Mitochondrial genomes of the genus Ephydatia Lamouroux, 1816: can palindromic elements be used in species-level studies?

Poriferan mitochondrial DNA (mtDNA), especially large intergenic regions, is a target for the insertion of repetitive hairpin-forming elements. These elements are responsible for the large mt genome size differences observed even among closely related sponge taxa. In this study, we present the new, nearly complete, mt genome sequence of Ephydatia fluviatilis and compare it with previously published mt genomes of freshwater sponges. Special emphasis was placed on comparison with the closely related species Ephydatia muelleri, thereby comparing the only two species of the genus Ephydatia on the western Balkan Peninsula. In particular, we analyzed repetitive palindromic elements within the mitochondrial intergenic regions. The genomic distribution of these repetitive elements was analyzed and their potential role in the evolution of mt genomes discussed. We show here that palindromic elements are widespread through the whole mt genome, including the protein coding genes, thus introducing genetic variability into mt genomes.     

Analysis of 3D genomic interactions identifies candidate host genes that transposable elements potentially regulate

Background

The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation.

Results

Using PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes.

Conclusions

4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.

     

Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.     

CRISPR Double Cutting through the Labyrinthine Architecture of 3D Genomes

The genomes are organized into ordered and hierarchical topological structures in interphase nuclei.Within discrete territories of each chromosome,topologically associated domains(TADs) play important roles in various nuclear processes such as gene regulation.Inside TADs separated by relatively constitutive boundaries,distal elements regulate their gene targets through specific chromatin-looping contacts such as long-distance enhancer-promoter interactions.High-throughput sequencing studies have revealed millions of potential regulatory DNA elements,which are much more abundant than the mere ~ 20,000 genes they control.The recently emerged CRISPRCas9 genome editing technologies have enabled efficient and precise genetic and epigenetic manipulations of genomes.The multiplexed and high-throughput CRISPR capabilities facilitate the discovery and dissection of gene regulatory elements.Here,we describe the applications of CRISPR for genome,epigenome,and 3D genome editing,focusing on CRISPR DNA-fragment editing with Cas9 and a pair of sgRNAs to investigate topological folding of chromatin TADs and developmental gene regulation.     

The landscape of transposable elements in the finished genome of the fungal wheat pathogen Mycosphaerella graminicola

Background

In addition to gene identification and annotation, repetitive sequence analysis has become an integral part of genome sequencing projects. Identification of repeats is important not only because it improves gene prediction, but also because of the role that repetitive sequences play in determining the structure and evolution of genes and genomes. Several methods using different repeat-finding strategies are available for whole-genome repeat sequence analysis. Four independent approaches were used to identify and characterize the repetitive fraction of the Mycosphaerella graminicola (synonym Zymoseptoria tritici) genome. This ascomycete fungus is a wheat pathogen and its finished genome comprises 21 chromosomes, eight of which can be lost with no obvious effects on fitness so are dispensable.

Results

Using a combination of four repeat-finding methods, at least 17% of the M. graminicola genome was estimated to be repetitive. Class I transposable elements, that amplify via an RNA intermediate, account for about 70% of the total repetitive content in the M. graminicola genome. The dispensable chromosomes had a higher percentage of repetitive elements as compared to the core chromosomes. Distribution of repeats across the chromosomes also varied, with at least six chromosomes showing a non-random distribution of repetitive elements. Repeat families showed transition mutations and a CpA → TpA dinucleotide bias, indicating the presence of a repeat-induced point mutation (RIP)-like mechanism in M. graminicola. One gene family and two repeat families specific to subtelomeres also were identified in the M. graminicola genome. A total of 78 putative clusters of nested elements was found in the M. graminicola genome. Several genes with putative roles in pathogenicity were found associated with these nested repeat clusters. This analysis of the transposable element content in the finished M. graminicola genome resulted in a thorough and highly curated database of repetitive sequences.

Conclusions

This comprehensive analysis will serve as a scaffold to address additional biological questions regarding the origin and fate of transposable elements in fungi. Future analyses of the distribution of repetitive sequences in M. graminicola also will be able to provide insights into the association of repeats with genes and their potential role in gene and genome evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1132) contains supplementary material, which is available to authorized users.     

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.     

MicroRNA genes derived from repetitive elements and expanded by segmental duplication events in mammalian genomes

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression by targeting mRNAs for translation repression or mRNA degradation. Many miRNAs are being discovered and studied, but in most cases their origin, evolution and function remain unclear. Here, we characterized miRNAs derived from repetitive elements and miRNA families expanded by segmental duplication events in the human, rhesus and mouse genomes. We applied a comparative genomics approach combined with identifying miRNA paralogs in segmental duplication pair data in a genome-wide study to identify new homologs of human miRNAs in the rhesus and mouse genomes. Interestingly, using segmental duplication pair data, we provided credible computational evidence that two miRNA genes are located in the pseudoautosomal region of the human Y chromosome. We characterized all the miRNAs whether they were derived from repetitive elements or not and identified significant differences between the repeat-related miRNAs (RrmiRs) and non-repeat-derived miRNAs in (1) their location in protein-coding and intergenic regions in genomes, (2) the minimum free energy of their hairpin structures, and (3) their conservation in vertebrate genomes. We found some lineage-specific RrmiR families and three lineage-specific expansion families, and provided evidence indicating that some RrmiR families formed and expanded during evolutionary segmental duplication events. We also provided computational and experimental evidence for the functions of the conservative RrmiR families in the three species. Together, our results indicate that repetitive elements contribute to the origin of miRNAs, and large segmental duplication events could prompt the expansion of some miRNA families, including RrmiR families. Our study is a valuable contribution to the knowledge of evolution and function of non-coding region in genome.     

Unique profile of ordered arrangements of repetitive elements in the C57BL/6J mouse genome implicating their functional roles

The entirety of all protein coding sequences is reported to represent a small fraction (~2%) of the mouse and human genomes; the vast majority of the rest of the genome is presumed to be repetitive elements (REs). In this study, the C57BL/6J mouse reference genome was subjected to an unbiased RE mining to establish a whole-genome profile of RE occurrence and arrangement. The C57BL/6J mouse genome was fragmented into an initial set of 5,321 units of 0.5 Mb, and surveyed for REs using unbiased self-alignment and dot-matrix protocols. The survey revealed that individual chromosomes had unique profiles of RE arrangement structures, named RE arrays. The RE populations in certain genomic regions were arranged into various forms of complexly organized structures using combinations of direct and/or inverse repeats. Some of these RE arrays spanned stretches of over 2 Mb, which may contribute to the structural configuration of the respective genomic regions. There were substantial differences in RE density among the 21 chromosomes, with chromosome Y being the most densely populated. In addition, the RE array population in the mouse chromosomes X and Y was substantially different from those of the reference human chromosomes. Conversion of the dot-matrix data pertaining to a tandem 13-repeat structure within the Ch7.032 genome unit into a line map of known REs revealed a repeat unit of ~11.3 Kb as a mosaic of six different RE types. The data obtained from this study allowed for a comprehensive RE profiling, including the establishment of a library of RE arrays, of the reference mouse genome. Some of these RE arrays may participate in a spectrum of normal and disease biology that are specific for mice.     

The repetitive component of the A genome of peanut (Arachis hypogaea) and its role in remodelling intergenic sequence space since its evolutionary divergence from the B genome

Background and Aims

Peanut (Arachis hypogaea) is an allotetraploid (AABB-type genome) of recent origin, with a genome of about 2·8 Gb and a high repetitive content. This study reports an analysis of the repetitive component of the peanut A genome using bacterial artificial chromosome (BAC) clones from A. duranensis, the most probable A genome donor, and the probable consequences of the activity of these elements since the divergence of the peanut A and B genomes.

Methods

The repetitive content of the A genome was analysed by using A. duranensis BAC clones as probes for fluorescence in situ hybridization (BAC-FISH), and by sequencing and characterization of 12 genomic regions. For the analysis of the evolutionary dynamics, two A genome regions are compared with their B genome homeologues.

Key Results

BAC-FISH using 27 A. duranensis BAC clones as probes gave dispersed and repetitive DNA characteristic signals, predominantly in interstitial regions of the peanut A chromosomes. The sequences of 14 BAC clones showed complete and truncated copies of ten abundant long terminal repeat (LTR) retrotransposons, characterized here. Almost all dateable transposition events occurred <3·5 million years ago, the estimated date of the divergence of A and B genomes. The most abundant retrotransposon is Feral, apparently parasitic on the retrotransposon FIDEL, followed by Pipa, also non-autonomous and probably parasitic on a retrotransposon we named Pipoka. The comparison of the A and B genome homeologous regions showed conserved segments of high sequence identity, punctuated by predominantly indel regions without significant similarity.

Conclusions

A substantial proportion of the highly repetitive component of the peanut A genome appears to be accounted for by relatively few LTR retrotransposons and their truncated copies or solo LTRs. The most abundant of the retrotransposons are non-autonomous. The activity of these retrotransposons has been a very significant driver of genome evolution since the evolutionary divergence of the A and B genomes.     

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55–83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.     

Les BIMEs: un exemple de séquences répétées chez les entérobactéries

Bacterial interspersed mosaic elements (or BIMEs) are repeated sequences identified in several enterobacterial genomes. BIMEs are a mosaic combination of small sequence motifs. It has been estimated that 500 BIMEs are scattered over the bacterial genome. BIMEs have been identified in several enterobacteria: Escherichia coli, Salmonella typhimurium, Klebsiella sp. and relatives of these bacteria. BIME function is not known, but their interactions with specific proteins (DNA polymerase I, gyrase and integration host factor) suggest that they could be involved in functional organization of bacterial chromosomes. Four other families of interspersed repetitive sequences have been shown to exist in a variety of bacterial genomes. Like BIMEs, these sequences are rather small, contain a region of dyad symmetry, and are found in extragenic locations. Unlike BIMEs, IRU (or ERIC), box C sequences and RSA sequences occur in enterobacteria but also in other Gram-negative bacteria.     

Identification and characterization of repetitive DNA in the genus Didelphis Linnaeus, 1758 (Didelphimorphia,Didelphidae) and the use of satellite DNAs as phylogenetic markers

Didelphis species have been shown to exhibit very conservative karyotypes, which mainly differ in their constitutive heterochromatin, known to be mostly composed by repetitive DNAs. In this study, we used genome skimming data combined with computational pipelines to identify the most abundant repetitive DNA families of Lutreolina crassicaudata and all six Didelphis species. We found that transposable elements (TEs), particularly LINE-1, endogenous retroviruses, and SINEs, are the most abundant mobile elements in the studied species. Despite overall similar TE proportions, we report that species of the D. albiventris group consistently present a less diverse TE composition and smaller proportions of LINEs and LTRs in their genomes than other studied species. We also identified four new putative satDNAs (sat206, sat907, sat1430 and sat2324) in the genomes of Didelphis species, which show differences in abundance and nucleotide composition. Phylogenies based on satDNA sequences showed well supported relationships at the species (sat1430) and groups of species (sat206) level, recovering topologies congruent with previous studies. Our study is one of the first attempts to present a characterization of the most abundant families of repetitive DNAs of Lutreolina and Didelphis species providing insights into the repetitive DNA composition in the genome landscape of American marsupials.