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.

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Grapevine leafroll ampeloviruses have been recently grouped into two major clades, one for Grapevine leafroll associated virus (GLRaV) 1 and 3 and another one grouping GLRaV-4 and its variants. In order to understand biological factors mediating differential ampelovirus incidences in vineyards, quantitative real-time polymerase chain reactions were performed to assess virus populations in three grapevine varieties in which different infection status were detected: GLRaV-3 + GLRaV-4, GLRaV-3 + GLRaV-4 strain 5, and GLRaV-4 alone. Specific primers based on the RNA-dependent RNA polymerase (RdRp) domains of GLRaV-3, GLRaV-4, and GLRaV-4 strain 5 were used. Absolute and relative quantitations of the three viruses were achieved by normalization of data to the concentration of the endogenous gene actin. In spring, the populations of GLRaV-4 and GLRaV-4 strain 5 were 1.7 × 104 to 5.0 × 105 genomic RNA copies/mg of petiole tissue whereas, for GLRaV-3, values were significantly higher, ranging from 5.6 × 105 and 1.0 × 107 copies mg–1. In autumn, GLRaV-4 and GLRaV-4 strain 5 populations increased significantly, displaying values for genome copies between 4.1 × 105 and 6.3 × 106 copies mg–1, whereas GLRaV-3 populations displayed a less pronounced boost but were still significantly higher, ranging from 4.1 × 106 to 1.6 × 107 copies mg–1. To investigate whether additional viruses may interfere in the quantifications the small RNA populations, vines were analyzed by Ion Torrent high-throughput sequencing. It allowed the identification of additional viruses and viroids, including Grapevine virus A, Hop stunt viroid, Grapevine yellow speckle viroid 1, and Australian grapevine viroid. The significance of these findings is discussed.

Three autochthonous grapevine varieties of Majorca (Spain) were analyzed for the presence of viruses listed by the international certification programs. Enzyme-Linked Immuno-Sorbent Assay (ELISA) screenings were performed in 193 vines from 46 vineyards included in a clonal selection. Virus-free vines were only 6.4%, 9.6% and 11.5%, in Manto Negro, Callet and Moll, respectively. Infections by grapevine leafroll associated viruses (GLRaVs) were ascertained in 71%, 78% and 60% of Manto Negro, Callet and Moll vines, respectively. Each variety was also highly infected by Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV). The percentage of plants displaying multiple infections was 58.4% in Manto Negro, 63.8% in Callet and 42.6% in Moll. Thus, it was very difficult to identify virusfree clones with suitable agronomic characteristics to be considered as a reference for the grape market. In order to obtain certified propagation material under such conditions of endemic viral infection, sanitation should be the main focus in clonal selection processes. However, the time and financial requirements for proper sanitation process bring to consideration the need to use, at least temporarily, standard multiplication material while certified clones are achieved.Additional key words: autochthonous varieties, grapevine certification, standard material virus incidence. Resumen Incidencia de las infecciones víricas en antiguos viñedos de tres variedades de vid autóctonas de Mallorca: consecuencias sobre las estrategias de la selección clonalEn este estudio se analizó la presencia de las virosis contempladas por las leyes internacionales en tema de certificación en tres variedades de vid autóctonas de Mallorca. Para ello se realizó el test ELISA (enzyme-linked immunosorbent assay) sobre 193 cepas procedentes de 46 viñedos incluidos en un programa de selección clonal. Las cepas que resultaron libres de los virus analizados fueron el 6,4% en Manto Negro, el 9,6% en Callet y el 11,5% en Moll. Los porcentajes de cepas infectadas por los virus asociados al síndrome del enrollado de la vid (GLRaVs) fueron 71% en Manto Negro, 78% en Callet y 60% en Moll. Se detectaron también altas tasas de infecciones para el virus del entrenudo corto infeccioso de la vid (GFLV) y el virus del jaspeado de la vid (GFkV) en las tres variedades. Los porcentajes de cepas sujetas a infecciones múltiples fueron 58,4% en Manto Negro, 63,8% en Callet y 42,6% en Moll. Por tanto fue difícil encontrar cepas que cumpliesen tanto los requisitos sanitarios para la certificación como aquellos agronómicos necesarios según las exigencias del mercado vitivinícola. En tales condiciones de infecciones víricas endémicas, el saneamiento debería ser el primer objetivo a seguir con el fin de obtener material de propagación certificado. Sin embargo, el tiempo y los recursos económicos necesarios para llevar a cabo el proceso de saneamiento conllevan la necesidad de considerar el uso temporal de material de propagación estándar hasta que se obtengan clones cer...

Grapevine Red Globe virus (GRGV) is a member of family Tymoviridae tentatively assigned to genus Maculavirus. There is evidence that the distribution of this virus may be widespread. In a recent report, the authors of the present study described the presence of GRGV in Spain. To further estimate its incidence, a survey was carried out that allowed the detection of the virus using RT‐PCR in a germplasm collection in northern Spain. In the present study, three isolates were selected to obtain full‐length genome sequences using high‐throughput sequencing (HTS) of RNA combined with Sanger sequencing. The GRGV complete genome consists of 6850 nucleotides, excluding the polyA tail. Two ORFs could be identified as the putative replicase and coat protein. Although comparison of the complete genomes provided no evidence for the presence of the additional ORF previously described, another ORF was identified within the coat protein ORF, but it probably lacks functional significance. Phylogenetic analysis supported the ascription of GRGV to genus Maculavirus. The profile of vsiRNA (virus‐derived small interfering RNA) distribution along the GRGV genomes suggested the presence of a subgenomic RNA corresponding to the coat protein ORF. To improve detection of GRGV, new primer sets were designed based on consensus sequences. HTS revealed a frequent co‐infection of GRGV and grapevine rupestris vein feathering virus (GRVFV). Biological indexing by grafting two GRGV‐infected vines on indicator plants and the visual inspection of symptoms in other infected plants revealed that the virus has a negligible impact on grapevine.