The power of synteny : deep evolutionary insights from comparative genomics

Synteny, or the order of genes in a given genome, is an emergent property of individuals and species that has only, with the implementation of next gen-sequencing, become available for evolutionary consideration. In this dissertation, I leverage syntenic information in concert with sequence data to draw connections between evolutionary mechanisms, species divergence, and trait innovation. In Chapter I, I review the major themes that ties my dissertation research together, highlighting important mechanisms at work in evolutionary complexity and introducing the system of which it will be a part. In Chapter II, I use a phylogenomic approach to better understand species relationships within the tribe. I utilize transcriptome sequences and genome derived synteny information to improve orthology detection over standard sequence similarity approaches and gain greater insight into the relationships of the tribe. I also implement differential fractionation rate orthology inference information to address gene tree-species tree incongruence. In Chapter III, as published in Abrahams et al., 2020, I utilize a microsynteny network and phylogenetic inference to investigate the origin and diversification of the MAM/IPMS gene family. I uncover unique MAM-like genes found at the orthologous locus in the Cleomaceae that shed light on the transition from IPMS to MAM. In the Brassicaceae, I identify six distinct MAM clades across Lineages I, II, and III. I characterize the evolutionary impact and consequences of local duplications, transpositions, whole genome duplications, and gene fusion events, generating several new hypotheses on the function and diversity of the MAM locus.

Genomic Imprinting at the Porcine PLAGL1 Locus and the Orthologous Locus in the Human

Implementation of genomic imprinting in mammals often results in cis-acting silencing of a gene cluster and monoallelic expression, which are important for mammalian growth and function. Compared with widely documented imprinting status in humans and mice, current understanding of genomic imprinting in pigs is relatively limited. The objectives of this study were to identify DNA methylation status and allelic expression of alternative spliced isoforms at the porcine PLAGL1 locus and assess the conservation of the locus compared to the orthologous human locus. DNA methylome and transcriptome were constructed using porcine parthenogenetic or biparental control embryos. Using methylome, differentially methylated regions between those embryos were identified. Alternative splicing was identified by differential splicing analysis, and monoallelic expression was examined using single nucleotide polymorphism sites. Moreover, topological boundary regions were identified by analyzing CTCF binding sites and compared with the boundary of human orthologous locus. As a result, it was revealed that the monoallelic expression of the PLAGL1 gene in porcine embryos via genomic imprinting was maintained in the adult stage. The porcine PLAGL1 locus was largely conserved in regard to maternal hypermethylation, tissue distribution of mRNA expression, monoallelic expression, and biallelic CTCF-binding, with exceptions on transcript isoforms produced by alternative splicing instead of alternative promoter usage. These findings laid the groundwork for comparative studies on the imprinted PLAGL1 gene and related regulatory mechanisms across species.

Knock-In of a 25-Kilobase Pair BAC-Derived Donor Molecule by Traditional and CRISPR/Cas9-Stimulated Homologous Recombination

ABSTRACTHere, we describe an expansion of the DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus. Driving our efforts was the desire to create a whole animal model that would replace 17 kbp of the mouseBcl2l11gene with the corresponding 25-kbp segment of humanBCL2L11, including a conditionally removable segment (2.9-kbp) of intron 2, a cryptic human exon immediately 3′ of this, and a native human exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic ES cell targeting, dual selection, and recombinase-driven cassette removal (traditional approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with CRISPR RNA guides andCas9into mouse zygotes (CRISPR approach). In both instances we were able to achieve humanization ofBcl2l11to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable,loxP-flanked, 2.9-kbp intronic segment.AUTHOR SUMMARYClustered regularly interspaced short palindromic repeat (CRISPR) technology can be used to place DNA sequences (designed in the laboratory) into the genomes of living organisms. Here, we describe a new method, whereby we have replaced an exceptionally large segment of the mouseBcl2l11gene with the corresponding segment of humanBCL2L11gene. The method represents an expansion of the DNA size limitations typically associated with the introduction of DNA sequences through traditional CRISPR methods.