The complete mitochondrial genome of the Kandelia obovata Sheue, H.Y.Liu & J.W.H.Yong (Rhizophoraceae)

Abstract

Kandelia obovata Sheue, H.Y.Liu & J.W.H.Yong is one of the most cold-resistant true mangrove species, and it is widely distributed from the South China Sea to southern Japan. In the current study, the complete mitochondrial genome sequence of K. obovata was assembled using Illumina reads. It is the first mitochondrial genome of the Kandelia genus within the family Rhizophoraceae to be sequenced. The mitochondrial genome size is 312,146 bp with a total of 49 predicted genes, including 29 protein-coding genes, 17 transfer RNA genes, and 3 ribosomal RNA genes. The overall GC content of the genome is 41.87%. A phylogenetic tree constructed using nine complete mitochondrial genomes revealed that K. obovata is more closely related to Bruguiera species. This study enriches the plastid genome of Kandelia, furnishing valuable genetic insights for the investigation of evolutionary and population genetics in Kandelia and other mangrove species.

Keywords:

• Kandelia obovata
• mangrove
• mitochondrial genome
• phylogenomic tree

Introduction

Mangrove plants are widely distributed in tropical and subtropical intertidal zones. They are of ecological value due to their specialized physiological properties such as vivipary, salt secretion, and aerial roots (Tomlinson 1986). Kandelia obovata Sheue, Liu & Yong(2023) (Figure 1) belongs to the genus Kandelia of the Rhizophoraceae family and that has only two species (Tomlinson 1986; Sheue et al. 2003). Kandelia species are geographically divided around the south of the South China Sea, and the populations growing in the north of the South China Sea are K. obovata (Sheue et al. 2003). Low temperature limits the distribution of mangrove plants to the subtropic regions, but K. obovata can be found as far north as southern Japan in the Northern Hemisphere, so it is often regarded as the most cold-tolerant mangrove species (Lu et al. 2021). Much attention has been drawn to genomics and transcriptomics of Kandelia due to their capacity to grow at lower temperatures, along with molecular and physiological mechanisms (Fei et al. 2022; Guo et al. 2022). A K. obovata nuclear genome of 209.87 Mb was assembled, covering 93.69% of the estimated 224 Mb genome, and genome annotation yielded 23,683 protein-coding genes with 95.5% BUSCO completeness (Qiao et al. 2020). Mitochondria is an indispensable organelle for energy in organisms, and it is involved in aerobic respiration and other essential biochemical processes such as energy transformation, the tricarboxylic acid cycle, oxidative phosphorylation, and calcium ion storage. However, little is known about the genomic characterization of K. obovata mitochondrial. A low mutation rate makes mitochondrial genomes ideal research models for plant classification, phylogenetic structure, and comparative genomic research.

Figure 1. The photograph of the tree (a) was taken by xiuming Xu in the field (guangxi, China). the photographs of flowers (b) and hypocotyl (c) were taken by chengcheng hou in the field (xiatanwei coastal wetland park, Fujian, China). K. obovata produces pencil-shape seedlings that germinate on the maternal plants.

Materials

For plant materials, young fresh K. obovata leaves were collected from Shenzhen Hekou Futian Mangrove Nature Reserve, north east of Shenzhen Bay, Shenzhen, China (22°53'N, 114°01'E). The specimens were preserved at the laboratory of the College of the Environment and Ecology, Xiamen University with the specimen code SZ4 (Miss. Xu, [email protected]).

Methods

Whole genome DNA was extracted using CTAB methods (Doyle 1991) and at least 5 μg of sheared concentrated DNA was used in size-selection procedures performed using the BluePippin system. A DNA library with an insert size of 350 bp was constructed using the library building kit and was sequenced using the Nova-PE150 platform. Sequencing produced a total of 12 Gb raw data. Clean data were obtained using Trimmomatic v0.40 (Bolger et al. 2014). The average resequencing depth of the mitochondrial genome was calculated and plotted the depth using the coverage plot program. (Ni et al. 2023). High-quality Illumina sequencing reads were used to assemble the mitochondrial genome using NOVOPlasty v.4.3.1 (Dierckxsens et al. 2017), with Bruguiera sexangula (GenBank accession NC_056359.1) used as the reference sequence. The simple sequence repeats (SSRs) distribution in the K. obovata mitochondrial genome was detected using MISA-web (Beier et al. 2017). Geseq (Tillich et al. 2017) was used to annotate the complete mitochondrial genome. Manual proofreading was conducted to ensure the reliability and accuracy of software annotation (Alverson et al. 2010).

To determine the phylogenetic position of K. obovata, complete mitochondrial genome sequences of K. obovata and several related mangrove species were used as datasets to construct a phylogenetic tree. Sequences of eight complete mitochondrial genomes were downloaded from NCBI GenBank (Asclepias syriaca L., Aegiceras corniculatum (Linn.) Blanco, Citrus sinensis (L.) Osbeck, Acacia ligulata A.Cunn. ex Benth., Sesuvium portulacastrum (L.) L., Arabidopsis thaliana (L.) Heynh., Bruguiera × rhynchopetala (W.C.Ko) N.C.Duke & X.J.Ge, and Bruguiera sexangular (Lour.) Poir.). The single-copy orthologues of eight protein sequences (cox1, nad3, nad7, cob, ccmFc, cox2, cox3, and rpl5) were identified via OrthoFinder2 v.2.2.7(Emms & Kelly, 2018). Each protein sequence was the aligned using MUSCLE v.5.1 (Edgar 2004) and the eight protein sequence alignment results were merged into one alignment result. A maximum likelihood phylogenetic tree was generated using IQ-TREE2 v.2.2.0.3 (Minh et al. 2020). The best-fitting evolution model was automatically selected (HIVw + F+G4) and phylogenetic construction was performed with the parameters -B 1000.

Results

The average resequencing depth was up to 3437X of the K. obovata mitochondrial genome (Supplementary Figure 1). The assembled K. obovata mitochondrial genome is 312,146 bp in length (Figure 2). It has a GC content of 41.87%, and its base composition is 29.30% A, 20.74% C, 21.13% G, and 28.83% T. It encodes a total of 49 genes, including 29 protein-coding genes, 17 transfer RNA genes, and 3 ribosomal RNA genes (rrn5, rrn18, and rrn26) (Figure 2). Notably, nad2, cox3, atp6, atp8, matR, sdh4, and rps7 were found to have two copies each. Complex I, Complex III, Complex IV, and Complex V were observed to possess four (nad2, nad3, and nad7), one (cob), four (cox1, cox2, and cox3), and six (atp1, atp4, atp6, and atp8) genes, respectively. The cytochrome-c biogenesis process involves ccmC, ccmFc, and ccmFn genes, while ribosomal proteins are encoded by rps7, rps10, rps12, rps14, and rpl5. Additionally, matR, sdh3, and sdh4 were identified as other types of protein-coding genes. Five of those genes contain introns, including three genes with one intron (cox2, ccmFc and nad2), and one gene with four introns (nad7) (Supplementary Figure 2). The nad7 gene has the longest intron (4315 bp), and ccmFc gene has the shortest intron (1112 bp). The total number of SSRs is 103 with an average density of 330.13 SSRs/Mb. Mononucleotide SSRs are the most abundant (91), followed by dinucleotides (18), then trinucleotides (4). The SSR motifs base composition exhibited strong AT-rich bias.

Figure 2. Representative map of the Kandelia obovata circular mitochondrial genome molecule. The colored squares distributed inside and outside the circle represent different mitochondrial genes.

A maximum likelihood phylogenetic tree was generated, with Asclepias syriaca as the outgroup (Figure 3). The relationships among nine plant species are revealed by the phylogenetic tree based on the mitochondrial genomes. K. obovata is more closely related to Bruguiera mangrove species.

Figure 3. Phylogenetic relationship between nine species based on eight orthologous protein-coding genes in the mitochondrial genome. The numbers above the branches represent bootstrap values. The labels next to the scientific name of each species indicate the GenBank accession numbers in the NCBI database. The following sequences were used: Asclepias syriaca NC_022796.1 (Straub et al. 2013), Aegiceras corniculatum NC_056358.1, Citrus sinensis NC_037463.1 (Yu et al. 2018), Acacia ligulata NC_040998.1 (Sanchez-Puerta et al. 2019), Sesuvium portulacastrum MN683736 (Li et al. 2020), Arabidopsis thaliana NC_037304.1 (Sloan et al. 2018), Bruguiera x rhynchopetala MT130511 (Zhang et al. 2020), and Bruguiera sexangular NC_056359.1 (Zhang et al. 2020).

Discussion and conclusion

Accurate sequencing technologies and improved bioinformatic methodologies have taken a breakthrough leap ahead in recent decades. Herein we report the complete mitochondrial genome of K. obovata for the first time, which was assembled based on 12 Gb Illumina sequencing data. We also collated a set of mitochondrial genome data to construct the phylogenetic tree relationships within Kandelia. Previous studies have shown that K. candel, Rhizophora stylosa, Rhizophora x lamarckii, and Rhizophora apiculata are the closest relatives to K. obovata, as revealed by the phylogenetic tree based on the chloroplast genomes (Ruang-Areerate et al. 2021; Xu et al. 2022;). However, high-quality mitochondrial genomes from closely related species of K. obovata are still lacking. Future research should focus on exploring more mitochondrial genomes from closely related species of K. obovata to enhance our understanding of its phylogenetic relationships. Overall, the complete mitochondrial genome of K. obovata could provide fundamental data to support further genetic comparisons and evolutionary studies on species in the Rhizophoraceae family.

Authors’ contributions

YS and XX designed the study. YS and XX collected samples and XX conducted the experiments. CH analyzed the data. YS, XX and CH wrote the manuscript.

Permission

Plants were collected and treated in accordance with the guidelines provided by the Regulations of the People’s Republic of China on the Protection of Wild Plants and the Regulations on laboratory safety management of Xiamen University.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings in this study are available. The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/156 nuccore/OP756530.1/under the accession no. NC_069222.1 and OP756530. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA909204, SRR22547472, and SAMN32069823, respectively.

Additional information

Funding

This work was supported by the Fieldwork Funds for graduate students of Xiamen University [2022FG022] and the Fundamental Research Funds for the Central Universities of China [No.20720210075].