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.

Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites.

Intergeneric crosses between Brassica species and Raphanus sativus have produced crops with prominent shoot and root systems of Brassica and R. sativus, respectively. It is necessary to discriminate donor genomes when studying cytogenetic stability in distant crosses to identify homologous chromosome pairing, and microsatellite repeats have been used to discriminate subgenomes in allopolyploids. To identify genome-specific microsatellites, we explored the microsatellite content in three Brassica species (B. rapa, AA, B. oleracea, CC, and B. nigra, BB) and R. sativus (RR) genomes, and validated their genome specificity by fluorescence in situ hybridization. We identified three microsatellites showing A, C, and B/R genome specificity. ACBR_msat14 and ACBR_msat20 were detected in the A and C chromosomes, respectively, and ACBR_msat01 was detected in B and R genomes. However, we did not find a microsatellite that discriminated the B and R genomes. The localization of ACBR_msat20 in the 45S rDNA array in ×Brassicoraphanus 977 corroborated the association of the 45S rDNA array with genome rearrangement. Along with the rDNA and telomeric repeat probes, these microsatellites enabled the easy identification of homologous chromosomes. These data demonstrate the utility of microsatellites as probes in identifying subgenomes within closely related Brassica and Raphanus species for the analysis of genetic stability of new synthetic polyploids of these genomes.

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.

Application of 5-Methylcytosine DNA Glycosylase to the Quantitative Analysis of DNA Methylation.

In higher eukaryotes DNA methylation is a prominent epigenetic mark important for chromatin structure and gene expression. Thus, profiling DNA methylation is important for predicting gene expressions associated with specific traits or diseases. DNA methylation is achieved by DNA methyltransferases and can be actively removed by specific enzymes in a replication-independent manner. DEMETER (DME) is a bifunctional 5-methylcytosine (5mC) DNA glycosylase responsible for active DNA demethylation that excises 5mC from DNA and cleaves a sugar-phosphate bond generating a single strand break (SSB). In this study, DME was used to analyze DNA methylation levels at specific epialleles accompanied with gain or loss of DNA methylation. DME treatment on genomic DNA generates SSBs in a nonsequence-specific fashion proportional to 5mC density, and thus DNA methylation levels can be easily measured when combined with the quantitative PCR (qPCR) method. The DME-qPCR analysis was applied to measure DNA methylation levels at the FWA gene in late-flowering Arabidopsis mutants and the CNR gene during fruit ripening in tomato. Differentially methylated epialleles were successfully distinguished corresponding to their expression levels and phenotypes. DME-qPCR is proven a simple yet effective method for quantitative DNA methylation analysis, providing advantages over current techniques based on methylation-sensitive restriction digestion.

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.

Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility.

There is little known about the function of rice hexokinases (HXKs) in planta. We characterized hxk5-1, a Tos17 mutant of OsHXK5 that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of hxk5-1 and reciprocal crosses between the wild-type and heterozygotes revealed that loss of HXK5 causes male sterility. Homozygous hxk5-1, produced via anther culture, and additional homozygous hxk5-2, hxk5-3 and hxk5-4 lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. In vitro pollen germination ability and in vivo pollen tube growth rate were significantly reduced in the hxk5 mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the hxk5 male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

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.

Transcriptional regulation of abscisic acid biosynthesis and signal transduction, and anthocyanin biosynthesis in 'Bluecrop' highbush blueberry fruit during ripening.

Highbush blueberry (Vaccinium corymbosum) fruit accumulate high levels of anthocyanins during ripening, which might be controlled by abscisic acid (ABA), a signal molecule in non-climacteric fruits. For an integrated view of the ripening process from ABA to anthocyanin biosynthesis, we analyzed the transcriptomes of 'Bluecrop' highbush blueberry fruit using RNA-Seq at three ripening stages, categorized based on fruit skin coloration: pale green at ca. 30 days after full bloom (DAFB), reddish purple at ca. 40 DAFB, and dark purple at ca. 50 DAFB. Mapping the trimmed reads against the reference sequences yielded 25,766 transcripts. Of these, 143 transcripts were annotated to encode five ABA biosynthesis enzymes, four ABA signal transduction regulators, four ABA-responsive transcription factors, and 12 anthocyanin biosynthesis enzymes. The analysis of differentially expressed genes between the ripening stages revealed that 11 transcripts, including those encoding nine-cis-epoxycarotenoid dioxygenase, SQUAMOSA-class MADS box transcription factor, and flavonoid 3',5'-hydroxylase, were significantly up-regulated throughout the entire ripening stages. In fruit treated with 1 g L-1 ABA, at least nine transcripts of these 11 transcripts as well as one transcript encoding flavonoid 3'-hydroxylase were up-regulated, presumably promoting anthocyanin accumulation and fruit skin coloration. These results will provide fundamental information demonstrating that ABA biosynthesis and signal transduction, and anthocyanin biosynthesis are closely associated with anthocyanin accumulation and skin coloration in highbush blueberry fruit during ripening.

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.

Global DNA Methylation in the Chestnut Blight Fungus Cryphonectria parasitica and Genome-Wide Changes in DNA Methylation Accompanied with Sectorization.

Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK) of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase), demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

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.

DEMETER plant DNA demethylase induces antiviral response by interferon signalling in animal cells.

DNA methylation is a prominent epigenetic modification in plants and animals regulated by similar mechanisms but the process of DNA demethylation is profoundly different. Unlike vertebrates that require a series of enzymatic conversions of 5-methylcytosine (5mC) into other bases for DNA demethylation, plants utilize the DEMETER (DME) family of 5mC DNA glycosylases to catalyze a direct removal of 5mC from DNA. Here we introduced Arabidopsis DME into human HEK-293T cells to allow direct 5mC excision, and observed that direct DNA demethylation activity was successfully implemented by DME expression. In addition, DME induced diverse cellular responses such as cell proliferation inhibition, cell cycle dysregulation and S phase arrest. Microarray and methylome analyses revealed that DME upregulated a number of genes including cell cycle components, heat shock proteins, and notably, various interferon-stimulated genes. Moreover, DME-mediated DNA demethylation activated endogenous repeat elements, which are likely to form dsRNAs as viral mimics and eventually trigger interferon cascades to establish the antiviral state. This work demonstrates that plant DNA demethylase catalyzes DNA demethylation with a bypass of initial base conversion steps, and the interferon signaling plays a pivotal role to alleviate genotoxic stresses associated with DME-induced DNA demethylation in mammalian cells.

Root Glucosinolate Profiles for Screening of Radish (Raphanus sativus L.) Genetic Resources.

Radish (Raphanus sativus L.), a root vegetable, is rich in glucosinolates (GLs), which are beneficial secondary metabolites for human health. To investigate the genetic variations in GL content in radish roots and the relationship with other root phenotypes, we analyzed 71 accessions from 23 different countries for GLs using HPLC. The most abundant GL in radish roots was glucoraphasatin, a GL with four-carbon aliphatic side chain. The content of glucoraphasatin represented at least 84.5% of the total GL content. Indolyl GL represented only 3.1% of the total GL at its maximum. The principal component analysis of GL profiles with various root phenotypes showed that four different genotypes exist in the 71 accessions. Although no strong correlation with GL content and root phenotype was observed, the varied GL content levels demonstrate the genetic diversity of GL content, and the amount that GLs could be potentially improved by breeding in radishes.

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.