Dates constitute the primary exportable agricultural product in Qatar and current efforts are being made to improve its production. Varieties with desirable morphological traits are usually maintained using clonal propagation leading to the establishment of uniform palm plantations. This practice reduces the genetic diversity among palm orchards and makes them more vulnerable to pests and diseases. The biggest limitation in date palm propagation from seeds is that half of the planted offspring are expected to be male individuals with no agricultural value. Previous cytological studies indicated the presence of heteromorphic sex chromosomes in date palm, but enzymatic approaches intended to anticipate gender determination have been unsuccessful. Single nucleotide polymorphism (SNP) analysis of female and male individuals from the commercial varieties Khalas, Deglet Noor and Medjool, allowed the identification of a sex-linked region that segregates with gender, and extends to 24 scaffolds. This sex locus was genetically mapped to the lower arm of linkage group 12, and was estimated to extend up to 13 Mb, or 2% of the genome. Our objective was to physically map the male/female determination region in date palm using a bacterial artificial chromosome (BAC) library. This library represents 12x genome coverage and clones have an average insert size of 125 kb. A first round of PCR screening of this library was developed using the sequence information from the 24 SNP-enriched scaffolds to generate PCR-based primers around gender segregating markers. Plasmid DNA from multiple BACs was barcoded, pooled and sequenced using Illumina MiSeq and 150 paired-end reads were then assembled and compared to the existing date palm reference genomes from Qatar and Saudi Arabia. Next generation sequencing of eighty-two BACs has allowed the mapping of nineteen markers to six large assembled contigs with an estimated average size of 496.4 kb spanning approximately 2.9 Mb. Preliminary analysis of the assembled sequences indicated that we have positively identified a genomic region highly enriched in gender-linked SNPs, with male and female alleles segregating in a 1:1 ratio. This is what we would have expected from an XY sex chromosome system. Multiple sequence gaps still exist within and between the sequenced region, and further analysis has indicated that many BAC end sequences correspond to repetitive elements that likely constitute a big portion of the unassembled DNA sequences. During the second phase of this study, we have designed and tested twenty-one new PCR markers that we expect will help to close the gaps, determine the minimum tilling path and obtain a complete sequence and physical map of the sex-determination region. Our ultimate goal is to detect the critical mutation that gave rise to male and female separation. This is the first time such level of detail in a dioecious plant will be achieved. Furthermore, standardization of a reliable sequencing and screening procedure will allow us to characterize other genomic regions associated with desirable commercial properties in date fruits. This in turn will provide a valuable tool for the development of marker-assisted selection programs and ultimately for the improvement of date palm production.


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