Objective: Acute corneal permeability change after instillation of benzalkonium chloride (BAC) was evaluated using a newly developed in vivo corneal transepithelial electric resistance (TER) measurement method. Method: Corneal TER was measured by Ag/AgCl electrodes placed in the anterior aqueous chamber and on the cornea of live rabbit eyes. TER was measured and TER change after instillation of 0.05% BAC solution was monitored. After TER measurement, cornea was excised and fixed for transmission and scanning electron microscopy. For the control study, physiologic saline was used instead of BAC. Results: The TER of normal rabbit cornea was 602.3 ± 195.0 Ωcm². TER decreased instantly after instillation of 0.05% BAC. In 5 s, TER decreased to 58.3 ± 5.2%. In 60 s, TER decreased to 18.5 ± 3.2%. At all time points, TER after instillation of 0.05% BAC was significantly lower than that of the control (p < 0.0001). Dissociation of tight junctions and the destruction of superficial cell membranes were observed under electron microscopy. Conclusion: Corneal epithelial change with increased permeability is rapid and intense after the instillation of highly concentrated BAC solution, accompanied by disorder of tight junctions and cell membranes of superficial cells. The newly developed in vivo corneal TER measurement method is suitable for assessing acute corneal change after drug instillation.

BackgroundJapanese cedar (Cryptomeria japonica) is an important tree for Japanese forestry. Male-sterile marker development in Japanese cedar would facilitate selection of male-sterile plus trees, addressing the widespread social problem of pollinosis and facilitating the identification of heterozygotes, which are useful for breeding.ResultsThis study used next-generation sequencing for single-nucleotide polymorphism discovery in libraries constructed from several organs, including male-sterile and male-fertile strobili. The single-nucleotide polymorphisms obtained were used to construct a high-density linkage map, which enabled identification of a locus on linkage group 9 strongly correlated with male-sterile trait. Expressed sequence tags corresponding to 11 marker loci from 5 isotigs were associated with this locus within 33.4-34.5 cM. These marker loci explained 100% of the phenotypic variation. Several homologs of these sequences are associated with male sterility in rice or Arabidopsis, including a pre-mRNA splicing factor, a DEAD-box protein, a glycosyl hydrolase, and a galactosyltransferase. These proteins are thus candidates for the causal male-sterile gene at the ms-1 locus. After we used a SNaPshot assay to develop markers for marker-assisted selection (MAS), we tested F2 progeny between male-sterile and wild-type plus trees to validate the markers and extrapolated the testing to a larger plus-tree population. We found that two developed from one of the candidates for the causal gene were suitable for MAS.ConclusionsMore than half of the ESTs and SNPs we collected were new, enlarging the genomic basis for genetic research on Japanese cedar. We developed two SNP markers aimed at MAS that distinguished individuals carrying the male-sterile trait with 100% accuracy, as well as individuals heterozygous at the male-sterile locus, even outside the mapping population. These markers should enable practical MAS for conifer breeding.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4581-5) contains supplementary material, which is available to authorized users.

BackgroundForest trees have ecological and economic importance, and Japanese cedar has highly valued wood attributes. Thus, studies of molecular aspects of wood formation offer practical information that may be used for screening and forward genetics approaches to improving wood quality.ResultsAfter identifying expressed sequence tags in Japanese cedar tissue undergoing xylogenesis, we designed a custom cDNA microarray to compare expression of highly regulated genes throughout a growing season. This led to identification of candidate genes involved both in wood formation and later cessation of growth and dormancy. Based on homology to orthologous protein groups, the genes were assigned to functional classes. A high proportion of sequences fell into functional classes related to posttranscriptional modification and signal transduction, while transcription factors and genes involved in the metabolism of sugars, cell-wall synthesis and lignification, and cold hardiness were among other classes of genes identified as having a potential role in xylem formation and seasonal wood formation.ConclusionsWe obtained 55,051 unique sequences by next-generation sequencing of a cDNA library prepared from cambial meristem and derivative cells. Previous studies on conifers have identified unique sequences expressed in developing xylem, but this is the first comprehensive study utilizing a collection of expressed sequence tags for expression studies related to xylem formation in Japanese cedar, which belongs to a different lineage than the Pinaceae. Our characterization of these sequences should allow comparative studies of genome evolution and functional genetics of wood species.

Genome-wide association studies (GWAS) are an alternative to bi-parental QTL mapping in long-lived perennials. In the present study, we examined the potential of GWAS in conifers using 367 unrelated plus trees of Cryptomeria japonica D. Don, which is the most widely planted and commercially important tree species in Japan, and tried to detect significant associations between wood property traits and quantity of male strobili on the one hand, and 1,032 single nucleotide polymorphisms (SNPs) assigned to 1,032 genes on the other. Association analysis was performed with the mixed linear model taking into account kinship relationships and subpopulation structure. In total, 6 SNPs were found to have significant associations with the variations in phenotype. These SNPs were not associated with the positions of known genes and QTLs that have been reported to date, thus they may identify novel QTLs. These 6 SNPs were all found in sequences showing similarities with known genes, although further analysis is required to dissect the ways in which they affect wood property traits and abundance of male strobili. These presumptive QTL loci provide opportunities for improvement of C. japonica, based on a marker approach. The results suggest that GWAS has potential for use in future breeding programs in C. japonica.