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Waterlogging is a growing threat to wheat production in high-rainfall areas. In this study, a doubled haploid (DH) population developed from a cross between Yangmai 16 (waterlogging-tolerant) and Zhongmai 895 (waterlogging-sensitive) was used to map quantitative trait loci (QTL) for waterlogging tolerance using a high-density 660K single-nucleotide polymorphism (SNP) array. Two experimental designs, waterlogging concrete tank (CT) and waterlogging plastic tank (PT), were used to simulate waterlogging during anthesis in five environments across three growing seasons. Waterlogging significantly decreased thousand-kernel weight (TKW) relative to non-waterlogged controls, although the degree varied across lines. Three QTL for waterlogging tolerance were identified on chromosomes 4AL, 5AS, and 7DL in at least two environments. All favorable alleles were contributed by the waterlogging-tolerant parent Yangmai 16. QWTC.caas-4AL exhibited pleiotropic effects on both enhancing waterlogging tolerance and decreasing plant height. Six high-confidence genes were annotated within the QTL interval. The combined effects of QWTC.caas-4AL and QWTC.caas-5AS greatly improved waterlogging tolerance, while the combined effects of all three identified QTL (QWTC.caas-4AL, QWTC.caas-5AS, and QWTC.caas-7DL) exhibited the most significant effect on waterlogging tolerance. Breeder-friendly kompetitive allele-specific PCR (KASP) markers (K_AX_111523809, K_AX_108971224, and K_AX_110553316) flanking the interval of QWTC.caas-4AL, QWTC.caas-5AS, and QWTC.caas-7DL were produced. These markers were tested in a collection of 240 wheat accessions, and three superior polymorphisms of the markers distributed over 67 elite cultivars in the test population, from the Chinese provinces of Jiangsu, Anhui, and Hubei. The three KASP markers could be used for marker-assisted selection (MAS) to improve waterlogging tolerance in wheat.
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