A SNP molecular marker for identifying cinnamyl aldehyde type of lindera and primers and application thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a SNP molecular marker for identifying cinnamyl aldehyde type cortex cinnamomi, a primer thereof and application. The SNP molecular marker is located at 9 sites between the 6th and 7th exons of a gene Cbur03G002680, including the 11th site, the 146th site, the 157th site, the 176th site, the 197th site, the 206th site, the 236th site, the 238th site and the 242nd site; the nucleic acid sequence of the gene between the 6th and 7th exons is shown as SEQ ID NO. 4, the accuracy of the SNP molecular marker for identifying cinnamyl aldehyde type cortex cinnamomi is more than 98.5%, the accurate prediction rate is 84.5%, the detection level accuracy is high, and the SNP molecular marker is easy to repeat.

Description

Technical Field

[0001] This invention belongs to the technical field of molecular biology, specifically relating to an SNP molecular marker for identifying cinnamaldehyde-type Aquilaria sinensis, its primers, and applications. Background Technology

[0002] Cinnamaldehyde is an aldehyde organic compound, a yellow, viscous liquid, naturally found in essential oils such as Sri Lankan cinnamon oil, cassia bark oil, and patchouli oil. Cinnamaldehyde has antibacterial, disinfectant, and preservative properties, particularly effective against fungi, and is used in the manufacture of insecticides, mosquito repellents, refrigerator deodorizers, and preservatives. It also inhibits adrenaline and promotes glucose-lipid synthesis, making it useful in blood sugar control drugs to enhance insulin's glucose-replacing properties and prevent diabetes. Cinnamaldehyde is also commonly used as a raw material and additive in food flavorings. *Cinnamomum burmannii* (Nees & T. Nees) Blume is a species of tree in the genus *Cinnamomum* of the family Lauraceae, widely distributed in southern my country, including Guangdong, Guangxi, Hunan, Jiangxi, Fujian, Guizhou, and Yunnan. A research team recently discovered *Cinnamomum burmannii* resources in Guizhou Province, primarily composed of cinnamaldehyde, with a significantly higher relative content and oil yield in its branches and leaves than cinnamon, cassia bark, and patchouli. Due to the large market potential and significant economic benefits of cinnamaldehyde, the development of cinnamaldehyde-type Aristolochia resources has a very broad application prospect.

[0003] Previous research by the inventors of this application showed that terpenoids are the main compounds found in the branches and leaves of *Cinnamomum camphora* in South China, including borneol-type, eucalyptol-type, caryophyllene, and phytol, all existing in the form of monoterpenes, diterpenes, and sesquiterpenes. These monoterpenoids are primarily synthesized via the 2-methylerythritol-4-phosphate (MEP) pathway in the upstream plasmid and the mevalonic acid (MVA) pathway in the cytoplasm, and then further synthesized in the downstream pathway involving various terpenoid synthases (TPS). Ma et al. (2021) first discovered a TPS gene, CbTPS1, in *Cinnamomum camphora* capable of synthesizing borneol. Ma et al. (2022) also found that CbTPS1, in addition to synthesizing borneol, also participates in the synthesis of other terpenoid compounds such as limonene and β-pinene.

[0004] Therefore, in order to develop a method suitable for detecting Aristolochia resources in South China and to accurately distinguish between cinnamaldehyde-type, borneol-type, and other chemically-type Aristolochia, this invention proposes the following effective method. Summary of the Invention

[0005] To address the above problems, the present invention aims to provide an SNP molecular marker for identifying cinnamaldehyde-type Cinnamomum cassia, its primers, and its applications.

[0006] The technical content of this invention is as follows:

[0007] This invention provides an SNP molecular marker for identifying cinnamaldehyde-type Cinnamomum cassia, wherein the SNP molecular marker is located at 9 sites between exons 6 and 7 of the gene Cbur03G002680, including sites 11, 146, 157, 176, 197, 206, 236, 238 and 242.

[0008] The non-coding sequence of the gene Cbur03G002680 is a full-length sequence formed by sequentially linking the sequences shown in SEQ ID NO.1-3;

[0009] The gene nucleic acid sequence between exons 6 and 7 is shown in SEQ ID NO. 4;

[0010] When the genotypes of the nine loci are T / C (site 1), C / C (site 2), T / G (site 3), A / A (site 4), A / A (site 5), C / C (site 6), T / T (site 7), G / G (site 8), and G / G (site 9), the candidate material is cinnamaldehyde-type Aquilaria sinensis.

[0011] When the genotypes of the nine loci are T / C (site 1), C / C (site 2), T / G (site 3), T / G (site 4), T / G (site 5), T / C (site 6), T / C (site 7), T / C (site 8), and T / C (site 9), the candidate material is camphor-type Aristolochia debilis, which can be discarded.

[0012] When the genotypes of the nine loci are T / C (site 1), C / C (site 2), T / G (site 3), T / G (site 4), T / G (site 5), T / C (site 6), T / C (site 7), T / C (site 8), and T / C (site 9), other chemically typed *Aristolochia debilis* can be discarded.

[0013] Samples with 1 to 2 sites that do not meet the requirements of this screening method among the above 9 sites can also be accurately determined to be cinnamaldehyde-type osmanthus.

[0014] The present invention also provides primers for identifying cinnamaldehyde-type Cinnamomum cassia based on the SNP molecular markers, including forward primers and reverse primers for the nine sites;

[0015] The nucleic acid sequences of the forward primers for the nine sites are shown in SEQ ID NO.5, and the nucleic acid sequences of the reverse primers are shown in SEQ ID NO.6.

[0016] The present invention also provides a kit for identifying cinnamaldehyde-type cinnamonwood, the kit comprising DNA (gene Cbur03G002680), the above-mentioned primers and PCR reaction reagents.

[0017] This invention also provides the application of the above-mentioned SNP molecular markers, the primers, or the kit in the identification of cinnamaldehyde-type Cinnamomum cassia.

[0018] This invention also provides a method for identifying cinnamaldehyde-type agarwood, comprising the following steps:

[0019] 1) Select materials from the *Cinnamomum camphora* variety and extract their DNA;

[0020] 2) Using the DNA obtained in step 1) as a template, perform PCR amplification using the primers described above to obtain the PCR amplification product;

[0021] 3) After purifying the PCR amplification products obtained in step 2), collect 260bp fragments for testing, using the same identification method as above:

[0022] When the genotypes of the nine loci are T / C (site 1), C / C (site 2), T / G (site 3), A / A (site 4), A / A (site 5), C / C (site 6), T / T (site 7), G / G (site 8), and G / G (site 9), the candidate material is cinnamaldehyde-type Aquilaria sinensis.

[0023] When the genotypes of the nine loci are T / C (site 1), C / C (site 2), T / G (site 3), T / G (site 4), T / G (site 5), T / C (site 6), T / C (site 7), T / C (site 8), and T / C (site 9), the candidate material is camphor-type Aristolochia debilis, which can be discarded.

[0024] When the genotypes of the nine loci are T / C (site 1), C / C (site 2), T / G (site 3), T / G (site 4), T / G (site 5), T / C (site 6), T / C (site 7), T / C (site 8), and T / C (site 9), other chemically typed *Aristolochia debilis* can be discarded.

[0025] Samples with 1 to 2 sites that do not meet the requirements of this screening method among the above 9 sites can also be accurately identified as cinnamaldehyde-type cinnamon ash.

[0026] Preferably, the reagents used for PCR amplification in step 2) include 15 μL 2×Taq PCR Master Mix, 1.0 μL genomic DNA, 1 μL forward primer (10 pmol / μL), 1 μL reverse primer (10 pmol / μL), and 12 μL ddH2O;

[0027] Preferably, the PCR amplification in step 2) uses the following reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 30 sec, 35 cycles; 72℃ final extension for 5 min; 16℃ for 1 min.

[0028] The applicant of this invention, through sequencing and gene mining of the *Cinnamomum camphora* genome, identified a homologous gene, Cbur03G002680, similar to the CbTPS1 gene. Using the full-length transcriptome of *Cinnamomum camphora* at different leaf development stages, Cbur03G002680 was found to be involved in the synthesis of several terpenoids, including borneol (Hou et al. 2023). It was also discovered that this gene may be involved not only in the synthesis of borneol but also in the synthesis of diterpenes and sesquiterpenes. Cinnamaldehyde synthesis mainly occurs through the metabolic pathway of plant flavonoids, specifically through the synthesis of cinnamyl-CoA from cinnamic acid followed by cinnamaldehyde. The key to distinguishing *Cinnamomum camphora* resources of the cinnamaldehyde type from other terpene-type chemotypes is to obtain terpene synthases with differentially encoded sequences.

[0029] The beneficial effects of this invention are as follows:

[0030] The SNP molecular markers for identifying cinnamaldehyde-type *Aquilaria sinensis* of this invention are obtained by screening SNP sites on the *Aquilaria sinensis* Cbur03G002680 gene. The accuracy rate for identifying cinnamaldehyde-type *Aquilaria sinensis* is above 98.5%, with a precise prediction rate of 84.5%, demonstrating high accuracy and reproducibility. By screening single nucleotide polymorphism (SNP) sites on the *Aquilaria sinensis* Cbur03G002680 gene from different provinces in southern China, and combining this with the applicant's results of trace extraction of essential oil chemical components, a precise and rapid molecular evaluation technology for high borneol content was developed (CN202011539774.3 A method for extracting organic compounds from plant tissues containing borneol). The detection method described in this invention is applicable to the detection of *Aquilaria sinensis* resources throughout South China, accurately distinguishing between cinnamaldehyde-type, borneol-type, and other chemically-type *Aquilaria sinensis*, thus possessing broad-spectrum applicability. The detection cost of this method is significantly lower than that of conventional gas chromatography or gas chromatography-mass spectrometry. This invention is an effective method for efficiently mining Cinnamomum cassia resources and obtaining special Cinnamomum cassia chemical type resources. Attached Figure Description

[0031] Figure 1 A schematic diagram showing the chemical composition of *Cinnamomum camphora* samples collected from seven provinces in southern China and the distribution of different populations.

[0032] Figure 2 The amplification electrophoresis gel images described in the examples (subjects Site 1-9);

[0033] Figure 3 The results of genome-wide association analysis (GWAS) were conducted on the whole-genome resequencing data of 140 *Cinnamomum camphora* specimens collected in South China and the cinnamaldehyde content. Detailed Implementation

[0034] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0035] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0036] Example 1

[0037] Screening and validation of SNP molecular markers related to borneol content in Cinnamomum camphora.

[0038] 1. Collection of Yinxiang materials

[0039] From 2018 to 2021, a survey of Cinnamomum camphora germplasm resources was conducted in Guangxi, Guizhou, Jiangxi, Hunan, Yunnan, Fujian, Guangdong, and other regions, collecting a total of 141 Cinnamomum camphora germplasm resources. Using a micro-extraction method and GC-MS detection, the inventors' self-developed metabolite micro-extraction technology (patent CN202011539774.3, a method for extracting organic compounds from plant tissues containing borneol), combined with a gas chromatography-mass spectrometry (GC / MS) system, was used to measure and detect the chemical types and volatile metabolite content of different Cinnamomum camphora germplasm resources, achieving an accuracy of 99%.

[0040] The chemical type distribution of the 141 collected *Cinnamomum camphora* samples is as follows: Figure 1 As shown, the Cinnamomum cassia populations in Guangdong, Guangxi, Hunan, Fujian and Jiangxi are dominated by borneol-type Cinnamomum cassia (with a relative borneol content of 14.8% to 49.6%), while the Cinnamomum cassia populations in Yunnan and Guizhou are dominated by cinnamaldehyde-type Cinnamomum cassia (with a relative borneol content close to 0%).

[0041] 2. Screening of SNPs related to cinnamaldehyde-type Aristolochia debilis

[0042] The 141 collected samples of *Aquilaria sinensis* were divided into two groups (grouped as needed): a screening group of 70 samples and a verification group of 71 samples. Each sample contained 30% cinnamaldehyde-type *Aquilaria sinensis*, 30% borneol-type *Aquilaria sinensis*, and 40% other chemically-type *Aquilaria sinensis* (including eucalyptol-type, caryophyllene-type, chlorophyll-type, and geraniol B-type, etc.).

[0043] The first screening group of 70 resources was selected using next-generation sequencing technology, and the specific method is as follows:

[0044] DNA was randomly fragmented into approximately 300 bp fragments using ultrasonic disruption (or enzyme digestion). These fragments underwent end repair, 3' end A addition, sequencing adapter pairing, purification, and PCR amplification to construct the sequencing library. After quality control, the library was sequenced using the Illumina platform. Library construction and sequencing were performed at Beijing BaiMike Biotechnology Co., Ltd. After sequencing data was processed, the raw data underwent quality control according to specific standards, removing sequences containing adapters and sequences with more than 50% low-quality base pairs. After removing low-quality and adapter sequences, the raw data was compared with the coding sequence (CDS, whose nucleic acid sequence is the full-length sequence formed by sequentially linking the sequences shown in SEQ ID NO. 1-3) of the gene Cbur03G002680 in the assembled Cinnamomum camphora reference genome of the inventors using Bowtie2 software in the Geneious software platform. The parameters for the consistent sequences were set to highest sensitivity / slow, and the consistent sites required that the number of aligned bases be at least 90%. The full genome sequence matrix of Cbur03G002680 of 71 samples was obtained using MAFFT software (Score matrix: 200PM / k=2). Then, the SNPs of the 71 sample sequences were found using the Finder Variation / SNP module in the Geneious software platform, and the parameter was set to the minimum variation frequency of 30% for each site.

[0045] Finally, based on the detection results obtained by the essential oil micro-extraction method described above, nine SNPs were found in the gene sequence (295 bp, whose nucleic acid sequence is shown in SEQ ID NO.4) located between exons 6 and 7. These SNPs are able to distinguish between cinnamaldehyde, borneol and other chemical types: they appear sequentially at site 111 (Site 1), site 146 (Site 2), site 157 (Site 3), site 176 (Site 4), site 197 (Site 5), site 206 (Site 6), site 236 (Site 7), site 238 (Site 8) and site 242 (Site 9).

[0046] Detailed explanations are shown in Table 1:

[0047] Table 1. Nine functional sites closely associated with the content traits of cinnamaldehyde, borneol, and other chemotypes.

[0048]

[0049] 3. Functional testing of SNPs in Cinnamaldehyde-type Cinnamaldehyde

[0050] This invention does not limit the method for extracting DNA from the stems and leaves of *Cinnamomum camphora*; either the kit method or the CTAB method can be used. In this study, the genomic DNA extraction kit (NanoMagBio) using magnetic beads was used to extract genomic DNA from the materials to be selected. After extracting DNA from the second group of 71 samples using this method, gel electrophoresis was performed first. The detection parameters were as follows: agarose gel concentration 1%, voltage 120V, electrophoresis time: 20min. 2μL of DNA sample was added to 2μL of 6×Loading Buffer. After electrophoresis, the gel was placed in a gel imaging analyzer for gel imaging. The main band was required to be bright and clear, without any dragging, and the main band size was approximately 10kb. Then, absorbance was measured. 2μL of DNA sample was taken, and the nucleic acid concentration was detected using a NanoDROP 8000 ultra-micro spectrophotometer. The DNA sample concentration (ng / μL) was required to be ≥30ng / μL, and the A260 / A280 value was required to be within the range of 1.8–2.0. After DNA extraction from 71 samples was qualified, PCR amplification of the sequence shown in SEQ ID NO.4 was carried out using Cinnamomum camphora DNA as a template and the primers were used to obtain PCR amplification products.

[0051] The amplification primers used are shown in Table 2:

[0052] Table 2 PCR amplification primers

[0053]

[0054] The PCR amplification system used is shown in Table 3:

[0055] Table 3. Reagent ratio information and PCR reaction conditions

[0056]

[0057] To ensure the specificity of PCR amplification, after PCR amplification, 2 μL of PCR product was taken for agarose gel electrophoresis (1% concentration). The specificity of the amplified products for each sample was determined by the banding pattern of the PCR products. Figure 2 (a. Spotting sequence ZZA02, FJNJ01, FJNJ03, FJFD05, FJSX05; b. Marker) as shown, corresponding to Figure 2 a) Results of 5 bands from left to right.

[0058] After PCR amplification, the obtained PCR amplification products are preferably purified, and 260bp amplification fragments are collected for sequencing. The sequencing is preferably bidirectional sequencing.

[0059] When the genotypes obtained from sequencing are T / C (site 1), C / C (site 2), T / G (site 3), A / A (site 4), A / A (site 5), C / C (site 6), T / T (site 7), G / G (site 8), and G / G (site 9), the candidate material is cinnamaldehyde-type Aquilaria sinensis.

[0060] When the genotypes are T / C (site 1), C / C (site 2), T / G (site 3), T / G (site 4), T / G (site 5), T / C (site 6), T / C (site 7), T / C (site 8), and T / C (site 9), the candidate material is camphor-type Aquilaria sinensis, which can be discarded.

[0061] When the genotypes are T / C (site 1), C / C (site 2), T / G (site 3), T / G (site 4), T / G (site 5), T / C (site 6), T / C (site 7), T / C (site 8), and T / C (site 9), other chemically derived *Aquilaria sinensis* materials can be discarded.

[0062] Samples with 1 to 2 sites that do not meet the requirements of this screening method among the above 9 sites can also be accurately determined to be cinnamaldehyde-type osmanthus.

[0063] The above breeding methods can help to quickly detect cinnamaldehyde-type Aquilaria sinensis, thus contributing to the development of the woody essential oil industry in South China.

[0064] For the functional testing of SNPs, GWAS analysis was further performed on all 141 selected *Cinnamomum camphora* samples. Using the EMMAX model, a total of 1043 SNPs related to the relative content of dextrorotatory borneol were obtained. The results are as follows: Figure 3 The three points circled in the diagram represent the nine amplification sites involved in this invention.

[0065] 4. Practical testing of high-quality resources of cinnamaldehyde-type Aquilaria sinensis.

[0066] Based on the DNA extraction and PCR amplification methods mentioned above, PCR amplification and sequencing were performed on the third group of 52 samples of *Cinnamomum camphora* collected from seven provinces in South China. Simultaneously, the relative content of the essential oil in *Cinnamomum camphora* leaves was obtained using the essential oil micro-extraction method and GC-MS detection mentioned above as the detection method for this molecular marker. The results are shown in the table below:

[0067] Table 4. Detection results of cinnamaldehyde-type agarwood using molecular markers

[0068]

[0069]

[0070]

[0071] As shown in Table 4, the evaluation results showed that 60 results were completely correct, 10 results were partially correct, and 1 result was incorrect. It can be seen that the accuracy of the results obtained by using the SNP molecular marker and corresponding evaluation method described in this invention is over 98.5%, the accurate prediction rate is 84.5%, the detection level accuracy is high, and it is easy to repeat.

Claims

1. The use of a SNP molecular marker for identifying a cinnamyl aldehyde type of Cortex Magnoliae Officinalis, characterized in that, The SNP molecular marker is 9 loci including loci 111, 146, 157, 176, 197, 206, 236, 238 and 242 located between the 6th and 7th exon of the gene Cbur03G002680 ​ the gene Cbur03G002680 the nucleotide sequence between the 6th and 7th exon of the gene of SEQ ID NO. 4; When the genotypes of the 9 loci are T / C, C / C, T / G, A / A, A / A, C / C, T / T, G / G and G / G respectively, the candidate material is cinnamyl cinnamic aldehyde type of Cinnamomum burmanii.

2. The use of a primer pair based on the SNP molecular marker of claim 1 for identifying cinnamyl aldehyde type of Cortex Magnoliae Officinalis, characterized in that, In the primer pair, the nucleotide sequence of the forward primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.

6.

3. An application of a reagent kit in the identification of cinnamaldehyde-type Aquilaria sinensis, characterized in that, The kit comprises the DNA of the gene of claim 1, the primer pair of claim 2 and PCR reaction reagents. Cbur03G002680 The kit comprises the DNA of the gene of claim 1, the primer pair of claim 2 and PCR reaction reagents.

4. A method of identifying cinnamaldehyde-type Cortex Cinnamomi, characterized by, The method comprises the following steps: 1) selecting Cinnamomum burmanii variety materials and extracting DNA therefrom; 2) using the DNA obtained in step 1) as a template and adopting the primer pair in claim 2 to perform PCR amplification to obtain a PCR amplification product; 3) purifying the PCR amplification product obtained in step 2), collecting the amplified fragments and performing sequencing, and judging whether the material is cinnamyl cinnamic aldehyde type Cinnamomum burmanii according to the genotype in claim 1.

5. The method for identifying cinnamaldehyde-type Cortex Linderae according to claim 4, characterized in that, The reagents used in the PCR amplification in step 2) comprise 15 μL 2×Taq PCR Master Mix, 1.0 μL genomic DNA, 1 μL forward primer with a concentration of 10 pmol / μL, 1 μL reverse primer with a concentration of 10 pmol / μL and 12 μL ddH2O.

6. The method for identifying cinnamaldehyde-type Cortex Linderae according to claim 4, characterized in that, The reaction program used in the PCR amplification in step 2) is 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 30 sec, 35 cycles, 72℃ final extension for 5 min, 16℃ for 1 min.

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