An improved Raphanus sativus cv. WK10039 genome localizes centromeres, uncovers variation of DNA methylation and resolves arrangement of the ancestral Brassica genome blocks in radish chromosomes.

KEY MESSAGE: This study presents an improved genome of Raphanus sativus cv. WK10039 uncovering centromeres and differentially methylated regions of radish chromosomes. Comprehensive genome comparison of radish and diploid Brassica species of U's triangle reveals that R. sativus arose from the Brassica B genome lineage and is a sibling species of B. nigra. Radish (Raphanus sativus L.) is a key root vegetable crop closely related to the Brassica crop species of the family Brassicaceae. We reported a draft genome of R. sativus cv. WK10039 (Rs1.0), which had 54.6 Mb gaps. To study the radish genome and explore previously unknown regions, we generated an improved genome assembly (Rs2.0) by long-read sequencing and high-resolution genome-wide mapping of chromatin interactions. Rs2.0 was 434.9 Mb in size with 0.27 Mb gaps, and the N50 scaffold length was 37.3 Mb (40-fold larger assembly compared to Rs1.0). Approximately 38% of Rs2.0 was comprised of repetitive sequences, and 52,768 protein-coding genes and 4845 non-protein-coding genes were predicted and annotated. The improved contiguity and coverage of Rs2.0, along with the detection of highly methylated regions, enabled localization of centromeres where R. sativus-specific centromere-associated repeats, full-length OTA and CRM LTR-Gypsy retrotransposons, hAT-Ac, CMC-EnSpm and Helitron DNA transposons, and sequences highly homologous to B. nigra centromere-specific CENH3-associated CL sequences were enriched. Whole-genome bisulfite sequencing combined with mRNA sequencing identified differential epigenetic marks in the radish genome related to tissue development. Synteny comparison and genomic distance analysis of radish and three diploid Brassica species of U's triangle suggested that the radish genome arose from the Brassica B genome lineage through unique rearrangement of the triplicated ancestral Brassica genome after splitting of the Brassica A/C and B genomes.

Comparative chloroplast genome analysis of Artemisia (Asteraceae) in East Asia: insights into evolutionary divergence and phylogenomic implications.

BACKGROUND: Artemisia in East Asia includes a number of economically important taxa that are widely used for food, medicinal, and ornamental purposes. The identification of taxa, however, has been hampered by insufficient diagnostic morphological characteristics and frequent natural hybridization. Development of novel DNA markers or barcodes with sufficient resolution to resolve taxonomic issues of Artemisia in East Asia is significant challenge. RESULTS: To establish a molecular basis for taxonomic identification and comparative phylogenomic analysis of Artemisia, we newly determined 19 chloroplast genome (plastome) sequences of 18 Artemisia taxa in East Asia, de novo-assembled and annotated the plastomes of two taxa using publicly available Illumina reads, and compared them with 11 Artemisia plastomes reported previously. The plastomes of Artemisia were 150,858-151,318 base pairs (bp) in length and harbored 87 protein-coding genes, 37 transfer RNAs, and 8 ribosomal RNA genes in conserved order and orientation. Evolutionary analyses of whole plastomes and 80 non-redundant protein-coding genes revealed that the noncoding trnH-psbA spacer was highly variable in size and nucleotide sequence both between and within taxa, whereas the coding sequences of accD and ycf1 were under weak positive selection and relaxed selective constraints, respectively. Phylogenetic analysis of the whole plastomes based on maximum likelihood and Bayesian inference analyses yielded five groups of Artemisia plastomes clustered in the monophyletic subgenus Dracunculus and paraphyletic subgenus Artemisia, suggesting that the whole plastomes can be used as molecular markers to infer the chloroplast haplotypes of Artemisia taxa. Additionally, analysis of accD and ycf1 hotspots enabled the development of novel markers potentially applicable across the family Asteraceae with high discriminatory power. CONCLUSIONS: The complete sequences of the Artemisia plastomes are sufficiently polymorphic to be used as super-barcodes for this genus. It will facilitate the development of new molecular markers and study of the phylogenomic relationships of Artemisia species in the family Asteraceae.

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals.

The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factor-induced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:ß-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor- and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.

Identification of candidate domestication regions in the radish genome based on high-depth resequencing analysis of 17 genotypes.

KEY MESSAGE: This study provides high-quality variation data of diverse radish genotypes. Genome-wide SNP comparison along with RNA-seq analysis identified candidate genes related to domestication that have potential as trait-related markers for genetics and breeding of radish. Radish (Raphanus sativus L.) is an annual root vegetable crop that also encompasses diverse wild species. Radish has a long history of domestication, but the origins and selective sweep of cultivated radishes remain controversial. Here, we present comprehensive whole-genome resequencing analysis of radish to explore genomic variation between the radish genotypes and to identify genetic bottlenecks due to domestication in Asian cultivars. High-depth resequencing and multi-sample genotyping analysis of ten cultivated and seven wild accessions obtained 4.0 million high-quality homozygous single-nucleotide polymorphisms (SNPs)/insertions or deletions. Variation analysis revealed that Asian cultivated radish types are closely related to wild Asian accessions, but are distinct from European/American cultivated radishes, supporting the notion that Asian cultivars were domesticated from wild Asian genotypes. SNP comparison between Asian genotypes identified 153 candidate domestication regions (CDRs) containing 512 genes. Network analysis of the genes in CDRs functioning in plant signaling pathways and biochemical processes identified group of genes related to root architecture, cell wall, sugar metabolism, and glucosinolate biosynthesis. Expression profiling of the genes during root development suggested that domestication-related selective advantages included a main taproot with few branched lateral roots, reduced cell wall rigidity and favorable taste. Overall, this study provides evolutionary insights into domestication-related genetic selection in radish as well as identification of gene candidates with the potential to act as trait-related markers for background selection of elite lines in molecular breeding.

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.

Construction of a reference genetic map of Raphanus sativus based on genotyping by whole-genome resequencing.

KEY MESSAGE: This manuscript provides a genetic map of Raphanus sativus that has been used as a reference genetic map for an ongoing genome sequencing project. The map was constructed based on genotyping by whole-genome resequencing of mapping parents and F 2 population. Raphanus sativus is an annual vegetable crop species of the Brassicaceae family and is one of the key plants in the seed industry, especially in East Asia. Assessment of the R. sativus genome provides fundamental resources for crop improvement as well as the study of crop genome structure and evolution. With the goal of anchoring genome sequence assemblies of R. sativus cv. WK10039 whose genome has been sequenced onto the chromosomes, we developed a reference genetic map based on genotyping of two parents (maternal WK10039 and paternal WK10024) and 93 individuals of the F2 mapping population by whole-genome resequencing. To develop high-confidence genetic markers, ~83 Gb of parental lines and ~591 Gb of mapping population data were generated as Illumina 100 bp paired-end reads. High stringent sequence analysis of the reads mapped to the 344 Mb of genome sequence scaffolds identified a total of 16,282 SNPs and 150 PCR-based markers. Using a subset of the markers, a high-density genetic map was constructed from the analysis of 2,637 markers spanning 1,538 cM with 1,000 unique framework loci. The genetic markers integrated 295 Mb of genome sequences to the cytogenetically defined chromosome arms. Comparative analysis of the chromosome-anchored sequences with Arabidopsis thaliana and Brassica rapa revealed that the R. sativus genome has evident triplicated sub-genome blocks and the structure of gene space is highly similar to that of B. rapa. The genetic map developed in this study will serve as fundamental genomic resources for the study of R. sativus.

Construction of a genetic map based on high-throughput SNP genotyping and genetic mapping of a TuMV resistance locus in Brassica rapa.

Brassica rapa is a member of the Brassicaceae family and includes vegetables and oil crops that are cultivated worldwide. The introduction of durable resistance against turnip mosaic virus (TuMV) into agronomically important cultivars has been a significant challenge for genetic and horticultural breeding studies of B. rapa. Based on our previous genome-wide analysis of DNA polymorphisms between the TuMV-resistant doubled haploid (DH) line VC40 and the TuMV-susceptible DH line SR5, we constructed a core genetic map of the VCS-13M DH population, which is composed of 83 individuals derived from microspore cultures of a F1 cross between VC40 and SR5, by analyzing the segregation of 314 sequence-characterized genetic markers. The genetic markers correspond to 221 SNPs and 31 InDels of genes as well as 62 SSRs, covering 1,115.9 cM with an average distance of 3.6 cM between the adjacent marker loci. The alignment and orientation of the constructed map showed good agreement with the draft genome sequence of Chiifu, thus providing an efficient strategy to map genic sequences. Using the genetic map, a novel dominant TuMV resistance locus (TuMV-R) in the VCS-13M DH population was identified as a 0.34 Mb region in the short arm of chromosome A6 in which four CC-NBS-LRR resistance genes and two pathogenesis-related-1 genes reside. The genetic map developed in this study can play an important role in the genetic study of TuMV resistance and the molecular breeding of B. rapa.

Comparative analysis of the radish genome based on a conserved ortholog set (COS) of Brassica.

KEY MESSAGE: This manuscript provides a Brassica conserved ortholog set (COS) that can be used as diagnostic cross-species markers as well as tools for genetic mapping and genome comparison of the Brassicaceae. A conserved ortholog set (COS) is a collection of genes that are conserved in both sequence and copy number between closely related genomes. COS is a useful resource for developing gene-based markers and is suitable for comparative genome mapping. We developed a COS for Brassica based on proteome comparisons of Arabidopsis thaliana, B. rapa, and B. oleracea to establish a basis for comparative genome analysis of crop species in the Brassicaceae. A total of 1,194 conserved orthologous single-copy genes were identified from the genomes based on whole-genome BLASTP analysis. Gene ontology analysis showed that most of them encoded proteins with unknown function and chloroplast-related genes were enriched. In addition, 152 Brassica COS primer sets were applied to 16 crop and wild species of the Brassicaceae and 57.9-92.8 % of them were successfully amplified across the species representing that a Brassica COS can be used as diagnostic cross-species markers of diverse Brassica species. We constructed a genetic map of Raphanus sativus by analyzing the segregation of 322 COS genes in an F2 population (93 individuals) of Korean cultivars (WK10039 × WK10024). Comparative genome analysis based on the COS genes showed conserved genome structures between R. sativus and B. rapa with lineage-specific rearrangement and fractionation of triplicated subgenome blocks indicating close evolutionary relationship and differentiation of the genomes. The Brassica COS developed in this study will play an important role in genetic, genomic, and breeding studies of crop Brassicaceae species.

Development of tools for the biochemical characterization of the symbiotic receptor-like kinase DMI2.

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

Effect of structural variation in the promoter region of RsMYB1.1 on the skin color of radish taproot.

Accumulation of anthocyanins in the taproot of radish is an agronomic trait beneficial for human health. Several genetic loci are related to a red skin or flesh color of radish, however, the functional divergence of candidate genes between non-red and red radishes has not been investigated. Here, we report that a novel genetic locus on the R2 chromosome, where RsMYB1.1 is located, is associated with the red color of the skin of radish taproot. A genome-wide association study (GWAS) of 66 non-red-skinned (nR) and 34 red-skinned (R) radish accessions identified three nonsynonymous single nucleotide polymorphisms (SNPs) in the third exon of RsMYB1.1. Although the genotypes of SNP loci differed between the nR and R radishes, no functional difference in the RsMYB1.1 proteins of nR and R radishes in their physical interaction with RsTT8 was detected by yeast-two hybrid assay or in anthocyanin accumulation in tobacco and radish leaves coexpressing RsMYB1.1 and RsTT8. By contrast, insertion- or deletion-based GWAS revealed that one large AT-rich low-complexity sequence of 1.3-2 kb was inserted in the promoter region of RsMYB1.1 in the nR radishes (RsMYB1.1nR), whereas the R radishes had no such insertion; this represents a presence/absence variation (PAV). This insertion sequence (RsIS) was radish specific and distributed among the nine chromosomes of Raphanus genomes. Despite the extremely low transcription level of RsMYB1.1nR in the nR radishes, the inactive RsMYB1.1nR promoter could be functionally restored by deletion of the RsIS. The results of a transient expression assay using radish root sections suggested that the RsIS negatively regulates the expression of RsMYB1.1nR, resulting in the downregulation of anthocyanin biosynthesis genes, including RsCHS, RsDFR, and RsANS, in the nR radishes. This work provides the first evidence of the involvement of PAV in an agronomic trait of radish.

Identification and profiling of novel microRNAs in the Brassica rapa genome based on small RNA deep sequencing.

BACKGROUND: MicroRNAs (miRNAs) are one of the functional non-coding small RNAs involved in the epigenetic control of the plant genome. Although plants contain both evolutionary conserved miRNAs and species-specific miRNAs within their genomes, computational methods often only identify evolutionary conserved miRNAs. The recent sequencing of the Brassica rapa genome enables us to identify miRNAs and their putative target genes. In this study, we sought to provide a more comprehensive prediction of B. rapa miRNAs based on high throughput small RNA deep sequencing. RESULTS: We sequenced small RNAs from five types of tissue: seedlings, roots, petioles, leaves, and flowers. By analyzing 2.75 million unique reads that mapped to the B. rapa genome, we identified 216 novel and 196 conserved miRNAs that were predicted to target approximately 20% of the genome's protein coding genes. Quantitative analysis of miRNAs from the five types of tissue revealed that novel miRNAs were expressed in diverse tissues but their expression levels were lower than those of the conserved miRNAs. Comparative analysis of the miRNAs between the B. rapa and Arabidopsis thaliana genomes demonstrated that redundant copies of conserved miRNAs in the B. rapa genome may have been deleted after whole genome triplication. Novel miRNA members seemed to have spontaneously arisen from the B. rapa and A. thaliana genomes, suggesting the species-specific expansion of miRNAs. We have made this data publicly available in a miRNA database of B. rapa called BraMRs. The database allows the user to retrieve miRNA sequences, their expression profiles, and a description of their target genes from the five tissue types investigated here. CONCLUSIONS: This is the first report to identify novel miRNAs from Brassica crops using genome-wide high throughput techniques. The combination of computational methods and small RNA deep sequencing provides robust predictions of miRNAs in the genome. The finding of numerous novel miRNAs, many with few target genes and low expression levels, suggests the rapid evolution of miRNA genes. The development of a miRNA database, BraMRs, enables us to integrate miRNA identification, target prediction, and functional annotation of target genes. BraMRs will represent a valuable public resource with which to study the epigenetic control of B. rapa and other closely related Brassica species. The database is available at the following link: http://bramrs.rna.kr [1].

Auxin response factor gene family in Brassica rapa: genomic organization, divergence, expression, and evolution.

Completion of the sequencing of the Brassica rapa genome enabled us to undertake a genome-wide identification and functional study of the gene families related to the morphological diversity and agronomic traits of Brassica crops. In this study, we identified the auxin response factor (ARF) gene family, which is one of the key regulators of auxin-mediated plant growth and development in the B. rapa genome. A total of 31 ARF genes were identified in the genome. Phylogenetic and evolutionary analyses suggest that ARF genes fell into four major classes and were amplified in the B. rapa genome as a result of a recent whole genome triplication after speciation from Arabidopsis thaliana. Despite its recent hexaploid ancestry, B. rapa includes a relatively small number of ARF genes compared with the 23 members in A. thaliana, presumably due to a paralog reduction related to repetitive sequence insertion into promoter and non-coding transcribed region of the genes. Comparative genomic and mRNA sequencing analyses demonstrated that 27 of the 31 BrARF genes were transcriptionally active, and their expression was affected by either auxin treatment or floral development stage, although 4 genes were inactive, suggesting that the generation and pseudogenization of ARF members are likely to be an ongoing process. This study will provide a fundamental basis for the modification and evolution of the gene family after a polyploidy event, as well as a functional study of ARF genes in a polyploidy crop species.

The effect of mHealth-based exercise on Insulin Sensitivity for patients with Hepatocellular carcinoma and insulin resistance (mISH): protocol of a randomized controlled trial.

BACKGROUND: The importance of insulin resistance is gaining increasing attention as it plays an important role in carcinogenesis in hepatocellular carcinoma (HCC). Although exercise is the most important intervention for lowering insulin resistance, it is not easy for HCC patients to maintain high compliance and do appropriate exercise. Mobile health (mHealth) with wearable devices can be the solution to carry out an adjusted and supervised exercise that can normalize insulin resistance in patients with HCC. We developed an HCC-specific application equipped with patient-centered exercise. In this paper, we present a randomized controlled trial protocol comparing an intervention group with a control group to determine whether mHealth-based exercise is effective in normalizing insulin sensitivity in HCC patients with insulin resistance after anticancer treatment. METHODS: An assessor unblinded open label randomized controlled trial (RCT) will be conducted for 80 participants with treatment-naïve or recurrent HCC who have received treatment and achieved complete response at the time of screening. They will be randomly assigned (1:1) to one of two groups: an intervention group (n = 40) and a control group (n = 40). The intervention group will carry out mHealth-based exercise for 6 months from baseline, whereas the control group will receive the usual follow-up care for the first 3 months and mHealth-based exercise for the next 3 months. Both groups will be assessed at baseline, 3 months, and 6 months from baseline. The primary outcome is the normalized rate of insulin resistance in each group at 3 months. Insulin resistance is estimated by calculating homeostatic model assessment for insulin resistance (HOMA-IR). The secondary outcomes are body composition, physical fitness level, physical activity, and quality of life at 3 months. DISCUSSION: This study is the first RCT to investigate the effect of mHealth-based home exercise with a wrist-wearable device on insulin sensitivity, physical fitness, and quality of life for HCC patients with insulin resistance. The result of this RCT will confirm not only safety and functional improvement but also biological effect when exercising using mHealth in HCC patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT04649671 . Registered on 2 December 2020. The World Health Organization Trial Registration Data Set is not registered.

Genome-wide discovery of DNA polymorphism in Brassica rapa.

Single nucleotide polymorphisms (SNPs) and/or insertion/deletions (InDels) are frequent sequence variations in the plant genome, which can be developed as molecular markers for genetic studies on crop improvement. The ongoing Brassica rapa genome sequencing project has generated vast amounts of sequence data useful in genetic research. Here, we report a genome-wide survey of DNA polymorphisms in the B. rapa genome based on the 557 bacterial artificial clone sequences of B. rapa ssp. pekinensis cv. Chiifu. We identified and characterized 21,311 SNPs and 6,753 InDels in the gene space of the B. rapa genome by re-sequencing 1,398 sequence-tagged sites (STSs) in eight genotypes. Comparison of our findings with a B. rapa genetic linkage map confirmed that STS loci were distributed randomly over the B. rapa whole genome. In the 1.4 Mb of aligned sequences, mean nucleotide polymorphism and diversity were theta = 0.00890 and pi = 0.00917, respectively. Additionally, the nucleotide diversity in introns was almost three times greater than that in exons, and the frequency of observed InDel was almost 17 times higher in introns than in exons. Information regarding SNPs/InDels obtained here will provide an important resource for genetic studies and breeding programs of B. rapa.

A collection of Ds insertional mutants associated with defects in male gametophyte development and function in Arabidopsis thaliana.

Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, since approximately 10% of the genes are expressed in the male gametophyte and approximately 9% in the female gametophyte. Here we describe the genetic and molecular characterization of 67 Ds insertion lines that show reduced transmission through the male gametophyte. About half of these mutations are male gametophytic-specific mutations, while the others also affect female transmission. Genomic sequences flanking both sides of the Ds element were recovered for 39 lines; for 16 the Ds elements were inserted in or close to coding regions, while 7 were located in intergenic/unannotated regions of the genome. For the remaining 16 lines, chromosomal rearrangements such as translocations or deletions, ranging between 30 and 500 kb, were associated with the transposition event. The mutants were classified into five groups according to the developmental processes affected; these ranged from defects in early stages of gametogenesis to later defects affecting pollen germination, pollen tube growth, polarity or guidance, or pollen tube-embryo sac interactions or fertilization. The isolated mutants carry Ds insertions in genes with diverse biological functions and potentially specify new functions for several unannotated or unknown proteins.

Genome-wide identification of NBS-encoding resistance genes in Brassica rapa.

Nucleotide-binding site (NBS)-encoding resistance genes are key plant disease-resistance genes and are abundant in plant genomes, comprising up to 2% of all genes. The availability of genome sequences from several plant models enables the identification and cloning of NBS-encoding genes from closely related species based on a comparative genomics approach. In this study, we used the genome sequence of Brassica rapa to identify NBS-encoding genes in the Brassica genome. We identified 92 non-redundant NBS-encoding genes [30 CC-NBS-LRR (CNL) and 62 TIR-NBS-LRR (TNL) genes] in approximately 100 Mbp of B. rapa euchromatic genome sequence. Despite the fact that B. rapa has a significantly larger genome than Arabidopsis thaliana due to a recent whole genome triplication event after speciation, B. rapa contains relatively small number of NBS-encoding genes compared to A. thaliana, presumably because of deletion of redundant genes related to genome diploidization. Phylogenetic and evolutionary analyses suggest that relatively higher relaxation of selective constraints on the TNL group after the old duplication event resulted in greater accumulation of TNLs than CNLs in both Arabidopsis and Brassica genomes. Recent tandem duplication and ectopic deletion are likely to have played a role in the generation of novel Brassica lineage-specific resistance genes.

MtGA2ox10 encoding C20-GA2-oxidase regulates rhizobial infection and nodule development in Medicago truncatula.

Gibberellin (GA) plays a controversial role in the legume-rhizobium symbiosis. Recent studies have shown that the GA level in legumes must be precisely controlled for successful rhizobial infection and nodule organogenesis. However, regulation of the GA level via catabolism in legume roots has not been reported to date. Here, we investigate a novel GA inactivating C20-GA2-oxidase gene MtGA2ox10 in Medicago truncatula. RNA sequencing analysis and quantitative polymerase chain reaction revealed that MtGA2ox10 was induced as early as 6 h post-inoculation (hpi) of rhizobia and reached peak transcript abundance at 12 hpi. Promoter::ß-glucuronidase fusion showed that the promoter activity was localized in the root infection/differentiation zone during the early stage of rhizobial infection and in the vascular bundle of the mature nodule. The CRISPR/Cas9-mediated deletion mutation of MtGA2ox10 suppressed infection thread formation, which resulted in reduced development and retarded growth of nodules on the Agrobacterium rhizogenes-transformed roots. Over-expression of MtGA2ox10 in the stable transgenic plants caused dwarfism, which was rescued by GA3 application, and increased infection thread formation but inhibition of nodule development. We conclude that MtGA2ox10 plays an important role in the rhizobial infection and the development of root nodules through fine catabolic tuning of GA in M. truncatula.

The radish genome database (RadishGD): an integrated information resource for radish genomics.

Radish (Raphanus sativus L.) is an important root vegetable crop in the family Brassicaceae, which provides diverse nutrients for human health and is closely related to the Brassica crop species. Recently, we sequenced and assembled the radish genome into nine chromosome pseudomolecules. In addition, we developed diverse genomic resources, including genetic maps, molecular markers, transcriptome, genome-wide methylation and variome data. In this study, we describe the radish genome database (RadishGD), including details of data sets that we generated and the web interface that allows access to these data. RadishGD comprises six major units that enable researchers and general users to search, browse and analyze the radish genomic data in an integrated manner. The Search unit provides gene structures and sequences for gene models through keyword or BLAST searches. The Genome browser displays graphic representations of gene models, mRNAs, repetitive sequences, genome-wide methylation and variomes among various genotypes. The Functional annotation unit offers gene ontology, plant ontology, pathway and gene family information for gene models. The Genetic map unit provides information about markers and their genetic locations using two types of genetic maps. The Expression unit presents transcriptional characteristics and methylation levels for each gene in 18 tissues. All sequence data incorporated into RadishGD can be downloaded from the Data resources unit. RadishGD will be continually updated to serve as a community resource for radish genomics and breeding research.

The complete chloroplast genome of Artemisia hallaisanensis Nakai (Asteraceae), an endemic medicinal herb in Korea.

We determined the complete chloroplast DNA sequence of Artemisia hallaisanensis Nakai, an endemic herbal species distributed on Jeju Island, Korea. The chloroplast DNA is 151,015 bp in length and encodes 4 rRNA, 30 tRNA, and 80 protein-coding genes. Phylogenetic analysis and sequence comparison of protein-coding genes with other Artemisa chloroplast DNAs revealed that the chloroplast genome of A. hallaisanensis is closely related to that of A. capillaris. Additionally, a unique 9 bp deletion in ycf1 gene is specific to A. hallaisanensis.

Draft genome sequence of wild Prunus yedoensis reveals massive inter-specific hybridization between sympatric flowering cherries.

BACKGROUND: Hybridization is an important evolutionary process that results in increased plant diversity. Flowering Prunus includes popular cherry species that are appreciated worldwide for their flowers. The ornamental characteristics were acquired both naturally and through artificially hybridizing species with heterozygous genomes. Therefore, the genome of hybrid flowering Prunus presents important challenges both in plant genomics and evolutionary biology. RESULTS: We use long reads to sequence and analyze the highly heterozygous genome of wild Prunus yedoensis. The genome assembly covers > 93% of the gene space; annotation identified 41,294 protein-coding genes. Comparative analysis of the genome with 16 accessions of six related taxa shows that 41% of the genes were assigned into the maternal or paternal state. This indicates that wild P. yedoensis is an F1 hybrid originating from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura, and it can be clearly distinguished from its confusing taxon, Yoshino cherry. A focused analysis of the S-locus haplotypes of closely related taxa distributed in a sympatric natural habitat suggests that reduced restriction of inter-specific hybridization due to strong gametophytic self-incompatibility is likely to promote complex hybridization of wild Prunus species and the development of a hybrid swarm. CONCLUSIONS: We report the draft genome assembly of a natural hybrid Prunus species using long-read sequencing and sequence phasing. Based on a comprehensive comparative genome analysis with related taxa, it appears that cross-species hybridization in sympatric habitats is an ongoing process that facilitates the diversification of flowering Prunus.