Tea tree liquid phase chip and application thereof

Abstract

The invention discloses a tea tree liquid phase chip and application thereof, and relates to the technical field of molecular detection. The invention discloses a tea tree liquid phase chip and application thereof, according to site screening requirements and probe design principles, the tea tree liquid phase chip comprises 5781 SNP sites, and tea tree resource genetic typing can be realized based on a target interval genome sequence liquid phase capture accurate positioning sequencing typing technology. The tea tree liquid chip can realize low-cost genetic typing, is mainly specific to tea trees, can realize variety identification and genetic relationship analysis of the tea trees, scientifically guides hybridization improvement work of the tea trees and assists protection and development of germplasm resources of the tea trees, and has relatively high application values in multiple fields of tea tree breeding. The tea tree liquid phase chip can be used for tea tree genetic diversity evaluation, germplasm resource and genetic relationship identification, genetic map construction and gene localization, whole genome association analysis and tea tree molecular marker assisted breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, in particular to a tea tree liquid phase chip and applications thereof. Background Art

[0002] Tea genetic resources are an important component of biodiversity, and their protection is crucial for promoting the sustainable development of the tea industry and meeting diverse human needs. Currently, tea genetic resources are abundant and possess many desirable traits. However, due to improved selection, local varieties are declining or even disappearing. To prevent further loss of tea genetic resources, it is necessary to protect the genetic resources of local varieties.

[0003] Genomic selection is an important method for genetic improvement of economic traits in tea plants, and genetic variation detection technology based on molecular markers is a very good molecular detection method. With the development of high-throughput sequencing and array technology, the cost of large-scale genotyping has been greatly reduced, and the selection of SNPs (single nucleotide polymorphisms) as genetic markers has become a trend. Currently, there are three main methods for large-scale SNP genotyping: sequencing genotyping methods, whole genome resequencing, and SNP array-based methods. SNP chips, also called SNP arrays, are used for SNPs genotyping and are widely used in genetic diversity analysis, whole genome association analysis, gene mapping, germplasm resource development, and DNA fingerprinting.

[0004] Currently, GoldenGate and Infinium assays are widely used in plant genetics research for SNP genotyping. Both assays are based on Illumina's solid-phase microarray technology, which involves direct hybridization of whole-genome amplified genomic DNA to a microarray of microbeads composed of locus-specific primers, followed by enzyme-based extension assays, sandwich-based immunohistochemistry, and final imaging using a dual-color confocal laser system. Liquid-phase microarrays enable specific capture of each target locus region and high-depth resequencing. The basic principle of liquid-phase microarrays is that a biotin-labeled molecular probe covering the target SNP is designed for each target locus. These probes hybridize and bind to the target genomic region in solution. The hybrid complex is then captured using magnetic beads. After elution, amplification, sequencing library construction, and high-throughput sequencing, the genotypes of all SNPs / InDel sites within the target region are determined.

[0005] Liquid-phase microarrays based on solution hybridization for targeted sequencing offer numerous advantages, including high flexibility in site selection and sample size, high accuracy, high throughput, and low cost. These advantages overcome the technical bottlenecks of traditional solid-phase microarrays, which suffer from poor flexibility, high cost, and difficulty in large-scale application. This invention proposes the development of a liquid-phase microarray for tea plants, with applications in assessing tea genetic diversity, identifying germplasm resources and kinship relationships, constructing genetic maps and mapping genes, performing genome-wide association studies, and molecular marker-assisted breeding for tea plants. Summary of the Invention

[0006] The present invention aims to provide a tea plant liquid phase chip and its application to address the aforementioned problems of the prior art. This tea plant liquid phase chip can achieve genotyping of tea plant resources based on precise positioning sequencing and typing technology using liquid phase capture of target genomic sequences. This technology can be applied to tea plant genetic diversity assessment, germplasm resource and kinship identification, genetic map construction and gene mapping, genome-wide association analysis, and molecular marker-assisted breeding of tea plants.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a SNP site combination for tea variety identification, including 5781 SNP sites located on the reference genome CSS_ChrLev_20200506 as shown in Table 1.

[0009] The present invention also provides the use of the above-mentioned SNP site combination in at least one of the following:

[0010] (1) Assessment of genetic diversity of tea plants;

[0011] (2) Identification of tea germplasm resources and phylogenetic relationships;

[0012] (3) Construction of tea plant genetic map and gene mapping;

[0013] (4) Genome-wide association analysis of tea trees;

[0014] (5) Molecular marker-assisted breeding of tea trees.

[0015] The present invention also provides the use of the above-mentioned SNP site combination in preparing a tea tree 5K liquid phase chip.

[0016] The present invention also provides a tea tree 5K 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.

[0017] The present invention also provides the use of the above-mentioned Tea Tree 5K liquid phase chip in at least one of the following:

[0018] (1) Assessment of genetic diversity of tea plants;

[0019] (2) Identification of tea germplasm resources and phylogenetic relationships;

[0020] (3) Construction of tea plant genetic map and gene mapping;

[0021] (4) Genome-wide association analysis of tea trees;

[0022] (5) Molecular marker-assisted breeding of tea trees.

[0023] The present invention also provides a method for identifying tea tree varieties, comprising the following steps:

[0024] (1) Obtaining genomic DNA of the sample to be tested;

[0025] (2) constructing a sequencing library based on the genomic DNA;

[0026] (3) performing probe hybridization reaction between the sequencing library and the above-mentioned tea tree 5K liquid phase chip;

[0027] (4) extracting genotyping information from the sequence captured by the liquid chip after sequencing to form a genotyping file;

[0028] (5) Construct fingerprint data of 5781 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.

[0029] The present invention discloses the following technical effects:

[0030] The present invention discloses a tea tree liquid phase chip and its application, which includes 5781 SNP sites according to the site screening requirements and probe design principles, and can realize tea tree resource genotyping based on the precise positioning sequencing typing technology of liquid phase capture of target interval genomic sequence. The tea tree liquid phase chip of the present invention can realize low-cost genotyping, which is mainly specific to tea trees, can realize variety identification and kinship analysis of tea trees, scientifically guide tea tree hybridization and improvement work, help the protection and development of tea tree germplasm resources, and has high application value in multiple fields of tea tree breeding. The tea tree liquid phase chip of the present invention can be used for tea tree genetic diversity assessment, germplasm resource and kinship identification, genetic map construction and gene positioning, whole genome association analysis, and tea tree molecular marker assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0032] Figure 1 The distribution map of SNP sites on chromosomes of tea tree 5K liquid phase array;

[0033] Figure 2 The MAF distribution of SNP sites in 256 tea germplasm resources of tea tree 5K liquid phase array;

[0034] Figure 3 This is an annotation information diagram of the SNP sites of the tea tree 5K liquid phase array;

[0035] Figure 4 This is a statistical chart of the site detection rates of 7 tea varieties;

[0036] Figure 5 This is a genetic similarity analysis diagram of 7 tea varieties;

[0037] Figure 6 This is the principal component analysis diagram of 532 tea germplasm resources. DETAILED DESCRIPTION

[0038] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] The present invention provides a tea tree 5K liquid phase chip, which is based on the tea tree genome variation database established by the tea tree germplasm resource innovation team of the Chinese Academy of Agricultural Sciences, combined with Designed and developed with a comprehensive technology system, this chip can be used in breeding applications for genotyping tea germplasm resources, molecular marker-assisted breeding, genetic similarity analysis, kinship analysis, variety protection, and variety authenticity verification; and in scientific research, it can be used for genetic evolution analysis, genetic map construction, and QTL location analysis.

[0045] The samples used to design the chip are derived from the whole-genome resequencing data of hundreds of tea germplasm resources (selected varieties, local varieties, wild resources, etc.) from all over the country and abroad. Medium and high-frequency SNP sites are screened out from them, which is conducive to the development of new tea variety breeding and the research and protection of tea germplasm resources.

[0046] Using 256 test samples, 5,116 marker segments were retained after optimization, totaling 36,357 SNP markers, and the reference genome was CSS_ChrLev_20200506. 5781 core SNP markers were further designed (see Table 1 for site information) to evenly cover the entire genome. The distribution map of the core sites is shown in Figure 1 The MAF distribution map of 256 tea germplasm resources is shown in Figure 2 , site annotation information diagram see Figure 3 .based on Technology can type the target site on the chip and the SNP sites near both sides. Each target site of the tea tree can capture an average of 7 high-quality SNP markers. The 5K liquid phase chip can obtain at least 35K high-quality SNP sites. Compared with traditional solid phase chips, the average genotyping cost of each target site is lower, which can realize large-scale genotyping of tea trees.

[0047] Table 1 SNP sites of tea tree 5K liquid phase array

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] Example 2

[0073] Seven clonal tea varieties were randomly selected, with 12 samples from each variety. The detection rates of the loci in the seven tea varieties were tested, and the genetic similarity between different samples within the variety was analyzed using the following method:

[0074] (1) DNA extraction: DNA was extracted from the tea tree samples using a high-throughput DNA extraction kit or the CTAB method.

[0075] (2) DNA quality inspection: DNA purity, integrity, and contamination were analyzed using 1% agarose gel electrophoresis; DNA concentration was accurately quantified using Qubit; and integrity was accurately tested using Agilent 2100. Acceptable quality inspection criteria included a total amount of no less than 4 μg, a sample concentration less than 40 ng / μL, good sample integrity, and no contamination from impurities.

[0076] (3) Library construction: Use an ultrasonic disruptor to randomly fragment the qualified sample DNA, recover the DNA fragments of the required length by electrophoresis, and add linkers to their ends to form a library.

[0077] (4) Sequencing library construction: The sample library is amplified using LM-PCR and purified to form a sequencing library, which can be used in probe hybridization experiments.

[0078] (5) Construction of hybrid capture library: 300 ng of the constructed sequencing library was taken, freeze-dried, added to the tea tree 5K 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 hybrid capture library.

[0079] (6) Hybridization capture library quality control: 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.

[0080] (7) Sequencing: Sequencing was performed using an Illumina sequencer and related reagents.

[0081] (8) Analysis: After obtaining the offline data, the GTAK best practice process is used to perform standard SNP calling on the data; the sequencing results are compared with the tea tree reference genome to obtain the genome genotyping data to be tested.

[0082] The statistical graph of the detection rate of 7 tea variety sites is shown in Figure 4 The genetic similarity between different samples within a variety is shown in Figure 5 .

[0083] Example 3

[0084] Genotyping of 532 tea samples was performed based on the tea 5K liquid phase array of Example 1, including 286 cultivated varieties, 175 natural resources, 70 wild materials, and 1 outgroup material (Camellia chrysantha). The genotyping method was the same as that of Example 2.

[0085] The test results showed that the detection rate of tea tree 5K liquid phase chip in the above materials was between 90.00% and 98.80%, with an average detection rate of 95.04%. Based on the genotyping data of each tea tree sample, principal component analysis ( Figure 6 ), the results showed that cultivated varieties, resources and wild materials had clustering trends, indicating that the tea tree 5K liquid phase chip can effectively distinguish cultivated varieties, resources, wild materials and out-group materials.

[0086] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A SNP locus combination for tea variety identification, characterized in that: Including 5781 SNP sites located on the reference genome CSS_ChrLev_20200506 as shown in the following table:

2. Use of the SNP locus combination according to claim 1 in at least one of the following: (1) Assessment of genetic diversity of tea plants; (2) Identification of tea germplasm resources and phylogenetic relationships; (3) Construction of tea plant genetic map and gene mapping; (4) Genome-wide association analysis of tea trees; (5) Molecular marker-assisted breeding of tea trees.

3. Use of the SNP locus combination as claimed in claim 1 in preparing a tea tree 5K liquid phase chip.

4. A tea tree 5K liquid phase chip, characterized in that: The method comprises a probe combination for identifying the genotype of each SNP site in the SNP site combination according to claim 1.

5. Use of the Tea Tree 5K liquid phase chip according to claim 4 in at least one of the following: (1) Assessment of genetic diversity of tea plants; (2) Identification of tea germplasm resources and phylogenetic relationships; (3) Construction of tea plant genetic map and gene mapping; (4) Genome-wide association analysis of tea trees; (5) Molecular marker-assisted breeding of tea trees.

6. A method for identifying tea tree varieties, characterized in that: The following steps are involved: (1) Obtaining genomic DNA of the sample to be tested; (2) constructing a sequencing library based on the genomic DNA; (3) performing probe hybridization reaction between the sequencing library and the tea tree 5K liquid phase chip according to claim 4; (4) extracting genotyping information from the sequence captured by the liquid chip after sequencing to form a genotyping file; (5) Construct fingerprint data of 5781 SNP sites of the samples to be tested; The fingerprints of the samples to be tested 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 is calculated.

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