Novel Set of Sex-Specific PCR-Based Markers Reveals New Hypothesis of Sex Differentiation in Date Palm

Abstract

The date palm (Phoenix dactylifera L.), 2n=36, is a dioecious long-lived monocotyledonous plant, with separate male and female trees. The dioecy represents the major challenge in development of breeding programs as it is impossible to distinguish tree gander till they flower approximately five to eight years after planting. Although, during the past two decades numerous attempts to develop molecular markers can discriminate among male and female trees in date palm. But, to date, sex-differentiation mechanism is still uncertain and there is no reliable way to determine the sex of date palm plants before reproductive age across all cultivars. Here, we employed an effective three novel gene-targeting marker approaches (SCoT, CDDP and ITAP) in additions to AFLP, in an attempt to develop a novel set of reliable sex-specific PCR-based markers can helping in early gender determination in Egyptian date palm trees. A set of 26 SCoT, 21 CDDP, 18 ITAP and 14 AFLP primers/primer combinations (PCs) were applied against twelve date palm genotypes belonging to three superior Egyptian date palm cultivars to identify any sex-specific markers. Four SCoT (SCoT1, SCoT24, SCoT26 and SCoT35), two CDDP (CDDP4 and CDDP6), one ITAP (ITAP-8/1) and one AFLP (AFLP-4/1) primer/PC exhibited differential fragments/bands between males and females. These differential bands were gel extracted and cloned for subsequent sequencing analysis. Three of the sequenced bands found to be contain more than one sequence. BLAST analysis results indicated that the eleven sequences generated from different gene-targeting marker systems (SCoT, CDDP and ITAP) revealed main similarity with master transcription factors, transcriptional activators/repressors and regulatory proteins involved in plant hormone signal transduction pathways, plant development and biosynthesis of secondary metabolites, playing important role in different types of abiotic and biotic stresses in date palm or oil palm. We speculate that kind of similarity is not just a coincidence. Our results reveals hypothesis that sex differentiation is a complex but well-organized process that involves endogenous and exogenous factors regulate and control the changes in gene expression, physiology, metabolism and architecture of the plant. These results represent the first case-study focusing on the applications of CDDP, ITAP and SCoT techniques as a novel gene targeting markers in sex-determination in date palm. Moreover, indicate that sex-differentiation process have to be addressed at system biology level for deep and better understanding. This developed sex-specific markers expected to be helpful in distinguish the gander in date palm at earliest stages.