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Key message: Transcriptome-based gene expression analysis identifies many critical salt-responsive genes in radish and facilitates further dissecting the molecular mechanism underlying salt stress response. Abstract: Salt stress severely impacts plant growth and development. Radish, a moderately salt-sensitive vegetable crop, has been studied for decades towards the physiological and biochemical performances under salt stress. However, no systematic study on isolation and identification of genes involved in salt stress response has been performed in radish, and the molecular mechanism governing this process is still indistinct. Here, the RNA-Seq technique was applied to analyze the transcriptomic changes on radish roots treated with salt (200 mM NaCl) for 48 h in comparison with those cultured in normal condition. Totally 8709 differentially expressed genes (DEGs) including 3931 up- and 4778 down-regulated genes were identified. Functional annotation analysis indicated that many genes could be involved in several aspects of salt stress response including stress sensing and signal transduction, osmoregulation, ion homeostasis and ROS scavenging. The association analysis of salt-responsive genes and miRNAs exhibited that 36 miRNA-mRNA pairs had negative correlationship in expression trends. Reverse-transcription quantitative PCR (RT-qPCR) analysis revealed that the expression profiles of DEGs were in line with results from the RNA-Seq analysis. Furthermore, the putative model of DEGs and miRNA-mediated gene regulation was proposed to elucidate how radish sensed and responded to salt stress. This study represents the first comprehensive transcriptome-based gene expression profiling under salt stress in radish. The outcomes of this study could facilitate further dissecting the molecular mechanism underlying salt stress response and provide a valuable platform for further genetic improvement of salt tolerance in radish breeding programs. [ABSTRACT FROM AUTHOR]

Simple sequence repeats (SSRs) are highly polymorphic and co-dominant markers, providing an important genomic resource for genetic research. Recently, large-scale transcriptome sequencing has become a reliable and efficient approach for the identification and development of new genic-SSR markers and has been successfully conducted in a few important plant species. However, SSR development based on transcriptome sequencing remains limited in radish ( Raphanus sativus L.). In the present study, from a total of 73,084 unigenes and 150,455 contigs which were assembled from 71.95 million Illumina sequence reads of a radish taproot library, a collection of 11,928 genic-SSR loci were successfully identified in 11,311 unigene sequences. Trinucleotide repeats were the most abundant repeat units, as in many other plants, with a frequency of 52 %. Furthermore, a total of 5,503 genic-SSR primers were developed, from which 1,052 SSR primers were synthesized, and a subset of 823 (78.23 %) primers could generate stable bands. Moreover, 67 selected informative genic-SSR markers were used to determine the genetic diversity of 32 radish genotypes, in which the polymorphism information content values ranged from 0.49 to 0.89. For effective cultivar identification, a novel strategy called manual cultivar identification diagram was employed. Thirty-two radish accessions were clearly separated by six genic-SSR markers. Additionally, the cross-species/genera transferability of these SSRs was further validated in nine relatives in Brassicaceae. These results suggested that the novel genic-SSR markers, as a basis for future genetic linkage and gene tagging analysis, could be very valuable in facilitating genetic mapping, marker-assisted selection and comparative genome analysis. [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]

Radish (Raphanus sativus L.) is an important worldwide root vegetable crop. Little information of the GRAS gene family was available in radish. Herein, a total of 51 GRAS family members were firstly identified from radish genome, and unevenly located onto nine radish chromosomes. Expression analysis of RsGRAS genes in taproot displayed that RsSCL15a and RsSHRc were highly expressed in the radish cambium, and its expression level was increased with the taproot thickening. Comparative transcriptome analysis revealed that the expression patterns of RsGRAS genes varied upon exposure to different abiotic stresses including heavy metals, salt and heat. The expression level of six RsGRAS genes including RsSHRc was increased under chilling stress in two radish genotypes with different cold tolerance. Further analysis indicated that RsGRAS genes could respond to cold stress rapidly and the expression of RsSHRc was up-regulated at different development stages (cortex splitting and thickening stages) under long-term cold treatment. Transient expression of RsSHRc gene in radish showed that RsSHRc possessed the reliable function of eliminating reactive oxygen species (ROS), inhibiting the formation of malondialdehyde (MDA) and promoting to accumulate proline under cold stress. Together, these findings provided insights into the function of RsGRAS genes in taproot development and chilling stress response in radish. • The GRAS gene family was comprehensively characterized in radish. • Expression analysis indicates RsSHRc might be involved in radish taproot thickening. • Transient expression analysis revealed RsSHRc gene could enhance cold tolerance of radish plants. [ABSTRACT FROM AUTHOR]

• Agrobacterium -mediated in planta transformation system was effectively established in radish. • RsWUSb might play an important role in shoot formation and development. • RsRR18-1-RsWUSb module regulates shoot development through CTK signaling-related pathway. The WUSCHEL-related homeobox (WOX) family genes are involved in cell division and differentiation throughout various stages of plant growth and development. Till now, the function of WOX genes in radish remains unclear. In this study, a total of 23 RsWOX genes were identified from the radish genome. RT-qPCR analysis showed that the expression level of RsWUSb was significantly increased with exogenous cytokinin treatment. Overexpression of RsWUSb in Arabidopsis resulted in apical shoot clustering. Notably, the RsWUSb -overexpressed radish plants were generated by Agrobacterium- mediated in planta transformation approach. Compared with the wide-type (WT) plants, the RsWUSb -OE plants displayed clustered apical shoots, more leaves, and weak apical dominance. Moreover, yeast one-hybrid (Y1H) and dual-luciferase assay (DLA) showed that the RsRR18-1 (RESPONSE REGULATOR 18-1) bound to the promoter of RsWUSb and upregulated its transcription. Furthermore, the expression of RsRR18-1 was significantly activated by exogenous cytokinin, suggesting that cytokinin signaling might regulate the formation and development of radish shoots via RsRR18-1-RsWUSb module. Collectively, these findings indicated that in planta transformation approach is a simple and effective method for generating transgenic plants and gene function validation in radish. [ABSTRACT FROM AUTHOR]

• An efficient transformation system with A. rhizogenes was established in radish. • RsMYB90 promoted anthocyanin accumulation in transgenic hairy roots. • RsMYB90 - RsUFGT module positively regulated the biosynthesis of anthocyanin. Radish (Raphanus sativus L.) is recalcitrant to regeneration and genetic transformation, which has severely limited its gene function verification and germplasm innovation. To overcome the limitation, we have developed a rapid and stable regeneration and transformation of hairy roots in radish to explore the potential function of the critical genes involved in the anthocyanin biosynthesis via the optimization of explant selection, bacteria solution concentration, acetosyrinone (AS) concentration and strain types. The single factor experimental designing method was applied and each treatment was conducted for three independent biological replicates. The hairy roots induction rate was more than 90% when using cotyledon with petiole or rootless seedling as explants, while only 53.33% hypocotyls could induce hairy roots. Phenotypic observation and molecular identification indicated that the highest positive transgenic rate of hairy roots could reach approximately 17.51%, when employed optimal optical density (OD 600) and AS concentration at 1.0 and 300 μM, respectively, as well as the best strain of MSU440. Subsequently, RsMYB90 was found to play a positive role in the anthocyanin accumulation through the established hairy root transgenic system, and a number of anthocyanin-related genes, such as RsUFGT, RsCHS and RsF3H , were up-regulated by RNA-seq and RT-qPCR analysis. In addition, yeast one-hybrid (Y1H) and dual-luciferase assays (DLA) showed that the RsMYB90 could bind to the promoter of RsUFGT to activate its expression. Taken together, the stable A. rhizogenes -mediated transformation system provides an avenue for gene function assay, genetic engineering and study of secondary metabolisms 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]