Sequence-based physical mapping of complex genomes by whole genome profiling.

We present whole genome profiling (WGP), a novel next-generation sequencing-based physical mapping technology for construction of bacterial artificial chromosome (BAC) contigs of complex genomes, using Arabidopsis thaliana as an example. WGP leverages short read sequences derived from restriction fragments of two-dimensionally pooled BAC clones to generate sequence tags. These sequence tags are assigned to individual BAC clones, followed by assembly of BAC contigs based on shared regions containing identical sequence tags. Following in silico analysis of WGP sequence tags and simulation of a map of Arabidopsis chromosome 4 and maize, a WGP map of Arabidopsis thaliana ecotype Columbia was constructed de novo using a six-genome equivalent BAC library. Validation of the WGP map using the Columbia reference sequence confirmed that 350 BAC contigs (98%) were assembled correctly, spanning 97% of the 102-Mb calculated genome coverage. We demonstrate that WGP maps can also be generated for more complex plant genomes and will serve as excellent scaffolds to anchor genetic linkage maps and integrate whole genome sequence data.

A hybrid BAC physical map of potato: a framework for sequencing a heterozygous genome.

BACKGROUND: Potato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so far been very limited. Here we present the methods of construction and the general statistics of the first two genome-wide BAC physical maps of potato, which were made from the heterozygous diploid clone RH89-039-16 (RH). RESULTS: First, a gel electrophoresis-based physical map was made by AFLP fingerprinting of 64478 BAC clones, which were aligned into 4150 contigs with an estimated total length of 1361 Mb. Screening of BAC pools, followed by the KeyMaps in silico anchoring procedure, identified 1725 AFLP markers in the physical map, and 1252 BAC contigs were anchored the ultradense potato genetic map. A second, sequence-tag-based physical map was constructed from 65919 whole genome profiling (WGP) BAC fingerprints and these were aligned into 3601 BAC contigs spanning 1396 Mb. The 39733 BAC clones that overlap between both physical maps provided anchors to 1127 contigs in the WGP physical map, and reduced the number of contigs to around 2800 in each map separately. Both physical maps were 1.64 times longer than the 850 Mb potato genome. Genome heterozygosity and incomplete merging of BAC contigs are two factors that can explain this map inflation. The contig information of both physical maps was united in a single table that describes hybrid potato physical map. CONCLUSIONS: The AFLP physical map has already been used by the Potato Genome Sequencing Consortium for sequencing 10% of the heterozygous genome of clone RH on a BAC-by-BAC basis. By layering a new WGP physical map on top of the AFLP physical map, a genetically anchored genome-wide framework of 322434 sequence tags has been created. This reference framework can be used for anchoring and ordering of genomic sequences of clone RH (and other potato genotypes), and opens the possibility to finish sequencing of the RH genome in a more efficient way via high throughput next generation approaches.

Physical mapping in highly heterozygous genomes: a physical contig map of the Pinot Noir grapevine cultivar.

BACKGROUND: Most of the grapevine (Vitis vinifera L.) cultivars grown today are those selected centuries ago, even though grapevine is one of the most important fruit crops in the world. Grapevine has therefore not benefited from the advances in modern plant breeding nor more recently from those in molecular genetics and genomics: genes controlling important agronomic traits are practically unknown. A physical map is essential to positionally clone such genes and instrumental in a genome sequencing project. RESULTS: We report on the first whole genome physical map of grapevine built using high information content fingerprinting of 49,104 BAC clones from the cultivar Pinot Noir. Pinot Noir, as most grape varieties, is highly heterozygous at the sequence level. This resulted in the two allelic haplotypes sometimes assembling into separate contigs that had to be accommodated in the map framework or in local expansions of contig maps. We performed computer simulations to assess the effects of increasing levels of sequence heterozygosity on BAC fingerprint assembly and showed that the experimental assembly results are in full agreement with the theoretical expectations, given the heterozygosity levels reported for grape. The map is anchored to a dense linkage map consisting of 994 markers. 436 contigs are anchored to the genetic map, covering 342 of the 475 Mb that make up the grape haploid genome. CONCLUSIONS: We have developed a resource that makes it possible to access the grapevine genome, opening the way to a new era both in grape genetics and breeding and in wine making. The effects of heterozygosity on the assembly have been analyzed and characterized by using several complementary approaches which could be easily transferred to the study of other genomes which present the same features.

A bacterial artificial chromosome-based genetic linkage map of the nematode Pristionchus pacificus.

To understand the evolution of developmental processes, nonmodel organisms in the nematodes, insects, and vertebrates are compared with established model systems. Often, these comparisons suffer from the inability to apply sophisticated technologies to these nonmodel species. In the nematode Pristionchus pacificus, cellular and genetic analyses are used to compare vulva development to that of Caenorhabditis elegans. However, substantial changes in gene function between P. pacificus and C. elegans limit the use of candidate gene approaches in studying P. pacificus mutations. To facilitate map-based cloning of mutations in P. pacificus, we constructed a BAC-based genetic linkage map. A BAC library of 13,440 clones was generated and completely end sequenced. By comparing BAC end and EST sequences between the "wild-type" strain P. pacificus var. California and the polymorphic strain P. pacificus var. Washington, 133 single-stranded conformational polymorphisms were identified. These markers were tested on a meiotic mapping panel of 46 randomly picked F(2) animals after a cross of the two strains, providing the first genetic linkage map of P. pacificus. A mapping strategy using two selected markers per chromosome was devised and the efficiency of this approach was illustrated by the mapping of the Ppa-unc-1/Twitchin gene.

TOPAAS, a tomato and potato assembly assistance system for selection and finishing of bacterial artificial chromosomes.

We have developed the software package Tomato and Potato Assembly Assistance System (TOPAAS), which automates the assembly and scaffolding of contig sequences for low-coverage sequencing projects. The order of contigs predicted by TOPAAS is based on read pair information; alignments between genomic, expressed sequence tags, and bacterial artificial chromosome (BAC) end sequences; and annotated genes. The contig scaffold is used by TOPAAS for automated design of nonredundant sequence gap-flanking PCR primers. We show that TOPAAS builds reliable scaffolds for tomato (Solanum lycopersicum) and potato (Solanum tuberosum) BAC contigs that were assembled from shotgun sequences covering the target at 6- to 8-fold coverage. More than 90% of the gaps are closed by sequence PCR, based on the predicted ordering information. TOPAAS also assists the selection of large genomic insert clones from BAC libraries for walking. For this, tomato BACs are screened by automated BLAST analysis and in parallel, high-density nonselective amplified fragment length polymorphism fingerprinting is used for constructing a high-resolution BAC physical map. BLAST and amplified fragment length polymorphism analysis are then used together to determine the precise overlap. Assembly onto the seed BAC consensus confirms the BACs are properly selected for having an extremely short overlap and largest extending insert. This method will be particularly applicable where related or syntenic genomes are sequenced, as shown here for the Solanaceae, and potentially useful for the monocots Brassicaceae and Leguminosea.

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.