SUPPORTING INFORMATIONS2 Title: Genetic landscape with sharp discontinuities shaped by complex demographic history in moose (Alces alces) Authors: Lovisa Wennerström, Nils Ryman, Jean-Luc Tison, Anna Hasslow, Love Dalén, and Linda Laikre Affiliation of first and senior authors (LW, NR, LL): Division of Population Genetics, Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden E-mail adresses: [email protected], [email protected], [email protected] Journal: Journal of Mammalogy Microsatellite genotyping and mtDNA sequencing Genomic DNA was isolated from frozen muscle tissue and 12 microsatellite loci that had previously been analyzed in moose were scored, including 5 loci used in our own previous work (Charlier et al. 2008): BM757, BM848, CSSM003, CSSM39, IDGA8, IDGVA29, MAF46, McM58, McM64, McM130, OarCP21, and RT30 (Swarbrick et al. 1992; Bishop et al. 1994; Hulme et al. 1994; Moore et al. 1994; Ede et al. 1995; Mezzelani et al. 1995; Wilson et al. 1997; Slate et al. 1998; Haanes et al. 2011; Kangas et al 2013). Laboratory work was conducted at the Center of Evolutionary Applications, University of Turku, Finland. DNA was extracted with a salt extraction method modified from Aljnabi and Martinez (1997). Samples were genotyped with markers divided into 2 multiplexes (MPs) and one single PCR (MP1: MAF46, McM58, McM130, CSSM003, RT30, OarCP21, CSSM39, MP2: McM64, BM848, BM757, IDGVA8, single: IDGVA29). To improve the microsatellite peak profiles, a GTTT-“PIGtail” was added to the 5’ end of each non-labelled primer (Brownstein et al. 1996). Amplifications were done in 3 (three) 8-µl reactions with c. 80 ng of DNA, 0.025 to 0.8 µM of each primer (one of which was fluorescently labeled) and 1X Qiagen multiplex PCR master mix (Qiagen Inc. Valencia, CA, USA). The PCR profile used was according to the manufacturer’s protocol with annealing temperatures of 58 °C, 61 °C, and 58 °C for MP1, MP2 and the single reaction, respectively. Amplifications were performed on PTC-100 (MJ Research) and AB 2720 (Applied Biosystems) thermal cyclers. The PCR products were pooled for electrophoresis (1.4 µl of MP1, 1 µl of MP2 and 1.6 µl of the single PCR) and diluted with 100 µl sterile water. 2 µl of the pooled and diluted PCR product was combined with GS600LIZ size standard (Applied Biosystems) and HiDi-formamide (Applied Biosystems). Samples were denatured at 98 °C for 3 minutes and the size of the fragments was determined by capillary electrophoresis on an ABI PrismTM 3130xl genetic analysis instrument. The genotypes were scored using GENEMAPPER v4.0 (Applied Biosystems) and following visual inspection, exported to a spreadsheet program for downstream analyses. mtDNA Genomic DNA was extracted from muscle tissue using Qiagen DNEasy blood and tissue kit. The 5’ end of the mtDNA with the first hypervariable part of the control region (CR1) was amplified using primers Alces-PRO1F (5’-CCCCACTATCAACACCCAAA-3’) and AlcesDL1R (5’-GTGGGCGATTTTAGRTGAGA-3´). PCR reactions were performed in 25μl volumes, containing 1μl DNA extract, 0.4µM of each primer, and an Illustra Hot Start mix RTG bead (GE Healthcare). The PCR thermal conditions started with 10 minutes denaturation at 95°C, followed by 3 cycles of denaturation for 30 seconds (sec) at 94°C, 30 sec annealing at 56°C, 60 sec extension at 72°C. This was followed by 32 cycles of 30 sec denaturation at 94°C, 30 sec annealing at 54°C, 60 sec extension at 72°C. PCR products were sequenced for both the forward and reverse strands using BigDye Kit v.3.1 (Applied Biosystems) and run on an ABI3130xl sequencer at the Swedish Museum of Natural History, Stockholm. Sequences were edited using Geneious (Kearse et al. 2012). Literature cited (not provided in the paper) ALJNABI S. M. AND I. MARTINEZ. 1997. 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