A tea tree liquid phase chip and its application

Abstract

This invention discloses a tea plant liquid phase chip and its applications, belonging to the field of molecular detection technology. The invention discloses a tea plant liquid phase chip and its applications, which, based on site screening requirements and probe design principles, includes 5781 SNP sites. It can achieve genotyping of tea plant resources using precise localization sequencing and genotyping technology based on liquid phase capture of target genomic sequences. This tea plant liquid phase chip of the invention enables low-cost genotyping, primarily due to its tea plant specificity. It can facilitate tea plant variety identification and phylogenetic analysis, scientifically guide tea plant hybridization and improvement work, contribute to the protection and development of tea plant germplasm resources, and has high application value in multiple fields of tea plant breeding. The tea plant liquid phase chip of the invention can be used for tea plant genetic diversity assessment, germplasm resource and phylogenetic identification, genetic map construction and gene localization, genome-wide association analysis, and marker-assisted breeding of tea plants.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, and in particular to a tea tree liquid phase chip and its application. Background Technology

[0002] Tea plant genetic resources are an important component of biodiversity, and protecting them is crucial for promoting the sustainable development of the tea industry and meeting diverse human needs. Currently, tea plant genetic resources are abundant and possess many excellent characteristics. However, due to improvement and selection, the number of local varieties has decreased or even disappeared. To prevent further loss of tea plant genetic resources, it is necessary to protect the genetic resources of local varieties.

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

[0004] Currently, GoldenGate and Infinium analyses are widely used in plant genetics research among gene chip technologies for SNP locus genotyping. Both methods are based on Illumina's solid-phase microbead chip technology. This technology involves directly hybridizing whole-genome amplified genomic DNA onto a microbead array composed of site-specific primers, followed by enzyme-based extension assays, sandwich-based immunohistochemical assays, and final imaging via a dual-color confocal laser system. This liquid-phase chip can specifically capture each target locus region and perform high-depth resequencing. The basic principle of the liquid-phase chip is that a biotin-labeled molecular probe covering the target SNP is designed for each test site. These probes hybridize with the target genomic region in solution, and then magnetic beads capture the hybridized conjugates. After elution, amplification, sequencing library construction, and high-throughput sequencing, the genotypes of all SNP / InDel loci within the target region are obtained.

[0005] Liquid-phase microarrays based on solution hybridization for targeted sequencing offer numerous advantages, including high flexibility in site selection, sample quantity flexibility, high accuracy, high throughput, and low cost, overcoming the technical bottlenecks of traditional solid-phase microarrays, which suffer from poor flexibility, high cost, and difficulty in large-scale application. This invention aims to develop a liquid-phase microarray for tea plants, applicable to areas such as tea plant genetic diversity assessment, germplasm resource and kinship identification, genetic map construction and gene localization, genome-wide association analysis, and marker-assisted breeding of tea plants. Summary of the Invention

[0006] The purpose of this invention is to provide a tea plant liquid phase chip and its applications to solve the problems existing in the prior art. This tea plant liquid phase chip can achieve genotyping of tea plant resources based on precise localization sequencing and typing technology using liquid phase capture of target region genomic sequences. This allows for applications in tea plant genetic diversity assessment, germplasm resource and kinship identification, genetic map construction and gene localization, genome-wide association analysis, and marker-assisted breeding of tea plants.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a combination of SNP loci for tea variety identification, including 5781 SNP loci located on the reference genome CSS_ChrLev_20200506 as shown in Table 1.

[0009] The present invention also provides the application of the above-described SNP site combinations in at least one of the following:

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

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

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

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

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

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

[0016] The present invention also provides a tea tree 5K liquid phase chip, comprising a probe array, the probe array being used to identify the genotype of each SNP site in the above-mentioned SNP site array.

[0017] This invention also provides the application of the above-mentioned tea tree 5K liquid phase chip in at least one of the following:

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

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

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

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

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

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

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

[0025] (2) Based on the genomic DNA, a sequencing library was constructed;

[0026] (3) The sequencing library was subjected to probe hybridization reaction with the above-mentioned tea tree 5K liquid phase chip;

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

[0028] (5) Construct fingerprint data of 5781 SNP sites of the sample to be tested; compare the fingerprints of the sample to be tested in pairs, count the total number of sites and the number of different sites, and calculate the site similarity of the sample to be tested.

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

[0030] This invention discloses a tea plant liquid phase chip and its applications. Based on site selection requirements and probe design principles, it includes 5781 SNP sites, enabling precise localization and sequencing genotyping of tea plant resources using liquid phase capture of target genomic sequences. This tea plant liquid phase chip allows for low-cost genotyping, is specifically specific to tea plants, and can be used for variety identification and phylogenetic analysis, scientifically guiding tea hybridization and improvement efforts, contributing to the protection and development of tea germplasm resources, and having high application value in multiple fields of tea breeding. The tea plant liquid phase chip of this invention can be used for tea plant genetic diversity assessment, germplasm resource and phylogenetic identification, genetic map construction and gene localization, genome-wide association analysis, and marker-assisted breeding of tea plants. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0033] Figure 2 MAF distribution map of SNP sites in 5K liquid phase chip of tea plant in 256 tea germplasm resources;

[0034] Figure 3 Annotation information diagram of SNP sites on a 5K liquid phase chip of tea plants;

[0035] Figure 4 A statistical graph showing the detection rate of loci for 7 tea tree varieties;

[0036] Figure 5 Genetic similarity analysis diagram for 7 tea tree varieties;

[0037] Figure 6 Principal component analysis diagram for 532 tea germplasm resources. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0043] Example 1

[0044] This invention provides a 5K liquid phase chip for tea plants, which is based on the tea plant genome variation database established by the Tea Plant Germplasm Resources Innovation Team of the Chinese Academy of Agricultural Sciences, combined with... This chip was designed and developed using a technological system. In breeding applications, it can be used for genotyping of tea germplasm resources, molecular marker-assisted breeding, genetic similarity analysis, kinship analysis, variety protection, and variety authenticity identification. In scientific research, it can be used for genetic evolution analysis, genetic map construction, and QTL mapping analysis.

[0045] The samples used for chip design come from whole-genome resequencing data of hundreds of tea germplasm resources (breeding varieties, local varieties, wild resources, etc.) from various regions at home and abroad. High-frequency SNP sites are screened out from these samples, which is beneficial 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 regions were retained after optimization, totaling 36,357 SNP markers. The reference genome was CSS_ChrLev_20200506. Further, 5,781 core SNP markers were designed (locus information is shown in Table 1), evenly covering the entire genome. The distribution map of the core loci is shown below. Figure 1 The distribution map of MAF in 256 tea germplasm resources is shown below. Figure 2 See the site annotation information diagram. Figure 3 .based on The technology can genotype target sites and SNP sites on both sides of the chip. On average, 7 high-quality SNP markers can be captured for each target site in tea trees. A 5K liquid phase chip can obtain at least 35K high-quality SNP sites. Compared with traditional solid phase chips, the average genotyping cost per target site is lower, which can realize large-scale genotyping of tea trees.

[0047] Table 1. SNP sites on the 5K liquid-phase microarray of tea trees.

[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 asexual tea cultivars were randomly selected, and twelve samples were taken from each cultivar. The detection rate of loci in the seven tea cultivars was detected, and the genetic similarity among different samples within the cultivar was analyzed. The method is as follows:

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

[0075] (2) DNA quality control: The purity, integrity, and contamination of DNA were analyzed using 1% agarose gel electrophoresis; the DNA concentration was accurately quantified using Qubit; and the integrity was precisely detected using Agilent 2100. The standard for passing quality control is a total amount of not less than 4 μg, a sample concentration of less than 40 ng / μL, good sample integrity, and no contaminants.

[0076] (3) Library construction: The qualified sample DNA is randomly fragmented using an ultrasonic disruptor, and the DNA fragments of the required length are recovered by electrophoresis. Adapters are added to the ends of the fragments to form a library.

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

[0078] (5) Hybrid capture library construction: Take 300ng of the constructed sequencing library, freeze-dry it, add tea tree 5K liquid phase chip and hybridization reagent, denature it and incubate it at 65℃ for 6 hours to complete the hybridization reaction; after washing the hybridization product with washing solution, perform another round of PCR to complete the construction of the hybrid capture library.

[0079] (6) Hybrid capture library quality control: Initial quantification was performed using Qubit2.0, and the effective concentration of the library was accurately quantified using qPCR to ensure library quality.

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

[0081] (8) Analysis: After obtaining the sequencing data, standard SNPs were called using the GTAK best practice process; the sequencing results were compared with the tea tree reference genome to obtain the genotyping data of the genome to be tested.

[0082] The statistical chart of the detection rate of 7 tea tree varietal sites is shown below. Figure 4 Genetic similarity among different samples within the same variety is shown in [reference needed]. Figure 5 .

[0083] Example 3

[0084] Genotyping of 532 tea plant samples was performed using the 5K liquid chromatography chip from Example 1, including 286 bred varieties, 175 resources, 70 wild materials, and 1 outgroup material (Camellia chrysantha). The genotyping method was the same as in Example 2.

[0085] The detection results showed that the detection rate of tea plant 5K liquid phase chip in the above materials was between 90.00% and 98.80%, with an average detection rate of 95.04%. Principal component analysis was performed based on the genotyping data of each tea plant sample. Figure 6 The results showed that bred varieties, resources, and wild materials exhibited clustering trends, indicating that the 5K liquid phase chip for tea trees can effectively distinguish bred varieties, resources, wild materials, and outgroup materials.

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

Claims

1. A 5K liquid phase chip for tea trees, characterized in that, It includes a probe array used to identify the genotype of each SNP locus in a combination of SNP loci; The SNP locus combinations include 5781 SNP loci located on the reference genome CSS_ChrLev_20200506, as shown in the table below: 。 2. The application of the tea tree 5K liquid phase chip as described in claim 1 in at least one of the following: (1) Assessment of genetic diversity in tea plants; (2) Identification of tea germplasm resources and kinship; (3) Construction of genetic map and gene localization of tea plant; (4) Genome-wide association analysis of tea plants; (5) Molecular marker-assisted breeding of tea trees.

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