Identification of Lignum Aquilariae Resinatum

Abstract

The present application relates to the identification of Aquilaria sinensis, and provides a SNP molecular marker for identifying Aquilaria sinensis and application thereof, and a nucleotide sequence, a probe, a kit, a preparation method and a detection method for identifying Aquilaria sinensis based on the SNP molecular marker. The use of these molecular biology techniques can efficiently, quickly, sensitively and accurately identify Aquilaria sinensis, and has great significance for port species inspection work and variety resource identification, germplasm resource protection.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically, to SNP molecular markers for the identification of Aquilaria sinensis and their applications. Background Technology

[0002] Aquilaria sinensis (Lour.) Spreng., commonly known as native agarwood, belongs to the phylum Angiospermum, class Dicotyledonae, order Myrtiflorae, family Thymelaeaceae, and genus Aquilaria. It is found in Guangdong, Hainan, Guangxi, and Fujian provinces of China, preferring to grow in sparse forests in sunny locations along roadsides and in low-altitude mountains and hills. The resin accumulated after the old stems are injured is commonly known as agarwood, which can be used as a raw material for fragrances and is also a highly effective medicine for treating stomach ailments. The bark fibers are flexible, white, and fine, making them suitable for high-grade paper and rayon. Aromatic oils can be extracted from the xylem, and the flowers can be used to make extracts. Morphological identification is a commonly used method for identifying native agarwood, but shape characteristics are easily affected by habitat, climate, and physiological conditions, often leading to subjective biases in identification. Therefore, accurate identification based solely on morphological differences is difficult.

[0003] In recent years, molecular biology techniques have been used for species identification in some rare species and in inspection and quarantine. How to use molecular biology techniques to establish an efficient, rapid, sensitive and accurate identification method will be one of the key technologies in the field of agarwood identification. Summary of the Invention

[0004] The first objective of this invention is to provide an SNP molecular marker for identifying Aquilaria sinensis, wherein the SNP molecular marker is a C at the 65th base of the sequence shown in SEQ ID NO.1, and if the 65th base is C, it is identified as Aquilaria sinensis.

[0005] The second objective of this invention is to apply the above-mentioned SNP molecular markers to the identification of Aquilaria sinensis.

[0006] The third objective of this invention is to provide a nucleotide sequence for identifying Aquilaria sinensis, the nucleotide sequence comprising SEQ ID NO.1, or cDNA or mRNA corresponding to SEQ ID NO.1, wherein the 65th base of the SEQ ID NO.1 sequence is C.

[0007] The fourth objective of this invention is to provide a probe comprising at least 10 consecutive nucleotides corresponding to the sequence of SEQ ID NO.1 and wherein the 65th base of the sequence is C.

[0008] More specifically, the probe sequence is selected from the sequence shown in SEQ ID NO.6.

[0009] The fifth objective of this invention is to provide a kit for identifying Aquilaria sinensis, comprising a reaction system for detecting nucleotides of SEQ ID NO.1 or containing the sequence of SEQ ID NO.1, said reaction system comprising one or more primers and / or probes for specifically detecting the presence or absence of a C base at position 65 of the sequence of SEQ ID NO.1.

[0010] The reaction system of the kit can be either a fluorescent PCR reaction system or a sequencing reaction system.

[0011] The primers or probes may be selected from those defined in SEQ ID NO.2 to 6.

[0012] The kit also includes a positive control, a negative control, and a blank control. In a specific example, DNA from *Aquilaria sinensis* leaves was used as a positive control, DNA from *Aquilaria agallocha* leaves was used as a negative control, and sterile double-distilled water was used as a blank control.

[0013] The sixth objective of this invention is to provide a method for preparing a kit for identifying Aquilaria sinensis, comprising the step of preparing a reaction system, wherein the reaction system contains one or more primers and / or probes for specifically detecting the presence or absence of a C base at position 65 of the sequence SEQ ID NO.1.

[0014] The present invention also provides a method for identifying Aquilaria sinensis, including extracting DNA from the plant to be tested and performing a PCR reaction, and further including a step of detecting whether the 65th base of the sequence SEQ ID NO.1 is C.

[0015] The presence or absence of C at position 65 of SEQ ID NO.1 can be determined by either fluorescent PCR or sequencing.

[0016] The aforementioned SNP molecular markers and applications, as well as the nucleotide sequences, probes, kits, preparation methods, and detection methods used to identify Aquilaria sinensis, can all be used to identify Aquilaria sinensis from Aquilaria sinensis, Aquilaria agallocha, or Aquilaria agallocha.

[0017] This invention establishes a molecular biological technique for identifying Aquilaria sinensis based on SNP sites, which can quickly distinguish Aquilaria sinensis from closely related species such as Aquilaria agallocha and Aquilaria yunnanensis. This is of great significance for species inspection at ports of entry, identification of varietal resources, and protection of germplasm resources. Attached Figure Description

[0018] Figure 1 This is a sequence comparison diagram of Aquilaria sinensis, Aquilaria agallocha, and Aquilaria yunnanensis.

[0019] Figure 2This is a fluorescent PCR amplification curve of Aquilaria sinensis.

[0020] Figure 3 This is a fluorescent PCR amplification curve of Malaysian agarwood.

[0021] Figure 4 This is a fluorescent PCR amplification curve of Yunnan agarwood. Detailed Implementation

[0022] The present invention will now be described in detail with reference to specific embodiments. These specific embodiments are merely limited enumerations without departing from the spirit of the present invention, and do not exclude other specific implementation schemes that can be generated by combining prior art with the present invention by those skilled in the art.

[0023] The following examples were conducted under standard experimental conditions, such as the operating procedures described in Sambrook et al. Molecular Cloning Laboratory Manual (New York: Gold Spring Harbor Laboratory Press, 1989), or according to the experimental conditions recommended by the manufacturer.

[0024] Example 1: Obtaining SNP molecular markers from Aquilaria sinensis

[0025] The 5.8, 18, and 28S rRNA genes on rDNA exhibit high conservation, meaning they possess extensive heterologous homology. Because the ITS region does not incorporate mature ribosomes, ITS fragments experience very little natural selection pressure during evolution, thus tolerating greater variation. In most eukaryotes, ITS exhibits extremely broad sequence polymorphism; even closely related species can show differences in their ITS sequences, revealing recent evolutionary characteristics. This characteristic makes ITS suitable for molecular identification of species and for analyzing phylogenetic relationships with significant differences between or within species. Since ITS sequence analysis substantially reflects base pair differences between genera and species, and because ITS sequences are small and easy to analyze, they are now widely used in phylogenetic studies between different species or closely related genera.

[0026] 1. Based on the ITS genes publicly available in the NCBI database, corresponding primers were designed for PCR amplification. The upstream primer (ITS): 5'-CGTAACAAGGTTTTCGTAGGTGAAC-3' (SEQ ID NO.2), the downstream primer (ITS): 5'-GCTACGTTCTTCATCGAT-3' (SEQ ID NO.3), and the following PCR reaction system and procedure were used. Genomic DNA from three Aquilaria species (Aquilaria sinensis (Lour.) Spreng.), Aquilaria malaccensis, and Aquilaria yunnanensis SCHuang) were used as templates for PCR amplification. The amplified products were detected by 1.5% agarose gel electrophoresis and sequenced after passing the UV gel imaging system.

[0027]

[0028]

[0029] Among them, Vazyme AceTaq DNA enzyme (catalog number p401-d1 250U)

[0030] The genomic DNA of the Aquilaria species was extracted using the Plant Genomic DNA Extraction Kit (Koning) from Hangzhou Baimai Biotechnology Co., Ltd. TM Use the xylem genomic DNA extraction kit (catalog number F2612) and follow the kit instructions. Store the extracted DNA at -20°C for later use.

[0031] 2. Sequence alignment and development of species-specific molecular markers

[0032] The ITS gene sequences of the three Aquilaria species obtained in step 1 were used to perform sequence alignment using DNAMAN software to identify suitable SNP sites. The sequence alignment results are as follows: Figure 1 As shown, analysis revealed that the 65th base of the sequence shown in SEQ ID NO.1 within the Aquilaria sinensis ITS gene is C, while it is T in other Aquilaria species. Therefore, the 65th base C in the sequence shown in SEQ ID NO.1 is the SNP site of Aquilaria sinensis (the position shown in the box in the sequence). The gene sequence of SEQ ID NO.1 is:

[0033]

[0034] Example 2: Application of SNP molecular markers in *Aquilaria sinensis* for identification by fluorescent PCR amplification.

[0035] This embodiment, based on the obtained SNP sites in *Aquilaria sinensis*, designed specific upstream primer F, downstream primer R, and probes for the gene sequences containing the SNP sites using PrimerPremier 5.0. The primers and probes were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. Fluorescent PCR amplification and detection were performed on *Aquilaria sinensis*, *Aquilaria agallocha*, and *Aquilaria yunnanensis* samples using the specific primers and probes. Specifically, the upstream primer F, downstream primer R, and probe are as follows:

[0036] Upstream primer F: 5'-GATGCGCGCTATGATTGAAC-3' (SEQ ID NO.4)

[0037] Downstream primer R: 5'-CGGTGTATGCCATGATGCA-3' (SEQ ID NO.5)

[0038] Probe: 5'-AGGGCATGATGCGCATGATGCT-3' (SEQ ID NO.6)

[0039] In one specific example, the 5' end of the probe is labeled with FAM reporter fluorescent dye, and the 3' end is labeled with BHQ1 quencher fluorescent dye.

[0040] Using genomic DNA from three Aquilaria species as templates, a 20 μL PCR reaction system included:

[0041]

[0042]

[0043] The amount of each reagent in the reaction system can be adjusted appropriately according to the specific situation or different total reaction volume. Alternatively, a commercially available real-time fluorescence PCR detection kit can be used to prepare the system. The reaction system should be prepared in accordance with the instructions.

[0044] Fluorescent PCR reaction procedure:

[0045]

[0046] The procedure includes the following steps: 1) Extracting genomic DNA from the plant to be tested; 2) Using the genomic DNA as a template, amplifying the ITS gene of *Aquilaria sinensis* by PCR using primers and probes; 3) Detecting the PCR amplification products using fluorescent PCR. If the amplification curve has a standard S-shaped curve and Ct≤36, then the plant to be tested is *Aquilaria sinensis*. *Aquilaria sinensis* leaf DNA was used as a positive control, *Aquilaria agallocha* leaf DNA as a negative control, and sterile double-distilled water as a blank control.

[0047] When the results of the negative, positive, and blank control tests in molecular biology are normal, and the tested sample shows obvious fluorescence amplification, the sample is identified as Aquilaria sinensis.

[0048] The agarwood, Malaysian agarwood, and Yunnan agarwood samples used in Example 2 are also a different group of samples from Example 1, identified using existing methods. The test results are as follows: Figures 2 to 4 As shown, only the fluorescent PCR amplification curve of the *Aquilaria sinensis* sample exhibited an S-shaped curve, with Ct ≤ 36, while other *Aquilaria sinensis* samples did not show an S-shaped curve. Furthermore, the results of the negative control, positive control, and blank control were normal. This indicates that the present invention has extremely high specificity for the *Aquilaria sinensis* ITS gene SNP site, and therefore can be used for rapid identification of *Aquilaria sinensis*. sequence list <110> Hangzhou Baimai Biotechnology Co., Ltd. Zhejiang Provincial Institute of Inspection and Quarantine Science and Technology <120> Identification of Aquilaria sinensis <130> 201701 <150> 2017108138776 <151> 2017-09-11 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 103 <212> DNA <213> Aquilaria sinensis <400> 1 cagggcggtg tatgccatga tgaacggtgg tgtgtgctta gcctgccgtc gttagagcat 60 catgcgcatc atgcccttga ggtatgtttc gtggacaacc ccg 103 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <400> 2 cgtaacaagg ttttcgtagg tgaac 25 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 gctacgttct tcatcgat 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 gatgcgcgct atgattgaac 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <400> 5 cggtgtatgc catgatgca 19 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <400> 6 agggcatgat gcgcatgatg ct 22

Claims

1. A kit for identifying agarwood, comprising a reaction system for detecting nucleotides of SEQ ID NO.1 or containing the sequence of SEQ ID NO.1, characterized in that, The reaction system contains primers and probes for specifically detecting the presence or absence of a C base at position 65 relative to the sequence of SEQ ID NO.

1. The upstream primer sequence is SEQ ID NO. 4, the downstream primer sequence is SEQ ID NO. 5, and the probe sequence is SEQ ID NO.

6.

2. The reagent kit according to claim 1, characterized in that, The kit also includes a positive control, a negative control, and a blank control.

3. A method for preparing a reagent kit for identifying Aquilaria sinensis, comprising the step of preparing a reaction system for nucleotide detection, characterized in that, The reaction system comprises primers and probes for specifically detecting the presence or absence of a C base at position 65 relative to the sequence of SEQ ID NO. 1, with the upstream primer sequence being SEQ ID NO. 4, the downstream primer sequence being SEQ ID NO. 5, and the probe sequence being SEQ ID NO.

6.

4. A method for identifying Aquilaria sinensis, comprising the steps of extracting DNA from the plant to be tested and a PCR reaction, characterized in that, It also includes a step of detecting the presence or absence of a C base at position 65 relative to the sequence of SEQ ID NO.1, wherein the upstream primer sequence used in the PCR reaction step is SEQ ID NO. 4, the downstream primer sequence is SEQ ID NO. 5, and the probe sequence is SEQ ID NO.

6.

5. The method according to claim 4, characterized in that, Positive and negative controls were set up, with DNA from *Aquilaria sinensis* leaves used as a positive control and DNA from *Aquilaria agallocha* leaves used as a negative control.

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