Kesara Anamthawat-Jónsson

Retrotransposon BARE-1 and Its Role in Genome Evolution in the Genus Hordeum

The replicative retrotransposon life cycle offers the potential for explosive increases in copy number and consequent inflation of genome size. The BARE-1 retrotransposon family of barley is conserved, disperse, and transcriptionally active. To assess the role of BARE-1 in genome evolution, we determined the copy number of its integrase, its reverse transcriptase, and its long terminal repeat (LTR) domains throughout the genus Hordeum. On average, BARE-1 contributes 13.7 × 103 full-length copies, amounting to 2.9% of the genome. The number increases with genome size. Two LTRs are associated with each internal domain in intact retrotransposons, but surprisingly, BARE-1 LTRs were considerably more prevalent than would be expected from the numbers of intact elements. The excess in LTRs increases as both genome size and BARE-1 genomic fraction decrease. Intrachromosomal homologous recombination between LTRs could explain the excess, removing BARE-1 elements and leaving behind solo LTRs, thereby reducing the complement of functional retrotransposons in the genome and providing at least a partial “return ticket from genomic obesity.”

Large Retrotransposon Derivatives: Abundant, Conserved but Nonautonomous Retroelements of Barley and Related Genomes

Retroviruses and LTR retrotransposons comprise two long-terminal repeats (LTRs) bounding a central domain that encodes the products needed for reverse transcription, packaging, and integration into the genome. We describe a group of retrotransposons in 13 species and four genera of the grass tribe Triticeae, including barley, with long, ∼4.4-kb LTRs formerly called Sukkula elements. The ∼3.5-kb central domains include reverse transcriptase priming sites and are conserved in sequence but contain no open reading frames encoding typical retrotransposon proteins. However, they specify well-conserved RNA secondary structures. These features describe a novel group of elements, called LARDs or large retrotransposon derivatives (LARDs). These appear to be members of the gypsy class of LTR retrotransposons. Although apparently nonautonomous, LARDs appear to be transcribed and can be recombinationally mapped due to the polymorphism of their insertion sites. They are dispersed throughout the genome in an estimated 1.3 × 103 full-length copies and 1.16 × 104 solo LTRs, indicating frequent recombinational loss of internal domains as demonstrated also for the BARE-1 barley retrotransposon.

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 × 104. 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.

Detection and characterization of 1B/1R translocations in hexaploid wheat.

Total genomic DNA from rye was labelled with biotin and used as a probe for in situ hybridization to show the sizes and translocation points of the rye chromosome segments in five wheat varieties which carry a translocation between wheat chromosome IB and the short arm of rye chromosome IR (1B/1R). All the translocation breakpoints were at, or very near to, the centromere. Using genomic DNA to block some cross-hybridization, little signal from hybridization between the rye probe and wheat chromosomes was observed despite the 78 per cent sequence homology between the species. The translocation was identified in cells at all stages of the cell cycle. Total genomic rye DNA used as a probe was also able to distinguish rye, triticale and wheat varieties carrying the translocation, by Southern hybridization. The techniques using genomic probes are useful for detecting, characterizing and following alien chromosomes or chromosome segments through breeding programmes, by both in situ and Southern hybridization.

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 vulgareL.) and other Hordeum species. Copia-like in its organization, it consists of 1.8-kb long terminal repeats bounding an internal domain of 5275bp 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 -1 integration to produce molecular markers for breeding, biodiversity, and mapping applications.

Genomic and genetic relationships among species of Leymus (Poaceae: Triticeae) inferred from 18S–26S ribosomal genes

The 18S-26S ribosomal genes in three closely related species of Leymus (Poaceae: Triticeae) were examined using fluorescence in situ hybridization (FISH) and restriction fragment length polymorphism (RFLP). Both approaches revealed a close relationship between L. arenarius (8x = 56, northern European) and L. racemosus (4x = 28, central Eurasian), whereas L. mollis (4x = 28, northern American/Pacific) was distinct. Each species had three homologous pairs of major rDNA loci: a1, a2, and a3 for L. arenarius; m1, m2, and m3 for L. mollis; and r1, r2, and r3 for L. racemosus. Leymus arenarius had in addition three minor loci, a4, a5, and a6. The major loci of L. arenarius and L. racemosus were identical, indicating that the former species could have originated from the latter, via interspecific hybridization and/or polyploidy. The rDNA-RFLPs further indicated relationships of these species to other species of Leymus (L. karellini, 8x = 56 and L. angustus, 12x = 84) and Psathyrostachys (P. fragilis, P. huashanica, P. juncea, and P. lanuginosa, which are all diploids). A phenogram constructed from 20 BamHI, EcoRI, and DraI rDNA fragments revealed closer relationship between the two genera, Leymus and Psathyrostachys, than that among species within a genus.

Natural hybridisation in birch: triploid hybrids between Betula nana and B. pubescens

Hybridisation between diploid (2n=28) dwarf birch Betula nana L. and tetraploid (2n=56) downy birch B. pubescens Ehrh. has occurred in natural populations in Iceland. About 10% of birch plants randomly collected are triploid (2n=42) hybrids. Ribosomal gene mapping on chromosomes and genomic in situ hybridisation confirms the hybridity. However, the triploid hybrids are not morphologically distinct, i.e. they are not different from diploid and tetraploid birch plants that have intermediate morphology. The triploid hybrids have evidently played an important role in driving bi-directional gene flow between these two species. This paper reviews the extent of interspecific hybridisation in selected birch woodland populations and discusses the significance of natural hybridisation and introgression in birch.

Wide hybridization between wheat (Triticum L.) and lymegrass (Leymus Hochst.)

A total of 240 F1 hybrids was made beween wheat (Triticum aestivum L. em. Thell. (2n = 6x = 42) and T. carthlicum Nevski (2n = 4x = 28)) and perennial lymegrass (North European Leymus arenarius (L.) Hochst. (2n = 8x = 56) and North American L. mollis (Trin.) Pilger (2n = 4x = 28)). The wide crosses yielded embryos in 20% of caryopses and 96% of the embryos developed into normal hybrid plants. The hybrids were vegetatively vigorous, with evidence of the Leymus rhizomatous habit. Those deriving from L. arenarius survived overwintering in Iceland, but the hybrids L. mollis did not, whereas in a milder environment, both showed perenniality. Cytogenetic analysis of root tip cells before the plants were treated with colchicine showed that 21 out of 28 hybrids investigated had chromosome mosaics, with a population of both amphihaploid and amphidiploid cells. This spontaneous doubling of somatic chromosomes occurred in all cross combinations, with the highest average frequency of diploid cells (28%) in T. carthlicum × L. arenarius crosses. A few selfed seeds have been obtained from a T. aestivum × L. arenarius hybrid. All the hybrids were treated twice with colchicine, but the treatment appeared to have little or no effect on the frequency of chromosome doubling in the hybrids deriving from T. aestivum. The frequency of diploid cells, however, increased significantly (e.g. to 80%) in the hybrids deriving from the T. carthlicum parent. Genomic in situ hybridization confirmed the hybridity of the plants and showed that the hybrids were amphiploids containing genomes of both wheat and lymegrass. In situ hybridization using ribosomal DNA probe differentiated chromosomes of L. mollis, L. arenarius from those of wheat. The hybrids are being backcrossed with lymegrass pollen, aiming to domesticate the wild, perennial species.

Differentiating pollen of Betula species from Iceland

Subfossil pollen from two co‐existing Betula species in Iceland, B. nana and B. pubescens, is frequently found in sediments and peat. Interpretation of the findings often depends on the ability to differentiate between the two species according to pollen size and structure. Fresh pollen samples were prepared from 70 individual trees/shrubs which had been identified to species by chromosome number. Grain diameters and pore depths were measured and ratios of grain diameter to pore depth (D/P ratios) were calculated. The mean grain diameters of pollen from diploid B. nana and tetraploid B. pubescens were 20.42 and 24.20 µm, whereas mean pore depths were 2.20 and 2.81 µm respectively. Mean D/P ratios were therefore 9.55 for B. nana and 8.85 for B. pubescens. The difference between species was statistically significant for all three pollen parameters. Grain diameter appeared to be the most useful parameter, as only about 20% of the samples were in the overlapping region of the species distributions. Pollen size (grain diameter) was also positively correlated to tree morphology, which was evaluated using species‐specific botanical characters. Pollen samples from different locations/populations in Iceland varied slightly in mean size and ratio. The size difference between pollen of B. nana and B. pubescens in this study is less than other papers have reported, which may be due to the effect of introgressive hybridisation between the two birch species in Iceland.

Characteristics of pollen from natural triploid Betula hybrids

Birch has a key role in the Holocene vegetation history of northern Europe and in sub‐arctic climates dwarf birch and tree birch co‐exist. In Iceland, triploid hybrids between diploid Betula nana (dwarf birch) and tetraploid B. pubescens (downy birch) are common and therefore likely to contribute to pollen deposition. Pollen from 22 triploid trees/shrubs from ten woodlands in Iceland was examined and its size and shape compared with pollen from the parental species. The mean diameter of pollen grains from the triploid hybrids was not statistically different from that of B. nana pollen, but was significantly smaller than the mean value of B. pubescens pollen. On the contrary, the size of the vestibulum was similar to that of B. pubescens, which was significantly greater than that of B. nana, and therefore the diameter‐pore depth ratio was lower than the values from either species. The pattern of size distribution within plants indicated that triploid hybrids might have produced two sizes of triporate pollen grains, but the small B. nana size was far more prevalent than the larger B. pubescens size. Several anomalies in pollen morphology were common among the hybrid pollen grains: four or more pores were the most frequent type of abnormality. Characteristics of the pollen of triploid Betula hybrids, especially structural anomalies, may provide a means to reveal periods of interspecific hybridisation in the analysis of sub‐fossil pollen.