Diversity of genome size, endopolyploidy and SCoT markers in 20 Trigonella (Fabaceae) species.

The Trigonella species possess medicinal, nutraceutical and pharmaceutical properties due to the presence of many bioactive compounds. Its therapeutic effects are mostly valuable in medicine, cosmetics and the functional food industry. Correct genetic characterisation of plant material is needed to increase the potential of Trigonella species by breeding and conservation programs. The aim of this study was to develop a reliable marker system to support the morphological and phytochemical analysis in Trigonella taxonomic research, species identification and characterization as well as determination of the interspecific variation within this genus along with relationships between species. For this purpose, flow cytometry and SCoT molecular markers were combined. Flow cytometric analyses revealed that Trigonella species possess very small and small genomes. The range of genome sizes was from 1.10 to 5.76 pg/2C, with most species possessing very small genomes (< 2.8 pg/2C). In seeds of 14 species endopolyploid nuclei were detected. Flow cytometric analysis of genome size enabled quick identification of four out of 20 species, while combined with endopolyploidy detection in seeds, facilitated distinction of the next seven species. ScoT molecular markers helped to identify closely related species with similar genome size and cell cycle activity. Therefore, flow cytometry was proposed as the first-choice method for quick accession screening, while the more detailed genetic classification was obtained using SCoT molecular markers.

Taxonomy and distribution of Taraxacum sect. Erythrosperma (Asteraceae) in Poland.

The dandelions from Taraxacumsect.Erythrosperma are taxonomically well distinguished and ecologically restricted to warm and sunlit habitats of steppes, dry and sandy grasslands, and distributed in temperate regions of Europe and Central Asia, with some being introduced to North America. Despite the long tradition of botanical research, the taxonomy and distribution of dandelions of T.sect.Erythrosperma is still underexplored in central Europe. In this paper, by combining traditional taxonomic studies supported by micromorphological, molecular and flow cytometry analyses as well as potential distribution modelling we shed light on taxonomical and phylogenetical relationships between members of T.sect.Erythrosperma in Poland. We also provide an identification key, species-checklist, detailed descriptions of morphology and occupated habitats as well as distribution maps for 14 Polish erythrosperms (T.bellicum, T.brachyglossum, T.cristatum, T.danubium, T.disseminatum, T.dissimile, T.lacistophyllum, T.parnassicum, T.plumbeum, T.proximum, T.sandomiriense, T.scanicum, T.tenuilobum, T.tortilobum). Finally, conservation assessments performed using the IUCN method and threat categories for all the examined species are proposed.

Chromosome Number and Genome Size Evolution in Brasolia and Sobralia (Sobralieae, Orchidaceae).

Despite the clear circumscription of tribe Sobralieae (Orchidaceae), its internal relationships are still dubious. The recently delimited genus Brasolia, based on previous Sobralia species, is now assumed to be paraphyletic, with a third genus, Elleanthus, nested in it. The morphology of these three genera is significantly different, indicating the necessity of new data for a better genera delimitation. Though morphology and molecular data are available, cytogenetics data for Sobralieae is restricted to two Sobralia and one Elleanthus species. Aiming to evaluate the potential of cytogenetic data for Brasolia-Elleanthus-Sobralia genera delimitation, we present chromosome number and genome size data for 21 and 20 species, respectively, and used such data to infer the pattern of karyotype evolution in these genera. The analysis allowed us to infer x = 24 as the base chromosome number and genome size of average 1C-value of 5.0 pg for the common ancestor of Brasolia-Elleanthus-Sobralia. The recurrent descending dysploidy in Sobralieae and the punctual genome upsize suggest a recent diversification in Sobralieae but did not allow differing between Brasolia and Sobralia. However, the basal position of tribe Sobralieae in the subfamily Epidendroideae makes this tribe of interest to further studies clarifying the internal delimitation and pattern of karyotype evolution.

Tetraploids expanded beyond the mountain niche of their diploid ancestors in the mixed-ploidy grass Festuca amethystina L.

One promising area in understanding the responses of plants to ongoing global climate change is the adaptative effect of polyploidy. This work examines whether there is a coupling between the distribution of cytotypes and their biogeographical niche, and how different niches will affect their potential range. The study uses a range of techniques including flow cytometry, gradient and niche analysis, as well as distribution modelling. In addition, climatic, edaphic and habitat data was used to analyse environmental patterns and potential ranges of cytotypes in the first wide-range study of Festuca amethystina-a mixed-ploidy mountain grass. The populations were found to be ploidy homogeneous and demonstrate a parapatric pattern of cytotype distribution. Potential contact zones have been identified. The tetraploids have a geographically broader distribution than diploids; they also tend to occur at lower altitudes and grow in more diverse climates, geological units and habitats. Moreover, tetraploids have a more extensive potential range, being six-fold larger than diploids. Montane pine forests were found to be a focal environment suitable for both cytotypes, which has a central place in the environmental space of the whole species. Our findings present polyploidy as a visible driver of geographical, ecological and adaptive variation within the species.

Genome Size Diversity in Rare, Endangered, and Protected Orchids in Poland.

Orchidaceae is one of the largest and the most widespread plant families with many species threatened with extinction. However, only about 1.5% of orchids' genome sizes have been known so far. The aim of this study was to estimate the genome size of 15 species and one infraspecific taxon of endangered and protected orchids growing wild in Poland to assess their variability and develop additional criterion useful in orchid species identification and characterization. Flow cytometric genome size estimation revealed that investigated orchid species possessed intermediate, large, and very large genomes. The smallest 2C DNA content possessed Liparis loeselii (14.15 pg), while the largest Cypripedium calceolus (82.10 pg). It was confirmed that the genome size is characteristic to the subfamily. Additionally, for four species Epipactis albensis, Ophrys insectifera, Orchis mascula, Orchis militaris and one infraspecific taxon, Epipactis purpurata f. chlorophylla the 2C DNA content has been estimated for the first time. Genome size estimation by flow cytometry proved to be a useful auxiliary method for quick orchid species identification and characterization.

The transferability of microsatellite loci from a homoploid to a polyploid hybrid complex: an example from fine-leaved Festuca species (Poaceae).

BACKGROUND: Microsatellite loci, or single sequence repeats (SSR), are widely used as powerful markers in population genetics. They represent an attractive tool for studying plants such as grasses, whose evolution is driven by hybridisation and polyploidization. However, the development of microsatellite markers has been challenging and time-consuming, especially for non-model organisms lacking available genome-wide sequence data. One straightforward and low-cost approach is to transfer the SSR loci developed for one species, or complex, to another closely-related one. This work evaluates the transferability of microsatellite loci from homoploid to allopolyploid complexes of fine-leaved Festuca species and to assess their use in two new species. The studied complex (F. amethystina-F. tatrae) is a useful model for research on the local adaptability of grasses with different ploidy levels. Since both species can be considered as rare or threatened (F. tatrae-as a mountain and narrow endemic species and F. amethystina-a mountain species with relict lowland populations), any tool enabling studies on genetic diversity and population genetics, such as SSR markers, could also be very useful in a conservation context. METHODS: The ploidy level within populations was estimated using flow cytometry. One diploid and one tetraploid population of F. amethystina and a diploid population of F. tatrae were chosen to test the transferability of SSR loci. Because our work describes the transfer of SSR nuclear markers designed originally for F. gautieri, a phylogenetic tree was prepared based on the ITS marker to assess the genetic distance between the studied complexes. The PCR products were separated on a high-resolution agarose gel, intended for SSR marker analysis. Appropriate solutions for the allotetraploid population and whole mixed-ploidy complex were implemented. RESULTS: Flow cytometry confirmed earlier data regarding DNA content in the investigated species and cytotypes. The phylogenetic ITS tree indicated a small genetic distance between F. gautieri complexes and the studied species. Ten microsatellite markers were successfully transferred. All markers were polymorphic. In total, 163 different alleles were scored from the 10 SSR loci. PCoA of accessions revealed well-separated groups corresponding to studied populations. Over 60% of the total variance is explained by differentiation within populations and one third among them. CONCLUSIONS: The transferred markers are valid tools for the study of population genetics and inheritance relationships within cytotypes and species and between them. The presented markers can be used to study inbreeding depression in the Festuca species, and variations in the degrees of genetic diversity between different cytotypes in mountain and lowland areas. Our findings can also be applied to study conservation strategies for ensuring biodiversity at the genetic level in polyploid complexes.

Field cress genome mapping: Integrating linkage and comparative maps with cytogenetic analysis for rDNA carrying chromosomes.

Field cress (Lepidium campestre L.), despite its potential as a sustainable alternative oilseed plant, has been underutilized, and no prior attempts to characterize the genome at the genetic or molecular cytogenetic level have been conducted. Genetic maps are the foundation for anchoring and orienting annotated genome assemblies and positional cloning of candidate genes. Our principal goal was to construct a genetic map using integrated approaches of genetic, comparative and cytogenetic map analyses. In total, 503 F2 interspecific hybrid individuals were genotyped using 7,624 single nucleotide polymorphism markers. Comparative analysis demonstrated that ~57% of the sequenced loci in L. campestre were congruent with Arabidopsis thaliana (L.) genome and suggested a novel karyotype, which predates the ancestral crucifer karyotype. Aceto-orcein chromosome staining and fluorescence in situ hybridization (FISH) analyses confirmed that L. campestre, L. heterophyllum Benth. and their hybrids had a chromosome number of 2n = 2x = 16. Flow cytometric analysis revealed that both species possess 2C roughly 0.4 picogram DNA. Integrating linkage and comparative maps with cytogenetic map analyses assigned two linkage groups to their particular chromosomes. Future work could incorporate FISH utilizing A. thaliana mapped BAC clones to allow the chromosomes of field cress to be identified reliably.

Micropropagation of Chaenomeles japonica: A Step towards Production of Polyphenol-rich Extracts Showing Antioxidant and Antimicrobial Activities.

A protocol for C. japonica micropropagation with a confirmation of genome size stability of the in vitro-propagated plantlets was developed. The highest number of shoots multiplied in vitro was obtained on Murashige & Skoog medium (MS) with 1.0 mg L-1 N6-benzyladenine plus 1.0 mg L-1 indole-3-acetic acid. The highest number of roots was observed for the shoots on MS with 15 g L-1 sucrose plus 1.0 mg L-1 indole-3-acetic acid. The acclimatization rate was significantly high. The qualitative HPLC analyses confirmed the presence of phenolic acids and flavonoids in the extracts. The extracts from both shoot cultures and the leaves from field-grown plants revealed antioxidant activity and they exhibited moderate antimicrobial activity. The conducted research confirmed the regeneration potential of genetically-stable plants of C. japonica under in vitro conditions, the ability of the plantlets to produce polyphenols as those present in field-grown plants, as well as their antioxidant potential.

DNA synthesis pattern, proteome, and ABA and GA signalling in developing seeds of Norway maple (Acer platanoides).

Mature seeds of Norway maple exhibit desiccation tolerance and deep physiological dormancy. Flow cytometry, proteomics, and immunodetection have been combined to investigate seed development of this species. DNA content analysis revealed that cell cycle/endoreduplication activity differs between seed organs and developmental stages. In the embryo axis, the proportion of the nuclei with the highest DNA content (4C) increases at the beginning of maturation (17 weeks after flowering; WAF), and then is stable until the end of maturation, to increase again after drying. In cotyledons, during maturation endopolyploid nuclei (8C) occur and the intensity of endoreduplication increases up to 21 WAF, and then is stable until development is completed. In dry mature seeds, the proportion of 4C nuclei is high, and reaches 36% in the embryo axis and 52% in cotyledons. Proteomic studies revealed that energy and carbon metabolism, fatty acid biosynthesis, storage and antioxidant proteins are associated with seed development. Study of the ABI5 protein, a transcription factor involved in ABA signalling, and the RGL2 protein, a repressor of the GA signalling indicates that the highest accumulation of these proteins occurs in fully-matured and dried seeds. It is suggested that this increase in accumulation can be associated with completion of maturation, mainly with desiccation and dormancy acquisition.

Morphometric traits in the fine-leaved fescues depend on ploidy level: the case of Festuca amethystina L.

BACKGROUND: Polyploid specimens are usually characterized by greater exuberance: they reach larger sizes and/or have a larger number of some organs. Festuca amethystina L. belongs to the section Aulaxyper. Based on morphological features, four subspecies of F. amethystina have been already identified. On the other hand, it has two cytotypes: diploid and tetraploid. The main aim of our study was to distinguish morphological differences between the cytotypes of F. amethystina, assuming that its phenotype differs significantly. METHODS: The nuclear DNA content was measured by flow cytometry in dry leaves from specimens originating from 13 populations of F. amethystina. Several macrometric and micrometric traits of stems, spikelets and leaf blades were taken into account in the comparative analysis of two cytotypes. RESULTS: In the case of cytotypes, specimens of tetraploids were larger than diploids. The conducted morphometric analysis of leaf cross-sections showed significant differences between the cytotypes. DISCUSSION: The research has confirmed for the first time that in the case of F. amethystina the principle of greater exuberance of polyploids is true. Differences between the cytotypes are statistically significant, however, they are not enough to make easy the distinction of cytotypes on the basis of the measurements themselves. Our findings favor the rule known in Festuca taxonomy as a whole, i.e. that the ploidy level can be one of the main classification criteria.

Morphology and genome size of Epipactis helleborine (L.) Crantz (Orchidaceae) growing in anthropogenic and natural habitats.

BACKGROUND: The process of apophytism or spreading native species to human-made habitats is one of the main elements in the creation of plant cover in anthropogenic areas. Lately, an increase of anthropogenic localities with valuable flora has been observed. Apophytes are also members of the family Orchidaceae, especially from the genus Epipactis. The aim of the study was to (i) determine and compare the phenotypic variation of E. helleborine (L.) Crantz plants in anthropogenic and natural habitats, (ii) compare the genome size of plants growing in natural and anthropogenic habitats. The results reported in this study may indicate that a habitat influences morphological characteristics of plant species. METHODS: Field studies were conducted on four native stands and four stands in anthropogenic areas of E. helleborine in Poland in years 2011-2013. Biometrical analyses were performed on shoots and flowers. The flowers were characterised by 25 biometric features and measured using a Nikon SMZ 800 binocular, microscopic Moticam-1SP cameras and the MIPlus07 programme (Conbest Co.). The nuclear DNA content was determined in fresh and young leaves of E. helleborine, collected from four natural and four anthropogenic populations. RESULTS: We observed that in anthropogenic populations: (i) shoots were higher than shoots from natural populations, (ii) flowers differed significantly in terms of ten biometric features between habitats, (iii) the genome size of some population differed significantly between plants growing in natural and anthropogenic habitats. DISCUSSION: According to some researchers, the presence of phenotypic variability and the occurrence of ecotypes are adaptation strategies of plants to environmental changes. In our opinion, in the case of the studied anthropogenic habitats (roadside) in which the E. helleborine populations grew, we can talk about ecofen due to the often repeated set of characteristic features, i.e., high shoots, long inflorescence and long, broad leaves. We agree, however, that it is difficult to isolate a taxonomic unit for ecofen due to the lack of experimental research.

Endoreduplication in the germinating embryo and young seedling is related to the type of seedling establishment but is not coupled with superoxide radical accumulation.

During germination, the embryo axis elongates and the radicle emerges through the surrounding structures of the seed. However, this elongation is not even along the axis, and it has been suggested that the region responsible for radicle protrusion is related to the type of subsequent seedling establishment. Eleven epigeal- and five hypogeal-type species were selected to study endoreduplication, a process coupled with cell elongation, in the radicle, hypocotyl-radicle transition zone, hypocotyl, and cotyledons of dry and germinating seeds, and in seedlings after radicle protrusion. Flow cytometry was used to establish the proportions of nuclei with different DNA contents, the mean C-value, and the (Σ>2C)/2C ratio. Additionally, a nitroblue tetrazolium chloride test was applied to the embryos/seedlings in the dry state and during and after germination to localize superoxide radical (O2(•-)) accumulation, which has been suggested to play a role in cell elongation. Endoreduplication intensity varied in different species, in the embryo/seedling regions, and with the type of seedling establishment. In most of the cases, it was highest in the transition zone of epigeal species and in the hypocotyl in hypogeal species. O2(•-) was invariably produced during germination in the radicle, and additionally in the transition zone at the time of radicle protrusion; thus, it was not coupled with endoreduplication, and most probably played a role in defence against biotic and abiotic environmental stresses. These results provide information to aid in the selection of the most suitable plant material for molecular research on germination and for monitoring seed priming.

Endoreduplication intensity as a marker of seed developmental stage in the Fabaceae.

Flow cytometry (FCM) can be used to study cell cycle activity in developing, mature and germinating seeds. It provides information about a seed's physiological state and therefore can be used by seed growers for assessing optimal harvest times and presowing treatments. Because an augmented proportion of 4C nuclei usually is indicative of high mitotic activity, the 4C/2C ratio is commonly used to follow the progress of seed development and germination. However, its usefulness for polysomatic (i.e., containing cells with different DNA content) seeds is questioned. Changes in cell cycle/endoreduplication activity in developing seeds of five members of the Fabaceae were studied to determine a more suitable marker of seed developmental stages for polysomatic species based on FCM measurements. Seeds of Phaseolus vulgaris, Medicago sativa, Pisum sativum, Vicia sativa, and Vicia faba var. minor were collected 20, 30, 40, 50, and 60 days after flowering (DAF), embryos were isolated and the proportion of nuclei with different DNA contents in the embryo axis and cotyledon was established. The ratios 4C/2C and (Σ>2C)/2C were calculated. Dried seeds were subjected to laboratory germination tests following international seed testing association (ISTA) rules. Additionally, the absolute nuclear DNA content was estimated in the leaves of the studied species. During seed development nuclei with DNA contents from 2C to 128C were detected; the endopolyploidy pattern depended on the species, seed organ and developmental stage. The cell cycle/endoreduplication parameters correlated negatively with genome size. The (Σ>2C)/2C ratio in the cotyledons reflected the seed developmental stage and corresponded with seed germinability. Therefore, this ratio is recommended as a marker in polysomatic seed research and production instead of the 4C/2C ratio, which does not consider the occurrence of endopolyploid cells.