Thomas Rosleff Sörensen

The genome of the recently domesticated crop plant sugar beet (Beta vulgaris)

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world’s annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714–758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant’s potential in energy biotechnology.

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction.

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

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.

Palaeohexaploid Ancestry for Caryophyllales Inferred from Extensive Gene-Based Physical and Genetic Mapping of the Sugar Beet Genome (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important crop plant that accounts for 30% of the worlds sugar production annually. The genus Beta is a distant relative of currently sequenced taxa within the core eudicotyledons; the genomic characterization of sugar beet is essential to make its genome accessible to molecular dissection. Here, we present comprehensive genomic information in genetic and physical maps that cover all nine chromosomes. Based on this information we identified the proposed ancestral linkage groups of rosids and asterids within the sugar beet genome. We generated an extended genetic map that comprises 1127 single nucleotide polymorphism markers prepared from expressed sequence tags and bacterial artificial chromosome (BAC) end sequences. To construct a genome-wide physical map, we hybridized gene-derived oligomer probes against two BAC libraries with 9.5-fold cumulative coverage of the 758 Mbp genome. More than 2500 probes and clones were integrated both in genetic maps and the physical data. The final physical map encompasses 535 chromosomally anchored contigs that contains 8361 probes and 22 815 BAC clones. By using the gene order established with the physical map, we detected regions of synteny between sugar beet (order Caryophyllales) and rosid species that involves 1400-2700 genes in the sequenced genomes of Arabidopsis, poplar, grapevine, and cacao. The data suggest that Caryophyllales share the palaeohexaploid ancestor proposed for rosids and asterids. Taken together, we here provide extensive molecular resources for sugar beet and enable future high-resolution trait mapping, gene identification, and cross-referencing to regions sequenced in other plant species.

Genome-wide identification and characterisation of R2R3-MYB genes in sugar beet ( Beta vulgaris )

BackgroundThe R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playing regulatory roles in plant-specific developmental processes, metabolite accumulation and defense responses. Although genome-wide analysis of this gene family has been carried out in some species, the R2R3-MYB genes in Beta vulgaris ssp. vulgaris (sugar beet) as the first sequenced member of the order Caryophyllales, have not been analysed heretofore.ResultsWe present a comprehensive, genome-wide analysis of the MYB genes from Beta vulgaris ssp. vulgaris (sugar beet) which is the first species of the order Caryophyllales with a sequenced genome. A total of 70 R2R3-MYB genes as well as genes encoding three other classes of MYB proteins containing multiple MYB repeats were identified and characterised with respect to structure and chromosomal organisation. Also, organ specific expression patterns were determined from RNA-seq data. The R2R3-MYB genes were functionally categorised which led to the identification of a sugar beet-specific clade with an atypical amino acid composition in the R3 domain, putatively encoding betalain regulators. The functional classification was verified by experimental confirmation of the prediction that the R2R3-MYB gene Bv_iogq encodes a flavonol regulator.ConclusionsThis study provides the first step towards cloning and functional dissection of the role of MYB transcription factor genes in the nutritionally and evolutionarily interesting species B. vulgaris. In addition, it describes the flavonol regulator BvMYB12, being the first sugar beet R2R3-MYB with an experimentally proven function.

Reliable in silico identification of sequence polymorphisms and their application for extending the genetic map of sugar beet (Beta vulgaris).

Molecular markers are a highly valuable tool for creating genetic maps. Like in many other crops, sugar beet (Beta vulgaris L.) breeding is increasingly supported by the application of such genetic markers. Single nucleotide polymorphism (SNP) based markers have a high potential for automated analysis and high-throughput genotyping. We developed a bioinformatics workflow that uses Sanger and 2nd-generation sequence data for detection, evaluation and verification of new transcript-associated SNPs from sugar beet. RNAseq data from one parent of an established mapping population were produced by 454-FLX sequencing and compared to Sanger ESTs derived from the other parent. The workflow established for SNP detection considers the quality values of both types of reads, provides polymorphic alignments as well as selection criteria for reliable SNP detection and allows painless generation of new genetic markers within genes. We obtained a total of 14,323 genic SNPs and InDels. According to empirically optimised settings for the quality parameters, we classified these SNPs into four usability categories. Validation of a subset of the in silico detected SNPs by genotyping the mapping population indicated a high success rate of the SNP detection. Finally, a total of 307 new markers were integrated with existing data into a new genetic map of sugar beet which offers improved resolution and the integration of terminal markers.

A De Novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny

Arabidopsis thaliana is the most important model organism for fundamental plant biology. The genome diversity of different accessions of this species has been intensively studied, for example in the 1001 genome project which led to the identification of many small nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). In addition, presence/absence variation (PAV), copy number variation (CNV) and mobile genetic elements contribute to genomic differences between A. thaliana accessions. To address larger genome rearrangements between the A. thaliana reference accession Columbia-0 (Col-0) and another accession of about average distance to Col-0, we created a de novo next generation sequencing (NGS)-based assembly from the accession Niederzenz-1 (Nd-1). The result was evaluated with respect to assembly strategy and synteny to Col-0. We provide a high quality genome sequence of the A. thaliana accession (Nd-1, LXSY01000000). The assembly displays an N50 of 0.590 Mbp and covers 99% of the Col-0 reference sequence. Scaffolds from the de novo assembly were positioned on the basis of sequence similarity to the reference. Errors in this automatic scaffold anchoring were manually corrected based on analyzing reciprocal best BLAST hits (RBHs) of genes. Comparison of the final Nd-1 assembly to the reference revealed duplications and deletions (PAV). We identified 826 insertions and 746 deletions in Nd-1. Randomly selected candidates of PAV were experimentally validated. Our Nd-1 de novo assembly allowed reliable identification of larger genic and intergenic variants, which was difficult or error-prone by short read mapping approaches alone. While overall sequence similarity as well as synteny is very high, we detected short and larger (affecting more than 100 bp) differences between Col-0 and Nd-1 based on bi-directional comparisons. The de novo assembly provided here and additional assemblies that will certainly be published in the future will allow to describe the pan-genome of A. thaliana.

Genetics of flowering time in grapevine

The project is dealing with the improvement of a universal pneumatic seeder which is suitable for sowing a wide range of different seeds in order to decrease particulate emissions of seed dressings. Aim of the project is to identify leakages in the pneumatic system in order to develop opportunities for the subsequent improvement of existing equipment to fulfill the high demands of user and environmental protection. Pneumatic seeders are characterized by a central, funnelshaped hopper. The batch feeder being an airlock and dosing feeder is located in the outlet of the seeder. The metered seed is fed into the air stream and transported to the sowing distributor by a conveying air stream.Due to climatic change, phenology traits are becoming increasingly important in grapevine breeding, since a premature flowering and ripening time could be observed for grapevine in the last decades. However, knowledge about these traits is still limited as they are genetically very complex and highly influenced by environmental factors. The analysis of the genetic basis of flowering time therefore will enable the development of tightly linked molecular markers useful for markerassisted selection of especially late flowering breeding lines.One recently occurred invasive insect pest has caught the attention for investigating biological control mechanisms and systems: The spotted wing drosophila (SWD, Drosophila suzukii Matsumura) is endemic in East China and Japan but has been introduced to the western hemisphere about 10 years ago and has been found in Europe since 2009. Nowadays, it has emerged to one of the most harmful pests to commercially grown fruit plants like stone fruits and nearly all kind of berries while it prefers ripe and overripe fruits. Our intention is to investigate the possible usage of natural antagonists for biological control. Therefore, we examine the natural load of parasites and pathogens (i.e. fungi, bacteria, viruses, microsporidia and protista) in fruit flies, isolate them and re-infect lab populations of D. suzukii for investigating the antagonistic potential. Furthermore, we will integrate the fruit pest codling moth (Cydia pomonella), which is an ongoing problem in apple orchards also because the pest develops resistance against commercially available insecticides. The long-term aim is to establish a stable system for pathogen detection that can be used for rapid identification of microbial antagonists in natural populations.Two main problems have to be considered in the development of control agents against the Fire Blight pathogen Erwinia amylovora: First, the ability of exponential growth leads to high cell densities in a short amount of time. Second, the most critical phase of Fire Blight infection occurs during blossoming, when the pathogen is transported to open flowers by various insects. To prevent infection, it is important to avoid invasion inside the plants tissue by interfering with growth of E. amylovora cells.The entomopathogenic fungus Isaria fumosorosea, formerly known as Paecilomyces fumosoroseus, has got a relatively wide host range. Within the scope of the EU project BIOCOMES investigations were done to validate the use of I. fumosorosea as a BCA against several pest insects. Under laboratory conditions it could be shown that I. fumosorosea seems to be a suitable BCA against Bemisia tabaci (silverleaf whitefly) and Spodoptera exigua (beet armyworm).Since the last two decades a number of fingerprinting methods have been developed to analyze microbial communities and their dynamics, including Terminal Restriction Fragment Length Polymorphism (T-RFLP), Length Heterogeneity-Polymerase Chain Reaction (LHPCR) and Automated Ribosomal Intergenic Spacer Analysis (ARISA). Because the latter provides a quick and cheap way together with high accuracy, we have chosen this method to investigate the fungal communities on grapevine, wood, leaves and berries.Saoussen Ben Tiba, Andreas Larem, Eva Fritsch, Karin Undorf-Spahn, Asma Laarif, Sami Fattouch, Johannes A. Jehle 1 Julius Kuhn-Institut, Institute for Biological Control, Darmstadt, Germany 2 Regional Center for Agriculture and Biological Agriculture, Chott Meriem, Tunisia 3 National Institute for applied technological Science Tunis (INSAT), Carthage University, Tunisia Email of corresponding author: saoussen.ben.tiba@jki.bund.deOne promising approach to a more environmentally friendly viticulture is growing fungus resistant cultivars with the novel cultivation method of the minimal pruning of trellis trained grapevines (MPTS). This practice reduces the tremendous amounts of fungicides needed to protect traditional cultivars and is expected to increase biodiversity compared to vineyards with traditional trellis trained grapevines (TS).Meiosis as the specialized cell division of sexual reproduction plays a crucial role in the exchange and reorganization of genetic material between two individuals by dividing the chromosome set in half and forming gametes. Even though in the last years major findings in the field of meiosis have been achieved, especially in plants, some key questions remain concealed. For a proper meiosis the initiation of double strand breaks (DSBs) during early prophase I is essential. Without DSBs no physical connection can occur between homologous chromosomes and recombination, pairing, and crossing over are excluded. So far in all analyzed eukaryotes SPO11, a meiosis specific transesterase, is the key enzyme inducing DSBs. But other than in animals and fungi where a single SPO11 is sufficient, plants need at least two different SPO11, referred to as SPO11-1 and SPO11-2, for proper meiosis. In Arabidopsis thaliana both have crucial functions and are essential in a functional form for the induction of meiotic DSBs as single knock out mutants are leading to near sterility by random chromosome distribution. Despite the same function of the homologs SPO111 and -2, the identity between both proteins is quite low. Homology of the orthologous SPO11 from different organisms is much higher. By exchanging SPO11-1 and -2 in Arabidopsis by their orthologs from various organisms we could demonstrate a species specific function of each SPO11, as a functional complementation of sterility could only be achieved with SPO11 from closely related species from the Brassicaceae. By exchanging non conserved regions between SPO11-1 and -2 of Arabidopsis we additionally could show a sequence specific function for each SPO11, as a functional rescue could not be achieved with all chosen regions. Interestingly, we could reveal a specific pattern of aberrant spliced isoforms for each SPO11 which are also sequence as well as species specific. By producing antibodies against AthSPO11-1 and -2 we were able to analyze for the first time the binding of SPO11-2 onto the DNA and perform co-immunolocalization studies with SPO11-1 and -2.European Stone Fruit Yellows (ESFY) is one of the most serious diseases in European fruit production. Infected Prunus cultivars yield poorly and lead to high economic losses. ESFY is caused by a specialized bacterium located in the phloem tissue of Prunus ssp., the Phytoplasma ‘Candidatus Phytoplasma prunorum’. It is spread by the phloemfeeding plum psyllid (Cacopsylla pruni) which acquires the bacterium by feeding on infected plants and is able to transmit it to healthy plants.Leaf rust caused by Puccinia triticina can cause yield losses up to 60 % and is the most common rust disease of wheat in the world. Vertical leaf rust resistance genes (Lr-genes) have been introduced in cultivars. Many of these resistances are broken down by virulent pathotypes. Horizontal resistances which are independent from races of a pathogen are known but show a quantitative characteristic which is carried by a few cultivars.