The Bioinformatics Exploration of Stevia rebaudiana
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2023
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Stevia rebaudiana (stevia) is a short-day perennial Asteraceae that is native to Brazil and Paraguay and is renowned for its sweet taste. In the undertaking of this project, a large Next Generation Sequencing (NGS) dataset was generated from twenty-three genetically unique stevia plants. Stevia leaf samples were collected three times over eleven months in 2019. The leaf samples were used to generate RNA-seq data and resulted in a cDNA library of over one billion Illumina small reads.
The dissertation work commenced with constructing the best possible transcriptome from S. rebaudiana leaf tissue. This effort resulted in a high-quality, non-redundant (to 95% similarity) catalog of S. rebaudiana genes replete with names and functional annotations and a BUSCO score of 96%. The high BUSCO score indicates that the final catalog of stevia RNAs is complete and representative. The resultant index of RNAs contains 66,022 complete RNA sequences that can be sorted by putative function, length, or expression level and contains ~23% more RNAs than the publicly available transcriptome published in 2021. The catalog of 66,022 RNA sequences also includes 9,275 novel RNAs that were missed in the previous construction of the stevia genome.
During an early stage of the de novo transcriptome, the presence of a Picornavirus, Bundaberg Bee Virus 4 (BBV4), was detected in some of the samples. This represents the first identification of BBV4 in Hawaii and the second in the world. This unexpected finding expanded my interest from stevia genetics to the virome of the stevia plants grown in Hawaii. The genome of BBV4 was extensively characterized, and the results have been accepted for publication in the European Journal of Biotechnology. Further effort was exerted to catalog the virome in the transcriptome, and the results provided fascinating insights into the viral landscape of stevia plants grown in Hawaii. Initially, the virome catalog was exclusive to Riboviria, as the data was generated from RNA-seq. However, the discovery of viral RNA from a DNA virus was identified in the dataset, and after that, the project's scope expanded to include DNA viruses. The revised direction resulted in a comprehensive catalog of over 40 viruses, 70 species of bacteriophages, and at least one mega-virus.
In summary, this project constructed the most complete transcriptome of S. rebaudiana to date, and cataloged 66,022 full stevia RNA sequences, of which, including 9,275 novel stevia RNAs that were identified for the first time. Furthermore, this project documented the first virome in the leaves of S. rebaudiana grown in Hawaii and comprehensively characterized the genome of one of the Picornaviruses, BBV4. This project employed many bioinformatics tools across multiple platforms to examine and analyze the dataset and obtained a rich body of genetic information of stevia plants and the virome associated with the plants. The ever-changing nature of bioinformatics necessitates ongoing engagement with new tools, resources, and methods, and this project exemplifies that sentiment.
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