Identification and Characterization of Seed Longevity Genes in Barley (Hordeum vulgare)

Plant diversity is being lost at an unprecedented rate. This makes the conservation of plant species to a global task of high priority. Seed conservation, both in situ and ex situ, is one of the best strategies for the conservation of plant diversity. For germplasm preservation in seed banks, seed longevity is of particular importance. Seed longevity constitutes a measure for the period a seed remains viable when stored under the optimum conditions for that species and/or cultivar. Seed longevity is induced during seed maturation and is involved in retaining germination ability that is gradually lost as a result of ageing. Hence, seed longevity is a major parameter controlling seed quality and an important survival trait in the soil seed bank helping the seed to adapt to changing environmental conditions. Until now, little is known about the genetic basis of differences in seed longevity because this trait is affected by numerous environmental effects during seed formation, harvest, and storage, and genetic variation between and within species is probably controlled by several genes. The work presented in this thesis aims to identify and characterize seed longevity loci/genes in barley (Hordeum vulgare) by combining a quantitative genetics approach with 'omics' technologies. This work is based on a previous Quantitative Trait Loci (QTL) study in recombinant inbred lines (RILs), derived from the short-lived Ethiopian spring barley landrace L94 and the long-lived Argentinian spring barley landrace Cebada Capa (CC). In total, four putative QTLs for seed longevity were identified on chromosomes 1 and 2, and near isogenic lines (NILs) were generated (Adimargono et al., in preparation). Using these near isogenic lines, the four putative QTLs could be confirmed and further mapping of Cebada Capa introgressions was achieved in the so-called L94 NILs using RNA-seq. For candidate and downstream target gene identification, a combined transcriptome and proteome analysis was performed. The analysis of mature, non-aged seeds of the two parental lines and the L94 NILs by RNA-seq and total seed proteomic profiling identified two putative candidate genes and one possible downstream target gene. The (NADP)-dependent Malic Enzyme (NADP-ME) AK248526.1 and the UDP-glycosyltransferase MLOC_11661. 1 were chosen as possible candidate genes for two of the four seed longevity QTLs, while the second NADP-ME MLOC_35785. 1 was chosen as a possible downstream target. To validate these putative candidate and downstream target genes, a T-DNA knock-out line of a homologous Arabidopsis gene, shown to have a seed longevity phenotype by Nguyen (2014), was complemented with the respective Cebada Capa and L94 alleles under the expression of different promoters. Both the NADP-ME MLOC_35785. 1 and the UDP-glycosyltransferase MLOC_11661. 1 were able to rescue the nadp-me1 seed longevity phenotype when being expressed under the control of the 35S- or the Ubi-promoter. In the latter case, only the CC-specific coding sequence had an effect. Since the identified candidate NADP-ME MLOC_35785. 1 may affect the redox status in normal and deteriorating seeds, the corresponding Arabidopsis mutant nadp-me1 was combined with the redox reporter roGFP2 to visualize the redox potential in seeds using confocal microscopy. First transformant generation (T1) lines, showing strong fluorescence in the cytosol, could be generated in this study and allow the measurement of the NADP+/NADPH redox potential in future studies.