Unravelling Novel Genetic Determinants of Anaerobic Germination Tolerance in Tamil Nadu Rice Landraces via GWAS

ABSTRACT While direct‐seeded rice (DSR) offers a sustainable and resource‐efficient alternative to conventional puddled transplanting, its widespread adoption is severely constrained by the susceptibility of elite cultivars to anaerobic stress during germination, leading to poor crop establishment and substantial yield losses. To address this critical agricultural bottleneck, we conducted a comprehensive genetic evaluation of 96 rice genotypes, predominantly comprising traditional landraces from Tamil Nadu, India, to identify superior genetic donors and elucidate the molecular architecture underlying anaerobic germination (AG) tolerance. Phenotypic screening under controlled anaerobic conditions identified shoot and coleoptile elongation as key adaptive traits conferring survival advantage, with landraces Kudavazhai and Poongar demonstrating exceptional AG tolerance and representing invaluable genetic resources for future breeding initiatives. Genomewide association studies (GWASs) employing a high‐density 50 K SNP array revealed 50 significant marker–trait associations distributed across 21 linkage disequilibrium (LD) blocks spanning 11 chromosomes. The reliability of these associations is strongly supported by extensive colocalization with previously characterized quantitative trait loci (QTLs) for AG and related stress tolerance traits, including the major QTL qAG9 on chromosome 9, as well as QTLs controlling coleoptile length ( qCL6b ), mesocotyl development and root architectural traits ( QRSA‐4 , QRV‐4 ). These genomic regions harbour well‐characterized AG‐tolerance genes, most notably OsTPP7 , which facilitates gibberellic acid‐independent starch mobilization, along with genes encoding alcohol dehydrogenase (ADH) and the metallothionein OsMT2B , collectively confirming their essential roles in anaerobic metabolic adaptation. Our investigation revealed an extensive repertoire of novel candidate genes critical for hypoxic survival, including energy metabolism regulators (ADP‐glucose pyrophosphorylase, fructose‐6‐phosphate‐2‐kinase and beta‐glucosidase), hormone biosynthesis and signalling components (ACC synthase mediating ethylene production, galactinol synthase governing raffinose family oligosaccharide synthesis), cellular stress signalling and detoxification systems (calmodulin‐like proteins, calcineurin B‐like proteins orchestrating Ca 2+ ‐mediated responses, glutathione homeostasis regulators and superoxide dismutase) and polyamine biosynthesis enzymes (S‐adenosylmethionine decarboxylase). Haplotype analysis of critical LD blocks (1.1, 5.1 and 10.1) revealed functionally significant allelic variations that directly correlate with phenotypic performance. Notably, specific haplotypes of the calcineurin B‐like protein gene ( Os10g0564800 ) within LD block 10.1 exhibited strong correlation with differential shoot elongation capacity, establishing its function as a molecular switch in Ca 2+ ‐mediated stress response cascades involving the calcium‐dependent protein kinase CIPK15. These findings reinforce the complex polygenic architecture of AG tolerance, which is orchestrated through coordinated molecular networks integrating anaerobic fermentation pathways, carbohydrate mobilization systems, reactive oxygen species detoxification mechanisms and hormonal regulatory circuits. Through this integrated approach combining GWAS mapping, comprehensive candidate gene identification and functional haplotype analysis, we provide a detailed molecular framework to understand anaerobic germination tolerance, identify elite germplasm resources with superior adaptive capacity and deliver specific allelic targets to accelerate precision breeding of flood‐resilient, high‐yielding DSR varieties for sustainable rice production systems.