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.

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.

Local adaptation to the native environment affects pyrethrin variability in Dalmatian pyrethrum populations.

The insecticidal compound pyrethrin is synthesized in Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch.Bip.; Asteraceae), a plant species endemic to the eastern Mediterranean. Pyrethrin is a mixture of six compounds, pyrethrin I and II, cinerin I and II, and jasmolin I and II. For this study we sampled 15 natural Dalmatian pyrethrum populations covering the entire natural distribution range of the species; Croatian coastal regions and the islands, inland Bosnia and Herzegovina and Montenegro. The plants were grown in a field experiment under uniform growing conditions to exclude a short-term response to environmental factors and instead observe variation in pyrethrin content and composition among and within populations due to genetic adaptation to the native environment. The drivers of local adaptation were explored by examining the role of bioclimatic factors as a cause of population differentiation. Pyrethrins were extracted by ultrasound-assisted extraction, and the extracts were analyzed by HPLC-UV-DAD. The populations differed significantly in the content and composition of pyrethrins. The highest levels of total pyrethrins (1.27% flower DW), were found in population P14 Budva and the significantly highest levels of pyrethrin I in population P14 Vranjske Njive, Podgorica (66.47% of total pyrethrin). Based on bioclimatic conditions of the sampling sites, populations were grouped into five bioclimatic groups (A, B, C, D, and E), which showed qualitative and quantitative variability in pyrethrin content. The most abundant bioclimatic group was bioclimatic group E, which was characterized by the highest average values for pyrethrin I (53.87% of total pyrethrin), total pyrethrin content (1.06% flower DW) and the ratio of pyrethrin I and II (1.85). The correlation analysis between the pyrethrin compounds and some of the bioclimatic variables (e. g., BIO03 Isothermality and BIO04 Temperature seasonality) showed their significant contribution in explaining the variation of pyrethrins in T. cinerariifolium. The differences in pyrethrin content and composition may be partly due to genetic adaptation to the ecological conditions of the native environment. The obtained data would enable the selection of source populations for breeding programs aimed at producing cultivars with desirable biochemical properties and adaptation to different bioclimatic conditions.

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.

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.

High diversity of natural Dalmatian pyrethrum based on pyrethrin composition at intra- and interpopulation level.

Seeds collected from 10 Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip. Asteraceae) populations naturally occurring along the Adriatic coast and islands, were used to establish a common garden with the aim of estimating the variation of pyrethrin compounds between and within populations. A recently optimized matrix solid phase dispersion (MSPD) method was used for the extraction of six pyrethrin compounds from dry flower heads of 200 Dalmatian pyrethrum individuals. Separation and quantification of pyrethrin compounds were performed by high performance liquid chromatography with diode array detector (HPLC-DAD). High variability in pyrethrin content and composition was observed within and between populations. Total pyrethrin content in individual samples varied from 0.10% to 1.35% of flower dry weight. On average, the lowest total pyrethrin content was observed in the population of Peljesac (0.22%) and the highest in the island populations of Mali Losinj and Zlarin (0.87%). The population of Mali Losinj had the most favourable pyrethrin profile, including the lowest variability in total pyrethrin content, the highest pyrethrin I (PI) content (up to 60.47%) and PI/PII ratio (up to 5.88), as well as the lowest pyrethrin II (PII) content. In contrast, the populations from Biokovo and Peljesac represented pyrethrin profiles with the lowest content of total pyrethrin, PI and PI/PII ratio. The Biokovo population also had the highest PII content (43.18% on average), while the Peljesac population had the highest jasmolin I (JI) and jasmolin II (JII) content. Four different chemotypes were determined by cluster analysis. Chemotype P1 is the most promising for future breeding programs and is characterized by the highest total pyrethrin content, PI content and PI/PII ratio, as important measures of insecticidal activity. Correlation analysis of each pyrethrin compound with 25 environmental variables and Principal Component Analysis (PCA) were carried out and the importance of optimum temperature and precipitation on pyrethrin quantity and quality was suggested. Total pyrethrin content was significantly correlated with six temperature and five precipitation parameters. Pyrethrin I, PII and cinerin II (CII) were significantly correlated mainly with temperature parameters; JII and cinerin I (CI) with precipitation parameters, while JI was correlated with temperature range and precipitation parameters. The mountainous Biokovo population was an outlier in the PCA analysis, mainly due to the extremely low values of temperature parameters at a higher altitude. Precipitation variables separated the central Adriatic islands inhabiting drier habitats from populations inhabiting habitats with greater precipitation. The high-resolution data from this study provide the opportunity for individual-based selection for breeding plants with the best pyrethrin profiles, as well as for increased adaptation to broader climatic conditions.

Assessment of the Origin and Diversity of Croatian Common Bean Germplasm Using Phaseolin Type, SSR and SNP Markers and Morphological Traits.

Landraces represent valuable genetic resources for breeding programmes to produce high-yielding varieties adapted to stressful environmental conditions. Although the common bean (Phaseolus vulgaris L.) is an economically important food legume for direct human consumption worldwide, common bean production in Croatia is based almost exclusively on landraces and there is no common bean breeding program. Information on phaseolin type and results of population structure and genetic diversity obtained by analysis of SSR and SNP markers, in combination with the morphological characterization of 174 accessions of 10 common bean landraces (morphotypes), enabled thorough classification of accessions. The accessions were classified into phaseolin type H1 ("S") of Mesoamerican origin and phaseolin types H2 ("H" or "C") and H3 ("T") of Andean origin. By applying distance- and model-based clustering methods to SSR markers, the accessions were classified into two clusters at K = 2 separating the accessions according to the centres of origin, while at K = 3, the accessions of Andean origin were further classified into two clusters of accessions that differed in phaseolin type (H2 and H3). Using SNP markers, model-based analysis of population structure was performed, the results of which were consistent with those of SSR markers. In addition, 122 accessions were assigned to 14 newly formed true-type morphogenetic groups derived from three different domestication events: (1) Mesoamerican (H1A) ("Biser", "Kukuruzar", "Tetovac", "Tresnjevac"), (2) Andean-indeterminate type (H2B1) ("Dan noc", "Sivi", "Puter", "Sivi prosarani", "Tresnjevac") and (3) Andean-determinate type (H3B2) ("Bijeli", "Dan noc", "Puter", "Tresnjevac", "Zelencec"). The rest of the accessions could represent putative hybrids between morphogenetic groups. The differences between the true-type groups of accessions were further analysed based on nine quantitative traits, and the subsets of traits that best distinguish among centres of origin (A: Mesoamerican, B: Andean) and genetic groups (H1A, H2B1, H3B2) were proposed.

Population structure and adaptive variation of Helichrysum italicum (Roth) G. Don along eastern Adriatic temperature and precipitation gradient.

Immortelle (Helichrysum italicum (Roth) G. Don; Asteraceae) is a perennial plant species native to the Mediterranean region, known for many properties with wide application mainly in perfume and cosmetic industry. A total of 18 wild H. italicum populations systematically sampled along the eastern Adriatic environmental gradient were studied using AFLP markers to determine genetic diversity and structure and to identify loci potentially responsible for adaptive divergence. Results showed higher levels of intrapopulation diversity than interpopulation diversity. Genetic differentiation among populations was significant but low, indicating extensive gene flow between populations. Bayesian analysis of population structure revealed the existence of two genetic clusters. Combining the results of FST - outlier analysis (Mcheza and BayeScan) and genome-environment association analysis (Samßada, LFMM) four AFLP loci strongly associated with the bioclimatic variables Bio03 Isothermality, Bio08 Mean temperature of the wettest quarter, Bio15 Precipitation seasonality, and Bio17 Precipitation of driest quarter were found to be the main variables driving potential adaptive genetic variation in H. italicum along the eastern Adriatic environmental gradient. Redundancy analysis revealed that the partitioning of genetic variation was mainly associated with the adaptation to temperature oscillations. The results of the research may contribute to a clearer understanding of the importance of local adaptations for the genetic differentiation of Mediterranean plants and allow the planning of appropriate conservation strategies. However, considering that the identified outlier loci may be linked to genes under selection rather than being the target of natural selection, future studies must aim at their additional analysis.

Towards the Well-Tempered Chloroplast DNA Sequences.

With the development of next-generation sequencing technology and bioinformatics tools, the process of assembling DNA sequences has become cheaper and easier, especially in the case of much shorter organelle genomes. The number of available DNA sequences of complete chloroplast genomes in public genetic databases is constantly increasing and the data are widely used in plant phylogenetic and biotechnological research. In this work, we investigated possible inconsistencies in the stored form of publicly available chloroplast genome sequence data. The impact of these inconsistencies on the results of the phylogenetic analysis was investigated and the bioinformatic solution to identify and correct inconsistencies was implemented. The whole procedure was demonstrated using five plant families (Apiaceae, Asteraceae, Campanulaceae, Lamiaceae and Rosaceae) as examples.

Divergent selection and genetic structure of Sideritis scardica populations from southern Balkan Peninsula as revealed by AFLP fingerprinting.

Sideritis scardica Giseb. is a subalpine/alpine plant species endemic to the central part of the Balkan Peninsula. In this study, we combined Amplified Fragment Length Polymorphism (AFLP) and environmental data to examine the adaptive genetic variations in S. scardica natural populations sampled in contrasting environments. A total of 226 AFLP loci were genotyped in 166 individuals from nine populations. The results demonstrated low gene diversity, ranging from 0.095 to 0.133 and significant genetic differentiation ranging from 0.115 to 0.408. Seven genetic clusters were revealed by Bayesian clustering methods as well as by Discriminant Analysis of Principal Components and each population formed its respective cluster. The exception were populations P02 Mt. Shara and P07 Mt. Vermio, that were admixed between two clusters. Both landscape genetic methods Mcheza and BayeScan identified a total of seven (3.10%) markers exhibiting higher levels of genetic differentiation among populations. The spatial analysis method Samßada detected 50 individual markers (22.12%) associated with bioclimatic variables, among them seven were identified by both Mcheza and BayeScan as being under directional selection. Four bioclimatic variables associated with five out of seven outliers were related to precipitation, suggesting that this variable is the key factor affecting the adaptive variation of S. scardica.

Genetic Diversity of Croatian Common Bean Landraces.

In Croatia, the majority of the common bean production is based on local landraces, grown by small-scale farmers in low input production systems. Landraces are adapted to the specific growing conditions and agro-environments and show a great morphological diversity. These local landraces are in danger of genetic erosion caused by complex socio-economic changes in rural communities. The low profitability of farms and their small size, the advanced age of farmers and the replacement of traditional landraces with modern bean cultivars and/or other more profitable crops have been identified as the major factors affecting genetic erosion. Three hundred accessions belonging to most widely used landraces were evaluated by phaseolin genotyping and microsatellite marker analysis. A total of 183 different multi-locus genotypes in the panel of 300 accessions were revealed using 26 microsatellite markers. Out of 183 accessions, 27.32% were of Mesoamerican origin, 68.31% of Andean, while 4.37% of accessions represented putative hybrids between gene pools. Accessions of Andean origin were further classified into phaseolin type II ("H" or "C") and III ("T"), the latter being more frequent. A model-based cluster analysis based on microsatellite markers revealed the presence of three clusters in congruence with the results of phaseolin type analysis.

Genetic Diversity and Demographic History of Wild and Cultivated/Naturalised Plant Populations: Evidence from Dalmatian Sage (Salvia officinalis L., Lamiaceae).

Dalmatian sage (Salvia officinalis L., Lamiaceae) is a well-known aromatic and medicinal Mediterranean plant that is native in coastal regions of the western Balkan and southern Apennine Peninsulas and is commonly cultivated worldwide. It is widely used in the food, pharmaceutical and cosmetic industries. Knowledge of its genetic diversity and spatiotemporal patterns is important for plant breeding programmes and conservation. We used eight microsatellite markers to investigate evolutionary history of indigenous populations as well as genetic diversity and structure within and among indigenous and cultivated/naturalised populations distributed across the Balkan Peninsula. The results showed a clear separation between the indigenous and cultivated/naturalised groups, with the cultivated material originating from one restricted geographical area. Most of the genetic diversity in both groups was attributable to differences among individuals within populations, although spatial genetic analysis of indigenous populations indicated the existence of isolation by distance. Geographical structuring of indigenous populations was found using clustering analysis, with three sub-clusters of indigenous populations. The highest level of gene diversity and the greatest number of private alleles were found in the central part of the eastern Adriatic coast, while decreases in gene diversity and number of private alleles were evident towards the northwestern Adriatic coast and southern and eastern regions of the Balkan Peninsula. The results of Ecological Niche Modelling during Last Glacial Maximum and Approximate Bayesian Computation suggested two plausible evolutionary trajectories: 1) the species survived in the glacial refugium in southern Adriatic coastal region with subsequent colonization events towards northern, eastern and southern Balkan Peninsula; 2) species survived in several refugia exhibiting concurrent divergence into three genetic groups. The insight into genetic diversity and structure also provide the baseline data for conservation of S. officinalis genetic resources valuable for future breeding programmes.

Genetic diversity and structure of Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir./Sch./Bip., Asteraceae) within the Balkan refugium.

Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir./Sch./Bip.) is an outcrossing, perennial insecticidal plant, restricted to the eastern Adriatic coast (Mediterranean). Amplified fragment-length polymorphisms (AFLP) were used to investigate the genetic diversity and structure within and among 20 natural plant populations. The highest level of gene diversity, the number of private alleles and the frequency down-weighted marker values (DW) were found in northern Adriatic populations and gradually decreased towards the southern boundary of the species range. Genetic impoverishment of these southern populations is most likely the result of human-related activities. An analysis of molecular variance (AMOVA) indicated that most of the genetic diversity was attributed to differences among individuals within populations (85.78%), which are expected due to the outcrossing nature of the species. A Bayesian analysis of the population structure identified two dominant genetic clusters. A spatial analysis of the genetic diversity indicated that 5.6% of the genetic differentiation resulted from isolation by distance (IBD), while 12.3% of the genetic differentiation among populations followed the pattern of isolation by environmental distance (IBED). Knowledge of the genetic diversity patterns of the natural populations and the mechanism behind these patterns is required for the exploitation and possible conservation management of this endemic and economically important species.