Разработка генетической диверсификации в сортах кизилов (Crataegus azarolus L.) естественно растущих в восточной части Медитерранееанской области Турции с использованием морфологических, помологических и интерпретирующих последовательностей повторов маркеров.

Introduction

The genus Crataegus L. (2n = 34), which is part of the Rosaceae family, is among the most significant genera in terms of species count. The genus Crataegus , commonly referred to as hawthorn, is a small shrub or tree [ 30 ]. Christensen [ 10 ] noted that Crataegus is the largest genus within the Maloideae subfamily, encompassing more than 250 species, which makes its taxonomy quite complex [ 21 ]. The Mediterranean region of Türkiye stands out as one of the key areas with abundant diverse types of wild edible fruits. For centuries, a multitude of wild edible fruits such as carob, myrtle, cactus pear, hawthorn, raspberry, blackberry, strawberry, fig, barberry, olive, and more, have thrived in this region. In addition, these plants exhibit strong adaptability to various soil types [ 48 ]. Türkiye is a key center for the diversity of wild-grown Crataegus species. Hawthorns naturally grow in mountainous regions, shrublands, and rocky areas throughout our country, without any cultural interventions. Consequently, it is found in almost every region of Türkiye, including the Aegean, Eastern Mediterranean, Southern Anatolia, Inner Anatolia, and Northeast Anatolia [ 16 ]. Hawthorn plants in these areas exhibit significant morphological diversity, especially in their leaves, flowers, and fruits. This variation is thought to result from birds and some mammals that consume the fruits and act as dispersal agents [ 62 ].

In various regions of Türkiye, it is recognized by local names including ‘halıç’, ‘haziran’, ‘yaban gülü’, ‘ekşi muşmula’, ‘aluç’, ‘alıç’, and ‘yemişen’ [ 6 ]. Yellow, red, and black serve as fundamental colors for ripe hawthorn fruits, although numerous intermediate shades exist as well. In Türkiye, C. azarolus L., C. tanecetifolia , and Crateagus x bornmuelleri Zabel display varying hues of yellow, while C. pentagyna , C. davisii Browicz, and C. caucasica C. Koch are characterized by black fruits. The remaining species bear fruits in shades of red, yellowish red, or various other tones of red [ 15 ]. Despite various uses of hawthorn, it hasn’t garnered as much attention as compared with other fruit species. Known primarily as a wild fruit, hawthorn is also used as an ornamental plant owing to its appealing plant shape and flowers [ 8 ]. Traditionally, its fruits are harvested from the wild types, but currently, there is a growing interest in economically cultivating hawthorn for consumers, leading to the establishment of hawthorn orchards in Türkiye.

The dried fruits and flowers of hawthorn, which are highly beneficial for human health, are used for conditions such as cough, throat inflammation, heart palpitations, weakness of heart activity, liver pain, atherosclerosis, and kidney diseases [ 9 ]. Hawthorn fruit is rich in macro- and micronutrients [ 45 ]. In addition, the significance of the hawthorn plant has increased further due to its use as rootstock for some stone fruit species [ 62 ].

The increase in the global population, coupled with the unregulated exploitation of plant resources to satisfy human demands, as well as habitat destruction due to land expansion, urban sprawl, and industrial activities, has contributed to the decline and swift depletion of plant germplasms [ 11 ]. Detecting genetic variation represents the initial step toward the efficient utilization and safeguarding of genetic resources [ 37 ]. Germplasms play a crucial role in supplying breeders with the necessary materials for the advancement of plant species [ 41 ]. Therefore, germplasms with wide diversity are preferred. For years, morphological, pomological, and molecular marker systems have been used to determine genetic diversity among different plants, including hawthorn [ 31 , 53 , 60 ]. Among these marker systems, molecular techniques provide more precise results than other methods because they are not influenced by environmental conditions [ 42 ] Given that certain traits contributing to plant resistance to abiotic and biotic conditions are governed by multiple genes [ 58 ], research that integrates morphological, pomological, and molecular markers is essential for the identification, conservation, and sustainability of plant genetic diversity [ 61 ]. Different molecular methods, such as cpDNA, ITS, iPBS, SSR, and ISSR markers, are widely used in studies determining the molecular relationships of hawthorn germplasm. SSR markers are commonly utilized due to their high polymorphism and reliability for assessing genetic variation in plants, iPBS markers are effective for retrotransposon-based genetic studies, while cpDNA and ITS markers provide insights into evolutionary relationships and maternal lineages [ 12 , 19 , 24 , 25 , 32 , 64 ]. ISSR markers are also widely used in genetic studies due to their ability to reveal high levels of polymorphism, making them valuable for assessing genetic diversity and population structure [ 46 ].

The Eastern Mediterranean region of Türkiye stands out as one of the most significant centers of diversity for naturally grown hawthorn, characterized by its extensive phenotypic variation. This diversity is largely attributed to the region’s unique ecological conditions, coupled with the biological variability arising from cross-pollination and generative propagation through both natural and anthropogenic processes. Despite the global recognition of the region’s genetic richness, comprehensive studies integrating morphological, pomological, and molecular analyses of wild hawthorn populations remain limited.

This study uniquely addresses this gap by conducting an in-depth assessment of 125 wild accessions of C. azarolus collected from the Eastern Mediterranean region of Türkiye. By employing an integrative approach that combines morphological, pomological, and molecular data, this research provides novel insights into the genetic and phenotypic relationships within the hawthorn germplasm. Unlike previous studies, which have primarily focused on isolated traits or smaller sample sizes, this work offers a holistic evaluation of the region’s hawthorn diversity. Moreover, the findings have significant implications for understanding the evolutionary dynamics, conservation strategies, and potential utilization of hawthorn genetic resources, thereby underscoring the critical role of the Eastern Mediterranean as a global biodiversity hotspot for this genus.

Materials and methods

Plant material

In this study, phenotypic and fruit variability were investigated among 125 accessions of C. azarolus naturally growing in the Centre Neighborhood of Hasanbeyli district in Osmaniye Province (25 accessions), Fevzipaşa Neighborhood of Islahiye district in Gaziantep Province (25 accessions), and Akbez (25 accessions), Çınarbaşı (25 accessions), and Sapanözü (25 accessions) Neighborhoods of Hassa district in Hatay Province, located within the Eastern Mediterranean region of Türkiye (Fig. 1 ). The formal identification of the samples was performed by Dr. Yazgan Tunç.

The location of the regions was recorded using a Global Positioning System (GPS, Garmin GPSMAP 67, Garmin Ltd., Olathe, Kansas, USA) device (Table 1 ). Appropriate distances were maintained between the accessions in each region to prevent the sampling and collection of clones from the selected accessions.

The characteristics evaluated

A total of 53 morphological and pomological traits were utilized to assess the phenotypic diversity of the hawthorn accessions (Table 2 ). For this purpose, a total of 50 leaves and 50 fruits randomly selected from each accession at harvest maturity were used.

Variables including leaf length, leaf width, leaf lobe depth, petiole length, petiole thickness, fruit length, fruit width, and fruit stalk length were measured using a digital caliper with 0.01 mm precision from the Insize brand (model no. 1108 − 300, Insize Co., ltd. Suzhou New District, 215009, China). A precision scale branded Kern (model no. PCB 250-3, KERN & Sohn GmbH, Balingen, Germany) with 0.01 precision was used to measure fruit and seed weight. Plant growth type, tree growth habit, plant shape, plant height, canopy size, foliage density of the plant, presence of thorns on the shoot, number of thorns on the shoot, thorn length of the shoot, shoot length, shoot growth type, suckering, branching, branch flexibility, leaf margin status, leaf lobes, leaf anthocyanin coloration, leaf glossiness, leaf upper surface color, leaf lower surface color, leaf upper surface pubescence, leaf lower surface pubescence, leaf apex shape, leaf density, fruit color, glossiness of fruit skin, density of fruit lenticels, texture of fruit surface, fruit taste, fruit general shape, fruit density, stone shape, stone hardness, fruit cavity of eye basin, fruit depth of eye basin, and fruit main color of flesh were visually examined and coded according to the hawthorn descriptor [ 1 , 33 , 36 , 56 , 60 ].

Fruit juice extract was obtained by crushing 100 g of hawthorn fruit, with the endocarp and seeds removed, in a juice extractor (SINBO SJ-3143, İstanbul, Türkiye) at room temperature for 5 min. Following centrifugation for 10 min at 5,000 × g (Elektromag M615 E, İstanbul, Türkiye), clear juice was obtained and promptly analyzed [ 4 ]. pH was measured using a digital pH meter (Hanna HI 98130; Woonsocket, RI/USA) [ 4 ]. A refractometer branded Atago 2383 Master-20 M (Tokyo, Japan) was utilized to measure the soluble solids content in degrees Brix [ 60 ]. The titratable acidity was determined through titration with 0.1 M NaOH at pH 7 and expressed as grams of malic acid per 100 ml of juice [ 4 ].

The ripening index was calculated using the formula presented in Eq. 1 [ 39 ].

Molecular analysis through inter simple sequence repeat (ISSR) markers

DNA was extracted from young hawthorn leaves using the CTAB method [ 17 ]. DNA concentrations were measured using a spectrophotometer (ONDA VIS-10 plus, ONDA Laboratori s.r.l., Perugia, Italy), DNAs were visualized on a 2% agarose gel using gel electrophoresis and a DNA ladder (100 bp, Thermo Fisher Scientific), and the extracted DNAs were stored at − 20 °C. For ISSR analysis, testing was performed with 25 different primers, and band formation was observed with 15 of these primers. PCR reactions consisted of 2 µL DNA (20 ng), 1.5 µL 10×PCR buffer, 0.2 µL Taq DNA polymerase (5 u/µL), 1 µL dNTP (2.5 mM), 1.5 µL MgCl2 (25 mM), 2 µL 10 mM ISSR primer, and 6.8 µL H2O, making a final volume of 15 µL for PCR products. The PCR cycling conditions followed those described by Uzun et al. [ 57 ]: initial denaturation at 94 °C for 2 min, followed by 38 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 1.15 min, and a final extension cycle at 72 °C for 7 min.

Statistical analysis

This study, which was conducted in 2022 and 2023, utilized the average of the two-year datasets. Multivariate analysis methods were employed to identify genetic similarities and differences within the hawthorn germplasm. To detect significant differences among the 125 accessions, the JMP ® Pro 17 [ 28 ] statistical software package was used to subject the accessions to one-way analysis of variance (ANOVA). Using the same program, the minimum, maximum, mean, and coefficient of variation (%CV) of the datasets were determined. Multivariate analysis methods among variables (correlation, principal component analysis, and cluster analysis based on UPGMA) were evaluated using the Origin Pro ® 2024b [ 44 ] statistical software package. Pearson correlation coefficient was used for correlation analysis. A 2D principal component scatter plot was created to visualize the distribution among accessions. In addition, to reveal similarities and differences among regions due to the collection of accessions from different regions, 2D principal component scatter plots were created. To mitigate potential errors resulting from scale differences, the mean of each variable was taken and normalized using Z scores before cluster analysis. Ward’s method with the Euclidean distance coefficient was utilized for cluster analysis [ 27 ].

In ISSR analyses, images obtained from agarose gel electrophoresis and imaging (Kodak, Eastman Kodak Company, Rochester, New York, United States) were scored as follows: presence of a band (1), absence of a band (0), or no amplification (9). The data were analyzed using NTSYS (version 2.11X) software [ 47 ]. Dendrograms for accessions were constructed according to a similarity matrix created using the Dice method and unweighted pair group method with arithmetic mean (UPGMA) clustering analysis [ 51 ]. In addition, the total number of bands, number of polymorphic bands, and polymorphism rate were determined for each marker. In addition, the polymorphism rate was calculated using the formula provided in Eq. 2 [ 60 ].

Results and discussion

Descriptive statistics, morphological, and pomological diversity among accessions

The minimum, maximum, mean, standard deviation (SD), and variation coefficient (CV%) values of ​​ the examined features are presented in detail in Table 2 . The examined accessions showed significant differences in morphological and pomological aspects (ANOVA, p < 0.05). According to the results obtained, the number of thorns on the shoot exhibited the highest coefficient of variation (CV = 97.46%), whereas pH showed the lowest coefficient of variation (CV = 8.53%) (Table 2 ). In addition, 51 out of 53 characters (96.23% in total) had CVs greater than 20.00%, indicating high variation among the accessions [ 34 ]. This situation suggests that the studied germplasm exhibits a high degree of heterozygosity, leading to genetic variability in the accessions at the studied location due to open pollination [ 23 ]. Traits with high CVs exhibit a wider range of feature quantities and thus offer a wider range of selection based on that feature [ 18 ]. Leaf length ranged between 21.27 (‘G53’) and 50.55 mm (‘G125’), leaf width ranged from 14.11 (‘G116’) to 49.66 mm (‘G84’), petiole length varied from 3.13 (‘G71’) to 23.73 mm (‘G4’), and petiole thickness ranged from 0.20 (‘G96’) to 0.99 mm (‘G15’). In a similar study conducted in Türkiye, the leaf length, leaf width, petiole length, and petiole thickness of hawthorn accessions varied from 20.93 to 48.53 mm, 13.07 to 46.33 mm, 2.73 to 19.67 mm, and 0.17 to 0.97 mm, respectively [ 60 ]; in Iran, it was reported that the variables leaf length, leaf width, and petiole length ranged from 19.00 to 53.11 mm, 17.44 to 51.08 mm, and 9.10 to 28.09 mm, respectively [ 33 ]; and also, leaf length, leaf width, and petiole length varied from 41.86 to 75.91 mm, 41.00 to 70.41 mm, and 16.66 to 39.60 mm, respectively [ 36 ]. The leaf length/width ratio ranged from 0.49 (‘G13’) to 3.48 (‘G81’). In a study conducted by Çalışkan et al. [ 9 ] in Türkiye, it was reported that this variable ranged between 1.16 and 1.24. Leaf lobe depth changed between 6.14 (‘G39’) and 20.99 mm (‘G115’). In a similar study conducted by Khadivi et al. [ 33 ] in Iran, this variable was found to vary between 5.25 and 21.79. The fruit length changed between 8.02 (‘G37’) and 27.99 mm (‘G68’), the fruit width between 7.3 (‘G114’) and 32.99 mm (‘G38’), the fruit weight between 0.97 (‘G15’) and 14.73 g (‘G86’), and stone weight between 0.22 (‘G17’) and 0.79 g (‘G112’). These values in Türkiye were reported to range from 10.77 to 17.32 mm, 10.33 to 20.90 mm, 0.69 to 3.13 g, and 0.15 to 0.32 g, respectively [ 60 ]; in Iran, from 10.99 to 15.25 mm, 9.89 to 15.32 mm, 0.70 to 2.04 g, and 0.27 to 0.56 g, respectively [ 36 ]. In another study on hawthorn in Iran, fruit length ranged from 13.00 to 18.20 mm, and fruit width varied between 15.53 and 22.5 mm [ 40 ]. The flesh/stone ratio ranged from 1.02 (‘G107’) to 64.95 (‘G124’). The fruit stalk length changed between 8.55 (‘G42’) and 44.7 mm (‘G51’). Khadivi et al. [ 33 ] reported the range of this variable between 12.54 and 40.25 mm in Iran. The pH varied from 3.02 (‘G75’) to 3.99 (‘G55’), the soluble solids content changed between 8.19 (‘G108’) and 21.98 °Brix (‘G1’), the titratable acidity ranged from 0.69 (‘G3’) to 2.29% (‘G37’), and the ripening index varied from 4.33 (‘G107’) to 29.90 (‘G3’). In a similar study conducted on hawthorn in Türkiye, the pH was reported to range from 3.0 to 3.6 [ 9 ], while in Tunisia, it varied between 3.2 and 4.2 [ 4 ], and in Iran, it was found to change between 3.46 and 3.86 [ 1 ]. The soluble solids content ranged from 9.00 to 18.00 °Brix in Türkiye [ 60 ] and from 16.33 to 24.00 °Brix in Iran [ 1 ]. In Tunisia, the titratable acidity was determined to be between 0.9 and 1.9% [ 4 ]. The ripening index was detected to change between 9.78 and 11.60 in Iran [ 40 ]. When our findings are evaluated together with the findings in the literature, although they are generally similar, there are minor differences. It is estimated that this is due to the different hawthorn accessions used. In addition, as highlighted in the scientific literature, the interaction between genotype and environmental conditions plays a significant role in shaping morphological traits. Variations in climate, soil type, and other environmental factors can lead to different phenotypic expressions, even among the same genotypes. For example, Schmid [ 49 ] and Orgogozo et al. [ 43 ] reported that environmental stressors, such as temperature fluctuations and soil pH can influence fruit size, leaf shape, and other morphological characteristics in hawthorn populations. These environmental factors, in combination with genetic diversity, contribute to the observed morphological variability.

Although the findings we obtained within the scope of fruit characteristics are generally similar to the studies in the literature, partial differences were detected. This difference is likely due to differences in the genetic material used, climate factors, and soil structure [ 23 ]. In hawthorn breeding programs, the most desirable fruit characteristics are greater fruit weight and a greater flesh-to-seed ratio [ 20 ].

The frequency distribution of qualitative morphological and pomological characteristics measured in the studied C. azarolus accessions are presented in detail in Table 3 . Accordingly, the important traits in the accessions showed the following dominance: spreading growth habit in tree growth (55 accessions), tall plant height (45 accessions), intermediate canopy size (58 accessions), intermediate suckering and branching (59 and 58 accessions, respectively), serrate leaf margin status (107 accessions), low pubescence on the upper leaf surface (95 accessions), intermediate pubescence on the lower leaf surface (50 accessions), high leaf density (46 accessions), and high fruit density (35 accessions). The fruit taste was sour in 26 accessions, sweet-sour in 19 accessions, and sweet in 80 accessions. In a similar study on hawthorn in Iran, Khadivi et al. [ 33 ] found that tree height, branch flexibility, trunk diameter, and canopy density were predominantly intermediate, while branching, branch density, tendency to suckering, and leaf density were predominantly high. Our findings are largely consistent with those of the other researchers. The observed differences are due to environmental conditions, plant growth behavior, and the varietal characteristics resulting from the heterozygous structure of the accessions [ 52 ]. The variations in the examined C. azarolus accessions are shown in Fig. 2 .

Correlations among variables

Correlation expresses the relationship between two variables. There are three main types of correlation: positive (an increase in one variable is associated with an increase in the other), negative (an increase in one variable is associated with a decrease in the other), and zero (there is no significant relationship between the two variables). Correlation analysis typically uses the Pearson correlation coefficient ( r ) to measure the relationship. This analysis aims to determine whether a relationship exists between two variables, assess the predictive power of one variable over another, and provide initial data for statistical modeling or regression analysis [ 54 ]. Attention should be paid to the fact that outliers can affect the correlation coefficient and lead to misleading results [ 61 ]. Therefore, quantitative morphological variables were used in the correlation analysis [ 35 ]. Basic correlations among quantitative morphological variables analyzed in accessions of C . azarolus are presented in detail in Fig. 3 . A statistically significant strong positive correlation ( r = 0.60*** ) was found between leaf width and leaf length. Our findings are consistent with the results of a similar study conducted in Iran [ 36 ]. The relationship between leaf width and leaf length indicates that the leaf structure of plants follows a certain pattern and develops similarly according to growth conditions. Wider leaves can generally be longer, which increases the plant’s capacity to capture light and supports photosynthesis [ 65 ]. Leaf width and length are key factors that affect the photosynthetic efficiency of plants. Wide and long leaves can better capture sunlight and provide a larger surface area, optimizing water evaporation, and thereby supporting the growth and development of the plant [ 13 ]. This correlation may also be related to certain genetic traits and adaptation strategies of plants. In specific species, the width and length of leaves may have evolved as a response to environmental conditions (such as light, water, and nutrient availability) [ 14 ]. In addition, the size of the leaves can affect the number and distribution of chloroplasts; wider and longer leaves can enhance chlorophyll production, thereby strengthening the photosynthetic processes. As a result, this situation has a positive impact on the overall health and productivity of the plant. A negative correlation ( r=-0.79*** ) occurred between leaf ratio length/width and leaf width. Positive correlations have been observed between petiole length and leaf lobes ( r = 0.20* ), and between petiole thickness and leaf lobe depth ( r = 0.18* ). A negative correlation has formed between the fruit length/width ratio and leaf lobe depth ( r=-0.19* ) as well as between fruit width ( r=-0.72*** ), while a positive correlation has occurred with fruit length ( r = 0.59*** ). A negative correlation has been found between stone weight and petiole thickness ( r=-0.18* ). There is a positive correlation between flesh/stone ratio and fruit weight ( r = 0.75*** ), while a negative correlation has occurred with stone weight ( r=-0.47*** ). A negative correlation has been detected between titratable acidity and fruit width ( r=-0.18* ). Negative correlations have emerged between ripening index and stone weight ( r=-0.19* ), pH ( r=-0.21* ), and titratable acidity ( r=-0.76*** ), while a positive correlation has been established with soluble solids content ( r = 0.53*** ). These correlations provide important insights into the morphological characteristics, photosynthetic processes, and environmental adaptations of plants. Understanding such relationships among plants can be beneficial for agricultural practices and plant breeding. The correlations can also shed light on the evolutionary adaptations of plants and how they respond to environmental conditions. This data can be used to optimize plant growth and development processes.

Principal component analysis (PCA)

In PCA, an eigenvalue greater than 1.0 indicates that the corresponding component explains a significant portion of the variability in the dataset. This suggests that the component accounts for more variance than an individual variable in the original dataset, highlighting its importance in understanding the structure and relationships within the data. Therefore, an eigenvalue exceeding 1.0 signifies that the component is noteworthy and plays a critical role in comprehending the overall structure of the dataset. Such components carry more information and meaning, ensuring that essential information is retained while reducing the dimensionality of the dataset [ 29 ]. In the context of PCA, the degree of significance for eigenvectors was found to be ≥ 0.29 for each variable (bold values), and the eigenvectors greater than 1.0 (the first 22 components) accounted for 72.47% of the total variance. A total of 6.76% of the variation was obtained by PC1, where the presence of thorns on the shoot (0.44), thorns length on the shoot (0.43), and number of thorns on the shoot (0.40) had the greatest values. PC2 explained 5.70% of the total variation, consisting of the highest values for ripening index (0.42), and fruit width (0.29). PC3 was correlated with leaf ratio length/width (0.38) for 5.10% of the total variance. This distribution showed that there was great diversity among the germplasms [ 18 ]. The findings we obtained within the scope of the study are partially similar to the results of similar studies conducted in Iran [ 33 , 36 ]. It is thought that the differences in the results are due to the different accesses used and the different areas studied. This result demonstrated significant morphological variation, indicating high genetic diversity among the accessions, and suggested that evaluating various morphological characteristics is essential for meaningful characterization. The traits mentioned above were the most effective in distinguishing and identifying the studied accessions. PCs were separated according to their significance. Accordingly, PC1, PC2, and PC3 were found to be ***p < = 0.001 (Table 4 ).

The PCA scatter plot is a powerful tool for visualizing high-dimensional data by reducing it to key components, which helps to elucidate relationships and distributions among data points. These plots facilitate the discovery of specific groups or clusters, enabling a clearer understanding of the underlying structure in the data. By providing a visual analysis of data distribution and density, PCA scatter plots reduce noise through the cleaned data, allowing for more accurate interpretation. They also enable the evaluation of the impact of components that reflect specific characteristics of the data, which can pave the way for the application of clustering or classification algorithms. In this context, the scatter plot illustrates the geometric distances between accessions, reflecting their phenotypes and highlighting potential genetic similarities and dissimilarities. In summary, PCA scatter plots are an essential method for understanding, visualizing, and analyzing complex data sets [ 38 ].

Accordingly, PCA was used to create a two-dimensional scatter plot to graphically illustrate the relationships among the accessions. The projection of the examined accessions on the PC1/PC2 plane based on the regression factor score values ​​is shown in detail in Fig. 4 . PC1 and PC2 represented 12.46% of the total variation. The accessions were distributed across the four regions of the scatter plot. 119 accessions were located within the 95% confidence ellipse, while 6 accessions (G10, G22, G24, G64, G98, and G122) remained outside the 95% confidence ellipse. Genotypes outside the 95% confidence ellipse in the scatter plot indicate that they exhibit different characteristics compared with the majority of the analyzed genotypes. This situation suggests that these genotypes are generally distant from the observed distribution concerning the examined traits, signaling a distinct difference or diversity [ 29 ]. These genotypes may possess special adaptive capabilities, exhibit different phenotypic traits, or respond to specific environmental conditions. In addition, this could enhance genetic diversity and provide interesting targets for particular research or applications. However, it is also important to evaluate the relationships and genetic similarities of these genotypes with other genotypes in the analysis, as this can assist in making strategic decisions for breeding or conservation programs.

Cluster analysis

A dendrogram was created using Ward’s method and Euclidean distance (Fig. 5 ). The resulting dendrogram divided the genotypes into two main groups, A and B, with each main group further divided into two subgroups. The A1 subgroup contained 26 accessions, the A2 subgroup contained 45 accessions, the B1 subgroup contained 28 accessions, and the B2 subgroup contained 26 accessions. The highest variation was detected in the A2 subgroup. The present study serves as a valuable reference for future research on the morphological and pomological characteristics of the species, aiming to identify the most favorable traits for their introduction into commercial production. Similar levels of diversity and similar intervals of variation for morphological and pomological characteristics were found in different hawthorns evaluated [ 2 , 5 , 26 , 36 , 55 , 59 ].

Population analysis

Within the scope of population analysis, PC1 and PC2 represented 58.87% of the total variance. In the population scatter plot, the five populations are divided into four clusters. The first cluster included Akbez/Hassa, the second cluster included Çınarbaşı/Hassa, the third cluster included Sapanözü/Hassa and Fevzipaşa/Islahiye, and the fourth cluster included the Centre/Hasanbeyli (Fig. 6 ). Population genetics is a fairly old field in biology, starting with the study of the diversity of hereditary traits in populations that influence an organism’s adaptation to its environment [ 22 ].

ISSR analysis

Fifteen different ISSR primers were used to detect the genetic diversity of the hawthorn accessions used in this study (Table 5 ). The total number of bands varied between 7 [(TCC) 5 RY] and 19 [DBDA(CA) 7 and (GA) 8 YG], and a total of 195 countable bands were obtained from these primers. Of the 195 countable bands obtained, 191 were determined to be polyformic and the number of polyformic bands was found to vary between 7 [(CT) 8 TG] and 19 [DBDA(CA) 7 ]. The average band lengths were between 80 and 2050 bp, the total number of bands per primer was 13, and the number of polymorphic bands was 12.73. The average polymorphism rate was 97.20%. A total of 54 scorable bands were obtained from seven different ISSR primers to determine the genetic diversity of the hawthorn germplasm in Iran, and the average polymorphism rate was 89%. Of these bands, 49 were polymorphic. In addition, the band length ranged from 300 to 2500 bp, and an average length of 950 bp was reported [ 3 ]. In another study conducted in Iran, six different ISSR primers were used, resulting in a total of 79 scorable bands. Of these bands, 71 were found to be polymorphic, and the average polymorphism rate was reported to be 89.9%. In addition, the band length ranged from 100 to 3000 bp [ 7 ]. In a study conducted in Türkiye, which aimed to determine the genetic diversity of hawthorn germplasm using thirteen different ISSR primers, a total of 101 scorable bands were obtained. Among these bands, 76 were found to be polymorphic, and the polymorphism rate was 75.24% [ 60 ].

Within the scope of the study, a dendrogram was created using the UPGMA method to determine the molecular diversity of hawthorn accessions (Fig. 7 ). The similarity index among the 125 hawthorn accessions ranged from 0.72 to 0.90. In the dendrogram, the accessions were divided into two main groups. Only group B was divided into two subgroups. In group A, the accessions ‘G70’ and ‘G3’ were included. In the B1 subgroup, the accessions ‘G65’, ‘G47’, and ‘G22’ were included. All the remaining accessions were placed in the B2 subgroup. In the study, the accessions ‘G13’ and ‘G28’ had the highest genetic similarity with a similarity index of 0.90. In this study, consisting of the same species, the accessions were both genetically similar to each other and showed significant differences. The reasons for this are the natural gene exchange occurring due to cross-pollination in nature and the ISSR marker systems targeting random regions in the genome [ 63 ]. In a similar study conducted in China, researchers using ISSR markers found that the similarity index among 92 different hawthorn genotypes ranged from 0.53 to 0.87 [ 50 ], whereas in Türkiye, it was detected to vary between 0.71 and 0.88 [ 60 ], in parallel to t our findings.

Conclusions

In this study, significant morphological, pomological, and molecular differences were detected between C. azarolus accessions. Superior accessions for commercial production of this species have been detected. Fruit quality and yield depend on several factors such as fruit color, fruit density, fruit weight, fruit main color of flesh, pH, soluble solids content, titratable acidity, and ripening index. According to the quantitative data set, identified twenty accessions (‘G21’, ‘G117’, ‘G104’, ‘G17’, ‘G11’, ‘G124’, ‘G23’, ‘G10’, ‘G125’, ‘G30’, ‘G2’, ‘G38’, ‘G115’, ‘G4’, ‘G111’, ‘G8’, ‘G35’, ‘G106’, ‘G97’, and ‘G52’, respectively) with superior qualities that can be used as parents in breeding programs or selected directly for cultivation.

Understanding genetic variation and relationships within C. azarolus populations is essential for maximizing the use of germplasm resources and designing effective breeding and plant protection strategies. ISSR markers revealed genetic relationships among populations, showing potential for interspecific crossings. The scarcity of commercial hawthorn varieties makes these findings critical for breeding programs.

Although C. azarolus holds ecological and socio-cultural value in the Eastern Toros forests of Türkiye, its natural populations are declining due to climate change, overhunting, illegal logging, and forest fires. This study highlights the need to preserve forests rich in C. azarolus to safeguard genetic diversity. In situ and ex situ conservation strategies, such as vegetative and seed propagation, hybridization, and open-pollinated progeny, are vital. Propagation via seeds is recommended for maintaining genetic diversity. Stopping habitat destruction and improving seed germination conditions are also crucial. This study provides detailed information on the morphological, pomological, and genetic structure of C. azarolus germplasms.

Acknowledgements

None.

Author contributions

All authors contributed to the study’s conception and design. YT, MY, and KUY experimented and collected data. YT analyzed data and wrote and edited the manuscript. AK and SG edited the manuscript. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not Applicable.

Research involving human participants and, or animals

Not applicable.

Informed consent

Not applicable.

Consent for publication

Not applicable.

Statement specifying permissions

For this study, we acquired permission to study C. azarolus issued by the Agricultural and Forestry Ministry of the Republic of Türkiye.

Competing interests

The authors declare no competing interests.

Statement on experimental research and field studies on plants

The either cultivated or wild-growing plants sampled comply with relevant institutional, national, and international guidelines and domestic legislation of Türkiye.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Yazgan Tunç, Email: yazgan.tunc@tarimorman.gov.tr.

Ali Khadivi, Email: a-khadivi@araku.ac.ir.

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Associated Data

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.