Agricultura Scientia 21: No 2 23-33(2024) https://doi.org/10.18690/agricsci.21.2.3 *Correspondence to: E-mail: tina.ternjak1@um.si 23 Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank Tina TERNJAK University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoče, Slovenia ABSTRACT The Slovenian Plant Gene Bank (SPGB) of the Faculty of Agriculture and Life Sciences houses approximately 250 accessions of stone fruit, with most of the material belonging to the species Prunus domestica L. and Prunus cerasifera Ehrh. The main objectives of this study using a set of 11 SSR primers were: 1. to determine the genetic structure of the traditional Slovenian in situ plum material in comparison to the ex situ the SPGB collection; 2. to identify unique material among the in situ collected accessions; 3. to gain insight into the genetic relationship between the two studied species. The genetic structure of 60 plum samples was analyzed using Principal Coordinate Analysis (PCoA) and Bayesian model-based analysis. PCoA separated the P. cerasifera and P. domestica accessions, while Bayesian model-based analysis revealed that many accessions of P. domestica and P. cerasifera shared a common ancestral history. The ex situ material showed greater genetic diversity as it was distributed over more populations than the in situ material. Promising in situ genotypes, especially from the Prekmurje and Lower Styria, were identified as valuable additions to enrich the existing collection. Keywords: plum, Prunus domestica L., Prunus cerasifera Ehrh., SSR markers, genetic structure INTRODUCTION Germplasm collections serve as an important reservoir of plant genetic resources as well as a source of diversity and are essential for successful crop improvement (Butac, 2020). As a result of careful collection, conservation and evaluation, these collections provide a valuable source of genes (e.g., wild genotypes, landraces, local populations, clones and lines bred from indigenous plant materials and ecotypes) that can be used in breeding programs or as cultivars with high tolerance to abiotic and biotic stresses, suitable for sustainable agricultural practices (Blazek, 2007; Butac et al., 2010; Dey et al., 2016). The Slovenian Plant Gene Bank (SPGB) of the Faculty of Agriculture and Life Sciences is part of the Slovenian Plant Gene Bank Program (SRGB), which aims to preserve, evaluate, regenerate and conserve indigenous Slovenian germplasm, including local ecotypes, populations and landraces of agricultural, medicinal and aromatic plants, as well as forest trees and other woody plants from Slovenian forests (Šiško, 2016). It was established in 2007 on an area of approximately 3 ha and is located in an isolated location according to FAO standards next to the Botanical Garden of the University of Maribor (Pivola). It is a permanent collection plantation intended both for the storage of accessions and for evaluation of accessions. Part of the collection, including vines, is located in the Meranovo viticulture center (Limbuš). The germplasm collection includes accessions from the following genera: Prunus, Rubus and Vitis. After 17 years of work on the collection, there are currently approximately 250 accessions belonging to stone fruit (e.g., plums, sweet and sour cherries, apricots, peaches, and almonds). Of these, around 170 plum accessions belong to the Prunus domestica L. and Prunus cerasifera Ehrh. species (Šiško, 2018 and unpublished data from the SPGB collection). The European plum, P. domestica (2n = 6x = 48), and the myrobalan plum, P. cerasifera (2n = 2x = 16) are species which belong to a group of European plums (Hartmann et al., 2009; Neumuller, 2011). The first species is used worldwide in fruit production due to the versatility of the fruit use (fresh, dried Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 24 or processed) (Milošević et al., 2023). It is also known to form different pomological groups (based on fruit use and morphological characteristics such as fruit shapes and color), including large-fruited European plums, prunes, egg plums, greengages, mirabelles, damsons, bullaces and St. Julien plums (Zhebentyayeva et al., 2019; Gaši et al., 2020). The use of fresh and dried fruits of P. cerasifera is limited to traditional use in Western Asia (Hanelt, 2001; Okie and Hancock, 2008), while in Europe, myrobalan is often used for processing (e.g., jams and chutneys) and for the spirit production (Topp et al., 2012). In addition, P. cerasifera is resilient species and adapted to a wide range of rural and urban areas (forest edges, open woodlands, along roads and rivers, abandoned orchards, around farm buildings, gardens and parks) (Hartmann et al., 2009; Popescu and Caudullo, 2016). It is widely used as an ornamental tree and as an important rootstock for other Prunus species (e.g., plum, peach, apricot and almond) (Sancin, 1988; Sedaghathoor et al., 2009; Das, 2011). The use of plant genetic resources, in particular when they are not present in dedicated conservation centers, is often limited by insufficient information, such as material identification, phenotypic and genetic diversity and pomological data (Milošević and Milošević, 2018). Comprehensive information on the phenotypic and genotypic characteristics of the material and various preservation strategies are crucial for the conservation process (Ramanatha and Hodgkin, 2002). Traditionally, plum material is characterized and identified based on morphological traits (Martínez-Gómez et al., 2005). However, this method of distinguishing genotypes has limitations, as some traits are unreliable (e.g., variations in growing conditions, plant age, and phenological stage) (Mehdi et al., 2012). To identify plant genetic relationships, structure and diversity, molecular marker technology has developed in recent decades to replace or complement morphological markers (Soriano, 2020). As part of a broader research endeavor (Ternjak et al., 2023), the main objective of this study was to determine the genetic structure of the traditional Slovenian in situ plum material in comparison to the ex situ material of SPGB collection, using SSR molecular markers. The focus was on identifying unique material among the accessions collected in situ that could be used to enrich the collection. In addition, we wanted to gain insight into the genetic relationship between the two studied species (P. domestica and P. cerasifera). MATERIALS AND METHODS Plant material The accessions examined in this research are part of a broader study investigating the genetic diversity and structure of three plum species: P. domestica, P. cerasifera, and P. spinosa (Ternjak et al., 2023). This paper focuses specifically on the analyzes and comparison of plum material belonging to P. domestica and P. cerasifera collected in Slovenia (in situ and ex situ). From 2018 to 2019, young and healthy leaves were collected from 60 plum accessions. Roughly half of the material (29 accessions) belonged to P. domestica species and 31 accessions to P. cerasifera. Sixteen accessions were collected ex situ in SPGB, while 44 accessions were collected in situ from different regions in Slovenia (Lower Styria, Prekmurje, Upper Carniola and Istria). Each sample was documented, and for the in situ accessions, the exact location of the tree was determined using latitude and longitude coordinates (WGS84 system). For the ex situ accessions, the location of the collection site was recorded together with the data on the origin of the material. Table 1 lists the data of the collected material, including name, species, status, origin and other passport data. Table 1: The data of the 60 genotypes (29 accessions of Prunus domestica L. and 31 accessions of Prunus cerasifera Ehrh.), including name, species, origin and other passport data as well as the membership values to the populations were analyzed with STRUCTURE (Pritchard et al., 2000) Sample No. Name Species Accession ID Conservation type Origin of the material Structure analysis K = 2 K = 7 37 Plum_green_37 P. domestica / ex situ SPGB* Brdce, Vojnik, Lower Styria K2 6 38 Plum_38 P. cerasifera / in situ Črnc, Brežice, Lower Styria K1 2 44 Bluish_plum_44 P. domestica / in situ Lendavske gorice, Prekmurje K1 2 45 Bluish_plum_45 P. domestica / in situ Lendavske gorice, Prekmurje K1 2 46 Bluish_plum_46 P. domestica / in situ Lendavske gorice, Prekmurje K1 2 Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 25 Sample No. Name Species Accession ID Conservation type Origin of the material Structure analysis K = 2 K = 7 51 Plum_51 P. cerasifera 6356 ex situ SPGB* Črnc, Brežice, Lower Styria K1 2 52 Plum_52 P. domestica 6085 ex situ SPGB* Črnc, Brežice, Lower Styria K1 2 55 Plum_55 P. cerasifera 6372 ex situ SPGB* Trnje pri Brežicah, Lower Styria K1 2 58 Plum_58 P. cerasifera 6370 ex situ SPGB* Trnje pri Brežicah, Lower Styria K1 Admixed 59 Plum_59 P. cerasifera 6391 ex situ SPGB* Trnje pri Brežicah, Lower Styria K1 4 60 Plum_60 P. cerasifera 6371 ex situ SPGB* Trnje pri Brežicah, Lower Styria K1 3 61 Plum_61 P. cerasifera / ex situ SPGB* Brezina, Brežice, Lower Styria K1 3 62 Plum_62 P. cerasifera 6392 ex situ SPGB* Trnje pri Brežicah, Lower Styria K1 3 63 Plum_63 P. cerasifera 6394 ex situ SPGB* Trnje pri Brežicah, Lower Styria K1 1 67 Bluish_plum_67 P. domestica 3576 ex situ SPGB* Maribor, Lower Styria K1 1 76 Plum_76 P. cerasifera 6350 ex situ SPGB* Črnc, Brežice, Lower Styria K1 1 79 Common_prune_Bistrica_79 P. domestica 6317 ex situ SPGB* NA K1 1 82 Plum_82 P. cerasifera 6347 ex situ SPGB* Črnc, Brežice, Lower Styria K1 1 84 Common_prune_Bistrica_84 P. domestica 6416 ex situ SPGB* Sromlje, Brežice, Lower Styria K1 1 92 Bluish_plum_92 P. domestica / in situ Maribor, Lower Styria K1 1 101 Bluish_plum_101 P. domestica / in situ Orehova vas, Lower Styria K1 1 102 Bluish_plum_102 P. domestica / in situ Orehova vas, Lower Styria K1 1 103 Bluish_plum_103 P. domestica / in situ Orehova vas, Lower Styria K1 1 104 Bluish_plum_104 P. domestica / in situ Orehova vas, Lower Styria K1 1 105 Bluish_plum_105 P. domestica / in situ Orehova vas, Lower Styria K1 1 106 Bluish_plum_106 P. domestica / in situ Orehova vas, Lower Styria K1 1 107 Common_prune_107 P. domestica / in situ Orehova vas, Lower Styria K1 1 108 Common_prune_108 P. domestica / in situ Orehova vas, Lower Styria K1 Admixed 109 Common_prune_109 P. domestica / in situ Orehova vas, Lower Styria K2 5 110 Common_prune_110 P. domestica / in situ Orehova vas, Lower Styria K2 5 111 Plum_111 P. cerasifera / in situ Orehova vas, Lower Styria K2 5 112 Bluish_plum_112 P. domestica / in situ Maribor, Lower Styria K2 5 113 Bluish_plum_113 P. domestica / in situ Limbuš, Lower Styria K2 5 114 Bluish_plum_114 P. domestica / in situ Limbuš, Lower Styria K2 5 116 Bluish_plum_116 P. domestica in situ Lovrenc na Pohorju, Lower Styria K2 5 Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 26 Sample No. Name Species Accession ID Conservation type Origin of the material Structure analysis K = 2 K = 7 117 Common_prune_117 P. domestica / in situ Miklavž na Dravskem polju, Lower Styria K2 5 118 Plum_118 P. cerasifera / in situ Maribor, Lower Styria K2 5 119 Plum_119 P. cerasifera / in situ Maribor, Lower Styria K2 Admixed 120 Plum_120 P. cerasifera / in situ Maribor, Lower Styria K2 5 121 Plum_121 P. cerasifera / in situ Maribor, Lower Styria K2 5 122 Plum_122 P. cerasifera / in situ Maribor, Lower Styria K2 5 129 Plum_yellow_129 P. cerasifera / in situ Koper, Istria K2 5 130 Plum_bluish_130 P. cerasifera / in situ Koper, Istria K2 5 131 Plum_red_131 P. cerasifera / in situ Koper, Istria K2 5 132 Plum_yellow_132 P. cerasifera / in situ Koper, Istria K2 5 133 Plum_yellow_133 P. cerasifera / in situ Izola, Istria K2 5 134 Plum_violet_134 P. cerasifera / in situ Strunjan, Istria K2 5 135 Plum_red_135 P. cerasifera / in situ Vas Dragonja, Istria K2 5 136 Plum_bluish_136 P. cerasifera / in situ Krkavče, Istria K2 5 137 Myrobalan_137 P. cerasifera / in situ Kasaze, Lower Styria K2 5 138 Myrobalan_138 P. cerasifera / in situ Kasaze, Lower Styria K2 5 139 Myrobalan_139 P. cerasifera / in situ Kasaze, Lower Styria K2 5 140 Mirabelle_140 P. domestica / in situ Kasaze, Lower Styria K2 5 145 Bluish_plum_145 P. domestica / in situ Ruše, Lower Styria 2 5 146 Bluish_plum_146 P. domestica / in situ Ruše, Lower Styria 2 5 147 Plum_147 P. cerasifera / ex situ Črnc, Brežice, Lower Styria 2 5 153 Spindel_plum_153 P. domestica / in situ Rašica, Upper Carniola 2 7 155 Plum_yellow_155 P. cerasifera / in situ Maribor, Lower Styria 2 5 156 Plum_red_156 P. cerasifera / in situ Maribor, Lower Styria 2 5 189 Bluish_plum_189 P. domestica / in situ Zakot, Brežice, Lower Styria 2 5 * SPGB (Slovenian Plant Gene Bank) Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 27 Molecular analyzes DNA isolation and molecular markers analyzes Total genomic DNA was extracted from the young leaf material following the CTAB protocol described by Doyle and Doyle (1987), with some modifications. Two separate extractions per sample were performed. The DNA concentration of each sample was estimated using a fluorimeter (Hoefer DQ 300, California, USA). The quality was also checked on a 3% agarose gel by electrophoresis (Bio-Rad, California, USA), and the products were visualized under UV light. All studied accessions were analyzed using a set of eleven 11 SSR primer pairs: UDP96-005, BPPCT034, EMPAS12, UCD- CH17, EMPAS06, EMPAS11, EMPAS14, BPPCT014, BPPCT025, CPSCT026 and CPPCT006, which were developed on different Prunus species. Information on marker selection, polymerase chain reaction (PCR) amplification procedures and fragment size analysis was previously published by Ternjak et al. (2023). Data analyzes The genetic structure among the studied accessions was analyzed using the Principal Coordinate Analysis (PCoA) and complemented with the Bayesian model-based clustering method. For the PCoA calculations, the microsatellite allele data were converted into a binary matrix. Dissimilarities were calculated with Sokal and Michener index and transformed into Euclidean distances using the 0.5 power transformation. Using DARwin 6.0.21 software (Perrier and Jacquemoud-Collet, 2006), each accession was assigned to a location in a two-dimensional space and the figure was constructed. The STRUCTURE V2.3.4 software package (Pritchard et al., 2000) was used to perform the Bayesian model-based clustering method. Ternjak et al. (2023) have already presented a detailed description of the settings and construction of the bar plots. The most relevant parameter K (number of populations) for the analyzed data was determined by calculating ΔK according to the method described by Evanno et al. (2005). This calculation was performed with the Structure Harvester V0.6.94 application (Earl and von Holdt, 2012). The individuals with a membership coefficient (ql) > 0.9 were assigned to a specific population, and those with a threshold value below the estimated membership were considered admixed. The STRUCTURE 2.3.4 software (Pritchard et al., 2000) was also used to compute the average distances (expected heterozygosity) between individuals within the same population. RESULTS AND DISCUSSION Principal Coordinate Analysis provided insight into the distribution of different plum groups and allowed us to obtain a global representation of diversity. In the analysis, the examined material was divided according to species and when considering the conservation type (in situ and ex situ) the material was distributed over the entire graph (Fig. 1). The accessions of P. cerasifera were located on the left side of the figure and formed a denser arrangement in space, while the accessions of P. domestica were located on the right side of the figure. The only exception was the ex situ accession Plum 52 on the left side of the figure, which clustered slightly closer to the P. cerasifera species. Although the study by Ternjak et al. (2023) considered Plum 52 to be P. domestica, as the flow cytometry results confirmed that the accession was hexaploid, the analyzed SSR profiles also showed similar behavior to other P. domestica accessions. However, morphological observations also revealed a similarity with the P. cerasifera species. In addition, genetic diversity assessment using three universal cpDNA primers showed that Plum 52 belonged to haplotype H4, which was shared by P. domestica and P. cerasifera (Ternjak et al., 2023). Possibly, accession Plum 52 is an example of a putative hybrid origin, which is not surprising as P. domestica can easily hybridize with P. cerasifera (Topp et al., 2012). The P. domestica material was more scattered on the right side of the figure, forming different clusters. On the upper right side of the figure, two clusters of the landrace Bluish plum could be observed. The Bluish plum accessions mostly collected in situ (except Bluish plum 67) were positioned according to their origin, with a larger cluster of Bluish plums from the Styria region and a smaller cluster of only three Bluish plum accessions (44,45 and 46) from the Prekmurje region. On the lower left side of the figure, four accessions were highlighted, material slightly different from the other clusters: Bluish plum (189), which was also collected in the Styria region, but is genetically distinct and was separated from the two Bluish plum clusters; Plum green (37), a small-fruited landrace with green color, yellow-oval-fruited landrace Spindel plum (153) and the accession Mirabelle 140, which belongs to the mirabelle pomological group. Mirabelles are a specialty of the Lorraine region in France, sweet small-fruited plums that are predominantly yellow to orange, often with red spots (Gaši et al., 2020). The PCoA analysis also identified a pomological group of common prunes which included in situ and ex situ material and clustered in the lowest part of the figure. Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 28 Figure 1: Principal Coordinate Analysis (PCoA) based on polymorphism at 11 SSR loci of the 60 plum genotypes. The accessions of P. cerasifera Ehrh. (Myrobalan cluster, grey color) formed a dense cluster, whereas the material of P. domestica L. (black color) is more scattered and consists of different clusters. The accessions marked with a full dot represent in situ material, while the accessions with an empty dot are ex situ material Bayesian model-based clustering analysis revealed the structural patterns and divided the analyzed material into ancestral populations. The maximum value for ΔK was K = 2 (975.15), dividing the material into two populations (Fig. 2, Fig. 3 and Table 2). Population K1 (bar plots in black color) accounted for 27 genotypes and consisted of 13 in situ and 14 ex situ accessions (Table 1). Population K2 (bar plots in grey color) accounted for 33 genotypes, mostly belonging to the in situ material (31), with two ex situ accessions (Table 1). This collected material has the potential to increase and enrich the SPGB genetic resources collection. Both populations were comprised of various accessions belonging to both studied species and different pomological groups. For the K = 2, no material was considered admixed. Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 29 Table 2: Table summarizing the results using Evanno et al. (2005) method, output of Structure Harvester V0.6.94 application (Earl and von Holdt, 2012) # K Reps Mean LnP(K) Stdev LnP(K) Ln'(K) |Ln''(K)| Delta K 1 10 -10143.72000 0.85739 NA NA NA 2 10 -7461.45000 2.21071 2682.27000 2155.77000 975.14990 3 10 -6934.95000 94.39210 526.50000 186.26000 1.97326 4 10 -6594.71000 223.41714 340.24000 36.47000 0.16324 5 10 -6290.94000 210.95657 303.77000 1477.68000 7.00466 6 10 -7464.85000 4418.45931 -1173.91000 2612.48000 0.59126 7 10 -6026.28000 4.16034 1438.57000 1386.09000 333.16733 8 10 -5973.80000 5.99574 52.48000 32.35000 5.39550 9 10 -5953.67000 18.74923 20.13000 125.85000 6.71228 10 10 -6059.39000 25.61642 -105.72000 NA NA Figure 2: Bar plot of the results of the Bayesian model-based clustering (K = 2) for 60 plum genotypes. Population K1 accessions are shown in black and Population K2 accessions are shown in grey. No accessions were admixed In terms of allelic variation within the two populations, STRUCTURE software revealed that the mean distances between individuals were the greatest in Population K1 (0.7410), while the distances were smaller in Population K2 (0.5213) (Table 3). Table 3: Average distances (expected heterozygosity) between individuals in the same population Population K1: 0.7410 Population K2: 0.5213 Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 30 Figure 3: Graphical method, as in Evanno et al. (2005), allowing the detection of the number of groups K for the 60 plum genotypes using ΔK The Evanno criterion, used to evaluate the genetic structure showed a weaker signal for K = 7 (333.17), dividing the material into seven populations (Fig. 4, Fig. 3 and Table 2). Population K1 split into four sub-clusters (Populations 1- 4), while Population K2 split into three sub-clusters (Populations 5-7) (Table 1). Most accessions (14) belonging to Population 1 (black) maintained the same profile or remained unchanged from the original Population K1 (Plum: 63, 76, 82; Bluish plum: 67, 92, 101, 102, 103, 104, 105, 106; Common prune: 79, 84 and 107). Seven accessions, Bluish plums: 44, 45, 46 separated and formed a new Population 2 (light grey) that also included accessions Plum: 38, 51, 52 and 55, while Population 3 (grey with black dots) contained three accessions (Plum: 60, 61 and 62). Population 4 (white) consisted of a single accession (Plum 59). Most accessions (30) were assigned to Population 5 (dark grey) and had the same profile or remained unchanged from the original Population K2. The other two populations contained only one accession, namely Population 6 (white with black stripes), Plum green (37) and Population 7 (black with white dots), Spindel plum (153). In the remaining three admixed accessions, the largest part of their genome 0.7 for Plum 58, 0.87 for Plum 119 and 0.7 for Common prune 108, was associated with populations 3, 6 and 7, respectively. The results of the STRUCTURE software for K = 7 have also highlighted some accessions or groups that stood out for their originality. For some of them, results were consistent with the PCoA analysis, e.g., Plum green (37) and Spindel plum (153). For the other accessions, Bayesian model- based clustering analysis showed that they belonged to a new ancestral population, that was not shown in previous analysis. For example, Plum 59 from Population 4, or accessions from the Population 3. Figure 4: Bar plot of the results of the Bayesian model-based clustering analysis (K = 7) for 60 plum genotypes. Population 1 accessions are shown in black, Population 2 accessions are shown in light grey, Population 3 accessions are shown in grey with black dots, Population 4 accessions are shown in white, Population 5 accessions are shown in dark grey, Population 6 accessions are shown in white with black stripes and Population 7 accessions are shown in black with white dots. Three accessions were admixed. Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 31 When comparing in situ and ex situ material at K = 7, the first was distributed among four ancestral populations (1, 2, 5 and 7), while the second was distributed among six populations (1, 2, 3, 4, 5 and 6) and thus showed higher genetic diversity. This was expected, as the material from the SPGB collection had been selected for its high diversity. Nevertheless, we found promising material among the accessions collected in situ that could be included to enrich the existing collection. For example, Bluish plum genotypes originating from the Prekmurje region and belonging to Population 2 (accessions 44, 45 and 46), as well as Bluish plum genotypes belonging to Population 5 and originating from Lower Styria, but both different from Bluish plum material in the current SPGB collection. In addition, we also discovered common prune genotypes belonging to Population 5, which also differed from the existing collection. Another example was already mentioned, Spindel plum (153) which was pointed out as a unique material by the both analyzes. When we studied the structure of P. domestica and P. cerasifera together with other wild relatives such as P. spinosa, the Bayesian model-based clustering analysis revealed P. domestica and P. cerasifera as independent groups (Ternjak et al., 2023). Interestingly, the present study, which focused exclusively on the analysis of P. domestica and P. cerasifera material, showed that many accessions of the two species belonged to the same population and thus share ancestral history. This is supported too by the results of genetic diversity assessment studies using chloroplast DNA markers reported by Bortiri et al. (2009), Reales et al. (2010), Horvath et al. (2011) and Ternjak et al. (2023), in which P. domestica and P. cerasifera clustered together, suggesting that P. cerasifera may have contributed to the maternal chloroplast DNA of P. domestica. CONCLUSIONS We aimed to determine the genetic structure of traditional Slovenian in situ plum material in comparison to the ex situ material of the SPGB collection, using SSR molecular markers. The combined results provided valuable insights into the genetic diversity and structure of the Slovenian plum genetic resources. PCoA clearly separated the P. cerasifera and P. domestica accessions, with P. cerasifera forming a dense cluster and the P. domestica accessions being more scattered and consisting of different clusters. An exception was accession Plum 52, which, although classified as P. domestica, genetic markers suggesting a hybrid origin with P. cerasifera. An exception was accession Plum 52, which, although classified as P. domestica, clustered near P. cerasifera. The combined results of the different analyzes based on genetic markers therefore suggest a hybrid origin with P. cerasifera. The ex situ material showed higher genetic diversity and was distributed among more populations compared to the in situ material, reflecting the selection for high diversity that was made when accessions were introduced into the SPGB collection. Nonetheless, promising in situ accessions, particularly from the Prekmurje and Lower Styria regions, were identified as valuable additions to enrich the existing collection. Certain accessions, such as Plum green (37) and Spindel plum (153), were highlighted as unique by both PCoA and Bayesian model-based clustering analysis. Bluish plum populations from different regions and common prune genotypes were identified as distinct from the ones in existing collections, suggesting potential new additions to the Slovenian germplasm collection. The analyzes revealed that many accessions of P. domestica and P. cerasifera share a common ancestral history. This finding is consistent with previous studies using chloroplast DNA markers, which also suggested that P. cerasifera may have contributed to the maternal chloroplast DNA of P. domestica. Overall, this study demonstrated the utility of integrating molecular markers and advanced clustering methods to uncover the complex genetic relationships and diversity within plum species to aid future breeding and conservation efforts. REFERENCES 1. Blazek, J. (2007). A survey of the genetic resources used in plum breeding. Acta Horticulturae, 734, 31-45. https://doi.org/10.17660/ActaHortic.2007.734.2 2. Bortiri, E., Oh, S.-H., Jiang, J., Baggett, S., Granger, A., Weeks, C., Buckingham, M., Potter, D., & Parfitt, D. (2009). Phylogeny and systematics of Prunus (Rosaceae) as determined by sequence analysis of ITS and the chloroplast trnL-trnF spacer DNA. Systematic Botany, 26, 797-807. https://doi.org/10.1043/0363-6445-26.4.797 3. Butac, M. (2020). Plum breeding. In A. Küden (Ed.), Prunus. IntechOpen. https://doi.org/10.5772/intechopen.92432 4. Butac, M., Zagrai, I., & Botu, M. (2010). Breeding of new plum cultivars in Romania. 9th IS on Plum and Prune Genetics, Breeding and Pomology 5. Das, B. (2011). Prunus diversity- early and present development: A review. International Journal of Biodiversity and Conservation, 3(14), 721-734. https://doi.org/10.5897/IJBCX11.003 6. Dey, K., Ghosh, A., Priya, B., Dutta, P., & Das, S. (2016). Impact of climate change and mitigation strategies on fruit production. Advances Life Sciences, 5(7), 2588-2596. 7. Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochemical Bulletin, 19(1), 11-15. 8. Earl, D. A., & von Holdt, B. M. (2012). STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetic Resources, 4(2), 359-361. https://doi.org/10.1007/s12686-011-9548-7 Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 32 9. Evanno, G., Regnaut, S., & Goudet, J. (2005). Detecting the number of clusters of individuals using the software structure: a simulation study. Molecular Ecology, 14(8), 2611-2620. https://doi.org/10.1111/j.1365-294X.2005.02553.x 10. Gaši, F., Sehic, J., Grahic, J., Hjeltnes, S. H., Ordidge, M., Benedikova, D., Blouin-Delmas, M., Drogoudi, P., Giovannini, D., Höfer, M., Kahu, K., Kovács, S., Lācis, G., Lateur, M., Toldam - Andersen, T. B., Ognjanov, V., & Nybom, H. (2020). Genetic assessment of the pomological classification of plum Prunus domestica L. accessions sampled across Europe. Genetic Resources and Crop Evolution, 67(5), 1137-1161. https://doi.org/10.1007/s10722-020-00901-y 11. Hanelt, P. (2001). European wild relatives of Prunus fruit crops. Springer-Verlag. 12. Hartmann, W., Neumüller, M., & Gradziel, T. (2009). Plum Breeding. In S. Mohan Jain and P. M. Priyadarshan (Eds.), Breeding Plantation Tree Crops: Temperate Species (pp. 161-231). Springer Science Business Media. https://doi.org/10.1007/978-0-387-71203- 1_6 13. Horvath, A., Balsemin, E., Barbot, J.-C., Christmann, H., Manzano, G., Reynet, P., Laigret, F., & Mariette, S. (2011). Phenotypic variability and genetic structure in plum (Prunus domestica L.), cherry plum (P. cerasifera Ehrh.) and sloe (P. spinosa L.). Scientia Horticulturae, 129(2), 283-293. https://doi.org/10.1016/j.scienta.2011.03.049 14. Martínez-Gómez, P., Sánchez-Pérez, R., Rubio, M., & Dicenta, F. (2005). Application of recent biotechnologies to Prunus tree crop genetic improvement. Journal of Food, Agriculture and Environment, 1(1), 52-63. 15. Mehdi, A., Reza, F., & Zabihollah, Z. (2012). Molecular and morphological discrimination of selected plum seedlings for rootstock breeding. Journal of Fruit and Ornamental Plant Resources., 20(1), 5-19. https://doi.org/https://doi.org/10.2478/v10290-012-0001- 0 16. Milošević, T., & Milošević, N. (2018). Plum (Prunus spp.) Breeding. In J. M. Al-Khayri, S. M. Jain, & D. V. Johnson (Eds.), Advances in Plant Breeding Strategies: Fruits (Vol. Volume 3, pp. 165-215). Springer Nature Switzerland AG. 17. Milošević, T., Milošević, N., & Mladenović, J. (2023). Diversity of plums belonging to P. domestica L., P. insititia L. and Prunus × rossica Erem. Tree vigour, yielding and fruit quality attributes. Scientia Horticulturae, 320. https://doi.org/https://doi.org/10.1016/j.scienta.2023.112220 18. Neumuller, M. (2011). Fundamental and applied aspects of plum (Prunus domestica) breeding. Fruit, Vegetable and Cereal Science and Biotechology, 5, 139-156. 19. Okie, W. R., & Hancock, J. F. (2008). Plums. In J. F. Hancock (Ed.), Temperate fruit crop breeding: Germplasm to genomics (pp. 337-358). Springer Netherlands. https://doi.org/10.1007/978-1-4020-6907- 9_11 20. Perrier, X., & Jacquemoud-Collet, J. P. (2006). DARwin software. Cirad, France. 21. Popescu, I., & Caudullo, G. (2016). Prunus cerasifera in Europe: distribution, habitat, usage and threats. In San-Miguel-Ayanz J., De Rigo D., Caudullo G., Houston D. T. , and M. A. (Eds.), European atlas of forest tree species (pp. 142). Publication Office of the European Union. 22. Pritchard, J. K., Stephens, M., & Donnelly, P. (2000). Inference of population structure using multilocus genotype data. Genetics, 155(2), 945. https://doi.org/10.1093/genetics/155.2.945 23. Ramanatha, R., & Hodgkin, T. (2002). Genetic diversity and conservation and utilization of plant genetic resources. Plant Cell Tissue and Organ Culture, 68(1), 1- 19. https://doi.org/10.1023/A:1013359015812 24. Reales, A., Sargent, D., Tobutt, K., & Rivera, D. (2010). Phylogenetics of Eurasian plums, Prunus L. Section Prunus (Rosaceae), according to coding and non-coding chloroplast DNA sequences. Tree Genetics & Genomes, 6, 37-45. https://doi.org/10.1007/s11295-009-0226-9 25. Sancin, V. (1988). Sadje z našega vrta. Založništvo tržaškega tiska. 26. Sedaghathoor, S., Ansari, R., Allahyari, M. S., & Nasiri, E. (2009). Comparison of morphological characteristics of some plum and prune cultivars of Iran. Scientific Research and Essays, 4(10), 992-996. https://doi.org/10.5897/SRE.9000293 27. Soriano, J. M. (2020). Molecular marker technology for crop improvement. Agronomy, 10(10), 5. https://doi.org/https://doi.org/10.3390/agronomy101014 62 28. Šiško, M. (2016). Identification of hypothetical duplicate accessions of plums (Prunus domestica L.) within the Slovene plant gene bank collection using molecular markers. Agricultura, 13(1-2), 57- 64. https://doi.org/10.1515/agricultura-2017-0007 29. Šiško, M. (2018). Predstavitev genske banke kmetijskih rastlin FKBV. Posvet o ohranjanju in trajnostni rabi rastlinskih genskih virov, Pivola. 30. Ternjak, T., Barreneche, T., Šiško, M., Ivančič, A., Šušek, A., & Quero-García, J. (2023). Genetic diversity and structure of Slovenian native germplasm of plum species (P. domestica L., P. cerasifera Ehrh. and P. spinosa L.). Frontiers in Plant Science, 14. https://doi.org/10.3389/fpls.2023.1150459 31. Topp, B. L., Russell, D. M., Michael, N., A., D. M., & Weisheng, L. (2012). Plum. In M. L. Badenes and D. H. Byrne (Eds.), Fruit breeding (pp. 571-621). Springer. 32. Zhebentyayeva, T., Shankar, V., Scorza, R., Callahan, A., Ravelonandro, M., Castro, S., DeJong, T., Saski, C. A., & Dardick, C. (2019). Genetic characterization ofworldwide Prunus domestica (plum) germplasm using sequence- based genotyping. Horticulture Research, 6(1), 12. https://doi.org/10.1038/s41438-018-0090-6 Comparison of Slovenian Traditional Plum Materials with Genetic Resources from the Slovenian Plant Gene Bank 33 Primerjava slovenskih tradicionalnih genotipov sliv z genskimi viri iz Slovenske rastlinske genske banke IZVLEČEK Rastlinska genska banka Fakultete za kmetijstvo in biosistemske vede hrani poleg drugih vrst tudi okrog 250 akcesij koščičarjev, pri čemer večina materiala pripada vrstama Prunus domestica L. in Prunus cerasifera Ehrh. Z uporabo 11 mikrosatelitskih lokusov smo v raziskavi želeli: 1. oceniti genetsko strukturo slovenskih tradicionalnih genotipov sliv nabranih in situ v primerjavi z ex situ materialom iz kolekcije genske banke koščičarjev; 2. prepoznati edinstven material med in situ nabranimi akcesijami; 3. raziskati genetske odnose med dvema proučevanima vrstama. Genetska struktura 60 genotipov sliv je bila analizirana s pomočjo Principalne koordinatne analize (PCoA) ter Bayesove analize. PCoA analiza je razdelila akcesije glede na pripadnost preučevanima vrstama (P. cerasifera oz. P. domestica), medtem ko je Bayesova analiza pokazala, da si številne akcesije tako iz vrste P. domestica, kot P. cerasifera delijo pripadnost znotraj specifične populacije. Material nabran ex situ je pokazal večjo genetsko raznolikost, saj je bil razdeljen na več populacij v primerjavi z materialom nabranim in situ. Med slednjim smo identificirali unikatne genotipe sliv, predvsem iz Prekmurja in Štajerske, ki bi jih bilo smiselno vključiti v obstoječo kolekcijo rastlinske genske banke. Ključne besede: sliva, Prunus domestica L., Prunus cerasifera Ehrh., mikrosatelitski markerji, genetska struktura