Corn genome breeding 45K chip and application thereof

Abstract

The invention discloses a corn genome breeding 45K chip and application thereof, the chip covers 44, 637 SNP loci and 27 InDel loci, and the SNP loci are uniformly distributed on 10 chromosomes and part of scaffold of a corn genome and have rich polymorphism. Compared with an existing corn chip, the SNP marker of the 45K chip is derived from re-sequencing results of 507 corn varieties, has better variety representativeness, and contains 925 key functional gene loci for close planting and high yield and 96 corn variety authenticity identification loci; the method can be more effectively used for functional gene identification, genetic diversity analysis, heterosis group division, whole genome selection analysis and variety authenticity identification.

Description

Technical Field

[0001] This invention belongs to the field of crop genome breeding technology, specifically involving a 45K chip for maize genome breeding and its application. Background Technology

[0002] The development of SNP markers is based on DNA sequencing. In the more than ten years since the advent of the 454 sequencer in 2005, second-generation sequencing technology has been continuously developed and improved, greatly increasing the efficiency of genome sequencing, significantly reducing sequencing costs, and completing the whole genome sequences of a large number of species, which has greatly promoted the progress of functional genomics research.

[0003] Maize (Zea mays L.), as a major food crop worldwide, plays a crucial role in global agricultural development and food security. In 2012, maize production surpassed rice production, becoming my country's largest food crop. However, there is still significant room for improvement in my country's maize yield. Therefore, in the short term, developing high-yield, densely planted maize varieties is a goal pursued by breeders. With the advent of Breeding 4.0 and the continuous reduction in sequencing costs, genome breeding applications have emerged, which can be considered a product of the high-throughput sequencing era.

[0004] This invention screened 42,445 SNP loci based on second-generation resequencing data from 507 maize inbred lines. To better apply this to maize genome breeding, it also designed functional loci for published functional genes, unpublished and validated QTL loci, and important loci related to high-yield traits in dense planting identified by t-tests based on collected genotype and phenotypic data. These include functional loci for genes affecting yield (YIGE1, KRN5b, ZmEXPB15, etc.), plant architecture (Br2, UPA1, etc.), and flowering time (ZmSPL13, etc.), as well as loci significantly influencing traits. These loci are evenly distributed across the 10 chromosomes and part of the scaffold of the maize genome, exhibiting rich polymorphism in the tested varieties. Therefore, the 45K chip developed in this invention has excellent application prospects in maize genome breeding and genome research. Summary of the Invention

[0005] This invention protects a 45K chip for maize genome breeding.

[0006] In another aspect, this invention protects the use of the maize genome breeding 45K chip in maize breeding, functional gene identification, genetic diversity analysis, whole genome selection analysis, and heterosis grouping.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] The loci detected in the 45K chip for maize genome breeding came from five parts: 1) resequencing data from 507 maize inbred lines, including 216 from the International Maize and Wheat Improvement Center, 210 from China, 60 from the United States, and 1 from Peru, representing very good global maize varieties; among them, 126 materials belonged to the NSS group, 32 to the SS group, and 201 to the tropical group. A total of 75,680,644 original variant SNP loci were identified, and 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. Functional annotation of these variant loci was performed using ANNOVAR software, and they were classified according to uniform distribution, 42,445 SNP loci were selected based on the principles of high minor allele frequency (MAF), low deletion rate, low heterozygosity, and potential variation sites that may affect gene function in functional annotation; 2) A large number of cloned genes in published literature were reviewed, and important SNPs and InDel loci affecting yield (YIGE1, KRN5b, ZmEXPB15, etc.), plant type (Br2, TSH4, etc.), and flowering time (ZmSPL13, ZmCCT9, etc.) were obtained through analysis; 3) Newly discovered and validated candidate QTL loci; 4) Loci with significant trait influence were identified by t-test analysis based on multi-year and multi-location phenotypic and genotypic data collected in the previous period; 5) 96 SNP loci for maize variety authenticity identification. Finally, 44,637 SNP sites and 27 InDel sites were obtained. Probes were designed for these SNP sites and InDel sites, thereby developing a 45K chip for maize genome breeding. The physical locations of the 44,637 SNP sites are shown in Table 1, and the physical locations of the 27 InDel sites are shown in Table 2.

[0009] Applications of the 45K chip in maize genome breeding include:

[0010] (1) Diagnosis of functional genes in maize;

[0011] (2) Analysis of genetic diversity in maize;

[0012] (3) Corn kinship identification;

[0013] (4) Classification of heterotic groups in maize;

[0014] (5) Genome-wide selection analysis;

[0015] (6) Identification of variety authenticity.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The maize genome breeding 45K chip loci developed in this invention 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. In addition, the chip contains 2123 loci affecting important maize traits and 96 SNP loci for authenticity identification in maize varieties, enabling more effective identification of functional genes and genetic background analysis of breeding materials. Attached Figure Description

[0018] Figure 1 A schematic diagram of the distribution of SNP sites on the whole genome in maize genome breeding using the 45K chip.

[0019] Figure 2 Distribution of minor allele frequencies (MAF) at SNP sites on the 45K chip for maize genome breeding and distribution of molecular marker polymorphism information (PIC).

[0020] Figure 3 A schematic diagram showing the results of applying the 45K chip to hybrid vigor grouping in maize genome breeding.

[0021] Figure 4 A schematic diagram showing the results of applying the 45K chip in maize genome breeding to identify maize kinship.

[0022] Figure 5 A schematic diagram showing the results of applying the 45K chip to maize genome selection in maize genome breeding.

[0023] Figure 6 A schematic diagram of the results of using a 45K chip for functional gene diagnosis in maize genome breeding. Detailed Implementation

[0024] 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.

[0025] Example 1

[0026] The steps for obtaining loci from the 45K chip in maize genome breeding are as follows:

[0027] 1) Using resequencing data from 507 maize inbred lines, including 216 from the International Maize and Wheat Improvement Center, 210 from China, 60 from the United States, and 1 from Peru, the data are highly representative of maize varieties worldwide. Among them, 126 materials belong to the NSS group, 32 materials belong to the SS group, and 201 materials belong to the tropical group. A total of 75,680,644 original variant SNP sites were identified.

[0028] 2) A total of 10,770,214 SNP sites with only two genotypes, a minor allele frequency greater than 0.05, and a quality score (a value used in SNP calling to quantify the reliability and accuracy of each SNP site) greater than or equal to 30 were retained. These variant sites were functionally annotated using ANNOVAR software. From these sites, 42,445 SNP sites were selected based on the following principles: uniform distribution, high minor allele frequency (MAF), low deletion rate, low heterozygosity, and variant sites that may affect gene function in the functional annotation (such as annotated information such as alternative splicing, premature termination, nonsynonymous mutation, etc.).

[0029] 3) Review a large number of published literatures on cloned genes and analyze them to obtain SNP and InDel sites that affect important traits such as yield (YIGE1, KRN5b, ZmEXPB15, etc.), plant type (Br2, TSH4, etc.) and flowering time (ZmSPL13, ZmCCT9, etc.).

[0030] 4) Unpublished and validated candidate QTL sites from the inventor's laboratory;

[0031] 5) Based on the phenotypic and genotypic data collected by the inventor's laboratory over many years and from multiple locations, the loci that significantly affect the trait were identified through t-test analysis;

[0032] Steps 3) through 5) identify a total of 925 loci for important traits;

[0033] 6) Added 96 SNP loci for authenticity identification of maize varieties (referencing the People's Republic of China Agricultural Industry Standard NY / T4022-2021);

[0034] Probe design was performed on these sites (parameters included: probe length 110 bp, GC content: 30-).

[0035] 70% (with ≤5 homologous regions), 44,664 loci were successfully evaluated. Specific locus information is shown in Tables 1 and 2.

[0036] As shown, the developed chip is named the Maize Genome Breeding 45K Chip. The distribution of all SNP loci across the entire genome on this chip is as follows: Figure 1As shown, it is generally distributed across all segments of the genome, and is also densely distributed in important functional gene regions; and according to Figure 2 The distribution of MAF and molecular marker polymorphism are shown.

[0037] The distribution of information (PIC) shows that the SNP sites have good polymorphism and are highly representative.

[0038] Table 1. Locations and genotypes of SNP sites in the maize B73_v4 genome from the 45K chip for maize genome breeding.

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[0280] Table 2. Location of InDel sites in the maize B73_v4 genome from the 45K chip for maize genome breeding.

[0281]

[0282] Example 2

[0283] The method for detecting the genotype of maize samples using a 45K chip for whole-genome breeding includes the following steps: 1. Extracting genomic DNA from the sample and constructing a sequencing library of the sample.

[0284] 1) Preparation of DNA from maize samples

[0285] DNA was extracted from the target sample using a DNA extraction kit or the CTAB method.

[0286] 2) Sample DNA quality inspection

[0287] The concentration of DNA in the samples was determined using qubit fluidometric quantitation (Thermo Fisher), 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.

[0288] 3) Sample DNA fragmentation

[0289] Take 12 μL of qualified DNA and place it in a 0.2 μL PCR tube. Place the PCR tube in an ultrasonic disruptor to randomly break the DNA into fragments of 200–400 bp.

[0290] 4) Sample end repair

[0291] Add 7 μL of GenoBaits End Repair Buffer and 1.2 μL of GenoBaits to the PCR tube.

[0292] End Repair Enzyme (provided by Shijiazhuang Borui Biotechnology Co., Ltd.), replenishes moisture to

[0293] 20 μL, mix well, centrifuge briefly, and incubate in a BIO-RAD S1000 PCR instrument at 25°C and 72°C for 20 minutes to complete the end repair of fragmented fragments and the addition of adenine (A).

[0294] 5) Sample sequencing adapter connection

[0295] Remove the PCR tube from the PCR instrument and add 2 μL of GenoBaits Ultra DNA ligase and 8 μL of...

[0296] GenoBaits Ultra DNA Ligase Buffer and 2 μL GenoBaits Adapter (provided by Shijiazhuang Borui Biotechnology Co., Ltd.) were added to a final volume of 40 μL and then placed on an ABI 9700 PCR instrument at 25°C for 30 minutes to complete the ligation of the sequencing adapter.

[0297] 6) Sample DNA purification

[0298] 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.

[0299] 7) Sample library amplification

[0300] Add 1.2 μL GenoBaits Primer Mix for ILM, 10 μL Beadswith Captured DNA, and 15 μL GenoBaits PCR Master Mix to the PCR tube from the previous step, and bring the volume to 30 μL with pure water; perform amplification using a BIO-RADS1000 PCR instrument with the following amplification program: 98℃ pre-denaturation for 45 s;

[0301] Denaturation at 98℃ for 15 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, for a total of 13 cycles; extension at 72℃ for 1 min. 2. Genotyping of all loci in the target plant was determined using the liquid-phase gene chip prepared in Example 1.

[0302] 1) DNA hybridization

[0303] 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) to concentrate to dryness at ≤30℃. 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.

[0304] 2) DNA capture

[0305] Add 50 μL of GenoBaits DNA Probe Beads to the reaction system 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 at room temperature with 100 μL GenoBaits Wash Buffer I, 150 μL GenoBaits Wash Buffer II, and 150 μL GenoBaits Wash Buffer III (provided by Shijiazhuang Borui Biotechnology Co., Ltd.). Resuspend the washed magnetic beads in 20 μL of Nuclease-Free Water.

[0306] 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 (provided by Shijiazhuang Borui Biotechnology Co., Ltd.) to prepare a post-PCR system. Use a BIO-RAD S1000 PCR instrument for library amplification. 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, for a total of 13 cycles; 72℃ extension for 1 min.

[0307] 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.

[0308] 3) Quality control of DNA hybridization capture library

[0309] 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.

[0310] 4) DNA hybridization capture library sequencing

[0311] The constructed DNA library was sequenced using the BGI DNBSEQ-T7 sequencer.

[0312] 5) Genotype data analysis

[0313] 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 maize reference genome (B73v4) using the default parameters of BWA (mem alignment method). Variation detection was performed using GATK4, a best-practice bioinformatics analysis tool, and probes were extracted to capture the genotyping information of the sequencing data, forming the final genotyping file.

[0314] Example 3

[0315] 45K chip for maize genome breeding applied to maize heterosis grouping:

[0316] Using the method described in Example 2, the maize genome breeding 45K chip was applied to detect the germplasm materials. After obtaining the genotyping results, the data was filtered and the gcta (version 1.94) software was used to calculate the eigenvalues ​​and eigenvectors. The PCA diagram was drawn using R to determine which heterotic group it belonged to, which can provide important information for the next step of hybridization.

[0317] Analysis showed that 53 materials belonged to the SS group, 129 to the NSS group, 201 to the TST group, 15 to the Reid group, 21 to the HG group, 4 to the P group, and 18 to the X group. Figure 3 The clustering results match the breeder's clustering information. Based on these results, this chip can be extended to all maize inbred line materials for heterosis clustering, assisting breeders in creating higher-quality hybrids.

[0318] Example 4

[0319] 45K chip for maize genome breeding applied to phylogenetic identification of maize materials:

[0320] Using the method described in Example 2, the maize genome breeding 45K chip was applied to detect germplasm materials. After obtaining the genotyping results, the phylogenetic relationships between pairs of materials were calculated, and the results were obtained from the heatmap. Figure 4 It can be seen that JING724 and JING725 are closely related, HYS and HZS are also closely related, while HZS and JB materials are not closely related.

[0321] Example 5

[0322] 45K chip applied to maize genome selection in maize genome breeding:

[0323] Using the method described in Example 2, 1200 CUBIC (Complete-diallel plus Unbalanced Breeding-derived Inter-Cross) materials were analyzed using a maize genome breeding 45K chip. After obtaining the genotyping results, 10-fold cross-validation was performed, repeated 100 times. Different indices of yield (EL, ED, ERN, KNPR, KWPE), plant architecture (PH, EH, TBN, TL), flowering time (DTT, DTA, DTS), and disease resistance (SCR, SLB, NLB) were predicted using rrBLUP and GBLUP models. The results (see [link to example]) are shown in [link to example]. Figure 5 Among yield traits, ERN (rows per ear) showed the highest predictive ability; among plant type traits, TL (tassel length) showed the highest predictive ability; among flowering time, DTT (tasseling time) showed the highest predictive ability; and among disease resistance traits, SCR (southern rust) showed the highest predictive ability. Of the 15 trait indicators, TL (tassel length) showed the highest predictive ability, reaching 0.729, close to the theoretical value. Therefore, the maize genome breeding 45K designed by the inventors can be well applied to the breeding of different traits in maize.

[0324] Example 6

[0325] 45K microarrays used in maize genome breeding for functional gene diagnosis:

[0326] Using the method described in Example 2, the genotypes of six germplasm inbred lines were detected by applying the maize genome breeding 45K chip, thereby diagnosing whether they contained superior alleles of relevant important genes.

[0327] The applicant used the chip to test six maize inbred lines. Genotypic analysis revealed that all six inbred lines contained the superior allele of the YIGE1 gene, while only two lines contained the superior allele of the Br2 gene. For detailed gene diagnostic results, please refer to [link to relevant documentation]. Figure 6 The results show that when materials numbered 1 and 2 are crossed, all nine genes shown in the diagram are superior alleles, thus achieving the goal of multi-gene aggregation. Similarly, crossing materials numbered 5 and 4, and then crossing the offspring with material numbered 1, yields materials with all nine genes being superior alleles, achieving multi-gene aggregation. Therefore, this chip can diagnose functional genes, laying a solid foundation for multi-gene aggregation.

[0328] 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 44,637 SNP sites and 27 InDel sites. The location information of the SNP and InDel sites on the maize B73_RefGen_v4 genome is shown in Table 1 and Table 2.

2. A set of nucleotide probes for detecting the molecular marker combination as described in claim 1.

3. A 45K chip for whole-genome breeding of maize, characterized in that... The gene chip is loaded with the nucleotide probe combination as described in claim 2.

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

5. The application of the marker combination of claim 1, the probe combination of claim 2, or the chip of claim 3 in the identification of functional genes in maize.

6. The application of the marker combination of claim 1, the probe combination of claim 2, or the chip of claim 3 in the analysis of maize genetic diversity.

7. The application of the marker combination of claim 1, the probe combination of claim 2, or the chip of claim 3 in the identification of maize kinship or the classification of heterotic groups.

8. The application of the marker combination of claim 1, the probe combination of claim 2, or the chip of claim 3 in whole-genome selection analysis of maize.

9. The application of the marker combination of claim 1, the probe combination of claim 2, or the chip of claim 3 in the identification of maize variety authenticity.

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