Corn susceptible gene, Indel marker for identifying disease-resistant haplotype of corn susceptible gene and application of Indel marker

Abstract

The invention provides a corn susceptible gene, an Indel marker for identifying a disease-resistant haplotype of the corn susceptible gene and application of the Indel marker, and belongs to the technical field of corn molecular breeding. The invention provides a corn susceptible gene ZmCIP6; the corn susceptible gene ZmCIP6 can reduce the resistance of corn plants to various leaf diseases of corn. The ZmCIP6 gene expression quantity or the ZmCIP6 protein content is reduced through gene editing, and the resistance of corn plants to various leaf diseases can be improved. The invention also provides the disease-resistant haplotype ZmCIP6-Hap1 of the maize susceptible gene ZmCIP6, and designs a primer pair for detecting the Indel molecular marker of the disease-resistant haplotype ZmCIP6-Hap1, so that materials with the disease-resistant haplotype in the existing maize varieties can be successfully detected. The invention provides technical support for corn molecular breeding to improve the resistance of corn plants to various leaf diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of corn molecular breeding, and in particular relates to a corn disease susceptibility gene and an Indel marker for identifying a disease resistance haplotype of the corn disease susceptibility gene and applications thereof. Background Art

[0002] Maize (Zeamays L.) is one of the most important crops for food, fuel and feed in the world. However, the yield and quality of maize are seriously threatened by diseases caused by pathogens. Therefore, breeding varieties with resistance to multiple maize diseases is an effective means and shortcut to solve the problem of maize being affected by multiple diseases. However, there are currently few genes in maize that have resistance to multiple diseases. Summary of the invention

[0003] The purpose of the present invention is to provide a corn disease susceptibility gene and an Indel marker for identifying the disease resistance haplotype of the corn disease susceptibility gene and applications thereof. The corn disease susceptibility gene ZmCIP6 of the present invention can reduce the resistance of corn to multiple diseases. By detecting the haplotype ZmCIP6-Hap1 with disease resistance advantage of the ZmCIP6 gene, the technical requirements for the breeding of corn varieties with resistance to multiple diseases can be met.

[0004] The present invention provides a maize disease susceptibility gene ZmCIP6, and the amino acid sequence of a disease susceptibility protein ZmCIP6 encoded by the maize disease susceptibility gene ZmCIP6 is shown in SEQ ID NO.1.

[0005] Preferably, the nucleotide sequence of the ZmCIP6 gene is shown as SEQ ID NO.2; the CDS nucleotide sequence of the maize susceptibility gene ZmCIP6 is shown as SEQ ID NO.3.

[0006] The present invention also provides the use of the maize disease susceptibility gene ZmCIP6 described in the above scheme for positively regulating the maize disease susceptibility gene in increasing maize disease sensitivity or constructing a disease-susceptible maize model.

[0007] The present invention also provides the use of the maize disease susceptibility gene ZmCIP6 described in the above scheme by negatively regulating the maize disease resistance and / or breeding of disease-resistant maize varieties.

[0008] The present invention also provides a disease-resistant haplotype ZmCIP6-Hap1 of the maize disease susceptibility gene ZmCIP6 described in the above scheme; the nucleotide sequence of the ZmCIP6-Hap1 is shown in SEQ ID NO.6.

[0009] The present invention also provides a primer pair for identifying the Indel molecular marker of the maize disease susceptibility gene disease resistance haplotype ZmCIP6-Hap1 described in the above scheme, comprising a first primer pair and / or a second primer pair; the first primer pair comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO.10 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.11; the second primer pair comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO.12 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.13.

[0010] The present invention also provides a kit comprising the primer pair described in the above scheme.

[0011] The present invention also provides the use of the disease-resistant haplotype ZmCIP6-Hap1 of the maize disease-susceptible gene ZmCIP6 described in the above scheme, the primer pair or the kit in molecular marker-assisted maize breeding.

[0012] Preferably, the application comprises one or more of identifying disease resistance of corn, improving disease resistance of corn and identifying corn materials with disease resistance.

[0013] The present invention also provides a method for identifying the haplotype of the maize disease susceptibility gene ZmCIP6, comprising the following steps: extracting genomic DNA of a maize material to be identified; using the genomic DNA of the maize material to be identified as a template, and using the first primer pair in the primer pair described in the above scheme to perform a first PCR amplification to obtain a first PCR amplification product; if the size of the first PCR amplification product is 356 bp, the maize disease susceptibility gene ZmCIP6 haplotype of the maize material is identified as ZmCIP6-Hap1; if the size of the first PCR amplification product is 490 bp, the maize material is identified as ZmCIP6-Hap1. The haplotype of the disease-susceptible gene ZmCIP6 is ZmCIP6-Hap2; alternatively, the genomic DNA of the corn material to be identified is used as a template, and a second PCR amplification is performed using the second primer pair in the primer pair described in the above scheme to obtain a second PCR amplification product; if the size of the second PCR amplification product is 480bp, the haplotype of the corn disease-susceptible gene ZmCIP6 of the corn material is identified as ZmCIP6-Hap1; if the size of the second PCR amplification product is 472bp, the haplotype of the corn disease-susceptible gene ZmCIP6 of the corn material is identified as ZmCIP6-Hap2.

[0014] The present invention provides a maize disease susceptibility gene ZmCIP6, and the amino acid sequence of the disease susceptibility protein ZmCIP6 encoded by the maize disease susceptibility gene ZmCIP6 is shown in SEQ ID NO.1. The maize disease susceptibility gene ZmCIP6 provided by the present invention can reduce the resistance of maize plants to various leaf diseases of maize. By reducing the expression amount of the ZmCIP6 gene or the ZmCIP6 protein content through gene editing, the resistance of maize plants to various leaf diseases of maize can be increased, and these leaf diseases include corn leaf blight, corn gray leaf spot and corn southern rust. At the same time, the present invention also provides a disease resistance haplotype ZmCIP6-Hap1 of the maize disease susceptibility gene ZmCIP6, and according to population variation data, a primer pair of Indel molecular markers for detecting the disease resistance haplotype ZmCIP6-Hap1 is designed, which can successfully detect materials with disease resistance haplotypes in existing maize varieties. The present invention provides technical support for maize molecular breeding to improve the resistance of maize plants to various leaf diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1 Schematic diagram for the construction of ZmCIP6 overexpression vector;

[0017] Figure 2 This is a diagram showing the results of DNA identification of the overexpression vector inserted into the ZmCIP6 transgenic material;

[0018] Figure 3 This is a diagram for identifying the expression level of CIP6-eYFP protein in ZmCIP6 transgenic materials;

[0019] Figure 4 The phenotype of ZmCIP6 transgenic material inoculated with corn leaf blight in the greenhouse; A is the phenotype picture of corn leaf blight, and B is the accumulation of the pathogen colonies of the transgenic positive material and negative material after inoculation;

[0020] Figure 5 The gray leaf spot and southern rust phenotypes of ZmCIP6 transgenic materials in the field; A is the gray leaf spot phenotype picture, B is the gray leaf spot disease level statistical analysis, C is the field southern rust phenotype picture, D is the southern rust disease level statistical analysis; statistical analysis was performed using t-test, *: p<0.05; **: p<0.01; ***: p<0.001;

[0021] Figure 6 The disease level statistics of corn leaf blight, corn gray leaf blight and corn southern rust of two haplotype materials in natural populations; A is the disease level statistics of corn leaf blight of two haplotype materials in natural populations; B is the disease level statistics of corn southern rust of two haplotype materials in natural populations; C is the disease level statistics of corn gray leaf blight of two haplotype materials in natural populations; t-test was used for statistical analysis, *: p<0.05; **: p<0.01; ***: p<0.001;

[0022] Figure 7 Schematic diagram of molecular markers for identifying two haplotypes; A is a schematic diagram of the marker1 primer pair CIP6-F1+CIP6-R1 amplifying two haplotypes, wherein the amplified size of ZmCIP6-Hap1 is 356bp, and the amplified size of ZmCIP6-Hap2 is 490bp; B is a schematic diagram of the marker2 primer pair CIP6-F2+CIP6-R2 amplifying two haplotypes, wherein the amplified size of ZmCIP6-Hap1 is 480bp, and the amplified size of ZmCIP6-Hap2 is 472bp;

[0023] Figure 8 Schematic diagram for the construction of Zmcip6-KO knockout vector;

[0024] Fig. 9 Schematic diagram of the knockout target of Zmcip6-KO; A is a schematic diagram of the locations of the two target sites targeting the ZmCIP6 gene; B is a schematic diagram of the knockout type sequencing of Zmcip6-KO1;

[0025] Fig.10 This is a gel image of Zmcip6-KO1 plant identification; A is a gel image of ZmCIP6 gene fragment amplification; B is a gel image of Cas9 gene fragment amplification;

[0026] Fig.11 This is the gray spot phenotype of the Zmcip6-KO1 knockout material in the field; A is the gray spot phenotype picture, and B is the statistical analysis of the gray spot disease level; the statistical analysis was performed using t-test, *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. DETAILED DESCRIPTION

[0027] The present invention provides a maize disease susceptibility gene ZmCIP6, wherein the amino acid sequence of the disease susceptibility protein ZmCIP6 encoded by the maize disease susceptibility gene ZmCIP6 is shown in SEQ ID NO.1, specifically:

[0028] MARKLRELLQKSGNRVCADCGAPDPKWASANIGVFICLKCSGVHRSLGTHVSKVLSVTLDQWTDDEINSMIEVGGNSYANAIYEAFLPEGYQKPHPDSSQEERADFIRSKYELQEFLKPSLRLVSSNKGSLEATSSRKHMDNSVSLSASFSSEAGMVEF IGILKVKVIRGTKLAVRDLMSSDPYVVLTLGQQKAKTSVSKRNLNPVWNEELKLSVPQHYGPLKLQVFDHDMLSKDDEMGDAEIDLQPMISAATAFGDPDLLADMQIGKWLRSPDNALARDSAVNVVGGKVKQEVSLRLQNVESGEVELELEWIPLNQ.

[0029] In the specific implementation of the present invention, the nucleotide sequence of the ZmCIP6 gene is shown in SEQ ID NO.2, which is located at positions 103104706 to 103107759 of chromosome 6 (chr6) of the reference genome, and the reference genome is: B73_V4 genome (B73 RefGen_v4). The nucleotide sequence shown in SEQ ID NO.2 is specifically:

[0030]

[0031] In the specific implementation of the present invention, the CDS nucleotide sequence of the maize susceptibility gene ZmCIP6 is shown in SEQ ID NO.3, specifically:

[0032] atggcgaggaagctgagagagttgctgcagaagagcggcaaccgcgtatgtgccgactgcggcgcacctgatcccaaatgggcatcggctaacattggagtattcatttgcctaaagtgttctggtgttcacagaagcctcgggacacatgtttcaaaggtgctgtcagtcacactagatcaatggacggatgatgagatcaactccatgatagaagttggtggaaactcttatgccaatgctatatatgaagcatttctgccagagggctatcagaagccgcatccagattctagccaagaagagagagctgatttcatcaggtcgaagtatgagttgcaagaattcctgaagccaagcctaaggttagtttccagcaataagggctctttagaggcgaccagctctcgaaagcatatggataattctgtgtctctttctgctagtttcagcagtgaggctggaatggttgagttcattggaatactaaaagttaaagtgataagagggactaaactagctgtaagagacctgatgagtagtgacccctatgttgtactgaccctaggacagcagaaagcaaaaacttctgtgagtaaacgtaatctgaatcctgtttggaatgaagagcttaagctgtcagttccccagcactacgggcctctgaaacttcaagtgttcgaccacgacatgctatcgaaggatgacgagatgggtgacgcagagattgacctgcagccgatgatcagtgcagctacagcgtttggcgaccctgatctgctcgcggacatgcagatcggcaagtggctcaggtccccagataatgcgctggcaagggacagcgccgtcaatgtcgttggtggcaaggtcaagcaggaggtctctctgaggctgcagaacgtggaatcgggggaggtggaactggagctggagtggataccactcaatcagtag。

[0033] In the present invention, the calcium ion-dependent lipid binding protein encoded by the maize disease susceptibility gene ZmCIP6 participates in the plant immune response.

[0034] The present invention also provides the use of the maize disease susceptibility gene ZmCIP6 described in the above scheme for positively regulating the maize disease susceptibility gene in increasing maize disease sensitivity or constructing a disease-susceptible maize model.

[0035] In the present invention, the diseased corn model includes a corn model susceptible to northern corn leaf blight, a corn model susceptible to southern corn rust or a corn model susceptible to gray corn leaf spot.

[0036] In the specific implementation of the present invention, the positive regulation includes overexpression. Overexpression of the maize disease susceptibility gene ZmCIP6 reduces the resistance of maize plants to diseases.

[0037] In one embodiment of the present invention, overexpression of the CDS sequence of the maize disease susceptibility gene ZmCIP6 reduces the resistance of maize plants to corn leaf blight, maize gray leaf spot and maize southern rust. In an embodiment of the present invention, the backbone plasmid of the recombinant plasmid overexpressing the CDS sequence of the maize disease susceptibility gene ZmCIP6 is pZZ01523-ZmUBIp-eYPF, and the insertion site is a SmaI restriction site. The recombinant plasmid was handed over to Weimi Biotechnology Co., Ltd. (hereinafter referred to as Weimi Company) and transformed into the maize inbred line KN5585 for overexpression.

[0038] The present invention also provides the use of the maize disease susceptibility gene ZmCIP6 described in the above scheme by negatively regulating the maize disease resistance and / or breeding of disease-resistant maize varieties.

[0039] In the specific implementation of the present invention, the disease resistance includes resistance to leaf diseases; the leaf diseases include one or more of corn leaf blight, corn southern rust and corn gray leaf spot.

[0040] In the specific implementation process of the present invention, the negative regulation includes gene editing of the corn disease susceptibility gene ZmCIP6 fragment, silencing the RNA expression of the corn disease susceptibility gene ZmCIP6 or reducing the ZmCIP6 protein or protein activity, thereby improving the resistance of corn plants to diseases. In one embodiment of the present invention, gene editing of ZmCIP6 using CRISPR / CAS9 technology can improve the resistance of plants to corn leaf blight, corn gray leaf spot and corn southern rust; the target sequence used to silence the RNA expression of the corn disease susceptibility gene ZmCIP6 or knock out the corn disease susceptibility gene ZmCIP6 is shown in SEQ ID NO.4 and SEQ ID NO.5; the nucleotide sequence described in SEQ ID NO.4 is specifically: gcagatatctggacatatta; the nucleotide sequence described in SEQ ID NO.5 is specifically: gctgtagctgcactgatcat.

[0041] The present invention also provides a disease-resistant haplotype ZmCIP6-Hap1 of the maize disease susceptibility gene ZmCIP6 described in the above scheme; the nucleotide sequence of the ZmCIP6-Hap1 is shown in SEQ ID NO.6, specifically:

[0042]

[0043] The genomic cDNA sequence of ZmCIP6-Hap1 is shown in SEQ ID NO.7, specifically:

[0044]

[0045] The CDS sequence of ZmCIP6-Hap1 is shown in SEQ ID NO.8, specifically:

[0046] atgacgaggaagctgagagagttgctgcagaagagcggcaaccgcgtgtgtgccgactgcggcgcacctgatcccaaatgggcatcggctaacattggagtattcatttgcctaaagtgttctggtgttcacagaagcctcgggacacatgtttcaaaggtgctgtcagtcacactagatcaatggacggatgatgagatcaactccatgatagaagttggtggaaactcttatgccaatgctatatatgaagcatttctgccagagggctatcagaagccgcatccagattctagccaagaagagagagctgatttcatcaggtcgaagtatgagttgcaagaattcctgaagccaagcctaaggttagtttccagcaataagggctctttagaggctaccagtaccagctctcgaaaacataaggacaatactgtgtctctttctgctagtttcagcagtgaggctggaatggttgagttcattggaatactaaaagttaaagtgataagagggactaagctagctgtaagagacctgatgagtagtgacccctacgttgtactgaccctaggacagcagaaagcaaaaacttctgtgagtaaacgtaatctgaatcctgtttggaatgaagagcttaagctgtcagttccccagcagtacgggcctctgaagcttcaagtgttcgaccatgacatgctatcgaaggatgacgagatgggcgacgcagagattgacctgcagccgatgatcagtgcagctacagcgtttggcgaccctgatctgctcgcggacatgcagatcggcaagtggctcaggtccccagataatgcgctggcaagggacagcgccgtcaatgtcgttggtggcaaggtcaagcaggaggtctctctgaggctgcagaacgtggaatcgggggaggtggaactggagctggagtggataccactcaatcagtag;

[0047] The amino acid sequence encoded by ZmCIP6-Hap1 is as shown in SEQ ID NO.9, specifically:

[0048] MTRKLRELLQKSGNRVCADCGAPDPKWASANIGVFICLKCSGVHRSLGTHVSKVLSVTLDQWTDDEINSMIEVGGNSYANAIYEAFLPEGYQKPHPDSSQEERADFIRSKYELQEFLKPSLRLVSSNKGSLEATSTSSRKHKDNTVSLSASFSSEAGMVEFIGILKVKVIRGTKLAVRDLMSSDPYVVLTLGQQKAKTSVSKRNLNPVWNEELKLSVPQQYGPLKLQVFDHDMLSKDDEMGDAEIDLQPMISAATAFGDPDLLADMQIGKWLRSPDNALARDSAVNVVGGKVKQEVSLRLQNVESGEVELELEWIPLNQ.

[0049] In the present invention, ZmCIP6-Hap1 is a disease-resistant haplotype, and the disease-resistant haplotype ZmCIP6-Hap1 of the present invention has certain disease-resistant advantages against corn leaf blight, corn southern rust and corn gray leaf blight. In the present invention, there are two main haplotypes in the interval from 2k upstream of ZmCIP6ATG to 2k downstream of the terminator, one is ZmCIP6-Hap1 represented by CIMBL11, and the other is ZmCIP6-Hap2 represented by B73. The two haplotypes have 3 Indel mutation sites, and the mutation site information is as follows: Indel1: located in the 3'UTR region of the ZmCIP6 gene. 134 bases are deleted at positions 103104845 to 103104978 of chromosome 6 based on the B73_V4 reference genome (B73RefGen_v4), and the deleted sequence is shown in SEQ ID NO.36 (DNA negative strand based on the B73_V4 reference genome). This sequence is deleted in the ZmCIP6-Hap1 material represented by CIMBL11; Indel2: the base G at position 103105967 of chromosome 6 based on the B73_V4 reference genome (B73 RefGen_v4) (DNA negative strand based on the B73_V4 reference genome) mutates to taccagt; Indel3: the base sequence gatttgatcagt at positions 103106095 to 103106106 of chromosome 6 based on the B73_V4 reference genome (B73 RefGen_v4) (SEQ ID NO.37) (DNA negative strand based on the B73_V4 reference genome) mutated to gactcgatttggtc (SEQ ID NO.38), with a size difference of 2bp.

[0050] Based on the above-mentioned Indel mutation site, a primer pair for detecting disease-resistant haplotypes as described in the following scheme was designed.

[0051] The present invention also provides a primer pair for identifying the Indel molecular marker of the maize disease susceptibility gene disease resistance haplotype ZmCIP6-Hap1 described in the above scheme, comprising a first primer pair and / or a second primer pair; the first primer pair comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO.10 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.11; the second primer pair comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO.12 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.13.

[0052] The Indel marker of the present invention can be applied to the disease-resistant molecular breeding of maize as an auxiliary molecular marker selection.

[0053] The present invention also provides a kit comprising the primer pair described in the above scheme.

[0054] The present invention also provides the use of the disease-resistant haplotype ZmCIP6-Hap1 of the maize disease-susceptible gene ZmCIP6 described in the above scheme, the primer pair or the kit in molecular marker-assisted maize breeding.

[0055] In the specific implementation process of the present invention, the application includes one or more of identifying corn disease resistance, improving corn disease resistance and identifying corn materials with disease resistance.

[0056] The present invention also provides a method for identifying the haplotype of maize disease susceptibility gene ZmCIP6, comprising the following steps:

[0057] Extracting genomic DNA of corn materials to be identified;

[0058] Using the genomic DNA of the corn material to be identified as a template, and using the first primer pair in the primer pair described in the above scheme to perform a first PCR amplification to obtain a first PCR amplification product;

[0059] If the size of the first PCR amplification product is 356 bp, the haplotype of the maize disease susceptibility gene ZmCIP6 of the maize material is identified as ZmCIP6-Hap1; if the size of the first PCR amplification product is 490 bp, the haplotype of the maize disease susceptibility gene ZmCIP6 of the maize material is identified as ZmCIP6-Hap2;

[0060] Alternatively, using the genomic DNA of the corn material to be identified as a template, a second PCR amplification is performed using the second primer pair in the primer pair described in the above scheme to obtain a second PCR amplification product;

[0061] If the size of the second PCR amplification product is 480 bp, the haplotype of the maize disease susceptibility gene ZmCIP6 of the maize material is identified as ZmCIP6-Hap1; if the size of the second PCR amplification product is 472 bp, the haplotype of the maize disease susceptibility gene ZmCIP6 of the maize material is identified as ZmCIP6-Hap2.

[0062] In the specific implementation process of the present invention, the PCR amplification system of the first PCR amplification or the second PCR amplification includes the following components in 20 μL: 100 ng DNA, 10 μL 2×Taq mix, 0.5 μL Seq-F, 0.5 μL Seq-R and the remainder ddH2O; the PCR amplification program of the first PCR amplification or the second PCR amplification is: 95°C, 3 min; 97°C, 15 s; 58°C, 30 s; 72°C, 45 s; 35 cycles; 72°C, 5 min; 25°C, 10 s; the detection instrument is Agilent 5200 fragment Analyzer.

[0063] To further illustrate the present invention, a maize susceptibility gene and an Indel marker for identifying the disease resistance haplotype of the maize susceptibility gene provided by the present invention and their applications are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1 Gene amplification and overexpression material construction of disease-susceptible gene ZmCIP6

[0065] In this example, a gene that reduces the resistance of corn plants to various leaf diseases was found, which was named ZmCIP6. In the corn inbred line B73, the nucleotide sequence of the ZmCIP6 gene is shown in SEQ ID NO.2. The CDS nucleotide sequence encoding the amino acid sequence of ZmCIP6 is shown in SEQ ID NO.3. The disease-susceptibility protein sequence encoded by the gene is shown in SEQ ID NO.1.

[0066] 1. Using the overexpression vector pZZ01523-ZmUBIp-eYPF as the backbone, the CDS sequence of ZmCIP6 was inserted between the UBI promoter and the eYFP tag by the Gibson method, and expressed by the UBI promoter ( Figure 1 );

[0067] 2. Extract total RNA from maize inbred line B73 and reverse transcribe to obtain cDNA. Design primers:

[0068] ZmCIP6-OE-F (SEQ ID NO. 14): gtcgacaaacgcactagtatcccgggatggcgaggaagctgagagag;

[0069] ZmCIP6-OE-R (SEQ ID NO. 15): tcctcgcccttgctcaccatcccgggctgattgagtggtatccactccag.

[0070] 3. Use the cDNA obtained by reverse transcription as a template for amplification to obtain the target fragment.

[0071] The PCR amplification system is as follows: Vazyme PhantaMax Super-Fidelity DNA polymerase is used for gene amplification to obtain PCR

[0072] The product, the reaction system is shown in Table 1 (total volume 50 μL).

[0073] Table 1 Reaction system

[0074] 2×PhantaBuffer 25μL dNTP 1μL ZmCIP6-OE-F 2μL ZmCIP6-OE-R 2μL Phantamax 1μL cDNA(B73) 1.5μL <![CDATA[ddH2O]]> 17.5μL

[0075] The reaction program was as follows: 95°C, 3 min; 95°C, 15 s; 58°C, 30 s; 72°C, 60 s; 72°C, 5 min; 35 cycles;

[0076] 4. pZZ01523-ZmUBIp-eYPF (published in reference: Gengshen C, Bao Z, Junqiang D, et al. Cloning Southern Corn Rust Resistant Gene RppK and Its Cognate Gene AvrRppK from Puccinia polysora [C] / / Proceedings of the 2023 Annual Academic Conference of the Chinese Society of Plant Pathology. 2023.) was digested with SmaI, and the amplified target fragment was inserted. The sequence of the inserted fragment is shown in SEQ ID NO.3. The overexpression vector pZZ01523-ZmUBIp-ZmCIP6-eYPF was obtained. It was handed over to Weimi Company and transformed into the corn inbred line KN5585 (provided by Weimi Company).

[0077] Example 2 Acquisition and identification of ZmCIP6 overexpressing plants

[0078] 1. PCR identification of T0 generation plants

[0079] The transgenic T0 generation transgenic plants were obtained from Weimi Company in Example 1.

[0080] PCR identification method: Take plant leaves and extract plant genomic DNA. Use ZmCIP6-OE-F1 and ZmCIP6-OE-R1 primer pairs for amplification. If a fragment of about 304 bp is obtained, the plant is considered to be a transgenic positive plant; if no amplification product is obtained, the plant is a negative plant.

[0081] ZmCIP6-OE-F1 (SEQ ID NO. 16): gtgcagctacagcgtttggc;

[0082] ZmCIP6-OE-R1 (SEQ ID NO. 17): ccggacacgctgaacttgtg.

[0083] The PCR system is 20 μL and includes the following components: DNA 100 ng, 2× Taq mix 10 μL, Seq-F 0.5 μL, Seq-R

[0084] 0.5 μL and the balance ddH2O; PCR amplification program: 95°C, 3 min; 97°C, 15 s; 58°C, 30 s; 72°C, 45 s; 35 cycles; 72°C, 5 min; 25°C, 10 s;

[0085] The two transgenic plants obtained were named ZmCIP6-OE1 and ZmCIP6-OE2. Figure 2 .

[0086] 2. Obtaining homozygous strains

[0087] The T0 generation plants are self-pollinated, the grains are harvested, and the grains are cultivated into plants, which are the T1 generation plants; transgenic plants are selected from the T1 generation plants, and the PCR identification method is the same as step 1; the T1 generation transgenic plants are self-pollinated, the grains are harvested, and the grains are cultivated into plants, which are the T2 generation plants; transgenic plants are selected from the T2 generation plants, and the PCR identification method is the same as step 1. If there is no separation, it means that the strain is homozygous.

[0088] Example 3 Acquisition and identification of Zmcip6-KO knockout plants

[0089] 1. Knockout vector construction

[0090] 1.1. Using the CRISPR knockout vector pCPB-ZmUbi-hspCas9 as the backbone (High-Throughput CRISPR / Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize. Plant Cell. 2020 May; 32(5):1397-1413.doi:10.1105 / tpc.19.00934.), the amplified U6 promoter and sgRNA were merged into the pU6-sgRNA1-pU6-sgRNA2 fragment by overlapping PCR. The pU6-sgRNA1-pU6-sgRNA2 fragment expression frame containing the specific target of ZmCIP6 was constructed on the HindIII-digested CPB-ZmUbi-hspCas9 vector by the Gibson method. The constructed knockout vector map is shown in the figure. Figure 8 shown.

[0091] 1.2. Design website according to CRISPR target ( http: / / crispr.hzau.edu.cn / CRISPR2 / ) ZmCIP6 Target1: gcagatatctggacatatta (SEQ ID NO.4), target2: gctgtagctgcactgatcat (SEQ ID NO.5). The target genomic location is as follows Fig. 9 shown.

[0092] 1.3. Amplification of U6-sgRNA expression cassette

[0093] According to the U6 promoter and sgRNA sequence, the U6 promoter and the sgRNA containing the target sequence were amplified, and the U6 and the sgRNA containing the target sequence were merged into a U6-sgRNA expression frame by overlapping, and then the two U6-sgRNA expression frames were synthesized into U6-sgRNA1-U6-sgRNA2 fragments by overlapping. The template is the SSV plasmid sequence in the literature (High-Throughput CRISPR / Cas9Mutagenesis Streamlines Trait Gene Identificationin Maize.Plant Cell.2020May;32(5):1397-1413.doi:10.1105 / tpc.19.00934.) Because the design is to knock out two targets, two U6-sgRNA expression frames need to be amplified.

[0094] 1.3.1.U6-sgRNA1 expression frame amplification:

[0095] 1.3.1.1PCR1:

[0096] U6-F1:tgcactgcacaagctgctgtttttgttagccccatcg (SEQ ID NO. 18);

[0097] U6-R1:ctatttctagctctaaaac taatatgtccagatatctgc aattcggtgcttgcggct(SEQ ID NO. 19).

[0098] The PCR amplification system is as follows: Vazyme PhantaMax Super-Fidelity DNA polymerase is used for gene amplification to obtain PCR products. The reaction system is shown in Table 1 (total volume 50 μL). The reaction program is as follows: 95°C, 3 min; 95°C, 15 s; 58°C, 30 s; 72°C, 60 s; 72°C, 5 min; 35 cycles;

[0099] 1.3.1.2.PCR2

[0100] sgRNA1-F1:agccgcaagcaccgaatt gcagatatctggacatatta gttttagagctagaaatag(SEQ ID NO.20);

[0101] sgRNA1-R1:caaaaacagcagcttaaaaaaagcaccgactcggtgccactttttc (SEQ ID NO. 21).

[0102] The PCR amplification system is as follows: Vazyme PhantaMax Super-Fidelity DNA polymerase is used for gene amplification to obtain PCR products. The reaction system is shown in Table 1 (total volume 50 μL). The reaction program is as follows: 95°C, 3 min; 95°C, 15 s; 58°C, 30 s; 72°C, 60 s; 72°C, 5 min; 35 cycles;

[0103] 1.3.1.3. The products of PCR1 and PCR2 were mixed in a ratio of 1:1 as templates for amplification of the U6-sgRNA1 expression frame; the primers were U6-F1 and sgRNA1-R1, and the PCR amplification system was: Vazyme PhantaMax Super-Fidelity DNA polymerase was used for gene amplification to obtain PCR products, and the reaction system was shown in Table 1 (total volume 50 μL). The reaction procedure was: 95°C, 3 min; 95°C, 15 s; 58°C, 30 s; 72°C, 60 s; 72°C, 5 min; 35 cycles;

[0104] 1.3.2.U6-sgRNA2 expression frame amplification:

[0105] 1.3.2.1PCR3:

[0106] U6-F2:gaaaaagtggcaccgagtcggtgctttttttaagctgctgtttttgttagcccca (SEQ IDNO.22);

[0107] U6-R2:ctatttctagctctaaaac atgatcagtgcagctacagc aattcggtgcttgcggct(SEQ ID NO. 23).

[0108] The PCR amplification system is as follows: Vazyme PhantaMax Super-Fidelity DNA polymerase is used for gene amplification to obtain PCR products. The reaction system is shown in Table 1 (total volume 50 μL). The reaction program is as follows: 95°C, 3 min; 95°C, 15 s; 58°C, 30 s; 72°C, 60 s; 72°C, 5 min; 35 cycles;

[0109] 1.3.2.2PCR4:

[0110] sgRNA2-F2:agccgcaagcaccgaatt gctgtagctgcactgatcat gttttagagctagaaatag(SEQ ID NO.24);

[0111] sgRNA2-R2: gtaaaacgacggccagtgccaagcttaaaaaaagcaccgactcggtgccac (SEQ ID NO. 25).

[0112] The PCR amplification system is as follows: Vazyme PhantaMax Super-Fidelity DNA polymerase is used for gene amplification to obtain PCR products. The reaction system is shown in Table 1 (total volume 50 μL). The reaction program is as follows: 95°C, 3 min; 95°C, 15 s; 58°C, 30 s; 72°C, 60 s; 72°C, 5 min; 35 cycles;

[0113] 1.3.2.3. The products of PCR3 and PCR4 were mixed in a ratio of 1:1 as templates for amplification of the U6-sgRNA2 expression frame; the primers were U6-F2 and sgRNA2-R2, and the PCR amplification system was: Vazyme PhantaMax Super-Fidelity DNA polymerase was used for gene amplification to obtain PCR products, and the reaction system was shown in Table 1 (total volume 50 μL). The reaction program was: 95°C, 3min; 95°C, 15s; 58°C, 30s; 72°C, 60s; 72°C, 5min; 35 cycles;

[0114] 1.4. Amplification of U6-sgRNA1-U6-sgRNA2 expression cassette fragment

[0115] The U6-sgRNA1 expression frame fragment obtained in 1.3.1.3 and the U6-sgRNA2 expression frame fragment obtained in 1.3.2.3 were mixed in a ratio of 1:1 as templates for amplification of the U6-sgRNA1-U6-sgRNA2 expression frame; the primers were U6-F1 and sgRNA2-R2, and the PCR amplification system was: Vazyme PhantaMax Super-Fidelity DNA polymerase was used for gene amplification to obtain PCR products, and the reaction system was shown in Table 1 (total volume 50 μL). The reaction program was: 95°C, 3min; 95°C, 15s; 58°C, 30s; 72°C, 60s; 72°C, 5min; 35 cycles;

[0116] 1.5. Carrier connection

[0117] pCPB-ZmUbi-hspCas9 was linearized by HindIII digestion, and the U6-sgRNA1-U6-sgRNA2 expression frame fragment was recovered. The linearized vector and fragment were mixed at a mass ratio of 1:3, 4 μl of T5 ligase mix was added, and the rest was supplemented with ddH2O to 20 μl. After 40 min of 37°C water bath, 20 μl was transferred to DH5α Escherichia coli competent cells, and the bacterial solution was spread on the kan + On the resistant LB plate, the positive single clone was picked to extract the plasmid and sent to Qingke Bio for sequencing. The sequencing analysis showed that the CRISPR knockout vector containing the U6-sgRNA1-U6-sgRNA2 expression frame was obtained. The vector map is shown in Figure 8 The plasmid was crossed with Weimi Company to transform the KN5585 background inbred line.

[0118] 2. Homozygous knockout and no Cas9 material obtained

[0119] 1) PCR identification method: Plant the T0 generation seeds provided by Weimi Company to obtain T1 generation plants, take plant leaves, and extract plant genomic DNA. Use knockout primers ZmCIP6-KO-F1 and ZmCIP6-KO-R1 for identification. If only a DNA fragment of 2348bp is obtained, the material without large fragment deletion is obtained, and sequencing is required to identify whether point mutations occur; if only a fragment of about 675bp is obtained, the material with homozygous large fragment deletion is obtained. If both sizes can be obtained, it is a heterozygous material.

[0120] 2) Cas9 gene detection: Use CAS9-F1 and CAS9-R1 primer pairs to detect the presence of Cas9 gene fragments in the above materials to determine whether transgenic elements exist. Use corn Actin gene detection primer pairs Actin-F1 and Actin-R1 to detect the quality of extracted DNA. If the Actin gene detection primer pair can amplify a DNA fragment of 452bp in size, it proves that the extracted DNA quality is good and can be used to detect the presence of Cas9 gene. If the Actin gene detection primer pair cannot amplify a DNA fragment of 452bp in size, it proves that the extracted DNA quality is poor and cannot be used to detect the presence of Cas9 gene. Based on the amplification of the Actin gene fragment, detect whether the Cas9 gene fragment exists. If a DNA fragment of 676bp in size is amplified, it proves that the plant contains transgenic elements. If a DNA fragment of 676bp in size cannot be amplified, it proves that the plant does not contain transgenic elements.

[0121] Actin-F1: ccaaatgggccctaagagaagttg (SEQ ID NO. 26); Actin-R1: aacgttgtcctcagtggggg (SEQ ID NO. 27); CAS9-F1: cggtgcttgttgtaggcgga (SEQ ID NO. 28); CAS9-R1: tgtgggataagggccgggat (SEQ ID NO. 27); NO.29); ZmCIP6-KO-F1: agcttctggtgctggtcttcttcc (SEQ ID NO.30); ZmCIP6-KO-R1: ctgcaggcacccggacaaactgaa (SEQ ID NO.31).

[0122] The PCR amplification system is: use Vazyme PhantaMax Super-Fidelity DNA polymerase for gene amplification to obtain PCR products. The reaction system is shown in Table 1 (total volume 50 μL). The reaction procedure is: 95°C, 3min; 95°C, 15s; 58°C, 30s; 72°C, 60s; 72°C, 5min; 35 cycles; According to the detection and sequencing results, a family with homozygous large fragment knockout and no Cas9 transgenic elements was obtained, named Zmcip6-KO1, and the wild type that was not knocked out isolated from the same family was used as the control Zmcip6-KO1-WT. The PCR identification results are shown in Fig.10 As shown in Figure 1, A is the gel image of ZmCIP6 gene fragment amplification, and it can be seen that the band of Zmcip6-KO1 plant is significantly smaller than that of wild type and KN5585; B is the amplification of Cas9 gene fragment, and it can be seen that Zmcip6-KO1 plant and its wild type and KN5585 plants do not contain Cas9 gene fragments. Fig. 9 As shown. The homozygous knockout and control materials obtained from the T1 generation transgenic plants were self-pollinated to obtain the T2 generation materials.

[0123] Example 4 Disease resistance test and identification of overexpression plants

[0124] The test plants were: T2 homozygous transgenic plants of ZmCIP6-OE1 plants and T2 homozygous transgenic plants of ZmCIP6-OE2 plants. The negative materials of T1 plants identified in step 6 of Example 2 were self-pollinated to obtain T2 negative materials as negative controls.

[0125] 1. Identification of protein expression levels in transgenic plants:

[0126] Total protein was extracted from the leaves of the tested plants, and maize Acitn protein was used as an internal reference. The accumulation level of ZmCIP6-eYFP protein was detected by western blot. Figure 3 .

[0127] The antibody used to detect ZmCIP6-eYFP protein is GFP mouse antibody; the antibody used to detect Actin protein is plant Actin mouse antibody.

[0128] 2. Identification of resistance to corn leaf blight

[0129] The identification of resistance to corn leaf blight was carried out in the laboratory of Huazhong Agricultural University.

[0130] The pathogen of the big spot disease is Exserohilum turcicum, which was obtained from field sampling in Yunnan.

[0131] Greenhouse conditions: 26°C, 14h light, 10h dark. The test plants were cultured to the 5-leaf and 1-heart stage. The pathogen of northern leaf spot was inoculated by foliar spraying. The spores of the pathogen of northern leaf spot were suspended in sterile water to obtain a spore concentration of 1×10 5 spore suspension of 100 ml / mL, add 8 μl of Tween-20 to every 100 ml of spore suspension. Then spray the spore suspension evenly on the leaves of the test plants to make the leaves completely soaked, then turn on the humidifier and keep the humidity above 90% for two days. Wait for the disease to occur in the infected parts of the corn, evaluate the degree of large spot disease on the leaves sprayed with spores and the accumulation of pathogens in the leaves to evaluate the resistance of the plants.

[0132] Detection of pathogen accumulation: Select leaves of the same size for each inoculated leaf, extract total RNA from the leaves and reverse transcribe to obtain cDNA. Use the corn Actin gene as an internal reference gene to detect the expression level of the pathogen Actin gene to indicate the accumulation of pathogens.

[0133] Primer pair for detecting the expression of Actin gene of pathogenic bacteria of northern leaf spot disease: qEtActin-F (SEQ ID NO.32): accccaagtccaaccgtgag; qEtActin-R (SEQ ID NO.33): cagaggcgtacagggacagg;

[0134] Primer pair used to detect the expression level of the maize gene ZmActin: qZmActin-F (SEQ ID NO.34): tacgagatgcctgatggtcaggtca; qZmActin-R (SEQ ID NO.35): tggagttgtacgtggcctcatggac.

[0135] Results Figure 4 Figure A shows the phenotype of the inoculated corn leaf blight pathogen, and Figure B shows the accumulation of corn leaf blight pathogen in the plant. The results show that compared with the negative material, the transgenic positive material can normally translate the ZmCIP6-eYFP protein, and the corresponding transgenic positive plant leaf blight area is higher than that of the negative material, and the accumulation of pathogens is also higher than that of the negative material. This shows that the resistance of the transgenic material to corn leaf blight is significantly reduced.

[0136] 3. Identification of resistance to southern corn rust and gray leaf spot

[0137] The identification of corn southern rust resistance was carried out at the transgenic experimental base of Huazhong Agricultural University in Nanbin Farm, Yazhou Bay, Sanya City, Hainan Province; the identification of corn gray leaf spot resistance was carried out at the transgenic experimental base in Wufeng Tujia Autonomous County, Yichang City, Hubei Province.

[0138] The pathogen of southern corn rust: Puccinia polysora Underw.; the pathogen of corn gray spot: Cercosporazeina Crous & U. Braun. The pathogen is naturally produced in the field.

[0139] The pathogens of southern corn rust and gray leaf spot are naturally occurring at the planting site. The adult stage is generally about 2 to 3 weeks after pollination to evaluate the disease status of individual plants in the field.

[0140] The disease rating standard is: 0 to 9. Level 0 means that the individual plant is not diseased; Level 1 means that the diseased part has not reached the leaves of the female ear of corn, and there are a few lesions on the leaves; Level 3 means that the diseased part has not reached the leaves of the female ear of corn, and there are a few lesions on the leaves; Level 5 means that the diseased part has reached the leaves of the female ear of corn, and there are a moderate number of lesions on the leaves; Level 7 means that the diseased part has reached the leaves of the female ear of corn, and there are a large number of lesions on the leaves; Level 9 means that the disease is spread all over the corn plant, and the leaves are basically covered with lesions. After the disease rating of the individual plants of the transgenic negative and positive materials was carried out, the t-test was used to test the resistance of the transgenic positive materials, with p < 0.05 as the threshold.

[0141] Results Figure 5 , A is a phenotype diagram of corn gray leaf spot in the field, B is a t-test test of the disease index of corn gray leaf spot in transgenic positive and negative materials. C is a phenotype diagram of corn southern rust in the field, and D is a t-test test of the disease index of corn southern rust in transgenic positive and negative materials.

[0142] The results showed that the disease index of maize southern rust and maize gray leaf spot in ZmCIP6 transgenic positive plants was higher than that in negative plants, indicating that the resistance of transgenic positive materials to maize southern rust and maize gray leaf spot was significantly reduced.

[0143] Example 5 Identification of the gray leaf spot resistance phenotype of Zmcip6-KO1 knockout material

[0144] The test plants are: Zmcip6-KO1 and its wild-type control material, T2 generation homozygous knockout Zmcip6-KO1 and its wild-type control material Zmcip6-KO1-WT obtained according to step 2 of Example 3.

[0145] 1. Identification of resistance to corn gray leaf spot

[0146] The identification of corn gray leaf spot resistance was carried out at the transgenic experimental base in Wufeng Tujia Autonomous County, Yichang City, Hubei Province.

[0147] Pathogen of corn gray spot disease: Corn Cercosporazeina (Cercosporazeina Crous & U.Braun) pathogen is naturally produced in the field.

[0148] The pathogen of corn gray spot disease originates from natural disease at the planting site. The adult stage is generally about 2 to 3 weeks after the pollination, and the disease status of individual plants in the field is rated.

[0149] The disease rating standard is: 0 to 9. Level 0 means no disease in individual plants; Level 1 means the diseased part has not reached the leaves at the female ear of corn, and there are a few lesions on the leaves; Level 3 means the diseased part has not reached the leaves at the female ear of corn, and there are a few lesions on the leaves; Level 5 means the diseased part has reached the leaves at the female ear of corn, and there are a moderate number of lesions on the leaves; Level 7 means the diseased part has reached the leaves at the female ear of corn, and there are a large number of lesions on the leaves; Level 9 means the disease is spread throughout the entire corn plant, and the leaves are basically covered with lesions. After rating the disease of individual plants for the homozygous knockout material Zmcip6-KO1 and the wild-type control material Zmcip6-KO1-WT, the t-test was used to judge the resistance of the transgenic positive material, with p<0.05 as the threshold. The results are as follows Fig.11 The results showed that the knockout material Zmcip6-KO1 was more resistant to corn gray leaf spot than the control material Zmcip6-KO1-WT, indicating that the knockout material had significantly increased resistance to corn gray leaf spot.

[0150] Example 6 Determination of the dominant haplotype of ZmCIP6

[0151] In order to determine the haplotype of gene ZmCIP6 in natural populations, this example is based on the published variation data of 523 related populations.

[0152] ( CNGBSequenceArchive(CNSA)FTPpublicservicehttps: / / ftp.cngb.org / pub / CNSA / data3 / CNP0001565 / zeamap / 02_Variants / ), the present embodiment analyzed and found that there are two main haplotypes in the interval from 2k upstream of ZmCIP6ATG to 2k downstream of the terminator, one is ZmCIP6-Hap1 represented by CIMBL11, and the other is ZmCIP6-Hap2 represented by B73. The difference information of the two haplotypes is as follows:

[0153] Indel1: Located in the 3'UTR region of the ZmCIP6 gene. A 134-base deletion is found at position 103104845-103104978 of chromosome 6 based on the B73_V4 reference genome (B73 RefGen_v4). The deletion sequence is gttgtagactatctttgcggttgtagaatcagtgtcggagcccgggcaccttgactggctcggcaactcggtacaatgatagtttgaggactcaatgacttgtta gtacaagtatgagactgttgtttgtatgc (DNA negative strand based on the B73_V4 reference genome, SEQ ID NO.36). This sequence is deleted in the ZmCIP6-Hap1 material represented by CIMBL11.

[0154] Indel2: The base G at position 103105967 on chromosome 6 (negative strand of DNA based on the B73_V4 reference genome) (B73 RefGen_v4) mutated to TACCAGT.

[0155] Indel3: The base sequence gatttgatcagt (SEQ ID NO.37) (DNA negative chain based on the B73_V4 reference genome) at 103106095-103106106 on chromosome 6 based on the B73_V4 reference genome (B73 RefGen_v4) mutated to gactcgatttggtc (SEQ ID NO.38), with a size difference of 2bp.

[0156] In order to analyze the differences between the two haplotypes in maize disease resistance, this example was based on the published disease resistance data of 523 natural populations ( Phenomics-a comprehensive databaseadaptedtothemaizemulti-omicsera. (cngb.org)https: / / db.cngb.org / zeamap / module / phenomics / ), combined with the ZmCIP6 haplotype typing of these natural populations for differential analysis; the analysis found that haplotype ZmCIP6-Hap1 was more resistant to disease than ZmCIP6-Hap2, such as Figure 6 As shown: A is a differential analysis of corn leaf blight; B is a differential analysis of corn southern rust; C is a differential analysis of corn gray leaf spot. In summary, the results show that this example found that the dominant haplotype ZmCIP6-Hap1 of ZmCIP6 is resistant to multiple corn diseases compared to the haplotype ZmCIP6-Hap2.

[0157] Example 7 Development of molecular markers for identifying the dominant haplotype of gene ZmCIP6

[0158] The analysis in Example 6 shows that the dominant haplotype ZmCIP6-Hap1 of the gene ZmCIP6 is significantly higher than ZmCIP6-Hap2 in terms of maize disease resistance, and is an important gene for breeding maize materials with resistance to multiple diseases. Therefore, the Indel variation was screened out according to the variation information analyzed in Example 6, and primers were designed according to the Indel differences as shown in Example 6 to detect the dominant haplotype of the gene ZmCIP6. Therefore, two markers for identifying this dominant haplotype were developed. The first marker (Marker1) was designed based on the Indel1 variation in the 3'UTR region of the ZmCIP6 gene, and its primers and extension sizes are as follows: the forward primer sequence is CIP6-F1 (SEQ ID NO.10): aaaagttgagggggagggtcc; the reverse primer sequence is CIP6-R1 (SEQ ID NO.11): gtgcagctacagcgtttggc; the amplification results are as shown in FIG. Figure 7 As shown in A, the amplified size of ZmCIP6-Hap1 is 356 bp; the amplified size of ZmCIP6-Hap2 is 490 bp.

[0159] The second marker (Marker2) was designed based on the Indel2 and Indel3 mutations in the ZmCIP6 gene. The primers and amplification size are as follows: the forward primer sequence is CIP6-F2 (SEQ ID NO.12): ctcactgctgaaactagcag; the reverse primer sequence is CIP6-R2 (SEQ ID NO.13): gagggctatcagaagccgca. The amplification results are shown in Figure 2. Figure 7 As shown in B, the amplified size of ZmCIP6-Hap1 is 480 bp; the amplified size of ZmCIP6-Hap2 is 472 bp.

[0160] The PCR amplification system reaction system was as follows (total volume 20 μL): DNA 100 ng, 2× Taq mix 10 μL, Seq-F 0.5 μL, Seq-R 0.5 μL and the balance ddH2O. The reaction program was: 95°C, 3 min; 97°C, 15 s; 58°C, 30 s; 72°C, 45 s; 35 cycles; 72°C, 5 min; 25°C, 10 s. The detection instrument was Agilent 5200 fragment analyzer.

[0161] If the results of Marker1 and / or Marker2 detection are ZmCIP6-Hap1, then this material is a material with the disease-resistant haplotype ZmCIP6-Hap1; if the results of both marker detections are ZmCIP6-Hap2, then this material is a material with the disease-susceptible haplotype ZmCIP6-Hap2.

[0162] The above results show that the present invention provides an effective detection kit for preliminary identification of corn materials with resistance to multiple diseases.

[0163] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A maize disease susceptibility gene ZmCIP6, characterized in that: The amino acid sequence of the disease susceptibility protein ZmCIP6 encoded by the maize disease susceptibility gene ZmCIP6 is shown in SEQ ID NO.

1.

2. The maize disease susceptibility gene ZmCIP6 according to claim 1, characterized in that: The nucleotide sequence of the ZmCIP6 gene is shown in SEQ ID NO.2; the CDS nucleotide sequence of the maize susceptibility gene ZmCIP6 is shown in SEQ ID NO.

3.

3. Use of the maize disease susceptibility gene ZmCIP6 described in claim 1 or 2 in positively regulating the maize disease susceptibility gene to increase maize disease susceptibility or constructing a disease-susceptible maize model.

4. Use of the maize disease susceptibility gene ZmCIP6 described in negative regulation claim 1 or 2 in improving maize disease resistance and / or breeding disease-resistant maize varieties.

5. The disease resistance haplotype ZmCIP6-Hap1 of the maize disease susceptibility gene ZmCIP6 according to claim 1 or 2; the nucleotide sequence of ZmCIP6-Hap1 is shown in SEQ ID NO.

6.

6. A primer pair for identifying the Indel molecular marker of the maize disease susceptibility gene disease resistance haplotype ZmCIP6-Hap1 according to claim 5, characterized in that: comprising a first primer pair and / or a second primer pair; The first primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO.10 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.11; The second primer pair includes a forward primer having a nucleotide sequence as shown in SEQ ID NO.12 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.

13.

7. A kit comprising the primer pair according to claim 6.

8. Use of the disease-resistant haplotype ZmCIP6-Hap1 of the maize disease susceptibility gene ZmCIP6 according to claim 5, the primer pair according to claim 6 or the kit according to claim 7 in molecular marker-assisted maize breeding.

9. The use according to claim 8, characterized in that: The application includes one or more of identifying corn disease resistance, improving corn disease resistance and identifying corn materials with disease resistance.

10. A method for identifying the haplotype of maize disease susceptibility gene ZmCIP6, characterized in that: The following steps are involved: Extracting genomic DNA of corn materials to be identified; Using the genomic DNA of the corn material to be identified as a template, and using the first primer pair in the primer pair of claim 6 to perform a first PCR amplification to obtain a first PCR amplification product; If the size of the first PCR amplification product is 356 bp, the haplotype of the maize disease susceptibility gene ZmCIP6 of the maize material is identified as ZmCIP6-Hap1; if the size of the first PCR amplification product is 490 bp, the haplotype of the maize disease susceptibility gene ZmCIP6 of the maize material is identified as ZmCIP6-Hap2; Alternatively, using the genomic DNA of the corn material to be identified as a template, a second PCR amplification is performed using the second primer pair in the primer pair of claim 6 to obtain a second PCR amplification product; If the size of the second PCR amplification product is 480 bp, the haplotype of the maize disease susceptibility gene ZmCIP6 of the maize material is identified as ZmCIP6-Hap1; if the size of the second PCR amplification product is 472 bp, the haplotype of the maize disease susceptibility gene ZmCIP6 of the maize material is identified as ZmCIP6-Hap2.

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