Dig-up Primers: A Pipeline for Identification of Polymorphic Microsatellites Loci within Assemblies of Related Species.

Simple sequence repeats (SSRs) have become one of the most popular molecular markers and are used in numerous fields, including conservation genetics, population genetic studies, and genetic mapping. Advances in next-generation sequencing technology and the growing amount of genomic data are driving the development of bioinformatics tools for SSR marker design. These tools work with different combinations of input data, which can be raw reads or assemblies, and with one or more input datasets. We present here a new strategy and implementation of a simple standalone pipeline that utilizes more than one assembly for the in silico design of PCR primers for microsatellite loci in more than one species. Primers are tested in silico to determine if they are polymorphic, eliminating the need to test time-consuming cross-species amplification in the laboratory. The end result is a set of markers that are in silico polymorphic in all analyzed species and have great potential for the identification of interspecies hybrids. The efficiency of the tool is demonstrated using two examples at different taxonomic levels and with different numbers of input assemblies to generate promising, high-quality SSR markers.

Revealing the Diversity and Complex Relationships of Croatian Olive Germplasm.

Olive growing in Croatia has a long tradition and is of great economic and social impact. The present study includes a set of 108 tree samples (88 samples corresponding to 60 presumed cultivars and 20 trees of unnamed ones) collected from 27 groves in the entire olive growing area, and is the most comprehensive survey to be conducted in Croatia. The genetic diversity, relationships, and structures of olive plants were studied using eight microsatellite loci. All loci were polymorphic and revealed a total of 90 alleles. A total of 74 different genotypes were detected that were subjected to further diversity and genetic relationship studies. The Fitch-Margoliash tree and Bayesian analysis of population structure revealed a complex relationship between the identified olive genotypes, which were clustered into three gene pools, indicating different origins of Croatian olive germplasms. Excluding the redundant germplasms, 44 different genotypes among the sampled trees of well-known cultivars and 16 new local germplasms were identified. In addition, we provide the etymology of 46 vernacular names, which confirms that the vast majority of traditional Croatian cultivars have common and widespread names. The results presented herein underline the importance of safeguarding local cultivars and conducting continuous surveys.

Chloroplast Genome Annotation Tools: Prolegomena to the Identification of Inverted Repeats.

The development of next-generation sequencing technology and the increasing amount of sequencing data have brought the bioinformatic tools used in genome assembly into focus. The final step of the process is genome annotation, which works on assembled genome sequences to identify the location of genome features. In the case of organelle genomes, specialized annotation tools are used to identify organelle genes and structural features. Numerous annotation tools target chloroplast sequences. Most chloroplast DNA genomes have a quadripartite structure caused by two copies of a large inverted repeat. We investigated the strategies of six annotation tools (Chloë, Chloroplot, GeSeq, ORG.Annotate, PGA, Plann) for identifying inverted repeats and analyzed their success using publicly available complete chloroplast sequences of taxa belonging to the asterid and rosid clades. The annotation tools use two different approaches to identify inverted repeats, using existing general search tools or implementing stand-alone solutions. The chloroplast sequences studied show that there are different types of imperfections in the assembled data and that each tool performs better on some sequences than the others.

Link between epigenetic diversity and invasive status of south-eastern European populations of phytopathogenic fungus Cryphonectria parasitica.

Epigenetic modifications may play an important role in invasion and adaptation of clonal and invasive populations to different environments. The aim of this study was to analyse epigenetic diversity and structure within and among populations of invasive pathogenic fungus Cryphonectria parasitica from south-eastern Europe, where one haplotype S12 dominates. The highest level of epigenetic diversity was found in haplotype S1, followed by S2, while the lowest level of epigenetic diversity was found in haplotype S12. Similar pattern of epigenetic diversity was detected in the control, genetically diverse Croatian population where S1 haplotype dominates. In four south-eastern European populations, the highest level of epigenetic diversity was observed in the Italian population, the oldest population in the studied area, while the lowest diversity was found in most recently established Bulgarian population. This relationship between epigenetic diversity and population age implies the important role of epigenetic modifications on the process of invasion. Our data suggest that epigenetic differences might affect the success of expansion of certain haplotype into new regions. Understanding the role of epigenetic processes in expansion and (pre)adaptation of fungal plant pathogens, besides fundamental knowledge, can contribute to development of strategies for control of fungal spread and pathogenesis.

Cryphonectria hypovirus 1-Induced Epigenetic Changes in Infected Phytopathogenic Fungus Cryphonectria parasitica.

Biotic stress caused by virus infections induces epigenetic changes in infected plants and animals, but this is the first report on methylation pattern changes in a fungus after mycovirus infection. As a model pathosystem for mycovirus-host interactions, we used Cryphonectria hypovirus 1 (CHV1) and its host fungus Cryphonectria parasitica, in which deregulation of methylation cycle enzymes upon virus infection was observed previously. Six CHV1 strains of different subtypes were transferred into three different C. parasitica isolates in order to assess the effect of different CHV1 strains and/or subtypes on global cytosine methylation level in infected fungus, using methylation-sensitive amplification polymorphism (MSAP). Infection with CHV1 affected the methylation pattern of the C. parasitica genome; it increased the number and diversity of methylated, hemi-methylated, and total MSAP markers found in infected fungal isolates compared to virus-free controls. The increase in methylation levels correlated well with the CHV1-induced reduction of fungal growth in vitro, indicating that C. parasitica genome methylation upon CHV1 infection, rather than being the defensive mechanism of the fungus, is more likely to be the virulence determinant of the virus. Furthermore, the severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended mostly on individual CHV1 strains and on the combination of host and virus genomes, rather than on the virus subtype. These novel findings broaden our knowledge about CHV1 strains which could potentially be used in human-aided biocontrol of chestnut blight, a disease caused by C. parasitica in chestnut forest ecosystems and orchards.

Asymmetric Introgression and Cryptic Natural Hybridization between Two Species of Teucrium Section Polium (Lamiaceae) on the Balkan Peninsula.

In this work, we analyzed the morphology and genetic structure of Teucrium montanum, T. capitatum and their hybrid T. × rohlenae from three syntopic populations. A morphometric study showed that the parents and their hybrids exhibited continuous morphological variation, with the hybrid positioned exactly between the parents. Genetic analysis revealed that plants morphologically identified as T. × rohlenae are fertile hybrids that produce hybrid swarms dominated by later-generation hybrids. This suggests that introgression, rather than speciation, is the more likely outcome of hybridization between these plant species. The extent and direction of gene flow between the two species differed markedly between the three syntopic localities. At the Trilj locality, it was clearly unidirectional, with T. capitatum playing the dominant role. At the Sicevo locality, gene flow was slightly asymmetric, favoring the genetic background of T. capitatum, while at the Sliven site, it was completely asymmetric in the opposite direction. The extreme case of unidirectional gene flow was observed at the Trilj locality where plants morphologically identified as T. montanum could not be genetically distinguished from T. capitatum. This suggests that interspecific hybridization occurred long ago, leading to introgression and cryptic hybrids, blurring of species boundaries and generating evolutionary noise.

Lack of pollinators selects for increased selfing, restricted gene flow and resource allocation in the rare Mediterranean sage Salvia brachyodon.

Range contraction and habitat fragmentation can cause biodiversity loss by creating conditions that directly or indirectly affect the survival of plant populations. Fragmented habitats can alter pollinator guilds and impact their behavior, which may result in pollen/pollinator limitation and selection for increased selfing as a mechanism for reproductive assurance. We used Salvia brachyodon, a narrowly distributed and endangered sage from eastern Adriatic, to test the consequences of range contraction and habitat fragmentation. Molecular data indicate a severe and relatively recent species range reduction. While one population is reproductively almost completely isolated, moderate gene flow has been detected between the remaining two populations. The high pollen-to-ovule ratio and the results of controlled hand pollination indicate that S. brachyodon has a mixed mating system. Quantitative and qualitative differences in the community and behaviour of flower visitors resulted in limited pollination services in one population where no effective pollinator other than pollen and nectar robbers were observed. In this population, self-pollination predominated over cross-pollination. Various environmental factors, in which plant-pollinator interactions play a pivotal role, have likely created selection pressures that have led to genetic and phenotypic differentiation and different resource allocation strategies among populations.

Phylogenetic relationships in Brassicaceae tribe Alysseae inferred from nuclear ribosomal and chloroplast DNA sequence data.

Numerous molecular systematic studies within Brassicaceae have resulted in a strongly improved classification of the family, as morphologically defined units at and above the generic level were often found to poorly reflect phylogenetic relationships. Here, we focus on tribe Alysseae, which despite its size (accounting for about 7% of all species) has only received limited coverage in previous phylogenetic studies. Specifically, we want to test phylogenetic hypotheses implied by current tribal and generic circumscriptions and to put diversification within tribe Alysseae into a temporal context. To this end, sequence data from the nrDNA ITS and two plastid regions (ndhF gene, trnL-F intergenic spacer) were obtained for 176 accessions, representing 16 out of 17 currently recognized genera of the tribe, and were phylogenetically analysed, among others, using a relaxed molecular clock. Due to large discrepancies with respect to published ages of Brassicaceae, age estimates concerning Alysseae are, however, burdened with considerable uncertainty. The tribe is monophyletic and contains four strongly supported major clades and Alyssum homalocarpum, whose relationships among each other remain uncertain due to incongruences between nuclear and plastid DNA markers. The largest genus of the tribe, Alyssum, is not monophyletic and contains, apart from A. homalocarpum, two distinct lineages, corresponding to sections Alyssum, Psilonema, Gamosepalum and to sections Odontarrhena and Meniocus, respectively. Clypeola, whose monophyly is supported only by the plastid data, is very closely related to and possibly nested within the second Alyssum lineage. Species of the genus Fibigia intermingle with those of Alyssoides, Clastopus, Degenia, and Physoptychis, rendering Fibigia polyphyletic. The monotypic genera Leptoplax and Physocardamum are embedded in Bornmuellera.

Chemical diversity of the natural populations of Dalmatian pyrethrum (Tanacetum cinerariifolium (TREVIR.) SCH.BIP.) in Croatia.

Dalmatian pyrethrum (Tanacetum cinerariifolium (TREVIR.) SCH.BIP.) is a plant species endemic to the east Adriatic coast. The bioactive substance of Dalmatian pyrethrum is a natural insecticide, pyrethrin, a mixture of six active components (pyrethrins I and II, cinerins I and II, and jasmolins I and II). The insecticidal potential of pyrethrin was recognized decades ago, and dried and ground flowers have traditionally been used in Croatian agriculture and households. A total of 25 Dalmatian pyrethrum populations from Croatia were studied to determine the pyrethrin content and composition, and to identify distinct chemotypes. The total pyrethrin content ranged from 0.36 to 1.30% (dry flower weight; DW) and the pyrethrin I/pyrethrin II ratio ranged from 0.64 to 3.33%. The statistical analyses revealed that the correlations between the percentage of pyrethrin I and of all the other components were significant and negative. The total pyrethrin content was positively correlated with the percentage of pyrethrin I and negatively correlated with cinerin II. The multivariate analysis of the chemical variability enabled the identification of five chemotypes among 25 Dalmatian pyrethrum populations. The chemical characterization of indigenous Dalmatian pyrethrum populations may serve as a good background for future breeding and agricultural exploitation.

Variation in Chloroplast Genome Size: Biological Phenomena and Technological Artifacts.

The development of bioinformatic solutions is guided by biological knowledge of the subject. In some cases, we use unambiguous biological models, while in others we rely on assumptions. A commonly used assumption for genomes is that related species have similar genome sequences. This is even more obvious in the case of chloroplast genomes due to their slow evolution. We investigated whether the lengths of complete chloroplast sequences are closely related to the taxonomic proximity of the species. The study was performed using all available RefSeq sequences from the asterid and rosid clades. In general, chloroplast length distributions are narrow at both the family and genus levels. In addition, clear biological explanations have already been reported for families and genera that exhibit particularly wide distributions. The main factors responsible for the length variations are parasitic life forms, IR loss, IR expansions and contractions, and polyphyly. However, the presence of outliers in the distribution at the genus level is a strong indication of possible inaccuracies in sequence assembly.

High diversity of indigenous populations of dalmatian sage (Salvia officinalis L.) in essential-oil composition.

Essential oils of 25 indigenous populations of Dalmatian sage (Salvia officinalis L.) that represent nearly half of native distribution area of the species were analyzed. Plantlets collected from wild populations were grown in the same field under the same environmental conditions and then sampled for essential-oil analysis. The yield of essential oil ranged from 1.93 to 3.70% with average of 2.83%. Among the 62 compounds detected, eight (cis-thujone, camphor, trans-thujone, 1,8-cineole, ß-pinene, camphene, borneol, and bornyl acetate) formed 78.13-87.33% of essential oils of individual populations. Strong positive correlations were observed between camphor and ß-pinene, ß-pinene and borneol, as well as between borneol and bornyl acetate. The strongest negative correlation was detected between camphor and trans-thujone. Principal component analysis (PCA) on the basis of eight main compounds showed that first main component separated populations with high thujone content, from those rich in camphor, while the second component separated populations rich in cis-thujone from those rich in trans-thujone. Cluster analysis (CA) led to the identification of three chemotypes of S. officinalis populations: cis-thujone; trans-tujone, and camphor/ß-pinene/borneol/bornyl acetate. We propose that differences in essential oils of 25 populations are mostly genetically controlled, since potential environmental factors were controlled in this study.

Development of new microsatellite markers for Salvia officinalis L. and its potential use in conservation-genetic studies of narrow endemic Salvia brachyodon Vandas.

Nine new microsatellite markers (SSR) were isolated from Salvia officinalis L. A total of 125 alleles, with 8 to 21 alleles per locus, were detected in a natural population from the east Adriatic coast. The observed heterozygosity, expected heterozygosity, and polymorphic information content ranged from 0.46 to 0.83, 0.73 to 0.93 and 0.70 to 0.92, respectively. New microsatellite markers, as well as previously published markers, were tested for cross-amplification in Salvia brachyodon Vandas, a narrow endemic species known to be present in only two localities on the Balkan Peninsula. Out of 30 microsatellite markers tested on the natural S. brachyodon population, 15 were successfully amplified. To obtain evidence of recent bottleneck events in the populations of both species, observed genetic diversity (H(E)) was compared to the expected genetic diversity at mutation-drift equilibrium (H(EQ)) and calculated from the observed number of alleles using a two-phased mutation model (TPM). Recent bottleneck events were detected only in the S. brachyodon population. This result suggests the need to reconsider the current threat category of this endemic species.

The complete chloroplast genome of dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip. (Asteraceae)), the source of the natural insecticide pyrethrin.

Tanacetum cinerariifolium is an endemic species of the eastern Adriatic coast that synthesizes the natural insecticide pyrethrin. We have characterized the complete chloroplast genome of the species and analyzed its phylogeny within the Asteraceae family. The complete chloroplast genome of T. cinerariifolium has a size of 150,136 bp, including a large single-copy (LSC) region of 82,717 bp, a small single-copy (SSC) region of 18,411bp, and a pair of inverted repeats (IRs) of 24,504 bp. The chloroplast genome of T. cinerariifolium encodes 108 genes, including 77 protein-coding genes (PCGs), 27 tRNA genes, and 4 rRNA genes. Phylogenetic analyses based on the complete chloroplast genomes placed T. cinerariifolium in a sister position to species of the genera Artemisia and Chrysanthemum.

Development of Microsatellite Markers for Tanacetum cinerariifolium (Trevis.) Sch. Bip., a Plant with a Large and Highly Repetitive Genome.

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch. Bip.) is an outcrossing plant species (2n = 18) endemic to the eastern Adriatic coast and source of the natural insecticide pyrethrin. Due to the high repeatability and large genome (1C-value = 9.58 pg) our previous attempts to develop microsatellite markers using the traditional method were unsuccessful. Now we have used Illumina paired-end whole genome sequencing and developed a specific procedure to obtain useful microsatellite markers. A total of 796,130,142 high-quality reads (approx. 12.5× coverage) were assembled into 6,909,675 contigs using two approaches (de novo assembly and joining of overlapped pair-end reads). A total of 31,380 contigs contained one or more microsatellite sequences, of which di-(59.7%) and trinucleotide (25.9%) repeats were the most abundant. Contigs containing microsatellites were filtered according to various criteria to achieve better yield of functional markers. After two rounds of testing, 17 microsatellite markers were developed and characterized in one natural population. Twelve loci were selected for preliminary genetic diversity analysis of three natural populations. Neighbor-joining tree, based on the proportion of shared alleles distances, grouped individuals into clusters according to population affiliation. The availability of codominant SSR markers will allow analysis of genetic diversity and structure of natural Dalmatian pyrethrum populations as well as identification of breeding lines and cultivars.

Phylogeographic structure of common sage (Salvia officinalis L.) reveals microrefugia throughout the Balkans and colonizations of the Apennines.

Studying the population-genetic and phylogeographic structures of a representative species of a particular geographical region can not only provide us with information regarding its evolutionary history, but also improve our understanding of the evolutionary processes underlying the patterns of species diversity in that area. By analysing eight highly polymorphic microsatellite loci and two chloroplast DNA regions, we have investigated the influence of Pleistocene climate fluctuations on the evolutionary history of Salvia officinalis L. (common sage). The populations with the highest genetic diversity were located in the central parts of the Balkan distribution range. A large group of closely related haplotypes was distributed throughout the Balkans and the central Apennines, while the private lineage occupied the southern Apennines. In addition, two highly differentiated lineages were scattered only over the Balkans. The results suggest that a single refugium of the studied species from the last glacial period was located in the central part of the range in the Balkans. Numerous microrefugia, probably spanning several glaciation cycles, were scattered across the Balkans, while colonisation of the Apennines from the Balkans occurred at least on two occasions.

Quantitatively Unraveling Hierarchy of Factors Impacting Virgin Olive Oil Phenolic Profile and Oxidative Stability.

A single phenolic group and even a compound play different roles in the sensory properties and stability of virgin olive oil (VOO), which in turn are strongly influenced by several factors. Understanding the causes of differences in phenolic compound composition and oxidative stability (OS) in VOOs is essential for targeted and timely harvest and processing while maintaining desired oil quality. The phenolic profile and OS of two monocultivar VOOs (Oblica and Leccino) grown in two geographical sites of different altitudes (coastal plain and hilly hinterland) were analyzed throughout the ripening period over two years. Concentration of secoiridoids was 30% higher in the Oblica than in the Leccino VOOs, which in turn had significantly higher values of OS. Both cultivars had more than twice as high concentrations of the two most abundant phenolic compounds, the dialdehyde form of decarboxymethyl oleuropein aglycone and the dialdehyde form of decarboxymethyl ligstroside aglycone, and OS values in a colder growing site of higher altitude. Among the studied monocultivar VOOs, the secoiridoid group did not behave equally during ripening. The hierarchy of different influencing factors was investigated using multivariate statistics and revealed: cultivar > geographical site > harvest period > growing season. In addition, the possibility of traceability of VOO using molecular markers was investigated by establishing SSR profiles of oils of the studied cultivars and comparing them with SSR profiles of leaves.

Gene flow between wild trees and cultivated varieties shapes the genetic structure of sweet chestnut (Castanea sativa Mill.) populations.

Gene flow between cultivated and wild gene pools is common in the contact zone between agricultural lands and natural habitats and can be used to study the development of adaptations and selection of novel varieties. This is likely the case in the northern Adriatic region, where centuries-old cultivated orchards of sweet chestnut (Castanea sativa Mill.) are planted within the natural distribution area of the species. Thus, we investigated the population structure of several orchards of sweet chestnuts. Furthermore, the genetic background of three toponymous clonal varieties was explored. Six genomic simple sequence repeat (gSSR) and nine EST-derived SSR (EST-SSR) loci were utilized in this research, and both grafted and non-grafted individuals were included in this study. Five closely related clones were identified, which represent a singular, polyclonal marron variety, found in all three cultivation areas. Furthermore, many hybrids, a result of breeding between cultivated and wild chestnuts, have been found. Analyzed semi-wild orchards defined by a diverse genetic structure, represent a hotspot for further selection and could result in creation of locally adapted, high-yielding varieties.

Phenotypic Variation in European Wild Pear (Pyrus pyraster (L.) Burgsd.) Populations in the North-Western Part of the Balkan Peninsula.

Leaves play a central role in plant fitness, allowing efficient light capture, gas exchange and thermoregulation, ensuring optimal growing conditions for the plant. Phenotypic variability in leaf shape and size has been linked to environmental heterogeneity and habitat characteristics. Therefore, the study of foliar morphology in plant populations can help us to identify the environmental factors that may have influenced the process of species diversification. In this study, we used European wild pear (Pyrus pyraster (L.) Burgsd., Rosaceae) as a model species to investigate the phenotypic variability of leaves under different environmental conditions. Using leaf morphometric data from 19 natural populations from the north-western part of the Balkan Peninsula, a high level of variability among and within populations were found. Leaf traits related to leaf size were more variable compared to leaf shape traits, with both influenced by geographic and environmental factors. Consequently, patterns of isolation by environment (IBE) and distance (IBD) were identified, with IBE showing a stronger influence on leaf variability. Multivariate statistical analysis revealed that European wild pear populations from the north-western part of the Balkan Peninsula can be divided into two morphological clusters, consistent with their geographical distance and environmental conditions. Our results confirm a high level of phenotypic variability in European wild pear populations, providing additional data on this poorly studied species, emphasizing phenotypic plasticity as a major driver in the adaptation of this noble hardwood species to rapid climate change.

Phytochemicals and Their Correlation with Molecular Data in Micromeria and Clinopodium (Lamiaceae) Taxa.

A study of the phytochemical and molecular characteristics of ten Micromeria and six Clinopodium taxa (family Lamiaceae) distributed in the Balkan Peninsula was carried out. The phytochemicals detected in essential oils by gas chromatography, mass spectrometry, and molecular data amplified fragment length polymorphism were used to study the taxonomic relationships among the taxa and the correlations between phytochemical and molecular data. STRUCTURE analysis revealed three genetic groups, while Bayesian Analysis of Population Structure grouped the studied taxa into 11 clusters nested in the groups obtained by STRUCTURE. Principal components analysis performed with the 21 most represented compounds in the essential oils yielded results that were partly consistent with those obtained by STRUCTURE and neighbour-joining analyses. However, their geographic distributions did not support the genetic grouping of the studied taxa and populations. The Mantel test showed a significant correlation between the phytochemical and genetic data (r = 0.421, p < 0.001). Genetic distance explained 17.8% of the phytochemical distance between populations. The current taxonomic position of several of the studied taxa is yet to be satisfactorily resolved, and further studies are needed. Such future research should include nuclear and plastid DNA sequences from a larger sample of populations and individuals.

New microsatellite markers for Salvia officinalis (Lamiaceae) and cross-amplification in closely related species.

PREMISE OF THE STUDY: Microsatellite primers were identified to study the genetic diversity, population genetic structure, and relationships of common sage and closely related species. METHODS AND RESULTS: Nine novel polymorphic trinucleotide microsatellite loci were identified for Salvia officinalis. A total of 127 alleles were observed. The observed and expected heterozygosity values ranged from 0.375 to 0.880 and from 0.624 to 0.931, respectively. Nine new and 11 previously published microsatellite primers were tested for cross-amplification. The species with the most successful cross-amplification was S. fruticosa. CONCLUSIONS: The nine new microsatellite markers will be useful in genetic studies of wild and cultivated populations of common sage and are potentially useful in genetic studies of closely related species.