Efficient protocols for CAPS-based mapping in Arabidopsis

Positional cloning continues to be an essential method for gene identification and characterisation. The introduction of PCR-based techniques such as Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Length Polymorphisms (SSLP) and Cleaved Amplified Polymorphic Sequences (CAPS) has greatly increased the efficiency of gene mapping in arabidopsis. To develop the CAPS marker approach further, we have altered several critical mapping parameters. Efficiency was improved by using a small volume of dry seed for DNA extraction instead of the commonly used vegetative tissue. Reproducibility of PCR reactions was enhanced by faster and reduced protocols for PCR and restriction enzyme digestion and optimisation of PCR conditions for over 50 CAPS primer pairs. Finally, the density of genetic markers was increased by providing polymorphic information for all CAPS markers in arabidopsis ecotypes Wassilewskija (Ws), Columbia (Col) and Cape Verde Islands (Cvi).