A critical look on CRISPR-based genome editing in plants.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing, derived from prokaryotic immunity system, is rapidly emerging as an alternative platform for introducing targeted alterations in genomes. The CRISPR-based tools have been deployed for several other applications including gene expression studies, detection of mutation patterns in genomes, epigenetic regulation, chromatin imaging, etc. Unlike the traditional genetic engineering approaches, it is simple, cost-effective, and highly specific in inducing genetic variations. Despite its popularity, the technology has limitations such as off-targets, low mutagenesis efficiency, and its dependency on in-vitro regeneration protocols for the recovery of stable plant lines. Several other issues such as persisted CRISPR activity in subsequent generations, the potential for transferring to its wild type population, the risk of reversion of edited version to its original phenotype particularly in cross-pollinated plant species when released into the environment and the scarcity of validated targets have been overlooked. This article briefly highlights these undermined aspects, which may challenge the wider applications of this platform for improving crop genetics.

Development of broad-spectrum insect-resistant tobacco by expression of synthetic cry1Ac and cry2Ab genes.

Efficacy of two newly synthesized cry1Ac and cry2Ab genes was checked in tobacco before their expression in cotton. Both genes were artificially synthesized and codon optimized with respect to cotton-preferred codon usage. These genes were cloned in a plant expression vector and then transformed into tobacco. Fifty-eight putative transgenic plants were recovered from the selected explants. Successful integration of both genes in plant genome was confirmed by PCR amplification. Expression of transgenes was confirmed by PCR amplification from total plant RNA. Detached leaf insect bioassays were conducted with Helicoverpa armigera and Spodoptera exigua larvae. About 12 % of the transgenic plants showed significantly high resistance to S. exigua. Significant mortality (62 %) of H. armigera was recorded within 24 h of bioassays. Both toxins showed synergistic effect in tobacco and broadened the spectrum of plant activity against insects.

Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences.

Several important biological processes are performed by distinct functional domains found on replication-associated protein (Rep) encoded by AC1 of geminiviruses. Two truncated forms of replicase (tAC1) gene, capable of expressing only the N-terminal 669 bp (5'AC1) and C-terminal 783 bp (3'AC1) nucleotides cloned under transcriptional control of the CaMV35S were introduced into cotton (Gossypium hirsutum L.) using LBA4404 strain of Agrobacterium tumefaciens to make use of an interference strategy for impairing cotton leaf curl virus (CLCuV) infection in transgenic cotton. Compared with nontransformed control, we observed that transgenic cotton plants overexpressing either N-terminal (5'AC1) or C-terminal (3'AC1) sequences confer resistance to CLCuV by inhibiting replication of viral genomic and ß satellite DNA components. Molecular analysis by Northern blot hybridization revealed high transgene expression in early and late growth stages associated with inhibition of CLCuV replication. Of the eight T(1) transgenic lines tested, six had delayed and minor symptoms as compared to nontransformed control lines which developed disease symptoms after 2-3 weeks of whitefly-mediated viral delivery. Virus biological assay and growth of T(2) plants proved that transgenic cotton plants overexpressing 5'- and 3'AC1 displayed high resistance level up to 72, 81%, respectively, as compared to non-transformed control plants following inoculation with viruliferous whiteflies giving significantly high cotton seed yield. Progeny analysis of these plants by polymerase chain reaction (PCR), Southern blotting and virus biological assay showed stable transgene, integration, inheritance and cotton leaf curl disease (CLCuD) resistance in two of the eight transgenic lines having single or two transgene insertions. Transgenic cotton expressing partial AC1 gene of CLCuV can be used as virus resistance source in cotton breeding programs aiming to improve virus resistance in cotton crop.

Pepper leaf curl Lahore virus requires the DNA B component of Tomato leaf curl New Delhi virus to cause leaf curl symptoms.

BACKGROUND: Begomoviruses are whitefly-transmitted geminiviruses with genomes that consist of either two components (known as DNA A and DNA B) or a single component (homologous to the DNA A component of bipartite begomoviruses). Monopartite begomoviruses are often associated with a symptom-modulating DNA satellite (collectively known as betasatellites). Both bipartite and monopartite begomoviruses with associated satellites have previously been identified in chillies showing leaf curl symptoms in Pakistan. RESULTS: A chilli plant (Capsicum annum) with chilli leaf curl disease symptoms was found to contain a begomovirus, a betasatellite and the DNA B component of Tomato leaf curl New Delhi virus (ToLCNDV). The begomovirus consisted of 2747 nucleotides and had the highest sequence identity (99%) with Pepper leaf curl Lahore virus (PepLCLV-[PK: Lah:04], acc. no. AM404179). Agrobacterium-mediated inoculation of the clone to Nicotiana benthamiana, induced very mild symptoms and low levels of viral DNA, detected in systemically infected leaves by PCR. No symptoms were induced in Nicotiana tabacum or chillies either in the presence or absence of a betasatellite. However, inoculation of PepLCLV with the DNA B component of ToLCNDV induced leaf curl symptoms in N. benthamiana, N. tabacum and chillies and viral DNA accumulated to higher levels in comparison to plants infected with just PepLCLV. CONCLUSIONS: Based on our previous efforts aimed at understanding of diversity of begomoviruses associated with chillies, we propose that PepLCLV was recently mobilized into chillies upon its interaction with DNA B of ToLCNDV. Interestingly, the putative rep-binding iterons found on PepLCLV (GGGGAC) differ at two base positions from those of ToLCNDV (GGTGTC). This is the first experimental demonstration of the infectivity for a bipartite begomovirus causing chilli leaf curl disease in chillies from Pakistan and suggests that component capture is contributing to the emerging complexity of begomovirus diseases in the region.

Codon optimization of cry1Ab gene for hyper expression in plant organelles.

With the advent of genetic manipulation techniques, it has become possible to clone and insert gene into the genome of crop plants to confer resistance to insects and pests. Resistance to insects has been demonstrating in transgenic plants either by triggering defense system of plants or by expressing heterologous cry genes for delta-endotoxins from Bacillus thuringiensis. In the present study, synthetic cry1Ab gene was developed with optimized chloroplast preferred codons and is expressed in tobacco plastid genome called plastome, following chloroplast transformation strategy, which is environment friendly technique to minimize out-crossing of transgenes to related weeds and crops. In addition, due to high polyploidy of plastid genome transformation of chloroplast permits the introduction of thousands of copies of foreign genes per plant cell, leading to extraordinarily high levels of foreign protein expression. The chloroplast transformation technology aims to insert stably into the plastome through homologous recombination into pre-decided position. To characterize the synthetic cry1Ab gene, chloroplast transformation vectors were developed and bombarded to the leaf cells of tobacco plants maintained under aseptic conditions. After bombardment, the drug resistant shoots were selected and regenerated on drug containing regeneration medium. Homoplasmic shoots were recovered after successive rounds of selection and regeneration. Proliferated plants were subjected to genomic DNA analysis by using polymerase chain reaction (PCR) technique where cry1Ab gene-specific primers were used. PCR positive plants were subjected to protein analysis, and functionally expressed proteins were detected using Immuno-Strips specific for cry1Ab/Ac gene products. Transgenic plants carrying cry1Ab gene were found expressing Bt toxins confirming that engineered gene could be expressed in other plants as well.

Aflatoxin Contamination of Milk Marketed in Pakistan: A Longitudinal Study.

A longitudinal one-year study was conducted to determine aflatoxin M1 levels in different types of milk marketed in Pakistan. Processed and raw liquid milk from 21 sources, two milk powder and six tea whitener brands were sampled on monthly basis from Islamabad. The aflatoxin M1 levels in liquid milk were lower (p < 0.05) in summer (April to July) compared with the levels in winter (January, November and December). The mean aflatoxin M1 levels were 254.9, 939.5, and 1535.0 ng/L in UHT, pasteurized, and raw milk, respectively (differing at p < 0.001). The mean toxin level in powdered milk after reconstitution was 522.1 ng/L. Overall, 12.9, 41.0, 91.9 and 50.0% of the UHT, pasteurized, raw and powdered milk samples, respectively, exceeded the Codex maximum tolerable limit of 500 ng of aflatoxin M1/L. It was estimated that consumers of raw and processed milk were exposed to 11.9 and 4.5 ng aflatoxin M1, respectively, per kg of body weight daily. The study indicates potential aflatoxin M1 exposure risks for the consumers of raw milk in the country. The levels of the toxin though comparatively lower in milk powder, requires attention as this type of milk is consumed by infants.

Evolution of Deeper Rooting 1-like homoeologs in wheat entails the C-terminus mutations as well as gain and loss of auxin response elements.

Root growth angle (RGA) in response to gravity controlled by auxin is a pertinent target trait for obtainment of higher yield in cereals. But molecular basis of this root architecture trait remain obscure in wheat and barley. We selected four cultivars two each for wheat and barley to unveil the molecular genetic mechanism of Deeper Rooting 1-like gene which controls RGA in rice leading to higher yield under drought imposition. Morphological analyses revealed a deeper and vertically oriented root growth in "NARC 2009" variety of wheat than "Galaxy" and two other barley cultivars "Scarlet" and "ISR42-8". Three new homoeologs designated as TaANDRO1-like, TaBNDRO1-like and TaDNDRO1-like corresponding to A, B and D genomes of wheat could be isolated from "NARC 2009". Due to frameshift and intronization/exonization events the gene structures of these paralogs exhibit variations in size. DRO1-like genes with five distinct domains prevail in diverse plant phyla from mosses to angiosperms but in lower plants their differentiation from LAZY, NGR and TAC1 (root and shoot angle genes) is enigmatic. Instead of IGT as denominator motif of this family, a new C-terminus motif WxxTD in the V-domain is proposed as family specific motif. The EAR-like motif IVLEM at the C-terminus of the TaADRO1-like and TaDDRO1-like that diverged to KLHTLIPNK in TaBDRO1-like and HvDRO1-like is the hallmark of these proteins. Split-YFP and yeast two hybrid assays complemented the interaction of TaDRO1-like with TOPLESS-a repressor of auxin regulated root promoting genes in plants-through IVLEM/KLHTLIPNK motif. Quantitative RT-PCR revealed abundance of DRO1-like RNA in root tips and spikelets while transcript signals were barely detectable in shoot and leaf tissues. Interestingly, wheat exhibited stronger expression of TaBDRO1-like than barley (HvDRO1-like), but TaBDRO1-like was the least expressing among three paralogs. The underlying cause of this expression divergence seems to be the presence of AuxRE motif TGTCTC and core TGTC with a coupling AuxRE-like motif ATTTTCTT proximal to the transcriptional start site in TaBDRO1-like and HvDRO1-like promoters. This is evident from binding of ARF1 to TGTCTC and TGTC motifs of TaBDRO1-like as revealed by yeast one-hybrid assay. Thus, evolution of DRO1-like wheat homoeologs might incorporate the C-terminus mutations as well as gain and loss of AuxREs and other cis-regulatory elements during expression divergence. Since root architecture is an important target trait for wheat crop improvement, therefore DRO1-like genes have potential applications in plant breeding for enhancement of plant productivity by the use of modern genome editing approaches.

Silicon carbide whisker-mediated embryogenic callus transformation of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants.

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (kmr) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T1, T2, and T3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T1 AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.

Geminivirus disease complexes: the threat is spreading.

Symptom-modulating DNA satellites associated with geminiviruses have come to our attention only recently but have proven to be widespread, associated with many diseases throughout the Old World, and economically significant, particularly in developing countries. Recent developments are elucidating the role played by these novel molecules in pathogenicity and in overcoming host plant defense. Further investigation into the promiscuous nature of these satellites and their ability to recruit further begomoviruses indicates that regions not yet affected by such begomovirus-satellite complexes are at great risk.

Engineering novel traits in plants through RNA interference.

RNA interference (RNAi) is a homology-dependent gene silencing technology that involves double-stranded RNA directed against a target gene or its promoter region. Using hairpin constructs, double-stranded RNA can be expressed in plants relatively easily, enabling this technology to be applied to a wide range of species to silence the expression of both specific endogenous genes and genes of invading pathogens. RNAi has also been used to engineer metabolic pathways to overproduce secondary products with health, yield or environmental benefits. The application of tissue-specific or inducible gene silencing, with the use of appropriate promoters, and the ability to silence several genes simultaneously should enhance our ability to create novel traits in plants.

Co-expression of YieF and PhoN in Deinococcus radiodurans R1 improves uranium bioprecipitation by reducing chromium interference.

Overexpression of the enzyme phosphatase (PhoN/PhoK) in the radiation-resistant bacterium Deinococcus radiodurans could be an efficient strategy for uranium remediation. However, the presence of other metals in nuclear wastes often interferes with uranium bioprecipitation. In our study, the uranium-precipitating ability of the PhoN-expressing D. radiodurans strain (Deino-phoN) significantly decreased by 45.4% in 13 h in the presence of chromium (VI); however, it was partially recovered after supplementation with chromium (III). Therefore, the reduction of chromium (VI) to chromium (III) was obtained by the co-expression of the YieF protein and PhoN in D. radiodurans (Deino-phoN-yieF). As a result, an increase in the chromium (VI) reduction (25.1%) rate was observed in 24 h. Furthermore, uranium precipitation also increased by 28.0%. For the decontamination of groundwater, we immobilized Deino-phoN-yieF cells using Polyvinyl alcohol (PVA)-sodium alginate (SA) beads, followed by incubation in a bioreactor. Approximately 99% of chromium (VI) and uranium (VI) was removed after 4 continuous cycles operated for a period of over 20 days at room temperature (25 °C). Therefore, Deino-phoN-yieF could be used as a potential biological agent for mixed radioactive nuclear waste remediation.

The hypersensitive response to tomato leaf curl New Delhi virus nuclear shuttle protein is inhibited by transcriptional activator protein.

The hypersensitive response (HR) is a common feature of plant disease resistance reactions and a type of programmed cell death (PCD). Many pathogens are able to modulate pathways involved in cell death. In contrast to animal viruses, inhibitors of PCD activity have not been identified for plant-infecting viruses. Previously, we have reported that the nuclear shuttle protein (NSP) of Tomato leaf curl New Delhi virus (ToLCNDV) induces an HR in Nicotiana tabacum and Lycopersicon esculentum plants when expressed under the control of the Cauliflower mosaic virus 35S promoter. However, HR is not evident in plants infected with ToLCNDV, suggesting that the virus encodes a factor (or factors) that counters this response. Analysis of all ToLCNDV-encoded genes pinpointed the transcriptional activator protein (TrAP) as the factor mediating the anti-HR effect. Deletion mutagenesis showed the central region of TrAP, containing a zinc finger domain and nuclear localization signal, to be important in inhibiting the HR. These results demonstrate that TrAP counters HR-induced cell death, the first such activity identified for a plant-infecting virus.

Silencing of the AV2 gene by antisense RNA protects transgenic plants against a bipartite begomovirus.

Whitefly-transmitted geminiviruses (genus Begomovirus) are phytopathogens that cause heavy losses to crops worldwide. Efforts to engineer resistance against these viruses are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a virion-sense gene (AV2) to develop resistance against Tomato leaf curl New Delhi virus, a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that tobacco plants transformed with an antisense construct targeting this gene are resistant to the virus. Following challenged with the virus, transgenic plants remained symptomless, although viral DNA could be detected in some plants by PCR. This is the first report of transgenic resistance against a bipartite begomovirus obtained by targeting a virion-sense gene. The relatively conserved nature of the gene suggests that the technology may be useful to develop broad-spectrum resistance which is required because of the fact that plants are often infected with multiple begomoviruses in the field.

RNAi-mediated male sterility of tobacco by silencing TA29.

The superior performance of F1 hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference (RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines for producing hybrid seed.

Development of agriculture biotechnology in Pakistan.

Agriculture plays an important role in the national economy of Pakistan, where most of the rapidly increasing population resides in rural areas and depends on agriculture for subsistence. Biotechnology has considerable potential for promoting the efficiency of crop improvement, food production, and poverty reduction. Use of modern biotechnology started in Pakistan since 1985. Currently, there are 29 biotech centers/institutes in the country. However, few centers have appropriate physical facilities and trained manpower to develop genetically modified (GM) crops. Most of the activities have been on rice and cotton, which are among the top 5 crops of Pakistan. Biotic (virus/bacterial/insect) and abiotic (salt) resistant and quality (male sterility) genes have already been incorporated in some crop plants. Despite acquiring capacity to produce transgenic plants, no GM crops, either produced locally or imported, have been released in the country. Pakistan is signatory to the World Trade Organization, Convention on Biological Diversity, and Cartagena protocols. Several legislations under the Agreement on Trade-Related Aspects of Intellectual Property Rights have been promulgated in the country. National Biosafety Guidelines have been promulgated in April 2005. The Plant Breeders Rights Act, Amendment in Seed Act-1976, and Geographical Indication for Goods are still passing through discussion, evaluation, and analysis phases. Meanwhile, an illegal GM crop (cotton) has already sneaked into farmer's field. Concerted and coordinated efforts are needed among various ministries for implementation of regulation and capacity building for import/export and local handling of GM crops. Pakistan could easily benefit from the experience of Asian countries, especially China and India, where conditions are similar and the agriculture sector is almost like that of Pakistan. Thus, the exchange of information and experiences is important among these nations.

Genetics and Genomics of Cotton Leaf Curl Disease, Its Viral Causal Agents and Whitefly Vector: A Way Forward to Sustain Cotton Fiber Security.

Cotton leaf curl disease (CLCuD) after its first epidemic in 1912 in Nigeria, has spread to different cotton growing countries including United States, Pakistan, India, and China. The disease is of viral origin-transmitted by the whitefly Bemisia tabaci, which is difficult to control because of the prevalence of multiple virulent viral strains or related species. The problem is further complicated as the CLCuD causing virus complex has a higher recombination rate. The availability of alternate host crops like tomato, okra, etc., and practicing mixed type farming system have further exaggerated the situation by adding synergy to the evolution of new viral strains and vectors. Efforts to control this disease using host plant resistance remained successful using two gene based-resistance that was broken by the evolution of new resistance breaking strain called Burewala virus. Development of transgenic cotton using both pathogen and non-pathogenic derived approaches are in progress. In future, screening for new forms of host resistance, use of DNA markers for the rapid incorporation of resistance into adapted cultivars overlaid with transgenics and using genome editing by CRISPR/Cas system would be instrumental in adding multiple layers of defense to control the disease-thus cotton fiber production will be sustained.

Geminivirus disease complexes: an emerging threat.

Small circular single-stranded DNA satellites have recently been isolated from plants infected with whitefly-transmitted monopartite begomoviruses. The satellites, named DNA beta, depend on the helper viruses for their proliferation and, in turn, are required for helper virus accumulation and symptom expression. They are highly diverse yet retain an overall conserved structure with respect to potential coding regions and regulatory elements. The begomovirus-satellite disease complexes are associated with economically important diseases, and have been isolated from vegetable and fibre crops, ornamental plants and weeds throughout Africa and Asia. Their widespread distribution and diversity, coupled to the global movement of plant material and the dissemination of the whitefly vector, suggests that these disease complexes pose a serious threat to tropical and sub-tropical agro-ecosystems worldwide.

A single complementary-sense transcript of a geminiviral DNA beta satellite is determinant of pathogenicity.

Small circular single-stranded DNA satellites, termed DNAbeta, have recently been found associated with some geminivirus infections. The DNA beta associated with Cotton leaf curl virus is responsible for symptom expression of a devastating disease in Pakistan. Mutagenesis of DNA beta revealed that the complementary-sense open reading frame (ORF) betaC1 is required for inducing disease symptoms in Nicotiana tabacum. An ORF present on the virion-sense strand betaV1 appeared to have no role in pathogenesis. Tobacco plants transformed with a betaC1 ORF under the control of the Cauliflower mosaic virus 35S promoter or with a dimeric DNA beta exhibited severe disease-like phenotypes, while plants transformed with a mutated version of betaC1 appeared normal. Northern blot analysis of RNA from the transgenic plants, using strand-specific probes, identified a single complementary-sense transcript. The transcript carries the full betaC1 ORF encoding a 118-amino acid product. It maps to the DNA beta at nucleotide position 186 to 563 and contains a polyadenylation signal 18 nt upstream of the stop codon. A TATA box is located 43 nt upstream of the start codon. Our results indicate that betaC1 protein is responsible for DNA beta-induced disease symptoms.

A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana.

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.

DNA barcoding of Bemisia tabaci complex (Hemiptera: Aleyrodidae) reveals southerly expansion of the dominant whitefly species on cotton in Pakistan.

BACKGROUND: Although whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan. METHODS/PRINCIPAL FINDINGS: Sequence diversity in the DNA barcode region (mtCOI-5') was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3' to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage "Pakistan". The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and "Pakistan" were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan. CONCLUSIONS: DNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region.