Dominance of Cotton leaf curl Multan virus-Rajasthan strain associated with third epidemic of cotton leaf curl disease in Pakistan.

Cotton (Gossypium hirsutum) is an economically potent crop in many countries including Pakistan, India, and China. For the last three decades, cotton production is under the constant stress of cotton leaf curl disease (CLCuD) caused by begomoviruses/satellites complex that is transmitted through the insect pest, whitefly (Bemisia tabaci). In 2018, we identified a highly recombinant strain; Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Raj), associated with the Cotton leaf curl Multan betasatellite-Vehari (CLCuMuBVeh). This strain is dominant in cotton-growing hub areas of central Punjab, Pakistan, causing the third epidemic of CLCuD. In the present study, we have explored the CLCuD diversity from central to southern districts of Punjab (Faisalabad, Lodhran, Bahawalpur, Rahimyar Khan) and the major cotton-growing region of Sindh (Tandojam), Pakistan for 2 years (2020-2021). Interestingly, we found same virus (CLCuMuV-Raj) and associated betasatellite (CLCuMuBVeh) strain that was previously reported with the third epidemic in the central Punjab region. Furthermore, we found minor mutations in two genes of CLCuMuV-Raj C4 and C1 in 2020 and 2021 respectively as compared to its isolates in 2018, which exhibited virus evolution. Surprisingly, we did not find these mutations in CLCuMuV-Raj isolates identified from Sindh province. The findings of the current study represent the stability of CLCuMuV-Raj and its spread toward the Sindh province where previously Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Shahdadpur virus (CLCuShV) have been reported. The findings of the current study demand future research on CLCuD complex to explore the possible reasons for prevalence in the field and how the virus-host-vector compatible interaction can be broken to develop resistant cultivars.

Deciphering the differential expression patterns of yield-related negative regulators in hexaploid wheat cultivars and hybrids at different growth stages.

BACKGROUND: Hexaploid bread wheat underwent a series of polyploidization events through interspecific hybridizations that conferred adaptive plasticity and resulted in duplication and neofunctionalization of major agronomic genes. The genetic architecture of polyploid wheat not only confers adaptive plasticity but also offers huge genetic diversity. However, the contribution of different gene copies (homeologs) encoded from different subgenomes (A, B, D) at different growth stages remained unexplored. METHODS: In this study, hybrid of elite cultivars of wheat were developed via reciprocal crosses (cytoplasm swapping) and phenotypically evaluated. We assessed differential expression profiles of yield-related negative regulators in these cultivars and their F1 hybrids and identified various cis-regulatory signatures by employing bioinformatics tools. Furthermore, the preferential expression patterns of the syntenic triads encoded from A, B, and D subgenomes were assessed to decipher their functional redundancy at six different growth stages. RESULTS: Hybrid progenies showed better heterosis such as up to 17% increase in the average number of grains and up to 50% increase in average thousand grains weight as compared to mid-parents. Based on the expression profiling, our results indicated significant dynamic transcriptional expression patterns, portraying the different homeolog-dominance at the same stage in the different cultivars and their hybrids. Albeit belonging to same syntenic triads, a dynamic trend was observed in the regulatory signatures of these genes that might be influencing their expression profiles. CONCLUSION: These findings can substantially contribute and provide insights for the selective introduction of better cultivars into traditional and hybrid breeding programs which can be harnessed for the improvement of future wheat.

Assessment of Genomic Diversity and Selective Pressures in Crossbred Dairy Cattle of Pakistan.

Improving the low productivity levels of native cattle breeds in smallholder farming systems is a pressing concern in Pakistan. Crossbreeding high milk-yielding holstein friesian (HF) breed with the adaptability and heat tolerance of Sahiwal cattle has resulted in offspring that are well-suited to local conditions and exhibit improved milk yield. The exploration of how desirable traits in crossbred dairy cattle are selected has not yet been investigated. This study aims to provide the first overview of the selective pressures on the genome of crossbred dairy cattle in Pakistan. A total of eighty-one crossbred, thirty-two HF and twenty-four Sahiwal cattle were genotyped, and additional SNP genotype data for HF and Sahiwal were collected from a public database to equate the sample size in each group. Within-breed selection signatures in crossbreds were investigated using the integrated haplotype score. Crossbreds were also compared to each of their parental breeds to discover between-population signatures of selection using two approaches: cross-population extended haplotype homozygosity and fixation index. We identified several overlapping genes associated with production, immunity, and adaptation traits, including U6, TMEM41B, B4GALT7, 5S_rRNA, RBM27, POU4F3, NSD1, PRELID1, RGS14, SLC34A1, TMED9, B4GALT7, OR2AK3, OR2T16, OR2T60, OR2L3, and CTNNA1. Our results suggest that regions responsible for milk traits have generally experienced stronger selective pressure than others.

Evaluation of BG, NPR1, and PAL in cotton plants through Virus Induced gene silencing reveals their role in whitefly stress.

Whitefly is one of the most hazardous insect pests that infests a wide range of host plants and causes huge damage to crop worldwide. In order to engineer plants resilient to whitefly stress, it is important to identify and validate the responsive genes by exploring the molecular dynamics of plants under stress conditions. In this study three genes BG, NPR1, and PAL genes have been studied in cotton for elucidating their role in whitefly stress response. Initially, insilico approach was utilized to investigate the domains and phylogeny of BG, NPR1 and PAL genes and found out that these genes showed remarkable resemblance in four cotton species Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii. In BG proteins the main functional domain was X8 belonging to glycohydro superfamily, in NPR1 two main functional domains were BTB_POZ at N terminal and NPR1_like_C at C terminal. In PAL functional domain PLN was found which belongs to Lyase class I superfamily. The promoter analysis of these genes displayed enrichment of hormone, stress and stimuli responsive cis elements. Through Virus Induced Gene Silencing (VIGS), these genes were targeted and kept under whitefly infestation. Overall, the whitefly egg and nymph production were observed 60-70% less on gene down regulated plants as compared to control plants. The qPCR-based expression analysis of certain stress-responsive genes showed that in BG down regulated plants the elevated expression of these whitefly responsive genes was detected, in NPR1 down regulated plants JAZ1 and HSP were found up regulated, ERF1 and WRKY40 didn't show significant differential expression, while MAPK6 was slightly down regulated. In PAL down regulated plants ERF1 and JAZ1 showed elevated expression while others didn't show significant alternation. Differential expression in gene down-regulated plants showed that whitefly responsive genes act in a complex inter signaling pathway and their expression impact each other. This study provides valuable insight into the structural and functional analysis of important whitefly responsive genes BG, NPR1, and PAL. The results will pave a path to future development of whitefly resilient crops.

Salicylic acid-driven innate antiviral immunity in plants.

Pathogenic viruses are a constant threat to all organisms, including plants. However, in plants, a small group of cells (stem cells) protect themselves from viral invasion. Recently, Incarbone et al. uncovered a novel salicylic acid (SA) and RNAi mechanism of stem cell resistance, broadening our understanding of RNAi-mediated antiviral plant immunity.

Gene drive in plants emerges from infancy.

Selfish genetic elements (SGEs) display biased transmission to offspring. However, their breeding potential has remained obscure. Wang et al. recently reported a natural gene-drive system that can be harnessed to prevent hybrid incompatibility and to develop a synthetic gene-drive (SGD) system for crop improvement.

Methyl-salicylate (MeSA)-mediated airborne defence.

Stressed plants emit a variety of chemicals into the environment, leading to increased pest resistance in neighbouring plants but the genetic and molecular mechanisms of the emissions remain obscure. Recently, Gong et al. identified novel methyl salicylate (MeSA)-mediated airborne defence that confers resistance to neighbouring plants against aphids and viruses.

A source of resistance against yellow mosaic disease in soybeans correlates with a novel mutation in a resistance gene.

Yellow mosaic disease (YMD) is one of the major devastating constraints to soybean production in Pakistan. In the present study, we report the identification of resistant soybean germplasm and a novel mutation linked with disease susceptibility. Diverse soybean germplasm were screened to identify YMD-resistant lines under natural field conditions during 2016-2020. The severity of YMD was recorded based on symptoms and was grouped according to the disease rating scale, which ranges from 0 to 5, and named as highly resistant (HR), moderately resistant (MR), resistant (R), susceptible (S), moderately susceptible (MS), and highly susceptible (HS), respectively. A HR plant named "NBG-SG Soybean" was identified, which showed stable resistance for 5 years (2016-2020) at the experimental field of the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, a location that is a hot spot area for virus infection. HS soybean germplasm were also identified as NBG-47 (PI628963), NBG-117 (PI548655), SPS-C1 (PI553045), SPS-C9 (PI639187), and cv. NARC-2021. The YMD adversely affected the yield and a significant difference was found in the potential yield of NBG-SG-soybean (3.46 ± 0.13a t/ha) with HS soybean germplasm NARC-2021 (0.44 ± 0.01c t/ha) and NBG-117 (1.12 ± 0.01d t/ha), respectively. The YMD incidence was also measured each year (2016-2020) and data showed a significant difference in the percent disease incidence in the year 2016 and 2018 and a decrease after 2019 when resistant lines were planted. The resistance in NBG-SG soybean was further confirmed by testing for an already known mutation (SNP at 149th position) for YMD in the Glyma.18G025100 gene of soybean. The susceptible soybean germplasm in the field was found positive for the said mutation. Moreover, an ortholog of the CYR-1 viral resistance gene from black gram was identified in soybean as Glyma.13G194500, which has a novel deletion (28bp/90bp) in the 5`UTR of susceptible germplasm. The characterized soybean lines from this study will assist in starting soybean breeding programs for YMD resistance. This is the first study regarding screening and molecular analysis of soybean germplasm for YMD resistance.

Genomic analysis of wolves from Pakistan clarifies boundaries among three divergent wolf lineages.

Among the three main divergent lineages of gray wolf (Canis lupus), the Holarctic lineage is the most widespread and best-studied, particularly in North America and Europe. Less is known about Tibetan (also called Himalayan) and Indian wolf lineages in southern Asia, especially in areas surrounding Pakistan where all three lineages are thought to meet. Given the endangered status of the Indian wolf in neighboring India and unclear southwestern boundary of the Tibetan wolf range, we conducted mitochondrial and genome-wide sequencing of wolves from Pakistan and Kyrgyzstan. Sequences of the mitochondrial D-loop region of 81 wolves from Pakistan indicated contact zones between Holarctic and Indian lineages across the northern and western mountains of Pakistan. Reduced-representation genome sequencing of 8 wolves indicated an east-to-west cline of Indian to Holarctic ancestry, consistent with a contact zone between these two lineages in Pakistan. The western boundary of the Tibetan lineage corresponded to the Ladakh region of India's Himalayas with a narrow zone of admixture spanning this boundary from the Karakoram Mountains of northern Pakistan into Ladakh, India. Our results highlight the conservation significance of Pakistan's wolf populations, especially the remaining populations in Sindh and Southern Punjab that represent the highly endangered Indian lineage.

A graft that crafts nontransgenic and genome-edited plants.

Grafting in plants facilitates the transmission of biomolecules across the union formation. Recently, Yang et al. demonstrated that inter- and intraspecific grafting in plants can be exploited for trafficking tRNA-tagged mobile reagents of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system from the transgenic rootstock to wild-type scion for genetic improvement in plants through targeted mutagenesis.

PASTE: a high-throughput method for large DNA insertions.

Prime editing (PE) enables precise genome editing at targeted locus without inducing double-stranded breaks (DSBs). Despite its precision, PE lacks the tendency to integrate large DNA fragments into the genome. Recently, Yarnall et al. reported clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 and an integrase-based system that conducts targeted integration of large DNA sequences (~36 kb) into the genome more efficiently.

Plant Virus-Derived Vectors for Plant Genome Engineering.

Advances in genome engineering (GE) tools based on sequence-specific programmable nucleases have revolutionized precise genome editing in plants. However, only the traditional approaches are used to deliver these GE reagents, which mostly rely on Agrobacterium-mediated transformation or particle bombardment. These techniques have been successfully used for the past decades for the genetic engineering of plants with some limitations relating to lengthy time-taking protocols and transgenes integration-related regulatory concerns. Nevertheless, in the era of climate change, we require certain faster protocols for developing climate-smart resilient crops through GE to deal with global food security. Therefore, some alternative approaches are needed to robustly deliver the GE reagents. In this case, the plant viral vectors could be an excellent option for the delivery of GE reagents because they are efficient, effective, and precise. Additionally, these are autonomously replicating and considered as natural specialists for transient delivery. In the present review, we have discussed the potential use of these plant viral vectors for the efficient delivery of GE reagents. We have further described the different plant viral vectors, such as DNA and RNA viruses, which have been used as efficient gene targeting systems in model plants, and in other important crops including potato, tomato, wheat, and rice. The achievements gained so far in the use of viral vectors as a carrier for GE reagent delivery are depicted along with the benefits and limitations of each viral vector. Moreover, recent advances have been explored in employing viral vectors for GE and adapting this technology for future research.

Biomechanical effects of high heeled shoes on neck pain among females: A narrative review.

Neck pain is the fourth leading cause of disability, and is the most common musculoskeletal disorder. High-heel shoes, one of the significant identities of females, cause pain in the neck as well as in feet and ankle regions. The current narrative review was planned to explore evidence to highlight the biomechanical factors of high-heel shoes as the source of neck pain, which mostly remains undiagnosed. PubMed and Google Scholar search engines were explored for full text of research articles published in English language from 2016 to 2021. Of the 82 studies initially found, 22(27%) were shortlisted for full-text assessment, and, of them, 6 (27.27%) were selected for detailed analysis. Despite other contributing factors, kinematics and kinetics should be considered primarily during neck pain management. Based on best available evidence, high heels increase the individual's height, but result in significant decrease in trunk flexion. Evidence also suggests that the type and width of heels do not affect as much as the height of the heels in the context of pain and functional issues in the cervical region.

Genome-Wide Analysis of WRKY Gene Family and Negative Regulation of GhWRKY25 and GhWRKY33 Reveal Their Role in Whitefly and Drought Stress Tolerance in Cotton.

The WRKY transcription factor family is marked by its significant responsiveness to both biotic and abiotic plant stresses. In the present study, the WRKY family of Gossypium hirsutum has been identified and classified into three groups based on the number of conserved WRKY domains and the type of zinc finger motif. This classification is further validated by conserved domain and phylogenetic analysis. Two members of the WRKY family, WRKY25 and WRKY33, have been targeted through VIGS in G. hirsutum. VIGS-infiltrated plants were evaluated under drought stress and whitefly infestation. It was observed that GhWRKY33-downregulated plants showed a decrease in whitefly egg and nymph population, and GhWRKY33 was found to be a strong negative regulator of whitefly and drought stress, while GhWRKY25 was found to be a moderate negative regulator of whitefly and drought stress. As the targeted genes are transcription factors influencing the expression of other genes, the relative expression of other stress-responsive genes, namely MPK6, WRKY40, HSP, ERF1, and JAZ1, was also analyzed through qRT-PCR. It was found elevated in GhWRKY33-downregulated plants, while GhWRKY25-downregulated plants through VIGS showed the elevated expression of ERF1 and WRKY40, a slightly increased expression of HSP, and a lower expression level of MPK6. Overall, this study provides an important insight into the WRKY TF family and the role of two WRKY TFs in G. hirsutum under drought stress and whitefly infestation. The findings will help to develop crops resilient to drought and whitefly stress.

Whole-Genome Resequencing Deciphers New Insight Into Genetic Diversity and Signatures of Resistance in Cultivated Cotton Gossypium hirsutum.

Cotton is an important crop that produces fiber and cottonseed oil for the textile and oil industry. However, cotton leaf curl virus disease (CLCuD) stress is limiting its yield in several Asian countries. In this study, we have sequenced Mac7 accession, a Gossypium hirsutum resistance source against several biotic stresses. By aligning with the Gossypium hirsutum (AD1) 'TM-1' genome, a total of 4.7 and 1.2 million SNPs and InDels were identified in the Mac7 genome. The gene ontology and metabolic pathway enrichment indicated SNPs and InDels role in nucleotide bindings, secondary metabolite synthesis, and plant-pathogen interaction pathways. Furthermore, the RNA-seq data in different tissues and qPCR expression profiling under CLCuD provided individual gene roles in resistant and susceptible accessions. Interestingly, the differential NLR genes demonstrated higher expression in resistant plants rather than in susceptible plants expression. The current resequencing results may provide primary data to identify DNA resistance markers which will be helpful in marker-assisted breeding for development of Mac7-derived resistance lines.

Development of modified Cry1Ac for the control of resistant insect pest of cotton, Pectinophora gossypiella.

Cotton has been one of the most important cash crops in Pakistan, but its production is adversely affected by biotic and abiotic stresses. Insect pests such as pink bollworm present a colossal vulnerability to such a financially important commodity. Bt toxins have been widely used to safeguard agricultural plants against notorious insect pests such as cotton bollworm and pink bollworm, and they have proven to be effective in reducing chewing insect pests. However, its efficacy has been challenged due to the development of resistance in insect pests against Bt toxins such as Cry1Ac and this poses a significant risk to the long-term adoption of these Bt crops. Resistance in insect pests against Bt toxin Cry1Ac is developed due to the mutations in the midgut receptors such as cadherin. In this study first 56 amino acids which also includes helix alpha-1 portion from N-terminus of the Cry1Ac were removed and the gene was commercially synthesized following codon optimization. Modified Cry1Ac was used to develop transgenic plants of Nicotiana tabacum and insect bioassays were conducted to check the efficacy of Cry1Ac through leaf bioassays. Cry1Ac, a modified Bt toxin, was produced pET-28a (+), and diet bioassays were performed using purified protein at various doses against Pectinophora gossypiella. Based on the insect mortality and LC50, the Cry1AcM3 form of the modified toxins was shown to be more potent than the other modified versions (Cry1AcM1, Cry1AcM2), with more than 80 % mortality against resistant pink bollworm at 1.25 g/mL and an LC50 of 0.48. The results suggest that modified toxin cry1Ac may be useful in controlling population of pink bollworm resistant against cry1Ac.

Transcriptome diversity assessment of Gossypium arboreum (FDH228) leaves under control, drought and whitefly infestation using PacBio long reads.

Alternative splicing (AS) and alternative polyadenylation (APA) are common mechanisms in eukaryotes to increase the complexity of transcriptomes and subsequently proteomes. Analysis of long reads transcriptomics data can result in the discovery of novel transcripts, splice sites, AS or APA events. Gossypium arboreum is an important cultivated cotton species and a putative contributor of the A sub-genome to the modern tetraploid cotton; and inherently tolerant to several biotic and abiotic stresses. Specifically, its variety 'FDH228' is considered to be an important resistance source. In this study, we sequenced the G. arboreum (var. FDH228) transcriptome using PacBio IsoSeq and illumina short read sequencing under three different conditions i.e. untreated/healthy, treated with biotic stress through whitefly infestation, and treated with abiotic stress via water deprivation, for the discovery and surveying of canonical and non-canonical AS, APA and transcript fusion events. We were able to obtain 15,419 unique transcripts from all samples representing 11,343 genes, out of which 10,832 were annotated and 520 were novel with respect to the published reference genome. These transcripts were grouped into different structural categories including 60 Antisense, 11,959 having a full-splice match, 999 with incomplete-splice match, 30 fusion transcripts, 177 genic, 479 intergenic, 771 novels in the catalog, and 944 Novel but not found in the catalog. Subsequently, randomly selected candidate transcripts were experimentally validated using qRT-PCR. Our comprehensive identification of canonical and non-canonical splicing events, and novel and fusion transcripts aids in the understanding of the resistance mechanisms for this specific germplasm.

Cotton Mi-1.2-like Gene: A potential source of whitefly resistance.

Whitefly (Bemisia tabaci) inflicts tremendous yield losses to cotton crops in many parts of the world by sap-sucking and transmitting viral diseases. The tomato-associated Mi-1.2 gene has been successfully deployed in tomato cultivars to attain whitefly resistance. In the current study, putative Mi-1.2-like orthologs were identified in five whitefly hosts and functionally validated through virus-inducing gene silencing (VIGS) in cotton plants. The expression profiling and qPCR results depicted differential regulation of the Mi-1.2-like gene in various tissue types and under different biotic and abiotic stresses, especially in whitefly susceptible and resistant cotton plants. The upregulation of the Mi-1.2-like gene (Gadrp RPP-13 Like gene) was observed at 24 h and 48 h post-whitefly exposure (PWFE) in whitefly resistant (FDH-228) and tolerant (Mac7) cotton plants as compared to susceptible plants of Coker-312. However, delayed expression was recorded at 72 h of PWFE in Coker-312 plants. In TRV based gene silencing experiment, silencing of the Mi-1.2-like gene, significantly enhanced the whitefly infestation on both whitefly-resistant and susceptible cotton genotypes. Based on these results, we conducted the evolutionary analysis of Mi-1.2-like orthologs among cotton, cassava, tomato, papaya, and cucumber hosts. This indicated that cotton associated Mi-1.2 like gene has a close relation with cassava and tomato. These results suggested that Mi-1.2-like R genes could be the potential candidate for deriving whitefly resistance response in cotton plants.

Omics-driven exploration and mining of key functional genes for the improvement of food and fiber crops.

The deployment of omics technologies has obtained an incredible boost over the past few decades with the advances in next-generation sequencing (NGS) technologies, innovative bioinformatics tools, and the deluge of available biological information. The major omics technologies in the limelight are genomics, transcriptomics, proteomics, metabolomics, and phenomics. These biotechnological advances have modernized crop breeding and opened new horizons for developing crop varieties with improved traits. The genomes of several crop species are sequenced, and a huge number of genes associated with crucial economic traits have been identified. These identified genes not only provide insights into the understanding of regulatory mechanisms of crop traits but also decipher practical grounds to assist in the molecular breeding of crops. This review discusses the potential of omics technologies for the acquisition of biological information and mining of the genes associated with important agronomic traits in important food and fiber crops, such as wheat, rice, maize, potato, tomato, cassava, and cotton. Different functional genomics approaches for the validation of these important genes are also highlighted. Furthermore, a list of genes discovered by employing omics approaches is being represented as potential targets for genetic modifications by the latest genome engineering methods for the development of climate-resilient crops that would in turn provide great impetus to secure global food security.