Functional marker of corn southern rust resistance gene and application thereof

Abstract

The application discloses a corn southern rust resistance gene function marker, the gene is located on the 6th chromosome of corn, from the main QTL site RppQ6.01 of the southern rust resistance of Zi 319, gene silencing test shows that ZmWRKY66 is the disease resistance gene of RppQ6.01, sequence analysis finds that there is an insertion / deletion of CACTA-TIR transposon in the promoter region of ZmWRKY66, two function markers are developed based on the DNA sequence of the transposon, and are named as CACTA-L and CACTA-R. In the application, the main QTL and gene position of the corn southern rust resistance are clear, the phenotype contribution rate is higher, the southern rust resistance of corn inbred line material can be significantly improved, the development and application of the two function markers make the corn seedling stage ZmWRKY66 gene typing detection convenient and fast, and the application is not affected by the environment, and a feasible technical approach is provided for the molecular assisted breeding of the corn southern rust resistance.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular biology, and more specifically, to functional markers of maize resistance to southern rust and their applications. Background Technology

[0002] Corn (Zea mays L.) is the most widely planted and highest-yielding grain and cash crop in my country. Southern Corn Rust (SCR) is an airborne fungal disease caused by *Puccinia polysora* Underw., which first discovered and named the fungus *Tripsacum dactyloides* L. in Alabama, USA, in 1891 by Underwoods. [1] This pathogen can also infect plants in the genera *Erianthus*, *Zea*, and *Teosinte*. [2] First reported in Sierra Leone, South Africa in 1949, this epidemic disease caused maize yield losses of up to 50%. [3] By 1952, SCR had spread to Tanganyika, Uganda, Nyasaland, and Kenya, as well as the east coast of Africa, including the Republic of Mauritius in the Indian Ocean, and began to pose a threat to maize production in Asia and Australia. [4] This disease was first discovered in Hainan Province, my country, in 1972. [5] In recent years, due to global warming, changes in major cultivated varieties, the widespread adoption of susceptible varieties, and the rapid evolution of physiological races, southern rust of maize has become a significant disease in tropical and subtropical maize-producing areas such as Hainan, Guangxi, Guangdong, Jiangsu, and Zhejiang, as well as the Huang-Huai-Hai maize-producing region in my country. [6] The promotion of disease-resistant varieties is the most effective way to control southern rust in maize. Therefore, screening for resistant germplasm resources to ensure the continuous and stable development of maize production is an urgent task in maize breeding.

[0003] In recent years, numerous studies on the mapping of resistance QTLs for maize southern rust have been reported by scholars both domestically and internationally. Multiple resistance QTLs have been detected using different populations. These studies have played a role in elucidating the genetic mechanism of maize southern rust. However, due to differences in population type and size, genetic background, genetic map density, and statistical analysis methods used in different studies, there are differences in the number, location, and effect of QTLs mapping the same trait. Moreover, the detected QTLs are usually distributed on different chromosomes, and the effect value of a single QTL is relatively small, thus limiting the application of the research results in breeding.

[0004] Breeders often utilize a limited number of resistance genes with relatively obvious resistance characteristics, resulting in an increasingly narrow genetic background for disease-resistant germplasm resources. In production practice, the emergence of new physiological races has led to the overcoming of existing resistant varieties, causing the continued spread of diseases and resulting in economic losses. [7] Therefore, screening new disease-resistant germplasm and integrating multiple disease-resistant genes and broad-spectrum disease-resistant genes into breeding materials can provide broad-spectrum resistance to diseases, meeting the requirements of disease-resistant breeding and production practices. Screening disease-resistant resources and discovering and locating new broad-spectrum disease-resistant genes or QTLs are the prerequisites and foundations for breeding disease-resistant varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a functional marker for the maize resistance gene ZmWRKY66 to southern rust.

[0006] Another object of the present invention is to provide the application of the aforementioned functional marker in screening or identifying maize germplasm resources resistant to southern rust.

[0007] Another object of the present invention is to provide a method for breeding maize germplasm resistant to southern rust.

[0008] To achieve the objectives of this invention, this invention first provides a functional marker for the maize southern rust resistance gene ZmWRKY66. This gene is located at the major QTL locus RppQ6.01 on maize chromosome 6, and the functional markers include CACTA-L and CACTA-R. The primers for the molecular markers are as follows:

[0009] The forward and reverse primer sequences of CACTA-L are SEQ ID NO.1 and 2, respectively;

[0010] The forward and reverse primer sequences of CACTA-R are SEQ ID NO.4 and 5, respectively.

[0011] Among them, the primers shown in SEQ ID NO.1 and SEQ ID NO.2 could not amplify the band in the highly resistant maize inbred line Qi 319; but a band of 1107 bp could be amplified in the highly susceptible maize inbred line Ye 478, with the nucleotide sequence shown in SEQ ID NO.3.

[0012] Using the primers shown in SEQ ID NO.4 and SEQ ID NO.5, no band could be amplified in the maize inbred line Qi 319, which is highly resistant to southern rust; however, a band of 995 bp could be amplified in the maize inbred line Ye 478, which is highly susceptible to southern rust, and the nucleotide sequence is shown in SEQ ID NO.6.

[0013] The present invention also provides the application of the molecular marker in screening or identifying maize germplasm resources resistant to southern rust.

[0014] The application includes the following steps:

[0015] 1) Extract genomic DNA from the plants to be tested;

[0016] 2) Using the genomic DNA of the plant to be tested as a template, PCR amplification reaction is performed using primers for amplifying the molecular markers described in claim 1 or 2;

[0017] 3) Detect PCR amplification products;

[0018] Beneficial effects of this study:

[0019] This invention marks the first time that the major QTL locus RppQ6.01 for maize resistance to southern rust and the resistance gene ZmWRKY66 have been located on maize chromosome 6. In conventional breeding methods, the identification of maize resistance to southern rust must wait until after the milk stage, which is time-consuming, labor-intensive, and inefficient. By detecting the major QTL locus for maize resistance to southern rust, susceptible plants can be eliminated at the seedling stage or using a small portion of the seed's endosperm, saving production costs and significantly improving selection efficiency.

[0020] In this invention, the major QTL loci and resistance genes for maize resistance to southern rust are clearly identified, with a high phenotypic contribution rate. They can significantly improve the resistance to southern rust in maize inbred lines. The development and application of the two functional markers make the genotyping detection of maize southern rust in seedlings convenient and rapid, unaffected by the environment, and provide a feasible technical approach for molecular-assisted breeding of maize for disease resistance. Attached Figure Description

[0021] Figure 1 The plant performance and disease index of maize inbred lines Qi 319 (disease-resistant parent) and Ye 478 (disease-susceptible parent) after artificial inoculation with southern rust were investigated.

[0022] Figure 2 This is the result of validating the major resistance QTL_RppQ6.01 using a chromosome segment substitution line (CSSL) population in Example 2 of this invention.

[0023] Figure 3 This is the result of fine mapping of the RppQ6.01 locus of resistance to southern maize rust using a chromosome segment substitution line (CSSL) population in Example 3 of the present invention.

[0024] Figure 4 This is the result of the analysis of the expression levels of three candidate genes at the RppQ6.01 site before and after inoculation with CL184 and Ye478 in Example 3 of the present invention.

[0025] Figure 5 The results of phenotypic identification before and after inoculation of the gene silencing material mediated by Pr-CMV-VIGS technology in Example 3 of this invention.

[0026] Figure 6 The diagram shows the structure of the ZmWRKY66 promoter in Example 3 of this invention, and the correlation analysis results between the CACTA-L and CACTA-R genotypes and the resistance phenotype of southern rust in Example 4. Detailed Implementation

[0027] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0028] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] Example 1: Localization of RppQ6.01, the major site of maize resistance to southern rust

[0030] 1.1 Materials and Methods

[0031] 1.1.1 Test Materials

[0032] Using Qi 319 and Ye 478 as parents, a population of 314 recombinant inbred lines (RILs) was constructed through single-seed transduction, and disease resistance QTLs were detected by combining genotype and phenotype.

[0033] 1.1.2 Disease resistance identification

[0034] To ensure a consistent inoculum, rust spores were collected in Nanning, Guangxi Province. After laboratory purification, they were artificially inoculated onto the susceptible inbred line Huangzao 4 in a greenhouse for cultivation and propagation. Once disease developed, fresh infected leaves carrying spores of *Puccinia polysora Underw.* were collected, manually rubbed or brushed, filtered, and then mixed thoroughly with 0.02% Tween (v / v) to prepare a solution with a concentration of 6 × 10⁻⁶. 4 A suspension of urediniospores per mL was used as material for artificial inoculation.

[0035] Both artificial inoculation and natural disease identification employed the same disease investigation methods. Inoculation with bacterial solution was carried out by spraying at the 8-9 leaf stage of corn, with an inoculation volume of 7-8 mL per corn plant. Simultaneously, field humidity was maintained to meet the requirements for pathogen invasion and plant disease development. Phenotypic identification was conducted when the corn reached the milk stage. During the investigation, the diseased area of ​​each identification material population was visually assessed. The key investigation area was the upper and lower three leaves of the corn ear. Based on the disease symptoms, each material was investigated and the disease severity was recorded. [7]Then, a comprehensive evaluation of disease resistance is conducted based on the severity of the disease (Table 1).

[0036] Table 1 Disease levels of southern rust in maize

[0037]

[0038] 1.1.3 Genotype Analysis and QTL Mapping

[0039] Our laboratory has previously completed resequencing of RIL populations constructed from the resistant parent Qi 319 and the susceptible parent Ye 478, as well as both parents. The relevant data and genotypes have been published. [8] .

[0040] Molecular marker linkage maps were constructed using QTL Icimapping V4.0 software. Genotype data were organized according to the software requirements, grouped according to LOD values ​​greater than 3.0, sorted using nntwoOpt, and processed using SaRF. [9] After constructing the molecular marker linkage map, QTL localization was performed using the IcIM additive mapping method. Missing phenotypes were removed during the localization process. The mapping step size was 0.20 cM. ​​QTLs were determined at the P=0.05 level through 1000 permutation tests.

[0041] 1.2 Results

[0042] 1.2.1 Linkage Graph Construction

[0043] Genotyping was performed on the RIL population and the parents (Qi 319 and Ye 478). A total of 137,699,000 reads were screened, with an average of 357,376 reads per individual. A total of 88,268 SNPs were developed, and 4,183 parent-specific SNPs were ultimately identified and anchored on the genetic linkage map. A high-density map covering all 10 maize chromosomes, with a total chromosome length of 1545.65 cM, was constructed. The average genetic distance between molecular markers was 0.37 cM, and the average physical distance was 0.51 Mb (B73_v3, Mb). [8] .

[0044] 1.2.2 Positioning of the main QTL for maize resistance to southern rust

[0045] Combining field phenotypic and genotypic data of RILs populations, a mixed linear model using QTL IciMapping V4.1 software was used to analyze the phenotypic and genotypic data of RILs populations from two sites in Nanning, Guangxi in 2017 and 2018. After 1000 iterations, at the P=0.05 level, the QTL mapping results are shown in Table 2. A major-effect QTL, _RppQ6.01, was detected on chromosome 6, which can explain 24.15% of the phenotypic contribution.

[0046] Table 2. QTL detection of resistance to southern rust in recombinant inbred lines.

[0047]

[0048] Example 2: Verification Analysis of RppQ 6.01 Based on CSSL Community

[0049] 2.1 Materials and Methods

[0050] 2.1.1 Test Materials

[0051] To effectively validate the mapping results of the RIL population and subsequent fine mapping, the superior maize inbred line Qi 319 was selected as the non-recurrent parent, and Ye 478 as the recurrent parent, to construct a BC5F3 CSSLs population covering the entire chromosome. From this population, six chromosome replacement lines covering chromosome 6 were selected, with the Ye 478 genotype as the genetic background and the Qi 319 gene fragment introduced into the line, for validation of disease resistance QTLs. Among them, the introduced line CL184 contained a QTL introduced into chromosome 6 for resistance to southern rust, and the F2 segregating population of CL183 × Ye 478 was used for fine mapping.

[0052] 2.1.2 Phenotypic Identification

[0053] Reference 1.1.2

[0054] 2.2 Results

[0055] Based on the QTL mapping results of recombinant inbred families, the gene for resistance to southern rust was initially located on chromosome 6. Therefore, replacement lines carrying the gene for resistance to southern rust were selected from 18 replacement lines covering chromosome 6 to eliminate genetic background interference and perform fine mapping. Genotyping revealed 6 replacement lines that essentially covered chromosome 6. Figure 2The average background recovery rate of the six chromosome segment substitution lines (BC5F3) was 98.3%–99.8% (Table 3). Phenotypic identification of the six chromosome segment substitution lines was performed; Table 3 shows the genotypic and phenotypic analysis of the six substitution lines. Field phenotypic identification showed that CL183 (Type 2) was resistant to southern rust, with clean leaves and a small number of spores; CL184 (Type 1) was second; the other substitution lines were susceptible to southern rust, with the plant surface covered with numerous yellowish-brown spore masses.

[0056] Fine mapping of RppQ6.01 was performed using CSSLs populations derived from Qi319 and Ye478. Genotypic and phenotypic data indicated that CL183 and CL184 carry a southern rust resistance gene located between Y6p2 and Y6p3, with a resistance grade of 4.5–5.5. In the RIL population, the QTL on chromosome 6 was located between 75.30 and 78.25 Mb (B73_RefGen_v3) (Table 3), which corroborated the insertion fragments from CL183 and CL184. Figure 2 ).

[0057] Table 3 Comparison of chromosome segment replacement lines and resistance to Ye 478

[0058]

[0059] In the table, "+ / +" indicates that the target section is consistent with the Qi 319 belt type; "- / -" indicates that the target section is consistent with the Ye 478 belt type.

[0060] Example 3: Fine mapping of RppQ6.01 and functional verification of candidate genes

[0061] 3.1 Materials and Methods

[0062] 3.1.1 Test Materials

[0063] Because CL183 contains some heterozygous fragments, CL184 was crossed with Ye 478 to form the F1 generation. The F1 generation was then self-crossed to obtain the F2 population. End-terminal SSR markers and primers showing polymorphism between parents were used. Existing regions were validated, and different types of homozygous and heterozygous exchange plants were screened. Homozygous lines were self-crossed and used for field phenotypic identification. After self-crossing heterozygous plants, the progeny family method was used to narrow down the region.

[0064] All the selected recombinant progeny were then planted with Ye 478 at a 1:1 ratio, artificially inoculated with P. polysora, and field identification was performed. Fine mapping was carried out using homozygous lines of the F8 generation of the secondary segregating population, which exhibited polymorphism among the parents (Table 4).

[0065] Table 4. Primer information for 14 pairs

[0066]

[0067] Note: The physical locations in the table refer to (Zm-B73-REFERENCE-NAM-5.0).

[0068] 3.1.2 Development of Polymorphic Markers

[0069] Indel markers were developed and designed by combining resequencing data from parental lines Qi319 and Ye478.

[0070] 3.1.3 Genotyping

[0071] When the maize plants reached the 5-leaf stage, a small number of fresh leaves were taken from each plant, and genomic DNA was extracted using the CTAB method. Indel markers were developed based on resequencing data from Qi 319 and Ye 478, and primer sequences were synthesized by BGI Genomics Co., Ltd. PCR reactions employed a falling-step amplification program, and the amplification products were separated by 8% polyacrylamide gel electrophoresis and silver staining.

[0072] The PCR amplification reaction used a 20 μL system with the following components: 6.4 μL ddH2O; 10 μL 2x 3G Taq Master Mix for PAGE (Red Dye); 0.8 μL each of forward and reverse primers (1.0 μM); and 2.0 μL DNA template (50 ng / μL). After mixing all reaction components, 20 μL of mineral oil was added to cover the mixture, and amplification was performed on a PCR instrument using the following program: 94℃ for 5 min; 94℃ for 30 s, 65℃ for 30 s (decreasing by 1℃ per cycle), 72℃ for 30 s, for a total of 10 cycles; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; 72℃ for 5 min.

[0073] 3.1.4 Screening and functional verification of candidate genes

[0074] Genes within this localization region were annotated based on the third-generation genome assembly information of Qi 319 and annotation information from the B73 reference genome database (https: / / www.ncbi.). Figure 3 ).

[0075] Samples were taken from CL184 and Ye478 at 0, 12, 24, 48 and 72 hpi after inoculation, and the expression levels of candidate genes after P. polysora induction were analyzed using RT-qRCR.

[0076] To assess the resistance level of candidate genes after infection with P. polysora, Pr-CMV-VIGS technology was used to silence candidate genes and verify their biological functions.

[0077] 3.1.5 Phenotypic Identification

[0078] Reference 1.1.2

[0079] 3.2 Results

[0080] 3.2.1 RppQ6.01 Fine Positioning

[0081] All selected recombinant progeny were planted with Ye 478 at a 1:1 ratio, artificially inoculated with *P. polysora*, and then field-tested. Field-testing results showed that, compared with the susceptible parent Ye 478, the disease severity of recombinant exchange plants Type 1, Type 4, Type 5, and Type 6 differed significantly from that of Ye 478 (P < 0.01), while there was no significant difference in disease severity for Type 2, Type 3, Type 7, and Type 8. In summary, based on the genotypic and phenotypic identification results of the recombinant exchange plant progeny, RppQ6.01 was finally located between the molecular markers Y6p76 and SCR08 on chromosome 6, with a physical distance of 0.73 Mb (Zm-B73-REFERENCE-NAM-5.0). Figure 3 Based on the third-generation genome assembly information of Qi 319 and the annotation information of the B73 reference genome database (https: / / www.ncbi.), this localization interval contains 3 candidate genes. Figure 3 The corresponding numbers are Zm00001eb271180 (ORF1), Zm00001eb271190 (ORF2, ZmWRKY66), and Zm00001eb271200 (ORF3).

[0082] To further identify candidate genes, samples were taken from CL184 and Ye478 at 0, 12, 24, 48, and 72 hpi after inoculation. RT-qCR was used to analyze the expression levels of candidate genes after P. polysora induction. Figure 4 The results showed that after induction by *P. polysora*, the expression level of Zm00001eb271180 in CL184 was irregular, while it decreased in Ye478, and the expression levels at 12, 24, and 72 hpi were significantly higher than those in CL184 (P < 0.01). After induction by *P. polysora*, the expression level of Zm00001eb271190 in the susceptible parent Ye478 was significantly upregulated at 24 hpi, and the expression levels at 24, 48, and 72 hpi were significantly higher than those in the resistant fragment substitution line CL184 (P < 0.01). However, after induction by *P. polysora*, the expression level of Zm00001eb271200 in Ye478 and CL184 showed no significant difference from 0 to 48 hpi, but at 72 hpi, the expression level of this gene in Ye478 was significantly reduced (P < 0.01).

[0083] 3.2.2 Functional validation of candidate genes

[0084] To assess the resistance levels of candidate genes after infection with *P. polysora*, gene silencing of three candidate genes, Zm00001eb271180, ZmWRKY66, and Zm00001eb271200, was performed using Pr-CMV-VIGS technology. The results showed that the control group, Pr CMV::LUC, produced a large number of spores on its leaves. Compared with the control, PrCMV::ZmWRKY66 plants showed a significantly reduced number of spores and lower susceptibility (P≤0.01), while Pr CMV::Zm00001eb271180 and Pr CMV::Zm00001eb271200 plants showed no significant difference in susceptibility compared to the control. Figure 5 In summary, silencing ZmWRKY66 enhances maize's resistance to *P. polysora* and reduces its susceptibility. This indicates that ZmWRKY66 is a disease resistance gene of RppQ6.01.

[0085] Example 4: ZmWRKY66 Sequence Analysis and Development of Functional Markers

[0086] 4.1 Materials and Methods

[0087] 4.1.1 Test Materials

[0088] Disease-resistant parent Qi 319 and disease-susceptible parent Ye 478

[0089] 4.1.2 Functional Tag Development

[0090] Based on the presence of a 9365bp CACTA-TIR transposon insertion in the promoter region of the susceptible parent Ye478, molecular markers CACTA-L and CACTA-R were developed on its left and right sides, respectively. Figure 6 ).

[0091] 4.1.3 Genotyping

[0092] When maize reached the 5-leaf stage, a small number of fresh leaves were taken from each plant, and genomic DNA was extracted using the CTAB method. The molecular markers CACTA-L and CACTA-R were developed based on sequencing data from the amplified promoter region of the susceptible parent Ye478. Primer sequences were synthesized by BGI Genomics Co., Ltd. PCR reactions used a falling-step amplification program, and the amplification products were separated and detected by 1% agarose gel electrophoresis.

[0093] The PCR amplification reaction was performed in a 20 μL system. The system components and amplification procedure are described in section 3.1.3.

[0094] 4.2 Results

[0095] RT-qPCR results showed that ZmWRKY66 was significantly induced by the pathogen. Considering that changes in gene expression may be related to promoter activity, it was speculated that the promoter sequence of ZmWRKY66 differed between the resistant parent Qi319 and the susceptible parent Ye478. Therefore, amplification analysis of the ZmWRKY66 promoter sequence was performed, and the results showed that compared with Qi319, Ye478 and B73 promoter regions (before the 1092bp ATG) contained a 9365bp CACTA-TIR transposon insertion. Figure 6 A, B). Molecular markers CACTA-L and CACTA-R were developed for the left and right sides of this transposon, respectively. The primers for the molecular markers are as follows:

[0096] The forward and reverse primer sequences of CACTA-L are SEQ ID NO.1 and 2, respectively;

[0097] The forward and reverse primer sequences of CACTA-R are SEQ ID NO.4 and 5, respectively.

[0098] Among them, the primers shown in SEQ ID NO.1 and SEQ ID NO.2 could not amplify the band in the maize inbred line Qi 319 which is highly resistant to southern rust; however, a band of 1107 bp could be amplified in the maize inbred line Ye 478 which is highly susceptible to southern rust, and the nucleotide sequence is shown in SEQ ID NO.3.

[0099] Using the primers shown in SEQ ID NO.4 and SEQ ID NO.5, no band could be amplified in the maize inbred line Qi 319, which is highly resistant to southern rust; however, a band of 995 bp could be amplified in the maize inbred line Ye 478, which is highly susceptible to southern rust, and the nucleotide sequence is shown in SEQ ID NO.6.

[0100] Example 5: Validation and analysis of molecular markers in maize southern rust resistant / susceptible inbred lines

[0101] 5.1 Materials and Methods

[0102] 5.1.1 Test Materials

[0103] Fourteen maize inbred lines resistant to southern rust and fourteen susceptible inbred lines were selected, and phenotypic identification of these 28 maize inbred lines has been completed.

[0104] 5.1.2 Genotyping

[0105] Reference 4.1.3

[0106] 5.2 Results

[0107] Phenotypic identification results of 28 maize inbred lines under three environments were combined with genotyping results of the 28 inbred lines using CACTA-L and CACTA-R markers for correlation analysis. The results showed that the CACTA-L and CACTA-R genotypes were significantly correlated with the phenotype of southern rust in maize (P<0.01). Figure 6 C).

[0108] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

[0109] References

[0110] 1.Underwood LM.Some new fungi chiefly from Alabama[J].Bull Torrey BotClub.1897,24(2):81-86. 2. Casela CR, Ferreira AS. 2002. Variability in isolates of Pucciniapolysora in Brazil. Fitopatol Bras, 27(4):414-416.

[0111] 3. Rhind D, Waterston JM, Deighton FC. Occurrence of Puccinia polysora Underw in West Africa[J].Nature.1952,169:631.

[0112] 4.Orian G.Occurrence of Puccinia polysora Underwood in the IndianOcean Area[J].Nature,1954,173:505-505.

[0113] 5. Duan Dingren, He Hongzhen. A multi-stalked rust fungus on maize in Hainan Island [J]. Acta Mycologica Sinica, 1984(02):125-126.

[0114] 6. Lu Weiting, Yu Huan, Cao Shengnan, Chen Changqing. The impact of climate warming in the Huang-Huai-Hai region on the growth process and yield of summer maize in the past 20 years [J]. Chinese Agricultural Science, 2015, 48(16):3132-3145.

[0115] 7. Wang Xiaoming. Series of lectures on maize diseases and pests (III): Identification and investigation techniques of maize disease and pest resistance. Crop Journal, 2005(06):53-55.

[0116] 8.Zhou Zhiqiang, Zhang Chaoshu, Zhou Yu.et al.Genetic dissection ofmaize plant architecture with anultra-high density bin map based on recombinant inbred lines[J].BMC GENOMICS, 2016,17:178-193.

[0117] 9.Doerge RW, Churchill GA.Permutation tests for multiple loci affecting a quantitative character[J].Genetics.1996,142(1):285-294.

Claims

1. The application of primers for a functional marker of a maize southern rust resistance gene in screening or identifying maize germplasm resources resistant to southern rust, wherein the primers are as follows: The forward and reverse primer sequences of CACTA-L are SEQ ID NO.1 and 2, respectively; The forward and reverse primer sequences of CACTA-R are SEQ ID NO.4 and 5, respectively.

2. The application according to claim 1, characterized in that, Includes the following steps: 1) Extract genomic DNA from the plants to be tested; 2) Using the genomic DNA of the plant to be tested as a template, PCR amplification reaction was performed using primers that amplify the functional markers; 3) Detection of PCR amplification products; wherein the primers for the functional markers are as follows: The forward and reverse primer sequences of CACTA-L are SEQ ID NO.1 and 2, respectively; The forward and reverse primer sequences of CACTA-R are SEQ ID NO.4 and 5, respectively.

3. The application according to claim 2, characterized in that, In step 3), 1% agarose gel electrophoresis was used to detect the PCR amplification products labeled CACTA-L and CACTA-R.

4. A method for breeding maize germplasm resistant to southern rust, characterized in that, The method is as follows: 1) Extract genomic DNA from the plants to be tested; 2) Using genomic DNA as a template, PCR amplification was performed using the primers shown in SEQ ID NO.1-2 and SEQ ID NO.4-5. The obtained PCR products were subjected to electrophoresis, and the band patterns of the electrophoretic bands were compared with the reference band patterns. 3) The reference band pattern is the band pattern obtained by PCR amplification and electrophoresis using the genomic DNA of the maize inbred line Qi 319, which is highly resistant to southern rust, as a template, and using the primer pairs shown in SEQ ID NO.1-SEQ ID NO.2 and the primer pairs shown in SEQ ID NO.4-SEQ ID NO.

5. 4) Evaluate the plants to be tested and select plants with the same band pattern as the reference band pattern as parents for breeding.

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