A kind of oat 2K SNP liquid phase chip and its application

Abstract

The present invention discloses an oat 2K SNP liquid phase chip and its application, and relates to the technical field of molecular biology. The present invention develops a high-throughput 2K SNP liquid phase chip for oat genotyping based on targeted capture sequencing technology. The liquid phase chip is determined after many years of multi-point multi-sample experiments, has good repeatability and stability, and is easy to promote and apply; it can be effectively applied to oat variety authenticity identification, seed purity analysis, hybrid breeding and fingerprint map construction. It provides scientific and technological support for oat variety management, quality inspection and intellectual property protection, lays a technical foundation for oat germplasm resource analysis, and is conducive to accelerating the breeding of excellent oat varieties.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, in particular to an oat 2K SNP liquid phase chip and application thereof. Background Art

[0002] Oats used in agricultural production belong to the genus Avena (Avena L., 2n=6x=42; AACCDD genome) of the Poaceae family. They are a globally cultivated dual-purpose crop for food and feed with important economic value. Their 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. The cultivated oat genome is large (~11G) and has a high content of repetitive sequences (~87%). With the rapid development of high-throughput sequencing technology and genome assembly technology, the release of high-quality diploid, tetraploid, and hexaploid oat genome sequence information has improved our understanding of the basic biology of oats, which is crucial for genetics, genomics, and molecular breeding research.

[0003] Variety breeding is the foundation for the development and growth of the oat seed industry, and variety molecular identification is an inevitable requirement for the healthy development of the seed industry. Molecular identification of oat varieties can be applied to variety approval and variety right protection, so that domestic and foreign seed markets and breeding utilization can be legally protected. Therefore, it is very necessary to formulate technical standards for SNP liquid chip identification of oat varieties. Since the implementation of the Seed Law of the People's Republic of China, the Ministry of Agriculture and Rural Affairs of China has aimed to improve the approval of crop varieties to meet the needs of modern agricultural development. The establishment of a national unified variety information database and supervision system can enhance the supervision of variety authenticity, strengthen market and enterprise spot checks, crack down on counterfeiting and counterfeiting, and protect seed quality and farmers' interests.

[0004] In terms of variety authenticity identification, the technology has developed from the initial electron microscope scanning, physical and chemical identification, and protein electrophoresis technology to image-level hyperspectral imaging technology and deep learning image processing technology, and then to the current molecular-level identification. Common molecular identification methods include SSR markers, competitive allele-specific PCR (KASP), SNP identification methods, and the application of MNP molecular markers. As the most widely distributed genetic marker in the genome, SNP has the characteristics of high density, high genetic stability, and easy automated analysis, making efficient and low-cost SNP genotype detection technology the first choice for shared technology and platform development. At present, the gene chip method is the most commonly used method for SNP marker genotyping, including SNP solid phase chips and SNP liquid phase chips. High-throughput SNP liquid phase chips are important tools for molecular breeding research such as high-throughput crop genotyping, germplasm resource identification, true and false seed identification, and fingerprint map construction. SNP molecular marker identification of crop varieties has become one of the important bases for the protection of new varieties. The Seed Law of the People's Republic of China stipulates that the same detection method shall not be used for re-inspection. Therefore, the combined use of multiple detection methods will meet the needs of rapid detection in the future. At present, there are no relevant technical regulations for the identification of oat germplasm resources, the authenticity and purity of varieties at home and abroad, which shows that there is still a lot of room for development in this field. At present, there are no relevant technologies for the identification of oat germplasm resources, the authenticity and purity of varieties at home and abroad. Developing an economical, high-density, high-throughput oat genotyping chip for oat variety identification will help better serve the industrial application of oats. Summary of the invention

[0005] The purpose of the present invention is to provide an oat 2K SNP liquid phase chip and its application to solve the problems existing in the above-mentioned prior art. The oat 2K liquid phase chip developed by the present invention based on targeted capture sequencing technology has sites that evenly cover the chromosome group, and has strong representativeness, high polymorphism, high specificity, and strong versatility, and is suitable for application in the authenticity identification of oat varieties.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a SNP site combination for oat variety identification, including SNP sites located in reference genomes sanfensan and OT3098 as shown in Table 1.

[0008] The present invention also provides the application of the above-mentioned SNP site combination in oat variety identification, seed purity analysis, hybrid breeding and fingerprint map construction.

[0009] The present invention also provides the use of the above-mentioned SNP site combination in preparing a 2K SNP liquid phase chip for oat variety identification.

[0010] The present invention also provides an oat 2K SNP liquid phase chip, comprising a probe combination, wherein the probe combination is used to identify the genotype of each SNP site in the above-mentioned SNP site combination.

[0011] The present invention also provides the application of the oat 2K SNP liquid phase chip in oat variety identification, seed purity analysis, hybrid breeding and fingerprint map construction.

[0012] The present invention also provides a method for identifying oat varieties, comprising the following steps:

[0013] (1) Obtaining genomic DNA of the sample to be tested;

[0014] (2) constructing a sequencing library based on the genomic DNA;

[0015] (3) performing probe hybridization reaction between the sequencing library and the oat 2K SNP liquid phase chip;

[0016] (4) extracting genotyping information after sequencing of the sequence captured by the liquid phase chip to form a genotyping file;

[0017] (5) Construct fingerprint data of 2429 SNP sites of the samples to be tested; compare the fingerprints of the samples to be tested in pairs, statistically compare the total number of sites and the number of differential sites, and calculate the site similarity of the samples to be tested.

[0018] The present invention discloses the following technical effects:

[0019] (1) The present invention adopts liquid phase probe capture technology to design and synthesize a set of liquid phase chips containing 2K SNP site information that can be used to detect oat background sites. The liquid phase chip can quickly and effectively track the genetic material of oats, which is conducive to accelerating the oat breeding process and improving the efficiency of new variety breeding.

[0020] (2) The material background of the data related to the SNP sites involved in the liquid phase chip of the present invention covers a wider range of oat varieties, with high representativeness and rich diversity information.

[0021] (3) The SNP sites involved in the liquid phase chip of the present invention are evenly distributed on the chromosome.

[0022] (4) Compared with other oat chips in the prior art, the present invention has the advantage of low cost for variety authenticity identification.

[0023] (5) The chip of the present invention is a liquid phase chip. Compared with a solid phase chip, the liquid phase chip has high detection accuracy, more effective information, high flexibility and diversity, and can effectively reduce detection costs and improve scientific research and breeding efficiency.

[0024] (6) The oat 2K liquid phase chip developed by the present invention can be applied to the authenticity identification of oat varieties, seed purity analysis, hybrid breeding and fingerprint map construction, which is conducive to accelerating the progress of oat research and breeding.

[0025] (7) SNP marker screening: This technology uses SNP markers for oat variety identification, rather than traditional molecular markers such as SSR or AFLP. SNP markers are highly stable and abundant, widely present in the genome, and have high genetic diversity, so they can more accurately identify oat varieties.

[0026] (8) Data analysis: The technical procedures for the authenticity of oat varieties using SNP liquid phase chip identification also include data analysis of SNP typing results. The oat variety can be determined by analyzing and comparing the genotype combinations of different SNP loci. This data analysis method can identify oat varieties more quickly and accurately, improving identification efficiency.

[0027] In summary, the present invention has developed a high-throughput SNP 2K liquid phase chip for oat genotyping based on targeted capture sequencing technology. The liquid phase chip is determined after many years of multi-point multi-sample experiments, has good repeatability and stability, and is easy to promote and apply; it can be effectively used for oat variety authenticity identification, seed purity analysis, hybrid breeding and fingerprint map construction. It provides scientific and technological support for oat variety management, quality inspection and intellectual property protection, lays a technical foundation for oat germplasm resource analysis, and is conducive to accelerating the breeding of excellent oat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a schematic diagram of the distribution of SNP sites of oat 2K SNP chip on different chromosomes;

[0030] Figure 2 It is a uniform distribution map of SNP sites;

[0031] Figure 3 It is the MAF distribution statistics chart;

[0032] Figure 4 It is a statistical diagram of the annotation information of SNP sites in the genome;

[0033] Figure 5 This is a cluster analysis diagram of genetic distance of 49 germplasm resources. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Example 1

[0040] The present invention provides an oat 2K SNP liquid phase chip, comprising an oat 2K SNP site probe, wherein the oat 2K SNP site probe is a single-stranded DNA synthesized according to an oat 2K SNP molecular marker, and the oat 2K SNP site includes the SNP molecular markers shown in Table 1. 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 to one probe.

[0041] The preparation process of the above oat 2K SNP liquid phase chip is as follows:

[0042] 1. Screening oat 2K SNP loci. Specifically, oat 2K SNP loci were screened and obtained by the following method:

[0043] 1.1 Based on 285 oat 15X resequencing data and 659 GBS data, the oat sanfensan genome (GenBank: GCA_023646675.1) was used as the main reference genome for site screening. BWA was used to detect SNP sites on the genome. The program automatically screened sites with the following parameters: MQ>=55; 30bp / 60bp unique alignment on the genome; GC 35-65; LD filtering; 120bp SNP<=3; 10M windows were used for uniform coverage. A total of 2200 SNP sites were screened.

[0044] 1.2 Use the oat OT3098 genome (GenBank: GCA_916181665.1) as the reference genome, perform supplementary design, and add GAP region probes. Screen according to the selection method of step 1.1. Ensure that the probes cover more than 98% of the region in both genes. A total of 229 SNP sites were screened.

[0045] 2. Design and prepare oat 2K SNP locus probe, the process is as follows:

[0046] 2.1 Determine the probe design principles:

[0047] The probe length is 120bp, the probe GC content is between 25% and 49%, the average GC content is about 45%, the number of homology regions is ≤3, and the selected region does not include SSR and GAP regions as much as possible; and the priority is: gene region>gene promoter region (2Kb)>gene downstream adjacent region (2Kb)>gene spacer region, combined with the principle of uniform chromosome distribution.

[0048] 2.2 Taking the SNP site obtained in step 1 as the center, select the 120 bp nucleotide sequence with the GC content closest to 45% in the left and right 120 bp as the probe.

[0049] 2.3 Based on the 120 bp nucleotide sequence obtained by screening in step 2.2, a single-stranded DNA with a biotin group modified at the 5' end was synthesized, which was called the oat 2K SNP site probe.

[0050] 3. The probe prepared in step 2 is coupled to the fluorescent microspheres, and a targeted capture reagent is added to obtain the oat 2K SNP liquid phase chip.

[0051] The oat 2K SNP liquid phase chip obtained in this example has the following distribution of SNP sites on different chromosomes: Figure 1 As shown by Figure 1It can be seen that the probes of the present invention are uniformly designed on different chromosomes, so that the capture sites cover the entire genome.

[0052] The uniform distribution of SNP sites in the oat 2K liquid phase chip is shown in the figure below. Figure 2 As shown by Figure 2 It can be seen that the 5Mb window coverage of the genome is 99.5%, and the overall coverage of the genome is good.

[0053] The distribution of minor allele frequency (MAF) of the oat 2K liquid phase chip is as follows: Figure 3 As shown by Figure 3 It can be seen that the MAF value is between 0.1 and 0.5. The statistical diagram of SNP annotation information in the genome is shown in Figure 4 .

[0054] Table 1 SNP sites of oat 2K liquid phase array

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[0065]

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[0069] Example 2

[0070] The application method of oat 2K SNP liquid phase array is as follows:

[0071] (1) DNA extraction: DNA was extracted from oat samples using a high-throughput DNA extraction kit or the CTAB method.

[0072] (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 qualified standards were a total amount of no less than 4 μg, a sample concentration of less than 40 ng / μL, good sample integrity, and no impurity contamination of DNA.

[0073] (3) Library construction: Use an ultrasonic disruptor to randomly fragment qualified sample DNA, recover DNA fragments of the required length by electrophoresis, and add linkers to their ends to form a library.

[0074] (4) Sequencing library construction: The sample library is amplified using LM-PCR and purified to obtain a sequencing library, which can be used in probe hybridization experiments.

[0075] (5) Construction of hybridization capture library: 300 ng of the constructed sequencing library was taken, freeze-dried, added to the oat 2K SNP liquid phase chip and hybridization reagent, denatured and incubated at 65°C for 6 hours to complete the hybridization reaction; after the hybridization product was washed with washing solution, another round of PCR was performed to complete the construction of the hybridization capture library.

[0076] (6) Hybridization capture library quality inspection: Qubit2.0 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.

[0077] (7) Sequencing: Sequencing was performed using an Illumina sequencer and related reagents.

[0078] (8) Analysis: After obtaining the data from the machine, use the GTAK best practice process to perform standard SNP calls on the data; compare the sequencing results with the oat reference genome to obtain the genome genotyping data to be tested.

[0079] Example 3

[0080] The oat 2K SNP liquid phase chip obtained in Example 1 has the following application cases:

[0081] (1) Take 49 oat leaves (see Table 2 for samples (including different replicate samples)) and store them in a -20°C refrigerator for DNA extraction.

[0082] (2) According to the oat 2K SNP liquid phase chip application method of Example 2, DNA extraction, library construction, sequencing and obtaining the final SNP data were performed.

[0083] (3) Variety authenticity identification:

[0084] The fingerprint data of 2429 SNP sites of the samples to be tested were constructed; the fingerprints of the samples to be tested were compared in pairs, the total number of sites and the number of differential sites were statistically compared, and the similarity of the sites of the samples to be tested was calculated. The calculation results of the similarity are shown in Tables 3 to 8. The genetic distance cluster analysis diagram of 49 germplasm resources is shown in Figure 5 .

[0085] Similarity (%) = number of consistent sites / total number of sites × 100%

[0086] Based on the 49 oat varieties tested, through similarity calculation, it was determined that when the site similarity is less than 88.00%, the two are excluded from being the same variety; when the site similarity is between 88% and 96%, it is uncertain whether the two are the same variety; when the site similarity is greater than 96.00%, it is not excluded that the two are the same variety.

[0087] Table 2 49 oat samples

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[0090] Table 3 Similarity calculation results between 49 oat samples and K107722 and Dingyan 2

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[0093] Table 4 Similarity calculation results between 49 oat samples and Xiaoyaomai and Beilehao

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[0096] Table 5 Similarity calculation results between 49 oat samples and Modern and 578

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[0099] Table 6 Similarity calculation results between 49 oat samples and Yanke 2 and Mengshaiyan 7

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[0102] Table 7 Similarity calculation results between 49 oat samples and Mengshaiyan No. 1 and Huazao No. 2

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[0105] Table 8 Similarity calculation results between 49 oat samples and Baiyan No. 8 (VI)

[0106]

[0107] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An application of a probe combination for detecting SNP site combinations in oat variety identification, seed purity analysis, hybrid breeding and fingerprint map construction, characterized in that: The SNP site combination includes SNP sites located in the reference genome sanfensan and OT3098 as shown in the following table:

2. Use of a probe combination for detecting a SNP site combination in the preparation of a 2K SNP liquid phase chip for oat variety identification, characterized in that: The SNP site combination includes SNP sites located in the reference genome sanfensan and OT3098 as shown in the following table:

3. An oat 2K SNP liquid phase chip, characterized in that: A probe combination is included, wherein the probe combination is used to identify the genotype of each SNP site in the SNP site combination; The SNP site combination includes SNP sites located in the reference genome sanfensan and OT3098 as shown in the following table:

4. An application of the oat 2K SNP liquid phase chip as claimed in claim 3 in variety identification, seed purity analysis, hybrid breeding and fingerprint map construction of oats.

5. A method for identifying oat varieties, characterized in that: The following steps are involved: (1) Obtaining genomic DNA of samples to be tested and standards; (2) constructing sequencing libraries based on the genomic DNA; (3) performing probe hybridization reaction on the sequencing library and the oat 2K SNP liquid phase chip according to claim 3; (4) extracting genotyping information after sequencing of the sequence captured by the liquid phase chip to form a genotyping file; (5) Construct fingerprint data of 2429 SNP loci of the samples to be tested; The fingerprints of the samples to be tested and the standards are compared in pairs, the total number of sites and the number of difference sites are statistically compared, and the site similarity of the samples to be tested and the standards is calculated; when the site similarity is <88.00%, it is excluded that the two are the same variety; when the site similarity is between 88% and 96%, it is uncertain whether the two are the same variety; when the site similarity is >96.00%, it is not excluded that the two belong to the same variety.

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