A Multi-Year, Multi-Cultivar Approach to Differential Expression Analysis of High- and Low-Protein Soybean (Glycine max).

Soybean (Glycine max (L.) Merr.) is among the most valuable crops based on its nutritious seed protein and oil. Protein quality, evaluated as the ratio of glycinin (11S) to ß-conglycinin (7S), can play a role in food and feed quality. To help uncover the underlying differences between high and low protein soybean varieties, we performed differential expression analysis on high and low total protein soybean varieties and high and low 11S soybean varieties grown in four locations across Eastern and Western Canada over three years (2018-2020). Simultaneously, ten individual differential expression datasets for high vs. low total protein soybeans and ten individual differential expression datasets for high vs. low 11S soybeans were assessed, for a total of 20 datasets. The top 15 most upregulated and the 15 most downregulated genes were extracted from each differential expression dataset and cross-examination was conducted to create shortlists of the most consistently differentially expressed genes. Shortlisted genes were assessed for gene ontology to gain a global appreciation of the commonly differentially expressed genes. Genes with roles in the lipid metabolic pathway and carbohydrate metabolic pathway were differentially expressed in high total protein and high 11S soybeans in comparison to their low total protein and low 11S counterparts. Expression differences were consistent between East and West locations with the exception of one, Glyma.03G054100. These data are important for uncovering the genes and biological pathways responsible for the difference in seed protein between high and low total protein or 11S cultivars.

Human-Soybean Allergies: Elucidation of the Seed Proteome and Comprehensive Protein-Protein Interaction Prediction.

The soybean crop, Glycine max (L.) Merr., is consumed by humans, Homo sapiens, worldwide. While the respective bodies of literature and -omics data for each of these organisms are extensive, comparatively few studies investigate the molecular biological processes occurring between the two. We are interested in elucidating the network of protein-protein interactions (PPIs) involved in human-soybean allergies. To this end, we leverage state-of-the-art sequence-based PPI predictors amenable to predicting the enormous comprehensive interactome between human and soybean. A network-based analytical approach is proposed, leveraging similar interaction profiles to identify candidate allergens and proteins involved in the allergy response. Interestingly, the predicted interactome can be explored from two complementary perspectives: which soybean proteins are predicted to interact with specific human proteins and which human proteins are predicted to interact with specific soybean proteins. A total of eight proteins (six specific to the human proteome and two to the soy proteome) have been identified and supported by the literature to be involved in human health, specifically related to immunological and neurological pathways. This study, beyond generating the most comprehensive human-soybean interactome to date, elucidated a soybean seed interactome and identified several proteins putatively consequential to human health.

Identifying new variation at the J locus, previously identified as e6, in long juvenile 'Paranagoiana' soybean.

KEY MESSAGE: A previously identified soybean maturity locus, E6, is discovered to be J, with the long juvenile allele in Paranagoiana now deemed j-x. Soybean grown at latitudes of ~20° or lower can produce lower grain yields due to the short days. This limitation can be overcome by using the long juvenile trait (LJ) which delays flowering under short day conditions. Two LJ loci have been mapped to the same location on Gm04, J and E6. The objective of this research was to investigate the e6 allele in 'Paranagoiana' and determine if E6 and J are the same locus or linked loci. KASP markers showed that e6 lines did not have the j-1 allele of LJ PI 159925. A population fixed for E1 but segregating for E6, with e6 introgressed from Paranagoiana, showed single gene control for flowering and maturity under short days. Sequencing Glyma.04G050200, the J gene, with long amplification Taq found that the e6 line 'Paranagoiana' contains a Ty1-copia retrotransposon of ~10,000 bp, inserted within exon 4. PCR amplification of the cDNA of Glyma.04G050200 also showed differences between the mRNA sequences (presence of insertion in j-x). Hence, we conclude that the loci E6 and J are one locus and deem this new variation found in Paranagoiana as j-x.

P-TarPmiR accurately predicts plant-specific miRNA targets.

microRNAs (miRNAs) are small non-coding ribonucleic acids that post-transcriptionally regulate gene expression through the targeting of messenger RNA (mRNAs). Most miRNA target predictors have focused on animal species and prediction performance drops substantially when applied to plant species. Several rule-based miRNA target predictors have been developed in plant species, but they often fail to discover new miRNA targets with non-canonical miRNA-mRNA binding. Here, the recently published TarDB database of plant miRNA-mRNA data is leveraged to retrain the TarPmiR miRNA target predictor for application on plant species. Rigorous experiment design across four plant test species demonstrates that animal-trained predictors fail to sustain performance on plant species, and that the use of plant-specific training data improves accuracy depending on the quantity of plant training data used. Surprisingly, our results indicate that the complete exclusion of animal training data leads to the most accurate plant-specific miRNA target predictor indicating that animal-based data may detract from miRNA target prediction in plants. Our final plant-specific miRNA prediction method, dubbed P-TarPmiR, is freely available for use at http://ptarpmir.cu-bic.ca . The final P-TarPmiR method is used to predict targets for all miRNA within the soybean genome. Those ranked predictions, together with GO term enrichment, are shared with the research community.

Species-specific microRNA discovery and target prediction in the soybean cyst nematode.

The soybean cyst nematode (SCN) is a devastating pathogen for economic and food security considerations. Although the SCN genome has recently been sequenced, the presence of any miRNA has not been systematically explored and reported. This paper describes the development of a species-specific SCN miRNA discovery pipeline and its application to the SCN genome. Experiments on well-documented model nematodes (Caenorhabditis elegans and Pristionchus pacificus) are used to tune the pipeline's hyperparameters and confirm its recall and precision. Application to the SCN genome identifies 3342 high-confidence putative SCN miRNA. Prediction specificity within SCN is confirmed by applying the pipeline to RNA hairpins from known exonic regions of the SCN genome (i.e., sequences known to not be miRNA). Prediction recall is confirmed by building a positive control set of SCN miRNA, based on a limited deep sequencing experiment. Interestingly, a number of novel miRNA are predicted to be encoded within the intronic regions of effector genes, known to be involved in SCN parasitism, suggesting that these miRNA may also be involved in the infection process or virulence. Beyond miRNA discovery, gene targets within SCN are predicted for all high-confidence novel miRNA using a miRNA:mRNA target prediction system. Lastly, cross-kingdom miRNA targeting is investigated, where putative soybean mRNA targets are identified for novel SCN miRNA. All predicted miRNA and gene targets are made available in appendix and through a Borealis DataVerse open repository ( https://borealisdata.ca/dataset.xhtml?persistentId=doi:10.5683/SP3/30DEXA ).

Large-scale data mining pipeline for identifying novel soybean genes involved in resistance against the soybean cyst nematode.

The soybean cyst nematode (SCN) [Heterodera glycines Ichinohe] is a devastating pathogen of soybean [Glycine max (L.) Merr.] that is rapidly becoming a global economic issue. Two loci conferring SCN resistance have been identified in soybean, Rhg1 and Rhg4; however, they offer declining protection. Therefore, it is imperative that we identify additional mechanisms for SCN resistance. In this paper, we develop a bioinformatics pipeline to identify protein-protein interactions related to SCN resistance by data mining massive-scale datasets. The pipeline combines two leading sequence-based protein-protein interaction predictors, the Protein-protein Interaction Prediction Engine (PIPE), PIPE4, and Scoring PRotein INTeractions (SPRINT) to predict high-confidence interactomes. First, we predicted the top soy interacting protein partners of the Rhg1 and Rhg4 proteins. Both PIPE4 and SPRINT overlap in their predictions with 58 soybean interacting partners, 19 of which had GO terms related to defense. Beginning with the top predicted interactors of Rhg1 and Rhg4, we implement a "guilt by association" in silico proteome-wide approach to identify novel soybean genes that may be involved in SCN resistance. This pipeline identified 1,082 candidate genes whose local interactomes overlap significantly with the Rhg1 and Rhg4 interactomes. Using GO enrichment tools, we highlighted many important genes including five genes with GO terms related to response to the nematode (GO:0009624), namely, Glyma.18G029000, Glyma.11G228300, Glyma.08G120500, Glyma.17G152300, and Glyma.08G265700. This study is the first of its kind to predict interacting partners of known resistance proteins Rhg1 and Rhg4, forming an analysis pipeline that enables researchers to focus their search on high-confidence targets to identify novel SCN resistance genes in soybean.

A Broad Review of Soybean Research on the Ongoing Race to Overcome Soybean Cyst Nematode.

Plant pathogens greatly impact food security of the ever-growing human population. Breeding resistant crops is one of the most sustainable strategies to overcome the negative effects of these biotic stressors. In order to efficiently breed for resistant plants, the specific plant-pathogen interactions should be understood. Soybean is a short-day legume that is a staple in human food and animal feed due to its high nutritional content. Soybean cyst nematode (SCN) is a major soybean stressor infecting soybean worldwide including in China, Brazil, Argentina, USA and Canada. There are many Quantitative Trait Loci (QTLs) conferring resistance to SCN that have been identified; however, only two are widely used: rhg1 and Rhg4. Overuse of cultivars containing these QTLs/genes can lead to SCN resistance breakdown, necessitating the use of additional strategies. In this manuscript, a literature review is conducted on research related to soybean resistance to SCN. The main goal is to provide a current understanding of the mechanisms of SCN resistance and list the areas of research that could be further explored.

GmSWEET29 and Paralog GmSWEET34 Are Differentially Expressed between Soybeans Grown in Eastern and Western Canada.

Over the past two decades soybeans grown in western Canada have persistently had lower seed protein than those grown in eastern Canada. To understand the discrepancy in seed protein content between eastern- and western-grown soybeans, RNA-seq and differential expression analysis have been investigated. Ten soybean genotypes, ranging from low to high in seed protein content, were grown in four locations across eastern (Ottawa) and western (Morden, Brandon, and Saskatoon) Canada. Differential expression analysis revealed 34 differentially expressed genes encoding Glycine max Sugars Will Eventually be Exported Transporters (GmSWEETs), including paralogs GmSWEET29 and GmSWEET34 (AtSWEET2 homologs) that were consistently upregulated across all ten genotypes in each of the western locations over three years. GmSWEET29 and GmSWEET34 are likely candidates underlying the lower seed protein content of western soybeans. GmSWEET20 (AtSWEET12 homolog) was downregulated in the western locations and may also play a role in lower seed protein content. These findings are valuable for improving soybean agriculture in western growing regions, establishing more strategic and efficient agricultural practices.

Novel QTL for Low Seed Cadmium Accumulation in Soybean.

Soybean is a valuable crop, used in animal feed and for human consumption. Selecting soybean cultivars with low seed cadmium (Cd) concentration is important for the purpose of minimizing the transfer of Cd into the human body. To ensure international trade, farmers need to produce soybean that meets the European Union (EU) Cd limit of 0.2 mg kg-1. In this study, we evaluated two populations of recombinant inbred lines (RILs), X5154 and X4050, for seed Cd accumulation. Linkage maps were constructed with 325 and 280 polymorphic simple sequence repeat (SSR) markers, respectively, and used to identify a novel minor quantitative trait locus (QTL) on chromosome 13 in the X4050 population between SSR markers Satt522 and Satt218. Based on a gene ontology search within the QTL region, seven genes were identified as candidates responsible for low seed Cd accumulation, including Glyma.13G308700 and Glyma.13G309100. In addition, we confirmed the known major gene, Cda1, in the X5154 population and developed KASP and CAPS/dCAPS allele-specific markers for efficient marker-assisted breeding for Cda1.