Plant & Animal Genome VI Conference Plant & Animal Genome VI Conference
P39
Sval|f Weibull AB, Nilsson-Ehle Laboratory, S-268 81 Sval|v, Sweden
DNA extraction is one of the major bottlenecks, in practical marker assisted
selection, because of the high throughput requirement of plant breeding
programmes. We have therefore developed a protocol for DNA preparation, from
fresh or frozen leaf samples including very few simple steps, carried out in a
microtitre dish and enabling the DNA to be used directly for PCR. Since the
whole procedure just includes a boiling alkali treatment followed by squashing
and neutralising, up to 8 000 samples can be handled in a single working day by
one person.
In our pilot study we first screened 1266 Hordeum vulgare individuals of
various generations with an STS primer for the presence of the BaYMV/BaMMV
resistance gene ym4. Then we conducted a control of LibertyLink,
transformation events on 1047 Brassica napus individuals using elite
event specific primers.
In both experiments our quick DNA extraction technique gave exactly the same
results as our standard extraction protocol. These results suggest that the
quick method is very useful for large scale molecular breeding or backcrossing
purposes, when thousands of samples have to be screened with linked markers.
Furthermore, allowing all reactions to take place in microtitre dishes makes
further automation possible (i.e. use of multipipettes, pipetting robot),
reducing the amount of chemicals and utensils and thereby the cost per analysis.