Torsten Wenke

Targeted Identification of Short Interspersed Nuclear Element Families Shows Their Widespread Existence and Extreme Heterogeneity in Plant Genomes

Plant genomes contain a large number of short interspersed nuclear elements, which so far have been detected only randomly. A software tool was created integrating weakly conserved structural motifs for the analysis of genome sequences. Thousands of sequences from many plant taxa were identified, giving insights into their variability, localization along chromosomes, and evolution. Short interspersed nuclear elements (SINEs) are non-long terminal repeat retrotransposons that are highly abundant, heterogeneous, and mostly not annotated in eukaryotic genomes. We developed a tool designated SINE-Finder for the targeted discovery of tRNA-derived SINEs. We analyzed sequence data of 16 plant genomes, including 13 angiosperms and three gymnosperms and identified 17,829 full-length and truncated SINEs falling into 31 families showing the widespread occurrence of SINEs in higher plants. The investigation focused on potato (Solanum tuberosum), resulting in the detection of seven different SolS SINE families consisting of 1489 full-length and 870 5′ truncated copies. Consensus sequences of full-length members range in size from 106 to 244 bp depending on the SINE family. SolS SINEs populated related species and evolved separately, which led to some distinct subfamilies. Solanaceae SINEs are dispersed along chromosomes and distributed without clustering but with preferred integration into short A-rich motifs. They emerged more than 23 million years ago and were species specifically amplified during the radiation of potato, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum). We show that tobacco TS retrotransposons are composite SINEs consisting of the 3′ end of a long interspersed nuclear element integrated downstream of a nonhomologous SINE family followed by successfully colonization of the genome. We propose an evolutionary scenario for the formation of TS as a spontaneous event, which could be typical for the emergence of SINE families.

The Ty1-copia families SALIRE and Cotzilla populating the Beta vulgaris genome show remarkable differences in abundance, chromosomal distribution, and age.

Long terminal repeat (LTR) retrotransposons are major components of plant genomes influencing genome size and evolution. Using two separate approaches, we identified the Ty1-copia retrotransposon families Cotzilla and SALIRE in the Beta vulgaris (sugar beet) genome. While SALIRE elements are similar to typical Ty1-copia retrotransposons, Cotzilla elements belong to a lineage called Sireviruses. Hallmarks of Cotzilla retrotransposons are the existence of an additional putative env-like open reading frame upstream of the 3′LTR, an extended gag region, and a frameshift separating the gag and pol genes. Detected in a c0t-1 DNA library, Cotzilla elements belong to the most abundant retrotransposon families in B. vulgaris and are relatively homogenous and evolutionarily young. In contrast, the SALIRE family has relatively few copies, is diverged, and most likely ancient. As revealed by fluorescent in situ hybridization, SALIRE elements target predominantly gene-rich euchromatic regions, while Cotzilla retrotransposons are abundant in the intercalary and pericentromeric heterochromatin. The analysis of two retrotransposons from the same subclass contrasting in abundance, age, sequence diversity, and localization gives insight in the heterogeneity of LTR retrotransposons populating a plant genome.

Diversity of a complex centromeric satellite and molecular characterization of dispersed sequence families in sugar beet (Beta vulgaris).

BACKGROUND AND AIMS The aim of this work was the identification and molecular characterization of novel sugar beet (Beta vulgaris) repetitive sequences to unravel the impact of repetitive DNA on size and evolution of Beta genomes via amplification and diversification. METHODS Genomic DNA and a pool of B. vulgaris repetitive sequences were separately used as probes for a screening of high-density filters from a B. vulgaris plasmid library. Novel repetitive motifs were identified by sequencing and further used as probes for Southern analyses in the genus Beta. Chromosomal localization of the repeats was analysed by fluorescent in situ hybridization on chromosomes of B. vulgaris and two other species of the section Beta. KEY RESULTS Two dispersed repetitive families pDvul1 and pDvul2 and the tandemly arranged repeat family pRv1 were isolated from a sugar beet plasmid library. The dispersed repetitive families pDvul1 and pDvul2 were identified in all four sections of the genus Beta. The members of the pDvul1 and pDvul2 family are scattered over all B. vulgaris chromosomes, although amplified to a different extent. The pRv1 satellite repeat is exclusively present in species of the section Beta. The centromeric satellite pBV1 by structural variations of the monomer and interspersion of pRv1 units forms complex satellite structures, which are amplified in different degrees on the centromeres of 12 chromosomes of the three species of the Beta section. CONCLUSIONS The complexity of the pBV1 satellite family observed in the section Beta of the genus Beta and, in particular, the strong amplification of the pBV1/pRv1 satellite in the domesticated B. vulgaris indicates the dynamics of centromeric satellite evolution during species radiation within the genus. The dispersed repeat families pDvul1 and pDvul2 might represent derivatives of transposable elements.

Analysis of a c0t-1 library enables the targeted identification of minisatellite and satellite families in Beta vulgaris

BackgroundRepetitive DNA is a major fraction of eukaryotic genomes and occurs particularly often in plants. Currently, the sequencing of the sugar beet (Beta vulgaris) genome is under way and knowledge of repetitive DNA sequences is critical for the genome annotation. We generated a c0t-1 library, representing highly to moderately repetitive sequences, for the characterization of the major B. vulgaris repeat families. While highly abundant satellites are well-described, minisatellites are only poorly investigated in plants. Therefore, we focused on the identification and characterization of these tandemly repeated sequences.ResultsAnalysis of 1763 c0t-1 DNA fragments, providing 442 kb sequence data, shows that the satellites pBV and pEV are the most abundant repeat families in the B. vulgaris genome while other previously described repeats show lower copy numbers. We isolated 517 novel repetitive sequences and used this fraction for the identification of minisatellite and novel satellite families. Bioinformatic analysis and Southern hybridization revealed that minisatellites are moderately to highly amplified in B. vulgaris. FISH showed a dispersed localization along most chromosomes clustering in arrays of variable size and number with exclusion and depletion in distinct regions.ConclusionThe c0t-1 library represents major repeat families of the B. vulgaris genome, and analysis of the c0t-1 DNA was proven to be an efficient method for identification of minisatellites. We established, so far, the broadest analysis of minisatellites in plants and observed their chromosomal localization providing a background for the annotation of the sugar beet genome and for the understanding of the evolution of minisatellites in plant genomes.

An abundant and heavily truncated non-LTR retrotransposon (LINE) family in Beta vulgaris

We describe a non-LTR retrotransposon family, BvL, of the long interspersed nuclear elements L1 clade isolated from sugar beet (Beta vulgaris). Characteristic molecular domains of three full-length BvL elements were determined in detail, showing that coding sequences are interrupted and most likely non-functionally. In addition, eight highly conserved endonuclease regions were defined by comparison with other plant LINEs. The abundant BvL family is widespread within the genus Beta, however, the vast majority of BvL copies are extremely 5′ truncated indicating an error-prone reverse transcriptase activity. The dispersed distribution of BvL copies on all sugar beet chromosomes with exclusion of most heterochromatic regions was shown by fluorescent in situ hybridization. The analysis of BvL 3′ end sequences and corresponding flanking regions, respectively, revealed the preferred integration of BvL into A/T-rich regions of the sugar beet genome, but no specific target sequences.

Development and application of SINE-based markers for genotyping of potato varieties

Potato variety discrimination based on morphological traits is laborious and influenced by the environment, while currently applied molecular markers are either expensive or time-consuming in development or application. SINEs, short interspersed nuclear elements, are retrotransposons with a high copy number in plant genomes representing a potential source for new markers. We developed a marker system for potato genotyping, designated inter-SINE amplified polymorphism (ISAP). Based on nine potato SINE families recently characterized (Wenke et al. in Plant Cell 23:3117–3128, 2011), we designed species-specific SINE primers. From the resulting 153 primer combinations, highly informative primer sets were selected for potato variety analysis regarding number of bands, quality of the banding pattern, and the degree of polymorphism. Fragments representing ISAPs can be separated by conventional agarose gel electrophoresis; however, automation with a capillary sequencer is feasible. Two selected SINE families, SolS-IIIa and SolS-IV, were shown to be highly but differently amplified in Solanaceae, Solaneae tribe, including wild and cultivated potatoes, tomato, and eggplant. Fluorescent in situ hybridization demonstrated the genome-wide distribution of SolS-IIIa and SolS-IV along potato chromosomes, which is the basis for genotype discrimination and differentiation of somaclonal variants by ISAP markers.

Inter-SINE Amplified Polymorphism (ISAP) for Rapid and Robust Plant Genotyping

The unambiguous differentiation of crop genotypes is often laborious or expensive. A rapid, robust, and cost-efficient marker system is required for routine genotyping in plant breeding and marker-assisted selection. We describe the Inter-SINE Amplified Polymorphism (ISAP) system that is based on standard molecular methods resulting in genotype-specific fingerprints at high resolution. These markers are derived from Short Interspersed Nuclear Elements (SINEs) which are dispersed repetitive sequences present in most if not all plant genomes and can be efficiently extracted from plant genome sequences. The ISAP method was developed on potato as model plant but is also transferable to other plant species.

Diversity studies in genetic resources of Solanum spp. (section Petota) by comparative application of ISAP markers

The Genebank at the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) harbours three different potato collections comprising 2846 wild potato accessions, 2757 accessions of cultivars, landraces and breeding material of Solanum tuberosum L. and 579 Solanum clones originating mainly from South and Central America (as of July 2016). The three collections are maintained via seeds, in vitro culture, cryopreservation or in the field. All four processes are labour and cost intensive. Therefore, methods facilitating the efficient management and maintenance of the collections are needed. We describe the experimental application of the retrotransposon-based Inter-SINE amplified polymorphism (ISAP) marker system for supporting the management of potato collections. Three ISAP primer combinations were evaluated on a set of 259 cultivated and 89 wild potato accessions focussing on three different aspects: diversity analysis, duplicate identification and phylogenetic studies. Compared to up to 29 microsatellites, ISAPs were similarly efficient in monitoring genetic diversity and detecting duplicated accessions. In addition, the three ISAP primer combinations could be applied to wild potato species, although their use in phylogenetic studies is limited due to their highly polymorphic nature. Generally, ISAPs are a very valuable tool in the maintenance of clonally maintained potato collections and for the verification of the identity of accessions.