Precision Genome Editing with CRISPR-Cas9.

The CRISPR/Cas9 system is a revolutionary technology for genome editing that allows for precise and efficient modifications of DNA sequences. The system is composed of two main components, the Cas9 enzyme and a guide RNA (gRNA). The gRNA is designed to specifically target a desired DNA sequence, while the Cas9 enzyme acts as molecular scissors to cut the DNA at that specific location. The cell then repairs the digested DNA, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in either indels or precise modifications of DNA sequences with broad implications in biotechnology, agriculture, and medicine. This chapter provides a practical approach for utilizing CRISPR/Cas9 in precise genome editing, including identifying the target gene sequence, designing gRNA and protein (Cas9), and delivering the CRISPR components to target cells.

Identification and expression analysis of SBP-Box-like (SPL) gene family disclose their contribution to abiotic stress and flower budding in pigeon pea (Cajanus cajan).

The SPL gene family (for Squamosa Promoter-binding like Proteins) represents specific transcription factors that have significant roles in abiotic stress tolerance, development and the growth processes of different plants, including initiation of the leaf, branching and development of shoot and fruits. The SPL gene family has been studied in different plant species; however, its role is not yet fully explored in pigeon pea (Cajanus cajan ). In the present study, 11 members of the CcSPL gene family were identified in C. cajan . The identified SPLs were classified into nine groups based on a phylogenetic analysis involving SPL protein sequences from C. cajan , Arabidopsis thaliana , Cicer arietinum , Glycine max , Phaseolus vulgaris , Vigna unguiculata and Arachis hypogaea . Further, the identification of gene structure, motif analysis, domain analysis and presence of cis -regulatory elements in the SPL family members were studied. Based on RNA-sequencing data, gene expression analysis was performed, revealing that CcSPL2.1, 3 and 13A were significantly upregulated for salt-tolerance and CcSPL14 and 15 were upregulated in a salt-susceptible cultivar. Real-time qPCR validation indicated that CcSPL3, 4, 6 and 13A were upregulated under salt stress conditions. Therefore, molecular docking was performed against the proteins of two highly expressed genes (CcSPL3 and CcSPL14 ) with three ligands: abscisic acid, gibberellic acid and indole-3-acetic acid. Afterward, their binding affinity was obtained and three-dimensional structures were predicted. In the future, our study may open avenues for harnessing CcSPL genes in pigeon pea for enhanced abiotic stress resistance and developmental traits.

Assessment of Resistance Induction in Mungbean against Alternaria alternata through RNA Interference.

A comprehensive survey of mungbean-growing areas was conducted to observe leaf spot disease caused by Alternaria alternata. Alternaria leaf spot symptoms were observed on the leaves. Diversity of 50 genotypes of mungbean was assessed against A. alternata and data on pathological traits was subjected to cluster analysis. The results showed that genotypes of mungbean were grouped into four clusters based on resistance parameters under the influence of disease. The principal component biplot demonstrated that all the disease-related parameters (% disease incidence, % disease intensity, lesion area, and % of infection) were strongly correlated with each other. Alt a 1 gene that is precisely found in Alternaria species and is responsible for virulence and pathogenicity. Alt a 1 gene was amplified using gene specific primers. The isolated pathogen produced similar symptoms when inoculated on mungbean and tobacco. The sequence analysis of the internal transcribed spacer (ITS) region, a 600 bp fragment amplified using specific primers, ITS1 and ITS2 showed 100% identity with A. alternata. Potato virus X (PVX) -based silencing vector expressing Alt a 1 gene was constructed to control this pathogen through RNA interference in tobacco. Out of 50 inoculated plants, 9 showed delayed onset of disease. Furthermore, to confirm our findings at molecular level semi-quantitative reverse transcriptase polymerase chain reaction was used. Both phenotypic and molecular investigation indicated that RNAi induced through the VIGS vector was efficacious in resisting the pathogen in the model host, Tobacco (Nicotiana tabacum). To the best of our knowledge, this study has been reported for the first time.

Genome-wide survey of HMA gene family and its characterization in wheat (Triticum aestivum).

Background: Abiotic stresses, particularly drought and heavy metal toxicity, have presented a significant risk to long-term agricultural output around the world. Although the heavy-metal-associated domain (HMA) gene family has been widely explored in Arabidopsis and other plants, it has not been thoroughly studied in wheat (Triticum aestivum). This study was proposed to investigate the HMA gene family in wheat. Methods: To analyze the phylogenetic relationships, gene structure, gene ontology, and conserved motifs, a comparative study of wheat HMA genes with the Arabidopsis genome was performed. Results: A total of 27 T. aestivum proteins belonging to the HMA gene family were identified in this study, with amino acid counts ranging from 262 to 1,071. HMA proteins were found to be grouped into three subgroups in a phylogenetic tree, and closely related proteins in the tree showed the same expression patterns as motifs found in distinct subgroups. Gene structural study elucidated that intron and exon arrangement differed by family. Conclusion: As a result, the current work offered important information regarding HMA family genes in the T. aestivum genome, which will be valuable in understanding their putative functions in other wheat species.

Sequence, Secondary Structure, and Phylogenetic Conservation of MicroRNAs in Arabidopsis thaliana.

MicroRNAs are small non-coding RNA molecules that are produced in a cell endogenously. They are made up of 18 to 26 nucleotides in strength. Due to their evolutionary conserved nature, most of the miRNAs provide a logical basis for the prediction of novel miRNAs and their clusters in plants such as sunflowers related to the Asteraceae family. In addition, they participate in different biological processes of plants, including cell signaling and metabolism, development, growth, and tolerance to (biotic and abiotic) stresses. In this study profiling, conservation and characterization of novel miRNA possessing conserved nature in various plants and their targets annotation in sunflower (Asteraceae) were obtained by using various computational tools and software. As a result, we looked at 152 microRNAs in Arabidopsis thaliana that had already been predicted. Drought tolerance stress is mediated by these 152 non-coding RNAs. Following that, we used local alignment to predict novel microRNAs that were specific to Helianthus annuus. We used BLAST to do a local alignment, and we chose sequences with an identity of 80% to 100%. MIR156a, MIR164a, MIR165a, MIR170, MIR172a, MIR172b, MIR319a, MIR393a, MIR394a, MIR399a, MIR156h, and MIR414 are the new anticipated miRNAs. We used MFold to predict the secondary structure of new microRNAs. We used conservation analysis and phylogenetic analysis against a variety of organisms, including Gossypium hirsutum, H. annuus, A. thaliana, Triticum aestivum, Saccharum officinarum, Zea mays, Brassica napus, Solanum tuberosum, Solanum lycopersicum, and Oryza sativa, to determine the evolutionary history of these novel non-coding RNAs. Clustal W was used to analyze the evolutionary history of discovered miRNAs.

Detection of SARS-CoV-2 by using real-time PCR nasopharyngeal swabs in suspected patients and their clinical medication.

BACKGROUND: The corona name derived from their crown like spike proteins attach with cell receptors. It belongs to coronaviradae family and nideovirales order, envelop virus, size range 65-125 â€‹nm and positive single standard RNA between 26.4 and 31.7 â€‹kb and contain 7096 amino acid. There are four subtypes that have been detected these are alpha, beta, gamma and delta. METHODOLOGY: The 267 covid-19 blood and nasopharyngeal samples were collected from Multan region. RNA extraction from nasopharyngeal samples and run the PCR. The blood samples use for clinical tests, Lactate dehydrogenase, serum ferritin level, D-Dimer, TG, cholesterol, thyphoidot, HDL, lymphocyte count and CRP. RESULTS: 127 (47.21%) out of 267 patients were covid-19 PCR positive and showed the amplification of ORF1ab, E, and N gene, while 140 individuals were covid-19 PCR negative and not showed the amplification of ORF1ab, E and N gene. The patients with negative Covid-19 PCR, the other analysis tests such as lactate dehydrogenase, HDL, ferritin, ESR, CBP, D-Dimer, Tg, cholesterol, CRP and CT scan. The patients effected covid-19 have higher values of D-Dimer, ESR, Neutrophils, LDH, CRP and ferritin level than normal ranges. However, the values of HDL, cholesterol and lymphocytes were decreased from the normal range.

Comprehensive genomics and expression analysis of eceriferum (CER) genes in sunflower (Helianthus annuus).

Sunflower occupies the fourth position among oilseed crops the around the world. Eceriferum (CER) is an important gene family that plays critical role in very-long-chain fatty acids elongation and biosynthesis of epicuticular waxes under both biotic and abiotic stress conditions. The aim of present study was to investigate the effect of sunflower CER genes during drought stress condition. Thus, comparative analysis was undertaken for sunflower CER genes with Arabidopsis genome to determine phylogenetic relationship, chromosomal mapping, gene structures, gene ontology and conserved motifs. Furthermore, we subjected the sunflower cultivars under drought stress and used qRT-PCR analysis to explore the expression pattern of CER genes during drought conditions. We identified thirty-seven unevenly distributed CER genes in the sunflower genome. The phylogenetic analysis revealed that CER genes were grouped into seven clades in Arabidopsis, Helianthus annuus, and Gossypium hirsutum. Expression analysis showed that genes CER10 and CER60 were upregulated in sunflower during drought conditions, indicating that these genes are activated during drought stress. The results obtained will serve to characterize the CER gene family in sunflower and exploit the role of these genes in wax biosynthesis under limited water conditions. KEY MESSAGE: Cuticular waxes protect the plants from drought stress, so we observed the expression of wax bio synthesis genes in recently sequences genome of Helianthus annuus. We observed that expression of wax biosynthesis genes CER10 and CER60 was upregulated when the plants were subjected to drought stress.

Comparative genomic analysis of MYB transcription factors for cuticular wax biosynthesis and drought stress tolerance in Helianthus annuus L.

Sunflower is an important oil-seed crop in Pakistan, it is mainly cultivated in the spring season. It is severely affected by drought stress resulting in lower yield. Cuticular wax acts as the first defense line to protect plants from drought stress condition. It seals the aerial parts of plants and reduce the water loss from leaf surfaces. Various myeloblastosis (MYB) transcription factors (TFs) are involved in biosynthesis of epicuticular waxes under drought-stress. However, less information is available for MYB, TFs in drought stress and wax biosynthesis in sunflower. We used different computational tools to compare the Arabidopsis MYB, TFs involved in cuticular wax biosynthesis and drought stress tolerance with sunflower genome. We identified three putative MYB genes (MYB16, MYB94 and MYB96) in sunflower along with their seven homologs in Arabidopsis. Phylogenetic association of MYB TFs in Arabidopsis and sunflower indicated strong conservation of TFs in plant species. From gene structure analysis, it was observed that intron and exon organization was family-specific. MYB TFs were unevenly distributed on sunflower chromosomes. Evolutionary analysis indicated the segmental duplication of the MYB gene family in sunflower. Quantitative Real-Time PCR revealed the up-regulation of three MYB genes under drought stress. The gene expression of MYB16, MYB94 and MYB96 were found many folds higher in experimental plants than control. The present study provided the first insight into MYB TFs family's characterization in sunflower under drought stress conditions and wax biosynthesis TFs.

A genome-wide comparative analysis of bZIP transcription factors in G. arboreum and G. raimondii (Diploid ancestors of present-day cotton).

Basic leucine zipper motif (bZIP) transcription factors (TFs) are involved in plant growth regulation, development, and environmental stress responses. These genes have been well characterized in model plants. In current study, a genome-wide analysis of bZIP genes was performed in Gossypium raimondii and Gossypium arboreum taking Arabidopsis thaliana as a reference genome. In total, 85 members of G. raimondii and 87 members of G. arboreum were identified and designated as GrbZIPs and GabZIPs respectively. Phylogenetic analysis clustered bZIP genes into 11 subgroups (A, B, C, D, F, G, H, I, S and X). Gene structure analysis to find the intro-exon structures revealed 1-14 exons in both species. The maximum number of introns were present in subgroup G and D while genes in subgroup S were intron-less except GrbZIP78, which is a unique characteristic as compared to other groups. Results of motif analysis predicted that all three species share a common bZIP motif. A detailed comparison of bZIPs gene distribution on chromosomes has shown a diverse arrangement of genes in both cotton species. Moreover, the functional similarity with orthologs was also predicted. The findings of this study revealed close similarity in gene structure of both cotton species and diversity in gene distribution on chromosomes. This study supports the divergence of both species from the common ancestor and later diversity in gene distribution on chromosomes due to evolutionary changes. Additionally, this work will facilitate the functional characterization of bZIP genes in cotton. Outcomes of this study represent foundation research on the bZIP TFs family in cotton and as a reference for other crops.

Relationship between psychological distress and resilience in rescue workers.

OBJECTIVES: To assess the relationship between psychological distress and resilience in rescue workers. Following hypothesis was formulated; there would be negative correlation between psychological distress and resilience in rescue workers.  METHOD: A correlational study was conducted from June-August 2015 in Rahim Yar Khan, Punjab, Pakistan. The sample of the present study consisted of 100 rescue workers. The age of the participants ranged from 23 to 40 year old with the mean age of 27.4±3.9 years. Demographic information form, Kessler psychological distress scale and adult resilience measure were administered on the participants to assess the level of psychological distress and resilience.   RESULTS: Pearson product moment coefficient of correlation was applied to analyze the relationship of psychological distress and resilience. Analysis of the result indicated that there is negative relationship between psychological distress and resilience (r= -0.203, p less than 0.01) in rescue workers. Further, contextual factors (r= -0.292, p less than 0.05) and its subcomponents including spiritual beliefs (r= -0.239, p less than 0.05) and cultural resources (r= -0.287, p less than 0.01) were also found to be inversely correlated with psychological distress.  CONCLUSION: The research evidenced that rescue workers were experiencing psychological distress Resilience factors should be considered while designing trainings to preserve mental health and to enhance the psychological well-being of rescue workers.

Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.

Study of rust resistance genes in wheat germplasm with DNA markers.

Wheat is a vital dietary component for human health and widely consumed in the world. Wheat rusts are dangerous pathogens and contribute serious threat to its production. In present study, PCR-Based DNA Markers were employed to check the rust resistance genes among 20 wheat genotypes and 22 markers were amplified. NTSYS-pc 2.2 was used to calculate genetic diversity and Nei and Li's coefficients ranged from 0.55 to 0.95. Cluster analysis was obtained using UPGMA (Unweighted Pair Group Method of Arithmetic Average) algorithm. Maximum no. of genes (23) was amplified from TW-760010 genotype whereas minimum no of genes (14) were amplified from TW-76005 genotype. The data gained from present study open up new ways to produce new varieties by breeding rust resistant germplasm to avoid the economic and food loss and varieties with improved characteristics.

Detection of single nucleotide polymorphisms in the conserved ESTs regions of Gossypium arboreum

Exploring genetic variation in Gossypium arboreum L. germplasm is useful as it contains many important genes conferring resistance to different stresses. In limited earlier studies, low level of genetic diversity was found by using conventional DNA marker systems which may impede future genome mapping studies. In the present investigation, we explored the extent of Single Nucleotide Polymorphisms (SNP) among 30 conserved regions of Expressed Sequence Tags (EST) of low copy genes between two genotypes of G. arboreum. A total of 27 SNPs including 21 substitutions and 6 Insertions and deletions (Indels) in 7804 bp were found between these genotypes with a frequency of one SNP per 371 bp and one Indel after every 1300 bp. Out of these SNPs, 52 percent were transitions, whilst 48 percent SNPs were transversion. In conclusion, SNPs are expedient markers that can explore polymorphism in highly conserved sequences where other markers are not effective.