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.

This report presents the outcome of the joint work of PhD students and senior researchers working with DNA-based biodiversity assessment approaches with the goal to facilitate others the access to definitions and explanations about novel DNA-based methods. The work was performed during a PhD course (SLU PNS0169) at the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. The course was co-organized by the EU COST research network DNAqua-Net and the SLU Research Schools Focus on Soils and Water (FoSW) and Ecology - basics and applications. DNAqua-Net (COST Action CA15219, 2016-2020) is a network connecting researchers, water managers, politicians and other stakeholders with the aim to develop new genetic tools for bioassessment of aquatic ecosystems in Europe and beyond. The PhD course offered a comprehensive overview of the paradigm shift from traditional morphology-based species identification to novel identification approaches based on molecular markers. We covered the use of molecular tools in both basic research and applied use with a focus on aquatic ecosystem assessment, from species collection to the use of diversity in environmental legislation. The focus of the course was on DNA (meta)barcoding and aquatic organisms. The knowledge gained was shared with the general public by creating Wikipedia pages and through this collaborative Open Access publication, co-authored by all course participants.

The Dinaric endemic plant species Moltkia petraea (Tratt.) Griseb. is often called a "living fossil" of ancient Tertiary flora, with great importance for Bosnia and Herzegovina’s biodiversity. Considering its narrow and limited distribution range, insufficient data on the molecular background of this species is given so far. Due to the presence of various secondary metabolites that interfere with the DNA, isolation of nucleic acids from plant cells is known to be challenging. Even in closely related species it is necessary to optimize DNA isolation protocol in order to obtain high quality PCR amplifiable DNA. We collected 91 samples from five populations in Herzegovina. Doyle and Doyle (1987) CTAB protocol was modified by adding vitamin C (ascorbic acid) to the cell lysis buffer to improve DNA yield and quality. trnL(UAA) intron and nrDNA (ITS1, ITS2) molecular markers were applied to demonstrate amplifiability of isolated DNA and elucidate the intra- and interpopulation genetic diversity. Our results suggest a successful PCR amplification for 81% of the analyzed samples. PCR-RFLP analysis of trnL(UAA) revealed that all individuals in five populations have the same haplotype based on the obtained enzymatic profile for three enzymes (TaqI, HinfI, HindII). Alignment and comparison of ITS sequences didn’t reveal any hypervariable portion that could be informative in elucidating the genetic diversity of M. petraea populations. Further studies with additional application of microsatellite loci, RAPD and AFLP methods are necessary in an attempt to get insights into the genetic diversity of M. petraea.

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. 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.