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

Two approaches to optimise AFLP fingerprinting for the rapid detection of genetic polymorphisms, i.e. reduced primer selectivity and bulked DNA analysis were examined. The efficiency of reduced primer selectivity to increase the detection frequency of genetic polymorphisms and to obtain more informative fingerprinting profiles was tested in six different crops. The number of selective nucleotides was reduced to six in onion, to five in barley, potato, lettuce and cabbage, and to four in flax. This allowed the rapid identification of several primer pairs that were able to discriminate between closely related germplasm. Reproducibility tests on replicate DNA samples indicated no major negative effects on the reliability of the fingerprinting profiles due to the use of less selective primers, although for onion purified DNA was needed to avoid irreproducible results. In barley, flax and onion, a less than fourfold increase in the number of fragments was observed when primer pairs were reduced by one selective nucleotide. This result was attributed to different tolerance levels for amplification mismatches between primer pairs of different selectivity. The efficiency of bulked DNA analysis to detect genetic polymorphisms was investigated in different mixtures of two barley DNA samples. AFLP's of varying intensity could still be recovered when the two DNA's were mixed in a 1:1 ratio. However, the frequency of recovered bands quickly dropped when in the mixtures the presence of the DNA carrying the fragments was decreased below 50%. The usefulness of the two approaches is discussed in relation to various aspects of genetic resources management.