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.

Classification and Expression Profile of the U-Box E3 Ubiquitin Ligase Enzyme Gene Family in Maize (Zea mays L.).

The U-box E3 (PUB) family genes encode the E3 ubiquitin ligase enzyme, which determines substrate specific recognition during protein ubiquitination. They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristic of PUB gene family in the important staple crop, maize (Zea mays L.). In this study, the PUB gene in maize was aimed to identify and classify through whole-genome screening. Phylogenetic tree, gene structure, conserved motif, chromosome location, gene duplication (GD), synteny, and cis-acting regulatory element of PUB member were analyzed. The expression profiles of ZmPUB gene family in maize during development and under abiotic stress and hormones treatment were analyzed by the RNA-seq data. A total of 79 PUB genes were identified in maize genome, and they were stratified into seven categories. There were 25 pairs of segmental duplications (SD) and 1 pair of tandem duplication (TD) identified in the maize PUB gene family. A close relationship was observed between the monocot plant maize and rice in PUB gene family. There were 94 kinds of cis-acting elements identified in the maize PUB gene family, which included 46 biotic- and abiotic-responsive elements, 19 hormone-responsive elements, 13 metabolic and growth-related elements. The expression profiles of maize PUB gene family showed characteristics of tissue specificity and response to abiotic stress and hormones treatment. These results provided an extensive overview of the maize PUB gene family.

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.

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.

Genome-wide association study of four yield-related traits at the R6 stage in soybean.

BACKGROUND: The 100-pod fresh weight (PFW), 100-seed fresh weight (SFW), 100-seed dry weight (SDW) and moisture content of fresh seeds (MCFS) at the R6 stage are crucial factors for vegetable soybean yield. However, the genetic basis of yield at the R6 stage remains largely ambiguous in soybean. RESULTS: To better understand the molecular mechanism underlying yield, we investigated four yield-related traits of 133 soybean landraces in two consecutive years and conducted a genome-wide association study (GWAS) using 82,187 single nucleotide polymorphisms (SNPs). The GWAS results revealed a total of 14, 15, 63 and 48 SNPs for PFW, SFW, SDW and MCFS, respectively. Among these markers, 35 SNPs were repeatedly identified in all evaluated environments (2015, 2016, and the average across the two years), and most co-localized with yield-related QTLs identified in previous studies. AX-90496773 and AX-90460290 were large-effect markers for PFW and MCFS, respectively. The two markers were stably identified in all environments and tagged to linkage disequilibrium (LD) blocks. Six potential candidate genes were predicted in LD blocks; five of them showed significantly different expression levels between the extreme materials with large PFW or MCFS variation at the seed development stage. Therefore, the five genes Glyma.16g018200, Glyma.16g018300, Glyma.05g243400, Glyma.05g244100 and Glyma.05g245300 were regarded as candidate genes associated with PFW and MCFS. CONCLUSION: These results provide useful information for the development of functional markers and exploration of candidate genes in vegetable soybean high-yield breeding programs.

Metabolomics Analysis of Soybean Hypocotyls in Response to Phytophthora sojae Infection.

Soybean is one of the most important economic and oil crops across the world. Phytophthora root rot (PRR), caused by Phytophthora sojae (P. sojae), is a major disease in most soybean-growing regions worldwide. Here, we investigated metabolic changes in hypocotyls of two soybean lines, Nannong 10-1 (resistant line, R) and 06-070583 (susceptible line, S), at two time points (12 and 36 hpi) after P. sojae infection and metabolic differences between the R line and the S line. In total, 90 differentially accumulated metabolites (DAMs) were identified after P. sojae infection; the levels of 50 metabolites differed between the R line and the S line. There are 28 DAMs that not only differentially accumulated between the R line and the S line but also differentially accumulated after P. sojae infection. Based on the changes of these DAMs in response to P. sojae infection in different lines and at different timepoints, and the differences in the contents of these DAMs between the R line and the S line, we speculated that DAMs, including sugars (monosaccharides and oligosaccharides), organic acids (oxalic acid, cumic acid), amino acid derivatives, and other secondary metabolites (mannitol, octanal, hypoxanthine, and daidzein etc.) may participate in the metabolic-level defense response of soybean to P. sojae. In this study, an integrated pathway-level analysis of transcriptomics (obtained by RNA-Seq) and metabolomics data illustrated the poor connections and interdependencies between the metabolic and transcriptional responses of soybean to P. sojae infection. This work emphasizes the value of metabolomic studies of plant-pathogen interactions and paves the way for future research of critical metabolic determinants of the soybean-P. sojae interaction.

Transcriptomics Analysis of Apple Leaves in Response to Alternaria alternata Apple Pathotype Infection.

Alternaria blotch disease of apple (Malus × domestica Borkh.), caused by the apple pathotype of Alternaria alternata, is one of the most serious fungal diseases to affect apples. To develop an understanding of how apples respond to A. alternata apple pathotype (AAAP) infection, we examined the host transcript accumulation over the period between 0 and 72 h post AAAP inoculation. Large-scale gene expression analysis was conducted of the compatible interaction between "Starking Delicious" apple cultivar and AAAP using RNA-Seq and digital gene expression (DGE) profiling methods. Our results show that a total of 9080 differentially expressed genes (DEGs) were detected (>two-fold and FDR < 0.001) by RNA-Seq. During the early phase of infection, 12 h post inoculation (HPI), AAAP exhibited limited fungal development and little change in the transcript accumulation status (950 DEGs). During the intermediate phase of infection, the period between 18 and 36 HPI, increased fungal development, active infection, and increased transcript accumulation were detected (4111 and 3838 DEGs detected at each time point, respectively). The majority of DEGs were detected by 72 HPI, suggesting that this is an important time point in the response of apples' AAAP infection. Subsequent gene ontology (GO) and pathway enrichment analyses showed that DEGs are predominately involved in biological processes and metabolic pathways; results showed that almost gene associated with photosynthesis, oxidation-reduction were down-regulated, while transcription factors (i.e., WRKY, MYB, NAC, and Hsf) and DEGs involved in cell wall modification, defense signaling, the synthesis of defense-related metabolites, including pathogenesis-related (PRs) genes and phenylpropanoid/cyanoamino acid /flavonoid biosynthesis, were activated during this process. Our study also suggested that the cell wall defensive vulnerability and the down-regulation of most PRs and HSP70s in "Starking Delicious" following AAAP infection might interpret its susceptible to AAAP.

A two-photon fluorescent turn-on probe for imaging of SO2 derivatives in living cells and tissues.

SO2 and its derivatives (bisulfite/sulfite) play crucial roles in several physiological processes. Therefore, development of reliable analytical methods for monitoring SO2 and its derivatives in biological systems is very significant. In this paper, a FRET-based two-photon fluorescent turn-on probe, A-HCy, was proposed for specific detection of SO2 derivatives through the bisulfite/sulfite-promoted Michael addition reaction. In this FRET system, an acedan (2-acetyl-6-dialkylaminonaphthalene) moiety was selected as a two-photon donor and a hemicyanine derivative served as both the quencher and the recognition unit for bisulfite/sulfite. A-HCy exhibited excellent selectivity and rapid response to HSO3(-) with a detection limit of 0.24 µM. More importantly, probe A-HCy was first successfully applied in two-photon fluorescence imaging of biological SO2 derivatives in living cells and tissues, suggesting its great potential for practical application in biological systems.