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The present study portrays the achievement of the genetic polymorphism surveying and the establishment of an ecotypes identification key on the basis of simple sequence repeats data. Seventy-two Tunisian fig ecotypes in situ and ex situ conserved were analyzed using six microsatellite loci. A total of 58 alleles and 124 genotypes were revealed and permitted to evidence high degree of genetic diversity mainly explained at the intra group level. Cluster analysis based on genetic distances proved that a typical continuous genetic diversity characterizes the local germplasm. In addition, the microsatellite multilocus genotyping has permitted to unambiguously distinguish 70 well-defined ecotypes (resolving power of 97.22%). Data are discussed in relation with the reliability of the used markers to check the conformity of the plant material and to rationally manage the conservation of this crop.