Molecular and morphological assessment of cucumber germplasm leading to the discovery of ToLCNDV resistance

Introduction

Cucumber is believed to have originated in India (De Candolle, 1886) [3] , which remains an important centre of diversity and primary gene pool for the species. It belongs to the genus Cucumis, which contains two major domesticated species: C. sativus (2n = 14) and C. melo (2n = 24). The cultivated cucumber and its wild progenitor, C. sativus var. hardwickii, form part of the Asiatic group distributed along the Himalayan foothills. Despite intensive global cultivation, modern cucumber cultivars often possess a narrow genetic base, increasing their vulnerability to rapidly evolving pathogens and emerging climatic stresses. Therefore, characterization and utilization of indigenous Indian germplasm are critical for broadening the genetic base and enhancing resilience. In recent decades, tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus belonging to the family Geminiviridae and transmitted by the whitefly Bemisiatabaci, has emerged as one of the most destructive viral pathogens affecting cucurbits. Since its initial detection in India in 1995 (Padidam et al., 1995) [18] , ToLCNDV has rapidly expanded its geographical distribution, causing epidemics across Asia, the Mediterranean basin, Africa, and parts of Europe. The virus poses a serious threat not only to cucumber but also to several other key crops such as tomato, pumpkin, zucchini, and bitter gourd. Under conducive Conditions, yield losses due to ToLCNDV can reach up to 100%, making it one of the most economically significant pathogens of cucurbits (Czosnek et al., 1997; Zaidi et al., 2016) [2] . Despite the severity of ToLCNDV outbreaks, no verified sources of resistance have been identified in cultivated cucumber. This absence of confirmed resistance poses a significant bottleneck for breeding programs aiming to develop durable ToLCNDV-resistant varieties. However, given its Indian origin and the rich genetic diversity within local landraces, wild relatives, and indigenous germplasm, cucumber offers significant potential to uncover novel alleles conferring resistance to ToLCNDV and other viral pathogens. Recent studies have emphasized that resistance to leaf curl and related begomoviruses is often found within Indian cucurbit germplasm (Saez et al., 2021), further underscoring the importance of systematic screening efforts. Therefore, identification and characterization of ToLCNDV resistance sources in Indian cucumber germplasm is an urgent research priority. Such efforts are essential not only for breeding disease-resistant cultivars but also for enhancing the overall genetic resilience of cucumber production systems. Integrating agro-morphological evaluation, molecular marker analysis, and field disease screening provides a comprehensive framework for identifying promising germplasm that can serve as valuable parental lines in future breeding programs.

Materials and Methods

Experimental site and plant material

The field experiment was conducted at the research farm of the Division of Vegetable Science, ICAR-Indian Agricultural Research Institute (IARI), New Delhi (28°35′ N, 77°12′ E; 228.6 m above mean sea level). The site has a semi-arid, sub-tropical climate with hot summers and mild winters. The experimental soil was loam in texture with an organic matter content of 0.75%. A total of 100 cucumber (Cucumis sativus L.) germplasm lines, maintained at the Division of Vegetable Science, IARI, were evaluated during the summer season of 2020. The trial was laid out in a Randomized Block Design (RBD) with three replications. Each genotype was grown in a single row per replication, and recommended agronomic practices were followed to ensure uniform and healthy crop establishment. No insecticides were applied during the crop cycle to facilitate natural infestation by whitefly (Bemisiatabaci), the vector of Tomato leaf curl New Delhi virus (ToLCNDV). During the cropping period, the field exhibited pronounced leaf curl symptoms, and ToLCNDV was confirmed as the predominant causal agent in a parallel diagnostic study conducted at ICAR-IARI. This ensured a uniform and reliable natural disease pressure, enabling robust evaluation of genotype responses to ToLCNDV under field conditions.

Assessment of horticultural traits and disease scoring for ToLCNDV

Observations on horticultural traits were recorded for all cucumber genotypes evaluated under a randomized block design. Data from ten randomly selected plants per genotype per replication were used to compute replication-wise means, which were subsequently utilized for statistical analyses. The study comprised three replications. Quantitative traits recorded included node to first male flower, node to first female flower, days to first female flower anthesis, number of fruits per plant, fruit length (cm), fruit diameter (cm), average fruit weight (g), vine length (cm), and yield per plant (g). For disease assessment, tomato leaf curl New Delhi virus (ToLCNDV) scoring was performed on selected plants across all three replications. Disease severity was assessed visually based on characteristic viral symptoms. Observations were recorded at 10-day intervals on three dates during the cropping period: 35, 45, and 55 days after sowing. The mean score across these three assessment points was used to classify each genotype's response to ToLCNDV. ToLCNDV symptom severity was evaluated using the 0-4 scoring scale described by Saez et al. (2020) for Cucurbita moschata, Cucumis melo, and C. sativus. Based on this scale, plants with a disease score of 0 or 1 were categorized as resistant, whereas plants scoring between 2 and 4 were classified as susceptible (Fig A ). The overall disease reaction of each genotype was further categorized using the vulnerability index (VI), calculated following Havey (1996) [7] :

Where, n: represents the number of plants in each disease category, nt: total number of plants screened and nc: denotes the number of categories.

DNA extraction and SSR based genotyping

Genomic DNA was extracted from fresh leaf samples collected from healthy, field-grown plants at the Delhi site using the standard CTAB method (Doyle and Doyle, 1990) [5] . DNA quantity and integrity were initially assessed on a 0.8% agarose gel. Further evaluation of DNA purity and precise concentration was performed using a Nano Drop spectrophotometer (Eppendorf). The DNA samples were subsequently diluted in TE buffer to obtain a working concentration of 25-30 ng µl⁻¹. A total of 427 genomic SSR primers specific to Cucumis sativus were screened for PCR amplification. The sequence information for SSR markers that yielded polymorphic amplicons is provided in Table S1. PCR amplification was carried out in a 10 µl reaction volume containing 50 ng genomic DNA, 1.0 U Taq DNA polymerase (HiMedia Laboratories, Mumbai, India), 1× PCR buffer with 1.5 mM MgCl₂, 10 pmol each of forward and reverse primers, and 100 µM dNTP mix (Thermo Scientific). Amplification was performed using a touchdown PCR protocol in an Eppendorf Mastercycler with the following conditions: an initial denaturation at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 1 min, primer annealing at 55-65 °C for 1 min (depending on primer-specific annealing temperature), and extension at 72 °C for 2 min; followed by a final extension at 72 °C for 10 min. Amplicons were held at 4 °C until retrieval and subsequently stored at -20 °C.

Electrophoresis and fragment detection

Amplified PCR products were mixed with 1 μl of 1× loading dye (bromophenol blue) and separated on 3% agarose gels prepared in 1× TAE buffer. Gels were stained with ethidium bromide, and DNA bands were visualized under UV light using a gel documentation system (Alpha Imager, Cell Biosciences, Santa Clara, CA). Amplicon sizes were scored manually using a 50 bp DNA ladder as the reference. A binary data matrix was generated based on amplicon size. Power Marker v3.25 (Liu and Muse, 2005) [14] was used to calculate polymorphism information content (PIC), major allele frequency, and to conduct cluster analysis. Genetic similarity indices were computed using Nei's coefficient (Nei and Li, 1979) [17] .

Statistical and molecular data analysis

Agro-morphological data were subjected to analysis of variance (ANOVA) using the online statistical package OPSTAT (http://14.139.232.166/opstat/). Mean and standard deviation values for each trait were computed using Microsoft Excel 2019. Diversity analysis and dendrogram construction for cucumber genotypes were performed using DAR win version 6.

Results

Analysis of variance and trait variability

Analysis of variance revealed highly significant differences among the cucumber genotypes for all nine horticultural traits, indicating substantial genetic variability. Traits including node to first male and female flower, days to first female flower anthesis, number of fruits per plant, fruit length, fruit diameter, average fruit weight, vine length, and fruit yield per plant showed wide and significant variation. Mean performance across the 100 genotypes also exhibited broad phenotypic variation. The node-to-first-male-flower averaged 4.78, with gynoecious lines such as DGC-102 and DGPC-58 producing no male flowers. The first female flower appeared between nodes 2.28 (769, AZMC-1) and 13.82 (DC-48), with a population mean of 7.02. Days to first female flower anthesis ranged from 26.58 (DGC-104) to 44.92 (Chaitali), with a mean of 37.94 days. Fruit number per plant ranged from 2.33 (DC-921) to 8.67 (DC-91), averaging 4.74. Fruit length varied from 5.65 cm (DC-70-26-1) to 15.27 cm (WBC-231) and fruit diameter from 1.76 cm (DC-70-26-1) to 4.92 cm (DC-209), with means of 10.76 cm and 3.43 cm, respectively. Average fruit weight ranged between 25.33 g and 158.82 g (mean 94.08 g). Vine length ranged from 60 cm (JLG) to 351.99 cm (DGC-103), with a mean of 139.22 cm. Fruit yield per plant showed extensive variation, ranging from 138.16 g to 991.19 g (DC-91), with a mean of 425.37 g. This range highlights considerable scope for selection in cucumber improvement.

Genetic parameters and trait associations

Genetic variability was further supported by high genotypic and phenotypic variances, particularly for fruit yield per plant (35046.02 and 38453.06), vine length (3631.94 and 3657.21), and average fruit weight (1265.04 and 1370.11). The highest GCV and PCV values were observed for node to first male flower (50.12% and 50.76%), fruit yield (44.00% and 46.09%), and vine length (43.28% and 43.43%). All traits exhibited high heritability, notably vine length (0.99), node to first male flower (0.97), and fruit yield per plant (0.91), indicating strong genetic control and the potential for effective selection (Table 1). Phenotypic correlation analysis showed that fruit yield per plant had strong positive associations with average fruit weight (0.689), fruit diameter (0.761), fruit length (0.748), and vine length (0.459). Average fruit weight correlated strongly with fruit diameter (0.761) and fruit length (0.748). Conversely, number of fruits per plant showed negative correlations with fruit weight (-0.388), fruit length (-0.327), and fruit diameter (-0.315). These results identify fruit weight, fruit length, and vine length as key yieldcontributing traits.

Genetic divergence and cluster analysis

Genetic divergence assessed through the Hierarchical cluster analysis and Mahalanobis D² statistic grouped the 100 cucumber genotypes into four distinct clusters (Fig. C), with Cluster II being the largest, followed by Cluster I. The wide inter-cluster distances, particularly between Clusters II and I, suggest their potential for use in hybridization programmes. These genetically diverse clusters can be exploited to generate high-yielding, transgressive segregants owing to their broad genetic bases.

Molecular diversity

Molecular diversity among 96 cucumber genotypes was assessed using 59 polymorphic SSR markers out of 427 screened. A total of 295 alleles were amplified, with allele numbers per locus ranging from 3 to 8 and a mean of 5.0. The highest number of alleles ( 8) was observed with primers UW039642, SSR02771, UW084969, and SSR13511. PIC values ranged from 0.22 (UW085401) to 0.75 (UW084908), with an average of 0.55, indicating good marker informativeness. Gene diversity ranged from 0.24 to 0.78, and heterozygosity varied from 0 to 0.68 (mean 0.06), the highest being recorded for UW08450.

Evaluation of cucumber germplasm for resistance to ToLCNDV

A total of 100 cucumber genotypes were systematically screened under natural field conditions during the rainy season, a period conducive to high whitefly populations and severe Tomato Leaf Curl New Delhi Virus (ToLCNDV) pressure. The vulnerability index (VI), used as a quantitative measure of disease severity, showed considerable variation among the genotypes, ranging from as low as 18.75% to as high as 97.92%. This wide phenotypic spectrum clearly indicates substantial genetic diversity in the panel with respect to ToLCNDV response. Among the evaluated materials, three genotypes DC-61 (18.75%), DC-70 (24.95%), and DC-91 (25.35%) consistently exhibited minimal symptom development, placing them in the highly resistant category. These genotypes maintained normal growth, flowered on time, and produced relatively healthy fruits despite intense disease pressure, highlighting their strong tolerance and potential underlying resistance mechanisms. In contrast, seven genotypes displayed moderate resistance, limiting disease progression to intermediate levels, whereas the majority 54 genotypes were categorized as moderately susceptible. These genotypes showed noticeable symptoms but still retained partial tolerance. The remaining thirty-six genotypes recorded VI values approaching the upper range and were classified as susceptible, exhibiting severe leaf curling, stunting, and significant yield reduction (Table 2). The presence of highly resistant accessions such as DC-61, DC-70, and DC-91 is particularly promising for resistance breeding programmes. Their stable performance under natural epiphytotic conditions makes them potential donor lines for introgressing ToLCNDV resistance into elite, highyielding cucumber backgrounds. Incorporating these resistant sources into hybridization and molecular breeding strategies could accelerate the development of durable ToLCNDV-resistant cucumber cultivars suited for cultivation across disease-prone regions.

Discussion

The evaluation of 100 cucumber genotypes under natural field conditions with high Tomato leaf curl New Delhi virus (ToLCNDV) pressure revealed substantial genetic variability for yield, yield components, and disease resistance. The high mean sums of squares for vine length, average fruit weight, and fruit yield per plant demonstrate the presence of considerable genetic divergence that can be effectively utilized in breeding programmes. As phenotypic variances consistently exceeded genotypic variances, a moderate influence of the environment on trait expression is evident. Comparable trends have been documented in earlier studies on cucumber by Kumar (2006) [11] , Munshi et al. (2007) [16] , and Singh et al. (2013) [25] , validating the robustness of the present findings. Traits such as node to first male flower, fruit yield per plant, and vine length exhibited high genotypic and phenotypic coefficients of variation, indicating a broad scope for improvement through phenotypic selection. The relatively narrow gap observed between GCV and PCV for most traits suggests a predominance of additive genetic variance and a minimal role of environmental fluctuation, thereby increasing the reliability of selection. These results are consistent with the observations of Kandasamy (2017) [9] and Deepa et al. (2018) [4] , who also emphasized the influence of additive gene action on key horticultural traits in cucumber. High broad-sense heritability recorded for all traits implies strong genetic control. Furthermore, traits such as vine length, fruit weight, node to first male and female flower, and fruit yield per plant exhibited both high heritability and high genetic advance as a percentage of the mean. This combination indicates additive gene action and suggests that these traits can be improved through direct selection. Similar conclusions were previously reported by Kumar (2006) [11] , Munshi et al. (2007) [16] , and Kandasamy (2017) [9] . The correlation analysis demonstrated that fruit yield per plant was significantly and positively associated with fruit weight, fruit diameter, fruit length, vine length, node to first female flower, and number of fruits per plant. These relationships indicate that simultaneous improvement of these component traits would likely enhance overall productivity. Conversely, the negative association between yield and days to first flowering and the node number of the first female flower underscores the importance of earliness in achieving higher yield potential. These trends corroborate earlier reports by Rao et al. (2004) [21] , Kumar et al. (2011), and Pal et al. (2017) [19] . Mahalanobis D²-based genetic divergence analysis grouped the 100 genotypes into four distinct clusters, with Cluster II being the largest, followed by Cluster I. The substantial inter-cluster distances, particularly between Clusters I and II, indicate considerable genetic divergence that can be exploited in hybridization to generate transgressive segregants. The weak correspondence between the cluster pattern and geographical origin suggests that cucumber diversity may be shaped more by selection pressure, localized adaptation, and gene flow than by geographic separation. Similar conclusions were drawn by Sharma and Sharma (2006) [24] and Hossain et al. (2010) [8] . Molecular characterization using 59 polymorphic SSR markers revealed substantial allelic richness, with 295 alleles identified (3-8 alleles per locus). The high mean PIC value, coupled with moderate to high gene diversity and heterozygosity, confirms the effectiveness of SSRs in capturing genetic variation in cucumber. These results are in agreement with previous SSR-based diversity studies in cucumber reported by Chiba et al. (2003) [1] . Screening for ToLCNDV resistance identified three genotypes, DC-61, DC-70 and DC-91, with consistently low vulnerability index (VI) values (18.75-25.35%), indicating a high degree of resistance. Seven genotypes exhibited moderate resistance, whereas the remaining accessions ranged from moderately susceptible to susceptible. The observed variation in disease response reveals substantial genetic diversity for ToLCNDV resistance within the indigenous germplasm. Notably, the resistant genotypes identified in this study represent valuable and novel sources of resistance in cucumber. These results are consistent with global observations that cucurbit germplasm showing resistance to ToLCNDV and related begomoviruses often originates from the Indian subcontinent

Acknowledgement

AcknowledgmentsThe authors highly acknowledge the Director, ICAR-IARI, New Delhi for providing the necessary facilities to carry out the research

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