Search

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. Nasl. z nasl. zaslona. Opis vira z dne 25. 1. 2023. Bibliografija: str. 10-12. Abstract.

Europski pitomi kesten plemenita je listača od koje imamo višestruku gospodarsku korist. Osim jestivih plodova i kvalitetnog drva, kestenove šume su još od davnina predstavljale važan izvor različitih sirovina. Međutim, intenzivnim korištenjem kestenovih šuma i nasada te pojavom raka kestenove kore, sredinom prošloga stoljeća došlo je do njihovog propadanja i sušenja. Iz tih je razloga u mnogim europskim zemljama pokrenut niz multidisciplinarnih projekta kojima je svrha očuvanje genskih resursa pitomog kestena i njegovog povrataka na nekadašnje značajno mjesto u ruralnim područjima i šumskim ekosustavima. U ovom radu dan je pregled najznačajnijih ­dugoročnih strategija usmjerenih na očuvanje genofonda ove plemenite listače na europskoj razini, kao i prijedlog smjernica za očuvanje genofonda ove vrste in situ i ex situ metodama na području Hrvatske., Sweet chestnut (Castanea sativa Mill.) is a noble, multi-purpose hardwood species. In addition to ­edible fruits and high-quality wood, chestnut forests have been used as a source of various resources since the antiquity. Intensive management of forests and stands, however, together with the emergence of chestnut blight, has led to their decline and dieback. As a result, in numerous European countries multidisciplinary projects were initialized, with goals of conserving the genetic resources of the ­species, as well as reestablishing its significance in rural and forest ecosystems. In this paper, an overview of the most important long-term gene conservation strategies is presented, with suggestions for ­guidelines for application of in situ and ex situ conservation methods in Croatia.

The genus Teucrium is a source of beneficial bioactive phytochemicals and its use is well known in the traditional medicine of many countries. Studies on the wild populations of Teucrium species are scarce. Croatian flora numbers 12 accepted species of this genus, including a recently discovered hybrid between T. montanum and T. polium. This study aimed at sampling of T. montanum and T. polium in Split-Dalmatia County in an area where the two species grow in sympatry and the occurrence of hybrids is more likely. We collected six populations of both T. polium and T. montanum and found potential hybrids at five sites. The goal of our future research is to confirm the hybrids at the genetic level. This work contributes to in situ conservation by providing new insights into the status of wild populations of Teucrium species, supports ex situ conservation by including seed accessions to the Collection of Medicinal and Aromatic Plants ; and enables their further characterization and evaluation for use in future breeding programs.

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.

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.

AFLP (Amplified Fragment Length Polymorphism) and SSR (Single Sequence Repeats ; microsatellites) markers are widely used in plant genetic studies and it is fair to say that the fundamental insights into plant population dynamics and structure have been gained from the analysis of either AFLP or SSR data, or both. The AFLP technique requires no prior knowledge of the genetic make-up of a species and produces a large number of polymorphic fragments that are generally scored as dominant markers. SSRs, on the other hand, are species specific, codominant, multiallelic markers. In this study, we wanted to compare some basic population genetic parameters calculated on the basis of both marker systems.

Željeli smo istražiti u kojoj mjeri današnje stanovništvo poznaje samoniklo jestivo bilje što raste na otoku, kojih se starinskih recepata Zlarinke sjećaju iz prošlih vremena te pripremaju li se još uvijek neka jela od samoniklog bilja. Težište je bilo upravo na mješavini od nekoliko (često i više od desetak) različitih biljaka koje se zajedno beru i pripremaju za hranu, a na Zlarinu su poznate pod nazivom diblje zelje. U razgovorima smo saznali da se za mišancu, ili po zlarinski diblje zelje, prikupljalo više bilja nego što smo mogli i pomisliti. Najzanimljivija su nam, isto tako, bila izgubljena imena bilja. Tko danas zna prepoznati mačja muda i kozju bradu ili tustopizdu i kurcoglava? S nestankom znanja o bilju, nestaje i nazivlje, nestaju riječi. Tustopizda je zlarinski naziv za bijelu babljaču (Urospermum picroides (L.) Scop. ex F.W.Schmidt ; Cichoriaceae), a kurcoglav za zlaticu (Ranunculus ficaria L. ; Ranunculaceae). Naslov knjige ukazuje na činjenicu da je bijelu babljaču na Zlarinu lako naći, dok zlatica nije toliko česta. O toponimijskoj sintagmi Babina Guzica i drugim toponimima motiviranim genitalijama akademik Petar Šimunović je u članku Metaforičnost u toponimiji zadarsko-šibenskih otoka nadahnuto napisao: „Životare oni tako na marginama našeg društvenog poimanja uljudnosti, pokriveni smokvinim listom.

European wild pear (Pyrus pyraster (L.) Burgsd.) is a rare, noble hardwood species belonging to the Rosaceae family, with high-quality wood and great importance for biodiversity and ecosystems. It is native in almost all of Europe, and along with P. caucasica Fed., is considered to be the ancestor of the cultivated European pear (P. communis L.). In this study, we evaluate the morphological variability of the wild pear in Croatia. The inter- and intrapopulation variability was determined on the basis of ten measured morphological characteristics of leaves, using descriptive and multivariate statistical methods. The conducted research included 12 populations from both continental and Mediterranean parts of Croatia, and each population was represented by ten trees. The differences between the trees within populations, as well as the differences between populations, were confirmed for all studied characteristics. Biogeographical structuring of populations with a high level of among-tree variation within the populations was identified. In general, populations from the Mediterranean and the continental regions differed in the majority of the studied characteristics, with the Mediterranean populations possessing generally smaller leaves. This study provided insights into the variability of the European wild pear populations in Croatia, which is an important part of the efforts towards producing guidelines for the breeding and conservation of genetic resources of this rare and endangered noble hardwood species.

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch. Bip.) is an endemic species of the eastern Adriatic from the family Asteraceae. The plant produces pyrethrin, a secondary metabolite that has been shown to an effective natural insecticide with little or no adverse effects on humans. To assess genetic diversity, samples were collected from 10 natural populations of Dalmatian pyrethrum along the Adriatic coast and on the islands representing the species' range in Croatia. Genetic diversity analysis was carried out using 12 newly developed microsatellite markers. A total of 121 alleles with an average of 10.08 alleles per locus were detected in the studied populations, indicating high genetic diversity. Allelic richness (Nar) varied from 2.86 (Biokovo) to 5.02 (Zlarin). A total of 36 private alleles were observed in 10 populations with the highest number observed in Konavle (7). Evidence of bottlenecks was found only in the Biokovo population. Analysis of molecular variance showed that most of the genetic diversity can be explained by differences between individuals within populations (87.03%). Bayesian analysis of population structure revealed the existence of two gene pools, the mainland gene pool and the island gene pool. At K = 3, the Biokovo population formed a separate gene pool. Spatial analysis revealed that only a small proportion of genetic differentiation was due to isolation by distance (0.7%), while 40.6% of genetic differentiation was due to isolation by environmental distance. The results of this research will contribute to the development of future breeding programmes and hopefully commercial varieties of T. cinerariifolium to revive Dalmatian pyrethrum production in Croatia and the region. Furthermore, these results will help to improve conservation strategies for the species.

Lavandin is an iconic plant of Island Hvar, Croatia, where it has been cultivated for decades and gradually brought prosperity to the Island. A story among local inhabitants says that it was brought from Island Vis a century ago. We investigated lavandin landraces from islands Hvar and Vis using Amplified Fragment Lenght Polymorphism (AFLP) markers. Two well-known cultivars of lavandin ('Grosso and 'Abrialis') and the parental species (Lavandula angustifolia Mill. and L. latifolia Medik.) were also included in the analysis. Our assumption was that we were investigating two landraces, but the Neighbor-Joining (NJ) cluster analysis revealed another one. The existence of three landraces was further confirmed by the analysis of genetic structure using BAPS and STRUCTURE softwares. The three detected landraces were ‘Bila’, ‘Budrovka’ and ‘Budrovka Sveti Nikola’. Our results suggest that ‘Bila’ and ‘ Budrovka Sveti Nikola’ are polyclonal landraces originating from different hybridization events. Detected intra-cultivar variability could be attributed to the accumulation of somatic mutations.

Due to the availability and affordability of large scale genotyping, identification of genomic regions affected by the natural or artificial selection in the populations of wild and domesticated plants became standard procedure in modern population genetics. Identification of these regions and their association with traits of interest helps to understand evolutionary history of the species. The structure of cultivated common bean populations strongly resembles the structure of wild populations due to occurrence of at least two independent domestication events that occurred independently in Central and South America. Long- termed spatial isolation of two gene pools preserved their differential patterns of adaptation. Basic structure in collection of Croatian common bean landraces corresponds to the general pattern: first division is between two clusters of Central and South American origin, and later than divides into two clusters identified by different phaseolin types. In this study we used the differences in allele frequencies between populations (estimated by Fst values) to identify the SNP loci under adaptive selection.

U okviru projekta ''Bioraznolikost i molekularno oplemenjivanje bilja'' provedena je molekularna karakterizacija divljih i kultiviranih maslina u Hrvatskoj upotrebom 12 SSR (Simple Sequence Repeates) markera s ciljem dobivanja molekularnih profila specifičnih za genotipove uključene u ovo istraživanje. Sorte maslina smo prikupili iz kolekcijskih i proizvodnih nasada, dok su divlje masline izdvojene iz devet populacija s područja otoka Brijuna, Paga, Dugog otoka, Šolte, Hvara, Visa, Lastova, Mljeta i poluotoka Pelješca. Rezultati su pokazali jedinstvene profile za mnoge sorte i divlje masline te izdvojili tri slučaja sinonimije. Dendrogram izrađen metodom neighbor- joining je razdvojio grupu divljih od kultiviranih genotipova. Sorte su se većinom grupirale u zajedno, ali su pokazale i povezanost s divljim maslinama. Dobiveni rezultati su istaknuto bogatstvo genetskih resursa masline u Hrvatskoj, dajući informacije za njihovo očuvanje.

Sweet chetsnut (Castanea sativa Mill.) has been cultivated in semi-wild orchards of Sub- Mediterranean Croatia for centuries. Two such historical cultivation area are Lovran and Lovrin on the Istrian peninsula, while the third one is located on the island Cres. From these three cultivation areas, both grafted and ungrafted individual trees were sampled. Six genomic simple sequence repeat (gSSR) and nine EST-derived SSR (EST-SSR) loci were used to explore population diversity and clonality, as well as the population genetic structure. Five closely related clones have been found within the grafted group. Within the ungrafted group, many hybrids resulting from spontaneous cross-pollination between the cultivated and wild chestnut have been found. Thus, the three analyzed populations have varying levels of diversity and clonality. The analyzed semi-wild orchards defined by a diverse genetic structure represent a hotspot for further selection and could result in the creation of locally adapted, high-yielding varieties of chestnut.

The number of available DNA sequences in public genetic databases is constantly increasing. In plants, this is particularly evident in the amount of available complete chloroplast genomes, which are widely used in phylogenetic research. Chloroplast DNA genomes are circular and most have a four-part structure caused by two copies of a large inverted repeat (IR). We investigated inconsistencies in publicly available chloroplast genome sequence data regarding how stored public data account for structure. Our results show that there is no standardization in the storage of chloroplast genome sequences with respect to the structure of inverted repeats, as sequences are stored in different orders. Furthermore, there are many sequences in the public data without annotated inverted repeats, although these repeats are expected. In reviewing specialized chloroplast annotation tools, we found that there is no uniform method for identifying inverted repeats. Each tool analyzed takes a different approach and covers different specific situations. These results show that there is a need to standardize formats when it comes to storing data of specific types such as chloroplast sequences. Our results suggest that the existing public chloroplast data should be revised in terms of standard storage format and missing data. In addition to stored data, we found that specialized chloroplast annotation tools need improvement regarding the detection of inverted repeats.

Kloroplastni genomi su važan tip podataka u biljnim genomskim istraživanjima jer se često koriste u filogenetskim istraživanjima. Napredak tehnologija sekvenciranja i metoda sastavljanja sekvenci genoma pridonosi povećanju broja javno dostupnih sekvenci kloroplastnog genoma. Broj sastavljenih cjelokupnih kloroplastnih sekvenci je barem za red veličine veći od sastavljenih jezgrenih DNA sekvenci. Stabilna genetska struktura i uniparentalno nasljeđivanje kloroplasta dovodi do niske stope mutacija tijekom evolucije, pa bi tako srodne vrste morale imati vrlo slične kloroplastne sekvence. Ova činjenica te dostupnost velike količine podataka cjelokupnih kloroplastnih genoma omogućila je analizu metoda identifikacije mogućih odstupanja u kloroplastnim genomskim podacima. Identifikacija odstupanja ukazuje na moguće greške prilikom sastavljanja sekvenci. Analizirali smo odstupanja između sekvenci koje bi trebale biti vrlo slične i provjerili jesu li ta odstupanja uzrokovana različitim metodama sastavljanja sekvenci. Dobiveni rezultati ukazuju na postojanje odstupanja u proučavanim kompletnim kloroplastnim sekvencama te potrebu dodatnih provjera postojećih javno dostupnih podataka.Stoga je moguće unaprijediti i postojeće metode sastavljanja sekvenci uključivanjem sličnih provjera.

Salvia officinalis L. occupies an important place in the Albanian market for aromatic medicinal plants due to the high content and composition of its essential oil and is the most economically important species for the country. The aim of this study was to investigate the genetic diversity and structure of eight populations of Dalmatian sage from Albania, using eight microsatellite markers (SSRs) previously developed for this species. All eight- microsatellite markers yielded unique and easily detectable amplified fragments that revealed a high level of polymorphism (average of 15 alleles per locus). Overall values of expected heterozygosity (HE) ranged from 0.608 to 0.818, with a mean of 0.721. The highest gene diversity was found in northern Adriatic populations, decreasing towards the southern limit of the species range. Multilocus estimates of Wright’s inbreeding coefficient within populations (FIS) showed no significant deviation from Hardy- Weinberg equilibrium. Analysis of molecular variance showed that most of the total genetic diversity was attributable to differences between individuals within populations (83.14 %) which is to be expected due to outcrossing nature of the species. Bayesian analysis of population structure revealed the existence of two gene pools, the northern gene pool, and the southern gene pool. At K = 3, the Dibër population (the easternmost population in the sample) formed a separate gene pool. As commercial collection of Dalmatian sage in the wild has a negative impact on biodiversity, these results will form the basis for of plant genetic resource conservation and plant breeding programmes for this valuable species.

In this study, morphometric methods and chemical analyses were used to quantify the extent of differences in phenotypic and nutritional traits between sweet chestnut (Castanea sativa Mill, Fagaceae) populations from different environmental conditions, where different management types are applied, high-forest and coppice. I order to do that, we collected samples from 160 trees originating from eight natural populations stretching 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, 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. Moreover, differences in morphological and chemical nut traits evaluated here corresponded mainly to management practices. Extensively managed coppice populations on average resulted in smaller nuts with higher water and magnesium (Mg) content. In contrast, high-forest stands grown from seeds were characterized by larger nuts with lower water and Mg content. However, it was noteworthy that the type of management did not affect the morphological and chemical nut diversity of the coppice populations and that they remained similar to those of the high-forest stands. Finally, we concluded that the differences between coppice and high-forest populations are due to several factors, including habitat changes resulting from the forms and intensities of management practices, physiological processes related to stem growth, and genetic diversity.

The olive tree (Olea europaea L.) is an important fruit tree species in all Mediterranean countries, including Croatia, which is characterized by a fair number of indigenous, locally grown varieties, and by a rich heritage of wild olive trees still found on numerous islands along the Adriatic coast. In the framework of the project "Biodiversity and Molecular Plant Breeding", the samples of 223 olive varieties and wild olives were collected from different locations in the olive growing area and molecularly characterized using 12 SSR (Simple Sequence Repeats) markers. The results showed a unique profile for most cultivars and wild trees, except for three cases of synonymy, probably attributable to misnaming. Overall, the Croatian germplasm showed high genetic variability. The neighbor-joining dendrogram separated a group of wild genotypes originated mostly from the middle and southern Dalmatian islands. Cultivated genotypes mostly clustered together but they also showed relationship with wild germplasm. The results obtained underlined the richness of the genetic resources of the olive tree in Croatia, providing information for the protection of genetic material and monovarietal oils.

Microsatellites (SSRs) are highly informative repetitive sequences of 2-6 base pairs, which are the most used molecular markers in assessing the genetic diversity of plant species. Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip) is an outcrossing diploid (2n = 18), endemic to the eastern Adriatic coast and source of the natural insecticide pyrethrin. Due to the high repetitiveness and large size of the genome (haploid genome size of 9, 58 pg) previous attempts to develop microsatellite markers using the standard methods were unsuccessful. A next-generation sequencing (NGS) approach was applied on genomic DNA extracted from fresh leaves of Dalmatian pyrethrum. The sequencing was conducted using NovaSeq6000 Illumina sequencer after which almost 400 million high- quality paired-end reads were obtained, with a read length of 150 base pairs. Short reads were assembled by combining two approaches ; (1) de- novo assembly and (2) joining of overlapped pair-end reads. In total, 6, 909, 675 contigs were obtained, with the contig average length of 249 base pairs. Of the resulting contigs, 31, 380 contained one or multiple microsatellite sequences, in total 35, 556 microsatellite loci were identified. Out of detected microsatellites, dinucleotide repeats were the most frequent, accounting for more than half of all microsatellites identifies (21, 212 ; 59.7%), followed by trinucleotide repeats (9, 204 ; 25.9%). Tetra-, penta- and hexanucleotides had similar frequency of 1, 822 (5.1%), 1, 472 (4.1%), and 1, 846 (5.2%), respectively. Contigs containing microsatellites were further filtered by SSR pattern type, transposon occurrences, assembly characteristics, GC content, and the number of occurrences against the draft genome of T. cinerariifolium published previously. After the selection process, 50 microsatellite loci were used for primer design. Designed primers were tested on samples from five distinct populations and 25 of them showed a high degree of polymorphism. The selected loci were then genotyped on 20 samples belonging to one population resulting in 17 microsatellite markers. Availability of codominant SSR markers will significantly improve the knowledge on population genetic diversity and structure as well as complex genetics and biochemistry of this species.

Background: Chestnut blight fungus Cryphonectria parasitica is a pathogenic ascomycete introduced from Eastern Asia to North America and Europe, where it causes the aforementioned disease, devastating to chestnut trees. The disease can be biologically controlled utilising Cryphonectria hypovirus 1 (CHV1), a mycovirus which changes the physiology of the host, reducing its virulence towards chestnut. Objectives: The objective of the study was to determine whether the decrease of fungal virulence is caused by epigenetic changes and/or stress induction in the fungus after CHV1 infection. Methods: Six CHV1 strains of different subtypes were transferred to three C. parasitica isolates in order to assess the effect of different CHV1 strains and/or subtypes on global cytosine methylation level in infected fungus, using methylation-sensitive amplification polymorphism (MSAP). Furthermore, the activity of stress enzymes catalase, glutathione S-transferase and superoxide dismutase was measured in CHV1-infected and uninfected fungal isolates. Results: Infection with CHV1 affected fungal stress enzymes’ activity and the methylation pattern of C. parasitica genome. The increase in methylation levels correlated well with the CHV1-induced reduction of fungal growth in vitro, indicating that C. parasitica genome methylation upon CHV1 infection, rather than being the defensive mechanism of the fungus, is more likely to be the virulence determinant of the virus. Furthermore, the severity of CHV1 effect on stress enzymes’ activity and methylation levels of infected C. parasitica isolates depended mostly on individual CHV1 strain and on the combination of host and virus genomes, rather than on the virus subtype.

Znanstveni centar izvrsnosti za bioraznolikost i molekularno oplemenjivanje bilja (ZCI CroP-Biodiv) je istraživačka mreža usmjerena na prijenos znanja i tehnologija sa svrhom izravnog doprinosa napretku istraživanja u poljoprivredi. Ciljevi Znanstvenog centra izvrsnosti su povećanje dobiti koja proizlazi iz upotrebe biljnih genetskih izvora, optimizacija protokola fenotipizacije i genotipizacije, te primjena novih pristupa u statističkoj analizi podataka. Krajnji je cilj poticanje suradnje i sinergije između hrvatskih sveučilišta i znanstvenih instituta na području poljoprivrede u svrhu utemeljenja istraživačke platforme koja će objediniti nova znanstvena saznanja i tehnološka postignuća u svrhu prevladavanja poteškoća u području oplemenjivanja bilja.

Common bean (Phaseolus vulgaris L.) is a species of great interest worldwide. It is grown in Europe, Asia and Africa, where it presents similarities to Andean and Mesoamerican domestication centers or forms hybrids between both genepools [1]. In Croatia the production is based on landraces which display high levels of diversity and are important reservoirs of natural genetic variations [2]. On the basis of results of our research, 135 out of 174 accessions were assigned to 14 morpho-genetic groups originating from distinct domestication events: (1) Mesoamerican ('Trešnjevac', 'Tetovac', 'Kukuruzar', 'Biser'), (2) Andean - indeterminate type ('Sivi', 'Puter', 'Maslina', 'Dan i noć', 'Visoki Trešnjevac'), and (3) Andean - determinate type ('Niski Trešnjevac', 'Dan i noć', 'Puter', Bijeli', 'Zelenčec'). The rest of the accessions could represent putative hybrids among morpho-genetic groups including the hybrids among the gene pools. Common bean is one of the most beneficial crop species while in diet it provides macro- and micronutrients. However, there is a substantial variability present in seed mineral content among genotypes. As reported by Palčić et al. (2018) [3] among investigated Croatian common bean landraces the highest nitrogen (N) content in seed dry weight was in 'Visoki Trešnjevac' (3.92%) while the lowest was in 'Zelenčec' (3.13%). Moreover, average N content of indeterminate types was significantly higher compared to determinate types. Phosphorus (P) values varied from 0.49% ('Puter') to 0.57% ('Biser') where determined P content in landraces of Andean gene pool was significantly lower compared to landraces of Mesoamerican gene pool. Significantly lower seed potassium content was determined in 'Niski Trešnjevac' (1.42%) and 'Zelenčec' (1.38%). Calcium content in all studied landraces varied from 0.34 ('Kukuruzar') to 0.42% ('Biser'), with no significant differences among the researched landraces. Also, there were no significant differences in magnesium content among landraces. Magnesium content varied from 0.17 to 0.2% ('Puter'), with the average of 0.18% ('Biser'). Concerning the investigated micronutrients content, we can conclude that the highest iron content was found in landrace 'Niski Trešnjevac' while the lowest was in 'Kukuruzar'. 'Kukuruzar' also had the lowest value of manganese (Mn) content (15.19 mg kg-1) and zinc (Zn) content (24.98 mg kg-1) while the highest Mn content was in 'Biser' (19.94 mg kg-1) whereas the highest Zn content was determined in 'Visoki Trešnjevac' landrace (27.84 mg kg-1). Genotyping was carried out using microsatellite and DArTseq-based SNP markers to be used in genome-wide association study (GWAS). Linkage disequilibrium (LD) based GWAS has gained popularity particularly for screening a great number of accessions and to associate markers with desired traits in crops [4]. The unified mixed-model approach [5] allows the correction for population structure and family relatedness in order to remove false positives. Mixed model Q matrix was based on population structure obtained by model-based clustering method [6], while K matrix was constructed by calculating Loiselle's kinship coefficients [7] in SPAGeDi [8]. Genome-wide scan for association between markers and nutrient content was performed using single marker approach as implemented in TASSEL [9] as well as multi-locus approach implemented in R package “mlmm” [10].

Viruses represent an important biotic stress and are known to induce epigenetic changes in plants and animals, but knowledge about their effect on fungal genome methylation levels is still scarce. To study epigenetic changes in a fungus infected with a mycovirus we used a model pathosystem for mycovirus-host interactions: Cryphonectria hypovirus 1 (CHV1) and its host, phythopathogenic fungus Cryphonectria parasitica. This system is very important since C. parasitica causes chestnut blight, a devastating disease of chestnut trees which can be biologically controlled with CHV1. In C. parasitica deregulation of methylation cycle enzymes upon virus infection was observed previously. In order to study if those changes are reflected on global cytosine methylation levels of infected fungus, we used methylation-sensitive amplification polymorphism (MSAP) method. Furthermore, to assess if there is a difference in the effects of different CHV1 strains and/or subtypes on different fungus genotypes, we used six CHV1 strains of different virus subtypes transferred into three different C. parasitica isolates. Following the infection, we have observed an increase in the number and diversity of methylated, hemi-methylated, and total MSAP markers compared to virus-free controls, indicating that CHV1 infection affects the methylation pattern of the C. parasitica genome. Since the increase in methylation levels correlated with previously observed CHV1-induced reduction of fungal growth in vitro, we hypothesize that CHV1-induced increase in C. parasitica genome methylation is likely to be the virulence determinant of the virus, rather than being the defensive mechanism of the fungus. Furthermore, CHV1 subtypes did not consistently show either strong or weak effect on all studied fungal isolates. Rather, the severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended both on individual CHV1 strain, and on the combination of host and virus genomes. These novel findings broaden our knowledge about CHV1 biology and practicality of different viral strains to be used in human-mediated biocontrol.

The Balkan Peninsula is one of the most important speciation and diversification centres in Europe. One of the morphologically and phylogenetically clearly recognized groups is the Campanula garganica complex. This complex exhibits amfi-Adriatic and Ionian distribution with the majority of species distributed along the western Balkan Peninsula. We here explore the phylogeography and diversification of the C. garganica complex based on nuclear (ITS), plastid (trnL-trnF) DNA sequences and AFLPs. The analysis of AFLP fingerprinting was performed on 39 populations in order to obtain further insights into phylogenetic relationships within the complex. In addition, phylogeographic data were complemented with habitat suitability models for present and last glacial maximum (LGM) period to infer the locations of putative LGM refugia. Phylogenetic study confirmed the monophyly of the complex, with poorly resolved phylogenetic relationship among taxa. The AFLP data clearly separated all investigated taxa revealing some new lineages, and indicated the existence of eight genetic groups. It also revealed hybridisation zones between C. fenestrellata and C. istriaca in the Northern Adriatic region, as well as the close relationship among C. garganica and C. retina from Italy, C. cephallenica and C. acarnaica from Greece while southern Adriatic populations of C. teutana and C. poscharskyana were connected to Albanian taxa C. aureliana and C. skanderbegii. The haplotype network detected 18 haplotypes and suggested that the taxa within the complex survived within different Balkan refugial areas which was consistent with the predictions of the LGM habitat suitability models. Constructed potential dispersal corridors during LGM between populations that share haplotypes indicate several highly isolated groups of populations with no apparent connectivity. The highest connectivity (both present and LGM) was identified between C. fenestrellata and C. istriaca populations in the North Adriatic and between C. portenschlagiana populations in the Central Adriatic. In summary, the obtained genetic structure of C. garganica complex supports connections between Balkan and Apennine Peninsulas probably during Pleistocene fluctuations of the sea level as well as the existence of several refugial areas in the Adriatic region.

Basil (Ocimum basilicum L.) is a widely known member of Lamiaceae family with a long tradition of cultivation. Cultivation for different market requirements led to great intraspecific variation at both morphological and biochemical level. The aim of this research was to assess diversity among basil accessions maintained at the Collection of Medicinal and Aromatic Plants as a part of the National Bank of Plant Genes. Eighty basil accessions belonging to five basil morphotypes (True basil, Small-leaf, Lettuce- leaf, Purple basil A and Purple basil B) and five chemotypes (High linalool, Linalool/trans-α- bergamotene, Linalool/methyl chavicol, Linalool/ trans- methyl cinnamate and High methyl chavicol) were analysed using AFLP markers. Sixteen AFLP primer combinations yielded a total of 2, 114 polymorphic markers. Analysis of molecular variance (AMOVA) revealed that most of the genetic diversity was attributable to differences among accessions within morphotypes and within chemotypes. Bayesian Analysis of Population Structure (BAPS) resulted in assignment of the investigated accessions to four clusters. The accessions belonging to ‘green’ morphotypes (True basil, Small-leaf, Lettuceleaf) were assigned to Clusters A or B and Purple basil A accessions to Clusters A or C. Purple basil B morphotype was genetically the most admixed, having accessions belonging to all four genetic clusters.

Genus Rubus L. (Rosaceae), with more than 700 species, is one of the largest genera in plant kingdom. Raspberry (Rubus idaeus L.) is diploid and belongs to subgenus Idaeobatus. Raspberry leaves are used in traditional medicine for treatment of various disorders most commonly related to menstruation, parturition, and ailments of the gastrointestinal tract and other purposes, while fruit is used as food. In this work we used AFLP markers to analyse intrapopulation diversity and interpopulation relationships of seven wild growing populations of R. idaeus from Serbia, that can be grouped into three spatial groups as well as into two altitude groups. Four AFLP combinations yield a total of 247 polymorphic bands in 133 R. idaeus specimens. Data analysis revealed similar levels of diversity across populations. Population from Mt. Ozren had both the highest diversity and the highest frequency down-weighted marker values. AMOVA analyses further showed high intrapopulation diversity, while population differentiation was low. The partition among spatial groups was not significant while the partition between altitude groups was highly significant. BAPS grouped the samples into three clusters of which to cluster C belong exclusively individuals originating from population Ozren. Clusters were almost identical with or without spatial coordinates. In analysed populations altitude had a stronger influence on differentiation than horizontal distance and that can be attributed to different phenology at different altitudes. Our results indicate that AFLP markers are reliable technique for assessing genetic diversity and relations between R. idaeus populations.

Salvia brachyodon Vandas is a narrowly endemic species restricted to only three known populations in the central Adriatic region and it is treated as a highly vulnerable species in Montenegro and statutorily strictly protected indigenous plant species in Croatia. It is a partially clonal species and reproduces both sexually through seeding and clonally from perennial underground stolons. The species is characterized by rich content of essential oil, of which chemical composition is rather similar to other closely related sage species including well-studied common sage. The studied population of S. brachyodon, located in Pelješac peninsula, Croatia is the largest of all known populations. In order to assess levels of genetic diversity, 687 individuals were georeferenced, sampled and genotyped using eight microsatellite markers. For additional assessment of trade-off between sexual and clonal reproduction, the ramets and inflorescence were counted. Population genetic analysis revealed high levels of genetic diversity with no significant departure from Hardy-Weinberg equilibrium. Although numerous small clones dominated population over only few large ones, clonal reproduction strongly prevail over sexual reproduction. Since different genets virtually do not intermingle with each other, population’s clonal architecture relies on phalanx strategy of ramets’ spatial distribution. Results revealed that clones of different sizes flower with similar intensity and consequently the larger clones contribute more to sexual reproduction than the smaller ones. The analysis of clone size distribution revealed that studied population was characterized by Repeated Seedling Recruitment (RSR) strategy of new genets recruitment, which assumed constant emergence and establishment of new seedling. Strong spatial segregation was detected among large and small clones implying specific colonization strategy of the population.

Campanula versicolor, commonly known as various-colored bell-flower, is a constituent of the C. pyramidalis complex, distributed in the southern Balkan Peninsula with a small disjunct range in the southeastern Italy. Morphologically, it is highly variable species, which lead to the description of sixteen taxa which can be related to it. However, in modern floristic literature and checklists those taxa are neglected and considered as synonyms of broadly defi ned C. versicolor. Our extensive population sampling covered the entire range of the taxonomically intricate C. versicolor s.l. additionally, populations of recently described taxa from Albania – Campanula korabensis, C. korabensis subsp. bicajensis and C. longipetiolata are included in this study as well, based on the description from protologues, type materials and personal observations. Reconstruction of relationships among the populations based on microsatellite DNA data revealed three genetic groups of populations, which are morphologically and geographically well differentiated. Based on the obtained results a new taxonomic treatment of C. versicolor s.l. was proposed, along with the key for the identification, description, distribution and habitat data.

Dalmatian sage (Salvia officinalis L.) and Spanish sage (S. lavandulifolia Vahl) are closely related, perennial species from the family Lamiaceae. Dalmatian sage is native to coastal regions of the western Balkans as well as to the southern Apennine Peninsula while the natural distribution area of Spanish sage is the Iberian Peninsula. Thus, the populations of Dalmatian and Spanish sage most probably survived in different refugia during the glacial periods according to the traditional interpretation of southern European peninsulas (Balkans, Apennines and Iberia) as being single refugia that served as a source for a postglacial recolonization. However, 'refugia within refugia' model implies the existence of multiple glacial refugia within each larger refugial area that may confound the interpretation of phylogeographic patterns. Microsatellite markers and chloroplast DNA sequences were used to examine and compare genetic diversity and population structure of Dalmatian and Spanish sage with the aim to assess the fine-scale geographical distribution of putative 'refugia within refugia'.

Immortelle (Helichrysum italicum /Roth/ G.Don) is a perennial thermophilous subshrub widely distributed in the Mediterranean basin. Immortelle essential oil is highly praised having a wide range of remarkable medicinal properties. In recent years, massive uncontrolled commercial exploitation of wild immortelle populations prompt the Ministry of Environmental and Nature Protection of the R. of Croatia to limit or even ban wild harvesting. Genetic diversity and population structure of 18 immortelle populations from Croatia were examined by using amplified fragment length polymorphism (AFLP) markers. By assuming that the populations were in Hardy- Weinberg equilibrium, Wright's index of genetic differentiation was significant but low suggesting extensive gene flow among populations. The analysis of molecular variance indicated that the most of the genetic diversity was attributable to differences among individuals within populations in concordance with the patterns expected for an outcrossing and moderately long-lived species. Bayesian Analysis of Population Structure (BAPS) resulted in assignment of the investigated samples in two clusters. Samples originating from populations sampled in northern Adriatic were assigned to Cluster A, while the Cluster B included the samples belonging to populations from the middle and southern Adriatic. The results were compared with those obtained from AFLP analyses of Dalmatian sage (Salvia officinalis L.) and Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip.) populations from Croatia in which the similar pattern of genetic diversity and population structure has been observed.

In order to better understand how the climate changes lead to the genetic biodiversity we conducted a phylogenetic study of the endemic south – eastern European plant genus Aurinia, a genus within the Alysseae tribe (Brassicaceae). The objective of the study was to determine the phylogeny of the genus and to delimit the spatial boundaries of all taxa in the genus. Consequently, the obtained data will provide insights into spatiotemporal diversification of the genus and offer an outline for the reconstruction of individual Aurinia phylogeographies.

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 central and southern Apennine Peninsulas and is commonly cultivated worldwide. We used eight microsatellite markers to investigate genetic diversity and population structure. A total of 1, 350 specimens from 50 locations across the Balkan Peninsula as well as from 12 locations form the Apennine Peninsula were sampled and genotyped. The model-based structure analysis revealed the presence of five geographically coherent genetic clusters that differed significantly in allelic richness, observed and expected heterozygosity. The highest level of allelic richness was found in populations located in the central part of the eastern Adriatic coast, while decreases in allelic richness were evident towards the northwestern Adriatic coast and southern and eastern regions of the Balkan Peninsula. Populations from Apennine Peninsula had substantially lower allelic richness than those from balkan Peninsula. The genetic relationships among the genetic clusters were evaluated based on net nucleotide distance. A Neighbor–Joining tree based on the net nucleotide distances was generated showing that the two Apennine clusters were markedly divergent from each other as well as from the three Balkan clusters suggesting their long-term genetic isolation that lead to a reduction in gene diversity.

Most of the common bean (Phaseolus vulagris L.) production in Croatia is based on local landraces, grown by small-scale farmers in low input production systems. The landraces are commonly known by their traditional names based primarily on seed coat pattern and colour. In order to assess the genetic diversity of Croatian common bean landraces, 174 accessions were analysed using a DNA marker for phaseolin type, microsatellite markers and a DArTseq method of SNP genotyping. The information on phaseolin type and the results of the distance- and model-based analysis of population structure coupled with the morphological characterization of the accessions enabled the thorough classification of the accessions, that was, for the most part, congruent. Out of 174 accessions, 135 were assigned to 14 newly formed morpho-genetic groups originating from three distinct domestication events: (1) Mesoamerican, (2) Andean - indeterminate type, and (3) Andean - determinate type. The rest of accessions could represent putative hybrids among morpho-genetic groups including the hybrids among the gene pools. This study would be a first step into a genome-wide association mapping using a panel of Croatian common bean landraces. In conjunction with a detailed data on bioactive nutrient content, a whole genome association mapping analysis will be carried out to identify quantitative trait loci (QTLs) related to bioactive nutrient content.

Cilj istraživanja bio je analizirati genetsku raznolikost i strukturu 299 primki graha u svrhu izrade sržnih kolekcija hrvatskih genetskih izvora graha. Sržne su kolekcije uspostavljene pomoću dva algoritma (PowerCore i MSTRAT) koji koriste maksimizacijsku (M) strategiju na temelju rezultata genetske analize pomoću 26 mikrosatelitnih biljega. Analize skupina na temelju matrice udaljenosti kao i na temelju modela ukazale su na postojanje triju skupina u skladu s rezultatima analize tipa fazeolina. Primke graha su svrstane u 10 skupina koje predstavljaju najčešće morfotipove (tradicijske kultivare) u Hrvatskoj, te u miješanu skupinu manje zastupljenih morfotipova. Utvrđeno je postojanje 245 jedinstvenih genotipova. Sržna kolekcija odabrana upotrebom algoritma PowerCore sastojala se od 40 genotipova predstavljajući tako najmanji podskup primki u kojem su sadržani svi aleli zapaženi u cjelokupnoj kolekciji. Algoritmom MSTRAT utvrđen je podskup od 20 genotipova koji je imao nesignifikantno manje alelno bogatstvo u odnosu na cjelokupnu kolekciju. Maksimiziranjem genetske raznolikosti u reduciranom skupu genotipova, sržna kolekcija od 20 primki će olakšati buduća istraživanja raznolikosti graha upotrebom fenotipizacije visoke propusnosti kao i genotipizacije pomoću sekvenciranja nove generacije.

Dalmatian sage (Salvia officinalis L.) and Spanish sage (Salvia lavandulifolia Vahl) are perennial, evergreen shrubs from the family Lamiaceae. Because of the essential oils both species have been treasured as medicinal herbs. The taxonomic position of S. lavandulifolia is still controversial since certain authors consider it as subspecies of the S. officinalis. The natural distribution area of S. officinalis is coastal region of the western Balkan and southern Apennine Peninsulas while S. lavandulifolia is native to Iberian Peninsula. Genetic diversity and phylogeographical pattern of Dalmatian and Spanish sage were analysed comparing two chloroplast DNA regions. A total of 29 chloroplast haplotypes were identified in 88 native populations. Twelve chloroplast haplotypes were found on the Balkan Peninsula (S. officinalis), six on the Apennine Peninsula (S. officinalis) and 13 on Iberian Peninsula (S. lavandulifolia). The two species did not share haplotypes while Balkan and Apennine populations of S. officinalis had two haplotypes in common. S. officinalis exhibited higher haplotype and nucleotide diversity than S. lavandulifolia.

To understand the role of epigenetic processes in plant adaptation, existing epigenetic variation of natural plant populations has been assessed and compared to genetic variation. Twenty-five Dalmatian sage (Salvia officinalis L.) populations, each consisting of 20 to 25 plants, were analysed using epigenetic Methylation-Sensitive Amplification Polymorphism markers (MSAP) and genetic AFLP markers. Epigenetic and genetic diversity was compared by analysing within-population diversities and pairwise genetic differentiation between populations. The importance of epigenetic processes in divergence and adaptation of Dalmatian sage was analysed by comparing the number of outlier epiloci/loci under divergent selection and by the assessment of the association between epiloci/loci and bioclimatic variables. The identification of FST outlier epiloci/loci was carried out using a Bayesian method while the spatial analysis was used to test the probability of presence of an epiallelic/allelic variant given the environmental conditions of the sampling locations.

Common bean (Phaseolus vulgaris L.) is economically the most important among five domesticated species from genus Phaseolus. In Croatia, common bean is a traditionally grown in low input production systems. The production is based on landraces displaying high levels of diversity. In order to assess the genetic diversity of Croatian common bean landraces, 174 accessions were analysed using a DArTseq method of SNP genotyping. Out of 17, 514 polymorphic markers, 8, 092 (46%) had high scoring reproducibility (>0.95), high call-rate (>0.90) and the minor-allele frequency higher than 5%. Genomic regions flanking SNPs were than aligned against the reference genome of P. vulgaris. From the 8, 092 SNP sequences, 6, 599 (82%) were aligned to 11 chromosomes of common bean. The average number of SNPs per chromosome was 599.91, ranging from 403 on chromosome 4 to 834 on chromosome 2. Mean number of SNPs per Mbp was 12.85 or on average one SNP every 77, 828 base pairs. The average expected heterozyogsity was 0.373, ranging from 0.096 to 0.500 while the polymorphism information content ranged from 0.091 to 0.373 with an average of 0.298. The model-based analysis of population structure was carried out using 923 SNPs in linkage equilibrium. The results were compared to those obtained by microsatellite markers. The SNP set will be used in genome- wide association studies (GWAS) concerning bioactive nutrient content.

The evolutionary origin and population structure of 189 accessions belonging to five Croatian common bean landraces was assessed by phaseolin genotyping and microsatellite marker analysis. All the accessions belonging to landraces ‘Tetovac’ and ‘Kukuruzar’ were of phaseolin type I (Mesoamerican ; “S”), those belonging to ‘Visoki Trešnjevac’ were of type II (Andean ; “H” or “C”), while the accessions of ‘Niski Trešnjevac’ and ‘Zelenčec’ were of type III (Andean ; “T”). Out of 189 accessions, 141 showed unique multi-locus genotype using 26 SSRs. Both distance- and modelbased clustering approaches revealed the presence of three clusters in congruence with the results of phaseolin genotyping.

Ljekovita kadulja (Salvia officinalis L.) je višegodišnji grm prirodno rasprostranjen u obalnom području na zapadu Balkanskog poluotoka kao i u središnjem i južnom dijelu Apeninskog poluotoka. Ukupno 1, 350 uzoraka s 50 lokacija na Balkanskom i 20 na Apeninskom poluotoku je analizirano pomoću osam mikrosatelitnih biljega. Analizom genetske strukture na temelju modela utvrđeno je postojanje pet zemljopisno jasno odijeljenih genetskih skupina koje su se signifikantno razlikovale u alelnom bogatstvu kao u zapaženoj i očekivanoj heterozigotnosti. Genetski odnosi između skupina su izračunati na temelju neto nukleotidne udaljenosti. Stablo dobiveno metodom sparivanja susjeda je pokazalo da se dvije apeninske genetske skupine znatno razlikuju kako između sebe tako i od triju balkanskih skupina ukazujući na dugotrajnu genetsku izolaciju koja je dovela do smanjenja genetske raznolikosti.

Ljekovita kadulja (Salvia officinalis L.) je višegodišnja, vazdazelena, grmolika biljka porodice Lamiaceae. Zbog svojih eteričnih ulja cijenjena je kao ljekovita biljka još od antičkih vremena, a i danas ima veliku gospodarsku važnost. Filogeografsko istraživanje je provedeno na temelju dvije dvije kloroplastne DNA regije uključujući cjelokupan prirodni areal vrste. Analizom 62 populacije uzorkovane s 50 lokacija na Balkanskom i 20 na Apeninskom poluotoku utvrđeno je postojanje 16 kloroplastnih haplotipova. Potvrđena je visoka razina genetske diferencijacije između populacija kao i postojanje signifikantnog filogeografskog obrasca. Na Balkanskom poluotoku je utvrđeno postojanje 14 haplotipova dok je na Apeninskom utvrđeno šest. Dva su haplotipa zapažena isključivo na Apeninskom poluotoku što ukazuje na postojanje neovisne evolucijske grane.

Dalmatinski buhač (Tanacetum cinerariifolium /Trevir./Sch. Bip.) je stranooplodna, diploidna samo-inkompatibilna i termofilna vrsta iz porodice Asteraceae. Endemska je vrsta istočne obale Jadranskog mora a najbrojnije populacije rastu na ekstremno degradiranim staništima s plitkim kamenitim tlima. Biljke dalmatinskog buhača proizvode prirodni insekticid piretrin, koji je trenutno ekonomski najvažniji insekticid biljnog podrijetla. Uzgoj i upotreba dalmatinskog buhača u hrvatskim kućanstvima i poljoprivrednim sustavima ima dugu tradiciju. Iako ima veliko povijesno i ekonomsko značenje podaci o genetskoj i biokemijskoj raznolikosti prirodnih populacija dalmatinskog buhača gotovo ne postoje. Stoga je značaj predloženog projekta u integraciji različitih znanstvenih područja kako bi se stekla nova saznanja vezana uz biokemijsku i genetsku raznolikost ove endemske i gospodarski važne biljne vrste. Ciljevi projekta su sljedeći: (I) razvitak mikrosatelitnih početnica za dalmatinski buhač ; (II) analiza genetske raznolikosti prirodnih populacija dalmatinskog buhača mikrosatelitnim i AFLP biljezima, (III) optimizacija metode ekstrakcije disperzijom matrice u čvrstoj fazi i njena primjena u kombinaciji s HPLC-DAD u identifikaciji i kvantifikaciji sadržaja i sastava piretrina istraživanih populacija, (IV) utvrđivanje biljega AFLP vezanih za lokuse za sintezu pojedinih aktivnih sastavnica piretrina pomoću pridružujućeg kartiranja (Association Mapping) što će olakšati korištenje prirodnih populacija u oplemenjivačkim programima temeljenima na odabiru pomoću biljega (MAS - Marker Assisted Selection).Nove tehnologije i metode predložene u ovom projektu pridonijet će razvoju novih pristupa u praćenju bioraznolikosti i očuvanju biljnih genetskih izvora kao i analizi biokemijskog sastava dalmatinskog buhača. Također, dobiveni će rezultati predstavljati dobar temelj za razvoj budućih oplemenjivačkih programa i uvođenje dalmatinskog buhača u poljoprivrednu proizvodnju

Cryphonectria parasitica is a phytopathogenic fungus introduced from Eastern Asia to North America and to Europe, where it causes chestnut blight, a devastating disease of chestnut trees. A hyperparasitic mycovirus, Cryphonectria hypovirus 1 (CHV1), can infect the fungus, causing the change in morphology and virulence of the affected mycelia, indicating physiological changes. In order to elucidate what changes occur after the infection we have transferred six CHV1 strains belonging to three different virus subtypes into three different, but isogenic, C. parasitica isolates. Using methylation-sensitive amplification polymorphism (MSAP) we assessed the methylation pattern changes of the C. parasitica genome following the infection. The most obvious change was the increase of the number and diversity of methylated, hemi-methylated, and total MSAP markers found in infected fungal isolates compared to virus-free controls. The increase in methylation levels correlated well with the CHV1-induced reduction of fungal growth in vitro, indicating that C. parasitica genome methylation upon CHV1 infection, rather than being the defensive mechanism of the fungus, is more likely to be the virulence determinant of the virus. The severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended mostly on individual CHV1 strain and on the combination of host and virus genomes, rather than on the virus subtype alone. Furthermore, fungal growth and glutathione S-transferase, catalase and superoxide dismutase activities have been monitored. These enzymes are involved in oxidative stress response and in most cases we observed increase in oxidative stress enzymes’ activity in hypovirulent mycelia coupled with reduced growth of the affected mycelia. However, no clear correlation between the severity of the infection symptoms and virus subtype was determined. The severity of symptoms and the effect on the fungus does not depend on the virus subtype, but rather on the combination of the particular CHV1 and C. parasitica genotype.

Mikrosatelitni biljezi su najčešće korišteni molekularni biljezi za analizu genetske raznolikosti i populacijske strukture biljnih vrsta. Standardna metoda za razvitak mikrosatelitnih biljega uključuje izradu genomne knjižnice obogaćene ponavljajućim motivima, te probir knjižnice hibridizacijom. Prethodni pokušaji razvitka mikrosatelitnih biljega za dalmatinski buhač (Tanacetum cinerariifolium /Trev./ Schultz Bip. ; Asteraceae) pomoću standardne metode nisu bili uspješni, vjerojatno zbog vrlo velikog genoma ove vrste. Stoga je u tu svrhu korištena metoda sekvenciranja nove generacije. Sekvenciranje pomoću sustava Illumina NextSeq provedeno je u svrhu generiranja uparenih očitanja duljine 150 parova baza (pb). Dobiveno je više od 6 milijuna očitanja visoke kakvoće od kojih je sastavljeno 35, 758 kontiga prosječne duljine od 348 pb. Pronađeno je ukupno 1, 169 mikrosatelitnih sekvenci, a najčešće su bile di- (77.33% ; 904 lokusa) i trinukleotidne (14.54% ; 170 lokusa). Izrađene su početnice za lančanu reakciju polimerazom koje omeđuju mikrosatelitna ponavljanja za 40 sekvenci. Novorazvijeni mikrosatelitni biljezi bit će od velike koristi u budućim istraživanjima populacijske genetike dalmatinskog buhača.

Genetska raznolikost 18 populacija sredozemnog smilja iz R. Hrvatske analizirana je pomoću biljega AFLP. Otkrivanje lokusa za koje se pretpostavlja da su pod selekcijom je provedeno na dva temeljna načina: utvrđivanjem lokusa koji pokazuju atipične vrijednosti parametra genetske diferencijacije (FST) kao i analizom korelacija između genetske raznolikosti i okolišnih svojstava. U svrhu utvrđivanja lokusa koji pokazuju atipične vrijednosti korištena je frekventistička kao i Bayesovska metoda. Odnos između genetske raznolikosti i okolišnih svojstava je analiziran upotrebom metode prostorne analize na temelju logističke regresije u svrhu procjene vjerojatnosti da jedinka posjeduje određeni genetski biljeg imajući na umu okolišne uvjete mjesta prikupljanja. Okolišni uvjeti mjesta prikupljanja su opisani na temelju 19 bioklimatskih svojstva pohranjenih u bazi WorldClim. Usporedbom rezultata triju navedenih metoda utvrđeno je devet biljega AFLP koji su jako povezani s bioklimatskim svojstvima od kojih su najvažnija: srednja temperatura najkišovitijeg tromjesečja kao i količina oborina najsušnijeg tromjesečja.

Ramanova spektroskopija je vibracijska spektroskopska metoda koja omogućava analizu spektra različitih materijala, uključujući i sekundarne biljne metabolite, ovisno o molekularnoj strukturi i vrsti kemijskih veza. Ova je tehnika prikladna za utvrđivanje i lokalizaciju različitih bioaktivnih spojeva kao što su terpeni, alkaloidi, fenolni spojevi i dr. Kod dalmatinskog buhača (Tanacetum cinerariifolium /Trevir./ Sch. Bip. ; Asteraceae) aktivna insekticidna tvar je piretrin kojeg čini šest sastavnica (piretrin I i II, cinerin I i II te jasmolin I i II). Utvrđivanje nazočnosti piretrina u ekstraktima cvjetnih glavica dalmatinskog buhača provodeno je pomoću Ramanove spektroskopije. Prosječne vrijednosti Ramanovih spektara analiziranih uzoraka ukazuju na nazočnost karotenoida u Ramanovom opsegu od 1000 do 1530 cm-1. Karakteristični signali visokog intenziteta koji ukazuju na nazočnost piretrina I i II uočeni su u Ramanovim spektrima svih uzoraka na pozicijama 1438, 1600 i 1634 cm-1, dok su signali niskog intenziteta uočeni na pozicijama 1256, 1295 i 1414 cm-1. Nazočnost pojedinih sastavnica piretrina u ekstraktima dokazana je na temelju spektara prethodno uočenih kod izoliranih sastavnica koje su korištene kao standardi. Ramanova spektroskopija na ispitivanim uzorcima pokazala se kao brza i nedestruktivna metoda. U svrhu utvrđivanja kvantitativnih razlika u sadržaju piretrina između prirodnih populacija dalmatinskog buhača dobiveni će se rezultati usporediti s rezultatima dobivenih standardnom metodom pomoću HPLC-a.

Epigenetic modifications, such as DNA methylation, may play an important role in the process of adaptation of natural populations to different environmental conditions, without involvement of genetic variation. Although the interest on natural epigenetic variation of non-model organisms recently increased, there are no studies on a population level concerning plant pathogenic fungi. The fungus Cryphonectria parasitica is an invasive pathogen of sweet chestnut that has spread throughout the chestnut-growing areas of Europe. A study based on microsatellite markers showed that in south-eastern Europe a single haplotype dominates the C. parasitica population. The aim of this study was to analyse the epigenetic diversity and structure within and among C. parasitica populations from south-eastern Europe, which are grouped together within the same microsatellite haplotypes. We compared general methylation patterns of different haplotypes - S12 (dominant haplotype), S1, and S2, from south-eastern Europe using the methylation sensitive amplified polymorphism (MSAP) approach. The highest level of epigenetic diversity was found in S1 haplotype (H' = 0.4095), followed by the S2 haplotype (H' = 0.3787), while the lowest level of diversity was found in S12 haplotype (H' = 0.345). MSAP analysis also showed the highest number of hemimethylated epiloci in all three haplotypes. In five populations in which S12 is the dominant haplotype, the highest level of epigenetic diversity among S12 isolates was observed in the Italian populations (the oldest populations in the researched area). Diversity is lower in the populations that have been invaded recently. Our data suggest that epigenetic plasticity, observed especially in the older populations, might improve C. parasitica success in expanding into new regions. This study presents a first insight into the epigenetic diversity within and among natural populations of an invasive fungal species.

In plants, epigenetic variations based on DNA methylation are often transmitted across generations. Epigenetic processes may cause significant heritable variation in phenotypic traits without changing the sequence and thus increase considerably the evolutionary potential of plants in response to abiotic and biotic stress. Thus, heritable epigenetic variation could influence the course of evolution in plants, as it can affect the processes of adaptation and divergence through selection of stable epigenetic variants without involvement of genetic variation. In order to understand the true importance of epigenetic processes, existing epigenetic variation of natural plant populations have been assessed and compared to genetic variation. Three Croatian endemic Salvia species including (1) Salvia officinalis L., (2) Salvia brachyodon Vandas, and (3) Salvia x auriculata Mill. have been used as a study system. The species were chosen for having contrasting range sizes and genetic diversity heavily influenced by different phenomena: clonality (S. brachyodon) and interspecies hybridization (S. x auriculata). S. officinalis is widely distributed along the eastern Adriatic coast while S. brachyodon is a stenoendemic plant of a very narrow range size. S. x auriculata is a natural hybrid between S. officinalis and S. fruticosa. Preliminary results based on the analysis of 25 S. officinalis populations showed that the proportion of epigenetic variance attributable to differences among populations was similar to the proportion of the among-population component of genetic variance. Systematic patterns of epigenetic variation were observed in relation to a number of bioclimatic variables related to amount of precipitation at sampling sites. The investigation of clonal S. brachyodon revealed the existence of substantial epigenetic variation among genetically identical plants (clones). Finally, hybrid S. x auriculata exhibited higher epigenetic diversity in comparison to parental species.

We reconstructed the relationships among populations of Campanula secundiflora s.l. and closely related and geographically close populations of C. austroadriatica and C. versicolor. Based on analyses of microsatellite DNA data, the investigated populations have high overall genetic diversity and abundant allelic variation over seven investigated loci. Bayesian model-based clustering identified four clearly differentiated genetic groups of populations. The genetic differentiation was reflected by morphological differentiation, allowing us to propose a new taxonomic treatment of the constituents of C. secundiflora s.l. The populations distributed in south-western Serbia and north-eastern Montenegro were included in C. secundiflora. A new species, Campanula montenegrina sp. nov., distributed in the continental part of Montenegro and the northern part of Albania is described

Numerous molecular systematic studies within Brassicaceae have resulted in a strongly improved classification of the family, as morphologically defined units at and above the generic level were often found to poorly reflect phylogenetic relationships. Recently the study of tribe Alysseae provided insight into the generic circumscriptions of one of the largest tribes within Brassicaceae, which harbours about 7% of the family’s species diversity. One of the identified monophyletic clades was the Fibigia clade encompassing several species poor genera, namely Alyssoides, Clastopus, Degenia, Fibigia and Physoptychis. The genera within the clade are mainly distributed within Irano-Turanian floristic region, with some taxa occurring in the Mediterranean region and the majority of species exhibiting a narrow geographic range (“narrow endemics”). Morphologically, species of this clade are characterized by being perennial herbs, subshrubs or shrubs with an indumentum of stellate hairs sometimes mixed with simple ones, relatively large yellow or purple flowers and large fruits with mostly winged seeds. Additionally, this clade encompasses greater fruit diversity than the rest of the tribe ; being dehiscent or indehiscent, inflated fruits or strongly compressed, with papery, leathery or thickened valves. The initial phylogenetic analysis revealed that the largest genus in the clade, Fibigia was not monophyletic and the relationships among genera were poorly resolved and burdened with incongruences among markers. In order to provide further insight into phylogenetic relationships we included additional taxa, encompassing the majority of species in the clade. To this end, we obtained sequence data from the nrDNA ITS and ndhF gene plastid region. The non-monophyly of Fibigia was confirmed and six lineages were identified. Most lineages were strongly supported and included only one species supporting the taxonomical recognition of additional monotypic genera. However, F. afghanica, F. membranacea and F. multicaulis were intermingled with genera Clastopus and Straussiella indicating complex phylogenetic relationships. Separation of genus Alyssoides into monotypic Alyssoides including A. utriculata and monotypic Lutzia with L. cretica was also confirmed.