Small RNA Identification, Genomic Characterization, And MicroRNA Paleo-historical Evolution In Brachypodium Distachyon (Schnable Et Al.) Beauv

The temperate grass Brachypodium is considered as a promising model of wheat and other energy crops due to its physical and genomic attributes:small genome size, rapid generation time, and highly efficient transformation. As a member of the Pooideae subfamily, Brachypodium shares the same ancetral genome with other grass crops such as rice, sorghum and maize, among which chromosomic synteny and paleo-duplication have been clearly identificated. Small RNAs are single-stranded RNAs of18-30nucleotide (nt) long. They play important roles in plant development, response to various stresses and maintenance of genome stability. There are two major categories of small RNAs in plants, microRNA (miRNA) and small interfering RNA (siRNA). MiRNAs regulate the expression of target genes mainly in the post-transcriptional and translational level, while siRNAs modify genes in epigenetics level. In this PhD dissertation, three aspects of studies were performed surrounding the Brachypodium small RNAs:1. To identify novel Brachypodium small RNAs (including miRNAs), we sequenced four small RNAs libraries from vegetative tissues, young spikes,8h light/16h dark treated2week old seedlings, and20h light/4h dark treated2week old seedlings. Bioinformatic analaysis identified a number of miRNA genes, nat-siRNAs and phased siRNAs. Some conserved miRNAs exhibited day length-specific expression patterns, mainly involved in development such as miR156-160-444and disease-resistance such as miR169-408-1432. Only a few novel miRNAs were found to be potentially involved in response to day length. The targets of these differential small RNAs were annotated as photosynthesis related genes and hence affect the growth and development of Brachypodium plants. However, a small number of nat-siRNAs were found to be involved in the development and photoperoid process in Brachypodium, while phased-siRNAs may play important roles in the flower development.2. To further characterize Brachypodium small RNAs in the genome, we performed a genome wide analysis of their structures and family expansion mechanims. We found that, similar to protein-coding genes, the miRNA families were also amplified via transposition in addition to whole genome duplication (WGD), segmental duplication and tandem duplication. Interestingly, tandem duplication seems to significantly contribute to the expansion of miRNA families probably due to their special hairpin structure. Moreover, with the mutation in mature miRNA sequence and the promoter sequences, the duplicated miRNA genes may undergo sub-/neo-functionalization where they may take on novel functions to mke plants better adapt to the environment in the evolution process.3. Grass species including Brachypodium, rice, sorghum and maize were derived from the same paleo-polyploid ancestor. We conducted a colinearity and paleo-duplication analysis in the genomes of the four grasses, in order to comprehend the evolutionary patterns and their functional evolution. We found that similar to transcript factors, miRNA genes were dosage sensitive, involving in the complicated regulatory network. About45%miRNA gene families were over-retained. This evidence is in support of the gene balance hypothesis that explains gene activities during the genome diploidization process. We found that the distribution of the duplicated miRNAs is biased among ancient chromosome pairs, displaying clear post-WGD subgenome dominance. We also found that, in contrast to the targets of the under-retained miRNA families, which were mainly involved in essential biological regulation processes and metabolic processes, the over-retained miRNA families targets were enriched functions for stress response.In summary, this work discovered a set of tissue-specific or day length specific miRNAs. It also explores the potential mechanisms for the miRNA family expansion in the grass genomes. Furthermore, the work analyzed the miRNA paleo-evolution patterns, and discovered the subgenome dominance of miRNA genome distribution which provides additional evidence for the gene dosage hypothesis. This work may lay a foundation for further study of miRNA functions in Brachypodium and other grass species.