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.

Fig 1. Morphologic and Phylogenetic analysis of different-colored mutants of Cymbidium sinense 'Dharma.'

(A) The UPGMA cluster of ten leaf-color mutants of Cymbidium sinense 'Dharma' based on AFLP markers; (B) Morphologic characteristics of six leaf-color mutants of Cymbidium sinense 'Dharma' studied in this work.

Fig 2. The variations in the contents of photosynthetic pigments between the wild type and leaf-color mutants.

(A) Chlorophyll content in wild-type and color-mutant leaves of Cymbidium sinense 'Dharma'(mg g^-1 FW); (B) The relative contents of chlorophyll synthesis precursors PBG, Proto IX, Mg-Proto, and Pchlide, and chlorophyll degradation metabolite Pheide a (Take each intermediate product content in the wild type as 100%, comparing it with its mutant correspondent). Values are means ±standard deviation (n > 10). Mean-Whitney U-test significant at *P<0.05 between the mutants and the wild type control.

Fig 3. The size distribution of assembled isotigs.

The x-axis represents the sequence length in base pairs. The y-axis represents the isotigs number.

Fig 4. Characteristics of homology search of the isotigs.

(A) Similarity distribution of the best Blast hits in Nr database; (B) Similarity distribution of the best Blast hits in Uniprot database; (C) Similarity distribution of the best Blast hits in Swissport database.

Fig 5. GO classification of unigenes of assembled isotigs.

Fig 6. KOG function classification of assembled isotigs.

Fig 7. Assembled isotigs involved in the chlorophyll biosynthesis pathway pathway of Cymbidium sinense 'Dharma'.

Abbreviation: HEMC, hydroxymethylbilane synthase; HEMD, uroporphyrinogen-III synthase; HEME, uroporphyrinogen decarboxylase; CHLH, magnesium chelatase; CHLD, magnesium chelatase subunit ChlD; CHLI, magnesium chelatase subunit ChlI; CHLM, magnesium-protoporphyrin O-methyltransferase; PORA, protochlorophyllide oxidoreductase A; CHLG, Chlorophyll synthase; CAO, chlorophyllide a oxygenase.

Fig 8. Assembled isotigs involved in the chlorophyll degradation pathway pathway of Cymbidium sinense 'Dharma'.

Abbreviation: NOL, chlorophyll b reductase; CLH, chlorophyllase; PAO, pheophorbide a oxygenase, RCCR, red Chl catabolite reductase.

Fig 9. Northern blotting analysis of the expression of identified genes in different tissues of Cymbidium sinense 'Dharma'.

Each lane contained 15 g total RNA isolated from roots, pseudobulbs and six-month old leaves, rRNA served as a loading control (bottom of the panel).

Fig 10. Gene expression analysis.

The qRT-PCR analysis of the expressions of two enzyme-encoding genes in chl degradation pathway (A) and five key enzyme-encoding genes in chl biosynthesis pathway (B). The y-axis indicates fold change in expression among the samples. The Lg(Relative Quantitation) of the genes in the wild-type leaves was calibrated as zero. RNA was extracted from six-month-old normal growth leaves. ACT gene served as the internal control. Error bars indicate the standard deviation of the mean (SD) (n = 3). Three biological replicates were analyzed, with similar results.