Repetitive DNA represents an important driver of sex chromosome differentiation. Yet, repetitive sequences tend to be misrepresented or overlooked in genomic studies. We analysed repetitive DNA landscape of sex chromosomes in several populations of a turquoise killifish Nothobranchius furzeri and its sister species N. kadleci (Teleostei: Nothobranchiidae), representatives of African annual killifishes with high rate of karyotype and sex chromosome evolution. We combined bioinformatic analyses of repeatome with molecular cytogenetic techniques such as comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, genes for ribosomal RNAs (rDNA) and bacterial artificial chromosomes (BACs) and immunostaining of SYCP3 and MLH1 proteins, which marked lateral elements of synaptonemal complexes and recombination sites, respectively. We revealed that N. furzeri and N. kadleci share the XY sex chromosome system, which is thus much older than previously assumed. Sex chromosomes are mostly heteromorphic as evidenced by distinct distribution of satellite DNAs and major rDNA. Yet, the heteromorphic XY sex chromosomes pair almost exclusively regularly in meiosis, which implies synaptic adjustment. Physical mapping of BACs identified inversions on Y chromosomes of the N. kadleci populations, akin to the pattern previously reported in N. furzeri. Yet, repetitive DNA landscape of X and Y sex chromosomes either diverged in parallel in populations of both species or it evolved in their common ancestor and thus predates the inversions. The observed differentiation via repeat repatterning thus cannot be explained by the classical sexually antagonistic model. Rather, we hypothesized that relaxed meiotic drive and recombination reduced by neutral processes could drive changes in repeatome and secondary inversions could be maintained by sexually antagonistic regulatory effects resulting from early evolution of dosage compensation..

The role of chromosome changes in speciation remains a debated topic, although demographic conditions associated with divergence should promote their appearance. We tested a potential relationship between chromosome changes and speciation by studying two Lake Whitefish (Coregonus clupeaformis) lineages that recently colonized postglacial lakes following allopatry. A dwarf limnetic species evolved repeatedly from the normal benthic species, becoming reproductively isolated. Lake Whitefish hybrids experience mitotic and meiotic instability, which may result from structurally divergent chromosomes. Motivated by this observation, we test the hypothesis that chromosome organization differs between Lake Whitefish species pairs using cytogenetics. While chromosome and fundamental numbers are conserved between the species (2n = 80, NF = 98), we observe extensive polymorphism of subtle karyotype traits. We describe intrachromosomal differences associated with heterochromatin and repetitive DNA, and test for parallelism among three sympatric species pairs. Multivariate analyses support the hypothesis that differentiation at the level of subchromosomal markers mostly appeared during allopatry. Yet we find no evidence for parallelism between species pairs among lakes, consistent with colonization effect or postcolonization differentiation. The reported intrachromosomal polymorphisms do not appear to play a central role in driving adaptive divergence between normal and dwarf Lake Whitefish. We discuss how chromosomal differentiation in the Lake Whitefish system may contribute to the destabilization of mitotic and meiotic chromosome segregation in hybrids, as documented previously. The chromosome structures detected here are still difficult to sequence and assemble, demonstrating the value of cytogenetics as a complementary approach to understand the genomic bases of speciation.

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BackgroundPikes represent an important genus (Esox) harbouring a pre-duplication karyotype (2n = 2x = 50) of economically important salmonid pseudopolyploids. Here, we have characterized the 5S ribosomal RNA genes (rDNA) in Esox lucius and its closely related E. cisalpinus using cytogenetic, molecular and genomic approaches. Intragenomic homogeneity and copy number estimation was carried out using Illumina reads. The higher-order structure of rDNA arrays was investigated by the analysis of long PacBio reads. Position of loci on chromosomes was determined by FISH. DNA methylation was analysed by methylation-sensitive restriction enzymes.ResultsThe 5S rDNA loci occupy exclusively (peri)centromeric regions on 30–38 acrocentric chromosomes in both E. lucius and E. cisalpinus. The large number of loci is accompanied by extreme amplification of genes (>20,000 copies), which is to the best of our knowledge one of the highest copy number of rRNA genes in animals ever reported. Conserved secondary structures of predicted 5S rRNAs indicate that most of the amplified genes are potentially functional. Only few SNPs were found in genic regions indicating their high homogeneity while intergenic spacers were more heterogeneous and several families were identified. Analysis of 10–30 kb-long molecules sequenced by the PacBio technology (containing about 40% of total 5S rDNA) revealed that the vast majority (96%) of genes are organised in large several kilobase-long blocks. Dispersed genes or short tandems were less common (4%). The adjacent 5S blocks were directly linked, separated by intervening DNA and even inverted. The 5S units differing in the intergenic spacers formed both homogeneous and heterogeneous (mixed) blocks indicating variable degree of homogenisation between the loci. Both E. lucius and E. cisalpinus 5S rDNA was heavily methylated at CG dinucleotides.ConclusionsExtreme amplification of 5S rRNA genes in the Esox genome occurred in the absence of significant pseudogenisation suggesting its recent origin and/or intensive homogenisation processes. The dense methylation of units indicates that powerful epigenetic mechanisms have evolved in this group of fish to silence amplified genes. We discuss how the higher-order repeat structures impact on homogenisation of 5S rDNA in the genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3774-7) contains supplementary material, which is available to authorized users.

Leuciscine cyprinids possess a nearly invariant diploid number (2n = 50) with an extremely uniform karyotype comprising of 8 pairs of metacentric, 13-15 pairs of submetacentric and 2-4 pairs of subtelocentric (st) to acrocentric (a) chromosomes. The largest pair is characteristically an st/a element-the 'leuciscine' cytotaxonomic marker. Previously, the interspecific homology of this chromosome pair could not be assessed owing to the inability to produce euchromatic or serial banding patterns. In the present study, we used laser-microdissection (15-20 copies of the marker chromosome) to construct a whole chromosome probe (WCP) from the marker chromosome of the roach Rutilus rutilus to ascertain the interspecific homology of marker chromosomes by cross-species in-situ hybridization. WCP was hybridized to chromosomes of widely distributed (Abramis brama, Alburnoides bipunctatus, Alburnus alburnus, Aspius aspius, Ballerus ballerus, B. sapa, Blicca bjoerkna, Chondrostoma nasus, Leucaspius delineatus, Leuciscus leuciscus, L. idus, R. rutilus, Scardinius erythrophthalmus, Squalius cephalus, and Vimba vimba) and Iberian endemic species (Achondrostoma oligolepis, Iberochondrostoma almacai, I. lusitanicum, Pseudochondrostoma duriense, S. alburnoides and S. pyrenaicus). Cross-species in-situ hybridization to chromosomes of Phoxinus phoxinus, a representative of leuciscine sister lineage, showed the same pattern as in all of the leuciscins. The probe consistently hybridized to the distal part of the short arm of the marker chromosome, indicating sequence homology.