Construction and Phenotypic Characterization of a Recombination Inbred Line (RIL) Population from a Melo-agrestis Melon Hybrid

Abstract

Melon (Cucumis melo L.) is an economically important horticultural crop worldwide, while its production is continuously endangered by powdery mildew (PM), a fungal disease mainly caused by Podosphaera xanthii, due to the insufficiency of disease resistant germplasms. Here, a melon recombinant inbred line (RIL) population that consisted of 188 independent individuals was obtained through the crossing of ‘SN-1’ (C. melon L. ssp. melo) and ‘YJM’ (C. melon L. ssp. agrestis), two parents with contrasting PM resistance, followed by 7-round selfings. Comprehensive phenotypic investigation revealed substantial variations in key agronomic traits among these RILs, such as stem diameters of 3.7~12.6 mm and internode lengths of 1.6~12.2 cm at the anthesis stage, as well as peduncle lengths of 0.5~9.5 cm and soluble solid content of 1.6~17.4% at the maturation stage. Particularly, 95 RILs, of which 60 and 35 belonged to thin-peel and netted types, respectively, were identified to be highly resistant to P. xnathii infection, providing new germplasms for melon improvement. Altogether, the generation of this melo-agrestis RIL population, together with the phenotypic observations, lays a solid foundation for mechanistic investigation of the traits with economic importance and could contribute to future breeding programs of melon cultivars with PM resistance.

1. Introduction

Melon (Cucumis melo L.), one of the most economically important vegetable crops, is widely cultivated all over the world, especially in temperate, subtropical and tropical regions [1,2]. Its wild relatives are widely distributed in Africa, Asia and Australia [3,4], and at least three independent domestication events, two in Africa and one in Asia, have been demonstrated for current cultivation types [5]. The maintenance and efficient utilization of these wild, semi-wild and cultivation germplasms are crucial considerations for the continuous genetic improvement of melon traits with economic importance, such as sugar content and disease resistance, worldwide [6].

During cultivation period, melon plants are commonly subjected to a variety of diseases, among which powdery mildew is considered as one of the most severe challenges [7]. Three fungal pathogens, namely Leveillula taurica, Golovinomyces orontii and Podosphaera xanthii, have been reported to infect Cucurbit family plants [8]. In China, melon powdery mildew (PM) is mainly caused by P. xanthii, and races 1 and 2F of this pathogen species are predominant [9]. Harmful impacts of PM on melon plants limit photosynthesis, growth and reproduction, thus leading to yield loss and low fruit quality [10]. To cope with this disease, multiple control strategies have been proposed in melon production. Chemical control, wherein chemical/organic fungicides and inorganic chemicals are often adopted, is the most widespread and effective method to manage melon PM [11]. For instance, in an early study carried out by Warkentin et al., sulfur and dinocap formulations were used for managing PM of field pea, and both fungicides are effective in reducing disease severity and increasing seed weight and final yield [12]. Chemical fungicides, such as hexaconazole and flutianil, have subsequently been developed to protect host crop plants from PM invasion [13,14]. Recently, Wang and colleagues reported that melon PM resistance is apparently improved via exogenous spraying of potassium bicarbonate (KHCO₃) due to the stimulated reactive oxidative species (ROS) and phenylpropanoid metabolic pathways [15]. However, high risk of drug resistance greatly limits the long-term protective efficacy of major fungicides against PM [16].

Biological control has attracted increasing attention for PM treatment and prevention of different crops [17]. For example, in a study regarding wheat PM caused by Blumeria graminis f. sp. tritici, Zhang et al. investigated the effects of Aspergillus chevalieri BYST01 on wheat PM, and the beneficial roles of this termite-associated fungus together with its main metabolite physcion have been revealed to effectively fight against mildew pathogens [18]. For cucurbit plants, biological agents, such as Bacillus bacteria, epiphytic fungi and other mycoparasitic microorganisms, have been used to control PM [7]. However, there are several disadvantages of this strategy, including environment dependency, strain specificity, limited residual activity, as well as compatibility and cost issues, constraining its efficacy and usage in PM management.

In contrast to the abovementioned strategies, the creation and breeding of melon germplasms/cultivars with PM resistance is considered a more effective and environment-friendly approach to control PM [19]. Different genetic populations have thus been constructed to both identify genes responsible for important agronomic traits, such as PM resistance, and develop trait-associated molecular markers, providing foundations of melon germplasm creation and new cultivar breeding [20]. In a study carried out by Tijskens et.al., a melon near-isogenic line (NIL) population derived from the crossing of ‘Shongwan Charmi’ and ‘Piel de Sapo’ has been prepared for genetically exploring postharvest hardness of fruits, and revealed that a small variation in asymptotic end value together with a low end value as to ascertain edibility could be used as a good indication of the usefulness of certain NILs for commercial application [21]. Recombinant inbred line (RIL) populations, which are often generated by several-round selfings of single-seeded progenies from two or more parents with significant genotypic and phenotypic variations, can serve as powerful tools to dissect the genetic bases of quantitative traits of interest as well [22]. For example, Branham et al. have developed an ‘MR-1’ x ‘Ananas Yokneam’ RIL population that is composed of 172 homozygous lines, and a high-density genetic map harboring 5663 small nucleotide polymorphisms (SNPs) has been drawn based on ‘DHL92’ reference genome [23]. With the aid of those SNP markers, four quantitative trait loci (QTLs) that are associated with melon resistance to Fusarium oxysporum f. sp. melonis race 1 have further been detected [23]. Recently, two major QTLs associated with melon resistance to downy mildew, a devastating disease caused commonly by Pseudoperonospora cubensis [(Berkeley & M. A. Curtis) Rostovzev], were unveiled in melon genome via systematically screening a RIL population [24]. In spite of its great potentials in functional genomics and cultivar breeding, the current usage of RIL populations remains largely limited for melon in comparison to field crops such as rice and maize.

In the present study, an RIL population was constructed by crossing ‘SN-1’, a netted melon inbred line with PM immunity that was used as the male parent, with ‘YJM’, a thin-peel inbred line with high-yield and -quality fruits that was used as the female parent. Phenotypic variations in a set of agronomic important traits, particularly PM resistance, were comprehensively characterized for the RIL population. These observations could, on one hand, benefit functional genomics studies of melon, and on the other hand, facilitate the breeding of new cultivars with PM resistance.

2. Materials and Methods

2.1. Plant Materials and Cultivation Conditions

A melon (Cucumis melo L.) RlL population consisted of 188 independent individuals that were used as plant materials in this study. This population was originated from the crossing of ‘SN-1’, a melo inbred line immune to PM, and ‘YJM’, an agrestis inbred line with high-yield and -quality fruits, followed by 7 generations of self-pollination. To phenotypically characterize the population, 20 seeds of each RIL were subjected to surface-sterilization by 1.5% (w/v) sodium hypochlorite and 3-h incubation in 55 °C ddH₂O [25], and thereafter, kept at 28 °C in darkness. After germination, the seeds were sown in nutrient soil (peat:vermiculite:pearlite = 2:1:1). At three-leaf stage, the seedlings were transplanted to a greenhouse in the experimental field of Shandong Agricultural University, Tai’an, Shandong Province, China. The growth conditions in the greenhouse were maintained at daytime temperature of 26~28 °C, nighttime temperature of 15~18 °C, and photoperiod of 10~12-h. Normal field management was carried out from February to June, 2025. Vegetative growth-related parameters were investigated at anthesis stage [~44 days after sowing (DAS)], and premature senescence, sex expression and powdery mildew resistance were evaluated at fruit-setting stage (~62 DAS). Fruit-related parameters were finally determined at maturation stage [~43 days after pollination (DAP)]. At least three biological repeats were performed for each parameter.

2.2. Evaluation for Vegetative Growth of RILs

Leaf-related parameters, including petiole length, as well as leaf length and width, were measured using a stainless ruler for the eleventh to thirteenth true leaves, which were counted from the bottom of each plant. Leaf lobe was investigated through a multi-person evaluation method, wherein five experienced persons were invited to independently observe and score RIL leaves. Based on the evaluation results, leaf shape was categorized into three different groups, entire, intermediate and deeply lobed ones, according to the previously described method [26]. Stem diameter of the fourth node and internode length of the fourth to sixth nodes, which were counted from the bottom of each plant, were measured with a digital vernier caliper and a stainless ruler, respectively. For the parameters of petiole length, leaf length, leaf width and internode length, the mean of values form different positions of the same plant was used for each biological repeat.

2.3. Assay for Premature Senescence, Sex Expression and Powdery Mildew Resistance of RILs

For premature senescence investigation, visual assessment was carried out for the overall appearance of melon plants, including leaf color, size and density by following Muchero et al.’s method [27]. The sex phenotype of each RIL line was determined by examining the morphology of its Pistillate flowers during the peak fruiting period. The parental line ‘SN-1’ is monoecious and produces separately male and female flowers. In contrast, the parental line ‘YJM’ is andromonoecious and yields both perfect and staminate flowers. Therefore, classification of the RILs was based solely on the type of their Pistillate flowers: lines that produced female flowers were classified as monoecious, while lines that produced bisexual flowers were classified as andromonoecious [28].

Disease resistance assays were conducted under field conditions in the abovementioned greenhouse. To maintain sufficient disease pressure, no fungicides were applied over cultivation period, and resistance performance of RIL plants were then evaluated based on natural infection by P. xanthii. At the time point of investigation, disease symptoms of individual plants were visually assessed. RILs showing no visible signs of white, powdery mycelia were classified as ‘resistant’ ones, while those exhibiting any symptoms were classified as ‘susceptible’ ones [29,30].

2.4. Evaluation for Fruit-Related Traits of RILs

For fruit-related trait evaluation, five fruits per RIL were sampled to measure peduncle length and diameter using a millimeter ruler and a digital vernier caliper, respectively. Soluble solid content was measured with a hand-held optical refractometer (ATAGO PAL-3 ATAGO CO., LTD, Tokyo, Japan) from fresh extracted juice. Fruit shape, as well as peel color and nets, was visually assessed according to the method described by Park et al. [31]. Briefly, ten experienced persons were invited to independently evaluate the three characteristics at fruit ripening stage: fruit shape was classified as elliptic, ovate, long-horn, or round; peel color was classified as yellow or dark green; and netting was classified based on its presence or absence.

2.5. Data Processing

All phenotypic and physiological data, including leaf length, leaf width, petiole length, internode length, stem diameter, pedicel length, pedicel diameter and soluble solid content (SSC), were recorded and organized using Microsoft Excel 2019. Subsequent statistical analysis and visualization were performed using GraphPad Prism 9.0. Descriptive statistics, including the mean, standard deviation (SD) and range (minimum and maximum values), were carried out for each trait, and the data distribution was further assessed using both the D’Agostino—Pearson test and the Shapiro–Wilk test. To evaluate the level of variation among the materials, a one-way analysis of variance (ANOVA) was conducted, and the coefficient of variation (CV, %) was also calculated for each quantitative trait to compare their relative levels of dispersion. A p-value no more than 0.05 was considered to indicate statistical significance.

3. Results

3.1. Variations of Leaf-Related Traits in the RIL Population

Leaf is crucial for final yield and fruit quality due to its profound involvement in photosynthesis [32]. We first measured petiole length of each RIL, and the values of this parameter in the population ranged from 8.2 cm (RIL ‘07-13’) to 30.5 cm (RIL ‘19-1’) with a mean of 18.72 cm and a coefficient of variation of 0.20 (Figure 1A and Table S1). Notably, petiole length was 18~22 cm for over 45% RILs, while only 20% RILs had petioles shorter than 14 cm or greater than 24 cm (Figure 1A and Table S1). The Shapiro–Wilk test suggested that the datasets of petiole length from RILs well fitted a normal distribution (p-value = 0.54), of which the skewness and kurtosis values were 0.256 and 0.072, respectively (Figure 1A).

Leaf length and width were subsequently investigated for RILs, and large variations were observed in both parameters. For leaf length, the values ranged from 11.1 cm, which was detected for RIL ‘16-8’, to 29.6 cm, which was represented by RIL ‘07-13’, with a mean of 20.2 cm and a coefficient of variation of 0.13 (Figure 1B and Table S1). For leaf width, we observed that the minimum and maximum values were 11.9 cm for RIL ‘33-4’ and 35.5 cm for RIL ‘27-14’, respectively, with a mean of 23.9 cm and a coefficient of variation of 0.14 (Figure 1C and Table S1). A normal distribution was further revealed for both parameters, of which the p-value, skewness and kurtosis values were 0.55 and 0.09, 0.14 and −0.38, and 0.36 and 0.51, respectively (Figure 1B,C).

We further evaluated leaf shapes of RILs based on their lobes, and three different groups were uncovered: (1) RILs with entire leaves, such as ‘04-5’, of which the number was 62 and accounted for 33.0% of the population; (2) RILs with intermediate leaves, such as ‘32-4’, of which the number was 58 and accounted for 30.9% of the population; and (3) RILs with deeply lobed leaves, such as ‘20-7’, of which the number was 68 and accounted for 36.1% of the population (Figure 1D,E).

3.2. Variations of Stem Diameter and Internode Length in the RIL Population

Stem diameter and internode length are commonly used as parameters of evaluating plant vigor that is greatly involved in the determination of fruit quality, size and final yield [33,34]. We first measured stem diameter of RIL plants, and the values of this parameter ranged from 4.85 mm (RIL ‘11-3’) to 11.27 mm (RIL ‘21-8’) with a mean of 7.83 mm and a coefficient of variation of 0.19 in the population (Figure 2A and Table S1). For internode length, we found that the values of this parameter in the population ranged from 1.9 cm (RIL ‘16-11’) to 10.7 cm (RIL ‘16-3’) with a mean of 5.02 and a coefficient of variation of 0.33 (Figure 2B and Table S1). The Shapiro–Wilk test for stem diameter and internode length revealed that the datasets of both parameters well fitted the normal distribution, with p-value, skewness and kurtosis values being 0.43, 0.33 and 0.58 for the former, as well as 0.25, 0.38 and 0.51 for the latter (Figure 2).

3.3. Sex Expression and Premature Senescence in the RIL Population

Floral sexuality is a critical consideration for the selection and breeding of new melon varieties [35]. We characterized sex expression in the population, and two sexual systems were observed: (1) monoecious RILs, which produced male and female flowers separately on the same plant and accounted for 46.4% of the population; (2) andromonoecious RILs, which bore both bisexual and male flowers on the same plant and accounted for 53.6% of the population (Figure 3A,B and Table S1). Premature senescence refers to the earlier initiation of deterioration events, such as leaf yellowing, vigor reduction and photosynthetic worsening, during the late stage of plant lifecycle upon either exogenous and endogenous stimuli, thus negatively impacting fruit quality and final yield [36]. We further assessed the occurrence of premature senescence in the population. The results showed that, during the fruit-setting stage, the occurrence of senescence-related events were discovered in 74 RILs to different extents, while at the same time, the left 114 RILs displayed normal growth and no apparent deterioration was observed (Figure 3C,D).

3.4. Variations of Fruit-Related Traits in the RIL Population

Peduncle, a stalk-like structure that connects the fruit to the main stem of plant, plays crucial roles in the support and storage of melon fruits [37]. We first investigated peduncle diameter in the population, and the minimum value was detected as 1.29 mm for RIL ‘15-1’, and the maximum value was detected as 8.03 mm for RIL ‘13-3’, with the mean of 5.19 and the coefficient of variation of 0.20 (Figure 4A and Table S1). For peduncle length, the minimum and maximum values of this parameter were revealed as 0.5 cm for RIL ‘10-3’ and 9.5 cm for RIL ‘27-14’, with the mean of 3.43 cm and the coefficient of variation of 0.41 (Figure 4B and Table S1). The Shapiro–Wilk test for peduncle diameter and length demonstrated that the datasets of both parameters were well in agreement with the normal distribution, with p-value, skewness, and kurtosis values being 0.06, 0.08 and 1.08 for peduncle diameter, as well as 0.34, 0.31 and 0.02 for peduncle length (Figure 4A,B). Correlation analysis between peduncle diameter and length unveiled a coefficient value of 0.12 (Table S1), implying that both traits might be inherited independently in the population. For soluble solid content, a large variation was observed in the population, with the minimum and maximum values being 1.6% for RIL ‘23-12’ and 17.4% for ‘03-5’, as well as the mean of 8.27% and the coefficient of variation of 0.29 (Figure 4C and Table S1). Statistical analysis by following the rules of the Shapiro–Wilk test showed that the datasets of this parameter just fitted a non-normal model with p-value of 0.06, and kurtosis and skewness values of 0.46 and 0.176, respectively (Figure 4C).

Fruit appearance-related parameters, including fruit shape, peel color and smoothness, were further evaluated in the population. We found that, based on the shape, RILs in the population were generally divided into four groups: (1) RILs with elliptic-shape fruits, of which the number was 88 and accounted for 46.8% of the population; (2) RILs with ovate-shape fruits, of which the number was 15 and accounted for 8.0% of the population; (3) RILs with long-horn-shape fruits, of which the number was 58 and accounted for 30.9% of the population; and (4) RILs with round-shape fruits, of which the number was 27 and accounted for 14.4% of the population (Figure 4E). Regarding peel color, we found that 31.8% of RILs in the population generated yellow-peeled fruits, and the left RILs generated fruits with dark-green peels (Figure 4F,G). Moreover, 75 RILs were observed to generate netted fruits, which accounted for 39.8% of the population, and the left 113 RILs generated thin-peel fruits, which accounted for 60.2% of the population (Figure 4H,I). Intriguingly, we found that the predominant proportion of fruits from dark-green peeled RILs simultaneously were netted, evidencing the possible coinheritance of both traits in the population.

3.5. Resistance of Different RILs to Powdery Mildew Infection

As one of the most important focus traits, we evaluated powdery mildew resistance of RILs in field conditions. The occurrence of powdery mildew, to different extents, were revealed for 93 RILs, which accounted for 49.5% of the population. In contrast, no mildew spots were observed in the left 95 RILs, demonstrating their good performance in the resistance to powdery mildew infection (Figure 5). These disease-resistant RILs could provide new alternative germplasms to breed melon cultivars with powdery mildew resistance.

4. Discussion

Recently, global market demand for new melon varieties, such as high-quality and disease-resistant ones, is continuously increased [38]. To satisfy these new targets of melon breeding, different genetic populations have been developed in the past decades [39]. Among these breeding populations, RIL populations are usually used as genetic resources for mapping genetic loci associated with agronomic important traits [40], while the usage of this strategy remained largely limited in germplasm creation and cultivar breeding of melon when compared to field crops. Here, an RIL population was constructed from the crossing of a high-quality and high-yield agrestis inbred line ‘YJM’ and a disease-resistant melo inbred line ‘SN-1’, and a set of agronomic important traits were systematically investigated in the agrestis-melo population. In general, we found that most of the traits that were explored in this study, such as stem diameter, as well as the length of internode, leaf petiole and fruit peduncle, well conformed to a normal distribution, revealing their genetically quantitative essence. Similar results have been reported in previous studies [41,42,43].

To maximize its productivity, high-density cultivation is recommended for melon plants, particularly under greenhouse conditions. Ideal melon cultivars suitable for high-density planting should exhibit specific architectural modifications, such as shorter leaf petioles and less lush growth, to optimize resource distribution between vegetative and reproductive growth [44]. In this population, we identified 14 RILs, of which petiole length was less than 14 cm, potentially being adopted in the breeding program of compact cultivars. In addition to petiole length, stem diameter and internode length are functionally interconnected traits that influence plant architecture and yield potential [45,46]. Here we identified a set of RILs displaying phenotypic variations with significant breeding potential in either stem diameter or internode length. For example, RIL ‘11-7’, of which stem diameter and internode length were 7.51 mm and 3.0 cm, respectively, possessed compact architecture ideal for breeding new cultivars used for greenhouse production. Another example was RIL ‘15-6’, of which stem diameter and internode length were 8.75 mm and 10.3 cm, respectively, was considered as a valuable germplasm for improving melon dwarf cultivars such as ‘X090’ [45].

Previous studies have demonstrated that properly retaining the fruit peduncle effectively reduces the loss of dry matter and nutrients, thus extending the storage period of melon fruits [47]. Meanwhile, for postharvest processing purpose, melon cultivars that generate fruits with relatively shorter and robust peduncles are preferred due to their easy picking, transportation and storage [19]. In this population, we identified 36 RILs that produced fruits with peduncle length greater than 5 cm, potentially being used for breeding new melon cultivars for long-term storage. Additionally, several RILs, such as ‘15-1’ and ‘09-5’, were found to yield fruits with both shorter (the length less than 3 cm) and more robust (the diameter greater than 6 mm) peduncles, providing new germplasm resources to breed melon cultivars suitable for processing. Soluble solid content, primarily composed of sugars, organic acids and soluble polysaccharides, is a key biochemical parameter for evaluating melon fruit quality, and a major driver of consumer preference and marketability [48]. We analyzed this parameter in the population, and a large range of variations was detected with the minimum, maximum and mean of 1.6%, 17.4% and 8.71%, respectively. These RILs with variable soluble solid contents provide a valuable genetic resource for improving the quality of melon fruits.

Peel color and smoothness, which are governed by complex biochemical processes, serve as critical morphological and physiological indicators of ripeness, postharvest quality and consumer preference [49]. Results from previous studies have unveiled a series of loci, such as ‘CmAPRR2’ and ‘CmSN’, genetically associated with both traits, respectively [50,51]. In the present population, we identified that, 71 out of 75 RILs with netted fruits had dark-green peel, reflecting the strong linkage of both traits. To unveil the molecular mechanism underlying this association could be helpful in developing novel cultivars with diverse combinations of peel characteristics.

Powdery mildew resistance is a critical consideration in melon breeding, particularly for that used for greenhouse cultivation [7]. Creation of melon germplasms with powdery mildew resistance has become an urgent need of farmers and thus attracted extensive attentions from researchers and breeders [19]. In the present study, 95 RILs displayed high resistance to powdery mildew, while the left 93 RILs were susceptible to mildew challenge, conforming to a 1:1 segregation ratio for this trait. This segregation ratio suggested that powdery mildew resistance in the population could be controlled by a single locus with two allelic forms, thus contributing to future marker development, gene cloning and precision breeding. Additionally, premature senescence, which negatively impacts agricultural productivity [52], was assessed in the population. We found that, out of 74 RILs exhibiting premature senescence, 57 were also susceptible to powdery mildew as well. This association could be explained at least partially by the reduced plant fitness upon the early initiation of senescence process. Similar results have been reported in field crops such as wheat [53] and rice [54], as well horticultural crops such as lily [55] and tomato [56].

5. Conclusions

An RIL population from melo-agrestis melon hybrid was successfully established and characterized via systematically evaluating a set of agronomic important traits, providing valuable phenotypic data resources for further mechanistic studies. Moreover, we obtained some RILs, such as ‘16-15’ and ‘05-1’, which displayed good performance in not only vegetative growth and fruit-related traits but powdery mildew resistance, potentially benefiting the genetic improvement of melon disease resistance in future breeding programs.