Seed dormancy and seed longevity: from genetic variation to gene identification

Abstract

Seed dormancy and seed longevity are the most important survival traits in the soil seed bank. Both traits are induced during seed maturation and evolved to assure seed survival during environmental conditions that cannot support the regular course of life. Seed dormancy is related to the timing of germination while seed longevity is involved in retaining germination ability that is gradually lost as a result of aging. The work presented in this thesis aims to identify genetic factors that regulate seed dormancy and seed longevity by generating and exploiting genetic variation.The exploitation of natural variation that is present in six recombinant inbred line populations for seed longevity revealed nine quantitative trait loci (QTLs) named Germination Ability After Storage (GAAS). Comparison between GAAS loci and seed dormancy Delay Of Germination (DOG) QTLs earlier established in the same populations led to the identification of collocating loci for which the allele effects were negatively correlated. The QTL effects of the GAAS loci and their colocation with the DOGs were validated in near isogenic lines (NILs). To our knowledge this is the first time that such a negative correlation between dormancy and longevity was reported, deep seed dormancy correlating with low seed longevity and vice versa. Detailed analysis on the collocating GAAS5 and DOG1 loci revealed that the DOG1-Cape Verde Islands (Cvi) allele both reduces seed longevity and increases seed dormancy. The three strongest GAAS loci have been submitted to proteome analyses to reveal molecular mechanisms as well as genetic factors that are involved in seed aging. During aging, dry seed proteomes are markedly changed in a genotype specific manner, implying that different mechanisms are involved in seed longevity conferred by the three NILs. The shared pathways revealed the importance of seed reserves (seed storage proteins, SSPs), antioxidant systems notably vitamin E, as well as the protection and maintenance of the translation machinery, and energy related pathways in seed longevity. The SSPs cruciferins and napins, the small ribosomal subunit RPS12C, and the NADP-dependent malic enzyme1 (NADP-ME1) were confirmed to significantly affect seed longevity by T-DNA knock-out mutants.The investigations on the negative correlation between seed dormancy and seed longevity were continued by fine-mapping the DOG2/GAAS1 locus. Genetic analyses showed that DOG2 and GAAS1 were likely two independent loci due to the differences in genetic behavior of DOG2 (maternal) and GAAS1 (co-dominant). The fine-mapping of DOG2 resulted in a 85 Kb-region containing 27 genes. Ethyl methanesulfonate-induced mutagenesis in the dormant NILDOG6-Shakdara background revealed six novel seed dormancy modifier mutants (sdm1 to sdm6). Sdm1 and sdm3-6 showed reduced seed longevity as a pleiotropic effect of the mutated seed dormancy gene. Mutants sdm2 and sdm4 exhibited abnormal seed mucilage. Mapping populations were generated and phenotyped in order to identify the underlying sdm genes by a mapping-by-sequencing approach. This work provides new insights in the seed dormancy/longevity relation and genes and processes involved.