History of medicinal plants usage goes back to 60 thousand years ago in Shanidar cave in Kurdistan. Among the oldest medicinal plants, stevia (Stevia rebaudiana) and fennel (Foeniculum vulgare var. vulgare) are used as flavoring and curative agents in food and pharmaceutical goods, due to possessing certain metabolites. These metabolites in fennel are essential oils and fatty acids stored in the seeds, and in stevia are steviol glycosides (SGs) stored in the leaves. To keep up with the increasing demand for fennel and stevia products, developing high yielding cultivars is a necessity. For this, understanding the existing diversity and genetic basis of desired metabolites is important. To do so, in the first part of this study, 50 Iranian fennel landraces in a field study were evaluated for their agro-morphological traits and five high yielding synthetic cultivars were developed. In the second part of this study, RNA seq and QTL analysis were used to find genes / genomic regions underlying biosynthesis of Rebaudioside D (Reb D) which is one of the most desired SGs. The results from the fennel experiment showed the fennel landraces were early, medium, or late maturities, with life spans of three to five years. During life spans of the landraces, a wide diversity for seed and essential oil production was observed, and in each maturity group high yielding landraces were identified. A single year analysis of total fatty acid content followed by GCMS analysis, indicated that some of these fennel landraces have the potential to be complementary sources of certain fatty acids, such as oleic and linoleic acids. The main compositions of fatty acids, measured in twelve of the landraces, were oleic acid and linoleic acid. Landraces with high oleic acid content originated from regions with a dry and warm climate, while landraces with high linoleic acid content originated from regions with a humid and cool climate. Understanding relationships between fatty acid profile and landrace origin climate may improve the efficiency of identifying landraces with specific fennel chemotypes. After observing the diversity among these 50 fennel landraces, five fennel synthetic cultivars with different maturity habits, three with the goal of high essential oil yield, and the other two with the goal of high seed yield under drought conditions, were developed. Evaluation of the five synthetic cultivars showed, in drought stress conditions, the five synthetic cultivars had a higher essential oil yield and seed yield than their parents. Given that fennel is also an orphan crop, and pollination control in fennel is really challenging, synthetic cultivar development is a viable breeding method for fennel, especially in early and medium maturity fennels. In the QTL analysis on stevia, a genetic linkage map was constructed using 2298 SNPs across 11 linkage groups and a total map distance of 2190 cM, for an average distance of 0.95 cM between markers. Using this linkage map and phenotypic data from three field locations, seven QTL on linkage group five for Reb D concentration and proportion, explaining 13.5 to 39.6% of variance were identified. Six of these QTL overlapped, and QTL peaks for three and two of them were the same positions. These regions can go under further investigation to narrow down the region to specific genes. Additionally, QTL for Reb A, stevioside, Reb B, and total steviol glycosides were identified. The RNA seq experiment on stevia identified 63 upregulated, and 44 downregulated transcripts as being differentially expressed between high and low Reb D genotypes. Five modules containing from 99 to 421 transcripts, with significant and positive correlations with Reb D concentration, were identified. The differentially expressed transcripts, modules and their hubgenes are interesting targets for future investigations on Reb D production in stevia.
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