Role of Fluorescence in situ Hybridization in Sequencing the Tomato Genome

Abstract

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.

Integration of Genetic, Physical, and Cytogenetic Maps for <i>Brassica rapa</i> Chromosome A7

“…Our study is similar to those that integrated genetic, physical, and cytogenetic maps of individual chromosomes using BAC-FISH in Sorghum Kim et al, 2002], rice [Cheng et al, 2001;Tang et al, 2007], maize [Danilova and Birchler, 2008], Brachypodium [Hasterok et al, 2004], Arabidopsis [Jackson et al, 1998[Jackson et al, , 2000Lysak et al, 2010], Brassica [Howell et al, 2002[Howell et al, , 2005[Howell et al, , 2008Kim et al, 2009], Solanum [Iovene et al, 2008;Tang et al, 2008Tang et al, , 2009Stack et al, 2009], and various genera of legumes [Kulikova et al, 2001;Pagel et al, 2004;Walling et al, 2006;Pedrosa-Harand et al, 2009]. In addition, our study is similar to those that used BAC-FISH across species (comparative chromosome painting or CCP) [reviewed in Lysak, 2009 andLysak et al, 2010].…”

“…Stack et al, 2009, regarding tomato BAC-FISH; and de Jong, pers. commun., regarding potato BAC-FISH].…”

“…BAC-FISH has played a role in sequencing the genomes of A. thaliana, Oryza sativa, Medicago truncatula [e.g. Fransz et al, 1998;Cheng et al, 2001;Kulikova et al, 2001] to determine not only if BACs are located on the expected chromosome, but also to prioritize sequencing efforts if BACs are near telomeres or pericentric heterochromatin as is being done with the tomato genome sequencing project [Stack et al, 2009]. Even if future genome sequencing technology no longer requires BAC libraries, markerbased FISH studies will still play an important role in studies of comparative genomics, evaluating recombination and chromosome behavior.…”

“…The only published BAC-FISH map integration studies in the asterids have been with tomatoes and potatoes (Solanaceae) [Iovene et al, 2008;Tang et al, 2008Tang et al, , 2009Stack et al, 2009]. However, among rosids, there have been numerous studies in legumes (Fabaceae) [Kulikova et al 2001;Walling et al, 2006;PedrosaHarand, 2009], cotton (Malvaceae) [Wang et al, 2007a[Wang et al, , b, c, 2008Guo et al, 2008], Populus (Salicaceae) [IslamFaridi et al, 2009], Poncirus (Rutaceae) [Moraes et al, 2008], cucurbits (Cucurbitaceae) [Han et al, 2009], and mustards (Brassicaceae).…”

FluorescenceIn SituHybridization and Optical Mapping to Correct Scaffold Arrangement in the Tomato Genome

“…Tomato SC spreads were prepared as described previously (Stack et al 2009; Stack and Anderson 2009). Briefly, primary microsporocytes in pachytene were squeezed out of anthers, and cell walls were removed with cytohelicase.…”