Role of Fluorescence in situ Hybridization in Sequencing the Tomato GenomeStack S.M.a, b · Royer S.M.a · Shearer L.A.a · Chang S.B.d · Giovannoni J.J.c · Westfall D.H.a · White R.A.c · Anderson L.K.a, b
aDepartment of Biology and bProgram in Molecular Plant Biology, Colorado State University, Fort Collins, Colo.; cUSDA-ARS Robert Holley Center and Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, N.Y., USA; dDepartment of Agronomy, National Taiwan University, Taipei, Taiwan
Stephen M. Stack
Department of Biology, Colorado State University
Fort Collins, CO 80523-1878 (USA)
Tel. +1 970 491 6802, Fax +1 970 491 0649
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The tomato (Solanum lycopersicum L.) genome is being sequenced by a consortium of laboratories in 10 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. It is estimated that approximately 90% of tomato’s nuclear genes can be characterized by limiting the sequencing effort to euchromatin while avoiding the problems involved in sequencing the repetitive DNA in heterochromatin. 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 the location of selected BACs on tomato chromosomes. While mitotic metaphase chromosomes are too short and compact for this purpose, long pachytene chromosomes are ideal. BACs localized in euchromatin can be used confidently as anchors for the assembly of BAC contigs that extend through the euchromatic length of each chromosome arm. Another important role for FISH is identification of BACs near telomeres and near borders with pericentric heterochromatin to indicate that sequencing should not extend much further. This role of FISH is enhanced by our ability to estimate base pair distances between localized BACs and these chromosomal features. Finally, it is noteworthy that when BAC-FISH is combined with chromosomal in situ suppression (CISS) hybridization to block repeats and localize single/low copy sequences, the great majority of BACs localize to single sites. This observation is consistent with tomato being an ancient diploid.
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