Tomato Genetics Research Articles

Genes, Genetics and Breeding of Tomato

Tomato (Solanum lycopersicum) is widely cultivated and is one of the most important vegetable crops in the world, with great economic significance [...]

Study of Combining Ability Analysis in Cherry Tomato (Solanum lycopersicum var. cerasiforme)

The study aimed to show the best effect of the general combining ability (GCA) and specific combining ability (SCA) for parents and their hybrids of the studied traits. The study included two main successive seasons. In the first season, cherry tomato pure lines were selected that were introduced from the Tomato Genetics Resource Center (TGRC) at University of California, Davis in USA, 5 pure lines were selected which were the most genetically Distant which are (LA4451, LA4753, LA3334, LA1221 and LA4689) and its code (1, 2, 3, 4 and 5) respectively and entered into Full Diallel crossing program to produce individual crosses. The second season included an experiment to compare genotypes (5 parents + 20 cross-hybrids. and its reciprocal hybrids) according to RCBD design with three replications. The results were as follows: parents 1 showed a general significant ability to combine in the desired direction in terms of fruit set percentage and fruit weight, while the superiority of parent 4 in terms of plant yield and total yield. The parent 5 was superior in the number of flowers in the inflorescence, the number of total flowers of the plant and the number of fruits, whereas the hybrid (5×1) showed superiority in the number of flowers in the inflorescence 35.667, number of total flowers 259.66, the percentage of the set 15.69 and the number of fruits 86.83. (1×4) appeared significant effect of (SCA) were shown significant and desirable in the traits average fruit weight 1.47, plant yield 0.55 and total yield 0.69.

BIOLOGICALLY ACTIVE COMPOUNDS TRANSFORM DURING THE RIPENING STAGES IN GREENHOUSE TOMATOES

Currently, the selection of tomatoes with a high content of biologically active substances and antioxidant properties at the large green and breaker stage is relevant since mature tomatoes cannot tolerate storage and transportation. For this purpose, 11 tomato genotypes, chosen in a preliminary study in 2018–2020 from the Genetic Collection of Plant Resources of Federal State Budgetary Scientific Institution Federal Scientific Vegetable Center (FSBSI FSVC), Moscow, Russia, and Tomato Genetics Resource Center (TGRC), the University of California, Davis, USA, for further studies during 2020–2022 for antioxidant pool changes: measuring the contents of chlorophyll, lycopene, β-carotene, ascorbic acid, and lutein. The experiment arranged in a randomized complete block design proceeded in the film unheated greenhouses. The results showed five promising tomato genotypes, i.e., VFN Hi Sugar, VS-420, Paul Robeson, Black Cherry, and VS-410. The genotype Black Cherry fruits with breaker ripeness contained 42% lycopene and 93% β-carotene, while the genotype Paul Robeson at the same stage contained 80% β-carotene. The three other tomato genotypes, viz., VFN Hi Sugar, VS-420, and Paul Robeson, also gave a higher content of ascorbic acid in the fruits at the breaker ripening stage.

A Genetic Map and Linkage Panel for the Large-fruited Fresh-market Tomato

Genetic maps saturated with genetic markers are useful for genetic research and crop breeding; however, the genetic map for the large-fruited fresh-market tomato (Solanum lycopersicum) has never been constructed, and the recombination frequency between DNA fragments is only partly understood for fresh-market tomato. We constructed a novel fresh-market tomato genetic map by using 3614 single nucleotide polymorphism (SNP) markers and a 93 F2 segregating progeny derived from a cross between two United States large-fruited fresh-market tomato lines. The average distance between markers was less than 1 cM, and substantial recombination densities between markers were observed across the approximate centromere locations. A linkage panel for large-fruited fresh-market tomato was also established using the combined dataset of the genetic map and 58 SNP-genotyped core tomato lines. The allelic information in the linkage panel will be a significant resource for both tomato genetics and future breeding approaches.

Estimating genotype-environment interactions for internal fruit quality traits in cherry tomatoes

Genotype-environment interactions (GEI) were assessed in 10 cherry tomato accessions in nine environments, including four artificial settings (0, 60 120, and 180 kg ha-1 of potassium) established on the experimental farms Montelindo (Palestina), Tesorito (Manizales), and CEUNP (Palmira) (Colombia). The plant material included 10 cherry tomato genotypes obtained from the germplasm bank at the Instituto Agronómico de Campinas and Tomato Genetics Resources Center (TGRC). A completely randomized block design with four blocks corresponding to the level of potassium fertilization was used (0, 60, 120, 180 kg ha-1); 0 kg ha-1 was the level reported for the soil. The effective size of the experiment unit was seven plants; the plot included the five central plants. A distance of 1.5 m between rows, 0.50 m between plants, and 2 m between blocks was used. The contents of soluble solids (°Brix), vitamin C (mg/100 g fresh weight), and lycopene (µg g-1 fresh weight) were assessed. The analysis of variance (ANOVA) showed significant differences (P<0.01) between the tomato genotypes, environments, and G×E interactions for the three assessed traits . The AMMI analysis identified similar and contrasting environments and determined the genotypes that contributed the most to the GEI. The environments with 120 and 180 kg ha-1 potassium favored the expression of vitamin C, while Palmira favored the lycopene content. The findings are useful for identifying optimal locations and elite genotypes that can be used as sources of variability in fruit quality improvement programs for cherry tomatoes.

High-Throughput Phenotyping Approach for Screening Major Carotenoids of Tomato by Handheld Raman Spectroscopy Using Chemometric Methods.

Our objective was to develop a rapid technique for the non-invasive profiling and quantification of major tomato carotenoids using handheld Raman spectroscopy combined with pattern recognition techniques. A total of 106 samples with varying carotenoid profiles were provided by the Ohio State University Tomato Breeding and Genetics program and Lipman Family Farms (Naples, FL, USA). Non-destructive measurement from the surface of tomatoes was performed by a handheld Raman spectrometer equipped with a 1064 nm excitation laser, and data analysis was performed using soft independent modelling of class analogy (SIMCA)), artificial neural network (ANN), and partial least squares regression (PLSR) for classification and quantification purposes. High-performance liquid chromatography (HPLC) and UV/visible spectrophotometry were used for profiling and quantification of major carotenoids. Seven groups were identified based on their carotenoid profile, and supervised classification by SIMCA and ANN clustered samples with 93% and 100% accuracy based on a validation test data, respectively. All-trans-lycopene and β-carotene levels were measured with a UV-visible spectrophotometer, and prediction models were developed using PLSR and ANN. Regression models developed with Raman spectra provided excellent prediction performance by ANN (rpre = 0.9, SEP = 1.1 mg/100 g) and PLSR (rpre = 0.87, SEP = 2.4 mg/100 g) for non-invasive determination of all-trans-lycopene in fruits. Although the number of samples were limited for β-carotene quantification, PLSR modeling showed promising results (rcv = 0.99, SECV = 0.28 mg/100 g). Non-destructive evaluation of tomato carotenoids can be useful for tomato breeders as a simple and rapid tool for developing new varieties with novel profiles and for separating orange varieties with distinct carotenoids (high in β-carotene and high in cis-lycopene).

Tomato proteomics: Tomato as a model for crop proteomics

The understanding of plant stress response is essential to develop crops that are capable of withstanding adverse conditions. The development of proteomics led to the characterization of many of the metabolic pathways involved in plant resistance and adaptation to abiotic stresses. Mass spectrometry has been a popular tool for the study of plant protein expression under special environmental conditions due to its high throughput capacity and sensitivity. Recent studies have applied proteomics methodologies, such as phosphoproteomics, to understand metabolic dynamics and regulations. Isobaric tags, such as iTRAQ, have been used to obtain more precise and less time-consuming quantitative analysis. Although these proteomic strategies have been successfully applied to studies with tomato (Solanum lycopersicum), a major challenge of crop proteomics is the lack of genetic functional information when compared to the model plant, Arabidopsis thaliana. Still, tomato has been the model crop for genetic and molecular research of the Solanaceae family due to its attributes of diploidy, easy genetic transformation, and many genetic resources. The necessity of better strategies to increase the genetics and proteomics resources of tomato is in high demand. Here, we explore the various proteomics methodologies used in studies on tomato plants, and we discuss the present challenges of crop proteomics data interpretation.

BIFURCATE FLOWER TRUSS: a novel locus controlling inflorescence branching in tomato contains a defective MAP kinase gene.

A mutant line, bifurcate flower truss (bif), was recovered from a tomato genetics programme. Plants from the control line produced a mean of 0.16 branches per truss, whereas the value for bif plants was 4.1. This increase in branching was accompanied by a 3.3-fold increase in flower number and showed a significant interaction with exposure to low temperature during truss development. The control line and bif genomes were resequenced and the bif gene was mapped to a 2.01 Mbp interval on chromosome 12; all coding region polymorphisms in the interval were surveyed, and five candidate genes displaying altered protein sequences were detected. One of these genes, SlMAPK1, encoding a mitogen-activated protein (MAP) kinase, contained a leucine to stop codon mutation predicted to disrupt kinase function. SlMAPK1 is an excellent candidate for bif because knock-out mutations of an Arabidopsis orthologue MPK6 were reported to have increased flower number. An introgression browser was used to demonstrate that the origin of the bif genomic DNA at the BIF locus was Solanum galapagense and that the SlMAPK1 null mutant is a naturally occurring allele widespread only on the Galápagos Islands. This work strongly implicates SlMAPK1 as part of the network of genes controlling inflorescence branching in tomato.

CRISPR: A Technical Breakthrough for Tomato Research

CRISPR is a contemporary biological technology that has attracted the interest of both the science community and the general public. This 4-page article discusses why tomato researchers are interested in CRISPR, what tomato traits have been targeted by CRISPR, and potential applications of CRISPR for tomato genetics and breeding. Written by Tong Geon Lee and published by the UF/IFAS Department of Horticultural Sciences, February 2018. http://edis.ifas.ufl.edu/hs1314

Review on Genetics and Breeding of Tomato (Lycopersicon esculentum Mill)

Tomato is considered as a member of the family Solanaceae. The botanical name of tomato is Lycopersicon esculentum Mill and is a diploid plant with 2n=2x=24 chromosomes. Great advances in tomato genetics have been achievable because of the understanding of mating systems and the possibility of controlled hybridization within and among species, the naturally occurring variability in the species, the occurrence of self-pollination that leads to the expression of recessive mutations, the lack of gene duplication, and the possibility to easily identify the 12 chromosomes. New methodological approaches like molecular mapping of important agronomical characters and the development of advanced-backcross and introgression lines have provided powerful tools for the improvement of the tomato crop and to understand the processes of domestication. The general breeding goals of tomato are fruit yield, fruit quality, and resistance to diseases and pests. Tomatoes are presented as a classical example for gene transfer from wild species into cultivated cultivars for improvement the qualitative traits. It was established that until now the achievements in tomato breeding are based on classical breeding-genetic methods and also, an essential change in the accelerated introduction of useful traits in the cultivars is not made. It is believable that conventional breeding wouldn’t allow the increase of productivity in the future. The significant progress in molecular genetics and use of molecular marker techniques are established. Therefore, the combined application of traditional breeding and contemporary plant biotechnology methods including selection based on molecular markers marker-Assisted Selection might be valuable tools for tomato breeding.

Selection of Reference Genes for Quantitative Real-Time RT-PCR Studies in Tomato Fruit of the Genotype MT-Rg1.

Quantitative real-time RT-PCR (qRT-PCR) has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed, and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 and APETALA2c during fruit development are comparable to previous reports using other tomato cultivars.

ROOTSTOCK-MEDIATED VARIATION IN TOMATO VEGETATIVE GROWTH UNDER LOW POTASSIUM OR PHOSPHOROUS SUPPLIES

Grafting provides a rapid and direct tool to transfer rootstock-mediated abiotic stress tolerance to commercial stress-sensitive cultivars. Nutrient deficiency is a widespread problem in some soils, inhibiting physiological processes and thus reducing crop yield. With the aim of selecting rootstocks conferring tolerance to potassium (K+) or phosphorous (P) deficits, a commercial tomato cultivar (‘Boludo F1’, ‘Monsanto’) was grafted onto 144 different rootstocks: six accessions from S. lycopersicum (‘Cerasiforme’) and S. pimpinellifolium, selected for drought tolerance (sourced from The World Vegetable Center, AVRDC); nine introgression lines from S. lycopersicum A— S. pennellii and A— S. habrochaites, selected for high root/shoot ratio, salinity and drought tolerances (sourced from The Tomato Genetics Resource Center, TGRC); and a population of 129 recombinant inbred lines (RILs) derived from a salt sensitive genotype of S. lycopersicum var. cerasiforme and a salt tolerant line from S. pimpinellifolium L. (sourced from Instituto Valenciano de Investigaciones Agrarias, IVIA). Plants were grown in semi-hydroponics or soil for 4-6 weeks (vegetative stage), under low K+ (1 mM) or low P (0.1 mM) supply. Shoot fresh weight was measured at the end of each experiment. There was a large rootstock-mediated variation in shoot biomass under each low nutrient condition. Some specific lines used as rootstocks performed better under low nutrient conditions than the self-grafted commercial cultivar ‘Boludo F1’. Furthermore, although some rootstocks seemed to perform well under different limiting conditions, no relation was found in shoot fresh weight for the two low nutrient conditions studied, suggesting that the mechanisms that control vegetative vigour seem to work differently depending on the specific nutrient deficiency. Results from this study will help in the future to identify genetic and physiological determinants that allow tomato roots to confer crop resistance to nutrient limitation as well as to improve fruit yield under individual and combined stresses.

Potential of a tomato MAGIC population to decipher the genetic control of quantitative traits and detect causal variants in the resequencing era.

Identification of the polymorphisms controlling quantitative traits remains a challenge for plant geneticists. Multiparent advanced generation intercross (MAGIC) populations offer an alternative to traditional linkage or association mapping populations by increasing the precision of quantitative trait loci (QTL) mapping. Here, we present the first tomato MAGIC population and highlight its potential for the valorization of intraspecific variation, QTL mapping and causal polymorphism identification. The population was developed by crossing eight founder lines, selected to include a wide range of genetic diversity, whose genomes have been previously resequenced. We selected 1536 SNPs among the 4 million available to enhance haplotype prediction and recombination detection in the population. The linkage map obtained showed an 87% increase in recombination frequencies compared to biparental populations. The prediction of the haplotype origin was possible for 89% of the MAGIC line genomes, allowing QTL detection at the haplotype level. We grew the population in two greenhouse trials and detected QTLs for fruit weight. We mapped three stable QTLs and six specific of a location. Finally, we showed the potential of the MAGIC population when coupled with whole genome sequencing of founder lines to detect candidate SNPs underlying the QTLs. For a previously cloned QTL on chromosome 3, we used the predicted allelic effect of each founder and their genome sequences to select putative causal polymorphisms in the supporting interval. The number of candidate polymorphisms was reduced from 12284 (in 800 genes) to 96 (in 54 genes), including the actual causal polymorphism. This population represents a new permanent resource for the tomato genetics community.

Marker-Assisted Selection in Tomato Breeding

The cultivated tomato, Solanum lycopersicum L., is the second most consumed vegetable crop after potato and unquestionably the most popular garden crop in the world. There are more varieties of tomato sold worldwide than any other vegetable crop. Most of the commercial cultivars of tomato have been developed through phenotypic selection and traditional breeding. However, with the advent of molecular markers and marker-assisted selection (MAS) technology, tomato genetics and breeding research has entered into a new and exciting era. Molecular markers have been used extensively for genetic mapping as well as identification and characterization of genes and QTLs for many agriculturally important traits in tomato, including disease and insect resistance, abiotic stress tolerance, and flower- and fruit-related characteristics. The technology also has been utilized for marker-assisted breeding for several economically important traits, in particular disease resistance. However, the extent to which MAS has been employed in public and private tomato breeding programs has not been clearly determined. The objectives of this study were to review the publically-available molecular markers for major disease resistance traits in tomato and assess their current and potential use in public and private tomato breeding programs. A review of the literature indicated that although markers have been identified for most disease resistance traits in tomato, not all of them have been verified or are readily applicable in breeding programs. For example, many markers are not validated across tomato genotypes or are not polymorphic within tomato breeding populations, thus greatly reducing their utility in crop improvement programs. However, there seems to be a considerable use of markers, particularly in the private sector, for various purposes, including testing hybrid purity, screening breeding populations for disease resistance, and marker assisted backcross breeding. Here we provide a summary of molecular markers available for major disease resistance traits in tomato and discuss their actual use in tomato breeding programs. It appears that many of the available markers may need to be further refined or examined for trait association and presence of polymorphism in breeding populations. However, with the recent advances in tomato genome and transcriptome sequencing, it is becoming increasingly possible to develop new and more informative PCR-based markers, including single nucleotide polymorphisms (SNPs), to further facilitate the use of markers in tomato breeding. It is also expected that more markers will become available via the emerging technology of genotyping by sequencing (GBS).

Single Nucleotide Polymorphism Discovery in Cultivated Tomato via Sequencing by Synthesis

Plant breeding is enhanced by the availability of molecular markers for rapid screening and selection in populations. Identifi cation of polymorphic loci in cultivated tomato (Solanum lycopersicum L.) has been hampered by limited genome sampling across cultivated types. Whole transcriptome sequencing of six accessions that span cultivated market classes was performed using sequencing by synthesis. A total of 291,915,037 quality fi ltered reads representing 17 Gb of sequence were generated. Assembly of the reads resulted in 30.6 to 34.9 Mb of sequence for each of the six accessions and provided representation of 55.3 to 59.6% of the predicted tomato gene set with a wide range of molecular function Gene Ontologies (GOs) represented. A computational pipeline was developed to identify single nucleotide polymorphisms (SNPs). When coupled with two Sanger-derived expressed sequence tag datasets and a reference genome, 62,576 nonredundant putative SNPs in tomato were identifi ed. The SNPs within the contigs were present within all of the GO molecular function categories. The computational pipeline had validation rates in SNP genotyping assays that ranged from 95 to 100%, and the utility of these SNPs for assessing genetic variation within cultivated and wild populations was demonstrated. Collectively, the transcript sequences and the annotated SNPs provide a resource to facilitate tomato genetics and breeding efforts.

The Rg1 allele as a valuable tool for genetic transformation of the tomato 'Micro-Tom' model system.

BackgroundThe cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps.ResultsHere we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 μM 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 μM 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization.ConclusionsThe genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.

On the Tomato Trail: In Search of Ancestral Roots

A profile of Roger Chetelat, the director of the C.M. Rick Tomato Genetics Resource Center at the University of California, Davis. Chetelat maintains one of the largest collections of tomato seeds in the world. Many of those seeds come from wild tomato species that Chetelat and his associates collect on field research trips to the dry coastal areas of Chile, Peru, and Ecuador. Wild tomatoes are tough, versatile organisms that have evolved resistance to virtually all common tomato diseases and pests and stubbornly tolerate extreme environmental conditions. Some boast extraordinarily high levels of sugars, beta carotene, vitamin C, lycopene, and antioxidants. Chetelat has dedicated his career to finding and preserving these genetic riches. Modern cultivated tomatoes are a frail, inbred lot. They all trace their origins to a single, wild tomato plant that underwent a random mutation sometime in prehistory. Because of this genetic fluke, that plant's fruits were plump, juicy, and many, many times larger than the output of its progenitors. Offspring from that tomato were taken away from the Andes and domesticated in what is present-day Mexico, becoming severed from their wild ancestors and the vast pool of genetic diversity that tomatoes had evolved over the millennia. Botanists call this a “bottleneck.” It leaves subsequent generations susceptible to disease and unable to adjust to rapid climate changes. The stored wild seeds at the Rick Center enable plant breeders to re-incorporate desirable wild traits into new tomato varieties, literally reconnecting them to their ancestral roots, ensuring that this vast reservoir of genetic diversity will be available when it is needed.

BREEDING THE TOMATO MICRO-TOM MODEL SYSTEM FOR ORNAMENTAL VALUE

BREEDING THE TOMATO MICRO-TOM MODEL SYSTEM FOR ORNAMENTAL VALUE

Structural changes, chemical composition and antioxidant activity of cherry tomato fruits (cv. Micro‐Tom) stored under optimal and chilling conditions

AbstractBACKGROUND:Cherry tomato fruits cv. Micro‐Tom, a model plant for tomato genetics, were analysed in order to determine the main structural and chemical changes under optimal and chilling storage conditions. The comparison of Micro‐Tom to standard tomato cultivars will give an insight into suitability of this dwarf cultivar as a model for studying the influence of low‐temperature conditions on tomato fruits.RESULTS:During chilling, fruit tissue was progressively destroyed due to the collapse of the deep layers of the pericarp. Chilling lowered the typical tomato kinetics of ripening in sugars (glucose, fructose, sucrose), organic acids (tartaric, malic, citric, ascorbic and succinic) and the antioxidants phenol and lycopene, while carotenoid synthesis seemed to be blocked. Glutathione level was elevated and the activity of ROS scavenging enzymes was altered. Essentially, Micro‐Tom fruits showed a quality evolution that was similar to that described for standard tomato cultivars.CONCLUSION:The cherry tomato cultivar Micro‐Tom could be used as a model for studying the influence of low temperature on biochemical and structural changes taking place during chilling injury conditions. Copyright © 2009 Society of Chemical Industry

Phenotypic and genetic characterization of the pistillate mutation in tomato

Many floral phenotypes have been described in decades of tomato genetics, but for very few of them the underlying genes have been identified so far. Because the increasing availability of genome sequence data will facilitate forward genetics in tomato, novel descriptive and map information will help the attribution of genes to phenotypes. In this contribution, we present our work on pistillate (pi), a genotype that directly recalls mutations affecting class B MADS-box genes, but that has not been further characterized after the first description. Plants homozygous for the pi allele appear with Mendelian proportions and, compared to wild-type, show delayed flowering, a frequently modified sympodial segment, higher occurrence of compound inflorescences, and reversion of the floral meristem to vegetative identity. In pi mutant flowers, the most striking aberration is the homeotic transformation of stamens into carpels. Ultrastructural analysis also reveals more or less subtle sepaloid features in the three inner floral whorls, mainly based on the presence, distribution and amount of glandular and non glandular trichomes. In the ovary, a 'flower within flower' phenotype was seldom observed; in one instance such phenotype was coupled with the setting of a parthenocarpic fruit, that reiterated the differentiation of a new flower. Mapping experiments positioned PI on the distal end of the long arm of chromosome 3. This position was not compatible with any class B or E MADS box gene; differently, the PI genetic window contained the FALSIFLORA (FA) gene, the tomato orthologue of LEAFY (LFY). The pi defects in flowering time and inflorescence development are in agreement with a direct involvement of the floral meristem identity gene. The class B- and E-like phenotypes shown by pi mutant plants are likely an indirect consequence because FA, as LFY, is reported as a positive regulator of homeotic MADS-box genes. Because fa mutant plants do not form complete flowers, the pi mutation deserves a particular interest, producing four-whorled, although modified, flowers useful to study the functional linkage between flower induction and flower organ identity specification.