Transcriptomic Analysis Of Petunia Hybrida In Response To Salt Stress
Villarino Pizarro, Gonzalo
Abiotic stresses, such as salinity and drought, are among the most limiting factors to crop yield. In sodic saline soils, sodium chloride (NaCl) disrupts normal plant growth and development. Many studies have used both forward and reverse genetic techniques to understand the complex interactions of plant systems with abiotic stress. These approaches have been invaluable in deciphering some mechanisms of plant salt stress tolerance. Salt tolerance research has also been an important part of basic plant biology, increasing the understanding in areas encompassing gene regulation, mineral nutrition, signaling components, ion transport and osmoregulation. To better understand the detrimental effects of NaCl, as well the fundamental questions associated with salt tolerance, transcript regulation in response to NaCl stress was undertaken using ultra-high-throughput RNA sequencing technology (RNA-seq). RNA-seq has quickly become the method of choice to perform transcriptomic analysis owing to many advantages over existing platforms. The transcriptomic research presented here was carried out in Petunia hybrida, a salt resistant Solanaceous plant that has also been an excellent model species in molecular genetic research regarding flower development and senescence, synthesis and regulation of volatiles, and so on. In chapter one, to bypass the absence of an available Petunia genome, a de-novo assembled Petunia transcriptome was reconstructed by assembling over one- hundred million Illumina cDNA reads with Trinity software. The de-novo assembled contigs represents the most in-depth transcriptome ever reported for a Petunia species, which can be used as an excellent tool for biological and bioinformatics in the absence of an available Petunia genome. The transcriptome has been made publically available on the SOL Genomics Network (SGN) http://solgenomics.net. Using this newly assembled reference transcriptome, more than 7,000 differentially expressed genes were identified within 24 h of acute NaCl stress. Genes related to regulation of reactive oxygen species, transport, and signal transduction as well as novel and undescribed transcripts were among those differentially expressed in response to salt stress. Gene ontology analyses revealed that plants by 24 h after acute NaCl undertook many changes occurring at the molecular level including genotoxicity, affecting transport and organelles due to the high concentration of Na+ ions. RNA-seq, despite the many advantages it offers, it is a relatively new methodology with developments and improvements to be made. At the end of chapter one a modification to the library preparation protocol is presented whereby cDNA samples were bar-coded with non-HPLC purified primers, without affecting the quality and quantity of the RNA-seq data. This methodological improvement could substantially reduce the cost of sample preparation for future high-throughput RNA sequencing experiments. In chapter two, root and leaf transcriptomic response to salt stress was investigated, utilizing the Petunia Genome Sequencing Project's draft Petunia axillaris genome v1.6.2. Having access to the P. axillaris draft genome enabled use of a more robust bioinformatic tool to perform a Whole Transcriptome Shotgun Sequencing experiment. This chapter expands upon chapter one by using the genome as a reference and also including root response to NaCl. Twenty-five candidate genes that were significantly induced at different time points under salt stress were identified for both leaves and roots. These genes, upon functional characterization, represent a good amenable number of genes for plant breeding or to genetically engineer plants to enhance salt tolerance. Lastly, a polymorphism analysis was conducted using Single Nucleotide Polymorphism (SNP) and Insertions/Deletions (INDELs) to explore the relationship between P. hybrida (used for the current dissertation work) and P. axillaris (Petunia reference genome). A large number of allelic variant was found, when comparing P. hybrida vs. reference genome, inducing early stop codon and transcript frame shift with disruptive effects. This chapter will be published as a short publication included within the 'Petunia Genome Publication', in which the genomes of the parental species of P. hybrida are being sequenced and annotated by an international consortium.
transcriptomics; RNA-seq; Salt stress
Mattson, Neil S.Mattson, Neil S.
Hanson, Maureen R; Scanlon, Michael J.; Nero, Debra; Hanson, Maureen R; Scanlon, Michael J.; Nero, Debra
Ph.D. of Horticultural Biology
Doctor of Philosophy
dissertation or thesis