Probe-Based qPCR Workflow Using Fast Nucleic Acid Sampling and Molecular Genotyping in a Dioecious Plant.

This chapter presents a protocol for probe-based quantitative polymerase chain reaction (qPCR) genotyping, focusing on male-specific genotype identification in plant samples. DNA is isolated from raw tissue or FTA cards and subjected to a preamplification enrichment reaction. The diluted product is then used as input for qPCR detection, employing specific probes to identify the presence or absence of the target genotype. This robust and high-throughput method offers increased specificity for genotyping various sample types, including crude nucleic acid extracts. An advantage of qPCR is that genotyping can be performed on various sample types, including from crudely prepared nucleic acid extracts used as input with inhibitor-tolerant polymerases. In this workflow, we describe an extraction and probe-based qPCR method used to detect male-specific genotype identification in plant sample DNA extracted from raw tissue or FTA imprinted cards.