|Stack, Stephen - COLORADO STATE|
|Royer, Susan - COLORADO STATE|
|Shearer, Lawrence - COLORADO STATE|
|Chang, Song-Bin - NATIONAL TAIWAN UNIV.|
|Westfall, Diane - COLORADO STATE|
|Anderson, Lorinda - COLORADO STATE|
Submitted to: Cytogenetics and Genome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 21, 2009
Publication Date: October 2, 2009
Citation: Stack, S., Royer, S., Shearer, L., Chang, S., Giovannoni, J.J., Westfall, D., Anderson, L. 2009. Role of fluorescence in situ hybridization (FISH) in sequencing the tomato genome. Cytogenetics and Genome Research. 124:339-350. Interpretive Summary: The tomato (Solanum lycopersicum) genome is being sequenced by a consortium of laboratories in ten countries. Seventy-seven percent of the tomato genome (DNA) is located in repeat-rich, gene-poor pericentric heterochromatin, while 23% of the genome is located in repeat-poor, gene-rich, distal euchromatin components of the tomato chromosomes, repsectively. It is estimated that if the sequencing effort can be limited to euchromatin, ~90% of tomato’s nuclear genes can be characterized without the problems involved in sequencing the majority of DNA in heterochromatin which contains relatively few genes. Sequencing is being performed on tomato nuclear DNA cloned into bacterial artificial chromosome (BAC) vectors. Fluorescence in situ hybridization (FISH) is used to help direct the sequencing effort by cytologically demonstrating that selected BACs are visibly located in euchromatin. Here we show the utility of FISH in localizing DNA to be sequenced in the euchromatic regions of tomato chromosomes.
Technical Abstract: Chromosomes at various stages of the cell cycle can be used for localization of DNA probes via Fluorescence in situ hybridization (FISH). While mitotic metaphase chromosomes are demonstrably too short and compact for this purpose, long pachytene chromosomes are ideal. BACs that hybridize to euchromatin are used with confidence as anchors for the assembly of contigs (series of overlapping BACs) that can be sequenced to describe super DNA pseudomolecules extending throughout the euchromatic length of whole chromosome arms. In addition, FISH is used to determine the order of nearby BACs and to identify BACs near telomeres and near borders with pericentric heterochromatin to alert sequencers that as contigs are extended, repeated sequences characteristic of heterochromatin will be encountered shortly. Because the DNA densities per unit length of euchromatin and heterochromatin in tomato pachytene chromosomes are known, the size of gaps between localized BACs and between localized BACs and chromosomal features, such as telomeres and euchromatin/heterochromatin borders, can be estimated in base pairs. Finally, it is noteworthy that when BAC-FISH is combined with chromosomal in situ suppression (CISS) hybridization to block repeats and localize single copy sequences, the great majority of BACs localize to single sites. This observation is consistent with tomato’s being an ancient diploid with few of the duplications and paralogous genes that should be common if a whole genome doubling event(s) had occurred in tomato’s currently interpretable history.