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Selection of reference genes to develop an RNA-based viability assay in response to thermotherapy.

Naweena Thapa: University of Florida

<div>Huanglongbing (HLB), caused by the bacterium <em>Candidatus</em> Liberibacter asiaticus (CLas), is one of the most destructive citrus diseases to threaten citrus industries worldwide. Thermotherapy is being tested in Florida as a potential management practice for HLB. To ensure thermotherapy’s effectiveness, a post-treatment viability assessment of CLas is important. Our overall objective is to develop an RNA-based viability assay for CLas to measure lethality of high temperatures. It will be evaluated by comparing the expression of CLas 16S rRNA, and other CLas transcripts if necessary, at different time-temperature treatments (55°C/0s; 55°C/60s; 55°C/90s; 55°C/120s; 60°C/30s; and untreated control). To study gene-expression, we used qRT-PCR, which is a robust, highly sensitive and reproducible method. Citrus gene expression will be used for normalization because citrus tissue remains viable post-treatment. We studied candidate citrus genes for stable expression throughout the treatments to select reference genes for the viability assay. Nine candidate reference genes were evaluated: <em>GAPDH</em>, <em>TUB</em>, <em>ACT2</em>, <em>GAPC2</em>, <em>FBOX</em>, <em>SAND</em>, <em>EF1α</em>, <em>UPL7</em>, and <em>UBC21</em>. geNorm and BestKeeper software were used to determine the most stable reference genes for the six different treatments, and among the nine genes, <em>GAPC2</em>, <em>EF1α</em>, and <em>GAPDH</em> were the best candidates. We chose <em>GAPC2</em> and <em>EF1α, </em>as reference genes for further gene expression studies.</div>