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Whole-genome shogun sequence (WGS) data generated by next-generation sequencing (NGS) platforms are a valuable resource for crop improvement. We produced 5–6 × WGS coverage of 44 Brassica oleracea breeding lines representing seven subspecies/morphotypes: cabbage, broccoli, cauliflower, kailan, kale, Brussels sprout, and kohlrabi to systematically evaluate the nuclear and chloroplast (Cp) diversity in the 44 B. oleracea breeding lines. We then exploited the impact of low-coverage NGS by evaluating nuclear genome diversity and assembly, annotation of complete chloroplast (Cp) genomes and 45S nuclear ribosomal DNA (45S nrDNA) sequences, and copy number variation for major repeats. Nuclear genome diversity analysis has revealed a total of 496 463 SNPs and 37 493 indels in the nuclear genome across the 44 accessions. Interestingly, some SNPs showed subspecies enrichment at certain chromosomal regions. The assembly of complete Cp genomes contained 153 361–153 372 bp with 37 variants including SNPs and indels. The 45S nrDNA transcription unit was 5 802 bp long with a total of 31 SNPs from the 44 lines. The phylogenetic tree inferred from the nuclear and Cp genomes coincided and clustered broccoli, cauliflower, and kailan in one group and cabbage, Brussels sprout, kale, and kohlrabi in another group. The morphotypes diverged during the last 0.17 million years. The Cp genome diversity reflected the unique cytoplasm of each subspecies, and revealed that the cytoplasm of many breeding lines was replaced and intermingled via inter-subspecies crosses during the breeding process instead. The polymorphic Cp markers provide a classification system for the cytoplasm types in B. oleracea. Furthermore, copy numbers of major transposable elements (TEs) showed high diversity among the 44 accessions, indicating that many TEs have become active recently. Overall, we demonstrated a comprehensive utilization of low-coverage NGS data and might shed light on the genetic diversity and evolution of diverse B. oleracea morphotypes.

Complete plastid genome (plastome) and ribosomal DNA (rDNA) sequences of three Rubus accessions (two Rubus longisepalus and one R. hirsutus) were newly assembled using Illumina whole-genome sequences. Rubus longisepalus Nakai and R. longisepalus var. tozawai, described as different varieties, have identical plastomes and rDNA sequences. The plastomes are 155,957 bp and 156,005 bp and the 45S rDNA transcription unit sizes are 5809 bp and 5811 bp in R. longisepalus and R. hirsutus, respectively. The 5S rDNA transcription unit is an identical 121 bp in three Rubus accessions. We developed three DNA markers to authenticate R. longisepalus and R. hirsutus based on plastome diversity. Phylogenomic analysis revealed that the Rubus species classified as two clades and R. longisepalus, R. hirsutus, and R. chingii are the most closely related species in clade 1.

Chromosomal mapping of repetitive elements is invaluable in understanding genome structure and evolution. Repetitive elements constitute ~ 80% of the allotetraploid ginseng (Panax ginseng) genome. Preparing sporophytic metaphase chromosomes of ginseng is laborious; therefore, it would be advantageous to maximize the information obtained from a single slide. Here, we investigated the suitability of simultaneous five-color fluorescence in situ hybridization using major ginseng repeats, namely PgDel1, PgDel2, PgTel, and Pg167TR. For Pg167TR, we generated two degenerate probe libraries (Pg167TRa and Pg167TRb) based on the chromosomal target coverage. We labeled the probes with dark-red, blue, red, orange, and green fluorochromes and used excitation/emission filter sets specific to each fluorochrome to detect fluorescence in situ hybridization signals. PgDel1 was distributed across all 24 chromosome pairs, except for the secondary constriction region of chromosome 16, whereas PgDel2 was distributed over 12 of the 24 pairs. PgTel was localized in the termini of chromosomes and in an intercalary region in chromosome 13. Pg167TRa and Pg167TRb were distributed among 22 chromosome pairs with loci polymorphisms. These results showed the utility of five-color fluorescence in situ hybridization for chromosomal mapping of five repeats to expedite karyotyping and facilitate genome evolution studies in ginseng and other plant species.

Background Ginseng (Panax ginseng Meyer) is one of the world's most valuable medicinal plants with numerous pharmacological effects. Ginseng has been cultivated from wild mountain ginseng collections for a few hundred years. However, the genetic diversity of cultivated and wild ginseng populations is not fully understood. Methods We developed 92 polymorphic microsatellite markers based on whole-genome sequence data. We selected five markers that represent clear allele diversity for each of their corresponding loci to elucidate genetic diversity. These markers were applied to 147 individual plants, including cultivars, breeding lines, and wild populations in Korea and neighboring countries. Results Most of the 92 markers displayed multiple-band patterns, resulting from genome duplication, which causes confusion in interpretation of their target locus. The five high-resolution markers revealed 3 to 8 alleles from each single locus. The proportion of heterozygosity (He) ranged from 0.027 to 0.190, with an average of 0.132, which is notably lower than that of previous studies. Polymorphism information content of the markers ranged from 0.199 to 0.701, with an average of 0.454. There was no statistically significant difference in genetic diversity between cultivated and wild ginseng groups, and they showed intermingled positioning in the phylogenetic relationship. Conclusion Ginseng has a relatively high level of genetic diversity, and cultivated and wild groups have similar levels of genetic diversity. Collectively, our data demonstrate that current breeding populations have abundant genetic diversity for breeding of elite ginseng cultivars.

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

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.

Miniature form transposable elements (mTEs) are ubiquitous in plant genomes and directly linked to gene regulation and evolution. With the advantage of completely sequenced genomes of Brassica rapa and Brassica oleracea, an open-source web portal called, BrassicaTED was developed. This database provides a user-friendly interface to explore invaluable information of mTEs in Brassica species and unique visualization and comparison tools. In this chapter, we describe an overview of this database construction and explain the utilities of data search, visualization, and analysis tools. In addition, we show the possible obstacles users may encounter when using this database.

Miniature inverted-repeat transposable elements (MITEs) are non-autonomous class II transposons which have been shown to influence genome evolution. Brassica nigra L. (B-genome) is one of three Brassica diploids cultivated primarily as an oil crop, which harbors novel alleles important for breeding. Two new high copy hAT MITE families (BniHAT-1 and BniHAT-2) from the B-genome were characterized and their prevalence assessed in the genomes of the related diploids, rapa L. (A) and Brassica oleracea L. (C). Both novel MITE families were present at high copy numbers in the B-genome with 434 and 331 copies of BniHAT-1 and BniHAT-2, respectively. Yet less than 20 elements were identified in the genome assemblies of the A, and C -genomes, supporting B-genome specific proliferation of these MITE families. Although apparently randomly distributed across the genome, 68 and 70% of the B-genome MITEs were present within 2 kb flanking regions of annotated genes suggesting they might influence gene expression and/or function. In addition, MITE derived microRNAs and transcription factor binding sites suggested a putative role in gene regulation. Age of insertion analysis revealed that the major proliferation of these elements occurred during 2–3 million years ago. Additionally, site-specific polymorphism analyses showed that 44% MITEs were undergoing active amplification into the B-genome. Overall, this study provides a comprehensive analysis of two high copy MITE families, which were specifically amplified in the B-genome, suggesting a potential role in shaping the Brassica B-genome.

The concept of U’s triangle, which revealed the importance of polyploidization in plant genome evolution, described natural allopolyploidization events in Brassica using three diploids [B. rapa (A genome), B. nigra (B), and B. oleracea (C)] and derived allotetraploids [B. juncea (AB genome), B. napus (AC), and B. carinata (BC)]. However, comprehensive understanding of Brassica genome evolution has not been fully achieved. Here, we performed low-coverage (2–6×) whole-genome sequencing of 28 accessions of Brassica as well as of Raphanus sativus [R genome] to explore the evolution of six Brassica species based on chloroplast genome and ribosomal DNA variations. Our phylogenomic analyses led to two main conclusions. (1) Intra-species-level chloroplast genome variations are low in the three allotetraploids (2~7 SNPs), but rich and variable in each diploid species (7~193 SNPs). (2) Three allotetraploids maintain two 45SnrDNA types derived from both ancestral species with maternal dominance. Furthermore, this study sheds light on the maternal origin of the AC chloroplast genome. Overall, this study clarifies the genetic relationships of U’s triangle species based on a comprehensive genomics approach and provides important genomic resources for correlative and evolutionary studies.

Summary Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane‐type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome‐scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics‐assisted breeding or metabolic engineering.

Repetitive DNA elements are ubiquitous in plant genomes. Although repeats provide relevant information for cytogenetic, evolutionary, and genomic studies, identifying and characterizing their sequence and chromosomal distribution are not always easily achieved through conventional methods. However, a high-throughput identification of genomic repeats can be obtained with short reads from next-generation sequencing data. Here, we identified the telomeric and two chromosome-specific repeats in Panax ginseng using low-coverage whole genome sequence data. The telomeric repeat sequence is same with the canonical angiosperm sequence, (TTTAGGG)n, and localized mostly in every chromosome termini, except for an additional interstitial location in chromosome 10. A dinucleotide (GA) microsatellite, PgGA15, with total genome representation (GR) of more than 33 kb localized in the long arm of chromosome 20. An 11-bp minisatellite, Pgms1, with more than 58 kb of GR localized in the long arm of chromosome 1. This study provides chromosome-specific markers for cytogenetic studies in P. ginseng.

Mahonia eurybracteata subsp. ganpinensis (H.Lev.) T.S.Ying & Boufford. is an evergreen shrub of Berberidaceae and has the potentials for horticultural and medicinal development. In the present pape...

Dehydrins (DHNs) are hydrophilic proteins with conserved lysine-rich K-segment, which belong to Group II of the late embryogenesis abundant (LEA) protein family. DHNs are considered as molecular chaperons playing important roles in abiotic stress tolerance. In this study, DHN genes were identified through genome-wide searches in five Cucurbitaceae species, including cucumber, wild cucumber, melon, watermelon, and bitter gourd. Three to five DHN genes were found in each of the five species, which were further divided into several protein architecture types based on the presence and order of the major conserved motifs such as K-, Y-, and S-segments. In silico expression profiling using RNA-Seq data revealed high expression of SK3-type DHN gene and low expression of other type DHN genes in cucumber and melon. In silico promoter analysis identified a number of cis-acting element-like sequences related to abiotic stress-response such as DRE and ABRE in 2-kb putative promoter sequences. DHN genes identified in this study will be valuable for understanding the stress response mechanism as well as assisting molecular breeding in Cucurbitaceae crops.

Ginseng represents a set of high-value medicinal plants of different species: Panax ginseng (Asian ginseng), Panax quinquefolius (American ginseng), Panax notoginseng (Chinese ginseng), Panax japonicus (Bamboo ginseng), and Panax vietnamensis (Vietnamese ginseng). Each species is pharmacologically and economically important, with differences in efficacy and price. Accordingly, an authentication system is needed to combat economically motivated adulteration of Panax products. We conducted comparative analysis of the chloroplast genome sequences of these five species, identifying 34-124 InDels and 141-560 SNPs. Fourteen InDel markers were developed to authenticate the Panax species. Among these, eight were species-unique markers that successfully differentiated one species from the others. We generated at least one species-unique marker for each of the five species, and any of the species can be authenticated by selection among these markers. The markers are reliable, easily detectable, and valuable for applications in the ginseng industry as well as in related research.

Background Prenyltransferases catalyze the sequential addition of isopentenyl diphosphate units to allylic prenyl diphosphate acceptors and are classified as either trans -prenyltransferases (TPTs) or cis -prenyltransferases (CPTs). The functions of CPTs have been well characterized in bacteria, yeast, and mammals compared to plants. The characterization of CPTs also has been less studied than TPTs. In the present study, molecular cloning and functional characterization of a CPT from a medicinal plant, Panax ginseng Mayer were addressed. Methods Gene expression patterns of PgCPT1 were analyzed by quantitative reverse transcription polymerase chain reaction. In planta transformation was generated by floral dipping using Agrobacterium tumefaciens . Yeast transformation was performed by lithium acetate and heat-shock for rer2Δ complementation and yeast-two-hybrid assay. Results The ginseng genome contains at least one family of three putative CPT genes. PgCPT1 is expressed in all organs, but more predominantly in the leaves. Overexpression of PgCPT1 did not show any plant growth defect, and its protein can complement yeast mutant rer2Δ via possible protein–protein interaction with PgCPTL2. Conclusion Partial complementation of the yeast dolichol biosynthesis mutant rer2Δ suggested that PgCPT1 is involved in dolichol biosynthesis. Direct protein interaction between PgCPT1 and a human Nogo-B receptor homolog suggests that PgCPT1 requires an accessory component for proper function.

We produced complete sequences and conducted comparative analysis of the maternally inherited chloroplast (cp) genomes and bi-parentally inherited 45S nuclear ribosomal RNA genes (nrDNA) from ten Araliaceae species to elucidate the genetic diversity and evolution in that family. The cp genomes ranged from 155,993 bp to 156,730 bp with 97.1–99.6% similarity. Complete 45S nrDNA units were about 11 kb including a 5.8-kb 45S cistron. Among 79 cp protein-coding genes, 74 showed nucleotide variations among ten species, of which infA, rpl22, rps19 and ndhE genes showed the highest Ks values and atpF, atpE, ycf2 and rps15 genes showed the highest Ka/Ks values. Four genes, petN, psaJ, psbF, and psbN, related to photosynthesis and one gene, rpl23, related to the ribosomal large subunit remain conserved in all 10 Araliaceae species. Phylogenetic analysis revealed that the ten species could be resolved into two monophyletic lineages, the Panax-Aralia and the Eleutherococcus-Dendropanax groups, which diverged approximately 8.81–10.59 million years ago (MYA). The Panax genus divided into two groups, with diploid species including P. notoginseng, P. vietnamensis, and P. japonicus surviving in Southern Asia and a tetraploid group including P. ginseng and P. quinquefolius Northern Asia and North America 2.89–3.20 MYA.

Most ginseng cultivars bear red berry and only one cultivar `Gumpoong` (GU) bears golden berry. GU is an elite cultivar bred by pedigree selection from a golden berry landrace (a mixed population) `Hwangsook` (HS). We developed three unique polymorphic markers from complete chloroplast genome sequences of GU and HS. A population of GU showed uniform band amplicon against three chloroplast markers whereas HS population displayed mixed genotypes for both GU and HS. Using the characteristics of mixed genotypes in HS population, we developed a convenient method to differentiate GU and HS population by application of pooled DNA template for PCR analysis (pooling method). The pooling method revealed that the GU pool was identical with GU genotype while the HS pool showed both GU and HS genotype. The pooling method is a cost and time effective method for accurate authentication of both golden berry ginseng cultivars. The method is useful to protect GU products from its tentative counterfeits from seeds to mature plant stages as well as processed root products.

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.

Plagiorhegma dubia Maxim. is a traditional Chinese medicinal herb from Plagiorhegma, Berberidaceae, which is distributed in the northeast of China, Korea, Russia. The complete chloroplast genome is 152,468 bp in length, with large single copy (LSC 82,257 bp) and small single copy (SSC 16,599 bp) regions separated by a pair of inverted repeats (IR 26 805 bp). The genome has a total of 113 genes including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis shows that P. dubia is closely related with Sinopodophyllum hexandrum and Epimedium species. The results are of great implication for the development and utilization of P. dubia and the phylogenetic researches on Berberidaceae.

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.

Mungbean (Vigna radiata), genus Vigna, is an economically important legume crop that is valued for its protein-rich dry seeds. However, bruchid infestation causes severe threat to seed storage in terms of deterioration in quantity along with nutritional quality. A new resistance source was found in Vigna umbellata, a species that is cross compatible with mungbean. Thus, the objective of this study is to identify the quantitative trait locus (QTL) controlling the bruchid resistance in RIL (recombinant inbred line) population developed from a hybridization between VRM (Gg) 1 (V. radiata) and TNAU RED (V. umbellata). The RIL population was screened for bruchid resistance using the following traits, viz. per cent of seed damage (SD), the total developmental period (TDP) and per cent of adult emergence (AE) in 2017 and 2018. The QTL analysis of these traits using a genetic map composed of 538 single nucleotide polymorphism (SNP) markers covering 11 chromosomes detected 12 QTLs in over the 2 years. Among them, the QTLs on chromosomes 05 and 08, designated qSD05 and qAE08, respectively, were stably detected in both years. qSD05 exhibited large effects in both years and mapped to 1.58-Mb genomic region of the mungbean reference genome. Genome mining of this QTL region identified the likely candidate genes involved in bruchid resistance. The outcomes and QTLs found in this study may provide useful information for fine mapping, marker-assisted selection (MAS), gene cloning and breeding for the resistance to bruchids.

Three Apiaceae species Ledebouriella seseloides, Peucedanum japonicum, and Glehnia littoralis are used as Asian herbal medicines, with the confusingly similar common name &ldquo, Bang-poong&rdquo, 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&ndash, 6 million years ago (Mya), while P. japonicum diverged from L. seseloides only 2&ndash, 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.

Indian barnyard millet (Echinochloa frumentacea) is a rich source of dietary fiber, minerals and protein. The lack of genetic resources has slowed the discovery of genes involved in its nutrient accumulation and climate resilience. Here, we present the first transcriptomes of E. frumentacea [97,065 transcripts, including 65,276 protein-coding transcripts, over 90% of which have been functionally annotated, and 31,789 long noncoding RNA (lncRNA) transcripts] and its wild relative E. crus-galli (93,725 transcripts, including 68,480 protein-coding transcripts, 89% of which have been annotated). Comparative transcriptome analysis identified 4159 protein-coding and 2258 lncRNA transcripts in Indian barnyard millet that showed either up- or down-regulated expression when compared with E. crus-galli, and 3489 protein-coding transcripts unique to Indian barnyard millet were also detected. Additional analysis revealed that photosynthesis is likely crucial in the drought adaptation of Indian barnyard millet. We then identified possible genes regulation responsible for drought tolerance and Fe and Zn accumulation. Moreover, based on the simple sequence repeat (SSR)-containing sequence, 30 SSR primer pairs were arbitrarily selected, synthesized and used to screen the 30 E. frumentacea accessions. Of these, 10 SSR primers were polymorphic. Collectively, our results enhance the knowledge of micronutrient accumulation and drought tolerance in Indian barnyard millet, as well as of the genetic diversity of Echinochloa species.

The woody Sonchus alliance consists primarily of woody species of the genus Sonchus (subgenus Dendrosonchus, family Asteraceae). Most members of the alliance are endemic to the oceanic archipelagos in the phytogeographic region of Macaronesia. They display extensive morphological, ecological, and anatomical diversity, likely caused by the diverse habitats on islands and rapid adaptive radiation. As a premier example of adaptive radiation and insular woodiness of species endemic to oceanic islands, the alliance has been the subject of intensive evolutionary studies. While phylogenetic studies suggested that it is monophyletic and its major lineages radiated rapidly early in the evolutionary history of this group, genetic mechanisms of speciation and genomic evolution within the alliance remain to be investigated. We first attempted to address chloroplast (cp) genome evolution by conducting comparative genomic analysis of three representative endemic species (Sonchus acaulis, Sonchus canariensis, and Sonchus webbii) from the Canary Islands. Despite extensive morphological, anatomical, and ecological differences among them, their cp genomes were highly conserved in gene order and content, ranging from 152,071 to 152,194 bp in total length. The number of repeat variations and six highly variable regions were identified as valuable molecular markers. Phylogenetic analysis of 32 species in the family Asteraceae revealed the phylogenetic position of the woody Sonchus alliance within the tribe Cichorieae and the sister relationship between the weedy Sonchus oleraceus and the alliance.

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.

Hibiscus syriacus (L.) (rose of Sharon) is one of the most widespread garden shrubs in the world. We report a draft of the H. syriacus genome comprised of a 1.75 Gb assembly that covers 92% of the genome with only 1.7% (33 Mb) gap sequences. Predicted gene modeling detected 87,603 genes, mostly supported by deep RNA sequencing data. To define gene family distribution among relatives of H. syriacus, orthologous gene sets containing 164,660 genes in 21,472 clusters were identified by OrthoMCL analysis of five plant species, including H. syriacus, Arabidopsis thaliana, Gossypium raimondii, Theobroma cacao and Amborella trichopoda. We inferred their evolutionary relationships based on divergence times among Malvaceae plant genes and found that gene families involved in flowering regulation and disease resistance were more highly divergent and expanded in H. syriacus than in its close relatives, G. raimondii (DD) and T. cacao. Clustered gene families and gene collinearity analysis revealed that two recent rounds of whole-genome duplication were followed by diploidization of the H. syriacus genome after speciation. Copy number variation and phylogenetic divergence indicates that WGDs and subsequent diploidization led to unequal duplication and deletion of flowering-related genes in H. syriacus and may affect its unique floral morphology.

The genus Brassica is an important resource for major agricultural products such as oils, vegetable and fodder. The Brassiceae tribe-specific whole-genome triplication that occurred ~15.9 million years ago influenced the speciation and morphological diversification that has been exploited in agriculture, making Brassica an excellent model system for studying polyploidization- mediated evolution. Genome sequencing and comparative genome analysis have revealed conserved structures and uncovered the genome evolution of Brassica species. While chromosome shuffling and asymmetric subgenome gene retention are widely reported in Brassica species, limited information is available about the dynamics of repetitive elements (REs), which are central to epigenetic mechanisms and thus play a pivotal role in plant genome adaptation and evolution. The assembled reference genome sequences of B. rapa (AA) and B. oleracea (CC), and their derived allotetraploid, B. napus (AACC), cover 58%, 86%, and 75% of their respective estimated genome sizes. The remaining non-assembled genome portions vary between these three genome sequences, and the major components remain hidden in each genome. Here, we review the dynamics of the major Brassica repeats that have played roles in speciation of the AA, CC, and AACC genomes. We show that 10 major Brassica repeats appear to occupy more than 50% of each respective unassembled genome sequence, yet represent less than 1% of assembled reference genome sequences. We have estimated their genome proportions using whole-genome Illumina reads and cytogenetic analyses in an attempt to understand the role of these repeats in genome evolution.

Cytoplasmic male-sterility (CMS) conferred by the CMS-S cytoplasm has been most commonly used for onion (Allium cepa L.) F1 hybrid seed production. We first report the complete mitochondrial genome sequence containing CMS-S cytoplasm in this study. Initially, seven contigs were de novo assembled from 150-bp paired-end raw reads produced from the total genomic DNA using the Illumina NextSeq500 platform. These contigs were connected into a single circular genome consisting of 316,363 bp (GenBank accession: KU318712) by PCR amplification. Although all 24 core protein-coding genes were present, no ribosomal protein-coding genes, except rps12, were identified in the onion mitochondrial genome. Unusual trans-splicing of the cox2 gene was verified, and the cox1 gene was identified as part of the chimeric orf725 gene, which is a candidate gene responsible for inducing CMS. In addition to orf725, two small chimeric genes were identified, but no transcripts were detected for these two open reading frames. Thirteen chloroplast-derived sequences, with sizes of 126-13,986 bp, were identified in the intergenic regions. Almost 10 % of the onion mitochondrial genome was composed of repeat sequences. The vast majority of repeats were short repeats of

Most chloroplast genes in Zanthoxylum schinifolium (Korean pepper) and Z. piperitum (Japanese pepper) are subject to neutral or purifying selection (Ka/Ks values 1); however, NAD(P)H dehydrogenase subunit G (ndhG) has a Ka/Ks value of 1.43, which may indicate positive selection. Here, we modeled the ZsNdhG and ZpNdhG structures by comparing them with the NuoJ subunit of respiratory complex I in Escherichia coli, revealing the locations of four amino acid differences between ZsNdhG and ZpNdhG. As these polymorphisms were located at the end of a membrane-spanning α-helix or in peptide loops external to the membrane, they are not expected to have major effects on the membrane-embedding properties of these proteins. However, we found that C-to-U RNA editing occurred at the ndhG-50 sites of ndhG (uCa to uUa, Ser to Leu) in both species, resulting in changes to an amino acid located in the middle of a membrane-spanning α-helix, which may maintain its hydrophobicity. RNA editing at the ndhG-50 site was conserved in many plant species, and the modeled structures of Anthoceros formosae NdhG and Spirodela polyrhiza NdhB provided further evidence that RNA editing increases the hydrophobicity of membrane-embedded proteins. We also speculate that the polar residues inside membrane-spanning α-helices serve to support the protein structure. This report represents the first RNA-editing site identified in Zanthoxylum and points to the importance of considering RNA editing when identifying positively selected genes based on Ka/Ks values.

Mungbean (Vigna radiata) and ricebean (V. umbellata) were utilized to obtain an inter-specific recombinant inbred line (RIL) population with the objective of detecting quantitative trait loci (QTL) associated with mungbean yellow mosaic virus (MYMV) resistance. To precisely map QTLs, accurate genetic linkage maps are essential. In the present study, genotyping-by-sequencing (GBS) platform was utilized to develop the genetic linkage map. The map contained 538 single nucleotide polymorphism (SNP) markers, consisted of 11 linkage groups and spanned for 1291.7 cM with an average marker distance of 2.40 cM. The individual linkage group ranged from 90.2 to 149.1 cM in length, and the SNP markers were evenly distributed in the genetic linkage map, with 30–79 SNP markers per chromosome. The QTL analysis using the genetic map and 2 years (2015 and 2016) of phenotyping data identified five QTLs with phenotypic variation explained (PVE) from 10.11 to 20.04%. Of these, a QTL on chromosome 4, designated as qMYMV4-1, was major and stably detected in the same marker interval in both years. This QTL region harbours possible candidate genes for controlling MYMV resistance. The linkage map and QTL/gene (s) for MYMV resistance identified in this study should be useful for QTL fine mapping and cloning for further studies.

The family Araliaceae contains many medicinal species including ginseng of which the whole genome sequencing analyses have been going on these days. To characterize the chromosomal distribution of 5S and 45S rDNAs and telomeric repeat in four ginseng related species of Aralia elata (Miq.) Seem., Dendropanax morbiferus H. Lev., Eleutherococcus sessiliflorus (Rupr. Et Maxim.) Seem., Kalopanax septemlobus (Thunb. ex A.Murr.) Koidz. Pre-labelled oligoprobe (PLOP)-fluorescence in situ hybridization (FISH) was carried out. The chromosome number of A. elata was 2n = 24, whereas that of the other three species of D. morbiferus, E. sessiliflorus, and K. septemlobus was 2n = 48, corresponding to diploid and tetraploid, respectively, based on the basic chromosome number x = 12 in Araliaceae. In all four species, one pair of 5S signals were detected in the proximal regions of the short arms of chromosome 3, whereas in K. septemlobus, the 5S rDNA signals localized in the subtelomeric region of short arm of chromosome 3, while all the 45S rDNA signals localized at the paracentromeric region of the short arm of chromosome 1. And the telomeric repeat signals were detected at the telomeric region of both short and long arms of most chromosomes. The PLOP-FISH was very effective compared with conventional FISH method. These results provide useful comparative cytogenetic information to better understand the genome structure of each species and will be useful to trace the history of ginseng genomic constitution.

Ginsenosides are dammarane-type or triterpenoidal saponins that contribute to the various pharmacological activities of the medicinal herb Panax ginseng. The putative biosynthetic pathway for ginsenoside biosynthesis is known in P. ginseng, as are some of the transcripts and enzyme-encoding genes. However, few genes related to the UDP-glycosyltransferases (UGTs), enzymes that mediate glycosylation processes in final saponin biosynthesis, have been identified. Here, we generated three replicated Illumina RNA-Seq datasets from the adventitious roots of P. ginseng cultivar Cheongsun (CS) after 0, 12, 24, and 48 h of treatment with methyl jasmonate (MeJA). Using the same CS cultivar, metabolomic data were also generated at 0 h and every 12–24 h thereafter until 120 h of MeJA treatment. Differential gene expression, phylogenetic analysis, and metabolic profiling were used to identify candidate UGTs. Eleven candidate UGTs likely to be involved in ginsenoside glycosylation were identified. Eight of these were considered novel UGTs, newly identified in this study, and three were matched to previously characterized UGTs in P. ginseng. Phylogenetic analysis further asserted their association with ginsenoside biosynthesis. Additionally, metabolomic analysis revealed that the newly identified UGTs might be involved in the elongation of glycosyl chains of ginsenosides, especially of protopanaxadiol (PPD)-type ginsenosides.

Fluorescence in situ hybridization (FISH) is used to visualize the distribution of DNA elements within a genome. Conventional methods for FISH take 1–2 days. Here, we developed a simplified, rapid FISH technique using pre-labeled oligonucleotide probes (PLOPs) and tested the procedure using 18 PLOPs from 45S and 5S rDNA, Arabidopsis-type telomere, and newly-identified Panax ginseng-specific tandem repeats. The 16 developed rDNA PLOPs can be universally applied to plants and animals. The telomere PLOPs can be utilized in most plants with Arabidopsis-type telomeres. The ginseng-specific PLOP can be used to distinguish P. ginseng from related Panax species. Differential labeling of PLOPs allowed us to simultaneously visualize different target loci while reducing the FISH hybridization time from ~16 h to 5 min. PLOP-FISH is efficient, reliable, and rapid, making it ideal for routine analysis, especially of newly sequenced genomes using either universal or specific targets, such as novel tandem repeats identified from whole-genome sequencing data.

The ginseng (Panax ginseng C.A. Meyer) is a perennial herbaceous plant that has been used in traditional oriental medicine for thousands of years. Ginsenosides, which have significant pharmacological effects on human health, are the foremost bioactive constituents in this plant. Having realized the importance of this plant to humans, an integrated omics resource becomes indispensable to facilitate genomic research, molecular breeding and pharmacological study of this herb. The first draft genome sequences of P. ginseng cultivar “Chunpoong” were reported recently. Here, using the draft genome, transcriptome, and functional annotation datasets of P. ginseng, we have constructed the Ginseng Genome Database http://ginsengdb.snu.ac.kr /, the first open-access platform to provide comprehensive genomic resources of P. ginseng. The current version of this database provides the most up-to-date draft genome sequence (of approximately 3000 Mbp of scaffold sequences) along with the structural and functional annotations for 59,352 genes and digital expression of genes based on transcriptome data from different tissues, growth stages and treatments. In addition, tools for visualization and the genomic data from various analyses are provided. All data in the database were manually curated and integrated within a user-friendly query page. This database provides valuable resources for a range of research fields related to P. ginseng and other species belonging to the Apiales order as well as for plant research communities in general. Ginseng genome database can be accessed at http://ginsengdb.snu.ac.kr /.

Lonicera spp. (Caprifoliaceae) are important not only as a common medicinal herb in East Asia but also as one of the most problematic invasive species in North America. In the present study, we performed a systemic analysis of genomic and chemical diversity among six Lonicera species occurring in Korea, L. japonica, L. maackii, L. insularis, L. sachalinensis, L. praeflorens, and L. vesicaria, using chloroplast DNA whole genome shotgun (WGS) sequencing and LC-MS analyses. The phylogenetic and phylochemical relationships did not coincide with each other, but partial consistency could be found among them. InDel-based cDNA marker for authentication was developed based on the genome sequences. Flavonoids, iridoids, and organic acids were identified in the LC-MS analyses, and their inter-species distribution and localization were also revealed.

Plant mitochondrial genomes (mtDNAs) vary in sequence structure. We assembled the Brassica oleracea var. capitata mtDNA using a mean coverage depth of 25X whole genome sequencing (WGS) and confirmed the presence of eight contigs/fragments by BLASTZ using the previously reported KJ820683 and AP012988 mtDNA as reference. Assembly of the mtDNA sequence reads resulted in a circular structure of 219,975 bp. Our assembled mtDNA, NCBI acc. no. KU831325, contained 34 protein-coding genes, 3 rRNA genes, and 19 tRNA genes with similarity to the KJ820683 and AP012988 reference mtDNA. No large repeats were found in the KU831325 assembly. However, KU831325 showed differences in the arrangement of bases at different regions compared to the previously reported mtDNAs. In the reference mtDNAs KJ820683 and AP012988, contig/fragment number 4 is partitioned into two contigs/fragments, 4a and 4b. However, contig/fragment number 4 was a single contig/fragment with 29,661 bp in KU831325. PCR and qRT-PCR using flanking markers from separate parts of contig/fragment number 4 confirmed it to be a single contig/fragment. In addition, genome re-alignment of the plastid genome and mtDNAs supported the presence of heteroplasmy and reverse arrangement of the heteroplasmic blocks within the other mtDNAs compared to KU831325 that might be one of the causal factors for its diversity. Our results thus confirm the existence of different mtDNAs in diverse B. oleracea subspecies.

Repetitive elements (REs) play major roles in genome organization, size, and evolution, but are often underrepresented in genome assemblies. The recent genome assembly of the allotetraploid Brassica napus genome revealed that 48% of the genome comprised REs, including transposons and tandem repeats. In the present work, we show the overall quantity and comparative analyses of major repeat families in both the assembled and unassembled portions of the reference B. napus genome. We surveyed the abundance, distribution, diversity, and dynamics of ten major REs in the B. napus genome, which represented less than 1% of the total 1130 Mb B. napus genome in the current assembly. However, in silico mapping of raw whole-genome sequence reads from nine B. napus accessions revealed about 11% of the genome as represented by these ten repeat families. Comparative analyses of these major repeats showed their evolutionary dynamics in the B. rapa (Ar), B. oleracea (Co), and B. napus (AnCn) genomes as well as a considerable inter- and intraspecies repeat diversity among different B. napus accessions. Cytogenetic mapping of these major repeats showed their genomic abundance and distribution, with some families having a conserved subgenomic distribution pattern in the B. napus genome. Finally, the impact of genetic changes to REs and their corresponding epigenetic readjustments during B. napus evolution are also discussed in this chapter.

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.

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 comple...

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.

Decoding complete genome sequences is prerequisite for comprehensive genomics studies. However, the currently available reference genome sequences of Brassica rapa (A genome), B. oleracea (C) and B. napus (AC) cover 391, 540, and 850 Mbp and represent 80.6, 85.7, and 75.2% of the estimated genome size, respectively, while remained are hidden or unassembled due to highly repetitive nature of these genome components. Here, we performed the first comprehensive genome-wide analysis using low-coverage whole-genome sequences to explore the hidden genome components based on characterization of major repeat families in the B. rapa and B. oleracea genomes. Our analysis revealed 10 major repeats (MRs) including a new family comprising about 18.8, 10.8, and 11.5% of the A, C and AC genomes, respectively. Nevertheless, these 10 MRs represented less than 0.7% of each assembled reference genome. Genomic survey and molecular cytogenetic analyses validates our insilico analysis and also pointed to diversity, differential distribution, and evolutionary dynamics in the three Brassica species. Overall, our work elucidates hidden portions of three Brassica genomes, thus providing a resource for understanding the complete genome structures. Furthermore, we observed that asymmetrical accumulation of the major repeats might be a cause of diversification between the A and C genomes.

Background Transposable elements are major evolutionary forces which can cause new genome structure and species diversification. The role of transposable elements in the expansion of nucleotide-binding and leucine-rich-repeat proteins (NLRs), the major disease-resistance gene families, has been unexplored in plants. Results We report two high-quality de novo genomes (Capsicum baccatum and C. chinense) and an improved reference genome (C. annuum) for peppers. Dynamic genome rearrangements involving translocations among chromosomes 3, 5, and 9 were detected in comparison between C. baccatum and the two other peppers. The amplification of athila LTR-retrotransposons, members of the gypsy superfamily, led to genome expansion in C. baccatum. In-depth genome-wide comparison of genes and repeats unveiled that the copy numbers of NLRs were greatly increased by LTR-retrotransposon-mediated retroduplication. Moreover, retroduplicated NLRs are abundant across the angiosperms and, in most cases, are lineage-specific. Conclusions Our study reveals that retroduplication has played key roles for the massive emergence of NLR genes including functional disease-resistance genes in pepper plants. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1341-9) contains supplementary material, which is available to authorized users.

Background: Panax ginseng has been used in traditional medicine to strengthen the body and mental well-being of humans for thousands of years. Many elite ginseng cultivars have been developed, and ginseng cultivation has become well established during the last century. However, heat stress poses an important threat to the growth and sustainable production of ginseng. Efforts have been made to study the effects of high temperature on ginseng physiology, but knowledge of the molecular responses to heat stress is still limited. Methods: We sequenced the transcriptomes (RNA-Seq) of two ginseng cultivars, Chunpoong (CP) and Yunpoong (YP), which are sensitive and resistant to heat stress, respectively, after 1- and 3-week heat treatments. Differential gene expression and gene ontology enrichment along with profiled chlorophyll contents were performed. Results: CP is more sensitive to heat stress than YP and exhibited a lower chlorophyll content than YP. Moreover, heat stress reduced the chlorophyll content more rapidly in CP than in YP. A total of 329 heat-responsive genes were identified. Intriguingly, genes encoding chlorophyll a/b–binding proteins, WRKY transcription factors, and fatty acid desaturase were predominantly responsive during heat stress and appeared to regulate photosynthesis. In addition, a genome-wide scan of photosynthetic and sugar metabolic genes revealed reduced transcription levels for ribulose 1,5-bisphosphate carboxylase/oxygenase under heat stress, especially in CP, possibly attributable to elevated levels of soluble sugars. Conclusion: Our comprehensive genomic analysis reveals candidate loci/gene targets for breeding and functional studies related to developing high temperature–tolerant ginseng varieties. Keywords: Chunpoong, Heat stress, Panax ginseng, RNA-Seq, Yunpoong

We performed chloroplast genome sequencing and comparative analysis of two Rutaceae species, Zanthoxylum schinifolium (Korean pepper tree) and Z. piperitum (Japanese pepper tree), which are medicinal and culinary crops in Asia. We identified more than 837 single nucleotide polymorphisms and 103 insertions/deletions (InDels) based on a comparison of the two chloroplast genomes and developed seven DNA markers derived from five tandem repeats and two InDel variations that discriminated between Korean Zanthoxylum species. Metabolite profile analysis pointed to three metabolic groups, one with Korean Z. piperitum samples, one with Korean Z. schinifolium samples, and the last containing all the tested Chinese Zanthoxylum species samples, which are considered to be Z. bungeanum based on our results. Two markers were capable of distinguishing among these three groups. The chloroplast genome sequences identified in this study represent a valuable genomics resource for exploring diversity in Rutaceae, and the molecular markers will be useful for authenticating dried Zanthoxylum berries in the marketplace.

Genome duplication and repeat multiplication contribute to genome evolution in plants. Our previous work identified a recent allotetraploidization event and five high-copy LTR retrotransposon (LTR-RT) families PgDel, PgTat, PgAthila, PgTork, and PgOryco in Panax ginseng. Here, using whole-genome sequences, we quantified major repeats in five Panax species and investigated their role in genome evolution. The diploids P. japonicus, P. vietnamensis, and P. notoginseng and the tetraploids P. ginseng and P. quinquefolius were analyzed alongside their relative Aralia elata. These species possess 0.8–4.9 Gb haploid genomes. The PgDel, PgTat, PgAthila, and PgTork LTR-RT superfamilies accounted for 39–52% of the Panax species genomes and 17% of the A. elata genome. PgDel included six subfamily members, each with a distinct genome distribution. In particular, the PgDel1 subfamily occupied 23–35% of the Panax genomes and accounted for much of their genome size variation. PgDel1 occupied 22.6% (0.8 Gb of 3.6 Gb) and 34.5% (1.7 Gb of 4.9 Gb) of the P. ginseng and P. quinquefolius genomes, respectively. Our findings indicate that the P. quinquefolius genome may have expanded due to rapid PgDel1 amplification over the last million years as a result of environmental adaptation following migration from Asia to North America.