Final Report Summary - SPIKE (Regulatory networks underpinning fertility of the lateral florets on the barley inflorescence (spike))
Cereal grains are staple food that provide more energy worldwide than any other kind of crop. In terms of quantity produced over the world, barley (Hordeum vulgare L.) has been ranked in the fourth position after corn, rice and wheat (1). The European Union is the first-word producer of barley (1). Therefore, the economic interest of the European Community in this crop is undeniable.
In this project, we have capitalized upon the recent and highly significant advances and resources in barley research (a new version of the barley genome is currently in press (2)) to improve the overall performance of the crop. Yield is one of the major concerns of cereal production and is the most important economic trait in barley breeding programs. Yield and yield stability are complex traits controlled by multiple genes and influenced by agronomic and environmental conditions (3). However one of the most important yield components, and one which is genetically tractable, is the architecture of the grain-bearing inflorescence (known as the ‘spike’). Barley spike is characteristically unbranched with the axis, or rachis, carrying a cluster of three single-flowered spikelets (one central and two laterals) alternately at each node along its length. The relative fertility of the lateral spikelets within each cluster leads to spikes that are two-rowed (only the central spikelet is fertile), six-rowed (the central and both lateral spikelets are fertile) of grains or show intermediate morphology (intermedium) between two and six row along the inflorescence (Figure 1). These striking architectural variations have the potential to contribute significantly to yield increases in barley, potentially producing up to three times as many grain per single inflorescence. Therefore, understanding the component genes and how they interact to condition the spike architecture could provide novel solutions to enhanced grain yield, a key global challenge.
Classical genetic studies identified five major SIX-ROWED SPIKE (VRS) (VRS1-5) genes to be responsible for the spike architecture and fertility in barley, four of which are now known to encode transcription factors (proteins that control the expression rate of a gene) (4, 5, 6, 7). In our lab, we have identified the remaining major VRS genes, VRS3. Being known the main genes that determine spikelet structure in barley, the aims of this project have been focused on VRS3. We have performed a detailed molecular characterisation of VRS3 gene in order to address a combination of descriptive, fundamental and applied objectives to explore the downstream genes involved in producing this striking morphological switch.
To accomplish these goals we have conducted specific expression analysis (RNA-seq) in developing spikes in the wild type (two-rowed) and vrs3 mutant (intermedium) at different developmental stages. Analysing the network of genes modulated by VRS3, we have found specific interaction with other genes that belong to particular molecular pathways that, in most cases, were reported to be involved in barley fertility for the first time.
We have also examined further the VRS3 expression tissue specificity. To fulfil this aim, we have performed in-situ hybridization experiments in the wild type (two-rowed) and the vrs3 mutant (intermedium) inflorescence tissues (in-situ) at different developmental stages. This technique has allowed us to localise specifically the VRS3 expression in the spike and the space-temporal changes in the VRS3 expression across the development. Likewise, we have characterised the spike morphology of these genotypes by using SEM (Scanning Electron Microscope) in order to identify the accurate point in the development in which the anatomical differences in the inflorescence between the wild type and the vrs3 mutant take place. Both experiments (in-situ and SEM) have been critical steps for understanding the interaction, regulation, and function of VRS3 gene.
Finally, during this project it has been initiated the production of transgenic barleys to test the function of some of the candidate genes downstream of VRS3 to warrant functional validation, and to investigate deeper the molecular pathway regulated by VRS3.
The results obtained in this project have provided a detailed mechanistic understanding of the genes involved in converting sterile into fertile lateral florets in barley. These outcomes have important agricultural impact as they have the realistic potential of manipulating the number of grains per spike and developing novel six-rowed barleys with evenly-filled grains and without a concomitant reduction in tillering (grain bearing stems).
As mentioned above, cereals are a dominant component of European agriculture. Barley is the second most important crop specie in Europe next to wheat, with acreage of over 28 million ha. Thus, boosting the number of grains per spike in this crop has an outstanding socio-economic impact in the European society and industry. Barley has great potential as a whole-grain health-promoting food of the future, given its high content of fibre. Consuming barley may help offset the adverse social and personal impact of several serious human health conditions i.e. coronary heart disease. By extending this scenario, the health sector could benefit from lower demand for their services and the tax payer could benefit from offsetting the costs of medical care. Lastly, as a consequence of an increasing in the barley consumption, the importance of this crop in the food sector may rise contributing to competitiveness of the economy.
Current six-row barley 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. Additionally, barley straw 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 is an important outcome. This basic scientific interest supply European and international researches with a list of candidate genes involved in the shift between two and six row barleys. In view of the above, this output will make a very significant contribution to EU industry and quality of life in the long term by benefiting breeders, commercial breeding organizations, farmers and ultimately consumers.
1. FAO (2016) FAOSTAT 20016. 2. Mascher M et al. (2017) (accepted in press). 3. Shi J et al (2009) Genetics 182: 851–861. 4. Komatsuda T. et al. (2007) Proc. Natl. Acad. Sci. USA104, 1424-1429. 5. Ramsay L et al. (2011) Nat. Genet.43 169–172. 6. Koppolu R (2013) et al Proc. Natl. Acad. Sci. USA 110, 13198–13203. 7. Yousseff H et al. (2017) Nat. Genet. 49,157-161.
In this project, we have capitalized upon the recent and highly significant advances and resources in barley research (a new version of the barley genome is currently in press (2)) to improve the overall performance of the crop. Yield is one of the major concerns of cereal production and is the most important economic trait in barley breeding programs. Yield and yield stability are complex traits controlled by multiple genes and influenced by agronomic and environmental conditions (3). However one of the most important yield components, and one which is genetically tractable, is the architecture of the grain-bearing inflorescence (known as the ‘spike’). Barley spike is characteristically unbranched with the axis, or rachis, carrying a cluster of three single-flowered spikelets (one central and two laterals) alternately at each node along its length. The relative fertility of the lateral spikelets within each cluster leads to spikes that are two-rowed (only the central spikelet is fertile), six-rowed (the central and both lateral spikelets are fertile) of grains or show intermediate morphology (intermedium) between two and six row along the inflorescence (Figure 1). These striking architectural variations have the potential to contribute significantly to yield increases in barley, potentially producing up to three times as many grain per single inflorescence. Therefore, understanding the component genes and how they interact to condition the spike architecture could provide novel solutions to enhanced grain yield, a key global challenge.
Classical genetic studies identified five major SIX-ROWED SPIKE (VRS) (VRS1-5) genes to be responsible for the spike architecture and fertility in barley, four of which are now known to encode transcription factors (proteins that control the expression rate of a gene) (4, 5, 6, 7). In our lab, we have identified the remaining major VRS genes, VRS3. Being known the main genes that determine spikelet structure in barley, the aims of this project have been focused on VRS3. We have performed a detailed molecular characterisation of VRS3 gene in order to address a combination of descriptive, fundamental and applied objectives to explore the downstream genes involved in producing this striking morphological switch.
To accomplish these goals we have conducted specific expression analysis (RNA-seq) in developing spikes in the wild type (two-rowed) and vrs3 mutant (intermedium) at different developmental stages. Analysing the network of genes modulated by VRS3, we have found specific interaction with other genes that belong to particular molecular pathways that, in most cases, were reported to be involved in barley fertility for the first time.
We have also examined further the VRS3 expression tissue specificity. To fulfil this aim, we have performed in-situ hybridization experiments in the wild type (two-rowed) and the vrs3 mutant (intermedium) inflorescence tissues (in-situ) at different developmental stages. This technique has allowed us to localise specifically the VRS3 expression in the spike and the space-temporal changes in the VRS3 expression across the development. Likewise, we have characterised the spike morphology of these genotypes by using SEM (Scanning Electron Microscope) in order to identify the accurate point in the development in which the anatomical differences in the inflorescence between the wild type and the vrs3 mutant take place. Both experiments (in-situ and SEM) have been critical steps for understanding the interaction, regulation, and function of VRS3 gene.
Finally, during this project it has been initiated the production of transgenic barleys to test the function of some of the candidate genes downstream of VRS3 to warrant functional validation, and to investigate deeper the molecular pathway regulated by VRS3.
The results obtained in this project have provided a detailed mechanistic understanding of the genes involved in converting sterile into fertile lateral florets in barley. These outcomes have important agricultural impact as they have the realistic potential of manipulating the number of grains per spike and developing novel six-rowed barleys with evenly-filled grains and without a concomitant reduction in tillering (grain bearing stems).
As mentioned above, cereals are a dominant component of European agriculture. Barley is the second most important crop specie in Europe next to wheat, with acreage of over 28 million ha. Thus, boosting the number of grains per spike in this crop has an outstanding socio-economic impact in the European society and industry. Barley has great potential as a whole-grain health-promoting food of the future, given its high content of fibre. Consuming barley may help offset the adverse social and personal impact of several serious human health conditions i.e. coronary heart disease. By extending this scenario, the health sector could benefit from lower demand for their services and the tax payer could benefit from offsetting the costs of medical care. Lastly, as a consequence of an increasing in the barley consumption, the importance of this crop in the food sector may rise contributing to competitiveness of the economy.
Current six-row barley 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. Additionally, barley straw 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 is an important outcome. This basic scientific interest supply European and international researches with a list of candidate genes involved in the shift between two and six row barleys. In view of the above, this output will make a very significant contribution to EU industry and quality of life in the long term by benefiting breeders, commercial breeding organizations, farmers and ultimately consumers.
1. FAO (2016) FAOSTAT 20016. 2. Mascher M et al. (2017) (accepted in press). 3. Shi J et al (2009) Genetics 182: 851–861. 4. Komatsuda T. et al. (2007) Proc. Natl. Acad. Sci. USA104, 1424-1429. 5. Ramsay L et al. (2011) Nat. Genet.43 169–172. 6. Koppolu R (2013) et al Proc. Natl. Acad. Sci. USA 110, 13198–13203. 7. Yousseff H et al. (2017) Nat. Genet. 49,157-161.
