| Abstract | Divlja loza (Vitis vinifera subsp. sylvestris Gmel Hegi) je dvodomna drvenasta penjačica i srodnik kultivirane loze (Vitis vinifera subsp. vinifera L.) koja je uglavnom hermafroditna. Prirodna sylvestris staništa su relativno netaknute šume u neposrednoj blizini vode. Sylvestris i vinifera čine genetski i taksonomski kontinuum što otežava njihovu identifikaciju. Morfološka i genetska svojstva te gospodarski i oplemenjivački potencijal divlje loze istražuju se intenzivno od početka 21. stoljeća kao izvor genetske varijabilnosti koji bi se mogao iskoristiti za oplemenjivanje kultivirane loze zbog pritiska štetnih organizama, stresova, genetske erozije te nepovoljnog učinka klimatskih promjena na sortiment kultivirane loze. Ovim istraživanjem provedena je detaljna morfološka i genetička karakterizacija te analiza gospodarski važnih svojstava 112 sylvestris jedinki sa sedam in situ populacija u Hrvatskoj te 67 F1 sjemenjaka sylvestris jedinki. Morfološka karakterizacija obuhvatila je mladicu, odrasli list i spol cvijeta putem 21 OIV deskriptora. Morfometrijske karakteristike grozda, bobica i sjemenki određene su direktnim mjerenjem na dostupnim ženskim jedinkama. Fenotipski opis bolesti pepelnice i plamenjače praćen je temeljem razvoja simptoma in vivo na jedinkama sjemenjaka te inokulacijom isječaka listova (leaf disk) sporama i praćenjem pod stereo mikroskopom. Genetske analize provedene su upotrebom 24 SSR lokusa, devet kloroplastnih SSR, četiri SSR lokusa za otpornost na bolesti te APT3 markerom za determinaciju spola cvijeta. Morfološkim analizama definiran je najčešći sylvestris profil. Utvrđeni su grozdovi malih dimenzija i većih vrijednosti ukupne kiselosti u soku kod sylvestris u odnosu na kultivare plemenite loze. Analizom sjemenki utvrđene su značajne razlike između sylvestris i vinifera sjemenki i potvrdila pouzdanost ovog svojstva u definiranju sylvestris profila. Mikrosatelitskim markerima utvrđen je visok polimorfizam istraživanog seta, ali i manja genetska raznolikost sylvestris u odnosu na vinifera i podloge za vinovu lozu uključujući i broj privatnih alela. Klaster metode (NJ, PCoA i Structure) jasno su razdvojile grupe sylvestris, vinifera i podloge za vinovu lozu, ali je utvrđeno preklapanje grupa sylvestris i vinifera što upućuje na protok gena u manjem obimu. Utvrđeno je 19% jedinki unutar populacija sylvestris koje taksonomski ne pripadaju podvrsti sylvestris, te su označene kao feralne. Potvrđena je strukturiranost sylvestris populacija koja prati geografsku distribuciju populacija, pri čemu južne, dalmatinske populacije imaju veću genetsku raznolikost (Imotski, Ho=0,72) od kontinentalnih (Lukovdol, Ho=0,54). Potvrđena je prisutnost dva klorotipa A i D, s dominacijom klorotipa A (64%). Puno roditeljstvo rekonstruirano je za 23% sjemenjaka pri čemu su oba roditelja bila unutar sylvestris populacija. Izdvojena grupa sjemenjaka sylvestris pokazala je različite razine fenotipske djelomične otpornosti na pepelnicu i plamenjaču. Analiza alelne različitosti na lokusima SC47-18 i UDV124 koji su povezani s Ren1 gen lokusom identificirala je sylvestris jedinke sjemenjaka i in situ koje nose rezistentne alele.
Primjena morfoloških i genetskih metoda pokazala se korisna u identifikaciji true to type sylvestris i razlikovanju od vinifera i loznih podloga. Identifikacija feralnih jedinki samo morfološkim analizama nije bila dovoljna već ju je bilo potrebno kombinirati s genetičkim metodama od kojih je posebno informativna bila metoda Structure. Rezultati upućuju na veću sličnost hrvatskih sylvestris populacija populacijama zapadne Europe, no pojedine populacije i svojstva bila su bliža istočnim i crnomorskim populacijama što odgovara geografskom položaju Hrvatske između ovih regija. |
| Abstract (english) | Wild grape (Vitis vinifera subsp. sylvestris Gmel Hegi) is dioecious woody liana, a relative of the cultivated grapevine (Vitis vinifera subsp. vinifera L.) which is mainly hermaphroditic. The natural habitats of sylvestris represent relatively unaltered forests in close proximity to water. Sylvestris and vinifera in natural habitats form a genetic and taxonomic continuum which makes their identification difficult. Morphological and genetic traits, productive and breeding potential of sylvestris have been intensively investigated since the beginning of the 21st century due to pest pressure, stress, genetic erosion and the adverse climate change effects on cultivated crops. This study aimed at detailed morphological and genetic characterization and analysis of economically important traits of 112 sylvestris individuals at seven in situ populations in Croatia and 67 F1 sylvestris seedlings. Morphological characterization encompassed descriptions of shoot, adult leaf and flower sex using 21 OIV descriptors. Morphometric characteristics of clusters, berries, and seeds were determined by direct measurement on female individuals available. Phenotypic description of powdery and downy mildew was carried out on sylvestris seedlings by observing development of symptoms in vivo and leaf disk inoculations with monitoring of fungal spores under a stereo microscope. Genetic analyzes were performed using 24 SSR markers, nine chloroplast SSR markers, four resistance related, R-SSR markers and DNA APT3 marker for flower sex determination. Morphological analyzes defined the most common sylvestris profile. Small clusters and higher values of total acidity in the juice of sylvestris were recorded compared to vinifera cultivars. Seed analysis revealed significant differences between sylvestris and vinifera and confirmed its reliability in defining sylvestris profile. Only the seed width was not significantly different parameter between sylvestris and vinifera. Seed morphology was less reliable in discriminating sylvestris from feral accessions.
Microsatellite markers revealed a high polymorphism of the studied set, but also a lower genetic diversity of sylvestris compared to vinifera and rootstocks, including the number of private alleles (PA). There was 12 PA in sylvestris cluster observed but PA showed sensitivity to the change in size of the sample set. Cluster methods (NJ, PCoA and Structure) clearly differentiated groups sylvestris, vinifera and rootstocks, but also revealed an overlaps between sylvestris and vinifera groups, indicating gene flow to some extent. Analyses identified about 19% of individuals within sylvestris populations that taxonomically do not belong to the subsp. sylvestris and were designated as feral. Croatian cultivars showed on average a lower level (9%) of admixed genotypes than the western European cultivars (24%), while the amount of admixed genome in Croatian sylvestris accessions originating from vinifera pool was similar between Croatian and the western European vinifera clusters. The structure of sylvestris populations was slightly fragmented but follows the geographical distribution of populations, with the southern Dalmatian populations having greater genetic diversity (Imotski, Ho = 0.72) than the continental ones (Psunj, Ho=0,60 and Lukovdol, Ho = 0.54). Inbreeding coeficient (F) in the studied set was negative indicating an excess of heterozygots and high genetic diversity of the studied set. Two sylvestris populations with negative F values (Imotski and Krka) showed highest number of feral individuals while other populations were positive for F values indicating higher level of inbreeding depression. The presence of two chlorotypes A and D from a previous study was confirmed, with a predominance of chlorotype A (64%). The full parentage was determined for 23% of the seedlings analysed with both parents originating from sylvestris populations. Parent-pair analysis have not showed any reliable parent-offspring trio, but allowed us to assume the higher hybridization events within a single sylvestris population and thus higher isolation between the studied populations. The subset of sylvestris seedlings showed different and partial levels of phenotypic resistance to powdery and downy mildew. Analysis of allelic diversity at the SC47-18 and UDV124 loci associated with the Ren1 gene locus
identified sylvestris seedlings and in situ individuals carrying resistant alleles. The highest number of R-alleles was detected on the SC47-18 marker. Individuals that showed resistance alleles, especially those having two R-alleles have shown a consistent resistance in biological tests and deserve to be additionaly studied (SjPak3, SjCer7, SjCer14, SjCer23).
Morphological and genetic methods applied in this study have proved to be useful in the identification of true to type sylvestris, to distinguish sylvestris from vinifera and rootstocks. The identification of feral individuals by morphological analyzes alone was not sufficient and requires combined analysis with genetic methods of which the Structure method proved to be particularly useful. The results indicate a greater similarity of Croatian sylvestris populations with the populations of the Western Europe, but some populations and individual traits showed affinity towards eastern and Black Sea populations, which corresponds to the geographical position of Croatia between these regions. |
