UC Berkeley

Abstract

Sex Chromosome Evolution in Insects

by

Shivani Mahajan

Doctor of Philosophy in Integrative Biology

University of California, Berkeley

Professor Doris Bachtrog, Chair

Sex chromosomes play a role in sex determination in several organisms, ranging from humans and other mammals to flies, and present unique characteristics that distinguish them from autosomes. However, the underlying evolutionary forces that drive sex chromosome evolution and the molecular processes and mechanisms shaping their unusual characteristics are poorly understood, even in well-studied organisms like Drosophila melanogaster and humans. In this dissertation, I studied several aspects of sex chromosome evolution, including the mechanism of dosage compensation, Y degeneration and gene content evolution of Y chromosomes, sequence evolution of recently formed neo-sex chromosomes, and changes of the chromatin landscape in species with newly evolved sex chromosomes, in both model and non-model organisms.

X and Y chromosomes are derived from a pair of ordinary autosomes. Y chromosomes do not recombine, which leads to their degeneration and causes them to lose genes. This creates a gene-dose imbalance for X-linked genes in males compared to females, and also compared to autosomes, and different organisms have evolved different mechanisms to compensate for this imbalance. While in humans one of the two X chromosomes in females is randomly inactivated in different cells, Drosophila melanogaster males hyper-transcribe genes on their single X chromosome. In the first chapter, I tested for dosage compensation in the order Strepsiptera, which is a sister group of Coleoptera (beetles). Using DNA-seq and RNA-seq data, I showed that the species Xenos vesparum shares an X chromosome with the flour beetle Tribolium that is fully dosage compensated. However, X. vesparum also contains a more recently evolved X chromosome that is autosomal in Tribolium, and which has evolved only partial dosage compensation.

Y chromosomes degenerate and contain very few genes. They also accumulate repetitive DNA, which makes their sequencing and assembly extremely difficult. In the second chapter I developed a bioinformatics pipeline to extract Y-linked coding sequences using DNA-seq and RNA-seq data from males and females of a species, without having to assemble the repetitive Y chromosome. I applied this pipeline to several Diptera flies, to characterize and study their Y gene content. I showed that there was no overlap between Y-linked genes in different Dipterans, and that different species had convergently acquired genes with testis-specific functions, highlighting the importance of male-specific selection in driving the evolution of Y gene content.

Species with newly evolved neo-sex chromosomes, such as Drosophila miranda, provide a unique opportunity to study sex chromosome evolution, since its neo-Y still retains significant sequence identity to its former homolog, the neo-X chromosome, and it also still contains thousands of genes. However, this also makes the neo-Y chromosome particularly difficult to assemble using short read technology alone, due to the inability to unambiguously assign sequencing reads to either the neo-X or the neo-Y, and also due to the repetitive nature of the neo-Y in general. In the third chapter, I used Single Molecule Sequencing (Pacbio) and Chromatin Conformation Capture along with Illumina whole genome shot-gun sequencing to build a high quality genome assembly for Drosophila miranda. I showed that the neo-Y chromosome has greatly increased in size by almost 3-fold, compared to the neo-X chromosome, due to the accumulation of repetitive sequences, but also due to the expansion of some male-specific genes on the neo-Y. This assembly provides the basis for future functional studies of sex chromosome evolution in this species.

A large proportion of the genome in Drosophila miranda is repetitive and heterochromatic (~43%). The different chromatin compartments are established during early embryonic development, but very little is known about how this happens at the molecular level, and what primary sequences target parts of the genome to establish a heterochromatic conformation. In the fourth chapter I studied heterochromatin establishment in D.miranda during early development using single embryo ChIP-seq. I showed that males experience a delay in the establishment of the heterochromatic histone mark H3K9me3 compared to females. I also investigated signatures of H3K9me3 spreading near euchromatic transposable element (TE)/repeat insertions and showed that this signal is more pronounced for TEs that are targeted by maternally inherited piRNAs, suggesting that they may play an important role in the establishment of heterochromatin.