DNA preparation. DNA of six ancient date seedlings from the current study and one (Methuselah) from the previous study (6) was analyzed. A set of 19 SSR was used for genotyping as described by Zehdi-Azouzi et al. (4). Gender was determined using date palm sex-linked microsatellite markers (11). Maternal lineages were traced back using the plastid intergenic spacer psbZ-trnf minisatellite (12, 38). Paternal lineages were studied through Y haplotypes using the three sex-linked SSRs (mPdIRDP80, mPdIRDP50, and mPdIRDP52) (11).

Total cellular DNA was extracted from lyophilized leaves using the TissueLyser and the DNeasy Plant Mini Kit (QIAGEN SA, Courtaboeuf, France) according to the manufacturer’s instructions. After purification, DNA concentrations were determined using a GeneQuant spectrometer (Amersham Pharmacia Biotech, France). The quality was checked by agarose minigel electrophoresis. The resulting DNA solutions were stored at −20°C.

Amplification and genotyping. Polymerase chain reactions were performed in an Eppendorf (AG, Hamburg, Germany) thermocycler. Reaction was performed in 20 μl and contained 10 ng of genomic DNA, 10× reaction buffer, 2 mM MgCl2, 200 μM deoxynucleotide triphosphates, 0.5 U polymerase, and 0.4 pmol of the forward primer labeled with a 5′M13 tail, 2 pmol of the reverse primer, and 2 pmol of the fluorochrome-marked M13 tail and MilliQ water. A touchdown polymerase chain reaction (PCR) was carried out with following parameters: denaturation for 2 min at 94°C, followed by six cycles of 94°C for 45 s, 60°C for 1 min, and 72°C for 1 min; then 30 cycles of 94°C for 45 s, 55°C for 1 min, and 72°C for 1.5 min; then 10 cycles of 94°C for 45 min, 53°C for 1 min, 72°C for 1.5 min; and a final elongation step at 72°C for 10 min. PCR products were analyzed using an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Allele size scoring was performed with GeneMapper software v3.7 (Applied Biosystems).

Genetic analyses. The ancient genotypes were compared to a reference matrix (90 genotypes) containing genotyping data on current date palm varieties covering the two genetic pools defined by Zehdi-Azouzi et al. (4) and including 35 samples from the eastern pool and 55 samples from the western pool (table S5). The number of alleles per group (NA), the number of alleles with a frequency higher than 5% (NA,P), and the observed (Ho), the expected (He) heterozygosities, and the fixation index values (FIS) were estimated using the GenAlEx 6.5 program (table S6). The allelic richness of each group was also calculated via the divBasic function implemented in the R package diversity (table S6) (39).

The hierarchical classifications were generated using PHYLIP package by calculating Cavalli-Sforza and Edwards distances (40) between ancient genotypes and current varieties (table S7). The obtained distance was used to construct the dendrogram using the neighbor-joining algorithm (41). The tree was drawn using DARwin software (42).

The membership probabilities of the ancient genotypes were identified by using a model-based clustering algorithm implemented in the computer program STRUCTURE v.2.3.4 (43). This algorithm identifies clusters (K) with different allele frequencies and assigns portions of individual genotypes to these clusters. It assumes the Hardy-Weinberg equilibrium and linkage equilibrium within clusters. The STRUCTURE algorithm was run without previous information on the geographic origin of the accessions using a model with admixture and correlated allele frequencies with 10 independent replicate runs for each K value (K value ranging from 1 to 6). For each run, we used a burn period of 10,000 iterations followed by 1 million iterations. The optimal number of clusters was assigned by using the run with the maximum likelihood validated with an ad hoc quantity based on the second-order rate of change in the log probability of data between different K values (fig. S3).The optimal alignment of the independent iterations was obtained by CLUMPP v.1.1 implemented in the Pophelper software v.1.0.10 (44); Pophelper v.1.0.10 (44) was also used to plot the results for the optimal K.

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