Camellia oleifera snp molecular marker combination and application thereof

Abstract

The application discloses a kind of oil tea SNP molecular marker combinations and application thereof.The oil tea SNP molecular marker combination includes at least one of 50,454 SNP molecular markers, and the physical position of the 50,454 SNP molecular markers is determined based on oil tea reference genome GCA_039905865.1 sequence alignment.The oil tea SNP molecular marker combination of the present application can be effectively used for molecular marker breeding of oil tea, and the oil tea SNP molecular marker combination of the present application is prepared to obtain the first oil tea whole genome liquid chip, which effectively reduces the cost of genetic diversity analysis, QTL positioning, GWAS analysis and other applications in scientific research of oil tea, solves the problem of no suitable product for molecular assisted breeding and whole genome selection breeding of oil tea, and speeds up the process of oil tea basic research and breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a combination of SNP molecular markers from Camellia oleifera and its applications. Background Technology

[0002] Camellia oleifera is one of the most important oilseed tree species, ranking alongside oil palm, olive, and coconut as one of the world's four major woody edible oilseed tree species. It is an indispensable pillar of the woody edible oil industry. For a long time, the long growth cycle of Camellia oleifera has led to relatively backward breeding techniques, slow progress in genetic improvement, and a lack of maturely developed genotyping products. Therefore, utilizing efficient modern molecular breeding techniques to develop an economical and suitable genotyping product can effectively solve the existing difficulties in Camellia oleifera breeding, significantly improve breeding efficiency, accelerate the selection of superior varieties, cultivate high-yielding, high-quality, and stress-resistant varieties, and ensure the healthy and sustainable development of the Camellia oleifera industry.

[0003] Single nucleotide polymorphisms (SNPs) are DNA sequence polymorphisms caused by single-base mutations at the genomic level. Their wide distribution, high stability, and ease of automated analysis make them ideal tools for studying genetic variation, molecular breeding, and germplasm resource assessment. Currently, gene chips used for SNP genotyping mainly include solid-phase chips and liquid-phase chips. Traditional solid-phase chips are based on complementary hybridization between probes and DNA sequences, using fluorescent colorimetric signals from labeled markers for genotyping. Liquid-phase chips, on the other hand, are based on targeted sequencing technology. A biotin-labeled probe covering the target SNP is designed for each genotype. These probes hybridize with the target genomic region in a liquid state to form a double strand. Then, streptavidin-coated magnetic beads adsorb the biotin-containing molecules. After elution, amplification, library construction, and next-generation sequencing, the genotyping results of the target locus and surrounding SNPs are obtained, offering advantages such as high detection accuracy and high throughput. It is currently widely used in genetic diversity analysis, genome-wide association analysis, genome-wide selection breeding, gene / QTL mapping, germplasm resource development evaluation, and DNA fingerprinting. Summary of the Invention

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

[0005] The present invention also proposes a liquid-phase chip for detecting the above-mentioned Camellia oleifera SNP molecular marker combinations.

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

[0007] This invention also proposes a screening method for the above-mentioned Camellia oleifera SNP molecular marker combinations.

[0008] This invention also proposes an application of the above-mentioned Camellia oleifera SNP molecular marker combination, liquid phase chip, or reagent kit.

[0009] This invention also proposes a breeding method for Camellia oleifera.

[0010] According to one aspect of the present invention, a combination of SNP molecular markers for Camellia oleifera is proposed, comprising at least one of 50,454 SNP molecular markers, wherein the physical locations of the 50,454 SNP molecular markers are determined by sequence alignment based on 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 invention, a liquid-phase chip is provided, the liquid-phase chip comprising a primer set and / or probes for detecting the above-described combinations of Camellia oleifera SNP molecular markers.

[0037] In a third aspect of the invention, a kit is provided comprising a primer set and / or probes for detecting the above-described Camellia oleifera SNP molecular marker combinations.

[0038] In a fourth aspect of the present invention, a method for screening the above-mentioned Camellia oleifera SNP molecular marker combinations is proposed, the screening method comprising the following steps:

[0039] (1) Sentieon software was used to compare and detect variants in the whole genome sequencing data of the camellia oleifera samples, and preliminary hard filtering was performed to obtain a file containing SNP variant information of all samples.

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

[0041] (3) Collect and organize the phenotypic data of the camellia oleifera samples; perform genome-wide association analysis on the phenotypic data to obtain the SNP loci associated with the traits; the phenotypic data includes oil content, fatty acid composition, disease resistance and fruit characteristics data;

[0042] (4) Based on the file containing SNP variation information of all samples, calculate the interpopulation genetic differentiation index for each SNP variation site of different Camellia oleifera varieties; the parameter is --weir-fst-pop, and filter according to the threshold of FST>0.8 to obtain Camellia oleifera variety-specific SNP sites.

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

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

[0045] (7) Probe design is performed on the target SNPs obtained in step (2), the trait-related SNPs obtained in step (3), the variety-specific SNPs of Camellia oleifera obtained in step (4), the SNPs of genes related to oil synthesis obtained in step (5), and the SNPs of genes related to pericarp thickness, fruit size, fertility and anthracnose resistance obtained in step (6). SNPs that meet the requirements of the probe are selected to obtain the combination of Camellia oleifera SNP molecular markers.

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

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

[0048] In some embodiments of the present invention, the comparison and variation detection of whole genome sequencing data of Camellia oleifera samples using Sentieon software specifically includes the following steps:

[0049] (1) The whole genome sequencing data of the Camellia oleifera sample was aligned to the Camellia oleifera reference genome GCA_039905865.1 using Sentieon software, and the positions were sorted and duplicate reads were marked.

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

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

[0052] In some embodiments of the present invention, the hard filtering criteria are 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 genome-wide association analysis includes the following steps: performing genome-wide association analysis using the R package rMVP; the R package rMVP includes a general linear model (GLM) and a mixed linear model (MLM); the kinship matrix and principal components are added as covariates to the model for calibration, and the significance threshold at the genome-wide level is 0.04-0.06.

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

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

[0056] In some embodiments of the present invention, the probe is approximately 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 for SNPs that meet the requirements includes the following steps: designing probes to remove probes that cannot be uniquely aligned on the genome or whose flanking sequences contain repetitive 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||ReadPosRankSum<-8.0||DP>6950.

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

[0061] In some embodiments of the present invention, the probe is approximately 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 for SNPs that meet the requirements includes the following steps: designing probes to remove probes that cannot be uniquely aligned on the genome or whose flanking sequences contain repetitive sequences.

[0064] In a fifth aspect of the invention, an application of at least one of the above-mentioned Camellia oleifera SNP molecular marker combinations, liquid phase chips, and kits is proposed, wherein the application is in the whole-genome selection breeding of Camellia oleifera.

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

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

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

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

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

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

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

[0072] S1. Genotyping of the samples to be tested is performed using at least one of the following: Camellia oleifera SNP molecular marker combination, liquid phase chip, and kit, to obtain genotyping results;

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

[0074] In some embodiments of the present invention, the variety of Camellia oleifera includes at least one of the following: ancient Camellia oleifera trees, Hunan Camellia oleifera, Hengdong Camellia oleifera, Jiangxi Camellia oleifera, Zhejiang Camellia oleifera, Hubei Camellia oleifera, Fujian Camellia oleifera, Guangxi Camellia oleifera, Youxian Camellia oleifera, narrow-leaved Camellia oleifera, small-fruited Camellia oleifera, and Bobai large-fruited Camellia oleifera.

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

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

[0077] In some embodiments of the present invention, the variety of Camellia oleifera includes at least one of the following: ancient Camellia oleifera trees, Hunan Camellia oleifera, Hengdong Camellia oleifera, Jiangxi Camellia oleifera, Zhejiang Camellia oleifera, Hubei Camellia oleifera, Fujian Camellia oleifera, Guangxi Camellia oleifera, Youxian Camellia oleifera, narrow-leaved Camellia oleifera, small-fruited Camellia oleifera, and Bobai large-fruited Camellia oleifera.

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

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

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

[0081] The SNP molecular marker combination of Camellia oleifera according to the present invention can be effectively used for molecular marker breeding of Camellia oleifera. The Camellia oleifera SNP molecular marker combination of the present invention was used to prepare the first whole-genome liquid chip for Camellia oleifera, effectively reducing the cost of genetic diversity analysis, QTL mapping, GWAS analysis, and other applications of Camellia oleifera in scientific research. It also solves the problem of lacking suitable products for molecular-assisted breeding and whole-genome selection breeding of Camellia oleifera, accelerating the progress of basic research and breeding in Camellia oleifera.

[0082] Meanwhile, the Camellia oleifera liquid phase chip prepared by the present invention has the characteristics of high universality. Camellia oleifera has complex ploidy, including not only diploid Camellia oleifera, but also tetraploid, hexaploid, and octaploid Camellia oleifera. The present invention can achieve an average detection rate of 98.98% in diploid Camellia oleifera, and an average detection rate of 85.78% in tetraploid, hexaploid, and octaploid Camellia oleifera, which can meet the detection needs of Camellia oleifera with different ploidy. Moreover, the Camellia oleifera liquid phase chip prepared by the present invention is rich in functional genes, with 7,554 loci added that are related to the yield, quality, disease resistance, fertility, and variety specificity of Camellia oleifera. This is beneficial for molecular marker-assisted selection, targeted improvement, multi-gene aggregation breeding, and the mining, identification, and functional analysis of important trait genes, and has great utilization value in the process of Camellia oleifera variety improvement and breeding.

[0083] Compared to traditional solid-phase microarray technology, liquid-phase microarray technology exhibits significant flexibility and efficiency. Liquid-phase microarrays eliminate the need for sample collection, allowing for detection even with small sample sizes, thus greatly shortening the detection cycle and accelerating the efficiency of scientific research and breeding processes. Furthermore, liquid-phase microarrays offer flexibility in site design; through continuous optimization and adjustment of existing products, sites can be added or removed from existing chips to change the chip density and meet the specific needs of different research or application scenarios. In addition to acquiring target SNP information, liquid-phase microarrays can also acquire all genetic variation sites within the target region (200-300 bp / target region), providing richer site information support for molecular breeding of Camellia oleifera. Attached Figure Description

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

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

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

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

[0088] Figure 4 This is a graph showing the detection results of the locus detection rate of camellia oil samples in Example 3 of the present invention;

[0089] Figure 5 This is a graph showing the average genotypic consistency rate of the technical replicate samples in Example 3 of the present invention.

[0090] Figure 6 This is a cluster analysis diagram of the 50K cGPS liquid phase chip for Camellia oleifera in Embodiment 4 of the present invention. Detailed Implementation

[0091] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0092] Example 1

[0093] This embodiment prepared a Camellia oleifera SNP molecular marker combination and a Camellia oleifera 50K liquid phase chip (50K cGPS liquid phase chip) designed based on the Camellia oleifera SNP molecular marker combination.

[0094] 1. The screening process for SNP molecular marker combinations in Camellia oleifera is as follows:

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

[0096] To obtain a rich variety of whole-genome SNP loci, 213 Camellia oleifera germplasm resources from different regions and varieties (including wild and cultivated varieties) in Hunan, Hubei, Jiangxi, and Zhejiang were collected. Specific information is shown in Table 2. Whole-genome resequencing was then performed on the obtained Camellia oleifera germplasm resources.

[0097] Table 2

[0098]

[0099] (2) Camellia oleifera whole genome resequencing

[0100] DNA extraction and whole-genome resequencing were performed on 213 collected Camellia oleifera samples. The specific steps included: (1) DNA extraction using magnetic beads. (2) Using the MGI library standard method, the qualified samples were used for DNA-seq sequencing library construction. (3) After the library passed the quality inspection, sequencing was performed 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 workflow is as follows:

[0102] 1) Sentieon was used to align reads to the corresponding Camellia oleifera reference genome (GCA_039905865.1), orient them by position, and mark duplicate reads. 2) Sentieon was used to detect variant sites for each sample, obtaining the gVCF for each sample. 3) Sentieon was used for joint-calling to perform joint analysis of the gVCF of all samples, obtaining the variant results for each individual in the population. To ensure SNP accuracy, the SNP sites obtained after joint analysis were initially hard-filtered (SNP hard-filter criteria: "QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0"), resulting in a vcf file containing SNP variant information for all samples.

[0103] (3) Site screening

[0104] 1) Whole genome loci

[0105] From the VCF file containing SNP variant information for all samples, the MAF value, detection rate, heterozygosity, and sequencing depth of the loci were calculated and statistically analyzed. Based on MAF ≥ 0.05, SNP detection rate ≥ 90%, heterozygosity ≤ 45%, and sequencing depth ≥ 10×, 2,146,275 target loci were identified. Probe design was performed on the selected target loci set. Specifically, probes were designed within a 100 bp range to the left and right of each target locus, with probe lengths of approximately 100 bp and GC content between 20% and 80%. Based on the probe design results, probes that could not be uniquely aligned to the genome or contained repetitive sequences in their flanking sequences were removed. Based on the principle of uniform distribution, loci with high MAF values ​​were preferentially retained, ultimately yielding 42,311 highly polymorphic SNP loci.

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

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

[0108] 3) Variety-specific loci

[0109] The interpopulation genetic differentiation index (FST) is a measure of population differentiation and genetic distance; a higher FST indicates greater diversity. FST ranges from 0 to 1, with values ​​closer to 1 indicating greater differentiation and selection between populations, and values ​​closer to 0 indicating less differentiation and selection. Based on the VCF file containing SNP variation information for all samples, different types of Camellia oleifera were classified. Each SNP variation site in *Camellia oleifera* var. *narrow-leaved* and *Camellia oleifera* var. *small-fruited* was calculated (single-point calculation) using the parameter `--weir-fst-pop`. Finally, filtering and probe design were performed according to an FST > 0.8 threshold, retaining sites where probe design was successful. A total of 251 variety-specific loci were obtained for *Camellia oleifera* var. *narrow-leaved* and 116 variety-specific loci for *Camellia oleifera* var. *small-fruited*, for a total of 367 loci.

[0110] 4) Genes related to lipid synthesis

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

[0112] 5) Differentially expressed genes in the transcriptome

[0113] Based on the VCF file containing SNP variation information for all samples, and according to the differentially expressed genes in the transcriptome obtained in previous studies, including four groups of traits: peel thickness, fruit size, fertility, and anthracnose resistance, the top 300 genes in each group were selected and retained. SNPs on these genes were screened as candidate sites, with priority given to sites with high polymorphism and successful probe design. 1-4 SNP sites were retained for each gene. In the end, 2,357 differentially expressed gene sites for peel thickness, 2,824 for fruit size, 2,893 for fertility, and 2,340 for anthracnose resistance were retained. After integrating all sites and removing duplicates, a total of 7,138 sites were obtained.

[0114] After integrating and deduplicating all the sites mentioned in 1)-5), a 50K liquid phase chip for Camellia oleifera was constructed, resulting in a final number of 50,454 sites (as shown in Table 1 of the instruction manual), with an average spacing of 55Kb. The distribution of the 50K sites on the chromosome is shown in the diagram. Figure 1 The MAF distribution histogram is shown below. Figure 2 .

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

[0116] The system of Camellia oleifera liquid phase chip was formed by synthesizing liquid phase capture probes through Huazhi Biotechnology Co., Ltd. and using precise localization sequencing and typing technology (cGPS) based on liquid phase capture of target region genome sequences.

[0117] cGPS is based on an optimized thermodynamic stability algorithm model. It designs probes for genomic sequences in different target regions and uses synthesized specific probes to capture and enrich multiple different target sequences located at different genomic positions through liquid-phase hybridization. Then, it 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 of using the Camellia oleifera 50K liquid phase chip

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

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

[0121] Camellia oleifera samples with different ploidy levels were selected as validation samples for the 50K liquid chromatography-mass spectrometry (LC-MS) chip development system. 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 analyses were conducted by Huazhi Biotechnology Co., Ltd., and the procedure is briefly described below:

[0123] (1) Take 200ng of qualified genomic DNA, use enzyme digestion reagent to digest the DNA into fragments of 100-500bp, and then add Taq enzyme for end repair.

[0124] (2) Use T4 ligase to ligate the adapter fragments to both ends of the DNA, and 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, blocking reagent, RNase inhibitor, and 50K liquid phase chip probe on a PCR instrument for hybridization reaction, run at 80℃ for 5 minutes, and incubate at 55℃ for 16-24 hours.

[0126] (4) The hybridization product was captured by 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 undergoes quality control filtering and other processing. FastP software is used to remove reads containing adapter contamination and low-quality reads. BWA software is used to align the sequencing data with the target genome, and GATK software is then used to analyze the mutation sites to obtain the genotyping results for the target loci. A flowchart is shown below. Figure 3 As 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, the genotyping detection of 14 Camellia oleifera samples (including 3 duplicate samples, of which 6 were diploid, 3 were tetraploid, 4 were hexaploid, and 1 was octoploid) was performed using the 50K liquid phase chip prepared in Example 1 (see Example 2 for specific operation method).

[0130] Sequencing and data analysis yielded the following results: Figure 4 As shown in the figure, the detection rate of loci in the 14 samples ranged from 84.21% to 99.03%, with an average detection rate of 91.42%. The detection rates varied among different ploidy species of Camellia oleifera, with diploid showing the highest detection rate at an average of 98.94%. Genotypic consistency was determined by comparing the genotypes of three replicate samples. Figure 5 As shown in the figure, the genotype concordance rate is between 99.22% and 99.66%, with an average concordance rate of 99.47%.

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

[0132] Example 4: Population Structure Analysis of Camellia oleifera 50K Liquid Phase Chip

[0133] The 50K liquid phase chip for Camellia oleifera prepared in Example 1 was used to identify the genotypes of 35 Camellia oleifera samples from different sources (the specific operation method is described in Example 2, and the sources of the 35 Camellia oleifera 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, the 50K liquid phase chip for Camellia oleifera can effectively distinguish Camellia oleifera tree species from different sources, and the classification effect is consistent with the actual classification.

[0135] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A liquid phase chip, characterized in that, The liquid-phase chip contains probes for detecting Camellia oleifera SNP molecular marker combinations; the Camellia oleifera SNP molecular marker combinations consist of 50,454 SNP molecular markers, the physical locations of which are determined by sequence alignment based on the Camellia oleifera reference genome GCA_039905865.1, and the specific site information is shown in Table 1 of the specification.

2. The application of the liquid phase chip according to claim 1 in any of the following: (1) Genome-wide association analysis of Camellia oleifera; (2) Germplasm resources and variety identification of Camellia oleifera; (3) Cluster analysis and phylogenetic identification of Camellia oleifera; (4) Molecular design breeding of Camellia oleifera.

3. A method for breeding Camellia oleifera, characterized in that, The process includes the following steps: using the liquid phase chip as described in claim 1 to detect the DNA of the Camellia oleifera to be tested, and selecting Camellia oleifera for subsequent breeding.

Images