Ian Bancroft

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

Analysis of the bread wheat genome using whole-genome shotgun sequencing

Bread wheat (Triticum aestivum) is a globally important crop, accounting for 20 per cent of the calories consumed by humans. Major efforts are underway worldwide to increase wheat production by extending genetic diversity and analysing key traits, and genomic resources can accelerate progress. But so far the very large size and polyploid complexity of the bread wheat genome have been substantial barriers to genome analysis. Here we report the sequencing of its large, 17-gigabase-pair, hexaploid genome using 454 pyrosequencing, and comparison of this with the sequences of diploid ancestral and progenitor genomes. We identified between 94,000 and 96,000 genes, and assigned two-thirds to the three component genomes (A, B and D) of hexaploid wheat. High-resolution synteny maps identified many small disruptions to conserved gene order. We show that the hexaploid genome is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. Several classes of genes involved in energy harvesting, metabolism and growth are among expanded gene families that could be associated with crop productivity. Our analyses, coupled with the identification of extensive genetic variation, provide a resource for accelerating gene discovery and improving this major crop.

Early allopolyploid evolution in the post-neolithic Brassica napus oilseed genome

The genomic origins of rape oilseed Many domesticated plants arose through the meeting of multiple genomes through hybridization and genome doubling, known as polyploidy. Chalhoub et al. sequenced the polyploid genome of Brassica napus, which originated from a recent combination of two distinct genomes approximately 7500 years ago and gave rise to the crops of rape oilseed (canola), kale, and rutabaga. B. napus has undergone multiple events affecting differently sized genetic regions where a gene from one progenitor species has been converted to the copy from a second progenitor species. Some of these gene conversion events appear to have been selected by humans as part of the process of domestication and crop improvement. Science, this issue p. 950 The polyploid genome of oilseed rape exhibits evolution through homologous gene conversion. Oilseed rape (Brassica napus L.) was formed ~7500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling, a process known as allopolyploidy. Together with more ancient polyploidizations, this conferred an aggregate 72× genome multiplication since the origin of angiosperms and high gene content. We examined the B. napus genome and the consequences of its recent duplication. The constituent An and Cn subgenomes are engaged in subtle structural, functional, and epigenetic cross-talk, with abundant homeologous exchanges. Incipient gene loss and expression divergence have begun. Selection in B. napus oilseed types has accelerated the loss of glucosinolate genes, while preserving expansion of oil biosynthesis genes. These processes provide insights into allopolyploid evolution and its relationship with crop domestication and improvement.

FCA, a Gene Controlling Flowering Time in Arabidopsis, Encodes a Protein Containing RNA-Binding Domains

A strong promoter of the transition to flowering in Arabidopsis is encoded by FCA. FCA has been cloned and shown to encode a protein containing two RNA-binding domains and a WW protein interaction domain. This suggests that FCA functions in the posttranscriptional regulation of transcripts involved in the flowering process. The FCA transcript is alternatively spliced with only one form encoding the entire FCA protein. Plants carrying the FCA gene fused to the strong constitutive 35S promoter flowered earlier, and the ratio and abundance of the different FCA transcripts were altered. Thus, FCA appears to be a component of a posttranscriptional cascade involved in the control of flowering time.

The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes

Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.

Comparative Genomics of Brassica oleracea and Arabidopsis thaliana Reveal Gene Loss, Fragmentation, and Dispersal after Polyploidy

We sequenced 2.2 Mb representing triplicated genome segments of Brassica oleracea, which are each paralogous with one another and homologous with a segmentally duplicated region of the Arabidopsis thaliana genome. Sequence annotation identified 177 conserved collinear genes in the B. oleracea genome segments. Analysis of synonymous base substitution rates indicated that the triplicated Brassica genome segments diverged from a common ancestor soon after divergence of the Arabidopsis and Brassica lineages. This conclusion was corroborated by phylogenetic analysis of protein families. Using A. thaliana as an outgroup, 35% of the genes inferred to be present when genome triplication occurred in the Brassica lineage have been lost, most likely via a deletion mechanism, in an interspersed pattern. Genes encoding proteins involved in signal transduction or transcription were not found to be significantly more extensively retained than those encoding proteins classified with other functions, but putative proteins predicted in the A. thaliana genome were underrepresented in B. oleracea. We identified one example of gene loss from the Arabidopsis lineage. We found evidence for the frequent insertion of gene fragments of nuclear genomic origin and identified four apparently intact genes in noncollinear positions in the B. oleracea and A. thaliana genomes.

Unraveling the Complex Trait of Crop Yield With Quantitative Trait Loci Mapping in Brassica napus

Yield is the most important and complex trait for the genetic improvement of crops. Although much research into the genetic basis of yield and yield-associated traits has been reported, in each such experiment the genetic architecture and determinants of yield have remained ambiguous. One of the most intractable problems is the interaction between genes and the environment. We identified 85 quantitative trait loci (QTL) for seed yield along with 785 QTL for eight yield-associated traits, from 10 natural environments and two related populations of rapeseed. A trait-by-trait meta-analysis revealed 401 consensus QTL, of which 82.5% were clustered and integrated into 111 pleiotropic unique QTL by meta-analysis, 47 of which were relevant for seed yield. The complexity of the genetic architecture of yield was demonstrated, illustrating the pleiotropy, synthesis, variability, and plasticity of yield QTL. The idea of estimating indicator QTL for yield QTL and identifying potential candidate genes for yield provides an advance in methodology for complex traits.

Sequence-Level Analysis of the Diploidization Process in the Triplicated FLOWERING LOCUS C Region of Brassica rapa

Strong evidence exists for polyploidy having occurred during the evolution of the tribe Brassiceae. We show evidence for the dynamic and ongoing diploidization process by comparative analysis of the sequences of four paralogous Brassica rapa BAC clones and the homologous 124-kb segment of Arabidopsis thaliana chromosome 5. We estimated the times since divergence of the paralogous and homologous lineages. The three paralogous subgenomes of B. rapa triplicated 13 to 17 million years ago (MYA), very soon after the Arabidopsis and Brassica divergence occurred at 17 to 18 MYA. In addition, a pair of BACs represents a more recent segmental duplication, which occurred ∼0.8 MYA, and provides an exception to the general expectation of three paralogous segments within the B. rapa genome. The Brassica genome segments show extensive interspersed gene loss relative to the inferred structure of the ancestral genome, whereas the Arabidopsis genome segment appears little changed. Representatives of all 32 genes in the Arabidopsis genome segment are represented in Brassica, but the hexaploid complement of 96 has been reduced to 54 in the three subgenomes, with compression of the genomic region lengths they occupy to between 52 and 110 kb. The gene content of the recently duplicated B. rapa genome segments is identical, but intergenic sequences differ.

Associative transcriptomics of traits in the polyploid crop species Brassica napus

Association genetics can quickly and efficiently delineate regions of the genome that control traits and provide markers to accelerate breeding by marker-assisted selection. But most crops are polyploid, making it difficult to identify the required markers and to assemble a genome sequence to order those markers. To circumvent this difficulty, we developed associative transcriptomics, which uses transcriptome sequencing to identify and score molecular markers representing variation in both gene sequences and gene expression, and correlate this with trait variation. Applying the method in the recently formed tetraploid crop Brassica napus, we identified genomic deletions that underlie two quantitative trait loci for glucosinolate content of seeds. The deleted regions contained orthologs of the transcription factor HAG1 (At5g61420), which controls aliphatic glucosinolate biosynthesis in Arabidopsis thaliana. This approach facilitates the application of association genetics in a broad range of crops, even those with complex genomes.

A Nuclear Protease Required for Flowering-Time Regulation in Arabidopsis Reduces the Abundance of SMALL UBIQUITIN-RELATED MODIFIER Conjugates

The Arabidopsis mutant early in short days4 (esd4) shows extreme early flowering and alterations in shoot development. We have identified ESD4 and demonstrate that it encodes a nuclear protein located predominantly at the periphery of the nucleus. ESD4 contains a segment of >200 amino acids with strong similarity to yeast and animal proteases that are specific for the protein modifier SMALL UBIQUITIN-RELATED MODIFIER (SUMO). ESD4 shows a similar function to these proteases in vitro and processes the precursor of Arabidopsis SUMO (AtSUMO) to generate the mature form. This activity of ESD4 is prevented by mutations that affect the predicted active site of the protease or the cleavage site of the AtSUMO precursor. In yeast, these proteases also recycle SUMO from conjugates, and this appears to be the major role of ESD4 in vivo. This is suggested because esd4 mutants contain less free AtSUMO and more SUMO conjugates than wild-type plants, and a transgene expressing mature SUMO at high levels enhanced aspects of the esd4 phenotype. ESD4 defines an important role for protein modification by AtSUMO in the regulation of flowering.