436 



Microsatellite multiplex panels for genetic studies 

 of gray snapper iLutjanus griseus) and 

 lane snapper iLut/anus synagns) 



Mark A. Renshaw 



Eric Saillant 



Siya Lem 



Philip Berry 



John R. Gold 



Email address for M A. Renshaw: mrenshaw@ag.tamu.edu 



Center for Biosystematlcs and Biodiversity 

 Texas A&fV\ University, TAMU-2258 

 College Station, Texas 77843-2258 



Microsatellites are codominantly 

 inherited nuclear-DNA markers 

 (Wright and Bentzen, 1994) that are 

 now commonly used to assess both 

 stock structure and the effective 

 population size of exploited fishes 

 (Turner et al., 2002; Chistiakov et 

 al., 2006; Saillant and Gold, 2006). 

 Multiplexing is the combination of 

 polymerase chain reaction (PCR) 

 amplification products from multiple 

 loci into a single lane of an electropho- 

 retic gel (Olsen et al., 1996; Neff et 

 al., 2000) and is accomplished either 

 by co-amplification of multiple loci 

 in a single reaction (Chamberlain et 

 al., 1988) or by combination of prod- 

 ucts from multiple single-locus PCR 

 amplifications (Olsen et al., 1996). 

 The advantage of multiplexing micro- 

 satellites lies in the significant reduc- 

 tion in both personnel time (labor) 

 and consumable supplies generally 

 required for large genotyping proj- 

 ects (Neff et al., 2000; Renshaw et 

 al., 2006). 



In this note, we report the devel- 

 opment of multiplex panels of mic- 

 rosatellites that will facilitate popu- 

 lation-level genetic studies of both 

 gray (Lutjanus griseus) and lane 

 (L. synagris) snappers. The overex- 

 ploitation of Gulf red snapper (L. 

 campechanus) in U.S. waters and the 

 increasing restrictions on both com- 

 mercial and recreational red snap- 

 per catches (Gillig et al., 2001) have 

 led to increased fishing pressure on 

 other snapper species (Fischer et al., 



2005), including both gray (Burton, 

 2001; Fischer et al, 2005) and lane 

 snappers (GMFMCM. Although nei- 

 ther species has yet been classified 

 as "overfished" or subject to "overfish- 

 ing," the increased exploitation of the 

 two species could jeopardize these 

 snapper resources in the future. In 

 this study, we optimized multiplex 

 panels for gray and lane snappers 

 from among microsatellite markers 

 designed originally for red snapper 

 by Gold et al. (2001) and vermillion 

 snapper (Rhomboplites aurorubens) 

 by Bagley and Geller (1998). 



Materials and methods 



Samples of gray and lane snappers 

 were obtained off the west coast of 

 Florida during April of 2004. Fin clips 

 and pieces of liver were preserved in 

 95% ethanol, brought to the labora- 

 tory, and stored at room temperature. 

 Genomic DNA was extracted by using 

 an alkaline-lysis method (Saillant et 

 al, 2002) and stored at -20°C. 



1 GMFMC (Gulf of Mexico Fishery 

 Management Council). 2005. Final 

 amendment to the FMPs for: reef fish 

 (Amendment 25) and coastal migratory 

 pelagics (Amendment 17) for extending 

 the charter vessel/headboat permit mor- 

 atorium (including SEIS/RIR/IRFA), 

 111 p. Gulf of Mexico Fishery Man- 

 agement Council, 3018 North U.S. 

 Highway 301, Suite 1000, Tampa, FL 

 33619-2272. 



Microsatellites were first evaluated 

 in single-locus (simplex) reactions in 

 order to determine the size range 

 and ease of scoring of PCR products 

 in each species. PCR amplifications 

 were performed in 11.5-;(L volumes 

 comprising 1.5 fiL of DNA (approxi- 

 mately 50 ng), 1 f(L of lOx reaction 

 buffer [50 mm KCl, 10 mm Tris, 1% 

 Triton-X 100], 0.75 U Taq DNA poly- 

 merase (Invitrogen, Carlsbad, CA), 

 200 f(m of each dNTP, 1 mm MgClg 

 and various quantities of PCR prim- 

 ers. One PCR primer of each pair 

 was labeled with one of three fluores- 

 cent dyes from set D (Applied Biosys- 

 tems, Foster City, CA): Fam, Hex, or 

 Ned. Fragment analysis was carried 

 out on an ABI Prism 377 automated 

 DNA sequencer (Applied Biosystems, 

 Foster City, CA). Allele size was 

 estimated by using the Genescan- 

 400HD [Rox] size standard (Applied 

 Biosystems, Foster City, CA); allele 

 size estimation was performed with 

 Genescan 3.1.2 (Applied Biosystems, 

 Foster City, CA) and allele calling 

 was performed in Genotyper, version 

 2.5 (Applied Biosystems, Foster City, 

 CA). Multiplex tests were performed 

 only on those microsatellites that am- 

 plified successfully and yielded PCR 

 products that were easy to score; loci 

 not meeting these criteria were elim- 

 inated from subsequent analyses. 



Initial multiplex PCR amplifica- 

 tions were performed by using the 

 three multiplex panels designed to 

 amplify 20 microsatellite loci in red 

 snapper (Renshaw et al., 2006). At 

 first, PCR primer concentrations fol- 

 lowed those outlined in Renshaw et 

 al. (2006), and changes were made 

 according to the relative success of 

 amplifications at each microsatellite 

 within a particular multiplex. Mic- 

 rosatellites that failed to amplify in 

 multiplex reactions were optimized 

 in simplex reactions. Additional PCR 



Manuscript submitted 14 September 2006 

 to the Scientific Editor's Office. 



Manuscript approved for publication 



9 November 2006 by the Scientific Editor. 



Fish. Bull. 105:436-439 (2007). 



