The High-Amylose Maize Line GEMS-0067 Is Associated with an Intron 8 Insertion in Sbe1a, Altered Native PAGE Zymogram Patterns, and Development of PCR Markers for Breeding Applications

The high-amylose maize (Zea mays L.) germplasm line GEMS-0067 carries the recessive amylose-extender (ae/sbe2b) allele together with modifier loci that elevate starch amylose content from approximately 55% to more than 70%. Previous quantitative trait locus (QTL) studies associated a major component of this modifier effect with the Sbe1a region on chromosome 5S; however, the underlying DNA polymorphism and practical breeding markers remained unresolved. To identify candidate variation, a targeted genomic amplicon-scanning strategy was applied across coding and non-coding regions of Sbe1a. Screening with 23 gene-specific primer pairs identified a unique fragment-length polymorphism spanning the intron 8-exon 9 boundary. Sequencing revealed a 6 bp TACATA insertion together with a downstream 2 bp GA insertion within intron 8. The polymorphism was present in GEMS-0067 and its ancestral source Guatemala 209 but absent from 33 surveyed USDA Germplasm Enhancement of Maize (GEM) releases. Native PAGE zymogram analysis identified a distinct starch-metabolizing enzyme activity band in the putative SBEI region that displayed altered migration in GEMS-0067 relative to reference genotypes. A PCR-CAPS marker based on the insertion was developed using MslI digestion and enabled clear codominant discrimination of homozygous and heterozygous individuals. These results establish a novel gene-based marker associated with the GEMS-0067 high-amylose modifier background and provide a practical tool for marker-assisted breeding.