Molecular and genetic networks determining row number in cultivated barley.

Wild barleys, the original domesticated forms and the majority of current elite UK cultivars produce two rows of grain bearing spikelets either side of the inflorescence, or spike. However, soon after the domestication, barleys with six rows of grain emerged that ultimately dominated early barley cultivation. Mutations in a single gene called SIX-ROWED SPIKE 1 (VRS1) have been identified as responsible for this important developmental switch. In two-rowed types, VRS1 mRNA is expressed in progenitor cells of the lateral spikelets which remain sterile presumably because VRS1 protein represses expression of genes that are required for development of 'lateral fertility'. Inactivating VRS1 via mutation would de-repress expression of lateral fertility genes, resulting in a six-rowed spike. While VRS1 is core to this process, we also know from historical studies with barley mutants that at least 11 different SIX-ROWED SPIKE genes influence the degree of fertility of the lateral spikelets. For example, we recently identified SIX-ROWED SPIKE 5 (VRS5), and showed that different versions of this gene (that we call 'alleles') are always paired with different versions of VRS1 in commercial two- and six- rowed barleys. This pairing is important because in lines that have the six-row version of VRS1 (denoted as vrs1), a two-row VRS5 allele (Vrs5) causes the development of small grain from the lateral spikelets. In contrast, the six-row version of VRS5 (vrs5) causes the lateral spikelets to develop fully, with important consequences on yield. This observation demonstrates that getting the correct combination of alleles at VRS genes is extremely important. While mutant studies have identified many VRS genes, we recently showed that natural variation in only four genes is associated with determining whether current elite UK barley cultivars are genetically optimal two- or six-row-types. As expected, one of these was VRS1 and another VRS5. We recently identified the third gene as VRS3 and are trying to identify the fourth, which does not coincide with the location of any of the eleven VRS mutants. In parallel, VRS4 has been identified by German collaborators.
While we now know these genes are intrinsically linked by their involvement in the developmental pathway that restores fertility to a nascent floral organ (i.e. the lateral spikelets) at the moment we have no idea if or how these components interact, what other genes/proteins are involved or how six-rowed types evolved over the 10,000 years since the domestication of the species. These are the issues we plan to address in this project. We believe that a better understanding of this fundamental developmental process will provide insights into how we can exploit variation in genes controlling plant morphology and architecture to ultimately improve plant yield.

The number of fertile rows of grain (i.e. two vs. six) on a barley inflorescence is determined by complex interactions between different SIX-ROWED SPIKE (VRS) genes. One of these genes, VRS1, is generally considered both necessary and sufficient for this developmental switch, with recessive alleles (denoted as vrs1) found in all six-row varieties. However up to 10 additional independent genetic loci affecting row-type have been characterised genetically. They all represent induced recessive mutations, generated in two-rowed accessions with an ancestral (or 'wild type (wt)') VRS1 gene. Spikes of these mutant lines have either complete or partial fertility restoration compared to sterile lateral florets of the wt spikes. We genetically mapped these VRS mutant alleles by SNP genotyping BC6 NILs in a two-row recurrent parent background. We also conducted a GWAS experiment for 'row-type' using a large collection of elite barley two- and six-row cultivars. The latter analysis identified four highly significant associations with row-type segregating in this germplasm set. As expected, one of these corresponded to VRS1. Two others corresponded to the proposed genetic locations of VRS3 and VRS5 genes, and one remains unassigned. We subsequently identified and validated VRS3 and VRS5 genes, while colleagues in Germany have identified VRS4. Of these four characterised genes, three are transcription factors and one is a chromatin remodelling factor. Here we propose to investigate how these components interact genetically, physically and phenotypically, and attempt to identify other genes in the network (i.e. interactors) that ultimately control lateral floret fertility. Finally, with breeders' support, we will explore the potential of novel six-row varieties in European agriculture.

The underlying thrust of the proposed research is to understand the development and architecture of the grain bearing inflorescence of the world's 4th largest cereal crop. Morphological and developmental changes have been central to yield increases in many crop species thus this research directly relates to the BBSRC priority of Crop Science (Food Security). As well as gaining a detailed mechanistic understanding of the genes involved in converting lateral florets from sterile into fertile, this project has the realistic potential of developing genetically novel six-rowed barleys with evenly-filled grains without a concomitant reduction in tillering. There may also be benefits of this research for two-row barleys of the 'deficiens' class that are emerging strongly in current markets where the lateral florets are virtually absent. There, an increase in tillering could potentially be effected without impacting inflorescence structure. The complexities of yield determination in the field mean that yield potential offered by such lines may not be realised in some environments. However, the range of lines produced (both by ourselves and by our commercial partner) will allow us to explore how it may be possible to optimize yield potential by manipulating these specific crop architectural traits. Existing NILs and newly developed lines will facilitate the proposed detailed genetic interaction and developmental studies required to promote the rapid exploitation of alternative six-row genes.

The Triticeae cereals are a dominant component of European agriculture. As a collaborator on this project, the immediate commercial beneficiary of this project will likely be Limagrain, a leading commercial sector organisation that breeds new barley and wheat varieties along with the farmers that grow new varieties in their fields (UK farm gate value >£500M). Current six-row lines do not have suitable grain quality for the malting industry - so, if it is possible to maintain malting quality in a novel six-row spring or winter phenotype, there may also be benefits to the malting and distilling sector. The European brewing (and Scotch whisky) industry is the largest in the world. Four of the seven largest brewers in the world are European, with their product directly / indirectly generating total government revenues estimated at ~$57.5 billion annually. Novel six-row barleys generated in an existing high quality two-row background may overcome some of the hurdles associated with breeding efforts that have repeatedly failed to generate a good quality (for malting) six-row type (with rare exception).

Barley is a key feedstock for the livestock industry and remains a traditional food in marginal environments that are unable to support the growth of wheat or maize. It has great potential as a whole-grain health-promoting food of the future, given its high content of sterols, stenols, arabinoxylans, and beta glucans, with the US FDA recently allowing barley products to claim a role in the 'reduction in risk' of coronary heart disease. Furthermore, barley straw (which may be enhanced through increased tillering) has a potentially expanding role in animal nutrition and in the second-generation bioenergy sector. A simple increase in yield in either two- or six-rowed types would be an important outcome.

The conduit through which almost all genetic advances in crop production must pass to release their benefits to the broader community is the plant breeding / biotech sector, and as such translational activities from basic science to application are crucial. A novel six-row barley would generate an unique selling proposition in the marketplace and a pathway to deployment through the involvement of Limagrain is an intrinsic component of this project proposal.