2025 Volume 94 Issue 4 Pages 417-428
Tomatoes, Solanum lycopersicum, are globally important crops, valued for their nutritional, economic, and scientific importance. However, domestication has narrowed their genetic diversity, limiting advancements in key traits, such as yield, quality, and stress resistance. Their wild relatives, such as Solanum pennellii, are vital genetic resources, harboring traits for enhanced fruit size, sugar accumulation, and stress tolerance. S. pennellii-derived introgression lines (ILs), developed by crossing the S. lycopersicum cultivar ‘M82’ with S. pennellii accession LA716, have been instrumental in dissecting complex traits controlled by quantitative trait loci (QTLs). This review highlights key IL-based studies on fruit development and quality, focusing on IL5-4, IL8-3, and IL12-1-1. IL5-4, harboring a chromosomal segment from S. pennellii when introgressed into chromosome 5 of S. lycopersicum, exhibits elevated Brix values because of enhanced vegetative biomass. IL8-3, which carries a chromosomal segment from S. pennellii when introgressed into chromosome 8 of S. lycopersicum, also demonstrates high Brix values, which is linked to the increased expression of cell wall invertase and sucrose synthase during fruit ripening. Additionally, IL5-4 and IL8-3 differ from ‘M82’ in their susceptibility to blossom-end rot (BER), a serious physiological disorder in tomatoes, making them valuable resources for investigating the mechanisms underlying the incidence of BER. IL12-1-1, a derivative of IL12-1, produces substantially larger fruits with higher mean weights and sizes, attributed to the increased cell division and elevated levels of auxin and cytokinin during early development. Candidate genes, including Solyc12g005250 and Solyc12g005310, within the chromosomal region of S. pennellii in IL12-1-1, have been proposed to contribute to these traits. In recent years, the use of advanced methods, such as genome-wide association studies (GWASs), has facilitated QTL identification. However, GWASs require comprehensive phenotypic data and analyses of diverse accessions, which are hindered by low genomic diversity in cultivated tomatoes. Conversely, IL-based QTL analysis remains a robust approach for uncovering genetic mechanisms and advancing tomato breeding programs.
The Solanaceae family is particularly noteworthy among angiosperms, encompassing a wide range of species, including economically vital crops such as potatoes (Solanum tuberosum), tomatoes (Solanum lycopersicum), eggplants (Solanum melongena), sweet and chili peppers (Capsicum spp.), and tobacco (Nicotiana spp.), as well as ornamental plants such as Petunia spp. and Solanum spp. (Särkinen et al., 2013). Among these, tomatoes are particularly prominent as one of the most extensively cultivated and consumed vegetable crops worldwide and are a vital source of micronutrients, including amino acids, vitamins, and antioxidants (Liu et al., 2016; Tieman et al., 2017). Reflecting their global importance, data from the Food and Agriculture Organization of the United Nations (<http://faostat.fao.org/>, Accessed: February 11, 2025) indicate that global tomato production in 2023 reached approximately 192.3 million tons, with major producers being China, India, Turkey, the United States, Egypt, and Italy.
In addition to their economic significance, tomatoes have gained recognition as a model system for studying fruit biology, offering valuable insights into the genetic and physiological mechanisms underlying this complex process (Schauer et al., 2006; The Tomato Genome Consortium, 2012). Domesticated tomatoes (S. lycopersicum) are believed to have evolved from their closest wild ancestor, Solanum pimpinellifolium, which produces small, round edible fruits (Blanca et al., 2015; Labate and Robertson, 2012; van der Knaap et al., 2014). During domestication, selection prioritizes traits that enhance adaptation to specific cultivation environments, resulting in the diversification of tomatoes into a wide range of types (Zhu et al., 2018). Although modern breeding and domestication have substantially increased agricultural productivity, these processes have also narrowed the genetic base of cultivated tomatoes, leading to reductions in flavor and nutrient content (Gur and Zamir, 2004; Klee and Tieman, 2013; Overy et al., 2005). In addition, cultivated tomatoes retain only a small fraction of the genetic diversity of their wild relatives (Eshed and Zamir, 1994a). Therefore, harnessing the genetic diversity of wild tomato relatives lost during domestication has emerged as a critical objective in modern breeding programs (Gur and Zamir, 2004; Tanksley and McCouch, 1997).
Several wild tomato species, including S. pimpinellifolium, Solanum neorickii, Solanum habrochaites, Solanum chmielewskii, and Solanum pennellii, exhibit phenotypes markedly different from those of S. lycopersicum, particularly in terms of fruit characteristics (Schauer et al., 2005). All wild species produce significantly smaller fruits than those produced by domesticated species, with S. pimpinellifolium being the only species to bear red-colored fruits (Schauer et al., 2005). Among these, S. pennellii is a distant relative of S. lycopersicum and is characterized by its highly specialized morphology, unique reproduction system, and diverse phytochemical profiles (Alseekh et al., 2013). Studies comparing S. lycopersicum and S. pennellii have revealed advantageous traits of S. pennellii, such as salinity, drought, and insect tolerance (Eshed and Zamir, 1994b). However, the genomes of wild relatives also harbor undesirable traits and numerous interacting quantitative trait loci (QTLs), which can complicate their use in breeding programs (Kanayama, 2017).
A set of 76 introgression lines (ILs) has been developed by crossing the S. lycopersicum cultivated tomato variety ‘M82’ with the S. pennellii accession LA716 to address these challenges (Eshed and Zamir, 1994a, 1995; Lippman et al., 2007). Each IL contains a single short chromosomal segment from S. pennellii introduced into the genetic background of S. lycopersicum (Lippman et al., 2007). A schematic representation of the chromosomes of IL5-4, IL8-3, and IL12-1-1, which are discussed in detail in this review, is presented in Figure 1. Consequently, the most undesirable traits from S. pennellii are eliminated, enabling the identification and analysis of QTLs on S. pennellii chromosomal segments as independent genetic factors (Kanayama, 2017). Since their development, ILs have been widely utilized to investigate traits such as disease resistance (e.g., Ruffel et al., 2005), abiotic stress tolerance (e.g., Frary et al., 2010), fruit size and composition (e.g., Causse et al., 2004), fruit color (e.g., Liu et al., 2003), and fruit yield (e.g., Semel et al., 2006), which have contributed significantly to elucidating the genetic basis underlying these complex traits. Owing to their utility in analyzing beneficial phenotypes and genes from closely related wild species, ILs have been developed for S. pennellii (LA716) and other wild species, such as S. pimpinellifolium (Tanksley and Nelson, 1996), Solanum parviflorum (Fulton et al., 2000), S. habrochaites (Finkers et al., 2007), and more recently, S. pennellii (LA5240) (Torgeman et al., 2024).
Schematic representation of chromosomes in the S. lycopersicum cultivar ‘M82’, S. pennellii accession LA716, and the tomato introgression lines (ILs) IL5-4, IL8-3, and IL12-1-1. Each IL carries a single short chromosomal segment from S. pennellii introduced into the genetic background of ‘M82’. The latest tomato reference genome (SL4.0) downloaded from the Sol Genomics Network database reveals that the introgressed segments in IL5-4, IL8-3, and IL12-1-1 span approximately 1.25 Mbp (156 genes), 3.56 Mbp (487 genes), and 0.18 Mbp (39 genes), respectively.
In this review, we focus on ILs derived from S. pennellii (LA716), which is the most extensively studied IL set. Notably, these ILs are available through the National BioResource Project Tomato, which was established in 2007 in Japan to collect, propagate, maintain, and distribute tomato bioresources, as well as promote functional genomic studies on tomatoes (Ariizumi et al., 2011). Specifically, we describe studies investigating sugar accumulation, stress tolerance, physiological disorders, and fruit size, highlighting the potential of these ILs in advancing tomato breeding and genetic research. Hereafter, S. pennellii is referred to as S. pennellii (LA716).
Brix in ripening fruits is a critical determinant of fruit quality, making its enhancement a key objective in tomato cultivation and breeding. Salinity and water stress in the root zone can increase fruit Brix by elevating the soluble sugar content; however, these approaches are also associated with a progressive reduction in fruit yield (Adams, 1991; Gao et al., 1998; Saito et al., 2008a, b). To produce high-Brix tomatoes without relying on salinity or water stress in the root zone, breeding and genetic studies focusing on quality of tomato fruit are essential. Certain wild tomato species are known to produce fruits with naturally high Brix values, and cultivation trials have demonstrated that a few ILs derived from S. pennellii exhibit higher Brix values than those of the cultivated variety ‘M82’ (Eshed and Zamir, 1994b, 1995; Matsumoto et al., 2021; Fig. 2).
Graphs depicting the Brix values of ripened fruits in 2018 under field (A) and greenhouse (B) conditions for 49 tomato introgression lines (ILs) obtained from National BioResource Project Tomato. Data represent the means ± standard error (n = 5–100). Significant differences between these ILs and the ‘M82’ cultivar were determined using Welch’s t-test, with the asterisk symbols * and ** indicating significance at P < 0.05 and P < 0.01, respectively. Data are cited from Matsumoto et al. (2021) with permission from the Japanese Society for Horticultural Science.
Among the QTLs associated with fruit Brix, Brix9-2-5, identified using one of the ILs (IL9-2-5), is a notable example in which map-based cloning with restriction fragment length polymorphism (RFLP) markers successfully led to the identification of the underlying gene (Fridman et al., 2000). Brix9-2-5, an S. pennellii QTL, was mapped to a flower- and fruit-specific invertase gene (LIN5), and high-resolution mapping further delimited it to a single nucleotide polymorphism (SNP) located within a 484 bp region spanning the exon–intron boundary of LIN5 (Fridman et al., 2000). A subsequent fine-mapping study revealed that a single SNP in LIN5 causes an amino acid substitution within the exon that substantially affects invertase activity (Fridman et al., 2004). Although not based on research using ILs, a genome-wide association study (GWAS) involving 398 modern, heirloom, and wild tomato accessions demonstrated that an amino acid substitution in LIN5, specifically the replacement of asparagine (Asn) with aspartic acid (Asp), is associated with increased sugar content in tomato fruits (Tieman et al., 2017). Biochemical analysis of IL9-2-5 revealed an enhanced capacity of the columella tissue to take up exogenous sucrose, indicating that the increased sugar content in IL9-2-5 fruit primarily results from improved sucrose uptake from the phloem (Baxter et al., 2005). Furthermore, reverse genetic evidence has been provided through the RNAi-mediated suppression of LIN5 expression in the S. lycopersicum cultivar ‘Moneymaker’, resulting in a notable reduction in fruit Brix values (Zanor et al., 2009).
In IL9-2-5, an increase in sucrose uptake from the phloem and high accumulation of glucose and sucrose in the peels of ripened fruits were identified as key factors contributing to an increase in Brix (Baxter et al., 2005). However, in other ILs that also exhibited increased Brix values, the enhancement was attributed to different mechanisms. One such line, IL8-3, has been shown to exhibit higher Brix values than those of ‘M82’ (Eshed and Zamir, 1995; Gur and Zamir, 2004). In this line, a portion of chromosome 8 in ‘M82’ has been replaced with the corresponding segment from S. pennellii (Eshed and Zamir, 1994a, 1995; Eshed et al., 1996). Subsequent studies have further demonstrated that the ripened fruit of IL8-3 consistently achieves high Brix values, primarily because of elevated hexose levels during the ripening stage (Ikeda et al., 2013). Research on the high sugar content of IL9-2-5 and IL8-3 revealed that starch content in fruits during the early developmental stage, specifically 10 to 20 days after flowering (DAF), is higher in these ILs than in ‘M82’, likely contributing to the higher Brix values observed in ripened fruits (Baxter et al., 2005; Ikeda et al., 2013). This finding can be attributed to the hydrolysis of starch accumulated in immature fruits into soluble sugars and their storage in ripe fruits (Dinar and Stevens, 1981; Schaffer and Petreikov, 1997).
ADP-glucose pyrophosphorylase (AGPase) is a key enzyme involved in starch synthesis (Ballicora et al., 2004; Petreikov et al., 2006). AGPase has been reported to regulate starch synthesis in tomato fruits (Guan and Janes, 1991; Petreikov et al., 2006), and ILs containing chromosomal segments from S. habrochaites with high AGPase activity exhibit elevated starch content in the fruit (Petreikov et al., 2006; Schaffer et al., 2000). Accordingly, AGPase activity in immature fruits has been measured in both IL9-2-5 and IL8-3, which have higher starch content than in ‘M82’ during the early developmental stage. Notably, although IL8-3 exhibits significantly higher AGPase activity in immature fruits than in those of ‘M82’, IL9-2-5 exhibits no difference in AGPase activity relative to that in ‘M82’ (Baxter et al., 2005; Ikeda et al., 2013). This finding suggests that although both IL9-2-5 and IL8-3 share the trait of elevated starch content in immature fruits, the mechanisms underlying starch metabolism in both these lines differ, leading to distinct sugar accumulation patterns. This difference is further supported by the observation that the high Brix values of IL9-2-5 are associated with increased glucose and sucrose levels without differences in fructose content (Baxter et al., 2005). In contrast, IL8-3 exhibits elevated levels of hexose, which is a product of starch degradation, while showing no differences in sucrose content compared with that in ‘M82’ (Ikeda et al., 2013). These findings highlight the distinct mechanisms underlying sugar accumulation in IL9-2-5 and IL8-3.
Metabolome and transcriptome analyses for IL8-3 have been performed throughout fruit developmental stages. Metabolome analysis revealed significant metabolic differences between ‘M82’ and IL8-3 in fruits at 20 DAF and the ripening stage (Ikeda et al., 2016). Tomatoes are climacteric fruits that undergo dramatic metabolic changes during ripening, such as increases in sugars, organic acids, and amino acids (Osorio et al., 2011). In contrast, the time point at 20 DAF marks the beginning of the cell expansion stage immediately following the cell division stage, a phase characterized by rapid fruit enlargement and active accumulation of metabolites (Tanksley, 2004). These findings suggest that genes functioning during these critical stages of metabolic fluctuations, i.e., at 20 DAF and ripening, may play a key role in the metabolic differences between cultivated and wild tomato species. It has also been proposed that these genes are likely located within the S. pennellii-introgressed region of IL8-3. Transcriptome analysis aimed at identifying these genes revealed higher expression levels of the cell wall invertase gene (LIN6) and sucrose synthase gene (TOMSSF) in IL8-3 than in ‘M82’ (Ikeda et al., 2016). Cell wall invertase plays a crucial role in sink activity by hydrolyzing sucrose into hexoses within the apoplast, whereas sucrose synthase contributes to sink activity by cleaving sucrose into fructose and UDP-glucose in the cytosol (Koch, 2004; Roitsch and González, 2004). Therefore, the expression of LIN6 and TOMSSF is believed to be closely associated with starch and hexose accumulation in IL8-3 (Ikeda et al., 2016).
The high sugar contents observed in the fruits of IL9-2-5 and IL8-3 have been attributed to differences in enzymatic activity within their fruits, which account for the disparity in their Brix values compared with those in ‘M82’. Similarly, IL5-4, wherein a portion of chromosome 5 from S. pennellii is introgressed into the genetic background of ‘M82’, has also been shown to exhibit higher Brix values in ripening fruits than in those of ‘M82’ (Eshed and Zamir, 1995; Matsumoto et al., 2021). It has been suggested that a higher proportion of vegetative tissue may significantly contribute to increased Brix values (Luengwilai et al., 2010). Consistently, IL5-4 exhibits greater leaf and stem fresh weights than those in ‘M82’ both on the day the first flower blooms and 15 days after the first flower blooms (Matsumoto et al., 2021). Therefore, the larger shoot mass of IL5-4 relative to that of ‘M82’ may be a key factor underlying the high Brix values observed in IL5-4 fruits (Matsumoto et al., 2021). Notably, similar to the sugar contents in the immature fruits of IL9-2-5 and IL8-3, no significant differences in sugar contents have been observed between the immature fruits of IL5-4 and ‘M82’, whereas differences become apparent at the ripening stage. This finding suggests that, as in IL9-2-5 and IL8-3, variations in starch accumulation in immature fruits may play a role. According to the latest version of the tomato reference genome (SL4.0) and annotation (ITAG4.0) available in the Sol Genomics Network database <https://solgenomics.net/>, the S. pennellii chromosome segment of IL5-4 spans approximately 1.25 Mbp and contains 156 genes. Further analysis of starch content, map-based cloning using DNA markers similar to those employed for IL9-2-5, and omics studies, such as those conducted for IL8-3, are expected to provide deeper insights into the mechanisms underlying the high sugar content of IL5-4 fruit.
Plants face various environmental stresses that affect their growth and productivity, including biotic factors, such as pathogens and herbivores, and abiotic factors, such as drought, heat, cold, nutrient deficiencies, and soil contamination with salts or toxic metals (Zhu, 2016). Among these, drought and salinity are particularly concerning, with projections indicating that over 50% of arable land will be affected by salinization by 2050 (Wang et al., 2003). Therefore, developing salt-tolerant crops is a major priority in plant breeding. S. pennellii exhibits notable salt tolerance, making it a valuable genetic resource for breeding efforts (Bolger et al., 2014; Frary et al., 2011). As salt tolerance in tomatoes is a complex trait governed by multiple genes, ILs incorporating S. pennellii chromosomal segments have been widely used to identify the relevant loci involved in NaCl tolerance (Foolad, 2004).
Among these ILs, IL8-3 enhances salt tolerance, particularly during germination, which is crucial for crop establishment. Comparative studies have revealed that IL8-3 maintains a higher germination rate than maintained by the cultivated variety ‘M82’ under saline conditions, and this was attributed to introgressed S. pennellii genes (Uozumi et al., 2012). Further genomic analysis identified four candidate genes associated with salt and drought stress: Solyc08g079430, Solyc08g079830, Solyc08g080590, and Solyc08g082210 (Bolger et al., 2014). Investigations using the Sol Genomics Network database confirmed that Solyc08g079430 is the only gene located within the chromosomal region linked to salt stress tolerance identified through map-based cloning with DNA markers in Uozumi et al. (2012). Solyc08g079430 encodes an amine oxidase, an enzyme involved in polyamine homeostasis that plays a key role in abiotic stress responses (Cona et al., 2006). The differential gene expression analysis of ‘M82’ and S. pennellii suggests that Solyc08g079430 may contribute to salt tolerance during germination by regulating polyamine metabolism (Bolger et al., 2014). However, further validation through genetic transformations or genome editing is required to confirm its precise role in salt stress tolerance.
The quality of tomato fruits grown under saline conditions is often compromised because of blossom-end rot (BER) (Cuartero and Fernández-Muñoz, 1999). BER is a physiological disorder that negatively affects the production of various fruits and vegetables, including tomatoes, peppers (Capsicum spp.), eggplants (S. melongena), and watermelons (Citrullus lanatus) (Saure, 2001; Taylor et al., 2004; Taylor and Locascio, 2004; White and Broadley, 2003). In tomatoes, BER manifests as necrosis and browning of the cells in the distal part of the fruit, even in immature fruits (Fig. 3A). BER progression begins with the leakage of intracellular substances, leading to plasmolysis and degradation of membrane proteins, followed by the formation of water-soaked symptoms in the distal part of the fruit. Ultimately, necrosis occurs, resulting in discoloration of the affected tissue (Ho and White, 2005; Saure, 2001). Fruits affected by BER mature earlier and are smaller than healthy fruits (Aktas et al., 2005). BER can also progress internally, causing browning and necrosis in the parenchymal tissue around the seeds and distal part of the fruit (Adams and Ho, 1992; Ho and White, 2005; Fig. 3B).
Blossom-end rot (BER) symptoms in tomato fruits. (A) BER develops within two weeks after fruit set, leading to necrosis and discoloration in the distal portion of fruits during their early stages of development. BER-affected fruits exhibit accelerated softening, premature ripening, reduced size, and water-soaked symptoms at their distal ends. (B) Necrotic lesions also form in the internal parenchymal tissue surrounding young seeds and the distal placenta. The photographs were taken of plants grown in pots under greenhouse conditions, and the fruit shown in (A) is IL5-4.
BER in tomatoes is strongly associated with low Ca availability, as it is known to occur relatively more frequently under low-Ca conditions. Affected fruits exhibit reduced Ca content, which can be mitigated by Ca application (Ho et al., 1993; Ho and White, 2005; White and Broadley, 2003). Ca plays a critical role in maintaining cell membrane integrity (Hepler, 2005; White and Broadley, 2003), and insufficient Ca in fruit tissues is generally considered a major factor contributing to BER. However, Saure (2014) and Matsumoto et al. (2021) reported cases of BER despite adequate Ca levels in the affected distal part of the fruit tissue. Rapid fruit enlargement owing to intense light or high temperatures has also been reported to reduce Ca transport to the distal part of fruit or increase Ca demand beyond the available supply, leading to BER (Ho et al., 1993; Ho and White, 2005). Notably, BER is more prevalent in processed tomatoes than in fresh-market tomatoes and occurs less frequently in cherry tomatoes and wild tomato species (Ho and White, 2005). Thus, when studying the occurrence of BER in tomatoes, it is essential to investigate not only the role of Ca, but also the effects of cultivation conditions and genetic factors on the occurrence of this disorder.
The initial stages of BER are characterized by the leakage of cellular contents through the plasma membrane, reportedly because of insufficient apoplastic free Ca ions (Ca2+) (Clarkson and Hanson, 1980; Hirschi, 2004; Kirkby and Pilbeam, 1984). Apoplastic Ca2+ is critical for stabilizing the plasma membrane by linking phospholipids and surface proteins (Clarkson and Hanson, 1980; Legge et al., 1982). Adequate apoplastic Ca2+ helps prevent plasma membrane damage and the leakage of cellular contents (Kirkby and Pilbeam, 1984; Picchioni et al., 1998). In tomatoes, overexpression of the Ca exchanger 1 (CAX1) gene, which encodes the H+/Ca2+ exchanger in Arabidopsis thaliana, increases BER incidence compared with that in wild-type plants. This transformation results in decreased apoplastic Ca2+ concentrations and the subsequent leakage of cellular contents, leading to BER development (de Freitas et al., 2011; Park et al., 2005). Conversely, suppressing the expression of the pectin methylesterase gene in transgenic plants increases apoplastic Ca2+ levels, thereby reducing the incidence of BER (de Freitas et al., 2012). These findings suggest that apoplastic Ca2+, rather than total or peel Ca content, significantly impacts BER incidence by stabilizing the plasma membrane.
Tomato ILs have also been used in BER research. For instance, IL5-4, in which a part of chromosome 5 from S. pennellii replaces the corresponding segment in ‘M82’, exhibits higher BER incidence than that in ‘M82’, whereas IL8-3, with a segment of chromosome 8 from S. pennellii, shows reduced BER incidence (Matsumoto et al., 2021; Uozumi et al., 2012). BER in tomatoes has been reported to occur within the first 15 DAF, when fruit expansion is most rapid, because of insufficient Ca transport to the distal portion of the fruit (Adams and Ho, 1992; Bradfield and Guttridge, 1984; Ho and White, 2005; Saure, 2001). Ikeda et al. (2017) investigated BER incidence in IL8-3 by comparing fruit expansion rates between IL8-3 and ‘M82’ during the critical period ranging from 5–20 DAF. Their findings indicated that IL8-3 fruits exhibit slower expansion rates than those exhibited by ‘M82’ fruits, particularly during crucial early growth stages up to 15 DAF, reducing the likelihood of Ca deficiencies in distal fruit tissues, thereby suppressing BER incidence.
As previously discussed, apoplastic Ca2+ and Ca2+ transporters have also been implicated in the development of BER. Ikeda et al. (2017) reported higher expression of Ca2+-related transporter genes, including CAX, Ca2+-ATPase, and the Na+/Ca2+ exchanger gene (NCX), in IL8-3 fruits at 10 DAF than those in ‘M82’ fruits, indicating enhanced Ca2+ transport activity in IL8-3. Although the specific relationship between individual genes and BER development remains unclear, apoplastic free Ca2+ plays a critical role in stabilizing the plasma membrane (Clarkson and Hanson, 1980; Legge et al., 1982). Among these genes, Ca2+-ATPase, which localizes to the plasma and vacuolar membranes and transports Ca2+ to the apoplast using ATP, may contribute to maintaining apoplastic Ca2+ levels and reducing BER incidence (Axelsen and Palmgren, 2001; White and Broadley, 2003). Thus, the elevated expression of Ca2+-ATPase in IL8-3 may be a key factor responsible for the relatively low BER incidence in this line (Ikeda et al., 2017).
In contrast, IL5-4, which exhibits a higher BER incidence than that in ‘M82’, is a promising resource for investigating the mechanisms underlying BER development. Although many studies have suggested that BER causes Ca deficiency in distal fruit tissues, an analysis of these tissues revealed no significant differences in their total and water-soluble Ca contents between IL5-4 and ‘M82’ (Matsumoto et al., 2021). These findings align with the hypothesis of Saure (2014) that Ca deficiency is a result rather than a cause of BER, as the depletion of apoplastic water-soluble Ca content is observed only after the appearance of BER symptoms. Further research on the localization of water-soluble Ca within fruits is necessary to validate this hypothesis, as its apoplastic distribution has been linked to BER incidence (Ho and White, 2005). The development of BER involves multiple stages, including abiotic stress-induced reactive oxygen species production, lipid peroxidation, and increased membrane permeability (Saure, 2014). However, for IL5-4, abiotic stress is unlikely to be the primary cause of the high incidence of BER, as both ‘M82’ and IL5-4 were grown under identical conditions. Instead, genetic factors derived from the S. pennellii chromosome may underlie the elevated BER incidence in IL5-4, highlighting its potential as a valuable resource for unraveling the genetic and physiological mechanisms of BER development.
Fruit size and shape are critical determinants of yield, quality, and consumer preference for many crops (Grandillo et al., 1999). As mentioned previously, the cultivated tomato (S. lycopersicum) is believed to have evolved from S. pimpinellifolium, a species known for producing small fruits (Blanca et al., 2015; Labate and Robertson, 2012; van der Knaap et al., 2014). Despite the remarkable diversity in fruit size achieved through domestication, the cellular changes and genetic networks driving this variation remain poorly understood (Mauxion et al., 2021). Tomato fruits undergo multiple developmental stages in their growth, reaching their full size at the mature green stage (Penchovsky and Kaloudas, 2023). The final fruit size is determined by the total number of cells and their sizes, with changes in either parameter impacting overall fruit size (Guo and Simmons, 2011).
Extensive research has been conducted to identify QTLs associated with fruit mass, leading to the identification of key QTLs, such as fruit weight (fw)1.1, fw2.1, fw2.2, fw3.1, fw3.2, and fw11.3, which regulate fruit size (Frary et al., 2000; Grandillo et al., 1999; Huang and van der Knaap, 2011). Among these QTLs, fw2.2 is a major locus associated with fruit mass, capable of altering fruit weight by up to 30% (Frary et al., 2000). This QTL was mapped to chromosome 2 of S. pennellii using IL2-5 (Alpert and Tanksley, 1996), and the first fruit mass gene cloned from vegetables or fruit crops (Chakrabarti et al., 2013). Tomato fruit development is characterized by two distinct phases: an initial phase of cell division, typically occurring within a few weeks of pollination, and a subsequent phase of cell expansion (Cong et al., 2002; Fig. 4A). Research has shown that the transcription of fw2.2 is associated with reduced cell division and is inversely correlated with the mitotic activity of the pericarp and placental cells during the early stages of fruit development (Frary et al., 2000). Therefore, fw2.2 functions as a negative regulator of cell division in developing fruits (Cong et al., 2002). Furthermore, it has been demonstrated that fw2.2 exerts its inhibitory effects on cell division by interacting with the regulatory subunit of casein kinase II at or near the plasma membrane (Cong and Tanksley, 2006).
Tomato fruit development and the cellular structure of the pericarp at 20 days after flowering (DAF). (A) Fruit development progresses through three distinct phases in ‘M82’: fruit set, cell division, and cell expansion, followed by the onset and progression of ripening. (B) Pericarp tissue structure in the fruits of the cultivar ‘M82’ and IL12-1-1. Scale bar: 100 μm. Images in (B) are sourced from Yada et al. (2022) with permission from the Japanese Society for Horticultural Science.
The fw3.2 locus, located on chromosome 3, has been identified as a significant QTL that impacts fruit mass (van der Knaap and Tanksley, 2003). Molecular characterization revealed that fw3.2 could be fine-mapped to a 51.4 kb region and was found to primarily regulate fruit weight with a minor impact on fruit shape (Zhang et al., 2012). Subsequent fine-mapping studies have identified the SlKLUH gene as the causal gene for fw3.2, which impacts fruit mass by increasing the number of cell layers and delaying fruit ripening (Chakrabarti et al., 2013). Additionally, an SNP in the promoter region of SlKLUH was significantly associated with variations in fruit mass (Chakrabarti et al., 2013). Further investigations have demonstrated that the upregulated expression of SlKLUH enhances cell proliferation in the pericarp within five days post-anthesis and regulates lipid metabolism, affecting fruit and seed weights (Li et al., 2021).
Similarly, fw11.3, located in the distal region of chromosome 11, affects fruit mass by modulating the number of carpels and locules (Lippman and Tanksley, 2001). Subsequent studies identified candidate genes and elucidated the potential mechanisms associated with this QTL by investigating related phenotypic traits (Huang and van der Knaap, 2011; Mu et al., 2017). However, the specific gene underlying fw11.3 has not yet been isolated, and limited research has been conducted on other loci, such as fw1.1, fw2.1, and fw3.1, resulting in a paucity of knowledge regarding their roles and associated mechanisms.
An analysis of tomato ILs has identified several QTLs associated with fruit yield, with IL7-5 and IL12-1 reported to harbor genes derived from S. pennellii that contribute to increased fruit size (Eshed and Zamir, 1995). Among the derivatives of IL12-1, the subline IL12-1-1 has been shown to produce fruits that have significantly larger mass, including greater mean fruit diameter, length, and weight, than those produced by the ‘M82’ (Yada et al., 2022). In tomato plants and many other plant species, fruit development progresses through three distinct phases: fruit set, cell division, and cell expansion (Gillaspy et al., 1993; Srivastava and Handa, 2005; Fig. 4A). The fruit set phase involves ovarian development and lays the foundation for subsequent cell division and fruit growth (Gillaspy et al., 1993). During the second phase, cell division establishes the final cell number that constitutes the mature fruit (Mapelli et al., 1978; Srivastava and Handa, 2005). In the final phase, cell expansion occurs, which enables the fruit to reach its maximum size (Srivastava and Handa, 2005). The cell division and expansion phases are considered critical determinants of the final size of tomato fruits. Notably, pericarp cell counts in IL12-1-1 fruits at 20 DAF were approximately 46.3% higher than in ‘M82’ fruits (Yada et al., 2022; Fig. 4B). Cell layer formation during the cell division phase occurs from 5 to 8 DAF, followed by randomly oriented cell divisions that continue from 10 to 18 DAF, with the process being largely completed by 20 DAF (Azzi et al., 2015; Cheniclet et al., 2005). Therefore, the increased fruit size observed in IL12-1-1 at the ripening stage may be attributed to enhanced cellular activity during cell division (Yada et al., 2022).
Phytohormones, including auxins, gibberellins, cytokinins, and ethylene, play critical roles in plant growth and development regulation (Gray, 2004; Srivastava and Handa, 2005). Auxins are particularly effective in initiating fruit setting and subsequent fruit development processes (Kanayama, 2017; Nishio et al., 2010; Vogel, 2006). During the first 30 days of fruit development, auxin levels steadily increase, promoting fruit growth by stimulating cell division (Ariizumi et al., 2013; Srivastava and Handa, 2005). Similarly, cytokinins have been shown to drive cell division following anthesis, with their concentrations increasing after flowering and peaking at approximately 5 DAF (Matsuo et al., 2012). A comparative analysis of auxin and cytokinin levels in fruits at 10 and 20 DAF revealed higher concentrations of these phytohormones in IL12-1-1 than those in ‘M82’, and this increase in phytohormone concentrations in IL12-1-1 may contribute to the higher cell counts observed in their pericarp tissues (Yada et al., 2022).
Based on the latest tomato reference genome and gene annotation data obtained from the Sol Genomics Network database, IL12-1-1 harbors approximately 0.18 Mbp of an S. pennellii chromosomal segment, which includes 39 genes. Among these, Solyc12g005250 and Solyc12g005310 were identified as candidate genes that potentially regulate fruit mass, as inferred from gene annotations in the database (Yada et al., 2022). Solyc12g005250 encodes a kinesin-like protein that impacts cell division and expansion in fruit crops (Yang et al., 2013). Solyc12g005310 is associated with auxin regulation, as it belongs to the GH3 family (Sravankumar et al., 2018). In tomatoes, 24 GH3 genes have been identified, with Solyc12g005310 designated as SlGH3-15 (Kumar et al., 2012; Sravankumar et al., 2018). This gene encodes a group II GH3 enzyme that functions as an IAA-amido synthetase and helps maintain auxin homeostasis by conjugating excess indole-3-acetic acid (IAA) to amino acids (Sravankumar et al., 2018; Staswick et al., 2005). In IL12-1-1, the expression levels of Solyc12g005250 are higher than those in ‘M82’ at both 10 and 20 DAF, and the expression of SlGH3-15 is elevated at 10 DAF, with the increased expression of these genes potentially contributing to the enhanced fruit mass observed in IL12-1-1 (Yada et al., 2022). However, to pinpoint the specific gene responsible for the increased fruit size in IL12-1-1, as has been identified for fw2.2 and fw3.1, reverse genetic approaches, such as creating transgenic lines overexpressing these candidate genes, are essential for validation.
Tomato ILs were developed by crossing the cultivated tomato variety ‘M82’ with S. pennellii, followed by the repeated backcrossing of progeny and selection using RFLP markers (Eshed and Zamir, 1994a). As discussed in this review, ILs have significantly contributed to the identification and dissection of complex traits controlled by QTLs, such as fruit yield, sugar content, and resistance to physiological disorders such as BER. They have also been instrumental in elucidating the genetic mechanisms underlying these traits and isolating key genes. The complete genome of the ‘Heinz 1706’ cultivar of S. lycopersicum has been sequenced (The Tomato Genome Consortium, 2012). With the advent and widespread adoption of next-generation sequencing (NGS) technologies, which enable the analysis of vast amounts of genomic data, the genomes of wild tomato species have been sequenced. Additionally, GWASs, which link phenotypic variation to genomic data across diverse populations, have been increasingly utilized in plant research. As noted in this review, an amino acid substitution in LIN5 associated with increased sugar content in tomato fruits, originally identified by Fridman et al. (2004), was also detected in a GWAS conducted by Tieman et al. (2017). These findings highlight that genes and genetic variants underlying QTLs can now be identified not only through approaches such as map-based cloning using ILs and DNA markers, but also through GWAS. However, as demonstrated by Tieman et al. (2017), achieving gene identification through GWAS requires extensive analysis of many accessions or cultivars and the acquisition of accurate and comprehensive phenotypic data from field experiments. Additionally, reverse genetic validation is indispensable for confirming the functional roles of any identified genes. To further investigate the genetic variants related to the increased fruit size observed in IL12-1-1, the authors conducted a preliminary analysis of genome-wide genotype data available for numerous accessions in the Sol Genomics Network database. However, many currently available tomato cultivars exhibit low genomic diversity, making it challenging to identify targeted genetic variants. Therefore, despite the advancements in NGS technologies, QTL analysis using ILs remains a valuable approach.
As discussed in this review, QTL Brix9-2-5 (LIN5), which affects fruit Brix, and fw3.2 (SlKLUH), which is associated with fruit size, contain SNPs within their respective genes that directly impact their phenotypes (Chakrabarti et al., 2013; Fridman et al., 2004; Tieman et al., 2017). Notably, Tieman et al. (2017) suggested that reintroducing an alternative LIN5 allele could effectively enhance the sugar content in tomatoes. In recent years, genome editing using clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has been increasingly applied to plants. A landmark achievement was reported on December 11, 2020, when the Japanese government approved the first genome-edited tomato with elevated levels of γ-aminobutyric acid, a compound widely recognized for its health benefits in humans (Waltz, 2022). This genome-edited tomato was the world’s first agricultural product developed using CRISPR/Cas9 technology to reach commercial markets (Ezura, 2022). Considering the acceptance of genome-edited products in Japan, targeted modifications of genes, such as LIN5 and SlKLUH, and other yet-to-be-identified QTL-related genes, could contribute to the development of tomato varieties with various improved traits, e.g., yield, quality, and resistance to physiological disorders. Therefore, it will be essential to pinpoint the causal genes underlying these QTLs and deepen our understanding of the physiological mechanisms regulating these traits.
