Maize genome-wide SNP site combination, probes, liquid phase chip and its application

Abstract

The present invention discloses a maize genome-wide SNP site combination, probes, liquid-phase microarrays, and their applications. Based on the genome of the maize inbred line T32, the present invention has developed a 10K maize genome-wide liquid-phase microarray. The targeted SNP sites uniformly cover the entire chromosome set, providing a more comprehensive coverage of SNP sites that are key functional molecular markers for most important traits. With a greater number of sites, the detection range is more comprehensive, with a higher detection rate and better reproducibility. The present invention's targeted capture probes based on the 10k SNP site information can quickly and effectively track maize genetic material, accelerating maize breeding and improving the efficiency of new variety selection.

Description

Technical Field

[0001] The present invention relates to the technical field of corn molecular biology, and in particular to a corn whole genome SNP site combination, a probe, a liquid phase chip and applications thereof. Background Art

[0002] Maize (Zea may L.), a member of the genus Zea in the Poaceae family, is a globally cultivated dual-purpose crop for food and feed, possessing significant economic value and a key focus for molecular breeding research. Its kernels are rich in dietary fiber, antioxidants, minerals, and vitamins, playing a vital role in lowering blood sugar and preventing cardiovascular disease, and have long been prized by consumers. With the rapid development of high-throughput sequencing and genome assembly technologies, the release of high-quality maize genome sequence information has advanced our understanding of maize's basic biology and is crucial for genetics, genomics, and molecular breeding research.

[0003] Single Nucleotide Polymorphism (SNP), as a genetic marker that is more widely distributed in the genome, has the characteristics of high density, high genetic stability and ease of automated analysis. It has developed into the most common molecular marker in the study of plant genetic variation. At present, the acquisition of variant genotypes within the whole genome is mainly achieved through resequencing and gene chips. The cost of obtaining variation information through whole genome or simplified genome sequencing (GBS) methods is too high, there are serious site omissions, the amount of sequencing data is relatively large, and the software and hardware requirements for data storage, analysis and calculation are high. The detection quality of variant sites is heavily dependent on the quality of the reference genome. SNP chips have a good detection rate and stability in sample site detection, and they have been developed and applied in crops such as corn, rice, and soybeans, and have made important research progress. Therefore, efficient and low-cost SNP genotyping technology has become the best choice for developing shared technologies and platforms.

[0004] In SNP chip technology, traditional solid-phase chips are based on complementary hybridization between probes and DNA sequences, and typing is performed through the fluorescent colorimetric signal of the marker. These chips can only type the SNP sites contained on the chip, and cannot add or delete SNPs. This leads to poor flexibility and high typing costs. Liquid-phase chips, on the other hand, generally include a biotin-labeled probe covering the target SNP, designed based on the principle of DNA complementarity, for each site to be tested. These probes hybridize to the target region of the genome in liquid form, forming double strands. The adsorption of streptavidin-coated magnetic beads and biotin-containing molecules can be utilized. After elution, amplification, and library construction, second-generation sequencing is performed to ultimately restore the genotype status of the target site and its surrounding SNPs, offering advantages over solid-phase chips.

[0005] Currently, solid-phase microarrays developed based on SNPs obtained through genome-wide association analysis, linkage analysis, and genome sequencing have been used to analyze maize population structure, principal component analysis, genome-wide association analysis, and quantitative trait loci. However, the vast majority of these microarrays are developed based on temperate maize germplasm genomes, while gene chips specifically targeting the genomic characteristics of tropical maize germplasm are still lacking. The SNPs targeted by these microarrays lack the necessary molecular markers, limiting their application in tropical maize genetic improvement. Therefore, new microarrays specifically targeting the genetic characteristics of tropical maize germplasm are needed to meet the needs of tropical maize germplasm resource identification. Summary of the Invention

[0006] Therefore, based on the above background, the present invention provides a corn whole-genome SNP site combination, probe, liquid phase chip and its application. The present invention develops a more comprehensive combination of key functional SNP molecular markers covering the chromosome set and encompassing almost all important traits, thereby developing a SNP site liquid phase chip with a more comprehensive detection range, which can meet the breeding needs of tropical corn.

[0007] The technical solution of the present invention is:

[0008] The whole-genome SNP site combination of corn consists of 10,160 SNP sites. The physical locations and typing information of the 10,160 SNP sites are shown in Table 1 below.

[0009] Based on the same inventive concept, the present invention applies the above-mentioned corn whole genome SNP site combination to the preparation of a corn whole genome liquid phase chip, and the corn whole genome liquid phase chip is used to identify the genotype of each site in the corn whole genome SNP site combination as described in claim 1.

[0010] Based on the same inventive concept, the present invention also provides a 10K targeted capture probe, comprising a corn 10K SNP site probe, wherein the corn 10K SNP site probe is a single-stranded DNA synthesized based on the corn whole genome SNP site combination.

[0011] Furthermore, the 5' end of the probe carries a biotin group, and the probe is coupled to a fluorescent microsphere via a C12 molecular arm and amino modification, with each fluorescent microsphere being coupled to one probe.

[0012] Based on the same inventive concept, the present invention also provides a maize whole-genome liquid phase chip, which includes the targeted capture probe according to claim 2, and the targeted capture probe is used to identify the genotype of the maize whole-genome SNP site according to claim 1.

[0013] Based on the same inventive concept, the present invention provides the application of the corn whole-genome SNP site combination, or the targeted capture probe, or the corn whole-genome liquid phase chip in any of the following applications, including:

[0014] ①Analysis of genetic diversity of maize;

[0015] ②Construction of molecular genetic map of maize;

[0016] ③ Genome-wide association analysis of maize;

[0017] ④ Authenticity identification of maize inbred lines;

[0018] ⑤ Molecular marker-assisted selection breeding of maize;

[0019] ⑥ Whole genome selection breeding of corn;

[0020] ⑦ Kinship identification;

[0021] ⑧ Germplasm resource identification;

[0022] ⑨Gene mapping;

[0023] ⑩Analysis of corn population structure.

[0024] Based on the same inventive concept, the present invention provides a detection method for corn genotyping, which comprises the following steps:

[0025] S1: Obtain genomic DNA of corn samples;

[0026] S2: Detecting the genomic DNA obtained in step S1 using the above-mentioned corn whole genome liquid phase chip;

[0027] S3: Analyze the detection results of step S2 to obtain the genotyping results of corn.

[0028] The beneficial effects achieved by adopting the technical solution of the present invention are:

[0029] The present invention has developed a maize 10K whole-genome liquid phase array. The SNP sites targeted by the array evenly cover the entire chromosome set, encompassing nearly all key functional SNP molecular markers associated with important traits. It has a larger and more comprehensive number of sites and a more comprehensive detection range. It can be applied not only to temperate maize, but also to tropical maize, with a higher detection rate and better repeatability.

[0030] The targeted capture probes involved in the 10k SNP site information of the present invention can quickly and effectively track the genetic material of corn, which is beneficial to accelerate the corn breeding process and improve the efficiency of new inbred line breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Attachment Figure 1 This is a statistical diagram of the distribution of SNP sites targeted by the maize 10k SNP capture probe on different chromosomes; that is, the statistical results of the number of target sites on different chromosomes; the horizontal axis is the chromosome, and the vertical axis is the number of SNP markers on each chromosome;

[0032] Attachment Figure 2 This is a map of uniform distribution of SNP sites (the probe coverage density is calculated with a 0.1 Mb length observation window on the chromosome).

[0033] This map was drawn based on the maize reference genome B73 V5.

[0034] Attachment Figure 3 This is a statistical graph of the detection rate and consistency distribution of repeated samples. This graph is based on the maize reference genome version V5. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] The maize reference genome data of the embodiment is derived from the maize inbred line B73.

[0037] The maize 10k SNP targeted capture probe of this embodiment includes a maize 10k SNP site probe mixture and a hybridization capture reagent.

[0038] The preparation process of the corn 10k SNP targeted capture probe is as follows:

[0039] 1) Screening of 10k SNP loci in maize;

[0040] Specifically, the maize 10k SNP loci were screened and obtained by the following method:

[0041] ① Based on the sequencing data of T32, the SNP sites on the genomes of the two maize samples were obtained by comparing them with the maize reference genome B73 (MaizeGDB). The above sites were screened according to the principle of MAF ≥ 0.05, NA < 5%, and heterozygosity < 5%, combined with the principle of uniform chromosome distribution, to obtain 166,594,907 SNP sites.

[0042] ② Based on 166,594,907 SNP sites, 10,041 sites were screened based on the principle of uniform chromosome distribution and linkage distance between markers less than 40Kb.

[0043] ③ Combining the located QTL / QTNs, 192 SNP sites with clear physical locations on the reference genome and unique copies found by BLAST were screened.

[0044] The above SNP sites constitute the maize 10k SNP sites of the present invention. After removing the overlapping sites, a total of 10160 SNP sites are obtained.

[0045] The physical locations and allele information of the 10,160 SNP sites are shown in Table 1 below.

[0046] Table 1: Physical location and typing information of SNP sites

[0047]

[0048]

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[0050]

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[0073] In the above table, chromosome indicates the chromosome ID where the SNP site is located, and * in the typing indicates deletion.

[0074] 2) Design and preparation of maize 10k SNP locus probes

[0075] The design process of the maize 10k SNP locus probe is as follows:

[0076] ① Determine the probe design principles:

[0077] The probe length is 120 bp, the probe GC content is between 25% and 69%, the average GC content is about 45%, the number of homology regions is ≤ 3, the selected regions do not contain SSR and GAP regions as much as possible, and the principle of uniform chromosome distribution is combined.

[0078] ② Centering the SNP site obtained in step 1), select a 120 bp nucleotide sequence with a GC content closest to 45% in the left and right 120 bp as a probe;

[0079] ③ Based on the 120 bp nucleotide sequence obtained by screening in step ②, a single-stranded DNA nucleotide sequence modified with a biotin group at the 5' end was synthesized to obtain a maize 10k SNP site probe;

[0080] Specifically, the probe obtained in step ③ of step 2) is coupled to a fluorescent microsphere through a C12 molecular arm and amino modification, and each fluorescent microsphere is coupled with one probe, which is finally added to the targeted capture reagent to obtain the corn whole genome targeted capture probe, i.e., the corn 10k SNP targeted capture probe.

[0081] The target capture reagents used include independently packaged universal blocking solution I,

[0082] Universal blocking solution II, TCGBS2× hybridization buffer,

[0083] TCGBS repeat sequence blocking solution Block,

[0084] TCGBS2×Beads Wash Buffer,

[0085] TCGBS WashBuffer I / Wash Buffer II / Wash Buffer III, TCGBS StringentWash Buffer.

[0086] The maize 10k SNP targeted capture probe obtained in this example targets the distribution of SNP sites on different chromosomes. Figure 1 As shown by Figure 1 It can be seen that the present invention combines the principles of genome coverage and high linkage of important genes to design probes covering different chromosomes, so that the capture sites cover the entire genome.

[0087] The distribution of the maize 10k targeted capture probe on the genome is shown in the figure below: Figure 2 As shown by Figure 2 It can be seen that the coverage rate of the 0.1Mb ​​window of the genome is 95%, and the overall coverage is good.

[0088] Ten samples were randomly selected for repeatability testing, and targeted capture genotyping of these 10 samples was performed using the corn 10k chip. For each sample, the ratio of sites with successful SNP typing to all SNP sites through sequencing was defined as the detection rate; for the same sample, the ratio of sites with consistent SNP typing in the two tests to all SNP sites was defined as consistency. The detection rate and consistency distribution are shown in Figure 2. Figure 3 As shown by Figure 3 It can be seen that the detection rate and consistency of sample sites are good.

[0089] Example 2:

[0090] The application steps of the maize 10k targeted capture probe are as follows:

[0091] (1) DNA extraction: DNA was extracted from the corn samples using a high-throughput DNA extraction kit or the CTAB method;

[0092] (2) DNA quality inspection: DNA purity, integrity, and contamination were analyzed using 1% agarose gel electrophoresis; DNA concentration was accurately quantified using Qubit; and integrity was accurately tested using Agilent 2100. The quality inspection criteria were a total amount of no less than 4 μg, a sample concentration less than 40 ng / μl, good sample integrity, and no impurity contamination.

[0093] (3) Library construction: Use an ultrasonic disruptor to randomly shear qualified sample DNA, recover DNA fragments of the desired length by electrophoresis, and add adapters to their ends to form a library;

[0094] (4) Sequencing library construction: The sample library is amplified using LM-PCR and purified to form a sequencing library, which can be used for probe hybridization experiments;

[0095] (5) Construction of hybridization capture library: 300 ng of the constructed sequencing library was taken, lyophilized, added with corn 10k SNP targeted capture probe and hybridization reagent, denatured and incubated at 65°C for 16 hours to complete the hybridization reaction; the hybridization product was washed with washing solution, and then 5 to 6 rounds of PCR were performed to complete the construction of the hybridization capture library;

[0096] (6) Hybridization capture library quality control: Qubit4.0 was used for preliminary quantification, and the effective concentration of the library was accurately quantified using qPCR to ensure the quality of the library;

[0097] (7) Sequencing: Sequencing was performed using a BGI high-throughput sequencer and related reagents;

[0098] (8) Analysis: After obtaining the data, the sequencing data was compared with the reference genome using BWA, and the standard SNP call of the data was performed using GTAK; thus, the genotyping data of the genome to be tested was obtained.

[0099] Example 3:

[0100] The application cases of the maize 10k SNP targeted capture probe obtained in Example 1 are as follows:

[0101] (1) Eight maize leaves were collected, including four inbred lines, namely T32, ZH6218, QR273, and B73, with two replicates of each inbred line. Specifically, they were numbered ZF1-1, ZF2-1, ZF3-1, and ZF4-1, which were replicates of ZF1-2, ZF2-2, ZF3-2, and ZF4-2, respectively. They were stored in a -20°C refrigerator for DNA extraction.

[0102] (2) Following the maize 10k SNP targeted capture probe application method of Example 2, DNA extraction, library construction, sequencing, and final SNP data were obtained;

[0103] (3) Calculate the detection rate. The product detection rate is an important indicator for measuring chip quality. The average detection rate of the 24 samples in this example was greater than 82%, and the detection site consistency was greater than 95%, indicating that the site design quality and probe capture efficiency of the targeted capture probe were good. In general, the maize 10k SNP targeted capture probe can be effectively used in maize genetic diversity analysis, molecular genetic map construction, whole-genome association analysis, inbred line authenticity identification, molecular marker-assisted selection breeding, and whole-genome selection breeding.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of a corn genome-wide SNP site combination in any of the following, characterized in that: The applications include: ①Analysis of genetic diversity of maize; ②Construction of molecular genetic map of maize; ③ Genome-wide association analysis of maize; ④ Authenticity identification of corn varieties; ⑤ Molecular marker-assisted selection breeding of maize; ⑥ Whole genome selection breeding of corn; ⑦ Kinship identification; ⑧ Germplasm resource identification; ⑨Gene mapping; ⑩Analysis of corn population structure; The corn genome-wide SNP site combination consists of 10,160 SNP sites; The maize reference genome is based on the B73 V5 version and is obtained through the MaizeGDB database; The physical locations and typing information of the 10,160 SNP sites are shown below: In the above table, chromosome indicates the chromosome ID where the SNP site is located, and * in the typing indicates deletion.

2. Use of the reagent for detecting the corn whole genome SNP site combination according to claim 1 in the preparation of a corn whole genome liquid phase chip, characterized in that: The corn whole genome liquid phase chip is used to identify the genotype of each site in the corn whole genome SNP site combination as claimed in claim 1.

3. A 10k targeted capture probe for identifying the genotype of each site in the corn genome-wide SNP site combination as claimed in claim 1, characterized in that: The invention comprises a corn 10K SNP site probe, wherein the corn 10K SNP site probe is a single-stranded DNA synthesized by combining the corn whole genome SNP sites according to claim 1.

4. The 10k targeted capture probe for identifying the genotype of each site in the whole genome SNP site combination of maize according to claim 1 according to claim 3, characterized in that: The 5' end of the probe carries a biotin group, and the probe is coupled to a fluorescent microsphere via a C12 molecular arm and amino modification, with each fluorescent microsphere being coupled to one probe.

5. A maize whole genome liquid phase chip, characterized in that: The maize whole genome liquid phase chip comprises the 10k targeted capture probe described in claim 3 for identifying the genotype of each site in the maize whole genome SNP site combination described in claim 1.

6. Use of the 10k targeted capture probe for identifying the genotype of each site in the corn whole genome SNP site combination according to claim 1 according to claim 3, or the corn whole genome liquid phase chip according to claim 5 in any of the following, characterized in that: The applications include: ①Analysis of genetic diversity of maize; ②Construction of molecular genetic map of maize; ③ Genome-wide association analysis of maize; ④ Authenticity identification of corn varieties; ⑤ Molecular marker-assisted selection breeding of maize; ⑥ Whole genome selection breeding of corn; ⑦ Kinship identification; ⑧ Germplasm resource identification; ⑨Gene mapping; ⑩Analysis of corn population structure.

7. A method for corn genotyping, characterized in that: It includes the following steps: S1: Obtain genomic DNA of corn samples; S2: Detecting the genomic DNA obtained in step S1 using the corn whole genome liquid phase chip according to claim 5; S3: Analyze the detection results of step S2 to obtain the genotyping results of corn.

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