Alexander V. Vershinin

Polymorphism of insertion sites of Ty1-copia class retrotransposons and its use for linkage and diversity analysis in pea.

Abstract A sample of 15 cultivars and 56 Pisum accessions from the JIC germplasm core collection has been studied using a modification of the SSAP (sequence-specific amplification polymorphisms) technique; the specific primer was designed to correspond to the polypurine tract (PPT) of PDR1, a Ty1-copia group retrotransposon of pea. Most of these SSAP products were shown to be PDR1 derived. The PDR1 SSAP markers are more informative than previously studied AFLP or RFLP markers and are distributed throughout the genome. Their pattern of variation makes them ideal for integrating genetic maps derived from related crosses. Data sets obtained with AFLP and PDR1 SSAP markers were used to construct neighbour-joining trees and for principal component analysis. These data sets give greater resolution than hitherto available for the characterisation of variation within Pisum, showing that the genus has three main groups: P. fulvum, P. abyssinicum and all other Pisum spp. P. abyssinicum is not a subgroup of cultivated P. sativum, as was previously thought, but has probably been domesticated independently. Modern cultivars are shown to form a single group within Pisum as a whole.

The genetic diversity and evolution of field pea (Pisum) studied by high throughput retrotransposon based insertion polymorphism (RBIP) marker analysis

BackgroundThe genetic diversity of crop species is the result of natural selection on the wild progenitor and human intervention by ancient and modern farmers and breeders. The genomes of modern cultivars, old cultivated landraces, ecotypes and wild relatives reflect the effects of these forces and provide insights into germplasm structural diversity, the geographical dimension to species diversity and the process of domestication of wild organisms. This issue is also of great practical importance for crop improvement because wild germplasm represents a rich potential source of useful under-exploited alleles or allele combinations. The aim of the present study was to analyse a major Pisum germplasm collection to gain a broad understanding of the diversity and evolution of Pisum and provide a new rational framework for designing germplasm core collections of the genus.Results3020 Pisum germplasm samples from the John Innes Pisum germplasm collection were genotyped for 45 retrotransposon based insertion polymorphism (RBIP) markers by the Tagged Array Marker (TAM) method. The data set was stored in a purpose-built Germinate relational database and analysed by both principal coordinate analysis and a nested application of the Structure program which yielded substantially similar but complementary views of the diversity of the genus Pisum. Structure revealed three Groups (1-3) corresponding approximately to landrace, cultivar and wild Pisum respectively, which were resolved by nested Structure analysis into 14 Sub-Groups, many of which correlate with taxonomic sub-divisions of Pisum, domestication related phenotypic traits and/or restricted geographical locations. Genetic distances calculated between these Sub-Groups are broadly supported by principal coordinate analysis and these, together with the trait and geographical data, were used to infer a detailed model for the domestication of Pisum.ConclusionsThese data provide a clear picture of the major distinct gene pools into which the genus Pisum is partitioned and their geographical distribution. The data strongly support the model of independent domestications for P. sativum ssp abyssinicum and P. sativum. The relationships between these two cultivated germplasms and the various sub-divisions of wild Pisum have been clarified and the most likely ancestral wild gene pools for domesticated P. sativum identified. Lastly, this study provides a framework for defining global Pisum germplasm which will be useful for designing core collections.

Retroelements, transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation

We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed, and plants regenerated from tissue culture that show mantling, an abnormality leading to flower abortion. Using PCR assays, reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of En/Spm transposons were cloned. Sequence analysis revealed the presence of a major family of LINEs in oil palm, with other diverged copies. Gypsy-like retrotransposons form a single homologous group, whereas En/Spm transposons are present in several diverged families. Southern analysis revealed their presence in low (LINEs) to medium (gypsy and En/Spm) copy numbers in oil palm, and in situ hybridization showed a limited number of distinct loci for each class of transposable element. No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected, but different levels of sequence methylation were observed. During tissue culture, McrBC digestion revealed the genome-wide reduction in DNA methylation, which was restored to near-normal levels in regenerated trees. HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent. The genomic organization of the transposable DNA elements in different oil palm species, accessions and individual regenerated trees was investigated revealing only minor differences. The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components.

Comparative analysis of the nucleosomal structure of rye, wheat and their relatives

Analysis of the structure of chromatin in cereal species using micrococcal nuclease (MNase) cleavage showed nucleosomal organization and a ladder with typical nucleosomal spacing of 175–185 bp. Probing with a set of DNA probes localized in the authentic telomeres, subtelomeric regions and bulk chromatin revealed that these chromosomal regions have nucleosomal organization but differ in size of nucleosomes and rate of cleavage between both species and regions. Chromatin from Secale and Dasypyrum cleaved more quickly than that from wheat and barley, perhaps because of their higher content of repetitive sequences with hairpin structures accessible to MNase cleavage. In all species, the telomeric chromatin showed more rapid cleavage kinetics and a shorter nucleosome length (160 bp spacing) than bulk chromatin. Rye telomeric repeat arrays were shortest, ranging from 8 kb to 50 kb while those of wheat ranged from 15 kb up to 175 kb. A gradient of sensitivity to MNase was detected along rye chromosomes. The rye-specific subtelomeric sequences pSc200 and pSc250 have nucleosomes of two lengths, those of the telomeric and of bulk nucleosomes, indicating that the telomeric structure may extended into the chromosomes. More proximal sequences common to rye and wheat, the short tandem-repeat pSc119.2 and rDNA sequence pTa71, showed longer nucleosomal sizes characteristic of bulk chromatin in both species. A strictly defined spacing arrangement (phasing) of nucleosomes was demonstrated along arrays of tandem repeats with different monomer lengths (118, 350 and 550 bp) by combining MNase and restriction enzyme digestion.

Molecular diversification of tandemly organized DNA sequences and heterochromatic chromosome regions in some triticeae species

The subtelomeric heterochromatin of rye (Secale cereale) chromosomes makes up 12–18% of the genome and consists largely of a small number of tandemly organized DNA sequence families. The genomic organization, chromosomal locations and the structural organization of monomer units of the major DNA sequences from these regions were investigated and compared in other Triticeae species from the generaSecale, Agropyron, Dasypyrum, Triticum andHordeum. Southern hybridization and polymerase chain reaction analysis established that all studied species preserve the tandem type of sequence organization but the copy number is altered drastically between species. In the pSc200 family, a fraction of the tandem arrays is present with a head-to-head orientation of dimers inS. cereale andS. montanum. Members of the same family are more heterogeneous and present as head-to-head monomers in theDasypyrum species andA. cristatum. In situ hybridization demonstrates different organization of the sequence families in the various species: pSc200 and pSc250 are concentrated in major blocks at the ends of most rye chromosome arms, whereas they are more dispersed and in smaller blocks inDasypyrum andAgropyron indicating that accumulation is not simply due to the sequence itself. In contrast to rye,D. villosum has large blocks of only pSc200 whereasD. breviaristatum shows greater amplification of pSc250. These data indicate that each repetitive family is an independent unit of evolution, and suggest that the twoDasypyrum species are not closely related. The data are discussed in terms of existing evolutionary models for repetitive DNA sequences. The contribution of random events, through molecular drive and selection, to the evolution of heterochromatic regions is considered.

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, collocalizing 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.

Diverse patterns of the tandem repeats organization in rye chromosomes.

Although the monomer size, nucleotide sequence, abundance and species distribution of tandemly organized DNA families are well characterized, little is known about the internal structure of tandem arrays, including total arrays size and the pattern of monomers distribution. Using our rye specific probes, pSc200 and pSc250, we addressed these issues for telomere associated rye heterochromatin where these families are very abundant. Fluorescence in situ hybridization (FISH) on meiotic chromosomes revealed a specific mosaic arrangement of domains for each chromosome arm where either pSc200 or pSc250 predominates without any obvious tendency in order and size of domains. DNA of rye-wheat monosomic additions studied by pulse field gel electrophoresis produced a unique overall blot hybridization display for each of the rye chromosomes. The FISH signals on DNA fibres showed multiple monomer arrangement patterns of both repetitive families as well as of the Arabidopsis-type telomere repeat. The majority of the arrays consisted of the monomers of both families in different patterns separated by spacers. The primary structure of some spacer sequences revealed scrambled regions of similarity to various known repetitive elements. This level of complexity in the long-range organization of tandem arrays has not been previously reported for any plant species. The various patterns of internal structure of the tandem arrays are likely to have resulted from evolutionary interplay, array homogenization and the generation of heterogeneity mediated by double-strand breaks and associated repair mechanisms.

Rye Chromosome Variability in Wheat–rye Addition and Substitution Lines

In a study of polymorphism and stability in rye chromosomes, three rye varieties and the sets of wheat-rye addition and substitution lines were compared using two non-homologous highly repetitive DNA families, pSc200 and pSc250. The rye varieties, Petkus, Imperial and Onohoiskaya, showed polymorphism for the presence and the size of the pSc200 in-situ hybridization signals on chromosome pairs, 2R, 4R and 7R, and the pSc250 signals on chromosomes, 5R, 6R and 7R. Chromosome 1R was heteromorphic within the Onohoiskaya variety. Differences in the distribution of chromosome polymorphisms imply that intervarietal changes to these highly repetitive DNA families occurred independently, despite their juxtaposition or even overlapping locations in subtelomeric heterochromatic regions. In the set of Saratovskaya 29 wheat/Onohoiskaya substitution lines, only chromosome 2R was altered relative to its counterpart in the parental rye variety due to amplification of the pSc250 signal on the long arm, although this did not exceed intervarietal polymorphism. In the set of Chinese Spring wheat/Imperial addition lines, only two Imperial chromosomes, 4R and 6R, were unchanged. We detected the loss of one or both rye homologous chromosomes, the loss of one arm, and the deletion of subtelomeric heterochromatin accompanied by the loss of the pSc200 signal. The results show that Saratovskaya 29/Onohoiskaya chromosome substitution lines possess increased chromosome stability compared with Chinese Spring/Imperial addition lines.

BARE-ID, a representative of a family of BARE-like elements of the barley genome

In our search for transposable elements in barley, Hordeum vulgare, we have isolated and cloned two BamHI-fragments of 4.7 and 4.2 kb in length containing very abundant DNA sequences. The 4.7 kb fragment is homologous to the extended region, including more than half of the 5′-LTR and some part of the coding domain of BARE-1, a member of copia-like retrotransposon family of barley. The 4.2 kb fragment, bearing homology to BARE-1 and the WIS-2 family isolated from wheat, is unique among studied retroelements of cereals because it consists of two inverted parts, each containing homology to the LTR and UTL of BARE-1. Functional motifs for reverse transcription, two TATA-boxes and two primer-binding sites, were found within the LTRs. The element contained within this fragment was generated by significant rearrangement of a BARE-like retrotransposon, which included inversion of the extended 5′-terminal region and deletion of the internal domain. Therefore this element is named BARE-ID (BARE-inverted, deleted). A family of BARE-like elements is amplified in the H. vulgare genome compared with wild barley species. The terminal inverted repeat of BARE-ID was used as a probe for examination of evolutionary diversity within genus Hordeum. Our data are basically in agreement with the modern classification system. However, they do not support the combination of H. vulgare and H. bulbosum into one group with the same type of genome. New data concerning the possible origin of the polyploid species, H. secalinum, confirm that retrotransposons are a useful tool for phylogenetic studies.