Application of medicinal plant dictamnus dasycarpus chloroplast genome in identifying dictamnus dasycarpus variety
By assembling the chloroplast genome of Dictamnus and constructing the phylogenetic tree of Rutaceae, the problem of accuracy in identification and classification of Dictamnus plants was solved, and the accurate identification and phylogenetic status analysis of Dictamnus varieties were achieved, supporting the protection and utilization of germplasm resources.
Patent Information
- Application Number
- CN202311802543.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-22
- Publication Date
- 2025-09-26
AI Technical Summary
Existing technologies make it difficult to accurately identify and classify Dictamnus plants, which affects their development and utilization, and traditional classification methods have deviations.
By assembling the chloroplast genome of Dictamnus chinensis and performing phylogenetic analysis, the chloroplast genome was used as a super DNA barcode for Dictamnus cultivars. By combining molecular systematics and phylogenetic genomics methods, a phylogenetic tree of Rutaceae was constructed to identify Dictamnus cultivars.
It provides more accurate identification of Dictamnus varieties and analysis of phylogenetic status, supports the protection and utilization of Dictamnus germplasm resources, and improves the accuracy of species identification and breeding.
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Figure CN120700175A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of gene technology, and in particular relates to an application of a chloroplast genome of a medicinal plant Dictamnus in variety identification and reconstruction of a Rutaceae phylogenetic tree. Background Art
[0002] Dictamnus dasycarpus Turcz. is a perennial herb belonging to the genus Dictamnus in the Rutaceae family. It is primarily found in northern my country. Dictamnus plants grow 30 to 90 cm tall, with a compact, neat shape. The plant possesses a distinctive fragrance, with leaves resembling a lemony scent. Its pinnately compound leaves are lustrous and emerald green, making it an ornamental plant. The root bark of Dictamnus dasycarpus Turcz. has the properties of clearing heat and dampness, dispelling wind and detoxifying. Research has shown that Dictamnus dasycarpus not only has medicinal value but also possesses health and beauty benefits. Rutaceae plants are widely distributed worldwide. In China alone, there are a total of 126 species native to and introduced from China, primarily found in southwestern and southern my country. Traditional taxonomic methods use morphological markers, pollen morphology, or reproductive characteristics to determine the relationships of Rutaceae plants. Other methods use differential chemical compounds to classify Rutaceae plants. However, due to long-term introductions and cross-pollination, these plants can lead to biased classification results, hindering the development and utilization of Rutaceae plants.
[0003] Chloroplasts are photosynthetic organelles in green plants and are found in plants, algae, and some protists. Electron microscopic observations have revealed the presence of DNA filaments in Chlamydomonas chloroplasts, demonstrating that chloroplasts possess autonomous genetic material. Chloroplast DNA is a double-stranded circular molecule that has been shown in most higher plants to have a highly conserved tetrad structure, consisting of two inverted reapet sequences (IRs), a large single copy region (LSC), and a small single copy region (SSC). Compared to nuclear genomes, chloroplasts have advantages such as a smaller genome size, no genetic recombination issues, and a moderate evolutionary rate. Chloroplast genome sequences between plants can be used to establish phylogenetic relationships between species, facilitating species identification and classification.
[0004] Therefore, assembling the chloroplast genome of Dictamnus and analyzing its phylogenetic status are of great significance for research on its identification and utilization, new cultivar protection, phylogeny, and breeding. Currently, research on the chloroplast genome of Dictamnus is still lacking, and using the plant chloroplast genome can provide more accurate evidence for the classification and identification of Dictamnus germplasm resources. Summary of the Invention
[0005] The purpose of the present invention is to provide an application of the chloroplast genome of the medicinal plant Dictamnus dasyphylus in variety identification and in reconstructing the phylogenetic tree of the Rutaceae family, which can analyze the phylogenetic status of Dictamnus dasyphylus in the Rutaceae family, identify Dictamnus dasyphylus varieties, and provide technical support for the protection of Dictamnus dasyphylus germplasm resources and species identification.
[0006] To solve the above-mentioned technical problems, the present invention is achieved as follows:
[0007] One of the purposes of the present invention is to provide an application of a chloroplast genome of a medicinal plant Dictamnus salsa in identifying Dictamnus varieties, wherein the nucleotide sequence of the chloroplast genome of Dictamnus salsa is shown as SEQ ID NO.1.
[0008] A second object of the present invention is to provide an application of the chloroplast genome of the medicinal plant Dictamnus serrata in reconstructing the phylogenetic tree of Rutaceae.
[0009] The method for identifying the above-mentioned medicinal plant Dictamnus species comprises the following steps:
[0010] (1) The total DNA of medicinal plant Dictamnus dasyphylla samples was extracted using the CTAB method;
[0011] (2) Using the whole genome shotgun method, a 350 bp library was constructed from the total DNA of the sample obtained in step (1), and the library was subjected to double-end sequencing using an Illumina NovaSeq sequencer;
[0012] (3) Low-quality data were filtered using SOAPnuke v1.3.0 software to obtain valid data;
[0013] (4) Assembly and assembling of the chloroplast genome of the medicinal plant Dictamnus;
[0014] (5) The assembled sample medicinal plant Dictamnus chloroplast genome nucleotide sequence was compared with the chloroplast genome nucleotide sequence of Rutaceae plant Rutaecarpa. When the similarity index reached above 99.9%, it was determined to be the medicinal plant Dictamnus chloroplast.
[0015] Furthermore, in step (3), the filtering criteria are to remove reads with a quality value ≤ 5 and a base number of 50%, remove reads containing adapter contamination, and remove reads with more than 5% N base content.
[0016] Furthermore, the steps of splicing and assembling the chloroplast genome of the medicinal plant Dictamnus in step (4) are as follows:
[0017] a. GetOrganelle v1.7.5 software was used for genome assembly. The chloroplast genome of the closely related reference plant Rutaecarpa in the NCBI database was used to perform blastn (version: BLAST 2.2.30+; parameter: -evalue 1×10 -5 ) comparison;
[0018] b. Use Gapcloser (Version: 1.12) to fill the gaps in the N sequence;
[0019] c. Use the annotation software CPGAVAS2 to perform gene annotation and draw the physical map of the Dictamnus chloroplast genome.
[0020] Statistics of the genome characteristics of white chloroplasts:
[0021] (1) The relative synonymous codon usage of 87 protein-coding gene sequences in the chloroplast genome of Dictyophore was statistically analyzed using Codon W 1.4.2 software. The amino acid codons were used as the horizontal axis and the RSCU value was used as the vertical axis. The ggplot software was used for visualization.
[0022] (2) The Shuffle-LAGAN model in the mVISTA online tool was used to align, annotate, and visually compare the chloroplast genome of Dictamnus dasyphylla with the chloroplast genomes of different Rutaceae species.
[0023] (3) Chloroplast genome data of different Rutaceae plants were downloaded from the NCBI database. The IRSCOPE online tool was used to analyze and compare the gene types and gene distribution in the IR / SC boundary region of Rutaceae plants to determine whether there was expansion or contraction of the genome boundary.
[0024] The present invention discloses the chloroplast genome of the medicinal plant Dictamnus for the first time, applies it to the phylogenetic study of Dictamnus, and analyzes the phylogenetic position of Dictamnus in the Rutaceae family.
[0025] This study used molecular systematics and phylogenetic genomics methods to establish the phylogenetic relationships of Rutaceae species through chloroplast genomes. On this basis, the whole chloroplast genome was used as a super DNA barcode for the Dictamnus genus to identify Dictamnus varieties, providing technical support for the protection of Dictamnus germplasm resources and species identification. It is of great significance for the breeding of typical Dictamnus plants and the evaluation, protection and utilization of genetic diversity. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The present invention will be described in detail below through specific examples. These examples are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art. As mentioned throughout the specification and claims, "including" or "comprising" is an open-ended term and is interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be determined by the appended claims. Unless otherwise specified, the various reagents and materials used in the present invention can be purchased from the market.
[0027] Figure 1 This is the annotated map of the chloroplast genome of Dictamnus edulis;
[0028] Figure 2 This is the phylogenetic tree of Rutaceae based on chloroplast genomes;
[0029] Figure 3 is the relative synonymous codon usage of the dill chloroplast genome;
[0030] Figure 4 It is the genome structure map of 8 species of Rutaceae;
[0031] Figure 5 It is a visual comparison of chloroplast genomes of seven species of Rutaceae;
[0032] Figure 6 This is a comparison map of the IR / SC boundaries of the chloroplast genomes of Rutaceae plants. DETAILED DESCRIPTION
[0033] 1. Materials and Methods
[0034] 1. Plant Total DNA Extraction
[0035] The present invention adopts the CTAB method to extract the total DNA of the plant sample and stores the extracted DNA in a -20°C refrigerator for future use.
[0036] 2. Total DNA Sequencing Method
[0037] The total DNA sample was mechanically sheared (ultrasound) to fragment the DNA, and then the fragments were purified, end-repaired, 3′-end A added, and sequencing adapters were connected. The fragment size was selected by agarose gel electrophoresis, and PCR amplification was performed to form a sequencing file. Ultra TM DNA Library Prep Kit for ), the constructed library was first subjected to library quality inspection, and the qualified library was sequenced using Illumina NovaSeq. The sequenced sequences were filtered for low-quality data using SOAPnuke (version: 1.3.0) software. The filtering criteria were to remove reads with an N base content exceeding 5%, remove reads with a low-quality base content (quality value ≤ 5) reaching 50%, and remove reads with adapter contamination, and finally obtain valid data (clean reads).
[0038] 3. Data Analysis Methods
[0039] GetOrganelle v1.7.5 software was used for genome assembly. The assembled genome was then aligned with the chloroplast genome of a closely related plant, Psoralea corylifolia, from the NCBI database using BLAST (version 2.2.30+; parameter: -evalue 1×10⁻¹). Gaps (including N sequences) were subsequently filled using Gapcloser (Version 1.12). GeSeq software was used to functionally annotate the Dictyophora chloroplast genome and visualize the complete map.
[0040] 4. Phylogenetic Analysis Methods
[0041] The assembled samples and the published chloroplast genome sequences of different Rutaceae species, Arabidopsis thaliana, and maize (outgroup) downloaded from GenBank were integrated. Multiple sequence alignment was performed using MAFFT software, and a phylogenetic tree was constructed using MEGA X software based on the neighbor-joining (NJ) method with a bootstrap setting of 1000.
[0042] 2. Results and Analysis
[0043] 1. Analysis of the Chloroplast Genome of Dictamnus chinensis
[0044] The chloroplast genome of D. difficile is 157139 bp in size and has a typical circular tetrad structure, including one LCS region (84478 bp), one SCC region (18587 bp) and two reverse complementary IRs regions (27037 bp). The total A+T content is 61.5% ( Figure 1 ).
[0045] A total of 132 genes were successfully annotated in the chloroplast genome of D. difficile ( Table 1 ), including 87 protein-coding genes (PCGs), 8 ribosomal RNAs (rRNAs), and 37 transfer RNAs (tRNAs). According to their functions, protein-coding genes in the chloroplast genome of D. difficile can be divided into the following four categories: the first category is genes related to self-replication, which are subdivided into three groups, namely, large subunit of ribosome, DNA dependent RNA polymerase, and small subunit of ribosome, with 13, 4, and 5 members, respectively; the second category is genes related to photosynthesis, which are subdivided into six groups, namely, subunits of ATP synthase, subunits of photosystem I, subunits of photosystem II, subunits of NADH-dehydrogenase, subunits of cytochrome b / f complex, and subunits of ribulose diphosphate oxygenase / carboxylase. rubisco), with 6, 15, 5, 12, 6 and 1 members respectively; the third category was other functional genes, including 1 each of acetyl-CoA carboxylase subunit, C-type cytochromsynthesis gene, outer membrane protein gene and protease gene; the fourth category was 6 genes of unknown function.
[0046] Table 1 Functional classification of genes encoding chloroplasts in Dictamnus
[0047]
[0048] 2. Phylogenetic Analysis of Dictamnus
[0049] The assembled Dictamnus chloroplast genome data was merged and aligned with published chloroplast genome sequences of Rutaceae species downloaded from GenBank. Twenty-four chloroplast genomes of species in the subgenus Cerasus have been published on GenBank, as detailed in Table 2. Two were selected as outgroups, as detailed in Table 3.
[0050] Table 2 Chloroplast genome sequences of Rutaceae species published in GenBank
[0051] Department name Generic name Species name GenBank ID Rutaceae Clausena excavata NC032685 Rutaceae Citrus reticulata MW147176 Rutaceae Atalantia kwangtungensis MH329190 Rutaceae Tetradium ruticarpum MT800757 Rutaceae Zanthoxylum motuoense MT772302 Rutaceae Murraya exotica MT747442 Rutaceae Orixa japonica MW574915
[0052] Table 3 Chloroplast genome sequences of outgroup species
[0053] Department name Generic name Species name GenBank ID Poaceae Zea mays NC001666 Brassicaceae Arabidopsis thaliana NC000932
[0054] Multiple sequence alignment was performed using MAFFT software, and a phylogenetic tree was constructed using MEGA X software based on the neighbor-joining (NJ) method with a bootstrap of 1000. Figure 2 The results showed that Dictamnus dasyphylla and Orixa japonica clustered into one branch, and the bootstrap support rate of the differentiation node was 100.
[0055] 3. Codon Bias in the Chloroplast Genome of Dictamnus
[0056] The relative synonymous codon usage frequency (RSCU) of the chloroplast genome of Dictamnus was statistically analyzed. There are 23,680 codons in the chloroplast genome coding sequence. The most common codon is leucine (Leu / L), with a total of 2,057 codons; the least common codon is cysteine (Cys / C), with a total of 288 codons. The highest RSCU value is 1.78 for glycine (Gly / G) encoded by GGG and GGA; the lowest RSCU value is 0.4 for phenylalanine (Phe / F) encoded by UUU. There are 32 codons with RSCU values greater than 1, which are the codons preferentially used in the chloroplast genome of Dictamnus. Figure 3 .
[0057] 4. Application in identification of Rutaceae plants
[0058] In this study, the chloroplast genome sequence of Dictamnus dasyphylla was compared with the chloroplast genomes of Rutaceae species downloaded from GenBank. The chloroplast genome structures of the eight species were very similar, with good colinearity, and no rearrangement or inversion was found. Figure 4 .
[0059] The chloroplast genomes of seven Rutaceae species were analyzed using the mVISTA program. Since the comparison was between different genera in the same family, most regions were highly divergent, such as trnK-UUU / trnS-GCU, atpF / atpL, and trnC-GCA / trnE-UUC. We also found some conserved regions, such as psbA, rpoB, psbD, rps14 / psbB, rbcL, ycf2, and rps12. Figure 5 .
[0060] 5. Characterization of the IR / SC boundary in the chloroplast genomes of Rutaceae plants
[0061] The characteristic analysis results of chloroplast genome 1R boundaries of 7 species of Rutaceae plants were visualized and constructed. Figure 6 The gene distribution order and gene structure of Rutaceae species are very similar, with the main differences occurring in the JLB (LSC / IRb) region, JSB (IRb / SSC) region, and JIR (IRa / LCS) region. Within the JLB region, the rps3 gene coding region of the Tetradium species Tripterygium wilfordii is spanned by the rps3 gene coding region, while the rp112 gene is completely located in the IRb region. The rp112 gene coding region spans the JLB region of the remaining seven Rutaceae species. The rps3 genes are all located in the LCS region. The rp112 gene of Guangdong wine cake ragweed has the largest expansion in the LSC and IRb regions, which is 266244bp, while the rp112 gene of Medog Zanthoxylum bungeanum has the smallest expansion, which is 174237bp. The white mulberry is in between the two, which is 183237bp. In the JSB region, the ndhF genes of citrus, stinking shanshan, Medog Zanthoxylum bungeanum and thyme span the IRb / SSC region, while the ndhF genes of white mulberry, three-leaved bitter and Guangdong wine cake ragweed are completely located in the SSC region. In the JSB region, the trnH genes of white mulberry and Medog Zanthoxylum bungeanum are between IRb / SSC, but the span difference is very small. The trnH genes of the other five species are all located in the LCS region. In the JSA (SSC / IRa) region, the structures of the seven Rutaceae species are consistent, and no obvious expansion or contraction has occurred. This indicates that although the chloroplast genomes of different species of Rutaceae have certain differences in gene distribution and gene types in the IR / SC boundary region, they are relatively conservative.
[0062] Although the present invention has been disclosed above by way of embodiments, they are not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.
Claims
1. Application of the chloroplast genome of the medicinal plant Dictamnus in identifying Dictamnus varieties, characterized in that: The nucleotide sequence of the chloroplast genome of Dictamnus salsa is shown in SEQ ID NO.
1.
2. Use of the chloroplast genome of the medicinal plant Dictamnus salsa according to claim 1 in reconstructing the phylogenetic tree of Rutaceae.
3. A method for identifying varieties of the medicinal plant Dictamnus, characterized in that: The steps include: (1) The total DNA of medicinal plant Dictamnus dasyphylla samples was extracted using the CTAB method; (2) Using the whole genome shotgun method, a 350 bp library was constructed from the total DNA of the sample obtained in step (1), and the library was subjected to double-end sequencing using an Illumina NovaSeq sequencer; (3) Low-quality data were filtered using SOAPnuke v1.3.0 software to obtain valid data; (4) Assembly and assembling of the chloroplast genome of the medicinal plant Dictamnus; (5) The assembled sample medicinal plant Dictamnus chloroplast genome nucleotide sequence was compared with the chloroplast genome nucleotide sequence of Rutaceae plant Rutaecarpa. When the similarity index reached above 99.9%, it was determined to be the medicinal plant Dictamnus chloroplast.
4. The method for identifying the medicinal plant Dictamnus var. In step (3), the filtering criteria were to remove reads with a quality value ≤ 5 and a base number of 50%, remove reads containing adapter contamination, and remove reads with more than 5% N base content.
5. The method for identifying the medicinal plant Dictamnus var. The steps of splicing and assembling the chloroplast genome of the medicinal plant Dictamnus in step (4) are as follows: a. Use GetOrganelle v1.7.5 software to assemble the genome, and perform blastn comparison with the chloroplast genome of the closely related reference plant Rutaecarpa in the NCBI database; b. Use Gapcloser to fill the gap in the N sequence; c. Use the annotation software CPGAVAS2 to perform gene annotation and draw the physical map of the Dictamnus chloroplast genome.