Plastid genome of Chenopodium petiolare from Trujillo, Peru.

OBJECTIVES: The Peruvian Andean region is an important center for plant domestication. However, to date, there have been few genetic studies on native grain, which limits our understanding of their genetic diversity and the development of new genetic studies for their breeding. Herein, we revealed the plastid genome of Chenopodium petiolare to expand our knowledge of its molecular markers, evolutionary studies, and conservation genetics. DATA DESCRIPTION: Total genomic DNA was extracted from fresh leaves (voucher: USM < PER > :MHN333570). The DNA was sequenced using Illumina Novaseq 6000 (Macrogen Inc., Seoul, Republic of Korea) and reads 152,064 bp in length, with a large single-copy region of 83,520 bp and small single-copy region of 18,108 bp were obtained. These reads were separated by a pair of inverted repeat regions (IR) of 25,218 bp, and the overall guanine and cytosine (GC) was 37.24%. The plastid genome contains 130 genes (111 genes were unique and 19 genes were found duplicated in each IR region), including 86 protein-coding genes, 36 transfer RNA-coding genes, eight ribosomal RNA-coding genes, and 25 genes with introns (21 genes with one intron and four genes with two introns). The phylogenetic tree reconstructed based on single-copy orthologous genes and maximum likelihood analysis indicated that Chenopodium petiolare is most closely related to Chenopodium quinoa.

The FLOWERING LOCUS T LIKE 2-1 gene of Chenopodium triggers precocious flowering in Arabidopsis seedlings.

The FLOWERING LOCUS T (FT) gene is the essential integrator of flowering regulatory pathways in angiosperms. The paralogs of the FT gene may perform antagonistic functions, as exemplified by BvFT1, that suppresses flowering in Beta vulgaris, unlike the paralogous activator BvFT2. The roles of FT genes in other amaranths were less investigated. Here, we transformed Arabidopsis thaliana with the FLOWERING LOCUS T like (FTL) genes of Chenopodium ficifolium and found that both CfFTL1 and CfFTL2-1 accelerated flowering, despite having been the homologs of the Beta vulgaris floral promoter and suppressor, respectively. The floral promotive effect of CfFTL2-1 was so strong that it caused lethality when overexpressed under the 35S promoter. CfFTL2-1 placed in an inducible cassette accelerated flowering after induction with methoxyphenozide. The spontaneous induction of CfFTL2-1 led to precocious flowering in some primary transformants even without chemical induction. The CqFT2-1 homolog from Chenopodium quinoa had the same impact on viability and flowering as CfFTL2-1 when transferred to A. thaliana. After the FTL gene duplication in Amaranthaceae, the FTL1 copy maintained the role of floral activator. The second copy FTL2 underwent subsequent duplication and functional diversification, which enabled it to control the onset of flowering in amaranths to adapt to variable environments.

The FLOWERINGLOCUS T like 2­1 gene of Chenopodium ficifolium andChenopodium quinoa acts as a strong activator of flowering in Arabidopsis, triggering flowering at cotyledon stage and causing lethality when overexpressed.

A chromosome-scale reference of Chenopodium watsonii helps elucidate relationships within the North American A-genome Chenopodium species and with quinoa.

Quinoa (Chenopodium quinoa), an Andean pseudocereal, attained global popularity beginning in the early 2000s due to its protein quality, glycemic index, and high fiber, vitamin, and mineral contents. Pitseed goosefoot (Chenopodium berlandieri), quinoa's North American free-living sister species, grows on disturbed and sandy substrates across the North America, including saline coastal sands, southwestern deserts, subtropical highlands, the Great Plains, and boreal forests. Together with South American avian goosefoot (Chenopodium hircinum) they comprise the American tetraploid goosefoot complex (ATGC). Superimposed on pitseed goosefoot's North American range are approximately 35 AA diploids, most of which are adapted to a diversity of niche environments. We chose to assemble a reference genome for Sonoran A-genome Chenopodium watsonii due to fruit morphological and high (>99.3%) preliminary sequence-match similarities with quinoa, along with its well-established taxonomic status. The genome was assembled into 1377 scaffolds spanning 547.76 Mb (N50 = 55.14 Mb, L50 = 5), with 94% comprised in nine chromosome-scale scaffolds and 93.9% Benchmarking Universal Single-Copy Orthologs genes identified as single copy and 3.4% as duplicated. A high degree of synteny, with minor and mostly telomeric rearrangements, was found when comparing this taxon with the previously reported genome of South American C. pallidicaule and the A-subgenome chromosomes of C. quinoa. Phylogenetic analysis was performed using 10,588 single-nucleotide polymorphisms generated by resequencing a panel of 41 New World AA diploid accessions and the Eurasian H-genome diploid Chenopodium vulvaria, along with three AABB tetraploids previously sequenced. Phylogenetic analysis of these 32 taxa positioned the psammophyte Chenopodium subglabrum on the branch containing A-genome sequences from the ATGC. We also present evidence for long-range dispersal of Chenopodium diploids between North and South America.

CgMYB1, an R2R3-MYB transcription factor, can alleviate abiotic stress in an annual halophyte Chenopodium glaucum.

MYB transcription factors (TFs) are important regulators of the stress response in plants. In the present study, we characterized the CgMYB1 gene in Chenopodium glaucum, a member of the R2R3-MYB TF family. CgMYB1 was located in the nucleus with an activating domain at the C terminus. The CgMYB1 gene could be induced by salt and cold stress in C. glaucum. Overexpressing CgMYB1 in Arabidopsis significantly enhanced salt and cold tolerance, probably by improving physiological performance and stress-related gene expression. Further analysis suggests that the positive response of CgMYB1 to abiotic stress may partially be attributed to the interaction between CgMYB1 and the CgbHLH001 promoter followed by activation of downstream stress-responsive genes, which mediates stress tolerance. Our findings should contribute to further understanding of the function of R2R3 MYB TF in response to abiotic stress.

Chromosome-Scale Genome Assembly of the Hexaploid Taiwanese Goosefoot "Djulis" (Chenopodium formosanum).

Djulis (Chenopodium formosanum Koidz.) is a crop grown since antiquity in Taiwan. It is a BCD-genome hexaploid (2n = 6x = 54) domesticated form of lambsquarters (C. album L.) and a relative of the allotetraploid (AABB) C. quinoa. As with quinoa, djulis seed contains a complete protein profile and many nutritionally important vitamins and minerals. While still sold locally in Taiwanese markets, its traditional culinary uses are being lost as diets of younger generations change. Moreover, indigenous Taiwanese peoples who have long safeguarded djulis are losing their traditional farmlands. We used PacBio sequencing and Hi-C-based scaffolding to produce a chromosome-scale, reference-quality assembly of djulis. The final genome assembly spans 1.63 Gb in 798 scaffolds, with 97.8% of the sequence contained in 27 scaffolds representing the nine haploid chromosomes of each sub-genome of the species. Benchmarking of universal, single-copy orthologs indicated that 98.5% of the conserved orthologous genes for Viridiplantae are complete within the assembled genome, with 92.9% duplicated, as expected for a polyploid. A total of 67.8% of the assembly is repetitive, with the most common repeat being Gypsy long terminal repeat retrotransposons, which had significantly expanded in the B sub-genome. Gene annotation using Iso-Seq data from multiple tissues identified 75,056 putative gene models. Comparisons to quinoa showed strong patterns of synteny which allowed for the identification of homoeologous chromosomes, and sub-genome-specific sequences were used to assign homoeologs to each sub-genome. These results represent the first hexaploid genome assembly and the first assemblies of the C and D genomes of the Chenopodioideae subfamily.

The high concentrations of abscisic, jasmonic, and salicylic acids produced under long days do not accelerate flowering in Chenopodium ficifolium 459.

The survival and adaptation of angiosperms depends on the proper timing of flowering. The weedy species Chenopodium ficifolium serves as a useful diploid model for comparing the transition to flowering with the important tetraploid crop Chenopodium quinoa due to the close phylogenetic relationship. The detailed transcriptomic and hormonomic study of the floral induction was performed in the short-day accession C. ficifolium 459. The plants grew more rapidly under long days but flowered later than under short days. The high levels of abscisic, jasmonic, and salicylic acids at long days were accompanied by the elevated expression of the genes responding to oxidative stress. The increased concentrations of stress-related phytohormones neither inhibited the plant growth nor accelerated flowering in C. ficifolium 459 at long photoperiods. Enhanced content of cytokinins and the stimulation of cytokinin and gibberellic acid signaling pathways under short days may indicate the possible participation of these phytohormones in floral initiation. The accumulation of auxin metabolites suggests the presence of a dynamic regulatory network in C. ficifolium 459.

Transcriptomic study of the night break in Chenopodium rubrum reveals possible upstream regulators of the floral activator CrFTL1.

The transition from vegetative to reproductive phases is the most fundamental and tightly controlled switch in the life of flowering plants. The short-day plant Chenopodium rubrum is a fast cycling annual plant lacking a juvenile phase. It can be induced to flowering at the seedling stage by exposure to a single period of darkness. This floral induction may then be cancelled by a short pulse of red light at midnight called night break (NB), which also inhibits the floral activator FLOWERING LOCUS T LIKE 1 (CrFTL1). We performed a comparative transcriptomic study between C. rubrum seedlings treated by NB and ones growing through uninterrupted night, and found about six hundred differentially expressed genes, including the B-BOX DOMAIN (BBX) genes. We focused on the CrBBX19 and BOLTING TIME CONTROL 1 (BTC1) genes, homologous to the upstream regulators of the BvFT2, a floral inducer in sugar beet. The transcription patterns of the two genes were compatible with their putative role as a sensor of the dark period length optimal for flowering (CrBBX19), and a signal of lights-on (CrBTC1), but the participation of other genes cannot be excluded. The expression profiles of CrBBX19 and the homolog of the core endogenous clock gene LATE ELONGATED HYPOCOTYL (LHY) were highly similar, which suggested their co-regulation.

Chenopodium ficifolium flowers under long days without upregulation of FLOWERING LOCUS T (FT) homologs.

MAIN CONCLUSION: Chenopodium ficifoliumflowered under long days despite much lower expression ofFLOWERING LOCUS Thomolog than under short days. Frequent duplications of the FLOWERING LOCUS T (FT) gene across various taxonomic lineages resulted in FT paralogs with floral repressor function, whereas others duplicates maintained their floral-promoting role. The FT gene has been confirmed as the inducer of photoperiodic flowering in most angiosperms analyzed to date. We identified all FT homologs in the transcriptome of Chenopodium ficifolium and in the genome of Chenopodium suecicum, which are closely related to diploid progenitors of the tetraploid crop Chenopodium quinoa, and estimated their expression during photoperiodic floral induction. We found that expression of FLOWERING LOCUS T like 1 (FTL1), the ortholog of the sugar beet floral activator BvFT2, correlated with floral induction in C. suecicum and short-day C. ficifolium, but not with floral induction in C. ficifolium with accelerated flowering under long days. This C. ficifolium accession was induced to flowering without the concomitant upregulation of any FT homolog.

Deciphering species relationships and evolution in Chenopodium through sequence variations in nuclear internal transcribed spacer region and amplified fragment-length polymorphism in nuclear DNA.

Evaluation of sequence variations in the internal transcribed spacer (ITS) region of 19 accessions, comprising of 11 accessions of Chenopodium quinoa, eight accessions of Chenopodium album and 165 retrieved sequences of different species of Chenopodium belonging to subfamily Chenopodioideae revealed a higher intraspecific genetic diversity in Himalayan C. album than that in C. quinoa. ITS and amplified fragment-length profiles of the accessions suggest the existence of accessions of Himalayan C. album as heteromorphs of the same species rather than a heterogenous assemblage of taxa. While the evolutionary relationship reconstructed from variations in 184 sequences of ITS region from species belonging to Chenopodiaceae, Amaranthaceae, Polygonaceae and Nelumbonaceae established a paraphyletic evolution of family Chenopodiaceae, it also revealed a monophyletic evolution of Chenopodieae I. The reconstruction also established five independent lineages of the subfamily Chenopodioideae with C. album as a sister clade of C. quinoa within the tribe Chenopodieae I. The results also indicate a much younger age for Himalayan chenopods (C. album) than the reported crown age of Chenopodieae I.

Natural History of a Satellite DNA Family: From the Ancestral Genome Component to Species-Specific Sequences, Concerted and Non-Concerted Evolution.

Satellite DNA (satDNA) is the most variable fraction of the eukaryotic genome. Related species share a common ancestral satDNA library and changing of any library component in a particular lineage results in interspecific differences. Although the general developmental trend is clear, our knowledge of the origin and dynamics of satDNAs is still fragmentary. Here, we explore whole genome shotgun Illumina reads using the RepeatExplorer (RE) pipeline to infer satDNA family life stories in the genomes of Chenopodium species. The seven diploids studied represent separate lineages and provide an example of a species complex typical for angiosperms. Application of the RE pipeline allowed by similarity searches a determination of the satDNA family with a basic monomer of ~40 bp and to trace its transformation from the reconstructed ancestral to the species-specific sequences. As a result, three types of satDNA family evolutionary development were distinguished: (i) concerted evolution with mutation and recombination events; (ii) concerted evolution with a trend toward increased complexity and length of the satellite monomer; and (iii) non-concerted evolution, with low levels of homogenization and multidirectional trends. The third type is an example of entire repeatome transformation, thus producing a novel set of satDNA families, and genomes showing non-concerted evolution are proposed as a significant source for genomic diversity.

The bHLH transcription factor CgbHLH001 is a potential interaction partner of CDPK in halophyte Chenopodium glaucum.

Plants have evolved different abilities to adapt to the ever-fluctuating environments for sessility. Calcium-dependent protein kinase (CDPK) is believed to play a pivotal role in abiotic stress signaling. So far, study on the specific substrates that CDPK recognized in response to adversity is limited. In the present study, we revealed a potential interaction between CDPK and a bHLH transcription factor under salt stress in Chenopodium glaucum. First, we identified a CgCDPK, which was up-regulated under salt and drought stress; then by Y2H screening, CgCDPK was detected to be involved in interaction with a bHLH TF (named as CgbHLH001), which also positively respond to salt and drought stress. Further computational prediction and experiments including GST-pulldown and BiFC assays revealed that potential interaction existed between CgCDPK and CgbHLH001, and they might interact on the plasma membrane. In addition, CgCDPK-overexpressed transgenic tobacco line could significantly accumulate transcripts of NtbHLH (a homolog of CgbHLH001 in N. tabacum), which provided another evidence of correlation between CgCDPK and CgbHLH001. Our results suggest that CgbHLH001 can interact with CgCDPK in signal transduction pathway in response to abiotic stress, which should provide new evidence for further understanding of the substrate specificity of plant CDPK signaling pathway.

Characterization of the genome of a phylogenetically distinct tospovirus and its interactions with the local lesion-induced host Chenopodium quinoa by whole-transcriptome analyses.

Chenopodium quinoa is a natural local lesion host of numerous plant viruses, including tospoviruses (family Bunyaviridae). Groundnut chlorotic fan-spot tospovirus (GCFSV) has been shown to consistently induce local lesions on the leaves of C. quinoa 4 days post-inoculation (dpi). To reveal the whole genome of GCFSV and its interactions with C. quinoa, RNA-seq was performed to determine the transcriptome profiles of C. quinoa leaves. The high-throughput reads from infected C. quinoa leaves were used to identify the whole genome sequence of GCFSV and its single nucleotide polymorphisms. Our results indicated that GCFSV is a phylogenetically distinct tospovirus. Moreover, 27,170 coding and 29,563 non-coding sequences of C. quinoa were identified through de novo assembly, mixing reads from mock and infected samples. Several key genes involved in the modulation of hypersensitive response (HR) were identified. The expression levels of 4,893 deduced complete genes annotated using the Arabidopsis genome indicated that several HR-related orthologues of pathogenesis-related proteins, transcription factors, mitogen-activated protein kinases, and defense proteins were significantly expressed in leaves that formed local lesions. Here, we also provide new insights into the replication progression of a tospovirus and the molecular regulation of the C. quinoa response to virus infection.

Differentially expressed genes of Chenopodium amaranticolor in response to cymbidium mosaic virus infection.

Cymbidium mosaic virus (CymMV)-induced expressed sequence tag (EST) clones from Chenopodium amaranticolor were identified. CymMV was mechanically inoculated onto C. amaranticolor, and local lesion symptoms were observed. Inoculated leaves were collected on serial days post inoculation (dpi) to identify activated or suppressed genes. mRNA isolation and suppression subtractive hybridization (SSH) were then performed to identify differentially expressed genes related to the local lesion response. Fifty-three ESTs, including genes related to defense and stress responses (e.g., lipoxygenase, jasmonate-induced protein, and heat shock protein), were generated. In addition, a large proportion of the ESTs were found to be involved in photosynthesis, as determined by their functional categories. Expression levels of several EST genes were observed using quantitative real-time reverse transcription-polymerase chain reaction, and the evaluated genes showed varying levels of expression during the experimental period. In this study, differentially expressed sequences via SSH were identified from CymMV-infected C. amaranticolor, and profiling and annotation were carried out to determine the expression pattern of CymMV and its interaction with C. amaranticolor.

Effects of sowing date on phenotypic plasticity of fitness-related traits in two annual weeds on the Songnen Plain of China.

BACKGROUND: Phenotypic plasticity of fitness-related traits is vital for plant species to adapt to variable environments. Chenopodium glaucum L. and Amaranthus retroflexus L. are two common weed species globally. Understanding the plasticity in life-history traits, especially in reproductive allocation, within and among these species is important for predicting their success and for managing them in different environments. METHODOLOGY/PRINCIPAL FINDINGS: Seeds of the two plant species were sown every 10 days from 26 Jun to 15 Aug. Life-history and fitness-related traits of both phenology and morphology were measured, and dry biomass of roots, stems, leaves, and reproductive tissues was determined at physiological maturity. Length of reproductive and total life period of the two species differed among six sowing-date treatments. Later germinating plants led to relatively reduced total life period, size, and earlier reproduction than earlier germinating plants. The ratio of reproductive biomass to total plant biomass increased with later planting dates in C. glaucum but declined in A. retroflexus. Mature plant height, crown diameter, and reproductive tissue biomass, and seed production of C. glaucum and A. retroflexus increased with delayed reproductive period. Both species displayed true plasticity in reproductive allocation. However, the sowing date had a far greater effect on rate of vegetative growth than on allocation to reproduction. CONCLUSIONS/SIGNIFICANCE: The fitness of both C. glaucum and A. retroflexus populations have an apparent increase when the period between germination and seed production is much longer. However, C. glaucum appears better adapted to later sowing than A. retroflexus. Controlling seedlings prior to reproduction will alleviate the negative effect not only in the present year but also in future years.

Chenopodium polyploidy inferences from Salt Overly Sensitive 1 (SOS1) data.

PREMISE OF THE STUDY: Single-copy nuclear loci can provide powerful insights into polyploid evolution. Chenopodium (Amaranthaceae) is a globally distributed genus composed of approximately 50-75 species. The genus includes several polyploid species, some of which are considered noxious agricultural weeds, and a few are domesticated crops. Very little research has addressed their evolutionary origin to date. We construct a phylogeny for Chenopodium based on two introns of the single-copy nuclear locus Salt Overly Sensitive 1 (SOS1) to clarify the relationships among the genomes of the allotetraploid and allohexaploid species, and to help identify their genome donors. METHODS: Diploid species were sequenced directly, whereas homeologous sequences of polyploid genomes were first separated by plasmid-mediated cloning. Data were evaluated in maximum likelihood and Bayesian phylogenetic analyses. KEY RESULTS: Homeologous sequences of polyploid species were found in four clades, which we designate as A-D. Two distinct polyploid lineages were identified: one composed of American tetraploid species with A and B class homeologs and a second composed of Eastern Hemisphere hexaploid species with B, C, and D class homeologs. CONCLUSIONS: We infer that the two polyploid lineages arose independently and that each lineage may have originated only once. The American diploid, C. standleyanum, was identified as the closest living diploid relative of the A genome donor for American tetraploids, including domesticated C. quinoa, and is of potential importance for quinoa breeding. The east Asian diploid species, C. bryoniifolium, groups with American diploid species, which suggests a transoceanic dispersal.

Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato.

Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.

Identification of genes encoding photoconvertible (Class I) water-soluble chlorophyll-binding proteins from Chenopodium ficifolium.

Photoconvertible water-soluble chlorophyll-binding proteins, called Class I WSCPs, have been detected in Chenopodiaceae, Amaranthaceae and Polygonaceae plant species. To date, Chenopodium album WSCP (CaWSCP) is the only cloned gene encoding a Class I WSCP. In this study, we identified two cDNAs encoding Chenopodium ficifolium Class I WSCPs, CfWSCP1, and CfWSCP2. Sequence analyses revealed that the open reading frames of CfWSCP1 and CfWSCP2 were 585 and 588 bp, respectively. Furthermore, both CfWSCPs contain cystein2 and cystein30, which are essential for the chlorophyll-binding ability of CaWSCP. Recombinant CfWSCP1 and CfWSCP2, expressed in Escherichia coli as hexa-histidine fusion proteins (CfWSCP1-His and CfWSCP2-His), formed inclusion bodies; however, we were able to solubilize these using a buffer containing 8 M urea and then refold them by dialysis. The refolded CfWSCP1-His and CfWSCP2-His could bind chlorophylls and exhibited photoconvertibility, confirming that the cloned CfWSCPs are further examples of Class I WSCPs.

A dark-light transition triggers expression of the floral promoter CrFTL1 and downregulates CONSTANS-like genes in a short-day plant Chenopodium rubrum.

The proper timing of flowering is essential for the adaptation of plant species to their ever-changing environments. The central position in a complex regulatory network is occupied by the protein FT, which acts as a florigen. We found that light, following a permissive period of darkness, was essential to induce the floral promoter CrFTL1 and to initiate flowering in seedlings of the short-day plant Chenopodium rubrum L. We also identified two novel CONSTANS-like genes in C. rubrum and observed their rhythmic diurnal and circadian expressions. Strong rhythmicity of expression suggested that the two genes might have been involved in the regulation of photoperiod-dependent processes, despite their inability to complement co mutation in A. thaliana. The CrCOL1 and CrCOL2 genes were downregulated by dark-light transition, regardless of the length of a preceding dark period. The same treatment activated the floral promoter CrFTL1. Light therefore affected CrCOL and CrFTL1 in an opposite manner. Both CrCOL genes and CrFTL1 displayed expression patterns unique among short-day plants. Chenopodium rubrum, the subject of classical physiological studies in the past, is emerging as a useful model for the investigation of flowering at the molecular level.

De novo foliar transcriptome of Chenopodium amaranticolor and analysis of its gene expression during virus-induced hypersensitive response.

BACKGROUND: The hypersensitive response (HR) system of Chenopodium spp. confers broad-spectrum virus resistance. However, little knowledge exists at the genomic level for Chenopodium, thus impeding the advanced molecular research of this attractive feature. Hence, we took advantage of RNA-seq to survey the foliar transcriptome of C. amaranticolor, a Chenopodium species widely used as laboratory indicator for pathogenic viruses, in order to facilitate the characterization of the HR-type of virus resistance. METHODOLOGY AND PRINCIPAL FINDINGS: Using Illumina HiSeq™ 2000 platform, we obtained 39,868,984 reads with 3,588,208,560 bp, which were assembled into 112,452 unigenes (3,847 clusters and 108,605 singletons). BlastX search against the NCBI NR database identified 61,698 sequences with a cut-off E-value above 10(-5). Assembled sequences were annotated with gene descriptions, GO, COG and KEGG terms, respectively. A total number of 738 resistance gene analogs (RGAs) and homology sequences of 6 key signaling proteins within the R proteins-directed signaling pathway were identified. Based on this transcriptome data, we investigated the gene expression profiles over the stage of HR induced by Tobacco mosaic virus and Cucumber mosaic virus by using digital gene expression analysis. Numerous candidate genes specifically or commonly regulated by these two distinct viruses at early and late stages of the HR were identified, and the dynamic changes of the differently expressed genes enriched in the pathway of plant-pathogen interaction were particularly emphasized. CONCLUSIONS: To our knowledge, this study is the first description of the genetic makeup of C. amaranticolor, providing deep insight into the comprehensive gene expression information at transcriptional level in this species. The 738 RGAs as well as the differentially regulated genes, particularly the common genes regulated by both TMV and CMV, are suitable candidates which merit further functional characterization to dissect the molecular mechanisms and regulatory pathways of the HR-type of virus resistance in Chenopodium.

Use of Chenopodium murale L. transgenic hairy root in vitro culture system as a new tool for allelopathic assays.

We investigated Chenopodium murale transgenic hairy root in vitro culture system as a new tool for allelopathic assays. Transgenic hairy roots were induced by Agrobacterium rhizogenes A4M70GUS from roots, cotyledons, leaves, and internodes of C. murale seedlings. Roots were found to be the best target explants, providing transformation efficiency of up to 11.1%. Established hairy root clones differed in their morphology and growth potential. Molecular characterization of these clones was carried out by PCR, RT-PCR and histochemical GUS analyses. No differences in rol gene expression were observed. Liquid culture system of characterized hairy root clones was maintained for over 2 years. Six hairy root clones were selected for assaying the allelopathic effect of their growth medium against germination and seedling elongation of wheat and lettuce test plants. The inhibitory potential varied depending on the hairy root clone. Some transgenic clones showed significantly higher inhibition compared to wild-type roots. These results revealed that hairy roots as an independent system synthesize some bioactive substances with allelopathic activity and exude them into the growth medium. Concentrations of caffeic, ferulic and p-coumaric acids (0.07-2.85 µmol/L) identified by HPLC analysis in the growth media were at least 1000 times lower than the inhibitory active concentration (5 mmol/L) of pure grade phenolic acids, suggesting that they have a limited role in the allelopathic phenomena of C. murale. The presented hairy root system appears to be a suitable tool for further investigation of the potential and nature of root-mediated allelopathic interference of C. murale.