Genetic Relatedness and Heterotic Grouping in MRIZP Elite Maize Inbred Lines Using SNP Markers from 25k SNP Array and RNA-seq Data

Knowledge of population structure and genetic relationship among inbred lines is essential for exploiting heterosis in maize breeding programs. This study evaluated the concordance between 25k Illumina® Infinum Maize SNP (Single Nucleotide Polymorphism) Array (25k SNP array)-derived and ribonucleic acid (RNA)-sequencing (seq)-derived markers in estimating genetic relatedness and population structure within the Maize Research Institute “Zemun Polje” (MRIZP) breeding program. A panel of 28 elite MRIZP maize inbred lines, along with two public lines, was analyzed. For the RNA-seq data, three alternative SNP datasets were generated based on heterozygous-site handling (ALL, HOM, and FINAL) to assess their impact on downstream genetic inference. These approaches had distinct effects on clustering resolution and genetic relationship structure. The FINAL dataset, in which heterozygous positions were recoded as missing values and re-filtered, was selected as the most balanced dataset for comparative analyses with 25k SNP array data. Only a limited number of overlapping SNP positions were identified between RNA-seq and 25k SNP array datasets (six in ALL, two in FINAL, and none in HOM), all located within coding regions. Despite this minimal overlap, distance-based analyses revealed partial concordance in genetic relationship patterns and population structure between platforms. Genetic distances estimated from 25k SNP array markers were consistent with pedigree records and provided more informative insights than pedigree data alone. Population structure inferred from 25k SNP array data showed high concordance with previously defined heterotic groups, correctly assigning 29 out of 30 lines (96.67%) to their expected clusters. In contrast, RNA-seq-derived SNPs showed moderate concordance with expected heterotic groupings (56.67%), indicating that transcriptome-derived markers capture part of the underlying breeding structure, but do not fully resolve heterotic group separation. Overall, these results support the 25k SNP array as a robust tool for assessing genetic relatedness and population structure in maize breeding programs, while RNA-seq-derived SNPs provide complementary but less reliable information for routine heterotic assignment.