Meilink Willem R M, de Visser Manon C, Theodoropoulos Anagnostis, Fahrbach Michael, Wielstra Ben
Institute of Biology Leiden Leiden University Leiden the Netherlands.
Naturalis Biodiversity Center Leiden the Netherlands.
Ecol Evol. 2025 Jun 21;15(6):e71642. doi: 10.1002/ece3.71642. eCollection 2025 Jun.
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 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.
我们介绍了多重竞争性等位基因特异性PCR(mxKASP):这是对“经典”KASP基因分型的一种改进,可用于确定二倍体生物的纯合性。mxKASP不是针对与单个标记相关的单核苷酸多态性(SNP),而是针对两个非同源标记。我们通过将mxKASP应用于蝾螈的平衡致死系统来证明其概念,在该系统中,已知个体要么具有:(1)1号染色体1A版本的零拷贝和1B版本的两个拷贝;(2)1A的一个拷贝和1B的一个拷贝;要么(3)1A的两个拷贝和1B的零拷贝。mxKASP能够在单个反应中成功扩增1A和1B标记(如果存在),从而推断个体的纯合性。我们用多重PCR方法独立验证了mxKASP的结果。我们认为mxKASP可用于快速、经济地确定二倍体生物中大量样本的纯合性。