The objective of this study was to validate the capability of SSR primers to determine the sex of date palm (cv. Madjoole) at an early stage of growth development. Out of eight SSR primers tested only seven SSR primers amplified clear bands successfully. The amplified SSR band sizes varied between 120 bp for mPdCIR015 and 335 bp for mPdCIR093. This range of SSR lengths has also been reported in previous studies. (Ben Abdallah et al. 2020) (Maryam et al. 2016) (Ahmed and Al-Qaradawi 2009). In our work, the relatively large range obtained in SSR bands increased the chance for a polymorphism between male and female date palm seedlings. Consequently, a high potentiality of SSR markers for sex determination is expected.
In total, 15 alleles were scored with a mean of 2.1 alleles per locus. However, in other work, mPdCIR078 showed a large number of allele/locus with 13 (Elmeer and Mattat 2012). The relative low number of alleles in this work is due to two reasons; the number of involved individuals in this study was low, and all of the female samples belonged to one genotype (cv. Madjoole).
Out of the eight SSR primers tested, only six were able to generate polymorphic and well-defined bands. On the contrary, one SSR failed to produce any fragments in this study, despite having yielded readable bands in other research (Zehdi et al. 2004).
In previous studies, mPdCIR048 showed inconsistent performance, whereas mpdCIR048 produced a specific locus (250/250 pb) in all male individuals. However, this locus was not present in any of the female samples, as reported by Maryam et al. (Maryam et al. 2016). Conversely, a different study by Zehdi et al. (Zehdi et al. 2004) found that mPdCIR048 failed to amplify any bands.
Male specific markers have been reported previously. In their study, Mohammed and Mohamed (Mohammed and Mohamed 2019) detected four male-specific loci using four RAPD markers, where distinctive bands were consistently visible in all male genotypes and absent in female samples. As well, DPM4 proved to be a 100% accurate SSR marker in determining the sex in the genus Phoenix, with a high ability to distinctly show the X and Y alleles in the sex chromosomes (Wang et al. 2020). This finding is in accordance with the results of a previous study, which identified that three SSR markers (mPdIRDP80, mPdIRDP50, and mPdIRDP52) were exclusively associated with male loci, providing confirmation of the presence of an XY chromosomal system in Phoenix dactylifera, as reported by Cherif et al. (Cherif et al. 2013). In our work, mPdCIR035 identified all six male plants with a specific homozygous locus (325 bp). These results are consistent with prior research that suggested a potential connection between SSR data and the sex of date palms, as indicated in the studies conducted by Ali et al. (Ali et al. 2018), Cherif et al. (Cherif et al. 2013), and Ismail (Ismail 2008).
In this study, mPdCIR015, mPdCIR035, mPdCIR078, and mPdCIR093 successfully distinguished the gender of young date palm seedlings at an early stage, categorizing them into two groups based on their sex. So, the ability of SSR markers to discriminate the sex of date palm (cv. Madjoole) was validated. The four SSR markers could be used efficiently to identify male and female individuals at an early stage for date palm. Among the four reproducible SSR markers, mPdCIR093 was recognized as a cv. Madjoole female specific marker. This marker needs to be used for further genotyping with other date palm cultivars in order to identify whether the (335 pb) loci is a Madjoole specific loci or not. This step could support using the mPdCIR093 marker solely, practically and routinely for female seedling cv. Madjoole selection at an early stage.