Primer Notes

This paper characterizes eight pairs of PCR primers that amplify microsatellite loci in green-rumped parrotlets, Forpus passerinus, Psittacidae. We examined polymorphism of these loci in this species and in five other parrot species. The green-rumped parrotlet is a neotropical member of one of the worldÕs most endangered bird families, the parrots, Psittacidae (Bennett & Owens 1997; Collar & Juniper 1992). Parrots are particularly worthy of attention as they are among the best flagship species in South America for stimulating conservation efforts. For this reason, and to investigate the population biology of F. passerinus, we developed microsatellite loci. High heterozygosity, neutrality, and ease of assay make microsatellite loci ideal tools for studies of behavioural ecology and population genetics (Queller et al. 1993). As primers developed in one species may amplify loci in related species, we investigated the utility of our loci for some other neotropical parrots (Primmer et al. 1996; Hughes et al. 1998). Loci could provide species and population identification, confirming that birds purported to come from captive breeding or sustainably harvested populations did originate from such environments (Beissinger & Bucher 1992; Derrickson & Snyder 1992). Such distinctions are critical to enforcement of the Exotic Wild Bird Conservation Act of 1992. Three partial genomic libraries were made in Lambda Zap Express (Stratagene, La Jolla, CA) (Hughes & Moralez Deloach 1997). We screened approximately 300 000 clones, sequenced several hundred positives, and developed primers for clones containing 38 uninterrupted repeats of the sequence AAT. Most positive clones contained loci with seven or fewer AAT repeats. Libraries were also screened for other trinucleotide, and dinucleotide, repeats, with less success. DNA amplification reactions (5 or 10 μl) contained about 10 ng of DNA, 50 mM KCl, 10 mM Tris-Cl pH 8.3, 1.5 mM MgCl2, 0.1% NP40, 250 μM each dNTP, and 500 nM each primer. Using the Ôtube controlÕ function of a Hybaid thermal cycler (where a thermistor monitors temperature in a dummy tube), reactions were cycled: 90 s at 92 ¡C, then 0 s at 92 ¡C, 5 s at 55 ¡C, 5 s at 72 ¡C, 30 times, and finally 90 s at 72 ¡C. When genotyping, 0.05 μl of 3.3 μM, S-labelled, dATP was included per reaction. We found microsatellite loci unusually difficult to develop in this species. It took three iterations of cloning and sequencing to find eight polymorphic loci (GenBank Accession nos AF035366ÐAF035373, Table 1). Five loci that contained six or seven AAT repeats in original clones were monomorphic; no other primer pairs were bought for loci containing fewer than eight repeats. Bird genomes are apparently under selection to be small (Hughes & Hughes 1995), and this may partly explain why microsatellite loci containing longer runs of repeats (those most likely to be polymorphic) are rare. However, the extreme paucity of these loci in F. passerinus is remarkable. We found that these loci are unexpectedly monomorphic in other species (Table 2). We attempted PCR amplification of these loci in five other species, using annealing temperatures 5 ¡C lower than in Table 1. Work in passerines suggests that about 50% of primers that amplify an appropriately sized product will be polymorphic (Primmer et al. 1996). Assuming that mutation rates are similar in psittacines as passerines, this result is consistent with small effective population size in the species tested.