A practical assembly guideline for genomes with various levels of heterozygosity.

Although current long-read sequencing technologies have a long-read length that facilitates assembly for genome reconstruction, they have high sequence errors. While various assemblers with different perspectives have been developed, no systematic evaluation of assemblers with long reads for diploid genomes with varying heterozygosity has been performed. Here, we evaluated a series of processes, including the estimation of genome characteristics such as genome size and heterozygosity, de novo assembly, polishing, and removal of allelic contigs, using six genomes with various heterozygosity levels. We evaluated five long-read-only assemblers (Canu, Flye, miniasm, NextDenovo and Redbean) and five hybrid assemblers that combine short and long reads (HASLR, MaSuRCA, Platanus-allee, SPAdes and WENGAN) and proposed a concrete guideline for the construction of haplotype representation according to the degree of heterozygosity, followed by polishing and purging haplotigs, using stable and high-performance assemblers: Redbean, Flye and MaSuRCA.

MarpolBase Expression: A Web-Based, Comprehensive Platform for Visualization and Analysis of Transcriptomes in the Liverwort Marchantia polymorpha.

The liverwort Marchantia polymorpha is equipped with a wide range of molecular and genetic tools and resources that have led to its wide use to explore the evo-devo aspects of land plants. Although its diverse transcriptome data are rapidly accumulating, there is no extensive yet user-friendly tool to exploit such a compilation of data and to summarize results with the latest annotations. Here, we have developed a web-based suite of tools, MarpolBase Expression (MBEX, https://marchantia.info/mbex/), where users can visualize gene expression profiles, identify differentially expressed genes, perform co-expression and functional enrichment analyses and summarize their comprehensive output in various portable formats. Using oil body biogenesis as an example, we demonstrated that the results generated by MBEX were consistent with the published experimental evidence and also revealed a novel transcriptional network in this process. MBEX should facilitate the exploration and discovery of the genetic and functional networks behind various biological processes in M. polymorpha and promote our understanding of the evolution of land plants.

OryzaGenome: Genome Diversity Database of Wild Oryza Species.

The species in the genus Oryza, encompassing nine genome types and 23 species, are a rich genetic resource and may have applications in deeper genomic analyses aiming to understand the evolution of plant genomes. With the advancement of next-generation sequencing (NGS) technology, a flood of Oryza species reference genomes and genomic variation information has become available in recent years. This genomic information, combined with the comprehensive phenotypic information that we are accumulating in our Oryzabase, can serve as an excellent genotype-phenotype association resource for analyzing rice functional and structural evolution, and the associated diversity of the Oryza genus. Here we integrate our previous and future phenotypic/habitat information and newly determined genotype information into a united repository, named OryzaGenome, providing the variant information with hyperlinks to Oryzabase. The current version of OryzaGenome includes genotype information of 446 O. rufipogon accessions derived by imputation and of 17 accessions derived by imputation-free deep sequencing. Two variant viewers are implemented: SNP Viewer as a conventional genome browser interface and Variant Table as a text-based browser for precise inspection of each variant one by one. Portable VCF (variant call format) file or tab-delimited file download is also available. Following these SNP (single nucleotide polymorphism) data, reference pseudomolecules/scaffolds/contigs and genome-wide variation information for almost all of the closely and distantly related wild Oryza species from the NIG Wild Rice Collection will be available in future releases. All of the resources can be accessed through http://viewer.shigen.info/oryzagenome/.

Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes.

BACKGROUND: Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. RESULTS: We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression evolution of changed-tissues DE genes was rapid in tissue specifically expressed genes and those rapidly evolved changed-tissues DE genes were regulated not by cis-eQTLs, but by complicated trans-eQTLs. CONCLUSIONS: Global DE genes and changed-tissues DE genes had contrasting characteristics. The two contrasting types of DE genes provide possible explanations for the previous controversial conclusions about the relationships between molecular evolution and expression evolution of genes in different species, and the relationship between expression breadth and expression conservation in evolution.

Gene expression profiles associated with early relapse during first remission induction in canine multicentric high-grade B-cell lymphoma.

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

A highly contiguous genome assembly of red perilla (Perilla frutescens) domesticated in Japan.

Perilla frutescens (Lamiaceae) is an important herbal plant with hundreds of bioactive chemicals, among which perillaldehyde and rosmarinic acid are the two major bioactive compounds in the plant. The leaves of red perilla are used as traditional Kampo medicine or food ingredients. However, the medicinal and nutritional uses of this plant could be improved by enhancing the production of valuable metabolites through the manipulation of key enzymes or regulatory genes using genome editing technology. Here, we generated a high-quality genome assembly of red perilla domesticated in Japan. A near-complete chromosome-level assembly of P. frutescens was generated contigs with N50 of 41.5 Mb from PacBio HiFi reads. 99.2% of the assembly was anchored into 20 pseudochromosomes, among which seven pseudochromosomes consisted of one contig, while the rest consisted of less than six contigs. Gene annotation and prediction of the sequences successfully predicted 86,258 gene models, including 76,825 protein-coding genes. Further analysis showed that potential targets of genome editing for the engineering of anthocyanin pathways in P. frutescens are located on the late-stage pathways. Overall, our genome assembly could serve as a valuable reference for selecting target genes for genome editing of P. frutescens.

Best Practices for Comprehensive Annotation of Neuropeptides of Gryllus bimaculatus.

Genome annotation is critically important data that can support research. Draft genome annotations cover representative genes; however, they often do not include genes that are expressed only in limited tissues and stages, or genes with low expression levels. Neuropeptides are responsible for regulation of various physiological and biological processes. A recent study disclosed the genome draft of the two-spotted cricket Gryllus bimaculatus, which was utilized to understand the intriguing physiology and biology of crickets. Thus far, only two of the nine reported neuropeptides in G. bimaculatus were annotated in the draft genome. Even though de novo assembly using transcriptomic analyses can comprehensively identify neuropeptides, this method does not follow those annotations on the genome locus. In this study, we performed the annotations based on the reference mapping, de novo transcriptome assembly, and manual curation. Consequently, we identified 41 neuropeptides out of 43 neuropeptides, which were reported in the insects. Further, 32 of the identified neuropeptides on the genomic loci in G. bimaculatus were annotated. The present annotation methods can be applicable for the neuropeptide annotation of other insects. Furthermore, the methods will help to generate useful infrastructures for studies relevant to neuropeptides.

Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida.

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here, we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a nonphotosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms and heterotrophic algae with parasitic lifestyle revealed that a combination of gene loss, the accumulation of genes involved in organic carbon degradation, a unique secretome, and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a free-living secondary heterotroph.

OryzaExpress: an integrated database of gene expression networks and omics annotations in rice.

Similarity of gene expression profiles provides important clues for understanding the biological functions of genes, biological processes and metabolic pathways related to genes. A gene expression network (GEN) is an ideal choice to grasp such expression profile similarities among genes simultaneously. For GEN construction, the Pearson correlation coefficient (PCC) has been widely used as an index to evaluate the similarities of expression profiles for gene pairs. However, calculation of PCCs for all gene pairs requires large amounts of both time and computer resources. Based on correspondence analysis, we developed a new method for GEN construction, which takes minimal time even for large-scale expression data with general computational circumstances. Moreover, our method requires no prior parameters to remove sample redundancies in the data set. Using the new method, we constructed rice GENs from large-scale microarray data stored in a public database. We then collected and integrated various principal rice omics annotations in public and distinct databases. The integrated information contains annotations of genome, transcriptome and metabolic pathways. We thus developed the integrated database OryzaExpress for browsing GENs with an interactive and graphical viewer and principal omics annotations (http://riceball.lab.nig.ac.jp/oryzaexpress/). With integration of Arabidopsis GEN data from ATTED-II, OryzaExpress also allows us to compare GENs between rice and Arabidopsis. Thus, OryzaExpress is a comprehensive rice database that exploits powerful omics approaches from all perspectives in plant science and leads to systems biology.

Identification of the sex-determining factor in the liverwort Marchantia polymorpha reveals unique evolution of sex chromosomes in a haploid system.

Sex determination is a central process for sexual reproduction and is often regulated by a sex determinant encoded on a sex chromosome. Rules that govern the evolution of sex chromosomes via specialization and degeneration following the evolution of a sex determinant have been well studied in diploid organisms. However, distinct predictions apply to sex chromosomes in organisms where sex is determined in the haploid phase of the life cycle: both sex chromosomes, female U and male V, are expected to maintain their gene functions, even though both are non-recombining. This is in contrast to the X-Y (or Z-W) asymmetry and Y (W) chromosome degeneration in XY (ZW) systems of diploids. Here, we provide evidence that sex chromosomes diverged early during the evolution of haploid liverworts and identify the sex determinant on the Marchantia polymorpha U chromosome. This gene, Feminizer, encodes a member of the plant-specific BASIC PENTACYSTEINE transcription factor family. It triggers female differentiation via regulation of the autosomal sex-determining locus of FEMALE GAMETOPHYTE MYB and SUPPRESSOR OF FEMINIZATION. Phylogenetic analyses of Feminizer and other sex chromosome genes indicate dimorphic sex chromosomes had already been established 430 mya in the ancestral liverwort. Feminizer also plays a role in reproductive induction that is shared with its gametolog on the V chromosome, suggesting an ancestral function, distinct from sex determination, was retained by the gametologs. This implies ancestral functions can be preserved after the acquisition of a sex determination mechanism during the evolution of a dominant haploid sex chromosome system.

A simple optimization can improve the performance of single feature polymorphism detection by Affymetrix expression arrays.

BACKGROUND: High-density oligonucleotide arrays are effective tools for genotyping numerous loci simultaneously. In small genome species (genome size: < approximately 300 Mb), whole-genome DNA hybridization to expression arrays has been used for various applications. In large genome species, transcript hybridization to expression arrays has been used for genotyping. Although rice is a fully sequenced model plant of medium genome size (approximately 400 Mb), there are a few examples of the use of rice oligonucleotide array as a genotyping tool. RESULTS: We compared the single feature polymorphism (SFP) detection performance of whole-genome and transcript hybridizations using the Affymetrix GeneChip Rice Genome Array, using the rice cultivars with full genome sequence, japonica cultivar Nipponbare and indica cultivar 93-11. Both genomes were surveyed for all probe target sequences. Only completely matched 25-mer single copy probes of the Nipponbare genome were extracted, and SFPs between them and 93-11 sequences were predicted. We investigated optimum conditions for SFP detection in both whole genome and transcript hybridization using differences between perfect match and mismatch probe intensities of non-polymorphic targets, assuming that these differences are representative of those between mismatch and perfect targets. Several statistical methods of SFP detection by whole-genome hybridization were compared under the optimized conditions. Causes of false positives and negatives in SFP detection in both types of hybridization were investigated. CONCLUSIONS: The optimizations allowed a more than 20% increase in true SFP detection in whole-genome hybridization and a large improvement of SFP detection performance in transcript hybridization. Significance analysis of the microarray for log-transformed raw intensities of PM probes gave the best performance in whole genome hybridization, and 22,936 true SFPs were detected with 23.58% false positives by whole genome hybridization. For transcript hybridization, stable SFP detection was achieved for highly expressed genes, and about 3,500 SFPs were detected at a high sensitivity (> 50%) in both shoot and young panicle transcripts. High SFP detection performances of both genome and transcript hybridizations indicated that microarrays of a complex genome (e.g., of Oryza sativa) can be effectively utilized for whole genome genotyping to conduct mutant mapping and analysis of quantitative traits such as gene expression levels.

Rice expression atlas in reproductive development.

Gene expression throughout the reproductive process in rice (Oryza sativa) beginning with primordia development through pollination/fertilization to zygote formation was analyzed. We analyzed 25 stages/organs of rice reproductive development including early microsporogenesis stages with 57,381 probe sets, and identified around 26,000 expressed probe sets in each stage. Fine dissection of 25 reproductive stages/organs combined with detailed microarray profiling revealed dramatic, coordinated and finely tuned changes in gene expression. A decrease in expressed genes in the pollen maturation process was observed in a similar way with Arabidopsis and maize. An almost equal number of ab initio predicted genes and cloned genes which appeared or disappeared coordinated with developmental stage progression. A large number of organ-/stage-specific genes were identified; notably 2,593 probe sets for developing anther, including 932 probe sets corresponding to ab initio predicted genes. Analysis of cell cycle-related genes revealed that several cyclin-dependent kinases (CDKs), cyclins and components of SCF E3 ubiquitin ligase complexes were expressed specifically in reproductive organs. Cell wall biosynthesis or degradation protein genes and transcription factor genes expressed specifically in reproductive stages were also newly identified. Rice genes homologous to reproduction-related genes in other plants showed expression profiles both consistent and inconsistent with their predicted functions. The rice reproductive expression atlas is likely to be the most extensive and most comprehensive data set available, indispensable for unraveling functions of many specific genes in plant reproductive processes that have not yet been thoroughly analyzed.

Chromatin Organization in Early Land Plants Reveals an Ancestral Association between H3K27me3, Transposons, and Constitutive Heterochromatin.

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes, and our knowledge of the evolution of chromatin-related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants circa 400 mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional genome organization in an early diverging land plant lineage. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and other repetitive sequences in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres. Although a large fraction of constitutive heterochromatin was marked by H3K9 methylation as in flowering plants, a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein-coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that repressed B compartments were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that, in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

SNEP: Simultaneous detection of nucleotide and expression polymorphisms using Affymetrix GeneChip.

BACKGROUND: High-density short oligonucleotide microarrays are useful tools for studying biodiversity, because they can be used to investigate both nucleotide and expression polymorphisms. However, when different strains (or species) produce different signal intensities after mRNA hybridization, it is not easy to determine whether the signal intensities were affected by nucleotide or expression polymorphisms. To overcome this difficulty, nucleotide and expression polymorphisms are currently examined separately. RESULTS: We have developed SNEP, a new method that allows simultaneous detection of both nucleotide and expression polymorphisms. SNEP involves a robust statistical procedure based on the idea that a nucleotide polymorphism observed at the probe level can be regarded as an outlier, because the nucleotide polymorphism can reduce the hybridization signal intensity. To investigate the performance of SNEP, we used three species: barley, rice and mice. In addition to the publicly available barley data, we obtained new rice and mouse data from the strains with available genome sequences. The sensitivity and false positive rate of nucleotide polymorphism detection were estimated based on the sequence information. The robustness of expression polymorphism detection against nucleotide polymorphisms was also investigated. CONCLUSION: SNEP performed well regardless of the genome size and showed a better performance for nucleotide polymorphism detection, when compared with other previously proposed methods. The R-software 'SNEP' is available at http://www.ism.ac.jp/~fujisawa/SNEP/.

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc.) Using a Hybrid Assembly Approach.

Satsuma (Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma ("Miyagawa Wase") was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives.

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information.

Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle Genomes.

Most indigenous citrus varieties are assumed to be natural hybrids, but their parentage has so far been determined in only a few cases because of their wide genetic diversity and the low transferability of DNA markers. Here we infer the parentage of indigenous citrus varieties using simple sequence repeat and indel markers developed from various citrus genome sequence resources. Parentage tests with 122 known hybrids using the selected DNA markers certify their transferability among those hybrids. Identity tests confirm that most variant strains are selected mutants, but we find four types of kunenbo (Citrus nobilis) and three types of tachibana (Citrus tachibana) for which we suggest different origins. Structure analysis with DNA markers that are in Hardy-Weinberg equilibrium deduce three basic taxa coinciding with the current understanding of citrus ancestors. Genotyping analysis of 101 indigenous citrus varieties with 123 selected DNA markers infers the parentages of 22 indigenous citrus varieties including Satsuma, Temple, and iyo, and single parents of 45 indigenous citrus varieties, including kunenbo, C. ichangensis, and Ichang lemon by allele-sharing and parentage tests. Genotyping analysis of chloroplast and mitochondrial genomes using 11 DNA markers classifies their cytoplasmic genotypes into 18 categories and deduces the combination of seed and pollen parents. Likelihood ratio analysis verifies the inferred parentages with significant scores. The reconstructed genealogy identifies 12 types of varieties consisting of Kishu, kunenbo, yuzu, koji, sour orange, dancy, kobeni mikan, sweet orange, tachibana, Cleopatra, willowleaf mandarin, and pummelo, which have played pivotal roles in the occurrence of these indigenous varieties. The inferred parentage of the indigenous varieties confirms their hybrid origins, as found by recent studies.

Draft Genome Sequence of Lactobacillus oryzae Strain SG293T.

We report the 1.86-Mb draft genome and annotation of Lactobacillus oryzae SG293(T) isolated from fermented rice grains. This genome information may provide further insights into the mechanisms underlying the fermentation of rice grains.

Draft Genome Sequence of Weissella oryzae SG25T, Isolated from Fermented Rice Grains.

Weissella oryzae was originally isolated from fermented rice grains. Here we report the draft genome sequence of the type strain of W. oryzae. This first report on the genomic sequence of this species may help identify the mechanisms underlying bacterial adaptation to the ecological niche of fermented rice grains.

Minimal erythema dose (MED) in normal canine skin by irradiation of narrow-band ultraviolet B (NB-UVB).

Narrow-band UVB (NB-UVB) is light over a very narrow band of wavelengths (around 311 nm) that is concentrated in the therapeutic range and minimally in the sunburn range. It has therefore become the phototherapy treatment of choice for skin diseases. The minimal erythema dose (MED) on canine skin for standardizing dosage schedules in NB-UVB treatment and histopathological analyses were performed in these dogs. In all 32 dogs tested, the MED ranged from 432 to 864 mJ/cm(2). There were no significant differences in MED among breeds, sex and age groups. Histopathology obtained from areas irradiated by MED showed only mild vascular dilatation. These findings might be valuable for the application of NB-UVB phototherapy to canine skin diseases.