Leveraging public transcriptome data for enhanced qPCR probe design

Authors: WALIULLAH, SUMYYA - Washington State University; Honaas, Loren; KALCSITS, LEE - Washington State University

Submitted to: Plant and Animal Genome Conference
Publication Type: Abstract Only
Publication Acceptance Date: 10/24/2017
Publication Date: 1/12/2018
Citation: Waliullah, S., Honaas, L.A., Kalcsits, L.A. 2018. Leveraging public transcriptome data for enhanced qPCR probe design. Plant and Animal Genome Conference. January 12, 2018, San Diego, California.

Interpretive Summary:

Technical Abstract: Quantitative real-time PCR (qPCR) is the gold standard for targeted gene expression analysis and is a critical validation tool for RNA-seq experiments. And as plant genomes continue to become available for economically and scientifically important plants, opportunities to leverage these genomic resources for closely related plants (i.e. cultivars, species, ecotypes) arise. However, qPCR (and RNA-Seq) rely on prior knowledge of gene sequences, thus genetic differences between sequenced plants and closely related plants of interest can cause loss of fidelity in gene expression measurements at multiple steps in an experiment. To avoid this pitfall, we used publicly available transcriptome data from Honey Crisp apple to discover cultivar specific variants and guide our qPCR primer design strategy to study genes potentially involved in the physiological fruit disorder, bitter pit. Bitter pit is a calcium-related physiological disorder that typically manifests as corky, dark and depressed spots on the fruit surface which cause high post­harvest losses in apple. Towards predictive and diagnostic tests, knowledge of molecular mechanism underlying BP development are essential. To check BP related gene expression we chose genes from a previous report that are implicated in the disorder. Using BLAST we queried a de novo transcriptome assembly of Honey Crisp with coding sequences of Golden Delicious genes or cultivar specific clones. We used the Honey Crisp best hits to design qPCR primers with 100% success - all primer pairs produced target specific amplicons with high efficiency. Furthermore, we were able to learn about cultivar specific gene variants and, by examining alignments to discover polymorphisms that would impact primer binding, opened the door to qPCR assays that are variant specific or have cross-cultivar applicability. This work is possible with open-source software and freely available public data, necessitating no upfront cost.