Corn whole genome SNP site combination, probe, liquid phase chip and application of corn whole genome SNP site combination, probe and liquid phase chip

Abstract

The invention discloses a corn whole genome SNP (Single Nucleotide Polymorphism) site combination, a probe, a liquid chip and application thereof, the corn 10K whole genome liquid chip is developed based on a genome of a corn inbred line T32, SNP sites targeted by the corn 10K whole genome liquid chip uniformly cover a chromosome group, and key functional molecular marker SNP site markers of most important characters are more comprehensively included; compared with the prior art, the method has the advantages that more sites are provided, the detection range is more comprehensive, the detection rate is higher, and the repeatability is better. The target capture probe involved based on 10k SNP site information can rapidly and effectively track genetic materials of corn, the corn breeding process is accelerated, and the new variety breeding efficiency is improved.

Description

Technical Field

[0001] The 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] Corn (Zea may L.) belongs to the genus Zea of ​​the Poaceae family. It is a food and feed crop grown worldwide, has important economic value, and is a key plant for molecular breeding research. Its seeds are rich in dietary fiber, antioxidants, minerals, and vitamins, and play an important role in lowering blood sugar and preventing cardiovascular diseases. They have long been cherished by consumers. With the rapid development of high-throughput sequencing technology and genome assembly technology, the release of high-quality corn genome sequence information has improved our understanding of the basic biology of corn, which is crucial for genetics, genomics, and molecular breeding research.

[0003] Single Nucleotide Polymorphism (SNP) is a genetic marker that is more widely distributed in the genome. It has the characteristics of high density, high genetic stability and easy 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 through resequencing and gene chips. The cost of obtaining variant information through whole genome or simplified genome sequencing (GBS) methods is too high, the site is seriously missing, 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 genotype detection technology has become the best choice for the development of shared technologies and platforms.

[0004] In SNP chip technology, traditional solid-phase chips are based on complementary hybridization of probes and DNA sequences, and typing is performed through fluorescent colorimetric signals of markers. They can only type the SNP sites contained on the chip, and cannot be added or deleted. They have poor flexibility and high typing costs. Liquid-phase chips generally include a biotin-labeled probe covering the target SNP designed for each site to be tested based on the principle of DNA complementarity. These probes hybridize with the target region of the genome in the liquid to form a double strand. The adsorption of streptavidin-coated magnetic beads and biotin-containing molecules can be used to perform second-generation sequencing after elution, amplification, and library construction, and finally restore the genotype status of the target site and its surrounding SNPs, which is more advantageous than solid-phase chips.

[0005] At present, solid-phase chips developed based on SNP sites obtained by whole-genome association analysis, linkage analysis and genome sequencing have been used for maize population structure, principal component analysis, whole-genome association analysis, quantitative trait loci positioning, etc. However, most chips are developed based on temperate maize germplasm genomes, while gene chips targeting tropical maize germplasm genome characteristics are still blank. The SNP sites targeted by gene chips lack necessary molecular markers, which limits the application of these chips in tropical maize genetic improvement. Therefore, it is necessary to develop new chips targeting tropical maize germplasm genetic characteristics 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 including almost all important traits, and thus develops 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, the whole genome SNP site combination of corn consists of 10160 SNP sites, and the physical locations and typing information of the 10160 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 according to 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, wherein the maize whole genome liquid phase chip comprises 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, the application including:

[0014] ①Analysis of genetic diversity of maize;

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

[0016] ③ Maize genome-wide association analysis;

[0017] ④ Authenticity identification of corn inbred lines;

[0018] ⑤ Maize molecular marker-assisted selection breeding;

[0019] ⑥ Whole genome selection breeding of corn;

[0020] ⑦ Kinship identification;

[0021] ⑧ Germplasm resource identification;

[0022] ⑨Gene location;

[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: using the above-mentioned corn whole genome liquid phase chip to detect the genomic DNA obtained in step S1;

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

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

[0029] The present invention has developed a maize 10K whole genome liquid phase chip, which evenly covers the chromosome group with the targeted SNP sites, encompasses almost all the key functional SNP molecular markers related to important traits, 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 the detection of tropical maize, and has a higher detection rate and better repeatability.

[0030] The targeted capture probe 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 selection. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0034] Attached Figure 3 This is a statistical diagram of the detection rate and consistency distribution of repeated samples. This figure 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 from the maize inbred line B73.

[0037] The corn 10k SNP targeted capture probe of this embodiment includes a corn 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 above two maize samples were obtained by comparing the maize reference genome B73 (MaizeGDB). According to the MAF≥0.05, NA<5%, heterozygosity<5%, and the principle of uniform chromosome distribution, 166,594,907 SNP sites were screened out.

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

[0043] ③ Combined with 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 loci constitute the maize 10k SNP loci of the present invention. After removing the overlapping sites, a total of 10160 SNP loci are obtained.

[0045] The physical locations and allele information of the 10160 SNP sites are shown in Table 1 below.

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

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

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

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

[0076] ① Determine the probe design principles:

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

[0078] ② Centering on 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 According to the 120bp 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 corn 10k SNP site probe;

[0080] Specifically, the probe obtained in step ③ of step 2) is coupled to the fluorescent microsphere through the C12 molecular arm and amino modification, each fluorescent microsphere is coupled with one probe, and finally added to the targeted capture reagent to obtain the corn whole genome targeted capture probe, that is, 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 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] 10 samples were randomly selected for repeatability testing, and the maize 10k chip was used to perform targeted capture genotyping on these 10 samples. For each sample, the ratio of the sites that successfully interpreted the SNP typing through sequencing to all SNP sites was defined as the detection rate; for the same sample, the ratio of the 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] Embodiment 2:

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

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

[0092] (2) DNA quality inspection: 1% agarose gel electrophoresis was used to analyze the purity, integrity, and contamination of DNA; Qubit was used to accurately quantify the DNA concentration; Agilent 2100 was used to accurately detect the integrity. The quality inspection passed the standard of total amount not less than 4μg, sample concentration less than 40ng / μl, good sample integrity, and no impurity contamination of DNA;

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

[0094] (4) Sequencing library construction: The sample library is amplified by LM-PCR and purified to obtain a sequencing library, which can be used in 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 the qPCR method 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 offline data, use BWA to compare the sequencing data with the reference genome, and use GTAK to perform standard SNP calls on the data; thereby obtaining the genotyping data of the genome to be tested.

[0099] Embodiment 3:

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

[0101] (1) Take 8 maize leaves, which contain 4 inbred lines, namely T32, ZH6218, QR273 and B73, and each inbred line is repeated twice. The specific numbers are ZF1-1, ZF2-1, ZF3-1, and ZF4-1, which are the repeats of ZF1-2, ZF2-2, ZF3-2, and ZF4-2 respectively; store them in a -20°C refrigerator for DNA extraction;

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

[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 embodiment is greater than 82%, and the consistency of the detection sites is greater than 95%, indicating that the site design quality and probe capture efficiency of the targeted capture probe are good. In general, the corn 10k SNP targeted capture probe can be effectively used in corn 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A combination of SNP sites in the whole genome of corn, characterized in that: The corn genome-wide SNP locus combination consists of 10160 SNP loci, and the physical locations and typing information of the 10160 SNP loci are as follows: In the above table, chromosome indicates the chromosome ID where the SNP site is located, and * in the typing represents deletion.

2. The use of 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, characterized in that It comprises a corn 10K SNP site probe, which is a single-stranded DNA synthesized according to the corn whole genome SNP site combination according to claim 1.

4. A targeted capture probe 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 corn whole genome liquid phase chip comprises the targeted capture probe according to claim 2, and the targeted capture probe is used to identify the genotype of the corn whole genome SNP site according to claim 1.

6. Use of the whole-genome SNP site combination of maize according to claim 1, or a targeted capture probe according to claim 3, or a whole-genome liquid phase chip of maize 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 Maize genome-wide association analysis; ④ Authenticity identification of corn varieties; ⑤ Maize molecular marker-assisted selection breeding; ⑥ Whole genome selection breeding of corn; ⑦ Kinship identification; ⑧ Germplasm resource identification; ⑨Gene location; ⑩Analysis of corn population structure.

7. A method for maize genotyping, characterized in that: It includes the following steps: S1: Obtain genomic DNA of corn samples; S2: using the corn whole genome liquid phase chip of claim 4 to detect the genomic DNA obtained in step S1; S3: Analyze the detection result of step S2 to obtain the genotyping result of corn.

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