Quantitative Differential Gene Expression with NGS to Uncover Regulators of Short-Day Induced Growth Cessation in Peach

Measuring differential gene expression in non-model species requires methodologies that do not depend on extensive a priori knowledge of the genome or transcriptome. Subtractive methods and custom arrays have been valuable tools for gene differential expression and gene discovery in non-model crops, nonetheless these methods can be time-consuming and insensitive and the genomic tools they generate are only informative for the individual experiment. Next generation sequencing (NGS) offers many improvements for global expression profiling and discovery of differentially expressed genes in non-model species.  Rapid advances in NGS technology (reduced cost and dramatically increased read numbers), now allow for direct sequencing and efficient global quantitative expression profiling with a depth that permits detection of relatively rare transcripts. Direct sequencing and transcript count data can be compared not just between samples from one experiment, but also among samples from different experiments.  Extracting transcript counts from NGS data sets does require some a priori knowledge of the transcriptome, an investment in bioinformatic infrastructure/support and the adoption of statistical models appropriate for evaluating expression changes between large data sets.

We recently performed a NGS differential gene expression experiment using the peach evergrowing mutant (which does not cease terminal growth under short days) and a sibling wild-type genotype. Terminal regions of wild-type and evergrowing peach were sampled at 0, 1, and 2 weeks following the transition to short days.  During this time, elongation growth of the wild-type was reduced and the mutant was unaffected. Pooled RNA samples from each genotype and time combination (six total) were extracted, quality checked and quantified.  Each sample was spiked with mRNA standards to assess the effect of transcript length and concentration on sequence recovery.  Samples were submitted to the ISU DNA Facility for cDNA library synthesis and 75 cycle sequencing by Illumina™ GAII technology.

Here we present our suggestions for using NGS for differential gene expression experiments gained from our experiences in peach.  We will also compare the differential gene discovery success to our previous experience with the subtractive suppressive hybridization in a similar experiment.