Whole-Genome Resequencing Reveals Extensive Natural Variation in the Model Green Alga Chlamydomonas reinhardtii.

We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits.

The Chlamydomonas genome project: a decade on.

The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis, and micronutrient homeostasis. Ten years since its genome project was initiated an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the omics era. Housed at Phytozome, the plant genomics portal of the Joint Genome Institute (JGI), the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of whole transcriptome sequencing (RNA-Seq) data. We present here the past, present, and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions, and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes.

A unified taxonomy for ciliary dyneins.

The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, 54 proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologs of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles, and orthologs has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.

The Chlamydomonas genome reveals the evolution of key animal and plant functions.

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

EST assembly supported by a draft genome sequence: an analysis of the Chlamydomonas reinhardtii transcriptome.

Clustering and assembly of expressed sequence tags (ESTs) constitute the basis for most genomewide descriptions of a transcriptome. This approach is limited by the decline in sequence quality toward the end of each EST, impacting both sequence clustering and assembly. Here, we exploit the available draft genome sequence of the unicellular green alga Chlamydomonas reinhardtii to guide clustering and to correct errors in the ESTs. We have grouped all available EST and cDNA sequences into 12,063 ACEGs (assembly of contiguous ESTs based on genome) and generated 15,857 contigs of average length 934 nt. We predict that roughly 3000 of our contigs represent full-length transcripts. Compared to previous assemblies, ACEGs show extended contig length, increased accuracy and a reduction in redundancy. Because our assembly protocol also uses ESTs with no corresponding genomic sequences, it provides sequence information for genes interrupted by sequence gaps. Detailed analysis of randomly sampled ACEGs reveals several hundred putative cases of alternative splicing, many overlapping transcription units and new genes not identified by gene prediction algorithms. Our protocol, although developed for and tailored to the C. reinhardtii dataset, can be exploited by any eukaryotic genome project for which both a draft genome sequence and ESTs are available.

Portrait of a species: Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, the first alga subject to a genome project, has been the object of numerous morphological, physiological, and genetic studies. The organism has two genetically determined mating types (plus and minus) and all stages of the simple life cycle can be evoked in culture. In the nearly 60 years since the first standard laboratory strains were isolated, numerous crosses and exchanges among laboratories have led to some confusion concerning strain genealogy. Here we use analyses of the nuclear internal transcribed spacer regions and other genetic traits to resolve these issues, correctly identify strains currently available, and analyze phylogenetic relationships with all other available similar chlamydomonad types. The presence of a 10-bp indel in ITS2 in some but not all copies of the nuclear ribosomal cistrons of an individual organism, and the changing ratios of these in crosses, provide a tool to investigate mechanisms of concerted evolution. The standard C. reinhardtii strains, plus C. smithii +, plus the new eastern North American C. reinhardtii isolates, comprise one morphological species, one biological species of high sexual intercompatibility, and essentially identical ITS sequences (except the tip of helix I of ITS2). However, variant RFLP patterns characterize strains from each geographic site.

Chlamydomonas reinhardtii genome project. A guide to the generation and use of the cDNA information.

The National Science Foundation-funded Chlamydomonas reinhardtii genome project involves (a) construction and sequencing of cDNAs isolated from cells exposed to various environmental conditions, (b) construction of a high-density cDNA microarray, (c) generation of genomic contigs that are nucleated around specific physical and genetic markers, (d) generation of a complete chloroplast genome sequence and analyses of chloroplast gene expression, and (e) the creation of a Web-based resource that allows for easy access of the information in a format that can be readily queried. Phases of the project performed by the groups at the Carnegie Institution and Duke University involve the generation of normalized cDNA libraries, sequencing of cDNAs, analysis and assembly of these sequences to generate contigs and a set of predicted unique genes, and the use of this information to construct a high-density DNA microarray. In this paper, we discuss techniques involved in obtaining cDNA end-sequence information and the ways in which this information is assembled and analyzed. Descriptions of protocols for preparing cDNA libraries, assembling cDNA sequences and annotating the sequence information are provided (the reader is directed to Web sites for more detailed descriptions of these methods). We also discuss preliminary results in which the different cDNA libraries are used to identify genes that are potentially differentially expressed.

The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats.

Chlamydomonas reinhardtii is a unicellular eukaryotic alga possessing a single chloroplast that is widely used as a model system for the study of photosynthetic processes. This report analyzes the surprising structural and evolutionary features of the completely sequenced 203,395-bp plastid chromosome. The genome is divided by 21.2-kb inverted repeats into two single-copy regions of approximately 80 kb and contains only 99 genes, including a full complement of tRNAs and atypical genes encoding the RNA polymerase. A remarkable feature is that >20% of the genome is repetitive DNA: the majority of intergenic regions consist of numerous classes of short dispersed repeats (SDRs), which may have structural or evolutionary significance. Among other sequenced chlorophyte plastid genomes, only that of the green alga Chlorella vulgaris appears to share this feature. The program MultiPipMaker was used to compare the genic complement of Chlamydomonas with those of other chloroplast genomes and to scan the genomes for sequence similarities and repetitive DNAs. Among the results was evidence that the SDRs were not derived from extant coding sequences, although some SDRs may have arisen from other genomic fragments. Phylogenetic reconstruction of changes in plastid genome content revealed that an accelerated rate of gene loss also characterized the Chlamydomonas/Chlorella lineage, a phenomenon that might be independent of the proliferation of SDRs. Together, our results reveal a dynamic and unusual plastid genome whose existence in a model organism will allow its features to be tested functionally.

CHLAMYDOMONAS AS A MODEL ORGANISM.

The unicellular green alga Chlamydomonas offers a simple life cycle, easy isolation of mutants, and a growing array of tools and techniques for molecular genetic studies. Among the principal areas of current investigation using this model system are flagellar structure and function, genetics of basal bodies (centrioles), chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. A genome project has begun with compilation of expressed sequence tag data and gene expression studies and will lead to a complete genome sequence. Resources available to the research community include wild-type and mutant strains, plasmid constructs for transformation studies, and a comprehensive on-line database.