Project description:De novo centromeres originate occasionally from non-centromeric regions of chromosomes, providing an excellent model system to study centromeric chromatin. The maize mini-chromosome Derivative 3-3 contains a de novo centromere, which was derived from a euchromatic site on the short arm of chromosome 9 that lacks traditional centromeric repeat sequences. Our previous study found that the CENH3 binding domain of this de novo centromere is only 288 kb with a high-density gene distribution with low-density of transposons. Here we applied next generation sequencing technology to analyze gene transcription, DNA methylation for this region. Our RNA-seq data revealed that active chromatin is not a barrier for de novo centromere formation. Bisulfite-ChIP-seq results indicate a slightly increased DNA methylation level after de novo centromere formation, reaching the level of a native centromere. These results provide insight into the mechanism of de novo centromere formation and subsequent consequences. RNA-seq was carried out using material from seedling and young leaves between control and Derivative 3-3. Bisulfite-ChIP-seq was carried out with anti-CENH3 antibodies using material from young leaves in Derivative 3-3.
Project description:We combined an iTRAQ-based proteome-level analysis with an RNA sequencing-based transcriptome-level analysis to detect the proteins and genes related to fruit peel colour development during two fruit development stages in the ‘Tunisia’ and ‘White’ pomegranate cultivars.
Project description:In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge, the necessity to erase and reset genomic methylation. In the male germline RNA-directed DNA methylation silences young active transposable elements (TEs). The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) are proposed to tether MIWI2 to nascent TE transcripts and instruct DNA methylation. The mechanism by which MIWI2 directs de novo TE methylation is poorly understood but central to the immortality of the germline. Here, we define the interactome of MIWI2 in foetal gonocytes that are undergoing de novo genome methylation and identify a novel MIWI2-associated factor, SPOCD1, that is essential for young TE methylation and silencing. The loss of Spocd1 in mice results in male specific infertility and does not impact on piRNA biogenesis nor localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein and its expression is restricted to the period of de novo genome methylation. We found SPOCD1 co-purified in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery as well as constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent TE transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through its association with SPOCD1. In summary, we have identified a novel and essential executor of mammalian piRNA-directed DNA methylation.
Project description:Rosa roxburghii Tratt belongs to the Rosaceae family, and the fruit is flavorful, economic, and high nutritious, providing health benefits. MYB proteins play key roles in R. roxburghii’ development and fruit quality. However, the available genomic and transcriptomic information are extremely deficient. Here, a normalized cDNA library was constructed using five tissues, stem, leaf, flower, young fruit, and mature fruit, with three repetitions, and sequenced using the Illumina HiSeq 3000 platform. De novo assembly was performed, and 470.66 million clean reads were obtained. In total, 63,727 unigenes, with an average GC content of 42.08%, were determined and 59,358 were annotated. In addition, 9,354 unigenes were assigned the Gene Ontology category, and 20,202 unigenes were assigned to 25 Eukaryotic Ortholog Groups. Additionally, 19,507 unigenes were classified into 140 pathways of the Kyoto Encyclopedia of Genes and Genomes database. Using the transcriptome, 163 unigenes associated with MYB were detected. Among these genes, there were total 75 genes which strikingly expressed in various tissues, including 10 R1 MYB, 42 R2R3 MYB, 1 R1R2R3 MYB, 3 MYB and 19 atypical MYB-like proteins. The expression levels of 12 MYB genes randomly selected for qRT-PCR analysis were consistent with the RNA-seq results. A total of 37,545 microsatellites were detected, with an average EST-SSR frequency of 0.59 (37,545/63,727). This transcriptome data will be valuable for identifying genes of interest and studying their expression and evolution.
Project description:De novo peptide sequencing is a fundamental research area in mass spectrometry (MS) based proteomics. However, those methods have often been evaluated using a couple of simple metrics that do not fully reflect their overall performance. Moreover, there has not been an established method to estimate the false discovery rate (FDR) and the significance of de novo peptide-spectrum matches (PSMs). Here we propose NovoBoard, a comprehensive framework to evaluate the performance of de novo peptide sequencing methods. The framework consists of diverse benchmark datasets (including tryptic, nontryptic, immunopeptidomics, and different species), and a standard set of accuracy metrics to evaluate the fragment ions, amino acids, and peptides of the de novo results. More importantly, a new approach is designed to evaluate de novo peptide sequencing methods on target-decoy spectra and to estimate their FDRs. Our results thoroughly reveal the strengths and weaknesses of different de novo peptide sequencing methods, and how their performances depend on specific applications and the types of data. Our FDR estimation also shows that some tools may perform better than the others in distinguishing between de novo PSMs and random matches, and can be used to assess the significance of de novo PSMs.
Project description:In order to understand the mechanism of single-cell C4 photosynthesis, we extracted total RNA from leaf tissues at young, intermediate, and mature stages. We then conducted gene expression profiling using RNA-seq and de novo transcriptome assembly. The gene expression data was normalized using Transcript Per Million, which was additionally adjusted by the Trimmed Mean of the M values (TPMTMM).