Camellia oleifera variety SSR molecular identity card and application thereof

Abstract

The invention discloses a camellia oleifera variety SSR molecular identity card and application thereof. Three pieces of diploid camellia oleifera genome data are analyzed by utilizing a bioinformatics method, SSR site search of the whole genome is respectively carried out, and the distribution mode of SSR sites in three species is clarified. A group of polymorphic genome SSR loci is developed, and different oil tea varieties are subjected to genetic typing by using an SSRSeq technology. Finally, a specific molecular fingerprint spectrum of each variety is generated. A large number of SSR markers developed by the invention and molecular fingerprints generated for oil-tea camellia varieties can provide powerful support for genetic research, breeding projects and variety authentication and identification of oil-tea camellia.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology and plant variety identification, in particular to an SSR molecular identity card of oil-tea camellia varieties and applications thereof. Background Art

[0002] Camellia oleifera generally refers to oil-rich species in the Theaceae family, such as common Camellia oleifera ( Camellia oleifera )、Vietnamese Tea( Camellia vietnamensis )、Zhejiang Red Camellia( Camellia chekiangoleosa ) etc. Camellia oleifera has been cultivated as a traditional woody oil crop in my country for more than 2,300 years (Lin et al ., 2022; Yao Xiaohua, 2016). Among them, common tea oil is the main cultivated species, and its seed oil content exceeds 30%, oleic acid content exceeds 80%, followed by linoleic acid (about 8%). Tea oil is rich in physiologically active substances and is a high-quality edible vegetable oil (Chai et al ., 2023; Wang et al ., 2024). In addition, Camellia oleifera fruit has important chemical and medicinal value due to its rich content of proteins, polysaccharides, saponins, functional peptides and other photoactive components (Qin et al ., 2024). Different tea varieties show significant differences in yield, quality, resistance and regional adaptability (Yang et al ., 2022; Yao Xiaohua, 2016), effective variety identification is crucial for the precise application of improved varieties and the protection of intellectual property rights.

[0003] Due to the large number of varieties and high phenotypic similarity, it is extremely difficult to scientifically and correctly identify oil-tea camellia varieties (Zhu et al ., 2022; Lin et al ., 2022). The DUS test required by the International Union for the Protection of New Varieties of Plants (UPOV) is the main method for identifying new varieties (Xu Yunbi, 2022). In addition, phenotypic-based machine learning technology has also been used to quickly identify oil tea varieties (Zhu et al ., 2022). However, phenotypic traits tend to be unstable and affected by environmental factors, cultivation practices, and growth stage. In addition, seedlings exhibit a limited number of phenotypic traits, making it more difficult to distinguish between similar varieties ( Figure 1 ). Therefore, there is an urgent need to develop a reliable, simple and stable method to identify oil-tea camellia varieties. In recent years, molecular marker technology has been widely used in genetic diversity research, molecular fingerprinting and variety identification due to its stability and reproducibility (Lin et al., 2022; Xu Yunbi et al., 2022). RAPD (Chen Yongzhong et al., 2005), ISSR (Yang Cuifang et al., 2013), AFLP (Xie Yiqing et al., 2014), SRAP (Peng Shaofeng et al., 2011), SSR (Cui et al ., 2018; Jia et al ., 2014) and SNP (Lin Ping et al., 2023; Yan et al ., 2022) and other molecular markers have been used in the study of genetic diversity and variety identification of Camellia oleifera. Among these markers, simple sequence repeats (SSRs), also known as microsatellites, have been widely used in genetic diversity research, variety identification, genetic map construction and association mapping studies of various plant species due to their high polymorphism information content, high allelic variation and good reproducibility (Testolin et al ., 2023), such as tea (Tan et al ., 2015), Chamomile (Sharan et al ., 2024), Persimmon (Xu et al ., 2023), thin-shelled pecan (Zhang et al ., 2020) etc.

[0004] In recent years, a number of polymorphic SSR markers have been developed in Camellia oleifera (Cui et al ., 2018; Dong et al ., 2022; Jia et al ., 2014). Chen et al. (2016) constructed a molecular fingerprint for 128 camellia oleifera varieties using 10 SSR markers. Yan et al. (2020) constructed a molecular fingerprint for 140 camellia oleifera germplasm resources using 24 fluorescent SSR markers. Despite these advances, the number of SSR markers in camellia oleifera is still insufficient to meet the needs of genetic research, and there is a lack of SSR loci widely distributed in the genome. In recent years, the release of the diploid camellia oleifera genome (Lin et al .,2022;Gong et al ., 2022; Shen et al ., 2022), laying the foundation for the large-scale development of SSR markers across the whole genome.

[0005] However, most of the main cultivated varieties of Camellia oleifera are hexaploid (2n = 6x = 90) with large genomes (about 9.0 Gb). Traditional methods are difficult to accurately assess the allele dosage of polyploid species, in which multiple gene copies can result in more than two bands at the SSR locus (Cui et al., 2022; Jia et al ., 2014; Yan et al ., 2022). To determine the allele dosage of polyploid species, the microsatellite DNA allele count-peak ratio method was established (Esselink et al ., 2004) and the “allele-phenotype” approach (Urrestarazu et al ., 2018). However, these methods cannot determine the exact genotype, resulting in uncertainty in allele dosage (Cui et al ., 2022). Recently, a high-throughput sequencing-based SSRSeq technology has provided a new approach for genetic research of polyploid species (Cui et al ., 2022; Li et al ., 2021). However, there are few reports on the application of SSRSeq in the construction of molecular fingerprints of polyploid species and variety identification research. Summary of the invention

[0006] The purpose of the present invention is to provide an SSR molecular identification card of a tea oil variety and its application.

[0007] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a core SSR molecular marker of the Camellia oleifera genome, numbered Coz06080 and Coz08114; The base repeating unit corresponding to marker Coz06080 is (TATG)6, and the primers used to amplify marker Coz06080 are shown in SEQ ID NO: 1-2; The base repeating unit corresponding to marker Coz08114 is (TTC)8, and the primers used to amplify marker Coz08114 are shown in SEQ ID NO:3-4.

[0008] In a second aspect, the present invention provides a combination of SSR molecular identification cards of oil-tea camellia varieties, which are a combination of SSR molecular markers of the oil-tea camellia genome, numbered Coz06080, Coz08114, Coz10144 and Coz14197; The base repeating unit corresponding to marker Coz10144 is (TTTC)7, and the primers used to amplify marker Coz10144 are shown in SEQ ID NO:5-6; The base repeating unit corresponding to marker Coz14197 is (TTC)5, and the primers used to amplify marker Coz14197 are shown in SEQ ID NO:7-8.

[0009] In a third aspect, the present invention provides primers for amplifying core markers, the primers for amplifying marker Coz06080 are shown in SEQ ID NOs: 1-2, and the primers for amplifying marker Coz08114 are shown in SEQ ID NOs: 3-4.

[0010] In a fourth aspect, the present invention provides primers for amplifying the SSR molecular ID card (i.e., the marker combination), the primers for amplifying marker Coz06080 are shown in SEQ ID NOs: 1-2, the primers for amplifying marker Coz08114 are shown in SEQ ID NOs: 3-4, the primers for amplifying marker Coz10144 are shown in SEQ ID NOs: 5-6, and the primers for amplifying marker Coz14197 are shown in SEQ ID NOs: 7-8.

[0011] In a fifth aspect, the present invention provides a detection reagent or a kit containing the primers.

[0012] In a sixth aspect, the present invention provides the application of the marker, the SSR molecular ID card (i.e., the marker combination), the primer, or the detection reagent or kit in the identification of camellia oleifera germplasm resources (identification of camellia oleifera varieties), improvement or molecular marker-assisted breeding.

[0013] In a seventh aspect, the present invention provides a method for constructing an SSR fingerprint of Camellia oleifera, comprising the following steps: 1) Extracting genomic DNA from the tea oil plant to be tested; 2) Using the extracted DNA as a template, perform PCR amplification using the above primers; 3) Analyze the PCR amplification products and construct the SSR fingerprint of Camellia oleifera.

[0014] The oil tea includes common oil tea ( Camellia oleifera var Nanyongensis), narrow-leaved tea ( C. lanceoleosa ) and Zhejiang safflower tea ( Camellia chekiangoleosa ).

[0015] Preferably, the PCR amplification system in step 2) is: 1 μL of template DNA, 0.4 μL of 10 μM upstream and downstream primers, 0.4 μL of 10 μM dNTPs, 2 μL of 10× buffer and 0.5 U of Taq enzyme, which is made up to 20 μL with ddH2O.

[0016] Preferably, the PCR reaction program in step 2) is: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; and finally extension at 72°C for 10 minutes.

[0017] Furthermore, in step 3), the number of repetitions of the base repeating unit in the amplified product is detected and analyzed, and information data on the number of repetitions of SSR markers of each variety is established. The obtained information list is the SSR fingerprint map of the oil-tea camellia variety.

[0018] The present invention uses bioinformatics methods to analyze the genome data of three diploid camellia oleifera (Gong et al .,2022; Lin et al ., 2022; Shen et al ., 2022), and carried out genome-wide SSR loci searches respectively, clarifying the distribution patterns of SSR loci in the three species. A set of polymorphic genomic SSR loci was developed, and SSRSeq technology was used to genotype different oil-tea camellia varieties. Finally, a specific molecular fingerprint map for each variety was generated. The large number of SSR markers developed by this invention and the molecular fingerprint maps generated for oil-tea camellia varieties will provide strong support for genetic research, breeding programs, and variety certification and identification of oil-tea camellia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a tea oil fruiting tree and its grafted seedling in a preferred embodiment of the present invention.

[0020] Figure 2 It is the percentage of dinucleotide and trinucleotide repeat types in the total number of SSRs in the genomes of the three species in the preferred embodiment of the present invention.

[0021] Figure 3 The distribution of 132 newly developed SSRs in the preferred embodiment of the present invention on the genome of C. oleifera var. Nanyongensis is shown in Figure 1. The green fonts represent 107 polymorphic SSRs, and the blue fonts represent 25 non-polymorphic SSRs.

[0022] Figure 4 The genotype of the SSR marker Coz06080 in different varieties in the preferred embodiment of the present invention. The repeating motif of Coz06080 is (TATG)6. The horizontal axis represents the number of times the motif is repeated. The vertical axis represents the density of each peak. "Geno" represents the genotype of a specific variety. For example, the genotype of GF20 is 4 / 4 / 4 / 5 / 6 / 7, which means that in the hexaploid GF20 variety, there are three alleles of (TATG)6 repeated four times of the Coz06080 marker, and one allele each of five times, six times, and seven times.

[0023] Figure 5 This is a UPGMA cluster diagram of the test samples in the preferred embodiment of the present invention, wherein the red vertical line marks the position of the genetic similarity coefficient of 0.385.

[0024] Figure 6 It is the "3D fingerprint" of 43 varieties constructed by two core SSR loci and two alternative SSR loci in the preferred embodiment of the present invention. Each column represents a variety. The primer numbers in red font represent the core loci, and those in black font represent the alternative loci. The letters in brackets next to the primer numbers represent the repeating motif of SSR. The numbers arranged vertically next to the SSR repeating motif represent the number of repetitions of the motif. Each colored spot represents the variety with an amplified product at the number of repetitions of the corresponding SSR locus motif, and different colors represent different allele dosages.

[0025] Figure 7 The results showed that the banding pattern of Coz1116 marker in the two varieties was consistent, but the allele dosage was different. DETAILED DESCRIPTION

[0026] The invention develops a new SSR marker for the oil-tea camellia genome and establishes a molecular fingerprint and a molecular ID card specific to the main hexaploid cultivar.

[0027] The present invention uses bioinformatics to analyze common camellia oleifera ( Camellia oleifera varNanyongensis), narrow-leaved tea ( Camellia lanceoleosa ) and Zhejiang safflower tea ( Camellia chekiangoleosa ) The distribution pattern of SSR sites in the genome; develop new genomic SSR markers, use the SSR-Seq method based on the second-generation sequencing technology that can detect polyploid allele dosage, carry out genetic research on oil-tea varieties, screen core SSR primer sets, and construct variety-specific molecular fingerprints and molecular ID cards.

[0028] The present invention clarifies Camellia oleifera var Nanyongensis, Camellia lanceoleosa and C. chekiangoleosa The frequency of SSR in the genomes of the three species ranged from 1 SSR / 4.40kb to 1 SSR / 4.22kb. Camellia oleifera var Nanyongensis and Camellia chekiangoleosa The SSR frequency is close to and slightly lower than C. lanceoleos .based on Camellia oleifera var Nanyongensis genome, a total of 14,854 SSR primers were designed, of which 5073 (34.16%) and 1921 (12.93%) primers were in Camellia lanceoleosa and C. chekiangoleosaThe results showed that the SSR primers were universal in the genome of Camellia oleifera. A total of 225 pairs of SSR primers were synthesized and verified, and a total of 132 pairs (58.67%) of SSR primers amplified the target bands, of which 107 SSR markers were polymorphic and 25 were not polymorphic. Thirty SSR markers were selected on 15 chromosomes, and PCR amplification and SSRSeq genotyping were performed on 45 Camellia oleifera samples. After genetic analysis, it was found that the number of alleles detected at each SSR locus (Na) ranged from 4 to 22, the polymorphism information content (PIC) value ranged from 0.07 to 0.90, the observed heterozygosity (Ho) ranged from 0.05 to 0.94, and the expected heterozygosity (He) ranged from 0.07 to 0.91. Cluster analysis showed that when the genetic similarity coefficient was 0.385, the 45 samples could be clustered into 5 categories, and some varieties from the same geographical area had a close genetic relationship. Finally, specific molecular fingerprints and molecular ID cards were constructed for 43 Camellia oleifera varieties using four highly polymorphic loci (Coz06080, Coz08114, Coz10144 and Coz14197).

[0029] The present invention has found Camellia oleifera var Nanyongensis, Camellia lanceoleosa and C. chekiangoleosa The distribution patterns of SSRs in the genomes of three oil-tea camellia species were studied, 132 new genomic SSR markers were developed, and 43 specific molecular fingerprints and molecular ID cards of the main oil-tea camellia varieties were constructed. The present invention is expected to promote genetic research and variety identification research of oil-tea camellia.

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] Example 1 Development of Camellia oleifera genome SSR molecular markers (i.e. Camellia oleifera variety SSR molecular ID card) 1 Materials and methods 1.1 Experimental materials and DNA extraction The present invention used 45 plant samples (Table 1), including 43 common camellia varieties, 1 Zhejiang red camellia plant and 1 small-fruited camellia ( Camellia meiocarpa Hu) single plant. Oil-tea fruiting trees and their grafted seedlings can be found in Figure 1Young leaves collected in spring were immediately frozen in liquid nitrogen and stored at −80°C. Genomic DNA was extracted using a plant DNA extraction kit (Tiangen, Beijing). DNA integrity was assessed using 1% agarose gel electrophoresis, and DNA concentration was determined using a Quawell Q5000 spectrophotometer (Quawell Technology, Inc., USA), diluted to 50 ng / μL, and stored at −80°C until use.

[0032] Table 1 Information of 45 samples tested

[0033] 1.2 Genomic SSR site mining The genome data of three diploid Camellia oleifera species were downloaded and analyzed. Camellia oleifera var. Nanyongensis(Lin et al ., 2022) Camellia lanceoleosa (Gong et al ., 2022) and C. chekiangoleosa (Shen et al ., 2022). Using the MISA program (http: / / pgrc.ipk-gatersleben.de / misa / misa.html), SSR identification criteria included at least 10 dinucleotide repeats and at least five tri- to hexanucleotide repeats, and two simple repeat sequences less than 100 bp apart were classified as composite SSRs. SSR primer design was completed using the scripts p3_in.pl, primer3_core, and p3_out.pl (Untergasser et al ., 2012).

[0034] 1.3 Development and validation of SSR markers 2-base repeat SSR may cause mixed bands, affecting the accuracy of the test results (Testolin et al ., 2023). Therefore, 3-base and 4-base repeat SSR loci were selected to design PCR primers. The universality of all primers among species was evaluated. The designed primers were mapped to Camellia lanceoleosa and C. chekiangoleosa 225 SSR loci were selected for experimental verification. Camellia chekiangoleosaPCR amplification was performed on the genomic DNA of a single strain. The PCR reaction was carried out in a 20 μL system, including 1 μL of template DNA, 0.4 μL of 10μM upstream and downstream primers, 0.4 μL of 10μM dNTPs, 2 μL of 10× buffer and 0.5 U of Taq enzyme, and ddH2O was used to make up to 20 μL. PCR program: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; and finally extension at 72°C for 10 minutes. The target bands were separated by 10% non-denaturing polyacrylamide gel electrophoresis, stained with 1 g / L AgNO3 solution for 10-15 minutes, developed with developer (20 g / L NaOH plus 10 mL formaldehyde) for 5-8 minutes, and imaged after washing twice with pure water. The SSR sites amplified to the target bands were selected for the next experiment. The successfully amplified SSRs were aligned to Camellia oleifera var. Nanyongensis genome and used TBtools-II software (Chen et al ., 2023) created a chromosomal distribution map of each marker, and selected two polymorphic SSRs on each chromosome, for a total of 30 SSRs for subsequent studies.

[0035] 1.4 Genotyping based on SSRSeq PCR products were detected by 1% agarose gel electrophoresis. et al .(2022) described the method, using 30 SSR loci to amplify genomic DNA of 8 samples, and performing high-throughput sequencing and SSR genotyping on the PCR products. Prepare a multiplex PCR primer mixture and use multiplex PCR for amplification. Evaluate the specificity and amplification efficiency of each pair of SSR primers in the multiplex PCR system by sequencing, and adjust and optimize the primer mixture. Use the optimized multiplex PCR primers to amplify all samples to be tested and construct a sequencing library. An index sequence compatible with the Illumina platform is introduced to the end of the library by PCR amplification. All sample index PCR amplification products are mixed in equal amounts. The final FastTarget TMThe sequencing library was purified by gel extraction. Sequencing was performed on the Illumina HiSeq platform at 2×150 / 2×250 bp. The sequencing data were merged using FLASH software (Magoc and Salzberg, 2011), and the successfully merged data were aligned with the reference sequence using BLAST+ (ftp: / / ftp.ncbi.nlm.nih.gov / blast / executables / blast+ / LATEST) to screen the effective read number. After alignment with the reference genome, the enrichment efficiency of the target fragment was calculated. The SSR motifs in the reads were identified, and the number of reads with different repeats of each motif was quantified. Genotyping was performed using SSRSeq_type software (http: / / bioinfo.geneskybiotech.com / software / ssrseq_type / v1.0).

[0036] 1.5 Data Processing PopGene software was used to calculate the data of each marker according to the dominant marker method, including observed heterozygosity (Ho), expected heterozygosity (He), number of alleles (Na), and allele frequency. The polymorphism information content (PIC) was calculated using the formula PIC = 1 - ∑ Pi^2, where Pi is the frequency of the i-th allele. Genetic distance and similarity coefficient were calculated, and the UPGMA method was used to perform cluster analysis on the 45 samples. Phylogenetic trees were generated using NTSYS 2.10e software.

[0037] 1.6 Fingerprint and Molecular ID Construction Clustering was performed in descending order of SSR marker PIC values, and the minimum SSR primer combination that could distinguish each sample was selected to construct a molecular fingerprint for each variety, and TBTools II software was used to draw the plot. A QR code generator (http: / / qr-batch.com / ) was used to construct the molecular ID cards of 43 oil-tea camellia varieties.

[0038] 2 Results and Analysis 2.1 SSR distribution characteristics exist Camellia oleifera var. Nanyongensis, Camellia lanceoleosa and Camellia chekiangoleosa A total of 640,983, 694,090 and 608,668 SSR loci were identified in the genomes of the two strains, with an occurrence frequency of 1 SSR per 4.40 kb, 4.22 kb and 4.38 kb, respectively (Table 2). Camellia oleifera var. Nanyongensis and C. chekiangoleosaThe SSR occurrence frequencies of Camellia lanceoleosa The frequency of SSRs was slightly higher in the genus SSR. Dinucleotide repeats were the main type, accounting for about 74.03% to 74.80% of all SSRs, followed by trinucleotide repeats, and hexanucleotide repeats were the least (Table 2). Among the dinucleotide repeats, AG / CT was the most abundant (46.61%~49.46%), followed by AT / AT (40.76%~43.16%), while CG / CG type was the least, accounting for only 0.14% to 0.24%. Among the trinucleotide types, AAT / ATT was the most common, accounting for 35.96% to 36.87%, followed by AAG / CTT and ACC / GGT types ( Figure 2 ). The frequency of occurrence of individual SSR motifs varies between species, such as AACAGT / ACTGTT in Camellia oleifera and Camellia chekiangoleosa 3.18% and 3.62% of the total hexanucleotide repeats in Camellia lanceoleosa In contrast, AGGCCC / CCTGGG Camellia oleifera and Camellia chekiangoleosa 0.04% and 0.06% of the total hexanucleotide repeats in Camellia lanceoleosa The medium to high percentage is 4.12%.

[0039] Table 2 Distribution of SSRs in the genomes of three Camellia oleifera species

[0040] 2.2 Development and validation of genomic SSR primers use Camellia oleifera var. Nanyongensis genome data. 14854 pairs of SSR primers were successfully designed using Primer 3 software. These primers were then compared with the C. lanceoleosa and Camellia chekiangoleosa The genomes of the two strains were compared, and 5073 (34.16%) and 1921 (12.93%) pairs of primers successfully matched to unique fragments of the two genomes, respectively. The expected amplified fragment length ranged from 60bp to 300 bp. Subsequently, 225 pairs of SSR primers were selected for synthesis and verification. The results showed that 15 pairs (6.67%) of primers did not amplify the target band, 78 pairs (34.67%) of primers amplified mixed bands or had poor amplification efficiency, and 132 pairs (58.67%) of primers had good amplification effects (Tables 5 to 7), of which 107 pairs of primers were polymorphic (Table 3). Then, these 132 pairs of SSR primers were compared to C. oleiferavar. Nanyongensis, 5 to 13 SSR loci were found on each chromosome. Figure 3 ).

[0041] 2.3 SSR typing and genetic analysis based on SSRSeq According to the distribution of 107 polymorphic SSR markers on 15 chromosomes ( Figure 3 ), two markers were selected on each chromosome, and 45 samples were genotyped using the SSRSeq method (Table 3, Figure 4 ). The results showed that the number of alleles of the 30 markers ranged from 4 to 22, with an average of 10.23; Ho ranged from 0.05 to 0.94 (average 0.55); He ranged from 0.07 to 0.91 (average 0.66); and PIC values ​​ranged from 0.07 to 0.90 (average 0.63). Based on the genotyping results of the 30 SSRs, the UPGMA method was used to cluster all the samples tested ( Figure 5 ), when the genetic similarity coefficient was 0.385, the 45 samples were clustered into 6 groups. Group I contained 37 samples, Group II contained 4 samples, and the remaining samples were clustered into one group each. Group I was further subdivided into 4 subgroups, each containing 7-12 samples ( Figure 5 ).

[0042] Table 3 Information of 30 SSR loci

[0043] 2.4 Construction of 3D molecular fingerprint Among the 30 polymorphic SSR loci, PIC values ​​of 22 loci were greater than 0.5. The highest PIC values ​​were Coz04051 and Coz09127, followed by Coz08117, Coz06083, Coz12178 and Coz14197. Cluster analysis of 43 varieties (excluding 1 Zhejiang red camellia and 1 small-fruited tea sample) was performed by increasing the number of SSR markers in the order of PIC values ​​from large to small. It was found that at least two markers, Coz06080 and Coz08114, were needed to effectively distinguish all the tested varieties. Therefore, these two SSR loci (Coz06080 and Coz08114) were determined as the core primer set. In view of the large number of Camellia oleifera varieties currently used in production (Lin Ping et al., 2023), and considering the limited ability of two SSR markers to identify varieties, two additional markers with high polymorphic information content and good typing effect, Coz10144 (located on chromosome 10) and Coz14197 (located on chromosome 14) were added as additional loci. Finally, two core loci and two additional loci were used to create a molecular fingerprint specific to each variety ( Figure 6). This molecular fingerprint is different from the typical fingerprint and contains three kinds of information: the presence or absence of the band, the size of the band and the allele dosage, so it is called a "3D fingerprint".

[0044] 2.5 Building a molecular ID card Based on the genotypes of the four SSRs, a molecular identity card of 24 digits or letters specific to each variety was constructed. Because most of the improved varieties of common oil-tea camellia are hexaploid, every six digits and letters represent the genotype of a marker. There are four SSR markers in total, and the primer sequence is Coz06080, Coz08114, Coz10144 and Coz14197. Taking the molecular identity card 44444877777M666678NNNNNN of the variety 'Changlin 53' as an example, the first six digits 444448 are the genotype of primer Coz06080, indicating that the repeating motif (TATG) 6 at this site is repeated 4 times, with 5 alleles, and the repeating motif (TTC) 8 is repeated 7 times, with 1 allele; the second group of six digits 77777M are the genotypes detected by primer Coz08114, indicating that the repeating motif (TTC) 8 at this site is repeated 7 times, with 5 alleles, and the repeating motif (TTC) 8 is repeated 8 times. The third group of six digits, 666678, are the genotypes detected by primer Coz10144, indicating that its repeating motif (TTTC) 7 is repeated 6 times and has 4 alleles, and those repeated 7 times and 8 times have 1 allele each; the last six digits, NNNNNN, are the genotypes of primer Coz14197, indicating that its repeating motif (TTC) 5 is repeated 14 times (N is ranked 14th in 26 letters) and has 6 alleles.

[0045] The molecular IDs of the 43 Camellia oleifera cultivars are shown in Table 4 .

[0046] Table 4 Molecular IDs of 43 Camellia oleifera varieties

[0047] 3 Discussion In recent years, some Camellia oleifera (Cui et al., 2018; Dong et al., 2022; Jia et al., 2014) and Camellia chekiangoleosa (Shi et al., 2013; Tian et al., 2022), however, the number of markers is insufficient to meet the needs of genetics and variety identification research. The three diploid Camellia oleifera reference genomes released in recent years provide an opportunity for rapid development of large-scale SSR loci. Camellia oleiferavar. Nanyongensis, Camellia lanceoleosa and Camellia chekiangoleosa The genomic data of the tea plant (Liu et al.) showed that the frequency of genomic SSRs was one SSR per 4.22 kb to 4.40 kb (Table 2). These frequencies were higher than those of tea plant (216.88 SSRs / Mb) ( et al., 2018), bamboo (1 SSR / 16 kb) (Zhao et al., 2015) and Carya sylvestris (1 SSR / 4.55 kb) (Zhang et al., 2020); lower than Camellia chrysantha (1 SSR / 3.84kb, excluding mononucleotide repeats) (Bai et al., 2022), walnut (1 SSR / 2.3 kb) (Wu et al., 2012) and jujube (1 SSR / 1.17 kb) (Fu et al., 2016). Meanwhile, the SSR density of the three species was significantly higher than Camellia oleifera var Cenruan No. 3 (48.34 SSRs / Mbp) (Wang et al., 2023). This discrepancy may be because Wang et al. (2023) only analyzed hexaploid C. oleifera Among the dinucleotide repeats, AG / TC is the most common, followed by AT / TA, which is consistent with the common Camellia oleifera (Cui et al., 2018) and Golden Camellia ( C. nitidissima Chi) (Bai et al., 2022) transcriptome findings, but consistent with Diospyros oleifera On the contrary, in the latter, AT / TA is more than AG / TC (Xu and al ., 2023). Among the trinucleotide types, AAT / ATT (35.96-36.87%), AAG / CTT (19.73-20.32%), and ACC / GGT (19.12-20.69%) were dominant, while in the transcriptome, ACC / GGT, AAG / CTT, and ATC / ATG were the main types (Cui et al., 2018;Li et al., 2017). Similarly, the main motifs in genomic and transcriptomic SSRs differ in rose ( Hybrid rose ) has also been reported (Qi et al., 2018). This may be due to base bias in coding and noncoding regions (Tóth et al., 2000).

[0048] exist C. oleiferavar. Nanyongensis, a total of 14,854 primer pairs were designed, and 5073 pairs (34.16%) and 1921 pairs (12.93%) of primers could be used to identify C. lanceoleosa and C. chekiangoleosa General, higher than Diospyros L. (13.60%) (Xu and others ., 2023). The high versatility may be due to the close evolutionary relationship between Camellia oleifera species. A total of 225 primer pairs were synthesized and verified, and 132 pairs (107 polymorphic sites and 25 low-polymorphic sites) were found to be able to amplify the target product, with a marker development success rate of 58.67%, which is higher than that of Camellia sinensis (32.00%, Liu et al., 2018) and the success rate of SSR development in the common Camellia oleifera transcriptome (49%) (Cui et al., 2018), but lower than guanidine ( Cyamopsis tetragonoloba , 86.4%) (Tribhuvan et al., 2019) and Cinnamomum yunnanensis ( Exbucklandia tonkinensis , 76.26%) (Huang and al., 2019). 107 polymorphic SSRs were found in C. oleifera There are 3 to 10 loci on each chromosome of var. Nanyongensis. Two SSR loci were further selected on each chromosome, with a total of 30 SSR markers, and the genomic DNA of 45 samples was amplified and genotyped using the SSRSeq technology. The results showed that the number of alleles of the tested primers ranged from 4 to 22, and the PIC values ​​ranged from 0.07 to 0.90, of which 22 loci (73.33%) showed high PIC values ​​(>0.5), which are suitable for genetic diversity research, genetic map construction, quantitative trait loci (QTL) positioning and variety identification research of Camellia oleifera.

[0049] SSR is an important tool for evolutionary and genetic research (Cui et al. ., 2022; Zhang et al. .,2020). As shown in Figure 5, HH1 ( C. chekiangoleosa ) is a diploid species and is genetically the most distant from other Camellia oleifera species (Yan and others ., 2022). However, in the clustering tree, XG ( C. meiocarpa Hu) is distributed in C. oleifera The results of the study of Li et al. (2017) and Chang and Ren (1992) indicate that there is a close genetic relationship between the two varieties. C. meiocarpa Hu was reclassified as C. oleiferavar. monosperma, indicating that it belongs to the subgenus Camellia oleifera. The clustering results of some varieties are consistent with their geographical origins, for example, GZY1 and GW1, CL27 and CL3 are genetically close, and they are all from Jiangxi Province (Table 1). Similarly, CL40 and ZL5 from Zhejiang Province also showed a close relationship (Table 1, Figure 5). Chen et al. (2016) used 10 SSR markers to study 128 Camellia oleifera varieties, and pointed out through cluster analysis that CL21 and CL40 may be the same variety. However, CL40 is from Zhejiang Province, while CL21 is from Jiangxi Province (Yao Xiaohua, 2016). In the present invention, CL40 is clustered in subgroup I with other 11 varieties, while CL21 is clustered in subgroup II with other 9 varieties, with a genetic similarity coefficient of 0.7893, indicating that they are different varieties ( Figure 5 In addition, varieties from Guangdong, Guangxi, Hunan, and Hubei provinces were scattered in the cluster diagram, a phenomenon that has also been reported in previous studies (Chen and others ., 2016). The cluster analysis results are affected by the original geographical distribution and family background (Guerrero and others ., 2021). One possible reason is that Camellia oleifera is a perennial cross-pollinating tree species with a cultivation history of more than 2,300 years (Lin and others ., 2022), during its long cultivation history, especially between 1970 and 1990, there was extensive resource exchange between different provinces, and the genetic background of the origin was maintained through hybridization with local varieties, reflecting the geographical origin and kinship in the cluster diagram. This also shows that using SSR markers to study the genetic diversity of Camellia oleifera is expected to reveal its transmission path between the origin and production areas.

[0050] Camellia oleifera Usually hexaploid, with a complex genetic background (Cui et al. ., 2022; Lin et al. ., 2022). The present invention adopts the SSRSeq method for SSR genotyping, and avoids non-specific amplification interference through sequencing, sequence assembly and alignment, ensuring that the amplified product is the real target product (Cui and others ., 2022;Li and al., 2021). In addition, it can accurately identify the number of repetitions of SSR motifs (allele dosage) and improve the identification ability of SSR markers. For example, marker Coz11161 showed 6, 7, and 8 TAG sequence repeats in XL27 and ZL2, and the allele number ratio in XL27 was 3:2:1, while in ZL2 it was 4:1:1 ( Figure 7 ). Therefore, even if different varieties have the same band, SSRSeq can detect the dosage of each allele and thus distinguish the corresponding varieties, which is difficult to achieve with traditional polyacrylamide gel electrophoresis and fluorescent SSR technology. Traditional SSR detection methods can only provide two-dimensional information (band size, presence or absence of bands), while the SSRSeq method can provide three-dimensional information (band size, presence or absence of bands and allele dosage). Therefore, we call the molecular fingerprint created using the SSRSeq method a 3D fingerprint ( Figure 6 ).

[0051] The authenticity and high purity of improved seedlings are the basis for ensuring long-term high-quality and high-yield orchards. However, it is difficult to distinguish which variety a seedling belongs to based on phenotype alone, which often results in losses when it bears fruit three to five years later. Molecular fingerprints can identify varieties at the seedling stage, thus providing a reference for growers and seedling management agencies (Iqbal and others ., 2021). A large number of studies have been conducted in agricultural and forestry crops (Harshitha and Sandal, 2022). Chen et al. (2016) used 10 fluorescent SSR markers to construct molecular fingerprints of 128 common Camellia oleifera varieties and achieved differentiation between different varieties. Yan et al. (2022) used 24 fluorescent SSR markers to distinguish five Camellia oleifera varieties and constructed molecular fingerprints of 140 Camellia oleifera germplasm resources. However, in Camellia oleifera (Chen and others ., 2016; Yan and others ., 2022), Salix psammophila (tetraploid) (Hao and others ., 2020), alleles may exist in multiple copies, making it difficult to accurately estimate allele dosage. In this paper, we used four SSR loci (two core loci Coz06080 and Coz08114 and two additional loci Coz10144 and Coz14197) to construct 3D fingerprints and molecular ID cards for 43 varieties. This method only requires one round of multiplex PCR and one SSRSeq assay, which greatly reduces the test cost, shortens the detection cycle, and improves the convenience and repeatability of variety identification. Therefore, SSRSeq technology and 3D fingerprinting may play an important role in variety identification and genetic research in plants, especially polyploid species.

[0052] 4 Conclusion This study analyzed the distribution of SSR loci in the genomes of three Camellia oleifera species and found that there were differences in SSR occurrence frequency and motif types among species. C. oleifera var Nanyongensis, a total of 14,854 pairs of SSR primers were designed, and 132 new SSR markers were successfully developed (Tables 5 to 7). Based on the SSRSeq technology, 30 SSR markers were used to carry out genetic analysis of the test samples. The number of alleles at these loci ranged from 4 to 22, and the PIC values ​​ranged from 0.07 to 0.90. Cluster analysis showed that some varieties from the same geographical area had a close genetic distance. Four SSRs (Coz06080, Coz08114, Coz10144 and Coz14197) were able to identify 43 hexaploid Camellia oleifera varieties, and 43 variety-specific 3D molecular fingerprints and molecular ID cards were further constructed. The present invention is expected to promote variety identification, seedling purity assessment and genetic research of Camellia oleifera.

[0053] Table 5 Information of 132 newly developed genome SSR markers

[0054] Table 6 Information of 132 newly developed genome SSR markers

[0055] Table 7 Information of 132 newly developed genome SSR markers

[0056] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

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Claims

1. The core SSR molecular markers of the Camellia oleifera genome are characterized by: The numbers are Coz06080 and Coz08114; The base repeating unit corresponding to marker Coz06080 is (TATG)6, and the primers used to amplify marker Coz06080 are shown in SEQ ID NO: 1-2; The base repeating unit corresponding to marker Coz08114 is (TTC)8, and the primers used to amplify marker Coz08114 are shown in SEQ ID NO:3-4.

2. SSR molecular identity card of Camellia oleifera varieties, characterized by: It is a combination of SSR molecular markers of the Camellia oleifera genome, numbered Coz06080, Coz08114, Coz10144 and Coz14197; The base repeating unit corresponding to marker Coz10144 is (TTTC)7, and the primers used to amplify marker Coz10144 are shown in SEQ ID NO:5-6; The base repeating unit corresponding to marker Coz14197 is (TTC)5, and the primers used to amplify marker Coz14197 are shown in SEQ ID NO:7-8; Among them, the marks Coz06080 and Coz08114 are the same as the marks Coz06080 and Coz08114 described in claim 1.

3. A primer for amplifying the marker according to claim 1, characterized in that: The primers used to amplify marker Coz06080 are shown in SEQ ID NOs: 1-2, and the primers used to amplify marker Coz08114 are shown in SEQ ID NOs: 3-4.

4. The primers used to amplify the SSR molecular ID card according to claim 2 are characterized in that: The primers used to amplify marker Coz06080 are shown in SEQ ID NOs: 1-2, the primers used to amplify marker Coz08114 are shown in SEQ ID NOs: 3-4, the primers used to amplify marker Coz10144 are shown in SEQ ID NOs: 5-6, and the primers used to amplify marker Coz14197 are shown in SEQ ID NOs: 7-8.

5. A detection reagent or kit containing the primers according to claim 3 or 4.

6. Use of the marker according to claim 1, the SSR molecular ID card according to claim 2, the primer according to claim 3 or 4, or the detection reagent or kit according to claim 5 in the identification, improvement or molecular marker-assisted breeding of Camellia oleifera germplasm resources.

7. A method for constructing a Camellia oleifera SSR fingerprint, characterized in that: The following steps are involved: 1) Extracting genomic DNA from the tea oil plant to be tested; 2) using the extracted DNA as a template, and performing PCR amplification using the primers described in claim 3 or 4; 3) Analyze PCR amplification products and construct SSR fingerprint of Camellia oleifera; The oil tea includes common oil tea ( Camellia oleifera var Nanyongensis), narrow-leaved tea ( C. lanceoleosa ) and Zhejiang safflower tea ( C. chekiangoleosa ).

8. The method according to claim 7, characterized in that Step 2) The PCR amplification system is: 1 μL template DNA, 0.4 μL each of 10 μM upstream and downstream primers, 0.4 μL of 10 μM dNTPs, 2 μL of 10× buffer and 0.5 U of Taq enzyme, and make up to 20 μL with ddH2O.

9. The method according to claim 7, characterized in that: Step 2) The PCR reaction program was as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, for 30 cycles; and finally extension at 72°C for 10 minutes.

10. The method according to any one of claims 7 to 9, characterized in that: Step 3) Detect and analyze the number of repetitions of the base repeating unit in the amplified product, establish information data on the number of repetitions of SSR markers of each variety, and the obtained information list is the SSR fingerprint map of the oil-tea variety.

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