Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement

Tomato represents an important source of fiber and nutrients in the human diet and is a central model for the study of fruit biology. To identify components of fruit metabolic composition, here we have phenotyped tomato introgression lines (ILs) containing chromosome segments of a wild species in the genetic background of a cultivated variety. Using this high-diversity population, we identify 889 quantitative fruit metabolic loci and 326 loci that modify yield-associated traits. The mapping analysis indicates that at least 50% of the metabolic loci are associated with quantitative trait loci (QTLs) that modify whole-plant yield-associated traits. We generate a cartographic network based on correlation analysis that reveals whole-plant phenotype associated and independent metabolic associations, including links with metabolites of nutritional and organoleptic importance. The results of our genomic survey illustrate the power of genome-wide metabolic profiling and detailed morphological analysis for uncovering traits with potential for crop breeding.     

Application of nontargeted metabolite profiling to discover novel markers of quality traits in an advanced population of malting barley

The process of breeding superior varieties for the agricultural industry is lengthy and expensive. Plant metabolites may act as markers of quality traits, potentially expediting the appraisal of experimental lines during breeding. Here, we evaluated the utility of metabolites as markers by assessing metabolic variation influenced by genetic and environmental factors in an advanced breeding setting and in relation to the phenotypic distribution of 20 quality traits. Nontargeted liquid chromatography–mass spectrometry metabolite profiling was performed on barley (Hordeum vulgare L.) grain and malt from 72 advanced malting barley lines grown at two distinct but climatically similar locations, with 2‐row and 6‐row barley as the main genetic factors. 27 420 molecular features were detected, and the metabolite and quality trait profiles were similarly influenced by genotype and environment; however, malt was more influenced by genotype compared with barley. An O2PLS model characterized molecular features and quality traits that covaried, and 1319 features associated with at least one of 20 quality traits. An indiscriminant MS/MS acquisition and novel data analysis method facilitated the identification of metabolites. The analysis described 216 primary and secondary metabolites that correlated with multiple quality traits and included amines, amino acids, alkaloids, polyphenolics and lipids. The mechanisms governing quality trait–metabolite associations were interpreted based on colocalization to genetic markers and their gene annotations. The results of this study support the hypothesis that metabolism and quality traits are co‐influenced by relatively narrow genetic and environmental factors and illustrate the utility of grain metabolites as functional markers of quality traits.     

Homeologous recombination in Solanum lycopersicoides introgression lines of cultivated tomato

A library of "introgression lines" containing Solanum lycopersicoides chromosome segments in the genetic background of cultivated tomato (Lycopersicon esculentum) was used to study factors affecting homeologous recombination. Recombination rates were estimated in progeny of 43 heterozygous introgressions and whole-chromosome substitution lines, together representing 11 of the 12 tomato chromosomes. Recombination within homeologous segments was reduced to as little as 0-10% of expected frequencies. Relative recombination rates were positively correlated with the length of introgressed segments on the tomato map. The highest recombination (up to 40-50% of normal) was observed in long introgressions or substitution lines. Double-introgression lines containing two homeologous segments on opposite chromosome arms were synthesized to increase their combined length. Recombination was higher in the double than in the single segment lines, despite a preference for crossovers in the region of homology between segments. A greater increase in homeologous recombination was obtained by crossing the S. lycopersicoides introgression lines to L. pennellii--a phylogenetically intermediate species--or to L. esculentum lines containing single L. pennellii segments on the same chromosome. Recombination rates were highest in regions of overlap between S. lycopersicoides and L. pennellii segments. The potential application of these results to breeding with introgression lines is discussed.     

A library of Solanum lycopersicoides introgression lines in cultivated tomato.

A set of introgression lines (ILs), containing individual chromosome segments from the wild nightshade Solanum lycopersicoides bred into the genetic background of cultivated tomato (Lycopersicon esculentum), has been developed. A primary group of 56 lines was selected for maximum representation of the S. lycopersicoides genome (approximately 96% of the total map units), homozygosity, and a minimum number of introgressed segments per line. A secondary set of 34 lines provides increased map resolution in certain regions. Approximately 34% of the lines were sterile in the homozygous condition, but could be maintained by heterozygotes. To facilitate identification of segregating ILs, restriction fragment length polymorphism probes were converted to higher throughput cleaved amplified polymorphic sequence markers, which supplement allozyme and morphological loci. Strong segregation distortion was observed in F2 progeny of heterozygous ILs, with an excess of L. esculentum alleles in most regions. For introgressions on distal chromosome 1L, a preferential transmission of S. lycopersicoides alleles was observed in the male germ line. Homozygous ILs generally yielded less seed from self pollination than corresponding heterozygotes, indicating that sterility effects were recessive. This IL library provides a novel resource for genetic studies of traits found in S. lycopersicoides.     

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     

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.     

An integrated view of quantitative trait variation using tomato interspecific introgression lines

Resolving natural phenotypic variation into genetic and molecular components is a major objective in biology. Over the past decade, tomato interspecific introgression lines (ILs), each carrying a single 'exotic' chromosome segment from a wild species, have exposed thousands of quantitative trait loci (QTL) affecting plant adaptation, morphology, yield, metabolism, and gene expression. QTL for fruit size and sugar composition were isolated by map-based cloning, while others were successfully implemented in marker-assisted breeding programs. More recently, integrating the multitude of IL-QTL into a single database has unraveled some unifying principles about the architecture of complex traits in plants.     

GISH analysis of meiotic chromosome pairing in Solanum lycopersicoides introgression lines of cultivated tomato.

Meiotic chromosome pairing was studied in introgression lines of cultivated tomato, Lycopersicon esculentum (= Solanum lycopersicum), containing 1 or 2 chromosome segments from the wild species Solanum lycopersicoides. Genomic in situ hybridization (GISH) was used to compare the relative lengths at diakinesis of the different introgressed segments and to measure the chiasmate arm frequency for the chromosome pair involved in the introgression(s). Longer segments generally produced stronger GISH signals than shorter segments. GISH signal intensity also depended on whether or not an introgressed segment encompassed the centromeric region. For example, a 29 cM segment that included the centromeric region produced a stronger GISH signal than a 42 cM segment that did not. In each line the chromosome arm containing the homeologous segment showed a reduction in chiasmate arm frequency that was most pronounced in lines with long segments. This reduction was accompanied by an increased chiasmate arm frequency on the other arm. Double introgression lines, heterozygous in repulsion phase for 2 introgressions on opposite chromosome arms, showed a lower frequency of chiasmata than single introgression lines. Pairing failure, indicated by the presence of univalents, was highest in the double introgression and whole chromosome substitution lines. These results are discussed with respect to observations of suppressed recombination in these stocks and potential practical implications for reducing linkage drag in breeding programs.     

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 morphological traits in a tomato recombinant inbred line population.

Quantitative trait loci (QTLs) influencing morphological traits were identified by restriction fragment length polymorphism (RFLP) analysis in a population of recombinant inbred lines (RILs) derived from a cross of the cultivated tomato Lycopersicon esculentum with a related wild species, Lycopersicon cheesmanii. One hundred and thirty-two RFLP loci spaced throughout the tomato genome were used as DNA probes on genomic DNA from 97 RIL families. Morphological traits, including plant height, plant fresh mass, number of branches, number of nodes, first flower-bearing node, and leaf length, were evaluated in two controlled environment trials in 1992 and 1993. QTLs were detected via regression analyses at multiple marker loci for each morphological trait. A total of 41 markers were significantly associated with the traits examined. Large additive effects were measured at many of these loci. QTLs for multiple traits were detected on chromosomes 3 (TG74) and 4 (CT188), suggesting the possible association of these chromosome segments with genes controlling growth and development in tomato. These chromosomal regions were also associated with multiple morphological traits in a L. esculentum x Lycopersicon pennellii cross. A total of 13% of the QTLs identified for traits common to both studies occupied similar map positions.     

Quantitative trait locus analysis of leaf dissection in tomato using Lycopersicon pennellii segmental introgression lines

Leaves are one of the most conspicuous and important organs of all seed plants. A fundamental source of morphological diversity in leaves is the degree to which the leaf is dissected by lobes and leaflets. We used publicly available segmental introgression lines to describe the quantitative trait loci (QTL) controlling the difference in leaf dissection seen between two tomato species, Lycopersicon esculentum and L. pennellii. We define eight morphological characteristics that comprise the mature tomato leaf and describe loci that affect each of these characters. We found 30 QTL that contribute one or more of these characters. Of these 30 QTL, 22 primarily affect leaf dissection and 8 primarily affect leaf size. On the basis of which characters are affected, four classes of loci emerge that affect leaf dissection. The majority of the QTL produce phenotypes intermediate to the two parent lines, while 5 QTL result in transgression with drastically increased dissection relative to both parent lines.     

General combining ability of most yield-related traits had a genetic basis different from their corresponding traits per se in a set of maize introgression lines

Evaluation of combining ability is a crucial process in hybrid breeding, and dissection of the genetic basis of combining ability will facilitate hybrid breeding. In this study, molecular markers significantly associated with general combining ability (GCA) of seven yield-related traits and the traits per se were detected in a set of maize introgression lines (ILs) under three environments. Totally 25 and 31 significant loci for GCA and the traits per se were commonly detected under multiple environments, respectively. Correlation analysis and comparison among these significant loci revealed that the genetic basis of GCA of these yield-related traits was generally different from that of the traits per se except for the trait of ear row number. In addition, GCA of the ILs was positively and significantly correlated to the total relative effects of significant GCA loci in the ILs in general, implying that the GCA loci identified in this study would be useful in molecular breeding. Correlation analysis also showed that the GCA of yield per plant was strongly correlated to the GCA of kernel number per row, ear length and 100-kernel-weight, thus these traits were more important in genetic improvement for GCA. Results in this study would provide useful information for hybrid breeding in maize.     

Identification of combining ability loci for five yield-related traits in maize using a set of testcrosses with introgression lines

Combining ability is essential for hybrid breeding in crops. However, the genetic basis of combining ability remains unclear and has been seldom investigated. Identifying molecular markers associated with this complex trait would help to understand its genetic basis and provide useful information for hybrid breeding in maize. In this study, we identified genetic loci of general combining ability (GCA) and specific combining ability (SCA) for five yield-related traits under three environments using a set of testcrosses with introgression lines (ILs). GCA or SCA of the five yield-related traits of the ILs was estimated by the performance of testcrosses with four testers from different heterotic groups. Genetic correlations between GCA of the traits and the corresponding traits per se were not significant or not strong, suggesting that the genetic basis between them is different. A total of 56 significant loci for GCA and 21 loci for SCA were commonly identified in at least two environments, and only 5 loci were simultaneously controlling GCA and SCA, indicating that the genetic basis of GCA and SCA is different. For all of the traits investigated, positive and significant correlations between the number of GCA loci in the ILs and the performance of the corresponding GCA of the ILs were detected, implying that pyramiding GCA loci would have positive effect on the performance of GCA. Results in this study would be useful for maize hybrid breeding.     

Association of barley photoperiod and vernalization genes with QTLs for flowering time and agronomic traits in a BC2DH population and a set of wild barley introgression lines

The control of flowering time has important impacts on crop yield. The variation in response to day length (photoperiod) and low temperature (vernalization) has been selected in barley to provide adaptation to different environments and farming practices. As a further step towards unraveling the genetic mechanisms underlying flowering time control in barley, we investigated the allelic variation of ten known or putative photoperiod and vernalization pathway genes between two genotypes, the spring barley elite cultivar ‘Scarlett’ (Hordeum vulgare ssp. vulgare) and the wild barley accession ‘ISR42-8’ (Hordeum vulgare ssp. spontaneum). The genes studied are Ppd-H1, VRN-H1, VRN-H2, VRN-H3, HvCO1, HvCO2, HvGI, HvFT2, HvFT3 and HvFT4. ‘Scarlett’ and ‘ISR42-8’ are the parents of the BC₂DH advanced backcross population S42 and a set of wild barley introgression lines (S42ILs). The latter are derived from S42 after backcrossing and marker-assisted selection. The genotypes and phenotypes in S42 and S42ILs were utilized to determine the genetic map location of the candidate genes and to test if these genes may exert quantitative trait locus (QTL) effects on flowering time, yield and yield-related traits in the two populations studied. By sequencing the characteristic regions of the genes and genotyping with diagnostic markers, the contrasting allelic constitutions of four known flowering regulation genes were identified as ppd-H1, Vrn-H1, vrn-H2 and vrn-H3 in ‘Scarlett’ and as Ppd-H1, vrn-H1, Vrn-H2 and a novel allele of VRN-H3 in ‘ISR42-8’. All candidate genes could be placed on a barley simple sequence repeat (SSR) map. Seven candidate genes (Ppd-H1, VRN-H2, VRN-H3, HvGI, HvFT2, HvFT3 and HvFT4) were associated with flowering time QTLs in population S42. Four exotic alleles (Ppd-H1, Vrn-H2, vrn-H3 and HvCO1) possibly exhibited significant effects on flowering time in S42ILs. In both populations, the QTL showing the strongest effect corresponded to Ppd-H1. Here, the exotic allele was associated with a reduction of number of days until flowering by 8.0 and 12.7%, respectively. Our data suggest that Ppd-H1, Vrn-H2 and Vrn-H3 may also exert pleiotropic effects on yield and yield-related traits.