Chromosome-scale assembly of the streamlined picoeukaryote Picochlorum sp. SENEW3 genome reveals Rabl-like chromatin structure and potential for C4 photosynthesis.

Genome sequencing and assembly of the photosynthetic picoeukaryotic Picochlorum sp. SENEW3 revealed a compact genome with a reduced gene set, few repetitive sequences, and an organized Rabl-like chromatin structure. Hi-C chromosome conformation capture revealed evidence of possible chromosomal translocations, as well as putative centromere locations. Maintenance of a relatively few selenoproteins, as compared to similarly sized marine picoprasinophytes Mamiellales, and broad halotolerance compared to others in Trebouxiophyceae, suggests evolutionary adaptation to variable salinity environments. Such adaptation may have driven size and genome minimization and have been enabled by the retention of a high number of membrane transporters. Identification of required pathway genes for both CAM and C4 photosynthetic carbon fixation, known to exist in the marine mamiellale pico-prasinophytes and seaweed Ulva, but few other chlorophyte species, further highlights the unique adaptations of this robust alga. This high-quality assembly provides a significant advance in the resources available for genomic investigations of this and other photosynthetic picoeukaryotes.

Massive Somatic and Germline Chromosomal Integrations of Polydnaviruses in Lepidopterans.

Increasing numbers of horizontal transfer (HT) of genes and transposable elements are reported in insects. Yet the mechanisms underlying these transfers remain unknown. Here we first quantify and characterize the patterns of chromosomal integration of the polydnavirus (PDV) encoded by the Campopleginae Hyposoter didymator parasitoid wasp (HdIV) in somatic cells of parasitized fall armyworm (Spodoptera frugiperda). PDVs are domesticated viruses injected by wasps together with their eggs into their hosts in order to facilitate the development of wasp larvae. We found that six HdIV DNA circles integrate into the genome of host somatic cells. Each host haploid genome suffers between 23 and 40 integration events (IEs) on average 72 h post-parasitism. Almost all IEs are mediated by DNA double-strand breaks occurring in the host integration motif (HIM) of HdIV circles. We show that despite their independent evolutionary origins, PDV from both Campopleginae and Braconidae wasps use remarkably similar mechanisms for chromosomal integration. Next, our similarity search performed on 775 genomes reveals that PDVs of both Campopleginae and Braconidae wasps have recurrently colonized the germline of dozens of lepidopteran species through the same mechanisms they use to integrate into somatic host chromosomes during parasitism. We found evidence of HIM-mediated HT of PDV DNA circles in no less than 124 species belonging to 15 lepidopteran families. Thus, this mechanism underlies a major route of HT of genetic material from wasps to lepidopterans with likely important consequences on lepidopterans.

Investigating bracovirus chromosomal integration and inheritance in lepidopteran host and nontarget species.

Bracoviruses (BVs) are domesticated viruses found in braconid parasitoid wasp genomes. They are composed of domesticated genes from a nudivrius, coding viral particles in which wasp DNA circles are packaged. BVs are viewed as possible vectors of horizontal transfer of genetic material (HT) from wasp to their hosts because they are injected, together with wasp eggs, by female wasps into their host larvae, and because they undergo massive chromosomal integration in multiple host tissues. Here, we show that chromosomal integrations of the Cotesia typhae BV (CtBV) persist up to the adult stage in individuals of its natural host, Sesamia nonagrioides, that survived parasitism. However, while reproducing host adults can bear an average of nearly two CtBV integrations per haploid genome, we were unable to retrieve any of these integrations in 500 of their offspring using Illumina sequencing. This suggests either that host gametes are less targeted by CtBVs than somatic cells or that gametes bearing BV integrations are nonfunctional. We further show that CtBV can massively integrate into the chromosomes of other lepidopteran species that are not normally targeted by the wasp in the wild, including one which is divergent by at least 100 million years from the natural host. Cell entry and chromosomal integration of BVs are thus unlikely to be major factors shaping wasp host range. Together, our results shed new light on the conditions under which BV-mediated wasp-to-host HT may occur and provide information that may be helpful to evaluate the potential risks of uncontrolled HT associated with the use of parasitoid wasps as biocontrol agents.

Assessing the Impact of a Viral Infection on the Expression of Transposable Elements in the Cabbage Looper Moth (Trichoplusia ni).

Most studies of stress-induced transposable element (TE) expression have so far focused on abiotic sources of stress. Here, we analyzed the impact of an infection by the AcMNPV baculovirus on TE expression in a cell line (Tnms42) and midgut tissues of the cabbage looper moth (Trichoplusia ni). We find that a large fraction of TE families (576/636 in Tnms42 cells and 503/612 in midgut) is lowly expressed or not expressed at all [≤ 4 transcripts per million (TPM)] in the uninfected condition (median TPM of 0.37 in Tnms42 and 0.46 in midgut cells). In the infected condition, a total of 62 and 187 TE families were differentially expressed (DE) in midgut and Tnms42 cells, respectively, with more up- (46) than downregulated (16) TE families in the former and as many up- (91) as downregulated (96) TE families in the latter. Expression log2 fold changes of DE TE families varied from -4.95 to 9.11 in Tnms42 cells and from -4.28 to 7.66 in midgut. Large variations in expression profiles of DE TEs were observed depending on the type of cells and on time after infection. Overall, the impact of AcMNPV on TE expression in T. ni is moderate but potentially sufficient to affect TE activity and genome architecture. Interestingly, one host-derived TE integrated into AcMNPV genomes is highly expressed in infected Tnms42 cells. This result shows that virus-borne TEs can be expressed, further suggesting that they may be able to transpose and that viruses may act as vectors of horizontal transfer of TEs in insects.

Genome-Wide Patterns of Bracovirus Chromosomal Integration into Multiple Host Tissues during Parasitism.

Bracoviruses are domesticated viruses found in parasitic wasp genomes. They are composed of genes of nudiviral origin that are involved in particle production and proviral segments containing virulence genes that are necessary for parasitism success. During particle production, proviral segments are amplified and individually packaged as DNA circles in nucleocapsids. These particles are injected by parasitic wasps into host larvae together with their eggs. Bracovirus circles of two wasp species were reported to undergo chromosomal integration in parasitized host hemocytes, through a conserved sequence named the host integration motif (HIM). Here, we used bulk Illumina sequencing to survey integrations of Cotesia typhae bracovirus circles in the DNA of its host, the maize corn borer (Sesamia nonagrioides), 7 days after parasitism. First, assembly and annotation of a high-quality genome for C. typhae enabled us to characterize 27 proviral segments clustered in proviral loci. Using these data, we characterized large numbers of chromosomal integrations (from 12 to 85 events per host haploid genome) for all 16 bracovirus circles containing a HIM. Integrations were found in four S. nonagrioides tissues and in the body of a caterpillar in which parasitism had failed. The 12 remaining circles do not integrate but are maintained at high levels in host tissues. Surprisingly, we found that HIM-mediated chromosomal integration in the wasp germ line has occurred accidentally at least six times during evolution. Overall, our study furthers our understanding of wasp-host genome interactions and supports HIM-mediated chromosomal integration as a possible mechanism of horizontal transfer from wasps to their hosts. IMPORTANCE Bracoviruses are endogenous domesticated viruses of parasitoid wasps that are injected together with wasp eggs into wasp host larvae during parasitism. Several studies have shown that some DNA circles packaged into bracovirus particles become integrated into host somatic genomes during parasitism, but the phenomenon has never been studied using nontargeted approaches. Here, we use bulk Illumina sequencing to systematically characterize and quantify bracovirus circle integrations that occur in four tissues of the Mediterranean corn borer (Sesamia nonagrioides) during parasitism by the Cotesia typhae wasp. Our analysis reveals that all circles containing a HIM integrate at substantial levels (from 12 to 85 integrations per host cell, in total) in all tissues, while other circles do not integrate. In addition to shedding new light on wasp-bracovirus-host interactions, our study supports HIM-mediated chromosomal integration of bracovirus as a possible source of wasp-to-host horizontal transfer, with long-term evolutionary consequences.

Draft nuclear genome and complete mitogenome of the Mediterranean corn borer, Sesamia nonagrioides, a major pest of maize.

The Mediterranean corn borer (Sesamia nonagrioides, Noctuidae, Lepidoptera) is a major pest of maize in Europe and Africa. Here, we report an assembly of the nuclear and mitochondrial genome of a pool of inbred males and females third-instar larvae, based on short- and long-read sequencing. The complete mitochondrial genome is 15,330 bp and contains all expected 13 and 24 protein-coding and RNA genes, respectively. The nuclear assembly is 1021 Mb, composed of 2553 scaffolds and it has an N50 of 1105 kb. It is more than twice larger than that of all Noctuidae species sequenced to date, mainly due to a higher repeat content. A total of 17,230 protein-coding genes were predicted, including 15,776 with InterPro domains. We provide detailed annotation of genes involved in sex determination (doublesex, insulin-like growth factor 2 mRNA-binding protein, and P-element somatic inhibitor) and of alpha-amylase genes possibly involved in interaction with parasitoid wasps. We found no evidence of recent horizontal transfer of bracovirus genes from parasitoid wasps. These genome assemblies provide a solid molecular basis to study insect genome evolution and to further develop biocontrol strategies against S. nonagrioides.

Formation of the CenH3-Deficient Holocentromere in Lepidoptera Avoids Active Chromatin.

Despite the essentiality for faithful chromosome segregation, centromere architectures are diverse among eukaryotes1,2 and embody two main configurations: mono- and holocentromeres, referring, respectively, to localized or unrestricted distribution of centromeric activity. Of the two, some holocentromeres offer the curious condition of having arisen independently in multiple insects, most of which have lost the otherwise essential centromere-specifying factor CenH33 (first described as CENP-A in humans).4-7 The loss of CenH3 raises intuitive questions about how holocentromeres are organized and regulated in CenH3-lacking insects. Here, we report the first chromatin-level description of CenH3-deficient holocentromeres by leveraging recently identified centromere components6,7 and genomics approaches to map and characterize the holocentromeres of the silk moth Bombyx mori, a representative lepidopteran insect lacking CenH3. This uncovered a robust correlation between the distribution of centromere sites and regions of low chromatin activity along B. mori chromosomes. Transcriptional perturbation experiments recapitulated the exclusion of B. mori centromeres from active chromatin. Based on reciprocal centromere occupancy patterns observed along differentially expressed orthologous genes of Lepidoptera, we further found that holocentromere formation in a manner that is recessive to chromatin dynamics is evolutionarily conserved. Our results help us discuss the plasticity of centromeres in the context of a role for the chromosome-wide chromatin landscape in conferring centromere identity rather than the presence of CenH3. Given the co-occurrence of CenH3 loss and holocentricity in insects,7 we further propose that the evolutionary establishment of holocentromeres in insects was facilitated through the loss of a CenH3-specified centromere.

The Impact of Centromeres on Spatial Genome Architecture.

The development of new technologies and experimental techniques is enabling researchers to see what was once unable to be seen. For example, the centromere was first seen as the mediator between spindle fiber and chromosome during mitosis and meiosis. Although this continues to be its most prominent role, we now know that the centromere functions beyond cellular division with important roles in genome organization and chromatin regulation. Here we aim to share the structures and functions of centromeres in various organisms beginning with the diversity of their DNA sequence anatomies. We zoom out to describe their position in the nucleus and ultimately detail the different ways they contribute to genome organization and regulation at the spatial level.

An attempt to select non-genetic variation in resistance to starvation and reduced chill coma recovery time in Drosophila melanogaster.

Phenotypic variance is attributed to genetic and non-genetic factors, and only the former are presumed to be inherited and thus suitable for the action of selection. Although increasing amounts of data suggest that non-genetic variability may be inherited, we have limited empirical data in animals. Here, we performed an artificial selection experiment using Drosophila melanogaster inbred lines. We quantified the response to selection for a decrease in chill coma recovery time and an increase in starvation resistance. We observed a weak response to selection in the inbred and outbred lines, with variability across lines. At the end of the selection process, differential expression was detected for some genes associated with epigenetics, the piRNA pathway and canalization functions. As the selection process can disturb the canalization process and increase the phenotypic variance of developmental traits, we also investigated possible effects of the selection process on the number of scutellar bristles, fluctuating asymmetry levels and fitness estimates. These results suggest that, contrary to what was shown in plants, selection of non-genetic variability is not straightforward in Drosophila and appears to be strongly genotype dependent.

Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C.

In chromosome conformation capture experiments (Hi-C), the accuracy with which contacts are detected varies due to the uneven distribution of restriction sites along genomes. In addition, repeated sequences or homologous regions remain indistinguishable because of the ambiguities they introduce during the alignment of the sequencing reads. We addressed both limitations by designing and engineering 144 kb of a yeast chromosome with regularly spaced restriction sites (Syn-HiC design). In the Syn-HiC region, Hi-C signal-to-noise ratio is enhanced and can be used to measure the shape of an unbiased distribution of contact frequencies, allowing to propose a robust definition of a Hi-C experiment resolution. The redesigned region is also distinguishable from its native homologous counterpart in an otherwise isogenic diploid strain. As a proof of principle, we tracked homologous chromosomes during meiotic prophase in synchronized and pachytene-arrested cells and captured important features of their spatial reorganization, such as chromatin restructuration into arrays of Rec8-delimited loops, centromere declustering, individualization, and pairing. Overall, we illustrate the promises held by redesigning genomic regions to explore complex biological questions.

Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle.

Duplication and segregation of chromosomes involves dynamic reorganization of their internal structure by conserved architectural proteins, including the structural maintenance of chromosomes (SMC) complexes cohesin and condensin. Despite active investigation of the roles of these factors, a genome-wide view of dynamic chromosome architecture at both small and large scale during cell division is still missing. Here, we report the first comprehensive 4D analysis of the higher-order organization of the Saccharomyces cerevisiae genome throughout the cell cycle and investigate the roles of SMC complexes in controlling structural transitions. During replication, cohesion establishment promotes numerous long-range intra-chromosomal contacts and correlates with the individualization of chromosomes, which culminates at metaphase. In anaphase, mitotic chromosomes are abruptly reorganized depending on mechanical forces exerted by the mitotic spindle. Formation of a condensin-dependent loop bridging the centromere cluster with the rDNA loci suggests that condensin-mediated forces may also directly facilitate segregation. This work therefore comprehensively recapitulates cell cycle-dependent chromosome dynamics in a unicellular eukaryote, but also unveils new features of chromosome structural reorganization during highly conserved stages of cell division.

3D organization of synthetic and scrambled chromosomes.

Although the design of the synthetic yeast genome Sc2.0 is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes may affect genome organization and potentially alter cellular functions. We report here the Hi-C-determined three-dimensional (3D) conformations of Sc2.0 chromosomes. The absence of repeats leads to a smoother contact pattern and more precisely tractable chromosome conformations, and the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploit the contact maps to detect rearrangements induced in SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) strains.

Total synthesis of a functional designer eukaryotic chromosome.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

Assembling DNA fragments by USER fusion.

Recent advances in DNA synthesis technology make it possible to design and synthesize DNA fragments of several kb in size. However, the process of assembling the smaller DNA fragments into a larger DNA segment is still a cumbersome process. In this chapter, we describe the use of the uracil specific excision reaction (USER)-mediated approach for rapid and efficient assembly of multiple DNA fragments both in vitro and in vivo (using Escherichia coli). For USER fusion in vitro assembly, each of the individual building blocks (BBs), 0.75 kb in size (that are to be assembled), was amplified using the appropriate forward and reverse primers containing a single uracil (U) and DNA polymerase. The overlaps between adjoining BBs were 8-13 base pairs. An equimolar of the amplified BBs were mixed together and treated by USER enzymes to generate complementary 3' single-strand overhangs between adjoining BBs, which were then ligated and amplified simultaneously to generate the larger 3-kb segments. The assembled fragments were then cloned into plasmid vectors and sequenced to confirm their identity. For USER fusion in vivo assembly in E. coli, USER treatment of the BBs was performed in the presence of a synthetic plasmid, which had 8-13 base pair overlaps at the 5'-end of the 5' BB and at the 3'-end of the 3' BB in the mixture. The USER treated product was then transformed directly into E. coli to efficiently and correctly reconstitute the recombinant plasmid containing the desired target insert. The latter approach was also used to rapidly assemble three different target genes into a vector to form a new synthetic plasmid construct.

Assembling large DNA segments in yeast.

As described in a different chapter in this volume, the uracil-specific excision reaction (USER) fusion method can be used to assemble multiple small DNA fragments (∼0.75-kb size) into larger 3-kb DNA segments both in vitro and in vivo (in Escherichia coli). However, in order to assemble an entire synthetic yeast genome (Sc2.0 project), we need to be able to assemble these 3-kb pieces into larger DNA segments or chromosome-sized fragments. This assembly into larger DNA segments is carried out in vivo, using homologous recombination in yeast. We have successfully used this approach to assemble a 40-kb chromosome piece in the yeast Saccharomyces cerevisiae. A lithium acetate (LiOAc) protocol using equimolar amount of overlapping smaller fragments was employed to transform yeast. In this chapter, we describe the assembly of 3-kb fragments with an overlap of one building block (∼750 base pairs) into a 40-kb DNA piece.

The Build-a-Genome course.

Build-a-Genome is an intensive laboratory course at Johns Hopkins University that introduces undergraduates to the burgeoning field of synthetic biology. In addition to lectures that provide a comprehensive overview of the field, the course contains a unique laboratory component in which the students contribute to an actual, ongoing project to construct the first synthetic eukaryotic cell, a yeast cell composed of man-made parts. In doing so, the students acquire basic molecular biology skills and gain a truly "graduate student-like experience" in which they take ownership of their projects, troubleshoot their own experiments, present at frequent laboratory meetings, and are given 24-h access to the laboratory, albeit with all the guidance they will need to complete their projects during the semester. In this chapter, we describe the organization of the course and provide advice for anyone interested in starting a similar course at their own institution.

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.

Uneven distribution of mating types among genotypes of Candida glabrata isolates from clinical samples.

In order to shed light on its basic biology, we initiated a population genetic analysis of Candida glabrata, an emerging pathogenic yeast with no sexual stage yet recognized. A worldwide collection of clinical strains was subjected to analysis using variable number of tandem repeats (VNTR) at nine loci. The clustering of strains obtained with this method was congruent with that obtained using sequence polymorphism of the NMT1 gene, a locus previously proposed for lineage assignment. Linkage disequilibrium supported the hypothesis of a mainly clonal reproduction. No heterozygous diploid genotype was found. Minimum-spanning tree analysis of VNTR data revealed clonal expansions and associated genotypic diversification. Mating type analysis revealed that 80% of the strains examined are MATa and 20% MATalpha and that the two alleles are not evenly distributed. The MATa genotype dominated within large clonal groups that contained only one or a few MATalpha types. In contrast, two groups were dominated by MATalpha strains. Our data are consistent with rare independent mating type switching events occurring preferentially from type a to alpha, although the alternative possibility of selection favoring type a isolates cannot be excluded.

Genomic polymorphism in the population of Candida glabrata: gene copy-number variation and chromosomal translocations.

The genomic sequence of the type strain of the opportunist human pathogen Candida glabrata (CBS138, ATCC 2001) is available since 2004. This allows the analysis of genomic structure of other strains by comparative genomic hybridization. We present here the molecular analysis of a collection of 183 C. glabrata strains isolated from patients hospitalized in France and around the world. We show that the mechanisms of microevolution within this asexual species include rare reciprocal chromosomal translocations and recombination within tandem arrays of repeated genes, and that these account for the frequent size heterogeneity between chromosomes across strains. Gene tandems often encode cell wall proteins suggesting a possible role in adaptation to the environment.

The asexual yeast Candida glabrata maintains distinct a and alpha haploid mating types.

The genome of the type strain of Candida glabrata (CBS138, ATCC 2001) contains homologs of most of the genes involved in mating in Saccharomyces cerevisiae, starting with the mating pheromone and receptor genes. Only haploid cells are ever isolated, but C. glabrata strains of both mating types are commonly found, the type strain being MAT alpha and most other strains, such as BG2, being MATa. No sexual cycle has been documented for this species. In order to understand which steps of the mating pathway are defective, we have analyzed the expression of homologs of some of the key genes involved as well as the production of mating pheromones and the organism's sensitivity to artificial pheromones. We show that cells of opposite mating types express both pheromone receptor genes and are insensitive to pheromones. Nonetheless, cells maintain specificity through regulation of the alpha1 and alpha2 genes and, more surprisingly, through differential splicing of the a1 transcript.