A liquid phase chip for oolong tea trees and its application

Abstract

This invention relates to the field of whole-genome gene chips, specifically to a liquid-phase chip for oolong tea trees and its applications. The chip's genotyping targets include 45,477 SNP loci, all derived from the genome of the oolong tea variety Tieguanyin. Locus information is shown in Table 1. This invention mines SNP loci from large-scale resequencing data, using the oolong tea tree genome as a reference, discovering and screening 45,477 SNP loci (approximately 40K) suitable for chip design. The designed liquid-phase chip enables genotyping, demonstrating high application value in multiple related fields, including oolong tea germplasm resource conservation, oolong tea variety breeding, and other tea tree resource breeding.

Description

Technical Field

[0001] This invention relates to the field of whole genome gene chips, specifically to a liquid phase chip for oolong tea trees and its applications. Background Technology

[0002] Since RAPD molecular markers were first used in tea plant genetic diversity research in 1997, various molecular markers and their derivative technologies, such as RFLP, ISSR, AFLP, and SSR, have been widely used in tea science research over the past 30 years. Among these, there are numerous reports of constructing tea plant microarrays using expression sequence tags (Chen Liang, Ma Chunlei, Yao Mingzhe, Wang Xinchao, Jin Jiqiang. A gene chip composed of tea plant expression sequence tags [P]. CN101942514A. 2011, 01, 12). However, at this stage, the tea plant reference genome had not yet been fully assembled, and all typing analysis techniques lacked in terms of throughput, scalability, and flexibility. Subsequently, with the completion of the genome assembly of tea plants such as Yun Kang 10, Tieguanyin, Shucha Zao, Longjing 43, and Huangdan, the application of tea plant molecular markers is gradually shifting towards high-throughput technologies such as SNPs with reference genomes.

[0003] Currently, liquid-phase microarrays have been widely used in fields such as species evolution analysis, germplasm resource evaluation, and DNA fingerprinting, achieving satisfactory results. Liquid-phase microarrays utilize resequencing technology and biotin-labeled probes designed based on the principle of DNA complementarity to achieve high-depth detection of each target locus. This technology boasts advantages of high accuracy and high throughput, and has become an important tool in fields such as genotype detection, germplasm resource evaluation, and molecular genetic mapping construction, demonstrating significant application potential in areas such as genome-wide selection.

[0004] As a distinctive tea category, oolong tea is mostly made from Wuyi varieties of the Theaceae family. However, all studies have shown that these varieties have unique characteristics in metabolism and genetic regulation. Therefore, conducting oolong tea resource breeding research and developing dedicated gene chips based on the reference genome of oolong tea varieties is of great practical significance. Summary of the Invention

[0005] The purpose of this invention is to provide a liquid phase chip for oolong tea trees and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A liquid phase chip for oolong tea trees is provided. The genotyping target of this chip includes 45,477 SNP loci, all of which are derived from the genome of the oolong tea variety Tieguanyin. The specific locus information is shown in Table 1 below.

[0008] Table 1 SNP locus information

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032] The value before the underline in the table is the chromosome number, and the value after the underline is the locus on that chromosome.

[0033] This invention also provides the application of the liquid phase chip in the mapping of associated genes, genetic diversity analysis, genome-wide association analysis, genome selection, variety identification, kinship identification, core germplasm screening, fingerprinting, or assisted breeding of traits in oolong tea trees and other types of tea trees.

[0034] The beneficial effects of this invention are reflected in:

[0035] This invention mines SNP sites from the oolong tea tree genome as a reference in large-scale resequencing data, discovering and screening 45,477 SNP sites (approximately 40K) that can be used for chip design. Genotyping can be achieved using the designed liquid-phase chip, which has high application value in multiple related fields such as oolong tea germplasm resource protection, oolong tea variety breeding, and other tea tree resource breeding.

[0036] The liquid-phase microarray for oolong tea trees involved in this invention is based on targeted capture sequencing technology. It can not only perform genotyping at target loci, but also accurately genotype SNPs within a certain range near the target loci. Therefore, it can obtain more SNP genotyping information than marker loci. Compared with traditional solid-phase microarrays or other microarrays developed for tea trees, it offers higher flexibility and applicability, effectively narrowing the genotyping range, reducing interference from irrelevant genomic information outside the oolong tea tree, and allowing for the addition of marker loci as needed. Furthermore, the liquid-phase microarray relies on a next-generation sequencing platform, resulting in low genotyping costs, low requirements for subsequent data processing techniques, and low computational resource consumption, providing a technical means for large-scale genotyping in oolong tea tree-related research. Attached Figure Description

[0037] Figure 1 This example shows the distribution of 45,000 SNP sites on the 40K liquid phase chip of oolong tea across the entire genome. The number of SNP sites contained within a 1M sliding window on each chromosome is also shown.

[0038] Figure 2 The Manhattan plot is obtained from the genetic distance analysis between oolong tea varieties and non-oolong tea varieties. Detailed Implementation

[0039] The technical solution of the present invention will now be clearly and completely described in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0040] 1. Design and fabrication of a 40K liquid phase chip for oolong tea processing

[0041] This invention uses 279 tea varieties for resequencing (Table 2), all of which are representative varieties that have passed review and identification or variety registration. These include 48 oolong tea varieties and 231 varieties unsuitable for oolong tea production. When analyzing the 1,048,576 SNP loci obtained from the resequencing of these varieties, screening was performed based on criteria of deletion rate <0.1, heterozygosity rate <0.2, and MAF value >0.05, while also considering the principle of even distribution of SNP loci on each chromosome. A total of 45,477 effective SNP loci suitable for microarray development were obtained.

[0042] Based on the location of these 45,477 SNP sites and their flanking sequences, Shijiazhuang Borui Biotechnology Co., Ltd. designed primers and synthesized probes using targeted capture sequencing technology. A total of 86,801 probes were synthesized. The chip was evaluated in the first and second rounds using 18 and 302 samples, respectively, and finally a 40K liquid phase chip containing 45,477 SNP sites for oolong tea was prepared.

[0043] Table 2 lists the tea varieties used for resequencing.

[0044]

[0045]

[0046] 2. Procedure for detecting DNA in tea plant samples using a 40K liquid chromatography-mass spectrometry chip for oolong tea

[0047] (1) Extraction of tea tree genomic DNA: Leaves were taken from tea trees and DNA was extracted using the CTAB method or a polysaccharide and polyphenol plant DNA genomic extraction kit (Beijing Huayueyang Biotechnology Co., Ltd.).

[0048] (2) DNA sample quality detection: Agarose gel electrophoresis with a mass fraction of 1% to 1.5% was used for detection. The electrophoresis results were judged using a gel imaging system (GelDocXRSystem, Bio-Rad, USA) to ensure DNA quality. The concentration and OD value of genomic DNA were measured using a NanoDrop2000c ultra-micro spectrophotometer to confirm that the purity and concentration of DNA could meet the needs of subsequent experiments. For samples with high concentrations, the DNA concentration should be adjusted to a suitable working concentration of 10 to 50 ng / μl.

[0049] (3) Tea tree phase chip detection: mainly includes DNA hybridization, DNA capture, DNA hybridization capture library quality inspection, DNA hybridization capture library sequencing and other processes. The specific operation is carried out in accordance with the standard process of oolong tea 40K liquid phase chip detection (https: / / en.molbreeding.com / technologies).

[0050] (4) Data Analysis: The raw sequencing data needs to be quality controlled using the bioinformatics analysis tool FastQC (www.bioinformatics.babraham.ac.uk / project). Then, using the analysis tool BWA (bio-bwa.sourceforge.net) under default parameters, the quality-controlled sequencing data is aligned to the Tieguanyin reference genome. Finally, using the tool GATK (software.broadinstitute.org / gatk) and Perl scripts, SNP identification and probe capture sequencing genotyping information extraction are performed on the sequencing data to form the final genotyping file. The distribution of its 45,000 SNP loci on the chromosome is as follows: Figure 1 As shown.

[0051] 3. Application of 40K liquid phase chip in the population structure analysis of oolong tea

[0052] Fresh leaf samples from 48 representative tea varieties were collected in March 2023 at the Fujian Provincial Tea Germplasm Resource Nursery, Shekou Base, Tea Research Institute, Fujian Academy of Agricultural Sciences. Genotyping was performed using a 40K liquid phase chip for oolong tea (specific genotyping methods are described in Part 2 above). The genotyping results underwent quality control, removing individuals with a minimum allele frequency <0.05, genotype deletion rate >0.1, and sample deletion rate >0.1, ultimately yielding 43,112 SNP markers and 46 individuals. Population structure analysis using the selected SNP loci detected outliers, effectively distinguishing oolong tea varieties from non-oolong tea varieties based on genetic structure. Furthermore, the interpopulation genetic distance between oolong tea and non-oolong tea varieties was calculated, with a value of 0.0644, indicating a moderate degree of genetic differentiation between the oolong tea and non-oolong tea variety populations. Figure 2 This demonstrates that this liquid chip, combined with population structure analysis and phylogenetic tree construction, can be used for assisted screening of oolong tea varieties. Based on this analysis, subsequent work such as material grouping in metabolomics differential analysis among groups has scientific value and direct guiding significance.

[0053] 4. Application of 40K liquid chromatography-mass spectrometry chip in the screening of core germplasm of oolong tea

[0054] Using CoreHunter, based on the experimental data above and 480 effective metabolites obtained by GC-MS detection, core germplasm screening for oolong tea was conducted. The results showed that, with a sampling rate of 25%, the core germplasm varieties Jin Guan Yin and Mei Zhan were selected. These two varieties are widely cultivated in major tea-producing areas across China and are recognized as superior oolong tea varieties with excellent comprehensive traits. Therefore, this research fully demonstrates that 40K liquid phase microarrays can play a significant role in the construction of core oolong tea germplasm.

[0055] 5. Advantages of 40K liquid phase chip for oolong tea

[0056] (1) The 279 samples used in this invention for chip design are tea tree varieties that have been approved, identified or registered in 14 provinces and cities across the country. They are widely sourced and highly representative, including 48 oolong tea varieties. They cover the unique mid-to-high frequency SNP sites of oolong tea special varieties, making them more suitable for oolong tea resource breeding research and conducive to the development of oolong tea breeding work and the protection of oolong tea germplasm resources.

[0057] (2) The tea tree reference genome used in the design of the chip in this invention is the well-known oolong tea variety Tieguanyin. The polymorphism and fit of the SNP sites are more in line with the breeding research of oolong tea germplasm resources. In application, it can reduce the error of introducing genetic information of other tea tree varieties. At the same time, it is also convenient to effectively superimpose the research results with other related studies on oolong tea resource breeding in the later stage, such as direct comparison of associated sites and functional candidate gene sequences without converting index numbers.

[0058] (3) Compared with traditional solid-phase chips, this invention can detect more SNP sites (40-45K) compared with solid-phase chips with the same number of probes. Moreover, the design is flexible, and additional marker sites of interest can be added at any time according to research needs.

[0059] (4) Compared with whole genome resequencing, the present invention has a significant price advantage, not only in terms of the price difference of the chip itself, but also in terms of saving the computing resources required for data processing compared with whole genome resequencing. It can perform large-scale typing of tea trees (especially long oolong tea varieties), thereby promoting the breeding of oolong tea resources.

[0060] (5) The 45,477 valid SNP sites contained in this invention have been evaluated for quality in more than 300 samples, and have significant advantages in stability and accuracy when applied.

[0061] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A liquid phase chip for oolong tea trees, characterized in that: The genotyping target of this chip includes 45,477 SNP loci with the genome of the Oolong tea variety Tieguanyin as a reference genome. The specific SNP locus information is shown in Table 1 of the instruction manual.

2. The application of the liquid phase chip of oolong tea tree as described in claim 1 in the genetic diversity analysis, variety identification or core germplasm screening of oolong tea tree traits.

Images