RNA-seq of coding RNA of moss Funaria hygrometrica samples from three haploid (gametophyte) stages and three diploid (sporophyte) stages
ABSTRACT: We experimentally generated a genome-wide gene expression data. To this end, we first collected samples of three important haploid developmental stages, specifically germinating spores (gametophyte_1), protonemata (gametophyte_2) and young gametophores (gametophyte_3) (four biological replicates each). We also collected three developmental stages of the diploid phase (sporophyte), specifically sporophytes shorter than 5mm (sporophyte_1), elongated needle-like sporophytes (sporophyte_2), and sporophytes with swollen capsules (sporophyte_3)
In diploid organisms, selfing reduces the efficiency of selection in removing deleterious mutations from a population. This need not be the case for all organisms. Some plants, for example, undergo an extreme form of selfing known as intragametophytic selfing, which immediately exposes all recessive deleterious mutations in a parental genome to selective purging. Here, we ask how effectively deleterious mutations are removed from such plants. Specifically, we study the extent to which deleteriou ...[more]
Project description:Bryophytes are the most basal of the extant land plants. A major feature of these plants is the biphasic alteration of generations between a dominant haploid gametophyte and a minor diploid sporophyte phase. To analyse the differences in the transcriptome of the early gametophyte (protonema) and early and mid-sporophyte phases of the moss Physcomitrella patens, microarray gene expression profiles were performed using dissected sporophyte tissue. Through further analysis the early and mid-sporophyte phases were compared. RNA isolated from the Gametophytic protonemal tissue was hybridised to six microarrays. Each microarray was hybridised with RNA from a separate biological replicate. Three of these microarrays were co-hybridised with RNA isolated from early sporophytes. With the third gametophyte biological replicate and early sporophyte replicate a dye swap was carried out. The remaining three microarrays hybridised with RNA from the gametophytes were co-hybridised with RNA from mid-sporophytic tissue. A dye swap was carried out on the sixth gametophyte replicate and third mid-sporophyte replicate.To meet the quality requirements for the microarray experiment, at least 400 sporophytes were used per extraction. Three or four RNA extracts were then pooled for further precipitation to maximise purity and concentration. Up to 1600 sporophytes were harvested to prepare sufficient RNA for each microarray replicate. In bioinformatic analysis the channels were split into individual channels and the early and mid-sporophyte were compared.
Project description:To explore possible interactive effects of UV-radiation, temperature and growth conditions, cultivated and field sporophytes of Saccharina latissima were exposed for 24h to UV-radiation at three different temperatures (2,7 & 12°C). Gene expression profiles under UV-radiation at different temperatures were assessed through microarray hybridizations, afterwards comparisons of gene expression profiles in field and culture sporophytes were carried out.
Project description:Sporophytes of Saccharina latissima were exposed for two weeks to 12 different combinations of photosynthetically active radiation and UVR at three different temperatures (2,7,12 C). Maximum quantum yield of photosystem II was determined twice a week during the experimental duration for observing the extent of photoinhibition. For investigating molecular mechanisms of acclimation to high photosynthetically radiation, and UVR gene expression profiles were assessed through microarray hybridizations.
Project description:The aim of this experiment was to compare transcript abundances in parthenotes (i.e. organisms derived by parthenogenetic development of gametes) of two life cycle mutants of the brown alga Ectocarpus siliculosus with transcript abundances in the wild type sporophyte and gametophyte generations. This is of interest because the two mutations, immediate upright (imm) and ouroboros (oro), cause partial and almost complete hometic conversion, respectively, of the sporophyte into the gametophyte. imm parthenotes exhibit gametophyte-like morphology during early development but remain sporophytes in functional terms (they do not produce gametes) whereas oro parthenotes behave as functional gametophytes and are morphologically indistinguishable from gametophytes apart from the appearance some minor sporophyte-like features very early in development in some individuals. To minimise genetic background effects the samples for this experiment were derived from a segregating population derived from an imm/IMM oro/ORO sporophyte. Four classes of gametophyte were derived from this sporophyte were IMM/ORO (wild type), imm/ORO, IMM/oro and imm/oro. Parthenomes were bulked to provide a wild type sporophyte sample, samples corresponding to the two individual mutants, plus the double mutant. A wild type gametophyte sample was also compared for comparison. Hybridisations with cDNA derived from these five samples were carried out using a NimbleGen expressed-sequence-tag-(EST-)based microarray carrying probes corresponding to 10,600 of the 16,256 genes identified in the Ectocarpus genome.
Project description:The aim of this experiment was to analyse the expression of two sets of genes identified as being putatively sporophyte-specific or gametophyte-specific by a suppressive subtraction hybridisation using cDNA from immature sporophytes and immature gametophytes of the Ectocarpus strain Esil32. The expression of these genes was analysed in the sporophyte and gametophyte generations of the life cycle (again using immature algae that had not yet produces zoidangia) and in the sporophyte generation of a mutant strain, immediate upright, that exhibits gametophyte-like characteristics during the sporophyte generation.
Project description:Effect of PAR and temperature stress on the gene expression Saccharina latissima. Total RNA of stress treatments (low PAR 2° and 17°C, high PAR 2° and 17°C) was hybridized against the control treatment (low PAR 12°C); hybridizations were carried out in 4 replicates.
Project description:Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis. For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a “species-specific transcriptome database” (SSTDB). In total 1397 protein clusters with 5865 unique peptide sequences were identified (13 decoy proteins out of 1410, protFDR 0.93% on protein cluster level). We show that using a “species-specific transcriptome database” for protein identification increases the number of identified peptides almost four times compared to using only the publically available viridiplantae database. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy.
Project description:The experiment was aimed at identification of genes whose expression is up- or down-regulated in tobacco plants in response to sulfur (S) deficiency. Comparison of response to S deficit of LA Burley 21 line and AB3 line (overexpressing UP9C in antisense orientation) was done to clarify the function of LSU/UP9 family genes.