Assembled Chloroplast Genome Research Articles

Characterization of the complete chloroplast genome assembly of Amorphophallus yunnanensis Engler, Pflanzenr (Araceae) from southwestern China

Southwestern China is a biodiversity hotspot due to its diverse topography and environment. Amorphophallus yunnanensis is a species of perennial herb that is mainly distributed throughout southwestern China. The genetic diversity and divergence in this species have not been assessed largely due to a lack of genomic resources. To help with the phylogeographic study, we sequenced and assembled the first complete chloroplast genome sequence of A. yunnanensis. The length of the chloroplast genome was 164,417 bp, with an average GC content of 36% (GenBank accession no. OR400247). The genome possessed a typical quadripartite structure, and the lengths of the large single-copy (LSC), small single-copy (SSC), and two inverted repeat (IR) regions were 92,149 bp, 15,182 bp, 28,543bp, and 28,543bp, respectively. A total of 128 genes were annotated across the genome, including 82 protein-coding genes, 8 rRNAs, and 38 tRNAs. The maximum likelihood (ML) phylogeny confirmed the phylogenetic position of Amorphophallus within Araceae, with the Amorphophallus species forming a single monophyletic clade with a high bootstrap value. The ML tree also indicated that A. yunnanensis was most closely related to A. coaetaneus. This newly sequenced chloroplast genome assembly will aid in future studies of genetic diversity, historical population dynamics, and geographic differentiation patterns of A. yunnanensis.

Assembly, annotation and analysis of the chloroplast genome of the Algarrobo tree Neltuma pallida (subfamily: Caesalpinioideae)

BackgroundNeltuma pallida is a tree that grows in arid soils in northwestern Peru. As a predominant species of the Equatorial Dry Forest ecoregion, it holds significant economic and ecological value for both people and environment. Despite this, the species is severely threatened and there is a lack of genetic and genomic research, hindering the proposal of evidence-based conservation strategies.ResultsIn this work, we conducted the assembly, annotation, analysis and comparison of the chloroplast genome of a N. pallida specimen with those of related species. The assembled chloroplast genome has a length of 162,381 bp with a typical quadripartite structure (LSC-IRA-SSC-IRB). The calculated GC content was 35.97%. However, this is variable between regions, with a higher GC content observed in the IRs. A total of 132 genes were annotated, of which 19 were duplicates and 22 contained at least one intron in their sequence. A substantial number of repetitive sequences of different types were identified in the assembled genome, predominantly tandem repeats (> 300). In particular, 142 microsatellites (SSR) markers were identified. The phylogenetic reconstruction showed that N. pallida grouped with the other Neltuma species and with Prosopis cineraria. The analysis of sequence divergence between the chloroplast genome sequences of N. pallida, N. juliflora, P. farcta and Strombocarpa tamarugo revealed a high degree of similarity.ConclusionsThe N. pallida chloroplast genome was found to be similar to those of closely related species. With a size of 162,831 bp, it had the classical chloroplast quadripartite structure and GC content of 35.97%. Most of the 132 identified genes were protein-coding genes. Additionally, over 800 repetitive sequences were identified, including 142 SSR markers. In the phylogenetic analysis, N. pallida grouped with other Neltuma spp. and P. cineraria. Furthermore, N. pallida chloroplast was highly conserved when compared with genomes of closely related species. These findings can be of great potential for further diversity studies and genetic improvement of N. pallida.

Complete chloroplast genome assembly and phylogenetic analysis of blackcurrant (Ribes nigrum), red and white currant (Ribes rubrum), and gooseberry (Ribes uva-crispa) provide new insights into the phylogeny of Grossulariaceae.

Blackcurrant (Ribes nigrum), red currant (R. rubrum), white currant (R. rubrum), and gooseberry (R. uva-crispa) belong to Grossulariaceae and are popular small-berry crops worldwide. The lack of genomic data has severely limited their systematic classification and molecular breeding. The complete chloroplast (cp) genomes of these four taxa were assembled for the first time using MGI-DNBSEQ reads, and their genome structures, repeat elements and protein-coding genes were annotated. By genomic comparison of the present four and previous released five Ribes cp genomes, the genomic variations were identified. By phylogenetic analysis based on maximum-likelihood and Bayesian methods, the phylogeny of Grossulariaceae and the infrageneric relationships of the Ribes were revealed. The four cp genomes have lengths ranging from 157,450 to 157,802 bp and 131 shared genes. A total of 3,322 SNPs and 485 Indels were identified from the nine released Ribes cp genomes. Red currant and white currant have 100% identical cp genomes partially supporting the hypothesis that white currant (R. rubrum) is a fruit color variant of red currant (R. rubrum). The most polymorphic genic and intergenic region is ycf1 and trnT-psbD, respectively. The phylogenetic analysis demonstrated the monophyly of Grossulariaceae in Saxifragales and the paraphyletic relationship between Saxifragaceae and Grossulariaceae. Notably, the Grossularia subgenus is well nested within the Ribes subgenus and shows a paraphyletic relationship with the co-ancestor of Calobotrya and Coreosma sections, which challenges the dichotomous subclassification of the Ribes genus based on morphology (subgenus Ribes and subgenus Grossularia). These data, results, and insights lay a foundation for the phylogenetic research and breeding of Ribes species.

A Comprehensive Analysis of Chloroplast Genome Provides New Insights into the Evolution of the Genus Chrysosplenium.

Chrysosplenium, a perennial herb in the family Saxifragaceae, prefers to grow in low light and moist environments and is divided into two sections of Alternifolia and Oppositifolia based on phyllotaxy. Although there has been some progress in the phylogeny of Chrysosplenium over the years, the phylogenetic position of some species is still controversial. In this study, we assembled chloroplast genomes (cp genomes) of 34 Chrysosplenium species and performed comparative genomic and phylogenetic analyses in combination with other cp genomes of previously known Chrysosplenium species, for a total of 44 Chrysosplenium species. The comparative analyses revealed that cp genomes of Chrysosplenium species were more conserved in terms of genome structure, gene content and arrangement, SSRs, and codon preference, but differ in genome size and SC/IR boundaries. Phylogenetic analysis showed that cp genomes effectively improved the phylogenetic support and resolution of Chrysosplenium species and strongly supported Chrysosplenium species as a monophyletic taxon and divided into three branches. The results also showed that the sections of Alternifolia and Oppositifolia were not monophyletic with each other, and that C. microspermum was not clustered with other Chrysosplenium species with alternate leaves, but with C. sedakowii into separate branches. In addition, we identified 10 mutational hotspot regions that could serve as potential DNA barcodes for Chrysosplenium species identification. In contrast to Peltoboykinia, the clpP and ycf2 genes of Chrysosplenium were subjected to positive selection and had multiple significant positive selection sites. We further detected a significant positive selection site on the petG gene between the two sections of Chrysosplenium. These evolutionary characteristics may be related to the growth environment of Chrysosplenium species. This study enriches the cp genomes of Chrysosplenium species and provides a reference for future studies on its evolution and origin.

Chloroplast genome analysis of three Acanthus species reveal the adaptation of mangrove to intertidal habitats

Acanthus is a distinctive genus that covers three species with different ecological niches including Acanthus mollis (arid terrestrial), Acanthus leucostachyus (damp forest) and Acanthus ilicifolius (coastal intertidal). It is an intriguing question how these species evolved from terrestrial to coastal intertidal. In the present study, we assembled chloroplast genomes of A. ilicifolius, A. leucostachyus and A. mollis, which exhibited typical quadripartite structures. The sizes were 150,758, 154,686 and 150,339 bp that comprised a large single copy (LSC, 82,963, 86,461 and 82,612 bp), a small single copy (SSC, 17,191, 17,511 and 17,019 bp), and a pair of inverted repeats (IRs, 25,302, 25,357 and 25,354 bp), respectively. Gene annotation revealed that A. ilicifolius, A. leucostachyus and A. mollis contained 113, 112 and 108 unique genes, each of which contained 79, 79 and 74 protein-coding genes, 30, 29 and 30 tRNAs, and 4 rRNA genes, respectively. Differential gene analysis revealed plenty of ndhs gene deletions in the terrestrial plant A. mollis. Nucleotide diversity analysis showed that the psbK, ycf1, ndhG, and rpl22 have the highest nucleotide variability. Compared to A. leucostachyus and A. mollis, seven genes in A. ilicifolius underwent positive selection. Among them, the atpF gene showed a strong positive selection throughout terrestrial to marine evolution and was important for adaptation to coastal intertidal habitats. Phylogenetic analysis indicated that A. ilicifolius has a closer genetic relationship with A. leucostachyus than A. mollis which further confirmed the evolutionary direction of Acanthus going from terrestrial to coastal intertidal zones.

Characterization of the complete chloroplast genome of Dalbergia congesta (Fabaceae), an endangered legume endemic to the Nilgiri Hills of Tamil Nadu, India

Reference-guided de novo assembly of the Dalbergia congesta chloroplast genome was carried out using whole-genome sequencing data. The newly generated chloroplast genome size had a total length of 156,048 bp and a GC content of 36.1%. The plastome showed the classical quadripartite structure with two inverted repeats regions (IRs; each 25,715 bp) separating the large single-copy region (LSC; 85,456 bp) from the small single-copy region (SSC; 19,162 bp). The plastid genome contained 111 unique genes, including 77 protein-coding genes (CDS), 30 tRNAs, and 4 rRNAs. The phylogenomic analyses based on whole chloroplast genome sequences recovered Dalbergia as a distinct clade of the Papilionoideae, with Dalbergia congesta having a sister relationship to a clade comprising D. fusca and D. cultrata. The newly available plastome sequence will facilitate future genetic and conservational research aiming to protect this economically important but highly threatened legume species.

The complete chloroplast genome of Vitex parviflora A.Juss. (Lamiaceae) and its comparison with Vitex species

ABSTRACT Vitex parviflora A.Juss. is a highly valuable construction timber and ornamental landscape species native in the islands of the Philippines and Timor. Here, we assembled and characterized the complete chloroplast genome of V. parviflora which yielded a circularized genome 154,024 bp long, and represented by a quadripartite structure consisting of long single-copy (84,848 bp), short single-copy (17,852 bp), and two inverted repeat (IR) regions (25,662 bp). In comparison with the other published Vitex plastome sequences, the V. parviflora genome has the smallest number of annotated genes comprising 86 protein-coding, 36 tRNA, and 8 rRNA genes. Notable are the two tRNA genes having three and four copies, respectively. Phylogenetic analysis is supported with 100% bootstrap value which resulted to V. parviflora grouping closer with V. yunnanensis, a species endemic to China, than with V. quinata which it is difficult to discriminate from using morphological markers. Differences in IR regions and the presence of pseudogenes varied considerably, suggesting that V. parviflora is clearly different from the closely related taxa from the phylogram. These findings will provide a good basis for chloroplast genome assembly of other Vitex species and be useful for resolving problematic taxonomic positions within the genera, as well as provide additional evidence for species delimitation.

Chloroplast genomes of four Carex species: Long repetitive sequences trigger dramatic changes in chloroplast genome structure.

The chloroplast genomes of angiosperms usually have a stable circular quadripartite structure that exhibits high consistency in genome size and gene order. As one of the most diverse genera of angiosperms, Carex is of great value for the study of evolutionary relationships and speciation within its genus, but the study of the structure of its chloroplast genome is limited due to its highly expanded and restructured genome with a large number of repeats. In this study, we provided a more detailed account of the chloroplast genomes of Carex using a hybrid assembly of second- and third-generation sequencing and examined structural variation within this genus. The study revealed that chloroplast genomes of four Carex species are significantly longer than that of most angiosperms and are characterized by high sequence rearrangement rates, low GC content and gene density, and increased repetitive sequences. The location of chloroplast genome structural variation in the species of Carex studied is closely related to the positions of long repeat sequences; this genus provides a typical example of chloroplast structural variation and expansion caused by long repeats. Phylogenetic relationships constructed based on the chloroplast protein-coding genes support the latest taxonomic system of Carex, while revealing that structural variation in the chloroplast genome of Carex may have some phylogenetic significance. Moreover, this study demonstrated a hybrid assembly approach based on long and short reads to analyze complex chloroplast genome assembly and also provided an important reference for the analysis of structural rearrangements of chloroplast genomes in other taxa.

Assembly, annotation, and comparative analysis of Ipomoea chloroplast genomes provide insights into the parasitic characteristics of Cuscuta species.

In the Convolvulaceae family, around 1650 species belonging to 60 genera are widely distributed globally, mainly in the tropical and subtropical regions of America and Asia. Although a series of chloroplast genomes in Convolvulaceae were reported and investigated, the evolutionary and genetic relationships among the chloroplast genomes of the Convolvulaceae family have not been extensively elucidated till now. In this study, we first reported the complete chloroplast genome sequence of Ipomoea pes-caprae, a widely distributed coastal plant with medical values. The chloroplast genome of I. pes-caprae is 161667 bp in length, and the GC content is 37.56%. The chloroplastic DNA molecule of I. pes-caprae is a circular structure composed of LSC (large-single-copy), SSC (small-single-copy), and IR (inverted repeat) regions, with the size of the three regions being 88210 bp, 12117 bp, and 30670 bp, respectively. The chloroplast genome of I. pes-caprae contains 141 genes, and 35 SSRs are identified in the chloroplast genome. Our research results provide important genomic information for the molecular phylogeny of I. pes-caprae. The Phylogenetic analysis of 28 Convolvulaceae chloroplast genomes showed that the relationship of I. pes-caprae with I. involucrata or I. obscura was much closer than that with other Convolvulaccae species. Further comparative analyses between the Ipomoea species and Cuscuta species revealed the mechanism underlying the formation of parasitic characteristics of Cuscuta species from the perspective of the chloroplast genome.

Characterization of the Complete Chloroplast Genome and Phylogenetic Implications of Euonymus microcarpus (Oliv.) Sprague

Euonymus microcarpus (Oliv.) Sprague, is a species of evergreen shrub of the genus Euonymus, family Celastraceae. Here, we extracted the genomic DNA from the leaves of E. microcarpus and constructed a paired-end library. The chloroplast genome of E. microcarpus was generated with the high-throughput sequencing by the illumina Hiseq X Ten platform and de novo assembly. The chloroplast genome had a quadripartite structure, containing a long single copy region with a size of 85,386 bp and a short single copy region with a size of 18,456 bp, separated by two inverted repeat regions of 26,850 bp. The chloroplast genome contained 133 genes identified in total, including 87 potential protein-coding genes, 38 transfer RNA genes, and eight ribosomal RNA genes. A total of 282 simple sequence repeats and 63 long repeats were found. Furthermore, the phylogenetic relationships inferred that E. microcarpus is sister to E. japonicus and E. schensianus. A comparison of the structure of the chloroplast genomes of eight Euonymus species suggests a nucleotide variability of the junction sites and a higher divergence of non-coding regions, compared to the coding regions. The original findings of the study serves as a good reference for chloroplast genome assembly and a valuable foundation for the genetic diversity and evolution of E. microcarpus.

Complete chloroplast genome sequence of Hibiscus trionum Linnaeus 1753 (Malvaceae)

Hibiscus trionum L. is an annual herbaceous plant belonging to the Malvaceae family. It is native to Central Africa, however, is now naturalized in Europe and Asia including Korea. Here, we report the complete chloroplast genome assembly of H. trionum. The complete chloroplast genome comprises 160,530 bp and is divided into four typical regions: a large single-copy region of 89,272 bp, a pair of inverse repeats of 26,152 bp each, and a small single-copy region of 18,954 bp. A total of 131 genes were identified in this chloroplast, of which 86 were protein-coding, 37 were tRNA, and 8 were rRNA genes. The results of this study will serve as a key reference for further research on Hibiscus speciation.

Comprehensive Comparative Analysis and Development of Molecular Markers for Dianthus Species Based on Complete Chloroplast Genome Sequences

Dianthus spp. is a genus with high economic and ornamental value in the Caryophyllaceae, which include the famous fresh-cut carnation and the traditional Chinese herbal medicine, D. superbus. Despite the Dianthus species being seen everywhere in our daily lives, its genome information and phylogenetic relationships remain elusive. Thus, we performed the assembly and annotation of chloroplast genomes for 12 individuals from seven Dianthus species. On this basis, we carried out the first comprehensive and systematic analysis of the chloroplast genome sequence characteristics and the phylogenetic evolution of Dianthus. The chloroplast genome of 12 Dianthus individuals ranged from 149,192 bp to 149,800 bp, containing 124 to 126 functional genes. Sequence repetition analysis showed the number of simple sequence repeats (SSRs) ranged from 75 to 80, tandem repeats ranged from 23 to 41, and pair-dispersed repeats ranged from 28 to 43. Next, we calculated the synonymous nucleotide substitution rates (Ks) of all 76 protein coding genes to obtain the evolution rate of these coding genes in Dianthus species; rpl22 showed the highest Ks (0.0471), which suggested that it evolved the swiftest. By reconstructing the phylogenetic relationships within Dianthus and other species of Caryophyllales, 16 Dianthus individuals (12 individuals reported in this study and four individuals downloaded from NCBI) were divided into two strongly supported sister clades (Clade A and Clade B). The Clade A contained five species, namely D. caryophyllus, D. barbatus, D. gratianopolitanus, and two cultivars (‘HY’ and ‘WC’). The Clade B included four species, in which D. superbus was a sister branch with D. chinensis, D. longicalyx, and F1 ‘87M’ (the hybrid offspring F1 from D. chinensis and ‘HY’). Further, based on sequence divergence analysis and hypervariable region analysis, we selected several regions that had more divergent sequences, to develop DNA markers. Additionally, we found that one DNA marker can be used to differentiate Clade A and Clade B in Dianthus. Taken together, our results provide useful information for our understanding of Dianthus classification and chloroplast genome evolution.

The complete chloroplast genome of Verbascum thapsus L. (Scrophulariaceae) and its phylogenetic affinities

Verbascum thapsus L. has extensive pharmacological effects, including antioxidative and antineoplastic action, memory improvement and neuroprotection. However, its phylogenetic position is not established in Scrophulariaceae. In this study, we reported the complete chloroplast genome sequence of V. thapsus L. for the first time and investigate its phylogenetic relationship in Scrophulariaceae. The assembled chloroplast genome is a circular 153,338 bp sequence, including a large single copy (LSC) region of 84,627 bp, a small single copy (SSC) region of 17,829 bp and a pair of inverted repeats (IRs) of 25,441 bp. The genome contains 135 genes, including 86 protein coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic tree showed that V. thapsus is closely associated with V. chinense and V. phoeniceum.

Sequencing of the complete chloroplast genome of Rubia yunnanensis Diels and its phylogenetic analysis

Rubia yunnanensis Diels, an important medicinal herb, is mainly distributed in Yunnan province, Southwest China. In this study, the complete chloroplast genome of R. yunnanensis was successfully sequenced. The assembled chloroplast genome was 155,108 bp in length with an overall GC content of 36.98%, including a pair of inverted repeat (IR) regions (26,573 bp, each), respectively, a large single-copy (LSC) region (84,848 bp) and a small single-copy (SSC) region (17,114 bp). The genome contained 131 genes, comprising 85 protein-coding genes, 37 tRNA genes, eight rRNA genes, and one pseudogene. The phylogenetic analysis indicated that R. yunnanensis was closely related to R. cordifolia.

The first genome for the Cape Primrose Streptocarpus rexii (Gesneriaceae), a model plant for studying meristem-driven shoot diversity.

Cape Primroses (Streptocarpus, Gesneriaceae) are an ideal study system for investigating the genetics underlying species diversity in angiosperms. Streptocarpus rexii has served as a model species for plant developmental research for over five decades due to its unusual extended meristem activity present in the leaves. In this study, we sequenced and assembled the complete nuclear, chloroplast, and mitochondrial genomes of S. rexii using Oxford Nanopore Technologies long read sequencing. Two flow cells of PromethION sequencing resulted in 32 billion reads and were sufficient to generate a draft assembly including the chloroplast, mitochondrial and nuclear genomes, spanning 776 Mbp. The final nuclear genome assembly contained 5,855 contigs, spanning 766 Mbp of the 929‐Mbp haploid genome with an N50 of 3.7 Mbp and an L50 of 57 contigs. Over 70% of the draft genome was identified as repeats. A genome repeat library of Gesneriaceae was generated and used for genome annotation, with a total of 45,045 genes annotated in the S. rexii genome. Ks plots of the paranomes suggested a recent whole genome duplication event, shared between S. rexii and Primulina huaijiensis. A new chloroplast and mitochondrial genome assembly method, based on contig coverage and identification, was developed, and successfully used to assemble both organellar genomes of S. rexii . This method was developed into a pipeline and proved widely applicable. The nuclear genome of S. rexii and other datasets generated and reported here will be invaluable resources for further research to aid in the identification of genes involved in morphological variation underpinning plant diversification.

The complete chloroplast genome of Epimedium campanulatum Ogisu (Berberidaceae), a rare plant species endemic to China

Epimedium L. is an important medicinal herbaceous genus that belongs to the family Berberidaceae. Epimedium campanulatum Ogisu is a plant species only inhabited in the northwestern part of Sichuan province, China. Here, we reported the complete chloroplast genome sequence, assembly, and characterization of E. campanulatum. The chloroplast genome of E. campanulatum was 157,343 bp in length, and a total of 112 unique genes were identified. Phylogenetic results revealed that E. campanulatum formed a sister relationship with the cluster of Epimedium ecalcaratum, Epimedium davidii, and Epimedium chlorandrum. Our findings provided valuable data for future taxonomic and phylogenetic research within the genus Epimedium.

Comparison of Magnoliaceae Plastomes: Adding Neotropical Magnolia to the Discussion.

Chloroplast genomes are considered to be highly conserved. Nevertheless, differences in their sequences are an important source of phylogenetically informative data. Chloroplast genomes are increasingly applied in evolutionary studies of angiosperms, including Magnoliaceae. Recent studies have focused on resolving the previously debated classification of the family using a phylogenomic approach and chloroplast genome data. However, most Neotropical clades and recently described species have not yet been included in molecular studies. We performed sequencing, assembly, and annotation of 15 chloroplast genomes from Neotropical Magnoliaceae species. We compared the newly assembled chloroplast genomes with 22 chloroplast genomes from across the family, including representatives from each genus and section. Family-wide, the chloroplast genomes presented a length of about 160 kb. The gene content in all species was constant, with 145 genes. The intergenic regions showed a higher level of nucleotide diversity than the coding regions. Differences were higher among genera than within genera. The phylogenetic analysis in Magnolia showed two main clades and corroborated that the current infrageneric classification does not represent natural groups. Although chloroplast genomes are highly conserved in Magnoliaceae, the high level of diversity of the intergenic regions still resulted in an important source of phylogenetically informative data, even for closely related taxa.

The complete chloroplast genome sequence of Ficus concinna (Moraceae) from Sichuan province

Ficus concinna is an important contributor to tropical forest biodiversity. Here, we provide the first report of the complete F. concinna chloroplast genome, thereby providing a basis for subsequent phylogenetic analyses of the Moraceae family. The final assembled chloroplast genome was 160,331 bp in size and included a 20,018 bp long small single-copy (SSC) region, an 88,541 bp long large single-copy (LSC) region, and two 25,886 bp inverted repeats (IRs). The total content of GC for this chloroplast genome was 42.6%, with respective frequencies of 33.6%, 35.9%, and 28.9% in the SSC, LSC, and IR regions, accordingly. Overall, the chloroplast genome was found to encode 131 genes, comprising 37 tRNAs, 86 protein-coding genes, and 8 rRNAs. Phylogenetic analyses revealed a close relationship between F. concinna and Ficus altissima, consistent with prior research results. Together, these data provide valuable insights regarding the evolution and conserved genetic features of F. concinna.

De novo assembly and characterisation of chloroplast genomes of broccoli cvs. Marathon and Green sprout using next generation sequencing

The genus Brassica (family Brassicaceae) includes nutritionally and economically important species such as Brassica napus, Brassica rapa, and Brassica oleracea. Many varieties of B. oleracea are available in various morphological forms including nutritive vegetables such as cauliflower (var. botrytis), Brussels sprouts (var. gemmifera), kales and collards (var. acephala), kohlrabi (var. gongylodes), cabbage (var. capitata), and broccoli (var. italica). Objective of the present study was to sequence chloroplast genomes of two cultivars of broccoli: Marathon and Green sprout. The sequencing was done by next generation sequencing. The analysis was performed using Velvet, Geneious, GeSeq, tRNAscan-SE, ARAGORN, OrganellarGenomeDRAW, IRscope and REPuter. The genomes of both cultivars showed highly similar quadripartite structure of 153,365 bp. The LSC (83,136 bp) and SSC (17,835 bp) regions were separated by a pair of IR (26,197 bp) region. In total, 114 unique genes were present in both species, including 80 protein-coding, 30 tRNA and 4 rRNA genes, while 18 genes were duplicated in IRs. The highest amino acid encoding frequency was found for Leucine whereas cysteine was the least encoding amino acid. The codon usage analyses confirmed high encoding efficacy of codons that ended at 3'-end with A/T. Repeat analyses of these genomes revealed 415 microsatellites and 36 oligonucleotide repeats. Microsatellites motifs were mostly comprised of A/T instead of C/G. The comparative analyses confirmed the presence of 17 substitutions between both cultivars. Overall, this study will increase knowledge about the chloroplast genomes of broccoli and will provide a resource for chloroplast genetic engineering of this important edible plant.

엽록체 유전체 바코딩기술 및 HPLC를 이용한 복분자딸기 식별법

Background: Rubus coreanus and R. occidentalis are commonly sold as, ‘Bokbunja’, in the Korean market. However, according to Korean Pharmacopoeia, ‘Bokbunja’ refers to the unripened fruits of R. coreanus. Therefore, authentication of R. coreanus and its related species is necessary to prevent adulteration and improper use.<BR>Methods and Results: Genomic DNA was extracted from leaves, stem and fruits of R. coreanus, fruits of R. occidentalis and leaves of R. longisepalus and R. hirsutus. The anthocyanin profile of R. coreanus and R. occidentalis was identified by high performance liquid chromatography/diode-array detection. We designed four universal barcoding primer sets with optimal restriction enzyme sites to identify polymerase chain reaction-restriction fragment length polymorphisms (PCRRFLPs) between R. coreanus, the newly assembled chloroplast genome of R. occidentalis, and two related species. In addition, two Insertion or Deletion (InDel) markers were also developed. These markers were successfully used to distinguish each species in nine samples.<BR>Conclusions: In this study, we sequenced and assembled the complete chloroplast genome of R. occidentalis and developed a DNA barcoding method to differentiate it from other related species. Thus, anthocyanin profiling, PCR-RFLPs, and InDel markers are useful authentication tools to distinguish R. coreanus from R. occidentalis.