Identifying the genetic basis of large-embryo mutants of barley affected at the Lys3 locus.

Lead Research Organisation: John Innes Centre
Department Name: Crop Genetics

Abstract

Grain quality in cereals is determined by the composition and relative sizes of the three main components of the grain: the endosperm, embryo (or germ) and the husk. The endosperm is starch-rich and it provides the bulk of dietary calories. In contrast, the embryo accounts for a small proportion of grain weight but it is nutrient-rich. The embryo contains several essential nutrients that are lacking or less abundant in the other grain components. The relative sizes of the embryo and endosperm appear to be tightly regulated in cereals but there are a few examples of grains with larger-than-normal embryos and consequently, improved grain quality. A good example is the giant embryo mutant of maize. This was originally selected for high oil content and consequently, improved nutritional value for animal feed. It was shown subsequently that the improved nutritional properties were due to the larger-than-normal embryos.
In barley, we have shown that a locus, Lys3 that controls grain lysine content also controls embryo size. Barley mutants affected at the Lys3 locus have embryos that are approximately twice the mass of normal embryos. Four independent mutant alleles of Lys3 have been identified in different labs and in different genetic backgrounds. As with the giant embryo mutants of maize, lys3 mutants of barley have also shown improved nutritional value as animal feed. Unfortunately, their uptake has been limited by reduced grain yield caused by reduced endosperm size and therefore low grain weight.
In addition to the affects of lys3 mutations on grain quality and organ size, we recently discovered an unexpected side-effect on the efficiency of barley transformation. At present, very few barley cultivars other than Golden Promise are susceptible to transformation mainly due to the inability of the excised immature embryos to regenerate shoots in culture. One of the lys3 mutants consistently gives transformation efficiencies equivalent to those of Golden Promise.
Our preliminary work suggests that although there is an orthologue of the giant embryo gene of maize in barley, it lies on a different chromosome from the Lys3 locus. Furthermore, no genes similar to the giant embryo gene have been found in the region of the barley genome containing Lys3 suggesting that the mechanism determining embryo size in the lys3 mutants may be novel. To understand the biological basis of the control of embryo size in barley and in the long term, to manipulate this most effectively and efficiently in cereal species generally, we need to identify the Lys3 gene. To do this, we propose to fine map the region of the barley genome containing Lys3. In addition we aim to transfer a lys3 mutant allele to elite barley cultivars thus generating tools for future barley improvement by genetic engineering.
We expect that the benefits of this study will be 1) to further our understanding of the determination of embryo size in barley and other species, 2) to suggest strategies for further nutritional enhancement of barley, retaining the positive aspects of the lys3 phenotype but without the detrimental effects on endosperm size and therefore yield, and 3) to understand of the role of lys3 in influencing shoot regeneration from embryos in culture and to extend the range of genotypes of barley and ultimately, of other species that are amenable to transformation.

Technical Summary

In this project, we propose to study a type of barley mutant, Lys3 that has larger-than-normal embryos and increased transformability. Our ultimate goal is to identify the Lys3 gene by positional cloning in order to study it at a molecular level. This knowledge may enable separation of the favourable traits (nutritional enhancements due to the large embryos) from the unfavourable traits (yield depression due to reduced endosperm size) of the lys3 phenotype. It will also enhance our understanding of the control of embryo size in cereal grains and has the added advantage of potentially decreasing the cultivar-specificity of transformation in barley and other cereals.
We have already made good progress towards Lys3 gene identification by mapping the location to a <5 cM region of chromosome 5H. The syntenous region in Brachypodium contains ~100 genes. We aim to refine our genetic map making use of next-generation sequencing data from the parents of our mapping populations to generate KASPar markers. We expect to be able to locate Lys3 to within 2-3 genes by mapping. Once candidate genes are identified, we will verify lys3 by comparison of allelic diversity in the four lys3 mutant lines compared to wild-types and by genetic manipulation (RNAi and/or complementation).
Our preliminary work has provided two lines of evidence that Lys3 impacts on the ability of embryos in culture to regenerate shoots. Firstly, all four independently-generated lys3 mutants show increased shoot regeneration. Secondly, after backcrossing to the untransformable cultivar Optic, four BC2 F2 lines that inherited the large-embryo phenotype also inherited the ability to regenerate. We intend to further introgress the lys3 mutation into Optic using marker-assisted selection to maximize the genome contribution of the recurrent parent and also to produce regenerable lines of the reference barley cultivar, Morex by the same means.

Planned Impact

Government policies recognise the need to improve grain quality whilst maintaining or enhancing yield. Thus, genetic improvements affecting critical nutritional attributes, such as oil, protein and micronutrient content, are encouraged. The existing giant-embryo mutants of maize (Illinois high-oil lines) and rice have demonstrated the potential nutritional benefits of altered embryo-to-endosperm size for food and feed. Some progress has also been made in improving barley for animal feed by incorporating the lys3 large-embryo mutation. However, these efforts have been hampered by lack of knowledge of the nature of the lys3 mutation and its specific effects on embryo vs. endosperm development. In a wider context, further understanding of the control of relative organ size in plants has broad implications for efforts to improve crop productivity and quality. This project is designed to provide a deeper understanding of the molecular basis of the lys3 mutation and in addition, its affect on the regeneration of plants from cultured embryos. Understanding and overcoming the cultivar-specificity of plant regeneration will enhance our ability to develop improved crops by the direct genetic engineering of elite cultivars. The proposed work is directly relevant to the BBSRC key strategic aims of 'advancing fundamental understanding of complex biological processes' and of 'helping to provide the skilled researchers needed for industrial R&D and academic research'.

Who will benefit from this research?

Plant breeders and scientists, farmers, consumers

How will they benefit from this research?

UK breeders will benefit from the increased understanding of a key parameter affecting grain nutritional value. This knowledge could lead, in the future, to separation of the negative aspects of the lys3 mutation (on endosperm size and hence yield) from the positive effects on grain composition. This would then provide genetic variation that is not presently available in barley and will provide UK growers with new market opportunities and farmers with improved animal feed stuff. In the longer term, therefore this work could enhance quality of life and health by leading to nutritionally improved barley for food and feed.

Scientists in both the academic and commercial private sectors will benefit from the barley DNA sequence information, which is an open-ended resource. The use of genetic/genomic information from other cereal systems to help define candidate genes will provide a paradigm for other similar work. Scientists will also benefit from access to elite barley varieties (and potentially in the longer term, other elite cereals) which are readily transformable. Whilst there is no direct commercial market for transgenic barley in the UK at present, this will indirectly benefit UK agriculture by providing more relevant germplasm with which to test gene modifications of potential benefit.

Our data and germplasm, after appropriate protection of IP, will be released into the public domain and will be available to industrialists and academics worldwide.

Publications

10 25 50
 
Description Many shrunken endosperm mutants of barley (Hordeum vulgare L.) have been described and several of these are known to have lesions in starch biosynthesis genes. Here we confirm that one type of shrunken endosperm mutant, lys3 (so called because it was first identified as a high-lysine mutant) has an additional phenotype: as well as shrunken endosperm it also has enlarged embryos. The lys3 embryos have a dry weight that is 50-150% larger than normal. Observations of developing lys3 embryos suggest that they undergo a form of premature germination and the mature lys3 grains show reduced dormancy. In many respects, the phenotype of barley lys3 is similar to that of rice GIANT EMBRYO mutants (affected in the OsGE gene). However, the barley orthologue of OsGE is located on a different chromosome from Lys3. Together these results suggest that the gene underlying Lys3 is unlikely to encode a starch biosynthesis protein but rather a protein influencing grain development. We recently cloned LYS3 and found that it encodes a transcription factor called Prolamin Binding Factor (PBF) that is expressed in grains only. Wheat and barley LYS3/PBF mutants have enlarged embryos suggesting that this gene suppresses embryo growth. The down-stream target genes of PBF in wheat are predicted to be involved in a wide range of biological processes including organ development and starch metabolism.
Exploitation Route not yet applicable
Sectors Agriculture, Food and Drink

 
Description UK Global Food Security Science Advisory Board
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
URL https://www.foodsecurity.ac.uk/
 
Description Bayer 
Organisation Bayer
Department Bayer CropScience Ltd
Country United Kingdom 
Sector Private 
PI Contribution Wheat genetics and genomics
Collaborator Contribution Wheat breeding and molecular knowledge
Impact joint projects
Start Year 2012
 
Description KWS 
Organisation KWS UK
Country United Kingdom 
Sector Private 
PI Contribution Genetics and genomics
Collaborator Contribution Breeder know how and germplasm
Impact joint projects
Start Year 2009
 
Description Limagrain 
Organisation Limagrain
Country France 
Sector Private 
PI Contribution Genetics and genomics
Collaborator Contribution Germplasm and breeder know-how
Impact Phd Studentship, field trails, among others
Start Year 2009
 
Description RAGT 
Organisation RAGT Seeds
Country United Kingdom 
Sector Learned Society 
PI Contribution Genetics and genomics
Collaborator Contribution Wheat germplasm and know how
Impact Shared projects
Start Year 2009
 
Description BBSRC Food Priming Partnership Workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact BBSRC Food Priming Partnership Workshop
Year(s) Of Engagement Activity 2018
 
Description Discussion Norman Lamb, MP 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Discussion Norman Lamb, MP
Year(s) Of Engagement Activity 2017
 
Description Discussion with Gov Office Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Discussion with Gov Office Science
Year(s) Of Engagement Activity 2017
 
Description JIC Open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact JIC Open Day
Year(s) Of Engagement Activity 2018