Identification and amplification of candidate genes for virus defence in common ash (Fraxinus excelsior L.)

Abstract

Across various European countries, Fraxinus excelsior L. is facing a decline caused by Hymenoscyphus fraxineus. The underlying mechanisms of the disease are still insufficiently understood in detail. Observations suggest that, in addition to the main pathogen, viral infections may also contribute to the decline of ash trees. We described homologues in the F. excelsior genome of formerly annotated virus resistance genes in model species and tested the applicability of primers on these gene fragments. Further, a prediction of cis-acting regulatory elements and transcription factor binding sites was accomplished, to determine the exact location of the promoters of these genes in the ash genome. After annotation and filtering the low-complexity regions, primers for use with genomic DNA were designed on sequences that showed a high degree of similarity and equality in length to translated regions and promoters of these regions. From the newly designed primers, those with the most favourable primer selection parameters were selected and tested on eight virus infected—four symptomatic and four asymptomatic—samples. The amplified products were analysed by agarose gel electrophoresis. From all producing one-band amplicons, 36 primer pairs amplifying translated, and 36 primers amplifying promoter regions were selected. Our study is the first step of a SNP discovery and marker development test. The further marker development on these sequences could be useful for the selection of ash individuals with various resistance to viral infections, providing a route for a more thorough understanding of the ash dieback–virus resistance interaction.