Evolution and dosage compensation of nucleolar organizing regions (NORs) mediated by mobile elements in turtles with female (ZZ/ZW) but not with male (XX/XY) heterogamety

Abstract

Understanding the evolution and regulation of nucleolar organizing regions (NORs) is important to elucidate genome structure and function. This is because ribosomal gene (rDNA) copy number and activity mediate protein biosynthesis, stress response, ageing, disease, dosage compensation and genome stability. Here, we found contrasting dosage compensation of sex-linked NORs in turtles with male and female heterogamety. Most taxa examined exhibit homomorphic rRNA gene clusters in a single autosome pair (determined by 28S rDNA fluorescence in situ hybridization), whereas NORs are sex-linked in Apalone spinifera, Pelodiscus sinensis and Staurotypus triporcatus. Full-dosage compensation upregulates the male X-NOR (determined via silver staining-AgNOR) in Staurotypus (who lacks Y-NOR) compared with female X-AgNORs. In softshell Apalone and Pelodiscus, who share homologous ZZ/ZW micro-chromosomes, their enlarged W-NOR is partially active (due to 28S rDNA invasion by R2 retroelements), whereas their smaller Z-NOR is silent in females but active in both male-Zs (presumably because the W-NOR meets cellular demands and excessive NOR activity is costly). We hypothesize that R2 disruption favoured W enlargement to add intact 28S-units, perhaps facilitated by reduced recombination during sex chromosome evolution. The molecular basis of the potentially adaptive female Z-silencing is likely intricate and perhaps epigenetic, as non-ribosomal Z genes are active in Apalone females. Yet, Emydura maquarii exhibit identical heteromorphism in their autosomal NOR (R2 invaded 28S-units and the small-autosome NOR is silent), suggesting that the softshell turtle pattern can evolve independent of sex chromosome evolution. Our study illuminates the complex sex chromosome evolution and dosage compensation of non-model systems that challenges classic paradigms.

Keywords: 18S/28S repetitive DNA; R2 retrotransposon retroelement transposable element; adaptive evolution of sex chromosome dosage compensation; gene regulation silencing; nucleolar organizing region; ribosomal RNA genes (rRNA, rDNA); silver-staining AgNOR; temperature-dependent (TSD) and ZZ/ZW XX/XY genotypic sex determination (GSD); turtle reptile vertebrates.

FIGURE 1

rDNA cluster mapping using 18S RNA gene probe (green) and 28S RNA gene (red) in Apalone female (a) and male (c), Pelodiscus female (e) and male (g) and Staurotypus female (i) and male (k). Pseudo‐yellow indicates colocalization of green (18S) and red (28S) probes. Additionally, silver‐stained interphase nuclei are included from Apalone (b, d), Pelodiscus (f, h) and Staurotypus (j, l). Boxes contain the enlarged sex chromosomes from each spread. F = female, M = male. Scale bar = 10 μm

FIGURE 2

FISH using R2 element probe (green) and 28S RNA gene (red) in Apalone spinifera female (a), Pelodiscus sinensis female (c) and Emydura macquarii (e). Enlarged image of W chromosome of Apalone (b) and Pelodiscus (d), and the autosome carrying the larger NOR of Emydura (f) showing DAPI staining, hybridization signal of R2 element, 28S RNA gene and both probe signals merged. White boxes contain silver‐stained W chromosomes and larger‐NOR autosome for comparison between active NOR position and R2 population. White arrows denote low R2 hybridization signal in rDNA cluster while black arrows denote the active fraction of the rDNA cluster. Scale bar = 10 μm

FIGURE 3

Activity of sex‐linked NORs in turtles detected via silver staining (AgNOR). Metaphase chromosomes from Apalone spinifera female (a) and male (b), Pelodiscus sinensis female (c) and male (d), and Staurotypus triporcatus female (e) and male (f). Red arrow heads point to Ag‐stained NORs. Scale bar = 10 μm

FIGURE 4

Ratio of nucleolus area to nucleus area in Apalone spinifera adults and embryos (a) and differences between stages and sexes (b). NS, not significant; **p < 0.01; ***p < 0.001