Upland cotton is an important allotetraploid crop that provides both natural fiber for the textile industry and edible vegetable oil for the food or feed industry. To better understand the genetic mechanism that regulates the biosynthesis of storage oil in cottonseed, we identified the genes harbored in the major quantitative trait loci/nucleotides (QTLs/QTNs) of kernel oil content (KOC) in cottonseed via both multiple linkage analyses and genome-wide association studies (GWAS). In ‘CCRI70′ RILs, six stable QTLs were simultaneously identified by linkage analysis of CHIP and SLAF-seq strategies. In ‘0-153′ RILs, eight stable QTLs were detected by consensus linkage analysis integrating multiple strategies. In the natural panel, thirteen and eight loci were associated across multiple environments with two algorithms of GWAS. Within the confidence interval of a major common QTL on chromosome 3, six genes were identified as participating in the interaction network highly correlated with cottonseed KOC. Further observations of gene differential expression showed that four of the genes, LtnD, PGK, LPLAT1, and PAH2, formed hub genes and two of them, FER and RAV1, formed the key genes in the interaction network. Sequence variations in the coding regions of LtnD, FER, PGK, LPLAT1, and PAH2 genes may support their regulatory effects on oil accumulation in mature cottonseed. Taken together, clustering of the hub genes in the lipid biosynthesis interaction network provides new insights to understanding the mechanism of fatty acid biosynthesis and TAG assembly and to further genetic improvement projects for the KOC in cottonseeds.
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