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Summary: High‐quality radish (Raphanus sativus) genome represents a valuable resource for agronomical trait improvements and understanding genome evolution among Brassicaceae species. However, existing radish genome assembly remains fragmentary, which greatly hampered functional genomics research and genome‐assisted breeding. Here, using a NAU‐LB radish inbred line, we generated a reference genome of 476.32 Mb with a scaffold N50 of 56.88 Mb by incorporating Illumina, PacBio and BioNano optical mapping techniques. Utilizing Hi‐C data, 448.12 Mb (94.08%) of the assembled sequences were anchored to nine radish chromosomes with 40 306 protein‐coding genes annotated. In total, 249.14 Mb (52.31%) comprised the repetitive sequences, among which long terminal repeats (LTRs, 30.31%) were the most abundant class. Beyond confirming the whole‐genome triplication (WGT) event in R. sativus lineage, we found several tandem arrayed genes were involved in stress response process, which may account for the distinctive phenotype of high disease resistance in R. sativus. By comparing against the existing Xin‐li‐mei radish genome, a total of 2 108 573 SNPs, 7740 large insertions, 7757 deletions and 84 inversions were identified. Interestingly, a 647‐bp insertion in the promoter of RsVRN1 gene can be directly bound by the DOF transcription repressor RsCDF3, resulting into its low promoter activity and late‐bolting phenotype of NAU‐LB cultivar. Importantly, introgression of this 647‐bp insertion allele, RsVRN1In‐536, into early‐bolting genotype could contribute to delayed bolting time, indicating that it is a potential genetic resource for radish late‐bolting breeding. Together, this genome resource provides valuable information to facilitate comparative genomic analysis and accelerate genome‐guided breeding and improvement in radish. [ABSTRACT FROM AUTHOR]

Summary: High‐density genetic map is a valuable tool for exploring novel genomic information, quantitative trait locus (QTL) mapping and gene discovery of economically agronomic traits in plant species. However, high‐resolution genetic map applied to tag QTLs associated with important traits and to investigate genomic features underlying recombination landscape in radish (Raphanus sativus) remains largely unexplored. In this study, an ultra‐high‐density genetic map with 378 738 SNPs covering 1306.8 cM in nine radish linkage groups (LGs) was developed by a whole‐genome sequencing‐based approach. A total of 18 QTLs for 11 horticulture traits were detected. The map‐based cloning data indicated that the R2R3‐MYB transcription factor RsMYB90 was a crucial candidate gene determining the taproot skin colour. Comparative genomics analysis among radish, Brassica rapa and B. oleracea genome revealed several genomic rearrangements existed in the radish genome. The highly uneven distribution of recombination was observed across the nine radish chromosomes. Totally, 504 recombination hot regions (RHRs) were enriched near gene promoters and terminators. The recombination rate in RHRs was positively correlated with the density of SNPs and gene, and GC content, respectively. Functional annotation indicated that genes within RHRs were mainly involved in metabolic process and binding. Three QTLs for three traits were found in the RHRs. The results provide novel insights into the radish genome evolution and recombination landscape, and facilitate the development of effective strategies for molecular breeding by targeting and dissecting important traits in radish. [ABSTRACT FROM AUTHOR]

Background: Abiotic stresses due to climate change pose a great threat to crop production. Heat shock transcription factors (HSFs) are vital regulators that play key roles in protecting plants against various abiotic stresses. Therefore, the identification and characterization of HSFs is imperative to dissect the mechanism responsible for plant stress responses. Although the HSF gene family has been extensively studied in several plant species, its characterization, evolutionary history and expression patterns in the radish (Raphanus sativus L.) remain limited. Results: In this study, 33 RsHSF genes were obtained from the radish genome, which were classified into three main groups based on HSF protein domain structure. Chromosomal localization analysis revealed that 28 of 33 RsHSF genes were located on nine chromosomes, and 10 duplicated RsHSF genes were grouped into eight gene pairs by whole genome duplication (WGD). Moreover, there were 23 or 9 pairs of orthologous HSFs were identified between radish and Arabidopsis or rice, respectively. Comparative analysis revealed a close relationship among radish, Chinese cabbage and Arabidopsis. RNA-seq data showed that eight RsHSF genes including RsHSF-03, were highly expressed in the leaf, root, cortex, cambium and xylem, indicating that these genes might be involved in plant growth and development. Further, quantitative real-time polymerase chain reaction (RT-qPCR) indicated that the expression patterns of 12 RsHSF genes varied upon exposure to different abiotic stresses including heat, salt, and heavy metals. These results indicated that the RsHSFs may be involved in abiotic stress response. Conclusions: These results could provide fundamental insights into the characteristics and evolution of the HSF family and facilitate further dissection of the molecular mechanism responsible for radish abiotic stress responses. [ABSTRACT FROM AUTHOR]

CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides are a class of small molecules involved in plant growth and development. Although radish (Raphanus sativus) is an important root vegetable crop worldwide, the functions of CLE peptides in its taproot formation remain elusive. Here, a total of 48 RsCLE genes were identified from the radish genome. RNA in situ hybridization showed that RsCLE22a gene was highly expressed in the vascular cambium. Overexpression of RsCLE22a inhibited root growth by impairing stem cell proliferation in Arabidopsis, and radish plants with exogenous supplementation of RsCLE22 peptide (CLE22p) showed a similar phenotype. The vascular cambial activity was increased in RsCLE22a -silenced plants. Transcriptome analysis revealed that CLE22p altered the expression of several genes involved in meristem development and hormone signal transduction in radish. Immunolocalization results showed that CLE22p increased auxin accumulation in vascular cambium. Yeast one-hybrid and dual-luciferase assays showed that the WUSCHEL-RELATED HOMEOBOX 4 (RsWOX4) binds to RsCLE22a promoter and activates its transcription. The expression level of RsWOX4 was related to vascular cambial activity and was regulated by auxin. Furthermore, a RsCLE22a–RsWOX4 module is proposed to regulate taproot vascular cambium activity through an auxin signaling-related pathway in radish. These findings provide novel insights into the regulation of root growth in a horticultural crop. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish. [ABSTRACT FROM AUTHOR]

Radish (Raphanus sativus L.), an important annual or biennial root vegetable crop, is widely cultivated in the world for its high nutritive value. Isolated microspore culture (IMC) is one of the most effective methods for rapid development of homozygous lines. Due to imperfection of the IMC technology system, it is particularly important to establish an efficient IMC system in radish. In this study, the effects of different factors on radish microspore embryogenesis were investigated with 23 genotypes. Buds with the largest population of late-uninucleate-stage microspores were most suitable for embryogenesis, with a ratio of petal length to anther length (P/A) in buds of about 3/4 ~ 1. Cold pretreatment was found to be genotype specific, and the highest microspore-derived embryoid (MDE) yield occurred for treatment of the heat shock of 48 h. In addition, the supplement of 0.75 g/L activated charcoal (AC) could increase the yield of embryoids. It was found that genotypes, bud size, as well as temperature treatments had significant effects on microspore embryogenesis. Furthermore, somatic embryogenesis–related kinase (SERK) genes were profiled by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, which indicated that they are involved in the process of MDE formation and plantlet regeneration. The ploidy of microspore-derived plants was identified by chromosome counting and flow cytometry, and the microspore-derived plants were further proved as homozygous plants through expressed sequence tags-simple sequence repeats (EST-SSR) and genetic-SSR markers. The results would facilitate generating the large-scale double haploid (DH) from various genotypes, and promoting further highly efficient genetic improvement in radish. [ABSTRACT FROM AUTHOR]

Lead (Pb), one of the most toxic heavy metals, can be absorbed and accumulated by plant roots and then enter the food chain resulting in potential health risks for human beings. The radish (Raphanus sativus L.) is an important root vegetable crop with fleshy taproots as the edible parts. Little is known about the mechanism by which radishes respond to Pb stress at the molecular level. In this study, Next Generation Sequencing (NGS)–based RNA-seq technology was employed to characterize the de novo transcriptome of radish roots and identify differentially expressed genes (DEGs) during Pb stress. A total of 68,940 assembled unique transcripts including 33,337 unigenes were obtained from radish root cDNA samples. Based on the assembled de novo transcriptome, 4,614 DEGs were detected between the two libraries of untreated (CK) and Pb-treated (Pb1000) roots. Gene Ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Pb stress are predominately involved in defense responses in cell walls and glutathione metabolism-related processes, while downregulated DEGs were mainly involved in carbohydrate metabolism-related pathways. The expression patterns of 22 selected genes were validated by quantitative real-time PCR, and the results were highly accordant with the Solexa analysis. Furthermore, many candidate genes, which were involved in defense and detoxification mechanisms including signaling protein kinases, transcription factors, metal transporters and chelate compound biosynthesis related enzymes, were successfully identified in response to heavy metal Pb. Identification of potential DEGs involved in responses to Pb stress significantly reflected alterations in major biological processes and metabolic pathways. The molecular basis of the response to Pb stress in radishes was comprehensively characterized. Useful information and new insights were provided for investigating the molecular regulation mechanism of heavy metal Pb accumulation and tolerance in root vegetable crops. [ABSTRACT FROM AUTHOR]

Basic leucine zipper (bZIP) transcription factors play crucial roles in various abiotic stress responses as well as anthocyanin accumulation. Anthocyanins are most abundant in colorful skin radish, which exhibit strong antioxidant activity that offers benefits for human health. Here, a total of 135 bZIP-encoding genes were identified from radish genome. Synteny analysis showed that 104 radish and 63 ArabidopsisbZIP genes were orthologous. Transcriptome analysis revealed that 10 RsbZIP genes exhibited high-expression levels in radish taproot (RPKM>10). Specifically, RsbZIP010 exhibited down-regulated expression under Cd, Cr and Pb stresses, whereas RsbZIP031 and RsbZIP059 showed significant down-regulation under heat and salt stresses, respectively. RT-qPCR analysis indicated that RsbZIP011 and RsbZIP102 were significantly up-regulated in the tissues of radish with high anthocyanin contents. Furthermore, the promoter sequences of 39 anthocyanin-related genes were found to contain G-box or ACE-box elements that could be recognized by bZIP family members. Taken together, several RsbZIPs might be served as critical regulators in radish taproot under Cd, Cr, Pb, heat and salt stresses. RsbZIP011 and RsbZIP102 were the potential participants in anthocyanin biosynthesis pathway of radish. These results facilitate further investigation on functional characterization of bZIP genes in response to abiotic stress and anthocyanin biosynthesis in radish. [ABSTRACT FROM AUTHOR]

Main conclusion Among 247 RsAP2/ERF identified, the majority of the 21 representatives were preferably expressed under drought and heat while suppressed under heavy metals, indicating their potential roles in abiotic stress responses and tolerance. APETALA2/Ethylene-Responsive factor (AP2/ERF) transcription factor (TF) is one of the largest gene families in plants that play a fundamental role in growth and development as well as biotic and/or abiotic stresses responses. Although AP2/ERFs have been extensively characterized in many plant species, little is known about this family in radish, which is an important root vegetable with various medicinal properties. The available genome provides valuable opportunity to identify and characterize the global information on AP2/ERF TFs in radish. In this study, a total of 247 ERF family genes were identified from the radish genome, and sequence alignment and phylogenetic analyses classified the AP2/ERF superfamily into five groups (AP2, ERF, DREB, RAV and soloist). Motif analysis showed that other than AP2/ERF domains, other conserved regions were selectively distributed among different clades in the phylogenetic tree. Chromosome location analysis showed that tandem duplication may result in the expansion of RsAP2/ERF gene family. The RT-qPCR analysis confirmed that a proportion of AP2/ERF genes were preferably expressed under drought and heat stresses, whereas they were suppressed under the ABA and heavy metal stresses. These results provided valuable information for further evolutionary and functional characterization of RsAP2/ERF genes, and contributed to genetic improvement of stress tolerances in radish and other root vegetable crops. • Totally 247 AP2-ERF genes were firstly genome-widely identified in radish. • The AP2/ERF superfamily could be classified into five groups. • Tandem duplications play critical role in the expansion of RsAP2/ERF genes. • A proportion of AP2/ERF genes were differentially expressed under abiotic stresses. [ABSTRACT FROM AUTHOR]

Background: Taproot is the main edible organ and ultimately determines radish yield and quality. However, the precise molecular mechanism underlying taproot thickening awaits further investigation in radish. Here, RNA-seq was performed to identify critical genes involved in radish taproot thickening from three advanced inbred lines with different root size. Results: A total of 2606 differentially expressed genes (DEGs) were shared between 'NAU-DY' (large acicular) and 'NAU-YB' (medium obovate), which were significantly enriched in 'phenylpropanoid biosynthesis', 'glucosinolate biosynthesis', and 'starch and sucrose metabolism' pathway. Meanwhile, a total of 16 differentially expressed miRNAs (DEMs) were shared between 'NAU-DY' and 'NAU-YH' (small circular), whereas 12 miRNAs exhibited specific differential expression in 'NAU-DY'. Association analysis indicated that miR393a-bHLH77, miR167c-ARF8, and miR5658-APL might be key factors to biological phenomenon of taproot type variation, and a putative regulatory model of taproot thickening and development was proposed. Furthermore, several critical genes including SUS1, EXPB3, and CDC5 were characterized and profiled by RT-qPCR analysis. Conclusion: This integrated study on the transcriptional and post-transcriptional profiles could provide new insights into comprehensive understanding of the molecular regulatory mechanism underlying taproot thickening in root vegetable crops. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play vital regulatory roles in plant growth and development. The phase transition from vegetative growth to flowering is crucial in the life cycle of plants. To date, miRNA-mediated flowering regulatory networks remain largely unexplored in radish. In this study, two small RNA libraries from radish leaves at vegetative and reproductive stages were constructed and sequenced by Solexa sequencing. A total of 94 known miRNAs representing 21 conserved and 13 non-conserved miRNA families, and 44 potential novel miRNAs, were identified from the two libraries. In addition, 42 known and 17 novel miRNAs were significantly differentially expressed and identified as bolting-related miRNAs. RT-qPCR analysis revealed that some miRNAs exhibited tissue- or developmental stage-specific expression patterns. Moreover, 154 target transcripts were identified for 50 bolting-related miRNAs, which were predominately involved in plant development, signal transduction and transcriptional regulation. Based on the characterization of bolting-related miRNAs and their target genes, a putative schematic model of miRNA-mediated bolting and flowering regulatory network was proposed. These results could provide insights into bolting and flowering regulatory networks in radish, and facilitate dissecting the molecular mechanisms underlying bolting and flowering time regulation in vegetable crops. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small noncoding RNAs that play pivotal roles in plant growth, development and stress response. Chromium (Cr) is one of common environmental contaminants possessing potential health hazards to living organisms. To date, little is known about the regulatory roles of miRNAs in response to Cr stress in radish. To systematically identify Cr-responsive miRNAs and their targets in radish, two sRNA libraries derived from Cr-free (CK) and Cr-treated (Cr200) roots were constructed. With Solexa sequencing, 81 known and 72 novel miRNAs were identified, from which 54 known and 16 novel miRNAs were significantly differentially expressed under Cr stress. Several target genes for Cr-responsive miRNAs encode different transcription factor (TF) families, including SPLs, MYBs, ERFs and bZIPs, might regulate corresponding HM-related transcriptional processes in plants. Notably, a few key responsive enzymes or proteins, including HMA, YSL1 and ABC transporter protein were involved in Cr uptake and homeostasis process. Furthermore, the expression patterns of some Cr-responsive miRNAs and their targets were validated by RT-qPCR. This study represents the first characterization of Cr-responsive miRNAs and their targets in radish. The outcomes of this study could provide novel insights into miRNA-mediated regulatory mechanisms underlying plant response to Cr stress in root vegetable crops. [ABSTRACT FROM AUTHOR]

Heat stress (HS) causes detrimental effects on plant morphology, physiology, and biochemistry that lead to drastic reduction in plant biomass production and economic yield worldwide. To date, little is known about HS-responsive genes involved in thermotolerance mechanism in radish. In this study, a total of 6600 differentially expressed genes (DEGs) from the control and Heat24 cDNA libraries of radish were isolated by high-throughput sequencing. With Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, some genes including MAPK, DREB, ERF, AP2, GST, Hsf, and Hsp were predominantly assigned in signal transductions, metabolic pathways, and biosynthesis and abiotic stress-responsive pathways. These pathways played significant roles in reducing stress-induced damages and enhancing heat tolerance in radish. Expression patterns of 24 candidate genes were validated by reverse-transcription quantitative PCR (RT-qPCR). Based mainly on the analysis of DEGs combining with the previous miRNAs analysis, the schematic model of HS-responsive regulatory network was proposed. To counter the effects of HS, a rapid response of the plasma membrane leads to the opening of specific calcium channels and cytoskeletal reorganization, after which HS-responsive genes are activated to repair damaged proteins and ultimately facilitate further enhancement of thermotolerance in radish. These results could provide fundamental insight into the regulatory network underlying heat tolerance in radish and facilitate further genetic manipulation of thermotolerance in root vegetable crops. [ABSTRACT FROM AUTHOR]

Microsporogenesis is an indispensable period for investigating microspore development and cytoplasmic male sterility (CMS) occurrence. Radish CMS line plays a critical role in elite F hybrid seed production and heterosis utilization. However, the molecular mechanisms of microspore development and CMS occurrence have not been thoroughly uncovered in radish. In this study, a comparative analysis of radish floral buds from a CMS line (NAU-WA) and its maintainer (NAU-WB) was conducted using next generation sequencing (NGS) technology. Digital gene expression (DGE) profiling revealed that 3504 genes were significantly differentially expressed between NAU-WA and NAU-WB library, among which 1910 were upregulated and 1594 were downregulated. Gene ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly enriched in extracellular region, catalytic activity, and response to stimulus. KEGG enrichment analysis revealed that the DEGs were predominantly associated with flavonoid biosynthesis, glycolysis, and biosynthesis of secondary metabolites. Real-time quantitative PCR analysis showed that the expression profiles of 13 randomly selected DEGs were in high agreement with results from Illumina sequencing. Several candidate genes encoding ATP synthase, auxin response factor (ARF), transcription factors (TFs), chalcone synthase (CHS), and male sterility (MS) were responsible for microsporogenesis. Furthermore, a schematic diagram for functional interaction of DEGs from NAU-WA vs. NAU-WB library in radish plants was proposed. These results could provide new information on the dissection of the molecular mechanisms underlying microspore development and CMS occurrence in radish. [ABSTRACT FROM AUTHOR]

• Totally 42 members of the Histone gene family were firstly identified in radish. • Seven RsCENH3 genes exhibited specifically high expression level in the radish bud. • RsCENH3 gene might be involved in promoting microspore development in radish. Histone, a predominant protein component of chromatin, participates in DNA packaging and transcriptional regulation. However, the available information of Histone gene family is limited in radish. In this study, a total of 42 Histone gene family members were identified from the radish genome. Sequence alignment and phylogenetic analyses classified the Histone family into three groups (H2A, H2B and H3). Motif analysis showed that the functions of some motifs shared by H3 subfamily genes were related to chromosome regulation and cell development activities, such as motif 5 containing Cks1 and PPR region. Analysis of intron/exon structure indicated that RsCENH3 (RsHistone 18) has the characteristics of variant Histone. Furthermore, several motifs, including the LTR, G-box and TC-elements, were found in the promoters of RsHistone genes, which involved in cell development or various abiotic stresses responses. Transcriptome analysis indicated that the RsHistone genes exhibited higher expression level in floral buds than in roots and leaves. Subcellular localization showed that the RsCENH3 was localized on the nucleus, and it was highly expressed in the floral bud of 3.0–4.0 mm in radish. These findings would provide valuable information for characterization and potential utilization of Histone genes, and facilitate the efficient induction of double haploid plants in radish. [ABSTRACT FROM AUTHOR]

• The identified 65 Aux/IAA genes show an uneven chromosomal distribution in radish. • Six Aux/IAA genes exhibit specifically high expression level in radish root tissues. • RsIAA33 gene might involve in promoting taproot thickening and development in radish. Auxin/indole–3–acetic acid (Aux/IAA) genes encode short lived nuclear proteins that cooperated with auxin or auxin response factor (ARF), which are involved in plant growth and developmental processes. However, it's still ambiguous how the Aux/IAA genes regulate the process governing taproot thickening in radish. Herein, 65 Aux/IAA genes were identified from the radish genome. Gene duplication analysis showed that two pairs of tandem duplication and 17 (27%) segmental duplication events were identified among Aux/IAA family genes in radish. Transcriptomic analysis revealed that most of Aux/IAA genes (52/65) exhibited differential expression pattern in different root tissues, and six root–specific genes were highly expressed in root cortex, cambium, xylem, and root tip in radish. RT–qPCR analysis showed that the expression level of RsIAA33 was the highest at cortex splitting stage (CSS), and early expanding stage (ES). Furthermore, amiRNA–mediated gene silencing of RsIAA33 indicated that it could inhibit the reproductive growth, thus promoting taproot thickening and development. These results would provide valuable information for elucidating the molecular function of Aux/IAA genes involved in taproot thickening in radish. [ABSTRACT FROM AUTHOR]

The CPA (cation proton antiporter) family plays an essential role during plant stress tolerance by regulating ionic and pH homeostasis of the cell. Radish fleshy roots are susceptible to abiotic stress during growth and development, especially salt stress. To date, CPA family genes have not yet been identified in radish and the biological functions remain unclear. In this study, 60 CPA candidate genes in radish were identified on the whole genome level, which were divided into three subfamilies including the Na+/H+ exchanger (NHX), K+ efflux antiporter (KEA), and cation/H+ exchanger (CHX) families. In total, 58 of the 60 RsCPA genes were localized to the nine chromosomes. RNA-seq. data showed that 60 RsCPA genes had various expression levels in the leaves, roots, cortex, cambium, and xylem at different development stages, as well as under different abiotic stresses. RT–qPCR analysis indicated that all nine RsNHXs genes showed up regulated trends after 250 mM NaCl exposure at 3, 6, 12, and 24h. The RsCPA31 (RsNHX1) gene, which might be the most important members of the RsNHX subfamily, exhibited obvious increased expression levels during 24h salt stress treatment. Heterologous over-and inhibited-expression of RsNHX1 in Arabidopsis showed that RsNHX1 had a positive function in salt tolerance. Furthermore, a turnip yellow mosaic virus (TYMV)-induced gene silence (VIGS) system was firstly used to functionally characterize the candidate gene in radish, which showed that plant with the silence of endogenous RsNHX1 was more susceptible to the salt stress. According to our results we provide insights into the complexity of the RsCPA gene family and a valuable resource to explore the potential functions of RsCPA genes in radish. [ABSTRACT FROM AUTHOR]