Botanical Studies (2011) 52: 145-152.
MOLECULAR 
 BIOLOGY
Allopolyploidization induced the activation of Ty1-copia retrotransposons in Cucumis hytivus, a newly formed Cucumis allotetraploid
Biao JIANG, Qun-Feng LOU, Dong WANG, Zhi-Ming WU, Wan-Ping ZHANG, and Jin-Feng CHEN*
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Southern Vegetable Crop Genetic Improvement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, PR. China
(Received April 20, 2010; Accepted September 17, 2010)
ABSTRACT. Allopolyploidy is an important process of species evolution that delivers a huge 'genomic shock' to the host genome. To investigate the effect of allopolyploidy on the expression of retrotransposons in a new synthetic allopolyploid, Cucumis hytivus, RT-PCR strategy was carried out to amplify reverse tran­scriptase (RT) genes from allotetraploid C. hytivus and its diploid parents, C. hystrix and C. sativus, using de­generate oligonucleotide primers corresponding to conserved RT domains of Tyl-copia retrotransposons. Only the allotetraploid yielded a specific product with expected size. After recovering and sequencing, 18 unique clones with significantly high heterogeneity were obtained. All of these clones were different from each other and could be divided into at least eight groups. The synonymous (dS) and nonsynonymous (dN) substitution analysis suggested that the RT sequences had been under constraint or purifying selection. A comparative maximum likelihood (ML) tree was constructed based on the deduced amino acids of the 18 RT sequences and those of other species acquired from the GenBank database. These results showed that the cloned sequences had high homology with both related and unrelated species, implying that they shared a common ancestor. The expression analysis of the cloned reverse transcriptase in the first four generations of allotetraploid further proved that the activation of retrotransposons was induced by allopolyploidization. These findings provide im­portant information for polyploid evolution and will be of great importance for further epigenetic studies.
Keywords: Allopolyploidization; Cucumis; Retrotransposon; Reverse transcriptase; RT-PCR.
Abbreviations: gPCR, Genomic PCR; IRAP, Inter-retrotransposon amplified polymorphism; LTR, Long ter­minal repeat; REMAP, Retrotransposons-microsatellite amplified polymorphism; RT, Reverse transcriptase; PCR, Polymerase chain reaction; UV, Ultraviolet light.
INTRODUCTION
Allopolyploidy is a well-known process in plant spe-ciation where two or more genomes are joined into the same nucleus through interspecific or intergeneric hybrid­ization followed by chromosome doubling. Many impor-tant crop plants, such as wheat, oat, cotton, canola, and tobacco are allopolyploids (Masterson, 1994). The process of allopolyloidy has thus played a key role in the origin of many species and has driven and shaped plant evolution. Newly-formed allopolyploids undergo a huge 'genomic shock' (McClintock, 1984), which triggers wide genomic and epigenetic changes in the early stages of allopoly-ploid formation (Wendel, 2000; Adams et al., 2003). Ge-
nomes of newly-formed allopolyploids exhibit obvious instabilities, including major structural, cytogenetic, and functional changes to the genome, which potentially lead to new phenotypes and to reproductive isolation (Song et al., 1995; Ozkan et al., 2001; Shaked et al., 2001; Kash-kush et al., 2002, 2003; Adams and Wendel, 2005; Chen and Ni, 2006).
Retrotransposon is ubiquitous in the plant kingdom (Flavell et al., 1992; Voytas et al., 1992). It contributes to increasing genome size and genome evolution in the plant kingdom (Kumar and Bennetzen, 1999; Feschotte et al., 2002). Retrotransposons are mostly inactive during normal plant development, but can be activated by biotic and abi­otic stresses (Grandbastien, 1998). Wide hybridization and polyploidy can also induce activation of retrotransposons. Transcriptional activation of retrotransposons has been reported in response to wide crosses in wheat, Arabidop-
*Corresponding author: E-mail: jfchen@njau.edu.cn.
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sis and rice (Kashkush et al., 2002, 2003; Liu et al., 2004; Madlung et al., 2005). Newly-formed allopolyploid species Spartina anglica and Gossypium, however, inherited all their parents' genomes, which indicated that retrotranspo-son activation did not occur in the allopolyploid genomes (Liu and Wendel, 2000; Liu et al., 2001; Baumel et al., 2002). Consequently, more elaborate research is needed to pierce what effects polyploidy has on retrotransposons in different species.
Cucumis hytivus (2n=4x=38) is a newly synthesized al­lotetraploid species obtained after successful interspecific hybridization and chromosome doubling of C. hystrix (2n=2x=24) and C. sativus cv. Beijingjietou (2n=2x=14) (Chen et al., 1997; Chen and Kirkbnde, 2000). Our pre­vious studies showed that allopolyploid formation in Cucumis can induce various changes, including flower­ing time, fruit shape, morphological traits, cytogenetic and rapid genetic variations (Chen et al., 1997, 2002, 2003, 2007). In Cucumis, numerous sequences of LTR retrotransposons were isolated and characterized from melon, and it was reported that the transcription of a co-pia retrotransposon Reme1 was induced by UV light, but not by wounding or by water stress (Ramallo et al., 2008). In our current study, we focused mainly on the effect of allopolyploidy on the transcription of Ty1-copia ret-rotransposons and analyzed the characterization of RT do­mains of active retrotransposons in the newly-synthesized allotetraploid species. In addition, we investigated the expression of activated retrotransposons in the first four generations of the allotetraploid.
MATERIALS AND METHODS
Plant materials
Plant materials consisted of the two diploid parents, cucumber cultivar [C. sativus cv. Beijingjietou (genome CC, 2n=2x=14)], a wild species [C. hystrix (genome HH, 2n=2x=24)], and the S1-S4 generations of the synthetic al­lotetraploid [C. hytivus (genome HHCC, 2n=4x=38)]. The primary allotetraploid (S0) was previously obtained from interspecific hybridization, through embryo rescue and chromosome doubling (Chen and Kirkbride, 2000). The S1-S4 generations constitute the first four self-pollinated generations. All the plants were grown in a plastic green­house under standard conditions.
DNA and RNA extraction
Total genomic DNA was extracted from young leaves of seedlings by the CTAB method described by Murray and Thompson (Murray and Thompson, 1980). Total RNA was isolated from 1 g of young leaves using the Trizol kit (Promega) and RNA quality was checked by running the samples on a formaldehyde agarose gel. The residual DNA was removed by DNase I (RNase-free) (TaKaRa), and RNA concentration was estimated using a spectrophotometer. The first cDNA strand was synthe­sized according to instructions of the Kit (TaKaRa).
Cloning of RT domain of retrotransposons
RT-PCR was used to amplify a conserved region in the RT domain of Ty1-copia retrotransposons from the allotetraploid C. hytivus and its diploid parents using the degenerative primers (5'ACNGCNTTPyPyTNCAPyGG3' and 5'APuCATPuTCPuTCNACPuTA3'), corresponding to the conserved RT peptide motifs of the Ty1-copia group retrotransposons TAFLH and YVDDM, respectively (Ku­mar et al., 1997). 1 μl cDNA solution was added into a 20 μl reaction mixture containing 50 pmol of each primer, 0.2 mmol/L of dNTP, 2.5 mmol/L of MgC2 and 1 U of Taq polymerase (TaKaRa). PCR amplification consisted of the following conditions: 3 min initial denaturation at 94°C; 35 cycles of 1min at 94°C, 1 min at 45°C, 1 min at 72°C; followed by 10 min at 72°C.
Purifying, cloning and sequencing
RT-PCR products were purified with Gel Extraction Mini kit (Bio Spin) from agarose gel and directly ligated to pGEM-T-Easy vector (Promega). The DH5a strain of competent cells was used as a bacterial host. Plasmids from selected colonies were isolated by standard alkaline lysis and digested with ECOR I. Sequencing of the clones containing anticipated product was performed by Shang­hai Bioasia Biological Engineering Technology & Service CO., Ltd.
Sequence and phylogenetic analyses
We tested the authenticity and homology of the clones by blastX at the NCBI website (http://www.ncbi.nlm. nih.gov/). These amino acid sequences were aligned us­ing ClustalW (Thompson et al., 1994) with other RT se­quences obtained from GenBank: G53226 in Arabidopsis thaliana; CAH56518 in Brassica juncea; CAD11841 in Brassica napus; CAD11830 in Brassica rapa; CAJ09747 in Camellia sinensis; CAJ41394 in Citrus sinensis; GU569971 in Cucumis hystrix; CAJ76068 in Cucumis melon; EU162122 in Cucumis sativus; BAB47218 in Dio-spyros kaki; ABS11056 in Mains x domestica; AAK55317 in Oryza sativa; AAA33849 in Platanus occidentalis; ABD19061 in Phelipanche ramose; ABD19073 in Pheli-panche tunetana; ABF57076 in Prunus mume; AAL36463 in Setaria adhaerans; AAC34606 in Solanum lycoper-sicum and AAK84849 in Zea mays. A protein maximum likelihood (ML) phylogenetic tree was constructed based on the deduced amino acid sequences of the RT region using TREE-PUZZLE 5.2 (Schmidt et al., 2002), as well as MEGA 4.0 software (Tamura et al., 2007). Values that support the internal branches within the ML tree were ob-tained from a total of 1000 puzzling steps.
Maximum likelihood (ML) estimates of nonsynony-mous substitutions (dN) and synonymous substitutions (dS) between pairwise alignment were obtained with PAML (Yang, 2000) using a codon-based model of se­quence evolution with dN, dS, and transition-tranversion bias as the data at each codon position (Goldman and Yang, 1994; Yang, 2000).
JIANG et al. — Allopolyploidization induced the activation of Tyl-copia retrotransposons in Cucumis allotetraploid 147
Genomic PCR and RT-PCR analysis
To verify the expression of the cloned reverse tran-scriptase sequences in the first four generations of allotet­raploid, genomic PCR (gPCR) and RT-PCR using specific primers were carried out respectively. We tried to design specific primers based on all the 13 intact clones. RT3 and RT10 belonging to different groups of retrotransposons in C. hytrix were chosen as target fragments for gPCR and RT-PCR. The PCR condition was followed as: 3 min at 94°C; 30 sec at 94°C, 30 sec at 55°C, 30 sec at 72°C, fol-lowed by 35 cycles. Actin gene was used as positive con-trol in the RT-PCR reactions.
other, we confirmed that these sequences were highly heterogeneous. The 18 sequences were conceptually trans­lated and where necessary edited for frameshift, based on published residue landmarks of plant retrotransposons' RT domains. The amino acid alignments had properly translated primer sequences at both ends. Among these 18 sequences, 5 (27.8%) contained premature stop codons and/or frameshift in their coding regions (RT1, RT5, RT7, RT12, and RT13). The remaining 13 sequences (72.2%) were not affected by either stop codons or frameshift. The homology ranged from 49.4% (RT4 and RT9) to 98.1% (RT3 and RT14) when amino acid sequences of these RTs
RESULTS
Identification of active retrotransposons in Cucumis
Transcriptional active retrotransposons were tested by RT-PCR from allotetraploid and its two diploid parents, C. hystrix and C. sativus. Only the allotetraploid yielded a specific product with expected size (260 bp) (Figure 1). The amplified fragment was recovered and cloned into a pGEM-T vector, and 20 clones were obtained. Sequence analysis revealed that 18 clones contained the RT domains of the retrotransposons (named as RT1 to RT18), while the remaining 2 didn't. These sequences have already been submitted to GenBank with the accession numbers of HMO036494-HMO036511.
RT sequence alignments
Comparing the 18 newly-isolated sequences with each
Figure 1. RT-PCR amplification of reverse transcriptase (RT) domains of Ty 1 -copia retrotransposons from genomes of al­lotetraploid and its two diploid parents. M: 2000 bp Marker; P1: Cucumis hystrix; P2: C. sativus; S: C. hytivus.
Table 1. Homology matrix of the 18 reverse transcriptase (RT) sequences of active retrotransposons amplified from allotetraploid C. hytivus.
RT1
RT2
RT3
RT4
RT5
RT6
RT7
RT8
RT9
RT10
RT11
RT12
RT13
RT14
RT15
RT16
RT17
RT2
82.2
RT3
60.4
63.5
RT4
86.4
83.8
61.7
RT5
67.8
68.8
60.5
66.5
RT6
53.3
57.0
53.6
53.2
54.8
RT7
86.0
89.5
63.2
85.7
65.0
54.4
RT8
57.3
57.3
57.3
58.4
58.0
52.9
57.6
RT9
51.7
53.6
53.2
49.4
51.7
90.1
53.2
51.3
RT10
86.4
81.6
59.8
85.3
67.7
52.5
83.1
56.9
49.0
RT11
67.4
68.4
63.9
65.4
91.0
55.5
65.0
56.1
54.4
66.2
RT12
53.6
55.9
56.3
53.6
56.7
51.5
55.1
68.8
51.5
53.2
58.2
RT13
59.1
59.4
56.8
57.1
57.1
52.5
59.0
56.1
48.7
54.1
57.9
56.3
RT14
61.0
63.5
98.1
62.4
60.9
53.6
63.9
57.6
53.2
60.5
62.8
55.9
58.6
RT15
57.6
54.9
55.6
53.4
53.0
52.9
56.0
57.6
51.7
53.4
54.1
52.1
51.9
56.4
RT16
69.7
69.9
63.2
66.9
90.6
55.1
66.5
56.9
52.9
66.9
94.4
59.7
59.8
63.2
54.5
RT17
58.6
60.4
57.7
57.7
60.4
57.3
59.2
59.8
55.7
57.4
59.2
55.0
61.5
57.7
58.1
58.9
RT18
89.4
83.8
62.4
91.7
65.0
52.9
86.1
59.5
51.3
86.1
66.9
54.8
56.8
62.0
55.6
66.2
59.2
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compared with each other (Table 1). Alignment of the deduced amino acid sequences showed that they had char­acteristic amino acid motifs at both ends of RT gene (5'-TAFLHG and 3'-YVDDM). The YGLKQ located in the central region of RT gene (Figure 2).
Phylogenetic analysis of RT sequences
To investigate the relationships among the RT domains representing the active Ty1-copia retrotransposons of C. hytivus and further comparison with those acquired from the GenBank database, a 50% majority consensus tree from maximum likelihood (ML) phylogenetic was constructed using the TREE-PUZZLE 5.2 (Schmidt et al., 2002) and MEGA 4.0 software (Tamura et al., 2007). Eight groups were clearly separated in the tree based on its branching patterns (Figure 3). Five groups (II, V, VI, VII, and VIII) were constituted; those containing a single sequence were considered independent groups due to their distant relations to all the other sequences. Figure 3 also showed that the sequence of group II was clustered with a D. kaki clone (BAB47218) as well as with a C. melon clone (CAJ76068). Group III seemed to be highly homologous with Ty1-copia RT sequences of C. sativus (EU162122) and C. hystrix (GU569971). The sequence of group V was in the same clade as Malus x domestica (ABS11056).
The numbers of synonymous and nonsynonymous sub­stitutions per site were estimated for three groups (I, III and IV), which contained at least two intact RT sequences (Table 2). The ratios of nonsynonymous to synonymous substitutions (dN/dS) were 0.12, 0.12 and 0.18 for group I, III and IV respectively, which suggested that the three groups had been under constraint or purifying selection.
Expression of RT gene in the first four genera­tions of allotetraploid and their diploid parents
Genomic PCR and RT-PCR were carried out to verify the expression of RT gene in the first four generations of allotetraploid and their diploid parents using specific prim­ers based on the RT3 and RT10 sequences. These sequenc­es belonged to different groups of retrotransposons in C. hystrix. gPCR results showed that RT3 existed in the S1-S4 generations and in one of their parents (C. hystrix). How­ever, RT-PCR results revealed that although it was inactive in C. hystrix, it was activated in the S1-S4 generations (Fig­ure 4A). In the meantime, though RT10 existed in both of the two diploid parents and in all the four generations of allotetraploid, it was inactive in the parents but activated in the allotetraploid (Figure 4B). Since all plants were grown in the same condition, it is conceivable that the expression change of RT3 was induced by allopolyploidization.
DISCUSSION
Retrotransposons are mostly inactive under normal conditions, but can be activated by biotic and abiotic
Table 2. Number of synonymous and nonsynonymous substi­tutions per site within the RT gene domain of two C. hytivus Ty1-copia retrotransposon groups.
Group
Synonymous substitutions (dS)
Nonsynonymous substitutions (dN)
Ratio (dN/dS)
I
0.078
0.0092
0.12
III
0.85±0.36
0.1±0.05
0.12
IV
3.84
0.36
0.18
Figure 2. Sequence alignment of deduced amino acid corresponding to the RT domains of transcriptional active retrotransposons from allotetraploid C. hytivus. The four shading levels indicate degree of residue conservation: black (100% conserved), dark gray (75% or greater conserved), light gray (50% or greater conserved), and no shading (<50% conserved). Gaps are indicated as (-) and stop codons are presented as (*). The amino acid of frameshift is underlined. The numbers of amino acid residues are displayed on the right hand of each sequence.
JIANG et al. ― Allopolyploidization induced the activation of Ty1-copia retrotransposons in Cucumis allotetraploid 149
stresses (Grandbastien, 1998). Allopolyploidization causes a huge 'genomic shock' to the host genome and can re­sult in activation of retrotransposon (McClintock, 1984; Kashkush et al., 2003). In wheat, it was reported that the Ty1-copia retrotransposon Wis 2-1A was activated by al-lopolyploidization (Kashkush et al., 2002). In contrast, in the young allopolyploid species Spartina anglica, the re­sult of IRAP and REMAP indicated that retrotransposons were not reactivated during the formation of allopolyploid (Baumel et al., 2002). To examine whether retrotranspo-sons present in the parental genomes have been activated in newly-synthesized allotetraploid C. hytivus, we used the RT-PCR strategy to amplify the reverse transcriptase (RT) region of Ty1-copia group retrotransposons from C. hytivus and its diploid parents, C. sativus and C. hystrix. The RT primers correspond to the two conserved amino acid domains of the Ty1-copia-like retrotransposons of plant species, TAFLHG (amino-terminal) and YVDDM (carboxy-terminal) of reverse transcriptase (Kumar et al., 1997). The allotetraploid amplified the expected fragment, while the diploid parents did not. This result showed that retrotransposons were activated in C. hytivus by allopo-lyploidization, since all the seedlings were grown under the same condition, thus excluding the variances induced by surrounding conditions. We also found in our previous studies that there was very little variation in the polymor­phic level between diploid and autotetraploid cucumbers as revealed by AFLP technique (Zhang et al., 2006). Ex­tensive genomic and epigenetic changes were detected, however, in the newly formed allotetraploid C. hytivus (Chen et al., 2007; Chen and Chen, 2008). Consequently,
we can presume to some extent that the expression variant detected here was induced by allopolyploidization. The synonymous (dS) and nonsynonymous substitution (dN) analysis showed that constraint or purifying selection acted on these elements, therefore supporting the suggestion that most of the retrotransposons obtained there were active.
Like other genes, retrotransposons can be transmit­ted from parent to the next generation. This is designated as vertical transmission (Doolittle et al., 1989), the main method for the genetic transmission of retrotransposons. Horizontal transmission also occurs via an asexual mode. The result of horizontal transmission leads to the wide distribution of retrotransposons in a variety of species (Doolittle et al., 1989). Phylogenetic analysis based on
Figure 3. The 50% majority consensus tree from maximum likelihood (MP) analyses based on the deduced amino acid se­quences corresponding to RT domain of active retrotransposons isolated from C. hytivus with other RT sequences by quartet-puzzling analysis (Schmidt et al., 2002). The tree was arbitrarily rooted using Phelipanche ramose (ABD19061) as an outgroup. Numbers above branches indicate bootstrap values.
Figure 4. Expression of RT genes in the first four generations of allotetraploid and their diploid parents. A, The expression of RT3 was studied by RT-PCR using specific primers. Actin was used as positive control. gPCR was carried out using the same con­dition with RT-PCR; B, The expression of RT10 was studied using the same method with RT3. P" Cucumis hystrix; P2: C. sati­vus; S1-S4: the first four generations of C. hytivus.
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nucleotide sequences of the reverse transcriptase provided evidence for the vertical transmission of retrotransposons in this study. The comparative phylogenetic analysis showed that the RT sequences obtained here had homol-ogy with related as well as unrelated species (Figure 3). Most of the RTs cluster together with sequences from Cucumis, such as C. sativus (EU162122), C. hytivus (GU569971) and C. melon (CAJ76068). In addition, some RTs showed a close phylogenetic relationship with S. ad-
haerans (AAL36463), D. kaki (BAB47218), P. ramose (ABD19061) as well as P. tunetana (ABD19073), which implied that they shared a common ancestor prior to spe-ciation. Thus retrotransposons have contributed to the ge-netic diversity and evolution of the host genome.
To verify the expression of the cloned reverse tran-scriptase in the first four generations of allotetraploid and their diploid parents, gPCR and RT-PCR were carried out using specific primers corresponding to RT3 and RT10. The results showed that RT3 initially existed in C. hystrix, that RT10 existed in both of the diploid parents, and that these two sequences could inherit into the first four gen­erations of allotetraploid. Although these were inactive in parent material, they were activated in all four genera­tions of allotetraploid. One might doubt that factors other than allopolyploidization induced the activation of Ty1-copia retrotransposons in C. hytivus. In order to minimize this doubt, all the seedlings in this study were carefully grown under the same conditions without artificial or environmental stresses. The results further prove that al-lopolyploidization affects the expression of Ty1-copia ret-rotransposons immediately after allopolyploid formation.
The transposition activity of retrotransposons converse­ly changed the expression of neighboring genes. For exam­ple, transcriptional activation of retrotransposon Wis 2-1A in wheat altered the expression of neighboring genes, lead­ing to the activation or silence of flanking genes (Kashkush et al., 2003). Our studies suggested that there was a direct link between the expression of retrotransposons and al-lopolyploidy, which provided a significant foundation for further polyploid evolution in Cucumis. In the future, we will focus on the influence of epigenetic changes on the activation of retrotransposons in early generations of the synthesized allotetraploid - C. hytivus.
Acknowledgements. The authors thank Dr. Ahmed Ma­lik, and Mr. Kere George, for reading of the manuscript. This research was partially supported by the Key Pro­gram (30830079) and the General Program 30700541 and
31071801 from the National Natural Science Foundation of China; National Basic Research Program of China (973 Program)(2009CB119000); the '863' Programs (2008AA10Z150, 20101110A108); the National Sup-porting Programs (2008BADB105) from the Ministry of Science and Technology of China; Ph. D. funding 20070307034 and 20090097110024 from the Ministry of Education of People's Republic of China.
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Botanical Studies, Vol. 52, 2011
異源多倍化誘導甜瓜屬新合成的異源四倍體Cucumis hytivus
中TyUcopia類逆轉座子的啟動
江  彪   婁群峰  王  東   吳志明   張萬萍   陳勁楓
中國南京作物遺傳與種質創新國家重點實驗室，
南方蔬菜遺傳改良重點開放實驗室，南京農業大學園藝學院
異源多倍化是作物進化的一個重要過程，是宿主基因組巨大的“基因組衝擊”。為了研究異源多倍
化對C. hytivus逆轉座子表達的影響，我們根據Ty 1-copia類逆轉座子逆轉錄酶的保守區設計簡並引物，
利用RT-PCR技術從異源四倍體C. hytivus及其二倍體親本C. hystrix和C. sativus中擴增逆轉錄酶基因，
結果僅在異源四倍體中擴增出目的片斷。回收測序後，獲得了 18個高度異質的克隆。這些克隆彼此不
同，至少能夠分為8個家族。同義（dS)和非同義（dN)替換分析表明這些序列受淨化選擇的作用。
根據這18個RT序列的氨基酸和從G en Bank資料庫中獲得的其它作物序列，利用最大似然法構建進化
樹，結果表明這些序列與其它作物存在高度的同源性，表明它們可能有共同起源。進一歩對所獲得的逆
轉錄酶序列在異源四倍體早期四個世代的表達分析證實，異源多倍化誘導了該類逆轉錄酶的表達。這些
發現為多倍體進化提供了重要資訊，並對進一歩表觀遺傳學研究具有重要意義。
關鍵詞：逆轉座子；逆轉錄酶；異源多倍化；RT-PCR ；甜瓜屬。