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

Abstract

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.

Identification of Sex-specific Molecular Markers in Barbel and Nile Carp using SCoT and ISSR Markers

“…However, it was also used to assess genetic diversity in animals such as camel (Al-Soudy et al, 2018) and fish (Marie and Allam, 2017). Also, SCoT marker was efficient to identify molecular sex marker in date palm (Adawy et al 2014 andSami 2015) and when used with fish it successfully discriminated between male and female of Brycinus nurse using bulked and individual samples (Mohamed et al 2019).…”

“…Interestingly, in the last decades, many molecular markers have been developed and successfully used in molecular sexing studies in many species including fish, such as random amplified polymorphic DNA (RAPD) (Wuertz et al, 2006;Chen et al, 2009;Durna, 2009;Xia et al, 2011;Silva et al, 2012;Al-Qurainy et al, 2018 andMohamed et al 2019), start codon targeted (SCoT) polymorphism Sami, 2015 andMohamed et al 2019) and inter simple sequence repeat (ISSR) (Wuertz et al, 2006;Adhikari et al, 2014 andMohamed et al 2019).…”

Karyotyping and Molecular Sexing in Brycinus nurse (Characiformes, Alestidae) from Nile River in Egypt

“…However, SCoT was used recently to study genetic diversity in other organisms such as fish (Marie and Allam, 2017) and camel (Al-Soudy et al, 2018). In addition, it was also used as a novel gene targeting marker in sex-determination in date palm (Mohamed and Sami, 2015) with no available reports about using it in molecular sexing studies in animals. Herein SCoT marker was used for the first time to investigate the molecular sex differences in fish and among ten primers tested to differentiate between males and females of B. nurse only one primer (SCoT-18) tested successfully generated a female specific band (720 bp) in the bulked DNA sample from females specimens which confirmed using individual DNA samples.…”

“…However it is difficult to distinguish between the sex chromosomes and autosomes of almost majority fish species based only on their size and shape (De Rosa et al, 2017). Interestingly, in the past few years, many molecular markers have been developed to investigate sex-specific molecular markers in fishes and many other species such as random amplified polymorphic DNA (RAPD) (Durna, 2009;Xia et al, 2011;Silva et al, 2012 andAl-Qurainy et al, 2018), start codon targeted (SCoT) (Mohamed and Sami, 2015) and inter simple sequence repeat (ISSR) (Wuertz et al, 2006 andAdhikari et al, 2014). Identification of sex specific DNA markers in fishes is very useful for investigating sex determination system, identifying sex chromosomes and sex-related genes.…”

Development of sex‐linked SSR marker in the genus Phoenix and validation in P. dactylifera

“…Considering the progress made to date and the prospect offered by biotechnology tools in date palm programs, the need for development and validation of markers for sex determination across the genus is highly desirable. Researchers have reported the development of DNA markers that were said to be useful for sex determination in date palm (Al‐Qurainy et al., 2018; Ali, Abubakari, Martin, & Krutovsky, 2018; Cherif et al., 2013; Dhawan, Kharb, Sharma, Uppal, & Aggarwal, 2013; Maryam et al., 2016; Mohamed & Sami, 2015; Torres et al., 2018) To the best of our knowledge, most of these published markers have not been sufficiently tested and validated to prove their usefulness for marker‐assisted selection (MAS).…”