Differential expression and evolutionary diversification of RNA helicases in Boechera sexual and apomictic reproduction.

In higher plants, sexual reproduction is characterized by meiosis of the first cells of the germlines, and double fertilization of the egg and central cell after gametogenesis. In contrast, in apomicts of the genus Boechera, meiosis is omitted or altered and only the central cell requires fertilization, while the embryo forms parthenogenetically from the egg cell. To deepen the understanding of the transcriptional basis underlying these differences, we applied RNA-seq to compare expression in reproductive tissues of different Boechera accessions. This confirmed previous evidence of an enrichment of RNA helicases in plant germlines. Furthermore, few RNA helicases were differentially expressed in female reproductive ovule tissues harboring mature gametophytes from apomictic and sexual accessions. For some of these genes, we further found evidence for a complex recent evolutionary history. This included a homolog of Arabidopsis thaliana FASCIATED STEM4 (FAS4). In contrast to AtFAS4, which is a single-copy gene, FAS4 is represented by three homologs in Boechera, suggesting a potential for subfunctionalization to modulate reproductive development. To gain first insights into functional roles of FAS4, we studied Arabidopsis lines carrying mutant alleles. This identified the crucial importance of AtFAS4 for reproduction, as we observed developmental defects and arrest during male and female gametogenesis.

RH17 restricts reproductive fate and represses autonomous seed coat development in sexual Arabidopsis.

Plant sexual and asexual reproduction through seeds (apomixis) is tightly controlled by complex gene regulatory programs, which are not yet fully understood. Recent findings suggest that RNA helicases are required for plant germline development. This resembles their crucial roles in animals, where they are involved in controlling gene activity and the maintenance of genome integrity. Here, we identified previously unknown roles of Arabidopsis RH17 during reproductive development. Interestingly, RH17 is involved in repression of reproductive fate and of elements of seed development in the absence of fertilization. In lines carrying a mutant rh17 allele, development of supernumerary reproductive cell lineages in the female flower tissues (ovules) was observed, occasionally leading to formation of two embryos per seed. Furthermore, seed coat, and putatively also endosperm development, frequently initiated autonomously. Such induction of several features phenocopying distinct elements of apomixis by a single mutation is unusual and suggests that RH17 acts in regulatory control of plant reproductive development. Furthermore, an in-depth understanding of its action might be of use for agricultural applications.

Gene Function Rather than Reproductive Mode Drives the Evolution of RNA Helicases in Sexual and Apomictic Boechera.

In higher plants, sexual and asexual reproductions through seeds (apomixis) have evolved as alternative strategies. Evolutionary advantages leading to coexistence of both reproductive modes are currently not well understood. It is expected that accumulation of deleterious mutations leads to a rapid elimination of apomictic lineages from populations. In this line, apomixis originated repeatedly, likely from deregulation of the sexual pathway, leading to alterations in the development of reproductive lineages (germlines) in apomicts as compared with sexual plants. This potentially involves mutations in genes controlling reproduction. Increasing evidence suggests that RNA helicases are crucial regulators of germline development. To gain insights into the evolution of 58 members of this diverse gene family in sexual and apomictic plants, we applied target enrichment combined with next-generation sequencing to identify allelic variants from 24 accessions of the genus Boechera, comprising sexual, facultative, and obligate apomicts. Interestingly, allelic variants from apomicts did not show consistently increased mutation frequency. Either sequences were highly conserved in any accession, or allelic variants preferentially harbored mutations in evolutionary less conserved C- and N-terminal domains, or presented high mutation load independent of the reproductive mode. Only for a few genes allelic variants harboring deleterious mutations were only identified in apomicts. To test if high sequence conservation correlates with roles in fundamental cellular or developmental processes, we analyzed Arabidopsis thaliana mutant lines in VASA-LIKE (VASL), and identified pleiotropic defects during ovule and reproductive development. This indicates that also in apomicts mechanisms of selection are in place based on gene function.

The low-copy nuclear gene Agt1 as a novel DNA barcoding marker for Bromeliaceae.

BACKGROUND: The angiosperm family Bromeliaceae comprises over 3.500 species characterized by exceptionally high morphological and ecological diversity, but a very low genetic variation. In many genera, plants are vegetatively very similar which makes determination of non flowering bromeliads difficult. This is particularly problematic with living collections where plants are often cultivated over decades without flowering. DNA barcoding is therefore a very promising approach to provide reliable and convenient assistance in species determination. However, the observed low genetic variation of canonical barcoding markers in bromeliads causes problems. RESULT: In this study the low-copy nuclear gene Agt1 is identified as a novel DNA barcoding marker suitable for molecular identification of closely related bromeliad species. Combining a comparatively slowly evolving exon sequence with an adjacent, genetically highly variable intron, correctly matching MegaBLAST based species identification rate was found to be approximately double the highest rate yet reported for bromeliads using other barcode markers. CONCLUSION: In the present work, we characterize Agt1 as a novel plant DNA barcoding marker to be used for barcoding of bromeliads, a plant group with low genetic variation. Moreover, we provide a comprehensive marker sequence dataset for further use in the bromeliad research community.