Empirical phenotyping and genome-wide association study reveal the association of panicle architecture with yield in Chenopodium quinoa.

Chenopodium quinoa manifests adaptability to grow under varying agro-climatic scenarios. Assessing quinoa germplasm's phenotypic and genetic variability is a prerequisite for introducing it as a potential candidate in cropping systems. Adaptability is the basic outcome of ecological genomics of crop plants. Adaptive variation predicted with a genome-wide association study provides a valuable basis for marker-assisted breeding. Hence, a panel of 72 quinoa plants was phenotyped for agro morphological attributes and association-mapping for distinct imperative agronomic traits. Inter simple sequence repeat (ISSR) markers were employed to assess genetic relatedness and population structure. Heatmap analysis showed three genotypes were early maturing, and six genotypes were attributed for highest yield. The SD-121-07 exhibited highest yield per plant possessing green, glomerulate shaped, compact density panicle with less leaves. However, SJrecm-03 yielded less exhibiting pink, intermediate shape, intermediate density panicles with less leaves. The phenotyping revealed strong correlation of panicle architecture with yield in quinoa. A genome-wide association study unraveled the associations between ISSR makers and agro-morphological traits. Mixed linear modes analysis yielded nine markers associated with eight traits at p ≤ 0.01. Moreover, ISSR markers significantly associated with panicle shape and leafiness were also associated with yield per plant. These findings contribute to the provision of authenticity for marker-assisted selection that ultimately would support quinoa breeding programs.

Selection and validation of reference genes for normalizing qRT‒PCR gene expression studies in Colletotrichum gloeosporioides and interaction with the guava plants.

Quantitative real-time PCR is used to quantify gene expression, even to detect low-level transcripts. It detects and quantifies the inoculum level of fungal pathogens in infected hosts. However, reliable expression profiling data require accurate transcript normalization against a stable reference gene. Hence, using stably expressed reference genes under variable conditions is paramount in gene expression analysis. In the current study, reference genes were selected and validated in Colletotrichum gloeosporioides, a guava canker and dieback pathogen. The reference gene selection and validation in C. gloeosporioides were evaluated for germinated conidia and mycelium (in vitro) and in infected guava (Psidium guajava) (interaction with host plant). The CgCAL gene was determined as a highly stable reference gene, followed by the CgTUB2 in C. gloeosporioides for germinating conidia and mycelium. However, the CgTUB2 gene was determined to be a highly stable reference gene, followed by the CgCAL for expression analysis during its interaction with the plant. Expression profiling revealed stable and constant relative expression patterns of selected reference genes for both PR genes by determining their relative transcript level. This study is the first to describe reference gene selection and validation to quantify target gene expression in C. gloeosporioides.

In silico characterization of differentially expressed short-read nucleotide sequences identified in dieback stress-induced transcriptomic analysis reveals their role as antimicrobial peptides.

We investigated the in silico characterization of short-length nucleotide sequences that were differentially expressed in dieback stress-induced transcriptomic analysis. They displayed homology with C-terminal flanking peptides and defensins-like proteins, revealing their antimicrobial activity. Their predicted fingerprints displayed protein signatures related to antimicrobial peptides. These short-length RGAs have been shown to possess structural motifs such as APLT P-type ATPase, casein kinase II (CK2), protein kinase 3, protein kinase C (PKC), and N-glycosylation site that are the attributes of disease resistance genes. The prediction of arginine and lysine residues in active binding sites in ligand docking analysis prophesied them as antimicrobial peptides due to their strong relation with antimicrobial activity. The in silico structural-functional characterization has predicted their role in resistance against microbial pathogens. Moreover, the predicted antimicrobial peptide regions showed their homology with the signature domain of PR-5-like protein and AMP family Thaumatin.

Phylogenomics resolves the etiology of dieback disease and deciphers Ceratocystis dalbergicans sp.nov., causal agent of Dalbergia sissoo decline.

Dalbergia sissoo is one of the most economically important trees in forestry, agroforestry, and horticulture. This tree species is severely threatened by dieback. Widespread dieback outbreaks and infestations have drastically destroyed billions of D. sissoo trees. Hence, we attempted to resolve the dieback etiology through phylogenomics associated with D. sissoo mortality. The Ceratocystis species was evaluated using morphologically investigated fungal isolates collected from dieback-affected tissue plants. Based on the symptomatology, we have differentiated dieback from Fusarium wilt and concluded that the Ceratocystis fimbriata sensu lato complex is causing shisham dieback in Pakistan. As the Ceratocystis species complex is a cryptic species complex, we used genomics and phylogenetic analysis for deciphering its evolutionary hierarchical order. The pathogen's operational taxonomy was unlocked with the help of phylogenomics, and it was discovered that isolates from D. sissoo represent a species distinct from the other species in the C. fimbriata sensu lato species complex. The name Ceratocystis dalbergicans sp. nov. has been given to the fungus causing dieback disease in D. sissoo.

Mutation introduced in DDTFR10/A gene of ethylene response element-binding protein (EREBP) family through CRISPR/Cas9 genome editing confers increased Fusarium wilt tolerance in tomato.

We investigated the role of the DDTFR10/A gene of the ethylene response element-binding protein (EREBP) family through the CRISPR/Cas9 genome editing approach. The associated role of this gene in tomato fruit ripening was known. The involvement of ripening-regulatory proteins in plant defense has been documented; therefore, to find the involvement of the DDTFR10/A gene in host susceptibility, we introduced the mutation in DDTFR10/A gene through CRISPR/cas9 in the genome of the tomato plant. The 50% biallelic and 50% homozygous mutations were observed in the T0 generation. The CRISPR/Cas9 edited plants showed 40% reduced symptoms of Fusarium wilt compared to control plants (non-edited). The DDTFR10/A gene expression in tomato plants was evaluated against biotic (Fusarium wilt) and abiotic (salinity) stresses, and the upregulated expression of this gene was found under both challenges. However, a comparative increase in DDTFR10/A gene expression was observed in tomato plants upon inoculation with Fusarium oxysporum f. sp. lycopersici. The phenotypic assay performed on edited tomato plants demonstrated the role of the DDTFR10/A gene in contributing toward susceptibility against Fusarium wilt. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01273-6.

Comparative metabolomic profiling and nutritional chemistry of Chenopodium quinoa of diverse panicle architecture and agroecological zones.

Chenopodium quinoa possesses remarkable nutritional value and adaptability to various agroecological conditions. Panicle architecture influences the number of spikelets and grains in a panicle, ultimately leading to productivity and yield. Therefore, this study aimed to investigate the metabolites, nutrients, and minerals in Chenopodium quinoa accessions of varying panicle architecture. Metabolic profiling using liquid chromatography-mass spectrometry (LC-MS) analysis identified seventeen metabolites, including flavonoids, phenolics, fatty acids, terpenoids, phenylbutenoid dimers, amino acids, and saccharides. Eight metabolic compounds were reported in this study for the first time in quinoa. Some metabolites were detected as differentially expressed. The compound (Z)-1-(2,4,5-trimethoxyphenyl) butadiene and chrysin were found only in SPrecm. Sodium ((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxtetrahydrofuran-2-yl) methyl hydrogen phosphate and elenolic acid were detected only in CHEN-33, and quercetin, 3-hydroxyphloretin-3'-C-glucoside, kurarinone, and rosmarinic acid were identified only in D-12175. Variable importance in projection (VIP) scores annotated ten metabolites contributing to variability. Mineral analysis using atomic absorption spectrophotometry indicated that the quantity of magnesium and calcium is high in D-12175. In comparison, SPrecm showed a high quantity of magnesium compared to CHEN-33, while CHEN-33 showed a high quantity of calcium compared to SPrecm. However, the proximate composition showed no significant difference among quinoa accessions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01398-2.

Identification of resistance gene analogs of the NBS-LRR family through transcriptome probing and in silico prediction of the expressome of Dalbergia sissoo under dieback disease stress.

Dalbergia sissoo is an important timber tree, and dieback disease poses a dire threat to it toward extinction. The genomic record of D. sissoo is not available yet on any database; that is why it is challenging to probe the genetic elements involved in stress resistance. Hence, we attempted to unlock the genetics involved in dieback resistance through probing the NBS-LRR family, linked with mostly disease resistance in plants. We analyzed the transcriptome of D. sissoo under dieback challenge through DOP-rtPCR analysis using degenerate primers from conserved regions of NBS domain-encoded gene sequences. The differentially expressed gene sequences were sequenced and in silico characterized for predicting the expressome that contributes resistance to D. sissoo against dieback. The molecular and bioinformatic analyses predicted the presence of motifs including ATP/GTP-binding site motif A (P-loop NTPase domain), GLPL domain, casein kinase II phosphorylation site, and N-myristoylation site that are the attributes of proteins encoded by disease resistance genes. The physicochemical characteristics of identified resistance gene analogs, subcellular localization, predicted protein fingerprints, in silico functional annotation, and predicted protein structure proved their role in disease and stress resistance.

Integrative Pathogenicity Assay and Operational Taxonomy-Based Detection of New Forma Specialis of Fusarium oxysporum Causing Datepalm Wilt.

Pathogenicity-associated genes are highly host-specific and contribute to host-specific virulence. We tailored the traditional Koch's postulates with integrative omics by hypothesizing that the effector genes associated with host-pathogenicity are determinant markers for virulence, and developed Integrative Pathogenicity (IP) postulates for authenticated pathogenicity testing in plants. To set the criteria, we experimented on datepalm (Phoenix dactylifera) for the vascular wilt pathogen and confirmed the pathogen based on secreted in xylem genes (effectors genes) using genomic and transcriptomic approaches, and found it a reliable solution when pathogenicity is in question. The genic regions ITS, TEF1-α, and RPBII of Fusarium isolates were examined by phylogenetic analysis to unveil the validated operational taxonomy at the species level. The hierarchical tree generated through phylogenetic analysis declared the fungal pathogen as Fusarium oxysporum. Moreover, the Fusarium isolates were investigated at the subspecies level by probing the IGS, TEF1-α, and Pgx4 genic regions to detect the forma specialis of F. oxysporum that causes wilt in datepalm. The phylogram revealed a new forma specialis in F. oxysporum that causes vascular wilt in datepalm.

Functional inhibition of the StERF3 gene by dual targeting through CRISPR/Cas9 enhances resistance to the late blight disease in Solanum tuberosum L.

BACKGROUND: Disease-resistant cultivars are the best solution to get their maximum yield potential and avoid fungicide application. There is no doubt about the contribution, and use of R genes (resistance genes) in resistance development in plants, while S genes (susceptibility genes) also hold a strong position in pathogenesis by resistance repression, and their loss of function contributes to enhanced resistance. Hence, we attempted to knock out the function of the StERF3 gene in potatoes through CRISPR/Cas9-based genome editing and investigated the CRISPR/Cas9 approach as strategic control against late blight disease in potato plants. METHODS AND RESULTS: The StERF3 gene was edited in late blight susceptible cv. Lady Rosetta. Full allelic edited plants were identified through DnpI, and N1aIV mediated restriction digestion and then further analyzed through Indel Detection by Amplicon Analysis. Sequence analysis of targeted plants for indel identification showed full allelic editing. The detached leaf assay of full allelic edited plants demonstrated the role of the StERF3 gene in susceptibility to late blight in potatoes. In planta disease assay also showed reduced, slowed, and delayed disease progression in StERF3-loss-of-function mutants compared to wild-type (control) plants. Less fungal biomass was quantified in knockouts through Real-time qPCR that supported less susceptibility of edited plants to late blight. Besides, relatively high expression of pathogens-related genes, StPR1, and StNPR1, were also observed in StERF3-loss-of-function mutants compared to the corresponding control. CONCLUSION: The results showed the functional inhibition of StERF3 genes using the CRISPR/Cas9 approach. The functional knockouts (StERF3 gene-edited potato plants) revealed enhanced resistance against Phytophthora infestans, thereby demonstrating the best strategic control for late blight disease in potato plants.

MSAL-Net: improve accurate segmentation of nuclei in histopathology images by multiscale attention learning network.

BACKGROUND: The digital pathology images obtain the essential information about the patient's disease, and the automated nuclei segmentation results can help doctors make better decisions about diagnosing the disease. With the speedy advancement of convolutional neural networks in image processing, deep learning has been shown to play a significant role in the various analysis of medical images, such as nuclei segmentation, mitosis detection and segmentation etc. Recently, several U-net based methods have been developed to solve the automated nuclei segmentation problems. However, these methods fail to deal with the weak features representation from the initial layers and introduce the noise into the decoder path. In this paper, we propose a multiscale attention learning network (MSAL-Net), where the dense dilated convolutions block captures more comprehensive nuclei context information, and a newly modified decoder part is introduced, which integrates with efficient channel attention and boundary refinement modules to effectively learn spatial information for better prediction and further refine the nuclei cell of boundaries. RESULTS: Both qualitative and quantitative results are obtained on the publicly available MoNuseg dataset. Extensive experiment results verify that our proposed method significantly outperforms state-of-the-art methods as well as the vanilla Unet method in the segmentation task. Furthermore, we visually demonstrate the effect of our modified decoder part. CONCLUSION: The MSAL-Net shows superiority with a novel decoder to segment the touching and blurred background nuclei cells obtained from histopathology images with better performance for accurate decoding.

Neurocognitive Improvement in Patients Undergoing Carotid Endarterectomy for Atherosclerotic Occlusive Carotid Artery Disease.

Objectives: To assess the improvement in neurocognitive functions after carotid endarterectomy (CEA) under local anesthesia (LA) in patients with carotid bifurcation occlusive disease. Place and duration of study: Department of Vascular Surgery, Combined Military Hospital Lahore from January 2013 to January 2015. Patients and Methods: A total of 79 patients with carotid artery occlusive disease, having no history of major stroke, depression, or dementia underwent CEA under LA. Cognitive functions were assessed 3 days before surgery and then 4 weeks and 12 weeks after the surgery using the Addenbrookes cognitive examination (ACE) score and General Practitioner Assessment of Cognition (GPCOG) Score. Results: In ACE score, Attention, Memory, Fluency, Language, and Visuospatial orientation improved by 33.3%, 30.7%, 21.4%, 38.4%, and 31.2%, respectively, by the end of 12 weeks. An overall improvement in neurocognition was 32% (P = 0.03). In GPCOG score, Orientation, Recall, and Memory improved by 33%, 20%, and 100%, respectively, with an overall improvement of 33.3% at the end of 12 weeks (P = 0.02). Conclusion: Both scoring systems show an overall improvement in neurocognition as well as improvements in all the subcategories in each system. Hence, we conclude statistically significant improvement in neurocognitive functions after CEA.