Multi-environment QTL mapping reveals genetic architecture of fruit cracking in a tomato RIL <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> × <Emphasis Type="Italic">S. pimpinellifolium</Emphasis> population

Key message

QTL and codominant genetic markers for fruit cracking have been identified in a tomato genetic map derived from a RIL population, providing molecular tools for marker-assisted breeding of this trait.

Abstract

In tomato, as well as in other fleshy fruits, one of the main disorders that widely limit quality and production is fruit cracking or splitting of the epidermis that is observed on the fruit skin and flesh at any stage of fruit growth and maturation. To elucidate the genetic basis of fruit cracking, a quantitative trait loci (QTL) analysis was conducted in a recombinant inbred line (RIL) population derived from a cross between tomato (Solanum lycopersicum) and the wild-relative species S. pimpinellifolium. The RIL population was evaluated for fruit cracking during three consecutive growing seasons. Construction of a high-density linkage map based on codominant markers, covering more than 1000 cM of the whole genome, led to the identification of both main and epistatic QTL controlling fruit cracking on the basis of a single-environment as well as multiple-environment analysis. This information will enhance molecular breeding for novel cracking resistant varieties and simultaneously assist the identification of genes underlying these QTL, helping to reveal the genetic basis of fruit cracking in tomato.

     

QTL mapping of fruit mineral contents provides new chances for molecular breeding of tomato nutritional traits

Key message

Agronomical characterization of a RIL population for fruit mineral contents allowed for the identification of QTL controlling these fruit quality traits, flanked by co-dominant markers useful for marker-assisted breeding.

Abstract

Tomato quality is a multi-variant attribute directly depending on fruit chemical composition, which in turn determines the benefits of tomato consumption for human health. Commercially available tomato varieties possess limited variability in fruit quality traits. Wild species, such as Solanum pimpinellifolium, could provide different nutritional advantages and can be used for tomato breeding to improve overall fruit quality. Determining the genetic basis of the inheritance of all the traits that contribute to tomato fruit quality will increase the efficiency of the breeding program necessary to take advantage of the wild species variability. A high-density linkage map has been constructed from a recombinant inbred line (RIL) population derived from a cross between tomato Solanum lycopersicum and the wild-relative species S. pimpinellifolium. The RIL population was evaluated for fruit mineral contents during three consecutive growing seasons. The data obtained allowed for the identification of main QTL and novel epistatic interaction among QTL controlling fruit mineral contents on the basis of a multiple-environment analysis. Most of the QTL were flanked by candidate genes providing valuable information for both tomato breeding for new varieties with novel nutritional properties and the starting point to identify the genes underlying these QTL, which will help to reveal the genetic basis of tomato fruit nutritional properties.

     

Identification of novel quantitative trait loci for increased lycopene content and other fruit quality traits in a tomato recombinant inbred line population

Epidemiological and clinical studies indicate that a steady dietary intake of bioavailable lycopene, a C₄₀ carotenoid and potent natural antioxidant, may be associated with a decreased incidence of prostate cancer in humans. Since fresh tomatoes and processed tomato products represent approximately 85% of the average human??s dietary lycopene intake, the identification of novel genetic factors which regulate high fruit lycopene content in tomato is imperative for the improvement of nutritional quality in this commercially valuable specialty crop. To understand the genetic control of the extraordinarily high fruit lycopene content in an accession (LA2093) of the tomato wild species Solanum pimpinellifolium, a quantitative trait locus (QTL) mapping study was conducted using a recombinant inbred line (RIL) population of a cross between LA2093 and a cultivated tomato (S. lycopersicum) breeding line, NCEBR-1. The parental lines, F1 progeny, and F7-F10 RIL populations were grown in replicated field trials in four successive years and evaluated for lycopene content as well as several other traits, including fruit fresh weight, soluble solids content, pH of puree, and plant maturity. The lycopene content of ripe fruit was estimated using three methods: high-performance liquid chromatography (HPLC), spectrophotometry, and colorimetric assays. Based on these measurements, QTL were identified and compared across generations. Among the QTL identified for lycopene, two QTL, located on chromosomes 7 and 12, had very large effects and were consistent across generations. The genomic intervals in which these two QTL reside do not correspond to known map positions of carotenoid biosynthetic genes, indicating that these QTL may represent novel alleles with potentially important implications for tomato breeding as well as increased understanding of carotenoid accumulation in tomato. Several QTL were also identified for fruit weight, soluble solids content and plant maturity. The potential implications of these results for tomato crop improvement are discussed.     

A 54-kDa polypeptide identified by 2D-PAGE and bulked segregant analysis underlies differences for pH values in tomato fruit

Fruit pH is an important quality attribute in tomato and it is defined during ripening. The aims of this work were to detect pericarp polypeptides associated with pH in an interspecific tomato BC₁ generation by 1D-PAGE and to identify those differentially expressed polypeptides by comparing 2D-PAGE protein profiles from bulked segregant analysis (BSA). Polypeptide patterns were resolved by 1D-PAGE in a BC₁ population obtained by crossing the cv. ‘Caimanta’ of Solanum lycopersicum (recurrent parental genotype) and the accession LA722 of S. pimpinellifolium (donor parental genotype). Putative QTL for fruit quality were detected by single point analysis. The presence of a 54-kDa band at the mature green stage (MG) carried by the wild genotype decreased the mean value of the pH trait. A BSA combined with 2D-PAGE was applied to the extreme phenotypes for pH in the BC₁ segregating population. Four differentially expressed spots were detected when the polypeptide patterns of the bulks were compared. The spots had the expected molecular mass (around 54-kDa), and they were present in the lower-pH bulk and absent in the higher-pH one. The spots were identified by MS MALDI-TOF and two of them showed homology with the ATP synthase CF₁ alpha subunit of S. lycopersicum. These results indicate that the association between the polypeptide marker and a fruit quantitative trait detected by 1D-PAGE not only would indicate genetic linkage but also could be directly related with the gene underlying the quantitative trait.     

QTL mapping for tuberous stem formation of kohlrabi (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">gongylodes</Emphasis> L.)

The tuberous stem of kohlrabi is an important quantitative trait, which affects its yield and quality. Genetic control of this trait has not yet been unveiled. To identify the QTLs controlling stem swelling of kohlrabi, a BC₁ population of 92 plants was developed from a cross of broccoli DH line GCP04 and kohlrabi var. Seine. A wide range of variation in tuberous stem diameter was observed among the mapping populations. We constructed a genetic map of nine linkage groups (LGs) with different types of markers, spanning a total length of 913.5 cM with an average marker distance of 7.55 cM. Four significant QTLs for radial enlargement of kohlrabi stem, namely, REnBo1, REnBo2, REnBo3, and REnBo4 were detected on C02, C03, C05, and C09, respectively, and accounted for the phenotypic variation of 59% for the stem diameter and 55% for the qualitative grading of tuberous stem in classes. Then, we confirmed the stability of identified QTLs using BC₁S₁ populations derived from the BC₁ plants having heterozygous alleles at the target QTL and homozygous kohlrabi alleles at the remaining QTLs. REnBo1and REnBo2 using 128 plants of BC₁68S₁ and 94 plants of BC₁43S₁, respectively, and REnBo3 and REnBo4 using 152 plants of BC₁57S₁ were detected at the same positions as the respective QTLs of the BC₁ population. Confirmation of QTLs in two successive generations indicates that the QTLs are persistent. The QTLs obtained in this study could be useful in marker-assisted selection of kohlrabi breeding, and to understand the genetic mechanisms of stem swelling and storage organ development in kohlrabi and other Brassica species.     

Phenotypic diversity and association mapping for fruit quality traits in cultivated tomato and related species

Association mapping has been proposed as an efficient approach to assist in the identification of the molecular basis of agronomical traits in plants. For this purpose, we analyzed the phenotypic and genetic diversity of a large collection of tomato accessions including 44 heirloom and vintage cultivars (Solanum lycopersicum), 127 S. lycopersicum var. cerasiforme (cherry tomato) and 17 Solanum pimpinellifolium accessions. The accessions were genotyped using a SNPlex? assay of 192 SNPs, among which 121 were informative for subsequent analysis. Linkage disequilibrium (LD) of pairwise loci and population structure were analyzed, and the association analysis between SNP genotypes and ten fruit quality traits was performed using a mixed linear model. High level of LD was found in the collection at the whole genome level. It was lower when considering only the 127 S. lycopersicum var. cerasiforme accessions. Genetic structure analysis showed that the population was structured into two main groups, corresponding to cultivated and wild types and many intermediates. The number of associations detected per trait varied, according to the way the structure was taken into account, with 0–41 associations detected per trait in the whole collection and a maximum of four associations in the S. lycopersicum var. cerasiforme accessions. A total of 40 associations (30 %) were co-localized with previously identified quantitative trait loci. This study thus showed the potential and limits of using association mapping in tomato populations.     

A major QTL (<Emphasis Type="Italic">qFT12.1</Emphasis>) allele from wild soybean delays flowering time

Soybean is highly sensitive to photoperiod. To improve the adaptability and productivity of soybean, it is essential to understand the molecular mechanisms regulating flowering time. To identify new flowering time QTLs, we evaluated a BC₃F₅ population consisting of 120 chromosome segment substitution lines (CSSLs) over 2 years under field conditions. CSSLs were derived from a cross between the cultivated soybean cultivar Jackson and the wild soybean accession JWS156-1, followed by continuous backcrossing using Jackson as the recurrent parent. Four QTLs (qFT07.1, qFT12.1, qFT12.2, and qFT19.1) were detected on three chromosomes. Of these, qFT12.1 showed the highest effect, accounting for 36.37–38.27% of the total phenotypic variation over 2 years. This QTL was further confirmed in the F₇ recombinant inbred line population (n?=?94) derived from the same cross (Jackson × JWS156-1). Analysis of the qFT12.1 BC₃F₅ residual heterozygous line RHL509 validated the allele effect of qFT12.1 and revealed that the recessive allele of qFT12.1 resulted in delayed flowering. Evaluating the qFT12.1 near-isogenic lines (NILs) under different growth conditions showed that NILs with the wild soybean genotype always showed later flowering than those with the cultivated soybean genotype. qFT12.1 was delimited to a 2703-kb interval between the markers BARCSOYSSR_12_0220 and BARCSOYSSR_12_0368 on chromosome 12. qFT12.1 may be a new flowering time gene locus in soybean.     

In Silico Identification and Experimental Validation of Insertion–Deletion Polymorphisms in Tomato Genome

Comparative analysis of the genome sequences of Solanum lycopersicum variety Heinz 1706 and S. pimpinellifolium accession LA 1589 using MUGSY software identified 145 695 insertion–deletion (InDel) polymorphisms. A selected set of 3029 candidate InDels (≥2 bp) across the entire tomato genome were subjected to PCR validation, and 82.4% could be verified. Of 2272 polymorphic InDels between LA 1589 and Heinz 1706, 61.6, 45.2, and 31.6% were polymorphic in 8 accessions of S. pimpinellifolium, 4 accessions of S. lycopersicum var. cerasiforme, and 10 varieties of S. lycopersicum, respectively. Genetic distance was 0.216 in S. pimpinellifolium, 0.202 in S. lycopersicum var. cerasiforme, and 0.108 in S. lycopersicum. The data suggested a reduction of genetic variation from S. pimpinellifolium to S. lycopersicum var. cerasiforme and S. lycopersicum. Cluster analysis showed that the 8 accessions of S. pimpinellifolium were in one group, whereas 4 accessions of S. lycopersicum var. cerasiforme and 10 varieties of S. lycopersicum were in the same group.     

Determining resistance to Pseudomonas syringae in tomato, a comparison of different molecular markers

Pseudomonas syringae pv. tomato (Pst) is the causal agent of bacterial speck disease in tomato. Resistance to Pst is determined by Pto, a single resistance gene that belongs to a multi-gene family clustered on chromosome 5. Pst-resistant phenotypes in cultivated tomato are determined by a semi-dominant allele of Solanum pimpinellifolium, which was introgressed into Solanum lycopersicum in the past century. Seed companies, which are continuously interested in producing resistant varieties, can benefit from genetic markers closely linked to the Pto locus in breeding programs based on marker-assisted selection. In this research, three sequence characterised amplified region markers have been developed for identification of resistant and susceptible genotypes of Solanum lycopersicum. A cleaved amplified polymorphic sequence marker has been adapted to a real-time polymerase chain reaction platform using high-resolution melting curve analysis. Application to a tomato population for breeding programs is described. Advantages and disadvantages of the different markers are discussed.     

Fine mapping of the Ph-3 gene conferring resistance to late blight (Phytophthora infestans) in tomato

Late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is a devastating disease for tomato and potato crops. In the past decades, many late blight resistance (R) genes have been characterized in potato. In contrast, less work has been conducted on tomato. The Ph-3 gene from Solanum pimpinellifolium was introgressed into cultivated tomatoes and conferred broad-spectrum resistance to P. infestans. It was previously assigned to the long arm of chromosome 9. In this study, a high-resolution genetic map covering the Ph-3 locus was constructed using an F₂ population of a cross between Solanum lycopersicum CLN2037B (containing Ph-3) and S. lycopersicum LA4084. Ph-3 was mapped in a 0.5 cM interval between two markers, Indel_3 and P55. Eight putative genes were found in the corresponding 74 kb region of the tomato Heinz1706 reference genome. Four of these genes are resistance gene analogs (RGAs) with a typical nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 domain. Each RGA showed high homology to the late blight R gene Rpi-vnt1.1 from Solanum venturii. Transient gene silencing indicated that a member of this RGA family is required for Ph-3-mediated resistance to late blight in tomato. Furthermore, this RGA family was also found in the potato genome, but the number of the RGAs was higher than in tomato.     

fw 2.2:a major QTL controlling fruit weight is common to both red- and green-fruited tomato species

We have shown that a major QTL for fruit weight (fw2.2) maps to the same position on chromosome 2 in the green-fruited wild tomato species, Lycopersicon pennellii and in the red-fruited wild tomato species, L. pimpinellifolium. An introgression line F₂ derived from L. esculentum (tomato) x L. pennellii and a backcross 1 (BC₁) population derived from L. esculentum x L. pimpinellifolium both place fw2.2 near TG91 and TG167 on chromosome 2 of the tomato highdensity linkage map. fw2.2 accounts for 30% and 47% of the total phenotypic variance in the L. pimpinellifolium and L. pennellii populations, respectively, indicating that this is a major QTL controlling fruit weight in both species. Partial dominance (d/a of 0.44) was observed for the L. pennellii allele of fw 2.2 as compared with the L. esculentum allele. A QTL with very similar phenotypic affects and gene action has also been identified and mapped to the same chromosomal region in other wild tomato accessions: L. cheesmanii and L. pimpinellifolium. Together, these data suggest that fw2.2 represents an orthologous QTL (i.e., derived by speciation as opposed to duplication) common to most, if not all, wild tomato species. High-resolution mapping may ultimately lead to the cloning of this key locus controlling fruit development in tomato.     

A novel tomato mutant,Solanum lycopersicum elongated fruit1 (Slelf1), exhibits an elongated fruit shape caused by increased cell layers in the proximal region of the ovary

Genes controlling fruit morphology offer important insights into patterns and mechanisms determining organ shape and size. In cultivated tomato (Solanum lycopersicum L.), a variety of fruit shapes are displayed, including round-, bell pepper-, pear-, and elongate-shaped forms. In this study, we characterized a tomato mutant possessing elongated fruit morphology by histologically analyzing its fruit structure and genetically analyzing and mapping the genetic locus. The mutant line, Solanum lycopersicum elongated fruit 1 (Slelf1), was selected in a previous study from an ethylmethane sulfonate-mutagenized population generated in the background of Micro-Tom, a dwarf and rapid-growth variety. Histological analysis of the Slelf1 mutant revealed dramatically increased elongation of ovary and fruit. Until 6 days before flowering, ovaries were round and they began to elongate afterward. We also determined pericarp thickness and the number of cell layers in three designated fruit regions. We found that mesocarp thickness, as well as the number of cell layers, was increased in the proximal region of immature green fruits, making this the key sector of fruit elongation. Using 262 F₂ individuals derived from a cross between Slelf1 and the cultivar Ailsa Craig, we constructed a genetic map, simple sequence repeat (SSR), cleaved amplified polymorphism sequence (CAPS), and derived CAPS (dCAPS) markers and mapped to the 12 tomato chromosomes. Genetic mapping placed the candidate gene locus within a 0.2?Mbp interval on the long arm of chromosome 8 and was likely different from previously known loci affecting fruit shape.     

QTL mapping and confirmation for tolerance of anaerobic conditions during germination derived from the rice landrace Ma-Zhan Red

Wide adoption of direct-seeded rice practices has been hindered by poorly leveled fields, heavy rainfall and poor drainage, which cause accumulation of water in the fields shortly after sowing, leading to poor crop establishment. This is due to the inability of most rice varieties to germinate and reach the water surface under complete submergence. Hence, tolerance of anaerobic conditions during germination is an essential trait for direct-seeded rice cultivation in both rainfed and irrigated ecosystems. A QTL study was conducted to unravel the genetic basis of tolerance of anaerobic conditions during germination using a population derived from a cross between IR42, a susceptible variety, and Ma-Zhan Red, a tolerant landrace from China. Phenotypic data was collected based on the survival rates of the seedlings at 21 days after sowing of dry seeds under 10 cm of water. QTL analysis of the mapping population consisting of 175 F_2:3 families genotyped with 118 SSR markers identified six significant QTLs on chromosomes 2, 5, 6, and 7, and in all cases the tolerant alleles were contributed by Ma-Zhan Red. The largest QTL on chromosome 7, having a LOD score of 14.5 and an R ² of 31.7 %, was confirmed using a BC₂F₃ population. The QTLs detected in this study provide promising targets for further genetic characterization and for use in marker-assisted selection to rapidly develop varieties with improved tolerance to anaerobic condition during germination. Ultimately, this trait can be combined with other abiotic stress tolerance QTLs to provide resilient varieties for direct-seeded systems.     

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and β-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.     

Cuticular wax variation in the tomato (Solanum lycopersicum L.), related wild species and their interspecific hybrids

The tomato (Solanum lycopersicum L.) is one of the world's most important vegetable crop species. Among the many tomato accessions available, only a few are tolerant to abiotic stresses, which are responsible for the majority of the crop losses worldwide. Wild tomato species are then secondary gene pool in the breeding of more resistant tomato cultivars. In the current study, the composition of leaf cuticular waxes from fourteen tomato accessions, including S. lycopersicum, Solanum pennellii, Solanum pimpinellifolium, and their interspecific hybrids was studied in order to select the most adequate chemotaxonomic markers. Total cuticular wax load of S. pennellii plants was much higher than in the other plant species. Hydrocarbons were usually the most abundant wax components, followed by minor quantities of triterpenes and other compounds. Interspecific hybrids showed intermediate wax characteristics. The amount and composition of surface waxes were not correlated with the abiotic stress tolerance in S. lycopersicum. The composition of the hydrocarbon fraction was the least variable both within a single accession and between all the plants studied. Based on the results, cuticular hydrocarbons are proposed as potential chemotaxonomic markers in the classification of tomato and related species.     

Mineral and Trace Elements Content in 30 Accessions of Tomato Fruits (Solanum lycopersicum L.,) and Wild Relatives (Solanum pimpinellifolium L., Solanum cheesmaniae L. Riley, and Solanum habrochaites S. Knapp & D.M. Spooner)

Tomato quality and its potential health benefits are directly related to its chemical composition. The characterization of nutritional properties of Solanum germplasm is essential to choose suitable donor parents for breeding programs. In this sense, wild species could be very useful for tomato fruit quality genetic improvement. With this objective, in this work, we characterize micronutrients content in Eulycopersicon germplasm (20 cultivars of S. lycopersicum L. and 10 accessions of wild relatives) analyzing mineral (Na, K, Ca, Mg) and trace elements (Cu, Fe, Zn, Mn) and applying multidimensional analysis (principal component and cluster analysis). The classification obtained and the comparison of cultivars performance showed that wild accessions belonging to S. cheesmaniae (L. Riley), S. pimpinellifolium L., and S. habrochaites S. Knapp & D.M. Spooner can be of great usefulness in breeding programs to improve mineral content characteristics of conventional S. lycopersicum varieties due to its higher mineral content.     

Inheritance of resistance to the two-spotted spider mite from L. pimpinellifolium (Jusl.) Mill. accession ‘TO-937’

The two-spotted spider mite (Tetranychus urticae Koch) is an important pest of tomato (Lycopersicon esculentum Mill.) crops in temperate regions as this spider mite has a very large capacity for population increase and causes severe tomato yield losses. There is no described tomato cultivar fully resistant to this pest, although resistant accessions have been reported within the green-fruited tomato wild species L. pennellii (Corr.) D’Arcy and L. hirsutum Humb. & Bonpl. We observed a L. pimpinellifolium (Jusl.) Mill. accession, ‘TO-937’, which seemed to be completely resistant to mite attacks and we crossed it with the susceptible L. esculentum cultivar. ‘Moneymaker’ to obtain a family of generations consisting of the two parents, the F₁, the F₂, the BC₁ to L. esculentum, and the BC₁ to L. pimpinellifolium. This family was evaluated for mite resistance in a polyethylene greenhouse using an experimental design in 60 small complete blocks distributed along 12 double rows. Each block consisted of five F₂ plants in one row and one plant of each of the two parents, the F₁, the BC₁ to L. esculentum, and the BC₁ to L. pimpinellifolium in the adjacent row. Plants at the 10–15 leaf stage were artificially infested by putting on them two pieces of French bean leaf heavily infested with T. urticae. After two months, evaluations of infestation were made by visual observation of mite nets and leaf damage. Plants that were free of signs of mite reproduction on the top half were considered as resistant, plants with silky nets only on their basal leaves, intermediate, and plants with mite reproduction on both basal and top canopies were scored as susceptible. Dominance for resistance appeared because all the ‘To-937’, BC₁ to L. pimpinellifolium, and F₁ plants were resistant. Not all ‘Moneymaker’ plants behaved as susceptible because 35% of plants were intermediate. In the BC₁ to L. pimpinellifolium and the F₂, most plants were scored as resistant, only 7 % BC₁ and 3 % F₂ plants were intermediate, and a single F₂ plant (0.3 %) was susceptible. With these figures, resistance seemed to be controlled by either four or two genes according to whether segregation in the BC₁ or in the F₂, respectively, were considered. These results could in part be explained because of appearance of negative interplot interference due to the high frequency of resistant genotypes within most of the generations. Therefore, the family was evaluated again but using a different experimental design. In the new experiment, 16 ‘TO-937’, 17 ‘Moneymaker’, 17 F₁, 37 BC₁ to L. pimpinellifolium, 38 BC₁ to L. esculentum, and 125 F₂ plants were included. Each of these test plants was grown besides a susceptible ‘Moneymaker’ auxilliary plant that served to keep mite population high and homogeneous in the greenhouse. Negative interplot interference was avoided with this design and all the ‘TO-937’, F₁, and BC₁ to L. pimpinellifolium plants were resistant, all ‘Moneymaker’ test plants were susceptible, and 52 % BC₁ to L. esculentum and 25 % F₂ plants were susceptible, which fitted very well with the expected for resistance governed by a single dominant gene. The simple inheritance mode found will favour sucessful introgression of mite resistance into commercial tomatoes from the very close relative L. pimpinellifolium.