The complete chloroplast genome of Aegle marmelos and its phylogenetic analysis

Abstract

Aegle marmelos (L.) Correa 1800, a plant belonging to the Rutaceae family, is extensively used in Tibetan medicine. We employed Illumina HiSeq reads to assemble the complete chloroplast (cp) genome of A. marmelos, which spans 144,538 bp. The genome comprises 114 genes, including 75 protein-coding genes, 31 tRNA genes, and 8 rRNA genes. It is characterized by four regions: The large single-copy (LSC) region (74,253 bp), the inverted repeat A (IRa) region (26,015 bp), the small single-copy (SSC) region (18,255 bp), and the inverted repeat B (IRb) region (26,015 bp). Phylogenomic analysis demonstrated a close relationship between A. marmelos and Citrus. The assembly of The cp genome in this study serves as a foundation for conservation efforts and phylogenetic investigations of A. marmelos, paving the way for future experimentation.

Keywords:

• Aegle marmelos
• chloroplast genome
• Rutaceae
• phylogenetic trees

Introduction

Aegle marmelos (L.) Correa, a 10-m-tall tree of the Rutaceae family (Figure 1), holds significant importance in Tibetan medicine. Our team captured local images of this species with a professional team. While It is indigenous to India, it is also found in Southeast Asia and has been introduced to Florida and Hawaii (Maity et al. 2009). A. marmelos is native to Xishuangbanna in Yunnan Province, as well as India, Burma, Laos, Vietnam, Cambodia, Thailand, Malaysia, and Indonesia, where it thrives in slightly dry, sloping forests at altitudes of 600–1000 m. All parts of the plant, including the roots, bark, leaves, flowers, fruits, seeds, and latex, are utilized in traditional medicinal systems (Patel et al. 2012). The plant possesses numerous pharmacological properties, with Its roots, bark, leaves, and flowers being employed in the treatment of intermittent fevers. The fleshy part of the fruit serves as a gastrointestinal medicine and dietary supplement. In Myanmar, young leaves are utilized to treat wounds, scabies, swelling, pain, and foot-and-mouth disease. Additionally, the juice derived from crushed leaves is employed to treat eye diseases. However, caution must be exercised by Pregnant women and those preparing for pregnancy, as the consumption of young industry from this species can lead to infertility and even miscarriage (Rana et al. 1997; Charoensiddhi and Anprung 2008). This review focuses on the investigation of the chloroplast (cp) genome of A. marmelos and its phylogenetic position within the Rutaceae family. We assembled and characterized the complete cp genome of A. marmelos, enhancing our understanding of its genetic information and facilitating species identification within the Rutaceae family.

Figure 1. Images of Aegle marmelos (L.) Correa 1800: (a) Leaves; (b) fruit; (c) whole plant. A. marmelos is a deciduous small tree, approximately 10 m tall, with leaflets featuring numerous oil glandular dots. The leaflets are broadly ovate or long elliptic, measuring 4–12 cm in length. terminal leaflets are larger and long-stalked, while lateral leaflets are subsessile with shallowly obtuse serrations. The fruit measures 10–12 cm in height and 6–8 cm in diameter, displaying a light greenish-yellow color, smooth texture, and a hard woody pericarp, approximately 3–4 mm thick. The fruit stalk is 4–6 cm long, and the seeds, numerous in quantity, are compressed ovoid with transparent gum. The testa is wooly, and cotyledons emerge during germination. These photos were captured by a local professional team in Xishuangbanna County, Yunnan Province, China.

Materials and methods

Plant material and DNA sequencing

This study was conducted in compliance with the Regulations on Biodiversity Conservation of Yunnan Province and received authorization from Xishuangbanna (Yunnan Province, China) and the Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences (Beijing, China). permission was obtained from the local forestry department for sample collection. Kunming Caizhi Biotechnology Co., Ltd. (Kunming, China) identified the A. marmelos specimen. A. marmelos impatiens samples were obtained from Mengla County, Xishuangbanna Prefecture, Yunnan Province, China (alt. 2683 m, E101°15′12″, N21°55′40″). The voucher specimen (specimen accession number: LQE-2022-0523) was deposited in the Institute of Chinese Meteria Medica, China Academy of Chinese Medical Sciences (contact: Xiangxiao Meng, [email protected]). Total DNA was extracted from silica-dried young leaves using a Plant genomic DNA kit (Tiangen Biotechnology (Beijing) Co., Ltd., Beijing, China).

Genome assembly and annotation

In this study, the chloroplast genome was annotated using GetOrganelle v.1.7.5 (Jin et al. 2020) and CPGAVAS2 (http://www.herbalgenomics.org/cpgavas2). the complete chloroplast genome assembly was performed using Illumina HiSeq reads, and the C. inopinata chloroplast genome (Genome Repository accession number: Gwhbk01000000) was confirmed through a BLAST search.

Phylogenetic analysis

A maximum-likelihood (ML) method was employed to reconstruct the phylogenetic relationships between A. marmelos and 14 other species. The chloroplast genomes were downloaded from the NCBI GenBank database, with Ruta graveolens (NC_045946) serving as an outgroup.

The cp genomes were aligned using MAFFT v. 7.471 (Katoh and Standley 2013) and trimmed. A maximum likelihood phylogenetic tree was constructed using RAxML v8.2.12 (Alexandros 2014), employing the GTRGAMMA model, 1,000 bootstrap replicates, and the rapid bootstrapping and search for best-scoring ML tree algorithm. The resulting phylogenetic tree (Figure 3) was modified using iTOL (Ivica and Bork 2019).

Result and discussion

Genome structure analysis

To ensure high-quality reads, the raw sequencing data (raw reads) from the offline machine were filtered to remove low-quality reads with adapters, resulting in clean reads and ensuring the accuracy of subsequent analyses. The genome assembly achieved 100% coverage (Figure S1).

DNA fragmentation was performed using an ultrasonic processor, yielding DNA fragments with an insert length of approximately 144,538 bp. The library preparation involved processes such as terminal repair, addition of base A, addition of sequence adapters, purification, and PCR amplification to obtain a 144,538 bp library. Novenol Co., Ltd. (Tianjin, China) and the Novena vaSeq System (Novena, Santiago, CA) performed whole-genome sequencing. The chloroplast genome was assembled using SPAdes version 3.13.0 with default settings, without employing a reference genome.

The complete chloroplast genome of A. marmelos (Genome Warehouse accession number: Gwhbkhk01000000) spans a length of 144,538 bp, with a GC content of 38.9%. It consists of four regions: two inverted repeat regions (IRa and IRb) of 26,015 bp each, separated by a large single-copy gene region (LSC) of 74,253 bp, and a small single-copy gene region (SSC) of 18,255 bp. The chloroplast genome contains a total of 114 genes, including 75 protein-coding genes, 31 tRNA genes, and 8 rRNA genes (Figure 2). Among these genes, nine were found to contain introns. Seven genes (rpoC1, petB, petD, rpl16, rpl2, ndhB, and ndhA) contain a single intron, while two genes (ycf3 and clpP) contain two introns (Figure S2).

Figure 2. Chloroplast reference genome of Aegle marmelos. The diagram illustrates the organization of the chloroplast genome. Starting from the center, the first circle represents the forward and reverse repeat sequences, indicated by red and green arcs, respectively. The second circle depicts a tandem repeat sequence, while the third circle highlights microsatellite sequences with short strips. The fourth circle displays the positions of the large single-copy gene region (LSC), small single-copy gene region (SSC), and inverted repeat regions (IRA and IRB). the fifth circle represents the GC content. The genes located outside the sixth circle are transcribed counterclockwise, while those inside are transcribed clockwise. Different functional groups of genes are color-coded, as indicated in the legend.

Figure 3. Showcases the phylogenetic analysis of 14 species and 1 taxonomic unit outgroup, utilizing the complete chloroplast genome sequence with the RAxML method. The following sequences were used for analysis: NC_063578 Atalantia buxifolia (Wang et al. 2017), NC_052719 Citrus aurantium (Lin et al. 2020), NC_024929 Citrus aurantiifolia (Su et al. 2014), NC_064135 Citrus juno (Zhong-Wang 2008), NC_053690 Citrus hindsii (Xu et al. 2019), NC_053733 Citrus japonica (Wu et al. 2019), NC_045403 Citrus polytrifolia (Li et al. 2019), NC_057088 Citrus trifoliata, Gwhbkhk01000000 Aegle marmelos, NC_032684 Murraya koenigii (Shivakumar et al. 2017), NC_032685 Clausena excavata (Natthawadi et al. 2018), NC_061742 Clausena lansium (Zhao et al. 2019), NC_032687 Glycosmis pentaphylla (Shivakumar et al. 2017), NC_060410 Glycosmis parviflora (Wen-Fang et al. 2013), and NC_045946 Ruta graveolens (Ling and Zhang 2019).

Phylogenetic analysis

Aegle marmelos belongs to the genus Aegle, and no similar structure is found within the genus. Therefore, a phylogenetic tree was constructed using 14 species from different genera within the same subfamily, along with one outgroup species from a different subfamily. The complete chloroplast genome sequence of A. marmelos provides valuable information for species conservation and phylogenetic studies of the Rutaceae family.

Authors’ contributions

Sijia Wang completed the article. Rushang Xiang uploaded the genomic data, using the database of the Chinese Academy of Traditional Chinese Medicine, Xiubo Liu and Jiaxin Lu analyzed the data in the article, Xiubo Liu created the graphs in the article, and Lingyang Kong and Zhanping Zhang conducted some literature searches. Wei Ma designed the experiments. All authors agreed to take responsibility for all aspects of the work.

Endangered species statement

This article adheres to the International Union for Conservation of Nature (IUCN) policy research involving endangered species and strictly adheres to the Convention on Biological Diversity and the Convention on Trade in Endangered Species of Wild Fauna and Flora (CITES) without damaging endangered species.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The genome sequence data obtained in this study are openly available in GenBank of NGDC (https://ngdc.cncb.ac.cn/) under the accession number Gwhbkhk01000000. The associated BioProject, Bio-Sample and GSA numbers are PRJCA011521, SAMC866117 and CRA007998, respectively.

Additional information

Funding

This work was supported by National Key Research and Development Project, Research and Demonstration of Collection, Screening and Breeding Technology of Ginseng and Other Genuine Medicinal Materials [Project No.2021YFD1600901]; Heilongjiang Touyan Innovation Team Program [Grant Number:2019 No. 5]; Science and Technology Innovation project of Chinese Academy of Traditional Chinese Medicine [CI2021A03710].