| Summary: | Soybean seeds are primary sources of natural tocopherols used by the food and pharmaceutical industries, owing to their beneficial impacts on human health. Selection for higher tocopherol contents in seeds along with other desirable traits is an important goal in soybean breeding. In order to identify the genomic loci and candidate genes controlling tocopherol content in soybean seeds, the bulked-segregant analysis technique was performed using a natural population of soybean consisting of 1525 accessions. We constructed the bulked-segregant analysis based on 98 soybean accessions that showed extreme phenotypic variation for the target trait, consisting of 49 accessions with extremely-high and 49 accessions with extremely-low tocopherol content. A total of 144 variant sites and 109 predicted genes related to tocopherol content were identified, in which a total of 83 genes were annotated by the gene ontology functions. Furthermore, 13 enriched terms (<i>p</i> < 0.05) were detected, with four of them found to be highly enriched: response to lipid, response to abscisic acid, transition metal ion transmembrane transporter activity, and double-stranded DNA binding. Especially, six candidate genes were detected at 41.8–41.9 Mb genomic hotspots on chromosome 5 based on ANNOtate VARiation analysis. Among the genes, only <i>Glyma.05G243400</i> carried a non-synonymous mutation that encodes a “translation elongation factor EF1A or initiation factor IF2gamma family protein” was identified. The haplotype analysis confirmed that <i>Glyma.05G243400</i> exhibited highly significant variations in terms of tocopherol content across multiple experimental locations, suggesting that it can be the key candidate gene regulating soybean seed tocopherols. The present findings provide novel gene resources related to seed tocopherols for further validation by genome editing, functional characterization, and genetic improvement targeting enhanced tocopherol composition in soybean molecular breeding.
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