The retrotransposon landscape of the Beta vulgaris genome: Evolutionary conservation and diversity

Retrotransposons are major components of plant genomes influencing their genome size, organization and evolution. In the frame of this work, retrotransposons of the Beta vulgaris genome have been identified by molecular methods and whole genome bioinformatics approaches. Neither belonging to the rosids nor asterids, B. vulgaris (cultivated beet including sugar beet, beet root and mangold) is taxonomically placed at a key position at the root of the core eudicots, and considerably different from traditional plant model species such as thale cress or rice. Its genome has been sequenced, and annotation is under way. In order to compare different evolutionary lineages of B. vulgaris retrotransposons, long terminal repeat (LTR) and non-LTR retrotransposon family have been analyzed in detail. Full-length members have been isolated and characterized by bioinformatics, Southern and fluorescent in situ hybridization. Hallmarks of the LTR retrotransposon family Cotzilla are an additional env-like open reading frame (ORF), homogeneity of the members and the very high abundance. Most family members are evolutionarily young, and have most likely been created during recent bursts of amplification during species radiation. In contrast, the non-LTR retrotransposon family BNR has fewer copies and is much more diverged. Although the BNR ORF2 resembles previously analyzed long interspersed nuclear elements (LINEs) of the L1 clade, its ORF1 sequence differs strongly. It lacks the zinc finger domain described for plant LINEs, but contains instead an RNA recognition motif (RRM) likely to have an RNA-binding function. Database searches revealed the presence of similar LINE families in higher plant genomes such as poplar, lotus and soybean. Comparing their reverse transcriptase regions with other retrotransposons, these BNR-like LINEs form a separate group of L1 LINEs designated as BNR subclade. Availability of the B. vulgaris genome sequence allowed retrotransposon analyses on a genome-wide scale. A Hidden Markov Model-based detection algorithm has been developed in order to retrieve retrotransposon information directly from the database. Nearly 6000 B. vulgaris reverse transcriptase sequences have been isolated and classified into LTR retrotransposons of the Ty3-gypsy and Ty1-copia type, and non-LTR retrotransposons of the LINE type. As a result, a comprehensive overview of the retrotransposon spectrum of the B. vulgaris genome has been generated. Since plant LINEs have been only rarely investigated, the B. vulgaris LINE composition was studied in detail. Out of 28 described LINE clades, only members of the L1 and RTE clades have been identified. Based on a minimal shared sequence identity of 60 %, they form at least 17 L1 families and one RTE family. Full-length members of all investigated L1 families have been analyzed regarding their sequence, structure and diversity. In order to transfer the algorithm tested in B. vulgaris to other angiosperm genomes, twelve additional plant genomes have been queried for LINE reverse transcriptases. Key finding is the presence of only two LINE clades (L1 and RTE) in the analyzed genomes of higher plants. Whereas plant L1 LINEs are highly diverse and form at least seven subclades with members across species borders, RTE LINEs are extremely homogenized and constitute most likely only a single family per genome. In summary, this work’s results help to gain an understanding of the different strategies of retrotransposon evolution in plants, whereas the generated data directly contributes to the B. vulgaris genome annotation project.Retrotransposons sind eine wesentliche Komponente von Pflanzengenomen, die sowohl die Größe und Organisation als auch die Evolution dieser Genome wesentlich beeinflussen können. Im Rahmen dieser Arbeit wurden verschiedene Gruppen von Retrotransposons des Beta vulgaris Genoms mittels molekularer und bioinformatischer Methoden identifiziert. Innerhalb der dikotyledonen Blütenpflanzen gehört B. vulgaris (kultivierte Rübe einschließlich Zuckerrübe, Roter Beete und Mangold) weder zu den Rosiden noch zu den Asteriden, sondern nimmt eine Schlüsselposition innerhalb der Kerneudikotyledonen ein. Somit zeigt das Rübengenom wesentliche Unterschiede zu traditionellen Modellpflanzen wie Arabidopsis thaliana oder Oryza sativa. Das Genom ist bereits sequenziert, die Annotation jedoch noch nicht abgeschlossen. Um verschiedene evolutionäre Linien von B. vulgaris Retrotransposons vergleichend zu untersuchen wurden insbesondere Long Terminal Repeat (LTR)- und Non-LTR-Retrotransposon-Familien detailliert analysiert. Vollständige Mitglieder wurden isoliert und mittels bioinformatischer Methoden, Southern- und Fluoreszenz-in situ-Hybridisierung untersucht. Die LTR-Retrotransposon-Familie Cotzilla ist durch einen zusätzlichen env-ähnlichen offenen Leserahmen (ORF), Homogenität ihrer Mitglieder und eine hohe Abundanz gekennzeichnet. Die meisten Cotzilla-Kopien sind evolutionär jung und wurden wahrscheinlich innerhalb eines kurzen Zeitraumes während der Artentstehung stark amplifiziert. Im Gegensatz zur Cotzilla-Familie besitzt die Non-LTR-Retrotransposon-Familie BNR weniger Kopien und ist wesentlich divergierter. Während der BNR-spezifische ORF2 starke Ähnlichkeiten zu anderen pflanzlichen Long Interspersed Nuclear Elements (LINEs) der L1-Klade aufweist, unterscheidet sich der BNR ORF1 von diesen sehr stark. Im Gegensatz zu bereits beschrieben pflanzlichen LINEs kodiert er kein Zinkfingermotiv, sondern substituiert dieses durch ein RNA-Erkennungsmotiv (RRM). Durch Datenbanksuche konnten BNR-ähnliche LINEs in den Genomen höherer Pflanzen wie Soja, Lotus und Pappel identifiziert werden. Ein Vergleich der entsprechenden Reversen Transkriptasen (RT) mit den RTs anderer Retrotransposons zeigt, dass die BNR-ähnlichen LINEs eine separate Gruppe innerhalb der L1 LINEs bilden. Diese wurde daher als BNR-Subklade definiert. Die Untersuchung von Retrotransposons auf Genomebene wurde durch die B. vulgaris Genomsequenz ermöglicht. Um Retrotransposon-Informationen direkt aus dem Genom zu extrahieren, wurde ein Hidden Markov Modell (HMM)-basierter Detektions-algorithmus entwickelt. Annähernd 6000 B. vulgaris Reverse Transkriptase-Sequenzen konnten identifiziert und in LTR-Retrotransposons des Ty3-gypsy- beziehungsweise des Ty1-copia-Typs und in Non-LTR-Retrotransposons des LINE-Typs klassifiziert werden. Somit wurde ein umfassender Überblick über die Bandbreite der B. vulgaris Retrotransposons arhalten. Da pflanzliche LINEs bisher nur wenig erforscht sind, wurde die B. vulgaris LINE Zusammensetzung genauer untersucht. Von 28 beschriebenen LINE-Kladen konnten nur Mitglieder der L1- und der RTE-Klade identifiziert werden. Basierend auf einer Identität von mindestens 60 % bilden die Sequenzen 17 L1 Familien und eine RTE Familie. Vollständige Mitglieder aller L1 Familien wurden hinsichtlich ihrer Sequenz, Struktur und Diversität analysiert. Um den in B. vulgaris getesteten HMM-basierten Algorithmus auf andere Angiospermengenome zu übertragen, wurden zwölf weitere Pflanzengenome auf das Vorhandensein von LINE-spezifischen Reversen Transkriptasen untersucht. Wesentlichstes Ergebnis ist der Nachweis von nur zwei LINE-Kladen (L1 und RTE) in höheren Pflanzen. Während pflanzliche L1 LINEs hochgradig divers sind und über Artgrenzen hinaus mindestens sieben Subkladen mit Vertretern verschiedener Pflanzen bilden, sind RTE LINEs extrem homogenisiert und stellen höchstwahrscheinlich nur eine einzelne Familie pro Genom einer Art dar. Zusammenfassend ermöglichen die Ergebnisse dieser Arbeit eine Erweiterung des Verständnisses der unterschiedlichen Evolutionsstrategien von Retrotransposons in Pflanzen. Zusätzlich tragen die gewonnen Daten zur Annotation des B. vulgaris Genoms bei

Using Lorentz forces to control the distribution of bubbles in a vertical tube filled with liquid metal

In this work, a method to increase the residence time of bubbles in tubes or pipes filled with liquid metal is investigated. Imposing a horizontal electric current and a perpendicular horizontal magnetic field generates an upward-directed Lorentz force. This force can counteract gravity and cause floating of bubbles. Even with homogeneous electric fields these float in the mean but fluctuate randomly within the swarm due to mutual interactions. In the present case the cylindrical shape of the container furthermore creates inhomogeneous electric currents and an inhomogeneous force distribution resulting in a macroscopic convection pattern stirring the bubbles and further homogenising the spatial distribution of the bubbles.LIMTECHDFG/HE 7529/1-

Dynamic stereo microscopy for studying particle sedimentation

We demonstrate a new method for measuring the sedimentation of a single colloidal bead by using a combination of optical tweezers and a stereo microscope based on a spatial light modulator. We use optical tweezers to raise a micron-sized silica bead to a fixed height and then release it to observe its 3D motion while it sediments under gravity. This experimental procedure provides two independent measurements of bead diameter and a measure of Faxén’s correction, where the motion changes due to presence of the boundary

Complete pan-plastome sequences enable high resolution phylogenetic classification of sugar beet and closely related crop wild relatives.

Funder: Universität Bielefeld (3146)BACKGROUND: As the major source of sugar in moderate climates, sugar-producing beets (Beta vulgaris subsp. vulgaris) have a high economic value. However, the low genetic diversity within cultivated beets requires introduction of new traits, for example to increase their tolerance and resistance attributes - traits that often reside in the crop wild relatives. For this, genetic information of wild beet relatives and their phylogenetic placements to each other are crucial. To answer this need, we sequenced and assembled the complete plastome sequences from a broad species spectrum across the beet genera Beta and Patellifolia, both embedded in the Betoideae (order Caryophyllales). This pan-plastome dataset was then used to determine the wild beet phylogeny in high-resolution. RESULTS: We sequenced the plastomes of 18 closely related accessions representing 11 species of the Betoideae subfamily and provided high-quality plastome assemblies which represent an important resource for further studies of beet wild relatives and the diverse plant order Caryophyllales. Their assembly sizes range from 149,723 bp (Beta vulgaris subsp. vulgaris) to 152,816 bp (Beta nana), with most variability in the intergenic sequences. Combining plastome-derived phylogenies with read-based treatments based on mitochondrial information, we were able to suggest a unified and highly confident phylogenetic placement of the investigated Betoideae species. Our results show that the genus Beta can be divided into the two clearly separated sections Beta and Corollinae. Our analysis confirms the affiliation of B. nana with the other Corollinae species, and we argue against a separate placement in the Nanae section. Within the Patellifolia genus, the two diploid species Patellifolia procumbens and Patellifolia webbiana are, regarding the plastome sequences, genetically more similar to each other than to the tetraploid Patellifolia patellaris. Nevertheless, all three Patellifolia species are clearly separated. CONCLUSION: In conclusion, our wild beet plastome assemblies represent a new resource to understand the molecular base of the beet germplasm. Despite large differences on the phenotypic level, our pan-plastome dataset is highly conserved. For the first time in beets, our whole plastome sequences overcome the low sequence variation in individual genes and provide the molecular backbone for highly resolved beet phylogenomics. Hence, our plastome sequencing strategy can also guide genomic approaches to unravel other closely related taxa

TE Hub: A community-oriented space for sharing and connecting tools, data, resources, and methods for transposable element annotation

Transposable elements (TEs) play powerful and varied evolutionary and functional roles, and are widespread in most eukaryotic genomes. Research into their unique biology has driven the creation of a large collection of databases, software, classification systems, and annotation guidelines. The diversity of available TE-related methods and resources raises compatibility concerns and can be overwhelming to researchers and communicators seeking straightforward guidance or materials. To address these challenges, we have initiated a new resource, TE Hub, that provides a space where members of the TE community can collaborate to document and create resources and methods. The space consists of (1) a website organized with an open wiki framework, https://tehub.org, (2) a conversation framework via a Twitter account and a Slack channel, and (3) bi-monthly Hub Update video chats on the platform’s development. In addition to serving as a centralized repository and communication platform, TE Hub lays the foundation for improved integration, standardization, and effectiveness of diverse tools and protocols. We invite the TE community, both novices and experts in TE identification and analysis, to join us in expanding our community-oriented resource

Larix species range dynamics in Siberia since the Last Glacial captured from sedimentary ancient DNA

Climate change is expected to cause major shifts in boreal forests which are in vast areas of Siberia dominated by two species of the deciduous needle tree larch (Larix). The species differ markedly in their ecosystem functions, thus shifts in their respective ranges are of global relevance. However, drivers of species distribution are not well understood, in part because paleoecological data at species level are lacking. This study tracks Larix species distribution in time and space using target enrichment on sedimentary ancient DNA extracts from eight lakes across Siberia. We discovered that Larix sibirica, presently dominating in western Siberia, likely migrated to its northern distribution area only in the Holocene at around 10,000 years before present (ka BP), and had a much wider eastern distribution around 33 ka BP. Samples dated to the Last Glacial Maximum (around 21 ka BP), consistently show genotypes of L. gmelinii. Our results suggest climate as a strong determinant of species distribution in Larix and provide temporal and spatial data for species projection in a changing climate

A comparative analysis of two conserved motifs in bacterial poly(A) polymerase and CCA-adding enzyme

Showing a high sequence similarity, the evolutionary closely related bacterial poly(A) polymerases (PAP) and CCA-adding enzymes catalyze quite different reactions—PAP adds poly(A) tails to RNA 3′-ends, while CCA-adding enzymes synthesize the sequence CCA at the 3′-terminus of tRNAs. Here, two highly conserved structural elements of the corresponding Escherichia coli enzymes were characterized. The first element is a set of amino acids that was identified in CCA-adding enzymes as a template region determining the enzymes' specificity for CTP and ATP. The same element is also present in PAP, where it confers ATP specificity. The second investigated region corresponds to a flexible loop in CCA-adding enzymes and is involved in the incorporation of the terminal A-residue. Although, PAP seems to carry a similar flexible region, the functional relevance of this element in PAP is not known. The presented results show that the template region has an essential function in both enzymes, while the second element is surprisingly dispensable in PAP. The data support the idea that the bacterial PAP descends from CCA-adding enzymes and still carries some of the structural elements required for CCA-addition as an evolutionary relic and is now fixed in a conformation specific for A-addition

Repeat Composition of CenH3-chromatin and H3K9me2-marked heterochromatin in Sugar Beet (Beta vulgaris)

Kowar T, Zakrzewski F, Macas J, et al. Repeat Composition of CenH3-chromatin and H3K9me2-marked heterochromatin in Sugar Beet (Beta vulgaris). BMC Plant Biology. 2016;16(1): 120.Background Sugar beet (Beta vulgaris) is an important crop of temperate climate zones, which provides nearly 30 % of the world’s annual sugar needs. From the total genome size of 758 Mb, only 567 Mb were incorporated in the recently published genome sequence, due to the fact that regions with high repetitive DNA contents (e.g. satellite DNAs) are only partially included. Therefore, to fill these gaps and to gain information about the repeat composition of centromeres and heterochromatic regions, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using antibodies against the centromere-specific histone H3 variant of sugar beet (CenH3) and the heterochromatic mark of dimethylated lysine 9 of histone H3 (H3K9me2). Results ChIP-Seq analysis revealed that active centromeres containing CenH3 consist of the satellite pBV and the Ty3-gypsy retrotransposon Beetle7, while heterochromatin marked by H3K9me2 exhibits heterogeneity in repeat composition. H3K9me2 was mainly associated with the satellite family pEV, the Ty1-copia retrotransposon family Cotzilla and the DNA transposon superfamily of the En/Spm type. In members of the section Beta within the genus Beta, immunostaining using the CenH3 antibody was successful, indicating that orthologous CenH3 proteins are present in closely related species within this section. Conclusions The identification of repetitive genome portions by ChIP-Seq experiments complemented the sugar beet reference sequence by providing insights into the repeat composition of poorly characterized CenH3-chromatin and H3K9me2-heterochromatin. Therefore, our work provides the basis for future research and application concerning the sugar beet centromere and repeat rich heterochromatic regions characterized by the presence of H3K9me2

The retrotransposon landscape of the Beta vulgaris genome: Evolutionary conservation and diversity

Retrotransposons are major components of plant genomes influencing their genome size, organization and evolution. In the frame of this work, retrotransposons of the Beta vulgaris genome have been identified by molecular methods and whole genome bioinformatics approaches. Neither belonging to the rosids nor asterids, B. vulgaris (cultivated beet including sugar beet, beet root and mangold) is taxonomically placed at a key position at the root of the core eudicots, and considerably different from traditional plant model species such as thale cress or rice. Its genome has been sequenced, and annotation is under way. In order to compare different evolutionary lineages of B. vulgaris retrotransposons, long terminal repeat (LTR) and non-LTR retrotransposon family have been analyzed in detail. Full-length members have been isolated and characterized by bioinformatics, Southern and fluorescent in situ hybridization. Hallmarks of the LTR retrotransposon family Cotzilla are an additional env-like open reading frame (ORF), homogeneity of the members and the very high abundance. Most family members are evolutionarily young, and have most likely been created during recent bursts of amplification during species radiation. In contrast, the non-LTR retrotransposon family BNR has fewer copies and is much more diverged. Although the BNR ORF2 resembles previously analyzed long interspersed nuclear elements (LINEs) of the L1 clade, its ORF1 sequence differs strongly. It lacks the zinc finger domain described for plant LINEs, but contains instead an RNA recognition motif (RRM) likely to have an RNA-binding function. Database searches revealed the presence of similar LINE families in higher plant genomes such as poplar, lotus and soybean. Comparing their reverse transcriptase regions with other retrotransposons, these BNR-like LINEs form a separate group of L1 LINEs designated as BNR subclade. Availability of the B. vulgaris genome sequence allowed retrotransposon analyses on a genome-wide scale. A Hidden Markov Model-based detection algorithm has been developed in order to retrieve retrotransposon information directly from the database. Nearly 6000 B. vulgaris reverse transcriptase sequences have been isolated and classified into LTR retrotransposons of the Ty3-gypsy and Ty1-copia type, and non-LTR retrotransposons of the LINE type. As a result, a comprehensive overview of the retrotransposon spectrum of the B. vulgaris genome has been generated. Since plant LINEs have been only rarely investigated, the B. vulgaris LINE composition was studied in detail. Out of 28 described LINE clades, only members of the L1 and RTE clades have been identified. Based on a minimal shared sequence identity of 60 %, they form at least 17 L1 families and one RTE family. Full-length members of all investigated L1 families have been analyzed regarding their sequence, structure and diversity. In order to transfer the algorithm tested in B. vulgaris to other angiosperm genomes, twelve additional plant genomes have been queried for LINE reverse transcriptases. Key finding is the presence of only two LINE clades (L1 and RTE) in the analyzed genomes of higher plants. Whereas plant L1 LINEs are highly diverse and form at least seven subclades with members across species borders, RTE LINEs are extremely homogenized and constitute most likely only a single family per genome. In summary, this work’s results help to gain an understanding of the different strategies of retrotransposon evolution in plants, whereas the generated data directly contributes to the B. vulgaris genome annotation project.Retrotransposons sind eine wesentliche Komponente von Pflanzengenomen, die sowohl die Größe und Organisation als auch die Evolution dieser Genome wesentlich beeinflussen können. Im Rahmen dieser Arbeit wurden verschiedene Gruppen von Retrotransposons des Beta vulgaris Genoms mittels molekularer und bioinformatischer Methoden identifiziert. Innerhalb der dikotyledonen Blütenpflanzen gehört B. vulgaris (kultivierte Rübe einschließlich Zuckerrübe, Roter Beete und Mangold) weder zu den Rosiden noch zu den Asteriden, sondern nimmt eine Schlüsselposition innerhalb der Kerneudikotyledonen ein. Somit zeigt das Rübengenom wesentliche Unterschiede zu traditionellen Modellpflanzen wie Arabidopsis thaliana oder Oryza sativa. Das Genom ist bereits sequenziert, die Annotation jedoch noch nicht abgeschlossen. Um verschiedene evolutionäre Linien von B. vulgaris Retrotransposons vergleichend zu untersuchen wurden insbesondere Long Terminal Repeat (LTR)- und Non-LTR-Retrotransposon-Familien detailliert analysiert. Vollständige Mitglieder wurden isoliert und mittels bioinformatischer Methoden, Southern- und Fluoreszenz-in situ-Hybridisierung untersucht. Die LTR-Retrotransposon-Familie Cotzilla ist durch einen zusätzlichen env-ähnlichen offenen Leserahmen (ORF), Homogenität ihrer Mitglieder und eine hohe Abundanz gekennzeichnet. Die meisten Cotzilla-Kopien sind evolutionär jung und wurden wahrscheinlich innerhalb eines kurzen Zeitraumes während der Artentstehung stark amplifiziert. Im Gegensatz zur Cotzilla-Familie besitzt die Non-LTR-Retrotransposon-Familie BNR weniger Kopien und ist wesentlich divergierter. Während der BNR-spezifische ORF2 starke Ähnlichkeiten zu anderen pflanzlichen Long Interspersed Nuclear Elements (LINEs) der L1-Klade aufweist, unterscheidet sich der BNR ORF1 von diesen sehr stark. Im Gegensatz zu bereits beschrieben pflanzlichen LINEs kodiert er kein Zinkfingermotiv, sondern substituiert dieses durch ein RNA-Erkennungsmotiv (RRM). Durch Datenbanksuche konnten BNR-ähnliche LINEs in den Genomen höherer Pflanzen wie Soja, Lotus und Pappel identifiziert werden. Ein Vergleich der entsprechenden Reversen Transkriptasen (RT) mit den RTs anderer Retrotransposons zeigt, dass die BNR-ähnlichen LINEs eine separate Gruppe innerhalb der L1 LINEs bilden. Diese wurde daher als BNR-Subklade definiert. Die Untersuchung von Retrotransposons auf Genomebene wurde durch die B. vulgaris Genomsequenz ermöglicht. Um Retrotransposon-Informationen direkt aus dem Genom zu extrahieren, wurde ein Hidden Markov Modell (HMM)-basierter Detektions-algorithmus entwickelt. Annähernd 6000 B. vulgaris Reverse Transkriptase-Sequenzen konnten identifiziert und in LTR-Retrotransposons des Ty3-gypsy- beziehungsweise des Ty1-copia-Typs und in Non-LTR-Retrotransposons des LINE-Typs klassifiziert werden. Somit wurde ein umfassender Überblick über die Bandbreite der B. vulgaris Retrotransposons arhalten. Da pflanzliche LINEs bisher nur wenig erforscht sind, wurde die B. vulgaris LINE Zusammensetzung genauer untersucht. Von 28 beschriebenen LINE-Kladen konnten nur Mitglieder der L1- und der RTE-Klade identifiziert werden. Basierend auf einer Identität von mindestens 60 % bilden die Sequenzen 17 L1 Familien und eine RTE Familie. Vollständige Mitglieder aller L1 Familien wurden hinsichtlich ihrer Sequenz, Struktur und Diversität analysiert. Um den in B. vulgaris getesteten HMM-basierten Algorithmus auf andere Angiospermengenome zu übertragen, wurden zwölf weitere Pflanzengenome auf das Vorhandensein von LINE-spezifischen Reversen Transkriptasen untersucht. Wesentlichstes Ergebnis ist der Nachweis von nur zwei LINE-Kladen (L1 und RTE) in höheren Pflanzen. Während pflanzliche L1 LINEs hochgradig divers sind und über Artgrenzen hinaus mindestens sieben Subkladen mit Vertretern verschiedener Pflanzen bilden, sind RTE LINEs extrem homogenisiert und stellen höchstwahrscheinlich nur eine einzelne Familie pro Genom einer Art dar. Zusammenfassend ermöglichen die Ergebnisse dieser Arbeit eine Erweiterung des Verständnisses der unterschiedlichen Evolutionsstrategien von Retrotransposons in Pflanzen. Zusätzlich tragen die gewonnen Daten zur Annotation des B. vulgaris Genoms bei