Oral Paper

         Hybrids and Hybridization

Development of a ddPCR Protocol for Detecting Homeolog Expression in Nicotiana Allopolyploids of Different Ages

Presenting Author
Trinity Tobin
Description
Allopolyploidy is when two or more sets of chromosome sets come together due to hybridization, creating homeologs. The genus Nicotiana has a variety of allopolyploids that stem from different polyploidization events, with these events giving rise to new distinct species. For example, N. sylvestris and N. tomentosiformis are the diploid progenitors of the allopolyploid N. tabacum, which originated 0.6 million years ago. In contrast, the offspring of N. sylvestris and N. obtusifolia (section Repandae) originated 4.3 million years ago. To analyze the differences in homeolog expression in allopolyploids of different ages, the diploid progenitors N. sylvestris, N. tomentosiformis, and N. obtusifolia were studied, as well as their allopolyploid offspring. Our question tests whether the differences we see in flower color in allopolyploids are due to variation in homeolog expression of flavonoid biosynthetic pathway genes. This pathway created the pigments cyanidin (pink), delphinidin (purple), and pelargonidin (red), as well as a group of colorless pigments called flavonols. We used droplet digital PCR (ddPCR) to develop a protocol to distinguish between homeologs using a single reaction to quantify expression of both homeologs. Droplet digital PCR separates the reaction into small droplets using oil. The PCR portion of the protocol amplifies our gene of interest in the droplets. If a droplet contains our gene of interest, it will fluoresce, allowing us to quantify the expression of a given gene based on how much transcript is present. Achieving this fluorescence would let us synchronously quantify the expression of both homeologs with a single ddPCR reaction. The goal of our ddPCR fluorescence is to have high fluorescence in the maternal homelog and lower fluorescence in the paternal homeologs. To do this, we designed primers that were more complementary to the maternal homeolog than the paternal homeolog, which will impact primer efficiency. Initially, we capitalized on SNPs between the maternal and paternal homeologs at the 3’ end of our primers, effecting the primer’s ability to anneal to the paternal homeolog efficiently. We found in our ddPCR relatively high fluorescence for the maternal homeolog and a large variation in the fluorescence in the paternal homeolog. To troubleshoot this, we created new primers that were identical on the 3’ end but had SNPs throughout the primer that would allow us to distinguish based on differences in primer melting temperatures. We hypothesize that this new method will decrease this variation of paternal homeolog expression while also achieving our original goal of distinguishing the homeologs in a single reaction.