The TIGR Maize Database

Maize is a staple crop of the grass family and also an excellent model for plant genetics. Owing to the large size and repetitiveness of its genome, we previously investigated two approaches to accelerate gene discovery and genome analysis in maize: methylation filtration and high C(0)t selection. These techniques allow the construction of gene-enriched genomic libraries by minimizing repeat sequences due to either their methylation status or their copy number, yielding a 7-fold enrichment in genic sequences relative to a random genomic library. Approximately 900 000 gene-enriched reads from maize were generated and clustered into Assembled Zea mays (AZM) sequences. Here we report the current AZM release, which consists of ∼298 Mb representing 243 807 sequence assemblies and singletons. In order to provide a repository of publicly available maize genomic sequences, we have created the TIGR Maize Database (). In this resource, we have assembled and annotated the AZMs and used available sequenced markers to anchor AZMs to maize chromosomes. We have constructed a maize repeat database and generated draft sequence assemblies of 287 maize bacterial artificial chromosome (BAC) clone sequences, which we annotated along with 172 additional publicly available BAC clones. All sequences, assemblies and annotations are available at the project website via web interfaces and FTP downloads

The sequence of rice chromosomes 11 and 12, rich in disease resistance genes and recent gene duplications

Background: Rice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals. Results: We have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes. Conclusion: Because the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently evolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as previously suggested

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing

<p>Abstract</p> <p>Background</p> <p>In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage.</p> <p>Results</p> <p>To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20–454 pyrosequencing runs of a cDNA library obtained from 2 week-old <it>Palomero Toluqueño </it>maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20–454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20–454 sequences and corresponding levels of gene expression.</p> <p>Conclusion</p> <p>A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.</p

The Dynamics Of LTR Retrotransposon Accumulation Across 25 Million Years Of Panicoid Grass Evolution

Sample sequence analysis was employed to investigate the repetitive DNAs that were most responsible for the evolved variation in genome content across seven panicoid grasses with 45-fold variation in genome size and different histories of polyploidy. In all cases, the most abundant repeats were LTR retrotransposons, but the particular families that had become dominant were found to be different in the Pennisetum, Saccharum, Sorghum and Zea lineages. One element family, Huck, has been very active in all of the studied species over the last few million years. This suggests the transmittal of an active or quiescent autonomous set of Huck elements to this lineage at the founding of the panicoids. Similarly, independent recent activity of Ji and Opie elements in Zea and of Leviathan elements in Sorghum and Saccharum species suggests that members of these families with exceptional activation potential were present in the genome(s) of the founders of these lineages. In a detailed analysis of the Zea lineage, the combined action of several families of LTR retrotransposons were observed to have approximately doubled the genome size of Zea luxurians relative to Zea mays and Zea diploperennis in just the last few million years. One of the LTR retrotransposon ampli?cation bursts in Zea may have been initiated by polyploidy, but the great majority of transposable element activations are not. Instead, the results suggest random activation of a few or many LTR retrotransposons families in particular lineages over evolutionary time, with some families especially prone to future activation and hyper-amplification

Gramene: a growing plant comparative genomics resource

Gramene (www.gramene.org) is a curated resource for genetic, genomic and comparative genomics data for the major crop species, including rice, maize, wheat and many other plant (mainly grass) species. Gramene is an open-source project. All data and software are freely downloadable through the ftp site (ftp.gramene.org/pub/gramene) and available for use without restriction. Gramene's core data types include genome assembly and annotations, other DNA/mRNA sequences, genetic and physical maps/markers, genes, quantitative trait loci (QTLs), proteins, ontologies, literature and comparative mappings. Since our last NAR publication 2 years ago, we have updated these data types to include new datasets and new connections among them. Completely new features include rice pathways for functional annotation of rice genes; genetic diversity data from rice, maize and wheat to show genetic variations among different germplasms; large-scale genome comparisons among Oryza sativa and its wild relatives for evolutionary studies; and the creation of orthologous gene sets and phylogenetic trees among rice, Arabidopsis thaliana, maize, poplar and several animal species (for reference purpose). We have significantly improved the web interface in order to provide a more user-friendly browsing experience, including a dropdown navigation menu system, unified web page for markers, genes, QTLs and proteins, and enhanced quick search functions

Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

<p>Abstract</p> <p>Background</p> <p>Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends.</p> <p>Results</p> <p>A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the <it>Sal</it>I MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary.</p> <p>Conclusion</p> <p>MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely understood at this time, MSLL technology flags both approximate boundaries and methylated genes that deserve additional investigation. MSLL and HMPR sequences provide a valuable resource for maize genome annotation, and are a uniquely valuable complement to any plant genome sequencing project. In order to make these results fully accessible to the community, a web display was developed that shows the alignment of MSLL, HMPR, and other gene-rich sequences to the BACs; this display is continually updated with the latest ESTs and BAC sequences.</p