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Polyploidy (whole-genome duplication, WGD) has played a major role in speciation and genome evolution in diverse organisms, including yeast (Kellis et al., 2004), vertebrates (Dehal & Boore, 2005; Hufton et al., 2008) and plants (Soltis & Soltis, 2000; Wendel, 2000; Osborn et al., 2003; Adams & Wendel, 2005; Chen & Ni, 2006; Chen, 2007; Jiao et al., 2011). Early estimates (based on chromosome number) of the frequency of polyploidy in angiosperms ranged from 30% to 80%. However, recent studies have revealed that all angiosperms have experienced one or more rounds of polyploidy. Angiosperms for which genome sequences are available exhibit evidence of at least one round of polyploidy (Arabidopsis Genome Initiative, 2000; Simillion et al., 2002; International Rice Genome Sequencing Project, 2005; Tuskan et al., 2006; Jaillon et al., 2007; Velasco et al., 2007; Wei et al., 2007; Ming et al., 2008; Huang et al., 2009; Paterson et al., 2009; Schmutz et al., 2010); genomic and transcriptomic data (including expressed sequence tags (ESTs)) across a diversity of angiosperms reveal multiple ancient polyploidization events (Ku et al., 2000; Cui et al., 2006). Indeed, ancient polyploidy events are associated with the emergence of both seed plants and angiosperms (Jiao et al., 2011), as well as with major large clades within angiosperms (Soltis & Soltis, 2000; Soltis et al., 2009; Fawcett & Van de Peer, 2010).
The most obvious genetic consequence of polyploidy is the simultaneous duplication of all genes, which may provide increased evolutionary potential for any given polyploid. Although genes duplicated via polyploidy can be subsequently silenced or lost (nonfunctionalization), the divergence of duplicated genes can provide genetic redundancy on which mutation and selection may act. These processes may lead to the possible acquisition of new gene functions (neofunctionalization) and the acquisition of divergent gene functions, such as tissue-specific gene expression (subfunctionalization); both neo- and subfunctionalization may be important sources of novelty and adaptability (Kirschner & Gerhart, 1998; Lynch & Conery, 2000; Force et al., 2005; He & Zhang, 2005). Studies of the expression patterns of duplicated genes in polyploids have been conducted using systems such as Arabidopsis (Comai et al., 2000; Lee & Chen, 2001; Madlung et al., 2002, 2005; Lawrence et al., 2004; Wang et al., 2004, 2006), cotton (Zhao et al., 1998; Liu et al., 2001; Adams et al., 2003; Hovav et al., 2008; Chaudhary et al., 2009; Flagel & Wendel, 2009), wheat (Feldman et al., 1997; Shaked et al., 2001; Kashkush et al., 2002, 2003; He et al., 2003; Bottley et al., 2006; Bottley & Koebner, 2008; Pumphrey et al., 2009), Brassica (Song et al., 1995; Lukens et al., 2004, 2006; Gaeta et al., 2007; Marmagne et al., 2010), Spartina (Baumel et al., 2001; Ainouche et al., 2004; Salmon et al., 2005; Fortune et al., 2007), Senecio (Hegarty et al., 2005, 2006, 2008; Hegarty & Hiscock, 2008) and Tragopogon (Tate et al., 2006; Buggs et al., 2009; Koh, 2010; Koh et al., 2010). Most of these approaches have been carried out at the genomic or transcript level using cDNA-amplified fragment length polymorphism (cDNA-AFLP) display, CAPS (cleaved amplified polymorphic sequences), reverse transcriptase-PCR or microarrays. Very few studies have examined the outcome of gene expression at the protein level in a polyploid and its parents (Bahrman & Thiellement, 1987; Albertin et al., 2005, 2006; Hu et al., 2011; Ng et al., 2012).
Proteomics is an important complement to genomics and studies of gene expression because proteins are the final products of genes and are more directly related to cellular metabolism and phenotypes. Indeed, the relationship between RNA transcripts and protein abundance is not direct because of post-transcriptional regulation and post-translational modifications (PTMs) (Gygi et al., 1999), making predictions about the proteome of a polyploid relative to its diploid progenitors difficult. Therefore, the application of proteomic approaches to polyploid systems will enhance our understanding of polyploid evolution and adaptation. Only a few studies have implemented proteomic analysis in polyploids. These include studies on Triticum (wheat; Bahrman & Thiellement, 1987; Islam et al., 2003), Brassica napus (Albertin et al., 2006), Gossypium (cotton; Hu et al., 2011) and Arabidopsis suecica (Ng et al., 2012). Several studies have shown additivity of the parental profiles in the proteomes of allopolyploids (Triticum, Gossypium, Arabidopsis) (Bahrman & Thiellement, 1987; Hu et al., 2011; Ng et al., 2012), as well as increased protein production in an autopolyploid (Brassica oleracea; Albertin et al., 2005); nonadditive patterns have been observed in wheat (Bahrman & Thiellement, 1987), cotton (Islam et al., 2003; Hu et al., 2011) and synthetic B. napus (Albertin et al., 2006). However, all of these studies employed two-dimensional electrophoresis (2-DE), which has limitations, including gel-to-gel variation and problems with quantification based on spot intensity. In addition, Triticum, Gossypium and Brassica are polyploid crops and may have experienced strong artificial selection during their evolutionary history, possibly masking other effects. Therefore, the use of robust methodology and the examination of a naturally occurring allopolyploid system will provide valuable new insights into the impact of hybridization and polyploidization on the proteome.
Two natural allotetraploids of Tragopogon have become models for polyploidy research (Soltis & Soltis, 1999; Soltis et al., 2004, 2012). Tragopogon mirus and T. miscellus were formed by genomic merger of the diploid species T. dubius and T. porrifolius, and T. dubius and T. pratensis, respectively. Polyploidization occurred in the northwestern USA after the introduction of the diploid progenitors from Europe in the early 1900s. The three diploids were not all reported from this region before 1928, and so T. mirus and T. miscellus cannot be more than c. 80 yr old (Ownbey, 1950; Soltis et al., 2004). Studies have revealed rapid homeolog loss, plus differential gene expression, in both polyploids relative to their parents, as well as chromosomal rearrangement and compensating aneuploidy (Tate et al., 2006, Tate et al. 2009; Lim et al., 2008; Buggs et al., 2009a,b, 2010, Buggs et al., 2011; Buggs et al. 2012; Koh, 2010; Koh et al., 2010; Chester et al., 2012). Here, we examined the proteomes of natural allopolyploid T. mirus and its diploid parents (T. dubius and T. porrifolius) on a per-cell basis using isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS technology. We included an artificial F1 diploid hybrid line between T. dubius and T. porrifolius and a synthetic (S1 generation) allotetraploid T. mirus line. Analysis of these materials will help to determine the relative contribution of hybridization and genome doubling to expression differences at the proteomic level in young polyploids. We sought to determine whether: (1) differences in protein expression are present among the diploid parents, artificial F1 hybrid, natural allopolyploids and synthetic polyploid, (2) hybridization or polyploidy has had a larger impact on changes in protein expression, and (3) any proteomic changes correlate with existing changes obtained from transcriptional analysis (Koh, 2010; Koh et al., 2010).
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Fig. S1 Example of cation exchange chromatography of iTRAQ labeled peptides derived from Tragopogon porrifolius, T. dubius, T. mirus, the diploid hybrid, and the synthetic allopolyploid.
Fig. S2 Novel expression in diploid F1 hybrids and protein posttranslational modifications (PTMs).
Table S1 Primer sequences using quantitative Real-time RT-PCR
Table S2 Number of proteins identified at critical false discovery rates from three analyses
Table S3 476 identified proteins from all three species
Table S4 Expression patterns in Tragopogon mirus inferred from quantitative Real-time PCR