'QTL-hotspot' is a genomic region on linkage group 04 (CaLG04) in chickpea (Cicer arietinum) that harbours major-effect quantitative trait loci (QTLs) for multiple drought-adaptive traits, and it therefore represents a promising target for improving drought adaptation. To investigate the mechanisms underpinning the positive effects of 'QTL-hotspot' on seed yield under drought, we introgressed this region from the ICC 4958 genotype into five elite chickpea cultivars. The resulting introgression lines (ILs) and their parents were evaluated in multi-location field trials and semi-controlled conditions. The results showed that the 'QTL-hotspot' region improved seed yield under rainfed conditions by increasing seed weight, reducing the time to flowering, regulating traits related to canopy growth and early vigour, and enhancing transpiration efficiency. Whole-genome sequencing data analysis of the ILs and parents revealed four genes underlying the 'QTL-hotspot' region associated with drought adaptation. We validated diagnostic KASP markers closely linked to these genes using the ILs and their parents for future deployment in chickpea breeding programs. The CaTIFY4b-H2 haplotype of a potential candidate gene CaTIFY4b was identified as the superior haplotype for 100-seed weight. The candidate genes and superior haplotypes identified in this study have the potential to serve as direct targets for genetic manipulation and selection for chickpea improvement.

Centre for Crop and Food Innovation State Agricultural Biotechnology Centre Murdoch University Murdoch Western Australia Australia

Centre of Excellence in Genomics and Systems Biology International Crops Research Institute for the Semi Arid Tropics Hyderabad India

Crops Physiology and Modeling International Crops Research Institute for the Semi Arid Tropics Hyderabad India

Department of Crop Physiology University of Agricultural Sciences Bengaluru India

Department of Genetics Osmania University Hyderabad India

Department of Information Technologies Faculty of Economics and Management Czech University of Life Sciences Prague Kamýcká 129 Prague Czech Republic

ICAR Indian Agricultural Research Institute Delhi India

ICAR Indian Institute of Pulses Research Kanpur India

Khalifa Center for Genetic Engineering and Biotechnology United Arab Emirates University Al Ain United Arab Emirates

Queensland Alliance for Agriculture and Food Innovation The University of Queensland Australia

RAK College of Agriculture Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya Gwalior India

The UWA Institute of Agriculture The University of Western Australia Perth Western Australia Australia

UAS Dharwad Regional Agricultural Research Station Vijayapura India

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