Norman J. Wickett

Ancestral polyploidy in seed plants and angiosperms

Whole-genome duplication (WGD), or polyploidy, followed by gene loss and diploidization has long been recognized as an important evolutionary force in animals, fungi and other organisms, especially plants. The success of angiosperms has been attributed, in part, to innovations associated with gene or whole-genome duplications, but evidence for proposed ancient genome duplications pre-dating the divergence of monocots and eudicots remains equivocal in analyses of conserved gene order. Here we use comprehensive phylogenomic analyses of sequenced plant genomes and more than 12.6 million new expressed-sequence-tag sequences from phylogenetically pivotal lineages to elucidate two groups of ancient gene duplications—one in the common ancestor of extant seed plants and the other in the common ancestor of extant angiosperms. Gene duplication events were intensely concentrated around 319 and 192 million years ago, implicating two WGDs in ancestral lineages shortly before the diversification of extant seed plants and extant angiosperms, respectively. Significantly, these ancestral WGDs resulted in the diversification of regulatory genes important to seed and flower development, suggesting that they were involved in major innovations that ultimately contributed to the rise and eventual dominance of seed plants and angiosperms.

Phylotranscriptomic analysis of the origin and early diversification of land plants

Significance Early branching events in the diversification of land plants and closely related algal lineages remain fundamental and unresolved questions in plant evolutionary biology. Accurate reconstructions of these relationships are critical for testing hypotheses of character evolution: for example, the origins of the embryo, vascular tissue, seeds, and flowers. We investigated relationships among streptophyte algae and land plants using the largest set of nuclear genes that has been applied to this problem to date. Hypothesized relationships were rigorously tested through a series of analyses to assess systematic errors in phylogenetic inference caused by sampling artifacts and model misspecification. Results support some generally accepted phylogenetic hypotheses, while rejecting others. This work provides a new framework for studies of land plant evolution. Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but inconsistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.

FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar

Annual plants grow vegetatively at early developmental stages and then transition to the reproductive stage, followed by senescence in the same year. In contrast, after successive years of vegetative growth at early ages, woody perennial shoot meristems begin repeated transitions between vegetative and reproductive growth at sexual maturity. However, it is unknown how these repeated transitions occur without a developmental conflict between vegetative and reproductive growth. We report that functionally diverged paralogs FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2), products of whole-genome duplication and homologs of Arabidopsis thaliana gene FLOWERING LOCUS T (FT), coordinate the repeated cycles of vegetative and reproductive growth in woody perennial poplar (Populus spp.). Our manipulative physiological and genetic experiments coupled with field studies, expression profiling, and network analysis reveal that reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days in the growing season. The basis for functional differentiation between FT1 and FT2 appears to be expression pattern shifts, changes in proteins, and divergence in gene regulatory networks. Thus, temporal separation of reproductive onset and vegetative growth into different seasons via FT1 and FT2 provides seasonality and demonstrates the evolution of a complex perennial adaptive trait after genome duplication.

Data access for the 1,000 Plants (1KP) project

The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.

A genome triplication associated with early diversification of the core eudicots.

BackgroundAlthough it is agreed that a major polyploidy event, gamma, occurred within the eudicots, the phylogenetic placement of the event remains unclear.ResultsTo determine when this polyploidization occurred relative to speciation events in angiosperm history, we employed a phylogenomic approach to investigate the timing of gene set duplications located on syntenic gamma blocks. We populated 769 putative gene families with large sets of homologs obtained from public transcriptomes of basal angiosperms, magnoliids, asterids, and more than 91.8 gigabases of new next-generation transcriptome sequences of non-grass monocots and basal eudicots. The overwhelming majority (95%) of well-resolved gamma duplications was placed before the separation of rosids and asterids and after the split of monocots and eudicots, providing strong evidence that the gamma polyploidy event occurred early in eudicot evolution. Further, the majority of gene duplications was placed after the divergence of the Ranunculales and core eudicots, indicating that the gamma appears to be restricted to core eudicots. Molecular dating estimates indicate that the duplication events were intensely concentrated around 117 million years ago.ConclusionsThe rapid radiation of core eudicot lineages that gave rise to nearly 75% of angiosperm species appears to have occurred coincidentally or shortly following the gamma triplication event. Reconciliation of gene trees with a species phylogeny can elucidate the timing of major events in genome evolution, even when genome sequences are only available for a subset of species represented in the gene trees. Comprehensive transcriptome datasets are valuable complements to genome sequences for high-resolution phylogenomic analysis.

Mechanisms of Functional and Physical Genome Reduction in Photosynthetic and Nonphotosynthetic Parasitic Plants of the Broomrape Family

The authors report the structure of ten plastid genomes from hemi- and holoparasitic plants and their closest nonparasitic relative. Structural plastome reconfiguration is associated with obligate parasitism. The extent of genome reduction after the loss of photosynthesis is governed by dispensable genes’ proximity to essential genes and the position in operons or by alternative gene function. Nonphotosynthetic plants possess strongly reconfigured plastomes attributable to convergent losses of photosynthesis and housekeeping genes, making them excellent systems for studying genome evolution under relaxed selective pressures. We report the complete plastomes of 10 photosynthetic and nonphotosynthetic parasites plus their nonparasitic sister from the broomrape family (Orobanchaceae). By reconstructing the history of gene losses and genome reconfigurations, we find that the establishment of obligate parasitism triggers the relaxation of selective constraints. Partly because of independent losses of one inverted repeat region, Orobanchaceae plastomes vary 3.5-fold in size, with 45 kb in American squawroot (Conopholis americana) representing the smallest plastome reported from land plants. Of the 42 to 74 retained unique genes, only 16 protein genes, 15 tRNAs, and four rRNAs are commonly found. Several holoparasites retain ATP synthase genes with intact open reading frames, suggesting a prolonged function in these plants. The loss of photosynthesis alters the chromosomal architecture in that recombinogenic factors accumulate, fostering large-scale chromosomal rearrangements as functional reduction proceeds. The retention of DNA fragments is strongly influenced by both their proximity to genes under selection and the co-occurrence with those in operons, indicating complex constraints beyond gene function that determine the evolutionary survival time of plastid regions in nonphotosynthetic plants.

De novo Characterization of the Gametophyte Transcriptome in Bracken Fern, Pteridium Aquilinum

BackgroundBecause of their phylogenetic position and unique characteristics of their biology and life cycle, ferns represent an important lineage for studying the evolution of land plants. Large and complex genomes in ferns combined with the absence of economically important species have been a barrier to the development of genomic resources. However, high throughput sequencing technologies are now being widely applied to non-model species. We leveraged the Roche 454 GS-FLX Titanium pyrosequencing platform in sequencing the gametophyte transcriptome of bracken fern (Pteridium aquilinum) to develop genomic resources for evolutionary studies.Results681,722 quality and adapter trimmed reads totaling 254 Mbp were assembled de novo into 56,256 unique sequences (i.e. unigenes) with a mean length of 547.2 bp and a total assembly size of 30.8 Mbp with an average read-depth coverage of 7.0×. We estimate that 87% of the complete transcriptome has been sequenced and that all transcripts have been tagged. 61.8% of the unigenes had blastx hits in the NCBI nr protein database, representing 22,596 unique best hits. The longest open reading frame in 52.2% of the unigenes had positive domain matches in InterProScan searches. We assigned 46.2% of the unigenes with a GO functional annotation and 16.0% with an enzyme code annotation. Enzyme codes were used to retrieve and color KEGG pathway maps. A comparative genomics approach revealed a substantial proportion of genes expressed in bracken gametophytes to be shared across the genomes of Arabidopsis, Selaginella and Physcomitrella, and identified a substantial number of potentially novel fern genes. By comparing the list of Arabidopsis genes identified by blast with a list of gametophyte-specific Arabidopsis genes taken from the literature, we identified a set of potentially conserved gametophyte specific genes. We screened unigenes for repetitive sequences to identify 548 potentially-amplifiable simple sequence repeat loci and 689 expressed transposable elements.ConclusionsThis study is the first comprehensive transcriptome analysis for a fern and represents an important scientific resource for comparative evolutionary and functional genomics studies in land plants. We demonstrate the utility of high-throughput sequencing of a normalized cDNA library for de novo transcriptome characterization and gene discovery in a non-model plant.

Phylogenomic analysis of transcriptome data elucidates co-occurrence of a paleopolyploid event and the origin of bimodal karyotypes in Agavoideae (Asparagaceae)

PREMISE OF THE STUDY The stability of the bimodal karyotype found in Agave and closely related species has long interested botanists. The origin of the bimodal karyotype has been attributed to allopolyploidy, but this hypothesis has not been tested. Next-generation transcriptome sequence data were used to test whether a paleopolyploid event occurred on the same branch of the Agavoideae phylogenetic tree as the origin of the Yucca-Agave bimodal karyotype. METHODS Illumina RNA-seq data were generated for phylogenetically strategic species in Agavoideae. Paleopolyploidy was inferred in analyses of frequency plots for synonymous substitutions per synonymous site (K(s)) between Hosta, Agave, and Chlorophytum paralogous and orthologous gene pairs. Phylogenies of gene families including paralogous genes for these species and outgroup species were estimated to place inferred paleopolyploid events on a species tree. KEY RESULTS K(s) frequency plots suggested paleopolyploid events in the history of the genera Agave, Hosta, and Chlorophytum. Phylogenetic analyses of gene families estimated from transcriptome data revealed two polyploid events: one predating the last common ancestor of Agave and Hosta and one within the lineage leading to Chlorophytum. CONCLUSIONS We found that polyploidy and the origin of the Yucca-Agave bimodal karyotype co-occur on the same lineage consistent with the hypothesis that the bimodal karyotype is a consequence of allopolyploidy. We discuss this and alternative mechanisms for the formation of the Yucca-Agave bimodal karyotype. More generally, we illustrate how the use of next-generation sequencing technology is a cost-efficient means for assessing genome evolution in nonmodel species.

The Parasitic Plant Genome Project: New Tools for Understanding the Biology of Orobanche and Striga

Abstract The Parasitic Plant Genome Project has sequenced transcripts from three parasitic species and a nonparasitic relative in the Orobanchaceae with the goal of understanding genetic changes associated with parasitism. The species studied span the trophic spectrum from free-living nonparasite to obligate holoparasite. Parasitic species used were Triphysaria versicolor, a photosynthetically competent species that opportunistically parasitizes roots of neighboring plants; Striga hermonthica, a hemiparasite that has an obligate need for a host; and Orobanche aegyptiaca, a holoparasite with absolute nutritional dependence on a host. Lindenbergia philippensis represents the closest nonparasite sister group to the parasitic Orobanchaceae and was included for comparative purposes. Tissues for transcriptome sequencing from each plant were gathered to identify expressed genes for key life stages from seed conditioning through anthesis. Two of the species studied, S. hermonthica and O. aegyptiaca, are economically important weeds and the data generated by this project are expected to aid in research and control of these species and their relatives. The sequences generated through this project will provide an abundant resource of molecular markers for understanding population dynamics, as well as provide insight into the biology of parasitism and advance progress toward understanding parasite virulence and host resistance mechanisms. In addition, the sequences provide important information on target sites for herbicide action or other novel control strategies such as trans-specific gene silencing. Nomenclature: Egyptian broomrape, Orobanche aegyptiaca (Pers.) (Syn. Phelipanche aegyptiaca) ORAAE; Lindenbergia philippensis (Cham. & Schltdl.) Benth. LINPH; yellowbeak owls-clover, Triphysaria versicolor (Fisch. & C.A. Mey) TRVEV; purple witchweed, Striga hermonthica, (Del.) Benth. STRHE.

Selecting superior de novo transcriptome assemblies: Lessons learned by leveraging the best plant genome

Whereas de novo assemblies of RNA-Seq data are being published for a growing number of species across the tree of life, there are currently no broadly accepted methods for evaluating such assemblies. Here we present a detailed comparison of 99 transcriptome assemblies, generated with 6 de novo assemblers including CLC, Trinity, SOAP, Oases, ABySS and NextGENe. Controlled analyses of de novo assemblies for Arabidopsis thaliana and Oryza sativa transcriptomes provide new insights into the strengths and limitations of transcriptome assembly strategies. We find that the leading assemblers generate reassuringly accurate assemblies for the majority of transcripts. At the same time, we find a propensity for assemblers to fail to fully assemble highly expressed genes. Surprisingly, the instance of true chimeric assemblies is very low for all assemblers. Normalized libraries are reduced in highly abundant transcripts, but they also lack 1000s of low abundance transcripts. We conclude that the quality of de novo transcriptome assemblies is best assessed through consideration of a combination of metrics: 1) proportion of reads mapping to an assembly 2) recovery of conserved, widely expressed genes, 3) N50 length statistics, and 4) the total number of unigenes. We provide benchmark Illumina transcriptome data and introduce SCERNA, a broadly applicable modular protocol for de novo assembly improvement. Finally, our de novo assembly of the Arabidopsis leaf transcriptome revealed ~20 putative Arabidopsis genes lacking in the current annotation.