Soybean seed isoflavones are a type of secondary metabolites that can provide health and nutrition benefits for humans. In our previous study, a stable quantitative trait locus (QTL) qIF05-1 controlling the seed isoflavone content in soybean was detected on chromosome (Chr.) 05 in a recombinant inbred line (RIL) population from a cross of Huachun 2×Wayao. In this study, the parental lines were re-sequenced using the Illumina Solexa System with deep coverage. A total of 63,099 polymorphic long insertions and deletions (InDels) (≥15 bp) were identified between the parents Huachun 2 and Wayao. The InDels were unevenly distributed on 20 chromosomes of soybean, varying from 1,826 in Chr. 12 to 4,544 in Chr. 18. A total of 10,002 long InDels (15.85% of total) were located in genic regions, including 1,139 large-effect long InDels which resulted in truncated or elongated protein sequences. In the qIF05-1 region, 68 long InDels were detected between the two parents. Using a progeny recombination experiment and genotype analysis, the qIF05-1 locus was mapped into a 102.2 kb genomic region, and this region contained 12 genes. By RNA-seq data analysis, genome sequence comparison and functional validation through ectopic expression in Arabidopsis thaliana, Glyma.05G208300 (described as GmEGL3), which is a basic helix-loop-helix (bHLH) transcription factor in plants, emerged as the most likely confirmed gene in qIF05-1. These long InDels can be used as a type of complementary genetic method for QTL fine mapping, and they can facilitate genetic studies and molecular-assisted selection breeding in soybean.
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The productivity of acid soil crops is severely impacted by the limited availability of phosphorus. Soybean (Glycine max) is an important grain and oil crop, known for its preference for phosphorus. Phosphorus deficiency significantly affect both the yield and quality of soybean. While the NAC (NAM, ATAF1/2, CUC2) transcription factor family has been recognized for its involvement in regulating plant responses to various biotic and abiotic stresses, its role in soybean under low phosphorus stress remains largely unexplored. In this study, we focused on the low-phosphorus-tolerant wild soybean variety BW69 as our material, with the objective of cloning and analyzing the expression patterns and functions of the low-phosphorus-tolerant gene GsNAC1. This investigation lays the foundation for a deeper understanding the mechanisms behind the regulation of GsNAC1 response to low phosphorus stress.
The full-length sequence of GsNAC1 was cloned from BW69, and the characteristics of its encoded amino acid sequence were explored by bioinformatics analysis. In addition, the tissue expression patterns of GsNAC1 were examined through quantitative real-time PCR (qRT-PCR). The subcellular localization of GsNAC1 was observed using laser confocal microscopy. Furthermore, soybean genetic transformation experiments were conducted for further phenotype analysis, and RNA-seq analysis was performed to identify differentially expressed genes (DEGs) related to low phosphorus stress.
The GsNAC1 gene was successfully cloned, with a full-length coding region of 876 bp. Phylogenetic analysis showed a 62.46% sequence similarity between GsNAC1 and AtATAF1, and no difference was observed with the GmNAC1 sequence from the Williams 82 reference genome. Subcellular localization experiments further revealed that GsNAC1 was localized in the nucleus. Using qRT-PCR, it was discovered that GsNAC1 is expressed in roots, stems, leaves, apes, flowers and pods, with the highest relative expression level found in the roots. Notably, GsNAC1 exhibited significant upregulation in response to low pH and low phosphorus conditions. To assess the phenotypic effects, we performed experiments using both hydroponic and soil cultivation methods under low phosphorus conditions. The transgenic lines showed notable increases in root/shoot ratio, total root length, root surface area, root volume, and phosphorus content compared to the wild type (WT). Transcriptome analysis revealed that GsNAC1 may enhance tolerance to low phosphorus stress by promoting the expression of genes such as GmALMT6, GmALMT27, GmPAP27, and GmWRKY21.
The expression of GsNAC1 was up-regulated by low pH and low phosphorus, and overexpression of GsNAC1 significantly enhanced the tolerance to low phosphorus stress in soybean, playing a promoting role in the response to low phosphorus stress. Besides, GsNAC1 may enhance the tolerance to low phosphorus stress in soybean by regulating the expression of downstream genes.
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Powdery mildew (PM), caused by the fungus Microsphaera diffusa, causes severe yield losses in soybean [Glycine max (L.) Merr.] under suitable environmental conditions. Identifying resistance genes and developing resistant cultivars may prevent soybean PM damage. In this study, analysis of F1, F2, and F8:11 recombinant inbred line (RIL) populations derived from the cross between Zhonghuang 24 (ZH24) and Huaxia 3 (HX3) indicated that adult-plant resistance (APR) to powdery mildew in the soybean cultivar (cv.) ZH24 was controlled by a single dominant locus. A high-density genetic linkage map of the RIL population was used for fine mapping. The APR locus in ZH24 was mapped to a 281-kb genomic region on chromosome 16. Using 283 susceptible plants of another F2 population, the candidate region was fine-mapped to a 32.8-kb genomic interval flanked by the markers InDel14 and Gm16_428. The interval harbored five genes, including four disease resistance (R)-like genes, according to the Williams 82.a2.v1 reference genome. Quantitative real-time PCR assays of candidate genes revealed that the expression levels of Glyma.16g214300 and Glyma.16g214500 were changed by M. diffusa infection and might be involved in disease defense. Rmd_B13 showed all-stage resistance (ASR) to PM in soybean cv. B13. An allelism test in the F2 segregating population from the cross of ZH24 × B13 suggested that the APR locus Rmd_ZH24 and the ASR locus Rmd_B13 may be allelic or tightly linked. These results provide a reference marker-assisted selection in breeding programs.
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