A molecular genetic approach to roebuck individual identification in the case of poaching in Serbia

Application of the molecular genetic methods in forensic cases dealing with wild animals has significantly increased recently. These techniques are practically used in order to help solving four key problems : determination of kind of the wild animal, geographic origin, kinship ties and individual identification. In this work the first case of introducing the examination of polimorphism of microsatelite genetic markers within forensic analysis in the cases of poaching in Serbia is presented. The objectives of this forensic analysis was to determine if the meat confiscated during house search of the suspect comes from roebuck origin (Capreolus capreolus), which remains had been found by a game warden in the field during closed season, where the suspect denied the offense, claiming that the meat comes from other roebuck that had been shot during the previous hunting season. DNK was isolated from the skin and fur samples taken from the roebuck corpse found in the woods, as well as from the frozen meat found in the suspect’s house. Both amplification and polimorphism examination of the eight microsatelite markers (ROE01, NVHRT21, NVHRT24, NVHRT48, NVHRT73, RT7 AND RT27) were carried out. In all the examined samples, the same pattern of variability of the tested microsatelites was determined, that is it was proved that DNK profiles of the samples taken from roebuck corpse were identical to DNK profile of the meat sample found in the suspect’s house. This result clearly indicates that all the examined biological samples originate from the same animal, and consequently represents forensically valid evidence in the case of roebuck poaching. [Projekat Ministarstva nauke Republike Srbije, br. III46002

Plant glacial refugia in Europe

Izmjene glacijala i interglacijala u razdoblju Kvartara utjecale su na geografsku rasprostranjenost vrsta u Europi danas. Vrste koje su bile bolje prilagođene na umjerenu klimu bile su ograničene na područja refugija u vrijeme glacijala dok su vrste koje su bile prilagođene na hladnu klimu bile ograničene na područja refugija u interglacijalima. Poseban utjecaj na geografski raspon vrsta imao je posljednji glacijalni maksimum. Glacijalni refugiji nalazili su se na jugu Europe (Apeninskom poluotoku, Pirinejskom poluotoku i Balkanu). Postojanje kriptičnih refugija za vrste umjerene klime koji su se nalazili sjevernije još uvijek je predmet mnogih znanstvenih rasprava. U ovom radu pokazana je mogućnost postojanja takvih refugija na primjeru obične bukve (Fagus sylvatica) i divlje jabuke (Malus sylvestris). Također, cilj ovog rada bio je dati uvid u kompleksne procese koji su se događali u refugijima tijekom posljednjeg glacijanog maksimuma i procese kolonizacije koji su uslijedili nakon njega. Naglašena je uloga glacijalnih refugija kao žarišta genske raznolikosti Europe te potreba za zaštitom tih područja. Potrebna su daljnja, detaljnija paleobotanička i genetička istraživanja kako bi se dobio detaljniji uvid u prošlost biljnih i drugih vrsta na Europskom kontinentu. Razumijevanje prošlosti glacijalnih refugija omogućit će bolja predviđanja utjecaja trenutnih klimatskih promjena na živi svijet.Changes between glacial and interglacial periods in Quaternary have influenced the geographical species distribution in Europe. Temperate-adapted species were confined to refugial areas during glacial periods while cold-adapted species were confined to refugial areas during the interglacial. Last glacial maximum had a significant influence on the today’s species distribution. Glacial refugia were located in the Iberian, Italian and Balkan Peninsula. The existence of cryptic, more northern glacial refugia for temperate species is still questioned by many scientists. In this paper, the possibility of existence for such refugia is demonstrated on examples of European beech (Fagus sylvatica) and wild apple (Malus sylvestris). The aim of this review was to give insights on the complex processes that happened in the glacial refugia during last glacial maximum and postglacial colonization. The role of glacial refugia as “hotspots” of genetic diversity in Europe and the need for their protection has been emphasized. Further paleobotanical and genetic research is needed to get a more precise insight in plant and other species’ history on European continent. Better understanding of the history of glacial refugia could give better predictions on influence of current climatic change on today’s species

ANALYSIS OF THE GENETIC DIVERSITY OF "LOVRAN MARRON" (Castanea sativa Mill.) USING MICROSATELLITE MARKERS

Maruni (maroni) su sorte europskog pitomog kestena (Castanea sativa Mill.) dobivene selekcijom, koje se od davnina uzgajaju radi proizvodnje krupnih i kvalitetnih plodova. Maruni su u Hrvatskoj sađeni na privatnim posjedima istočnih padina Učke, u okolici Lovrana i poznati su pod nazivom "lovranski marun". Do sada nije bilo znanstvenih istraživanja lovranskog maruna te nije poznato s kojim su biljnim materijalom nasadi podignuti, odnosno koliko je različitih genotipova zastupljeno. Ta saznanja ključna su za sve daljnje korake koje treba poduzeti kako bi se očuvali postojeći genetski izvori. Cilj ovoga istraživanja bila je analiza genetske raznolikosti stabala lovranskog maruna u postojećim nasadima, korištenjem mikrosatelitnih biljega. Istraživanje je rađeno na uzorku od 72 stabla, korištenjem 5 mikrosatelitnih biljega. Analiza je pokazala prisutnost 11 multilokusnih genotipova, što govori u prilog raznovrsnosti i bogatstvu svojti pitomog kestena na lovranskom području, koje još uvijek nisu taksonomski određene, a vode se pod kolektivnim nazivom "lovranski marun". Većina uzorkovanih stabala, 58, pripada istom genotipu, što se može tumačiti statičnošću u smislu introdukcije novih svojti na istraživano područje i forsiranjem, tj. ekstenzivnim uzgojem.Marrons are varieties of the European chestnut (Castanea sativa Mill.) obtained through selection, which have been grown since antiquity for the production of large and high quality fruits. In Croatia, marrons were planted on private properties on the eastern slopes of the Učka mountain, in the environs of Lovran, and are hence known as the "Lovran marron". There has been no scientific research of the Lovran marron to date, and it is unknown which plant material was used to raise the plantations, or how many different genotypes are represented. Those insights are crucial for any further steps to be undertaken in order to conserve the existing genetic resources. The aim of this study was to analyze the genetic diversity of the Lovran marron trees in the existing plantations, by using microsatellite markers. The study was conducted on a sample of 72 trees, using 5 microsatellite markers (Table 1). The analysis demonstrated the presence of 11 multilocus genotypes, pointing to the diversity and abundance of sweet chestnut taxa in the Lovran area, which have not yet been taxonomically defined and bear the collective name of the "Lovran marron". The majority of analyzed trees, specifically 58 individuals, had a uniform genetic structure and areassigned to the MG01 cultivar, which is therefore the most represented cultivar in the researched area, i.e. the one most often grown. However, not all trees are uniform, which is proven by the fact that the remaining 14 analyzed trees belong to 10 different gene pools. Of the 14 trees, 2 had not been grafted, but are found in the plantations together with the grafted marrons and are genetically specific as is to be expected. The remaining 12 grafted trees belong to 9 gene pools. Out of those, 5 trees share common alleles on all loci and are assigned the MG02 cultivar, whereas 7 trees were genetically unique and classified into 7 different cultivars (Tables 2, 3, 4 and Figure 1). Consequently, with regard to the "Lovran marron" operational taxonomic unit grown in the area of the Municipality of Lovran, although it is not taxonomically specified, on the basis of the genetic diversity analysis conducted using 5 microsatellite markers, it can be said to include several different genotypes, or cultivars, one of which (MG01) is present at a much higher frequency than others

Genetic characterization of alpine sheep breeds

The aim of this study was to characterize from the genetic point of view eight alpine sheep breeds reared in Italy (Bergamasca, Biellese, Schwarzbraunes Bergschaf and Tiroler Bergschaf), Germany (Brillenschaf and Weisses Bergschaf) and Slovenia (Bov\u161ka and Jezerzko-Sol\u10davska) through the use of microsatellite molecular markers. Allelic richness was rather high in each breed highlighting a considerable genetic diversity. However, the study evidenced a significant departure from Hardy-Weinberg equilibrium in all analyzed breeds caused by a heterozygote deficiency. Such lack seems to be caused by a rather high level of inbreeding. The genetic differentiation among breeds was rather low (F ST = 0.064) but significant. The neighbour-joining tree obtained from Reynolds\u2019 genetic distance estimates, showed the presence of three groups formed by the three Bergschaf breeds, the Italian Bergamasca and Biellese and the two Slovenian breeds together with the German Brillenschaf. Such grouping is in accordance with the breeds\u2019 region of origin and with their known history. Concluding, microsatellite resulted to be a useful tool to investigate breed variability and to characterize alpine sheep breeds. Obtained findings suggest the need to set up a conservation plan aiming to safeguard and increase the genetic variability of the studied breeds compromised by the high level of inbreeding. Microsatellites genotyping could help to monitor breed variability and to organize matings

Research of haplotypes HLA-B27 in patients with spondyloarthropathies in Croatian population

U ovom radu analizirali smo alele 5 mikrosatelita HLA (D6S248, D6S2674, D6S2811, STR_MICA i D6S273) među HLA-B27 pozitivnim nesrodnim osobama (N=94), bolesnicima s psorijatičnim artritisom (PsA) (N=22) i bolesnicima s juvenilnim spondiloartropatijama (jSpA) (N=29). U sve testirane skupine najučestaliji aleli bili su: D6S248-291pb, D6S2674-131pb, D6S2811-98pb i STR_MICA-A4, dok je na lokusu D6S273 najčešći alel među bolesnicima s jSpA i kontroli bio alel D6S273-4, a meĊu bolesnicima s PsA alel D6S273-5. Analizom bolesnika s PsA utvrdili smo da je alel D6S273-3 podložan za bolest, dok aleli D6S2811-126pb, D6S273-4, kao i haplotipska veza HLA-B*27/D6S273-4 pokazuju zaštitnu ulogu za razvoj bolesti. Među bolesnicima s jSpA otkrivena je smanjena učestalost alela D6S2674-131pb i dviju haplotipskih veza HLA-B*27/D6S2674-131 i HLA-B*27/D6S2811-98 što govori u prilog njihovoj zaštitnoj ulozi za razvoj bolesti, dok je za alel STR_MICA-A9 i haplotipsku vezu HLA-B*27/D6S248-291 utvrđena statistički značajno povišena učestalost što upućuje na zaključak da su podložni za razvoj jSpA.In this study we analyzed the alleles of 5 HLA microsatellites (D6S248, D6S2674, D6S2811, STR_MICA and D6S273) among HLA-B27 positive, healthy individuals (N=94), patients with psoriatic arthritis (PsA) (N=22) and patients with juvenile spondyloarthropaties (jSpA) (N=29). The most common alleles in all tested groups were: D6S248-291pb, D6S2674-131pb, D6S2811-98pb and STR_MICA-A4 alleles, while at the D6S273 locus, the most common allele among patients with jSpA and controls was D6S273-4 allele and among patients with PsA D6S273-5 allele. The analysis of patients with PsA determined that D6S273-3 allele is associated with an increased risk for the disease, while D6S2811-126pb and D6S273-4 alleles, as well as HLA-B*27/D6S273-4 haplotypic association show a protective role for the development of the disease. A decreased frequency of D6S2674-131pb allele and HLA-B*27/D6S2674-131 and HLA-B*27/D6S2811-98 haplotypic associations was discovered among patients with jSpA which implies their protective role for the development of the disease. On the other hand, a statistically significant increase in frequency was determined for STR_MICA-A9 allele and the HLA-B*27/D6S248-291 haplotypic association which leads to the conclusion that they are involved in the development of jSpA

Definition of HLA-DRB haplotypes by microsatellite D6S2878 analysis in a Croatian population

U ovom radu analizirali smo alele mikrosatelita D6S2878, u svrhu određivanja haplotipova HLA-DRB, unutar skupine od 53 nesrodne zdrave osobe. Broj alela mikrosatelita D6S2878 u jednom haplotipu ovisi o tome koliki je broj gena DRB (aktivnih i pseudogena) u pojedinom haplotipu HLA. Analizom haplotipova HLA-DR1, DR10 te haplotipova DR52 uočili smo po dva alela mikrosatelita D6S2878. Kraći alel D6S2878 u haplotipu HLA-DR1 (alel D6S2878-136pb) odgovarao je alelu na pseudogenu DRB6, a duži alelima na lokusu DRB1 (alel D6S2878-154pb i alel D6S2878-161pb). U haplotipu HLA-DR52 alel D6S2878 koji je odgovarao alelima lokusa DRB1 (aleli D6S2878 dužine od 172pb do 206pb) bio je kraći od alela za lokus DRB3 (aleli D6S2878 dužine od 180pb do 214pb). Na haplotipovima HLA-DR51 i HLADR53 uočili smo po 3 alela mikrosatelita D6S2878. Kod haplotipova HLA-DR51 najkraći alel D6S2878 odgovarao je pseudogenu DRB6 (aleli D6S2878-144pb, 152pb, 154pb i 174pb) dok su duži aleli (od 176pb do 204pb) odgovarali alelima lokusa DRB1, odnosno aleli D6S2878 dužine od 180pb do 220pb alelima lokusa DRB5. Pseudogen DRB7 u haplotipovima HLA-DR53 imao je najkraće alele D6S2878 od 135pb, dok su dužine alela D6S2878 od 170pb do 194pb odgovarale alelima DRB1, a aleli D6S2878 od 176pb do 182pb alelima DRB4. Kod alela DRB1*08 uočen je samo jedan alel D6S2878 i to dužine od 170pb i 176pb.In this study, we analyzed alleles at D6S2878 microsatellite among 53 unrelated subjects in order to define HLA-DRB haplotypes. Number of D6S2878 alleles detected in one haplotype depends on the number of DRB genes (active and pseudogenes) contained by a specific haplotype. Analysis of HLA-DR1, and HLA-DR52 haplotypes revealed two D6S2878 alleles. Shorter allele in HLA-DR1 haplotype (D6S2878-136bp) matched the allele on pseudogene DRB6, while longer matched the alleles on DRB1 locus (D6S2878- 154bp and D6S2878-161bp). In HLA-DR52 haplotype D6S2878 allele which matched alleles on DRB1 locus (172bp-206bp) was shorter than allele which matched DRB3 locus (180bp-214bp). We observed three D6S2878 microsatellite alleles HLA-DR51 and HLADR53 haplotypes. The shortest allele in HLA-DR51 haplotype matched the allele on pseudogene DRB6 (D6S2878-144bp, 152bp, 154bp and 174bp), while the longer alleles (176bp-204pb) corresponded to alleles at DRB1 locus or in the case of alleles of the lenght 180bp-220bp to the alleles at DRB5 locus. Pseudogene DRB7 in HLA-DR53 haplotypes had the shortest D6S2878 allele (D6S2878-135bp), while alleles D6S2878 of the lenght 170bp-194bp matched alleles at DRB1 locus and alleles D6S2878 of the lenght 176bp-182bp matched alleles at DRB4 locus. Allele DRB1*08 displayed only one D6S2878 allele which could be either 170bp or 176bp long

Microsatellite instability and loss of heterozygosity in glioblastoma

U ovom radu govorit ću o dvije genske promjene u glioblastoma i njihovom utjecaju na proces kancerogeneze. Riječ je o gubitku heterozigotnosti (LOH) i mikrosatelitnoj nestabilnosti (MSI). Cilj je iznijeti teorijsku pozadinu i sumirati neka od dosadašnjih istraživanja u području glioblastoma za navedene nestabilnosti. Naime, njihova pojava i učestalost varira u promatranim istraživanjima, no svakako doprinose genomskoj nestabilnosti. Daju uvid u genske promjene uključene u razvoj (i vraćanje) glioblastoma. Funkciju MSI teško je definirati i pretpostavlja se da je manje važan čimbenik pri razvoju glioblastoma, dok je LOH dokazano bitan čimbenik. Također, obje promjene služe kao markeri za bolje predviđanje progresije bolesti i ishoda preživljavanja. Može se zaključiti kako ove pojave i slični mehanizmi predstavljaju područje interesa u budućim istraživanjima glioblastoma.This paper is based on two genetic changes in glioblastoma and their impact on the process of carcinogenesis: the loss of heterozygosity (LOH) and microsatellite instability (MSI). The aim is to present theoretical background and summarize some of the current research in the field of glioblastoma for specified instabilities. Their occurrence and frequency varies in observed studies, but they surely contribute to genomic instability. They provide insight into the genetic changes involved in the development (and recurrence) of glioblastoma. MSI function is difficult to determine and it is assumed to be a less important factor for developing glioblastoma, while LOH has proven to be a significant factor. In addition, both can be used as markers to predict disease progression and mortality rate. Therefore, these phenomena and similar mechanisms represent a promising area for future glioblastoma research

Microsatellite instability and loss of heterozygosity in glioblastoma

U ovom radu govorit ću o dvije genske promjene u glioblastoma i njihovom utjecaju na proces kancerogeneze. Riječ je o gubitku heterozigotnosti (LOH) i mikrosatelitnoj nestabilnosti (MSI). Cilj je iznijeti teorijsku pozadinu i sumirati neka od dosadašnjih istraživanja u području glioblastoma za navedene nestabilnosti. Naime, njihova pojava i učestalost varira u promatranim istraživanjima, no svakako doprinose genomskoj nestabilnosti. Daju uvid u genske promjene uključene u razvoj (i vraćanje) glioblastoma. Funkciju MSI teško je definirati i pretpostavlja se da je manje važan čimbenik pri razvoju glioblastoma, dok je LOH dokazano bitan čimbenik. Također, obje promjene služe kao markeri za bolje predviđanje progresije bolesti i ishoda preživljavanja. Može se zaključiti kako ove pojave i slični mehanizmi predstavljaju područje interesa u budućim istraživanjima glioblastoma.This paper is based on two genetic changes in glioblastoma and their impact on the process of carcinogenesis: the loss of heterozygosity (LOH) and microsatellite instability (MSI). The aim is to present theoretical background and summarize some of the current research in the field of glioblastoma for specified instabilities. Their occurrence and frequency varies in observed studies, but they surely contribute to genomic instability. They provide insight into the genetic changes involved in the development (and recurrence) of glioblastoma. MSI function is difficult to determine and it is assumed to be a less important factor for developing glioblastoma, while LOH has proven to be a significant factor. In addition, both can be used as markers to predict disease progression and mortality rate. Therefore, these phenomena and similar mechanisms represent a promising area for future glioblastoma research