A SNP molecular marker closely linked to corn southern rust resistance gene RPPM and application thereof

Abstract

This invention discloses a SNP molecular marker closely linked to the maize southern rust resistance gene RPPM and its application. The SNP molecular marker is located at position 1682035 on chromosome 10 of the maize reference genome Zea_mays.AGPv4, with a polymorphism of C / A. This invention provides an SNP molecular marker linked to the maize southern rust resistance gene RPPM. The marker is co-dominant and features high specificity, high sensitivity, high resolution, and high genotyping quality. It can rapidly determine whether breeding materials possess southern rust resistance based on the resistance genotype, has broad applicability, and can be used for marker-assisted breeding to improve resistance to southern rust.

Description

Technical Field

[0001] This invention belongs to the field of agricultural molecular biology, specifically relating to an SNP molecular marker closely linked to the maize southern rust resistance gene RPPM and its application. Background Technology

[0002] Corn rust is widespread in the Huang-Huai-Hai summer maize region, and its severity is increasing year by year. In recent years, in particular, large-scale outbreaks of corn rust have caused enormous damage to maize production. Severely affected varieties suffer premature leaf death, and the disease can even spread to the entire plant. Leaves wither due to nutrient depletion, or stems collapse. Many varieties with poor resistance experience significant yield losses, potentially reducing yields by 20%-30%, and in severe cases, by more than 50%. Currently, the main type of corn rust is Southern Rust, but Common Rust also occurs, and many maize fields suffer from a mixed outbreak of both. The most economical and effective way to solve this problem, especially from a breeding perspective, is to discover and utilize disease-resistant genes for breeding, developing new maize varieties carrying resistance genes.

[0003] Currently, breeding for resistance to southern rust in maize mainly relies on conventional breeding, which depends on phenotypic selection. Phenotypic investigation and identification primarily rely on investigating the phenotype exhibited by the breeding samples during growth. This method depends on visual recognition of morphological characteristics and biological traits; the judgment criteria are often difficult to quantify precisely, are highly subjective, and have low detection sensitivity and resolution. It is also susceptible to environmental and cultivation conditions, resulting in poor accuracy and stability; it is time-consuming and lacks timeliness; and it requires significant investment of manpower and resources, leading to high costs. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes an SNP molecular marker closely linked to the maize southern rust resistance gene RPPM and its application.

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

[0006] The present invention also proposes a reagent kit.

[0007] This invention also proposes a gene chip.

[0008] This invention also proposes the application of the above-mentioned SNP molecular markers, primer sets, kits and / or gene chips.

[0009] This invention also proposes a method for identifying or assisting in the identification of southern rust resistance in maize.

[0010] This invention also proposes a method for maize breeding.

[0011] According to a first aspect of the present invention, a SNP molecular marker closely linked to the maize southern rust resistance gene RPPM is proposed, wherein the SNP molecular marker is located at position 1682035 on chromosome 10 of the maize reference genome Zea_mays.AGPv4, and has a polymorphism of C / A.

[0012] According to a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular markers is provided.

[0013] In some embodiments of the present invention, the primer set includes specific primers, wherein the specific primer sequences include Primer X and Primer Y.

[0014] In some embodiments of the present invention, the specific primer sequences are shown in SEQ ID NO.4 and SEQ ID NO.5.

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

[0016] According to some embodiments of the present invention, the primer set further includes universal primers, the nucleotide sequences of which are shown in SEQ ID NO.6.

[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 aforementioned primer set.

[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 proposed:

[0020] (1) Application in the genotyping of the maize southern rust resistance gene RPPM;

[0021] (2) Application in detecting the maize resistance gene RPPM for southern rust;

[0022] (3) Application in the identification and screening of maize with resistance to southern rust;

[0023] (4) Application in molecular marker-assisted breeding of maize;

[0024] (5) Application in maize breeding;

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

[0026] According to a sixth aspect of the present invention, a method for identifying or assisting in the identification of southern rust resistance in maize is provided, the method comprising the following steps:

[0027] S1. Extracting genomic DNA from maize material;

[0028] S2. Perform polymorphism detection of the SNP molecular marker on the genomic DNA extracted in step S1, and determine whether the maize material has resistance to southern rust based on the genotype.

[0029] In some embodiments of the present invention, if the genotype obtained by the SNP molecular marker detection is AA, then the maize material has resistance to southern rust; if the genotype obtained by the SNP molecular marker detection is CC, then the maize material does not have resistance to southern rust; if the genotype obtained by the SNP molecular marker detection is AC, then the maize material has resistance to southern rust.

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

[0031] In some embodiments of the present invention, 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; 94℃ for 20 s, 57℃ for 60 s, 33 cycles.

[0035] According to a seventh aspect of the present invention, a maize breeding method is proposed, comprising the following steps: using the above-mentioned SNP molecular marker detection method to detect the maize southern rust resistance gene RPPM, and selecting maize with southern rust resistance for subsequent breeding.

[0036] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention provides an SNP molecular marker linked to the maize southern rust resistance gene RPPM. This marker is a co-dominant marker, characterized by high specificity, high sensitivity, high resolution, and high genotyping quality. The marker is unaffected by environmental conditions, can be used with seeds or any type of plant tissue, and provides accurate, repeatable, and stable detection results. Data from different testing laboratories and different data can be compared and verified, exhibiting universal comparability. It allows for rapid determination of southern rust resistance in breeding materials based on resistance genotypes, demonstrating broad applicability and potential for marker-assisted breeding for southern rust resistance improvement. The detection method of the present invention combines KASP detection technology, resulting in a simple, highly automated, high-throughput, and fast detection method with low reagent consumption and low cost. The detection results are accurate, repeatable, and stable, and data from different testing laboratories can be compared and verified, exhibiting universal comparability. This allows for widespread application and selection in breeding.

[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 typical result diagram of the molecular marker ZM900033 typing in Example 1 of the present invention;

[0041] Figure 3 This is a typical result diagram of the molecular marker ZM900034 typing in Example 1 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] Embodiment of the present invention: A SNP molecular marker closely linked to the maize southern rust resistance gene RPPM.

[0044] The design process of this molecular marker, such as Figure 1 As shown, based on publicly available germplasm resources, a maize southern rust resistance gene germplasm resource database was constructed using resequencing data from 67 core germplasm resources. Through extensive literature searches and data analysis, the southern rust resistance gene RPPM was determined to be located on chromosome 10 of the maize southern rust resistance gene genome, with a position interval of 1600459-1705143. The reference genome version of the maize southern rust resistance gene is Zea_mays.AGPv4. Sequence extraction was performed using the obtained data, and SNP polymorphism analysis was conducted on the gene sequences. Finally, multiple SNP sites were obtained, and approximately 150 bp of flanking sequences were extracted. By designing and synthesizing primer sequences for the markers, and then screening them, molecular markers ZM900033 and ZM900034 were obtained. ZM900033 exhibits a T / C variant at position 1608571 on chromosome 10 of the Zea_mays.AGPv4 reference genome, while ZM900034 exhibits a C / A variant at position 1682035 on chromosome 10 of the Zea_mays.AGPv4 reference genome. The specific SNP molecular markers closely linked to the maize southern rust resistance gene are verified as follows:

[0045] 1 Primer Design

[0046] For the molecular markers ZM900033 and ZM900034 obtained from the above screening, KASP primer markers were designed based on the reference genome Zea_mays.AGPv4 of the maize southern rust resistance gene using the online primer design website BatchPrimer3 (http: / / probes.pw.usda.gov / batchprimer3 / ). Each marker group had three primers, with FAM and HEX fluorescent sequences attached to the 5' ends of two specific primers, respectively. After the design was completed, genome-wide copy number analysis was performed on the primer sequences, and the final high-quality single-copy KASP marker site design information is shown in Table 1 below. The primers were synthesized by Sangon Biotech.

[0047] Using the two molecular markers designed based on the KASP reaction principle, high-throughput detection of RPPM mutants of the southern rust resistance gene in maize materials is possible. When the genotype detected by the ZM900033 molecular marker is CC, it indicates that the maize material has resistance to southern rust; when the detected genotype is TT, it indicates that the maize material does not have resistance to southern rust; and when the detected genotype is TC, it is a heterozygous genotype, indicating that the maize material has resistance to southern rust.

[0048] When the genotype obtained by the ZM900034 molecular marker is AA, it indicates that the maize material has resistance to southern rust; when the genotype obtained is CC, it indicates that the maize material does not have resistance to southern rust; when the genotype obtained is AC, it is a heterozygous genotype, indicating that the maize material has resistance to southern rust.

[0049] Table 1 Marking Information

[0050]

[0051]

[0052] 2 Sample Testing

[0053] DNA extraction: Genomic DNA was extracted from maize using a simplified CTAB method.

[0054] KASP Reaction Assay: The KASP reaction assay was performed on the Douglas Arraytape genotyping platform. The reaction system is shown in Table 2. The PCR amplification system was automatically assembled using NEXAR. The ArrayTape genotyping platform includes NEXAR for PCR amplification system assembly, SOELLEX for PCR amplification, ARAYA for fluorescence signal scanning, and INTELLICS for data analysis.

[0055] Table 2. PCR amplification system for KASP marker genotyping

[0056]

[0057] The PCR amplification reaction conditions were as follows: NEXAR was used for PCR amplification, and the amplification conditions were as follows: 94℃ for 15 min; 94℃ for 20 s, 65℃-57℃ (annealing temperature decreased by 0.8℃ per cycle) for 60 s, 10 cycles; 94℃ for 20 s, 57℃ for 60 s, 33 cycles.

[0058] Signal scanning and genotyping: After the PCR reaction was completed, the fluorescence signal of the reaction system was scanned using ARAYA; then genotyping and data analysis were performed using INTELLICS.

[0059] 3-labeled classification data

[0060] Based on the above detection method, KASP reaction verification was performed on 67 corn samples to test the markers ZM900033 and ZM900034.

[0061] A typical KASP marker genotyping diagram is shown below. Figure 2-3As shown in the figure, in the KASP marker genotyping detection, the sample genotypes were divided into three clusters: the X cluster, the Y cluster, and the heterozygous genotype cluster. The X cluster indicates that the sample contains a homozygous X allele at this KASP marker locus (marked in red, in the upper left corner of the genotyping graph); the Y cluster indicates that the sample contains a homozygous Y allele at this KASP marker locus (marked in blue, in the lower right corner of the genotyping graph); and the heterozygous genotype cluster indicates that the sample contains both X and Y heterozygous alleles at this KASP marker locus (marked in purple).

[0062] The quality verification results showed that the two homozygous and heterozygous clusters of the KASP marker ZM900034 were well-generated and compact, with single-copy loci. The detection rate was higher than 98%, and the consistency with the phenotype was high. The genotyping quality of the KASP marker can fully meet the requirements for accurate detection of the maize southern rust resistance gene RPPM. Therefore, ZM900034 was selected for subsequent experimental verification.

[0063] 4. Specificity and Practicality Testing

[0064] To test the specificity and practicality of the marker ZM900034 in this invention, 64 improved strains of the RPPM gene were selected for field germplasm experiments and genotyping according to the above detection method.

[0065] Table 3

[0066]

[0067]

[0068] The results are shown in Table 3. As can be seen from the table, the genotype and phenotype identification are basically consistent. The marker ZM900034 of this invention has high specificity in detecting maize resistance to southern rust and can quickly and accurately identify whether the test material is resistant to southern rust.

[0069] The reagents and consumables used in this invention with the Douglas Arraytape genotyping platform were all purchased from LGC Ltd., UK. Advantages of KASP marker detection based on the Douglas Arraytape platform: KASP marker detection based on the Douglas Arraytape platform achieves 90% automation, significantly reducing laboratory manpower and human error. High throughput: 122,880 data points can be obtained in 8 hours, 10 times that of traditional 96-well plate SNP genotyping methods. Low reaction volume (only 0.8 μL / reaction): Compared with traditional 96-well plate SNP genotyping methods, reagent and consumable costs are reduced by 70%-90%.

[0070] 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 primer set for identifying and screening maize with resistance to southern rust, characterized in that, The primer set includes specific primers and universal primers. The sequences of the specific primers are shown in SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequences of the universal primers are shown in SEQ ID NO.

6.

2. The primer set according to claim 1, characterized in that, The specific primers are respectively linked to FAM and HEX fluorescent adapter sequences.

3. A reagent kit, characterized in that, The kit includes the primer set as described in any one of claims 1-2.

4. A gene chip, characterized in that, The gene chip includes the primer set as described in any one of claims 1-2.

5. Any of the following applications of the primer set according to any one of claims 1-2, the kit according to claim 3, or the gene chip according to claim 4: (1) Application in the identification and screening of maize with resistance to southern rust; (2) Application in marker-assisted breeding of maize for resistance to southern rust; (3) Application in breeding of maize with resistance to southern rust; (4) Application in the preparation of breeding products related to maize resistance to southern rust.

6. A method for identifying or assisting in the identification of southern rust resistance in maize, characterized in that, The method includes the following steps: S1. Extracting genomic DNA from maize material; S2. Using the primer set described in claim 1, the genomic DNA extracted in step S1 is detected, and the genotype is used to determine whether the maize material has resistance to southern rust.

7. A method for breeding maize, characterized in that, The method includes the following steps: using the method described in claim 6, selecting maize with resistance to southern rust for subsequent breeding.

Images