Factors Influencing Somatic Embryo Maturation in Sugi (Japanese Cedar, Cryptomeria japonica (Thunb. ex L.f.) D. Don).
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ABSTRACT:
SUBMITTER: Maruyama TE
PROVIDER: S-EPMC8145757 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.
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Project description: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.
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Project description:Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.
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Project description: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.
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Project description:Sugi (Cryptomeria japonica D. Don) is an important conifer used for afforestation in Japan. As the genome of this species is 11 Gbps, it is too large to assemble within a short timeframe. Transcriptomics is one approach that can address this deficiency. Here we designed a workflow consisting of three stages to de novo assemble transcriptome using Oases and Trinity. The three transcriptomic stage used were independent assembly, automatic and semi-manual integration, and refinement by filtering out potential contamination. We identified a set of 49,795 cDNA and an equal number of translated proteins. According to the benchmark set by BUSCO, 87.01% of cDNAs identified were complete genes, and 78.47% were complete and single-copy genes. Compared to other full-length cDNA resources collected by Sanger and PacBio sequencers, the extent of the coverage in our dataset was the highest, indicating that these data can be safely used for further studies. When two tissue-specific libraries were compared, there were significant expression differences between male strobili and leaf and bark sets. Moreover, subtle expression difference between male-fertile and sterile libraries were detected. Orthologous genes from other model plants and conifer species were identified. We demonstrated that our transcriptome assembly output (CJ3006NRE) can serve as a reference transcriptome for future functional genomics and evolutionary biology studies.
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2024-01-22 | GSE77156 | GEO