Primer development methodology for RT-qPCR genetic studies of Lodgepole Pine Dwarf Mistletoe (Arceuthobium americanum)

The genus Arceuthobium (Dwarf Mistletoe) of the family Santalaceae, is a group of dioecious parasitic flowering plants with a unique, explosive, seed dispersal system. Arceuthobium americanum infects Lodgepole Pine (Pinus contorta var.latifolia) in North Western America and is considered as a pest in the forestry industry. Mistletoe is also an ecologically important organism, promoting wildlife habitat, and biodiversity. Current literature on Arceuthobium shows incomplete understanding of molecular mechanisms underlying explosive seed dispersal, and critical mechanisms linking parasitic infection and mortality of the Lodgepole Pinehost. Experimentation using Real-time quantitative polymerase chain reaction (RT-qPCR) measures the expression of genes, giving insight into the molecular mechanisms.Determination of stably expressed genes, known as reference genes, is an important step for conducting reproducible experiments and to obtain reliable RT-qPCR expression data. The objective of this thesis is to create an in silico methodology to develop primer pairs to target reference genes as a basis to study Dwarf Mistletoe using RT-qPCR. Highly conserved genes, such as Ubiquitin, Actin, EF-a, and GAPDH, are targeted using sequences from same family Santalaceae plant Viscum album, model organism Arabidopsis thaliana, as well as Solanum lycopersicum, Coffea canephora, Paeonia suffruticosa, Zea mays, Kalanchoe dalgremontiana, Triticum aestiyum, and Nicotiana benthamiana. Algorithms designed to develop primer pairs, Primer-3, Beacon Designer 8, and Primer-BLAST, were used to determine primers in published sequence sof the Arceuthobium oxycedriaquaporin gene, a gene of interest in seed dispersal in A. americanum. In experimental validation, the primer pair candidates taken from literature produced no significant results in RT-qPCR. A primer pair produced from primer development algorithm Primer-BLAST using the Arceuthobium oxycedriaquaporin gene sequence provided consistent and reliable RT-qPCR data. Although a reliable reference gene was not developed, the research presented in this thesis establishes methodological steps for insilico RT-qPCR primer design, it demonstrates that genetic sequences from organisms within the same genus can be used for RT-qPCR primer design, and it provides a primer pair for future studies of the A. americanum aquaporin gene using RT-qPCR.