Microsatellite Marker Genotyping Protocol

The 16 microsatellite markers described in Puppo et al. [31 (link)] were amplified using the same multiplex primer combinations, fluorescent dyes, and PCR conditions from Puppo et al. [23 (link), 31 (link)]. Genotyping was done in an ABI3130xl automatic sequencer (Applied Biosystems, Inc., Foster City, CA; USA) using an internal size standard (Genescan-500 LIZ; Applied Biosystems, Inc.). GeneMapper ver. 4.0 was used for allele scoring (Applied Biosystems, Inc.). A total of 96 individuals were genotyped two times to evaluate scoring consistency. After scoring, only markers showing data for a majority of the samples were used for further analysis. This led to the exclusion of one marker (6493). Puppo et al. [23 (link), 31 (link)] did not find any significant deviation from the Hardy-Weinberg equilibrium, genotyping errors or a high amount of null alleles for any of the markers, so no further exclusions were necessary.

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Curto M., Puppo P., Kratschmer S, & Meimberg H. (2017). Genetic diversity and differentiation patterns in Micromeria from the Canary Islands are congruent with multiple colonization dynamics and the establishment of species syngameons. BMC Evolutionary Biology, 17, 198.

Publication 2017

ABI3130xl Genescan-500 LIZ

Alleles Fluorescent dyes Microsatellite markers Primer

Corresponding Organization : BOKU University

Other organizations : Universidade do Porto

Top 5 similar protocols

Variable analysis

independent variables

Microsatellite markers

dependent variables

Allele scoring

control variables

Multiplex primer combinations
Fluorescent dyes
PCR conditions
Internal size standard (Genescan-500 LIZ)
GeneMapper ver. 4.0 for allele scoring

positive controls

A total of 96 individuals were genotyped two times to evaluate scoring consistency

negative controls

Puppo et al. [23, 31] did not find any significant deviation from the Hardy-Weinberg equilibrium, genotyping errors or a high amount of null alleles for any of the markers, so no further exclusions were necessary

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