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The Selective Amplification of Start Codon Polymorphic Loci (SASPL) has been developed as a new PCR-based molecular marker. SASPL was validated for the analysis of varietal diversity on ten olive varieties. Validation included in vitro comparison against RAPD, SCoT and SAMPL markers. Assessment of these techniques included primer selectivity, genome coverage and the ability to target genic regions through in silico PCR analysis. Candidate PCR fragments were further sequenced to annotate non-identified genes in olive. Eight SASPL primers were compared to 24 RAPD, 39 SCoT and 12 SAMPL primers. The TA produced by the RAPD, SCoT, SAMPL and the eight SASPL primers were 359, 642, 571 and 269 amplicons, respectively. The highest average number of TA was revealed by SAMPL (47.6), average of PA (18.1) and genetic similarity (GS) (96%) among the olive varieties. On the other hand, SASPL analysis provided higher average number of TA (33.6), average of PA (16.2) and GS (93%) than SCoT and RAPD. The highest average of (PIC) (0.2909) was exhibited by SASPL analysis and the lowest average (0.2038) was revealed by SCoT. The highest number of UB (111) was revealed by SCoT and the lowest UB (43) was obtained by SASPL. Across the four marker types, variety Maraki was characterized by the highest number of unique markers (74). Meanwhile, the variety Manzanillo was characterized by the lowest number of unique markers (8). In addition, in the in silico analysis SASPL exhibited the highest chromosomal coverage (0.59%) and targeted genes (1090) using the lowest number of primers. Additionally, the average area covered by the SASPL primers (354kb) was larger than SCoT and SAMPL. RAPD analysis provided the lowest potential, chromosomal coverage (0.04%) and number of targeted genes (17) compared to SASPL, SCoT and SAMPL analysis. The total coverage of the genome, revealed by combined data was higher (1.21%) than that of each technique separately. Meanwhile, the difference between the actual and the total genomic regions covered by the combined data was about 652kb. Our results suggested that the newly developed SASPL marker is the most adequately and each of the studied marker target different genomic areas, while some areas are shared. Two SCoT and one RAPD fragment were sequenced and showed a high similarity to genes of high physiological functions; such as cyclic plant-specific DNA-binding transcription factor, SANT domain and Copia-type retrotransposon.

Selective Amplification of Start codon Polymorphic Loci (SASPL): a new PCR-based molecular marker in olive