IJUP08 - Universidade do Porto

Authenticity evaluation and molecular characterization of
Portuguese varieties of Juglans regia using microsatellite
markers
E.M. Cabral 1 , M.A. Faria 1 and M.B.P.P. Oliveira 1
1 REQUIMTE, Serviço de Bromatologia, Faculty of Pharmacy, University of Porto, Portugal.
Persian walnut is cultivated mostly in Asia, Europe and America. Europe accounts for 23%
of walnut overall production. In 2005, Portugal was responsible for an area of cultivation
of 3155 ha producing 4565 t of nuts. The main cultivated varieties are of French and
American origin, however, the Portuguese cultivars Arco and Rego have a significant
expression. Worldwide cultivars have been characterized at the molecular level however
there is a complete lack of this kind of studies for Portuguese varieties. Several
methodologies have been used to evaluate the genetic diversity and relationships among
cultivars of walnut, including isozymes, restriction fragment length polymorphism (RFLP),
random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR), and
simple sequence repeats (SSR). These studies reflect the need for accurate cultivar
identification and for the verification of paternities and genealogy. SSRs are the markers of
choice for genotyping since they are highly polymorphic, co-dominant and relatively easy
to use.
In this study 21 cultivars of walnut collected in an experimental field of Direcção Regional
de Agricultura e Pescas do Centro (DRAPC) (including three Portuguese varieties Arco,
Rego and Samil) were analyzed at 14 microsatellite loci [1,2]. DNA was obtained through
the CTAB method [3]. The genetic relations were evaluated and a database of
microsatellite profiles was constructed to be used in nut and nut based products
authenticity. In order to obtain DNA extracts directly from the nuts, two other methods of
DNA extraction (Wizard ® Plus Minipreps DNA Purification System, Promega and
GenElute Plant Genomic DNA Miniprep Kit, SIGMA) were tested and compared with the
CTAB method.
Microsatellite markers used showed useful in distinguishing all cultivars under study, with
the exceptions of Franquette and Ronde de Montignac. These results are in agreement with
the bibliography that considers these markers the most appropriate to this kind of studies.
The genotypes of the Portuguese varieties were compared with the ones of international
varieties using the neighbor-joining algorithm and with those results it’s possible to
associate the variety Rego with the group of varieties of French origin and Samil with the
North-American ones.
References:
[1] Foroni, I. et al. (2007), Identification of ‘Sorrento’ walnut using simple sequence repeats
(SSRs), Genet Resour Crop Evol, 54, 1081-1094.
[2] Dangl, G.S. et al. (2005), Characterization of 14 microsatellite markers for genetic analysis
and cultivar identification of walnut, J Amer Soc Hort Sci, 130, 348-354.
[3] Wang, X-D., Wang, Z-P., Zou, Y-P. (1996), An improved procedure for the isolation of nuclear
DNA from leaves of wild grapevine dried with silica gel, Plant Mol. Biol. Rep., 14, 369-373.
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