| Camellia reticulata is one of the most economically important Camellia species, well-known for its beautifull flowers and high quality seed oil. C. reticulata is native to Southwest China and possesses considerably environmental adaptability to the mountainous regions with poor soil as well as unique climates. It plays important roles in flower and oil industry and tourism. Unfortunately, little genomic information has led to poor knowledge of functional genes related to oil production, flower coloration and flowering time control. This situation has also hampered the exploitation of abundant molecular markers and novel gene resources towards modern breeding efforts of this economic plant. For the first time, we employed the Illumina techonology to sequence the transcriptome of C. reticulata. The results are summarized as follows:1. Using Illumina sequencing platform, we obtained approximately 311.3 million clean reads from the five tissues of C. reticulata. A total of 141,460 unigenes were assembled with the length of ～96.1 million nucleotides. Systematic evaluation indicates a good quality of the transcriptome assembly, which is suitable for further studies such as expressed sequence tag simple sequence repeats (EST-SSRs) mining and pathway analysis.2. By similarity searches against public databases,69,922 unigenes were annotated as putative protein-coding sequences. On average, the annotated genes had more than three counterparts in C. reticulata transcriptome compared to Aribidopsis thaliana and Vitis vinifera, suggesting that whole genome duplication events might occur in C. reticulata.3. Expression levels of unigenes were assessed by aligning the RNA-Seq reads from each library to the assembly. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the reliability of RNA-Seq expression analysis, showing a high degree of consistency between these two methods. A total of 94,450 unigenes were positively expressed (FPKM≥1) in at least one of the five sequenced tissues. Of them,22,229 unigenes were defined as differentially expressed genes (|fold change|≥4 and false discovery rate≤0.001) across these five tissues.4. We observed that the flowering transition of C. reticulata often occurs at the apex of branch around mid-April when the day length is more than 12 hours. This suggests that the photoperiodic flowering pathway may be involved in flowering time control of C. reticulata. We identified an almost full set of gene homologs involved in the photoperiodic flowering pathway except for CONSTANS (CO) and CYCLING-DOF-FACTOR-1 (CDF1). Many genes involved in this pathway had multiple copies. Different expression patterns of these duplicated genes were observed across tissues, such as the differential expression of SOC1_a, SOC1_b and SOC1 c.5. We totally identified 93 unigenes for the triacylglycerol biosynthesis (TAGBS) pathway. Many FA desaturease genes, including SAD_a, FAD2_a, FAD2_b, FAD3, FAD8_b, and FAD8_c were up-regulated in flowers or flower buds. The flowers of C. reticulata often bloom during winter when the temperatures are quite low. Thus, high expression of these desaturase genes may explain the freezing tolerance of C. reticulata flowers and flower buds in winter. While FAD2_a had a lower expression level in seeds than in mature leaves, SAD a and FATA had several folds higher expression level in seeds than in mature leaves. These observations suggest that SADa and FATA may be key genes responsible for monounsaturated fatty acid (MUFAs) production in C. reticualta seeds. In seeds, up-regulation of SADa and FATA genes and down-regulation of FAD2_a gene in the TAGBS pathway may control the ratio of MUFA to polyunsaturated fatty acid (PUFAs) in seed oil.6. Flavonoids and carotenoids, two major groups of colorful pigments, have been proposed to involve in flower coloration of Camellia species. Homology analysis identified 56 and 33 unigenes as candidate genes related to the flavonoid biosynthesis (FlaBS) and carotenoid biosynthesis (CrtBS) pathways, respectively. Systematic analysis of both sequence and expression levels revealed the extent of variation within these genes in C. reticulata. For example, MYBF1, a transcription regulator gene of the FlaBS pathway, was found with great sequence variation and alteration of expression patterns, probably resulting in functionally evolutionary differentiation. MYBA1_a and some anthocyanin-specific biosynthetic genes in the FlaBS pathway were highly expressed in both flower buds and flowers, showing important roles of anthocyanin biosynthesis in flower development. Several genes that are related to formation of pigment compounds in CrtBS pathway (e.g.lutein, zeaxanthin, carotene), such as LUT1, LUT2, LUT5, and LYC, were expressed at relatively low levels in C. reticulata. Our results indicate that the flavonoid biosynthesis pathway may play key roles in flower coloration rather than carotenoids biosynthesis pathway in C. reticulata. The analysis of expression profiles suggest that ABA biosynthesis genes may play important roles in developmental processes of flowers. Collectively, these findings have provided an in-depth understanding of the expression patterns and potential functions of these candidate genes.7. We developed a new pipeline, CandiSSR, to easily and efficiently identify putative polymorphic SSRs based on the multiple assembled sequences from a given genus or species. Using CandiSSR, a total of 835 polymorphic SSRs were identified in the transcriptomes of C. reticulata and its closely related relatives. In addition, using MISA a total of 40,823 EST-SSRs were identified specifically in C. reticulata.Overall, the obtained transcriptome has considerably increased the number of sequences deposited in the public databases for C. reticulata. This large dataset has provided a good opportunity to develop EST-SSRs and explore the candidate genes involved in TAGBS, photoperiodic flowering, FlaBS and CrtBS pathways. The datasets and results reported here will contribute to further basic and applied researches on this economically important Camellia plant. |
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