SNP molecular marker related to resistance to head smut of corn and application thereof

Abstract

The application discloses a SNP molecular marker related to corn head smut resistance and application thereof. The molecular marker is DNdCAPs837, which is located at the 837th site of a corn chromosome 4 ZmMYB42 Gene, and the mutation base is G / C. Using corn genome total DNA as a template, a dCAPS primer pair is used for PCR amplification, the amplification product is 224bp, the 27th base is a mutation site, the site of a susceptible material is C, and the site of a resistant material is G, which can be cut into two fragments of 197bp and 27bp by KpnI restriction endonuclease. Based on the detection result of the molecular marker, the head smut resistance of a plant can be identified. The molecular marker provided by the application can be used for breeding of a disease-resistant plant, and provides a new technical means and method basis for screening and creating a new material resistant to head smut, and has important value in the field of plant breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant breeding, in particular to a SNP molecular marker related to the resistance of corn smut and application thereof. BACKGROUND

[0002] Corn is an important food, feed and ethanol raw material crop, which has important significance for economic development. Corn smut mainly harms the tassel and ear of corn, and can cause absolute production once it occurs, which causes serious harm to corn yield. Therefore, it is of great significance to mine corn smut resistance related genes, and then screen and create new corn varieties with smut resistance to improve the resistance of corn smut.

[0003] Under stress, plants undergo a series of physiological changes. During evolution, plants have formed complex adaptive mechanisms at the levels of morphology, physiology and biochemistry, cells and molecules to protect themselves from harm. Among them, transcription factors such as MYB are involved in cell morphogenesis, stress signal perception and transduction, and hormone response and other physiological and biochemical processes. Such transcription factors bind to the downstream target gene promoter region cis-acting elements through DBD, regulate the expression of downstream target genes, and thus transduce the corresponding regulatory signals. There are few reports on MYB genes in corn smut resistance. SUMMARY

[0004] The purpose of the present application is to provide a SNP molecular marker related to the resistance of corn smut and application thereof.

[0005] The present application mines corn smut resistance related ZmMYB42 genes, analyzes the sequence variation of the coding region in different corn inbred lines, and then performs association analysis with the smut resistance of the inbred lines, thereby mining associated loci and developing molecular markers, providing a way and reference for breeding new corn varieties with smut resistance in the future, providing a basis for exploring the molecular regulation mechanism of corn smut resistance, and having practical significance for further improving corn yield and quality. In the research process, a SNP molecular marker related to the resistance of corn smut was obtained, which can be used for molecular marker assisted breeding of corn lines with smut resistance.

[0006] In order to achieve the purpose of the present application, in the first aspect, the present application provides a SNP molecular marker related to the resistance of corn smut, which contains a nucleotide sequence with polymorphism G / C at position 27 from 3'-5' end of the sequence of corn ZmMYB42 gene as shown in SEQ ID NO: 1; corn germplasm resources with polymorphism G have stronger smut resistance than corn germplasm resources with polymorphism C.

[0007] Further, the cornZmMYB42 The gene name of the gene in Maize GDB database (https: / / maizegdb.org / ) is Zm00001d053220 .

[0008] In a second aspect, the present application provides a primer pair for amplifying the SNP molecular marker, comprising an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3.

[0009] In a third aspect, the present application provides a detection reagent or kit containing the primer pair.

[0010] In a fourth aspect, the present application provides any of the following applications of the SNP molecular marker, the primer pair, or the detection reagent or kit:

[0011] 1) for identifying the smut resistance ability of a plant;

[0012] 2) for identifying, improving or marker-assisted breeding of smut resistance plant germplasm;

[0013] 3) for early prediction of smut resistance plant material.

[0014] Further, the plant is corn.

[0015] In a fifth aspect, the present application provides a method for identifying the smut resistance of corn, comprising: detecting the above-mentioned SNP molecular marker on the corn to be tested, and judging the smut resistance of the corn to be tested according to the detection result.

[0016] Further, the genomic DNA of the corn to be tested is extracted, and the primer pair as shown in SEQ ID NO: 2-3 or a detection reagent or kit containing the primer pair is used for PCR amplification, and the smut resistance of the corn to be tested is judged according to the amplification result.

[0017] Preferably, the PCR amplification reaction system is:

[0018]

[0019] The PCR amplification procedure is:

[0020]

[0021] Further, judging the smut resistance of the corn to be tested according to the amplification result comprises: performing KpnI restriction endonuclease enzyme digestion on the amplification product, and the corn to be tested with two characteristic bands of 197 bp and 27 bp in the enzyme digestion product has higher smut resistance than the corn to be tested with one characteristic band in the enzyme digestion product.

[0022] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0023] This invention is based on maize smut resistance-related genes. ZmMYB42 By analyzing different maize inbred lines ZmMYB42 Sequence variations in the CDS region of the gene were correlated with phenotypic indices of head smut resistance in inbred lines. Association sites were identified, restriction enzyme sites were analyzed, and restriction endonucleases were selected to develop a gene associated with head smut resistance in plants. ZmMYB42 The relevant SNP molecular markers, by detecting the polymorphism of these SNP molecular markers in plants, can be used to detect plant resistance to head smut. The SNP molecular markers provided by this invention can be applied to improve maize germplasm resources, breed head smut-resistant maize varieties, and increase maize yield and quality, which is of great value to the field of maize cultivation. Attached Figure Description

[0024] Figure 1 Provided for Embodiment 1 of the present invention ZmMYB42 Gene nucleotide diversity.

[0025] Figure 2 The results of genotyping of the molecular marker DNdCAPS837 provided in Example 2 of this invention are shown. M: Trans 2k, wells 1-9 are PCR products of Huangzaosi, Chang7-2, Dong237, Diangu11A, Dong46, Hai268, Liao3162, PH09B, and Dan6263, respectively; wells 10-15 are KpnI digestion products of Ji1037, Qi319, Shen5003, L237, KW5G321, and H1208, respectively.

[0026] Figure 3 The results of verifying the molecular marker DNdCAPS837 provided in Example 3 of this invention in some self-crossing lines are shown below. Note: M: Trans 2K; the remaining lanes are for the verification material DNA, and the materials that can be digested by restriction enzymes into 27bp+197bp are L237, Ji1037, PH4CV, KX, T4312, Shen5003, H1208, Qi205, Lv983, Jing388, KW5G321, Liao7990, Xin444, Ji818, Jia33, He604, Liao2345, and Liao3162. Detailed Implementation

[0027] This invention provides a molecular marker associated with the MYB gene, which is associated with maize resistance to head smut, and its application.

[0028] The present invention adopts the following technical solution:

[0029] In a first aspect, the present application provides a molecular marker related to the maize anti-smut gene MYB, which is a SNP molecular marker related to the maize smut resistance, and is located at the 837th base of the 4th chromosome of maize ZmMYB42 , and the mutation base is G / C, and the molecular marker is named as DNdCAPs837.

[0030] Further, the ZmMYB42 gene is named as Zm00001d053220 in the Maize GDB database.

[0031] Further, the molecular marker comprises a nucleic acid of the nucleotide sequence shown in SEQ ID NO:1, and a mutation exists at the 27th base from the 3'-5' end, and the mutation base is G / C (N is G or C).

[0032] Further, the molecular marker has stronger smut resistance in plants with G at the site than in plants with C at the site.

[0033] In a second aspect, the present application provides a primer pair, which comprises the nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:3. When the primer is designed, the 25th base in the sequence is replaced by T instead of C, so as to be consistent with the enzyme cutting site GGTAC / C of KpnI. After PCR amplification, the mutation site base is G in the disease-resistant material, which is consistent with the enzyme cutting site of the restriction enzyme KpnI, and two characteristic bands are generated after enzyme cutting. The mutation site base is C in the disease-susceptible material, and the restriction enzyme KpnI cannot recognize the enzyme cutting site, and the enzyme cutting product is only one characteristic band.

[0034] The upstream primer is 5'-CAACCTGGACCTCTGCATCAGC-3' (SEQ ID NO:2);

[0035] The downstream primer is 5'-TGGTCCTGAGCCCCAGGAAGTGGTA-3' (SEQ ID NO:3).

[0036] In a third aspect, the present application provides a kit comprising the molecular marker or the primer pair.

[0037] In a fourth aspect, the present application provides the use of the molecular marker or the primer pair or the kit in identifying the smut resistance of plants.

[0038] The present application further provides the use of the molecular marker or the primer pair or the kit in any one of the following:

[0039] (1) cultivating a transgenic plant resistant to smut;

[0040] (2) Molecular marker-assisted breeding of plants;

[0041] (3) Improve plant germplasm resources.

[0042] Furthermore, the plant in question is corn.

[0043] Fifthly, the present invention provides a method for identifying resistance to corn smut, the method comprising: detecting the polymorphism of the molecular marker in a plant sample to be tested, and determining the resistance of the plant sample to be tested to corn smut based on the detection results.

[0044] Furthermore, the method includes: extracting genomic DNA from the plant sample to be tested, performing PCR amplification using the primer pair or the kit, and determining the plant's resistance to head smut based on the amplification results.

[0045] Furthermore, the determination of the plant's resistance to head smut based on the amplification results includes: digesting the amplification product with KpnI restriction endonuclease to obtain two fragments, 197 bp and 27 bp, which indicate that the plant has higher resistance to head smut.

[0046] The following examples 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 examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0047] Example 1: Corn ZmMYB42 Gene sequence variation analysis

[0048] 1. ZmMYB42 The gene in the Maize GDB database is named Zm00001d053220 The genome is located on chromosome 4, with a total length of 898 bp, and its sequence is contained in sequence SEQ ID NO:4. Its CDS sequence is 783 bp long, encoding 260 amino acids. ZmMYB42 A gene consists of two exons and one intron.

[0049] This invention designed primers (Table 1) to amplify the DNA sequences of maize inbred lines with different resistance to head smut, and used Snap Gene software to analyze the results. ZmMYB42 The gene CDS region was extracted and analyzed using DNA SPv 6.0 software. ZmMYB42 Sequence variation analysis was performed on the CDS region of the gene, with a sliding box of 100 bp and a step size of 25 bp. The results showed that the maximum π value of nucleotide polymorphism in the sequence was 0.02191, within the range of 483-582 bp, indicating that this segment was rich in variation and had higher polymorphism than other exon regions.

[0050] Table 1 ZmMYB42 Gene sequence primer

[0051]

[0052] 2、 ZmMYB42 Gene haplotype analysis

[0053] The present application detects 16 SNPs in 124 inbred lines CDS regions of different resistance levels in the eastern North China, and the SNPs are located at positions 200, 251, 286, 294, 322, 464, 496, 505, 545, 552, 569, 609, 631, 697, 725, and 837, and the Indel sites are located at two positions of 271-276 and 711-713 (Table 2 and Table 3). According to the type and number of nucleotide variations, the polymorphic sites are typed, and 15 haplotypes are detected by using DNA SPv6.0 software, the haplotype polymorphism is 0.899, the main haplotypes are HAP1, HAP3, HAP4, HAP5, HAP8, HAP10, HAP14, accounting for 72.8% of the test materials, and the remaining haplotypes belong to a small amount of variation. Among them, HAP5 contains 32 test inbred lines, including 4 inbred lines resistant to smut, W9706, 7884-7Ht, 7884 and A4-227; The remaining 28 test materials include 6 inbred lines with moderate resistance to smut, K10, Jia28, M60, Shenyang 8078, HR0110 and DN-1-2; 17 inbred lines susceptible to smut including KWS49, Jia33 and D22; 5 inbred lines with high susceptibility, including 8902, Tie T0403, M502, Jing 724 and HD568. It is preliminarily considered that the HAP5 haplotype is an excellent haplotype (Table 4).

[0054] Table 2 ZmMYB42 Gene CDS region SNP position

[0055]

[0056] Table 3 ZmMYB42 Gene CDS region Indel position

[0057]

[0058] Table 4 ZmMYB42 Haplotype corresponding inbred line

[0059]

[0060] 3、 ZmMYB42 Gene amino acid level change analysis

[0061] This invention discovered in maize inbred lines with different resistance levels ZmMYB42 There is a non-synonymous mutation SNP in the CDS region of the gene. The mutation site is located at SNP837 (G>C), which causes an amino acid mutation in exon 2, which may change the structure and physiological function of the protein.

[0062] 4. ZmMYB42 Gene nucleotide diversity analysis

[0063] This invention uses DNA SPv 6.0 software to... Figure 1 Sequence variation analysis was performed on the CDS region of the gene, using a sliding frame of 100 bp and a step size of 25 bp. The results showed that the maximum π value of nucleotide polymorphism in the sequence was 0.02191, within the range of 483-582 bp, indicating that this segment was rich in variation and had higher polymorphism than other exon regions. ZmMYB42 ).

[0064] Example 2 ZmMYB42 Development of dCAPS functional markers

[0065] 1. ZmMYB42 Association analysis between candidate genes and resistance to maize inbred lines to head smut

[0066] This invention combines the results of head smut resistance identification of maize inbred lines and uses TASSEL 5.0 software to... ZmMYB42 SNPs in the gene's CDS region were associated with disease resistance phenotypes. The results are shown in Table 5. At the 0.05 level, SNP273, SNP276, SNP277, SNP402, SNP702, SNP703, SNP704, SNP716, SNP883, and SNP884 were significantly associated with disease resistance (P<0.05), while SNP837 was highly significantly associated with disease resistance (P<0.01), with the largest phenotypic contribution rate of 18.11%.

[0067] Table 5 ZmMYB42 Results of correlation analysis between gene sequence variation and resistance to maize inbred lines to head smut

[0068]

[0069] 2. ZmMYB42 Design of dCAPS functional markers

[0070] This invention is based on Figure 2The results of the genetic CDS region association analysis and the amino acid level change results, select the SNP837 (G / C) site which is extremely significantly related (P<0.01) to the corn inbred line smut resistance and has the largest phenotype contribution rate to develop the dCAPS marker. The optimal enzyme cutting site and restriction enzyme are analyzed by using the online website dCAPS Finder to develop the functional marker. The marker converted from the site SNP837 (G / C) is named as DNdCAPs837, the full-length of PCR amplification is 224bp, the 27th base is the mutation site, the site of the susceptible material is C, and the site of the resistant material is G, which can be cut into two fragments of 197bp and 27bp by KpnI restriction enzyme Figure 3 ).

[0071] Example 3 Verification of DNdCAPS837 functional marker

[0072] In the application, 254 corn inbred lines with different disease resistance levels are selected to verify the DNdCAPs837 marker, and the specific enzyme cutting results are shown in ZmMYB42 The genotype of the material which can be cut into 197 and 27bp bands by restriction enzyme is resistant, and the genotype which cannot be cut is susceptible. Among them, the materials which can be cut in the resistance identification are high resistance (HR) inbred lines Ji1037, Xin444, T4312, H1208 and KX, a total of 5 materials; 4 materials which can be cut in the resistance identification are resistant, respectively L237, KW5G321, Lia7990 and Lia3162; 5 materials which can be cut in the resistance identification are medium resistant, respectively PH4CV, Zi205, Lv983, Jing388 and Ji818; Jia33, He604 and Lia2345 reach the susceptible level in the resistance identification; Sh5003 shows high susceptibility in the resistance identification. In summary, 18 materials of the 254 inbred lines show the genotype of resistant smut, among which 14 families have the phenotype of medium resistant smut and above, the genotype and phenotype coincidence rate is 77.78%, and the DNdCAPs837 can effectively identify the SNP837 (G / C) site.

[0073] In summary, the application shows that the molecular marker DNdCAPs837 developed based on the corn ​ The molecular marker DNdCAPs837 developed based on the corn

[0074] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.

Claims

1. SNP molecular markers associated with resistance to maize head smut, characterized in that, The SNP molecular marker is the nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence shown in SEQ ID NO:1 has a G / C polymorphism at the 27th base from the 3′-5′ end.

2. A primer pair for amplifying the SNP molecular marker of claim 1, characterized in that, The primer pair consists of the upstream primer shown in SEQ ID NO:2 and the downstream primer shown in SEQ ID NO:

3.

3. A detection reagent or kit containing the primer pair described in claim 2.

4. Any of the following applications of the primer pair of claim 2 or the detection reagent or kit of claim 3: 1) Used to identify resistance to corn smut; 2) Used for identification of maize smut-resistant germplasm resources or for marker-assisted breeding; 3) Used for early prediction of resistance to corn smut; The SNP molecular marker is the nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence shown in SEQ ID NO:1 has a G / C polymorphism at the 27th base from the 3′-5′ end; maize with the SNP molecular marker polymorphism of G has stronger resistance to head smut than maize with polymorphism of C.

5. A method for identifying resistance to corn smut, characterized in that, The method includes: detecting the SNP molecular marker as described in claim 1 on the corn to be tested, and determining the corn's resistance to head smut based on the detection results; Maize with the SNP molecular marker polymorphism G exhibits stronger resistance to head smut compared to maize with the polymorphism C.

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