Genetic diversity of cultivated flax (Linum usitatissimum L.) germplasm assessed by retrotransposon-based markers.

Retrotransposon segments were characterized and inter-retrotransposon amplified polymorphism (IRAP) markers developed for cultivated flax (Linum usitatissimum L.) and the Linum genus. Over 75 distinct long terminal repeat retrotransposon segments were cloned, the first set for Linum, and specific primers designed for them. IRAP was then used to evaluate genetic diversity among 708 accessions of cultivated flax comprising 143 landraces, 387 varieties, and 178 breeding lines. These included both traditional and modern, oil (86), fiber (351), and combined-use (271) accessions, originating from 36 countries, and 10 wild Linum species. The set of 10 most polymorphic primers yielded 141 reproducible informative data points per accession, with 52% polymorphism and a 0.34 Shannon diversity index. The maximal genetic diversity was detected among wild Linum species (100% IRAP polymorphism and 0.57 Jaccard similarity), while diversity within cultivated germplasm decreased from landraces (58%, 0.63) to breeding lines (48%, 0.85) and cultivars (50%, 0.81). Application of Bayesian methods for clustering resulted in the robust identification of 20 clusters of accessions, which were unstratified according to origin or user type. This indicates an overlap in genetic diversity despite disruptive selection for fiber versus oil types. Nevertheless, eight clusters contained high proportions (70-100%) of commercial cultivars, whereas two clusters were rich (60%) in landraces. These findings provide a basis for better flax germplasm management, core collection establishment, and exploration of diversity in breeding, as well as for exploration of the role of retrotransposons in flax genome dynamics.

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.

Genetic variability in sunflower (Helianthus annuus L.) and in the Helianthus genus as assessed by retrotransposon-based molecular markers.

The inter-retrotransposon amplified polymorphism (IRAP) protocol was applied for the first time within the genus Helianthus to assess intraspecific variability based on retrotransposon sequences among 36 wild accessions and 26 cultivars of Helianthus annuus L., and interspecific variability among 39 species of Helianthus. Two groups of LTRs, one belonging to a Copia-like retroelement and the other to a putative retrotransposon of unknown nature (SURE) have been isolated, sequenced and primers were designed to obtain IRAP fingerprints. The number of polymorphic bands in H. annuus wild accessions is as high as in Helianthus species. If we assume that a polymorphic band can be related to a retrotransposon insertion, this result suggests that retrotransposon activity continued after Helianthus speciation. Calculation of similarity indices from binary matrices (Shannon's and Jaccard's indices) show that variability is reduced among domesticated H. annuus. On the contrary, similarity indices among Helianthus species were as large as those observed among wild H. annuus accessions, probably related to their scattered geographic distribution. Principal component analysis of IRAP fingerprints allows the distinction between perennial and annual Helianthus species especially when the SURE element is concerned.

Parasitism and the retrotransposon life cycle in plants: a hitchhiker's guide to the genome.

LTR (long terminal repeat) retrotransposons are the main components of higher plant genomic DNA. They have shaped their host genomes through insertional mutagenesis and by effects on genome size, gene expression and recombination. These Class I transposable elements are closely related to retroviruses such as the HIV by their structure and presumptive life cycle. However, the retrotransposon life cycle has been closely investigated in few systems. For retroviruses and retrotransposons, individual defective copies can parasitize the activity of functional ones. However, some LTR retrotransposon groups as a whole, such as large retrotransposon derivatives and terminal repeats in miniature, are non-autonomous even though their genomic insertion patterns remain polymorphic between organismal accessions. Here, we examine what is known of the retrotransposon life cycle in plants, and in that context discuss the role of parasitism and complementation between and within retrotransposon groups.

A movable feast: diverse retrotransposons and their contribution to barley genome dynamics.

Cellular genes comprise at most 5% of the barley genome; the rest is occupied primarily by retrotransposons. Retrotransposons move intracellularly by a replicative mechanism similar to that of retroviruses. We describe the major classes of retrotransposons in barley, including the two nonautonomous groups that were recently identified, and detail the evidence supporting our current understanding of their life cycle. Data from analyses of long contiguous segments of the barley genome, as well as surveys of the prevalence of full-length retrotransposons and their solo LTR derivatives in the genus Hordeum, indicate that integration and recombinational loss of retrotransposons are major factors shaping the genome. The sequence conservation and integrative capacity of barley retrotransposons have made them excellent sources for development of molecular marker systems.

Comparison of the utility of barley retrotransposon families for genetic analysis by molecular marker techniques.

The Sequence-Specific Amplification Polymorphism (S-SAP) method, and the related molecular marker techniques IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism), are based on retrotransposon activity, and are increasingly widely used. However, there have been no systematic analyses of the parameters of these methods or of the utility of different retrotransposon families in producing polymorphic, scorable fingerprints. We have generated S-SAP, IRAP, and REMAP data for three barley (Hordeum vulgare L.) varieties using primers based on sequences from six retrotransposon families (BARE-1, BAGY-1, BAGY-2, Sabrina, Nikita and Sukkula). The effect of the number of selective bases on the S-SAP profiles has been examined and the profiles obtained with eight MseI+3 selective primers compared for all the elements. Polymorphisms detected in the insertion pattern of all the families show that each can be used for S-SAP. The uniqueness of each transposition event and differences in the historic activity of each family suggest that the use of multiple retrotransposon families for genetic analysis will find applications in mapping, fingerprinting, and marker-assisted selection and evolutionary studies, not only in barley and other Hordeum species and related taxa, but also more generally.

Envelope-class retrovirus-like elements are widespread, transcribed and spliced, and insertionally polymorphic in plants.

Retrotransposons and retroviruses share similar intracellular life cycles and major encoded proteins, but retrotransposons lack the envelope (env) critical for infectivity. Retrotransposons are ubiquitous and abundant in plants and active retroviruses are known in animals. Although a few env-containing retroelements, gypsy-like Athila, Cyclops, and Calypso and copia-like SIRE-1, have been identified in plants, the general presence and functionality of the domain remains unclear. We show here that env-class elements are present throughout the flowering plants and are widely transcribed. Within the grasses, we show the transcription of the env domain itself for Bagy-2 and related retrotransposons, all members of the Athila group. Furthermore, Bagy-2 transcripts undergo splicing to generate a subgenomic env product as do those of retroviruses. Transcription and the polymorphism of their insertion sites in closely related barley cultivars suggests that at least some are propagationally active. The putative ENV polypeptides of Bagy-2 and rice Rigy-2 contain predicted leucine zipper and transmembrane domains typical of retroviral ENVs. These findings raise the prospect of active retroviral agents among the plants.

Active retrotransposons are a common feature of grass genomes.

A large fraction of the genomes of grasses, members of the family Graminae, is composed of retrotransposons. These elements resemble animal retroviruses in their structure and possess a life cycle similar to theirs that includes transcription, translation, and integration of daughter copies. We have investigated if retrotransposons are generally transcribed in the grasses and other plants, and whether the various families of elements are translationally and integrationally active in multiple grass species. A systematic search of 7.8 x 10(5) publicly available expressed sequence tags from plants revealed widespread retrotransposon transcripts at a frequency of one in 1,000. Monocot retrotransposons found relatively more expressed sequence tags from non-source species than did those of dicots. Antibodies were raised to the capsid protein, GAG, of BARE-1, a transcribed and translated copia-like retrotransposon of barley (Hordeum vulgare). These detected immunoreactive proteins of sizes identical to those of the BARE-1 GAG and polyprotein, respectively, in other species of the tribe Triticeae as well as in oats (Avena sativa) and rice (Oryza sativa). Retrotransposon-based markers showed integrational polymorphisms for BARE-1 in different subfamilies of the Graminae. The results suggest that grasses share families of transcriptionally, translationally, and integrationally active retrotransposons, enabling a comparative and integrative approach to understanding the life cycle of retrotransposons and their impact on the genome.

Application of BARE-1 retrotransposon markers to the mapping of a major resistance gene for net blotch in barley.

Net blotch, which is caused by the fungus Pyrenophoral teres Drechs. f. teres Smedeg., presents a serious problem for barley production worldwide, and the identification and deployment of sources of resistance to it are key objectives for many breeders. Here, we report the identification of a major resistance gene, accounting for 65% of the response variation, in a cross between the resistant line C19819 and the susceptible cv. Rolfi. The resistance gene was mapped to chromosome 6H with the aid of two recently developed systems of retrotransposon-based molecular markers, REMAP and IRAP. A total of 239 BARE-1 and Sukkula retrotransposon markers were mapped in the cross, and the 30-cM segment containing the locus with significant resistance effect contained 26 of the markers. The type and local density of the markers should facilitate future map-based cloning of the resistance gene as well as manipulation of the resistance through backcross breeding.

A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion.

Organisms with large genomes contain vast amounts of repetitive DNA sequences, much of which is composed of retrotransposons. Amplification of retrotransposons has been postulated to be a major mechanism increasing genome size and leading to "genomic obesity." To gain insights into the relation between retrotransposons and genome expansion in a large genome, we have studied a 66-kb contiguous sequence at the Rar1 locus of barley in detail. Three genes were identified in the 66-kb contig, clustered within an interval of 18 kb. Inspection of sequences flanking the gene space unveiled four novel retroelements, designated Nikita, Sukkula, Sabrina, and BAGY-2 and several units of the known BARE-1 element. The retroelements identified are responsible for at least 15 integration events, predominantly arranged as multiple nested insertions. Strikingly, most of the retroelements exist as solo LTRs (Long Terminal Repeats), indicating that unequal crossing over and/or intrachromosomal recombination between LTRs is a common feature in barley. Our data suggest that intraelement recombination events deleted most of the original retrotransposon sequences, thereby providing a possible mechanism to counteract retroelement-driven genome expansion.

Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence.

The replicative spread of retrotransposons in the genome creates new insertional polymorphisms, increasing retrotransposon numbers and potentially both their share of the genome and genome size. The BARE-1 retrotransposon constitutes a major, dispersed, active component of Hordeum genomes, and BARE-1 number is positively correlated with genome size. We have examined genome size and BARE-1 insertion patterns and number in wild barley, Hordeum spontaneum, in Evolution Canyon, Lower Nahal Oren, Mount Carmel, Israel, along a transect presenting sharply differing microclimates. BARE-1 has been sufficiently active for its insertional pattern to resolve individuals in a way consonant with their ecogeographical distribution in the canyon and to distinguish them from provenances outside the canyon. On both slopes, but especially on the drier south-facing slope, a simultaneous increase in the BARE-1 copy number and a decrease in the relative number lost through recombination, as measured by the abundance of solo long terminal repeats, appear to have driven the BARE-1 share of the genome upward with the height and dryness of the slope. The lower recombinational loss would favor maintenance of more full-length copies, enhancing the ability of the BARE-1 family to contribute to genome size growth. These local data are consistent with regional trends for BARE-1 in H. spontaneum across Israel and therefore may reflect adaptive selection for increasing genome size through retrotransposon activity.

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.

Structure, functionality, and evolution of the BARE-1 retrotransposon of barley.

The BARE-1 retrotransposon is a major, active component of the genome of barley (Hordeum vulgare L.) and other Hordeum species. Copia-like in its organization, it consists of 1.8-kb long terminal repeats bounding an internal domain of 5275 bp which encodes a predicted polyprotein of 1301 residues. The polyprotein contains the key residues, structural motifs, and conserved regions associated with retroviral and retrotransposon GAG, aspartic proteinase, integrase, reverse transcriptase, and RNaseH polypeptides. BARE-1 is actively transcribed and translated. As part of our effort to understand the evolution and function of BARE-1, we have examined its copy number and localization. Full-length members of the BARE-1 family constitute 2.8% of the barley genome. Globally, they are dispersed throughout the genome, excepting the centromeric, telomeric, and NOR regions. Locally, BARE-1 occurs more commonly in repetitive DNA than in coding regions, forming clusters of nested insertions. Both barley and other Hordeum genomes contain a high proportion of BARE-1 solo LTRs. New techniques have been developed which exploit the insertion site polymorphism generated by BARE-1 integration to produce molecular markers for breeding, biodiversity, and mapping applications.

The core domain of retrotransposon integrase in Hordeum: predicted structure and evolution.

Propagation of long terminal repeat (LTR)-bearing retrotransposons and retroviruses requires integrase (IN, EC 2.7.7.-), encoded by the retroelements themselves, which mediates the insertion of cDNA copies back into the genome. An active retrotransposon family, BARE-1, comprises approximately 7% of the barley (Hordeum vulgare subsp. vulgare) genome. We have generated models for the secondary and tertiary structure of BARE-1 IN and demonstrate their similarity to structures for human immunodeficiency virus 1 and avian sarcoma virus INs. The IN core domains were compared for 80 clones from 28 Hordeum accessions representative of the diversity of the genus. Based on the structural model, variations in the predicted, aligned translations from these clones would have minimal structural and functional effects on the encoded enzymes. This indicates that Hordeum retrotransposon IN has been under purifying selection to maintain a structure typical of retroviral INs. These represent the first such analyses for plant INs.

Gypsy-like retrotransposons are widespread in the plant kingdom.

Retrotransposons propagate via an RNA intermediate which is then reverse-transcribed and packaged into virus-like particles. They are either copia- or gypsy-like in coding domain order and sequence similarity, the gypsy-like elements sharing their organization with the retroviruses but lacking retroviral envelope domains. Copia-like retrotransposons, or at least their reverse transcriptase domains, appear broadly distributed in higher plants, but gypsy-like elements have been reported only for scattered species. The authors have exploited the difference in domain order between these groups to amplify and clone segments bridging the reverse transcriptase-integrase region of specifically gypsy-like retrotransposons. Species representative of the diversity of higher plants yielded products whose sequences establish that gypsy-like transposons are dispersed throughout the plant genomes. This class of plant elements has been named romani retrotransposons. The presence of both types ubiquitously in the fungi, plants and animals support their existence as ancient distinct lineages and subsequent, vertical radiation.

BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNA processing sites.

The BARE-1 copia-like retrotransposon constitutes nearly 7% of the barley (Hordeum vulgare L.) genome as a family of more than 2 x 10(4) mostly full-length copies dispersed on all chromosomes. BARE-1 elements are transcribed in barley tissues from promoters within the LTR (long terminal repeat). The predicted, translated polyprotein contains conserved domains for GAG, aspartic proteinase, integrase, reverse-transcriptase, and RNase H. Here, we have used inverse PCR with LTR-based primers to establish the consensus sequences for the terminal region of the LTR, the external dinucleotides of the cDNA integration intermediate, and the minus- and plus-strand priming sites. These key functional entities are well-conserved in the BARE-1 family, including wheat Wis2, but differ from those of other plant retrotransposons. The target site duplication was established as 5 bp. Of the 13 integration sites identified here, 8 were other BARE-1 elements and 1 another retrotransposon; 59% of the total 17 identified BARE-1 insertion sites are retrotransposons. This nested insertion pattern may represent a basic feature of plant retrotransposons.

The genome sizes of Hordeum species show considerable variation.

Hordeum, distributed worldwide in temperate zones, is the second largest genus in the tribe Triticeae and includes diploid, tetraploid, and hexaploid species. We determined, by DAPI staining and flow cytometry, the nuclear DNA content for 35 accessions of the genus Hordeum, from a total of 19 species, including specimens of 2 cultivars and 2 landraces of Hordeum vulgare ssp. vulgare as well as samples of 12 Hordeum vulgare ssp. spontaneum populations. Genome sizes ranged from 5.69 to 9.41 pg for the G1 nuclei of the diploids, and from 13.13 to 18.36 pg for those of the tetraploids. This constitutes a 1.7-fold variation for the diploids, contrasting with a 4% variation previously reported. For H. vulgare ssp. vulgare (barley), the accessions examined differed by 18%. These variations in genome size cannot be correlated with meiotic pairing groups (I, H, X, Y) or with proposed phylogenetic relationships within the genus. Genome size variation between barley accessions cannot be related to status as cultivated or wild, or to climatic or geological gradients. We suggest these data may indicate rapid but sporadic changes in genome size within the genus. Key words : barley, Hordeum, Triticeae, genome size, flow cytometry.

The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays.

The BARE-1 retrotransposon occurs in more than 10(4) copies in the barley genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing motifs typical of retrotransposon promoters. These, the presence of predicted priming sites for reverse transcription, and the high conservation for all key functional domains of the coding region suggest that copies within the genome could be active retrotransposons. In view of this, we looked for transcription of BARE-1 within barley tissues and examined the promoter function of the BARE-1 LTR. We demonstrate here that BARE-1-like elements are transcribed in barley tissues, and that the transcripts begin within the BARE-1 LTR downstream of TATA boxes. The LTR can drive expression of reporter genes in transiently transformed barley protoplasts. This is dependent on the presence of a TATA box functional in planta as well. Furthermore, we identify regions within the LTR responsible for expression within protoplasts by deletion analyses of LTR-luc constructs. Similarities between promoter regulatory motifs and regions of the LTR were identified by comparisons to sequence libraries. The activity of the LTR as a promoter, combined with the abundance of BARE-1 in the genome, suggests that BARE-1 may retain the potential for propagation in the barley genome.

Retrotransposon BARE-1 is a major, dispersed component of the barley (Hordeum vulgare L.) genome.

The barley BARE-1 is a transcribed, copia-like retroelement with well-conserved functional domains, an active promoter, and a copy number of at least 3 x 10(4). We examined its chromosomal localization by in situ hybridization. The long terminal repeat (LTR) probe displayed a uniform hybridization pattern over the whole of all chromosomes, excepting paracentromeric regions, telomeres, and nucleolar organizer (NOR) regions. The integrase probe showed a similar pattern. The 5'-untranslated leader (UTL) probe, expected to be the most rapidly evolving component, labeled chromosomes in a dispersed and non-uniform manner, concentrated in the distal regions, possibly indicating a targe site preference.

BARE-1, a copia-like retroelement in barley (Hordeum vulgare L.).

Retroviruses and retrotransposons make up the broad class of retroelements replicating and transposing via reverse transcriptase. Retroelements have recently been found to be ubiquitous in the plants. We report here the isolation, sequence and analysis of a retroelement from barley (Hordeum vulgare L.) with all the features of a copia-like retrotransposon. This is named BARE-1 (for BArley RetroElement 1), the first such element described for barley. BARE-1 is 12,088 bp, with long terminal repeats (LTRs) of 1829 bp containing perfect 6 bp inverted repeats at their ends and flanked by 4 bp direct repeats in the host DNA. Between the long terminal repeats is an internal domain with a derived amino acid sequence of 1285 residues, bearing homology to the gag, pro, int and rt domains of retroviruses and both plant and non-plant copia-like retrotransposons. Cultivated barley contains about 5000 elements in the genome similar to the BARE-1 putative gag domain, but ten-fold more hybridizing to rt or LTR probes. The particular BARE-1 element reported here appears to be inactive, as the putative protein-coding domain is interrupted by four stop codons and a frameshift. In addition, the 3' LTR is 4% divergent from the 5' LTR and contains a 3135 bp insertion. Nevertheless, we have recently detected transcripts hybridizing to BARE-1 on northern blots, presumably from active copies. Analysis of BARE-1 expression and function in barley is currently underway.