Genome-wide characterization of Rice Black Streaked Dwarf Virus-responsive genes in rice

Background: Rice black streaked dwarf virus (RBSDV) is an important pathogen disease in rice and gramineous planting regions all over the world. The major phenotypes of RBSDV infected rice were dwarf with dark green leaves, which generally resulted in seriously loss of yield. The RBSDV known so far is preoperatively transmitted to rice in a persistent manner by small brown planthopper (SBPH), instead of transmitting to offspring through ovary. Results: To identify RBSDV responsive genes and to explore and clarify the molecular mechanisms involved in plant-virus interaction, digital gene expression profile (DGE) analysis was performed by high-throughput sequencing using wild-type (WT) and RBSDV infected rice leaves (IRL) as materials. A total of 165,975 and 165,940 unique tags were obtained in IRL and WT libraries, respectively. In comparison with the control, 896 differentially expressed genes (DEGs) were obtained, of which 500 DEGs were up regulated and 396 DEGs were down regulated. Functional analysis showed that DEGs mainly classified into10 groups, including metabolism, stress pathogen and defense, signal transduction, Transporter, transcription and post-transcription, cell structure and division etc. To further validate reliability and authenticity of the data, 10 DEGs were randomly picked and Real-time RT-PCR was carried out, and the expression trends of 7 genes were in line with the RNA-seq results. By searching the RBSDV related miRNA database of rice, 10 targeted genes of 6 significantly changed miRNAs were also identified in these DEGs. Conclusions: The data derived from RNA-seq were valid and credible. Through this research, a series of candidate RBSDV-responsive genes were obtained, and special signal transduction and metabolism pathways were built and pulled out in rice. This study provided further insight into the molecular mechanisms during compatible and incompatible interactions between viruses and their host plants.

Screening and analysis on the differentially expression genes between diploid and autotetraploid watermelon by using of digital gene expression profile

Synthetic polyploids are key breeding materials for watermelon. Compared with diploid watermelon, the tetraploid watermelon often exhibit wide phenotypic differences and differential gene expression. Digital gene expression (DGE) profile technique was performed in this study to present gene expression patterns in an autotetraploid and its progenitor diploid watermelon, and deferentially expressed genes (DEGs) related to the abiotic and biotic stress were also addressed. Altogether, 4,985 DEGs were obtained in the autotetraploid against its progenitor diploid, and 66.02% DEGs is up-regulated. GO analysis shows that these DEGs mainly distributed in ‘metabolic process’, ‘cell’ and ‘catalytic activity’. KEGG analysis revealed that these DEGs mainly cover ‘metabolic pathways’, ‘secondary metabolites’ and ‘ribosome’. Moreover, 134 tolerance related DEGs were identified which cover osmotic adjustment substance, protective enzymes/protein, signaling proteins and pathogenesis-related proteins. This study present the differential expression of stress related genes and global gene expression patterns at background level in autotetraploid watermelons. These new evidences could supplement the molecular theoretical basis for the better resistance after the genome doubling in the gourd family.

Combinatorial labeling of single cells for gene expression cytometry

We present a technically simple approach for gene expression cytometry combining next-generation sequencing with stochastic barcoding of single cells. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We applied the technology to dissect the human hematopoietic system and to characterize heterogeneous response to in vitro stimulation. High sensitivity is demonstrated by detection of low-abundance transcripts and rare cells. Under current implementation, the technique can analyze a few thousand cells simultaneously and can readily scale to 10,000s or 100,000s of cells.

Identification of genes associated with low furanocoumarin content in grapefruit

Some furanocoumarins in grapefruit (Citrus paradisi) are associated with the so-called grapefruit juice effect. Previous phytochemical quantification and genetic analysis suggested that the synthesis of these furanocoumarins may be controlled by a single gene in the pathway. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of fruit tissues was performed to identify the candidate gene(s) likely associated with low furanocoumarin content in grapefruit. Fifteen tentative differentially expressed fragments were cloned through the cDNA-AFLP analysis of the grapefruit variety Foster and its spontaneous low-furanocoumarin mutant Low Acid Foster. Sequence analysis revealed a cDNA-AFLP fragment, Contig 6, was homologous to a substrate-proved psoralen synthase gene, CYP71A22, and was part of citrus unigenes Cit.3003 and Csi.1332, and predicted genes Ciclev10004717m in mandarin and orange1.1g041507m in sweet orange. The two predicted genes contained the highly conserved motifs at one of the substrate recognition sites of CYP71A22. Digital gene expression profile showed the unigenes were expressed only in fruit and seed. Quantitative real-time PCR also proved Contig 6 was down-regulated in Low Acid Foster. These results showed the differentially expressed Contig 6 was related to the reduced furanocoumarin levels in the mutant. The identified fragment, homologs, unigenes, and genes may facilitate further furanocoumarin genetic study and grapefruit variety improvement.