Admixture of divergent genomes facilitates hybridization across species in the family Brassicaceae.

Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species are rare in nature due to reproductive barriers but how such hurdles can be overcome is largely unknown. Here we report the hybrid genome structure of xBrassicoraphanus, a synthetic allotetraploid of Brassica rapa and Raphanus sativus. We performed cytogenetic analysis and de novo genome assembly to examine chromosome behaviors and genome integrity in the hybrid. Transcriptome analysis was conducted to investigate expression of duplicated genes in conjunction with epigenome analysis to address whether genome admixture entails epigenetic reconfiguration. Allotetraploid xBrassicoraphanus retains both parental chromosomes without genome rearrangement. Meiotic synapsis formation and chromosome exchange are avoided between nonhomologous progenitor chromosomes. Reconfiguration of transcription network occurs, and less divergent cis-elements of duplicated genes are associated with convergent expression. Genome-wide DNA methylation asymmetry between progenitors is largely maintained but, notably, B. rapa-originated transposable elements are transcriptionally silenced in xBrassicoraphanus through gain of DNA methylation. Our results demonstrate that hybrid genome stabilization and transcription compatibility necessitate epigenome landscape adjustment and rewiring of cis-trans interactions. Overall, this study suggests that a certain extent of genome divergence facilitates hybridization across species, which may explain the great diversification and expansion of angiosperms during evolution.

Epigenome editing: targeted manipulation of epigenetic modifications in plants.

BACKGROUND: Epigenetic modifications play important roles in diverse cellular processes such as X chromosome inactivation, cell differentiation, development and senescence. DNA methylation and histone modifications are major epigenetic modifications that regulate chromatin structure and gene expression without DNA sequence changes. Epigenetic alterations may induce phenotypic changes stable enough for mitotic or meiotic inheritance. Moreover, the reversibility of epigenetic marks makes the manipulation of chromatin and epigenetic signature an attractive strategy for therapeutic and breeding purposes. Targeted epigenetic manipulation, or epigenome editing, at the gene of interest commonly utilizes specific epigenetic modifiers fused with a targeting module of the conventional genome editing system. OBJECTIVE: This review aims to summarize essential epigenetic components and introduce currently available epigenetic mutants and the corresponding epialleles in plants. Furthermore, advances in epigenome editing technology are discussed while proposing its potential application to plant breeding. CONCLUSIONS: Epimutations associated with useful traits may provide a valuable resource for crop development. It is important to explore epimutations in a variety of crop species while understanding the fundamental aspects of epigenetic regulation of agronomically important traits such as yield, quality, disease resistance and stress tolerance. In the end, plant breeding programs through epigenome editing may help not only to expand the use of limited genetic resources but also to alleviate consumers' concerns about genetically manipulated crops.

Dynamic changes in DNA methylation occur in TE regions and affect cell proliferation during leaf-to-callus transition in Arabidopsis.

Plant somatic cells can be reprogrammed into pluripotent cell mass, called callus, through a two-step in vitro tissue culture method. Incubation on callus-inducing medium triggers active cell proliferation to form a pluripotent callus. Notably, DNA methylation is implicated during callus formation, but a detailed molecular process regulated by DNA methylation remains to be fully elucidated. Here, we compared genome-wide DNA methylation profiles between leaf and callus tissues in Arabidopsis using whole-genome bisulphite-sequencing. Global distribution of DNA methylation showed that CHG methylation was increased, whereas CHH methylation was reduced especially around transposable element (TE) regions during the leaf-to-callus transition. We further analysed differentially expressed genes around differentially methylated TEs (DMTEs) during the leaf-to-callus transition and found that genes involved in cell cycle regulation were enriched and also constituted a coexpression gene network along with pluripotency regulators. In addition, a conserved DNA sequence analysis for upstream cis-elements led us to find a putative transcription factor associated with cell fate transition. CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) was newly identified as a regulator of plant regeneration, and consistently, the cca1lhy mutant displayed altered phenotypes in callus proliferation. Overall, these results suggest that DNA methylation coordinates cell cycle regulation during callus formation, and CCA1 may act as a key upstream coordinator at least in part in the processes.

Expanded transcriptomic view of strawberry fruit ripening through meta-analysis.

Strawberry is an important fruit crop and a model for studying non-climacteric fruit ripening. Fruit ripening and senescence influence strawberry fruit quality and postharvest storability, and have been intensively studied. However, genetic and physiological differences among cultivars preclude consensus understanding of these processes. We therefore performed a meta-analysis by mapping existing transcriptome data to the newly published and improved strawberry reference genome and extracted meta-differentially expressed genes (meta-DEGs) from six cultivars to provide an expanded transcriptomic view of strawberry ripening. We identified cultivar-specific transcriptome changes in anthocyanin biosynthesis-related genes and common changes in cell wall degradation, chlorophyll degradation, and starch metabolism-related genes during ripening. We also identified 483 meta-DEGs enriched in gene ontology categories related to photosynthesis and amino acid and fatty acid biosynthesis that had not been revealed in previous studies. We conclude that meta-analysis of existing transcriptome studies can effectively address fundamental questions in plant sciences.

Reduced fertility caused by meiotic defects and micronuclei formation during microsporogenesis in xBrassicoraphanus.

BACKGROUND: Hybridization and polyploidization events are important driving forces in plant evolution. Allopolyploids formed between different species can be naturally or artificially created but often suffer from genetic instability and infertility in successive generations. xBrassicoraphanus is an intergeneric allopolyploid obtained from a cross between Brassica rapa and Raphanus sativus, providing a useful resource for genetic and genomic study in hybrid species. OBJECTIVE: The current study aims to understand the cause of hybrid sterility and pollen abnormality in different lines of synthetic xBrassicoraphanus from the cytogenetic perspective. METHODS: Alexander staining was used to assess the pollen viability. Cytogenetic analysis was employed to monitor meiotic chromosome behaviors in pollen mother cells (PMCs). Origins of parental chromosomes in xBrassicoraphanus meiocytes were determined by genome in situ hybridization analysis. RESULTS: The xBrassicoraphanus lines BB#4 and BB#6 showed high rates of seed abortion and pollen deformation. Abnormal chromosome behaviors were observed in their PMCs, frequently forming univalents and inter-chromosomal bridges during meiosis. A positive correlation also exists between meiotic defects and the formation of micronuclei, which is conceivably responsible for unbalanced gamete production and pollen sterility. CONCLUSION: These results suggest that unequal segregation of meiotic chromosomes, due in part to non-homologous interactions, is responsible for micronuclei and unbalanced gamete formation, eventually leading to pollen degeneration and inferior fertility in unstable xBrassicoraphanus lines.

Meiotic Chromosome Stability and Suppression of Crossover Between Non-homologous Chromosomes in xBrassicoraphanus, an Intergeneric Allotetraploid Derived From a Cross Between Brassica rapa and Raphanus sativus.

Hybridization and polyploidization are major driving forces in plant evolution. Allopolyploids can be occasionally formed from a cross between distantly related species but often suffer from chromosome instability and infertility. xBrassicoraphanus is an intergeneric allotetraploid (AARR; 2n = 38) derived from a cross between Brassica rapa (AA; 2n = 20) and Raphanus sativus (RR; 2n = 18). xBrassicoraphanus is fertile and genetically stable, while retaining complete sets of both B. rapa and R. sativus chromosomes. Precise control of meiotic recombination is essential for the production of balanced gametes, and crossovers (COs) must occur exclusively between homologous chromosomes. Many interspecific hybrids have problems with meiotic division at early generations, in which interactions between non-homologous chromosomes often bring about aneuploidy and unbalanced gamete formation. We analyzed meiotic chromosome behaviors in pollen mother cells (PMCs) of allotetraploid and allodiploid F1 individuals of newly synthesized xBrassicoraphanus. Allotetraploid xBrassicoraphanus PMCs showed a normal diploid-like meiotic behavior. By contrast, allodiploid xBrassicoraphanus PMCs displayed abnormal segregation of chromosomes mainly due to the absence of homologous pairs. Notably, during early stages of meiosis I many of allodiploid xBrassicoraphanus chromosomes behave independently with few interactions between B. rapa and R. sativus chromosomes, forming many univalent chromosomes before segregation. Chromosomes were randomly assorted at later stages of meiosis, and tetrads with unequal numbers of chromosomes were formed at completion of meiosis. Immunolocalization of HEI10 protein mediating meiotic recombination revealed that COs were more frequent in synthetic allotetraploid xBrassicoraphanus than in allodiploid, but less than in the stabilized line. These findings suggest that structural dissimilarity between B. rapa and R. sativus chromosomes prevents non-homologous interactions between the parental chromosomes in allotetraploid xBrassicoraphanus, allowing normal diploid-like meiosis when homologous pairing partners are present. This study also suggests that CO suppression between non-homologous chromosomes is required for correct meiotic progression in newly synthesized allopolyploids, which is important for the formation of viable gametes and reproductive success in the hybrid progeny.

Revealing biomass heterosis in the allodiploid xBrassicoraphanus, a hybrid between Brassica rapa and Raphanus sativus, through integrated transcriptome and metabolites analysis.

BACKGROUND: Heterosis is biologically important but the molecular basis of the phenomenon is poorly understood. We characterized intergeneric hybrids between B. rapa cv. Chiifu and R. sativus cv. WK10039 as an extreme example of heterosis. Taking advantage of clear heterosis phenotypes and the genetic distance between parents, we performed transcriptome and metabolite analysis to decipher the molecular basis of heterosis. RESULTS: The heterosis was expressed as fresh weight in the field and as inflorescence stem length in the glass house. Flowering time, distributed as a normal segregating population, ranged from the early flowering of one parent to the late flowering of the other, in contrast to the homogeneous flowering time in a typical F1 population, indicating unstable allelic interactions. The transcriptome and metabolome both indicated that sugar metabolism was altered, suggesting that the change in metabolism was linked to the heterosis. Because alleles were not shared between the hybridized genomes, classic models only partly explain this heterosis, indicating that other mechanisms are involved. CONCLUSION: The differential expression of genes for primary and secondary metabolism, along with the altered metabolite profiles, suggests that heterosis could involve a change in balance between primary and secondary metabolism.

A genetic characterization of Korean waxy maize (Zea mays L.) landraces having flowering time variation by RNA sequencing.

Maize is the second-most produced crop in the Korean peninsula and has been continuously cultivated since the middle of the 16th century, when it was originally introduced from China. Even with this extensive cultivation history, the diversity and properties of Korean landraces have not been investigated at the nucleotide sequence level. We collected 12 landraces with various flowering times and performed RNA-seq in the early vegetative stage. The transcriptomes of 12 Korean landraces have been analyzed for their genetic variations in coding sequence and genetic relationships to other maize germplasm. The Korean landraces showed specific genetic characteristics and were closely related to a Chinese inbred line. Flowering-time related gene profiles pointed to multiple causes for the variation of flowering time within Korean landraces; the profiles revealed significant positive and negative correlations among genes, allowing us to infer possible mechanisms for flowering time variation in maize. Our results demonstrate the value of transcriptome-based genetic and gene expression profiles for information on possible breeding resources, which is particularly needed in Korean waxy landraces.

The catalytic core of DEMETER guides active DNA demethylation in Arabidopsis.

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.

ROS1-Dependent DNA Demethylation Is Required for ABA-Inducible NIC3 Expression.

DNA methylation plays an important role in diverse developmental processes in many eukaryotes, including the response to environmental stress. Abscisic acid (ABA) is a plant hormone that is up-regulated under stress. The involvement of DNA methylation in the ABA response has been reported but is poorly understood. DNA demethylation is a reverse process of DNA methylation and often induces structural changes of chromatin leading to transcriptional activation. In Arabidopsis (Arabidopsis thaliana), active DNA demethylation depends on the activity of REPRESSOR OF SILENCING 1 (ROS1), which directly excises 5-methylcytosine from DNA. Here we showed that ros1 mutants were hypersensitive to ABA during early seedling development and root elongation. Expression levels of some ABA-inducible genes were decreased in ros1 mutants, and more than 60% of their proximal regions became hypermethylated, indicating that a subset of ABA-inducible genes are under the regulation of ROS1-dependent DNA demethylation. Notable among them is NICOTINAMIDASE 3 (NIC3) that encodes an enzyme that converts nicotinamide to nicotinic acid in the NAD+ salvage pathway. Many enzymes in this pathway are known to be involved in stress responses. The nic3 mutants display hypersensitivity to ABA, whereas overexpression of NIC3 restores normal ABA responses. Our data suggest that NIC3 is responsive to ABA but requires ROS1-mediated DNA demethylation at the promoter as a prerequisite to transcriptional activation. These findings suggest that ROS1-induced active DNA demethylation maintains the active state of NIC3 transcription in response to ABA.

MYB1 transcription factor is a candidate responsible for red root skin in radish (Raphanus sativus L.).

Root skin color is one of the economically important traits in radish (Raphanus sativus), and the pigmentation in red skin varieties is largely attributable to anthocyanin accumulation. Pelargonidin was found as a major anthocyanin pigment accumulated in the sub-epidermal layer of red radish roots. In the 20 F2 population generated from the F1 with red root skins, root skins with red and white colors segregated in a 3:1 ratio. Additionally, a test cross between a red F3 individual and a white skin individual gave rise to 1:1 segregation of red and white, indicating that the root skin color of radish is determined by a single locus and red color is dominant over white. We performed association mapping for root skin color using SNPs obtained from RNA-seq analysis. Segregation analysis on the 152 F3 test-cross population revealed an RsMyb1 transcription factor as a candidate gene to determine root skin color. A PCR marker based on the polymorphism within 2 kb of RsMyb1 was developed and tested on 12 and 152 individuals from F2 and F3 test cross populations, respectively, and red and white root skin colors were completely distinguished corresponding to the genotypes. Expression levels of RsMyb1 in red or purple root cultivars were significantly higher than in white root cultivars. These findings suggest that RsMyb1 is a crucial determinant for anthocyanin biosynthesis in radish roots, and the molecular marker developed in this study will be useful for marker-assisted selection for red skin individuals at early seedling stages.

New reference genome sequences of hot pepper reveal the massive evolution of plant disease-resistance genes by retroduplication.

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.

De Novo Transcriptome Analysis to Identify Anthocyanin Biosynthesis Genes Responsible for Tissue-Specific Pigmentation in Zoysiagrass (Zoysia japonica Steud.).

Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars 'Anyang-jungji' (AJ) and 'Greenzoa' (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information.

AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis.

DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3'-phosphor-α, ß-unsaturated aldehyde and 3'-phosphate by successive ß- and δ-eliminations, respectively. The kinetic studies revealed that these 3'-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants.

Excision of 5-hydroxymethylcytosine by DEMETER family DNA glycosylases.

In plants and animals, 5-methylcytosine (5mC) serves as an epigenetic mark to repress gene expression, playing critical roles for cellular differentiation and transposon silencing. Mammals also have 5-hydroxymethylcytosine (5hmC), resulting from hydroxylation of 5mC by TET family-enzymes. 5hmC is abundant in mouse Purkinje neurons and embryonic stem cells, and regarded as an important intermediate for active DNA demethylation in mammals. However, the presence of 5hmC in plants has not been clearly demonstrated. In Arabidopsis, the DEMETER (DME) family DNA glycosylases efficiently remove 5mC, which results in DNA demethylation and transcriptional activation of target genes. Here we show that DME and ROS1 have a significant 5hmC excision activity in vitro, although we detected no 5hmC in Arabidopsis, suggesting that it is very unlikely for plants to utilize 5hmC as a DNA demethylation intermediate. Our results indicate that both plants and animals have 5mC in common but DNA demethylation systems have independently evolved with distinct mechanisms.