Janet Higgins

The FLOWERING LOCUS T-Like Gene Family in Barley (Hordeum vulgare)

The FLOWERING LOCUS T (FT) gene plays a central role in integrating flowering signals in Arabidopsis because its expression is regulated antagonistically by the photoperiod and vernalization pathways. FT belongs to a family of six genes characterized by a phosphatidylethanolamine-binding protein (PEBP) domain. In rice (Oryza sativa), 19 PEBP genes were previously described, 13 of which are FT-like genes. Five FT-like genes were found in barley (Hordeum vulgare). HvFT1, HvFT2, HvFT3, and HvFT4 were highly homologous to OsFTL2 (the Hd3a QTL), OsFTL1, OsFTL10, and OsFTL12, respectively, and this relationship was supported by comparative mapping. No rice equivalent was found for HvFT5. HvFT1 was highly expressed under long-day (inductive) conditions at the time of the morphological switch of the shoot apex from vegetative to reproductive growth. HvFT2 and HvFT4 were expressed later in development. HvFT1 was therefore identified as the main barley FT-like gene involved in the switch to flowering. Mapping of HvFT genes suggests that they provide important sources of flowering-time variation in barley. HvFTI was a candidate for VRN-H3, a dominant mutation giving precocious flowering, while HvFT3 was a candidate for Ppd-H2, a major QTL affecting flowering time in short days.

Comparative genomics of flowering time pathways using Brachypodium distachyon as a model for the temperate grasses.

Brachypodium distachyon (Brachypodium) is a model for the temperate grasses which include important cereals such as barley, wheat and oats. Comparison of the Brachypodium genome (accession Bd21) with those of the model dicot Arabidopsis thaliana and the tropical cereal rice (Oryza sativa) provides an opportunity to compare and contrast genetic pathways controlling important traits. We analysed the homologies of genes controlling the induction of flowering using pathways curated in Arabidopsis Reactome as a starting point. Pathways include those detecting and responding to the environmental cues of day length (photoperiod) and extended periods of low temperature (vernalization). Variation in these responses has been selected during cereal domestication, providing an interesting comparison with the wild genome of Brachypodium. Brachypodium Bd21 has well conserved homologues of circadian clock, photoperiod pathway and autonomous pathway genes defined in Arabidopsis and homologues of vernalization pathway genes defined in cereals with the exception of VRN2 which was absent. Bd21 also lacked a member of the CO family (CO3). In both cases flanking genes were conserved showing that these genes are deleted in at least this accession. Segmental duplication explains the presence of two CO-like genes in temperate cereals, of which one (Hd1) is retained in rice, and explains many differences in gene family structure between grasses and Arabidopsis. The conserved fine structure of duplications shows that they largely evolved to their present structure before the divergence of the rice and Brachypodium. Of four flowering-time genes found in rice but absent in Arabidopsis, two were found in Bd21 (Id1, OsMADS51) and two were absent (Ghd7, Ehd1). Overall, results suggest that an ancient core photoperiod pathway promoting flowering via the induction of FT has been modified by the recruitment of additional lineage specific pathways that promote or repress FT expression.

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.

Dissecting the genome of the polyploid crop oilseed rape by transcriptome sequencing

Polyploidy complicates genomics-based breeding of many crops, including wheat, potato, cotton, oat and sugarcane. To address this challenge, we sequenced leaf transcriptomes across a mapping population of the polyploid crop oilseed rape (Brassica napus) and representative ancestors of the parents of the population. Analysis of sequence variation and transcript abundance enabled us to construct twin single nucleotide polymorphism linkage maps of B. napus, comprising 23,037 markers. We used these to align the B. napus genome with that of a related species, Arabidopsis thaliana, and to genome sequence assemblies of its progenitor species, Brassica rapa and Brassica oleracea. We also developed methods to detect genome rearrangements and track inheritance of genomic segments, including the outcome of an interspecific cross. By revealing the genetic consequences of breeding, cost-effective, high-resolution dissection of crop genomes by transcriptome sequencing will increase the efficiency of predictive breeding even in the absence of a complete genome sequence.

Use of mRNA-seq to discriminate contributions to the transcriptome from the constituent genomes of the polyploid crop species Brassica napus

BackgroundPolyploidy often results in considerable changes in gene expression, both immediately and over evolutionary time. New phenotypes often arise with polyploid formation and may contribute to the fitness of polyploids in nature or their selection for use in agriculture. Oilseed rape (Brassica napus) is widely used to study the process of polyploidy both in artificially resynthesised and natural forms. mRNA-Seq, a recently developed approach to transcriptome profiling using deep-sequencing technologies is an alternative to microarrays for the study of gene expression in a polyploid.ResultsIllumina mRNA-Seq is comparable to microarray analysis for transcript quantification but has increased sensitivity and, very 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 separate A and C genome versions for each of the 94,558 original unigenes. We aligned reads from B. napus to this cured reference, finding 38% more reads mapping from resynthesised lines and 28% more reads mapping from 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 between the A and 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.ConclusionmRNA-Seq is the method of choice for measuring transcript abundance in polyploids due to its ability to measure the contributions of homoeologues to gene expression. The identification of large numbers of differentially expressed genes in both a newly resynthesised polyploid and natural B. napus confirms that there are both immediate and long-term alterations in the expression of homoeologous gene pairs following polyploidy.

Arabidopsis Reactome: A Foundation Knowledgebase for Plant Systems Biology

New ways of capturing and representing biological knowledge are needed to enable individual researchers to remain abreast of relevant discoveries and to permit computational approaches for interpreting the large volumes of diverse data generated by modern biological research. Here, we describe a

Seedling development traits in Brassica napus examined by gene expression analysis and association mapping.

BackgroundAn optimal seedling development of Brassica napus plants leads to a higher yield stability even under suboptimal growing conditions and has therefore a high importance for plant breeders. The objectives of our study were to (i) examine the expression levels of candidate genes in seedling leaves of B. napus and correlate these with seedling development as well as (ii) detect genome regions associated with gene expression levels and seedling development traits in B. napus by genome-wide association mapping.ResultsThe expression levels of the 15 candidate genes examined in the 509 B. napus inbreds showed an averaged standard deviation of 5.6 across all inbreds and ranged from 3.2 to 8.8. The gene expression differences between the 509 B. napus inbreds were more than adequate for the correlation with phenotypic variation of seedling development. The average of the absolute value correlations of the correlation coefficients of 0.11 were observed with a range from 0.00 to 0.39. The candidate genes GER1, AILP1, PECT, and FBP were strongly correlated with the seedling development traits. In a genome-wide association study, we detected a total of 63 associations between single nucleotide polymorphisms (SNPs) and the seedling development traits and 31 SNP-gene associations for the candidate genes with a P-value < 0.0001. For the projected leaf area traits we identified five different association hot spots on the chromosomes A2, A7, C3, C6, and C7.ConclusionA total of 99.4% of the adjacent SNPs on the A genome and 93.0% of the adjacent SNPs on the C genome had a distance smaller than the average range of linkage disequilibrium. Therefore, this genome-wide association study is expected to result on average in 14.7% of the possible power. Compared to previous studies in B. napus, the SNP marker density of our study is expected to provide a higher power to detect SNP-trait/-gene associations in the B. napus diversity set. The large number of associations detected for the examined 14 seedling development traits indicated that these are genetically complex inherited. The results of our analyses suggested that the studied genes ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit (RBC) on the chromosomes A4 and C4 and fructose-1,6-bisphosphatase precursor (FBP) on the chromosomes A9 and C8 are cis-regulated.

Network and biosignature analysis for the integration of transcriptomic and metabolomic data to characterize leaf senescence process in sunflower

BackgroundIn recent years, high throughput technologies have led to an increase of datasets from omics disciplines allowing the understanding of the complex regulatory networks associated with biological processes. Leaf senescence is a complex mechanism controlled by multiple genetic and environmental variables, which has a strong impact on crop yield. Transcription factors (TFs) are key proteins in the regulation of gene expression, regulating different signaling pathways; their function is crucial for triggering and/or regulating different aspects of the leaf senescence process. The study of TF interactions and their integration with metabolic profiles under different developmental conditions, especially for a non-model organism such as sunflower, will open new insights into the details of gene regulation of leaf senescence.ResultsWeighted Gene Correlation Network Analysis (WGCNA) and BioSignature Discoverer (BioSD, Gnosis Data Analysis, Heraklion, Greece) were used to integrate transcriptomic and metabolomic data. WGCNA allowed the detection of 10 metabolites and 13 TFs whereas BioSD allowed the detection of 1 metabolite and 6 TFs as potential biomarkers. The comparative analysis demonstrated that three transcription factors were detected through both methodologies, highlighting them as potentially robust biomarkers associated with leaf senescence in sunflower.ConclusionsThe complementary use of network and BioSignature Discoverer analysis of transcriptomic and metabolomic data provided a useful tool for identifying candidate genes and metabolites which may have a role during the triggering and development of the leaf senescence process. The WGCNA tool allowed us to design and test a hypothetical network in order to infer relationships across selected transcription factor and metabolite candidate biomarkers involved in leaf senescence, whereas BioSignature Discoverer selected transcripts and metabolites which discriminate between different ages of sunflower plants. The methodology presented here would help to elucidate and predict novel networks and potential biomarkers of leaf senescence in sunflower.

Heterarchy of transcription factors driving basal and luminal cell phenotypes in human urothelium

Cell differentiation is affected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq was used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq, as having altered accessibility during differentiation, were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including forkhead box A1 (FOXA1), P63, GRHL2, CTCF and GATA-binding protein 3 (GATA3). In addition, co-occurrence of GATA3 motifs was observed within subsets of differentiation-specific peaks containing P63 or FOXA1. Changes in abundance of GRHL2, GATA3 and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally regulated programme, urothelial differentiation operates as a heterarchy, wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes.

microRNAs associated with early neural crest development in Xenopus laevis

BackgroundThe neural crest (NC) is a class of transitory stem cell-like cells unique to vertebrate embryos. NC cells arise within the dorsal neural tube where they undergo an epithelial to mesenchymal transition in order to migrate and differentiate throughout the developing embryo. The derivative cell types give rise to multiple tissues, including the craniofacial skeleton, peripheral nervous system and skin pigment cells. Several well-studied gene regulatory networks underpin NC development, which when disrupted can lead to various neurocristopathies such as craniofrontonasal dysplasia, DiGeorge syndrome and some forms of cancer. Small RNAs, such as microRNAs (miRNAs) are non-coding RNA molecules important in post-transcriptional gene silencing and critical for cellular regulation of gene expression.ResultsTo uncover novel small RNAs in NC development we used high definition adapters and next generation sequencing of libraries derived from ectodermal explants of Xenopus laevis embryos induced to form neural and NC tissue. Ectodermal and blastula animal pole (blastula) stage tissues were also sequenced. We show that miR-427 is highly abundant in all four tissue types though in an isoform specific manner and we define a set of 11 miRNAs that are enriched in the NC. In addition, we show miR-301a and miR-338 are highly expressed in both the NC and blastula suggesting a role for these miRNAs in maintaining the stem cell-like phenotype of NC cells.ConclusionWe have characterised the miRNAs expressed in Xenopus embryonic explants treated to form ectoderm, neural or NC tissue. This has identified novel tissue specific miRNAs and highlighted differential expression of miR-427 isoforms.