SNP (Single Nucleotide Polymorphism) molecular marker for detecting size of camellia oleifera fruit and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for detecting the size of an oil-tea camellia fruit and application of the SNP molecular marker, the size of the oil-tea camellia fruit can be quickly and accurately identified through the SNP molecular marker for detecting the size of the oil-tea camellia fruit, and early screening of high-yield oil-tea camellia germplasm is realized; the method can shorten the camellia oleifera breeding period and improve the camellia oleifera yield, economic benefit and commercial value, lays a breeding theoretical foundation for people to plant high-yield large-fruit camellia oleifera in the future, also provides reference for related research of other camellia oleifera fruit sizes, and has important theoretical guidance significance for the camellia oleifera industry in China.

Description

Technical Field

[0001] The invention belongs to the field of agricultural molecular biology, and in particular relates to a SNP molecular marker for detecting the size of oil-tea camellia fruit and an application thereof. Background Art

[0002] Camellia drupifera is a woody oil crop that is widely distributed in the middle and lower reaches of the Yangtze River and has a very long history of cultivation. Gaozhou Camellia drupifera has many characteristics that far exceed ordinary Camellia drupifera: large fruit, high oil content in kernels, rich active ingredients in oil, high polyphenol content, fast growth, strong stress resistance, etc. However, the size of its fruits varies greatly, and there is a large variation at the population level. Breeding large-fruit genotypes is an important way to increase the yield of native Camellia drupifera in Guangdong. How to screen out large-fruit genotypes in the early stage (seedling stage) is a key link that urgently needs to be broken through in the breeding of high-yield and high-quality Gaozhou Camellia drupifera varieties.

[0003] Fruit size is a complex quantitative trait controlled by multiple genes and is only expressed in the late stages of growth and development. Early screening based on phenotype is not possible, which limits the application and promotion of large-fruited and high-yield varieties. Therefore, it is urgent to provide a method for rapid identification of oil-tea camellia fruit size using molecular markers. 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 for detecting the size of oil-tea camellia fruit.

[0005] The present invention also provides a primer set for detecting the above SNP molecular markers.

[0006] The present invention also provides a kit.

[0007] The invention also provides a gene chip.

[0008] The present invention also proposes the application of the above molecular marker, primer set, kit and / or gene chip.

[0009] The present invention also provides a method for detecting the above SNP site.

[0010] The invention also provides a method for detecting the size of oil-tea camellia fruit.

[0011] According to a first aspect of the present invention, a SNP molecular marker for detecting the size of Camellia oleifera fruit is provided, wherein the SNP molecular marker comprises at least one, at least two or at least three of the following (1)-(3):

[0012] (1) a first SNP molecular marker, wherein the first SNP molecular marker is located at nucleotide 124124891 of chromosome 12 of the Camellia oleifera reference genome, and the nucleotide base at the site is C or T;

[0013] (2) a second SNP molecular marker, wherein the second SNP molecular marker is located at nucleotide 65965458 of chromosome 3 of the Camellia oleifera reference genome, and the nucleotide base at the site is C or G;

[0014] (3) a third SNP molecular marker, wherein the third SNP molecular marker is located at nucleotide 141688545 of chromosome 5 of the Camellia oleifera reference genome, and the nucleotide base at the site is T or C;

[0015] The Camellia oleifera reference genome is a diploid wild Camellia oleifera (https: / / github.com / Hengfu-Yin / CON_genome_data) reference genome.

[0016] According to a second aspect of the present invention, a primer set for amplifying the above-mentioned SNP molecular marker is proposed.

[0017] In some embodiments of the present invention, the primer set comprises:

[0018] A first primer set for detecting a first molecular marker, the first primer set comprising a forward primer sequence shown in SEQ ID NO: 1 and a reverse primer sequence shown in SEQ ID NO: 2;

[0019] A second primer set for detecting a second molecular marker, the second primer set comprising a forward primer sequence shown in SEQ ID NO: 3 and a reverse primer sequence shown in SEQ ID NO: 4;

[0020] A third primer set for detecting a third molecular marker, the third primer set comprising a forward primer sequence shown in SEQ ID NO:5 and a reverse primer sequence shown in SEQ ID NO:6.

[0021] According to a third aspect of the present invention, a kit is provided, the kit comprising the above-mentioned primer set.

[0022] According to a fourth aspect of the present invention, a gene chip is provided, wherein the gene chip comprises the above-mentioned primer set.

[0023] According to a fifth aspect of the present invention, the use of the above-mentioned SNP molecular marker, primer set, kit and / or gene chip in any one of the following 1)-9) is proposed:

[0024] 1) Detect the size of Camellia oleifera fruit;

[0025] 2) Identification and selection of Camellia oleifera with different fruit sizes;

[0026] 3) Breeding tea with fruit size ranging from 120-150g;

[0027] 4) Breeding tea with fruit size ranging from 110-120g;

[0028] 5) Breeding tea with fruit size ranging from 90-110g;

[0029] 6) Breeding tea with fruit size ranging from 0-90g;

[0030] 7) Molecular marker-assisted breeding of Camellia oleifera;

[0031] 8) Camellia oleifera breeding;

[0032] 9) Prepare camellia oleifera breeding products.

[0033] In some embodiments of the present invention, the application is application in Camellia oleifera genotyping.

[0034] According to a sixth aspect of the present invention, a method for detecting the size of tea fruit using the above-mentioned SNP molecular markers is proposed, the method comprising the following steps:

[0035] S1. Extract genomic DNA from Camellia oleifera;

[0036] S2. Performing polymorphism detection of the SNP molecular markers on the genomic DNA extracted in step S1, and determining the fruit size of the tested Camellia oleifera according to the detection result.

[0037] When the SNP molecular marker is the first SNP molecular marker, the second SNP molecular marker and the third SNP molecular marker, if the SNP molecular marker genotypes detected are TT, CC, TT, the size range of the tea oil fruit is 120-150g, if the SNP molecular marker genotypes detected are TT, GG, TT, the size range of the tea oil fruit is 110-120g, if the SNP molecular marker genotypes detected are TC, GC, TC, the size range of the tea oil fruit is 90-110g, if the SNP molecular marker genotypes detected are CC, GG, CC, the size range of the tea oil fruit is 20-90g.

[0038] In some embodiments of the present invention, in step S1, the genomic DNA is extracted from Camellia oleifera using a simplified CTAB method (cetyltrimethylammonium bromide method).

[0039] In some embodiments of the present invention, in step S2, sequencing is used to detect the SNP site.

[0040] A method for breeding camellia oleifera, comprising the following steps: using the above-mentioned SNP molecular marker method to detect the fruit size of camellia oleifera, selecting camellia oleifera with a fruit size range of 20-90g, camellia oleifera with a fruit size range of 90-110g, camellia oleifera with a fruit size range of 110-120g or camellia oleifera with a fruit size range of 120-150g for subsequent breeding.

[0041] The SNP molecular marker for detecting the size of oil-tea camellia fruit according to the embodiment of the present invention has at least the following beneficial effects: the size of oil-tea camellia fruit can be quickly and accurately identified by using the SNP molecular marker for detecting the size of oil-tea camellia fruit of the present invention. It can realize the early screening of high-yield oil-tea camellia germplasm, shorten the oil-tea camellia breeding cycle and improve the tea oil yield, economic benefits and commercial value, lay a theoretical foundation for breeding for people to plant high-yield large-fruit oil-tea in the future, and also provide a reference for other related research on the size of oil-tea camellia fruit, which has important theoretical guiding significance for my country's oil-tea industry.

[0042] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0044] Figure 1 This is a diagram showing the results of measuring the single fruit size of different strains of Gaozhou Camellia oleifera at maturity in Example 1 of the present invention;

[0045] Figure 2 This is a structure analysis diagram of the analysis of the genetic composition results of different families of Gaozhou oil-tea tea in Example 1 of the present invention;

[0046] Figure 3 It is a phylogenetic tree diagram between samples in the genetic composition analysis of different families of Gaozhou oil-tea tea in Example 1 of the present invention;

[0047] Figure 4 This is a PCA principal component analysis diagram of the genetic composition analysis results of different families of Gaozhou oil-tea camellia in Example 1 of the present invention;

[0048] Figure 5 The Manhattan plot of the genome association analysis in Example 1 of the present invention;

[0049] Figure 6 Draw a GWAS-QQ graph with a confidence interval P value in Example 1 of the present invention;

[0050] Figure 7The gel electrophoresis diagram of the products detected by the specific primers of the Gaozhou oil-tea tea fruit size-associated molecular markers SNP Cd12g124124891C>T, SNP Cd3g65965458G>C and SNP Cd5g141688545C>T in Example 1 of the present invention, and the marker corresponding to the product band is 100 bp;

[0051] Figure 8 This is a diagram showing the sequencing verification results of Cd12g124124891C>T in Example 1 of the present invention;

[0052] Fig. 9 This is a diagram showing the sequencing verification results of Cd3g65965458G>C in Example 1 of the present invention;

[0053] Fig.10 This is a diagram of the sequencing verification results of Cd5g141688545C>T in Example 1 of the present invention. DETAILED DESCRIPTION

[0054] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels.

[0055] The present invention is a SNP molecular marker for detecting the size of oil-tea camellia fruit.

[0056] 1. Acquisition of SNP molecular markers

[0057] (1) Material selection

[0058] The Gaozhou oil-tea material used in the present invention comes from the Zhongkai oil-tea base in Huizhou. During the best fruit picking period, 10 to 15 representative fruits were selected from each plant (lineage), and a total of 169 Gaozhou oil-tea fruits were obtained. Figure 1 As shown in Figure 2, the genetic composition of different Gaozhou oil-tea camellia families is as follows: Figure 2-4 shown.

[0059] 2. Sample processing and phenotypic analysis

[0060] Fruit size measurement includes transverse diameter, longitudinal diameter, and fruit weight, and the average single fruit size and weight are calculated separately.

[0061] The 169 samples obtained by harvesting were weighed using an analytical balance to calculate the average single fruit weight (accuracy 0.1g); the transverse and longitudinal diameters of the samples were measured using a ruler, and the frequency distribution table of the obtained fruit sizes is shown in Table 1.

[0062] Table 1

[0063] Fruit size (g) Number frequency 20，90 157 0.93 90，110 7 0.04 110，120 4 0.02 120，150 1 0.01

[0064] 3. Genotype data collection and analysis

[0065] The genotype data of all samples were obtained by sequencing, and the specific steps are as follows:

[0066] (1) Extraction of genomic DNA from Gaozhou tea leaves using the modified CTAB method

[0067] Take 1-2 young and healthy leaves from different strains, use color-changing silica gel to fully dry for 2-3 days, add liquid nitrogen to grind into powder, add 800μL of 1×CTAB extract, shake and mix, and then bathe at 65℃ for 30 minutes, mixing continuously. Then centrifuge at 12000g for 6min at room temperature using a common centrifuge (Sigma, 1-14, Germany), and take the supernatant. Add an equal volume of chloroform: isoamyl alcohol (24:1) solution and mix several times, centrifuge at 12000g for 6min at room temperature using a common centrifuge (Sigma, 1-14, Germany), take the supernatant, and repeat 1-2 times. Finally, take the supernatant, add 1M sodium acetate and an equal volume of anhydrous ethanol, and precipitate overnight. After centrifugation, wash the obtained precipitate twice with 70% ethanol, dry it, add 50-100μL of TE buffer and store it for later use.

[0068] (2) Construction of simplified genomic ddRAD library

[0069] DNA from different Gaozhou Camellia oleifera strains was taken and simplified genomic library was constructed according to the following steps (library construction reagents were purchased from BGI, and the construction method also referred to the library construction method provided by BGI):

[0070] 1) Purifying the intact DNA extracted in step (1) above by magnetic beads to remove impurities and small fragments;

[0071] 2) Adjust the volume and concentration of each qualified DNA sample to 10 μL and 200 ng of DNA;

[0072] 3) Add 10 μL of pre-mixed double enzyme digestion (NEB EcoRI+MseI, the amount added depends on the enzyme activity) to each sample DNA with adjusted concentration and volume, and mix thoroughly with a 200 μL pipette tip. Then put it into the PCR instrument for enzyme digestion, set the temperature to 37℃ for 8 hours, 65℃ for 20 minutes, and keep warm at 12℃;

[0073] 4) Take 5 μL of each digestion product and perform agarose gel electrophoresis. If there is no obvious total DNA main band and the digestion product shows a complete diffuse band in the lane, it can be judged that the digestion is complete;

[0074] 5) After adding the EcoR I-terminal adapter with a specific label (barcode) to the digested product of each sample, add the ligation mixture containing the universal Mse I adapter (connection uses T4 DNA ligase produced by NEB), and mix thoroughly with a 200μL pipette tip. Then put it into the PCR instrument for digestion treatment, set the temperature to 16℃ for 8 hours, 65℃ for 20 minutes, and keep warm at 12℃;

[0075] 6) Purify with equal volume of magnetic beads to remove excess adapter sequences;

[0076] 7) After the ligation is completed, equal volumes of the sample digestion / ligation products with different barcodes are mixed, and then the gel is excised and recovered under agarose gel electrophoresis (Omega gel recovery kit), and the screening fragment range is 350-550bp.

[0077] 8) Amplify the recovered library to reach the concentration required for sequencing.

[0078] 9) The library was sequenced using the Illumina Novaseq platform, the sequencing mode was PE 150, and the average sequencing amount of a single sample was 0.5G raw data.

[0079] (3) Simplified genomic data analysis of different strains of Gaozhou Camellia oleifera in the examples

[0080] The data were processed using the software Stacks 2 (Rochette et al. 2019) to obtain a high-quality SNP genotype dataset, including the following steps:

[0081] 1) Use the process_radtags module to process the raw data, including sample separation, quality control, and checking the integrity of restriction sites.

[0082] 2) Obtain sample variation data. Establish an index for the Camellia oleifera genome library, and use the alignment tool BWA to align the high-quality short reads of each strain after filtering to the reference genome. Use the Mseack module of Stacks 2 to identify the SNPs sites of each strain, and use cstacks to further merge the location information into a directory file containing all site information. Then use sstacks to search the site information of each sample from the directory file created by cstacks.

[0083] 3) Use the population module to filter and export the SNP dataset.

[0084] 4. Association analysis of fruit size genomes of different strains of Gaozhou oil-tea tea in the examples

[0085] The association statistical analysis of fruit size data and SNP data was performed using the software PLINK v1.09, including the following steps:

[0086] 1) Organize the map and ped files including pedigree and phenotype values.

[0087] 2) The association analysis of continuous traits was performed using a logistic regression model taking into account covariates, and the threshold of the Bonferroni multiple test was set to 0.01.

[0088] 3) The R package GAPIT was used to visualize the significantly associated sites. The gene sequences and gene annotations of these molecular marker sites were extracted. The results are as follows: Figure 5-6 As shown, the fruit size-associated molecular markers SNPCo12g124124891C>T, SNP Co3g65965458G>C and SNP Co5g141688545C>T were finally screened. The fruit size and site information of Camellia oleifera corresponding to different SNP markers are shown in Table 2. The reference genome corresponding to the site is the wild Camellia oleifera diploid (https: / / github.com / Hengfu-Yin / CON_genome_data) reference genome.

[0089] Table 2

[0090]

[0091] 5. Design and synthesis of primers for amplifying the above SNPs

[0092] For the significantly associated SNP sites obtained from the above screening, specific amplification primers were designed using the extracted gene sequences in Premier Primer 5.0. The primer sequences are shown in Table 3.

[0093] Table 3

[0094]

[0095] 6. Specificity and practicality verification

[0096] Twelve Camellia oleifera materials that were completely different from the above 169 Camellia oleifera materials were typed using the above three SNP molecular markers and the primers used to amplify the above molecular markers were used for typing and size verification. The typing method was as follows:

[0097] (1) Collection of Gaozhou Camellia oleifera leaf samples and DNA extraction

[0098] The tea leaf samples to be tested were Gaozhou tea leaves from the Xinfeng base. Twelve samples were randomly selected and DNA was extracted using the CTAB method described above.

[0099] (2) Amplification of nucleotide fragments containing SNP sites

[0100] The genomic DNA in each Gaozhou tea leaf sample to be tested obtained by the aforementioned extraction was used as a template and the primer set described in Table 3 was used to amplify the target fragment.

[0101] (3) Sequencing to identify SNP genotypes

[0102] The PCR product obtained in the above steps was first detected by 1.5% agarose gel electrophoresis, and the result was a single specific band. Figure 7 , and the sequencing was performed according to the above method. The sequencing results of SNP 1 are as follows Figure 8 The sequencing results of SNP 2 are shown in Fig. 9 The sequencing results of SNP 3 are shown in Fig.10 As shown, the results obtained from sequencing were consistent with the phenotype.

[0103] The typing and phenotypic test results of the 12 oil-tea camellia varieties are shown in Table 4.

[0104] Table 4

[0105]

[0106]

[0107] The test results are shown in Table 4. It can be seen from the table that the SNP molecular markers of the scheme of the present invention can be accurately used to detect the size of oil-tea camellia fruits, and can realize the rapid breeding of oil-tea camellia varieties with fruits of different sizes. The SNP loci of the scheme of the present invention are linked molecular markers, and in practical applications, it is best to verify three pairs of molecular markers with each other.

[0108] Genome-wide association study (GWAS) is an effective method for studying genetic variation of complex traits and diseases. Its core is to study the association between molecular variation and target phenotypic traits. Especially in recent years, with the continuous development of high-throughput sequencing, as well as the development of various bioinformatics technologies and statistical methods, it has been widely used in the study of the genetic mechanism of complex traits. Genome-wide association study (GWAS) relies on the association between genetic variation and traits in the linkage disequilibrium detection population, and then screens out genetic variation with significant influence by statistically analyzing the size of the association between genotype and phenotype. The general analysis process of genome-wide association study (GWAS) is to collect samples and phenotype records, genotyping, population stratification analysis, association analysis, single nucleotide polymorphism (SNP) annotation and candidate gene screening, linkage disequilibrium analysis and haplotype analysis.

[0109] At present, there is a lack of Gaozhou tea genomic resources, and no complex trait association analysis at the genome level has been carried out. Based on the independently completed high-quality tea oleifera genome, the applicant collected different varieties of Gaozhou tea oleifera with rich variations in fruit size. Through simplified genome sequencing, high-confidence fruit size-associated molecular markers were screened, and identification primers were developed to achieve early identification of large-fruit tea oleifera.

[0110] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, 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 for detecting the size of Camellia oleifera fruit, characterized in that: The SNP molecular markers include at least one, at least two or at least three of the following (1)-(3): (1) a first SNP molecular marker, wherein the first SNP molecular marker is located at nucleotide 124124891 of chromosome 12 of the Camellia oleifera reference genome, and the nucleotide base at the site is C or T; (2) a second SNP molecular marker, wherein the second SNP molecular marker is located at nucleotide 65965458 of chromosome 3 of the Camellia oleifera reference genome, and the nucleotide base at the site is C or G; (3) a third SNP molecular marker, wherein the third SNP molecular marker is located at nucleotide 141688545 of chromosome 5 of the Camellia oleifera reference genome, and the nucleotide base at the site is T or C; The Camellia oleifera reference genome is a wild Camellia oleifera diploid reference genome.

2. A primer set for amplifying the SNP molecular marker as described in claim 1.

3. The primer set according to claim 2, characterized in that The primer set comprises: A first primer set for detecting a first molecular marker, the first primer set comprising a forward primer sequence shown in SEQ ID NO: 1 and a reverse primer sequence shown in SEQ ID NO: 2; A second primer set for detecting a second molecular marker, the second primer set comprising a forward primer sequence shown in SEQ ID NO: 3 and a reverse primer sequence shown in SEQ ID NO: 4; A third primer set for detecting a third molecular marker, the third primer set comprising a forward primer sequence shown in SEQ ID NO:5 and a reverse primer sequence shown in SEQ ID NO:

6.

4. A kit, characterized in that: The kit comprises the primer set according to claim 2 or 3.

5. A gene chip, characterized in that: The gene chip comprises the primer set according to claim 2 or 3.

6. Use of at least one of the SNP molecular marker according to claim 1, the primer set according to claim 2 or 3, the kit according to claim 4, and the gene chip according to claim 5 in any one of the following 1) to 9): 1) Detect the size of Camellia oleifera fruit; 2) Identification and selection of Camellia oleifera with different fruit sizes; 3) Breeding tea with fruit size ranging from 120-150g; 4) Breeding tea with fruit size ranging from 110-120g; 5) Breeding tea with fruit size ranging from 90-110g; 6) Breeding tea with fruit size ranging from 0-90g; 7) Molecular marker-assisted breeding of Camellia oleifera; 8) Camellia oleifera breeding; 9) Prepare camellia oleifera breeding products.

7. A method for detecting the size of Camellia oleifera fruit using the SNP molecular marker as claimed in claim 1, characterized in that: The method comprises the following steps: S1. Extract genomic DNA from Camellia oleifera; S2. Performing polymorphism detection of the SNP molecular markers on the genomic DNA extracted in step S1, and determining the fruit size of the tested Camellia oleifera according to the detection result.

8. The method according to claim 7, characterized in that When the SNP molecular marker is the first SNP molecular marker, the second SNP molecular marker and the third SNP molecular marker, if the SNP molecular marker genotypes detected are TT, CC, TT, the size range of the tea oil fruit is 120-150g, if the SNP molecular marker genotypes detected are TT, GG, TT, the size range of the tea oil fruit is 110-120g, if the SNP molecular marker genotypes detected are TC, GC, TC, the size range of the tea oil fruit is 90-110g, if the SNP molecular marker genotypes detected are CC, GG, CC, the size range of the tea oil fruit is 20-90g.

9. The method according to claim 7, characterized in that: The simplified CTAB method was used to extract genomic DNA from Camellia oleifera.

10. A method for breeding oil-tea camellia, characterized in that: The method comprises the following steps: using the method described in any one of claims 7 to 9, selecting oil tea with a fruit size range of 20-90g, oil tea with a fruit size range of 90-110g, oil tea with a fruit size range of 110-120g or oil tea with a fruit size range of 120-150g for subsequent breeding.

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