A combination of maize molecular markers, a liquid-phase chip and its applications

Abstract

This invention discloses a maize molecular marker ensemble, a liquid-phase chip, and their applications. The maize molecular marker ensemble comprises 60,364 molecular markers, the physical locations of which are determined by sequence alignment based on the maize reference genome B73RefGen_v4. The maize molecular marker ensemble provided by this invention can be widely used for the detection and application of different types of maize materials, with high site detection rate and reliable genotyping results.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a maize molecular marker combination, a liquid-phase chip, and their applications. Background Technology

[0002] Corn is a widely cultivated crop and occupies a pivotal position among agricultural crops. For a long time, traditional breeding, represented by hybridization breeding, has provided a large number of superior varieties. However, because traditional breeding is basically based on empirical breeding and phenotypic selection, and has disadvantages such as long breeding cycles and low efficiency, its development potential can no longer meet the needs of the corn market.

[0003] In recent years, various molecular markers, such as restriction fragment length polymorphism (RFLP), simple sequence repeats (SSR), random amplified polymorphic DNA markers (RAPD), and single nucleotide polymorphisms (SNP), have been used in maize research to assess the genetic characteristics of populations or germplasm, locate quantitative trait loci (QTLs), and facilitate the selection of breeding materials. Compared with other molecular markers, SNPs are characterized by high density, high genetic stability, and ease of analysis. Genome microarrays are a breeding tool based on high-throughput molecular marker technology that can cover SNP loci across the entire genome. They feature wide coverage, high sensitivity, high throughput, and high precision. SNP microarrays offer advantages such as convenient detection, low cost, high throughput, and simple analysis, making them an important tool in the field of molecular breeding. Utilizing whole-genome breeding microarrays for comprehensive evaluation of maize germplasm resource genotyping, genetic diversity, and functional gene discovery helps accelerate the discovery of superior maize germplasm and the identification of novel functional genes. Currently, there are several corn SNP chips on the market, such as MaizeSNP50 Bead Chip, MaizeSNP600K, Maize6H-60KSNP, and Maize 55K. These are all based on solid-phase chip design, which has the disadvantages of high detection cost and low flexibility, making it difficult to use on a large scale in corn breeding.

[0004] With the rapid development of maize breeding and the seed industry, the identification of varieties, germplasm resources, and variety purity has become increasingly difficult. Previously, a series of national and industry standards for maize variety identification and authenticity verification were approved, such as the "Technical Regulations for Maize Variety Identification: SSR Marker Method" (NY / T 1432-2014), the "Maize Variety Purity Identification: SSR Molecular Marker Method" (NY / T3750-2020), the "Detection of Authenticity and Purity of Major Crop Varieties: SSR Molecular Markers - Maize" (GB / T 39914-2021), the "Maize Variety Authenticity Identification: SNP Marker" (NY / T4022-2021), and the "Plant Variety Identification: MNP Marker Method" (GB / T38551-2020), providing strong technical support and basis for maintaining maize variety innovation and purity verification. However, the marker types in these standards are inconsistent, making it impossible to achieve unified testing data. Therefore, an efficient, accurate, universal, and comprehensive method for identifying maize varieties, germplasm resources, and variety purity is needed.

[0005] To overcome these problems, it is necessary to develop a high-density, high-throughput, high-accuracy, low-cost, multifunctional liquid-phase chip with a wide range of applications 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 prior art. To this end, the present invention proposes a combination of maize molecular markers.

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

[0008] The present invention also proposes a liquid phase chip.

[0009] The present invention also proposes a reagent kit.

[0010] This invention also proposes an application of the above-mentioned maize molecular marker combination, primer set and / or probe, liquid phase chip or reagent kit.

[0011] This invention also proposes a method for detecting combinations of molecular markers in maize.

[0012] This invention also proposes a method for breeding maize.

[0013] According to one aspect of the present invention, a maize molecular marker combinatorial system is proposed, comprising 60,364 molecular markers, the physical locations of which are determined by sequence alignment based on the maize reference genome B73 RefGen_v4, and the specific site information is shown in Table 1 below.

[0014] Table 1

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[0024]

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[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 regions.

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

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

[0040] In a second aspect of the invention, primer sets and / or probes for detecting the above-mentioned maize molecular marker combinations are proposed.

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

[0042] In some embodiments of the present invention, the probe is approximately 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 a third aspect of the invention, a liquid-phase chip is provided, the liquid-phase chip comprising the primer set and / or probes described above for detecting combinations of maize molecular markers.

[0045] In a fourth aspect of the invention, a kit is provided comprising the primer set and / or probe described above for detecting combinations of maize molecular markers.

[0046] In a fifth aspect of the invention, an application of at least one of the above-mentioned maize molecular marker combinations, liquid phase chips, and kits is proposed, said application being an application in maize genotyping.

[0047] In some embodiments of the present invention, the application is an application in maize gene mapping.

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

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

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

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

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

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

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

[0055] In some embodiments of the present invention, the application is in the construction of maize genetic maps and QTL mapping.

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

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

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

[0059] In some embodiments of the present invention, the application is in maize multigene aggregation breeding.

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

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

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

[0063] S1. Genotyping of the sample to be tested is performed using at least one of the primer set and / or probe, liquid phase chip and kit to obtain genotyping results;

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

[0065] In some embodiments of this invention, the detection method is based on liquid-phase probe capture sequencing genotyping technology. This invention uses high-throughput sequencing technology for genotyping of maize materials, offering advantages such as high throughput, large data output in a single run, and the ability to simultaneously cover the detection of nearly a thousand materials. It is also compatible with mainstream second-generation sequencing platforms such as Illumina and MGI, demonstrating broad platform adaptability.

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

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

[0068] (1) The maize molecular marker combination of this invention is based on 1,218 maize resequencing data from Hapmap3, which are rich in diversity both domestically and internationally. It selects highly representative, specific, polymorphic, and universally applicable loci, with abundant functional sites. These include 39 functional markers related to important traits such as maize stalk rot, southern rust, and drought resistance; 65 genes (271 loci) related to maize yield, disease resistance (southern rust, gray spot, stalk rot, ear rot), and abiotic stress; and 625 GWAS loci related to important traits such as maize yield, flowering time, and plant growth and development. This can be used for the discovery and utilization of important trait genes, marker-assisted breeding, and functional analysis. Meanwhile, the maize molecular marker combination of the present invention also includes molecular markers from multiple national or industry standards, such as the "Technical Specification for Maize Variety Identification - SSR Marker Method" (NY / T 1432-2014), the "Maize Variety Purity Identification - SSR Molecular Marker Method" (NY / T 3750-2020), the "Maize Authenticity and Purity Detection of Major Crop Varieties - SSR Molecular Marker Method" (GB / T 39914-2021), the "Maize Variety Authenticity Identification - SNP Marker" (NY / T4022-2021), and the "Maize in Plant Variety Identification - MNP Marker Method" (GB / T38551-2020), etc.; it contains molecular markers for EDV identification and fingerprint identification, and can be used for maize variety identification, variety purity identification, variety authenticity identification, EDV identification, and kinship identification.

[0069] (2) The maize molecular marker combination (60,364 molecular markers in the 50K region of maize) proposed in this invention can be widely used for the detection and application of different types of maize materials. The site detection rate is high and the typing results are reliable. The average detection rate of the reference genome constructed variety (B73) is 99.73%, and the genotypic consistency rate of the repeated samples is as high as 99.99%. It is a high-throughput, high-accuracy, low-cost, multifunctional liquid phase chip with a wide range of applications. It can be widely used for the detection and application of different types of maize materials. Attached Figure Description

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

[0071] Figure 1 This shows the intersection of the three chip sites in Embodiment 1 of the present invention;

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

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

[0074] Figure 4 This is a flowchart of the cGPS sequencing library construction process in Embodiment 2 of the present invention;

[0075] Figure 5 This is a graph showing the genotype detection rate results of 50 maize samples in Example 3 of this invention;

[0076] Figure 6 This is a graph showing the genotypic consistency rate detection results of duplicate samples from the maize 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 genotypic consistency rate detection results of duplicate samples of the B73 material in Example 3 of this invention;

[0079] Figure 9 This is a cluster analysis diagram of 96 maize samples in Example 4 of the present invention. Detailed Implementation

[0080] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort 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 label combination

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

[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 loci (the specific loci are shown in Table 1). The method for obtaining the maize molecular marker combination is as follows:

[0085] (1) Genome-wide site screening:

[0086] Based on 1,218 maize resequencing data from both domestic and international sources using Hapmap3, highly reliable SNP locus information was obtained through sequencing data analysis and used as the source of resequencing data. SNP loci meeting the following population quality indicators—Minimum Allele Frequency (MAF) ≥ 0.05, deletion rate ≤ 0.2, and heterozygosity ≤ 0.5—were selected as the initial candidate locus set. Probe design was then performed on the selected target locus set. Specifically, probes were designed within a 100 bp radius to the left and right of each target locus, with probe lengths of approximately 100 bp and GC content between 20% and 80%. Based on the probe design results, probes that could not be uniquely aligned to the genome or contained repetitive sequences in their flanking sequences were removed. Based on the principle of uniform distribution, loci with high MAF values ​​were preferentially retained, ultimately obtaining SNP / Indel markers with high 100K polymorphism.

[0087] To construct a reliable maize molecular marker set with high universality compared to previous mainstream domestic and international chips, the aforementioned 100K locus set was aligned with 49,585 and 61,224 markers from Illumina MaizeSNP50BeadChip and Maize6H-60K, respectively, already aligned to the maize reference genome B73RefGen_v4. High-quality, highly polymorphic SNP loci in Illumina MaizeSNP50BeadChip and Maize6H-60K were screened, ultimately yielding 51,043 high-quality, highly polymorphic SNP / Indel markers. Illumina MaizeSNP50BeadChip accounted for 54.01%, and Maize6H-60K accounted for 48.08% (locus alignment results are shown in the figure). Figure 1 (As shown). The maize 50K molecular markers obtained through screening exhibit high polymorphism and high versatility in mainstream microarrays both domestically and internationally.

[0088] (2) Functional loci

[0089] Extensive literature review and screening of reported functional loci, GWAS loci, and functional genes yielded 39 functional markers associated with 19 genes related to maize stalk rot, southern rust, and drought resistance (Table 2); 271 loci associated with 65 genes related to traits such as maize yield, biotic stress (resistance to southern rust, gray leaf spot, stalk rot, and ear rot), and abiotic stress (cold tolerance and drought tolerance); and 625 GWAS loci associated with morphological characteristics such as maize yield, flowering time, and plant growth (Table 3). Probes were designed within a 100 bp radius to the left and right of each target locus, with probe lengths of approximately 100 bp and GC content between 20% and 80%. Based on the probe design results, probes that could not be uniquely aligned to the genome or contained repetitive sequences in their flanking sequences were removed.

[0090] Table 2 Agronomic trait types and marker counts

[0091] Properties Number of tags 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 resistant 4 ZmLRT, ZmVPP1

[0092] Table 3 Agronomic trait types and marker counts

[0093] Properties Number of tags Yield 103 Growth and development related 347 Flowering period 314 Resistance to Southern Rust 10 Anti-gray spot disease 7 Resistant to stem rot 25 resistance to ear rot 39 Cold-resistant 11 drought resistant 39

[0094] (3) National and industry standard sites

[0095] Based on the flanking sequence information of the markers 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 authenticity identification sites for maize varieties were obtained. Probes were designed for the sites using the above-mentioned probe design principles, and all probes were successfully designed.

[0096] Based on the published SSR marker primer sequences for maize in the "Technical Specification for Maize Variety Identification - SSR Marker Method" (NY / T 1432-2014), "Maize Variety Purity Identification - SSR Molecular Marker Method" (NY / T 3750-2020), and "Detection of Authenticity and Purity of Major Crop Varieties - SSR Molecular Markers - Maize" (GB / T 39914-2021), the sequences were aligned to the maize reference genome B73 RefGen_v4, resulting in 39 target regions. The specific locations of SSRs were predicted using MISA software, yielding 49 SSR markers. Probes were designed for these sites using the aforementioned probe design principles, and all probes were successfully designed. Specific information is shown in Table 4.

[0097] Table 4. 49 SSR markers predicted by MISA

[0098]

[0099]

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

[0101] Table 5 details the national and industry standard markers covered by the chip and their quantities. The maize 60K molecular marker suite contains a wealth of national and industry standards for maize, enabling the integration and sharing of multiple standard data. It provides an efficient, accurate, universal, and comprehensive method for maize variety identification, variety authenticity verification, and variety purity verification.

[0102] Table 5. Details and Quantity of National and Industry Standard Site Markers

[0103]

[0104] After integrating and deduplicating all the loci mentioned in 1)-3), based on the quality indicators of these loci, a total of 60,364 molecular markers with strong representativeness, high polymorphism, good universality, and uniform distribution on chromosomes were finally screened. Among them, there are 39 functional markers related to 19 genes such as maize stalk rot, southern rust, and drought resistance; 271 loci of 65 genes related to traits such as maize yield, biotic stress (resistance to southern rust, gray spot, stalk rot, and ear rot), and abiotic stress (cold resistance and drought resistance); and 625 GWAS loci related to morphological traits such as maize yield, flowering time, and plant growth; 899 MNP intervals for maize variety identification, totaling 8,581 markers; and 96 SNP markers for maize variety authenticity identification and 49 SSR markers for maize variety purity identification and maize variety identification. Together, they form the maize 60K molecular marker assemblage (locus information is shown in Table 1 of the instruction manual).

[0105] 2. Fabrication of a 50K corn-based liquid phase chip

[0106] The selected molecular markers (including 51,730 SNP markers, 4 indel markers, 49 SSR markers, and 899 MNP regions (8,581 SNP markers)) were used by Huazhi to develop a maize 50K liquid-phase microarray using their independently developed liquid-phase probe precise localization sequencing and genotyping technology (cGPS). cGPS liquid-phase microarray technology is based on an optimized thermodynamic stability algorithm model for designing characteristic probes for target region sequences. Synthesized specific probes are used to capture and enrich multiple different target sequences located at different genomic positions through liquid-phase hybridization. Then, libraries are constructed and next-generation sequencing is performed on the captured and enriched target regions to obtain the genotypes of the marker sites within the target regions.

[0107] The density distribution of sites on a 50K liquid-phase chip for corn is shown in the figure below. Figure 2 As shown in the figure, the average coverage on the chromosomes is 99.78%, the average spacing is 40Kb, and the 50K loci are evenly distributed on each chromosome. The MAF value distribution map is shown below. Figure 3 As shown in the figure, the maize 50K chip locus has high polymorphism, with an average minor allele frequency (MAF value) of 0.33.

[0108] Example 2: Genotyping Procedure for Maize 60K Molecular Marker Combinations

[0109] This embodiment provides a method for genotyping maize 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] DNA was extracted from the samples using a magnetic bead method, and the DNA samples underwent quality testing. Quality testing included determining DNA concentration using a Qubit quantitative PCR instrument and assessing DNA integrity using 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0112] (2) GPS Library Construction and Quality Control

[0113] GPS library construction and quality control were performed using commercially available sequencing library kits. The principles and procedures for cGPS library construction are as follows: Figure 4 As shown, the steps are briefly described below:

[0114] 1) DNA samples were digested with a fragmentation enzyme to repair the enzyme ends, and an A base was added to the 3' end. Fragment size was detected by agarose gel electrophoresis. 2) Sequencing adapters and DNA fragments were ligated using T4 ligase. The ligation products were purified using magnetic beads. The concentration of the purified products was detected using a Qubit real-time fluorescence instrument, and fragment size was detected by agarose gel electrophoresis. 3) PCR amplification was performed on the purified ligation products. Fragment selection was performed using magnetic beads. The concentration of the selected fragments was detected using a Qubit real-time fluorescence instrument, and fragment size was detected by agarose gel electrophoresis. 4) 200 ng of the constructed library was added, along with probes and hybridization reagents, and incubated at 50°C for 16-24 hours to complete the hybridization reaction. Target regions were captured using magnetic beads. The captured products were washed with washing buffer to remove non-specific binding fragments, and then another round of PCR amplification was performed. The library concentration was detected using a Qubit real-time fluorescence instrument, and fragment size was detected by agarose gel electrophoresis. Once the concentration and fragment size were within acceptable limits, the cGPS sequencing library construction was complete.

[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 reads obtained from sequencing are filtered to obtain high-quality clean reads. FASTP software is used for quality control of the processed data. 2) Contamination Detection: Based on the fastq files of the filtered clean reads, 10,000 sequences are randomly selected from the fastq files of each sample using seqtk software. The sequences are then aligned to the NCBI NT database using blastn for contamination assessment. 3) Reference Genome Alignment: The sequencing reads are aligned to the reference genome using BWA software, and their positions are sorted to obtain the sorted BAM files.

[0118] 4) Mutation detection:

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

[0120] 5) Target locus genotyping: Genotyping is determined based on the proportion of supporting reads for different alles at the locus. If the proportion of supporting reads is ≥0.8 or ≤0.2, the locus is classified as homozygous; if the proportion is between 0.2 and 0.8, it is classified as heterozygous. An internally written Perl script processes the original VCF variant result file to obtain a 28-transformed VCF variant result file. Finally, high-throughput genotyping results for each target SNP in a specific individual are obtained, achieving high-throughput SNP genotyping.

[0121] Example 3: Evaluation of the Genotyping Effect of Maize 60K Molecular Marker Combinations

[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 B73 samples (reference genome construction varieties) of the maize "B73 RefGen_v4" reference genome. The genotyping procedure of the maize 60K molecular marker combination is shown in Example 2.

[0123] Sequencing and data analysis yielded the following results: Figure 5-8 As shown, from Figure 5The results show that the detection rate of loci in the 50 corn samples ranged from 96.60% to 99.73%, with an average detection rate of 97.61%. Figure 6 The results show that the genotypic similarity rate of the three technical replicate samples ranged from 99.45% to 99.99%, with an average similarity rate of 99.75%. Figure 7-8 As can be seen, the detection rate of B73 sample sites in the maize “B73RefGen_v4” reference genome is between 99.69% and 99.75%, with an average detection rate of 99.72%, and the genotypic consistency rate of the technically replicated 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 genotyping results when used for genotyping of different maize samples.

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

[0126] Genotyping of 96 maize materials was performed using the maize 60K molecular marker combination prepared in Example 1 and the genotyping procedure of the maize 60K molecular marker combination provided in Example 2. The genetic distance matrix of the 96 maize materials was calculated and cluster analysis was performed using Plink software.

[0127] The results are as follows Figure 9 As shown in the figure, the 96 test materials were mainly divided into 5 subgroups, consistent with known results. The results indicate that the maize 60K molecular marker combination prepared in this invention is highly representative and can be used to determine the phylogenetic relationships, evolutionary relationships, and structural composition of different materials.

[0128] Example 5: Application of Maize 60K Molecular Marker Combinations in Maize Variety Difference Analysis

[0129] To verify whether the 60K molecular marker combination of maize can be used for differential analysis of maize varieties, two maize samples were tested using 96 authenticity identification markers specified by national standards. Among them, 95 marker genotypes were completely identical, with a consistency of 98.95%. The maize samples that were determined by national standards to be uncertain whether they belonged to the same variety were then tested.

[0130] Genotyping of two maize samples was performed using the maize 60K molecular marker combination prepared in Example 1 and the genotyping procedure in Example 2. 57,517 loci were detected in both samples, with 2,036 loci showing genotypic inconsistency, resulting in a consistency of 96.46%. This indicates that the maize 60K molecular marker combination can effectively distinguish differences between different maize varieties, and its detection results are more effective than SNP markers for variety authenticity identification in distinguishing differences between maize varieties.

[0131] Example 6: Application of functional trait loci for drought resistance, stem rot, and southern rust.

[0132] Genotyping of 24 maize samples (provided by Gansu Dunhuang Seed Industry Group Co., Ltd.) was performed using the maize 60K molecular marker combination prepared in Example 1 and the genotyping procedure in Example 2. The traits of the samples were analyzed based on the genotyping results of functional trait loci reported in the microarray. The detection rates of drought resistance, stalk rot, and southern rust functional trait loci were high. Of the 24 samples, 3 were drought resistant, 13 were resistant to stalk rot, 16 were resistant to southern rust, and 3 were neither resistant to stalk rot, southern rust, nor drought resistant. The results are shown in Table 6, and the analysis results are consistent with the sample phenotypes. This indicates that drought resistance, stalk rot, and southern rust traits can be accurately detected.

[0133] Table 6. Phenotypic Data Statistics of Varieties

[0134]

[0135]

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

[0137] Genotyping of nine maize materials was performed using the maize 60K molecular marker combination prepared in Example 1 and the genotyping procedure in Example 2. One material was the recurrent parent A, and the other eight materials were recurrent offspring. Based on the genotypic data of the nine materials, the genotypic similarity rate between the eight recurrent offspring and the recurrent parent A was analyzed.

[0138] Table 7. Parental comparison test results

[0139] Material 1 Material 2 Genotype concordance rate 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. As can be seen from Table 7, the similarity rate between the eight maize materials and the recurrent parent A was 89.66% to 99.24%, indicating that the maize 60K molecular marker combination can accurately detect the kinship between the recurrent parent and its hybrid offspring, and can be used to assist in the selection of maize breeding materials and applied in subsequent breeding processes.

[0141] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A liquid phase chip, characterized in that, The liquid-phase chip contains probes for detecting maize molecular marker assemblies; the maize molecular marker assemblies consist of 60,364 molecular markers; the physical locations of the 60,364 molecular markers are determined by sequence alignment based on the maize reference genome B73 RefGen_v4, and the specific site information is shown in Table 1 of the specification.

2. The liquid phase chip according to claim 1, characterized in that, The probe has a length of 80-120 bp.

3. The liquid phase chip according to claim 1, characterized in that, The GC content of the probe is between 20% and 80%.

4. The application of the liquid phase chip according to any one of claims 1-3 in any of the following: (1) Construction of maize fingerprint map (2) Identification of maize germplasm resources; (3) Identification of maize kinship; (4) Construction of maize genetic map and QTL mapping; (5) Maize genome-wide association analysis; (6) Marker-assisted selection breeding of maize.

5. A method for breeding maize, characterized in that, The method includes the following steps: using a liquid phase chip as described in any one of claims 1-3 to detect the DNA of the maize to be tested, and selecting maize for subsequent breeding.

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