Study on the Evolution of the Grande Retrotransposon in the Zea Genus

José García-Martínez1 and José A. Martínez-Izquierdo

Departamento de Genética Molecular, Instituto de Biología Molecular de Barcelona, Barcelona, Spain


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
The study of Grande retrotransposon (RTN) variation reported here comprises the intrinsic element variability and the changes that element insertion provokes in the Zea genome, including its abundance among species. Sequence analysis of a defined long-terminal repeat (LTR) region from Grande RTN revealed a high level of sequence divergence since no identical sequences were found among the 65 clones examined that belong to different Zea species or maize inbred lines. Average diversity values within accessions ranged from 0.17 to 0.37 substitutions per nucleotide. Phylogenetic analysis revealed a lack of concordance between the phylogenetic tree obtained from LTR sequences and the conventional taxonomic tree, suggesting that different subfamilies of Grande elements existed before Zea speciation. When sequence-specific amplification polymorphism (SSAP) marker data, which combines genomic and RTN variation, are used, the derived trees reflect the established species phylogeny and allow, as well, differentiating among some maize lines. Finally, the evaluation of Grande abundance, using different element probes in all the Zea species but Z. luxurians, revealed around 5,700 copies per haploid genome in all the diploid species examined, indicating a similar expansion process of Grande in all the Zea genomes. This number of copies represents in all cases around a 3% of the genome, which implies that Grande RTN is an important component of the maize genome. The copy number ratio LTR/gag is around 2 in all the species analyzed, indicating that overwhelming majority of elements have internal region. Thus, mechanisms such as homologous recombination between LTRs of a single RTN, which would remove the internal region and one LTR, leaving behind a single recombinant LTR, seems not to be active in maize for Grande RTN.

Key Words: Grande retrotransposon • copy number • phylogenetics • SSAP markers • maize • teosinte


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Retrotransposons (RTNs) are mobile elements that replicate via an RNA intermediate and constitute a conspicuous fraction of eukaryotic genome (International Human Genome Sequencing Consortium 2001). They move to new genome sites by converting their transcribed RNA into extrachromosomal DNA by the reverse transcriptase (RT) enzyme before reinsertion into the genome (Boeke and Corces 1989). There are two major types of RTNs, the retroviral type or long-terminal repeat (LTR) retrotransposons, simply called retrotransposons, and the non-LTR retrotransposons, such as LINEs (long interspersed nuclear elements) and SINEs (short interspersed nuclear elements) (for reviews see Kumar and Bennetzen 1999; Bennetzen 2000). The LTR retrotransposons encode a number of proteins, specified by three major genes named gag, pol, and int. These RTNs are further subdivided into Ty1-copia and Ty3-gypsy groups, which differ both in the order of the encoded enzymes RT and integrase and in their degree of sequence similarity (Xiong and Eickbush 1990). LTR retrotransposons are widespread and abundant in plant genomes (Flavell et al. 1992; Voytas et al. 1992; Suoniemi, Tanskanen, and Schulman 1998; Kumekawa, Ohtsubo, and Ohtsubo 1999) and can constitute a very important fraction of large genomes (Pearce et al. 1996; SanMiguel et al. 1996).

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.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Plant Material and DNA Extraction
We used 5-day-old to 10-day-old plants from Zea species Z. perennis and Z. diploperennis and from Z. mays subspecies Z. m. mexicana, Z.m. parviglumis, Z. m. mays, and Z. m. mays inbred lines B73, W64A, Mo17, and Palomero Toluqueño.

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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FIG. 1. Schematic representation of a Grande LTR. The origins of the typical LTR regions U3 (with borders defined by the beginning of the LTR and the transcription start site) and U5 (with borders defined by the polyadenylation site and the end of the LTR) are indicated by arrowheads. Thick arrows indicate the location of the primers used in LTR amplification by PCR. Thin arrowheads show the ends of the amplified fragment, which size is given in base pairs. The sequence and approximate location of the putative TATA box are indicated

 
Samples were amplified for 35 cycles at the following cycling conditions: denaturation at 94°C for 30 s, annealing at 58°C for 1 min, and extension at 72°C for 1 min. A first denaturation step at 94°C for 2 min was performed and a final extension step for 2 min.

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 ({pi}) (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 {gamma}-[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.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
LTR Sequence Variability
We have analyzed sequence variation within Grande retrotransposon by cloning and sequencing internal LTR fragments amplified by PCR from several Zea DNA accessions. The sequence analyzed was a 485-bp fragment, which encompasses at least part of the LTR U3 region (fig. 1). The amplified region contains putative regulatory motives, such as the TATA box, whose sequence TATATAA shows 87.5% of identity with the consensus TATA box sequence of plant genes (Joshi 1987). The absence of identical sequences among 65 analyzed corresponding to six Zea accessions reflects high sequence variability, as expected for some LTR sequences of retrotransposons. The LTR DNAs sequenced showed different levels of variability among different Zea accessions and among clones belonging to the same accession along the sequence. These aspects can be observed in two alignments (fig. 2), which correspond to the less variable set of sequences (Z. diploperennis) and the most variable set of sequences (Palomero Toluqueño maize), respectively. Only two parts of each alignment are shown, the first (73 to 102) is one of the less-conserved areas among all the accessions, and the second (302/308 to 331/337) is one of the most-conserved areas. Thus, in Z. diploperennis, only three polymorphic sites are observed in the latter zone versus 14 seen in Palomero Toluqueño. Similar pattern of variability is also observed in the first region, although all the sites are polymorphic for Palomero Toluqueño in opposition to only 16 polymorphic sites seen in the same area for Z. diploperennis sequences.



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FIG. 2. Alignment of highly variable and conserved regions of Grande LTR PCR-amplified sequences. Alignment of Grande LTR PCR-amplified sequences of Zea diploperennis and Palomero Toluqueño maize line, showing the lowest and highest sequence polymorphism among Zea accessions, respectively. Only partial sequences, corresponding to the most variable and conserved parts of amplified LTRs within each alignment, are shown

 
The sequence heterogeneity found in Grande LTRs was analyzed by calculating some genetic variability parameters, as shown in table 1. The {pi} (average number of nucleotide differences per site) and k (average number of nucleotide differences between any pair of sequences) support the variability observed in the sequence alignments. As can be shown in table 1, values of nucleotide divergence between pairs of sequences in each Zea species or maize lines ranged from 0.17 to 0.37, presenting an average number of nucleotide differences between any pair of sequences for each line that ranges from 65 to 117. Z. diploperennis has the lowest variability value, whereas Z. m. mays Palomero Toluqueño is the most variable DNA accession, showing the highest proportion of polymorphic sites. Finally, the Tajima's D statistic indicates that the hypothesis of neutral evolution could explain the polymorphism found among the sequences analyzed for each accession.


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Table 1 Genetic Variability Parameters of Grande1 LTR-Sequences from Different Zea DNA Accessions.

 
LTR Phylogenies
When all LTR sequences (65) were considered, a phylogenetic tree was obtained by the Neighbor-Joining method (Saitou and Nei 1987) using the F84 genetic distance (Felsenstein 2001). In this tree, it is possible to observe a certain grouping of the sequences in some branches of the phylogenetic tree (fig. 3). Two different groups of sequences can be observed in the tree, both containing sequences from all the Zea accessions. One group contains the majority and more closely related sequences and the other group contains the most divergent sequences. The latter branch (node marked by an asterisk) has a bootstrap value of 90, which strongly supports the grouping. When phylogenetic trees are constructed from each Zea accession, every individual tree reproduces the topology of the global tree well (data not shown). However, there is no correspondence between the LTR global tree and the Zea species taxonomy. Some Grande LTR sequences in the global tree from a particular genome are more closely related to other sequences from different genomes than to sequences from the same genome. This is true not only for sequences from maize lines but also for the other Zea subspecies and species. The incongruence between LTR-based and Zea species trees could be due to the existence of different Grande RTN lineages and to dissimilar evolutionary rates of the different elements. The latter can produce long-branch attraction artifacts in the tree. In order to check for the presence of long-branch attraction artifacts in the data, all LTR sequences excluding the most divergent ones were reanalyzed. The topology of the new tree (data not shown) is similar to the global tree in that LTR sequences appeared again interspersed in the different tree branches, not grouped by the Zea accession to which the sequences belong.



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FIG. 3. Global phylogenetic tree of Zea Grande LTR sequences. The tree was obtained by the Neighbor-Joining method (Saitou and Nei 1987), using Felsenstein's F84 distance. The asterisk indicates the node (with a bootstrap value of 90) that separates some of the most divergent sequences. Codes for Zea DNA accessions are as follows: B (Zea mays mays, inbred line B73), W (Z. m. mays, inbred line W64A), MO (Z. m. mays, inbred line Mo17), PT (Z. m. mays, Palomero Toluqueño), ZMM (Z. m. mexicana), and ZD (Zea diploperennis). Bootstrap values higher than 50% are shown. Horizontal distances are proportional to evolutionary distances according to the scale shown on the bottom

 
The LTR amplified sequences were also aligned accession by accession. From each of these alignments, a consensus sequence was obtained per accession, using in each position the nucleotide present in at least 50% of the individual sequences. The alignment of these consensus strings shows that less than 13% of positions are polymorphic (fig. 4). When these consensus sequences are used in turn for constructing a phylogenetic tree by the Neighbor-Joining method (fig. 5), a grouping of the branches corresponding to the Z. m. mays lines can be observed. High bootstrap values (showed on the branches) support this phylogenetic cluster. From the alignment presented in figure 4, a new consensus string was obtained that may reflect the closest sequence to the functional Grande ancestor.



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FIG. 4. Alignment of Grande LTR consensus sequences from DNA accessions of the Zea genus. LTR-amplified sequences from the six different Zea accessions (see caption of fig. 3) were aligned accession by accession, and a consensus sequence was obtained. The global consensus sequence (written at the top in bold letters), as well as the consensus for each accession, has been generated using in each position the nucleotide present in more than 50% of the sequences. Dots represent consensus nucleotides, and only nucleotides that differ are shown. Dashes represent the absence of nucleotides in those positions. Codes for ambiguities are as follows: M (A or C), R (A or G), W (A or T), S (C or G), Y (C or T), K (G or T), H (A, C or T), D (A, G or T), B (G, C or T), V (G, A or C), and N (G, A, T or C). The putative TATA box is highlighted

 


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FIG. 5. Phylogenetic tree of Zea Grande LTR consensus sequences. The tree was obtained using the Neighbor-Joining method (Saitou and Nei 1987) from the consensus sequence for each accession (shown in fig. 4). Numbers indicate the bootstrap values

 
Grande SSAP-Based Phylogenies
SSAP-PCR reactions on Zea DNAs were performed using LTR-G1 oligonucleotide from the 5' termini of Grande retrotransposon LTRs as specific and outward primer and PstI adapter complementary inward primers. When two or more additional selective nucleotides were added at the 3' end of the PstI adapter complementary primers, too few SSAP bands were produced to detect enough polymorphism for phylogenetic analysis (data not shown). On the other hand, the selective M+N primers produced too many SSAP bands (data not shown). PCR amplifications using four different primers (P+G, P+A, P+T, and P+C) with only one additional selective nucleotide and the LTR-G1 primer generated a convenient and distinguishable number of discrete SSAP bands (fig. 6). All four primer combinations yielded a similar number of bands that were sufficient to detect considerable levels of polymorphism among the six Zea DNA accessions tested. In fact, few common markers, defined as a sizeable DNA band obtained with a primer combination, can be observed in the gel autoradiogram among all the Zea accessions (fig. 6).



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FIG. 6. Grande1 retrotransposon SSAP. To generate SSAP bands, four different primer combinations were used. The outward Grande LTR specific primer, LTR-G1, was used in combination with every one of the four selective primers P+G, P+A, P+T, and P+C, respectively. Lane numbers indicate (1) Zea diploperennis, (2 to 5) Z. mays mays lines B73, W64A, Mo17, and Palomero Toluqueño, respectively, and (6) Z. mays mexicana

 
Considering the area of the film where the Grande SSAP bands were clearly distinguishable, 80 bands per accession were selected, involving 320 markers (80 x 4 primer combinations) and 1,920 data points (320 x 6 accessions). Four presence/absence matrixes of SSAP bands (one per primer combination) were constructed (not shown), and unbiased genetic distances of Nei (1978) were generated using the TFGA version 1.3 program (Miller 1997). The highest values (ranging from 1.1239 to 0.9163) corresponded to comparisons of Z. diploperennis with maize lines (Z. m. mays inbred lines), and the lowest values (ranging from 0.2389 to 0.2977) were obtained for comparisons between maize lines. From these distances, four different UPGMA trees with node bootstrap values from 1,000 replicates (one for each primer combination) were constructed (fig. 7). Other methods of phylogenetic reconstruction yielded similar topologies and bootstrap values higher than 50% in most of the cases. Unlike LTR phylogenies, all four Grande SSAP-derived trees clearly reproduce the Zea genus taxonomic subdivision at the subspecies level and, in some cases, at the maize lines level. Thus, the four inbred lines of Z. m. mays appear in all cases grouped in the same cluster, which can be seen as a sister group of the subspecies Z. m. mexicana. In three UPGMA dendrograms, Palomero Toluqueño maize is clearly located in a different branch than the other three maize lines (fig. 7). Finally, the most divergent lineage is that corresponding to Z. diploperennis, belonging to a different section than maize within the Zea genus (Doebley 1990).



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FIG. 7. Phylogenetic trees for Zea Grande SSAP bands. The four trees obtained by the UPGMA method correspond to PCR amplifications with LTR-G1 primer (see fig. 6 caption) in combination with primers P+G (A), P+A (B), P+T (C), and P+C (D), respectively. Numbers indicate the bootstrap values (from 1,000 replicates) associated to the nodes. Abbreviations for accessions are as in the caption of figure 3

 
Grande Copy Number Determination in the Zea Genus
The contribution of Grande retrotransposon to the genome size was explored in all the Zea species, except for Z. luxurians. Those examined were Z. diploperennis, Z. perennis, and Z. mays, including the subspecies mexicana, parviglumis (Balsas' teosinte race), and mays (maize). The subspecies Z. m. mays contributed four lines: B73, W64A, Mo17, and Palomero Toluqueño. Estimates of the copy number of Grande in the different Zea accessions were obtained by genomic reconstruction using slot-blot hybridization as described (Aledo et al. 1995). The probes were multiple PCR-generated DNAs corresponding to each of the following domains of Grande retrotransposon: LTR, gag, RT, integrase, ORF23, and noncod (table 2). All these probes were initially used to estimate Grande retrotransposon copy number in Z. diploperennis and maize B73 inbred line (table 2). Only probes coming from LTR, gag, and noncod regions were used for copy number determination in the remainder accessions, due to the lower variability data observed in Grande retrotransposon copy number compared with RT, integrase, and ORF23 (table 2). ORF23 resulted in more dispersion of data, and RT and integrase overestimated the number of Grande elements, most likely due to hybridization to other retrotransposon RT and integrase domains, which was probably the result of their high sequence similarity in those domains with other RTNs belonging to different families present in the Zea genus (SanMiguel et al. 1996). Except for Z. perennis, the only tetraploid species of the Zea genus, copy number values ranged from 10,900 to 11,600, 5,400 to 6,100, and 5,300 to 6,000 for the LTR, gag, and noncod domains, respectively. In complete retrotransposon elements, two LTRs per internal re-gion are expected as a consequence of retrotransposon mechanisms of replication and transposition, unless mechanisms such as intrachromosomal homologous recombination produce solo LTRs as a result of removal of the internal region and one out of the two LTRs of RTN elements. If this is so, one expects LTR/internal region ratios higher than 2, as have been reported for BARE-1 retrotransposon in Hordeum species (Vicient et al. 1999). Unlike this case, LTR/internal region ratios for Grande RTN are very close to 2 in all the Zea accessions, including Z. perennis (table 2). In Z. perennis, the estimated copy number values per haploid genome were around 21,800, 11,600, and 12,400 for LTR, gag, and noncod regions, respectively. The ratio LTR/gag copy number values in Z. perennis (the only tetraploid species in the genus) are also very close to 2 (1.9) as in diploid accessions. The average copy number of Grande retrotransposon, which is 13.7-kb long, is 5,700 copies. Assuming an average value for the haploid genome size of the Zea diploid species analyzed in this work as 2.5 x 109 bp (Laurie and Bennet 1985), Grande contributes 3.12% to the genome of diploid Zea accessions. For the tetraploid Z. perennis, with a genome size of 5.28 x 109 bp and an average of 11,600 copies of Grande per haploid genome, the retrotransposon occupies 3.01% of the genome, suggesting that the same amplification event of Grande retrotransposon has occurred in all the Zea species.


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Table 2 Grande Retrotransposon Copy Number in the Zea Genus.

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
Sequence Heterogeneity of Grande LTRs
The efficient amplification of Grande LTR sequences from all the Zea genomes used in this work (excluding Z. perennis and Z. luxurians) indicates the abundance of Grande RTN in those genomes and/or the relatively high conservation of the sequences corresponding to the primers used in the PCR amplification. However, a high degree of sequence variability was estimated among the 65 LTR PCR-amplified sequences corresponding to the six accessions under analysis (table 1). These nucleotide diversity values, ranging between 0.17 and 0.37 substitutions per nucleotide, are in the same order than in the tobacco RTN Tnt1 (Casacuberta, Vernhettes, and Grandbastien 1995). This sequence variability found in some regions of Grande retrotransposon LTRs is in agreement with the degree of sequence heterogeneity found in those zones of LTRs from plant retrotransposons (SanMiguel and Bennetzen 1998; Vernhettes, Grandbastien, and Casacuberta 1998) and with the evolutionary dynamics for noncoding regions that have less evolutionary constrains to sequence changes (i.e., mutations and indels). As has been theoretically proposed by Charlesworth (1986), sequence heterogeneity should be related to element copy number within individuals in a species, since each element has a given probability of acquiring mutations. There is also direct experimental confirmation that this correlation holds between closely related species (Pearce et al. 1996). Our results of high sequence heterogeneity in the retrotransposon LTRs between closely related Zea species (even maize inbred lines) are in agreement with those theories and evidences, taking into account the high copy number values for Grande RTN determined in these genomes (see later discussion). From the Tajima's (1989) test for neutrality, which indicates that all values are not statistically significant (table 1), we can conclude that the hypothesis of neutral mutation could explain the sequence polymorphism found, as is usual for noncoding regions.

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.


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
The 65-nucleotide sequences reported in this paper will appear in the EMBL nucleotide sequence database under the accession numbers AJ312443 to AJ312507. The EMBL number for the alignment of those sequences is ALIGN_000045. The EMBL numbers for the six alignments of sequences from individual accessions are B73, ALIGN_000060; Mo17, ALIGN_000061; PT, ALIGN_000062; W64A, ALIGN_000063; ZD, ALIGN_000064; and ZMM, ALIGN_000065. The corresponding phylogenetic trees (not shown in the manuscript) are available upon request.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 
This work was supported by the following grants to J.A.M.I.: ERBIO4OCT960508 from EU Biotechnology Program and BIO97-0770 (CICYT), PB97-1255 (DGICYT), and BIO99-1175 (CICYT) from the Spanish MEC. The authors are indebted to E. Barrio for the critical reading of the manuscript. J.G.M. was the recipient of a Postdoctoral Fellowship from EU Biotechnology Program (ERBIO4OCT960508).


    Footnotes
 
1 Present address: Departament de Bioquímica i Biología Molecular, Universitat de València, Valencia, Spain. Back

Pierre Capy, Associate Editor Back

E-mail: jamgmj{at}cid.csic.es. Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 Literature Cited
 

    Aledo, R., R. Raz, A. Monfort, C. M. Vicient, P. Puigdomenech, and J. A. Martínez-Izquierdo. 1995. Chromosome localization and characterization of a family of long interspersed repetitive DNA elements from the genus Zea. Theor. Appl. Genet. 90:1094-1100.[ISI]

    Bennetzen, J. F. 2000. Transposable element contributions to plant gene and genome evolution. Plant Mol. Biol. 42:251-269.[CrossRef][ISI][Medline]

    Bennetzen, J. L., and E. A. Kellogg. 1997. Do plants have a one-way ticket to genome obesity? Plant Cell 9:1509-1514.[Free Full Text]

    Boeke, J. D., and V. G. Corces. 1989. Transcription and reverse transcription of retrotransposons. Annu. Rev. Microbiol. 42:403-434.[CrossRef]

    Casacuberta, J. M., S. Vernhettes, and M. A. Grandbastien. 1995. Sequence variability within tobacco retrotransposon Tnt1 population. EMBO J. 14:2670-2678.[Abstract]

    Charlesworth, B. 1986. Genetic divergence between transposable elements. Genet. Res. 48:111-118.[ISI][Medline]

    Chen, M., P. SanMiguel, A. C. De Oliveira, S.-S. Woo, H. Zhang, R. A. Wing, and J. L. Bennetzen. 1997. Microcolinearity in sh2-homologous regions of the maize, rice, and sorghum genomes. Proc. Natl. Acad. Sci. USA 94:3431-3435.[Abstract/Free Full Text]

    Doebley, J. 1990. Molecular systematic of Zea (Gramineae). Maydica 35:143-150.[ISI]

    Ellis, T. H. N., S. J. Poyser, M. R. Knox, A. V. Vershinin, and M. J. Ambrose. 1998. Polymorphism of insertion sites of Ty1-copia class retrotransposons and its use for linkage and diversity analysis in pea. Mol. Gen. Genet. 260:9-19.[CrossRef][ISI][Medline]

    Felsenstein, J. 2001. PHYLIP: phylogeny inference package (version 3.6). University of Washington, Seattle.

    Flavell, A. J., E. Dunbar, R. Anderson, S. R. Pearce, R. Hartley, and A. Kumar. 1992. Ty1-copia group retrotransposons are ubiquitous and heterogeneous in higher plants. Nucleic Acid Res. 20:3639-3644.[Abstract]

    Friesen, N., A. Brandes, and J. S. Heslop-Harrison. 2001. Diversity, origin, and distribution of retrotransposons (gypsy and copia) in conifers. Mol. Biol. Evol. 18:1176-1188.[Abstract/Free Full Text]

    Gomez-Orte, E. 2002. Estudio del gen 23 del retrotransposon Grande de maíz. Ph.D. Thesis, University of Barcelona, Spain.

    Gribbon, B. M., S. R. Pearce, R. Kalendar, A. H. Schulman, L. Paulin, P. Jack, A. Kumar, and A. J. Flavell. 1999. Phylogeny and transpositional activity of Ty1-copia group retrotransposons in cereal genomes. Mol. Gen. Genet. 261:883-891.[CrossRef][Medline]

    International Human Genome Sequencing Consortium. 2001. Initial sequencing and analysis of the human genome. Nature 409:860-921.[CrossRef][ISI][Medline]

    Joshi, C. P. 1987. An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res. 15:6643-6653.[Abstract]

    Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein Molecules. Pp. 21–132 in H. N. Munro, ed. Mammalian protein metabolism, Vol. 3. Academic Press, New York.

    Kumar, A., and J. L. Bennetzen. 1999. Plant retrotransposons. Annu. Rev. Genet. 33:479-532.[CrossRef][ISI][Medline]

    Kumar, A., and H. Hirochika. 2001. Applications of retrotransposons as genetic tools in plant biology. Trends Plant Sci. 6:127-134.[CrossRef][ISI][Medline]

    Kumekawa, N., E. Ohtsubo, and H. Ohtsubo. 1999. Identification and phylogenetic analysis of gypsy-type retrotransposons in the plant kingdom. Genes Genet. Syst. 74:83-91.[CrossRef][ISI][Medline]

    Laurie, D. A., and M. D. Bennet. 1985. Nuclear DNA content in the genera Zea and Sorghum. Intergeneric, interspecific and intraspecific variation. Heredity 55:307-313.[ISI]

    Martínez-Izquierdo, J. A., J. García-Martínez, and C. M. Vicient. 1997. What makes Grande1 retrotransposon different? Genetica 100:15-28.[CrossRef][ISI][Medline]

    Matsuoka, Y., Y. Vigouroux, M. M. Goodman, G. J. Sanchez, E. Buckler, and J. Doebley. 2002. A single domestication for maize shown by multilocus microsatellite genotyping. Proc. Natl. Acad. Sci. USA 99:6080-6084.[Abstract/Free Full Text]

    Meyers, B. C., S. V. Tingey, and M. Morgante. 2001. Abundance, distribution and transcriptional activity of repetitive elements in the maize genome. Genome Res. 11:1660-1676.[Abstract/Free Full Text]

    Miller, M. P. 1997. Tools for population genetic analyses (TFPGA) v. 1.3: a Windows program for the analysis of allozyme and molecular population genetic data. Distributed by the author, Department of Biological Sciences, Northern Arizona University.

    Monfort, A., C. M. Vicient, R. Raz, P. Puigdomenech, and J. A. Martínez-Izquierdo. 1995. Molecular analysis of a putative transposable retroelement from the Zea genus with internal clusters of tandem repeats. DNA Res. 2:255-261.[Medline]

    Nei, M. 1972. Genetic distance between populations. Am. Nat. 106:283-292.[CrossRef][ISI]

    Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583-590.[Abstract/Free Full Text]

    Nei, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York.

    Ohtsubo, H., N. Kumekawa, and E. Ohtsubo. 1999. RIRE2, a novel gypsy-type retrotransposon from rice. Genes Genet. Syst. 74:83-91.[CrossRef][ISI][Medline]

    Pearce, S. R., G. Harrison, D. Li, J. S. Heslop-Harrison, A. Kumar, and A. J. Flavell. 1996. The Ty1-copia group retrotransposons in Vicia species: copy number, sequence heterogeneity and chromosomal localization. Mol. Gen. Genet. 250:305-315.[CrossRef][ISI][Medline]

    Puchta, H., and B. Hohn. 1991. A transient assay in plant cells reveals a positive correlation between extrachromosomal recombination rates and length of homologous overlap. Nucleic Acids Res. 19:2693-2700.[Abstract]

    Rozas, J., and R. Rozas. 1997. DnaSP version 2.0: a novel software package for extensive molecular population genetic analysis. Comput. Appl. Biosci. 13:307-311.[Abstract]

    Saitou, N., and M. Nei. 1987. The Neighbor-Joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]

    SanMiguel, P., and J. L. Bennetzen. 1998. Evidence that a recent increase in maize genome size was caused by the massive amplification of intergene retrotransposons. Ann. Bot. 82:(Suppl. A): 37-44.[Abstract/Free Full Text]

    SanMiguel, P., A. Tikhonov, and Y. K. Jin, et al. (11 co-authors). 1996. Nested retrotransposons in the intergenic regions of the maize genome. Science 274:765-768.[Abstract/Free Full Text]

    Shirasu, K., A. H. Schulman, T. Lahaye, and P. Schulze-Lefert. 2000. A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion. Genome Res. 10:893-894.[Free Full Text]

    Suoniemi, A., J. Tanskanen, and A. H. Schulman. 1998. Gypsy-like retrotransposons are widespread in the plant kingdom. Plant J. 13:699-705.[CrossRef][ISI][Medline]

    Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585-595.[Abstract/Free Full Text]

    Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. ClustalW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract]

    Vernhettes, S., M. A. Grandbastien, and J. M. Casacuberta. 1998. The evolutionary analysis of Tnt1 retrotransposon in Nicotiana species reveals the high variability of its regulatory sequences. Mol. Biol. Evol. 15:827-836.[Abstract]

    Vicient, C. M. 1995. Caracterizacion molecular de Grande1, un nuevo retrotransposon del genero Zea. Ph.D. Thesis, University of Barcelona, Spain.

    Vicient, C. M., M. J. Jääskeläinen, R. Kalendar, and A. H. Schulman. 2001. Active retrotransposons are a common feature of grass genomes. Plant Physiol. 125:1283-1292.[Abstract/Free Full Text]

    Vicient, C. M., A. Suoniemi, K. Anamthawat-Jónsson, J. Tanskanen, A. Beharav, E. Nevo, and A. H. Schulman. 1999. Retrotransposon BARE-1 and its role in genome evolution in the genus Hordeum. Plant Cell 11:1769-1784.[Abstract/Free Full Text]

    Voytas, D. F., M. P. Cummings, A. Konieczny, F. M. Ausubel, and S. R. Rodermel. 1992. Copia-like retrotransposons are ubiquitous among plants. Proc. Natl. Acad. Sci. USA 89:7124-7128.[Abstract]

    Waugh, R., K. McLean, A. J. Flavell, S. R. Pearce, A. Kumar, B. B. T. Thomas, and W. Powell. 1997. Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol. Gen. Genet. 253:687-694.[CrossRef][ISI][Medline]

    Wright, S. 1978. Evolution and the genetics of populations, Vol. 4. Variability within and among natural populations. University of Chicago Press, Chicago.

    Xiong, Y., and T. H. Eickbush. 1990. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 9:3353-3362.[Abstract]

Accepted for publication January 20, 2003.





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