John Fosu-Nyarko

The genes/enzymes involved in supplying carbon to the developing head in wheat are not well characterized. The activities of cell wall invertases (IVR1s) are known to be important in supplying sugars to pollen during the early stages of head development. Recently multiple IVR1 isoforms were identified on the wheat genome (Webster et al 2012; Funct Plant Biol 39: 569 - 579; GRDC-GRS10028) and were found to be located on a number of chromosome arms. These findings in addition to the existence of IVR1 inhibitors, provides evidence to suggest a complex regulatory network exists, which controls the flow of sugars to maturing pollen. A reduction in supply of sugars to pollen in response to water stress during the early stages of maturation leads to sterility. A large water stress experiment was carried out to analyse the transcriptome using RNASeq and RNA from developing wheat heads. RNASeq performed on tissues extracted from developing heads, extending from immature floret formation through to anthesis, enabled temporal profiling of transcript expression in response to water stress and non-limited water conditions. The IWGSC wheat genome survey sequence data was used as the reference to assign chromosome arm locations of those transcripts showing expression in the RNASeq data. Mapping individual transcripts to an IWGSC survey sequence contig facilitated downstream functional and biological characterisation to identify key genes involved in the supply of carbon to developing wheat heads.

Genetic analyses of controlled crosses in wheat have identified a region on chromosome 3B (short arm) that controls variation in water stress tolerance. The region, near the Sr2 resistance locus, is covered by a series of well studied BAC clones that are being sequenced as part of a large 3B sequencing program in France (led by Catherine Feuillet, INRA, and the International Wheat Genome Sequencing Consortium). A 1Mb region from this part of the wheat genome (ctg506) was assembled based on sequence data from overlapping BAC clones. The sequencing of the BAC clones combined standard Sanger sequencing (5-8 x coverage) and Solexa/Illumina short read sequencing. Annotation of the sequence identified the IVR1 gene considered to be important in conferring drought and frost tolerance to wheat when the stress occurs early in head development. Alignment to the rice and Brachypodium genome sequences indicated very little synteny and detailed analyses provided a sequence based model accounting for the evolutionary instability in this region of the genome.

Root lesion nematodes (Pratylenchus spp., RLNs) are major pests of most crops, and reduce yields of wheat in Western Australia by up to 15%, with Australia-wide losses of more than $36 million per annum. The aim of this project is to investigate the use of RNA interference (RNAi) as an approach to confer resistance to RLNs. RNAi is a well established technology that can be used to silence specific genes in animals and plants. Exposure to artificially introduced double-stranded RNA (dsRNA) leads to the silencing of endogenous genes with homologous sequence. RNAi can silence genes in Caenorhabditis elegans, and some success has been reported in root-knot nematodes. There is no evidence yet that RNAi works for RLNs. RLNs are migratory endoparasitic nematodes, and so mobility is an important aspect of parasitism. In this study, we are investigating genes involved in locomotion in RLNs via RNAi. We have shown that P.thornei and P.zeae are indeed amenable to RNAi. Exposure to dsRNA for locomotion specific genes by 14 hours soaking in medium containing M9 buffer with 50 mM octopamine, 3 mM spermidine and 0.05% gelatin led to locomotion impairment in both of these species. In addition, dsRNA originating from P.thornei also led to abnormalities in the closely related species P.zeae and vice versa, indicating that inter-species gene knockdown is possible. The outcome of this study is economically significant as no reported natural resistance genes have broad effectiveness against RLNs. Bioengineered crops expressing dsRNA that silence essential target genes to interrupt the parasitic process represents a potential approach to develop novel, broadly applicable and durable RLN-resistance in crop plants .