Aster spathulifolius Maxim. a leaf transcriptome provides an overall functional characterization, discovery of SSR marker and phylogeny analysis.

Aster spathulifolius Maxim. is belongs to the Asteraceae family, which is distributed only in Korea and Japan. The species is traditionally a medicinal plant and is economically valuable in the ornamental field. On the other hand, the Aster genus, among the Asteraceae family, lacks genomic resources and its molecular functions. Therefore, in our study the high-throughput RNA-sequencing transcriptome data of A. spathulifolius were obtained to identify the molecular functions and its characterization. The de novo assembly produced 98660 uniqueness with an N50 value of 1126bp. Total unigenes were procure to analyze the functional annotation against databases like non-redundant protein, Pfam, Uniprot, KEGG and Gene ontology. The overall percentage of functional annotation to the nr database (43.71%), uniprotein database (49.97%), Pfam (39.94%), KEGG (42.3%) and to GO (30.34%) were observed. Besides, 377 unigenes were found to be involved in the terpenoids pathway and 666 unigenes were actively engaged in other secondary metabolites synthesis, given that 261 unigenes were within phenylpropanoid pathway and 81 unigenes to flavonoid pathway. A further prediction of stress resistance (9,513) unigenes and transcriptional factor (3,027) unigenes in 53 types were vastly regulated in abiotic stress respectively in salt, heat, MAPK and hormone signal transduction pathway. This study discovered 29,692 SSR markers that assist the genotyping approaches and the genetic diversity perspectives. In addition, eight Asteraceae species as in-group together with one out-group were used to construct the phylogenetic relationship by employing their plastid genome and single-copy orthologs genes. Among 50 plastid protein-coding regions, A. spathulifolius is been closely related to A. annua and by 118 single copy orthologs genes, O. taihangensis is more neighboring species to A. spathulifolius. Apart from this, A. spathulifolius and O. taihangensis, genera have recently diverged from other species. Overall, this research gains new insights into transcriptome data by revealing and exposing the secondary metabolite compounds for drug development, the stress-related genes for producing resilient crops and an ortholog gene of A. spathulifolius for the robustness of phylogeny reconstruction among Asteraceae genera.

Complete Chloroplast Genome Sequence and Phylogenetic Analysis of Aster tataricus.

We sequenced and analyzed the complete chloroplast genome of Aster tataricus (family Asteraceae), a Chinese herb used medicinally to relieve coughs and reduce sputum. The A. tataricus chloroplast genome was 152,992 bp in size, and harbored a pair of inverted repeat regions (IRa and IRb, each 24,850 bp) divided into a large single-copy (LSC, 84,698 bp) and a small single-copy (SSC, 18,250 bp) region. Our annotation revealed that the A. tataricus chloroplast genome contained 115 genes, including 81 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. In addition, 70 simple sequence repeats (SSRs) were detected in the A. tataricus chloroplast genome, including mononucleotides (36), dinucleotides (1), trinucleotides (23), tetranucleotides (1), pentanucleotides (8), and hexanucleotides (1). Comparative chloroplast genome analysis of three Aster species indicated that a higher similarity was preserved in the IR regions than in the LSC and SSC regions, and that the differences in the degree of preservation were slighter between A. tataricus and A. altaicus than between A. tataricus and A. spathulifolius. Phylogenetic analysis revealed that A. tataricus was more closely related to A. altaicus than to A. spathulifolius. Our findings offer valuable information for future research on Aster species identification and selective breeding.

Unmasking cryptic biodiversity in polyploids: origin and diversification of Aster amellus aggregate.

Background and Aims: The origin of different cytotypes by autopolyploidy may be an important mechanism in plant diversification. Although cryptic autopolyploids probably comprise the largest fraction of overlooked plant diversity, our knowledge of their origin and evolution is still rather limited. Here we study the presumed autopolyploid aggregate of Aster amellus, which encompasses diploid and hexaploid cytotypes. Although the cytotypes of A. amellus are not morphologically distinguishable, previous studies showed spatial segregation and limited gene flow between them, which could result in different evolutionary trajectories for each cytotype. Methods: We combine macroevolutionary, microevolutionary and niche modelling tools to disentangle the origin and the demographic history of the cytotypes, using chloroplast and nuclear markers in a dense population sampling in central Europe. Key Results: Our results revealed a segregation between diploid and hexaploid cytotypes in the nuclear genome, where each cytotype represents a monophyletic lineage probably homogenized by concerted evolution. In contrast, the chloroplast genome showed intermixed connections between the cytotypes, which may correspond to shared ancestral relationships. Phylogeny, demographic analyses and ecological niche modelling supported an ongoing differentiation of the cytotypes, where the hexaploid cytotype is experiencing a demographic expansion and niche differentiation with respect to its diploid relative. Conclusions: The two cytotypes may be considered as two different lineages at the onset of their evolutionary diversification. Polyploidization led to the occurrence of hexaploids, which expanded and changed their ecological niche.

Ecophysiological constraints of Aster tripolium under extreme thermal events impacts: Merging biophysical, biochemical and genetic insights.

Cold and heat waves are phenomenon that occurs in higher frequency and intensity due to global climate changes. Commonly cultivated crop species are crucially affected by extreme weather events, and therefore alternative crops - such as halophytes - gain in agricultural interest. While halophytes are potentially able to cope with temperature extremes on the long term exposure, effects of temporary events such as cold and heat waves are not yet described. In order to unveil the effects of these altered thermal environments, Aster tripolium plants were subjected to cold (9/5 °C) and heat (42/38 °C) waves regimes during 3 days and its photochemical and biochemical traits evaluated. In the potential cash crop A. tripolium cold waves induced the gene expression of dehydrins in order to counteract desiccation and thus to prevent oxidative stress. Regulatory proteins on the RNA maturation level (Maturase K) were highly expressed. Heat stress induced the gene expression of the cystein protease gene; most likely to degrade misfolded proteins temporary. Both thermal treatments decreased the photosynthetic efficiency and capacity, driven by a loss in the connectivity between PSII antennae. Nevertheless the light absorption capacity was unaffected due to an increased RC closure net rate. Cold wave-treated individuals showed a decrease in the carotenoid pigmentation, except auroxanthin. In cold wave treated individuals the overall peroxidase activity was significantly increased. Data suggest that exposure to both, cold and heat wave treatment decreased the ecophysiological capacity of A. tripolium.

Absence of gene flow between diploids and hexaploids of Aster amellus at multiple spatial scales.

The potential for gene exchange across ploidy levels has long been recognized, but only a few studies have explored the rate of gene flow among different cytotypes. In addition, most of the existing knowledge comes from contact zones between diploids and tetraploids. The purpose of this paper was to investigate relationships between diploid and hexaploid individuals within the Aster amellus aggregate. A. amellus is known to occur in diploid and hexaploid cytotypes in Europe, with a complex contact zone in central Europe. Patterns of genetic diversity were investigated using seven microsatellite loci at three different spatial scales: (1) in the single known mixed-ploidy population; (2) in populations at the contact zone and (3) in a wider range of populations across Europe. The results show clear separation of the cytotypes at all three spatial scales. In addition, analysis of molecular variance strongly supported a model predicting a single origin of the hexaploids, with no or very limited gene flow between the cytotypes. Some hexaploid individuals found in the mixed-ploidy population, however, fell into the diploid cluster. This could suggest recurrent polyploid formation or occasional cross-pollination between cytotypes; however, there are strong post-zygotic breeding barriers between the two cytotypes, making the latter less plausible. Overall, the results suggest that the cytotypes could represent two cryptic species. Nevertheless, their formal separation is difficult as they cannot be distinguished morphologically, occupy very similar habitat conditions and have largely overlapping distribution ranges. These results show that polyploid complexes must be treated with caution as they can hide biological diversity and can have different adaptation potentials, evolving independently.

Cytotype distribution at a diploid-hexaploid contact zone in Aster amellus (Asteraceae).

BACKGROUND AND AIMS: The present study aims to assess the diversity and distribution of cytotypes of Aster amellus in central and eastern Europe, contributing with data to improve understanding of the evolutionary dynamics of the contact zone between diploids and hexaploids of this polyploid complex. METHODS: Large-scale cytotype screening of 4720 individuals collected in 229 populations was performed using 4',6-diamidino-2-phenylindole (DAPI) flow cytometry. Fine-scale cytotype screening was performed in the mixed-ploidy population. Reproductive variables, such as number of florets per flower head, seed set and seedling emergence, as well as ploidy level of seeds and seedlings were recorded in this population. KEY RESULTS: The diploid-hexaploid contact zone is large and complex, reaching the Czech Republic in the west, Austria in the south, Poland in the north-east and Romania in the extreme east of the surveyed areas. Most populations presented only one cytotype, either diploid or hexaploid. In several areas of the contact zone both cytotypes were found to grow in parapatry. One mixed-ploidy population of diploids and hexaploids was detected for the first time, but no signs of hybridization were detected. In this population, diploids had a significantly lower reproductive success, and significantly higher production of intercytotype offspring, being in reproductive disadvantage in comparison with hexaploids. CONCLUSIONS: The contact zone of diploid and hexaploid A. amellus in central and eastern Europe seems to be highly dynamic and diffuse, with both primary and secondary contacts being possible. The obtained results suggest the origin of hexaploids through diploids, overall supporting previous hypotheses that this species is autopolyploid. Data from the only mixed-ploidy population detected so far suggest that the minority cytotype exclusion is an important evolutionary mechanisms driving the prevalence of single-cytotype populations, and thus contributing to the current distributional patterns of the cytotypes of A. amellus.

The response of Aster amellus (Asteraceae) to mycorrhiza depends on the origins of both the soil and the fungi.

PREMISE OF STUDY: Previous studies testing pairwise interactions between plants, arbuscular mycorrhizal fungi (AMF) and soil have shown that the effectiveness of such interactions depends on the origins of the plants, soil, and AMF. Surprisingly, no study has yet looked at the three-way interaction between plants, soil, and AMF originating from the same and from different sites. Such knowledge could elucidate the determinants of local adaptations of plants and thus might help in various revegetation attempts. METHODS: Six populations of the obligately mycorrhizal plant species Aster amellus from two isolated regions were combined with the soil and the AMF ecotype from their sites and plant growth were monitored over 16 months. KEY RESULTS: For each combination of soil and native AMF, plants grown with their native AMF in their native soil had higher aboveground biomass, invested more to aboveground biomass and had higher numbers of fl ower heads than the other plants. The specifi city of the relationship among plant populations, AMF, and soil was also observed for percentage of root colonization. CONCLUSIONS: The study extends our knowledge of the specifi c interaction between plants and AMF by demonstrating that the positive effect of native AMF occurs only when the plants are also grown in their native soil. Thus, when attempting to facilitate plant growth, we need to consider the origin of the plants, the soil, and the AMF, because all three components are adapted to each other.

Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus.

Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six-membered tetracyclic skeleton and 3-oxo-4-monomethyl structure. To clarify its biosynthetic process, an oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the enzyme was determined in lanosterol synthase-deficient yeast. The cyclase yielded ca. 90% shionone and small amounts of ß-amyrin, friedelin, dammara-20,24-dienol, and 4-epishionone and was designated as a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from ß-amyrin synthase lineages and acquired characteristic species- and product-specificities.

Quinic acids from Aster caucasicus and from transgenic callus expressing a beta-amyrin synthase.

Several different classes of secondary metabolites, including flavonoids, triterpenoid saponins and quinic acid derivatives, are found in Aster spp. (Fam. Asteraceae). Several Aster compounds revealed biological as well as pharmacological activities. In this work, a phytochemical investigation of A. caucasicus evidenced the presence of quinic acid derivatives, as well as the absence of triterpene saponins. To combine in one species the production of different phytochemicals, including triterpenes, an Agrobacterium-mediated transformation of A. caucasicus was set up to introduce A. sedifolius beta-amyrin synthase (AsOXA1)-encoding gene under the control of the constitutive promoter CaMV35S. The quali-quantitative analysis of transgenic calli with ectopic expression of AsOXA1 showed, in one sample, a negligible amount of triterpene saponins combined with higher amount of quinic acid derivatives as compared with the wild type callus.

Consequences of near and far between-population crosses for offspring fitness in a rare herb.

Crosses between plants from different populations may result in heterosis or outbreeding depression. However, despite its importance for conservation, little is known about the spatial scale over which these effects may arise. To investigate the consequences of between-population crosses at two distinct spatial scales, we conducted reciprocal crosses between four populations from two regions in the rare perennial herb Aster amellus. We assessed seed set and offspring fitness in a common garden experiment. Overall, between-population crosses within regions (10 km) resulted in 8% lower seed set than within-population crosses, while between-region crosses (70 km) resulted in 17% higher seed set than within-population crosses. Moreover, offspring from between-population crosses produced 18% more flower heads than offspring from within-population crosses. We conclude that hybridisation between A. amellus plants from different populations did not lead to immediate outbreeding depression and, thus, could represent a valid conservation option to increase genetic diversity. Moreover, our results suggest that the distance between populations affects the outputs of between-population crosses and therefore needs to be taken into account when promoting gene flow between populations.

Enhanced triterpene saponin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel beta-amyrin synthase (AsOXA1) gene.

Triterpene saponins are a group of bioactive compounds abundant in the genus Medicago, and have been studied extensively for their biological and pharmacological properties. In this article, we evaluated the effects of the ectopic expression of AsOXA1 cDNA from Aster sedifolius on the production of triterpene saponins in barrel medic (Medicago truncatula Gaertn.). AsOXA1 cDNA encodes beta-amyrin synthase, a key enzyme involved in triterpene saponin biosynthesis. One of the four transgenic lines expressing AsOXA1 accumulated significantly larger amounts of some triterpenic compounds in leaf and root than did control plants. In particular, the leaf exhibited significantly higher levels of bayogenin, medicagenic acid and zanhic acid. The amounts of medicagenic acid and zanhic acid, which represent the core of the M. truncatula leaf saponins, were 1.7 and 2.1 times higher, respectively, than the amounts extracted from the control line. In root, the production of bayogenin, hederagenin, soyasapogenol E and 2beta-hydroxyoleanolic acid was increased significantly. The increase in the total amounts of triterpenic compounds observed in the leaves of transgenic lines correlated with the AsOXA1 expression level. Interestingly, the plants expressing AsOXA1 showed, under different growth conditions, improved nodulation when compared with the control line. Nodulation enhancement was also accompanied by a significant change in the soyasapogenol B content. Our results indicate that the ectopic expression of AsOXA1 in barrel medic leads to a greater accumulation of triterpene saponins and enhanced root nodulation.

Niche differentiation between diploid and hexaploid Aster amellus.

The maintenance of separated diploid and polyploid populations within a contact zone is possible due to both prezygotic and postzygotic isolation mechanisms. Niche differentiation between two cytotypes may be an important prezygotic isolating mechanism and can be studied using reciprocal transplant experiments. We investigated niche differentiation between diploid and hexaploid Aster amellus in their contact zone in the Czech Republic. Diploid populations are confined to habitats with low productivity, whereas hexaploid populations occur in habitats with both low and high productivity. Thus, we chose three diploid populations and six hexaploid populations, three in each of the two different habitat types. We analyzed habitat characteristics and carried out reciprocal transplant experiments in the field using both seeds and adult plants. Sites of diploid and hexaploid populations differed significantly in vegetation and soil properties. The mean number of juveniles was higher at sites of home ploidy level than at sites of foreign ploidy level, suggesting niche differentiation between the two cytotypes. On the other hand, transplanted adult plants survived at all sites and juvenile plants were able to establish at some sites of the foreign cytotype. Furthermore, the mean number of juveniles, survival, and flowering percentages were higher at home sites than at foreign sites, indicating local adaptation. We conclude that niche differentiation between the two cytotypes and local adaptation within each cytotype may contribute to the maintenance of diploid and hexaploid populations of A. amellus in their contact zone. Moreover, further factors, such as differences in flowering phenology and exclusion of minority cytotypes, should also be considered.

Differences in AM fungal root colonization between populations of perennial Aster species have genetic reasons.

We tested the hypothesis whether differences between plant populations in root colonization by arbuscular mycorrhizal (AM) fungi could be caused by genetic differentiation between populations. In addition, we investigated whether the response to AM fungi differs between plants from different populations and if it is affected by the soil in which the plants are cultivated. We used Aster amellus, which occurs in fragmented dry grasslands, as a model species and we studied six different populations from two regions, which varied in soil nutrient concentration. We found significant differences in the degree of mycorrhizal colonization of plant roots between regions in the field. To test if these differences were due to phenotypic plasticity or had a genetic basis, we performed a greenhouse experiment. The results suggested that Aster amellus is an obligate mycotrophic plant species with a high dependency upon mycorrhiza. Plant biomass was affected only by soil, and not by population or the interaction between the population and the soil. Mycorrhizal colonization was significantly affected by all three factors (soil, population, interaction of soil and population). Plants from the population originating from the soil with lower nutrient availability developed more mycorrhiza even when grown in soil with higher nutrient availability. The correspondence between mycorrhizal colonization of plants in the field and in both soils in the pot experiment suggests that the observed differences in root colonization have a genetic basis.

Identification of parental genomes and genomic organization in Aster microcephalus var. ovatus.

The karyotype of diploid Aster iinumae is morphologically similar to that of diploid Aster ageratoides var. ageratoides, however, its chromosome size is apparently smaller (S-type chromosomes versus L-type chromosomes, respectively). The hybrid origin of tetraploid Aster microcephalus var. ovatus (LS-type chromosomes) has previously been suggested by cytogenetics and chloroplast DNA (cp DNA) data. The cp DNA phylogeny also implies that the S-type chromosome is apomorphic, which means that genome size reduction occurred on the evolutionary way to A. iinumae. In this study, we have demonstrated that the chromosome size difference does not depend on the intensity of chromosome condensation but on the DNA content. The simultaneous genomic in situ hybridization (GISH) results show the similarity between S-type chromosomes of A. iinumae and A. microcephalus var. ovatus, and between L-type chromosomes of A. ageratoides and A. microcephalus var. ovatus, which provide additional evidence for A. microcephalus var. ovatus being a tetraploid amphidiploid produced by hybridization between S-type chromosomes and L-type chromosomes. The distribution patterns of Ty1-copia-like retrotransposons were similar in L- and S-type chromosomes. The copies of this retrotransposon dispersed uniformly on all chromosomes, and it is not yet apparent how the Ty1-copia-like retrotransposon affects the size difference between them.

Co-regulation of brassinosteroid biosynthesis-related genes during xylem cell differentiation.

To understand the regulatory mechanisms of brassinosteroid (BR) biosynthesis in specific plant developmental processes, we first investigated the accumulation profiles of BRs and sterols in xylem differentiation in a Zinnia culture. The amounts of many substances in the late C28 sterol biosynthetic pathway to campesterol (CR), such as episterol and 24-methylenecholesterol, as well as those in the BR-specific biosynthetic pathway from CR to brassinolide (BL), were elevated in close association with tracheary element differentiation. Among them, 6-deoxotyphasterol (6-deoxoTY) accumulated to unusually high levels within cells cultured in tracheary element-inductive medium, while castasterone (CS) was not elevated either within or outside cells. To identify the molecular basis of this co-up-regulation of BRs and C28 sterols, we isolated Zinnia genes for the key enzymes of BR biosynthesis, ZeSTE1, ZeDIM, ZeDWF4, ZeCPD1 and ZeCPD2. RNA gel blot analysis of these genes indicated a coordinated increase in transcripts for ZeSTE1, ZeDIM, ZeDWF4 and ZeCPD1, and a tracheary element differentiation-specific increase in transcripts for ZeDWF4 and ZeCPD1. In situ hybridization experiments of ZeDWF4 and ZeCPD1 mRNAs revealed their preferential accumulation in procambium cells, immature xylem cells and xylem parenchyma cells. These results suggest that BR biosynthesis during tracheary element differentiation may be regulated by the coordinated regulation of broad sterol biosynthesis and specific regulation of BR biosynthesis, which occurs in part by elevated transcript levels of genes encoding BR biosynthetic enzymes, specifically ZeDWF4 and ZeCPD1. These data provide new insights into the regulation of BR biosynthesis and BR signaling during plant development.

Distribution and ecology of cytotypes of the Aster amellus aggregates in the Czech Republic.

BACKGROUND AND AIMS: Polyploidy is viewed as an important mechanism of sympatric speciation, but only a few studies have documented patterns of distribution and ecology of different cytotypes in their contact zone. Aster amellus agg. (Asteraceae) is one of the species with documented multiple ploidy levels. The aim of this study was to determine spatial distribution and ecology of two cytotypes, diploid (2n = 18) and hexaploid (2n = 54), of Aster amellus agg. at their contact zone in the Czech Republic. METHODS: Root-tip squashes and flow cytometry were used to determine the ploidy of 2175 individuals from 87 populations. To test whether some differences in ecology between the two ploidy levels exist, in each locality relevés were recorded and abiotic conditions of the sites were studied by estimating potential direct solar radiation, Ellenberg indicator values and above-ground biomass. KEY RESULTS: Together with diploid and hexaploids, minorite cytotypes (triploid, pentaploid and nonaploid) were found. No significant ecological differences between diploid and hexaploid cytotypes were found. In spite of this, no population consisting of both of the two basic cytotypes was found. CONCLUSIONS: The results of this study show that the contact zone of diploid and hexaploid cytotypes in the Czech Republic is much more diffuse than indicated in previous records. Although populations of both cytotypes occur in close proximity (the closest populations of different cytotypes were 500 m apart), each individual population consists of only one basic ploidy level. This was unexpected since there are no clear differences in abiotic conditions between populations. Taken together with the absence of an intermediate tetraploid cytotype and with reference to published world distributional patterns of different ploidy levels, this suggests a secondary contact zone. Detailed genetic study is, however, necessary to confirm this.

Genetic relationships among Aster species by multivariate analysis and AFLP markers.

Fourteen species of Aster were characterized for morphological traits of commercial interest, chromosome number and genetic diversity by AFLP markers to exploit this material not only for breeding purposes but also as source of pharmaceuticals. The variation observed among the Aster species for five morphological traits was summarized by means of discriminant analysis. Three significant canonical variables, accounting for about 96% of total variance, were mainly correlated with capitulum diameter, number of ligulae and leaf length. The morphological variation appeared related to ploidy level of the species. A diploid chromosome number ranging from 10 to 18 was evident in seven species whereas the other species are polyploids with variable chromosome numbers up to 52. The pollen production and stainability were quite variable among Aster species. Furthermore, seven species produced big pollen grains besides normally-sized ones, at a frequency ranging from 1 to 12%, possibly due to 2n pollen production. The possibility that evolution of Aster genus could be driven by 2n gametes and sexual polyploidization is discussed. Implications of 2n gametes for Aster breeding are also mentioned. AFLP analysis of the fourteen Aster species based on six primer combinations revealed a total number of 421 polymorphic amplified DNA fragments. The genetic similarities estimated between the Aster species, based on both shared and unique amplification products ranged from 0.335 to 0.604 across all species and revealed a geographically based clustering within the genus. The highest similarity was detected between A. alpinus and A. amellus with Eurasian origin.