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SWEET (Sugars Will Eventually be Exported Transporter) genes play essential roles in various biological processes, including phloem loading, sugar efflux, plant development and stress response. In this study, a total of 33 RsSWEET gene members were identified in the radish genome. They could be divided into four subfamilies and are distributed on eight radish chromosomes. Cis-acting regulatory element analysis indicated that these RsSWEET genes were potentially involved in the radish growth and development and stress response process, including circadian control and light response and responses to numerous stresses, including low-temperature and drought stress. Transcriptome data analysis revealed that a number of RsSWEET genes exhibited specific expression patterns in different tissues and developmental stages of radish. Moreover, several RsSWEET genes (e.g., RsSWEET2a, RsSWEET3a, RsSWEET16b and RsSWEET17) showed differential expression profiles under various abiotic stresses, including cold, heat, salt, Cd and Pb stress. Remarkably, the RsSWEET17 was specifically expressed in the cambium of radish. RsSWEET17 was heterologously expressed in yeast strain EBY.VW4000, which suggested that it has the ability to transport sugar. Notably, RsSWEET17-overexpressing Arabidopsis plants exhibited excessive root length, greater fresh weight and higher soluble sugar content (SSC) accumulation compared with wild-type (WT) plants, indicating that RsSWEET17 might positively regulate radish taproot development by strategically manipulating sugar accumulation. Collectively, these results clarify the molecular mechanisms underlying RsSWEET-mediated sugar accumulation and root growth and development in radish. [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]

In most higher plants, sucrose is the significant form of carbohydrate for long-distance transportation. Sucrose transporters/sucrose carriers (SUTs/SUCs) are involved in the loading and unloading of sucrose in phloem and play an important role in the growth and development of plants. In this study, 12 RsSUC genes were first identified from the radish genome, and their phylogenetic relationships, gene structure, and conserved motifs were further analyzed. RT-qPCR results indicated that RsSUC genes exhibited various expression patterns in different tissues and development stages of the radish. Overexpression of RsSUC1b in Arabidopsis significantly improved the uptake efficiency of exogenous sucrose, and promoted leaves and lateral root growth. In addition, the transgenic plants flowered significantly earlier than wild-type (WT) plants, and the soluble sugar contents (SSCs) including sucrose, glucose, and fructose in the mature leaves and pods were increased. It could be inferred that RsSUC1b is a plasma membrane sucrose transporter and plays a vital role in sucrose transportation and sugar accumulation during plant growth and development. These findings provided novel insights into the biological function of RsSUC genes and facilitate dissecting the molecular mechanism underlying sugar transport during radish development. [ABSTRACT FROM AUTHOR]

Radish is a typical self-incompatible crop. The rapid and accurate identification of S haplotypes can circumvent the blindness of the hybrid combination process, which is critical in radish heterosis utilization and the breeding of new varieties. In this study, based on the gene sequence which encodes the S-locus receptor kinase (SRK) of radish, and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, the S haplotypes were identified among 79 cultivated radish genotypes. The PCR results indicated that 79 radish genotypes could be divided into 48 Class I, 13 Class II, and 17 Class I/II S haplotypes. Sequence alignment confirmed that the Class I materials contained 19 S haplotypes, of which three haplotypes ('NAU-S53', 'NAU-S54' and 'NAU-S55') were identified for the first time in radish. After digestion using the Hinf I restriction endonuclease, the SRK domain of DNA fragments of different genotypes showed high polymorphism. Homozygous materials S haplotypes could be quickly distinguished by the differences in the digested bands. Molecular identification of the S haplotype was highly consistent with the field pollination and pollen tube germination results. These results would provide an important approach for the rapid identification of radish S haplotypes and the efficient utilization of self-incompatibility in heterosis breeding. [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]

Cytoplasmic male sterility (CMS) is a maternally inherited trait, which becomes an important way in heterosis breeding to simplify the seed production procedures and reduce the production cost. Cytological observation of pollen development stages showed a clear difference between the newly developed CMS line and its maintainer; the profiling comparisons between floral buds of CMS line and its maintainer were conducted using differential display reverse transcription PCR (DDRT-PCR) technology. Thirty genes were up-regulated and sixty genes were down-regulated in newly developed CMS line when compared with its maintainer. These genes were involved in cell wall biosynthesis and regulation, transporter and ion channel, flower development and protein metabolism, etc. Expression patterns of six genes encoding RsCAM6, RsGPI, RsPMEI, RsRac, RsCHS, and RsSTP9 were verified by RT-PCR and qRT-PCR in different development stage of floral buds and different organs of CMS line and its maintainer. The expression level of RsRac was higher in stamens and microspores of CMS line than in maintainer. In comparison, the expression levels of the other five genes in CMS line were lower compared with maintainer. This expression profile suggests that these genes played important roles in the development of the pollen and may be closely related to male sterility. The results observed in this study will contribute to understanding the mechanism of pollen abortion during CMS in radish. [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]

SUMMARY Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes‐mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red‐violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots‐to‐shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2‐ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2‐ipt combination. Then, Wus2‐ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene‐edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome‐editing genetic improvement of other vegetable species. [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]

Highlights: [•] RGAs-based TRAP markers were firstly developed in radish. [•] RGA-based TRAP markers were firstly applied in analyzing genetic diversity in radish. [•] A MCID was firstly made in radish based on the polymorphic TRAP markers. [•] 30 radish genotypes were discriminated from each other by the MCID. [•] Cluster groups from TRAP marker genotype analysis matched well with responses to TuMV. [ABSTRACT FROM AUTHOR]

• A total of 28 RsZIP genes were identified and classified into 18 subgroups. • Four tandem and six WGD/segmental duplication events exist in radish genome. • Four RsZIP genes were induced at 2 h under Pb and Cd treatment. • RsIRT1d and RsIRT1e confer yeast Pb but no Cd tolerance. Radish is an important root vegetable crop susceptible to heavy metal (HM) stresses. Zinc- and iron-regulated transporter-like proteins play vital roles in transporting metal ion to enhance HM tolerance or decrease detoxification in plants. In this study, 27 out of 28 RsZIP genes were unevenly located on eight chromosomes with exception of chromosome 3 (Chr.3). The evolutionary pattern divided the whole RsZIP proteins into 18 subgroups. Furthermore, four tandem and six WGD/segmental duplication events were identified, while 24 pairs containing 16 RsZIP and 13 AtZIP genes were orthologous. Moreover, the expression levels of 17 RsZIP genes including RsZIP10b,c and RsIRT1a,b were significantly decreased under 200 mg/L CdCl 2 and 1000 mg/L Pb(NO 3) 2 treatment. RT-qPCR analysis indicated that four RsZIP genes (RsIRT1d, RsIRT1e, RsIAR1 and RsZIP10d) were rapidly induced at 2 h under Pb and Cd treatment, while both RsZIP1 and RsZIP10b were downregulated. The results of yeast functional complementation indicated that RsIRT1d and RsIRT1e confer yeast Pb but no Cd tolerance, suggesting that they might play an important role in the transport of metal ions. These results could provide valuable insights into the characteristics of ZIP family genes and facilitate further exploring the molecular mechanism underlying HM stress responses in radish and other root vegetable crops. [ABSTRACT FROM AUTHOR]

Highlights • MeJA and SA have positive effects on GLS accumulation in radish taproot. • Differential expression of genes was observed under MeJA, SA and ABA treatments. • Myrosinase activity and SF content were highly sensitive to SA. • SF is correlated with myrosinase activity and GRA content. Abstract Radish is an important root vegetable crop. The effects of environmental stresses on glucosinolate (GLS) contents were extensively investigated in Brassicaceae species. However, little is known about the accumulation of GLS and sulforaphane (SF) in hormone-treated radish taproot. In this study, the effects of methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) on the accumulation of GLS and SF in radish taproot were investigated. The GLS content increased considerably in response to MeJA treatment. Moreover, MeJA also up-regulated the expression level of several transcription factors and GLS biosynthesis related genes. The effects of SA on GLS content were less obvious, while ABA suppressed GLS biosynthesis. Additionally, the SF content increased under treatments with MeJA, SA and ABA. Furthermore, changes in myrosinase activity and glucoraphanin (GRA) content after the application of MeJA, SA and ABA were consistent with the changes in SF contents. The results indicated that the MeJA represents one of the most effective plant growth regulators (PGRs) for increasing the GLS content in radish taproot by up-regulating the expression levels of a few GLS biosynthetic related genes. These findings also implied that SF formation is correlated with myrosinase activity and GRA content in radish taproot. [ABSTRACT FROM AUTHOR]

Radish is rich in anthocyanin pigments which are beneficial both to plants and humans. Although the anthocyanin synthesis has been widely investigated, limited information is available on the mechanism underlying transport of these pigments. Recent research indicates that Multi-drug and toxic extrusion proteins (MATE) are possible anthocyanin transporters. In the present study, a total of 51 deferentially expressed transcripts (DETs) were annotated as MATEs, with 21 and 30 transcripts up-regulated at the cortex splitting and taproot thickening stages, respectively. The RsMATE proteins comprised of 490–510 residues and phylogeny analysis clustered RsMATE2, RsMATE3, RsMATE7, RsMATE8 and RsMATE9 with anthocyanin MATE transporters, implying that they might be involved in the transport of anthocyanin in radish. RT-qPCR revealed that transcript levels of RsMATE2 , RsMATE3 , RsMATE7 , RsMATE8 and RsMATE9 were significantly expressed in all colored tissues at both pre-cortex splitting and cortex splitting stages. The expression pattern indicated RsMATE5 is involved in both anthocyanin transport and other secondary metabolites due to its commensurate expression in both the colored and non-colored radish. Microscopic analysis of the spatial distribution of anthocyanin in radish was also performed, and provided evidence that anthocyanin is differentially translocated in plant tissues. Additionally, reciprocal grafting indicated that long distance transport is involved in the sequestration of anthocyanin in radish. These results show that anthocyanin type MATE transporter genes might be involved in the mutual movement of anthocyanin across cell membranes in conjunction with other mechanisms, which provided fundamental information for clarification of mechanism underlying anthocyanin accumulation and facilitated development of nutritionally enhanced taproots in radish. [ABSTRACT FROM AUTHOR]

Nitrite reductase (NiR, EC1.7.7.1) is a key enzyme that catalyzes the second step of nitrite reduction to ammonium in the process of nitrate assimilation in plants. To study the molecular mechanism underlying nitrate accumulation during plant development, the molecular characterization of the NiR gene ( RsNiR ) and its expression patterns under different nitrate concentrations and induction periods of nitrate treatments in leaf, root, petiole, stem and lateral root of radish ( Raphanus sativus L.) were investigated in this study. Based on our radish transcriptome database, the genomic DNA sequence of RsNiR was isolated with four exons and three introns. The cDNA length was 1756 bp containing an open reading frame (ORF) of 1524 bp. The deduced protein consists of 506 amino acids, and showed highest identity with NiRs from Arabidopsis . Moreover, using the genomic DNA walking approach, the 843-bp 5’-flanking region upstream of RsNiR was isolated, which contained several basic cis -regulatory elements including TATA-box, CAAT-box and nitrate-responsive cis -element (NRE). With the increasing nitrate concentration, the NR activity (NRA), nitrate contents and RsNiR expression levels exhibited increasing trends in leaf and root, reached a maximum at 30 mM nitrate treatment and then decreased. In contrast, there was no obvious relationship among RsNiR expression level, NRA and nitrate content under different induction periods at 30 mM nitrate. These results could provide fundamental insight into clarification of the molecular regulation mechanism underlying nitrate assimilation in radish. [ABSTRACT FROM AUTHOR]

Abstract: Real-time quantitative reverse transcription PCR (RT-qPCR) is a rapid and reliable method for gene expression studies. Normalization based on reference genes can increase the reliability of this technique; however, recent studies have shown that almost no single reference gene is universal for all possible experimental conditions. In this study, eight frequently used reference genes were investigated, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin2/7 (ACT), Tubulin alpha-5 (TUA), Tubulin beta-1 (TUB), 18S ribosomal RNA (18SrRNA), RNA polymerase-II transcription factor (RPII), Elongation factor 1-b (EF-1b) and Translation elongation factor 2 (TEF2). Expression stability of candidate reference genes was examined across 27 radish samples, representing a range of tissue types, cultivars, photoperiodic and vernalization treatments, and developmental stages. The eight genes in these sample pools displayed a wide range of Ct values and were variably expressed. Two statistical software packages, geNorm and NormFinder showed that TEF2, RPII and ACT appeared to be relatively stable and therefore the most suitable for use as reference genes. These results facilitate selection of desirable reference genes for accurate gene expression studies in radish. [Copyright &y& Elsevier]

• 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]