Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using twelve and fourteen AFLP EcoRI/MseI primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in wheat nullitetrasomic stocks and 22.3% in wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome specific AFLP markers in wheat nullitetrasomic stocks and 174 in wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and sixteen AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, reamplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley-derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, eleven primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. We also examined 21 wheat nullitetrasomic stocks using seven methylation sensitive PstI/Mse I primer combinations. 21.3% of the scored hypomethylated AFLP fragments in wheat nullitetrasomic stocks could be mapped to specific chromosomes. Out of four pairs of sequence-specific primers designed from the cloned wheat chromosome-specific PstI/MseI AFLP fragments, one primer pair amplified a fragment marking the expected chromosome. From these experiments we postulate that conversion of AFLPs to sequence-specific PCR markers in wheat is a promising, feasible, yet not efficient method so far.