Project description:We show there is minimal genome-wide chromatin rearrangements (as measured by DNA accessibility) during tissue differentiation in C. elegans transgenic expression of E. coli DAM (DNA Adenine Methyltransferase) from tissue-specific promoters followed by mapping of methylated sites using a procedure that captures 20bp sequences flanking methylated sites for Illumina sequencing PD3994 = transgenic line expressing DAM from myo-3 promoter PD3995 = transgenic line expressing DAM from rol-6 promoter PD3997 = transgenic line expressing DAM from vit-2 promoter N2dam = wildtype (N2) line; its genomic DNA was in vitro DAM-methylated parsed = Solexa reads in which linker sequences were successfully parsed out by the authors WS170DAMtags_ALL.txt = the set of all in_silico-generated DAM tags from C. elegans genome version WS170
Project description:Sugarcane is a very efficient crop to produce ethanol. In recent years, extensive efforts have been made in order to increase sugarcane yields. To reach this goal, molecular biology tools have been used comprehensively, identifying genes, pathways and genetic polymorphisms. However, some important molecular components, like microRNAs, have not been deeply investigated. MicroRNAs are an important class of endogenous small, noncoding RNAs that regulate gene expression at the post-transcription level and play fundamental roles in diverse aspects of animal and plant biology. Plant genomes harbor numerous miRNA genes that regulate many protein-coding genes to influence key processes ranging from development, metabolism, and responses to abiotic and biotic stresses. There is wide range of pests and diseases that affect sugarcane, yet the mechanisms that regulate pathogen interactions with sugarcane have not been thoroughly investigated. To gain knowledge on the physiological responses to pathogens mediated by microRNAs in sugarcane, we screened the transcriptoma of sugarcane plants infected with Acidovorax avenae subsp avenae, the causal agent of red stripe disease in sugarcane, and detected several microRNAs modulated in the presence of the pathogen. Furthermore, we validated with qPCR a number of microRNA expression patterns observed by bioinformatics analysis. In addition, we observed high expression levels of several star microRNAs, in numbers larger than the mature microRNAs in some cases. Interestingly, sof-miR408 was consistently down-regulated in the presence of several pathogens, but not in the presence beneficial microbes. This result indicates that the sugarcane senses pathogenic or beneficial microorganisms differentially and triggers specific epigenetic regulatory mechanisms accordingly
Project description:To accelerate genetic studies in sugarcane, an Axiom Sugarcane100K single nucleotide polymorphism (SNP) array was designed and customized in this study. Target enrichment sequencing 300 sugarcane accessions selected from the world collection of sugarcane and related grass species yielded more than four million SNPs, from which a total of 31,449 single dose (SD) SNPs and 68,648 low dosage (33,277 SD and 35,371 double dose) SNPs from two datasets respectively were selected and tiled on Affymetrix Axiom SNP array. Most of selected SNPs (91.77%) were located within genic regions (12,935 genes), with an average of 7.1 SNPs/gene according to sorghum gene models. This newly developed array was used to genotype 469 sugarcane clones, including one F1 population derived from cross between Green German and IND81-146, one selfing population derived from CP80-1827, and 11 diverse sugarcane accessions as controls. Results of genotyping revealed a high polymorphic SNP rate (77.04%) among the 469 samples. Three linkage maps were constructed by using SD SNP markers, including a genetic map for Green German with 3,482 SD SNP markers spanning 3,336 cM, a map for IND81-146 with 1,513 SD SNP markers spanning 2,615 cM, and a map for CP80-1827 with 536 SD SNP markers spanning 3,651 cM. Quantitative trait loci (QTL) analysis identified a total of 18 QTLs controlling Sugarcane yellow leaf virus resistance segregating in the two mapping populations, harboring 27 disease resistant genes. This study demonstrated the successful development and utilization of a SNP array as an efficient genetic tool for high throughput genotyping in highly polyploid sugarcane.
Project description:According to the key words, the gene set, including oxidation-reduction, RNA silence, disease resistance, phytohormone, phosphorylation, dephosphorylation, transcription factor, receptor, kinase, ubiquitination and RNA binding,etc. from sugarcane and the whole CDS sequence from smut genome, was achieved and used as targets in the present microarray assay. Based on smut infection samples from smut-susceptible sugarcane genotype YC71-374 and smut-resistant sugarcane genotype NCo376, the hybridization was conducted and validated by real-time fluorescent quantitative PCR. It would provide a basic data for the study on sugarcane-smut interaction mechanism, which referred to sugarcane smut resistance and smut pathogenesis.
Project description:Sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs) are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out. Sixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements. Individual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNA's that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome.
Project description:During B cell development the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin k light chain (Igk) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vk transcription. To investigate whether pre-BCR signaling modulates Vk accessibility through enhancer-mediated Igk locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the k enhancers robustly interact with the ~3.2 Mb Vk region and its flanking sequences. Analyses in wild-type, Btk and Slp65 single and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igk locus flanking sequences and increases interactions of the 3â??k enhancer with Vk genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vk genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vk genes, which are often marked by transcription factor E2a. We conclude that the k enhancers interact with the Vk region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vk region, whereby the two enhancers play distinct roles. We performed genome-wide expression profiling of FACS-purified B220+CD19+ pre-B cell fractions from wild-type (WT), Btk and Slp65 single and double deficient VH81x transgenic Rag1-/- mice (n=4 of each genotype). In these experiments non-VH81x transgenic Rag1-/- pro-B cells served as controls (n=3).
Project description:Small RNAs are a class of noncoding RNAs which are of great importance in gene expression regulatory networks. Different families of small RNAs are generated via distinct biogenesis pathways. One such family specific to plants is that of phased, secondary siRNAs (phasiRNAs); these require RDR6, DCL4, and microRNA (miRNA) trigger for their biogenesis. Protein-encoding genes are an important source of phasiRNAs. The model legume Medicago truncatula generates phasiRNAs from many PHAS loci, and we aimed to investigate their biogenesis and mechanism by which miRNAs trigger these molecules. We modulated miRNA abundances in transgenic tissues showing that the abundance of phasiRNAs correlates with the levels of both miRNA triggers and the target, precursor transcripts. We identified sets of phasiRNAs or PHAS loci that predominantly and substantially increase in response to miRNA overexpression. In the process of validating targets from miRNA overexpression tissues, we found that in the miRNA-mRNA target pairing, the 3’ terminal nucleotide (the 22nd position), but not the 10th position, is important for phasiRNA production. Mutating the single 3’ terminal nucleotide dramatically diminishes phasiRNA production. Ectopic expression of Medicago NB-LRR-targeting miRNAs in Arabidopsis showed that only a few NB-LRRs are capable of phasiRNA production; our data indicate that this is due to target inaccessibility determined by sequences flanking target sites. We propose that target accessibility is an important component of miRNA-target interactions that could be utilized in target prediction, and the evolution of mRNA sequences flanking miRNA target sites may be impacted.
Project description:The soldier fly is an endemic pest of sugarcane in Australia. Small numbers of larvae can cause significant damage to roots and reduce the crop yields. Little is known about the composition and function of the soldier fly salivary gland, its secretions, and their roles in insect-plant interactions. In this study, we performed transcriptome analysis of the salivary glands of starved and sugarcane root-fed soldier fly larvae. A total of 31,119 highly expressed assembled contigs were identified in the salivary glands and almost 50% of them showed high levels of similarity to known proteins in Nr databases. Of all the obtained contigs, only 9,727 sequences contain an open reading frame of over 100 amino acids. Around 31% of contigs were predicted to encode secretory proteins, including some digestive and detoxifying enzymes and potential effectors. Some known salivary secreted peptides such as serine protease, cysteine proteinase inhibitors, antimicrobial peptides and venom proteins were among the top 100 highly expressed genes. Differential gene expression analysis revealed significant modulation of 850 transcripts in salivary glands upon exposure to plant roots or starvation stress. Here, we identified some venom proteins which were significantly upregulated in the salivary glands of soldier fly larvae exposed to sugarcane roots. In other insects and nematodes some of these proteins have been used to manipulate host plant defense systems and facilitate the invasion of the host plant. These findings provide a further insight into the identification of potential effector proteins involved in soldier fly- sugarcane interactions.
Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).