The Complete Chloroplast Genome of Erythrina variegata L. (Papilionoideae, Fabaceae)

Abstract

ABSTRACT

Erythrina variegata L. 1754, a thorny deciduous tree of Fabaceae, contains various chemical compounds such as alkaloids, flavonoids, and triterpenoids and exhibits anti‐depressant, anti‐inflammatory, and antidiabetic activities. However, genomic data of E. variegata are limited. In this study, the complete chloroplast genome of E. variegata was sequenced and characterized using Illumina sequencing platform. The chloroplast genome of E. variegata was 152,351 bp in length and consisted of a large single copy (82,907 bp), a small single copy (26,309 bp), and two inverted repeat regions (16,826 bp). There were 79 protein‐coding genes, 30 transfer RNA genes, and four ribosomal RNA genes. Comparative analysis revealed high conservation of chloroplast genomes among Erythrina species regarding genome size, structure, and gene content. The phylogenetic study also indicated a close relationship between E. variagata and E. sanwicensis. This study provides initial plastome data for further genomic studies examining E. variegata and related species in Fabaceae.

Introduction

Chloroplast is an essential organelle of angiosperms that is responsible for photosynthesis (Shanker et al. 2022). Chloroplast has its own genomes (cpDNA) that are usually 120–160 kb in length and encode 80 protein-coding genes, 30 transfer RNA genes, and four ribosomal RNA genes (Daniell et al. 2016). The genomic information of the chloroplast is crucial for elucidating the evolutionary history of angiosperms. For example, the backbone phylogenetic tree of land plants and related species was established based on the plastome data (Gitzendanner et al. 2018). Additionally, the sequences of cpDNA were used for mining molecular markers, exploring population genetics, and developing biotechnology (Kumar and Ling 2021; Li, Zheng, and Huang 2020; Olejniczak et al. 2016; Song et al. 2023). It is clear that chloroplast genome data are precious for genomic studies of angiosperms.

Erythrina L. is a genus of Fabaceae and contains 124 species distributed in tropical and subtropical regions (POWO 2024). Members of Erythrina possess various metabolites for flavonoids, alkaloids, and triterpenoids; therefore, they exhibit anti-inflammatory, antidiabetic, estrogenic, antifungal, antidepressant, anticancer, and antibacterial activities (Susilawati et al. 2023). For example, the lectin extracted from seeds of Erythrina senegalensis exhibited antimicrobial activities (Enoma et al. 2023). Similarly, the bark of Erythrina suberosa had cytotoxic and antimicrobial features (Ahmed et al. 2020). A similar pattern was also found in E. variegata of which the bark contained alkaloids, phenols, and flavonoids and showed an enhancement of memory, anti-inflammatory, and antidiabetic activities (Biradar et al. 2022; Santhiya et al. 2016). In various African countries, Erythrina abyssinica was used as a traditional herbal medicine for treatment of malaria, bacterial infection, skin injury, diarrhea, tuberculosis, diabetes, and meningitis (Obakiro et al. 2021). Another example is the traditional use of Erythrina velutina in Brazil for insomnia, toothache, headache, cough, and fever (Adetunji et al. 2024). Last but not least, traditional medicines made of Erythrina variegata plant were used for anti-asthmatic, antiepileptic, antiseptic, and toothache treatments in India (Karunanithi, Rajkishore, and Radharamalingam 2017). Despite reported pharmacological features, only three chloroplast genomes of Erythrina species have been reported (Choi et al. 2022; McAssey et al. 2023; Oyebanji et al. 2020). The current genomic data of Erythrina genus is deficient to elucidate its evolutionary history. Therefore, in this study, we sequenced and characterized the complete chloroplast genome of Erythrina variegata to enlarge the genomic data of Erythrina genus and to provide essential information to trace the evolutionary pattern of this interesting genus in further studies. Additionally, phylogenetic relationships between E. variegata and related species in Fabaceae were reconstructed.

Materials and Methods

The fresh leaves of Erythrina vagiegata were collected at Tra Vinh University (9°55′25.1″ N/106°20′47.5″ E) and dried with silica gel beads (Figure 1). The dried leaves were stored at −81°C for further experiments. The sample was deposited to School of Agriculture and Aquaculture, Tra Vinh University under voucher number of SAA-TVU-2024-0110147 (contact person: Dr. Nguyen Nhat Nam, [email protected]). The total genomic DNA was extracted using DNeasy Plant Pro Kits (Qiagen, USA) following the manufacturer's instructions. The quality of DNA samples was checked using NanoDrop One Microvolume UV–Vis Spectrophotometer (Thermo Fisher Scientific, USA) and gel electrophoresis. The high-quality DNA sample (having a concentration of at least 100 ng/μL and a clear band on the 1% agarose gel) was applied for the Nextseq 550 sequencing system (Illumina, USA) to generate 150 bp paired-end reads.

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The raw sequencing data were checked and filtered using fastp v0.23.4 with default options for trimming and filtering processes (Chen et al. 2018). Then, NOVOPlasty v4.5.3 was used to assemble the complete chloroplast genome of E. variegata with the chloroplast genome of E. herbacea (GenBank accession number OL672880) as a reference and default settings (Dierckxsens, Mardulyn, and Smits 2016). The gene content of the newly completed cpDNA was annotated using Geseq (Tillich et al. 2017) and then checked using Geneious Prime v2024.2 () and tRNAscan-SE 2.0 (Chan et al. 2021). The map of the cpDNA and illustration of trans- and cis-splicing genes were generated using OGDRAW v1.3.1 and CPGView, respectively (Greiner, Lehwark, and Bock 2019; Liu et al. 2023).

For phylogenetic analysis, the complete chloroplast genomes of 13 species were downloaded from the GenBank database with Robinia pseudoacacia (GenBank accession number KJ468102) serving as an outgroup according to a previous phylogenetic study of papilionoid legume (Table 1) (Choi et al. 2022). A total of 78 protein-coding regions (except infA gene which is pseudogenized and lost in the surveyed taxa) were extracted and aligned using Muscle5 embedded in Geneious Prime v2024.2 (Edgar 2022). The aligned sequences were subjected to jModeltest2 v2.1.10 to identify the best substitution model for elucidating the phylogeny with default settings and Best option for Base tree search (Darriba et al. 2012). Consequently, the model of GTR + I + G (Akaike Information Criterion) was chosen for running phylogenetic analysis using maximum likelihood (ML) and Bayesian inference (BI) methods. The ML method was conducted using IQ-TREE 2 with substitution model of GTR + I + G, ultrafast option for bootstrap analysis with 1000 replicates and other default settings (Minh et al. 2020). Meanwhile, the BI method was performed using MrBayes v3.2.7 with GTR + I + G substitution model and 1,000,000 generations (Ronquist et al. 2012). Additionally, 25% of samples generated by MrBayes were discarded by default. The phylogenetic trees were illustrated using Figtree v1.4.4 ().

TABLE 1 List of species used for phylogenetic analysis.

Species	Accession number	Tribe	Subfamily	Family
Robinia pseudoacacia	KJ468102	Robinieae	Papilionoideae	Fabaceae
Indigofera tinctoria	KJ468098	Indigofereae	Papilionoideae	Fabaceae
Platycyamus regnellii	OL672877	Millettieae	Papilionoideae	Fabaceae
Cullen corylifolium	MK069582	Psoraleeae	Papilionoideae	Fabaceae
Campylotropis macrocarpa	MG867566	Desmodieae	Papilionoideae	Fabaceae
Phyllodium pulchellum	MN614126	Desmodieae	Papilionoideae	Fabaceae
Hylodesmum podocarpum	MT120798	Desmodieae	Papilionoideae	Fabaceae
Psophocarpus tetragonolobus	MN966643	Phaseoleae	Papilionoideae	Fabaceae
Phaseolus acutifolius	OL672884	Phaseoleae	Papilionoideae	Fabaceae
Apios americana	KF856618	Phaseoleae	Papilionoideae	Fabaceae
Erythrina sandwicensis	OQ870895	Phaseoleae	Papilionoideae	Fabaceae
Erythrina herbacea	OL672880	Phaseoleae	Papilionoideae	Fabaceae
Erythrina crista-galli	MN966629	Phaseoleae	Papilionoideae	Fabaceae
Erythrina variegata	PQ469842	Phaseoleae	Papilionoideae	Fabaceae

Results and Discussion

The complete chloroplast genome of E. variegata (average coverage 618×) was 152,351 bp in length and had a quadripartite structure (Figure 2). This genome consisted of four regions, including a large single copy (LSC), a small single copy (SSC), and two inverted repeat (IR) regions, of which the lengths were 82,907 bp, 16,826 bp, and 26,309 bp, respectively. Additionally, the cpDNA of E. variegata was composed of 79 protein-coding genes, 30 transfer RNA genes, and four ribosomal RNA genes (Table 2). There were 19 duplicated genes in the IR regions, including rrn4.5, rrn5, rrn16, rrn23, trnA-UGC, trnL-CAA, trnI-GAU, trnI-CAU, trnN-GUU, trnR-ACG, trnV-GAC, rpl2, rpl22, rps7, rps12, rps19, ndhB, ycf1, and ycf2. Additionally, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, trnV-UAC, rpl2, rpl16, rps16, rpoC1, petB, petD, atpF, ndhA, and ndhB had one intron whereas pafI and clpP1 had two introns (Figure 3A). The rps12 gene was a trans-splicing gene, of which exon 2 and exon 3 were located in the IR regions (Figure 3B).

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TABLE 2 Gene composition of Erythrina variegata chloroplast genome.

Groups of genes	Name of genes
Ribosomal RNAs	rrn4.5a, rrn5a, rrn16a, rrn23a
Transfer RNAs	trnA-UGCa,b, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-UCCb, trnG-GCC, trnH-GUG, trnI-GAUa,b, trnK-UUUb, trnL-CAAa, trnL-UAAb, trnL-UAG, trnfM-CAU, trnI-CAUa, trnM-CAU, trnN-GUUa, trnP-UGG, trnQ-UUG, trnR-ACGa, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GACa, trnV-UACb, trnW-CCA, trnY-GUA
Large units of ribosome	rpl2a,b, rpl14, rpl16b, rpl20, rpl22a, rpl23, rpl32, rpl33, rpl36
Small units of ribosome	rps2, rps3, rps4, rps7a, rps8, rps11, rps12a,c, rps14, rps15, rps16b, rps18, rps19a
RNA polymerase	rpoA, rpoB, rpoC1b, rpoC2
Translational initiation factor	infA
Subunit of photosystem I	psaA, psaB, psaC, psaI, psaJ, pafIc, pafII
Subunit of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, pbfI, psbM, psbT, psbZ
Subunit of cytochrome	petA, petBb, petDb, petG, petL, petN
Subunit of ATP synthases	atpA, atpB, atpE, atpFb, atpH, atpI
Large unit of Rubisco	rbcL
Subunit of NADH dehydrogenase	ndhAb, ndhBa,b, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
Maturase	matK
Envelope membrane protein	cemA
Subunit of acetyl-CoA	accD
C-type cytochrome synthesis gene	ccsA
ATP-dependent protease subunit P	clpP1 c
Hypothetical proteins and conserved reading frames	ycf1a, ycf2a

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Comparative analysis revealed high conservation in chloroplast genomes of examined Erythrina species regarding gene content and order, genome structure, GC content, and genome size (Table 3). Although an identical number of genes was found, the cpDNAs of four Erythrina taxa were slightly different in length. Specifically, the biggest cpDNA belonged to E. variegata whereas E. crista-galli had the smallest cpDNA. Additionally, the size of LSC, SSC, and IR regions ranged from 82,389 bp to 82,907 bp, from 16,756 bp to 16,826 bp, and from 26,303 bp to 26,360 bp respectively. Among four Erythrina cpDNA, the junction between LSC and IR regions was mostly located in rps19 genes. Only E. sandwicensis had the LSC/IR junction in the intergenic spacer between rps19 and rpl22 genes. In the case of the boundary between the SSC and IR regions, three species exhibited the overlap of ycf1 and ndhF genes. In contrast, the SSC/IR junction of E. crista-galli was located within ycf1 gene (Table 3).

TABLE 3 Features of chloroplast genomes among Erythrina species.

Species	Erythrina sandwicensis	Erythrina herbacea	Erythrina crista-galli	Erythrina variegata
Accession number	OQ870895	OL672880	MN966629	PQ469842
Total length (bp)	152,399	151,821	151,751	152,351
Total % GC	35.1	35.3	35.3	35.1
LSC length (bp)	82,865	82,403	82,389	82,907
LSC % GC	32.4	32.7	32.8	32.5
SSC length (bp)	16,814	16,784	16,756	16,826
SSC % GC	28.3	28.7	28.8	28.4
IR length (bp)	26,360	26,317	26,303	26,309
IR % GC	41.3	41.4	41.4	41.4
Protein-coding gene	79	79	79	79
tRNAs	30	30	30	30
rRNAs	4	4	4	4
LSC/IR junction	IGS (rps19-rpl22)	rps19 (68 bp)	rps19 (68 bp)	rps19 (67 bp)
SSC/IR junction	Overlap (ycf1–1295 bp/ndhF—28 bp)	Overlap (ycf1–1280 bp/ndhF—2 bp)	ycf1 (1249 bp)	Overlap (ycf1–1280 bp/ndhF—2 bp)

Previous studies examining chloroplast genomes in Fabaceae demonstrated the conservation of genomic features within the genera. For example, comparative chloroplast genome analyses of six Hedysarum and 22 Campylotropis confirmed the quadripartite structure or loss of one IR region, gene content, and genome size within these genera (Feng et al. 2022; Juramurodov et al. 2023). Similarly, another comparative genomic analysis of 35 Dalbergia species disclosed the similarity of genome features, including junctions between LSC, SSC, and IR regions, the numbers of protein-coding genes, tRNA genes, and rRNA genes, and codon usage (Hong et al. 2022). These previous results demonstrated the high conservation of chloroplast genomes among the genera of Fabaceae. In the current study, the phenomenon of conservation was also found; however, only four Erythrina species were observed. Therefore, further studies examining 124 members of Erythrina species should be conducted to explore the evolutionary history of this interesting genus of Fabaceae.

The ML and BI methods resulted in the same topology of phylogenetic trees (Figure 4). The monophyly of Erythrina species was highly supported (bootstrap value = 100 and posterior probability = 1). Within the Erythrina genus, E. variegata had a close relationship with E. sanwicensis. However, members of the Phaseoleae tribe exhibited a polyphyletic trend, in which Phaseolus acutifolius formed a clade with Cullen corylifolium of the Psoraleeae tribe. Similarly, Apios americana was sister to groups of Desmodieae, Phaseoleae, and Psoraleeae tribes. The non-monophyletic patterns of members in Papilionoideae were also found in previous studies based on genomic and transcriptomic data (Choi et al. 2022; Zhao et al. 2021). These results raised a question about the tribal classification of Papilionoideae and require more data in further studies to fulfill the taxonomic treatments within Fabaceae.

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Conclusion

In the current study, we reported the complete chloroplast genome of Erythrina variegata using the next-generation sequencing method. Comparative genomic analysis revealed the similarities and differences among four Erythrina species regarding genome size, genome structure, GC content, and gene content. Additional phylogenetic analysis provided an initial relationship between E. variegata and related taxa based on 78 protein-coding regions of cpDNA. The outcomes of this study provided essential information for further genomic studies of E. variegata and other members of Fabaceae.

Author Contributions

Nguyen Nhat Nam: conceptualization (equal), data curation (lead), formal analysis (equal), writing – original draft (lead). Nguyen Ngoc Trai: data curation (supporting), methodology (supporting), software (supporting), visualization (equal), writing – review and editing (supporting). Nguyen Phuong Thuy: conceptualization (equal), methodology (supporting), software (supporting), visualization (equal). Le Quoc Duy: methodology (supporting), visualization (equal), writing – review and editing (supporting). Pham Nguyen Tuong Van: methodology (supporting), visualization (equal), writing – review and editing (supporting). Tan Tai Nguyen: conceptualization (equal), investigation (equal), writing – review and editing (supporting). Hoang Dang Khoa Do: conceptualization (equal), formal analysis (equal), methodology (lead), software (lead), writing – review and editing (supporting).

Acknowledgments

The authors thank Nguyen Tat Thanh University for supporting high-performing computing devices. The authors would like to express their sincere gratitude to Tra Vinh University for providing the opportunity to carry out this work.

Conflicts of Interest

The authors declare no conflicts of interest. No permission is needed to collect the samples of E. variegata for research purposes at Tra Vinh University.

Data Availability Statement

The complete chloroplast genome of Erythrina variegata was submitted to GenBank under accession number PQ469842. The raw sequencing data were also deposited to NCBI under the accession number PRJNA1183432.

References

Adetunji, T. L., M. A. Acho, V. O. Samuel, C. R. Ohoro, and M. Ramulondi. 2024. “Erythrina Velutina Willd.: A Review of Its Traditional Uses, Phytochemistry, Pharmacology, and Toxicology.” Journal of Ethnopharmacology 319, no. 2: [eLocator: 117273]. [DOI: https://dx.doi.org/10.1016/j.jep.2023.117273].