BackgroundThe gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis.ResultsThe wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1β-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp.ConclusionsThe comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD enzymes of the GA pathway in wheat and barley will provide the basis for a better understanding of GA-regulated development in these species. This analysis revealed the existence of a novel, endosperm-specific GA 1-oxidase in wheat and a related GA 3,18-dihydroxylase enzyme in barley that may play important roles during grain expansion and development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0520-7) contains supplementary material, which is available to authorized users.

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.

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Low oxidative stability, off-flavor and rancidity are the major drawbacks of soybean oil. Modification of the fatty acid composition of soybean [Glycine max (L.) Merrill] oil can improve its quality and value for processors and acceptability among consumers. Mutation breeding of soybean was therefore initiated with the objective of identifying stable soybean mutants with altered fatty acid composition for improved oxidative stability and nutritional quality. Seeds of soybean cultivar 'MACS 450' were treated with c-radiation and/or ethyl methane sulfonate (EMS). The harvest of M 1 plants was evaluated for fatty acid composition by gas chromatography. Highly significant variation in all the fatty acids except palmitic acid was observed. Treatment of EMS in higher concentrations as well as combined treatment of both the mutagens, i.e., c-radiation and EMS were effective in increasing the variability for the fatty acid content in soybean oil. The variability was skewed towards high levels of oleic (35-42%) and low levels of linolenic acid (3.77-5.00%). M 3 and M 4 generations of desirable variants were analyzed for the stability of the mutated trait . Only high oleic variants were stable in M 3 and M 4 generations. Based on fatty acid values, oxidative stability index (OSI), nutritional quality index (NQI) and ratio of essential fatty acids (x 6 / x 3 ) were calculated for the control and M 2 , M 3 and M 4 generations. The x 6 /x 3 ratio in all the high oleic mutants was within the World Health Organization (WHO) recommended value (5-10%). A significant positive correlation between OSI and oleic acid content (P \ 0.001) indicated improved oxidative stability of the oil while retaining nutritional quality. These high oleic lines could be utilized further in breeding programs for improvement of soybean oil quality.

Wheat contains abundant xylan in cell walls of all tissues, but in endosperm, there is an unusual form of xylan substituted only by arabinose (arabinoxylan; AX) that has long chains and low levels of feruloylation, a fraction of which is extractable in water (WE-AX). WE-AX acts as soluble dietary fibre but also gives rise to viscous extracts from grain, a detrimental trait for some nonfood uses of wheat. Here, we show that a glycosyl transferase family 43 wheat gene abundantly expressed in endosperm complements the Arabidopsis irx9 mutant and so name the three homoeologous genes TaIRX9b. We generated wheat lines with a constitutive knockout of TaIRX9b by stacking loss-of-function alleles for these homeologues from a mutagenized hexaploid wheat population resulting in decreases in grain extract viscosity of 50%-80%. The amount and chain length of WE-AX molecules from grain of these triple-stack lines was decreased accounting for the changes in extract viscosity. Imaging of immature wheat grain sections of triple-stacks showed abolition of immunolabelling in endosperm with LM11 antibody that recognizes epitopes in AX, but also showed apparently normal cell size and shape in all cell types, including endosperm. We identified differentially expressed genes from endosperm of triple-stacks suggesting that compensatory changes occur to maintain this endosperm cell wall integrity. Consistent with this, we observed increased ferulate dimerization and increased crosslinking of WE-AX molecules in triple-stacks. These novel wheat lines lacking functional TaIRX9b therefore provide insight into control of wheat endosperm cell walls.

The composition of proanthocyanidins in the testa (seed coat) of bread wheat was analyzed by thiolysis of PA oligomers from developing grain and found to consist of (+)‐catechin monomers, with a small amount of (+)‐gallocatechin. The average chain length of soluble PA stayed relatively constant between 10 and 20 days post‐anthesis, whereas that of unextractable PA increased over the same period, suggesting that increases in chain length might account for the insolubility of PAs from mature wheat grain. We carried out RNA‐Seq followed by differential expression analysis from dissected tissues of developing grain from red‐ and white‐grained near‐isogenic lines differing in the presence of an active R gene that encodes a MYB transcription factor involved in control of PA biosynthesis. In addition to genes already identified encoding chalcone synthase, chalcone isomerase, flavanone 3‐hydroxylase, and dihydroxyflavonoid 4‐reductase, we showed that wheat genes encoding phenylalanine ammonia lyase, flavonoid 3′,5′‐hydroxylase, leucoanthocyanidin reductase, and a glutathione S ‐transferase (the orthologue of maize Bronze‐2 ) were more highly expressed in the red NIL. We also identified candidate orthologues of other catalytic and regulatory components of flavonoid biosynthesis in wheat.