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Radish is an important root vegetable crop with high nutritional, economic, and medicinal value. Lignin is an important secondary metabolite possessing a great effect on plant growth and product quality. To date, lignin biosynthesis-related genes have been identified in some important plant species. However, little information on characterization of critical genes involved in plant lignin biosynthesis is available in radish. In this study, a total of 71,148 transcripts sequences were obtained from radish root, of which 66 assembled unigenes and ten candidate genes were identified to be involved in lignin monolignol biosynthesis. Full-length cDNA sequences of seven randomly selected genes were isolated and sequenced from radish root, and the assembled unigenes covered more than 80% of their corresponding cDNA sequences. Moreover, the lignin content gradually accumulated in leaf during the developmental stages, and it increased from pre-cortex to cortex splitting stage, followed by a decrease at thickening stage and then increased at mature stage in root. RT-qPCR analysis revealed that all these genes except RsF5H exhibited relatively low expression level in root at thickening stage. The expression profiles of Rs4CL5, RsCCoAOMT1, and RsCOMT genes were consistent with the changes of root lignin content, implying that these candidate genes may play important roles in lignin formation in radish root. These findings would provide valuable information for identification of lignin biosynthesis-related genes and facilitate dissection of molecular mechanism underlying lignin biosynthesis in radish and other root vegetable crops. [ABSTRACT FROM AUTHOR]

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]

microRNAs (miRNAs) are a class of single-stranded endogenous non-coding RNAs that play critical roles in plant growth, development, and environmental stress responses. Temperature is one of the major physical parameters disturbing cellular homeostasis and causing leaf etiolation in plants. Previous studies have reported that several conserved and novel miRNAs were responsive to heat stress in plants. However, the characterization of miRNAs responsive to heat stress in radish remains poorly understood. To better understand miRNAs and their target genes under heat stress, two small RNA libraries were constructed from heat-treated (Heat24) and heat-untreated (CK) radish roots. Using Solexa system, totally, 26 known and 19 novel miRNAs were identified as differentially expressed under heat stress. Expression patterns of a set of heat-responsive miRNAs were validated by quantitative real-time PCR (qRT-PCR). Furthermore, 422 sliced targets for 25 known miRNAs were identified by degradome sequencing technology, and most of the identified targets are involved in multiple biological processes including transcriptional regulation and response to biotic and abiotic stresses. Moreover, some miRNAs and their corresponding targets, which are related to the accumulation of heat stress transcription factors and heat shock proteins, played important roles in thermo-tolerance in radish. These findings could enhance the understanding of molecular mechanisms underlying miRNAs and their targets in regulating plant responses to heat stress. [ABSTRACT FROM AUTHOR]

microRNAs (miRNAs), endogenous non-coding RNAs of approximately 21-24 nucleotides, are important regulators of transcriptional and post-transcriptional gene expression. These regulators play a key role during plant growth and development, including embryogenesis, which is crucial to the life cycle of most plant species. However, although embryogenesis-associated miRNAs have been isolated in a few species, the diversity of these regulatory miRNAs remains largely unexplored, especially in radish. In this study, two small RNA libraries were constructed from radish ovules before and after fertilization. Both libraries were sequenced by next generation sequencing (NGS) technology. This analysis identified 144 conserved and 34 non-conserved miRNAs (representing 43 known miRNA families) and 38 novel miRNAs (representing 28 miRNA families). Comparative analysis revealed that 29 known and 10 novel miRNA families were differentially expressed during embryogenesis. QRT-PCR analysis confirmed miRNA expression patterns and revealed tissue-specific and/or developmental stage-dependent expression for some miRNAs. Moreover, potential target predictions indicated that most of these targets were transcription factors involved in regulating plant growth, development and metabolism. Notably, target transcripts such as squamosa promoter-binding protein, auxin response factor and agamous-like MADS-box protein participated in radish embryogenesis. [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]

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]

High temperature is one of the most important abiotic factors influencing plant growth and development. Radish is a cool season vegetable crop sensitive to higher temperatures. Heat injuries may affect plant growth, and interfere with formation and thickening of the fleshy taproot. To characterize the heat-stress response in radish, 30-day-old radish seedlings were exposed to a temperature of 40 °C. Leaf samples were then collected from the seedlings at 0 h, 12 h and 24 h after temperature exposure. Proteins extracted from leaves were analyzed with two-dimensional electrophoresis (2-DE), and differentially expressed protein spots were identified by mass spectrometry (MS). In total, 11 differentially expressed proteins were identified successfully by MALDI-TOF MS. Of these, four were heat shock proteins (HSPs), four were related to energy and metabolism, two were related to redox homeostasis, and one was related to signal transduction. These proteins were analyzed further for mRNA levels, corresponding to differential levels of gene expression. The result showed that gene expression profiles at the transcriptional level were not completely consistent with those at translational levels. The differentially expressed heat stress response proteins identified, like small heat shock proteins together with energy and metabolism-related proteins, provide new insights into the molecular basis of plant responses to high temperature stresses in radish. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a widespread soil pollutant and poses a significant threat to human health via the food chain. Large phenotypic variations in Cd concentration of radish roots and shoots have been observed. However, the genetic and molecular mechanisms of Cd accumulation in radish remain to be elucidated. In this study, a genetic linkage map was constructed using an F mapping population derived from a cross between a high Cd-accumulating cultivar NAU-Dysx and a low Cd-accumulating cultivar NAU-Yh. The linkage map consisted of 523 SRAP, RAPD, SSR, ISSR, RAMP, and RGA markers and had a total length of 1,678.2 cM with a mean distance of 3.4 cM between two markers. All mapped markers distributed on nine linkage groups (LGs) having sizes between 134.7 and 236.8 cM. Four quantitative trait loci (QTLs) for root Cd accumulation were mapped on LGs 1, 4, 6, and 9, which accounted for 9.86 to 48.64 % of all phenotypic variance. Two QTLs associated with shoot Cd accumulation were detected on LG1 and 3, which accounted for 17.08 and 29.53 % of phenotypic variance, respectively. A major-effect QTL, qRCd9 (QTL for root Cd accumulation on LG9), was identified on LG 9 flanked by NAUrp011_754 and EM5me6_286 markers with a high LOD value of 23.6, which accounted for 48.64 % of the total phenotypic variance in Cd accumulation of F lines. The results indicated that qRCd9 is a novel QTL responsible for controlling root Cd accumulation in radish, and the identification of specific molecular markers tightly linked to the major QTL could be further applied for marker-assisted selection (MAS) in low-Cd content radish breeding program. [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]

Key message : The radish WRKY gene family was genome-widely identified and played critical roles in response to multiple abiotic stresses. Abstract: The WRKY is among the largest transcription factors (TFs) associated with multiple biological activities for plant survival, including control response mechanisms against abiotic stresses such as heat, salinity, and heavy metals. Radish is an important root vegetable crop and therefore characterization and expression pattern investigation of WRKY transcription factors in radish is imperative. In the present study, 126 putative WRKY genes were retrieved from radish genome database. Protein sequence and annotation scrutiny confirmed that RsWRKY proteins possessed highly conserved domains and zinc finger motif. Based on phylogenetic analysis results, RsWRKYs candidate genes were divided into three groups (Group I, II and III) with the number 31, 74, and 20, respectively. Additionally, gene structure analysis revealed that intron-exon patterns of the WRKY genes are highly conserved in radish. Linkage map analysis indicated that RsWRKY genes were distributed with varying densities over nine linkage groups. Further, RT-qPCR analysis illustrated the significant variation of 36 RsWRKY genes under one or more abiotic stress treatments, implicating that they might be stress-responsive genes. In total, 126 WRKY TFs were identified from the R. sativus genome wherein, 35 of them showed abiotic stress-induced expression patterns. These results provide a genome-wide characterization of RsWRKY TFs and baseline for further functional dissection and molecular evolution investigation, specifically for improving abiotic stress resistances with an ultimate goal of increasing yield and quality of radish. [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]

Increasing evidence has revealed that microRNA (miRNA)-mediated gene regulation plays a significant role in response to heavy metal stresses. However, there is little information available about the expression patterns or roles of miRNAs under lead toxicity stress in plants. The radish is an important root vegetable crop with a fleshy taproot as the edible part. It was of vital importance to investigate the response mechanisms and explore the regulatory network at the molecular level under the heavy metal stresses in radish. In the present study, using high-throughput sequencing and degradome analysis, a genome-wide identification of radish miRNA and their targets under the exposure of Pb stress was conducted. A total of 74 known and 173 potential novel miRNAs were successfully identified from two radish root libraries of one untreated control (CK) and one Pb-stressed (Pb500). Of these, 25 known and nine novel miRNAs were significantly differentially expressed and identified as Pb-responsive miRNAs. Degradome analysis revealed that 1,979 miRNA-mRNA target transcripts could potentially be cleaved. Gene Ontology (GO) analysis revealed that these target transcripts were predominately involved in the regulation of transcription, defense responses, and binding related terms. The identified target genes for Pb-responsive miRNAs were mainly involved in stress-related signal sensing and transduction, specific metal uptake and homeostasis mechanisms. Additionally, the expression patterns of 20 Pb-responsive miRNAs and six target genes were validated by quantitative real-time PCR (qRT-PCR). These results provide fundamental insights into the miRNA-mediated regulatory networks and molecular mechanisms underlying plant responsiveness to Pb stresses. [ABSTRACT FROM AUTHOR]

Cytoplasmic male sterility (CMS) is a maternally inherited trait that is widely utilized in hybrid seed production. In this study, the Rs- Rf gene and its allele, the Rs- rf gene, were isolated from a radish CMS restorer line and maintainer, respectively. The Rs- Rf gene was identified as a member of the pentatricopeptide repeat family. The results of reverse transcription (RT)-PCR and quantitative RT-PCR analyses of the floral buds at different development stages were used to characterize the expression features of Rs- Rf and orf138, a unique transcribed gene in the mitochondrion for its role in determining male sterility in the cytoplasm. orf138 was up-regulated in the CMS line and F hybrid at three bud developmental stages, including meiosis and the tetroid and microspore stages, but it was not expressed in the maintainer and restorer line, suggesting that the Rs- Rf gene was not able to block orf138 transcription. As in the F plant, the transcription of Rs- Rf was gradually up-regulated in the restorer line with progressive stages of bud development, but it was not transcribed in the CMS line and maintainer. Different restriction sites for SspI between the allele of Rs- Rf and Rs- rf were also identified, and a guideline for using the functional marker to distinguish the genotypes of individuals with Rs- Rf was developed. Due to its co-dominant character, this marker could be used to discriminate the heterozygous and homozygous fertility-restoring allele. Using this functional marker, we established a system of molecular marker-assisted development of elite CMS lines in radish. This system will contribute greatly to candidate maintainer identification and accelerate the process of elite CMS line development in late-bolting radish breeding programs. [ABSTRACT FROM AUTHOR]