Identification of cross-pollinated and self-pollinated progeny in alfalfa through RAPD nulliplex loci analysis

Alfalfa (Medicago sativa L.) is the most important forage crop in the world, but with current breeding practices improvements in yield potential have been limited during the last 40 yr. The development of hybrid alfalfa cultivars would likely improve forage yield potential, but an efficient method to estimate outcrossing rates would be needed to confirm percentage hybrid seed. The objective of this research was to demonstrate the applicability of a RAPD nulliplex analysis to estimate outcrossing/selfing rates in autotetraploid alfalfa. Marker selection is based on polymorphism between a seed parent and its bulk open-pollination progeny. Polymorphic markers identify the seed parent as nulliplex (aaaa) for those particular loci, which allows the individual open-pollinated progeny from that seed parent to be classified as F₁ or S₁ progeny based on the presence/absence of these markers. Two alfalfa genotypes served as seed parents in crossing studies which differed in pollination control and number of pollen parents. Random amplified polymorphic DNA (RAPD) nulliplex analysis was conducted on these two seed parents and their progeny. Controlled crossing and selfing provided independent control over S₁s and F₁s. Five polymorphic markers were identified for both seed parents and these markers were sufficient to identify S₁ and F₁ progeny. The percentage S₁ seed was 7, 27, and 100% for crossing with emasculation, crossing without emasculation and self-pollination, respectively. In conclusion, RAPD nulliplex loci analysis can provide accurate identification of F₁ and S₁ progeny and an estimation of outcrossing rates in autotetraploid alfalfa.