| Rosa rugosa is famous as an embellishment traditional flowers In the world, which were widely used for an excellent landscaping plant material or soil and water conservation, also as food and industrial raw materials for many years. Color is an important feature of ornamental plants, Rosa rugosa is known for its potpourri but lacks the various pattern and rich color as Rosa chinensis.’Zi zhi’ rose has two mutations, the color of their petals have changed to pink and white, which are ideal experimental material for research on flower color formation in rose. In this article, ’Zi zhi’ rose and the other two mutations’Fen Zizhi’rose and’Bai Zizhi’ rose were chosen as the test materials. HPLC-ESI-MSn technology was used to test the the quality and quantity analysis of anthocyanin composition in different flower developmental stages. The samples were mixed for transcriptome analysis through Illumina/Solexa RNA-Seq method in full opening stage, then 5 DEGS were selected, In conjunction with Real-time PCR experiment to test the model of the selected genes, we were try to figure out the molecular mechanism of color formation in Rosa rugosa, which can lay foundation for getting novel flower colors in roses. The main findings are as follows:1. The quality and quantity analysis of anthocyanin composition in petals of the 3 rose cultivars were tested by HPLC-ESI-MSn technology.The results showed that three kinds of anthocyanins were found in’Zizhi’rose and’Fen Zizhi’rose, only two kinds of anthocyanins contained in’Bai Zizhi’rose. Besides Cyanidin glycosides are contained in three samples, Paeoniflorin are found in’Zizhi’rose and’Fen Zizhi’rose, besides seven flavones and flavonol glycosides were detected from the three samples, Kaempferol and Quercetin are mainly contented in every sample.Quercetin-3-sophoroside is the highest Quercetin in each sample.2. The mixed mRNA samples contained full opening petals of three rose cultivars, were used for high-throughput parallel sequencing on Illumina HiSeqTM 2000 platform. De novo assemblies yielded 103,364,222 reads, then we assembly 51,332 All-unigene according to discard dirty raw reads. The length greater than 1,000 bp was 12,098 about 23.57% of All-unigene. In order to identify the functions of the 51,332 All-unigene sequences, which were discard dirty raw reads, we performed Blast X alignments (E-value< 1.0e-5) against the known databases of NR、NT、 Swiss-Prot、KEGG、COG、and GO. About 40,018(77.96%) All-Unigene of this data were annotation to one or more of the database,36,089 (70.32%) All-Unigene were annotation to NR;12,592 (24.53%) All-Unigene were annotation to COG by 25 Categories; 25,975 (50.6%) All-Unigene were annotation to GO by Molecular ftinction、 Cellular component or Biological process; 20,054 All-Unigene (39.07%) were annotation to 128 KEGG pathways, the metabolic pathways related to Color synthetic are three, one is phenylpropanoid biosynthesis include 316(1.58%) All-unigene, the other two pathways are flavonoid biosynthesis include 191 (0.95%) All-unigene and anthocyanin biosynthesis include 15 (0.07%) All-unigene.3. All-unigenes sequencesare aligned by Blast X (E-value< 1.0e-5) to protein databases, about 36,032 Unigene were annotiaction to CDS. The length greater than 1,000 bp was 6136(17%).4. From the results of DEGs analysis (FDR<0.001 and |log2Ratio|>1), we found that there were significantly differences between two any libraries of the three samples, white flower and pink flower contain 431 DEGs, red flower and pink flower contain 1319 DEGS, and red flower and white flower contain 1324 DEGs.5. Based on the database of rose petal transcriptome and digital gene expression profiling technology,5 candidate DEGs, Unigene23269(ir4CL), CL4955(RrCHS), Unigene6173(RrChI), Unigene\729(RrDFR) and CL1274(RrUGT) were selected from 3 pathways related to flower colour formation.The pathways are phenylpropanoid biosynthesis, flavonoid biosynthesis and anthocyanin biosynthesis,.6. According to Real-time PCR,5 candidate DEGs were selected to study the expression patterns combined with the database from HPLC-ESI-MSn technology during the flower developmental stages. The results showed that Rr4CL gene has the highest expression in the flower-budding stage of ’Fen Zizhi’ rose, besides Kaempferol keep the highest expression than the other two samples, we can speculate that Rr4CL is related to the color formation of’Fen Zizhi’rose. RrDFR has the obvious specificity in the three samples during flower development stages. In budding stage, the expression level of RrDFR were high in ’Zizhi’ rose and ’Fen Zizhi’rose, but in ’Bai Zizhi’ rose, the expression of RrDFR gene was 2.01% account of ’Zizhi’ rose. In half opening stage, the expression level of RrDFR were quickly decreased in 3 rose cultivars, the expression of RrDFR gene was 0.18% and 41.85% account of budding stage in ’Zizhi’ rose and’Fen Zizhi’rose respectively. In full opening stage, the expression of RrDFR gene was 0.46% and3.64% account of budding stage in ’Zizhi’ rose and ’Fen Zizhi’ rose respectively. In withering stage, the expression of RrDFR gene was 0.15% and 0.22% account of budding stage in ’Zizhi’rose and ’Fen Zizhi’ rose respectively.Besides,RrDFR almost expressed very low in budding stage of ’Bai Zizhi’ rose.It can illustrate that anthocyanin may have finished accumulation during flower-bud stage, also the low expression level of RrDFR gene may related to the color formation of’Bai Zizhi’rose. |
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