Departamento de Genética Molecular, Instituto de Biología Molecular de Barcelona, Barcelona, Spain
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Abstract |
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Key Words: Grande retrotransposon copy number phylogenetics SSAP markers maize teosinte
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Introduction |
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Retrotransposons are, due to their contribution to genome size and insertional mutagenesis potential, main players in the evolution of plant genomes and genes (Kumar and Bennetzen 1999). The study of RTN sequence variability will allow us to know the evolution of that particular RTN and to compare its phylogeny with the known host phylogeny. However, sequence diversity varies along the different functional regions of the RTN. It is well known that several boxes of RT, integrase, and other functional motifs from the RTN internal region are relatively well conserved over genera and kingdoms (Xiong and Eickbush 1990). On the other hand, LTR is one of the most variable regions (Vernhettes, Grandbastien, and Casacuberta 1998), often defining different families of RTNs by the percentage of nucleotide identity (SanMiguel and Bennetzen 1998). Most of the articles recently published on the evolution of copia-like and gypsy-like RTNs, from several plant genera, have been based on RT sequences (Pearce et al. 1996; Vernhettes, Grandbastien, and Casacuberta 1998; Kumekawa, Ohtsubo, and Ohtsubo 1999; Friesen, Brandes, and Heslop-Harrison 2001).
The inherent sequence variability of RTN elements and the variation of the flanking host sequences have allowed the development of a series of markers based on RTN, which offer several advantages over other molecular markers in assessing genetic diversity (reviewed in Kumar and Hirochika 2001). When sequence-specific amplification polymorphism (SSAP), an amplified fragment length polymorphism (AFLP)like technique, is done, using one of the PCR primers with a specific RTN sequence (Waugh et al. 1997) seems to be better for estimating phylogenetic relationships among plant genomes than, for example, AFLP-markers (Ellis et al. 1998; Gribbon et al. 1999).
Grande1 (or simply called Grande) retrotransposon, a gypsy-type of RTN, was initially found in the teosinte Zea diploperennis (Martínez-Izquierdo, García-Martínez, and Vicient 1997). Grande is interspersed along all maize chromosomes (Aledo et al. 1995) and has a 7-kb long 3' region downstream the integrase domain. The large size of Grande (13.8 kb) is manly due to the long 3' region since LTRs are only 0.6 kb long (Martínez-Izquierdo, García-Martínez, and Vicient 1997). That region contains two tandem arrays of repeats and several ORFs, ORF 23 being the only one transcribed in maize tissues (Martínez-Izquierdo, García-Martínez, and Vicient 1997). Zea is a genus that belongs to the Andropogoneae tribe of the Poaceae (Gramineae) family. The Zea intrageneric phylogeny was initially established by using isozyme and chloroplast DNA analysis (Doebley 1990), which has been recently confirmed by microsatellite genotyping (Matsuoka et al. 2002). According to that phylogeny, the Zea genus has two sections, luxuriantes and Zea. The former includes three species, the teosintes Z. diploperennis, Z. luxurians, and Z. perennis. The section Zea includes the only species Z. mays with four subspecies, mays (maize) and three teosinte subspecies, mexicana, parviglumis, and huehuetenangensis. Z. m. parviglumis has been shown to be the sole progenitor of maize (Z. m. mays) from a single domestication event (Matsuoka et al. 2002).
In this report, we study the evolution of Grande RTN in the Zea genus by three main approaches. First, we analyze the sequence variation of element LTRs in six different representative genomes of Zea. Second, we look at the host flanking sequences of Grande by applying SSAP technique, which, in turn, is used to develop molecular markers. Finally, we assess the contribution of Grande elements on Zea genomes. We show high LTR sequence heterogeneity, which correlates with the high number of Grande elements found, accounting for around 3% of the Zea genomes. We also report that great majority of the elements have the internal region between the two LTRs. Finally, phylogenetic trees derived from Grande-LTR-SSAP markers reflect the established Zea genus phylogeny.
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Materials and Methods |
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Plant genomic DNA was extracted from leaves using DNeasy Plant mini kit (QIAGEN). The yields were of 20 to 30 mg of pure DNA per 100 mg of fresh tissue.
LTR Amplification, Cloning, and Sequencing
We used 100 to 200 ng of genomic DNA for PCR amplification of Grande LTR (see fig. 1). Amplifications were performed in a 50 µl final volume containing 0.25 µM of each primer (U3-1u, 5'-ATATCCCCCGGGTCCACTA-3'; R-1l, 5'-GTTCGGGCCGCTTAGAGAT-3'), corresponding to positions 14 to 32 and 497 to 479, respectively, of Grande 5' LTR (GenBank accession number X97604), 0.2 mM dNTPs, 2.5 mM MgCl2, and 2.6 U of Expand High Fidelity System polymerase (Roche Diagnosis).
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The broad band obtained by PCR was purified and directly cloned into pGEM-T vector (Promega). Single bacterial clones were sequenced using PRISM sequencing kit (Applied Biosystems) by means of an automatic sequenator (ABI PRISM 377, Applied Biosystems).
Sequence Analyses
Sequences were aligned using the ClustalW program (Thompson, Higgins, and Gibson 1994). DNADIST of the Felsenstein's PHYLIP package (Felsenstein 2001) with the F84 distance option was used to generate distance matrixes (considering the observed base composition). The Tree-Puzzle program was used to establish the transition/transversion rates. Phylogenetic trees were constructed using the Neighbor-Joining method (Saitou and Nei 1987). Bootstrap values for the tree nodes were obtained using the SEQBOOT program (included in the PHYLIP package) with 1,000 replicates.
Different genetic variability parameters, such as nucleotide diversity () (Nei 1987), average number of nucleotide differences between any pair of aligned sequences (k), and Tajima's D statistic for neutrality test (D) were calculated using DnaSP 2.2 package (Rozas and Rozas 1997).
SSAP Band Analysis
Genomic DNA (0.2 to 0.5 mg) from each accession (a total of six) was digested with PstI and MseI for 3 h at 37°C in 1 x RL buffer (10 mM Tris-acetate pH 7.5, 10 mM magnesium acetate, 50 mM potassium acetate, and 5mM DTT), followed by ligation to adaptors. For this, each digested genomic DNA was added to 5 µl of ligation mix (5 pmol PstI adaptor [5'-CTCGTAGACTGCGTACATGCA-3' plus 5'-TGTACGCAGTCTAC-3'], 50 pmol MseI adaptor [5'-GACGATGAGTCCTGAG-3' plus 5'-TACTCAGGACTCAT-3'], 1.2 mM ATP, 1 x RL buffer, and 1.4 U T4 DNA ligase). The ligation mix was incubated at 37°C for 3 h and then at 4°C overnight. The resulting mix was diluted four to five times and then PCR preamplified using primers complementary to the adaptor sequences (P, 5'-GACTGCGTACATGCAG-3' and M, 5'-GATGAGTCCTGAGTA-3'). PCR reactions were performed with one initial cycle at 94°C (2 min), followed by 28 cycles at 94°C (30 s), 60°C (1 min), and 72°C (1 min); in 20 µl PCR buffer, containing 3 µl of template DNA, 30 ng P and M oligonucleotides, 0.2 mM dNTPs, 2.5 mM Cl2Mg, and 0.4 U of Taq polymerase. The preamplification mix was diluted by adding 150 to 200 µl of sterile distilled water and stored at 4°C. Before performing specific amplification reactions, the specific primer, LTR-G1 (5'-CTTGGGCCTTTCGTGAG-3'), was end-labeled by combining 0.1 µl -[33P] ATP (3000 Ci/mmol), 0.1 µl 10 x T4 buffer (0.25 mM Tris-HCl pH 7.5, 0.1 M MgCl2, 50 mM DTT, 5mM spermidine), 0.134 µl of LTR-G1 oligonucleotide (50 ng/µl), 0.25 U T4 kinase (0.03 µl), and 0.636 µl sterile distilled H2O. Each amplification reaction contained 1 µl labeled LTR-G1 primer, 0.4 µl dNTPs (5 mM), 2 µl 10 x PCR buffer, 1.2 µl MgCl2 (25 mM), 0.6 µl P+N selective primer (50 ng/µl), 0.1 µl Taq polymerase (0.5 U), 11.2 µl distilled H2O, and 3.5 µl preamplified DNA. P+N selective primers had the same sequence as the P primer given above plus additional selective nucleotides at the 3' end. The primers used were P+G, P+A, P+T, and P+C.
Cycling conditions of selective PCRs were as follows: 1 x (94°C [30 s], 65°C [30 s], and 72°C [1 min]), 12 x (94°C [30 s], 65°C [30 s], and 72°C [1 min]), with the annealing temperature decreasing 0.7°C per cycle, and 23 x (94°C [30 s], 56°C [30 s], and 72°C [1 min]).
After PCR, 20 µl of electrophoresis loading buffer (98% formamide, 10 mM EDTA pH 8.0, 0.05% bromophenol blue, and 0.05% xylene cyanol) was added to each tube. Samples were denatured by incubation at 95°C for 5 min and kept on ice until loaded (3 to 5 µl aliquots) into the wells of denaturing polyacrylamide gel (5% acrylamide/bis-acrylamide [19:1], 7 M urea, and 1 x TBE). Before loading the samples, the gel was prerun for 30 min at 95 W. Electrophoresis was then performed for 2 to 3 h at 95 W constant power. The gel was dried directly onto a glass plate and exposed to Kodak X-Omat film for 1 to 4 days at room temperature.
SSAP Data Analysis
A total of 80 clear and distinguishable Grande-SSAP bands were considered in order to construct four presence/absence matrixes (one for each primer combination). These matrixes were used to calculated genetic distances between accessions (species, subspecies, and inbred lines) using TFGA version 1.3 program (Miller 1997). Data are described as follows: each band is considered as a locus with two possible alleles (presence or absence) within six populations (six different DNA accessions, in this work) from a diploid organism and codominant marker. Two different options were tested to calculate genetic distances: unbiased Nei (1972, 1978) distances and modified Roger's distance (Wright 1978). The obtained distances were used to establish phylogenetic relationships among accessions by the UPGMA and Neighbor-Joining methods of generating phylogenetic trees. Node confidence was tested by performing bootstrap analysis with 1,000 replicates.
Copy Number Determination by Slot Blot Hybridization
DNA was blotted onto the nylon filters by vacuum filtration through the slot wells of a manifold device (Hoeffer PR600, Amersham Pharmacia Biotech), applying 50, 100, and 500 ng of genomic DNA samples from each Zea accession (including all the Zea species but Z. luxurians). Different amounts (0.1, 0.5, and 2.5 ng) of the PCR-amplified fragments from different Grande regions (LTR, gag [gag domain], RT [reverse transcriptase], int [integrase domain], ORF23, and noncod [noncoding region between ORF23 and 3' LTR]) were also loaded into the slot wells and used as controls in each filter. DNA was fixed into filters by incubation at 80°C for 2 h. The same Grande amplified fragments used as controls were used as hybridization probes: LTR (485 bp), gag (406 bp), RT (309 bp), int (338 bp), ORF23 (339 bp), noncod (468 bp). All the probes were labeled by random priming (Boerhinger Mannhein).
Filters were hybridized in 0.25 mM Na2HPO4, 7% SDS, 1 mM EDTA, pH 7.2, overnight at 65°C. Hybridized filters were consecutively washed with 2 x SSC (SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS (10 min, at room temperature), twice in 2 x SSC, 0.1% SDS (15 min, 65°C), twice in 0.5 x SSC, 0.1% SDS (15 min, 65°C), and twice in 0.1 x SSC, 0.1% SDS (15 min, 65°C). Bound DNA radiation on filters was quantified by exposure of filters to an imaging plate for 15 min to 6 h followed by scanning in a PhosphorImager (Personal Molecular Imager FX System, BIORAD).
Genomic copy number was calculated on the basis of the hybridization signal of the genomic DNA compared with the control DNA of the different Grande fragments on the slot blots as follows: copies ng-1 = genomic PSL ng-1 x fragment copies x fragment PSL-1 (PSL stands for photo-stimulated luminescence units, the output unit for exposure of the PosphorImager screens). Copies per ng were converted to copies per genome using the Zea genome size (data available from literature) (Laurie and Bennet 1985). A replicate for every amount of genomic DNA as well as amplified fragment was used to determine copy number for each accession.
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Results |
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Discussion |
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The nucleotide variability is not randomly distributed along the amplified sequences of Grande LTRs. Two of the most variable zones, due to punctual mutations and/or indels, are defined by nucleotides 75 to 101 (fig. 2) and 343 to 387 (fig. 4) and located upstream and downstream, respectively, to the putative TATA box (fig. 4). In the latter region, nucleotide mutations are almost equally frequent with indels. The most conserved zones are placed at the beginning and the end of the amplified fragments and around the putative TATA box (fig. 4). Interestingly, some motifs, such as the putative TATA box, are extraordinarily well conserved even in the most variable Zea genome analyzed (Palomero Toluqueño maize). Mutations within these regions could affect the transcriptional activity of the element and consequently its retrotransposition. Conservation in equivalent sequences that are involved in transcription has been reported for Tnt1 RTNs (Casacuberta, Vernhettes, and Grandbastien 1995). In the Tnt1 LTR U3 region, the so-called B boxes that are transcriptional activators exhibit the lowest mutation frequency within the transcribed elements. However, those B boxes are among the most variable regions within the LTR sequences corresponding to the Tnt1 genomic population, including active or inactive elements. In the case of Grande LTRs, the extremely well conserved putatively regulatory motifs, such as the TATA box, are congruent with recent transcriptional activity of Grande. Although there is no evidence, in historical times, of transposition events of Grande elements, there is evidence on transcriptional activity. This evidence includes some ESTs corresponding to Grande sequences recently found in different EST data collections (Meyers, Tingey, and Morgante 2001; Vicient et al. 2001), promoter activity of one of the B73 maize amplified fragments in transient expression experiments in maize tissues (unpublished data), and very recent results showing Grande RT-PCR transcripts corresponding to gag and integrase domains (Gomez-Orte 2002). The high heterogeneity of Grande LTR sequences outside the TATA box and other important regulatory motifs could reflect the antiquity of Grande in plant genomes. As a matter of fact, RIRE2, a gypsy-like RTN from rice, shows significant sequence similarity, especially in the pol domain, but no significant similarity was found between LTRs of the two RTNs. Interestingly, the similarity between Grande and RIRE2 goes downstream of the pol domain, where exceptional ORFs such as ORF3' from RIRE2 and ORF24 Grande, only described in these two RTNs, show significant similarity at the nucleotide and amino acid levels (Ohtsubo, Kumekawa, and Ohtsubo 1999). Therefore, the relatively high similarity of protein domains and the absence of similarity of LTRs between RIRE2 and Grande is compatible with long-term divergence of these two RTNs from a common ancestor before that Zea and Oryza genera separated, more than 50 MYA (Chen et al. 1997), assuming a higher rate of sequence variation for LTRs than for internal domains.
Phylogenetic Relationships: Grande LTR Sequences Versus SSAP Data
The high variability of the Grande LTR sequences from Zea is well shown by the phylogenetic trees from each genome accession as well as by the global tree, which showed similar topology to individual trees. In the global phylogenetic tree, one can see a certain grouping of the sequences without correlation with the classical Zea species and subspecies phylogenetic relationships. Since this incongruence is not due to the presence of long-branch attraction artifacts in the tree, which can be secondary to dissimilar evolutionary rates of most divergent elements, those results are compatible with the existence of different subfamilies or lineages of Grande elements before the speciation process of the Zea genus. A similar situation was also observed in the Tnt1 retrotransposon in Nicotiana species (Vernhettes, Grandbastien, and Casacuberta 1998), showing a high variability of its LTR sequences.
When a consensus sequence is obtained for each of the six Zea accessions analyzed (fig. 4) and the six consensus sequences are used for phylogenetic reconstruction, the depicted tree (fig. 5) better reflects the conventional Zea taxonomy than the global tree of LTR sequences. In fact, Z. diploperennis appears in a different branch than the other species included in the analysis, corresponding to the four accessions of maize (Z. m. mays) lines and its subspecies Z. m. mexicana. However, some maize lines are closer to Z. m. mexicana and even to Z. diploperennis than to other maize lines. For these reasons, we conclude that Grande retrotransposon LTR sequences are not good markers for phylogenetic studies on the host species analyzed.
Partly based in the AFLP technique, the SSAP analysis allows measuring the variability flanking a specific known sequence, such as a retrotransposon LTR (Waugh et al. 1997). SSAP yields, as row data, a binary matrix with two possibilities: presence or absence of a specific band. Whereas LTR-sequence variability only take into account intrinsic variability of retrotransposons, SSAP data also measure variability at other levels, in addition to RTN sequences, such as retrotransposon insertion polymorphism and genome sequence variability at the restriction site of the enzyme used in the digestion of genomic DNA. RTN-based SSAP technique, versus AFLP, has been particularly useful in estimating phylogenetic relationships in Pisum, which has allowed establishment of two events of domestication in the pea (Ellis et al. 1998). In the Zea genus, phylogenetic analysis using Grande LTR-based SSAP data, reconstructs the established taxonomy at the species and subspecies levels (Doebley 1990; Matsuoka et al. 2002), as can be seen in all UPGMA trees of figure 7. In these trees, the four maize lines (Z. m. mays species) are more closely related to its cospecies Z. m. mexicana than to a different species of the Zea genus, such as Z. diploperennis. Moreover, in three out four trees, the three maize inbred lines are in a different branch than Palomero Toluqueño maize, and this is, in turn, closer to Z. m. mexicana (fig. 7). In fact, Palomero Toluqueño is a Mexican race of maize with 12% of Z. m. mexicana germplasm (Matsuoka et al. 2002). These results, coming from only one gel and four PCR selective primers, show the usefulness of Grande LTR-based SSAP data for phylogenetic studies. These SSAP data, which also use host genome sequences in addition to RTN sequences, allow the reconstruction of the previously established intrageneric Zea phylogeny and the distinction among some maize lines.
Abundance and Structure of Grande Retrotransposon in the Zea Genus
The consistency of Grande copy number data (table 2) indicates that probes for estimation of RTN copy number used in this work, a set of labeled PCR-amplified products with a couple of primers from different RTN regions, are better than some of the probes regularly used in RTN copy determination. PCR amplified probes recognized all the sequence variability present between the two PCR primers, and they are preferred over cloned probes which represent only one element sequence and could result in underestimation of copy number, especially in heterogeneous or abundant RTNs. This was the case, for instance, when cloned probes from Grande1-7 were used for copy number determination (Aledo et al. 1995). On the other hand, the use of several different functional domains of the RTN as probes facilitates not only a more accurate copy number determination but also a better knowledge of the common structure for most elements of the RTN under study. In the case of Grande and for the Zea diploid species analyzed, copy number estimation per haploid genome was around 11,000 for LTR and 5,700 for the different internal region probes. The consistency in the number of copies is congruent with the similar number of SSAP bands per primer combination observed in all the accessions considered in this study (fig. 5). The Grande copy number value in Z. perennis, the only tetraploid species in the Zea genus (2n = 40 instead of 20 for the remainder species), was approximately two times higher than the estimated copy number for diploid accessions. These data suggest that colonization of the Z. perennis genome by Grande retrotransposon was prior to the polyploidization process that occurred in this species.
The efficient amplification of Grande LTR and internal domain sequences from all the Zea species used in this work (except Z. luxurians) corroborates the presence of Grande retrotransposon in the Zea genus previously reported by Southern hybridization experiments in all the Zea species (including Z. luxurians) using as probes Z. diploperennis clones Grande1-7 or Grande1-4 (Monfort et al. 1995; Vicient 1995). Grande has been also found in the Tripsacum genus with lower copy number as detected by Southern hybridization (Vicient 1995) and PCR experiments (unpublished data), confirming that massive amplification of Grande in Zea genomes occurred before the speciation process had started in the Zea genus and after the separation of Zea and Tripsacum genera. This conclusion is also supported by the fact that some of the most abundant RTNs in Zea, such as Huck, Opie, Zeon-1, and Grande, although present in Tripsacum, have a much lower copy number than in Zea (Vicient 1995; Meyers, Tingey, and Morgante 2001).
Grande copy numbers of 5,700 (Zea diploid species) or 11,600 (tetraploid Z. perennis) per haploid genome and 13.7-kb long elements (Martínez-Izquierdo, García-Martínez, and Vicient 1997) makes up around 3% of the Zea genome, which constitutes an important fraction of the genome for a single retrotransposon and places Grande in the medium/high copy number ranking among known RTNs (Martínez-Izquierdo, García-Martínez, and Vicient 1997; Kumar and Bennetzen 1999). A high genome share for Grande was already suggested for maize (SanMiguel et al. 1996) and more recently, Meyers, Tingey, and Morgante 2001 have reported that about 3.9% of the sequences of a library of randomly sheared genomic DNA from maize match with Grande sequences.
Retrotransposon copy number seems to be positively correlated with genome size across and within phyla. In the absence of disadvantageous effects on the host, RTN replication dynamics should lead to one-way "fattening" of the host genomes (Bennetzen and Kellogg 1997), unless mechanisms of removing RTNs could counteract that repetitive element amplification in the genome. Such mechanisms as unequal homologous recombination between the two LTRs or between two distant RTN elements have been described in yeast and Drosophila (reviewed in Bennetzen 2000). In the former case, the homologous recombination between the two LTRs results in a solo LTR and the loss of the RTN internal region. Recent reports in plants have shown that homologous recombination between the two LTRs from individual elements of BARE-1 retrotransposon has been an active mechanism in Hordeum genomes (Vicient et al. 1999), where high copy number LTR/internal region ratios were found. Ratios of 7 to 40 were determined, for instance, for the integrase probe. These authors suggest that this mechanism acts like a "partial return ticket from genomic obesity" caused by the genomic explosive growth of retrotransposons. Our results indicate that the great majority of Grande elements in the Zea genus have two LTRs per internal region (table 2) and in all of the internal domains analyzed, namely, gag, RT, int, ORF 23, and noncod. These data suggest that the mechanism of unequal homologous recombination between the two-element LTRs is not operating for Grande RTN in the Zea genus. The positive correlation seen in plants between recombination rates and the length of homologous sequence overlap (Puchta and Hohn 1991) could explain the very different outcome in solo LTR copy number of Hordeum BARE-1 versus Zea Grande retrotransposons, considering that BARE-1 LTRs are three times larger than Grande LTRs. Nevertheless, the scarceness of solo LTRs corresponding to maize RTNs, including the most abundant ones that correspond to intergene RTNs, is a general fact, irrespective of the LTR size (Bennetzen 2000). The scarceness of solo LTRs among maize intergene RTNs has been explained by the low recombination activity that the methylated and presumably heterochromatic blocks, where those RTNs are located, undergo (Bennetzen 2000). However, most of the intergene LTR retrotransposons found in the Rar1 locus of barley, including BARE-1, exist as solo LTRs (Shirasu et al. 2000). We conclude that the length of the LTRs or the presence of RTNs in large blocks are not the primary causes of the homologous recombination activity between retrotransposon LTRs that could be genome-dependent and/or RTN-dependent.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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E-mail: jamgmj{at}cid.csic.es.
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