Distinct patterns of satDNA distribution in holocentric chromosomes of spike-sedges (Eleocharis, Cyperaceae).

Eleocharis R. Br. (Cyperaceae) species are known for having holocentric chromosomes, which enable rapid karyotype differentiation. High intra- and interspecific variations in chromosome numbers and genome sizes are documented for different Eleocharis species, frequently accompanied by fluctuations in the repetitive DNA fraction. However, a lack of detailed analysis has hampered a better understanding of the interplay between holocentricity and repetitive DNA evolution in this genus. In our study, we confirmed the holocentricity of Eleocharis chromosomes by immunostaining against the kinetochore protein KNL1 and the cell-cycle dependent posttranslational modifications histone H2AThr121ph and H3S10ph. We further studied the composition and chromosomal distribution of the main satellite DNA repeats found in the newly sequenced species E. maculosa, E. geniculata, E. parodii, E. elegans, and E. montana. Five of the six satellites discovered were arranged in clusters, while EmaSAT14 was distributed irregularly along the chromatid length in a line-like manner. EmaSAT14 monomers were present in a few copies in few species across the Eleocharis phylogenetic tree. Nonetheless, they were accumulated within a restricted group of Maculosae series, subgenus Eleocharis. The data indicates that the amplification and line-like distribution of EmaSAT14 along chromatids may have occurred recently within a section of the genus.

How diverse a monocentric chromosome can be? Repeatome and centromeric organization of Juncus effusus (Juncaceae).

Juncus is the largest genus of Juncaceae and was considered holocentric for a long time. Recent findings, however, indicated that 11 species from different clades of the genus have monocentric chromosomes. Thus, the Juncus centromere organization and evolution need to be reassessed. We aimed to investigate the major repetitive DNA sequences of two accessions of Juncus effusus and its centromeric structure by employing whole-genome analyses, fluorescent in situ hybridization, CENH3 immunodetection, and chromatin immunoprecipitation sequencing. We showed that the repetitive fraction of the small J. effusus genome (~270 Mbp/1C) is mainly composed of Class I and Class II transposable elements (TEs) and satellite DNAs. Three identified satellite DNA families were mainly (peri)centromeric, with two being associated with the centromeric protein CENH3, but not strictly centromeric. Two types of centromere organization were discerned in J. effusus: type 1 was characterized by a single CENH3 domain enriched with JefSAT1-155 or JefSAT2-180, whereas type 2 showed multiple CENH3 domains interrupted by other satellites, TEs or genes. Furthermore, while type 1 centromeres showed a higher degree of satellite identity along the array, type 2 centromeres had less homogenized arrays along the multiple CENH3 domains per chromosome. Although the analyses confirmed the monocentric organization of J. effusus chromosomes, our data indicate a more dynamic arrangement of J. effusus centromeres than observed for other plant species, suggesting it may constitute a transient state between mono- and holocentricity.

Differential Repeat Accumulation in the Bimodal Karyotype of Agave L.

The genus Agave presents a bimodal karyotype with x = 30 (5L, large, +25S, small chromosomes). Bimodality within this genus is generally attributed to allopolyploidy in the ancestral form of Agavoideae. However, alternative mechanisms, such as the preferential accumulation of repetitive elements at the macrochromosomes, could also be important. Aiming to understand the role of repetitive DNA within the bimodal karyotype of Agave, genomic DNA from the commercial hybrid 11648 (2n = 2x = 60, 6.31 Gbp) was sequenced at low coverage, and the repetitive fraction was characterized. In silico analysis showed that ~67.6% of the genome is mainly composed of different LTR retrotransposon lineages and one satellite DNA family (AgSAT171). The satellite DNA localized at the centromeric regions of all chromosomes; however, stronger signals were observed for 20 of the macro- and microchromosomes. All transposable elements showed a dispersed distribution, but not uniform across the length of the chromosomes. Different distribution patterns were observed for different TE lineages, with larger accumulation at the macrochromosomes. The data indicate the differential accumulation of LTR retrotransposon lineages at the macrochromosomes, probably contributing to the bimodality. Nevertheless, the differential accumulation of the satDNA in one group of macro- and microchromosomes possibly reflects the hybrid origin of this Agave accession.

Aiming off the target: recycling target capture sequencing reads for investigating repetitive DNA.

BACKGROUND AND AIMS: With the advance of high-throughput sequencing, reduced-representation methods such as target capture sequencing (TCS) emerged as cost-efficient ways of gathering genomic information, particularly from coding regions. As the off-target reads from such sequencing are expected to be similar to genome skimming (GS), we assessed the quality of repeat characterization in plant genomes using these data. METHODS: Repeat composition obtained from TCS datasets of five Rhynchospora (Cyperaceae) species were compared with GS data from the same taxa. In addition, a FISH probe was designed based on the most abundant satellite found in the TCS dataset of Rhynchospora cephalotes. Finally, repeat-based phylogenies of the five Rhynchospora species were constructed based on the GS and TCS datasets and the topologies were compared with a gene-alignment-based phylogenetic tree. KEY RESULTS: All the major repetitive DNA families were identified in TCS, including repeats that showed abundances as low as 0.01 % in the GS data. Rank correlations between GS and TCS repeat abundances were moderately high (r = 0.58-0.85), increasing after filtering out the targeted loci from the raw TCS reads (r = 0.66-0.92). Repeat data obtained by TCS were also reliable in developing a cytogenetic probe of a new variant of the holocentromeric satellite Tyba. Repeat-based phylogenies from TCS data were congruent with those obtained from GS data and the gene-alignment tree. CONCLUSIONS: Our results show that off-target TCS reads can be recycled to identify repeats for cyto- and phylogenomic investigations. Given the growing availability of TCS reads, driven by global phylogenomic projects, our strategy represents a way to recycle genomic data and contribute to a better characterization of plant biodiversity.

Addressing Long-Standing Questions with Advanced Approaches: The 4th B Chromosome Conference.

B chromosomes (Bs) are enigmatic accessory genomic elements extensively characterized in diverse eukaryotes. Since their discovery in the beginning of the 20th century, B chromosomes have been the subject of investigation in laboratories all around the world. As a consequence, scientific meetings have dealt with B chromosomes, including the most specific and important conference in the field, "The B Chromosome Conference." The 4th B Chromosome Conference (4BCC) took place in Botucatu, Brazil, in 2019 and was an excellent opportunity to discuss the latest developments in the B chromosome research field. B chromosome science has advanced from classical and molecular cytogenetics to genomics and bioinformatics approaches. The recent advances in next-generation sequencing technologies and high-throughput molecular biology protocols have led Bs to be the subject of massive data analysis, thus enabling the investigation of structural and functional issues not considered before. Although extensive progress has been made, questions are still remaining to be answered. The advances in functional studies based on RNA, epigenetics, and gene ontologies open the perspective to a better understanding of the complex biology of B chromosomes.

Together But Different: The Subgenomes of the Bimodal Eleutherine Karyotypes Are Differentially Organized.

Bimodal karyotypes are characterized by the presence of two sets of chromosomes of contrasting size. Eleutherine bulbosa (2n = 12) presents a bimodal karyotype with a large chromosome pair, which has a pericentric inversion in permanent heterozygosity with suppressed recombination, and five pairs of three to four times smaller chromosomes. Aiming to understand whether high copy number sequence composition differs between both chromosome sets, we investigated the repetitive DNA fraction of E. bulbosa and compared it to the chromosomal organization of the related Eleutherine latifolia species, not containing the pericentric inversion. We also compared the repetitive sequence proportions between the heteromorphic large chromosomes of E. bulbosa and between E. bulbosa and E. latifolia to understand the influence of the chromosome inversion on the dynamics of repetitive sequences. The most abundant repetitive families of the genome showed a similar chromosomal distribution in both homologs of the large pair and in both species, apparently not influenced by the species-specific inversions. The repeat families Ebusat1 and Ebusat4 are localized interstitially only on the large chromosome pair, while Ebusat2 is located in the centromeric region of all chromosomes. The four most abundant retrotransposon lineages are accumulated in the large chromosome pair. Replication timing and distribution of epigenetic and transcriptional marks differ between large and small chromosomes. The differential distribution of retroelements appears to be related to the bimodal condition and is not influenced by the nonrecombining chromosome inversions in these species. Thus, the large and small chromosome subgenomes of the bimodal Eleutherine karyotype are differentially organized and probably evolved by repetitive sequences accumulation on the large chromosome set.

Correction: Marques, A. et al. Evolution of Plant B Chromosome Enriched Sequences. Genes 2018, 9, 515.

The authors wish to make the following modification in their review [...].

Evolution of Plant B Chromosome Enriched Sequences.

B chromosomes are supernumerary chromosomes found in addition to the normal standard chromosomes (A chromosomes). B chromosomes are well known to accumulate several distinct types of repeated DNA elements. Although the evolution of B chromosomes has been the subject of numerous studies, the mechanisms of accumulation and evolution of repetitive sequences are not fully understood. Recently, new genomic approaches have shed light on the origin and accumulation of different classes of repetitive sequences in the process of B chromosome formation and evolution. Here we discuss the impact of repetitive sequences accumulation on the evolution of plant B chromosomes.

Decondensation of chromosomal 45S rDNA sites in Lolium and Festuca genotypes does not result in karyotype instability.

Fragile sites (FSs) in plants have been described for species like Lolium and other grasses. Whereas in humans FSs were shown to be involved in genome instabilities; the consequences of FSs expression in plants are not known yet. To evaluate whether FSs cause karyotype instabilities, we assessed the frequency of micronuclei and lagging chromosomes in meristematic cells, the stability of the DNA content, and the occurrence of neocentromeres in the presumed chromosomal fragments of Lolium perenne, Lolium multiflorum, Festuca arrundinacea, and two Festulolium hybrids. The cell cycle analysis along with flow cytometric genome size measurements showed high stability in all genomes evaluated. Neocentromeric activity was neither observed in the presumed fragments nor in any other chromosomal region, then this is not the mechanism responsible by the stability. However, Fluorescence in situ hybridization (FISH) with a 45S ribosomal DNA (rDNA) probe in combination with YOYO staining of metaphasic chromosomes showed that many extended nucleolus organizing region (NOR) form very thin YOYO-positive chromatin fibers connecting the acentric 'fragment' with the centromere-containing chromosome region. The obtained data indicate that the expression of FSs does not result in genome instabilities or neocentromere formation. The FS-containing 45S rDNA carrying chromatin fibers undergo a cell cycle and gene activity-dependent dynamic decondensation process.

Centromeric and non-centromeric satellite DNA organisation differs in holocentric Rhynchospora species.

Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.

Restructuring of Holocentric Centromeres During Meiosis in the Plant Rhynchospora pubera.

Centromeres are responsible for the correct segregation of chromosomes during mitosis and meiosis. Holocentric chromosomes, characterized by multiple centromere units along each chromatid, have particular adaptations to ensure regular disjunction during meiosis. Here we show by detecting CENH3, CENP-C, tubulin, and centromeric repeats that holocentromeres may be organized differently in mitosis and meiosis of Rhynchospora pubera Contrasting to the mitotic linear holocentromere organization, meiotic centromeres show several clusters of centromere units (cluster-holocentromeres) during meiosis I. They accumulate along the poleward surface of bivalents where spindle fibers perpendicularly attach. During meiosis II, the cluster-holocentromeres are mostly present in the midregion of each chromatid. A linear holocentromere organization is restored after meiosis during pollen mitosis. Thus, a not yet described case of a cluster-holocentromere organization, showing a clear centromere restructuration between mitosis and meiosis, was identified in a holocentric organism.

Developmental programmed cell death during asymmetric microsporogenesis in holocentric species of Rhynchospora (Cyperaceae).

Members of the Cyperaceae family exhibit an asymmetric microsporogenesis that results in the degeneration of three out of four meiotic products. Efforts have been made previously to describe the resulting structure, named the pseudomonad, but mechanisms concerning the establishment of cell domains, nuclear development, and programmed cell death are largely unknown. Using the Rhynchospora genus as a model, evidence for cell asymmetry, cytoplasmic isolation, and programmed cell death was obtained by a combination of electron microscopic, cytochemical, immunocytochemical, in situ hybridization, and flow cytometric methods. Degenerative cells were identified at the abaxial region, with the cytoskeleton marking their delimitation from the functional domain after meiosis. After attempting to initiate cell division with an unreplicated genome and abnormal spindle assembly, these cells exhibited a gradual process of cytoplasmic contraction associated with hypermethylation of cytosines and differential loss of DNA. These results indicate that the asymmetric tetrad establishes a functional cell, where one nucleus is preferentially selected to survive. Degenerative haploid cells are then eliminated in a multistep process associated with mitotic disorder, non-random elimination of repetitive DNA, vacuolar cell death, and DNA fragmentation.

Fragile sites of 45S rDNA of Lolium multiflorum are not hotspots for chromosomal breakages induced by X-ray.

Sites of 45S rDNA of Lolium are regions denominated fragile sites (FSs), constituting regions slightly stained with DAPI due to increased DNA unpacking in metaphasic chromosomes. Considered to be fragile regions in the genome, the FSs might be more responsive to induced breaks and result in chromosomal fragments and rearrangements, unless repairing mechanisms such as recombination or de novo telomere formation play a role at the break site of the DNA. Thus, this study aimed at investigating if SFs from Lolium are hotspots for the occurrence of breakages induced by X-ray and if they are regions favorable to synthesize new telomeres, using Hordeum vulgare as a comparative model. Lolium multiflorum and H. vulgare seedlings were irradiated with 20 and 50 Gy X-ray and evaluated one day following the irradiation and at 7-days intervals for a 28-days period, using FISH technique with 45S rDNA and Arabidopsis-type telomere probes in order to investigate the presence of chromosomal breakages and new telomere formation. H. vulgare did not survive after a few days of irradiation due to the increased rate of abnormalities. L. multiflorum also exhibited chromosomal abnormalities following the exposure, yet over the 28-days trial it had a decrease in the chromosomal damage rate and formation of de novo telomere has not been detected along this time. Despite being considered to be fragile regions in the genome, the 45S rDNA sites of Lolium are not hotspots to chromosomal breakages after the induction of breakages.

Tissue-specific genome instability in synthetic interspecific hybrids of Pennisetum purpureum (Napier grass) and Pennisetum glaucum (pearl millet) is caused by micronucleation.

Genome instability is observed in several species hybrids. We studied the mechanisms underlying the genome instability in hexaploid hybrids of Napier grass (Pennisetum purpureum R.) and pearl millet (Pennisetum glaucum L.) using a combination of different methods. Chromosomes of both parental genomes are lost by micronucleation. Our analysis suggests that genome instability occurs preferentially in meristematic root tissue of hexaploid hybrids, and chromosome elimination is not only caused by centromere inactivation. Likely, beside centromere dysfunction, unrepaired DNA double-strand breaks result in fragmented chromosomes in synthetic hybrids.