1. Introduction
Tomato (
Solanum lycopersicum L.) is considered one of the most important vegetable crops grown worldwide due to its economic and health importance. According to a statement by the FAO in 2018 (
http://faostat.fao.org/), the world production of tomato was 182,258,016 tons, which was harvested from 4,762,129 hectares. The importance of tomato fruits is related to its considerable level of antioxidants, including lycopene pigment, vitamins such as Vit C and several minerals, which can reduce the progression of various types of dangerous human diseases such as prostate, colon and breast cancers [
1]. Recently, the salinity of soil and water became a severe universal problem restricting the growth, productivity and quality of most crops [
2,
3,
4]. In the coming few decades, the percentage of affected land will increase due to a reduction in the quantity and quality of available irrigation water and global climate change [
5]. Nearly twenty percent of the global cultivated lands and 33% of the irrigated agricultural lands suffer from high salinity [
6].
Two theories have explained plant growth inhibition by salinity, either through ion toxicity or disruption of osmotic functions [
7]. It was reported that salinity reduce many physiological processes in plants including reduce water absorption [
8], inhibit nutrient uptake [
9], reduction in photosynthetic rate [
10] and reduce yield [
6]. Numerous commercial cultivars of tomato range from moderately sensitive to sensitive to saline stress. Creating a new irrigation system or producing phenotypes resistant to adverse conditions are two principal strategies for controlling salinity stress. Many previous techniques were evaluated to reduce the adverse effect of salinity, such as the using of compost and vermicompost [
11,
12], application of plant growth-promoting bacteria [
13], aeration of soil by the improvement of root growth, photosynthetic rate, water infiltration rates, as well as nutrient absorption [
14,
15], foliar application of indole-3-acetic acid [
16] and root and foliar application of salicylic acid [
17].
Hence, it is essential to develop novel effective methods to reduce the salinity stress of tomato. One of these treatments for reducing the adverse effect of salinity of tomato is grafting on resistance rootstocks. Moreover, grafting is a sustainable technique and environmentally friendly. Recently, the grafting procedure has been commonly used to cover soil-borne diseases and abiotic stress conditions such as salinity, drought, chilling stress, the poison of heavy metal and alkalinity of soil [
18]. The benefits of grafting to enhance tomato plant growth and production under salinity stress were recorded [
19]. Some cultivars, such as the local Egyptian cultivar, Edkawi, show greater salt tolerance by demonstrating greater stability in growth with increasing salinity. Moreover, high salt tolerance has been reported for various wild tomatoes, such as
Lycopersicon peruvianum and
Solanum pennelli, which are salt-tolerant relatives of cultivated ones [
20].
Molecular evaluation of the genetic diversity among crop germplasm is a key for breeding and conservation of genetic resources. It is especially helpful as a general guide for selecting parents for breeding hybrids [
21]. The conservation of numerous collections of plant genetic resources represents the backbone of plant breeding programs; therefore, this genetic variability is considered the raw material for the crop breeding business, which relies on choices to evolve new superior genotypes. Molecular markers have been widely employed during the last decades for each assessment of original material and exploration of valuable plant phenotypes.
The conventional plant taxonomy relies on shared phenological, biochemical and ecological characteristics. In the past few years, many powerful molecular marker techniques were developed and applied in various research studies. The development of those techniques is principally the result of invasiveness in genomic studies. It has been initiated as a modern trend to develop a group of gene-targeting markers more powerful and effective than Random markers [
22]. As a result of the massive availability of genomic databases, the progress in developing efficient markers positioned inside or close to specific genes has become more straightforward [
3]. These gene-targeting markers have several applications, such as genetic diversity, molecular ecology, molecular phylogeny, genetic resources conservation and developmental biology. In 2009, Collard and Mackill developed two of the foremost effective gene-targeting molecular marker techniques in plants. These techniques are called Start Codon Targeted Polymorphism (SCoT) and Conserved DNA-Derived Polymorphism (CDDP).
The SCoT technique theory depended on the conserved regions flanking the start codon (ATG) in plant genes. The SCoT system was highly reproducible because of the utilization of an 18-mer single primer with a relatively high annealing temperature (50 °C) [
23]. SCoT is dominant, like the inter-simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) techniques. During the last decade, SCoT was effectively used in different applications such as genetic analyses, QTL-mapping and bulk segregant analysis. SCoT markers showed particularly significant success in diversity studies and fingerprinting in potato, grape, peanut and medicinal plants [
24].
Meanwhile, the CDDP technique was developed to produce DNA markers based on sequence-mining of short conserved amino acid parts within plant proteins. CDDP employed a singular primer within a length ranging from 15 to 19-mer and a Ta of 50 °C [
25]. In fact, gene-targeted marker systems such as CDDP and SCoT were evolved to join the traditional practices of random marker systems with powerful workflow through combining promoter or gene sequences in their primers [
26]. These characteristics of gene-targeted markers give it some advantages, such as improved resolution and good reproducibility.
In nature, plants evolve their non-enzymatic and enzymatic antioxidation mechanisms to counteract the adverse effects of salinity. The enzymatic antioxidation mechanisms include various enzymes such as peroxidase (POD), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR). In contrast, the non-enzymatic antioxidation mechanisms include compounds such as proline and vitamin C [
27]. Thus, the previous compounds could be serving as an indicator of salinity stress.
Therefore, the current study aimed to evaluate growth performance, yield parameters, bioactive compounds, plant hormones (peroxidase (POD), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR)), antioxidant enzymes and proline of different wild genotypes and commercial tomato hybrids under different salinity stress conditions. Additionally, to perform a molecular phylogeny and characterization of nine tomato cultivars/hybrids using two robust gene-targeting markers toward assessing their salinity tolerance.
4. Discussion
For the first time in this study, the genetic diversity between wild accessions and commercial cultivars/hybrids of tomato in terms of their salinity tolerance was analyzed using two modern functional markers (SCoT and CDDP). Genome-wide conserved regions across diverse plant species have sped the development of several functional markers such as CDDP and SCoT. These marker systems employ longer primers (15- to 19-mer) with higher annealing temperatures, which gives them more reproducibility and reliability than other random marker systems such as DAF or RAPD. Furthermore, they focus on coding regions, which makes them preferable over random markers in genome mapping applications [
29]. Collard and Mackill-b [
30] used conserved regions within groups of well-known plant gene families mainly involved in response to abiotic and biotic stresses or plant developmental stages to design CCDP primers. Conclusively, several reports advised employing the CDDP and SCoT markers in genetic analyses because they were developed based on functional regions of the genome with great expectations to be highly useful in crop improvement programs [
39].
Based on the obtained results, the two marker systems successfully differentiated between the nine tomato genotypes, although the percentage of polymorphism for ScoT was higher than CDDP (49.3% and 28%, respectively). Although, these results indicated that SCoT was more capable of discriminating between the tested genotypes than CDDP. Simultaneously, in terms of salinity tolerance, the CDDP was found to be more precise to cluster the five accessions (LA1995, LA2711, LA2485, LA3845, and Bark) characterized by better performance under salinity condition. This relatively high degree of precision may be because CDDP utilizes conserved regions of well-known plant gene families mainly involved in response to abiotic and biotic stresses. Our obtained results agree with the findings of two independent studies on durum wheat and chickpea, which also reported that CDDP markers proved superior over SCoT in studying genetic diversity across different durum or chickpea accessions [
40,
41]. Also, Poczai et al. [
42] reported that the CDDP marker had higher reproducibility than other traditional arbitrary markers. The technique could easily generate functional markers related to a given plant phenotype.
The tomato plant is classified as a moderate salt-tolerant crop. There are many considerable variations among its genotypes. Plant growth is an ideal index for evaluating various abiotic stresses on the plant. In the current study, shoot and root measured growth parameters of tomato plants decreased with increasing salt stress. Similar results have been obtained in previous studies [
5,
43]. Our results, in
Figure 3, indicated that root growth was negatively affected by salt stress, which affects foliage growth, in
Figure 2, plus water and ion uptake [
44]. All tested grafted rootstocks showed higher values of plant and root growth compared with the commercial hybrid. These rootstocks have the capability to enhance the salt tolerance of grafted tomato plants.
The harmful effects of saline stress on vegetative and root growth of plants might be due to the presence of some ion toxicity such as Na
+ and Cl
−, which create an ionic imbalance inside the plant cells (Chaichi et al. 2017 and our results in
Table 10), restriction of plant growth [
45] and a reduction in photosynthesis [
46]. In this study, the high concentration of Na
+ recorded in the shoots of tomato plants under salt stress may be due to an increase in Na+ accumulation in cellular vacuoles to achieve osmotic balance and allow the ideal photosynthesis operation of the tomato leaves [
47].
In the current study, the highest number of fruits, mean fruit weight, and total yield per plant was obtained with the control treatment (non-saline water), while the lowest values were obtained with the highest salt treatments (sub.3). Tomato fruit yield can be reduced either by a decrease in fruit weight or the number of fruit [
44]. The reduction of yield could be explained by the fact that high NaCl concentration decreases water potential in plants, which reduces water flow into fruit and limits the rate of fruit expansion [
48]. Moreover, decreased mean fruit weight could also be explained by an accumulation of Na
+ in plant tissue as supported by our results in
Table 10 and Incrocci et al. [
49]. LA3845 and LA4285 grafted rootstocks have higher total yield compared with control grafted rootstock (Bark).
Our results presented in
Table 8 and previous studies have exposed that tomato fruit quality such as TSS, vitamin C and firmness is affected in fruit from tomato plants irrigated with saline water [
44]. Despite the decrease of tomato yield irrigated with saline water, tomato fruit quality increased under saline conditions [
5]. Total soluble solids content in tomato fruits is considered to be one of the most critical factors influencing tomato quality. Our findings in
Figure 5B showed that TSS increased with increasing NaCl levels. Previous works have been reported that high levels of NaCl increased TSS content in tomato fruits [
2,
50]. This result could be due to lower fruit water content, as irrigation high salt level leads to increases in percentage of TSS [
51] or the adaptation of tomato plants to saline stress by increasing TSS in tomato fruits [
43].
The firmness of tomato fruits increased significantly (
Figure 5C) with increasing water salinity. The same result was observed by El-Mogy et al. [
2] and Abdelgawad et al. [
5], which supports the results of this study. Increases in firmness with increasing salt stress could either be due to that salinity firming tomato skin resulting in increases in its thickness [
52] or the presence of smaller cells with thicker walls in the pericarp of tomato fruits grown under salt stress [
53].
Ascorbic acid is considered to be one of the most important antioxidant compounds for human health. Our results in
Table 8 support the hypothesis that vitamin C increased with high salinity levels (100 and 200 mM NaCl). The same trend was reported by Marín et al. [
54], who found that the increase of nutrient solution salinity resulted in a rise in vitamin C content in red pepper fruit.
In the current study, the content of N, P, K, Ca, Mg, Fe and Zn in tomato shoots was lower under salinity stress (
Table 9 and
Table 10). On the other hand, in
Table 5, Na content was higher in plants irrigated with saline water (100 and 200 mM NaCl). Chaichi et al. [
55] and Zhu et al. [
56] indicated that the N, K, Ca and Mg contents in the aerial part of tomato plant were decreased at the high salinity level compared to the lower salinity level. It was reported that a shortage of mineral absorbance is negatively affecting the metabolic functions in plants [
57]. The results in
Figure 2 and
Figure 3 show a reduction in tomato plant growth (shoots and roots) and yield is due to the reduction of mineral absorbance under salinity stress. The higher Na content in plants that received saline water could be due to the accumulation of this element inside vacuoles to decrease cell water potential [
47]. Gibberellin (GA3) controls several important physiochemical processes inside plants, such as cambium activity, xylem fiber expansion and secondary growth [
58].
In general, abiotic stresses including salinity, drought and wounding increase abscisic acid ABA biosynthesis. As in this study (
Table 11), previous research has shown that ABA concentration is increased in tomato shoots under saline stress [
59]. This result might be due to ABA’s role in preventing water loss via transpiration and maintaining the water content of plant leaf [
58].
In this study, peroxidase activity (POD) increased with increasing salt stress (
Table 11). This result agrees with Rahnama and Ebrahimzadeh [
60], suggesting that POD was increased significantly at 100 mM NaCl in potato seedling leaves. It has been well known that ascorbate peroxidase (APX) is one of the major antioxidant enzymes involved in the ascorbate-glutathione cycle, which has a vital role in protecting the plants against salt stress and acting as reactive oxygen species (ROS) scavengers [
61]. Our results in
Table 11 indicated that APX activity in plant shoots was increased at high salt levels. The same results have been obtained in previous studies such as Mittova et al. [
62] and Murshed et al. [
63] in tomato. The increasing APX activity in tomato plant shoots under salt stress is one of the plant defense mechanisms that can recover the damage in tissue metabolism and the toxic levels of H
2O
2 [
64].
Abiotic stress, including salinity, is also responsible for ROS’s overproduction, which induces oxidative stress in plants [
65]. Dehydroascorbate reductase (DHAR) is one of the ROS scavengers in plants; produced under salinity stress to recover ROS overproduced. Our findings in
Table 11 showed that DHAR activity increased with increasing water salinity at a 100 and 200 mM NaCl concentration. This finding is supported by previous studies [
66,
67].
Non-enzymatic low molecular metabolites such as proline are acting as ROS scavengers. Accumulation of porline in plant tissues under salinity stress is counteracting ROS toxicity [
68]. In this study, proline content in plant shoots was significantly increased with increasing salinity stress (
Table 11). Our results agree with Kahlaoui et al. [
69], who found that proline content in tomato increased with increasing salt levels.
Our results suggest that grafting is an alternative technique for enhancing salt tolerance in tomato plants. Under saline stress conditions, plant growth and fruit yield of most graft combinations were significantly higher than that of the commercial hybrid Barak on its rootstock.