Abstract:Heterosis utilization is the main way to significantly improve crop yield， which helps to solve the contradiction between the increasing population and limited arable land. Soybean， which identified as an important grain， oil and feed crop in the world， the utilization of heterosis in it has been used for more than 30 years. Among them， the three-line hybrid breeding system based on cytoplasmic male sterility is the main way to utilize the heterosis in soybean. At present， more than 40 hybrid soybean varieties have been approved and popularized in production. Furthermore， hybrid soybean is in the stage of advancing from pilot to industrialization. Therefore， this paper reviewed the genetic basis and breeding application of cytoplasmic male sterility in soybean. The discovery and utilization of various cytoplasmic male sterility lines， the genetic and molecular mechanism of sterility traits， the map-based clone of restorer-of-fertility gene and inhibitor of fertility restorer genes were systematically described. Based on the description and analysis of current situation， the challenges and solutions of three-line hybrid soybean breeding were put forward， and the innovation of three-line hybrid soybean technology was prospected， aiming to provide new methods and new ideas for the molecular basic and application research of soybean heterosis.

Abstract:Frogeye leaf spot is a global soybean disease caused by Cercospora sojina K. Hara， and its prevalence in major soybean production areas has resulted in significant yield losses. Cercospora sojina mutates rapidly and evolves multiple physiological races with different pathogenic specificity to overcome the resistance of cultivars. With the rapid development of molecular biotechnology recently， research focuses have been shifted in traditional breeding of resistance varieties to generate germplasm with broad-spectrum resistance， uncover its pathogenic mechanism and delimit resistance genes against soybean frogeye leaf spot. This article provides a systematic review on the identification of physiological races and pathogenicity of Cercospora sojina， inheritance of resistance and resistant breeding. We also explore the precise identification of disease phenotype， analysis of pathogenic mechanism， fine mapping of resistance gene， and resistant breeding， which provides reference for further research on soybean resistance to frogeye leaf spot.

Abstract:Soybean mosaic virus （SMV） occurs in nearly all soybean-growing areas worldwide and it causes severe yield loss and seed quality reduction in soybean. This review summarizes the recent progress in identification of SMV resistance genes and linked molecular markers， and discusses pyramiding of multiple molecular markers to facilitate breeding of soybean varieties resistant to SMV， and analyzes physical position of SMV resistance genes， for example R_SC3（w）、R_SC14-r and R_SC18 etc. Many candidate genes （such as GsCAD1， GmCAL and GmMLRK1） were proposed based on sequence variation， quantitative real-time polymerase chain reaction （qRT-PCR）， virus induced gene silencing （VIGS） and CRISPR （clustered regularly interspersed short palindromic repeats）/Cas9. Collectively， this study highlights the molecular mechanism of SMV-soybean interactions， e.g. the progress of the Rsv3 gene conferring SMV resistance， and provides perspectives for future studies via applying the markers or mechanism for SMV resistance breeding.

Abstract:Wild soybean （G. soja Sieb. & Zucc.） is the ancestor species of cultivated soybean （Glycine max ［L.］ Merr.）. A large number of genes or allelic variants have been lost in the process of domestication， thus leading to genetic bottlenecks in variety selection and improvement of current cultivated soybean. Wild soybean is widely distributed in China and with rich genetic diversity， harboring elite genes for high protein content， resistance to diseases and pests， drought tolerance， salt and alkali tolerance， etc. Reintroducing elite allelic variants into modern cultivated soybean varieties using molecular design breeding techniques can effectively broaden the genetic diversity. In this paper， we review the distribution and morphological characteristics， and the important functional genes or loci that control some important agronomic traits in wild soybean in recent years， including hundred grain weight， flowering time and maturity time， protein and oil content， disease resistance， insect resistance， saline-alkali resistance， drought resistance， etc. We also discuss the potential of these genes in future soybean breeding， expecting to provide insight and strategy for improving new varieties in soybean.

Abstract:MADS-box is an important transcription factor in plants， and its family members have a typical MIKC structure， highly conserved N-terminal MADS， and less conserved I domains and C-terminals. MADS-box genes are widely expressed in the roots， stems， leaves， flowers， buds and other trophic tissue parts of plants， and are involved in regulating the flowering time， flower development， seed development and abiotic stress response. In recent years， researches have shown that the expression patterns of different MADS-box genes are distinct， and their functions are also quite different. This review introduces the structure and classification of soybean MADS-box gene family， and summarizes the research progress of related members of MADS-box genes in ABCDE model， such as SVP， SOC1， FLC and other genes in the flower development. Finally， the research on soybean MADS-box is prospected， which provides an essential information for further utilization of such transcription factor genes for soybean genetic improvement and germplasm innovation in the future.

Abstract:Soybean serves as an essential food and oil crop， and plays a crucial role in people's livelihoods. However， in recent years， domestic soybean supply has been insufficient， and there is a high dependency on imports. This situation has seriously impacted the domestic soybean market and brought some hidden dangers to the national food security. Hence， enhancing yield and improving seed quality are major goals in current soybean breeding programs in China. At present， a number of key genes controlling important traits in soybean have been cloned and analyzed， which provides important theoretical support for molecular design breeding. The traditional breeding is time-consuming and low efficiency. Gene editing technology provides a new way and tool for biological breeding， which can accelerate the breeding process. Gene editing technologies， represented by CRISPR/Cas9， have rapidly developed into important tools for studying soybean gene functions， genetic modifications， and improving agronomic traits. This article provides an overview of gene editing technology types， features， and their utilization in plants. It also reviews the latest research progress of gene editing technology in enhancing agronomic traits related to soybean yield， quality， stress resistance， disease resistance， flowering time， symbiotic nitrogen fixation， fertility and other traits， providing a theoretical basis and reference for soybean gene editing breeding. Furthermore， this paper also discusses the challenges of gene editing technology in soybean genetic improvement and presents its promising future applications.

Abstract:Based on “the Third National Campaign of Crop Germplasm Census and Collection”， soybean germplasm resources were collected from different cities in Sichuan province. 192 soybean germplasm resources were identified the morphological features and major agronomic traits in the field. These soybean germplasm resources were collected from sixteen cities and forty-one counties， with enrichments at five cities including Ya'an， Leshan， Guangyuan， Bazhong and Luzhou. This study indicated that the coefficient of variation for 12 morphological traits ranged from 10.11% to 57.62%， with the highest coefficient of variation for seed number per plant and the lowest coefficient of variation for SPAD value. The results of correlation analysis showed that there was extremely significant positive correlation among plant height， stem diameter， branch number， pod number per plant and seed number per plant， while 100-seed weight was negatively correlated with branch number and pod number per plant. The results suggested that three principal components that showed a cumulative contribution rate of 76.29%， reflecting most of the morphological features by principal component analysis. 192 soybean germplasm resources were divided into four groups by cluster analysis， which had the characteristics of big 100-seed weight， high pod number， high SPAD value and long petiole length， and seven elite soybean germplasm resources were selected. Additionally， soybean germplasm resources showed good genetic diversity in terms of seed color and seed types in Sichuan province. In summary， this study revealed the genetic diversity of soybean germplasm resources in Sichuan province. Ultimately， this study offers a theoretical basis for improving soybean breeding in terms of high yield parental lines and special soybean varieties.

Abstract:Saline-alkali soil is one of the main types of marginal soils. Using the marginal land for agricultural cultivation is an effective way to alleviate the shortage of farming land. In order to screen soybean germplasm resources showing salt tolerance to improve soybean yield in saline soils， 392 samples from different geographic regions at home and abroad were treated with 150 mmol/L NaCl at the seedling stage. Each single plant was identified and genotyped using 10 SSR markers linked to salt tolerant genes， in order to perform molecular-assisted identification and genetic diversity analysis. Similarity coefficient analysis， cluster analysis and other methods were applied to comprehensively evaluate the soybean germplasm resources. Fifty-eight soybean germplasm resources were identified， including 14 showing high tolerance， such as Chidou 1 hao and Dongnong 69， and 44 showing salt-tolerant， such as Heinong 51 and Heihe 35. Although genotyping these 58 samples， Suinong 1 hao， Hefeng 50 and Dongda 2 hao carried the most salt tolerant allele variations， all of which were 6， and the average identification efficiency was 43.45% and the average accuracy was 68.46%， of which the molecular markers Satt201 had the highest identification efficiency of 60.34% and the highest accuracy of 96.55%. Cluster analysis showed that the similarity coefficients among the 58 soybean germplasm resources ranged from 0.5385 to 0.9231， with an average value of 0.6974 and a correlation coefficient of 0.6240， indicating that most of the 58 soybean germplasm resources were genetically close， and the genetic diversity was relatively low， and that the 58 soybean germplasm resources were not clustered geographically， but most of them were geographically identical or had the same place of origin in a taxon or subgroup. However， we didn’t detect the correlation between genetic similarity and geographically collection sites. The distant germplasm resources can be selected as parents， to breed new salt-tolerant soybean varieties.

Abstract:The amino acids are rich in soybean grains and are important components of the grain quality.In this study， 264 soybean genotypes were analyzed using high-performance liquid chromatography （HPLC） to determine the levels of four free amino acids： arginine， glycine， glutamic acid， and lysine in dried soybean grains. Among the free amino acids， arginine was detected with the highest content， followed by glutamic acid， and glycine. Three soybean varieties with high levels of these four free amino acids were identified， including Haimen Yang 104， Liaoxian Bean 12， and Guanyun Dasiyu. By genome-wide association analysis combined with the phenotypes and genotypes of four free amino acids in soybean natural population， 27 significant SNPs co-located by four amino acids in soybean were identified in two years， among which there were two SNPs for arginine， including S17_19067780 and S17_19067789， and 19 SNPs for glycine，including S01_53974257， S08_38878988，et al， one SNP（S18_53291599） for glutamic acid， five SNPs for lysine，including S08_18555689， S08_18567542， et al. The candidate genes related to high amino acid content in soybean were inferred to be Glyma.17G177400 and Glyma.17G177600 for arginine， and Glyma.11G157000 and Glyma.11G161700 for glycine. Glutamic acid are Glyma.18G244200 and Glyma.18G244700， lysine are Glyma.08G227600 and Glyma.08G228100.The results of this study provide a theoretical basis for improving soybean varieties and assisting soybean molecular breeding.

Abstract:Soybean male sterile lines play vital value for developing hybrid soybean varieties， while the traditional three-line hybridization system has problems due to a limited number of restorer lines. The environmental sensitive genic male sterile （EGMS） line can switch fertility under different conditions. In this research， the phenotypes and mutation sites of the three independent mutants ms3 （Washington）， ms3 （Flanagan） and ms3 （Plainview） were further studied. Phenotypic identification of sterility， high-throughput sequencing， molecular marker design and mutation site verification， analysis of the effect of mutation on MS3 protein structure， ms3 （Plainview） anther semi-thin section experiments were carried out separately. These results showed that most of the pollens are aborted， only sporadic pollen grains with irregular shape were stained by I₂-KI dye in the anthers of the three mutants. High-throughput sequencing results showed that ms3 （Washington） and ms3 （Flanagan） had a large fragment insertion in the PHD coding region of the third exon of the gene MS3， eventually compromising the PHD domain of MS3 protein， which was named ms3-1. In ms3 （Plainview）， there is one base pair deletion in the first exon of MS3， resulting in frameshift mutation encoding only 40 amino acids. The loss-of-function allele was named ms3-2. Through semi-thin section analysis of ms3 （Plainview）， its tapetum layer and pollen development were abnormal at the middle and late anther development stage. In summary， this study provided materials and basis for future application and genetic modification of ms3 and MS3 in soybean breeding.

Abstract:Saline-alkaline lands， which are found with large area in China， are considered as potential exploitable land resource inagriculture. Breeding of new soybean cultivars showing salt-alkaline tolerance in these regions is a way to improve the soybean production in China. Therefore， identification of the negative regulatory genes to saline-alkaline stress is of interest， and editing of them in elite varieties is able to create new soybean cultivars with salt-alkaline tolerance. Previously， we found that the gene Glyma.02g271000 （named as GmDUF247-1） was down-regulated under mixed salt-alkaline stress condition. GmDUF247-1 was annotated with one DUF247 （Domain of Unknown Function 247） domain and one transmembrane domain， and the GmDUF247-1-GFP fusion protein was observed on the cell membrane when transiently expressing in tobacco leaves. GmDUF247-1 was detected with the highest transcripts in roots， and its expression was down-regulated under mixed saline-alkaline treatment. Through generating the hairy root composite plants， the transgenic plants overexpressing GmDUF247-1 showed more wilted leaves， lower survival rates， shorter roots and shoots if compared with these lines expressing the empty vector. Based on the haplotype analysis of GmDUF247-1 in soybean population， eight SNPs and four InDels in the promoter region were detected， implying alterations potentially in the binding of growth- and stress-related transcription. Three haplotypes were identified based on the coding sequence variations， and the haplotype GmDUF247-1^H1 was detected with artificial selection. In summary， we provided the preliminary results on negative regulation of GmDUF247-1 under mixed salt-alkaline stress treatment， which will facilitate to systematically characterize its function and elucidate its potential in breeding new soybean cultivars with superior tolerance to mixed salt-alkaline stress.

Abstract:Soybean （Glycine max （L.） Merr.） is a self-pollinating crop， and producing hybrids through artificial emasculation is time-consuming and expensive. Unlocking the function of the male sterility gene is a prerequisite for harnessing heterosis in soybean. Up to now， only a few loci of male sterility had been reported in soybean， and progress in molecular cloning and functional characterization of related genes lagged behind. Advances in biotechnology and soybean genetic transformation system enabled the possibility to employ reverse genetics methodology for studying the male sterility genes. The transcriptomic data indicated that the small G protein encoding gene GmARFA1a was regulated simultaneously by the male sterility gene MS1 （Male Sterile 1） and MS2. Data from the public library indicated that GmARFA1a expression was the highest in unopened soybean flowers， qRT-PCR data demonstrated that GmARFA1a was preferentially expressed in stamen before flowering. The pollen germination experiment and seed setting statistics found that the decrease of pollen viability of Gmarfa1a mutant resulted in the significant inhibition of seed setting Collectively， this study identified the GmARFA1a gene and uncovered its function on male fertility. It will not only enhance our understanding of the GmARFA1a and ARF gene families but also lay the foundation for further study the function of GmARFA1a genes and the utilization of heterosis in soybean.

Abstract:This study used RNA-seq data from physiological race 3 of soybean cyst nematode， Heinong 37 （susceptible） and Dongnong L10 （resistant）， to screen for the differentially expressed gene GmSBPC. Bioinformatics analysis was conducted on the spatial structure， protein physicochemical properties， hydrophilicity， and hydrophobicity of the protein encoded by this gene. Cloning GmSBPC using cDNA from the root system of disease resistant Dongnong L10. Transform the recombinant vector containing pCAMBIA1302-GmSBPC into Escherichia coli DH5α，Agrobacterium GV3101 （psoup-p19） for subcellular localization analysis. Recombinant pCAMBIA3300-GmSBPC was transferred to Agrobacterium tumefaciens K599 for soybean hairy root infection. Planting Dongnong L10 （resistant） and Dongnong 50 （susceptible） in nematode soil， soybean cyst nematode stress treatment was performed on roots， stems， and leaves at 0， 3， 6， 9， 12， and 15 days for qRT-PCR analysis of gene expression patterns. The results indicate that the GmSBPC protein encodes 146 amino acids and is an insoluble protein， α spiral zone accounts for 28.08%， extended structure accounts for 15.75%， and irregular curl accounts for 56.16%. The subcellular localization results indicate that the gene is located in the nucleus. Overexpression of hairy roots reduces the number of nematodes per unit area compared to wild-type soybeans. The gene expression pattern of the roots of Dongnong 50 and Dongnong L10 under soybean cyst nematode stress was initially increased and then decreased， with the overall expression level being higher in Dongnong L10 roots than in Dongnong 50 roots. The highest expression level was observed in Dongnong L10 roots after 12 days， which is the second instar larval stage of soybean infection by nematodes. Therefore， it is determined that this gene has a response to nematode stress， and it is speculated that this gene is involved in the stress response of soybean cyst nematodes. These research results contribute to further exploring the physiological function of SBPC gene in soybean resistance to cyst nematodes.

Abstract:Due to industrial development and increasing pollution from domestic wastes，heavy metal concentrations in crops are exceeding standards，posing a serious threat to human health. Aluminum-activated malate transporters （ALMT） encode a class of anion channel proteins that play important roles in the transmembrane transport of plant organic acids. In order to investigate the function of GmALMT33 gene in response to Cd stress in soybean， the GmALMT33 gene was cloned from soybean using RT-PCR in this study. The CDS region of the gene is 1622 bp in lengh， encodes 553 amino acids，and contains the ALMT structural domain. qRT-PCR results showed that the expression level of GmALMT33 was the highest in the roots of soybeans，and the expression of this gene showed a tendency of first increasing and then decreasing after cadmium stress. We constructed the plant expression vector pCPB-GmALMT33 and genetically transformed tobacco and soybean hairy roots. Phenotypic analysis of the transgenic plants showed that under cadmium stress （66 μmol/L CdCl₂），the leaf blades of the transgenic tobacco were yellowed and greenish，and the degree of browning at the edges was significantly lower than that of wild-type tobacco. The transgenic soybean hairy root complex plants showed significantly weaker degree of reddish-brown toxicity symptoms in stalks and leaf veins than the trans-space vector plants，which indicated that the GmALMT33 gene improved the toxicity symptoms of transgenic soybean hairy root complex.After 7 d of cadmium stress treatment，the SOD，APX activity and soluble sugar content of transgenic tobacco leaves were higher than those of the wild-type control， and the MDA content was lower than that of the control. After 0 d，1 d and 3 d of cadmium stress treatment，the SOD，APX activity and soluble sugar content of roots and leaves of transgenic soybean hairy root complex were higher than those of the trans-space vector control，and the MDA content was lower than that of the control. GmALMT33 gene improved the cadmium tolerance ability of plants. This study provides a basis for further exploring the mechanism of action of GmALMT33 gene and provides a new gene for soybean stress tolerance breeding.

Abstract:Fresh flavor is the key factor of the taste quality of vegetable soybean， and its formation is closely related to the organic acids. It is of great practical significance to analyze the synthesis mechanism of organic acids in quality improvement of vegetable soybean. In this study， the soybean hairy root system was used to explore the functions of candidate genes GmALMT8， GmIF7GT5 and GmAP， which are significantly related to malic acid content， in regulating malic acid content. The transcripts of GmALMT8 gene and the content of malic acid in GmALMT8-OE hairy roots were significantly higher than those in control hairy roots without inserts， while the content of malic acid in GmIF7GT5 and GmAP transformed hairy roots had no significant change.In view of the reported malic acid transport function of ALMT family genes， it is speculated that GmALMT8 gene in soybean may have a similar function and play an important role in regulating malic acid content. In order to verify whether the change of malic acid content in GmALMT8-OE hairy roots is caused by the change of GmALMT8 gene expression， this study overexpressed GmALMT8 gene in Arabidopsis thaliana by dipping flower method. Similar to the results of malic acid determination in positive hairy roots， overexpression of GmALMT8 significantly increased the malic acid content in seeds of T₂ transgenic Arabidopsis thaliana.These results approved that soybean GmALMT8 gene had biological function in regulating malic acid content， which enriched the theoretical research of soybean organic acid and provided reference value for high-quality breeding of vegetable soybean.