NOTE Renshaw et al.: Microsatellite multiplex panels for genetic studies of Lut/onus griseus and Lut/anus synogns 



437 



primers {Ra 1, Ra 2, and Ra 4) designed by Bagley and 

 Geller (1998) for vermillion snapper microsatellites were 

 tested in simplex reactions as described above and la- 

 beled with one fluorescent dye to allow integration into 

 one of the three multiplex panels. 



Multiplex PCR amplifications initially followed the 

 Touchdown I and II protocols outlined in Renshaw et al. 

 (2006). Touchdown PCR protocols enable the amplifica- 

 tion of multiple loci with different optimal annealing 

 temperatures. The former (Touchdown I) protocol in- 

 volved a one-half degree drop in annealing temperature 

 with each subsequent cycle, for a total of 12 cycles, fol- 

 lowed by 30 cycles at a bottom annealing temperature 

 that was 6°C below the initial annealing temperature; 

 the latter (Touchdown II) protocol featured a three-step 

 drop in annealing temperature with seven cycles at an 

 initial annealing temperature, followed by seven cycles 

 at a lower annealing temperature, followed by 28 cycles 



at a bottom annealing temperature that was 4-6°C 

 below the initial annealing temperature. Changes were 

 made to optimize multiplex panels individually for each 

 species. These changes included 1) removal of various 

 primers and addition of others, and 2) modification of 

 annealing temperatures and species-specific differences 

 in primer concentrations. 



Once multiplex or simplex protocols were developed for 

 the two species, DNA from 28 individuals of each spe- 

 cies was assayed across all optimized loci. Genetic vari- 

 ability of microsatellites in each species was measured 

 by the number of alleles, expected heterozygosity (gene 

 diversity), and observed heterozygosity. Fisher's exact 

 test was used to evaluate loci for significant departures 

 from Hardy-Weinberg equilibrium expectations. Three 

 common causes for genotyping errors — null alleles, 

 large allele dropout, and stuttering — were assessed with 

 Micro-Checker (Van Oosterhout et al., 2004). 



