and Fusarium verticillioides are the most serious. Pathogens mainly infect the root system and stem base of corn, causing rot at the stem base, affecting the normal growth of corn, and ultimately leading to reduced yield. Stalk rot develops rapidly and causes serious damage. Once corn is infected, the yield will decrease by 10%-30% in mild cases, and more than 50% in severe cases. At present, chemical agents are often used in production to prevent and control stalk rot. The large-scale use of pesticides not only pollutes the environment, but also causes drug resistance in pathogens, increasing the difficulty of preventing and controlling stalk rot.

[0003] Compared with chemical control, breeding corn stalk rot-resistant varieties has the characteristics of safety and environmental protection, and has outstanding advantages in stalk rot control. In recent years, related technologies have cloned or located multiple genes related to stalk rot resistance, providing a theoretical basis for the breeding of corn stalk rot-resistant varieties. The identification of corn stalk rot resistance requires artificial inoculation of pathogens, which is easily affected by the environment and the measurement data may be biased. At present, there are few molecular markers that can be used for the breeding of corn stalk rot-resistant varieties, and these molecular markers require cumbersome gel electrophoresis detection, low degree of automation and low throughput, which greatly limits the breeding process of corn stalk rot-resistant varieties. Therefore, the development of corn stalk rot resistance-linked KASP molecular markers can quickly and high-throughput screen disease-resistant materials, accelerate the breeding process of new varieties, and is of great significance to reducing the yield losses caused by corn stalk rot. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a SNP molecular marker linked to the maize stalk rot resistance gene ZmAuxRP1.

[0005] The present invention also provides a primer set for detecting the above SNP molecular markers.

[0006] The present invention also provides a kit.

[0007] The invention also provides a gene chip.

[0008] The present invention also proposes the application of the above-mentioned SNP molecular marker, primer set, kit and / or gene chip.

[0009] The present invention also provides a method for detecting the above-mentioned SNP molecular marker.

[0010] According to the first aspect of the present invention, a SNP molecular marker linked to the maize stalk rot resistance gene ZmAuxRP1 is provided, wherein the SNP molecular marker is located at base position 267362668 of chromosome 1 of the reference genome Maize B73 genome sequences AGPv4, and the polymorphism is C / G.

[0011] According to the second aspect of the present invention, the primer set for amplifying the above-mentioned SNP molecular marker comprises a specific primer and a universal primer, wherein the specific primer sequence comprises Primer X and Primer Y.

[0012] In some embodiments of the present invention, the specific primers include nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0013] According to some embodiments of the present invention, the SNP molecular marker further includes a universal primer sequence such as a nucleotide sequence shown in SEQ ID NO.3.

[0014] In some embodiments of the present invention, the specific primers are connected to FAM and HEX fluorescent linker sequences respectively.

[0015] According to a third aspect of the present invention, a kit is provided, the kit comprising the above-mentioned primer set.

[0016] According to a fourth aspect of the present invention, a gene chip is provided, wherein the gene chip comprises the above-mentioned primer set.

[0017] According to a fifth aspect of the present invention, any of the following applications of the above-mentioned SNP molecular marker, primer set, kit or gene chip:

[0018] (1) Application in genotyping of maize stalk rot resistance gene ZmAuxRP1;

[0019] (2) Application in detecting the maize stalk rot resistance gene ZmAuxRP1;

[0020] (3) Application in identification and screening of corn with resistance to stalk rot;

[0021] (4) Application in molecular marker-assisted breeding of maize;

[0022] (5) Application in corn breeding;

[0023] (6) Application in the preparation of corn breeding products.

[0024] According to a sixth aspect of the present invention, a method for detecting corn stalk rot resistance using the above-mentioned SNP molecular markers comprises the following steps:

[0025] S1. Extract genomic DNA from corn;

[0026] S2. Performing polymorphism detection of the SNP molecular marker on the genomic DNA extracted in step S1, and determining the stalk rot resistance of the tested corn according to the detection result.

[0027] In some embodiments of the present invention, only the base corresponding to the primer Primer X is detected, and the tested corn material is determined to contain the ZmAuxRP1 stalk rot resistance gene and has no stalk rot resistance; if only the base corresponding to the primer Primer Y is detected, the tested corn material is determined to contain the ZmAuxRP1 stalk rot resistance gene and has stalk rot resistance; if the bases corresponding to Primer X and Primer Y are detected at the same time, the tested corn material is determined to be a heterozygous genotype, carrying the ZmAuxRP1 stalk rot resistance gene and having stalk rot resistance.

[0028] In some embodiments of the present invention, preferably, in step S1, the genomic DNA is extracted from corn using a simplified CTAB method (cetyltrimethylammonium bromide method).

[0029] In some embodiments of the present invention, preferably, in step S2, the SNP molecular markers are detected using KASP (competitive allele-specific PCR) technology.

[0030] A corn breeding method comprises the following steps: using the above genotype detection method to select samples with stalk rot resistance for subsequent breeding.

[0031] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention utilizes specific SNP molecular markers linked to corn stalk rot resistance and combines them with KASP detection technology, without the need for agarose gel electrophoresis, to rapidly, accurately, efficiently, and with high throughput identify corn stalk rot-resistant materials, thereby accelerating the breeding process of new corn stalk rot-resistant varieties.

[0032] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0034] Figure 1 A flow chart for the molecular marker development in Example 1 of the present invention;

[0035] Figure 2 The typing results of the marker Zm900003_K01 in Example 1 of the present invention in the donor and recipient samples;

[0036] Figure 3 These are the typing results of marker Zm900003_K01 in Example 1 of the present invention in 8 samples. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels.

[0038] Embodiment of the present invention: A SNP molecular marker linked to the maize stalk rot resistance gene ZmAuxRP1

[0039] The design process of the molecular marker, such as Figure 1 As shown, relevant studies have shown that the ZmAuxRP1 gene has resistance to Fusarium graminearum stalk rot and can be used to improve corn stalk rot resistant varieties. By comparing the resequencing data of the target gene and its upstream and downstream sequences in resistant varieties, SNP sites were screened, SNP sites and flanking sequences were extracted, and the marker primer sequences were designed and synthesized, and then the markers were screened and tested, as follows:

[0040] 1 Primer design

[0041] By comparing the resequencing data of the ZmCCT gene and its upstream and downstream sequences in the resistant materials, the SNP sites linked to the gene were mined, and it was found that there was a SNP mutation at position 267362668 of chromosome 1 in the reference genome Maize B73 genome sequences AGPv4, and the polymorphism was C / G. The 100bp flanking sequence before and after the site was extracted, and the primers were designed using the BatchPrimer3 primer design website. The marker Zm900003_K01 consists of 3 primers, as shown in Table 1, of which the 5' ends of the 2 specific primers were connected to the FAM and HEX fluorescent linker sequences respectively, and 1 universal primer. The primers were commissioned to Shanghai Bioengineering to synthesize.

[0042] Using molecular markers designed based on the KASP reaction principle and single-base differences in materials, high-throughput genotype detection can be performed on samples. If only the fluorescent signal corresponding to Primer X is detected in the PCR product, the base of the detection site is C, and the test material does not contain the ZmAuxRP1 stem rot resistance gene; if only the fluorescent signal corresponding to Primer Y is detected, the base of the detection site is G, and the test material contains the ZmAuxRP1 stem rot resistance gene; if the fluorescent signals corresponding to Primer X and Primer Y are detected at the same time, the base of the detection site is C:G, and the test material is a heterozygous genotype and carries the ZmAuxRP1 stem rot resistance gene.

[0043] Table 1 Marking information

[0044]

[0045] 2. Sample testing

[0046] DNA extraction: Genomic DNA was extracted from maize leaves using a simplified CTAB method, which included the following steps:

[0047] (1) Approximately 30 mg of leaves were placed in a 1.3 mL 96-well plate and placed in a freeze dryer and vacuumed for 12 h or more;

[0048] (2) After vacuuming, two steel beads were added to each well using a bead separator, and the well was covered with a silicone membrane and placed in a high-throughput grinder for 1 min. After grinding, the well was placed in a deep-well plate centrifuge for instant centrifugation to centrifuge the ground tissue to the bottom of the well;

[0049] (3) Use the TECAN pipetting workstation to add 700 μL of CTAB extract to each well, shake and mix, and then place in a 65°C water bath for about 1-1.5 hours. Take out 1.3 mL of the 96-well plate every 20 minutes and shake it several times on a vortex shaker;

[0050] (4) After the incubation period, take out 1.3 mL of the 96-well plate, place it in a deep-well plate refrigerated centrifuge, and centrifuge at 4000 rpm for 10 min;

[0051] (5) Use the TECAN pipetting workstation to transfer 380 μL of supernatant from each well to a new 1.3 mL 96-well plate, add an equal volume of chloroform, mix thoroughly by inversion, let stand for 2 min, and place in a deep-well plate refrigerated centrifuge at 4000 rpm for 10 min;

[0052] (6) After centrifugation, use the TECAN pipetting workstation to extract 250 μL of the supernatant into a 0.8 mL 96-well plate pre-added with 250 μL of isopropanol, vortex to mix, and place it in a -20°C refrigerator to precipitate for 1 h or more;

[0053] (7) Take out 0.8 mL of the 96-well plate and place it in a deep-well plate refrigerated centrifuge at 4000 rpm for 15 min;

[0054] (8) Discard the supernatant and add 250 μL of 70% ethanol to each well using a TECAN pipetting workstation. Vortex the mixture several times at 5000 rpm and centrifuge for 15 min.

[0055] (9) Discard the supernatant and dry it in an oven at 65°C for 30 min;

[0056] (10) Add 200 μL of sterile ultrapure water to each well and leave to dissolve at room temperature overnight.

[0057] KASP reaction test: KASP reaction test 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 in the PCR amplification reaction is calculated as follows: 20ng-50ng of sample DNA was dried and then 0.0013μL of each of the two specific primers of 100μM, 0.0033μL of 100μM universal primer, 0.3945μL of 2×KASP Master Mix, and 0.3996μL of ultrapure water were added. PCR amplification was completed in a water bath thermal cycler. The Touchdown PCR reaction conditions were: 94℃ pre-denaturation for 15min; the first amplification reaction was denaturation at 94℃ for 20s, annealing and extension at 65℃~57℃ for 60s, 10 cycles, and the temperature of annealing and extension was reduced by 0.8℃ in each cycle; the second amplification reaction was denaturation at 94℃ for 20s, annealing and extension at 57℃ for 60s, and 30 cycles. After the reaction is completed, the fluorescence data of the KASP reaction product is read using the Arraytape scanning system, and the results of the fluorescence scanning are automatically converted into graphics.

[0058] Table 2 Reaction system for KASP detection

[0059]

[0060]

[0061] 3 Marker typing data

[0062] According to the above detection method, the marker Zm900003_K01 was used to verify the KASP reaction of the stem rot improved donor PHV37 and the stem rot improved recipient GS692 with known genotypes, with 5 replicates for each sample.

[0063] Test results such as Figure 2 As shown, the detection result of the stem rot improvement donor PHV37 at the target site was base G, which contained the ZmAuxRP1 stem rot resistance gene; the detection result of the stem rot improvement recipient GS692 at the target site was base C, which did not contain the ZmAuxRP1 stem rot resistance gene.

[0064] 4 Specificity and practicality of detection

[0065] In order to detect the specificity and practicality of the marker Zm900003_K01 in the present invention, 8 commonly used germplasm resources with known genotypes were selected for identification according to the above detection method, and each sample was repeated 3 times.

[0066] The results of marker typing are as follows Figure 3 As shown, the marker Zm900003_K01 detected 2 samples of C:C homozygous genotype, which did not contain the ZmAuxRP1 stem rot resistance gene; detected 5 samples of G:G homozygous genotype, which contained the ZmAuxRP1 stem rot resistance gene; detected 1 sample of C:G heterozygous genotype, which contained the ZmAuxRP1 stem rot resistance gene heterozygous, and the results were in line with expectations. The SNP molecular marker Zm900003_K01 linked to corn stalk rot resistance of the present invention can be used for the screening and detection of corn Fusarium graminearum stalk rot resistance materials.

[0067] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for amplifying genes related to corn stalk rot resistance ZmAuxRP1 The application of a primer set of linked SNP molecular markers in detecting corn stalk rot resistant plants is characterized in that: The primer set includes specific primers and universal primers, the specific primers include nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; the universal primer nucleotide sequence is shown in SEQ ID NO.3; the stem rot is Fusarium graminearum stem rot; when the primer set only detects the fluorescent signal corresponding to the sequence SEQ ID NO.1, it indicates that the detected plant does not have resistance to stem rot; when the primer set only detects the fluorescent signal corresponding to the sequence SEQ ID NO.2, it indicates that the detected plant has resistance to stem rot; when the primer set simultaneously detects the fluorescent signals corresponding to the sequences SEQ ID NO.1 and SEQ ID NO.2, it indicates that the detected plant has resistance to stem rot; the specific primers are respectively connected to FAM and HEX fluorescent linker sequences.

2. Any of the following uses of the primer set described in claim 1: (1) Application in molecular marker-assisted breeding of maize; (2) Application in corn breeding; (3) Application in the preparation of corn breeding products.

3. Use of a kit for detecting corn stalk rot-resistant plants, characterized in that: The kit comprises the primer set as claimed in claim 1; the stem rot disease is Fusarium graminearum stem rot disease.

4. An application of a gene chip in detecting plants resistant to corn stalk rot, characterized in that: The gene chip comprises the primer set as claimed in claim 1; the stem rot disease is Fusarium graminearum stem rot disease.

5. Any of the following uses of the kit described in claim 3 or the gene chip described in claim 4: (1) Application in molecular marker-assisted breeding of maize; (2) Application in corn breeding; (3) Application in the preparation of corn breeding products.

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