Department of Crop Genetics, John Innes Centre, Norwich, United Kingdom
Correspondence: E-mail: alexander.vershinin{at}bbsrc.ac.uk.
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Abstract |
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Key Words: transposable elements Pisum evolution molecular markers recombination domestication retrotransposon activity
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Introduction |
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The differences between closely related genomes are often associated with repetitive DNAs (Britten and Kohne 1968; Walker 1968). Mobile genetic elements (or transposable elements, TEs) are a major component of plant genomes where they may comprise more than 50% of the nuclear DNA (Kunze, Saedler, and Lonning 1997; SanMiguel and Bennetzen 1998; Bennetzen 2000a) and contribute to diversity through both insertion site polymorphism and small structural rearrangement (Bennetzen 2000a). Transposable elements are classified into two groups according to their transposition mechanism and mode of propagation (Finnegan 1992): retrotransposons (class I elements) transpose via an RNA intermediate, whereas transposons (class II elements) move by excision and reintegration ("cut-paste"). The ubiquity and distribution of these TEs suggest that they should be potentially useful as diagnostic tools conforming to the requirement for abundant and reliable markers.
The relationship between transposable elements and chromosomal rearrangement in plants was first shown by McClintock (1946); deletions, insertions, frameshifts, inversions, duplications, translocations, and the generation of intron-like sequences have all been associated with TEs (Kunze, Saedler, and Lonning 1997). Because of their ability to move from place to place within a genome, or to produce new copies of themselves at any genomic location, TEs possess an extraordinary potential for the alteration of genome structure and adjacent gene function (Nekrutenko and Li 2001). These characteristics have promoted the idea that TEs are important players in plant evolution (Wessler, Bureau, and White 1995).
The genus Pisum L. has characteristics that make it particularly interesting for the application of TE-based markers to the analysis of intragenus genetic diversity. Pea (Pisum sativum L.) is an Old World legume crop thought to have been among the first cultivated in the Middle East, about 10,000 years ago (Blixt 1972; Zohary 1996). The modern gene pool of cultivated Pisum is diverse, reflecting this early domestication and subsequent widespread cultivation. However, in spite of the extensive phenotypic and genetic variability, existing taxonomic classifications distinguish few Pisum species, from two (Ben-Ze'ev and Zohary 1973) to six (Govorov 1937).
Pisum has a large genome (about 4,000 Mb), which is stable in size between species (Greilhuber and Ebert 1994). Pea is one of a few plant species in which the representatives of different TE groups have been isolated and characterized, and as an inbreeding species, it contrasts with maize that has extensively characterized retroelement families (SanMiguel et al. 1996). We have selected three elements from different TE groups for this study: PDR1 is a Ty1-copia group retrotransposon present in about 200 copies per haploid genome; it is about 4 kb in length, and its LTRs (long terminal repeats), at 156 bp (Lee et al. 1990), are exceptionally short. In contrast, Cyclops has the typical pol region of the Ty3/gypsy group and is present in about 5,000 copies (Chavanne et al. 1998). Cyclops elements are approximately 12 kb long, including very long LTRs of about 1,500 bp. The reading frame of the pol region of the canonical Cyclops element is disrupted by several mutations, suggesting that it is nonfunctional. The third element we have studied was Pis1, a representative of pea class II mobile elements (Shirsat 1988), which has the copy number comparable to that of Cyclops as determined in pilot sequence-specific amplification polymorphism (SSAP) experiments. Its terminal inverted repeats contain the sequence (5'-CACTA-3') of the En/Spm superfamily, and it is abundant in the genome.
DNA markers have been powerful in phylogenetic diversity analysis and several methods have been shown to be efficient for comparison of different plant genotypes (Powell et al. 1996; Russell et al. 1997; Pejic et al. 1998), including pea (Lu et al. 1996). Amplified fragment length polymorphism (AFLP) and SSAP have a high multiplex ratio (Lu et al. 1996; Powell et al. 1996; Waugh et al. 1997; Ellis et al. 1998) and offer a distinct advantage when genome coverage is a major issue. For an extensively inbreeding system, such as pea, dominance of the markers is not such an important consideration and it has been shown that SSAP markers produced by TEs are more informative than AFLP and restriction fragment length polymorphism (RFLP) markers (Ellis et al. 1998). This SSAP approach reveals insertion site polymorphism and sequence variation in the flanking DNA or primer binding site.
We have applied the SSAP approach to study the genetic structure and evolutionary history of Pisum. Our analysis is based on a wide range of Pisum accessions selected from John Innes Centre germplasm collection, which contains more than 3,000 accessions. We aimed to: (1) characterize the phylogenetic diversity of Pisum and to compare the patterns revealed by TEs with large differences in abundance and contrasting transposition mechanisms; (2) examine the relative contribution of different molecular mechanisms to genetic diversity within Pisum; and (3) compare the genetic diversity associated with the domestication of two domesticated species, P. sativum and P. abyssinicum, Braun.
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Materials and Methods |
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Template Preparation, Polymerase Chain Reaction Condition, Adapters, and Primers
The structure of TEs with the position of restriction sites and primers used in this study is shown in figure 1. The short PDR1 long terminal repeats (LTRs) allowed the use of the polypurine tract (PPT) primer and digestion of the genomic DNA (about 0.5 µg) by one restriction enzyme, TaqI. For Cyclops SSAP, the primer was designed to the very 3'-end of the LTR, and DNA was cut by two restriction enzymes, EcoRV and SfoI, the latter to avoid the production of internal Cyclops fragments from the 5' LTR.
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Data Analysis
A table of presence/absence for each band in an SSAP profile was generated. Bands with the same migration were considered to be identical (Casa et al. 2002), whereas others were treated as independent insertions. The total number of pairwise mismatches was used to calculate pairwise distances from the SSAP band scores. The resulting matrix was used as input for principal component analysis (PCA) using the GenStat 5 package and analysis of molecular variance (AMOVA) (Excoffier, Smouse, and Quattro 1992). Phylogenetic trees were generated from this matrix using the Neighbor-Joining (NJ) algorithm (Saitou and Nei 1987) of the Neighbor program in the PHYLIP package (Felsenstein 1993). Neighbor-Joining dendrograms were also constructed from pairwise ST values between accession groups generated by AMOVA. Trees were plotted using Treeview. The genetic variation was measured in terms of gene diversity (Nei 1987) as Hmean.
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Results |
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The PDR1 family has a copy number well suited to SSAP analysis and genome mapping because no preamplification is required in the PCR and we can examine a high proportion of the total number of insertion sites. Therefore, we selected this family for the analysis of extended sets of main Pisum lineages (step 2) and produced NJ trees for each species, together with corresponding reference accessions (fig. 2). In the NJ tree of the P. fulvum extended set (fig. 2A), all P. fulvum accessions are quite well separated from other groups and are characterized by long branches, suggesting remote common ancestry. Within P. fulvum group one can distinguish three sub-groups with shorter distances between branch points. A very different pattern of branching was found in the extended set of P. abyssinicum (fig. 2B). Of the 32 accessions, 30 are placed on one major branch with extremely short distances between accessions, but two accessions are slightly separated from all others. While on the basis of morphology JI1937 is typical for P. abyssinicum, JI2674 carries some P. sativum morphological characteristics and probably represents P. abyssinicum introgressed with P. sativum.
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Analysis of the extended sets of different Pisum species revealed a high number of new markers for each species (table 1) that were absent in initial reference accessions of given species. However, for P. fulvum and particularly P. elatius, a significant proportion of these new markers were absent from the initial reference set while for P. abyssinicum and P. sativum there were a very few markers of this type.
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Principal component analysis (PCA) of these data, obtained with the final set of accessions (step 3), identified three major groups, P. fulvum, P. abyssinicum, and a P. elatiusP. humileP. sativum complex for both PDR1 and Cyclops (fig. 3A and 3B). The percentages of the total variation, explained by the first two axes for Cyclops and PDR1, were 64% and 67%, respectively. Within the P. elatiusP. humileP. sativum complex, two subclusters of accessions were evident. One subcluster (group 1) comprised eight P. elatius and one P. humile accession, and the other (group 2) consisted of four P. elatius and all P. sativum accessions. For the Pis1 data (fig. 3C) the P. fulvum accessions were closer to the accessions of P. elatius complex, but P. abyssinicum was as distinct as for the retroelement data. In this data set, the first two axes made up 56.5% of the total variation, with nearly 45% in PCA1.
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Phylogenetic trees constructed with the NJ algorithm, from pairwise ST values, were similar for the three TE data sets (fig. 5). However, the P. fulvum branch was noticeably shorter in the tree generated with Pis1-derived markers than in the one generated with the retrotransposon data, suggesting a higher proportion of P. fulvum Pis1-markers shared with other species. The P. elatiusP. sativum branch has the same length in all trees; however, the shape of the branch in the Pis1 tree is different when compared to the retrotransposon trees; for these, the position of P. elatius is closer to the point of divergence from P. fulvum and P. abyssinicum. These differences in the Pis1 tree are consistent with its lower value for the variance among accession groups and are likely reflect differences in the evolutionary history of these elements and their relative contribution in genetic diversity.
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Discussion |
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At the level of SSAP analysis we cannot distinguish between different mechanisms that rearrange TE flanking DNA. Transposon (Pis1 in our analysis) propagates by a cut-and-paste mechanism; this and other rearrangements of flanking DNA would result in the replacement of one SSAP band by another. Retrotransposition based on amplification and the insertion of new copies will produce new SSAP markers in addition to preexisting ones. If the DNA that flanking different TEs is affected by rearrangement to a similar extent, and if each transposition event is unique, then the relative proportion of unique and species-specific markers for each element will reflect the relative contribution of that element's transposition to the observed diversity. The longer P. fulvum branches in the NJ tree constructed from the AMOVA analysis of retrotransposon markers, as compared to transposon markers (fig. 5), results from a higher value of the variance among the accession groups. This implies that there is a higher proportion of retrotransposon markers (rather than transposon markers) that are restricted to P. fulvum. This tendency is well supported by the data of table 3, where P. fulvum, P. elatius, and P. sativum shared a similar high level of polymorphism, but in P. sativum alone the proportion of species-specific and unique bands was close to zero. The PDR1 markers were the most variable in P. fulvum and P. elatius, more than twofold higher than the Pis1 markers, a finding consistent with retrotransposition in these two lineages.
Despite the high level of polymorphism, element copy number is remarkably constant. The evolutionary history of the reverse transcriptase gene in 11 plant species, including pea, has indicated strong patterns of purifying selection (Navarro-Quezada and Schoen 2002) with high rates of element loss balanced by transposition. Our data suggest that segregation and drift could be effective mechanisms accounting for the loss of insertion sites.
Recombination Is a Dominant Feature of Pisum Evolution and Domestication
Virtually all SSAP bands are polymorphic (table 3), with unique and species-specific markers making up only a small proportion of them. This situation is consistent with the possibility that introgression, segregation, and small rearrangement, rather than transposition itself, are the dominant modes of diversity generation, even for the most ancient Pisum lineages, P. fulvum and P. elatius. Comparative sequence analysis has revealed a much higher degree of diversity at the microstructural level than was predicted by genetic mapping studies of closely related plant species (Bennetzen 2000b; Bennetzen and Ramakrishna 2002). Sequencing of the bz genomic region of two maize lines revealed dramatic differences between them in both retrotransposon clusters and genes, demonstrating that genetic microcolinearity can be violated even within the same species (Fu and Dooner 2002).
The taxonomy of Pisum has been much disputed (Govorov 1937; Blixt 1972; Ben-Ze'ev and Zohary 1973; Zohary 1996). Conventionally one cultivated species P. sativum and three wild taxa, P. elatius, P. humile, and P. fulvum, are recognized. Based on the analysis of morphology, ecology, cytogenetics, and hybrid performance, Ben Ze'ev and Zohary (1973) concluded that P. fulvum is a fully divergent species, whereas P. humile, P. elatius, and P. sativum form a single-species complex comprised of two main races, weedy forms (elatius and humile), and cultivated derivatives (sativum); P. fulvum together with P. elatius were recognized as the ancient lineages (Govorov 1937; Blixt 1972; Ben-Ze'ev and Zohary 1973; Zohary 1996).
Phylogenetic relationships revealed by SSAP analysis are generally congruent with those derived from traditional taxonomic studies. The NJ trees constructed for extended sets of each of the major Pisum lineages illustrate very well the main characteristics of Pisum phylogeny summarized in the combined NJ tree (fig. 4). The P. fulvum lineage with long internal branches forms a distinct group; P. abyssinicum also forms a distinct group, but with extremely short internal branches and, correspondingly low within-lineage diversity. The P. elatius accessions are widely distributed, and several of them are intermingled with P. humile and P. sativum accessions. Some authors considered P. fulvum as species having insignificant intraspecific differentiation of morphological features (Govorov 1937). In contrast, the distribution pattern of our molecular markers produced by different TEs among P. fulvum accessions did not show any monomorphic bands and Hmean of P. fulvum is closer to the P. elatius and P. sativum values than to the P. abyssinicum value (table 2). A similar disparity between phenotypic and molecular estimation of diversity has been reported for other species, such as maize (Burstin and Charcosset 1997; Smith et al. 1997) and tomato (Noli, Salvi, and Tuberosa 1997).
The close relationships among P. elatius, P. humile, and P. sativum have been corroborated by PCA analysis (fig. 3), where Pis1-derived markers placed these species in one broad cluster. PDR1 and Cyclops markers subdivided this cluster into two groups. Group 1 comprises eight accessions of P. elatius plus both P. humile accessions, whereas group 2 consists of four P. elatius accessions and all accessions of P. sativum. Analysis of the P. elatius accessions showed that the two groups carried contrasting alleles associated with domestication at the following loci: Np (neoplastic pods), Gty (gritty testa), and Dp (dehiscent pod). Thus P. elatius group 1 can be considered the wild forms of P. elatius (and P. humile), whereas P. elatius accessions from group 2 likely represent the section of P. elatius from which the antecedents of cultivated P. sativum were drawn, or alternatively that have had recent introgression with P. sativum.
One of the most discussed problems in Pisum taxonomy is the position and status of P. abyssinicum, namely whether this lineage has diverged far enough from other taxa to be considered a separate species or whether it should be placed within P. sativum as a subgroup or ecotype. Govorov (1937) found slightly differentiated morphological features among different P. abyssinicum accessions, as for P. fulvum, and judged that these were sufficiently distinct to consider this lineage a separate cultivated species. However, more often, P. abyssinicum has been regarded as an ecotype or subgroup of P. sativum (Makasheva 1984, p.40). All our data demonstrate the clear distinction of P. abyssinicum from all other lineages. The transposon and retrotransposon markers showed the same level of resolution in PCA (fig. 2), and, for both, the P. abyssinicum branch was the longest in the NJ trees generated by AMOVA (fig. 4). P. abyssinicum accessions are very similar, so for the final set of 52 accessions, we selected fewer P. abyssinicum accessions than accessions from other species (except P.humile). The high genetic homogeneity and exceptional distinction of this taxon was also revealed for 48 loci controlling morphological characters and allozymes (Weeden and Wolko 2001). We conclude that P. abyssinicum is distinct from all other lineages, including P. sativum. The extreme homogeneity of P. abyssinicum can be explained by the possibility that this taxon has gone through a bottleneck, and that this passage may have been associated with a hybridization event. One of the consequences of a bottleneck for population is a low percentage of polymorphic loci (Ledig et al. 1999). Although P. abyssinicum showed a very low percentage of polymorphic markers compared to other pea species (table 3), their frequency distribution also has the U-shape typical of almost all natural populations. The bottleneck may be associated with the cultivation of this taxon, which is endemic to the extremely hot parts of Ethiopia (Lamprecht 1974).
Pisum abyssinicum has a low number of polymorphic, species-specific, and unique markers, as well as a low proportion of total markers from the Pisum gene pool (table 3, fig. 6A), suggesting that few progenitors gave rise to this lineage and that P. fulvum was a major contributor (fig. 6). In contrast, in P. sativum the low proportion of unique and species-specific markers is combined with a high level of total and polymorphic markers, similar to those in P. fulvum and P. elatius (table 3). This finding is consistent with the origin of P. sativum from the hybridization of significantly more progenitors, with the major contribution from P. elatius (fig. 6A). The absence of common markers shared exclusively by P. abyssinicum and P. sativum strongly supports the idea that both species were brought into cultivation independently, and it is not consistent with the widely accepted view of P. abyssinicum as an ecotype or subspecies of P. sativum (Makasheva 1984, p.40).
Although Pisum is well known as an inbreeder, a significant level of heterogeneity is maintained within pea species (table 2). Our data highlight extensive introgression and intermixing among all lineages. Even the highly homogeneous P. abyssinicum appears to have a hybrid origin. We estimate 14.3% of markers as common among lineages (fig. 6B); however, even this fraction of insertion sites is not fixed, but its polymorphism is shared between lineages. Most of the genome exists in a state of presenceabsence polymorphism. Despite the many observed differences between the main lineages, shared polymorphism is common, so the term species complex can be applied to Pisum. We conclude that, as expected for a species complex, recombination, introgression, and segregation between pea inbred lineages is common, although this may be rare per plant generation.
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Acknowledgements |
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Footnotes |
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