1999 Volume 74 Issue 2 Pages 55-59
Segregation of AFLP markers was frequently severely distorted when initial attempts were made to develop a molecular-based linkage map of sugi (Cryptomeria japonica). Only 27 out of 46 AFLP markers detected with combinations of three and four selective nucleotide primers (EcoRI + 3 and MseI + 4) fitted a 1:1 segregation ratio (α > 0.01). Greater selectivity was sought using M + 5 primers, derived by extending the original selective M + 4 primers with each possible additional nucleotide. More than 60% of all the fragments detected with M + 4 primers were amplified by at least two kinds of M + 5 primers. The results suggested that single fragments in the E + 3/M + 4 analysis were really fragment complexes in many cases. Of the 19 AFLP markers showing distortion, 13 were amplified by two to four of the M + 5 primers, but the level of distortion shown by these markers was reduced (or eliminated), when M + 5 primers were used. Thus, fragment complexes may be a major cause of segregation distortion. On the other hand, most of markers with a segregation that fitted the 1:1 ratio were amplified by a single M + 5 primer. In addition, E + 3/M + 4 primers detected a much greater frequency of polymorphism than E + 3/M + 5 primers in sugi. Therefore, we would recommend the use of both E + 3/M + 4 and E + 3/M + 5 primers for efficient AFLP mapping.