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The complex quantitative nature of drought-related traits is a major constraint to breed tolerant wheat varieties. Pairs of near-isogenic lines (NILs) with a common genetic background but differing in a particular locus could turn quantitative traits into a Mendelian factor facilitating our understanding of genotype and phenotype interactions. In this study, we report our fast track development and evaluation of NILs from C306 × Dharwar Dry targeting a wheat 4BS QTL hotspot in C306, which confers drought tolerance following the heterogeneous inbreed family (HIF) analysis coupled with immature embryo culture-based fast generation technique. Molecular marker screening and phenotyping for grain yield and related traits under post-anthesis water stress (WS) confirmed four isoline pairs, viz., qDSI.4B.1-2, qDSI.4B.1-3, qDSI.4B.1-6, and qDSI.4B.1-8. There were significant contrasts of responses between the NILs with C306 QTL (+NILs) and the NILs without C306 QTL (−NILs). Among the four confirmed NIL pairs, mean grain yield per plant of the +NILs and −NILs showed significant differences ranging from 9.61 to 10.81 and 6.30 to 7.56 g, respectively, under WS condition, whereas a similar grain yield was recorded between the +NILs and −NILs under well-watered condition. Isolines of +NIL and −NIL pairs showed similar chlorophyll content (SPAD), assimilation rate (A), and transpiration rate (Tr) at the beginning of the stress. However, the +NILs showed significantly higher SPAD (12%), A (66%), stomatal conductance (75%), and Tr (97%) than the −NILs at the seventh day of stress. Quantitative RT-PCR analysis targeting the MYB transcription factor gene  Triticum aestivum  MYB 82 ( TaMYB 82), within this genomic region which was retrieved from the wheat reference genome TGACv1, also revealed differential expression in +NILs and –NILs under stress. These results confirmed that the NILs can be invaluable resources for fine mapping of this QTL, and also for cloning and functional characterization of the gene(s) responsible for drought tolerance in wheat.

Multiple Near-Isogenic Lines Targeting a QTL Hotspot of Drought Tolerance Showed Contrasting Performance Under Post-anthesis Water Stress