Genetic analysis and molecular validation of gene conferring petal spot phenotype in interspecific crosses of cotton.

Cotton (Gossypium species) has received considerable interest from the geneticists, cytologists and evolutionary biologists since the last more than a century. Here, we explore the genetics of petal spot in the interspecific derivatives involving tetraploid and diploid cottons; and confirm the location of gene governing petal spot phenotype on chromosome A7 by demonstrating co-segregation of SSR marker NAU 2186 with petal spot phenotype. The presence of petal spot was observed to be dominant over its absence. Petal spot inheritance showed significant deviation from the expected Mendelian ratio in all the segregating populations indicating segregation distortion. The distortion was biased towards the hirsutum parent which has important implications from introgression point of view. We also report a strong association between petal spot and petal margin coloration phenotypes. Extant American cotton varieties generally lack petal spot and margin coloration phenotypes. These petal characteristics can serve as morphological markers during germplasm characterization.

Nanoparticle-Shielded dsRNA Delivery for Enhancing RNAi Efficiency in Cotton Spotted Bollworm Earias vittella (Lepidoptera: Nolidae).

The spotted bollworm Earias vittella (Lepidoptera: Nolidae) is a polyphagous pest with enormous economic significance, primarily affecting cotton and okra. However, the lack of gene sequence information on this pest has a significant constraint on molecular investigations and the formulation of superior pest management strategies. An RNA-seq-based transcriptome study was conducted to alleviate such limitations, and de novo assembly was performed to obtain transcript sequences of this pest. Reference gene identification across E. vittella developmental stages and RNAi treatments were conducted using its sequence information, which resulted in identifying transcription elongation factor (TEF), V-type proton ATPase (V-ATPase), and Glyceraldehyde -3-phosphate dehydrogenase (GAPDH) as the most suitable reference genes for normalization in RT-qPCR-based gene expression studies. The present study also identified important developmental, RNAi pathway, and RNAi target genes and performed life-stage developmental expression analysis using RT-qPCR to select the optimal targets for RNAi. We found that naked dsRNA degradation in the E. vittella hemolymph is the primary reason for poor RNAi. A total of six genes including Juvenile hormone methyl transferase (JHAMT), Chitin synthase (CHS), Aminopeptidase (AMN), Cadherin (CAD), Alpha-amylase (AMY), and V-type proton ATPase (V-ATPase) were selected and knocked down significantly with three different nanoparticles encapsulated dsRNA conjugates, i.e., Chitosan-dsRNA, carbon quantum dots-dsRNA (CQD-dsRNA), and Lipofectamine-dsRNA conjugate. These results demonstrate that feeding nanoparticle-shielded dsRNA silences target genes and suggests that nanoparticle-based RNAi can efficiently manage this pest.

Inheritance and molecular tagging of genes introgressed from Gossypium arboreum to G. hirsutum for leafhopper tolerance.

Cotton cultivation is conquered by transgenic Bt upland cotton hybrids in India. Bt gene does not provide resistance against sucking insect pests. Due to the inherent vulnerability of extant Bt cotton hybrids to sap-sucking insect pests including leafhopper, upland cotton cultivation is seriously threatened by surging populations of these pests. Consistent and extensive screening of upland cotton germplasm over the years has revealed absence of adequate resistance against leafhopper. Here, we report introgression of leafhopper tolerance from a diploid A-genome cotton species, Gossypium arboreum into G. hirsutum. The dominance of leafhopper tolerance was observed over its susceptibility. Genetic analysis revealed that tolerance to leafhopper was inherited in a simple Mendelian fashion and was controlled by two genes, either singly or in combination. Using bulked segregant analysis, two simple-sequence repeat markers, namely NAU 922 and BNL 1705, located on chromosomes A5 and A11 respectively, were tagged with leafhopper tolerance. To the best of our knowledge, this is the first report of molecular tagging of leafhopper tolerance introgressed from G. arboreum into G. hirsutum. A significant negative association was observed between leaf trichome density and leafhopper nymph population.

Molecular mapping of CLCuD resistance introgressed from synthetic cotton polyploid in upland cotton.

Cotton leaf curl disease (CLCuD), caused by a geminivirus complex, is the most serious disease of upland cotton in northwest India and Pakistan. It results in substantial losses in cotton yield and fibre quality. Due to continuous appearance of new viral strains, all the established CLCuD resistant stocks, extant and obsolete cultivars of upland cotton have become susceptible. Therefore, it became crucial to explore the novel sources of CLCuD resistance, as development of CLCuD resistant varieties is the most practical approach to manage this menace. Here, for the first time, we report introgression and mapping of CLCuD resistance from a 'synthetic cotton polyploid' to upland cotton. A backcross population (synthetic polyploid / Gossypium hirsutum Acc. PIL 43/G. hirsutum Acc. PIL 43) was developed for studying inheritance and mapping of CLCuD resistance. Dominance of CLCuD resistance was observed over its susceptibility. Two dominant genes were found to confer resistance to CLCuD. Molecular analysis through genotyping-by-sequencing revealed that chromosomes A01 and D07 harboured one CLCuD resistance gene each.

Transcriptome analysis unravels RNAi pathways genes and putative expansion of CYP450 gene family in cotton leafhopper Amrasca biguttula (Ishida).

Cotton Leafhopper, Amrasca biguttula is an important pest of cotton and okra in the Indian subcontinent. Presently limited genomic/transcriptomic information is available for this insect in any of open source databases. The present study reports the first assembled and annotated de novo transcriptome of cotton leafhopper. Out of 75,551 transcripts, 39,613 CDS (Coding Sequence) were predicted with 35,282 showing positive blast hits with NCBI nr database. The Gene ontology (GO) analysis annotated 7431 CDS  with KEGG pathway categorizing these CDS into 22 different functional groups. The majority of CDS were annotated in signal transduction and transport catabolism pathways. The sequence data was screened for RNAi pathway genes and presence of 37 transcripts associated with this process confirmed the existence of robust RNAi machinery. The role of core RNAi machinery genes (Dicer-2, Ago-2, Piwi and Staufen) has been validated through dsRNA feeding studies. The data resource has also been used to identify potential RNAi targets and genes associated with insecticide detoxification specifically CYP 450 family. The current study provides a useful sequence resource which can be used to initiate molecular studies in this insect with emphasis on insecticide resistance, RNAi and functional genomics.

Collagen Nanoparticle-Mediated Brain Silymarin Delivery: An Approach for Treating Cerebral Ischemia and Reperfusion-Induced Brain Injury.

Silymarin is a bioactive constituent isolated from milk thistle (Silybum marinum). Since its discovery, silymarin has been considered a gold standard drug in treating ailments related to the liver, resulting from alcohol consumption and viral hepatitis. This hepatoprotective nature of silymarin arises out of antioxidative and tissue-regenerating properties of silymarin. However, several recent studies have established the neuroprotective link of silymarin, too. Thus, the current investigation was aimed at exploring the neuroprotective effect of nanosilymarin (silymarin encapsulated inside collagen-based polymeric nanoparticulate drug delivery system). The study aimed at bringing out the role of nanoparticles in enhancing the therapeutic effect of silymarin against neuronal injury, originating out of oxidative-stress-related brain damages in focal cerebral ischemia. Collagen-based micellar nanoparticles were prepared and stabilized using 3-ethyl carbodiimide-hydrochloride (EDC-Hcl) and malondialdehyde (MDA) as crosslinkers. Nanoparticles were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and Fourier transform infrared (FT-IR) spectroscopy techniques, and the size of nanoparticles was found to be around 48 nm. Male albino Wistar rats were pretreated with three different doses of nanosilymarin of 10, 100, and 1,000 µg/kg b.wt and a dose of free silymarin of 100 mg/kg b.wt intraperitoneally (i.p.) for 7 days. Focal cerebral ischemia was induced using the middle cerebral artery occlusion (MCAO) model on the eighth day for 1 h followed by 24 h reperfusion. The animals were then evaluated for neurobehavioral, infarct analysis, biochemical, histopathological, and immunohistochemical studies. All the above parameters showed remarkable improvement in nanosilymarin-treated groups in comparison to the silymarin-treated group. Nanoparticle encapsulation of drug enhanced neuroprotection by increasing drug bioavailability and targeting. Thus, the present study concluded with satisfactory results, showing the critical role played by nanoparticles in improving the neuroprotection at very low drug doses.

Genetic analysis of some morphological traits in synthetic x naturally polyploid cotton derivatives.

Cotton has received attention of geneticists since more than a century. Gossypium hirsutum, the predominantly cultivated cotton species worldwide, has a narrow genetic base. It is important to broaden its genetic base through introgression of novel alleles from related species. Here, we report the development and characterization of a backcross population derived from the hybridization of a 'synthetic' (derived by crossing and chromosomal doubling of nonprogenitor Gossypium species) and natural tetraploid upland cotton. 'Synthetic' was observed to be male-sterile and thus, was used as the female parent. A total of 7434 flowers were pollinated to obtain 1868 BC1F1 seeds by direct and reciprocal crosses. Characterization of the experimental plant material was conducted in the field for several morphological traits such as pubescence on the stem, leaf, petiole and bract, presence/absence of petal spot, petal margin colouration and stamen filament colouration. Genetic analysis revealed that petal margin colouration phenotype was governed by a single dominant gene, whereas the petal spot and filament colouration phenotypes manifested segregation distortion. None of the BC1F1 plants was devoid of trichomes thus demonstrating that presence of trichomes is dominant over their absence. Modern upland cotton cultivars are usually devoid of petal spot, petal margin colouration and stamen filament colouration. These floral anthocyanin pigmentation characteristics, if fixed in the cotton germplasm, may serve as diagnostic features for the identification of cultivars during DUS testing as well as in the maintenance breeding programmes.

Author Correction: Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Collagen-curcumin nanocomposites showing an enhanced neuroprotective effect against short term focal cerebral ischemia.

The effectiveness of curcumin in treating cerebral ischemia has been reported in recent studies. However, its mode of action is still not defined. The objective of the present study is to formulate collagen-curcumin nanocomposites which will work effectively against cerebral ischemia/reperfusion injury. Ischemic injury is followed by inflammatory damage and oxidative stress, which together contribute a lot in the pathogenesis of cerebral ischemia and may be considered a good target for treatment. The present study focused on examining the effectiveness of collagen-curcumin nanocomposites stabilized by increasing the degree of crosslinking in reducing oxidative stress associated with brain injury resulting from cerebral ischemia. The collagen nanoparticles were prepared by conjugating collagen on the surface of Tween©80 micelles, and further stabilizing them using crosslinkers. The effectiveness of the prepared nanocomposite was validated by performing infarct analysis followed by biochemical, behavioral, histopathological and immunohistochemical studies. The outcomes of this study are promising for the use of collagen-curcumin nanocomposites in showing neuroprotective potential in treating ischemic injury.

Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae).

Phenacoccus solenopsis is one of the major polyphagous crop pests in India. Inadequate genomic or transcriptomic resources have limited the molecular studies in this insect despite its huge economic importance. The existing molecular sequence resources of this insect were supplemented through RNA sequencing, de novo transcriptome assembly and analysis, which generated 12, 925 CDS from 23,643 contigs with an average size of 1077.5 bp per CDS and 85.1% positive BLAST hits with NCBI Non redundant (nr) database. Twenty three genes involved in RNAi machinery identified through BLASTx search against NCBI nr database suggested the existence of robust RNAi in mealybug. RNAi in P. solenopsis was demonstrated through knockdown of IAP (Inhibitor of Apoptosis), AQP (Aquaporin), CAL (Calcitonin), VATPase (V-type proton ATPase subunit F 1), bursicon, chitin synthase, SNF7 and α-amylase by injecting sequence specific dsRNA of respective genes in adult female. Additionally, feeding RNAi has been demonstrated in 2nd instar nymph through dsRNA uptake in plant. The knockdown of core RNAi machinery genes such as Dicer, Argonaute and Staufen significantly hampered RNAi efficiency in this insect. However, downregulation of dsRNases improved RNAi efficiency. Sequential studies for understanding RNAi in P. solenopsis using transcriptome sequences have also been reported. The present study provides a base for future research on developing RNAi as strategy for management of this pest.

RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae).

BACKGROUND: Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this insect, RNA was sequenced; de novo transcriptome assembly and analysis were performed. The RNAseq data was used to identify reference and RNAi pathway genes in this insect. Additionally, feeding RNAi was demonstrated in T. tabaci for the first time. RESULTS: From the assembled transcriptome, 27,836 coding sequence (CDS) with an average size of 1236 bp per CDS were identified. About 85.4% of CDS identified showed positive Blast hits. The homologs of most of the core RNAi machinery genes were identified in this transcriptome. To select reference genes for reverse-transcriptase real-time quantitative PCR (RT-qPCR) experiments, 14 housekeeping genes were identified in the transcriptome and their expression was analyzed by (RT-qPCR). UbiCE in adult, 28s in nymphs and SOD under starvation stress were identified as the most stable reference genes for RT-qPCR. Feeding dsSNF7 and dsAQP caused 16.4- and 14.47-fold reduction in SNF7 and AQP mRNA levels respectively, when compared to their levels in dsGFP fed control insects. Feeding dsSNF7 or dsAQP also caused 62 and 72% mortality in T. tabaci. Interestingly, simultaneous feeding of dsRNAs targeting SNF7 or AQP and one of the RNAi pathway genes (Dicer-2/Aubergine/Staufen) resulted in a significant reduction in RNAi of target genes. These data suggest the existence of robust RNAi machinery in T. tabaci. CONCLUSION: The current research is the first report of the assembled, analyzed and annotated RNAseq resource for T. tabaci, which may be used for future molecular studies in this insect. Reference genes validated across stages and starvation stress provides first-hand information on stable genes in T. tabaci. The information on RNAi machinery genes and significant knockdown of the target gene through dsRNA feeding in synthetic diet confirms the presence of efficient RNAi in this insect. These data provide a solid foundation for further research on developing RNAi as a method to manage this pest.

Reference Gene Selection in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) and Their Normalization Impact on Gene Expression in RNAi Studies.

Phenacoccus solenopsis, the cotton mealybug (Hemiptera: Pseudococcidae), is one of the major cotton pests in India. Scanty information is available on molecular studies in this insect due to limited genomic or transcriptomic sequence data. With advancement in sequencing technology, enormous genomic and sequencing data are being generated, and RNAi studies are being undertaken in insects, which require reverse transcription quantitative polymerase chain reaction evaluation. These gene expression studies require normalization of mRNA levels with reference genes to account for sample variability. To supplement the molecular studies in this insect, candidate reference genes were identified and evaluated for their expression stability across various developmental stages and starvation stress. Fourteen candidate reference genes including several commonly used ones were investigated across five different stages and under starvation stress using four different statistical algorithms (NormFinder, genNorm, BestKeeper, and RefFinder). Based on this analysis, GST (third, fourth, and adult stage), Actin (Crawler, second instar), TFIID (starvation stress), SDHA, and 28s were identified as best reference genes for expression studies in mealybug.

Selection of housekeeping genes and demonstration of RNAi in cotton leafhopper, Amrasca biguttula biguttula (Ishida).

Amrasca biguttula biguttula (Ishida) commonly known as cotton leafhopper is a severe pest of cotton and okra. Not much is known on this insect at molecular level due to lack of genomic and transcriptomic data. To prepare for functional genomic studies in this insect, we evaluated 15 common housekeeping genes (Tub, B-Tub, EF alpha, GADPH, UbiCF, RP13, Ubiq, G3PD, VATPase, Actin, 18s, 28s, TATA, ETF, SOD and Cytolytic actin) during different developmental stages and under starvation stress. We selected early (1st and 2nd), late (3rd and 4th) stage nymphs and adults for identification of stable housekeeping genes using geNorm, NormFinder, BestKeeper and RefFinder software. Based on the different algorithms, RP13 and VATPase are identified as the most suitable reference genes for quantification of gene expression by reverse transcriptase quantitative PCR (RT-qPCR). Based on RefFinder which comprehended the results of three algorithms, RP13 in adults, Tubulin (Tub) in late nymphs, 28S in early nymph and UbiCF under starvation stress were identified as the most stable genes. We also developed methods for feeding double-stranded RNA (dsRNA) incorporated in the diet. Feeding dsRNA targeting Snf7, IAP, AQP1, and VATPase caused 56.17-77.12% knockdown of targeted genes compared to control and 16 to 48% mortality of treated insects when compared to control.

Dose and time dependent efficacy alteration of different defoliants on seed cotton yield.

Field experiments were conducted during 2012 and 2013 to determine the effect of select defoliants i.e dropp ultra and ethrel, their optimal dose and suitable time of application on earliness and seed cotton yield in 3 American cotton cultivars. MRC7361BGII (3055.2 kg ha(-1)) and MRC7017BGII (2913.0 kg ha(-1)) resulted in significantly improved seed cotton yield as compared to F1 861 (2346.0 kg ha(-1)). Dropp ultra? @ 200 ml ha(-1) revealed highest yield (3018.7 kg ha(-1)) followed by ethrel @ 2000 ppm (2820.1 kg ha(-1)) and control (2730.0 kg ha(-1)) , while statistically least yield (2516.9 kg ha(-1)) was observed with higher dose of dropp ultra @ 225 ml ha(-1). Defoliants applied 150 days after sowing (DAS) resulted in significantly better yield (2853.3 kg ha(-1)) as compared to the early application at 140 DAS (2689.5 kg ha(-1) owing to improvement in open bolls and boll weight. Pooled data indicated that dropp ultra @ 200 ml ha(-1) has potential to promote crop earliness, better boll opening and their retention by keeping vegetative and reproductive growth in harmony to enhance seed cotton yield.

Efficacy evaluation of selected herbicides on weed control and productivity evaluation of Bt cotton in Punjab.

Field experiments were conducted during Kharif 2012 and 2013 to evaluate the efficacy of different herbicides for weed management in cotton. Highest seed cotton yield (3537.3 kg ha(-1)) was recorded in weed free plots followed by pendimethalin @1.0 kg a.i ha(-1) as Pre.em.+quizalofopethyl @50 g a.i ha(-1) post-em at 2-4 weed leaf stage + one hoeing (3318.9 kg ha") owing to improved number of bolls per plant and boll weight. Statistically least yield was recorded underweedy check (1435.4 kg ha(-1)). Application of pyrithiobac sodium could not express any visible toxic effect on crop indicating its selectivity for cotton, although none of the tested new chemicals i.e., pyrithiobac sodium@ 62.5g a.i ha(-1) and quizalofopethyl @50g a.i ha(-1) when applied alone could not outperform the existing recommended chemicals for weed management. Yield losses to the extent of 6.2-59.4% were recorded due to weed competition. Weed control efficiency (WCE) was highest under weed free check (86.8%) followed by pendimethalin @1.0 kg a.i ha(-1) as Pre. em.+quizalofopethyl @50g a.i ha(-1), at 2-4 weed leaf stage + one hoeing (73.7%), whereas minimum values were for weedy check (24.7%). Though net returns (r94660 ha(-1)) were highest for weed free check but higher B:C ratio (2:11) was observed for pendimethalin @1.0 kg a.i ha(-1) as Pre em.+quizalofopethyl @50 g a.i ha(-1) post-em at 2-4 weed leaf stage+one hoeing. Therefore, for reasons such as labor shortage besides their timely availability, using these herbicides in combination with cultural practices could be the practical solution foreconomically efficient and effective weed management.

Productivity potential and monetary evaluation of Bt cotton hybrids under varied agronomic manipulations in semi-arid conditions.

Field experiments comprising three Btcotton hybrids in main, two plant geometries in sub and three nutrient levels in sub sub plots of Split Plot design replicated thrice were conducted at PAU, Regional Research Station, Faridkot during Kharif 2010 and 2011. MRC7361 recorded significantly the highest SCY (3121.6 kg ha(-1)) followed by Bioseed 6488 (2649.5 kg ha(-1)) and RCH134 (2107.4 kg ha(-1)). Statistically improved bolls per plant and boll weight primarily helped MRC7361 in achieving the highest yield. Owing to better fertilizer use efficiency (5.47) and water productivity (649.3 gm(-3)), highest net returns of < or = 101047 ha(-1) were observed with MRC7361 as compared to Bioseed 6488 (< or = 83343 ha(-1)) and RCH134 (< or = 63014 ha(-1)). Bolls per plant and benefit cost ratio was significantly enhanced under wider geometry of 67.5 x 90 cm. Application of 225 kg N, 45 kg P ha(-1) produced significantly higher SCY (2825.9 kg ha(-1)) owing to improved sympods and bolls per plant than 150 kg N, 30 kg P ha(-1) (2374.9 kg ha(-1)) and 187.5 kg N, 37.5 kg P ha(-1) (2677.6 kg ha(-1)). As a result of this, statistically highest net returns of < or = 89552 ha(-1) were obtained with application of 225 kg N, 45 kg Pha(-1). Water productivity improved significantly with each successive increase in the level of nutrients. Contrarily, fertilizer use efficiency exhibited reverse trend with statistically highest value (5.08) for the lowest level of nutrients.