Dissecting Heterotic Potential in Ethiopian Barley (Hordeum vulgare L.) using Transcriptomics Approaches

Barley (Hordeum vulgare L.) is a staple cereal crop with significant economic and nutritional importance, yet its productivity is often limited by various abiotic and biotic stresses. In the face of climate change and rapid population growth, there is a pressing need for plant breeding technologies that can substantially increase crop yields, including heterosis. Heterosis, or hybrid vigor, refers to the phenomenon where offspring from two genetically distinct parents outperform both parents in traits such as yield, yield components, and resistance to abiotic and biotic stresses. Despite its importance, the molecular mechanisms underlying heterosis in crops remain unclear. This study was designed to (i) develop F₁ hybrids from Ethiopian barley germplasm and assess their phenotypic performance on the field, (ii) assess the level of heterosis in the F₁ hybrids relative to their parental lines, (iii) identify differentially expressed genes between the F₁ hybrids and their homozygous parents, and (iv) characterize DEG for additive or non-additive expression patterns and their functions. We developed F1 hybrids from 15 genetically diverse Ethiopian barley lines by using a simple nested association mapping (NAM) mating design. Both the F1 hybrids and their parental lines were evaluated under field conditions, and six phenotypic traits were collected: days to 50% flowering, plant height, spike length, number of seeds per spike, awn length and thousand seed weight. Total RNA was extracted from leaves of both the F1 hybrids and their parental lines at the fourth leaf stage, and mRNA was then isolated from the total RNA and sequenced using Oxford Nanopore Technologies (ONT) to enable transcriptomic analysis. Phenotypic data analysis revealed significant differences between the F1 hybrids and their parental lines for all traits, except awn length. Estimates of heterosis in F1 hybrids relative to their parents showed both positive and negative mid-parent heterosis across the evaluated traits. The highest mid-parent heterosis was observed in the F1 hybrid 64065a × HB-1307 (35.84%) for number of seeds per spike, while the lowest was recorded in the F1 hybrid 64068a × HB-1307 (-2.62%) for days flowering. Genetic correlation analysis between parental genetic distances and mid-parent heterosis showed positive and significant associations for spike length and thousand seed weight. Transcriptomic analysis of the long-read RNA-seq revealed many differentially expressed genes (DEGs) between the F1 hybrid and their homozygous parental lines; most of differentially expressed genes were up-regulated in most of F1 hybrids as compared to their homozygous parent, and non-additive gene expression pattern was prevalent. Gene Ontology (GO) enrichment analysis of the DEG revealed significant enrichment of terms related to stress responses in the F₁ hybrids. Our results provide useful phenotypic and transcriptome resources for further understanding the molecular basis of heterosis in barley and related crops.