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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Plant, Soil and Nutrition Research » Research » Publications at this Location » Publication #322421

Research Project: Genetic and Genomic Basis of Vegetable and Fruit Biology, Quality and Nutrient Content

Location: Plant, Soil and Nutrition Research

Title: Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome

item SHEARER, LINDSAY - Colorado State University
item ANDERSON, LORINDA - Colorado State University
item DE JONG, HANS - University Of Wageningen
item SMIT, SANDRA - University Of Wageningen
item GOICOECHEA, JOSE LUIS - Boyce Thompson Institute
item Giovannoni, James
item STACK, STEPHEN - Colorado State University

Submitted to: Genes, Genomes, and Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/12/2014
Publication Date: 12/21/2014
Citation: Shearer, L., Anderson, L., De Jong, H., Smit, S., Goicoechea, J., Giovannoni, J.J., Stack, S. 2014. Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome. Genes, Genomes, and Genomics. 4:1395-1405.

Interpretive Summary: The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome–fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.

Technical Abstract: Modern biological analyses are often assisted by recent technologies making the sequencing of complex genomes both technically possible and feasible. We recently sequenced the tomato genome that, like many eukaryotic genomes, is large and complex. Current sequencing technologies allow the development of what might best be termed as complete puzzle pieces that still require a method for properly ordering and organizing them into a more complete “picture’ of the genome. The tomato genome was organized through the use of available high resolution maps on which marker sequences were anchored to the corresponding genome sequences so as to order and align the fragments of sequence resulting from DNA sequencing into sequenced molecules corresponding to the 12 tomato chromosomes. Such ordering and alignments are only as good as the genetic maps on which they are based. Here we demonstrate through hybridization and alternative (optical) mapping techniques that much of the original sequence order of the tomato genome is incorrect. This is especially in region of the genome where there is little genetic recombination and the genetic map is thus less robust. Fortunately, this is also reflects regions of the genome with the least genes (centromeres and paracentric heterochromatin) but dies point to an issue with genome assembly quality that is likely to persist through many eukaryotic genomes as most are ordered based on genetic maps.