The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.

The tree of life is highly reticulate, with the history of population divergence emerging from populations of gene phylogenies that reflect histories of introgression, lineage sorting and divergence. In this study, we investigate global patterns of oak diversity and test the hypothesis that there are regions of the oak genome that are broadly informative about phylogeny.We utilize fossil data and restriction-site associated DNA sequencing (RAD-seq) for 632 individuals representing nearly 250 Quercus species to infer a time-calibrated phylogeny of the world's oaks. We use a reversible-jump Markov chain Monte Carlo method to reconstruct shifts in lineage diversification rates, accounting for among-clade sampling biases. We then map the > 20 000 RAD-seq loci back to an annotated oak genome and investigate genomic distribution of introgression and phylogenetic support across the phylogeny.Oak lineages have diversified among geographic regions, followed by ecological divergence within regions, in the Americas and Eurasia. Roughly 60% of oak diversity traces back to four clades that experienced increases in net diversification, probably in response to climatic transitions or ecological opportunity.The strong support for the phylogeny contrasts with high genomic heterogeneity in phylogenetic signal and introgression. Oaks are phylogenomic mosaics, and their diversity may in fact depend on the gene flow that shapes the oak genome.

Chlamydomonas reinhardtii is a widely used reference organism in studies of photosynthesis, cilia, and biofuels. Most research in this field uses a few dozen standard laboratory strains that are reported to share a common ancestry, but exhibit substantial phenotypic differences. In order to facilitate ongoing Chlamydomonas research and explain the phenotypic variation, we mapped the genetic diversity within these strains using whole-genome resequencing. We identified 524,640 single nucleotide variants and 4812 structural variants among 39 commonly used laboratory strains. Nearly all (98.2%) of the total observed genetic diversity was attributable to the presence of two, previously unrecognized, alternate haplotypes that are distributed in a mosaic pattern among the extant laboratory strains. We propose that these two haplotypes are the remnants of an ancestral cross between two strains with ;2% relative divergence. These haplotype patterns create a fingerprint for each strain that facilitates the positive identification of that strain and reveals its relatedness to other strains. The presence of these alternate haplotype regions affects phenotype scoring and gene expression measurements. Here, we present a rich set of genetic differences as a community resource to allow researchers to more accurately conduct and interpret their experiments with Chlamydomonas.

SignificanceForested ecosystems provide many ecological, economic, and societal benefits, but those benefits are threatened by climate change. Conservation strategies often assume that plants are currently growing in conditions well-suited to their growth, survival, and reproduction, regardless of whether this assumption is valid. We show that an ecosystem-foundational species in California, valley oak (Quercus lobata), is already mismatched to current temperature and will likely experience further declines in growth rates as temperatures rise over the next century. Given this mismatch, new approaches to climate change management are needed. By using genomic information and identifying genotypes with faster growth rates under warmer temperatures, we present an approach to mitigate negative consequences of rising temperatures for species that may already be experiencing maladaptation.

The genus Quercus, which emerged ∼55 million years ago during globally warm temperatures, diversified into ∼450 extant species. We present a high-quality de novo genome assembly of a California endemic oak, Quercus lobata, revealing features consistent with oak evolutionary success. Effective population size remained large throughout history despite declining since early Miocene. Analysis of 39,373 mapped protein-coding genes outlined copious duplications consistent with genetic and phenotypic diversity, both by retention of genes created during the ancient γ whole genome hexaploid duplication event and by tandem duplication within families, including numerous resistance genes and a very large block of duplicated DUF247 genes, which have been found to be associated with self-incompatibility in grasses. An additional surprising finding is that subcontext-specific patterns of DNA methylation associated with transposable elements reveal broadly-distributed heterochromatin in intergenic regions, similar to grasses. Collectively, these features promote genetic and phenotypic variation that would facilitate adaptability to changing environments.

Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.

Engineering a microbial strain for production sometimes entails metabolic modifications that impair essential physiological processes for growth or production. Restoring these functions may require amending a variety of non-obvious physiological networks, and thus, rational design strategies may not be practical. Here we demonstrate that growth and production may be restored by evolution that repairs impaired metabolic function. Furthermore, we use genomics, metabolomics and proteomics to identify several underlying mutations and metabolic perturbations that allow metabolism to repair. Previously, high titers of butanol production were achieved by Escherichia coli using a growth-coupled, modified Clostridial CoA-dependent pathway after all native fermentative pathways were deleted. However, production was only observed in rich media. Native metabolic function of the host was unable to support growth and production in minimal media. We use directed cell evolution to repair this phenotype and observed improved growth, titers and butanol yields. We found a mutation in pcnB which resulted in decreased plasmid copy numbers and pathway enzymes to balance resource utilization. Increased protein abundance was measured for biosynthetic pathways, glycolytic enzymes have increased activity, and adenosyl energy charge was increased. We also found mutations in the ArcAB two-component system and integration host factor (IHF) that tune redox metabolism to alter byproduct formation. These results demonstrate that directed strain evolution can enable systematic adaptations to repair metabolic function and enhance microbial production. Furthermore, these results demonstrate the versatile repair capabilities of cell metabolism and highlight important aspects of cell physiology that are required for production in minimal media.

The genus Quercus, which emerged ~55 million years ago during globally warm temperatures, diversified into ~450 species. We present a high-quality de novo genome assembly of a California endemic oak, Quercus lobata, revealing features consistent with oak evolutionary success. Effective population size remained large throughout history despite declining since the early Miocene. Analysis of 39,373 mapped protein-coding genes outlined copious duplications consistent with genetic and phenotypic diversity, both by retention of genes created during the ancient γ whole genome hexaploid duplication event and by tandem duplication within families, including the numerous resistance genes and also unexpected candidate genes for an incompatibility system involving multiple non-self-recognition genes. An additional surprising finding is that subcontext-specific patterns of DNA methylation associated with transposable elements reveal broadly-distributed heterochromatin in intergenic regions, similar to grasses (another highly successful taxon). Collectively, these features promote genetic and phenotypic variation that would facilitate adaptability to changing environments.