Tea tree 40K assisted breeding liquid phase chip and application thereof

Abstract

The invention discloses a tea tree 40K assisted breeding liquid phase chip and application thereof, and belongs to the field of whole genome gene chips. Genetic typing SNP loci of the chip are all from 30X high-depth re-sequencing genome data of the core germplasm of the Chinese tea tree. According to the invention, SNP loci taking a Chinese tea 102 tea tree genome as a reference are mined from large-scale re-sequencing data, 44, 915 SNP loci capable of being used for chip design are found and screened, the loci are good in representativeness, strong in specificity, high in polymorphism and high in detection throughput, detection of nearly thousands of materials can be covered at the same time, and the SNP loci are suitable for a mainstream sequencing platform. The designed liquid chip can realize high-throughput genotyping, and has application value in multiple related fields of tea tree germplasm resource protection, tea tree variety breeding and other tea tree resource breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a liquid phase chip for tea tree 40K-assisted breeding and its application. Background Technology

[0002] Traditional molecular marker technologies for tea plants, such as SSR, AFLP, and ISSR, have played a crucial role in tea plant genetic research. However, these technologies generally suffer from drawbacks such as low marker density, incomplete genome coverage, and limited polymorphism detection capabilities. Furthermore, they are costly, complex to operate, and have poor reproducibility when used for large-scale sample testing. With the deepening development of tea plant genomics research and the significant reduction in sequencing costs, genome-wide SNP markers have gradually become the mainstream technology for tea plant molecular genetic research. As the most abundant type of genetic variation in the genome, SNPs have advantages such as high distribution density, good genetic stability, and a high degree of standardization in detection, providing a more reliable technical means for the precise identification of tea plant germplasm resources and molecular breeding.

[0003] SNP liquid-phase microarray technology, as a next-generation genotyping platform, has demonstrated significant advantages in the field of crop genetics and breeding. Based on the detection principle of single nucleotide polymorphism (SNP) sites, this technology achieves simultaneous high-throughput detection of a large number of SNP sites through the hybridization reaction of specific probes with target DNA sequences, combined with fluorescent labeling and flow cytometry. Compared with traditional molecular markers, SNP liquid-phase microarrays offer advantages such as high detection accuracy, good reproducibility, high automation, and cost-effectiveness. In major crops such as rice, maize, and wheat, SNP liquid-phase microarrays have been successfully applied to research on germplasm resource genetic diversity evaluation, variety fingerprinting, heterosis prediction, and genome-wide association studies of important agronomic traits, providing a powerful molecular tool for modern crop breeding. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to overcome the technical defects of existing tea tree molecular marker technology, such as low marker density, insufficient sample representativeness, and high detection cost.

[0005] The present invention solves the above-mentioned technical problems through the following technical means: This invention proposes a 40K-assisted breeding liquid phase chip for tea trees, which contains 44,915 SNP loci. The SNP loci information is located based on the tea tree reference genome "Zhongcha 102", and the specific loci information is shown in Table 2.

[0006] These 44,915 SNP loci were all derived from 30X high-depth resequencing genome data of core germplasm of Chinese tea trees.

[0007] Preferably, the SNP site is obtained by the following method: (1) Whole genome site screening; (2) Site probe design and screening; (3) Site optimization screening.

[0008] Preferably, the whole-genome site screening criteria include: setting quality control standards of minimum allele frequency greater than or equal to 0.3, site deletion frequency less than 0.1, and sequencing depth greater than or equal to 5×, and removing insertion / deletion sites (indels) and retaining only biallelic SNP sites.

[0009] Preferably, the site probe design and screening criteria include: designing a probe sequence with a length of 110 bp, and setting screening criteria of ≤5 homologous regions and 30-70% GC content.

[0010] Preferably, the site optimization screening criteria include: for regions with a gap greater than 1 Mb, 190 additional sites with a MAF ≥ 0.1 are selected.

[0011] This invention also proposes the application of the above-mentioned 40K-assisted breeding liquid phase chip for tea plant germplasm resource genetic diversity analysis, population structure analysis, genetic and evolutionary analysis, kinship identification, genome-wide association analysis, or genome selection.

[0012] This invention also proposes the application of the above-mentioned 40K tea tree assisted breeding liquid phase chip in the location of associated genes, variety identification, core germplasm screening, fingerprint map construction, or assisted breeding of traits in tea trees and other types of tea trees.

[0013] This invention also proposes the application of the above-mentioned 40K-assisted breeding liquid phase chip in the genetic evaluation of tea germplasm resources, variety identification, phylogenetic analysis, linkage map construction, and quantitative trait gene localization.

[0014] The beneficial effects of this invention are as follows: 1. This invention develops a liquid chromatography-mass spectrometry (LC-MS) chip for auxiliary breeding based on a 30X high-depth resequencing dataset of core Chinese tea germplasm. The chip's data encompasses various types of tea germplasm resources, including wild relatives, local species, and cultivated varieties. The samples are diverse in origin, have deep sequencing depth, and exhibit good representativeness and reliability. This chip can be widely applied in multiple fields, such as identifying phylogenetic relationships in tea germplasm resources, phylogenetic analysis, genetic map construction, genome-wide association studies (GWAS), varietal authenticity identification, genotyping of hybrid offspring, and genetic background analysis of breeding materials, providing crucial technical support for molecular breeding and the conservation and utilization of tea germplasm resources.

[0015] 2. High marker density and comprehensive genome coverage. The genotyping target of this chip includes 44,915 SNP loci across the entire tea plant genome, which are evenly distributed throughout the tea plant genome (e.g., ...). Figure 1As shown in the figure, it covers all 15 chromosomes, with an average distance of about 69 kb between loci. It is evenly distributed on the tea tree genome, providing high-resolution genomic information and significantly improving the accuracy and reliability of genetic analysis.

[0016] 3. Highly representative samples with wide applicability. Developed based on 30X high-depth resequencing data of core tea tree germplasm nationwide, covering wild, local, and cultivated varieties, it demonstrates excellent detection results and broad applicability to different types of tea tree materials.

[0017] 4. High throughput and cost-effectiveness. Based on liquid-phase capture sequencing technology, hundreds of samples can be detected simultaneously in a single experiment. Compared with traditional labeling technologies, the detection efficiency is significantly improved, and the cost per sample is greatly reduced.

[0018] 5. Excellent technical stability and high reproducibility. Standardized experimental procedures and a rigorous quality control system ensure the accuracy and consistency of test results, providing reliable technical support for tea molecular breeding and germplasm resource research.

[0019] 6. The present invention develops a high-density tea tree SNP liquid phase breeding chip, which is constructed based on the high-depth resequencing data of core tea tree germplasm across the country. It has technical characteristics such as abundant marker sites, uniform genome coverage, high polymorphism level, and good detection stability, and can meet the technical needs of large-scale tea tree germplasm resource evaluation, molecular breeding and genetic research.

[0020] 7. This invention mines SNP sites from the genome of the "Zhongcha 102" tea tree using large-scale resequencing data as a reference, discovering and screening 44,915 SNP sites suitable for microarray design. These sites exhibit good representativeness, high specificity, high polymorphism, and high detection throughput, capable of simultaneously covering the detection of nearly a thousand samples, and are suitable for mainstream sequencing platforms. The designed liquid-phase microarray can achieve high-throughput genotyping, with application value in multiple related fields such as tea germplasm resource conservation, tea variety breeding, and other tea resource breeding.

[0021] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0022] Figure 1 This is a diagram showing the distribution of SNP sites on the genome using a 500kb window size in the chromosome sliding statistical analysis of Embodiment 1 of the present invention. Figure 2 This is an application of the 40K-assisted breeding liquid-phase chip system for developmental analysis of tea trees in Embodiment 2 of the present invention (Note: the red area represents the Assam variety, and the yellow area represents the tea variety). Figure 3This is an IBD heatmap analysis of the kinship of tea trees in Example 3 of the present invention. (Note: The PI-HAT value (kinship coefficient) ranges from 0 to 1, with higher values ​​indicating closer kinship between samples. Shuchazao is the male parent, Longjing 43 is the female parent, and samples 1-5 are hybrid offspring. The PI-HAT value between the hybrid offspring and the parents is >0.5, confirming the parent-child relationship.) Detailed Implementation To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0023] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0024] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0025] Example 1: 1. Collection of tea tree germplasm resources We collected 1,076 tea germplasm resources from 16 major tea-producing provinces in China, covering wild relatives, local species, and cultivated tea varieties. Based on ddRAD-seq sequencing technology, we conducted a preliminary analysis of the genetic diversity of the germplasm resources and constructed a core germplasm set of 160 Chinese tea accessions based on this genetic diversity. C. crassicolumna (Thick axis tea), C. gymnogyna (Bald House Tea) C. kwangsiensis (Guangxi Tea) C. tachangensis (Tea from a major factory) C. taliensis (Dali Tea) C. grandibracteata (Large-budded tea) and tea tree cultivars C. sinensis var. dehungensis (A variant of Dehong tea) C. sinensis var. pubilimba (White Hair Tea Variety) C. sinensis var. assamica (Assam variant) C. sinensis var. sinensis (Tea variety).

[0026] Table 1: 160 core germplasm sets of Chinese tea trees used for resequencing

[0027] 2. Tea plant whole genome resequencing The 160 tea germplasm resources collected were subjected to whole-genome resequencing. The specific steps included: (1) DNA was extracted using the CTAB method; (2) DNA that passed quality inspection was used to construct a sequencing library using the BGI library standard method; (3) After the library passed quality inspection, 2×150bp paired-end sequencing was performed using the DNBSEQ-T7 sequencer of the BGI sequencing platform, with a sequencing depth of 30× and no less than 90Gb of clean data for each sample.

[0028] Mutation detection: (1) The raw data was filtered for quality using the fastp (v0.20.0) sliding window method; (2) The filtered high-quality data was aligned to the reference genome of the tea variety “Zhongcha 102” using the bwa (0.7.12-r1039) mem program. The alignment parameters were set to the default bwa mem settings. The sam file was sorted and converted to a bam file using picard 1.107 software; (3) SNP detection was performed using GATK software. The filtering criteria were as follows: Fisher strand bias test (FS) ≤60, haplotype score (HaplotypeScore) ≤13.0, alignment quality (MQ) ≥40, quality depth (QD) ≥2, read position rank sum (ReadPosRankSum) ≥-8.0, alignment quality rank sum (MQRankSum) >-12.5, sequencing depth (DP) ≥4 reads.

[0029] 3. Development of liquid phase breeding chips for tea trees (1) Whole genome locus screening: Based on the 30× high-depth resequencing data of the core germplasm of Chinese tea tree, whole genome candidate loci were screened. The quality control standards were set as follows: minimum allele frequency greater than or equal to 0.3, locus deletion frequency less than 0.1, and sequencing depth greater than or equal to 5×. Insertion and deletion sites (indels) were removed, and only biallelic SNP sites were retained. Finally, 4,041,035 high-quality SNP polymorphic sites were screened as candidate sites for the development of liquid phase breeding chips for tea trees.

[0030] (2) Site probe design and screening: Based on the SNP site, a probe sequence with a length of 110bp was designed. The screening criteria were set as follows: the number of homologous regions ≤ 5 and the GC content 30-70%. Finally, 839,677 high-quality SNP sites that can be used for chip development were obtained.

[0031] (3) Site optimization screening: Selection clearance signals were detected by XP-EHH analysis, and the top 2% selection clearance regions between wild closely related species and Assam variety, and between Assam variety and tea variety were identified, resulting in a total of 3,527 bed regions. These regions were intersected with the 839,677 sites screened above, and 10,005 sites were evenly selected from the intersection sites. At the same time, in order to ensure that the probes are evenly distributed on the chromosome, we also selected 34,720 sites located in non-bed regions that were successfully evaluated. Finally, for regions with a gap of more than 1Mb, 190 sites with MAF≥0.1 were additionally selected, for a total of 44,915 sites for probe synthesis and testing. The 44,915 SNP sites were located based on the tea reference genome "Zhongcha 102", and the specific site information is shown in Table 2.

[0032] 4. Procedure for detecting tea tree DNA samples using a 40K SNP liquid chromatography-mass spectrometry chip. (1) Extraction of tea plant genomic DNA: Fresh leaves were taken from tea plant samples, and tea plant genomic DNA was automatically extracted using a high-throughput automated nucleic acid extraction platform, or manually extracted using the CTAB method. The extraction process was strictly carried out in accordance with standard operating procedures to ensure the integrity and purity of the DNA.

[0033] (2) DNA sample quality testing: DNA integrity was assessed using agarose gel electrophoresis at a concentration of 1%–1.5% (w / w). The electrophoresis results were reviewed using a gel imaging system (GelDoc XR System, Bio-Rad, USA) to ensure no significant DNA degradation. The concentration and OD value of the genomic DNA were measured using a NanoDrop 2000c ultra-micro spectrophotometer to confirm that the DNA purity (OD260 / 280 ratio between 1.8 and 2.0) and concentration met the requirements for subsequent experiments. For samples with insufficient concentration, the DNA concentration needed to be adjusted to a suitable working concentration of 50–200 ng / μl.

[0034] (3) Tea tree genome chip detection: GenoBaits hybridization capture method: A quantitative amount of DNA (500 ng) is taken and fragmented using restriction endonucleases. After end repair, the fragmented DNA is ligated with A-tails. The A-tailed DNA fragments are ligated to sequencing adapters using ligase, and the library is then purified using carboxyl-modified magnetic beads. The ligation product is added to barcoded sequencing primers and a high-fidelity PCR reaction system for PCR amplification. Different barcodes are used to distinguish different samples. After purification with carboxyl-modified magnetic beads, the amplified product is ready for probe hybridization experiments. The hybridization product is lyophilized, and then probes and hybridization reagents are added. After denaturation, the mixture is incubated at 65°C for 2 hours to complete the hybridization reaction. After washing with washing buffer, a second round of PCR is performed to complete the construction of the hybridization capture library.

[0035] GenoPlexs Multiplex PCR: Multiplex PCR panel mix and multiplex PCR amplification enzyme system are added to quantitative DNA, and the mixture is placed on a PCR instrument to complete the PCR reaction. The PCR product is purified using carboxyl magnetic beads, and then barcoded sequencing primers and a high-fidelity PCR reaction system are added again for PCR amplification. Different barcodes are used to distinguish different samples. The amplified product after purification with carboxyl magnetic beads completes the multiplex PCR capture and library construction.

[0036] (4) Library construction and sequencing: Targeted sequencing libraries for DNA samples were constructed using the methods described above. After library construction, preliminary quantification was performed using Qubit 2.0, followed by accurate quantification of the effective concentration of the library using qPCR to ensure library quality. Once the library passed the testing, it proceeded to the sequencing stage.

[0037] (5) Data Analysis: The raw sequencing data needs to undergo bioinformatics analysis, mainly including: data quality control (removal of adapters and low-quality data), alignment with the reference genome, variant detection and annotation, etc. Using the analysis tool BWA (bio-bwa.sourceforge.net) under default parameters, the quality-controlled sequencing data is aligned to the tea reference genome. Finally, the tool GATK is used to identify SNPs and extract genotyping information from the sequencing data, forming the final genotyping file. The annotated variant information can be used for applications in genetic evaluation of tea germplasm resources, variety identification, phylogenetic analysis, linkage map construction, quantitative trait gene mapping, genome-wide association analysis, or genome selection.

[0038] Application of 40K-assisted breeding liquid phase chip in tea tree Example 2: Application of 40K-assisted breeding liquid-phase chip system for developmental analysis of tea trees The application process of the 40K assisted breeding liquid phase chip for tea trees obtained in Example 1 is as follows.

[0039] (1) Take 40 samples of tea tree leaves of different varieties for DNA extraction; (2) DNA extraction, library construction, sequencing and final SNP data were performed according to the experimental procedure of 40K-assisted breeding liquid chip for tea trees; (3) Calculation of detection rate: The product detection rate is an important indicator for measuring chip quality. In plants, the detection rate is generally measured by the ratio of the number of detected sites to the number of developed sites. The average detection rate of the 40 samples of this product is 97.80%. The specific detection rate results (partial samples) are shown in Table 3.

[0040] Table 3 Sample Detection Rate Results

[0041] (4) Phylogenetic analysis: Based on the obtained genotyping data, a phylogenetic tree was constructed using IQ-TREE software to analyze the kinship, evolutionary relationships, and genetic structure among different materials. Results (e.g.) Figure 2 The results show that the chip can effectively distinguish between the original tea variety and the Assam variety, and the grouping effect is highly consistent with the actual botanical classification, proving that the selected SNP sites have high representativeness and resolution, and providing reliable technical support for the identification of tea germplasm resources and molecular-assisted breeding.

[0042] Example 3: Kinship Identification The application process of the 40K assisted breeding liquid phase chip for tea trees obtained in Example 1 is as follows.

[0043] (1) Fresh leaf samples of Shuchazao, Longjing 43 and their five hybrid offspring, along with 37 representative tea tree varieties, were taken for DNA extraction. (2) DNA extraction, library construction, sequencing and final SNP data were performed according to the experimental procedure of 40K-assisted breeding liquid chip for tea trees; (3) Calculation of detection rate: The product detection rate is an important indicator for measuring chip quality. In plants, the detection rate is generally measured by the ratio of the number of detected sites to the number of developed sites. The average detection rate of the 44 samples of this product is 97.82%. The specific detection rate results are shown in Table 4.

[0044] Table 4 Sample Detection Rate Results

[0045]

[0046] (4) Kinship analysis: IBD (Identity by Descent) analysis was performed on the genotyping data using PLINK software. Results (e.g.) Figure 3 The results showed that the PI-HAT values ​​between the hybrid offspring and the parents were all greater than 0.5, which verified the high representativeness and resolution of the selected SNP sites and provided reliable molecular marker technology support for tea tree parentage identification.

[0047] Table 2: SNP locus information

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[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liquid phase chip for 40K-assisted breeding of tea trees, characterized in that, The chip contains 44,915 SNP sites; the SNP site information is located based on the tea tree reference genome "Zhongcha 102", and the specific site information is shown in Table 2.

2. The tea tree 40K-assisted breeding liquid phase chip according to claim 1, characterized in that, The SNP sites were obtained through the following methods: whole-genome site screening, site probe design and screening, and site optimization screening.

3. The tea tree 40K-assisted breeding liquid phase chip according to claim 2, characterized in that, The whole-genome site screening criteria include: setting quality control standards of minimum allele frequency greater than or equal to 0.3, site deletion frequency less than 0.1, and sequencing depth greater than or equal to 5×, and removing insertion and deletion sites, retaining only biallelic SNP sites.

4. The tea tree 40K-assisted breeding liquid phase chip according to claim 2, characterized in that, The site probe design and screening criteria include: designing probe sequences with a length of 110 bp, and setting screening criteria of ≤5 homologous regions and 30-70% GC content.

5. The tea tree 40K-assisted breeding liquid phase chip according to claim 2, characterized in that, The site optimization screening criteria include: for regions with gaps, 190 additional sites with MAF ≥ 0.1 are selected.

6. The application of the 40K-assisted breeding liquid phase chip for tea trees as described in any one of claims 1 to 5 in the analysis of genetic diversity, population structure, and genetic and evolutionary analysis of tea tree germplasm resources.

7. The application of the 40K-assisted breeding liquid phase chip for tea trees according to any one of claims 1 to 5 in tea tree kinship identification, genome-wide association analysis or genome selection.

8. The application of the 40K-assisted breeding liquid phase chip for tea trees as described in any one of claims 1 to 5 in the location of associated genes, variety identification, and core germplasm screening of traits in tea trees and other types of tea trees.

9. The application of the tea tree 40K assisted breeding liquid phase chip according to any one of claims 1 to 5 in the construction of tea tree fingerprint maps or assisted breeding.

10. The application of the 40K-assisted breeding liquid phase chip for tea trees as described in any one of claims 1 to 5 in the genetic evaluation of tea tree germplasm resources, variety identification, phylogenetic analysis, linkage map construction, and quantitative trait gene localization.

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