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• YS is an emerging physiological disorder negatively affecting lettuce production. • YS reduces photosynthesis and disrupts homeostasis of chemical elements. • YS is a heritable trait which manifestation is affected by environmental conditions. • YS-linked molecular markers can facilitate development of YS-resistant lines. Lettuce (Lactuca sativa L.) is an important leafy vegetable. An unknown malady that causes yellow spot (YS) blemishes on leaves has recently appeared in commercial fields in California, potentially reducing lettuce yield and/or quality. YS occurred on all morphological types of cultivated lettuce regardless of their color, with the highest (0.9) and lowest (0.09) rate in non-leaf (stem and oilseed) and leaf lettuce, respectively. The spots ranging in diameter from 0.5 to 3.1 mm (depending on genotype) were mostly observed on the adaxial leaf surface of mature or nearly mature plants. YS symptoms began as light-yellow circular spots that later became more intensely yellow and eventually turned necrotic. The YS affected area displayed decreased chlorophyll fluorescence and photosynthesis that were not caused by the effect of fungi, bacteria, viruses, insects, or foliar nematodes. Among 31 tested chemical elements, four macronutrients (P, Ca, Mg, and S) and eight microminerals (B, Cd, Cu, Li, Mn, Ni, Si, and Sn) had significantly changed concentration in the YS affected leaves. The trait exhibited a relatively high broad-sense inheritability (H2 = 47.9% ∼ 68.4%) in four experiments conducted at two locations in three years. The genome-wide association study (GWAS) involving 479 accessions revealed the effect of eight loci (qYS2.1, qYS3.1, qYS3.2, qYS4.1, qYS5.1, qYS5.2, qYS6.1 , and qYS7.1) defined by 10 significant SNP markers. A total of 162 genes were predicted in the identified chromosomal regions, with four oxidation-reduction (redox) catalyzers being potentially the most promising candidate genes involved in the regulation of this physiological disorder. [ABSTRACT FROM AUTHOR]