DOI: 10.5061/dryad.4mw6m90ck
title: Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci

description: 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. 
description: 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. 
description: <strong>NFU_GRZ.zip</strong> 
 NFU_GRZ_absolute.tiff X and Y chromosomes
 used to estimate the length difference between the sex chromosomes in the
 <em>N</em>. <em>furzeri</em> 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 <em>N</em>.
 <em>furzeri</em> population GRZ 
 NFU_GRZ_measurements.tiff 
 Schematic representation of measurements performed in the
 <em>N</em>. <em>furzeri</em> population
 GRZ 
 <strong>NFU_MZCS121_1.zip</strong>
 The sample of the <em>N</em>.
 <em>furzeri</em> 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 <em>N</em>. <em>furzeri</em>
 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
 <em>N</em>. <em>furzeri</em> population MZCS121,
 sample 1 
 NFU_MZCS121_1_measurements.tiff Schematic
 representation of measurements performed in the <em>N</em>.
 <em>furzeri</em> population MZCS121, sample 1
 
 <strong>NFU_MZCS121_2.zip</strong>
 The sample of the <em>N</em>.
 <em>furzeri</em> 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 <em>N</em>. <em>furzeri</em>
 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
 <em>N</em>. <em>furzeri</em> population MZCS121,
 sample 2 
 NFU_MZCS121_2_measurements.tiff Schematic
 representation of measurements performed in the <em>N</em>.
 <em>furzeri</em> population MZCS121, sample 2
 
 <strong>NFU_MZCS222.zip</strong> 
 NFU_MZCS222_absolute.tiff X and Y
 chromosomes used to estimate the length difference between the sex
 chromosomes in the <em>N</em>. <em>furzeri</em>
 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
 <em>N</em>. <em>furzeri</em> population
 MZCS222 
 NFU_MZCS222_measurements.tiff Schematic
 representation of measurements performed in the <em>N</em>.
 <em>furzeri</em> population MZCS222 
 
 <strong>NKA_MZCS91.zip</strong> 
 NKA_MZCS91_absolute.tiff X and Y
 chromosomes used to estimate the length difference between the sex
 chromosomes in the <em>N</em>. <em>kadleci</em>
 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
 <em>N</em>. <em>kadleci</em> population
 MZCS91 
 NKA_MZCS91_measurements.tiff Schematic
 representation of measurements performed in the <em>N</em>.
 <em>kadleci</em> population MZCS91 
 
 <strong>NKA_MZCS430.zip</strong> 
 NKA_MZCS430_absolute.tiff X and Y
 chromosomes used to estimate the length difference between the sex
 chromosomes in the <em>N</em>. <em>kadleci</em>
 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 <em>N</em>. <em>kadleci</em> population
 MZCS430 
 NKA_MZCS430_measurements.tiff Schematic
 representation of measurements performed in the <em>N</em>.
 <em>kadleci</em> population MZCS430