Transcriptomic Analysis and Specific Expression of Transcription Factor Genes in the Root and Sporophyll of Dryopteris fragrans (L.) Schott.

Background: Dryopteris fragrans, which is densely covered with glandular trichomes, is considered to be one of the ferns with the most medicinal potential. The transcriptomes from selected tissues of D. fragrans were collected and analyzed for functional and comparative genomic studies. The aim of this study was to determine the transcriptomic characteristics of wild D. fragrans sporangium in tissues from the SR (root), SL (sporophyll), and TRL (sporophyll with glandular trichomes removed). Results: Cluster analysis identified genes that were highly expressed in an organ-specific manner according to read mapping, feature counting, and normalization. The functional map identified gene clusters that can uniquely describe the function of each tissue. We identified a group of three tissue-specific transcription factors targeting the SL, SR, and TRL. In addition, highly expressed transcription factors (TFs) were found in each tissue-specific gene cluster, where ERF and bHLH transcription factors were the two types showing the most distinct expression patterns between the three different tissues. The specific expression of transcription factor genes varied between the different types of tissues. The numbers of transcription factors specifically expressed in the roots and sporophylls were 60 and 30, respectively, while only seven were found for the sporophylls with glandular trichomes removed. The expression of genes known to be associated with the development of glandular trichomes in flowering plants, including MIXTA, ATML1, and MYB106, were also validated and are discussed. In particular, a unigene encoding MIXTA was identified and exhibited the highest expression level in SL in D. fragrans. Conclusions: This study is the first report of global transcriptomic analysis in different tissues of D. fragrans, and the first to discuss these findings in the context of the development of homologous glandular trichomes. These results set the stage for further research on the development, stress resistance, and secondary metabolism of D. fragrans glandular trichomes.

Complete chloroplast genome sequence of Dryopteris fragrans (L.) Schott and the repeat structures against the thermal environment.

Dryopteris fragrans (L.) Schott is a fern growing on the surface of hot rocks and lava. It is exposed to sunlight directly and bears local hot environment. We sequenced the complete nucleotide sequence of its chloroplast (cp) genome. The cp genome was 151,978 bp in length, consisting of a large single-copy region (85,332 bp), a small single-copy region (31,947 bp) and a pair of inverted repeats (17,314 bp). The cp genome contained 112 genes and 345 RNA editing sites in protein-coding genes. Simple sequence repeats (SSRs) and long repeat structure pairs (30-55 bp) were identified. The number and percent of repeat structures are extremely high in ferns. Thermal denaturation experiments showed its cp genome to have numerous, dispersed and high GC percent repeat structures, which conferred the strongest thermal stability. This repeat-heavy genome may provide the molecular basis of how D. fragrans cp survives its hot environment.

Global transcriptome analysis and characterization of Dryopteris fragrans (L.) Schott sporangium in different developmental stages.

BACKGROUND: Dryopteris fragrans (D. fragrans) is a potential medicinal fern distributed in volcanic magmatic rock areas under tough environmental condition. Sporangia are important organs for fern reproduction. This study was designed to characterize the transcriptome characteristics of the wild D. fragrans sporangia in three stages (stage A, B, and C) with the aim of uncovering its molecular mechanism of growth and development. RESULTS: Using a HiSeq 4000, 79.81 Gb clean data (each sample is at least 7.95 GB) were obtained from nine samples, with three being supplied from each period, and assembled into 94,705 Unigenes, among which 44,006 Unigenes were annotated against public protein databases (NR, Swiss-Prot, KEGG, COG, KOG, GO, eggNOG and Pfam). Furthermore, we observed 7126 differentially expressed genes (DEG) (Fold Change > 4, FDR < 0.001), 349,885 SNP loci, and 10,584 SSRs. DEGs involved in DNA replication and homologous recombination were strongly expressed in stage A, and several DEGs involved in cutin, suberin and wax biosynthesis had undergone dramatic changes during development, which was consistent with morphological observations. DEGs responsible for secondary metabolism and plant hormone signal transduction changed clearly in the last two stages. DEGs homologous to those known genes associated with the development of reproductive organs of flowering plants have also been validated and discussed, such as AGL61, AGL62, ONAC010. In particular, a Unigene encoding TFL1, an important flower-development regulator in flowering plants, was identified and exhibited the highest expression level in stage B in D. fragrans sporangia. CONCLUSIONS: This study is the first report on global transcriptome analysis in the development of sporangia of wild D. fragrans. DEGs related to development and homologous to flower-seed development in flowering plants were discussed. All DEGs involved in DNA replication and homologous recombination were consistent with morphological observations of paraffin slices. The results of this study provide rare resources for further investigation of the D. fragrans sporangium development, stress resistance and secondary metabolism.

Seasonal dynamics of total flavonoid contents and antioxidant activity of Dryopteris erythrosora.

The seasonal dynamics of the total flavonoid contents in various parts of Dryopteris erythrosora, a traditional Chinese medicinal fern, and their antioxidant activity were investigated. The total flavonoids content in various parts of D. erythrosora showed an obvious seasonal dynamic change. The total flavonoid contents in stems (from 4.3% to 12.5%) were much higher than that in leaves with an average content of 2.01%. In spring, the total flavonoid contents in stems were relatively low, but increased rapidly from summer to winter. However, the seasonal dynamics of total flavonoid contents in leaves showed different model. The total flavonoid contents in the stems showed a negative correlation with that in the leaves from January to July. The correlation coefficient of about -0.7 was obtained. The antioxidant activity of the extracts also altered in proportion to the change of total flavonoid contents. In general, the extracts from stems always showed highest antioxidant potentials and it was suggested that the stems can be used as crude medicine.

Microsatellites reveal substantial among-population genetic differentiation and strong inbreeding in the relict fern Dryopteris aemula.

BACKGROUND AND AIMS: A previous study detected no allozyme diversity in Iberian populations of the buckler-fern Dryopteris aemula. The use of a more sensitive marker, such as microsatellites, was thus needed to reveal the genetic diversity, breeding system and spatial genetic structure of this species in natural populations. METHODS: Eight microsatellite loci for D. aemula were developed and their cross-amplification with other ferns was tested. Five polymorphic loci were used to characterize the amount and distribution of genetic diversity of D. aemula in three populations from the Iberian Peninsula and one population from the Azores. KEY RESULTS: Most microsatellite markers developed were transferable to taxa close to D. aemula. Overall genetic variation was low (H(T) = 0.447), but was higher in the Azorean population than in the Iberian populations of this species. Among-population genetic differentiation was high (F(ST) = 0.520). All loci strongly departed from Hardy-Weinberg equilibrium. In the population where genetic structure was studied, no spatial autocorrelation was found in any distance class. CONCLUSIONS: The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy-Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa.

Reproductive and competitive interactions among gametophytes of the allotetraploid fern Dryopteris corleyi and its two diploid parents.

BACKGROUND AND AIMS: Several models predict that the establishment of polyploids within diploid populations is enhanced by non-random mating (i.e. selfing and assortative mating) of cytotypes and by a higher relative fitness of polyploids. This report assesses the role that antheridiogens (i.e. maleness-inducing pheromones) and intercytotype differences in growth rate have on polyploid performance. METHODS: Three buckler-fern species were studied: the allotetraploid Dryopteris corleyi and its diploid parents, D. aemula and D. oreades. In one experiment, gametophytes of these species were cultured under rich growth conditions to compare the timing of gametangia production. The substrata on which these gametophytes had grown were used as antheridiogen sources in a second experiment. The three species were combined as source and target of antheridiogen (i.e. nine species pairs). Timing of antheridia production and gametophyte size were determined after those antheridiogen treatments. KEY RESULTS: Under rich growth conditions the allotetraploid produced archegonia earlier than those of diploid parents. Female gametophytes of the three species produced antheridiogens that inhibited growth and favoured maleness both within and among species. Gametophyte size was similar in the three species but antheridia formed earlier in the allotetraploid. CONCLUSIONS: Unisexuality, promoted by non-specific antheridiogens, enhances random mating both within and among species. The resulting hybridization can favour the reproductive exclusion of the allopolyploid in sites where it is outnumbered by diploids. However, the earlier production of gametangia in the allotetraploid favours assortative mating and may thus counterbalance reproductive exclusion.

Microsite-limited recruitment controls fern colonization of post-agricultural forests.

Assessing the relative roles of dispersal limitation and environmental effects in population dynamics and community assembly is fundamental to understanding patterns of species distribution and diversity. In forests growing on abandoned agricultural lands, both legacies of vegetation disturbance and changes in the abiotic environment shape the diversity and composition of recovering communities. Here I specify how interactions among historical, environmental, and biological factors influence species distributions, focusing on three fern species with contrasting distributions across forests of different history in central New York, USA: Dryopteris carthusiana, Dryopteris intermedia, and Polystichum acrostichoides. Using population surveys, spore-trap and spore-bank studies, and a three-year field experiment, I compare demographic rates among species and between forest types to determine which life history stages limit colonization and which traits explain species distributions. Adult plants of all three species were larger and more likely to produce spores in post-agricultural forests than in adjacent, uncleared stands. Though lower population densities led to fewer spores in post-agricultural soils, spore availability still exceeded recruitment by four to five orders of magnitude. Sowing additional spores had relatively little effect, while microhabitat conditions had the greatest impact on establishment rates. Given similar microsites, the two forest types had equal rates of establishment, but some forest-floor features preferentially occupied by juvenile plants were less frequent in post-agricultural stands. The availability of suitable sites for establishment, created by small-scale heterogeneity on forest floors, thus limits both the growth of fern populations and the colonization of new habitats. In fact, reduced microtopographic variation in post-agricultural forests may represent a greater hindrance to plant establishment than changes in mean environmental conditions. Among the three fern species, establishment rates differed as species distributions would predict, with the strongest colonizer consistently having the highest rates and the slowest colonizer the lowest. Rather than random or trait-mediated dispersal, the different distributions of these species reflect life history traits that determine establishment rates and thus colonization ability. This case study demonstrates that ecological interactions based on the unique life histories of individual species can override dispersal in determining species distributions.

Exogenous and endogenous growth regulators on apogamy in Dryopteris affinis (Lowe) Fraser-Jenkins sp. affinis.

This work showed for the first time the relationship between the effect of exogenous auxins and gibberellins on apogamy in Dryopteris affinis (Lowe) Fraser-Jenkins sp. affinis and its endogenous contents during early apogamic events. The addition of NAA (0.53 and 5.37 microM) or GA(3) 2.8 microM to an MS solid medium significantly increased apogamous sporophyte formation. BA induced brown callus that regenerated sporophytes in a hormone-free medium. The endogenous contents of GA(1), GA(3), GA(4), GA(7), GA(9) and IAA were determined by GC-MS in gametophytes cultured on MS solid medium, before and during early stages of apogamous embryo development. The accumulation of both GA(9) and IAA before embryo development was evident as high levels of GA(4) in the earliest analysed stage of embryo development and high levels of GA(3) in elongating shoots were found. The role of gibberellins on apogamy was also supported by data showing a decrease in the percentage of gametophytes developing embryos because of the addition of flurprimidol to the culture medium.

Preprophase band loses its function as a cytokinetic apparatus in mitosis of neck canal mother cell.

Preprophase bands in the neck canal mother cell and the central cell of the archegonium of the fern Dryopteris crassirhizoma are observed with immunofluorescence microscopy. No phragmoplast is found during mitosis of the neck canal mother cell; however, the phragmoplast develops very well in the central cell. The neck canal mother cell undergoes karyokinesis but not cytokinesis and finally produces only one binucleate neck canal cell. However, the central cell undergoes cytokinesis and produces an egg cell and a ventral canal cell. These observations suggest that the preprophase band in the neck canal mother cell loses its function as a cytokinetic apparatus and becomes an evolutionary vestige in the development of the archegonium.

Effect of storage method on spore viability in five globally threatened fern species.

Spore germination of five globally threatened fern species [Culcita macrocarpa C. Presl, Dryopteris aemula (Aiton) O. Kuntze, D. corleyi Fraser-Jenkins, D. guanchica Gibby and Jermy and Woodwardia radicans (L.) Sm.] was determined after 1, 6 or 12 months of storage in glass vials (dry storage) or on agar (wet storage) at -20, 5 or 20 degrees C. In all species, storage technique, storage temperature and the technique-temperature interaction all had a significant effect on germination percentage. In most cases, the germination percentage was best maintained by wet storage at 5 or 20 degrees C. In the case of the hygrophilous species C. macrocarpa and W. radicans, 6 or 12 months' dry storage killed most spores. Only Woodwardia radicans germinated in the dark during wet storage at 20 degrees C. Wet storage at 5 degrees C prevented dark germination, and reduced bacterial and fungal contamination. Wet storage at -20 degrees C killed all or most spores in all species. In the three Dryopteris species, the differences among the storage conditions tested were smaller than in C. macrocarpa and W. radicans, and the decline in spore viability during storage was less marked, with high germination percentages being observed after 12 months of dry storage at all three temperatures. Dry storage, which has lower preparation time and space requirements than wet storage, was generally more effective at the lower temperatures (-20 or 5 degrees C).