Hidi formamid

All the primers used for sequencing are listed in the Additional file 2: Table S2. All PCR reaction conditions are specified in the Additional file 3. The obtained products were purified using the PCR Purification Kit (Qiagen), as described in the manual. Sequencing reaction was performed using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing products were purified using DyeEx 2.0 Spin Kit (Qiagen) according to the manufacturer’s instructions. The purified products were diluted with 18 μl HiDi-Formamid (Applied Biosystems), incubated at 95°C for 3 min and chilled on ice for 3 min. Sequencing was performed using ABI310 Genetic Analyser (Applied Biosystems), and data were collected using ABI Prism 310 Data Collection Software.

19 microsatellites were chosen from previous studies: PP8E11 (Chr2), PP6E09 (Chr3), D1EnsmusG22992 (Chr3),PP10E08 (Chr4), D5Mit149 (Chr5), D6Mit309 (Chr6), PP10A02 (Chr8), D9Mit54 (Chr9), D9Mit330 (Chr9), PP3A02 (Chr10), PP4A02 (Chr11), D13Mit61 (Chr13), D14Mit203 (Chr14), E1EnsmusG46849 (Chr15), D15Mit98 (Chr15), PP7B08 (Chr16), PP8A05 (Chr17), D19Mit39 (Chr19) and, PP2A01 (ChrX) [37 (link),38 ]. Forward primers were labeled with FAM or HEX dye on the 5’end and the reaction conditions were as follow: denaturation, 95°C for 15 min, 28 cycles with 0.30s at 95°C, 1.30 min at 60°C and 1.30 min at 72°C, final elongation 10 min at 72°C. Primers (Additional file 1: Table S1) were pooled in 5 different reactions with a final reaction volume of 5 μl using Multiplex PCR kit (Qiagen). PCR products were then diluted 1/20 in Millipore water. 1 μL of this dilution was added to a mix of 0.1 μL ROX Standard (Applied Biosystems) and 10 μL HiDi Formamid. Heating for the denaturation step was 2 min at 90°C followed by 5 min at 20°C. Microsatellites were scored using GeneMapper (Applied Bioscience).