Date palm (Phoenix dactylifera L.) is the major fruit tree grown in arid areas in the Middle East and North Africa. In the last century, dates were introduced to new regions including the USA. Date palms are traditionally propagated through offshoots. Expansion of modern date palm groves led to the development of Tissue Culture propagation methods that generate a large number of homogenous plants, have no seasonal effect on plant source and provide tools to fight the expansion of date pests and diseases. The disadvantage of this procedure is the occurrence of off-type trees which differ from the original cultivar. In the present project we focused on two of the most common date palm off-types: (1) trees with reduced fruit setting, in which most of the flowers turn into three-carpel parthenocarpic fruits. In a severe form, multi-carpel flowers and fruitlets (with up to six or eight carpels instead of the normal three-carpel flowers) are also formed. (2) dwarf trees, having fewer and shorter leaves, very short trunk and are not bearing fruits at their expected age, compared to the normal trees. Similar off-types occur in other crop species propagated by tissue culture, like banana (mainly dwarf plants) or oil palm (with a common 'Mantled' phenotype with reduced fruit setting and occurrence of supernumerary carpels). Some off-types can only be detected several years after planting in the fields. Therefore, efficient methods for prevention of the generation of off-types, as well as methods for their detection and early removal, are required for date palms, as well as for other tissue culture propagated crops. This research is aimed at the understanding of the mechanisms by which off-types are generated, and developing markers for their early identification. Several molecular and genomic approaches were applied. Using Methylation Sensitive AFLP and bisulfite sequencing, we detected changes in DNA methylation patterns occurring in off-types. We isolated and compared the sequence and expression of candidate genes, genes related to vegetative growth and dwarfism and genes related to flower development. While no sequence variation were detected, changes in gene expression, associated with the severity of the "fruit set" phenotype were detected in two genes - PdDEF (Ortholog of rice SPW1, and AP3 B type MADS box gene), and PdDIF (a defensin gene, highly homologous to the oil palm gene EGAD). We applied transcriptomic analyses, using high throughput sequencing, to identify genes differentially expressed in the "palm heart" (the apical meristem and the region of embryonic leaves) of dwarf vs. normal trees. Among the differentially expressed genes we identified genes related to hormonal biosynthesis, perception and regulation, genes related to cell expansion, and genes related to DNA methylation. Using Representation Difference Analyses, we detected changes in the genomes of off-type trees, mainly chloroplast-derived sequences that were incorporated in the nuclear genome and sequences of transposable elements. Sequences previously identified as differing between normal and off-type trees of oil palms or banana, successfully identified variation among date palm off-types, suggesting that these represent highly labile regions of monocot genomes. The data indicate that the date palm genome, similarly to genomes of other monocot crops as oil palm and banana, is quite unstable when cells pass through a cycle of tissue culture and regeneration. Changes in DNA sequences, translocation of DNA fragments and alteration of methylation patterns occur. Consequently, patterns of gene expression are changed, resulting in abnormal phenotypes. The data can be useful for future development of tools for early identification of off-type as well as for better understanding the phenomenon of somaclonal variation during propagation in vitro.
Targeted epigenomic changes to the maize methylome resulting from tissue culture
