Harnessing Rf3/Rf4-Mediated WA-CMS Fertility Restoration for Hybrid Rice Breeding in India's North Eastern Hilly Region: Integrating Fertility Restoration, Heterosis, and Parental Genetic Divergence

Abstract Hybrid rice has driven substantial yield gains across India, yet no hybrid built on north-eastern Indian landraces has reached large-scale cultivation, despite the region's distinct agro-ecology and growing exposure to climate variability. Closing this gap requires restorer lines that combine reliable fertility restoration, adequate genetic diversity from the cytoplasmic male sterile (CMS) parents they will be paired with, and strong combining ability, three requirements rarely evaluated together. Here, 33 candidate restorer lines and 5 CMS lines (IR 68888A, IR 58025A, RTN 10A, RTN 12A, and APMS 6A) were screened for the fertility-restoration genes Rf3 and Rf4 using gene-based/linked markers, profiled for genetic diversity with SSR markers, and evaluated as 165 test crosses in a modified line x tester design. Rf4 was more prevalent than Rf3 , and gene-based markers predicted realised spikelet fertility more reliably than gene-linked markers; applying a standard four-class fertility-restoration scheme classified 26 of the 33 lines (78.8%) as effective restorers (Fully or Partial), though most lines shifted class depending on the specific CMS tester used, showing that restoration is CMS-line-dependent rather than fixed. SSR-based clustering (STRUCTURE, UPGMA, PCoA) consistently resolved two major genetic groups, clearly separating Ratnagiri-derived CMS lines from north-eastern restorer germplasm, but this genetic distance only weakly predicted hybrid yield performance. Line x tester analysis showed non-additive gene action dominated yield and its component traits (σ²sca > σ²gca; dominance variance > additive variance), and combining this with commercial-heterosis screening identified IR 68888A/Sakur Dhan (156.67%), IR 68888A/MTU 1010 (148.57%), and AMPS-6A/DRR Dhan 44 (141.43%) as the top hybrids over the check Shahsarang. Classifying all test crosses by maturity duration showed that six of these eight top-heterosis hybrids were Mid-early (121–130 days) and none were Late, meaning the best-performing hybrids are also best positioned to escape terminal-season stress and enable double-cropping, linking this breeding result directly to climate-resilient food security in the north-eastern hill region