Chloroplast genome of anthurium andraeanum and its application in species identification

Abstract

The application belongs to the technical field of genes, and particularly relates to a chloroplast genome of Viburnum awabuki and application of the chloroplast genome in species identification and reconstruction of a phylogenetic tree of Viburnum. The nucleotide sequence of the chloroplast genome of Viburnum awabuki is shown in SEQ ID NO. 1. The sequence of the chloroplast genome of Viburnum awabuki is reported for the first time, so as to fill the blank of the existing technical gene database without the sequence of the chloroplast genome of Viburnum awabuki, and the sequence can be used for identification of Viburnum awabuki, and provides technical support for protection of Viburnum germplasm resources and species identification.

Description

Technical Field

[0001] This invention belongs to the field of gene technology, specifically relating to the chloroplast genome of Viburnum macrocephalum and its application in species identification and reconstruction of the phylogenetic tree of the Viburnum genus. Background Technology

[0002] viburnum ( Viburnum keteleeri ) belongs to the genus Viburnum in the family Viburnaceae. Viburnum The Viburnum macrocephalum (also known as the Chinese magnolia) is a deciduous shrub or small tree, and one of my country's traditional and precious ornamental plants. It is highly valued for its large, white inflorescences and beautiful tree shape, and is widely used in landscaping, scenic area construction, and the floriculture industry. In addition to its significant horticultural value, the Viburnum macrocephalum is also an important material for research on Viburnum resources, germplasm innovation, and the breeding of new varieties, thus attracting widespread attention in the fields of plant taxonomy, horticulture, and plant breeding.

[0003] The genus *Viburnum* is rich in species, with over 150 species worldwide, widely distributed in temperate and subtropical regions of the Northern Hemisphere. Due to the high similarity among species within the genus in leaf morphology, inflorescence structure, and fruit characteristics, and the tendency for convergent evolution among different species, accurate identification relying solely on traditional morphological characteristics is often insufficient. In the processes of germplasm resource surveys, resource preservation, seedling production, and new variety protection, problems such as difficulties in species identification, name confusion, and unclear germplasm origins frequently arise, seriously affecting germplasm resource evaluation, genetic improvement, and intellectual property protection. Therefore, establishing a rapid, accurate, and stable molecular identification method for *Viburnum macrocephalum* is of great significance.

[0004] With the development of molecular biology techniques, DNA barcoding has become an important tool for plant classification and identification. Currently, DNA barcodes widely used for plant identification mainly include rbcL, matK, and ITS sequences. However, due to the low degree of genetic differentiation among closely related species in the *Viburnum* genus, conventional DNA barcodes contain a limited number of information sites, often lacking sufficient resolution in species identification and phylogenetic analysis, making it difficult to meet the practical needs of accurate identification and germplasm resource management in *Viburnum*. Therefore, developing novel molecular markers and identification systems with higher resolution has become an important research direction.

[0005] The chloroplast genome, characterized by structural conservation, genetic stability, uniparental inheritance, abundant information loci, and a moderate evolutionary rate, provides far more information than traditional DNA barcodes. In recent years, it has been widely used in plant phylogenetic analysis, germplasm resource evaluation, and species identification research. The complete chloroplast genome is considered a highly discriminative "super-barcode," exhibiting a significant advantage, particularly in the identification of closely related species. Therefore, obtaining the complete chloroplast genome sequence of *Viburnum macrocephalum* and establishing an identification system based on the chloroplast genome is of significant theoretical and practical value for the accurate identification of *Viburnum macrocephalum* and its closely related species in the *Viburnum* genus, promoting germplasm resource protection and utilization, and carrying out new variety protection and intellectual property management. Summary of the Invention

[0006] The purpose of this invention is to provide the chloroplast genome of Viburnum macrocephalum to fill the gap in the existing gene databases that do not have the chloroplast genome sequence of Viburnum macrocephalum, which can be used to identify Viburnum macrocephalum and provide technical support for the protection of Viburnum germplasm resources and species identification.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: This invention is the first to obtain and disclose the genome of the chloroplasts of Viburnum macrocephalum, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The above-mentioned application of the Viburnum chloroplast genome in identifying Viburnum species, reconstructing the phylogenetic tree of the Viburnum genus, and screening Viburnum-specific SNP loci.

[0009] In addition, this invention also discloses a method for identifying species of *Viburnum macrocephalum*, the method comprising: (1) Total DNA was extracted from the samples using the mCTAB method; (2) Perform paired-end sequencing on the extracted total DNA; (3) Chloroplast genome splicing and assembly; (4) The nucleotide sequence of the assembled sample chloroplast genome is compared with the nucleotide sequence of the chloroplast genome of the viburnum. When the similarity is 100%, it is identified as viburnum. The nucleotide sequence of the chloroplast genome of the viburnum is shown in SEQ ID NO.1.

[0010] The present invention has the following advantages: This invention discloses for the first time the chloroplast genome of *Viburnum macrocephalum* and applies it to a phylogenetic study of the plant, elucidating its phylogenetic position within the *Viburnum* genus. The results of this study are of great significance for the future breeding and conservation of *Viburnum* species.

[0011] This study used molecular systematics and phylogenetic genomics methods to establish phylogenetic relationships among Viburnum species through chloroplast genomes. Based on this, the whole chloroplast genome was used as a super DNA barcode for Viburnum to identify species, providing technical support for the protection and species identification of Viburnum germplasm resources. This is of great significance for the breeding, genetic diversity evaluation, protection and utilization of Viburnum plants. Attached Figure Description

[0012] Figure 1 This is a diagram of the genome structure of the chloroplasts in Viburnum macrocephalum.

[0013] Figure 2 This is a phylogenetic tree of Viburnum plants constructed based on the complete chloroplast genome. Note: * indicates that the bootstrap support of the corresponding node is 100. Detailed Implementation

[0014] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0015] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0017] I. Materials and Methods 1. Extraction of total plant DNA Total DNA was extracted from fresh leaves of *Viburnum keteleeri* using a modified CTAB (mCTAB) method. DNA integrity was assessed by 1% agarose gel electrophoresis, and DNA purity and concentration were determined using a NanoDrop 2000 micro-volume spectrophotometer and a Qubit quantitative PCR instrument. After passing the tests, the DNA samples were stored at -20°C for later use.

[0018] 2. Plant total DNA sequencing methods Approximately 1 μg of high-quality total DNA was collected and randomly fragmented to approximately 350 bp using a Covaris sonicator. End repair, A-tailing, adapter ligation, and PCR amplification enrichment were performed according to the Illumina standard library construction protocol to construct sequencing libraries. Library quality was assessed using an Agilent 2100 Bioanalyzer, and quantification was performed by qPCR. Paired-end sequencing (PE150) was performed on the Illumina NovaSeq 6000 platform, yielding approximately 5 Gb of raw sequencing data per sample.

[0019] 3. Chloroplast genome assembly and annotation The raw sequencing data were quality controlled using Trimmomatic v0.39 software to remove adapter sequences, low-quality sequences (Q < 20), and short sequences less than 36 bp in length, obtaining high-quality clean reads. De novo chloroplast genome assembly was performed using NOVOPlasty v4.3.1 software, with the rbcL gene sequence of a closely related chloroplast as the seed sequence.

[0020] Preliminary chloroplast genome annotation was performed using the CPGAVAS2 online platform, followed by correction using PGA software and manual checking and revision using Geneious Prime 2021 software to ensure the accuracy of gene boundaries, start codons, stop codons, and intron structures. Finally, the genome structure of *Viburnum macrocephalum* chloroplasts was drawn using OGDRAW software.

[0021] 4. Chloroplast genome structure analysis of Viburnum macrocephalum The genome length, lengths of large single copy regions (LSCs), small single copy regions (SSCs), and inverted repeat regions (IRs) of *Viburnum macrocephalum* chloroplasts were statistically analyzed, along with their genome composition. The number and distribution characteristics of protein-coding genes (PCGs), transfer RNA genes (tRNAs), and ribosomal RNA genes (rRNAs) were also analyzed.

[0022] 5. Phylogenetic Analysis Methods Downloaded published chloroplast genome sequences of Viburnum species from the NCBI GenBank database, and selected those from the Elderberry genus ( Sambucus Plants were considered as outgroups. Multiple sequence alignment was performed using MAFFT v7.313 software, and low-quality alignment regions were removed using trimAlv1.2 software.

[0023] The optimal nucleotide substitution model was selected using ModelFinder software based on the Bayesian Information Criterion (BIC). A maximum likelihood (ML) phylogenetic tree was constructed using IQ-TREE v1.6.8 software, with 1000 bootstrap iterations. The phylogenetic tree was visualized and analyzed using FigTree v1.4.2 software.

[0024] 6. Molecular identification method for Viburnum macrocephalum The obtained chloroplast genome of *Viburnum macrocephalum* was compared and analyzed with the chloroplast genomes of other *Viburnum* species. Identification was performed comprehensively based on phylogenetic position, chloroplast genome structural characteristics, and species-specific single nucleotide polymorphisms (SNPs). The complete chloroplast genome was used as a super-barcode to achieve rapid and accurate identification of *Viburnum macrocephalum* and other *Viburnum* species.

[0025] II. Results and Analysis 1. Chloroplast genome analysis of Viburnum macrocephalum After analysis, see Figure 1 The chloroplast genome of *Viburnum macrocephalum* exhibits a typical angiosperm tetrad structure, with a total length of 158,662 bp. It consists of a large single copy region (LSC), a small single copy region (SSC), and two inverted repeat regions (IR). The large single copy region is 87,347 bp long, the small single copy region is 18,199 bp long, and both inverted repeat regions are 26,558 bp long. A total of 133 genes were annotated from the entire chloroplast genome, including 86 protein-coding genes, 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes.

[0026] Further analysis revealed that the chloroplast genome of *Viburnum macrocephalum* possesses a relatively stable gene structure and sequence. Among them, the ycf1 gene, located at the IRb / SSC boundary region, reaches a length of 1499 bp, the longest among the compared *Viburnum* species. Simultaneously, its IR region is 26558 bp long, while its SSC region is 18199 bp long, exhibiting structural characteristics of IR region expansion and SSC region contraction, respectively. This feature can serve as an important reference for the molecular identification of the *Viburnum macrocephalum* chloroplast genome.

[0027] 2. Phylogenetic analysis of Viburnum macrocephalum The assembled Viburnum chloroplast genome sequence was integrated with 94 published Viburnum chloroplast genome sequences in the GenBank database, and three Sambucus species were selected as outgroups. Sequence alignment was performed using MAFFT software (Table 1). The optimal nucleotide substitution model was selected using ModelFinder software based on the Bayesian Information Criterion (BIC), and a phylogenetic tree was constructed using IQ-TREE software based on Maximum Likelihood (ML). Figure 2 ).

[0028] Table 1. Chloroplast genome sequences of Viburnum species published in GenBank.

[0029]

[0030]

[0031] The phylogenetic tree constructed based on the complete chloroplast genome sequence showed a clear topology, with each major branch receiving high support. The results indicate that *Viburnum macrocephalum* can form stable phylogenetic relationships with other *Viburnum* species and establish independent lineages within the phylogenetic tree. Among these, *Viburnum macrocephalum* and *Viburnum shaanxiense* (…) Viburnum schensianum ) and pruning viburnum ( Viburnum burejaeticum The branches consisting of these groups are sister groups, and the bootstrap support of their differentiation nodes is 100. Compared with traditional DNA barcodes, the complete chloroplast genome can provide more phylogenetic information sites, which can effectively improve the accuracy and reliability of phylogenetic relationship analysis in Viburnum plants.

[0032] Further analysis revealed that the chloroplast genomes of Viburnum species are generally conserved, but some degree of genetic differentiation still exists among different species. Some different samples of the same species from public databases failed to cluster completely into monophyletic groups, suggesting that species boundaries within the Viburnum genus may not be fully defined, or that interspecific hybridization or chloroplast trapping may occur. In contrast, *Viburnum macrocephalum* maintains a stable position in the phylogenetic tree and can be effectively distinguished from other Viburnum species.

[0033] The above results indicate that the complete chloroplast genome can serve as a high-resolution "super-barcode," enabling accurate identification of Viburnum macrocephalum and other Viburnum species. This provides a reliable molecular basis for the classification, phylogenetic studies, germplasm resource evaluation, and new variety protection of Viburnum species.

[0034] Therefore, in the actual identification process, when a Viburnum plant sample suspected to be *Viburnum macrocephalum* is identified, it is compared with the whole genome data of the chloroplasts of *Viburnum macrocephalum*. The pairwise alignment function of Geneious (R10.2.3) software is used for detailed comparison. When the similarity index is 100%, it is determined to be a *Viburnum macrocephalum* variety. The nucleotide sequence of the chloroplast genome of *Viburnum macrocephalum* is shown in the sequence listing SEQ ID NO.1.

[0035] 3. Molecular identification method for Viburnum macrocephalum To verify the application effect of this invention in the identification of Viburnum species, the obtained Viburnum chloroplast genome was compared and analyzed with 50 published Viburnum chloroplast genomes in the GenBank database, resulting in a total of 51 chloroplast genome datasets. Using the complete chloroplast genome as a super-barcode, whole-genome sequence comparison analysis was conducted, and specific molecular markers capable of distinguishing Viburnum from other Viburnum species were screened.

[0036] Comparative analysis results show that the overall structure of the chloroplast genome of Viburnum species is highly conserved, but certain sequence variations still exist. Coding regions are more conserved than non-coding regions, with highly variable regions mainly distributed in the LSC and SSC regions. Regions such as trnC-GCA–petN–psbM, trnD–trnY–trnA–trnM-CAU–psbD, ycf4–cemA, and trnN-GUU–rpl32–trnL-UAG exhibit high levels of sequence variation. Among them, the two highly variable regions, trnC-GCA–petN–psbM and trnN-GUU–rpl32–trnL-UAG, show significantly different sequence characteristics in Viburnum compared to other Viburnum species, demonstrating their potential as candidate molecular markers and serving as important candidate regions for molecular identification of Viburnum species.

[0037] Further analysis of single nucleotide polymorphisms (SNPs) in 51 chloroplast genomes identified 293 *Viburnum macrocephalum*-specific SNP sites (Table 2). These specific SNP sites can serve as important criteria for the molecular identification of *Viburnum macrocephalum*. When used in conjunction with complete chloroplast genomes, they can enable rapid and accurate identification of *Viburnum macrocephalum* and other *Viburnum* species.

[0038] Table 2. Genome-specific single nucleotide polymorphism (SNP) sites in Viburnum chloroplasts

[0039]

[0040]

[0041]

[0042] In summary, this invention utilizes the complete chloroplast genome of Viburnum macrocephalum and its specific SNP sites to establish a molecular identification system for Viburnum macrocephalum, which can be widely applied in the fields of classification and identification of Viburnum species, germplasm resource evaluation, seedling authenticity identification, new variety protection, and germplasm resource preservation.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A Zoa leaf chloroplast genome characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The application of the Viburnum chloroplast genome as described in claim 1 in the identification of Viburnum species.

3. The application of the Viburnum chloroplast genome as described in claim 1 in reconstructing the phylogenetic tree of the Viburnum genus.

4. The application of the Viburnum chloroplast genome as described in claim 1 in screening Viburnum-specific SNP sites.

5. A method for identifying a variety of anemones, characterized by, The identification method includes: (1) Total DNA was extracted from the samples using the mCTAB method; (2) Perform paired-end sequencing on the extracted total DNA; (3) Chloroplast genome splicing and assembly; (4) The nucleotide sequence of the assembled sample chloroplast genome is compared with the nucleotide sequence of the chloroplast genome of the viburnum. When the similarity index is 100%, it is identified as viburnum. The nucleotide sequence of the chloroplast genome of the viburnum is shown in SEQ ID NO.1.

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