Project description:We profiled the gene regulatory landscape of Brassica napus reproductive development using RNA sequencing. Comparative analysis of this nascent allotetraploid across the plant lifecycle revealed the contribution of each subgenome to plant reproduction. Global mRNA profiling across reproductive development revealed lower accumulation of C subgenome transcripts relative to the A subgenome. Subgenome-specific transcriptional networks identified distinct transcription factor families enriched in each of the A and C subgenome in early seed development. Analysis of a tissue specific transcriptome of early seed development revealed transcription factors predicted to be regulators encoded by the A subgenome are expressed primarily in the seed coat whereas regulators encoded by the C subgenome were expressed primarily in the embryo. Whole genome transcription factor networks identified BZIP11 as an essential regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in knockdown of predicted target genes, and a reproductive-lethal phenotype. Our data indicate that subgenome bias are characteristic features of the B. napus seed throughout its development, and that such bias might not be universal across the embryo, endosperm, and seed coat of the developing seed. We also find that examining transcriptional networks spanning both the A and C genomes of the whole B. napus seed can identify valuable targets for seed development research. We suggest that-omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses.
Project description:High temperature stress results in yield loss and alterations to seed composition during seed filling in oilseed rape (Brassica napus). However, the mechanism underlying this heat response is poorly understood. In this study, we employed a microarray analysis with silique walls and seeds from the developing siliques (20 days after flowering) of Brassica napus that had undergone heat stress.
Project description:High temperature stress results in yield loss and alterations to seed composition during seed filling in oilseed rape (Brassica napus). However, the mechanism underlying this heat response is poorly understood. In this study, we employed a microarray analysis with silique walls and seeds from the developing siliques (20 days after flowering) of Brassica napus that had undergone heat stress. Two-condition experiment, control vs heat stress, 2 time points
Project description:Illumina mRNA-Seq is comparable to microarray analysis for transcript quantification but has increased sensitivity and, importantly, the potential to distinguish between homoeologous genes in polyploids. Using a novel curing process, we adapted a reference sequence that was a consensus derived from ESTs from both Brassica A and C genomes to one containing A and C genome versions for each of the 94,558 original unigenes. We aligned reads from Brassica napus to this cured reference, finding 38% more reads mapping in resynthesised lines and 28% in natural lines. Where the A and C versions differed at single nucleotide positions, termed inter-homoeologue polymorphisms (IHPs), we were able to apportion expression in the polyploid to the A or C genome homoeologues. 43,761 unigenes contained at least one IHP, with a mean frequency of 10.5 per kb unigene sequence. 6,350 of the unigenes with IHPs were differentially expressed between homoeologous gene pairs in resynthesised B. napus. 3,212 unigenes showed a similar pattern of differential expression across a range of natural B. napus crop varieties and, of these, 995 were in common with resynthesised B. napus. Functional classification showed over-representation in gene ontology categories not associated with dosage-sensitivity.
Project description:Association Genetics can quickly and efficiently delineate regions of the genome that control traits and provide markers to accelerate breeding by marker-assisted selection. The requirements for many markers and a genome sequence to order those markers have limited its exploitation in crops. To harness this approach for use in a broad range of crops, even those with complex genomes, we developed an approach based on transcriptome sequencing to exploit markers representing variation in both gene sequences and gene expression. We term this approach Associative Transcriptomics. Applying it successfully in Brassica napus, as an example, we identified that genomic deletions including orthologues of the transcription factor controlling aliphatic glucosinolate biosynthesis in Arabidopsis thaliana, HAG1 (At5g61420), underlie two QTL for glucosinolate content of seeds.
Project description:The purposes of this study are to compare euploid B. napus cv. “Oro” and the C1 nullisomics transcriptome profiling (RNA-seq) and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods: Leaves mRNA profiles of 30-day-old euploid B. napus cv. “Oro” and the C1 nullisomics were generated by deep sequencing, in triplicate, using Illumina. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods. qRT–PCR validation was performed. Results: After the high-throughput sequencing, each sample generated 7.2G Clean data on average and the 44.6-53.8 million clean reads were generated. Our study represents detailed analysis of leaves transcriptomes in euploid B. napus and nullisomic, with biologic replicates, generated by RNA-seq technology for comparative investigations of expression profiles. Our results show that Dose complementary effect exists between highly homologous genes, and partial loss of C subgenomic chromosome will lead to increased expression of A genome.