TP-ARMS: A Cost-Effective PCR-Based Genotyping System for Precision Breeding of Small InDels in Crops

Accurate genotyping of small insertions and deletions (InDels; <5 bp) remains technically challenging in routine molecular breeding, largely due to the limited resolution of agarose gel electrophoresis and the labor-intensive nature of polyacrylamide-based assays. Here, we present the Tri-Primer Amplification Refractory Mutation System (TP-ARMS), a simple and cost-effective PCR-based strategy that enables high-resolution genotyping of small InDels using standard agarose gels. The TP-ARMS employs a universal reverse primer in combination with two allele-specific forward primers targeting insertion and deletion alleles, respectively. This design allows clear discrimination of homozygous and heterozygous genotypes using a two-tube PCR workflow. The method showed complete concordance with Sanger sequencing in detecting 1-5 bp InDels across multiple crop species, including rice (Oryza sativa) and quinoa (Chenopodium quinoa). In addition, using a TP-ARMS reduced experimental time by approximately 90% compared with PAGE-based approaches and avoided the high equipment and DNA quality requirements of fluorescence-based assays. The practical applicability of the TP-ARMS was demonstrated in breeding populations, including efficient genotyping of a 3-bp InDel in OsNRAMP5 associated with cadmium accumulation and a 6-bp promoter InDel in OsSPL10 underlying natural variation in rice trichome density across 370 accessions. Collectively, the TP-ARMS provides a robust, scalable, and low-cost solution for precise small InDel genotyping, with broad applicability in marker-assisted breeding and functional genetic studies.