Genetic diversity, quantitative genetic traits, intrapopulation genetic variability, interapopulation genetic diversity, levels and patterns ofquantitative genetic differentiation, genotype by environment interaction,molecular genetic analysis, nuclear and chloroplast microsatellite DNA markers

Genetska raznolikost (varijabilnost, diverzitet) temeljni je dio ukupne biološke raznolikosti, a predstavlja bogatstvo različitih alela odnosno gena narazinama jedinki, populacija odnosno vrsta. Veća razina genetske raznolikosti omogućava populacijama veći kapacitet prilagođavanja putem prirodneselekcije i zbog toga je važan preduvjet njihova opstanka u promjenjivomokolišu. Stoga su upoznavanje i očuvanje genetske raznolikosti izuzetnovažne aktivnosti za dugoročni opstanak vrsta šumskog drveća, posebice onihkoje su nositelji ekosustava.Utvrđivanje razine, obrasca i uzroka genetske raznolikosti kod vrsta šumskog drveća moguće je putem dvaju metoda, a to su: 1. analizom kvantitativnih fenotipskih svojstava u genetičkim testovima i 2. analizom DNK biljega. Glavni cilj ovoga rada bio je utvrditi razinu i obrazac genetske raznolikosti populacija hrasta lužnjaka u Hrvatskoj korištenjem obje dostupne metode.Na tri različite lokacije osnovani su genetički testovi s potomstvom hrasta lužnjaka iz 16 sjemenskih i jedne gospodarske sastojine, kojereprezentiraju cjelokupan areal ove vrste u Hrvatskoj. Provedene su izmjere i ocjenjivanje raznovrsnih kvantitativnih fenotipskih svojstava: visinskog rasta,visinskog prirasta, preživljenja, zimske retencije lišća, intenziteta zaraze hrastovom pepelnicom, intenziteta oštećenosti biljaka kasnim proljetnim mrazem i fenologije listanja. Analizom varijance utvrđena je statistička značajnost ispitivanih izvora varijabilnosti (blokova, populacija, familija unutar populacija, interakcije blokova s populacijama i familijama), te su izračunati kvantitativni genetički parametri: nasljednost (individualna (h2i) i familijska (h2f)), koeficijent aditivne genetske varijacije (CVA) i parametar kvantitativne genetskediferencijacije (QST). Determinacija obrasca genetske diferencijacijeprovedena je multivarijatnom regresijskom stabalnom analizom (engl.Multivariate Regression Tree analysis MRT).Vrijednosti CVA i nasljednosti bile su kod većine svojstava niske, štoupućuje na nisku razinu unutarpopulacijske genetske varijabilnostiistraživanih populacija. Međutim vjerojatno je da su niske vrijednostigenetičkih parametara bile uzrokovane visokom varijancom ostatka tj.neaditivnom genetskom i okolišnom varijancom uključujući i varijancueksperimentalne pogreške. Stoga, izračunati genetički parametri nisuIIdovoljno pouzdani za konačan zaključak o razini unutarpopulacijske aditivnegenetske varijabilnosti hrasta lužnjaka u Hrvatskoj.Izračunati QST parametari ukazuju na značajnu kvantitativnu genetskudiferencijaciju istraživanih populacija hrasta lužnjaka i to vrlo vjerojatnouslijed djelovanja prirodne selekcije, na što je ukazala visina vrijednostiparametara.Determinacija obrasca kvantitativne genetske diferencijacije pokazala jekako su se populacije razdvojile s obzirom na relativnu humidnost i toplinustaništa iz kojeg potječu. Populacije iz suših i toplijih staništa imale su boljekarakteristike za ispitivana svojstva u pokusnim nasadima Jastrebarsko iKoška, jer su klimatske prilike u tim pokusnim nasadima tijekom analiziranogperioda više odgovarale originalnim stanišnim prilikama tih populacija. Sdruge strane, populacije koje potječu i iz relativno humidnijih i iz relativnoaridnijih staništa imale su bolje karakteristike visinskog rasta i preživljenja upokusnom nasadu Vrbanja.Genetska udaljenost između populacija izračunata analizama jezgrinihmikrosatelitnih biljega ukazuje kako većina populacija nije genetski značajnoudaljena. Ipak, utvrđeno je da su populacije HR 609 i HR 387 nešto višegenetski udaljene od ostalih. Kod tri populacije (HR: 609, 387 i 577) uočen jenedostatak očekivane heterozigotnosti, te visok FIS parametar, ukazujući nakrižanje u srodstvu.Analizom molekularne varijance (AMOVA) utvrđena je visokaunutarpopulacijska raznolikost, te statistički značajna komponenta varijanceuzrokovana razlikama između populacija, ukazujući na postojanje neutralnegenetske diferencijacije. Ipak, međupopulacijska diferencijacija zauzimala jesamo 1,47% ukupne raznolikosti, što ukazuje na snažan utjecaj migracijagena između populacija. Uz proces prirodne migracije gena (posebicepeludom) vrlo je vjerojatan i antropogeni utjecaj prijenosom šumskogreprodukcijskog materijala.Prostorna udaljenost ima signifikantan udio u raspodjeli genetičkeraznolikosti. Ukupno 19,6% genetske diferencijacije između analiziranihpopulacija može se objasniti njihovom prostornom udaljenošću (IBD), asrodnost jedinki se gubi nakon 240 km međusobne udaljenosti. Ipak,faktorijalnom analizom korespodencije nije utvrđena prava zemljopisnarazdvojenost populacija.Analizom kloroplastnih mikrosatelitnih biljega ukupna procijenjenaraznolikost svih populacija bila je velika. Utvrđena raznolikost izmeđupopulacija analizom molekularne varijance (AMOVA) bila je statističkivisoko značajna i veća od unutarpopulacijske raznolikosti. Također, jeustanovljeno postojanje 66 haplotipova u istraživanim populacijama, koji susvrstani u tri rodoslovne linije: linija L2 koja ujedno sadržava i najveći brojhaplotipova prostire se od istoka prema zapadu, linija L1 najviše jezastupljena u zapadnom dijelu rasprostranjenja hrasta lužnjaka u Hrvatskoj,dok L3 zauzima središnji dio areala.Genetic diversity (i.e. variability) is the basic part of the biological diversityand represents reaches of the alleles and genes of individuals, populations andspecies. Higher level of genetic diversity in populations able them highercapacity for adjustment through natural selection, and therefore it is animportant part for their survival in a changing environment. Therefore, learnand preserve genetic diversity is extremely important for long-term survivalof forest tree species, especially those which are the carriers of ecosystem.Determination of level, pattern and causes of genetic diversity in forest treesspecies is possible by two methods: 1. the analysis of quantitative phenotypictraits in genetic field trials, and 2. by analysis of DNA markers. The mainobjective of this study was to determine the level and pattern of geneticdiversity of pedunculate oak populations in Croatia using both availablemethods.Three genetic field trials (Jastrebarsko, Koška and Vrbanja) were establishedon different locations with progeny from 16 seed stands and one normalmanaged stand, representing the distribution range of pedunculated oak inCroatia. Measurements of quantitative phenotypic traits such as height,survival, winter leaf retention, intensity of infection with powdery mildew,intensity of late spring frost damages and phenology were conducted todeterminate genetic diversity and differentiation.Statistical significance of analyses of variance was calculated for sources ofvariability: blocks, populations, family within populations, interaction ofblocks with populations and families. Based on percentage of variancecomponents from ANOVA, heritability (individual (h2i) and family (h2f))coefficient of additive genetic variation (CVA) and parameter of quantitativegenetic differentiation (QST) were calculated. Multivariate regression treeanalysis (MRT) was used to determine the pattern of genetic differentiation.Heritability (individual (h2i) and family (h2f)) and coefficient of additivegenetic variation (CVA) were low for almost all quantitative traits indicating alow level of intrapopulation genetic variability. However, low additivegenetic variation was probably caused by high variance residual in ANOVA Considering that calculated genetic parameters are not reliable enough forfinal conclusion about level of intrapopulation genetic variability inpopulations of pedunculated oak in Croatia.High QST parameter reveals significant differences between populations,which confirm their genetic differentiation probably caused by naturalselection. Statistical significance level for interaction effect of populationswithin blocks indicates a certain level of phenotypic plasticity for analyzedpopulations. Populations were clustered according to relative humidity andtemperature of habitat of origin.Populations originating from more arid habitat showed higher phenotypiccharacteristics for survival and height in trials Jastrebarsko and Koška,probably because climatic in this field trials were favorable for populationoriginating from more aridic habitat. Populations originating from morehumid habitats and also from relatively arid habitats had the higher survivaland height in field trial Vrbanja.Analysis with nuclear microsatellite markers showed a low level of geneticdiversity between populations. Populations HR 609 and HR 387 are morediverse form other populations. Three populations (HR: 609, 387 and 577)have high FIS parameter indicating inbreeding.Analysis of molecular variance (AMOVA) determine high level ofintrapopulation diversity, also a statistically significance percentage ofvariance between populations indicate a neutral genetic differentiation.However, established interpopulation differences were only 1.47% of overalldifferences, referring to high migration of genes between populations. Besidethe natural gene flow (especially by pollen), the highly plausible isanthropogenic influence by moving of forest reproductive material.Total of 19.6% of genetic differentiation among populations can be explainedwith isolation by distance (IBD). Relatedness of individuals is lost after 240km mutual distance.Analysis with chloroplast microsatellite markers gave high overall diversityof populations. Diversity between populations in AMOVA was statisticalyhighly significant, higher than intrapopulation diversity. Also, 66 haplotypeswas determined, origination in three lines: line L2, with highest number ofhaplotypes extends from east to west, line L1 prevails in western part ofdistribution range, while line L3 is concentrated in central part

Genetic diversity, quantitative genetic traits, intrapopulation genetic variability, interapopulation genetic diversity, levels and patterns ofquantitative genetic differentiation, genotype by environment interaction,molecular genetic analysis, nuclear and chloroplast microsatellite DNA markers

Genetska raznolikost (varijabilnost, diverzitet) temeljni je dio ukupne biološke raznolikosti, a predstavlja bogatstvo različitih alela odnosno gena narazinama jedinki, populacija odnosno vrsta. Veća razina genetske raznolikosti omogućava populacijama veći kapacitet prilagođavanja putem prirodneselekcije i zbog toga je važan preduvjet njihova opstanka u promjenjivomokolišu. Stoga su upoznavanje i očuvanje genetske raznolikosti izuzetnovažne aktivnosti za dugoročni opstanak vrsta šumskog drveća, posebice onihkoje su nositelji ekosustava.Utvrđivanje razine, obrasca i uzroka genetske raznolikosti kod vrsta šumskog drveća moguće je putem dvaju metoda, a to su: 1. analizom kvantitativnih fenotipskih svojstava u genetičkim testovima i 2. analizom DNK biljega. Glavni cilj ovoga rada bio je utvrditi razinu i obrazac genetske raznolikosti populacija hrasta lužnjaka u Hrvatskoj korištenjem obje dostupne metode.Na tri različite lokacije osnovani su genetički testovi s potomstvom hrasta lužnjaka iz 16 sjemenskih i jedne gospodarske sastojine, kojereprezentiraju cjelokupan areal ove vrste u Hrvatskoj. Provedene su izmjere i ocjenjivanje raznovrsnih kvantitativnih fenotipskih svojstava: visinskog rasta,visinskog prirasta, preživljenja, zimske retencije lišća, intenziteta zaraze hrastovom pepelnicom, intenziteta oštećenosti biljaka kasnim proljetnim mrazem i fenologije listanja. Analizom varijance utvrđena je statistička značajnost ispitivanih izvora varijabilnosti (blokova, populacija, familija unutar populacija, interakcije blokova s populacijama i familijama), te su izračunati kvantitativni genetički parametri: nasljednost (individualna (h2i) i familijska (h2f)), koeficijent aditivne genetske varijacije (CVA) i parametar kvantitativne genetskediferencijacije (QST). Determinacija obrasca genetske diferencijacijeprovedena je multivarijatnom regresijskom stabalnom analizom (engl.Multivariate Regression Tree analysis MRT).Vrijednosti CVA i nasljednosti bile su kod većine svojstava niske, štoupućuje na nisku razinu unutarpopulacijske genetske varijabilnostiistraživanih populacija. Međutim vjerojatno je da su niske vrijednostigenetičkih parametara bile uzrokovane visokom varijancom ostatka tj.neaditivnom genetskom i okolišnom varijancom uključujući i varijancueksperimentalne pogreške. Stoga, izračunati genetički parametri nisuIIdovoljno pouzdani za konačan zaključak o razini unutarpopulacijske aditivnegenetske varijabilnosti hrasta lužnjaka u Hrvatskoj.Izračunati QST parametari ukazuju na značajnu kvantitativnu genetskudiferencijaciju istraživanih populacija hrasta lužnjaka i to vrlo vjerojatnouslijed djelovanja prirodne selekcije, na što je ukazala visina vrijednostiparametara.Determinacija obrasca kvantitativne genetske diferencijacije pokazala jekako su se populacije razdvojile s obzirom na relativnu humidnost i toplinustaništa iz kojeg potječu. Populacije iz suših i toplijih staništa imale su boljekarakteristike za ispitivana svojstva u pokusnim nasadima Jastrebarsko iKoška, jer su klimatske prilike u tim pokusnim nasadima tijekom analiziranogperioda više odgovarale originalnim stanišnim prilikama tih populacija. Sdruge strane, populacije koje potječu i iz relativno humidnijih i iz relativnoaridnijih staništa imale su bolje karakteristike visinskog rasta i preživljenja upokusnom nasadu Vrbanja.Genetska udaljenost između populacija izračunata analizama jezgrinihmikrosatelitnih biljega ukazuje kako većina populacija nije genetski značajnoudaljena. Ipak, utvrđeno je da su populacije HR 609 i HR 387 nešto višegenetski udaljene od ostalih. Kod tri populacije (HR: 609, 387 i 577) uočen jenedostatak očekivane heterozigotnosti, te visok FIS parametar, ukazujući nakrižanje u srodstvu.Analizom molekularne varijance (AMOVA) utvrđena je visokaunutarpopulacijska raznolikost, te statistički značajna komponenta varijanceuzrokovana razlikama između populacija, ukazujući na postojanje neutralnegenetske diferencijacije. Ipak, međupopulacijska diferencijacija zauzimala jesamo 1,47% ukupne raznolikosti, što ukazuje na snažan utjecaj migracijagena između populacija. Uz proces prirodne migracije gena (posebicepeludom) vrlo je vjerojatan i antropogeni utjecaj prijenosom šumskogreprodukcijskog materijala.Prostorna udaljenost ima signifikantan udio u raspodjeli genetičkeraznolikosti. Ukupno 19,6% genetske diferencijacije između analiziranihpopulacija može se objasniti njihovom prostornom udaljenošću (IBD), asrodnost jedinki se gubi nakon 240 km međusobne udaljenosti. Ipak,faktorijalnom analizom korespodencije nije utvrđena prava zemljopisnarazdvojenost populacija.Analizom kloroplastnih mikrosatelitnih biljega ukupna procijenjenaraznolikost svih populacija bila je velika. Utvrđena raznolikost izmeđupopulacija analizom molekularne varijance (AMOVA) bila je statističkivisoko značajna i veća od unutarpopulacijske raznolikosti. Također, jeustanovljeno postojanje 66 haplotipova u istraživanim populacijama, koji susvrstani u tri rodoslovne linije: linija L2 koja ujedno sadržava i najveći brojhaplotipova prostire se od istoka prema zapadu, linija L1 najviše jezastupljena u zapadnom dijelu rasprostranjenja hrasta lužnjaka u Hrvatskoj,dok L3 zauzima središnji dio areala.Genetic diversity (i.e. variability) is the basic part of the biological diversityand represents reaches of the alleles and genes of individuals, populations andspecies. Higher level of genetic diversity in populations able them highercapacity for adjustment through natural selection, and therefore it is animportant part for their survival in a changing environment. Therefore, learnand preserve genetic diversity is extremely important for long-term survivalof forest tree species, especially those which are the carriers of ecosystem.Determination of level, pattern and causes of genetic diversity in forest treesspecies is possible by two methods: 1. the analysis of quantitative phenotypictraits in genetic field trials, and 2. by analysis of DNA markers. The mainobjective of this study was to determine the level and pattern of geneticdiversity of pedunculate oak populations in Croatia using both availablemethods.Three genetic field trials (Jastrebarsko, Koška and Vrbanja) were establishedon different locations with progeny from 16 seed stands and one normalmanaged stand, representing the distribution range of pedunculated oak inCroatia. Measurements of quantitative phenotypic traits such as height,survival, winter leaf retention, intensity of infection with powdery mildew,intensity of late spring frost damages and phenology were conducted todeterminate genetic diversity and differentiation.Statistical significance of analyses of variance was calculated for sources ofvariability: blocks, populations, family within populations, interaction ofblocks with populations and families. Based on percentage of variancecomponents from ANOVA, heritability (individual (h2i) and family (h2f))coefficient of additive genetic variation (CVA) and parameter of quantitativegenetic differentiation (QST) were calculated. Multivariate regression treeanalysis (MRT) was used to determine the pattern of genetic differentiation.Heritability (individual (h2i) and family (h2f)) and coefficient of additivegenetic variation (CVA) were low for almost all quantitative traits indicating alow level of intrapopulation genetic variability. However, low additivegenetic variation was probably caused by high variance residual in ANOVA Considering that calculated genetic parameters are not reliable enough forfinal conclusion about level of intrapopulation genetic variability inpopulations of pedunculated oak in Croatia.High QST parameter reveals significant differences between populations,which confirm their genetic differentiation probably caused by naturalselection. Statistical significance level for interaction effect of populationswithin blocks indicates a certain level of phenotypic plasticity for analyzedpopulations. Populations were clustered according to relative humidity andtemperature of habitat of origin.Populations originating from more arid habitat showed higher phenotypiccharacteristics for survival and height in trials Jastrebarsko and Koška,probably because climatic in this field trials were favorable for populationoriginating from more aridic habitat. Populations originating from morehumid habitats and also from relatively arid habitats had the higher survivaland height in field trial Vrbanja.Analysis with nuclear microsatellite markers showed a low level of geneticdiversity between populations. Populations HR 609 and HR 387 are morediverse form other populations. Three populations (HR: 609, 387 and 577)have high FIS parameter indicating inbreeding.Analysis of molecular variance (AMOVA) determine high level ofintrapopulation diversity, also a statistically significance percentage ofvariance between populations indicate a neutral genetic differentiation.However, established interpopulation differences were only 1.47% of overalldifferences, referring to high migration of genes between populations. Besidethe natural gene flow (especially by pollen), the highly plausible isanthropogenic influence by moving of forest reproductive material.Total of 19.6% of genetic differentiation among populations can be explainedwith isolation by distance (IBD). Relatedness of individuals is lost after 240km mutual distance.Analysis with chloroplast microsatellite markers gave high overall diversityof populations. Diversity between populations in AMOVA was statisticalyhighly significant, higher than intrapopulation diversity. Also, 66 haplotypeswas determined, origination in three lines: line L2, with highest number ofhaplotypes extends from east to west, line L1 prevails in western part ofdistribution range, while line L3 is concentrated in central part

Kvantitativna genetska diferencijacija populacija hrasta lužnjaka (Quercus robur L.) u pokusnom nasadu »Jastrebarski lugovi«

Hrast lužnjak, kao jedna od triju najrasprostranjenijih vrsta drveća u Republici Hrvatskoj, ima važnu ulogu u ekonomsko-socijalnom i ekološkom smislu. Poznavanje genetske strukture tako značajne vrste šumskoga drveća temeljni je preduvjet njezina očuvanja za budućnost. Jer to, poznavanje genetske strukture, omogućuje naprednije korištenje šumskoga reprodukcijskoga materijala hrasta lužnjaka radi povećanja prilagodljivosti populacija na stresne okolišne prilike. Analiza kvantitativnih (poligenih, metričkih) fenotipskih svojstava u genetičkim testovima jedna je od metoda utvrđivanja genetske strukture vrsta šumskoga drveća. Iz šesnaest sjemenskih i jedne gospodarske sastojine koje reprezentiraju cjelokupan areal hrasta lužnjaka u Hrvatskoj, u rujnu i listopadu 2006. godine skupljen je sjemenski materijal. S dvogodišnjim potomstvom 2008. godine osnovan je genetički pokusni nasad u gospodarskoj jedinici »Jastrebarski lugovi«. Mjeren je visinski rast i određivani su preživljavanje i visinski prirast u trima uzastopnim godinama (2010, 2011. i 2012). Analiza varijance provedena je procedurom PROC MIXED u statističkom paketu SAS te su izračunate komponente varijance i statistička značajnost slučajnih efekata, tj. izvora varijabilnosti (blokovi, populacije, familije unutar populacija, interakcija blokova i populacija te interakcija blokova s familijama unutar populacija). Izračunate komponente varijance poslužile su za izračunavanje parametra QST kojim se određuje razina međupopulacijske genetske diferencijacije. Multivarijantna regresijska stabalna analiza (engl. MRT) korištena je za određivanje obrasca genetske diferencijacije populacija u nasadu, a s obzirom na okolišne (klimatske) varijable njihovih izvornih matičnih sastojina. Parametar QST bio je visok kod sva tri analizirana svojstva, što jasno upućuje na međupopulacijsku genetsku diferencijaciju hrvatskih populacija hrasta lužnjaka. Utvrđeni obrazac adaptivne genetske diferencijacije potvrđuje ekotipski obrazac, odnosno najvjerojatnije je prirodna selekcija u matičnim sastojinama (ponajprije s obzirom na vlažnost i toplinu staništa) uzrokovala genetsku izdiferenciranost lokalnih populacija

Genome sequence diversity of SARS-CoV-2 in Serbia: insights gained from a 3-year pandemic study

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19-positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the following positions: 5′-untranslated region (5’UTR:241); N protein (N:RG203KR); NSP3 protein (NSP3:F106F); NSP4 protein (NSP4:T492I); NSP6 protein (NSP6: S106/G107/F108 - triple deletion), and NSP12b protein (NSP12b:P314L). This national-level study is the most comprehensive in terms of sequencing and genomic surveillance of SARS-CoV-2 during the pandemic in Serbia, highlighting the importance of establishing and maintaining good national practice for monitoring SARS-CoV-2 and other viruses circulating worldwide.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19- positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the following positions: 5′-untranslated region (5’UTR:241); N protein (N:RG203KR); NSP3 protein (NSP3:F106F); NSP4 protein (NSP4:T492I); NSP6 protein (NSP6: S106/G107/F108 - triple deletion), and NSP12b protein (NSP12b:P314L). This national-level study is the most comprehensive in terms of sequencing and genomic surveillance of SARS-CoV-2 during the pandemic in Serbia, highlighting the importance of establishing and maintaining good national practice for monitoring SARS-CoV-2 and other viruses circulating worldwide

Genetic diversity, quantitative genetic traits, intrapopulation genetic variability, interapopulation genetic diversity, levels and patterns ofquantitative genetic differentiation, genotype by environment interaction,molecular genetic analysis, nuclear and chloroplast microsatellite DNA markers

Genetska raznolikost (varijabilnost, diverzitet) temeljni je dio ukupne biološke raznolikosti, a predstavlja bogatstvo različitih alela odnosno gena narazinama jedinki, populacija odnosno vrsta. Veća razina genetske raznolikosti omogućava populacijama veći kapacitet prilagođavanja putem prirodneselekcije i zbog toga je važan preduvjet njihova opstanka u promjenjivomokolišu. Stoga su upoznavanje i očuvanje genetske raznolikosti izuzetnovažne aktivnosti za dugoročni opstanak vrsta šumskog drveća, posebice onihkoje su nositelji ekosustava.Utvrđivanje razine, obrasca i uzroka genetske raznolikosti kod vrsta šumskog drveća moguće je putem dvaju metoda, a to su: 1. analizom kvantitativnih fenotipskih svojstava u genetičkim testovima i 2. analizom DNK biljega. Glavni cilj ovoga rada bio je utvrditi razinu i obrazac genetske raznolikosti populacija hrasta lužnjaka u Hrvatskoj korištenjem obje dostupne metode.Na tri različite lokacije osnovani su genetički testovi s potomstvom hrasta lužnjaka iz 16 sjemenskih i jedne gospodarske sastojine, kojereprezentiraju cjelokupan areal ove vrste u Hrvatskoj. Provedene su izmjere i ocjenjivanje raznovrsnih kvantitativnih fenotipskih svojstava: visinskog rasta,visinskog prirasta, preživljenja, zimske retencije lišća, intenziteta zaraze hrastovom pepelnicom, intenziteta oštećenosti biljaka kasnim proljetnim mrazem i fenologije listanja. Analizom varijance utvrđena je statistička značajnost ispitivanih izvora varijabilnosti (blokova, populacija, familija unutar populacija, interakcije blokova s populacijama i familijama), te su izračunati kvantitativni genetički parametri: nasljednost (individualna (h2i) i familijska (h2f)), koeficijent aditivne genetske varijacije (CVA) i parametar kvantitativne genetskediferencijacije (QST). Determinacija obrasca genetske diferencijacijeprovedena je multivarijatnom regresijskom stabalnom analizom (engl.Multivariate Regression Tree analysis MRT).Vrijednosti CVA i nasljednosti bile su kod većine svojstava niske, štoupućuje na nisku razinu unutarpopulacijske genetske varijabilnostiistraživanih populacija. Međutim vjerojatno je da su niske vrijednostigenetičkih parametara bile uzrokovane visokom varijancom ostatka tj.neaditivnom genetskom i okolišnom varijancom uključujući i varijancueksperimentalne pogreške. Stoga, izračunati genetički parametri nisuIIdovoljno pouzdani za konačan zaključak o razini unutarpopulacijske aditivnegenetske varijabilnosti hrasta lužnjaka u Hrvatskoj.Izračunati QST parametari ukazuju na značajnu kvantitativnu genetskudiferencijaciju istraživanih populacija hrasta lužnjaka i to vrlo vjerojatnouslijed djelovanja prirodne selekcije, na što je ukazala visina vrijednostiparametara.Determinacija obrasca kvantitativne genetske diferencijacije pokazala jekako su se populacije razdvojile s obzirom na relativnu humidnost i toplinustaništa iz kojeg potječu. Populacije iz suših i toplijih staništa imale su boljekarakteristike za ispitivana svojstva u pokusnim nasadima Jastrebarsko iKoška, jer su klimatske prilike u tim pokusnim nasadima tijekom analiziranogperioda više odgovarale originalnim stanišnim prilikama tih populacija. Sdruge strane, populacije koje potječu i iz relativno humidnijih i iz relativnoaridnijih staništa imale su bolje karakteristike visinskog rasta i preživljenja upokusnom nasadu Vrbanja.Genetska udaljenost između populacija izračunata analizama jezgrinihmikrosatelitnih biljega ukazuje kako većina populacija nije genetski značajnoudaljena. Ipak, utvrđeno je da su populacije HR 609 i HR 387 nešto višegenetski udaljene od ostalih. Kod tri populacije (HR: 609, 387 i 577) uočen jenedostatak očekivane heterozigotnosti, te visok FIS parametar, ukazujući nakrižanje u srodstvu.Analizom molekularne varijance (AMOVA) utvrđena je visokaunutarpopulacijska raznolikost, te statistički značajna komponenta varijanceuzrokovana razlikama između populacija, ukazujući na postojanje neutralnegenetske diferencijacije. Ipak, međupopulacijska diferencijacija zauzimala jesamo 1,47% ukupne raznolikosti, što ukazuje na snažan utjecaj migracijagena između populacija. Uz proces prirodne migracije gena (posebicepeludom) vrlo je vjerojatan i antropogeni utjecaj prijenosom šumskogreprodukcijskog materijala.Prostorna udaljenost ima signifikantan udio u raspodjeli genetičkeraznolikosti. Ukupno 19,6% genetske diferencijacije između analiziranihpopulacija može se objasniti njihovom prostornom udaljenošću (IBD), asrodnost jedinki se gubi nakon 240 km međusobne udaljenosti. Ipak,faktorijalnom analizom korespodencije nije utvrđena prava zemljopisnarazdvojenost populacija.Analizom kloroplastnih mikrosatelitnih biljega ukupna procijenjenaraznolikost svih populacija bila je velika. Utvrđena raznolikost izmeđupopulacija analizom molekularne varijance (AMOVA) bila je statističkivisoko značajna i veća od unutarpopulacijske raznolikosti. Također, jeustanovljeno postojanje 66 haplotipova u istraživanim populacijama, koji susvrstani u tri rodoslovne linije: linija L2 koja ujedno sadržava i najveći brojhaplotipova prostire se od istoka prema zapadu, linija L1 najviše jezastupljena u zapadnom dijelu rasprostranjenja hrasta lužnjaka u Hrvatskoj,dok L3 zauzima središnji dio areala.Genetic diversity (i.e. variability) is the basic part of the biological diversityand represents reaches of the alleles and genes of individuals, populations andspecies. Higher level of genetic diversity in populations able them highercapacity for adjustment through natural selection, and therefore it is animportant part for their survival in a changing environment. Therefore, learnand preserve genetic diversity is extremely important for long-term survivalof forest tree species, especially those which are the carriers of ecosystem.Determination of level, pattern and causes of genetic diversity in forest treesspecies is possible by two methods: 1. the analysis of quantitative phenotypictraits in genetic field trials, and 2. by analysis of DNA markers. The mainobjective of this study was to determine the level and pattern of geneticdiversity of pedunculate oak populations in Croatia using both availablemethods.Three genetic field trials (Jastrebarsko, Koška and Vrbanja) were establishedon different locations with progeny from 16 seed stands and one normalmanaged stand, representing the distribution range of pedunculated oak inCroatia. Measurements of quantitative phenotypic traits such as height,survival, winter leaf retention, intensity of infection with powdery mildew,intensity of late spring frost damages and phenology were conducted todeterminate genetic diversity and differentiation.Statistical significance of analyses of variance was calculated for sources ofvariability: blocks, populations, family within populations, interaction ofblocks with populations and families. Based on percentage of variancecomponents from ANOVA, heritability (individual (h2i) and family (h2f))coefficient of additive genetic variation (CVA) and parameter of quantitativegenetic differentiation (QST) were calculated. Multivariate regression treeanalysis (MRT) was used to determine the pattern of genetic differentiation.Heritability (individual (h2i) and family (h2f)) and coefficient of additivegenetic variation (CVA) were low for almost all quantitative traits indicating alow level of intrapopulation genetic variability. However, low additivegenetic variation was probably caused by high variance residual in ANOVA Considering that calculated genetic parameters are not reliable enough forfinal conclusion about level of intrapopulation genetic variability inpopulations of pedunculated oak in Croatia.High QST parameter reveals significant differences between populations,which confirm their genetic differentiation probably caused by naturalselection. Statistical significance level for interaction effect of populationswithin blocks indicates a certain level of phenotypic plasticity for analyzedpopulations. Populations were clustered according to relative humidity andtemperature of habitat of origin.Populations originating from more arid habitat showed higher phenotypiccharacteristics for survival and height in trials Jastrebarsko and Koška,probably because climatic in this field trials were favorable for populationoriginating from more aridic habitat. Populations originating from morehumid habitats and also from relatively arid habitats had the higher survivaland height in field trial Vrbanja.Analysis with nuclear microsatellite markers showed a low level of geneticdiversity between populations. Populations HR 609 and HR 387 are morediverse form other populations. Three populations (HR: 609, 387 and 577)have high FIS parameter indicating inbreeding.Analysis of molecular variance (AMOVA) determine high level ofintrapopulation diversity, also a statistically significance percentage ofvariance between populations indicate a neutral genetic differentiation.However, established interpopulation differences were only 1.47% of overalldifferences, referring to high migration of genes between populations. Besidethe natural gene flow (especially by pollen), the highly plausible isanthropogenic influence by moving of forest reproductive material.Total of 19.6% of genetic differentiation among populations can be explainedwith isolation by distance (IBD). Relatedness of individuals is lost after 240km mutual distance.Analysis with chloroplast microsatellite markers gave high overall diversityof populations. Diversity between populations in AMOVA was statisticalyhighly significant, higher than intrapopulation diversity. Also, 66 haplotypeswas determined, origination in three lines: line L2, with highest number ofhaplotypes extends from east to west, line L1 prevails in western part ofdistribution range, while line L3 is concentrated in central part

The Treatment of Hypercholesterolemia in a Patient with Panhypopituitarism and Familial Hypercholesterolemia: A Case Report

Cilj: Prikazati pacijenta s porodičnom hiperkolesterolemijom (engl. familial hypercholesterolemia; FH) i panhipopituitarizmom kod kojeg je unatoč trojnoj hipolipemijskoj terapiji ciljna vrijednost LDL kolesterola gotovo postignuta tek nakon uvođenja nadomjesne terapije somatotropinom koja inače nije neophodna za odrasle osobe s panhipopituitarizmom. Prikaz slučaja: 54-godišnji pacijent upućen je na endokrinološku evalvaciju radi hipogonadizma i hiperlipidemije. Pacijentu je 2005. godine učinjena perkutana koronarna intervencija s postavljanjem stenta u desnu koronarnu arteriju te je 2016. i 2017. godine zbog ateroskleroze provedeno endovaskularno liječenje stenoza femoralnih arterija. Pacijent boluje od arterijske hipertenzije, dislipidemije, šećerne bolesti tipa 2 te debljine 3. stupnja (ITM 43,5 kg/m2). Obradom erektilne disfunkcije i poliurije 2018. godine ustanovljen je panhipopituitarizam. Pacijent je redovito uzimao preporučenu nadomjesnu hormonsku terapiju (hidrokortizon, levotiroksin, dezmopresin, testosteron undekanoat) uz kroničnu terapiju (metformin, fenofibrat, atorvastatin, ezetimib, klopidogrel, nebivolol, lerkanidipin). Unatoč maksimalnoj dozi visokopotentnog statina i ezetimiba utvrđena je izrazito povišena razina LDL kolesterola (8,0 mmol/L), ukupnog kolesterola (10,5 mmol/L), triglicerida (3,8 mmol/L) te snižena vrijednost HDL kolesterola (0,6 mmol/L). S obzirom na anamnestičke podatke i laboratorijske nalaze te prema bodovnom sustavu Duch Lipid Clinical Network koji je iznosio 12, vrlo je vjerojatna dijagnoza porodične hiperkolesterolemije te je, uz dosadašnju hipolipemijsku terapiju, preporučeno liječenje PCSK-9 inhibitorom. Tri mjeseca nakon uvođenja PCSK9i u terapiju značajno su se reducirali LDL kolesterol (2,3), ukupni kolesterol (4,0) i trigliceridi (1,8) te porasle vrijednosti HDL-a (0,9). S obzirom na pretpostavljeni povoljan učinak nadomjesne terapije hormonom rasta, preporučen je somatotropin kojega je u daljnjem tijeku liječenja pacijent, uz dosadašnju terapiju, uzimao te se na sljedećem kontrolnom pregledu vrijednost LDL-a dodatno reducirala (1,6), uz povoljan učinak i na ostale lipidne parametre (ukupni kolesterol 3,5, trigliceridi 2,5, HDL 0,8, ne-HDL 2,7). Zaključak: Istraživanja koja se bave odnosom nadomjesne terapije hormonom rasta i dislipidemije, malobrojna su te se u liječenju dislipidemije preporučuje trojna hipolipemijska terapija. U našega pacijenta, koji boluje od FH i panhipopituitarizma, primjenom kombinacije statina, ezetimiba, PCSK9i te somatotropina gotovo su postignute ciljne vrijednosti LDL-a, što može sugerirati da su potrebna dodatna istraživanja u ovom području kako bi se definirale terapijske smjernice.Aim: To present a patient with FH (familial hypercholesterolemia) and panhypopituitarism in whom, despite triple hypolipidemic therapy, the target LDL cholesterol was approximately reached only after the introduction of somatotropin replacement therapy, which is not otherwise necessary for adults with panhypopituitarism. Case report: A 54-year-old patient was referred for endocrinological evaluation at the Rijeka Clinical Hospital. In 2005, the patient underwent percutaneous coronary intervention with stent placement in the right coronary artery, endovascular treatment of femoral artery stenosis, he also suffers from arterial hypertension, dyslipidemia, type 2 diabetes and grade 3 obesity (BMI 43.5 kg/m2). In the treatment of erectile dysfunction and polyuria in 2018, panhypopituitarism was established. The patient regularly took the recommended hormone replacement therapy (hydrocortisone, levothyroxine, desmopressin, testosterone undecanoate) in addition to chronic therapy (metformin, fenofibrate, atorvastatin, ezetimibe, clopidogrel, nebivolol, lercanidipine). Despite the maximum dose of high-potency statin and ezetimibe, markedly elevated levels of LDL cholesterol (8.0 mmol/L), total cholesterol (10.5 mmol/L), triglycerides (3.8 mmol/L) and decreased HDL cholesterol (0.6 mmol/L) were found. Given the anamnestic data, laboratory findings and the Duch Lipid Clinical Network scoring system, which was 12, the diagnosis of familial hypercholesterolemia was very likely and, in addition to current hypolipemia therapy, the treatment with PCSK-9 inhibitor was recommended. Three months after the introduction of PCSK9i into therapy, LDL cholesterol (2.3), total cholesterol (4.0) and triglycerides (1.8) were significantly reduced, and HDL values (0.9) increased. Given the presumed beneficial effect of growth hormone replacement therapy, somatotropin was recommended, which the patient took in the course of further treatment, and the LDL value was further reduced at the next follow-up examination (1.6), with a beneficial effect on other lipid parameters. (total cholesterol 3.5, triglycerides 2.5, HDL 0.8, non-HDL 2.7). Conclusion: There are few studies that deal with the relationship between growth hormone replacement therapy and dyslipidemia, and triple hypolipidemic therapy is recommended in the treatment of dyslipidemia. In our patient, who has FH and panhypopituitarism, the use of a combination of statins, ezetimibe, PCSK9i and somatotropin has almost reached the target values of LDL, which may suggest that further research is needed in this area to define therapeutic guidelines

Candidate gene SNP variation in floodplain populations of pedunculate oak (Quercus robur L.) near the species' southern range margin: Weak differentiation yet distinct associations with water availability

Populations residing near species' low-latitude range margins (LLMs) often occur in warmer and drier environments than those in the core range. Thus, their genetic composition could be shaped by climatic drivers that differ from those occurring at higher latitudes, resulting in potentially adaptive variants of conservation value. Such variants could facilitate the adaptation of populations from other portions of the geographical range to similar future conditions anticipated under ongoing climate change. However, very few studies have assessed standing genetic variation at potentially adaptive loci in natural LLM populations. We investigated standing genetic variation at single nucleotide polymorphisms (SNPs) located within 117 candidate genes and its links to putative climatic selection pressures across 19 pedunculate oak (Quercus roburL.) populations distributed along a regional climatic gradient near the species' southern range margin in southeastern Europe. These populations are restricted to floodplain forests along large lowland rivers, whose hydric regime is undergoing significant shifts under modern rapid climate change. The populations showed very weak geographical structure, suggesting extensive genetic connectivity and gene flow or shared ancestry. We identified eight (6.2%) positiveF(ST)-outlier loci, and genotype-environment association analyses revealed consistent associations between SNP allele frequencies and several climatic variables linked to water availability. A total of 61 associations involving 37 SNPs (28.5%) from 35 annotated genes provided important insights into putative functional mechanisms in our system. Our findings provide empirical support for the role of LLM populations as sources of potentially adaptive variation that could enhance species' resilience to climate change-related pressures