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Functional Genomics and Molecular Breeding of Soybeans

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A special issue of Agronomy (ISSN 2073-4395). This special issue belongs to the section "Crop Breeding and Genetics".

Deadline for manuscript submissions: 31 October 2024 | Viewed by 10442

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Special Issue Editors

Prof. Dr. Fang Huang

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Guest Editor

National Center for Soybean Improvement, State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing 210095, China
Interests: soybean molecualr biology; soybean molecular genetics and breeding for yield and quality

Prof. Dr. Lin Zhao

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Guest Editor Assistant

Key Laboratory of Soybean Biology of Ministry of Education China, Northeast Agricultural University, Harbin 150030, China
Interests: soybean molecualr biology; genetic improvement of soybean growth period

Prof. Dr. Xiaobo Wang

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Guest Editor Assistant

School of Agronomy, Anhui Agricultural University, Hefei 230036, China
Interests: soybean molecular breeding; functional genomics; gene editing

Special Issue Information

Dear Colleagues,

Soybean (Glycine max (L.) Merr.) is one of the most important grain and oil crops. With the extensive exploitation of soybean gene resources, research on soybean functional genomics using genomic information and phenotypic group information has become increasingly important. With the continuous development of biotechnology, modern breeding techniques, such as whole genome selection breeding and genome editing breeding, are changing rapidly, and the selection of excellent soybean varieties has shifted towards soybean molecular design breeding. This Special Issue will collect cutting-edge research on soybean functional genomics and molecular breeding to further promote soybean molecular design and breeding.

Prof. Dr. Fang Huang
Guest Editor

Prof. Dr. Lin Zhao
Prof. Dr. Xiaobo Wang
Guest Editor Assistant

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Keywords

soybean
functional genomics
molecular breeding

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Published Papers (8 papers)

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18 pages, 5160 KiB

Open AccessArticle

A Soybean Pyrroline-5-Carboxylate Dehydrogenase GmP5CDH1 Modulates Plant Growth and Proline Sensitivity

by Shupeng Dong, Zhuozhuo Mao, Zhongyi Yang, Xiao Li, Dezhou Hu, Fei Wu, Deyue Yu and Fang Huang

Agronomy 2024, 14(10), 2411; https://doi.org/10.3390/agronomy14102411 (registering DOI) - 18 Oct 2024

Abstract

Soybean [Glycine max (L.) Merr.], as a globally commercialized crop, is an important source of protein and oil for both humans and livestock. With more frequent extreme weather disasters, abiotic stress has become one of the critical factors restricting soybean production. Proline (Pro) is a well-known substance in plants that responds to abiotic stress. To identify potential effector genes involved in soybean resistance to abiotic stress, we focused on the pyrroline-5-carboxylate dehydrogenase (P5CDH) which is a key enzyme in the degradation process of Pro. Through homologous sequence alignment, phylogenetic tree, and predicted expression, we chose GmP5CDH1 (Glyma.05G029200) for further research. Tissue-specific expression assay showed that GmP5CDH1 had higher expression levels in soybean seed and cotyledon development. Subcellular localization assay revealed that GmP5CDH1 was a nuclear-membrane-localized protein. As the result of the predicted cis-acting regulatory element indicates, the expression level of GmP5CDH1 was induced by low temperature, drought, salt stress, and ABA in soybean. Next, we constructed transgenic Arabidopsis overexpressing GmP5CDH1. The results showed that GmP5CDH1 also strongly responded to exogenous Pro, and overcame the toxicity of abiotic stress on plants by regulating the endogenous concentration of Pro. The interaction between GmP5CDH1 and GmSAM1 was validated through yeast two-hybrid, LUC fluorescence complementary, and BIFC. In conclusion, overexpression of a soybean pyrroline-5-carboxylate dehydrogenase GmP5CDH1 regulates the development of Arabidopsis thaliana by altering proline content dynamically under salt stress, especially improving the growth of plants under exogenous Pro. Full article

(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)

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Figure 1
Bioinformatics analysis of P5CDH genes family in soybean. (A) Phylogenetic tree assay of seven species. (B) Gene structure assay of seven species. Different colored squares represent different protein motifs. (C) Predicted expression pattern for P5CDH genes family in soybean. Full article ">Figure 2
Genomic sequence assay of GmP5CDH1. (A) The genome sequence structure diagram of GmP5CDH1. The black horizontal line represents the length of 100 bases. (B) Conserved structural domain of GmP5CDH1. (C) Protein sequence alignment of P5CDH gene family in three leguminous crops and Arabidopsis. The protein sequence of the Aldedh domain is shown in red underline. (D) Predicted cis-acting regulatory element structure of GmP5CDH1. Full article ">Figure 3
The expression pattern of GmP5CDH1. (A) Tissue-specific analysis of GmP5CDH1 in soybean. The Y-axis is divided into two parts, with the bottom value ranging from 1 to 3, accounting for 70% of the Y-axis, and the top value ranging from 3 to 18, accounting for 30%. (B) GmP5CDH1 was induced by low temperature (B1), drought (B2), salt stress (B3), and ABA (B4). (C) Subcellular localization assay of GmP5CDH1 in Nicotiana benthamiana leaf. GFP, green fluorescent protein; BF, brightfield; Chlorophyll, chlorophyll autofluorescence (red); Scale bars, 20 µm. Significant differences according to two-sided Student’s t-test (0.01 < * p < 0.05, ** p < 0.01). Full article ">Figure 4
Germination rate (A) and amino acid content (B) of transgenic Arabidopsis seeds. Significant differences according to two-sided Student’s t-test (0.01 < * p < 0.05, ** p < 0.01). Full article ">Figure 5
The phenotype of transgenic Arabidopsis under salt stress. (A,B,E) Morphology (A), root length (B), and fresh weight (E) of transgenic Arabidopsis under salt stress. (C) Endogenous Pro content in plants under salt stress. (D) Expression level of GmP5CDH1 in plants under salt stress. Significant differences according to two-sided Student’s t-test (** p < 0.01). Full article ">Figure 6
The phenotype of transgenic Arabidopsis under different exogenous Pro concentrations. (A) Germination rate of transgenic plants. (B–D) Phenotype (C), root length (B), and fresh weight (D) of transgenic plants. Endogenous Pro content (E) and expression level of GmP5CDH1 (F) in plants treatment with 0 mM, 2 mM, and 150 mM concentrations of Pro. Significant differences according to two-sided Student’s t-test (** p < 0.01). Full article ">Figure 7
GmP5CDH1 interacts with GmSAM1. (A) Transformed yeast cells were grown on synthetic defined (SD) medium/—Trp–Leu/X-α-Gal and SD medium/—Trp–Leu–His–Ade/X-α-Gal. pGADT7-T+ pGBKT7-53 represents the positive control; pGADT7-T+ pGBKT7-lam represents the negative control. (B) LUC fluorescence complementary of GmP5CDH1 in Nicotiana benthamiana leaf. GmP5CDH1-C-LUC+N-LUC, C-LUC+GmSAM1-N-LUC, and C-LUC+ N-LUC constructs represent the negative control. (C) Bimolecular fluorescence complementation (BiFC) assay was conducted to confirm the interaction location. YFPc + GmP5CDH1-YFPn and GmSAM1-YFPc + YFPn constructs were used as controls. YFP, yellow fluorescent protein; BF, brightfield; Chlorophyll, chlorophyll autofluorescence (red). Scale bars, 50 μm. Full article ">

13 pages, 2756 KiB

Open AccessArticle

Resistance Analysis of a Soybean Cultivar, Nongqing 28 against Soybean Cyst Nematode, Heterodera glycines Ichinohe 1952

by Changjun Zhou, Yanfeng Hu, Yingpeng Han, Gang Chen, Bing Liu, Jidong Yu, Yaokun Wu, Jianying Li, Lan Ma and Jian Wei

Agronomy 2024, 14(9), 1964; https://doi.org/10.3390/agronomy14091964 - 30 Aug 2024

Viewed by 588

Abstract

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, 1952, is one of the most destructive plant-parasitic nematodes in soybean production worldwide. The use of resistant soybean is the most effective alternative for its management. However, SCN-resistant soybean cultivars with increased yield and favorable agronomic traits remain limited in the market. Here, we developed a new SCN-resistant soybean cultivar Nongqing 28 from the cross of the female parent cultivar An 02-318 and a male parent line F2 (Hei 99-980 × America Xiaoheidou). Resistance evaluation suggested that Nongqing 28 displayed stable resistance to SCN race 3 in pot assays and the 5-year field experiments, including inhibition of SCN development and reduction in female and cyst numbers. The average yields of Nongqing 28 were 2593 kg/ha and 2660 kg/ha in the 2-year regional trails and the 1-year production trials, with a yield increase of 6.2% and 8.1% compared with the local cultivar Nengfeng 18, respectively. The average seed fat contents in Nongqing 28 reached 21.26%. Additionally, RNA-seq analysis revealed that the resistance of Nongqing 28 to SCN infection is involved in pathogen perception and defense activation, such as reactive oxygen species burst, calcium-mediated defense signaling, hormonal signaling, the MAPK signaling cascade, and phenylpropanoid biosynthesis. In summary, this study provides a detailed characterization of a novel SCN-resistant soybean cultivar with high oil and yield potential. Full article

(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)

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Pedigree chart of soybean cultivar Nongqing 28. An 02-318 derived from Hartwig is resistant to soybean cyst nematode (SCN), but Hei 99-980 and America Xiaoheidou are susceptible to SCN. Full article ">Figure 2
The phenotypes and seeds of soybean cultivar Nongqing 28 in maturity stage. Full article ">Figure 3
The comparable development of soybean cyst nematode on roots of Nongqing28, PI 437654, and Hefeng 50 at 5 and 10 days post inoculation (dpi). * denotes J2, and ** denotes J3. Scale bar = 500 µm. Full article ">Figure 4
Transcriptome response of Nongqing 28 to soybean cyst nematode at 10 days post inoculation (dpi). (A) The number of differentially expressed genes (DEGs). (B) Volcano plots indicating all soybean genes of Nongqing 28 at 10 dpi. The red and green dots represent up- and downregualted genes. (C) Top 30 enriched Gene Ontology (GO) biological processes (BP) of the upregulated DEGs. (D) Top 20 most enriched KEGG terms of upregulated DEGs. Full article ">Figure 5
Validation of DEGs in Nongqing 28 and Hefeng 50 at 10 dpi by quantity reverse transcription RT-qPCR. The expression levels of target genes are relative to the level of expression of soybean genes in the non-inoculated control plants using GmUBQ3 as reference gene. The data are presented as the means of three replicates ± SE. Asterisks indicate a statistically significant difference between soybeans genes in SCN-infected plants and the non-infected control plants (Student’s t-test, * p < 0.5). Full article ">

17 pages, 4630 KiB

Open AccessArticle

Identifications of Seed Vigor-Related QTLs and Candidate Genes Combined Cultivated Soybean with Wild Soybean

by Shengnan Ma, Haojie Feng, Yiran Sun, Lin Yu, Chunshuang Tang, Yanqiang Zhao, Liansong Xue, Jinhui Wang, Chunyan Liu, Dawei Xin, Qingshan Chen and Mingliang Yang

Agronomy 2024, 14(2), 332; https://doi.org/10.3390/agronomy14020332 - 6 Feb 2024

Cited by 1 | Viewed by 1395

Abstract

Soybean (Glycine max) is an economically important cash crop and food source that serves as a key source of high-quality plant-derived protein and oil. Seed vigor is an important trait that influences the growth and development of soybean plants in an agricultural setting, underscoring a need for research focused on identifying seed vigor-related genetic loci and candidate genes. In this study, a population consisting of 207 chromosome segment substitution lines (CSSLs) derived from the crossing and continuous backcrossing of the Suinong14 (improved cultivar, recurrent parent) and ZYD00006 (wild soybean, donor parent) soybean varieties was leveraged to identify quantitative trait loci (QTLs) related to seed vigor. The candidate genes detected using this approach were then validated through RNA-seq, whole-genome resequencing, and qPCR approaches, while the relationship between specific haplotypes and seed vigor was evaluated through haplotype analyses of candidate genes. Phenotypic characterization revealed that the seed vigor of Suinong14 was superior to that of ZYD00006, and 20 total QTLs were identified using the selected CSSLs. Glyma.03G256700 was also established as a seed vigor-related gene that was upregulated in high-vigor seeds during germination, with haplotypes for this candidate gene also remaining consistent with observed soybean seed vigor. The QTLs identified herein can serve as a foundation for future marker-assisted and convergent breeding efforts aimed at improving seed vigor. In addition, future molecular and functional research focused on Glyma.03G256700 has the potential to elucidate the signaling network and key regulatory mechanisms that govern seed germination in soybean plants. Full article

(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)

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Phenotypic analyses of seed vigor-associated traits in Suinong14 and ZYD00006. (A) Suinong14 and ZYD00006 seed vigor phenotypes after germination. (B) Suinong14 exhibited higher IR, GI, GP, and GR values than ZYD00006. Data are means ± SE of three replicate analyses; ** p < 0.01, Student’s t-test. Full article ">Figure 2
Fine mapping of segregants exhibiting divergent seed vigor phenotypes. Black and striped sections correspond to ZYD00006 chromosomal fragments, whereas black sections with black and striped areas respectively indicating homozygous and heterozygous areas. Wild soybean chromosomal segment distributions in these CSSLs were utilized to narrow seed vigor-associated gene candidates to a 293.7 kb region between the BARCSOYSSR_03_1647 and BARC-900569-00953 markers. Full article ">Figure 3
Comparisons of the transcriptomes of germinated Suinong14 and ZYD00006 seeds. (A,B) Volcano plots, (C) the Venn of genes differentially expressed between ZYD-12 h vs. ZYD-24 h and SN-12 h vs. SN-24 h, (D) heatmap of identified DEGs. Full article ">Figure 4
Sequencing-based seed vigor-related candidate gene identification. (A) SNPs distributed in the 44.88 Mb–45.18 Mb region of chromosome 03 in the Suinong14 and ZYD00006 varieties were analyzed using a sliding window approach with a window size of 1 kb. The number of SNPs and InDels within this window is indicated with the corresponding colors, ranging from 0 (grey) to 30 (red), with white indicating the absence of any SNPs and InDels. (B) A heat map for candidate genes containing SNPs or InDels. The color key (blue to red) represents gene expression (fragments per kilobase per million mapped reads, FPKM). (C) Relative Glyma.03G253900 and Glyma.03G256700 expression levels during Suinong14 and ZYD00006 seed germination were established by qPCR, employing the 2−ΔCt method to calculate relative expression. GmUNK1 (Gm12g020500) was selected as a normalization control. Data are means ± SE of three replicate analyses; ** p < 0.01, ns: non-significant, Student’s t-test. Full article ">Figure 5
Analyses of Glyma.03G256700 and its expression in different soybean accessions. (A) YFP-fused Glyma.03G256700 was assessed for subcellular localization. BF: brightfield; scale bar: 25 μm. (B) Maximum-likelihood phylogenetic analysis of the Glyma.03G256700 in Glycine max, Triticum aestivum, Oryza sativa, Zea mays, Lotus corniculatus, Arabidopsis thaliana, and Medicago truncatula as generated using MEGA7. (C) Glyma.03G256700 expression was analyzed at 12 h and 24 h post-germination in 10 CSSLs each exhibiting high and low levels of seed vigor. Data are means ± SE of three replicate analyses; ** p < 0.01, ns: non-significant, Student’s t-test. (D–G) The seed vigor phenotypes after germination in five with enhanced seed vigor (CSSL-003, CSSL-012, CSSL-120, CSSL-159 and CSSL-168) and five with poorer seed vigor (CSSL-031, CSSL-054, CSSL-067, CSSL-106, and CSSL-167). Full article ">Figure 6
Haplotype analyses of Glyma.03G256700. (A) Seed vigor phenotypes were analyzed for 310 soybean varieties (229 improved cultivars, 71 landraces, and 10 wild varieties), different letters represent significant differences (p < 0.05), while the same letters indicate no significant differences, Student’s t-test. (B) Glyma.03G256700 haplotype analyses in these 310 soybean accessions. (C) Hap1 and Hap2 seed vigor phenotypes, data are means ± SE of three replicate analyses; ** p < 0.01, Student’s t-test. Full article ">

21 pages, 4659 KiB

Open AccessArticle

Genome-Wide Identification, Characterization, and Expression Analysis of the Amino Acid Permease Gene Family in Soybean

by Yuan Zhang, Le Wang, Bao-Hua Song, Dan Zhang and Hengyou Zhang

Agronomy 2024, 14(1), 52; https://doi.org/10.3390/agronomy14010052 - 23 Dec 2023

Viewed by 1517

Abstract

Amino acid permeases (AAPs) play important roles in transporting amino acids in plant species, leading to increased low-nitrogen tolerance, grain yield, or protein content. However, very few AAPs have been characterized in soybean (Glycine max). In this study, we scanned the soybean reference genome and identified a total of 36 AAP genes (named GmAAP). The GmAAPs were phylogenetically divided into three evolutionary clades, with the genes in the same clades sharing similar gene structures and domain organization. We also showed that seventeen GmAAP genes on ten chromosomes were in collinearity, likely due to whole-genome duplication. Further analysis revealed a variety of cis-acting regulatory elements (such as hormone response elements (ABRE, ERE, GARE, P-box, and TGA-element), stress response elements (LTR, MBS, MYB-related components, TC-rich repeats, TCA-element, and WUN-motif), the tissue expression element (GCN4-motif), and the circadian regulatory element (circadian) present in the 2 kb region of the GmAAP promoter region, demonstrating functional diversity and expression specificity. RNA-Seq data and quantitative real-time PCR identified five GmAAPs showing differential expression under nitrogen limitation, including GmAAP3, GmAAP5, and GmAAP8 showing downregulation while GmAAP14, GmAAP29 showed upregulation, suggesting their involvement in low-nitrogen stress response. These results provide comprehensive information on soybean AAP genes in nitrogen stress, and provide putative candidates with possible roles in enhancing amino acid delivery to seeds for yield improvement. Full article

(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)

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12 pages, 1969 KiB

Open AccessArticle

Soybean LEAFY COTYLEDON 1: A Key Target for Genetic Enhancement of Oil Biosynthesis

by Sehrish Manan, Khulood Fahad Alabbosh, Abeer Al-Andal, Waqas Ahmad, Khalid Ali Khan and Jian Zhao

Agronomy 2023, 13(11), 2810; https://doi.org/10.3390/agronomy13112810 - 13 Nov 2023

Cited by 2 | Viewed by 1510

Abstract

Soybean is an important oilseed crop that is used as a feed for livestock and has several industrial uses. Lipid biosynthesis and accumulation primarily occur during seed development in plants. This process is regulated by several transcription factors and interconnected biochemical pathways. This study investigated the role of glycine max LEAFY COTYLEDON 1 (GmLEC1) in soybean seed development and the accumulation of storage reserves. The overexpression of GmLEC1 significantly increased the amount of triacylglycerol (TAG) in transgenic Arabidopsis seeds compared to the wild-type and an atlec1 mutant. Similarly, the high expression of GmLEC1 led to a 12% increase in TAG content in transgenic soybean hairy roots compared to the control. GmLEC1 also altered the fatty acid composition in transgenic Arabidopsis seeds and soybean hairy roots. Additionally, the overexpression of GmLEC1 resulted in a reduction in starch accumulation in seeds and vegetative tissues, as well as changes in cotyledon and seed morphology. The cotyledons of the atlec1 mutant displayed abnormal trichome development, and the seeds were smaller and less tolerant to desiccation. A complementation assay in Arabidopsis restored normal cotyledon phenotype and seed size. The main downstream targets of LEC1 are GL2 and WRI1, which were found to participate in fatty acid biosynthesis and trichome formation through the regulation of phytohormones and various transcription factors involved in seed development and maturation. The findings of this study suggest that GmLEC1 controls seed development and regulates the accumulation of seed storage compounds. Furthermore, these results demonstrate that GmLEC1 could be a reliable target for the genetic improvement of oil biosynthesis in soybean. Full article

(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)

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(A) Phylogenetic relationship between GmLEC1 and LEC1 homologs from other crops. Red triangle represent the GmLEC1 used in this study (B) Tissue-specific expression of GmLEC1 (SAM: Shoot apical meristem). (C) Expression of GmLEC1 in soybean seeds at various developmental stages. Developmental stages were classified according to seed weight. Full article ">Figure 2
Comparison of plant developmental characteristics of atlec1, wild-type, and GmLEC1/atlec1 transgenic plants. (A) Four-day-old cotyledons of atlec1 have trichomes on the upper surface, while wild-type and GmLEC1/atlec1 complemented cotyledons showed true cotyledon morphology with no trichrome. (B) Mature atlec1 mutant seeds have vague characteristics, such as being shrunken and darker in color compared to the wild type. (C) Iodine staining of starch granules in four-week-old leaves of atec1, wild-type, and GmLEC1/atlec1 transgenic plants. The dark color of the atlec1 leaves indicates more accumulated starch in the leaves. (D) Content of starch in mature dry seeds. ** p < 0.01 by Student’s t-test (n = 3). Full article ">Figure 3
Composition of fatty acids (A) and total TAG content (B) of wild-type and GmLEC1 overexpressed seeds. Composition of fatty acids (C) and total TAG content (D) of WS-2 (wild-type), atlec1, and genetically complemented GmLEC1/atlec1 seeds. (Palmitic acid:C16:0; Stearic acid:C18:0; Oleic acid:C18:1; Linoleic acid:C18:2; Alpha linolenic acid:C18:3; Eicosenoic acid:C20:1). ** p < 0.01 and * p < 0.05 by Student’s t-test (n = 3). Full article ">Figure 4
GmLEC1 expression in soybean hairy roots. (A) Soybean cotyledons were used to produce hairy roots. (B) Lipids staining after TLC analysis (St: standard/positive control, GUS: control). (C) Composition of TAGs from hairy roots. (D) Amount of TAGs. Representative data are from three biological replicates and expressed as the mean SD. ** p < 0.01 and * p < 0.05 by Student’s t-test (n = 3). Full article ">Figure 5
Quantitative RT-PCR of a few genes involved in fatty acid/TAG biosynthesis to confirm the role of GmLEC1 in lipid biosynthesis. Representative data are from three biological replicates and expressed as the mean SD. The sequence of all genes is provided in <a href="#app1-agronomy-13-02810" class="html-app">Supplementary Data S2</a>. Full article ">

12 pages, 3075 KiB

Open AccessCommunication

Development of a Set of Polymorphic DNA Markers for Soybean (Glycine max L.) Applications

by Man-Wah Li, Xin Wang, Ching-Ching Sze, Wai-Shing Yung, Fuk-Ling Wong, Guohong Zhang, Gyuhwa Chung, Ting-Fung Chan and Hon-Ming Lam

Agronomy 2023, 13(11), 2708; https://doi.org/10.3390/agronomy13112708 - 27 Oct 2023

Cited by 1 | Viewed by 1643

Abstract

Soybean (Glycine max L.) is gaining in importance due to its many uses, including as a food crop and a source of industrial products, among others. Increasing efforts are made to accelerate soybean research and develop new soybean varieties to meet global demands. Soybean research, breeding, identification, and variety protection all rely on precise genomic information. While DNA markers are invaluable tools for these purposes, the older generations, especially those developed before the advent of genome sequencing, lack precision and specificity. Thankfully, advancements in genome sequencing technologies have generated vast amounts of sequence data over the past decade, allowing precise and high-resolution analyses. However, making sense of the genomic information requires a certain level of professional training and computational power, which are not universally available to researchers. To address this, we generated a set of PCR-based DNA markers out of the existing genomic data from 228 popular soybean varieties that offer precise, unambiguous genomic information and can be easily adapted in various applications. A standard operating procedure (SOP) was also designed for these markers and validated on diverse soybean varieties to ensure their reproducibility. This user-friendly universal panel of DNA markers, along with the SOP, will facilitate soybean research and breeding programs through simple applications. Full article

(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)

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Gel images illustrating the expected amplified band patterns of the 100 markers. Agarose gel images of 88 random SNP markers and 12 trait-associated markers are shown. The left and right lanes of each gel image are the amplified band pattern of the reference genotype and of the alternative genotype, respectively. Full article ">Figure 2
Distributions and allele frequencies of the selected markers on the 20 chromosomes of the soybean genome. Grey horizontal bars, soybean chromosomes; green vertical lines, physical positions of random SNP markers; purple vertical lines, physical positions of trait-associated markers; orange bars above the chromosomes, frequencies (with scale above the corresponding chromosome numbers on the left) of the reference alleles in the 228 re-sequenced soybean accessions. The chromosomes and markers were illustrated using the R package “ChromoMap” (version 4.1.1) [<a href="#B42-agronomy-13-02708" class="html-bibr">42</a>]. Full article ">Figure 3
Validation of soybean trait-associated marker examples selected from the panel. (A) Markers associated with hilum color. Pigmentation indicates the color of the hilum. U: unpigmented; P: pigmented. (B) Markers associated with salt tolerance. Salt tolerance levels were retrieved from a previous study [<a href="#B21-agronomy-13-02708" class="html-bibr">21</a>]. Asterisk, common outer amplicon; filled triangle, amplified fragment of the salt tolerance allele; open triangle, amplified fragment of the salt-sensitive allele. Full article ">

13 pages, 1875 KiB

Open AccessArticle

Increased Accumulation of Recombinant Proteins in Soybean Seeds via the Combination Strategy of Polypeptide Fusion and Suppression of Endogenous Storage Proteins

by Jing Yang, Yuanyu Zhang, Guojie Xing, Jia Wei, Lu Niu, Qianqian Zhao, Qinan Cai, Xiaofang Zhong and Xiangdong Yang

Agronomy 2023, 13(11), 2680; https://doi.org/10.3390/agronomy13112680 - 25 Oct 2023

Viewed by 1356

Abstract

Soybean seeds show great potential as a safe and cost-effective host for the large-scale production of biopharmaceuticals and industrially important macromolecules. However, the yields of desired recombinant proteins in soybean seeds are usually lower than the economic threshold for their potential commercialization. Our previous study demonstrated that polypeptide fusion such as maize γ-zein or elastin-like polypeptide (ELP) could significantly increase the accumulation of foreign proteins. In the present study, a recombination strategy of polypeptide fusions (γ-zein or ELP) and suppression of intrinsic storage proteins (glycinin or conglycinin) via RNA interference was further exploited to improve the yield of the target protein in soybean seeds. Transgenic soybean plants harboring both polypeptide-fused green fluorescent protein (GFP) and glycinin/conglycinin RNAi expression cassettes were generated and confirmed by molecular analysis. The results showed that on both the glycinin and conglycinin suppression backgrounds, the average accumulation levels of recombinant zein-GFP and GFP-ELP proteins were significantly increased as compared to that of their counterparts without such suppressions in our previous study. Moreover, zein-GFP and GFP-ELP accumulation was also remarkably higher than unfused GFP on the glycinin suppression background. However, no significant differences were detected in the glycinin or conglycinin suppression backgrounds for the same polypeptide fusion constructs, though suppression of one of the storage proteins in soybean seeds led to a significant increase in the other. Additionally, the increases in the recombinant protein yield did not affect the total protein content and the protein/oil ratio in soybean seeds. Taken together, the results indicate that both the fusion of the foreign protein with polypeptide tags together with the depletion of endogenous storage proteins contributed to a higher accumulation of the recombinant proteins without affecting the total protein content or the protein/oil ratio in soybean seeds. Full article

(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)

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Figure 1
Schematic maps of the vector constructs used for soybean genetic transformation. All these constructs contained the glycinin (Gy) or conglycinin (Cgy) RNAi cassette and gfp (zein- or elp-fused, and unfused) expression cassettes. BCSP and Pgy1, soybean seed-specific promoters of β-conglycinin alpha subunit and glycinin Gy1 subunit genes, respectively; GFP, green fluorescent protein; zein, maize 27 kDa γ-zein; ELP, a 13.56 kDa elastin-like polypeptide; D, enterokinase cleavage site (DDDDK); K, endoplasmic reticulum (ER) localization signal; nos, nopaline synthase terminator; Gyi-S and Gyi-R, tandem RNAi fragments based on glycinin subunit-encoding genes (Gy1–Gy5); Cgyi-S and Cgyi-R, tandem RNAi fragments based on conglycinin subunit-encoding genes (Cgy1–Cgy3); ocs, octopine synthase terminator; pdk, a pdk intron spacer from Flaveria trinervia; LB, left border; RB, right border. Full article ">Figure 2
Expression analysis of the foreign genes in transgenic soybean plants. (a) Quantification of green fluorescent protein (gfp) expression in immature T3 transgenic seeds at 45 days after flowering (DAF) using qRT-PCR (n = 3). Variability in the mRNA levels of the foreign genes was observed among the independent transgenic plants, and only the plants with similar expression of the transgenes were selected and presented. (b–f) Western blotting analysis of the GFP expression in T3 soybean seeds at 45 DAF. The bands represent the unfused GFP (26.8 kDa), maize 27 kDa γ-zein fused GFP (zein-GFP, 51.8 kDa), and elastin-like polypeptide fused GFP (GFP-ELP, 40.5 kDa). Ct+, GFP standard; Nt, non-transformed plant. Full article ">Figure 3
Expression analysis of endogenous storage proteins glycinin and conglycinin in transgenic soybean seeds carrying the Gy or Cgy RNAi cassettes (glycinin-suppressed, Gyless, or conglycinin-suppressed, Cgyless). qRT–PCR analysis of glycinin (a) and conglycinin (b) gene expression. Nt, non-transformed plants; B66, Gyless/GFP plants; G45, Gyless/zein–GFP plants; A58, Gyless/GFP-ELP plants; F24, Cgyless/zein–GFP plants; J41, Cgyless/GFP–ELP plants. (c) Protein expression profiles analyzed using SDS-PAGE. M, protein marker; Nt, non-transformed plants; DG44, zein–GFP expressing plants without suppression; DE9, GFP-ELP expressing plants without suppression; G45, G54, and G59, Gyless/zein–GFP plants; F24 and F32, Cgyless/zein–GFP plants; A44, A58, and A59, Gyless/GFP–ELP plants; J41, J47, and J53, Cgyless/GFP–ELP plants. Full article ">

15 pages, 7945 KiB

Open AccessArticle

Genome-Wide Identification of the Phytocyanin Gene Family and Its Potential Function in Salt Stress in Soybean (Glycine max (L.) Merr.)

by Li Wang, Jinyu Zhang, Huici Li, Gongzhan Zhang, Dandan Hu, Dan Zhang, Xinjuan Xu, Yuming Yang and Zhongwen Huang

Agronomy 2023, 13(10), 2484; https://doi.org/10.3390/agronomy13102484 - 27 Sep 2023

Cited by 2 | Viewed by 1466

Abstract

Phytocyanins (PCs), plant-specific blue copper proteins, are crucial for various biological processes during plant development. However, a comprehensive characterization of the soybean PC gene family (GmPC) is lacking. In this study, we performed genome-wide screening of soybean PC genes, and 90 PC genes were identified in the soybean genome. Further analysis revealed that the GmPC family was categorized into four subfamilies (stellacyanins, GmSCs; uclacyanins, GmUCs; plantacyanins, GmPLCs; and early nodulin-like proteins, GmENODLs). In-depth analysis revealed that each specific GmPC subfamily exhibited similar characteristics, with segmental duplications playing a major role in expanding the members of GmPC. Additionally, synteny and evolutionary constraint analyses suggested that GmPCs have undergone strong selective pressure for purification during the evolution of soybeans. The promoter cis-regulatory elements analysis of GmPCs suggested that GmPCs might play a crucial role in various stress responses. The expression patterns of GmPCs exhibited tissue-specific variations. Moreover, 23 of the GmPCs may be involved in soybean’s response to salt stress. In all, our study presents a systematic overview of GmPC, which not only provides a valuable foundation for further functional investigations of GmPCs, but also offers new insights into the mechanism of soybean salt tolerance. Full article

(This article belongs to the Special Issue Functional Genomics and Molecular Breeding of Soybeans)

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Title: Characterization analysis of GmTCP15 A key regulator of light signaling pathways in soybean
Author: Wu
Highlights: 1.GmTCP15 regulates soybean development by connecting light signals. 2.Prediction of GmTCP15 promoter regulatory elements reveals the mechanism of light and hormone signal regulation, advancing soybean research. 3.GmTCP15 gene expresses in all tissues at different developmental stages, with higher expression during the nutrient growth stage. 4.Genetic polymorphism analysis of GmTCP15 can identify high-quality soybean varieties.

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