Haplotype-based nested association mapping for net form net blotch response in Australian barley

Haplotype-based nested association mapping conducted in Australian barley identified 30 haploblocks for resistance/susceptibility to net form of net blotch. Stacking multiple resistance haplotypes significantly reduced disease severity. Net form net blotch (NFNB), caused by Pyrenophora teres f. teres, is a major fungal disease affecting barley, leading to significant yield losses globally. Improving the sustainability of barley production requires identifying genetic sources that confer effective resistance across genetic backgrounds and diverse environments. Considerable progress has been made through mapping studies conducted internationally, but many have largely focused on unadapted germplasm such as landraces or used foreign pathotypes. To support breeding outcomes in Australia, characterization of resistance alleles that are effective against local pathotypes is critical, along with investigating resistances that may already be present in breeding germplasm as this could reduce the time to deployment of resistant cultivars. In this study, we applied the local genomic estimated breeding value approach to perform haplotype mapping using a multi-reference parent nested association mapping (MR-NAM) population developed using resistant breeding lines as donor parents. The population was evaluated across three seasons in Queensland to investigate the genetic architecture of NFNB resistance and genotype-by-environment interactions. We identified 30 haploblocks, with seven representing novel genomic regions not previously associated with NFNB resistance. Three haploblocks on chromosomes 2H, 6H, and 7H were consistently associated with resistance across all environments, suggesting that they likely harbor stable resistance loci. Haplotype stacking analysis demonstrated that combining multiple resistance haplotypes significantly reduced disease severity, with lines carrying 6-10 resistance haplotypes showing progressive improvements in NFNB resistance. The distribution and effectiveness of these key haplotypes across environments, and in diverse genetic backgrounds adapted to Australia (Commander, Compass and La Trobe), highlight the potential to harness them in ongoing pre-breeding and breeding programs.