Determining Zygosity With Multiplex Kompetitive Allele‐Specific PCR ( mxKASP ) Genotyping

Ecology and Evolution, vol. 15, pp. e71642

Abstract

ABSTRACT We introduce multiplex Kompetitive Allele‐Specific PCR (mxKASP): a modification of “classical” KASP genotyping that allows zygosity to be determined in diploid organisms. Rather than targeting a SNP associated with a single marker, mxKASP targets two non‐homologous markers. We show proof of concept by applying mxKASP to the balanced lethal system in Triturus newts, in which individuals are known to possess either: (1) zero copies of the 1A version of chromosome 1 and two copies of the 1B version; (2) one copy of 1A and one copy of 1B; or (3) two copies of 1A and zero copies of 1B. mxKASP is successful in amplifying both a 1A and a 1B marker in a single reaction (if present), allowing the zygosity of individuals to be inferred. We independently confirm our mxKASP results with a multiplex PCR approach. We argue that mxKASP can be applied to rapidly and economically determine zygostity in diploid organisms, for a large number of samples at once.

Authors 5

1. Willem R. M. Meilink corresponding Aachen Institute of Biology Leiden Leiden University Leiden the Netherlands Naturalis Biodiversity Center Leiden The Netherlands

   Leiden University · Naturalis Biodiversity Center

   Affiliation as printed

   Institute of Biology Leiden Leiden University Leiden the Netherlands

   Naturalis Biodiversity Center Leiden the Netherlands

2. Manon Chantal de Visser Aachen Institute of Biology Leiden Leiden University Leiden the Netherlands Naturalis Biodiversity Center Leiden The Netherlands

   Leiden University · Naturalis Biodiversity Center

   Affiliation as printed

   Blijdorp Conservation & Science Center Royal Rotterdam Zoological & Botanical Gardens Rotterdam the Netherlands

   Institute of Biology Leiden Leiden University Leiden the Netherlands

   Naturalis Biodiversity Center Leiden the Netherlands

3. Anagnostis Theodoropoulos Aachen Institute of Biology Leiden Leiden University Leiden the Netherlands Naturalis Biodiversity Center Leiden The Netherlands

   Leiden University · Naturalis Biodiversity Center

   Affiliation as printed

   Institute of Biology Leiden Leiden University Leiden the Netherlands

   Naturalis Biodiversity Center Leiden the Netherlands

4. Michael Fahrbach
   Affiliation as printed

   Niedernhaller Str. 8/2 Criesbach Germany

5. Ben Wielstra Aachen Institute of Biology Leiden Leiden University Leiden the Netherlands Naturalis Biodiversity Center Leiden The Netherlands

   Leiden University · Naturalis Biodiversity Center

   Affiliation as printed

   Institute of Biology Leiden Leiden University Leiden the Netherlands

   Naturalis Biodiversity Center Leiden the Netherlands