Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated “Tyba” and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle–dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences.

We studied the karyotypes of four Brazilian Cestrum species (C. amictum, C. intermedium, C. sendtnerianum and C. strigilatum) using conventional Feulgen staining, C-Giemsa and C-CMA 3 /DAPI banding, induction of cold-sensitive regions (CSRs) and fluorescent in situ hybridization (FISH) with rDNA probes. We found that the karyotypes of all four species was 2n = 2x = 16, with, except for the eighth acrocentric pair, a predominance of meta-and submetacentric chromosomes and various heterochromatin classes. Heterochromatic types previously unreported in Cestrum as neutral C-CMA 3 0 /DAPI 0 bands, CMA 3 + bands not associated with NORs, and C-Giemsa/CSR/DAPI -bands were found. The heterochromatic blocks varied in size, number, position and composition. The 45S rDNA probe preferentially located in the terminal and subterminal regions of some chromosomes, while 5S rDNA appeared close to the centromere of the long arm of pair 8. These results suggest that karyotype differentiation can occur mainly due to changes in repetitive DNA, with little modification in the general composition of the conventionally stained karyotype.

The Triatominae are distributed in Brazilian regions and are the vector of Chagas disease. This group is also characterized by lower karyotype variability, occurrence of holokinetic chromosomes and inverted meiosis of the sex chromosomes. In this study, we determined the karyotype of four species of the genus Triatoma Laporte, 1832 using chromosome measurement, chromosome banding and FISH with a 45S rDNA probe. All samples showed 2n = 20A+XY with chromosomes sorted by size in decreasing order. Heterochromatin of Triatoma infestans melanosoma Lent, Jurberg, Galvão, Carcavallo, 1994 is distributed among eight autosomes and the sex chromosomes. In Triatoma brasiliensis Neiva, 1911, T. matogrossensis Leite, Barbosa, 1953 and T. rubrovaria Blanchard, 1834, heterochromatin was restricted to the Y chromosome, which was characterized as DAPI+ (weak signal). FISH using a 45S rDNA probe of Drosophila melanogaster Meigen, 1830 showed differences in the number and location of hybridization sites. T. brasiliensis and T. rubrovaria showed the signal on one autosome pair. T. matogrossensis showed signals on both sex chromosomes, and T. infestans melanosoma only on the X chromosome. Conventional banding analysis suggests a closer relationship between T. brasiliensis, T. matogrossensis and T. rubrovaria, except with regard to 45S rDNA location in T. matogrossensis, and a more differentiated karyotype in T. infestans melanosoma

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.

Like other eukaryotes, the nuclear genome of plants consists of DNA with a small proportion of low-copy DNA (genes and regulatory sequences) and very abundant DNA sequence motifs that are repeated thousands up to millions of times in the genomes including transposable elements (TEs) and satellite DNA. Retrotransposons, one class of TEs, are sequences that amplify via an RNA intermediate and reinsert into the genome, are often the major fraction of a genome. Here, we put research on retrotransposons into the larger context of plant repetitive DNA and genome behaviour, showing features of genome evolution in a grass genus, Brachiaria, in relation to other plant species. We show the contrasting amplification of different retroelement fractions across the genome with characteristics for various families and domains. The genus Brachiaria includes both diploid and polyploid species, with similar chromosome types and chromosome basic numbers x = 6, 7, 8 and 9. The polyploids reproduce asexually and are apomictic, but there are also sexual species. Cytogenetic studies and flow cytometry indicate a large variation in DNA content (C-value), chromosome sizes and genome organization. In order to evaluate the role of transposable elements in the genome and karyotype organization of species of Brachiaria, we searched for sequences similar to conserved regions of TEs in RNAseq reads library produced in Brachiaria decumbens. Of the 9649 TE-like contigs, 4454 corresponded to LTR-retrotransposons, and of these, 79.5 % were similar to members of the gypsy superfamily. Sequences of conserved protein domains of gypsy were used to design primers for producing the probes. The probes were used in FISH against chromosomes of accesses of B. decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola. Probes showed hybridization signals predominantly in proximal regions, especially those for retrotransposons of the clades CRM and Athila, while elements of Del and Tat exhibited dispersed signals, in addition to those proximal signals. These results show that the proximal region of Brachiaria chromosomes is a hotspot for retrotransposon insertion, particularly for the gypsy family. The combination of high-throughput sequencing and a chromosome-centric cytogenetic approach allows the abundance, organization and nature of transposable elements to be characterized in unprecedented detail. By their amplification and dispersal, retrotransposons can affect gene expression; they can lead to rapid diversification of chromosomes between species and, hence, are useful for studies of genome evolution and speciation in the Brachiaria genus. Centromeric regions can be identified and mapped, and retrotransposon markers can also assisting breeders in the developing and exploiting interspecific hybrids.

No abstract

No abstract

Eleocharis subarticulata is recorded as the third species of Cyperaceae with a reduced chromosome number ( n = 3), following reports on Rhynchospora tenuis ( n = 2) and Fimbristylis umbellaris ( n = 3). For Eleocharis, the numbers recorded to date vary from 2 n = 10 to 2 n = c. 196, with x = 5 as the possible basic number. The karyotype of E. subarticulata was studied using conventional staining (mitosis and meiosis), C-CMA 3 /DAPI banding, and FISH with 45S rDNA and telomere probes. The chromosomes showed no primary constrictions, as expected in the holocentric chromosomes of Cyperaceae. The meiotic behaviour was abnormal, with a single multivalent ring of six chromosomes at metaphase I, resulting from multiple translocations. At anaphase I six chromatids migrated to each pole, evidencing the inverted meiosis, and these groups were also visible at metaphase II. The C-CMA 3 /DAPI banding technique showed only four terminal GC-rich blocks. FISH with 45S rDNA probes revealed four terminal signals, probably associated with GC-rich blocks. The telomeric probe located terminal signals in all the chromosomes, besides a hybridization site in the middle of the large pair. The occurrence of ectopic telomeric sites has not been described previously for plants with holokinetic karyotypes and with reduced chromosome numbers. These data reinforce the hypothesis of the reduction in chromosome number by multiple translocations.