Durum wheat (Triticum turgidum L. ssp. durum) is a tetraploid wheat species grown
primarily in the North American Great Plains, Mediterranean Europe, Northern Africa,
Mexico and Australia. An important limiting factor for durum production in Mediterranean
environments like South Australia is water deficit immediately prior to and during anthesis,
adversely affecting durum productivity and quality. Investigating water deficit response
mechanisms and genotypic differences within a crop species is an important strategy for
understanding the basis of water-deficit stress response and for selection of elite genotypes
with improved stress tolerance. In plants, microRNAs (miRNAs), which are a class of small
non coding RNAs, have been identified as important regulators of plant development and
abiotic stress responses. While the miRNA transcriptome under water limiting conditions has
been investigated in many crop species, it is poorly characterised in durum wheat.
In this study, glasshouse experiments over two years evaluated 20 durum wheat
genotypes for their variation in various morphological, physiological and yield responses to
pre-anthesis water-deficit stress. Four Australian durum varieties with contrasting stress
sensitivities were identified. High-throughput Illumina sequencing of 96 small RNA libraries
constructed from the flag leaf and head tissues of these four genotypes detected 110
conserved miRNAs and 159 novel candidate miRNA hairpins. Statistical analysis of
sequencing reads revealed the differential expression profiles of durum miRNAs associated
with water-deficit stress treatment, tissue type and genotype. Most importantly, several
conserved and novel miRNAs showed inverted regulatory profiles between the stress tolerant
and sensitive varieties. Subsequent genome-wide in silico analysis identified 2055 putative
targets for conserved durum miRNAs, and 131 targets for four novel durum miRNAs possibly
contributing to genotypic stress tolerance. Predicted mRNA targets of the stress responsive
miRNAs encode various transcription factors, binding proteins, and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and
metabolic processes, suggesting the extensive involvement of miRNA-target regulatory
modules in water-deficit stress adaptation. Quantitative PCR profiling further characterised 50
target genes and 12 miRNAs with stress responsive and/or genotype-dependent expression
profiles. A 5′ RLM-RACE approach subsequently validated the regulation of nine targets by
water-deficit stress responsive miRNAs, providing the first experimental evidence that target
mRNAs are genuinely cleaved by miRNAs in durum wheat. Characterisation of the individual
miR160/Auxin Response Factors regulatory module further revealed their expression profile
over different time points during water-deficit stress.
The present study provides a comprehensive and comparative description of the
miRNA transcriptome and their targets in durum wheat varieties with contrasting waterdeficit
stress tolerance, providing new insights into the functional roles of miRNA-guided
RNAi mechanisms. Results derived from this work could contribute to future research on the
characterisation of individual miRNA regulatory modules and their specific biological
functions, exploiting the potential of Triticum turgidum miRNA in developing RNAiimproved
crops with stress tolerance.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2016
