Seed size and shape are important traits determining yield and quality in soybean. However, the genetic mechanism and genes underlying these traits remain largely unexplored. In this regard, this study used two related recombinant inbred line (RIL) populations (ZY and K3N) evaluated in multiple environments to identify main and epistatic-effect quantitative trait loci (QTLs) for six seed size and shape traits in soybean. A total of 88 and 48 QTLs were detected through composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively, and 15 QTLs were common among both methods; two of them were major (R² > 10%) and novel QTLs (viz., qSW-1-1_ZN and qSLT-20-1_K3N). Additionally, 51 and 27 QTLs were identified for the first time through CIM and MCIM methods, respectively. Colocalization of QTLs occurred in four major QTL hotspots/clusters, viz., “QTL Hotspot A”, “QTL Hotspot B”, “QTL Hotspot C”, and “QTL Hotspot D” located on Chr06, Chr10, Chr13, and Chr20, respectively. Based on gene annotation, gene ontology (GO) enrichment, and RNA-Seq analysis, 23 genes within four “QTL Hotspots” were predicted as possible candidates, regulating soybean seed size and shape. Network analyses demonstrated that 15 QTLs showed significant additive x environment (AE) effects, and 16 pairs of QTLs showing epistatic effects were also detected. However, except three epistatic QTLs, viz., qSL-13-3_ZY, qSL-13-4_ZY, and qSW-13-4_ZY, all the remaining QTLs depicted no main effects. Hence, the present study is a detailed and comprehensive investigation uncovering the genetic basis of seed size and shape in soybeans. The use of a high-density map identified new genomic regions providing valuable information and could be the primary target for further fine mapping, candidate gene identification, and marker-assisted breeding (MAB).

Seed-flooding stress is one of the major abiotic constraints severely affecting soybean yield and quality. Understanding the molecular mechanism and genetic basis underlying seed-flooding tolerance will be of greatly importance in soybean breeding. However, very limited information is available about the genetic basis of seed-flooding tolerance in soybean. The present study performed Genome-Wide Association Study (GWAS) to identify the quantitative trait nucleotides (QTNs) associated with three seed-flooding tolerance related traits, viz., germination rate (GR), normal seedling rate (NSR) and electric conductivity (EC), using a panel of 347 soybean lines and the genotypic data of 60,109 SNPs with MAF > 0.05. A total of 25 and 21 QTNs associated with all three traits were identified via mixed linear model (MLM) and multi-locus random-SNP-effect mixed linear model (mrMLM) in three different environments (JP14, HY15, and Combined). Among these QTNs, three major QTNs, viz., QTN13, qNSR-10 and qEC-7-2, were identified through both methods MLM and mrMLM. Interestingly, QTN13 located on Chr.13 has been consistently identified to be associated with all three studied traits in both methods and multiple environments. Within the 1.0 Mb physical interval surrounding the QTN13, nine candidate genes were screened for their involvement in seed-flooding tolerance based on gene annotation information and available literature. Based on the qRT-PCR and sequence analysis, only one gene designated as GmSFT (Glyma.13g248000) displayed significantly higher expression level in all tolerant genotypes compared to sensitive ones under flooding treatment, as well as revealed nonsynonymous mutation in tolerant genotypes, leading to amino acid change in the protein. Additionally, subcellular localization showed that GmSFT was localized in the nucleus and cell membrane. Hence, GmSFT was considered as the most likely candidate gene for seed-flooding tolerance in soybean. In conclusion, the findings of the present study not only increase our knowledge of the genetic control of seed-flooding tolerance in soybean, but will also be of great utility in marker-assisted selection and gene cloning to elucidate the mechanisms of seed-flooding tolerance.

In plants, lesion mimic mutants (LMMs) reveal spontaneous disease-like lesions in the absence of pathogen that constitutes powerful genetic material to unravel genes underlying programmed cell death (PCD), particularly the hypersensitive response (HR). However, only a few LMMs are reported in soybean, and no related gene has been cloned until now. In the present study, we isolated a new LMM named spotted leaf-1 (spl-1) from NN1138-2 cultivar through ethyl methanesulfonate (EMS) treatment. The present study revealed that lesion formation might result from PCD and excessive reactive oxygen species (ROS) accumulation. The chlorophyll content was significantly reduced but antioxidant activities, viz., superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the malondialdehyde (MDA) contents, were detected higher in spl-1 than in the wild-type. According to segregation analysis of mutant phenotype in two genetic populations, viz., W82×spl-1 and PI378692×spl-1, the spotted leaf phenotype of spl-1 is controlled by a single recessive gene named lm1. The lm1 locus governing mutant phenotype of spl-1 was first identified in 3.15 Mb genomic region on chromosome 04 through MutMap analysis, which was further verified and fine mapped by simple sequence repeat (SSR) marker-based genetic mapping. Genetic linkage analysis narrowed the genomic region (lm1 locus) for mutant phenotype to a physical distance of ~76.23 kb. By searching against the Phytozome database, eight annotated candidate genes were found within the lm1 region. qRT-PCR expression analysis revealed that, among these eight genes, only Glyma.04g242300 showed highly significant expression levels in wild-type relative to the spl-1 mutant. However, sequencing data of the CDS region showed no nucleotide difference between spl-1 and its wild type within the coding regions of these genes but might be in the non-coding regions such as 5′ or 3′ UTR. Hence, the data of the present study are in favor of Glyma.04g242300 being the possible candidate genes regulating the mutant phenotype of spl-1. However, further validation is needed to prove this function of the gene as well as its role in PCD, which in turn would be helpful to understand the mechanism and pathways involved in HR disease resistance of soybean.

Soybean (Glycine max L.) is the most important legume and oilseed crop. As a leguminous crop, it plays an irreplaceable role towards the sustainable agricultural system with biological nitrogen fixation. However, its production can be dramatically decreased by the occurrence of water stress. Water stress including drought and flooding induces the morpho-physiological and biochemical changes at different growth stages, which negatively affects the adaptability and yield of soybean. Genetic diversity that ensures productivity in challenging environment exists within germplasm, their wild relatives and species that are adapted to the water stress. The discovery of gene mapping, QTLs associated with root traits, slow canopy wilting, nitrogen fixation and flooding tolerance have accomplished significant progress in breeding programs. Identification of drought-responsive genes and transcription factors such as WRKY, DREBs, ERFs, ZIP, ZFP, MYB and NAC are valuable to ameliorate the water stress in soybean. Understanding the genetic mechanism using transcriptomic and proteomic approaches would be the ultimate choice for mitigating the water stress. Integration of well-designed soybean breeding program coupled with omic techniques would pave the way for developing drought and flooding resilient soybean cultivars.

Waterlogging is one of the key abiotic factors that severely impedes the growth and productivity of soybeans on a global scale. To develop soybean cultivars that are tolerant to waterlogging, it is a prerequisite to unravel the mechanisms governing soybean responses to waterlogging. Hence, we explored the morphological, physiological, biochemical, and transcriptional changes in two contrasting soybean introgression lines, A192 (waterlogging tolerant, WT) and A186 (waterlogging sensitive, WS), under waterlogging. In comparison to the WT line, waterlogging drastically decreased the root length (RL), shoot length (ShL), root fresh weight (RFW), shoot fresh weight (ShFW), root dry weight (RDW), and shoot dry weight (ShDW) of the WS line. Similarly, waterlogging inhibited soybean plant growth by suppressing the plant’s photosynthetic capacity, enhancing oxidative damage from reactive oxygen species, and decreasing the chlorophyll content in the WS line but not in the WT line. To counteract the oxidative damage and lipid peroxidation, the WT line exhibited increased activity of antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), as well as higher levels of proline content than the WS line. In addition, the expression of antioxidant enzyme genes (POD1, POD2, FeSOD, Cu/ZnSOD, CAT1, and CAT2) and ethylene-related genes (such as ACO1, ACO2, ACS1, and ACS2) were found to be up-regulated in WT line under waterlogging stress conditions. In contrast, these genes showed a down-regulation in their expression levels in the stressed WS line. The integration of morpho-physiological, biochemical, and gene expression analyses provide a comprehensive understanding of the responses of WT and WS lines to waterlogging conditions. These findings would be beneficial for the future development of soybean cultivars that can withstand waterlogging.

Seed-flooding stress is one of major abiotic constraints that adversely affects soybean production worldwide. Identifying tolerant germplasms and revealing the genetic basis of seed-flooding tolerance are imperative goals for soybean breeding. In the present study, high-density linkage maps of two inter-specific recombinant inbred line (RIL) populations, named NJIRNP and NJIR4P, were utilized to identify major quantitative trait loci (QTLs) for seed-flooding tolerance using three parameters viz., germination rate (GR), normal seedling rate (NSR), and electrical conductivity (EC). A total of 25 and 18 QTLs were detected by composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively, and 12 common QTLs were identified through both methods. All favorable alleles for the tolerance are notably from the wild soybean parent. Moreover, four digenic epistatic QTL pairs were identified, and three of them showed no main effects. In addition, the pigmented soybean genotypes exhibited high seed-flooding tolerance compared with yellow seed coat genotypes in both populations. Moreover, out of five identified QTLs, one major region containing multiple QTLs associated with all three traits was identified on Chromosome 8, and most of the QTLs within this hotspot were major loci (R² > 10) and detectable in both populations and multiple environments. Based on the gene expression and functional annotation information, 10 candidate genes from QTL “hotspot 8-2” were screened for further analysis. Furthermore, the results of qRT-PCR and sequence analysis revealed that only one gene, GmDREB2 (Glyma.08G137600), was significantly induced under flooding stress and displayed a TTC tribasic insertion mutation of the nucleotide sequence in the tolerant wild parent (PI342618B). GmDREB2 encodes an ERF transcription factor, and the subcellular localization analysis using green fluorescent protein (GFP) revealed that GmDREB2 protein was localized in the nucleus and plasma membrane. Furthermore, overexpression of GmDREB2 significantly promoted the growth of soybean hairy roots, which might indicate its critical role in seed-flooding stress. Thus, GmDREB2 was considered as the most possible candidate gene for seed-flooding tolerance.