Cell-type-specific transcriptomics uncovers spatial regulatory networks in bioenergy sorghum stems.

Bioenergy sorghum is a low-input, drought-resilient, deep-rooting annual crop that has high biomass yield potential enabling the sustainable production of biofuels, biopower, and bioproducts. Bioenergy sorghum's 4-5 m stems account for ~80% of the harvested biomass. Stems accumulate high levels of sucrose that could be used to synthesize bioethanol and useful biopolymers if information about cell-type gene expression and regulation in stems was available to enable engineering. To obtain this information, laser capture microdissection was used to isolate and collect transcriptome profiles from five major cell types that are present in stems of the sweet sorghum Wray. Transcriptome analysis identified genes with cell-type-specific and cell-preferred expression patterns that reflect the distinct metabolic, transport, and regulatory functions of each cell type. Analysis of cell-type-specific gene regulatory networks (GRNs) revealed that unique transcription factor families contribute to distinct regulatory landscapes, where regulation is organized through various modes and identifiable network motifs. Cell-specific transcriptome data was combined with known secondary cell wall (SCW) networks to identify the GRNs that differentially activate SCW formation in vascular sclerenchyma and epidermal cells. The spatial transcriptomic dataset provides a valuable source of information about the function of different sorghum cell types and GRNs that will enable the engineering of bioenergy sorghum stems, and an interactive web application developed during this project will allow easy access and exploration of the data (https://mc-lab.shinyapps.io/lcm-dataset/).

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Transformation and gene editing in the bioenergy grass Miscanthus.

BACKGROUND: Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. RESULTS: We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. CONCLUSIONS: We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop.

Three founding ancestral genomes involved in the origin of sugarcane.

BACKGROUND AND AIMS: Modern sugarcane cultivars (Saccharum spp.) are high polyploids, aneuploids (2n = ~12x = ~120) derived from interspecific hybridizations between the domesticated sweet species Saccharum officinarum and the wild species S. spontaneum. METHODS: To analyse the architecture and origin of such a complex genome, we analysed the sequences of all 12 hom(oe)ologous haplotypes (BAC clones) from two distinct genomic regions of a typical modern cultivar, as well as the corresponding sequence in Miscanthus sinense and Sorghum bicolor, and monitored their distribution among representatives of the Saccharum genus. KEY RESULTS: The diversity observed among haplotypes suggested the existence of three founding genomes (A, B, C) in modern cultivars, which diverged between 0.8 and 1.3 Mya. Two genomes (A, B) were contributed by S. officinarum; these were also found in its wild presumed ancestor S. robustum, and one genome (C) was contributed by S. spontaneum. These results suggest that S. officinarum and S. robustum are derived from interspecific hybridization between two unknown ancestors (A and B genomes). The A genome contributed most haplotypes (nine or ten) while the B and C genomes contributed one or two haplotypes in the regions analysed of this typical modern cultivar. Interspecific hybridizations likely involved accessions or gametes with distinct ploidy levels and/or were followed by a series of backcrosses with the A genome. The three founding genomes were found in all S. barberi, S. sinense and modern cultivars analysed. None of the analysed accessions contained only the A genome or the B genome, suggesting that representatives of these founding genomes remain to be discovered. CONCLUSIONS: This evolutionary model, which combines interspecificity and high polyploidy, can explain the variable chromosome pairing affinity observed in Saccharum. It represents a major revision of the understanding of Saccharum diversity.

Genome biology of the paleotetraploid perennial biomass crop Miscanthus.

Miscanthus is a perennial wild grass that is of global importance for paper production, roofing, horticultural plantings, and an emerging highly productive temperate biomass crop. We report a chromosome-scale assembly of the paleotetraploid M. sinensis genome, providing a resource for Miscanthus that links its chromosomes to the related diploid Sorghum and complex polyploid sugarcanes. The asymmetric distribution of transposons across the two homoeologous subgenomes proves Miscanthus paleo-allotetraploidy and identifies several balanced reciprocal homoeologous exchanges. Analysis of M. sinensis and M. sacchariflorus populations demonstrates extensive interspecific admixture and hybridization, and documents the origin of the highly productive triploid bioenergy crop M. × giganteus. Transcriptional profiling of leaves, stem, and rhizomes over growing seasons provides insight into rhizome development and nutrient recycling, processes critical for sustainable biomass accumulation in a perennial temperate grass. The Miscanthus genome expands the power of comparative genomics to understand traits of importance to Andropogoneae grasses.

Identification and characterization of a novel stay-green QTL that increases yield in maize.

Functional stay-green is a valuable trait that extends the photosynthetic period, increases source capacity and biomass and ultimately translates to higher grain yield. Selection for higher yields has increased stay-green in modern maize hybrids. Here, we report a novel QTL controlling functional stay-green that was discovered in a mapping population derived from the Illinois High Protein 1 (IHP1) and Illinois Low Protein 1 (ILP1) lines, which show very different rates of leaf senescence. This QTL was mapped to a single gene containing a NAC-domain transcription factor that we named nac7. Transgenic maize lines where nac7 was down-regulated by RNAi showed delayed senescence and increased both biomass and nitrogen accumulation in vegetative tissues, demonstrating NAC7 functions as a negative regulator of the stay-green trait. More importantly, crosses between nac7 RNAi parents and two different elite inbred testers produced hybrids with prolonged stay-green and increased grain yield by an average 0.29 megagram/hectare (4.6 bushel/acre), in 2 years of multi-environment field trials. Subsequent RNAseq experiments, one employing nac7 RNAi leaves and the other using leaf protoplasts overexpressing Nac7, revealed an important role for NAC7 in regulating genes in photosynthesis, chlorophyll degradation and protein turnover pathways that each contribute to the functional stay-green phenotype. We further determined the putative target of NAC7 and provided a logical extension for the role of NAC7 in regulating resource allocation from vegetative source to reproductive sink tissues. Collectively, our findings make a compelling case for NAC7 as a target for improving functional stay-green and yields in maize and other crops.

Breeding progress and preparedness for mass-scale deployment of perennial lignocellulosic biomass crops switchgrass, miscanthus, willow and poplar.

Genetic improvement through breeding is one of the key approaches to increasing biomass supply. This paper documents the breeding progress to date for four perennial biomass crops (PBCs) that have high output-input energy ratios: namely Panicum virgatum (switchgrass), species of the genera Miscanthus (miscanthus), Salix (willow) and Populus (poplar). For each crop, we report on the size of germplasm collections, the efforts to date to phenotype and genotype, the diversity available for breeding and on the scale of breeding work as indicated by number of attempted crosses. We also report on the development of faster and more precise breeding using molecular breeding techniques. Poplar is the model tree for genetic studies and is furthest ahead in terms of biological knowledge and genetic resources. Linkage maps, transgenesis and genome editing methods are now being used in commercially focused poplar breeding. These are in development in switchgrass, miscanthus and willow generating large genetic and phenotypic data sets requiring concomitant efforts in informatics to create summaries that can be accessed and used by practical breeders. Cultivars of switchgrass and miscanthus can be seed-based synthetic populations, semihybrids or clones. Willow and poplar cultivars are commercially deployed as clones. At local and regional level, the most advanced cultivars in each crop are at technology readiness levels which could be scaled to planting rates of thousands of hectares per year in about 5 years with existing commercial developers. Investment in further development of better cultivars is subject to current market failure and the long breeding cycles. We conclude that sustained public investment in breeding plays a key role in delivering future mass-scale deployment of PBCs.

A mosaic monoploid reference sequence for the highly complex genome of sugarcane.

Sugarcane (Saccharum spp.) is a major crop for sugar and bioenergy production. Its highly polyploid, aneuploid, heterozygous, and interspecific genome poses major challenges for producing a reference sequence. We exploited colinearity with sorghum to produce a BAC-based monoploid genome sequence of sugarcane. A minimum tiling path of 4660 sugarcane BAC that best covers the gene-rich part of the sorghum genome was selected based on whole-genome profiling, sequenced, and assembled in a 382-Mb single tiling path of a high-quality sequence. A total of 25,316 protein-coding gene models are predicted, 17% of which display no colinearity with their sorghum orthologs. We show that the two species, S. officinarum and S. spontaneum, involved in modern cultivars differ by their transposable elements and by a few large chromosomal rearrangements, explaining their distinct genome size and distinct basic chromosome numbers while also suggesting that polyploidization arose in both lineages after their divergence.

Evolutionary divergence of phytochrome protein function in Zea mays PIF3 signaling.

Two maize phytochrome-interacting factor (PIF) basic helix-loop-helix (bHLH) family members, ZmPIF3.1 and ZmPIF3.2, were identified, cloned and expressed in vitro to investigate light-signaling interactions. A phylogenetic analysis of sequences of the maize bHLH transcription factor gene family revealed the extent of the PIF family, and a total of seven predicted PIF-encoding genes were identified from genes encoding bHLH family VIIa/b proteins in the maize genome. To investigate the role of maize PIFs in phytochrome signaling, full-length cDNAs for phytochromes PhyA2, PhyB1, PhyB2 and PhyC1 from maize were cloned and expressed in vitro as chromophorylated holophytochromes. We showed that ZmPIF3.1 and ZmPIF3.2 interact specifically with the Pfr form of maize holophytochrome B1 (ZmphyB1), showing no detectable affinity for the Pr form. Maize holophytochrome B2 (ZmphyB2) showed no detectable binding affinity for PIFs in either Pr or Pfr forms, but phyB Pfr from Arabidopsis interacted with ZmPIF3.1 similarly to ZmphyB1 Pfr. We conclude that subfunctionalization at the protein-protein interaction level has altered the role of phyB2 relative to that of phyB1 in maize. Since the phyB2 mutant shows photomorphogenic defects, we conclude that maize phyB2 is an active photoreceptor, without the binding of PIF3 seen in other phyB family proteins.

Transcriptional responses indicate maintenance of photosynthetic proteins as key to the exceptional chilling tolerance of C4 photosynthesis in Miscanthus × giganteus.

Miscanthus × giganteus is exceptional among C4 plants in its ability to acclimate to chilling (≤14 °C) and maintain a high photosynthetic capacity, in sharp contrast to maize, leading to very high productivity even in cool temperate climates. To identify the mechanisms that underlie this acclimation, RNA was isolated from M × giganteus leaves in chilling and nonchilling conditions and hybridized to microarrays developed for its close relative Zea mays. Among 21 000 array probes that yielded robust signals, 723 showed significant expression change under chilling. Approximately half of these were for annotated genes. Thirty genes associated with chloroplast membrane function were all upregulated. Increases in transcripts for the lhcb5 (chlorophyll a/b-binding protein CP26), ndhF (NADH dehydrogenase F, chloroplast), atpA (ATP synthase alpha subunit), psbA (D1), petA (cytochrome f), and lhcb4 (chlorophyll a/b-binding protein CP29), relative to housekeeping genes in M. × giganteus, were confirmed by quantitative reverse-transcription PCR. In contrast, psbo1, lhcb5, psbA, and lhcb4 were all significantly decreased in Z. mays after 14 days of chilling. Western blot analysis of the D1 protein and LHCII type II chlorophyll a/b-binding protein also showed significant increases in M. × giganteus during chilling and significant decreases in Z. mays. Compared to other C4 species, M. × giganteus grown in chilling conditions appears to counteract the loss of photosynthetic proteins and proteins protecting photosystem II typically observed in other species by increasing mRNA levels for their synthesis.

A detailed gene expression study of the Miscanthus genus reveals changes in the transcriptome associated with the rejuvenation of spring rhizomes.

BACKGROUND: The Miscanthus genus of perennial C4 grasses contains promising biofuel crops for temperate climates. However, few genomic resources exist for Miscanthus, which limits understanding of its interesting biology and future genetic improvement. A comprehensive catalog of expressed sequences were generated from a variety of Miscanthus species and tissue types, with an emphasis on characterizing gene expression changes in spring compared to fall rhizomes. RESULTS: Illumina short read sequencing technology was used to produce transcriptome sequences from different tissues and organs during distinct developmental stages for multiple Miscanthus species, including Miscanthus sinensis, Miscanthus sacchariflorus, and their interspecific hybrid Miscanthus × giganteus. More than fifty billion base-pairs of Miscanthus transcript sequence were produced. Overall, 26,230 Sorghum gene models (i.e., ~ 96% of predicted Sorghum genes) had at least five Miscanthus reads mapped to them, suggesting that a large portion of the Miscanthus transcriptome is represented in this dataset. The Miscanthus × giganteus data was used to identify genes preferentially expressed in a single tissue, such as the spring rhizome, using Sorghum bicolor as a reference. Quantitative real-time PCR was used to verify examples of preferential expression predicted via RNA-Seq. Contiguous consensus transcript sequences were assembled for each species and annotated using InterProScan. Sequences from the assembled transcriptome were used to amplify genomic segments from a doubled haploid Miscanthus sinensis and from Miscanthus × giganteus to further disentangle the allelic and paralogous variations in genes. CONCLUSIONS: This large expressed sequence tag collection creates a valuable resource for the study of Miscanthus biology by providing detailed gene sequence information and tissue preferred expression patterns. We have successfully generated a database of transcriptome assemblies and demonstrated its use in the study of genes of interest. Analysis of gene expression profiles revealed biological pathways that exhibit altered regulation in spring compared to fall rhizomes, which are consistent with their different physiological functions. The expression profiles of the subterranean rhizome provides a better understanding of the biological activities of the underground stem structures that are essentials for perenniality and the storage or remobilization of carbon and nutrient resources.

A framework genetic map for Miscanthus sinensis from RNAseq-based markers shows recent tetraploidy.

BACKGROUND: Miscanthus (subtribe Saccharinae, tribe Andropogoneae, family Poaceae) is a genus of temperate perennial C4 grasses whose high biomass production makes it, along with its close relatives sugarcane and sorghum, attractive as a biofuel feedstock. The base chromosome number of Miscanthus (x = 19) is different from that of other Saccharinae and approximately twice that of the related Sorghum bicolor (x = 10), suggesting large-scale duplications may have occurred in recent ancestors of Miscanthus. Owing to the complexity of the Miscanthus genome and the complications of self-incompatibility, a complete genetic map with a high density of markers has not yet been developed. RESULTS: We used deep transcriptome sequencing (RNAseq) from two M. sinensis accessions to define 1536 single nucleotide variants (SNVs) for a GoldenGate™ genotyping array, and found that simple sequence repeat (SSR) markers defined in sugarcane are often informative in M. sinensis. A total of 658 SNP and 210 SSR markers were validated via segregation in a full sibling F1 mapping population. Using 221 progeny from this mapping population, we constructed a genetic map for M. sinensis that resolves into 19 linkage groups, the haploid chromosome number expected from cytological evidence. Comparative genomic analysis documents a genome-wide duplication in Miscanthus relative to Sorghum bicolor, with subsequent insertional fusion of a pair of chromosomes. The utility of the map is confirmed by the identification of two paralogous C4-pyruvate, phosphate dikinase (C4-PPDK) loci in Miscanthus, at positions syntenic to the single orthologous gene in Sorghum. CONCLUSIONS: The genus Miscanthus experienced an ancestral tetraploidy and chromosome fusion prior to its diversification, but after its divergence from the closely related sugarcane clade. The recent timing of this tetraploidy complicates discovery and mapping of genetic markers for Miscanthus species, since alleles and fixed differences between paralogs are comparable. These difficulties can be overcome by careful analysis of segregation patterns in a mapping population and genotyping of doubled haploids. The genetic map for Miscanthus will be useful in biological discovery and breeding efforts to improve this emerging biofuel crop, and also provide a valuable resource for understanding genomic responses to tetraploidy and chromosome fusion.

Rapid genotyping of soybean cultivars using high throughput sequencing.

Soybean (Glycine max) breeding involves improving commercially grown varieties by introgressing important agronomic traits from poor yielding accessions and/or wild relatives of soybean while minimizing the associated yield drag. Molecular markers associated with these traits are instrumental in increasing the efficiency of producing such crosses and Single Nucleotide Polymorphisms (SNPs) are particularly well suited for this task, owing to high density in the non-genic regions and thus increased likelihood of finding a tightly linked marker to a given trait. A rapid method to develop SNP markers that can differentiate specific loci between any two parents in soybean is thus highly desirable. In this study we investigate such a protocol for developing SNP markers between multiple soybean accessions and the reference Williams 82 genome. To restrict sampling frequency reduced representation libraries (RRLs) of genomic DNA were generated by restriction digestion followed by library construction. We chose to sequence four accessions Dowling (PI 548663), Dwight (PI 597386), Komata (PI200492) and PI 594538A for their agronomic importance as well as Williams 82 as a control.MseI was chosen to digest genomic DNA based on predictions that it will cut sparingly in the mathematically defined high-copy-number regions of the genome. All RRLs were sequenced on the Illumina genome analyzer. Reads were aligned to the Glyma1 reference assembly and SNP calls made from the alignments. We identified from 4294 to 14550 SNPs between the four accessions and the Williams 82 reference. In addition a small number of SNPs (1142) were found by aligning Williams 82 reads to the reference assembly (Glyma1) suggesting limited genetic variation within the Williams 82 line. The SNP data allowed us to estimate genetic diversity between the four lines and Williams 82. Restriction digestion of soybean genomic DNA with MseI followed by high throughput sequencing provides a rapid and reproducible method for generating SNP markers.

Rapid, organ-specific transcriptional responses to light regulate photomorphogenic development in dicot seedlings.

The dicotyledon seedling undergoes organ-specific photomorphogenic development when exposed to light. The cotyledons open and expand, the apical hook opens, and the hypocotyl ceases to elongate. Using the large and easily dissected seedlings of soybean (Glycine max 'Williams 82'), we show that genes involved in photosynthesis and its regulation dominate transcripts specific to the cotyledon, even in etiolated seedlings. Genes for cell wall biosynthesis and metabolism are expressed at higher levels in the hypocotyl, while examination of genes expressed at higher levels in the hook region (including the shoot apical meristem) reveals genes involved in cell division and protein turnover. The early transcriptional events in these three organs in response to a 1-h treatment of far-red light are highly distinctive. Not only are different regulatory genes rapidly regulated by light in each organ, but the early-responsive genes in each organ contain a distinctive subset of known light-responsive cis-regulatory elements. We detected specific light-induced gene expression for the root phototropism gene RPT2 in the apical hook and also phenotypes in Arabidopsis (Arabidopsis thaliana) rpt2 mutants demonstrating that the gene is necessary for normal photomorphogenesis in the seedling apex. Significantly, expression of the RPT2 promoter fused to a ß-glucuronidase reporter gene shows differential expression across the hook region. We conclude that organ-specific, light-responsive transcriptional networks are active early in photomorphogenesis in the aerial parts of dicotyledon seedlings.

Transcriptomic analyses of xylan degradation by Prevotella bryantii and insights into energy acquisition by xylanolytic bacteroidetes.

Enzymatic depolymerization of lignocellulose by microbes in the bovine rumen and the human colon is critical to gut health and function within the host. Prevotella bryantii B(1)4 is a rumen bacterium that efficiently degrades soluble xylan. To identify the genes harnessed by this bacterium to degrade xylan, the transcriptomes of P. bryantii cultured on either wheat arabinoxylan or a mixture of its monosaccharide components were compared by DNA microarray and RNA sequencing approaches. The most highly induced genes formed a cluster that contained putative outer membrane proteins analogous to the starch utilization system identified in the prominent human gut symbiont Bacteroides thetaiotaomicron. The arrangement of genes in the cluster was highly conserved in other xylanolytic Bacteroidetes, suggesting that the mechanism employed by xylan utilizers in this phylum is conserved. A number of genes encoding proteins with unassigned function were also induced on wheat arabinoxylan. Among these proteins, a hypothetical protein with low similarity to glycoside hydrolases was shown to possess endoxylanase activity and subsequently assigned to glycoside hydrolase family 5. The enzyme was designated PbXyn5A. Two of the most similar proteins to PbXyn5A were hypothetical proteins from human colonic Bacteroides spp., and when expressed each protein exhibited endoxylanase activity. By using site-directed mutagenesis, we identified two amino acid residues that likely serve as the catalytic acid/base and nucleophile as in other GH5 proteins. This study therefore provides insights into capture of energy by xylanolytic Bacteroidetes and the application of their enzymes as a resource in the biofuel industry.

Genomic and small RNA sequencing of Miscanthus x giganteus shows the utility of sorghum as a reference genome sequence for Andropogoneae grasses.

BACKGROUND: Miscanthus x giganteus (Mxg) is a perennial grass that produces superior biomass yields in temperate environments. The essentially uncharacterized triploid genome (3n = 57, x = 19) of Mxg is likely critical for the rapid growth of this vegetatively propagated interspecific hybrid. RESULTS: A survey of the complex Mxg genome was conducted using 454 pyrosequencing of genomic DNA and Illumina sequencing-by-synthesis of small RNA. We found that the coding fraction of the Mxg genome has a high level of sequence identity to that of other grasses. Highly repetitive sequences representing the great majority of the Mxg genome were predicted using non-cognate assembly for de novo repeat detection. Twelve abundant families of repeat were observed, with those related to either transposons or centromeric repeats likely to comprise over 95% of the genome. Comparisons of abundant repeat sequences to a small RNA survey of three Mxg organs (leaf, rhizome, inflorescence) revealed that the majority of observed 24-nucleotide small RNAs are derived from these repetitive sequences. We show that high-copy-number repeats match more of the small RNA, even when the amount of the repeat sequence in the genome is accounted for. CONCLUSIONS: We show that major repeats are present within the triploid Mxg genome and are actively producing small RNAs. We also confirm the hypothesized origins of Mxg, and suggest that while the repeat content of Mxg differs from sorghum, the sorghum genome is likely to be of utility in the assembly of a gene-space sequence of Mxg.

The plant B3 superfamily.

The plant-specific B3 superfamily encompasses well-characterized families, such as the auxin response factor (ARF) family and the LAV family, as well as less well understood families, such as RAV and REM. In Arabidopsis, there are 118 B3 genes, and in rice there are 91 B3 genes. The B3 domain is present in genes from gymnosperms, mosses and green algae, indicating that the B3 domain evolved on the plant lineage before multicellularity. The aim of this review is to phylogenetically characterize the members of the B3 family in Arabidopsis and rice and to review the function of the B3 genes that have been studied to date.

Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey.

BACKGROUND: Extensive computational and database tools are available to mine genomic and genetic databases for model organisms, but little genomic data is available for many species of ecological or agricultural significance, especially those with large genomes. Genome surveys using conventional sequencing techniques are powerful, particularly for detecting sequences present in many copies per genome. However these methods are time-consuming and have potential drawbacks. High throughput 454 sequencing provides an alternative method by which much information can be gained quickly and cheaply from high-coverage surveys of genomic DNA. RESULTS: We sequenced 78 million base-pairs of randomly sheared soybean DNA which passed our quality criteria. Computational analysis of the survey sequences provided global information on the abundant repetitive sequences in soybean. The sequence was used to determine the copy number across regions of large genomic clones or contigs and discover higher-order structures within satellite repeats. We have created an annotated, online database of sequences present in multiple copies in the soybean genome. The low bias of pyrosequencing against repeat sequences is demonstrated by the overall composition of the survey data, which matches well with past estimates of repetitive DNA content obtained by DNA re-association kinetics (Cot analysis). CONCLUSION: This approach provides a potential aid to conventional or shotgun genome assembly, by allowing rapid assessment of copy number in any clone or clone-end sequence. In addition, we show that partial sequencing can provide access to partial protein-coding sequences.