Supernumerary (B) chromosomes are dispensable genomic elements occurring frequently among grasshoppers. Most B chromosomes are enriched with repetitive DNAs, including satellite DNAs (satDNAs) that could be implicated in their evolution. Although studied in some species, the specific ancestry of B chromosomes is difficult to ascertain and it was determined in only a few examples. Here we used bioinformatics and cytogenetics to characterize the composition and putative ancestry of B chromosomes in three grasshopper species, Rhammatocerus brasiliensis, Schistocerca rubiginosa, and Xyleus discoideus angulatus. Using the RepeatExplorer pipeline we searched for the most abundant satDNAs in Illumina sequenced reads, and then we generated probes used in fluorescent in situ hybridization (FISH) to determine chromosomal position. We used this information to infer ancestry and the events that likely occurred at the origin of B chromosomes. We found twelve, nine, and eighteen satDNA families in the genomes of R. brasiliensis, S. rubiginosa, and X. d. angulatus, respectively. Some satDNAs revealed clustered organization on A and B chromosomes varying in number of sites and position along chromosomes. We did not find specific satDNA occurring in the B chromosome. The satDNAs shared among A and B chromosomes support the idea of putative intraspecific ancestry from small autosomes in the three species, i.e., pair S11 in R. brasiliensis, pair S9 in S. rubiginosa, and pair S10 in X. d. angulatus. The possibility of involvement of other chromosomal pairs in B chromosome origin is also hypothesized. Finally, we discussed particular aspects in composition, origin, and evolution of the B chromosome for each species.

Neo-sex chromosome systems arose independently multiple times in evolution, presenting the remarkable characteristic of repetitive DNAs accumulation. Among grasshoppers, occurrence of neo-XY was repeatedly noticed in Melanoplinae. Here we analyzed the most abundant tandem repeats of R. bergii (2n = 22, neo-XY♂) using deep Illumina sequencing and graph-based clustering in order to address the neo-sex chromosomes evolution. The analyses revealed ten families of satDNAs comprising about ~1% of the male genome, which occupied mainly C-positive regions of autosomes. Regarding the sex chromosomes, satDNAs were recorded within centromeric or interstitial regions of the neo-X chromosome and four satDNAs occurred in the neo-Y, two of them being exclusive (Rber248 and Rber299). Using a combination of probes we uncovered five well-defined cytological variants for neo-Y, originated by multiple paracentric inversions and satDNA amplification, besides fragmented neo-Y. These neo-Y variants were distinct in frequency between embryos and adult males. The genomic data together with cytogenetic mapping enabled us to better understand the neo-sex chromosome dynamics in grasshoppers, reinforcing differentiation of neo-X and neo-Y and revealing the occurrence of multiple additional rearrangements involved in the neo-Y evolution of R. bergii. We discussed the possible causes that led to differences in frequency for the neo-Y variants between embryos and adults. Finally we hypothesize about the role of DNA satellites in R. bergii as well as putative historical events involved in the evolution of the R. bergii neo-XY.

Satellite DNAs (satDNAs) are organized in repetitions directly contiguous to one another, forming long arrays and composing a large portion of eukaryote genomes. These sequences evolve according to the concerted evolution model, and homogenization of repeats is observed at the intragenomic level. Satellite DNAs are the primary component of heterochromatin, located primarily in centromeres and telomeres. Moreover, satDNA enrichment in specific chromosomes has been observed, such as in B chromosomes, that can provide clues about composition, origin and evolution of this chromosome. In this study, we isolated and characterized a satDNA in A and B chromosomes of Abracris flavolineata by integrating cytogenetic, molecular and genomics approaches at intra- and inter-population levels, with the aim to understand the evolution of satDNA and composition of B chromosomes. AflaSAT-1 satDNA was shared with other species and in A. flavolineata, was associated with another satDNA, AflaSAT-2. Chromosomal mapping revealed centromeric blocks variable in size in almost all chromosomes (except pair 11) of A complement for both satDNAs, whereas for B chromosome, only a small centromeric signal occurred. In distinct populations, variable number of AflaSAT-1 chromosomal sites correlated with variability in copy number. Instead of such variability, low sequence diversity was observed in A complement, but monomers from B chromosome were more variable, presenting also exclusive mutations. AflaSAT-1 was transcribed in five tissues of adults in distinct life cycle phases. The sharing of AflaSAT-1 with other species is consistent with the library hypothesis and indicates common origin in a common ancestor; however, AflaSAT-1 was highly amplified in the genome of A. flavolineata. At the population level, homogenization of repeats in distinct populations was documented, but dynamic expansion or elimination of repeats was also observed. Concerning the B chromosome, our data provided new information on the composition in A. flavolineata. Together with previous results, the sequences of heterochromatic nature were not likely highly amplified in the entire B chromosome. Finally, the constitutive transcriptional activity suggests a possible unknown functional role, which should be further investigated.

Supernumerary chromosomes (B chromosomes) have been an intriguing subject of study. Our understanding of the molecular differentiation of B chromosomes from an interpopulation perspective remains limited, with most analyses involving chromosome banding and mapping of a few sequences. To gain insights into the molecular composition, origin, and evolution of B chromosomes, we conducted cytogenetic and next-generation sequencing analysis of the repeatome in the grasshopper Abracris flavolineata across various populations. Our results unveiled the presence of B chromosomes in two newly investigated populations and described new satellite DNA sequences. While we observed some degree of genetic connection among A. flavolineata populations, our comparative analysis of genomes with and without B chromosomes provided evidence of two new B chromosome variants. These variants exhibited distinct compositions of various repeat classes, including transposable elements and satellite DNAs. Based on shared repeats, their chromosomal location, and the C-positive heterochromatin content on the B chromosome, these variants likely share a common origin but have undergone distinct molecular differentiation processes, resulting in varying degrees of heterochromatinization. Our data serve as a detailed example of the dynamic and differentiated nature of B chromosome molecular content at the interpopulation level, even when they share a common origin.

B chromosomes are additional elements in a normal karyotype that has been considered frequently as parasitic and dispensable. These elements do not recombine with A chromosomes, could be accumulated and present non-mendelian patterns of inheritance. In most of the cases they comprise an enrichment of heterochromatic repetitive DNA, such as Satellite DNAs (satDNA). To contribute to understanding about origin, composition and evolutionary patterns of B chromosome in the grasshopper Abracris flavolineata, in this work we characterized through Illumina sequencing and graph-based clustering the most abundant tandem repeats of A. flavolineata, karyotype 2n = 22,X0+B?.The analyses revealed the occurrence of ten families of satDNAs comprising about 6.279% of the male genome harboring one B chromosome. For the X chromosome and autosomes, distinct patterns of hybridization was observed between the satDNAs showing variability among them, like scattered blocks on euchromatic areas or specific ones on heterochromatic regions of the chromosomes. Five of the satDNAs recovered were distributed on centromeric and both terminal regions of the B chromosome, reinforcing the hypothesis of B chromosome origin through isochromosome formation. Two satDNA families were located only in centromere, while three families were not present in B chromosome. CL220 satDNA was the unique family shared between the B chromosome and only one A element, the chromosome 1, supporting strongly the hypothesis of its origin from this autosomal pair, also noticed previously by U2 snDNA mapping. In addition, this analysis provides new information regarding the composition and evolution of B chromosome of A. flavolineata. Furthermore, the non-spreading and specific location of satDNAs and other repetitive sequences restrict to telomeres and centromere reinforces the possible idea that this B chromosome could harbor unknown euchromatic sequences along chromosomal arms and do not present intense amplification of heterochromatic ones, as have been commonly described for other species.