Changes in annual transcriptome dynamics of a clone of Japanese cedar (Cryptomeria japonica D. Don) planted under different climate conditions.

Environmental responses are critical for plant growth and survival under different climate conditions. To elucidate the underlying biological mechanisms of environmental responses in Japanese cedar (Cryptomeria japonica D. Don), the annual transcriptome dynamics of common clonal trees (Godai1) planted at three different climate sites (Yamagata, Ibaraki, and Kumamoto Prefectures) were analyzed using microarrays. Both principal component analysis (PCA) and hierarchical clustering of the microarray data indicated the transition to dormant transcriptome status occurred earlier and the transition to active growth status later in the colder region. Interestingly, PCA also indicated that the transcriptomes of trees grown under three different conditions were similar during the growth period (June to September), whereas the transcriptomes differed between sites during the dormant period (January to March). In between-site comparisons, analyses of the annual expression profiles of genes for sites 'Yamagata vs. Kumamoto', 'Yamagata vs. Ibaraki', and 'Ibaraki vs. Kumamoto' identified 1,473, 1,137, and 925 targets exhibiting significantly different expression patterns, respectively. The total of 2,505 targets that exhibited significantly different expression patterns in all three comparisons may play important roles in enabling cuttings to adapt to local environmental conditions. Partial least-squares regression analysis and Pearson correlation coefficient analysis revealed that air temperature and day length were the dominant factors controlling the expression levels of these targets. GO and Pfam enrichment analyses indicated that these targets include genes that may contribute to environmental adaptation, such as genes related to stress and abiotic stimulus responses. This study provided fundamental information regarding transcripts that may play an important role in adaptation to environmental conditions at different planting sites.

Effects of day length- and temperature-regulated genes on annual transcriptome dynamics in Japanese cedar (Cryptomeria japonica D. Don), a gymnosperm indeterminate species.

Seasonal phenomena in plants are primarily affected by day length and temperature. The shoot transcriptomes of trees grown in the field and a controlled-environment chamber were compared to characterize genes that control annual rhythms and the effects of day length- and temperature-regulated genes in the gymnosperm Japanese cedar (Cryptomeria japonica D. Don), which exhibits seasonally indeterminate growth. Annual transcriptome dynamics were clearly demonstrated by principal component analysis using microarray data obtained under field-grown conditions. Analysis of microarray data from trees grown in a controlled chamber identified 2,314 targets exhibiting significantly different expression patterns under short-day (SD) and long-day conditions, and 2,045 targets exhibited significantly different expression patterns at 15°C (LT; low temperature) versus 25°C. Interestingly, although growth was suppressed under both SD and LT conditions, approximately 80% of the SD- and LT-regulated targets differed, suggesting that each factor plays a unique role in the annual cycle. The top 1,000 up-regulated targets in the growth/dormant period in the field coincided with more than 50% of the SD- and LT-regulated targets, and gene co-expression network analysis of the annual transcriptome indicated a close relationship between the SD- and LT-regulated targets. These results indicate that the respective effects of day length and temperature interact to control annual transcriptome dynamics. Well-known upstream genes of signaling pathways responsive to environmental conditions, such as the core clock (LHY/CjLHYb and CCA1/CjLHYa) and PEBP family (MFT) genes, exhibited unique expression patterns in Japanese cedar compared with previous reports in other species, suggesting that these genes control differences in seasonal regulation mechanisms between species. The results of this study provide new insights into seasonal regulation of transcription in Japanese cedar.

Identification of novel putative causative genes and genetic marker for male sterility in Japanese cedar (Cryptomeria japonica D.Don).

BACKGROUND: Japanese 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. RESULTS: This 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. CONCLUSIONS: More 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.

Species characteristics and intraspecific variation in growth and photosynthesis of Cryptomeria japonica under elevated O3 and CO2.

In order to predict the effects of future atmospheric conditions on forest productivity, it is necessary to clarify the physiological responses of major forest tree species to high concentrations of ozone (O3) and carbon dioxide (CO2). Furthermore, intraspecific variation of these responses should also be examined in order to predict productivity gains through tree improvements in the future. We investigated intraspecific variation in growth and photosynthesis of Cryptomeria japonica D. Don, a major silviculture species in Japan, in response to elevated concentrations of O3 (eO3) and CO2 (eCO2), separately and in combination. Cuttings of C. japonica were grown and exposed to two levels of O3 (ambient and twice-ambient levels) in combination with two levels of CO2 (ambient and 550 µmol mol-1 in the daytime) for two growing seasons in a free-air CO2 enrichment experiment. There was no obvious negative effect of eO3 on growth or photosynthetic traits of the C. japonica clones, but a positive effect was observed for annual height increments in the first growing season. Dry mass production and the photosynthetic rate increased under eCO2 conditions, while the maximum carboxylation rate decreased. Significant interaction effects of eO3 and eCO2 on growth and photosynthetic traits were not observed. Clonal effects on growth and photosynthetic traits were significant, but the interactions between clones and O3 and/or CO2 treatments were not. Spearman's rank correlation coefficients between growth traits under ambient conditions and for each treatment were significantly positive, implying that clonal ranking in growth abilities might not be affected by either eO3 or eCO2. The knowledge obtained from this study will be helpful for species selection in afforestation programs, to continue and to improve current programs involving this species, and to accurately predict the CO2 fixation capacity of Japanese forests.

Clock genes and diurnal transcriptome dynamics in summer and winter in the gymnosperm Japanese cedar (Cryptomeria japonica (L.f.) D.Don).

BACKGROUND: The circadian clock and diurnal dynamics of the transcriptome are presumed to play important roles in the regulation of physiological, biological and developmental processes synchronized with diurnal and annual cycles of plant environments. However, little is known about the circadian clock and its regulation in gymnosperms, including conifers. Here we present the diurnal transcriptome dynamics of Japanese cedar (Cryptomeria japonica (L.f.) D.Don) in both active (summer) and dormant (winter) periods. RESULTS: Microarray analysis revealed significant differences in transcripts between summer and winter, and diurnal transcriptome dynamics only in the summer. About 7.7% of unique genes (556 out of 7,254) on the microarray were periodically expressed in summer. Expression patterns of some genes, especially light-related genes, did not show significant oscillation in Japanese cedar, thus differing from those reported in angiosperms. Gene network analysis of the microarray data revealed a network associated with the putative core clock genes (CjLHYa, CjLHYb, CjTOC1, CjGI and CjZTL), which were also isolated, indicating their importance in the diurnal regulation of the transcriptome. CONCLUSION: This study revealed the existence of core clock genes and diurnal rhythms of the transcriptome in summer in Japanese cedar. Dampening of diurnal rhythms in winter indicated seasonal change in the rhythms according to environmental conditions. The data also revealed genes that showed different expression patterns compared to angiosperms, suggesting a unique gene regulatory network in conifers. This study provides fundamental data to understand transcriptional regulatory mechanisms in conifers.

Ribosomal DNA haplotype distribution of Bursaphelenchus xylophilus in Kyushu and Okinawa islands, Japan.

Ribosomal DNA region sequences (partial 18S, 28S and complete ITS1, 5.8S, and ITS2) of the pinewood nematode (Bursaphelenchus xylophilus) were obtained from DNA extracted directly from wood pieces collected from wilted pine trees throughout the Kyushu and Okinawa islands, Japan. Either a 2569bp or 2573bp sequence was obtained from 88 of 143 samples. Together with the 45 rDNA sequences of pinewood nematode isolates previously reported, there were eight single nucleotide polymorphisms and two indels of two bases. Based on these mutations, nine haplotypes were estimated. The haplotype frequencies differed among regions in Kyushu island (northwest, northeast and center, southeast, and southwest), and the distribution was consistent with the invasion and spreading routes of the pinewood nematode previously estimated from past records of pine wilt and wood importation. There was no significant difference in haplotype frequencies among the collection sites on Okinawa island.