Development and characterization of microsatellite markers in Castanea sativa (Mill.)

Thirty-three simple sequence repeat (SSR) markers were isolated and characterized in Castanea sativa (Mill.) from the cultivar Garrone Nero. For the identification of SSR loci, primers were designed on each side flanking the repeat region and they were initially tested on 5 chestnut samples using chemiluminescence detection. Twenty four loci where shown to be polymorphic and the number of alleles detected per locus varied from 2 to 7. Fourteen loci were chosen for the analysis of 20 cultivars grown in North Italy using the semi-automatic system ABI PRISM 377. These 14 markers showed a high level of genetic polymorphism with a total of 90 alleles; the number of alleles ranged from 4 to 10 per locus, with an average level of 6.4. The mean expected and observed heterozygosity were 0.724 (range: 0.649-0.835) and 0.793 (range: 0.350-0.950) respectively. The estimated frequency of null alleles showed a positive value for 3 loci, but except for 1 locus, the values were very low. The total value for the probability of identity was 7.04 x 10(-11). Paternity exclusion probability was very high (0.999), sufficiently high to study pollen flow.