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Abstract To identify the genetic determinants of domestication and productivity of Asian water buffaloes (Bubalus bubalis), 470 genomes of domesticated river and swamp buffaloes along with their putative ancestors, the wild water buffaloes (Bubalus arnee) are sequenced and integrated. The swamp buffaloes inherit the morphology of the wild buffaloes. In contrast, most river buffaloes are unique in their morphology, but their genomes cluster with the wild buffaloes. The levels of genomic diversity in Italian river and Indonesian swamp buffaloes decrease at opposite extremes of their distribution range. Purifying selection prevented the accumulation of harmful loss‐of‐function variants in the Indonesian buffaloes. Genes that evolved rapidly (e.g., GKAP1) following differential selections in the river and swamp buffaloes are involved in their reproduction. Genes related to milk production (e.g., CSN2) and coat color (e.g., MC1R) underwent strong selections in the dairy river buffaloes via soft and hard selective sweeps, respectively. The selective sweeps and single‐cell RNA‐seq data revealed the luminal cells as the key cell type in response to artificial selection for milk production of the dairy buffaloes. These findings show how artificial selection has been driving the evolutionary divergence and genetic differentiation in morphology and productivity of Asian water buffaloes.

Recent advances allow the deployment of cluster regularly interspaced short palindromic repeats (CRISPR)-associated endonucleases (Cas) system for the targeted mutagenesis in the genome with accuracy and precision for trait improvement in crops. CRISPR-Cas systems have been extensively utilized to induce knockout or frameshift mutations in the targeted sequence of mostly negative regulating genes for wheat improvement. However, most of the reported work has been done in non-commercial varieties of wheat and introgression of edited alleles into breeding population comes with the penalty of unwanted linkage-drag. Wheat yield is controlled by various genes such as positive and negative regulators. The TaD27 gene is described as a negative regulator of shoot branching or tillering and involved in the biosynthesis of strigolactones. In this study, we developed Tad27 knockout mutant lines of an elite wheat cultivar that showed a twofold increase in the number of tillers and 1.8-fold increase in the number of grains per plant. Subsequently, enhancing the grain yield without any morphological penalty in the architecture of the plants. The co-transformation of regeneration enhancing growth regulator, Growth Regulating Factor 4 (GRF4) and its cofactor GRF-Interacting Factor 1 (GIF1), under single T-DNA cassette improved the regeneration efficiency up to 6% of transgenic events from mature embryos of wheat. Our results indicate that the CRISPR-mediated targeted mutagenesis confers the potential to knockout yield-related negative regulators in elite cultivars of wheat that can substantially enhance grain yield per plant and this strategy can be harnessed for the improvement of future wheat.

Abstract 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.

Abstract Nucleotide-binding site (NBS) domain genes are one of the superfamily of resistance genes involved in plant responses to pathogens. The current study identified 12,820 NBS-domain-containing genes across 34 species covering from mosses to monocots and dicots. These identified genes are classified into 168 classes with several novel domain architecture patterns encompassing significant diversity among plant species. Several classical (NBS, NBS-LRR, TIR-NBS, TIR-NBS-LRR, etc.) and species-specific structural patterns (TIR-NBS-TIR-Cupin_1-Cupin_1, TIR-NBS-Prenyltransf, Sugar_tr-NBS etc.) were discovered. We observed 603 orthogroups (OGs) with some core (most common orthogroups; OG0, OG1, OG2, etc.) and unique (highly specific to species; OG80, OG82, etc.) OGs with tandem duplications. The expression profiling presented the putative upregulation of OG2, OG6, and OG15 in different tissues under various biotic and abiotic stresses in susceptible and tolerant plants to cotton leaf curl disease (CLCuD). The genetic variation between susceptible (Coker 312) and tolerant (Mac7) Gossypium hirsutum accessions identified several unique variants in NBS genes of Mac7 (6583 variants) and Coker312 (5173 variants). The protein–ligand and proteins-protein interaction showed a strong interaction of some putative NBS proteins with ADP/ATP and different core proteins of the cotton leaf curl disease virus. The silencing of GaNBS (OG2) in resistant cotton through virus-induced gene silencing (VIGS) demonstrated its putative role in virus tittering. The presented study will be further helpful in understanding the plant adaptation mechanism.

Abstract Indicine cattle, also referred to as zebu (Bos taurus indicus), play a central role in pastoral communities across a wide range of agro-ecosystems, from extremely hot semiarid regions to hot humid tropical regions. However, their adaptive genetic changes following their dispersal into East Asia from the Indian subcontinent have remained poorly documented. Here, we characterize their global genetic diversity using high-quality whole-genome sequencing data from 354 indicine cattle of 57 breeds/populations, including major indicine phylogeographic groups worldwide. We reveal their probable migration into East Asia was along a coastal route rather than inland routes and we detected introgression from other bovine species. Genomic regions carrying morphology-, immune-, and heat-tolerance-related genes underwent divergent selection according to Asian agro-ecologies. We identify distinct sets of loci that contain promising candidate variants for adaptation to hot semi-arid and hot humid tropical ecosystems. Our results indicate that the rapid and successful adaptation of East Asian indicine cattle to hot humid environments was promoted by localized introgression from banteng and/or gaur. Our findings provide insights into the history and environmental adaptation of indicine cattle.

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.

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.

Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVYN, PVYO, and PVYNTN. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.

Abstract Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F1 generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.

Abstract Globally, Whitefly (Bemisia tabaci) is one of the most important insect pests of crops that causes huge economical losses. The current study was designed to exclusively screen the B. tabaci species in the cotton field of Pakistan during 2017–2020 and have to conduct comparative analysis of B. tabaci species in Asia where Asia II 1 has been reported. A total of 5142 B. tabaci sequences of mitochondrial cytochrome oxidase 1 (mtCO1) from Asian countries were analyzed to determine the species and their distribution in the region. Our analysis over time and space showed that Asia II 1 has gradually dominated over Asia 1 in Punjab Province and over both Asia 1 and MEAM1 in Sindh Province. Asia has been divided into three regions i.e., South Asia (2524 sequences), Southeast Asia (757 sequences) and East Asia (1569 sequences) and dominance of different species of B. tabaci has been determined by calculating the relative percentage of each species. Interestingly, Asia II 1 has been found dominant in the neighboring region (northern zone) of India and also being dominant in its central zone. The dominance of Asia II 1 in Pakistan and northern India explains whitefly epidemic being reported in recent years.

Background: Many studies have demonstrated the utility of machine learning (ML) methods for genomic prediction (GP) of various plant traits, but a clear rationale for choosing ML over conventionally used, often simpler parametric methods, is still lacking. Predictive performance of GP models might depend on a plethora of factors including sample size, number of markers, population structure and genetic architecture. Methods: Here, we investigate which problem and dataset characteristics are related to good performance of ML methods for genomic prediction. We compare the predictive performance of two frequently used ensemble ML methods (Random Forest and Extreme Gradient Boosting) with parametric methods including genomic best linear unbiased prediction (GBLUP), reproducing kernel Hilbert space regression (RKHS), BayesA and BayesB. To explore problem characteristics, we use simulated and real plant traits under different genetic complexity levels determined by the number of Quantitative Trait Loci (QTLs), heritability (h2 and h2e), population structure and linkage disequilibrium between causal nucleotides and other SNPs. Results: Decision tree based ensemble ML methods are a better choice for nonlinear phenotypes and are comparable to Bayesian methods for linear phenotypes in the case of large effect Quantitative Trait Nucleotides (QTNs). Furthermore, we find that ML methods are susceptible to confounding due to population structure but less sensitive to low linkage disequilibrium than linear parametric methods. Conclusions: Overall, this provides insights into the role of ML in GP as well as guidelines for practitioners.

Domestic buffaloes (Bubalus bubalis), known as water buffaloes, play a key role as versatile multipurpose agricultural animals in the Asiatic region. Pakistan, with the second-largest buffalo population in the world, holds a rich domestication history of buffaloes. The overall trends in buffalo production demand the genomic characterization of Pakistani buffalo breeds. To this end, the resequencing data of Pakistani breeds, along with buffalo breeds from 13 other countries, were retrieved from our previous study. This dataset, which contained 34,671,886 single-nucleotide polymorphisms (SNPs), was analyzed through a pipeline that was developed to compare possible allele differences among breeds at each SNP position. In contrast, other available tools only check for positional SNP differences for breed-specific markers. In total, 1918, 1549, 404, and 341 breed-specific markers were identified to characterize the Nili, Nili–Ravi, Azakheli, and Kundi breeds of Pakistani buffalo, respectively. Sufficient evidence in the form of phenotypic data, principal component analysis, admixture analysis, and linkage analysis showed that the Nili breed has maintained its distinct breed status despite sharing a close evolutionary relationship with the Nili–Ravi breed of buffalo. In this era of genome science, the conservation of these breeds and the further validation of the given selection markers in larger populations is a pressing need.

The development of portable and sensitive biosensors for the label-free detection of DNA has influenced fundamental biological research as well as advanced applications. Here, we report the novel microchip-based electronic devices for the efficient and sensitive detection of a crop virus i.e. Cotton leaf curl Khokran virus-Burewala strain (CLCuKoV-Bur), predominant cotton infecting virus worldwide. Three-dimensional copper nanostructures reinforced graphene nanohybrid (Cu Ns@GO) is developed via a biological synthesis approach. The hybrid consists of the typical graphene sheets, embellished with copper nanoparticles of 10–15 nm, to impart conducting and metallic character. This nanohybrid is applied as the active interface of microchips, and the surface charge of +38 mV enables successful anchoring of virus DNA as a probe to the interface. The hybridization events are manipulated as the change in the electron transport between sheets of graphene, leading to a corresponding decrease in conductance of the devices. Termed as Cu Ns@GO-microchip, the devices can detect the presence of viral DNA down to the detection limit of 200 pM. We further investigate specificity patterns and the non-complementary DNAs i.e. Cotton leaf curl Multan betasatellite, Cotton leaf curl Multan alphasatellite, and Maize insect resistance1-cystein protease genes show a negligible response generating only 10–20% of the signal. We apply this strategy to the virus-infected cotton field samples and using the devices, the level of virus infectivity can be discerned. This study suggests the potential of nanotechnology-based microchips for designing benchmark recognition interfaces, for the direct and facile monitoring of agricultural pathogens and other bio-threats.

Improving the crop traits is highly required for the development of superior crop varieties to deal with climate change and the associated abiotic and biotic stress challenges. Climate change-driven global warming can trigger higher insect pest pressures and plant diseases thus affecting crop production sternly. The traits controlling genes for stress or disease tolerance are economically imperative in crop plants. In this scenario, the extensive exploration of available wild, resistant or susceptible germplasms and unraveling the genetic diversity remains vital for breeding programs. The dawn of next-generation sequencing technologies and omics approaches has accelerated plant breeding by providing the genome sequences and transcriptomes of several plants. The availability of decoded plant genomes offers an opportunity at a glance to identify candidate genes, quantitative trait loci (QTLs), molecular markers, and genome-wide association studies that can potentially aid in high throughput marker-assisted breeding. In recent years genomics is coupled with marker-assisted breeding to unravel the mechanisms to harness better better crop yield and quality. In this review, we discuss the aspects of marker-assisted breeding and recent perspectives of breeding approaches in the era of genomics, bioinformatics, high-tech phonemics, genome editing, and new plant breeding technologies for crop improvement. In nutshell, the smart breeding toolkit in the post-genomics era can steadily help in developing climate-smart future food crops.

Coronavirus disease 2019 (COVID-19) has been a pandemic disease reported in almost every country and causes life-threatening, severe respiratory symptoms. Recent studies showed that various environmental selection pressures challenge the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectivity and, in response, the virus engenders new mutations, leading to the emergence of more virulent strains of WHO concern. Advance prediction of the forthcoming virulent SARS-CoV-2 strains in response to the principal environmental selection pressures like temperature and solar UV radiation is indispensable to overcome COVID-19. To discover the UV-solar radiation-driven genomic adaption of SARS-CoV-2, a curated dataset of 2,500 full-grade genomes from five different UVindex regions (25 countries) was subjected to in-depth downstream genome-wide analysis. The recurrent variants that best respond to UV-solar radiations were extracted and extensively annotated to determine their possible effects and impacts on gene functions. This study revealed 515 recurrent single nucleotide variants (rcntSNVs) as SARS-CoV-2 genomic responses to UV-solar radiation, of which 380 were found to be distinct. For all discovered rcntSNVs, 596 functional effects (rcntEffs) were detected, containing 290 missense, 194 synonymous, 81 regulatory, and 31 in the intergenic region. The highest counts of missense rcntSNVs in spike (27) and nucleocapsid (26) genes explain the SARS-CoV-2 genomic adjustment to escape immunity and prevent UV-induced DNA damage, respectively. Among all, the most commonly observed rcntEffs were four missenses (RdRp-Pro327Leu, N-Arg203Lys, N-Gly204Arg, and Spike-Asp614Gly) and one synonymous (ORF1ab-Phe924Phe) functional effects. The highest number of rcntSNVs found distinct and were uniquely attributed to the specific UVindex regions, proposing solar-UV radiation as one of the driving forces for SARS-CoV-2 differential genomic adaptation. The phylogenetic relationship indicated the high UVindex region populating SARS-CoV-2 as the recent progenitor of all included samples. Altogether, these results provide baseline genomic data that may need to be included for preparing UVindex region-specific future diagnostic and vaccine formulations.

Potato (Solanum tuberosum L.) is an important staple food around the world, and potato virus Y (PVY) is a major constraint for potato production. The VPg protein of PVY interacts with the translation initiation factor eIF4E of the host that works as a susceptibility factor during infection. The interaction between eIF4E and VPg was disrupted by CRISPR/Cas9. The homozygous conserved region of eIF4E of the potato variety “Kruda” was mutated by CRISPR/Cas9. Tracking of insertion, deletion, and conversion events was performed by Sanger sequencing with ∼15% editing efficiency. Truncated and mutated eIF4E proteins were unable to interact with VPg, and the virus was not able to exploit the host machinery for replication and systemic spreading. Mutated eIF4E lines showed enhanced resistance to PVYO strain. DAS-ELISA and RT-PCR were used for validation of the observed resistance. PVY resistance in tetraploid lines via CRISPR/Cas9 provides a route to develop novel resistant potato cultivars.

Abstract Bt cotton expressing Cry1Ac is being cultivated in Pakistan. It has been observed that pink bollworm may have developed resistance against single Bt gene (Cry1Ac). For durable resistance, insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct was developed. There was a need to characterize NIBGE-1601 event for intellectual property rights protection. The Presence of NIBGE Cry1Ac and NIBGE Cry2Ab genes was checked in NIBGE-1601 cotton plants through PCR, while there was no amplification using primers specific for Monsanto events (MON531, MON15985, MON1445). Using genome walking technology, NIBGE-601 event has been characterized. Event-specific primers of NIBGE-1601 were designed and evaluated to differentiate it from other cotton events mentioned above. NIBGE-1601 event detection primers are highly specific, therefore, can detect NIBGE 1601 event at different conditions using single or multiplex PCR. In the qualitative PCR, using NIBGE-1601 event specific primers, 0.05 ng was the limit of detection for NIBGE-1601double gene cotton genomic DNA. Thus event characterization and development of event-specific diagnostics will help in breeding new cotton varieties resistant to cotton bollworms.

The ensuing heat stress drastically affects wheat plant growth and development, consequently compromising its grain yield. There are many thermoregulatory processes/mechanisms mediated by ion channels, lipids, and lipid-modifying enzymes that occur in the plasma membrane and the chloroplast. With the onset of abiotic or biotic stresses, phosphoinositide-specific phospholipase C (PI-PLC), as a signaling enzyme, hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which is further phosphorylated into phosphatidic acid (PA) as a secondary messenger and is involved in multiple processes. In the current study, a phospholipase C (PLC) signaling pathway was investigated in spring wheat (Triticum aestivum L.) and evaluated its four AtPLC5 overexpressed (OE)/transgenic lines under heat and osmotic stresses through 32Pi radioactive labeling. Naturally, the wheat harbors only a small amount of PIP2. However, with the sudden increase in temperature (40°C), PIP2 levels start to rise within 7.5 min in a time-dependent manner in wild-type (Wt) wheat. While the Phosphatidic acid (PA) level also elevated up to 1.6-fold upon exposing wild-type wheat to heat stress (40°C). However, at the anthesis stage, a significant increase of ∼4.5-folds in PIP2 level was observed within 30 min at 40°C in AtPLC5 over-expressed wheat lines. Significant differences in PIP2 level were observed in Wt and AtPLC5-OE lines when treated with 1200 mM sorbitol solution. It is assumed that the phenomenon might be a result of the activation of PLC/DGK pathways. Together, these results indicate that heat stress and osmotic stress activate several lipid responses in wild-type and transgenic wheat and can explain heat and osmotic stress tolerance in the wheat plant.

Although Pakistan has rich biodiversity, many groups are poorly known, particularly insects. To address this gap, we employed DNA barcoding to survey its insect diversity. Specimens obtained through diverse collecting methods at 1,858 sites across Pakistan from 2010–2019 were examined for sequence variation in the 658 bp barcode region of the cytochrome c oxidase 1 (COI) gene. Sequences from nearly 49,000 specimens were assigned to 6,590 Barcode Index Numbers (BINs), a proxy for species, and most (88%) also possessed a representative image on the Barcode of Life Data System (BOLD). By coupling morphological inspections with barcode matches on BOLD, every BIN was assigned to an order (19) and most (99.8%) were placed to a family (362). However, just 40% of the BINs were assigned to a genus (1,375) and 21% to a species (1,364). Five orders (Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera) accounted for 92% of the specimens and BINs. More than half of the BINs (59%) are so far only known from Pakistan, but others have also been reported from Bangladesh (13%), India (12%), and China (8%). Representing the first DNA barcode survey of the insect fauna in any South Asian country, this study provides the foundation for a complete inventory of the insect fauna in Pakistan while also contributing to the global DNA barcode reference library.

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.

Cotton (Gossypium hirsutum L.) is the most essential fiber crop and is widely cultivated within the tropical regions of the world. It has a major impact on socio-economical get-together in Pakistan. Unfortunately, it has been tormented by cotton leaf curl disease (CLCuD) that is produced by distinct species of begomoviruses (Family; Geminiviridae) like Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bu) associated with specific betasatellite—Cotton leaf curl Multan betasatellite (CLCuMuB). Host resistance against CLCuD has not been achieved yet. The present study employed the use of synthetic genes considering the phenomenon of pathogen derived resistance (PDR). The coat protein (CP) and replication associated protein (Rep) genes sequence from CLCuKoV-Bu were codon optimized, synthesized and used to control CLCuKoV-Bu and associated betasatellite related to CLCuD. The Nicotiana benthamiana plants were Agrobacterium mediated transformed with synthetic CP (CPsyn) and synthetic Rep (Repsyn) under the control of Cauliflower mosaic virus 35S promoter. The transgenic plants harboring CPsyn and Repsyn genes were infiltrated with CLCuKoV-Bu alone and CLCuKoV-Bu- CLCuMuB inoculums showed relatively milder symptoms as compared with wild sorts and low virus concentration as checked by southern hybridization. The results confirmed that CPsyn, Repsyn genes may be utilized for the resistance of CLCuKoV-Bu and associated betasatellites related to CLCuD and PDR mechanism may also be developed for the control of CLCuD.

Abstract Background Whiteflies (Bemisia tabaci) are phloem sap-sucking pests that because of their broad host range and ability to transmit viruses damage crop plants worldwide. B. tabaci are now known to be a complex of cryptic species that differ from each other in many characteristics such as mode of interaction with viruses, invasiveness, and resistance to insecticides. Asia II 1 is an indigenous species found on the Indian sub-continent and south-east Asia while the species named as Middle East Asia Minor 1 (MEAM1), likely originated from the Middle-East and has spread worldwide in recent decades. The purpose of this study is to find genomic differences between these two species. Results Sequencing of the nuclear genome of Asia II 1 with Illumina HiSeq and MiSeq generated 198.90 million reads that covers 88% of the reference genome. The sequence comparison with MEAM1 identified 2,327,972 SNPs and 202,479 INDELs. In Total, 1294 genes were detected with high impact variants. The functional analysis revealed that some of the genes are involved in virus transmission including 4 genes in Tomato yellow leaf curl virus (TYLCV) transmission, 96 in Tomato crinivirus (ToCV) transmission, and 14 genes in insecticide resistance. Conclusions These genetic differences between Asia II 1 and MEAM1 may underlie the major biological differences between the two species such as virus transmission, insecticide resistance, and range of host plants. The present study provides new genomic data and information resources for Asia II 1 that will not only contribute to the species delimitation of whitefly, but also help in conceiving future research studies to develop more targeted management strategies against whitefly.

Genome- or gene-editing (abbreviated here as ‘GEd’) presents great opportunities for crop improvement. This is especially so for the countries in the Asia-Pacific region, which is home to more than half of the world’s growing population. A brief description of the science of gene-editing is provided with examples of GEd products. For the benefits of GEd technologies to be realized, international policy and regulatory environments must be clarified, otherwise non-tariff trade barriers will result. The status of regulations that relate to GEd crop products in Asian countries and Australasia are described, together with relevant definitions and responsible regulatory bodies. The regulatory landscape is changing rapidly: in some countries, the regulations are clear, in others they are developing, and some countries have yet to develop appropriate policies. There is clearly a need for the harmonization or alignment of GEd regulations in the region: this will promote the path-to-market and enable the benefits of GEd technologies to reach the end-users.

Bovine and buffalo are important livestock species that have contributed to human lives for more than 1000 years. Improving fertility is very important to reduce the cost of production. In the current review, we classified reproductive traits into three categories: ovulation, breeding, and calving related traits. We systematically summarized the heritability estimates, molecular markers, and genomic selection (GS) for reproductive traits of bovine and buffalo. This review aimed to compile the heritability and genome-wide association studies (GWASs) related to reproductive traits in both bovine and buffalos and tried to highlight the possible disciplines which should benefit buffalo breeding. The estimates of heritability of reproductive traits ranged were from 0 to 0.57 and there were wide differences between the populations. For some specific traits, such as age of puberty (AOP) and calving difficulty (CD), the majority beef population presents relatively higher heritability than dairy cattle. Compared to bovine, genetic studies for buffalo reproductive traits are limited for age at first calving and calving interval traits. Several quantitative trait loci (QTLs), candidate genes, and SNPs associated with bovine reproductive traits were screened and identified by candidate gene methods and/or GWASs. The IGF1 and LEP pathways in addition to non-coding RNAs are highlighted due to their crucial relevance with reproductive traits. The distribution of QTLs related to various traits showed a great differences. Few GWAS have been performed so far on buffalo age at first calving, calving interval, and days open traits. In addition, we summarized the GS studies on bovine and buffalo reproductive traits and compared the accuracy between different reports. Taken together, GWAS and candidate gene approaches can help to understand the molecular genetic mechanisms of complex traits. Recently, GS has been used extensively and can be performed on multiple traits to improve the accuracy of prediction even for traits with low heritability, and can be combined with multi-omics for further analysis.

Prediction of growth-related complex traits is highly important for crop breeding. Photosynthesis efficiency and biomass are direct indicators of overall plant performance and therefore even minor improvements in these traits can result in significant breeding gains. Crop breeding for complex traits has been revolutionized by technological developments in genomics and phenomics. Capitalizing on the growing availability of genomics data, genome-wide marker-based prediction models allow for efficient selection of the best parents for the next generation without the need for phenotypic information. Until now such models mostly predict the phenotype directly from the genotype and fail to make use of relevant biological knowledge. It is an open question to what extent the use of such biological knowledge is beneficial for improving genomic prediction accuracy and reliability. In this study, we explored the use of publicly available biological information for genomic prediction of photosynthetic light use efficiency (ΦPSII) and projected leaf area (PLA) in Arabidopsis thaliana. To explore the use of various types of knowledge, we mapped genomic polymorphisms to Gene Ontology (GO) terms and transcriptomics-based gene clusters, and applied these in a Genomic Feature Best Linear Unbiased Predictor (GFBLUP) model, which is an extension to the traditional Genomic BLUP (GBLUP) benchmark. Our results suggest that incorporation of prior biological knowledge can improve genomic prediction accuracy for both ΦPSII and PLA. The improvement achieved depends on the trait, type of knowledge and trait heritability. Moreover, transcriptomics offers complementary evidence to the Gene Ontology for improvement when used to define functional groups of genes. In conclusion, prior knowledge about trait-specific groups of genes can be directly translated into improved genomic prediction.

Basmati rice is famous around the world for its flavor, aroma, and long grain. Its demand is increasing worldwide, especially in Asia. However, its production is threatened by various problems faced in the fields, resulting in major crop losses. One of the major problems is bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xoo hijacks the host machinery by activating the susceptibility genes (OsSWEET family genes), using its endogenous transcription activator like effectors (TALEs). TALEs have effector binding elements (EBEs) in the promoter region of the OsSWEET genes. Out of six well-known TALEs found to have EBEs in Clade III SWEET genes, four are present in OsSWEET14 gene’s promoter region. Thus, targeting the promoter of OsSWEET14 is very important for creating broad-spectrum resistance. To engineer resistance against bacterial blight, we established CRISPR-Cas9 mediated genome editing in Super Basmati rice by targeting 4 EBEs present in the promoter of OsSWEET14. We were able to obtain four different Super Basmati lines (SB-E1, SB-E2, SB-E3, and SB-E4) having edited EBEs of three TALEs (AvrXa7, PthXo3, and TalF). The edited lines were then evaluated in triplicate for resistance against bacterial blight by choosing one of the locally isolated virulent Xoo strains with AvrXa7 and infecting Super Basmati. The lines with deletions in EBE of AvrXa7 showed resistance against the Xoo strain. Thus, it was confirmed that edited EBEs provide resistance against their respective TALEs present in Xoo strains. In this study up to 9% editing efficiency was obtained. Our findings showed that CRISPR-Cas9 can be harnessed to generate resistance against bacterial blight in indigenous varieties, against locally prevalent Xoo strains.

This study explores the effects of monetary policy on firms’ business fixed investment spending through the interest rate and broad credit channels of monetary policy transmission mechanism in Pakistan. Due to the problem of endogeneity, Generalized Method of Moments (GMM) two step estimation technique is applied on neo-classical investment model by using disaggregated firm level data of manufacturing sector of Pakistan over the period 1974-2010. The results suggest the relevance of both the interest rate and broad credit channels in Pakistan. Firms’ investment found to be negatively affected by the monetary contraction while positively influenced by cash flow and the sales. Small firms explored to be more sensitive to the monetary tightening as compared to large firms indicating that monetary policy exerts heterogeneous effects. Results highlight the importance of considering the financial conditions of the firms in formulation of monetary policy.

Spinach is a vegetable crop which is widely grown over a large area especially in Punjab province of Pakistan. Leaf curling and enations on spinach plant collected shown to be associated with the begomovirus Pedilanthus leaf curl virus (PeLCV) and Shahdadpur strain of Cotton leaf curl Multan betasatellite (CLCuMBSha). Defective molecules of half and quarter size derived from monopartite begomoviruses are usually generated by the deletion of virion-sense sequences. Characterization of defective molecules of PeLCV from spinach revealed that the molecules of half the size are derived from the deletion of complementary-sense genes while quarter size molecule appears to have evolved by further deletion. This is the first report of the begomovirus-betasatellite complex on spinach and unusual defective molecules derived from deletion of complementary-sense genes in Pakistan.

This study investigates that either the firms in manufacturing sector of Pakistan are facing external financial constraints or not along with that this study also explores investment cash flow sensitivity in different regimes. We used a panel data of 500 manufacturing sector firms over the period of 1974 to 2010 and the sample is divided into different time periods on the basis of different political regimes, pre and post financial sector reform era in the history of Pakistan. Generalized Method of Moments, one step and two step estimation techniques have been applied for analysis. Results obtained for the full sample indicates that overall firms are not facing external financial constraint. The analysis of financial constrains in different political regimes reveals that merely from 1978 to 1988 firms were facing external financial constraints whereas empirical results for pre and post financial sector reform periods reveals that firms were facing tight external financial constraints in pre financial sector reform era as compared to post financial sector reform era.

Abstract The first epidemic of cotton leaf curl disease (CLCuD) in early 1990’s in the Indian subcontinent was associated with several distinct begomoviruses along with a disease-specific betasatellite. Resistant cotton varieties were introduced in late 1990’s but soon resistance was broken and was associated with a single recombinant begomovirus named Burewala strain of Cotton leaf curl Kokhran virus that lacks a full complement of a gene encoding a transcription activator protein (TrAP). In order to understand the ongoing changes in CLCuD complex in Pakistan, CLCuD affected plants from cotton fields at Vehari were collected. Illumina sequencing was used to assess the diversity of CLCuD complex. At least three distinct begomoviruses characterized from the first epidemic; Cotton leaf curl Multan virus, Cotton leaf curl Kokhran virus and Cotton leaf curl Alabad virus, several distinct species of alphasatellites and cotton leaf curl Multan betasatellite were found associated with CLCuD. These viruses were also cloned and sequenced through Sanger sequencing to confirm the identity of the begomoviruses and that all clones possessed a full complement of the TrAP gene. A new strain of betasatellite was identified here and named CLCuMuBVeh. The implications of these findings in efforts to control CLCuD are discussed.

Cotton leaf curl disease (CLCuD) caused by viruses of genus Begomovirus is a major constraint to cotton (Gossypium hirsutum) production in many cotton-growing regions of the world. Symptoms of the disease are caused by Cotton leaf curl Multan betasatellite (CLCuMB) that encodes a pathogenicity determinant protein, βC1. Here, we report the identification of interacting regions in βC1 protein by using computational approaches including sequence recognition, and binding site and interface prediction methods. We show the domain-level interactions based on the structural analysis of G. hirsutum SnRK1 protein and its domains with CLCuMB-βC1. To verify and validate the in silico predictions, three different experimental approaches, yeast two hybrid, bimolecular fluorescence complementation and pull down assay were used. Our results showed that ubiquitin-associated domain (UBA) and autoinhibitory sequence (AIS) domains of G. hirsutum-encoded SnRK1 are involved in CLCuMB-βC1 interaction. This is the first comprehensive investigation that combined in silico interaction prediction followed by experimental validation of interaction between CLCuMB-βC1 and a host protein. We demonstrated that data from computational biology could provide binding site information between CLCuD-associated viruses/satellites and new hosts that lack known binding site information for protein–protein interaction studies. Implications of these findings are discussed.

This paper basically examines implementation of Basel Accords in Pakistan and investigates to what extent; these accords have positive or negative impact on economy. Two types of capital, Tier 1 and Tier 2 are examined under Basel II for the first time for a developing country. Both have impact on capital management but their mechanisms are different. For this purpose, two models, capital buffer model and provisioning models are used for the estimation through GMM one step and two steps for the period of 2001-2012 for 47 Pakistani commercial banks. Results show that Basel II Capital accord is pro-cyclical. Sample is divided into two regimes. Tier 1 capital is positively related and Tier 2 is negatively related to loan loss provisions in the pre Basel regime while the results are reversed in the post Basel regime. Business cycle fluctuations are positively related to loan loss provisions in the pre Basel regime and negatively related in the post Basel regime providing the evidence that new capital regulation is pro-cyclical.

New risk based capital requirement have pro-cyclical effect and causes negative externalities in the economy. During recession, on one side, quality of loan portfolio deteriorates and probability of default increases resulting into increased level of provisions and write off’s and reduced capital level. This causes an increase in capital requirements which becomes more expensive. Weaker banks fail to access new capital and ultimately reduce the credit supply. On the other side, banks are required to maintain the minimum capital which results into credit supply contraction and hits the bank’s profitability leading to a situation called Credit Crunch. This situation may prolong recession. During the crisis, developing countries are more affected than developed countries and this debate is entirely new in Pakistan. This research empirically investigates the pro-cyclical effect of new capital regulation under Basel II using panel data of 47 Pakistani Banks from 2001-2012. Particularly this paper examines the capital management mechanisms using capital buffers, using Generalized Method of Moments (GMM) one step and two step estimation techniques on dynamic panel data model. The results gives evidence that capital buffer are counter-cyclical except in case of specialized banks because of difference in operations. The findings also suggest that adjustment costs, cost of raising capital and bankruptcy costs are major determines of holding capital buffer. Analysis confirms too big to fail hypothesis. Form the results, it is concluded that capital buffer are counter-cyclical, consistent with the hypothesis. The findings suggest the banks to adopt Basel III Accord.

Cotton leaf curl Multan virus (CLCuMuV) is one of the major casual agents of cotton leaf curl disease. Previous studies show that two indigenous whitefly species of the Bemisia tabaci complex, Asia II 1 and Asia II 7, are able to transmit CLCuMuV, but the molecular mechanisms underlying the transmission are poorly known. In this study, we attempted to identify the whitefly proteins involved in CLCuMuV transmission. First, using a yeast two-hybrid system, we identified 54 candidate proteins of Asia II 1 that putatively can interact with the coat protein of CLCuMuV. Second, we examined interactions between the CLCuMuV coat protein and several whitefly proteins, including vacuolar protein sorting-associated protein (Vps) twenty associated 1 (Vta1). Third, using RNA interference, we found that Vta1 positively regulated CLCuMuV acquisition and transmission by the Asia II 1 whitefly. In addition, we showed that the interaction between the CLCuMuV coat protein and Vta1 from the whitefly Middle East-Asia Minor (MEAM1), a poor vector of CLCuMuV, was much weaker than that between Asia II 1 Vta1 and the CLCuMuV coat protein. Silencing of Vta1 in MEAM1 did not affect the quantity of CLCuMuV acquired by the whitefly. Taken together, our results suggest that Vta1 may play an important role in the transmission of CLCuMuV by the whitefly.

Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting with plant proteins. In the old world, this group of viruses are usually associated with betasatellite that induces severe disease symptoms by encoding a protein, βC1, which is a pathogenicity determinant. Here, we show that βC1 encoded by Cotton leaf curl Multan betasatellite (CLCuMB) requires Gossypium hirsutum calmodulin-like protein 11 (Gh-CML11) to infect cotton. First, we used the in silico approach to predict the interaction of CLCuMB-βC1 with Gh-CML11. A number of sequence- and structure-based in-silico interaction prediction techniques suggested a strong putative binding of CLCuMB-βC1 with Gh-CML11 in a Ca+2-dependent manner. In-silico interaction prediction was then confirmed by three different experimental approaches: The Gh-CML11 interaction was confirmed using CLCuMB-βC1 in a yeast two hybrid system and pull down assay. These results were further validated using bimolecular fluorescence complementation system showing the interaction in cytoplasmic veins of Nicotiana benthamiana. Bioinformatics and molecular studies suggested that CLCuMB-βC1 induces the overexpression of Gh-CML11 protein and ultimately provides calcium as a nutrient source for virus movement and transmission. This is the first comprehensive study on the interaction between CLCuMB-βC1 and Gh-CML11 proteins which provided insights into our understating of the role of βC1 in cotton leaf curl disease.

Cotton is a commercial and economically important crop that generates billions of dollars in annual revenue worldwide. However, cotton yield is affected by a sap-sucking insect Bemisia tabaci (whitefly), and whitefly-borne cotton leaf curl disease (CLCuD). The causative agent of devastating CLCuD is led by the viruses belonging to the genus Begomovirus (family Geminiviridae), collectively called cotton leaf curl viruses. Unfortunately, the extensively cultivated cotton (Gossypium hirsutum) species are highly susceptible and vulnerable to CLCuD. Yet, the concomitant influence of whitefly and CLCuD on the susceptible G. hirsutum transcriptome has not been interpreted. In the present study we have employed an RNA Sequencing (RNA-Seq) transcriptomics approach to explore the differential gene expression in susceptible G. hirsutum variety upon infection with viruliferous whiteflies. Comparative RNA-Seq of control and CLCuD infected plants was done using Illumina HiSeq 2500. This study yielded 468 differentially expressed genes (DEGs). Among them, we identified 220 up and 248 downregulated DEGs involved in disease responses and pathogen defense. We selected ten genes for downstream RT-qPCR analyses on two cultivars, Karishma and MNH 786 that are susceptible to CLCuD. We observed a similar expression pattern of these genes in both susceptible cultivars that was also consistent with our transcriptome data further implying a wider application of our global transcription study on host susceptibility to CLCuD. We next performed weighted gene co-expression network analysis that revealed six modules. This analysis also identified highly co-expressed genes as well as 55 hub genes that co-express with ≥ 50 genes. Intriguingly, most of these hub genes are shown to be downregulated and enriched in cellular processes. Under-expression of such highly co-expressed genes suggests their roles in favoring the virus and enhancing plant susceptibility to CLCuD. We also discuss the potential mechanisms governing the establishment of disease susceptibility. Overall, our study provides a comprehensive differential gene expression analysis of G. hirsutum under whitefly-mediated CLCuD infection. This vital study will advance the understanding of simultaneous effect of whitefly and virus on their host and aid in identifying important G. hirsutum genes which intricate in its susceptibility to CLCuD.

The primary goal of cattle genomics is the identification of genome-wide polymorphism associated with economically important traits. The bovine genome sequencing project was completed in 2009. Since then, using massively parallel sequencing technologies, a large number of Bos taurus cattle breeds have been resequenced and scanned for genome-wide polymorphisms. As a result, a substantial number of single nucleotide polymorphisms (SNPs) have been discovered across European Bos taurus genomes, whereas extremely less number of SNPs are cataloged for Bos indicus breeds. In this study, we performed whole-genome resequencing, reference-based mapping, functional annotation and gene enrichment analysis of 20 sires representing eleven important Bos indicus (indicine) breeds of Pakistan. The breeds sequenced here include: Sahiwal, Red Sindhi, Tharparkar and Cholistani (tropically adapted dairy and dual purpose breeds), Achai, Bhagnari, Dajal and Lohani (high altitude adapted dual and drought purpose breeds); Dhanni, Hisar Haryana and Gabrali (dairy and light drought purpose breeds). A total of 17.4 billion QC passed reads were produced using BGISEQ-500 next generation sequencing platform to generate 9 to 27-fold genome coverage (average ~16×) for each of the 20 sequenced sires. A total of 67,303,469 SNPs were identified, of which 3,850,365 were found novel and 1,083,842 insertions-deletions (InDels) were detected across the whole sequenced genomes (491,247 novel). Comparative analysis using coding region SNPs revealed a close relationship between the best milking indicine breeds; Red Sindhi and Sahiwal. On the other hand, Bhagnari and Tharparkar being popular for their adaptation to dry and extremely hot climates were found to share the highest number of SNPs. Functional annotation identified a total of 3,194 high-impact (disruptive) SNPs and 745 disruptive InDels (in 275 genes) that may possibly affect economically important dairy and beef traits. Functional enrichment analysis was performed and revealed that high or moderate impact variants in wingless-related integration site (Wnt) and vascular smooth muscle contraction (VSMC) signaling pathways were significantly over-represented in tropically adapted heat tolerant Pakistani-indicine breeds. On the other hand, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) signaling pathways were found over-represented in highland adapted Pakistani-indicine breeds. Similarly, the ECM-receptor interaction and Jak-STAT signaling pathway were significantly enriched in dairy and beef purpose Pakistani-indicine cattle breeds. The Toll-like receptor signaling pathway was significantly enriched in most of the Pakistani-indicine cattle. Therefore, this study provides baseline data for further research to investigate the molecular mechanisms of major traits and to develop potential genomic markers associated with economically important breeding traits, particularly in indicine cattle.

Morphological study of 1,795 spiders from sites across Pakistan placed these specimens in 27 families and 202 putative species. COI sequences >400 bp recovered from 1,782 specimens were analyzed using neighbor-joining trees, Bayesian inference, barcode gap, and Barcode Index Numbers (BINs). Specimens of 109 morphological species were assigned to 123 BINs with ten species showing BIN splits, while 93 interim species included representatives of 98 BINs. Maximum conspecific divergences ranged from 0-5.3% while congeneric distances varied from 2.8-23.2%. Excepting one species pair (Oxyopes azhari-Oxyopes oryzae), the maximum intraspecific distance was always less than the nearest-neighbor (NN) distance. Intraspecific divergence values were not significantly correlated with geographic distance. Most (75%) BINs detected in this study were new to science, while those shared with other nations mainly derived from India. The discovery of many new, potentially endemic species and the low level of BIN overlap with other nations highlight the importance of constructing regional DNA barcode reference libraries.

The first decade of genetic engineering primarily focused on quantitative crop improvement. With the advances in technology, the focus of agricultural biotechnology has shifted toward both quantitative and qualitative crop improvement, to deal with the challenges of food security and nutrition. Potato (Solanum tuberosum L.) is a solanaceous food crop having potential to feed the populating world. It can provide more carbohydrates, proteins, minerals, and vitamins per unit area of land as compared to other potential food crops, and is the major staple food in many developing countries. These aspects have driven the scientific attention to engineer potato for nutrition improvement, keeping the yield unaffected. Several studies have shown the improved nutritional value of potato tubers, for example by enhancing Amaranth Albumin-1 seed protein content, vitamin C content, β-carotene level, triacylglycerol, tuber methionine content, and amylose content, etc. Removal of anti-nutritional compounds like steroidal glycoalkaloids, acrylamide and food toxins is another research priority for scientists and breeders to improve potato tuber quality. Trait improvement using genetic engineering mostly involved the generation of transgenic products. The commercialization of these engineered products has been a challenge due to consumer preference and regulatory/ethical restrictions. In this context, new breeding technolgies like TALEN (transcription activator-like effector nucleases) and CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated 9) have been employed to generate transgene-free products in a more precise, prompt and effective way. Moreover, the availability of potato genome sequence and efficient potato transformation systems have remarkably facilitated potato genetic engineering. Here we summarize the potato trait improvement and potential application of new breeding technologies (NBTs) to genetically improve the overall agronomic profile of potato.

Cotton leaf curl is devastating disease of cotton characterized by leaf curling, vein darkening and enations. The disease symptoms are induced by DNA satellite known as Cotton leaf curl Multan betasatellite (CLCuMuB), dominant betasatellite in cotton but another betasatellite known as Chili leaf curl betasatellite (ChLCB) is also found associated with the disease. Grafting experiment was performed to determine if host plant resistance is determinant of dominant population of betasatellite in cotton (several distinct strains of CLCuMuB are associated with the disease). Infected scion of Gossypium hirsutum collected from field (the source) was grafted on G. arboreum, a diploid cotton species, resistant to the disease. A healthy scion of G. hirsutum (sink) was grafted at the top of G. arboreum to determine the movement of virus/betasatellite to upper susceptible scion of G. hirsutum. Symptoms of disease appeared in the upper scion and presence of virus/betasatellite in the upper scion was confirmed via molecular techniques, showing that virus/betasatellite was able to move to upper scion through resistant G. arboreum. However, no symptoms appeared on G. arboreum. Betasatelites were cloned and sequenced from lower scion, upper scion and G. arboreum which show that the lower scion contained both CLCuMuB and ChLCB, however only ChLCB was found in G. arboreum. The upper scion contained CLCuMuB with a deletion of 78 nucleotides (nt) in the non-coding region between A-rich sequence and βC1 gene and insertion of 27 nt in the middle of βC1 ORF. This study may help in investigating molecular basis of resistance in G. arboreum.

The modern cattle was domesticated from aurochs, sharing its physiological traits into two subspecies Bos taurus and Bos indicus. MicroRNAs (miRNAs) are a class of non-coding short RNAs of ~22nt which have a key role in the regulation of many cellular and physiological processes in the animal. The current study was aimed to predict and annotate the potential mutations in indicine miRNAs throughout the genome using de novo and homology-based in silico approaches. Genome-wide mapping was performed in available indicine assembly by the homology-based approach and 768 miRNAs were recovered out of 808 reported taurine miRNAs belonging to 521 unique mature miRNA families. While 42 precursors were dropped due to lack of secondary miRNA structure, increasing stringency or decreasing similarity between the two genomes' miRNA. Increasing tendency of miRNAs incidence was observed on chr5, chr7, chr8, chr12 and chr21 with 19 polycistronic miRNA within 1-kilobase distance throughout the indicine genome. Notably, 12 miRNAs showed copy number variation. Eighteen miRNAs showed a mutation in their mature sequences in which eight were found in their seed region. Whilst in de novo based approach, 12 novel potential miRNAs on Y chromosome in indicine cattle along with a new miRNA (bind-miR-1264) on chrX were found. The final data set is annotated and explains the impending target genes that are responsible for enhanced immunity, heat tolerance and disease tolerance regulation in indicine. The study conforms to better understanding and perceptive approach towards indicine genome.

Samples of two Ageratum conyzoides, one Sonchus oleraceus and one turnip (Brassica rapa var. rapa) exhibiting virus-like symptoms were collected from Pakistan and Nepal. Full-length begomovirus clones were obtained from the four plant samples and betasatellite clones from three of these. The begomovirus sequences were shown to be isolates of Ageratum enation virus (AEV) with greater than 89.1% nucleotide sequence identity to the 26 AEV sequences available in the databases. The three betasatellite sequences were shown to be isolates of Ageratum yellow leaf curl betasatellite (AYLCB) with greater than 90% identity to the 18 AYLCB sequences available in the databases. The AEV sequences were shown to fall into two distinct strains, for which the names Nepal (consisting of isolates from Nepal, India, and Pakistan—including the isolates identified here) and India (isolates occurring only in India) strains are proposed. For the clones obtained from two AEV isolates, with their AYLCB, infectivity was shown by Agrobacterium-mediated inoculation to Nicotiana benthamiana, N. tabacum, Solanum lycopersicon and A. conyzoides. N. benthamiana plants infected with AEV alone or betasatellite alone showed no symptoms. N. benthamiana plants infected with AEV with its associated betasatellite showed leaf curl symptoms. The findings show that AEV is predominantly a virus of weeds that has the capacity to infect crops. AYLCB appears to be the common partner betasatellite of AEV and is associated with diseases with a range of very different symptoms in the same plant species. The inability to satisfy Koch’s postulates with the cloned components of isolate SOL in A. conyzoides suggests that the etiology may be more complex than a single virus with a single betasatellite.

Viruses of the genus Begomovirus (family Geminiviridae) are economically important phytopathogens that are transmitted plant-to-plant by the whitefly Bemisia tabaci. Most Old World (OW) begomoviruses are monopartite and many of these interact with symptoms and host range determining betasatellites. Tomato leaf curl New Delhi virus (ToLCNDV) is one of only a few OW begomoviruses with a bipartite genome (components known as DNA A and DNA B). Four genes [AV2, coat protein (CP), transcriptional-activator protein (TrAP), and AC4] of ToLCNDV were mutated and the effects of the mutations on infectivity, symptoms and the ability to maintain Cotton leaf curl Multan betasatellite (CLCuMuB) were investigated. Infectivity and virus/betasatellite DNA titer were assessed by Southern blot hybridization, PCR, and quantitative PCR. The results showed TrAP of ToLCNDV to be essential for maintenance of CLCuMuB and AV2 to be important only in the presence of the DNA B. AC4 was found to be important for the maintenance of CLCuMuB in the presence of, but indispensable in the absence of, the DNA B. Rather than being required for maintenance, the CP was shown to possibly interfere with maintenance of the betasatellite. The findings show that the interaction between a bipartite begomovirus and a betasatellite is more complex than just trans-replication. Clearly, multiple levels of interactions are present and such associations can cause additional significant losses to crops although the interaction may not be stable.

MicroRNAs (miRNAs) are small 20–24nt molecules that have been well studied over the past decade due to their important regulatory roles in different cellular processes. The mature sequences are more conserved across vast phylogenetic scales than their precursors and some are conserved within entire kingdoms, hence, their loci and function can be predicted by homology searches. Different studies have been performed to elucidate miRNAs using de novo prediction methods but due to complex regulatory mechanisms or false positive in silico predictions, not all of them express in reality and sometimes computationally predicted mature transcripts differ from the actual expressed ones. With the availability of a complete genome sequence of Gossypium arboreum, it is important to annotate the genome for both coding and non-coding regions using high confidence transcript evidence, for this cotton species that is highly resistant to various biotic and abiotic stresses. Here we have analyzed the small RNA transcriptome of G. arboreum leaves and provided genome annotation of miRNAs with evidence from miRNA/miRNA∗ transcripts. A total of 446 miRNAs clustered into 224 miRNA families were found, among which 48 families are conserved in other plants and 176 are novel. Four short RNA libraries were used to shortlist best predictions based on high reads per million. The size, origin, copy numbers and transcript depth of all miRNAs along with their isoforms and targets has been reported. The highest gene copy number was observed for gar-miR7504 followed by gar-miR166, gar-miR8771, gar-miR156, and gar-miR7484. Altogether, 1274 target genes were found in G. arboreum that are enriched for 216 KEGG pathways. The resultant genomic annotations are provided in UCSC, BED format.

Viral diseases have been a major limiting factor threating sustainable potato (Solanum tuberosum L.) production in Pakistan. Surveys were conducted to serologically quantify the incidence of RNA viruses infecting potato; Potato virus X (PVX), Potato virus Y (PVY), Potato virus S (PVS), Potato virus A (PVA), Potato virus M (PVM) and Potato leaf roll virus (PLRV) in two major potato cultivars (Desiree and Cardinal). The results suggest the prevalence of multiple viruses in all surveyed areas with PVY, PVS and PVX dominantly widespread with infection levels of up to 50% in some regions. Co-infections were detected with the highest incidence (15.5%) for PVX and PVS. Additionally the data showed a positive correlation between co-infecting viruses with significant increase in absorbance value (virus titre) for at least one of the virus in an infected plant and suggested a synergistic interaction. To test this hypothesis, glasshouse grown potato plants were challenged with multiple viruses and analyzed for systemic infections and symptomology studies. The results obtained conclude that multiple viral infections dramatically increase disease epidemics as compared to single infection and an effective resistance strategy in targeting multiple RNA viruses is required to save potato crop.

Cotton leaf curl disease (CLCuD) in Pakistan and northwestern India is caused by monopartite begomoviruses in association with an essential, disease-specific satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Following a recent upsurge in CLCuD problems in Sindh province (southern Pakistan), sequences of clones of CLCuMB were obtained from Sindh and Punjab province (central Pakistan), where CLCuD has been a problem since the mid-1980s. The sequences were compared to all sequences of CLCuMB available in the databases. Analysis of the sequences shows extensive sequence variation in CLCuMB, most likely resulting from recombination. The range of sequence variants differ between Sindh, the Punjab and northwestern India. The possible significance of the findings with respect to movement of the CLCuD between the three regions is discussed. Additionally, the lack of sequence variation within the only coding sequence of CLCuMB suggests that the betasatellite is not involved in resistance breaking which became a problem after 2001 in the Punjab and subsequently also in northwestern India.

A collection of cultivated and non-cultivated species of cotton (Gossypium spp.) has been maintained for the last four decades in Multan, Pakistan. This geographical location has been observed as a hotspot for the evolution of begomoviruses and satellites associated with cotton leaf curl disease (CLCuD). Recent studies showed that begomoviruses responsible for the CLCuD epidemic in the 1990s, and that almost disappeared from the CLCuD complex in 2000s, have been observed again in CLCuD-infected cotton fields. To identify host species that acted as probable reservoirs for these viruses, we characterized begomoviruses and satellites in non-cultivated cotton species G. raimondii, G. thurberi and G. mustelinum and identified several species of CLCuD associated begomoviruses and satellites. Further, phylogenetic analysis indicated that the identified begomoviruses and beta/alphasatellites are closely related to the ones associated with the most recent CLCuD complex. qPCR indicated that the comparative level of virus significantly decreased in the presence of alphasatellites. Our results indicated that non-cultivated cotton species have been continuously challenged by diverse begomoviruses and associated satellites and act as reservoirs for CLCuD associated begomoviruses. These results provide novel insights into understanding the spread of begomoviruses and associated satellites in New World cotton species introduced into the Old World.