Molecular and Cytogenetic Characterization of Watermelon Using DNA Markers and FISH

We did fluorescent in situ hybridization (FISH) in cultivated watermelon var. lanatus (PI 270306) and its wild counterpart var. citroides (PI 244018), using 18S-28S rDNA and 5SrDNA probes. Well separated somatic chromosomes were prepared from root meristems, using enzyme digestion technique for in situ hybridization following the standard protoplast technique to prepare somatic chromosome spread. We have observed two different organizational features in these two species.  In lanatus, we have identified two major 18S-28S rDNA sites and these are located on two different homologous pairs of chromosomes.  One 5S rDNA site, on the other hand, was observed and located on a pair of homologous pairs of chromosomes.  A two-color FISH (dual FISH) showed the 5S rDNA site was located interstitially and was syntenic to one of the 18S-28S rDNA sites .  As revealed by the interphase FISH ,  the 18S-28S rDNA and the 5S rDNA loci are spaced out and may not tightly linked to each other.     In contrary, only one 18S-28S rDNA site and two 5S rDNA sites were observed in Citroides accession (PI 244018).  To our knowledge, it is unusual in plant species, where the number of 18S-28S rDNA sites is always higher than the 5S rDNA site.  A dual FISH showed that all three rDNA sites were on three different pairs of homologous chromosomes . During the evolutionary process the 18S-28S rDNA site has been duplicated in lanatus and one of the 5S rDNA sites might have lost in this species. These results indicated that some structural rearrangements might have occurred during the evolution of lanatus.  Further, meiocytes analysis of pollen mother cell involving the rDNA FISH with 18S-28S rDNA and 5S rDNA probes would shed light on structural rearrangements in lanatus.  The current research also explored additional insights such as extent of diversity at the methylation level among the watermelon cultivars. DNA profiles were generated using Methylation-Sensitive AFLP Assay for 47 watermelon heirlooms. Results indicated that methylation specific diversity (43%) in US watermelon heirlooms is higher than the diversity (19.8%) as estimated by several investigators using conventional DNA markers.