Clone differentiation and varietal identification by means of SSR, AFLP, SAMPL and M‐AFLP in order to assess the clonal selection of grapevine: the case study of Manto Negro, Callet and Moll, autochthonous cultivars of Majorca

Abstract

Clonal selection is the most worldwide spreading method to improve the performance of wine grapevine (Vitis vinifera) cultivars. In the special case of autochthonous varieties with only local interest, such as Manto Negro, Callet and Moll in Majorca (Spain), good knowledge of their genotypic resources is helpful to assess the development of viticultural and enological potentialities. In this study, 94 vines (including Manto Negro, Callet, Moll and wrongly identified samples) were analysed by means of genetic markers. Several varietal identification mistakes related to the clonal selection in Majorca were detected by the amplification of 33 simple sequence repeats (SSRs) or microsatellite loci, mainly because of the close genetic relationships between Manto Negro, Callet, Moll and other varieties. A very low degree of intravarietal genetic diversity, possibly related to high incidence of virus infections, was shown in all three varieties. However, analysis by amplified fragment length polymorphism (AFLP), selective amplification of microsatellite loci (SAMPL) and microsatellite-amplified fragment length polymorphism (M-AFLP) was suitable for clone genetic discrimination. More than 900 scorable bands were obtained by nine primer combinations. The most efficient system to detect intravarietal genetic differences was M-AFLP, which generated the highest number of polymorphic bands. The use of these markers allowed clustering vines in homogeneous groups, providing essential information about sanitation strategies in order to obtain certified propagation material.

Clones Identification and Genetic Characterization of Garnacha Grapevine by Means of Different PCR-Derived Marker Systems

“…Genomic DNA (500 ng) was digested and ligation was performed in 4 h at 37°C using EcoRl (or Pstl) and Msel enzymes (5 U each), 1 U of T4 ligase, 50 pmol of Mseladaptor, 5 pmol of £coRI-adaptor (or Pstl) in 1 x RL buffer (20 mM Tris-acetate, 20 mM magnesium acetate, 100 mM potassium acetate, 5 mM DTT, 2.5 mg BSA) added with ATP to a final concentration of 10 mM [36]. Then, 5 ul of seven-fold diluted digested and ligated DNA was preamplified in 20 ul reaction mixture containing 75 ng of EcoRl + N (or Pstl + N) and Msel + N primers, lx PCR buffer (50 mM MgCl, 1.5 mM MgCl 2 , 10 mM Tris-HCl), 10 mM dNTPs and 1 U of Taq DNA polymerase [39]. The Eco, Pst and Mse primers were called in tables and figures as E, P and M, respectively.…”

“…In order to detect intravarietal genetic variability, AFLP, SAMPL and M-AFLP molecular markers were used [39]. Analyses were performed by Starlet liquid handling workstations (Hamilton), ABI-3130XL capillary sequencer (Applied Biosystems) and ALFexpress-II semi-automated gel sequencer (Amersham).…”

“…An aliquot of genomic DNA was also assayed by electrophoresis on 1% agarose gels [41]. The DNA was quantified on the base of fluorometric determination using the PicoGreen dsDNA quantitation assay (Invitrogen) by the FLx800 TBI Fluorometer (Bio-Tek) [39]. …”

“…In this study, genetic variability of Italian, Spanish and French Garnacha was investigated using AFLP, M-AFLP, SSR and SAMPL molecular markers [39]. The purpose of this project was to study the utility of these marker types in the Garnacha genotypes (clones) discrimination and about the quantification of molecular variability of this important grapevine cultivar.…”

Virus interference on local scale viticulture: the case of Moll variety from Majorca (Spain)

“…Factor vineyard can include in its complexity many parameters related to soil, management and microclimate. Cretazzo et al (2010c) showed that Moll vines presented a high genetic similarity among appellations and vineyards of Majorca. Therefore, it is expected that grape plant effects on vine parameters are more linked to virus infection than to intravarietal genetic variability.…”