Promoter variation and transcript divergence in Brassicaceae lineages of FLOWERING LOCUS T.

Brassica napus (AACC, 2n = 38), an oil crop of world-wide importance, originated from interspecific hybridization of B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18), and has six FLOWERING LOCUS T (FT) paralogues. Two located on the homeologous chromosomes A2 and C2 arose from a lineage distinct from four located on A7 and C6. A set of three conserved blocks A, B and C, which were found to be essential for FT activation by CONSTANS (CO) in Arabidopsis, was identified within the FT upstream region in B. napus and its progenitor diploids. However, on chromosome C2, insertion of a DNA transposable element (TE) and a retro-element in FT upstream blocks A and B contributed to significant structural divergence between the A and C genome orthologues. Phylogenetic analysis of upstream block A indicated the conserved evolutionary relationships of distinct FT genes within Brassicaceae. We conclude that the ancient At-α whole genome duplication contributed to distinct ancestral lineages for this key adaptive gene, which co-exist within the same genus. FT-A2 was found to be transcribed in all leaf samples from different developmental stages in both B. rapa and B. napus, whereas FT-C2 was not transcribed in either B. napus or B. oleracea. Silencing of FT-C2 appeared to result from TE insertion and consequent high levels of cytosine methylation in TE sequences within upstream block A. Interestingly, FT-A7/C6 paralogues were specifically silenced in winter type B. napus but abundantly expressed in spring type cultivars under vernalization-free conditions. Motif prediction indicated the presence of two CO protein binding sites within all Brassica block A and additional sites for FT activation in block C. We propose that the ancestral whole genome duplications have contributed to more complex mechanisms of floral regulation and niche adaptation in Brassica compared to Arabidopsis.

A hypomethylated population of Brassica rapa for forward and reverse epi-genetics.

BACKGROUND: Epigenetic marks superimposed on the DNA sequence of eukaryote chromosomes provide agility and plasticity in terms of modulating gene expression, ontology, and response to the environment. Modulating the methylation status of cytosine can generate epialleles, which have been detected and characterised at specific loci in several plant systems, and have the potential to generate novel and relatively stable phenotypes. There have been no systematic attempts to explore and utilise epiallelic variation, and so extend the range of phenotypes available for selection in crop improvement. We developed an approach for generating novel epialleles by perturbation of the DNA methylation status. 5- Azacytidine (5-AzaC) provides selective targeting of 5 mCG, which in plants is associated with exonic DNA. Targeted chemical intervention using 5-AzaC has advantages over transgenic or mutant modulation of methyltransferases, allowing stochastic generation of epialleles across the genome. RESULTS: We demonstrate the potential of stochastic chemically-induced hypomethylation to generate novel and valuable variation for crop improvement. Systematic analysis of dose-response to 5-AzaC in B. rapa guided generation of a selfed stochastically hypomethylated population, used for forward screening of several agronomic traits. Dose-response was sigmoidal for several traits, similar to that observed for chemical mutagens such as EMS. We demonstrated transgenerational inheritance of some phenotypes. BraRoAZ is a unique hypomethylated population of 1000 E2 sib lines. When compared to untreated controls, 5-Aza C-treated lines exhibited reduced immuno-staining of 5mC on pachytene chromosomes, and Methylation Sensitive Amplified Polymorphism (MSAP) profiles that were both divergent and more variable. There was coincident phenotypic variation among these lines for a range of seed yield and composition traits, including increased seed protein content and decreased oil content, as well as decreased erucic acid and corresponding increases in linoleic and/or palmitic acid. Each 5-AzaC-treated line represents a unique combination of hypomethylated epialleles. CONCLUSIONS: The approach and populations developed are available for forward and reverse screening of epiallelic variation and subsequent functional and inheritance studies. The generation of stochastically hypomethylated populations has utility in epiallele discovery for a wide range of crop plants, and has considerable potential as an intervention strategy for crop improvement.

Brassica ASTRA: an integrated database for Brassica genomic research.

Brassica ASTRA is a public database for genomic information on Brassica species. The database incorporates expressed sequences with Swiss-Prot and GenBank comparative sequence annotation as well as secondary Gene Ontology (GO) annotation derived from the comparison with Arabidopsis TAIR GO annotations. Simple sequence repeat molecular markers are identified within resident sequences and mapped onto the closely related Arabidopsis genome sequence. Bacterial artificial chromosome (BAC) end sequences derived from the Multinational Brassica Genome Project are also mapped onto the Arabidopsis genome sequence enabling users to identify candidate Brassica BACs corresponding to syntenic regions of Arabidopsis. This information is maintained in a MySQL database with a web interface providing the primary means of interrogation. The database is accessible at http://hornbill.cspp.latrobe.edu.au.