Search

Colletotrichum acutatum spp. infects various economical crops worldwide and causes massive loss on their yields. Among those, Capsicum spp., which known as chili pepper, is on a critical issue by those pathogens. Due to the lack of their genetic markers in Korea, the unidentifiable various species of C. acutatum obstructs the mechanism studies of these pathogens and the selection of disease resistant breed lines. Therefore, we screened RGB images of the colonization progresses of pathogens to identify the species of Ca40042, K1, NN, AS2, and SW1 by time and temperature. Cultivated pathogens such as Ca40042, K1, and SW1 were detectable on quantified shape and color data of images from specific temperature conditions, while other pathogens were difficult to recognize. Although several limitations exist in identification results of current experiment, but also, we can expect this method can suggest the possibility to replace the genetic marker methods which is now unavailable in Korea.

A new small-type pink-colored Phalaenopsis cultivar, ‘Tiny Bell’, was released by the National Institute of Horticultural and Herbal Science, Rural Development Administration (Korea) in 2018. ‘Tiny Bell’ was derived in 2010 from crossing P. ‘D07PN16’, multi-floral small-type cultivar with pink-colored flowers, and P. ‘D03PN22’, a small-type cultivar with light pink-colored flowers. In the preliminary selection stage in 2014, the cultivar known as ‘10531-53’ was chosen among 120 individual progenies, based on phenotypic standards such as flower color and shape, inflorescence number, and leaf attitude. The stability and uniformity of the cultivar was confirmed through the first and second characteristics tests from 2014 to 2018, and the cultivar was thereafter named ‘Tiny Bell’. ‘Tiny Bell’ has violet pink (RHS, PVGN80A) flowers with a white edge. The dimensions of the f lowers are 3.89 cm ( length) and 4.01 cm (width). T he i nf ol rescence type is a r aceme with m any flowers (19.7 blooms per inflorescence). The leaves of ‘Tiny Bell’ grow horizontally and are 13.1 cm in length and 5.26 cm in width. This new cultivar of Phalaenopsis ‘Tiny Bell’ is well suited as a small-sized potted orchid.

Pre-harvest sprouting (PHS) leads to serious economic losses because of reductions in yield and quality. To analyze the quantitative trait loci (QTLs) for PHS resistance in japonica rice, PHS rates on panicles were measured in 160 recombinant inbred lines (RILs) derived from a cross between the temperate japonica varieties Odae (PHS resistant) and Unbong40 (PHS susceptible) under two different environmental conditions—field (summer) and greenhouse (winter) environments. Genome re-sequencing of the parental varieties detected 266,773 DNA polymorphisms including 248,255 single nucleotide polymorphisms and 18,518 insertions/deletions. We constructed a genetic map comprising 239 kompetitive allele-specific PCR and 49 cleaved amplified polymorphic sequence markers. In the field environment, two major QTLs, qPHS-3FD and qPHS-11FD, were identified on chromosomes 3 and 11, respectively, whereas three major QTLs, qPHS-3GH, qPHS-4GH, and qPHS-11GH, were identified on chromosomes 3, 4, and 11, respectively, in the greenhouse environment. qPHS-11GH and qPHS-11FD had similar locations on chromosome 11, suggesting the existence of a gene conferring stable PHS resistance effects under different environmental conditions. The QTLs identified in this study can be used to improve the PHS resistance of japonica varieties, and they may improve our understanding of the genetic basis of PHS resistance.

Anthracnose is caused by Colletotrichum species and is one of the most virulent fungal diseases affecting chili pepper (Capsicum) yield globally. However, the noble genes conferring resistance to Colletotrichum species remain largely elusive. In this study, we identified CbAR9 as the causal locus underlying the large effect quantitative trait locus CcR9 from the anthracnose-resistant chili pepper variety PBC80. CbAR9 encodes a nucleotide-binding and leucine-rich repeat (NLR) protein related to defense-associated NLRs in several other plant species. CbAR9 transcript levels were induced dramatically after Colletotrichum capsici infection. To explore the biological function, we generated transgenic Nicotiana benthamiana lines overexpressing CbAR9, which showed enhanced resistance to C. capsici relative to wild-type plants. Transcript levels of pathogenesis-related (PR) genes increased markedly in CbAR9-overexpressing N. benthamiana plants. Moreover, resistance to anthracnose and transcript levels of PR1 and PR2 were markedly reduced in CbAR9-silenced chili pepper fruits after C. capsici infection. Our results revealed that CbAR9 contributes to innate immunity against C. capsici.

Anthracnose is caused by Colletotrichum species and is one of the most virulent fungal diseases affecting chili pepper (Capsicum) yield globally. However, the noble genes conferring resistance to Colletotrichum species remain largely elusive. In this study, we identified CbAR9 as the causal locus underlying the large effect quantitative trait locus CcR9 from the anthracnose-resistant chili pepper variety PBC80. CbAR9 encodes a nucleotide-binding and leucine-rich repeat (NLR) protein related to defense-associated NLRs in several other plant species. CbAR9 transcript levels were induced dramatically after Colletotrichum capsici infection. To explore the biological function, we generated transgenic Nicotiana benthamiana lines overexpressing CbAR9, which showed enhanced resistance to C. capsici relative to wild-type plants. Transcript levels of pathogenesis-related (PR) genes increased markedly in CbAR9-overexpressing N. benthamiana plants. Moreover, resistance to anthracnose and transcript levels of PR1 and PR2 were markedly reduced in CbAR9-silenced chili pepper fruits after C. capsici infection. Our results revealed that CbAR9 contributes to innate immunity against C. capsici.

Chili pepper (Capsicumannuum) is an important fruit and spice used globally, but its yield is seriously threatened by anthracnose. Capsicum baccatum is particularly valuable as it carries advantageous disease resistance genes. However, most of the genes remain to be identified. In this study, we identified the C. baccatum-specific gene CbCN, which encodes a truncated nucleotide-binding and leucine-rich repeat protein in the anthracnose resistant chili pepper variety PBC80. The transcription of CbCN was greater in PBC80 than it was in the susceptible variety An-S after Colletotrichum acutatum inoculation. In order to investigate the biological function of CbCN, we generated transgenic tobacco lines constitutively expressing CbCN. Notably, CbCN-overexpressing transgenic plants exhibited enhanced resistance to C. acutatum compared to wild-type plants. Moreover, the expression of pathogenesis-related (PR) genes was remarkably increased in a CbCN-overexpressing tobacco plants. In order to confirm these results in chili pepper, we silenced the CbCN gene using the virus-induced gene silencing system. The anthracnose resistance and expressions of PR1, PR2, and NPR1 were significantly reduced in CbCN-silenced chili peppers after C. acutatum inoculations. These results indicate that CbCN enhances the innate immunity against anthracnose caused by C. acutatum by regulating defense response genes.

A pink big-lip Phalaenopsis cultivar ‘Lovely Angel’ was developed by the National Institute of Horticultural & Herbal Science, Rural Development Administration (Korea) in 2017. ‘Lovely Angel’ was derived by artificial crossing between white-colored cultivar P. ‘Timonthy’ and pink-colored flower with big-lip cultivar P. World Class ‘Big Foot’ in 2009. In 2013, 09056-12 was selected as excellent growth and flowering characteristics, such as flower color, shape, and inflorescence number. The first and second characteristic tests were conducted from 2014 to 2017 to evaluate the stability and uniformity, named thereafter as ‘Lovely Angel’. ‘Lovely Angel’ has violet-pink (RHS, PVG81C) flowers with stripes and big purple lips (RHS, PG78B). The incurved florets measure 6.1 and 5.6 cm in length and width, respectively. The number of florets per inflorescence is 20.6, which is suitably large and abundant, and the inflorescence length of ‘Lovely Angel’ is 31.8 cm. The leaves of ‘Lovely Angel’ grow semi-pendulously and are 18.7 cm in length and 6.8cm in width. The new cultivar ‘Lovely Angel’ can be mainly available for small-type potted flowers.

High-throughput molecular markers with high genotyping accuracy will be helpful for genetic analysis, mapping of interesting genes, and rice breeding program. To develop high-throughput and cost-effective molecular markers for Korean japonica rice varieties, which are closely-related genetically, we designed kompetitive allele-specific polymerase chain reaction (KASP) assays from the sequence data of 13 Korean japonica rice varieties. Of the 504 new KASP assays, 371 (73.6%) showed polymorphisms among the tested varieties. In addition to the 400 previously developed KASP markers, this resulted in 771 KASP markers being applicable for Korean japonica rice varieties. These KASP markers were used to map the quantitative trait loci (QTLs) for rice bakanae disease (BD) resistance. From the results of QTL mapping and determination of the mortality rate of BD in two F2:F3 populations, a major QTL, qFfR1-1, and a novel QTL, qFfR6, were revealed on chromosome 1 in the Junam/Nampyeong F2:F3 population and on chromosome 6 in the Saenuri/Nampyeong F2:F3 population, respectively. Further, the insertion/deletion markers in the qFfR1-1 region were developed to select BD-resistant japonica rice varieties. The 771 developed KASP markers will accelerate the molecular breeding in Korean japonica rice varieties, and the detected QTLs will be helpful in identifying candidate genes for BD resistance.

Background Internode elongation is an important agronomic trait in rice that determines culm length, which is related to lodging, panicle exsertion, and biomass. sui4 (shortened uppermost internode 4) mutants show reduced internode length and a dwarf phenotype due to shortened internodes; the uppermost internode is particularly severely affected. The present study was performed to identify the molecular nature and function of the SUI4 gene during internode elongation. Results Our previous study showed that the SUI4 gene was mapped to a 1.1-Mb interval on chromosome 7 (Ji et al. 2014). In order to isolate the gene responsible for the sui4 phenotype, genomic DNA resequencing of sui4 mutants and wild-type plants and reciprocal transformation of wild-type and mutant alleles of the putative SUI4 gene was performed. The data revealed that the causative mutation of sui4 was a T to A nucleotide substitution at the microRNA172 binding site of Os07g0235800, and that SUI4 is a new allele of the previously reported gene SUPERNUMERARY BRACT (SNB), which affects flower structure. In order to understand the effect of this mutation on expression of the SUI4/SNB gene, SUI4/SNB native promoter-fuzed GUS transgenics were examined, along with qRT-PCR analysis at various developmental stages. In sui4 mutants, the SUI4/SNB gene was upregulated in the leaves, culms, and panicles, especially when internodes were elongated. In culms, SUI4/SNB was expressed in the nodes and the lower parts of elongating internodes. In order to further explore the molecular nature of SUI4/SNB during internode elongation, RNA-seq and qRT-PCR analysis were performed with RNAs from the culms of sui4 mutants and wild-type plants in the booting stage. The data showed that in sui4 mutants, genes deactivating bioactive gibberellins and cytokinin were upregulated while genes related to cell expansion and cell wall synthesis were downregulated. Conclusion In summary, this paper shows that interaction between SUI4/SNB and microRNA172 could determine internode elongation during the reproductive stage in rice plants. Due to a mutation at the microRNA172 binding site in sui4 mutants, the expression of SUI4/SNB was enhanced, which lowered the activities of cell expansion and cell wall synthesis and consequently resulted in shortened internodes. Electronic supplementary material The online version of this article (10.1186/s12284-019-0324-8) contains supplementary material, which is available to authorized users.

Main conclusion A new imaging platform was constructed to analyze drought-tolerant traits of rice. Rice was used to quantify drought phenotypes through image-based parameters and analyzing tools. Abstract Climate change has increased the frequency and severity of drought, which limits crop production worldwide. Developing new cultivars with increased drought tolerance and short breeding cycles is critical. However, achieving this goal requires phenotyping a large number of breeding populations in a short time and in an accurate manner. Novel cutting-edge technologies such as those based on remote sensors are being applied to solve this problem. In this study, new technologies were applied to obtain and analyze imaging data and establish efficient screening platforms for drought tolerance in rice using the drought-tolerant mutant osphyb. Red–Green–Blue images were used to predict plant area, color, and compactness. Near-infrared imaging was used to determine the water content of rice, infrared was used to assess plant temperature, and fluorescence was used to examine photosynthesis efficiency. DroughtSpotter technology was used to determine water use efficiency, plant water loss rate, and transpiration rate. The results indicate that these methods can detect the difference between tolerant and susceptible plants, suggesting their value as high-throughput phenotyping methods for short breeding cycles as well as for functional genetic studies of tolerance to drought stress.

Data phenotyping traits on soybean seeds such as shape and color has been obscure because it is difficult to define them clearly. Further, it takes too much time and effort to have sufficient number of samplings especially length and width. These difficulties prevented seed morphology to be incorporated into efficient breeding program. Here, we propose methods for an image acquisition, a data processing, and analysis for the morphology and color of soybean seeds by high-throughput method using images analysis. As results, quantitative values for colors and various types of morphological traits could be screened to create a standard for subsequent evaluation of the genotype. Phenotyping method in the current study could define the morphology and color of soybean seeds in highly accurate and reliable manner. Further, this method enables the measurement and analysis of large amounts of plant seed phenotype data in a short time, which was not possible before. Fast and precise phenotype data obtained here may facilitate Genome Wide Association Study for the gene function analysis as well as for development of the elite varieties having desirable seed traits.

Plant phenomics is an area of biology dealing with the analysis of phenotypic traits in plants. It can be cointegrated with other omics like functional genomics, transcriptomics, and metabolomics etc. Phenotypic traits are generated by images of RGB, hyperspectral, near-infrared, thermal, fluorescence imaging and so on. Characterized phenotypes can be revealed in various morphological and physiological measurements of size, growth pattern, biomass and color in plants. The image-base automated plant phenotyping is described as a high throughput plant facility. Despite its advantages like nondestructive phenotyping it has its own limitations such as plant’s complex architectures and environmental conditions at the time of image capture especially in the field. Phenomics generates a large number of images and metadata through phenotyping instruments, so there is a need for proper data processing and managements. Standardized data storage and sharing is also necessary for meaningful data acquisition along with statistical analysis. Processes of data management are largely consisted of data collection, storage, documentation, along with improvement of data quality. In future, plant phenomics must be developed efficiently to store, analyze, protect and share the acquired data. Modern high throughput plant phenotyping could be used effectively in plant improvement programs.

Faster and more efficient breeding cycle is not an option to deal with unpredictable and fast global climate changes. Phenomics for collecting huge number of individuals in accurate manner could be an answer to solve this problem. We collected image data to measure plant height and manual data for shoot length to be compared. QTLs clustered of plant height and shoot length were detected in 2-week old seedlings, which was consistent with many other reports using various genetic resources in matured stage. Further, these traits are highly correlated with yield by pleiotropism or tight linkage of those traits. It implies the “phenome-assisted selection” can be applied for yield trait in rice in the very early stage to shorten the breeding cycle significantly in fast but low-cost manner.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, has become a serious threat in rice-cultivating regions worldwide. In the present study, quantitative trait locus (QTL) mapping was performed using F2 and F3 plants derived after crossing a BD-resistant and a BD-susceptible Korean japonica rice variety, ‘Samgwang’ and ‘Junam’, respectively. Resequencing of ‘Junam’ and ‘Samgwang’ genomes revealed 151,916 DNA polymorphisms between the two varieties. After genotyping 188 F2 plants, we constructed a genetic map comprising 184 markers, including 175 kompetitive allele-specific PCR markers, eight cleaved amplified polymorphic sequence (CAPS) markers, and a derived CAPS (dCAPS) marker. The degree of BD susceptibility of each F2 plant was evaluated on the basis of the mortality rate measured with corresponding F3 progeny seedlings by in vitro screening. Consequently, qFfR9, a major QTL, was discovered at 30.1 centimorgan (cM) on chromosome 9 with a logarithm of the odds score of 60.3. For the QTL interval, 95% probability lay within a 7.24–7.56 Mbp interval. In this interval, we found that eight genes exhibited non-synonymous single nucleotide polymorphisms (SNPs) by comparing the ‘Junam’ and ‘Samgwang’ genome sequence data, and are possibly candidate genes for qFfR9; therefore, qFfR9 could be utilized as a valuable resource for breeding BD-resistant rice varieties.

Table S3. qRT-PCR primer sequences (DOCX 14 kb)

Table S1. List of SNPs in the SUI4 mapped region of Dongjin plants and sui4 mutants. (DOCX 16 kb)

Methylated cytosines are associated with gene silencing. The ten-eleven translocation (TET) hydroxylases, which oxidize methylated cytosines to 5-hydroxymethylcytosine (5hmC), are essential for cytosine demethylation. Gene silencing and activation are critical for intestinal stem cell (ISC) maintenance and differentiation, but the potential role of TET hydroxylases in these processes has not yet been examined. Here, we generated genome-wide maps of the 5hmC mark in ISCs and their differentiated progeny. Genes with high levels of hydroxymethylation in ISCs are strongly associated with Wnt signaling and developmental processes. We found Tet1 to be the most abundantly expressed Tet gene in ISCs; therefore, we analyzed intestinal development in Tet1-deficient mice and determined that these mice are growth-retarded, exhibit partial postnatal lethality, and have significantly reduced numbers of proliferative cells in the intestinal epithelium. In addition, the Tet1-deficient intestine displays reduced organoid-forming capacity. In the Tet1-deficient crypt, decreased expression of Wnt target genes such as Axin2 and Lgr5 correlates with lower 5hmC levels at their promoters. These data demonstrate that Tet1-mediated DNA hydroxymethylation plays a critical role in the epigenetic regulation of the Wnt pathway in intestinal stem and progenitor cells and consequently in the self-renewal of the intestinal epithelium.

Temperate japonica rice varieties exhibit wide variation in the phenotypes of several important agronomic traits, including disease resistance, pre-harvest sprouting resistance, plant architecture, and grain quality, indicating the presence of genes contributing to favorable agronomic traits. However, gene mapping and molecular breeding has been hampered as a result of the low genetic diversity among cultivars and scarcity of polymorphic DNA markers. Single nucleotide polymorphism (SNP)-based kompetitive allele-specific PCR (KASP) markers allow high-throughput genotyping for marker-assisted selection and quantitative trait loci (QTL) mapping within closely related populations. Previously, we identified 740,566 SNPs and developed 771 KASP markers for Korean temperate japonica rice varieties. However, additional markers were needed to provide sufficient genome coverage to support breeding programs. In this study, the 740,566 SNPs were categorized according to their predicted impacts on gene function. The high-impact, moderate-impact, modifier, and low-impact groups contained 703 (0.1%), 20,179 (2.7%), 699,866 (94.5%), and 19,818 (2.7%) SNPs, respectively. A subset of 357 SNPs from the high-impact group was selected for initial KASP marker development, resulting in 283 polymorphic KASP markers. After incorporation of the 283 markers with the 771 existing markers in a physical map, additional markers were developed to fill genomic regions with large gaps between markers, and 171 polymorphic KASP markers were successfully developed from 284 SNPs. Overall, a set of 1225 KASP markers was produced. The markers were evenly distributed across the rice genome, with average marker density of 3.3 KASP markers per Mbp. The 1225 KASP markers will facilitate QTL/gene mapping and marker-assisted selection in temperate japonica rice breeding programs.

Bakanae disease (BD) has emerged as a serious threat in almost all rice cultivation regions worldwide. Nampyeong is a Korean japonica rice variety known to be resistant to BD. In this study, quantitative trait locus (QTL) mapping was performed with F2 and F3 plants derived from a cross between the Nampyeong variety and a susceptible Korean japonica line, DongjinAD. First, resequencing of Nampyeong and DongjinAD was performed, which identified 171,035 single nucleotide polymorphisms (SNPs) between the two parental varieties. Using these SNPs, 161 cleaved amplified polymorphic sequence (CAPS) markers and six derived CAPS markers were developed; then, a genetic map was constructed from the genotypes of 180 plants from the DongjinAD/Nampyeong F2 plants. The total length of the constructed genetic map was 1386 cM, with an average interval of 8.9 cM between markers. The BD mortality rates of each F3 family were measured by testing 40 F3 progenies using in vitro seedling screening method. QTL analysis based on the genetic map and mortality rate data revealed a major QTL, qFfR1, on rice chromosome 1. qFfR1 was located at 89.8 cM with a logarithm of the odds (LOD) score of 22.7. Further, there were three markers at this point: JNS01033, JNS01037, and JNS01041. A total of 15 genes were identified with annotations related to defense against plant diseases among the 179 genes in the qFfR1 interval at 95% probability, thereby providing potential candidate genes for qFfR1. qFfR1 and its closely linked markers will be useful in breeding rice varieties resistant to BD.

Inchan Choi, Keeman Lee, Mrinal Juddoo and A.R. MasriABSTRACTThis paper described an experimental investigations of combustion instability mode in a lean premixed dual swirl combustor for micro-gasturbine system. When such the instability occurs, a strong coupling between pressure oscillations and unsteady heat release excites a self-sustained acoustic wave which results in a loud, annoyed sound and may also lead a structural damage to the combustion chamber. The detailed period of flame behavior and heat release in combustion instability mode have been examined with high speed OH and CH-PLIF system and CH* chemiluminescence measurement, flame tomography with operated at 10 kHz and 6 kHz each. Experiment results suggest that unstable flame behavior has a specific frequency with 200 Hz and this frequency is accords with about 1/2 sub-harmonic of combustor resonance frequency, not funda-mental frequency. This is very interesting phenomenon that have not reported yet from other previous works. Therefore, when a thermo-acoustic instability with Rayleigh criterion occurs, the fact that the period of heat release and flame behavior are different each other was proposed for the first time through this work.

Bakanae disease (BD), caused by the fungal pathogen Fusarium fujikuroi, has become a serious threat in rice-cultivating regions worldwide. In the present study, quantitative trait locus (QTL) mapping was performed using F2 and F3 plants derived after crossing a BD-resistant and a BD-susceptible Korean japonica rice variety, &lsquo, Samgwang&rsquo, and &lsquo, Junam&rsquo, respectively. Resequencing of &lsquo, genomes revealed 151,916 DNA polymorphisms between the two varieties. After genotyping 188 F2 plants, we constructed a genetic map comprising 184 markers, including 175 kompetitive allele-specific PCR markers, eight cleaved amplified polymorphic sequence (CAPS) markers, and a derived CAPS (dCAPS) marker. The degree of BD susceptibility of each F2 plant was evaluated on the basis of the mortality rate measured with corresponding F3 progeny seedlings by in vitro screening. Consequently, qFfR9, a major QTL, was discovered at 30.1 centimorgan (cM) on chromosome 9 with a logarithm of the odds score of 60.3. For the QTL interval, 95% probability lay within a 7.24&ndash, 7.56 Mbp interval. In this interval, we found that eight genes exhibited non-synonymous single nucleotide polymorphisms (SNPs) by comparing the &lsquo, genome sequence data, and are possibly candidate genes for qFfR9, therefore, qFfR9 could be utilized as a valuable resource for breeding BD-resistant rice varieties.

Early seedling establishment in rice (Oryza sativa L.), which is measured by primary/secondary tiller, shoot length, biomass, root-related traits, and leaf area index, is an important trait because it helps to compete for light, air, and water for better tolerating various abiotic stresses. Consequently, it can affect the yield. However, there are not many research studies on this subject. Furthermore, previous studies have only measured the target traits once. However, this does not reflect the variation of growth rate during the seedling stage. Thus, two data points, two weeks and four weeks after planting, were used in the current study. As a result, two QTL regions were detected for the growth differences via plant height and green area (reflecting tillering). We expect that these results can be utilized by breeders to evaluate and select vigorous seedlings for their breeding programs.

The development and severity of inflammatory bowel diseases and other chronic inflammatory conditions can be influenced by host genetic and environmental factors, including signals derived from commensal bacteria. However, the mechanisms that integrate these diverse cues remain undefined. Here we demonstrate that mice with an intestinal epithelial cell (IEC)-specific deletion of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3(ΔIEC) mice) exhibited extensive dysregulation of IEC-intrinsic gene expression, including decreased basal expression of genes associated with antimicrobial defence. Critically, conventionally housed HDAC3(ΔIEC) mice demonstrated loss of Paneth cells, impaired IEC function and alterations in the composition of intestinal commensal bacteria. In addition, HDAC3(ΔIEC) mice showed significantly increased susceptibility to intestinal damage and inflammation, indicating that epithelial expression of HDAC3 has a central role in maintaining intestinal homeostasis. Re-derivation of HDAC3(ΔIEC) mice into germ-free conditions revealed that dysregulated IEC gene expression, Paneth cell homeostasis and intestinal barrier function were largely restored in the absence of commensal bacteria. Although the specific mechanisms through which IEC-intrinsic HDAC3 expression regulates these complex phenotypes remain to be determined, these data indicate that HDAC3 is a critical factor that integrates commensal-bacteria-derived signals to calibrate epithelial cell responses required to establish normal host-commensal relationships and maintain intestinal homeostasis.