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Cotton is globally known for its high-priority cellulose-rich natural fiber. In addition to providing fiber for the textile industry, it is an important source material for edible oil, livestock feed, and fuel products. Global warming and the growing population are the major challenges to the world’s agriculture and the potential risks to food security. In this context, improving output traits in cotton is necessary to achieve sustainable cotton production. During the last few years, high throughput omics techniques have aided in identifying crucial genes associated with traits of cotton fiber, seed, and plant architecture which could be targeted with more precision and efficiency through the CIRPSR/Cas-mediated genome editing technique. The various CRISPR/Cas systems such as CRISPR/Cas9, CRISPR/nCas9, and CRISPR/Cas12a have been employed to edit cotton genes associated with a wide range of traits including fiber length, flowering, leaf colour, rooting, seed oil, plant architecture, gossypol content, somatic embryogenesis, and biotic and abiotic stresses tolerance, highlighting its effectiveness in editing the cotton genome. Thus, CRISPR/Cas-mediated genome editing has emerged as a technique of choice to tailor crop phenotypes for better yield potential and environmental resilience. The review covers a comprehensive analysis of cotton phenotypic traits and their improvement with the help of the latest genome editing tools to improve fiber, food, feed, and fuel-associated genes of cotton to ensure food security.

Abstract Cotton is one of the most important fiber crops that plays a vital role in the textile industry. Its production has been unstable over the years due to climate change induced biotic stresses such as insects, diseases, and weeds, as well as abiotic stresses including drought, salinity, heat, and cold. Traditional breeding methods have been used to breed climate resilient cotton, but it requires a considerable amount of time to enhance crop tolerance to insect pests and changing climatic conditions. A promising strategy for improving tolerance against these stresses is genetic engineering. This review article discusses the role of genetic engineering in cotton improvement. The essential concepts and techniques include genome editing via clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (CRISPR-Cas9), overexpression of target genes, downregulation using RNA interference (RNAi), and virus-induced gene silencing (VIGS). Notably, the Agrobacterium-mediated transformation has made significant contributions to using these techniques for obtaining stable transgenic plants.

Abstract Dalbergia sissoo Roxb. (Shisham) is a timber-producing species of economic, cultural, and medicinal importance in the Indian subcontinent. In the past few decades, Shisham's dieback disease caused by the fungus Botryodiplodia theobromae has become an evolving issue in the subcontinent endangering its survival. To gain insights into this issue, a standard transcriptome assembly was deployed to assess the response of D. sissoo at the transcriptomic level under the stress of B. theobromae infection. For RNA isolation, the control and infected leaf tissue samples were taken from 1-year-old greenhouse-grown D. sissoo plants after 20 days of stem-base spore inoculation. cDNA synthesis was performed from these freshly isolated RNA samples that were then sent for sequencing. About 18.14 Gb (Giga base) of data was generated using the BGISEQ-500 sequencing platform. In terms of Unigenes, 513,821 were identified after a combined assembly of all samples and then filtering the abundance. The total length of Unigenes, their average length, N50, and GC-content were 310,523,693 bp, 604 bp, 1,101 bp, and 39.95% respectively. The Unigenes were annotated using 7 functional databases i.e., 200,355 (NR: 38.99%), 164,973 (NT: 32.11%), 123,733 (Swissprot: 24.08%), 142,580 (KOG: 27.75%), 139,588 (KEGG: 27.17%), 99,752 (GO: 19.41%), and 137,281 (InterPro: 26.72%). Furthermore, the Transdecoder detected 115,762 CDS. In terms of SSR (Simple Sequence Repeat) markers, 62,863 of them were distributed on 51,508 Unigenes and on the predicted 4673 TF (Transcription Factor) coding Unigenes. A total of 16,018 up- and 19,530 down-regulated Differentially Expressed Genes (DEGs) were also identified. Moreover, the Plant Resistance Genes (PRGs) had a count of 9230. We are hopeful that in the future, these identified Unigenes, SSR markers, DEGs and PRGs will provide the prerequisites for managing Shisham dieback disease, its breeding, and in tree improvement programs.

Abstract Background Fatty acid desaturases (FADs) are involved in regulating plant fatty acid composition by adding double bonds to growing hydrocarbon chain. Apart from regulating fatty acid composition FADs are of great importance, and are involved in stress responsiveness, plant development, and defense mechanisms. FADs have been extensively studied in crop plants, and are broadly classed into soluble and non-soluble fatty acids. However, FADs have not yet been characterized in Brassica carinata and its progenitors. Results Here we have performed comparative genome-wide identification of FADs and have identified 131 soluble and 28 non-soluble FADs in allotetraploid B. carinata and its diploid parents. Most soluble FAD proteins are predicted to be resided in endomembrane system, whereas FAB proteins were found to be localized in chloroplast. Phylogenetic analysis classed the soluble and non-soluble FAD proteins into seven and four clusters, respectively. Positive type of selection seemed to be dominant in both FADs suggesting the impact of evolution on these gene families. Upstream regions of both FADs were enriched in stress related cis-regulatory elements and among them ABRE type of elements were in abundance. Comparative transcriptomic data analysis output highlighted that FADs expression reduced gradually in mature seed and embryonic tissues. Moreover, under heat stress during seed and embryo development seven genes remained up-regulated regardless of external stress. Three FADs were only induced under elevated temperature whereas five genes were upregulated under Xanthomonas campestris stress suggesting their involvement in abiotic and biotic stress response. Conclusions The current study provides insights into the evolution of FADs and their role in B. carinata under stress conditions. Moreover, the functional characterization of stress-related genes would exploit their utilization in future breeding programs of B. carinata and its progenitors.

Abstract Cotton has enormous economic potential providing high-quality protein, oil, and fibre. A large increase in cotton output is necessary due to the world's changing climate and constantly expanding human population. In the past, conventional breeding techniques were used to introduce genes into superior cotton cultivars to increase production and to improve quality. The disadvantages of traditional breeding techniques are their time-consuming, reliance on genetic differences that are already present, and considerable backcrossing. To accomplish goals in a short amount of time, contemporary plant breeding techniques, in particular modern genome editing technologies (GETs), can be used. Numerous crop improvement initiatives have made use of GETs, such as zinc-finger nucleases, transcription-activator-like effector nucleases, clustered regularly interspaced palindromic repeats (CRISPR), and CRISPR-associated proteins systems (CRISPR/Cas)-based technologies. The CRISPR/Cas system has a lot of potential because it combines three qualities that other GETs lack: simplicity, competence, and adaptability. The CRISPR/Cas mechanism can be used to improve cotton tolerance to biotic and abiotic stresses, alter gene expression, and stack genes for critical features with little possibility of segregation. The transgene clean strategy improves CRISPR acceptability addressing regulatory issues associated with the genetically modified organisms (GMOs). The research opportunities for using the CRISPR/Cas system to address biotic and abiotic stresses, fibre quality, plant architecture and blooming, epigenetic changes, and gene stacking for commercially significant traits are highlighted in this article. Furthermore, challenges to use of CRISPR technology in cotton and its potential for the future are covered in detail.

Abstract Background Plant aquaporins are critical genetic players performing multiple biological functions, especially climate resilience and water-use efficiency. Their genomic diversity across genus Oryza is yet to be explored. Results This study identified 369 aquaporin-encoding genes from 11 cultivated and wild rice species and further categorized these into four major subfamilies, among which small basic intrinsic proteins are speculated to be ancestral to all land plant aquaporins. Evolutionarily conserved motifs in peptides of aquaporins participate in transmembrane transport of materials and their relatively complex gene structures provide an evolutionary playground for regulation of genome structure and transcription. Duplication and evolution analyses revealed higher genetic conservation among Oryza aquaporins and strong purifying selections are assisting in conserving the climate resilience associated functions. Promoter analysis highlighted enrichment of gene upstream regions with cis-acting regulatory elements involved in diverse biological processes, whereas miRNA target site prediction analysis unveiled substantial involvement of osa-miR2102-3p, osa-miR2927 and osa-miR5075 in post-transcriptional regulation of gene expression patterns. Moreover, expression patterns of japonica aquaporins were significantly perturbed in response to different treatment levels of six phytohormones and four abiotic stresses, suggesting their multifarious roles in plants survival under stressed environments. Furthermore, superior haplotypes of seven conserved orthologous aquaporins for higher thousand-grain weight are reported from a gold mine of 3,010 sequenced rice pangenomes. Conclusions This study unveils the complete genomic atlas of aquaporins across genus Oryza and provides a comprehensive genetic resource for genomics-assisted development of climate-resilient rice cultivars.

Begomoviruses are contagious and severely affect commercially important fiber and food crops. Cotton leaf curl Multan virus (CLCuMuV) is one of the most dominant specie of Begomovirus and a major constraint on cotton yield in Pakistan. Currently, the field of plant genome editing is being revolutionized by the CRISPR/Cas system applications such as base editing, prime editing and CRISPR based gene drives. CRISPR/Cas9 system has successfully been used against biotic and abiotic plant stresses with proof-of-concept studies in both model and crop plants. CRISPR/Cas12 and CRISPR/Cas13 have recently been applied in plant sciences for basic and applied research. In this study, we used a novel approach, multiplexed crRNA-based Cas12a toolbox to target the different ORFs of the CLCuMuV genome at multiple sites simultaneously. This method successfully eliminated the symptoms of CLCuMuV in Nicotiana benthamiana and Nicotiana tabacum. Three individual crRNAs were designed from the CLCuMuV genome, targeting the specific sites of four different ORFs (C1, V1 and overlapping region of C2 and C3). The Cas12a-based construct Cas12a-MV was designed through Golden Gate three-way cloning for precise editing of CLCuMuV genome. Cas12a-MV construct was confirmed through whole genome sequencing using the primers Ubi-intron-F1 and M13-R1. Transient assays were performed in 4 weeks old Nicotiana benthamiana plants, through the agroinfiltration method. Sanger sequencing indicated that the Cas12a-MV constructs made a considerable mutations at the target sites of the viral genome. In addition, TIDE analysis of Sanger sequencing results showed the editing efficiency of crRNA1 (21.7%), crRNA2 (24.9%) and crRNA3 (55.6%). Furthermore, the Cas12a-MV construct was stably transformed into Nicotiana tabacum through the leaf disc method to evaluate the potential of transgenic plants against CLCuMuV. For transgene analysis, the DNA of transgenic plants of Nicotiana tabacum was subjected to PCR to amplify Cas12a genes with specific primers. Infectious clones were agro-inoculated in transgenic and non-transgenic plants (control) for the infectivity assay. The transgenic plants containing Cas12a-MV showed rare symptoms and remained healthy compared to control plants with severe symptoms. The transgenic plants containing Cas12a-MV showed a significant reduction in virus accumulation (0.05) as compared to control plants (1.0). The results demonstrated the potential use of the multiplex LbCas12a system to develop virus resistance in model and crop plants against begomoviruses.

Stripe rust is a continuous threat to wheat crop all over the world. It causes considerable yield losses in wheat crop every year. Continuous deployment of adult plant resistance (APR) genes in newly developing wheat cultivars is the most judicious strategy to combat this disease. Herein, we dissected the genetics underpinning stripe rust resistance in Pakistani wheat germplasm. An association panel of 94 spring wheat genotypes was phenotyped for two years to score the infestation of stripe rust on each accession and was scanned with 203 polymorphic SSRs. Based on D´ measure, linkage disequilibrium (LD) exhibited between loci distant up to 45 cM. Marker-trait associations (MTAs) were determined using mixed linear model (MLM). Total 31 quantitative trait loci (QTLs) were observed on all 21 wheat chromosomes. Twelve QTLs were newly discovered as well as 19 QTLs and 35 previously reported Yr genes were validated in Pakistani wheat germplasm. The major QTLs were QYr.uaf.2AL and QYr.uaf.3BS (PVE, 11.9%). Dissection of genes from the newly observed QTLs can provide new APR genes to improve genetic resources for APR resistance in wheat crop.

Cotton being the major fiber crop across the world is exposed to numerous biotic and abiotic stresses. Genetic transformation of cotton is vital to meet the world's food, feed and fiber demands. Genetic manipulation by randomly transferring the genes emanate variable gene expression. Targeted gene insertion by latest genome editing tools results in predictable expression of genes at a specified location. Gene stacking technology emerged as an adaptive strategy to combat biotic and abiotic stresses by integrating 2-3 genes simultaneously and at a specific site to avoid variable gene expression at diverse locations. This study explains the development of cotton's founder transformants to be used as a base line for multiple gene stacking projects. We introduced Cre and PhiC31 mediated recombination sites to specify the locus of incoming genes. CRISPR-Cas9 gene was integrated for developing CRISPR based founder lines of cotton. Cas9 gene along with gRNA was integrated to target Rep (replication) region of cotton leaf curl virus. Replication region of virus was specifically targeted to diminish further proliferation and preventing the virus to develop new strains. To successfully develop these primary transformants, a model transformation system has been optimized with the red color visualization (DS-Red). Following red color transformation system, three baselines with recombination specified site (Rec), targeted replication region (Rep) and Cas9 founder lines have been developed. These founder transformants are useful for developing recombinase mediated and CRISPR/Cas9 based originator lines of cotton. Moreover, these transformants will set up a base system for all the recombinase mediated gene stacking projects.

The regeneration of the high-yielding multilocular types has not been attempted, although successful regeneration and transformation in brassica have been done. Here, we report efficient regeneration and transformation protocols for two B. rapa genotypes; UAF11 and Toria. The B. rapa cv UAF11 is a multilocular, non-shattering, and high-yielding genotype, while Toria is the bilocular type. For UAF11 8 shoots and for Toria 7 shoots, explants were observed on MS supplemented with 3 mg/L BAP + 0.4 mg/L NAA + 0.01 mg/L GA3 + 5 mg/L AgNO3 + 0.75 mg/L Potassium Iodide (KI), MS salt supplemented with 1 mg/L IBA and 0.37 mg/L KI produced an equal number of roots (3) in UAF11 and Toria. For the establishment of transformation protocols, Agrobacterium-mediated floral dip transformation was attempted using different induction media, infection time, and flower stages. The induction medium III yielded a maximum of 7.2% transformants on half-opened flowers and 5.2% transformants on fully opened flowers in UAF11 and Toria, respectively, with 15 min of inoculation. This study would provide the basis for the improvement of tissue culture and transformation protocols in multilocular and bilocular Brassica genotypes.

Genetic diversity is the base of any genetic improvement breeding program aimed at stress breeding. The variability among breeding materials is of primary importance in the achievements of a good crop production. Herein, 105 wheat genotypes were screened against drought stress using factorial completely randomized design at seedling stage to determine the genetic diversity and traits association conferring drought tolerance. Analysis of variances revealed that all the studied parameters differed significantly among all genotypes, indicating the significance genetic variability existed among all genotypes for studied indices. The 10 best performance genotypes G1, G6, G11, G16, G21, G26, G39, G44, G51, and G61 were screened as drought tolerant, while five lowest performance genotypes G3, G77, G91, G98, and G105 were screened as drought susceptible. Root length, chlorophyll a, chlorophyll b, and carotenoid contents were significantly correlated among themselves which exhibited the importance of these indices for rainfed areas in future wheat breeding scheme. Shoot length exhibited non-significant and negative association with other studied traits, and its selection seems not to be a promising criteria for this germplasm for drought stress. Best performance genotypes under drought stress conditions will be useful in future wheat breeding program and early selection will be effective for developing high yielding and drought tolerant wheat varieties.

Fish is a fundamentally healthy food, loaded with essential nutrients, high protein content, vitamin D, and omega-three fatty acid. Mislabeling is a common problem in the fish industry that causes an imbalance in prices and fluctuation in the market. DNA barcoding is a potential technique for authentication of mislabeled and misidentified fish species. In this study, 11 freshwater and 6 marine fish species were used for DNA barcoding and further authentication using the mitochondrial cytochrome b (Cyt b) gene. Cyt b was amplified using PCR, producing an average read length of 1,141 bp. The obtained sequences were compared to the National Center for Biotechnology Information database (NCBI) using the Basic Local Alignment Search Tool (BLAST). The average AT content (55.20%) was higher than the average GC content (44.78%) in marine and freshwater fish species. The mean genetic Kimura 2-parameter distances for species, genus, families, and orders were 0.311, 0.308, 0.023, and 0.337, respectively. Phylogenetic tree analysis revealed that most of the freshwater fish species clustered together due to the fact that they were in the same order or family, while the marine fish species clustered distantly. Single nucleotide polymorphism (SNP) analysis of all species in the study revealed distinct features regarding unique sites. All fish species could be identified based on their unique SNP profiles. Based on SNP data, DNA sequence based QR codes were developed for accurate identification of fish species. This is the first study to develop DNA-based QR barcodes for proper authentication of species during the chain of custody using simple technology.

Abstract Background The Flo2 gene is a member of a conserved gene family in plants. This gene has been found to be related to thousand grain weight (TGW) in rice. Its orthologs in hexaploid wheat were cloned, and the haplotype variation in TaFlo2-A1 was tested for association with TGW. Results The cloned sequences of TaFlo2-A1, TaFlo2-B1 and TaFlo2-D1 contained 23, 23 and 24 exons, respectively. The deduced proteins of TaFlo2-A1 (1734 aa), TaFlo2-B1 (1698 aa) and TaFlo2-D1 (1682 aa) were highly similar (>94%) and exhibited >77% similarity with the rice FLO2 protein. Like the rice FLO2 protein, four tetratricopeptide repeat (TPR) motifs were observed in the deduced TaFLO2 protein. An 8-bp InDel (−10 to −17 bp) in the promoter region and five SNPs in first intron of TaFlo2-A1 together formed two haplotypes, TaFlo2-A1a and TaFlo2-A1b, in bread wheat. TaFlo2 was located on homeologous group 2 chromosomes. TaFlo2-A1 was inferred to be located on deletion bin ‘2AL1–0.85-1.00’. The TaFlo2-A1 haplotypes were characterized in the Chinese Micro Core Collection (MCC) and Pakistani wheat collection using the molecular marker TaFlo2-Indel8. TaFlo2-A1 was found to be associated with TGW but not with grain number per spike (GpS) in both the MCC and Pakistani wheat collections. The frequency of TaFlo2-A1b (positive haplotype) was low in commercial wheat cultivars; thus this haplotype can be selected to improve grain weight without negatively affecting GpS. The expression level of TaFlo2-A1 in developing grains at 5 DAF (days after flowering) was positively correlated with TGW in cultivars carrying the positive haplotype. Conclusion This study will likely lead to additional investigations to understand the regulatory mechanism of the Flo2 gene in hexaploid wheat. Furthermore, the newly developed molecular marker ‘TaFlo2-InDel8’ could be incorporated into the kit of wheat breeders for use in marker-assisted selection.

Whitefly (Bemisia tabaci)-transmitted Geminiviruses cause serious diseases of crop plants in tropical and sub-tropical regions. Plants, animals, and their microbial symbionts have evolved complex ways to interact with each other that impact their life cycles. Blocking virus transmission by altering the biology of vector species, such as the whitefly, can be a potential approach to manage these devastating diseases. Virus transmission by insect vectors to plant hosts often involves bacterial endosymbionts. Molecular chaperonins of bacterial endosymbionts bind with virus particles and have a key role in the transmission of Geminiviruses. Hence, devising new approaches to obstruct virus transmission by manipulating bacterial endosymbionts before infection opens new avenues for viral disease control. The exploitation of bacterial endosymbiont within the insect vector would disrupt interactions among viruses, insects, and their bacterial endosymbionts. The study of this cooperating web could potentially decrease virus transmission and possibly represent an effective solution to control viral diseases in crop plants.

The decrease in water resources is a serious threat to food security world-wide. In this regard, a genome-wide association study (GWAS) was conducted to identify grain yield and quality-related genes/loci under normal and water-deficit conditions. Highly significant differences were exhibited among genotypes under both conditions for all studied traits. Water-deficit stress caused a reduction in grains yield and an increase in grains protein contents (GPC) and gluten contents (GLC). Population structure divided the 96 genotypes into four sub-populations. Out of 72 significant marker-trait associations (MTAs), 28 and 44 were observed under normal and water-deficit stress conditions, respectively. Pleiotropic loci (RAC875_s117925_244, BobWhite_c23828_341 and wsnp_CAP8_c334_304253) for yield and quality traits were identified on chromosomes 5A, 6B and 7B, respectively, under normal conditions. Under a water-deficit condition, the pleiotropic loci (Excalibur_c48047_90, Tdurum_contig100702_265 and BobWhite_c19429_95) for grain yield per plant (GYP), GPC and GLC were identified on chromosomes 3A, 4A and 7B, respectively. The pleiotropic loci (BS00063551_51 and RAC875_c28721_290) for GPC and GLC on chromosome 1B and 3A, respectively, were found under both conditions. Besides the validation of previously reported MTAs, some new MTAs were identified for flag leaf area (FLA), thousand grain weight (TGW), GYP, GPC and GLC under normal and water-deficit conditions. Twenty SNPs associated with the traits were mapped in the coding DNA sequence (CDS) of the respective candidate genes. The protein functions of the identified candidate genes were predicted and discussed. Isolation and characterization of the candidate genes, wherein, SNPs were mapped in CDS will result in discovering novel genes underpinning water-deficit tolerance in bread wheat.