Mitochondrial genome recombination in somatic hybrids of Solanum commersonii and S. tuberosum.

Interspecific somatic hybridization has been performed in potato breeding experiments to increase plant resistance against biotic and abiotic stress conditions. We analyzed the mitochondrial and plastid genomes and 45S nuclear ribosomal DNA (45S rDNA) for the cultivated potato (S. tuberosum, St), wild potato (S. commersonii, Sc), and their somatic hybrid (StSc). Complex genome components and structure, such as the hybrid form of 45S rDNA in StSc, unique plastome in Sc, and recombinant mitogenome were identified. However, the mitogenome exhibited dynamic multipartite structures in both species as well as in the somatic hybrid. In St, the mitogenome is 756,058 bp and is composed of five subgenomes ranging from 297,014 to 49,171 bp. In Sc, it is 552,103 bp long and is composed of two sub-genomes of 338,427 and 213,676 bp length. StSc has 447,645 bp long mitogenome with two subgenomes of length 398,439 and 49,206 bp. The mitogenome structure exhibited dynamic recombination mediated by tandem repeats; however, it contained highly conserved genes in the three species. Among the 35 protein-coding genes of the StSc mitogenome, 21 were identical for all the three species, and 12 and 2 were unique in Sc and St, respectively. The recombinant mitogenome might be derived from homologous recombination between both species during somatic hybrid development.

Genome structure and diversity among Cynanchum wilfordii accessions.

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.

The complete mitochondrial genome of Panax ginseng (Apiales, Araliaceae): an important medicinal plant.

Ginseng (Panax ginseng C. A. Meyer) is a multifunctional medicinal herb used worldwide and is an economically important high-value crop in Korea. Here, we presented the mitochondrial genome of P. ginseng landrace 'Jakyung', which is one of the most common cultivars cultivated in Korean farms. The complete mitochondrial genome sequence was 464,661 bp in length and had a single circular form. The ginseng mitochondrial genome encoded 72 unique genes, including 45 protein-coding genes, 24 tRNA genes, and three rRNA genes. Nucleotide composition analysis revealed a GC content of 45.1%, with a slightly higher A + T bias (A, 27.1%; T, 27.8%; G, 22.5%; C, 22.6%). Phylogenetic analysis showed that P. ginseng was closely related to Daucus carota in the Apiales. This complete mitochondrial genome sequence of P. ginseng provides valuable genetic information for further studies of this important medicinal plant.

Complete Plastome of Three Korean Asarum (Aristolochiaceae): Confirmation Tripartite Structure within Korean Asarum and Comparative Analyses.

The genus Asarum (Aristolochiaceae) is a well-known resource of medicinal and ornamental plants. However, the taxonomy of Korean Asarum is ambiguous due to their considerable morphological variations. Previously, a unique plastome structure has been reported from this genus. Therefore, we investigated the structural change in the plastomes within three Korean Asarum species and inferred their phylogenetic relationships. The plastome sizes of Asarum species assembled here range from 190,168 to 193,356 bp, which are longer than a typical plastome size (160 kb). This is due to the incorporation and duplication of the small single copy into the inverted repeat, which resulted in a unique tripartite structure. We first verified this unique structure using the Illumina Miseq and Oxford Nanopore MinION platforms. We also investigated the phylogeny of 26 Aristolochiaceae species based on 79 plastid protein-coding genes, which supports the monophyly of Korean Asarum species. Although the 79 plastid protein-coding gene data set showed some limitations in supporting the previous classification, it exhibits its effectiveness in delineating some sections and species. Thus, it can serve as an effective tool for resolving species-level phylogeny in Aristolochiaceae. Last, we evaluated variable sites and simple sequence repeats in the plastome as potential molecular markers for species delimitation.

Inheritance of chloroplast and mitochondrial genomes in cucumber revealed by four reciprocal F1 hybrid combinations.

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.

Plastid Genomes of the Early Vascular Plant Genus Selaginella Have Unusual Direct Repeat Structures and Drastically Reduced Gene Numbers.

The early vascular plants in the genus Selaginella, which is the sole genus of the Selaginellaceae family, have an important place in evolutionary history, along with ferns, as such plants are valuable resources for deciphering plant evolution. In this study, we sequenced and assembled the plastid genome (plastome) sequences of two Selaginella tamariscina individuals, as well as Selaginella stauntoniana and Selaginella involvens. Unlike the inverted repeat (IR) structures typically found in plant plastomes, Selaginella species had direct repeat (DR) structures, which were confirmed by Oxford Nanopore long-read sequence assembly. Comparative analyses of 19 lycophytes, including two Huperzia and one Isoetes species, revealed unique phylogenetic relationships between Selaginella species and related lycophytes, reflected by structural rearrangements involving two rounds of large inversions that resulted in dynamic changes between IR and DR blocks in the plastome sequence. Furthermore, we present other uncommon characteristics, including a small genome size, drastic reductions in gene and intron numbers, a high GC content, and extensive RNA editing. Although the 16 Selaginella species examined may not fully represent the genus, our findings suggest that Selaginella plastomes have undergone unique evolutionary events yielding genomic features unparalleled in other lycophytes, ferns, or seed plants.

Complete chloroplast genome sequence of an orchid hybrid Cymbidium sinense (♀) × C. goeringii (♂).

The complete chloroplast genome sequence of the Cymbidium hybrid, C. sinense (♀) × C. goeringii (♂) was assembled in this study. The circular genome was 150,149 bp in length with an overall GC content of 37.1% and consisted of a pair of 25,691 bp inverted repeats, and two single-copy regions that were 84,987 bp and 13,780 bp, respectively. Gene annotation analysis identified 109 genes including 75 protein-coding genes, 30 transfer RNA, and 4 ribosomal RNA genes. Phylogenetic analysis showed its closest relationship to Cymbidium sinense, reflecting a maternal inheritance of chloroplasts.

Genome Assembly and Annotation of Soft-Shelled Adlay (Coix lacryma-jobi Variety ma-yuen), a Cereal and Medicinal Crop in the Poaceae Family.

Coix lacryma-jobi, also called adlay or Job's tears, is an annual herbal plant belonging to the Poaceae family that has been cultivated as a cereal and medicinal crop in Asia. Despite its importance, however, genomic resources for better understanding this plant species at the molecular level and informing improved breeding strategies remain limited. To address this, we generated a draft genome of the C. lacryma-jobi variety ma-yuen (soft-shelled adlay) Korean cultivar, Johyun, by de novo assembly, using PacBio and Illumina sequencing data. A total of 3,362 scaffold sequences, 1.28 Gb in length, were assembled, representing 82.1% of the estimated genome size (1.56 Gb). Genome completeness was confirmed by the presence of 91.4% of the BUSCO angiosperm genes and mapping ratio of 98.3% of Illumina paired-end reads. We found that approximately 77.0% of the genome is occupied by repeat sequences, most of which are Gypsy and Copia-type retrotransposons, and evidence-based genome annotation predicts 39,574 protein-coding genes, 85.5% of which were functionally annotated. We further predict that soft-shelled adlay diverged from a common ancestor with sorghum 9.0-11.2 MYA. Transcriptome profiling revealed 3,988 genes that are differentially expressed in seeds relative to other tissues, of which 1,470 genes were strongly up-regulated in seeds and the most enriched Gene Ontology terms were assigned to carbohydrate and protein metabolism. In addition, we identified 76 storage protein genes including 18 seed-specific coixin genes and 13 candidate genes involved in biosynthesis of benzoxazinoids (BXs) including coixol, a unique BX compound found in C. lacryma-jobi species. The characterization of those genes can further our understanding of unique traits of soft-shelled adlay, such as high seed protein content and medicinal compound biosynthesis. Taken together, our genome sequence data will provide a valuable resource for molecular breeding and pharmacological study of this plant species.

Mitochondrial plastid DNA can cause DNA barcoding paradox in plants.

The transfer of ancestral plastid genomes into mitochondrial genomes to generate mitochondrial plastid DNA (MTPT) is known to occur in plants, but its impacts on mitochondrial genome complexity and the potential for causing a false-positive DNA barcoding paradox have been underestimated. Here, we assembled the organelle genomes of Cynanchum wilfordii and C. auriculatum, which are indigenous medicinal herbs in Korea and China, respectively. In both species, it is estimated that 35% of the ancestral plastid genomes were transferred to mitochondrial genomes over the past 10 million years and remain conserved in these genomes. Some plastid barcoding markers co-amplified the conserved MTPTs and caused a barcoding paradox, resulting in mis-authentication of botanical ingredients and/or taxonomic mis-positioning. We identified dynamic and lineage-specific MTPTs that have contributed to mitochondrial genome complexity and might cause a putative barcoding paradox across 81 plant species. We suggest that a DNA barcoding guidelines should be developed involving the use of multiple markers to help regulate economically motivated adulteration.

Dynamic Chloroplast Genome Rearrangement and DNA Barcoding for Three Apiaceae Species Known as the Medicinal Herb "Bang-Poong".

Three Apiaceae species Ledebouriella seseloides, Peucedanum japonicum, and Glehnia littoralis are used as Asian herbal medicines, with the confusingly similar common name "Bang-poong". We characterized the complete chloroplast (cp) genomes and 45S nuclear ribosomal DNA (45S nrDNA) sequences of two accessions for each species. The complete cp genomes of G. littoralis, L. seseloides, and P. japonicum were 147,467, 147,830, and 164,633 bp, respectively. Compared to the other species, the P. japonicum cp genome had a huge inverted repeat expansion and a segmental inversion. The 45S nrDNA cistron sequences of the three species were almost identical in size and structure. Despite the structural variation in the P. japonicum cp genome, phylogenetic analysis revealed that G. littoralis diverged 5-6 million years ago (Mya), while P. japonicum diverged from L. seseloides only 2-3 Mya. Abundant copy number variations including tandem repeats, insertion/deletions, and single nucleotide polymorphisms, were found at the interspecies level. Intraspecies-level polymorphism was also found for L. seseloides and G. littoralis. We developed nine PCR barcode markers to authenticate all three species. This study characterizes the genomic differences between L. seseloides, P. japonicum, and G. littoralis; provides a method of species identification; and sheds light on the evolutionary history of these three species.

Sequencing and Comparative Analysis of the Chloroplast Genome of Angelica polymorpha and the Development of a Novel Indel Marker for Species Identification.

The genus Angelica (Apiaceae) comprises valuable herbal medicines. In this study, we determined the complete chloroplast (CP) genome sequence of A. polymorpha and compared it with that of Ligusticum officinale (GenBank accession no. NC039760). The CP genomes of A. polymorpha and L. officinale were 148,430 and 147,127 bp in length, respectively, with 37.6% GC content. Both CP genomes harbored 113 unique functional genes, including 79 protein-coding, four rRNA, and 30 tRNA genes. Comparative analysis of the two CP genomes revealed conserved genome structure, gene content, and gene order. However, highly variable regions, sufficient to distinguish between A. polymorpha and L. officinale, were identified in hypothetical chloroplast open reading frame1 (ycf1) and ycf2 genic regions. Nucleotide diversity (Pi) analysis indicated that ycf4⁻chloroplast envelope membrane protein (cemA) intergenic region was highly variable between the two species. Phylogenetic analysis revealed that A. polymorpha and L. officinale were well clustered at family Apiaceae. The ycf4-cemA intergenic region in A. polymorpha carried a 418 bp deletion compared with L. officinale. This region was used for the development of a novel indel marker, LYCE, which successfully discriminated between A. polymorpha and L. officinale accessions. Our results provide important taxonomic and phylogenetic information on herbal medicines and facilitate their authentication using the indel marker.

Assembly of the Mitochondrial Genome in the Campanulaceae Family Using Illumina Low-Coverage Sequencing.

Platycodongrandiflorus (balloon flower) and Codonopsislanceolata (bonnet bellflower) are important herbs used in Asian traditional medicine, and both belong to the botanical family Campanulaceae. In this study, we designed and implemented a de novo DNA sequencing and assembly strategy to map the complete mitochondrial genomes of the first two members of the Campanulaceae using low-coverage Illumina DNA sequencing data. We produced a total of 28.9 Gb of paired-end sequencing data from the genomic DNA of P.grandiflorus (20.9 Gb) and C.lanceolata (8.0 Gb). The assembled mitochondrial genome of P.grandiflorus was found to consist of two circular chromosomes; the master circle contains 56 genes, and the minor circle contains 42 genes. The C.lanceolata mitochondrial genome consists of a single circle harboring 54 genes. Using a comparative genome structure and a pattern of repeated sequences, we show that the P.grandiflorus minor circle resulted from a recombination event involving the direct repeats of the master circle. Our dataset will be useful for comparative genomics and for evolutionary studies, and will facilitate further biological and phylogenetic characterization of species in the Campanulaceae.

Authentication of Herbal Medicines Dipsacus asper and Phlomoides umbrosa Using DNA Barcodes, Chloroplast Genome, and Sequence Characterized Amplified Region (SCAR) Marker.

Dried roots of Dipsacus asper (Caprifoliaceae) are used as important traditional herbal medicines in Korea. However, the roots are often used as a mixture or contaminated with Dipsacus japonicus in Korean herbal markets. Furthermore, the dried roots of Phlomoides umbrosa (Lamiaceae) are used indiscriminately with those of D. asper, with the confusing Korean names of Sok-Dan and Han-Sok-Dan for D. asper and P. umbrosa, respectively. Although D. asper and P. umbrosa are important herbal medicines, the molecular marker and genomic information available for these species are limited. In this study, we analysed DNA barcodes to distinguish among D. asper, D. japonicus, and P. umbrosa and sequenced the chloroplast (CP) genomes of D. asper and D. japonicus. The CP genomes of D. asper and D. japonicus were 160,530 and 160,371 bp in length, respectively, and were highly divergent from those of the other Caprifoliaceae species. Phylogenetic analysis revealed a monophyletic group within Caprifoliaceae. We also developed a novel sequence characterised amplified region (SCAR) markers to distinguish among D. asper, D. japonicus, and P. umbrosa. Our results provide important taxonomic, phylogenetic, and evolutionary information on the Dipsacus species. The SCAR markers developed here will be useful for the authentication of herbal medicines.

The complete chloroplast genome of Cnidium officinale Makino.

Cnidium officinale (Ligusticum officinale) is an important herbal medicine. To facilitate species identification, we determined the complete chloroplast genome of C. officinale using the Illumina MiSeq platform. The genome was 148,518 bp in length, comprising a large single copy (LSC) region of 93,977 bp, a small single copy (SSC) region of 17,607 bp, and two inverted repeat regions (IRa and IRb) of 18,467 bp each. The genome contains 113 unique genes, including 79 protein-coding genes, four ribosomal RNAs (rRNAs), and 30 transfer RNAs (tRNAs). Phylogenetic analysis revealed that C. officinale is most closely related to L. tenuissium, with high bootstrap values.

The complete chloroplast genome of Cuscuta pentagona Engelm.

Cuscuta pentagona is a parasitic plant whose seeds are often mixed with the seeds of medicinal Cuscuta species. To facilitate the identification of C. pentagona seeds, we generated the complete chloroplast genome sequence of C. pentagona Engelm. using the Illumina MiSeq platform. The complete chloroplast genome is 86,380 bp long, with a 50,958 bp LSC region, a 7022 bp SSC region, and two inverted repeat (IRa and IRb) regions comprising 14,200 bp. The chloroplast genome consists of 85 unique genes, 57 protein-coding genes, four ribosomal RNA (rRNA) genes, and 24 transfer RNA (tRNA) genes. Two gene families, NADH oxidoreductases and RNA polymerase-related genes, are absent in this genome. Phylogenetic analysis revealed that C. pentagona is closely related to C. reflexa and C. exaltata, with strong support values.

Complete chloroplast genome of Sanguisorba × tenuifolia Fisch. ex Link.

Sanguisorba × tenuifolia Fisch. ex Link is an important herbal medicine. To facilitate species identification, we sequenced its complete chloroplast genome using the Illumina MiSeq platform. The data show that the chloroplast genome of S. × tenuifolia is 155,403 bp in size, comprising an 85,525 bp large single-copy (LSC) region, a 18,726 bp small single-copy (SSC) region, and two inverted repeats (IR) regions, IRa and IRb (each 25,576 bp). The genome contains 112 unique genes, including 79 protein-coding genes, four ribosomal RNAs genes, and 30 transfer RNAs genes. Phylogenetic analysis revealed that S. × tenuifolia is most closely related to Hagenia abyssinica.

Complete chloroplast genome of Actaea heracleifolia (Kom.) J. Compton.

Dried rhizomes of Actaea heracleifolia, used as a traditional Korean herbal medicine, are frequently adulterated with other plant species. For accurate species identification, we sequenced the complete chloroplast genome of A. heracleifolia using Illumina MiSeq. A. heracleifolia harbours a 159,578 bp chloroplast genome comprising a large single-copy region (88,770 bp), small single-copy region (18,070 bp) and two inverted repeat (IR) regions (IRa and IRb; each 26,519 bp). The chloroplast genome contains 112 unique genes, including 78 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. Phylogenetic analysis revealed that A. heracleifolia was closely related to Gymnaconitum gymnandrum.

The complete chloroplast genome sequence of Coix lacryma-jobi L. (Poaceae), a cereal and medicinal crop.

Coix lacryma-jobi is a cereal and medicinal crop belonging to the Poaceae family. This study characterized complete chloroplast genome sequence of a Korean cultivar Johyun of C. lacryma-jobi var. ma-yuen through the de novo hybrid assembly with Illumina and PacBio genomic reads. The chloroplast genome is 140,863 bp long and composed of large single copy (82,827 bp), small single copy (12,522 bp), and a pair of inverted repeats (each 22,757 bp). A total of 123 genes including 87 protein-coding genes, 32 tRNA genes, and four rRNA genes were predicted in the genome. Phylogenetic analysis confirmed a close relationship of C. lacryma-jobi with species in the Panicoideae subfamily of the Poaceae family.

Complete chloroplast genome and 45S nrDNA sequences of the medicinal plant species Glycyrrhiza glabra and Glycyrrhiza uralensis.

Glycyrrhiza uralensis and G. glabra, members of the Fabaceae, are medicinally important species that are native to Asia and Europe. Extracts from these plants are widely used as natural sweeteners because of their much greater sweetness than sucrose. In this study, the three complete chloroplast genomes and five 45S nuclear ribosomal (nr)DNA sequences of these two licorice species and an interspecific hybrid are presented. The chloroplast genomes of G. glabra, G. uralensis and G. glabra × G. uralensis were 127,895 bp, 127,716 bp and 127,939 bp, respectively. The three chloroplast genomes harbored 110 annotated genes, including 76 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The 45S nrDNA sequences were either 5,947 or 5,948 bp in length. Glycyrrhiza glabra and G. glabra × G. uralensis showed two types of nrDNA, while G. uralensis contained a single type. The complete 45S nrDNA sequence unit contains 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 26S rRNA. We identified simple sequence repeat and tandem repeat sequences. We also developed four reliable markers for analysis of Glycyrrhiza diversity authentication.