Fine mapping of a locus presumably involved in hybrid inviability (HIs-1) between flowering cherry cultivar Cerasus × yedoensis 'Somei-yoshino' and its wild relative C. spachiana.

Flowering cherry is an extremely renowned ornamental tree, consisting of a variety of species and cultivars. Because cherry species have no strict barriers for interspecific hybridization before fertilization, identification of the gene underlying post-zygotic hybrid inviability will help breeders identify specimens for breeding and help us understand speciation mechanisms. In this study, we mapped the genetic linkages and physical genome sequences for a presumed hybrid inviability locus (HIs-1) that we observed in the seedlings crossed between Cerasus × yedoensis 'Somei-yoshino' and its wild relative C. spachiana. By the surveying linkage maps of 'Somei-yoshino' and C. spachiana, we identified correlation with seedling inviability only in linkage group 4 (LG4) of the 'Somei-yoshino' map. When we produced a finer-scaled map of HIs-1 in LG4, we found that HIs-1 is located between two microsatellite (SSR) markers with a 3.8 cM span. Using eight SSR markers based on peach genome sequences, we further refined the candidate region for HIs-1. This region was located between Pp04C001 and Pp04C007 markers, spanning 240 Kb of the peach genome, in which 45 transcribed genes had been estimated. From these candidate genes, it will be feasible to identify molecular mechanisms involved in cherry hybrid inviability.

Genetic structure of island populations of Prunus lannesiana var. speciosa revealed by chloroplast DNA, AFLP and nuclear SSR loci analyses.

The wild flowering cherry Prunus lannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an "isolation by distance" pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.

Genetic structure of Cerasus jamasakura, a Japanese flowering cherry, revealed by nuclear SSRs: implications for conservation.

The genetic resources of a particular species of flowering cherry, Cerasus jamasakura, have high conservation priority because of its cultural, ecological and economic value in Japan. Therefore, the genetic structures of 12 natural populations of C. jamasakura were assessed using ten nuclear SSR loci. The population differentiation was relatively low (F (ST), 0.043), reflecting long-distance dispersal of seeds by animals and historical human activities. However, a neighbor-joining tree derived from the acquired data, spatial analysis of molecular variance and STRUCTURE analysis revealed that the populations could be divided into two groups: one located on Kyusyu Island and one on Honshu Island. Genetic diversity parameters such as allelic richness and gene diversity were significantly lower in the Kyushu group than the Honshu group. Furthermore, STRUCTURE analysis revealed that the two lineages were admixed in the western part of Honshu Island. Thus, although the phylogeographical structure of the species and hybridization dynamics among related species need to be evaluated in detail using several marker systems, the Kyusyu Island and Honshu Island populations should be considered as different conservation units, and the islands should be regarded as distinct seed transfer zones for C. jamasakura, especially when rapid assessments are required.

Distribution of S-alleles in island populations of flowering cherry, Prunus lannesiana var. speciosa.

We surveyed the distribution of S-alleles in natural island populations of Prunus lannesiana var. speciosa sampled from seven sites on the Izu Peninsula and six Izu islands, Japan. The S-genotypes of sampled individuals were determined by Southern analysis of RFLPs generated by restriction enzyme digestion of genomic DNA, using cDNA of the S-RNase gene as a probe. All individuals were heterozygous, as expected under gametophytic self-incompatibility (GSI). Sixty-three S-alleles were observed in the species, but 12 private to the Izu Peninsula population seemed to be derived from related species, giving a total of 75. The estimated number of S-alleles in each population ranged from 26 to 62, and was inversely correlated with the respective population's distance from the Izu Peninsula, the closest point in the mainland to the islands. This geographical cline in the estimated numbers of S-alleles suggests that gene flow to and from the distant island populations was less frequent, and that the studied species has migrated from the mainland to the Izu islands. The genetic relationship at the S-locus among populations also gave an "isolation by distance" pattern. The genetic differentiation at the S-locus among the populations was very low (F(ST) = 0.014, p < 0.001). The number of S-alleles in the species did not seem to depend on genetic differences associated with population subdivisions. This might be due to the greater effective migration rates of S-alleles, as expected under balancing selection in GSI.

A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags.

A consensus map for sugi (Cryptomeria japonica) was constructed by integrating linkage data from two unrelated third-generation pedigrees, one derived from a full-sib cross and the other by self-pollination of F1 individuals. The progeny segregation data of the first pedigree were derived from cleaved amplified polymorphic sequences, microsatellites, restriction fragment length polymorphisms, and single nucleotide polymorphisms. The data of the second pedigree were derived from cleaved amplified polymorphic sequences, isozyme markers, morphological traits, random amplified polymorphic DNA markers, and restriction fragment length polymorphisms. Linkage analyses were done for the first pedigree with JoinMap 3.0, using its parameter set for progeny derived by cross-pollination, and for the second pedigree with the parameter set for progeny derived from selfing of F1 individuals. The 11 chromosomes of C. japonica are represented in the consensus map. A total of 438 markers were assigned to 11 large linkage groups, 1 small linkage group, and 1 nonintegrated linkage group from the second pedigree; their total length was 1372.2 cM. On average, the consensus map showed 1 marker every 3.0 cM. PCR-based codominant DNA markers such as cleaved amplified polymorphic sequences and microsatellite markers were distributed in all linkage groups and occupied about half of mapped loci. These markers are very useful for integration of different linkage maps, QTL mapping, and comparative mapping for evolutional study, especially for species with a large genome size such as conifers.

Photosynthetic acclimation to dynamic changes in environmental conditions associated with deciduous overstory phenology in Daphniphyllum humile, an evergreen understory shrub.

Photoprotective responses during photosynthetic acclimation in Daphniphyllum humile Maxim, an evergreen understory shrub that grows in temperate deciduous forests, were examined in relation to changes in light availability and temperature caused by the seasonal dynamics of canopy leaf phenology. Gradual increases in irradiance in the understory from summer to autumn as overstory foliage senesced were accompanied by increased concentrations of xanthophyll cycle pigments (VAZ) in understory leaves. The chlorophyll (Chl) a/b ratio in understory leaves also increased from summer to autumn, reflecting the change in ratio of the light-harvesting antenna to the reaction center. However, low temperatures following overstory leaf fall reduced Rubisco activity. In contrast, the photosynthetic capactiy of leaves of D. humile growing at the forest border, which was higher in summer than that of leaves of understory plants, decreased in autumn. In autumn, Fv/Fm ratios decreased and concentrations of zeaxanthin (Z) and especially antheraxanthin (A) increased in leaves of both forest-border and understory plants. Although VAZ was twice as high in leaves of forest-border than of understory plants, NPQ was similar in both. We conclude that leaves of understory plants are able to acclimate to seasonal changes in light and temperature by varying their photosynthetic and photoprotective functions, thereby taking advantage of the favorable light conditions caused by overstory leaf fall.

Seasonal changes in the xanthophyll cycle and antioxidants in sun-exposed and shaded parts of the crown of Cryptomeria japonica in relation to rhodoxanthin accumulation during cold acclimation.

Xanthophyll rhodoxanthin, which is present in sun-exposed needles of certain gymnosperms in winter, may have a photoprotective role during long-term cold acclimation. To examine how cold acclimation processes vary within tree crowns and to examine putative correlations between xanthophyll cycle pigments (VAZ), rhodoxanthin and the water-water cycle in photoprotection, we monitored seasonal changes in the activities of two key antioxidant enzymes (ascorbate peroxidase (APX) and glutathione reductase (GR)), pigment composition and chlorophyll fluorescence parameters in sun and shade needles of crowns of the gymnosperm Cryptomeria japonica D. Don. Although APX and GR activities in both sun and shade needles were higher in winter than in summer when assayed at 20 degrees C, differences between seasons were less pronounced when enzymatic activities in summer and winter were assayed at 20 and 5 degrees C, respectively. These results suggest that increases in the potential activity of antioxidant enzymes in winter is an adaptation that helps counterbalance reductions in absolute enzyme activity caused by low temperature, and thus allows the photoprotective capacity of the water-water cycle in C. japonica to be maintained at a roughly constant value throughout the year. In shade needles, the concentration of VAZ increased in winter, but no rhodoxanthin accumulated. Photosynthetic activity was maintained in winter. In sun needles, however, the electron transport rate (ETR) and photochemical quenching (q(P)) decreased to their lowest values in December, just before the accumulation of rhodoxanthin, which coincided with the highest amount of VAZ. Changes in rhodoxanthin concentration mirrored changes in VAZ concentration from January to March. Winter values of ETR and q(P) were comparable with summer values after accumulation of rhodoxanthin, indicating that rhodoxanthin may play a more important role than the VAZ cycle in protecting the photosynthetic apparatus from photodamage in winter. Photosynthetic activity may be modulated, as a result of the interception of light by rhodoxanthin, to match the extent to which absorbed light energy can be utilized in winter when the VAZ cycle is unable to operate effectively because of low temperatures.

Horizontal and vertical variations in photosynthetic capacity in a Pinus densiflora crown in relation to leaf nitrogen allocation and acclimation to irradiance.

We measured horizontal and vertical gradients of light (rPPFD) along four first-order branches of a Pinus densiflora Sieb. & Zucc. crown, and compared variations in specific leaf area (SLA), needle nitrogen concentration (N), chlorophyll concentration (Chl) and photosynthetic capacity (i.e., maximum rate of carboxylation (V(cmax))) along the two axes. The horizontal gradient of rPPFD along first-order branches was similar in magnitude to the vertical gradient of rPPFD from the upper to the lower crown. None of the measured parameters (i.e., SLA, N, Chl and Vcmax) were strictly proportional to rPPFD, although they were more or less correlated with light when data obtained for all of the crown were pooled (r(2) = 0.31-0.80). The slope of rPPFD against N on an area basis (Narea) for a branch in the middle of the crown orientated northward was significantly greater than the slope for a similar branch orientated southward. Horizontal variations were unrelated to age effects because measurements were all on 1-year-old needles. We conclude that factors other than light (i.e., orientation) may influence N allocation within branches. There was considerably less variation in the relationship of Vcmax to Narea (r2 = 0.58) than in the relationship of Vcmax to rPPFD (r2 = 0.41). Fractional N distribution among components of the photosynthetic machinery was constant within the crown. Together with the relationships between rPPFD and N on a mass basis (r2 = 0.80) and SLA and Vcmax (r2 = 0.60), these findings suggest that most light acclimation in P. densiflora occurs through changes in needle morphology (e.g., SLA) during development.

Comparative analysis of expressed sequence tags of conifers and angiosperms reveals sequences specifically conserved in conifers.

To identify and characterize lineage-specific genes of conifers, two sets of ESTs (with 12791 and 5902 ESTs, representing 5373 and 3018 gene transcripts, respectively) were generated from the Cupressaceae species Cryptomeria japonica and Chamaecyparis obtusa. These transcripts were compared with non-redundant sets of genes generated from Pinaceae species, other gymnosperms and angiosperms. About 6% of tentative unique genes (Unigenes) of C. japonica and C. obtusa had homologs in other conifers but not angiosperms, and about 70% had apparent homologs in angiosperms. The calculated GC contents of orthologous genes showed that GC contents of coniferous genes are likely to be lower than those of angiosperms. Comparisons of the numbers of homologous genes in each species suggest that copy numbers of genes may be correlated between diverse seed plants. This correlation suggests that the multiplicity of such genes may have arisen before the divergence of gymnosperms and angiosperms.

Isolation and characterization of cDNAs that encode homologs of a pathogenesis-related protein allergen from Cryptomeria japonica.

Many plant pathogenesis-related (PR) proteins are allergenic. We isolated three cDNAs, Cry j 3.1, Cry j 3.2, and Cry j 3.3, that encoded homologs of Jun a 3, a PR protein allergen in Juniperus ashei, from a cDNA library derived from the pollen of Cryptomeria japonica. The predicted amino acid sequences encoded by the three cDNAs were more than 85% identical to each other and about 57% identical to the sequence of Jun a 3. The Cry j 3 genes seemed to form a small multigene family in the genome of C. japonica. Expression of Cry j 3 was strong in roots and in female and male strobili; expression was weaker in cotyledons, leaves, stems, and pollen grains.