Amplified PCR products were hybridized twice with di- (GA, GT, AT, GC), tri- (CAA, ATT, GCC), and tetranucleotide (GATA, CATA, ATAG) repeats bounded to Hybond N+ filters (GE Healthcare, Salt Lake City, Utah, USA). Genomic DNA was digested with the RsaI enzyme, ligated to the MluI adapter (5′-CTCTTGCTTACGCGTGGACTA-3′) and amplified through PCR. pinea individual using the DNeasy Plant Kit (QIAGEN, Valencia, California, USA) following the manufacturer's protocol. Briefly, we extracted ∼2 μg of total genomic DNA from the needles of a single P. In approach A, an enriched genomic library for di- (GA, GT, AT, GC), tri- (CAA, ATT, GCC), and tetranucleotide (GATA, CATA, ATAG) repeats was constructed following 3. METHODS AND RESULTSįor the isolation of nSSRs, two approaches were followed. pinea, an ecologically important widespread Mediterranean conifer with high nutritional value seeds ( 7), which confers them great commercial value. For this reason, an effort has been made to isolate and optimize new nuclear microsatellites (nSSRs) for P. This feature has been very rarely observed in other species with similar life history traits ( 11) and requires a deeper knowledge of the existing population genetic variation.
Italian stone pine ( Pinus pinea L.) represents such a case in fact, an almost complete lack of variation was observed across the entire species range using chloroplast microsatellite markers (cpSSRs) ( 11), fatty acids ( 6), and allozyme markers ( 4). The finding of widely distributed, obligatory sexually reproducing species that are genetically impoverished is then surprising. Genetic variation is considered a prerequisite for adaptation to new environmental conditions. Conclusions: Despite low polymorphism, these new markers will be useful to resolve population structure and hold potential for seed origin identification and traceability.
Nine out of the 12 microsatellites were successfully transferred to P. All loci were in linkage equilibrium, and the frequency of null alleles was very low (≤1% in 332 out of 396 locus/population combinations). Hardy–Weinberg equilibrium expectations were not met in only ∼10% of the possible locus/population combinations. Low level of genetic variability was confirmed with average expected heterozygosity of 0.11.
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