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Diversity and evolution of Pong-like elements in Bambusoideae subfamily
Biochemical Systematics and Ecology 38 (2010) 750–758
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Diversity and evolution of Pong-like elements in Bambusoideae subfamily Hao Zhong 2, Mingbing Zhou 1, Chuanmei Xu, Ding-Qin Tang* The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, LinAn 311300, Zhejiang Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2010 Accepted 26 June 2010
The PIF/IS5 is a recently discovered superfamily of DNA transposons which include Ponglike elements and PIF-like elements and has been successively detected in the genomes of many flowering plants, fungi and diverse animals. Here we present the first comprehensive characterization and analysis of Pong-like elements in Bambusoideae subfamily. Eighty-two Pong-like elements were cloned and sequenced from 44 representative species of Bambusoideae. Phylogenetic analysis of 82 distinct Pong-like elements sequences showed that Pong-like elements were widespread, diverse and abundant in Bambusoideae. A molecular phylogeny of Bambusoideae was established by using the internal transcribed spacer sequence of nuclear ribosomal DNA (ITS) information. The comparison between ITS and Pong-like elements based trees reveals obviously incongruent. The results suggest that 1) there are multiple Pong-like element families in Bambusoideae; 2) a single Pong-like element family could be present in multiple bamboo species; 3) Pong-like elements from the same family from different bamboo species could be more similar than elements from different families in the same bamboo species or closely related species. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Bambusoideae Diversity Evolution Pong-like elements
1. Introduction The bamboo subfamily (Bambusoideae) is a division of the grasses (Poaceae) and is further divided into nine subtribes comprising 77 genera and about 1030 species worldwide (Soderstrom and Ellis, 1987; Dransfield and Widjaja, 1995). Bamboo can be classified as woody, herbaceous or liana according to growth characteristics, and grows predominantly in tropical and subtropical regions although a few species grow in temperate and frigid zones (Chen et al., 2003). In China, there are 48 genera and nearly 500 species, among which Phyllostachys pubescens accounts for over two-thirds of commercially planted bamboo because of its high economic value. Many different cultivars or forms of Ph. pubescens with diverse phenotypes have been produced during its long cultivation history (Fu, 2001), and fewer AFLP and SSR diversity was observed among these cultivars and forms (Lin et al., 2009; Tang et al., 2010). Additionally, long term in vitro culture induces stable changes in leaf color in Bambusa oldhamii in our lab. The mechanisms of variations are still not clear. One of the possible mechanisms to explain variation may be the activation of transposable elements (Bennetzen, 2000). Transposable elements are sequences of DNA that can move around to different positions within the genome of a single cell, which are divided into class I and class II according to mechanism of transposition. Class I elements (retrotransposons) transpose by means of an RNA intermediate in a reaction involving several enzymes of reverse transcriptase and integrase. In contrast, class II elements (DNA transposons) move directly via DNA and the transposition reaction is catalyzed by a transposase encoded by autonomous DNA transposons (Bennetzen, 2000; Feschotte et al., 2002).
* Corresponding author. Tel.: þ86 57163748811; fax: þ86 57163732738. E-mail address: [email protected] (D.-Q. Tang). 1 These authors contributed equally to this work. 2 Who now works in China National Bamboo Researcher Center. 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.06.010
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Miniature inverted-repeat transposable elements (MITEs) are reminiscences of class II nonautonomous transposable elements with high copy number and intra-family homogeneity in size and sequence (Wessler et al., 1995). The mPing was the first active MITE identified in rice (Jiang et al., 2003; Kikuchi et al., 2003), and Pong transposon has been identified as the most likely source of mPing transposase (Jiang et al., 2003). Many transposases from nematodes, fungus, animals and plants with homologous motifs of Pong in rice were named Pong-like transposable elements (Zhang et al., 2004). Pong-like elements contain ORF1 and ORF2, of which ORF2 most likely encodes the transposase because it contains an apparent DDE motif, a signature consisting of three acidic residues found in the catalytic domains of some eukaryotic and prokaryotic transposases (Rezsohazy et al., 1993; Mahillon and Chandler, 1998; Jiang et al., 2003). The domain of amino acids surrounding the acidic triad is relatively well-conserved and thus has served to establish the evolutionary relationships of Pong-like transposon elements in plants (Zhang et al., 2004). In this study we performed first comprehensive analysis Pong-like superfamily of transposases in Bambusoideae by PCR with newly designed degenerate primers. Phylogenetic analyses of 82 Pong-like transposase fragments from 44 representative species of bamboo indicated that multiple divergent lineages of Pong-like transposases coexisted within a single plant species. Comparison with the phylogenetic data of ITS information shows that there are multiple Pong-like element families in Bambusoideae, and a single family could be present in multiple bamboo species, and Pong-like elements from the same family from different species could be more similar than elements from different families in the same bamboo species or closely related species. 2. Materials and methods 2.1. Bamboo materials and DNA extraction According to taxonomy of bamboo (Soderstrom and Ellis, 1987; Geng and Wang, 1996; Li, 1997; Das et al., 2008), we selected 44 representative species from 38 genera of 6 subtribes mainly distributing in China. In the intra-species level, we also selected 9 cultivars or forms derived from Ph. pubescens, which showing morphological differences in stem shape and color, and leaf color (Lin et al., 2009). Bamboo species and its cultivars or forms examined along with their taxonomic classification and voucher are listed in Table 1. Oryza sativa ssp. japonica cv. Nipponbare was collected for cloning of ITS fragment used as outgroup of phylogenic tree based on bamboo ITS sequences. Young leaves were sampled from each species (cultivars or forms) and desiccated for DNA extraction. Genomic DNA was extracted using the method of hexadecyltrimethylammonium bromide (CTAB) (Doyle and Doyle, 1987) with a few modifications. 2.2. PCR amplification and sequencing of Pong-like transposases Degenerate primers (Pong-5: GGCWCCATYGAYTGTATGCAC, Pong-3: YTCGTCYTCVACYATCATRTTGTG) used to detect the Ponglike elements were derived from the regions encoding amino acid residues motifs GTIDCMH and NMIVEDE conserved in Pong-like transposases of most flowering plants. PCR amplifications were performed with 50–100 ng of genomic DNA, 0.2 mmol/L primer pair (synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd), 2 units ExTaq Polymerase, 1 PCR buffer (10 mmol/L Tris–HCl, pH 8.3 at 25 C; 50 mmol/L KCl), 1.5 mmol/L MgCl2, and 0.2 mmol/L of dNTP each (TaKaRa Japan) in a final reaction volume of 20 mL. Cycling temperature parameters were: 1 cycle at 94 C for 5 min, 35 cycles at 94 C for 60 s, 57 C for 40 s, 72 C for 1 min, and 1 cycle at 72 C for 5 min. ITS have been proved to be better marker for systematic analysis for bamboo species (Guo and Li, 2004; Yang et al., 2008). Bamboo and O. sativa ITS sequences were amplified using primers ITS5 and ITS4 were used to amplify the whole ITS region as described by White et al. (1990). The thermal cycling comprised 30 cycles at 94 C for 1.5 min, at 55 C for 2 min primer annealing, at 72 C for 1 min, followed by a final extension step at 72 C for 7 min. Both Pong-like element and ITS PCR amplification products were resolved by 1% agarose gels electrophoresis, purified using the EZ-10 Spin Column DNA Gel Extraction Kit (Biobasic Inc.) and ligated into the pUC18 Vectors (TaKaRa, Japan) for cycle sequencing using BigDye terminator V1.3 system (PE Applied Biosystems) on an ABI PRISM 3100-Avant according to the manufacturer’s instructions. For Pong-like element detection, one positive clone of Pong-like element for each species was selected for sequencing. For intra-species of Ph. pubescens in order to identify as many Pong-like transposases as possible many independent positive clones were selected from each of its cultivars or forms for sequencing until at least three repeat clones were detected for every species. For ITS regions identified, one positive clone from 44 representative species and O. sativa was selected for sequencing. The resulting 82 Pong-like transposase sequences and 45 ITS sequences were deposited in the NCBI GenBank database (accession numbers GU350795–GU350876 and GQ464804–GQ464847, GU205182). Each element was denominated using the first letter of the genus name and the first letter of the species name followed by the clone number, for example: Bm-1 for Bambusa multiplex Pong-like element clone 1. Pong-like transposases which were obtained by database searches were named according to the species initials followed by NCBI accession no. by Zhang et al. (2004). 2.3. Database searching and phylogenetic analysis Database searches for the known Pong-like elements were performed with BLAST servers available from the NCBI nr, gss, est and wgs databases (http://www.ncbi.nlm.nih.gov) as well as the TIGR rice genomic database (http://tigrblast.tigr.org/eukblast/index). Nucleotide sequences of Pong-like elements from database and PCR amplification were conceptually translated into amino acid sequences. The translated amino acid sequences of Pong-like elements and ITS nucleotide sequences were aligned using CLUSTALW (Thompson et al., 1994) with default parameters, respectively.
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Table 1 Species whose Pong-like element sequences were analyzed in this study, and GenBank accession numbers. Taxa
Abb
Source
ITS
Pong-like elements
Subtribe Melocanninae Melocanna baccifera Cephalostachyum pergracile Monocladus saxatilis Schizostachyum funghomii Pseudostachyum polymorphum
Mb Cp Ms Sf Pp
Guangdong, China Yunnan, China Fujian, China Yunnan, China Yunnan, China
GQ464827 GQ464810 GQ464829 GQ464842 GQ464837
GU350795 GU350796 GU350797 GU350798 GU350799
Subtribe Bambusinae Bambusa multiplex Bambusa chungii Bambusa arundinacea Dendrocalamopsis oldhami Gigantochloa levis Thyrsostachys oliveri Neosinocalamus affinis Dendrocalamus minor Melocalamus arrectus
Bm Bc Ba Do Gl To Na Dm Ma
Yunnan, China Yunnan, China Ghana Guangdong, China Fujian, China Fujian, China Yunnan, China Zhejiang, China Yunnan, China
GQ464807 GQ464806 GQ464805 GQ464815 GQ464820 GQ464846 GQ464830 GQ464816 GQ464826
GU350800 GU350801 GU350802 GU350803 GU350804 GU350805 GU350806 GU350807 GU350808
Subtribe Shibataeeae Hibanobambusa tranguillans Indosasa shibataeoides Brachystachyum densiflorum Phyllostachys pubescens Ph pubescens cv. Anjiensis Ph pubescens cv. Gracilis Ph pubescens cv. Heterocycla Ph pubescens cv. Luteosulcata Ph pubescens cv. Obliquinoda Ph pubescens cv. Tao Kiang Ph pubescens cv. Tubaeformis Ph pubescens cv. Ventricosa Ph pubescens cv. Viridisulcata Shibataea chinensis Semiarundinaria fastuosa Qiongzhuea tumidinoda Sinobambusa tootsik Chimonobambusa marmorea Chimonobambusa quadrangularis
Ht Is Bd Ph PhY PhG PhP PhL PhO PhK PhT PhV PhW Sc Se Qt St Cm Cq
Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China
GQ464822 GQ464825 GQ464809 GQ464833 – – – – – – – – – GQ464844 GQ464843 GQ464838 GQ464845 GQ464811 GQ464812
GU350809 GU350810 GU350811 GU350812–GU350816 GU350821–GU350824 GU350825–GU350828 GU350829–GU350830 GU350831–GU350835 GU350836–GU350839 GU350840–GU350843 GU350844–GU350846 GU350847–GU350848 GU350849–GU350850 GU350851 GU350852 GU350853 GU350854 GU350855 GU350856
Subtribe Chusqueeae Chusquea coronalis
Ca
Yunnan, China
GQ464814
GU350857
Subtribe Arudinarieae Fargesia fungosa Himalayacalamus intermedia Yushania uniramosa Pseudosasa japonica Acidosasa gigantea Pleioblastus gramineus Pleioblastus chino Bashania fargesii Indocalamus latifolius Oligostachyum sulcatum Gelidocalamus annulatus Menstruocalamus sichuanensis Sasa fortunei Sasa sinica Sasa veitchii Chimonocalamus pallens Drepanostachyum luodianense
Ff Hi Yu Pj Ag Pg Pc Bf Il Os Ge Mo Sa Ss Sv Ch Dl
Zhejiang, China Shizuoka, Japan Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China
GQ464818 GQ464823 GQ464847 GQ464836 GQ464804 GQ464835 GQ464834 GQ464808 GQ464824 GQ464831 GQ464819 GQ464828 GQ464839 GQ464840 GQ464841 GQ464813 GQ464817
GU350858 GU350859 GU350860 GU350861 GU350862 GU350863 GU350864 GU350865 GU350866 GU350867 GU350868 GU350869 GU350870 GU350871 GU350872 GU350873 GU350874
Subtribe Guaduinae Otatea acuminata Guadua angustifolia
Oa Ga
Mexico Ecuador
GQ464832 GQ464821
GU350875 GU350876
The best-fit model of nucleic substitution of Pong-like element and ITS sequences was tested by software Modeltest 3.7 (Posada and Crandall, 1998) with Akaike information criterion (Sullivan and Joyce, 2005). GTR model (GTR þ I þ G) was the optimum for Pong-like elements and TIMef þ I þ G was the optimum for ITS sequences. The phylogenetic relationships among the bamboo Pong-like elements were investigated from either the nucleic consensus sequences or their in silico translation into protein sequences, while the phylogenetic relationships among Bambusoideae were investigated from the ITS nucleic sequences. All
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phylogenetic trees were generated using neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) with PAUP software v4.0b10 (Swofford, 2002) and Bayesian inference (BI) with MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), and reswapped with NEAREST NEIGHBOR INTERCHANGE by using Molphy v2.3b3 (Adachi and Hasegawa, 1996). Rearranged topologies were very similar to those initially generated by four methods, at least for the groupings discussed in this study. 3. Results 3.1. Identification and polymorphism of transposases of Pong-like elements in the Bambusoideae subfamily A pair of degenerate primers was designed to amplify a fragment of the transposase gene containing “DDE” domain of Pong-like transposase (Zhang et al., 2004). PCR with the degenerate primers yielded one uniform size fragment of approximately 520-bp for each bamboo species (Fig. 1). Taken together, 82 amplification fragments from 44 representative species and 9 Ph. pubescens cultivars or forms were obtained and sequenced. At the amino acid level, the bamboo sequences showed between 67.2% and 79.1% identity with an identified typical Ponglike element from O. sativa (OsBK000586, accession number BK000586) (data not shown). At least one representative species were selected from six subtribes (Melocanninae, Bambusinae, Shibataeeae, Chusqueeae, Arundinarieae and Guaduinae) for detecting the conserved functional domains regions. Several blocks of highly conserved residues were consistent with those of Pong-like transposases which most likely comprise the catalytic domain. The conserved amino acid residues of DDE motif were identified in all fragments. First D is present at the position 4, second D present at the position 94 and the E present at the position 171 of the consensus amino acid sequence (Fig. 2). The data confirmed that the all PCR products contained the DDE region of the Pong-like transposase genes. Almost all 82 sequences of Pong-like transposases we cloned in bamboo species were unique. Pairwise comparison showed 57.8–99.6% identity at the nucleotide sequences level and 54.9–100% identity at the amino acid level (supplementary Tables 1, 2). Comparison within each subtribe revealed 90.6% identity among the Guaduinae, 71.7–86.7% among the Melocanninae, 63.3– 97.1% among the Bambusinae, 62.7–100% among the Shibataeeae, and 61.8–100% among the Arundinarieae at amino acid level (supplementary Table 2). The results indicate Pong-like transposases are highly polymorphic in bamboo species tested. 3.2. Phylogenetic position of bamboo Pong-like elements in flowering plants Up to date, at least three subfamilies of Pong-like elements have been reported in flowering plants, i.e., clade O, clade P and clade Q, of which Clade O and Clade P can be further classed into subclade O1, O2 and subclade P1, P2, respectively (Zhang et al., 2004). In order to place the above characterized bamboo Pong-like elements in this phylogenetic tree, several elements representing each of the three subfamilies previously reported (Zhang et al., 2004) were aligned with bamboo Ponglike elements. Three Pong-like transposase clusters similar to the clades mentioned above are shown and are defined as the largest well-supported monophyletic group of sequences obtained from phylogenetic trees generated by four distinct methods (NJ, MP, ML and BI, all bootstrap values >63%, Fig. 3). Most of bamboo Pong-like elements clustered in two branches of tree. First, significant portion of bamboo Pong-like elements grouped into clade Q (bootstrap value of 78%). Second, the three bamboo Pong-like elements grouped into subclade O2 (bootstrap value of 100%). It was noted that there were three bamboo Pong-like elements which could not grouped into any clades defined by Zhang et al. (2004) and clustered as new clade BaS (bootstrap value of 82%). 3.3. Evolution of bamboo Pong-like elements To analyze of phylogenetic relationships among Bambusoideae Pong-like elements, phylogenetic tree was generated from amino acid sequences of the 44 representative species that were selected to represent the different lineages within each subtribe with one PIF-like element from a fungus (Filobasisiella neoformans) as the outgroup (Fig. 4A). Meanwhile the analysis of ITS sequences allowed the construction of a parallel tree showing the phylogenetic relationship among the same 44 bamboo species with the ITS sequence from O. sativa as outgroup (Fig. 4B). The ITS tree supported the existence of six subtribes, i.e.
Fig. 1. Detection of Pong-like transposase genes in bamboo subfamily by using PCR with degenerate primers. Representative examples are shown from 10 species. M: 100-bp ladder. 1. Sasa fortunei, 2. Bashania fargesii, 3. Acidosasa gigantean, 4. Himalayacalamus intermedia, 5. Chimonobambusa marmoreal, 6. Semiarundinaria fastuosai, 7. Dendrocalamus minor, 8. Indosasa shibataeoides, 9. Hibanobambusa tranguillans, 10. Schizostachyum funghomii.
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Fig. 2. Structure and conserved coding regions of the amplification fragments of Pong-like elements. Multiple alignments of conceptually translated amplification fragments from bamboo Pong-like elements are shown. Horizontal arrows indicate the annealing position of the dPCR primers, and DD37E motifs in transposases are indicated by asterisks. A consensus sequence of OsBK000586, the Pong-like elements in rice is shown above alignments for comparison. Sequences were named according to the species initials followed by their clone number (Table 1 for abbreviations).
Melocanninae, Bambusinae, Shibataeeae, Chusqueeae, Arundinarieae, Guaduinae. In contrast, 44 Pong-like elements from Bambusoideae grouped into 4 clusters (QBaI, QBaII BaS and O2) which indicated that there are four Pong-like element families in Bambusoideae and a single family could be present in multiple species. It is also noted that Pong-like elements from the same family from different species could be more similar than elements from different families in the same bamboo subtribes or closely related species. For example, Ht-1 Pong-like element (Hibanobambusa tranguillans) was 100% identity with Ss-1 Pong-like element (Sasa sinica) at the amino-acid level, while they are from subtribes of Shibataeeae and Arundinarieae, respectively. In
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Fig. 3. Phylogenetic position of bamboo Pong-like elements in the plant Pong-like elements family. The phylogenetic tree was generated by multiple alignments of 95 catalytic DDE motifs of Pong-like transposase fragments including 82 from 44 representative species of Bambusoideae subfamily isolated by PCR and 13 obtained by database searches which were marked by circles. Main bootstrap values (1000 replicates) are shown. Groupings defining lineages and sublineages of plant Pong-like transposases are emphasized by capital letters and different colors. Sequences were named according to the species initials followed by the number of the clone (see Table 1 for abbreviations), Os ¼ Oryza sativa (rice), Lj ¼ Lotus japonicas, At ¼ Arabidopsis thaliana, Bo ¼ Brassica oleracea, Sb ¼ Sorghum bicolor.
contrast, Pong-like elements from the same bamboo subtribes or closely related species could be more divergent than that from different subtribes species or distant related species, e.g. two diverse Pong-like transposases subclades were present in Melocanninae group, Pp-1, Ms-1 and Mb-1 from subclade QBa I shared 72.4%–75.3% identity with Sf-1 and Cp-1 from the clade QBaII. The phenomenon was farther supported by the analysis of phylogenetic relationships among intra-species Pong-like transposases. Another phylogenetic tree was constructed from 39 amino acid sequences from nine different cultivars or forms of Ph. pubescens (Fig. 4C). All Pong-like transposases from cultivars or forms of Ph. pubescens belong to clade Q. They could further be clustered into three subclades names as QPhI, QPhII and QPhIII. Pong-like elements from the same cultivars (such as PhK-1 and PhK-2 from Ph. pubescens cv. Heterocycla) demonstrated a 43.1% lower level of identity than that from different cultivars (such as PhO-1 from Ph pubescens cv. Obliquinoda). This revealed that Pong-like elements have evolved relatively independent of taxonomy of bamboo and even one individual may display more than one subclade of Pong-like elements in its genome. 4. Discussion 4.1. Pong-like elements were widespread and abundant in Bambusoideae subfamily The PIF/IS5 is a recently discovered superfamily of DNA transposons which include Pong-like elements and PIF-like elements and has been successively detected in the genomes of flowering plants, fungi and animals (Le et al., 2001; Zhang et al., 2001, 2004; Kapitonov and Jurka, 2004). Here we present the first comprehensive characterization and analysis of Pong-like elements in Bambusoideae subfamily. Of all 9 subtribes in Bambusoideae subfamily, 6 subtribes including 44 species
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Fig. 4. Phylogenetic analysis of Pong-like transposase fragments and ITS sequences within the Bambusoideae subfamily. Sequences were named according to the species initials followed by the number of the clone (see Table 1 for abbreviations). Main bootstrap values (1000 replicates) are shown. Groupings defining lineages and sublineages of bamboo Pong-like transposase are emphasized by capital letters and different colors, : Shibataeeae; : Arundinarieae; : Melocanninae; : Bambusinae; -: Guaduinae; : Chusqueeae. A. Phylogenetic relationships among bamboo Pong-like transposase fragments. The phylogenetic tree was generated by multiple alignments of 44 Pong-like transposase fragments obtained by PCR from representative bamboo species. B. Phylogenetic relationships of ITS sequences from the same representative bamboo species, obtained as above. C. Phylogenetic relationships among 39 Pong-like transposase fragments isolated from 10 Ph. pubescens cultivars or forms, obtained as above.
of 38 genera mainly distributed in China were selected for testing materials. These tested bamboo species covered type of woody (most species) and liana (Melocalamus arrectus, etc.), distributed in as south to equatorial America (Guadua angustifolia) and north to temperate regions (Phyllostachys species in Beijing) (Clayton et al., 2006). All the species studied displayed Pong-like elements in their genome by degenerate primers PCR amplification. Almost all the sequences of cloned products were unique and diverse with 57.8–99.6% identity at the nucleotide sequences level, of which three sequences were
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species-specific of bamboo (Clade BaS). Intra-species level, 39 Pong-like elements from Ph. pubescens and its cultivars or forms were identified. Sequencing of 5 PCR products from Ph. pubescens showed as different Pong-like elements. All the results show that Pong-like elements are indeed abundant and heterogeneous in the Bambusoideae subfamily. Genome-wide analysis identified total 80 Pong-like elements in O. sativa (Zhang et al., 2004). The DNA contents of bamboo were 2.45–5.3 pg DNA/2C, and the temperate bamboo (Phyllostachys species) falling within the range 4.17–5.3 pg (Gielis et al., 1997). The genome size of Ph. pubescens was estimated to be about 2034 Mb, approximately 5.4 times that of the diploid cultivated rice (Gui et al., 2007). Given that amplification of transposable elements is largely responsible for the huge differences in plant genome size (Bennetzen, 2002; Feschotte et al., 2002), it is reasonable to assume that even larger families of Pong-like transposable elements will be found once bamboo genomes are sequenced. 4.2. Evolution of Pong-like transposable elements in Bambusoideae subfamily The significant portion of Pong-like elements from Bambusoideae subfamily in this study could be clustered into clade Q and several into subclade O2. Clade Q has been proved to be ancient origin in plant Pong-like elements family (Zhang et al., 2004). The available fossil evidence and the surviving basal lineages suggested the Bambusoideae were evolved in Gondwanaland during the upper Cretaceous of more than 65 Myr ago (Guo et al., 2002). The results suggest that most Pong-like elements from Bambusoideae subfamily are likely transposable elements of ancient origin. We constructed a comparative ITS-based phylogeny of Bambusoideae subfamily which agrees with the previous analyses based on morphologic and molecular characters (Soderstrom and Ellis, 1987; Li, 1997; Das et al., 2008; Yang et al., 2008). The phylogenetic tree constructed with the Pong-like element sequences was obviously incongruent with the ITS-based phylogeny. The incongruence was particularly reflected by the presence of near identical Pong-like elements from distantly-related species and the presence of very diverse Pong-like elements within the same cultivars of Ph. pubescens. There are at least four alternative hypotheses to explain the result. First, Pong-like elements analyzed from 44 representative bamboo species in this research are most likely paralogous not orthologous because of high heterogeneity (supplementary Tables 1, 2). Second, bamboo Pong-like elements originated from different ancestors (Fig. 4A, C). In every clade, divergent evolution of different ancestors might be different. Ancestral duplication and/or polymorphism may create paralogous copies of transposable elements that evolve distinctly in different genomes (Davis and Wurdack, 2004; Mower et al., 2004). Third, usually, transposable elements suffer more tough natural selection than other sequences due to their potential impact of transposition on the host genome. There are at least three deleterious effects of transposable elements in the host genome including mutations resulting from insertions into genes or regulatory sequences (Finnegan, 1992), chromosomal rearrangements caused by ectopic recombination between elements in non-homologous insertion sites (Montgomery et al., 1987) and direct costs due to its transposition activity (Brookfield, 1991). So the sequences of transposable elements provide a more robust target for natural selection than other sequences (Pritham, 2009). When transposon activity is suppressed, such elements could be eliminated from the population by stochastic loss or vertical extinction, a process that may occur in one species but perhaps not in a closely related one (Hartl et al., 1997). Fourth, horizontal transfer might be another potential explanation about the incongruence. The scenario of horizontal transfer remains a remote speculation, until a common transmission vector is identified (Capy et al., 1998; Won and Renner, 2003; Bergthorsson et al., 2004; Richardson and Palmer, 2007). Although hundreds of Pong-like element sequences were identified by database search there is so far no clear indication for any horizontal movement of Pong-like elements among plants (Zhang et al., 2004). In this study, 82 Pong-like element sequences were isolated from bamboo and phylogenetic analysis of these sequences could not prove beyond doubt the occurrence of horizontal transfer. Large-scale sequencing of bamboo species would allow to the evolution of Pong-like elements to be studied in more details. Acknowledgments We are grateful to Ma Naixun of Research Institute of Subtropical Forestry and Xingchun Lin of Key Lab for Modern Silvicultural Technology of Zhejiang Province for their advices on sketching the sampling strategies. This work was supported by a special grant from the National Natural Science Foundation of China (grant nos. 30371181 and 30771753) and Talents Program of Natural Science Foundation of Zhejiang Province (grant no. R303420). Appendix. Supplementary information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bse.2010.06.010. References Adachi, J., Hasegawa, M., 1996. MOLPHY: Programs for Molecular Phylogenetics Based on Maximum Likelihood 2.3. The Institute of Statistical Mathematics, Tokyo. Bennetzen, J.L., 2000. Transposable element contributions to plant gene and genome evolution. Plant Mol. Biol. 42, 251–269. Bennetzen, J.L., 2002. Mechanisms and rates of genome expansion and contraction in flowering plants. Genetica 115, 29–36. Bergthorsson, U., Richardson, A.O., Young, G.J., Goertzen, L.R., Palmer, J.D., 2004. Massive horizontal transfer of mitochondrial genes from divers land plant donors to the basal angiosperm Amborella. Proc. Natl. Acad. Sci. U.S.A. 101, 17747–17752.
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Diversity and evolution of Pong-like elements in Bambusoideae subfamily Hao Zhong 2, Mingbing Zhou 1, Chuanmei Xu, Ding-Qin Tang* The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, LinAn 311300, Zhejiang Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2010 Accepted 26 June 2010
The PIF/IS5 is a recently discovered superfamily of DNA transposons which include Ponglike elements and PIF-like elements and has been successively detected in the genomes of many flowering plants, fungi and diverse animals. Here we present the first comprehensive characterization and analysis of Pong-like elements in Bambusoideae subfamily. Eighty-two Pong-like elements were cloned and sequenced from 44 representative species of Bambusoideae. Phylogenetic analysis of 82 distinct Pong-like elements sequences showed that Pong-like elements were widespread, diverse and abundant in Bambusoideae. A molecular phylogeny of Bambusoideae was established by using the internal transcribed spacer sequence of nuclear ribosomal DNA (ITS) information. The comparison between ITS and Pong-like elements based trees reveals obviously incongruent. The results suggest that 1) there are multiple Pong-like element families in Bambusoideae; 2) a single Pong-like element family could be present in multiple bamboo species; 3) Pong-like elements from the same family from different bamboo species could be more similar than elements from different families in the same bamboo species or closely related species. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Bambusoideae Diversity Evolution Pong-like elements
1. Introduction The bamboo subfamily (Bambusoideae) is a division of the grasses (Poaceae) and is further divided into nine subtribes comprising 77 genera and about 1030 species worldwide (Soderstrom and Ellis, 1987; Dransfield and Widjaja, 1995). Bamboo can be classified as woody, herbaceous or liana according to growth characteristics, and grows predominantly in tropical and subtropical regions although a few species grow in temperate and frigid zones (Chen et al., 2003). In China, there are 48 genera and nearly 500 species, among which Phyllostachys pubescens accounts for over two-thirds of commercially planted bamboo because of its high economic value. Many different cultivars or forms of Ph. pubescens with diverse phenotypes have been produced during its long cultivation history (Fu, 2001), and fewer AFLP and SSR diversity was observed among these cultivars and forms (Lin et al., 2009; Tang et al., 2010). Additionally, long term in vitro culture induces stable changes in leaf color in Bambusa oldhamii in our lab. The mechanisms of variations are still not clear. One of the possible mechanisms to explain variation may be the activation of transposable elements (Bennetzen, 2000). Transposable elements are sequences of DNA that can move around to different positions within the genome of a single cell, which are divided into class I and class II according to mechanism of transposition. Class I elements (retrotransposons) transpose by means of an RNA intermediate in a reaction involving several enzymes of reverse transcriptase and integrase. In contrast, class II elements (DNA transposons) move directly via DNA and the transposition reaction is catalyzed by a transposase encoded by autonomous DNA transposons (Bennetzen, 2000; Feschotte et al., 2002).
* Corresponding author. Tel.: þ86 57163748811; fax: þ86 57163732738. E-mail address: [email protected] (D.-Q. Tang). 1 These authors contributed equally to this work. 2 Who now works in China National Bamboo Researcher Center. 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.06.010
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Miniature inverted-repeat transposable elements (MITEs) are reminiscences of class II nonautonomous transposable elements with high copy number and intra-family homogeneity in size and sequence (Wessler et al., 1995). The mPing was the first active MITE identified in rice (Jiang et al., 2003; Kikuchi et al., 2003), and Pong transposon has been identified as the most likely source of mPing transposase (Jiang et al., 2003). Many transposases from nematodes, fungus, animals and plants with homologous motifs of Pong in rice were named Pong-like transposable elements (Zhang et al., 2004). Pong-like elements contain ORF1 and ORF2, of which ORF2 most likely encodes the transposase because it contains an apparent DDE motif, a signature consisting of three acidic residues found in the catalytic domains of some eukaryotic and prokaryotic transposases (Rezsohazy et al., 1993; Mahillon and Chandler, 1998; Jiang et al., 2003). The domain of amino acids surrounding the acidic triad is relatively well-conserved and thus has served to establish the evolutionary relationships of Pong-like transposon elements in plants (Zhang et al., 2004). In this study we performed first comprehensive analysis Pong-like superfamily of transposases in Bambusoideae by PCR with newly designed degenerate primers. Phylogenetic analyses of 82 Pong-like transposase fragments from 44 representative species of bamboo indicated that multiple divergent lineages of Pong-like transposases coexisted within a single plant species. Comparison with the phylogenetic data of ITS information shows that there are multiple Pong-like element families in Bambusoideae, and a single family could be present in multiple bamboo species, and Pong-like elements from the same family from different species could be more similar than elements from different families in the same bamboo species or closely related species. 2. Materials and methods 2.1. Bamboo materials and DNA extraction According to taxonomy of bamboo (Soderstrom and Ellis, 1987; Geng and Wang, 1996; Li, 1997; Das et al., 2008), we selected 44 representative species from 38 genera of 6 subtribes mainly distributing in China. In the intra-species level, we also selected 9 cultivars or forms derived from Ph. pubescens, which showing morphological differences in stem shape and color, and leaf color (Lin et al., 2009). Bamboo species and its cultivars or forms examined along with their taxonomic classification and voucher are listed in Table 1. Oryza sativa ssp. japonica cv. Nipponbare was collected for cloning of ITS fragment used as outgroup of phylogenic tree based on bamboo ITS sequences. Young leaves were sampled from each species (cultivars or forms) and desiccated for DNA extraction. Genomic DNA was extracted using the method of hexadecyltrimethylammonium bromide (CTAB) (Doyle and Doyle, 1987) with a few modifications. 2.2. PCR amplification and sequencing of Pong-like transposases Degenerate primers (Pong-5: GGCWCCATYGAYTGTATGCAC, Pong-3: YTCGTCYTCVACYATCATRTTGTG) used to detect the Ponglike elements were derived from the regions encoding amino acid residues motifs GTIDCMH and NMIVEDE conserved in Pong-like transposases of most flowering plants. PCR amplifications were performed with 50–100 ng of genomic DNA, 0.2 mmol/L primer pair (synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd), 2 units ExTaq Polymerase, 1 PCR buffer (10 mmol/L Tris–HCl, pH 8.3 at 25 C; 50 mmol/L KCl), 1.5 mmol/L MgCl2, and 0.2 mmol/L of dNTP each (TaKaRa Japan) in a final reaction volume of 20 mL. Cycling temperature parameters were: 1 cycle at 94 C for 5 min, 35 cycles at 94 C for 60 s, 57 C for 40 s, 72 C for 1 min, and 1 cycle at 72 C for 5 min. ITS have been proved to be better marker for systematic analysis for bamboo species (Guo and Li, 2004; Yang et al., 2008). Bamboo and O. sativa ITS sequences were amplified using primers ITS5 and ITS4 were used to amplify the whole ITS region as described by White et al. (1990). The thermal cycling comprised 30 cycles at 94 C for 1.5 min, at 55 C for 2 min primer annealing, at 72 C for 1 min, followed by a final extension step at 72 C for 7 min. Both Pong-like element and ITS PCR amplification products were resolved by 1% agarose gels electrophoresis, purified using the EZ-10 Spin Column DNA Gel Extraction Kit (Biobasic Inc.) and ligated into the pUC18 Vectors (TaKaRa, Japan) for cycle sequencing using BigDye terminator V1.3 system (PE Applied Biosystems) on an ABI PRISM 3100-Avant according to the manufacturer’s instructions. For Pong-like element detection, one positive clone of Pong-like element for each species was selected for sequencing. For intra-species of Ph. pubescens in order to identify as many Pong-like transposases as possible many independent positive clones were selected from each of its cultivars or forms for sequencing until at least three repeat clones were detected for every species. For ITS regions identified, one positive clone from 44 representative species and O. sativa was selected for sequencing. The resulting 82 Pong-like transposase sequences and 45 ITS sequences were deposited in the NCBI GenBank database (accession numbers GU350795–GU350876 and GQ464804–GQ464847, GU205182). Each element was denominated using the first letter of the genus name and the first letter of the species name followed by the clone number, for example: Bm-1 for Bambusa multiplex Pong-like element clone 1. Pong-like transposases which were obtained by database searches were named according to the species initials followed by NCBI accession no. by Zhang et al. (2004). 2.3. Database searching and phylogenetic analysis Database searches for the known Pong-like elements were performed with BLAST servers available from the NCBI nr, gss, est and wgs databases (http://www.ncbi.nlm.nih.gov) as well as the TIGR rice genomic database (http://tigrblast.tigr.org/eukblast/index). Nucleotide sequences of Pong-like elements from database and PCR amplification were conceptually translated into amino acid sequences. The translated amino acid sequences of Pong-like elements and ITS nucleotide sequences were aligned using CLUSTALW (Thompson et al., 1994) with default parameters, respectively.
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Table 1 Species whose Pong-like element sequences were analyzed in this study, and GenBank accession numbers. Taxa
Abb
Source
ITS
Pong-like elements
Subtribe Melocanninae Melocanna baccifera Cephalostachyum pergracile Monocladus saxatilis Schizostachyum funghomii Pseudostachyum polymorphum
Mb Cp Ms Sf Pp
Guangdong, China Yunnan, China Fujian, China Yunnan, China Yunnan, China
GQ464827 GQ464810 GQ464829 GQ464842 GQ464837
GU350795 GU350796 GU350797 GU350798 GU350799
Subtribe Bambusinae Bambusa multiplex Bambusa chungii Bambusa arundinacea Dendrocalamopsis oldhami Gigantochloa levis Thyrsostachys oliveri Neosinocalamus affinis Dendrocalamus minor Melocalamus arrectus
Bm Bc Ba Do Gl To Na Dm Ma
Yunnan, China Yunnan, China Ghana Guangdong, China Fujian, China Fujian, China Yunnan, China Zhejiang, China Yunnan, China
GQ464807 GQ464806 GQ464805 GQ464815 GQ464820 GQ464846 GQ464830 GQ464816 GQ464826
GU350800 GU350801 GU350802 GU350803 GU350804 GU350805 GU350806 GU350807 GU350808
Subtribe Shibataeeae Hibanobambusa tranguillans Indosasa shibataeoides Brachystachyum densiflorum Phyllostachys pubescens Ph pubescens cv. Anjiensis Ph pubescens cv. Gracilis Ph pubescens cv. Heterocycla Ph pubescens cv. Luteosulcata Ph pubescens cv. Obliquinoda Ph pubescens cv. Tao Kiang Ph pubescens cv. Tubaeformis Ph pubescens cv. Ventricosa Ph pubescens cv. Viridisulcata Shibataea chinensis Semiarundinaria fastuosa Qiongzhuea tumidinoda Sinobambusa tootsik Chimonobambusa marmorea Chimonobambusa quadrangularis
Ht Is Bd Ph PhY PhG PhP PhL PhO PhK PhT PhV PhW Sc Se Qt St Cm Cq
Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China
GQ464822 GQ464825 GQ464809 GQ464833 – – – – – – – – – GQ464844 GQ464843 GQ464838 GQ464845 GQ464811 GQ464812
GU350809 GU350810 GU350811 GU350812–GU350816 GU350821–GU350824 GU350825–GU350828 GU350829–GU350830 GU350831–GU350835 GU350836–GU350839 GU350840–GU350843 GU350844–GU350846 GU350847–GU350848 GU350849–GU350850 GU350851 GU350852 GU350853 GU350854 GU350855 GU350856
Subtribe Chusqueeae Chusquea coronalis
Ca
Yunnan, China
GQ464814
GU350857
Subtribe Arudinarieae Fargesia fungosa Himalayacalamus intermedia Yushania uniramosa Pseudosasa japonica Acidosasa gigantea Pleioblastus gramineus Pleioblastus chino Bashania fargesii Indocalamus latifolius Oligostachyum sulcatum Gelidocalamus annulatus Menstruocalamus sichuanensis Sasa fortunei Sasa sinica Sasa veitchii Chimonocalamus pallens Drepanostachyum luodianense
Ff Hi Yu Pj Ag Pg Pc Bf Il Os Ge Mo Sa Ss Sv Ch Dl
Zhejiang, China Shizuoka, Japan Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China Zhejiang, China
GQ464818 GQ464823 GQ464847 GQ464836 GQ464804 GQ464835 GQ464834 GQ464808 GQ464824 GQ464831 GQ464819 GQ464828 GQ464839 GQ464840 GQ464841 GQ464813 GQ464817
GU350858 GU350859 GU350860 GU350861 GU350862 GU350863 GU350864 GU350865 GU350866 GU350867 GU350868 GU350869 GU350870 GU350871 GU350872 GU350873 GU350874
Subtribe Guaduinae Otatea acuminata Guadua angustifolia
Oa Ga
Mexico Ecuador
GQ464832 GQ464821
GU350875 GU350876
The best-fit model of nucleic substitution of Pong-like element and ITS sequences was tested by software Modeltest 3.7 (Posada and Crandall, 1998) with Akaike information criterion (Sullivan and Joyce, 2005). GTR model (GTR þ I þ G) was the optimum for Pong-like elements and TIMef þ I þ G was the optimum for ITS sequences. The phylogenetic relationships among the bamboo Pong-like elements were investigated from either the nucleic consensus sequences or their in silico translation into protein sequences, while the phylogenetic relationships among Bambusoideae were investigated from the ITS nucleic sequences. All
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phylogenetic trees were generated using neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) with PAUP software v4.0b10 (Swofford, 2002) and Bayesian inference (BI) with MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), and reswapped with NEAREST NEIGHBOR INTERCHANGE by using Molphy v2.3b3 (Adachi and Hasegawa, 1996). Rearranged topologies were very similar to those initially generated by four methods, at least for the groupings discussed in this study. 3. Results 3.1. Identification and polymorphism of transposases of Pong-like elements in the Bambusoideae subfamily A pair of degenerate primers was designed to amplify a fragment of the transposase gene containing “DDE” domain of Pong-like transposase (Zhang et al., 2004). PCR with the degenerate primers yielded one uniform size fragment of approximately 520-bp for each bamboo species (Fig. 1). Taken together, 82 amplification fragments from 44 representative species and 9 Ph. pubescens cultivars or forms were obtained and sequenced. At the amino acid level, the bamboo sequences showed between 67.2% and 79.1% identity with an identified typical Ponglike element from O. sativa (OsBK000586, accession number BK000586) (data not shown). At least one representative species were selected from six subtribes (Melocanninae, Bambusinae, Shibataeeae, Chusqueeae, Arundinarieae and Guaduinae) for detecting the conserved functional domains regions. Several blocks of highly conserved residues were consistent with those of Pong-like transposases which most likely comprise the catalytic domain. The conserved amino acid residues of DDE motif were identified in all fragments. First D is present at the position 4, second D present at the position 94 and the E present at the position 171 of the consensus amino acid sequence (Fig. 2). The data confirmed that the all PCR products contained the DDE region of the Pong-like transposase genes. Almost all 82 sequences of Pong-like transposases we cloned in bamboo species were unique. Pairwise comparison showed 57.8–99.6% identity at the nucleotide sequences level and 54.9–100% identity at the amino acid level (supplementary Tables 1, 2). Comparison within each subtribe revealed 90.6% identity among the Guaduinae, 71.7–86.7% among the Melocanninae, 63.3– 97.1% among the Bambusinae, 62.7–100% among the Shibataeeae, and 61.8–100% among the Arundinarieae at amino acid level (supplementary Table 2). The results indicate Pong-like transposases are highly polymorphic in bamboo species tested. 3.2. Phylogenetic position of bamboo Pong-like elements in flowering plants Up to date, at least three subfamilies of Pong-like elements have been reported in flowering plants, i.e., clade O, clade P and clade Q, of which Clade O and Clade P can be further classed into subclade O1, O2 and subclade P1, P2, respectively (Zhang et al., 2004). In order to place the above characterized bamboo Pong-like elements in this phylogenetic tree, several elements representing each of the three subfamilies previously reported (Zhang et al., 2004) were aligned with bamboo Ponglike elements. Three Pong-like transposase clusters similar to the clades mentioned above are shown and are defined as the largest well-supported monophyletic group of sequences obtained from phylogenetic trees generated by four distinct methods (NJ, MP, ML and BI, all bootstrap values >63%, Fig. 3). Most of bamboo Pong-like elements clustered in two branches of tree. First, significant portion of bamboo Pong-like elements grouped into clade Q (bootstrap value of 78%). Second, the three bamboo Pong-like elements grouped into subclade O2 (bootstrap value of 100%). It was noted that there were three bamboo Pong-like elements which could not grouped into any clades defined by Zhang et al. (2004) and clustered as new clade BaS (bootstrap value of 82%). 3.3. Evolution of bamboo Pong-like elements To analyze of phylogenetic relationships among Bambusoideae Pong-like elements, phylogenetic tree was generated from amino acid sequences of the 44 representative species that were selected to represent the different lineages within each subtribe with one PIF-like element from a fungus (Filobasisiella neoformans) as the outgroup (Fig. 4A). Meanwhile the analysis of ITS sequences allowed the construction of a parallel tree showing the phylogenetic relationship among the same 44 bamboo species with the ITS sequence from O. sativa as outgroup (Fig. 4B). The ITS tree supported the existence of six subtribes, i.e.
Fig. 1. Detection of Pong-like transposase genes in bamboo subfamily by using PCR with degenerate primers. Representative examples are shown from 10 species. M: 100-bp ladder. 1. Sasa fortunei, 2. Bashania fargesii, 3. Acidosasa gigantean, 4. Himalayacalamus intermedia, 5. Chimonobambusa marmoreal, 6. Semiarundinaria fastuosai, 7. Dendrocalamus minor, 8. Indosasa shibataeoides, 9. Hibanobambusa tranguillans, 10. Schizostachyum funghomii.
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Fig. 2. Structure and conserved coding regions of the amplification fragments of Pong-like elements. Multiple alignments of conceptually translated amplification fragments from bamboo Pong-like elements are shown. Horizontal arrows indicate the annealing position of the dPCR primers, and DD37E motifs in transposases are indicated by asterisks. A consensus sequence of OsBK000586, the Pong-like elements in rice is shown above alignments for comparison. Sequences were named according to the species initials followed by their clone number (Table 1 for abbreviations).
Melocanninae, Bambusinae, Shibataeeae, Chusqueeae, Arundinarieae, Guaduinae. In contrast, 44 Pong-like elements from Bambusoideae grouped into 4 clusters (QBaI, QBaII BaS and O2) which indicated that there are four Pong-like element families in Bambusoideae and a single family could be present in multiple species. It is also noted that Pong-like elements from the same family from different species could be more similar than elements from different families in the same bamboo subtribes or closely related species. For example, Ht-1 Pong-like element (Hibanobambusa tranguillans) was 100% identity with Ss-1 Pong-like element (Sasa sinica) at the amino-acid level, while they are from subtribes of Shibataeeae and Arundinarieae, respectively. In
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Fig. 3. Phylogenetic position of bamboo Pong-like elements in the plant Pong-like elements family. The phylogenetic tree was generated by multiple alignments of 95 catalytic DDE motifs of Pong-like transposase fragments including 82 from 44 representative species of Bambusoideae subfamily isolated by PCR and 13 obtained by database searches which were marked by circles. Main bootstrap values (1000 replicates) are shown. Groupings defining lineages and sublineages of plant Pong-like transposases are emphasized by capital letters and different colors. Sequences were named according to the species initials followed by the number of the clone (see Table 1 for abbreviations), Os ¼ Oryza sativa (rice), Lj ¼ Lotus japonicas, At ¼ Arabidopsis thaliana, Bo ¼ Brassica oleracea, Sb ¼ Sorghum bicolor.
contrast, Pong-like elements from the same bamboo subtribes or closely related species could be more divergent than that from different subtribes species or distant related species, e.g. two diverse Pong-like transposases subclades were present in Melocanninae group, Pp-1, Ms-1 and Mb-1 from subclade QBa I shared 72.4%–75.3% identity with Sf-1 and Cp-1 from the clade QBaII. The phenomenon was farther supported by the analysis of phylogenetic relationships among intra-species Pong-like transposases. Another phylogenetic tree was constructed from 39 amino acid sequences from nine different cultivars or forms of Ph. pubescens (Fig. 4C). All Pong-like transposases from cultivars or forms of Ph. pubescens belong to clade Q. They could further be clustered into three subclades names as QPhI, QPhII and QPhIII. Pong-like elements from the same cultivars (such as PhK-1 and PhK-2 from Ph. pubescens cv. Heterocycla) demonstrated a 43.1% lower level of identity than that from different cultivars (such as PhO-1 from Ph pubescens cv. Obliquinoda). This revealed that Pong-like elements have evolved relatively independent of taxonomy of bamboo and even one individual may display more than one subclade of Pong-like elements in its genome. 4. Discussion 4.1. Pong-like elements were widespread and abundant in Bambusoideae subfamily The PIF/IS5 is a recently discovered superfamily of DNA transposons which include Pong-like elements and PIF-like elements and has been successively detected in the genomes of flowering plants, fungi and animals (Le et al., 2001; Zhang et al., 2001, 2004; Kapitonov and Jurka, 2004). Here we present the first comprehensive characterization and analysis of Pong-like elements in Bambusoideae subfamily. Of all 9 subtribes in Bambusoideae subfamily, 6 subtribes including 44 species
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Fig. 4. Phylogenetic analysis of Pong-like transposase fragments and ITS sequences within the Bambusoideae subfamily. Sequences were named according to the species initials followed by the number of the clone (see Table 1 for abbreviations). Main bootstrap values (1000 replicates) are shown. Groupings defining lineages and sublineages of bamboo Pong-like transposase are emphasized by capital letters and different colors, : Shibataeeae; : Arundinarieae; : Melocanninae; : Bambusinae; -: Guaduinae; : Chusqueeae. A. Phylogenetic relationships among bamboo Pong-like transposase fragments. The phylogenetic tree was generated by multiple alignments of 44 Pong-like transposase fragments obtained by PCR from representative bamboo species. B. Phylogenetic relationships of ITS sequences from the same representative bamboo species, obtained as above. C. Phylogenetic relationships among 39 Pong-like transposase fragments isolated from 10 Ph. pubescens cultivars or forms, obtained as above.
of 38 genera mainly distributed in China were selected for testing materials. These tested bamboo species covered type of woody (most species) and liana (Melocalamus arrectus, etc.), distributed in as south to equatorial America (Guadua angustifolia) and north to temperate regions (Phyllostachys species in Beijing) (Clayton et al., 2006). All the species studied displayed Pong-like elements in their genome by degenerate primers PCR amplification. Almost all the sequences of cloned products were unique and diverse with 57.8–99.6% identity at the nucleotide sequences level, of which three sequences were
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species-specific of bamboo (Clade BaS). Intra-species level, 39 Pong-like elements from Ph. pubescens and its cultivars or forms were identified. Sequencing of 5 PCR products from Ph. pubescens showed as different Pong-like elements. All the results show that Pong-like elements are indeed abundant and heterogeneous in the Bambusoideae subfamily. Genome-wide analysis identified total 80 Pong-like elements in O. sativa (Zhang et al., 2004). The DNA contents of bamboo were 2.45–5.3 pg DNA/2C, and the temperate bamboo (Phyllostachys species) falling within the range 4.17–5.3 pg (Gielis et al., 1997). The genome size of Ph. pubescens was estimated to be about 2034 Mb, approximately 5.4 times that of the diploid cultivated rice (Gui et al., 2007). Given that amplification of transposable elements is largely responsible for the huge differences in plant genome size (Bennetzen, 2002; Feschotte et al., 2002), it is reasonable to assume that even larger families of Pong-like transposable elements will be found once bamboo genomes are sequenced. 4.2. Evolution of Pong-like transposable elements in Bambusoideae subfamily The significant portion of Pong-like elements from Bambusoideae subfamily in this study could be clustered into clade Q and several into subclade O2. Clade Q has been proved to be ancient origin in plant Pong-like elements family (Zhang et al., 2004). The available fossil evidence and the surviving basal lineages suggested the Bambusoideae were evolved in Gondwanaland during the upper Cretaceous of more than 65 Myr ago (Guo et al., 2002). The results suggest that most Pong-like elements from Bambusoideae subfamily are likely transposable elements of ancient origin. We constructed a comparative ITS-based phylogeny of Bambusoideae subfamily which agrees with the previous analyses based on morphologic and molecular characters (Soderstrom and Ellis, 1987; Li, 1997; Das et al., 2008; Yang et al., 2008). The phylogenetic tree constructed with the Pong-like element sequences was obviously incongruent with the ITS-based phylogeny. The incongruence was particularly reflected by the presence of near identical Pong-like elements from distantly-related species and the presence of very diverse Pong-like elements within the same cultivars of Ph. pubescens. There are at least four alternative hypotheses to explain the result. First, Pong-like elements analyzed from 44 representative bamboo species in this research are most likely paralogous not orthologous because of high heterogeneity (supplementary Tables 1, 2). Second, bamboo Pong-like elements originated from different ancestors (Fig. 4A, C). In every clade, divergent evolution of different ancestors might be different. Ancestral duplication and/or polymorphism may create paralogous copies of transposable elements that evolve distinctly in different genomes (Davis and Wurdack, 2004; Mower et al., 2004). Third, usually, transposable elements suffer more tough natural selection than other sequences due to their potential impact of transposition on the host genome. There are at least three deleterious effects of transposable elements in the host genome including mutations resulting from insertions into genes or regulatory sequences (Finnegan, 1992), chromosomal rearrangements caused by ectopic recombination between elements in non-homologous insertion sites (Montgomery et al., 1987) and direct costs due to its transposition activity (Brookfield, 1991). So the sequences of transposable elements provide a more robust target for natural selection than other sequences (Pritham, 2009). When transposon activity is suppressed, such elements could be eliminated from the population by stochastic loss or vertical extinction, a process that may occur in one species but perhaps not in a closely related one (Hartl et al., 1997). Fourth, horizontal transfer might be another potential explanation about the incongruence. The scenario of horizontal transfer remains a remote speculation, until a common transmission vector is identified (Capy et al., 1998; Won and Renner, 2003; Bergthorsson et al., 2004; Richardson and Palmer, 2007). Although hundreds of Pong-like element sequences were identified by database search there is so far no clear indication for any horizontal movement of Pong-like elements among plants (Zhang et al., 2004). In this study, 82 Pong-like element sequences were isolated from bamboo and phylogenetic analysis of these sequences could not prove beyond doubt the occurrence of horizontal transfer. Large-scale sequencing of bamboo species would allow to the evolution of Pong-like elements to be studied in more details. Acknowledgments We are grateful to Ma Naixun of Research Institute of Subtropical Forestry and Xingchun Lin of Key Lab for Modern Silvicultural Technology of Zhejiang Province for their advices on sketching the sampling strategies. This work was supported by a special grant from the National Natural Science Foundation of China (grant nos. 30371181 and 30771753) and Talents Program of Natural Science Foundation of Zhejiang Province (grant no. R303420). Appendix. Supplementary information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bse.2010.06.010. References Adachi, J., Hasegawa, M., 1996. MOLPHY: Programs for Molecular Phylogenetics Based on Maximum Likelihood 2.3. The Institute of Statistical Mathematics, Tokyo. Bennetzen, J.L., 2000. Transposable element contributions to plant gene and genome evolution. 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