Understanding the processes that regulate plant sink formation and development at the molecular level will contribute to the areas of crop breeding, food production and plant evolutionary studies. We report the annotation and analysis of the draft genome sequence of the radish Raphanus sativus var. hortensis (long and thick root radish) and transcriptome analysis during root development. Based on the hybrid assembly approach of next-generation sequencing, a total of 383 Mb (N50 scaffold: 138.17 kb) of sequences of the radish genome was constructed containing 54,357 genes. Syntenic and phylogenetic analyses indicated that divergence between Raphanus and Brassica coincide with the time of whole genome triplication (WGT), suggesting that WGT triggered diversification of Brassiceae crop plants. Further transcriptome analysis showed that the gene functions and pathways related to carbohydrate metabolism were prominently activated in thickening roots, particularly in cell proliferating tissues. Notably, the expression levels of sucrose synthase 1 (SUS1) were correlated with root thickening rates. We also identified the genes involved in pungency synthesis and their transcription factors.

/pdf/the-radish-genome-and-comprehensive-gene-expression-profile-32ypuruly1.pdf

The radish genome and comprehensive gene expression profile of tuberous root formation and development

Next generation DNA sequencing technologies are driving increasingly rapid, affordable and high resolution analyses of plant transcriptomes through sequencing of their associated cDNA populations; an analytical platform commonly referred to as RNA-sequencing (RNA-seq). Since entering the arena of whole genome profiling technologies only a few years ago, RNA-seq has proven itself to be a powerful tool with a remarkably diverse range of applications, from detailed studies of biological processes at the cell type specific level, to providing insights into fundamental questions in plant biology on an evolutionary time scale. Applications include generating genomic data for heretofore unsequenced species, thus expanding the boundaries of what had been considered ‘model organisms’, elucidating structural and regulatory gene networks, revealing how plants respond to developmental cues and their environment, allowing a better understanding of the relationships between genes and their products, and uniting the ‘omics’ fields of transcriptomics, proteomics and metabolomics into a now common systems biology paradigm. We provide an overview of the breadth of such studies and summarize the range of RNA-seq protocols that have been developed to address questions spanning cell-type specific based transcriptomics, transcript secondary structure and gene mapping.

/pdf/catalyzing-plant-science-research-with-rna-seq-1v5dz92ojo.pdf

Catalyzing plant science research with RNA-seq.

In plants, miR390 directs the production of tasiRNAs from TRANS-ACTING SIRNA3 (TAS3) transcripts to regulate AUXIN RESPONSIVE FACTOR (ARF) genes, critical for auxin signaling; these tasiRNAs are known as tasiARFs. To understand the evolution of this miR390-TAS3-ARF pathway, we characterized homologs of these three genes from thousands of plant species, from bryophytes to angiosperms. We found the lower-stem region of MIR390 genes, critical for accurate DICER-LIKE1 processing, is conserved in sequence in seed plants. We propose a model for the transition of functional tasiRNA sequences in TAS3 genes occurred at the emergence of vascular plants, in which the two miR390 target sites of TAS3 genes showed distinct pairing patterns. Based on the cleavability of miR390 target sites and the distance between target site and tasiARF, we inferred a potential bidirectional processing mechanism exists for some TAS3 genes. We also demonstrated a tight mutual selection between tasiARF and its target genes and that ARGONAUTE7, the partner of miR390, was specified later than other factors in the pathway. All these data illuminate the evolutionary path of the miR390-TAS3-ARF pathway in land plants and demonstrate the significant variation that occurs in this functionally important and archetypal regulatory circuit.

/pdf/the-emergence-evolution-and-diversification-of-the-mir390-3h615rug2s.pdf

The Emergence, Evolution, and Diversification of the miR390-TAS3-ARF Pathway in Land Plants.

Natural disasters such as drought, extreme temperatures, and flooding can severely impact crop production. Understanding the metabolic response of crops threatened with these disasters provides insights into biological response mechanisms that can influence survival. In this study, a comparative analysis of GC-MS and (1)H NMR results was conducted for wild-type and tolerant rice varieties stressed by up to 3 days of submergence and allowed 1 day of postsubmergence recovery. Most metabolomics studies are conducted using a single analytical platform. Each platform, however, has inherent advantages and disadvantages that can influence the analytical coverage of the metabolome. In this work, a more thorough analysis of the plant stress response was possible through the use of both (1)H NMR and GC-MS. Several metabolites, such as S-methyl methionine and the dipeptide alanylglycine, were only detected and quantified by (1)H NMR. The high dynamic range of NMR, as compared with that of the GC-TOF-MS used in this study, provided broad coverage of the metabolome in a single experiment. The sensitivity of GC-MS facilitated the quantitation of sugars, organic acids, and amino acids, some of which were not detected by NMR, and provided additional insights into the regulation of the TCA cycle. The combined metabolic information provided by (1)H NMR and GC-MS was essential for understanding the complex biochemical and molecular response of rice plants to submergence.

Comparison of GC-MS and NMR for metabolite profiling of rice subjected to submergence stress

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.

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

The tuberous root of radish is an important vegetable, but insufficient transcriptomic and genomic data are currently available to understand the molecular mechanisms underlying tuberous root formation and development. High-throughput transcriptomic sequencing is essential to generate a large transcript sequence data set for gene discovery and molecular marker development. In this study, a total of 107.3 million clean reads were generated using Illumina paired-end sequencing technology. De novo assembly generated 61,554 unigenes with an average length of 820 bp. Based on a sequence similarity search with known proteins or nucleotides, 85.51 % (52,634), 90.18 % (55,507) and 54 % (33,242) consensus sequences showed homology with sequences in the Nr, Nt and Swiss-Prot databases, respectively. Of these annotated unigenes, 21,109 and 17,343 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. A total of 27,809 unigenes were assigned to 123 pathways in the Kyoto Encyclopedia of Genes and Genomes database. Analysis of transcript differences between libraries from the early and late seedling developmental stages demonstrated that starch and sucrose metabolism and phenylpropanoid biosynthesis may be the dominant metabolic events during tuberous root formation and plant hormones probably play critical roles in regulation of this developmental process. In total, 14,641 potential EST-SSRs were identified among the unigenes, and 12,733 primer pairs for 2,511 SSR were obtained. Summarily, this study gave us a clue to understand the radish tuberous root formation and development, and also provided us with a valuable sequence resource for novel gene discovery and marker-assisted selective breeding in radish. De novo assembled and characterized the radish tuberous root transcriptome; explored the mechanism of radish tuberous root formation; development of EST-SSR markers in radish.

Transcriptome analysis of the roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish

Vernalization is a key process for premature bolting. Although many studies on vernalization have been reported, the molecular mechanism of vernalization is still largely unknown in radish. In this study, we sequenced the transcriptomes of radish seedlings at three different time points during vernalization. More than 36 million clean reads were generated for each sample and the portions mapped to the reference genome were all above 67.0%. Our results show that the differentially expressed genes (DEGs) between room temperature and the early stage of vernalization (4,845) are the most in all treatments pairs. A series of vernalization related genes, including two FLOWERING LOCUS C (FLC) genes, were screened according to the annotations. A total of 775 genes were also filtered as the vernalization related candidates based on their expression profiles. Cold stress responsive genes were also analyzed to further confirm the sequencing result. Several key genes in vernalization or cold stress response were validated by quantitative RT-PCR (RT-qPCR). This study identified a number of genes that may be involved in vernalization, which are useful for other functional genomics research in radish.

/pdf/genome-wide-transcriptome-profiling-of-radish-raphanus-37ft8xl9gb.pdf

Genome-wide transcriptome profiling of radish (Raphanus sativus L.) in response to vernalization

We aimed to identify Turnip mosaic virus (TuMV) resistance genes in Chinese cabbage by analysing the TuMV resistance of 43 P1 (resistant), 88 P2 (susceptible), 26 F1, 104 B1 (F1 × P1), 108 B2 (F1 × P2) and 509 F2 individuals. All parents and progeny populations were mechanically inoculated with TuMV-C4. Both F1 and B1 populations showed TuMV resistance. Resistant: susceptible ratios in the B2 and F2 populations were 1 : 1 and 3 : 1, respectively. TuMV resistance in P1 was controlled by a dominant gene, TuRBCS01. Bulked segregation analysis was performed to identify simple sequence repeat or insertion or deletion markers linked to TuRBCS01. Data from 108 B2 individuals with resistant or susceptible phenotypes were analysed using mapmaker/exp 3.0. Polymorphic marker sequences were blast searched on http://brassicadb.org/brad/. TuRBCS01 was found to be linked to eight markers: SAAS_mDN192117a_159 (3.3 cM), SAAS_mDN192117b_196 (4.0 cM), SAAS_mDN192403_148 (13.0 cM), SAAS_mGT084561_233 (6.8 cM), BrID10723 (3.3 cM), mBr4041 (3.3 cM), SAAS_mBr4055_194 (2.6 cM) and mBr4068 (4.0 cM). Further, TuRBCS01 was mapped to a 1.98-Mb region on chromosome A04 between markers BrID10723 and SAAS_mBr4055_194.

Identification and mapping of a novel Turnip mosaic virus resistance gene TuRBCS01 in Chinese cabbage (Brassica rapa L.)

High-throughput small RNA sequencing and degradome analysis were used in this study to thoroughly investigate the role of miRNA-mediated regulatory network in tuberous root development of radish. Samples from the early seedling stage (RE) and the cortex splitting stage (RL) were used for the construction of six small RNA libraries and one degradome library. A total of 518 known and 976 novel miRNAs were identified, of which, 338 known and 18 novel miRNAs were expressed in all six libraries, respectively. A total of 52 known and 57 novel miRNAs were identified to be significantly differentially expressed between RE and RL, and 195 mRNAs were verified to be the targets of 194 miRNAs by degradome sequencing. According to the degradome analysis, 11 differentially expressed miRNAs had miRNA-mRNA targets, and 13 targets were identified for these 11 miRNAs. Of the 13 miRNA-mRNA targets, 4 genes (RSG11079.t1, RSG11844.t1, RSG16775.t1, and RSG42419.t1) were involved in hormone-mediated signaling pathway, 2 gens (RSG11079.t1 and RSG16775.t1) were related to post-embryonic root development, and 1 gene (RSG23799.t1) was involved in anatomical structure morphogenesis, according to the GO function analysis for biological process. Target Genes participated in these processes are important candidates for further studies. This study provides valuable information for a better understanding of the molecular mechanisms involved in radish tuberous root formation and development.

Identification of miRNAs and their targets in regulating tuberous root development in radish using small RNA and degradome analyses.

The invention discloses a method for improving the lateral branch rooting survival rate and the seed reproduction quality of radish. The method comprises the following steps: a second-stage lateral branch is taken as a cutting branch, after the cut-off lateral branch heals naturally, the cut-off lateral branch is soaked in an indolebutyric acid solution for 10 seconds and then inserted in a seedbed substrate composed of coco coir, vermiculite and mushroom dreg; a mixed solution containing Huabao No.1, Huabao No.2 and naphthylacetic acid is sprayed on the branch so that the branch can be acclimated and then survives; when new leaves come out, transplanting is performed by use of an underground simple culture tank filled with planting soil which is composed of coco coir, vermiculite, mushroom dreg and decomposed cow dung; after the cutting lateral branch survives, ventilation and light illumination are realized, Huabao No.1 and Huabao No.2 are sprayed, and after seeds are mature, the seeds are harvested. The method for improving the lateral branch rooting survival rate and the seed reproduction quality of the radish has the advantages that the method of rooting by use of the second-stage cutting lateral branch of the radish is adopted, a new method is provided for solving the problem of seed reproduction material loss of the radish due to the infringement of soft rot, the plants cultured by use of the method are well developed in root system, high in transplanting survival rate, and stable in heritability, and furthermore, the seed reproduction can be saved.

Xu Wenling

Papers

Transcriptome analysis of the roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish

Genome-wide transcriptome profiling of radish (Raphanus sativus L.) in response to vernalization

Identification and mapping of a novel Turnip mosaic virus resistance gene TuRBCS01 in Chinese cabbage (Brassica rapa L.)

Identification of miRNAs and their targets in regulating tuberous root development in radish using small RNA and degradome analyses.

Method for improving lateral branch rooting survival rate and seed reproduction quality of radish