Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber

Abstract

Monoecious species provide a comprehensive system to study the developmental programs underlying the establishment of female and male organs in unisexual flowers. However, molecular resources for most monoecious non-model species are limited, hampering our ability to study the molecular mechanisms involved in flower development of these species. The objective of this study was to identify differentially expressed genes during the development of male and female flowers of the monoecious species Quercus suber, an economically important Mediterranean tree. Total RNA was extracted from different developmental stages of Q. suber flowers. Non-normalized cDNA libraries of male and female flowers were generated using 454 pyrosequencing technology producing a total of 962,172 high-quality reads with an average length of 264 nucleotides. The assembly of the reads resulted in 14,488 contigs for female libraries and 10,438 contigs for male libraries. Comparative analysis of the transcriptomes revealed genes differentially expressed in early and late stages of development of female and male flowers, some of which have been shown to be involved in pollen development, in ovule formation and in flower development of other species with a monoecious, dioecious, or hermaphroditic sexual system. Moreover, we found differentially expressed genes that have not yet been characterized and others that have not been previously shown to be implicated in flower development. This transcriptomic analysis constitutes a major step toward the characterization of the molecular mechanisms involved in flower development in a monoecious tree with a potential contribution toward the knowledge of conserved developmental mechanisms in other species.

Keywords: EST; Quercus suber; RNA-seq; cork oak; flower development; monoecious; pyrosequencing; transcriptomics.

Figure 1

Quercus suber female and male flowers in different developmental stages used in RNA-seq. (A) Early and (B) late stages of female flower development used in pools 1F and 2F, respectively. (C) Early and (D) late stages of male flower development used in pools 1M and 2M, respectively. (Cf) female bud enclosed by protective scales; (Df) female reddish bud with open scales; (Ef) elongation of the spike axe and the emergency of the first pair of flowers; (Ff) female flower showing distinct, erect, yellow stigmas with curved pinkish/brownish tips; (Ff2) flower with shining yellow and viscous pattern stigmas in clear divergent position; (Gf) female flower with closed stigmas that lost the receptivity, exhibiting a dark brown color. (Cm) catkin with red round shape due to the tight clustering of the flowers; (Dm) elongated cluster of male flowers; (Dm2) pendent catkin with some individualized flowers; (Em) male flowers with the anthers individualized; (Fm) flowers with individualized green/yellow anthers where pollen shedding begins; (Gm) catkin with male flowers in full anthesis.

Figure 2

Functional classification of Quercus suber unigenes. Four EST projects were generated from four-specific RNA pools, two for female flowers (1F and 2F) and two for male flowers (1M and 2M), covering either early (1F and 1M) or the late (2F and 2M) developmental stages. The four individually EST projects were assembled into the 1F_1M_2F_2M library and the deduced aminoacid sequences of this library were annotated using InterProScan. The Gene Ontology terms (GOs) for each translated amino acid sequence were used to classify the transcript products within the category of (A) cellular component, (B) molecular function, and (C) biological process sub-ontologies.

Figure 3

Relative expression of differentially expressed male and female genes chosen to validate RNAseq results. QsAMS, QsLAP3, QsLAP5, QsLAP6 were selected as male candidate genes, whereas QsAt4g27290, QsCYP78A9, QsPG1, and QsSTIG1 as female candidate genes. Transcript abundance was determined using qPCR, and normalized to QsPP2AA3 using cDNA synthesized from distinct pools of RNA covering either early (1F and 1M) or late (2F and 2M) stages of male or female flower development. Reactions were performed in three biological and technical replicates. Error bars indicate standard deviation (SD).

Figure 4

Description of the Quercus suber unique and differentially expressed genes. (A) Venn diagram indicating the number of exclusive and shared transcripts of early and late developmental stages of Quercus suber flower. Four EST projects were generated from four-specific RNA pools, two for female flowers (1F and 2F) and two for male flowers (1M and 2M), covering either early (1F and 1M) or the late (2F and 2M) developmental stages. The four individually EST projects were assembled into the 1F_1M_2F_2M library and the exclusive transcripts were identified using the Venny application (Oliveros, 2007). (B–J) Differentially expressed genes were clustered using the Self-organizing Trees algorithm (SOTA), euclidean distance (Dopazo and Carazo, ; Herrero et al., 2001) and the default settings of the MeV, MultiExperiment Viewer program (http://www.tm4.org/mev.html).