Analiza unutarsortne varijabilnosti sorte Kleščec bijeli (V. vinifera L.) AFLP markerima

Vinova loza višegodišnja je kultura koja se razmnožava vegetativno i trenutno na svijetu postoji nekoliko tisuća sorata. Sorta 'Kleščec bijeli' (Vitis vinifera L.) se uzgaja na ograničenim površinama na samo nekoliko lokacija među ostalim i u okolici Križevaca. U prošlosti je 'Kleščec bijeli' bio zapostavljen i pogrešno poistovjećivan s njemačkom sortom Knipperle to jest Ortlieber. Nedavno je otkriveno da je 'Kleščec bijeli' jedinstvenog genotipa i nova hrvatska autohtona sorta (Pejić i sur., 2013.) te je pokrenut projekt njezine revitalizacije, odnosno zdravstvene i genetičke selekcije klonskih kandidata. Dugogodišnjim vegetativnim razmnožavanjem unutar sorata vinove loze može doći do pojave spontanih mutacija koje ostaju fiksirane unutar genoma (Meneghetti i sur., 2012.). Odabir trsova koji svojim karakteristikama odstupaju od prosjeka sorte okosnica je klonske selekcije koja u kombinaciji s odabirom bezvirusnih trsova osigurava provjereni i sortno čisti sadni materijal. Cilj ovog rada je provesti analizu unutarsortne varijabilnosti bezvirusnih klonskih kandidata sorte 'Kleščec bijeli' koji su korišteni kao izvorni trsovi za podizanje matičnog nasada sorte. U istraživanju su korišteni mladi listovi 20 klonskih kandidata sorte 'Kleščec bijeli' čija je DNA izolirana nakon liofilizacije koristeći PeqGold Plant kit (Peqlab GmbH, Njemačka). Nakon toga napravljena je AFLP analiza sa 4 kombinacije početnica (Vos i sur. 1995.). Nakon PCR reakcija fragmenti su elektroforetski razdvojeni ABI 3130 genetičkim analizatorom i veličine fragmenata očitane su pomoću GeneMapper 4.0 softverskog paketa (AppliedBiosystems, SAD). Iz dobivenih veličina fragmenta napravljena je binarna matrica, izračunat "Simple Matching" koeficijent sličnosti i utvrđena razina unutarsortne varijabilnosti sorte 'Kleščec bijeli'.Grapevine is a perennial crop that is propagated vegetatively and currently there exists several thousands of varieties in the world. The variety ‘Kleščec bijeli’ (Vitis vinifera L.) is grown on limited areas in only several locations, among others around Križevci. In the past, ‘Kleščec bijeli’ has been neglected and considered to be a synonym with the German variety ‘Knipperle’ which is also known as ‘Ortlieber’. It was recently discovered that the variety ‘Kleščec bijeli’ is a unique genotype and can be considered as a new Croatian autochthonous variety (Pejić et al., 2013.) and a project was initiated to revitalize the variety using sanitary and genetic selection of clonal candidates. After many years of vegetative propagation within the grape varieties spontaneous mutations may occur and due to the nature of propagation they remain fixed in the genome (Meneghetti et al., 2012.). Selecting the vines whose characteristics deviate from the average of the variety is the backbone of clonal selection, which combined with the selection of virus-free vines provides verified and varietal purity of planting material. The aim of this study is to analyze the intravarietal variability of the clonal candidates of ‘Kleščec bijeli’, which are free from economically important viruses, and were used as mother vines for a mother block of the variety. In this study young leaves of 20 virus-free clonal candidates of ‘Kleščec bijeli’ were used. DNA was isolated after freeze-drying using the PeqGold Plant kit (Peqlab GmbH, Germany). AFLP analysis was performed using 4 primer combinations (Vos et al. 1995.). After the PCR reaction, fragments were separated electrophoretically using an ABI 3130 genetic analyzer and the fragment sizes were determined using a GeneMapper 4.0 software package (AppliedBiosystems, USA). Resulting fragment sizes were used for the construction of a binary matrix which was used for the calculation of a "Simple Matching" similarity coefficient which was used to evaluate the intravarietal variability of ‘Kleščec bijeli’