Genomic insights into fine-scale recombination variation in adaptively diverging threespine stickleback fish (Gasterosteus aculeatus)

Meiotic recombination is one of the major molecular mechanisms generating genetic diversity and influencing genome evolution. By shuffling allelic combinations, it can directly influence the patterns and efficacy of natural selection. Studies in various organisms have shown that the rate and placement of recombination varies substantially within the genome, among individuals, between sexes and among different species. It is hypothesized that this variation plays an important role in genome evolution. In this PhD thesis, I investigated the extent and molecular basis of recombination variation in adaptively diverging threespine stickleback fish (Gasterosteus aculeatus) to further understand its evolutionary implications. I used both ChIP-sequencing and whole genome sequencing of pedigrees to empirically identify and quantify double strand breaks (DSBs) and meiotic crossovers (COs). Whole genome sequencing of large nuclear families was performed to identify meiotic crossovers in 36 individuals of diverging marine and freshwater ecotypes and their hybrids. This produced the first genome-wide high-resolution sex-specific and ecotype-specific map of contemporary recombination events in sticklebacks. The results show striking differences in crossover number and placement between sexes. Females recombine nearly 1.76 times more than males and their COs are distributed all over the chromosome while male COs predominantly occur near the chromosomal periphery. When compared among ecotypes a significant reduction in overall recombination rate was observed in hybrid females compared to pure forms. Even though the known loci underlying marine-freshwater adaptive divergence tend to fall in regions of low recombination, considerable female recombination is observed in the regions between adaptive loci. This suggests that the sexual dimorphism in recombination phenotype may have important evolutionary implications. At the fine-scale, COs and male DSBs are nonrandomly distributed involving ‘semi-hot’ hotspots and coldspots of recombination. I report a significant association of male DSBs and COs with functionally active open chromatin regions like gene promoters, whereas female COs did not show an association more than expected by chance. However, a considerable number of COs and DSBs away from any of the tested open chromatin marks suggests possibility of additional novel mechanisms of recombination regulation in sticklebacks. In addition, we developed a novel method for constructing individualized recombination maps from pooled gamete DNA using linked read sequencing technology by 10X Genomics®. We tested the method by contrasting recombination profiles of gametic and somatic tissue from a hybrid mouse and stickleback fish. Our pipeline faithfully detects previously described recombination hotspots in mice at high resolution and identify many novel hotspots across the genome in both species and thereby demonstrate the efficiency of the novel method. This method could be employed for large scale QTL mapping studies to further understand the genetic basis of recombination variation reported in this thesis. By bridging the gap between natural populations and lab organisms with large clutch sizes and tractable genetic tools, this work shows the utility of the stickleback system and provides important groundwork for further studies of heterochiasmy and divergence in recombination during adaptation to differing environments

Population genetics of the highly polymorphic RPP8 gene family

Plant nucleotide-binding domain and leucine-rich repeat containing (NLR) genes provide some of the most extreme examples of polymorphism in eukaryotic genomes, rivalling even the vertebrate major histocompatibility complex. Surprisingly, this is also true in Arabidopsis thaliana, a predominantly selfing species with low heterozygosity. Here, we investigate how gene duplication and intergenic exchange contribute to this extraordinary variation. RPP8 is a three-locus system that is configured chromosomally as either a direct-repeat tandem duplication or as a single copy locus, plus a locus 2 Mb distant. We sequenced 48 RPP8 alleles from 37 accessions of A. thaliana and 12 RPP8 alleles from Arabidopsis lyrata to investigate the patterns of interlocus shared variation. The tandem duplicates display fixed differences and share less variation with each other than either shares with the distant paralog. A high level of shared polymorphism among alleles at one of the tandem duplicates, the single-copy locus and the distal locus, must involve both classical crossing over and intergenic gene conversion. Despite these polymorphism-enhancing mechanisms, the observed nucleotide diversity could not be replicated under neutral forward-in-time simulations. Only by adding balancing selection to the simulations do they approach the level of polymorphism observed at RPP8. In this NLR gene triad, genetic architecture, gene function and selection all combine to generate diversity

Meiotic Recombination: The Essence of Heredity.

The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans

Molecular characterization of XerS/difSL site-specific recombination system in Streptococcus suis

L'état circulaire du chromosome bactérien pose un problème particulier lors de la réplication. Un nombre impair d'événements de recombinaison homologue donne des chromosomes dimères concaténés qui ne peuvent pas être divisés en cellules filles. Pour résoudre ce problème, les bactéries ont mis au point un mécanisme de résolution des dimères basé sur un système de recombinaison spécifique au site. Ceci est effectué par le système Xer/dif. Dans ce système, les protéines Xer effectuent une réaction de recombinaison dans le site dif au niveau du septum cellulaire immédiatement avant la division cellulaire. Dans la plupart des bactéries, cette réaction est effectuée par deux recombinases, XerC et XerD. Cependant, Streptococcus suis, un agent pathogène zoonotique important utilise un système de recombinaison différent, constitué d'une seule enzyme recombinase appelée XerS, qui catalyse la réaction de recombinaison dans un site dif non conventionnel. Pour caractériser le mode de clivage de XerS, des expériences EMSA ont été réalisées en utilisant des fragments de PCR marqués par HEX et des "suicide substrates". Nos données suggèrent que 1.) XerS est capable de lier la séquence entière de difSL; 2.) XerS lie plus efficacement le côté gauche des mutants difSL incomplets que le côté droit; 3.) XerS coupe les brins supérieur et inférieur du site difSL, avec une réaction plus efficace au bas. 4.) Modifications des nucléotides de la région la plus externe ou de la région centrale changent les préférences de clivage. 5.) XerS n'a montré aucune activité spécifique sur un autre site dif non conventionnel des Firmicutes, 6.) XerS interagit avec la sous-unité FtsK-y. L'ensemble des résultats présentés permet de mieux comprendre le fonctionnement de la recombinaison XerS dans le système de recombinase unique de Streptococcus et comment cette recombinaison est régulée par des facteurs de l'hôte.The circular state of the bacterial chromosome presents a specific problem during replication. An odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. To solve this problem, bacteria have developed a mechanism of dimer resolution based on site-specific recombination system. This is performed by the Xer/dif system. In this system, the Xer proteins perform a recombination reaction in the dif site at the cell septum immediately prior to cell division. In most bacteria this reaction is performed by two recombinases, XerC and XerD. However, an important zoonotic pathogen; Streptococcus suis harbors a different recombination system, composed by a single recombinase enzyme called XerS, that catalyzes the recombination reaction in an unconventional dif site; difSL. A region characterized by two imperfect inverted repeat regions that flank a central region of 11 bp.To characterize the mode of cleavage of XerS, EMSA experiments were performed by using HEX-labelled PCR fragments and “nicked suicide substrates”. Our data suggests that; 1.) XerS is able to bind the entire difSL sequence; 2.) XerS binds more efficiently the left half side on incomplete difSL mutants than the right half side; 3.) XerS cleaves both the top and bottom strands of the difSL site, with a more efficient reaction at the bottom strand; 4.) Nucleotides at the outermost region of a T rich region seem to be determinant for binding selectivity and modifications of the extra spacing between the inverted repeat arms as well as length modifications of the central region change cleavage preference. 5.) XerS did not show any specific activity on another unconventional dif site in Firmicutes, as tested on difH. 6.) XerS interacts with FtsK-y subunit. This research aims to understand how XerS recombination works in the single recombinase system of Streptococcus and how this recombination is regulated by host factors. Exploration of these recombinases will provide a better understanding of the mechanisms of DNA exchange and genome stability in bacteria. It can also increase our knowledge of the evolution and speciation of recombinogenic bacteria

Properties and Action of Tn3 Resolvase

Under standard in vitro reaction conditions resolvase-mediated recombination is strictly intramolecular, acting only on supercoiled substrates with 2 directly repeated res sites to generate singly linked catenated products. These unique properties are not explicable by random collision of the res sites, making the mechanism of site synapsis an intriguing problem. This study has attempted to distinguish between a number of proposed models for site synapsis. A wide range of multiple re3 site constructs were made and the resolution characteristics examined. The results cannot be accounted for by the 'tracking' model for synapsis, based on 1-dimensional diffusion of resolvase along the DNA intervening between the 2 res sites. The resolution characteristics of these multiple res site plasmids are explicable by a 'pairing model' which arises as a natural consequence of the '2-step' model for site synapsis; the bias against non-adjacent events can be explained if there is a tendency to maximise the number of synapsed sites and if there are conformational difficulties in forming more than one non-adjacent synapse. The bias can be relieved by increasing the number of intervening sites; non-adjacent events can occur at a frequency equal to adjacent events when the recombining sites are separated by 2 intervening sites on either side. Inverted sites have been shown to cause 'shadowing' in the same way as directly repeated sites. Methylation protection experiments have shown resolvase recognises and binds to each of the 6 half sites of res in essentially the same way, making major groove interactions with bases of the concensus sequence. Photofootprinting results also imply a similar mode of resolvase binding at each subsite and a similar although not identical pattern of changes in the base stacking and helix geometry at the centres of each of the subsites. It is proposed that these changes represent resolvase-induced bending of the res site DNA. These resolvase-induced changes in base photoreactivity are also detected in in vivo photofootprinting experiments

A Complex Genomic Rearrangement Involving the Endothelin 3 Locus Causes Dermal Hyperpigmentation in the Chicken

Dermal hyperpigmentation or Fibromelanosis (FM) is one of the few examples of skin pigmentation phenotypes in the chicken, where most other pigmentation variants influence feather color and patterning. The Silkie chicken is the most widespread and well-studied breed displaying this phenotype. The presence of the dominant FM allele results in extensive pigmentation of the dermal layer of skin and the majority of internal connective tissue. Here we identify the causal mutation of FM as an inverted duplication and junction of two genomic regions separated by more than 400 kb in wild-type individuals. One of these duplicated regions contains endothelin 3 (EDN3), a gene with a known role in promoting melanoblast proliferation. We show that EDN3 expression is increased in the developing Silkie embryo during the time in which melanoblasts are migrating, and elevated levels of expression are maintained in the adult skin tissue. We have examined four different chicken breeds from both Asia and Europe displaying dermal hyperpigmentation and conclude that the same structural variant underlies this phenotype in all chicken breeds. This complex genomic rearrangement causing a specific monogenic trait in the chicken illustrates how novel mutations with major phenotypic effects have been reused during breed formation in domestic animals

Diverse outcomes of homologous recombination in the human Y chromosome

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.Includes bibliographical references.Mammalian sex chromosomes began diverging from an ordinary pair of autosomes roughly 300 million years ago. Inversions in the evolving Y chromosome sequentially suppressed recombination with the X chromosome. While pseudoautosomal regions in the human Y chromosome still participate regularly in allelic homologous recombination, the male-specific region of the Y (MSY) - the only haploid portion of the nuclear genome - does not. It does, however, engage in non-allelic homologous recombination. In this thesis, I examine modes and outcomes of non-allelic homologous recombination in the MSY. The predictions presented here are based on the double-strand break repair model of recombination between homologous chromosomes, in which a double-strand break (DSB) is the common precursor to crossing over and gene conversion. First, I show that massive MSY-specific palindromes, which maintain arm-to-arm sequence identity via gene conversion, are also the targets of crossing over. Crossover events in palindromes can lead to isochromosome formation and diverse reproductive disorders including sex reversal, male infertility, and Turner syndrome. Second, I demonstrate that a region of the MSY - thought to be recombinationally suppressed with the X chromosome - does undergo extensive X-Y gene conversion. This region encompasses hotspots of ectopic crossover events that lead to X-Y translocations associated with sex reversal syndromes. Although sequences in the MSY engage in productive recombination via gene conversion, alternative resolution of DSBs by crossing over can produce evolutionary "dead ends".by Julian H. Lange.Ph.D

The Cyclically Seasonal Drosophila subobscura Inversion O Originated From Fragile Genomic Sites and Relocated Immunity and Metabolic Genes

Chromosome inversions are important contributors to standing genetic variation in Drosophila subobscura. Presently, the species is experiencing a rapid replacement of high-latitude by low-latitude inversions associated with global warming. Yet not all low-latitude inversions are correlated with the ongoing warming trend. This is particularly unexpected in the case of O because it shows a regular seasonal cycle that peaks in summer and rose with a heatwave. The inconsistent behavior of O across components of the ambient temperature suggests that is causally more complex than simply due to temperature alone. In order to understand the dynamics of O, high-quality genomic data are needed to determine both the breakpoints and the genetic content. To fill this gap, here we generated a PacBio long read-based chromosome-scale genome assembly, from a highly homozygous line made isogenic for an O chromosome. Then we isolated the complete continuous sequence of O by conserved synteny analysis with the available reference genome. Main findings include the following: (i) the assembled O inversion stretches 9.936 Mb, containing > 1,000 annotated genes; (ii) O had a complex origin, involving multiple breaks associated with non-B DNA-forming motifs, formation of a microinversion, and ectopic repair in trans with the two homologous chromosomes; (iii) the O breakpoints carry a pre-inversion record of fragility, including a sequence insertion, and transposition with later inverted duplication of an Attacin immunity gene; and (iv) the O inversion relocated the major insulin signaling forkhead box subgroup O (foxo) gene in tight linkage with its antagonistic regulatory partner serine/threonine-protein kinase B (Akt1) and disrupted concerted evolution of the two inverted Attacin duplicates, reattaching them to dFOXO metabolic enhancers. Our findings suggest that O exerts antagonistic pleiotropic effects on reproduction and immunity, setting a framework to understand its relationship with climate change. Furthermore, they are relevant for fragility in genome rearrangement evolution and for current views on the contribution of breakage versus repair in shaping inversion-breakpoint junctions

Investigating the regulation of cohesin dynamics during meiotic prophase in C. elegans

The physical linking of sister chromatids after S-phase is known as sister chromatid cohesion (SCC) and is largely provided by the cohesin complex. The coordinated loss of SCC at anaphase onset is essential for correct chromosome segregation, a process mediated by proteolysis of the kleisin subunit of cohesin. In addition, during mitotic prophase of most organisms a large portion of cohesin is removed from chromosomes by a non-proteolytic pathway that depends on the conserved protein WAPL. Cohesin is also known to reload and SCC is re-established in response to DSBs after mitotic S-phase. During meiosis, SCC is similarly established during S-phase, but is then released in two steps during the sequential meiotic divisions. Also, unlike mitosis, multiple cohesin complexes with divergent functions exist in meiosis. Whether cohesin is dynamically associated with chromosomes during meiotic prophase and how this may be regulated was not known. Thus, the key aims of this project were to determine if WAPL mediates cohesin removal during meiotic prophase, and to find out if meiotic cohesin complexes display turnover on prophase chromosomes of the nematode C. elegans. Alternative meiosis-specific kleisins (REC-8, COH-3, and COH-4) define the different complexes present during worm meiosis. I show here that WAPL-1 limits the association of COH-3/4 complexes with meiotic prophase chromosomes, which severely limits their cohesive function. REC-8 complexes on the other hand are not affected much by WAPL-1. I show that loss of WAPL-1 affects the structure of axial elements and disrupts chromosome segregation and DSB repair. I also demonstrate by FRAP live imaging that there is significant turnover of cohesin on meiotic prophase chromosomes. Dynamic turnover of COH-3 is much greater that REC-8, as predicted by the different sensitivity to WAPL-1 of REC-8 and COH-3 cohesin complexes. These findings demonstrate that cohesin is actively removed and reloaded during meiotic prophase. Dysregulation of these processes could be relevant for human fertility, since SCC exhaustion over time is thought to contribute to the decline in fertility with increased maternal age.Open Acces