Allan W. Dickerman

The genome of woodland strawberry ( Fragaria vesca )

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.

Identification of Genes Required for Embryo Development in Arabidopsis

A long-term goal of Arabidopsis research is to define the minimal gene set needed to produce a viable plant with a normal phenotype under diverse conditions. This will require both forward and reverse genetics along with novel strategies to characterize multigene families and redundant biochemical pathways. Here we describe an initial dataset of 250 EMB genes required for normal embryo development in Arabidopsis. This represents the first large-scale dataset of essential genes in a flowering plant. When compared with 550 genes with other knockout phenotypes, EMB genes are enriched for basal cellular functions, deficient in transcription factors and signaling components, have fewer paralogs, and are more likely to have counterparts among essential genes of yeast (Saccharomyces cerevisiae) and worm (Caenorhabditis elegans). EMB genes also represent a valuable source of plant-specific proteins with unknown functions required for growth and development. Analyzing such unknowns is a central objective of genomics efforts worldwide. We focus here on 34 confirmed EMB genes with unknown functions, demonstrate that expression of these genes is not embryo-specific, validate a strategy for identifying interacting proteins through complementation with epitope-tagged proteins, and discuss the value of EMB genes in identifying novel proteins associated with important plant processes. Based on sequence comparison with essential genes in other model eukaryotes, we identify 244 candidate EMB genes without paralogs that represent promising targets for reverse genetics. These candidates should facilitate the recovery of additional genes required for seed development.

The Xylem and Phloem Transcriptomes from Secondary Tissues of the Arabidopsis Root-Hypocotyl

The growth of secondary xylem and phloem depends on the division of cells in the vascular cambium and results in an increase in the diameter of the root and stem. Very little is known about the genetic mechanisms that control cambial activity and the differentiation of secondary xylem and phloem cell types. To begin to identify new genes required for vascular cell differentiation and function, we performed genome-wide expression profiling of xylem and phloem-cambium isolated from the root-hypocotyl of Arabidopsis (Arabidopsis thaliana). Gene expression in the remaining nonvascular tissue was also profiled. From these transcript profiles, we assembled three sets of genes with expression significantly biased toward xylem, phloem-cambium, or nonvascular tissue. We also assembled three two-tissue sets of genes with expression significantly biased toward xylem/phloem-cambium, xylem/nonvascular, or phloem-cambium/nonvascular tissues. Localizations predicted by transcript profiles were supported by results from promoter-reporter and reverse transcription-polymerase chain reaction experiments with nine xylem- or phloem-cambium-biased genes. An analysis of the members of the phloem-cambium gene set suggested that some genes involved in regulating primary meristems are also regulators of the cambium. Secondary phloem was implicated in the synthesis of auxin, glucosinolates, cytokinin, and gibberellic acid. Transcript profiles also supported the importance of class III HD ZIP and KANADI transcription factors as regulators of radial patterning during secondary growth, and identified several members of the G2-like, NAC, AP2, MADS, and MYB transcription factor families that may play roles as regulators of xylem or phloem cell differentiation and activity.

Origin, Evolution, and Genotyping of Emergent Porcine Epidemic Diarrhea Virus Strains in the United States

ABSTRACT Coronaviruses are known to infect humans and other animals and cause respiratory and gastrointestinal diseases. Here we report the emergence of porcine epidemic diarrhea virus (PEDV) in the United States and determination of its origin, evolution, and genotypes based on temporal and geographical evidence. Histological lesions in small intestine sections of affected pigs and the complete genomic sequences of three emergent strains of PEDV isolated from outbreaks in Minnesota and Iowa were characterized. Genetic and phylogenetic analyses of the three U.S. strains revealed a close relationship with Chinese PEDV strains and their likely Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, which can be classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the divergent time based on the complete genomic sequences is consistent with the actual time difference, approximately 2 to 3 years, of the PED outbreaks between China (December 2010) and the United States (May 2013). The finding that the emergent U.S. PEDV strains share unique genetic features at the 5′-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission. The data from this study have important implications for understanding the ongoing PEDV outbreaks in the United States and will guide future efforts to develop effective preventive and control measures against PEDV. IMPORTANCE The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States. The sudden emergence of porcine epidemic diarrhea virus (PEDV), a coronavirus, for the first time in the United States causes significant economic and public health concerns. Since its recognition in May 2013, PEDV has rapidly spread across the United States, resulting in high mortality in piglets in more than 17 States now. The ongoing outbreaks of Middle East respiratory syndrome coronavirus in humans from countries in or near the Arabian Peninsula and the historical deadly nature of the 2002 outbreaks of severe acute respiratory syndrome coronavirus create further anxiety over the emergence of PEDV in the United States due to the lack of scientific information about the origin and evolution of this emerging coronavirus. Here we report the detailed genetic characterization, origin, and evolution of emergent PEDV strains in the United States. The results provide much needed information to devise effective preventive and control strategies against PEDV in the United States.

A Robust Species Tree for the Alphaproteobacteria

The branching order and coherence of the alphaproteobacterial orders have not been well established, and not all studies have agreed that mitochondria arose from within the Rickettsiales. A species tree for 72 alphaproteobacteria was produced from a concatenation of alignments for 104 well-behaved protein families. Coherence was upheld for four of the five orders with current standing that were represented here by more than one species. However, the family Hyphomonadaceae was split from the other Rhodobacterales, forming an expanded group with Caulobacterales that also included Parvularcula. The three earliest-branching alphaproteobacterial orders were the Rickettsiales, followed by the Rhodospirillales and then the Sphingomonadales. The principal uncertainty is whether the expanded Caulobacterales group is more closely associated with the Rhodobacterales or the Rhizobiales. The mitochondrial branch was placed within the Rickettsiales as a sister to the combined Anaplasmataceae and Rickettsiaceae, all subtended by the Pelagibacter branch. Pelagibacter genes will serve as useful additions to the bacterial outgroup in future evolutionary studies of mitochondrial genes, including those that have transferred to the eukaryotic nucleus.

Identifying essential genes in Arabidopsis thaliana.

Eight years after publication of the Arabidopsis genome sequence and two years before completing the first phase of an international effort to characterize the function of every Arabidopsis gene, plant biologists remain unable to provide a definitive answer to the following basic question: what is the minimal gene set required for normal growth and development? The purpose of this review is to summarize different strategies employed to identify essential genes in Arabidopsis, an important component of the minimal gene set in plants, to present an overview of the datasets and specific genes identified to date, and to discuss the prospects for future saturation of this important class of genes. The long-term goal of this collaborative effort is to facilitate basic research in plant biology and complement ongoing research with other model organisms.

Genome Sequences of Three Agrobacterium Biovars Help Elucidate the Evolution of Multichromosome Genomes in Bacteria

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.

The Arabidopsis SeedGenes Project

The SeedGenes database (http://www.seedgenes.org) presents molecular and phenotypic information on essential, non-redundant genes of Arabidopsis that give a seed phenotype when disrupted by mutation. Experimental details are synthesized for efficient use by the community and organized into two major sections in the database, one dealing with genes and the other with mutant alleles. The database can be queried for detailed information on a single gene to create a SeedGenes Profile. Queries can also generate lists of genes or mutants that fit specified criteria. The long-term goal is to establish a complete collection of Arabidopsis genes that give a knockout phenotype. This information is needed to focus attention on genes with important cellular functions in a model plant and to assess from a genetic perspective the extent of functional redundancy in the Arabidopsis genome.

Diversity of gut bacteria of Reticulitermes flavipes as examined by 16S rRNA gene sequencing and amplified rDNA restriction analysis.

The phylogenetic species richness of the bacteria in the gut of the termite Reticulitermes flavipes was examined using near full-length 16S rRNA gene sequencing and amplified rDNA restriction analysis (ARDRA). We amplified the genes by polymerase chain reaction (PCR) directly from a mixed population of termite gut bacteria and isolated them using cloning techniques. Sequence analysis of 42 clones identified a broad taxonomic range of ribotypes from six phyla within the domain Bacteria: Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and the recently proposed “Endomicrobia.” Analysis of the sequence data suggested the presence of a termite specific bacterial lineage within Bacteroidetes. The ARDRA data included 261 different ARDRA profiles of 512 clones analyzed. These data suggest the gut flora in R. flavipes is extremely diverse.

A Bifunctional Locus ( BIO3 - BIO1 ) Required for Biotin Biosynthesis in Arabidopsis

We identify here the Arabidopsis (Arabidopsis thaliana) gene encoding the third enzyme in the biotin biosynthetic pathway, dethiobiotin synthetase (BIO3; At5g57600). This gene is positioned immediately upstream of BIO1, which is known to be associated with the second reaction in the pathway. Reverse genetic analysis demonstrates that bio3 insertion mutants have a similar phenotype to the bio1 and bio2 auxotrophs identified using forward genetic screens for arrested embryos rescued on enriched nutrient medium. Unexpectedly, bio3 and bio1 mutants define a single genetic complementation group. Reverse transcription-polymerase chain reaction analysis demonstrates that separate BIO3 and BIO1 transcripts and two different types of chimeric BIO3-BIO1 transcripts are produced. Consistent with genetic data, one of the fused transcripts is monocistronic and encodes a bifunctional fusion protein. A splice variant is bicistronic, with distinct but overlapping reading frames. The dual functionality of the monocistronic transcript was confirmed by complementing the orthologous auxotrophs of Escherichia coli (bioD and bioA). BIO3-BIO1 transcripts from other plants provide further evidence for differential splicing, existence of a fusion protein, and localization of both enzymatic reactions to mitochondria. In contrast to most biosynthetic enzymes in eukaryotes, which are encoded by genes dispersed throughout the genome, biotin biosynthesis in Arabidopsis provides an intriguing example of a bifunctional locus that catalyzes two sequential reactions in the same metabolic pathway. This complex locus exhibits several unusual features that distinguish it from biotin operons in bacteria and from other genes known to encode bifunctional enzymes in plants.