Abstract
The paper reports the results of a project of recovery and molecular characterization aimed at identifying the local vine varieties cultivated on the archipelago of Malta. A total of 58 accessions from the two major islands, Malta and Gozo, were sampled within the framework of a clonal and sanitary selection program. DNA analyses were conducted using 13 SSR markers and the profiles obtained compared with the CRA-VIT archive. A total of 28 different genotypes were identified, eight of which correspond to imported varieties: Ansonica, Axina de tres bias, Calabrese, Gold, Koenigin der weingaerten, Muscat fleur d’oranger, Muscat Hamburg, and Negroamaro. Reference SSR profiles have been produced for the most important local varieties, Girgentina (Insolja tal-Girgenti), Gellewza, and Gennarua (Insolja ta’ Gennaru).
- identification
- genetic variability
- Girgentina
- Gellewza
- Gennarua
The geopolitical situation of Malta has favored the arrival of grapevines from various countries. In addition, the cultivation practices adopted by Maltese growers have led to the production of seedlings giving rise to new cultivars. In 1922, Dr. John Borg published a list of 120 cultivars growing on the archipelago, excluding recently introduced varieties, but including the name used in the original country for cultivars of ascertained identity and the names used by Maltese growers (Borg 1922). Of these 120 cultivars, 36 were deemed as being local seedlings or varieties. These materials were all grown prior to the arrival of phylloxera (Daktulosphaira vitifoliae Fitch, 1856), which greatly reduced the number of genotypes that would henceforth be cultivated in Malta. In 1963, a report by Prof. H.P. Olmo was commissioned by the Maltese Government on varietal assortment within vineyards covering 700 to 800 ha and recorded the following: on the island of Malta, 95% cv. Girgentina, 2% cv. Gellewza, and 3% others; on Gozo, 92% cv. Gennarua, 1% cv. Gellewza, and 7% others (Olmo 1963). Many of the cultivars previously listed (Borg 1922) were no longer available or were not chosen when vineyards were replanted, but some may still exist as isolated plants or in private collections.
By 2002, the viticultural area had decreased to <500 ha, with ~75% of the ampelographic assortment represented by the two most widespread varieties, Girgentina (50%) and Gellewza (>20%), while the remaining cultivars were mainly French varieties such as Chardonnay, Cabernet Sauvignon, Merlot, and Sauvignon blanc. By 2008, vineyards covered 910 ha, with Girgentina comprising ~27% and Gellewza ~12% (data provided by Viticulture and Oenology Unit, Ministry for Resources and Rural Affairs, Malta).
From 2003 to 2005, the Maltese Ministry of Agriculture, within the framework of Project Twinning MT2002/IB-AG-04 “Capacity Building in Malta–Viticulture Unit” (2004) funded by the European Union, promoted the recovery and characterization of old Vitis vinifera genotypes still found in the islands and representing the historical ampelographic assortment of the archipelago. Fifty-eight accessions were collected in the two main islands of Malta and Gozo, encompassing both wine and table cultivars: only 13 of the 58 have a local name; the remaining, represented by one or a few isolated vines without a precise denomination, have been allotted an acronym corresponding to the vineyard and vine number or to the locality in which they were collected. The accurate identification of the existing grape germplasm is important for evaluating, preserving, and using the national and regional genetic resources. With this aim, DNA analyses were performed on the recovered plants with simple sequence repeat (SSR) markers. The molecular profiles were compared with the CRA-VIT (Centro di Ricerca per la Viticoltura, Conegliano) database and others published in the literature. The level of genetic diversity present in these materials and the existence of parent/progeny relationships were also evaluated. In addition, a brief description is given of the three principal Maltese varieties: Girgentina, Gellewza, and Gennarua.
Materials and Methods
Plant material and SSR analysis.
Fifty-eight grapevine accessions were analyzed (Table 1⇓). DNA was extracted from young leaves following a protocol similar to that described previously (Crespan et al. 1999). Genotyping was performed with 13 SSR loci: VVS2 (Thomas and Scott 1993); VVMD5, VVMD7, VVMD27, and VVMD28 (Bowers et al. 1996, 1999); VrZAG62 and VrZAG79 (Sefc et al. 1999); ISV2, ISV3, ISV4, and VMCNG4b9 (Crespan 2003); VVS1 (Thomas and Scott 1993); and VMC1e12 (Welter et al. 2007). The first 11 SSR represent the basic set routinely used at CRA-VIT for cultivar identification and were used to overlap with the CRA-VIT database to facilitate comparison. Multiplex PCR of two or three SSR loci were suitably arranged based on expected allele lengths. The PCR reaction mixture (25 μL final volume) contained 20 ng total DNA, 10 μL HotMasterMix (Eppendorf, Milan, Italy) (2.5 x) and 5 to 10 pmole of each primer. The PCR was performed in an AB 9700 thermal cycler (Applied Biosystems, Monza, Italy) with the following steps: 1 min 30 sec at 94°C, 35 cycles at 94°C for 30 sec, 55°C for 30 sec, 65°C for 30 sec, 65°C for 7 min, and a final step of at least 10 min at 8°C to stop the reaction. On the basis of signal intensity on agarose gel, 0.75 to 1.5 μL amplified DNA was used for electrophoresis onto a sequencing gel (5% polyacrylamide, 1 x TBE, 7 M urea). Amplification products of cultivars with alleles of known molecular size were used as references for allele sizing. Allele bands were revealed by silver staining and visually scored at least twice, as reported previously (Crespan and Milani 2001).
Data analysis.
Molecular data significance was computed with Identity 1.0 software (Wagner and Sefc 1999). Relatedness among varieties was estimated using both the genetic similarity coefficient after Dice (1945) and genetic dissimilarity coefficient, calculated using the co-logarithm of the proportion of shared alleles (Bowcock et al. 1994, Dangl et al. 2001). Cluster analyses were performed with the UPGMA method using Numerical Taxonomy and Multivariate Analysis System (NTSYS-pc), version 2.10 (Rohlf 2002).
Results and Discussion
The results from the 58 sampled accessions revealed 28 different genotypes (Table 2⇓) that could be divided in three groups: group A, 34 accessions corresponding to nine unique genotypes having local names, some proposed here as reference for the variety; group B, 10 accessions corresponding to eight well-known imported varieties; and group C, 14 accessions corresponding to 11 unknown genotypes.
Group A.
A total of 11 accessions (number 2, 4, 11, 12, 23, 31, 33, 40, 47, 50, and 56) had a molecular profile ascribable to Girgentina and another 11 accessions (1, 6, 13, 14, 20, 27, 38, 46, 49, 55, and 57) to that of Gellewza, the two most common local cultivars on the island of Malta. Girgentina is a white wine variety; the name (also found as Insolja tal-Girgenti) probably originated from the village of Girgenti on Malta or could have some connection with the city of Agrigento (historically Girgenti) in Sicily. The vine is vigorous and productive, with pruning preferable to long canes. The adult leaf is medium-large, pentagonal, with five to seven lobes, V-shaped petiolar sinus, overlapping leaf sinuses, and hairs absent on the underside of the leaf blade. The bunch is large, long, cylindrical-composite, with more than three wings; the berry is elliptic and greenish yellow. The sugar level is generally modest, as is the acidity, especially tartaric acid, which produces a light wine with a neutral taste (unpublished data, Project Twinning, 2004).
Gellewza is the most important local red vine variety, characterized by good vigor and high production. The adult leaf is medium-large, pentagonal-orbicular, with three to five lobes, V-shaped petiolar sinus, overlapping leaf sinuses, and hairs absent or very sparse on the lower leaf blade. The bunch is long, cylindrical-conical, with one to three wings, loose; the berry is also elliptic and the pulp has an aromatic taste. The total anthocyanin index is modest, yet, due to a high percentage of malvidin, resulting wines may not be richly colored but are satisfactorily stable. Gellewza is not normally suitable for aged wines but is used in light-red and semisparkling wines and in rose wines (unpublished data, Project Twinning, 2004).
Three samples collected on the island of Gozo (26, 28, and 30) have been identified as Gennarua, while the homonymous accession in an ex situ collection in Malta (number 7) differed from the reference accession. As no matches were found between its SSR profile and the compared databases, it has been labeled as “unknown G1” and included in group C. Gennarua is an old variety grown on the island of Gozo, where it is the prevalent local white cultivar. Gennarua is productive and vigorous, with a medium-sized bunch that is short and very compact. The berry is yellow with rusty blotches when mature, small and ellipse shaped. The pulp is juicy with modest sugar and acid (unpublished data, Project Twinning, 2004).
The same SSR profile has been found for accessions Midbiela (8) and Insolia nera (35). Single plants have been indicated with the names Katlana and Bezzul in-Naghag, also listed earlier (Borg 1922). The earlier list also contains Marsusa; however, the three accessions of Marsusa sampled (17, 49, and 51) differ from one another, and the black-berried accession (49) is identical to Gellewza. It is unclear which of the remaining two could have been the Marsusa listed earlier (Borg 1922). Of the three accessions 25, 32, and 44, which have been confirmed as identical, only the first has been named by the grower, Argentina; however, this variety is different from the Argentina in the CRA-VIT collection and from South American varieties such as Torrontés (Agüero et al. 2003). This variety has been found in three different localities (Msida, Tarxien, and Siggiewi), which could indicate that it had been traditionally more widespread and of some importance.
Group B.
By comparison with the CRA-VIT and other published databases, eight varieties have been identified, which are retraceable to 10 accessions and were surely imported into Malta. Five of these accessions are table grapes: Axina de tres bias (21), Koenigin der weingaerten (54), Muscat fleur d’oranger (9), Muscat Hamburg (37), and Gold (16). The latter is a cross produced by Prof. Olmo, which was identified through a cross-check of our SSR data with previously published data (Ibáñez et al. 2009) (eight SSR loci in common). Three are winegrape cultivars of southern Italy: Ansonica (15) and Calabrese (3, 5, and 10) from Sicily and Negroamaro (29) from Apulia.
Group C.
The other 14 accessions, corresponding to 11 varieties, have yet to be identified and have been classified as unknown genotypes (Table 2⇑). These are mainly represented by single plants, only in one case by three (unknown G2) and two accessions (unknown G10). The challenge posed by these data is based on the evidence that unidentified varieties still exist on the two islands. Investigations may be conducted through morphologic description, in search of a link with previous known varieties, using historical sources and ampelographic descriptions. Other possibilities for identification could arise from the European Project GrapeGen06 (www1.montpellier.inra.fr/grapegen06), a significant development within Work Package 5 (true-to-type confirmation) and which is preliminarily based on research on the varietal SSR profiles of interest in the molecular archives of the Project partners. The aim of these cross-checks is essentially two-fold: to assign a name to the unknown variety or to verify whether varieties considered specific to certain zones are cultivated elsewhere, perhaps under different names.
Further studies on the genotypes of groups A and C.
We focused our attention on the 20 unique genotypes in groups A and C and estimated the molecular variability with the classical measurements relative to the number of alleles found in the sample, the probability of finding null alleles, the heterozygosis observed and expected, and the polymorphic index content (PIC). There were a total of 78 alleles, with an average of six alleles per locus; the average expected heterozygosis was 0.7175, while the average observed heterozygosis was decidedly higher at 0.8461. This data, together with the abundant literature, support the hypothesis that human and natural selection favored heterozygosis within the same grapevine genotype (Sefc et al. 2000). Even the probability of identity, notwithstanding the very limited number of genotypes considered, was high at 2.528 x 10−10. These values also show a marked heterogeneity in the sampled genotypes.
Two dendrograms were initially constructed using Dice’s coefficient or the proportion of shared alleles to attempt to elucidate the possible relationships between the 20 varieties. The clusters obtained were identical, with very similar cophenetic correlation coefficients of 0.86219 and 0.8328, respectively (good fit). With the aim of obtaining further clarifications on the possible links between the 20 genotypes and other cultivars present in the archipelago, a third dendrogram was constructed using the proportion of shared alleles, including in the analysis the eight group B varieties identified as imported and also Muscat of Alexandria, an ancient variety throughout the Mediterranean basin. The value of the cophenetic coefficient reduced to 0.7624 (poor fit), but as the clusters of the 20 genotypes analyzed separately in the previous elaborations had not changed, this last tree is reported (Figure 1⇓). There are four groups: the first contains the majority of the genotypes sampled in Malta, including the three principal varieties of the archipelago and those very likely imported from Sicily (i.e., Ansonica and Calabrese). The second group includes all the well-known muscat varieties, which constitute a closely related family (Crespan and Milani 2001). In particular, Muscat Hamburg is a progeny of Schiava grossa x Muscat of Alexandria (Crespan 2003), Muscat fleur d’oranger is from Muscat blanc à petits grains and Chasselas (Schneider et al. 2008), Gold is a cross by Prof. Olmo and has Muscat Hamburg as an ancestor (VIVC, http://www.vivc.de/index.php), and lastly Koenigin der weingaerten is a cross with parents that include Perle von Csaba, another muscat variety, in its turn a progeny of Muscat Ottonel (VIVC). Also in this second group, but set apart, is Negroamaro, an ancient non-muscat variety from Apulia. The third and fourth groups each contain just one genotype, both classified well apart from the others: Bezzul in-Naghag and accession 45 (unknown G9). Viewing the molecular data in detail, accession 45 is characterized by an allele of 133 bp at locus ISV2, which to this point has only been found in rootstocks, and we therefore hypothesize that this could be a hybrid (Crespan et al. 2009). This hypothesis is also supported by ampelographic observations and by the resistance of this accession to oidium and peronospora, so we suppose that it could be one of the hybrid varieties Olmo had introduced to the experimental research station at Buskett. Our SSR data provided no particular information for Bezzul in-Naghag.
Another peculiarity that could be useful in confirming the identity of the unknown material is that accession 43 (unknown G8) has a rare allele of 224 bp at VVMD5, to this point only found in varieties of the Middle East (Armenia, Turkey, Afghanistan, C. Crespan, unpublished data, 2010).
Conclusions
The recovery and molecular characterization of cultivated vines on the archipelago of Malta has highlighted an interesting varietal richness, shown by the identification of 28 different genotypes. At least eight varieties are the result of importation, mostly from southern Italy; of the other 20, only three—Girgentina, Gellewza, and Gennarua—are ancient and widespread varieties, as indicated by the names supplied by vinegrowers. Other varieties, although they have been assigned a name, are lesser known and in most cases during sampling were represented by single plants. Yet another 11 genotypes remain nameless and represent a varietal heritage that calls for further exploration.
- Received October 2009.
- Revision received January 2010.
- Accepted February 2010.
- Copyright © 2010 by the American Society for Enology and Viticulture