Richard Flavell

Insights into corn genes derived from large-scale cDNA sequencing

We present a large portion of the transcriptome of Zea mays, including ESTs representing 484,032 cDNA clones from 53 libraries and 36,565 fully sequenced cDNA clones, out of which 31,552 clones are non-redundant. These and other previously sequenced transcripts have been aligned with available genome sequences and have provided new insights into the characteristics of gene structures and promoters within this major crop species. We found that although the average number of introns per gene is about the same in corn and Arabidopsis, corn genes have more alternatively spliced isoforms. Examination of the nucleotide composition of coding regions reveals that corn genes, as well as genes of other Poaceae (Grass family), can be divided into two classes according to the GC content at the third position in the amino acid encoding codons. Many of the transcripts that have lower GC content at the third position have dicot homologs but the high GC content transcripts tend to be more specific to the grasses. The high GC content class is also enriched with intronless genes. Together this suggests that an identifiable class of genes in plants is associated with the Poaceae divergence. Furthermore, because many of these genes appear to be derived from ancestral genes that do not contain introns, this evolutionary divergence may be the result of horizontal gene transfer from species not only with different codon usage but possibly that did not have introns, perhaps outside of the plant kingdom. By comparing the cDNAs described herein with the non-redundant set of corn mRNAs in GenBank, we estimate that there are about 50,000 different protein coding genes in Zea. All of the sequence data from this study have been submitted to DDBJ/GenBank/EMBL under accession numbers EU940701–EU977132 (FLI cDNA) and FK944382-FL482108 (EST).

Full-length messenger RNA sequences greatly improve genome annotation

BackgroundAnnotation of eukaryotic genomes is a complex endeavor that requires the integration of evidence from multiple, often contradictory, sources. With the ever-increasing amount of genome sequence data now available, methods for accurate identification of large numbers of genes have become urgently needed. In an effort to create a set of very high-quality gene models, we used the sequence of 5,000 full-length gene transcripts from Arabidopsis to re-annotate its genome. We have mapped these transcripts to their exact chromosomal locations and, using alignment programs, have created gene models that provide a reference set for this organism.ResultsApproximately 35% of the transcripts indicated that previously annotated genes needed modification, and 5% of the transcripts represented newly discovered genes. We also discovered that multiple transcription initiation sites appear to be much more common than previously known, and we report numerous cases of alternative mRNA splicing. We include a comparison of different alignment software and an analysis of how the transcript data improved the previously published annotation.ConclusionsOur results demonstrate that sequencing of large numbers of full-length transcripts followed by computational mapping greatly improves identification of the complete exon structures of eukaryotic genes. In addition, we are able to find numerous introns in the untranslated regions of the genes.

Features of Arabidopsis genes and genome discovered using full-length cDNAs

Arabidopsis is currently the reference genome for higher plants. A new, more detailed statistical analysis of Arabidopsis gene structure is presented including intron and exon lengths, intergenic distances, features of promoters, and variant 5′-ends of mRNAs transcribed from the same transcription unit. We also provide a statistical characterization of Arabidopsis transcripts in terms of their size, UTR lengths, 3′-end cleavage sites, splicing variants, and coding potential. These analyses were facilitated by scrutiny of our collection of sequenced full-length cDNAs and much larger collection of 5′-ESTs, together with another set of full-length cDNAs from Salk/Stanford/Plant Gene Expression Center/RIKEN. Examples of alternative splicing are observed for transcripts from 7% of the genes and many of these genes display multiple spliced isoforms. Most splicing variants lie in non-coding regions of the transcripts. Non-canonical splice sites constitute less than 1% of all splice sites. Genes with fewer than four introns display reduced average mRNA levels. Putative alternative transcription start sites were observed in 30% of highly expressed genes and in more than 50% of the genes with low expression. Transcription start sites correlate remarkably well with a CG skew peak in the DNA sequences. The intergenic distances vary considerably, those where genes are transcribed towards one another being significantly shorter. New transcripts, missing in the current TIGR genome annotation and ESTs that are non-coding, including those antisense to known genes, are derived and cataloged in the Supplementary Material. They identify 148 new loci in the Arabidopsis genome. The conclusions drawn provide a better understanding of the Arabidopsis genome and how the gene transcripts are processed. The results also allow better predictions to be made for, as yet, poorly defined genes and provide a reference for comparisons with other plant genomes whose complete sequences are currently being determined. Some comparisons with rice are included in this paper.

Herbaceous energy crop development: recent progress and future prospects.

Oil prices and government mandates have catalyzed rapid growth of nonfossil transportation fuels in recent years, with a large focus on ethanol from energy crops, but the food crops used as first-generation energy crops today are not optimized for this purpose. We show that the theoretical efficiency of conversion of whole spectrum solar energy into biomass is 4.6-6%, depending on plant type, and the best year-long efficiencies realized are about 3%. The average leaf is as effective as the best PV solar cells in transducing solar energy to charge separation (ca. 37%). In photosynthesis, most of the energy that is lost is dissipated as heat during synthesis of biomass. Unlike photovoltaic (PV) cells this energetic cost supports the construction, maintenance, and replacement of the system, which is achieved autonomously as the plant grows and re-grows. Advances in plant genomics are being applied to plant breeding, thereby enabling rapid development of next-generation energy crops that capitalize on theoretical efficiencies while maintaining environmental and economic integrity.

High resolution genetic mapping by genome sequencing reveals genome duplication and tetraploid genetic structure of the diploid Miscanthus sinensis.

We have created a high-resolution linkage map of Miscanthus sinensis, using genotyping-by-sequencing (GBS), identifying all 19 linkage groups for the first time. The result is technically significant since Miscanthus has a very large and highly heterozygous genome, but has no or limited genomics information to date. The composite linkage map containing markers from both parental linkage maps is composed of 3,745 SNP markers spanning 2,396 cM on 19 linkage groups with a 0.64 cM average resolution. Comparative genomics analyses of the M. sinensis composite linkage map to the genomes of sorghum, maize, rice, and Brachypodium distachyon indicate that sorghum has the closest syntenic relationship to Miscanthus compared to other species. The comparative results revealed that each pair of the 19 M. sinensis linkages aligned to one sorghum chromosome, except for LG8, which mapped to two sorghum chromosomes (4 and 7), presumably due to a chromosome fusion event after genome duplication. The data also revealed several other chromosome rearrangements relative to sorghum, including two telomere-centromere inversions of the sorghum syntenic chromosome 7 in LG8 of M. sinensis and two paracentric inversions of sorghum syntenic chromosome 4 in LG7 and LG8 of M. sinensis. The results clearly demonstrate, for the first time, that the diploid M. sinensis is tetraploid origin consisting of two sub-genomes. This complete and high resolution composite linkage map will not only serve as a useful resource for novel QTL discoveries, but also enable informed deployment of the wealth of existing genomics resources of other species to the improvement of Miscanthus as a high biomass energy crop. In addition, it has utility as a reference for genome sequence assembly for the forthcoming whole genome sequencing of the Miscanthus genus.

Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions.

Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5′ and 3′ flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5′ upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.

Progress in upscaling Miscanthus biomass production for the European bio-economy with seed-based hybrids

Field trials in Europe with Miscanthus over the past 25 years have demonstrated that interspecies hybrids such as M. × giganteus (M × g) combine both high yield potentials and low inputs in a wide range of soils and climates. Miscanthus hybrids are expected to play a major role in the provision of perennial lignocellulosic biomass across much of Europe as part of a lower carbon economy. However, even with favourable policies in some European countries, uptake has been slow. M × g, as a sterile clone, can only be propagated vegetatively, which leads to high establishment costs and low multiplication rates. Consequently, a decade ago, a strategic decision to develop rapidly multiplied seeded hybrids was taken. To make progress on this goal, we have (1) harnessed the genetic diversity in Miscanthus by crossing and progeny testing thousands of parental combinations to select several candidate seed‐based hybrids adapted to European environments, (2) established field scale seed production methods with annual multiplication factors >1500×, (3) developed the agronomy for establishing large stands from seed sown plug plants to reduce establishment times by a year compared to M × g, (4) trialled a range of harvest techniques to improve compositional quality and logistics on a large scale, (5) performed spatial analyses of yield potential and land availability to identify regional opportunities across Europe and doubled the area within the bio‐climatic envelope, (6) considered on‐farm economic, practical and environmental benefits that can be attractive to growers. The technical barriers to adoption have now been overcome sufficiently such that Miscanthus is ready to use as a low‐carbon feedstock in the European bio‐economy.

Contrasting geographic patterns of genetic variation for molecular markers vs. phenotypic traits in the energy grass Miscanthus sinensis

Species and hybrids of Miscanthus are a promising energy crop, but their outcrossing mating systems and perennial life cycles are serious challenges for breeding programs. One approach to accelerating the domestication of Miscanthus is to harness the tremendous genetic variation that is present within this genus using phenotypic data from extensive field trials, high‐density genotyping and sequencing technologies, and rapidly developing statistical methods of relating phenotype to genotype. The success of this approach, however, hinges on detailed knowledge about the population genetic structure of the germplasm used in the breeding program. We therefore used data for 120 single‐nucleotide polymorphism and 52 simple sequence repeat markers to depict patterns of putatively neutral population structure among 244 Miscanthus genotypes grown in a field trial near Aberystwyth (UK) and delineate a population of 145 M. sinensis genotypes that will be used for association mapping and genomic selection. Comparative multivariate analyses of molecular marker and phenotypic data for 17 traits related to phenology, morphology/biomass, and cell wall composition revealed significant geographic patterns in this population. A longitudinal cline accounted for a substantial proportion of molecular marker variation (R2 = 0.60, P = 3.4 × 10−15). In contrast, genetic variation for phenotypic traits tended to follow latitudinal and altitudinal gradients, with several traits appearing to have been affected by divergent selection (i.e., QST >> FST). These contrasting geographic trends are unusual relative to other plants and provide opportunities for powerful studies of phenotype–genotype associations and the evolutionary history of M. sinensis.

In Planta Biocatalysis Screen of P450s Identifies 8-Methoxypsoralen as a Substrate for the CYP82C Subfamily, Yielding Original Chemical Structures

An in vivo plant screen that allows for the analysis of exogenously applied substrates against transgenic Arabidopsis lines overexpressing individual cytochrome P450s has been developed. By deploying this screen with a subset of 91 P450s, we have identified an original substrate for members of the CYP82C subfamily. The therapeutic compound 8-methoxypsoralen was hydroxylated by plants overexpressing CYP82C2 or CYP82C4, forming 5-hydroxy-8-methoxypsoralen. Additionally, plants further modified this product to create a glycosylated compound, likely the compound 5-O-beta-D-glucopyranosyl-8-methoxypsoralen. The discovery of adducts of therapeutic compounds demonstrates the potential of this biocatalysis screening approach to create compounds that may be of pharmacological value. Additionally, this platform provides a means to expand the general knowledge base of P450 enzyme/substrate combinations and may provide valuable tools for a vast array of biocatalytic and bioremediation processes.

Role of Model Plant Species

The use of model or reference species has played a major role in furthering detailed understanding of mechanisms and processes in the plant kingdom over the past 25 years. Species which have been adopted as models for dicotyledons and monocotyledons include arabidopsis and rice and more recently brachy-podium,Such models are diploids, have few and small chromosomes, well developed genetics, rapid life cycles, are easily transformed and have extensive sets of technical resources and databases curated by international resource centres. The study of crop genomics today is deeply rooted in earlier studies on model species. Genomes of model species share reasonable genetic synteny with key crop plants which facilitates the discovery of genes and association of genes with phenotypes. While some mechanisms and processes are conserved across the plant kingdom and so can be revealed by studies on any model species,others have diverged during evolution and so are revealed by studying only a closely related model species.Examples of processes that are conserved across the plant kingdom and others that have diverged and therefore need to be understood by studying a more closely related model species are described.