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- Supporting Information
Gene duplication is a major mechanism generating new templates for evolutionary innovation in eukaryotes (Ohno, 1970; Lynch & Conery, 2000). Gene duplicates may originate from single gene duplications such as tandem and proximal duplications, or from large-scale duplications (Maere et al., 2005). Tandem duplicates are adjacent and mainly result from unequal crossing-over, whereas in proximal duplications duplicates are separated by other genes and may result from unequal crossing-over or transposon activities (Wang et al., 2012). Large-scale gene duplications, including whole-genome duplications (WGDs) and segmental duplications (i.e. duplications of a chromosomal region; Koszul & Fischer, 2009), are frequent in the history of angiosperm genomes. A hexaploidization event (γ triplication) occurred near the origin of eudicots (Jiao et al., 2012). It was followed by lineage-specific duplications in some taxa, such as two tetraploidization events in Arabidopsis thaliana (α and β; Blanc et al., 2003; Bowers et al., 2003) and a hexaploidization in Solanaceae (‘T’ triplication; The Tomato Genome Consortium, 2012). Within monocots, two WGD events were characterized in sequenced Poaceae genomes. The ρ WGD occurred c. 50–70 million yr ago (Ma) (Paterson et al., 2004; Salse et al., 2008) and the σ WGD occurred earlier in the monocot lineage (Tang et al., 2010). In addition, a recent WGD occurred in Zea mays c. 5–12 Ma (Schnable et al., 2009). Recently, the genome of banana (Musa acuminata), a monocotyledon from the order Zingiberales, was sequenced, using DH-Pahang, a doubled haploid (523 Mb; 2n = 22) derived from a seedy diploid of the subspecies malaccensis (D'Hont et al., 2012). This subspecies contributed one of the three M. acuminata genomes of the sterile triploid cultivar Cavendish (AAA genome), which accounts for half of world-wide banana production (Lescot, 2011). Analyses of the banana genome revealed three rounds of WGD (α, β and γ) that were not shared with the Poales or the Arecales (palms) (D'Hont et al., 2012). The α and β WGDs were estimated to have occurred within a short time frame c. 65 Ma, whereas the γ WGD was dated to c. 100 Ma. The availability of the banana genome sequence offers the opportunity to study the evolution of banana gene families in the context of the three WGDs.
Following duplication, paralogous genes can have different fates. They can become pseudogenes or be lost, and it is now well established that, over evolutionary time, most of WGD duplicate genes are lost through fractionation (Lockton & Gaut, 2005). This process has a major impact on the evolution of plant genes, as some of them are preferentially retained after WGD or are found preferentially in a singleton state (Freeling, 2009). In addition, it has been observed that functional categories of genes that were more likely to be retained after WGD were less likely to be retained after tandem duplication and vice versa (Freeling, 2009; Woodhouse et al., 2011; Rodgers-Melnick et al., 2012). In banana, the most preferentially retained gene categories after WGD included transcription factors, signal transduction genes and translational elongation genes, similar to findings in A. thaliana (Blanc & Wolfe, 2004; Maere et al., 2005; D'Hont et al., 2012). Retention of these gene categories has been explained by the gene balance hypothesis (Birchler et al., 2001; Papp et al., 2003; Freeling & Thomas, 2006) which states that genes encoding products that are in a balanced interacting relationship, such as those encoding members of a protein complex or involved in multiple steps in regulatory cascades, will tend to be dosage sensitive because changes in the stoichiometry of individual components will be detrimental. These genes are thus more prone to be co-retained after WGD (Birchler & Veitia, 2007). Other models for duplicate gene retention include neofunctionalization, where one of the duplicates acquires a new function, and subfunctionalization, where the two copies share the function of the ancestral gene (Force et al., 1999).
To analyse gene family evolution, we focused on a key pathway for banana fruit ripening, the ethylene biosynthesis and signalling pathway (Supporting Information Fig. S1). Banana fruits are climacteric; they are characterized by drastic changes in ethylene production with an increased respiration burst during ripening (Burg & Burg, 1965; Liu et al., 1999). In addition, export bananas are ripened by exogenous application of ethylene. In A. thaliana, ethylene is perceived by a family of five ethylene receptors (ethylene response 1 (ETR1), ETR2, ethylene response sensor 1 (ERS1), ERS2 and ethylene-insensitive 4 (EIN4); reviewed by Shakeel et al., 2013). Ethylene receptors act as negative regulators of signalling through constitutive activation of the Ser/Thr kinase constitutive triple response 1 (CTR1; Kieber et al., 1993). The response-to-antagonist 1 (RAN1) protein is a copper transporter that is essential for biogenesis of ethylene receptors (Binder et al., 2010) and reversion-to-ethylene sensitivity 1 (RTE1) is involved in the function of the ETR1 receptor (Resnick et al., 2008). In the presence of ethylene, receptors inactivate CTR1, thus relieving suppression on downstream signalling components. The EIN2 protein, an endoplasmic reticulum-bound protein (Alonso et al., 1999), is processed and its C-terminal domain migrates into the nucleus (Qiao et al., 2012; Wen et al., 2012). There, it activates the EIN3/EIN3-like (EIL) transcription factors which, in turn, initiate the ethylene transcriptional responses by binding to specific elements in promoter regions of genes encoding ethylene response factors (ERFs). Additional regulation of ethylene signalling occurs at the post-transcriptional level: EIN3 protein levels are regulated through EIN3-binding F-box (EBF) proteins which are components of Skp, Cullin, F-box containing (SCF) complexes (Guo & Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). In banana, genes encoding the two main enzymes of the ethylene biosynthesis pathway (1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO)) were identified based on cDNA amplification (two ACO and four ACS genes; Liu et al., 1999; Inaba et al., 2007). In addition, three ERS genes (Yan et al., 2011), one CTR1-like gene (Hu et al., 2012), five EIL genes (Mbéguié-A-Mbéguié et al., 2008), two EBF genes (Chen et al., 2011; Kuang et al., 2013) and 15 ERF genes (Xiao et al., 2013) were identified but no complete inventory of these gene families could be performed.
Here, we identified all members of 10 gene families of the banana ethylene pathway using genome-scale approaches. We analysed their evolutionary patterns with a specific focus on the EIL and EBF gene families which play a central role in the control of ethylene signalling. Our results showed expansion of several ethylene gene families after banana WGD. Based on expression data, the co-expansion of EBF genes and of a specific subgroup of EIL genes is partly associated with functional redundancy; however, subfunctionalization also occurred in both families.