Prunus species , (2) investigate the possible involvement of P. cerasifera and P. spinosa species in P. domestica origin, (3) study the genetic relationships and variability among the most typical P. domestica accessions present in Slovenia. Ten haplotypes of cpDNA were identified and clustered into three groups according to the Neighbor-Joining analysis (NJ). All 11 SSR primer pairs were polymorphic, revealing 116 unique genotypes. A total of 328 alleles were detected with an average value of 29.82 alleles per locus, showing relatively high diversity. Bayesian analysis of genetic structure was used to identify two ancestral populations in the analyses of all three species as well as in a separate set consisting of P. domestica material only. Principal Coordinate Analysis (PCoA) showed that accessions clustered largely in agreement with Bayesian analysis. Neighbor-Joining analysis grouped 71 P. domestica accessions into three clusters with many subgroups that exhibited complex arrangement. Most accessions clustered in agreement with traditional pomological groups, such as common prunes, mirabelle plums and greengages. In this study, the analyses revealed within P. domestica pool valuable local landraces, such as traditional prunes or bluish plums, which seem to be highly interesting from a genetic point of view. Moreover, complementary approaches allowed us to distinguish between the three species and to gain insights into the origin of plum. The results will be instrumental in understanding the diversity of Slovenian plum germplasm, improving the conservation process, recovering local genotypes and enriching existing collections of plant genetic resources.Slovenia has particular climatic, soil, geographic and historical conditions that lead to long tradition of plum cultivation and use. In this work, a set of 11 SSR and three universal cpDNA markers, as well as flow cytometry, were used to (1) evaluate the genetic diversity of 124 accessions of the three  It is thought that the main existing European plum species, P. domestica L. (European plum)    .Prunus group, which includes the hexaploid European plum, P. domestica (2n = 6x = 48), and P. insititia, which was considered a distinct species by some authors in the past and is now classified as a subspecies of Prunus domestica (P. domestica subsp. insititia C. K. Schneider). European plum cultivars have been generally classified into different pomological groups based on morphological characteristics as well as fruit use, including large-fruited European plums, prunes, egg plums, greengages, mirabelles, damsons, bullaces and St. Julien plums (According to en plums . Prunes en plums . Traditien plums . Damsonsen plums .P. cerasifera (2n = 2x = 16), the diploid cherry plum, contains wild and cultivated genotypes used as ornamentals, for their fruits, or as rootstocks. Another important species closely related to the cultivated plum is the tetraploid P. spinosa (2n = 4x = 32). Some authors consider this wild species as one of the possible ancestors of P. domestica. There are still numerous questions about the genetic origin of P. domestica. Most authors support the theory that hexaploid P. domestica is an interspecific hybrid between diploid P. cerasifera and tetraploid P. spinosa , also known in the wider area of the Balkan Peninsula. Later, other varieties of European plum spread, and in the last two decades the selection was also enriched by some varieties of Chinese-Japanese origin  and western  parts of the country, plum drying was seasonal, but a very important source of income for rural population, especially women from small farm households . The tra century . The pre century .In Styria (NE Slovenia), the sweet and tasty fruits of another important landrace were used. The so-called ‘plavkica’  is an early flowering, round, small fruited, dark blue or violet-blue plum, with greenish to yellowish colour of the flesh that fully or partly adheres to the stone. Written information about this landrace is very scarce. Plum pox virus disease (Sharka) has greatly affected plum cultivation as well as the distribution of the species in its natural habitat, resulting in plum germplasm depletion  representing the Slovenian plum gene pool. Based on historical and economic importance, the most frequently present traditional plum genotypes in Slovenia probably belong to the species P. domestica. Therefore, our work focused on this species with particular interest on Bluish plum and common prune. In the present study, we used a set of SSR and cpDNA markers to: 1) evaluate the relationship among the three Prunus species  2) examine the possible involvement of P. cerasifera and P. spinosa species in the origin of P. domestica 3) investigate the genetic relationships and structure among P. domestica accessions collected in situ in the Slovenian landscape.The present research was set up to investigate the status of traditional plum germplasm as well as the genetic relationships and variability among accessions belonging to three 22.1in situ: in the wild, along roads, in home gardens, on abandoned estates and in orchards. The material was collected mostly throughout Slovenia with the emphasis on the north-eastern part, which is considered one of the typical plum-growing regions in Slovenia (ex situ collections: 38 from the Slovene Plant Gene Bank (SPGB) of the Faculty of Agriculture and Life Sciences (Maribor), 21 from the Plum French National Collection (PFNC) of the ‘Centre de Ressources Biologiques - CRB Prunus Juglans’, French National Research Institute for Agriculture, Food and Environment, (INRAE) (Nouvelle Aquitaine Bordeaux), and eleven from different botanical gardens or private collections from Slovenia (Maribor and Ljubljana) and Austria (Vienna and Graz). Of the 124 studied accessions, a set of 14 internationally known cultivars was used as reference. The accessions from the PFNC Collection were added in order to compare the genetic resources of Slovenian plum with a sample representing a large diversity within the species. This collection has been well described morphologically in INRAE multispecies integrative information system dedicated to plant and fungi pests , as well as studied with molecular markers by different authors  based on data from the existing collections and our morphological descriptions.The study included 124 accessions Table S1Slovenia , while t authors . Prior tex situ collections and according to The studied material was also classified according to its status, e.g., wild genotype, landrace, or improved variety. Based on passport data from the existing 2.2Total genomic DNA was isolated following the CTAB protocol described by 2.3Trifolium repens L. as an internal standard with a 2C nuclear DNA content of 2.07 pg  using a linear scale. Histograms were analysed using Flomax software 2.0 . The absolute amount of DNA in a sample was calculated according to the protocols proposed by Relative DNA content was estimated by flow cytometry using DAPI  staining in Otto buffers. Samples were prepared according to  2.07 pg . Ploidy The ploidy level was already determined for the accessions from the PFNC Collection , accordingly some of the material was used as reference . For each reference accession, the absolute amount of DNA was calculated and the values were compared between all accessions.2.4Prunus sp.  analyses . These unus sp. .Hinf I, Taq I, Alu I) and with two for VL and HK . Analyses were performed by BioGEVES platform  following the protocol described by Amplified fragments were digested with three primer pair–restriction enzyme combinations for K1K2  . Polymerase chain reactions (PCR) were performed using HotStarTaq®Plus Master Mix Kit , and the annealing temperature was adjusted accordingly for each marker (approximately 5°C bellow the Tm). Amplifications were performed in a Biometra TProfessional thermal cycler , and the quality of the fluorescently-labelled PCR products was checked. Fragment size analysis was performed using a Beckman CEQ 8000 DNA sequencer  according to the manufacturer’s instructions. Alleles appear as peaks in CEQ 8000 DNA sequencer and are compared to the standards during scoring to determine their size. Allele fragments were scored for each accession. First, we started with P. cerasifera accessions since they are diploids and therefore easier to evaluate. Then, each SSR was characterized by analyzing the peak profiles for all accessions. Following this approach, the accessions of the other two species were evaluated considering the corresponding profile observed in P. cerasifera. When P. cerasifera accessions had more than two peaks within the theoretical range of allele size expected for a given SSR, we assumed that the corresponding primer pair amplified two loci. We considered this information when scoring the alleles of the polyploid species. Sometimes for the polyploid material the number of peaks did not match the ploidy level, in these cases, allele dosage was based on the height of the peak. Examples of electropherograms with recorded allele fragments are provided in supplementary material  . In addiPrunus species discussed in this work, calculation of classical genetic diversity parameters is not possible because of the ambiguous allele dosage. Hence, distribution of SSR alleles in the studied Prunus species was estimated as follows: number of detected alleles, alleles found in all species, and alleles common only to P. domestica, P. cerasifera or P. spinosa.Due to the polyploid nature of the Bayesian model-based analysis was applied using the software package STRUCTURE V2.3.4  to invesP. domestica accessions with the same parameters.The most relevant number of clusters (K) for the analysed data was estimated by calculating ΔK based on the method of Evanno  implemenP. domestica accessions using DARwin 6.0.21 software  was used to confirm the genetic structure of the three studied species and independently of the software . This stsoftware Table S2 species .First, dissimilarities (30.000 bootstraps) were calculated with Sokal and Michener index (simple matching):ijd represents dissimilarity between units i and j, u is the number of unmatched variables and m is the number of matched variables. Information about the presence of the allele is rather confused in polyploids because allele frequencies are not clear. Therefore, the presence and absence modalities must be considered of equal weight, which minimises the loss of information (P. domestica accessions. Unweighted Neighbor-Joining (NJ) method was used to generate a tree with DARwin 6.0.21 software  amplified polymorphic patterns. After enzymatic digestion, ten polymorphic sites with 22 scorable bands were detected and ten haplotypes were identified (H1-H10) (P. domestica) with the exception of Blackthorn accession (42), of unknown ploidy level. Most of the reference accessions displayed affiliation to haplotype H1. Haplotypes H5 and H6 included solely accessions of hexaploid plum (P. domestica), whereas H2 and H3 were specific to the diploid cluster (P. cerasifera). Haplotype H4 was shared by these two Prunus species, with P. cerasifera (22 accessions) being the most abundant compared to the five P. domestica accessions. Haplotypes H7 to H10 were found only in P. spinosa accessions.The combination of three universal cpDNA primers resulted in distinctive haplotypes representing the studied accessions. Among the analysed material, eight accessions did not show clear PCR amplifications. All seven-primer pair–restriction enzyme combinations (K1K2-(H1-H10) Table S5P. domestica haplotypes (H1 and H6) and haplotype H2 found only in P. cerasifera. Haplotype H5 consisted of only one P. domestica accession: ‘Pitestean’ (69), which was not positioned with the other hexaploid plums in the first sub-cluster. The cluster II grouped together all P. spinosa accessions, which were further subdivided into two sub-clusters, comprising haplotypes H7-H8 and H9-H10, respectively. In the cluster III, diploid plums were mainly represented . H3 was represented by only one P. cerasifera accession whereas sub-cluster H4 was associated with diploid plums and five hexaploid accessions: local Plum (52), ‘Empress’ (80), ‘Čačanska lepotica’ (87), ‘Quetsche du Carmel’ (169) and ‘Krikon’ (179).The Neighbor-Joining tree Figure 13.2.2Based on 11 SSR markers, the analysis revealed 116 unique genotypes. Identification of genotypes based on SSR profiles revealed the presence of three duplicate groups accounting for 6.5% (8) of the studied accessions: Plum (150) and ‘Spindel plum’ (153); common prune accessions 108 and 110; and a group of four Bluish plum accessions .The 11 primer pairs used were polymorphic and amplified a single locus with the exception of UDP96-005, EMPAS12 and BPPCT014, which amplified two loci. The allele range for all primers extended from 131-173 (UCD-CH17) to 133-219 bp (BPPCT025), whereas the microsatellite markers , which amplified two loci each, had a range of alleles of 94-174 bp, 94-156 bp and 187-283 bp, respectively Table 2.P. domestica, followed by P. spinosa and P. cerasifera. The distribution of unique/specific alleles was the richest for P. domestica accessions (28.6%), while P. cerasifera (12.7%) and P. spinosa (8.6%) had lower values. We also calculated alleles found in only two species: P. domestica and P. spinosa shared 83 alleles; 49 alleles were found in P. domestica and P. cerasifera, while P. cerasifera and P. spinosa had only 20 alleles in common  to 40 (BPPCT025) alleles per locus. The total number of alleles for all accessions was 328 with an average of 29.82 alleles per locus Table 2.3.3(P. cerasifera) were assigned to Cluster 1 (green), with the exception of the hexaploid accession Plum (52). Sixty-nine P. domestica accessions were assigned to Cluster 2 (blue), which were mainly identified as hexaploid, with the exception of the accessions: ‘Stanley’ (73), and Plum (144), which were found to be diploid in flow cytometry analysis. In addition, twelve genotypes showed mixed ancestry . Among the admixed accessions, the highest number of accessions (10) belonged to P. spinosa with the remaining two genotypes representing a diploid accession, Plum (162) and one hexaploid, ‘Krikon’ (179).Bayesian analysis was used to examine the structural patterns of the three species. Regarding the most likely number of clusters (K) identified using the Evanno method, the maximum value for ΔK was two 1213.8, Table S6P. cerasifera, formed a denser arrangement in space. In contrast, Cluster 2, corresponding to P. domestica accessions, was more dispersed. The admixed accessions (mainly P. spinosa) were distributed between the two clusters but closer to Cluster 1 (P. cerasifera).Before performing the Principal Coordinate Analysis, dissimilarities were estimated using two distinctive indices: Sokal and Michener index and Jaccard coefficient. Comparison of the results showed no significant differences between the two indices, and only the results of the first index are presented. PCoA analysis revealed three main clusters Figure 3P. domestica but actually belonged to the diploid species P. cerasifera.Based on the results of various analyses , thirty-one accessions were identified, among the material collected in Slovenia, that were assumed to be hexaploid 3.4P. domestica collected in Slovenia.One of the main objectives of this study was to assess the genetic structure of P. domestica accessions with Bayesian analysis, two ancestral populations were identified using the ΔK method  and ‘HZW typ Mare’ (89) , ‘Hanita’ (75), ‘Ruth Gerstetter’ (81) and ‘Topper’ (86)]; eleven accessions from the SPGB collection, the Botanical Garden of the University of Maribor, the Botanical Garden of the Karl-Franzens University of Graz, and twelve accessions from the PFNC collection. The latter are reference accessions representing different pomological groups (such as mirabelles and greengages), some French landraces , and genotypes such as damson ‘Krikon’ (179). Seventeen accessions with estimated membership coefficients below 0.9 were considered admixed, including five accessions from Bluish plum group . The average distance (expected heterozygosity) between individuals was higher in Cluster 2 (0.8627) than in Cluster 1 (0.6617).When investigating patterns of structure among K method Table S7Examining the structure of the plum collection for K ranging between 3 and 6 . It was more dispersed and divided into several sub-clusters, demonstrating a higher genetic diversity. The accessions located between the two clusters corresponded to the admixed accessions identified by the Bayesian analysis.PCoA confirmed the results obtained with Structure for K=2 Figure 5. domestica accessions  clustered together with a local accession Plum (52). At the end of the first part of this sub-cluster, a pair of duplicate genotypes, Plum (150) with ‘Spindel plum’ (153), was found. The second half of the first sub-cluster consisted mainly of well-known improved cultivars: ‘Ruth Gerstetter’ (81), which appeared to be genetically close to the landrace ‘Californian Prune’ (66), ‘Pitestean’ (69), and ‘Topper’ (86). Associated with the latter were an improved variety ‘Plum Blue’ (53) maintained in the SPGB and a very old variety from Croatia, Plum (85), which is a prune type. The Slovenian small fruited genotype ‘Plum Green’ (37) was connected to this sub-cluster, although with a low bootstrap value. The second sub-cluster of Cluster I consisted of common prunes, collected in situ in Slovenia, well-known cultivars such as ‘Bistrica’  or ‘Quetsche commune’ (168), or derived from clonal selection [‘HZW Meschenmoser’ (74) and ‘HZW Typ Mare’ (89)] or from crosses involving common prunes .Neighbor-Joining analysis allowed us to evaluate the genetic relationships between 71 PPrunus insititia accession ‘Tersen’ (178), originating from Sweden and maintained in the PFNC collection, and the last Bluish plum (189), collected in eastern Slovenia. Cluster III contained several very well-known modern cultivars such as ‘Elena’ (68), ‘Čačanska lepotica’ (87), ‘President’ (88), ‘Jojo’ (127) and ‘Stanley’ (73).The Cluster II included all accessions of the Bluish plum group, separated into two groups according to the results of Bayesian analysis. One corresponded to Structure Cluster 1 and the other combined the accessions previously identified as admixed in the genetic structure analysis. The first group included material from different locations in the Styria region, associated to them, but clearly separated, was accession ‘Kriecherl Innerhofer’ (194) from Austria. The second group, much smaller, consisted of three accessions collected in the Prekmurje region. Related to both groups were found a 44.1P. domestica accessions/germplasm, but we discovered that approximately a third of the collected material actually belonged to P. cerasifera. This surprising result might be related to the fact that the Slovenian term ‘ringlo’, traditionally used for greengages, can also refer to any plum that is round, which is a typical characteristic of P. cerasifera fruits. When we carried out the initial prospection, we did not aim at species identification, but at categorizing accessions based on fruit description or other interesting agronomic traits.In this study we tried to collect mainly P. spinosa (4), P. domestica (3), P. cerasifera (2), and, as mentioned above, one haplotype was shared by the latter two species. P. cerasifera (15), P. spinosa (12) and P. domestica (5). On the other hand, P. domestica haplotypes (8), which is consistent with the fact that they studied the highest number of P. domestica accessions (166). The most predominant haplotype in our study contained mainly P. domestica accessions. P. domestica could be related to historical events , human interference and a narrow genetic base of material when plum was introduced into Europe. However, the relatively small number of accessions in our study could also explain this observation. Concerning P. spinosa, P. spinosa clustered together (8), four were linked to haplotypes of P. domestica and P. cerasifera. Because several identical accessions were examined in both studies (21) and the same primer pair–restriction enzyme combinations were used for cpDNA marker analysis, this discrepancy is rather unexpected. For 17 accessions, we confirmed the same trend for the corresponding haplotypes in both studies. However, three P. spinosa  accessions did not form individual haplotypes as was the case in Assessment of genetic diversity using PCR-RFLP analyses showed that the total number of haplotypes (10) was lower compared to similar studies: P. domestica accessions, P. domestica, P. cerasifera and P. spinosa species. Our results showed lower diversity than those of P. spinosa and P. cerasifera species.When studying SSR markers polymorphism, we obtained an average value of 29.82 alleles per locus, similar to the value of 29.25 alleles for eight SSR loci reported by P. domestica, which is not surprising given its polyploid nature as well as the largest number of accessions studied. The number of P. spinosa accessions for this study was the lowest of all three species, nevertheless the number and range of alleles were higher compared to P. cerasifera. This demonstrates the originality of the collected P. spinosa material.When comparing all three species, the number, range of alleles, and distribution of unique/specific alleles were highest for P. spinosa accession Blackthorn (42), shared haplotype H1 with P. domestica material. Furthermore, the results of a Neighbor-Joining analysis for the three species (data not shown) indicated that Blackthorn (42) clustered together with a group of Bluish plum accessions . However, Bayesian analysis classified this accession as admixed, together with other P. spinosa accessions. Hence, genetic relationship analyses suggest that Blackthorn (42) accession could be either a Bluish plum or an interspecific hybrid. Secondly, ‘Krikon’ (179) is another accession that exhibited a putative hybrid origin. When all species were examined together (data not shown), ‘Krikon’ clustered with the P. cerasifera and P. spinosa material. Interestingly, P. cerasifera accession from the UC Davis collection and a P. spinosa accession from Sweden.In addition, cpDNA analysis complemented with SSR markers allowed us to reveal some examples of complex relationships between the three species. Firstly, the supposedly 4.2P. cerasifera and P. spinosa species in the origin of P. domestica. Different analyses provided somehow contradictory information, which is not surprising given the unresolved question of the genetic origin of P. domestica. Assessment of genetic diversity using chloroplast DNA markers showed that P. spinosa clearly clustered separately, whereas P. domestica and P. cerasifera clustered together. The haplotypes of P. domestica and P. cerasifera appeared to be genetically closer compared to the haplotypes of P. spinosa, suggesting that P. cerasifera may have contributed to the maternal chloroplast DNA of P. domestica. This was also discussed by P. spinosa in the genome of P. domestica, sharing almost twice as many alleles as P. domestica with P. cerasifera, which is consistent with results of P. domestica and P. cerasifera as independent groups, while the accessions of P. spinosa showed admixed ancestry shared with both species.The combined results presented in this study allowed us to investigate the potential involvement of 4.3In this study, different approaches allowed us to establish genetic relationships among the local plum material collected in Slovenia and the traditional plum groups, such as common prunes, greengages and mirabelles.P. domestica accessions. Duplicates, represented 11.3% of the studied P. domestica accessions formed two distinct ancestral populations, which was also reported for plum by The STRUCTURE results were confirmed by the representation of PCoA analysis based on a genetic distance matrix. The studied th century (Prunus insititia ‘Tersen’ (178), which also originates from Sweden, clustered with several Bluish plum accessions. The fruits of ‘Tersen’ are small, blue-black in colour, and traditionally used for jams. Finally, ‘Kriecherl Innerhofer’ (194) is a clonal selection within subsp. insititia, a small, round, bluish-purple coloured landrace from Styria region in Austria. It is traditionally used for the production of plum brandy. Because its geographic origin, morphological appearance, and use overlap with our Bluish plum landraces, we suspected that they might be genetically close, which was confirmed by our analyses. The SSR analyses divided the Bluish plum accessions into two distinct populations, which were established in agreement with the region of origin. Similar to the first population, the Bluish plum accession (189) was also collected in the same region (Styria), although approximately 75 km of air distance apart and 100 m difference in elevation. It was in the same cluster with other Bluish plum populations, but separated from them and connected with Prunus insititia ‘Tersen’ (178). Since it seems to be a rather original material, it should be further investigated, by studying a larger sample at the same location. While Plum pox virus, Monilia laxa and Monilia fructigena. Perhaps this is the reason why they are slowly disappearing from the natural environment. Nevertheless, it might be interesting to test genetically well-identified Bluish plum accessions for tolerance to these diseases. There is also a possibility that Slovenian fruit producers and local farmers have tried to introduce more modern cultivars in recent years and somehow ‘abandoned’ the traditional material.The landraces belonging to the Bluish plum group are historically and genetically important plums that are slowly disappearing from the Slovenian environment. Since there is very scarce information about this plum group , we have tried to find reference material, corresponding to the morphological description of this varietal group in order to link them with some known genetic material. The French landrace ‘Perdrigon’ is a small, round-fruited, bluish-purple plum that was cultivated in Slovenia in the 19 century . However century . On the P. domestica accessions (H1), which was also pointed out by Another important locally collected group of plums are the common prunes, which clearly formed a unique group and seemed to include similar, but not identical accessions. When structure was examined (K=3), common prunes originating from different parts of Europe, such as Slovenia, France or Germany, formed a separate cluster, which was also confirmed by the Neighbor-Joining analysis. This result corroborates the fact that common prunes are widespread in Europe, with a large number of types that are distributed under different names such as ‘Hauszwetsche’ (Germany), ‘Quetsche Commune’ (France), ‘Casalinga’ , ‘Besztercei’ (Hungary) and ‘Vinete romanesti’ (Romania) . Linked P. domestica germplasm, as shown by the highest fixation indexes (FST) reported for these two groups. The Neighbor-Joining analysis divided the greengage and mirabelle accessions into two distinct groups associated with Cluster I. In the first group, ‘Plum green’ (56) from Slovenia was strongly linked to all French greengages. Although it was registered under a different name, genetic analyses and morphological characteristics confirmed that this plum belonged to the greengages. The second group of Cluster I was formed by two mirabelles from Slovenia: Mirabelle (140) and Myrobolan (141). The latter was initially thought to be diploid P. cerasifera, hence the name, but flow cytometry combined with the other genetic analyses confirmed that it actually belonged to the hexaploid mirabelle group. These two accessions showed strong affinity to French mirabelles, confirming historical reports , ‘Čačanska lepotica’ (87), ‘Jojo’ (127), located close to their genitor ‘Stanley’ (73). Since the latter is a well-known international cultivar belonging to ferences , ‘Čačansferences , as showian area . This re5P. spinosa, P. cerasifera and P. domestica. Our data disregard the possibility that P. domestica was entirely derived either from P. cerasifera or from P. spinosa. We were able to confirm that P. cerasifera may have contributed to the maternal lineage of P. domestica, and we suggest the possible involvement of P. spinosa in the P. domestica genome. Regarding the origin of P. domestica we managed to draw some parallels with other studies, however, our data were not conclusive enough to definitely resolve between different hypotheses. To address this question, further research should include genomics strategies including sequencing of cpDNA on a larger sampling of the three species covering their probable centres of origin and domestication. In addition, investigation of the genetic diversity using S-locus genotyping (The combined results provide insight into the relationship between the three plum species, notyping  could prP. domestica accessions collected in situ, demonstrating the authenticity of the collected material. It was surprising to discover that approximately a third of the material initially collected as P. domestica actually belonged to P. cerasifera. This shows the genetic complexity of plum species and highlights the importance of combining different tools and marker systems to evaluate relationships between the studied species.Our study revealed very low redundancy that occurred only in Even though prospection of Slovenian plum germplasm was preliminary, we found valuable material collected from a relatively small area. For example, Bluish plum accessions forming two distinct populations were collected within a radius of about 100 km. It would be interesting to cover a wider area, and try to collect different genotypes and study in depth a larger variability of this local population. Another traditionally important group that could be included in a more comprehensive study is the common prunes. Moreover, this type of research could be a next step for a comprehensive evaluation of plant genetic resources in the Balkan Peninsula, which harbors potentially valuable and unique plum genotypes.Finally, the obtained molecular data and the characterization of the collected material will be crucial in order to understand the diversity of Slovenian germplasm, to improve the conservation process, to recover local genotypes and to enrich the current SPGB collection. Furthermore, a complete agro-morphological study would be essential to evaluate prospective material in an attempt to integrate valuable genotypes into breeding programs. In addition, a thorough evaluation of commercial value for use in plum production would also be required.The original contributions presented in the study are included in the article/AI, MŠ, AŠ and TT designed the initial research, TB and JQ-G suggested complementary research and methods in data analysis. AI, MŠ, AŠ and TT conducted the field work and sampling. TT carried out SSR molecular analyses. TT and TB evaluated the SSR analyses results. TT analyzed the data. TB and JQ-G contributed to the data analysis. TT, TB and JQ-G, wrote the manuscript, AI, MŠ, AŠ edited and reviewed the original manuscript. All authors contributed to the article and approved the submitted version.


FST > 0.33) confirmed the high genetic separation among them.Viral symptoms, such as yellowing, leaf deformation, mottling, vein clearing, and reduced yield, were observed in cucurbits in Iran. This study aimed to detect the main suspected causal agent, cucurbit aphid-borne yellows virus (CABYV), in Iran and analyze the genetic diversity among isolates. Two hundred samples were collected from different growing areas between 2019 and 2022. PCR amplification was performed on the P3 and P4 genes. The sequences of 18 Iranian isolates were obtained and deposited in GenBank. Recombination, phylogenetic, and population genetics studies were then carried out for the complete genome and all ORFs sequences, together with other isolates in GenBank. The nucleotide identities of the overlapped ORF3/4 sequences of Iranian isolates were 94.8 to 99.5% among themselves, and with other tested isolates ranging from 94.3 to 99.3%. Phylogenetic trees based on the complete genome and the overlapped ORF3/4 showed two major clades, namely Asian and Mediterranean, and the new isolates from Iran were positioned in both clades. The obtained results also suggest that all the genes and two clades of CABYV populations were under negative selection pressure. Furthermore, rare gene flow between these two clades ( Citrullus lanatus), cucumber (Cucumis sativus), melon (Cucumis melo), and zucchini (Cucurbita pepo). These cucurbit species are predominantly grown during summer season, particularly in the Mediterranean. Plant viruses pose a severe threat to the cucurbit crops, causing over US$ 30 billion worth of damage annually [Cucurbitaceae is a prominent family of vegetables that is extensively cultivated in specific regions worldwide. It comprises several economically significant species, including watermelon . ORFs P0 and P1 encode a suppressor of post-transcriptional gene silencing (PTGS) and serine proteases with genome-linked viral proteins (VPgs), respectively, both of which are translated from the RNA genome. ORFs P1-P2 are produced by ribosomal frameshift, acting as RNA-dependent RNA polymerase. ORFs 3, 4, and 5 are translated from sub-genomic RNAs, encoding P3, P4, and P3-P5 proteins, respectively. P3 is a coat protein (CP), P4 is a movement protein (MP), and P3-P5 is involved in transmission by aphids, acting as a readthrough protein .Cucumis [The analysis of the full-length genome sequences of 33 CABYV isolates showed that they formed several distinct phylogenetic groups, which were associated with different geographic regions and collection time periods . A studyCucumis . SubsequCucumis . The genCucumis . High-thCucumis . A recenCucumis . Next-generation sequencing (NGS) was used for the first time to detect CABYV in melons showing yellow symptom in South Korea . CABYV hUnderstanding the genetic diversity and distribution of plant viruses is crucial for developing effective disease management strategies and ensuring global food security ,19. The A total of 200 samples were collected from most cucurbit growing areas in west and northwest of Iran, including Zanjan, Hamedan, Kurdistan, Ardabil, North Azerbaijan, and West Azerbaijan provinces. These samples were collected during the growing season of 2019–2023 from cucurbit, cucumber, squash, and melon plants which showed leaf yellowing and thickening symptoms. Each sample was assigned a unique number based on the time and date of collection and immediately frozen at −80 °C for subsequent total RNA extraction.The suspected samples exhibiting viral symptoms were subjected to RNA extraction using a modification of the CTAB method as described before . The extp value threshold of 0.05, to scan for any phylogenetic anomalies in the alignments. Anomalies detected by fewer than five algorithms were disregarded. The parent and recombinant relationship of the two recombinant strain clusters were confirmed by separating the alignments into regions that did, or did not, contain the recombinant regions, and comparing trees generated from those part sequences.In May 2023, all 76 isolates with complete genome sequences available in the NCBI GenBank were retrieved and then aligned using the ClustalW algorithm in the MEGA X program. The 5′ and 3′-untranslated regions were removed to create the “complete genome” alignment, which had a length of approximately 5690 base pairs. The complete coat protein sequences of 76 GenBank isolates, as well as new isolates obtained in this study, were added to the ORF3/CP alignment . We usedhttp://www.ncbi.nlm.nih.gov/BLAST/ (accessed on 11 May 2023)). Sequence alignment was performed with MEGA X v.10.2.4 software [The identity analysis of complete CABYV sequences in GenBank databases from around the world were conducted using NCBI-BLAST  method based on Tamura 3-parameter’s model  with gamHd), the number of polymorphic sites (S), the total number of mutations (η), the average number of nucleotide differences (k), average pairwise nucleotide diversity (π = Pi), and ω (dN/dS). The neutral selection hypothesis was also investigated using Tajima’s D [D* and F* [KS*, Z*, Snn, and FST (fixation index), were conducted to estimate inter-population diversity with 1000 replicates [FST value of higher than 0.33 suggests limited gene flow and further genetic isolation among tested populations [https://datamonkey.org (accessed on 19 June 2023)) to evaluate dS, dN, identifying codons, and clades under natural selection.This study utilized the DnaSP version 6.10.01 program  to examijima’s D  and Fu a* and F*  statisti* and F* . Additioplicates . The FSTulations . The frePolemovirus species, namley poinsettia latent virus (PnLV), which was used as an outgroup. The age of internal nodes was evaluated to estimate these divergence times, and the TimeTree was reconstructed using the fast-dating RelTime-ML computational method under the Tamura 3-parameter model [The study established the congruence between the lineages of Poleroviruses and their hosts. The divergence times of the CABYV population, along with eight other Poleroviruses, known to have a close genetic relationship, were determined. The six other Poleroviruses were chickpea chlorotic stunt virus (CpCSV), faba bean polerovirus 1 (FBPV-1), pepo aphid-borne yellows virus (PABYV), melon aphid-borne yellows virus (MABYV), Pumpkin polerovirus, suakwa aphid-borne yellows virus (SABYV), and a er model , which wer model . The defer model . A discrLuffa acutangula), interveinal yellowing was common, while bitter gourd (Momordica charantia) displayed yellowing with green vein-banding or yellow mosaic pattern, and watermelon mostly exhibited yellow mosaic pattern. Melons typically exhibited interveinal chlorosis or yellow mosaic pattern. These symptoms were often similar to nutrient deficiencies, and various factors such as environmental conditions, virus strain, and plant variety can also affect their expression. Some cucurbits infected by a virus may not display any symptoms at all.During the cucurbit-growing season in Iran, many regions in the west and northwest of Iran, including the Zanjan, Hamedan, Kurdistan, Ardabil, North Azerbaijan, and West Azerbaijan provinces, were visited, and various symptoms consistent with those caused by CABYV infection were observed in the cultivated cucurbits, including yellowing, leaf deformation, mottling, vein clearing, and reduced yield. Melons in different regions showed stunting as well as the additional symptoms mentioned previously. On ridge gourd  were recombinants originating from single recombination events . Using tThe analysis of new Iranian sequences revealed that they belong to the complete ORF3 and partial ORF4 of CABYV genome, with an encoded length of approximately 199 amino acids and varying similarities. Upon aligning the nucleotide sequences, it was observed that the identities between the new Iranian isolates and other tested isolates ranged from 94.3% to 99.3%, with the most significant differences observed in isolates from Thailand , USA (MK055337), South Korea , Japan (GQ221224), and China (EU000535). The obtained sequences from CP genes in this study showed a high sequence identity (94.8% to 99.5%) among themselves.Additionally, the alignment of deduced amino acid sequences indicated that the identities between the Iranian isolates and other isolates ranged from 96.2% to 99.5%, with the most amino acid differences observed in isolates from Thailand and South Korea. The pairwise nucleotide sequence identity matrix is presented as a matrix. The sequencing analysis results revealed that fragments ORF3 and ORF4 accounted for approximately 21% of the complete genome of CABYV.Phylogenetic trees generated using different models in the NJ method displayed similar topologies, identifying two major clades based on the complete genome  and ORF3The genetic diversity of CABYV populations was examined using various parameters, including nucleotide diversity (π), haplotype diversity (Hd), segregating sites (S), and the average number of nucleotide differences per site (η) for different regions of the virus genome  and new isolates from Iran. The highest nucleotide diversity was observed in ORF0, ORF3 + 5, and the complete genome of CBYV, with π values of 0.06814, 0.06633, and 0.06459, respectively. The π values for Asian and Mediterranean groups based on the complete genome and ORF3  were <0.3, with the lowest values obtained from the ORF3  analysis (0.01115 and 0.01432). The results also indicated that ORF3  was the most conserved region, with the lowest Hd, S, η, and k values among all the ORFs analyzed. Moreover, the analysis of 44 and 50 isolates’ complete genome and ORF3  sequences revealed 43 and 26 distinct haplotypes, respectively, indicating the presence of genetic diversity in CBYV populations . These fp-value threshold of (p ≤ 0.1)  ratio was estimated to be less than 1. The ORF0 analysis revealed the highest ω value of 0.52, while the complete genome and ORF3  showed ω values of 0.4294 and 0.2855, respectively. The dN/dS (ω) values for Asian and Mediterranean groups based on ORF3  were 0.1733 and 0.4606, respectively . Overallp ≤ 0.1) . The popp ≤ 0.1) . These fp ≤ 0.1) .D and Fu and Li’s D* and F* statistical tests. The results of the molecular variation analysis suggest that non-significantly positive values were obtained for the complete genomes, ORFs, and phylogroups in all of the statistical tests. For the French population, significantly negative values were calculated for the MP gene in all three statistical tests and only for Tajima’s D test for the CP gene. Among all the analyses, non-significantly negative values were obtained for all complete genomes, ORFs, and phylogroups except for the Mediterranean population, which showed non-significantly positive values for the complete genome in all three statistical tests . The FST values between the two clades (Asian and Mediterranean) were estimated to be 0.758 and 0.718 (>0.33) based on the complete genome and ORF3 , respectively, indicating a high level of genetic differentiation between these two clades , were distinct from other populations around the world  with significant o clades . Furtherhe world .Polerovirus were grouped into two main clusters, with one cluster encompassing CABYV, MABYV, Pumpkin polerovirus, SABYV, and PABYV, and with CpCSV and FBPV-1 grouped in another cluster. As shown in Polemovirus genus was selected as an outgroup.Based on the obtained phylogenetic TimeTree, the species of Some viral symptoms, including vein clearing, yellowing, and mosaic pattern, were observed extensively in a wide range of plants in many different regions. Generally, the symptoms observed in pumpkin, zucchini, and melon were much more pronounced, severe, and clearer than in other hosts. However, mosaic symptoms with yellowing were observed to be less severe in pumpkin than in other cucurbits in the fields. Vein clearing, which often leads to leaf distortion, was observed more often in cucumber than in other hosts and was even absent in pumpkin and sweet pumpkin plants. Overall, the lowest and mildest severities were observed in cucumber plants, possibly indicating the sensitivity and resistance of plant cultivars or virus host restriction. This study presented a unique occurrence in which both Asian and Mediterranean strains of CABYV were found in a same country, and have a relatively similar incidence, as indicated in the phylogenetic trees of 16 selected Iranian isolates. Some regions had a higher prevalence of these symptoms, probably due to a particular variant of the virus dominating in that area. However, symptom expressions caused by both strains on cucurbits were observed to be similar and indistinguishable in Iranian fields. Likewise, the Asian isolates were reported to induce yellowing and mosaic symptoms in melon cultivated in South Korea  while thViral symptoms were variable in different growth stages, from early to mid-summer and from late summer to early fall. Viral symptoms were more frequently observed in late growth stages, which may be due to the higher likelihood of mixed infection and the presence of multiple viruses in samples collected at the end of the growth season. Vectors actively transmitting the virus in autumn, a season with a relatively low temperature, could be another reason for infection in the late growth season leading to the highest incidence. In the spring, viral symptoms were already noticeable in pumpkin farms. In the summer, the lowest and weakest symptoms begin to show, while in the autumn, viral symptoms were severe and had a high percentage of occurrence, indicating that viral symptoms were milder and even disappeared under high temperature conditions.In order to identify the occurrence of viral CABYV infection in cucurbit crops, population analysis based on coat protein can play a prominent role. Recombination and mutation can affect the evolution of plant virus populations, therefore identifying haplotypes and relationships between CABYV populations based on the CP can shed light on population evolution. The genetic diversity of CABYV populations may be influenced by specific hosts and/or geographic origin ,32. In tEcballium elaterium (a wild cucurbit) to be under positive selection but not in the melon population. Furthermore, the FST values among E. elaterium and melon subpopulations from different fields ranged from 0.35 to 0.61, suggesting that the genetic differentiation of the isolates could be attributed to host adaptation [An observation on CABYV isolates collected in Murcia and Castilla-La Mancha, Spain, identified three codons in the ORF2 of aptation . Our phyHd was 0.960–1.000, indicating high levels of haplotype diversity in this study. Overall, ω was estimated to be <1 for CABYV populations, which suggests that all genes and clades of CABYV populations are under negative selection  indicate genetic separation between the Asian and Mediterranean clades. The Snn value (1.000) further confirms that the two clades are distinct from other populations around the world (The CP gene (ORF3) showed lower genetic variation in all parameters, such as the number of mutations and π value. The CP region is known as the most conserved region among all ORFs, and therefore, this analysis demonstrated the capability of CP sequences to play a main role in resolving CABYV phylogeny . Althougelection . Howeverhe world . Using fIn conclusion, understanding the genetic diversity of viruses is crucial for developing strategies for virus control. Therefore, in this study, we analyzed the CABYV population and genetic diversity. Our research aimed to reveal patterns of population genetic structure of CABYV in this region. Based on previous studies and our analysis, we confirmed the existence of Asian and Mediterranean clades in this region, and we identified three phylogroups based on the complete genome.


Bactrocera jarvisi is an endemic Australian fruit fly species (Diptera: Tephritidae). It occurs commonly across tropical and subtropical coastal Australia, from far-northern Western Australia, across the ‘Top End’ of the Northern Territory, and then down the Queensland east coast. Across this range, its distribution crosses several well documented biogeographic barriers. In order to better understand factors leading to the divergence of Australian fruit fly lineages, we carried out a population genetic study of B. jarvisi from across its range using genome-wide SNP analysis, utilising adult specimens gained from trapping and fruit rearing. Populations from the Northern Territory (NT) and Western Australia were genetically similar to each other, but divergent from the genetically uniform east-coast  population. Phylogenetic analysis demonstrated that the NT population derived from the QLD population. We infer a role for the Carpentaria Basin as a biogeographic barrier restricting east-west gene flow. The QLD populations were largely panmictic and recognised east-coast biogeographic barriers play no part in north-south population structuring. While the NT and QLD populations were genetically distinct, there was evidence for the historically recent translocation of flies from each region to the other. Flies reared from different host fruits collected in the same location showed no genetic divergence. While a role for the Carpentaria Basin as a barrier to gene flow for Australian fruit flies agrees with existing work on the related B. tryoni, the reason(s) for population panmixia for B. jarvisi (and B. tryoni) over the entire Queensland east coast, a linear north-south distance of >2000km, remains unknown. Bactrocera Macquart  ,41 10,41B. tryoni, Popa-Baez et al . Th. Th49]. ez et al , also foene flow ,22. The ene flow , and curene flow ,51. TogeB. jarvisi populations is evident, we still detected instances of high gene flow between NT and QLD in the data. Specifically, the Mackay sample is made up of ‘NT’ genotypes and a few individuals from Darwin and Mataranka in the NT have a QLD genotype. The most parsimonious explanation for this anomaly is human-mediated translocation. It is interesting to note that the whole Mackay sample was made up of introduced NT types with little evidence for introgression with local QLD types. While our study did not have the scope to address such issues, questions arising regarding non-random mating among lineages and competitive exclusion between a local and introduced lineage should be investigated.While the influence of the Carpentaria barrier in constraining dispersal of B. jarvisi samples along the east coast (QLD/NSW), a pattern also seen in B. tryoni [B. jarvisi and B. tryoni, that human movement of infested fruit is sufficiently extensive to account for the lack of genetic differentiation across >2000km and to hide the historical genetic signatures of multiple, well documented biogeographic barriers. While we consider this unlikely in the well-managed Australian horticultural system, particularly for B. jarvisi which is a lesser pest, the hypothesis is unfortunately very difficult to test. Fortunately, this is not the case for the corollary of the hypothesis. If human-mediated movement does explain the lack of genetic structure for pest species such as B. tryoni and B. jarvisi, then it is logical that a non-pest species would not be exposed to human-mediated dispersal and so should show a genetic structure more consistent with historical biogeography, current landscapes, and host-species’ distributions. To better understand the drivers of Dacini population genetic structuring, we consider population genetic studies on wide-spread, non-pest species to be a priority.There was a noted lack of genetic differentiation among . tryoni , with th. tryoni ,52–54. WRhagoletis pomonella between co-occurring hosts, hawthorn (the native host) and domestic apples, has been reported with a reduction in gene flow and sympatric divergence [B. jarvisi, while consistent with family structuring, provided no evidence for sympatric, host-associated divergence in the species.Population level divergence of some tephritid fruit flies through adaptation to novel hosts has been documented ,14,55. Fvergence ,14,55. HB. jarvisi and its native host, cocky apple, has long been known [B. jarvisi populations, even in the presence of alternate commercial hosts such as mango [B. jarvisi were dependent on cocky apple for the primary maintenance of populations, it could be argued that samples collected from cocky apple should be of the same genetic type as those samples collected from nearby but different host(s). However, such similarity or relatedness was not found among the flies from Walkamin Research Centre (FNQ) that were reared from four different host fruits: cocky apple, mango, guava and plum. Kinship analysis revealed no close relatedness between the flies reared from cocky apple and other hosts in sympatry.The close association between en known ,56,57 anas mango ,27,58. IB. jarvisi. We determined that: B. jarvisi exhibits significant genetic structure between QLD and NT populations, although there is evidence for recent translocation in both directions; a lack of genetic differentiation among the east-coast populations; and no evidence of host association divergence. Our study suggests that while the biogeographic barrier of the Carpentaria basin may be playing a role in the diversification of B. jarvisi in northern Australia, there is no evidence for the biogeographic barriers along the east coast of QLD influencing the genetic structure of B. jarvisi. Whether human-mediated dispersal could account for the lack of genetic differentiation across >2000k and several well-established biogeographic barriers is questionable. To test this a corollary of the human-mediated dispersal hypothesis, we should target a population genetic study on a non-pest species That is less likely to be moved by humans. Within Australia, the wild tobacco fly, B. cacuminata (Hering), is an abundant, widely distributed non-pest species suitable for such a study.This is the first study of the genetic structure of S1 Fig.(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigEach colour of the tip labels (sample IDs) corresponds with the location colours given in (DOCX)Click here for additional data file.S1 Table.(DOCX)Click here for additional data file.


Macrophomina phaseolina causes charcoal rot, which can significantly reduce yield and seed quality of soybean and dry bean resulting from primarily environmental stressors. Although charcoal rot has been recognized as a warm climate-driven disease of increasing concern under global climate change, knowledge regarding population genetics and climatic variables contributing to the genetic diversity of M. phaseolina is limited. This study conducted genome sequencing for 95 M. phaseolina isolates from soybean and dry bean across the continental United States, Puerto Rico, and Colombia. Inference on the population structure using 76,981 single nucleotide polymorphisms (SNPs) revealed that the isolates exhibited a discrete genetic clustering at the continental level and a continuous genetic differentiation regionally. A majority of isolates from the United States (96%) grouped in a clade with a predominantly clonal genetic structure, while 88% of Puerto Rican and Colombian isolates from dry bean were assigned to a separate clade with higher genetic diversity. A redundancy analysis (RDA) was used to estimate the contributions of climate and spatial structure to genomic variation . Climate significantly contributed to genomic variation at a continental level with temperature seasonality explaining the most variation while precipitation of warmest quarter explaining the most when spatial structure was accounted for. The loci significantly associated with multivariate climate were found closely to the genes related to fungal stress responses, including transmembrane transport, glycoside hydrolase activity and a heat-shock protein, which may mediate climatic adaptation for M. phaseolina. On the contrary, limited genome-wide differentiation among populations by hosts was observed. These findings highlight the importance of population genetics and identify candidate genes of M. phaseolina that can be used to elucidate the molecular mechanisms that underly climatic adaptation to the changing climate. Understanding the genetic diversity of plant pathogen populations and the factors influencing it allow for inferences about their evolutionary potential and identification of the molecular bases of adaptation. Plant pathogens are often genetically structured in different agricultural landscapes as a result of geographic and environmental differences . Among dMacrophomina phaseolina is a seed- and soil-borne fungal pathogen that infects more than 500 host species (Glycine max) and dry bean (Phaseolus vulgaris)  . During nt death . These snt death . Charcoant death , with thnt death . In the nt death . Diseasent death , with cont death .Macrophomina phaseolina is haploid, reproduces asexually, and overwinters in soil and crop residue as abundant, melanized microsclerotia that serve as the primary inoculum to initiate infection in subsequent seasons  can be confounded with discrete processes such as admixture and long-distance migration (human-mediated migration) which are commonly observed in plant pathogens . In addiMacrophomina phaseolina is recognized for its different ecological roles as an endophyte, saprotroph, and latent or opportunistic pathogen with broad geographic distribution . Specifically, the contribution of discrete vs continuous genetic differentiation was assessed by inferring M. phaseolina genetic groups while accounting for geographic isolation by distance. The hypotheses tested were M. phaseolina populations differentiated i) between countries and ii) between hosts within the United States The second aim was to investigate whether climatic variables contribute to patterns of adaptive genetic variation in M. phaseolina. Using RDA, the hypotheses were i) specific climatic variables contribute to genetic variation, ii) climatic variables independently contribute to patterns of genetic variation when accounting for underlying spatial and population structure, and iii) loci in strong association with multivariate climate can be identified and have roles in driving local adaptation to climate.The broad geographic distribution and population dynamics of M. phaseolina isolates collected across the United States, Puerto Rico, and Colombia, including 52 soybean isolates, 40 dry bean isolates, two strawberry isolates, and one Ethiopian mustard isolate  phylogenetic tree based on the 76,981 SNPs was constructed. Five genetic clusters were identified across the United States (n = 3), Colombia and Puerto Rico (n = 2). Furthermore, a pattern of hierarchical structure differentiating the United States and Colombian-Puerto Rican isolates was observed. The ML tree provided strong support (100% bootstrap) for two main clades, hereafter referred to as US and COLPR, and five well-supported clades within the main clades  within the US clade and two subclades (COLPR1 and COLPR2) within the COLPR clade. The PCA clustered isolates in five distinct groups in agreement with phylogenetic analysis, with little evidence of within group differentiation . The firM. phaseolina is genetically structured at a broader subcontinental regional extent.US1A isolates represented the predominant group in the United States, with most isolates collected in the East North Central and Central regions in the states of Michigan (29), followed by Wisconsin (11), Indiana (5), Tennessee (5) and Kentucky (2). Cluster US1B was represented by isolates from Mississippi (2) and South Carolina (1). US2 isolates represented the second largest group in the United States and were mostly collected in the Northern Great Plains  and South [Texas (2) and Georgia (2)] regions. Also, within this cluster were isolates from Wisconsin (1), Michigan (4), and Kentucky (1). In contrast, the COLPR1 cluster grouped most isolates from Colombia (11) and Puerto Rico (4) while COLPR2 grouped isolates from Colombia (5), one isolate from Puerto Rico, and three isolates from the United States. No evidence of population structure by states was found, which indicated that states do not represent genetic groups and M. phaseolina Macpha1 reference genome was reconstructed using the set of high-quality SNPs. The Macpha1 reference genome was considered as a suitable outgroup based on its European and Arabidopsis thaliana origin. The phylogenetic reconstruction with Macpha1 as a root revealed the COLPR2 clade as an outgroup to all other clades, while the United States clades were reconstructed as terminal clades  and a model-free matrix factorization (TESS3) framework. Spatial analysis of population structure incorporates geographic distance in the estimation of ancestry coefficients . The genetic structure of the 95 isolates was explained better by a spatial model of admixture between discrete genetic groups, where isolation by distance was accounted for rather than the non-spatial model. This was indicated by the increase in predictive accuracy in the conStruct spatial models for all tested values of K  and COLPR (COLPR1 COLPR2) clades were associated to isolation by distance or climatic differences rather than different discrete ancestry.M. phaseolina within and among clades and genetic clusters, we estimated gene diversity (He) and median pairwise genetic distance for each of the clades and genetic clusters. Pairwise genetic distance showed that COLPR isolates had greater genetic distances among isolates than those in the US clade, with a gene diversity (He) significantly higher in the COLPR clade (0.236) than the US clade . Among clusters, the COLPR2 cluster has the highest genetic diversity, considering both gene diversity and pairwise genetic distance, followed by COLPR1, US1B, US2, and the US1A cluster has the lowest values  and evenness of distribution of genotypes, the number of multilocus genotypes (MLG) was calculated for each clade and genetic cluster. A MLG was defined as a unique combination of SNPs. Given the large number of 76,981 SNPs and genotyping error rate from NGS data, it is unlikely that a true clone will be represented by an MLG. Thus, to better represent clones, closely related genotypes were collapsed into multilocus lineages (MLLs) based on a Prevosti’s genetic distance threshold of 0.0001 (8 SNPs). Of the 95 isolates, 79 had unique genotypes (MLGs) corresponding to 34 MLLs . eMLG anGenotypic richness was highest in the COLPR clade (15 eMLLs) as compared to the US clade (10.7 eMLLs). Among genetic clusters, the COLPR1 cluster had the highest number of eMLLs, followed by US2, US1B, COLPR2 and US1A. This indicates genotypic richness is highest in COLPR1 and lowest in the US1A genetic cluster, in which more than 80% of the isolates were clonal , CF. Altp = 0.002). Gene diversity in Puerto Rico (0.163) was intermediate and not significantly different from the United States  or Colombia . Pairwise genetic distances, corrected genotypic diversity indices and evenness calculated for each country follow the same pattern of gene diversity  compared with the United States (0.104)  and Nei’s GST . Populations were significantly differentiated among clades, genetic clusters, as well as within genetic clusters  and among genetic clusters  and US1B (GST = 0.54–0.69) clusters had the lowest GST when compared with any other cluster. Differentiation was lowest between COLPR1 – COLPR2 (GST = 0.50) clusters, and US1A – US1B (GST = 0.54) and highest between COLPR1 – US1A (GST = 0.80), COLPR1 – US2 (GST = 0.81) and US1A – US2 (GST = 0.99).To test the hypothesis that genetic clusters of ST = 0.63–0.69). The high values of GST in all pairwise comparisons suggest very high differentiation and little migration between genetic clusters. However, US1A – US2 GST estimation, which is notably high, was limited in power due to the low levels of gene diversity (Hexp) within these clusters. Across the 76,981 loci, there were only 76 and 255 polymorphic loci within US1A and US2 clusters, respectively. Thus, low gene diversity (Hexp) in US1A and US2 subpopulations likely resulted in overestimation of GST in pairwise comparisons of US1A and US2 with all other clusters.All other pairwise comparisons had similar intermediate levels of differentiation when compared to any other genetic cluster (GA) (M. phaseolina main populations (US and COLPR clades). The observed IA distributions for each population were compared to IA distributions for simulated populations with no linkage, 25%, 50%, 75% and 100% linkage. A predominantly clonal mode of reproduction was inferred in the US and COLPR populations of M. phaseolina. The simulated distributions and the different populations were significantly different from each other using ANOVA . The distribution of the standardized IA for the US population fell within the 75%–100% range of the linkage simulation  was calculated across pairs of loci for each of the clades. LD extends across a much larger distance in the US clade than in the COLPR clade, decaying over the first thousand base pairs, while in the COLPR clade LD decayed over the first hundreds of bases. LD half-decay distance, calculated as the average physical distance over which r2 decays to half of its initial value was ∼4,000 bp for US clade and ∼800 bp for COLPR clade   was usedmulation . This inR clades . To furtPR clade . This inM. phaseolina genetic clusters a redundancy analysis (RDA) was employed. Four climatic variables were identified as significantly predictive of genetic variation using the simple RDA model with forward variable selection. Temperature seasonality (TSsd) was the strongest predictor, explaining 28% of the variation, followed by precipitation of warmest quarter (Pwq), precipitation seasonality (PScv) and mean temperature of warmest quarter (mTwq) (To test the hypothesis that climate variation contributes to genetic variation across r (mTwq) . Importar (mTwq) . Spatialr (mTwq) . Notablyr (mTwq) , indicatr (mTwq) .M. phaseolina. Moreover, the large fraction of variation common to climate, population structure and space, emphasizes the importance of accounting for confounded effects in genotype-environment associations, particularly when inferring causal associations.Partial redundancy analysis (pRDA) was used to estimate the partial contribution of each set of explanatory variables  while removing the effect of the remaining variable sets . Variance partitioning with pRDA, using the LD-filtered set of 11,421 SNPs, revealed that climate , neutral population structure (isolate PC scores for the first three axes of a PCA using intergenic SNPs) and space (dbMEM3 variable identified by forward selection) together significantly explained 72% of the total SNP variance. Nearly half of this variance was uniquely attributable to neutral genetic structure (32%), climate (4%), or space (1%), while the other half of the SNP variation was explained jointly between the three sets of variables . The effp < 0.001) and the first three RDA axes explained most of the genomic variation associated with climate.To identify loci that are potentially involved in local adaptation to climatic conditions, SNPs strongly associated with climatic variables were identified using RDA with and without accounting for population structure. Neutral population structure was used as it uniquely contributed the most to genetic variation. The RDA models, whether accounting for population structure  or not (simple RDA), were globally significant  was modelled with 100% homology confidence to the transient receptor potential (TRP) NOMPC  mechanotransduction channel protein in Drosophila melanogaster , as revealed in variance partitioning, the candidate loci obtained with population structure correction represent a conservative set subjected to a reduction in the detection of SNPs truly associated with climate. In the simple RDA model, without correcting for population structure, 91 candidate unlinked SNPs were identified . Only twM. phaseolina in the continental United States, Puerto Rico and Colombia collected from soybean and dry bean fields and the contributions of climatic factors to patterns of genomic diversity among populations. We found that five distinct genetic clusters of M. phaseolina evolved across the United States, Colombia and Puerto Rico and evidence suggests migration between genetic clusters and countries. To date, population genetic studies in M. phaseolina have performed their analyses at the resolution of microsatellites molecular markers and have provided important information on genetic diversity, host and geographic associations in the United States  from the COLPR clusters, leading to a recent bottleneck in the US populations. The high probability assignments observed in US clusters may be consistent with the expected strong recent genetic drift in bottlenecked populations . In thiscurrence . Similarcurrence .M. phaseolina populations in this region. Overall, we believe the genomic signals of discrete and continuous structure that differentiate M. phaseolina populations could be reflective of a complex demographic and evolutionary history. Therefore, alternative demographic scenarios, including one of multiple independent introductions, should be considered in future studies ideally applying demographic modelling with a broad geographic and temporal distribution of isolates.Diversity was found to be further reduced in US1A genetic cluster as compared to all other clusters. Low diversity and high differentiation are signatures of genetic drift but also selection. If reduced diversity in the US1A genetic cluster was consistent with a clonal expansion following a bottleneck, the divergence and marked low diversity could reflect both genetic drift and selection. Genetic drift is expected to have substantial effects on pathogen populations, because migrations resulting in founder effects and reduced population sizes associated with pathogens survival in soil . AdditioM. phaseolina populations. Genetic diversity between countries also supported this hypothesis. Whereas Colombian isolates were significantly more diverse than United States isolates, diversity in Puerto Rico was intermediate and not significantly different from United States or Colombia. These findings may be consistent with the idea of Middle or South America as putative centers of origin for M. phaseolina and with its introduction to North America as part of historical crop migrations. For example, common bean Middle American origin, domestication centers in Middle America and South America  diversity and potentially involved in a host jump from native Solanum species  from widespread geographic locations found in M. phaseolina is in line with a markedly clonal population structure  or spatially structured environmental variables . This ste stress . Similarrevisiae  while Hsand cold .753,275-ankyrin repeat protein  were included as host outgroups. Cultures were routinely grown on potato dextrose agar  medium.A total of 95 n fields . Isolaten fields . There wn fields . Forty in fields . IsolateM. phaseolina cluster. A full heuristic search using the first ten most parsimonious trees and the Neighbor-joining tree as starting trees with 100 random sequence additions was performed in PAUP v4.0b10 . The broth was incubated for 7–9 days at room temperature. Mycelia were harvested, lyophilized for 24 h and ground using a FastPrep FP120 homogenizer . Genomic DNA was extracted from the lyophilized tissue using a modified SDS-based method; briefly, 50 mg of ground mycelia were mixed in lysis buffer (3% SDS (w/v); 100 mM Tris-HCl, pH 8.0; 50 mM EDTA, pH 8.0) followed by phenol/chloroform DNA extraction. The identity of all isolates was confirmed by multigene DNA analysis of the Internal Transcribed Spacer regions for the nuclear rDNA operon (ITS), part of the Translation Elongation Factor (TEF-1α) gene region, and part of the actin (ACT) gene region according to . Maximum v4.0b10 , to find v4.0b10 .M. phaseolina reference genome Macpha1  using bwa-mem . The libraries were prepared using the Illumina TruSeq Nano DNA Library Preparation Kit HT. The resulting sequences were quality assessed using FastQC  and clea bwa-mem . The ison France . The mapn France . Alignmen France . After mSingle nucleotide polymorphisms (SNPs) of all 95 isolates were predicted using the Genome Analysis Toolkit (GATK) v4.0 . InitialM. phaseolina isolates in RAxML-NG v1.01  reference genome was used as outgroup taxon. Maximum likelihood analysis was run in RAxML-NG v1.01 using the “-all” option with automatic bootstrap replicates and the GTR + G + ASC_LEWIS substitution model.The population structure was inferred according to the results from both model-based and model-free clustering methods and phylogenetic inference. The phylogenetic tree was inferred from the full set of high-quality SNPs among the 95 NG v1.01 . The RAxNG v1.01  in R 4.0NG v1.01 . To infeBayesian clustering of allele frequencies was implemented in conStruct . To asseFor each clade and genetic cluster, gene diversity  was calcST analog, GST  based on the quasi-Euclidean distance matrix was conducted in poppr v2.9.0  generated among 76, 981 loci and 48 samples (corresponding to the median population size of the two clusters). The observed and simulated IA values were tested for normality using the Shapiro-Wilk’s normality test and an analysis of variance (ANOVA) was conducted to test for significant differences among the distributions. Pairwise comparisons between the IA simulated distributions and for each population were tested for difference with Tukey’s HSD test in R. The extent of clonality was correlated to clonal (100%), mostly clonal (75%), semiclonal  or sexual (0%) modes of reproduction. Linkage disequilibrium (LD) decay rate was estimated using the physical distance over which LD decays to half its initial value, as measured by the squared correlation coefficient (r2). The linkage disequilibrium decay was calculated for each clade using the correlation coefficient (r2) in TASSEL v5 . We used data at a resolution of 2.5 min (∼21.5 km2), because it corresponds with our sampling design (single isolate samples rather than populations) being at a field or county scale. Coarser resolutions could combine multiple sampling locations into a single spatial grid and finer resolutions (30-s or <30-s), while this may be important for structuring patterns of genetic variation within populations, these data are less suitable for our sampling design and focus on regional to continental-wide patterns. We reduced the number of climatic variables from 19 to five to account for collinearity among them (|r| > 0.7) and to represent our hypothesis about the most important factors potentially driving selection. Diseases caused by M. phaseolina are more prevalent during hot and dry conditions, therefore temperature and precipitation variables were included. The selected climatic variables were: BIO18 = Precipitation of Warmest Quarter, BIO15 = Precipitation Seasonality (Coefficient of Variation), BIO17 = Precipitation of Driest Quarter, BIO10 = Mean Temperature of Warmest Quarter and BIO4 = Temperature Seasonality (standard deviation *100). Each bioclimatic variable was scaled, centered, and evaluated for inclusion using forward selection with 10,000 permutations using adespatial R package  and reduce spurious GEA, distance-based Moran’s eigenvector maps (dbMEM) were generated using sample coordinates in the quickMEM R function . The dbMr2 > 0.9) dataset of 11,421 SNPs. The independent contribution of each set of explanatory variables: climate, neutral population structure or space, was assessed while removing the effect of the remaining variable sets using partial RDA. In outlier loci identification, using a partial RDA is recommended to reduce the number of false-positive detections particularly in scenarios of multilocus adaptation when selective agents are unknown  and climatic variables hypothesized to drive selection were evaluated using a multivariate method, redundancy analysis (RDA, as implemented by . RDA sim unknown . On the  unknown . Candida unknown . A simpl