Blight Tolerance Research Articles

Canonical Correlation Analysis and Grouping of Bread Wheat Genotypes Based on Morphophenological Traits and Fusarium Head Blight Tolerance Indexes

Canonical Correlation Analysis and Grouping of Bread Wheat Genotypes Based on Morphophenological Traits and Fusarium Head Blight Tolerance Indexes

Pathogen-induced expression of a blight tolerance transgene inAmerican chestnut.

American chestnut (Castanea dentata) is a susceptible host of the invasive necrotrophic fungus Cryphonectria parasitica, which causes chestnut blight disease. The fungal pathogen attacks chestnut stems by invading wounded tissue and secreting oxalate. This process leads to the death of infected host cells and the formation of cankers, eventually girdling stems and killing the tree above the infections. To reduce damage caused by fungal oxalate, American chestnut has been genetically engineered to express a wheat oxalate oxidase (OxO). This enzyme degrades the oxalate produced by the pathogen and confers elevated tolerance to Cryphonectria parasitica infection. We report new lines of transgenic American chestnut that have been developed with the win3.12 inducible promoter from poplar (Populus deltoides) driving OxO expression. This promoter is responsive to both wounding and pathogen infection, with a low level of baseline expression. Targeted expression of OxO to wounded and infected tissue is sought as an alternative to constitutive expression for potential metabolic resource conservation and transgene stability over the long lifetime of a tree and over successive generations of breeding. Transgenic Castanea dentata lines harbouring the win3.12‐OxO construct were evaluated for transgene expression patterns and tolerance to chestnut blight infection. OxO transcript levels were low in uninfected plants, but robust infection‐induced expression levels were observed, with one transgenic line reaching levels comparable to those of previously characterized CaMV35S‐OxO lines. In chestnut blight infection bioassays, win3.12‐OxO lines showed elevated disease tolerance similar to blight‐resistant Chinese chestnut (Castanea mollissima) controls.

Genome-Wide Association Mapping of Late Blight Tolerance Trait in Potato (Solanum tuberosum L.).

Uncovering the genetic basis and optimizing the late blight tolerance trait in potatoes (Solanum tuberosum L.) are crucial for potato breeding. Late blight disease is one of the most significant diseases hindering potato production. The traits of late blight tolerance were evaluated for 284 potato cultivars to identify loci significantly associated with the late blight tolerance trait. Of all, 37 and 15 were the most tolerant to disease, and 107 and 30 were the most susceptible. A total of 22,489 high-quality single-nucleotide polymorphisms and indels were identified in 284 potato cultivars. All the potato cultivars were clustered into eight subgroups using population structure analysis and principal component analysis, which were consistent with the results of the phylogenetic tree analysis. The average genetic diversity for all 284 potato cultivars was 0.216, and the differentiation index of each subgroup was 0.025–0.149. Genome-wide linkage disequilibrium (LD) analysis demonstrated that the average LD was about 0.9 kb. A genome-wide association study using a mixed linear model identified 964 loci significantly associated with the late blight tolerance trait. Fourteen candidate genes for late blight tolerance traits were identified, including genes encoding late blight tolerance protein, chitinase 1, cytosolic nucleotide-binding site–leucine-rich repeat tolerance protein, protein kinase, ethylene-responsive transcription factor, and other potential plant tolerance-related proteins. This study provides novel insights into the genetic architecture of late blight tolerance traits and will be helpful for late blight tolerance in potato breeding.

Registration of ‘ND Grano’ durum wheat

AbstractDurum wheat [Triticum turgidum L. ssp. durum (Desf.)] is mainly used for the production of pasta, couscous, and bulgur. Spring durum wheat cultivar ‘ND Grano’ (Reg. no. CV‐1186, PI 687795) was released in 2017 and is adapted to North Dakota and surrounding regions. It was developed by the durum breeding program at North Dakota State University and released by the North Dakota Agricultural Experiment Station. ND Grano was tested as D09557 in the yield trials and has the pedigree D00752/DH01146. In field trials, ND Grano showed high grain yield potential, disease resistance, excellent end‐use qualities, and low cadmium uptake. Based on data from 34 location‐years of testing in the Uniform Regional Durum Nursery, the average grain yield of ND Grano was 4,008 kg ha–1, compared with 3,867 kg ha–1 for ‘Divide’ and 3,978 kg ha–1 for ‘Joppa’. ND Grano has very strong dough mixing characteristics and a high gluten index. Fusarium head blight (FHB) tolerance of ND Grano is similar to Divide. ND Grano is also resistant to predominant races of stem rust.

Determining the tolerance of the Turkish hazelnut cultivars (Coryllus avellana) against the pathovar Xanthomonas arboricola pv. corylina (causing agent of fireblight)

Objective: This study was conducted to define the tolerance of various hazelnut cultivars against Xanthomonas arboricola pv. corylina causing agent of bacterial blight. 
 Material and Methods: The Turkish hazelnut cultivars (Acı, Çakıldak, Foşa, İncekara, Kalınkara, Kan, Kargalak, Kuş, Mincane, Palaz, Sivri, Tombul, Uzunmusa, Yassı Badem and Yuvarlak Badem) were used as plant material. AT8/Z strain of Xanthomonas arboricola pv. corylina isolated from the field was tested. Parameters measured to detect bacterial blight tolerance were infected leaf percentage and leaf lesion rate. 
 Results: İncekara showed the minimum percentage of infected leaves (%17.57) and Yassı Badem reflected the highest (%77.51). Yassı Badem had the highest leaf lesion degree (3.00) and İncekara the lowest (1.34). The results suggested that Yassı Badem cultivar was the most susceptible and İncekara the most resistant to the pathogen. The highest antimicrobial activity was represented by Mincane with leaf diameter of 20 mm.
 Conclusion: In this study, resistance to Xanthomonas arboricola pv. corylina pathogen tolerance of Turkish hezelnut cultivars grown in our country was determined. It is thought that the results obtained from the study will shed light on the breeding studies of resistant varieties against this pathogen in the future.

Intentional introgression of a blight tolerance transgene to rescue the remnant population of American chestnut

AbstractIn contrast to many current applications of biotechnology, the intended consequence of the American Chestnut Research & Restoration Project is to produce trees that are well‐adapted to thrive not just in confined fields or orchards, but throughout their natural range. Our primary focus is on disease tolerance, but we believe it will also be critically important that optimal restoration trees should have robust genetic diversity and resilience, which can be supplied by a full complement of their wild‐type genes. Chestnut restoration offers a unique case study because many restoration or intervention options have been attempted: doing nothing, planting non‐native chestnut species, planting hybrids, mutagenesis (exposing seeds to high levels of radiation to induce random mutations), backcross breeding, and now genetic engineering. Any of these techniques may be advantageous independently or in combinations, depending on the specific goals of land managers or restoration practitioners, but genetic engineering offers a unique opportunity to enhance blight tolerance while minimizing other changes to the genome.

Transformation of American Chestnut (Castanea dentata (Marsh.) Borkh) Using RITA® Temporary Immersion Bioreactors and We Vitro Containers

American chestnut (Castanea dentata (Marsh.) Borkh) was almost completely wiped out by the fungal pathogen, Cryphonectria parasitica (Murrill) M.E. Barr. Another invasive pathogen, Phytophthora cinnamomi Rands, is devastating American chestnuts in the southern region of the United States. An alternative approach for controlling these pathogens is to use genetic engineering or gene editing. We successfully transformed American chestnut with a detoxifying enzyme, oxalate oxidase, to enhance blight tolerance and more recently with the Cast_Gnk2-like gene, which encodes for an antifungal protein, to be tested for P. cinnamomi putative tolerance. Eight somatic embryo lines were transformed using three methods of selection: semisolid medium in Petri plates, liquid medium in RITA® temporary immersion bioreactors, or liquid medium in We Vitro containers. No significant differences were found between the treatments. These methods will allow for further testing of transgenes and the development of enhanced pathogen resistance in chestnut. It can serve as a model for other tree species threatened by invasive pests and pathogens.

Combining ability of carrot genitors for root yield traits and leaf blight tolerance

The market demand for uniformity and productivity of commercial carrot roots has prioritized hybrid materials over open-pollinated varieties. In this sense, the objective of this work was to estimate the combining ability of carrot genitors for root productivity and resistance to leaf scorch. The experiments were conducted in Gama, DF, in the agricultural years 2012/13 and 2013/14. We evaluated 33 carrot hybrids, originated from crosses between three male-sterile populations, with 11 male-fertile S2 lines, all the genitors being of tropical origin. At 90 days after sowing, the severity of the leaf blight disease was estimated in the plots. At 100 days after sowing, harvesting was performed and root yield characters were evaluated. Analysis of variance and partial diallel analysis were performed for each year and jointly for both years. It was found that additive and non-additive genes are important in the manifestation of root yield and leaf blight resistance traits in carrot hybrids. The male-sterile parents with higher overall combining ability for root productivity are strains LM-649 and LM-650 and, among the male-fertile, strain LM-555-2-2. The best hybrids for root yield and leaf blight resistance are LM-649 × LM-555-11-1, LM-650 × LM-555-7-1 and LM-650 × LM-554-8-1.

A review on gene pyramiding of agronomic, biotic and abiotic traits in rice variety development

Rice Oryza sativa L is a staple food crop, and its seeds are the most important component part of the agronomic trait of the cereal crop, rich in nutrient and of economic value to human and even livestock. But, it is often threatened by various abiotic and biotic conditions that reduce the yield, because of high incidences of infectious disease agents and non-pathogenic conditions respectively. Pyramiding of the requisite resistance and tolerance genes into single elite high yielding variety of rice, confers wider spectrum of stress management, resulting to development of single multiline variety of rice. Marker-assisted selection utilizes DNA marker-linked primers for blast resistant gene (RM8225; Piz , RM6836; Piz , Pi2 , Pi9 ), bacteria leaf blight (RM224; Xa-4 , RM122; xa-5 , RG136; xa-13 , RM21; Xa-21 ) and drought tolerance (RM236; qDTY 2.2 , RM520; qDTY 3.1 , RM511; qDTY 12.1 ) in pedigree, backcross and recurrent selection breeding methods. The objectives are to create awareness on the environmental safety of host-resistance, significance of single multiline resistance variety, effect of the interaction of stress conditions and associated simple sequence repeat (SSR) linked markers.

Registration of ‘ND Riveland’ Durum Wheat

‘ND Riveland’ (Reg. no. CV‐1154, PI 687796) spring durum wheat [Triticum turgidum L. ssp. durum (Desf.) Husnot] was developed by the North Dakota Agricultural Experiment Station in cooperation with the USDA‐ARS and released on 5 July 2017. The original cross DT764/DH01146 was made in fall 2005. ND Riveland was developed using the pedigree breeding method and was bulked as an F4:5 line in summer 2009. ND Riveland was tested as D09690 in the Uniform Regional Durum Nursery and the North Dakota Durum State Variety Trials from 2013 to 2017. High grain yield potential, disease resistance, excellent end‐use qualities, and low cadmium uptake were the basis for ND Riveland's release. ND Riveland had higher yield than the predominant North Dakota cultivars ‘Divide’ and ‘Joppa’, as well as very strong dough mixing characteristics and a high gluten index. ND Riveland has improved Fusarium head blight tolerance compared with Divide and is resistant to predominant races of stem rust and the leaf rust.

Developing Blight-Tolerant American Chestnut Trees.

An invasive fungal pathogen has reduced the American chestnut (Castanea dentata), once a keystone tree species within its natural range in the eastern United States and Canada, to functional extinction. To help restore this important canopy tree, blight-tolerant American chestnut trees have been developed using an oxalate oxidase-encoding gene from wheat. This enzyme breaks down oxalate, which is produced by the pathogen and forms killing cankers. Expressing oxalate oxidase results in blight tolerance, where the tree and the fungus can coexist, which is a more evolutionarily stable relationship than direct pathogen resistance. Genetic engineering (GE) typically makes a very small change in the tree's genome, potentially avoiding incompatible gene interactions that have been detected in some chestnut hybrids. The GE American chestnut also retains all the wild American chestnut's alleles for habitat adaptation, which are important for a forest ecosystem restoration program.

Functional annotation and characterization of hypothetical protein involved in blister blight tolerance in tea (Camellia sinensis (L) O. Kuntze)

Tea [Camellia sinensis (L) O. Kuntze], worldwide known source of popular non-alcoholic beverage, is severely affected by various biotic and abiotic stresses. Among these, blister blight (BB) disease caused by the obligate biotrophic fungus Exobasidium vexan is responsible for significant depletion of its yield and quality. Our comparative NGS transcriptomic analysis although elucidated global gene expression pattern of BB defense transitions, yet left with 12,022 transcripts categorized as ‘hypothetical proteins (HPs)’. In this study, efforts were made for assigning functions to HPs derived from RNA-Seq data and successfully identified novel putative candidates involved in BB defense in tea. Domain and family-based characterization identified 9390 HPs representing 2867 protein families and 953 super families. Of these, 213 HPs were assigned with novel putative defense related functional categories (LRR, WRKY, NAC, chitinases and peroxidases). Further, sub-cellular localization (cytosolic,133 HPs; transmembranic, 80 HPs) with abundance of HPs exhibiting of acidic (133) and basic (80) nature suggests their wider functional range. 36 HPs upregulated in tolerant genotype having significant interactions with defense responsive candidates in Protein–Protein Interaction Network analysis, possibly suggests their key regulatory role in BB defense. Interestingly, 12 stereo-dynamically stable structures [LRR (5), NAC (4), WRKY, Chitinase & Peroxidase (1 each)] of HP’s were successfully modelled based on their conserved signature sequences and empirically validated using qRT-PCR analysis, therefore, can be potential novel candidates possibly involved in signal transduction and pathogen recognition during BB defense in tea. Futuristically, novel genes identified in this study can be potentially utilized to expedite genetic improvement efforts in tea. The approach successfully employed in tea can also be adopted for assigning molecular function to the HPs in other plant species.

Effects of transgenic American chestnut leaf litter on growth and survival of wood frog larvae

Biotechnology offers a new approach for the restoration of tree species affected by exotic pathogens; however, nontarget impacts of this novel strategy on other organisms have not been comprehensively assessed. We evaluated the effect of transgenic American chestnut (Castanea dentata) leaf litter on the growth and survival of larval wood frogs (Lithobates sylvaticus), a forest‐dwelling amphibian species widely sympatric with American chestnut, that forage almost entirely on periphyton and litter detritus that accumulate in temporary vernal pools in forests. We reared wood frog larvae on Castanea leaf litter (American chestnut genetically engineered for blight tolerance, nontransgenic American chestnut, Chinese chestnut [Castanea mollissima], and an American–Chinese chestnut hybrid) and litter from two non‐Castanea, nontransgenic “control” tree species, coupled with two levels of supplementary food. We observed no differences in growth or survival of wood frog larvae reared on transgenic versus nontransgenic American chestnut leaves. Without supplementary food, wood frog larvae provided leaves from American chestnut (both types) developed faster and grew larger than those exposed to other leaf litter treatments. Results of this study provide preliminary evidence that (1) American chestnut may have formerly been an important source of food for forest‐dwelling amphibians and (2) transgenic American chestnut litter generated as part of chestnut restoration efforts is unlikely to present direct novel risks to developing amphibian larvae in the forest environment.

Diversity analysis by SSR markers and morphological markers among Stemphylium blight tolerance genotypes with high yielding cultivars of Lentil (Lens Culinaris Medik)

Twelve Simple Sequence Repeat (SSR) markers along with twelve morphological markers were used to assess the diversity among thirteen screened genotypes both moderate resistant and susceptible against stemphylium blight of indigenous microsperma germplasm (i.e. pilosae) type of Lentil (Lens culinaris Medik.) at BCKV, West Bengal. Polymorphic Information Content (PIC) of SSR primers ranged from 0.77 (SSR 317-2) to 0.15 (PBA LC 0233). The maximum Jaccard’s similarity was recorded between L-4769 and Ranjan (approximately 100%) whereas minimum was between K-75 and VL 126 (0.11). Genotypes K-75 also showed less similarity with DL 14-2 and VL 151 (0.17). Another popular variety WBL 77 showed minimum similarity with moderately resistant genotypes VL 151 and DL 14-2 (0.17). While using morphological characters as the basis of diversity analysis, Euclidean coefficient revealed that high yielding cultivar Subrata was maximum diverse with genotype VL 151(7.12). Asha also showed diversity with VL 151(6.31) whereas VL 151 had dissimilarity coefficient of 6.21 with K-75. Precoz and VL 126 has dissimilarity coefficient of 6.19. On the basis of above findings it could be said that diverse parental pairs VL 151 and K 75 and VL 151 and Subrata / Asha could be used in hybridization programme or biotic stress breeding programme for developing Stemphylium resistant lentil cultivars in future.

Identification of quantitative trait loci (QTL) associated with sheath blight tolerance in rice

Sheath blight, caused by the pathogen Rhizoctonia solani Kuhn, is one of the most serious diseases of rice and leads to severe yield losses worldwide. A recombinant inbred line (RIL) population consisting of 122 lines was constructed from a cross between Danteshwari a high yielding popular rice cultivar but moderately susceptible to water stress, susceptible to sheath blight and Dagad Deshi a tall deep rooted poor yielder and tolerant to water stress and sheath blight. Five traits, namely infected tillers per hill, lesion size (total coalescing lesions) and individual lesion (one spot) and lesion's length and width) were used to evaluate sheath blight resistance. Using the RIL population and 162 molecular markers, 11 quantitative trait loci (QTLs) were identified for the five traits. Identification of map position was accomplished by identifying BAC or PAC clones that simultaneously contained a hit from the microsatellite/HvSSR marker further helped us to generate a high resolution molecular marker map of the identified QTL region. Identified QTLs were located on seven chromosomes. A major QTL for infected tillers per hill on chromosome 1 was identified with phenotypic variance of 18.02%. Six QTL's with positive additive effect and one with negative additive effects was identified indicating alleles at these loci are being contributed by either of the parents respectively.

Participatory Variety Selection: A Tool to Understand Farmers′ Potato Variety Selection Criteria

Abstract Although the Ethiopian potato program has released more than 30 new varieties, selected primarily for yield potential and late blight tolerance, the majority of farmers still choose to grow older ‘local varieties’. Cognizant of this, the current study aimed to identify traits that farmers consider most important when deciding which varieties to grow. The study was conducted at two sites in northwest Ethiopia, representing two major agroecological zones, in two distinct growing seasons, using a participatory variety selection approach. Diseasefree seed tubers of nine local and three new varieties were planted. Twenty-three traits were found to influence the varieties that farmers selected and seven of them differed between agroecological zones. Gender differences were also observed for three of the 23 traits at Laigaint (submoist cool agroecology). Of the 23 traits, 12 were considered very important in one or both of the agroecological zones as well as in at least one growing season. At present eight of these 12 traits are not given high priority by the Ethiopian potato variety selection program. The results show that farmers′ selection criteria are very diverse, that no variety possesses all desirable characteristics, and that there is a need for selecting varieties adapted to different agroecologies and growing seasons. This study further suggests that taking farmer interests into account is important for a highly heterozygous, clonally-propagated crop like potato to be accepted by farmers.

Dual gene expression cassette is superior than single gene cassette for enhancing sheath blight tolerance in transgenic rice

Sheath blight, caused by the necrotrophic fungal pathogen Rhizoctonia solani, is a serious and destructive disease of the rice. In order to improve sheath blight resistance, we developed three different kinds of transgenic rice lines. The first transgenic line overexpresses the rice chitinase gene (OsCHI11); the second contains the Arabidopsis NPR1 (AtNPR1) gene and, the third has pyramided constructs with both the genes (OsCHI11 and AtNPR1). This is a comparative study between the single-gene transgenic lines and the double gene transgenic in terms of their ability to activate the plant defense system. Rice plants of each individual construct were screened via PCR, Southern hybridization, activity assays, and expression analysis. The best transgenic lines of each construct were chosen for comparative study. The fold change in qRT-PCR and activity assays revealed that the pyramided transgenic rice plants show a significant upregulation of defense-related genes, PR genes, and antioxidant marker genes as compared to the single transgene. Simultaneous co-expression of both the genes was found to be more efficient in tolerating oxidative stress. In R. solani (RS) toxin assay, mycelial agar disc bioassay, and in vivo plant bioassay, pyramided transgenic plant lines were more competent at restricting the pathogen development and enhancing sheath blight tolerance as compared to single gene transformants.

Enhancement of sheath blight tolerance in transgenic rice by combined expression of tobacco osmotin (ap24) and rice chitinase (chi11) genes

Agrobacterium-mediated transformation was used to introduce pathogenesis-related protein genes into scutellum-derived callus of rice (Oryza sativa L. subsp. indica ‘White Ponni’) as a means of increasing resistance to sheath blight disease caused by Rhizoctonia solani Kuhn. Transformation with a tobacco osmotin (ap24) gene driven by the Cauliflower mosaic virus 35S promoter (P35S) yielded six single-copy transgenic lines. Homozygous T2 plants of all lines accumulated high levels of the ap24 transcript and the osmotin protein and reduced the percent disease index (PDI) of sheath blight disease from 100% down to 49–77%. Transformation with the binary plasmid pNSP2, which harbored the P35S-ap24 gene and a maize ubiquitin promoter (PUbi1)-driven rice chitinase (chi11) gene in the same T-DNA, yielded three single-copy transgenic lines: CO1, CO2, and CO3. Homozygous T2 plants of all three lines accumulated high levels of the chi11 transcript and the chitinase protein. Accumulation of ap24 transcript and osmotin protein was high in homozygous CO1 and CO2, but very low in CO3. At 7 d post infection with R. solani, the transgenic rice lines C18a (with chi11 alone), O8 (with ap24 alone), and CO1, CO2, and CO3 (with chi11 + ap24) reduced the sheath blight PDI from 100% to 64.2, 57.2, 43.3, 50.2, and 58.6%, respectively. At 15 d post infection, the sheath blight disease PDI was significantly lower in the CO1 and CO2 transgenic rice lines, which expressed both chi11 and ap24, than in those which expressed either chi11 or ap24 individually.

Introgression of sheath blight disease tolerance from the transgenic rice event Pusa Basmati1-CG27 to the variety White Ponni through backcross breeding

Sheath blight disease caused by Rhizoctonia solani is a major constraint for rice production. In order to obtain transgenic resistance against sheath blight disease, an indica rice variety Pusa Basmati1 (PB1) was transformed with constitutively express chitinase (chi11) and β-1,3-glucanase (GLU) genes. A single-copy transgenic PB1-CG27 showed a high level of tolerance to R. solani. Here, we report the introgression of sheath blight tolerance from PB1-CG27 into a non-basmati rice variety, White Ponni (WP) through backcross breeding. In each of the four backcross generations, the presence of the transgene was checked by hygromycin phosphotrans-ferase (hph) and βglucuronidase (gus) gene-specific foreground selection markers present in the T-DNA of the PB1-CG27 event. RAPD marker-based background selection was done to choose a BC4F1 plant that was similar to the WP recurrent parent. Morphological characters of the introgressed BC4F4 plants matched those of the recurrent WP parent. RT-PCR and western blot analyses confirmed the constitutive expression of the chi11 and GLU genes in the introgressed homozygous BC4F3 plants. Bioassay with R. solani a high level disease tolerance (43% disease reduction) revealed over the control WP plants.

Capacidade de combinação de genitores de cenoura para caracteres de produtividade de raízes e tolerância à queima-das-folhas

RESUMO A demanda de mercado por uniformidade e produtividade de raízes comerciais de cenoura tem priorizado materiais híbridos em detrimento às variedades de polinização aberta. Neste sentido, objetivou-se com este trabalho estimar as capacidades de combinação de genitores de cenoura para os caracteres produtividade de raiz e resistência à queima-das-folhas. Os experimentos foram realizados no Gama, DF, nos anos agrícolas de 2012/13 e 2013/14. Foram avaliados 33 híbridos de cenoura, originados de cruzamentos entre três populações macho-estéreis, com 11 linhagens S2 macho-férteis, sendo todos os genitores de origem tropical. Aos 90 dias após a semeadura, foi estimada a severidade da doença queima-das-folhas, nas parcelas. Aos 100 dias após a semeadura, foi realizada a colheita e foram avaliados os caracteres de produtividade de raízes. Foram realizadas análises de variância e análise dialélica parcial, para cada ano e, conjunta, para os dois anos. Verificou-se que genes de efeitos aditivos e não aditivos são importantes na manifestação dos caracteres de produtividade de raízes e de resistência à queima-das-folhas, em híbridos de cenoura. Os genitores macho-estéreis com maior capacidade geral de combinação para produtividade de raízes são as linhagens LM-649 e LM-650 e, dentre os macho-férteis, a linhagem LM-555-2-2. Os melhores híbridos para produtividade de raízes e para resistência à queima-das-folhas são LM-649 × LM-555-11-1, LM-650 × LM-555-7-1 e LM-650 × LM-554-8-1.