Maize dh breeding chip and application

Abstract

The application relates to the technical field of molecular plant breeding, and particularly discloses a maize DH breeding chip and application, the genotyping of the maize DH breeding chip comprises 11,001 SNP sites, 36 InDel sites and 4 transgenic event markers; the physical positions of the 11,001 SNP sites and the 36 InDel sites are determined based on whole genome sequence alignment of a maize reference genome B73v4; wherein, the 11,001 SNP sites are shown in Table 1; the 36 InDel sites are shown in Table 2, and the information of the 4 transgenic event markers is shown in Table 3. The maize DH breeding liquid phase chip can be used for DH line identification, DH hybrid advantage group division and transgene identification of herbicide tolerance, pest resistance and stress resistance of maize DH.

Description

Technical Field

[0001] This invention relates to the field of molecular plant breeding technology, specifically to maize DH breeding chips and their applications. Background Technology

[0002] Maize (Zea mays L.) plays a crucial role in global agricultural development and food security as a major food crop. In 2012, maize production surpassed rice production, becoming my country's largest food crop. However, my country's maize production still lags significantly behind that of the United States. Therefore, developing high-yield, densely planted maize varieties is a key goal for breeders in the short term.

[0003] Obtaining stable pure lines is a crucial step in maize breeding. Double haploid (DH) breeding is a method that uses induction lines (or anther culture to induce haploid plants) and then doubles the chromosome set to restore the plant to a normal chromosome number. Compared to traditional breeding methods, this method significantly shortens the time required to reach homozygosity, requiring only one generation, thus enabling faster fixation of the target trait.

[0004] DH technology enables breeders to obtain the desired homozygous inbred lines in a shorter time, thereby accelerating the selection, application, and hybridization of high-yielding new varieties. If a complete set of haploid technology and a year-round DH line creation model are applied, a "high-speed rail" rapid breeding model can be achieved within one year, from basic materials to pure line testing and evaluation.

[0005] Therefore, a breeding chip that can quickly identify DH lines is needed. Summary of the Invention

[0006] The purpose of this invention is to provide a maize DH breeding chip and its application. This liquid phase chip can be used to identify DH lines, classify DH heterotic groups, and identify herbicide-resistant, insect-resistant, and anti-retroviral genes in maize DH.

[0007] This invention is achieved through the following technical solution:

[0008] A molecular marker combinatorial for maize DH genotyping, the molecular marker combinatorial comprising 11,001 SNP sites, 36 InDel sites and 4 transgenic event markers;

[0009] The physical locations of the 11,001 SNP sites and 36 InDel sites were determined based on whole-genome sequence alignment of the maize reference genome B73v4; the 11,001 SNP sites are shown in Table 1; the 36 InDel sites are shown in Table 2; and the information on the 4 transgenic event markers is shown in Table 3.

[0010] The 11,001 SNP loci and 36 InDel loci selected in this invention were obtained based on representative maize varieties and literature reviews. They include loci that affect important maize traits and SNP loci for identifying the authenticity of maize varieties. This allows for more effective identification of maize functional genes and genetic background analysis of breeding materials. Ultimately, it enables the identification of DH lines, the classification of DH heterosis groups, and the classification of heterosis groups in maize.

[0011] The transgenic event markers of the present invention have functions including herbicide tolerance, insect resistance, and stress resistance. That is, the liquid phase chip prepared based on the site of the present invention can determine whether corn contains transgenic events nCX-1, GAB3, KN13, and KN19 by gene detection, and then determine whether the corn has traits such as herbicide tolerance, insect resistance, and stress resistance.

[0012] The application of the above molecular marker combinations in gene breeding chips; gene breeding chips include liquid phase chips, etc.

[0013] A maize DH breeding chip, wherein the genotyping of the maize DH breeding chip includes the aforementioned 11,001 SNP sites, 36 InDel sites and 4 transgenic event markers.

[0014] Furthermore, the maize DH breeding chip also includes probes designed based on gene sequences covering 11,001 SNP sites, 36 InDel sites, and 4 transgenic event markers. The probe sequences have a GC content of 30-70%, and the maximum length of the covered SSRs is less than 120 bp, with a length of 110 bp.

[0015] A method for designing a maize DH breeding chip includes the following steps:

[0016] S1. Loci for identifying maize DH materials were obtained through t-test analysis of second-generation resequencing data of maize inbred lines, literature, and genotype and phenotypic data collected in the laboratory.

[0017] S2. By evaluating the sites obtained in step S1, 11,001 SNP sites and 36 InDel sites were selected.

[0018] S3. Nucleotide probes were designed based on 11,001 SNP sites, 36 InDel sites and 4 transgenic event markers selected, and a liquid phase chip for maize DH breeding was prepared.

[0019] Specifically, in step S1, the process of obtaining loci through second-generation resequencing data of maize inbred lines is as follows:

[0020] Using the resequencing results of 507 maize inbred lines, original variant sites were identified; these original variant sites were then filtered and screened sequentially, resulting in 8,981 SNP sites.

[0021] Specifically, in step S1, the process of obtaining loci through t-test analysis of genotype and phenotype data collected from literature and the laboratory is as follows:

[0022] A total of 2,123 SNPs with significant p-values ​​were obtained from t-test analysis of genotypes and phenotypes of 507 maize inbred lines and loci related to disease resistance and abiotic stress traits (waterlogging tolerance, salt tolerance, drought resistance, etc.) collected from published literature.

[0023] Application of a maize DH breeding chip in DH line identification.

[0024] Application of a maize DH breeding chip in the classification of maize DH heterosis groups.

[0025] Application of a maize DH breeding chip in transgenic monitoring.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] 1. The maize DH breeding liquid phase chip loci of the present invention are derived from the resequencing results of 507 maize varieties and loci related to maize disease resistance and abiotic stress (waterlogging tolerance, salt tolerance, drought resistance, etc.) reported in numerous literatures. The probe sources are more varieties and the types of polymorphic loci are more diverse, which can be better applied to DH line identification, DH hybrid vigor group classification and herbicide resistance, insect resistance, and anti-retroviral gene identification.

[0028] 2. The gene chip of this invention adopts the latest marker genotyping by target sequencing (GBTS) technology, which has the advantages of low detection cost, high flexibility, high accuracy and high detection sensitivity. Attached Figure Description

[0029] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0030] Figure 1 This is a schematic diagram showing the distribution of the 11,001 SNP sites screened in Example 1 of the present invention across the entire genome;

[0031] Figure 2 This is a recombination heatmap of the liquid phase chip applied to DH identification materials in Embodiment 3 of the present invention;

[0032] Figure 3 This is a schematic diagram of the liquid phase chip used for heterosis grouping in DH series in Embodiment 4 of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The illustrative embodiments and descriptions of this invention are for illustrative purposes only and are not intended to limit the invention. The embodiments described below are some, but not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known structures, materials, or methods are not specifically described to avoid obscuring the invention. Unless otherwise specified, the materials, instruments, and reagents used in the following embodiments are commercially available. Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art.

[0035] Example 1:

[0036] The preparation of sites for maize DH breeding liquid phase chip acquisition includes the following steps:

[0037] Step 1: Site acquisition for preparing liquid-phase microarrays for maize DH breeding:

[0038] 1) Using the resequencing results of 507 maize inbred lines, original variant sites were identified. Of these, 216 were from the International Maize and Wheat Improvement Center, 210 from China, 60 from the United States, and 1 from Peru, representing a very high degree of representativeness of maize varieties worldwide. A total of 75,680,644 original variant sites (SNPs + InDel) were identified. These original variant sites underwent filtering and selection. Filtering: 10,770,214 sites with only two genotypes, a minor allele frequency greater than 0.05, and a quality fraction greater than or equal to 30 were retained. Selection: Functional annotation was performed on these variant sites using ANNOVAR software. From these, 8,981 SNP sites were selected based on the principles of uniform distribution, high minor allele frequency (MAF), low deletion rate, low heterozygosity, and the likelihood of being a variant site affecting gene function according to functional annotation.

[0039] 2) A total of 2,123 SNP loci with significant p-values ​​were obtained from a large number of published literatures that collected loci related to disease resistance and abiotic stress (waterlogging tolerance, salt tolerance, drought resistance, etc.) traits and t-test analysis of genotypes and phenotypes of 507 inbred lines.

[0040] The chip of the present invention contains sites containing traits related to disease resistance and abiotic stress, but is not intended for the detection of these traits. The purpose of the chip is for the identification of DH materials.

[0041] 3) 96 SNP loci for maize variety authenticity identification (referencing the People's Republic of China Agricultural Industry Standard NY / T4022-2021).

[0042] 4) Four genetically modified events.

[0043] By evaluating the loci obtained in steps 1)-3) (evaluation parameters: GC content: 30-70%, number of homologous regions ≤ 5), and after multiple optimizations, 11,001 SNP loci, 36 InDel loci and 4 transgenic events were selected.

[0044] The distribution of all SNP loci across the entire genome is as follows: Figure 1 As shown, it is distributed in all segments of the whole genome, and is densely distributed in important functional gene regions. It can meet the needs of DH line purity identification, key gene diagnosis, DH recombination map construction, DH line diversity analysis, DH line breeding value assessment, and can also be used for maize authenticity identification.

[0045] The 11,001 SNP sites are shown in Table 1, the 36 InDel sites are shown in Table 2, and the 4 transgenic events are shown in Table 3.

[0046] Table 1

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[0085] Table 2

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[0109] Table 3

[0110] nCX-1 Herbicide tolerant 7 172978251 173072614 GAB3 Insect-resistant and herbicide-tolerant 7 172712372 172712430 KN13 Resilience 5 178508732 178508748 KN19 Resilience 7 168318107 168318112

[0111] Among them, the sequences of events nCX-1 and GAB3 can be directly found. The nucleotide sequences of the left and right transgenic events of KN13 are shown in SEQ ID NO.1-SEQ ID NO.2, respectively, and the nucleotide sequences of the left and right transgenic events of KN19 are shown in SEQ ID NO.3-SEQ ID NO.4, respectively. Uppercase letters represent maize genome sequences, and lowercase letters represent exogenous DNA sequences (sequences specific to superior haplotypes, which cannot extend bands in B73). The sequence with the suffix "RB-genome" is the left transgenic event sequence (5' to 3'), and the sequence with the suffix "LB-genome" is the right transgenic event sequence (5' to 3').

[0112] KN13-RB-genome (SEQ ID NO.1)

[0113] ACAGCATCAGTCGCCCCACCGTCGCCTCCGCCTCCGCCATTTGCAGATCAGTAGATTACAGTGACGACTTCGCTCAACCGTAGTCCGAGACCGAGAGCGAgaaacgacaatctgatccaagctcaagctgctctagcattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattac

[0114] >KN13-LB-genome(SEQ ID NO.2)

[0115] agatcccccgaattaattcggcgttaattcagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatACTAAGGGTTCGCTTTGAAGGAACCTGCAAGCAAGCGAAGTGGAACACGGAATACAACTGAAATGGGCTCGTTTGCAGCAGACGCGGCCCATTATTTGGT

[0116] >KN19-RB-genome(SEQ ID NO.3)

[0117] AGCTATAAGATGAATCTTAAACCTATAATGAGTACGAAGATAGAGATGGATATGCCTCAGTCATAGGAATAGGGTGTCCAAATCCAGTGAGGAATTAGCAtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgatccaagctcaagctgctctagcattcgccattcaggctgcgcaactgttgggaag

[0118] >KN19-LB-genome(SEQ ID NO.4)

[0119] agatcccccgaattaattcggcgttaattcagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacacccaaatatTGACACCCTTAGTATGTATTTGTATTTGGAATTCTCTATTGACACCTTTGGTGGTCTGGCCTATGCGAGTGGTCACGTACGACGCCAACGCCGGCGACCA.

[0120] Step 2: Synthesize the probe

[0121] Based on the principle of base complementarity, probes were synthesized for detecting 11,001 SNP sites, 36 InDel sites, and 4 transgenic events.

[0122] Site probe design method: Synthesize probes to detect 11,001 SNP sites and 36 InDel sites; the GC content of the sequence is 30-70% (relaxed to 20-80% for some important sites), the maximum length of the covered SSR is less than 120bp, the maximum upper limit of the number of specific similar fragments on the reference genome is less than 5, the maximum distance from the designed region is less than 10bp, and design a 110bp probe sequence.

[0123] Transgenic probe design method: Design probes at the junction of maize reference sequence and exogenous DNA sequence, as well as at the exogenous DNA sequence. The GC content of the sequence should be 30-70% (relaxed to 20-80% for some important sites), the maximum length of the covered SSR should be less than 120bp, the maximum upper limit of the number of specific similar fragments on the reference genome should be less than 1, and the maximum distance from the designed region should be less than 10bp. Design a 110bp probe sequence.

[0124] Probes for detecting 11,001 SNP sites and 36 InDel sites were loaded onto a chip to obtain maize DH breeding liquid phase chip one; probes for detecting 11,001 SNP sites, 36 InDel sites and 4 transgenic events were loaded onto a chip to obtain maize DH breeding liquid phase chip two.

[0125] The liquid-phase chip loci in this embodiment are derived from the resequencing results of 507 maize varieties and numerous SNPs or InDel loci related to important agronomic traits, quality, and stress resistance reported in the literature. The probe sources are more extensive, and the probe sequences are designed based on the B73 genome (B73 RefGen_v4) updated using third-generation sequencing technology, resulting in more accurate probe sequences. Compared to existing DH breeding chips with insufficient polymorphic loci, this chip has moderate specifications, low cost, and can be better applied to DH line identification.

[0126] Example 2:

[0127] A method for detecting maize samples using liquid chromatography-mass spectrometry (LC-MS) in maize DH breeding includes the following steps:

[0128] 1. Extract genomic DNA from the sample and construct a library of the sample:

[0129] 1) Preparation of DNA from maize samples:

[0130] DNA is extracted from the target sample using a DNA extraction kit or the CTAB method.

[0131] 2) Sample DNA quality control:

[0132] The DNA concentration of the test samples was determined using a Qubit Fluorometric Quantitation (Thermo Fisher) instrument, and the integrity of the DNA was detected by 1% agarose gel electrophoresis. Samples that passed the tests were stored at 4°C for future use.

[0133] 3) Sample DNA fragmentation:

[0134] Take 12 μL of qualified DNA and place it in a 0.2 μL PCR tube. Place the tube in an ultrasonic disruptor to randomly break the DNA into fragments of 200–400 bp.

[0135] 4) Sample end repair

[0136] Add 7 μL of GenoBaits End Repair Buffer (GenoBaits, i.e., Shijiazhuang Borui Biotechnology Co., Ltd.) and 1.2 μL of GenoBaits End Repair Enzyme to the tube, add water to 20 μL, mix well, centrifuge briefly, and incubate at 25°C for 20 minutes (82°C hot lid) and 72°C (82°C hot lid) for 20 minutes to complete the end repair and A addition process of the fragmented fragments.

[0137] 5) Sample sequencing adapter connection

[0138] Remove the tube from the PCR instrument and add 2 μL of GenoBaits Ultra DNA Ligase and 8 μL of GenoBaits Ultra DNA Ligase.

[0139] Add 2 μL of buffer and 2 μL of GenoBaits Adapter, bring the total volume to 40 μL, and then place the mixture on an ABI 9700 PCR instrument at 25°C for 30 minutes to complete the ligation of the sequencing adapter.

[0140] 6) Sample DNA purification

[0141] Add 20 μL of GenoPrep DNA Clean Beads to the ligation product to purify it. After purification, use magnetic beads to screen the fragments and retain the ligation products with insert fragments of 200-300 bp.

[0142] 7) Sample library amplification

[0143] Add 5 μL of sequencing adapter with barcode sequence, 1 μL of P5 adapter, and 10 μL of LevoBaits PCR Master Mix to the PCR tube from the previous step, and bring the volume to 20 μL with pure water. Amplify using a BIO-RAD S1000 PCR instrument. The amplification program is: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 30 s, 65℃ annealing for 30 s, and 72℃ extension for 40 s. Repeat steps 2-4 for a total of 8 cycles. Finally, extend at 72℃ for 4 min. Different barcodes are used to distinguish different samples.

[0144] 8) Sample library purification

[0145] Add 24 μL of GenoPrepDNA Clean Beads to the second round of PCR products, pipette and mix well, then place the 0.2 μL PCR tube on a magnetic rack until the solution is clear. Discard the supernatant and wash the magnetic beads once with 80% ethanol. Elute the library DNA with Tris-HCl at pH 8.0.

[0146] 2. The genotypes of all loci in the target plant were determined using the liquid-phase gene chip prepared in Example 1:

[0147] 1) DNA hybridization

[0148] Take 500 ng of the constructed genomic DNA sequencing library, add 5 μL of GenoBaits Block I and 2 μL of GenoBaits Block II and 3 μL of liquid chromatography-mass spectrometry (LC-MS), place it on an Eppendorf Concentrator plus vacuum concentrator (Eppendorf), concentrate to dryness at ≤30°C, and centrifuge at 12000 rpm for 1 minute. Add 16 μL of GenoBaits 2x Hyb Buffer, 4 μL of GenoBaits Hyb Buffer Enhancer, and 12 μL of Nuclease-Free Water to the dry powder tube, mix well with a pipette, and incubate at 95°C for 10 minutes on a BIO-RAD S1000 PCR instrument. Vortex to mix well and incubate at 65°C for 2 hours on a BIO-RAD S1000 PCR instrument to complete the probe hybridization reaction.

[0149] 2) DNA capture

[0150] Add 50 μL of GenoBaits DNA Probe Beads to the reaction mixture from the previous hybridization step, pipette up and down 10 times, and incubate at 65°C for 45 minutes in a BIO-RAD S1000 PCR instrument to allow the magnetic beads to bind to the probe. Wash the probe-bound magnetic beads with 100 μL GenoBaits Wash Buffer I and 150 μL GenoBaits Wash Buffer II at 65°C, then wash them again at room temperature with 100 μL GenoBaits Wash Buffer I, 150 μL GenoBaits Wash Buffer II, and 150 μL GenoBaits Wash Buffer III. Resuspend the washed magnetic beads in 20 μL Nuclease-Free Water.

[0151] Add 10 μL of resuspended DNA (with magnetic beads) to a new 0.2 mL PCR tube, then add 15 μL GenoBaits PCR Master Mix and 1.2 μL GenoBaits Primer Mix to prepare a post-PCR system. Perform library amplification using a BIO-RADS1000 PCR instrument. The amplification program is as follows: 98℃ pre-denaturation for 45 s, 98℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 30 s; repeat steps 2-4 for a total of 13 cycles; extend at 72℃ for 1 min.

[0152] Add 45 μL to the post-PCR product DNA Clean Beads were pipetted and mixed thoroughly. A 0.2 mL PCR tube was then placed on a magnetic rack until the solution became clear. The supernatant was discarded, and the magnetic beads were washed twice with 80% ethanol. The library DNA was then eluted with 35 μL of 10 mM Tris-HCl at pH 8.0. This completed the probe hybridization and capture process.

[0153] 3) Quality control of DNA hybridization capture library

[0154] The DNA concentration of the library was determined using Qubit Fluorometric Quantitation (Thermo Fisher), and then agarose gel electrophoresis was used to detect whether the fragment size of the library DNA was between 300 and 400 bp.

[0155] 4) DNA hybridization capture library sequencing:

[0156] The constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

[0157] 5) Genotype data analysis:

[0158] After the sequencing data underwent quality control using Fastp (version 0.20.0, parameters: -n 10-q 20-u 40), the sequencing data was aligned to the reference genome using the default parameters of BWA (mem alignment method). Variation detection was performed using GATK's best practice bioinformatics analysis tools, and a self-written Perl script was used to extract the genotyping information from the probe-captured sequencing data to form the final genotyping file.

[0159] Example 3:

[0160] Recombination heatmap of maize DH breeding liquid phase chip applied to DH identification materials:

[0161] Using the method described in Example 2, the genotypes of DH line materials were detected using the maize DH breeding liquid phase chip (maize DH breeding liquid phase chip one or maize DH breeding liquid phase chip two) prepared in Example 1, thereby determining their recombination status. Based on the detected genotype results, a recombination heatmap was plotted, as shown below. Figure 2 As shown. Figure 2 The image shows the recombination of 200 DH-based materials, with each column representing a sample and each row representing a SNP site.

[0162] from Figure 2 As can be seen, the genotypes at different loci on different chromosomes of each material are derived from parent A (shown in blue in the figure) or parent B (shown in red in the figure).

[0163] Example 4:

[0164] The application of DH breeding liquid phase chip in maize for identifying maize heterosis groups and selecting superior maize inbred lines:

[0165] Using the method described in Example 2, the maize DH breeding liquid phase chip (maize DH breeding liquid phase chip one or maize DH breeding liquid phase chip two) prepared in Example 1 was used for genotyping to analyze DH line diversity and phylogenetic relationships. The results are as follows: Figure 3 As shown, determining which hybrid vigor group a material belongs to provides information for creating new materials.

[0166] from Figure 3 It can be seen that the tested DH materials mainly come from the SS and NSS groups. By utilizing the heterosis between the two groups, i.e., selecting materials from the two groups, new germplasm materials can be created. At the same time, the figure also shows that when two materials partially overlap or are very close together, it indicates that the two materials are closely related; conversely, if they are far apart, the two materials are more distantly related.

[0167] Example 5:

[0168] The DH breeding liquid phase chip for maize is used to detect transgenic events, in order to determine whether maize contains transgenic events nCX-1, GAB3, KN13, and KN19.

[0169] Using the method described in Example 2, the DH material of the maize DH breeding liquid phase chip (maize DH breeding liquid phase chip II) prepared in Example 1 was used for genotyping.

[0170] In this embodiment, for the two stress resistance genes KN13 and KN19, one homozygous positive (KN13_P, KN19_P) and one heterozygous positive (KN13_H, KN19_H) sample were selected. For nCX-1 and GAB3, three homozygous positive (P1, P2, P3) and three heterozygous positive (H1, H2, H3) samples were selected, along with seven transgenic negative samples (N1-N7). Genotyping was performed using a maize DH breeding liquid phase chip. The results are shown in Table 4.

[0171] Table 4

[0172]

[0173] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting molecular marker combinations in a maize haploid-diploid gene breeding chip, characterized in that, The molecular marker combination includes 11,001 SNP sites, 36 InDel sites, and 4 transgene event markers; The physical locations of the 11,001 SNP sites and 36 InDel sites were determined based on whole-genome sequence alignment of the maize reference genome B73v4; the 11,001 SNP sites are shown in Table 1; the 36 InDel sites are shown in Table 2; and the information on the four transgenic event markers is shown in Table 3. Table 1 Table 2 Table 3 2. A chip for mono- and diploid breeding of maize, characterized in that, The maize haploid and diploid breeding chip includes probes for detecting 11,001 SNP sites, 36 InDel sites, and 4 transgenic event markers as described in claim 1.

3. The corn mono- and diploid breeding chip according to claim 2, characterized in that, The probe sequence has a GC content of 30-70%, and the maximum length of the covered SSR is less than 120bp, with a length of 110bp.

4. The application of the maize monoploid and diploid breeding chip as described in claim 2 in the identification of maize monoploid and diploid systems.

5. The application of the maize haploid and diploid breeding chip as described in claim 2 in the identification of maize haploid and diploid hybrid dominant groups.

6. The application of the maize haploid and diploid breeding chip as described in claim 2 in the monitoring of maize haploid and diploid transgenic organisms.

7. The application of the maize monoploid and diploid breeding chip as described in claim 3 in the identification of maize monoploid and diploid systems.

8. The application of the maize haploid and diploid breeding chip as described in claim 3 in the classification of maize haploid and diploid hybrid dominant groups.

9. The application of the maize haploid and diploid breeding chip as described in claim 3 in the monitoring of maize haploid and diploid transgenic organisms.

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