Samples derived for M78, M81, M267 and M269 were not included in further analyses; those derived for M89 were subsequently analyzed for the M9, M45, M170, M172, M173 and M201 multiplex set; and ancestral ones for the M2, M33, M34, M60 and M96 multiplex set.
The PCR proceeded as follows: 15 min at 95 °C; 30 cycles of 30 s at 94 °C, 90 s at 53 °C, 1 min at 72 °C for multiplex set 1, or 35 cycles of 30 s at 94 °C, 90 s at 58 °C and 1 min at 72 °C for multiplex sets 2 and 3; and a final extension step of 30 min at 60 °C.
Forward primers were labeled with FAM, HEX (Integrated DNA Technologies, Coralville, Iowa, USA), or NED (Applied Biosystems) (see Table 1 for multiplex sets, specific concentration of primers, and fluorescent label).
The remaining 304 primer pairs, which generated unambiguous and stable PCR amplicons, were therefore selected and assembled into 41 multiplex sets of six to eight markers each (Supplementary Table S1).
Seventeen polymorphic markers were selected for further analysis and combined into four multiplex PCRs with Multiplex Manager version 1.0 (Holleley and Geerts, 2009; PCR multiplex sets 1 4 in Table 2).
Two markers (ZF01-196 and ZF01-025) were included in multiple multiplex sets to verify repeatability between the different PCR sets.
The optimized multiplex sets show reproducible amplification pattern with low genotyping error rate and should be therefore very useful in analyses of both experimental as well as natural populations of crickets.
New markers were divided into two PCR multiplex sets (Table 1) that were combined again after PCR for electrophoresis and fragment analysis.
Clark, L. A. et al. Chromosome-specific microsatellite multiplex sets for linkage studies in the domestic dog.
