The complete chloroplast genome of Malvaviscus penduliflorus (Malvaceae)

Abstract

In this study, we assembled the complete chloroplast (cp) genome of Malvaviscus penduliflorus using high-throughput Illumina sequencing reads. The resulting plastome assembly displayed a typical quadripartite structure with a total length of 160,332 bp, containing a pair of inverted repeat regions (IRs) of 26,313 bp separated by a large single-copy region (LSC) of 88,750 bp and a small single-copy region (SSC) of 18,956 bp. The M. penduliflorus cp genome contained 128 genes, and its overall GC content was 36.96%. Phylogenetic analysis among M. penduliflorus and five other Malvaceae species demonstrated that M. penduliflorus was closely related to Urena procumbens and Hibiscus cannabinus. The M. penduliflorus cp genome presented in this study will lay a good foundation for further genetic and genomic studies of the genus Malvaviscus as well as Malvaceae.

Keywords:

• Chloroplast genome
• malvaceae
• malvaviscus penduliflorus
• phylogenetic analyses

1. Introduction

Malvaviscus penduliflorus Moc. & Sessé ex DC. 1824 is an evergreen shrub with 30–60 cm, belonging to the Malvaceae family. It grows well in the environment of high temperature, humidity, or sufficient sunlight, which has good heat resistance, barren resistance, and drought resistance. It is widely distributed in tropical regions of the world, such as Cuba, Mexico, and Colombia. In the field of medical treatment, the extracts of M. penduliflorus have been used to treat high blood pressure, liver diseases, fever prevention, and so on (Delange et al. 2012).

The chloroplast genome of angiosperms provides a powerful tool for species tree estimation, population genetic analysis, chloroplast gene function and chloroplast metabolism analysis, and investigation of species adaptation to extreme environments (Mehmood et al. 2020a, 2020b, 2020c). However, the complete chloroplast (cp) genome of M. penduliflorus has not been reported to date, which hampers a deeper understanding of its genomic characterization and evolutionary history.

In this study, we first assembled the cp genome of M. penduliflorus and confirmed its phylogenetic position in Malvaceae. This presented cp genome will provide valuable genomic resource for comparative genomic and genetic studies of the genus Malvaviscus as well as Malvaceae.

2. Materials

Fresh and mature leaves from an adult plant of M. penduliflorus were taken from Chenghua District, Chengdu, China (30.7000°N, 104.1794°E) (Figure 1A), which were collected according to the Regulations of the People’s Republic of China on the Protection of Wild Plants. A specimen was deposited at the museum of Chengdu University of Traditional Chinese Medicine (Dr. Rong Liu, [email protected]) under the voucher number CDU20201201008.

Figure 1. (A) an Individual plant of M. penduliflorus taken from Chenghua District, Chengdu, China (photoed by Dr. Rong Liu). (B) The detailed genome map of M. penduliflorus cp genome. The large single copy (LSC), small single copy (SSC) region and two inverted repeat regions (IRA and IRB), and GC content (light gray) are shown in the inside track. Gene models including protein-coding genes, tRNA genes, and rRNA genes are shown with various colored boxes in the outer track. (C) Maximum likelihood species tree based on the coding sequences of 75 genes that are presented in all the six cp genomes of Malvaceae species, including M. penduliflorus, U. procumbens (Wang et al. 2021), H. cannabinus (Cheng et al. 2020), A. officinalis (Zhang et al. 2017), M. verticillata (Li et al. 2020), and G. hirsutum (Lee et al. 2006), using B. ceiba (Gao et al. 2018) as outgroup. Bootstrap support values were denoted on each internal node, indicating that M. penduliflorus clustered with U. procumbens and H. cannabinus with 100% bootstrap support.

3. Methods

For whole-genome DNA sequencing, we extracted total genomic DNA using a modified CTAB method (Doyle and Doyle 1987). Paired-end Illumina libraries with an insertion size of 350 bp were constructed in accordance with the manufacturer’s instructions and sequenced on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA), resulting in a total of 8.24 Gb Illumina reads. Subsequently, we de novo assembled the cp genome of M. penduliflorus using NOVOPlasty (Dierckxsens et al. 2017) using the following parameters: k-mer = 39 and genome range 120,000–200,000, and annotated the plastome assembly using Plann (Huang and Cronk 2015) with the Urena procumbens cp genome (GenBank accession number: NC_054171; Wang et al. 2021) annotation as the reference. The obvious annotation errors were corrected by Geneious (Kearse et al. 2012). We performed phylogentic analysis among M. penduliflorus and five other Malvaceae species, including U. procumbens, Hibiscus cannabinus (NC_045873; Cheng et al. 2020), Althaea officinalis (NC_034701; Zhang et al. 2017), Malva verticillata (NC_059767; Li et al. 2020), and Gossypium hirsutum (NC_007944; Lee et al. 2006), using Bombax ceiba (NC_037494; Gao et al. 2018) as outgroup. The coding sequences of protein-coding genes present in all the six cp genomes of Malvaceae species were aligned by MAFFT-LINSI v7.313 (Katoh and Standley 2013). The resulting alignments were concatenated into a supermatrix, and a maximum likelihood phylogenetic tree was constructed by RAxML v8.2.11 (Stamatakis 2006) under the GTRGAMMA model with 500 bootstrap replicates. Further, the cp genomes of M. penduliflorus, U. procumbens, H. cannabinus, A. officinalis, and Malva verticillata were compared using DnaSP v5.10 (Librado and Rozas 2009) with the window size and step size of 800 and 200 bp, respectively.

4. Results

Genome sequencing M. penduliflorus resulted in a total of 7.55 Gb Illumina short-read data. The cp genome assembly of M. penduliflorus (GenBank accession number: NC_066439 and ON464180) exhibited a circular structure with a total length of 160,332 bp, containing a pair of inverted repeat regions (IRs) of 26,313 bp separated by a large single-copy region (LSC) of 88,750 bp and a small single-copy region (SSC) of 18,956 bp (Figure 1B). The average coverage depth of M. penduliflorus cp genome was 760× (Supplementary Figure 1). This cp genome contained 138 genes, comprising of 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Among them, 11 protein-coding genes and 8 tRNA genes had a single intron, and three protein-coding genes (rps12, ycf3, and clpP) had two introns. The structure of trans-spliced gene rps12 was verified and shown in Supplementary Figure 2. The overall GC content was 36.96%, and the GC content was 45.76%, 38.18%, and 30.49% for the first, second, and third codons of protein-coding regions, respectively. The 83 protein-coding genes encoded a total of 25,693 codons, and leucine and cystine were the most (10.47%) and least (1.16%) frequently used amino acids. Phylogenetic analysis based on 75 protein-coding genes presented in all the plastomes of M. penduliflorus and five other Malvaceae species showed that M. penduliflorus was closely clustered with U. procumbens and H. cannabinus with 100% bootstrap support (Figure 1C). In addition, comparison of five cp genomes (Supplementary Figure 3) identified several highly variable regions with high nucleotide diversity values (> 0.2), including the genic regions of 7 protein-coding genes (ndhE, ndhG, ndhI, ndhA, ndhH, rps15, and ycf1), 5 tRNA genes (trnN-GUU, trnR-ACG, trnA-UGC, trnI-GAU, and trnV-GAC), and 4 rRNA genes (rrn5, rrn4.5, rrn23, and rrn16). These hotspot regions can be served as potential molecular markers for the phylogenetic analysis of Malvaceae species.

5. Discussion and conclusion

In this study, we first presented the cp genome of M. penduliflorus with a total length of 160,332 bp, a total of 138 genes and an overall GC content of 36.96%. We found that M. penduliflorus was closely clustered with U. procumbens with full support. This genome is the first reported cp genome in the genus Malvaviscus. This cp genome will provide useful genetic resource for further genetic and genomic studies of the genus Malvaviscus and Malvaceae. In the future, increasing number of cp genomes of Malvaceae will provide deeper insights into the evolution of this ecologically and economically important family.

Authors’ contributions

Rong Liu and Dan Wang conceived and supervised the project. Rong Liu and Li Xie analyzed the data and wrote the manuscript. All of the authors have read and approved the final manuscript and have agreed to be accountable for all aspects of the work.

Acknowledgments

The authors are really grateful to the open raw genome data form public database.

Disclosure statement

No potential conflict of interest was reported by the author(s). The coauthors do not have any conflict of interest to declare. The authors alone are responsible for the content and composition of the paper. Additionally, fresh and mature leaves from an adult plant of M. penduliflorus were taken with the permission of the community management department.

Data availability statement

The genome sequence data supporting the findings of this study are openly available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov/) under the accession no. NC_066439. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA885282, SRR21748890, and SAMN31083560, respectively.

Additional information

Funding

This work was supported by the opening project of a key laboratory of coarse cereal processing, Ministry of Agriculture and rural affairs (No: 2020CC015), the project of Sichuan’s education department (No: JG2021- 1107) as well as the Natural Science Project of Sichuan Nursing Vocational College (No: 2023ZRZ03).