Plant Genome Editing and the Relevance of Off-Target Changes.

Site-directed nucleases (SDNs) used for targeted genome editing are powerful new tools to introduce precise genetic changes into plants. Like traditional approaches, such as conventional crossing and induced mutagenesis, genome editing aims to improve crop yield and nutrition. Next-generation sequencing studies demonstrate that across their genomes, populations of crop species typically carry millions of single nucleotide polymorphisms and many copy number and structural variants. Spontaneous mutations occur at rates of ∼10-8 to 10-9 per site per generation, while variation induced by chemical treatment or ionizing radiation results in higher mutation rates. In the context of SDNs, an off-target change or edit is an unintended, nonspecific mutation occurring at a site with sequence similarity to the targeted edit region. SDN-mediated off-target changes can contribute to a small number of additional genetic variants compared to those that occur naturally in breeding populations or are introduced by induced-mutagenesis methods. Recent studies show that using computational algorithms to design genome editing reagents can mitigate off-target edits in plants. Finally, crops are subject to strong selection to eliminate off-type plants through well-established multigenerational breeding, selection, and commercial variety development practices. Within this context, off-target edits in crops present no new safety concerns compared to other breeding practices. The current generation of genome editing technologies is already proving useful to develop new plant varieties with consumer and farmer benefits. Genome editing will likely undergo improved editing specificity along with new developments in SDN delivery and increasing genomic characterization, further improving reagent design and application.

Whole-genome characterization and comparative genomics of a novel freshwater cyanobacteria species: Pseudanabaena punensis.

Cyanobacteria are emerging as a potential source of novel, beneficial bioactive compounds. However, some cyanobacteria species can harm water quality and public health through the production of toxins. Therefore, surveying the occurrence and generating genomic resources of cyanobacteria producing harmful compounds could help develop the control methods necessary to manage their growth and limit the release contaminants into the water bodies. Here, we describe a novel strain, Pseudanabaena punensis isolated from the open ends of pipelines supplying freshwater. This isolate was characterized morphologically, biochemically and by whole-genome sequence analysis. We also provide genomic information for P. punensis to help understand and highlight the features unique to this isolate. Morphological and genetic (analysis using 16S rRNA and rbcL genes) data were used to assign this novel strain to phylogenetic and taxonomic groups. The isolate was identified as a filamentous and non-heterocystous cyanobacteria. Based on morphological and 16S rRNA phylogeny, this isolate shares characteristics with the Pseudanabaenaceae family, but remains distinct from well-characterized species suggesting its polyphyletic assemblage. The whole-genome sequence analysis suggests greater genomic and phenotypic plasticity. Genome-wide sequence and comparative genomic analyses, comparing against several closely related species, revealed diverse and important genes associated with synthesizing bioactive compounds, multi-drug resistance pathway, heavy metal resistance, and virulence factors. This isolate also produces several important fatty acids with potential industrial applications. The observations described in this study emphasize both industrial applications and risks associated with the freshwater contamination, and therefore genomic resources provided in this study offer an opportunity for further investigations.

Fine-mapping and characterization of qSCN18, a novel QTL controlling soybean cyst nematode resistance in PI 567516C.

KEY MESSAGE: The qSCN18 QTL from PI 56756C was confirmed and fine-mapped to improve soybean resistance to the SCN population HG Type 2.5.7 using near-isogenic lines carrying recombination crossovers within the QTL region. The QTL underlying resistance was fine-mapped to a 166-Kbp region on chromosome 18, and the candidate genes were selected based on genomic analyses. Soybean cyst nematode (SCN, Heterodera glycines, Ichinohe) is the most devastating pathogen of soybean. Understanding the genetic basis of SCN resistance is crucial for managing this parasite in the field. Two major loci, rhg1 and Rhg4, were previously characterized as valuable resources for SCN resistance. However, their continuous use has caused shifts in the virulence of SCN populations, which can overcome the resistance conferred by these two major loci. Reduced effectiveness became a major concern in the soybean industry due to continuous use of rhg1 for decades. Thus, it is imperative to identify sources of SCN resistance for durable SCN management. A novel QTL qSCN18 was identified in PI567516C. To fine-map qSCN18 and identify resistance genes, a large backcross population was developed. Nineteen near-isogenic lines (NILs) carrying recombination crossovers within the QTL region were identified. The first phase of fine-mapping narrowed the QTL region to 549-Kbp, whereas the second phase confined the region to 166-Kbp containing 23 genes. Two flanking markers, MK-1 and MK-6, were developed and validated to detect the presence of the qSCN18 resistance allele. Haplotype analysis clustered the fine-mapped qSCN18 region from PI 567516C with the cqSCN-007 locus previously mapped in the wild soybean accession PI 468916. The NILs were developed to further characterize the causal gene(s) harbored in this QTL. This study also confirmed the previously identified qSCN18. The results will facilitate marker-assisted selection (MAS) introducing the qSCN18 locus from PI 567516C into high-yielding soybean cultivars with durable resistance to SCN.

Soybean transporter database: A comprehensive database for identification and exploration of natural variants in soybean transporter genes.

Transporters, a class of membrane proteins that facilitate exchange of solutes including diverse molecules and ions across the cellular membrane, are vital component for the survival of all organisms. Understanding plant transporters is important to get insight of the basic cellular processes, physiology, and molecular mechanisms including nutrient uptake, signaling, response to external stress, and many more. In this regard, extensive analysis of transporters predicted in soybean and other plant species was performed. In addition, an integrated database for soybean transporter protein, SoyTD, was developed that will facilitate the identification, classification, and extensive characterization of transporter proteins by integrating expression, gene ontology, conserved domain and motifs, gene structure organization, and chromosomal distribution features. A comprehensive analysis was performed to identify highly confident transporters by integrating various prediction tools. Initially, 7541 transmembrane (TM) proteins were predicted in the soybean genome; out of these, 3306 non-redundant transporter genes carrying two or more transmembrane domains were selected for further analysis. The identified transporter genes were classified according to a standard transporter classification (TC) system. Comparative analysis of transporter genes among 47 plant genomes provided insights into expansion and duplication of transporter genes in land plants. The whole genome resequencing (WGRS) and tissue-specific transcriptome datasets of soybean were integrated to investigate the natural variants and expression profile associated with transporter(s) of interest. Overall, SoyTD provides a comprehensive interface to study genetic and molecular function of soybean transporters. SoyTD is publicly available at http://artemis.cyverse.org/soykb_dev/SoyTD/.

Construction and comparison of three reference-quality genome assemblies for soybean.

We report reference-quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single-nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan-gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40-42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.

Epigenetics and epigenomics: underlying mechanisms, relevance, and implications in crop improvement.

Epigenetics is defined as changes in gene expression that are not associated with changes in DNA sequence but due to the result of methylation of DNA and post-translational modifications to the histones. These epigenetic modifications are known to regulate gene expression by bringing changes in the chromatin state, which underlies plant development and shapes phenotypic plasticity in responses to the environment and internal cues. This review articulates the role of histone modifications and DNA methylation in modulating biotic and abiotic stresses, as well as crop improvement. It also highlights the possibility of engineering epigenomes and epigenome-based predictive models for improving agronomic traits.

Soybean TILLING-by-Sequencing+ reveals the role of novel GmSACPD members in unsaturated fatty acid biosynthesis while maintaining healthy nodules.

Developing soybean lines with high levels of stearic acid is a primary goal of the soybean industry. Most high-stearic-acid soybeans carry different GmSACPD-C mutated alleles. However, due to the dual role of GmSACPD-C in seeds and nodule development, all derived deleterious GmSACPD-C mutant alleles are of extremely poor agronomic value because of defective nodulation. The soybean stearoyl-acyl carrier protein desaturase (GmSACPD) gene family is composed of five members. Comparative genomics analysis indicated that SACPD genes were duplicated and derived from a common ancestor that is still present in chlorophytic algae. Synteny analysis showed the presence of segment duplications between GmSACPD-A/GmSACPD-B, and GmSACPD-C/GmSACPD-D. GmSACPD-E was not contained in any duplicated segment and may be the result of tandem duplication. We developed a TILLING by Target Capture Sequencing (Tilling-by-Sequencing+) technology, a versatile extension of the conventional TILLING by sequencing, and successfully identified 12, 14, and 18 ethyl methanesulfonate mutants at the GmSACPD-A, GmSACPD-B, and GmSACPD-D genes, respectively. Functional analysis of all identified mutants revealed an unprecedented role of GmSACPD-A, GmSACPD-B, and GmSACPD-D in unsaturated fatty acid biosynthesis without affecting nodule development and structure. This discovery will positively impact the development of high-stearic-acid lines to enhance soybean nutritional value without potential developmental tradeoffs.

Identification of new loci for salt tolerance in soybean by high-resolution genome-wide association mapping.

BACKGROUND: Salinity is an abiotic stress that negatively affects soybean [Glycine max (L.) Merr.] seed yield. Although a major gene for salt tolerance was identified and consistently mapped to chromosome (Chr.) 3 by linkage mapping studies, it does not fully explain genetic variability for tolerance in soybean germplasm. In this study, a genome-wide association study (GWAS) was performed to map genomic regions for salt tolerance in a diverse panel of 305 soybean accessions using a single nucleotide polymorphism (SNP) dataset derived from the SoySNP50K iSelect BeadChip. A second GWAS was also conducted in a subset of 234 accessions using another 3.7 M SNP dataset derived from a whole-genome resequencing (WGRS) study. In addition, three gene-based markers (GBM) of the known gene, Glyma03g32900, on Chr. 3 were also integrated into the two datasets. Salt tolerance among soybean lines was evaluated by leaf scorch score (LSS), chlorophyll content ratio (CCR), leaf sodium content (LSC), and leaf chloride content (LCC). RESULTS: For both association studies, a major locus for salt tolerance on Chr. 3 was confirmed by a number of significant SNPs, of which three gene-based SNP markers, Salt-20, Salt14056 and Salt11655, had the highest association with all four traits studied. Also, additional genomic regions on Chrs. 1, 8, and 18 were found to be associated with various traits measured in the second GWAS using the WGRS-derived SNP dataset. CONCLUSIONS: A region identified on Chr. 8 was identified to be associated with all four traits and predicted as a new minor locus for salt tolerance in soybean. The candidate genes harbored in this minor locus may help reveal the molecular mechanism involved in salt tolerance and to improve tolerance in soybean cultivars. The significant SNPs will be useful for marker-assisted selection for salt tolerance in soybean breeding programs.

Whole-genome re-sequencing reveals the impact of the interaction of copy number variants of the rhg1 and Rhg4 genes on broad-based resistance to soybean cyst nematode.

Soybean cyst nematode (SCN) is the most devastating plant-parasitic nematode. Most commercial soybean varieties with SCN resistance are derived from PI88788. Resistance derived from PI88788 is breaking down due to narrow genetic background and SCN population shift. PI88788 requires mainly the rhg1-b locus, while 'Peking' requires rhg1-a and Rhg4 for SCN resistance. In the present study, whole genome re-sequencing of 106 soybean lines was used to define the Rhg haplotypes and investigate their responses to the SCN HG-Types. The analysis showed a comprehensive profile of SNPs and copy number variations (CNV) at these loci. CNV of rhg1 (GmSNAP18) only contributed towards resistance in lines derived from PI88788 and 'Cloud'. At least 5.6 copies of the PI88788-type rhg1 were required to confer SCN resistance, regardless of the Rhg4 (GmSHMT08) haplotype. However, when the GmSNAP18 copies dropped below 5.6, a 'Peking'-type GmSHMT08 haplotype was required to ensure SCN resistance. This points to a novel mechanism of epistasis between GmSNAP18 and GmSHMT08 involving minimum requirements for copy number. The presence of more Rhg4 copies confers resistance to multiple SCN races. Moreover, transcript abundance of the GmSHMT08 in root tissue correlates with more copies of the Rhg4 locus, reinforcing SCN resistance. Finally, haplotype analysis of the GmSHMT08 and GmSNAP18 promoters inferred additional levels of the resistance mechanism. This is the first report revealing the genetic basis of broad-based resistance to SCN and providing new insight into epistasis, haplotype-compatibility, CNV, promoter variation and its impact on broad-based disease resistance in plants.

Avenues of the membrane transport system in adaptation of plants to abiotic stresses.

Abiotic stress imposed by many factors such as: extreme water regimes, adverse temperatures, salinity, and heavy metal contamination result in severe crop yield losses worldwide. Plants must be able to quickly respond to these stresses in order to adapt to their growing conditions and minimize metabolic losses. In this context, transporter proteins play a vital role in regulating stress response mechanisms by facilitating movement of a variety of molecules and ions across the plasma membrane in order to maintain fundamental cellular processes such as ion homeostasis, osmotic adjustment, signal transduction, and detoxification. Aquaporins play a crucial role in alleviating abiotic stress by transporting water and other small molecules to maintain cellular homeostasis. Similarly, other transporter families such as CDF, ZIP, ABC, NHX, HKT, SWEETs, TMTs, and ion channels also contribute to abiotic stress tolerance. Hormones and other signaling molecules are necessary to coordinate responses across different tissues and to precisely regulate molecular trafficking. The present review highlights the current understanding of how membrane transporters orchestrate stress responses in plants. It also provides insights about the importance of these sensing and adaptive mechanisms for ensuring improved sustainable crop production during unfavorable conditions. Finally, this review discusses future prospects for the use of computational tools in constructing signaling networks to improve our understanding of the behavior of transporters under abiotic stress.

Dissecting genomic hotspots underlying seed protein, oil, and sucrose content in an interspecific mapping population of soybean using high-density linkage mapping.

The cultivated [Glycine max (L) Merr.] and wild [Glycine soja Siebold & Zucc.] soybean species comprise wide variation in seed composition traits. Compared to wild soybean, cultivated soybean contains low protein, high oil, and high sucrose. In this study, an interspecific population was derived from a cross between G. max (Williams 82) and G. soja (PI 483460B). This recombinant inbred line (RIL) population of 188 lines was sequenced at 0.3× depth. Based on 91 342 single nucleotide polymorphisms (SNPs), recombination events in RILs were defined, and a high-resolution bin map was developed (4070 bins). In addition to bin mapping, quantitative trait loci (QTL) analysis for protein, oil, and sucrose was performed using 3343 polymorphic SNPs (3K-SNP), derived from Illumina Infinium BeadChip sequencing platform. The QTL regions from both platforms were compared, and a significant concordance was observed between bin and 3K-SNP markers. Importantly, the bin map derived from next-generation sequencing technology enhanced mapping resolution (from 1325 to 50 Kb). A total of five, nine, and four QTLs were identified for protein, oil, and sucrose content, respectively, and some of the QTLs coincided with soybean domestication-related genomic loci. The major QTL for protein and oil were mapped on Chr. 20 (qPro_20) and suggested negative correlation between oil and protein. In terms of sucrose content, a novel and major QTL were identified on Chr. 8 (qSuc_08) and harbours putative genes involved in sugar transport. In addition, genome-wide association using 91 342 SNPs confirmed the genomic loci derived from QTL mapping. A QTL-based haplotype using whole-genome resequencing of 106 diverse soybean lines identified unique allelic variation in wild soybean that could be utilized to widen the genetic base in cultivated soybean.

Mapping and confirmation of loci for salt tolerance in a novel soybean germplasm, Fiskeby III.

KEY MESSAGE: The confirmation of a major locus associated with salt tolerance and mapping of a new locus, which could be beneficial for improving salt tolerance in soybean. Breeding soybean for tolerance to high salt conditions is important in some regions of the USA and world. Soybean cultivar Fiskeby III (PI 438471) in maturity group 000 has been reported to be highly tolerant to multiple abiotic stress conditions, including salinity. In this study, a mapping population of 132 F2 families derived from a cross of cultivar Williams 82 (PI 518671, moderately salt sensitive) and Fiskeby III (salt tolerant) was analyzed to map salt tolerance genes. The evaluation for salt tolerance was performed by analyzing leaf scorch score (LSS), chlorophyll content ratio (CCR), leaf sodium content (LSC), and leaf chloride content (LCC) after treatment with 120 mM NaCl under greenhouse conditions. Genotypic data for the F2 population were obtained using the SoySNP6K Illumina Infinium BeadChip assay. A major allele from Fiskeby III was significantly associated with LSS, CCR, LSC, and LCC on chromosome (Chr.) 03 with LOD scores of 19.1, 11.0, 7.7 and 25.6, respectively. In addition, a second locus associated with salt tolerance for LSC was detected and mapped on Chr. 13 with an LOD score of 4.6 and an R 2 of 0.115. Three gene-based polymorphic molecular markers (Salt-20, Salt14056 and Salt11655) on Chr.03 showed a strong predictive association with phenotypic salt tolerance in the present mapping population. These molecular markers will be useful for marker-assisted selection to improve salt tolerance in soybean.

Comparative genome analysis to identify SNPs associated with high oleic acid and elevated protein content in soybean.

The objective of this study was to determine the genetic relationship between the oleic acid and protein content. The genotypes having high oleic acid and elevated protein (HOEP) content were crossed with five elite lines having normal oleic acid and average protein (NOAP) content. The selected accessions were grown at six environments in three different locations and phenotyped for protein, oil, and fatty acid components. The mean protein content of parents, HOEP, and NOAP lines was 34.6%, 38%, and 34.9%, respectively. The oleic acid concentration of parents, HOEP, and NOAP lines was 21.7%, 80.5%, and 20.8%, respectively. The HOEP plants carried both FAD2-1A (S117N) and FAD2-1B (P137R) mutant alleles contributing to the high oleic acid phenotype. Comparative genome analysis using whole-genome resequencing data identified six genes having single nucleotide polymorphism (SNP) significantly associated with the traits analyzed. A single SNP in the putative gene Glyma.10G275800 was associated with the elevated protein content, and palmitic, oleic, and linoleic acids. The genes from the marker intervals of previously identified QTL did not carry SNPs associated with protein content and fatty acid composition in the lines used in this study, indicating that all the genes except Glyma.10G278000 may be the new genes associated with the respective traits.

Genetic mapping and haplotype analysis of a locus for quantitative resistance to Fusarium graminearum in soybean accession PI 567516C.

KEY MESSAGE: A major novel quantitative disease resistance locus, qRfg_Gm06, for Fusarium graminearum was genetically mapped to chromosome 6. Genomic-assisted haplotype analysis within this region identified three putative candidate genes. Fusarium graminearum causes seed, root rot, and seedling damping-off in soybean which contributes to reduced stands and yield. A cultivar Magellan and PI 567516C were identified with low and high levels of partial resistance to F. graminearum, respectively. Quantitative disease resistance loci (QDRL) were mapped with 241 F7:8 recombinant inbred lines (RILs) derived from a cross of Magellan × PI 567516C. Phenotypic evaluation for resistance to F. graminearum used the rolled towel assay in a randomized incomplete block design. The genetic map was constructed from 927 polymorphic single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers. One major QDRL qRfg_Gm06 was detected and mapped to chromosome 6 with a LOD score of 20.3 explaining 40.2% of the total phenotypic variation. This QDRL was mapped to a ~400 kb genomic region of the Williams 82 reference genome. Genome mining of this region identified 14 putative candidate disease resistance genes. Haplotype analysis of this locus using whole genome re-sequencing (WGRS) of 106 diverse soybean lines narrowed the list to three genes. A SNP genotyping Kompetitive allele-specific PCR (KASP) assay was designed for one of the genes and was validated in a subset of the RILs and all 106 diverse lines.

Molecular mapping and genomics of soybean seed protein: a review and perspective for the future.

KEY MESSAGE: Genetic improvement of soybean protein meal is a complex process because of negative correlation with oil, yield, and temperature. This review describes the progress in mapping and genomics, identifies knowledge gaps, and highlights the need of integrated approaches. Meal protein derived from soybean [Glycine max (L) Merr.] seed is the primary source of protein in poultry and livestock feed. Protein is a key factor that determines the nutritional and economical value of soybean. Genetic improvement of soybean seed protein content is highly desirable, and major quantitative trait loci (QTL) for soybean protein have been detected and repeatedly mapped on chromosomes (Chr.) 20 (LG-I), and 15 (LG-E). However, practical breeding progress is challenging because of seed protein content's negative genetic correlation with seed yield, other seed components such as oil and sucrose, and interaction with environmental effects such as temperature during seed development. In this review, we discuss rate-limiting factors related to soybean protein content and nutritional quality, and potential control factors regulating seed storage protein. In addition, we describe advances in next-generation sequencing technologies for precise detection of natural variants and their integration with conventional and high-throughput genotyping technologies. A syntenic analysis of QTL on Chr. 15 and 20 was performed. Finally, we discuss comprehensive approaches for integrating protein and amino acid QTL, genome-wide association studies, whole-genome resequencing, and transcriptome data to accelerate identification of genomic hot spots for allele introgression and soybean meal protein improvement.

Soybean (Glycine max) SWEET gene family: insights through comparative genomics, transcriptome profiling and whole genome re-sequence analysis.

BACKGROUND: SWEET (MtN3_saliva) domain proteins, a recently identified group of efflux transporters, play an indispensable role in sugar efflux, phloem loading, plant-pathogen interaction and reproductive tissue development. The SWEET gene family is predominantly studied in Arabidopsis and members of the family are being investigated in rice. To date, no transcriptome or genomics analysis of soybean SWEET genes has been reported. RESULTS: In the present investigation, we explored the evolutionary aspect of the SWEET gene family in diverse plant species including primitive single cell algae to angiosperms with a major emphasis on Glycine max. Evolutionary features showed expansion and duplication of the SWEET gene family in land plants. Homology searches with BLAST tools and Hidden Markov Model-directed sequence alignments identified 52 SWEET genes that were mapped to 15 chromosomes in the soybean genome as tandem duplication events. Soybean SWEET (GmSWEET) genes showed a wide range of expression profiles in different tissues and developmental stages. Analysis of public transcriptome data and expression profiling using quantitative real time PCR (qRT-PCR) showed that a majority of the GmSWEET genes were confined to reproductive tissue development. Several natural genetic variants (non-synonymous SNPs, premature stop codons and haplotype) were identified in the GmSWEET genes using whole genome re-sequencing data analysis of 106 soybean genotypes. A significant association was observed between SNP-haplogroup and seed sucrose content in three gene clusters on chromosome 6. CONCLUSION: Present investigation utilized comparative genomics, transcriptome profiling and whole genome re-sequencing approaches and provided a systematic description of soybean SWEET genes and identified putative candidates with probable roles in the reproductive tissue development. Gene expression profiling at different developmental stages and genomic variation data will aid as an important resource for the soybean research community and can be extremely valuable for understanding sink unloading and enhancing carbohydrate delivery to developing seeds for improving yield.

Genetic variants in root architecture-related genes in a Glycine soja accession, a potential resource to improve cultivated soybean.

BACKGROUND: Root system architecture is important for water acquisition and nutrient acquisition for all crops. In soybean breeding programs, wild soybean alleles have been used successfully to enhance yield and seed composition traits, but have never been investigated to improve root system architecture. Therefore, in this study, high-density single-feature polymorphic markers and simple sequence repeats were used to map quantitative trait loci (QTLs) governing root system architecture in an inter-specific soybean mapping population developed from a cross between Glycine max and Glycine soja. RESULTS: Wild and cultivated soybean both contributed alleles towards significant additive large effect QTLs on chromosome 6 and 7 for a longer total root length and root distribution, respectively. Epistatic effect QTLs were also identified for taproot length, average diameter, and root distribution. These root traits will influence the water and nutrient uptake in soybean. Two cell division-related genes (D type cyclin and auxin efflux carrier protein) with insertion/deletion variations might contribute to the shorter root phenotypes observed in G. soja compared with cultivated soybean. Based on the location of the QTLs and sequence information from a second G. soja accession, three genes (slow anion channel associated 1 like, Auxin responsive NEDD8-activating complex and peroxidase), each with a non-synonymous single nucleotide polymorphism mutation were identified, which may also contribute to changes in root architecture in the cultivated soybean. In addition, Apoptosis inhibitor 5-like on chromosome 7 and slow anion channel associated 1-like on chromosome 15 had epistatic interactions for taproot length QTLs in soybean. CONCLUSION: Rare alleles from a G. soja accession are expected to enhance our understanding of the genetic components involved in root architecture traits, and could be combined to improve root system and drought adaptation in soybean.

Core clock, SUB1, and ABAR genes mediate flooding and drought responses via alternative splicing in soybean.

Circadian clocks are a great evolutionary innovation and provide competitive advantage during the day/night cycle and under changing environmental conditions. The circadian clock mediates expression of a large proportion of genes in plants, achieving a harmonious relationship between energy metabolism, photosynthesis, and biotic and abiotic stress responses. Here it is shown that multiple paralogues of clock genes are present in soybean (Glycine max) and mediate flooding and drought responses. Differential expression of many clock and SUB1 genes was found under flooding and drought conditions. Furthermore, natural variation in the amplitude and phase shifts in PRR7 and TOC1 genes was also discovered under drought and flooding conditions, respectively. PRR3 exhibited flooding- and drought-specific splicing patterns and may work in concert with PRR7 and TOC1 to achieve energy homeostasis under flooding and drought conditions. Higher expression of TOC1 also coincides with elevated levels of abscisic acid (ABA) and variation in glucose levels in the morning and afternoon, indicating that this response to abiotic stress is mediated by ABA, endogenous sugar levels, and the circadian clock to fine-tune photosynthesis and energy utilization under stress conditions. It is proposed that the presence of multiple clock gene paralogues with variation in DNA sequence, phase, and period could be used to screen exotic germplasm to find sources for drought and flooding tolerance. Furthermore, fine tuning of multiple clock gene paralogues (via a genetic engineering approach) should also facilitate the development of flooding- and drought-tolerant soybean varieties.

Identification of two additional members of the tRNA isopentenyltransferase family in Physcomitrella patens.

The Physcomitrella patens genome has seven genes apparently coding for the isopentenyltransferase type of tRNA-modifying enzyme, while other organisms have one or two. The predicted sequences have parts that differ significantly from other isopentenyltransferases. Only one of the seven (PpIPT1) has earlier been shown to be expressed. We now report expression of two more, PpIPT4 and PpIPT5. The cloned genes were able to functionally complement a yeast mutant lacking tRNA isopentenyltransferase. Sequencing showed they are related to the earlier studied PpIPT1. The sequences of the three differ mainly from each other in a tRNA-binding area and the 5'-end subcellular targeting motif area. This indicates that, after arising through gene duplication, they have evolved to enable partly different functions.

Development of High-Quality Nuclei Isolation to Study Plant Root-Microbe Interaction for Single-Nuclei Transcriptomic Sequencing in Soybean.

Single-nucleus RNA sequencing (sNucRNA-seq) is an emerging technology that has been rapidly adopted and demonstrated to be a powerful tool for detailed characterization of each cell- and sub cell-types in complex tissues of higher eukaryotes. sNucRNA-seq has also been used to dissect cell-type-specific transcriptional responses to environmental or developmental signals. In plants, this technology is being utilized to identify cell-type-specific trajectories for the study of several tissue types and important traits, including the single-cell dissection of the genetic determinants regulating plant-microbe interactions. The isolation of high-quality nuclei is one of the prerequisite steps to obtain high-quality sNucRNA-seq results. Although nuclei isolation from several plant tissues is well established, this process is highly troublesome when plant tissues are associated with beneficial or pathogenic microbes. For example, root tissues colonized with rhizobium bacteria (nodules), leaf tissue infected with bacterial or fungal pathogens, or roots infected with nematodes pose critical challenges to the isolation of high-quality nuclei and use for downstream application. Therefore, isolation of microbe-free, high-quality nuclei from plant tissues are necessary to avoid clogging or interference with the microfluidic channel (e.g., 10× Genomics) or particle-templated emulsion that are used in sNucRNA-seq platforms. Here, we developed a simple, effective, and efficient method to isolate high-quality nuclei from soybean roots and root nodules, followed by washing out bacterial contamination. This protocol has been designed to be easily implemented into any lab environment, and it can also be scaled up for use with multiple samples and applicable to a variety of samples with the presence of microbes. We validated this protocol by successfully generating a barcoded library using the 10× Genomics microfluidic platform from tissue subjected to this procedure. This workflow was developed to provide an accessible alternative to instrument-based approaches (e.g., fluorescent cell sorting) and will expand the ability of researchers to perform experiments such as sNucRNA-seq and sNucATAC-seq on inherently heterogeneous plant tissue samples.