René Klein Lankhorst

Complete genome sequence of Lactobacillus plantarum WCFS1

The 3,308,274-bp sequence of the chromosome of Lactobacillus plantarum strain WCFS1, a single colony isolate of strain NCIMB8826 that was originally isolated from human saliva, has been determined, and contains 3,052 predicted protein-encoding genes. Putative biological functions could be assigned to 2,120 (70%) of the predicted proteins. Consistent with the classification of L. plantarum as a facultative heterofermentative lactic acid bacterium, the genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, all of which appear to belong to the class of potentially highly expressed genes in this organism, as was evident from the codon-adaptation index of individual genes. Moreover, L. plantarum encodes a large pyruvate-dissipating potential, leading to various end-products of fermentation. L. plantarum is a species that is encountered in many different environmental niches, and this flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 25 complete PTS sugar transport systems. Moreover, the chromosome encodes >200 extracellular proteins, many of which are predicted to be bound to the cell envelope. A large proportion of the genes encoding sugar transport and utilization, as well as genes encoding extracellular functions, appear to be clustered in a 600-kb region near the origin of replication. Many of these genes display deviation of nucleotide composition, consistent with a foreign origin. These findings suggest that these genes, which provide an important part of the interaction of L. plantarum with its environment, form a lifestyle adaptation region in the chromosome.

The Tomato Sequencing Project, the First Cornerstone of the International Solanaceae Project (SOL)

The genome of tomato (Solanum lycopersicum) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, The Netherlands, France, Japan, Spain, Italy and the United States) as part of a larger initiative called the ‘International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation’. The goal of this grassroots initiative, launched in November 2003, is to establish a network of information, resources and scientists to ultimately tackle two of the most significant questions in plant biology and agriculture: (1) How can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet? (2) How can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? The Solanaceae and closely related species such as coffee, which are included in the scope of the SOL project, are ideally suited to address both of these questions. The first step of the SOL project is to use an ordered BAC approach to generate a high quality sequence for the euchromatic portions of the tomato as a reference for the Solanaceae. Due to the high level of macro and micro-synteny in the Solanaceae the BAC-by-BAC tomato sequence will form the framework for shotgun sequencing of other species. The starting point for sequencing the genome is BACs anchored to the genetic map by overgo hybridization and AFLP technology. The overgos are derived from approximately 1500 markers from the tomato high density F2-2000 genetic map (http://sgn.cornell.edu/). These seed BACs will be used as anchors from which to radiate the tiling path using BAC end sequence data. Annotation will be performed according to SOL project guidelines. All the information generated under the SOL umbrella will be made available in a comprehensive website. The information will be interlinked with the ultimate goal that the comparative biology of the Solanaceae—and beyond—achieves a context that will facilitate a systems biology approach.

The genome of the stress-tolerant wild tomato species Solanum pennellii

Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm.

Cross-Species Bacterial Artificial Chromosome–Fluorescence in Situ Hybridization Painting of the Tomato and Potato Chromosome 6 Reveals Undescribed Chromosomal Rearrangements

Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC–FISH as a unique tool for comparative genetic studies across Solanum species.

FISH mapping and molecular organization of the major repetitive sequences of tomato

This paper presents a bird’s-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA]4, a microsatellite that also forms part of the pericentromeres, together with [GA]8, [GATA]4 and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.

Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice

Apomixis in plants is a form of clonal reproduction through seeds. A BAC clone linked to apomictic reproduction in Paspalum simplex was used to locate the apomixis locus on meiotic chromosome preparations. Fluorescent in situ hybridisation revealed the existence of a single locus embedded in a heterochromatin-poor region not adjacent to the centromere. We report here for the first time information regarding the sequencing of a large DNA clone from the apomixis locus. The presence of two genes whose rice homologs were mapped on the telomeric part of the long arm of rice chromosome 12 confirmed the strong synteny between the apomixis locus of P.simplex with the related area of the rice genome at the map level. Comparative analysis of this region with rice as representative of a sexual species revealed large-scale rearrangements due to transposable elements and small-scale rearrangements due to deletions and single point mutations. Both types of rearrangements induced the loss of coding capacity of large portions of the “apomictic” genes compared to their rice homologs. Our results are discussed in relation to the use of rice genome data for positional cloning of apomixis genes and to the possible role of rearranged supernumerary genes in the apomictic process of P.simplex.

Large-Scale Gene Discovery in the Septoria Tritici Blotch Fungus Mycosphaerella graminicola with a Focus on In Planta Expression

The foliar disease septoria tritici blotch, caused by the fungus Mycosphaerella graminicola, is currently the most important wheat disease in Europe. Gene expression was examined under highly different conditions, using 10 expressed sequence tag libraries generated from M. graminicola isolate IPO323 using seven in vitro and three in planta growth conditions. To identify fungal clones in the interaction libraries, we developed a selection method based on hybridization with the entire genomic DNA of M. graminicola, to selectively enrich these libraries for fungal genes. Assembly of the 27,007 expressed sequence tags resulted in 9,190 unigenes, representing 5.2 Mb of the estimated 39-Mb genome size of M. graminicola. All libraries contributed significantly to the number of unigenes, especially the in planta libraries representing different stages of pathogenesis, which covered 15% of the library-specific unigenes. Even under presymptomatic conditions (5 days postinoculation), when fungal biomass is less than 5%, this method enabled us to efficiently capture fungal genes expressed during pathogenesis. Many of these genes were uniquely expressed in planta, indicating that in planta gene expression significantly differed from in vitro expression. Examples of gene discovery included a number of cell wall-degrading enzymes, a broad set of genes involved in signal transduction (n=11) and a range of ATP-binding cassette (n=20) and major facilitator superfamily transporter genes (n=12) potentially involved in protection against antifungal compounds or the secretion of pathogenicity factors. In addition, evidence is provided for a mycovirus in M. graminicola that is highly expressed under various stress conditions, in particular, under nitrogen starvation. Our analyses provide a unique window on in vitro and in planta gene expression of M. graminicola.

Solanum lycopersicum cv. Heinz 1706 chromosome 6: distribution and abundance of genes and retrotransposable elements

We studied the physical and genetic organization of chromosome 6 of tomato (Solanum lycopersicum) cv. Heinz 1706 by combining bacterial artificial chromosome (BAC) sequence analysis, high-information-content fingerprinting, genetic analysis, and BAC-fluorescent in situ hybridization (FISH) mapping data. The chromosome positions of 81 anchored seed and extension BACs corresponded in most cases with the linear marker order on the high-density EXPEN 2000 linkage map. We assembled 25 BAC contigs and eight singleton BACs spanning 2.0 Mb of the short-arm euchromatin, 1.8 Mb of the pericentromeric heterochromatin and 6.9 Mb of the long-arm euchromatin. Sequence data were combined with their corresponding genetic and pachytene chromosome positions into an integrated map that covers approximately a third of the chromosome 6 euchromatin and a small part of the pericentromeric heterochromatin. We then compared physical length (Mb), genetic (cM) and chromosome distances (microm) for determining gap sizes between contigs, revealing relative hot and cold spots of recombination. Through sequence annotation we identified several clusters of functionally related genes and an uneven distribution of both gene and repeat sequences between heterochromatin and euchromatin domains. Although a greater number of the non-transposon genes were located in the euchromatin, the highly repetitive (22.4%) pericentromeric heterochromatin displayed an unexpectedly high gene content of one gene per 36.7 kb. Surprisingly, the short-arm euchromatin was relatively rich in repeats as well, with a repeat content of 13.4%, yet the ratio of Ty3/Gypsy and Ty1/Copia retrotransposable elements across the chromosome clearly distinguished euchromatin (2:3) from heterochromatin (3:2).