Analysis of Temporal and Tissue Specific Humulus lupulus Isoforms by Long-Read Sequencing.

LIEBELT, DONNA JEAN. Analysis of Temporal and Tissue Specific Humulus lupulus Isoforms by Long-Read Sequencing (Under the direction of Dr. Colleen Doherty). The phytochemical composition in plants is dependent on environmental factors, both directly and through daily and seasonal rhythms. We attribute the value of many consumer products and pharmaceuticals to plant phytochemicals. As influential environmental factors continue to change, including the global rise in temperatures, and the feedback this has on extreme weather events, we must understand the role environmental factors have in signaling regulatory pathways within specialty crops. This understanding has the potential to aid decisions in agricultural management and plant breeding. Humulus lupulus (hops) can be a model perennial specialty crop for studying the effects of the environment on metabolites as breeders consider composition carefully, and they are grown in diverse climates and regions. Hops, while most known for their role in beer production, are also a rich source of nutraceuticals, including antioxidants, antimicrobials, and essential oils. Though their metabolite profiles are extensive, there are restrictions in the tools available to further examine biochemical pathways due to a lack of detailed annotations. One way to address this challenge is to use long-read sequencing such as single-molecule real-time (SMRT) sequencing. SMRT sequencing, a third-generation sequencing technology developed by Pacific Biosciences optimized to capture entire transcripts. Like any new technology of its kind, the bioinformatic analysis and compatible software for isoform processing are continuously evolving. PacBio's SMRT Analysis software and compatible packages for post-processing are enormously valuable in analyzing Iso-Seq and isoform data; however, they can be complicated and challenging to understand and troubleshoot for biological scientists without a history in coding and bioinformatics. With the advancement of sequencing applications as well as the rise in implementation of interdisciplinary teams, inexperienced researchers can either expect to have to learn some level of bioinformatics to analyze the data they generate or pay to outsource the analysis. Basic processes to analyze the data have been established. However, due to the diversity of available software, operating systems, and hardware, it is difficult for a non-bioinformatician to understand the end-to-end workflow. Herein we provide a beginner-friendly end-to-end workflow for Iso-Seq and isoform analysis. The workflow commentary can be used to both understand the coding required as well as the biological relevance of these steps, bridging the divide between them. With this workflow applied, we analyzed long-read isoforms in Humulus lupulus Teamaker variety leaves, roots, and shoots sampled at dawn and dusk. We produced 373,555 full-length reads, which accounted for only ~40% of the total reads detected when mapped to the current Teamaker genome assembly. Over 5,000 isoforms are time-of-day or tissue-specific, and nearly 4,000 unique isoforms that are dependent on both tissue type and time of day. Future directions include analyzing cone tissues, as well as environmental factors such as temperature. While temperatures are steadily increasing, night temperatures are warming faster than day temperatures. Daily temperatures cycles are known to entrain endogenous rhythms in plants. The decreasing difference between day and night temperatures poses an interesting question of how plant composition will be affected by a change in this signal. As proof of principle, we examine the effects of three-degree warmer nights on the metabolite composition of broccoli and radish sprouts. Further exploration could reveal plant varieties and environmental conditions that maximize the desired phytochemical profiles to benefit human health. Additionally, we could gain insights into the impact of changing environments on the regulation of phytochemicals, which could be applied to other crop systems targeted for the production of pharmaceuticals and other consumer products.