Accumulation of retrotransposons contributes to W chromosome differentiation in the willow beauty Peribatodes rhomboidaria (Lepidoptera: Geometridae)

Abstract

The W chromosome of Lepidoptera is typically gene-poor, repeat-rich and composed of heterochromatin. Pioneering studies investigating this chromosome reported an abundance of mobile elements. However, the actual composition of the W chromosome varies greatly between species, as repeatedly demonstrated by comparative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH). Here we present an analysis of repeats on the W chromosome in the willow beauty, Peribatodes rhomboidaria (Geometridae), a species in which CGH predicted an abundance of W-enriched or W-specific sequences. Indeed, comparative analysis of male and female genomes using RepeatExplorer identified ten putative W chromosome-enriched repeats, most of which are LTR or LINE mobile elements. We analysed the two most abundant: PRW LINE-like and PRW Bel-Pao. The results of FISH mapping and bioinformatic analysis confirmed their enrichment on the W chromosome, supporting the hypothesis that mobile elements are the driving force of W chromosome differentiation in Lepidoptera. As the W chromosome is highly underrepresented in chromosome-level genome assemblies of Lepidoptera, this recently introduced approach, combining bioinformatic comparative genome analysis with molecular cytogenetics, provides an elegant tool for studying this elusive and rapidly evolving part of the genome.

Figure 1

Boxplot of estimated genome percentages of the two most abundant putative W repeats based on RepeatExplorer results from 3 females and 3 males of Peribatodes rhomboidaria. Median, interquartile range, and smallest and largest values in 1.5 × interquartile range are shown.

Figure 2

PRW LINE-like RepeatExplorer graph. Graph layout for cluster 32 generated by RepeatExplorer. Nodes represent individual illumina reads and edges their sequence overlap.

Figure 3

PRW Bel-Pao RepeatExplorer graph. Graph layout for cluster 79 generated by RepeatExplorer. Nodes represent individual illumina reads and edges their sequence overlap.

Figure 4

PCR amplification from genomic DNA of both sexes of Peribatodes rhomboidaria. Although the products are present in all samples, they are visibly stronger in females. Legend (from left): M—marker (100 bp DNA ladder, Invitrogen), PRW LINE-like male, PRW LINE-like female (540 bp), PRW Bel-Pao male, PRW Bel-Pao female (2118 bp; note a smaller band of 1905 bp carrying the deletion).

Figure 5

FISH mapping of PRW LINE-like on pachytene nuclei of Peribatodes rhomboidaria. (A–C) Female chromosomes with DAPI-positive W chromosome (arrow) accumulating hybridization signals and showing multiple scattered signals on other chromosomes, also seen in males (D). (A,D) Merged image of DAPI staining and probe (green), (B) DAPI staining, (C) probe (green). Bar = 10 μm.

Figure 6

FISH mapping of PRW Bel-Pao on pachytene nuclei of Peribatodes rhomboidaria. (A–C) Female chromosomes with DAPI-positive W chromosome (arrow) showing strong hybridization signals, while no signals are visible on other chromosomes and on male pachytene chromosomes (D). (A,D) Merged image of DAPI staining and probe (red), (B) DAPI staining, (C) probe (red)—detail of the W chromosome. Bar = 10 μm.