Genetic diversity in European Pisum germplasm collections.

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.

Analysis of plant diversity with retrotransposon-based molecular markers.

Retrotransposons are both major generators of genetic diversity and tools for detecting the genomic changes associated with their activity because they create large and stable insertions in the genome. After the demonstration that retrotransposons are ubiquitous, active and abundant in plant genomes, various marker systems were developed to exploit polymorphisms in retrotransposon insertion patterns. These have found applications ranging from the mapping of genes responsible for particular traits and the management of backcrossing programs to analysis of population structure and diversity of wild species. This review provides an insight into the spectrum of retrotransposon-based marker systems developed for plant species and evaluates the contributions of retrotransposon markers to the analysis of population diversity in plants.

Effects of ascertainment bias and marker number on estimations of barley diversity from high-throughput SNP genotype data.

The capability of molecular markers to provide information of genetic structure is influenced by their number and the way they are chosen. This study evaluates the effects of single nucleotide polymorphism (SNP) number and selection strategy on estimates of germplasm diversity and population structure for different types of barley germplasm, namely cultivar and landrace. One hundred and sixty-nine barley landraces from Syria and Jordan and 171 European barley cultivars were genotyped with 1536 SNPs. Different subsets of 384 and 96 SNPs were selected from the 1536 set, based on their ability to detect diversity in landraces or cultivated barley in addition to corresponding randomly chosen subsets. All SNP sets except the landrace-optimised subsets underestimated the diversity present in the landrace germplasm, and all subsets of SNP gave similar estimates for cultivar germplasm. All marker subsets gave qualitatively similar estimates of the population structure in both germplasm sets, but the 96 SNP sets showed much lower data resolution values than the larger SNP sets. From these data we deduce that pre-selecting markers for their diversity in a germplasm set is very worthwhile in terms of the quality of data obtained. Second, we suggest that a properly chosen 384 SNP subset gives a good combination of power and economy for germplasm characterization, whereas the rather modest gain from using 1536 SNPs does not justify the increased cost and 96 markers give unacceptably low performance. Lastly, we propose a specific 384 SNP subset as a standard genotyping tool for middle-eastern landrace barley.

Ty1-copia retrotransposon-based SSAP marker development in cashew (Anacardium occidentale L.).

The most popular retrotransposon-based molecular marker system in use at the present time is the sequence-specific amplification polymorphism (SSAP) system . This system exploits the insertional polymorphism of long terminal repeat (LTR) retrotransposons around the genome. Because the LTR sequence is used to design primers for this method, its successful application requires sequence information from the terminal region of the mobile elements . In this study, two LTR sequences were isolated from the cashew genome and used successfully to develop SSAP marker systems. These were shown to have higher levels of polymorphism than amplified fragment length polymorphic markers for this species.

Retrotransposon-based molecular markers for linkage and genetic diversity analysis in wheat.

Retrotransposon-based molecular markers have been developed to study bread wheat ( Triticum aestivum) and its wild relatives. SSAP (Sequence-Specific Amplification Polymorphism) markers based on the BARE-1/ Wis-2-1A retrotransposons were assigned to T. aestivum chromosomes by scoring nullisomic-tetrasomic chromosome substitution lines. The markers are distributed among all wheat chromosomes, with the lowest proportion being assigned the D wheat genome. SSAP markers for BARE-1/ Wis-2-1A and three other wheat retrotransposons, Thv19, Tagermina and Tar1, are broadly distributed on a wheat linkage map. Polymorphism levels associated with these four retrotransposons vary, with BARE-1/ Wis-2-1A and Thv19 both showing approximately 13% of bands polymorphic in a mapping population, Tagermina showing approximately 17% SSAP band polymorphism and Tar1 roughly 18%. This suggests that Tagermina and Tar1 have been more transpositionally active in the recent evolutionary past, and are potentially the more useful source of molecular markers in wheat. Lastly, BARE-1 / Wis-2-1A markers have also been used to characterise the genetic diversity among a set of 35 diploid and tetraploid wheat species including 26 Aegilops and 9 Triticum accessions. The SSAP-based diversity tree for Aegilops species agrees well with current classifications, though the Triticum tree shows several significant differences, which may be associated with polyploidy in this genus.

The evolution of Ty1-copia group retrotransposons in gymnosperms.

A diverse collection of Ty1-copia group retrotransposons has been characterized from the genome of Picea abies (Norway spruce) by degenerate PCR amplification of a region of the reverse transcriptase gene. The occurrence of these retrotransposable elements in the gymnosperms was investigated by Southern blot hybridization analysis. The distribution of the different retrotransposons across the gymnosperms varies greatly. All of the retrotransposon clones isolated are highly conserved within the Picea (spruce) genus, many are also present in Pinus (pine) and/or Abies (fir) genera, and some share strongly homologous sequences with one or more of cedar, larch, Sequoia, cypress, and Ginkgo. Further subclones of one of the most strongly conserved retrotransposon sequences, Tpa28, were obtained from Ginkgo and P. abies. Comparisons of individual sequence pairs between the two species show nucleotide cross-homologies of around 80%-85%, corresponding to nucleotide substitution rates similar to those of nuclear protein-coding genes. Analysis of Tpa28 consensus sequences reveals that strong purifying selection has acted on this retrotransposon in the lineages connecting Ginkgo and Picea. Collectively, these data suggest, first, that the evolution of the Ty1-copia retrotransposon group in the gymnosperms is dominated by germ line vertical transmission, with strong selection for reverse transcriptase sequence, and, second, that extinction of individual retrotransposon types has been comparatively rare in gymnosperm species lineages compared with angiosperms. If this very high level of sequence conservation is a general property of the retrotransposons, then their extreme sequence diversity implies that they are extremely ancient, and the major element lineages seen today may have arisen early in eukaryote evolution. The data are also consistent with horizontal transmission of particular retrotransposons between species, but such a mechanism is unnecessary to explain the results.

Pea Ty1-copia group retrotransposons: transpositional activity and use as markers to study genetic diversity in Pisum.

The variation in transposition history of different Ty1-copia group LTR retrotransposons in the species lineages of the Pisum genus has been investigated. A heterogeneous population of Ty1-copia elements was isolated by degenerate PCR and two of these (Tps12 and Tps19) were selected on the basis of their copy number and sequence conservation between closely related species for further in-depth study of their transpositional history in Pisum species. The insertional polymorphism of these elements and the previously characterised PDR1 element was studied by sequence-specific amplification polymorphism (SSAP). Each of these elements reveals a unique transpositional history within 55 diverse Pisum accessions. Phylogenetic trees based on the SSAP data show that SSAP markers for individual elements are able to resolve different species lineages within the Pisum genus. Finally, the SSAP data from all of these retrotransposon markers were combined to reveal a detailed picture of the intra and interspecies relationships within Pisum.

Rapid isolation of plant Ty1-copia group retrotransposon LTR sequences for molecular marker studies.

The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identified a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers specific for three pea LTRs have been used to reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.

Phylogeny and transpositional activity of Ty1-copia group retrotransposons in cereal genomes.

The Ty1-copia group retrotransposon populations of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) have been characterised by degenerate PCR and sequence analysis of fragments of the reverse transcriptase genes. The barley population is comprised of a highly heterogeneous set of retrotransposons, together with a collection of sequences that are closely related to the BARE-1 element. Wheat also contains a highly diverse Ty1-copia retrotransposon population, together with a less prominent BARE-1 subgroup. These data have been combined with previously published Gramineae sequences to construct a composite phylogenetic tree for this class of retrotransposons in cereal grasses. The analysis indicates that the ancestral Gramineae genome contained a heterogeneous population of Ty1-copia group retrotransposons, the descendants of which have proliferated to differing degrees in present-day species. Lastly, the level of recent transpositional activity of two Ty1-copia elements has been estimated by measuring their insertional polymorphism within species. Both transposons are highly polymorphic within all species tested. These data suggest that transposition proficiency may be a common and evolutionarily stable feature of the Ty1-copia group retrotransposons of cereal grasses.

Quantitative genetic analysis of copia retrotransposon activity in inbred Drosophila melanogaster lines.

The rates of transcription and transposition of retrotransposons vary between lines of Drosophila melanogaster. We have studied the genetics of differences in copia retrotransposon activity by quantitative trait loci (QTL) mapping. Ninety-eight recombinant inbred lines were constructed from two parental lines exhibiting a 10-fold difference in copia transcript level and a 100-fold difference in transposition rate. The lines were scored for 126 molecular markers, copia transcript level, and rate of copia transposition. Transcript level correlated with copia copy number, and the difference in copia copy number between parental lines accounted for 45.1% of copia transcript-level difference. Most of the remaining difference was accounted for by two transcript-level QTL mapping to cytological positions 27B-30D and 50F-57C on the second chromosome, which accounted for 11.5 and 30.4%, respectively. copia transposition rate was controlled by interacting QTL mapping to the region 27B-48D on the second and 61A-65A and 97D-100A on the third chromosome. The genes controlling copia transcript level are thus not necessarily those involved in controlling copia transposition rate. Segregation of modifying genes, rather than mutations, might explain the variability in copia retrotransposon activity between lines.

Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis.

Two assays based upon PCR detection of a polymorphic PDR1 retrotransposon insertion in Pisum sativum have been developed. Both methods involve PCR with primers derived from the transposon and flanking DNA. The first method uses a dot assay for PCR product detection which could be fully automated for handling thousands of samples. The second method, which is designed to handle lower numbers, requires a single PCR and gel lane per sample. Both methods yield co-dominant markers, with presence and absence of the transposon insertion independently scorable, and both could in principle be applied to any transposable element in any plant species.

Characterization and genomic organization of Ty1-copia group retrotransposons in rye (Secale cereale).

The genomic organisation of the Ty1-copia retrotransposons in rye (Secale cereale) has been studied. We have used the polymerase chain reaction (PCR) to amplify sequences from a conserved domain of the reverse transcriptase gene of the Ty1-copia retrotransposons in this species. Sequence analysis of 26 of these PCR products shows them to be a highly heterogeneous population, a feature that is common in plants. Slot blot analysis shows that there are about 100,000 individual Ty1-copia retrotransposons in rye. In situ hybridization of a heterogeneous probe, representing the whole population of rye Ty1-copia retrotransposon sequences, to chromosome spreads of triticale (xTriticosecale), a rye-wheat hybrid, shows that these sequences are present throughout all the rye chromosomes but absent from the centromeric regions and, in particular, from the terminal heterochromatin. Southern analysis of oat, barley, wheat, and rye, using as a probe R9, one of the rye sequences that is closely similar to the BARE-1 element of barley, shows that close relatives of this retrotransposon subgroup are present in all these species in high copy number. Northern analysis on RNAs from seedlings shows that the BARE-1 subgroup is transcribed in all these cereal plants but in variable amounts: high in barley, moderate in wheat and rye, and extremely low in oat.

Genetic and molecular analysis of smooth, a quantitative trait locus affecting bristle number in Drosophila melanogaster.

A semi-lethal, sterile allele of the smooth locus (2-91.5), sm3, was discovered in an artificial selection line for low abdominal bristle number that had been started from a P-M dysgenic cross. The fitness effects and extremely low bristle number phenotype of the allele could not be separated by recombination from a P-element insertion at cytological location 56E, and precise excision of the P-element at this site was associated with reversion to wild type. The smooth gene was cloned using the P-element insertion as a tag. The gene encodes a 2.6-kb transcript derived from 10 exons and covers a genomic region of at least 80 kb. The Drosophila smooth gene shares substantial sequence identity with a group of RNA binding proteins, with the closest relationship being to the human heterogeneous nuclear ribonucleoprotein L gene. The smooth gene is by definition an abdominal bristle number quantitative trait locus, but further work is required to discern whether naturally occurring allelic variation at this locus is a source of genetic variation for abdominal bristle number in natural populations.

Germ line transposition of the copia retrotransposon in Drosophila melanogaster is restricted to males by tissue-specific control of copia RNA levels.

Germ line transposition rates of the retrotransposon copia were directly measured in males and females of an inbred Drosophila melanogaster line, 2b3, which is highly polymorphic for copia insertion sites. The elevated germ line transposition rate of copia in this line (3-8 x 10(-3) per generation per element) is confined to males, with transposition in females being undetectable under the conditions of the experiment but at most 50-fold lower than the rate for males. To determine the molecular basis of this effect, copia RNA levels were measured in whole bodies and germ lines of male and female flies of both the unstable 2b3 line and a stable line, Oregon RC-iso, which shows normal rates of copia transposition. Both male and female 2b3 flies contain much more copia RNA than flies of the stable line. However, 2b3 male germinal tissues contain much higher levels of copia RNA than the equivalent female tissues. The highest copia expression is detected in maturing primary spermatocytes. Our data show that high rates of germ line copia transposition are restricted to males by tissue-specific control of RNA levels and suggest that transposition of copia only occurs in fly tissues containing more than a relatively high threshold level of copia RNA.

Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP).

Retrotransposons are present in high copy number in many plant genomes. They show a considerable degree of sequence heterogeneity and insertional polymorphism, both within and between species. We describe here a polymerase chain reaction (PCR)-based method which exploits this polymorphism for the generation of molecular markers in barley. The method produces amplified fragments containing a Bare-1-like retrotransposon long terminal repeat (LTR) sequence at one end and a flanking host restriction site at the other. The level of polymorphism is higher than that revealed by amplified fragment length polymorphism (AFLP) in barley. Segregation data for 55 fragments, which were polymorphic in a doubled haploid barley population, were analysed alongside an existing framework of some 400 other markers. The markers showed a widespread distribution over the seven linkage groups, which is consistent with the distribution of the Bare-1 class of retrotransposons in the barley genome based on in situ hybridisation data. The potential applicability of this method to the mapping of other multicopy sequences in plants is discussed.

The evolution of Ty1-copia group retrotransposons in eukaryote genomes.

The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila, the organisms in which they were first discovered, and more recently in higher plant and vertebrate species. Their properties, such as copy number, sequence homogeneity, transcriptional and transpositional activity vary greatly between these different hosts. We will try to resolve these apparent discrepancies between these properties, explain any fundamental differences in the biology of the Ty1-copia group between hosts, and propose a general model for LTR retrotransposon evolution.

The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implications for genome evolution.

Retrotransposons make up a major fraction--sometimes more than 40%--of all plant genomes investigated so far. We have isolated the reverse transcriptase domains of the Ty1-copia group elements from several species, ranging in genome size from some 100 Mbp to 23,000 Mbp, and determined the distribution patterns of these retrotransposons on metaphase chromosomes and within interphase nuclei by DNA:DNA in situ hybridization. With some exceptions, the reverse transcriptase domains were distributed over the length of the chromosomes. Exclusion from rDNA sites and some centromeres (e.g., slash pine, 23,000 Mbp, or barley, 5500 Mbp) is frequent, whereas many species exclude retrotransposons from other sites of heterochromatin (e.g., intercalary and centromeric sites in broad bean). In contrast, in the plant Arabidopsis thaliana, widely used for plant molecular genetic studies because of its small genome (c. 100 Mbp), the Ty1-copia group reverse transcriptase gene domains are concentrated in the centromeric regions, colocalizing with the 180 bp satellite sequence pAL1. Unlike the pAL1 sequence, however, the Ty1-copia signal is also detectable as weaker, diffuse hybridization along the lengths of the chromosomes. Possible mechanisms for evolution of the contrasting distributions are discussed. Understanding the physical distribution of retrotransposons and comparisons of the distribution between species is critical to understanding their evolution and the significance for generation of the new patterns of variability and in speciation.

The Ty1-copia group of retrotransposons in plants: genomic organisation, evolution, and use as molecular markers.

The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato (Solanum tuberosum), faba beans (Vicia faba), Vicia melanops, Vicia sativa, barley (Hordeum vulgare), rye (Secale cereale), and onion (Allium cepa). Extreme heterogeneity in the sequence of the Ty1-copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty1-copia group retrotransposons for the genomes of S. tuberosum, L. esculentum, A. cepa, S. cereale, and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba, S. cereale, and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1-copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants is presented.