Genetic Signature Controlling Root System Architecture in Diverse Spring Wheat Germplasm.

Roots are the main sensing organ, initiating multiple signaling pathways in response to abiotic factors, including nutrients, drought, and salt stress. A focus on improving the root system architecture is a key strategy to mitigate these stresses in wheat crop. In the present study, a diversity panel comprising indigenous landraces and historical cultivars from Pakistan was characterized for the root system architecture (RSA) and important loci were identified using a genome-wide association study (GWAS). RSA of the diversity panel was characterized 30 days after sowing in brunch tubes, and root images were taken. A high-throughput root imaging analysis using Rhizovision software was performed by setting the scale to extract the eight RSA traits and four plant biomass-related traits. GWAS identified 323 association signals for 12 root and biomass traits present on all wheat chromosomes, while the most important and reliable genetic loci (based on pleotropic loci and candidate genes) were identified on chromosomes 2A, 2B, 5A, 5D, 6A, 7B, and 7D for RSA. SNP annotation and transcriptome profiling identified nine candidate genes regulating the RSA and plant biomass traits, including ROOTLESS WITH UNDETECTABLE MERISTEM1, MYB TRANSCRIPTION FACTOR4, BRASSINOSTEROID INSENSITIVE1, SLENDER RICE1, AUXIN-RESPONSIVE FACTOR25, SCARECROW, NARROW LEAF2, PIN-FORMED1 AND PHOSPHATE TRANSCRIPTION FACTOR1. This study provided pre-breeding information for deep-rooting genotypes and associated markers that will accelerate the incorporation of such traits in breeding.

Comprehensive insights into the regulatory mechanisms of lncRNA in alkaline-salt stress tolerance in rice.

BACKGROUND: Alkaline-salt is one of the abiotic stresses that slows plant growth and developmental processes and threatens crop yield. Long non-coding RNAs (lncRNAs) are endogenous RNA found in plants that engage in a variety of cellular functions and stress responses. METHOD: lncRNAs act as competing endogenous RNAs (ceRNA) and constitute a new set of gene control. The precise regulatory mechanism by which lncRNAs function as ceRNAs in response to alkaline-salt stress remains unclear. We identified alkaline-salt responsive lncRNAs using transcriptome-wide analysis of two varieties including alkaline-salt tolerant [WD20342 (WD)] and alkaline-salt sensitive [Caidao (CD)] rice cultivar under control and alkaline-salt stress treated [WD20342 (WDT, and Caidao (CDT)] conditions. RESULTS: Investigating the competitive relationships between mRNAs and lncRNAs, we next built a ceRNA network involving lncRNAs based on the ceRNA hypothesis. Expression profiles revealed that a total of 65, 34, and 1549 differentially expressed (DE) lncRNAs, miRNAs, and mRNAs were identified in alkaline-salt tolerant WD (Control) vs. WDT (Treated). Similarly, 75 DE-lncRNAs, 34 DE-miRNAs, and 1725 DE-mRNAs (including up-regulated and down-regulated) were identified in alkaline-salt sensitive CD (Control) vs. CDT (Treated), respectively. An alkaline-salt stress ceRNA network discovered 321 lncRNA-miRNA-mRNA triplets in CD and CDT, with 32 lncRNAs, 121 miRNAs, and 111 mRNAs. Likewise, 217 lncRNA-miRNA-mRNA triplets in WD and WDT revealed the NONOSAT000455-osa_miR5809b-LOC_Os11g01210 triplet with the highest degree as a hub node with the most significant positive correlation in alkaline-salt stress response. CONCLUSION: The results of our investigation indicate that osa-miR5809b is dysregulated and plays a part in regulating the defense response of rice against alkaline-salt stress. Our study highlights the regulatory functions of lncRNAs acting as ceRNAs in the mechanisms underlying alkaline-salt resistance in rice.

Evaluation of sugarcane promising clones based on the morphophysiological traits developed from fuzz.

Sugarcane is one of the critical commercial crops and principal sources of ethanol and sugar worldwide. Unfavorable conditions and poor seed setting rates hinder variety development in sugarcane. Countries like Pakistan directly import fuzz (true seed) and other propagation material from the USA, China, Brazil, etc. In this study, we imported fuzz from China, developed 29 genotypes germinating in the glasshouse, and evaluated at field conditions along with two local checks (CPF-251 and HSF-240). Morphophysiological data were recorded, including plant height (PH), cane length (CL), internodal length (IL), tiller number (TN), brix percentage (B), cane diameter (CD), chlorophyll a (Chl. a), chlorophyll b (Chl. b), and total chlorophyll (T. Chl). Results showed highly significant (p < 0.001) differences among the sugarcane accessions for all the studied traits. High broad-sense heritability (81.89% to 99.91%) was recorded for all the studied parameters. Genetic Advance (GA) ranges from 4.6% to 65.32%. The highest GA was observed for PH (65.32%), followed by CL (63.28%). Chlorophyll leaching assay was also performed at different time points (0, 50, 100, 150, and 200 min). All the genotypes showed the same leaching trend at all times, and better performing genotypes showed less leaching compared to poor performing, indicating the high amount of cutin and wax on the leaf surface. Correlation analysis showed that PH, CL, IL, and TN had significant associations. Principal components analysis (PCA) further confirms these results. Based on PCA and correlation results, PH, CL, IL, and TN can be utilized as a selection criterion for sugarcane improvement. Genotypes such as NS-4a, NS-5, NS-6, NS-8, NS-9, and NS-15 are recommended for future breeding programs related to sugarcane variety development.

CRISPR/Cas9 mediated disruption of Inositol Pentakisphosphate 2-Kinase 1 (TaIPK1) reduces phytic acid and improves iron and zinc accumulation in wheat grains.

Introduction: Phytic acid (PA) is an important antinutrient agent present in cereal grains which reduces the bioavailability of iron and zinc in human body, causing malnutrition. Inositol pentakisphosphate 2- kinase 1 (IPK1) gene has been reported to be an important gene for PA biosynthesis. Objective: A recent genome editing tool CRISPR/Cas9 has been successfully applied to develop biofortified rice by disrupting IPK1 gene, however, it remained a challenge in wheat. The aim of this study was to biofortify wheat using CRISPR/Cas9. Methods: In this study, we isolated 3 TaIPK1 homeologs in wheat designated as TaIPK1.A, TaIPK1.B and TaIPK1.D and found that the expression abundance of TaIPK1.A was stronger in early stages of grain filling. Using CRISPR/Cas9, we have disrupted TaIPK1.A gene in cv. Borlaug-2016 with two guide RNAs targeting the 1st and 2nd exons. Results: We got several genome-edited lines in the T0 generation at frequencies of 12.7% and 10.8%. Sequencing analysis revealed deletion of 1-23 nucleotides and even an addition of 1 nucleotide in various lines. Analysis of the genome-edited lines revealed a significant decrease in the PA content and an increase in iron and zinc accumulation in grains compared with control plants. Conclusion: Our study demonstrates the potential application of CRISPR/Cas9 technique for the rapid generation of biofortified wheat cultivars.

Role of Wheat Phosphorus Starvation Tolerance 1 Genes in Phosphorus Acquisition and Root Architecture.

The wheat plant requires elevated phosphorus levels for its normal growth and yield, but continuously depleting non-renewable phosphorus reserves in the soil is one of the biggest challenges in agricultural production worldwide. The Phosphorus Starvation Tolerance 1 (PSTOL1) gene has been reported to play a key role in efficient P uptake, deeper rooting, and high yield in rice. However, the function of the PSTOL1 gene in wheat is still unclear. In this study, a total of 22 PSTOL1 orthologs were identified in the wheat genome, and found that wheat PSTOL1 orthologs are unevenly distributed on chromosomes, and these genes were under strong purifying selection. Under different phosphorus regimes, wheat PSTOL1 genes showed differential expression patterns in different tissues. These results strengthen the classification of Pakistan-13 as a P-efficient cultivar and Shafaq-06 as a P-inefficient cultivar. Phenotypic characterization demonstrated that Pakistan-13 wheat cultivar has significantly increased P uptake, root length, root volume, and root surface area compared to Shafaq-06. Some wheat PSTOL1 orthologs are co-localized with phosphorus starvation's related quantitative trait loci (QTLs), suggesting their potential role in phosphorus use efficiency. Altogether, these results highlight the role of the wheat PSTOL1 genes in wheat P uptake, root architecture, and efficient plant growth. This comprehensive study will be helpful for devising sustainable strategies for wheat crop production and adaptation to phosphorus insufficiency.

Evaluation of Green Super Rice Lines for Agronomic and Physiological Traits under Salinity Stress.

Rice (Oryza sativa) is an important staple food crop worldwide, especially in east and southeast Asia. About one-third of rice cultivated area is under saline soil, either natural saline soils or irrigation with brackish water. Salinity stress is among the devastating abiotic stresses that not only affect rice growth and crop productivity but also limit its cultivation area globally. Plants adopt multiple tolerance mechanisms at the morphological, physiological, and biochemical levels to tackle salinity stress. To identify these tolerance mechanisms, this study was carried out under both a controlled glass house as well as natural saline field conditions using 22 green super rice (GSR) lines along with two local varieties ("IRRI 6 and Kissan Basmati"). Several morpho-physiological and biochemical parameters along with stress-responsive genes were used as evaluation criteria under normal and salinity stress conditions. Correlation and Principal Component Analysis (PCA) suggested that shoot-related parameters and the salt susceptible index (SSI) can be used for the identification of salt-tolerant genotypes. Based on Agglomerative Hierarchical Cluster (AHC) analysis, two saline-tolerant ("S19 and S20") and saline-susceptible ("S3 and S24") lines were selected for further molecular evaluation. Quantitative RT-PCR was performed, and results showed that expression of 1-5-phosphoribosyl -5-5-phosphoribosyl amino methylidene amino imidazole-4-carboxamide isomerase, DNA repair protein recA, and peptide transporter PTR2 related genes were upregulated in salt-tolerant genotypes, suggesting their potential role in salinity tolerance. However, additional validation using reverse genetics approaches will further confirm their specific role in salt tolerance. Identified saline-tolerant lines in this study will be useful genetic resources for future salinity breeding programs.

Genome-wide and molecular characterization of the DNA replication helicase 2 (DNA2) gene family in rice under drought and salt stress.

Rice plants experience various biotic (such as insect and pest attack) and abiotic (such as drought, salt, heat, and cold etc.) stresses during the growing season, resulting in DNA damage and the subsequent losses in rice production. DNA Replication Helicase/Nuclease2 (DNA2) is known to be involved in DNA replication and repair. In animals and yeast DNA2 are well characterized because it has the abilities of both helicase and nuclease, it plays a crucial role in DNA replication in the nucleus and mitochondrial genomes. However; they are not fully examined in plants due to less focused on plants damage repair. To fill this research gap, the current study focused on the genome-wide identification and characterization of OsDNA2 genes, along with analyses of their transcriptional expression, duplication, and phylogeny in rice. Overall, 17 OsDNA2 members were reported to be found on eight different chromosomes (2, 3, 4, 6, 7, 9, 10, and 11). Among these chromosomes (Chr), Chr4 contained a maximum of six OsDNA2 genes. Based on phylogenetic analysis, the OsDNA2 gene members were clustered into three different groups. Furthermore, the conserved domains, gene structures, and cis-regulatory elements were systematically investigated. Gene duplication analysis revealed that OsDNA2_2 had an evolutionary relationship with OsDNA2_14, OsDNA2_5 with OsDNA2_6, and OsDNA2_1 with OsDNA2_8. Moreover, results showed that the conserved domain (AAA_11 superfamily) were present in the OsDNA2 genes, which belongs to the DEAD-like helicase superfamily. In addition, to understand the post-transcriptional modification of OsDNA2 genes, miRNAs were predicted, where 653 miRNAs were reported to target 17 OsDNA2 genes. The results indicated that at the maximum, OsDNA2_1 and OsDNA2_4 were targeted by 74 miRNAs each, and OsDNA2_9 was less targeted (20 miRNAs). The three-dimensional (3D) structures of 17 OsDNA2 proteins were also predicted. Expression of OsDNA2 members was also carried out under drought and salt stresses, and conclusively their induction indicated the possible involvement of OsDNA2 in DNA repair under stress when compared with the control. Further studies are recommended to confirm where this study will offer valuable basic data on the functioning of DNA2 genes in rice and other crop plants.

Neutrophil-to-Lymphocyte Ratio, derived Neutrophil-to-Lymphocyte Ratio, Platelet-to-Lymphocyte Ratio and Lymphocyte-to-Monocyte Ratio as risk factors in critically ill COVID-19 patients, a single centered study.

BACKGROUND: A lot remains anonymous about the characteristics and laboratory findings that may evaluate poor outcomes in patients with Coronavirus disease 2019.The aim of this study was to determine the relationship of change in the peripheral blood factors of Neutrophil-to-Lymphocyte Ratio, derived-Neutrophil-to-Lymphocyte Ratio, Lymphocyte-to-Monocyte Ratio, and Platelet-to-Lymphocyte Ratio in hospitalized patients with COVID-19 and its severity. METHODS: Cross-sectional analytical study was performed at Department of Haematology in Pak Emirates Military Hospital affiliated with Army Medical College, Rawalpindi, Pakistan from March-July 2020. We included 735 patients confirmed by real-time reverse transcriptase polymerase-chain-reaction test for subacute respiratory syndrome corona virus-2 of all ages, irrespective of gender and were classified in groups of severe and non-severe groups. RESULTS: Data of blood and baseline characteristics were compared in between the two groups and found to be significant (p-value <0.001). The median age was 46.3 years, and 82 cases were only females. Receiver operator curve demonstrated larger area under the curve of NLR, d-NLR, and PLR and showed them as independent diagnostic biomarkers which were significantly associated with the severity of illness. Binary logistic regression performed in the form of forest plot also showed these factors were significantly linked with the severity (p-value <0.001). CONCLUSION: NLR, d-NLR, and PLR along with pre-existing co morbidities can be used as an independent biomarker for the poor clinical outcome of COVID-19 illness.