Isolation-by-distance (IBD) pattern among bilberry (Vaccinium myrtillus L.) populations has previously been reported for this species in northern Europe. However, the number of molecular studies conducted on bilberry, using everything from isoenzymes, RAPDs to microsatellite markers, are very few and far between. Considering that Bosnia and Herzegovina (B&H) is a country rich with diverse fruit genetic resources, conducting a genetic characterization of the naturally occurring V. myrtillus populations could yield valuable data for the conservation and utilization of this resource. This study entailed genotyping samples collected from three bilberry populations located in Fojnica, Kladanj, and Srebrenica municipalities using seven polymorphic microsatellite or SSR (simple sequence repeats) markers. The obtained molecular data was used to calculate the correlation between the physical distance of the individual B&H populations and a parameter of the genetic differentiation (pairwise Fst). The results of the correlation analyses revealed an absence of a significant isolation-by-distance pattern among the three examined B&H bilberry populations. In addition, the most pronounced genetic differentiation was detected between the Srebrenica and each of the two remaining B&H populations. At the same time, the values for pFst were significant, albeit much lower, between the Fojnica and Kladanj populations. Bilberries from the sampled Srebrenica population appear to be distinct from the other B&H populations, possibly due to the different genetic origin of this population.

Background and purpose: Available data in research literature suggest that the Western Balkan countries hold a rich diversity of caddisflies. Assessment and biomonitoring of such rich diversity could be facilitated through DNA-based high-throughput approaches like DNA metabarcoding that depend on the availability of comprehensive reference libraries. Materials and methods: We assessed the status of the COI barcode sequence data for a total of 112 caddisflies species in the investigated region by determining the gaps in representative sequences in the Barcode of Life Data System (BOLD) and examining the accuracy of available records using the Barcode, Audit and Grade System (BAGS). Results: Results revealed a considerable underrepresentation of surveyed geographic region in BOLD records for the target insect group. Moreover, the large majority of the species records were rated “discordant” (72.80% grade E), and only 15.20% were classified as “consolidated concordance or basal concordance” (3.20% grade A and 12.00% B). Approximately 3.20% of the records pertaining to species occurring in multiple BINs (Barcode Index Number) and 8.80% were poorly represented (i.e., less than three specimens, grade D). A fraction of the species graded discordant were deemed concordant after detailed inspection of individual data, decreasing by 14.07%. Conclusions: The assessment of the current state of BOLD entries indicated that DNA barcoding is still not widely applied in Albania, Bosnia and Herzegovina, Montenegro, North Macedonia, Serbia, and Slovenia, emphasizing that Croatia has the most barcoded caddisflies species. The finding that available BOLD Trichopteran records for investigated countries were mainly graded as “discordant” indicates the need for better quality control of reference libraries.

Abstract Banat Naked Neck is the most important indigenous breed of chickens in Serbia. Marginalized until recently, it is becoming increasingly popular due to its adaptability and good productivity in alternative production systems. However, its history and the current breeding model pose challenges for breed preservation and future improvement. This study aimed to assess the genetic diversity and structure of four subpopulations of Banat Naked Neck from different districts in Serbia (West Backa, North Banat, South Banat and Kolubara) using D-loop mitochondrial DNA sequences and a set of 30 microsatellite markers. Seven haplotypes in the phylogenetic analysis of D-loop mitochondrial DNA suggested maternal origin related to the Indian subcontinent, while haplotype and nucleotide diversity averaged 0.731 ± 0.053 and 0.0067 ± 0.0018, respectively. Microsatellite genotyping showed an average detected number of alleles per locus of 5.129 ± 0.237, while the observed and expected heterozygosity averaged 0.560 ± 0.018 and 0.631 ± 0.014, respectively. Genetic differentiation estimated through FST was 0.051 (p < .001). Two clusters in STRUCTURE analysis showed possible separation of two older subpopulations (South Banat and Kolubara) from the two more recent ones (West Backa and North Banat). This first comprehensive study of genetic diversity serves as the basis for future preservation, use and improvement of the Banat Naked Neck breed.

Silene sendtneri Boiss. (Caryophyllaceae) is the Dinaric endemic plant species with white, decorative and scented flowers. Previous studies on this endemic species were based on morphology and effects on seed germination after the treatment with salicylic acid. However, no molecular genetic studies have been conducted on this species so far. This paper presents preliminary results of the usefulness of microsatellite loci created for cosmopolitan species in assessing the genetic diversity of endemic plant species. A total of 100 specimens were collected from 18 localities in the mountain regions of Treskavica, Igman, Bjelašnica and Ozren in Bosnia and Herzegovina. No S. sendtneri individuals were found at the mountain Trebević. We tested cross-amplification success and a polymorphism level for the set of microsatellite markers (Sil01, Sil03, Sil16, Sil31, Sil35) designed for the cosmopolitan species Silene nutans. In 100 analyzed individuals of S. sendtneri, Sil31 and Sil35 did not amplify, Sil01 was monomorphic and the remaining two loci showed a high level of allelic diversity. Our findings suggest that caution should therefore be exercised in selecting microsatellite markers designed for cosmopolitan plant species in the analyses of endemic species of the same genus since different genetic factors affect the amplification success and polymorphism of the given loci. Attention should be given to the number of detected and effective alleles and their ratio, the success of locus amplification concerning the complete set of markers used, and the ratio of polymorphs to the total number of observed loci.

The process of travertine formation and carbonate deposition in the rivers is unique, delicate, and depends on the activity of algae and mosses. Although diatoms have been used extensively in hydrobiological studies, the comparative analysis data on diatom communities of the travertine barriers in karstic rivers are still scarce. ‡ § | ¶ ‡ ‡ # § ¤,« »,˄ ˄,» »,˄ © Kamberović J et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The study aimed to detect the diatom composition on travertine barriers in the Una River, the large karstic river in Bosnia and Herzegovina. An integrated classical morphological identification approach with metabarcoding was applied on eight samples across the river length profile. Morphological analyses were performed using both light and scanning electron microscopes. Subsequent DNA metabarcoding of the chloroplastic gene 312bp rbcL was done. The DADA2 pipeline was used for the bioinformatic treatment of the demultiplexed MiSeq reads to infer Amplicon Sequence Variants (ASVs). ASVs were taxonomically assigned using the Diat.barcode v7 reference database. A total of 126 species were identified using the morphological approach, while 133 ASVs were taxonomically assigned to 58 unique taxa with the molecular approach. Diatom community structures in terms of molecular and morphological approaches were congruent with 49 shared species. Species from genera Gomphonema, Navicula and Encyonema were less assigned in molecular analysis. The most abundant taxa in the Una River are alkaliphilous, belonging to the genera Gomphonema, Nitzshia and Navicula. Although specific for their extremely good chemical status, the travertine barriers of the Una River are largely inhabited with meso-eutraphentic taxa.

Bosnia and Herzegovina has valuable natural resources with a high percentage of endemic and autochthonous species (Kučinić et al. 2008, Đug and Drešković 2012). The freshwater fauna of Trichoptera in this area is under-investigated, with a lack of morphological description of different life stages and DNA barcode data. Public data show 58,993 barcode entries for Trichoptera in the Barcode of Life Data Systems (BOLD) submitted from 92 countries, and none from Bosnia and Herzegovina (B&H) (BOLD 2021). Previous research in Bosnia and Herzegovina has provided the first DNA barcode for the endemic species Rhyacophila bosnica, stored in GeneBank, under accession number MK211322 by a domestic institution (Kalamujić Stroil et al. 2018). A few DNA barcodes of adult individuals of Trichoptera from Bosnia and Herzegovina were found in BOLD. However, these specimens were collected on B&H territory, but analyzed, processed, and stored by foreign institutions. To change the current state of DNA barcoding of Trichoptera in Bosnia and Herzegovina, we aimed to employ this approach in investigating caddisflies in selected habitats in the Sarajevo Canton. Our fieldwork was done in all five protected areas (spring of the Bosna River, Bijambare, Trebević, Skakavac, and Bentbaša) in which larvae samples were collected according to ‡,§ ‡,§ ‡,§ ‡,§ ‡ © Destanović D et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. the AQEM sampling methodology. The standard animal DNA barcode was successfully obtained using degenerated primers LCO1490JJ and HCO2198-JJ (Astrin and Stüben 2008). Out of 684 collected individuals (313 Trebević, 130 spring of the Bosna River, 117 Bijambare, 71 Bentbaša, 53 Skakavac), a subset of specimens were sequenced. We uncovered 14 different taxa, 11 genera and six families (Limnephilidae, Glossosomatidae, Rhyacophilidae, Goeridae, Hydropsychidae, Polycentropodidae). The preliminary data of Trichoptera composition in the Sarajevo Canton indicated species richness. Based on our sequential data, a new subspecies was discovered in two investigated areas (Valladolid et al. 2020), proving that Trichoptera species diversity in our country is far from entirely uncovered. The benefit and power of the DNA barcoding approach are that it can pinpoint the areas of vast and unknown species diversity more economically, both financially and temporarily, than the morphological approach. Therefore, we believe that it is critical to support the development of DNA barcoding for the bioassessment of freshwater ecosystems in Bosnia and Herzegovina. Several problems prevented us from exploiting sequential data to the fullest. Despite a general notion among scientists that European Trichoptera species are well covered in the BOLD database, most of the sequences we obtained were absent from the database. Secondly, we recognized that morphological data about the larval developmental stage of B&H Trichoptera species are largely missing. The unified, updated, and complete data on this order of insects is urgently needed. However, insufficient financial support by governmental institutions and lack of systematic approach to barcoding the wildlife of Bosnia and Herzegovina hampers this process. Further attempts to collaborate with the stakeholders can be crucial with profound and substantial implications for biomonitoring of aquatic macroinvertebrates in general. New approaches, such as novel DNA barcoding-based methodology can fill an important gap in our knowledge of Balkan caddisflies haplotypes, lineages, and their diversification and distribution patterns.

Chaetopteryx villosa (Fabricius, 1798) is a caddisfly species distributed throughout Europe, except in the Balkan and Apennine Peninsula. However, phylogenetically close species belonging to the C. villosa group are widespread throughout entire Europe. Species of this group (C. villosa, C. gessneri, C. fusca, C. sahlbergi, C. atlantica, C. bosniaca, C. vulture, and C. trinacriae) have distinct distributions with some overlaps. Adult forms of these species are morphologically similar, whereas larval morphology is only known for some species. There are also indications of species hybridization (e.g., C. villosa x fusca). Presumably, the molecular approach for the species determination of this group would be highly beneficial. In the BOLD database, there are 154 specimens with COI-5P barcodes of C. villosa species. Out of the remaining species, C. sahlbergi has 27 specimens with a barcode, C. fusca 20, C. gessneri 5, C. bosniaca 5, and C. atlantica 1, whereas sequences from the species C. vulture and C. trinacriae are missing. Therefore, we tested the power of discrimination of the COI-5P marker in the C. villosa group, as the most common barcoding markers for species identification in animals. Only sequences from public records originating from experienced research groups or taxonomists and containing a specimen photograph were taken as input. A total of 75 ‡,§ ‡,§ ‡,§ ‡,§ ‡,§ © Destanović D et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. sequences from the BOLD database were obtained. Out of these sequences, 11 belonged to C. fusca, 5 to C. gessneri, 52 to C. villosa, 5 to C. bosniaca, and 2 to C. sahlbergi. For the generation of overview trees, COI-5P barcodes of Rhyacophila fasciata and Rh. nubila were used as outgroups. All sequences were trimmed at 5’ and 3’ ends, resulting in a final alignment length of 516 base pairs. Multiple sequence alignments and editing were done in the MEGA-X software. Analysis of nucleotide polymorphism was done in DNASP6 software. MEGA-X was used to calculate the pairwise distance and overall mean pdistance, and to construct the overview trees. Analysis of DNA polymorphism revealed 14 haplotypes of C. villosa, 3 haplotypes of C. fusca, 2 haplotypes of C. gessneri, and one for species C. bosniaca and C. sahlbergi. There were no significant interspecific and intraspecific differences among haplotypes based on pairwise distances. The p-distance between one of the haplotypes of C. fusca and C. villosa was 0.000, whereas the p-distance among haplotypes of C. villosa varied from 0.001 to about 0.055. The mean overall p-distance among haplotypes of all species equaled 0.03. No species-specific clusters were observed when phylogenetic trees were constructed except for C. gessneri, regardless of the method used (i.e., NJ, UPGMA, ML, ME, or MP). To minimize the possibility of species misidentification, we used only records submitted by NTNU-Norwegian University of Science and Technology (Norway), SNSB-Zoologische Staatssammlung Muenchen (Germany), Zoologisches Forschungsmuseum Alexander Koenig (Germany), University of Oulu, Zoological Museum (Finland), prof Hans Malicky and prof Mladen Kučinić. No records identified as hybrids were included in the analyses. With the exception of C. gessneri, COI-5P marker failed to separate the species of the C. villosa group. However, it is highly unlikely that poor species determination was the basis for such a result. To enable the comprehensive and unbiased evaluation of the relationships within this group, data coverage in BOLD database for most of the studied species should be enhanced, encompassing different geographical distribution of samples. Further studies are needed to detect the array of molecular markers suitable for the species delineation in a complex group such as C. villosa.

The diploid Celina/QTee® (‘Colorée de Juillet’ × ‘Williams’), one of the most promising pear cultivars developed by the Norwegian breeding program Graminor, was launched in 2010. In Norway, the flowering is medium to late, while the fruits ripen in the beginning of September. The fruits are attractive with an intense red blush (50%) on a green background. Although, ‘Celina’ is cultivated in the most climatically suitable regions for fruit cultivation, present in Norway, unfavorable environmental conditions for pear pollination can have a very negative effect on fruit set and consequent yield. The aim of this study was to determine the S-alleles of ‘Celina’, as well as its frequently used pollinizers, and, through paternity testing of ‘Celina’ seeds, give a recommendation regarding the most important pollinizers of this pear cultivar. In order to accomplish this, ‘Celina’ and its potential pollinizers were all S-genotyped. After harvest, seeds collected from ‘Celina’ fruit in 2017 and 2018 were genotyped using eleven microsatellite markers. Genomic DNA was also extracted from leaf material collected from ‘Celina’, as well as from five pear cultivars used as pollinizers in the three examined orchards, and analyzed using the same marker set. Subsequently a simple sequence repeat (SSR) database was constructed and used for gene assignment analyses with the aim of quantifying pollen donor contribution from individual pollinizers. The obtained results indicate that ‘Anna’, the only examined pollinizer that was fully cross-compatible with ‘Celina’, together with ‘Fritjof’, the genotype which had the highest flowering overlap with ‘Celina’, proved to be the most successful pollinizers across all seasons and orchards. Although both cultivars were ubiquitous in the examined orchards, either as planted trees or as branches introduced during the flowering period, they were the most abundant pollinizers in only one orchard each. It is therefore possible to conclude that pollinizer abundance has a secondary significance in pollinizer success within investigated ‘Celina’ orchards.

The main goal of any DNA extraction procedure is to ensure reliable and reproducible results in a simple, fast and inexpensive manner. When it comes to plant tissues, this goal is challenging to achieve due to the presence of a variety of metabolites that interfere with DNA during isolation and downstream analysis. In this study, we compared the efficiency of three methods for DNA extraction from plum kernels: 1) the standard CTAB Soltis method which is the most common protocol for DNA extraction from various plant tissues (seeds, young leafs, mature leafs, root); 2) CTAB-based method originally described for DNA isolation from medicinal plants with high levels of secondary metabolites; 3) and one of various commercially available kits. The usefulness of the obtained DNA was evaluated by SSR analysis with seven microsatellite markers. Although the latter two extraction protocols retrieved genomic DNA that gave positive PCR results, only DNA isolated by kit produced full SSR profile

UDK: 582.675.1:575(497.6) Helleborus multifidus Vis. is endemic Illyric-Adriatic species with distribution range in Italy, Slovenia, Croatia, Bosnia and Herzegovina, Montenegro and Albania. Although few studies reported different taxonomic categories for H. multifidus, this one is the first using molecular-genetic markers (trnL region and matK of chloroplast DNA and nuclear ITS1 and ITS2 region) for genetic characterization of H. multifidus presented at three localites in Bosnia and Herzegovina. The results revealed that PCR-RFLP on trnL intron was not informative for testing inter- or intrapopulation diversity. Contrary, analysis of matK, ITS1 and ITS2 sequences showed differences between populations from Trebinje region and Kupreško polje, pointing to the need to include additional analyses in order to confirm these findings.