Transcriptome Characterization of Cymbidium sinense 'Dharma' Using 454 Pyrosequencing and Its Application in the Identification of Genes Associated with Leaf Color Variation

Abstract

The highly variable leaf color of Cymbidium sinense significantly improves its horticultural and economic value, and makes it highly desirable in the flower markets in China and Southeast Asia. However, little is understood about the molecular mechanism underlying leaf-color variations. In this study, we found the content of photosynthetic pigments, especially chlorophyll degradation metabolite in the leaf-color mutants is distinguished significantly from that in the wild type of Cymbidium sinense 'Dharma'. To further determine the candidate genes controlling leaf-color variations, we first sequenced the global transcriptome using 454 pyrosequencing. More than 0.7 million expressed sequence tags (ESTs) with an average read length of 445.9 bp were generated and assembled into 103,295 isotigs representing 68,460 genes. Of these isotigs, 43,433 were significantly aligned to known proteins in the public database, of which 29,299 could be categorized into 42 functional groups in the gene ontology system, 10,079 classified into 23 functional classifications in the clusters of orthologous groups system, and 23,092 assigned to 139 clusters of specific metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes. Among these annotations, 95 isotigs were designated as involved in chlorophyll metabolism. On this basis, we identified 16 key enzyme-encoding genes in the chlorophyll metabolism pathway, the full length cDNAs and expressions of which were further confirmed. Expression pattern indicated that the key enzyme-encoding genes for chlorophyll degradation were more highly expressed in the leaf color mutants, as was consistent with their lower chlorophyll contents. This study is the first to supply an informative 454 EST dataset for Cymbidium sinense 'Dharma' and to identify original leaf color-associated genes, which provide important resources to facilitate gene discovery for molecular breeding, marketable trait discovery, and investigating various biological process in this species.

Comparative Metabolomic Analysis Reveals Distinct Flavonoid Biosynthesis Regulation for Leaf Color Development of Cymbidium sinense ‘Red Sun’

“…The regulation of plant leaf color is a complex process. Most of the previous studies analyzed the mechanism of leaf color formation by methods of physiology, cytology and molecular biology [1], but the mechanism of leaf color regulation from the perspective of small molecular metabolites needs to be studied further. In this study, based on UPLC-MS/MS, the changes of metabolites in the process of leaf color change of C. sinense 'Red Sun' were qualitatively and quantitatively analyzed, and the unique pattern of flavonoid-related metabolites in the process of leaf color change was constructed for the first time.…”

“…The most important phenotypic feature of C. sinense is that it has a very rich variation in leaf color, which improves its ornamental and economic values. C. sinense contains a number of varieties based on the location and color of leaf variegation, including chimera art, claw art, crown art, crane art, middle penetration art, spot art and treasure art and so on [1]. At present, the leaf variegation varieties of C. sinense are basically yellow-green leaves, but there are few reports about red leaves.…”

“…Compared with flower color, the influencing factors of leaf color are more complex. Generally, the greening of leaves is mainly due to the absolute proportion of chlorophyll, and the formation of yellow leaves is mostly due to the degradation of chlorophyll, leaving the color of carotenoids to dominate in leaves [1,2]. Anthocyanins are responsible for the red color of leaves, which is helpful for plants against various biotic and abiotic stresses [3,4].…”

An AGAMOUS-like factor is associated with the origin of two domesticated varieties in Cymbidium sinense (Orchidaceae)

“…All the data were normalized against the expression of reference gene ACTIN, as previously described 44 . The transcript levels for these genes were summarized from three replicates.…”

Biochemical and Comparative Transcriptome Analyses Reveal Key Genes Involved in Major Metabolic Regulation Related to Colored Leaf Formation in Osmanthus fragrans ‘Yinbi Shuanghui’ during Development

“…Studies on leaf color mutant of Cymbidium sinense 'Dharma' have demonstrated that the gene encoding key enzyme of chlorophyll degradation is expressed at a higher level, which is consistent with its lower chlorophyll content. It suggests that leaf color variation may be due to the excessive degradation of chlorophyll [16]. In addition, the upregulated expression of genes involved in flavonoid biosynthesis and the downregulation of genes involved in carotenoid biosynthesis may be related to the yellow-leaf phenotype [17].…”