Combination of Sanger and target-enrichment markers supports revised generic delimitation in the problematic 'Urera clade' of the nettle family (Urticaceae).

Urera Gaudich, s.l. is a pantropical genus comprising c. 35 species of trees, shrubs, and vines. It has a long history of taxonomic uncertainty, and is repeatedly recovered as polyphyletic within a poorly resolved complex of genera in the Urticeae tribe of the nettle family (Urticaceae). To provide generic delimitations concordant with evolutionary history, we use increased taxonomic and genomic sampling to investigate phylogenetic relationships among Urera and associated genera. A cost-effective two-tier genome-sampling approach provides good phylogenetic resolution by using (i) a taxon-dense sample of Sanger sequence data from two barcoding regions to recover clades of putative generic rank, and (ii) a genome-dense sample of target-enrichment data for a subset of representative species from each well-supported clade to resolve relationships among them. The results confirm the polyphyly of Urera s.l. with respect to the morphologically distinct genera Obetia, Poikilospermum and Touchardia. Afrotropic members of Urera s.l. are recovered in a clade sister to the xerophytic African shrubs Obetia; and Hawaiian ones with Touchardia, also from Hawaii. Combined with distinctive morphological differences between Neotropical and African members of Urera s.l., these results lead us to resurrect the previously synonymised name Scepocarpus Wedd. for the latter. The new species epiphet Touchardia oahuensis T.Wells & A.K. Monro is offered as a replacement name for Touchardia glabra non H.St.John, and subgenera are created within Urera s.s. to account for the two morphologically distinct Neotropical clades. This new classification minimises taxonomic and nomenclatural disruption, while more accurately reflecting evolutionary relationships within the group.

Ancient DNA analysis of food remains in human dental calculus from the Edo period, Japan.

Although there are many methods for reconstructing diets of the past, detailed taxon identification is still challenging, and most plants hardly remain at a site. In this study, we applied DNA metabarcoding to dental calculus of premodern Japan for the taxonomic identification of food items. DNA was extracted from 13 human dental calculi from the Unko-in site (18th-19th century) of the Edo period, Japan. Polymerase chain reaction (PCR) and sequencing were performed using a primer set specific to the genus Oryza because rice (Oryza sativa) was a staple food and this was the only member of this genus present in Japan at that time. DNA metabarcoding targeting plants, animals (meat and fish), and fungi were also carried out to investigate dietary diversity. We detected amplified products of the genus Oryza from more than half of the samples using PCR and Sanger sequencing. DNA metabarcoding enabled us to identify taxa of plants and fungi, although taxa of animals were not detected, except human. Most of the plant taxonomic groups (family/genus level) are present in Japan and include candidate species consumed as food at that time, as confirmed by historical literature. The other groups featured in the lifestyle of Edo people, such as for medicinal purposes and tobacco. The results indicate that plant DNA analysis from calculus provides information about food diversity and lifestyle habits from the past and can complement other analytical methods such as microparticle analysis and stable isotope analysis.

Islands as a crossroad of evolutionary lineages: A case study of Centaurea sect. Centaurea (Compositae) from Sardinia (Mediterranean Basin).

The Mediterranean Basin is a biodiversity hotspot, where islands play a key role because of their high biological diversity, degree of endemicity and human pressure. One of these islands, Sardinia, is a good evolutionary laboratory, especially for the study of complex genera, such as Centaurea. In particular, endemic species of Centaurea sect. Centaurea from Sardinia provides an interesting case study of plant evolution on continental islands. We attempted to clarify the processes leading to the diversification of Centaurea species on Sardinia using bi-parentally inherited nuclear markers and maternally inherited plastid markers. Our plastid results revealed the presence of five lineages of sect. Centaurea on the island. Three of them were defined as three species: C. ferulacea, C. filiformis and C. horrida. The other two lineages highlighted the complex evolutionary history of the two polyploids C. corensis and C. magistrorum. Multiple colonization events from the mainland involving the C. deusta and C. paniculata lineages among others, have led to the diversity of sect. Centaurea on Sardinia. One colonization event likely followed a southern path via the land connection between the mainland, the Calabrian Plate and Sardinia. A second pathway likely followed a northern connection, probably through the Tuscan Archipelago. Implications of these findings on conservation efforts for Centaurea endemics on Sardinia are also discussed.

Effects of genetic variation and environmental factors on bergenin in Rodgersia sambucifolia Hemsl.

ETHNOPHARMACOLOGICAL RELEVANCE: Bergenin is a well-known active compound that exhibits antioxidant, antiarrhythmic, hepatoprotective, and anti-inflammatory activities. However, the resource reserve of Rodgersia sambucifolia, one of the main raw materials for extracting bergenin, have sharply declined, and the bergenin content in different germplasms differs vastly, resulting in a serious shortage of the market supply of bergenin. AIM OF THE STUDY: To investigate the influence of genetic diversity and environmental factors on bergenin content in Rodgersia sambucifolia. MATERIALS AND METHODS: Fifty Rodgersia sambucifolia samples with a growth period of 2-3 years were collected from different areas across China and the bergenin content was determined via HPLC. Meanwhile the total genomic DNA was extracted and ISSR was performed. The bergenin content as measured using HPLC and the environmental data gathered from the meteorological stations and field work were combined and analyzed using correlation tests in XLSTAT 2018 to detect the key factors affecting bergenin content. The genetic UPGMA tree constructed based on genetic distances of the 50 samples and the chemical dendrogram constructed according to the distance between the bergenin content were compared to determine the correlation between genetic and chemical differentiation. RESULTS: Among the 50 individuals, bergenin content varied from 2.83 to 12.54%, with the highest content being 4.43-fold that of the lowest content. The survey of the 50 individuals produced a total of 193 amplified bands, 187 of which were polymorphic (96.89%). In the study, bergenin content was positively correlated with annual mean temperature (AMT) (r = 0.583, P < 0.0001) and 1-12 month monthly mean temperature (MMT) (P < 0.0001). A comparison of the genetic dendrogram with the AHC dendrogram found no corresponding relationship between them. Mantel correlation analyses also showed that there was no significant correlation between them (r = 0.144). CONCLUSIONS: There were large differences in bergenin content among different germplasms that were not correlated with the high genetic variation in Rodgersia sambucifolia but were significantly correlated with environmental factors, such as temperature. This study lays the foundation for subsequent superior germplasm selection and artificial breeding of Rodgersia sambucifolia to improve the bergenin content and meet market demands.

Incidence of viruses infecting pepper in Thailand.

This study was conducted to determine the incidence, diversity and distribution of viruses infecting pepper (Capsicum spp.) in the central, northern and northeastern parts of Thailand. During a survey in 2016 - 2019, a total of 2,149 leaf samples from symptomatic and asymptomatic peppers were collected randomly from farmer's fields, and preliminary tested by an enzyme-linked immunosorbent assay (ELISA) using 7 antibodies specific for cucumber mosaic virus (CMV), chilli veinal mottle virus (ChiVMV), tomato necrotic ringspot virus (TNRV), tobacco mosaic virus (TMV), potato virus Y (PVY), tomato spotted wilt virus (TSWV), and begomoviruses. Our data revealed that the incidence of the viruses infecting pepper in Thailand was high, accounting for nearly 70% (1,482 infected samples). The highest viral incidence was found in the central part (96%), followed by the north (74.4%) and the northeastern (52.8%), respectively. Begomoviruses, CMV, ChiVMV, and TNRV were detected in the samples at varying rates, whereas PVY, TMV, and TSWV were not detected. Of these, the most frequently found virus was Begomoviruses accounting for nearly 33%, with the highest rate (ca. 82%) in the central Provinces of Thailand. In addition, of the 1,482 infected samples, mixed infections among the four viruses were also found in 616 samples (ca. 42%), and CMV + ChiVMV (approximately 11%) was the most common mixed infection. This is the first report describing an occurrence of viruses in pepper of Thailand, and the results obtained have revealed that viruses infecting pepper are widespread, which may pose a threat to pepper production in Thailand.

Development of nucleic acid isolation by non-silica-based nanoparticles and real-time PCR kit for edible vegetable oil traceability.

For efficient extraction of amplifiable DNA from edible vegetable oils, we developed a novel DNA extraction approach based on the non-silica-based dipolar nanocomposites. The nanoparticle comprises a hydrophilic polymethyl methacrylate core with abundant capillaries, hydrophilic vesicles decorated with molecules having DNA affinity and a coating hydrophobic polystyrene layer. The nanoparticles are soluble in oil, adsorb the DNA from the aqueous phase and gave a high DNA recovery ratio. All DNA extracts from fully refined vegetable oil soybean, peanut, rapeseed, and cottonseed oils, including their blends, were sufficiently pure to be amplified by real-time PCR targeting the chloroplast ribulose-1,5-bisphosphate gene (rbcL), therefore, the species of origin and their ratios in mixed vegetable oils blended from two or three oil-species could be determined. These results indicate that the novel DNA isolation and real-time PCR kit is a simple, sensitive and efficient tool for the species identification and traceability in refined vegetable oils.

Thioredoxin o-mediated reduction of mitochondrial alternative oxidase in the thermogenic skunk cabbage Symplocarpus renifolius.

Thermogenesis in plants involves significant increases in their cyanide-resistant mitochondrial alternative oxidase (AOX) capacity. Because AOX is a non-proton-motive ubiquinol oxidase, the dramatic drop in free energy between ubiquinol and oxygen is dissipated as heat. In the thermogenic skunk cabbage (Symplocarpus renifolius), SrAOX is specifically expressed in the florets. Although SrAOX harbours conserved cysteine residues, the details of the mechanisms underlying its redox regulation are poorly understood. In our present study, the two mitochondrial thioredoxin o cDNAs SrTrxo1 and SrTrxo2, were isolated from the thermogenic florets of S. renifolius. The deduced amino acid sequences of the protein products revealed that SrTrxo2 specifically lacks the region corresponding to the α3-helix in SrTrxo1. Expression analysis of thermogenic and non-thermogenic S. renifolius tissues indicated that the SrTrxo1 and SrAOX transcripts are predominantly expressed together in thermogenic florets, whereas SrTrxo2 transcripts are almost undetectable in any tissue. Finally, functional in vitro analysis of recombinant SrTrxo1 and mitochondrial membrane fractions of thermogenic florets indicated its reducing activity on SrAOX proteins. Taken together, these results indicate that SrTrxo1 is likely to play a role in the redox regulation of SrAOX in S. renifolius thermogenic florets.

Filter paper-based spin column method for cost-efficient DNA or RNA purification.

We describe herein a method of recharging used commercial spin columns or assembling homemade spin columns using filter paper as binding material for cost-effective, low throughput nucleic acid purification. The efficiency of filter paper-based spin columns was evaluated for purification of nucleic acids from various sources. Following protocols of commercial kits, we found filter paper to be a useful binding material for purification of nucleic acids, including plant genomic DNA, plant total RNA, PCR products, and DNA from agarose gels. However, filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Protocols for the use of filter paper recharged spin columns or homemade spin columns for low throughput purification of plant genomic DNA and total RNA with unused commercial kit buffers or less expensive homemade buffers are presented.

Genome-wide analyses of miniature inverted-repeat transposable elements reveals new insights into the evolution of the Triticum-Aegilops group.

The sequence drafts of wild emmer and bread wheat facilitated high resolution, genome-wide analysis of transposable elements (TEs), which account for up to 90% of the wheat genome. Despite extensive studies, the role of TEs in reshaping nascent polyploid genomes remains to be fully understood. In this study, we retrieved miniature inverted-repeat transposable elements (MITEs) from the recently published genome drafts of Triticum aestivum, Triticum turgidum ssp. dicoccoides, Aegilops tauschii and the available genome draft of Triticum urartu. Overall, 239,126 MITE insertions were retrieved, including 3,874 insertions of a newly identified, wheat-unique MITE family that we named "Inbar". The Stowaway superfamily accounts for ~80% of the retrieved MITE insertions, while Thalos is the most abundant family. MITE insertions are distributed in the seven homologous chromosomes of the wild emmer and bread wheat genomes. The remarkably high level of insertions in the B sub-genome (~59% of total retrieved MITE insertions in the wild emmer genome draft, and ~41% in the bread wheat genome draft), emphasize its highly repetitive nature. Nearly 52% of all MITE insertions were found within or close (less than 100bp) to coding genes, and ~400 MITE sequences were found in the bread wheat transcriptome, indicating that MITEs might have a strong impact on wheat genome expression. In addition, ~40% of MITE insertions were found within TE sequences, and remarkably, ~90% of Inbar insertions were located in retrotransposon sequences. Our data thus shed new light on the role of MITEs in the diversification of allopolyploid wheat species.

Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications.

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

Flow cytometric seed screen data are consistent with models of chromosome inheritance in asymmetrically compensating allopolyploids.

Angiosperms have evolved a mechanism of double fertilization, which results in the production of a separate embryo (new individual) and endosperm (nutritive tissue). The flow cytometric seed screen (FCSS) was developed to infer plant reproduction modes based on endosperm-to-embryo DNA content ratio (Pind ). A ratio of 1.5 indicates sexual reproduction, whereas higher values of ≥2.0 are consistent with apomixis. Although FCSS has been successfully applied to the study of sexual and asexual plants, the limits of FCSS and particularly its potential for determination of reproduction modes in hemisexual plants have not been explored. Here, we evaluated the application of FCSS to the study of reproduction modes in two asymmetrically compensating allopolyploids (ACAs), Onosma arenaria and Rosa canina. These two species are characterized by the presence of asexually inherited univalent-forming and sexually inherited bivalent-forming chromosome sets. They both use asymmetric meiosis, which eliminates univalent-forming chromosome sets from the male gamete and retains them in the female gamete. Different chromosomal behavior in male and female meiosis in these plants is reflected in different theoretically derived Pind values, which deviate from a sexual 1.5 value. Here, we determined Pind FCSS-based values in seeds of ACAs, and compared the results to sexual species. As expected, we determined that the mean Pind is 1.51, 1.52, and 1.52 in the sexual plants, that is, Capsella bursa-pastoris, Crataegus monogyna, and O. pseudoarenaria, respectively. In the ACAs, different mean Pind values were determined for O. arenaria (1.61) and R. canina (1.82). These values are consistent with the theoretical Pind values determined based on models of chromosome inheritance. This study highlights the precision of flow cytometry in determining DNA content and it's utility in screening reproduction modes. Additionally, it advocates for more in-depth investigations into rapid screening of accessions where the Pind ratio has deviated from the 1.5 value typical of sexual species, which may indicate meiotic irregularities.

Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device.

Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of "bench-top" based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (µSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.

Molecular phylogeny of bladderworts: A wide approach of Utricularia (Lentibulariaceae) species relationships based on six plastidial and nuclear DNA sequences.

The carnivorous plant genus Utricularia L. (bladderwort) comprises about 240 species distributed worldwide and is traditionally classified into two subgenera (Polypompholyx and Utricularia) and 35 sections, based mainly on general and trap morphology. It is one out of the largest carnivorous genera, representing ca. 30% of all carnivorous plant species, and is also the most widely distributed. According to previous phylogenetic studies, most infrageneric sections are monophyletic, but there are several incongruences considering their relationships and also the dissenting position of some species as a result of a too few (mostly one or two) molecular markers analyzed. Thus, here we present a multilocus phylogeny for Utricularia species with a wide taxonomic sampling (78 species and 115 accessions) based on six plastid (rbcL, matK, rpl20-rps12, rps16, trnL-F) and nuclear DNA (ITS region) sequences. The aim is to reconstruct a well-resolved tree to propose evolutionary and biogeographic hypotheses for the radiation of lineages with inferences about the divergence times of clades using a molecular clock approach.

Rapid allopolyploid radiation of moonwort ferns (Botrychium; Ophioglossaceae) revealed by PacBio sequencing of homologous and homeologous nuclear regions.

Polyploidy is a major speciation process in vascular plants, and is postulated to be particularly important in shaping the diversity of extant ferns. However, limitations in the availability of bi-parental markers for ferns have greatly limited phylogenetic investigation of polyploidy in this group. With a large number of allopolyploid species, the genus Botrychium is a classic example in ferns where recurrent polyploidy is postulated to have driven frequent speciation events. Here, we use PacBio sequencing and the PURC bioinformatics pipeline to capture all homeologous or allelic copies of four long (∼1 kb) low-copy nuclear regions from a sample of 45 specimens (25 diploids and 20 polyploids) representing 37 Botrychium taxa, and three outgroups. This sample includes most currently recognized Botrychium species in Europe and North America, and the majority of our specimens were genotyped with co-dominant nuclear allozymes to ensure species identification. We analyzed the sequence data using maximum likelihood (ML) and Bayesian inference (BI) concatenated-data ("gene tree") approaches to explore the relationships among Botrychium species. Finally, we estimated divergence times among Botrychium lineages and inferred the multi-labeled polyploid species tree showing the origins of the polyploid taxa, and their relationships to each other and to their diploid progenitors. We found strong support for the monophyly of the major lineages within Botrychium and identified most of the parental donors of the polyploids; these results largely corroborate earlier morphological and allozyme-based investigations. Each polyploid had at least two distinct homeologs, indicating that all sampled polyploids are likely allopolyploids (rather than autopolyploids). Our divergence-time analyses revealed that these allopolyploid lineages originated recently-within the last two million years-and thus that the genus has undergone a recent radiation, correlated with multiple independent allopolyploidizations across the phylogeny. Also, we found strong parental biases in the formation of allopolyploids, with individual diploid species participating multiple times as either the maternal or paternal donor (but not both). Finally, we discuss the role of polyploidy in the evolutionary history of Botrychium and the interspecific reproductive barriers possibly involved in these parental biases.

A simple microsatellite-based method for hazelnut oil DNA analysis.

Molecular food traceability requires continuous updates to identify more robust, efficient and affordable methodologies to guarantee food quality and safety and especially consumers' health. Available commercial kits are often unsatisfactory and require modifications to successfully detect single components on complex and transformed food matrices. Here we report a simple method for molecular traceability of cold-pressed hazelnut oil based on microsatellite DNA markers. Different genomic extraction methodologies were tested and a total genome pre-amplification step was applied on PCR-negative samples. PCR-capillary electrophoresis using nine microsatellites demonstrates the accuracy of the fingerprint analysis even for filtered oil.

Isolation of Wheat Genomic DNA for Gene Mapping and Cloning.

DNA is widely used in plant genetic and molecular biology studies. In this chapter, we describe how to extract DNA from wheat tissues. The tissue samples are ground to disrupt the cell wall. Then cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) is used to disrupt the cell and nuclear membranes to release the DNA into solution. A reducing agent, ß-mercaptoethanol, is added to break the disulfide bonds between the cysteine residues and to help remove the tanins and polyphenols. A high concentration of salt is employed to remove polysaccharides. Ethylenediaminetetraacetic acid (EDTA) stops DNase activity by chelating the magnesium ions. The nucleic acid solution is extracted with chloroform-isoamyl alcohol (24:1) or 6 M ammonium acetate. The DNA in aqueous phase is precipated with ethanol or isopropanol, which makes DNA less hydrophilic in the presence of sodium ions (Na+).

Insight into Central Asian flora from the Cenozoic Tianshan montane origin and radiation of Lagochilus (Lamiaceae).

The Tianshan Mountains play a significant role in the Central Asian flora and vegetation. Lagochilus has a distribution concentration in Tianshan Mountains and Central Asia. To investigate generic spatiotemporal evolution, we sampled most Lagochilus species and sequenced six cpDNA locations (rps16, psbA-trnH, matK, trnL-trnF, psbB-psbH, psbK-psbI). We employed BEAST Bayesian inference for dating, and S-DIVA, DEC, and BBM for ancestral area/biome reconstruction. Our results clearly show that the Tianshan Mountains, especially the western Ili-Kirghizia Tianshan, as well as Sunggar and Kaschgar, was the ancestral area. Ancestral biome was mainly in the montane steppe zone of valley and slope at altitudes of 1700-2700 m, and the montane desert zone of foothill and front-hill at 1000-1700 m. Here two sections Inermes and Lagochilus of the genus displayed "uphill" and "downhill" speciation process during middle and later Miocene. The origin and diversification of the genus were explained as coupled with the rapid uplift of the Tianshan Mountains starting in late Oligocene and early Miocene ca. 23.66~19.33 Ma, as well as with uplift of the Qinghai-Tibetan Plateau (QTP) and Central Asian aridification.

Molecular characterization, expression analysis and heterologous expression of two translationally controlled tumor protein genes from Cucumis sativus.

The translationally controlled tumor protein (TCTP) is a family of abundant and ubiquitous proteins involved in several important primary functions. Cucumbers harbor two TCTP genes, CsTCTP1 and CsTCTP2; however, their functional roles remain unclear. In this study, we isolated CsTCTP1 and CsTCTP2 (XP-004134215 and XP-004135602, respectively) promoters, full-length cDNA and genomic sequences from Cucumis sativus. Bioinformatics analysis, containing cis-acting elements, structural domains, phylogenetic tree and conserved motifs, suggested the conservation and divergence of CsTCTP1 and CsTCTP2, thus providing knowledge regarding their functions. Expression analysis indicated that CsTCTP1 and CsTCTP2 exhibited tissue-specific expression and were regulated by biotic or abiotic stresses in C. sativus. Furthermore, CsTCTP1 and CsTCTP2 were regulated by ABA and may be associated with the TOR (target of rapamycin) signaling pathway. In a prokaryotic expression analysis, CsTCTP1 and CsTCTP2 showed positive responses to salt and heat stresses and a negative response to drought and HgCl2 stresses. TCTP may exert multiple functions in various cellular processes.

Use of genotyping-by-sequencing to determine the genetic structure in the medicinal plant chamomile, and to identify flowering time and alpha-bisabolol associated SNP-loci by genome-wide association mapping.

BACKGROUND: Chamomile (Matricaria recutita L.) has a long history of use in herbal medicine with various applications, and the flower heads contain numerous secondary metabolites which are medicinally active. In the major crop plants, next generation sequencing (NGS) approaches are intensely applied to exploit genetic resources, to develop genomic resources and to enhance breeding. Here, genotyping-by-sequencing (GBS) has been used in the non-model medicinal plant chamomile to evaluate the genetic structure of the cultivated varieties/populations, and to perform genome wide association study (GWAS) focusing on genes with large effect on flowering time and the medicinally important alpha-bisabolol content. RESULTS: GBS analysis allowed the identification of 6495 high-quality SNP-markers in our panel of 91 M. recutita plants from 33 origins (2-4 genotypes each) and 4 M. discoidea plants as outgroup, grown in the greenhouse in Gatersleben, Germany. M. recutita proved to be clearly distinct from the outgroup, as was demonstrated by different cluster and principal coordinate analyses using the SNP-markers. Chamomile genotypes from the same origin were mostly genetically similar. Model-based cluster analysis revealed one large group of tetraploid genotypes with low genetic differentiation including 39 plants from 14 origins. Tetraploids tended to display lower genetic diversity than diploids, probably reflecting their origin by artificial polyploidisation from only a limited set of genetic backgrounds. Analyses of flowering time demonstrated that diploids generally flowered earlier than tetraploids, and the analysis of alpha-bisabolol identified several tetraploid genotypes with a high content. GWAS identified highly significant (P < 0.01) SNPs for flowering time (9) and alpha-bisabolol (71). One sequence harbouring SNPs associated with flowering time was described to play a role in self-pollination in Arabidopsis thaliana, whereas four sequences harbouring SNPs associated with alpha-bisabolol were identified to be involved in plant biotic and abiotic stress response in various plants species. CONCLUSIONS: The first genomic resource for future applications to enhance breeding in chamomile was created, andanalyses of diversity will facilitate the exploitation of these genetic resources. The GWAS data pave the way for future research towards the genetics underlying important traits in chamomile, the identification of marker-trait associations, and development of reliable markers for practical breeding.