CN119753217A - Corn whole genome SNP locus combination, probe, liquid phase chip and application thereof - Google Patents

Abstract

The present invention discloses a corn whole genome SNP site combination, a probe, a liquid phase chip and its application. The present invention develops a corn 10K whole genome liquid phase chip based on the genome of the corn inbred line T32. The SNP sites targeted by the present invention evenly cover the chromosome group, which is more comprehensive and includes most of the important traits, key functional molecular markers, SNP site markers; it has a larger number of sites, a more comprehensive detection range, a higher detection rate, and better repeatability. 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 conducive to accelerating the corn breeding process and improving the efficiency of new variety breeding.

Description

Corn whole genome SNP locus combination, probe, liquid phase chip and application thereof

Technical Field

The invention relates to the technical field of corn molecular biology, in particular to a corn whole genome SNP locus combination, a probe, a liquid phase chip and application thereof.

Background

Corn (Zea mays l.) belongs to the genus poaceae, is a grain and feed crop that is planted globally, has important economic value, and is a key plant for molecular breeding research. The seeds are rich in dietary fibers, antioxidants, minerals and vitamins, have important functions on reducing blood sugar and preventing cardiovascular diseases, and have long been at a premium for consumers. Along with the rapid development of high-throughput sequencing technology and genome assembly technology, high-quality corn genome sequence information is released, so that the understanding of corn basic biology is improved, and the method is of great importance to genetics, genomics, molecular breeding research and the like.

As a genetic marker with wider distribution in genome, single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) has the characteristics of high density, high genetic stability, easy automatic analysis and the like, and has been developed into the most common molecular marker in plant genetic variation research. Currently, genome-wide variant genotype acquisition is largely by resequencing and gene chip. The cost for obtaining mutation information by a whole genome or simplified genome sequencing (GBS) method is too high, site deletion is serious, the sequencing data volume is relatively large, and the software and hardware requirements for data storage and analysis calculation are high. The quality of detection of the mutation site is strongly dependent on the quality of the reference genome. SNP chips have good detection rate and stability in the aspect of sample site detection, and are developed and applied to crops such as corn, rice, soybean and the like, and important research progress is achieved. Therefore, efficient, low-cost SNP genotyping techniques are the best option for developing sharing techniques and platforms.

In SNP chip technology, traditional solid phase chip is based on the complementary hybridization of probe and DNA sequence, and the fluorescent color signal of the marker is used for parting, and only SNP sites contained on the chip can be parting, so that the SNP chip cannot be added or deleted, has poor flexibility and high parting cost. The liquid chip generally comprises a Biotin (Biotin) mark and target SNP covering probe designed for each to-be-detected site according to the DNA complementation principle, the probes are hybridized with a genome target region in a liquid state to form double chains, and the adsorption effect of streptavidin coated magnetic beads and molecules with Biotin can be utilized, and the second generation sequencing is carried out after elution, amplification and library establishment, so that the genotype state of the target site and the SNP around the target site is reduced finally, and the liquid chip has more advantages compared with the solid chip.

At present, a jade solid-phase chip developed based on SNP loci obtained by means of whole genome association analysis, linkage analysis, genome sequencing and the like is used for corn population structure, principal component analysis, whole genome association analysis, quantitative trait locus positioning and the like, but most of chips are developed based on temperate corn germplasm genomes, gene chips aiming at the characteristics of tropical corn germplasm genomes still belong to the blank, and the SNP loci aimed by the gene chips lack necessary molecular markers, so that the application of the chips in genetic improvement of tropical corn is limited. Therefore, a new chip is developed aiming at the genetic characteristics of the germplasm of the tropical corn so as to meet the requirements of germplasm resource identification of the tropical corn.

Disclosure of Invention

Therefore, based on the background, the invention provides a corn whole genome SNP locus combination, a probe, a liquid phase chip and application thereof, the invention develops a more comprehensive combination of key functional SNP molecular markers which cover a chromosome group and comprise almost all important characters, and the SNP locus liquid phase chip with a more comprehensive detection range is developed, so that the breeding work requirement of tropical corn can be met.

The technical scheme of the invention is as follows:

A maize whole genome SNP locus combination consisting of 10160 SNP loci, the physical location and typing information of the 10160 SNP loci being as follows in table 1.

Based on the same inventive concept, the invention applies the whole-genome SNP locus combination of the corn to the preparation of a whole-genome liquid chip of the corn, wherein the whole-genome liquid chip of the corn is used for identifying the genotypes of all loci in the whole-genome SNP locus combination of the corn according to claim 1.

Based on the same inventive concept, the invention also provides a 10K targeting capture probe, which comprises a corn 10K SNP locus probe, wherein the corn 10K SNP locus probe is single-stranded DNA synthesized according to the combination of the corn genome SNP loci.

Further, the 5' end of the probe is provided with a biotin group, and the probe is coupled to fluorescent microspheres through a C12 molecular arm and amino modification, and each fluorescent microsphere is coupled with one probe.

Based on the same inventive concept, the invention also provides a corn whole genome liquid phase chip comprising the targeted capture probe of claim 2 for identifying the genotype of the corn whole genome SNP locus as defined in claim 1.

Based on the same inventive concept, the invention provides the application of the corn whole genome SNP locus combination, or the targeting capture probe, or the corn whole genome liquid phase chip in any one of the following applications, wherein the application comprises:

① Corn genetic diversity analysis;

② Constructing a corn molecular genetic map;

③ Corn whole genome association analysis;

④ Identifying the authenticity of the maize inbred line;

⑤ Corn molecular marker assisted selective breeding;

⑥ Selecting and breeding the whole genome of corn;

⑦ Identifying the genetic relationship;

⑧ Germplasm resource identification;

⑨ Gene localization;

⑩ And (5) analyzing the corn colony structure.

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

s1, obtaining genome DNA of a corn sample;

s2, detecting the genome DNA obtained in the step S1 by using the whole genome liquid chip of the corn;

and S3, analyzing the detection result in the step S2 to obtain the genotyping result of the corn.

The technical scheme of the invention has the beneficial effects that:

The invention develops a corn 10K whole genome liquid phase chip, aiming at SNP loci, uniformly covers a chromosome group, contains almost all key functional SNP molecular markers related to important characters, has more and more comprehensive locus quantity and more comprehensive detection range, can be applied to temperature zone corn, is also suitable for detecting tropical corn, and has higher detection rate and better repeatability;

The targeting capture probe related to the 10k SNP locus information can rapidly and effectively track the genetic material of corn, is beneficial to accelerating the corn breeding process and improves the breeding efficiency of new inbred lines.

Drawings

FIG. 1 is a distribution statistical chart of SNP loci aimed at by a corn 10k SNP targeting capture probe on different chromosomes, namely, statistics results of the number of target loci on different chromosomes, wherein the abscissa is the chromosome, and the ordinate is the number of SNP markers of each chromosome;

FIG. 2 is a SNP site uniformity distribution map (probe coverage density was calculated on a chromosome with a length of 0.1Mb as an observation window).

The figure is plotted against the maize reference genome B73V 5.

FIG. 3 is a statistical plot of the detection rate and consistency distribution of repeated samples. The figure is plotted against the maize reference genome V5 version.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the following examples thereof, it being understood that the specific examples described herein are for illustration only and are not intended to be limiting.

The maize reference genome data of the examples was from maize inbred B73.

The corn 10k SNP targeting capture probe of the embodiment comprises a corn 10k SNP locus probe mixed solution and a hybridization capture reagent.

The preparation process of the corn 10k SNP targeting capture probe is as follows:

1) Screening corn 10k SNP loci;

Specifically, the corn 10k SNP locus is obtained by screening by the following method:

① According to the sequencing data of T32, comparing the corn reference genome B73 (MaizeGDB) to obtain SNP loci on the genome of the 2 corn samples, and screening 166594907 SNP loci on the loci according to MAF not less than 0.05, NA <5%, heterozygosity rate <5% and chromosome uniform distribution principle.

② And screening to obtain 10041 loci according to the principle that 166594907 SNP loci are uniformly distributed on the combined chromosome and the linkage distance between markers is smaller than 40 Kb.

③ In combination with the localized QTL/QTNs, screening resulted in 192 SNP sites with well-defined physical locations on the reference genome, and BLAST found unique copies.

The SNP loci form the 10k SNP loci of the corn, and 10160 SNP loci are obtained after the overlapping loci are removed.

The physical positions and allele information of the 10160 SNP sites are shown in table 1 below.

TABLE 1 physical position and typing information of SNP loci

In the above table, the chromosome represents the chromosome ID where the SNP site is located, and the loss is represented by the x in typing.

2) Design and preparation of corn 10k SNP locus probe

The design process of the corn 10k SNP locus probe comprises the following steps:

① Determining a probe design principle:

The probe length is 120bp, the GC content of the probe is 25% -69%, the average GC content is about 45%, the number of homologous regions is less than or equal to 3, the selected region does not contain SSR and GAP regions to the maximum extent, and the chromosome uniform distribution principle is combined.

② Taking the SNP locus obtained in the step 1) as a center, and screening a 120bp nucleotide sequence with the GC content closest to 45% in about 120bp as a probe;

③ Synthesizing a single-stranded DNA nucleotide sequence with biotin group modification at the 5' end according to the 120bp nucleotide sequence obtained by screening in the step ② to obtain a corn 10k SNP locus probe;

Specifically, the probe obtained in ③ step in step 2) is coupled to fluorescent microspheres through a C12 molecular arm and amino modification, each fluorescent microsphere is coupled with one probe, and finally the probe is added into a targeted capture reagent, so that the corn whole genome targeted capture probe, namely the corn 10k SNP targeted capture probe, is obtained.

The target capture reagent comprises a general sealing liquid I which is packaged independently,

General blocking solution II, TCGBS2 Xhybrid Buffer,

TCGBS repeat sequence blocking solution Block,

TCGBS2×Beads Wash Buffer、

TCGBS WashBuffer I/Wash Buffer II/Wash Buffer III、TCGBS Stringent Wash Buffer。

The distribution of the SNP loci on different chromosomes of the corn 10k SNP targeting capture probes obtained by the embodiment is shown in figure 1, and as can be seen from figure 1, the probes covering different chromosomes are designed by combining the principle of high genome coverage and important gene linkage degree, so that the capture loci cover the whole genome.

The distribution diagram of the corn 10k targeting capture probe on the genome is shown in fig. 2, and as can be seen from fig. 2, the coverage rate of the genome window of 0.1Mb is 95%, and the whole coverage is good.

10 Samples were randomly selected for reproducibility testing, and the 10 samples were genotyped for targeted capture using the maize 10k chip. For each sample, the ratio of sites successfully judging the classification of the snp to all the snp sites by sequencing is defined as the detection rate, and the ratio of sites with consistent classification of the snp to all the snp sites detected for 2 times in the same sample is defined as the consistency. The detection rate and consistency distribution are shown in fig. 3, and as can be seen from fig. 3, the detection rate and consistency of the sample sites are good.

Example 2:

The application of the corn 10k targeted capture probe comprises the following steps:

(1) DNA extraction, namely extracting DNA from a corn sample to be detected by adopting a high-throughput DNA extraction kit or a CTAB method;

(2) DNA quality inspection, namely analyzing the purity and the integrity of DNA and whether the DNA is polluted or not by using 1% agarose gel electrophoresis, accurately quantifying the concentration of the DNA by using Qubit and accurately detecting the integrity by using Agilent 2100. The standard of qualification of quality inspection is DNA with the total amount not less than 4 mug, the sample concentration less than 40 ng/. Mu.l, good sample integrity and no impurity pollution;

(3) Randomly breaking qualified sample DNA by using an ultrasonic breaker, and electrophoretically recovering DNA fragments with required length, and adding a connector at the tail end of the DNA fragments to form a library;

(4) The sequencing library construction, namely amplifying a sample library by utilizing LM-PCR and purifying to obtain a sequencing library, which can be used for probe hybridization experiments;

(5) The construction of a hybridization capture library, namely, taking 300ng of the sequencing library which is completed, adding a corn 10k SNP targeting capture probe and a hybridization reagent after freeze-drying, and carrying out denaturation and incubation at 65 ℃ for 16 hours to complete hybridization reaction;

(6) Quality inspection of the hybridization capture library, namely, performing preliminary quantification by utilizing Qubit4.0, and accurately quantifying the effective concentration of the library by using a qPCR method so as to ensure the quality of the library;

(7) Sequencing, namely sequencing by using a BGI high-throughput sequencer and related reagents;

(8) Analyzing, namely comparing the sequencing data with a reference genome by using BWA after obtaining the machine-down data, and using GTAK to carry out standard call SNP of the data so as to obtain genome genotyping data to be detected.

Example 3:

the corn 10k SNP targeted capture probe obtained in example 1 was used as follows:

(1) Taking 8 parts of corn leaves, wherein the corn leaves totally comprise 4 parts of inbred lines, namely T32, ZH6218, QR273 and B73, each inbred line is repeated 2 times, and specific numbers are ZF1-1, ZF2-1, ZF3-1 and ZF4-1 which are the repetition of ZF1-2, ZF2-2, ZF3-2 and ZF4-2 respectively;

(2) DNA extraction, library construction, on-machine sequencing and obtaining final SNP data were performed according to the corn 10k SNP targeting capture probe application method of example 2;

(3) And calculating the detection rate. The average detection rate of 24 samples in the embodiment is more than 82%, the consistency of detection sites is more than 95%, which indicates that the site design quality of the target capture probe and the capture efficiency of the probe are good. In general, the corn 10k SNP targeting capture probe can be effectively applied to corn genetic diversity analysis, molecular genetic map construction, whole genome association analysis, inbred line authenticity identification, molecular marker assisted selective breeding and whole genome selective breeding.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (7)

1. A corn whole genome SNP site combination, characterized in that the corn whole genome SNP site combination consists of 10160 SNP sites, and the physical locations and typing information of the 10160 SNP sites 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. Use of the corn whole genome SNP site combination as described in 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 described in claim 1. 3. A 10k targeted capture probe, characterized in that it comprises 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 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 through a C12 molecular arm and amino modification, and each fluorescent microsphere is coupled with a probe. 5. A corn 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 corn genotyping, characterized in that it comprises 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.