Rudolf Appels

Philippa Borrill obtained her PhD from the John Innes Centre (2014). She was subsequently awarded a BBSRC Anniversary Future Leader Fellowship in 2015 which she holds in the Crop Genetics Department at the John Innes Centre. Dr Borrill’s research focuses on understanding the gene networks which control agronomic traits, with a particular interest in senescence which is an important biological determinant of yield and grain nutrient content in cereal crops. Dr Borrill is also involved in developing novel resources to support wheat research. As part of the International Wheat Genome Sequencing Consortium she has contributed to the development of an improved wheat reference genome sequence. This work has facilitated the in-depth understanding of how gene expression varies in polyploids with multiple sub-genomes. Previously Dr Borrill contributed to the creation of sequenced mutant populations in wheat (www.wheat-tilling.com) and led the development of the www.wheat-expression.com atlas and the open access www.wheat-training.com website.

This presentation provides a report on two major investments in wheat molecular biology, namely the sequencing of chromosome 7A and the Australia - China Centre for Wheat Improvement (ACCWI). The sequencing of chromosome 7A project (GRDC/BioPlatforms Australia funded) is supported by the International Wheat genome Sequencing Consortium (IWGSC) and has established the physical assembly of BAC clones prepared from flow sorted ditelocentric chromosomes. The Hiseq sequencing of BAC pools (AGRF) is approximately two thirds completed. The BAC pools being sequenced comprise contigs of 1 - 2 Mb genomic DNA which were assembled using the SNAPshot DNA fingerprinting of individual BACs with 5 restriction endonucleases. Anchoring of the sequence assemblies is being achieved using the 9K and 90K SNP chip based molecular genetic maps as well as the standard deletion maps for chromosome 7A and the published 7A genome sequence for Triticum urartu . The new ACCWI will act as the conduit through which in - depth genome sequence information for chromosome 7A as well as the other chromosomes (through the IWGSC) can be integrated into broader molecular breeding initiatives aimed at increasing the accuracy and efficiency with which wheat phenotypes can be defined. The ACCWI core phenotype targets will focus on grain yield and quality with the characterization of individual quality parameters being undertaken through the use of mass spectroscopy to generate grain protein profiles/signatures, the technology of which is dependent on genome sequences to define genes. The ACCWI - strategy for linking grain protein profiles/signatures to agronomic traits of interest to marketers of grains and breeders will be discussed.

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.

Mixing of lupin flour to wheat in bread making provides a number of health benefits and has major effects on bread properties including protein extractability. The study investigated wheat and lupin protein interaction as influenced by baking process and modification of protein extractability, which is important for the final quality of bread. MALDI-TOF mass spectrometry and 2 dimensional gel electrophoresis followed by MS/MS peptide sequencing were used to study bread protein matrix and wheat and lupin flour proteins. The results demonstrated that many of the wheat and lupin proteins including high molecular weight glutenins remained unchanged in baking as per their elctrophoretic behavior. However, some of the proteins of wheat and lupin became unextractable from the bread, indicating lupin-wheat protein interaction during baking. For the lupin proteins, most of the α-conglutins could be readily extracted from the lupin-wheat bread even at low salt and non-reducing/non-denaturing extraction conditions. In contrast, most of the β-conglutins lost extractability suggesting that they were cross-linked to the wheat gluten network and trapped into the bread matrix. The higher thermal stability of α-conglutins compared to β-conglutins is speculated to account for this difference.

In the current study, we established a fast method to accurately measure the number of cysteine residues in high molecular weight glutenin subunits. An alkylation regent, 4-vinylpyridine (4-vp), was used to treat the proteins during extraction. For every cysteine residue in a protein, such treatment increases its molecular mass value by 105.14 Da, which can be accurately determined by MALDI-TOF equipment. Based on the changes of the molecular mass value after 4-vp treatment, the number of cysteine residue can be reliably determined. We found that this method is also useful in studying non-glutenin proteins such as lupin seed storage proteins. This method is particularly valuable when the number of cysteine residue is of importance.

Nutritional qualities of lupin suggest bread rich in lupin has the potential to provide a number of health benefits. Mixing of lupin flour to wheat has major effects on bread properties including the extractability of proteins that is crucial to define the ultimate attributes of food. Present study investigated the lupin and wheat proteome dynamics as affected by the baking of lupin–wheat bread by using 2-D electrophoresis and direct mass spectrometry. Many of the proteins from both lupin and wheat remained unchanged in baked lupin–wheat bread, while the others were incorporated into the bread matrix and could not be extracted. Most of the alpha-conglutins could be readily extracted from the lupin–wheat bread even at milder extraction. In contrast, most of the beta- conglutins lost extractability. The structural attribute that correlates with this difference in behaviour is the greater in thermal stability of alpha-conglutins relative to Beta- conglutins. Most of the beta and gamma conglutins showed relatively higher peptide sequence coverage of corresponding matched proteins compare to alpha conglutin due to lack of information regarding this group of protein in the database.

Two separate 1Mb regions of the wheat genome comprising extensive stretches of repetitive sequences were assembled based on sequence data from overlapping BAC clones located in a section of the chromosome 3B of particular interest with regards to disease resistance. The sequencing of the 24 BAC clones combined standard Sanger sequencing (5-8 x coverage) and Solexa/Illumina short read sequencing (>300 x coverage). In addition, available BAC end and 454-based sequences were utilized to provide a good quality reference sequence that was annotated with a particular focus on the repetitive elements. Alignment to the rice and Brachypodium genome sequences identified a repetitive locus that is conserved between these genomes and the wheat genome. The conserved repetitive protein kinase like genes at this conserved locus showed a good alignment to the bacterial effector protein AvrPto (Weiman et al. 2007).

Background: Molecular markers and genetic linkage maps are pre-requisites for molecular breeding, but no genetic map is currently available for subterranean clover (Trifolium subterraneum L.), a diploid and inbreeding pasture legume. A total of 2,646 microsatellite or simple sequence repeat (SSR) markers either available in the public domain or unpublished obtained from Kazusa DNA Research Institute, were screened on four genotypes (Denmark, DGI007, Woogenellup and Daliak) that are parents of two F2 mapping populations. Results: A total of 343 SSR loci could be mapped into 8 linkage groups with 6–31 loci per linkage group and 27 SSRs shared between the two populations. Phenotyping data obtained for flowering time, isoflavonoid content (formononetin, genistein, biochanin A), hardseedness, leaf marks, calyx pigmentation and hairiness of stem were analyzed together with genotyping data. For each trait, 1–3 QTLs were identified; the phenotypic variation explained by these QTLs was in the range of 5.5–59.8%. In addition, alignment of linkage groups was possible with the genetic maps of red clover (Trifolium pratense L.), white clover (Trifolium repens L.)and Medicago truncatula Gaertn. using shared loci. Segmental duplications were identified within subterranean clover genome and between the legume species. Candidate genes for isoflavone content were identified. Conclusions: The present study reports on comparative genomics among three clover species as well as Medicago truncatula following the construction of the first genetic map for subterranean clover. It demonstrates the utility of a comparative genomics approach for molecular mapping of QTL controlling seed hardness, flowering time and specifically isoflavone content traits, as well as establishing relationships between subterranean clover and genomes of red clover, white clover and model legume M. truncatula. The comparative mapping carried out demonstrated the value of the map to identify candidate genes controlling important traits such as isoflavone content.

Terminal drought is an increasing risk for wheat production in many parts of the world, especially those with mediterranean type climate. The improvement of drought tolerance has been identified as a research priority. Under terminal drought, the impact of stem carbohydrate remobilization makes significant contributions to grain filling. Our study showed that the stem water soluble carbohydrate (WSC, main components fructan) level is not, on its own, a reliable criterion for the drought tolerance, the performance of the key enzymes involved in the WSC remobilization, which varied among different varieties, were critical in the terminal drought response. The genes involved in the mobilization of stem WSC, 1-FEHs and 6-FEHs (fructan exohydrolases), have been studied in detail to define their role in contributing to water deficit tolerance. The study includes the isolation of mutations where genes of interest have been deleted.