Efficiency of reduced primer selectivity and bulked DNA analysis for the rapid detection of AFLP† polymorphisms in a range of crop species

Abstract

Two approaches to optimise AFLP fingerprinting forthe rapid detection of genetic polymorphisms, i.e.reduced primer selectivity and bulked DNA analysiswere examined. The efficiency of reduced primerselectivity to increase the detection frequency ofgenetic polymorphisms and to obtain more informativefingerprinting profiles was tested in six differentcrops. The number of selective nucleotides was reducedto six in onion, to five in barley, potato, lettuceand cabbage, and to four in flax. This allowed therapid identification of several primer pairs that wereable to discriminate between closely relatedgermplasm. Reproducibility tests on replicate DNAsamples indicated no major negative effects on thereliability of the fingerprinting profiles due to theuse of less selective primers, although for onionpurified DNA was needed to avoid irreproducibleresults. In barley, flax and onion, a less thanfourfold increase in the number of fragments wasobserved when primer pairs were reduced by oneselective nucleotide. This result was attributed todifferent tolerance levels for amplificationmismatches between primer pairs of differentselectivity.The efficiency of bulked DNA analysis to detectgenetic polymorphisms was investigated in differentmixtures of two barley DNA samples. AFLP's of varyingintensity could still be recovered when the two DNA'swere mixed in a 1:1 ratio. However, the frequency ofrecovered bands quickly dropped when in the mixturesthe presence of the DNA carrying the fragments wasdecreased below 50%.The usefulness of the two approaches is discussed inrelation to various aspects of genetic resourcesmanagement.