Selection of reference genes for quantitative real-time PCR in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.     

Differential expression of two <Emphasis Type="Italic">CONSTANS-LIKE1</Emphasis> genes in potato

Although the CONSTANS gene and its CONSTANS-LIKE1 (COL1) orthologs are known to control the photoperiod-dependent floral transition in many plant species, the role of these genes in Solanum development has not been sufficiently elucidated. Previously we characterized two forms of CONSTANS-LIKE1 genes, sCOL1 and lCOL1, in potato (Solanum tuberosum ssp. tuberosum). To prove that these genes were functional, we followed their expression in potato cv. Early Rose with the real-time PCR technique. Both sCOL1 and lCOL1 displayed characteristic day-night patterns of expression under long-day and short-day conditions. The profiles and amplitudes of expression dramatically differed in two genes, with the maximum sCOL1 expression exceeding that of lCOL1 by an order of magnitude.     

Inverse PCR for subtyping of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> carrying IS<Emphasis Type="Italic">Aba</Emphasis>1

Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla_OXA-51-like, bla_OXA-23-like, and bla_ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes — bla_ampC, bla_OXA-66-like, and csuD — were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1.     

Illumina-sequencing based transcriptome study of coat color phenotypes in domestic goats

This study performed a comprehensive expression profiling of genes expressed in the skin of goats with three different coat colors by Illumina Sequencing. A total of 91 significantly expressed genes were detected when comparing gray skin to white skin library and these included 74 up-regulated and 17 down-regulated genes in gray skin. There were 67 differentially expressed genes between brown skin and white skin libraries, 23 of which were up-regulated and 44 were down-regulated in brown skin. When we compared brown and gray libraries, 154 differentially expressed genes were found, of which 33 showed higher expression and 121 showed lower expression in brown skin. To our surprise, MC1R, MITF, TYR and KIT showed no significant difference in expression between the goats with three skin colors, whereas ASIP was detected in white skin but not in dark skins. In this study, PMEL, TRPM1, TYRP1 and DCT were significantly up-regulated in brown goat skin compare with gray and white skins. PMEL showed higher expression in gray goat skin compared with white goat skin, whereas there were no significant differences in the expression of TYRP1, TRPM1 and DCT between gray and white skin samples. In addition, ELOVL3 showed higher expression in gray goat skin than in brown and white skins, whereas there was no significant differences in the expression of ELOVL3 between brown and white skin samples. These results expand our understanding of the complex molecular mechanisms of skin physiology and melanogenesis in goat and provide a foundation for future studies.     

Down-regulation of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">DND1</Emphasis> orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.