Dissecting the genetic foundation of heterosis for rice complex traits using GWAS, TWAS and mGWAS

Heterosis has been extensively studied and plays an important role in agricultural production. However, heterosis genes are very limited. Therefore, we conducted genome-wide, metabolome-based, and transcriptome-wide association studies (GWAS, TWAS, and mGWAS); quantitative trait locus (QTL) and expression QTL (eQTL) mapping; and bulked segregant analysis. We used these methods to identify heterosis genes through four heterosis indicators, metabolites, and gene expression levels in 210 recombinant inbred lines (RILs) and 278 RIL-derived immortalized F 2 (IMF 2 ) individuals. After indirect validation in 1439 rice hybrids, we identified five candidate genes for general combining ability (GCA) via GWAS, TWAS, mGWAS in RILs, as well as seven candidate genes for specific combining ability (SCA), mid-parent heterosis (MPH) and better-parent heterosis (BPH) via GWAS and eQTL mapping in IMF 2 , where GCA-based OsPHR2 and OsSYT-5 were identified in two environments. The reported BPH gene OsDPE1 and the known grain weight gene OsMYB3 were found to be significantly associated with all four heterosis metrics. eQTL networks, genotype-stratified GWAS, and haplotype analysis revealed a three-gene ( Ghd7, DTH7 , and OsSPL17 ) core combination that regulates yield per plant and grains per panicle coordinately. A multi-omics model for genomic selection in fivefold cross-validation showed high accuracy of 0.90 and predicted that 3,112 hybrids (14.18%) would outyield the elite hybrid Shanyou 63. A strong positive correlation was observed between no. of superior heterosis locus genotypes and the four heterosis metrics. This study not only provides novel insights into the genetic basis of heterosis but also delivers practical genomic resources.