Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

Complete chloroplast genome of the Jewel Orchid,

Anoectochilus formosanus (Orchidaceae) and its relatives

Minh Phuong Nguyen1; https://orcid.org/0000-0003-1768-5555

Thi Huong Trinh1; https://orcid.org/0000-0002-5940-6588

Khuong Duy Dao2, 3; https://orcid.org/0009-0005-1607-2871

Phuc Loi Luu2, 4; https://orcid.org/0000-0001-8045-718X

Viet The Ho1*; https://orcid.org/0000-0003-4863-0530

1 Faculty of Biology and Environment, Ho Chi Minh City University of Industry and Trade, 140 Le Trong Tan, Tan Phu

district, Ho Chi Minh City 700000, Vietnam; phuongnguyen@huit.edu.vn, trinhth@huit.edu.vn, thehv@huit.edu.vn

(*Correspondence)

2 Tam Anh Research Institute (TamRI), 2B Pho Quang Street, Ward 2, Tan Binh District, Ho Chi Minh City 700000,

Vietnam; loilp@tamri.vn, duydk@tamri.vn

3 Faculty of Biology & Biotechnology, The University of Science, Viet Nam National University Ho Chi Minh City, 227

Nguyen Van Cu, Ward 4, District 5, Ho Chi Minh City 700000, Vietnam; duydk@tamri.vn

4 Mathematics Department, Faculty of Fundamental Sciences, University of Medicine and Pharmacy at Ho Chi Minh

City (UMP), 217 Hong Bang Street, Ward 11, District 5, Ho Chi Minh City 700000, Vietnam; luuphucloi@ump.edu.vn

Received 31-VIII-2023. Corrected 06-II-2024. Accepted 16-V-2024.

ABSTRACT

Introduction: Anoectochilus formosanus is a highly valuable herb known for its efficacy in treating a wide range

of diseases. However, the current methods used to differentiate this species from others within the same genus

are not effective due to the high similarity in morphological characteristics and DNA barcode sequences among

these species.

Objective: To characterize the chloroplast (cp) genome to distinguish A. formosanus at species or isolation levels.

Methods: The complete cp genome was sequenced using next-generation sequencing technology, annotated, and

compared with published cp genomes of various species within the Anoectochilus genus.

Results: The complete cp genome of A. formosanus is 152 658 bp in size, consisting of a large and small copy of

82 692 bp and 17 346 bp, respectively, separated by inverted repeats of 26 310 bp. Within the cp genome, there are

a total of 141 genes, including 92 protein-coding genes, 10 rRNA genes, and 39 tRNA genes. This genome con-

tains a total of 80 simple sequence repeats, with 50 long repeats. Through phylogenetic analysis, a close relation-

ship was observed between A. formosanus in Vietnam and A. formosanus samples from China (NC_061756.1).

However, genomic comparisons highlighted differences between the two cp genomes, specifically in their reverse

repeat sequences.

Conclusion: These findings reveal distinct variations in the cp genome of A. formosanus in Vietnam, offering

valuable insights into the taxonomy, plant identification, breeding, and conservation programs related to this

herb in Vietnam.

Key words: Anoectochilus formosanus; chloroplast genome; medicinal plant; next generation sequencing; simple

sequence repeat.

https://doi.org/10.15517/rev.biol.trop..v72i1.56423

BIOTECHNOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

INTRODUCTION

Anoectochilus formosanus is a medicinal

plant that belongs to the Orchidaceae fam-

ily (Shiau et al., 2002). It has been utilized for

centuries as a traditional medicine due to its

powerful healing properties. In recent times,

there has been a growing interest in scientific

research to explore the pharmacological poten-

tial of this plant. Several studies have demon-

strated that extracts from A. formosanus exhibit

substantial antioxidant activity and effectively

reduce the production of inflammatory (Ho

et al., 2018; Lin et al., 1993; Shiau et al., 2002).

Moreover, the extract has shown promising

anti-hyperglycemic effects in diabetic rats by

significantly reducing blood sugar levels and

improving insulin resistance, indicating its

potential therapeutic application in diabetes

treatment in mice (Shih et al., 2002; Tang et

al., 2018). Additionally, it possesses various

pharmacological activities, such as antioxidant,

anti-inflammatory, anticancer, and neuropro-

tective effects (Nguyen et al., 2023b; Wang et

al., 2002). The exceptional medicinal value

of this herb can be attributed to its chemical

composition, which includes phenolic acids,

flavonoids, diarylpentanoid, kinsenone, and

polysaccharides (Chiang & Lin, 2017; Wang et

al., 2002; Xu et al., 2022).

A. formosanus is highly sought after but

faces limited supply, raising concerns among

conservationists and researchers regarding

overharvesting. The species is on the brink

of extinction due to multiple factors, includ-

ing habitat destruction, overexploitation, illegal

trade, and climate change (Jiang et al., 2015,

Kumar & Gale, 2020; Ma et al., 2010; Zhang

et al., 2013). A previous study using ISSR and

AFLP markers revealed that A. formosanus

populations exhibit relatively low genetic diver-

sity, indicating their susceptibility to environ-

mental changes and overexploitation (Lin et al.,

2007). These findings underscore the urgent

need for conservation efforts and the adoption

of sustainable practices to safeguard A. formosa-

nus populations from further depletion and the

risk of extinction.

Traditionally, morphological identification

has been utilized for the conservation-oriented

identification of jewel orchids. However, this

RESUMEN

Genoma completo del cloroplasto de la orquídea joya, Anoectochilus formosanus (Orchidaceae) y sus afines

Introducción: Anoectochilus formosanus es una orquídea herbácea muy valiosa, conocida por su eficacia en el

tratamiento de una amplia gama de enfermedades. Sin embargo, los métodos actuales utilizados para diferenciar

esta especie de otras dentro del mismo género no son efectivos debido a la gran similitud en las características

morfológicas y las secuencias de códigos de barras de ADN entre estas especies.

Objetivo: Caracterizar el genoma del cloroplasto (cp) para distinguir A. formosanus a nivel de especie o de

aislamiento.

Métodos: El genoma completo del cp se secuenció utilizando tecnología de secuenciación de nueva generación, se

anotó y se comparó con los genomas del cp publicados de varias especies del género Anoectochilus.

Resultados: El genoma del cp completo de A. formosanus tiene un tamaño de 152 658 pb y consta de una copia

grande y pequeña de 82 692 pb y 17 346 pb, respectivamente, separadas por repeticiones invertidas de 26 310 pb.

Dentro del genoma del cp, hay un total de 141 genes, incluidos 92 genes codificadores de proteínas, 10 genes de

ARNr y 39 genes de ARNt. Este genoma contiene un total de 80 repeticiones de secuencia simple, con 50 repe-

ticiones largas. Mediante análisis filogenético, se observó una estrecha relación entre A. formosanus de Vietnam

y muestras de A. formosanus de China (NC_061756.1). Sin embargo, las comparaciones genómicas resaltaron

diferencias entre los dos genomas del cp, específicamente en sus secuencias de repetición invertida.

Conclusión: Estos hallazgos revelan distintas variaciones en el genoma del cp de A. formosanus en Vietnam, lo

que ofrece información valiosa sobre la taxonomía, la identificación de plantas, la reproducción y los programas

de conservación relacionados con esta hierba en Vietnam.

Palabras clave: Anoectochilus formosanus; genoma del cloroplasto; planta medicinal; secuenciación de próxima

generación; repetición de secuencia simple.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

approach has limitations, particularly when

closely related species share similar morpho-

logical characteristics (Suetsugu et al., 2022;

Tran et al., 2022). Certain orchid species exhibit

comparable leaf colors and patterns, creat-

ing challenges for differentiation. Furthermore,

some orchid species display considerable varia-

tion in morphological features, including leaf

shape and size, which further complicates

identification (Huynh et al. 2019; Nguyen et

al., 2023a). Due to the significant overlap in

their morphological traits, the reliability of

relying solely on morphological identification

for jewel orchids, especially for closely related

species with similar morphological character-

istics, is questionable (Bhattacharjee & Chow-

dhery, 2013; Hu et al., 2016; Ong & Lee, 2019;

Suetsugu et al., 2022).

In recent years, DNA barcodes have gained

significant popularity for the identification and

classification of various plant species, includ-

ing orchids. However, this method has cer-

tain limitations, especially when dealing with

closely related species that share similar DNA

sequences. Ho et al. (2021) discovered that

commonly used DNA barcoding markers, such

as rbcL and matK, have limited and variable

discriminatory power in distinguishing closely

related jewel orchid species. Furthermore, some

orchid species exhibit highly variable DNA

sequences, which can further complicate their

identification through DNA barcoding (Huynh

et al., 2019). Additionally, there is considerable

overlap in the DNA sequences of certain jewel

orchid species, rendering DNA barcoding an

unreliable method (Chen & Shiau, 2015; Ho et

al., 2021; Zhang et al., 2019).

The accessibility and affordability of next-

generation sequencing (NGS) technology have

revolutionized the characterization of com-

plete chloroplast (cp) genomes, proving to be

an effective method for identifying and clas-

sifying orchids (Konhar et al., 2019; Tang et

al., 2021; Yang et at., 2013). The cp genome,

inherited maternally from the parent, evolves

slowly, making it a valuable tool for study-

ing molecular evolution, population genetics,

phylogenetics, botany, and genomic evolution

(Yang et at., 2013). Ho and colleagues identi-

fied the differences in cp genomes of Ludisia

discolor accession collected in Vietnam with

three assessions from China (Ho et al., 2023).

These findings emphasize the potential of NGS

technology in identifying and classifying jade

orchids based on their cp genome. To our best

knowledge, only one cp genome of A. formo-

sanus with China origin has been published

with NC_061756.1 accession number in the

National Center for Biotechnology Informa-

tion (NCBI) genbank. Although this medicinal

plant is considered as endemic of Vietnam but

not such information available. In our spe-

cific study, we utilized NGS to sequence the cp

genome of A. formosanus samples collected in

Vietnam, comparing the results with published

sequences to identify distinctive genomic fea-

tures. This information holds valuable implica-

tions for the taxonomy, botanical identification,

breeding, and conservation programs of A.

formosanus in Vietnam.

MATERIALS AND METHODS

Sample collection, DNA extraction,

library construction, and sequencing: Sam-

ples of A. formosanus were provided by the

Biotechnology Center of Ho Chi Minh City

(HCMBIOTECH), the voucher sample is kept

at this center for conservation. Total DNA was

extracted from fresh leaves using the Isolate II

Plant DNA Kit (Bioline, UK). DNA quality and

quantity were determined by 1 % gel electro-

phoresis and Nanodrop, respectively (Thermo-

Scientific, Delaware, USA).

100-1 000 ng DNA that had undergone

quality control, was fragmented using acoustic

disruption with Covaris S220, followed by final

repair, dA tailing, adapter ligation, and purifi-

cation. The purified DNA was then selected for

the appropriate size before being PCR amplified

for library construction. Preliminary quanti-

fication and library dilution were carried out

using Qubit3.0, followed by the use of Agilent

2 100 to determine the insert size and nucleic

acid concentration of the resulting library sam-

ple. The effective concentration of each sample

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

library in the mixture was determined by qPCR

(Realtime PCR 7 500-Applied Biosystem, USA)

before sequencing to ensure the accuracy of the

sample concentration and the reliability of the

sequencing data.

Base calling was accomplished using the

RTA software integrated with the Illumina

Novaseq 6 000 sequencer, which converted the

four fluorescence signals obtained from the

Charge-Coupled Device (CCD) to binary bcl

data in real-time. The bcl data were then trans-

formed to a fastq file using bcl2fastq (v.2.17),

which is part of the software package provided

by Illumina. Concurrent demultiplexing of the

data was carried out based on the index infor-

mation. Primary analysis was conducted using

the built-in High-Content Screening (HCS)

sequencer software (Ogier & Dorval, 2012) to

determine whether the read data passed the

quality filter, based on the signal quality of the

first 25 cycles. The quality of the raw reads was

initially assessed using FastQC in the Galaxy

portal (The Galaxy Community, 2022) and

then submitted to the Sequence Read Archive

(SRA) database in NCBI (2009) under the

PRJNA982609 project.

Chloroplast genome assembly and

annotation: To eliminate adapter content,

the sequence data were processed using Trim

Galore (v.0.6.7) on Galaxy (Li et al., 2023),

which is a tool built into The Galaxy Server.

Reference-based assembly was performed using

the HISAT2 (v.2.2.1) tool, aligning the data

against the A. formosanus reference sequence

with NCBI accession number NC_061756 to

generate BAM files. The Pilon (v.1.21) tool was

then employed with the assembly and mapping

information to identify and correct any poten-

tial problems with the assembly and to generate

a FASTA file for further analysis. To annotate

and localize protein-coding genes, rRNA, and

tRNA in the cp genome, the Chloroplot pro-

gram (Zheng et al., 2020) was utilized.

Comparative analysis among species in

Anoectochilus genus: To align the cp genome

sequences, the MAFFT (v.7) program (Katoh &

Standley, 2013) was employed using the param-

eters according to Katoh et al. (2019). The

resulting alignment was then used to identify

DNA polymorphisms, and nucleotide diver-

sity (Pi) was calculated using DnaSP (v.6.12.03)

software (Rozas et al., 2017). The genetic dif-

ferences between cp genomes were determined

using the Kimura two-parameter algorithm of

MEGA X software, based on the P-value (P

distance) calculated from the evolutionary dis-

tance between sequences.

The comparison of cp genomes among

related species was carried out using the Shuﬄe-

LAGAN mode of the VISTA program (Frazer et

al., 2004). The junctions of LSC/IRB/SSC/IRA

were visualized using IRscope (Amiryousefi et

al., 2018) based on the cp genome annotations

of these related species available in Genbank.

The MIcroSAttelite (MISA) identification tool

was employed to identify SSR motifs, with ten

repeat parameters for mononucleotides, six

for dinucleotides, five for trinucleotides, four

for tetra-nucleotides, and three for penta- and

hexa-nucleotides, as described by Beier et al.

(2017). The REPuter software was used to

determine long repeat regions with repeat size

≥ 30 bp and a minimum of 90 % identity, iden-

tifying four types of repeats, namely forward

(F), reverse (R), complement (C), and retro-

grade (P), as reported by Kurtz et al. (2001).

Phylogenetic analysis: The NCBI Gen-

bank (NCBI, 1982) was used to retrieve addi-

tional complete cp genome sequences of various

species of jewel orchid from the Anoectochilus

genus. Unverified sequences were excluded

from the analysis, and only one sequence was

randomly chosen for further analysis in cases

where multiple sequences were available for a

particular species. The following eight com-

plete cp genome sequences were obtained:

NC_03895.1 A. emeiensis; NC_066958 A.

burmannicus; NC_061758 A. roxburghii;

NC_061756 A. formosanus; NC_054353 .1 A.

zhejiangensis; MW589501.1 A. hainanensis;

MW589500.1 A. chapaensis; and NC_033895.1

A. emeiensis.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

The MAFFT alignment were utilized to

establish the phylogenetic relationships between

the cp genomes. A phylogenetic tree of nine cp

genomes was constructed using the Neighbour

Joining (NJ) method and Maximum Likeli-

hood (ML) methods representing distance and

discrete character methods (Kang et al., 2017)

with 5 000 bootstrap replicates by using MEGA

X software. The Dendrobium sinense cp genome

(OM792979.1), a common ornamental orchid,

was used as an outgroup. The Kimura 2-param-

eter nucleotide substitution model, which is

commonly employed to estimate genetic differ-

ences resulting from nucleotide substitutions

(Nishimaki & Sato, 2019), was applied to the

phylogenetic trees.

RESULTS

Genome, sequence assembly, and fea-

tures of chloroplast: A total of 6.9 GB of 150

bp paired-end data was generated, resulting in

19 211 194 reads with a Phred scores of 95.26 %

of reads are greater than Q20. The GC content

of the plastome was approximately 37 %. Upon

assembly, the cp genome map exhibited a con-

served circular structure with a total length of

152 658 bp. The genome comprised four dis-

tinct parts, including a Large Single Copy (LSC)

region spanning 82 692 bp, a Small Single Copy

(SSC) region spanning 17 346 bp, and two

Inverted Repeat (IR) regions spanning 26 310

bp each. These IR regions were separated by the

LSC and SSC regions (Fig. 1).

Fig. 1. The cp genome map of Vietnam A. formosanus, generated with Chloroplot program, displays the genes transcribed

in clockwise and counterclockwise directions, depicted outside and inside of the circle, respectively. The LSC, SSC, IRA, and

IRB are labeled as the primary parts of the cp genome. The inner circle’s dark and light grey colors represent the GC and AT

content, respectively.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

Sequence annotation and comparison of

cp genomes: A total of 142 genes were anno-

tated in the obtained cp genome, including 92

protein-coding genes, 10 rRNA genes, and 39

tRNA genes. To compare the gene composition

of the cp genome, we examined eight other

Anoectochilus species, whose data had been

previously published on NCBI and are present-

ed in Table 1. The cp genome of A. formosanus

exhibited slight differences compared to the

other cp genomes, particularly in the number

of coding genes and tRNA genes. Surprisingly,

several variations were observed between the

A. formosanus specimen from Vietnam and the

one from China (NC_061765.1).

The divergence among the nine cp genomes

ranged from 0.000 to 0.005, as shown in Table 2.

The Dnasp program identified a total of 1 464

polymorphic sites from the aligned cp sequenc-

es of the Anoectochilus genus. The nucleotide

diversity value (Pi) was calculated to be 0.0036.

Repeat structure and Simple sequence

repeats: Among the nine jewel orchid species,

a total of 657 SSRs were detected, ranging from

68 SSRs in A. calcareus to 80 SSRs in VN_A_

formosanus, with an average of approximately

73 SSRs per cp genome (Fig. 2). Six types of

SSR motifs were identified: A, T, C, AT, TA,

and TTC. The most abundant mononucleotide

motifs were T and A, accounting for 64.2 %

(422 SSRs) and 28.0 % (184 SSRs), respectively.

Interestingly, only one PolyC motif was pres-

ent, while no polyG motif was detected in the

genome. Short polyA and polyT repeats are

commonly observed as SSRs in cp genomes,

whereas polyG or polyC repeats are rare (Lei et

al., 2016). Additionally, dinucleotide motifs (AT

and TA) and a trinucleotide motif (TTC) were

identified in relatively low frequencies.

The REPuter program was used to analyse

the nine cp sequences and assess the abun-

dance of four oligonucleotide repeat types:

Table 1

Size comparison of plastome features of nine Anoectochilus species.

Accession code Scientific name Genome

size (bp)

LSC size

(bp)

SSC size

(bp)

IRB size

(bp)

IRB size

(bp)

Coding

genes rRNA tRNA

VN_A_formosanus A. formosanus 152 658 82 692 17 346 23 610 23 610 92 10 39

NC_033895.1 A. emeiensis 152 650 82 670 17 342 26 319 26 319 93 8 46

NC_066958.1 A. burmannicus 152 868 82 733 17 473 26 331 26 331 89 8 38

NC_061758.1 A. roxburghii 152 821 82 693 17 488 26 320 26 320 91 8 37

NC_061756.1 A. formosanus 151 414 81 879 16 909 26 313 26 313 90 8 37

NC_054353.1 A. zhejiangensis 152 509 82 660 17 201 26 324 26 324 90 8 38

MW589501.1 A. hainanensis 152 645 82 881 17 626 26 069 26 069 90 8 38

MW589500.1 A. chapaensis 152 395 82 630 17 125 26 320 26 320 90 8 38

MT041259.1 A. calcareus 151 864 82 083 17 141 26 320 36 320 92 10 39

Table 2

Estimates of evolutionary divergence among cp genome sequences of nine Anoectochilus species

No Accession number Species 12345678

1 VN_A_formosanus A. formosanus

2 NC_033895.1 A. emeiensis 0.000

3 NC_066958.1 A. burmannicus 0.002 0.002

4 NC_061758.1 A. roxburghii 0.002 0.002 0.001

5 NC_061756.1 A. formosanus 0.002 0.002 0.001 0.001

6 NC_054353.1 A. zhejiangensis 0.006 0.005 0.005 0.005 0.005

7 MW589501.1 A. hainanensis 0.005 0.005 0.005 0.005 0.005 0.003

8 MW589500.1 A. chapaensis 0.005 0.005 0.005 0.005 0.005 0.003 0.000

9 MT041259.1 A. calcareus 0.005 0.005 0.004 0.004 0.005 0.005 0.004 0.005

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

forward (F), palindromic (P), reverse (R), and

complementary (C). The number and type of

repeat elements exhibited significant variation

among the nine cp genomes (Table 3), ranging

from 38 units in A. chapaensis to 53 units in

A. zhejiangensis.

IR contraction and expansion: Despite

the high conservation of genomic structure

and size in the nine Anoectochilus cp genomes,

the IR/SC boundary regions exhibited notable

differences (Fig. 3). Several genes, including

rpl22, ndhF, and ycf1, varied in length. Intrigu-

ingly, ndhF was absent in the IRb/SSC border

in only the A. hainanensis cp genome, indicat-

ing that the loss of this gene likely occurred

independently among jewel orchid species in

the Anoectochilus genus. Although the rpl22

gene was present in all LSC/IRb borders, this

gene was observed only in 4/9 cp genomes in

the IRa region, including A. burmannicus, A.

hainanensis, A. chapaensis, and A. zhejiangensis.

Fig. 2. The different simple sequence repeat types in the cp genomes of nine Anoectochilus species and D. sinense as an

outgroup.

Table 3

Number of repeated sequences in nine Anoectochilus cp genomes.

No Accession Forward vs.

Forward

Forward vs.

Complement

Forward vs.

Reverse

Forward vs.

Reverse Complement

1 VN_A_formosanus 8 7 13 22

2 NC_033895.1 17 0 24 9

3 NC_066958.1 17 2 19 12

4 NC_061758.1 12 5 14 18

5 NC_061756.1 10 7 13 20

6 NC_054353.1 10 3 23 17

7 MW589501.1 18 0 19 12

8 MW589500.1 17 1 10 10

9 MT041259.1 21 2 7 20

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We also noted that rpl2 gene is only appeared in

A. formosanus from Vietnam.

In order to align the cp genome of the

nine Anoectochilus species, the NC_061756.1

cp genome was employed as a reference in

sequence alignment with mMISTA software

(Fig. 4). Overall, the size and gene order of

the nine cp genomes analysed were found

to be conserved.

Phylogenetic relationship: The phyloge-

netic analysis of the nine Anoectochilus cp

genomes revealed a distinct clustering pattern

among jewel orchid species. The cp genomes

are divided into two main clades with high

bootstrap values (Fig. 5). In both phylogenetic

trees built by Neighbor- Joining phylogeny

(Fig. 5A) and Maximum Likelihood method

(Fig. 5B), A. formosanus accession from Viet-

nam and A. formosanus accession from China

(NC_061756.1) are formed a sub clade which

are sisters with another clade forming by three

accessions namely A. emeiensis (NC_033895.1),

A. roxburghii (NC_061758.1) and A. zheji-

angensis (NC_054353.1). Nevertheless, the

is slight difference between two phyloge-

nies where A. hainanensis (MW589501.1) is

grouped with other three accessions, namely

A. burmannicus (NC_066958.1), A. chapaensis

(MW589500.1) and A. calcareus (MT041259.1)

with the high bootstrap value at 99 to form the

second main clade using NJ method (Fig. 5A).

Whereas, by using ML method A. hainanensis

(MW589501.1) is clustered in different clade

which are closer to two A. formosanus acces-

sions (Fig. 5B).

DISCUSSION

Genetic variation in cp genomes: Orchi-

daceae is a large family of flowering plants

that comprises numerous endangered, rare,

and threatened species, including A. formo-

sanus. Due to human excavation and habitat

Fig. 3. The LSC, IR, and SSC border regions were compared among the nine Anoectochilus cp genomes. Genes located at

the IR/SC borders are represented by boxes above or below the main lines, with the numbers above the gene indicating the

distance in bp from the gene terminal to the boundary region.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

destruction, the presence of this species is high-

ly vulnerable. Gaining a deeper understanding

of the genetic composition of this plant could

greatly contribute to improved management

and conservation programs. Recently, chloro-

plast genomes have provided valuable infor-

mation for understanding genetic diversity,

phylogenetics, and speciation in land plants

(Henriquez et al., 2022; Liu et al., 2022; Ren

et al., 2021). In this study, we sequenced and

annotated the complete cp genome of A. for-

mosanus for the first time in Vietnam. The

assembled plastome exhibits the typical struc-

ture of a plant cp genome, consisting of four

parts. The GC content, an important parameter

for plant identification as higher GC content

aids in protecting the cp genome structure, was

found to be similar to that of other species in

the Anoectochilus genus in our study (Nguyen

et al., 2023a).

We found the variations among the cp

genomes of A. formosanus collected from Viet-

nam and the same species from China (acces-

sion: NC_061756.1) in terms of genome size,

gene numbers, and sequence repeat motifs.

This information is not surprised since previous

studies have reported notable variations among

accessions within species (Ren et al., 2021;

Fig. 4. Sequence identity plot compared nine cp genomes with NC_061756.1 as a reference by using mVISTA. A cut-off of

70 % identity was used for the plots, and the Y-scale represents the percent identity from 50 to 100 %.

Fig. 5. Phylogenetic tree of nine Anoectochilus jewel orchid species using Neighbor-Joining method (A) and using the

Maximum Likelihood method (B). The cp sequences D. sinense was used as outgroup. Numbers near branches are bootstrap

values.

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

Xu et al., 2022). The nucleotide diversity value

(Pi) in this study was relatively low compared

to other species in the orchid family such as

Paphiopedilum (Liu et al., 2022). The low levels

of nucleotide diversity could be due to humans’

selection pressure on the plant (Kanaka et al.,

2023). The obtained data provides insights

into the typical structure and content of the cp

genomes of A. formosanus in Vietnam. These

differences among cp genomes contribute to

our understanding of the genetic structure

within the Anoectochilus genus.

Early studies have reported the abundance

of repeated motifs in cp genomes, which are

associated with various types of genome rear-

rangements, recombination, and large inver-

sions, making them valuable for phylogenetic

studies (Ho et al. 2023; Lei et al. 2016; Zhang

et al., 2022). In our study, we observed variable

numbers of repeats among the cp genomes,

consistent with findings in a study on 13 Aroi-

deae species where repeat numbers were found

to be unrelated to genome size and phylogenetic

position of the species (Henriquez et al. 2022).

The A and T motifs were commonly identi-

fied in the nine Anoectochilus cp genomes,

aligning with a previous study by Nguyen et

al. (2023a). Microsatellites present in the cp

genome are inherited from a single parent and

are frequently utilized as molecular markers in

evolutionary studies, such as assessing genetic

diversity and species identification. The new

and specific microsatellites from A. formosanus

accession with Vietnam origin detected in our

study hold promise for evolutionary investiga-

tions in the A. formosanus genus, as well as

aiding in the identification and conservation

of different species within this genus. These cp

SSRs (simple sequence repeats) are informa-

tive molecular markers for evaluating genetic

relationships due to their high polymorphism

and copy number variation. The identified cp

SSR loci could prove highly valuable for genetic

diversity studies and may enhance the effective-

ness of interspecific discrimination, potentially

in combination with other nuclear genomic

SSRs (Zarei et al., 2022).

In general, cp genomes exhibit high con-

servation in terms of gene content and organi-

zation. However, variations are often observed

in the IR regions, indicating the involvement

of contraction and expansion events in shaping

cp genomes. Our data show a large variation

in the presence and absence of ndhF and rpl22

genes in the SSC/IR junctions. The loss of the

ndhF gene was also reported in other orchid

cp genomes (Lin et al., 2017) and other plants

such as Taxillus (Li et al., 2017), Buchnera

americana (Frailey et al., 2018). This loss could

be attributed to plant evolution towards photo-

synthetic adaptation (Scobeyeva et al., 2021).

On the other hand, previous studies reported

that rpl22 is one of the genes with the highest

deletion rate in plant cp genomes. Daniell et

al. (2016) reported the loss of this gene in 57

cp genomes of 26 plant genera, and up to 127

deletions were detected after comparing 2 511

cp genomes (Mohanta et al., 2020). Interest-

ingly, our study found that rpl2 gene was absent

in China A. formosanus (NC_061756.1) which

would facilitate the development of molecular

markers to differentiate between these two A.

formosanus accessions.

Phylogenetic relationship: Previous

attempts to determine phylogenetic relation-

ships using a limited set of genetic mark-

ers were insufficient in accurately establishing

these relationships, especially when dealing

with closely related species. Specifically, when

applying DNA barcoding techniques, misclas-

sification of species in Anoectochilus genus

has been frequent (Gao et al., 2009). Despite

incorporating multiple DNA barcode regions,

including rbcL, matK, rpoB1, rpoB2, rpoC1,

rpoC2, ITS1, ITS2, and ITS, Huynh et al. (2019)

faced challenges in effectively distinguishing

between A. formosanus and A. roxburghii which

is often misidentified as A. formosanus in tra-

ditional medicine (Ye et al., 2017). The limited

discriminatory power of DNA barcode regions

can be attributed to the minimal variation

observed among these sequences across spe-

cies, with only a few single nucleotide poly-

morphisms detected (Chen & Shiau, 2015;

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e56423, enero-diciembre 2024 (Publicado May. 21, 2024)

Huynh et al., 2019). The phylogenetic analysis

in this study reveals that the two samples of A.

formosanus formed a distinct subclade separate

from A. roxburghii. This finding aligns with a

previous study by Nguyen et al. (2023a), which

also classified A. formosanus and A. roxburghii

into different clades. Consequently, the analysis

of complete cp genomes provides a suitable

approach for resolving contentious phylogenet-

ic relationships among different species, even at

lower taxonomic levels.

In this study we sequenced and charac-

terized the complete cp sequence of A. for-

mosanus, an endangered jewel orchid species

collected from Vietnam. Comparative analysis

of this cp genome with closely related species

revealed distinct features, including variations

in genome size, gene numbers, and sequence

repeat motifs. The obtained data provides valu-

able insights into the typical structure and

content of cp genomes in A. formosanus from

Vietnam. These differences among cp genomes

contribute to our understanding of the genetic

structure within the Anoectochilus genus. Fur-

thermore, the identification of unique repeat

motifs and highly divergent regions in the

cp genome of Vietnam A. formosanus holds

potential for developing molecular markers.

These markers can be utilized in future studies

focusing on taxonomy and conservation efforts

for this precious herb in Vietnam.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENT

This work was supported by the Ho Chi

Minh City University of Industry and Trade-

Vietnam (Formerly Ho Chi Minh City Univer-

sity of Food Industry) through the HUFI fund

for Science and Technology under the Contract

No. 157/HD-DCT.

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