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The phytohormone auxin governs crucial developmental decisions throughout the plant life cycle. Auxin signaling is effectuated by auxin response factors (ARFs) whose activity is repressed by Aux/IAA proteins under low auxin levels, but relieved from repression when cellular auxin concentrations increase. ARF3/ETTIN (ETT) is a conserved noncanonical Arabidopsis thaliana ARF that adopts an alternative auxin-sensing mode of translating auxin levels into multiple transcriptional outcomes. However, a mechanistic model for how this auxin-dependent modulation of ETT activity regulates gene expression has not yet been elucidated. Here, we take a genome-wide approach to show how ETT controls developmental processes in the Arabidopsis shoot through its auxin-sensing property. Moreover, analysis of direct ETT targets suggests that ETT functions as a central node in coordinating auxin dynamics and plant development and reveals tight feedback regulation at both the transcriptional and protein-interaction levels. Finally, we present an example to demonstrate how auxin sensitivity of ETT-protein interactions can shape the composition of downstream transcriptomes to ensure specific developmental outcomes. These results show that direct effects of auxin on protein factors, such as ETT-TF complexes, comprise an important part of auxin biology and likely contribute to the vast number of biological processes affected by this simple molecule.

Sequencing a genome and identifying genetic markers lays the groundwork for genome-wide association studies, but can be difficult to achieve for polyploid species. Harper et al . present an approach for performing association studies using genetic maps and markers generated from transcriptome sequencing data alone and apply it to the polyploid crop 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.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

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. Sequencing of the hexaploid bread wheat genome shows that it is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. The bread — and barley — of life Two groups in this issue report the compilation and analysis of the genome sequences of major cereal crops — bread wheat and barley — providing important resources for future crop improvement. Bread wheat accounts for one-fifth of the calories consumed by humankind. It has a very large and complex hexaploid genome of 17 Gigabases. Michael Bevan and colleagues have analysed the genome using 454 pyrosequencing and compared it with diploid ancestral and progenitor genomes. The authors discovered significant loss of gene family members upon polyploidization and domestication, and expansion of gene classes that may be associated with crop productivity. Barley is one of the earliest domesticated plant crops. Although diploid, it has a very large genome of 5.1 Gigabases. Nils Stein and colleagues describe a physical map anchored to a high-resolution genetic map, on top of which they have overlaid a deep whole-genome shotgun assembly, cDNA and RNA-seq data to provide the first in-depth genome-wide survey of the barley genome.

Background Next generation sequencing (NGS) technologies are providing new ways to accelerate fine-mapping and gene isolation in many species. To date, the majority of these efforts have focused on diploid organisms with readily available whole genome sequence information. In this study, as a proof of concept, we tested the use of NGS for SNP discovery in tetraploid wheat lines differing for the previously cloned grain protein content (GPC) gene GPC-B1 . Bulked segregant analysis (BSA) was used to define a subset of putative SNPs within the candidate gene region, which were then used to fine-map GPC-B1 . Results We used Illumina paired end technology to sequence mRNA (RNAseq) from near isogenic lines differing across a ~30-cM interval including the GPC-B1 locus. After discriminating for SNPs between the two homoeologous wheat genomes and additional quality filtering, we identified inter-varietal SNPs in wheat unigenes between the parental lines. The relative frequency of these SNPs was examined by RNAseq in two bulked samples made up of homozygous recombinant lines differing for their GPC phenotype. SNPs that were enriched at least 3-fold in the corresponding pool (6.5% of all SNPs) were further evaluated. Marker assays were designed for a subset of the enriched SNPs and mapped using DNA from individuals of each bulk. Thirty nine new SNP markers, corresponding to 67% of the validated SNPs, mapped across a 12.2-cM interval including GPC-B1 . This translated to 1 SNP marker per 0.31 cM defining the GPC-B1 gene to within 13-18 genes in syntenic cereal genomes and to a 0.4 cM interval in wheat. Conclusions This study exemplifies the use of RNAseq for SNP discovery in polyploid species and supports the use of BSA as an effective way to target SNPs to specific genetic intervals to fine-map genes in unsequenced genomes.

In Triticeae endosperm (e.g. wheat and barley), starch granules have a bimodal size distribution (with A- and B-type granules) whereas in other grasses the endosperm contains starch granules with a unimodal size distribution. Here, we identify the gene, BGC1 (B-GRANULE CONTENT 1), responsible for B-type starch granule content in Aegilops and wheat. Orthologues of this gene are known to influence starch synthesis in diploids such as rice, Arabidopsis, and barley. However, using polyploid Triticeae species, we uncovered a more complex biological role for BGC1 in starch granule initiation: BGC1 represses the initiation of A-granules in early grain development but promotes the initiation of B-granules in mid grain development. We provide evidence that the influence of BGC1 on starch synthesis is dose dependent and show that three very different starch phenotypes are conditioned by the gene dose of BGC1 in polyploid wheat: normal bimodal starch granule morphology; A-granules with few or no B-granules; or polymorphous starch with few normal A- or B-granules. We conclude from this work that BGC1 participates in controlling B-type starch granule initiation in Triticeae endosperm and that its precise effect on granule size and number varies with gene dose and stage of development.

The symbiotic infection of root cells by nitrogen-fixing rhizobia during nodulation requires the transcription factor Nodule Inception (NIN). Our root hair transcriptomic study extends NIN's regulon to include Rhizobium Polar Growth and genes involved in cell wall modification, gibberellin biosynthesis, and a comprehensive group of nutrient (N, P, and S) uptake and assimilation genes, suggesting that NIN's recruitment to nodulation was based on its role as a growth module, a role shared with other NIN-Like Proteins. The expression of jasmonic acid genes in nin suggests the involvement of NIN in the resolution of growth versus defense outcomes. We find that the regulation of the growth module component Nodulation Pectate Lyase by NIN, and its function in rhizobial infection, are conserved in hologalegina legumes, highlighting its recruitment as a major event in the evolution of nodulation. We find that Nodulation Pectate Lyase is secreted to the infection chamber and the lumen of the infection thread. Gene network analysis using the transcription factor mutants for ERF Required for Nodulation1 and Nuclear Factor-Y Subunit A1 confirms hierarchical control of NIN over Nuclear Factor-Y Subunit A1 and shows that ERF Required for Nodulation1 acts independently to control infection. We conclude that while NIN shares functions with other NIN-Like Proteins, the conscription of key infection genes to NIN's control has made it a central regulatory hub for rhizobial infection.

Summary Winter, spring and biennial varieties of Brassica napus that vary in vernalization requirement are grown for vegetable and oil production. Here, we show that the obligate or facultative nature of the vernalization requirement in European winter oilseed rape is determined by allelic variation at a 10 Mbp region on chromosome A02. This region includes orthologues of the key floral regulators FLOWERING LOCUS C (BnaFLC.A02) and FLOWERING LOCUS T (BnaFT.A02). Polymorphism at BnaFLC.A02 and BnaFT.A02, mostly in cis‐regulatory regions, results in distinct gene expression dynamics in response to vernalization treatment. Our data suggest allelic variation at BnaFT.A02 is associated with flowering time in the absence of vernalization, while variation at BnaFLC.A02 is associated with flowering time under vernalizing conditions. We hypothesize selection for BnaFLC.A02 and BnaFT.A02 gene expression variation has facilitated the generation of European winter oilseed rape varieties that are adapted to different winter climates. This knowledge will allow for the selection of alleles of flowering time regulators that alter the vernalization requirement of oilseed rape, informing the generation of new varieties with adapted flowering times and improved yields.

Background Polyploidy 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. Results Illumina 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. Conclusion mRNA-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.

Cost-effective analysis of allelic variation can be problematic for polyploid crops. By sequencing leaf transcriptomes from a mapping population of oilseed rape and its progenitors, Bancroft et al . provide a general strategy to construct linkage maps for comparative genome analysis. 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 1 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.