Molecular methodologies for improved genetic diversity assessment in cucumber and melon

Genetic markers have been used successfully in germptasm management to catalogue accession relationships and develop strategies for their management. Molecular marker technology has been applied to the assessment of genetic diversity of Cucumis species. The interpretation of relationships among accessions is made more meaningful if marker-based diversity assessments are standardized by uniform marker arrays (SMA) and historically based reference accessions (RA). In this study, previously mapped random amplified polymorphic DNA (RAPD) markers were converted to more stable sequence characterized amplified region (SCARs) markers and tested in multiplexing reactions (duplex and triplex) to increase assessment efficiency. Efficiency of PCR analysis can be increased by the use of liquid dispensing machines (LDM) that operate on a 96-well format. The efficiency of one such machine was compared to manual dispensing in preparation of PCR. The effectiveness (based on band intensity) of LDM and manual PCR preparation was equivalent. When compared to manual preparation, the efficiency of PCR preparation is increased using the LDM if more than four plates are run per day. Although a single SCAR marker can function well when used alone or in combination with another SCAR marker, the complementary use of markers in multiplex reactions decreases as the number of markers used in the polymerase chain reaction (PCR) increases. The kinetics of the PCR and its optimization play a major role in the success of multiplexing.