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 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 X and Y 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, similar to the pattern previously reported in N. furzeri. Yet, the 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 sexual 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 evolution of dosage compensation. 

Supporting data contain additional information on sex chromosome measurements, figures with measured sex chromosomes and schemes depicting measured chromosome segments, for each population.

 Physical maps of sex chromosomes were reconstructed based on metric analysis of the distribution of markers detected by means of cytogenetics:

 (i) Absolute length of the X and Y was used to estimate the length difference between the sex chromosomes

 The absolute length of the sex chromosomes was measured with the Freehand Line tool using ImageJ software (https://imagej.nih.gov/ij/). The scale was calibrated on the 10 µm scale bar.

 (ii) Relative length and position of the markers of interest (i.e., BAC clones 201Bd03, 220Bc03; 18S rDNA; Nfu-SatC; CGH; p/q arm) was used for their localization on the X and Y chromosomes

The measurements were made independently for each cytogenetic marker using the ImageJ software with the Levan plugin [Sakamoto and Zacaro, 2009] which enables one to analyse karyotype characteristics using relative length data. The relative length and position of the marker were measured either on the left or right chromatid of the chromosome (depending on the quality of the chromatid and visualized FISH signal). Due to the appearance of chromosomes (bending, spiralization, etc.) the measurement lines were made using the Freehand Line tool. The tip of a short (p) arm was set as a zero point and chromosome units were measured as depicted in deposited files *_measurements.tiff (where * stands for species and population IDs). Note that Levan requires an even number of segments. Thus, additional data points were added where needed to meet this requirement (for details see attached files *_measurements.tiff). As noted by the authors of Levan, when measuring the chromosome units as described here, presented morphology classification (i.e., A.R., Morphology) in the output should be ignored.

For the measurements, we preferentially used results from sequential hybridization experiments in which the mitotic metaphases were reprobed, i.e. hybridized repeatedly with several sets of probes. In the files *_absolute.tiff and *_relative.tiff, the reprobed chromosomes were labelled based on markers used : “seq” (BAC+rDNA+satC), BAC+satC, BAC+rDNA, BAC+CGH, rDNA+CGH. The XY or XX chromosomes from male and female individual nuclei, respectively, are labelled by roman numerals. As the chromosomes experienced partial damage after each round of hybridization, we superposed the figures from reprobings and used the first, least damaged, DAPI layer from the BAC-FISH (denoted as “overlay”). If needed, we also used data from one round FISH experiments with only one probe (i.e., BAC clone 201Bd03, Nfu-satC). The white lines on both sides of a chromosome delimit the centromeric region which was used as a chromosomal landmark.

Similar to other cytogenetic markers, measurements of relative positions for borders of hybridization signals  BAC 201Bd03 and 220Bc03 were determined and their chromosomal location was calculated as the mean of the border values. Note that, unlike other markers, the 18S rDNA was detected using antibody signal amplification and the size of the cluster is thus probably overestimated. The position of CGH hybridization signals was determined only for X chromosomes in all populations but NFU MZCS121 sample 2, as the Y chromosome signals were considerably reduced. 

NFU_GRZ.zip

• NFU_GRZ_absolute.tiff
  • X and Y chromosomes used to estimate the length difference between the sex chromosomes in the N. furzeri population GRZ
• NFU_GRZ_relative.tiff
  • X and Y chromosomes used to determine relative positions of markers of interest, namely BAC clone 201Bd03, 18S rDNA, Nfu-SatC, CGH and p and q arm in the N. furzeri population GRZ
• NFU_GRZ_measurements.tiff
  • Schematic representation of measurements performed in the N. furzeri population GRZ

NFU_MZCS121_1.zip

The sample of the N. furzeri MZCS121 with the rDNA cluster localized only on the X chromosome

• NFU_MZCS121_1_absolute.tiff
  • X and Y chromosomes used to estimate the length difference between the sex chromosomes in the N. furzeri population MZCS121, sample 1
• NFU_MZCS121_1_relative.tiff
  • X and Y chromosomes used to determine relative positions of markers of interest, namely BAC clone 201Bd03, 18S rDNA, Nfu-SatC and p and q arm in the N. furzeri population MZCS121, sample 1
• NFU_MZCS121_1_measurements.tiff
  • Schematic representation of measurements performed in the N. furzeri population MZCS121, sample 1

NFU_MZCS121_2.zip

The sample of the N. furzeri MZCS121 with the rDNA cluster localized on both X and Y chromosomes

• NFU_MZCS121_2_absolute.tiff
  • X and Y chromosomes used to estimate the length difference between the sex chromosomes in the N. furzeri population MZCS121, sample 2
• NFU_MZCS121_2_relative.tiff
  • X and Y chromosomes used to determine relative positions of markers of interest, namely BAC clone 201Bd03, 18S rDNA, Nfu-SatC, CGH and p and q arm in the N. furzeri population MZCS121, sample 2
• NFU_MZCS121_2_measurements.tiff
  • Schematic representation of measurements performed in the N. furzeri population MZCS121, sample 2

NFU_MZCS222.zip

• NFU_MZCS222_absolute.tiff
  • X and Y chromosomes used to estimate the length difference between the sex chromosomes in the N. furzeri population MZCS222
• NFU_MZCS222_relative.tiff
  • X and Y chromosomes used to determine relative positions of markers of interest, namely BAC clone 201Bd03, 18S rDNA, Nfu-SatC, CGH and p and q arm in the N. furzeri population MZCS222
• NFU_MZCS222_measurements.tiff
  • Schematic representation of measurements performed in the N. furzeri population MZCS222

NKA_MZCS91.zip

• NKA_MZCS91_absolute.tiff
  • X and Y chromosomes used to estimate the length difference between the sex chromosomes in the N. kadleci population MZCS91
• NKA_MZCS91_relative.tiff
  • X and Y chromosomes used to determine relative positions of markers of interest, namely BAC clone 201Bd03, 18S rDNA, Nfu-SatC, CGH and p and q arm in the N. kadleci population MZCS91
• NKA_MZCS91_measurements.tiff
  • Schematic representation of measurements performed in the N. kadleci population MZCS91

NKA_MZCS430.zip

• NKA_MZCS430_absolute.tiff
  • X and Y chromosomes used to estimate the length difference between the sex chromosomes in the N. kadleci population MZCS430
• NKA_MZCS430_relative.tiff
  • X and Y chromosomes used to determine relative positions of markers of interest, namely BAC clones 201Bd03 and 220Bc03, 18S rDNA, Nfu-SatC, CGH and p and q arm in the N. kadleci population MZCS430
• NKA_MZCS430_measurements.tiff
  • Schematic representation of measurements performed in the N. kadleci population MZCS430