Tucker, MatthewChen, WeiweiEbrahimi Khaksefidi, Reyhaneh2024-10-282024-10-282024https://hdl.handle.net/2440/143015In cereals, the inflorescence influences flower formation, grain number, and yield. The formation of an inflorescence and the regulation of its morphology are key topics for both basic plant science and crop improvement. The barley inflorescence, known as the spike, contains spikelets directly attached to the main axis. The fertile spikelet forms one basic reproductive unit that produces seed. MicroRNAs (miRNAs) play a critical role in the regulation of gene expression both transcriptionally and post-transcriptionally and are integral to various plant biological processes. Despite known functions in model plants like Arabidopsis thaliana and rice (Oryza sativa), the comprehensive role of miRNAs in barley inflorescence development remains unclear. My research aimed to identify crucial miRNAs influencing barley inflorescence development at three key stages: spikelet initiation / Double Ridge (DR), floral organ differentiation / Awn Primordium (AP), and floral organ growth / Green Anther (GA). These stages were carefully chosen to represent critical points in inflorescence development. Small RNA and degradome sequencing were conducted across these stages with seedlings serving as a vegetative phase control (Chapter 2). I identified 208 known miRNAs across 142 miRNA families and 494 novel miRNAs. Notably, miRNAs showed stage-specific patterns with miR408-3p and miR5083 significantly expressed during the DR stage; miR168-3p and the miR2118 family during the AP stage; and miR2275b and miR1432-5p during the GA stage. These patterns suggest a complex regulatory role of miRNAs and highlight their potential influence on barley inflorescence development. Functional analysis of miRNA detected via small RNA-seq led to further functional experiments including overexpressing miR397a using the 35S promoter and knocking out its putative target gene, HvLaccase (HvLAC), using CRISPR/Cas9 (Chapter 3). The resulting transgenic plants showed phenotypic alterations including multiple spikes per tiller, increased tiller number, male sterility, and reduced plant height. Similarly, the hvlac mutants displayed semi-sterility, reduced plant height, and increased tiller number, suggesting a modification in HvLAC function. Our findings indicate that the role of hvu-miR397a is partially conserved in barley and Arabidopsis, with additional functions in barley related to male fertility. Another focus was understanding the role of miR156-meidated HvSPL14 (SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE 14) gene expression (Chapter 4) that was detected in the degradome sequencing. In rice, a point mutation in SPL14 (also known as IPA1) increases branch numbers and enhances crop yield. Despite initial success in generating Hvspl14 mutants, the publication of similar findings (Thiel et al., 2021) led us to discontinue this aspect of our project, though we report detailed insights into the SPL family and barley Hvspl14 mutants for future reference. In conclusion, our results provide a comprehensive view of potential miRNA-target networks that regulate barley inflorescence development. The findings underscore the importance of post-transcriptional mechanisms for enhancing our understanding of inflorescence morphogenesis and for crop improvement.enBarleyInflorescence DevelopmentMicroRNAThesis