Transposon-mediated gene exploration in barley and other cereals

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Characterization of mutant alleles provides valuable insights into the genetics of normal physiological functions. Mutagenesis via T-DNA insertion, RNA interference and transposon tagging enable researchers to more readily study specific genes and their functions. Transposon -mediated insertional mutagenesis has advantages over other approaches for determining gene function especially where genetic transformation is not routine. Important genes involved in development were identified using several gene-tagging strategies involving Ac/Ds-mediated insertional mutagenesis. During the transposon movement, the transposon inserts into a new chromosomal location and may cause interruption in gene’s normal function. Some epialleles undergo paramutation which is an interaction between alleles and leads to a heritable expression change of one allele. Transposons have also served as building blocks for epigenetic phenomena, both at the level of single genes and across larger chromosomal regions. Studies are being conducted for the understanding these silencing mechanisms. Using transposon-based strategy, we have created over 400 unique transposon insertion lines (TNPs) in barley. Barley is a model genome system for Triticeae. Because barley is diploid, observation of the segregation of phenotypic variation in tagged mutants in successive generations is simpler than in its close polyploid relatives, wheat and oats. The system was tested in barley for several generations by reactivating Ds transposons and development of primary, secondary, tertaiary and quaternary transposon insertion lines. We also established that in barley, Ds transposon have tendency to move into genic regions in high frequency. The system is now ready for its use in functional genomic studies. The long-term objective of my research program is to advance molecular knowledge related to the mechanisms of seed dormancy and stress tolerance in cereals. From our TNP repository, we have identified transposon mutants of the DNA silencing pathway with reduced germination. There is emerging evidence that epigenetic may contribute to grain development and seed dormancy. Our preliminary characterization of transposon mutants indicate that DNA silencing pathway may also be involved in regulation of seed dormancy. Our recent data indicate that a key gene of RdDM pathway, AGO4 acts as negative regulator of dormancy. However, it is not known whether specific dormancy related genes are subjected to silencing through RdDM in small grain cereals. This may be due to DNA methylation triggered by small RNAs which needs further investigation. I propose to determine, the unknown role of (DCL3) in grain dormancy in barley through transposon mutagenesis. Data from these experiments will advance fundamental knowledge on the molecular mechanisms involved in the regulation of seed dormancy and germination.