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Generalization of the polymorphism information content (PIC) index to represent marker informativeness (MI) for a three-generation F2 design requires that two additional sources of non-informativeness be added to the PIC formula: the probability of matings between like-heterozygous F1 individuals, of which one is non-informative; and that of matings between like-heterozygous F1 individuals, which are both fully informative but where line of origin of the same alleles is reciprocal. Given the dense marker-maps currently available for some species, this F22 informativeness parameter constitutes the natural criterion for marker selection in F2 designs, and two computer programs to predict MI from grandparental marker-genotypes were developed for an F2 population originating from two divergent selection lines of outbred mice (F ~ 0.2). A total of 403 markers had been genotyped for the F0 grandparents (n = 31), and 14 markers had also been genotyped in the complete pedigree including 559 F2 individuals. One program was based on assumptions of random-mating (RM), while the other (PED) accounted for the pedigreed mating structure. For the 403 markers, the correlation between MI from RM and from PED was 0.95, and the average deviation between the two predictions was 0.005 MI units (MI ranged from 0 to 1). Correlations between predicted and realized MI for the 14 fully genotyped markers were 0.97 for PED and 0.94 for RM, while the corresponding average of deviations between predicted and actual values were 0.01 and 0.04, respectively. Absolute deviations from realized MI never exceeded 0.09 and 0.16 for PED and RM, respectively. Simulated optimization of the mating system to maximize average MI of 28 markers on one chromosome led to improvements in the range of 15±20% average MI (0.07±0.09 MI units). The degree of relative advantage conferred by the F2 generalization of the PIC index over the traditional index was found to be of minor significance.