robust-dna-isolation-high-throughput-sequencing-library-construction

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Donald Danforth Plant Science Center

CLV3/ESR (CLE) proteins are important signaling peptides in plants. The short CLE peptide (12-13 amino acids) is cleaved from a larger pre-propeptide and functions as an extracellular ligand. The CLE family is large and has resisted attempts at classification because the CLE domain is too short for reliable phylogenetic analysis and the pre-propeptide is too variable. We used a model-based search for CLE domains from 57 plant genomes and used the entire pre-propeptide for comprehensive clustering analysis. In total, 1628 CLE genes were identified in land plants, with none recognizable from green algae. These CLEs form 12 groups within which CLE domains are largely conserved and pre-propeptides can be aligned. Most clusters contain sequences from monocots, eudicots and Amborella trichopoda, with sequences from Picea abies, Selaginella moellendorffii and Physcomitrella patens scattered in some clusters. We easily identified previously known clusters involved in vascular differentiation and nodulation. In addition, we found a number of discrete groups whose function remains poorly characterized. Available data indicate that CLE proteins within a cluster are likely to share function, whereas those from different clusters play at least partially different roles. Our analysis provides a foundation for future evolutionary and functional studies.

Comprehensive identification and clustering of CLV3/ESR-related (CLE) genes in plants finds groups with potentially shared function.

An extensive survey of the standing genetic variation in natural populations is among the priority steps in developing a species into a model system. In recent years, green foxtail (Setaria viridis), along with its domesticated form foxtail millet (S. italica), has rapidly become a promising new model system for C4 grasses and bioenergy crops, due to its rapid life cycle, large amount of seed production and small diploid genome, among other characters. However, remarkably little is known about the genetic diversity in natural populations of this species. In this study, we survey the genetic diversity of a worldwide sample of more than 200 S. viridis accessions, using the genotyping-by-sequencing technique. Two distinct genetic groups in S. viridis and a third group resembling S. italica were identified, with considerable admixture among the three groups. We find the genetic variation of North American S. viridis correlates with both geography and climate and is representative of the total genetic diversity in this species. This pattern may reflect several introduction/dispersal events of S. viridis into North America. We also modelled demographic history and show signal of recent population decline in one subgroup. Finally, we show linkage disequilibrium decay is rapid (<45 kb) in our total sample and slow in genetic subgroups. These results together provide an in-depth understanding of the pattern of genetic diversity of this new model species on a broad geographic scale. They also provide key guidelines for on-going and future work including germplasm preservation, local adaptation, crossing designs and genomewide association studies.

Population genetics of Setaria viridis, a new model system.

The role of polyploidy, particularly allopolyploidy, in plant diversification is a subject of debate. Whole-genome duplications precede the origins of many major clades (e.g., angiosperms, Brassicaceae, Poaceae), suggesting that polyploidy drives diversification. However, theoretical arguments and empirical studies suggest that polyploid lineages may actually have lower speciation rates and higher extinction rates than diploid lineages. We focus here on the grass tribe Andropogoneae, an economically and ecologically important group of C4 species with a high frequency of polyploids. A phylogeny was constructed for ca. 10% of the species of the clade, based on sequences of four concatenated low-copy nuclear loci. Genetic allopolyploidy was documented using the characteristic pattern of double-labeled gene trees. At least 32% of the species sampled are the result of genetic allopolyploidy and result from 28 distinct tetraploidy events plus an additional six hexaploidy events. This number is a minimum, and the actual frequency could be considerably higher. The parental genomes of most Andropogoneae polyploids diverged in the Late Miocene coincident with the expansion of the major C4 grasslands that dominate the earth today. The well-documented whole-genome duplication in Zea mays ssp. mays occurred after the divergence of Zea and Sorghum. We find no evidence that polyploidization is followed by an increase in net diversification rate; nonetheless, allopolyploidy itself is a major mode of speciation.

Allopolyploidy, diversification, and the Miocene grassland expansion.

The Food and Agriculture Organization (FAO) predicts that food production must rise 70% over the next 40 years to meet the demands of a growing population that is expected to reach nine billion by the year 2050. Many facets of basic plant science promoted by the Botanical Society of America are important for agriculture; however, more explicit connections are needed to bridge the gap between basic and applied plant research. This special issue, Speaking of Food: Connecting Basic and Applied Plant Science, was conceived to showcase productive overlaps of basic and applied research to address the challenges posed by feeding billions of people and to stimulate more research, fresh connections, and new paradigms. Contributions to this special issue thus illustrate some interactive areas of study in plant science-historical and modern plant-human interaction, crop and weed origins and evolution, and the effects of natural and artificial selection on crops and their wild relatives. These papers provide examples of how research integrating the basic and applied aspects of plant science benefits the pursuit of knowledge and the translation of that knowledge into actions toward sustainable production of crops and conservation of diversity in a changing climate.

Speaking of food: connecting basic and applied plant science.

Polyploidy and reticulate evolution are often a complication for discovering phylogenetic relationships between genera and species. Despite the huge economic importance of sugarcane (Saccharum officinarum-Poaceae, Andropogoneae), the limits of the genus Saccharum and its species are complex and largely unresolved, involving both polyploidy and reticulate evolution. This study aimed to assess the phylogenetic relationships of Saccharum s.l., including Erianthus and Tripidium, as well as investigate the taxonomic circumscription of the South American species of the genus.

Phylogenetic analysis of Saccharum s.l. (Poaceae; Andropogoneae), with emphasis on the circumscription of the South American species.

Brachypodium distachyon has emerged as a powerful model system for studying the genetics of flowering plants. Originally chosen for its phylogenetic proximity to the large-genome cereal crops wheat and barley, it is proving to be useful for more than simply providing markers for comparative mapping. Studies in B. distachyon have provided new insight into the structure and physiology of plant cell walls, the development and chemical composition of endosperm, and the genetic basis for cold tolerance. Recent work on auxin transport has uncovered mechanisms that apply to all angiosperms other than Arabidopsis. In addition to the areas in which it is currently used, B. distachyon is uniquely suited for studies of floral development, vein patterning, the controls of the perennial versus annual habit, and genome organization.

Brachypodium distachyon as a Genetic Model System.

Many major clades of angiosperms have several whole genome duplications (polyploidization events) in their distant past, suggesting that polyploidy drives or at least permits diversification. However, data on recently diverged groups are more equivocal, finding little evidence of elevated diversification following polyploidy. The discrepancy may be attributable at least in part to methodology. Many studies use indirect methods, such as chromosome numbers, genome size, and Ks plots, to test polyploidy, although these approaches can be misleading, and often lack sufficient resolution. A direct test of diversification following polyploidy requires a sequence-based approach that traces the history of nuclear genomes rather than species. These methods identify the point of coalescence of ancestral genomes, but may be misleading about the time and thus the extent of diversification. Limitations of existing methods mean that the connection between polyploidy and diversification has not been rigorously tested and remains unknown.

Has the connection between polyploidy and diversification actually been tested?

Species delimitation is a vital issue concerning evolutionary biology and conservation of biodiversity. However, it is a challenging task for several reasons, including the low interspecies variability of markers currently used in phylogenetic reconstructions and the occurrence of reticulate evolution and polyploidy in many lineages of flowering plants. The first phylogeny of the grass genus Eriochrysis is presented here, focusing on the New World species, in order to examine its relationships to other genera of the subtribe Saccharinae/tribe Andropogoneae and to define the circumscriptions of its taxonomically complicated species. Molecular cloning and sequencing of five regions of four low-copy nuclear genes (apo1, d8, ep2-ex7 and ep2-ex8, kn1) were performed, as well as complete plastome sequencing. Trees were reconstructed using maximum parsimony, maximum likelihood, and Bayesian inference analyses. The present phylogenetic analyses indicate that Eriochrysis is monophyletic and the Old World E. pallida is sister to the New World species. Subtribe Saccharinae is polyphyletic, as is the genus Eulalia. Based on nuclear and plastome sequences plus morphology, we define the circumscriptions of the New World species of Eriochrysis: E. laxa is distinct from E. warmingiana, and E. villosa is distinct from E. cayennensis. Natural hybrids occur between E. laxa and E. villosa. The hybrids are probably tetraploids, based on the number of paralogues in the nuclear gene trees. This is the first record of a polyploid taxon in the genus Eriochrysis. Some incongruities between nuclear genes and plastome analyses were detected and are potentially caused by incomplete lineage sorting and/or ancient hybridization. The set of low-copy nuclear genes used in this study seems to be sufficient to resolve phylogenetic relationships and define the circumscriptions of other species complexes in the grass family and relatives, even in the presence of polyploidy and reticulate evolution. Complete plastome sequencing is also a promising tool for phylogenetic inference.

Multilocus phylogeny and phylogenomics of Eriochrysis P. Beauv. (Poaceae-Andropogoneae): Taxonomic implications and evidence of interspecific hybridization.

Genome sequencing: Long reads for a short plant.

Development of an abscission zone (AZ) is needed for dispersal of seeds, and AZ loss was a critical early step in plant domestication. The AZ forms in different tissues in different species of plants, but whether the AZ is developmentally similar wherever it occurs is unknown. AZ development in Setaria viridis was studied as a representative of the previously uncharacterized subfamily Panicoideae.

Abscission zone development in Setaria viridis and its domesticated relative, Setaria italica.

Cphotosynthesis is perhaps one of the best examples of convergent adaptive evolution with over 25 independent origins in the grasses (Poaceae) alone. The availability of high quality grass genome sequences presents new opportunities to explore the mechanisms underlying this complex trait using evolutionary biology-based approaches. In this study, we performed genome-wide cross-species selection scans in Clineages to facilitate discovery of Cgenes. The study was enabled by the well conserved collinearity of grass genomes and the recently sequenced genome of a Cpanicoid grass, Dichanthelium oligosanthes This method, in contrast to previous studies, does not rely on any a priori knowledge of the genes that contribute to biochemical or anatomical innovations associated with Cphotosynthesis. We identified a list of 88 candidate genes that include both known and potentially novel components of the Cpathway. This set includes the carbon shuttle enzymes pyruvate, phosphate dikinase, phosphoenolpyruvate carboxylase and NADP malic enzyme as well as several predicted transporter proteins that likely play an essential role in promoting the flux of metabolites between the bundle sheath and mesophyll cells. Importantly, this approach demonstrates the application of fundamental molecular evolution principles to dissect the genetic basis of a complex photosynthetic adaptation in plants. Furthermore, we demonstrate how the output of the selection scans can be combined with expression data to provide additional power to prioritize candidate gene lists and suggest novel opportunities for pathway engineering.

Cross species selection scans identify components of C4 photosynthesis in the grasses.

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species.

Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

Chloroplast genomes are now produced in the hundreds for angiosperm phylogenetics projects, but current methods for annotation, alignment and tree estimation still require some manual intervention reducing throughput and increasing analysis time for large chloroplast systematics projects.

Verdant: automated annotation, alignment and phylogenetic analysis of whole chloroplast genomes.

Comparisons between Cand Cgrasses often utilize Cspecies from the subfamilies Ehrhartoideae or Pooideae and Cspecies from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the Cpanicoid grass Dichanthelium oligosanthes from the independent Clineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of Cand Cgrasses.

The draft genome of the Cpanicoid grass species Dichanthelium oligosanthes.

Independent evolution of derived complex characters provides a unique opportunity to assess whether and how similar genetic changes correlate with morphological convergence. Bilaterally symmetrical corollas have evolved multiple times independently from radially symmetrical ancestors and likely represent adaptations to attract specific pollinators. On the other hand, losses of bilateral corolla symmetry have occurred sporadically in various groups, due to either modification of bilaterally symmetrical corollas in late development or early establishment of radial symmetry.

Repeated and diverse losses of corolla bilateral symmetry in the Lamiaceae.

High-throughput phenotyping.

Setaria viridis is a rapid-life-cycle model panicoid grass. To identify genes that may contribute to inflorescence architecture and thus have the potential to influence grain yield in related crops such as maize, we conducted an N-nitroso-N-methylurea (NMU) mutagenesis of S. viridis and screened for visible inflorescence mutant phenotypes. Of the approximately 2,700 Mfamilies screened, we identified four recessive sparse panicle mutants (spp1-spp4) characterized by reduced and uneven branching of the inflorescence. To identify the gene underlying the sparse panicle1 (spp1) phenotype, we performed bulked segregant analysis and deep sequencing to fine map it to an approximately 1 Mb interval. Within this interval, we identified disruptive mutations in two genes. Complementation tests between spp1 and spp3 revealed they were allelic, and deep sequencing of spp3 identified an independent disruptive mutation in SvAUX1 (AUXIN1), one of the two genes in the ∼1 Mb interval and the only gene disruption shared between spp1 and spp3. SvAUX1 was found to affect both inflorescence development and root gravitropism in S. viridis. A search for orthologous mutant alleles in maize confirmed a very similar role of ZmAUX1 in maize, which highlights the utility of S. viridis in accelerating functional genomic studies in maize.

Article	Year
Comprehensive identification and clustering of CLV3/ESR-related (CLE) genes in plants finds groups with potentially shared function. The New phytologist\| PubMed ID: 27911469	2017
Population genetics of Setaria viridis, a new model system. Molecular ecology\| PubMed ID: 25185718	2014
Allopolyploidy, diversification, and the Miocene grassland expansion. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 25288748	2014
Speaking of food: connecting basic and applied plant science. American journal of botany\| PubMed ID: 25326609	2014
Phylogenetic analysis of Saccharum s.l. (Poaceae; Andropogoneae), with emphasis on the circumscription of the South American species. American journal of botany\| PubMed ID: 25667078	2015
Brachypodium distachyon as a Genetic Model System. Annual review of genetics\| PubMed ID: 26393966	2015
Has the connection between polyploidy and diversification actually been tested? Current opinion in plant biology\| PubMed ID: 26855304	2016
Multilocus phylogeny and phylogenomics of Eriochrysis P. Beauv. (Poaceae-Andropogoneae): Taxonomic implications and evidence of interspecific hybridization. Molecular phylogenetics and evolution\| PubMed ID: 26947710	2016
Genome sequencing: Long reads for a short plant. Nature plants\| PubMed ID: 27251714	2015
Abscission zone development in Setaria viridis and its domesticated relative, Setaria italica. American journal of botany\| PubMed ID: 27257006	2016
Cross species selection scans identify components of C4 photosynthesis in the grasses. Journal of experimental botany\| PubMed ID: 27436281	2017
Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins ... The New phytologist\| PubMed ID: 27634188	2017
Verdant: automated annotation, alignment and phylogenetic analysis of whole chloroplast genomes. Bioinformatics (Oxford, England)\| PubMed ID: 27634949	2017
The draft genome of the Cpanicoid grass species Dichanthelium oligosanthes. Genome biology\| PubMed ID: 27793170	2016
Repeated and diverse losses of corolla bilateral symmetry in the Lamiaceae. Annals of botany\| PubMed ID: 28334152	2017
High-throughput phenotyping. American journal of botany\| PubMed ID: 28400413	2017
Sparse panicle1 is required for inflorescence development in Setaria viridis and maize. Nature plants\| PubMed ID: 28418381	2017

Elizabeth A. Kellogg

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