Assembly of the 81.6 Mb centromere of pea chromosome 6 elucidates the structure and evolution of metapolycentric chromosomes.

Centromeres in the legume genera Pisum and Lathyrus exhibit unique morphological characteristics, including extended primary constrictions and multiple separate domains of centromeric chromatin. These so-called metapolycentromeres resemble an intermediate form between monocentric and holocentric types, and therefore provide a great opportunity for studying the transitions between different types of centromere organizations. However, because of the exceedingly large and highly repetitive nature of metapolycentromeres, highly contiguous assemblies needed for these studies are lacking. Here, we report on the assembly and analysis of a 177.6 Mb region of pea (Pisum sativum) chromosome 6, including the 81.6 Mb centromere region (CEN6) and adjacent chromosome arms. Genes, DNA methylation profiles, and most of the repeats were uniformly distributed within the centromere, and their densities in CEN6 and chromosome arms were similar. The exception was an accumulation of satellite DNA in CEN6, where it formed multiple arrays up to 2 Mb in length. Centromeric chromatin, characterized by the presence of the CENH3 protein, was predominantly associated with arrays of three different satellite repeats; however, five other satellites present in CEN6 lacked CENH3. The presence of CENH3 chromatin was found to determine the spatial distribution of the respective satellites during the cell cycle. Finally, oligo-FISH painting experiments, performed using probes specifically designed to label the genomic regions corresponding to CEN6 in Pisum, Lathyrus, and Vicia species, revealed that metapolycentromeres evolved via the expansion of centromeric chromatin into neighboring chromosomal regions and the accumulation of novel satellite repeats. However, in some of these species, centromere evolution also involved chromosomal translocations and centromere repositioning.

On the Origin of Neo-Sex Chromosomes in the Neotropical Dragonflies Rhionaeschna bonariensis and R. planaltica (Aeshnidae, Odonata).

Odonata have holokinetic chromosomes. About 95% of species have an XX/X0 sex chromosome system, with heterogametic males. There are species with neo-XX/neo-XY sex chromosomes resulting from an X chromosome/autosome fusion. The genus Rhionaeschna includes 42 species found in the Americas. We analyzed the distribution of the nucleolar organizer region (NOR) using FISH with rDNA probes in Rhionaeschna bonariensis (n = 12 + neo-XY), R. planaltica (n = 7 + neo-XY), and Aeshna cyanea (n = 13 + X0). In R. bonariensis and A. cyanea, the NOR is located on a large pair of autosomes, which have a secondary constriction in the latter species. In R. planaltica, the NOR is located on the ancestral part of the neo-X chromosome. Meiotic analysis and FISH results in R. planaltica led to the conclusion that the neo-XY system arose by insertion of the ancestral X chromosome into an autosome. Genomic in situ hybridization, performed for the first time in Odonata, highlighted the entire neo-Y chromosome in meiosis of R. bonariensis, suggesting that it consists mainly of repetitive DNA. This feature and the terminal chiasma localization suggest an ancient origin of the neo-XY system. Our study provides new information on the origin and evolution of neo-sex chromosomes in Odonata, including new types of chromosomal rearrangements, NOR transposition, and heterochromatin accumulation.

Extraordinary Sequence Diversity and Promiscuity of Centromeric Satellites in the Legume Tribe Fabeae.

Satellite repeats are major sequence constituents of centromeres in many plant and animal species. Within a species, a single family of satellite sequences typically occupies centromeres of all chromosomes and is absent from other parts of the genome. Due to their common origin, sequence similarities exist among the centromere-specific satellites in related species. Here, we report a remarkably different pattern of centromere evolution in the plant tribe Fabeae, which includes genera Pisum, Lathyrus, Vicia, and Lens. By immunoprecipitation of centromeric chromatin with CENH3 antibodies, we identified and characterized a large and diverse set of 64 families of centromeric satellites in 14 species. These families differed in their nucleotide sequence, monomer length (33-2,979 bp), and abundance in individual species. Most families were species-specific, and most species possessed multiple (2-12) satellites in their centromeres. Some of the repeats that were shared by several species exhibited promiscuous patterns of centromere association, being located within CENH3 chromatin in some species, but apart from the centromeres in others. Moreover, FISH experiments revealed that the same family could assume centromeric and noncentromeric positions even within a single species. Taken together, these findings suggest that Fabeae centromeres are not shaped by the coevolution of a single centromeric satellite with its interacting CENH3 proteins, as proposed by the centromere drive model. This conclusion is also supported by the absence of pervasive adaptive evolution of CENH3 sequences retrieved from Fabeae species.

A reference genome for pea provides insight into legume genome evolution.

We report the first annotated chromosome-level reference genome assembly for pea, Gregor Mendel's original genetic model. Phylogenetics and paleogenomics show genomic rearrangements across legumes and suggest a major role for repetitive elements in pea genome evolution. Compared to other sequenced Leguminosae genomes, the pea genome shows intense gene dynamics, most likely associated with genome size expansion when the Fabeae diverged from its sister tribes. During Pisum evolution, translocation and transposition differentially occurred across lineages. This reference sequence will accelerate our understanding of the molecular basis of agronomically important traits and support crop improvement.

Satellite DNA in Vicia faba is characterized by remarkable diversity in its sequence composition, association with centromeres, and replication timing.

Satellite DNA, a class of repetitive sequences forming long arrays of tandemly repeated units, represents substantial portions of many plant genomes yet remains poorly characterized due to various methodological obstacles. Here we show that the genome of the field bean (Vicia faba, 2n = 12), a long-established model for cytogenetic studies in plants, contains a diverse set of satellite repeats, most of which remained concealed until their present investigation. Using next-generation sequencing combined with novel bioinformatics tools, we reconstructed consensus sequences of 23 novel satellite repeats representing 0.008-2.700% of the genome and mapped their distribution on chromosomes. We found that in addition to typical satellites with monomers hundreds of nucleotides long, V. faba contains a large number of satellite repeats with unusually long monomers (687-2033 bp), which are predominantly localized in pericentromeric regions. Using chromatin immunoprecipitation with CenH3 antibody, we revealed an extraordinary diversity of centromeric satellites, consisting of seven repeats with chromosome-specific distribution. We also found that in spite of their different nucleotide sequences, all centromeric repeats are replicated during mid-S phase, while most other satellites are replicated in the first part of late S phase, followed by a single family of FokI repeats representing the latest replicating chromatin.

Karyotype Variability and Inter-Population Genomic Differences in Freshwater Ostracods (Crustacea) Showing Geographical Parthenogenesis.

Transitions from sexual to asexual reproduction are often associated with polyploidy and increased chromosomal plasticity in asexuals. We investigated chromosomes in the freshwater ostracod species Eucypris virens (Jurine, 1820), where sexual, asexual and mixed populations can be found. Our initial karyotyping of multiple populations from Europe and North Africa, both sexual and asexual, revealed a striking variability in chromosome numbers. This would suggest that chromosomal changes are likely to be accelerated in asexuals because the constraints of meiosis are removed. Hence, we employed comparative genomic hybridization (CGH) within and among sexual and asexual populations to get insights into E. virens genome arrangements. CGH disclosed substantial genomic imbalances among the populations analyzed, and three patterns of genome arrangement between these populations: 1. Only putative ribosomal DNA (rDNA)-bearing regions were conserved in the two populations compared indicating a high sequence divergence between these populations. This pattern is comparable with our findings at the interspecies level of comparison; 2. Chromosomal regions were shared by both populations to a varying extent with a distinct copy number variation in pericentromeric and presumable rDNA-bearing regions. This indicates a different rate of evolution in repetitive sequences; 3. A mosaic pattern of distribution of genomic material that can be explained as non-reciprocal genetic introgression and evidence of a hybrid origin of these individuals. We show an overall increased chromosomal dynamics in E. virens that is complementary with available phylogenetic and population genetic data reporting highly differentiated diploid sexual and asexual lineages with a wide variety of genetic backgrounds.

TAREAN: a computational tool for identification and characterization of satellite DNA from unassembled short reads.

Satellite DNA is one of the major classes of repetitive DNA, characterized by tandemly arranged repeat copies that form contiguous arrays up to megabases in length. This type of genomic organization makes satellite DNA difficult to assemble, which hampers characterization of satellite sequences by computational analysis of genomic contigs. Here, we present tandem repeat analyzer (TAREAN), a novel computational pipeline that circumvents this problem by detecting satellite repeats directly from unassembled short reads. The pipeline first employs graph-based sequence clustering to identify groups of reads that represent repetitive elements. Putative satellite repeats are subsequently detected by the presence of circular structures in their cluster graphs. Consensus sequences of repeat monomers are then reconstructed from the most frequent k-mers obtained by decomposing read sequences from corresponding clusters. The pipeline performance was successfully validated by analyzing low-pass genome sequencing data from five plant species where satellite DNA was previously experimentally characterized. Moreover, novel satellite repeats were predicted for the genome of Vicia faba and three of these repeats were verified by detecting their sequences on metaphase chromosomes using fluorescence in situ hybridization.