The complete chloroplast genome of Hedychium flavum Roxb., an ornamental, edible and medicinal plant

Abstract

Hedychium flavum Roxb. 1820 is a perennial herb mainly distributed in China, India, Myanmar and Thailand with ornamental, edible and medicinal value. It is extensively cultivated as a source of aromatic essential oils, ornamental plant, food flavorings and vegetables, and folk medicine. In this study, we sequence the complete chloroplast genome of H. flavum by de novo assembly. The assembled genome has a typical quadripartite circular structure with 163,909 bp in length, containing a large single-copy region (LSC, 88,589 bp), a small single-copy region (SSC, 15,762 bp), and two inverted repeat regions (IRs, 29,779 bp). The cp genome contains 133 genes, including 87 protein-coding genes, 38 tRNA genes and 8 rRNA genes. Phylogenetic analysis based on the complete cp genome shows a close affinity of H. flavum and H. neocarneum with 100% bootstrap support. This study will provide useful genetic resource for further phylogenetic analysis of the genus Hedychium and Zingiberaceae.

Keywords:

• Hedychium flavum
• chloroplast genome
• phylogenetic analysis
• Zingiberaceae

Introduction

The genus Hedychium (Zingiberaceae), commonly known as ginger lily, comprises approximately 93 species, which are mainly distributed in China, India, and Southeast Asia (Tian et al. 2023; The plant list 2012). The Hedychium species are herbs with thick, fleshy and aromatic rhizomes and extensively cultivated for their various uses in fragrance, paper, ornamental, cosmetics, medicine, and food industries (Sakhanokho et al. 2013; Hartati et al. 2014; Tian et al. 2020). Hedychium plants are rich in essential oil extracted from leaves, flowers and rhizomes and widely used in high-quality perfumes and traditional medicine for the treatment of asthma, bronchitis, diarrhea, gastric diseases, flu, nausea, snake bites, and leishmaniasis (Basak et al. 2010; Hartati et al. 2014). Moreover, some species of Hedychium were grown for their edible flowers (He 2000).

Hedychium flavum Roxb. 1820 (Roxburgh 1820), a perennial tuberous plant, is distributed in China, Thailand, India and Myanmar (Wu and Larsen 2000). It is extensively cultivated for its aromatic essential oil or as an ornamental plant. (Wu and Larsen 2000; Tian et al. 2020). H. flavum has large aromatic flowers forming a terminal spike with an orange spot in the petals (Figure 1) and blooming from August to September (Wu and Larsen 2000; Huang et al. 2023). Its flowers can remain open for 5-6 days after being cut and placed in bottle, making it an aromatic cut flower with great breeding prospects (Gao et al. 2002; Pan and Zhang 2013). Furthermore, the rhizomes (commonly known as yehansu) and flowers are widely used in Chinese folk medicine and has remarkable efficacy in treating various diseases, like coughs, stomach pain, bruises, rheumatism and headache (Ai 2013; Zhou et al. 2018). Meanwhile, their extracted essential oils have a variety of pharmacological effects such as antibacterial, anticancer, insecticidal, anti-inflammatory, and tyrosinase inhibitory properties, with the essential oils of the flowers also being used in cosmetics. (Sakhanokho et al. 2013; Tian et al. 2020, 2023). The rhizomes and young tender shoots of H. flavum are used as vegetables and food flavorings agent (Devi et al. 2014).

Figure 1. Species reference image of H. flavum with following distinguished characters: terminal spikes, imbricate bracts, yellow flowers, yellow obcordate labellum with orange at base, and long filament ca. 3 cm. (photo taken by Yong-Hong Zhang in Songming County, yunan province, China).

Previous studies on H. flavum are mainly focused on the utilization as a medicinal, edible, and ornamental plant (Pan and Zhang 2013; Tian et al. 2020, 2023). Until now, no genomic information on H. flavum has been reported. In this study, we sequenced and characterized the complete chloroplast genome of H. flavum to provide useful genetic resource for future molecular study of this species.

Materials and methods

The fresh leaves of H. flavum were collected from a healthy H. flavum individual, growing in Songming County (25°18'27′'N,102°53'14′'E) Yunnan province. The voucher specimens (LMF06) were deposited in the Herbarium of Yunnan Normal University (YNUB, website: https://life.ynnu.edu.cn/, Contact: Jian-Lin Hang, Email: [email protected]).

The total genomic DNA was extracted from fresh leaves using a modified CTAB (cetyl trimethylammonium bromide) method (Allen et al. 2006). The constructed library was sequenced using the Illumina Novaseq 6000 platform (Illumina Inc., San Diego, CA, USA). All raw reads were filtered to obtain clean reads with default parameter using NGS QC Toolkit v2.3.3 (Patel and Jain 2012). Based on the clean data, the plastome was de novo assembled using the software NOVOPlasty v4.2 (Dierckxsens et al. 2017). Geneious v2022.2.2 (Kearse et al. 2012) was used for annotation based on the H. neocarneum (GenBank accession No. MT473709) chloroplast genome (Li et al. 2021) and then manually corrected. The annotated chloroplast genome sequence was deposited into GenBank under the accession numbers of OQ308833, and the original reads were also deposited in GenBank. The circular genome map was drawn using CPGView program (Liu et al. 2023).

To explore the phylogenetic relationship of H. flavum with its closely related species, a phylogenetic tree was constructed using complete chloroplast genomes of 35 species from Zingiberaceae and three species from Musaceae (as outgroups) downloaded from NCBI GenBank. Total of 39 chloroplast genomes were aligned by the MAFFT v7.490 software (Katoh and Standley 2013). Subsequently, nucleotide substitution models were estimated using MEGA 11 (Tamura et al. 2021) and the model of GTR + G + I was chosen. The maximum likelihood (ML) tree was reconstructed by using RAxML v8.2.11 (Stamatakis 2014). Bootstrap values were calculated from 1,000 replicates to evaluate the reliability of each node.

Results

General feature of the chloroplast genome

The newly assembled plastid genome of H. flavum is 163,909 bp in length. The clean reads (24,029,978) were subjected to assembly to produce a circular molecule of complete chloroplast with about an average 731.4 coverage, which indicated that the genome assembly is reliable (Figure S1). The chloroplast genome of H. flavum had a typical quadripartite structure (Figure 2). It is divided into four distinct regions: a large single-copy (LSC) region of 88,589 bp, a small single-copy (SSC) region of 15,762 bp, and a pair of inverted repeats (IR) region of 29,779 bp. The GC content in whole cp genome, LSC region, SSC region, and IR regions were 36.1%, 33.9%, 29.6% and 41.1%, respectively.

Figure 2. The chloroplast genome of H. flavum visualized by CPGView. The genome map includes six tracks. From the inward to outward, the first track depicts the dispersed repeats connected by red (forward direction) and green (reverse direction) arcs, respectively. The second track shows the tandem repeats as short blue bars. The third track shows the short tandem repeats or microsatellite sequences as short bars. The small single-copy (SSC), inverted repeat (IRa and IRb), and large single-copy (LSC) regions are shown on the fourth track. The GC content along the genome is plotted on the fifth track. The outermost track shows the genes which are color-coded based on their functional classification. The outer and inner genes are transcribed in the clockwise and counterclockwise directions, respectively. The functional classification of the genes is shown in the left bottom corner.

A total of 133 genes were annotated in the assembled chloroplast genome, including 87 protein-coding genes, 38 transfer RNA genes and eight ribosomal RNA genes. There are 11 cis-splicing genes including rps16, atpF, rpoC1, petB, ycf3, clpP, petD, rpl6, rpl2, ndhB, and ndhA (Figure S2A) and one trans-splicing genes rps12 (Figure S2B). Most of the genes occurred in a single copy, while 20 genes occurred in double copies (rps19, trnH-GUG, rpl2, rpl23, trnL-CAU, ycf2, trnL-CAA, ndhB, rps7, rps12, trnV-GAC, rrn16, trnI-GAU, trnA-UGC, rrn23, rrn4.5, rrn5, trnR-ACG, trnN-GUU, and ycf1). There were 9 genes with one intron (rps16, atpF, rpoC1, petB, petD, rpl6, rpl2, ndhB, and ndhA), and 3 genes with two introns (rps12, ycf3, and clpP).

Phylogenetic analysis of H. flavum in the family Zingiberaceae

Based on the chloroplast genome dataset, we generated a well-supported phylogenetic tree (Figure 3). The inferred phylogenetic tree indicated that the selected species from the Zingiberaceae were clustered within a lineage distinct from the outgroups. Within Zingiberaceae, the genus Hedychium forms a monophyly, and is closely related to the genus Roscoea. The phylogenetic tree also shows a close affinity of H. flavum and H. neocarneum with 100% bootstrap support.

Figure 3. The maximum likelihood tree of H. flavum and its related relatives based on the complete chloroplast genomes. Bootstrap values were shown next to the nodes. The following sequences were used: Alpinia chinensis NC_050165 (Mei et al. 2020), A. katsumadae NC_048461 (Li et al. 2020), A. kwangsiensis MZ066612 (Zhang et al. 2021), Amomum compactum NC_036992 (Wu et al. 2018), A. krervanh NC_036935 (Wu et al. 2017), Boesenbergia kingii MW326451 (Liang and Chen 2021), B. rotunda NC_066450 (Liew et al. 2022), Curcuma flaviflora NC_028729 (Zhang et al. 2016), C. kwangsiensis NC_046685 (Gui et al. 2020), C. phaeocaulis NC_045242, C. wenyujin NC_045241 (Kim et al. 2021), C. yunnanensis NC_050037 (Liang et al. 2020), Globba lancangensis MT473704, G. marantina MT473705, G. multiflora MT473706, G. schomburgkii MK262735 (Li et al. 2021), H. coccineum MT473708 (Li et al. 2021), H. coronarium MW769779 (Yang et al. 2021), H. neocarneum MT473709 (Li et al. 2021), H. spicatum NC_047248 (Unpublished), H. villosum NC_060763 (Yang et al. 2021), Kaempferia elegans NC_040852, K. galanga NC_040851 (Li et al. 2019), Lanxangia tsao-ko MK926774 (Ma and Lu 2020), Musa aurantiaca NC_058957 (Feng et al. 2020), M. balbisiana NC_028439 (Niu et al. 2018), M. itinerans NC_035723 (Zhang et al. 2019), Roscoea cautleyoides NC_070285 (Unpublished), R. debilis NC_070286 (Unpublished), R. forrestii NC_070287 (Unpublished), R. humeana NC_046582 (Zhu et al. 2019), Wurfbainia longiligularis NC_044774 (Cui et al. 2019), W. neoaurantiaca NC_057208 (Li et al. 2019), W. villosa NC_044746 (Cui et al. 2019), Zingiber mioga NC_057615 (Unpublished), Z. recurvatum MT473712 (Li et al. 2021), Z. striolatum NC_065159 (Tian et al. 2023), Z. teres NC_062457 (Unpublished).

Discussion and conclusion

In the present study, the chloroplast genome of H. flavum was sequenced, assembled, and annotated for the first time. The results showed that the genome size, overall GC content, genome quadripartite structure, and gene composition in the H. flavum chloroplast genome were highly similar to chloroplast genome of other species of Hedychium (Li et al. 2019; Yang et al. 2021). These results suggested that the chloroplast genomes of Hedychium species were highly conserved at the genus level. The IR regions have a higher GC content (41.1%) than LSC (33.9%) and SSC (29.6%). The higher GC content in the IR region may be due to the presence of rRNA genes, while the SSC region contains most of the NADH genes (Cai et al. 2006).

Phylogenetic analysis demonstrated that the genus Hedychium is monophyletic, which is consistent with the results of phylogenetic analysis based on nuclear ITS, chloroplast matK/trnL-trnF sequences, and single nucleotide polymorphism (SNP) matrix of chloroplast genome (Wood et al. 2000; Ngamriabsakul et al. 2004; Li et al. 2021). Based on the phylogenetic study of chloroplast genome SNP matrix, there is a close relationship between Hedychium and Globba (Li et al. 2021). However, our study showed that Hedychium is closely related to Roscoea, which is inconsistent with the previous findings. Phylogenetic analysis also showed a close affinity of H. flavum and H. neocarneum. However, due to the limited sequence of Hedychium cp genomes, the phylogenetic relationship in the genus requires further study. This study provides the cp genome information of H. flavum, which would contribute to the species identification and phylogenetic analysis within Hedychium and Zingiberaceae.

Author’s contribution statement

Yong-Hong Zhang designed the research and revised the manuscript. Mei-Fei Li analyzed data and prepared a preliminary manuscript. Tao Xiao analyzed data and revised the manuscript. All authors read and approved the final manuscript, and agreed to be accountable for all aspects of the work.

Ethic statement

This research does not involve ethical issues. H. flavum does not belong to the China Species Red List. The collection of plant material carried out in accordance with guidelines provided by the authors’ institution (Yunnan Normal University) and national regulations.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The data that support the findings of this study are openly available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov/) with accession number OQ308833. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA941112, SRR23703126, SAMN33590324, respectively. The voucher specimens were deposited in the Herbarium of Yunnan Normal University (YNUB, Website: https://life.ynnu.edu.cn/, Contact: Jian-Lin Hang, Email: [email protected]) with the voucher number: LMF06, which is identified by Dr. Yong- Hong Zhang.

Additional information

Funding

This study is supported by the National Natural Science Foundation of China. [31760048].