Fluorescence-based absent allele-specific amplification (FAASA) for high-throughput detection of absent alleles

Authors: RUNNING, KATHERINE - North Dakota State University; SENEVIRATNE, SUDESHI - North Dakota State University; Zhang, Zengcui; SINGH, GURMINDER - North Dakota State University; Fiedler, Jason; Faris, Justin

Submitted to: Bio-protocol
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: Molecular markers are snippets of DNA fragments associated with specific genes that can be used to monitor the presence and movement of the target genes from parents to offspring. Plant breeders use molecular markers to ensure that selected lines contain the target genes for desirable traits to make their varieties highly productive. Sometimes, it is necessary to remove a gene entirely if it poses a detrimental effect such as disease susceptibility. However, it can be difficult to determine the absence of a gene in a molecular assay, because it cannot always be distinguished from a failed experiment. Here, we developed a unique molecular method called fluorescence-based absent allele-specific amplification (FAASA). The method employs the monitoring of an endogenous plant gene, which is always present, to ensure the validity of the marker assay for detection of the target gene. This allows the user to distinguish between the results of a failed experiment and the absence of the target gene, which is the desired outcome. The FAASA method will allow plant breeders to quickly and efficiently eliminate disease susceptibility genes from elite lines for variety production.

Technical Abstract: In wheat and other crops, some genes display presence/absence variation, and it is occasionally beneficial to select for the absent allele to remove a functional gene. However, current high-throughput genotyping methods used to detect the absence of genes tend to be inconsistent and inconclusive. Kompetitive allele-specific PCR (KASP) and PCR allele competitive extension (PACE) are two well-established methods for allele-specific polymerase chain reaction (AS-PCR) assays, each using fluorescence resonance energy transfer (FRET) to generate a signal for each allele, typically targeting biallelic single nucleotide polymorphisms. KASP and PACE methods are more difficult to apply to alleles with presence/absence variation because the lack of amplification of the absent allele is indistinguishable from a failed PCR. Here, we present a multiplex fluorescence-based absent allele-specific amplification (FAASA) method using the PACE marker system (compatible with KASP markers) to detect absent alleles using a primer mix consisting of one target-specific primer pair (TSP) and one core gene-specific primer pair (CGSP). The forward primer of each pair is tagged with a 5’ terminal tail complementary to dye-labeled oligonucleotides in commercially available FRET cassettes. Lines that amplify only the core gene do not carry the target gene while lines that amplify both the core gene and the target carry alleles of both the core gene and target. The inclusion of the CGSPs allows researchers to confidently distinguish lines with absent alleles of the target from lines with failed PCR reactions, which can happen due to various reasons, including inadequate DNA quality or quantity.