Resistances to begomoviruses, including bipartite tomato mottle virus and monopartite tomato yellow leaf curl virus (TYLCV), have been introgressed to cultivated tomato (Solanum lycopersicum) from wild tomato accessions. A major gene, Ty-2 from S. habrochaites f. glabratum accession “B6013,” that confers resistance to TYLCV was previously mapped to a 19-cM region on the long arm of chromosome 11. In the present study, approximately 11,000 plants were screened and nearly 157 recombination events were identified between the flanking markers C2_At1g07960 (82.5 cM, physical distance 51.387 Mb) and T0302 (89 cM, 51.878 Mb). Molecular marker analysis of recombinants and TYLCV evaluation of progeny from these recombinants localized Ty-2 to an approximately 300,000-bp interval between markers UP8 (51.344 Mb) and M1 (51.645 Mb). No recombinants were identified between TG36 and C2_At3g52090, a region of at least 115 kb, indicating severe recombination suppression in this region. Due to the small interval, fluorescence in situ hybridization analysis failed to clarify whether recombination suppression is caused by chromosomal rearrangements. Candidate genes predicted based on tomato genome annotation were analyzed by RT-PCR and virus-induced gene silencing. Results indicate that the NBS gene family present in the Ty-2 region is likely not responsible for the Ty-2-conferred resistance and that two candidate genes might play a role in the Ty-2-conferred resistance. Several markers very tightly linked to the Ty-2 locus are presented and useful for marker-assisted selection in breeding programs to introgress Ty-2 for begomovirus resistance.

Chromosomal rearrangements are relatively rare evolutionary events and can be used as markers to study karyotype evolution. This research aims to use such rearrangements to study chromosome evolution in Solanum. Chromosomal rearrangements between Solanum crops and several related wild species were investigated using tomato and potato bacterial artificial chromosomes (BACs) in a multicolour fluorescent in situ hybridization (FISH). The BACs selected are evenly distributed over seven chromosomal arms containing inversions described in previous studies. The presence/absence of these inversions among the studied Solanum species were determined and the order of the BAC-FISH signals was used to construct phylogenetic trees.Compared with earlier studies, data from this study provide support for the current grouping of species into different sections within Solanum; however, there are a few notable exceptions, such as the tree positions of S. etuberosum (closer to the tomato group than to the potato group) and S. lycopersicoides (sister to S. pennellii). These apparent contradictions might be explained by interspecific hybridization events and/or incomplete lineage sorting. This cross-species BAC painting technique provides unique information on genome organization, evolution and phylogenetic relationships in a wide variety of species. Such information is very helpful for introgressive breeding.

We have analysed the structural homology in euchromatin regions of tomato, potato and pepper with special attention for the long arm of chromosome 2 (2L). Molecular organization and colinear junctions were delineated using multi-color BAC FISH analysis and comparative sequence alignment. We found large-scale rearrangements including inversions and segmental translocations that were not reported in previous comparative studies. Some of the structural rearrangements are specific for the tomato clade, and differentiate tomato from potato, pepper and other Solanaceous species. Although local gene vicinity is largely preserved, there are many small-scale synteny perturbations. Gene adjacency in the aligned segments was frequently disrupted for 47% of the ortholog pairs as a result of gene and LTR retrotransposon insertions, and occasionally by single gene inversions and translocations. Our data also suggests that long distance intra-chromosomal rearrangements and local gene rearrangements have evolved frequently during speciation in the Solanum genus, and that small changes are more prevalent than large-scale differences. The occurrence of sonata and harbinger transposable elements and other repeats near or at junction breaks is considered in the light of repeat-mediated rearrangements and a reconstruction scenario for an ancestral 2L topology is discussed.

Tomato yellow leaf curl disease, a devastating disease of Solanum lycopersicum (tomato), is caused by a complex of begomoviruses generally referred to as Tomato yellow leaf curl virus (TYLCV). Almost all breeding for TYLCV resistance has been based on the introgression of the Ty-1 resistance locus derived from Solanum chilense LA1969. Knowledge about the exact location of Ty-1 on tomato chromosome 6 will help in understanding the genomic organization of the Ty-1 locus. In this study, we analyze the chromosomal rearrangement and recombination behavior of the chromosomal region where Ty-1 is introgressed. Nineteen markers on tomato chromosome 6 were used in F2 populations obtained from two commercial hybrids, and showed the presence of a large introgression in both. Fluorescence in situ hybridization (FISH) analysis revealed two chromosomal rearrangements between S. lycopersicum and S. chilense LA1969 in the Ty-1 introgression. Furthermore, a large-scale recombinant screening in the two F2 populations was performed, and 30 recombinants in the Ty-1 introgression were identified. All recombination events were located on the long arm beyond the inversions, showing that recombination in the inverted region was absent. Disease tests on progenies of informative recombinants with TYLCV mapped Ty-1 to the long arm between markers MSc05732-4 and MSc05732-14, an interval overlapping with the reported Ty-3 region, which led to the indication that Ty-1 and Ty-3 may be allelic. With this study we prove that FISH can be used as a diagnostic tool to aid in the accurate mapping of genes that were introgressed from wild species into cultivated tomato.

The genetic complexity in the genus Musa has been subject of study in many breeding programs worldwide. Parthenocarpy, female sterility, polyploidy in different cultivars and limited amount of genetic and genomic information make the production of new banana cultivars difficult and time consuming. In addition, it is known that part of the cultivars and related wild species in the genus contain numerous chromosomal rearrangements. In order to produce new cultivars more effectively breeders must better understand the genetic differences of the potential crossing parents for introgression hybridization, but extensive genetic information is lacking. As an alternative to achieve information on genetic collinearity we make use of modern chromosome map technology known as high-resolution fluorescent in situ hybridization (FISH). This article presents the technical aspects and applications of such a technology in Musa species. The technique deals with BAC clone positioning on pachytene chromosomes of Calcutta 4 (Musa acuminata ssp. burmanicoides, A genome group, section Eumusa) and M. velutina (section Rodochlamys). Pollen mother cells digestion with pectolytic enzymes and maceration with acetic acid were optimized for making cell spread preparations appropriate for FISH. As an example of this approach we chose BAC clones that contain markers to known resistance genes and hybridize them for establishing their relative positions on the two species. Technical challenges for adapting existing protocols to the banana cells are presented. We also discuss how this technique can be instrumental for validating collinearity between potential crossing parents and how the method can be helpful in future mapping initiatives, and how this method allows identification of chromosomal rearrangements between related Musa species and cultivars.

We studied the physical and genetic organization of chromosome 6 of tomato (Solanum lycopersicum) cv. Heinz 1706 by combining bacterial artificial chromosome (BAC) sequence analysis, high-information-content fingerprinting, genetic analysis, and BAC-fluorescent in situ hybridization (FISH) mapping data. The chromosome positions of 81 anchored seed and extension BACs corresponded in most cases with the linear marker order on the high-density EXPEN 2000 linkage map. We assembled 25 BAC contigs and eight singleton BACs spanning 2.0 Mb of the short-arm euchromatin, 1.8 Mb of the pericentromeric heterochromatin and 6.9 Mb of the long-arm euchromatin. Sequence data were combined with their corresponding genetic and pachytene chromosome positions into an integrated map that covers approximately a third of the chromosome 6 euchromatin and a small part of the pericentromeric heterochromatin. We then compared physical length (Mb), genetic (cM) and chromosome distances (microm) for determining gap sizes between contigs, revealing relative hot and cold spots of recombination. Through sequence annotation we identified several clusters of functionally related genes and an uneven distribution of both gene and repeat sequences between heterochromatin and euchromatin domains. Although a greater number of the non-transposon genes were located in the euchromatin, the highly repetitive (22.4%) pericentromeric heterochromatin displayed an unexpectedly high gene content of one gene per 36.7 kb. Surprisingly, the short-arm euchromatin was relatively rich in repeats as well, with a repeat content of 13.4%, yet the ratio of Ty3/Gypsy and Ty1/Copia retrotransposable elements across the chromosome clearly distinguished euchromatin (2:3) from heterochromatin (3:2).

The genome of tomato (Solanum lycopersicum L.) 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 the larger “International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation” initiative. The tomato genome sequencing project uses an ordered bacterial artificial chromosome (BAC) approach to generate a high‐quality tomato euchromatic genome sequence for use as a reference genome for the Solanaceae and euasterids. Sequence is deposited at GenBank and at the SOL Genomics Network (SGN). Currently, there are around 1000 BACs finished or in progress, representing more than a third of the projected euchromatic portion of the genome. An annotation effort is also underway by the International Tomato Annotation Group. The expected number of genes in the euchromatin is ∼40,000, based on an estimate from a preliminary annotation of 11% of finished sequence. Here, we present this first snapshot of the emerging tomato genome and its annotation, a short comparison with potato (Solanum tuberosum L.) sequence data, and the tools available for the researchers to exploit this new resource are also presented. In the future, whole‐genome shotgun techniques will be combined with the BAC‐by‐BAC approach to cover the entire tomato genome. The high‐quality reference euchromatic tomato sequence is expected to be near completion by 2010.

This paper presents a bird's-eye view of the major repeats and chromatin types of tomato. Using fluorescence in-situ hybridization (FISH) with Cot-1, Cot-10 and Cot-100 DNA as probes we mapped repetitive sequences of different complexity on pachytene complements. Cot-100 was found to cover all heterochromatin regions, and could be used to identify repeat-rich clones in BAC filter hybridization. Next we established the chromosomal locations of the tandem and dispersed repeats with respect to euchromatin, nucleolar organizer regions (NORs), heterochromatin, and centromeres. The tomato genomic repeats TGRII and TGRIII appeared to be major components of the pericentromeres, whereas the newly discovered TGRIV repeat was found mainly in the structural centromeres. The highly methylated NOR of chromosome 2 is rich in [GACA](4), a microsatellite that also forms part of the pericentromeres, together with [GA](8), [GATA](4) and Ty1-copia. Based on the morphology of pachytene chromosomes and the distribution of repeats studied so far, we now propose six different chromatin classes for tomato: (1) euchromatin, (2) chromomeres, (3) distal heterochromatin and interstitial heterochromatic knobs, (4) pericentromere heterochromatin, (5) functional centromere heterochromatin and (6) nucleolar organizer region.

Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC-FISH as a unique tool for comparative genetic studies across Solanum species.

Within the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver high-quality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.