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Abstract The chloroplast (cp.) genome, also known as plastome, plays crucial roles in plant survival, adaptation, and evolution. The stable genetic structure of cp. genomes provides an ideal system for investigating species evolution. We sequenced three complete cp. genome sequences of Capsicum species and analyzed them using sequences of various Capsicum species retrieved from the NCBI database. The cp. genome of Capsicum species maintains a well-preserved quadripartite structure consisting of two inverted repeats (IRs) flanked by a large single copy (LSC) region and a small single copy (SSC) region. The sizes of cp. genome sequences ranged from 156,583 bp (C. lycianthoides) to 157,390 bp (C.pubescens). A total of 127–132 unique genes, including 83–87 protein-coding, 36–37 tRNA, and eight rRNA genes, were predicted. Comparison of cp. genomes of 10 Capsicum species revealed high sequence similarity in genome-wide organization and gene arrangements. Fragments of trnT-UGU/trnL-UAA, ccsA, ndhD, rps12, and ycf1 were identified as variable regions, and nucleotide variability of LSC and SSC was higher than that of IR. Phylogenetic speciation analysis showed that the major domesticated C. annuum species were the most extensively divergent species and closely related to C. tovarii and C. frutescens. Analysis of divergent times suggested that a substantial range of speciation events started occurring ~ 25.79 million years ago (Mya). Overall, comparative analysis of cp. genomes of Capsicum species not only offers new insights into their genetic variation and phylogenetic relationships, but also lays a foundation for evolutionary history, genetic diversity, conservation, and biological breeding of Capsicum species.

Abstract The Araliaceae contain many valuable species in medicinal and industrial aspects. We performed intensive phylogenomics using the plastid genome (plastome) and 45S nuclear ribosomal DNA sequences. A total of 66 plastome sequences were used, 13 of which were newly assembled in this study, 12 from new sequences, and one from existing data. While Araliaceae plastomes showed conserved genome structure, phylogenetic reconstructions based on four different plastome datasets revealed phylogenetic discordance within the Asian Palmate group. The divergence time estimation revealed that splits in two Araliaceae subfamilies and the clades exhibiting phylogenetic discordances in the Asian Palmate group occurred at two climatic optima, suggesting that global warming events triggered species divergence, particularly the rapid diversification of the Asian Palmate group during the Middle Miocene. Nucleotide substitution analyses indicated that the Hydrocotyloideae plastomes have undergone accelerated AT-biased mutations (C-to-T transitions) compared with the Aralioideae plastomes, and the acceleration may occur in their mitochondrial and nuclear genomes as well. This implies that members of the genus Hydrocotyle, the only aquatic plants in the Araliaceae, have experienced a distinct evolutionary history from the other species. We also discussed the intercontinental disjunction in the genus Panax and proposed a hypothesis to complement the previously proposed hypothesis. Our results provide the evolutionary trajectory of Araliaceae and advance our current understanding of the evolution of Araliaceae species.

Abstract Agastache rugosa, also known as Korean mint, is a perennial plant from the Lamiaceae family that is traditionally used for various ailments and contains antioxidant and antibacterial phenolic compounds. Molecular breeding of A. rugosa can enhance secondary metabolite production and improve agricultural traits, but progress in this field has been delayed due to the lack of chromosome-scale genome information. Herein, we constructed a chromosome-level reference genome using Nanopore sequencing and Hi-C technology, resulting in a final genome assembly with a scaffold N50 of 52.15 Mbp and a total size of 410.67 Mbp. Nine pseudochromosomes accounted for 89.1% of the predicted genome. The BUSCO analysis indicated a high level of completeness in the assembly. Repeat annotation revealed 561,061 repeat elements, accounting for 61.65% of the genome, with Copia and Gypsy long terminal repeats being the most abundant. A total of 26,430 protein-coding genes were predicted, with an average length of 1,184 bp. The availability of this chromosome-scale genome will advance our understanding of A. rugosa’s genetic makeup and its potential applications in various industries.

Background: The genus Panax in the Araliaceae family has been used as traditional medicinal plants worldwide and is known to biosynthesize ginsenosides and phytosterols. However, genetic variation between Panax species has influenced their biosynthetic pathways is not fully understood. Methods: Simultaneous analysis of transcriptomes and metabolomes obtained from adventitious roots of two tetraploid species (Panax ginseng and P. quinquefolius) and two diploid species (P. notoginseng and P. vietnamensis) revealed the diversity of their metabolites and related gene expression profiles. Results: The transcriptome analysis showed that 2,3-OXIDOSQUALENE CYCLASEs (OSCs) involved in phytosterol biosynthesis are upregulated in the diploid species, while the expression of OSCs contributing to ginsenoside biosynthesis is higher in the tetraploid species. In agreement with these results, the contents of dammarenediol-type ginsenosides were higher in the tetraploid species relative to the diploid species. Conclusion: These results suggest that a whole-genome duplication event has influenced the triterpene biosynthesis pathway in tetraploid Panax species during their evolution or ecological adaptation. This study provides a basis for further efforts to explore the genetic variation of the Panax genus.

Abstract Long terminal repeat retrotransposons (LTR-RTs) make up a considerable portion of plant genomes. New insertions of these active LTR-RTs modify gene structures and functions and play an important role in genome evolution. Therefore, identifying active forms of LTR-RTs could uncover the effects of these elements in plants. Extrachromosomal linear DNA (eclDNA) forms during LTR-RT replication; therefore, amplification LTRs of eclDNAs followed by sequencing (ALE-seq) uncover the current transpositional potential of the LTR-RTs. The ALE-seq protocol was validated by identification of Tos17 in callus of Nipponbare cultivar. Here, we identified two active LTR-RTs belonging to the Oryco family on chromosomes 6 and 9 in rice cultivar Dongjin callus based on the ALE-seq technology. Each Oryco family member has paired LTRs with identical sequences and internal domain regions. Comparison of the two LTR-RTs revealed 97% sequence identity in their internal domains and 65% sequence identity in their LTRs. These two putatively active Oryco LTR-RT family members could be used to expand our knowledge of retrotransposition mechanisms and the effects of LTR-RTs on the rice genome.

Abstract Background Cynanchum wilfordii (Cw) and Cynanchum auriculatum (Ca) have long been used in traditional medicine and as functional food in Korea and China, respectively. They have diverse medicinal functions, and many studies have been conducted, including pharmaceutical efficiency and metabolites. Especially, Cw is regarded as the most famous medicinal herb in Korea due to its menopausal symptoms relieving effect. Despite the high demand for Cw in the market, both species are cultivated using wild resources with rare genomic information. Results We collected 160 Cw germplasm from local areas of Korea and analyzed their morphological diversity. Five Cw and one Ca of them, which were morphologically diverse, were sequenced, and nuclear ribosomal DNA (nrDNA) and complete plastid genome (plastome) sequences were assembled and annotated. We investigated the genomic characteristics of Cw as well as the genetic diversity of plastomes and nrDNA of Cw and Ca. The Cw haploid nuclear genome was approximately 178 Mbp. Karyotyping revealed the juxtaposition of 45S and 5S nrDNA on one of 11 chromosomes. Plastome sequences revealed 1226 interspecies polymorphisms and 11 Cw intraspecies polymorphisms. The 160 Cw accessions were grouped into 21 haplotypes based on seven plastome markers and into 108 haplotypes based on seven nuclear markers. Nuclear genotypes did not coincide with plastome haplotypes that reflect the frequent natural outcrossing events. Conclusions Cw germplasm had a huge morphological diversity, and their wide range of genetic diversity was revealed through the investigation with 14 molecular markers. The morphological and genomic diversity, chromosome structure, and genome size provide fundamental genomic information for breeding of undomesticated Cw plants.

[This corrects the article DOI: 10.1371/journal.pone.0264576.].

Peucedanum hakuunense Nakai is one of the rare species in the Korean Peninsula. This study characterized the complete plastid genome (plastome) sequence of P. hakuunense by de novo assembly with next-generation sequencing data. The complete plastome of P. hakuunense is 147,426 bp in length with a typical quadripartite structure comprising a large single-copy region of 91,915 bp, a small single-copy region of 17,425 bp, and two inverted repeat regions of 19,043 bp in length. The plastome of P. hakuunense is composed of 85 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The phylogenetic analysis revealed that two Peucedanum species formed an independent subclade, sister to the subclade of Angelica species within the tribe Selineae.

Abstract Ilex is a monogeneric plant group (containing approximately 600 species) in the Aquifoliaceae family and one of the most commonly used medicinal herbs. However, its taxonomy and phylogenetic relationships at the species level are debatable. Herein, we obtained the complete chloroplast genomes of all 19 Ilex types that are native to Hong Kong. The genomes are conserved in structure, gene content and arrangement. The chloroplast genomes range in size from 157,119 bp in Ilex graciliflora to 158,020 bp in Ilex kwangtungensis. All these genomes contain 125 genes, of which 88 are protein-coding and 37 are tRNA genes. Four highly varied sequences (rps16-trnQ, rpl32-trnL, ndhD-psaC and ycf1) were found. The number of repeats in the Ilex genomes is mostly conserved, but the number of repeating motifs varies. The phylogenetic relationship among the 19 Ilex genomes, together with eight other available genomes in other studies, was investigated. Most of the species could be correctly assigned to the section or even series level, consistent with previous taxonomy, except Ilex rotunda var. microcarpa, Ilex asprella var. tapuensis and Ilex chapaensis. These species were reclassified; I. rotunda was placed in the section Micrococca, while the other two were grouped with the section Pseudoaquifolium. These studies provide a better understanding of Ilex phylogeny and refine its classification.

Abstract Both genomes in chloroplasts and mitochondria of plant cell are usually inherited from maternal parent, with rare exceptions. To characterize the inheritance patterns of the organelle genomes in cucumber (Cucumis sativus var. sativus), two inbred lines and their reciprocal F1 hybrids were analyzed using an next generation whole genome sequencing data. Their complete chloroplast genome sequences were de novo assembled, and a single SNP was identified between the parental lines. Two reciprocal F1 hybrids have the same chloroplast genomes with their maternal parents. Meanwhile, 292 polymorphic sites were identified between mitochondrial genomes of the two parental lines, which showed the same genotypes with their paternal parents in the two reciprocal F1 hybrids, without any recombination. The inheritance patterns of the chloroplast and mitochondria genomes were also confirmed in four additional cucumber accessions and their six reciprocal F1 hybrids using molecular markers derived from the identified polymorphic sites. Taken together, our results indicate that the cucumber chloroplast genome is maternally inherited, as is typically observed in other plant species, whereas the large cucumber mitochondrial genome is paternally inherited. The combination of DNA markers derived from the chloroplast and mitochondrial genomes will provide a convenient system for purity test of F1 hybrid seeds in cucumber breeding.

Liriope platyphylla F.T. Wang & Tang and Ophiopogon japonicus (Thunb.) Ker Gawl are morphologically similar perennial herbaceous plants belonging to the Asparagaceae family used as evergreen ornamental plants as well as for medicinal purposes. Complete plastid genomes (plastomes) and 45S nuclear ribosomal DNA (45S rDNA) sequences were obtained from two L. platyphylla accessions and one O. japonicus accessions using low-coverage, whole-genome data. Phylogenomic analysis using plastomes or 45S rDNA of the three accessions showed identical topology. Comparative analysis of the three accessions and representatives of 28 other Asparagaceae species revealed that L. platyphylla and O. japonicus are the most closely related species in the family Asparagaceae. The plastomes were 156,754–157,071 bp and the 45S rDNA transcriptional unit was 5906–5789 bp, providing super-barcoding information. Plastome sequences contained 817 single-nucleotide polymorphisms (SNPs) and 173 insertions or deletions (InDels) at the interspecific level and 88 SNPs and 56 InDels at the intraspecific level. 45S rDNA sequences contained 70 SNPs and 3 InDels at the interspecific level and 12 SNPs and 1 InDel at the intraspecific level. We developed four dominant and two codominant DNA markers based on the plastome diversity for authentication of L. platyphylla and O. japonicus and successfully applied these to ingredients distributed in the herbal medicine market.

Background: Panax species are important herbal medicinal plants in the Araliaceae family. Recently, we reported the complete chloroplast genomes and 45S nuclear ribosomal DNA sequences from seven Panax species, two (P. quinquefolius and P. trifolius) from North America and five (P. ginseng, P. notoginseng, P. japonicus, P. vietnamensis, and P. stipuleanatus) from Asia. Methods: We conducted phylogenetic analysis of these chloroplast sequences with 12 other Araliaceae species and comprehensive comparative analysis among the seven Panax whole chloroplast genomes. Results: We identified 1,128 single nucleotide polymorphisms (SNP) in coding gene sequences, distributed among 72 of the 79 protein-coding genes in the chloroplast genomes of the seven Panax species. The other seven genes (including psaJ, psbN, rpl23, psbF, psbL, rps18, and rps7) were identical among the Panax species. We also discovered that 12 large chloroplast genome fragments were transferred into the mitochondrial genome based on sharing of more than 90% sequence similarity. The total size of transferred fragments was 60,331 bp, corresponding to approximately 38.6% of chloroplast genome. We developed 18 SNP markers from the chloroplast genic coding sequence regions that were not similar to regions in the mitochondrial genome. These markers included two or three species-specific markers for each species and can be used to authenticate all the seven Panax species from the others. Conclusion: The comparative analysis of chloroplast genomes from seven Panax species elucidated their genetic diversity and evolutionary relationships, and 18 species-specific markers were able to discriminate among these species, thereby furthering efforts to protect the ginseng industry from economically motivated adulteration. Keywords: Araliaceae evolution, Chloroplast genome, dCAPS markers, Ginseng authentication, Panax species

The genus Artemisia is an important source of medicines in both traditional and modern pharmaceutics, particularly in East Asia. Despite the great benefits of herbal medicine, quality assessment methods for these medicinal herbs are lacking. The young leaves from Artemisia species are generally used, and most of the species have similar morphology, which often leads to adulteration and misuse. This study assembled five complete chloroplast genomes of three Artemisia species, two accessions of A. gmelinii and A. capillaris, and one A. fukudo. Through comparative analysis, we revealed genomic variations and phylogenetic relationships between these species and developed seven InDel-based barcode markers which discriminated the tested species from each other. Additionally, we analyzed specialized metabolites from the species using LC-MS and suggested chemical markers for the identification and authentication of these herbs. We expect that this integrated and complementary authentication method would aid in reducing the misuse of Artemisia species.

The diversity of secondary metabolites of individual plants results from multiple enzymatic processes in planta and various environmental factors, such as temperature, moisture, and soil conditions. Chemical composition analysis of plants can lead to a new method to understand relationship among comparable plants along with biological classification such as genetic and anatomical method. In this study, the chemical diversity of nine different Lauraceae species was investigated, and the plant samples were chemically analyzed and classified. Multivariate analysis methods, such as PLS-DA, were used to select important metabolites distinguishing the nine Lauraceae species. The selected metabolites were identified through preparative LC-MS or MS/MS fragment pattern analysis. In addition, the chemical dendrogram for the nine Lauraceae species was interpreted through molecular network analysis and compared with the genetic dendrogram. This approach enabled us to compare the complete chemical compositions of multiple plant samples to identify relationships among plants.

Background: Various Panax ginseng cultivars exhibit a range of diversity for morphological and physiological traits. However, there are few studies on diversity of metabolic profiles and genetic background to understand the complex metabolic pathway in ginseng. Methods: To understand the complex metabolic pathway and related genes in ginseng, we tried to conduct integrated analysis of primary metabolite profiles and related gene expression using five ginseng cultivars showing different morphology. We investigated primary metabolite profiles via gas chromatography–mass spectrometry (GC-MS) and analyzed transcriptomes by Illumina sequencing using adventitious roots grown under the same conditions to elucidate the differences in metabolism underlying such genetic diversity. Results: GC-MS analysis revealed that primary metabolite profiling allowed us to classify the five cultivars into three independent groups and the grouping was also explained by eight major primary metabolites as biomarkers. We selected three cultivars (Chunpoong, Cheongsun, and Sunhyang) to represent each group and analyzed their transcriptomes. We inspected 100 unigenes involved in seven primary metabolite biosynthesis pathways and found that 21 unigenes encoding 15 enzymes were differentially expressed among the three cultivars. Integrated analysis of transcriptomes and metabolomes revealed that the ginseng cultivars differ in primary metabolites as well as in the putative genes involved in the complex process of primary metabolic pathways. Conclusion: Our data derived from this integrated analysis provide insights into the underlying complexity of genes and metabolites that co-regulate flux through these pathways in ginseng.

Liriope platyphylla is herbaceous perennial plant belonging to the Asparagaceae family and widely used both as ornamental plant and herbal medicine. The complete chloroplast genome of L. platyphylla was 156,754 bp in length, which was composed of four distinct parts; a large single copy (LSC) of 85,118 bp, a small single copy (SSC) of 18,680 bp, and a pair of inverted repeat regions (IRa and IRb) of 26,478 bp. A total of 130 genes including 83 protein-coding genes, 39 tRNA genes and 8 rRNA genes were identified. The phylogenetic tree showed that L. platyphylla has a close relationship with other Nolinoideae plants, especially with Maianthemum dilatatum and Nolina atopocarpa.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Wolfiporia cocos have been used as diuretic, sedative, and tonic medicine. Although its medicinal effects were reported, its genomic information is rare. In this paper, we report the complete mitochondrial genome of Wolfiporia cocos which is a 136,067 bp circular structure with 5 protein-coding genes, 25 tRNA genes, and 2 rRNA genes. Additional six protein-coding genes (cob, cox1, cox2, cox3, nad2, nad6) were truncated or pseudogenized by internal stop codons. Phylogenetic analysis of the mitochondrial genome of W. cocos showed the distinct relationship with other five mitochondrial genomes of Polyporales.

Rehmannia glutinosa is a plant used as traditional medicine for its various tonic effects in Korea and China. In this study, chloroplast genomes of two R. glutinosa were completed by de novo assembly using whole-genome Illumina sequence data. The length of chloroplast genomes of R. glutinosa collected from China and Korea was 153,680 bp and 153,499 bp, respectively. A total of 114 coding regions were predicted in both R. glutinosa including 80 protein-coding genes, 4 rRNA genes, and 30 tRNA genes. We identified abundant intra-species diversity of 87 InDels and 147 SNPs, among three R. glutinosa chloroplast genome sequences including one from GenBank. The phylogenetic analysis showed that these R. glutinosa were closely clustered with related Rehmannia species, separated from other Orobanchaceae and Scrophulariaceae species.

Panax ginseng C.A. Meyer is a traditional medicinal herb that produces bioactive compounds such as ginsenosides. Here, we investigated the diversity of ginsenosides and related genes among five genetically fixed inbred ginseng cultivars (Chunpoong [CP], Cheongsun [CS], Gopoong [GO], Sunhyang [SH], and Sunun [SU]). To focus on the genetic diversity related to ginsenoside biosynthesis, we utilized in vitro cultured adventitious roots from the five cultivars grown under controlled environmental conditions. PCA loading plots based on secondary metabolite composition classified the five cultivars into three groups. We selected three cultivars (CS, SH, and SU) to represent the three groups and conducted further transcriptome and gas chromatography-mass spectrometry analyses to identify genes and intermediates corresponding to the variation in ginsenosides among cultivars. We quantified ginsenoside contents from the three cultivars. SH had more than 12 times the total ginsenoside content of CS, with especially large differences in the levels of panaxadiol-type ginsenosides. The expression levels of genes encoding squalene epoxidase (SQE) and dammarenediol synthase (DDS) were also significantly lower in CS than SH and SU, which is consistent with the low levels of ginsenoside produced in this cultivar. Methyl jasmonate (MeJA) treatment increased the levels of panaxadiol-type ginsenosides up to 4-, 13-, and 31-fold in SH, SU, and CS, respectively. MeJA treatment also greatly increased the quantity of major intermediates and the expression of the underlying genes in the ginsenoside biosynthesis pathway; these intermediates included squalene, 2,3-oxidosqualene, and dammarenediol II, especially in CS, which had the lowest ginsenoside content under normal culture conditions. We conclude that SQE and DDS are the most important genetic factors for ginsenoside biosynthesis with diversity among ginseng cultivars.

Background: Panax ginseng Meyer is a traditional medicinal plant famous for its strong therapeutic effects and serves as an important herbal medicine. To understand and manipulate genes involved in secondary metabolic pathways including ginsenosides, transcriptome profiling of P. ginseng is essential. Methods: RNA-seq analysis of adventitious roots of two P. ginseng cultivars, Chunpoong (CP) and Cheongsun (CS), was performed using the Illumina HiSeq platform. After transcripts were assembled, expression profiling was performed. Results: Assemblies were generated from ∼85 million and ∼77 million high-quality reads from CP and CS cultivars, respectively. A total of 35,527 and 27,716 transcripts were obtained from the CP and CS assemblies, respectively. Annotation of the transcriptomes showed that approximately 90% of the transcripts had significant matches in public databases. We identified several candidate genes involved in ginsenoside biosynthesis. In addition, a large number of transcripts (17%) with different gene ontology designations were uniquely detected in adventitious roots compared to normal ginseng roots. Conclusion: This study will provide a comprehensive insight into the transcriptome of ginseng adventitious roots, and a way for successful transcriptome analysis and profiling of resource plants with less genomic information. The transcriptome profiling data generated in this study are available in our newly created adventitious root transcriptome database (http://im-crop.snu.ac.kr/transdb/index.php) for public use.

Lonicera japonica is a traditional medicinal plant well known for its anti-inflammatory effect. The complete chloroplast genome sequence of L. japonica collected from Korea was obtained by de novo assembly using whole genome sequence data. The chloroplast genome is 155,060 bp in length, containing 88,853 bp in a large single copy (LSC), 18,653 bp in a small single copy (SSC) and 23,777 bp in a pair of inverted repeats (IRs). A total of 112 genes including 78 protein-coding genes and 34 structural RNA genes were identified. The sequence comparison of two L. japonica collected from Korea and China revealed 48 single nucleotide polymorphisms (SNPs) and 45 insertions/deletions (InDels). In addition, phylogenetic analysis represented intraspecific diversity within L. japonica species collected in Korea and China.

Hosta capitata (Koidz.) Nakai is an herbaceous perennial plant belonging to the Asparagaceae family and has become a popular ornamental plant. In this study, the chloroplast genome sequence of H. capitata was completed by de novo assembly with whole genome sequence data. The chloroplast genome of H. capitata is 156,416 bp in length, which is composed of a large single-copy (LSC) of 84,788 bp, a small single-copy (SSC) of 18,206 bp, and a pair of inverted repeat regions (IRa and IRb) of 26,711 bp, as four distinct parts. In total, 114 genes were identified including 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. The phylogenetic analysis revealed that H. capitata has a close relationship with other Hosta species, H. minor and H. ventricosa, but is farther than the distance between them.

Magic Lily (Lycoris squamigera), belonging to the Amaryllidaceae family, is cultivated for ornamental and medicinal purposes. To characterize its genomic information, we obtained the complete chloroplast genome sequence of L. squamigera by assembling Illumina whole genome sequence data. The complete chloroplast genome is 158,482 bp in length which is composed of four unique regions, a large single copy region (LSC) of 86,454 bp, a small single copy region (SSC) of 18,500 bp, and a pair of inverted repeats (IR) of 26,764 bp. The genome annotation predicted 159 genes including 105 protein-coding genes, 46 tRNA genes, and 8 rRNA genes. Phylogenetic tree analysis revealed that L. squamigera clustered with Allium species belonging to the Amaryllidaceae family.

We report complete sequences of chloroplast (cp) genome and 45S nuclear ribosomal DNA (45S nrDNA) for 11 Panax ginseng cultivars. We have obtained complete sequences of cp and 45S nrDNA, the representative barcoding target sequences for cytoplasm and nuclear genome, respectively, based on low coverage NGS sequence of each cultivar. The cp genomes sizes ranged from 156,241 to 156,425 bp and the major size variation was derived from differences in copy number of tandem repeats in the ycf1 gene and in the intergenic regions of rps16-trnUUG and rpl32-trnUAG. The complete 45S nrDNA unit sequences were 11,091 bp, representing a consensus single transcriptional unit with an intergenic spacer region. Comparative analysis of these sequences as well as those previously reported for three Chinese accessions identified very rare but unique polymorphism in the cp genome within P. ginseng cultivars. There were 12 intra-species polymorphisms (six SNPs and six InDels) among 14 cultivars. We also identified five SNPs from 45S nrDNA of 11 Korean ginseng cultivars. From the 17 unique informative polymorphic sites, we developed six reliable markers for analysis of ginseng diversity and cultivar authentication.

Cacao (Theobroma cacao L.) is considered as economic importance crop playing a major role as source of chocolate industries for production of both chocolate candy and cocoa butter. Despite the advantages of cacao in industrial sector, understanding the global structure of cacao chloroplast genome plays a crucial role in explaining phylogenetic relationships and cultivar identification. This study aimed to perform phylogenetic analysis of cacao genotypes and develop DNA barcode markers using cacao chloroplast sequences. Chloroplast genome sequencing of two cacao genotypes (DR-1 and Sca-12) was conducted by the Illumina Miseq platform. Phylogenetic analysis of 12 cacao genotypes including two genotypes sequenced in this study (DR-1 and Sca-12) and ten genotypes previously sequenced (HQ336404, EET-64, ICS-01, ICS-06, ICS-39, Pentagonum, Sca-6, Stahel, Amelonado, and Criollo-22) showed a clear separation between bulk and fine types. This result demonstrated the usefulness of chloroplast sequences in revealing phylogenetic relatedness. Based on comparative chloroplast genome analysis of two cacao genotypes, DR-1 and Sca-12, three insertion/deletion (InDel) markers named as Theca_indel01, Theca_indel02, and Theca_indel03 which designed from the regions of trnA-UGC-rrn23, trnK-UUU-rps16, and rps16 intron, respectively were successfully developed to reveal barcode system for cacao genetic discrimination. Overall, these findings would provide valuable insight into the chloroplast genome structure of cacao plant, as well as information about the benefits of chloroplast sequences for constructing phylogenetic relationships and developing DNA barcode markers. [ABSTRACT FROM AUTHOR]