Molecular and genetic networks determining row number in cultivated barley.

Lead Research Organisation: The James Hutton Institute
Department Name: Cell & Molecular Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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 alting 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.
 
Description In BB/K01613X/1, we have been exploring the molecular mechanisms and interactions underlying the roles of HvVRS3 and HvVRS5 in relation to other major row-type genes.

Genetic and Developmental Analyses. Detailed ontological characterisation of spikelet meristem formation and differentiation in vrs single and double mutants resolved lateral awn initiation as the first morphological event associated with lateral spikelet (LS) fertility and established the critical developmental window for LS commitment to fertility. We revealed novel or improved genetic routes to fully six-rowed phenotypes, namely vrs1vrs3, vrs4vrs5 and vrs1vrs3vrs5, a significant advance and agreeing well with our project aims. Unexpectedly, we discovered that combined row-type gene function is required to establish the species-specific unbranched spike form and spikelet meristem determinacy. We found that central spikelets (CS) in vrs3vrs4 occasionally developed into secondary inflorescences, revealing that both VRS3 and VRS4 repress branch meristem identity in spikelet meristems (Figure 1A). Further, the basal rachis nodes in both vsr3vrs4 and vrs3vrs5 double mutants were remarkably indeterminate, forming multispikelet branches from a single spikelet meristem and ectopic spikelets alongside established nodes (Fig. 1B). Taken together, impaired VRS3 function along with mutant vrs4 or vrs5 alleles can enhance branching potential in conjunction with lateral fertility, highlighting deeper parallels between basal branching and indeterminacy in the barley spike with branching of axillary buds in the maize shoot and inflorescence through ZmTb1-ZmGT1 (VRS5 and VRS1 orthologues) and ZmRA2 (VRS4 homologue). However, crucially, spike determinacy in barley is mediated through targets other than VRS1 (unlike the situation in maize with GT1) since vrs1, and vrs1 doubles with vrs3, vrs4 or vrs5 do not show similar indeterminacy phenotypes. Our data suggest that VRS3 has a broader impact potentially acting on several pathways, one upstream of VRS4 and (potentially) VRS5, driving the transition from indeterminate inflorescence to determinate spikelet and floral meristems, thus forming the species-specific unbranched barley spike independently of VRS1.
We also took advantage of our isogenetic panel to analyse the effect of variation in row-type genes on axillary bud outgrowth (tillering behaviour). During early growth, we observed that significantly more tiller outgrowth in vrs3, vrs4 and vrs5 mutants (high-tillering) compared to BW, showing that these row-type genes also repress shoot branching. Later, the high tillering group ceases to produce more branches while BW continued to generate tillers. We conclude that the activity of axillary buds in the high tillering group is triggered earlier than in wild type but does not maintain performance throughout development. vrs3vrs4 double mutants showed an additive increase in tillering suggesting that these genes may function independently to repress tiller outgrowth. Interestingly, the vrs1 mutant was actually low tillering in dramatic contrast to the high tillering gt1 phenotype in maize, and vrs5 high-tillering was epistatic to low-tillering vrs1, again supporting that this gene function and regulation is divergent between maize and barley.

Molecular Networks. We compared Vrs1, Vrs2, Vrs3, Vrs4 and Vrs5 transcript abundance by qRT-PCR during spikelet development. Our data confirm that VRS4 is a strong activator of Vrs1 and that VRS3 positively regulates both Vrs1 and Vrs5. Vrs1 expression was not abolished in the vrs5 knock-out mutant background, in striking contrast to the control of ZmGT1 expression by ZmTB1, entirely consistent with our genetic analyses (Figure 2; manuscripts in prep). We have complemented these studies with fully replicated deep RNA-seq on 4-6 mm (awn primordium stage) and 9-11 mm (awn initials extend and floral organs differentiate) from WT, vrs3 and vrs5 isogenic lines, which supports our qRT-PCR observations and identifies further potential downstream targets. In addition, in-situ hybridisations on developing inflorescences show Vrs3 expression throughout the developing spike, specifically in rachis nodal plexus and spikelet axils, while Vrs5 is restricted to sub-domains of the developing lateral spikelet meristems (similar to Vrs1 and Vrs4), suggesting that VRS3 is expressed where spike branches and supernumerary spikelets originate in double mutants. We have generated transgenic plants containing HA-, GFP- and RFP-tagged VRS3 and VRS5 under the control of their native promoters (T1 plants are currently growing for analysis) and have developed peptide antibodies against both VRS3 and VRS5. Unfortunately, Y-2-H experiments showed that VRS3 and VRS5 are robustly self-activating, precluding interaction analysis in the Y-2-H system. However we plan to cross the differentially tagged VRS3 and VRS5 transgenics to determine co-localisation and/or interaction by FRET as well as through Co-IP. Finally, we do not definitively know what the precise activity of VRS3 is in barley. Finally, we do not definitively know what the precise activity of VRS3 is in barley though the rice orthologue has been shown to be a Histone H3K9me2/me3 demethylase. We have generated and checked protein expression vectors and conducted initial studies in E.coli, and showed that the protein is expressed. We are currently attempting to prove that VRS3 is also a Histone H3K9me2/me3 demethylase and should hopefully confirm this within a matter of days/weeks.

Agronomic Potential. We constructed lines containing all heterozygous and homozygous combinations of (recessive) vrs1, vrs3 and vrs5 mutants. In replicated field trials we observed a significant increase in the size of the lateral grains in the presence of six-row (mutant) alleles at all three loci, supporting the hypothesis that 'super-six rows' comprised of multiple vrs mutant alleles may provide a higher yielding 6-row genetic model. These studies confirmed the genetic and environmental sensitivity of the penetrance of the six row phenotype, particularly when recessive vrs alleles were present in different configurations and dosage, and in different genetic backgrounds (suggesting the presence of additional genetic modifiers that influence the phenotype). This was also reflected in field studies where elaboration of the phenotype was more severe in a winter barley background, possibly emphasising the interaction between FT signalling, floral induction and reproductive architecture, and similar to the influence of flowering-time genes on the development of novel spikelet arrangements, specifically paired spikelets in wheat (Ppd-1 - Boden et al 2015, Nature Plants).
Relevant Papers currently in preparation (target submission before march 2017)

Bull H, Casao MC, Zwirek. M et al (2016) Identification and characterisation of the barley row-type gene, VRS3 (research paper published in Nature Communications.
Zwirek M et al (2016). Multiple row type loci lead to increased lateral fertility in cultivated barley (research paper). In preparation (Target Journal Genes &Development).

Since the grant was completed we have now identified another component (SHORT LATERAL SPIKELETS1) of the row type network and have conducted detailed multiple-tissue specific RNA-seq in wild type in mutant plants. The data are currently being analysed in [reparation for publication
.
Exploitation Route Six row barley has the potential toa increase the number of grain per inflorescence to threefold more that two row barley. This yield benefit seldom materialises to this extent because the extra grain is smaller and becasue fewer grain bearing stems develop in six rowed genotypes. We want to understand the interplay between these traits. If we can, we may be able to use the information to boost grain yield and at the same time understand the factors involved in switching sterile inflorescences into fertile inflorescences and this will have implications for other areas of plant biology and breeding.
Sectors Agriculture, Food and Drink

 
Description The award started in February 2014 and its premature to issue major findings. However we have established collaborations with Thorsten Schnurbusch at IPK in Germany and Takao Komatsuda at NIAS in Japan who have similar interests and track records. We are have used the momentum afforded by this award to attract a marie curie postdoctoral fellow to work on aspects of transcriptional control. We wrote a further application to BBSRC Response mode to take this work forward which was unfortunately unsuccessful. We have collaborated with the the Plant breeding company 'Limagrain' to breed vrs3 mutant alleles into spring and winter barley backgrounds and conducted preliminary evaluations in the field. We see an interesting link between phenotypic plasticity and seasonal growth habit that could be the subject of future investigation. We have also shown that including vrs3 in a six-row VRS1 and VRS5 background increases lateral grain size and shape and generates more uniform grain - a key processing characteristic that may have some future application. We published a nice paper on the VRS3 mutant in Nature Communications in 2017, and VRS1 in Plant Physiology in 2017. We have a follow up paper on SHORT LATERAL SPIKELETS1 - another gene in the row-type network - in preparation.
First Year Of Impact 2016
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Row-type 
Organisation IPK Gatersleben
Country Germany 
Sector Private 
PI Contribution Collaborative research on a joint interest project to do with gene discovery and transcriptomics in barley row type determination.
Collaborator Contribution Plant growth, tissue sampling, gene identification (in parallel with us) RNA isolation
Impact Paper in preparation
Start Year 2018
 
Description Awaydays Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Monika Zwirek: Oral presentation at the annual Barley Away days, Birnam. Title: "Molecular mechanisms Underlying Row-Type Determination in Barley"
Year(s) Of Engagement Activity 2016
 
Description BBC Scotland 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interview with BBC Scotland about Barley Research
Year(s) Of Engagement Activity 2015
 
Description Cereals in Practice 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Cereals in Practice is an annual event held jointly with SRUC and attracts mainly the farming community throughout Scotland and NE England. Annual attendance is around the 200-300 mark but varies according to the weather as its an outdoor event. Showcases current research outputs and new varieties.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016
 
Description Edinburgh 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact • Edinburgh University, Invited Departmental Seminar, June 2015
Year(s) Of Engagement Activity 2015
 
Description Fascination of Plants and Family Fun Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact EPSO's Fascination of Plants day in Dundee is combined with an event called Family fun day that we run annually at the Botanic Gardens. It has a wide range of events - including plant sales from 'friends of the gardens', displays, activities, games, educational events (e.g. the genetics garden) and other activities.
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description General Board Meeting of EPSO 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact PI attended the European Plant Biology Association Annual meeting in Prague in June 2016. Two day meeting where strategies for European Plant Science funding were discussed. Great networking opportunities across Europe.
Year(s) Of Engagement Activity 2016
URL http://www.epsoweb.org/
 
Description IBGS poster 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Monika Zwirek: Poster presentation at the IBGS conference, Minneapolis, USA. Title: "Mechanisms Underlying Row-Type Determination in cultivated Barley"
Year(s) Of Engagement Activity 2016
 
Description ITMI 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact • International Triticeae Mapping Initiative Conference Fargo, June 2012(session organiser and chair)
Year(s) Of Engagement Activity 2012
 
Description Invited Presentation at Plant Animal and Genome Conference 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Delivered a talk about our research on the vrs3 row-type gene, it's role in lateral spikelet fertility and it's interactions with other row-type genes.
Year(s) Of Engagement Activity 2017
URL https://pag.confex.com/pag/xxv/meetingapp.cgi
 
Description Landward 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview on BBC Landward program about new breeding technologies in relation to barley and potatoes
Year(s) Of Engagement Activity 2015
 
Description Mutant Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact • Barley Mutant Workshop, IPK Gatersleben, June 2014 (organiser and session chair)
Year(s) Of Engagement Activity 2014
 
Description Open Doors Day school visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact PI lead an ethics workshop on GM technology with classes from Braeview Academy, a local secondary school.
Year(s) Of Engagement Activity 2016
 
Description Plant Genetics and Breeding Technologies, Vienna 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact • Molecular Mapping and marker assisted selection Vienna, Feb 2012 (session organiser, speaker, chair)
• Plant Genetics and Breeding Technologies, Vienna, Feb 2013 (session organiser, speaker and chair)
Year(s) Of Engagement Activity 2012,2013
 
Description Plant Power Day at the Botanic Gardens 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Scientific displays and interactive activities which showcase the plant science ongoing at the University of Dundee. The PI organised this event in collaboration with the Botanic Gardens. Display to showcase research on cereal architecture.
Living genetics and cereal gardens. Botany trails.
Year(s) Of Engagement Activity 2016
URL https://www.dundee.ac.uk/news/2016/fun-in-dundee-botanic-garden-for-plant-power-day.php
 
Description Poster Presentation at the International Barley Genetics Symposim 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The PDRA employed on the grant presented a poster at the International Barley Symposium in Minnesota in June 2016. This five day long conference happens only every four years and is a critical event for the barley research community.
Year(s) Of Engagement Activity 2016
URL http://ibgs2016.org/
 
Description Public engagement 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Open day at the botanic gardenss with displays, activities, demonstrations etc.
Year(s) Of Engagement Activity 2014,2015,2016
 
Description Rank Prize Symposium II 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact • Rank Prize symposium, Grasmere, 18-21 May 2015 (organiser, speaker and session chair)
Year(s) Of Engagement Activity 2015
 
Description Research Seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact General subject area stimulated discussion and potentially led to further collaborations

I was invited to give another talk on a different topic at a subsequent meeting.
Year(s) Of Engagement Activity 2014
 
Description Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Monika Zwirek: Oral presentation for the Division of Plant Science Seminar, JHI. Title: "Molecular mechanisms Underlying Row-Type Determination in Barley"
Year(s) Of Engagement Activity 2016
 
Description UK-Brazil 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact - Mechanism influencing row number in cultivated barley, UK-Brazil workshop entitled "Environmental and metabolic control of plant growth and development" Campinas, Brazil, March 2015 (Monika Zwirek)
Year(s) Of Engagement Activity 2015
 
Description UoD talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Monika Zwirek University of Dundee talk: Oral presentation for the Division of Plant Science Seminar, JHI. Title: "Mechanisms Underlying Row-Type Determination in cultivated Barley"
Year(s) Of Engagement Activity 2017
 
Description outreach 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Monika Zwirek: Guide at 'Cereals in Practice'
Year(s) Of Engagement Activity 2016
 
Description workshop talk 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Monika Zwirek: Oral presentation at the "RNA-seq afternoon" meeting, JHI. Title: "The application of RNA-seq to the comprehensive analysis of barley inflorescence transcriptome".
Year(s) Of Engagement Activity 2016