Corn nitrogen utilization rate related 5K breeding chip

Abstract

The invention discloses a corn nitrogen utilization rate related 5K breeding chip, the chip comprises 5,923 SNP (Single Nucleotide Polymorphism) sites and 43 InDel sites, and the SNP sites are uniformly distributed on 10 chromosomes and part of scaffold of a corn genome and have rich polymorphism in all test varieties. Compared with an existing corn chip, the corn nitrogen utilization rate related 5K breeding chip is lower in cost, the background SNP marker of the corn nitrogen utilization rate related 5K breeding chip is derived from re-sequencing results of 507 corn varieties, and the corn nitrogen utilization rate related 5K breeding chip has better variety representativeness; the chip comprises 476 nitrogen utilization efficiency related sites and 96 maize variety authenticity identification SNPs, and can be more effectively used for analysis of germplasm material nitrogen utilization efficiency related genes, functional gene identification, variety authenticity identification and the like.

Description

Technical Field

[0001] This invention belongs to the field of gene chip technology, specifically relating to a 5K breeding chip related to nitrogen use efficiency in maize and its application. Background Technology

[0002] Nitrogen is an essential mineral nutrient element for plant growth and development, playing a crucial role in the synthesis of key macromolecules such as nucleic acids, proteins, and hormones. It is also one of the major limiting factors for global food security and environmental protection; nitrogen deficiency restricts plant growth and productivity. To ensure crop yields, my country uses a massive amount of nitrogen fertilizer; however, the nitrogen fertilizer use efficiency of Chinese crops is only 40.2%. The excessive application of nitrogen-based fertilizers causes serious damage to the ecological environment. Against this backdrop, improving crop nitrogen use efficiency (NUE) is particularly critical. Nitrogen use efficiency refers to the yield obtained per unit of nitrogen applied.

[0003] Maize (Zea mays L.), as a major food crop worldwide, plays a pivotal role in global agricultural development and food security. In 2012, maize production surpassed rice production, becoming my country's largest food crop. As one of the most widely cultivated crops globally, advances in maize research on nitrogen absorption, allocation, and utilization have a profound impact on promoting sustainable agricultural development. Therefore, developing a maize chip related to nitrogen use efficiency is of great significance to the seed industry. Summary of the Invention

[0004] The purpose of this invention is to provide a 5K breeding chip related to nitrogen use efficiency in maize.

[0005] To achieve the above objectives, the present invention adopts the following technical measures:

[0006] The loci used in this invention come from three sources: 1) Second-generation resequencing data from 507 maize inbred lines (216 from the International Maize and Wheat Improvement Center, 210 from China, 60 from the United States, and 1 from Peru), representing a high degree of representativeness of maize varieties worldwide, identified 75,680,644 original variant loci (SNPs + InDel). Of these, 10,770,214 loci with only two genotypes, a minor allele frequency greater than 0.05, and a quality fraction greater than or equal to 30 were retained. From these, 3,373 SNP loci with high minor allele frequency, low deletion rate, low heterozygosity, uniform distribution, and unique locus annotation information were selected. 2) Loci related to nitrogen use efficiency, yield, and other traits collected from numerous published studies, significant SNP loci obtained through t-test analysis of genotype and phenotype, and significant loci identified through GWAS, totaling 2,454. 3) 96 SNPs for maize variety authenticity identification. A total of 5,923 SNP sites and 43 InDel sites were finally obtained. Site evaluation of these SNP and InDel sites yielded a total of 5,966 sites, enabling the development of a 5K breeding chip for maize nitrogen use efficiency. The physical locations and variation information of the 5,923 SNPs are shown in Table 1, and the physical location information of the 43 InDel sites is shown in Table 2.

[0007] Compared with the prior art, the present invention has the following advantages:

[0008] The nitrogen use efficiency-related breeding chip loci of this invention are derived from the resequencing results of 507 maize varieties, SNPs and InDel loci related to nitrogen use efficiency and yield that have been reported in numerous literatures, and significant SNP loci related to nitrogen use efficiency obtained through GWAS analysis. The probes are derived from more varieties, and the probe sequences are designed based on the B73 genome (B73 RefGen_v4) updated by third-generation sequencing technology, making the probe sequences more accurate.

[0009] In addition, the 5K breeding chip for maize nitrogen use efficiency includes 96 SNP loci for maize variety authenticity identification and 476 SNP loci related to nitrogen use efficiency, which can better perform nitrogen use efficiency related analysis, screen maize germplasm with high nitrogen use efficiency, and analyze the genetic background of breeding materials, etc. Attached Figure Description

[0010] Figure 1 A schematic diagram showing the distribution of SNP sites on chromosomes in a 5K breeding chip related to nitrogen use efficiency in maize.

[0011] Figure 2 This chip was used in Example 3 for functional gene diagnosis of nitrogen utilization-related phenotypes.

[0012] Figure 3 This is a graph showing the optimal material selection for nitrogen utilization using this chip in Example 4.

[0013] Figure 4 This is a PCA grouping diagram of materials with high and low nitrogen utilization using this chip in Example 5. Detailed Implementation

[0014] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0015] Example 1

[0016] The steps for obtaining the sites related to nitrogen use efficiency in maize using 5K breeding chips are as follows:

[0017] 1) Using the resequencing results of 507 maize inbred lines, original variant sites were identified. Among them, 216 maize ...

[0018] 2) 10,770,214 loci with only two genotypes, a minor allele frequency greater than 0.05 and a quality fraction greater than or equal to 30 were retained. From these, 3,373 SNP loci were selected based on the principles of uniform distribution, high minor allele frequency (MAF), low deletion rate, low heterozygosity, and special annotation information.

[0019] 3) We reviewed the functional sites of genes related to nitrogen use efficiency in published literature and obtained a total of 476 important SNPs and InDel sites, such as NRT1.1A, NRT1.1B, MYB32, etc., through genome-wide association analysis (GWAS). The set of these sites is shown in Table 3.

[0020] 4) Added 96 SNP loci for authenticity identification of maize varieties (referencing the People's Republic of China Agricultural Industry Standard NY / T4022-2021);

[0021] These loci were evaluated (evaluation parameters: GC content: 30-70%, number of homologous regions ≤ 5). After multiple optimizations, 5,966 loci were successfully evaluated and used for microarray synthesis, thus developing a maize nitrogen use efficiency-related 5K breeding microarray. The distribution of all SNP loci in the maize nitrogen use efficiency-related 5K breeding microarray across the entire genome is shown below. Figure 1As shown, it is distributed in all segments of the whole genome, and is densely distributed in important functional gene regions, which can meet the needs of identifying functional genes related to nitrogen use efficiency in maize and the identification of maize varieties.

[0022] Table 1. Locations and genotypes of SNP loci in the maize B73_v4 genome from 5K breeding chips related to nitrogen use efficiency in maize.

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] Table 2. Location of the InDel locus in the maize B73_v4 genome from a 5K breeding chip related to maize nitrogen use efficiency.

[0057]

[0058] Table 3. Locations of nitrogen use efficiency-related loci in the maize B73_v4 genome from the 5K breeding chip.

[0059]

[0060] Example 2

[0061] A method for detecting maize samples using a 5K breeding chip related to maize nitrogen use efficiency includes the following steps:

[0062] 1. Extract genomic DNA from the sample and construct a library of the sample.

[0063] 1) Preparation of DNA from maize samples

[0064] DNA is extracted from the target sample using a DNA extraction kit or the CTAB method.

[0065] 2) Sample DNA quality inspection

[0066] The DNA concentration of the test samples was determined using a qubit fluidometric quantitation (Thermo Fisher) instrument, and the integrity of the DNA was detected by 1% agarose gel electrophoresis. Samples that passed the test were stored at 4°C for future use.

[0067] 3) Sample DNA fragmentation

[0068] Take 10 μL of qualified DNA and place it in a 0.2 mL PCR tube. Place the tube in an ultrasonic disruptor to randomly break the DNA into fragments of 200–400 bp.

[0069] 4) Sample end repair

[0070] Add 8 μL of GenoBaits End Repair Buffer and 1.2 μL of GenoBaits End Repair Buffer to the PCR tube.

[0071] Repair Enzyme (provided by Shijiazhuang Borui Biotechnology Co., Ltd.), add water to 20 μL, mix well, briefly centrifuge, and incubate in a BIO-RAD S1000 PCR instrument at 25°C for 20 minutes (82°C hot lid), then at 72°C.

[0072] (82℃ hot cover) Incubate for 20 minutes to complete the end repair and A-addition process of the broken fragments.

[0073] 5) Sample sequencing adapter connection

[0074] Remove the PCR tube from the PCR instrument and add 2 μL of GenoBaits Ultra DNA ligase and 8 μL of...

[0075] GenoBaits Ultra DNA Ligase Buffer and 2 μL GenoBaits Adapter (provided by Shijiazhuang Borui Biotechnology Co., Ltd.) were added, and the volume was increased to 40 μL. The mixture was then placed on an ABI 9700 PCR instrument and reacted at 25°C.

[0076] The sequencing adapter connection was completed in 30 minutes.

[0077] 6) Sample DNA purification

[0078] Add 20 μL of GenoPrep DNAClean 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.

[0079] 7) Sample library amplification

[0080] 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 as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 8 cycles; 72℃ extension for 4 min. Different barcodes are used to distinguish different samples.

[0081] 8) Sample library purification

[0082] 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.

[0083] 2. The genotypes of all loci in the target plant were determined using the liquid-phase gene chip prepared in Example 1.

[0084] 1) DNA hybridization

[0085] Take 500 ng of the constructed genomic DNA sequencing library, add 5 μL of GenoBaits Block I and 2 μL of GenoBaits Block II (provided by Shijiazhuang Borui Biotechnology Co., Ltd.), and place it on an Eppendorf Concentrator plus vacuum concentrator (Eppendorf) at ≤30℃ to concentrate to dryness. Add 16 μL of GenoBaits 2×Hyb Buffer, 4 μL of GenoBaits Hyb Buffer Enhancer (provided by Shijiazhuang Borui Biotechnology Co., Ltd.), and 12 μL of Nuclease-Free Water to the dry powder tube, mix well with a pipette, and incubate at 95℃ for 10 minutes on a BIO-RAD S1000 PCR instrument. Then, remove the PCR tube and add 3 μL of the synthesized probe (the concentration of the probe is 60 ng / μL), vortex to mix well, and incubate at 65℃ for 2 hours on a BIO-RAD S1000 PCR instrument to complete the probe hybridization reaction.

[0086] 2) DNA capture

[0087] 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 with 150 μL GenoBaits Wash Buffer I, 150 μL GenoBaits Wash Buffer II, and 150 μL GenoBaits Wash Buffer III at room temperature. Resuspend the washed magnetic beads in 20 μL Nuclease-Free Water.

[0088] Add 10 μL of resuspended DNA (with magnetic beads) to a new 0.2 mL PCR tube, then add...

[0089] Prepare a post-PCR system using 15 μL GenoBaits PCR Master Mix and 1.2 μL GenoBaits Primer Mix, and amplify the library using a BIO-RAD S1000 PCR instrument. The amplification program is: 98℃ pre-denaturation for 45 s.

[0090] 98℃ denaturation for 15s, 60℃ annealing for 30s, 72℃ extension for 30s, for a total of 13 cycles; 72℃ extension for 1min.

[0091] Add 45 μL to the post-PCR product DNA Clean Beads were pipetted and mixed thoroughly. Then, a 0.2 mL PCR tube was 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. The probe hybridization capture was completed.

[0092] 3) Quality control of DNA hybridization capture library

[0093] 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.

[0094] 4) DNA hybridization capture library sequencing

[0095] The constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

[0096] 5) Genotype data analysis

[0097] 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, probes were extracted to capture the genotyping information of the sequencing data, and the final genotyping file was generated.

[0098] Example 3

[0099] The application of 5K breeding chips related to nitrogen use efficiency in maize for functional gene diagnosis of nitrogen use efficiency-related phenotypes:

[0100] Using the method described in Example 2, a 5K breeding chip related to maize nitrogen use efficiency was applied to detect the target materials and obtain the genotyping results. Based on the nitrogen use efficiency-related loci listed in Table 3, the target materials were diagnosed. If the material contained the superior allele of that locus, it was coded as 1; otherwise, if the material did not contain the superior allele of that locus, it was coded as 0. The functional gene diagnostic results are shown in [Table 3]. Figure 2 Each row represents one material, and each column represents one nitrogen utilization-related gene. The graph shows whether each material contains superior alleles of different nitrogen utilization-related genes. For example, material L237 contains superior alleles of 7 genes: MYB32, MYB85, NAC85, NACTF131, NACTF32, NRT1.1A, and NRT1.1B. Furthermore, among the 29 materials, only L471 and L360 contain the superior allele of the NRT7 gene.

[0101] Example 4

[0102] A 5K breeding chip related to nitrogen use efficiency in maize is used for screening materials with optimal nitrogen use efficiency.

[0103] Using the method described in Example 2, a 5K breeding chip related to maize nitrogen use efficiency was applied to detect the target materials and obtain the genotyping results. Based on the superior alleles of each gene's functional locus, the target materials were diagnosed. Then, for each material's genotyping results, the number of optimal alleles in each material was calculated. Finally, the number of superior alleles in all materials was compared, thereby selecting the material with the highest number of favorable alleles (e.g., maize). Figure 3 ).from Figure 3As can be seen, the material in the top right corner contains the most favorable alleles, therefore it is the best material.

[0104] Example 5

[0105] The application of a 5K breeding chip related to nitrogen use efficiency in maize for grouping nitrogen use efficiency materials:

[0106] Using the method described in Example 2, a 5K breeding chip for maize nitrogen use efficiency was applied to group 30 known high nitrogen use efficiency materials and 30 low nitrogen use efficiency materials, obtaining the PCA results of the material groupings (e.g., ...). Figure 4 The criteria for defining high nitrogen use efficiency materials are: ear weight (g) / (240kg / ha) > 0.016, and the criteria for defining low nitrogen use efficiency materials are: ear weight (g) / (240kg / ha) < 0.011. Figure 4 As can be seen, the two groups of materials with high and low nitrogen utilization rates can be well grouped by the genotypes obtained from the 5K breeding chip, verifying the chip's usability.

[0107] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. For those skilled in the art, modifications to these embodiments without contributing any inventive step can be made as needed after reading this specification, and such modifications should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A combination of molecular markers characterized in that, The molecular markers include 5,923 SNP sites and 43 InDel sites, and the position information of the SNP and InDel sites on the maize B73_RefGen_v4 genome is shown in Table 1 and Table 2.

2. A nucleotide probe combination for detecting the molecular marker combination of claim 1.

3. A 5K breeding chip related to corn nitrogen use efficiency, characterized in that, The gene chip is loaded with the nucleotide probe combination of claim 2.

4. Use of the marker combination of claim 1 or the probe combination of claim 2 or the chip of claim 3 in maize breeding.

5. Use of the marker combination of claim 1 or the probe combination of claim 2 or the chip of claim 3 in maize nitrogen utilization functional gene identification.

6. Use of the marker combination of claim 1 or the probe combination of claim 2 or the chip of claim 3 in screening of maize nitrogen utilization functional gene favorable allele materials.

7. Use of the marker combination of claim 1 or the probe combination of claim 2 or the chip of claim 3 in maize variety authenticity identification.

Images