Background: Hard fescue (Festuca brevipila Tracey, 2n=6x=42) is a cool season turfgrass with a ne leaf texture that performs well under low-input management. Breeding and genetics studies of F. brevipila have been limited due to the complexity of its hexaploid genome. To advance our knowledge of F. brevipila genomics, we used PacBio isoform sequencing to develop a reference transcriptome for this species. Results: Here, we report the F. brevipila reference transcriptome generated from root, crown, leaf, and in orescence tissues. We obtained 59,510 full-length transcripts, of which 38,556 were non-redundant full-length transcripts. The longest and shortest transcripts were 11,487 and 58 bp, respectively. Distribution of synonymous distances among paralogs within F. brevipila suggested highly similar subgenomes that are di cult to distinguish from sequencing errors. To evaluate the phylogenetic relationships among F. brevipila and close relatives, we sequenced three additional transcriptomes using closely related species on an Illumina platform. The results of our phylotranscriptomic analysis supported the close relationships among F. brevipila (6x), Festuca ovina (4x), Festuca ovina subsp. ovina (2x), and Festuca valesiaca (2x), with high levels of discordance among gene trees. Conclusions: Overall, the F. brevipila PacBio Isoseq reference transcriptome provided the foundation for transcriptome studies and allowed breeders a resource for gene discovery in this important turfgrass species.

Festuca ovina is a fine fescue that is used as a low-input turfgrass. The ploidy 11 levels of F. ovina accessions held by the USDA National Plant Germplasm System (NPGS) are 12 unknown, limiting the use of the germplasm in breeding programs. The objective of this study 13 was to determine DNA content and estimate ploidy of these 127 accessions. Among the 14 accessions, we identified a wide range of ploidy levels from diploid to octoploid. We also found 15 the accessions with higher ploidy levels usually had larger seed size. These results will be 16 informative to plant breeders and researchers using germplasm from the F. ovina collection and 17 point to challenges in maintaining polyploid, outcrossing germplasm seed stocks in common 18 nurseries. 20Fine fescues (Festuca spp. L.) are a diverse group of grasses characterized by fine leaf 22 texture. Fine fescues are native to Eurasia but have been introduced and naturalized to many 23