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Plan upravljanja istraživačkim podacima za istraživački projekt Hrvatske zaklade za znanost: Livadni procjepak (Chouardia litardierei, Hyacinthaceae) kao istraživački sustav ekološke divergencije (IP-2020-02-8099), za 2. projektno razdoblje (01.01.2022. - 30.06.2023.).

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.

Amethyst meadow squill (Chouardia litardierei, Hyacinthaceae) is a plant species characterized by exceptional ecological plasticity. Its populations are found in ecologically highly contrasting environments, from salt marshes near seashores, across periodically flooded karst fields, to arid mountain slopes in high altitudes. Nowadays, knowledge of the molecular mechanisms underlying the adaptive divergence and speciation processes can be gained by exploring complex interactions among environment, phenotype, epigenotype, and genotype of such a species as C. litardierei, and the availability of the species' genome sequence is an absolute prerequisite. To tackle this task, by implementing long-read PacBio sequencing followed by Hi-C scaffolding, we have obtained a high-quality assembly of the C. litardierei genome. The species is characterized by n=13 chromosomes and a large genome of an estimated size of 4.1 Gb. Pacific Bioscience HiFi technology providing longer reads of high quality was used for generating genomic sequences. In total, 6.526.182 Hi-Fi reads were obtained with a total size of 94.54 Gb and an average read length of 14.5 Kb. Reads were de-novo assembled into contigs by haplotype-resolved de novo assembler software hifiasm (v. 0.16.1-r375). Chromosomal scaffolding was obtained by high coverage Illumina Hi-C reads (1.72 billion reads, 432.3 Gb of data representing 100X coverage) using the Juicer, 3d-dna, and Juicebox scaffolding tools. The final assembly counted 13 main scaffolds, representing the expected number of chromosomes in a total length of 3, 33 Gb, while all scaffolds had a length of 3, 70 Gb. The average genome GC content has 42.9%. BUSCO analysis confirmed the high completeness of the assembly since only 2.6% of the liliopsida_odbio dataset was missing. As expected, a high proportion of various repeats in the genome was found. RepeatMasker screening against the curated libraries for Viridiplantae repeats (Dfam and Repbase) revealed 17.6% retrotransposons and 1.2% DNA transposons, while de-novo identification of repeats confirmed 55.6% retrotransposons, 2.6% DNA transposons, and 8.1% unknown repeats. The MAKER gene calling pipeline has generated 34, 000 reliable protein-coding gene models. Comparison with the complete garlic (Allium sativa L.) genome from the same order of Asparagales revealed a low level of conserved synteny.

Valuable knowledge of the mechanisms of the speciation, one of the evolution's fundamental processes, could be gained by exploring complex interactions among environment, phenotype, epigenotype, and genotype in a species characterized by exceptional ecological plasticity. For gathering as much information as possible on this subject, amethyst meadow squill (Chouardia litardierei, Hyacinthaceae), a species characterized by extreme ecological plasticity, was chosen as a study system. In-depth analysis of various aspects of local adaptation and divergence processes requires the availability of a studied species' draft genome assembly, so that (epi)genome annotations essential for interpretations of ecotypes characteristics can be performed. Here we report a high-quality genome sequence of Chouardia litardierei. The species has n=13 chromosomes and is characterized by a large genome with an estimated size of 4.1 Gb. Genome sequences were generated using Pacific Bioscience HiFi technology. 6, 526, 182 Hi-Fi reads with a total size of 94.54 Gb and an average read length of 14.5 Kb were obtained. Sequences were assembled into contigs using the hifiasm software (v. 0.16.1-r375). Chromosomal assemblies were generated with Illumina Hi-C reads (1.72 billion reads, 432.3 Gb) using the Juicer, 3d-dna, and Juicebox scaffolding tools. The final assembly comprised 13 main scaffolds, thus being in agreement with the expected chromosome number. The average GC content is 42.9%. BUSCO analysis confirmed the high completeness of the assembly, as only 2.6% of the liliopsida_odb10 dataset was missing. The high proportion of genome repeats was confirmed. RepeatMasker screening against the curated libraries for Viridiplantae repeats (Dfam and Repbase) revealed 17.6% retrotransposons, 1.2% DNA transposons, and 1.4% simple repeats. De novo identification of repeats found 55.6% retrotransposons, 2.6% DNA transposons, and 8.1% unknown repeats. In total, 80.9% of the genome is masked with repetitive elements. The gene calling pipeline has generated 30, 000 reliable protein- coding gene models.

Chouardia (Hyacinthaceae) is a genus restricted to Dinaric Alps that was recently described and segregated from Scilla . It comprises two species, C. litardierei (Breist.) Speta, known as a type species of periodically flooded karst fields across western parts of the Balkan Peninsula, and C. lakusicii (Šilić) Speta, found in dry, stony, calcareous grasslands and rock crevices of Montenegro, southern parts of Bosnia and Herzegovina, and Croatia. Chouardia lakusicii, as a distinct taxon, was described after vague morphological and phenological characterization of a limited number of individuals from only a few populations. Consequently, it is possible these assumed species present merely divergent ecotypes of the same polymorphic taxon. In addition, based on the ecological preferences, the third ecotype can be recognized as well, the one growing in the coastal grasslands of Northern Dalmatia, Croatia. As a part of substantially larger research activities that aim at deciphering the genomic background of the adaptive divergence process, a comprehensive common garden experiment that included almost 540 individuals (60 individuals from each of three selected populations from each ecotype) was set. During the 2022 vegetation season, we have measured numerous phenological and morphological characters. Based on preliminary analysis, strong contrasts among populations were observed. However, for the majority of these characters, differences were ecotype-independent, meaning they cannot be used as reliable descriptive elements. Here we present the obtained results and discuss their importance for further research on Chouardia as a study system for ecological divergence.

Panoramic images captured using laser scanning technologies, which principally produce point clouds, are readily applicable in colorization of point cloud, detailed visual inspection, road defect detection, spatial entities extraction, diverse maps creation etc. This paper underlines the importance of images in modern surveying technologies and different GIS projects at the same time having regard to their anonymization in accordance with GDPR. Namely, it is a legislative requirement that faces of persons and license plates of vehicles in the collected data are blurred. The objective of this paper is to present a novel architecture of the solution for a particular object blurring. The methodology was tested on four data sets counting 5000, 10 000, 15 000 and 20 000 panoramic images respectively. Percentage of accuracy, i.e. successfully detected and blurred objects of interest, was higher than 97 % for each data set.

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.

Akutni pankreatitis ima incidencu u stalnom porastu, a varijabilnost težine bolesti i mortaliteta je izražena. Na nastanak, težinu i ishod ovog oboljenja u određenoj meri može uticati polimorfizam gena koji kodiraju pankreasne proteine i medijatore zapaljenja. Cilj ove studije je utvrđivanje povezanosti najznačajnijih varijacija gena koji kodiraju katjonski tripsinogen, inhibitor pankreasnog tripsina, transmembranski regulator sprovođenja kod cistične fibroze i faktore nekroze tumora α i β sa težinom kliničke slike ili smrtnog ishoda kod pacijenata sa akutnim pankreatitisom. Studijom je uključeno 98 pacijenata sa akutnim pankreatitisom, a dizajnirana je kao retrospektivna, tipa slučaj-kontrola. Dijagnoza i klasifikacija pacijenata prema težini bolesti su zasnovani na Atlanta klasifikacionom sistemu iz 1992. godine. Genotipizacija je sprovedena PCR-RFLP metodom. U ispitanom uzorku nije identifikovan nijedan pacijent sa PRSS1 polimorfizmom. Prisustvo SPINK1101A>G varijacije uočeno je kod samo jednog pacijenta, koji je imao tešku formu bolesti. Osobe ženskog pola obolele od akutnog pankreatitisa imaju veći rizik za razvoj teške forme bolesti ukoliko su nosioci bar jednog CFTR IVS8 9T alela (RR za 9T/9T+9T/non9T vs. non9T/non9T: 2.115; 95% CI: 1.241-3.605). Uticaj M470V na težinu i mortalitet bolesti, kao i veza sa CFTR IVS8 poly-T varijacijom, nije pokazan. Nijedan od pacijenata sa akutnim pankreatitisom nije bio nosilac CFTR R117H varijacije. Nosioci najmanje jednog varijantnog alela TNF-α -308A imaju skoro deset puta manji rizik za smrtni ishod akutnog pankreatitisa, a naročito u poznijim godinama.4 Takođe, primećena je povezanost teškog oblika bolesti sa prethodno prisutnim komorbiditetima. Kod žena obolelih od akutnog pankreatitisa prisustvo CFTR IVS8 9T alela povezano je sa težom formom bolesti. Pacijenti oboleli od akutnog pankreatitisa imaju veću šansu za smrtni ishod ako su nosioci TNF-α-308G/G alela. Povezanost varijacija PRSS1 i SPINK1 gena sa težinom i ishodom akutnog pankreatitisa nije pokazana. The incidence of acute pancreatitis steadily increases, and the severity and mortality of the disease is markedly variable. To a certain extent, the development, severity and the outcome of acute pancreatitis are affected by genetic polymorphism of pancreatic proteins and inflammatory mediators. The aim of this study was to determine the correlation between the most significant variations of genes coding for cationic trypsinogen, pancreatic secretory trypsin inhibitor, cystic fibrosis transmembrane conductance regulator and tumor necrosis factors α and β and the severity or mortality in acute pancreatitis patients. The study was designed as a retrospective, case-control study and included 98 patients with acute pancreatitis. The diagnosis and classification of patients according to the severity of the disease were based on the 1992 Atlanta Classification System. The genotyping was carried out using the PCR-RFLP method. PRSS1 polymorphism was not detected in our investigated sample. SPINK1 101A>G variation was observed in only one patient, who was experiencing severe form of the disease. In females with acute pancreatitis, the risk of developing a severe form of the disease was increased if at least one CFTR IVS8 9T allele was present (RR for 9T / 9T + 9T / non9T vs. non9T / non9T: 2.115; 95% CI: 1.241-3.605). The effect of M470V on the severity and mortality of the disease, as well as its association with the CFTR IVS8 poly-T variation, has not been shown. None of the patients with acute pancreatitis was the carrier of CFTR R117H variation. Carriers of at least one variant TNF-α-308A allele have nearly ten times lower risk of fatal outcome of acute pancreatitis, especially at an advanced age. Also, there was association observed between severe form of acute pancreatis and previously present comorbidities. In females with acute pancreatitis, the presence of CFTR IVS8 9T allele is associated with severe form of the disease. Acute pancreatitis patients carrying TNF-α-308G/G allele have higher mortality risk. The association of PRSS1 and SPINK1 polymorphism with the severity and outcome of acute pancreatitis has not been confirmed.

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'.