Morphological and Molecular Insights into Genetic Variability and Heritability in Four Strawberry (Fragaria × ananassa) Cultivars

Abstract

Strawberry (Fragaria × ananassa Duch.) is a widely cultivated and economically important fruit crop with increasing consumer demand worldwide. Nowadays, in Uzbekistan, strawberry cultivation surpasses that of many other fruits and vegetables in terms of production volume. However, most genetic studies have focused on a limited set of cultivars, leaving a substantial portion of varietal diversity unexplored. This study aimed to evaluate the genetic variability and heritability among selected strawberry cultivars, as well as correlations between certain valuable agronomic traits, using molecular and statistical approaches. Polymorphism analysis was performed, using 67 gene-specific SSR markers, through PCR, and allele variations were observed in 46.3% of the markers analyzed. Among them, 31 markers displayed polymorphic bands, identifying fifty alleles, with one to four alleles per marker. Phylogenetic analysis was performed using MEGA 11 software, while statistical evaluations included AMOVA (GenAIEx), correlation (OriginPro), and descriptive statistics based on standard agronomic methods. Additionally, the degree of cross-compatibility and pollen viability among the cultivars were studied, and their significance for cultivar hybridization was analyzed. The highest fruit weight was observed in the Cinderella cultivar (26.2 g), and a moderate negative correlation (r = −0.688) was found between fruit number and fruit weight. These findings demonstrate the potential of molecular tools for assessing genetic diversity and provide valuable insights for breeding programs aimed at developing improved strawberry cultivars with desirable agronomic traits.

1. Introduction

Strawberry (Fragaria × ananassa Duch.) is one of the most popular and economically important soft-fruit crops, cultivated worldwide for its attractive aroma, sweet taste, and high nutritional value [1]. Currently, strawberries are commercially cultivated in nearly 80 countries worldwide. China is the leading producer, followed by the United States, Mexico, Turkey, and Spain. Strawberry production has been steadily increasing, particularly in regions such as Asia, North and Central America, and North Africa. This growth is closely linked to the rising global demand for strawberries [2,3]. In Uzbekistan as well, strawberry cultivation has gained economic prominence, surpassing many other fruit and vegetable crops in production volume and market value.

From a botanical perspective, F. × ananassa originated from a natural hybridization event between Fragaria chiloensis (native to South America) and Fragaria virginiana (native to North America) [4]. The resulting species is an allo-octoploid species (2n = 8× = 56) with a complex genomic structure. Genomic studies suggest the involvement of four diploid progenitor species, F. vesca, F. iinumae, F. viridis, and F. nipponica [5], although recent phylogenetic evidence has questioned the latter two as direct ancestors [6,7,8,9]. The haploid genome size of F. × ananassa is estimated at approximately 813.4 Mb, with each subgenome contributing around 170 Mb [10]. This complex polyploid nature leads to high heterozygosity and extensive allelic diversity among subgenomes, posing both challenges and opportunities in genetic studies [11,12,13,14].

The genus Fragaria comprises at least 22 wild species, ranging from diploid (2n = 2× = 14) to decaploid (2n = 10× = 70), and is classified under the tribe Potentilleae and the family Rosaceae [15]. Among these, F. vesca (2n = 14) is widely used as a model species due to its relatively small genome (~240 Mb), short life cycle, ease of vegetative propagation, and high responsiveness to genetic transformation [16]. The first complete genome sequence of F. vesca was published by Shulaev et al. [17], and high-quality genome assemblies for F. × ananassa have subsequently also been developed [4].

In breeding initiatives concerning Fragaria × ananassa, pivotal agronomic characteristics such as fruit mass, morphology, pigmentation, and palatability serve as major focal points for selection. The manifestation of these characteristics is contingent upon both the genetic constitution and the prevailing environmental parameters, and these characteristice significantly influence market preferences and consumer demand. Consequently, the evaluation of morphological and genetic heterogeneity among cultivars is essential for informing breeding methodologies and for the development of superior strawberry varieties that exhibit enhanced quality and adaptability [18].

Despite the agronomic and nutritional significance of strawberries, most studies have focused on a narrow selection of commercial cultivars, leaving the broader genetic diversity underexplored. Furthermore, postharvest losses remain a major challenge due to the crop’s sensitivity to biotic and abiotic stresses [19,20]. The reproductive biology of strawberries, primarily via vegetative runners, also poses limitations in expanding genetic variability. Commercial cultivars are classified into two main types: short-day (June-bearing) and day-neutral (everbearing), with flowering and yield being highly influenced by photoperiod and temperature conditions [21].

Thus, it is recommended to characterize strawberry plants morphologically based on traits expressed at different vegetative stages. The most suitable stages for morphological description are leaf emergence, flowering, and fruiting. Among these, the fruiting stage is considered the most convenient for a comprehensive and effective characterization of the cultivar under study. This aligns with the phenological staging provided by the BBCH scale for strawberry development, which supports systematic observation at these key growth phases [22].

In this study, the cultivars Seolhyang, Cinderella, Alba, and Vitaberry were selected primarily based on their marketability, distinctive fruit characteristics, and contrasting morphological traits. Their selection is supported by previous reports highlighting their commercial significance in Korea and several European countries, as well as their suitability for breeding purposes and demonstrated phenotypic variability.

Additionally, in agronomic evaluations, the Cinderella cultivar exhibited the highest average fruit weight; however, a moderate negative correlation was observed between the number of fruits and their weight. Although Cinderella produces fewer fruits, it yields high-quality produce due to the large size and uniformity of the berries. Therefore, this cultivar is considered a valuable parent in breeding programs for transmitting the trait of large fruit size [23].

Fruit weight in strawberry cultivars is known to have high heritability and is primarily determined by genetic factors. In contrast, total yield is largely influenced by environmental conditions. The genetic correlation between fruit weight and total yield is minimal or non-existent, indicating that these traits should be evaluated independently in breeding strategies. Therefore, the present study aims to clarify the phylogenetic relationships among diverse strawberry cultivars, using molecular markers and statistical methods, while also investigating the key morphological and agronomic traits relevant to strawberry breeding and improvement programs.

2. Materials and Methods

2.1. Plant Materials and Experimental Site

The study utilized four strawberry (Fragaria × ananassa Duch.) cultivars, Seolhyang, Cinderella, Alba, and Vitaberry, originally imported from Dongsang city, South Korea by “O’rtachirchiq Nursery Garden Service Cluster” LLC. Upon their arrival in Uzbekistan, the plants underwent a 10-day quarantine inspection at the Scientific Research Institute of Plant Quarantine and Protection. Experimental planting was carried out in open fields in early March and in greenhouse conditions in late August (

Table 1. Origins and key biological traits of the analyzed strawberry cultivars.

№	Research Samples	Country of Origin	Biological Characteristics
1	Seolhyang	Republic of Korea	High yield
2	Cinderella	Republic of Korea	Aesthetically attractive, pineapple-flavored, white-colored, and sweet
3	Alba	Italy	Relatively large fruit
4	Vitaberry	Republic of Korea	Sour-tasting fruit

).

Field trials were conducted from 2024 to 2025 at the Institute of Genetics and Experimental Plant Biology, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (41°22′05.3″ N, 69°24′17.9″ E; altitude 450 m), and at the “O’rtachirchiq Nursery Garden Service Cluster” LLC, Tashkent region, Uzbekistan (41°02′34″ N, 69°21′27″ E; altitude 380 m). The region is characterized by a subtropical continental climate with hot, dry summers (30–35 °C) and moderately cold winters (0–5 °C), with annual precipitation around 300–400 mm.

2.2. Experimental Design and Morphological Evaluation Method

The evaluation of strawberry cultivars was conducted under open-field conditions using a randomized complete block design (RCBD) with four replications. Prior to planting, the field was plowed to a depth of 30 cm and leveled. Each planting bed measured 0.7 × 2.5 m, with a planting arrangement with 15 cm between plants within a row and 20 cm between rows.

During the growing season, regular monitoring was conducted, and fruits were harvested every three days at full ripeness. Ripeness was determined based on the uniformity of skin color and attainment of full fruit size.

For each cultivar, 10 plants were randomly selected per replication (totaling 40 plants per cultivar), and morphological and reproductive traits were evaluated according to the UPOV (International Union for the Protection of New Varieties of Plants) and Bioversity International/IPGRI descriptor guidelines for Fragaria × ananassa.

1

Vegetative Traits:

• Plant height (cm), measured from the crown to the top of the highest leaf, using a measuring tape.
• Plant width (cm), measured horizontally at the widest point of the plant canopy.
• Number of leaves per plant, counted as fully developed trifoliate leaves.

2

Reproductive Traits:

• Number of flowers per plant, counted at the peak flowering stage.
• Number of fruits per plant, cumulatively counted throughout the fruiting period.

3

Fruit Traits:

• Fruit length and diameter (cm), measured using a digital caliper; average values were calculated from 10 randomly selected fruits per plant.
• Average fruit weight (g), determined by weighing 10 fruits per plant on a precision digital scale and calculating the mean.
• Skin color, visually evaluated based on a standardized color scale.
• Fruit shape index, calculated as the ratio of fruit length to diameter.

All measurements were performed using precision instruments (digital calipers and electronic balances) to ensure accuracy. The collected data were organized in spreadsheets and subjected to statistical analysis to evaluate inter-cultivar variability and identify promising genotypes for breeding applications.

2.3. Genomic DNA Extraction and PCR Amplification

Genomic DNA was extracted using the CTAB method, with modifications [24]. DNA quality and concentration were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). PCR was conducted using a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) in 10 µL reaction volumes: 2 µL 5× Screen Mix, 1 µL DNA, 1 µL primers, and 6 µL DNAse-free water. The PCR protocol involved initial denaturation at 94 °C for 2 min; 35 cycles of denaturation (94 °C, 20 s), annealing (55–60 °C, 30 s), extension (72 °C, 30 s), and a final extension at 72 °C for 5 min.

PCR products were visualized on 1.5–2% agarose gel using the GelDoc Go Imaging System (Bio-Rad Laboratories Pte Ltd, Singapore). Amplicon sizes were calculated with GelAnalyzer 23.1 software based on HyperLadder 50 bp standards. A phylogenetic tree was constructed in MEGA 11.

2.4. Marker Informativeness and Polymorphism Assessment

Polymorphism information content (PIC) and heterozygosity (He) were calculated for each SSR marker according to PowerMarker V3.0 [25]. PIC provides an estimate of the discriminatory power of a marker, while He reflects the degree of genetic variation within the population. PIC values were calculated for each polymorphic marker according to Botstein et al. (1980) [26], using the following formula:

$$PIC=1-\sum _{i=1}^n{P}_i^2-\sum _{i=1}^{n-1}\sum _{j=i+1}^{n}2P_i^2{P}_j^2$$

where pi—the frequency of the ith allele at a given locus, and n—the total number of alleles. Heterozygosity was calculated, by the following formula, according to Nei (1973) [27]:

$$He=1-\sum _{i=1}^n{P}_i^2$$

where pi—the frequency of the ith allele at a given locus, and n—the total number of alleles.

2.5. Statistical Analysis

To assess differences among strawberry cultivars based on morphological and reproductive traits, standard statistical methods were applied. The experiment was arranged in a randomized complete block design (RCBD) with four replications. Descriptive statistics, including the mean, standard deviation (SD), and coefficient of variation (CV%), were calculated for each trait.

Analysis of variance (ANOVA) was conducted to determine the significance of differences among cultivars for the studied traits. This analysis was performed based on the methodology proposed by Maxwell and Delaney [28]. When significant differences were observed, post hoc tests were applied to compare the mean values among cultivars.

To identify the interrelationships among traits, Pearson’s correlation coefficients were calculated between morphological, yield-related, and reproductive parameters, using OriginPro 8.5 software [29]. These correlations provide valuable insights for designing effective breeding strategies.

To investigate the distribution of genetic diversity both within and among cultivars, analysis of molecular variance (AMOVA) was performed using GenAlEx 6.5 software [30]. The AMOVA model partitioned the total variation into components attributable to differences within and among cultivars. The significance of the results was assessed based on 999 permutations. Additionally, the fixation index (F_ST) was calculated to evaluate the degree of genetic differentiation among the cultivars.

2.6. Pollen Fertility Analysis

To evaluate the reproductive potential and suitability of the studied strawberry cultivars for use as male parents in future breeding programs, pollen fertility analysis was conducted. This assessment provided insight into the reproductive stability and viability of each cultivar.

Fully developed flowers at the anthesis stage were selected from each cultivar. From each replication, five flowers were collected, resulting in a total of 20 flowers analyzed per cultivar.

Fresh pollen grains were carefully extracted from the anthers and stained with a 1% acetocarmine solution. A drop of the stained pollen suspension was placed on a microscope slide and covered with a cover slip. To enhance stain absorption, the slides were gently heated. Observations were conducted using a light microscope at 400× magnification.

Fertile (viable) pollen grains were identified by their uniform, intense staining and intact morphology. In contrast, sterile (non-viable) pollen appeared shrunken, fragmented, or poorly stained. For each flower, at least 200 pollen grains were counted. Analyses were performed using a Leica CM E microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica EC3 camera (Leica Microsystems, Wetzlar, Germany).

2.7. Intercrossing of Strawberry Cultivars and Pollen Fertility

The hybridization process was carried out in the spring months, from March to May. The anthers were carefully removed from the maternal flowers, and special isolation bags were applied. After 3–4 days, pollen from the selected paternal cultivar was applied to the stigma of the maternal flower, i.e., transferred through artificial pollination. To ensure the reliability of the research results, the pollination procedures for each hybrid combination were carried out in accordance with predetermined methodological guidelines. In particular, attention was given to maintaining a similar number of pollinations for each combination, ranging from 21 to 28. This approach allowed data to be obtained under equal conditions for all combinations and ensured an adequate sample size for statistical analysis.

3. Results

3.1. Morphological Description

It is advisable to morphologically characterize the strawberry plant based on the traits that appear during its various vegetative phases. For morphological description, the following phases are considered the most optimal: leaf emergence, flowering, and fruit formation. The fruit formation phase is the most favorable for a complete and effective characterization of the utilized variety [31].

Since the progression of each phase was significant in our research, morphological descriptions were carried out at all stages under greenhouse conditions and are presented in a general form. In particular, the morphological characteristics of the four strawberry cultivars—Seolhyang, Cinderella, Alba, and Vitaberry—revealed significant differences in their vegetative and reproductive traits (Figure 1).

The Seolhyang cultivar exhibited a moderately dense and upright plant architecture with compact bush formation [32]. The mean plant spread was 48 cm, and each mother plant produced at least 3–4 stolons, giving rise to 3–4 daughter plants. Leaves were trifoliate, dark green, and medium-sized (10.5 × 8 cm), with a smooth adaxial and a slightly rough abaxial surface. The petiole length averaged 13–14 cm. Each plant developed 16–18 leaves. Inflorescences bore 15–18 flowers with 5–7 white petals and 12 sepals per flower. The average flower size was 3.5 × 4.0 cm. Each flower contained 24–26 stamens (anthers), accompanied by a high number of ovules. Fruits were produced in 4–5 successive cycles during autumn. The berries were large, conical, red-colored, and sweet-tasting, with an average weight of 25.1 g (Figure 1A1–A3,

Table 2. Morphological and agronomic traits of four different strawberry cultivars.

No.	Samples	$\overline{\mathit{x}}\pm \mathbf{S}\overline{\mathit{x}}$	Minimum	Maximum	SD	V
Plant Width (cm)
1.	Seolhyang	48.2 ± 2.67	41	55	5.97	12.3%
2.	Cinderella	45.4 ± 2.79	41	54	6.22	13.7%
3.	Alba	53.8 ± 0.80	52	56	1.78	3.32%
4.	Vitaberry	47.8 ± 2.06	42	54	4.60	9.63%
Number of Leaves per Plant
1.	Seolhyang	17.0 ± 0.31	16	18	0.70	4.15%
2.	Cinderella	11.8 ± 0.37	11	13	0.83	7.0%
3.	Alba	13.6 ± 0.60	12	15	1.34	9.86%
4.	Vitaberry	14 ± 1.14	11	17	2.54	18.2%
Number of Flowers per Plant
1.	Seolhyang	16.2 ± 0.58	15	18	1.30	8.04%
2.	Cinderella	15.2 ± 0.37	14	16	0.83	5.50%
3.	Alba	19.2 ± 0.66	17	21	1.48	7.72%
4.	Vitaberry	21.4 ± 1.07	18	24	2.40	11.2%
Number of Fruits per Plant
1.	Seolhyang	15.4 ± 0.51	14	17	1.14	7.40%
2.	Cinderella	14.8 ± 0.37	14	16	0.83	5.65%
3.	Alba	19 ± 0.63	17	21	1.41	7.44%
4.	Vitaberry	21 ± 1.22	17	24	2.73	13.0%
Fruit Length (cm)
1.	Seolhyang	4.32 ± 0.14	3.8	4.6	0.31	7.32%
2.	Cinderella	4.56 ± 0.07	4.5	4.7	0.15	3.46%
3.	Alba	4.5 ± 0.14	4.1	4.8	0.31	7.02%
4.	Vitaberry	3.74 ± 0.12	3.5	4.2	0.27	7.32%
Fruit Diameter (cm)
1.	Seolhyang	3.12 ± 0.07	2.87	3.35	0.15	5.06%
2.	Cinderella	3.02 ± 0.07	2.87	3.15	0.15	5.23%
3.	Alba	2.97 ± 0.12	2.55	3.27	0.27	9.22%
4.	Vitaberry	2.78 ± 0.07	2.62	3.1	0.15	5.68%
Fresh Fruit Weight (g)
1.	Seolhyang	25.1 ± 1.04	22.3	27.2	2.32	9.25%
2.	Cinderella	26.2 ± 0.36	25.3	27.3	0.82	3.13%
3.	Alba	24.0 ± 1.26	20.4	27.6	2.81	11.7%
4.	Vitaberry	18.9 ± 0.66	16.5	20.3	1.47	7.80%

).

The next cultivar, Cinderella, had compact, upright bushes with moderate pubescence and a horizontal spread of 45.0 cm. More than four stolons were formed by each mother plant, resulting in the development of daughter plants. Leaves were dark green, medium-sized (7.5 × 8.0 cm), and trifoliate, with petioles 12.0–15.0 cm long. The upper surface was smooth, while the lower surface was slightly rough. The number of leaves on each plant was 11.0–13.0. There were 14.0–16.0 flowers, each consisting of 6–7 white petals and 12 sepals. Flowers measured an average of 3.5 × 4.0 cm in diameter, and the number of stamens per flower ranged from 24 to 26. This variety also bears fruit year-round under greenhouse conditions. The fruits are ovate in shape, exhibiting a pale pink hue at the apex that gradually shifts to white toward the base. The flesh is white. They are sweet in taste, with a distinctive pineapple flavor. The average fresh fruit weight is 26.2 g (Figure 1B1–B3, Table 2).

In Alba, the plants presented moderately thick, upright, and compact bushes with a horizontal spread of 53.8 cm. The mother plant produced more than four stolons, each giving rise to daughter plants. Leaves were trifoliate and medium-sized (7.0 × 7.5 cm), with a petiole length of 9–10 cm. The upper surface was smooth, and the lower surface was slightly rough with serrations. Leaf numbers ranged from 12–15. The flowers (15–18 per plant) had 6–7 white petals and 12 green sepals, averaging 3.2 × 4.2 cm. In this cultivar, an average of 27 to 31 stamens was recorded per flower. Fruits were large, ovoid, dark red, sweet, and pineapple-flavored, with an average weight of 24.0 g (Figure 1C1–C3, Table 2).

Another cultivar, the Vitaberry cultivar, displayed upright and compact bushes with sparse pubescence and a horizontal spread similar to Alba (53.8 cm). Leaves were trifoliate, sized 6.5 × 8 cm, and supported by 13–14 cm long petioles. Plants produced 11–17 leaves per plant. Flowers numbered between 18.0 and 24.0 per plant, with 6–7 white petals and 12 sepals. The average flower size was 3.5 × 4.0 cm. Up to 27 stamens were observed per flower. Fruits were red, conical, and sweet, with an average weight of 18.9 g (Figure 1D1–D3, Table 2).

Variations in key morphological and yield-related traits such as plant width, number of flowers, number of fruits, and mean fruit weight among the studied cultivars are comprehensively illustrated in the comparative diagram (Figure 2), offering a clear visual summary of phenotypic divergences relevant to cultivar differentiation and selection.

The cultivar Alba displayed the widest plant spread (53.8 cm), while Vitaberry showed the highest number of flowers (21.4 per plant). Vitaberry also produced the highest number of fruits (21.0 per plant), but Cinderella had the highest average fruit weight (26.2 g), suggesting variation in trait combinations that may influence cultivar performance under specific agro-ecological conditions (Figure 2).

Based on morphological characterization, among the four varieties studied, the Alba variety stood out as a marketable variety, with a large number of flowers and fruits, and large fruit size. The relatively short stem width of the Cinderella variety and its shorter petioles and fewer leaves compared to other varieties, but relatively large freshly harvested fruit, indicate that this variety is adapted to drought conditions.

3.2. Statistical Evaluation of Results

According to the results, the Alba cultivar exhibited the widest plant spread (53.8 ± 0.80 cm), indicating a uniform plant structure with low variability (CV = 3.32%). In contrast, Cinderella showed the narrowest spread (45.4 ± 2.79 cm), along with higher variability (CV = 13.7%). Seolhyang produced the highest number of leaves per plant (17.0 ± 0.31), followed by Alba (13.6 ± 0.60), while Vitaberry had a relatively lower leaf count (14.0 ± 1.14).

Flower production was highest in Vitaberry (21.4 ± 1.07), suggesting strong reproductive potential, followed by Alba (19.2 ± 0.66), whereas Cinderella (15.2 ± 0.37) and Seolhyang (16.2 ± 0.58) were lower in flower count. In terms of fruit number, Vitaberry and Alba again demonstrated the highest values (21 ± 1.22 and 19 ± 0.63, respectively), indicating potential for high yield.

Fruit size characteristics showed that Cinderella produced the longest fruits (4.56 ± 0.07 cm), while Seolhyang had the largest fruit diameter (3.12 ± 0.07 cm). The fruit weight data revealed that Cinderella recorded the heaviest fruits (26.2 ± 0.36 g), followed closely by Seolhyang (25.1 ± 1.04 g), Alba (24.0 ± 1.26 g), and Vitaberry (18.9 ± 0.66 g), indicating varietal differences in fruit development potential (Table 2).

To better understand the interrelationships among phenotypic traits, Pearson correlation coefficients were calculated between variables such as plant width, leaf number, flower count, fruit number, fruit dimensions, and fruit weight.

In this study, the morpho-biological characteristics of four strawberry cultivars were thoroughly examined and classified (Figure 3). A weak overall correlation (r = 0.057) was observed between plant width and number of leaves, although in Alba this association was negatively correlated (r = −0.268). The number of flowers and fruits showed a weak correlation (r = 0.075), with cultivar-specific differences: Seolhyang (r = −0.813), Cinderella (r = −0.285), Alba (r = −0.201), and Vitaberry (r = 0.57).

A moderate positive correlation (r = 0.31) was found between the crown width (cm) of strawberry plants and the number of fruits.

Plant width exhibited a moderate correlation with fruit number (r = 0.308), while fruit length and fruit weight were moderately correlated (r = 0.564), as were fruit diameter and weight (r = 0.550). Overall, a negative correlation (r = −0.688) between fruit number and fruit weight was observed, reflecting the general trend. However, in some cultivars, particularly Alba, a positive correlation (r = 0.501) was noted, indicating that this cultivar deviates from the general trend, possibly due to its genotypic characteristics or specific growth conditions.

Additional negative correlations were detected between plant width and fruit weight (r = −0.021), leaf number and fruit weight (r = −0.019), and plant width and fruit diameter (r = −0.149). Similarly, flower number and fruit length were negatively correlated (r = −0.632), as were flower number and fruit weight (r = −0.688). These findings suggest that high fruit yield does not always correspond to larger fruit size or weight.

3.3. Molecular Analysis Results

PCR analysis was carried out using 67 gene-specific marker pairs, of which 31 markers showed polymorphism, 26 were monomorphic, and 10 did not produce amplification. Polymorphism was observed in 46.3% of the studied samples. The 31 polymorphic markers revealed a total of fifty alleles, with each marker detecting between one and four alleles. The polymorphic markers had Polymorphic Information Content (PIC) values ranging from 0.304 to 0.375, while heterozygosity (He) values ranged from 0.1638 to 0.500 (Figure 4; Table S1). Lee et al. (2018) [32] analyzed sugar accumulation in Fragaria × ananassa and their transcripts. In their study, genes such as FaGPT1, FaTMT1, FaHXK1, FaPHS1, FaINVA-3, and FacxINV2-1 were highly expressed in cultivars with high sugar content, while FapGlcT, FaTMT2-1, FaPHS2-1, FaSUSY1-1, and FaSUSY1-2 showed higher expression levels in cultivars with low sugar content. Overall, genes involved in sugar transport or sugar synthesis were more highly expressed in high-sugar cultivars.

In our study, PCR analyses based on these markers also revealed associations with key sugar metabolism genes, including FaTMT2-1, FaSUT2-2, FaSPSB, and FaSUSY2-1. These markers are involved in sugar transport, sucrose synthesis, and degradation pathways. Additionally, several ABA-responsive genes (such as FaASR and FaHVA22) were also identified.

Phylogenetic analysis using MEGA 11 software grouped the cultivars into two main clades. Seolhyang, showing the greatest genetic distance (0.190), stood apart from the others. Cinderella and Vitaberry were closely related (distance = 0.117 and 0.045), while Alba showed intermediate similarity (0.063) (Figure 5).

3.4. AMOVA Analysis

In the current investigation, the application of analysis of molecular variance (AMOVA) elucidated that 97% of the overall genetic variation was localized within cultivars, while merely 3% was ascribed to variation across cultivars (

Table 3. Summary of the molecular variance (AMOVA) partitioned within and among cultivars.

No.	Source	Df	Sum of Squares	Mean Squares	Estimated Variation	Total Variation (%)
1.	Among cultivars	2	21.438	10.719	0.339	3%
3.	Within cultivars	8	75.500	9.438	9.438	97%
	Total	10	96.938	20.157	9.777	100%

). This distribution underscores that the majority of genetic diversity in Fragaria × ananassa is organized at the intra-population level, highlighting significant variation among individual genotypes within the same cultivar, rather than between distinct cultivars.

The fixation index (F_ST) was determined to be 0.035 (p = 0.289), indicating low, yet discernible, genetic differentiation among the four cultivars. The inbreeding coefficient (F_IS) was marginally negative (−0.022; p = 0.532), suggesting a slight predominance of heterozygosity, which may be attributable to clonal propagation or random mating practices. The overall fixation index (F_IT) was minimal (0.014; p = 0.385), reflecting negligible total genetic divergence.

Notably, certain loci demonstrated elevated F_ST values (up to 0.394), which may indicate genomic regions experiencing divergent selection pressures. In spite of these isolated instances of differentiation, the mean F_ST value was recorded at 0.090, and the estimated gene flow (Nm = 6.813) suggested a substantial degree of allelic exchange among cultivars. This gene flow reinforces the concept of considerable genetic coherence throughout the examined germplasm, even in the presence of pronounced morphological discrepancies.

3.5. Pollen Viability Assessment and Reproductive Potential

Pollen viability plays also an important role in the fertilization efficiency and fruit productivity of the strawberry plant [33]. High pollen viability ensures successful fertilization, promoting proper seed development and enhancing fruit set and quality. Therefore, assessing pollen viability across different cultivars and hybrids is of significant importance in breeding and propagation programs [34].

The results revealed cultivar-specific differences in pollen fertility. The Cinderella cultivar exhibited the highest viability, with 86.8 ± 0.7% viable pollen grains out of a total of 1501 counted. This high rate of viability indicates a strong fertilization capacity and highlights Cinderella as a promising pollen donor for breeding programs (Figure 6).

Conversely, Seolhyang and Vitaberry demonstrated relatively lower pollen viability, at 78.5 ± 1.2% and 77.0 ± 1.1%, respectively (

Table 4. Pollen fertility characteristics of four strawberry cultivars.

No.	Research Samples	Number of Analyzed Pollens	Pollen Fertility, %
			$\overline{\mathit{x}}\pm \mathbf{S}\overline{\mathit{x}}$	Range	S	V
1	Seolhyang	1301	78.55 ± 1.23	74.71–93.33	5.03	6.40
2	Cinderella	1501	86.88 ± 2.48	67.31–96.91	2.48	12.77
3	Alba	931	79.81 ± 1.18	76.77–83.16	5.13	6.43
4	Vitaberry	1497	77.09 ± 0.67	72.83–83.09	2.99	3.89

). While Cinderella had the highest mean viability, it also exhibited a relatively high coefficient of variation (CV = 12.77%), likely due to the large sample size and the broad range of staining intensity (from 67.31% to 96.91%), indicating variability in pollen development.

Pollen viability is known to be influenced by both genetic factors and environmental conditions, such as temperature, humidity, and cultivation practices. Adverse environmental conditions—such as excessive heat during anthesis or imbalanced soil moisture—can reduce cytoplasmic activity in pollen grains, thus impairing fertilization potential. Despite such constraints, the observed cross-compatibility among the cultivars was high, suggesting that fertilization success was not significantly hampered. This underscores the suitability of Cinderella as a robust male parent in future hybridization strategies due to its combination of high pollen viability and reproductive compatibility.

3.6. Intercrossing of Strawberry Cultivars

During the process of intercrossing strawberry cultivars, the forms selected as parental lines are evaluated based on their valuable agronomic and quality traits. Cultivars obtained through hybridization demonstrate high genetic combinability, the heterosis effect, and strong adaptability to various environmental factors. Therefore, in modern breeding programs, creating F₁ hybrids through genetically compatible parental lines is considered a priority direction, in contrast to clonal propagation.

In the course of the research, both direct and reciprocal crosses were conducted using different strawberry cultivars. As a result, 12 different direct and reciprocal hybrid combinations were successfully obtained. The results of the crosses showed very high efficiency, with overall hybridization rates ranging from 83.3% to 100%. Notably, when Cinderella was used as the female parent and crossed with Seolhyang, 100% hybrid fruit formation was achieved.

Combinations with relatively lower success rates (83.3–90.9%) included: Alba × Seolhyang, Vitaberry × Alba, Alba × Vitaberry, Vitaberry × Seolhyang, Seolhyang × Alba, Cinderella × Alba, Cinderella × Vitaberry, Vitaberry × Cinderella, and Seolhyang × Vitaberry (

Table 5. Fruit setting rate and hybrid fruit production in crosses among four strawberry cultivars.

No.	Research Samples	Number of Crosses	Number of Obtained Hybrid Fruits	Crossing Rate, %
1.	Seolhyang × Cinderella	23	22	95.6
2.	Cinderella × Seolhyang	25	25	100.0
3.	Seolhyang × Vitaberry	22	20	90.9
4.	Vitaberry × Seolhyang	24	21	87.5
5.	Seolhyang × Alba	24	21	87.5
6.	Alba × Seolhyang	24	20	83.3
7.	Cinderella × Alba	23	20	86.9
8.	Alba × Cinderella	24	22	91.6
9.	Cinderella × Vitaberry	27	24	88.8
10.	Vitaberry × Cinderella	26	23	88.4
11.	Alba × Vitaberry	23	20	86.9
12.	Vitaberry × Alba	25	21	84.0

).

4. Discussion

Modern strawberry breeding focuses primarily on improving fruit quality, size, and yield potential to meet growing consumer and market demands [35,36,37]. Among these attributes, fruit size and color are central to quality assessment due to their immediate visual appeal.

This study evaluated four commercial strawberry cultivars, Seolhyang, Cinderella, Alba, and Vitaberry, for their morphological, agronomic, and molecular diversity. Given the allo-octoploid nature of Fragaria × ananassa and its high heterozygosity, substantial phenotypic plasticity and genetic variability were observed [4,5,6,7,8].

Morphological assessments revealed that Alba and Vitaberry produced the highest number of flowers and fruits per plant, underlining their yield-enhancing potential. However, the observed moderate negative correlation between fruit number and weight (r = −0.688) suggests a trade-off between yield quantity and fruit size. Notably, Cinderella, despite producing fewer fruits, showed the highest average fruit weight (26.2 g), positioning it as a promising donor for the enhancement of fruit size in breeding programs. These observations are in line with previous reports highlighting the heritability and genetic control of fruit weight [23,38,39].

Further, moderate positive correlations between plant spread and fruit number (r = 0.308) and between fruit diameter and weight (r = 0.550) imply that certain vegetative traits may serve as proxies for reproductive output. However, the negative association between flower number and fruit weight emphasizes the need for careful trait prioritization during selection, as high flower numbers do not guarantee marketable yield.

Among the 67 SSR markers tested, 31 were polymorphic, revealing a total of 50 alleles, with 1–4 alleles per locus. Although this is lower than the 7.3–16.1 allele range reported in other F. × ananassa studies [40,41,42], it may be attributed to the complex polyploid genome structure and limitations of the gel-based genotyping method used. A more sensitive approach, such as capillary electrophoresis, could potentially reveal higher allelic diversity. Previous studies on diploid and octoploid Fragaria species have also shown wide variation in allele numbers [43,44,45], reflecting differences in ploidy and genome complexity.

Polymorphic Information Content (PIC) values ranged from 0.304 to 0.375, indicating moderate marker informativeness. Additionally, the detection of functionally relevant loci—such as those linked to sugar metabolism (FaTMT2-1, FaSUT2-2) and abiotic stress response (FaASR, FaHVA22)—highlights the potential for these markers in marker-assisted selection strategies.

Phylogenetic clustering grouped the cultivars into two main clusters, with Seolhyang showing the greatest genetic divergence, likely due to its distinct breeding history. Cinderella and Vitaberry were closely clustered, suggesting either shared ancestry or convergent selection.

The AMOVA analysis revealed that 97% of genetic variation resided within cultivars, with only 3% attributed to variation among them. This is consistent with previous findings in clonally propagated strawberries, where high intra-cultivar diversity predominates due to somatic mutations and residual heterozygosity [46,47,48,49]. The low overall F_ST value (0.035) indicates limited differentiation among the cultivars, although some loci exhibited higher divergence (F_ST up to 0.394), suggesting localized genomic regions under selection.

Similar patterns have been reported, in which theoretical population clusters explained only 15.2% of total variation [38]. Another study observed that in F. chiloensis, most diversity (85.1%) was found within populations [39]. In contrast, a study on wild strawberries from the Tibetan Plateau reported up to 92.1% among-population variance, underscoring the role of ecological gradients in shaping diversity patterns [47].

Pollen viability, a critical trait for reproductive success, is influenced by both genetic factors and environmental factors such as temperature, humidity, and soil moisture [50]. Suboptimal conditions, e.g., excess heat or poor soil moisture, can impair cytoplasmic activity, reducing fertilization potential. Conversely, optimal conditions enhance pollen tube growth and fertilization efficiency.

Consistent with these principles, Cinderella showed the highest pollen viability (86.8%), and its crosses with Seolhyang resulted in 100% fruit set, confirming its robust reproductive capacity. Cultivars with pollen viability exceeding 80% are generally considered ideal for hybridization, as they ensure high fertilization and fruiting success [51]. These findings emphasize the importance of parental genotype and pollen quality in hybrid development and underscore Cinderella’s value in breeding programs.

Altogether, the integration of morphological, molecular, and reproductive analyses provides a comprehensive framework for identifying high-value genotypes for breeding. The findings support the use of Cinderella as a fruit-size donor, Vitaberry and Alba as yield donors, and Seolhyang as a genetically distinct source for broadening the gene pool. Such insights are essential for developing resilient, high-quality strawberry cultivars tailored to diverse agro-ecological conditions.

5. Conclusions

This study assessed the morphological traits, reproductive potential, and molecular genetic diversity of four strawberry cultivars, revealing significant phenotypic and genotypic variability crucial for breeding and genetic resource management.

Cinderella showed high fruit weight, pollen fertility, and cross-compatibility, making it a promising parent for improving fruit size and reproductive success. Alba and Vitaberry demonstrated strong floral and fruit productivity, suggesting their usefulness in enhancing yield potential.

SSR marker analysis detected polymorphism at 34.8% of loci, highlighting notable genetic heterogeneity. Functionally relevant alleles linked to sugar metabolism (FaTMT2-1, FaSUT2-2) and stress response (FaASR, FaHVA22) were identified. Phylogenetic analysis grouped the cultivars into two main clusters, with Seolhyang showing the greatest genetic divergence. AMOVA indicated that most genetic variation (97%) exists within cultivars, while a low F_ST value (0.035) confirmed limited inter-cultivar differentiation and high allelic exchange.

These findings support the integration of morphological, physiological, and molecular data for selecting genetically diverse, agronomically valuable parental lines. The results provide a strong foundation for developing new cultivars with improved quality and fertility and environmental adaptability. Future studies should include wild relatives and local landraces, incorporating genomic tools to accelerate strawberry breeding.