Whole-Genome Sequence of Erwinia persicina B64, Which Causes Pink Soft Rot in Onions.

Erwinia persicina B64 was isolated from rotten onions in cold-storage facilities. Here, we report the complete genome sequence of E. persicina B64, which contains 5,070,450 bp with 55.17% GC content. The genome of this isolate is composed of one chromosome and two plasmids.

Re-exploration of U's Triangle Brassica Species Based on Chloroplast Genomes and 45S nrDNA Sequences.

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.

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.

Expression profiles of BrMYB transcription factors related to glucosinolate biosynthesis and stress response in eight subspecies of Brassica rapa.

Brassica rapa is a polyploid species with phenotypically diverse cultivated subspecies. Glucosinolates (GSLs) are secondary metabolites that contribute to anticarcinogenic activity and plant defense in Brassicaceae. Previously, complete coding sequences of 13 BrMYB transcription factors (TFs) related to GSL biosynthesis were identified in the B. rapa genome. In the present study, we investigated GSL content and expression levels of these BrMYB TFs in 38 accessions belonging to eight subspecies of B. rapa. Twelve identified GSLs were detected and were classified into three chemical groups based on patterns of GSL content and expression profiles of the BrMYB TFs. GSL content and BrMYB TF expression levels differed among genotypes, including B. rapa subspecies pekinensis, chinensis and rapa. BrMYB28.3, BrMYB51.1 and BrMYB122.2 positively regulated GSL content in 38 accessions. Furthermore, expression levels of BrMYB28s and BrMYB34.3 increased under most abiotic and biotic stress treatments. The three BrMYB51 paralogs also showed drastically increased expression levels after infection with Pectobacterium carotovorum. The results of the present study improve our understanding of the functional diversity of these 13 BrMYB TFs during the evolution of polyploid B. rapa.

Comparative transcriptome analysis reveals whole-genome duplications and gene selection patterns in cultivated and wild Chrysanthemum species.

KEY MESSAGE: Comparative transcriptome analysis of wild and cultivated chrysanthemums provides valuable genomic resources and helps uncover common and divergent patterns of genome and gene evolution in these species. Plants are unique in that they employ polyploidy (or whole-genome duplication, WGD) as a key process for speciation and evolution. The Chrysanthemum genus is closely associated with hybridization and polyploidization, with Chrysanthemum species exhibiting diverse ploidy levels. The commercially important species, C. morifolium is an allohexaploid plant that is thought to have originated via the hybridization of several Chrysanthemum species, but the genomic and molecular evolutionary mechanisms remain poorly understood. In the present study, we sequenced and compared the transcriptomes of C. morifolium and the wild Korean diploid species, C. boreale. De novo transcriptome assembly revealed 11,318 genes in C. morifolium and 10,961 genes in C. boreale, whose functions were annotated by homology searches. An analysis of synonymous substitution rates (Ks) of paralogous and orthologous genes suggested that the two Chrysanthemum species commonly experienced the Asteraceae paleopolyploidization and recent genome duplication or triplication before the divergence of these species. Intriguingly, C. boreale probably underwent rapid diploidization, with a reduction in chromosome number, whereas C. morifolium maintained the original chromosome number. Analysis of the ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) between orthologous gene pairs indicated that 107 genes experienced positive selection, which may have been crucial for the adaptation, domestication, and speciation of Chrysanthemum.

The complete mitochondrial genome of Wonwhang (Pyrus pyrifolia).

This is a de novo assembly and annotation of a complete mitochondrial genome from Pyrus pyrifolia in the family Rosaceae. The complete mitochondrial genome of P. pyrifolia was assembled from PacBio RSII P6-C4 sequencing reads. The circular genome was 458,873 bp in length, containing 39 protein-coding genes, 23 tRNA genes and three rRNA genes. The nucleotide composition was A (27.5%), T (27.3%), G (22.6%) and C (22.6%) with GC content of 45.2%. Most of protein-coding genes use the canonical start codon ATG, whereas nad1, cox1, matR and rps4 use ACG, mttB uses ATT, rpl16 and rps19 uses GTG. The stop codon is also common in all mitochondrial genes. The phylogenetic analysis showed that P. pyrifolia was clustered with the Malus of Rosaceae family. Maximum-likelihood analysis suggests a clear relationship of Rosids and Asterids, which support the traditional classification.

MultiSyn: A Webtool for Multiple Synteny Detection and Visualization of User's Sequence of Interest Compared to Public Plant Species.

Information on multiple synteny between plants and/or within a plant is key information to understand genome evolution. In addition, visualization of multiple synteny is helpful in interpreting evolution. So far, some web applications have been developed to determine and visualize multiple homology regions at once. However, the applications are not fully convenient for biologists because some of them do not include the function of synteny determination but visualize the multiple synteny plots by allowing users to upload their synteny data by determining the synteny based only on BLAST similarity information, with some algorithms not designed for synteny determination. Here, we introduce a web application that determines and visualizes multiple synteny from two types of files, simplified browser extensible data and protein sequence file by MCScanX algorithm, which have been used in many synteny studies.

Elucidating the triplicated ancestral genome structure of radish based on chromosome-level comparison with the Brassica genomes.

KEYMESSAGE: This study presents a chromosome-scale draft genome sequence of radish that is assembled into nine chromosomal pseudomolecules. A comprehensive comparative genome analysis with the Brassica genomes provides genomic evidences on the evolution of the mesohexaploid radish genome. Radish (Raphanus sativus L.) is an agronomically important root vegetable crop and its origin and phylogenetic position in the tribe Brassiceae is controversial. Here we present a comprehensive analysis of the radish genome based on the chromosome sequences of R. sativus cv. WK10039. The radish genome was sequenced and assembled into 426.2 Mb spanning >98 % of the gene space, of which 344.0 Mb were integrated into nine chromosome pseudomolecules. Approximately 36 % of the genome was repetitive sequences and 46,514 protein-coding genes were predicted and annotated. Comparative mapping of the tPCK-like ancestral genome revealed that the radish genome has intermediate characteristics between the Brassica A/C and B genomes in the triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between radish and other Brassica species provided genomic evidences that the current form of nine chromosomes in radish was rearranged from the chromosomes of hexaploid progenitor. Overall, this study provides a chromosome-scale draft genome sequence of radish as well as novel insight into evolution of the mesohexaploid genomes in the tribe Brassiceae.

The complete chloroplast genome sequence of medicinal plant, Artemisia argyi.

Artemisia argyi, called wormwood, is widely distributed in northeastern Asia. The complete chloroplast genome sequence of A. argyi was generated by de novo assembly using whole genome next generation sequences. The complete chloroplast genome sequence of A. argyi is 151 192 bp in size. It is composed of a large single-copy (LSC), a small single-copy (SSC) and two inverted repeat (IR) regions of 82 930 bp, 18 344 bp and 24 959 bp, respectively. Overall GC contents of the genome were 37.46%. The A. argyi chloroplast genome has a total of 114 genes including 80 protein-coding genes, 30 tRNA genes and four rRNA genes. Phylogenetic analysis based on the chloroplast genome demonstrated that A. argyi is most closely related to Artemisia montana.

Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.).

The onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops in the world. Although a considerable amount of onion transcriptome data has been deposited into public databases, the sequences of the protein-coding genes are not accurate enough to be used, owing to non-coding sequences intermixed with the coding sequences. We generated a high-quality, annotated onion transcriptome from de novo sequence assembly and intensive structural annotation using the integrated structural gene annotation pipeline (ISGAP), which identified 54,165 protein-coding genes among 165,179 assembled transcripts totalling 203.0 Mb by eliminating the intron sequences. ISGAP performed reliable annotation, recognizing accurate gene structures based on reference proteins, and ab initio gene models of the assembled transcripts. Integrative functional annotation and gene-based SNP analysis revealed a whole biological repertoire of genes and transcriptomic variation in the onion. The method developed in this study provides a powerful tool for the construction of reference gene sets for organisms based solely on de novo transcriptome data. Furthermore, the reference genes and their variation described here for the onion represent essential tools for molecular breeding and gene cloning in Allium spp.

Cell-autonomous-like silencing of GFP-partitioned transgenic Nicotiana benthamiana.

We previously reported the novel partitioning of regional GFP-silencing on leaves of 35S-GFP transgenic plants, coining the term "partitioned silencing". We set out to delineate the mechanism of partitioned silencing. Here, we report that the partitioned plants were hemizygous for the transgene, possessing two direct-repeat copies of 35S-GFP. The detection of both siRNA expression (21 and 24 nt) and DNA methylation enrichment specifically at silenced regions indicated that both post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) were involved in the silencing mechanism. Using in vivo agroinfiltration of 35S-GFP/GUS and inoculation of TMV-GFP RNA, we demonstrate that PTGS, not TGS, plays a dominant role in the partitioned silencing, concluding that the underlying mechanism of partitioned silencing is analogous to RNA-directed DNA methylation (RdDM). The initial pattern of partitioned silencing was tightly maintained in a cell-autonomous manner, although partitioned-silenced regions possess a potential for systemic spread. Surprisingly, transcriptome profiling through next-generation sequencing demonstrated that expression levels of most genes involved in the silencing pathway were similar in both GFP-expressing and silenced regions although a diverse set of region-specific transcripts were detected.This suggests that partitioned silencing can be triggered and regulated by genes other than the genes involved in the silencing pathway.

A multistep screening method to identify genes using evolutionary transcriptome of plants.

We introduced a multistep screening method to identify the genes in plants using microarrays and ribonucleic acid (RNA)-seq transcriptome data. Our method describes the process for identifying genes using the salt-tolerance response pathways of the potato (Solanum tuberosum) plant. Gene expression was analyzed using microarrays and RNA-seq experiments that examined three potato lines (high, intermediate, and low salt tolerance) under conditions of salt stress. We screened the orthologous genes and pathway genes involved in salinity-related biosynthetic pathways, and identified nine potato genes that were candidates for salinity-tolerance pathways. The nine genes were selected to characterize their phylogenetic reconstruction with homologous genes of Arabidopsis thaliana, and a Circos diagram was generated to understand the relationships among the selected genes. The involvement of the selected genes in salt-tolerance pathways was verified by reverse transcription polymerase chain reaction analysis. One candidate potato gene was selected for physiological validation by generating dehydration-responsive element-binding 1 (DREB1)-overexpressing transgenic potato plants. The DREB1 overexpression lines exhibited increased salt tolerance and plant growth when compared to that of the control. Although the nine genes identified by our multistep screening method require further characterization and validation, this study demonstrates the power of our screening strategy after the initial identification of genes using microarrays and RNA-seq experiments.

Identification and mapping of a novel dominant resistance gene, TuRB07 to Turnip mosaic virus in Brassica rapa.

KEY MESSAGE: A novel dominant resistance gene, TuRB07, was found to confer resistance to an isolate of TuMV strain C4 in B. rapa line VC1 and mapped on the top of chromosome A06. The inheritance of resistance to Turnip mosaic virus in Brassica rapa was investigated by crossing the resistant line, VC1 with the susceptible line, SR5, and genotyping and phenotyping diverse progenies derived from this cross. Both a doubled haploid population, VCS3M-DH, an F2 and two BC1 (F1 × VC1 and F1 × SR5) populations were created. Population tests revealed that the resistance to the TuMV C4 isolate in B. rapa is controlled by a single dominant gene. This resistance gene, TuRB07 was positioned on the top of linkage group A06 of the B. rapa genome through bulk segregation analysis and fine mapping recombinants in three doubled haploid- and one backcross population using microsatellite markers developed from BAC end sequences. Within the region between the two closely linked markers flanking TuRB07, H132A24-s1, and KS10960, in the Chiifu reference genome, two genes encoding nucleotide-binding site and leucine-rich repeat proteins with a coiled-coil motif (CC-NBS-LRR), Bra018862 and Bra018863 were identified as candidate resistance genes. The gene Bra018862 is truncated, but the gene Bra018863 has all the domains to function. Furthermore, the analysis of structural variation using resequencing data of VC1 and SR5 revealed that Bra018863 might be a functional gene because the gene has no structural variation in the resistant line VC1 when compared with Chiifu, whereas at the other NBS-LRR genes large deletions were identified in the resistant line. Allelic differences of Bra018863 were found between VC1 and SR5, supporting the notion that this gene is a putative candidate gene for the virus resistance.

Mapping quantitative trait loci for tissue culture response in VCS3M-DH population of Brassica rapa.

KEY MESSAGE: Quantitative trait loci (QTL) controlling callus induction and plant regeneration were identified in the VCS3M-DH population of Brassica rapa. Quantitative trait loci (QTL) controlling callus induction and plant regeneration were identified in the VCS3M-DH population of Brassica rapa. The VCS3M-DH population showed wide and continuous variation in callus induction and shoot regeneration. Significant coefficient correlations were detected between these two parameters. Broad-sense heritability (h (2)) for the two traits was around 0.7, indicating genetic regulation of regeneration ability in this population. In the composite interval mapping analysis, two QTLs for callus induction ability, qCi2 and qCi7, were mapped on chromosome A02 and A07, explaining 28.6 % of phenotypic variation. For plant regeneration, four QTLs, qPr6-1 qPr6-2, qPr7, and qPr9 were identified on chromosome A06, A07, and A09, which in total explained 50.1 % of phenotypic variation. Furthermore, 15 putative candidate genes were found on the interval of the six QTLs, which were related to various plant hormones, MADS-box genes, and serine/threonine related genes. These results provide important information to identify genes related to tissue culture ability in B. rapa.

The p19 protein of Grapevine Algerian latent virus is a determinant of systemic infection of Chenopodium quinoa.

A previous study showed that both Grapevine Algerian latent virus (GALV) and Tomato bushy stunt virus (TBSV) systemically infect Nicotiana benthamiana, but GALV causes systemic infection whereas TBSV causes only local lesions in Chenopodium quinoa (C. quinoa). We recently isolated GALV strain Naju (GALV-N) from Limonium sinense and TBSV strain Sacheon (TBSV-S) from tomato. Both viruses belong to the genus Tombusvirus and have a similar genome organization. To identify determinants of systemic infection of GALV-N in C. quinoa in the current study, we generated infectious clones and capsid protein (CP)-deletion clones for the two viruses and confirmed that CP of GALV-N is required for systemic infection of C. quinoa due to its primary structural role in virus assembly. Through the use of chimeras, we identified a viral factor in addition to CP that contributes to systemic infection by GALV-N. Inactivation of the p19 demonstrated that host-specific activities of p19 are necessary for efficient systemic infection of C. quinoa by GALV-N. Our study is the first report to determine the viral factors required for systemic infection of GALV in C. quinoa.

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP.

Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3-9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana.

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host's defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3-9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

A single amino acid change in HC-Pro of soybean mosaic virus alters symptom expression in a soybean cultivar carrying Rsv1 and Rsv3.

It is generally believed that infidelity of RNA virus replication combined with R-gene-driven selection is one of the major evolutionary forces in overcoming host resistance. In this study, we utilized an avirulent soybean mosaic virus (SMV) mutant to examine the possibility of emergence of mutant viruses capable of overcoming R-gene-mediated resistance during serial passages. Interestingly, we found that the emerged progeny virus induced severe rugosity and local necrotic lesions in Jinpumkong-2 (Rsv1 + Rsv3) plants, while SMV-G7H provoked a lethal systemic hypersensitive response. Genome sequence analysis of the emerged progeny virus revealed that the mutation in CI that had caused SMV-G7H to lose its virulence was restored to the original sequence, and a single amino acid was newly introduced into HC-Pro, which means that the symptom alteration was due to this single amino acid mutation in HC-Pro. Our results suggest that SMV HC-Pro functions as a symptom determinant in the SMV-soybean pathosystem.

Molecular variability and genetic structure of the population of soybean mosaic virus based on the analysis of complete genome sequences.

The complete genomes of 30 Soybean mosaic virus (SMV) isolates and strains were sequenced in this study. Together with fourteen previously reported sequences, we analyzed the genetic structure of the SMV population. Analyses of genetic diversity showed that different genomic regions of SMV are under different evolutionary constraints and that there was no significant genetic differentiation between East Asian and North American populations of SMV. Phylogenetic analyses revealed a significant correlation between phylogeny of the cylindrical inclusion (CI) gene of SMV and SMV resistance gene 3 (Rsv3)-relating pathogenicity of SMV, suggesting CI might be a pathogenic determinant in Rsv3-mediated disease response. Interestingly, recombination analyses identified 19 'clear' recombination events in the SMV population. Furthermore, as several resistance-breaking strains were identified as recombinants, it appears that recombination might contribute to overcome host resistance in SMV-soybean pathosystem. Our finding suggests that recombination as well as mutation is an important evolutionary process in the genetic diversification of SMV population.