A SNP molecular marker combination related to corn ear rot resistance, gene chip and its application

Abstract

The present invention provides a SNP molecular marker combination related to corn ear rot resistance, a gene chip and its application, relating to the field of plant molecular breeding technology, including 18 SNP molecular markers, which are shown as SNP1 to SNP18. The present invention reveals the genetic variation characteristics of corn ear rot from the haplotype level. The developed SNP molecular marker combination can be used for the accurate identification of ear rot genotypes of corn germplasm resources and provides a new tool for molecular marker-assisted selection breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular breeding, and in particular to a SNP molecular marker combination related to corn ear rot resistance, a gene chip and applications thereof. Background Art

[0002] Corn ear rot is a fungal disease that can reduce corn yield by 10%-20%. Furthermore, rotting corn ears contain a range of mycotoxins, including aflatoxins, deoxynivalenol, nivalenol, zearalenone, and fumonisins. Breeding corn varieties with disease-resistant genes to effectively reduce the incidence of the disease has become a key research focus to ensure corn yields and improve food safety and economic value.

[0003] However, the traditional phenotypic evaluation in the current identification of corn ear rot resistance is susceptible to environmental interference, has a long cycle, and the single-molecule marker detection system may cause false negatives due to primer failure or allele loss, thus affecting its identification accuracy and efficiency.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a SNP molecular marker combination related to corn ear rot resistance, accurately locate resistance-associated sites through whole-genome association analysis, and then develop a multi-locus collaborative SNP molecular marker combination based on linkage disequilibrium, significantly improve the identification accuracy of functional sites of micro-effect genes, and ultimately provide molecular tool support for scalable application for genomic design and breeding of corn resistant to ear rot.

[0006] A second object of the present invention is to provide a product for identifying or assisting in identifying corn ear rot resistance.

[0007] A third object of the present invention is to provide the use of the above-mentioned SNP molecular marker combination or the above-mentioned product in identifying or assisting in identifying corn ear rot resistance.

[0008] A fourth object of the present invention is to provide a method for identifying or assisting in identifying corn ear rot resistance.

[0009] A fifth object of the present invention is to provide application of the above method in corn breeding.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0011] In a first aspect, the present invention provides a SNP molecular marker combination associated with corn ear rot resistance, wherein the SNP molecular marker combination includes 18 SNP molecular markers, wherein the 18 SNP molecular markers are shown as SNP1 to SNP18; the physical location information of SNP1 to SNP18 is as follows:

[0012] The genomic position of SNP1 is Chr5: 151557168 bp;

[0013] The genomic position of SNP2 is Chr5: 151559871 bp;

[0014] The genomic position of SNP3 is Chr5: 151572304 bp;

[0015] The genomic position of SNP4 is Chr5: 151662991 bp;

[0016] The genomic position of SNP5 is Chr5: 151663004 bp;

[0017] The genomic position of SNP6 is Chr5: 151663059 bp;

[0018] The genomic position of SNP7 is Chr5: 151663067 bp;

[0019] The genomic position of SNP8 is Chr5: 151663110 bp;

[0020] The genomic position of SNP9 is Chr5: 151663706 bp;

[0021] The genomic position of SNP10 is Chr5: 151663867 bp;

[0022] The genomic position of SNP11 is Chr5: 151664144 bp;

[0023] The genomic position of SNP12 is Chr5: 151664608 bp;

[0024] The genomic position of SNP13 is Chr5: 151664744 bp;

[0025] The genomic position of SNP14 is Chr5: 151665600 bp;

[0026] The genomic position of SNP15 is Chr5: 151669601 bp;

[0027] The genomic position of SNP16 is Chr5: 151669704 bp;

[0028] The genomic position of SNP17 is Chr5: 151670968 bp;

[0029] The genomic position of SNP18 is Chr5: 151671184 bp;

[0030] The physical location information of the 18 SNP molecular markers was determined based on the whole genome sequence V4.0 of B73.

[0031] In a second aspect, the present invention provides a product for identifying or assisting in identifying corn ear rot resistance, comprising probes and / or primers for detecting the above-mentioned SNP molecular marker combination.

[0032] Furthermore, the product is a gene chip; the gene chip is a solid phase chip.

[0033] Furthermore, the product is a test kit.

[0034] In a third aspect, the present invention provides the use of the above-mentioned SNP molecular marker combination or the above-mentioned product in identifying or assisting in identifying corn ear rot resistance.

[0035] Furthermore, the identification of corn ear rot resistance includes genome-wide association analysis of corn ear rot resistance.

[0036] In a fourth aspect, the present invention provides a method for identifying or assisting in identifying corn ear rot resistance, comprising detecting the genotype of the above-mentioned SNP molecular marker combination in a corn sample to be tested, and identifying or assisting in identifying corn ear rot resistance based on the genotype of the corn sample to be tested;

[0037] When the haplotype sequence composed of SNP1 to SNP18 is as shown in SEQ ID NO. 1, the corn has an ear rot resistance phenotype.

[0038] Furthermore, the corn is an inbred line of corn.

[0039] In a fifth aspect, the present invention provides application of the above method in corn breeding.

[0040] Furthermore, the corn breeding includes breeding for resistance to corn ear rot.

[0041] The present invention provides a SNP molecular marker combination related to corn ear rot resistance, which reveals the genetic variation characteristics of corn ear rot at the haplotype level. The developed SNP molecular marker combination can be used for accurate identification of ear rot genotypes of corn germplasm resources and provide a new tool for molecular marker-assisted selection breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 This is an association analysis diagram of the mixed linear model analysis markers and corn ear rot resistance provided in Example 1 of the present invention;

[0044] Figure 2 The candidate gene expression profile provided in Example 2 of the present invention;

[0045] Figure 3 This is the haplotype typing result of 18 SNP sites provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0046] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0047] Generally, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein and its technology are those well-known and commonly used in this area.Unless otherwise indicated, the methods and techniques of the present invention are generally according to those well-known in the art, and are carried out as described in various general and more specific references, which are cited and discussed throughout this specification.Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in this area, or as described herein.The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein and its laboratory procedures and technology are those well-known and commonly used in this area.

[0048] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] Based on the information of 35,985 SNP sites and ear rot phenotypic data in the parents of 484 important corn varieties, the present invention carried out genome-wide association analysis, obtained SNP markers significantly associated with ear rot, combined with transcriptome data to predict functional genes, and then screened out a group of haplotype markers tightly linked to corn ear rot-associated sites through linkage disequilibrium analysis. This group of haplotype molecular markers can be used for the identification and improvement of corn ear rot.

[0050] Based on this, the present invention provides a SNP molecular marker combination associated with corn ear rot resistance, wherein the SNP molecular marker combination includes 18 SNP molecular markers, wherein the 18 SNP molecular markers are shown as SNP1 to SNP18; the physical location information of SNP1 to SNP18 is as follows:

[0051] The genomic position of SNP1 is Chr5: 151557168 bp;

[0052] The genomic position of SNP2 is Chr5: 151559871 bp;

[0053] The genomic position of SNP3 is Chr5: 151572304 bp;

[0054] The genomic position of SNP4 is Chr5: 151662991 bp;

[0055] The genomic position of SNP5 is Chr5: 151663004 bp;

[0056] The genomic position of SNP6 is Chr5: 151663059 bp;

[0057] The genomic position of SNP7 is Chr5: 151663067 bp;

[0058] The genomic position of SNP8 is Chr5: 151663110 bp;

[0059] The genomic position of SNP9 is Chr5: 151663706 bp;

[0060] The genomic position of SNP10 is Chr5: 151663867 bp;

[0061] The genomic position of SNP11 is Chr5: 151664144 bp;

[0062] The genomic position of SNP12 is Chr5: 151664608 bp;

[0063] The genomic position of SNP13 is Chr5: 151664744 bp;

[0064] The genomic position of SNP14 is Chr5: 151665600 bp;

[0065] The genomic position of SNP15 is Chr5: 151669601 bp;

[0066] The genomic position of SNP16 is Chr5: 151669704 bp;

[0067] The genomic position of SNP17 is Chr5: 151670968 bp;

[0068] The genomic position of SNP18 is Chr5: 151671184 bp;

[0069] The physical location information of the 18 SNP molecular markers was determined based on the whole genome sequence V4.0 of B73.

[0070] The present invention reveals the genetic variation characteristics of corn ear rot at the haplotype level. The developed SNP molecular marker combination can be used for accurate identification of ear rot genotypes of corn germplasm resources and provide a new tool for molecular marker-assisted selection breeding.

[0071] According to another aspect of the present invention, a product for identifying or assisting in identifying corn ear rot resistance is provided, comprising probes and / or primers for detecting the above-mentioned SNP molecular marker combination.

[0072] Genotyping of related populations using solid-phase microarrays is less expensive than resequencing.

[0073] In some specific embodiments, the product is a gene chip; the gene chip is a solid phase chip.

[0074] In some specific embodiments, the product is a kit.

[0075] According to another aspect of the present invention, there is also provided the use of the above-mentioned SNP molecular marker combination or the above-mentioned product in identifying or assisting in identifying corn ear rot resistance.

[0076] In some specific embodiments, the identifying corn ear rot resistance comprises genome-wide association analysis of corn ear rot resistance.

[0077] According to another aspect of the present invention, a method for identifying or assisting in identifying corn ear rot resistance is also provided, comprising detecting the genotype of the above-mentioned SNP molecular marker combination in a corn sample to be tested, and identifying or assisting in identifying corn ear rot resistance based on the genotype of the corn sample to be tested;

[0078] When the haplotype sequence composed of SNP1 to SNP18 is as shown in SEQ ID NO. 1, the corn has an ear rot resistance phenotype.

[0079] Identifying the genotype of corn ear rot-related sites to judge the situation of corn ear rot is more efficient and direct. At the same time, the haplotype markers developed by the present invention for identifying ear rot resistance can be used for further development of corn chip functional sites to lay the foundation for breeding varieties with multiple excellent site aggregations.

[0080] In some specific embodiments, the corn is an inbred line of corn.

[0081] According to another aspect of the present invention, there is also provided application of the above method in corn breeding.

[0082] In some specific embodiments, the corn breeding includes breeding for resistance to corn ear rot.

[0083] The research materials of the present invention are parental inbred lines of major domestic varieties, and the studied genes / locus can be directly applied to variety screening and improvement.

[0084] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0085] Example 1 Genome-wide association analysis of corn ear rot and verification of the chr5_151663110 locus

[0086] 1. Phenotype acquisition

[0087] In 2024, 484 core inbred lines from various corn-producing regions across China were planted in Fengcheng, Liaoning Province, during the mid-season corn-producing region of East North China. The planting density was 4,500 plants per mu (approximately 15 plants per inbred line), with three biological replicates per row. A completely randomized block design was used. After the plants developed natural disease, the ear rot disease grade of the 1,452 rows of corn inbred lines was assessed. Eight uniformly distributed plants were surveyed per row. The Xianyu 335 parent, PH6WC, was planted every ten rows to correct for inter-plot variability.

[0088] 2. Genotype identification

[0089] Leaves of 484 maize inbred lines were sampled, and DNA was extracted using the alkaline lysis method. The high-density gene chip Maize 50K (50K SNP) was used to identify the genotypes of the 484 inbred lines mentioned above according to the standard Illumina Infinium gene chip detection process, and 35,985 SNP markers covering the entire genome were obtained.

[0090] 3. Correlation Analysis

[0091] Using TASSEL 5.0 software, we set the minimum allele frequency to 0.05 for quality control. Principal component analysis (PCA) and a kinship matrix were used to obtain covariate data for genome-wide association studies (GWAS). First, we used the PCA results as the population structure for general linear model analysis. The QQ plot (quantile-quantile plot) showed that the points starting at 0 were consistently above the diagonal, indicating false positives and suggesting that the general linear model was not appropriate. We then attempted to use a mixed linear model (MLM) to analyze the association between markers and corn ear rot disease grade:

[0092] y=Xβ+Qυ+Zμ+ε,

[0093] Where y is the phenotypic observation, X is the SNP genotype, β is the SNP effect, Q is the population structure matrix, υ is the population structure effect, Z is the kinship matrix, μ is the random effect, and ε is the residual.

[0094] like Figure 1 As shown, the Manhattan plot (left) and the QQ plot (right), where the left figure shows a scatter plot of the association between ear rot and SNP sites in the GWAS output results of ear rot disease grade (ER, Ear Rot) using MLM (the horizontal axis is the physical position of the SNP site on chromosomes 1-10, and the vertical axis is the statistical significance intensity, the specific value is -log10 (p-value). The larger the value, the smaller the p-value, indicating that the SNP site is more likely to be associated with ear rot. The right figure is a QQ plot. The points starting from 0 are basically on the diagonal line. After being greater than 3, the points begin to deviate from the straight line and rise, indicating that both false positives and false negatives are well controlled, indicating that the MLM model is suitable.

[0095] 4. Functional gene prediction

[0096] According to the results of the mixed linear model, the SNP at position 151663110 on chromosome 5 was significantly associated with ear rot (p-value = 3.61×10 -5 Based on the reference genome sequence of the inbred line B73, two genes Zm00001d016235 and Zm00001d016236 that may be associated with ear rot were found within 100 kb upstream and downstream of the SNP. According to published RNA-seq data, Zm00001d016235 is not expressed under normal conditions, while Zm00001d016236 is expressed in the stems, leaves, embryos and pollen of the plant. The expression profiles of the candidate genes are shown in Figure 2. Figure 2The former has no functional annotation, while the latter is annotated as encoding galacturonosyltransferase, a key enzyme in the pectin biosynthesis pathway. In maize, it participates in the synthesis of cell wall pectin polysaccharides, which may affect the cell wall's ability to resist pathogen infection and is likely related to ear rot resistance.

[0097] 5. Site Verification

[0098] Based on the typing results of the SNP at position 151663110 on chromosome 5, the 484 inbred lines were divided into two categories. As shown in Table 1, the ear rot disease grade of the two inbred lines was significantly different (p-value = 8.31 × 10 -5 ), indicating that this locus is associated with ear rot. The genotype identification of this locus can be used to judge and improve the ear rot of inbred lines to a certain extent.

[0099] Table 1 Results of t-test for SNP_151663110 typing

[0100]

[0101] Example 2 Development of haplotype markers for the ear rot resistance associated loci of Example 1

[0102] (1) The SNP genotype information of the 1 Mb region upstream and downstream of the chr5_151663110 locus of 540 maize inbred lines from the maize association population was downloaded from the Maizego website (http: / / www.maizego.org / ), totaling 4878 SNP variant sites.

[0103] (2) After saving the 4878 SNP gene information from the 540 inbred lines as hapmap format files, the linkage disequilibrium analysis was performed using the TASSEL5 software to calculate the correlation coefficient r with the chr5_151663110 locus. 2 (The value is between 0 and 1, indicating the degree of linkage between the two SNPs. When it is equal to 1, it means that the two SNPs are completely linked) SNP sites with a value of 1 = 1, as shown in Table 2, a total of 17 SNP sites that are completely linked to this site were obtained.

[0104] Table 2

[0105]

[0106] 3) The genotypes of the above 18 SNP sites (including SNP_151663110) in 540 inbred lines were extracted and haplotype analysis was performed using Haploview software to obtain two haplotypes, Hap1 and Hap2 ( Figure 3), where Hap2 is closely linked to the corn ear rot-associated locus (chr5_151663110) and the ear rot resistance genotype (G). The SNP locus information is as follows:

[0107] The genomic position of SNP1 is Chr5: 151557168 bp;

[0108] The genomic position of SNP2 is Chr5: 151559871 bp;

[0109] The genomic position of SNP3 is Chr5: 151572304 bp;

[0110] The genomic position of SNP4 is Chr5: 151662991 bp;

[0111] The genomic position of SNP5 is Chr5: 151663004 bp;

[0112] The genomic position of SNP6 is Chr5: 151663059 bp;

[0113] The genomic position of SNP7 is Chr5: 151663067 bp;

[0114] The genomic position of SNP8 is Chr5: 151663110 bp;

[0115] The genomic position of SNP9 is Chr5: 151663706 bp;

[0116] The genomic position of SNP10 is Chr5: 151663867 bp;

[0117] The genomic position of SNP11 is Chr5: 151664144 bp;

[0118] The genomic position of SNP12 is Chr5: 151664608 bp;

[0119] The genomic position of SNP13 is Chr5: 151664744 bp;

[0120] The genomic position of SNP14 is Chr5: 151665600 bp;

[0121] The genomic position of SNP15 is Chr5: 151669601 bp;

[0122] The genomic position of SNP16 is Chr5: 151669704 bp;

[0123] The genomic position of SNP17 is Chr5: 151670968 bp;

[0124] The genomic location of SNP18 is Chr5: 151671184 bp

[0125] When the haplotype composed of the above SNP markers in ascending order based on position is GGGACCTGGGCGCCCAGC (SEQID NO.1), it is a functional genotype for ear rot resistance. The physical position of the SNP site is determined based on the whole genome sequence version 4.0 of B73.

[0126] Example 3 Verification of the haplotype markers of Example 2

[0127] In 2024, the same inbred lines were planted in Xinxiang, Henan Province, during the mid-season in the Huanghuaihai corn-producing region. Three biological replicates were also set up in a completely randomized block design. After the plants became naturally diseased, the ear rot disease grade of the maize inbred lines was investigated and counted. Eight uniform plants were surveyed in each row, and the MY73 parent T1932 was planted every 10 rows to correct for differences between plots.

[0128] 2. Based on the haplotype markers of Example 2, the inbred lines were divided into two categories. As shown in Table 3, the ear rot disease grades of the two inbred lines were significantly different (p-value = 2.35 × 10 -3 ), indicating that the haplotype marker is associated with ear rot. The genotype identification of the haplotype marker can be used to judge and improve the ear rot of inbred lines to a certain extent.

[0129] Table 3 Haplotype t-test results

[0130]

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a reagent for detecting a combination of SNP molecular markers associated with corn ear rot resistance in identifying or assisting in identifying corn ear rot resistance, characterized in that: The SNP molecular marker combination consists of 18 SNP molecular markers, and the 18 SNP molecular markers are shown as SNP1 to SNP18; the physical location information of SNP1 to SNP18 is as follows: The genomic position of SNP1 is Chr5: 151557168 bp; The genomic position of SNP2 is Chr5: 151559871 bp; The genomic position of SNP3 is Chr5: 151572304 bp; The genomic position of SNP4 is Chr5: 151662991 bp; The genomic position of SNP5 is Chr5: 151663004 bp; The genomic position of SNP6 is Chr5: 151663059 bp; The genomic position of SNP7 is Chr5: 151663067 bp; The genomic position of SNP8 is Chr5: 151663110 bp; The genomic position of SNP9 is Chr5: 151663706 bp; The genomic position of SNP10 is Chr5: 151663867 bp; The genomic position of SNP11 is Chr5: 151664144 bp; The genomic position of SNP12 is Chr5: 151664608 bp; The genomic position of SNP13 is Chr5: 151664744 bp; The genomic position of SNP14 is Chr5: 151665600 bp; The genomic position of SNP15 is Chr5: 151669601 bp; The genomic position of SNP16 is Chr5: 151669704 bp; The genomic position of SNP17 is Chr5: 151670968 bp; The genomic position of SNP18 is Chr5: 151671184 bp; The physical location information of the 18 SNP molecular markers is determined based on the whole genome sequence V4.0 of B73. When the haplotype sequence composed of SNP1 to SNP18 in sequence is as shown in SEQ ID NO.1, the corn has an ear rot resistance phenotype.

2. A method for identifying or assisting in identifying resistance to corn ear rot, characterized in that: The method comprises detecting the genotype of the SNP molecular marker combination according to claim 1 in a corn sample to be tested, and identifying or assisting in identifying corn ear rot resistance according to the genotype of the corn sample to be tested; When the haplotype sequence composed of SNP1 to SNP18 is as shown in SEQ ID NO. 1, the corn has an ear rot resistance phenotype.

3. The method according to claim 2, characterized in that The corn is an inbred line of corn.

4. Application of the method according to claim 2 or 3 in corn breeding, characterized in that: The corn breeding is corn ear rot resistance breeding.

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