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.

Dissecting nematode resistance regions in soybean revealed pleiotropic effect of soybean cyst and reniform nematode resistance genes.

Reniform nematode (RN, Rotylenchulus reniformis Linford & Oliveira) has emerged as one of the most important plant parasitic nematodes of soybean [Glycine max (L.) Merr.]. Planting resistant varieties is the most effective strategy for nematode management. The objective of this study was to identify quantitative trait loci (QTL) for RN resistance in an exotic soybean line, PI 438489B, using two linkage maps constructed from the Universal Soybean Linkage Panel (USLP 1.0) and next-generation whole-genome resequencing (WGRS) technology. Two QTL controlling RN resistance were identified-the soybean cyst nematode (SCN, Heterodera glycines) resistance gene GmSNAP18 at the rhg1 locus and its paralog GmSNAP11. Strong association between resistant phenotype and haplotypes of the GmSNAP11 and GmSNAP18 was observed. The results indicated that GmSNAP11 possibly could have epistatic effect on GmSNAP18, or vice versa, with the presence of a significant correlation in RN resistance of rhg1-a GmSNAP18 vs. rhg1-b GmSNAP18. Most importantly, our preliminary data suggested that GmSNAP18 and GmSNAP11 proteins physically interact in planta, suggesting that they belong to the same pathway for resistance. Unlike GmSNAP18, no indication of GmSNAP11 copy number variation was found. Moreover, gene-based single nucleotide polymorphism (SNP) markers were developed for rapid detection of RN or SCN resistance at these loci. Our analysis substantiates synergic interaction between GmSNAP11 and GmSNAP18 genes and confirms their roles in RN as well as SCN resistance. These results could contribute to a better understanding of evolution and subfunctionalization of genes conferring resistance to multiple nematode species and provide a framework 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.

Genome Editing in Plants: Exploration of Technological Advancements and Challenges.

Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility. In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs. Successful examples where CRISPR/Cas approach has been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. The review highlights the challenges to implement the genome editing in polyploid crop plants like wheat, canola, and sugarcane. Challenges for plants difficult to transform and germline-specific gene expression have been discussed. We have also discussed the notable progress with multi-target editing approaches based on polycistronic tRNA processing, Csy4 endoribonuclease, intron processing, and Drosha ribonuclease. Potential to edit multiple targets simultaneously makes it possible to take up more challenging tasks required to engineer desired crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome-editing will also be discussed. The present review also provides a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis. The information provided here will be useful for the efficient exploration of technological advances in genome editing field for the crop improvement programs.

Genome reorganization of the GmSHMT gene family in soybean showed a lack of functional redundancy in resistance to soybean cyst nematode.

In soybeans, eighteen members constitute the serine hydroxymethyltransferase (GmSHMT) gene family, of which the cytosolic-targeted GmSHMT08c member has been reported to mediate resistance to soybean cyst nematode (SCN). This work presents a comprehensive study of the SHMT gene family members, including synteny, phylogeny, subcellular localizations, haplotypes, protein homology modeling, mutational, and expression analyses. Phylogenetic analysis showed that SHMT genes are divided into four classes reflecting their subcellular distribution (cytosol, nucleus, mitochondrion, and chloroplast). Subcellular localization of selected GmSHMT members supports their in-silico predictions and phylogenetic distribution. Expression and functional analyses showed that GmSHMT genes display many overlapping, but some divergent responses during SCN infection. Furthermore, mutational analysis reveals that all isolated EMS mutants that lose their resistance to SCN carry missense and nonsense mutations at the GmSHMT08c, but none of the Gmshmt08c mutants carried mutations in the other GmSHMT genes. Haplotype clustering analysis using the whole genome resequencing data from a collection of 106 diverse soybean germplams (15X) was performed to identify allelic variants and haplotypes within the GmSHMT gene family. Interestingly, only the cytosolic-localized GmSHMT08c presented SNP clusters that were associated with SCN resistance, supporting our mutational analysis. Although eight GmSHMT members respond to the nematode infestation, functional and mutational analysis has shown the absence of functional redundancy in resistance to SCN. Structural analysis and protein homology modeling showed the presence of spontaneous mutations at important residues within the GmSHMT proteins, suggesting the presence of altered enzyme activities based on substrate affinities. Due to the accumulation of mutations during the evolution of the soybean genome, the other GmSHMT members have undergone neofunctionalization and subfunctionalization events.

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.

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.

Genomic-assisted phylogenetic analysis and marker development for next generation soybean cyst nematode resistance breeding.

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is a serious soybean pest. The use of resistant cultivars is an effective approach for preventing yield loss. In this study, 19,652 publicly available soybean accessions that were previously genotyped with the SoySNP50K iSelect BeadChip were used to evaluate the phylogenetic diversity of SCN resistance genes Rhg1 and Rhg4 in an attempt to identify novel sources of resistance. The sequence information of soybean lines was utilized to develop KASPar (KBioscience Competitive Allele-Specific PCR) assays from single nucleotide polymorphisms (SNPs) of Rhg1, Rhg4, and other novel quantitative trait loci (QTL). These markers were used to genotype a diverse set of 95 soybean germplasm lines and three recombinant inbred line (RIL) populations. SNP markers from the Rhg1 gene were able to differentiate copy number variation (CNV), such as resistant-high copy (PI 88788-type), low copy (Peking-type), and susceptible-single copy (Williams 82) numbers. Similarly, markers for the Rhg4 gene were able to detect Peking-type (resistance) genotypes. The phylogenetic information of SCN resistance loci from a large set of soybean accessions and the gene/QTL specific markers that were developed in this study will accelerate SCN resistance breeding programs.

A Sweetpotato Geranylgeranyl Pyrophosphate Synthase Gene, IbGGPS, Increases Carotenoid Content and Enhances Osmotic Stress Tolerance in Arabidopsis thaliana.

Sweetpotato highly produces carotenoids in storage roots. In this study, a cDNA encoding geranylgeranyl phyrophosphate synthase (GGPS), named IbGGPS, was isolated from sweetpotato storage roots. Green fluorescent protein (GFP) was fused to the C-terminus of IbGGPS to obtain an IbGGPS-GFP fusion protein that was transiently expressed in both epidermal cells of onion and leaves of tobacco. Confocal microscopic analysis determined that the IbGGPS-GFP protein was localized to specific areas of the plasma membrane of onion and chloroplasts in tobacco leaves. The coding region of IbGGPS was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana to obtain transgenic plants. High performance liquid chromatography (HPLC) analysis showed a significant increase of total carotenoids in transgenic plants. The seeds of transgenic and wild-type plants were germinated on an agar medium supplemented with polyethylene glycol (PEG). Transgenic seedlings grew significantly longer roots than wild-type ones did. Further enzymatic analysis showed an increased activity of superoxide dismutase (SOD) in transgenic seedlings. In addition, the level of malondialdehyde (MDA) was reduced in transgenics. qRT-PCR analysis showed altered expressions of several genes involved in the carotenoid biosynthesis in transgenic plants. These data results indicate that IbGGPS is involved in the biosynthesis of carotenoids in sweetpotato storage roots and likely associated with tolerance to osmotic stress.