Wheat solid-phase gene chip and application thereof

Abstract

The invention belongs to the field of genomics and solid-phase gene chip preparation, and particularly relates to a wheat solid-phase gene chip and application thereof. The chip provided by the invention contains the SNP marker covering the whole genome of wheat and the transgenic element marker, realizes the detection effects of high throughput, high detection rate, high reproducibility, convenient operation and high accuracy, and can be applied to identification of authenticity, specificity and genetic relationship of wheat varieties. According to application scenes such as variety and substantive derived variety identification, new variety protection, transgenic detection, molecular marker-assisted breeding, whole genome selective breeding and the like, a large number of SNP locus typing and transgenic detection results can be obtained at the same time through one-time detection.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology, molecular genetics, genomics, bioinformatics, and chip preparation. Specifically, it relates to a wheat solid-phase gene chip and its application. Background Art

[0002] DNA molecular markers refer to genetic markers based on nucleotide sequence variations among individuals. Compared with morphological markers, molecular markers based on DNA sequence differences have the characteristics of being less affected by the environment, having a large number, wide distribution, high polymorphism, and rich variations in natural populations, which provide great convenience for the selection of breeding parents and variety identification. Since the development of DNA molecular markers to date, they have gone through various updates. Early ones such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) markers, to the currently widely used simple sequence repeat markers (SSR) and single nucleotide polymorphism markers (single nucleotide polymorphism, SNP). SNP polymorphic markers refer to DNA sequence polymorphisms caused by single-base variations (mainly transitions and transversions) on the genomic DNA sequence. Currently, SNP and SSR molecular markers are the only two recommended marker methods in the DNA fingerprint database construction in the BMT molecular testing guidelines of the International Union for the Protection of New Varieties of Plants (UPOV) and the General Rules for DNA Fingerprinting Methods for Plant Variety Identification in China. Among them, the SSR technology has the advantages of co-dominant inheritance, high polymorphism, good repeatability and stability. However, it has limitations such as a limited number of markers, low throughput, high misreading rate, high cost, and difficulties in data compatibility and integration. The third-generation SNP molecular markers have co-dominant inheritance and have the characteristics of a large number, wide chromosomal distribution, dimorphism, high compatibility, high accuracy, and easy data integration. Moreover, they can combine with gene chip technology to achieve technical advantages such as multi-locus and large sample size, making up for the technical shortcomings of SSR markers such as high misreading rate, insufficient sample throughput, and incompatibility. Therefore, it is regarded as the most important and promising molecular marker.

[0003] Solid-phase chips are a SNP gene detection technology means with high throughput, high accuracy, high detection efficiency, and high stability, especially suitable for high-throughput SNP genotyping detection on a scale of over ten thousand, and are widely used by important large seed companies or well-known biological detection companies globally. The high-density solid-phase gene chip detection technology independently developed by Lasso Biology uses microsphere array technology to immobilize hundreds of thousands of oligonucleotide probes on micron-sized microspheres and fixes the microspheres on a silicon-based chip etched by light. Through ingenious probe sequence design and fluorescence labeling, these microspheres can accurately identify the SNP information at each locus. At the same time, each locus has 15 - 30 microspheres for repeated detection, greatly enhancing the detection accuracy.

[0004] SNP markers combined with high-density solid-phase gene chips can be used in agriculture for applications such as molecular marker-assisted breeding (MAS), genome-wide selection breeding (GS), transgenic detection, pedigree tracing, DNA fingerprint variety identification, and genetic resource background research. They can also be used for basic research such as genome-wide association analysis, discovery of excellent trait genes, QTL analysis, selection signal analysis, construction of genetic linkage maps, and population structure analysis.

[0005] The solid-phase breeding chip combines SNP markers with high-density solid-phase gene chip technology. Its popularization and application are expected to further reduce the cost of molecular breeding and help China's seed industry enter the 3.0 era of molecular breeding. Wheat is an important food crop in China, related to national food security. The development of wheat chips will help further consolidate China's dominant position in wheat breeding, safeguard national food security, and also play a certain demonstrative role in the research and development of solid-phase chips for other species.

[0006] Existing technologies such as the wheat-rye liquid-phase SNP chip disclosed in CN114395642B still have various limitations. It is a liquid-phase chip with high costs, easy to be contaminated, and requires regular cleaning and maintenance. Its SNP marker screening is based on a single chip platform, resulting in insufficient coverage of highly polymorphic sites with MAF > 0.3. In addition, transgenic detection requires separate PCR verification, increasing the detection cost by at least more than 30%. And the probe dot matrix is a planar spotting technology with a density limited to 40,000 / cm², restricting the efficiency of genome-wide detection.

[0007] Therefore, at present, the genome-wide SNP molecular marker combination for wheat and the supporting solid-phase gene chip still need to be further optimized and improved to meet the growing needs of wheat genetic research and breeding. Summary of the Invention

[0008] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention aims to provide a wheat solid-phase gene chip, which effectively solves the above problems through technological innovations such as dual-platform dynamic calibration screening, quadruple transgenic synchronous detection, and microsphere array-lithographic etching, and is realized through the following technical solutions.

[0009] In one embodiment, the present invention provides a wheat SNP molecular marker combination, which consists of SNP markers covering the wheat whole genome and transgenic element markers. Each SNP marker contains allelic variations of two different bases and is used to detect the allelic variation changes at this position. Each transgenic element marker is used to detect whether the element exists in the genome of the tested sample. The SNP markers are screened through the following steps. Step 1. Preparation of the wheat BAAFS 90K SNP array chip: Select representative wheat varieties, extract wheat genomic DNA, and screen markers from the Illumina wheat 90k chip with data read accuracy score values between 0.60 and 1.00. The higher the score, the more reliable the data. Eliminate sites that lack repeatability, stability, reproducibility, do not conform to genetic laws, have irregular clustering, or have no polymorphism. Eliminate sites with weak overall signals, a missing rate greater than 5%, a heterozygosity rate greater than 10%, linkage, or no mapping information. Finally, screen out high-quality, high-resolution, single-copy, non-linked, and relatively evenly distributed SNP markers. Screen out high-quality and highly polymorphic SNP markers from the AFFY wheat 660k chip. Eliminate sites that lack repeatability, stability, reproducibility, have irregular clustering, or do not conform to genetic laws. Eliminate unreliable markers with a high missing rate, weak overall signals, a heterozygosity rate greater than 10%, and a minor allele frequency (MAF) less than 0.2. Finally, screen out high-quality, high-resolution, and relatively evenly distributed sites. Combine the publicly published wheat functional SNP markers with the SNP markers screened from the Illumina wheat 90k chip and the AFFY wheat 660k chip respectively above. After sequence evaluation and pre-experiments, finally form the wheat BAAFS90K SNP array chip. Step 2. Determine the target site SNPs: Extract the genomic DNA of the test materials using the high-salt low-pH method, and measure the DNA concentration and purity with a UV spectrophotometer. Scan the known wheat representative varieties using the BAAFS 90K SNP array chip in Step 1, analyze the SNP site genotype data of the samples. The chip experiment adopts the Affymetrix Axiom® 2.0 experimental standard operating procedure to obtain the raw data, and use the Axiom Analysis Suite software for genotyping to obtain the SNP site genotyping data of the samples to be tested. The chip analysis process is as follows: (1) Transfer plates and quantify according to the experimental sample layout table and dilution table. (2) Denature and amplify the DNA samples. (3) Fragment and precipitate the amplified products. (4) Dry, resuspend, quantify, and perform quality inspection on the fragmented amplified products. (5) Hybridize and wash / dye the samples. (6) Scan the sample plate. According to the evaluation results of the Axiom Analysis Suite software, using Chinese Spring as a reference, site reliability verification was carried out, unreliable sites were deleted, and sites with obvious boundaries in the clustering results of three genotypes and containing two homozygous genotypes at the same time, with a deletion rate lower than 5%, high resolution, and evenly distributed on chromosomes were selected as extended sites. Candidate core sites were screened based on physical genetic distance and minimum allele frequency (MAF, Minor Allele Frequency), and sites without polymorphism, sites with only one homozygous genotype, sites with only one homozygous genotype and one heterozygous genotype, sites with a high data deletion rate, sites with a heterozygosity higher than 10%, sites with off-target mutations, and sites with poor genotyping results were removed. SNP markers that are unique and single-copy were screened out from the remaining markers.

[0010] In another embodiment, the present invention provides a set of probes for detecting SNP markers and transgenic elements in the wheat genome. The transgenic elements are labeled as Wx012, ubiquitin promoter, CaMV35S promoter, and NOS terminator.

[0011] In another embodiment, the present invention provides a wheat solid-phase gene chip, which includes the above-mentioned probes and the probes are fixed on the chip for detecting corresponding SNP molecular markers.

[0012] In one embodiment, the present invention provides the application of the wheat probe combination and the wheat solid-phase gene chip in wheat variety identification. The method includes using the probe combination or the solid-phase chip to detect the DNA of the wheat sample to be tested, comparing the SNP site information obtained by the detection with the SNP site information of the known standard variety, calculating the site similarity, and judging whether the sample to be tested and the standard variety are the same variety according to the site similarity.

[0013] In one embodiment, the present invention provides the application of the wheat probe combination and the wheat solid-phase gene chip in wheat variety-specific identification. By detecting the differences in the SNP markers and the transgenic element markers of different wheat varieties, the specific genetic characteristics of different varieties are clarified, thereby realizing the specific identification of wheat varieties.

[0014] In one embodiment, the present invention provides the application of the wheat probe combination and the wheat solid-phase gene chip in wheat genetic relationship identification. Based on the genetic information of the SNP markers detected in different wheat samples, the genetic distance or similarity coefficient is calculated, and a genetic relationship map is constructed to infer the genetic relationship between different wheat varieties.

[0015] In one embodiment, the present invention provides the application of a wheat probe combination and a wheat solid-phase gene chip in the identification of wheat varieties and their essentially derived varieties. By analyzing the genetic variation of the SNP markers of the variety to be tested and comparing it with the original variety, it is determined whether it is an essentially derived variety. If the gene combination of the original variety is retained on more than 92% of the SNP markers and there are specific variations on the key SNP markers, it can be determined as a suspected essentially derived variety.

[0016] In one embodiment, the present invention provides the application of a wheat probe combination and a wheat solid-phase gene chip in transgenic detection. The genomic DNA of a wheat sample is detected using the probe combination. If the signal corresponding to the transgenic element marker is detected, it indicates that the sample contains the corresponding transgenic element and is a transgenic wheat.

[0017] In one embodiment, the present invention provides the application of a wheat probe combination and a wheat solid-phase gene chip in marker-assisted breeding. During the wheat breeding process, the SNP markers related to the target traits of the breeding materials are detected with the help of this probe combination or solid-phase chip, and the materials carrying the markers of excellent traits are selected as breeding materials.

[0018] In one embodiment, the present invention provides the application of a wheat probe combination and a wheat solid-phase gene chip in genomic selection breeding. Based on the SNP molecular marker combination, the whole genome of wheat breeding materials is detected, and a prediction model is established by combining phenotypic data to predict the genetic value of the breeding materials, so as to accurately select individuals with excellent genetic potential in the early generations and achieve genomic selection breeding.

[0019] The beneficial effects achieved by the present invention: Adopting a dual-platform (Illumina 90K and Affy 660K) joint screening strategy, introducing double quality control standards of "screening in the score value range of 0.60 - 1.00" and "MAF≥0.2 threshold filtering", the intersection markers of the two are reversely aligned with the Chinese Spring reference genome, and the sites with off-target variations are removed, constructing a wheat high-density SNP molecular marker combination, which has the characteristics of high polymorphism, large throughput, high accuracy, simple gene typing, and covering the whole wheat genome compared with single-platform screening. The SNP markers cover 13,121 genes, accounting for about 10% of the genes in the hexaploid wheat genome. It can not only meet the application requirements of variety authenticity identification, variety and its essentially derived variety identification, genetic relationship identification, specificity identification, screening of similar varieties, construction of genetic linkage maps, association analysis, QTL mapping, marker-assisted breeding, genomic selection breeding, genomic mapping, cluster analysis, new variety protection, etc., but also meet the breeding and research requirements of phenotypic trait functional marker detection, functional gene mining, etc.

[0020] The present invention has developed a solid-phase gene chip for detecting wheat whole-genome SNP molecular marker combinations, which has high locus throughput, high detection rate, high reproducibility, convenient operation, high accuracy, and high timeliness. Moreover, for the first time, an internal reference gene and Wx01 two internal reference genes and ubiquitin promoters, CaMV35S promoters, NOS terminators are integrated into a four-in-one detection system. Among them, Wx012 the probe is designed in the 5'UTR conserved region, and the synchronous verification of endogenous genes and transgenic elements is achieved through melting curve analysis, solving the technical problem of high false negative rate in traditional detection. The transgenic element markers and SNP markers covering the wheat whole genome are integrated into the same SNP molecular marker combination and prepared on the same chip, enabling the acquisition of both a large number of SNP locus genotyping results and transgenic detection results in one detection, which is simple and efficient, and the variety discrimination efficiency reaches 99.2%.

[0021] The present invention adopts a "microsphere array-lithographic etching" composite process. Each probe dot matrix is composed of 15-30 silica microspheres with a diameter of 3μm (CN114395642B uses a planar spotting technique). The probe density reaches 65,000 / cm² through silicon-based micropore directional fixation (CN114395642B is 40,000 / cm²), the signal consistency deviation is <5%, and the throughput is increased by 62.5%. Among them, the transgenic detection probe adopts an optimized melting curve design, Wx012 the internal reference gene probe and CaMV35S the promoter probe have a Tm value difference controlled within 2°C to ensure the accuracy of synchronous detection. Description of the Drawings

[0022] Figure 1 : Screening of the optimal locus combination and variety discrimination ability.

[0023] Figure 2 : Chromosome distribution of 50,000 SNP loci for variety identification.

[0024] Figure 3 : MAF statistics of 50,000 SNP loci for variety identification.

[0025] Figure 4 : Distribution of 53,813 SNP molecular markers in the wheat genome.

[0026] Figure 5 : SNP fingerprint clustering map of 194 approved wheat standard samples constructed using the BAAFS wheat 65K wheat SNP solid-phase gene chip. Detailed Embodiments

[0027] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

[0028] In the following embodiments, the experimental materials, reagents, instruments and methods used are all conventional experimental materials, reagents, instruments and methods in the art and can be obtained through commercial channels without special instructions.

[0029] Example 1. Preparation of BAAFS 90K SNP array chip This example is used to demonstrate the preparation process of the BAAFS 90K SNP array chip independently developed by the Beijing Academy of Agriculture and Forestry Sciences used in the present invention. The Affymetrix Axiom chip platform includes the BAAFS 90K SNP array chip, the Axiom array plate, the complete Axiom kit, the automated target preparation workstation jointly developed by Affymetrix and Beckman Coulter, and the GeneTitan multi-channel instrument (Affymetrix, USA). Chip platform: Axiom 2.0 kit, including denaturing reagent, amplification reagent, fragmentation and precipitation reagent, resuspension and hybridization reagent, washing and staining reagent, etc. (Affymetrix, USA); 20bp DNA Ladder (Takara); Nuclease free water (Tiangen), agarose gel, etc. Specifically as follows: Genomic DNA of 96 materials was extracted by the CTAB method and dissolved in TE for storage. The quality and concentration of DNA were measured by ultraviolet spectrophotometer and agarose gel electrophoresis. It was required that the concentration of DNA samples was above 50 ng / μL, the OD 260 / 280 should be between 1.7 - 2.1, and the total amount of DNA should be greater than 2 μg. The 96 representative varieties were genotyped using the Illumina wheat 90k iSelect BeadChips whole-genome chip (with 81,587 SNP loci) and the AFFY wheat 660k chip respectively. Candidate SNP markers were screened according to the principles of repeatability, stability, polymorphism, missing rate, characteristics of three types of clustering maps of heterozygosity and homozygosity, heterozygosity rate, etc. Markers with score values between 0.60 and 1.00 were screened out from the Illumina wheat 90k chip (81,587 SNP markers); loci without repeatability and stability, not conforming to genetic laws, and without polymorphism were excluded; loci with weak overall signals, missing rate greater than 5%, heterozygosity rate greater than 10%, linkage, and no mapping information were excluded. Finally, high-quality, high-resolution, single-copy, non-linked, and evenly distributed SNP markers were screened out. High-quality polymorphic SNP markers were screened out from the AFFY wheat 660k chip; loci without repeatability, with non-standard three types of clustering maps of heterozygosity and homozygosity, and not conforming to genetic laws were excluded; unreliable, with high missing rate, weak overall signals, heterozygosity rate greater than 10%, and MAF less than 0.2 markers were excluded. Finally, high-quality, high-resolution, and evenly distributed loci were screened out. The publicly published wheat functional SNP markers were integrated with the SNP markers screened from the Illumina wheat 90k chip and the AFFY wheat 660k chip respectively to form a marker set.

[0030] The sequences of 50 bp upstream and downstream of the 103,905 candidate SNP markers screened out were submitted to Affymetrix (https: / / securefileexchange.affymetrix.com), and then based on the Axiom technology, they were evaluated and scored according to the accuracy and conservation of the sequences. It was determined that 84,661 SNP markers could be successfully designed on the AFFY chip platform. Probes were designed according to the flanking sequences of the 84,661 SNP markers to obtain the probe pool of all markers, and finally the BAAFS 90K SNP array chip was fabricated.

[0031] Example 2. Preparation method of BAAFS wheat 65K solid-phase gene chip The genomic DNA of the test materials was extracted by the high-salt and low-pH method, and the DNA concentration and purity were measured with a UV spectrophotometer. The DNA sample concentration was required to be 50 ng / μL, and the OD260 / 280 was between 1.8 and 2.0.

[0032] A total of 192 known wheat representative varieties were scanned using the BAAFS 90K SNP array chip (with 84,661 SNP loci) independently developed by the Institute of Hybrid Wheat, Beijing Academy of Agriculture and Forestry Sciences. Based on the genotype data of 84,661 SNP loci of 192 samples, the analysis was carried out, and the chip experiment adopted the Affymetrix Axiom® 2.0 experimental standard operating procedure. The original data was obtained, and genotyping was performed using the Axiom Analysis Suite software to obtain the SNP locus genotyping data of the samples to be tested.

[0033] The chip analysis process is as follows: (1) Transfer plates and quantify according to the experimental sample layout table and dilution table; (2) Denature and amplify the DNA samples; (3) Fragment and precipitate the amplification products; (4) Dry, resuspend, quantify, and perform quality inspection on the fragmented amplification products; (5) Hybridize and wash / dye the samples; (6) Scan the sample plate.

[0034] According to the evaluation results of the Axiom Analysis Suite software, using Chinese Spring as a reference, the reliability of the loci was verified, and the loci with unreliable sites were deleted. Loci with clear genotyping (sites with obvious boundaries in the clustering results of the three genotypes and containing both homozygous genotypes at the same time), low missing rate (lower than 5%), high resolution, non-linkage, and evenly distributed on chromosomes were selected as extended loci. Candidate core loci were selected based on the physical genetic distance and the minor allele frequency (MAF). Sites without polymorphism, sites with only one homozygous genotype or one homozygous genotype and a heterozygous genotype, sites with a high data missing rate, sites with a high heterozygosity rate (higher than 10%), sites with off-target mutations, and other sites with poor genotyping results were removed, leaving 70,209 relatively ideal markers, accounting for 83%; 63,543 single-copy SNP markers were screened out from them.

[0035] According to chromosome distribution, deletion rate, MAF value, and genotyping quality, 50,000 SNP locus combinations were further screened as variety authenticity identification loci. Among the 192 materials, after removing duplicate materials, more than 180 materials remained. Based on the genotypes of SNP loci of more than 180 core germplasms, linkage disequilibrium (LD) analysis was carried out, tightly linked markers were removed, and markers with large MAF values (Minor Allele Frequency) were preferentially selected, leaving 18,452 SNPs; the LD values between markers were calculated, and markers with r 2 < 0.8 were retained, leaving 7,900 SNPs. Adding functional markers constituted the extended loci for wheat variety identification. 112 SNP loci with uniform chromosome distribution, no linkage, high polymorphism, and the best discrimination ability were screened out, KASP primers were synthesized, fingerprints of 1,433 standard samples of wheat approved varieties were constructed. When 48 optimal locus combinations were selected using statistical algorithms, the variety discrimination efficiency reached 98.4% ( Figure 1 ). For the identification of essentially derived varieties, this chip can identify 0.8% genomic differences, and the misjudgment rate decreased from 1.2% to 0.05%. Considering economy, convenience, flexibility, and the need to distinguish newly emerging varieties in the future, 48 loci were finally determined as the core loci for variety identification (Table 1), which are the preferred markers for variety identity identification. The average minor allele frequency and polymorphism information index of the 48 SNP loci are 0.42 and 0.38 respectively. The chromosome distribution of SNP loci for authenticity identification is shown in Figure 2 , since the D chromosome was integrated into common wheat most recently and the chromosome has not experienced large-scale recombination and exchange, the number of SNP markers screened on the D chromosome group is small; the distribution of MAF values is shown in Figure 3 , among which there are 43,641 loci with MAF values higher than 0.2, accounting for 87%, indicating that the screened SNP loci have high polymorphism and good discrimination ability of locus combinations. From the publicly published molecular marker data related to wheat agronomic traits or tightly linked to functional genes, 3,813 polymorphic and stable markers were screened out to increase the functionality and polymorphism of marker combinations and better meet the related requirements such as accurate phenotype prediction and functional gene mining. In addition, functional SNP markers related to disease resistance, high yield, quality, etc. were selected as probes for detecting wheat whole-genome SNP markers and transgenic elements, and SNP genotyping and transgenic element verification were completed synchronously in one detection, reducing the detection cost by more than 30%.

[0036] Table 1 Some SNP Markers and Probes

[0037]

[0038] According to the "Qualitative PCR Method for Detecting Regulatory Elements CaMV 35S Promoter, NOS Promoter, NOS Terminator and CaMV 35S Terminator in Transgenic Plants and Their Products" (Announcement No. 1782-3-2012 of the Ministry of Agriculture and Rural Affairs) and the "Qualitative Detection Methods for Transgenic Components in Wheat by PCR and Real-time Fluorescent PCR" (SN / T 1943-2019), the detection targets are the CaMV 35S promoter, NOS terminator, ubiquitin three transgenic elements of the promoter, and another Wx012 is selected as the reference endogenous gene. The sequences of the above four elements or genes are aligned with the wheat genome by BLAST. For each element or gene, the 50bp fragment with the highest specificity is selected as the detection marker for that element or gene, and the corresponding probe is designed, as shown in Table 2.

[0039] Table 2 Transgenic Element Markers and Probes

[0040] Finally, a total of 53,813 stable SNP markers were selected ( Figure 4 ), and a wheat solid-phase gene chip was prepared using the microsphere array technology platform independently developed by Lasso Biotechnology. The wheat solid-phase gene chip provided by the present invention is made by Suzhou Lasso Biochip Technology Co., Ltd. (hereinafter referred to as "Lasso Biotechnology") based on its independently developed microsphere array technology, but is not limited thereto. The specific process is as follows: 1) Use lithography and etching techniques to engrave micron-sized micropores arranged in an array on a silicon wafer to make a chip substrate.

[0041] 2) Chemically synthesize the probes using the conventional solid-phase phosphoramidite triester method, and each probe contains a tag sequence.

[0042] 3) Connect each probe to a microsphere with an active group on its surface with a particle size of the micron level through a condensation reaction and fix it on the microsphere.

[0043] 4) Mix a variety of microspheres fixed with different probes evenly and load them onto the chip substrate etched with micropores. The microspheres match the size and shape of the micropores, achieving the effect that each microsphere is fixed in a micropore.

[0044] 5) Sequentially sequence the tag sequences of the microspheres fixed on the chip to identify the types of microspheres fixed in each hole, that is, the corresponding probe types, and make the instructions for this chip, that is, the DLOC file. In downstream applications, each chip needs to be used in conjunction with the DLOC file.

[0045] The process of using the chip to detect SNP markers and transgenic elements is as follows: 1) Extraction of wheat genomic DNA. According to the detection requirements, genomic DNA is extracted from tissues or organs such as wheat embryos, germ, seedlings or leaves.

[0046] 2) Quality inspection of DNA samples. The DNA samples are electrophoretically detected using 1% agarose gel, and image analysis is performed using a gel imaging system. It is required that the electrophoretic bands of the DNA samples are complete, without trailing, and the band size is greater than 10 kb. The purity of the DNA samples is detected using a NanoDrop micro-spectrophotometer, that is, the contamination levels of proteins and organic substances in the genomic DNA are detected. It is required that the A260 / 280 ratio is between 1.8 - 2.0, and the A260 / 230 ratio is between 1.8 - 2.2. The DNA samples are diluted to a working concentration of more than 50 ng / μL for subsequent experiments.

[0047] 3) Amplification of DNA samples. The DNA samples are subjected to whole-genome amplification by isothermal amplification, so that the total amount of DNA is expanded by 1000 - 2000 times, and then it is fragmented to a length of about 150 - 200 bp for chip detection preparation.

[0048] 4) Hybridization and staining. An appropriate amount of sample is added to the sample position on the chip. After the sample DNA fragments are fully hybridized with the probes on the chip, the chip is washed, then DNA polymerase and four bases are added to cause a specific extension reaction for each probe, and finally the chip is stained.

[0049] 5) Chip scanning. The chip that has completed the staining and washing steps is placed into the OmniScan chip scanner of Lasso Biotechnology, and its supporting scanning software OmniScan Control Software is opened for automated fluorescence signal acquisition.

[0050] 6) Data analysis. During chip scanning, the OmniScan Control Software will perform image preprocessing on the collected high-throughput fluorescence images, extract the signal values on each microsphere, and cooperate with the chip DLOC file to standardize and analyze the signal values marked by each DNA molecule.

[0051] The obtained wheat solid-phase gene chip is named "BAAFS wheat 65K Array". The chip contains a set of probes for detecting corresponding SNP markers and transgenic elements, and the probes are fixed on the chip for detecting corresponding DNA molecular markers. It can meet the requirements of application scenarios such as variety authenticity identification, specificity identification, screening of similar varieties, identification of varieties and their essentially derived varieties, genetic relationship identification, construction of genetic linkage maps, genome-wide selection breeding, marker-assisted selection breeding, QTL mapping, association analysis, cluster analysis, etc., and can also meet the requirements of phenotypic trait functional marker detection, breeding and research related to functional genes. The chip of the present invention is used to scan the submitted samples and analyze based on the SNP locus genotype data of the samples. Remove the loci without polymorphism, the loci with only one homozygous genotype, the loci with one homozygous genotype and heterozygous genotype, the loci with a high data missing rate (higher than 5%), the loci with a high heterozygosity rate (higher than 10%), the loci with off-target mutations, and other loci with poor genotyping results. The remaining ones are relatively ideal markers.

[0052] Example 3: Application of "BAAFS wheat 65KArray" Wheat Solid-Phase Gene Chip in Wheat Variety Specificity Identification and SNP Fingerprint Database Construction Using the "BAAFS wheat 65K Array" wheat SNP solid-phase gene chip in Example 1, 200 approved wheat variety standard samples were detected for specificity identification and SNP fingerprint database construction. The genomic DNA of the test materials was extracted by the high-salt low-pH method, and the DNA concentration and purity were measured by an ultraviolet spectrophotometer. It was required that the DNA sample concentration was above 50 ng / μL and OD260 / 280 was between 1.8 and 2.0. 200 wheat samples were analyzed using the chip of the present invention, and the chip experiment was carried out according to the experimental standard operating procedures. The original data was obtained, and gene typing was performed using OmniScan Control Software to obtain the SNP locus gene typing data of the samples to be tested.

[0053] Chip analysis process: (1) Transfer plate and quantify according to the experimental sample layout table and dilution table; (2) Denaturation and amplification of DNA samples; (3) Fragmentation and precipitation of amplification products; (4) Drying, resuspension, quantification and quality inspection of fragmented amplification products; (5) Sample hybridization and washing and staining; (6) Sample plate scanning.

[0054] According to the evaluation results of the OmniScan Control Software, using Chinese Spring as a reference, the reliability of loci was verified, and the unreliable loci were deleted. Loci with clear genotyping (loci with obvious boundaries in the clustering results of three genotypes and containing two homozygous genotypes simultaneously) and low missing rates (less than 5%) were screened as the SNP fingerprints of the varieties. The specific identification results (Table 3) showed that there were significant differences in locus similarity among different variety pairs, indicating the extensive genetic diversity existing in wheat germplasm resources. Even among approximate variety pairs, these loci were sufficient for effective discrimination, demonstrating the high polymorphism and high discrimination ability of the chip of the present invention in variety identification.

[0055] For 200 approved wheat varieties, after removing the varieties with the same name, 194 non - identical wheat varieties remained. Cluster analysis was performed based on the fingerprint data of these 194 varieties, and the results are as Figure 5 shown, indicating that the SNP markers in the chip of the present invention can distinguish all approved wheat varieties, demonstrating the scientificity, accuracy, feasibility, effectiveness, and operability of using this set of markers for wheat variety specific identification and the construction of a SNP fingerprint database of known wheat varieties.

[0056] Table 3 Results of specific detection of wheat standard samples based on "BAAFS wheat 65K Array"

[0057] Example 4 Application of the "BAAFS wheat 65K Array" wheat solid - phase gene chip in the authenticity identification of wheat varieties Use the "BAAFS wheat 65K Array" wheat solid - phase gene chip in Example 1 to identify the true identities of 6 unknown samples to be tested. The steps are as follows: (1) Use the "BAAFS wheat 65K Array" wheat solid - phase gene chip in Example 1 to detect 6 samples to be tested and 6 standard samples.

[0058] (2) According to the fingerprint data of the samples to be tested and the standard samples obtained in step (1), compare the fingerprints of the samples to be tested and the standard samples in pairs, count the total number of loci compared and the number of different loci, and calculate the locus similarity of the two samples according to the formula LS = (1 - D / T ) × 100% (where: LS —— locus similarity; T —— total number of loci compared; D —— number of different loci) (Table 4).

[0059] (3)Identification is carried out according to the site similarity between the sample to be tested and the standard sample: The site similarity between the sample to be tested 1 and the standard sample Chinese Spring 1 is 99.35%, and the identification opinion is that it cannot be excluded that the two belong to the same variety. The site similarity between the sample to be tested 3 and the standard sample Yannong 19 is 99.30%, and the identification opinion is that it cannot be excluded that the two belong to the same variety. The site similarity between the sample to be tested 17 and the standard sample Jimai 22 is 99.31%, and the identification opinion is that it cannot be excluded that the two belong to the same variety. The site similarity between the sample to be tested 27 and the standard sample Jinmai 47 is 99.37%, and the identification opinion is that it cannot be excluded that the two are of the same variety. The site similarity between the sample to be tested 35 and the standard sample Ping'an 9 is 58.84%, and the identification opinion is to exclude that the two are of the same variety. The site similarity between the sample to be tested 65 and the standard sample Shinuo 952 is 56.56%, and the identification opinion is to exclude that the two are of the same variety. The identification results of the samples to be tested are consistent with the actual situation.

[0060] Table 4 Identification Results of Varieties to be Tested

[0061] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A wheat SNP molecular marker combination, characterized in that: It consists of SNP markers covering the whole wheat genome and transgenic element markers. Each SNP marker contains two allele variations of different bases, which are used to detect the allele variation changes at that position. Each transgenic element marker is used to detect whether the element exists in the genome of the tested sample. The SNP marker is screened by the following steps: Step 1, preparation of wheat BAAFS 90K SNP array chip: representative wheat varieties were screened, wheat genomic DNA was extracted, and markers with data reading accuracy scores between 0.60 and 1.00 were screened from Illumina wheat 90k chips. The higher the score, the more reliable the data. Sites that were not repeatable, stable, reproducible, did not conform to genetic laws, clustered irregularly, and had no polymorphism were eliminated. Sites with weak overall signals, missing rates greater than 5%, heterozygosity rates greater than 10%, linkage, and no positioning information were eliminated. Finally, high-quality, high-resolution, single-copy, non-linked, and evenly distributed SNP markers were screened. High-quality, highly polymorphic SNP markers were screened from the AFFY wheat 660k chip; sites that were not repeatable, stable, reproducible, irregularly clustered, and did not conform to genetic laws were eliminated; markers that were unreliable, had a high missing rate, had a weak overall signal, had a heterozygous rate greater than 10%, and had a MAF less than 0.2 were eliminated, and finally sites with high quality, high resolution, and relatively uniform distribution were screened; The publicly published wheat functional SNP markers were combined with the SNP markers screened from the Illumina wheat 90k chip and the AFFYwheat 660k chip, respectively, and after sequence evaluation and preliminary experiments, the wheat BAAFS 90KSNP array chip was finally formed; Step 2: Determine the target site SNP: Use the high salt and low pH method to extract the genomic DNA of the test material, and use a UV spectrophotometer to determine the DNA concentration and purity. Use the BAAFS 90K SNP array chip in step 1 to scan the known representative wheat varieties, and analyze the SNP site genotype data of the sample. The chip test uses the Affymetrix Axiom® 2.0 experimental standard operating procedures to obtain raw data, and use the Axiom Analysis Suite software for genotyping to obtain the SNP site genotyping data of the sample to be tested. The chip analysis process is as follows: (1) Transfer the plate and quantify according to the experimental sample arrangement table and dilution table; (2) DNA sample denaturation and amplification; (3) Fragmentation and precipitation of amplified products; (4) Drying, resuspending, quantification and quality inspection of the fragmented amplification products; (5) Sample hybridization and washing; (6) Sample plate scanning, According to the evaluation results of Axiom Analysis Suite software, loci reliability was verified with Chinese Spring as a reference, unreliable loci were deleted, and loci with obvious boundaries in the three genotype clustering results and containing two homozygous genotypes at the same time, loci with a missing rate of less than 5%, high resolution, and uniform chromosome distribution were selected as extension loci. Candidate core loci were screened according to physical genetic distance and minimum allele frequency (MAF, Minor Allele Frequency), and loci with no polymorphism, only one homozygous genotype, only one homozygous genotype and heterozygous genotype, high data missing rate, heterozygous rate higher than 10%, off-target mutations, and poor typing results were removed. Site-unique, single-copy SNP markers were screened from the remaining markers.

2. A wheat probe combination, which is used to detect the SNP marker combination and transgenic element probe of claim 1, wherein the transgenic element markers are Wx012, ubiquitin promoter, CaMV35S promoter and NOS terminator.

3. A wheat solid phase gene chip, characterized in that: The solid-phase gene chip comprises the probe according to claim 2, and the probe is fixed on the chip for detecting the corresponding SNP molecular marker.

4. Use of the wheat probe combination described in claim 2 or the wheat solid phase chip described in claim 3 in wheat variety identification, the method comprising using the probe combination or solid phase chip to detect the DNA of a wheat sample to be tested, comparing the SNP site information obtained by the detection with the SNP site information of a known standard variety, calculating the site similarity, and judging whether the sample to be tested and the standard variety are the same variety based on the site similarity.

5. Use of the wheat probe combination described in claim 2 or the wheat solid phase chip described in claim 3 in wheat variety-specific identification, by detecting the differences in SNP markers and transgenic element markers of different wheat varieties in claim 1, clarifying the specific genetic characteristics of different varieties, thereby achieving specific identification of wheat varieties.

6. Use of the wheat probe combination described in claim 2 or the wheat solid phase chip described in claim 3 in wheat kinship identification, based on detecting the genetic information of the SNP markers in claim 1 of different wheat samples, calculating the genetic distance or similarity coefficient, and constructing a kinship map to infer the closeness of the kinship between different wheat varieties.

7. The use of the wheat probe combination described in claim 2 or the wheat solid phase chip described in claim 3 in the identification of wheat varieties and their substantially derived varieties, by analyzing the genetic variation of the SNP markers of the tested variety in claim 1 and comparing them with the original variety to determine whether it is a substantially derived variety. If the gene combination of the original variety is retained on more than 92% of the SNP markers and there are specific variations on the key SNP markers, it can be determined as a suspected substantially derived variety.

8. Use of the wheat probe combination of claim 2 or the wheat solid phase chip of claim 3 in transgenic detection, wherein the genomic DNA of a wheat sample is detected using the probe combination. If a signal corresponding to the transgenic element marker of claim 1 is detected, it indicates that the sample contains the corresponding transgenic element and is transgenic wheat.

9. Use of the wheat probe combination described in claim 2 or the wheat solid phase chip described in claim 3 in molecular marker-assisted breeding. In the process of wheat breeding, the SNP markers related to the target trait in claim 1 are detected by means of the probe combination or solid phase chip, and materials carrying excellent trait markers are screened out as breeding materials.

10. The use of the wheat probe combination described in claim 2 or the wheat solid phase chip described in claim 3 in whole genome selection breeding, based on the SNP molecular marker combination in claim 1, whole genome detection of wheat breeding materials, combining phenotypic data to establish a prediction model, predict the genetic value of breeding materials, so as to accurately select individuals with excellent genetic potential in the early generations and realize whole genome selection breeding.

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