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Sweet pepper (Capsicum annuum L.) is an annual herbaceous plant of the Solanaceae family, which is one of the most important agricultural crops due to its exceptional economic importance, the nutritional value of its fruits and its confirmed antioxidant activity. The aim of this work was to determine the influence of the application of calcite-based biostimulants during the growing season on the germination of sweet pepper seeds. In the 2019 growing season, the calcite-based biostimulants Eco Green and Zeogreen+P were foliar applied on two sweet pepper cultivars (bell fruit type – ‘Šorokšari’ and capia fruit type – ‘Kurtovska kapija’). At the end of the growing season, 300 seeds from each cultivar were collected (100 seeds from plants treated with each of the two biostimulants and 100 seeds from plants that had not been treated during the growing season - control) and placed for germination under controlled conditions at a constant temperature of 25 oC and a light regime of 16/8 h day/night. At the end of the experimental period, the seed germination parameters were calculated. It was found that the application of the calcite-based biostimulant Eco Green had a statistically significant positive effect on the germinability (G, %), mean germination rate (MR), the uncertainty of the germination process (U) and germination index (GI) in both cultivars and on mean germination time (MGT, day) in the cultivar ‘Kurtovska kapija’. The calcite-based biostimulant Zeogreen had no statistically significant positive effect on the germination parameters, except for the germinability (G, %) in the cultivar ‘Kurtovska kapija’.

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.

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 Sićevo 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.

Abstract Southeast Europe (SEE) is a very important maize-growing region, comparable to the Corn belt region of the United States, with similar dent germplasm (dent by dent hybrids). Historically, this region has undergone several genetic material swaps, following the trends in the US, with one of the most significant swaps related to US aid programs after WWII. The imported accessions used to make double-cross hybrids were also mixed with previously adapted germplasm originating from several more distant OPVs, supporting the transition to single cross-breeding. Many of these materials were deposited at the Maize Gene Bank of the Maize Research Institute Zemun Polje (MRIZP) between the 1960s and 1980s. A part of this Gene Bank (572 inbreds) was genotyped with Affymetrix Axiom Maize Genotyping Array with 616,201 polymorphic variants. Data were merged with two other genotyping datasets with mostly European flint (TUM dataset) and dent (DROPS dataset) germplasm. The final pan-European dataset consisted of 974 inbreds and 460,243 markers. Admixture analysis showed seven ancestral populations representing European flint, B73/B14, Lancaster, B37, Wf9/Oh07, A374, and Iodent pools. Subpanel of inbreds with SEE origin showed a lack of Iodent germplasm, marking its historical context. Several signatures of selection were identified at chromosomes 1, 3, 6, 7, 8, 9, and 10. The regions under selection were mined for protein-coding genes and were used for gene ontology (GO) analysis, showing a highly significant overrepresentation of genes involved in response to stress. Our results suggest the accumulation of favorable allelic diversity, especially in the context of changing climate in the genetic resources of SEE.

In Spain, several local studies have highlighted the likely presence of unknown olive cultivars distinct from the approximately 260 ones previously described in the literature. Furthermore, recent advancements in identification techniques have significantly enhanced in terms of efficacy and precision. This scenario motivated a new nationwide prospecting effort aimed at recovering and characterizing new cultivated germplasm using high-throughput molecular markers. In the present study, the use of 96 EST-SNP markers allowed the identification of a considerable amount of new material (173 new genotypes) coming from areas with low intensification of production in different regions of Spain. As a result, the number of distinct national genotypes documented in the World Olive Germplasm Bank of IFAPA, Córdoba (WOGBC-ESP046) increased to 427. Likewise, 65 and 24 new synonymy and homonymy cases were identified, respectively. This rise in the number of different national cultivars allowed to deepen the knowledge about the underlying genetic structure. The great genetic variability of Spanish germplasm was confirmed, and a new hot spot of diversity was identified in the northern regions of La Rioja and Aragon. Analysis of the genetic structure showed a clear separation between the germplasm of southern and northern-northeastern Spain and indicated a significantly higher level of admixture in the latter. Given the expansion of modern olive cultivation with only a few cultivars, this cryptic germplasm is in great danger of disappearing. This underlines the fact that maintaining as many cultivars as possible will increase the genetic variability of the olive gene pool to meet the future challenges of olive cultivation.

Teucrium montanum L. and T. capitatum L. are two plant species with overlapping distribution in the Balkan Peninsula, especially in Croatia, where several occurrences of their putative hybrid species T. × rohlenae have been recorded. Next-generation sequencing of both species and de novo assembly was carried out resulting in 120 contigs for T. montanum and 1685 contigs for T. capitatum assembled. The Dig-up primers pipeline was used for SSR mining of both assemblies, applying different criteria that resulted in 112 SSR candidates for testing. A subset of 41 SSRs was selected and after two rounds of testing, twelve SSRs were developed and characterized. A total of 232 alleles were detected with 5 to 29 alleles per locus, based on the test sample. The genetic diversity analysis of three Teucrium taxa from a single location revealed a higher level of diversity in T. montanum than in T. capitatum with intermediate values for the hybrid species. The NeighborNet diagram and genetic structure analysis grouped the taxa into two separate clusters, the first of which consisted exclusively of T. montanum, while the other was composed of intermixed T. capitatum and T. × rohlenae individuals. The availability of SSR markers for two Teucrium taxa will allow in-depth analysis of genetic diversity and structure, as well as molecular identification of their putative hybrids in the future.

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.

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.

Abstract 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.

Marginal populations are usually smaller and more isolated and grow in less favourable conditions than those at the distribution centre. The variability of these populations is of high importance, as it can support the adaptations needed for the conditions that they grow in. In this research, the morphological variability of eight Tatar maple (Acer tataricum L. subsp. tataricum) populations was analysed. Tatar maple is an insect-pollinated and wind-dispersed shrub/tree, whose northwestern distribution edge is in southeastern Europe. Morphometric methods were used to analyse the variability of the populations using leaf and fruit morphology. The research revealed significant differences between and within populations. Furthermore, differences in the distribution of the total variability were noted, which suggest that different evolutionarily factors affect different plant traits. Correlation analysis confirmed a weak dependency between the vegetative and generative traits. In addition, no evidence was found for the presence of isolation by environment (IBE). However, the Mantel test for isolation by distance (IBD) was significant for the leaf morphometric traits and non-significant for the fruit morphometric traits. Being the marginal leading-edge populations, they are younger and were less likely to have had time for adaptation to local environments, which would have resulted in the development of IBE. Overall, edge populations of Tatar maple were characterised by great morphological variability, which helps these populations in their response to the intensive selective pressures they face in their environment.

Croatian pedunculate oak (Quercus robur L.) populations represent southern range peripheral populations, often viewed as sources of valuable diversity and drought-resistant ecotypes. At the same time, they endure stronger selection pressures as a result of climate change. The leaves of 20 individuals per population (17) were sampled in a field trial and analyzed using 10 nuclear and 9 chloroplast SSRs to determine the level of intrapopulation genetic variability and genetic differentiation. Analysis with nSSRs revealed deviation from HWE in seven populations. AMOVA showed a high intra-population diversity (98.53%) and a small but statistically significant inter-population differentiation. Isolation by distance explained 19.6% of differentiation. Average FST between populations was low (0.013) compared with usual values for peripheral populations. Populations were rich in cpSSR haplotypes, confirming the hotspot of diversity caused by the encounter of recolonization routes. Unbiased haplotype diversity (HE) from 9 chloroplast SSRs and 325 individuals was (HE = 0.440). Sixty-six different haplotypes were grouped in three maternal lineages by both a median-joining network and a neighbor-joining algorithm. AMOVA for cpSSRs showed statistically significant diversity among populations (70.23%), suggesting genetic differentiation, but also a probable anthropogenic effect. AMOVA of nSSRs within and between lineages showed that original recolonization patterns of nuclear diversity were subsequently erased by gene flow.

Abstract 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 F ST - outlier analysis (Mcheza and BayeScan) and genome-environment association analysis (Samβada, 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.

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.

Due to climate extremes and limited natural resources, especially water, we can expect increased demand in the future for species that can better tolerate climate extremes such as drought. One potentially valuable horticultural species is the endemic species of the Dinaride Mountains Micromeria croatica (Pers.) Schott (family Lamiaceae). It grows in the crevices of carbonate rocks, extending from an altitude of 150 m to more than 2000 m. This study aims to provide additional insight into the genetic and morphological diversity of this endemic species, focusing on valuable horticultural traits. To achieve this goal, morphological and molecular analyses were performed on ten natural populations. Through STRUCTURE and PCoA analyses, ten M. croatica populations were placed into western and eastern genetic groups, with several individuals from western populations assigned to the eastern group and vice versa. These atypical individuals assigned to the new genetic group by BAPS analysis indicate gene flow between western and eastern populations. Similarly, an analysis of molecular variance revealed fewer genetic differences than within studied populations. Both PCA and CANDISC analysis based on eleven morphological traits largely confirmed the existence of two slightly different genetic groups. Two populations containing plants with the most flowers per shoot, one with white-flowered individuals, one with the roundest leaves, and one with the narrowest leaves proved to be the most horticulturally valuable. The genetic and morphological variability found should be a sufficient basis for the potential selection of M. croatica populations and individuals for horticultural purposes.

Essential oils from seven populations of Dalmatian sage from the North of Albania were extracted and analyzed according European Pharmacopeia 7.0. The yield differed from 1.95 to 2.75% v/w. The GC-FID-MS analysis of the sage oil revealed 42 constituents. Camphor (20.50-29.74%), followed by α-thujone (19.87-24.29%), 1,8-cineole (6.88-12.64%) and β-thujone (4.00-9.14%) were predominant constituents in all analyzed populations, representing 55.70-68.00% of the total components. All of the analyzed sage populations from northern Albania comply with ISO 6571/2008 standard and European Pharmacopeia 7.0. for Dalmatian sage essential oil content, while only four populations met the requirements of the ISO 9909/1997 standard for Dalmatian sage essential oil’s composition.

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.

Common bean (Phaseolus vulgaris L.) exhibits a wide range of seed coat colors and this morphological trait is widely used in cultivar identification and assessment of diversity within this species. With an advancement in technology and informatics, new methods of assessing seed color are emerging in addition to traditionally used visual observation. Due to a great variety of color measuring techniques, the evaluation of the agreement between methods is needed prior to using the methods interchangeably. Seed coat color in terms of CIE L*a*b* color coordinates of 100 common bean accessions belonging to five mono-colored landraces was assessed using two methods, colorimeter and Computer vision. The percentage difference between the two methods across all samples for L* color coordinate was 5.81%, for a* color coordinate 23.32% and for b* color coordinate 44.44%. According to Bland-Altman difference plot there is a considerable lack of agreement between the two methods. However, using stepwise discriminant analysis revealed that colorimeter method correctly classified 97% of accessions into their respective landrace, while the classification success of the Computer vision was 99%.

Abstract Grass pea (Lathyrus sativus L.) is an annual legume species, phylogenetically close to pea (Pisum sativum L.), that may be infected by Fusarium oxysporum f. sp. pisi (Fop), the causal agent of fusarium wilt in peas with vast worldwide yield losses. A range of responses varying from high resistance to susceptibility to this pathogen has been reported in grass pea germplasm. Nevertheless, the genetic basis of that diversity of responses is still unknown, hampering its breeding exploitation. To identify genomic regions controlling grass pea resistance to fusarium wilt, a genome‐wide association study approach was applied on a grass pea worldwide collection of accessions inoculated with Fop race 2. Disease responses were scored in this collection that was also subjected to high‐throughput based single nucleotide polymorphisms (SNP) screening through genotyping‐by‐sequencing. A total of 5,651 high‐quality SNPs were considered for association mapping analysis, performed using mixed linear models accounting for population structure. Because of the absence of a fully assembled grass pea reference genome, SNP markers’ genomic positions were retrieved from the pea's reference genome v1a. In total, 17 genomic regions were associated with three fusarium wilt response traits in grass pea, anticipating an oligogenic control. Seven of these regions were located on pea chromosomes 1, 6, and 7. The candidate genes underlying these regions were putatively involved in secondary and amino acid metabolism, RNA (regulation of transcription), transport, and development. This study revealed important fusarium wilt resistance favorable grass pea SNP alleles, allowing the development of molecular tools for precision disease resistance breeding.

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.

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.

Lavandin (Lavandula × intermedia Emeric ex Loisel.) was brought to the Island of Hvar (Croatia) in the 1920s, coinciding with the beginning of large-scale cultivation of lavandin in France. Although the cultivation of lavandin and the production of essential oils are of great importance worldwide, the genetic diversity of lavandin has been little studied. We performed an AFLP-based genetic analysis that included the landraces ‘Bila’ and ‘Budrovka’ and two lavandin cultivars from France ‘Grosso’ and ‘Abrialis’, as well as the parental species of the hybrid (L. angustifolia and L. latifolia). Distance-based cluster analysis revealed the existence of the third landrace, named ‘Budrovka Sveti Nikola’. This result was confirmed by the model-based cluster analyses implemented in STRUCTURE and BAPS, where the optimal number of clusters was three. ‘Budrovka’ clearly separated from all other samples, while ‘Bila’ and ‘Budrovka Sveti Nikola’ showed some degree of admixture, indicating ancestral polyclonality. The landrace ‘Bila’ showed higher polymorphism than ‘Budrovka’ and ‘Budrovka Sveti Nikola’. Analysis of molecular variance (AMOVA) showed that genetic diversity (56.63%) was higher within landraces than among (43.37%). This research will provide a basis for conservation of the Island landraces and will help in the establishment of a high-quality regional brand.

Olea europaea ‘Galega vulgar’ variety is a blend of West and Central Mediterranean germplasm with cultivated-wild admixture characteristics. ‘Galega vulgar’ is known for its high rusticity and superior-quality olive oil, being the main Portuguese variety with high impact for bioeconomy. Nevertheless, it has been replaced by higher-yielding and more adapted to intensive production foreign varieties. To clarify the potential ancestral origin, genetic diversity evolution, and existing genetic relationships within the national heritage of ‘Galega vulgar’, 595 trees, belonging to ancient and centenary age groups and prospected among ten traditional production regions, were characterized using 14 SSR markers after variety validation by endocarp measurements. Ninety-five distinguishable genets were identified, revealing the presence of a reasonable amount of intra-genetic and morphological variability. A minimum spanning tree, depicting the complete genealogy of all identified genets, represented the ‘Galega vulgar’ intra-varietal diversity, with 94% of the trees showing only a two-allele difference from the most frequent genet (C001). Strong correlations between the number of differentiating alleles from C001, the clonal size, and their net divergence suggested an ancestral monoclonal origin of the ‘Galega vulgar’, with the most frequent genet identified as the most likely origin of all the genets and phenotypic diversification occurring through somatic mutations. Genetic erosion was detected through the loss of some allele combinations across time. This work highlights the need to recover the lost diversity in this traditional olive variety by including ancient private genets (associated with potential adaptation traits) in future breeding programs and investing in the protection of these valuable resources in situ by safeguarding the defined region of origin and dispersion of ‘Galega vulgar’. Furthermore, this approach proved useful on a highly diverse olive variety and thus applicable to other diverse varieties due either to their intermediate nature between different gene pools or to the presence of a mixture of cultivated and wild traits (as is the case of ‘Galega vulgar’).

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.

Winter savory (Satureja montana L.) is an important medicinal, aromatic, and honey plant. In Croatia, it is widely distributed along the Adriatic coast, where it is frequently exposed to droughts. First, the winter savory natural population with the highest germination across different drought treatments after hydropriming was selected. Nine hundred seeds from each of the three natural populations (P1, P2, and P3) were hydroprimed (dH2O) for 48 h. The seeds were then germinated in drought treatments with different concentrations of polyethylene glycol (PEG 6000) (−0, −0.2, −0.4, −0.8, −1.2, −1.6, −2, −2.5, −3.0 MPa). Since P1 showed the best results in germination parameters, it was used for the second phase of the experiment, where the effect of hormonal priming (100 and 400 ppm GA3, 48 h in the dark) and osmopriming (0.2% and 0.6% w/v KNO3, 72 h in the dark) on seed germination and seedling morphological parameters of the selected winter savory population under drought stress conditions (−0.8 and −2.5 MPa) was evaluated. Although winter savory grows in dry areas, this study showed that extremely dry conditions (−3.0 and −2.5 MPa) negatively affected seed germination, but this effect can be mitigated by priming treatments, especially with the hormonal priming (GA3 400 ppm).

Micronutrient malnutrition is one of the main public health problems in many parts of the world. This problem raises the attention of all valuable sources of micronutrients for the human diet, such as common bean (Phaseolus vulgaris L.). In this research, a panel of 174 accessions representing Croatian common bean landraces was phenotyped for seed content of eight nutrients (N, P, K, Ca, Mg, Fe, Zn, and Mn), and genotyped using 6,311 high-quality DArTseq-derived SNP markers. A genome-wide association study (GWAS) was then performed to identify new genetic sources for improving seed mineral content. Twenty-two quantitative trait nucleotides (QTN) associated with seed nitrogen content were discovered on chromosomes Pv01, Pv02, Pv03, Pv05, Pv07, Pv08, and Pv10. Five QTNs were associated with seed phosphorus content, four on chromosome Pv07, and one on Pv08. A single significant QTN was found for seed calcium content on chromosome Pv09 and for seed magnesium content on Pv08. Finally, two QTNs associated with seed zinc content were identified on Pv06 while no QTNs were found to be associated with seed potassium, iron, or manganese content. Our results demonstrate the utility of GWAS for understanding the genetic architecture of seed nutritional traits in common bean and have utility for future enrichment of seed with macro– and micronutrients through genomics-assisted breeding.

Basil is one of the most widespread aromatic and medicinal plants, which is often grown in drought- and salinity-prone regions. Often co-occurrence of drought and salinity stresses in agroecosystems and similarities of symptoms which they cause on plants complicates the differentiation among them. Development of automated phenotyping techniques with integrative and simultaneous quantification of multiple morphological and physiological traits enables early detection and quantification of different stresses on a whole plant basis. In this study, we have used different phenotyping techniques including chlorophyll fluorescence imaging, multispectral imaging, and 3D multispectral scanning, aiming to quantify changes in basil phenotypic traits under early and prolonged drought and salinity stress and to determine traits which could differentiate among drought and salinity stressed basil plants. Ocimum basilicum “Genovese” was grown in a growth chamber under well-watered control [45–50% volumetric water content (VWC)], moderate salinity stress (100 mM NaCl), severe salinity stress (200 mM NaCl), moderate drought stress (25–30% VWC), and severe drought stress (15–20% VWC). Phenotypic traits were measured for 3 weeks in 7-day intervals. Automated phenotyping techniques were able to detect basil responses to early and prolonged salinity and drought stress. In addition, several phenotypic traits were able to differentiate among salinity and drought. At early stages, low anthocyanin index (ARI), chlorophyll index (CHI), and hue (HUE2D), and higher reflectance in red (RRed), reflectance in green (RGreen), and leaf inclination (LINC) indicated drought stress. At later stress stages, maximum fluorescence (Fm), HUE2D, normalized difference vegetation index (NDVI), and LINC contribute the most to the differentiation among drought and non-stressed as well as among drought and salinity stressed plants. ARI and electron transport rate (ETR) were best for differentiation of salinity stressed plants from non-stressed plants both at early and prolonged stress.

Almond-leaved willow (Salix triandra L., Salicaceae) is a dioecious shrub, rarely a small tree that grows under various environmental conditions. We examined the population structure of 12 populations of almond-leaved willow using nine leaf morphological traits and specific leaf area. Populations were selected from a range of habitats, from continental to the sub-Mediterranean zone, to examine the influence of environmental conditions (climate and altitude) and geographic distance on leaf variability. Significant differences were confirmed among all populations for all traits, with significant correlations between geographic location of populations and morphological traits, and between environmental conditions and morphological traits. Large-leaved populations were found in continental and sub-Mediterranean climates, while small-leaved populations were found in higher elevations and smaller karstic rivers. In addition, populations from floodplains showed greater variability than populations from the karstic habitats, indicating a positive influence of lowland habitats and possible underlying differences in gene pool size. In conclusion, we found that environmental conditions and geographical distances in addition to genetic drift, are the main influences on the variability in almond-leaved willow, with the species showing a high level of plasticity and adaptation to local environmental conditions.

Olive, the emblematic Mediterranean fruit crop, owns a great varietal diversity, which is maintained in ex situ field collections, such as the World Olive Germplasm Bank of Córdoba (WOGBC), Spain. Accurate identification of WOGBC, one of the world’s largest collections, is essential for efficient management and use of olive germplasm. The present study is the first report of the use of a core set of 96 EST-SNP markers for the fingerprinting of 1273 accessions from 29 countries, including both field and new acquired accessions. The EST-SNP fingerprinting made possible the accurate identification of 668 different genotypes, including 148 detected among the new acquired accessions. Despite the overall high genetic diversity found at WOGBC, the EST-SNPs also revealed the presence of remarkable redundant germplasm mostly represented by synonymy cases within and between countries. This finding, together with the presence of homonymy cases, may reflect a continuous interchange of olive cultivars, as well as a common and general approach for their naming. The structure analysis revealed a certain geographic clustering of the analysed germplasm. The EST-SNP panel under study provides a powerful and accurate genotyping tool, allowing for the foundation of a common strategy for efficient safeguarding and management of olive genetic resources.

Intra-host populations of plant RNA viruses are genetically diverse. Due to frequent reinfections, these populations often include phylogenetically distant variants that may have different biological properties. Random selection of variants, which occurs during host-to-host virus transmission, may affect isolate pathogenicity. Accurate characterization of genetic variants in intra-host virus populations is therefore epidemiologically important. In routine molecular characterization of Citrus tristeza virus (CTV) isolates, common practice is to analyze only a few cDNA clones per isolate. In the present study, based on the characterization of CTV populations displaying different levels of genetic diversity, we evaluated if analyzing large numbers of clones increased diversity parameters. A sequential sampling approach, based on analysis of genetic richness. is proposed for determining the minimal sample size required to obtain reliable information on levels of CTV genetic diversity.

Sweet chestnut (Castanea sativa Mill., Fagaceae) is one of the oldest cultivated tree species in the Mediterranean, providing multiple benefits, and, since it has edible seeds, it represents an interesting model species for the research of morphological and chemical variability. In this study, morphometric methods and chemical analyses were used to quantify the extent of differences in phenotypic and nutritional traits between eight natural populations of sweet chestnut from different environmental conditions, where different management types are applied, high-forest and coppice. The samples were collected from the Prealps in Italy to the western part of Bosnia and Herzegovina. In total, 31 nut and kernel morphometric and nutritional traits were studied on 160 trees, and various multivariate statistical analyses were used to study intra- and interpopulation variations. Both analyses, morphometric and chemical, revealed a similar pattern of diversity, with morphological and chemical variability not associated with geographic or environmental variables. In addition, we found significant correlations between morphometric and chemical data. High phenotypic variability was determined both among and within the studied populations, and all populations had a similar level of diversity. The results of the analysis of morphological and chemical diversity can have many practical applications for the management, production, and conservation of the sweet chestnut genetic resources for nut production.

Common or English yew (Taxus baccata L., Taxaceae) is a conifer species, native to Europe, northern Africa, Asia Minor and Caucasus. It is a dioecious, wind-pollinated and animal-dispersed tree, known for its high-quality wood and medicinal properties, albeit poisonous. The species is rare and has been legally protected at the European and national levels. In addition, its low population density and disjunct character of distribution have reinforced the need for its protection as regeneration is mostly lacking. The aim of this study was to phenotypically characterise the north-western Balkan yew populations. Phenotypic diversity was examined for seven populations, using morphometric analysis of nine phenotypic traits of needles. Descriptive and multivariate statistical analyses were conducted to evaluate the inter- and intrapopulation variability. In addition, to test correlations between geographic, climatic and phenotypic data, Mantel test was used. We identified a geographic structure across studied populations that exhibited high levels of variability on intra- and interpopulation levels. Two groups of populations have been defined and are consistent with previously described genetic divergent lineages from separate refugia. In addition, a significant correlation between phenotypic and geographic data were revealed, i.e., isolation by distance (IBD). However, the Mantel test revealed no significant correlation between morphometric and environmental data. In conclusion, our data reveal that the historical events and persistent IBD acted in combination to produce the morphological patterns observed in common yew populations in the north-western part of the Balkan Peninsula. Finally, we suggested conservation measures to be implemented on a stand level, with habitat preservation as the main goal. In addition, ex situ conservation should be considered, both in the form of collections and urban planting, as both provide additional gene pool reserves.

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.

Pyrethrin is a specialized metabolite of Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), Asteraceae, known worldwide as an effective bioinsecticide. It consists of six active compounds: Pyrethrin I and II, cinerin I and II, and jasmolin I and II. Pyrethrin accumulates mainly in the flower heads and its content depends on numerous factors, such as the flower developmental stage. This study aims to investigate the accumulation patterns of six pyrethrin compounds in the flower heads of Dalmatian pyrethrum over the eight developmental stages (FS1 to FS8), and to make a comparison in six natural populations. Ultrasound assisted extraction was used to extract the pyrethrin, while qualitative and quantitative analysis was performed by High performance liquid chromatography. The accumulation patterns of different pyrethrin compounds were generally similar and also synchronous between different populations, while the pyrethrin I/pyrethrin II ratio showed irregular patterns. In all populations studied, the highest increase of all compounds was observed from FS1 to FS2. Their concentration continuously increased, reaching the highest values at FS4 stage (2–5 rows of open disc flowers), and generally decreased gradually towards FS6 or FS7 and then stagnated until the FS8 stage. Despite the very similar accumulation pattern of pyrethrin compounds in the different populations, the significant differences in their content suggest a different genetic background. Knowledge of the dynamics of pyrethrin compounds accumulation across flower development stages is valuable for determining the optimal harvest time of pyrethrum flower heads.

Background and Purpose: Natural stands of pedunculate oak in Croatia have been delineated in seed areas, zones and regions. The current bylaw recommends that the transfer of reproductive material remains limited within zones, but that it is permitted within areas. Clonal seed orchards (CSOs) of pedunculate oak were established to increase genetic quality of seed and to acquire a more regular seed yield than in natural stands. In total 150 plus trees were selected within three seed regions. The selection included a number of favourable traits of tree size and stem quality. Three CSOs were planted with grafted plus-trees. We aimed to establish whether these orchards encompass enough genetic diversity to potentially produce genetically improved and sufficiently diverse reproductive material. We also wanted to assess neutral genetic differentiation between these orchards and compare it with the genetic diversity obtained from chloroplast DNA markers, depicting conserved lineages from recolonization routes. We wanted to investigate spatial genetic structure in the area of our research and provide additional information on the transfer of forest reproductive material. Materials and Methods: Leaves were collected from all clones in the CSOs. Total genomic DNA was extracted and clones were analysed with eight nuclear and ten chloroplast microsatellite markers. Spatial autocorrelation analysis was performed with nuclear microsatellite data and original plus trees’ coordinates, for each CSO separately, to determine whether shared favourable traits among the selected plus trees in smaller distances are the results of relatedness, which the sampling strategy tried to avoid. Results: We found 28 chloroplast haplotypes belonging to four maternal lineages, and significant differentiation between CSOs, indicating origin from different refuges. Nuclear microsatellites’ diversity in the CSOs is quite high and comparable to diversity found within a recent study of Croatian natural populations. Nuclear microsatellites did not show genetic differentiation between CSOs, i.e. between the seed regions and seed zones they represent. No genetic differentiation was found with nuclear microsatellites among haplotypic lineages. We found no genetic structure within the analysed regions. Conclusions: Lack of differentiation between CSOs found with nuclear microsatellites confirms the permission for transfer of reproductive material between zones within the seed area 1 - Lowland Forests. If original differentiation between chloroplast haplotypic lineages was present after recolonization, it was erased by subsequent gene flow. Lack of genetic structure, with nuclear microsatellites within regions indicates successful sampling strategy.

Almond-leaved pear (Pyrus spinosa Forssk., Rosaceae) is a scientifically poorly researched and often overlooked Mediterranean species. It is an insect-pollinated and animal-dispersed spiny, deciduous shrub or a small tree, with high-quality wood and edible fruits. The aim of the study was to assess the phenotypic diversity of almond-leaved pear in the eastern Adriatic region. The examination of phenotypic diversity was based on a morphometric analysis of 17 populations using ten phenotypic traits of leaves. Varieties of multivariate statistical analyses were conducted to evaluate the within- and among-population diversity. In addition, the Mantel tests were used to test the correlations between geographic, environmental, and phenotypic differences among populations. High phenotypic variability was determined both among and within the studied populations. Leaf-size-related traits proved to be the most variable ones, in contrast to more uniform leaf shape traits. Furthermore, three groups of populations were detected using multivariate statistical analyses. The first group included trees from northern- and southernmost populations characterized by high annual precipitation. However, the trees from the second and third group were highly overlapped without a clear geographical pattern. In addition, we revealed that both environmental and geographical interactions proved to be responsible for the patterns of phenotypic variation between almond-leaved pear populations, indicating significant isolation by environment (IBE) and isolation by distance (IBD) patterns. Overall, our results provide useful information about phenotypic diversity of almond-leaved pear populations for further conservation, breeding, and afforestation programs.

Common bean is one of the most cultivated and consumed grain legumes worldwide, showing a high level of genetic diversity. Here is presented a detailed review of development and mapping of simple sequence repeats (SSRs, microsatellite markers) in the common bean. In the last 25 years, common bean has been the subject of numerous genetic studies, in which the identification and use of SSRs were conducted, and lead to the development of genetic maps. First genetic maps of common bean have been developed in the 1990s and were based on different molecular markers, and included domestication genes and important agronomic traits. Later, SSRs allowed the genetic mapping of more narrow crosses that are often of interest in plant breeding. Most genetic maps have been correlated with the core map established in the recombinant inbred population BAT93 x Jalo EEP558, and includes different markers, RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphism), and SSRs in particular. More than 2,000 SSR markers are available for the common bean and they are an important tool to evaluate the genetic diversity of common bean landraces. SSRs are also useful to evaluate intra-specific diversity within the genus Phaseolus.

Hop (Humulus lupulus L.) is used in the brewing industry as a source of compounds responsible for the bitterness, aroma, and preservative properties of beer. In this study, we used microsatellite markers to investigate genetic diversity and genetic differentiation of wild hop populations sampled in the northwestern part of Croatia. Analysis of 12 microsatellite loci revealed high diversity and weak population differentiation among wild hop populations. A total of 152 alleles were determined with an average of 12.67 alleles per locus. Observed heterozygosity ranged from 0.689 to 0.839 (average 0.767) and expected heterozygosity ranged from 0.725 to 0.789 (average 0.760). A total of 38 private alleles were detected. The data suggest that H. lupulus populations are not affected by recent bottlenecks. The degree of genetic differentiation among populations was low and not significant for most pairwise FST values, except for the pair of geographically most distant populations. The results did not indicate the existence of genetic structure among the sampled populations. The high genetic diversity and low differentiation among populations, combined with the absence of isolation by distance, indicate the existence of substantial gene flow among wild hop populations. Therefore, extensive sampling per population is clearly required to assess the genetic diversity of hop populations. Sampling strategies involving sampling across a large number of localities represented by only a few samples could lead to erroneous conclusions.

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.

Sweet pepper (Capsicum annuum L.) is one of the most important vegetable crops in the world because of the nutritional value of its fruits and its economic importance. Calcium (Ca) improves the quality of sweet pepper fruits, and the application of calcite nanoparticles in agricultural practice has a positive effect on the morphological, physiological, and physicochemical properties of the whole plant. The objectives of this study were to investigate the effect of commercial calcite nanoparticles on yield, chemical, physical, morphological, and multispectral properties of sweet pepper fruits using a combination of conventional and novel image-based nondestructive methods of fruit quality analysis. In the field trial, two sweet pepper cultivars, i.e., Šorokšari and Kurtovska kapija, were treated with commercial calcite nanoparticles (at a concentration of 3% and 5%, calcite-based foliar fertilizer (positive control), and water (negative control) three times during vegetation). Sweet pepper fruits were harvested at the time of technological and physiological maturity. Significant differences were observed between pepper cultivars as well as between harvests times. In general, application of calcite nanoparticles reduced yield and increased fruit firmness. However, different effects of calcite nanoparticles were observed on almost all properties depending on the cultivar. In Šorokšari, calcite nanoparticles and calcite-based foliar fertilizers significantly increased N, P, K, Mg, Fe, Zn, Mn, and Cu at technological maturity, as well as P, Ca, Mg, Fe, Zn, Mn, Cu, and N at physiological maturity. However, in Kurtovska kapija, the treatments increased only Ca at technological maturity and only P at physiological maturity. The effect of treatments on fruit morphological properties was observed only at the second harvest. In Šorokšari, calcite nanoparticles (3% and 5%) increased the fruit length, minimal circle area, and minimal circle radius, and it decreased the fruit width and convex hull compared to the positive and negative controls, respectively. In Kurtovska kapija, calcite nanoparticles increased the fruit width and convex hull compared to the controls. At physiological maturity, lower anthocyanin and chlorophyll indices were found in Kurtovska kapija in both treatments with calcite nanoparticles, while in Šorokšari, the opposite effects were observed.

Selection for wheat (Triticum aestivum L.) grain quality is often costly and time-consuming since it requires extensive phenotyping in the last phases of development of new lines and cultivars. The development of high-throughput genotyping in the last decade enabled reliable and rapid predictions of breeding values based only on marker information. Genomic selection (GS) is a method that enables the prediction of breeding values of individuals by simultaneously incorporating all available marker information into a model. The success of GS depends on the obtained prediction accuracy, which is influenced by various molecular, genetic, and phenotypic factors, as well as the factors of the selected statistical model. The objectives of this article are to review research on GS for wheat quality done so far and to highlight the key factors affecting prediction accuracy, in order to suggest the most applicable approach in GS for wheat quality 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”, “Trešnjevac”), (2) Andean—indeterminate type (H2B1) (“Dan noć”, “Sivi”, “Puter”, ”Sivi prošarani”, “Trešnjevac”) and (3) Andean—determinate type (H3B2) (“Bijeli”, “Dan noć”, “Puter”, “Trešnjevac”, “Zelenčec”). 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.

Previous studies have reported promising differences in the quality of kernels from farmers' maize populations collected in a Portuguese region known to produce maize-based bread. However, several limitations have been identified in the previous characterizations of those populations, such as a limited set of quality traits accessed and a missing accurate agronomic performance evaluation. The objectives of this study were to perform a more detailed quality characterization of Portuguese farmers' maize populations; to estimate their agronomic performance in a broader range of environments; and to integrate quality, agronomic, and molecular data in the setting up of decision-making tools for the establishment of a quality-oriented participatory maize breeding program. Sixteen farmers' maize populations, together with 10 other maize populations chosen for comparison purposes, were multiplied in a common-garden experiment for quality evaluation. Flour obtained from each population was used to study kernel composition (protein, fat, fiber), flour's pasting behavior, and bioactive compound levels (carotenoids, tocopherols, phenolic compounds). These maize populations were evaluated for grain yield and ear weight in nine locations across Portugal; the populations' adaptability and stability were evaluated using additive main effects and multiplication interaction (AMMI) model analysis. The phenotypic characterization of each population was complemented with a molecular characterization, in which 30 individuals per population were genotyped with 20 microsatellites. Almost all farmers' populations were clustered into the same quality-group characterized by high levels of protein and fiber, low levels of carotenoids, volatile aldehydes, α- and δ-tocopherols, and breakdown viscosity. Within this quality-group, variability on particular quality traits (color and some bioactive compounds) could still be found. Regarding the agronomic performance, farmers' maize populations had low, but considerably stable, grain yields across the tested environments. As for their genetic diversity, each farmers' population was genetically heterogeneous; nonetheless, all farmers' populations were distinct from each other's. In conclusion, and taking into consideration different quality improvement objectives, the integration of the data generated within this study allowed the outline and exploration of alternative directions for future breeding activities. As a consequence, more informed choices will optimize the use of the resources available and improve the efficiency of participatory breeding activities.

Common bean (Phaseolus vulgaris L.) is among the most important grain legumes for human consumption worldwide. Portugal has a potentially promising common bean germplasm, resulting from more than five centuries of natural adaptation and farmers' selection. Nevertheless, limited characterization of this resource hampers its exploitation by breeding programs. To support a more efficient conservation of the national bean germplasm and promote its use in crop improvement, we performed, for the first time, a simultaneous molecular marker (21 microsatellites and a DNA marker for phaseolin-type diversity analysis) and seed and plant morphological characterization (14 traits) of 175 accessions from Portuguese mainland and islands traditional bean-growing regions. A total of 188 different alleles were identified and an average pairwise Cavalli-Sforza and Edwards' chord genetic distance of 0.193 was estimated among accessions. To relate the Portuguese germplasm with the global common bean diversity, 17 wild relatives and representative accessions from the Andean and Mesoamerican gene pools were evaluated at the molecular level. No correlation was detected between the variability found and the geographic origin of accessions. Structure analysis divided the collection into three main clusters. Most of the Portuguese accessions grouped with the race representatives and wild relatives from the Andean region. One third of the national germplasm had admixed genetic origin and might represent putative hybrids among gene pools from the two original centers of domestication in the Andes and Mesoamerica. The molecular marker-based classification was largely congruent with the three most frequent phaseolin haplotype patterns observed in the accessions analyzed. Seed and plant morphological characterization of 150 Portuguese common bean accessions revealed a clear separation among genetic structure and phaseolin haplotype groups of accessions, with seed size and shape and the number of locules per pod the most discriminant traits. Additionally, we used molecular and morphological data to develop a series of smaller core collections that, by maximizing the genetic and morphological diversity of the original collection, represents the Portuguese common bean germplasm with minimum repetitiveness. A core collection with 37 accessions contained 100% of the genetic variation found in the entire collection. This core collection is appropriate for a more detailed characterization and should be explored, as a priority, in national and international common bean breeding efforts. Furthermore, the identified intermediate accessions (with admixed genetic origin) may have novel genetic combinations useful in future bean breeding.

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.

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.

The plant height of wheat is an important quantitative trait, controlled by several genes with strong effect. However, in worldwide wheat breeding, only several of those genes have been used. Rht8 (Reduced Height Gene) is especially important in agro-climatic conditions of South-East Europe. Because of its close linkage with dwarfing gene Rht8, microsatellite marker gwm261 has been accepted as the diagnostic molecular marker for gene Rht8. In this study, allelic variability at the locus Xgwm261 for 122 Croatian and foreign wheat cultivars by means of microsatellite marker gwm261 was determined. A 192 base pairs allele at the locus Xgwm261 was found for 84 Croatian cultivars. The genetic heritage of Croatian cultivars at the locus Xgwm261 is the consequence of new parental components usage, carriers of short plant and early maturity attributes and the consequent selection of progeny with these traits during breeding process. The results of this research will be helpful in characterization of domestic wheat cultivars, as well as in more accurate selection of parents for hybridization purposes.