The maize genome as a model for efficient sequence analysis of large plant genomes

The genomes of flowering plants vary in size from about 0.1 to over 100 gigabase pairs (Gbp), mostly because of polyploidy and variation in the abundance of repetitive elements in intergenic regions. High-quality sequences of the relatively small genomes of Arabidopsis (0.14 Gbp) and rice (0.4 Gbp) have now been largely completed. The sequencing of plant genomes that have a more representative size (the mean for flowering plant genomes is 5.6 Gbp) has been seen as a daunting task, partly because of their size and partly because of the numerous highly conserved repeats. Nevertheless, creative strategies and powerful new tools have been generated recently in the plant genetics community, so that sequencing large plant genomes is now a realistic possibility. Maize (2.4-2.7 Gbp) will be the first gigabase-size plant genome to be sequenced using these novel approaches. Pilot studies on maize indicate that the new gene-enrichment, gene-finishing and gene-orientation technologies are efficient, robust and comprehensive. These strategies will succeed in sequencing the gene-space of large genome plants, and in locating all of these genes and adjacent sequences on the genetic and physical maps.     

Setaria genome sequencing: an overview

The genus Setaria includes two important C₄ Panicoid grass species, namely S. italica (cultivated) and S. viridis (weed; wild ancestor), which together represent an appropriate model system for architectural, physiological, evolutionary, and genomic studies in related grasses. It is a diploid, inbreeder, self-fertile annual cereal grass having short life cycle and minimal growth requirements. There close relatedness to biofuel crops like switch grass and napier grass further signifies their importance. Further, foxtail millet is an important food and fodder grain crop grown in arid and semi-arid regions in many parts of the world. Therefore, an increasing interest in these species has led to a gradual accumulation and development of genomic data and genetic resources. Setaria genome sequencing is an outcome of such endeavors. These sequencing efforts uncovered several distinctive attributes of Setaria genome that may help in understanding its physiology, evolution and adaptation. This will not only aid in comparative genomics studies of Setaria and related crops including bioenergy grasses but also help in rapid advancements of genomics information for developing varieties with superior traits either through marker-assisted selection (MAS) or using transgenic approaches in these crops.     

Translational genomics for plant breeding with the genome sequence explosion

The use of next‐generation sequencers and advanced genotyping technologies has propelled the field of plant genomics in model crops and plants and enhanced the discovery of hidden bridges between genotypes and phenotypes. The newly generated reference sequences of unstudied minor plants can be annotated by the knowledge of model plants via translational genomics approaches. Here, we reviewed the strategies of translational genomics and suggested perspectives on the current databases of genomic resources and the database structures of translated information on the new genome. As a draft picture of phenotypic annotation, translational genomics on newly sequenced plants will provide valuable assistance for breeders and researchers who are interested in genetic studies.     

Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.     

Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions

Reference genes are standards for quantifying gene expression through quantitative real-time PCR (qRT-PCR); however, the variation observed in their expression levels is the major hindrance towards realising their effective use. Hence, a systematic validation of reference genes is required to ensure proper normalization. However, no such study has been conducted in foxtail millet [Setaria italica (L.)], which has recently emerged as a model crop for genetic and genomic studies. In the present study, 8 commonly used reference genes were evaluated, including 18S ribosomal RNA, elongation factor-1α, Actin2, alpha tubulin, beta tubulin, translation factor, RNA polymerase II and adenine phosphoribosyl transferase. Expression stability of candidate internal control genes was investigated under salinity and dehydration treatments. The results obtained suggested a wide range of Ct values and variable expression of all reference genes. geNorm and NormFinder analysis had revealed that Act2 and RNA POL II are suitable reference genes for salinity stress-related studies and EF-1α and RNA POL II are appropriate internal controls for dehydration stress-related expression analyses. These qualified reference genes has also been validated for relative quantification of 14-3-3 expression analysis which demonstrated their applicability. Thus, this is the first report on selection and validation of superior reference genes for qRT-PCR in foxtail millet under different abiotic stress conditions.     

Complete genome sequence of the plant growth-promoting endophyte Burkholderia phytofirmans strain PsJN

Burkholderia phytofirmans PsJN(T) is able to efficiently colonize the rhizosphere, root, and above-ground plant tissues of a wide variety of genetically unrelated plants, such as potatoes, canola, maize, and grapevines. Strain PsJN shows strong plant growth-promoting effects and was reported to enhance plant vigor and resistance to biotic and abiotic stresses. Here, we report the genome sequence of this strain, which indicates the presence of multiple traits relevant for endophytic colonization and plant growth promotion.     

Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82

Ralstonia solanacearum strain Po82, a phylotype IIB/sequevar 4 strain, was found to be pathogenic to both solanaceous plants and banana. Here, we report the complete genome sequence of Po82 and its comparison with seven published R. solanacearum genomes.     

A model of the statistical power of comparative genome sequence analysis

Comparative genome sequence analysis is powerful, but sequencing genomes is expensive. It is desirable to be able to predict how many genomes are needed for comparative genomics, and at what evolutionary distances. Here I describe a simple mathematical model for the common problem of identifying conserved sequences. The model leads to some useful rules of thumb. For a given evolutionary distance, the number of comparative genomes needed for a constant level of statistical stringency in identifying conserved regions scales inversely with the size of the conserved feature to be detected. At short evolutionary distances, the number of comparative genomes required also scales inversely with distance. These scaling behaviors provide some intuition for future comparative genome sequencing needs, such as the proposed use of “phylogenetic shadowing” methods using closely related comparative genomes, and the feasibility of high-resolution detection of small conserved features.     

Draft genome sequence of the plant growth-promoting bacterium Bacillus siamensis KCTC 13613T

Bacillus siamensis KCTC 13613(T), a novel halophilic Bacillus species isolated from a salted Thai food, produced antimicrobial compounds against plant pathogens and promoted plant growth by volatile emission. We determined the 3.8-Mb genome sequence of B. siamensis KCTC 13613(T) to reveal the plant-beneficial effect at the genomic level.     

Complete chloroplast genome sequence of an orchid model plant candidate: Erycina pusilla apply in tropical Oncidium breeding

Oncidium is an important ornamental plant but the study of its functional genomics is difficult. Erycina pusilla is a fast-growing Oncidiinae species. Several characteristics including low chromosome number, small genome size, short growth period, and its ability to complete its life cycle in vitro make E. pusilla a good model candidate and parent for hybridization for orchids. Although genetic information remains limited, systematic molecular analysis of its chloroplast genome might provide useful genetic information. By combining bacterial artificial chromosome (BAC) clones and next-generation sequencing (NGS), the chloroplast (cp) genome of E. pusilla was sequenced accurately, efficiently and economically. The cp genome of E. pusilla shares 89 and 84% similarity with Oncidium Gower Ramsey and Phalanopsis aphrodite, respectively. Comparing these 3 cp genomes, 5 regions have been identified as showing diversity. Using PCR analysis of 19 species belonging to the Epidendroideae subfamily, a conserved deletion was found in the rps15-trnN region of the Cymbidieae tribe. Because commercial Oncidium varieties in Taiwan are limited, identification of potential parents using molecular breeding method has become very important. To demonstrate the relationship between taxonomic position and hybrid compatibility of E. pusilla, 4 DNA regions of 36 tropically adapted Oncidiinae varieties have been analyzed. The results indicated that trnF-ndhJ and trnH-psbA were suitable for phylogenetic analysis. E. pusilla proved to be phylogenetically closer to Rodriguezia and Tolumnia than Oncidium, despite its similar floral appearance to Oncidium. These results indicate the hybrid compatibility of E. pusilla, its cp genome providing important information for Oncidium breeding.     

Complete genome sequence of a beneficial plant root-associated bacterium, Pseudomonas brassicacearum

To shed light on the genetic equipment of the beneficial plant-associated bacterium Pseudomonas brassicacearum, we sequenced the whole genome of the strain NFM421. Its genome consists of one chromosome equipped with a repertoire of factors beneficial for plant growth. In addition, a complete type III secretion system and two complete type VI secretion systems were identified. We report here the first genome sequence of this species.     

Complete genome sequence of the metabolically versatile plant growth-promoting endophyte Variovorax paradoxus S110

Variovorax paradoxus is a microorganism of special interest due to its diverse metabolic capabilities, including the biodegradation of both biogenic compounds and anthropogenic contaminants. V. paradoxus also engages in mutually beneficial interactions with both bacteria and plants. The complete genome sequence of V. paradoxus S110 is composed of 6,754,997 bp with 6,279 predicted protein-coding sequences within two circular chromosomes. Genomic analysis has revealed multiple metabolic features for autotrophic and heterotrophic lifestyles. These metabolic diversities enable independent survival, as well as a symbiotic lifestyle. Consequently, S110 appears to have evolved into a superbly adaptable microorganism that is able to survive in ever-changing environmental conditions. Based on our findings, we suggest V. paradoxus S110 as a potential candidate for agrobiotechnological applications, such as biofertilizer and biopesticide. Because it has many associations with other biota, it is also suited to serve as an additional model system for studies of microbe-plant and microbe-microbe interactions.     

Setaria viridis and Setaria italica, model genetic systems for the Panicoid grasses

Setaria italica and its wild ancestor Setaria viridis are diploid C(4) grasses with small genomes of ～515 Mb. Both species have attributes that make them attractive as model systems. Setaria italica is a grain crop widely grown in Northern China and India that is closely related to the major food and feed crops maize and sorghum. A large collection of S. italica accessions are available and thus opportunities exist for association mapping and allele mining for novel variants that will have direct application in agriculture. Setaria viridis is the weedy relative of S. italica with many attributes suitable for genetic analyses including a small stature, rapid life cycle, and prolific seed production. Setaria sp. are morphologically similar to most of the Panicoideae grasses, including major biofuel feedstocks, switchgrass (Panicum virgatum) and Miscanthus (Miscanthus giganteus). They are broadly distributed geographically and occupy diverse ecological niches. The cross-compatibility of S. italica and S. viridis also suggests that gene flow is likely between wild and domesticated accessions. In addition to serving as excellent models for C(4) photosynthesis, these grasses provide novel opportunities to study abiotic stress tolerance and as models for bioenergy feedstocks.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Poplar genome sequence: functional genomics in an ecologically dominant plant species

In addition to their value for wood products, members of the genus Populus (poplars) provide a range of ecological services, including carbon sequestration, bioremediation, nutrient cycling, biofiltration and diverse habitats. They are also widely used model organisms for tree molecular biology and biotechnology. The sequencing of the poplar genome to an approximately 6x depth adds to a long list of important attributes for research. These include facile transformation, vegetative propagation, rapid growth, modest genome size and extensive expressed sequence tags. Here, we discuss how the genome sequence and transformability of poplar, together with its high levels of genetic and ecological diversity, are enabling new insights into the genetic programs controlling ontogeny, ecological adaptation and environmental physiology of trees.     

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.     

Insights into plant cell wall degradation from the genome sequence of the soil bacterium Cellvibrio japonicus

The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus, we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron, the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40(T). Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans, consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.     

The genome sequence of Podospora anserina, a classic model fungus

The completed genome sequence of the coprophilous fungus Podospora anserina increases the sampling of fungal genomes. In line with its habitat of herbivore dung, this ascomycete has an exceptionally rich gene set devoted to the catabolism of complex carbohydrates.     

Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.