Kristi Collura

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

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. 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. 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.BackgroundRice 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.ResultsWe 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.ConclusionBecause 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.

Sequencing, mapping, and analysis of 27,455 maize full-length cDNAs.

Full-length cDNA (FLcDNA) sequencing establishes the precise primary structure of individual gene transcripts. From two libraries representing 27 B73 tissues and abiotic stress treatments, 27,455 high-quality FLcDNAs were sequenced. The average transcript length was 1.44 kb including 218 bases and 321 bases of 5′ and 3′ UTR, respectively, with 8.6% of the FLcDNAs encoding predicted proteins of fewer than 100 amino acids. Approximately 94% of the FLcDNAs were stringently mapped to the maize genome. Although nearly two-thirds of this genome is composed of transposable elements (TEs), only 5.6% of the FLcDNAs contained TE sequences in coding or UTR regions. Approximately 7.2% of the FLcDNAs are putative transcription factors, suggesting that rare transcripts are well-enriched in our FLcDNA set. Protein similarity searching identified 1,737 maize transcripts not present in rice, sorghum, Arabidopsis, or poplar annotated genes. A strict FLcDNA assembly generated 24,467 non-redundant sequences, of which 88% have non-maize protein matches. The FLcDNAs were also assembled with 41,759 FLcDNAs in GenBank from other projects, where semi-strict parameters were used to identify 13,368 potentially unique non-redundant sequences from this project. The libraries, ESTs, and FLcDNA sequences produced from this project are publicly available. The annotated EST and FLcDNA assemblies are available through the maize FLcDNA web resource (www.maizecdna.org).

Dynamic Evolution of Oryza Genomes Is Revealed by Comparative Genomic Analysis of a Genus-Wide Vertical Data Set

Oryza (23 species; 10 genome types) contains the worlds most important food crop — rice. Although the rice genome serves as an essential tool for biological research, little is known about the evolution of the other Oryza genome types. They contain a historical record of genomic changes that led to diversification of this genus around the world as well as an untapped reservoir of agriculturally important traits. To investigate the evolution of the collective Oryza genome, we sequenced and compared nine orthologous genomic regions encompassing the Adh1-Adh2 genes (from six diploid genome types) with the rice reference sequence. Our analysis revealed the architectural complexities and dynamic evolution of this region that have occurred over the past ∼15 million years. Of the 46 intact genes and four pseudogenes in the japonica genome, 38 (76%) fell into eight multigene families. Analysis of the evolutionary history of each family revealed independent and lineage-specific gain and loss of gene family members as frequent causes of synteny disruption. Transposable elements were shown to mediate massive replacement of intergenic space (>95%), gene disruption, and gene/gene fragment movement. Three cases of long-range structural variation (inversions/deletions) spanning several hundred kilobases were identified that contributed significantly to genome diversification.

The physical and genetic framework of the maize b73 genome

Maize is a major cereal crop and an important model system for basic biological research. Knowledge gained from maize research can also be used to genetically improve its grass relatives such as sorghum, wheat, and rice. The primary objective of the Maize Genome Sequencing Consortium (MGSC) was to generate a reference genome sequence that was integrated with both the physical and genetic maps. Using a previously published integrated genetic and physical map, combined with in-coming maize genomic sequence, new sequence-based genetic markers, and an optical map, we dynamically picked a minimum tiling path (MTP) of 16,910 bacterial artificial chromosome (BAC) and fosmid clones that were used by the MGSC to sequence the maize genome. The final MTP resulted in a significantly improved physical map that reduced the number of contigs from 721 to 435, incorporated a total of 8,315 mapped markers, and ordered and oriented the majority of FPC contigs. The new integrated physical and genetic map covered 2,120 Mb (93%) of the 2,300-Mb genome, of which 405 contigs were anchored to the genetic map, totaling 2,103.4 Mb (99.2% of the 2,120 Mb physical map). More importantly, 336 contigs, comprising 94.0% of the physical map (∼1,993 Mb), were ordered and oriented. Finally we used all available physical, sequence, genetic, and optical data to generate a golden path (AGP) of chromosome-based pseudomolecules, herein referred to as the B73 Reference Genome Sequence version 1 (B73 RefGen_v1).

Transposable element distribution, abundance and role in genome size variation in the genus Oryza

BackgroundThe genus Oryza is composed of 10 distinct genome types, 6 diploid and 4 polyploid, and includes the worlds most important food crop – rice (Oryza sativa [AA]). Genome size variation in the Oryza is more than 3-fold and ranges from 357 Mbp in Oryza glaberrima [AA] to 1283 Mbp in the polyploid Oryza ridleyi [HHJJ]. Because repetitive elements are known to play a significant role in genome size variation, we constructed random sheared small insert genomic libraries from 12 representative Oryza species and conducted a comprehensive study of the repetitive element composition, distribution and phylogeny in this genus. Particular attention was paid to the role played by the most important classes of transposable elements (Long Terminal Repeats Retrotransposons, Long interspersed Nuclear Elements, helitrons, DNA transposable elements) in shaping these genomes and in their contributing to genome size variation.ResultsWe identified the elements primarily responsible for the most strikingly genome size variation in Oryza. We demonstrated how Long Terminal Repeat retrotransposons belonging to the same families have proliferated to very different extents in various species. We also showed that the pool of Long Terminal Repeat Retrotransposons is substantially conserved and ubiquitous throughout the Oryza and so its origin is ancient and its existence predates the speciation events that originated the genus. Finally we described the peculiar behavior of repeats in the species Oryza coarctata [HHKK] whose placement in the Oryza genus is controversial.ConclusionLong Terminal Repeat retrotransposons are the major component of the Oryza genomes analyzed and, along with polyploidization, are the most important contributors to the genome size variation across the Oryza genus. Two families of Ty3-gypsy elements (RIRE2 and Atlantys) account for a significant portion of the genome size variations present in the Oryza genus.

Construction, alignment and analysis of twelve framework physical maps that represent the ten genome types of the genus Oryza

We describe the establishment and analysis of a genus-wide comparative framework composed of 12 bacterial artificial chromosome fingerprint and end-sequenced physical maps representing the 10 genome types of Oryza aligned to the O. sativa ssp. japonica reference genome sequence. Over 932 Mb of end sequence was analyzed for repeats, simple sequence repeats, miRNA and single nucleotide variations, providing the most extensive analysis of Oryza sequence to date.

BAC-end Sequence Analysis and a Draft Physical Map of the Common Bean ( Phaseolus vulgaris L.) Genome

Common bean (Phaseolus vulgaris L.) is a legume that is an important source of dietary protein in developing countries throughout the world. Utilizing the G19833 BAC library for P. vulgaris from Clemson University, 89,017 BAC-end sequences were generated giving 62,588,675 base pairs of genomic sequence covering approximately 9.54% of the genome. Analysis of these sequences in combination with 1,404 shotgun sequences from the cultivar Bat7 revealed that approximately 49.2% of the genome contains repetitive sequence and 29.3% is genic. Compared to other legume BAC-end sequencing projects, it appears that P. vulgaris has higher predicted levels of repetitive sequence, but this may be due to a more intense identification strategy combining both similarity-based matches as well as de novo identification of repeats. In addition, fingerprints for 41,717 BACs were obtained and assembled into a draft physical map consisting of 1,183 clone contigs and 6,385 singletons with ~9x coverage of the genome.

Comparative Physical Mapping Between Oryza sativa (AA Genome Type) and O. punctata (BB Genome Type)

A comparative physical map of the AA genome (Oryza sativa) and the BB genome (O. punctata) was constructed by aligning a physical map of O. punctata, deduced from 63,942 BAC end sequences (BESs) and 34,224 fingerprints, onto the O. sativa genome sequence. The level of conservation of each chromosome between the two species was determined by calculating a ratio of BES alignments. The alignment result suggests more divergence of intergenic and repeat regions in comparison to gene-rich regions. Further, this characteristic enabled localization of heterochromatic and euchromatic regions for each chromosome of both species. The alignment identified 16 locations containing expansions, contractions, inversions, and transpositions. By aligning 40% of the punctata BES on the map, 87% of the punctata FPC map covered 98% of the O. sativa genome sequence. The genome size of O. punctata was estimated to be 8% larger than that of O. sativa with individual chromosome differences of 1.5–16.5%. The sum of expansions and contractions observed in regions >500 kb were similar, suggesting that most of the contractions/expansions contributing to the genome size difference between the two species are small, thus preserving the macro-collinearity between these species, which diverged ∼2 million years ago.

Detailed analysis of a contiguous 22-Mb region of the maize genome.

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on ∼1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.

An integrated physical, genetic and cytogenetic map of Brachypodium distachyon, a model system for grass research.

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent vaildation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation.