A comprehensive evaluation of 515 Korean wheat germplasms, including cultivars, experimental lines, and landraces, was conducted over 2 years under upland field conditions to characterize major agronomic and grain traits. Allelic variation at 13 key functional loci was assessed using Kompetitive Allele-Specific PCR (KASP) and PCR-based markers. The winter-type vrn-A1 with a single copy (CNV=1; 40.2%) advanced heading by approximately 5 days compared to multiple-copy genotypes, and winter-type vrn-B1 (88.5%) advanced heading by 2 days compared to the spring-type. The photoperiod-insensitive alleles Ppd-B1a (5.6%) and Ppd-D1a (76.3%) advanced heading by 3 and 4 days, respectively, with a combined effect of up to 6 days. Semi-dwarfing alleles Rht-B1b and Rht-D1b showed reduced culm lengths of 2.1 cm and 4.7 cm, respectively, and the Rht-B1a/Rht-D1b genotype was 6 cm shorter than Rht-B1a/Rht-D1a. The Pina-D1a/Pinb-D1a genotype had the lowest kernel hardness value (32.2), whereas Pina-D1b/Pinb-D1a had the highest (60.5). The thousand kernel weight ranged from 36.1 mg to 42.5 mg depending on the allelic combinations of TaCwi-A1, TaGW2-6A, and TaSus2-2B. Cultivars and experimental lines were clearly distinguished from landraces based on phenotype-based clustering, with the majority of cultivars (81.6%) and experimental lines (68.3%) grouped into cluster III. In contrast, landraces were predominantly distributed in clusters I (55.1%) and II (29.2%). Random forest analysis identified four genes, Ppd-D1, Pina-D1, Pinb-D1, and WAPO-A1, as major contributors to cluster classification. Cluster III was highly enriched with alleles favorable for earliness (Ppd-D1a, 98.3%) and grain hardness (Pina-D1b and Pinb-D1b, 57.9%). WAPO-A1b, an allele associated with an increased spikelet number per spike, was more frequently observed in clusters I (94.6%) and II (79.1%) than in cluster III (58.4%).

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