A reference transcriptome for the early-branching fern Botrychium lunaria enables fine-grained resolution of population structure

Abstract Ferns are the second most dominant group of land plants after angiosperms. Extant species occupy an extensive range of habitats and contribute significantly to ecosystem functioning. Despite the importance of ferns, most taxa are poorly covered by genomic resources. The genus Botrychium belongs to one of the earliest divergent lineages of vascular plants and has a global distribution with 35 species, half of which are polyploids. Here, we establish a reference transcriptome for Botrychium lunaria, a diploid species with an extremely large genome of 27.5 Gbp. We assembled 25,721 high-quality transcripts with an average length of 1,332 bp based on deep RNA-sequencing of a single individual. We sequenced an additional 11 transcriptomes of individuals from two populations in Switzerland, including the population of the reference individual. Based on read mapping to reference transcript sequences, we identified 374,510 single nucleotide polymorphisms (SNPs) segregating among individuals for an average density of 14 SNPs per kb. The transcriptome-wide markers provided unprecedented resolution of the population genetic structure revealing substantial variation in heterozygosity among individuals. We also constructed a phylogenomic tree of 73 taxa representating all fern orders to ascertain the placement of the genus Botrychium. The high-quality transcriptomic resources enable powerful population and phylogenomic studies in an important group of ferns. Significance statementFerns pose substantial puzzles in terms of lifestyles, genome organization and population structure. Progress has been significantly hampered by the lack of genomic resources. Here, we present a reference transcriptome for Botrychium lunaria, a phylogenetically early-branching fern with an extremely large genome of 27.5 Gbp. We show that the new transcriptome improves phylogenetic resolution among early-branching ferns. Based on an additional 11 transcriptomes of the same species, we identify unexpected variation in population-level heterozygosity.