Functional marker of corn southern rust resistance gene and application of functional marker

Abstract

The invention discloses a corn southern rust resistance gene functional marker, the gene is located on the sixth chromosome of corn and is from a southern rust resistance major QTL site RppQ6.01, a gene silencing test shows that ZmWRKY66 is an anti-disease gene of RppQ6.01, sequence analysis finds that insertion / deletion of a CACTA-TIR transposon exists in a promoter region of ZmWRKY66, and the gene is a gene with a gene sequence shown in the specification. Two functional markers are developed on the basis of the DNA sequence of the transposon, and are named as CACTA-L and CACTA-R. The corn southern rust resistance main effect QTL and the gene position are clear, the phenotype contribution rate is high, the southern rust resistance character of a corn inbred line material can be remarkably improved, the corn seedling stage ZmWRKY66 genetic typing detection becomes convenient and rapid and is not affected by the environment through development and application of the two functional markers, and the corn seedling stage ZmWRKY66 genetic typing detection method has the advantages that the corn southern rust resistance main effect QTL and the gene position are clear. And a feasible technical approach is provided for molecular assisted breeding of southern rust resistance of corn.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and molecular biology, and in particular to functional markers of maize southern rust resistance genes and their application. Background Art

[0002] Corn (Zea mays L) is the largest food and cash crop in my country with the highest yield and largest planting area. Southern Corn Rust (SCR) is an airborne fungal disease caused by Puccinia polysora Underw, which was first discovered and named by Underwoods in 1891 on Tripsacum dactyloides L in Alabama, USA. [1] The pathogen can also infect Erianthus, Zea and Teosinte. [2] First reported in 1949 in Sierra Leone, South Africa, the disease caused an epidemic that reduced maize production by 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 threaten corn production in Asia and Australia. [4] In 1972, the disease was first discovered in Hainan Province, my country. [5] In recent years, due to global warming, the replacement of main cultivars, the large-scale promotion of susceptible varieties and the rapid evolution of physiological subspecies, southern corn rust has become an important disease in tropical and subtropical corn producing areas such as Hainan, Guangxi, Guangdong, Jiangsu, Zhejiang and Huanghuaihai corn producing areas in my country. [6] The promotion of disease-resistant varieties is the most effective way to control southern corn rust. Therefore, screening resistant germplasm resources to ensure the sustainable and stable development of corn production is an urgent task for current corn breeding.

[0003] In recent years, domestic and foreign scholars have reported many studies on the positioning of QTLs for southern rust resistance in maize. Multiple resistance QTLs have been detected using different populations. These studies have played a certain role in clarifying the genetic mechanism of southern rust in maize. However, due to the differences in population types and sizes, genetic backgrounds, genetic map densities and statistical analysis methods used in different studies, there are differences in the number, position and effect of QTLs located for the same trait; and the QTLs detected are usually distributed on different chromosomes, and the effect value of a single QTL is small, which limits the application of the research results in breeding.

[0004] Breeders mostly use a few resistance genes with relatively obvious resistance for breeding. The genetic background of disease-resistant germplasm resources is becoming increasingly narrow. In production practice, the emergence of new physiological subspecies has led to the existing disease-resistant varieties being overcome, resulting in the continued prevalence of diseases and causing economic losses. [7] Therefore, screening new disease-resistant germplasm and aggregating multiple disease-resistant genes and broad-spectrum disease-resistant genes in breeding materials as much as possible can provide broad-spectrum resistance to diseases and meet the requirements of disease-resistant breeding and production practice. Screening disease-resistant resources and discovering and locating new broad-spectrum disease-resistant genes or QTLs are the premise and basis for breeding disease-resistant varieties. Summary of the invention

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

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

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

[0008] In order to achieve the purpose of the present invention, the present invention first provides a functional marker of a maize southern rust resistance gene ZmWRKY66, wherein the gene is located at the southern rust major effect QTL site RppQ6.01 on maize chromosome 6, and the functional marker includes CACTA-L and CACTA-R. The primers of the molecular marker are as follows:

[0009] The forward and reverse primer sequences of CACTA-L are SEQ ID NOs. 1 and 2, respectively;

[0010] The forward primer and reverse primer sequences of CACTA-R are SEQ ID NOs. 4 and 5, respectively.

[0011] Among them, using the primers shown in SEQ ID NO.1 and SEQ ID NO.2, no band can be amplified in the highly resistant maize inbred line Qi 319; a band of 1107 bp can be amplified in the highly susceptible maize inbred line Ye 478, and the nucleotide sequence is shown in SEQ ID NO.3;

[0012] Using the primers shown in SEQ ID NO.4 and SEQ ID NO.5, no band can be amplified in the maize inbred line Qi 319 with high resistance to southern rust; a band of 995 bp can be amplified in the inbred line Ye 478 with high sensitivity to southern rust, and the nucleotide sequence is shown in SEQ ID NO.6.

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

[0014] The application comprises the following steps:

[0015] 1) Extracting genomic DNA from the plant to be tested;

[0016] 2) using the genomic DNA of the plant to be tested as a template and using primers for amplifying the molecular marker according to claim 1 or 2 to perform a PCR amplification reaction;

[0017] 3) Detection of PCR amplification products;

[0018] Beneficial effects of this study:

[0019] The present invention locates for the first time the major QTL locus RppQ6.01 of maize resistance to southern rust and the disease resistance gene ZmWRKY66 on maize chromosome 6. In conventional breeding methods, the identification of maize resistance to southern rust must wait until the maize milky stage, which is time-consuming and labor-intensive and has low selection efficiency. By detecting the major QTL locus of maize resistance to southern rust, susceptible plants can be eliminated at the seedling stage or using a small part of the seed endosperm, which not only saves production costs but also greatly improves selection efficiency.

[0020] The major QTL loci and disease-resistant genes of corn southern rust resistance in the present invention are clear, the phenotypic contribution rate is high, and the southern rust resistance traits of corn inbred line materials can be significantly improved. The development and application of two functional markers makes the typing and detection of southern rust genes in corn seedlings convenient and rapid, and unaffected by the environment, providing a feasible technical approach for molecular-assisted breeding of corn disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0024] Figure 4 These are the results of the expression analysis of three candidate genes at the RppQ6.01 locus before and after CL184 and Ye478 inoculation in Example 3 of the present invention.

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

[0026] Figure 6 Schematic diagram of the ZmWRKY66 promoter structure in Example 3 of the present invention, and the results of the correlation analysis between the CACTA-L and CACTA-R genotypes and the resistance phenotype of southern rust in Example 4. DETAILED DESCRIPTION

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

[0028] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0029] Example 1: Location of the major locus RppQ6.01 for maize resistance to southern rust

[0030] 1.1 Materials and methods

[0031] 1.1.1 Test materials

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

[0033] 1.1.2 Disease resistance identification

[0034] To ensure the uniformity of the fungus source, rust spores were collected in Nanning, Guangxi, purified in the laboratory, and artificially inoculated on the susceptible inbred line Huang Zao Si in the greenhouse for cultivation and proliferation. After the disease occurred, fresh susceptible leaves carrying multiple piles of Puccinia polysora Underw spores were collected, manually scrubbed or brushed with a small brush, filtered, and 0.02% Tween (v / v) was added, fully mixed, and prepared into a concentration of 6×10 4 A summer spore suspension with spores / mL was used as the material for artificial inoculation.

[0035] The same disease investigation method is used for both artificial inoculation identification and natural disease identification. When corn grows to the 8-9 leaf stage, spray the bacterial solution with a sprayer for inoculation, and the inoculation amount for each corn plant is 7-8mL of bacterial solution. At the same time, ensure that the field humidity conditions meet the requirements of pathogen invasion and plant disease. Phenotypic identification is carried out when corn enters the milky stage. During the investigation, the diseased area of ​​each identification material group is visually inspected. The key parts of the investigation are the upper and lower 3 leaves of the corn cob. According to the symptoms of the disease, each material is investigated and the disease level is recorded [7], and then conduct a comprehensive evaluation of disease resistance according to the disease level (Table 1).

[0036] Table 1 Disease levels of southern corn rust

[0037]

[0038] 1.1.3 Genotype analysis and QTL mapping

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

[0040] The molecular marker linkage map was constructed using QTL Icimapping V4.0 software. Genotype data were sorted according to the software requirements, grouped under the condition that LOD was greater than 3.0, sorted using nntwoOpt, and sorted using SaRF. [9] After the molecular marker linkage map was constructed, the IcIM additive mapping method was used for QTL localization. The missing phenotypes were removed during the localization process. The mapping step was 0.20 cM. ​​The QTL was determined at the P = 0.05 level through 1000 permutation tests.

[0041] 1.2 Results

[0042] 1.2.1 Linkage map construction

[0043] The RILs population and parents (Qi 319, Ye 478) were genotyped. 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 4183 SNPs that were specific between the parents were finally screened and anchored on the genetic linkage map. A high-density map covering 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 physical distance was 0.51 Mb (B73_v3, Mb) [8] .

[0044] 1.2.2 Mapping of major QTLs for maize resistance to southern rust

[0045] Combining the field phenotypic data and population genotypic data of RILs populations, the mixed linear model of QTL IciMapping V4.1 software was used to analyze the phenotypic data and corresponding genotypic data of RILs populations at two points in Nanning, Guangxi in 2017 and 2018. After 1000 iterations, at the P=0.05 level, the positioning results of the QTLs were shown in Table 2. A major effect QTL_RppQ6.01 was detected on chromosome 6, which can explain 24.15% of the phenotypic contribution rate.

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

[0047]

[0048] Example 2: Validation analysis of RppQ6.01 based on CSSL population

[0049] 2.1 Materials and methods

[0050] 2.1.1 Test materials

[0051] In order to effectively verify the positioning results of the RIL population and the subsequent fine positioning, the excellent 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 them, the six chromosome segment replacement lines covering chromosome 6 with the Ye 478 genotype as the genetic background and the introduction of the Qi 319 gene fragment were selected for the verification of disease resistance QTL. Among them, the introduction line CL184 has an introduction fragment of the southern rust resistance QTL on chromosome 6, and the F2 segregation population of CL183×Ye 478 is used for fine positioning.

[0052] 2.1.2 Phenotypic identification

[0053] Reference 1.1.2

[0054] 2.2 Results

[0055] According to the results of QTL positioning of recombinant inbred families, the southern rust resistance gene was initially located on chromosome 6. Therefore, the replacement lines carrying the southern rust resistance gene were selected from the 18 replacement lines covering chromosome 6 to eliminate genetic background interference and conduct fine positioning. Through genotype detection, six chromosome segment replacement lines that basically covered chromosome 6 were selected ( Figure 2), the average background recovery rate of the six chromosome segment replacement lines (BC5F3) was 98.3% to 99.8% (Table 3). The phenotypes of the six chromosome segment replacement lines were identified. Table 3 shows the genotypes and phenotypic analysis of the six replacement 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 replacement lines were susceptible to southern rust, with a large number of yellow-brown spores covering the plant surface.

[0056] Fine mapping of RppQ6.01 was performed using CSSLs populations derived from Qi 319 and Ye 478. Genotypic and phenotypic data showed that CL183 and CL184 carried southern rust resistance genes, which were located between Y6p2 and Y6p3, with disease resistance levels of 4.5 to 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 was mutually verified with the introduced fragments of CL183 and CL184 ( Figure 2 ).

[0057] Table 3 Comparison of resistance between chromosome segment substitution lines and Ye 478

[0058]

[0059] In the table, “+ / +” means the target segment is consistent with the QI 319 belt type; “- / -” means the target segment is consistent with the YE 478 belt type.

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

[0061] 3.1 Materials and methods

[0062] 3.1.1 Test materials

[0063] Since CL183 contains some heterozygous fragments, CL184 and Ye 478 were selected for hybridization and F1 was formed. The F1 was self-pollinated to obtain the F2 population. The SSR markers at both ends and the primers screened for polymorphism between the parents were used to verify the existing intervals and screen different types of homozygous and heterozygous exchange plants. The homozygous lines were self-pollinated and kept for field phenotypic identification. After the heterozygous plants were self-pollinated, the progeny family method was used to narrow the positioning interval.

[0064] All the recombinant offspring screened were further planted with Ye 478 at a ratio of 1:1, and artificially inoculated with P. polysora for field identification. Fourteen pairs of primers with polymorphisms between the parents were used to perform fine mapping on the homozygous lines of the F8 generation of the secondary segregation population (Table 4).

[0065] Table 4 Information of 14 pairs of primers

[0066]

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

[0068] 3.1.2 Polymorphic marker development

[0069] Indel markers were developed and designed by combining the resequencing data of the parental lines Qi 319 and Ye 478.

[0070] 3.1.3 Genotyping

[0071] When corn grows to the 5-leaf stage, a small amount of fresh leaves are taken from each plant and genomic DNA is extracted using the CTAB method. The development of Indel markers is based on the data of Qi 319 and Ye 478 resequencing, and the primer sequences are synthesized by BGI Biotechnology Co., Ltd. The PCR reaction adopts a touchdown amplification procedure, and the amplified products are separated by 8% polyacrylamide gel electrophoresis and silver staining is used for color development.

[0072] The PCR amplification reaction used a 20 μL system, and the system components were as follows: 6.4 μL of ddH20; 10 μL of 2x 3G Taq Master Mixfor PAGE (Red Dye); 0.8 μL of each forward and reverse primer (1.0 μM); 2.0 μL of DNA template (50 ng / μL). After mixing the reaction components, add 20 μL of mineral oil to cover, and amplify on a PCR instrument. The amplification program was as follows: 94°C for 5 min; 94°C for 30 s, 65°C for 30 s (1°C drop for each cycle), 72°C for 30 s, for a total of 10 cycles; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, for a total of 30 cycles; 72°C for 5 min.

[0073] 3.1.4 Screening and functional verification of candidate genes

[0074] The genes in this positioning interval were annotated according to the third-generation genome assembly information of Qi 319 and the annotation information of the B73 reference genome database (https: / / www.ncbi. Figure 3 ).

[0075] Samples were taken at 0, 12, 24, 48 and 72 hpi after inoculation of CL184 and Ye 478, and the expression levels of candidate genes after P. polysora induction were analyzed using RT-qPCR.

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

[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 the recombinant offspring screened were planted with Ye 478 at a ratio of 1:1, and after artificial inoculation with P. polysora, field identification was carried out. The field identification results showed that compared with the susceptible parent Ye 478, the disease levels of recombinant exchange plants Type 1, Type 4, Type 5 and Type 6 were significantly different from those of Ye 478 (P < 0.01), while the disease levels of Type 2, Type 3, Type 7 and Type 8 were not significantly different. In summary, based on the genotype and phenotypic identification results of the recombinant exchange plant offspring, RppQ6.01 was finally located between the molecular markers Y6p76 and SCR08 on chromosome 6, with a physical distance of 0.73Mb (Zm-B73-REFERENCE-NAM-5.0)( Figure 3 According to the third-generation genome assembly information of Qi 319 and the annotation information of the B73 reference genome database (https: / / www.ncbi.nlm.nih.gov / pubmed / 2969669), this localization interval contains 3 candidate genes ( Figure 3 ), respectively Zm00001eb271180 (ORF1), Zm00001eb271190 (ORF2, ZmWRKY66), and Zm00001eb271200 (ORF3).

[0082] To further identify the candidate genes, samples were taken at 0, 12, 24, 48, and 72 hpi after inoculation of CL184 and Ye 478, and the expression levels of the candidate genes after P. polysora induction were analyzed using RT-qPCR ( Figure 4 ). The results showed that after being induced by P.polysora, the expression of Zm00001eb271180 in CL184 changed irregularly, while the expression level in Ye 478 decreased, and the expression levels at 12, 24 and 72 hpi were significantly higher than those in CL184 (P<0.01). Zm00001eb271190 was significantly upregulated in the susceptible parent Ye 478 at 24 hpi after being induced by P.polysora, and the expression level at 24, 48 and 72 hpi was significantly higher than that in the resistant fragment substitution line CL184 (P<0.01). However, there was no significant difference in the expression level of Zm00001eb271200 between Ye 478 and CL184 from 0 to 48 hpi after being induced by P.polysora. At 72 hpi after induction, the expression level of this gene in Ye 478 was significantly reduced (P<0.01).

[0083] 3.2.2 Functional verification of candidate genes

[0084] To evaluate the resistance level of candidate genes after infection with P. polysora, the three candidate genes Zm00001eb271180, ZmWRKY66 and Zm00001eb271200 were silenced using Pr-CMV-VIGS technology. The results showed that a large number of spores were produced on the leaves of the control group Pr CMV::LUC, the number of spores in the PrCMV::ZmWRKY66 plants was reduced compared with the control, and the disease susceptibility was significantly reduced (P≤0.01), while there was no significant difference in the disease susceptibility of the Pr CMV::Zm00001eb271180 and Pr CMV::Zm00001eb271200 plants compared with the control ( Figure 5 ). In summary, silencing ZmWRKY66 can enhance the resistance of maize to P. polysora and reduce its susceptibility to the disease. In summary, ZmWRKY66 is the 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] The resistant parent Qi 319 and the susceptible parent Ye 478

[0089] 4.1.2 Feature Flag Development

[0090] Based on the presence of a 9365 bp 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 corn grows to the 5-leaf stage, a small amount of fresh leaves are taken from each plant and genomic DNA is extracted using the CTAB method. The development of molecular markers CACTA-L and CACTA-R is based on the sequencing data of the promoter region sequence amplification of the susceptible parent Ye478, and the primer sequence is synthesized by BGI Biotechnology Co., Ltd. The PCR reaction adopts a touchdown amplification procedure, and the amplified products are separated and detected by 1% agarose gel electrophoresis.

[0093] The PCR amplification reaction uses a 20 μL system. The system components and amplification procedures refer to 3.1.3.

[0094] 4.2 Results

[0095] RT-qPCR results showed that ZmWRKY66 was significantly induced by pathogens. Considering that the change in gene expression may be related to promoter activity, it is speculated that there are differences in the promoter sequence of ZmWRKY66 between the disease-resistant parent Qi 319 and the susceptible parent Ye 478. Therefore, the ZmWRKY66 promoter sequence was amplified and analyzed. The results showed that compared with Qi 319, the promoter regions of Ye 478 and B73 (1092 bp before ATG) contained 9365 bp of CACTA-TIR transposon insertions ( Figure 6 A, B). For this transposon, molecular markers CACTA-L and CACTA-R were developed on its left and right sides, respectively. The primers for molecular markers are as follows:

[0096] The forward and reverse primer sequences of CACTA-L are SEQ ID NOs. 1 and 2, respectively;

[0097] The forward primer and reverse primer sequences of CACTA-R are SEQ ID NOs. 4 and 5, respectively.

[0098] Among them, using the primers shown in SEQ ID NO.1 and SEQ ID NO.2, no band can be amplified in the maize inbred line Qi 319 with high resistance to southern rust; a band of 1107 bp can be amplified in the maize inbred line Ye 478 with high sensitivity 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 can be amplified in the maize inbred line Qi 319 with high resistance to southern rust; a band of 995 bp can be amplified in the inbred line Ye 478 with high sensitivity 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 southern rust-resistant inbred lines and 14 maize southern rust-susceptible inbred lines were selected, and the phenotypic identification of the 28 maize inbred lines has been completed.

[0104] 5.1.2 Genotyping

[0105] Reference 4.1.3

[0106] 5.2 Results

[0107] The phenotypic identification results of 28 maize inbred lines under three environments were combined with the genotyping results of 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 phenotypes of maize southern rust (P<0.01)( Figure 6 C).

[0108] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made thereto on the basis of the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention all fall 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 ofPuccinia polysora in Brazil.Fitopatol Bras,27(4):414-416.

[0111] 3. Rhind D, Waterston JM, Deighton FC. Occurrence of Puccinia polysora Underwives in West Africa[J].

[0112] Nature.1952,169:631.

[0113] 4.Orian G.Occurrence of Puccinia polysora Underwood in the IndianOcean Area[J].Nature,1954,173:505-505.

[0114] 5. Duan Dingren, He Hongzhen. Multiple piles of Puccinia on corn in Hainan Island[J]. Acta Mycologica Sinica, 1984(02):125-126.

[0115] 6. Lu Weiting, Yu Huan, Cao Shengnan, Chen Changqing. Effects 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.

[0116] 7. Wang Xiaoming. Lecture series on corn pests and diseases (III): identification and investigation technology of corn resistance to pests and diseases. Crop Journal, 2005(06):53-55.

[0117] 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.

[0118] 9.Doerge RW, Churchill GA.Permutation tests for multiple loci affecting a quantitative character[J].Genetics.1996,142(1):285-294.

Claims

1. A functional marker of a maize southern rust resistance gene, the gene is ZmWRKY66 located at the southern rust resistance major QTL site RppQ6.01 on maize chromosome 6, and the functional markers include CACTA-L and CACTA-R The primers for each functional marker are as follows: The forward and reverse primer sequences of CACTA-L are SEQ ID NOs. 1 and 2, respectively; The forward primer and reverse primer sequences of CACTA-R are SEQ ID NOs. 4 and 5, respectively.

2. The molecular marker according to claim 1, characterized in that Using the primers shown in SEQ ID NO.1 and SEQ ID NO.2, no band can be amplified in the maize inbred line Qi 319 with high resistance to southern rust; a band of 1107 bp can be amplified in the maize inbred line Ye 478 with high sensitivity to southern rust, and the nucleotide sequence is shown in SEQ ID NO.3; No band could be amplified using the primers shown in SEQ ID NO.4 and SEQ ID NO.5 in the maize inbred line Qi 319 with high resistance to southern rust; a band of 995 bp could be amplified in the inbred line Ye 478 with high sensitivity to southern rust, and the nucleotide sequence was as shown in SEQ ID NO.

6.

3. Use of the functional marker according to claim 1 or 2 in identifying the major QTL locus RppQ6.01 for maize resistance to southern rust.

4. Use of the functional marker according to claim 1 or 2 in screening or identifying maize germplasm resources resistant to southern rust.

5. The use according to claim 4, characterized in that: The steps include: 1) Extracting genomic DNA from the plant to be tested; 2) using the genomic DNA of the plant to be tested as a template and primers for amplifying the molecular marker to perform a PCR amplification reaction; 3) Detecting 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 NOs. 1 and 2, respectively; The forward primer and reverse primer sequences of CACTA-R are SEQ ID NOs. 4 and 5, respectively.

6. The use according to claim 5, characterized in that: In step 3), 1% agarose gel electrophoresis was used to detect the PCR amplification products labeled CACTA-L and CACTA-R.

7. A kit for identifying or assisting in identifying the southern rust resistance trait of corn, characterized in that: The kit comprises primers for amplifying molecular markers CACTA-L and CACTA-R; wherein the primers for the molecular markers are as follows: the forward primer and reverse primer sequences of CACTA-L are SEQ ID NOs. 1 and 2, respectively; the forward primer and reverse primer sequences of CACTA-R are SEQ ID NOs. 4 and 5, respectively.

8. A method for cultivating corn germplasm resistant to southern rust, characterized in that: Here’s how: 1) Extracting genomic DNA from the plant 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, and the resulting PCR products were subjected to electrophoresis, and the band pattern of the electrophoresis band was compared with the reference band pattern; 3) The reference band pattern is the band pattern obtained by electrophoresis using genomic DNA of the highly southern rust-resistant corn inbred line Qi 319 as a template, 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 for PCR amplification; 4) Evaluate the plants to be tested, and select plants with the same or more than 80% similar band type to the reference band type as parents for breeding.

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