Project description:Polyploidization, the increase in genome copies, is considered a major driving force for speciation. We have recently provided the first direct in planta evidence for polyspermy induced polyploidization. Capitalizing on a novel sco1-based polyspermy assay, we here show that polyspermy can selectively polyploidize the egg cell, while rendering the genome size of the ploidy-sensitive central cell unaffected. This unprecedented result indicates that polyspermy can bypass the triploid block, which is an established postzygotic polyploidization barrier. In fact, we here show that most polyspermy-derived seeds are insensitive to the triploid block suppressor admetos. The robustness of polyspermy-derived plants is evidenced by the first transcript profiling of triparental plants and our observation that these idiosyncratic organisms segregate tetraploid offspring within a single generation. Polyspermy-derived triparental plants are thus comparable to triploids recovered from interploidy crosses. Our results expand current polyploidization concepts and have important implications for plant breeding.
Project description:Hybridizations of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. Unreduced diploid gametes generated by the omission of second division1 (osd1) mutant induce the triploid block, similar as tetraploid (4x) plants. We recently found that mutations in NRPD1, encoding the largest subunit of the plant-specific RNA Polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates small RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. Strikingly however, mutations in other components of the RdDM pathway like RDR2 and NRPE1 fail to suppress the triploid block when inherited in the osd1 background, but have a suppressive effect as 4x mutants. In this study, we aimed at understanding the cause for this discrepancy. We found that the ability of mutants in the RdDM pathway to suppress the triploid block depends on their degree of inbreeding. While nrpd1 is able to suppress in the first homozygous generation, mutants in RDR2, NRPE1, and DRM2 require at least one additional round of inbreeding to exert a suppressive effect. Our data thus reveal that loss of RdDM function differs in its effect in early and late generations and that Pol IV acts at an early stage of triploid block establishment.
Project description:Polyploidy, the presence of more than two sets of chromosomes within the nucleus, is a common phenomenon among plants that has shaped genome organization and is thought to be a major driver of speciation. The triploid block acts as a reproductive barrier that prevents successful backcrosses of newly formed polyploids with their progenitors. It is established in the endosperm, an ephemeral tissue that nurtures the developing embryo. Here we show that paternal 21/22 nucleotide epigenetically activated small interfering RNAs (easiRNAs) in Arabidopis thaliana are responsible for the establishment of the triploid block associated seed abortion. This dramatic phenotype is overcome when crosses are performed with a paternal mutant in the plant specific RNA polymerase IV (Pol IV). Loss of Pol IV reduces both easiRNAs and hetsiRNAs in the pollen grain. Seeds derived from crosses between wild type tetraploid fathers and diploid mothers have reduced levels of hetsiRNAs and cytosine methylation at transposable elements (TEs), indicating that they have suffered an excess of epigenetic reprogramming due to an excess of paternally derived easiRNAs. Paternal Pol IV mutants are able to buffer that easiRNA excess and rescue the abortion phenotype by rescuing both the cytosine methylation level at TEs and the 24 nt hetsiRNA accumulation levels. This mechanism highlights the elegant and important TE regulation mechanism controlled by the plant specific Pol IV that takes place pre-fertilization and regulates paternal easiRNAs and maternal hetsiRNAs, and determines the post-fertilization epigenetic stability and seed viability.
Project description:Dosage compensation restores a balanced network of gene expression between autosomes and sex chromosomes in males (XY) and females (XX). In mammals, this is achieved by doubling the expression of X-linked genes in both sexes, together with X inactivation in females. X up-regulation may be controlled by DNA sequence based and/or epigenetic mechanisms that double the X output potentially in response to an autosomal counting factor. Human triploids with either one or two active X chromosomes (Xa) provide a mean to test X chromosome expression in the presence of three sets of autosomes, which will help understand the underlying mechanisms of X up-regulation. We measured whole genome gene expression in human triploid cell cultures with either one or two active X. We found that overall X-linked gene expression is not tripled in the presence of three sets of autosomes. However, in triploid cells with a single active X chromosome, its expression is adjusted upward, presumably by an epigenetic mechanism that senses the active X-autosome ratio. Six human XXX triploid fibroblast clones with either one or two active X, three XYY triploid fibroblast cultures, and two male (XY) and two female (XaXi) control diploid fibroblast cultures were selected for RNA extraction and hybridization on Affymetrix whole genome expression arrays (HG-U133 2.0 plus chip). Probe labeling, array hybridization and scanning were done by the University of Washington Microarray Center.
Project description:To avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.
Project description:To examain effects of triploidy on gene expression in mouse cells, we have employed whole genome microarray expression profiling. To study the effects of aneuploidy in mouse cells, we isorated diploid, triploid and tetraploid cells from p53-/- mouse embryonic cells Gene expression in diploid cells (RRI2, 15 and 2-1), triploid cells (RRI2-5, 2-12 and 2-15) and tetraploid cells (RRI2-2, 2-4 and 2-6).