RNA-Seq analysis reveals expression regulatory divergence of W-linked genes between two contrasting chicken breeds

The regulation of gene expression is a complex process involving organism function and phenotypic diversity, and is caused by cis- and trans- regulation. While prior studies identified the regulatory pattern of the autosome rewiring in hybrids, the role of gene regulation in W sex chromosomes is not clear due to their degradation and sex-limit expression. Here, we developed reciprocal crosses of two chicken breeds, White Leghorn and Cornish Game, which exhibited broad differences of gender-related traits, and assessed the expression of the genes on W chromosome to disentangle the contribution of cis- and trans-factors to expression divergence. We found that there was not appear to be an association between female fecundity and W chromosome gene expression, that 44% of expressed genes had divergent expression between breeds in both tissues, with only 17% of them showing greater expression in White Leghorn. We observed that the proportion of trans-acting genes in W chromosome was higher than cis-regulatory divergence. There were most parental divergence expression genes in muscle, also more heterosis compared with other two tissues. A strong dominant impact of Cornish alleles in brain, while obvious crosses-specific regulatory patterns appeared in liver. Taken together, this work describes the regulatory divergence of W-linked genes between two contrasting breeds and indicates sex chromosomes have a unique regulation and expression mechanism.

The hidden structural variability in avian genomes

Structural variants (SVs) are DNA mutations that can have relevant effects at micro- and macro-evolutionary scales. The detection of SVs is largely limited by the type and quality of sequencing technologies adopted, therefore genetic variability linked to SVs may remain undiscovered, especially in complex repetitive genomic regions. In this study, we used a combination of long-read and linked-read genome assemblies to investigate the occurrence of insertions and dele-tions across the chromosomes of 14 species of birds-of-paradise and two species of estrildid finches including highly repetitive W chro-mosomes. The species sampling encompasses most genera and representatives from all major clades of birds-of-paradise, allowing comparisons between individuals of the same species, genus, and family. We found the highest densities of SVs to be located on the microchromosomes and on the female-specific W chromosome. Genome assemblies of multiple individuals from the same species allowed us to compare the levels of genetic variability linked to SVs and single nucleotide polymorphisms (SNPs) on the W and other chromosomes. Our results demonstrate that the avian W chromosome harbours more genetic variability than previously thought and that its structure is shaped by the continuous accumulation and turn-over of transposable element insertions, especially endogenous retroviruses.

Dynamic Patterns of Sex Chromosome Evolution in Neognath Birds: Many Independent Barriers to Recombination at the ATP5F1A Locus

Avian sex chromosomes evolved after the divergence birds and crocodilians from their common ancestor, so they are much younger than the better-studied chromosomes of mammals. It has long been recognized that there may have been several stages to the evolution of avian sex chromosomes. For example, the CHD1 undergoes recombination in paleognaths but not neognaths. Genome assemblies have suggested there may be variation in the timing of barriers to recombination among Neognathae, but there remains little understanding of the extent of this variability. Here, we look at partial sequences of ATP5F1A, which is on the avian Z and W chromosomes. It is known that recombination of this gene has independently ceased in Galliformes, Anseriformes, and at least five neoavian orders, but whether there are other independent cessations of recombination among Neoaves is not understood. We used a combination of data extracted from published chromosomal-level genomes with data collected using PCR and cloning to identify Z and W copies in 22 orders. Our results suggest there may be at least 19 independent cessations of recombination within Neognathae, and 3 clades that may still be undergoing recombination (or have only recently ceased recombination). Analyses of ATP5F1A protein sequences revealed an increased amino acid substitution rate for W chromosome gametologs, suggesting relaxed purifying selection on the W chromosome. Supporting this hypothesis, we found that the increased substitution rate was particularly pronounced for buried residues, which are expected to be more strongly constrained by purifying selection. This highlights the dynamic nature of avian sex chromosomes, and that this level of variation among clades means they should be a good system to understand sex chromosome evolution.

Chromosome-level genome assembly reveals female-biased genes for sex determination and differentiation in the human blood fluke Schistosoma japonicum

Schistosomiasis is a neglected tropical disease of humans caused by blood flukes of the genus Schistosoma – the only dioecious parasitic flatworms. Although aspects of sex determination, differentiation and reproduction have been studied in some Schistosoma species, almost nothing is understood for Schistosoma japonicum - the causative agent of schistosomiasis japonica. This relates mainly to a lack of high-quality genomic and transcriptomic resources for this species. As current draft genomes for S. japonicum are highly fragmented, we assembled here a chromosome-level reference genome (seven autosomes, the Z-chromosome and partial W-chromosome), achieving a substantially enhanced gene annotation. Utilising this genome, we discovered that the sex chromosomes of S. japonicum and its congener S. mansoni independently suppressed recombination during evolution, forming four and two ‘strata’, respectively. By exploring the W-chromosome and sex-specific transcriptomes, we identified 35 W-linked genes and 257 female-preferentially transcribed genes (FTGs) and identified a signature for sex determination and differentiation in S. japonicum. These FTGs cluster within autosomes or the Z-chromosome and exhibit a highly dynamic transcription profile during the pairing of female and male schistosomules (advanced juveniles), representing a critical phase for the maturation of the female worms, suggesting distinct layers of regulatory control of gene transcription at this stage of development. Collectively, these data provide a valuable resource for further functional genomic characterisation of S. japonicum, shed light on the evolution of sex chromosomes in this highly virulent human blood fluke and provide a pathway to identify novel targets for development of intervention tools against schistosomiasis.

Genome-wide association mapping uncovers sex-associated copy number variation markers and female hemizygous regions on the W chromosome in Salix viminalis

Background Sex chromosomes are in some species largely undifferentiated (homomorphic) with restricted sex determination regions. Homomorphic but different sex chromosomes are found in the closely related genera Populus and Salix indicating flexible sex determination systems, ideal for studies of processes involved in sex chromosome evolution. We have performed genome-wide association studies of sex and analysed sex chromosomes in a population of 265 wild collected Salix viminalis accessions and studied the sex determining locus. Results A total of 19,592 markers were used in association analyses using both Fisher’s exact tests and a single-marker mixed linear model, which resulted in 48 and 41 sex-associated (SA) markers respectively. Across all 48 SA markers, females were much more often heterozygous than males, which is expected if females were the heterogametic sex. The majority of the SA markers were, based on positions in the S. purpurea genome, located on chromosome 15, previously demonstrated to be the sex chromosome. Interestingly, when mapping the genotyping-by-sequencing sequence tag harbouring the two SA markers with the highest significance to the S. viminalis genomic scaffolds, five regions of very high similarity were found: three on a scaffold that represents a part of chromosome 15, one on a scaffold that represents a part of chromosome 9 and one on a scaffold not anchored to the genome. Based on segregation differences of the alleles at the two marker positions and on differences in PCR amplification between females and males we conclude that females had multiple copies of this DNA fragment (chromosome 9 and 15), whereas males only had one (chromosome 9). We therefore postulate that the female specific sequences have been copied from chromosome 9 and inserted on chromosome 15, subsequently developing into a hemizygous W chromosome linked region. Conclusions Our results support that sex determination in S. viminalis is controlled by one locus on chromosome 15. The segregation patterns observed at the SA markers furthermore confirm that S. viminalis females are the heterogametic sex. We also identified a translocation from chromosome 9 to the W chromosome.

Evidences of Z- and W-Linked Regions on the Genome of Fenneropenaeus chinensis

Fenneropenaeus chinensis is a commercially cultured shrimp in China. F. chinensis adults show significant sexual dimorphism, with larger females than males. However, sex determination (SD) of F. chinensis has not yet been elucidated. Clarification of the sex-determining system of F. chinensis could enrich our knowledge of the sex differentiation mechanism in crustaceans and facilitate the study of sex-controlling technologies. Here, we studied the sex-determining system of F. chinensis using the fixation index (FST) between the sexes to detect the genetic differentiation in resequencing data of multiple males and females. We located the candidate sex chromosome in the genome of F. chinensis and concluded the female heterogametic (ZW) SD system. We also assembled female-specific sequences, which could be used as molecular markers to identify the sex of F. chinensis. However, the differentiation of the F. chinensis Z and W chromosome is limited. RNA-seq data detected many genes with male-biased expression in the Z-specific region, which possibly could further intensify the divergency between the Z and W chromosomes.

Degenerated, Undifferentiated, Rearranged, Lost: High Variability of Sex Chromosomes in Geometridae (Lepidoptera) Identified by Sex Chromatin

Sex chromatin is a conspicuous body that occurs in polyploid nuclei of most lepidopteran females and consists of numerous copies of the W sex chromosome. It is also a cytogenetic tool used to rapidly assess the W chromosome presence in Lepidoptera. However, certain chromosomal features could disrupt the formation of sex chromatin and lead to the false conclusion that the W chromosome is absent in the respective species. Here we tested the sex chromatin presence in 50 species of Geometridae. In eight selected species with either missing, atypical, or normal sex chromatin patterns, we performed a detailed karyotype analysis by means of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The results showed a high diversity of W chromosomes and clarified the reasons for atypical sex chromatin, including the absence or poor differentiation of W, rearrangements leading to the neo-W emergence, possible association with the nucleolus, and the existence of multiple W chromosomes. In two species, we detected intraspecific variability in the sex chromatin status and sex chromosome constitution. We show that the sex chromatin is not a sufficient marker of the W chromosome presence, but it may be an excellent tool to pinpoint species with atypical sex chromosomes.

The avian W chromosome is a refugium for endogenous retroviruses with likely effects on female-biased mutational load and genetic incompatibilities

It is a broadly observed pattern that the non-recombining regions of sex-limited chromosomes (Y and W) accumulate more repeats than the rest of the genome, even in species like birds with a low genome-wide repeat content. Here, we show that in birds with highly heteromorphic sex chromosomes, the W chromosome has a transposable element (TE) density of greater than 55% compared to the genome-wide density of less than 10%, and contains over half of all full-length (thus potentially active) endogenous retroviruses (ERVs) of the entire genome. Using RNA-seq and protein mass spectrometry data, we were able to detect signatures of female-specific ERV expression. We hypothesize that the avian W chromosome acts as a refugium for active ERVs, probably leading to female-biased mutational load that may influence female physiology similar to the ‘toxic-Y’ effect in Drosophila males. Furthermore, Haldane's rule predicts that the heterogametic sex has reduced fertility in hybrids. We propose that the excess of W-linked active ERVs over the rest of the genome may be an additional explanatory variable for Haldane's rule, with consequences for genetic incompatibilities between species through TE/repressor mismatches in hybrids. Together, our results suggest that the sequence content of female-specific W chromosomes can have effects far beyond sex determination and gene dosage. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.

Amplified Fragments of an Autosome-Borne Gene Constitute a Significant Component of the W Sex Chromosome of Eremias velox (Reptilia, Lacertidae)

Heteromorphic W and Y sex chromosomes often experience gene loss and heterochromatinization, which is frequently viewed as their “degeneration”. However, the evolutionary trajectories of the heterochromosomes are in fact more complex since they may not only lose but also acquire new sequences. Previously, we found that the heterochromatic W chromosome of a lizard Eremias velox (Lacertidae) is decondensed and thus transcriptionally active during the lampbrush stage. To determine possible sources of this transcription, we sequenced DNA from a microdissected W chromosome sample and a total female DNA sample and analyzed the results of reference-based and de novo assembly. We found a new repetitive sequence, consisting of fragments of an autosomal protein-coding gene ATF7IP2, several SINE elements, and sequences of unknown origin. This repetitive element is distributed across the whole length of the W chromosome, except the centromeric region. Since it retained only 3 out of 10 original ATF7IP2 exons, it remains unclear whether it is able to produce a protein product. Subsequent studies are required to test the presence of this element in other species of Lacertidae and possible functionality. Our results provide further evidence for the view of W and Y chromosomes as not just “degraded” copies of Z and X chromosomes but independent genomic segments in which novel genetic elements may arise.

Sex chromosome diversification in the smooth snake Coronella austriaca (Reptilia, Serpentes)

The smooth snake Coronella austriaca is a widespread Palearctic colubrid species. The species has been the subject of several molecular and phylogeographic studies which highlighted the occurrence of distinct genetic lineages in different areas of the species distribution, but scarce cytogenetic data are currently available on the species. In this paper we present a molecular and karyological study performed with several banding, staining methods and NOR-FISH on samples of C. austriaca from different geographical areas (Italy and Greece) of the species distribution. The molecular and phylogenetic analysis unambiguously placed the studied samples in different clades with a clear geographical pattern. The karyotype of the two female samples studied was composed of 2n = 36 chromosomes with 16 macro- and 20 microchromosomes and a mix of plesiomorphic and derivate chromosome features. All macrochromosomes were biarmed with the exception of pair 5 that was telocentric. NORs were detected on a microchromosome pair. In both females, the pair 4 was heteromorphic (and completely heterochromatic after C-banding in the Italian female), representing the first report of a ZZ/ZW sex chromosome system with female heterogamety in C. austriaca. In addition, the W chromosome showed a different morphology between the two female studied (submetacentric and subtelocentric), highlighting the occurrence of a chromosomal diversification among distinct geographical areas of the species distribution and further supporting that the species contains different diverging evolutionary clades.