Genetic Diversity and Similarity of Asian and European Pears (Pyrus Spp.) Revealed by Genome Size and Morphological Traits Prediction

ABSTRACT Pears (Pyrus spp.) mainly are divided into two main groups of Asian and European species with a wide diversity or similarity in fruit and morphological traits. In this study, flow cytometry was performed to evaluate the genome size of 37 cultivars and genotypes of Asian, European, and Iranian indigenous pears. Besides, the relationship between cytological and morphological traits was investigated for the first time. Morphological traits were measured based on the International Union for the Protection of New Varieties of Plants (UPOV) descriptor. There were positive relationships between genome size, the fruit pedicel length, and the length to fruit diameter ratio. Although, as the genome size was increased, the fruit diameter and size were decreased. Increasing 1.6 pg of the genome size causes a reduction of one cm in the pear fruit diameter. Moreover, with a larger genome size by 1.1 pg, there was an increase in fruit pedicel by one cm. The obtained results revealed that most of the Iranian native genotypes were similar in shape to European pears, but some of them were also oriented to Asian pear cultivars. Most studied cultivars originated between different regions were diploid (2n = 34) and their genome size varied between 0.99 and 1.99 pg. The average genome size of European and Iranian pears was 1.29 pg and Asian’s was 1.32 pg. The principal component analysis (PCA) result showed that the Asian and European genotypes were distinctly separated. Also, according to the cluster analysis, the indigenous cultivars of Iran can be located in the European pear group species. There was a relationship between the East Asian and European pear origins because of the possible gene flow between two major pear culture regions in the past. In general, Iran can be considered as an important region and pathway of gene flow transfer and a rich center of genetic variation of pears. Therefore, this study provided essential information and a useful tool for genetic diversity or similarity assessment of pears through genome size and some morphological traits prediction. Further research on wider fruit tree species in the different fruit-growing regions of the world will warrant the use of genome size as a novel prediction tool for the important morphological traits with specific commercial breeding objectives.


Introduction
Pears (Pyrus spp.) with 34 chromosome number (2n = 2x = 34) is a major fruit of the Rosaceae family after apple, which is originated from Europe and Asia (Postman et al., 2015;Westwood, 1993). Its studying the cytology of two European pear cultivars `Dargazi' and `Khoj' that usually used as pear rootstock in the pear orchards in Iran. Also, because Iran considered an important region and pathway of gene flow transfer and a rich center of genetic variation of pears, further researches are suggested to continue on more pear genotypes and cultivars originated from Asia, Europe, and Iranian native pears (Arzani, 2017;Kadkhodaei et al., 2017). Therefore, this study was performed to provide additional information about genetic diversity and similarities among pears through genome size and some morphological traits prediction. Besides, this research aimed to assess the differences and possible relationship between morphological traits and genome size of some European, Asian, and indigenous pears in Iran.

Plant Materials
This study was conducted on 37 pears (Table 1) cultivars and genotypes of Pyrus including 25 European (P. communis L.) and 12 Asian (including ten P. serotina Rehd., one P. betulifolia, and one P. calleryana) pears based on a completely randomized design (CRD). Samples were taken from European and Asian pear collection orchards at Tarbiat Modares University (TMU) including several genotypes and indigenous cultivars of Iran (Arzani, 2002a(Arzani, , 2002b. Also, several samples were taken from the Horticultural Science Research Institute (HSRI) European pear collection orchards located in Karaj, Iran. The names of the collected samples are presented in Table 1.

Morphological Traits
Some important morphological characteristics, including fruit length, fruit diameter, leaf length, leaf width, and petiole length of pear were evaluated based on the International Union for the Protection of New Varieties of Plants (UPOV) descriptor (UPOV, 2000). Leaf-blade base, Leaf-blade apex, Leaf base margin, fruit size, and symmetry were measured based on rating and coding as represented. Furthermore, other morphological traits were measured with a digital caliper (model INSIZE, Japan). Note that morphological traits were assessed using 15 randomly selected mature fruits and 15 fully expanded leaves from each genotype and cultivars. Besides, fruits were assessed at the commercial maturity harvest index mainly based on the fruit background color, total soluble solids (TSS), and flesh firmness. Also, the outside canopy shoots with fully expanded leaves were considered for morphological leaf assessments.

Methodology and Calculation of Flow Cytometric Data
Flow cytometry was employed to estimate the genome size (2 Cx DNA value) of each genotype, via the propidium iodide (PI) staining method. The pear plant was compared with standard plant tomato (Solanum lycopersicum cv. `Stupicke'; 2 C DNA = 1.96 pg), corn (Zea mays CE-777; 2 C DNA = 5.43 pg), or soybean (Glycine max cv. `Polanka'; 2 C DNA = 2.5 pg ((Doležel et al., 1994((Doležel et al., , 1992. For this purpose, about 1 cm 2 of young and fully developed fresh leaves of tomato, soybean, and corn were used as the standard plants. Also, about 2 cm 2 of young and fully developed fresh leaves were sampled for cytological assessment of the studied pear cultivars. Samples were chopped with a sharp razor blade, and then mixed in 1 ml woody plant extraction buffer (WPB) (Loureiro et al., 2007). The Glycine max standard was consulted for the studied genotypes including G12, G15, G17, G27, G28, G29, and G32. Zea mays were considered as a standard for G16 genotype, and Solanum lycopersicum for the other studied genotypes. The obtained mixture was passed through Partech 30 mm nylon mesh filter (Munster, Germany) to remove cell debris. The amount of 50 µg ml −1 RNase was added to eliminate the RNA and prevent PI from joining RNA (Tarkesh Esfahani et al., 2016); the same amount of PI fluorescent color was also added to color the nuclear DNA. The nuclei suspension was analyzed, through BD FACS Canto TM KE Flow Cytometer using the BD FACSDiva TM Software (BD Biosciences, Bedford, MA, USA)( Figure 2). The obtained histograms were analyzed by the Flowing Software version 2.5.0 (Cell Imaging Core specification, Turku Center for Biotechnology, Finland). Afterward, the analyzed data were gated, using Partec FloMax ver. 2.4e (Partec, Münster, Germany). Moreover, areas of the gating range were specified for obtaining histograms. To calculate the genome size in Mbp, each 1 pg of the genome value was considered to be 978 Mbp (Doležel et al., 2003). The size of the monoploid genome (1 Cx) of DNA values in a chromosomal complex set (x) is a somatic diploid cell since the plants are most often diploid, 2 Cx DNA. We used the following formula to calculate the 2 Cx DNA of a pear plant (Doležel and Bartoš, 2005). Also, the absolute DNA amount of a sample was calculated based on the values of the G1 peak means Doležel et al., 2007) as follows: Sample 2 CxDNA (pg) = (Sample G1 peak mean/Standard G1 peak mean) × Standard 2 C DNA (pg).

Data Analysis
The obtained data were initially checked for normality and analyzed using SAS (Ver. 9.3, SAS Institute, Cary, NC). Oneway analysis of variance was carried out to determine significant differences between phenotypic and genome size data. The results were statistically evaluated by analysis of variance (ANOVA) and expressed as mean ± standard error (SE). Means comparisons were made using Duncan's multiple range test (DMRT); differences were considered statistically significant at P≤ 0.01 and P≤ 0.05. Also, morphological traits and genome size data were used for principal component analysis (PCA) and cluster analysis. Before PCA, cluster, and bivariate correlation analyses, the data were normalized by a specific reference sample. PCA and cluster analysis was conducted via Minitab software (Ver. 17). Furthermore, linear regression analysis was carried out to find out the relationship between 2Cx DNA values and some morphological traits using Minitab version 17.

Morphological Variability
In this study, thirty-seven genotypes of pears were selected and sampled, which were originally derived from different parts of the world (Table 1, Figure 1). ANOVA showed there were significant differences between the genotypes for all of the studied parameters (Table 2). Fruit diameter ranged from 2.00 cm to 7.87 cm. The highest fruit diameter was recorded for G7 (7.87 cm), and the lowest value for G29 (2.00 cm) and G27 (2.12 cm) genotypes (Table 3). Bashiri et al. (2017) suggested larger fruit diameter resulted in more desirable fruit shape and performance. Also, in the present research, among the studied genotypes and cultivars, the minimum fruit length was calculated in G27 (2.10 cm), G29 (2.03 cm), and G32 (2.13 cm), and the maximum in G1 (9.98 cm), G7 (9.80 cm) and G33 (9.88 cm), respectively. The largest and smallest length-to-diameter ratio was observed in G33 (1.67) and G32 (0.81), respectively, both of which were European origin cultivars. The fruit pedicel length was also different within the studied genotypes, and the maximum and minimum lengths were measured in G17 (7.47 cm) and G4 (1.47 cm), respectively (Table 3). The largest length and width of the leaves belonged to G27 (10.63 cm) and G25 (8.07 cm) which originated from Asia. Among the studied samples, G9 (5.80 cm) showed the highest and G32 (1.23 cm) the lowest values for the leaf petiole length (Table 4).

Genome Size
Flow cytometric histograms of 37 pear genotypes are shown in Figure 2, in which left peaks were referred for G1 pears and right peaks were referred to either standard plants including tomato (Solanum lycopersicum cv. `Stupicke'; 2C DNA = 1.96 pg), corn (Zea mays CE-777; 2C DNA = 5.43 pg), or soybean (Glycine max cv. `Polanka'; 2C DNA = 2.5 pg). As shown in Figure 2, the difference in genome size was observed among pear genotypes. Most cultivars were diploid (2 n = 2x = 34), but the triploid can be seen among the studied genotypes, so there was a variable amount of 2Cx. The highest amounts of DNA in the nucleus genome were found in G15 (`Astaneh' cultivar) with 1.99 pg in genome size (Table 3). According to Nikzad Gharehaghaji et al. (2014a), and also based on the results obtained in this study, three genotypes G15, G16, and G17 are most likely triploid. In the studied Asian pear cultivars, the nucleus was observed to be 2Cx = 1.25 pg on average, and it seems that all of them were diploid. Also, PCA analysis showed that most of the studied Asian pear cultivars and a small number of Iranian cultivars were positioned in one group and European cultivars, and most of the native cultivars of Iran were put in the other main group (Figure 3). It has been reported that PCA was used for genetic recognition and population structure (Ma et al., 2012). Also, in our study thirty-seven genotypes in cluster analysis were classified into 3 groups that had the least genetic differences (Figure 4). Notably, for cluster analysis, phenotypic and genome size traits were used. G28 and G29 were also placed between the Asian pears group. Some cultivars are native to Iran, and European cultivars were also included in the same group (Figure 4). According to the cluster analysis, pears were divided into two main European and Asian groups. Most pears have characteristics of both groups, which indicated that Iran is probably an area with a diversity of the Pyrus genus and has played a key role in linking European and Asian pears (Figure 1). There were significant relationships between the amount of 2Cx DNA and some morphological traits ( Figure 5) such as fruit diameter (P ≤ 0.01), fruit length-to-diameter ratio (P ≤ 0.05), fruit pedicel length (P ≤ 0.01), and fruit size (P ≤ 0.01).

Discussion
The present research was conducted on 37 genotypes of pears originated from Asia, Europe, and Iranian native pears. Also, the geographic position of Iran, as well as the country origins of the studied genotype/cultivars, are shown along with the Silk Road map (Figure 1). Iran played an important location link between European and Asian pear genotypes. Consequently, this country could be one of the important centers of pear genetic diversity. During the period of trade and commercialization, the transfer of culture between ancient China and Central Asia, the Silk Road was historically the most concentrated center for the exchange of agricultural products and technology (Wei et al., 2008) as well as pear germplasm (Nikzad Gharehaghaji et al., 2014c, 2014b. In this way, Iran may be the first country importer in the world for P. communis from Europe and imported P. serotina and P. pryifolia genotypes from East Asian countries (Arzani, 2002a(Arzani, , 2002b. The obtained results can be valid for the European and Japanese species, besides the related results of this study indicated that the ancient Iranian cultivars have been influenced during the evolution by the Japanese and European species. Table 1 shows an average of 633 Mbp in genome size for the studied genotypes. DNA amount (1C) in Pyrus genus were reported 588 Mbp for P. calleryana (Dickson et al., 1992), 563.50 Mbp for P. elaeagrifolia (Jedrzejczyk and Sliwinska, 2010), 607.60 Mbp P. communis, and 607.60 Mbp P. piraster (Pustahija et al., 2013). Dickson et al. (1992) reviewed two pear cultivars including Pyrus communis L. `Bartlett' and Pyrus calleryana `Decne', and reported the amount of 2Cx DNA for this Pyrus at 1.11 and 1.26 pg, respectively. The findings of the present research were consistent with those of Dickson et al. (1992). Also, in the present research, the study of phenotypic traits leads to valuable results, including information about germplasm as well as identification of superior genotypes as reported previously by the other researchers (Arzani, 2017;Bernard et al., 2018;Wang and Arzani, 2019).
In the last half-century, gene flow has been known as a part of the evolutionary path for new cultivars. The gene flow is now known as the success of the allele transfer from one population to another, which is also different among species, populations, and individuals over time. In this study, we found such gene flow in some studied genotypes and cultivars. It has been also reported that gene flow plays an important role in the evolution of the population of animals and plants (Ellstrand, 2014;Ellstrand and Rieseberg, 2016;Yakimowski and Rieseberg, 2014). Interstitial hybridization naturally occurs in flowering plants and affects their evolution path (Field et al., 2011).
According to the results of cluster analysis, most cultivated pears of Asian cultivars are diploid (2n = 34) (Figure 4.), and there is a small number of reported polyploidy (triploid and tetraploid) such as P. bretschneideri (Zielinski and Thompson, 1967) or as reported for the German and Romanian pear germplasm (Puskás et al., 2016) and Oregon pear collection in the USA (Postman et al., 2015). Iketani et al. (1998) showed that the Eastern and Western species of Pyrus evolved independently. In the present research, we identified a difference between the leaves of different studied genotypes. Previously, the variation in some pear species of leaf traits such as leaf length, width, shape, and leaf area was reported among different pear genotypes (Bashiri et al., 2017;Najafzadeh and Arzani, 2015;Paganová, 2009;Zarei et al., 2019). In a study conducted on morphological and molecular markers of four species of Pyrus, the mean length and diameter of fruit and pedicel length of fruit in P. communis genotypes were reported to be 7. 64, 5.86, and 2.71 and in P. pyrifolia 8.02, 7.91, and 3.62 cm, respectively. The leaf length, width, and petiole length were also reported 6.36, 4.31, and 3.87 in P. pyrifolia and 9.64, 6.19, and 2.54 cm for P. communis, which is quite similar to our data obtained in the present research (Jalilian et al., 2018). Pyrus syriaca Boiss., which is a wild and drought-tolerant pear species, was studied for pomology and morphology and the maximum leaf length was reported between 22.27-70.75, and the leaf width between 9.18-35.20 mm (Khadivi et al., 2020). Also, 138 pear genotypes from central Zagros mountains were studied, with the minimum and maximum fruit lengths at 1.4 and 10.2, and the minimum and maximum fruit diameters at 1.26 and 7.6. The mean pedicel length was 3.96 and the mean fruit symmetry was 1.63, which was consistent with our obtained and presented results (Bashiri et al., 2017).
We found a linear relationship between genome size with traits such as fruit diameter, fruit size, fruit pedicel length, and length-to-diameter ratio. Previous studies also showed a significant relationship between genome size and some phenotypic traits as well as geographical locations in different plant species, such as Lathyrus sativus (Karimzadeh et al., 2011), Diphasiastrum sp. (Hanusova et al., 2014), and Iranian endemic muskmelon . Besides, Basak et al. (2019) reported that genome size was not affected by the environment in the turnip. Although, genome size was directly and significantly correlated with cell size and stomatal density in some angiosperm species (Beaulieu et al., 2008). Also, Sarikhani Khorami et al. (2018) stated that genome size can be a strong and valuable predictor of nut and kernel weight, nut size of walnut genotypes, and total yield. In this research, we found that by decreasing 1.6 pg genome sizes in the pear, there is an increase of one cm in fruit diameter. In other words, fruit diameter inversely regressed upon genome size. Subsequently, with increasing genome size, fruit size decreased ( Figure 5). The genome size grew in response to a larger length-to-diameter ratio and fruit pedicel. Therefore, by enlarging genome size to about 1.1 pg, one centimeter is added to fruit pedicel length ( Figure 5).
In the present research, we employed cytological studies to detect the differences within the species and between the studied pear species. It has been reported that cytology studies are important in the Figure 5. Linear regression between genome size (2Cx DNA) and fruit diameter, fruit size, fruit length to diameter ratio, and pedicel length of the studied European, Asian, and indigenous Iranian pear genotypes. The Pearson correlation coefficient between genome size and fruit diameter was −0.306**, fruit size −0.209*, fruit length to diameter ratio 0.217*, and pedicel 0.464**. *and**correlation is significant at P ≤ 0.05 and P ≤ 0.01 respectively.
populations of one species due to their differences, each one exhibits a particular genomic that are in agreement with the environment in which they are being used Karimzadeh et al., 2011;Abedi et al., 2015). Flow cytometry can be employed to determine ploidy levels, estimate the size of the genome in plants, and examine the cell cycle in humans and animals (Doležel and Bartoš, 2005;Doležel et al., 2007). Also, the application of flow cytometry to detect the polyploidy induction and its effects on morphology, physiology, anatomy, and secondary metabolites have been reported on a wide variety of different plants (Majdi et al., 2010, Karimzadeh et al., 2011Abedi et al., 2015;Tavan et al., 2015;Javadian et al., 2017). Genetic diversity is assessed by various factors such as phenotypes, and biochemical, cytological, and molecular characteristics (Govindaraj et al., 2015). In the present study, the pear genome was about 633 Mbp on average which is five times smaller than the human genome (6153 Mbp) as reported by Doležel et al. (2003). Besides, in the present research, results showed that the genome size of G15, G16, and G17 were larger than other studied genotypes. According to the results, they are more likely to be triploid. Although Puskás et al. (2016) after using flow cytometric analysis on 124 german and Romanian genotypes, identified 30 triploids in which stated that triploid cultivars will not consider as useful for commercial or breeding programs. Also, Postman et al. (2015) concluded that the most of studied pear cultivars in the Oregon collection orchard are diploid with the few triploid cultivars in which are not recommended for any commercial cultivation. In our research, the native Iranian cultivars showed only a few exceptions, G12 (689.49 Mbp) and G14 (694.38 Mbp), which have relatively large genomes, but the native Iranian pear cultivars were expected to have a relatively small genome. The second, the G13 (484.11 Mbp), the variety has a tiny genome like G2 (493.89 Mbp). Therefore, genome size and morphological traits can be determining factors in establishing relationships in pear genotypes.
Extensive results of plant materials in different climatic areas in one region and the neighboring regions have also been historically documented, such as the movement of pears from Italy to neighboring countries and Eastern and Central Europe (Baccichet et al., 2020). The results obtained in the present research showed that given Iran's location on the Silk Road ( Figure  1), as an important link between Asia and Europe in the past, it played a major role to link Europe and Asian genotypes. Consequently, this country could be an important center of pear genetic diversity.
In conclusion, we observed high genetic diversity in European and Asian pear genotypes and also the similarity in the traits of some studied genotypes. The obtained results revealed that most of the Iranian native genotypes were similar in shape to European pears, but some of them were also oriented to Asian pear cultivars. Most studied cultivars that originated between different regions were diploid (2n = 34) with a varied range of genome size. The results based on PCA and cluster analysis showed that the Asian and European genotypes were distinctly separated and the indigenous cultivars of Iran were located in the European pear group species. There was a relationship between the East Asian and European pear origins because of the possible gene flow between two major pear culture regions in the past. In general, Iran can be considered as an important region and pathway of gene flow transfer and a rich center of genetic variation of pears. Therefore, this study provided essential information and a useful tool for genetic diversity or similarity assessment of pears through genome size and some morphological traits prediction. Besides, related research on wider fruit tree species in the different fruit-growing regions of the world will warrant the use of genome size as a novel prediction tool for the important morphological traits for the further breeding program with the specific commercial objectives.