Evaluation of Reference Genes for Expression Studies in Ash (Fraxinus spp.)

Abstract Ash (Fraxinus spp.) is a dominant tree species in North America, in both managed and natural landscapes. However, due to the rapid invasion by the emerald ash borer (Agrilus planipennis), an exotic invasive insect pest, millions of North American ash trees have been killed. Real-time quantitative polymerase chain reaction (RTq-PCR) is widely used for validating transcript levels in gene expression studies for which a good reference gene is mandatory. In the current study, we evaluated the stability of ten reference genes in at least five different tissues (phloem, roots, shoots, immature leaves, and mature leaves), and two developmental stages (young and old) among three ash species including the resistant Asian Manchurian ash (F. mandshurica) and two susceptible North American ash species (green—F. pennsylvanica and white—F. americana). Of the examined genes, the transla-tion elongation factor alpha (eEF1α) was observed to be most stable and thus is recommended for RTq-PCR based gene expression studies in Fraxinus species. To our knowledge, this is the first report on the stability of reference genes across ash species (in different tissues and during development)