A more efficient digital gene expression (DGE) method to produce sequencing data by multiplexing from a large plant population of winter oilseed rape (Brassica napus L.) was created. Subsequently, DGE analysis to identify candidate regulatory genes associated with complex traits during seedling development was performed. Seedling vigour is an important trait due to its influence on seedling establishment before winter and the consequent effects on yield and yield stability. The multiplexed-DGE analysis was proved to be a cost-effective method for large-scale quantitative transcriptome analysis, using next-generation Illumina (Solexa) sequencing. The normalised DGE data with barcode multiplexed indexing could then be successfully applied to weighted gene co-expression network analysis (WGCNA) together with an integration of phenotypic and metabolic hormone data. A bulked-segregant analysis (BSA)-DGE approach was used in this study. For each of the investigated traits, 20 best and 20 worst performing individuals, respectively, were assigned to bulks with significant phenotypic variance. The bulked DH lines segregate strongly for numerous complex quantitative traits, e.g. development, vigour, flowering, and yield components. A number of significantly differentially expressed genes could be identified. The regulatory gene CLV1 (CLAVATA 1) was found to be differentially expressed for traits such as shoot fresh weight and plant height at flowering for year 2006. This suggests its role as regulatory component of the molecular network controlling shoot meristem activity in winter oilseed rape. In addition, the genes NIP (NOD26-LIKE INTRINSIC PROTEIN), LHCB3 (LIGHT HARVESTING CHLOROPHYLL B BINDING PROTEIN 3), TT7 (TRANSPARENT TESTA 7) and CRU2 (CRUCIFERIN 2) were significantly differentially expressed for the seed yield trait for years 2006 and 2007. Interestingly, NIP, which belongs to the aquaporin superfamily and is plant-specific, was found to be differentialy expressed at both 8 and 12 DAS. The expression of NIP in WOSR indicates a wide range of function that may include a greater range in selectivity. The LHCB3 gene is a component of the main light harvesting chlorophyll a/b-protein complex of Photosystem II (PSII) and is therefore very important during the photosynthetic process. CRU2 belongs to an important group of seed storage proteins in Brassica napus, the 12S globulin complex, and is synthesized during seed development. In addition, a total of fifteen Top Hub Brassica Unigenes for A (B. rapa) and C (B. oleracea) genomes were identified as candidate regulatory genes for complex trait interaction during seedling development. Annotation of these Top Hub Unigenes with Arabidopsis thaliana genome (by BLASTX, TAIR 10 and agriGO) was performed to determine gene functions and they could be related to leaf and shoot system development, cytokinin pathway and circadian clock. The transcription activators GRF1 (GROWTH REGULATING FACTOR 1), UBP15 (UBIQUITIN-SPECIFIC PROTEASE 15), and VPS2 (VACUOLAR PROTEIN SORTING 2) were found to play a key role in leaf and shoot system development. Furthermore, GF14 (G BOX FACTOR 14-3-3 OMEGA) also known as GRF2 was identified within the co-expression network interacting almost all aspects of plant growth and development, including hormonal metabolism and gene transcription. These results gave an insight into the genetic regulation of complex traits, providing some candidate genes and allowing to some extent an interpretation of their regulatory role. The DGE data presented in this study comprise a novel basis for further genetical genomics approaches, e.g. eQTL, gene isolation, functional validation, and development of gene expression markers (GEMs).
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