GO Classification and Enrichment Analysis of DEGs.
In order to evaluate the observed differences in gene expression profiles toward specific functions, we performed gene ontology (GO) analysis using Singular Enrichment Analysis (SEA) tool in agriGO usingGlycine max as reference. A total of 2525 (1001 up-regulated and 1524 down-regulated), 3212 (1030 up-regulated and 2182 down-regulated), 3060 (860 up-regulated and 2200 down-regulated) and 2325 DEGs (496 up-regulated, and 1829 down-regulated) found in DTL, DSL, DSP & DTP, respectively were grouped in one of the three classes: biological processes (BP), cellular components (CC), molecular function (MF)(Fig 4). Moreover, the DEGs with unknown functional annotation (uncharacterized genes) can be considered as novel genes as they were not associated with any GO category (Table S6). Among the DEGs identified in DTL, 106, 24 and 64 were associated with BP, CC and MF terms, respectively (Fig. 4A and Table S2 ), while 107, 27 and 80 DEGs were identified with BP, CC and MF, respectively in case of DSL(Fig. 4B Table S3 ). However, in DTP total of 116 (BP), 71 (MF) and 34 (CC) GO terms were found, and for DSP 94, 76 and 40 GO terms were found for BP, MF and CC, respectively (Fig. 4C & 4D; Table S4 & S5 ). In all four soybean genotypes, a large number of DEGs were related with the terms viz., metabolic process, cellular process, signaling-organism process, catalytic activity, cell, cell part, membrane, membrane part, and binding (Fig. 4). These result suggests vital role of these GO terms in drought tolerance at germination stage in soybean.
To visualize difference of specific GO terms between the DTP vs DSP and DTL vs DSL genotypes, we used up- and down-regulated enriched GO categories (FDR < 0.05) from DTP vs DSP and DTL vs DSL to REVIGO analysis (Table S2-S5 ). GO terms associated with constantly expressed DEGs of DTP vs DSP and DTL vs DSL genotypes for BP, MF, and CC were shown in Fig. 5 & 6 . Scatter plots displayed highly significant GO terms of BP i.e., water transport (GO:0006833), carbohydrate transport (GO:0008643), response to oxidative stress (GO:0006979), hormone-mediated signaling pathway (GO:0009755), Ca+-mediated signaling (GO:0019722), secondary metabolite synthesis (GO:0044550), chaperone-mediated protein folding (GO:0061077), defense response (GO:0006952) and auxin transport (GO:0060918) were upregulated in tolerant genotypes (DTP & DTL)(Fig. 5A & 6A), whereas reverse trend was observed in drought-sensitive genotypes viz., DSP and DTL (Fig. 5A & 6A ). However, significant GO term viz., signal transduction (GO:0007165) was uniquely found in tolerant genotypes. In case of MF, GO terms such as GO:0016209 (antioxidant activity), GO:0009055 (electron carrier activity), GO:0003824 (catalytic activity), GO:0004601 (peroxidase activity), GO:0043169 (cation binding ), GO:0005509 (calcium ion binding), GO:0020037 (heme binding), GO:0043565 (sequence-specific DNA binding), transferase activity-transferring acyl groups (GO:0016746), transcription factor activity (GO:0003700) and GO:0016762 (xyloglucan-xyloglucosyl transferase activity) was highly expressed in the tolerant genotypes but reverse in sensitive genotypes (Fig. 5B & 6B) . In the CC category, plant-type vacuole membrane (GO:0000325), apoplast (GO:0048046), extracellular matrix (GO:0031012), and cell wall (GO:0005618) GO terms were upregulated in tolerant genotypes, whereas the reverse trend was observed in sensitive genotypes(Fig. 5C & 6C) .