Primary, Secondary Metabolites and Molecular Characterization of Hawthorn ( Crataegus spp.) Genotypes

: In this study, the molecular, biochemical and agro-morphological characterization of genotypes belonging to hawthorn species collected from Bolu province of Turkey was performed. Inter-priming binding sites (iPBS) markers based on retrotransposons were used for the ﬁrst time in the molecular properties of hawthorn genotypes in the world. The marker system provided very useful information for revealing the genetic variation of the genotypes. Six iPBS markers ampliﬁed 68 fragments, of which 65 were polymorphic (95.59%) with an average of 10.83 polymorphic bands per primer. The polymorphism and resolving power per primers ranged from 0.12 to 0.42 and from 0.78 to 8.11 with the average being 0.32 and 5.95, respectively. Pomological properties of Crataegus tanacetifolia , such as fruit pomology and core weight were determined to higher than those of Crataegus monogyna . Citric acid was determined as the most predominant organic acid, followed by malic and succinic acid in the genotypes of both species. The highest citric acid content (26.745 mg 100 g − 1 ) was noted for 14BL09 genotype. The vit. C content was recorded ranging from 2.681 to 9.621 mg 100 g − 1 . Catechin, chlorogenic, ca ﬀ eic and rutin contents were varied between 4.140–51.393 mg, 2.254–42.361 mg, 0.624–4.407 mg, and 1.241–10.029 mg per 100 g of fruits, respectively. As a result, it has been determined that twenty-ﬁve genotypes belonging to di ﬀ erent hawthorn species are important genetic resources to be evaluated in horticultural breeding studies in terms of their physical and biochemical contents.


Introduction
Wild edible fruits are, in general, naturally grown in rural areas throughout the world and represent important dietary sources of rural peoples because they are rich in vitamins, minerals, dietary fibers, etc. The stem, leaf, flowers, roots and the fruits of wild edible fruits have also a high potential for traditional medicinal uses [1][2][3][4][5].
Hawthorn is taxonomically classified within the Rosaceae family, Maloidae subfamily, Crataegeae section and Crataegus genus [6]. Among 50 species of hawthorn located in the northern hemisphere, more than 20 hawthorn species have been reported to be grown in Turkey [7,8]. Crataegus monogyna Jacq. subsp. azarelle (GRIS.) FRANCO is the most common naturally grown hawthorn species [9].

Plant Materials
Samples were collected from the area located between 30 • 32 and 32 • 36 E, 40 • 06 and 41 • 01 N latitudes in Bolu provinces in Turkey. High mountainous and dense forest structure of the area has led to the diversity of microclimate areas. Hawthorn species are widely wild-grown in this region.
The fruit and leaf samples taken from each hawthorn tree were labeled and put into boxes placed in appropriate containers and immediately transferred to the laboratory. Fruit and leaf samples to be used for biochemical analysis were kept in the ultra-low temperature chest freezers at −80 • C until analysis.

Extraction of Organic Acids
In this study, about 200 g of each sample was fragmented and 15 g from each fragmented sample was transferred to a centrifuge tube, then diluted 1:3 with distilled water. Twenty-five mL of 0.009 N H 2 SO 4 was added to samples and then the samples were homogenized with a crusher (Heidolph Silent Crusher M, Berlin, Germany) and mixed for an hour with a shaker (Heidolph Unimax 1010, Berlin, Germany). After centrifugation at 15,000× g for 15 min, the supernatant was passed through coarse filter paper, twice in 0.45 µm membrane filter (Millex-HV Hydrophilic PVDF, Millipore, Taufkirchen, Germany), and last in the SEP-PAK C18 cartridge. The concentration of organic acids was determined by HPLC using an Aminex column (HPX-87H, 300 mm × 7.8 mm, Bio-Rad) fitted on an 1100 series HPLC (Agilent Technologies, Waldbronn, Germany). Organic acids were detected at both 254 nm and 280 nm wavelengths. As the mobile phase, 0.009 N H 2 SO 4 was passed through a 0.45 µm filter membrane [24].

Extraction of Phenolics
About 10 g sample out of 200 g of fragmented sample for each sample was transferred to a centrifuge tube, mixed homogeneously, then diluted 1:1 with distilled water and centrifuged at 15,000× g for 15 min. The supernatant was passed through a 0.45 µm Millex-HV Hydrophilic PVDF membrane filter, then injected into the HPLC system (gradient). The chromatographic separation in Agilent 1100 series HPLC took place in a DAD detector (Agilent, Waldbronn, Germany) with 250 mm × 4.6 mm, 4 m ODS column (HiChrom, New Jersey, USA). The following solvents in water with a flow rate of 1 mL/min and 20 µL injection volume was used for spectral measurements taken at both 254 nm and 280 nm: as mobile phase solvent A, methanol-acetic acid-water (10:2:88) and Solvent B, methanol-acetic acid-water (90:2:8) [25].

Determination of Pomological Properties of Fruits
Some pomological properties of hawthorn genotypes were investigated. Average fruit weight of 10 fruits taken randomly from each genotype (with 0.1 g sensitive scales), fruit height, fruit width (with 0.01 mm sensitive caliper), seed weight, pH (with pH meter), soluble solid content (SSC) (by hand refractometer), titratable acidity (TA) (titration method) and color values (with minolta color meter) were determined.

Molecular Characterization
A modified version of the CTAB-based method as described in the DArT protocol [26] was employed to extract the genomic DNA of the hawthorn genotypes. Approximately 100 mg of leaf tissue of each genotype was ground with the help of a mortar and pestle in liquid nitrogen and immediately transferred into a 1.5 mL microfuge tube. DNA extraction was completed with extraction-lysis buffer. DNA (Agilent Technologies Inc., Waldbronn, Germany) was dissolved in 100 µL sterile ultra-pure water. The DNA concentration was estimated by the DS-11 FX Series Spectrophotometer (Labgene Scientific SA, Châtel-Saint-Denis, Switzerland) and adjusted to 20 ng/µL with sterile ultra-pure water for PCR assays.
To evaluate genetic diversity among genotypes, the genomic DNA was subjected to PCR amplification of all iPBS primers designed Kalendar et al. [22]. Annealing temperatures for iPBS markers were performed as recommended by Kalendar et al. [22]. PCR products were subjected to electrophoresis in 1.2% (w/v) agarose gel over 2 h and imagined by a G: Box F3 Gel Documentation System after ethidium bromide staining (Syngene, Cambridge, UK).
All bands obtained by iPBS analyses were scored as the presence (1) or absence (0) at positions for the construction of a binary data matrix. Each primer-sample combination was repeated at least two different amplifications and only reproducible bands were evaluated. Neighbor-joining cluster analysis was conducted using R [27] and MEGA7 [28] software. To evaluate the suitability of iPBS markers to Agronomy 2020, 10, 1731 4 of 18 molecular analysis of the genotypes, the performance of the markers was measured using resolving power (RP) and the polymorphic information content (PIC) as proposed by Rolden-Ruiz et al. [29].

Statistical Analysis
Descriptive statistics, normal distribution tests, correlation analysis, and one-way variance analysis were performed with the SAS 9.4 program (Statistical Analysis System, Raleigh, NC, USA). The Duncan test was used as a multiple comparison test to express the differences between the averages. In R software, the principal component analysis was used for all variables with the ggplot2 and factoextra packages [30].

Organic Acid Contents
The contents of oxalic, citric, tartaric, malic, succinic, fumaric, and ascorbic have been varied over a broad range, as shown in Table 1. The principal component analysis on the distribution of genotypes according to their organic acid content revealed 67.8% variation ( Figure 1). Sorkun [31] determined the ranges of malic, citric, tartaric, and ascorbic acid contents as 641.61 (30-K1)-1132.86 mg 100 g −1 (30-M2), 320.64 (30-K1)-831.73 mg 100 g −1 (30-M2), 29.11 (30-M1)-392.89 mg 100 g −1 (30-S2), 7.25 (30-M1)-60.02 mg 100 g −1 (30-S1), respectively, which, similar in ranges of our determinations. Gündogdu et al. [13] studied many different hawthorn species, and determined the differences among species such as their fruits' contents, which were 2.650 g 100 g −1 , 1.953 g 100 g −1 , 0.780 g 100 g −1 , 1.080 g 100 g −1 , 0.027 g 100 g −1 , 1.721 g 100 g −1 in Crataegus monogyna of oxalic, citric, tartaric, succinic, fumaric, and malic values, respectively. The content values obtained in this study are higher when compared with the findings of the other studies. Liu et al. [32] reported that malic acid and citric acid contents were varied between 0.32-1.12 g 100 g −1 and 1.97-8.38 g 100 g −1 in different hawthorn species, respectively. When the data presented by the researchers were compared with our findings, the amounts of malic acid and citric acid were higher than those of other researchers. This situation may caused by climatic factors, diversity in genetic factors, geographical location, day and night temperature difference, precipitation amount and soil characteristics. When the organic acid contents of the recipients were examined, difficulties became to compare due to the insufficiency of the studies on this subject. This situation reveals the importance and novelty of our study.

Phenolic Compound Contents
Phenolic substances are effective in most of the physiological events in fruits. Anthocyanins, which are phenolic substances, play an important role in the formation of the unique colors of fruits and vegetables. The variation rate of 48.9% was determined as a result of the principal component analysis to determine the distribution of genotypes according to phenolic compound content ( Figure  2). In this study, broad ranges of concentrations of gallic, catechin, chlorogenic, caffeic, syringic, pcoumaric, ferulic, o-coumaric, protocatechuic, vanilic, rutin, and quercetin were detected in fruits of hawthorn genotypes (Table 2). Sorkun [31] reported that the average total phenolic content of all genotypes was 9391 μg GAE g −1 , the highest value in terms of total phenolic content was found in the genotype 30-M2 (10991 μg GAE g −1 ) and mostly mahogany-black genotypes contain high amounts of total phenolic and especially 30-M2, 30-M3, and 30-M5 genotypes had more total phenolics than other genotypes studied. When the total phenolic content of hawthorn fruit is compared with other fruits and vegetables, it has been found that hawthorn fruit contains a high amount of total phenolic substance [33]. In a research conducted in China, 11 major phenolic compounds were determined in 22 hawthorn cultivars and hyperoside (0.1-0.8 mg g −1 dry mass [DM]), isoquercitrin (0.1-0.3 mg/g −1 DM), chlorogenic acid (0.2-1.6 mg g −1 DM), epicatechin (0.9-11.7 mg g −1 DM), PA B2 (0.7-12.4 mg g −1 DM), PA dimer II (0.1-1.5 mg g −1 DM), PA trimer I (0.1-2.7 mg g −1 DM), PA trimer II (0.7-6.9 mg g −1 DM), PA trimer III (0.01-1.2 mg g −1 DM) and a PA dimer-hexoside (trace-1.1 mg g −1 DM) were reported [34]. The differences between the findings of other researchers and our results might be caused by the cultural practices (fertilization, pruning, etc.), climate and soil characteristics of studied areas.

Phenolic Compound Contents
Phenolic substances are effective in most of the physiological events in fruits. Anthocyanins, which are phenolic substances, play an important role in the formation of the unique colors of fruits and vegetables. The variation rate of 48.9% was determined as a result of the principal component analysis to determine the distribution of genotypes according to phenolic compound content ( Figure 2). In this study, broad ranges of concentrations of gallic, catechin, chlorogenic, caffeic, syringic, p-coumaric, ferulic, o-coumaric, protocatechuic, vanilic, rutin, and quercetin were detected in fruits of hawthorn genotypes ( Table 2). Sorkun [31] reported that the average total phenolic content of all genotypes was 9391 µg GAE g −1 , the highest value in terms of total phenolic content was found in the genotype 30-M2 (10991 µg GAE g −1 ) and mostly mahogany-black genotypes contain high amounts of total phenolic and especially 30-M2, 30-M3, and 30-M5 genotypes had more total phenolics than other genotypes studied. When the total phenolic content of hawthorn fruit is compared with other fruits and vegetables, it has been found that hawthorn fruit contains a high amount of total phenolic substance [33]. In a research conducted in China, 11 major phenolic compounds were determined in 22 hawthorn cultivars and hyperoside (0.1-0.8 mg g −1 dry mass [DM]), isoquercitrin (0.1-0.3 mg/g −1 DM), chlorogenic acid (0.2-1.6 mg g −1 DM), epicatechin (0.9-11.7 mg g −1 DM), PA B2 (0.7-12.4 mg g −1 DM), PA dimer II (0.1-1.5 mg g −1 DM), PA trimer I (0.1-2.7 mg g −1 DM), PA trimer II (0.7-6.9 mg g −1 DM), PA trimer III (0.01-1.2 mg g −1 DM) and a PA dimer-hexoside (trace-1.1 mg g −1 DM) were reported [34]. The differences between the findings of other researchers and our results might be caused by the cultural practices (fertilization, pruning, etc.), climate and soil characteristics of studied areas.

Agro-Morphological Properties
Some pomological and biochemical properties of Crataegus monogyna and Crataegus tanacetifolia were determined. When genotypes were compared based on physical properties, genotype 14BL05 was found to be superlative. The 14BL07 genotype was found to have lower values than other genotypes in terms of pomological measurements of fruits ( Table 3). The fruits of genotypes of Crataegus tanacetifolia were superior due to their physical properties. The 14BL05, 14BL09, 14BL11,14BL12, and 14BL13 genotypes were found to be promising in terms of fruit weight, width, and height ( Table 3). As shown in Table 4, 14BL01, 14BL03, 14BL10, 14BL23, 14BL24, and 14BL25 genotypes have high values, as in pH amount and SSC. According to the principal component analysis, the variation rate between the genotypes in terms of pomological properties, pH, SSC, and acidity content was found to be 65.8% (Figure 3). In this study, fruit weight, fruit width, fruit height, seed weight, fruit volume, fruit stalk length, fruit stalk thickness, SSC ratio, pH amount and TA value varied over a wide range, as shown in Tables 3 and 4. The darkest fruit color (L* = 58.111) was measured in the 14BL19 genotype while the lightest color was measured in the 14BL10 (L* = 23.984) genotype ( Table 5). The highest a* value was found to be 41.939 in the 14BL02 genotype and the lowest a* value was −2.810 in the 14BL16 genotype. In the hawthorn genotypes examined, the highest b* value was found to be 46.566 for the 14BL12 genotype and the lowest b* value was 7.060 for the 14BL10 genotype. When investigating hue value, the highest value among genotypes was determined to 94.483 for the 14BL16 genotype while the lowest value was 13.541 for the 14BL10 genotype (Table 5). Table 3. Fruit weight (g), fruit width and length (mm), seed weight (g) and fruit volume (mL) of hawthorn genotypes.     Sorkun [31] determined that the L value was 20.09-21.00 in dark-colored mahogany-black genotypes, 28.67 in red 30-K1 genotype, and 67.80-68.50 in the yellow fruited genotypes. The highest a * value was also recorded as 31.95 in the 30-K1 genotype. The fruit weight, fruit width, fruit height, seed weight, ratio of SSC, pH value, and TA contents of hawthorn genotypes located in Uşak province of Turkey reportedly ranged from 4.03 g to 0.96 g, from 19.94 mm to 12.53 mm, from 17.43 mm to 10.48 mm, from 0.98 g to 0.23 g, from 17.40 to 9.12%, from 4.12 to 2.48 and from 2.85% to 0.58%, respectively [35]. In some pomological and biochemical properties of hawthorn genotypes, studies determined by different researchers show similarities with our results [36,37]. The slight differences in results might be derived from genotype, geographical location, ecological factors, soil properties.

Molecular Characterization
In this study, the reproducible and evaluable band profiles were obtained with six primers iPBS2074, iPBS2257, iPBS2388, iPBS2232, iPBS2239, and iPBS2415 (Table 6) and PCR studies of all samples were performed with these primers. In total, 68 loci, out of which 65 were polymorphic (95.59%), were obtained from PCR amplification with six iPBS markers for hawthorn genotypes. The iPBS marker system produced divergent fragments, providing a considerable variability among the genotypes belonging to different hawthorn species (Figure 4). The number of amplified fragments with iPBS markers ranged from 6 (iPBS2257) to 14 (iPBS2074), providing a ratio of 11.3 bands per primer. Sorkun [31] determined that the L value was 20.09-21.00 in dark-colored mahogany-black genotypes, 28.67 in red 30-K1 genotype, and 67.80-68.50 in the yellow fruited genotypes. The highest a * value was also recorded as 31.95 in the 30-K1 genotype. The fruit weight, fruit width, fruit height, seed weight, ratio of SSC, pH value, and TA contents of hawthorn genotypes located in Uşak province of Turkey reportedly ranged from 4.03 g to 0.96 g, from 19.94 mm to 12.53 mm, from 17.43 mm to 10.48 mm, from 0.98 g to 0.23 g, from 17.40 to 9.12%, from 4.12 to 2.48 and from 2.85% to 0.58%, respectively [35]. In some pomological and biochemical properties of hawthorn genotypes, studies determined by different researchers show similarities with our results [36,37]. The slight differences in results might be derived from genotype, geographical location, ecological factors, soil properties.

Molecular Characterization
In this study, the reproducible and evaluable band profiles were obtained with six primers iPBS2074, iPBS2257, iPBS2388, iPBS2232, iPBS2239, and iPBS2415 (Table 6) and PCR studies of all samples were performed with these primers. In total, 68 loci, out of which 65 were polymorphic (95.59%), were obtained from PCR amplification with six iPBS markers for hawthorn genotypes. The iPBS marker system produced divergent fragments, providing a considerable variability among the genotypes belonging to different hawthorn species (Figure 4). The number of amplified fragments with iPBS markers ranged from 6 (iPBS2257) to 14 (iPBS2074), providing a ratio of 11.3 bands per primer.  MirAli et al. [15] examined the genetic relationship of hawthorn genotypes belonging to different species (Crataegus monogyna, Crataegus sinaica, Crataegus aronia, and Crataegus azarolus) in Syria by using ISSR and CAPS markers. In the phylogenetic tree based on 20 ISSR primers used in the study, genotypes of Crataegus monogyna were collected in a cluster and the genotypes of the other species were clustered in the second branch. CAPS primers were insufficient to differentiate between species and genotypes. Keleş [38] performed a molecular analysis of 78 genotypes belonging to C. tanacetifolia, C. orientalis subsp. orientalis, C. meyeri, and C. monogyna Jacq. var monogyna and found that 14 ISSR primers produced 101 polymorphic bands with a polymorphism rate of 97.42%.
PIC and RP index values were estimated for iPBS marker systems as shown in Table 1. The highest PIC value of 0.42 (iPBS2239) and the lowest PIC value 0.12 (iPBS2257) with a mean of PIC per primer 0.32 were obtained from iPBS markers. The mean of RP values, a parameter for the discriminatory potential of the primers, was 5.95 for iPBS markers. iPBS2395 marker produced the highest RP value for iPBS2239, while iPBS2257 marker yielded the lowest RP value recorded as 0.78. The genotypes on the phylogenetic tree from neighbor-joining cluster analysis of iPBS markers data ( Figure 5) were grouped into two major clusters, which comprised the genotypes of each species based on genetic similarity. MirAli et al. [15] examined the genetic relationship of hawthorn genotypes belonging to different species (Crataegus monogyna, Crataegus sinaica, Crataegus aronia, and Crataegus azarolus) in Syria by using ISSR and CAPS markers. In the phylogenetic tree based on 20 ISSR primers used in the study, genotypes of Crataegus monogyna were collected in a cluster and the genotypes of the other species were clustered in the second branch. CAPS primers were insufficient to differentiate between species and genotypes. Keleş [38] performed a molecular analysis of 78 genotypes belonging to C. tanacetifolia, C. orientalis subsp. orientalis, C. meyeri, and C. monogyna Jacq. var monogyna and found that 14 ISSR primers produced 101 polymorphic bands with a polymorphism rate of 97.42%.
PIC and RP index values were estimated for iPBS marker systems as shown in Table 1. The highest PIC value of 0.42 (iPBS2239) and the lowest PIC value 0.12 (iPBS2257) with a mean of PIC per primer 0.32 were obtained from iPBS markers. The mean of RP values, a parameter for the discriminatory potential of the primers, was 5.95 for iPBS markers. iPBS2395 marker produced the highest RP value for iPBS2239, while iPBS2257 marker yielded the lowest RP value recorded as 0.78. The genotypes on the phylogenetic tree from neighbor-joining cluster analysis of iPBS markers data ( Figure 5) were grouped into two major clusters, which comprised the genotypes of each species based on genetic similarity. To date, several DNA marker methods such as SSR, ISSR, RAPD, and CAPS have been applied to generate genetic polymorphism among hawthorn genotypes [14,15,[39][40][41]. However, no record of detection of genetic differences in hawthorn genotypes using iPBS retrotransposon markers was found. This is the first study in which this marker system is used in the molecular characterization of To date, several DNA marker methods such as SSR, ISSR, RAPD, and CAPS have been applied to generate genetic polymorphism among hawthorn genotypes [14,15,[39][40][41]. However, no record of detection of genetic differences in hawthorn genotypes using iPBS retrotransposon markers was found. This is the first study in which this marker system is used in the molecular characterization of hawthorn genotypes. The analysis showed 100% polymorphism for many primers and it was determined that markers based on retrotransposons showed better results in polymorphism formation than previous studies.
The results from molecular analyses showed that iPBS markers provide useful information for generating genetic variation at the intra-and interspecies level in hawthorn genotypes which can be used for breeding programs.

Conclusions
In this study, biochemical, agro-morphological and molecular characterization of 25 genotypes belonging to two hawthorn species was performed. As a result of the research, the 14BL05 genotype was prominent according to the physical properties of fruits. The 14BL01 and 14BL10 genotypes have higher values of SSC than other genotypes. The 14BL05 genotype was determined as having higher titratable acidity content than others. The 14BL09 and 14BL16 genotypes were advantageous for organic acids while the 14BL09 and 14BL01 genotypes were advantageous for phenolic compounds. The determined phenolic compounds in hawthorn fruits are important for both human health and human nutrition. Moreover, these compounds are effective in plant physiology. Our results could add some more information on specific phenolic compounds found in hawthorn fruits into the literature. Molecular characterization of the genotypes belonging to two hawthorn species revealed that all genotypes clustered into two groups and iPBS markers could be used for hawthorn genotypes to obtain a high genetic variation. The results from molecular analyses showed that iPBS markers were useful for hawthorn breeding, which requires genetic variation in hawthorn genotypes. As a result of this research, it is suggested that these genotypes will be used in future breeding in terms of fruit breeding.