Genomic insights into local adaptation of upland cotton in China and Pakistan.

KEY MESSAGE: Different kinship and resistance to cotton leaf curl disease (CLCuD) and heat were found between upland cotton cultivars from China and Pakistan. 175 SNPs and 82 InDels loci related to yield, fiber quality, CLCuD, and heat resistance were identified. Elite alleles found in Pakistani accessions aided local adaptation to climatic condition of two countries. Adaptation of upland cotton (Gossypium hirsutum) beyond its center of origin is expected to be driven by tailoring of the genome and genes to enhance yield and quality in new ecological niches. Here, resequencing of 456 upland cotton accessions revealed two distinct kinships according to the associated country. Fiber quality and lint percentage were consistent across kinships, but resistance to cotton leaf curl disease (CLCuD) and heat was distinctly exhibited by accessions from Pakistan, illustrating highly local adaption. A total of 175 SNP and 82 InDel loci related to yield, fiber quality, CLCuD and heat resistance were identified; among them, only two overlapped between Pakistani and Chinese accessions underscoring the divergent domestication and improvement targets in each country. Loci associated with resistance alleles to leaf curl disease and high temperature were largely found in Pakistani accessions to counter these stresses prevalent in Pakistan. These results revealed that breeding activities led to the accumulation of unique alleles and helped upland cotton become adapted to the respective climatic conditions, which will contribute to elucidating the genetic mechanisms that underlie resilience traits and help develop climate-resilient cotton cultivars for use worldwide.

New genetic resources for aphid resistance were identified from a newly developed wheat mutant library.

Reports on development of resilient wheat mutants to aphid infestation-causing heavy losses to wheat production in many parts of the world, are scanty. The present study aimed to identify genetic diversity of wheat mutants in terms of varying degree of resistance to aphid infestation which can help protect wheat crop, improve yields and enhance food security. Resistance response to aphid infestation was studied on newly developed 33 wheat mutants, developed through irradiating seed of an elite wheat cultivar "Punjab-11" with gamma radiations, during three normal growing seasons at two sites. Data on various traits including aphid count per plant, biochemical traits, physiological traits and grain yield was recorded. Meteorological data was also collected to unravel the impact of environmental conditions on aphid infestation on wheat plants. Minimum average aphid infestation was found on Pb-M-2725, Pb-M-2550, and Pb-M-2719 as compared to the wild type. High yielding mutants Pb-M-1323, Pb-M-59, and Pb-M-1272 supported the moderate aphid infestation. The prevailing temperature up to 25 °C showed positive correlation (0.25) with aphid count. Among biochemical traits, POD (0.34), TSP (0.33), TFA (0.324) exhibited a high positive correlation with aphid count. In addition, CAT (0.31), TSS (0.294), and proline content (0.293) also showed a positive correlation with aphid count. However, all physiological traits depicted negative correlation with aphid count, while, a very weak correlation (0.12) was found between mean aphid count and grain yield. In PCA biplots, the biochemical variables clustered together with aphid count, while physiological variables grouped with grain yield. Biochemical parameters contributed most, towards first dimension of the PCA (48.6%) as compared to the physiological variables (13%). The FAMD revealed that mutant lines were major contributor towards total variation; Pb-M-1027, Pb-M-1323, Pb-M-59 were found to be the most diverse lines. The PCA revealed that biochemical parameters played a significant role in explaining variations in aphid resistance, emphasizing their importance in aphid defense mechanisms. The identified mutants can be utilized by the international wheat community for getting insight into the molecular circuits of resistant mechanism against aphids as well as for designing new KASP markers. This study also highlights the importance of considering both genetic and environmental factors in the development of resilient wheat varieties and pave the way for further investigations into the molecular mechanisms underpinning aphid resistance in wheat.

Vanillin derivatives as antiamnesic agents in scopolamine-induced memory impairment in mice.

Amnesia is a major health problem prevalent in almost every part of the world specifically in old age peoples. Vanillin analogues have played an important role in the field medicines. Some of them have been documented to be promising inhibitors of cholinesterases and could therefore, be used as antidepressant, anti-Alzheimer and as neuroprotective drugs. In this connection, the present study was designed to synthesize new vanillin analogues (SB-1 to SB-6) of varied biological potentials. The synthesized compounds were investigated as inhibitors against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes and as scavengers of DPPH and ABTS free radicals followed by behavioural antiamnesic evaluation in mice. The compounds; SB-1, SB-3, SB-4 and SB-6 more potently inhibited AChE with IC50 values of 0.078, 0.157, 0.108, and 0.014 µM respectively. The BChE was more potently inhibited by SB-3 with IC50 of 0.057 µM. Moreover, all of the tested compounds exhibited strong antioxidant potentials with promising results of SB-3 against DPPH with IC50 of 0.305 µM, while SB-5 was most active against ABTS with IC50 of 0.190 µM. The in-vivo studies revealed the improvement in memory deficit caused by scopolamine. Y-Maze and new object recognition test showed a considerable decline in cognitive dysfunctions. In Y-Maze test the spontaneous alteration of 69.44 ± 1% and 84.88 ± 1.35% for SB-1 and 68.92 ± 1% and 80.89 ± 1% for SB-3 at both test doses were recorded while during the novel object recognition test the Discrimination Index percentage of SB-1 was more pronounced as compared to standard drug. All compounds were found to be potent inhibitors of AChE, BChE, DPPH, and ABTS in vitro however, SB-1 and SB-3 were comparatively more potent. SB-1 was also more active in reclamation of memory deficit caused by scopolamine. SB-1 and SB-3 may be considered as excellent drug candidates for treating amnesia subjected to toxicological evaluations in other animal models.

RVE2, a new regulatory factor in jasmonic acid pathway, orchestrates resistance to Verticillium wilt.

Verticillium dahliae, one of the most destructive fungal pathogens of several crops, challenges the sustainability of cotton productivity worldwide because very few widely-cultivated Upland cotton varieties are resistant to Verticillium wilt (VW). Here, we report that REVEILLE2 (RVE2), the Myb-like transcription factor, confers the novel function in resistance to VW by regulating the jasmonic acid (JA) pathway in cotton. RVE2 expression was essentially required for the activation of JA-mediated disease-resistance response. RVE2 physically interacted with TPL/TPRs and disturbed JAZ proteins to recruit TPL and TPR1 in NINJA-dependent manner, which regulated JA response by relieving inhibited-MYC2 activity. The MYC2 then bound to RVE2 promoter for the activation of its transcription, forming feedback loop. Interestingly, a unique truncated RVE2 widely existing in D-subgenome (GhRVE2D) of natural Upland cotton represses the ability of the MYC2 to activate GhRVE2A promoter but not GausRVE2 or GbRVE2. The result could partially explain why Gossypium barbadense popularly shows higher resistance than Gossypium hirsutum. Furthermore, disturbing the JA-signalling pathway resulted into the loss of RVE2-mediated disease-resistance in various plants (Arabidopsis, tobacco and cotton). RVE2 overexpression significantly enhanced the resistance to VW. Collectively, we conclude that RVE2, a new regulatory factor, plays a pivotal role in fine-tuning JA-signalling, which would improve our understanding the mechanisms underlying the resistance to VW.

Targeted genome editing in polyploids: lessons from Brassica.

CRISPR-mediated genome editing has emerged as a powerful tool for creating targeted mutations in the genome for various applications, including studying gene functions, engineering resilience against biotic and abiotic stresses, and increasing yield and quality. However, its utilization is limited to model crops for which well-annotated genome sequences are available. Many crops of dietary and economic importance, such as wheat, cotton, rapeseed-mustard, and potato, are polyploids with complex genomes. Therefore, progress in these crops has been hampered due to genome complexity. Excellent work has been conducted on some species of Brassica for its improvement through genome editing. Although excellent work has been conducted on some species of Brassica for genome improvement through editing, work on polyploid crops, including U's triangle species, holds numerous implications for improving other polyploid crops. In this review, we summarize key examples from genome editing work done on Brassica and discuss important considerations for deploying CRISPR-mediated genome editing more efficiently in other polyploid crops for improvement.

Detecting Cotton Leaf Curl Virus Resistance Quantitative Trait Loci in Gossypium hirsutum and iCottonQTL a New R/Shiny App to Streamline Genetic Mapping.

Cotton leaf curl virus (CLCuV) causes devastating losses to fiber production in Central Asia. Viral spread across Asia in the last decade is causing concern that the virus will spread further before resistant varieties can be bred. Current development depends on screening each generation under disease pressure in a country where the disease is endemic. We utilized quantitative trait loci (QTL) mapping in four crosses with different sources of resistance to identify single nucleotide polymorphism (SNP) markers associated with the resistance trait to allow development of varieties without the need for field screening every generation. To assist in the analysis of multiple populations, a new publicly available R/Shiny App was developed to streamline genetic mapping using SNP arrays and to also provide an easy method to convert and deposit genetic data into the CottonGen database. Results identified several QTL from each cross, indicating possible multiple modes of resistance. Multiple sources of resistance would provide several genetic routes to combat the virus as it evolves over time. Kompetitive allele specific PCR (KASP) markers were developed and validated for a subset of QTL, which can be used in further development of CLCuV-resistant cotton lines.

Engineering Abiotic Stress Tolerance in Crop Plants through CRISPR Genome Editing.

Environmental abiotic stresses challenge food security by depressing crop yields often exceeding 50% of their annual production. Different methods, including conventional as well as genomic-assisted breeding, mutagenesis, and genetic engineering have been utilized to enhance stress resilience in several crop species. Plant breeding has been partly successful in developing crop varieties against abiotic stresses owning to the complex genetics of the traits as well as the narrow genetic base in the germplasm. Irrespective of the fact that genetic engineering can transfer gene(s) from any organism(s), transgenic crops have become controversial mainly due to the potential risk of transgene-outcrossing. Consequently, the cultivation of transgenic crops is banned in certain countries, particularly in European countries. In this scenario, the discovery of the CRISPR tool provides a platform for producing transgene-free genetically edited plants-similar to the mutagenized crops that are not extensively regulated such as genetically modified organisms (GMOs). Thus, the genome-edited plants without a transgene would likely go into the field without any restriction. Here, we focused on the deployment of CRISPR for the successful development of abiotic stress-tolerant crop plants for sustaining crop productivity under changing environments.

Domestication of newly evolved hexaploid wheat-A journey of wild grass to cultivated wheat.

Domestication of wheat started with the dawn of human civilization. Since then, improvement in various traits including resistance to diseases, insect pests, saline and drought stresses, grain yield, and quality were improved through selections by early farmers and then planned hybridization after the discovery of Mendel's laws. In the 1950s, genetic variability was created using mutagens followed by the selection of superior mutants. Over the last 3 decades, research was focused on developing superior hybrids, initiating marker-assisted selection and targeted breeding, and developing genetically modified wheat to improve the grain yield, tolerance to drought, salinity, terminal heat and herbicide, and nutritive quality. Acceptability of genetically modified wheat by the end-user remained a major hurdle in releasing into the environment. Since the beginning of the 21st century, changing environmental conditions proved detrimental to achieving sustainability in wheat production particularly in developing countries. It is suggested that high-tech phenotyping assays and genomic procedures together with speed breeding procedures will be instrumental in achieving food security beyond 2050.

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.

Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton.

Allotetraploid cotton is an economically important natural-fiber-producing crop worldwide. After polyploidization, Gossypium hirsutum L. evolved to produce a higher fiber yield and to better survive harsh environments than Gossypium barbadense, which produces superior-quality fibers. The global genetic and molecular bases for these interspecies divergences were unknown. Here we report high-quality de novo-assembled genomes for these two cultivated allotetraploid species with pronounced improvement in repetitive-DNA-enriched centromeric regions. Whole-genome comparative analyses revealed that species-specific alterations in gene expression, structural variations and expanded gene families were responsible for speciation and the evolutionary history of these species. These findings help to elucidate the evolution of cotton genomes and their domestication history. The information generated not only should enable breeders to improve fiber quality and resilience to ever-changing environmental conditions but also can be translated to other crops for better understanding of their domestication history and use in improvement.

Identification of induced mutations in hexaploid wheat genome using exome capture assay.

Wheat is a staple food crop of many countries. Improving resilience to biotic and abiotic stresses remain key breeding targets. Among these, rust diseases are the most detrimental in terms of depressing wheat production. In the present study, chemical mutagenesis was used to induce mutations in the wheat variety NN-Gandum-1. This cultivar is moderately resistant to leaf and yellow rust. The aim of mutagenesis was to improve resistance to the disease as well as to study function of genes conferring resistance to the disease. In the present investigation, a 0.8% EMS dose was found optimum for supporting 45-55% germination of NN-Gandum-1. A total of 3,634 M2 fertile plants were produced from each of the M1 plant. Out of these, 33 (0.91%) and 20 plants (0.55%) showed absolute resistance to leaf and yellow rust, respectively. While 126 (3.46%) and 127 plants (3.49%) exhibited high susceptibility to the leaf and yellow rust, respectively. In the M4 generation, a total of 11 M4 lines (nine absolute resistant and two highly susceptible) and one wild type were selected for NGS-based exome capture assay. A total of 104,779 SNPs were identified that were randomly distributed throughout the wheat sub genomes (A, B and D). Induced mutations in intronic sequences predominated. The highest total number of SNPs detected in this assay were mapped to chr.2B (14,273 SNPs), which contains the highest number of targeted base pairs in the assay. The average mutation density across all regions interrogated was estimated to be one mutation per 20.91 Mb. The highest mutation frequency was found in chr.2D (1/11.7 kb) and the lowest in chr.7D (1/353.4 kb). Out of the detected mutations, 101 SNPs were filtered using analysis criteria aimed to enrich for mutations that may affect gene function. Out of these, one putative SNP detected in Lr21 were selected for further analysis. The SNP identified in chimeric allele (Lr21) of a resistant mutant (N1-252) was located in a NBS domain of chr.1BS at 3.4 Mb position. Through computational analysis, it was demonstrated that this identified SNP causes a substitution of glutamic acid with alanine, resulting in a predicted altered protein structure. This mutation, therefore, is a candidate for contributing to the resistance phenotype in the mutant line. Based on this work, we conclude that the wheat mutant resource developed is useful as a source of novel genetic variation for forward-genetic screens and also as a useful tool for gaining insights into the important biological circuits of different traits of complex genomes like wheat.

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.

Identification of Marker-Trait Associations for Lint Traits in Cotton.

Harvesting high quality lint, a long-awaited breeding goal-accomplished partly, can be achieved by identifying DNA markers which could be used for diagnosing cotton plants containing the desired traits. In the present studies, a total of 185 cotton genotypes exhibiting diversity for lint traits were selected from a set of 546 genotypes evaluated for fiber traits in 2009. These genotypes were extensively studied for three consecutive years (2011-2013) at three different locations. Significant genetic variations were found for average boll weight, ginning out turn (GOT), micronaire value, staple length, fiber bundle strength, and uniformity index. IR-NIBGE-3701 showed maximum GOT (43.63%). Clustering of genotypes using Ward's method was found more informative than that of the clusters generated by principal component analysis. A total of 382 SSRs were surveyed on 10 Gossypium hirsutum genotypes exhibiting contrasting fiber traits. Out of these, 95 polymorphic SSR primer pairs were then surveyed on 185 genotypes. The gene diversity averaged 0.191 and the polymorphic information content (PIC) averaged 0.175. Unweighted pair group method with arithmetic mean (UPGMA), principal coordinate analysis (PCoA), and STRUCTURE software grouped these genotypes into four major clusters each. Genetic distance within the clusters ranged from 0.0587 to 0.1030. A total of 47 (25.41%) genotypes exhibited shared ancestry. In total 6.8% (r2 ≥ 0.05) and 4.4% (r2 ≥ 0.1) of the marker pairs showed significant linkage disequilibrium (LD). A number of marker-trait associations (in total 75) including 13 for average boll weight, 18 for GOT percentage, eight for micronaire value, 18 for staple length, three for fiber bundle strength, and 15 for uniformity index were calculated. Out of these, MGHES-51 was associated with all the traits. Most of the marker-trait associations were novel while few validated the associations reported in the previous studies. High frequency of favorable alleles in cultivated varieties is possibly due to fixation of desirable alleles by domestication. These favorable alleles can be used in marker assisted breeding or for gene cloning using next generation sequencing tools. The present studies would set a stage for harvesting high quality lint without compromising the yield potential-ascertaining natural fiber security.

Comparative sequence analysis of citrate synthase and 18S ribosomal DNA from a wild and mutant strains of Aspergillus niger with various fungi.

UNLABELLED: A mutation was induced in Aspergillus niger wild strain using ethidium bromide resulting in enhanced expression of citric acid by three folds and 112.42 mg/mL citric acid was produced under optimum conditions with 121.84 mg/mL of sugar utilization. Dendograms of 18S rDNA and citrate synthase from different fungi including sample strains were made to assess homology among different fungi and to study the correlation of citrate synthase gene with evolution of fungi. Subsequent comparative sequence analysis revealed strangeness between the citrate synthase and 18S rDNA phylogenetic trees. Furthermore, the citrate synthase movement suggests that the use of traditional marker molecule of 18S rDNA gives misleading information about the evolution of citrate synthase in different fungi as it has shown that citrate synthase gene transferred independently among different fungi having no evolutionary relationships. Random amplified polymorphic DNA (RAPD-PCR) analysis was also employed to study genetic variation between wild and mutant strains of A. niger and only 71.43% similarity was found between both the genomes. Keeping in view the importance of citric acid as a necessary constituent of various food preparations, synthetic biodegradable detergents and pharmaceuticals the enhanced production of citric acid by mutant derivative might provide significant boost in commercial scale viability of this useful product. ABBREVIATIONS: CS - Citrate synthase, CA - Citric acid, RAPD - Random amplified polymorphic DNA, TAF - Total amplified fragments, PAF - Polymorphic amplified fragments, CAF - Common amplified fragments.

QTL mapping for physiology, yield and plant architecture traits in cotton (Gossypium hirsutum L. ) grown under well-watered versus water-stress conditions

Increasing scarcity of irrigation water is a major threat to sustainable production of cotton (Gossypium hirsutum L.). Identifying genomic regions contributing to abiotic stress tolerance will help develop cotton cultivars suitable for water-limited regions through molecular marker-assisted breeding. A molecular mapping F2 population was derived from an intraspecific cross of the drought sensitive G. hirsutum cv. FH-901 and drought tolerant G. hirsutum cv. RH-510. Field data were recorded on physiological traits (osmotic potential and osmotic adjustment); yield and its component traits (seedcotton yield, number of bolls/plant and boll weight); and plant architecture traits (plant height and number of nodes per plant) for F2, F2:3 and F2:4 generations under well-watered versus water-limited growth conditions. The two parents were surveyed for polymorphism using 6500 SSR primer pairs. Joinmap3.0 software was used to construct linkage map with 64 polymorphic markers and it resulted into 35 markers mapped on 12 linkage groups. QTL analysis was performed by composite interval mapping (CIM) using QTL Cartographer2.5 software. In total, 7 QTLs (osmotic potential 2, osmotic adjustment 1, seedcotton yield 1, number of bolls/plant 1, boll weight 1 and plant height 1) were identified. There were three QTLs (qtlOP-2, qtlOA-1, and qtlPH-1) detected only in water-limited conditions. Two QTLs (qtlSC-1 and qtlBW-1) were detected for relative values. Two QTLs (qtlOP-1 and qtlBN-1) were detected for well-watered treatment. Significant QTLs detected in this study can be employed in MAS for molecular breeding programs aiming at developing drought tolerant cotton cultivars.

Detection of single nucleotide polymorphisms in the conserved ESTs regions of Gossypium arboreum

Exploring genetic variation in Gossypium arboreum L. germplasm is useful as it contains many important genes conferring resistance to different stresses. In limited earlier studies, low level of genetic diversity was found by using conventional DNA marker systems which may impede future genome mapping studies. In the present investigation, we explored the extent of Single Nucleotide Polymorphisms (SNP) among 30 conserved regions of Expressed Sequence Tags (EST) of low copy genes between two genotypes of G. arboreum. A total of 27 SNPs including 21 substitutions and 6 Insertions and deletions (Indels) in 7804 bp were found between these genotypes with a frequency of one SNP per 371 bp and one Indel after every 1300 bp. Out of these SNPs, 52 percent were transitions, whilst 48 percent SNPs were transversion. In conclusion, SNPs are expedient markers that can explore polymorphism in highly conserved sequences where other markers are not effective.