Scenario: Let’s say you are starting a small breeding program that does not justify large investments. You already have access to SNP markers that are widely validated in publications and used by competitors. What is the cheapest and most practical way to start genotyping?

The answer lies in using equipment you likely already have: a standard real-time PCR machine. Compared to modular, high-throughput SNP detection platforms, a real-time PCR setup is much more affordable, flexible, and easy to implement in small breeding programs.

Two practical options for small breeding programs — both compatible with a real-time PCR machine:
1️⃣ HRM (High Resolution Melting)
HRM is a SNP genotyping method that detects tiny DNA differences by looking at how the DNA separates when heated (this is demonstrated by the melting curve).
After PCR amplification, small sequence differences cause subtle changes in the melting curve, allowing you to distinguish alleles.

Advantages of HRM:
Cost-effective — requires only primers and a DNA dye, cheaper than KASPAR
Fast results — typically 1–2 hours per run
Excellent for validating a few key SNP trait markers
Ideal for screening a limited number of breeding lines
Limitations of HRM:
❌ Less suitable for large numbers of markers or samples
❌ Requires careful primer design to avoid ambiguous melting curves
❌ Cannot multiplex many SNPs in one reaction

2️⃣ KASP (Kompetitive Allele-Specific PCR)
KASP is a fluorescence-based SNP genotyping method using allele-specific primers. It allows robust discrimination between alleles and is widely used in plant breeding.
Advantages of KASP:
✔ Highly robust and reproducible
✔ Scalable to hundreds of samples and multiple SNPs
✔ Flexible for markers that are difficult to assay with HRM
✔ Can also run on a real-time PCR machine, avoiding the need for expensive modular equipment.

Key message: Both HRM and KASP can be run on a standard real-time PCR machine, making them ideal for small breeding programs. Start with HRM to validate your SNPs cost-effectively, and scale to KASP as your program grows to handle more samples or markers, gaining techical reproducibility and flexibility.

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How to cost-effectively genotype a few trait SNPs when running a small breeding program on a budget?