Camellia oleifera SNP molecular marker combination and application thereof

Abstract

The invention discloses a camellia oleifera SNP (Single Nucleotide Polymorphism) molecular marker combination and application thereof. The camellia oleifera SNP molecular marker combination comprises at least one of 50 SNP molecular markers and 454 SNP molecular markers, and the physical positions of the 50 SNP molecular markers and the 454 SNP molecular markers are determined on the basis of sequence alignment of a camellia oleifera reference genome GCA039905865.1. The camellia oleifera SNP molecular marker combination can be effectively used for molecular marker breeding of camellia oleifera, the camellia oleifera SNP molecular marker combination is used for preparing a first type of whole genome liquid phase chip of camellia oleifera, and the application cost of genetic diversity analysis, QTL positioning, GWAS analysis and the like in scientific research of camellia oleifera is effectively reduced; the problem that no applicable product exists in camellia oleifera molecular auxiliary breeding and whole genome selective breeding is solved, and the process of camellia oleifera basic research and breeding is accelerated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a camellia oleifera SNP molecular marker combination and application thereof. Background Art

[0002] Camellia oleifera is one of the important oil-bearing tree species. It is known as one of the four major woody edible oil tree species in the world along with oil palm, olive and coconut. It is an indispensable pillar of the woody edible oil industry. For a long time, the breeding technology of camellia oleifera has been relatively backward due to its long growth cycle, and the genetic improvement has progressed slowly. In addition, there is a lack of mature genotyping products available for camellia oleifera. Therefore, using efficient modern molecular breeding technology to develop an economical and suitable genotyping product can effectively solve the existing difficulties in camellia oleifera breeding, effectively improve the efficiency of camellia oleifera breeding, accelerate the process of camellia oleifera breeding, cultivate high-yield, high-quality and stress-resistant varieties, and ensure the healthy and sustainable development of the camellia oleifera industry.

[0003] Single nucleotide polymorphism (SNP) refers to a DNA sequence polymorphism caused by a single base mutation at the genome level. It has the characteristics of wide distribution, high stability and easy to realize automated analysis, making it an ideal tool for studying genetic variation, molecular breeding and germplasm resource evaluation. At present, the gene chips used for SNP site typing mainly include solid phase chips and liquid phase chips. The traditional solid phase chip is based on the complementary hybridization of the probe and the DNA sequence, and typing is performed through the fluorescent color development signal of the marker. The liquid phase chip is based on the principle of targeted sequencing technology. For each site to be tested, a biotin-labeled probe covering the target SNP is designed. These probes hybridize with the target region of the genome in the liquid to form a double strand, and then use the adsorption effect of streptavidin-coated magnetic beads and biotin-containing molecules. After elution, amplification, and library construction, the second-generation sequencing is performed to finally obtain the genotyping results of the target site and its surrounding SNPs. It has the advantages of high detection accuracy and high throughput. It is currently widely used in genetic diversity analysis, whole genome association analysis, whole genome selection breeding, gene / QTL positioning, germplasm resource development evaluation and DNA fingerprinting identification. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a SNP molecular marker combination for Camellia oleifera.

[0005] The present invention also provides a liquid phase chip for detecting the above-mentioned SNP molecular marker combination of oil-tea camellia.

[0006] The present invention also provides a kit for detecting the above-mentioned SNP molecular marker combination of Camellia oleifera.

[0007] The present invention also provides a method for screening the above-mentioned oil-tea camellia SNP molecular marker combination.

[0008] The present invention also provides an application of the above-mentioned camellia oleifera SNP molecular marker combination, liquid phase chip or kit.

[0009] The invention also provides a breeding method for oil-tea camellia.

[0010] According to one aspect of the present invention, a Camellia oleifera SNP molecular marker combination is proposed, including at least one of 50,454 SNP molecular markers, the physical positions of the 50,454 SNP molecular markers are determined based on sequence alignment of the Camellia oleifera reference genome GCA_039905865.1, and the specific site information is shown in Table 1 below.

[0011] Table 1

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] In a second aspect of the present invention, a liquid phase chip is provided, wherein the liquid phase chip comprises a primer set and / or a probe for detecting the above-mentioned Camellia oleifera SNP molecular marker combination.

[0037] In a third aspect of the present invention, a kit is provided, which comprises a primer set and / or a probe for detecting the above-mentioned Camellia oleifera SNP molecular marker combination.

[0038] In a fourth aspect of the present invention, a screening method for the above-mentioned oil-tea camellia SNP molecular marker combination is provided, and the screening method comprises the following steps:

[0039] (1) Use Sentieon software to align and detect variations in the whole genome sequencing data of Camellia oleifera samples, perform preliminary hard filtering, and obtain a file containing SNP variation information of all samples;

[0040] (2) mining and screening the file containing the SNP variation information of all samples to obtain the target site SNP; the mining and screening parameters are: Maf ≥ 0.05, detection rate ≥ 90%, heterozygosity ≤ 45%, Depth ≥ 10×;

[0041] (3) collecting and collating phenotypic trait data of oil-tea camellia samples; performing genome-wide association analysis on the phenotypic trait data to obtain trait-associated SNP sites; the phenotypic trait data include oil content, fatty acid composition, disease resistance, and fruit characteristics data;

[0042] (4) Based on the file containing the SNP variation information of all samples, the inter-population genetic differentiation index is calculated for each SNP variation site of different oil-tea camellia varieties; the parameter is --weir-fst-pop, and the threshold value of FST>0.8 is used for filtering to obtain oil-tea camellia variety-specific SNP sites;

[0043] (5) extracting SNP sites of genes related to lipid synthesis based on the file containing SNP variation information of all samples;

[0044] (6) Based on the file containing the SNP variation information of all samples, SNP sites of genes related to fruit skin thickness, fruit size, fertility and anthracnose resistance are extracted;

[0045] (7) Design probes for the target site SNP obtained in step (2), the trait-associated SNP site obtained in step (3), the oil-tea camellia variety-specific SNP site obtained in step (4), the SNP site of the gene related to oil synthesis obtained in step (5), and the SNP site of the gene related to peel thickness, fruit size, fertility and anthracnose resistance obtained in step (6), screen the SNPs whose probes meet the requirements, and obtain the oil-tea camellia SNP molecular marker combination.

[0046] In some embodiments of the present invention, the variety of the tea oil sample includes at least one of ancient tea oil trees, Hunan tea oil, Hengdong tea oil, Jiangxi tea oil, Zhejiang tea oil, Hubei tea oil, Fujian tea oil, Guangxi tea oil, Youxian tea oil, narrow-leaved tea oil, small-fruit tea oil, and Bobai large-fruit tea oil.

[0047] In some embodiments of the present invention, the oil tea includes diploid oil tea, tetraploid oil tea, hexaploid oil tea and octoploid oil tea.

[0048] In some embodiments of the present invention, using Sentieon software to compare and detect variations in whole genome sequencing data of Camellia oleifera samples specifically comprises the following steps:

[0049] (1) Using Sentieon software, the whole genome sequencing data of the Camellia oleifera sample was aligned to the Camellia oleifera reference genome GCA_039905865.1, and position sorting and marking of duplicate reads were performed;

[0050] (2) Use Sentieon software to detect variant sites in each sample and obtain the gVCF of each sample;

[0051] (3) Use Sentieon for joint-calling and jointly analyze the gVCF of all samples to obtain the variation results of each individual in the population.

[0052] In some embodiments of the present invention, the standard of hard filtering is as follows: QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0.

[0053] In some embodiments of the present invention, the whole genome association analysis includes the following steps: performing whole genome association analysis using the R package rMVP; the R package rMVP contains a general linear model (GLM) and a mixed linear model (MLM); adding the kinship matrix and principal components as covariates to the model for correction, and the significant threshold at the whole genome level is 0.04-0.06.

[0054] In some embodiments of the present invention, the oil-tea camellia varieties include narrow-leaved camellia and small-fruited camellia.

[0055] In some embodiments of the present invention, the length of the probe is 80-120 bp.

[0056] In some embodiments of the invention, the probe is about 100 bp in length.

[0057] In some embodiments of the present invention, the GC content of the probe is between 20% and 80%.

[0058] In some embodiments of the present invention, screening probes for SNPs that meet the requirements includes the following steps: designing probes to remove probes that cannot be uniquely aligned on the genome and that contain repetitive sequences in their flanking sequences.

[0059] In some embodiments of the present invention, the filtering criteria are as follows: QD<2.0||FS>60.0||MQ<35.0||MQRankSum<-12.5||Read PosRankSum<-8.0||DP>6950.

[0060] In some embodiments of the present invention, the length of the probe is 80-120 bp.

[0061] In some embodiments of the invention, the probe is about 100 bp in length.

[0062] In some embodiments of the present invention, the GC content of the probe is between 20% and 80%.

[0063] In some embodiments of the present invention, screening probes for SNPs that meet the requirements includes the following steps: designing probes to remove probes that cannot be uniquely aligned on the genome and that contain repetitive sequences in their flanking sequences.

[0064] In the fifth aspect of the present invention, an application of at least one of the above-mentioned Camellia oleifera SNP molecular marker combination, liquid phase chip and kit is proposed, and the application is application in whole genome selection breeding of Camellia oleifera.

[0065] In some embodiments of the present invention, the application is application in genome-wide association analysis of Camellia oleifera.

[0066] In some embodiments of the present invention, the application is application in the identification of germplasm resources and varieties of Camellia oleifera.

[0067] In some embodiments of the present invention, the application is application in cluster analysis and kinship identification of Camellia oleifera.

[0068] In some embodiments of the present invention, the application is an application in mining and identifying important traits of Camellia oleifera.

[0069] In some embodiments of the present invention, the application is application in genotyping detection of Camellia oleifera.

[0070] In some embodiments of the present invention, the application is application in molecular design breeding of Camellia oleifera.

[0071] In some embodiments of the present invention, the application can be implemented by the following method:

[0072] S1. Perform genotyping on the sample to be tested using at least one of a Camellia oleifera SNP molecular marker combination, a liquid phase chip, and a kit to obtain a genotyping result;

[0073] S2. Analyze the genotyping results obtained in step S1.

[0074] In some embodiments of the present invention, the varieties of tea oil include at least one of ancient tea oil trees, Hunan tea oil, Hengdong tea oil, Jiangxi tea oil, Zhejiang tea oil, Hubei tea oil, Fujian tea oil, Guangxi tea oil, Youxian tea oil, narrow-leaved tea oil, small-fruit tea oil, and Bobai large-fruit tea oil.

[0075] In some embodiments of the present invention, the oil tea includes diploid oil tea, tetraploid oil tea, hexaploid oil tea and octoploid oil tea.

[0076] In the sixth aspect of the present invention, a breeding method for oil-tea camellia is proposed, comprising the following steps: using at least one of the above-mentioned oil-tea camellia SNP molecular marker combination, liquid phase chip and kit to detect the DNA of the oil-tea camellia to be tested, and selecting the oil-tea camellia for subsequent breeding.

[0077] In some embodiments of the present invention, the varieties of tea oil include at least one of ancient tea oil trees, Hunan tea oil, Hengdong tea oil, Jiangxi tea oil, Zhejiang tea oil, Hubei tea oil, Fujian tea oil, Guangxi tea oil, Youxian tea oil, narrow-leaved tea oil, small-fruit tea oil, and Bobai large-fruit tea oil.

[0078] In some embodiments of the present invention, the oil tea includes diploid oil tea, tetraploid oil tea, hexaploid oil tea and octoploid oil tea.

[0079] In some embodiments of the present invention, the detection is performed based on liquid phase probe capture sequencing typing technology.

[0080] The present invention has at least the following beneficial effects:

[0081] The SNP molecular marker combination of the oil-tea camellia solution of the present invention can be effectively used in the molecular marker breeding of oil-tea camellia. The SNP molecular marker combination of the oil-tea camellia solution of the present invention is used to prepare the first whole genome liquid phase chip of oil-tea camellia, which effectively reduces the cost of genetic diversity analysis, QTL positioning, GWAS analysis and other applications of oil-tea camellia in scientific research, solves the problem of no applicable products for molecular assisted breeding and whole genome selection breeding of oil-tea camellia, and accelerates the progress of basic research and breeding of oil-tea camellia.

[0082] At the same time, the oil tea liquid phase chip prepared by the scheme of the present invention has the characteristics of strong universality. The ploidy of oil tea is complex. In addition to diploid oil tea, there are also tetraploid, hexaploid and octoploid oil tea. The average detection rate of the present invention in diploid oil tea detection can reach 98.98%, and the average detection rate in tetraploid, hexaploid and octoploid oil tea can reach 85.78%, which can meet the detection needs of oil teas with different ploidy. And the oil tea liquid phase chip prepared by the scheme of the present invention is rich in functional genes, and 7,554 loci related to the yield, quality, disease resistance, fertility and variety specificity of oil tea are added, which is conducive to molecular marker-assisted selection, directional improvement, multi-gene aggregation breeding, and important trait gene mining and identification and functional analysis, etc., and has great utilization value in the process of oil tea variety improvement and variety selection.

[0083] Compared with traditional solid-phase chip technology, liquid chip technology has shown remarkable flexibility and efficiency. Liquid chips do not need to wait for samples to be collected, and a small amount of samples can be tested, which greatly shortens the detection cycle and accelerates the efficiency of scientific research and breeding processes. At the same time, liquid chips are flexible in site design. Through continuous optimization and adjustment of existing products, sites can be added or subtracted based on existing chips, thereby changing the chip density to meet the specific needs of different research or application scenarios. In addition, in addition to obtaining target SNP information, liquid chips can also obtain all genetic variation sites within the target interval (200-300bp / target area), providing richer site information support for oil tea molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0085] Figure 1 This is the chromosome density distribution map of the 50K cGPS liquid phase chip site in Example 1 of the present invention;

[0086] Figure 2 is the MAF distribution histogram in Example 1 of the present invention;

[0087] Figure 3 This is a schematic diagram of the cGPS liquid chip process detection in Example 2 of the present invention;

[0088] Figure 4 This is a graph showing the detection rate of the oil-tea camellia sample sites in Example 3 of the present invention;

[0089] Figure 5 This is a graph showing the average consistency rate of genotype detection results of technical repeated samples in Example 3 of the present invention;

[0090] Figure 6 This is a cluster analysis diagram of the Camellia oleifera 50K cGPS liquid phase chip in Example 4 of the present invention. DETAILED DESCRIPTION

[0091] The following will be clearly and completely described in conjunction with the embodiments of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0092] Example 1

[0093] In this embodiment, a camellia oleifera SNP molecular marker combination and a camellia oleifera 50K liquid phase chip (50K cGPS liquid phase chip) designed according to the camellia oleifera SNP molecular marker combination were prepared.

[0094] 1. The screening process of the oil-tea camellia SNP molecular marker combination is as follows:

[0095] (1) Collection of Camellia oleifera germplasm resources

[0096] In order to obtain rich and diverse whole-genome SNP sites, 213 oil-tea camellia germplasm resources of different varieties (including wild varieties and main cultivated varieties) from different regions such as Hunan, Hubei, Jiangxi, and Zhejiang were collected. The specific information is shown in Table 2. The obtained oil-tea camellia germplasm resources were subjected to whole-genome resequencing.

[0097] Table 2

[0098]

[0099] (2) Whole-genome resequencing of Camellia oleifera

[0100] The 213 oil-tea camellia samples collected were subjected to DNA extraction and whole genome resequencing. The specific steps included: (1) DNA was extracted using the magnetic bead method. (2) The MGI library standard method was used to construct the DNA-seq sequencing library. (3) After the library passed the quality inspection, it was sequenced using the BGI sequencing platform (MGI), with a sequencing strategy of PE150 and a sequencing depth of 10×.

[0101] Sentieon was used to align and detect variants in 213 resequencing data. The analysis process is as follows:

[0102] 1) Use Sentieon to align reads to the Camellia oleifera reference genome (GCA_039905865.1), position sorting and marking duplicate reads; 2) Use Sentieon to detect variant sites for each sample and obtain the gVCF of each sample. 3) Use Sentieon for joint-calling, jointly analyze the gVCF of all samples, and obtain the variation results of each individual in the population. To ensure the accuracy of SNP, the SNP sites obtained after the joint analysis are initially hard filtered (SNP hard filtering standard: "QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0") to obtain a vcf file containing the SNP variation information of all samples.

[0103] (3) Site screening

[0104] 1) Genome-wide loci

[0105] The MAF value, detection rate, heterozygosity rate and sequencing depth of the site were calculated and counted from the vcf file containing the SNP variation information of all samples. According to MAF ≥ 0.05, SNP site detection rate ≥ 90%, heterozygosity rate ≤ 45%, and sequencing depth ≥ 10×, 2,146,275 target sites were screened. Probe design was performed on the screened target site set. Specifically, probes were designed within 100 bp of all target sites. The probe length was about 100 bp and the GC content was between 20% and 80%. According to the probe design results, probes that could not be uniquely aligned on the genome and contained repetitive sequences in the flanking sequences were removed. Based on the principle of uniform distribution, sites with high MAF values ​​were retained, and 42,311 SNP sites with high polymorphism were finally obtained.

[0106] 2) Candidate loci obtained by GWAS (Genome-wide association study) analysis

[0107] The phenotypic data of the resequencing samples were collected and sorted, including four traits: oil content, fatty acid composition, disease resistance and fruit characteristics. Based on the vcf file containing the SNP variation information of all samples, the R package rMVP was used for genome-wide association analysis, which included two models: general linear model (GLM) and mixed linear model (MLM). The kinship matrix and principal components (PCA1, PCA2, PCA3) were added to the model as covariates for correction to reduce the influence of kinship and population structure. The significant threshold at the genome-wide level was 0.05. GWAS analysis (genome-wide association analysis) was performed on the phenotypes of the four traits of Camellia oleifera, and significant trait association sites were obtained. Probes were designed for them, and the successfully designed sites were retained. Finally, 5 significant association sites for oil content, 17 significant association sites for fatty acid composition, 13 significant association sites for disease resistance, and 32 significant association sites for fruit characteristics were obtained, totaling 67.

[0108] 3) Variety-specific loci

[0109] The interpopulation genetic differentiation index (FST) is a measure of population differentiation and genetic distance. The larger the differentiation index, the greater the difference. Fst is between 0 and 1. The closer to 1, the greater the degree of differentiation between the two populations, and the higher the degree of selection. The closer to 0, the smaller the degree of differentiation between the two populations, and the lower the degree of selection. Based on the above vcf file containing the SNP variation information of all samples, different types of Camellia oleifera were classified, and each SNP variation site of Camellia oleifera and Camellia oleifera was selected for calculation (single point calculation). The parameter was --weir-fst-pop. Finally, according to the threshold of FST>0.8, the filtering and probe design were carried out, and the sites with successful probe design were retained. A total of 251 Camellia oleifera variety-specific sites and 116 Camellia oleifera variety-specific sites were obtained, totaling 367.

[0110] 4) Genes related to lipid synthesis

[0111] Genes related to oil synthesis that have been published in the literature were collected. After searching, comparing and deduplicating, 452 genes related to oil synthesis were retained. Based on the above vcf file containing the SNP variation information of all samples, SNPs were screened as candidate sites on these genes. Sites with high polymorphism and successful probe design were retained. 1-5 sites were retained for each gene, and a total of 632 SNP sites related to oil synthesis on 323 genes were obtained.

[0112] 5) Transcriptome differentially expressed genes

[0113] Based on the above vcf file containing SNP variation information of all samples, according to the transcriptome differentially expressed genes obtained in previous studies, including four groups of traits, namely, fruit peel thickness, fruit size, fertility and anthracnose resistance, the top 300 genes in each group were selected for retention, and SNPs were screened on these genes as candidate sites. Sites with high polymorphism and successful probe design were retained, and 1-4 SNP sites were retained for each gene. Finally, 2,357 gene sites for fruit peel thickness, 2,824 gene sites for fruit size, 2,893 gene sites for fertility, and 2,340 gene sites for anthracnose resistance were retained. All sites were integrated and deduplicated, totaling 7,138 sites.

[0114] All the sites in 1)-5) above were integrated and deduplicated to form the Camellia oleifera 50K liquid phase chip. The number of sites was finally 50,454 (as shown in Table 1 of the manual), with an average spacing of 55 Kb. The distribution map of 50K sites on chromosomes is shown in Figure 1 , MAF distribution histogram see Figure 2 .

[0115] 2. Camellia 50K liquid phase chip (50K cGPS liquid phase chip)

[0116] The liquid phase capture probes were synthesized by Huazhi Biotechnology Co., Ltd., and the precise positioning sequencing typing technology (cGPS) based on liquid phase capture of the target region genome sequence was used to form a Camellia oleifera liquid phase chip system.

[0117] Based on an optimized thermodynamic stability algorithm model, cGPS designs probes for genomic sequences in different target regions, uses synthetic specific probes to perform liquid phase hybridization capture and enrichment of multiple different target sequences located at different genomic locations, and then constructs sequencing libraries and performs high-throughput sequencing on the captured and enriched target genomic sequences to obtain the genotypes of all SNP / InDel sites in the target region.

[0118] Example 2 Method for using the Camellia oleifera 50K liquid phase chip

[0119] This embodiment provides a method for using the Camellia oleifera liquid phase chip prepared in the above embodiment 1, which specifically includes the following contents:

[0120] 1. Extraction and detection of Camellia oleifera genomic DNA

[0121] Camellia oleifera samples with different ploidy were selected as samples for the validation of the 50K liquid phase chip development system, and genomic DNA was extracted from fresh leaves using the magnetic bead method. The integrity and purity of the genomic DNA were analyzed by 1% agarose gel electrophoresis, and the concentration was accurately quantified using Qubit.

[0122] 2. cGPS experiments and analysis were conducted by Huazhi Biotechnology Co., Ltd. The process is briefly described as follows:

[0123] (1) Take 200 ng of genomic DNA that has passed the quantitative quality inspection, cut the DNA into 100-500 bp fragments using an enzyme cutting reagent, and then add Taq enzyme for end repair;

[0124] (2) Use T4 ligase to connect the adapter fragments to both ends of the DNA, use fragment sorting magnetic beads to purify the ligation products and amplify the library to complete the library construction;

[0125] (3) Place the qualified library with blocking reagent, RNase inhibitor, and 50K liquid phase chip probe on a PCR instrument for hybridization reaction, run at 80°C for 5 minutes, and incubate at 55°C for 16-24 hours;

[0126] (4) The hybridization product was captured using streptavidin, the captured library was amplified and enriched, and PE150 sequencing was performed using the BGI sequencing DNBSEQ-T7 platform;

[0127] (5) The raw data after high-throughput sequencing is processed by quality control filtering, and fastp software is used to remove reads containing adapter contamination and low-quality reads. BWA software is used to compare with the target genome, and then GATK software is used to analyze the variant sites of the sequencing results to obtain the genotyping results of the target sites. The process diagram is shown in the figure. Figure 3 shown.

[0128] Example 3 Genotyping Quality Evaluation of Camellia oleifera 50K Liquid Phase Chip

[0129] To verify the genotyping effect of the Camellia oleifera 50K liquid phase chip, 14 Camellia oleifera samples (including 3 duplicate samples, including 6 diploids, 3 tetraploids, 4 hexaploids, and 1 octoploid) were subjected to genotyping detection using the 50K liquid phase chip prepared in Example 1 (see Example 2 for the specific operation method).

[0130] After sequencing and data analysis, the results are as follows Figure 4 As shown in the figure, the detection rate of the 14 samples ranged from 84.21% to 99.03%, with an average detection rate of 91.42%. There were differences in the detection rates of different ploidy oil teas, with the diploid detection rate being the highest, with an average detection rate of 98.94%. By comparing the genotype results of the three repeated samples, the genotype consistency rate results are shown in Figure 5 As shown in the figure, it can be seen that the genotype consistency rate is between 99.22% and 99.66%, and the average consistency rate is 99.47%.

[0131] The evaluation results of the genotyping effect of the 50K liquid phase chip show that the Camellia oleifera 50K liquid phase chip prepared in Example 1 of the present invention can meet the requirements of the detection of Camellia oleifera samples with different ploidy, has good stability, and the typing results are accurate and reliable.

[0132] Example 4 Analysis of the structure of the Camellia oleifera 50K liquid phase chip population

[0133] The genotype of 35 tea oil samples from different sources was identified using the tea oil 50K liquid phase chip prepared in Example 1 (the specific operation method is shown in Example 2, and the sources of the 35 tea oil samples are known). The genotyping results of the 35 samples were extracted, and the genetic distance matrix was calculated using the IBS method of Plink software and cluster analysis was performed to construct a phylogenetic tree, which can determine the kinship, evolutionary relationship and composition structure between different materials.

[0134] The results are as follows Figure 6 As shown in the figure, it can be seen that the Camellia oleifera 50K liquid phase chip can effectively distinguish Camellia oleifera species from different sources, and the classification effect is consistent with the actual classification.

[0135] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A SNP molecular marker combination for Camellia oleifera, characterized in that: It includes at least one of 50,454 SNP molecular markers, the physical positions of the 50,454 SNP molecular markers are determined based on sequence alignment of the Camellia oleifera reference genome GCA_039905865.1, and the site information is specifically shown in Table 1 in the specification.

2. A liquid phase chip, characterized in that: The liquid phase chip comprises a primer set and / or a probe for detecting the oil-tea camellia SNP molecular marker combination as claimed in claim 1.

3. A kit, characterized in that: The kit comprises a primer set and / or a probe for detecting the oil-tea camellia SNP molecular marker combination as claimed in claim 1.

4. A SNP molecular marker combination for Camellia oleifera according to claim 1, characterized in that: The screening method comprises the following steps: (1) Use Sentieon software to align and detect variations in the whole genome sequencing data of Camellia oleifera samples, perform preliminary hard filtering, and obtain a file containing SNP variation information of all samples; (2) mining and screening the file containing the SNP variation information of all samples to obtain the target site SNP; the mining and screening parameters are: Maf ≥ 0.05, detection rate ≥ 90%, heterozygosity ≤ 45%, Depth ≥ 10×; (3) collecting and collating phenotypic trait data of oil-tea camellia samples; performing genome-wide association analysis on the phenotypic trait data to obtain trait-associated SNP sites; the phenotypic trait data include oil content, fatty acid composition, disease resistance, and fruit characteristics data; (4) Based on the file containing the SNP variation information of all samples, the inter-population genetic differentiation index is calculated for each SNP variation site of different oil-tea camellia varieties; the parameter is --weir-fst-pop, and the threshold value of FST>0.8 is used for filtering to obtain oil-tea camellia variety-specific SNP sites; (5) extracting SNP sites of genes related to lipid synthesis based on the file containing SNP variation information of all samples; (6) Based on the file containing the SNP variation information of all samples, SNP sites of genes related to fruit skin thickness, fruit size, fertility and anthracnose resistance are extracted; (7) Design probes for the target site SNP obtained in step (2), the trait-associated SNP site obtained in step (3), the oil-tea camellia variety-specific SNP site obtained in step (4), the SNP site of the gene related to oil synthesis obtained in step (5), and the SNP site of the gene related to peel thickness, fruit size, fertility and anthracnose resistance obtained in step (6), screen the SNPs whose probes meet the requirements, and obtain the oil-tea camellia SNP molecular marker combination.

5. The screening method according to claim 4, characterized in that The varieties of the tea oil samples include at least one of ancient tea oil trees, Hunan tea oil, Hengdong tea oil, Jiangxi tea oil, Zhejiang tea oil, Hubei tea oil, Fujian tea oil, Guangxi tea oil, Youxian tea oil, narrow-leaved tea oil, small-fruit tea oil, and Bobai large-fruit tea oil.

6. The screening method according to claim 4, characterized in that The criteria for hard filtering are as follows: QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.

0.

7. The screening method according to claim 4, characterized in that The length of the probe is 80-120 bp.

8. The screening method according to claim 4, characterized in that The GC content of the probe is between 20% and 80%.

9. Use of one of the oil-tea camellia SNP molecular marker combination according to claim 1, the liquid phase chip according to claim 2, and the kit according to claim 3 in any of the following: (1) Whole genome selection breeding of Camellia oleifera; (2) Genome-wide association analysis of Camellia oleifera; (3) Germplasm resources and variety identification of Camellia oleifera; (4) Cluster analysis and identification of genetic relationships of Camellia oleifera; (5) Discovery and identification of important traits of Camellia oleifera; (6) Genotyping of Camellia oleifera; (7) Molecular design breeding of Camellia oleifera.

10. A method for breeding oil-tea camellia, characterized in that: The method comprises the following steps: using the oil-tea camellia SNP molecular marker combination as claimed in claim 1, the liquid phase chip as claimed in claim 2 and one of the kits as claimed in claim 3 to detect the DNA of the oil-tea camellia to be tested, and selecting the oil-tea camellia for subsequent breeding.

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