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Persistence and prevalence of microbial diseases (pandemics, epidemics) is the most alarming threats to the human resulting in huge health and economic losses. Rapid detection and understanding of the disease dynamics by molecular biotechnology tools allow for robust reporting, treatment and control of diseases. As per WHO, the optimal diagnostic approach should be quick, specific, sensitive, without a stringed instrument, and low cost. The drawbacks of traditional detection techniques promote the use of CRISPR-mediated nucleic acid detection methods such as SHERLOCK as detection method. It takes advantage of the unexpected in vitro features of CRISPR-Cas system to develop field-deployable sensitive detection tools. Previously, CRISPR-mediated diagnostic methods have extensively been reviewed particularly for SARS-COV-2 detection, but it fails to provide the insight into advances of this technique. This study is the first attempt to review the advances of SHERLOCK approach as diagnostic tool for viral diseases detection. Variations of SHERLOCK mechanism for improved efficiency are discussed. Particularly integrated SHERLOCK approaches in terms of extraction-free assay and Bluetooth-enabled detection are reviewed to access their feasibility for the development of simpler and cost-effective diagnostic toolkits. Insight in to perks and limitations of diagnostic methods indicates its potential as ultimate diagnostic instrument for disease management.

Fruit production is an important part of the gross domestic product for many countries around the world especially to those who have a strong focus on agriculture. However, long-term maintenance and yield stability of fruit production may be threatened by the ongoing climate change and its consequences like extended drought periods, heavy rain events, and floodings. Genome editing, with its progressive technological developments, offers opportunities to adapt relevant fruit plant species to new climatic conditions. Among modern genome editing techniques, CRISPR/Cas, in particular, has the potential to support breeding for those fruit plant species with extended breeding cycles, e.g., perennial fruits. In this review, we discuss CRISPR/Cas and other genome editing techniques in detail and how these techniques can be applied to support the breeding of fruit plant species for adaptation to changing climates. The chronological history of CRISPR/Cas9 systems, their associated computational tools, genomic data sources, transformation methods along with their delivery vehicles, quality improvement, environmental-stress resiliency, limitations, and future perspectives will also be discussed with respect to securing future global fruit production.

A genome-wide association study (GWAS) was performed using a high-density infinium 90K SNP array. We identified a total markers traits associations (MTAs) (p ?0.000) for the following plant traits; days taken to 50% heading(DH), days to 50% maturity (DM), plant height (Ph) cm, flag leaf area cm2 (FLA), tillers number per plant, spike length (SL) cm and grain yield per plant (GP) g. Most of the SNPs were identified in the A and B genome as compared to the D genome. The significant associated SNPs were mainly distributed on the chromosome 2B, 3B, 5A, and 5B. Nine SNPs on chromosome 5A, 2B and 2D were identified having pleiotropic effects The correlation analysis showed a significant positive association among SL, NT, GP. Which depicted that these traits are promising for breeding high yielding wheat cultivars. This study provided useful information of the valuable genetic loci for marker-assisted breeding.

Understanding genetic architecture of a crop germplasm is necessary for designing a successful breeding program. Herein, we evaluated a large collection of 500 spring wheat accessions for 2 crop seasons to dissect the genetics of 11 yield components and their direct and indirect contributions to grain yield (GY). The genetic estimates of broad sense heritability (h2), genetic advance (GA), phenotypic correlation (rp), genotypic correlation (rg), and path coefficient analysis were performed. Significant genetic variation was observed for all yield traits suggesting that GY can be improved by exploiting the studied yield traits. Phenotypic coefficient of variation (Vp) was greater than genotypic coefficient of variation (Vg) for all studied traits. Higher broad sense h2 and GA were observed for grains per plant (GpP), spikes per plant (SpP), spikelets per spike (Spt/S) and grain yield (GY). The GY exhibited significant and positive correlation with all studied traits except with spikelet density (SptD). The GpP and TGW exhibited positive direct effect on increasing grain yield. Taking together, SpP, GpS, TGW and SDW are the major contributors to improving genetic yield potential of bread wheat with spring growth habit. The negative correlation between GY and SptD was dissected in path coefficient analysis as negative indirect effect of SptD on GY through reduced GpS and TGW. Our study provides new insights on the association of GpP, TGW and SDW in bread wheat. The GpP, TGW and SDW are influenced by SpP, Gpt/S, G/Spt, SptD, AL and PH with indirect effects on GY. To improve yield potential in wheat, the traits with direct effects (GpP, TGW and SDW) and indirect effects (e.g., SptD) can be used as selection criteria.

Among different abiotic stresses, drought stress is the leading cause of impaired plant growth and low productivity worldwide. It is therefore essential to understand the process of drought tolerance in plants and thus to enhance drought resistance. Accumulating evidence indicates that phytohormones are essential signaling molecules that regulate diverse processes of plant growth and development under drought stress. Plants can often respond to drought stress through a cascade of phytohormones signaling as a means of plant growth regulation. Understanding biosynthesis pathways and regulatory crosstalk involved in these vital compounds could pave the way for improving plant drought tolerance while maintaining overall plant health. In recent years, the identification of phytohormones related key regulatory genes and their manipulation through state-of-the-art genome engineering tools have helped to improve drought tolerance plants. To date, several genes linked to phytohormones signaling networks, biosynthesis, and metabolism have been described as a promising contender for engineering drought tolerance. Recent advances in functional genomics have shown that enhanced expression of positive regulators involved in hormone biosynthesis could better equip plants against drought stress. Similarly, knocking down negative regulators of phytohormone biosynthesis can also be very effective to negate the negative effects of drought on plants. This review explained how manipulating positive and negative regulators of phytohormone signaling could be improvised to develop future crop varieties exhibiting higher drought tolerance. In addition, we also discuss the role of a promising genome editing tool, CRISPR/Cas9, on phytohormone mediated plant growth regulation for tackling drought stress.

Acacia species are very important tree species in tropical and subtropical countries of the World for their economic and medicinal benefits. Precise identification of Acacia is very important to distinguish the invasive species from rare species however, it is difficult to differentiate Acacia species based on morphological charcters. In addition, precise identification is also important for wood charcterization in the forest industry as these species are declining due to illegal logging and deforestation. To overcome thsese limitations of morphological identification, DNA barcoding is being used as an efficient and quick approach for precise identification of tree species. In this study, we selected two chloroplast and plastid base DNA markers (rbcL and matK) for the identification of five selected tree species of Acacia (A. albida, A. ampliceps, A. catechu, A. coriacea and A. tortilis). The genomic DNA of the selected Acacia species was extracted, amplified through PCR using specific primers and subsequently sequenced through Sanger sequencing. In matK DNA marker the average AT nucleotide contents were higher (59.46%) and GC contents were lower (40.44%) as compared to the AT (55.40%) and GC content (44.54%) in rbcL marker. The means genetic distance K2P between the Acacia species was higher in matK (0.704%) as compared to rbcL (0.230%). All Acacia species could be identified based on unique SNPs profile. Based on SNP data profiles, DNA sequence based scannable QR codes were developed for accurate identification of Acacia species. The phylogenetic analysis based on both markers (rbcL and matK) showed that both A. coriacea and A. tortilis were closely related with each other and clustered in the same group while other two species A. albida and A. catechu were grouped together. The specie A. ampliceps remained ungrouped distantly, compared with other four species. These finding highlights the potential of DNA barcoding for efficient and reproducible identification of Acacia species.

To evaluate the genetic background of lodging traits, six populations of two wheat crosses were sown under different nitrogen levels by using split plot design. Generation mean analysis was used to estimate the gene action for lodging related traits. Results showed genetic variability and significant differences for all traits. Allelic and non-allelic gene actions were operative for different traits with different magnitude under both normal and lodging condition. Duplicate types of espistasis were observed for grain yield per plant, 1000- kernel weight, number of grains per spike and number of tillers. The stem diameter, height of basal nodes and basal inter-nodal distance depicted the additive, additive × dominance and dominance gene action. The aforementioned results showed that plant height, stem diameter, height of basal nodes and basal internodal distance were more prone traits for lodging and selection should be practiced in later generations with increasing homozygosity.

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.

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

Background: The COBL gene encodes a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. Recently identified COBRA genes supposed as a key regulator of the orientation of cell expansion in the root indicating that Cobra gene family members are likely to be important new players at the plasma membrane-cell wall interface. Methods and Results: By performing a database search and domain prediction, we identified five genes named as TaCOBL 1, TaCOBL 2, TaCOBL 3, TaCOBL 4 and TaCOBL 5, and selected for further analysis. Chromosomal locations of each gene were drawn on karyotype. Structure of genes, promoter analysis and phylogenetic analysis were done using bioinformatics tools and databases. Set of novel SNPs were also predicted. Gene ontologies were analyzed, and pathways involving cobra genes were predicted. Whole genes of 3kb to 4kb were successfully amplified. Five set of primers were designed targeting TaCOBL 1, TaCOBL 2, TaCOBL 3 TaCOBL 4 and TaCOBL 5 genes. Expression of COBL genes were checked in root and shoot by using qPCR. The qRT-PCR revealed that expression TaCOBL genes can be regulated under abiotic stress such drought stress. Conclusion: The comprehensive annotation and expression profiling of COBL genes performed in this study enhanced our understanding and these genes were found to play a significant role in drought stress. Our findings lay the groundwork for further functional investigation of COBL genes mechanism.

The COBL genes encode a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. Recently identified COBRA genes are supposed as a key regulator of the orientation of cell expansion in the root indicating that COBRA gene family members are likely to be important players at the plasma membrane-cell wall interface. Five COBL gene namely, TaCOBL 1, TaCOBL 2, TaCOBL 3, TaCOBL 4 and TaCOBL 5 were identified using database search and domain predictions. Chromosomal location of each gene was mapped on karyotype. Structure of genes, promoter analysis and phylogenetic analysis were performed using different bioinformatics tools. Set of novel SNPs were also predicted. Gene ontologies were analyzed, and the processes and pathways were identified in which COBRA genes were involved. The molecular weight all the cobra proteins was in range of 50–75 KDa with 429–461 amino acid residues. The COBL genes were mapped on homeologous groups 2, 4, 5, 6 and 7. Gene ontology analysis revealed that TaCOBL genes were involved in cellulose microfibril organization, mucilage biosynthetic process involved in seed coat development, plant-type cell wall biogenesis plant-type cell wall cellulose biosynthetic process, seed coat development and seed development. Three drought responsive cis-elements (WRKY, ABRE and DRE) were found nearby COBL genes The qRT-PCR revealed TaCOBL genes are drought responsive and can be further explored to understand their role in drought tolerance in wheat. The comprehensive annotation and expression profiling of COBL genes revealed that all five COBL genes are drought response. The promoter cis-regulatory element analysis revealed that COBL genes had stress related WRKY, ABRE and DRE cis-regulatory elements. This evidence suggest that TaCOBL genes are involved in drought stress tolerance.

Cotton is considered as a major cash crop of the world. It earns huge foreign exchange by its valuable products; fiber, lint, cotton seed oil, hull and a lot more. Being an important fiber crop, it earns huge foreign exchange by contributing to textile and seed oil industry. This review summarizes cotton biology, its diversity and domestication, genome assembly, constraints in its production and methods to improve cotton plant to fulfill the need of textile and oil industry. But cotton is facing enormous biotic and abiotic stresses with insect pests being most prominent. Massive destruction caused by insects needs to be controlled for maintaining fruitful cotton crop production. Conventional breeding approaches are limited to improving single trait and integrate stable genes within plant genome in approximately 7 - 8 years. Improved biotechnological procedures have paved new pathways to target genes specifically and improve cotton germplasm in lesser time than conventional breeding.

For HMW-GS, a total of 16 different subunits and 30 allelic haplotypes were identified. The expression of HMW-GS ranged from 3 to 5 per genotype instead of 6 per genotype, revealing the phenomenon of gene silencing. The 2* was the most frequent subunit for Glu-A1 locus and was found in 52.45% lines. Eight subunits were identified for Glu-B1 locus. Five subunit pairs i.e. 6+8, 7+8, 7+9, 13+16 and 17+18 were present in the germplasm. Some single subunits i.e. 6, 7, 8 and 20 were also found. Subunit pair 17+18 was the most frequent with a partial frequency of 32.17%. For Glu-D1, four subunit pairs i.e. 2+12, 5+10, 2+11 and 5+11 were observed. The 5+10 was the most frequent subunit for Glu-D1 and was present in 65.03% lines. The 2+12 subunit pair was identified in 34.97% lines. For Glu3 loci 18 LMW subunits were identified. For Glu-A3, five subunits i.e. b, c, d, e and g were identified. Eight subunits i.e. b, c, d, f, g, h, i and j were identified for Glu-B3 locus. Four subunits i.e. a, b, c and l were identified for Glu-D3 locus.

Advancements in genome manipulation have transformed the whole face of synthetic biology. Technologies like CRISPR/Cas based gene editing offer myriad ways to tweak genomes and help plant scientists to fulfil what they have been wishing for since the advent of molecular biology during the mid-nineties. The past decade witnessed an enormous adoption of this technology in almost all branches of biology from agriculture to medicine and environment. After successfully deploying gene editing to modify simple traits, scientists are now embarked on more ambitious adventures in genomics to combat challenges of food security in the wake of increasing population and climate change adversaries. The chapter outlines use of gene editing in tailoring crops beyond simple traits aiming to harvest the desired diversity lost during domestication and manipulating complex traits which evolved over evolutionary timescale like photosynthesis, nitrogen fixation, and epigenetics.

Since the inception of CRISPR technology, an unprecedented progress has been observed in the field of genome editing (GE) in animals and plants with equal success. Undoubtedly, the technology is very simple, cost-effective, time-saving, and precise compared to old techniques such as MNs, ZFNs, and TALENs. Although, off-targeting has been a serious issue with CRISPR technology but it is the most promising technique hitherto. A number of approaches have been used to address the issue of off-targeting such as use of modified gRNA and other variants of CRISPR/Cas. In this chapter, current scenario of CRISPR technology will be discussed along with its pros and cons. Moreover, the strategies to address the issues associated with CRISPR technology will be discussed with future perspectives of the technology.

Cotton is an important natural fiber crop and is known as “white gold” due to its economic importance. Its seed is also a source of oil, proteins, and feed for the livestock. Sustaining cotton production has been a major challenge owing to the multitude of biotic and abiotic stresses. Among these, the infestation of insect pests and diseases are the most devastating yield depressing factors. Keeping insect population under control heavily relies on the application of chemicals (pesticides) which may raise serious environmental concerns. Production of insecticidal proteins in the cotton plants through different genetic means opened a new era of insect-pest management. On the basis of number of transgenes introduced, three different generations of the Bt-cotton (cotton expressing genes coding for different insect toxins from the soil bacterium Bacillus thuringiensis) have been developed. The first generation of Bt cotton carried a single Bt gene, while the second- and third-generation Bt cottons contained double and triple genes for a better control over lepidopteran insects. In addition to Bt genes, genes conferring tolerance to different herbicides have also been introduced in cotton for controling weeds more effectively. Cultivation of Bt cotton has been instrumental in controlling bollworms; however, the emergence of resistance in insects against the Bt toxins has been reported in few regions worldwide. In this chapter, current state of the art knowledge on transgenic cotton including generations of Bt cotton, mechanism of action, risk assessment, and prospects has been discussed.

The ever-growing human population and climate shifts are creating a dire need to the development of crop plants with high yield and improved nutritional quality. Conventional breeding tools cannot satisfy the future needs. Increased agricultural production through advanced technologies is urgently required to increase yield and easy access to nutritious food worldwide. Providentially, next-generation genome editing technologies like CRISPR/Cas system pave the way toward a new horizon for crop plant improvement and subsequently transform classical plant breeding. In this chapter, applications of CRISPR/Cas-based genome editing for yield and quality improvement are highlighted.

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.

Acacia species are very important tree species in tropical and subtropical countries of the World for their economic and medicinal benefits. Precise identification of Acacia is very important to distinguish the invasive species from rare species however, it is difficult to differentiate Acacia species based on morphological charcters. In addition, precise identification is also important for wood charcterization in the forest industry as these species are declining due to illegal logging and deforestation. To overcome thsese limitations of morphological identification, DNA barcoding is being used as an efficient and quick approach for precise identification of tree species. In this study, we selected two chloroplast and plastid base DNA markers (rbcL and matK) for the identification of five selected tree species of Acacia (A. albida, A. ampliceps, A. catechu, A. coriacea and A. tortilis). The genomic DNA of the selected Acacia species was extracted, amplified through PCR using specific primers and subsequently sequenced through Sanger sequencing. In matK DNA marker the average AT nucleotide contents were higher (59.46%) and GC contents were lower (40.44%) as compared to the AT (55.40%) and GC content (44.54%) in rbcL marker. The means genetic distance K2P between the Acacia species was higher in matK (0.704%) as compared to rbcL (0.230%). All Acacia species could be identified based on unique SNPs profile. Based on SNP data profiles, DNA sequence based scannable QR codes were developed for accurate identification of Acacia species. The phylogenetic analysis based on both markers (rbcL and matK) showed that both A. coriacea and A. tortilis were closely related with each other and clustered in the same group while other two species A. albida and A. catechu were grouped together. The specie A. ampliceps remained ungrouped distantly, compared with other four species. These finding highlights the potential of DNA barcoding for efficient and reproducible identification of Acacia species.

Genome editing is considered as the most widely used approach of the present era. It had become a basic need of the current micro and molecular biological experiments. Gene engineering finds its widespread applications in medical, industry and agricultural sector. Unlike previous genetic engineering practices to insert or delete a part of genetic material at random place, genome editing allows the precise manipulation of DNA at a specific location. Zinc Finger Nucleases (ZFNs), Transcription Activator like Effector Nucleases (TALENs), Clustered Regularly Interspresed Short Palindromic repeats (CRISPR/Cas system) and meganucleases (recombinases) are the prime tools for editing an organism’s genome. Genome editing tools have an advantage to selectively delete or to integrate specific genes at specific loci. Use of recombinases for specifying site has further reduced time to integrate genes site specifically. Site specific gene stacking by the use of recombinases coupled with ZFNs, TALENs, or CRISPR/Cas genes have paved new pathways to target genes site specifically and to improve germplasm in lesser time than conventional breeding approaches.

Precise assessment of diversity in available breeding germplasm helps to preempt epidemics and abrupt environmental changes. Spring wheat germplasm consisting of 84 accessions including cultivars, breeding lines and landraces from various origins was scanned with 44 SSRs. For allele frequencies, allelic patterns, heterozygosity and polymorphism the selected population was divided in three subpopulations: (i) pre-green revolution (pre-1965), (ii) post-green revolution (post-1965), (iii) post-veery (post-2000). Alleles produced in pre-1965, post-1965 and post-2000 subpopulations were 115, 144 and 131, respectively. Mean PIC values for pre-1965, post-1965 and post-2000 subpopulations were 0.48, 0.52 and 051, respectively. Allelic patterns showed no locally common alleles in any of the subpopulation. The pre-1965 subpopulation had also no private allele, however, average number of private alleles decreased from post-1965 to post-2000 subpopulation. In case of effective alleles and Shannon’s information index trend was increasing from pre-1965 to post-1965 and then decreasing from post-1965 to post-2000. The decreasing trend alarms the reduced genetic diversity in wheat varieties developed after 2000. PCA and cluster analysis didn’t clearly differentiated subpopulations, though pre-1965 genotypes showed higher genetic distance from post-1965 and post-2000 subpopulations. The decreasing measures of genetic diversity in post-2000 wheat genotypes should be a concern for wheat breeders, therefore, all sources of broadening genetic diversity should be exploited.

The revolutionary technology of CRISPR/Cas systems and their extraordinary potential to address fundamental questions in every field of biological sciences has led to their developers being awarded the 2020 Nobel Prize for Chemistry. In agriculture, CRISPR/Cas systems have accelerated the development of new crop varieties with improved traits—without the need for transgenes. However, the future of this technology depends on a clear and truly global regulatory framework being developed for these crops. Some CRISPR-edited crops are already on the market, and yet countries and regions are still divided over their legal status. CRISPR editing does not require transgenes, making CRISPR crops more socially acceptable than genetically modified crops, but there is vigorous debate over how to regulate these crops and what precautionary measures are required before they appear on the market. This article reviews intended outcomes and risks arising from the site-directed nuclease CRISPR systems used to improve agricultural crop plant genomes. It examines how various CRISPR system components, and potential concerns associated with CRISPR/Cas, may trigger regulatory oversight of CRISPR-edited crops. The article highlights differences and similarities between GMOs and CRISPR-edited crops, and discusses social and ethical concerns. It outlines the regulatory framework for GMO crops, which many countries also apply to CRISPR-edited crops, and the global regulatory landscape for CRISPR-edited crops. The article concludes with future prospects for CRISPR-edited crops and their products.

Whitefly-transmitted geminiviruses (begomoviruses) cause heavy losses to many food and fiber crops in Pakistan. Many weeds also show symptoms typical of begomoviruses. Ageratum (Ageratum conyzoides) is a common perennial weed in Pakistan, growing along irrigation canals, that often shows symptoms, such as yellow vein and mosaic, suggesting infection by a begomovirus. To confirm this, symptomatic and asymptomatic ageratum plants were collected from three locations in the Punjab Province of Pakistan, and total DNA was isolated, subjected to agarose gel electrophoresis, transferred to a nylon membrane, and Southern blotted. Total DNA isolated from cotton infected with Cotton leaf curl virus (CLCuV), tomato infected with Tomato leaf curl virus from Pakistan (TLCV-Pak), tobacco infected with African cassava mosaic virus (ACMV) from Nigeria, and healthy tobacco were included as controls. A full-length clone of CLCuV DNA A was labeled with [32P]dCTP by oligo-labeling and hybridized at medium stringency. The probe detected characteristic geminivirus DNA forms in symptomatic ageratum and plants infected with CLCuV, TLCV-Pak, and ACMV, while no signal was detected in asymptomatic ageratum from the field or healthy tobacco. To confirm infection by a begomovirus, degenerate primers WTGF (5′-GATTGTACGCGTCCDCCTTTAATTT GAAYBGG-3′), designed in the rep gene of begomoviruses, and WTGR (5′-TANACGCGTGGC TTCKRTACATGGCCTDT-3′), designed in the coat protein gene of DNA A of begomoviruses, were used in polymerase chain reaction (PCR). Degenerate primers (PBLv2040 and PCRc1) also were used in PCR (2). A product of expected size (≈1.4 kb) was obtained with DNA A primers from symptomatic ageratum, while no product was obtained with DNA B primers in the same sample. Previously we were unable to detect a DNA component equivalent to begomovirus DNA B in cotton showing symptoms of cotton leaf curl disease (1). We recently reported a novel circular DNA molecule that was approximately half as long as the full-length DNA A (CLCuV DNA-1) associated with CLCuV that share homology to plant nanoviruses (1). The supercoiled replicative form of viral DNA isolated from infected ageratum plants indicated the presence of smaller molecules, as was found in cotton leaf curl disease, suggesting that a nanovirus-like molecule might be associated with ageratum yellow vein disease. A duplicate blot of samples used in Southern hybridization with the DNA A probe was prepared, and a probe of the full-length clone of the nanovirus-like molecule (CLCuV DNA-1) was prepared as described for DNA A. The probe detected characteristic nanovirus DNA forms in ageratum with yellow vein symptoms and cotton infected with CLCuV, while no signal was detected in plants infected with TLCV-Pak or ACMV, healthy tobacco, or asymptomatic ageratum. Abutting primers PB2-F and PB2R (1), designed based on the CLCuV DNA-1 sequence, were unable to amplify a PCR product from ageratum with yellow vein symptoms, suggesting the nanovirus-like molecule associated with ageratum yellow vein disease is distinct from CLCuV DNA-1. Our results show that yellow vein disease of ageratum in Pakistan is associated with a begomovirus infection and single-stranded circular DNA molecule with similarity to CLCuV DNA-1. References: (1) S. Mansoor et al. Virology 259:190, 1999. (2) M. R. Rojas et al., Plant Dis. 77:340, 1993.