Alternate isoforms are important contributors to phenotypic diversity across eukaryotes. Although short-read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length transcripts, preventing the identification of many alternate isoforms. Long-read sequencing technologies have made it possible to sequence full-length alternative transcripts, accurately characterizing alternative splicing events, alternate transcription start and end sites, and differences in UTR regions. Here, we use Pacific Biosciences (PacBio) long-read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five organs in threespine stickleback fish (Gasterosteus aculeatus), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly in which gene annotations are based on short-read RNA sequencing and predictions from coding sequence of other species. This suggests some of the existing annotations may be inaccurate or alternative transcripts may not be fully characterized. Using Iso-Seq we detected thousands of novel isoforms, indicating many isoforms are absent in the current Ensembl gene annotations. In addition, we refined many of the existing annotations within the genome. We noted many improperly positioned transcription start sites that were refined with long-read sequencing. The Iso-Seq-predicted transcription start sites were more accurate and verified through ATAC-seq. We also detected many alternative splicing events between sexes and across organs. We found a substantial number of genes in both somatic and gonadal samples that had sex-specific isoforms. Our study highlights the power of long-read sequencing to study the complexity of transcriptomes, greatly improving genomic resources for the threespine stickleback fish.

Allele-specific gene expression evolves rapidly across gametologs on heteromorphic sex chromosomes (X and Y or Z and W). Current models of sex chromosome evolution suggest this occurs through the rapid accumulation of mutations within regulatory regions. However, these patterns have not been demonstrated empirically due to the limited number of Y chromosome assemblies available to survey sequence evolution outside of coding regions. The threespine stickleback (Gasterosteus aculeatus) Y chromosome is an ideal model to test hypotheses of rapid regulatory evolution due to its intermediate state of divergence from the X chromosome. A large number of Y-linked gametologs still exist across three differently aged evolutionary strata to test these hypotheses. We found that putative enhancer regions (accessible chromatin regions defined through ATAC-seq) on the Y chromosome exhibited elevated substitution rates when compared to intergenic regions and synonymous sites within coding regions. This strongly suggests that many regulatory regions are under positive selection on the Y chromosome. This divergence was correlated with X-biased gametolog expression, indicating the loss of expression from the Y chromosome may be favored by selection. Our findings provide evidence that Y-linked regulatory regions exhibit signs of positive selection quickly after the suppression of recombination, supporting recent theoretical models that show the rapid divergence of regulatory regions may be favored to mask deleterious mutations on the Y chromosome.