Genomic information is critical to elucidating the evolutionary history and the genetic basis of diverse traits. However, despite over 300 plant genomes having been published, the majority of them were from seed plants and very little is known about seed-free plants. Our group is leading the effort to not only close this large “genome gap”, but also leverage omics data to understand the unique biology of seed-free plants. To date, we have completed the first genomes for ferns, hornworts, and Isoetales, and have more in the pipeline.

Ferns. In 2018, our group led an international collaboration to complete the first genomes of ferns (Li et al., 2018). These genomes, from Azolla and Salvinia, not only provided an exciting glimpse into fern genome structure and evolution, but also clarified the origins of gene families and pathways that coincide with the emergence of seed plants. We have continued to play a lead role in advancing fern genomics. This year we published the genome of the tree fern Alsophila spinulosa (Huang et al., 2022). This genome is notable because Alsophila is homosporous (in contrast to the heterosporous Azolla and Salvinia) and the size, at 6 Gb, is more typical of ferns. We discovered a remarkably preserved synteny, despite resulting from an ancient whole-genome duplication over 100 million years ago. Such a slow rate of genome fractionation is unprecedented and might explain the large genome sizes found in ferns. In parallel, we have also been working closely with Open Green Genomics (a JGI-funded project) and aim to have four more fern genomes completed by the end of 2022.

Lycophytes. There are three extant and deeply diverged lineages in lycophytes: Selaginellales, Isoetales, and Lycopodiales. For years, genomic data had only been available for Selaginella in Selaginellales. Recently, we made important progress in lycophyte genomics with the completion of Isoetes taiwanensis (Isoetales) genome (Wickell et al., 2021). Our interest in I. taiwanensis is primarily in its Crassulacean acid metabolism (CAM) photosynthesis, which is unusual given the aquatic lifestyle. From our time-course RNA-sequencing data, we found that I. taiwanensis may have recruited the lesser-known “bacterial-type” phosphoenolpyruvate (PEP) carboxylase, along with the “plant-type” exclusively used in other CAM and C4 plants for carboxylation of PEP. Our findings suggest the existence of more evolutionary paths to CAM than previously recognized. For the remaining order Lycopodiales, we have finalized the genomes of Diphasiastrum complanatum and Huperzia asiatica, which, together with those of Selaginella and Isoetes, will give us a much more holistic view of the lycophyte genomic landscape.

Hornworts. Hornworts are one of the three lineages of bryophytes, whose phylogenetic position among land plants has been highly debated. In 2020 we published the first hornwort genomes from two Anthoceros species (Li et al., 2020). Our genomic analyses provided support for the monophyly of bryophytes and help address several long-standing questions on early land plant evolution. Since then, we have massively expanded the sampling, with 10 new high-quality genomes from every hornwort family—hornworts are now the best sampled plant phylum to date. This genomic framework is paving the way to establish hornworts as a new model lineage.

Collections of cultures and genomes. We have established a diverse hornwort culture collection that includes 24 isolates from 10 genera representing all 5 families, some of which have already been fully sequenced. The cultures are axenic and derived from a single spore or thallus. Importantly, this culture collection contains 6 pyrenoid-present and 8 pyrenoid-absent species, which together cover 3 independent transitions over the hornwort phylogeny. In addition, Peter Schafran (NSF Plant Genome Postdoc) is now expanding this collection with separate male/female isolates to study hornwort sex chromosomes. 

Genetic tools. With funding from the NSF EDGE program (to me and Joyce Van Eck at BTI), we have been developing gene delivery and gene editing methods. In collaboration with Eftychis Frangedakis (Cambridge), Keiko Sakakibara (Tokyo), and Peter Szövényi (Zurich), we published the first Agrobacterium-mediated transformation protocol for hornworts (Frangedakis et al. 2021). In parallel, we have been testing a biolistic delivery method, which we believe is more efficient and better suited for CRISPR-Cas9 gene-editing. In one bombardment, we can now reliably obtain ~900 transiently transformed events from 0.3 g of tissue. CRISPR mutagenesis experiments are also underway. Finally, a new growth medium for hornworts was developed that yields >4X tissue mass compared to the original formula (Gunadi et al., 2022).

With these resources and tools in hand, we are delving into two unique features of hornworts: cyanobacteria symbiosis and pyrenoid-based carbon-concentrating mechanism. 

Plant symbiosis with nitrogen-fixing cyanobacteria is a unique form of mutualistic association that has independently evolved in diverse lineages including a few species of bryophytes, ferns, cycads, and one small genus of flowering plants. Compared to other nitrogen-fixing microbes, cyanobacteria are generally less dependent on the plant host, and therefore could be an ideal partner for engineering symbiotic nitrogen fixation into crop plants. However, our current understanding of plant-cyanobacteria symbioses is rudimentary. The phylogenetic diversity of cyanobionts has been largely unexplored, and very few studies have investigated variation in the symbiotic interaction. In addition, most genetic research has solely focused on the model cyanobiont Nostoc punctiforme, and the plant genes involved in symbiosis remain unknown.