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

Area of Science:

• Genetics
• Molecular Biology
• Evolutionary Biology

Background:

• Zygosity determination is crucial in diploid organisms for genetic studies.
• Traditional genotyping methods can be time-consuming and costly for large sample sizes.
• The balanced lethal system in *Triturus* newts presents a unique challenge for zygosity assessment.

Purpose of the Study:

• To introduce multiplex Kompetitive Allele-Specific PCR (mxKASP) as a novel method for zygosity determination.
• To demonstrate the efficacy of mxKASP in diploid organisms using a real-world example.
• To provide a rapid and economical genotyping solution for large-scale genetic analyses.

Main Methods:

• Modification of classical KASP genotyping to target two non-homologous markers simultaneously.
• Application of mxKASP to the balanced lethal system in *Triturus* newts.
• Independent validation of mxKASP results using a multiplex PCR approach.

Main Results:

• mxKASP successfully amplified both target markers (1A and 1B) in a single reaction.
• Zygosity of *Triturus* newt individuals was accurately inferred based on marker amplification patterns.
• mxKASP results were confirmed by an independent multiplex PCR method.

Conclusions:

• mxKASP is a powerful modification of KASP genotyping for determining zygosity in diploid organisms.
• The method offers a rapid, economical, and scalable approach for high-throughput genetic analysis.
• mxKASP has broad applicability for various genetic studies requiring efficient zygosity assessment.