Project

The Centre of Excellence for Biodiversity and Molecular Plant Breeding (CroP-BioDiv)

Goal: The Centre of Excellence for Biodiversity and Molecular Plant Breeding is a research driven network aimed to deliver both knowledge and outputs in direct support for agricultural research progress. The objectives of the CoE include: (1) Increase of benefits arising out of use of plant genetic resources for food and agriculture (2) Identification of key plant traits through field experiments and laboratory analysis and optimize protocols for phenotyping, (3) Optimalization of genotyping protocols introducing common standardized lab analysis procedures and (4) Implementation of novel statistical methodology. The research will be conducted in eight model species chosen to represent economically most important as well as the most promising crops in Croatia: maize, wheat, soybean, grape vine, olive, Brassicas, common bean and Dalmatian pyrethrum. The main aim is to promote cooperation and synergy among all Croatian universities and research institutes dedicated to research and development of agriculture in order to establish the novel research platform that will incorporate recent scientific and technological advancements to cope with and respond to impending challenges in plant breeding sector.

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Petra Štambuk
added 3 research items
Grapevine leaves are photosynthetically the most active green organs providing carbohydrates that are of utmost importance for the regular vine’s metabolism and growth. Moreover, leaves are the pioneers of fungal infections caused by B. cinerea, E. necator, and P. viticola. Plant response to these microorganisms mostly depends on the content and composition of phenolic compounds abundantly found in the leaf’s outer tissues (epidermis, cuticle, trichomes). In order to obtain a satisfactory quantity of phenolic compounds, an experiment was conducted towards optimizing a solid–liquid extraction method. Variables were as follows: the type of organic solvent, the sample weight, the extraction temperature, and the extraction time. The optimal conditions were obtained by applying the response surface methodology. Therefore, by using acetonitrile as the organic solvent, conducting a single-step extraction at the temperature of 48 °C during the time period of 2 h and 25 min with a solid-to-solvent ratio of 1:56 g mL−1 (178 mg of leaves powder and 10 mL of extraction solvent) the optimal content of phenolic compounds was obtained. This protocol is reliable, fast, and relatively easy to perform for the determination of the abovementioned compounds.
The Plasmopara viticola pathogen causes one of the most severe grapevine diseases, namely downy mildew. The response to P. viticola involves both visible symptoms and intricate metabolomic alterations, particularly in relation to volatile organic compounds, and depends on the degree of resistance of a particular variety. There are numerous native grapevine varieties in Croatia, and they vary in susceptibility to this oomycete. As previously reported, in vitro leaf disc bioassay and polyphenolic compound analysis are complementary methods that can be used to separate native varieties into various resistance classes. This research used the Solid Phase Microextraction-Arrow Gas Chromatography-Mass Spectrometry method to identify the early alterations in the VOCs in the leaves after P. viticola inoculation. Based on the absolute peak area of sesquiterpenes, some discrepancies between the sampling terms were noticed. The presence of certain chemical compounds such as humulene, ylangene, and α-farnesene helped distinguish the non-inoculated and inoculated samples. Although specific VOC responses to P. viticola infection of native varieties from various resistance classes could not be identified, the response of less susceptible native varieties and resistant controls was associated with an increase in the absolute peak area of several compounds, including geranylacetone, ß-ocimene, and (E)-2-hexen-1-ol.
In the era of sustainable grapevine production, there is a growing demand to define differences between Vitis vinifera varieties in susceptibility to downy mildew. Croatia, as a country with a long tradition of grapevine cultivation, preserves a large number of native grapevine varieties. A leaf disc bioassay has been conducted on 25 of them to define their response to downy mildew, according to the International Organisation of Vine and Wine (OIV) descriptor 452-1, together with the stress response of the leaf discs using chlorophyll fluorescence and multispectral imaging with 11 parameters included. Time points of measurement were as follows: before treatment (T0), one day post-inoculation (dpi) (T1), two dpi (T2), three dpi (T3), four dpi (T4), six dpi (T5), and eight dpi (T6). Visible changes in form of developed Plasmopara viticola (P. viticola) sporulation were evaluated on the seventh day upon inoculation. Results show that methods applied here distinguish varieties of different responses to downy mildew. Based on the results obtained, a phenotyping model in the absence of the pathogen is proposed, which is required to confirm by conducting more extensive research.
Zlatko Šatović
added a research item
Oplemenjivanje bilja dugotrajna je sofisticirana djelatnost koju obavlja osoblje sa specijaliziranim znanjima iz više znanstvenih područja i zahtijeva skupu infrastrukturu. Konačni su ishod oplemenjivanja bilja novi kultivari (sorte) poljoprivrednog bilja koji nastaju tijekom višegodišnjih ciklusa odabira. Novi, prinosniji kultivari, poboljšane kakvoće proizvoda, prilagođeni okolišnim i edafskim uvjetima ciljanog područja te otporni na biotske i abiotske stresove, temelj su razvitka poljoprivredne proizvodnje. Oplemenjivanje bilja kao djelatnost - kao i istraživanja iz područja biljne genetike, u Republici Hrvatskoj imaju dugu tradiciju. Suvremeno komercijalno oplemenjivanje bilja provodi se u privatnim tvrtkama i na javnim ustanovama koje hrvatskoj poljoprivredi osiguravaju veliku dodanu vrijednost. Udio je domaćih kultivara u strukturi sjetve najvažnijih poljoprivrednih kultura pozamašan i gospodarski vrlo značajan. Posljednjih godina hrvatske oplemenjivačke kuće plasiraju svoje sjeme te otvaraju sestrinske tvrtke i u inozemstvu. Ta istraživačka i gospodarska djelatnost prepoznata je i uvrštena i u Strategiju pametne specijalizacije Republike Hrvatske. Pojam „molekularno oplemenjivanje bilja” odnosi se na primjenu dijagnostičkih molekularnih metoda te pratećih biometričkih metoda i računalnih alata koji omogućuju ubrzanje i povećanje preciznosti konvencionalnog oplemenjivačkog procesa bez izravnih manipulacija u genomu. Sadržaj ove znanstvene monografije obuhvaća pregled problema klasičnog oplemenjivanja bilja i sadašnje stanje primjene molekularnih metoda koje se danas obilno koriste u bazičnim i primijenjenim istraživanjima. Uz teoretsku osnovu različitih pristupa koji se koriste u molekularnom oplemenjivanju bilja u monografiji su navedene i brojne studije slučaja u kojima se opisuju rezultati konkretnih istraživanja provedenih u okviru Znanstvenog centra izvrsnosti za bioraznolikost i molekularno oplemenjivanje bilja (ZCI CroP-BioDiv). Ova je monografija prvenstveno namijenjena studentima diplomskih i poslijediplomskih studija iz područja biotehničkih i prirodnih znanosti. Želja nam je potaknuti mlade istraživače i oplemenjivače na uvođenje i razvoj novih strategija i tehnologija u oplemenjivanju bilja koje integriraju upotrebu biljnih genetskih izvora sa suvremenim metodama fenotipizacje i genotipizacije. Ovo djelo posvećujemo svim našim nekadašnjim, sadašnjim i budućim oplemenjivačima čiji su trud i rezultati rada nedovoljno prepoznati i cijenjeni, a putem novih kultivara i domaćeg sjemena daju velik doprinos poljoprivredi i ukupnom gospodarstvu Hrvatske.
Filip Varga
added a research item
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.
Smiljana Goreta Ban
added 6 research items
Shallots are a perennial plant from the Alliaceae family, classified with the common onion under the name of the Allium cepa Aggregatum group. The term shallot is also used for diploid and triploid viviparous onions, known as Allium × proliferum (Moench) Schrad and Allium × cornutum Clementi ex Vis., respectively. In this study, we compared the dry matter, pyruvic acid content, sugar content, flavonoid content, antioxidant capacity and mineral composition of 34 shallot accessions falling into three shallot species (Allium × cornutum, Allium × proliferum and A. cepa Aggregatum). Shallot accessions belonging to the A.× cornutum and A. × proliferum groups are characterized by high dry matter content (around 25%), of which a little less than 50% is formed of inulin-type sugars, polysaccharides, considered an excellent prebiotic with beneficial effects on human health. On the other hand, accessions belonging to the A. cepa Aggregatum group have lower dry matter content and, as a result, lower pungency (measured as pyruvic acid content), making them more suitable for fresh consumption by a broader range of consumers, but, at the same time, abundant in phenolic compounds, especially quercetin and isorhamnetin glycosides. We also observed a greater biodiversity among accessions within the A. cepa Aggregatum group in all the analyzed physico-chemical parameters compared to the other shallot groups. The investigated shallot accessions have an excellent in vitro antioxidant capacity, as well as excellent nutritional properties.
Zlatko Šatović
added a research item
Common bean (Phaseolus vulgaris L.) is the most widespread legume in Croatia and its production is based on landraces of great morphological diversity. Landraces morphologically differ the most in the seed coat color and size. Because plant emergence and crop establishment represent the most sensitive stage in crop development, the aim of this study was to determine whether the seed coat color and seed size of Croatian common bean landraces affect the water absorption rate, seed germination, emergence and phenotypic characteristics of the seedlings. In this study seeds of four common bean landraces with different seed color and size, ‘Biser’ (white-colored, small-seeded), ‘Bijeli’ (white-colored, large-seeded), ‘Kornjača’ (dark-colored, small-seeded) and ‘Trešnjevac’ (dark-colored, large-seeded) were evaluated in three different experiments: (i) water uptake, (ii) seed germination and (iii) emergence and seedling phenotypic characterization. The results show that white-colored seeds have a higher absorption rate and release more electrolytes compared to dark-colored seeds of similar size (and weight). The germination results show that white-colored and smaller seeds germinate faster compared to dark-colored and large seeds. On the other hand, the white-colored landrace ‘Bijeli’ took the longest time to emerge, probably due to cell damage that occurred during the fast initial water absorption. Using multispectral imaging, chlorophyll fluorescence imaging and stomatal conductance analysis, the seedlings of the dark-colored and large-seeded landraces were found to contain more photosynthetic pigments and have higher light absorption. In contrast, seedlings of white-colored and large seeds have lower stomatal conductance and transpiration and higher photochemical efficiency (despite possible cell damage during water absorption and germination). Results suggest that dark-colored seeds could survive better under unfavorable soil conditions without absorbing water, swelling, emergence or molding than white-colored seeds. Despite all this, white-colored common bean landraces remain popular in human diets and are often grown on small-scale farms.
Filip Varga
added 4 research items
Pyrethrin is a leading plant biopesticide that is highly effective against numerous pests and has little or no negative impact on human health and the environment. It is a specialized metabolite from Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch. Bip. ; Asteraceae), a plant species native to the eastern Adriatic coast. Pyrethrin consists of six compounds, pyrethrin I and II, cinerin I and II, and jasmolin I and II, with pyrethrin I and II accounting for the largest proportion of total pyrethrins. In order to determine the intraspecific biochemical variability and to investigate the influence of different ecological conditions on the accumulation of the individual compounds, 15 natural Dalmatian pyrethrum samples were collected. Ultrasound-assisted extraction was used for the pyrethrin extraction, followed by their qualitative and quantitative analysis on HPLC-UV- DAD. Total pyrethrin content ranged from 0.82 % to 1.27% of flower dry weight (DW). Populations were clustered in five bioclimatic groups ; A (Northern Adriatic populations and one coastal population from central Adriatic), B (two central Adriatic island populations), C (two central Adriatic island populations), D (Southern Adriatic island populations), E (inland Southern Adriatic populations, populations from Bosnia and Herzegovina and Montenegro), each characterized by a unique chemical profile. The highest average values for pyrethrin I (53.07% of total pyrethrin), total pyrethrin content (1.06 % DW) and the ratio of pyrethrin I and II (1.85) were found in the bioclimatic group E. The observed variability was partially explained by significant correlation found between the levels of some pyrethrin compounds and bioclimatic variables (e.g., BIO03 Isothermality and BIO04 Temperature seasonality). The results will support the selection of populations for breeding programs aimed at producing cultivars with desirable biochemical properties and adaptation to different bioclimatic conditions.
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.
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.
Zlatko Šatović
added a research item
Studying the population-genetic and phylogeographic structures of a representative species of a particular geographical region can not only provide us with information regarding its evolutionary history, but also improve our understanding of the evolutionary processes underlying the patterns of species diversity in that area. By analysing eight highly polymorphic microsatellite loci and two chloroplast DNA regions, we have investigated the influence of Pleistocene climate fluctuations on the evolutionary history of Salvia officinalis L. (common sage). The populations with the highest genetic diversity were located in the central parts of the Balkan distribution range. A large group of closely related haplotypes was distributed throughout the Balkans and the central Apennines, while the private lineage occupied the southern Apennines. In addition, two highly differentiated lineages were scattered only over the Balkans. The results suggest that a single refugium of the studied species from the last glacial period was located in the central part of the range in the Balkans. Numerous microrefugia, probably spanning several glaciation cycles, were scattered across the Balkans, while colonisation of the Apennines from the Balkans occurred at least on two occasions.
Maja Jukić Špika
added a research item
The influence of different irrigation regimes on olive fruit morphological parameters and on the quantity and quality (marketable indices, phenolic content, fatty acid composition, and sensory profile) of virgin olive oil (VOO) obtained from the Croatian cultivar Oblica, grown on an extremely rocky and dry reclaimed karst soil, was studied over three years. Four treatments were applied: rain-fed and three treatments calculated as 50%, 75%, and 100% of the crop’s irrigation requirement (Irr). Principal component analysis separated growing seasons (GS) that differed in precipitation. In the 2016 season, which had a low number of fruits per kilogram and provided a higher amount of balanced VOO with medium to intense bitterness and pungency (rain-fed treatment), the oil yield increased by irrigation (Irr 75 and Irr 100) up to 18%, while unchanged phenolics, bitterness, and pungency were observed for the VOOs obtained. In the drier GS (2017), which under rain-fed conditions had high fruit per kg, smallest fruit sizes, and lowest oil yield, and in which the VOOs had high phenolic content and intense sensory taste attributes, fruit weight, fruit sizes, and oil yield increased by 35% in all irrigation treatments, while phenols, bitterness, and pungency decreased, balancing the sensory profile of the VOOs. The results obtained here led us to conclude that the irrigation of young olives resulted in a positive effect, with the indication that an abundant water supply is more effective in drought conditions.
Zlatko Šatović
added a research item
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.
Filip Varga
added a research item
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.
Gabriela Vuletin Selak
added a research item
Two bacteria belonging to the Pseudomonas and Pantoea genera were isolated from olive knots. Both bacterial strains were omnipresent in this study’s olive orchard with high susceptibility of the autochthonous olive genotypes indicating coevolution of bacteria with host plants. Genomes of two endemic bacteria show conserved core genomes and genome plasticity. The Pseudomonas ST1 genome has conserved virulence-related genes including genes for quorum sensing, pilus, and flagella biosynthesis, two copies of indole acetic acid biosynthesis (IAA) operons, type I-VI secretions systems, and genes for alginate and levan biosynthesis. Development of knots depends only on the presence of the Pseudomonas ST1 strain which then allows Pantoea paga strain co-infection and cohabitation in developed knots. The two bacteria are sensitive to a large number of antimicrobials, antibiotics, H2O2, and Cu (II) salts that can be efficiently used in propagation of bacterial free olive cultivars.
Filip Varga
added 2 research items
Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.) is a plant species that occurs naturally in the eastern Mediterranean. It is of immense economic importance as it synthesizes and accumulates the phytochemical compound pyrethrin. Pyrethrin consists of several monoterpene esters (pyrethrin I and II, cinerin I and II and jasmolin I and II), which have insecticidal and repellent activity through their synergistic action. In this study, 15 natural Dalmatian pyrethrum populations were sampled along their natural range in Croatia, Bosnia and Herzegovina and Montenegro to characterize and compare their pyrethrin profiles and to define the bioclimatic factors associated with the accumulation of each pyrethrin compound. Pyrethrins were extracted from the dried flower heads of Dalmatian pyrethrum using ultrasound-assisted extraction and the amount of each compound was quantified using high-performance liquid chromatography coupled to DAD-UV /VIS. The biochemical data were subjected to analysis of variance, correlation analysis and multivariate analysis. Quantitative variability within and among populations was found, with population P15 Vranjske Njive, Podgorica having the significantly highest pyrethrin I content (66.47% of total pyrethrin content), while the highest levels of total pyrethrin were found in P14 Budva (1.27% of flower dry weight ; DW), followed by P08 Korčula (1.15% DW). Based on the environmental conditions at the sampling sites of the populations, five bioclimatic groups were distinguished, referred to as A, B, C, D, and E, each with unique chemical profile. The first group (A) consisted of the northern Adriatic population P01 Vrbnik, Krk and the population P06 Sevid - the coastal population of the central Adriatic, and generally differed significantly from the other bioclimatic groups by higher average jasmolin II values (2.13% of total pyrethrin). The second group (B) consisted of two central Adriatic island populations (P02 Telašćica, Dugi otok and P03 Žman, Dugi otok), while the remaining central Adriatic island populations were grouped in bioclimatic group C, which was characterized by the significantly highest average pyrethrin II (48.52% of total pyrethrin) and cinerin II (5.31% DW) content. The South Adriatic inland populations P10 Srđ and P11 Trebinje (Bosnia and Herzegovina), and the populations from Montenegro (P12 Grahovo, P13 Lovćen, P14 Budva and P15 Vranjske Njive, Podgorica) formed bioclimatic group E. This bioclimatic group was characterized by the highest average values for pyrethrin I (53.07 % of total pyrethrin), total pyrethrin content (1.06 % DW) and the ratio of pyrethrin I and II (1.85). Slightly lower values (although not significant) for the latter traits were detected in bioclimatic group D (southern Adriatic island populations P07 Vis, P08 Korčula and P09 Mljet). A weak but significant correlation was found between the levels of some pyrethrin compounds and bioclimatic variables (e.g., BIO03 Isothermality and BIO04 Temperature Seasonality), which explains part of the variability observed in the populations studied. This suggests the interconnection between bioclimatic variables and biochemical profiles either through the selection of adapted genotypes or through the ability of species to alter the expression of biochemical traits in response to environmental changes.
Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip. ; Asteraceae), a source of the commercially dominant plant insecticide pyrethrin, is a species endemic to the eastern Adriatic. Genetic diversity of T. cinerariifolium was previously studied using amplified fragment length polymorphism (AFLP) markers. However, microsatellite markers (single sequence repeats - SSRs) are more informative because they are codominant, highly polymorphic, locus-specific, and more reproducible, and thus are most often used to assess genetic diversity of plant species. Dalmatian pyrethrum is an outcrossing diploid (2n = 18) whose large genome size and high repeatability have prevented the success of the traditional approach to SSR markers development. The advent of next-generation sequencing combined with the specifically developed method recently enabled the development of, to the author's best knowledge, the first set of SSRs for genomic characterization of Dalmatian pyrethrum, which is essential from the perspective of plant genetic resources conservation. To evaluate the effectiveness of the developed SSR markers in genetic differentiation of Dalmatian pyrethrum populations, a preliminary genetic diversity study was conducted on 30 individuals from three geographically distinct natural populations in Croatia (northern Adriatic island of Mali Lošinj, southern Adriatic island of Čiovo, and Mount Biokovo) based on 12 SSR loci. Analysis of molecular variance (AMOVA) by randomization test with 10, 000 permutations was performed in Arlequin 3.5. The average number of alleles per locus, observed and expected heterozygosity, probability of deviations from Hardy-Weinberg equilibrium, and inbreeding coefficient were calculated using GENEPOP 4.4. Genetic distance based on the proportion of common alleles (DPSA) was calculated using MICROSAT. Cluster analysis using the neighbor-joining method with 1, 000 bootstraps was performed with PHYLIP to generate a dendrogram. The results of the AMOVA analysis showed that the total SSR diversity was 23% within and 77% between the three populations. A slight deviation from Hardy-Weinberg equilibrium was observed in the Mali Lošinj population. Allele richness ranged from 2.92 to 3.92, with the highest number of private alleles observed in the Mali Lošinj population (17). The average observed DPSA between 30 individuals was 0.557. The highest DPSA (0.875) was observed between several pairs of Dalmatian pyrethrum individuals from the Mali Lošinj and Mt. Biokovo populations, and the lowest between two individuals from the Čiovo population. Neighbor-joining tree, based on DPSA, grouped individuals into clusters according to their population affiliation. The separation of Mt. Biokovo clade was supported (bootstrap value 58%), which is consistent with the previous study on AFLP markers, where isolated populations from Mt. Biokovo differed from the rest of the populations. The developed SSR markers are an effective tool for assessing the genetic diversity and structure of natural Dalmatian pyrethrum populations. These preliminary results are encouraging for a future comprehensive study with a larger sample size across the species' range. Combined with the biochemical data, these highly informative markers could help identify potential genotypes of interest for future development of breeding lines and cultivars that are both resistant to environmental stress and high in pyrethrins.
Filip Varga
added a research item
Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch. Bip.) is an outcrossing plant species (2n = 18) endemic to the eastern Adriatic coast and source of the natural insecticide pyrethrin. Due to the high repeatability and large genome (1C-value = 9.58 pg) our previous attempts to develop microsatellite markers using the traditional method were unsuccessful. Now we have used Illumina paired-end whole genome sequencing and developed a specific procedure to obtain useful microsatellite markers. A total of 796,130,142 high-quality reads (approx. 12.5× coverage) were assembled into 6,909,675 contigs using two approaches (de novo assembly and joining of overlapped pair-end reads). A total of 31,380 contigs contained one or more microsatellite sequences, of which di-(59.7%) and trinucleotide (25.9%) repeats were the most abundant. Contigs containing microsatellites were filtered according to various criteria to achieve better yield of functional markers. After two rounds of testing, 17 microsatellite markers were developed and characterized in one natural population. Twelve loci were selected for preliminary genetic diversity analysis of three natural populations. Neighbor-joining tree, based on the proportion of shared alleles distances, grouped individuals into clusters according to population affiliation. The availability of codominant SSR markers will allow analysis of genetic diversity and structure of natural Dalmatian pyrethrum populations as well as identification of breeding lines and cultivars.
Filip Varga
added a research item
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.
Jerko Gunjaca
added 2 research items
Combining experimental studies on grain yield variability with crop model simulations in maize could assist in choosing the optimum maturity group for a certain location, counteracting the effect of climate change. However, studies considering specificities in Southeast Europe are lacking. The objectives were to put various environmental covariates including stress degree days (SDD) into FAO maturity settings to determine the impact of climate change on maize growing in Southeast Europe and to compare trends for grain yields over twenty years of maize experimental and simulation data grouped in five FAO maturity groups (FAO 200–FAO 600). Pre-registration yield trials of maize planted in one location in Croatia grown from 1996 to 2015 were used to determine “potential yield”. Correlation coefficients between 12 climate covariates and grain yield (GY) across the maturity groups revealed the tightest negative associations between SDD and GY that were weakened by later-maturity groups. Similar trends in GY were obtained by both experimental and simulation data, highlighting FAO 600 as a nearly no yield-reducing FAO group over the two decades. Our results indicate that choosing early maize hybrids in Southeast Europe does not seem to be an optimum option in the future, since these hybrids are more sensitive to omnipresent heat stress than late hybrids.
Development of new cultivars and agronomic improvements are key factors of increasing in future grain yield in maize grown in environments affected by climate change. Assessment of value for cultivation and use (VCU) reflects the results of latest breeding efforts showing yield trends, whereby external environmental covariates were rarely used. This study aimed to analyze several environmental effects including stress degree days (SDD) on grain yields in Croatian VCU trials in three maturity groups using linear mixed model for the estimation of fixed and random effects. Best linear unbiased predictions (BLUPs) of location-year interaction showed no pattern among maturity groups. SDD showed mostly non-significant coefficients of regression on location BLUPs for yield. Analyzing location BLUPs, it was shown that the effect became consistently stronger with later maturity, either positive or negative. The effects of management might play more critical role in maize phenology and yield formation compared with climate change, at least in suboptimum growing conditions often found in Southeast Europe. To facilitate more robust predictions of the crop improvement, the traditional forked approach dealing with G×E by breeders and E×M by agronomists should be integrated to GxExM framework, to assess the full gradient of combinations forming the adaptation landscape.
Zoe Andrijanić
added a research item
Soybean is generally grown as a rainfed crop worldwide and very often is exposed to drought and high temperatures. The objectives of this study were to determine the performance and stability of 32 elite soybean genotypes for seed protein and oil contents across six dry and eight normal environments and to determine the influence of environment on the relationship between the two traits. In the combined ANOVA, genotypes (G), environments (E) and G × E were significant for both traits with protein content being more sensitive to environmental changes than oil content. Mean seed protein content decreased by 4.5% under drought conditions compared to normal ones and ranged from 2.3% to 7.1% for individual genotypes. At the same time drought caused a slight increase in seed oil content of +1.2%, with a range of −1.3% to +4.5% for individual genotypes. Genotype stability in terms of regression coefficient (b) and coefficient of variation (CV) was in moderate to weak negative correlation with mean genotype performance for protein content, while no correlation was observed between genotype stability and mean genotype performance for oil content. Protein and oil content were significantly negatively correlated in normal environments (r = −0.33), while no correlation between the traits was observed in dry environments (r = −0.02)
Maja Jukić Špika
added a research item
A single phenolic group and even a compound play different roles in the sensory properties and stability of virgin olive oil (VOO), which in turn are strongly influenced by several factors. Understanding the causes of differences in phenolic compound composition and oxidative stability (OS) in VOOs is essential for targeted and timely harvest and processing while maintaining desired oil quality. The phenolic profile and OS of two monocultivar VOOs (Oblica and Leccino) grown in two geographical sites of different altitudes (coastal plain and hilly hinterland) were analyzed throughout the ripening period over two years. Concentration of secoiridoids was 30% higher in the Oblica than in the Leccino VOOs, which in turn had significantly higher values of OS. Both cultivars had more than twice as high concentrations of the two most abundant phenolic compounds, the dialdehyde form of decarboxymethyl oleuropein aglycone and the dialdehyde form of decarboxymethyl ligstroside aglycone, and OS values in a colder growing site of higher altitude. Among the studied monocultivar VOOs, the secoiridoid group did not behave equally during ripening. The hierarchy of different influencing factors was investigated using multivariate statistics and revealed: cultivar > geographical site > harvest period > growing season. In addition, the possibility of traceability of VOO using molecular markers was investigated by establishing SSR profiles of oils of the studied cultivars and comparing them with SSR profiles of leaves
Petra Štambuk
added a research item
Since grapevine is highly susceptible to various pathogens, enormous amounts of pesticides are applied each season to achieve profitable production. One of the most destructive grapevine diseases is downy mildew, and their interaction has been in the spotlight for more than a decade. When it comes to a metabolome level, phenolic compounds are relevant to investigate due to their involvement in the plant immune system and known antifungal properties. Croatian grapevine germplasm is highly heterogeneous due to its long history of cultivation in diversified geographical regions. Since it has been found that native varieties react differently to the infection of Plasmopara viticola, the intention of this study is to define if the chemical background of the leaves, i.e., polyphenolic composition, is responsible for these dissimilarities. Therefore, the leaves of 17 genotypes, among which 14 were native and 3 were controls, were analyzed using high-performance liquid chromatography (HPLC) in four terms: before inoculation and 24, 48, and 96 h post inoculation (hpi). During this early phase, significant differences were found neither between the terms nor between the non-inoculated and inoculated samples, except for resveratrol-3-O-glucoside. By applying principal component analysis (PCA) using initial leaf polyphenolic composition, varieties of V. vinifera were clearly separated into three different groups corresponding to their International Organization of Vine and Wine (OIV) classes of susceptibility to P. viticola. Results obtained in this research suggest that the initial constitutive polyphenolic composition of the cultivar leaves has a crucial influence on their susceptibility to P. viticola, and this finding can be used to improve the success of grapevine breeding programs toward downy mildew resistance.
Filip Varga
added a research item
Pyrethrin is a specialized metabolite of Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), Asteraceae, known worldwide as an effective bioinsecticide. It consists of six active compounds: Pyrethrin I and II, cinerin I and II, and jasmolin I and II. Pyrethrin accumulates mainly in the flower heads and its content depends on numerous factors, such as the flower developmental stage. This study aims to investigate the accumulation patterns of six pyrethrin compounds in the flower heads of Dalmatian pyrethrum over the eight developmental stages (FS1 to FS8), and to make a comparison in six natural populations. Ultrasound assisted extraction was used to extract the pyrethrin, while qualitative and quantitative analysis was performed by High performance liquid chromatography. The accumulation patterns of different pyrethrin compounds were generally similar and also synchronous between different populations, while the pyrethrin I/pyrethrin II ratio showed irregular patterns. In all populations studied, the highest increase of all compounds was observed from FS1 to FS2. Their concentration continuously increased, reaching the highest values at FS4 stage (2–5 rows of open disc flowers), and generally decreased gradually towards FS6 or FS7 and then stagnated until the FS8 stage. Despite the very similar accumulation pattern of pyrethrin compounds in the different populations, the significant differences in their content suggest a different genetic background. Knowledge of the dynamics of pyrethrin compounds accumulation across flower development stages is valuable for determining the optimal harvest time of pyrethrum flower heads.
Domagoj Šimić
added a research item
The multiple-stress effects on plant physiology and gene expression are being intensively studied lately, primarily in model plants such as Arabidopsis, where the effects of six stressors have simultaneously been documented. In maize, double and triple stress responses are obtaining more attention, such as simultaneous drought and heat or heavy metal exposure, or drought in combination with insect and fungal infestation. To keep up with these challenges, maize natural variation and genetic engineering are exploited. On one hand, quantitative trait loci (QTL) associated with multiple-stress tolerance are being identified by molecular breeding and genome-wide association studies (GWAS), which then could be utilized for future breeding programs of more resilient maize varieties. On the other hand, transgenic approaches in maize have already resulted in the creation of many commercial double or triple stress resistant varieties, predominantly weed-tolerant/insect-resistant and, additionally, also drought-resistant varieties. It is expected that first generation gene-editing techniques, as well as recently developed base and prime editing applications, in combination with the routine haploid induction in maize, will pave the way to pyramiding more stress tolerant alleles in elite lines/varieties on time.
Gabriela Vuletin Selak
added a research item
The olive tree (Olea europaea L.) is a wind-pollinated crop that exhibits an extreme alternate bearing habit. To improve fruit set, several methods have been used to determine the most successful compatible combinations of cultivars. In this study, priority is given to seed paternity analysis based on simple sequence repeats (SSRs), microsatellite markers used for the identification of potential pollen donors of cultivar ‘Oblica’ in a mixed olive orchard during two consecutive years. Seven microsatellite primers were successfully used to examine the paternity of olive embryos from ‘Oblica’ mother trees. Embryos were considered as a product of self-fertilization if only maternal alleles were present, but not a single case of self-fertilization was found among all the embryos analyzed. Two dominant pollen donors were not the closest nor the cultivars with the highest number of trees in the orchard, suggesting that cross-compatibility may have a key role in determining pollen donor success. In our earlier studies, pollen tube growth and fertilization success correlated with fruit set when controlled crosses between cultivars were performed; however, some discrepancy might appear compared to paternity analyses when mother trees have a free choice among different pollen sources from cultivars growing in their surroundings.
Filip Varga
added a research item
Seeds collected from 10 Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip. Asteraceae) populations naturally occurring along the Adriatic coast and islands, were used to establish a common garden with the aim of estimating the variation of pyrethrin compounds between and within populations. A recently optimized matrix solid phase dispersion (MSPD) method was used for the extraction of six pyrethrin compounds from dry flower heads of 200 Dalmatian pyrethrum individuals. Separation and quantification of pyrethrin compounds were performed by high performance liquid chromatography with diode array detector (HPLC-DAD). High variability in pyrethrin content and composition was observed within and between populations. Total pyrethrin content in individual samples varied from 0.10% to 1.35% of flower dry weight. On average, the lowest total pyrethrin content was observed in the population of Pelješac (0.22%) and the highest in the island populations of Mali Lošinj and Zlarin (0.87%). The population of Mali Lošinj had the most favourable pyrethrin profile, including the lowest variability in total pyrethrin content, the highest pyrethrin I (PI) content (up to 60.47%) and PI/PII ratio (up to 5.88), as well as the lowest pyrethrin II (PII) content. In contrast, the populations from Biokovo and Pelješac represented pyrethrin profiles with the lowest content of total pyrethrin, PI and PI/PII ratio. The Biokovo population also had the highest PII content (43.18% on average), while the Pelješac population had the highest jasmolin I (JI) and jasmolin II (JII) content. Four different chemotypes were determined by cluster analysis. Chemotype P1 is the most promising for future breeding programs and is characterized by the highest total pyrethrin content, PI content and PI/PII ratio, as important measures of insecticidal activity. Correlation analysis of each pyrethrin compound with 25 environmental variables and Principal Component Analysis (PCA) were carried out and the importance of optimum temperature and precipitation on pyrethrin quantity and quality was suggested. Total pyrethrin content was significantly correlated with six temperature and five precipitation parameters. Pyrethrin I, PII and cinerin II (CII) were significantly correlated mainly with temperature parameters; JII and cinerin I (CI) with precipitation parameters, while JI was correlated with temperature range and precipitation parameters. The mountainous Biokovo population was an outlier in the PCA analysis, mainly due to the extremely low values of temperature parameters at a higher altitude. Precipitation variables separated the central Adriatic islands inhabiting drier habitats from populations inhabiting habitats with greater precipitation. The high-resolution data from this study provide the opportunity for individual-based selection for breeding plants with the best pyrethrin profiles, as well as for increased adaptation to broader climatic conditions.
Josip Tadic
added 5 research items
In the face of climate change, water deficit and increasing soil salinity pose an even greater challenge to olive cultivation in the Mediterranean basin. Due to its tolerance to abiotic stresses, wild olive (Olea europaea subsp. europaea var. sylvestris) presents a good candidate in breeding climate-resilient olive varieties. In this study, the early response of the native Croatian wild olive genotype (WOG) to salinity was evaluated and compared with that of well-known cultivars (cv.) Leccino and Koroneiki. Potted olive plants were exposed either to 150 mM NaCl or 300 mM mannitol for 3 weeks to distinguish between the osmotic and ionic components of salt stress. To determine the impact of the plant age on salinity, 1-, 2-, and 3-year-old WOG plants were used in the study. The growth parameters of both the cultivars and WOG of different ages decreased in response to the mannitol treatment. In contrast to cv. Leccino, the NaCl treatment did not significantly affect the growth of cv. Koroneiki or WOG of any age. The contents of Na + and Cl − were considerably higher in the salt-treated WOG, regardless of age, compared with the cultivars. However, while both treatments significantly reduced the K + content of cv. Koroneiki, that nutrient was not significantly affected in either cv. Leccino or WOG. Unlike the cultivars and older WOG, the NaCl treatment caused a significant decline of photosynthetic pigments in the 1-year-old WOG. The cultivars and WOG of different ages experienced a similar drop in the chlorophyll a content under the isotonic mannitol treatment. The absence of lipid peroxidation, modulation of superoxide dismutase, and guaiacol peroxidase activity were noted in all WOG ages under both stressors. These data suggest that WOG resilience to salinity is associated with its large leaf capacity for Na + and Cl − accumulation, K + retention, and its adaptable antioxidative mechanisms. The results are promising with regard to obtaining a new olive cultivar with better resilience to soil salinity.
Zlatko Šatović
added 2 research items
With the development of next-generation sequencing technology and bioinformatics tools, the process of assembling DNA sequences has become cheaper and easier, especially in the case of much shorter organelle genomes. The number of available DNA sequences of complete chloroplast genomes in public genetic databases is constantly increasing and the data are widely used in plant phylogenetic and biotechnological research. In this work, we investigated possible inconsistencies in the stored form of publicly available chloroplast genome sequence data. The impact of these inconsistencies on the results of the phylogenetic analysis was investigated and the bioinformatic solution to identify and correct inconsistencies was implemented. The whole procedure was demonstrated using five plant families (Apiaceae, Asteraceae, Campanulaceae, Lamiaceae and Rosaceae) as examples.
Hop (Humulus lupulus L.) is used in the brewing industry as a source of compounds responsible for the bitterness, aroma, and preservative properties of beer. In this study, we used microsatellite markers to investigate genetic diversity and genetic differentiation of wild hop populations sampled in the northwestern part of Croatia. Analysis of 12 microsatellite loci revealed high diversity and weak population differentiation among wild hop populations. A total of 152 alleles were determined with an average of 12.67 alleles per locus. Observed heterozygosity ranged from 0.689 to 0.839 (average 0.767) and expected heterozygosity ranged from 0.725 to 0.789 (average 0.760). A total of 38 private alleles were detected. The data suggest that H. lupulus populations are not affected by recent bottlenecks. The degree of genetic differentiation among populations was low and not significant for most pairwise FST values, except for the pair of geographically most distant populations. The results did not indicate the existence of genetic structure among the sampled populations. The high genetic diversity and low differentiation among populations, combined with the absence of isolation by distance, indicate the existence of substantial gene flow among wild hop populations. Therefore, extensive sampling per population is clearly required to assess the genetic diversity of hop populations. Sampling strategies involving sampling across a large number of localities represented by only a few samples could lead to erroneous conclusions.
Zlatko Šatović
added a research item
Olea europaea ‘Galega vulgar’ variety is a blend of West and Central Mediterranean germplasm with cultivated-wild admixture characteristics. ‘Galega vulgar’ is known for its high rusticity and superior-quality olive oil, being the main Portuguese variety with high impact for bioeconomy. Nevertheless, it has been replaced by higher-yielding and more adapted to intensive production foreign varieties. To clarify the potential ancestral origin, genetic diversity evolution, and existing genetic relationships within the national heritage of ‘Galega vulgar’, 595 trees, belonging to ancient and centenary age groups and prospected among ten traditional production regions, were characterized using 14 SSR markers after variety validation by endocarp measurements. Ninety-five distinguishable genets were identified, revealing the presence of a reasonable amount of intra-genetic and morphological variability. A minimum spanning tree, depicting the complete genealogy of all identified genets, represented the ‘Galega vulgar’ intra-varietal diversity, with 94% of the trees showing only a two-allele difference from the most frequent genet (C001). Strong correlations between the number of differentiating alleles from C001, the clonal size, and their net divergence suggested an ancestral monoclonal origin of the ‘Galega vulgar’, with the most frequent genet identified as the most likely origin of all the genets and phenotypic diversification occurring through somatic mutations. Genetic erosion was detected through the loss of some allele combinations across time. This work highlights the need to recover the lost diversity in this traditional olive variety by including ancient private genets (associated with potential adaptation traits) in future breeding programs and investing in the protection of these valuable resources in situ by safeguarding the defined region of origin and dispersion of ‘Galega vulgar’. Furthermore, this approach proved useful on a highly diverse olive variety and thus applicable to other diverse varieties due either to their intermediate nature between different gene pools or to the presence of a mixture of cultivated and wild traits (as is the case of ‘Galega vulgar’).
Ivana Plavšin
added a research item
Genotype-by-environment interaction (GEI) is often a great challenge for breeders since it makes the selection of stable or superior genotypes more difficult. In order to reduce drawbacks caused by GEI and make the selection for wheat quality more effective, it is important to properly assess the effects of genotype, environment, and GEI on the trait of interest. In the present study, GEI patterns for the selected quality and mixograph traits were studied using the Additive Main Effects and Multiplicative Interaction (AMMI) model. Two biparental wheat populations consisting of 145 and 175 RILs were evaluated in six environments. The environment was the dominant source of variation for grain protein content (GPC), wet gluten content (WGC), and test weight (TW), accounting for approximately 40% to 85% of the total variation. The pattern was less consistent for mixograph traits for which the dominant source of variation has been shown to be trait and population-dependent. Overall, GEI has been shown to play a more important role for mixograph traits compared to other quality traits. Inspection of the AMMI2 biplot revealed some broadly adapted RILs, among which, MG124 is the most interesting, being the prevalent “winner” for GPC and WGC, but also the “winner” for non-correlated trait TW in environment SB10.
Smiljana Goreta Ban
added a research item
This study examines genetic diversity and structure of a Croatian garlic germplasm collection using 13 simple sequence repeat (SSR) markers. A total of 71 alleles were observed across 64 accessions representing 3 Croatian regions (Istria, Dalmatia and continental Croatia) and 16 foreign landraces, with an average of 5.46 alleles per locus. Among the 80 accessions analysed, 61 distinct multilocus genotypes (MLG) were identified, of which 51 represented unique genotypes and the remaining accessions were divided into 10 MLG groups, comprising potential duplicates or redundant genotypes. Model-based Bayesian and hierarchical UPGMA clustering approaches revealed five major groups within the collection which partially correlated with geographical origin. The analysis of molecular variance (AMOVA) showed that the majority (87.71%) of the total molecular diversity is within the Croatian groups of accessions, even though a significant share (12.29%) of diversity derived from genetic diversity among groups. These results support regional structuring, as well as the existence of significant diversity within local populations. This study is the first comprehensive report on an extensive evaluation of genetic resources of garlic maintained by Croatia with the aim of setting the course for future preservation strategies with particular emphasis on the value of diversity in the context of climate change both on macro and micro levels.
Smiljana Goreta Ban
added a research item
The present study was conducted to establish a protocol for the regeneration of virus-free garlic plants through somatic embryogenesis of two Croatian garlic ecotypes. Basal parts of cloves from mother plants were cultured on a full Murashige and Skoog (MS) or modified MS medium (¼ of KNO3 and NH4NO3 and 2xMgSO4) containing 0.1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) or 1 mg L−1 2,4-D + 0.5 mg L−1 kinetin (Kin) and representing four different treatments. Plants were regenerated in MS medium containing 0.1 mg L−1 2,4-D and rooted in a medium containing 0.05 mg L−1 1-naphthaleneacetic acid (NAA) + 0.005 mg L−1 6-(γ,γ-dimethylallylamino)purine (2iP). The presence of viruses (i.e., sanitary status) of the mother plants and regenerants was checked by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The mother plants were infected with onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV). In addition, the presence of garlic common latent virus (GCLV) was confirmed in four mother plants. Embryogenic callus developed in all four treatments with success ranging from 55% to 81% depending on treatment and ecotype. Plant conversion was significantly higher in somatic embryos developed in media containing 0.1 mg L−1 2,4-D than those developed in media containing 1 mg L−1 2,4-D + 0.5 mg L−1 Kin. Virus elimination success ranged from 13.3% up to 62.5% depending on garlic ecotype and treatment. The overall rate of virus elimination by somatic embryogenesis for both treatments and ecotypes were 20.7%, 22.9%, and 30.5% for OYDV, GCLV, and LYSV, respectively. Based on these results, somatic embryogenesis has been shown to be equally or more successful in eliminating garlic viruses compared to other in vitro methods.
Zlatko Šatović
added a research item
Basil is one of the most widespread aromatic and medicinal plants, which is often grown in drought- and salinity-prone regions. Often co-occurrence of drought and salinity stresses in agroecosystems and similarities of symptoms which they cause on plants complicates the differentiation among them. Development of automated phenotyping techniques with integrative and simultaneous quantification of multiple morphological and physiological traits enables early detection and quantification of different stresses on a whole plant basis. In this study, we have used different phenotyping techniques including chlorophyll fluorescence imaging, multispectral imaging, and 3D multispectral scanning, aiming to quantify changes in basil phenotypic traits under early and prolonged drought and salinity stress and to determine traits which could differentiate among drought and salinity stressed basil plants. Ocimum basilicum "Genovese" was grown in a growth chamber under well-watered control [45-50% volumetric water content (VWC)], moderate salinity stress (100 mM NaCl), severe salinity stress (200 mM NaCl), moderate drought stress (25-30% VWC), and severe drought stress (15-20% VWC). Phenotypic traits were measured for 3 weeks in 7-day intervals. Automated phenotyping techniques were able to detect basil responses to early and prolonged salinity and drought stress. In addition, several phenotypic traits were able to differentiate among salinity and drought. At early stages, low anthocyanin index (ARI), chlorophyll index (CHI), and hue (HUE2 D ), and higher reflectance in red (R Red ), reflectance in green (R Green ), and leaf inclination (LINC) indicated drought stress. At later stress stages, maximum fluorescence (F m ), HUE2 D , normalized difference vegetation index (NDVI), and LINC contribute the most to the differentiation among drought and non-stressed as well as among drought and salinity stressed plants. ARI and electron transport rate (ETR) were best for differentiation of salinity stressed plants from non-stressed plants both at early and prolonged stress.
Maja Jukić Špika
added a research item
The authenticity and typicity of monocultivar oils and knowledge of the changes that environmental olive growing conditions bring to naturally present antioxidants and sensory attributes of virgin olive oils (VOO) are important for quality and safety improvement. This study delivers a comprehensive evaluation of the factors affecting phenolics, fatty acid composition and sensory characteristics of cultivars Oblica and Leccino VOOs throughout ripening season at two distinct olive growing environments during three consecutive crop years, and ranks the importance of each factor. Specified parameters were significantly influenced by olive growing environmental conditions. At the colder location of higher altitude, both cultivars gained higher amount of stearic, linoleic and linolenic fatty acids, as well as a higher proportion of phenolic compounds, but lower amounts of oleic fatty acid. At the warmer location of lower altitude, both cultivars had oils with lower level of fruitiness, bitterness and pungency. Analysis of the main components showed that VOOs were primarily differentiated by the cultivar, then main groups were divided with regard to the growing site, while harvest period affected the biosynthesis of natural VOOs antioxidants but had the least impact. These results reveal that the composition of fatty acids, phenolic content and sensory profile are predominantly characteristics of a cultivar.
Zlatko Šatović
added a research item
Selection for wheat (Triticum aestivum L.) grain quality is often costly and time-consuming since it requires extensive phenotyping in the last phases of development of new lines and cultivars. The development of high-throughput genotyping in the last decade enabled reliable and rapid predictions of breeding values based only on marker information. Genomic selection (GS) is a method that enables the prediction of breeding values of individuals by simultaneously incorporating all available marker information into a model. The success of GS depends on the obtained prediction accuracy, which is influenced by various molecular, genetic, and phenotypic factors, as well as the factors of the selected statistical model. The objectives of this article are to review research on GS for wheat quality done so far and to highlight the key factors affecting prediction accuracy, in order to suggest the most applicable approach in GS for wheat quality traits.
Zlatko Šatović
added 2 research items
Micronutrient malnutrition is one of the main public health problems in many parts of the world. This problem raises the attention of all valuable sources of micronutrients for the human diet, such as common bean (Phaseolus vulgaris L.). In this research, a panel of 174 accessions representing Croatian common bean landraces was phenotyped for seed content of eight nutrients (N, P, K, Ca, Mg, Fe, Zn, and Mn), and genotyped using 6,311 high-quality DArTseq-derived SNP markers. A genome-wide association study (GWAS) was then performed to identify new genetic sources for improving seed mineral content. Twenty-two quantitative trait nucleotides (QTN) associated with seed nitrogen content were discovered on chromosomes Pv01, Pv02, Pv03, Pv05, Pv07, Pv08, and Pv10. Five QTNs were associated with seed phosphorus content, four on chromosome Pv07, and one on Pv08. A single significant QTN was found for seed calcium content on chromosome Pv09 and for seed magnesium content on Pv08. Finally, two QTNs associated with seed zinc content were identified on Pv06 while no QTNs were found to be associated with seed potassium, iron, or manganese content. Our results demonstrate the utility of GWAS for understanding the genetic architecture of seed nutritional traits in common bean and have utility for future enrichment of seed with macro– and micronutrients through genomics-assisted breeding.
Landraces represent valuable genetic resources for breeding programmes to produce high-yielding varieties adapted to stressful environmental conditions. Although the common bean (Phaseolus vulgaris L.) is an economically important food legume for direct human consumption worldwide, common bean production in Croatia is based almost exclusively on landraces and there is no common bean breeding program. Information on phaseolin type and results of population structure and genetic diversity obtained by analysis of SSR and SNP markers, in combination with the morphological characterization of 174 accessions of 10 common bean landraces (morphotypes), enabled thorough classification of accessions. The accessions were classified into phaseolin type H1 (“S”) of Mesoamerican origin and phaseolin types H2 (“H” or “C”) and H3 (“T”) of Andean origin. By applying distance- and model-based clustering methods to SSR markers, the accessions were classified into two clusters at K = 2 separating the accessions according to the centres of origin, while at K = 3, the accessions of Andean origin were further classified into two clusters of accessions that differed in phaseolin type (H2 and H3). Using SNP markers, model-based analysis of population structure was performed, the results of which were consistent with those of SSR markers. In addition, 122 accessions were assigned to 14 newly formed true-type morphogenetic groups derived from three different domestication events: (1) Mesoamerican (H1A) (“Biser”, “Kukuruzar”, “Tetovac”, “Trešnjevac”), (2) Andean—indeterminate type (H2B1) (“Dan noć”, “Sivi”, “Puter”, ”Sivi prošarani”, “Trešnjevac”) and (3) Andean—determinate type (H3B2) (“Bijeli”, “Dan noć”, “Puter”, “Trešnjevac”, “Zelenčec”). The rest of the accessions could represent putative hybrids between morphogenetic groups. The differences between the true-type groups of accessions were further analysed based on nine quantitative traits, and the subsets of traits that best distinguish among centres of origin (A: Mesoamerican, B: Andean) and genetic groups (H1A, H2B1, H3B2) were proposed.
Smiljana Goreta Ban
added a research item
Shallots are an edible Alliaceous crop representing a group of genetically and morphologically different species. Shallot species determination is rather complex due to the high variability in phenotypes within a single species. Flower morphology has been successfully employed in shallot species determination; however, shallot florogenesis depends upon many genetic and environmental factors. There is a need for more accessible morphological descriptors used in shallot species determination, since flowering in shallot may not be consistent. In this study, we investigated the discriminating power of shallot vegetative and bulb morphology descriptors. European Cooperative Programme for Plant Genetic Resources morphology descriptors were used for describing 35 Croatian shallot accessions. The proposed methodology based on vegetative and bulb morphological descriptors could be used for shallot species discrimination. Additionally, two subtypes of A. cepa Aggregatum group were identified in this study: the first being the shallot type (1) and a potato onion type (2), which differed based on bulb morphology descriptors (bulb shape, bulb skin color, and a number of bulblets).
Petra Štambuk
added a research item
Downy mildew is one of the most destructive diseases affecting viticulture, caused by Plasmopara viticola Berl. and De Toni, an obligate biotrophic oomycete whose life cycle is mostly maintained in any grapevine green tissue. Infection of grapevine begins in spring with dispersed sporangium produced from oospores and lasts until the end of summer. The most common way for controlling this disease is by repeated fungicide applications which causes the development of resistant strains, residual toxicity, and pathogen pressure. Moreover, it is harmful to the environment and human health, but also economically expensive. In the absence of treatments, and with favourable weather conditions, downy mildew can devastate the crop in one season causing a serious economic loss. Unlike the innate disease tolerance present in several wild American and Asian Vitis species, there are different levels of susceptibility among cultivated Vitis vinifera L. varieties. In the era of “omics” approaches it became possible to detailly investigate the early host response mechanism and metabolomic background of resistant or partially resistant varieties. Phenolics compounds are used as biomarkers of resistance due to their higher concentration in resistant or tolerant genotypes compared to susceptible ones upon the downy mildew infection. With the introduction of deep sequencing, resistance proteins can be identified and their resistance gene analogues which is of utterly importance for using non-vinifera germplasm in breeding programs. The aim of this review is to congregate the most contemporary knowledge about the downy mildew biology and the complete background of grapevine’s subsequent response.
Smiljana Goreta Ban
added 5 research items
The interactive effects of cultivar, collecting period, and geographical location on the content and composition of biophenols and macro and micronutrients in olive (Olea europaea L.) leaf were investigated. Leaves of six cultivars were collected at three periods in two locations in Croatia. The leaves of Istarska bjelica cultivar had the greatest biophenol (oleuropein) potential, especially those sampled in January and in March at the location of Pag. All the cultivars yielded leaves with the highest concentration of biophenols in March, which coincided with the pruning period. Except for high oleuropein concentration in Istarska bjelica, flavonoids were found to be most useful for differentiating olive leaves according to cultivar. Verbascoside turned out to be the most potent differentiator of collecting periods, while phosphorus and zinc turned out to be most useful for differentiating locations. Despite different agroecological conditions at the two locations, cultivar exhibited a significant effect on olive leaf nutrient composition, which was certainly causally related to that of the biophenols. The results obtained showed that it is possible to plan more well-timed and efficient exploitation of biophenols from olive leaf based on the knowledge about the interactive effects of the three studied factors.
In order to investigate the potential of various olive cultivars and leaf sampling times for phytochemical farming practice in Croatia, phenolic and mineral composition was determined in olive leaves of four Croatian cultivars and Italian cultivar Leccino collected at three occasions, in October 2017, January 2018, and March 2018. Istarska bjelica turned out to have the largest phytochemical potential among the investigated cultivars due to steady high oleuropein concentrations found in its leaves. The concentration of main phenolic components in Istarska bjelica leaves changed only slightly during the sampling period, suggesting the possibility of its higher capability for low air temperatures stress resistance and different metabolic response compared to the other studied cultivars. Low air temperatures increased the oleuropein level and antioxidant activity in leaves of Leccino, Oblica, Levantinka, and Drobnica cultivars, which may be of crucial phytochemical farming interest. Each of the investigated olive cultivars was characterized by a specific leaf mineral nutrient composition, which could have had a specific role in their interplay with phenols.
Potato onions (Allium cepa var aggregatum G. Don) are multiplying or aggregating onions, very similar to shallots and have been historically cultivated throughout Europe. Currently in Northern Europe they are maintained in home gardens and ex situ field collections. Potato onions are primarily vegetatively propagated, however in Estonia, near Lake Peipsi, this species has been propagated by seed since the seventeenth century. There is increasing interest in Northern Europe in utilizing this germplasm in organic and/or sustainable farming systems. The genetic diversity and relationship between and within European potato onion collections is unclear. From historical records it is known that cultivation, exchange and trade of potato onion has occurred throughout Europe for hundreds of years. This study utilised molecular markers to assess genetic diversity, duplication of genotypes and relationships among and between Nordic, Baltic, Czech and Croatian potato onion collections. Of 264 accessions, 80 catalogued as unique had identical genotypes with one or more other accessions, and are putative duplicates. The genetic diversity within two Estonian sexually propagated accessions was comparable to that found in all of the vegetatively propagated accessions. Accessions from the Nordic countries grouped together genetically, as did Latvian and Lithuanian accessions. Croatian accessions were genetically separated. These genetic relationships suggest historical movement of potato onion germplasm in North-Eastern Europe. The results, in conjunction with other passport and characterization data, can assist in the development of potato onion core collections, facilitating the conservation and utilization of valuable potato onion genetic resources.
Mario Franić
added 2 research items
Chlorophyll fluorescence (ChlF) parameters are reliable early stress indicators in crops, but their relations with yield are still not clear. The aims of this study are to examine genetic correlations between photosynthetic performance of JIP-test during flowering and grain yield (GY) in maize grown under two heat scenarios in the field environments applying quantitative genetic analysis, and to compare efficiencies of indirect selection for GY through ChlF parameters and genomic selection for GY. The testcrosses of 221 intermated recombinant inbred lines (IRILs) of the IBMSyn4 population were evaluated in six environments at two geographically distinctive locations in 3 years. According to day/night temperatures and vapor pressure deficit (VPD), the two locations in Croatia and Turkey may be categorized to the mild heat and moderate heat scenarios, respectively. Mild heat scenario is characterized by daytime temperatures often exceeding 33°C and night temperatures lower than 20°C while in moderate heat scenario the daytime temperatures often exceeded 33°C and night temperatures were above 20°C. The most discernible differences among the scenarios were obtained for efficiency of electron transport beyond quinone A (QA) [ET/(TR-ET)], performance index on absorption basis (PIABS) and GY. Under the moderate heat scenario, there were tight positive genetic correlations between ET/(TR-ET) and GY (0.73), as well as between PIABS and GY (0.59). Associations between the traits were noticeably weaker under the mild heat scenario. Analysis of quantitative trait loci (QTL) revealed several common QTLs for photosynthetic and yield performance under the moderate heat scenario corroborating pleiotropy. Although the indirect selection with ChlF parameters is less efficient than direct selection, ET/(TR-ET) and PIABS could be efficient secondary breeding traits for selection under moderate heat stress since they seem to be genetically correlated with GY in the stressed environments and not associated with yield performance under non-stressed conditions predicting GY during flowering. Indirect selection through PIABS was also shown to be more efficient than genomic selection in moderate heat scenario.
Excess of cadmium (Cd) in soil leads to a number of adverse effects which challenge agricultural production. Aims of this study were to investigate variations in Cd content in leaves (Zea mays L.) of different maize genotypes and to detect effects of Cd on photosynthesis through chlorophyll a fluorescence. Pot vegetation experiments with four maize genotypes and four different soil Cd levels were repeated for two years. Chlorophyll fluorescence, photosynthetic pigments and inductively coupled plasma (ICP) analysis for ear-leaf Cd and zinc (Zn) and soil Cd were carried out. Significant differences between genotypes were found for leaf Cd, where higher Cd soil concentrations resulted in higher Cd leaf concentrations. Cd uptake into maize leaves increased with increasing Cd levels in soil, which was genotype-dependent and higher and lower Cd accumulating groups were formed. Changes in chlorophyll fluorescence caused by elevated Cd levels in soil were mostly visible as changes in dissipation energy, yields of primary photosystem II photochemistry and electron transport. Decrease of reaction centers per antenna chlorophyll and increased variable fluorescence at J step (VJ) resulted in decrease of performance indexes in the highest Cd concentration. Decreases in chlorophyll fluorescence parameters suggest reduced functionality of reaction centers and problems in re-oxidation of primary quinone acceptor (QA).
Zlatko Šatović
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By performing a high-resolution spatial-genetic analysis of a partially clonal Salvia brachyodon population, we elucidated its clonal architecture and seedling recruitment strategy. The sampling of the entire population was based on a 1 × 1 m grid and each sampled individual was genotyped. Population-genetic statistics were combined with geospatial analyses. On the population level, the presence of both sexual and clonal reproduction and repeated seedling recruitment as the prevailing strategy of new genets establishment were confirmed. On the patch level, a phalanx clonal architecture was detected. A significant negative correlation between patches’ sizes and genotypic richness was observed as young plants were not identified within existing patches of large genets but almost exclusively in surrounding areas. The erosion of the genetic variability of older patches is likely caused by the inter-genet competition and resulting selection or by a random die-off of individual genets accompanied by the absence of new seedlings establishment. This study contributes to our understanding of how clonal architecture and seedling recruitment strategies can shape the spatial-genetic structure of a partially clonal population and lays the foundation for the future research of the influence of the population’s clonal organization on its sexual reproduction.
Zoe Andrijanić
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There are numerous soybean [Glycine max (L.) Merr.] breeding programs in Europe focused on development of elite non genetically modified (GM) cultivars for fast growing market of GM-free proteins for animal feed. Due to low variability of visual descriptors and mostly unknown pedigrees, divergent parents’ selection for crosses is a great challenge. Another challenge is cultivar distinction and protection of plant breeders’ rights of ever-increasing number of cultivars. By using 42 microsatellite (SSR) markers, we performed characterization of 97 commercial soybean cultivars and experimental lines developed at various research and breeding institutions in Europe (86) and in North and South America (11) in order to assess their genotype distinction power as well as utility for estimating genetic diversity and genetic structure. A set of 27 most polymorphic SSR markers was sufficient to discriminate all 97 genotypes. Discrimination of, by pedigree very related cultivars, was somewhat difficult due to the low polymorphism but still possible. Cluster analysis showed that European germplasm is mainly distributed into clusters reflecting breeding programs and maturity groups. Performed genetic characterization provides an insight into genetic structure of European soybean germplasm and might serve as a starting point for future breeding decisions. Disclosed SSR data of the analyzed commercial European germplasm can serve for genetic fingerprinting purpose as well as for foundation of public soybean cultivar database.
Smiljana Goreta Ban
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The aim of this study was to define optimal harvesting date of garlic cv. 'Istarski crveni' by comparing the amount of volatile compounds as one of quality indicators. In total 17 volatile compounds were determined in tested garlic samples by headspace solid-phase microextraction coupled to gas chromatography with flame ionization and mass spectrometric detection (HS-SPME-GC-FID-MS) regardless of harvesting date. Major volatiles diallyl trisulfide, diallyl sulfide, methyl allyl disulfide and diallyl disulfide representing 98.5 % of all the determined compounds have shown no significant difference between harvest dates. Significantly higher content of the two minor volatiles, allyl mercaptane and thieno[2,3-b]thiophene, was found in the last and second when compared to the first harvest date. Principal components PC1 and PC2 explained 78% of the total variance and pointed to certain relations between harvest dates and volatiles. The technological maturity had probably been reached at the first harvesting date since similar amounts of volatiles were found during the observed period.
Zlatko Šatović
added a research item
Essential oils from seven populations of Dalmatian sage from the North of Albania were extracted and analyzed according European Pharmacopeia 7.0. The yield differed from 1.95 to 2.75% v/w. The GC-FID-MS analysis of the sage oil revealed 42 constituents. Camphor (20.50-29.74%), followed by α-thujone (19.87-24.29%), 1,8-cineole (6.88-12.64%) and β-thujone (4.00-9.14%) were predominant constituents in all analyzed populations, representing 55.70-68.00% of the total components. All of the analyzed sage populations from northern Albania comply with ISO 6571/2008 standard and European Pharmacopeia 7.0. for Dalmatian sage essential oil content, while only four populations met the requirements of the ISO 9909/1997 standard for Dalmatian sage essential oil’s composition.
Zlatko Šatović
added a research item
The high-biodiversity phenomenon of the Balkan Peninsula is a result of the past and present topographic, climatic and geological conditions, as well as human impact. The Dinaric Alps, as a part of the Balkan Peninsula, harbour a high number of endemic species, some of them comprising a small number of populations, which are often endangered. We investigated spatial distribution, community composition, site ecology, genetic diversity and conservation of Degenia velebitica, a stenoendemic species of the north-western Dinaric Alps. Our results showed that D. velebitica is nowadays restricted to three localities with the area of occupancy of 48,560 m2, harbouring approximately 37,000 individuals. Degenia velebitica stands are differentiated into three well-characterized, floristically homogenous syntaxa, very distinct from the surrounding vegetation, suggesting their azonal occurrence and restriction to spatially highly fragmented microsites exposed to stormy winds. Spatial distances of populations, differences in D. velebitica community composition and site ecology are mirrored in genetic variation patterns of the populations, such as high-frequency down-weighted marker values in the north-western populations and high gene diversity in the south-eastern group of populations. The fact that we could not find a single D. velebitica individual at the locus classicus indicates the existence of the ever-growing fragmentation. The analyses of genetic structure using AFLP data recognized two main genetic groups of populations as evolutionary significant units that should be considered when planning protection measures. According to our IUCN Red List reassessment, D. velebitica should be treated as a critically endangered species that requires immediate conservation actions.
Ana Nimac
added 2 research items
Sea fennel (Crithmum maritimum L.) is a perennial plant tolerant to the soil salinity and could be used as an alternative culture on salty soils. For successful sea fennel cultivation, it is necessary to test germinability of the seed. In this study, germination tests have been carried out on a population of sea fennel from Lopar, island of Rab, Croatia. Th e aim was to examine the eff ect of seed priming on seed germination, radicle length, radicle surface area, average diameter of radicle and radicle volume of sea fennel under diff erent salt concentrations. Seeds were primed with sodium chloride (NaCl) (50 mM) or distilled water (dH 2 O) during seven days aft er which its germinability and early seedling growth was tested on the germination paper treated with diff erent concentrations of NaCl (50, 100 and 150 mM) and dH 2 O as control. Seeds that were primed with dH 2 O and 50 mM NaCl solution showed the better performance than non-primed seeds; priming alleviated negative eff ects of low NaCl concentration.
Zlatko Šatović
added 3 research items
Background and Purpose: Natural stands of pedunculate oak in Croatia have been delineated in seed areas, zones and regions. The current bylaw recommends that the transfer of reproductive material remains limited within zones, but that it is permitted within areas. Clonal seed orchards (CSOs) of pedunculate oak were established to increase genetic quality of seed and to acquire a more regular seed yield than in natural stands. In total 150 plus trees were selected within three seed regions. The selection included a number of favourable traits of tree size and stem quality. Three CSOs were planted with grafted plus-trees. We aimed to establish whether these orchards encompass enough genetic diversity to potentially produce genetically improved and sufficiently diverse reproductive material. We also wanted to assess neutral genetic differentiation between these orchards and compare it with the genetic diversity obtained from chloroplast DNA markers, depicting conserved lineages from recolonization routes. We wanted to investigate spatial genetic structure in the area of our research and provide additional information on the transfer of forest reproductive material. Materials and Methods: Leaves were collected from all clones in the CSOs. Total genomic DNA was extracted and clones were analysed with eight nuclear and ten chloroplast microsatellite markers. Spatial autocorrelation analysis was performed with nuclear microsatellite data and original plus trees’ coordinates, for each CSO separately, to determine whether shared favourable traits among the selected plus trees in smaller distances are the results of relatedness, which the sampling strategy tried to avoid. Results: We found 28 chloroplast haplotypes belonging to four maternal lineages, and significant differentiation between CSOs, indicating origin from different refuges. Nuclear microsatellites’ diversity in the CSOs is quite high and comparable to diversity found within a recent study of Croatian natural populations. Nuclear microsatellites did not show genetic differentiation between CSOs, i.e. between the seed regions and seed zones they represent. No genetic differentiation was found with nuclear microsatellites among haplotypic lineages. We found no genetic structure within the analysed regions. Conclusions: Lack of differentiation between CSOs found with nuclear microsatellites confirms the permission for transfer of reproductive material between zones within the seed area 1 - Lowland Forests. If original differentiation between chloroplast haplotypic lineages was present after recolonization, it was erased by subsequent gene flow. Lack of genetic structure, with nuclear microsatellites within regions indicates successful sampling strategy.
Pre-breeding and elite breeding are two steps in creating high yielding sunflower hybrids that differ in well established procedures and selection methods. However, a methodology that bridge efficient use of introgression lines as product of pre-breeding procedures and their crossing to elite inbreed lines, is not yet very well established. Therefore, the development of cost- and time-efficient methods for the determination of best parent heterosis and the use of best inbred lines in crosses with introgression lines for obtaining high-yielding and stable hybrids is highly desirable. In this regard, sixteen Cytoplasmic Male Sterile (CMS) inbred lines (A) derived from four heterogeneous interspecific lines originating from three annual: H. debilis silvestris (DEB-SIL), H. praecox runyoni (PRA-RUN), H. deserticola (DES) and one perennial H. resinosus (RES) wild species were evaluated. Seven agronomic traits were measured over a period of 2 years and 38 DNA loci were analysed, in order to compare four different methods for the estimation of best parent heterosis (BPH). New inbred lines were characterized by Principal Component Analysis (PCA) of morphological traits and Principal Coordinate Analysis (PCoA) of molecular marker data. Line × tester mating design was used to evaluate General Combining Ability (GCA), while Genetic Distance (GC) estimated by markers was evaluated as a predictor of BPH by Locally Weighted Sequential Smoothing (LOESS). Analysis of combining ability is one of the most important tools breeders use to identify superior inbred lines on the basis of their performance in hybrid combinations. Results obtained in this research show that PCA of morphological and PCoA of molecular marker data on parental lines are generally in agreement with GCA effects for examined traits. GD versus BPH relationships indicate that intermediate to high GD between parental lines was optimal for best heterotic effects of most traits. In this study, we show that the combination of the PCA of morphological data, PCoA of molecular marker data and GD between parental lines is fast and affordable, giving the most important information for parental choice of introgression and elite lines in sunflower breeding programs.
The red raspberry (Rubus idaeus L.) is one of the most important fruit species in Serbia; there are currently approximately 11000 ha under cultivation. Wild red raspberry populations can be an important source of genes for breeding new raspberry varieties. This study was carried out to determine the genetic variability of wild populations, as well as their relationship with the most common cultivated cultivars using amplified fragment length polymorphism (AFLP) markers. In this study were included 128 individuals from seven wild populations and seven cultivar specimens from a nursery ('Mekeer', 'Willamette', 'Polka', 'Polana', 'Loganberry', 'Tayberry', 'Black Jewel'). Four AFLP combinations yielded a total of 247 polymorphic bands in 135 R. idaeus specimens. Analysis of AFLP markers showed that the cultivars are clearly distinct from wild populations. Population P4 (Mt. Ozren) had the highest gene diversity, the highest number of private markers and the highest rarity index. Analysis of molecular variance among and within wild populations showed that the most of the genetic diversity was attributable to differences between individuals within populations. A Mantel test showed that differences in altitude play the crucial role in structuring of the genetic diversity of red raspberry populations. Altitude contributed more to the population differentiation than spatial distance. Populations of wild raspberry from the territory of Serbia are characterized by high diversity, and should be protected and used as a resource in selection and breeding processes for the production of new varieties, with improved properties in terms of better yield and stronger resistance to external factors.
Filip Varga
added 3 research items
Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.) produces pyrethrin which is currently the most important natural insecticide. Due to the growing interest for its use in organic agriculture, the demand for this valuable plant is increasing. However, poor seed germination and inaccessibility of high quality planting material is one of the main constrains for the introduction of Dalmatian pyrethrum into agricultural production. Priming treatments are known to be able to induce rapid and more uniform seed germination, seedling emergence and plant growth and their effi ciency was tested on Dalmatian pyrethrum seed. Seed of four natural Dalmatian pyrethrum populations was subjected to hydropriming or osmopriming with 0.2% KNO 3 with the aim of determining their eff ect on germination parameters (germinability, mean germination time, mean germination rate, coeffi cient of variation of the germination time, uncertainty of the germination process and synchrony of the germination process). Th e signifi cantly highest(P<0.05) germinability was recorded for the Biokovo population (27.00%), followed by the population from Šolta (19.67%), Konavle (17.67%) and population from Šibenik (6.67%). Hydropriming for 24h signifi cantly increased the seed germination and germination index (P<0.05). Th e treatment with 0.2% KNO 3 improved germinability and germination index when compared to control, however, the diff erences were not signifi cant (P>0.05). As a low-cost, simple and practical, hydropriming can be recommended as an eff ective method to promote germination performance of Dalmatian pyrethrum seed.
An ethnobotanical survey was carried out in the Knin area (northern Dalmatia, Croatia) with the aim of recording traditional plant use by the local (native) people and contributing to the knowledge of plant biodiversity in the investigated area. Ethnobotanical data were collected by interviewing 40 local people at 17 locations. Data on 123 plant taxa (on average 21 taxa per interview) and 122 unique medicinal and 18 food uses in the local community were recorded. Prevalent medicinal uses of the recorded taxa were the treatment of digestive, respiratory, cardiovascular disorders, and injuries. The most commonly used plant parts gathered were leaves (35%) and flowers (20%). The most commonly collected plants in the investigated area were: Urtica dioica, Thymus longicaulis, Sambucus nigra, and Hypericum perforatum.
The mixture of six components (pyrethrin I and II, cinerin I and II, and jasmolin I and II) constituting natural insecticide pyrethrin is responsible for the insecticidal activity of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.). In order to obtain deeper knowledge of its insecticidal potential, the extraction of mentioned components from plant samples was carried out for the first time, by matrix solid-phase dispersion. The influence of different factors such as the type of sorbent, sorbent-sample ratio, the elution solvent and its volume, addition of co-sorbent and drying agent, affecting extraction yield was evaluated. The pyrethrin components were extracted under optimized conditions: florisil (0.5 g) as the sorbent, mixed with 0.25 g of pyrethrum flower heads, 5 mL of acetone-ethyl acetate (1:1, v/v) as the elution solvent and 0.4 g of Na2SO4 as the drying agent. All six components in the extracts were qualitatively and quantitatively determined by HPLC-DAD. Analytical method provides high degree of linearity with r2≻0.9974, except for pyrethrin II (0.9961). Also, the method showed good precision (intra- and inter-day) with a relative standard deviation below 14 %, and recoveries mainly above 80 %. In order to demonstrate the applicability of the developed method, three Dalmatian pyrethrum natural populations from Croatia were analyzed.
Igor Palcic
added 4 research items
The present study investigated the effect of boron (B) foliar application on the dynamic changes in the concentration of phenols, especially oleuropein, and volatile compounds of olive leaves during a 90-day period. The trial was conducted in a greenhouse on one year old olive plantlets, sprayed with B at 41.62 mM against a water treated control. It was revealed that total phenols and oleuropein concentration in old and in young olive leaves increased in B treated leaves 90 days after application. Furthermore, B enhanced the concentration of particular terpene and norisoprenoid volatiles in olive leaf infusion. The obtained results may give a new perspective in biochemical farming practice, with a possibility to increase the amounts of phenols and terpenes in olive leaves intended for medical uses, and to enhance olive plant resistance to pathogens and insects.
In modern agriculture, besides providing high and stable yields, it is imperative to produce products with a high nutritive quality. The goal of this study was to determine the effect of different fertilization regimes on the macro- and micronutrients in beetroot. A 3-year field trial was set up according to a Latin square method with four types of fertilization (unfertilized control, 50 t stable manure ha-1, and 500 and 1,000 kg NPK 5-20-30 ha-1). The mineral content was determined as follows (mg 100 g-1 in fresh weight of beetroot): 14-29 P, 189-354 K, 18-34 Ca, 17-44 Mg, 0.67-1.83 Fe, 0.41-0.65 Mn and 0.28-0.44 Zn. The highest beetroot P content was determined for the treatment with stable manure, especially in a year with dry climatic conditions. The highest beetroot K content was determined for the treatment with 1,000 kg NPK 5-20-30 ha-1, but at the same time for the same treatment, a general decreasing trend of micronutrient content was determined, due to the possible antagonistic effect of added potassium. For better mineral status of beetroot, application of combined mineral and organic fertilizers supplemented with additional foliar application of micronutrients can be suggested.
BACKGROUND The aim of this study was to examine the effect of foliar (Mn_fol) and soil Zeolite‐Mn (Mn_ZA) application on leaf mineral, total phenolic and oleuropein content, and mycorrhizae colonization of self‐rooted cv. Leccino plantlets grown on calcareous soil. RESULTS The dissolution of zeolite was 97% when citric acid was applied at 0.05 mM dm‐3, suggesting that organic acids excreted by roots can dissolve modified zeolite (Mn_ZA) making Mn available for plant uptake. The leaf Mn concentration was the highest for Mn_fol treatment at 90 DAT (172 mg kg‐1) and 150 DAT (70 mg kg‐1) compared to other treatments. Mn_ZA soil application increased leaf Mn concentration at 150 DAT compared to control and NPK treatment. The oleuropein leaf content was highest for Mn_fol compared to other treatments at 90 DAT and lowest at 150 DAT. Arbuscular mycorrhizal colonization was higher for Mn_fol treatment at 150 DAT compared to all other treatments. CONCLUSION Changes in the arbuscular colonization percentage and oleuropein content may be connected to stress conditions provoked by high leaf Mn concentration in Mn_fol treatment at 90 DAT. Mn_ZA application increased leaf Mn concentration at 150 DAT compared to control and NPK treatments. It can be assumed that the dominant mechanism in Mn uptake from modified zeolite is Mn_ZA dissolution through root exudates. This article is protected by copyright. All rights reserved.