The number of four-seed pods and 100-grain weight are important yield components in soybean. Typically, they are negatively correlated traits. Generally, soybean varieties with a high 100-grain weight and a higher number of three-or four-seed pods are more likely to obtain a high yield. It is difficult to select for double-excellent traits that meet the needs of farmers and the market using conventional breeding methods.The purpose of this study was to mine genes associated with the number of fourseed pods and 100-grain weight in soybean. Whole-transcriptome sequencing was performed on four specific chromosome segment substitution lines (HWMN, HWFN, LWMN and LWFN) combined with the distribution of blocks imported from wild soybean DNA fragments and gene annotation. The material was sampled in each of the eight development stages. Among them, globular embryo formation, heart embryo formation, and cotyledon primordium formation were used to mine differentially expressed genes (DEGs). A total of 3792 DEGs were identified, and 25 expression patterns were obtained by the K-means rapid clustering method. GO enrichment analysis of DEGs was performed by Agrigo, and a total of 43 GO terms were enriched. Through annotation analysis, 126 DEGs associated with seed size and number were obtained. Combined with analysis of the introduced DNA fragments of wild soybean ZYD00006, 19 genes that eventually aligned on those blocks were obtained.Six representative genes were selected as candidate genes, namely,
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