Marker-assisted introgression of five QTLs controlling fruit quality traits into three tomato lines revealed interactions between QTLs and genetic backgrounds

The evaluation of organoleptic quality of tomato fruit requires physical, chemical and sensory analyses, which are expensive and difficult to assess. Therefore, their practical use in phenotypic selection is difficult. In a previous study, the genetic control of several traits related to organoleptic quality of fresh-market tomato fruit was investigated. Five chromosome regions strongly involved in organoleptic quality attributes were then chosen to be introgressed into three different recipient lines through marker-assisted selection. A marker-assisted backcross (MABC) strategy was performed, as all the favorable alleles for quality traits were provided by the same parental tomato line, whose fruit weight (FW) and firmness were much lower than those of the lines commonly used to develop fresh market varieties. Three improved lines were obtained after three backcrossing and two selfing generations. The implementation of the MABC scheme is described. The three improved lines were crossed together and with the recipient lines in a half-diallel mating scheme, and the simultaneous effect of the five quantitative trait locus (QTL) regions was compared in different genetic backgrounds. Significant effects of the introgressed regions and of the genetic backgrounds were shown. Additive effects were detected for soluble solid and reducing sugar content in two genetic backgrounds. A partially dominant effect on titratable acidity was detected in only one genetic background. In contrast, additive to dominant unfavorable effects of the donor alleles were detected for FW and locule number in the three genetic backgrounds. Recessive QTL effects on firmness were only detected in the two firmest genetic backgrounds. Comparison of the hybrids in the half-diallel gave complementary information on the effects of: (1) the alleles at the selected regions, (2) the genetic backgrounds and (3) their interaction. Breeding efficiency strongly varied according to the recipient parent, and significant interactions between QTLs and genetic backgrounds were shown for all of the traits studied.     

Identification and verification of QTLs for agronomic traits using wild barley introgression lines

A set of 39 wild barley introgression lines (hereafter abbreviated with S42ILs) was subjected to a QTL study to verify genetic effects for agronomic traits, previously detected in the BC₂DH population S42 (von Korff et al. 2006 in Theor Appl Genet 112:1221–1231) and, in addition, to identify new QTLs and favorable wild barley alleles. Each line within the S42IL set contains a single marker-defined chromosomal introgression from wild barley (Hordeum vulgare ssp. spontaneum), whereas the remaining part of the genome is exclusively derived from elite spring barley (H. vulgare ssp. vulgare). Agronomic field data of the S42ILs were collected for seven traits from three different environments during the 2007 growing season. For detection of putative QTLs, a two-factorial mixed model ANOVA and, subsequently, a Dunnett test with the recurrent parent as a control were conducted. The presence of a QTL effect on a wild barley introgression was accepted, if the trait value of a particular S42IL was significantly (P < 0.05) different from the control, either across all environments and/or in a particular environment. A total of 47 QTLs were localized in the S42IL set, among which 39 QTLs were significant across all tested environments. For 19 QTLs (40.4%), the wild barley introgression was associated with a favorable effect on trait performance. Von Korff et al. (2006 in Theor Appl Genet 112:1221–1231) mapped altogether 44 QTLs for six agronomic traits to genomic regions, which are represented by wild barley introgressions of the S42IL set. Here, 18 QTLs (40.9%) revealed a favorable wild barley effect on the trait performance. By means of the S42ILs, 20 out of the 44 QTLs (45.5%) and ten out of the 18 favorable effects (55.6%) were verified. Most QTL effects were confirmed for the traits days until heading and plant height. For the six corresponding traits, a total of 17 new QTLs were identified, where at six QTLs (35.3%) the exotic introgression caused an improved trait performance. In addition, eight QTLs for the newly studied trait grains per ear were detected. Here, no QTL from wild barley exhibited a favorable effect. The introgression line S42IL-107, which carries an introgression on chromosome 2H, 17–42 cM is an example for S42ILs carrying several QTL effects simultaneously. This line exhibited improved performance across all tested environments for the traits days until heading, plant height and thousand grain weight. The line can be directly used to transfer valuable Hsp alleles into modern elite cultivars, and, thus, for breeding of improved varieties.     

QTL analysis of fruit antioxidants in tomato using Lycopersicon pennellii introgression lines

Antioxidants present in fruits and vegetables may help prevent some chronic diseases such as cancer, arthritis, and heart disease. Tomatoes provide a major contribution to human dietary nutrition because of their widespread consumption in fresh and processed forms. A tomato introgression line population that combines single chromosomal segments introgressed from the wild, green fruited species Lycopersicon pennellii in the background of the domesticated tomato, Lycopersicon esculentum, was used to identify quantitative trait loci (QTL) for nutritional and antioxidant contents. The concentration of ascorbic acid, total phenolics, lycopene and β-carotene, and the total antioxidant capacity of the water-soluble fraction (TACW) were measured in the ripe fruits. A total of 20 QTL were identified, including five for TACW (ao), six for ascorbic acid (aa), and nine for total phenolics (phe). Some of these QTL (ao6-2, ao6-3, ao7-2, ao10-1, aa12-4, phe6-2, and phe7-4) increased levels as compared to the parental line L. esculentum. For lycopene content, we detected four QTL, but none increased levels relative to L. esculentum. The two QTL (bc6-2 and bc6-3) detected for β-carotene increased its levels. The traits studied displayed a strong environmental interaction as only 35% of the water-soluble antioxidant QTL (including TACW, ascorbic, and phenolic contents) were consistent over at least two seasons. Also, only two QTL for phenolics were observed when plants were grown in the greenhouse and none was detected for ascorbic or TACW. The analysis demonstrates that the introgression of wild germplasm may improve the nutritional quality of tomatoes; however regulation appears to be complex with strong environmental effects. M. Cecilia Rousseaux and Carl M. Jones contributed equally to this work     

Application of metabolite profiling to the identification of traits in a population of tomato introgression lines

Naturally occurring variation in wild species can be used to increase the genetic diversity of cultivated crops and improve agronomic value. Populations of introgression lines carrying wild species alleles afford an opportunity to identify traits associated with the introgressed regions, and facilitate characterization of the biochemistry and genetics underlying these phenotypes. Understanding plant metabolic pathways and the interactions between genes, phenotype, and environment is fundamental to functional genomics. Successful analysis of the complex network of plant metabolism requires analytical methods able to record information on as many metabolites as possible. Metabolite profiling is used to provide a snapshot of the metabolome in samples which differ in a known factor such as genetic background. Differences between the metabolite profiles can identify those metabolites/metabolic pathways affected by the introgression and allow genetic maps for metabolic alterations to be established. A Time-of-Flight Mass Spectrometry method is presented, with associated data reduction, used for profiling aqueous metabolites fom tomato. Analysis of ripe fruits of two tomato species, Lycopersicon esculentum and L. pennellii, showed differences in the amounts of many metabolites, including organic acids and sugars. Six introgression lines, L. pennellii introgressions within L. esculentum, were also examined and showed that Principal Component Analysis can reveal subtle differences in metabolism of the introgressed lines when compared to their parents.     

Monitoring genome-wide changes in gene expression in response to endogenous cytokinin reveals targets in Arabidopsis thaliana

Cytokinins have been implicated in developmental and growth processes in plants including cell division, chloroplast biogenesis, shoot meristem initiation and senescence. The regulation of these processes requires changes in cytokinin-responsive gene expression. Here, we induced the expression of a bacterial isopentenyl transferase gene, IPT, in transgenic Arabidopsis thaliana seedlings to study the regulation of genome-wide gene expression in response to endogenous cytokinin. Using MPSS (massively parallel signature sequencing) we identified 823 and 917 genes that were up- and downregulated, respectively, following 24 h of IPT induction. When comparing the response to cytokinin after 6 and 24 h, we identified different clusters of genes showing a similar course of regulation. Our study provides researchers with the opportunity to rapidly assess whether genes of interest are regulated by cytokinins.     

Identification and stability of QTLs for fruit quality traits in apple

Breeding for fruit quality traits is complex due to the polygenic (quantitative) nature of the genetic control of these traits. Therefore, to improve the speed and efficiency of genotype selection, attention in recent years has focused on the identification of quantitative trait loci (QTLs) and molecular markers associated with these QTLs. However, despite the huge potential of molecular markers in breeding programmes, their implementation in practice has been limited by the lack of information on the stability of QTLs across different environments and within different genetic backgrounds. Here, we present the results from a comprehensive analysis of the inheritance of fruit quality traits within a population derived from a cross between the apple cultivars ‘Telamon’ and ‘Braeburn’ over two successive seasons. A total of 74 different QTLs were identified for all the major fruit physiological traits including fruit height, diameter, weight and stiffness, flesh firmness, rate of flesh browning, acidity, the oBrix content and harvest date. Seventeen of these QTLs were ‘major’ QTLs, accounting for over 20% of the observed population variance of the trait. However, only one third (26) of the identified QTLs were stable over both harvest years, and of these year-stable QTLs only one was a major QTL. A direct comparison with published QTL results obtained using other populations (King et al., Theor Appl Genet 102:1227–1235, 2001; Liebhard et al., Plant Mol Biol 52:511–526, 2003) is difficult because the linkage maps do not share a sufficient number of common markers and due to differences in the trait evaluation protocols. Nonetheless, our results suggest that for the six fruit quality traits which were measured in all populations, nine out of a total of 45 QTLs were common or stable across all population × environments combinations. These results are discussed in the framework of the development and application of molecular markers for fruit quality trait improvement.     

Identification of growth processes involved in QTLs for tomato fruit size and composition

Many quantitative trait loci (QTLs) for quality traits havebeen located on the tomato genetic map, but introgression offavourable wild alleles into large fruited species is hamperedby co-localizations of QTLs with antagonist effects. The aimof this study was to assess the growth processes controlledby the main QTLs for fruit size and composition. Four nearlyisogenic lines (NILs) derived from an intraspecific cross betweena tasty cherry tomato (Cervil) and a normal-tasting large fruittomato (Levovil) were studied. The lines carried one (L2, L4,and L9) or five (Lx) introgressions from Cervil on chromosomes1, 2, 4, and 9. QTLs for fruit size could be mainly associatedwith cell division processes in L2 and L9, whereas cell expansionwas rather homogeneous among the genotypes, except Cervil forwhich the low expansion rate was attributed to low cell plasticity.The link between endoreduplication and fruit size remained unclear,as cell or fruit sizes were positively correlated with the cellDNA content, but not with the endoreduplication factor. QTLsfor fruit composition reflected differences in water accumulationrather than in sugar accumulation, except in L9 for which theup-regulation of sucrose unloading and hexose transport and/orstarch synthesis was suggested. This may explain the increasedamount of carbon allocated to cell structures in L9, which couldbe related to a QTL for fruit texture. In Lx, these effectswere attenuated, except on fruit size and cell division. Finally,the region on top of chromosome 9 may control size and compositionattributes in tomato, by a combination of QTL effects on celldivision, cell wall synthesis, and carbon import and metabolism. Key words: Cell division and expansion, endoreduplication, fruit quality, near isogenic line, osmotic regulation, quantitative trait locus, Solanum lycopersicum, starch, sugar and acid contentsReceived 22 July 2008; Revised 17 October 2008 Accepted 20 October 2008     

Suppression of telomere-binding protein gene expression represses seed and fruit development in tomato

Tomato (Solanum lycopersicum L.) plants were transformed with an antisense construct of a cDNA encoding tomato telomere-binding protein (LeTBP1) to describe the role of a telomere-binding protein at the whole plant level. Fruit size decreased corresponding to the degree of suppression of LeTBP1 expression. This inhibition of fruit development was likely due to a decrease in the number of seeds in the LeTBP1 antisense plants. Pollen fertility and pollen germination rate decreased in accordance with the degree of suppression of LeTBP1 expression. Ovule viability was also reduced in the LeTBP1 antisense plants. Although plant height was somewhat reduced in the antisense plants compared to the control plants, the number and weight of leaves were unaffected by LeTBP1 suppression. The number and morphology of flowers were also normal in the antisense plants. These indicate that reduced fertility in the antisense plants is not an indirect effect of altered vegetative growth. LeTBP1 expression was sensitive to temperature stress in wild-type plants. We conclude that LeTBP1 plays a critical role in seed and fruit development rather than vegetative growth and flower formation.     

QTLs for drought escape and tolerance identified in a set of random introgression lines of rice

A large set of 254 introgression lines in an elite indica genetic background were evaluated for grain yield (GY) and related traits under the irrigated (control) and drought (stress) conditions in two consecutive years for genetic dissection of adaptive strategies of rice to water stress. A total of 36 quantitative trait loci (QTLs) affecting heading date (HD), plant height (PH), GY and yield components were identified and most QTLs showed pronounced differential expression either qualitatively or quantitatively in response to drought. These QTLs could be grouped into three major types based on their behaviors under control and stress conditions. Type I included 12 QTLs that expressed under both the stress and non-stress conditions. Type II comprised 17 QTLs that expressed under irrigation but not under stress. Type III included seven QTLs that were apparently induced by stress. The observation that the Lemont (japonica) alleles at all HD QTLs except QHd5 resulted in early heading under stress appeared to be responsible for the putative adaptation of Lemont to drought by escaping, whereas the Teqing (indica) alleles at most PH/GY QTLs were consistently associated with increased yield potential and trait stability and thus contributed to DT. Our result that most DT QTLs were non-allelic with QTLs for drought escaping suggests that the two adaptive strategies in the parental lines are under possible negative regulation of two largely non-overlapping genetic systems.     

A genetic map of candidate genes and QTLs involved in tomato fruit size and composition

In order to screen for putative candidate genes linked to tomato fruit weight and to sugar or acid content, genes and QTLs involved in fruit size and composition were mapped. Genes were selected among EST clones in the TIGR tomato EST database (http://www.tigr.org/tdb/tgi/lgi/) or corresponded to genes preferentially expressed in the early stages of fruit development. These clones were located on the tomato map using a population of introgression lines (ILs) having one segment of Lycopersicon pennellii (LA716) in a L. esculentum (M82) background. The 75 ILs allowed the genome to be segmented into 107 bins. Sixty-three genes involved in carbon metabolism revealed 79 loci. They represented enzymes involved in the Calvin cycle, glycolysis, the TCA cycle, sugar and starch metabolism, transport, and a few other functions. In addition, seven cell-cycle-specific genes mapped into nine loci. Fourteen genes, primarily expressed during the cell division stage, and 23 genes primarily expressed during the cell expansion stage, revealed 24 and 26 loci, respectively. The fruit weight, sugars, and organic acids content of each IL was measured and several QTLs controlling these traits were mapped. Comparison between map location of QTLs and candidate gene loci indicated a few candidate genes that may influence the variation of sugar or acid contents. Furthermore, the gene/QTL locations could be compared with the loci mapped in other tomato populations.     

Temporary effect of postharvest UV-C irradiation on gene expression profile in tomato fruit

To obtain an overall view on gene expression during the early stage (24 h) of tomato fruit in response to postharvest UV-C irradiation (4 kJ/m(2)), we performed a microarray analysis by using Affymetrix Tomato Genechip. The results showed that 274 and 403 genes were up- or down-regulated, respectively, more than two folds in postharvest tomato fruit irradiated with UV-C as compared with that in control fruit. The up-regulated genes mainly involve in signal transduction, defense response and metabolism. Conversely, genes related to cell wall disassembly, photosynthesis and lipid metabolism were generally down-regulated. These results opened ways to probe into the molecular mechanisms of the effects of postharvest UV-C irradiation on increased disease resistance, delayed softening, better quality maintenance and prolonged postharvest life in tomato fruit.     

Coupling virus-induced gene silencing to exogenous green fluorescence protein expression provides a highly efficient system for functional genomics in Arabidopsis and across all stages of tomato fruit development

Since the advent of the postgenomic era, efforts have focused on the development of rapid strategies for annotating plant genes of unknown function. Given its simplicity and rapidity, virus-induced gene silencing (VIGS) has become one of the preeminent approaches for functional analyses. However, several problems remain intrinsic to the use of such a strategy in the study of both metabolic and developmental processes. The most prominent of these is the commonly observed phenomenon of "sectoring" the tissue regions that are not effectively targeted by VIGS. To better discriminate these sectors, an effective marker system displaying minimal secondary effects is a prerequisite. Utilizing a VIGS system based on the tobacco rattle virus vector, we here studied the effect of silencing the endogenous phytoene desaturase gene (pds) and the expression and subsequent silencing of the exogenous green fluorescence protein (gfp) on the metabolism of Arabidopsis (Arabidopsis thaliana) leaves and tomato (Solanum lycopersicum) fruits. In leaves, we observed dramatic effects on primary carbon and pigment metabolism associated with the photobleached phenotype following the silencing of the endogenous pds gene. However, relatively few pleiotropic effects on carbon metabolism were observed in tomato fruits when pds expression was inhibited. VIGS coupled to gfp constitutive expression revealed no significant metabolic alterations after triggering of silencing in Arabidopsis leaves and a mild effect in mature green tomato fruits. By contrast, a wider impact on metabolism was observed in ripe fruits. Silencing experiments with an endogenous target gene of interest clearly demonstrated the feasibility of cosilencing in this system; however, carefully constructed control experiments are a prerequisite to prevent erroneous interpretation.     

Developmental changes in tomato fruit composition in response to water deficit and salinity

Processing tomato ( Lycopersicon esculentum Mill. cv. UC82B) plants were subjected to moderate levels of water deficit and salinity (Na₂SO₄/CaCl₂) in sand culture. Fruit water content and the relative contributions of organic and inorganic constituents to fruit solute potential (_Ψ) and soluble solids content were determined throughout development. Fruit _Ψ averaged –0.63, –0.86 and –0.77 MPa in the control, salinity and water deficit plants, respectively. Reduced net water import and maintenance of solute accumulation, irrespective of water import, accounted for the reductions in _Ψ of stressed fruits. Mineral ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl⁻ and SO^2-₄) contributed –0.31 MPa to _Ψ in salinized fruit, compared with –0.19 MPa in control and water deficit treatments. Changes in net carbon accumulation were not observed among treatments, despite considerable differences in fruit K⁺ status. Starch accumulation in immature fruit was increased and hexose accumulation was decreased by both salinity and water deficit. Maximum starch levels were negatively correlated with total fruit _Ψ, but were independent of fruit K⁺. Organic acid levels were generally higher throughout development in salinized plants, relative to control plants, and correlated with increased inorganic cation rather than anion accumulation in these fruits.     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

Effects of the Lycopersicon chmielewskii sucrose accumulator gene (sucr) on fruit yield and quality parameters following introgression into tomato

A gene controlling fruit sucrose accumulation, sucr, was introgressed from the wild tomato species Lycopersicon chmielewskii into the genetic background of a hexose-accumulating cultivated tomato, L. esculentum. During introgression, the size of the L. chmielewskii chromosomal segment containing sucr was reduced by selection for recombination between RFLP markers for the sucr gene and flanking loci. The effects of sucr on soluble solids content, fruit size, yield and other fruit parameters were studied in the genetic background of the processing tomato cultivar Huntl00. In a segregating BC₅F₂ generation, the smallest introgression containing sucr-associated markers was necessary and sufficient to confer high-level sucrose accumulation, the effects of which were completely recessive. Fruit of sucr/sucr genotypes were smaller than those of +/sucr or +/+ genotypes at all stages of development. The timing of sugar accumulation and total sugar concentration were unaffected by sugar composition. No differences in total fruit biomass (fresh weight of red and green fruit) at harvest were observed between the genotypes, and sucrose accumulators produced greater numbers of fruit than hexose accumulators in one family. However, the proportion of ripe fruit at harvest, and hence yield of ripe fruit, as well as average ripe fruit weight and seed set were reduced in sucr/sucr genotypes. Sucrose accumulation was also associated with increased soluble solids content, consistency, serum viscosity, predicted paste yield and acidity, and decreased color rating. In the first backcross to L. chmielewskii, hexose accumulators (+/sucr) had larger fruit than sucrose accumulators (sucr/sucr), while no difference in soluble solids was detected.     

Gene expression profiling of individual cases reveals consistent transcriptional changes in alcoholic human brain

Chronic alcohol exposure induces lasting behavioral changes, tolerance, and dependence. This results, at least partially, from neural adaptations at a cellular level. Previous genome-wide gene expression studies using pooled human brain samples showed that alcohol abuse causes widespread changes in the pattern of gene expression in the frontal and motor cortices of human brain. Because these studies used pooled samples, they could not determine variability between different individuals. In the present study, we profiled gene expression levels of 14 postmortem human brains (seven controls and seven alcoholic cases) using cDNA microarrays (46,448 clones per array). Both frontal cortex and motor cortex brain regions were studied. The list of genes differentially expressed confirms and extends previous studies of alcohol responsive genes. Genes identified as differentially expressed in two brain regions fell generally into similar functional groups, including metabolism, immune response, cell survival, cell communication, signal transduction and energy production. Importantly, hierarchical clustering of differentially expressed genes accurately distinguished between control and alcoholic cases, particularly in the frontal cortex.