Genetic dissection of ten photosynthesis-related traits based on InDel- and SNP-GWAS in soybean.

KEY MESSAGE: A total of 416 InDels and 112 SNPs were significantly associated with soybean photosynthesis-related traits. GmIWS1 and GmCDC48 might be related to chlorophyll fluorescence and gas-exchange parameters, respectively. Photosynthesis is one of the main factors determining crop yield. A better understanding of the genetic architecture for photosynthesis is of great significance for soybean yield improvement. Our previous studies identified 5,410,112 single nucleotide polymorphisms (SNPs) from the resequencing data of 219 natural soybean accessions. Here, we identified 634,106 insertions and deletions (InDels) from these 219 accessions and used these InDel variations to perform principal component and linkage disequilibrium analysis of this population. The genome-wide association study (GWAS) were conducted on six chlorophyll fluorescence parameters (chlorophyll content, light energy absorbed per reaction center, quantum yield for electron transport, probability that a trapped exciton moves an electron into the electron transport chain beyond primary quinone acceptor, maximum quantum yield of photosystem II primary photochemistry in the dark-adapted state, performance index on absorption basis) and four gas-exchange parameters (intercellular carbon dioxide concentration, stomatal conductance, net photosynthesis rate, transpiration rate) and revealed 416 significant InDels and 112 significant SNPs. Based on GWAS results, GmIWS1 (encoding a transcription elongation factor) and GmCDC48 (encoding a cell division cycle protein) with the highest expression in the mapping region were determined as the candidate genes responsible for chlorophyll fluorescence and gas-exchange parameters, respectively. Further identification of favorable haplotypes with higher photosynthesis, seed weight and seed yield were carried out for GmIWS1 and GmCDC48. Overall, this study revealed the natural variations and candidate genes underlying the photosynthesis-related traits based on abundant phenotypic and genetic data, providing valuable insights into the genetic mechanisms controlling photosynthesis and yield in soybean.

CALCIUM-DEPENDENT PROTEIN KINASE38 regulates flowering time and common cutworm resistance in soybean.

Photoperiod-sensitive plants such as soybean (Glycine max) often face threats from herbivorous insects throughout their whole growth period and especially during flowering; however, little is known about the relationship between plant flowering and insect resistance. Here, we used gene editing, multiple omics, genetic diversity and evolutionary analyses to confirm that the calcium-dependent protein kinase GmCDPK38 plays a dual role in coordinating flowering time regulation and insect resistance of soybean. Haplotype 2 (Hap2)-containing soybeans flowered later and were more resistant to the common cutworm (Spodoptera litura Fabricius) than those of Hap3. gmcdpk38 mutants with Hap3 knocked out exhibited similar flowering and resistance phenotypes as Hap2. Knocking out GmCDPK38 altered numerous flowering- and resistance-related phosphorylated proteins, genes, and metabolites. For example, the S-adenosylmethionine synthase GmSAMS1 was post-translationally upregulated in the gmcdpk38 mutants. GmCDPK38 has abundant genetic diversity in wild soybeans and was likely selected during soybean domestication. We found that Hap2 was mostly distributed at low latitudes and had a higher frequency in cultivars than in wild soybeans, while Hap3 was widely selected at high latitudes. Overall, our results elucidated that the two distinct traits (flowering time and insect resistance) are mediated by GmCDPK38.

GmFtsH25 overexpression increases soybean seed yield by enhancing photosynthesis and photosynthates.

Increasing plant photosynthetic capacity is a promising approach to boost yields, but it is particularly challenging in C3 crops, such as soybean (Glycine max (L.) Merr.). Here, we identified GmFtsH25, encoding a member of the filamentation temperature-sensitive protein H protease family, as a major gene involved in soybean photosynthesis, using linkage mapping and a genome-wide association study. Overexpressing GmFtsH25 resulted in more grana thylakoid stacks in chloroplasts and increased photosynthetic efficiency and starch content, while knocking out GmFtsH25 produced the opposite phenotypes. GmFtsH25 interacted with photosystem I light harvesting complex 2 (GmLHCa2), and this interaction may contribute to the observed enhanced photosynthesis. GmFtsH25 overexpression lines had superior yield traits, such as yield per plant, compared to the wild type and knockout lines. Additionally, we identified an elite haplotype of GmFtsH25, generated by natural mutations, which appears to have been selected during soybean domestication. Our study sheds light on the molecular mechanism by which GmFtsH25 modulates photosynthesis and provides a promising strategy for improving the yields of soybean and other crops.

Ectopic expression of GmRNF1a encoding a soybean E3 ubiquitin ligase affects Arabidopsis silique development and dehiscence.

MAIN CONCLUSION: A soybean E3 ubiquitin ligase, GmRNF1a, may affect pod dehiscence and seed development through MADS family genes. These results would be useful for the study of soybean pod and seed development. Pod dehiscence is one of the critical causes of yield loss in cultivated soybeans, and it is of great significance to understand the molecular mechanisms underlying pod dehiscence in soybeans. In this study, we identified a new RING family member of the E3 ubiquitin ligase, GmRNF1a, which was observed to interact with the MADS-box protein GmAGL1 to regulate siliques dehiscence. Tissue-specific gene expression analysis revealed that GmRNF1a was mainly expressed in flowers and pods in soybean. The subcellular localization assay showed the nuclear and cytoplasmic localization of GmRNF1a. In addition, it was found that GmRNF1a exhibits higher promoter activity in soybean hairy roots as well as in Arabidopsis leaves, flowers, and siliques. Heterologous expression of GmRNF1a in Arabidopsis showed that the transgenic Arabidopsis siliques had a faster maturation rate and cracked earlier than the wild-type plants. The functional and nucleotide diversity analysis suggests that GmRNF1a might play an important role in pod maturation and dehiscence and has been strongly selected for during soybean domestication.

Downregulation of a gibberellin 3ß-hydroxylase enhances photosynthesis and increases seed yield in soybean.

Seed yield, determined mainly by seed numbers and seed weight, is the primary target of soybean breeding. Identifying the genes underlying yield-related traits is of great significance. Through joint linkage mapping and a genome-wide association study for 100-seed weight, we cloned GmGA3ox1, a gene encoding gibberellin 3ß-hydroxylase, which is the key enzyme in the gibberellin synthesis pathway. Genome resequencing identified a beneficial GmGA3ox1 haplotype contributing to high seed weight, which was further confirmed by soybean transformants. CRISPR/Cas9-generated gmga3ox1 mutants showed lower seed weight, but promoted seed yield by increasing seed numbers. The gmga3ox1 mutants reduced gibberellin biosynthesis while enhancing photosynthesis. Knockout of GmGA3ox1 resulted in the upregulation of numerous photosynthesis-related genes, particularly the GmRCA family encoding ribulose-1,5-bispho-sphate carboxylase-oxygenase (Rubisco) activases. The basic leucine zipper transcription factors GmbZIP97 and GmbZIP159, which were both upregulated in the gmga3ox1 mutants and induced by the gibberellin synthesis inhibitor uniconazole, could bind to the promoter of GmRCAß and activate its expression. Analysis of genomic sequences with over 2700 soybean accessions suggested that GmGA3ox1 is being gradually utilized in modern breeding. Our results elucidated the important role of GmGA3ox1 in soybean yield. These findings reveal important clues for future high-yield breeding in soybean and other crops.

Identification of superior haplotypes in a diverse natural population for breeding desirable plant height in soybean.

KEY MESSAGE: Plant height of soybean is associated with a haplotype block on chromosome 19, which classified 211 soybean accessions into five distinct groups showing significant differences for the target trait. Genetic variation is pivotal for crop improvement. Natural populations are precious genetic resources. However, efficient strategies for the targeted utilization of these resources for quantitative traits, such as plant height (PH), are scarce. Being an important agronomic trait associated with soybean yield and quality, it is imperative to unravel the genetic mechanisms underlying PH in soybean. Here, a genome-wide association study (GWAS) was performed to identify single nucleotide polymorphisms (SNPs) significantly associated with PH in a natural population of 211 cultivated soybeans, which was genotyped with NJAU 355 K Soy SNP Array and evaluated across six environments. A total of 128 SNPs distributed across 17 chromosomes were found to be significantly associated with PH across six environments and a combined environment. Three significant SNPs were consistently identified in at least three environments on Chr.02 (AX-93958260), Chr.17 (AX-94154834), and Chr.19 (AX-93897200). Genomic regions of ~ 130 kb flanking these three consistent SNPs were considered as stable QTLs, which included 169 genes. Of these, 22 genes (including Dt1) were prioritized and defined as putative candidates controlling PH. The genomic region flanking 12 most significant SNPs was in strong linkage disequilibrium (LD). These SNPs formed a single haplotype block containing five haplotypes for PH, namely Hap-A, Hap-B, Hap-C, Hap-D, and Hap-E. Deployment of such superior haplotypes in breeding programs will enable development of improved soybean varieties with desirable plant height.

Soybean CALCIUM-DEPENDENT PROTEIN KINASE17 Positively Regulates Plant Resistance to Common Cutworm (Spodoptera litura Fabricius).

Soybean is frequently attacked by herbivorous pests throughout the growth period. Exploring anti-insect genes to improve insect resistance in soybean is an important soybean breeding goal. Here, we cloned and characterized the gene for a quantitative trait locus (QTL) related to insect resistance, Glyma.06g189600, which encodes CALCIUM-DEPENDENT PROTEIN KINASE17 (GmCDPK17) in soybean. The pairwise sequence alignment analysis revealed that the presumed protein of GmCDPK17 shares 52.06% similarity with that of GmCDPK38, a known negative regulatory gene of insect resistance in soybean. Ectopic expression of GmCDPK17 and GmCDPK38 restored the phenotypes of the Arabidopsis insect-susceptible mutant cpk10 and insect-resistant mutant cpk28, respectively. Moreover, transgenic hairy roots of the soybean cultivar Jack were generated by Agrobacterium-mediated transformation. Overexpression of GmCDPK17 increased soybean hairy root resistance to common cutworm (CCW), while RNA interference of the gene decreased soybean hairy root resistance to CCW. Sequencing data from the cultivated and wild soybeans were used to analyze the genetic diversity of GmCDPK17. This gene was subjected to domestication selection. Six and seven haplotypes (Haps) were identified in cultivated and wild soybeans, respectively. The resistance Hap1 is not widely used in cultivated soybeans and is mainly distributed at low latitudes. Accessions with resistance haplotypes of the GmCDPK17 and GmCDPK38 genes showed high resistance to CCW. Altogether, we revealed a novel positive regulatory insect resistance gene, GmCDPK17, which may further improve insect resistance in soybean.

Genetic dissection of yield-related traits via genome-wide association analysis across multiple environments in wild soybean (Glycine soja Sieb. and Zucc.).

MAIN CONCLUSION: A total of 41 SNPs were identified as significantly associated with five yield-related traits in wild soybean populations across multiple environments, and the candidate gene GsCID1 was found to be associated with seed weight. These results may facilitate improvements in cultivated soybean. Crop-related wild species contain new sources of genetic diversity for crop improvement. Wild soybean (Glycine soja Sieb. and Zucc.) is the progenitor of cultivated soybean [Glycine max (L.) Merr.] and can be used as an essential genetic resource for yield improvements. In this research, using genome-wide association study (GWAS) in 96 out of 113 wild soybean accessions with 114,090 single nucleotide polymorphisms (SNPs) (with minor allele frequencies ≤ 0.05), SNPs associated with five yield-related traits were identified across multiple environments. In total, 41 SNPs were significantly associated with the traits in two or more environments (significance threshold P ≤ 8.76 × 10-6), with 29, 7, 3, and 2 SNPs detected for 100-seed weight (SW), maturity time (MT), seed yield per plant (SY) and flowering time (FT), respectively. BLAST search against the Glycine soja W05 reference genome was performed, 20 candidate genes were identified based on these 41 significant SNPs. One candidate gene, GsCID1 (Glysoja.04g010563), harbored two significant SNPs-AX-93713187, with a non-synonymous mutation, and AX-93713188, with a synonymous mutation. GsCID1 was highly expressed during seed development based on public information resources. The polymorphisms in this gene were associated with SW. We developed a derived cleaved amplified polymorphic sequence (dCAPS) marker for GsCID1 that was highly associated with SW and was validated as a functional marker. In summary, the revealed SNPs/genes are useful for understanding the genetic architecture of yield-related traits in wild soybean, which could be used as a potential exotic resource to improve cultivated soybean yields.

Knockdown of GmVQ58 encoding a VQ motif-containing protein enhances soybean resistance to the common cutworm (Spodoptera litura Fabricius).

Plants have evolved complex defense mechanisms to withstand insect attack. Identification of plant endogenous insect resistance genes is of great significance for understanding plant-herbivore interactions and improving crop insect resistance. Soybean (Glycine max (L.) Merr.) is an important crop that is often attacked by the common cutworm (CCW) (Spodoptera litura Fabricius). In this study, based on our transcriptomic data, the gene GmVQ58, encoding a FxxxVQxxTG (VQ) motif-containing protein, was cloned and characterized. This gene showed the highest expression in the leaves and roots and was up-regulated significantly after CCW attack. Constitutive expression of GmVQ58 rescued the susceptibility of an Arabidopsis mutant to CCW, and interference of GmVQ58 in soybean hairy roots enhanced the resistance to CCW. Furthermore, GmVQ58 was localized to the nucleus and physically interacted with the transcription factor GmWRKY32. The expression of two defense-related genes, GmN:IFR and GmVSPß, was up-regulated in GmVQ58-RNAi lines. Additionally, the promoter region of GmVQ58 was likely selected during domestication, resulting in different expression patterns in cultivated soybeans relative to wild soybeans. These results suggest that silencing GmVQ58 confers soybean resistance to CCW.

Overexpression of a soybean YABBY gene, GmFILa, causes leaf curling in Arabidopsis thaliana.

BACKGROUND: YABBY genes play important roles in the growth and polar establishment of lateral organs such as leaves and floral organs in angiosperms. However, the functions of YABBY homologous genes are largely unknown in soybean. RESULTS: In this study, we identified GmFILa encoding a YABBY transcription factor belonging to FIL subfamily. In situ mRNA hybridization analysis indicated that GmFILa had specific expression patterns in leaf as well as in flower bud primordia. Ectopic expression of GmFILa in Arabidopsis thaliana altered the partial abaxialization of the adaxial epidermises of leaves. Besides, GmFILa transgenic plants also exhibited longer flowering period and inhibition of shoot apical meristem (SAM) development compared to the wild type plants. Digital expression data and quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that the expression of GmFILa was induced by biotic and abiotic stresses and hormone treatments. Transcriptome analysis suggested that overexpressing GmFILa yielded 82 significant differentially expressed genes (DEGs) in Arabidopsis leaves, which can be classified into transcription factors, transporters, and genes involved in growth and development, metabolism, signal transduction, redox reaction and stress response. CONCLUSIONS: These results not only demonstrate the roles of GmFILa involved in leaf adaxial-abaxial polarity in Arabidopsis, but also help to reveal the molecular regulatory mechanism of GmFILa based on the transcriptomic data.

Genome-wide association study, haplotype analysis, and genomic prediction reveal the genetic basis of yield-related traits in soybean (Glycine max L.).

Identifying the genetic components underlying yield-related traits in soybean is crucial for improving its production and productivity. Here, 211 soybean genotypes were evaluated across six environments for four yield-related traits, including seed yield per plant (SYP), number of pods per plant number of seeds per plant and 100-seed weight (HSW). Genome-wide association study (GWAS) and genomic prediction (GP) analyses were performed using 12,617 single nucleotide polymorphism markers from NJAU 355K SoySNP Array. A total of 57 SNPs were significantly associated with four traits across six environments and a combined environment using five Genome-wide association study models. Out of these, six significant SNPs were consistently identified in more than three environments using multiple GWAS models. The genomic regions (±670 kb) flanking these six consistent SNPs were considered stable QTL regions. Gene annotation and in silico expression analysis revealed 15 putative genes underlying the stable QTLs that might regulate soybean yield. Haplotype analysis using six significant SNPs revealed various allelic combinations regulating diverse phenotypes for the studied traits. Furthermore, the GP analysis revealed that accurate breeding values for the studied soybean traits is attainable at an earlier generation. Our study paved the way for increasing soybean yield performance within a short breeding cycle.

Genome-Wide DNA Methylation Analysis of Soybean Curled-Cotyledons Mutant and Functional Evaluation of a Homeodomain-Leucine Zipper (HD-Zip) I Gene GmHDZ20.

DNA methylation is a major, conserved epigenetic modification that influences many biological processes. Cotyledons are specialized tissues that provide nutrition for seedlings at the early developmental stage. To investigate the patterns of genomic DNA methylation of germinated cotyledons in soybean (Glycine max) and its effect on cotyledon development, we performed a genome-wide comparative analysis of DNA methylation between the soybean curled-cotyledons (cco) mutant, which has abnormal cotyledons, and its corresponding wild type (WT) by whole-genome bisulfite sequencing. The cco mutant was methylated at more sites but at a slightly lower level overall than the WT on the whole-genome level. A total of 46 CG-, 92 CHG-, and 9723 CHH- (H = A, C, or T) differentially methylated genes (DMGs) were identified in cotyledons. Notably, hypomethylated CHH-DMGs were enriched in the gene ontology term "sequence-specific DNA binding transcription factor activity." We selected a DMG encoding a homeodomain-leucine zipper (HD-Zip) I subgroup transcription factor (GmHDZ20) for further functional characterization. GmHDZ20 localized to the nucleus and was highly expressed in leaf and cotyledon tissues. Constitutive expression of GmHDZ20 in Arabidopsis thaliana led to serrated rosette leaves, shorter siliques, and reduced seed number per silique. A yeast two-hybrid assay revealed that GmHDZ20 physically interacted with three proteins associated with multiple aspects of plant growth. Collectively, our results provide a comprehensive study of soybean DNA methylation in normal and aberrant cotyledons, which will be useful for the identification of specific DMGs that participate in cotyledon development, and also provide a foundation for future in-depth functional study of GmHDZ20 in soybean.

QTL Mapping for Protein and Sulfur-Containing Amino Acid Contents Using a High-Density Bin-Map in Soybean (Glycine max L. Merr.).

Soybean provides essential protein and amino acids for humans and animals, while sulfur-containing amino acids (SAA), including methionine (Met) and cysteine (Cys), are very limited. In this study, we constructed a high-density bin-map with 3420 bin markers using 676 857 SNPs of a recombinant-inbred line (RIL) population derived from a cross between Kefeng no. 1 and Nannong 1138-2. Quantitative trait loci (QTL) mapping was performed for Cys, Met, SAA, and the protein content using this high-density bin-map. Twenty-five QTLs linked to these four traits were identified, and four genomic regions located on chromosomes (Chr) 07, 08, 15, and 20 were overlapped by multiple QTLs. Among them, bin 115-124 located on Chr 15 was associated with all four traits and was a novel locus with a high LOD value. These findings will provide a basis for nutritional quality improvement using marker-assisted selection breeding and clarify the genetic mechanisms of SAA and protein in soybean.

Identification of Loci and Candidate Genes Responsible for Pod Dehiscence in Soybean via Genome-Wide Association Analysis Across Multiple Environments.

Pod dehiscence (shattering) is the main cause of serious yield loss during the soybean mechanical harvesting process. A better understanding of the genetic architecture and molecular mechanisms of pod dehiscence is of great significance for soybean breeding. In this study, genome-wide association analysis (GWAS) with NJAU 355K SoySNP array was performed to detect single nucleotide polymorphisms (SNPs) associated with pod dehiscence in an association panel containing 211 accessions across five environments. A total of 163 SNPs were identified as significantly associated with pod dehiscence. Among these markers, 136 SNPs identified on chromosome 16 were located in the known QTL qPDH1. One, one, three, eleven, three, one, three, three and one SNPs were distributed on chromosome 1, 4, 6, 8, 9, 11, 17, 18, and 20, respectively. Favorable SNPs and six haplotypes were identified based on ten functional SNPs; among those Hap2 and Hap3 were considered as optimal haplotypes. In addition, based on GWAS results, the candidate gene Glyma09g06290 was identified. Quantitative real-time PCR (qRT-PCR) results and polymorphism analysis suggested that Glyma09g06290 might be involved in pod dehiscence. Furthermore, a derived cleaved amplified polymorphic sequences (dCAPS) marker for Glyma09g06290 was developed. Overall, the loci and genes identified in this study will be helpful in breeding soybean accessions resistant to pod dehiscence.

Genome-Wide Analysis of Soybean LATERAL ORGAN BOUNDARIES Domain-Containing Genes: A Functional Investigation of GmLBD12.

Plant-specific () genes play critical roles in various plant growth and development processes. However, the number and characteristics of genes in soybean [ (L.) Merr.] remain unknown. Here, we identified 90 homologous genes in the soybean genome that phylogenetically clustered into two classes (I and II). The majority of the genes were evenly distributed across all 20 soybean chromosomes, and 77 (81.11%) of them were detected in segmental duplicated regions. Furthermore, the exon-intron organization and motif composition for each were analyzed. A close phylogenetic relationship was identified between the soybean genes and 41 previously reported genes of different plants in the same group, providing insights into their putative functions. Expression analysis indicated that more than half of the genes were expressed, with the two gene classes showing differential tissue expression characteristics; in addition, they were differentially induced by biotic and abiotic stresses. To further explore the functions of genes in soybean, was selected for functional characterization. GmLBD12 was mainly localized to the nucleus and showed high expression in root and seed tissues. Overexpressing in (L.) Heynh resulted in increases in lateral root (LR) number and plant height. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that was induced by drought, salt, cold, indole acetic acid (IAA), abscisic acid (ABA), and salicylic acid SA treatments. This study provides the first comprehensive analysis of the soybean gene family and a valuable foundation for future functional studies of genes.