Award details

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

Reference	BB/L023229/1
Principal Investigator / Supervisor	Professor Cristobal Uauy
Co-Investigators / Co-Supervisors	Professor Wendy Harwood
Institution	John Innes Centre
Department	Crop Genetics
Funding type	Research
Value (£)	73,151
Status	Completed
Type	Research Grant
Start date	01/10/2014
End date	30/09/2017
Duration	36 months

Abstract

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.

Summary

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.

Impact Summary

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 willprovide 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.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Crop Science, Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/L023156/1 Identifying the genetic basis of large-embryo mutants of barley affected at the Lys3 locus.

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