Construction of SNP fingerprints and genetic diversity analysis of radish (Raphanus sativus L.).

Radish (Raphanus sativus L.) is a vegetable crop with economic value and ecological significance in the genus Radish, family Brassicaceae. In recent years, developed countries have attached great importance to the collection and conservation of radish germplasm resources and their research and utilization, but the lack of population genetic information and molecular markers has hindered the development of the genetic breeding of radish. In this study, we integrated the radish genomic data published in databases for the development of single-nucleotide polymorphism (SNP) markers, and obtained a dataset of 308 high-quality SNPs under strict selection criteria. With the support of Kompetitive Allele-Specific PCR (KASP) technology, we screened a set of 32 candidate core SNP marker sets to analyse the genetic diversity of the collected 356 radish varieties. The results showed that the mean values of polymorphism information content (PIC), minor allele frequency (MAF), gene diversity and heterozygosity of the 32 candidate core SNP markers were 0.32, 0.30, 0.40 and 0.25, respectively. Population structural analysis, principal component analysis and genetic evolutionary tree analysis indicated that the 356 radish materials were best classified into two taxa, and that the two taxa of the material were closely genetically exchanged. Finally, on the basis of 32 candidate core SNP markers we calculated 15 core markers using a computer algorithm to construct a fingerprint map of 356 radish varieties. Furthermore, we constructed a core germplasm population consisting of 71 radish materials using 32 candidate core markers. In this study, we developed SNP markers for radish cultivar identification and genetic diversity analysis, and constructed DNA fingerprints, providing a basis for the identification of radish germplasm resources and molecular marker-assisted breeding as well as genetic research.

Expression analysis of argonaute, Dicer-like, and RNA-dependent RNA polymerase genes in cucumber (Cucumis sativus L.) in response to abiotic stress.

Posttranscriptional control of gene expression can be achieved through RNA interference when the activities of Dicer-like (DCL), argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins are significant. In this study, we analysed the expression of seven AGO, five DCL and eight RDR genes in cucumber under cold, heat, hormone, salinity and dehydration treatments using quantitative reverse-transcription PCR (qRT-PCR). All CsAGO, CsDCL and CsRDR genes were differentially expressed under abiotic stress treatment. In response to abiotic stress treatment, most genes were expressed at higher levels in flowers or stems than in other organs, whereas some CsAGOs (CsAGO1c, CsAGO6 and CsAGO7) and CsRDRs (CsRDR1d and CsRDR2) were highly expressed in roots during dehydration treatment. The expression patterns indicate that most CsDCLs, CsAGOs and CsRDRs respond to abiotic stress, and stems or flowers are the most sensitive organs, followed by roots. This is the first report of expression analysis of all CsDCL, CsAGO and CsRDR family genes in cucumber under abiotic stress, which provides basic information and insights into the putative roles of these genes in abiotic stress. The results of this study should serve as a basis for further functional characterization of these gene families in cucumber and related Cucurbitaceae species.

Application of RNA interference methodology to investigate and develop SCMV resistance in maize.

Specific fragments of the sugarcane mosaic virus (SCMV) coat protein gene (cp) were amplified by reverse transcriptionpolymerase chain reaction and used to construct a marker free small interfering RNA complex expression vector against SCMV. In planta transformation was performed on maize (Zea mays) inbred line 8112 mediated by Agrobacterium tumefaciens. PCR and Southern blot analyses demonstrated successful integration of the cp segment into the 8112 genome. The in planta transformation frequency was 0.1%, and the cotransformed frequency with the cp and bar genes was 0.034%. Real-time quantitative PCR of samples from different transgenic plant organs showed that the expression of the cp gene fragment in transgenic plants was variable and that the highest expression level occurred in the tassels and leaves and the lowest expression occurred in the roots. Real-time quantitative PCR was also used to measure how gene expression in transgenic T2 generation plants inoculated with SCMV changes over time. The results showed that the hairpin RNA structure transcribed from the cp gene interfered with SCMV infection and transgenic maize lines were not equally effective in preventing SCMV infection. Our findings provide a valuable tool for controlling plant viruses using RNA interference and the posttranslational gene silencing approach.

Identification and characterization of the RCI2 gene family in maize (Zea mays).

Rare-cold-inducible (RCI2) genes are structurally conserved members that encode small, highly hydrophobic proteins involved in response to various abiotic stresses. Phylogenetic and functional analyses of these genes have been conducted in Arabidopsis, but an extensive investigation of the RCI2 gene family has not yet been carried out in maize. In the present study, 10 RCI2 genes were identified in a fully sequenced maize genome. Structural characterization and expression pattern analysis of 10 ZmRCI2s (Zea mays RCI2 genes) were subsequently determined. Sequence and phylogenetic analyses indicated that ZmRCI2s are highly conserved, and most of them could be grouped with their orthologues from other organisms. Chromosomal location analysis indicated that ZmRCI2s were distributed unevenly on seven chromosomes with two segmental duplication events, suggesting that maize RCI2 gene family is an evolutionarily conserved family. Putative stress-responsive cis-elements were detected in the 2-kb promoter regions of the 10 ZmRCI2s. In addition, the 10 ZmRCI2s showed different expression patterns in maize development based on transcriptome analysis. Further, microarray and quantitative real-time PCR (qRT-PCR) analysis showed that each maize RCI2 genes were responsive to drought stress, suggesting their important roles in drought stress response. The results of this work provide a basis for future cloning and application studies of maize RCI2 genes.

Identification and expression analysis of primary auxin-responsive Aux/IAA gene family in cucumber (Cucumis sativus).

Aux/IAA is an important gene family involved in many aspects of growth and development. Aux/IAA proteins are short-lived nuclear proteins that are induced primarily by various phytohormones. In this study, 29 Aux/IAA family genes (CsIAA01-CsIAA29) were identified and characterized in cucumber, including gene structures, phylogenetic relationships, conserved protein motifs and chromosomal locations. These genes show distinct organizational patterns of their putative motifs. The distributions of the genes vary: except for five CsIAA genes in cucumber that were not located, seven CsIAA genes were found on scaffold, while the other 17 CsIAA genes were distributed on seven other chromosomes. Based on a phylogenetic analysis of the Aux/IAA protein sequences from cucumber, Arabidopsis and other plants, the Aux/IAA genes in cucumber were categorized into seven subfamilies. To investigate whether the expression of CsIAA genes is associated with auxin induction, their transcript levels were monitored in seedlings treated with IAA (indole-3-acetic acid), and their expression patterns were analysed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that 11/29 CsIAA genes were expressed in leaves whether treated with IAA or not and the time course of processing and compared with the control, five CsIAA genes showed low expression only after 60 min treatment with IAA, while 11 genes showed no expression. These results provide useful information for further functional analysis of Aux/IAA gene family in cucumber.

Systematic analysis of sequences and expression patterns of drought-responsive members of the HD-Zip gene family in maize.

BACKGROUND: Members of the homeodomain-leucine zipper (HD-Zip) gene family encode transcription factors that are unique to plants and have diverse functions in plant growth and development such as various stress responses, organ formation and vascular development. Although systematic characterization of this family has been carried out in Arabidopsis and rice, little is known about HD-Zip genes in maize (Zea mays L.). METHODS AND FINDINGS: In this study, we described the identification and structural characterization of HD-Zip genes in the maize genome. A complete set of 55 HD-Zip genes (Zmhdz1-55) were identified in the maize genome using Blast search tools and categorized into four classes (HD-Zip I-IV) based on phylogeny. Chromosomal location of these genes revealed that they are distributed unevenly across all 10 chromosomes. Segmental duplication contributed largely to the expansion of the maize HD-ZIP gene family, while tandem duplication was only responsible for the amplification of the HD-Zip II genes. Furthermore, most of the maize HD-Zip I genes were found to contain an overabundance of stress-related cis-elements in their promoter sequences. The expression levels of the 17 HD-Zip I genes under drought stress were also investigated by quantitative real-time PCR (qRT-PCR). All of the 17 maize HD-ZIP I genes were found to be regulated by drought stress, and the duplicated genes within a sister pair exhibited the similar expression patterns, suggesting their conserved functions during the process of evolution. CONCLUSIONS: Our results reveal a comprehensive overview of the maize HD-Zip gene family and provide the first step towards the selection of Zmhdz genes for cloning and functional research to uncover their roles in maize growth and development.

Genome-wide analysis of BURP domain-containing genes in maize and sorghum.

BURP domain-containing genes comprise a large plant-specific family, yet the functions are very poorly understood, especially in maize (Zea mays) and sorghum (Sorghum vulgare). In this study, 26 BURP family genes in maize (ZmBURP01-15) and sorghum (SbBURP01-11) were identified including the gene structure, phylogenetic relationship, conserved protein motifs and chromosome locations. These genes have diverse exon-intron structures and distinct organization of putative motifs. The distributions of the genes vary: 15 ZmBURP genes are located in maize on five chromosomes, and 11 SbBURP genes in sorghum are on six chromosomes. Based on the phylogenetic analysis of BURP protein sequences from maize, sorghum and other plants, the BURP genes in maize and sorghum were categorized into five subfamilies (RD22-like, PG1ß-like, BURP VI, BURP VII and BURP VIII). Transcript level analysis of ZmBURP genes revealed the expression patterns of BURP genes in maize under diffferent stress conditions. The results suggested that only eight ZmBURP genes were responsive to at least one of the stress treatments applied. Among these genes, seven genes (ZmBURP04, ZmBURP05, ZmBURP08, ZmBURP09, ZmBURP12, ZmBURP14, ZmBURP15) were responsive to ABA and cold respectively, two genes (ZmBURP06 and ZmBURP14) were responsive to NaCl. The results presented here provide useful information for further functional analysis of the BURP gene family in maize and sorghum.

Bacterially expressed dsRNA protects maize against SCMV infection.

RNA interference (RNAi) is a sequence-specific, posttranscriptional gene silencing (PTGS) process in plants that is mediated by dsRNA homologous to the silenced gene(s). In this study, we report an efficient method to produce dsRNA using a bacterial expression system. Two fragments of the Sugarcane Mosaic Virus (SCMV) CP (coat protein) gene were amplified by RT-PCR, and cloned into the inverted-repeat cloning vector pUCCRNAi. The two recombinant plasmids were transformed individually into E. coli HT115, an RNase-III deficient strain, and dsRNA was induced by isopropyl-ß-D: -thiogalactopyranoside (IPTG). The crude extracts of E. coli HT115 containing large amounts of dsRNA were applied to plants as a spray and the experiment confirmed a preventative efficacy. Our findings demonstrated that spraying crude dsRNA-containing extracts inhibited SCMV infection, and the dsRNA derived from an upstream region (CP1) was more effective than was dsRNA derived from a downstream region (CP2) of the SCMV CP gene. The results provide a valuable tool for plant viral control using dsRNA and the PTGS approach.