Genetic and Phenotypic Characterization of Soybean Landraces Collected from the Zhejiang Province in China.

The soybean is an important feed, industrial raw material, and food crop in the world due to its rich components. There is a long history of soybean cultivation with different types and rich resources in the Zhejiang province of China. It is important to understand genetic diversity as well as phenotypic variation for soybean breeding. The objective of this study was to analyze both genetic and phenotypic characteristics of the 78 soybean landraces collected, and to explore a potential advantage of germplasm resources for further application. These 78 autumn-type soybean landraces have been propagated, identified, and evaluated in both 2021 and 2022. There were agronomic, quality, and genetic variations according to the comprehensive analyses. There was a good consistency between seed size and seed coat color. There were significant differences of seed protein, fat, and sugar contents based upon the seed coat color. These soybean landraces were genotyped using 42 simple sequence repeat markers and then clustered into two groups. The two groups had a consistency with the seed coat color. This study gave us a combined understanding of both the phenotypic variation and the genetic diversity of the soybean landraces. Therefore, the reasonable crossing between different soybean types is highly recommended.

Genome-Wide Comparison of Structural Variations and Transposon Alterations in Soybean Cultivars Induced by Spaceflight.

Space mutation causes genetic and phenotypic changes in biological materials. Transposon activation is an adaptive mechanism for organisms to cope with changes in the external environment, such as space mutation. Although transposon alterations have been widely reported in diverse plant species, few studies have assessed the global transposon alterations in plants exposed to the space environment. In this study, for the first time, the effects of transposon alterations in soybean caused by space mutation were considered. A new vegetable soybean variety, 'Zhexian 9' (Z9), derived from space mutation treatment of 'Taiwan 75' (T75), was genetically analyzed. Comparative analyses of these two soybean genomes uncovered surprising structural differences, especially with respect to translocation breakends, deletions, and inversions. In total, 12,028 structural variations (SVs) and 29,063 transposable elements (TEs) between T75 and Z9 were detected. In addition, 1336 potential genes were variable between T75 and Z9 in terms of SVs and TEs. These differential genes were enriched in functions such as defense response, cell wall-related processes, epigenetics, auxin metabolism and transport, signal transduction, and especially methylation, which implied that regulation of epigenetic mechanisms and TE activity are important in the space environment. These results are helpful for understanding the role of TEs in response to the space environment and provide a theoretical basis for the selection of wild plant materials suitable for space breeding.

Transcriptomic and Metabolomic Analyses Reveal a Potential Mechanism to Improve Soybean Resistance to Anthracnose.

Anthracnose, caused by Colletotrichum truncatum, leads to large-scale reduction in quality and yield in soybean production. Limited information is available regarding the molecular mechanisms of resistance to anthracnose in soybean. We conducted a transcriptomic and targeted metabolomic analysis of pods from two soybean lines, "Zhechun No. 3" (ZC3) and ZC-2, in response to C. truncatum infection. Factors contributing to the enhanced resistance of ZC-2 to anthracnose compared with that of ZC3, included signal transduction (jasmonic acid, auxin, mitogen-activated protein kinase, and Ca2+ signaling), transcription factors (WRKY and bHLH), resistance genes (PTI1, RPP13, RGA2, RPS6, and ULP2B), pathogenesis-related genes (chitinase and lipid transfer protein), and terpenoid metabolism. Targeted metabolomic analysis revealed that terpenoid metabolism responded more promptly and more intensely to C. truncatum infection in ZC-2 than in ZC3. In vitro antifungal activity and resistance induction test confirmed that jasmonic acid, auxin signaling and terpenoids played important roles in soybean resistance to anthracnose. This research is the first study to explore the molecular mechanisms of soybean resistance to anthracnose. The findings are important for in-depth analysis of molecular resistance mechanisms, discovery of resistance genes, and to expedite the breeding of anthracnose-resistant soybean cultivars.

A Sequence Variation in GmBADH2 Enhances Soybean Aroma and Is a Functional Marker for Improving Soybean Flavor.

The vegetable soybean (Glycine max L. Merr.) plant is commonly consumed in Southeast Asian countries because of its nutritional value and desirable taste. A "pandan-like" aroma is an important value-added quality trait that is rarely found in commercial vegetable soybean varieties. In this study, three novel aromatic soybean cultivars with a fragrant volatile compound were isolated. We confirmed that the aroma of these cultivars is due to the potent volatile compound 2-acetyl-1-pyrroline (2AP) that was previously identified in soybean. A sequence comparison of GmBADH1/2 (encoding an aminoaldehyde dehydrogenase) between aromatic and non-aromatic soybean varieties revealed a mutation with 10 SNPs and an 11-nucleotide deletion in exon 1 of GmBADH2 in Quxian No. 1 and Xiangdou. Additionally, a 2-bp deletion was detected in exon 10 of GmBADH2 in ZK1754. The mutations resulted in a frame shift and the introduction of premature stop codons. Moreover, genetic analyses indicated that the aromatic trait in these three varieties was inherited according to a single recessive gene model. These results suggested that a mutated GmBADH2 may be responsible for the aroma of these three aromatic soybean cultivars. The expression and function of GmBADH2 in aromatic soybean seeds were confirmed by qRT-PCR and CRISPR/Cas9. A functional marker developed on the basis of the mutated GmBADH2 sequence in Quxian No. 1 and Xiangdou was validated in an F2 population. A perfect association between the marker genotypes and aroma phenotypes implied that GmBADH2 is a major aroma-conferring gene. The results of this study are potentially useful for an in-depth analysis of the molecular basis of 2-AP formation in soybean and the marker-assisted breeding of aromatic vegetable soybean cultivars.

Development and Application of an In Vitro Method to Evaluate Anthracnose Resistance in Soybean Germplasm.

Anthracnose caused by Colletotrichum truncatum is a major fungal disease of soybean, especially vegetable soybean (edamame). Studies of this disease have mainly focused on resistance evaluation, but the primary methods used-in vivo inoculation of pods or plants under greenhouse or field conditions-have limitations with respect to accuracy, stability, scale, and environmental safety. In this study, we developed a method for inoculating pods in vitro by soaking in a mycelial suspension. We optimized the crucial components, including the mycelial suspension concentration (40 to 60 mg mL-1), the maturity of the sampled pods (15 days after flowering), and the post-inoculation incubation period (5 days). Application of the mycelial suspension by soaking rather than spraying improved the efficiency of inoculation and made large-scale evaluation possible. Using this method, we evaluated 589 soybean germplasm resources (275 cultivars, 233 landraces, and 81 wild accessions). We identified 25 highly resistant cultivars, 11 highly resistant landraces, but only one highly resistant wild accession. Our results will aid future research on soybean anthracnose resistance, including gene discovery, the elucidation of molecular mechanisms, and the breeding of resistant cultivars.

Comparative Analysis and Development of a Flavor Fingerprint for Volatile Compounds of Vegetable Soybean Seeds Based on Headspace-Gas Chromatography-Ion Mobility Spectrometry.

Evaluating the volatile compounds and characteristic fingerprints of the core cultivars of vegetable soybean would provide useful data for improving their aroma in the breeding programs. The present study used headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) to evaluate the volatile compounds of vegetable soybean seeds at a specific growth stage. In total, 93 signal peaks were identified, 63 compounds qualitatively, with 14 volatile flavor compounds providing multiple signals. The 63 volatile compounds consisted of 15 esters, 15 aldehydes, 13 alcohols, 15 ketones, one acid, and four other compounds. The peak intensity of most of the volatile compounds varied greatly between the core cultivars. The alcohols and aldehydes determined the basic volatile flavor of the vegetable soybean seeds. Volatile flavors were determined by their respective esters, ketones, or other components. Characteristic fingerprints were found in some core vegetable soybean cultivars. Four cultivars (Xiangdou, ZHE1754, Zhexian 65018-33, and Qvxian No. 1) had pleasant aromas, because of their higher content of 2-acetyl-1-pyrroline (2-AP). A principal component analysis (PCA) was used to distinguish the samples based on the signal intensity of their volatile components. The results showed that the composition and concentration of volatile compounds differed greatly between the core cultivars, with the volatile flavor compounds of soybeans being determined by the ecotype of the cultivar, the direction of breeding selection, and their geographical origin. Characteristic fingerprints of the cultivars were established by HS-GC-IMS, enabling them to be used to describe and distinguish cultivars and their offspring in future breeding studies.

Transcriptome analysis identifies differentially expressed genes in the progenies of a cross between two low phytic acid soybean mutants.

Phytic acid (PA) is a major antinutrient that cannot be digested by monogastric animals, but it can decrease the bioavailability of micronutrients (e.g., Zn and Fe). Lowering the PA content of crop seeds will lead to enhanced nutritional traits. Low-PA mutant crop lines carrying more than one mutated gene (lpa) have lower PA contents than mutants with a single lpa mutant gene. However, little is known about the link between PA pathway intermediates and downstream regulatory activities following the mutation of these genes in soybean. Consequently, we performed a comparative transcriptome analysis using an advanced generation recombinant inbred line with low PA levels [2mlpa (mips1/ipk1)] and a sibling line with homozygous non-mutant alleles and normal PA contents [2MWT (MIPS1/IPK1)]. An RNA sequencing analysis of five seed developmental stages revealed 7945 differentially expressed genes (DEGs) between the 2mlpa and 2MWT seeds. Moreover, 3316 DEGs were associated with 128 metabolic and signal transduction pathways and 4980 DEGs were annotated with 345 Gene Ontology terms related to biological processes. Genes associated with PA metabolism, photosynthesis, starch and sucrose metabolism, and defense mechanisms were among the DEGs in 2mlpa. Of these genes, 36 contributed to PA metabolism, including 22 genes possibly mediating the low-PA phenotype of 2mlpa. The expression of most of the genes associated with photosynthesis (81 of 117) was down-regulated in 2mlpa at the late seed developmental stage. In contrast, the expression of three genes involved in sucrose metabolism was up-regulated at the late seed developmental stage, which might explain the high sucrose content of 2mlpa soybeans. Furthermore, 604 genes related to defense mechanisms were differentially expressed between 2mlpa and 2MWT. In this study, we detected a low PA content as well as changes to multiple metabolites in the 2mlpa mutant. These results may help elucidate the regulation of metabolic events in 2mlpa. Many genes involved in PA metabolism may contribute to the substantial decrease in the PA content and the moderate accumulation of InsP3-InsP5 in the 2mlpa mutant. The other regulated genes related to photosynthesis, starch and sucrose metabolism, and defense mechanisms may provide additional insights into the nutritional and agronomic performance of 2mlpa seeds.

Genome-Wide Variation Analysis of Four Vegetable Soybean Cultivars Based on Re-Sequencing.

Vegetable soybean is a type of value-added specialty soybean, served as a fresh vegetable or snack in China. Due to the difference from other types, it is important to understand the genetic structure and diversity of vegetable soybean for further utilization in breeding programs. The four vegetable cultivars, Taiwan-75, Zhexiandou No. 8, Zhexian No. 9 and Zhexian No. 10 are popular soybean varieties planted in Zhejiang province, and have large pods and intermediate maturity. The clustering showed a close relationship of these four cultivars in simple sequence repeat analysis. To reveal the genome variation of vegetable soybean, these four improved lines were analyzed by whole-genome re-sequencing. The average sequencing depth was 7X and the coverage ratio of each cultivar was at least more than 94%. Compared with the reference genome, a large number of single-nucleotide polymorphisms, insertion/deletions and structure variations were identified with different chromosome distributions. The average heterozygosity rate of the single-nucleotide polymorphisms was 11.99% of these four cultivars. According to the enrichment analysis, there were 23,371 genes identified with putative modifications, and a total of 282 genes were related to carbohydrate metabolic processes. These results provide useful information for genetic research and future breeding, which can facilitate the selection procedures in vegetable soybean breeding.

Quantitative proteomic analyses of two soybean low phytic acid mutants to identify the genes associated with seed field emergence.

BACKGROUND: Seed germination is essential to crop growth and development, and ultimately affects its harvest. It is difficult to breed soybeans low in phytic acid with a higher seed field emergence. Although additional management and selection could overcome the phytate reduction, the mechanisms of seed germination remain unknown. RESULTS: A comparative proteomic analysis was conducted between two low phytic acid (LPA) soybean mutants (TW-1-M and TW-1), both of which had a deletion of 2 bp in the GmMIPS1 gene. However, the TW-1 seeds showed a significantly lower field emergence compared to the TW-1-M. There were 282 differentially accumulated proteins (DAPs) identified between two mutants at the three stages. Among these DAPs, 80 were down-accumulated and 202 were up-accumulated. Bioinformatic analysis showed that the identified proteins were related to functional categories of oxidation reduction, response to stimulus and stress, dormancy and germination processes and catalytic activity. KEGG analysis showed that these DAPs were mainly involved in energy metabolism and anti-stress pathways. Based upon the conjoint analysis of DAPs with the differentially expressed genes (DEGs) previously published among three germination stages in two LPA mutants, 30 shared DAPs/DEGs were identified with different patterns, including plant seed protein, beta-amylase, protein disulfide-isomerase, disease resistance protein, pyrophosphate-fructose 6-phosphate 1-phosphotransferase, cysteine proteinase inhibitor, non-specific lipid-transfer protein, phosphoenolpyruvate carboxylase and acyl-coenzyme A oxidase. CONCLUSIONS: Seed germination is a very complex process in LPA soybean mutants. The TW-1-M and TW-1 showed many DAPs involved in seed germination. The differential accumulation of these proteins could result in the difference of seed field emergence between the two mutants. The high germination rate in the TW-1-M might be strongly attributed to reactive oxygen species-related and plant hormone-related genes. All these findings would help us further explore the germination mechanisms in LPA crops.

Salt-Responsive Transcriptome Profiling of Suaeda glauca via RNA Sequencing.

BACKGROUND: Suaeda glauca, a succulent halophyte of the Chenopodiaceae family, is widely distributed in coastal areas of China. Suaeda glauca is highly resistant to salt and alkali stresses. In the present study, the salt-responsive transcriptome of Suaeda glauca was analyzed to identify genes involved in salt tolerance and study halophilic mechanisms in this halophyte. RESULTS: Illumina HiSeq 2500 was used to sequence cDNA libraries from salt-treated and control samples with three replicates each treatment. De novo assembly of the six transcriptomes identified 75,445 unigenes. A total of 23,901 (31.68%) unigenes were annotated. Compared with transcriptomes from the three salt-treated and three salt-free samples, 231 differentially expressed genes (DEGs) were detected (including 130 up-regulated genes and 101 down-regulated genes), and 195 unigenes were functionally annotated. Based on the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) classifications of the DEGs, more attention should be paid to transcripts associated with signal transduction, transporters, the cell wall and growth, defense metabolism and transcription factors involved in salt tolerance. CONCLUSIONS: This report provides a genome-wide transcriptional analysis of a halophyte, Suaeda glauca, under salt stress. Further studies of the genetic basis of salt tolerance in halophytes are warranted.

The acid phosphatase-encoding gene GmACP1 contributes to soybean tolerance to low-phosphorus stress.

Phosphorus (P) is essential for all living cells and organisms, and low-P stress is a major factor constraining plant growth and yield worldwide. In plants, P efficiency is a complex quantitative trait involving multiple genes, and the mechanisms underlying P efficiency are largely unknown. Combining linkage analysis, genome-wide and candidate-gene association analyses, and plant transformation, we identified a soybean gene related to P efficiency, determined its favorable haplotypes and developed valuable functional markers. First, six major genomic regions associated with P efficiency were detected by performing genome-wide associations (GWAs) in various environments. A highly significant region located on chromosome 8, qPE8, was identified by both GWAs and linkage mapping and explained 41% of the phenotypic variation. Then, a regional mapping study was performed with 40 surrounding markers in 192 diverse soybean accessions. A strongly associated haplotype (P = 10(-7)) consisting of the markers Sat_233 and BARC-039899-07603 was identified, and qPE8 was located in a region of approximately 250 kb, which contained a candidate gene GmACP1 that encoded an acid phosphatase. GmACP1 overexpression in soybean hairy roots increased P efficiency by 11-20% relative to the control. A candidate-gene association analysis indicated that six natural GmACP1 polymorphisms explained 33% of the phenotypic variation. The favorable alleles and haplotypes of GmACP1 associated with increased transcript expression correlated with higher enzyme activity. The discovery of the optimal haplotype of GmACP1 will now enable the accurate selection of soybeans with higher P efficiencies and improve our understanding of the molecular mechanisms underlying P efficiency in plants.

GmNAC5, a NAC transcription factor, is a transient response regulator induced by abiotic stress in soybean.

GmNAC5 is a member of NAM subfamily belonging to NAC transcription factors in soybean (Glycine max (L.) Merr.). Studies on NAC transcription factors have shown that this family functioned in the regulation of shoot apical meristem (SAM), hormone signalling, and stress responses. In this study, we examined the expression levels of GmNAC5. GmNAC5 was highly expressed in the roots and immature seeds, especially strongly in immature seeds of 40 days after flowering. In addition, we found that GmNAC5 was induced by mechanical wounding, high salinity, and cold treatments but was not induced by abscisic acid (ABA). The subcellular localization assay suggested that GmNAC5 was targeted at nucleus. Together, it was suggested that GmNAC5 might be involved in seed development and abiotic stress responses in soybean.

Neuroprotection by the soy isoflavone, genistein, via inhibition of mitochondria-dependent apoptosis pathways and reactive oxygen induced-NF-κB activation in a cerebral ischemia mouse model.

Recently, the treatment of stroke has focused on antioxidant therapies, where oxidative stress is implicated. The preventive and therapeutic potential of plant compounds on ischemic stroke has been intensively studied because many of them contain antioxidant properties. Genistein, one of the active ingredients in soybean, possesses many bioactivities. In this study, we investigated the potential neuroprotective effects of genistein and its possible mechanism of action in a cerebral ischemia mouse model. Mice were pretreated with genistein (2.5, 5, and 10mg/kg) or vehicle orally once daily for 14 consecutive days before transient middle cerebral artery occlusion was performed. Genistein at doses of 2.5-10mg/kg significantly reduced the infarct volume, improved the neurological deficit and prevented cell apoptosis after ischemia. In addition, genistein pretreatment was shown to inhibit the ischemia-induced reactive oxygen species (ROS) production, enhance the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), and decrease levels of malondialdehyde (MDA) in stroke mice. Moreover, genistein reversed the mitochondria dysfunction after ischemia, as evidenced by decreasing mitochondria ROS levels, preventing cytochrome C release to the cytoplasm and inhibiting caspase-3 activation. Western blotting showed ischemia activated the ROS-dependent nuclear factor-κB (NF-κB) signaling pathway, and genistein suppressed phosphorylation and activation of the NF-κB p65 subunit, as well as the phosphorylation and degradation of the inhibitor protein of κBα (IκBα). Our findings suggested that genistein has a neuroprotective effect in transient focal ischemia, which may involve regulation of mitochondria-dependent apoptosis pathways and suppression of ROS-induced NF-κB activation.

[Transgenic technology and soybean quality improvement].

Soybean is an important source of edible oil, protein and protein diet. The breeding process of high quality soybean can be accelerated via employment of transgenic technology, by which the key genes for soybean quality traits could be directly manipulated. Thus, various soybean varieties could be bred to fulfill different needs for specific consumers. Here, we reviewed the contribution of transgenic technology to improvement of soybean qualities in recent years. We also introduce some newly developed safe transgenic technologies and hope this information could relieve some concerns on the GM food.