Genetic mapping of a major dominant gene for resistance to Ralstonia solanacearum in eggplant.

Resistance of eggplant against Ralstonia solanacearum phylotype I strains was assessed in a F(6) population of recombinant inbred lines (RILs) derived from a intra-specific cross between S. melongena MM738 (susceptible) and AG91-25 (resistant). Resistance traits were determined as disease score, percentage of wilted plants, and stem-based bacterial colonization index, as assessed in greenhouse experiments conducted in Réunion Island, France. The AG91-25 resistance was highly efficient toward strains CMR134, PSS366 and GMI1000, but only partial toward the highly virulent strain PSS4. The partial resistance found against PSS4 was overcome under high inoculation pressure, with heritability estimates from 0.28 to 0.53, depending on the traits and season. A genetic map was built with 119 AFLP, SSR and SRAP markers positioned on 18 linkage groups (LG), for a total length of 884 cM, and used for quantitative trait loci (QTL) analysis. A major dominant gene, named ERs1, controlled the resistance to strains CMR134, PSS366, and GMI1000. Against strain PSS4, this gene was not detected, but a significant QTL involved in delay of disease progress was detected on another LG. The possible use of the major resistance gene ERs1 in marker-assisted selection and the prospects offered for academic studies of a possible gene for gene system controlling resistance to bacterial wilt in solanaceous plants are discussed.

Bacterial wilt resistance in tomato, pepper, and eggplant: genetic resources respond to diverse strains in the Ralstonia solanacearum species complex.

Bacterial wilt, caused by strains belonging to the Ralstonia solanacearum species complex, inflicts severe economic losses in many crops worldwide. Host resistance remains the most effective control strategy against this disease. However, wilt resistance is often overcome due to the considerable variation among pathogen strains. To help breeders circumvent this problem, we assembled a worldwide collection of 30 accessions of tomato, eggplant and pepper (Core-TEP), most of which are commonly used as sources of resistance to R. solanacearum or for mapping quantitative trait loci. The Core-TEP lines were challenged with a core collection of 12 pathogen strains (Core-Rs2) representing the phylogenetic diversity of R. solanacearum. We observed six interaction phenotypes, from highly susceptible to highly resistant. Intermediate phenotypes resulted from the plants' ability to tolerate latent infections (i.e., bacterial colonization of vascular elements with limited or no wilting). The Core-Rs2 strains partitioned into three pathotypes on pepper accessions, five on tomato, and six on eggplant. A "pathoprofile" concept was developed to characterize the strain clusters, which displayed six virulence patterns on the whole set of Core-TEP host accessions. Neither pathotypes nor pathoprofiles were phylotype specific. Pathoprofiles with high aggressiveness were mainly found in strains from phylotypes I, IIB, and III. One pathoprofile included a strain that overcame almost all resistance sources.

Advanced backcross QTL analysis of a Lycopersicon esculentum x L. pennellii cross and identification of possible orthologs in the Solanaceae.

In this study, the advanced backcross QTL (AB-QTL) mapping strategy was used to identify loci for yield, processing and fruit quality traits in a population derived from the interspecific cross Lycopersicon esculentum E6203 x Lycopersicon pennellii accession LA1657. A total of 175 BC(2) plants were genotyped with 150 molecular markers and BC(2)F(1) plots were grown and phenotyped for 25 traits in three locations in Israel and California, U.S.A. A total of 84 different QTLs were identified, 45% of which have been possibly identified in other wild-species-derived populations of tomato. Moreover, three fruit-weight/size and shape QTLs ( fsz2b.1, fw3.1/ fsz3.1 and fs8.1) appear to have putative orthologs in the related solanaceous species, pepper and eggplant. For the 23 traits for which allelic effects could be deemed as favorable or unfavorable, 26% of the identified loci had L. pennellii alleles that enhanced the performance of the elite parent. Alleles that could be targeted for further introgression into cultivated tomato were also identified.

Fine mapping of quantitative trait loci for improved fruit characteristics from Lycopersicon chmielewskii chromosome 1.

The near-isogenic line (NIL) TA1150 contains a 56-cM introgression from Lycopersicon chmielewskii chromosome 1 and has several interesting phenotypic characteristics including fruit with orange color, high levels of soluble solids, thick pericarp, small stem scars, and good firmness. A set of overlapping recombinant lines (subNILs) was developed and field tested to fine map the quantitative trait loci (QTL) controlling these traits. The results indicated that the solids, pericarp thickness, and firmness QTL are distinct from the color locus. Several of the QTL mapped in this study, including the soluble-solids QTL, probably correspond to QTL mapped in other wild species of tomato. However, analysis of a set of TA523 subNILs containing complementary introgressions from Lycopesicon hirsutum chromosome 1 suggests that this wild species may contain a different locus for improved soluble solids. Thus, it might be possible to combine the L. chmielewskii and L. hirsutum alleles for these loci in a single line with the potential for extremely highly soluble solids. The TA1150 subNIL TA1688 contains the smallest introgression of the solids locus (approximately 19 cM), as well as the pericarp thickness and firmness QTL, with a yield that was equivalent to two of the three control lines. Isolation of recombinant subNILs from TA1688 should break the linkage between orange color and high solids and provide a small introgressed segment for marker-assisted breeding and genetic improvement of processing tomato.

QTL analysis of morphological traits in eggplant and implications for conservation of gene function during evolution of solanaceous species.

An interspecific F(2) population from a cross between cultivated eggplant, Solanum melongena, and its wild relative, S. linnaeanum, was analyzed for quantitative trait loci (QTL) affecting leaf, flower, fruit and plant traits. A total of 58 plants were genotyped for 207 restriction fragment length polymorphism (RFLP) markers and phenotyped for 18 characters. One to eight loci were detected for each trait with a total of 63 QTL identified. Overall, 46% of the QTL had allelic effects that were the reverse of those predicted from the parental phenotypes. Wild alleles that were agronomically superior to the cultivated alleles were identified for 42% of the QTL identified for flowering time, flower and fruit number, fruit set, calyx size and fruit glossiness. Comparison of the map positions of eggplant loci with those for similar traits in tomato, potato and pepper revealed that 12 of the QTL have putative orthologs in at least one of these other species and that putative orthology was most often observed between eggplant and tomato. Traits showing potential orthology were: leaf length, shape and lobing; days to flowering; number of flowers per inflorescence; plant height and apex, leaf and stem hairiness. The functionally conserved loci included a major leaf lobing QTL ( llob6.1) that is putatively orthologous to the potato leaf ( c) and/or Petroselinum ( Pts) mutants of tomato, two flowering time QTL ( dtf1.1, dtf2.1) that also have putative counterparts in tomato and four QTL for trichomes that have potential orthologs in tomato and potato. These results support the mounting evidence of conservation of gene function during the evolution of eggplant and its relatives from their last common ancestor and indicate that this conservation was not limited to domestication traits.

Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato.

The efficiency of the binary bacterial artificial chromosome (BIBAC) vector for Agrobacterium-mediated stable transfer of high molecular weight DNA into plants was tested in tomato. Several variables affecting transformation efficiency were examined including insert size, Agrobacterium genetic background, and the presence of additional copies of the virG, virE1 and virE2 genes. It was found that a helper plasmid containing extra copies of virG was an absolute requirement for obtaining tomato transformants with the BIBAC. MOG101 with the virG helper plasmid was found to be the most efficient strain for transfer of high molecular weight DNA (150 kb). Selected high molecular weight DNA transformants were advanced several generations (up to the R4) to assess T-DNA stability. This analysis showed that the T-DNA was stably maintained and inherited through several meioses regardless of whether it was in the hemizygous or homozygous state. Expression of a selectable marker gene within the T-DNA was also examined through several generations and no gene silencing was observed. Thus, the BIBAC is a useful system for transfer of large DNA fragments into the plant genome.

The broad-spectrum tospovirus resistance gene Sw-5 of tomato is a homolog of the root-knot nematode resistance gene Mi.

We used a positional cloning approach to isolate the Sw-5 disease resistance locus of tomato. Complementation experiments with overlapping cosmid clones enabled us to demonstrate that Sw-5 is a single gene locus capable of recognizing several tospovirus isolates and species. Analysis of the predicted Sw-5 protein suggests that it is a cytoplasmic protein, with a potential nucleotide binding site (NBS) domain and a C-terminal end consisting of leucine-rich repeats (LRRs). Based on its structural features, Sw-5 belongs to the class of NBS-LRR resistance genes that includes the tomato Mi, 12, and Prf genes; the Arabidopsis RPM1 gene; and the plant potato virus X resistance gene Rx. The overall similarity between the Sw-5 and Mi proteins of tomato suggests that a shared or comparable signal transduction pathway leads to both virus and nematode resistance in tomato. The similarity also supports the hypothesis that Sw-5 provides resistance via a hypersensitive response. Sw-5 is a member of a loosely clustered gene family in the telomeric region of chromosome 9. Members of this family map to other regions of chromosome 9 and also to chromosome 12, where several fungal, virus, and nematode genes have been mapped, suggesting that paralogs of Sw-5 may have evolved to provide different resistance specificities.

fw2.2: a quantitative trait locus key to the evolution of tomato fruit size.

Domestication of many plants has correlated with dramatic increases in fruit size. In tomato, one quantitative trait locus (QTL), fw2.2, was responsible for a large step in this process. When transformed into large-fruited cultivars, a cosmid derived from the fw2.2 region of a small-fruited wild species reduced fruit size by the predicted amount and had the gene action expected for fw2.2. The cause of the QTL effect is a single gene, ORFX, that is expressed early in floral development, controls carpel cell number, and has a sequence suggesting structural similarity to the human oncogene c-H-ras p21. Alterations in fruit size, imparted by fw2.2 alleles, are most likely due to changes in regulation rather than in the sequence and structure of the encoded protein.

Stable transfer of intact high molecular weight DNA into plant chromosomes.

In conjunction with an enhanced system for Agrobacterium-mediated plant transformation, a new binary bacterial artificial chromosome (BIBAC) vector has been developed that is capable of transferring at least 150 kb of foreign DNA into a plant nuclear genome. The transferred DNA appears to be intact in the majority of transformed tobacco plants analyzed and is faithfully inherited in the progeny. The ability to introduce high molecular weight DNA into plant chromosomes should accelerate gene identification and genetic engineering of plants and may lead to new approaches in studies of genome organization.

Telomere-homologous sequences occur near the centromeres of many tomato chromosomes.

Several bacteriophage lambda clones containing interstitial telomere repeats (ITR) were isolated from a library of tomato genomic DNA by plaque hybridization with the cloned Arabidopsis thaliana telomere repeat. Restriction fragments lacking highly repetitive DNA were identified and used as probes to map 14 of the 20 lambda clones. All of these markers mapped near the centromere on eight of the twelve tomato chromosomes. The exact centromere location of chromosomes 7 and 9 has recently been determined, and all ITR clones that localize to these two chromosomes map to the marker clusters known to contain the centromere. High-resolution mapping of one of these markers showed cosegregation of the telomere repeat with the marker cluster closest to the centromere in over 9,000 meiotic products. We propose that the map location of interstitial telomere clones may reflect specific sequence interchanges between telomeric and centromeric regions and may provide an expedient means of localizing centromere positions.

Molecular mapping of the centromeres of tomato chromosomes 7 and 9.

The centromeres of two tomato chromosomes have been precisely localized on the molecular linkage map through dosage analysis of trisomic stocks. To map the centromeres of chromosomes 7 and 9, complementary telo-, secondary, and tertiary trisomic stocks were used to assign DNA markers to their respective chromosome arms and thus to localize the centromere at the junction of the short and long arms. It was found that both centromeres are situated within a cluster of cosegregating markers. In an attempt to order the markers within the centric clusters, genetic maps of the centromeric regions of chromosomes 7 and 9 were constructed from F2 populations of 1620 Lycopersicon esculentum x L. pennellii (E x P) plants and 1640 L. esculentum x L. pimpinellifolium (E x PM) plants. Despite the large number of plants analyzed, very few recombination events were detected in the centric regions, indicating a significant suppression of recombination at this region of the chromosome. The fact that recombination suppression is equally strong in crosses between closely related (E x PM) and remotely related (E x P) parents suggests that centromeric suppression is not due to DNA sequence mismatches but to some other mechanism. The greatest number of centromeric markers was resolved in the L. esculentum x L. pennellii F2 population. The centromere of chromosome 7 is surrounded by eight cosegregating markers: three on the short arm, five on the long arm. Similarly, the centric region of chromosome 9 contains ten cosegregating markers including one short arm marker and nine long arm markers. The localization of centromeres to precise intervals on the molecular linkage map represents the first step towards the characterization and ultimate isolation of tomato centromeres.

An examination of factors affecting the efficiency ofAgrobacterium-mediated transformation of tomato.

An improved protocol forAgrobacterium-mediated transformation of the tomato cultivar Moneymaker was developed by examining the effects of six different factors on the efficiency of transformation. Explant size, explant orientation, gelling agent and plate sealant were found to affect transformation efficiency. Two other factors, type of explant (hypocotyl or cotyledon) and frequency of transfer to fresh selective regeneration medium, did not have any effect on transformation efficiency. By combining the best treatments for each factor, an average transformation efficiency of 10.6% was obtained for Moneymaker.

A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death.

Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide. Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase. Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5. Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion. Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region. After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion. Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants. Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants. The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato. These results suggest that Pto and Fen participate in the same signal transduction pathway.

Map-based cloning of a protein kinase gene conferring disease resistance in tomato.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.