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

Area of Science:

• Plant genetics
• Molecular biology
• Biotechnology

Background:

• Accurate plant genotyping is crucial for breeding and research.
• Traditional methods can be labor-intensive and require high-purity DNA.
• Need for high-throughput, specific genotyping adaptable to various sample types.

Purpose of the Study:

• To present a robust protocol for probe-based quantitative polymerase chain reaction (qPCR) genotyping.
• To enable male-specific genotype identification in plant samples.
• To demonstrate the method's efficacy with various sample types, including crude extracts.

Main Methods:

• DNA isolation from raw tissue or FTA cards.
• Preamplification enrichment reaction of isolated DNA.
• Probe-based quantitative polymerase chain reaction (qPCR) for genotype detection.

Main Results:

• Successful male-specific genotype identification in plant DNA.
• High specificity achieved for genotyping various sample types.
• Method is robust even with crudely prepared nucleic acid extracts.

Conclusions:

• The described probe-based qPCR protocol is a high-throughput and specific method for plant genotyping.
• It is effective for male-specific genotype identification using diverse DNA sources.
• The protocol's adaptability to crude extracts enhances its practical application in plant science.