Maize molecular marker combination, liquid phase chip and application of corn molecular marker combination and liquid phase chip

Abstract

The invention discloses a corn molecular marker combination, a liquid phase chip and application of the corn molecular marker combination and the liquid phase chip. The corn molecular marker combination comprises 60 and 364 molecular markers, and the physical positions of the 60 and 364 molecular markers are determined by sequence alignment based on a corn reference genome B73RefGenv4. The corn molecular marker combination provided by the scheme of the invention can be widely applied to detection and application of different types of corn materials, the site detection rate is high, and the typing result is reliable.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a corn molecular marker combination, a liquid phase chip and applications thereof. Background Art

[0002] Corn is a widely planted crop and plays an important role in the crop industry. Traditional breeding, represented by hybrid breeding, has provided a large number of excellent varieties for a long time. However, since traditional breeding is basically empirical breeding and phenotypic selection, and has shortcomings such as long breeding cycle and low efficiency, its development potential can no longer meet the needs of the corn market.

[0003] In recent years, in the field of corn research, various molecular markers such as restriction fragment length polymorphism (RFLP), simple sequence repeat (SSR), random amplified polymorphic DNA marker (RAPD) and single nucleotide polymorphism (SNP) have been used to evaluate the genetic characteristics of populations or germplasm, locate quantitative trait loci (QTL), and promote the selection of breeding materials. Compared with other molecular markers, SNP has the characteristics of high density, high genetic stability and easy analysis; genome chip is a breeding tool based on high-throughput molecular marker technology, which can cover SNP sites of the entire genome, with the characteristics of wide coverage, high sensitivity, high throughput and high precision. SNP chip has the advantages of convenient detection, low price, high throughput and simple analysis, and is an important tool in the field of molecular breeding. The use of whole genome breeding chips to conduct a comprehensive evaluation of genotype analysis, genetic diversity and functional gene discovery of corn germplasm resources will help accelerate the discovery of excellent germplasm resources and the identification of new functional genes. There are many corn SNP chips on the market, such as MaizeSNP50 Bead Chip, MaizeSNP600K, Maize6H-60KSNP, Maize 55K, etc. They are all based on solid phase chip design, with high detection cost and low flexibility, making them difficult to use on a large scale in corn breeding.

[0004] With the rapid development of corn breeding and seed industry, the identification of varieties, germplasm resources and variety purity has become more difficult. Previously, a series of national standards and industry standards for corn variety identification and corn variety authenticity identification have been approved, such as "SSR Marker Method for Corn Variety Identification Technical Regulations" (NY / T 1432-2014), "SSR Molecular Marker Method for Corn Variety Purity Identification" (NY / T3750-2020), "SSR Molecular Marker Detection of Authenticity and Purity of Major Crop Varieties-Corn" (GB / T 39914-2021), "SNP Marker for Authenticity Identification of Corn Varieties" (NY / T4022-2021), "MNP Marker Method for Plant Variety Identification" (GB / T38551-2020), etc., which provide strong technical support and basis for maintaining corn variety innovation and variety purity identification. However, the marker types in these standards are not unified, and it is impossible to achieve unified detection data. Therefore, an efficient, accurate, universal and comprehensive method for corn variety, germplasm resources and variety purity identification is needed.

[0005] To overcome these problems, it is necessary to develop a high-density, high-throughput, high-accuracy, low-cost, multifunctional, and widely applicable liquid phase chip to cover sufficient genomic variations and ensure specific and broad applications at a reasonable cost. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a corn molecular marker combination.

[0007] The present invention also provides a primer set and / or probe for detecting the above-mentioned corn molecular marker combination.

[0008] The invention also provides a liquid phase chip.

[0009] The present invention also provides a kit.

[0010] The present invention also provides an application of the above-mentioned corn molecular marker combination, primer set and / or probe, liquid phase chip or kit.

[0011] The invention also provides a method for detecting the combination of corn molecular markers.

[0012] The invention also provides a corn breeding method.

[0013] According to one aspect of the present invention, a maize molecular marker combination is proposed, including 60,364 molecular markers, the physical positions of the 60,364 molecular markers are determined based on sequence alignment of the maize reference genome B73 RefGen_v4, and the site information is specifically shown in Table 1 below.

[0014] Table 1

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037] In some embodiments of the present invention, the 60,364 molecular markers consist of 51,734 molecular markers, 49 SSR molecular markers, and 8,581 molecular markers in 899 MNP intervals.

[0038] In some embodiments of the present invention, in Table 1, if there is a polymorphism of (A or T or G or C) number / , the content in the brackets is the SSR repeated base, and the number indicates the number of repetitions.

[0039] In some embodiments of the present invention, the number may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11...

[0040] In the second aspect of the present invention, a primer set and / or probe for detecting the above-mentioned maize molecular marker combination is proposed.

[0041] In some embodiments of the present invention, the length of the probe is 80-120 bp.

[0042] In some embodiments of the invention, the probe is about 100 bp in length.

[0043] In some embodiments of the present invention, the GC content of the probe is between 20% and 80%.

[0044] In the third aspect of the present invention, a liquid phase chip is proposed, wherein the liquid phase chip comprises the above-mentioned primer set and / or probe for detecting the maize molecular marker combination.

[0045] In a fourth aspect of the present invention, a kit is provided, wherein the kit comprises the above-mentioned primer set and / or probe for detecting the maize molecular marker combination.

[0046] In a fifth aspect of the present invention, an application of at least one of the above-mentioned maize molecular marker combination, liquid phase chip and kit is proposed, and the application is application in maize genotyping.

[0047] In some embodiments of the present invention, the application is application in gene localization in corn.

[0048] In some embodiments of the present invention, the application is application in the construction of corn fingerprint.

[0049] In some embodiments of the present invention, the application is an application in the identification of corn germplasm resources.

[0050] In some embodiments of the present invention, the application is application in corn genetic diversity assessment.

[0051] In some embodiments of the present invention, the application is application in corn variety purity identification.

[0052] In some embodiments of the present invention, the application is application in identification of maize kinship.

[0053] In some embodiments of the present invention, the application is application in corn EDV identification.

[0054] In some embodiments of the present invention, the application is application in authenticity identification of corn varieties.

[0055] In some embodiments of the present invention, the application is application in corn genetic map construction and QTL positioning.

[0056] In some embodiments of the present invention, the application is an application in whole genome association analysis of maize.

[0057] In some embodiments of the present invention, the application is application in molecular marker-assisted selection breeding of corn.

[0058] In some embodiments of the present invention, the application is application in targeted improvement of corn.

[0059] In some embodiments of the present invention, the application is application in corn multi-gene aggregation breeding.

[0060] In some embodiments of the present invention, the application is application in whole genome selection of maize.

[0061] In some embodiments of the present invention, the application is application in molecular design breeding of corn.

[0062] According to a sixth aspect of the present invention, a method for detecting a combination of molecular markers in maize is provided, comprising the following steps:

[0063] S1. Perform genotyping on the sample to be tested using at least one of a primer set and / or a probe, a liquid phase chip, and a kit to obtain a genotyping result;

[0064] S2. Analyze the genotyping results obtained in step S1.

[0065] In some embodiments of the present invention, the detection method is based on liquid phase probe capture sequencing typing technology. The present invention uses high-throughput sequencing technology to identify the genotype of corn materials, which has the advantages of high detection throughput, large amount of one-time output data, and can cover the detection of nearly a thousand materials at the same time. It is also applicable to mainstream second-generation sequencing platforms such as Illumina and MGI, and has wide platform adaptability.

[0066] In the seventh aspect of the present invention, a corn breeding method is proposed, comprising the following steps: using the above method to detect the DNA of the corn to be tested, and selecting corn for subsequent breeding.

[0067] The present invention has at least the following beneficial effects:

[0068] (1) The maize molecular marker combination of the present invention is based on 1,218 maize resequencing data with rich diversity at home and abroad from Hapmap3, and selects sites with strong representativeness, strong specificity, high polymorphism and good versatility. It has rich functional sites, covering 39 functional markers related to important traits such as maize stalk rot, southern rust and drought resistance; it includes 65 genes related to maize yield, disease resistance (southern rust, gray spot, stalk rot, ear rot), abiotic stress and other traits, a total of 271 sites; it contains GWAS sites related to important traits such as maize yield, flowering period, plant growth and development, a total of 625 sites. It can be used for the mining and utilization of important trait genes, molecular marker-assisted breeding and functional analysis. At the same time, the corn molecular marker combination of the present invention also includes molecular markers of multiple national or industry standards, such as "Technical Procedures for Corn Variety Identification SSR Marker Method" (NY / T 1432-2014), "Corn Variety Purity Identification SSR Molecular Marker Method" (NY / T 3750-2020), "Main Crop Variety Authenticity and Purity SSR Molecular Marker Detection Corn" (GB / T 39914-2021), "Corn Variety Authenticity Identification SNP Marker" (NY / T4022-2021) and "Corn in the MNP Marker Method for Plant Variety Identification" (GB / T38551-2020) and other national / industry standards; it contains EDV identification molecular markers and fingerprint identification molecular markers, which can be used for corn variety identification, variety purity identification, variety authenticity identification, EDV identification and kinship identification.

[0069] (2) The corn molecular marker combination (60,364 molecular markers in the 50K interval of corn) of the present invention can be widely used in the detection and application of different types of corn materials, with a high site detection rate and reliable typing results; the average detection rate of the reference genome construction variety (B73) is 99.73%, and the genotype consistency rate of repeated samples is as high as 99.99%; it is a high-throughput, high-accuracy, low-cost, multifunctional, and widely used liquid phase chip, which can be widely used in the detection and application of different types of corn materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0071] Figure 1 The intersection of the three chip sites in Example 1 of the present invention;

[0072] Figure 2 The distribution map of the maize 60K locus on the maize chromosome in Example 1 of the present invention;

[0073] Figure 3 This is the MAF distribution diagram of the corn 50K liquid phase chip in Example 1 of the present invention;

[0074] Figure 4 This is a flow chart of the construction of the cGPS sequencing library in Example 2 of the present invention;

[0075] Figure 5 This is a graph showing the genotype detection rate of 50 corn materials in Example 3 of the present invention;

[0076] Figure 6 This is a diagram showing the detection results of the genotype consistency rate of repeated samples of the corn 50K liquid phase chip in Example 3 of the present invention;

[0077] Figure 7 This is a graph showing the genotype detection rate of the B73 material in Example 3 of the present invention;

[0078] Figure 8 This is a graph showing the genotype consistency rate test results of repeated samples of the B73 material in Example 3 of the present invention;

[0079] Fig. 9 This is a cluster analysis diagram of 96 corn materials in Example 4 of the present invention. DETAILED DESCRIPTION

[0080] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0081] Example 1 Preparation of a corn 50K liquid phase chip and 60K molecular marker combination

[0082] This embodiment provides a corn 50K liquid phase chip and a 60K molecular marker combination.

[0083] 1. Maize 60K Molecular Marker Combination

[0084] The maize 60K molecular marker combination consists of 60,364 molecular markers, including 51,734 molecular markers, 49 SSR markers and 8,581 MNP interval loci (the specific loci are shown in Table 1). The method for obtaining the maize molecular marker combination is as follows:

[0085] (1) Whole-genome site screening:

[0086] Based on 1,218 corn resequencing data from home and abroad using Hapmap3, the sequencing data were analyzed to obtain highly reliable SNP site information as the source of resequencing data. The SNP sites that meet the minimum allele frequency MAF ≥ 0.05, deletion rate ≤ 0.2, and heterozygosity rate ≤ 0.5 in the screening population quality indicators are used as the initial screening candidate site set, and probes are designed for the screened target site set. Specifically, probes are designed within 100 bp around all target sites, with a probe length of about 100 bp and a GC content between 20% and 80%. According to the probe design results, probes that cannot be uniquely aligned on the genome and contain repetitive sequences in the flanking sequences are removed. Based on the principle of uniform distribution, sites with high MAF values ​​are selected for retention, and finally 100K SNP / Indel markers with high polymorphism are obtained.

[0087] In order to construct a reliable maize molecular marker combination that is highly compatible with previous mainstream chips at home and abroad, the above 100K site set was compared with 49,585 and 61,224 markers of Illumina MaizeSNP50BeadChip and Maize6H-60K that have been aligned to the maize reference genome B73RefGen_v4, and high-quality, highly polymorphic SNP sites on IlluminaMaizeSNP50BeadChip and Maize6H-60K were screened. Finally, 51,043 high-quality, highly polymorphic SNP / Indel markers were obtained. Illumina MaizeSNP50BeadChip accounted for 54.01%, and Maize6H-60K accounted for 48.08% (site alignment results are shown in Figure 2). Figure 1 The maize 50K molecular markers screened have high polymorphism and high versatility in mainstream chips at home and abroad.

[0088] (2) Functional sites

[0089] A large number of literatures were consulted to collect and screen the reported functional sites, GWAS sites, and functional genes, and 39 functional markers related to 19 genes such as corn stalk rot, southern rust, and drought resistance were obtained (as shown in Table 2); 271 sites of 65 genes related to corn yield, biotic stress (southern rust resistance, gray spot resistance, stalk rot resistance, ear rot resistance), abiotic stress (cold resistance, drought resistance) and other traits, and 625 GWAS sites related to corn yield, flowering period, plant growth and other shapes (as shown in Table 3). Probes were designed within 100 bp of all target sites, with a probe length of about 100 bp and a GC content between 20% and 80%. According to the probe design results, probes that could not be uniquely aligned on the genome and contained repeated sequences in the flanking sequences were removed.

[0090] Table 2 Agronomic trait types and marker numbers

[0091] Characteristics Number of markers Related genes Stem rot 18 QRfg3, Rpi1, Rgsr8.1, RpiQI319-1, RpiQI319-2, RpiW21-1, RpiW21-2, ZmAuxRP1, ZmCCT Southern Rust 17 RppC, RppCML496, RppD, RppK, RppM, RppP25, RppQ, RppS Drought tolerant 4 ZmLRT、ZmVPP1

[0092] Table 3 Agronomic trait types and marker numbers

[0093] Characteristics Number of markers Yield 103 Growth and development related 347 Flowering period 314 Resistant to southern rust 10 Gray spot resistance 7 Resistant to stem rot 25 Resistant to ear rot 39 Cold resistant 11 Drought tolerant 39

[0094] (3) National and industry standard sites

[0095] According to the marker flanking sequence information published in "SNP Markers for Authenticity Identification of Maize Varieties" (NY / T 4022-2021), the sequences were aligned to the reference genome B73 RefGen_v4, and 96 maize variety authenticity identification sites were obtained. Probes were designed for the sites using the above-mentioned probe design principles, and all probes were successfully designed.

[0096] According to the maize SSR marker primer sequences published in the Technical Specifications for Identification of Maize Varieties by SSR Marker Method (NY / T 1432-2014), the SSR Molecular Marker Method for Identification of Maize Varieties by Purity Identification (NY / T 3750-2020), and the SSR Molecular Marker Detection of Authenticity and Purity of Major Crop Varieties in Maize (GB / T 39914-2021), the sequences were aligned to the maize reference genome B73 RefGen_v4 to obtain 39 target intervals. The specific positions of SSRs were predicted using MISA software to obtain 49 SSR markers. Probes were designed for the loci using the above-mentioned probe design principles, and all probes were successfully designed. The specific information is shown in Table 4.

[0097] Table 4 49 SSR markers predicted by MISA

[0098]

[0099]

[0100] According to the maize MNP marker primer sequences published in the "MNP Marker Method for Plant Variety Identification" (GB / T38551-2020), the sequences were aligned to the maize reference genome B73RefGen_v4 to obtain 899 target intervals, and 8,581 high-quality, highly polymorphic sites in the intervals were extracted as MNP candidate markers. Probes were designed for the sites using the above-mentioned probe design principles, and all probes were successfully designed.

[0101] The details and quantity of national and industry standard loci markers covered by the chip are shown in Table 5. The corn 60K molecular marker combination contains a wealth of national and industry standards for corn, which can realize the integration and sharing of multiple standard data, and provide an efficient, accurate, universal and comprehensive method for corn variety identification, variety authenticity identification and variety purity identification.

[0102] Table 5 National and industry standard site marker details and quantity

[0103]

[0104] All the sites in 1)-3) above were integrated and deduplicated, and according to the quality indicators of these sites, 60,364 molecular markers with strong representativeness, high polymorphism, good versatility and uniform distribution on chromosomes were finally screened, including 39 functional markers related to 19 genes such as corn stalk rot, southern rust and drought resistance, 271 sites of 65 genes related to corn yield, biotic stress (southern rust resistance, gray spot resistance, stalk rot resistance, ear rot resistance), abiotic stress (cold resistance, drought resistance) and 625 GWAS sites related to corn yield, flowering period, plant growth and other shapes; 899 intervals of MNP for corn variety identification, a total of 8,581 markers, including 96 SNP markers for corn variety authenticity identification, 49 SSR markers for corn variety purity identification and corn variety identification, together forming a corn 60K molecular marker combination (site information is shown in Table 1 of the manual).

[0105] 2. Preparation of corn 50K liquid phase chip

[0106] The selected molecular markers (including 51,730 SNP markers, 4 indel markers, 49 SSR markers, and 899 MNP intervals (8,581 SNP markers)) were developed into a 50K liquid phase chip for corn using the liquid phase probe precise positioning sequencing typing technology (Genotyping by Pinpoint Sequencing of liguid captured target, cGPS) independently developed by Huazhi. The cGPS liquid phase chip technology is based on the optimized thermodynamic stability algorithm model to design characteristic probes for the target interval sequence, and uses synthetic specific probes to perform liquid phase hybridization capture and enrichment of multiple different target sequences located at different genomic positions, and then constructs the library and performs second-generation sequencing on the captured and enriched target intervals, so as to obtain the genotype of the marker site in the target interval.

[0107] The density distribution of the maize 50K liquid phase chip sites is shown in the figure below. Figure 2 As shown in the figure, it can be seen that the average coverage on the chromosome is 99.78%, the average spacing is 40Kb, and the 50K sites are evenly distributed on each chromosome. Figure 3 As shown in the figure, it can be seen that the maize 50K chip site has a high polymorphism, and the average minor allele frequency (MAF value) is 0.33.

[0108] Example 2 Genotyping process of maize 60K molecular marker combination

[0109] This example provides a method for genotyping corn samples using the maize 60K molecular marker combination prepared in Example 1. The steps are as follows:

[0110] (1) Extraction and quality control of genomic DNA

[0111] The sample DNA was extracted by magnetic bead method and the quality of DNA samples was tested. The quality test included measuring DNA concentration with Qubit fluorescence quantification instrument and testing DNA integrity with 1% agarose gel electrophoresis. Samples that passed the quality test were used for library preparation.

[0112] (2) GPS library construction and quality control

[0113] A commercially available sequencing library kit was used for GPS library construction and quality control. The principle and process of cGPS library construction are as follows: Figure 4 The steps are briefly described as follows:

[0114] 1) Use fragmentase to digest the DNA sample, repair the end of the digestion, add A base to the 3' end, and use agarose gel electrophoresis to detect the fragment size. 2) Use T4 ligase to connect the sequencing adapter and DNA fragment, and use magnetic beads to purify the connection product. The concentration of the purified product is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis. 3) PCR amplification of the purified connection product, and the amplified product is screened for fragments using magnetic beads. The concentration of the product after fragment screening is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis. 4) Take 200ng of the completed library, add probes and hybridization reagents, and incubate at 50℃ for 16-24 hours to complete the hybridization reaction. Use magnetic beads to capture the target segment, use cleaning solution to clean the captured product, remove non-specific binding fragments, and then perform another round of PCR amplification. Use Qubit fluorescence quantification instrument to detect the library concentration, and agarose gel electrophoresis to detect the fragment size. After the concentration and fragment size are qualified, the construction of cGPS sequencing library is completed.

[0115] The prepared library was sequenced using a BGI sequencer with a high-throughput sequencing strategy of PE150.

[0116] (3) Bioinformatics analysis

[0117] 1) Raw data filtering (sequencing data quality control): The raw sequencing sequences (Raw Reads) obtained by sequencing are filtered to obtain high-quality Clean Reads, and the data is quality controlled using FASTP software. 2) Contamination detection: Based on the fastq files of the Clean Reads after sample filtering, 10,000 sequences are randomly selected from the fastq files of each sample using seqtk software, and the sequences are aligned to the NCBI NT database using blastn for contamination assessment. 3) Reference genome alignment: Sequencing Reads are aligned to the reference genome using BWA software, and position sorting is performed to obtain the bam file after sample sorting.

[0118] 4) Mutation Detection:

[0119] a. Use HaplotypeCaller in gatk software to detect variant sites for each sample and obtain the gVCF file of each sample. b. Use CombineGVCFs in gatk software to analyze and obtain the variant result file of the test material. c. Use GenotypeGVCFs in gatk software to perform genotyping on the gVCF file of the population and obtain the original vcf variant result file.

[0120] 5) Genotyping of target sites: The site is judged based on the ratio of the number of supporting reads of different Alles. When the mutation reads support ratio is ≥0.8 or ≤0.2, the site is judged as a homozygous genotype. When the mutation reads support ratio is between 0.2-0.8, it is judged as a heterozygous genotype. The original vcf mutation result file is processed using an internally written perl script to obtain a 28-converted vcf mutation result file. Finally, the genotyping results of each target SNP in a specific individual are obtained with high throughput, achieving high-throughput SNP genotyping.

[0121] Example 3 Evaluation of the genotyping effect of the maize 60K molecular marker combination

[0122] To verify the genotyping effect of the maize 60K molecular marker combination, the maize 60K molecular marker combination prepared in Example 1 was used to perform genotyping on 50 maize samples (including 3 duplicate samples) and 6 maize "B73 RefGen_v4" reference genome B73 samples (reference genome construction varieties). The genotyping process of the maize 60K molecular marker combination is shown in Example 2.

[0123] After sequencing and data analysis, the results are as follows Figure 5-8 As shown, from Figure 5It can be seen that the detection rate of 50 corn samples is between 96.60% and 99.73%, and the average detection rate is 97.61%. Figure 6 As can be seen, the genotype consistency of the three technical replicate samples ranged from 99.45% to 99.99%, with an average consistency of 99.75%. Figure 7-8 It can be seen that the detection rate of B73 sample sites in the corn "B73RefGen_v4" reference genome is between 99.69% and 99.75%, the average detection rate is 99.72%, and the genotype consistency rate of technical replicate samples is 99.99%.

[0124] The results showed that the maize 60K molecular marker combination had a high detection rate of target sites, good stability, and accurate and reliable typing results when used for genotyping of the materials to be tested, and could be fully used for genotyping of different maize samples.

[0125] Example 4 Application of Maize 60K Molecular Marker Combination in Maize Population Structure Analysis

[0126] The maize 60K molecular marker combination prepared in Example 1 and the genotyping process of the maize 60K molecular marker combination provided in Example 2 were used to perform genotyping on 96 maize materials, and the genetic distance matrix of the 96 maize materials was calculated using Plink software and cluster analysis was performed.

[0127] The results are as follows Fig. 9 As shown in the figure, it can be seen that the 96 test materials are mainly divided into 5 subgroups, which is consistent with the known results. The results show that the maize 60K molecular marker combination prepared by the present invention has high representativeness and can be used to determine the kinship, evolutionary relationship, composition structure, etc. of different materials.

[0128] Example 5 Application of the 60K molecular marker combination in corn variety difference analysis

[0129] To verify whether the maize 60K molecular marker combination can be used for differential analysis of maize varieties, two sets of 96 variety authenticity identification markers specified by the national standard were tested, of which 95 marker genotypes were completely consistent with a consistency of 98.95%. The national standard determined that it was uncertain whether the maize samples were of the same variety for testing.

[0130] The genotypes of two corn samples were identified using the corn 60K molecular marker combination prepared in Example 1 and the genotyping process of Example 2. 57,517 loci were detected between the two samples, of which 2,036 loci had inconsistent genotypes with a consistency of 96.46%, indicating that the corn 60K molecular marker combination can effectively distinguish the differences between different corn varieties, and its detection results can better distinguish the differences between corn varieties than the variety authenticity identification SNP markers.

[0131] Example 6 Application of drought tolerance, stem rot, and southern rust functional trait loci

[0132] The genotype identification of 24 corn samples (provided by Gansu Dunhuang Seed Group Co., Ltd.) was performed using the maize 60K molecular marker combination prepared in Example 1 and the genotyping process of Example 2. The traits of the samples were analyzed according to the genotyping results of the functional trait loci reported in the chip. The detection rates of the functional trait loci for drought resistance, stem rot, and southern rust were high. Among the 24 samples, 3 samples were drought-resistant, 13 samples were resistant to stem rot, 16 samples were resistant to southern rust, and 3 samples were neither resistant to stem rot and southern rust nor drought-resistant. The results are shown in Table 6, and the analysis results are consistent with the sample phenotypes. It is shown that the traits of drought resistance, stem rot, and southern rust can be accurately detected.

[0133] Table 6 Phenotypic data statistics of varieties

[0134]

[0135]

[0136] Example 7 Application of Maize 60K Molecular Marker Combination in Maize Breeding

[0137] The maize 60K molecular marker combination prepared in Example 1 and the genotyping process of Example 2 were used to perform genotyping on 9 maize materials, of which 1 material was the recurrent parent A and the other 8 materials were recurrent offspring. Based on the genotype data of the 9 materials obtained, the genotype consistency rate between the 8 recurrent offspring and the recurrent parent A was analyzed.

[0138] Table 7 Parent comparison test results

[0139] Material 1 Material 2 Genotype consistency YM-1 A 91.22％ YM-2 A 90.37％ YM-3 A 89.66％ YM-4 A 98.80％ YM-5 A 98.71％ YM-6 A 97.96％ YM-7 A 99.24％ YM-8 A 98.98％

[0140] The results are shown in Table 7. It can be seen from Table 7 that the consistency rate between the 8 corn materials and the recurrent parent A is 89.66% to 99.24%, indicating that the corn 60K molecular marker combination can accurately detect the relationship between the recurrent parent and its hybrid offspring, can assist in the selection of corn breeding materials, and be used in subsequent breeding processes.

[0141] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A maize molecular marker combination, characterized in that: It includes 60,364 molecular markers, and the physical positions of the 60,364 molecular markers are determined based on sequence alignment of the corn reference genome B73 RefGen_v4. The site information is specifically shown in Table 1 in the specification.

2. A primer set and / or probe for detecting the maize molecular marker combination as claimed in claim 1.

3. The primer set and / or probe according to claim 2, characterized in that: The length of the probe is 80-120 bp.

4. The primer set and / or probe according to claim 2, characterized in that: The GC content of the probe is between 20% and 80%.

5. A liquid phase chip, characterized in that: The liquid phase chip comprises the primer set and / or probe according to any one of claims 2 to 4.

6. A kit, characterized in that: The kit comprises the primer set and / or probe according to any one of claims 2 to 4.

7. Use of one of the maize molecular marker combination according to claim 1, the primer set and / or probe according to any one of claims 2 to 4, the liquid phase chip according to claim 5, and the kit according to claim 6 in any one of the following: (1) Maize genotyping; (2) Maize gene localization; (3) Construction of corn fingerprint (4) Identification of maize germplasm resources; (5) Assessment of maize genetic diversity; (6) Identification of corn variety purity; (7) Identification of maize genetic relationships; (8) Identification of corn EDV; (9) Authenticity identification of corn varieties; (10) Construction of maize genetic map and QTL location; (11) Genome-wide association analysis of maize; (12) Maize molecular marker-assisted selection breeding; (13) Targeted corn improvement; (14) Corn multi-gene aggregation breeding; (15) Whole genome selection breeding of corn; (16) Molecular design breeding of corn.

8. A method for detecting the corn molecular marker combination according to claim 1, characterized in that: The following steps are involved: S1. Perform genotyping on the sample to be tested using at least one of the primer set and / or probe according to any one of claims 2 to 4, the liquid phase chip according to claim 5, and the kit according to claim 6 to obtain a genotyping result; S2. Analyze the genotyping results obtained in step S1.

9. The method according to claim 8, characterized in that The method is based on liquid phase probe capture sequencing typing technology.

10. A corn breeding method, characterized in that: The method comprises the following steps: using the method as claimed in claim 8 or 9 to detect the DNA of the corn to be detected, and selecting corn for subsequent breeding.

Images