Soybean (Glycine max) germination is highly sensitive to neutral salt stress. Although sodium chloride (NaCl) and sodium sulfate (Na2SO4) co-exist in nature, their distinct phytotoxic mechanisms remain severely under-investigated. In this study, 50 germplasm accessions were systematically screened, identifying R014 as highly salt-tolerant and R120 as highly sensitive. Phenotypic and dynamic antioxidant monitoring (0–72 h) established 48 h as the critical tolerance window, revealing that Na2SO4 induces complex physical damage (crystallization) and osmotic injury, with its ionic toxicity significantly exceeding that induced by NaCl. Crucially, R014 effectively maintained peak activities of antioxidant enzymes (SOD, POD, CAT) to combat these specific stressors. By integrating deep RNA sequencing with weighted gene co-expression network analysis (WGCNA) using 48 h radicle data, significant transcriptomic reprogramming was revealed. WGCNA robustly isolated 35 functional modules, located five key phenotypic clusters, and defined three major hub genes (Glyma.11G101900, Glyma.17G185000, and Glyma.20G247850) that regulate calcium signaling. Verified by qRT-PCR, this study suggests the differential physiological and molecular architectural characteristics between chloride and sulfate toxicities, providing precisely targeted genetic loci for the breeding of salt-tolerant soybean.

Soybean is an important crop for food, oil and feed production in China, and improving its yield is a major national goal. Salt stress severely restricts soybean production. XTH genes participate in plant growth and stress adaptation, yet the functions of most soybean XTH members are unclear. In this study, we cloned the soybean GmXTH-like26 gene previously identified via transcriptome sequencing, and successfully constructed its overexpression vector and CRISPR/Cas9 gene-editing vector. Subcellular localization analysis confirmed that GmXTH-like26 is localized to the cell wall. The gene was transformed into soybean via the Agrobacterium-mediated method. Under 100 mM NaCl stress, the GmXTH-like26-overexpressing lines exhibited markedly enhanced salt tolerance at both germination and seedling stages compared with the control group. Physiological and biochemical assays showed that the overexpression plants had higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), lower malondialdehyde (MDA) content and higher chlorophyll content under salt stress, while the gene-edited lines displayed the opposite trends. These results indicate that GmXTH-like26 improves salt tolerance in soybean by reducing reactive oxygen species accumulation and effectively enhances the resistance of soybean to salt stress.

Phytophthora root and stem infection by Phytophthora sojae is a global and devastating disease of soybeans. Selecting disease-resistant varieties is the most economical and effective measure for controlling this disease. Delving into the disease resistance and defense molecular mechanisms can lay a theoretical foundation for solving this problem. Here, we screened the soybean genome and identified 78 GmDof genes distributed on nineteen chromosomes. Subcellular localization analysis revealed that the majority of GmDof proteins were located in the cell nucleus. Phylogenetic analysis categorized these genes into nine subfamilies. Gene structure analysis showed that all GmDofs contained 0 to 2 introns, and most of them did not have introns. Motif and conserved domain analysis showed that all GmDofs contained a common motif (motif-1) and a typical conserved C2-C2 domain. The prediction of cis-acting elements in promoter regions revealed numerous cis-regulatory elements responsible for stress responses, plant growth and development, plant hormone responses, and light responses. RNA-seq and quantitative real-time PCR results showed that GmDof63 (Glyma.16G145000) was specifically expressed at high levels after P. sojae infection. GmDof63 was strongly induced by SA and ETH treatments. The soybean seedlings overexpressing GmDof63 displayed enhanced resistance to P. sojae infection compared with the wild-type soybean seedlings. Further experiments indicated that the expression levels of pathogenesis-related protein genes PR1a, PR4, PR5a, and PR10 were significantly up-regulated in GmDof63-overexpressing transgenic soybean seedlings. Taken together, these findings reveal the mechanism by which GmDof63 directly or indirectly regulates the expression of PR genes to modulate the soybean response to P. sojae infection.

Salinized soil can significantly hinder soybean growth, leading to a reduction in overall yield. To address this issue, identifying key genes related to salt tolerance in soybeans is essential for improving their resistance to salinity and ensuring sustainable development of soybean production. While current research predominantly focuses on salt tolerance during the seedling stage, there is still a lack of comprehensive studies on the genes involved in salt tolerance during the germination stage. This study established the optimal screening criteria by phenotyping the salt-tolerant variety R063 and the salt-sensitive variety W82 during the germination stage under salt stress. RNA-seq analysis was performed on 24 samples from both varieties at 36 and 48 hours under two different salt concentrations (0 and 150 mM/L NaCl). Differential expression analysis revealed that the salt-tolerant variety R063 exhibited the fewest differentially expressed genes (DEGs) compared to its control after 48 hours of salt stress. A total of 305 DEGs were commonly identified between the salt-tolerant variety R063 and the salt-sensitive variety W82 under salt stress at both time points. Additionally, 187 DEGs were commonly identified between R063 under salt stress and its corresponding control group across the two time points. Gene ontology (GO) enrichment analysis revealed that the differentially expressed genes were significantly enriched in ADP binding, monooxygenase activity, oxidoreductase activity, defense response, and protein phosphorylation signaling pathways. The weighted gene co-expression network analysis (WGCNA) method was employed to identify modules strongly correlated with salt tolerance during soybean germination. Candidate genes associated with soybean sprouting salt tolerance were identified by evaluating the connectivity and expression profiles of genes within these modules. These findings provide a theoretical foundation for further elucidating the molecular mechanisms underlying salt tolerance during soybean germination and present new genetic resources for studying this trait.

Phytophthora root and stem rot caused by Phytophthora sojae (P. sojae) is a globally prevalent oomycete disease. The use of resistant cultivars is an effective and environmentally friendly strategy to manage this disease. It is important to understand the molecular mechanisms underlying the response of Glycine max (soybean) to P. sojae infection. In this study, we demonstrated that an isoflavonoid-specific prenyltransferase gene (GmPT10d, Glyma.10G070300) was significantly upregulated in the soybean cultivar Williams 82 with high resistance to P. sojae infection. Transgenic soybean seedlings overexpressing GmPT10d exhibited enhanced resistance to P. sojae, and those subjected to RNA interference showed increased susceptibility to the pathogen. Yeast-one-hybrid and electrophoretic mobility shift assays revealed that GmARF15 could directly bind to the promoter of GmPT10d. Further analysis of the GmARF15 function showed that transgenic soybean seedlings overexpressing GmARF15 also exhibited enhanced resistance to P. sojae. Transactivation assay, luciferase assay, and qPCR analysis showed that GmARF15 could promote the expression of GmPT10d. Further analysis indicated that elevated salicylic acid levels were associated with increased expression of GmARF15 and GmPT10d. Taken together, these findings reveal a regulatory mechanism by which GmARF15 enhances soybean resistance to P. sojae, potentially by promoting the expression of GmPT10d through the salicylic acid signaling pathway.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors are a plant-specific family and play roles in plant growth, development, and responses to biotic and abiotic stresses. However, little is known about the functions of the TCP transcription factors in the soybean cultivars with tolerance to salt stress. In this study, TCP9-like, a TCP transcription factor, was identified in the soybean cultivars exposed to salt stress. The expression of TCP9-like gene in the roots of salt-tolerant soybean cultivars was higher than that in salt-sensitive cultivars treated with NaCl. The overexpression of TCP9-like enhanced the salt tolerance of the salt-sensitive soybean cultivar 'DN50'. In T2 generation, the plants with TCP9-like overexpression had significantly lower Na+ accumulation and higher K+ accumulation than the WT plants exposed to 200 or 250 mmol/L NaCl. The K+/Na+ ratio in the plants overexpressing TCP9-like was significantly higher than that in WT plants treated with 200 mmol/L NaCl. Meanwhile, the overexpression of TCP9-like up-regulated the expression levels of GmNHX1, GmNHX3, GmSOS1, GmSOS2-like, and GmHKT1, which were involved in the K+/Na+ homeostasis pathway. The findings indicated that TCP9-like mediated the regulation of both Na+ and K+ accumulation to improve the tolerance of soybean to salt stress.

Soybean (Glycine max (Linn.) Merr.) annual leguminous crop is cultivated all over the world. The occurrence of diseases has a great impact on the yield and quality of soybean. In this study, based on the RNA-seq of soybean variety M18, a complete CDS (Coding sequence) GmPR1L of the pathogenesis-related protein 1 family was obtained, which has the ability to resist fungal diseases. The overexpression vector and interference expression vector were transferred into tobacco NC89, and the resistance of transgenic tobacco (Nicotiana tabacum L.) to Botrytis cinerea infection was identified. The results show that: Compared with the control, the activities of related defense enzymes SOD (Superoxide dismutase), POD (Peroxidase), PAL (L-phenylalanine ammonia-lyase) and PPO (Polyphenol oxidase) in the over-expressed transgenic tobacco OEA1 and OEA2 increased to different degrees, and increased significantly at different infection time points. The activities of defense enzymes in the interfering strains IEA1 and IEA2 were significantly lower than those in the control strains. The results of resis-tance level identification showed that the disease spot rate of OEA1 was significantly lower than that of the control line, and the disease spot rate of OEA2 was significantly lower than that of the control line. The plaque rate of the interfering expression line IEA1-IEA2 was significantly higher than that of the control line. It is preliminarily believed that the process related protein GmPR1L can improve the resistance of tobacco to B. cinerea.

Polyamines play an important regulatory role during plant growth and development and adversity stress, and polyamine oxidase (PAO) is involved in polyamine catabolism. In this study, an up-regulated polyamine oxidase gene GmPAO1 was obtained by transcriptome sequencing analysis and screening at soybean seedling stages. Also, its expression pattern and function were analyzed. The identification results of transgenic GmPAO1 soybean posi-tive lines showed that the relative expression level of GmPAO1 in the overexpressed lines was increased under salt stress. With increasing stress concentration, the seed germination rate decreased. However, the seed germination rate of the overexpressed lines was significantly higher than that of the control lines, and the phenotypic character of the root systems was also better than that of the control lines. The measurement of superoxide dismutase (SOD) and peroxidase (POD) activities and malondialdehyde and hydrogen peroxide contents revealed that the overexpressed soybean lines significantly increased the SOD and POD activities, significantly reducing the malondialdehyde content. Although the hydrogen peroxide content in the transformed plants gradually increased, the hydrogen peroxide content in the overexpression lines was still lower than that in the gene editing lines. Based on this, it was preliminarily judged that GmPAO1 can improve soybean salt tolerance.

Pathogenesis-related proteins, often used as molecular markers of disease resistance in plants, can enable plants to obtain systemic resistance. In this study, a gene encoding a pathogenesis-related protein was identified via RNA-seq sequencing analysis performed at different stages of soybean seedling development. Because the gene sequence showed the highest similarity with PR1L sequence in soybean, the gene was named GmPR1-9-like (GmPR1L). GmPR1L was either overexpressed or silenced in soybean seedlings through Agrobacterium-mediated transformation to examine the resistance of soybean to infection caused by Cercospora sojina Hara. The results revealed that GmPR1L-overexpressing soybean plants had a smaller lesion area and improved resistance to C. sojina infection, whereas GmPR1L-silenced plants had low resistance to C. sojina infection. Fluorescent real-time PCR indicated that overexpression of GmPR1L induced the expression of genes such as WRKY, PR9, and PR14, which are more likely to be co-expressed during C. sojina infection. Furthermore, the activities of SOD, POD, CAT, and PAL were significantly increased in GmPR1L-overexpressing soybean plants after seven days of infection. The resistance of the GmPR1L-overexpressing lines OEA1 and OEA2 to C. sojina infection was significantly increased from a neutral level in wild-type plants to a moderate level. These findings predominantly reveal the positive role of GmPR1L in inducing resistance to C. sojina infection in soybean, which may facilitate the production of improved disease-resistant soybean cultivars in the future.

(1) Corn is the most widely planted food crop, feed crop, and economic crop in the world, and plays an important role in agricultural production and national economy development. The copine gene, also known as the BONZAI gene, encodes a Ca2+-dependent phospholipid membrane binding protein that is widely present in eukaryotes. It has been found that the copine protein is a negative regulator of disease resistance regulation and plays a key role in plants' disease resistance response. In this study, the Agrobacterium-tumefacien-mediated method was used to successfully obtain T2 generation ZmBON3-gene-overexpressing plants and gene-edited plants. Related phenotypes and molecular identification showed that the disease resistance of overexpression plants was significantly reduced, and the disease resistance of gene-edited plants was significantly increased, which verified that the ZmBON3 gene was a negative regulatory gene. By detecting the physiological indexes related to defense, it was found that the content of H2O2 and the enzyme active water of CAT, POD, SOD, and PAL in ZmBON3-gene-edited plants was higher than those in the control plants and ZmBON3-gene-overexpressing plants, and the content of H2O2 and CAT, POD, and SOD in ZmBON3-gene-overexpressing plants was significantly higher than that in the control plants and ZmBON3-gene-overexpressing plants. The enzyme activity of PAL was the lowest. By detecting the expression of key genes of defense-related signaling pathways, it was found that ZmBON3 may be involved in the related defense processes mediated by the R gene, SA pathway, JA pathway, and ABA pathway. In addition, ZmBON3-geneedited plants showed obvious dwarf phenomenon at the seedling stage, but this did not affect the ear length, axis diameter, ear row number, and grain color.

Soybean is an important crop for both food and oil, providing high quality protein and oil for humans and animals. Drought stress has an significant effect on the yield and quality of soybean. In the early vegetative stage of soybean, the developed root system can effectively improve the tolerance of soybean plant to drought. Xyloglucan endoglycosylase/hydrolase (XTH) is a cell wall remodeling enzyme involved in cell wall expansion and degradation. However, there are few studies on XTH’s regulation of root growth and response to drought stress in soybean seedlings.The objectives of this study were to clone GmXTH1 gene and determine the effects of over and interference-expression of GmXTH1 on soybean root development at seedling stage. A 1,015 bp full-length of the GmXTH1 gene was cloned from soybean (Glycine max L. Merril) cv. RM18 with a 840 bp open reading frame (ORF) of a putative protein. The over and interference expression vectors of GmXTH1 were transformed into soybean using Agrobacterium method. Transformed strains OEA1, OEA3, IEA4, IEA5 were identified by molecular detection methods. The roots of transformed strains OEA1and OEA3 with GmXTH1 gene overexpression are clearly developed and the root phenotypic attributes, including main root length, primary root number, lateral root length, root surface area, root volume, root dry weight, root fresh weight are higher than the control. In contrast, root attributes of IEA4 and IEA5 with GmXTH1 gene interference expression were significantly lower than those of the control, suggesting that the expression of GmXTH1 gene has an important effect on the soybean root growth and development at seedling stage. Under drought stress conditions, the activities of Superoxide dismutase (SOD), Peroxidase(POD)and Catalase(CAT)in the transgenic strains with an overexpression of the GmXTH1 gene were significantly increased, and the root activity, leaf relative water content and total chlorophyll content were ranked in order of OEAs, CK, and IEAs. In conclusion, GmXHT1 plays an important role in the growth of soybean root at seedling stage. Overexpression of GmXTH1 gene can promote root development of soybean seedling. The results of activities of protective enzymes (SOD, POD, CAT), root activity, leaf relative water content and total chlorophyll content under drought stress indicated that transgenic strains OEA1 and OEA3 with overexpressing GmXTH1 gene were resistant to drought stress, suggesting that GmXTH1 may play a positive role in responding to drought stress at soybean seedling stage.

Isoflavones are secondary metabolites present in seeds of soybean [ Glycine max (L.) Merr.] which have been recognized their benefit to human health. In this study, QTL mapping for soybean isoflavone gylcones including daidzin, glycitin and genistin and total isoflavones content was performed in population of 178 F2:6 recombinant inbred lines (RILs) which was generated from cross between varieties Jinong17 and Jinong18. A genetic linkage map covering 1248 cM was constructed using the simple sequence repeat (SSR) molecular markers. The results revealed 22 isoflavone- related QTLs, 5 for daidzin, 7 for genistin, 6 for glycitin, and 4 for total isoflavone content. Seven of these represent new QTLs. Twenty candidate genes were identified, including eight laccases with presumed role in lignin biosynthesis, and the transcriptional regulator BANYULS and all three components of the MYB-bHLH-WD40 (MBW) complex that regulate its expression. These findings suggest that alterations in lignin and proanthocyanidin metabolism influence isoflavone accumulation in seeds. These leads might be helpful in the efforts to breed new soybean varieties with improved isoflavone composition and content.

大豆异黄酮是有助于人类身体健康的一类次级代谢产物,大豆查尔酮还原酶(chalcone reductase,CHR)基因是控制大豆异黄酮主要组分大豆苷元生物合成的关键酶之一.为拓宽大豆CHR研究范围,促进异黄酮代谢机理研究,进而推进大豆品种改良,本研究利用基因工程技术对大豆CHR2-1基因进行克隆及生物信息学分析,并且构建植物表达载体pCAMBIA3301-CHR2-1,通过农杆菌介导法将目的基因整合到东农50受体大豆基因组中,PCR筛选鉴定获得大豆转化植株.结果表明:GmCHR2-1基因编码的蛋白属于非分泌型蛋白,可能在细胞质中发挥主要功能,属于非跨膜类蛋白并不在细胞中发生迁移;该蛋白存在22个潜在磷酸化位点,其中苏氨酸(Thr)4个、丝氨酸(Ser)18个;该蛋白由α-螺旋(40.63％)、延伸链(14.29％)、β-转角(4.13％)和无规则卷曲(40.95％)4个部分组成.PCR鉴定表明,转化植株基因组包含草丁膦抗性基因Bar、终止子NOS和启动子35S位点,初步确定已将GmCHR2-1转入到东农50大豆品种基因组中,并获得T2阳性植株转化苗12株.

The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors is one of the superfamilies of plant-specific transcription factors involved in plant growth, development, and biotic and abiotic stress. However, there is no report on the research of the TCP transcription factors in soybean response to Phytophthora sojae. In this study, Agrobacterium-mediated transformation was used to introduce the CRISPR/Cas9 expression vector into soybean cultivar "Williams 82" and generated targeted mutants of GmTCP19L gene, which was previously related to involve in soybean responses to P. sojae. We obtained the tcp19l mutants with 2-bp deletion at GmTCP19L coding region, and the frameshift mutations produced premature translation termination codons and truncated GmTCP19L proteins, increasing susceptibility to P. sojae in the T2-generation. These results suggest that GmTCP19L encodes a TCP transcription factor that affects plant defense in soybean. The new soybean germplasm with homozygous tcp19l mutations but the BAR and Cas9 sequences were undetectable using strip and PCR methods, respectively, suggesting directions for the breeding or genetic engineering of disease-resistant soybean plants.

Heterosis is an important biological phenomenon and is widely applied to increase agricultural productivity. However, the underlying molecular mechanisms of heterosis are still unclear. Here we constructed three combinations of reciprocal hybrids of soybean, and subsequently used MethylRAD-seq to detect CCGG and CCWGG (W = A or T) methylation in the whole genome of these hybrids and their parents at the middle development period of contemporary seed. We were able to prove that changes in DNA methylation patterns occurred in immature hybrid seeds and the parental variation was to some degree responsible for differential expression between the reciprocal hybrids. Non-additive differential methylation sites (DMSs) were also identified in large numbers in hybrids. Interestingly, most of these DMSs were hyper-methylated and were more concentrated in gene regions than the natural distribution of the methylated sites. Further analysis of the non-additive DMSs located in gene regions exhibited their participation in various biological processes, especially those related to transcriptional regulation and hormonal function. These results revealed DNA methylation reprogramming pattern in the hybrid soybean, which is associated with phenotypic variation and heterosis initiation.

Heterosis plays an important role in the breeding of commercial crops, but the molecular mechanism behind this phenomenon is still unclear. To better understand the molecular basis of heterosis in soybean, the correlation between DNA methylation status and heterosis in soybean was tested. From three soybean varieties used as parents, six hybrid combinations were generated. Twelve traits of the nine materials were investigated to study the relationship between DNA methylation level differences and heterosis. In this study, MethylRAD technique was used to evaluate the genomic methylation status among the six reciprocal soybean F1 hybrids and their parents in the immature grains. Results showed that the total site number of methylation and methylation levels of the F1 hybrids with strong heterosis were in a status between the two parents and the ones with weak heterosis were generally below that of two parents. Thus, it was beneficial to promote heterosis when the numbers of methylation sites and methylation levels were between two parents. Among three types of methylation variations, the type that parental demethylated and the progeny methylated (type A) may be relative to improve heterosis of some traits. The type A of methylation variations was significantly positively correlated with the mid-parent heterosis (MPH) of the fat content (r = 0.98**), the branch number per plant (r = 0.84), and also was significantly positively correlated with the BPH of the branch number per plant (r = 0.82*) and the stems thick (r = 0.81*). These results implied that both the methylation and demethylation in hybrids relative to their parents may control the expression of the genes that were associated with partial phenotypic variation in hybrids.

大豆疫霉根腐病是由大豆疫霉菌(Phytophthora sojae)引起的危害大豆生长的严重病害.课题组前期研究表明具有P-loop结构域的GmPR10(Gene Bank accession no.FJ960440)和具有P-loop、Bet v1结构域的Gly m 4l(Gene Bank accession no.HQ913577.1)抑制大豆疫霉菌生长,并且过表达GmPR10和Gly m 4l的转基因大豆植株可以提高对大豆疫霉根腐病的抗性.为研究GmPR10和Gly m 4l抑菌机理,本研究利用点突变技术,获得了GmPR10的P-loop结构域突变体(Gly48/Thr48和Gly51/Arg51)、Gly m 4l的P-loop结构域突变体(Gly49/Ile49和Lys55/Pro55)、GmPR10和Gly m 4l的P-loop结构域以及Gly m 4l的Bet v1结构域缺失突变体,并纯化回收相应突变体蛋白,进行体外抑制大豆疫霉菌试验.结果 表明,突变或缺失P-loop,Bet v1结构域的GmPR10和Gly m 4l失去了抑制大豆疫霉菌(Race 1)生长的能力,说明P-loop、Bet v 1结构域对GmPR10和Gly m 4l行使抑菌功能至关重要.

Phytophthora root and stem rot, a destructive disease of soybean [Glycine max (L.) Merr.], is caused by the oomycete Phytophthora sojae. However, how the disease resistance mechanisms of soybean respond to P. sojae infection remains unclear. Previously, we showed that GmWRKY31, which interacts with a sucrose non-fermenting-1(SNF1)-related protein kinase (SnRK), enhances resistance to P. sojae in soybean. Here, we report that the membrane-localized SnRK GmSnRK1.1 is involved in the soybean host response to P. sojae. The overexpression of GmSnRK1.1 (GmSnRK1.1-OE) increased soybean resistance to P. sojae, and the RNA interference (RNAi)-mediated silencing of GmSnRK1.1 (GmSnRK1.1-R) reduced resistance to P. sojae. Moreover, the activities and transcript levels of the antioxidant enzymes superoxide dismutase and peroxidase were markedly higher in the GmSnRK1.1-OE transgenic soybean plants than in the wild type (WT), but were reduced in the GmSnRK1.1-R plants. Several isoflavonoid phytoalexins related genes GmPAL, GmIFR, Gm4CL and GmCHS were significantly higher in “Suinong 10” and GmSnRK1.1-OE lines than these in “Dongnong 50,” and were significantly lower in GmSnRK1.1-R lines. In addition, the accumulation of salicylic acid (SA) and the expression level of the SA biosynthesis-related gene were significantly higher in the GmSnRK1.1-OE plants than in the WT and GmSnRK1.1-R plants, moreover, SA biosynthesis inhibitor treated GmSnRK1.1-R lines plants displayed clearly increased pathogen biomass compared with H2O-treated plants after 24 h post-inoculation. These results showed that GmSnRK1.1 positively regulates soybean resistance to P. sojae, potentially functioning via effects on the expression of SA-related genes and increased accumulation of SA.

In this experiment,the extraction method of soybean isoflavones was optimized,and a high performance liquid chromatography (HPLC) standard measurement system for the determination of isoflavones for laboratory extraction were established.The extraction conditions were as follows:Skimmed soy sample powder added 10 mL of chromatographic grade methanol at a volume of 80％,and placed at room temperature for 2 h.After the installation of ultrasonic power 200 W,80℃ water bath for 12 h,centrifuged at 12 000 r· min-1 for 15 min,taken the supernatant by 0.45 μm microporous membrane filtration,and to be stored at 4℃.The optimal HPLC method was as follows:The mobile phase was methanol-water (volume ratio 30∶70),column temperature set at 40℃,the wave length was 254 nm,the flow rate was 1 mL· min-1,the sample volume was 10 μL,set injection time for 26 min,and calculated by peak area method.The results showed that the optimized HPLC method was efficient and accurate in extracting soybean isoflavones.It could provide technical reference for efficient extraction of soy isoflavones in laboratory.

In order to well understand the molecular basis of heterosis in soybean, the methylation-sensitive amplification polymorphism (MSAP) method based on capillary electrophoresis was used to estimate levels and patterns of cytosine methylation in 15-day post-emergence leaves of four parental lines [Jilin 47 (no. 19), EXP (no. 12), Jilin 38 (no. 3) and Yi 3 (no. 6)] and 12 hybrids [Jilin 38 × Yi 3(3 ×  6), Jilin 38 ×  EXP(3 ×  12), Jilin 38 ×  Jilin 47(3 ×  19), Yi 3 ×  Jilin 38(6 ×  3), Yi 3 ×  EXP(6 ×  12), Yi 3 ×  Jilin 47(6 ×  19), EXP × Jilin 38(12 × 3), EXP × Yi 3(12 ×  6), EXP ×  Jilin 47(12 ×  19), Jilin 47 ×  Jilin 38(19 ×  3), Jilin 47 × Yi 3(19 × 6), Jilin 47 × EXP(19 × 12)]. In addition, 12 traits of the hybrids and their parents were also analyzed to understand the relationship between DNA methylation variation and heterosis. MSAP results showed that the total relative methylation level of all hybrids was lower than the corresponding middle parent value, indicating that the methylation degree was decreasing. And may express a variety of genes related to the phenotypic variation of hybridization. Moreover, the hemi-methylation levels of Jilin 38 × Jilin 47 and Yi 3 × Jilin 47 hybrids and full-methylation levels of EXP × Yi 3 and EXP × Jilin 47 hybrids was significant higher than the corresponding mid-parent values. In addition, the heredity of methylation from parents in hybrids is more than the variations, in which there were four types appeared great higher: A1, B4, B8, and D2. Furthermore, the results of relationship between genetic variation in DNA methylation and heterosis showed that the hypo-methylation had a promoting effect to increase node number, and the hype-methylation of hybrids was helpful to add to stem thick. Our results may provide new insights into well understanding the molecular mechanisms of heterosis at the epigenetic level in soybean.