Impaginato 287 Adv. Hort. Sci., 2020 34(3): 287­300 DOI: 10.13128/ahsc­7415 Genetic diversity, population structure, and relationships among wild and domesticated almond (Prunus spp.) germplasms revealed by ISSR markers S. Rahimi­Dvin 1, A. Gharaghani 1, 2 (*), A. Pourkhaloee 3 1 Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran. 2 Drought Research Center, College of Agriculture, Shiraz University, Shiraz, Iran. 3 Department of Horticultural Science, College of Agriculture, Vali‐e‐Asr University of Rafsanjan, Rafsanjan, Iran. Key words: cluster analysis, gene diversity, gene flow, population structure, wild almond. Abstract: The use of diverse almond genetic resources to expand the genetic bases of commercial cultivars is important for almond breeders. Iran is within the center of origin for almond and enjoys a huge diversity of wild species and local cultivars of this important nut crop. Despite some reports, there is still a critical need to collect comprehensive information on the genetic diversity of almond germplasm in Iran. This study was conducted to evaluate the genetic diversity, structure, and relationships among a total of 75 individuals from 10 populations of 4 wild and cultivated almond species by using 12 inter­simple sequence repeat (ISSR) primer pairs. A total number of 353 DNA fragments were obtained of which 352 were polymorphic (99.69%). The average of poly­ morphism information content (PIC), marker index (MI), and resolving power (Rp) were 0.932, 27.211, and 7.882, respectively which indicated high discrimi­ natory power of markers. Gene flow between wild and cultivated gene pools is shown to be moderate to high (Nm = 2.7607), which verifies the hypothesis of low genetic differentiation among populations. Cluster analysis based on unweighted pair­group, classified individuals into 7 major gene pools which showed the entire provenances were divided into 7 main groups. Overall high levels of genetic diversity were confirmed and useful information obtained on the differentiation and genetic structure of the studied almond germplasms. Future evaluation on morphological and physiological aspects, is necessary to identify the most promising individuals to be used directly in afforestation, landscape development as well as nut and oil production or indirectly in future almond and stone fruits breeding programs. 1. Introduction Almond [Prunus dulcis (L.) Batsch] belongs to the Rosaceae family and (*) Corresponding author: agharghani@shirazu.ac.ir Citation: RAHIMI­DVIN S., GHARAGHANI A., POURKHA­ LOEE A., 2020 ­ Genetic diversity, population structure, and relationships among wild and domesticated almond (Prunus spp.) germplasms revealed by ISSR markers. ­ Adv. Hort. Sci., 34(3): 287­300 Copyright: © 2020 Rahimi­Dvin S., Gharaghani A., Pourkhaloee A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 3 November 2019 Accepted for publication 25 June 2020 AHS Advances in Horticultural Science https://www.researchgate.net/profile/Ali_Pourkhaloee https://www.researchgate.net/institution/Vali-e-Asr_University_Of_Rafsanjan https://www.researchgate.net/institution/Vali-e-Asr_University_Of_Rafsanjan https://www.researchgate.net/institution/Vali-e-Asr_University_Of_Rafsanjan http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(3): 287­300 288 is one of the most important nut crops in the world which is known for its high nutritional value. Almond domestication occurred nearly 5000 years ago in the Fertile Crescent (Velasco et al., 2016). United States of America, Spain, Iran, Italy, Turkey, Tunisia, Morocco, Syria, Greece and Australia are the ten major producers of almond (Ardjmand et al., 2014). Iran is the fifth world producer of almond (Gharaghani et al., 2017) which produced approxi­ mately 2.99% of the total world production of culti­ vated almonds (Sorkheh et al., 2016). Due to the narrow genetic background of com­ mercial cultivars, breeding programs of almond face many challenges. In modern plant breeding, native plants are considered as valuable gene pools for crossing programs which can be used to introduce new traits into commercial relatives. Wild almond species are found in the mountains and deserts of Central Asia from western China to Iran and Turkey (Rahemi et al., 2012). Wild almond species could be valuable gene pools for breeding purposes due to late bloom, early maturity, adaption to drought and salinity, resistance to winter lower temperatures, reduced insect infestation and fungal attacks (Gharaghani et al., 2017). Thus, knowledge about genetic diversity of wild genetic resources of almond is an essential prerequisite for involvement of native germplasm in almond breeding programs. On the other hand, assessment of genetic diversity and pop­ ulation structure is necessary to evaluate the existing levels of genetic variability and its patterns of distrib­ ution among the local populations, which is consid­ ered as a guarantee for conservation management of natural populations (Cohen et al., 1991; Sreekanth et al., 2012). Iran is a center for genetic diversity of almond and nearly twenty wild species of almond have been reported from arid and semi­arid regions of this country (Sorkheh et al., 2009). Different regions of Iran have variable environmental conditions includ­ ing subtropical climate in the south, temperate in the north, and extended deserts in the middle which helps the distribution of wild species such as almonds. Wild almond germplasm forms the main part of distributed plant species in the mountainous and plain sub­regions of ecological zones in the Zagros of Iran where the annual precipitation rate is more than100 mm (Sabeti, 1994). Almond stands of the Irano­Turanian region have been observed in Badamak, Mohammadabad Maskun and Badameshk forests in Fars, Kerman and South Khorasan province of Iran, respectively (Talebi et al., 2013). Fars and Charmahal­o­Bakhtiari provinces cover parts of the central and southern Zagros where Prunus scoparia (Spach) C.K. Schneid., P. elaeagnifolia Spach., and P. eburnean Spach. are widely distributed (Gharaghani et al., 2017). Owing to some traits such as leaf shedding during hot seasons, and the remarkable capability of roots in water absorption, some of the wild almond species can resist draught (Madam et al., 2011). P. scoparia is a potentially multi­purpose wild almond species in Iran which has the potential to become the crop of choice for soil stabilization and landscape in arid and semi­arid areas (Mozaffarian, 2005). It has been used as a dwarfing rootstock for almond for centuries (Gharaghani and Eshghi, 2015). P. scoparia is a potential source of vegetable oil for human nutrition and health with relatively higher oxidative stability, higher unsaturated to saturated fatty acids ratio, cal­ culated oxidisability value, total tocopherols and phe­ nolics contents, and unsaponifiable matter contents, than those of olive oil (Farhoosh and Tavakoli, 2008). Zedu gum is exuded from the bark of P. scoparia, and its kernel oil are used in Iranian traditional medicine (Zargari, 1997). Zedu gum is also being used as emul­ sifier in cosmetic and textile industries (Rahimi et al., 2013). These species lie among the rare trees which naturally grow in barren soils (Ali et al., 2015). P. elaeagnifolia has been used as a rootstock for plum (Gholami et al., 2010) in drought conditions in Iran and some other species have also been used as root­ stocks for almond and peach by ancient Iranians in arid lands (Denisov, 1988). Grafting nectarine on wild almond trees as rootstock has also been reported by Alberghina in 1978. Wild almond species have also been used to afforest barren lands and to protect vegetative cover (Mardani, 2006). DNA­based molecular markers are important tools to study genetic variation in population genetics. Various molecular markers including random ampli­ fied polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), Inter­simple sequence repeat (ISSR), and single nucleotide polymorphism (SNP) have been previously used to describe genetic diversity and structure in the genus Prunus (Martins et al., 2003; Shiran et al., 2007; Sorkheh et al. , 2007; Wu et al. , 2008; Bouhadida et al., 2009; Rahemi et al., 2012). Among different DNA markers, ISSRs have greater reliability and reproducibility in comparison with RAPD system, as well as the lower cost of the analyses than AFLP Rahimi‐Dvin et al. ‐ Almond germoplasm identification by ISSR markers 289 and SSR (Rodrigues et al., 2013). Moreover, ISSR markers seem to be especially useful to study closely related individuals which show low levels of polymor­ phism (Zietkiewicz et al., 1994). Local wild species make up an excellent source of genetic diversity which can be used for crop improve­ ment and breeding programs (Khadivi­Khub and Anjam, 2014). Despite some reports, there is still a critical need to collect more information on the genetic diversity of almond germplasm in Iran. Because of high value of P. scoparia, it is important that the necessary steps be taken to comprehensive­ ly evaluate, utilize and ensure the conservation of this unique wild species. The purpose of our study was to study the genetic diversity and population structure of a collection of wild and cultivated almond populations in Iran using ISSR markers. The emphasis of this study is on the populations of P. sco‐ paria collected from different sites in central and southern Zagros regions, experiencing less natural precipitation and higher temperature comparing to other natural habitats of this species in Iran. These special climatic condition made these wild popula­ tions a promising source of genes evolved for drought and high temperature tolerance, which will be very valuable in facing harsh effects of climate change. We also sought to compare the diversity and illustrate the relationships of these populations with some populations of three other almond species including P. elaeagnifolia, P. eburnea and P. dulcis (common almond) from the same geographical region, to put more shed on the possible gene flow among them as well as to detect the footprint of these species in the genetic background of cultivated almond. The results of this study are useful for con­ servation of these wild stands as well as for decision making on direct or indirect utilization of them for afforestation, nut and oil production, landscape pur­ pose and through breeding programs. 2. Materials and Methods Field sampling To detect higher genetic diversity (resulting from cross­pollination in natural habitat of the plant mate­ rials used herein) as well as the feasibility of plant materials collection (seeds instead of leaf samples) we chose to use raised seedling populations instead of natural populations in this study. In total, seeds of 72 wild almond trees were sampled from southern and central regions of Zagros Mountain in Iran during late spring to early summer of 2014. These regions are placed in Fars and Chaharmahal­and­Bakhtiari provinces. The studied genotypes belong to P. sco‐ paria and P. eburnean in section Spartioides Spach. as well as P. elaeagnifolia and P. dulcis in section Euamygdalus Spach (Kester and Gradziel, 1996). In addition, seeds of 5 almond cultivars were sampled. Characteristics of the populations (collection sites, latitude, longitude, altitude, etc.) are listed in Table 1. Plant materials and DNA extraction The seeds of all species were mechanically scari­ fied and then soaked in water for 24 h. They were mixed with perlite and stratified at 4±1°C for 45 days. After stratification, nuts were directly sown in 5 kg pots filled with a mixture of fine sand, soil and leaf mold. The pots were then transferred to the green­ house with an average temperature of 26±3°C under daylight illumination conditions i.e. 800 μmol m­2 s­1 about 10 hours. In December 2017 a total of 75 seedlings (each seedling represents an individual tree in natural habitat) comprised 10 populations (3 to 11 individuals per population) were selected. Stem pieces (200 mg) for each individual were collected into aluminum foil, immediately snap­frozen in liquid nitrogen and stored at ­80°C until the DNA was extracted. Total genomic DNA was isolated following the cetyltrimethylammonium bromide (CTAB) proto­ col with minor modifications (Doyle and Doyle, 1987). DNA quantity and quality were determined by spectrophotometry and visual comparison of DNA electrophoresed on 1% agarose gel. ISSR genotyping In total, 12 ISSR primer pairs were selected based on literature review (Carvalho et al., 2002; Martins et al., 2003; Dje et al., 2006; Zhao et al., 2007; Oliveira et al., 2010; Moulin et al., 2012; Ahmed et al., 2013; Muraseva et al., 2018) and synthesized (by Metabion, Germany). Polymorphism of markers was first tested in a subset of samples and then polymerase chain reac­ tion (PCR) conditions were optimized. The list of primers and their information are presented in Table 2. The PCR mix contained 10 ng template DNA, 10 pmol of primer in a final 20 μl reaction volume. Conditions of the PCR amplification were as follows: 94°C (3 min), then 35 cycles at 94°C (45 s) / 38­61°C (varied for each primer according to Table 2) (45 s) / 72°C (1 min) and final extension at 72°C for 7 min. The amplified products were separated by 1% (w/v) agarose gel electrophoresis in 1× TBE buffer at con­ stant voltage (100) for 45 min, stained with Adv. Hort. Sci., 2020 34(3): 287­300 290 Table 1 ­ List of the studied genotypes with indication of their regions and geographical coordinates of the collection sites Species No. Population E N Altitude (m) Prunus scoparia 1 Shiraz 1 52 34.558 29 37.082 1535 2 Shiraz 2 52 35.822 29 44.275 1820 3 Shiraz 3 52 35.784 29 44.287 1816 4 Shiraz 4 52 35.811 29 44.269 1819 5 Shiraz 5 52 34.601 29 37.103 1569 6 Shiraz 6 52 34.449 29 37.465 1565 7 Shiraz 7 52 33.704 29 37.946 1549 8 Shiraz 8 52 34.586 29 39.490 1670 9 Shiraz 9 52 32.642 29 40.263 1728 10 Shiraz 10 52 35.789 29 44.353 1821 11 Shiraz 11 52 35.785 29 44.351 1818 P. scoparia 12 Nourabad 3 51 20.875 30 4.695 1179 13 Nourabad 4 51 39.730 29 48.513 934 14 Nourabad 5 51 32.377 30 1.167 1086 15 Nourabad 6 51 23.931 30 0.745 1285 16 Nourabad 7 51 39.823 29 48.605 946 17 Nourabad 8 51 21.011 30 6.513 1183 18 Nourabad 9 51 31.694 30 1.252 1067 19 Nourabad 10 51 58.427 30 01 08.4 1592 P. scoparia 20 Marvdasht 1 52 54.983 30 3.162 1728 21 Marvdasht 2 52 54.800 30 6.249 1730 22 Marvdasht 3 53 00.807 30 6.800 1812 23 Marvdasht 4 53 12.117 30 5.742 1837 24 Marvdasht 5 53 10.524 30 1.617 1828 25 Marvdasht 6 53 12.643 29 59.116 1803 26 Marvdasht 7 53 14.080 29 57.712 1765 27 Marvdasht 8 53 6.493 29 48.754 1667 28 Marvdasht 9 53 6.535 29 48.816 1663 29 Marvdasht 10 53 8.662 29 47.443 1640 P. scoparia 30 Firuzabad 2 52 32.509 29 8.712 1725 31 Firuzabad 3 52 34.486 28 58.157 1503 32 Firuzabad 4 52 33.874 28 57.406 1530 33 Firuzabad 5 52 32.400 28 55.816 1445 34 Firuzabad 6 52 38.719 29 5.885 1917 35 Firuzabad 7 52 23.202 28 53.222 1524 36 Firuzabad 8 52 38.310 29 3.808 1732 37 Firuzabad 9 52 32.801 29 9.124 1763 38 Firuzabad 10 52 41.274 28 13.319 1275 39 Firuzabad 11 ­­­­­ ­­­­­ 1578 P. scoparia 40 Mian Jangal Fasa 1 52 46.117 29 26.327 1481 41 Mian Jangal Fasa 2 52 50.130 29 19.653 1526 42 Mian Jangal Fasa 3 ­­ ­­ 2187 43 Mian JangalFasa 4 53 24.351 29 9.542 1729 44 Mian Jangal Fasa 5 53 23.894 29 9.939 1754 45 Mian Janga lFasa 6 53 26.033 29 7.617 1720 46 Mian Jangal Fasa 7 53 26.054 29 7.632 1716 47 Mian Jangal Fasa 8 53 24.138 29 9.082 1756 48 Mian Jangal Fasa 9 53 22.759 29 10.821 1815 49 Mian Jangal Fasa 10 53 19.277 29 12.351 1825 to be continued... Rahimi‐Dvin et al. ‐ Almond germoplasm identification by ISSR markers 291 power (Rp) of each primer was calculated as Rp = ∑Ib, where Ib shows the informative fragments. The Ib may be shown on a scale of 0/1 by the following for­ mula; Ib = 1 ­ (2 × |0.5 ­ pi|) where pi is the propor­ tion of populations containing the ith band (Prevost and Wilkinson, 1999). Based on ISSR bands identified in the individuals, some basic parameters for genetic diversity including the total number of bands (TNB), the number of poly­ morphic bands (NPB), the percentage of polymorphic bands (PPB), mean Nei’s gene diversity index (H), Shannon’s information index (I), the observed number of alleles per locus (Na), the effective number of alle­ les per locus (Ne), the level of gene flow (Nm), popu­ lation diversity (Hs), the total gene diversity (Ht), inter­population differentiation (Gst), genetic identity and genetic distance were calculated for each popula­ tion using software POPGENE 1.32 (Yeh et al., 1999). Private bands (referring to the bands found only with­ in one population) and major allele frequency were estimated by power marker software. To illustrate the relationship among populations, SimplySafe (EURx, Poland) and photographed with UV light (Nade Gel Documentation and Analysis System JS­6800, China). The size of produced frag­ ments was defined according to size marker (Fermentas, Germany). Data analysis Marker results (reproducible distinct bands with high resolution) were dominantly scored in a data matrix. The matrix was used for calculation of popu­ lation genetic variation indices. The informativeness of primer pairs in genotyping and subsequent evaluation of genetic diversity and population structure was compared using the poly­ morphism information content (PIC), effective multi­ plex ratio (EMR), marker index (MI), and resolving power (Rp). For each primer, the polymorphic infor­ mation content (PIC) was estimated by PowerMarker v3.25 (Liu and Muse, 2005). Marker index for each primer was calculated as a product of polymorphic information content and effective multiplex ratio: MI = EMR * PIC (Varshney et al., 2007). The resolving Table 1 ­ List of the studied genotypes with indication of their regions and geographical coordinates of the collection sites Species No. Population E N Altitude (m) P. scoparia 50 Eqlid 3 52 40.003 30 15.126 1701 51 Eqlid 4 52 38.310 30 16.346 1742 52 Eqlid 5 52 36.405 30 18.062 1800 53 Eqlid 6 52 35.080 30 22.196 2321 54 Eqlid 7 52 23.051 30 19.324 1843 55 Eqlid 8 52 23.764 30 19.060 1750 56 Eqlid 9 52 24.085 30 18.382 1715 P. scoparia 57 Lordegan 1 51 11.609 31 33.619 1752 58 Lordegan 2 51 13.020 31 34.354 1962 59 Lordegan 3 ­­ ­­ 1948 60 Lordegan 4 ­­ ­­ 1963 P. elaeagnifolia 61 P. elaeagnifolia 1 52 35.806 29 44.098 1801 62 P. elaeagnifolia 2 52 35.746 29 44.158 1804 63 P. elaeagnifolia 3 ­­ ­­ 2128 64 P. elaeagnifolia 4 ­­ ­­ 2570 65 P. elaeagnifolia 5 52 34.880 30 22.732 2458 66 P. elaeagnifolia 6 52 34.898 30 22.748 2455 P. eburnea 67 P. eburnea2 52 22.173 30 19.865 1912 68 P. eburnea3 53 24.368 29 09.592 1732 69 P. eburnea4 ­­ ­­ 2280 70 P. eburnea5 52 24.585 30 18.376 1711 71 P. eburnea7 52 22.130 30 19.854 1902 72 P. eburnea8 53 24.101 29 9.071 1763 P. dulcis 73 Mamaei ­­­ ­­­ 1910 74 Ferragnes ­­­ ­­­ 1910 75 Badam talk ­­­ ­­­ 1910 292 Adv. Hort. Sci., 2020 34(3): 287­300 an unweighted pair group method with arithmetic mean (UPGMA) dendrogram was constructed based on Nei’s genetic distance using POPGENE 1.32 (Yeh et al., 1999). The dendrogram was generated using TreeView program. To further understand the relationships among populations, a Bayesian clustering­based structure analysis was performed on the entire data set using STRUCTURE 2.3.4 (Pritchard et al., 2000) to reveal the number of genetic pools. Two runs of analysis using the admixture model were performed. Initial runs were performed with a burn­in length of 50000 and 750000 MCMC (Markov Chain Monte Carlo) repli­ cates for 10 times at each K from 1 to 10. The proba­ ble number of groups was estimated. The second run was 100000 for burn­in length and 300000 for MCMC replicates, 10 times for each K. To estimate the best K value. The Evanno test was performed on STRUC­ TURE results using ‘‘Structure Harvester’’ (Evanno et al., 2005). The results were summarized in a bar plot using DISTRUCT (Rosenberg, 2004). 3. Results Informativeness of markers The mean of PIC values was analyzed for all loci to evaluate markers efficiency. PIC value ranged from 0.845 (primer 4) to 0.973 (primer 1). The mean PIC value for all loci was 0.932. The highest EMR value of 38 (primer 1) and the lowest of 20 (primer 12), with an average EMR value of 29.25 per primer were obtained. The highest (36.97) and the lowest (19) MI values were observed with primers 1 and 12, respec­ tively. The mean MI value was 27.211 per primer. The highest Rp value was observed with primer 1 (13.183) and the lowest with primer 4 (3.518) with an average Rp of 7.882 per primer (Table 2). Genetic diversity The 75 individuals of wild and domesticated almond assigned to 10 populations and were ampli­ fied with 12 selected primers (Table 2). A total of 353 bands were scored with an average band number of 29.33 per primer across 75 individuals. Among the 353 bands, 352 bands (99.69%) were polymorphic. The percentage of polymorphic bands (PPB) varied from 96.29% for primer 3 to 100% for the other primers (Table 2). At the population level, PPB ranged from 18.70% in P. dulcis to 58.07% in P. scoparia (Shiraz and Firuzabad populations) with a mean value of 49.66% (Table 3). The Na ranged from 19.63 for ISSR 3 to 20.00 for other ISSRs. Across the populations, Na ranged from 11.870 for P. dulcis to 15.807 for P. scoparia (Shiraz and Firuzabad populations). The Ne ranged from 11.453 for ISSR 4 to 14.254 for ISSR 5 with an average of 13.003 alleles per locus. Across the populations, Ne Y = (C, T); D = (A, G, T); V = (A, C, G); B = (C, G, T); R = (A, G) Rp= resolving power; PIC= polymorphism information content; MI= marker index; EMR effective multiplex ratio; MAF= major allele frequency. Table 2 ­ ISSR primers used in this study and their results Primers Primer sequences (5’­3’) Tm Total number of alleles (a) Number of polymorphic alleles (b) % Polymorphism (b/a)*100 PIC MI EMR MAF Rp 1 GAC AGA CAG ACA GAC A 48 38 38 100 0.973 36.97 38.00 0.120 13.183 2 GTG CGT GCG TGC GTG C 58 30 30 100 0.952 28.56 30.00 0.173 4.932 3 GTG GTGGTGGTGGTG­ 61 27 26 96.29 0.948 23.72 25.03 0.186 8.132 4 CTC TCT CTC TCT CTC TTG 54 27 27 100 0.845 22.68 27.00 0.373 3.518 5 GAG AGA GAG AGA GAG 50 23 23 100 0.953 21.85 23.00 0.173 9.160 6 CAC CACCAC GC 38 33 33 100 0.947 31.02 33.00 0.200 9.946 7 ACA CAC ACA CAC ACA 54 23 23 100 0.855 19.55 23.00 0.360 4.889 8 GAA GAAGAAGAAGAA­ 50 28 28 100 0.958 26.60 28.00 0.173 8.172 9 GTC GTCGTCGTCGTCGTC 61 32 32 100 0.873 27.84 32.00 0.333 4.692 10 GAG AGA GAG AGA CC 44 35 35 100 0.961 33.60 35.00 0.160 12.177 11 BDB ACA ACAACAACAA­ 49 37 37 100 0.956 35.15 37.00 0.160 9.531 12 YHY GTG TGT GTG TG 42 20 20 100 0.959 19.00 20.00 0.133 6.252 Min. ­­­ ­­­ 20 20 96.29 0.845 19.00 20.00 0.120 3.518 Max. ­­­ ­­­ 38 38 100 0.973 36.97 38.00 0.373 13.183 Means ­­­ ­­­ 29.41 29.33 99.69 0.932 27.21 29.25 0.212 7.882 Total ­­­ ­­­ 353 352 ­­­ ­­­ 326.54 351.03 2.544 ­­­ Rahimi‐Dvin et al. ‐ Almond germoplasm identification by ISSR markers 293 ranged from 11.496 for P. dulcis to 13.161 for P. sco‐ paria (Lordegan population) (Tables 3 and 4). Across the populations, the highest values of I (0.2816) and H (0.1838) indexes were observed for P. scoparia (Eqlid populations). However, P. dulcis showed the lowest I (0.1190) and H (0.0831) values (Table 3). However, for studied accessions, the average values of Na and Ne were 14.966 and 12.722, respectively. Genetic similarity and cluster analysis among popula‐ tions The dendrogram derived from UPGMA cluster analysis was generated for all populations (Fig. 1). Among seven distinct groups obtained by dendro­ gram, four groups represent populations of P. sco‐ paria. The group I consisted of two populations of P. scoparia (Shiraz and Mian Jangal­e­Fasa) collected from Fars province. The group II was composed of the other three populations of P. scoparia( Nourabad, Marvdasht, and Firuzabad) sampled from Fars province. The Eqlid population of P. scoparia Table 3 ­ Genetic diversity within the populations of almond in Iran exhibited by inter simple sequence repeat (ISSR) Population No. Observed no. of alleles (Na) Effective no. of alleles (Ne) Shannon’s in formation index (I) Nei’s genetic diversity (H) Percentage of polymorphic loci (PPB) No. bands No. private Bands Shiraz 11 15.807 12.853 0.2749 0.1774 58.07 205 7 Nourabad 8 15.212 12.657 0.2570 0.1664 52.12 184 3 Marvdasht 10 15.581 12.723 0.2653 0.1708 55.81 197 6 Firuzabad 10 15.807 12.812 0.2737 0.1759 58.07 205 11 Mian Jangal Fasa 10 15.666 12.879 0.2738 0.1774 56.66 200 8 Eqlid 7 15.524 12.968 0.2816 0.1838 55.24 195 12 Lordegan 4 14.419 13.161 0.2652 0.1817 44.19 157 1 P. elaeagnifolia 6 14.703 12.577 0.2453 0.1607 47.03 167 6 P. eburnea 6 15.071 13.099 0.2778 0.1854 50.71 179 8 P. dulcis 4 11.870 11.496 0.1190 0.0831 18.70 66 0 Mean 7.5 14.966 12.722 0.2533 0.1662 49.66 Fig. 1 ­ UPGMA dendrogram on the basis of Nei’s (1978) evalua­ tion of genetic distance among 10 populations of almond. Table 4 ­ A summary of genetic parameters across inter­simple sequence repeat loci Locus Na Ne I h Ht Hs Gst Nm 1 20.00 13.759 0.3872 0.2418 0.2412 0.2034 0.1568 2.6884 2 20.00 11.847 0.2547 0.1408 0.1427 0.1197 0.1615 2.5953 3 19.630 13.564 0.3529 0.2244 0.2273 0.1959 0.1385 3.1111 4 20.00 11.453 0.2178 0.1165 0.1183 0.0974 0.1771 2.3231 5 20.00 14.245 0.4103 0.2623 0.2538 0.2088 0.1774 2.3181 6 20.00 13.447 0.3652 0.2247 0.2234 0.1939 0.1319 3.2900 7 20.00 12.437 0.3061 0.1780 0.1775 0.1522 0.1428 3.0022 8 20.00 13.268 0.3392 0.2095 0.2092 0.1726 0.1751 2.3559 9 20.00 11.649 0.2255 0.1237 0.1234 0.1063 0.1387 3.1053 10 20.00 14.034 0.3857 0.2474 0.2426 0.2125 0.1244 3.5201 11 20.00 12.712 0.2869 0.1739 0.1761 0.1426 0.1903 2.1274 12 20.00 13.630 0.3593 0.2261 0.2216 0.1904 0.1408 3.0504 Mean 19.90 13.003 0.3242 0.1974 0.1964 0.1663 0.1546 2.7906 Na= Observed number of alleles; Ne= Effective number of alleles; I= Shannon’s Information index; Nei’s genetic diversity; Ht= Total gene diversity; Hs= Population diversity; Gst= Inter­population differentiation; Nm= Estimate of gene flow. Adv. Hort. Sci., 2020 34(3): 287­300 294 sampled in north of Fars province was separated in the group III. The group IV comprised Lordegan popu­ lation of P. scoparia sampled from Charmahal and Bakhtiari province in central Zagros region. Groups V, VI and VII included populations of P. eburnea, P. dul‐ cis, and P. elaeagnifolia, respectively. The Nei’s genetic distance ranged from 0.0077 to 0.0452 and genetic identity ranged from 0.9558 to 0.9923 (Table 5). The genetic identity between Nourabad and Firuzabad populations of P. scoparia was 0.9923 having the closest genetic relationship; however, the farthest genetic identity was 0.9558 between Lordegan population of P. scoparia and P. elaeagnifolia populations. Population structure The amount of gene flow (Nm) among popula­ tions was 2.7607, showing the moderate to high gene flow among populations studied herein. The genetic diversity within populations (Hs) and the total genetic diversity (Ht) of the species were 0.1663 and 0.1964, respectively (Table 6). The genetic differentiation among the populations (Gst) was 0.15 which shows that 15% of the total genetic variability was among populations and 85% was within populations. The results of the structure analysis with ISSR markers are presented in figure 2. The structure plot suggested a lack of definite structure although Table 5 ­ Genetic identity (above diagonal) and genetic distance (below diagonal) estimates between populations across all loci based on Nei (1978) Population Shiraz Nourabad Marvdasht Firuzabad Mian Jangal Eqlid Lordegan Prunus elaeagnifolia Prunus eburnea Prunus dulcis Shiraz **** 0.9862 0.9898 0.9899 0.9896 0.9773 0.9774 0.9693 0.9715 0.9722 Nourabad 0.0139 **** 0.9910 0.9923 0.9882 0.9806 0.9806 0.9789 0.9792 0.9756 Marvdasht 0.0102 0.0091 **** 0.9900 0.9841 0.9868 0.9756 0.9677 0.9709 0.9721 Firuzabad 0.0102 0.0077 0.0101 **** 0.9894 0.9835 0.9783 0.9714 0.9777 0.9789 MianJangalFasa 0.0104 0.0119 0.0161 0.0107 **** 0.9749 0.9779 0.9675 0.9725 0.9737 Eqlid 0.0230 0.0196 0.0132 0.0167 0.0254 **** 0.9730 0.9600 0.9625 0.9728 Lordegan 0.0229 0.0196 0.0247 0.0219 0.0224 0.0273 **** 0.9558 0.9718 0.9567 P. elaeagnifolia 0.0312 0.0214 0.0328 0.0290 0.0330 0.0409 0.0452 **** 0.9713 0.9609 P. eburnea 0.0289 0.0210 0.0296 0.0225 0.0279 0.0382 0.0286 0.0291 **** 0.9560 P. dulcis 0.0282 0.0247 0.0283 0.0213 0.0267 0.0275 0.0442 0.0399 0.0450 **** Fig. 2 ­ Population structure of almond populations for K = 2 and K = 4, showing a high degree of genotypic admixture among individuals. On the horizontal axis, the following population are illustrated: (1): Prunus scoparia (Shiraz); (2): Prunus scoparia (Nourabad); (3): Prunus scoparia (Marvdasht); (4): Prunus scoparia (Firuzabad); (5): Prunus scoparia (MianJangalFasa); (6): Prunus scoparia (Eqlid); (7): Prunus scoparia (Lordegan); (8): Prunus elaeagnifolia; (9): Prunus eburnea; (10): Prunus dulcis. Table 6 ­ Assessment of the genetic variability among ten populations designated based on the ISSR analysis Ht Total gene diversity Hs population diversity Gst Inter­population differentiation Nm Estimate of gene flow Average 0.1964 0.1663 0.1533 2.7607 Standard deviation 0.0236 0.0168 ­­­ ­­­­­ Evanno’s test indicated that the most informative number of populations was K = 2 and K = 4. Using the defined strategies of DNA purification with the selected primers, good patterns could be attained for the different accessions under study. Instance of patterns of amplification attained by ISSR Rahimi‐Dvin et al. ‐ Almond germoplasm identification by ISSR markers 295 in various accessions of almond are shown in figure 3. 4. Discussion and Conclusions Informativeness of markers Due to highly variable nature and less investment in time and money than other marker systems, ISSR markers are widely used in population genetic stud­ ies (Harris, 1999). Moreover, Matesanz et al. (2011) reported that because of high polymorphism, only a few ISSR loci (as few as five to seven primer pairs) are enough to obtain reliable information on genetic diversity of populations. The efficiency of a molecular marker system in distinguishing genotypes depends largely upon the polymorphism it can discover (Guo et al., 2014). On the basis of high PIC values, MI, and Rp we conclude that ISSR markers used in this study were informative in the assessment of genetic diversity of almond accessions. The high PIC values with a mean of 0.932 show that all primers are informative, and this can be related to high genetic variation among accessions used in this research. Similar results were reported for sour cherry and Prunus mira (Najafzadeh et al., 2014; Tian et al., 2015). The variation may have been contributed by gene flow, natural hybridization, propagation by seed and human selection (Sefc et al., 2000). The Rp and MI measurements show distribution and number of alleles (bands) within the studied genotypes. Bands that are scored in the half of geno­ types would possess optimal discriminatory power and with an increase in the number of bands, the Rp of a particular primer pair will be increased (Kayis et al., 2010). Therefore, primers with the highest PIC, EMR, MI, and Rp values (ISSR1, ISSR10, and ISSR11) were generally the most effective in distinguishing between accessions and could be further used in almond genetic diversity studies. The similar results are reported in, Prunus genus, sweet cherry, and sour cherry (Yılmaz et al., 2009; Ganopoulos et al., 2011; Najafzadeh et al., 2014). Genetic diversity Information on genetic diversity and structure of wild almond populations is essential for their conser­ vational programs. Moreover, narrow genetic back­ ground of the commercial cultivars of the genus Prunus restricts their cultivation in new regions with different environmental conditions. Therefore, genetic diversity among populations of this genus can be used to broaden the genetic background of com­ mercial scion and rootstock cultivars and to over­ come their distribution across different regions (Gradziel et al., 2001). Genetic variation depends on many factors such as mating system, genetic drift, gene flow, human activities, long­term evolutionary history, natural selection, and breeding systems (Schaal et al., 1998; Hamrick and Godt, 1996).Populations of domesticat­ ed almonds used in this study possess restricted number of individuals and often are reproduced veg­ etatively. Thus, cultivated almonds shows lower lev­ els of genetic diversity than the other species. However, higher genetic diversity was observed in certain individuals and populations of the wild almonds. This phenomenoncould be expounded by the fact that are propagated sexually whereas indi­ viduals of cultivated almonds aremainly reproduced asexually. The PPB is a major genetic diversity index that showed high levels of genetic diversity among almond genotypes. Similar great genetic diversity Fig. 3 ­ a) ISSR banding pattern generated using primer 11. Lane 1­14, Eqlid 7, Mian Jangal Fasa 6, Marvdasht 6, Eqlid 4, Nourabad 4, Firuzabad 2, P. elaeagnifolia 4, Nourabad 5, P. elaeagnifolia 2, Nourabad 10, Shiraz 8, Nourabad 9, Shiraz 2, Shiraz 6. b) ISSR banding pattern generated using primer 10. Lane 1­15, Shiraz 3, Shiraz 4, Marvdasht 8, Nourabad 7, Lordegan 1, Mamaei, Marvdasht 4, Shiraz 9, Badam talk, Shiraz 7, Mian Jangal Fasa 3, Mian Jangal Fasa 1, Eqlid 9, Marvdasht 3, Mian Jangal Fasa 4. M. 10 kb DNA ladder. Adv. Hort. Sci., 2020 34(3): 287­300 296 was reported by Sorkheh et al. (2017) in wild almond. The obtained PPB in this study was higher than val­ ues reported by Kumar et al. (2009) and Shuxia (2011) with Prunus armeniaca (96.5%) and Prunus persica (93%), respectively. Also, high genetic varia­ tion was shown by Rahemi et al. (2012) in wild almond, being similar to our results. One of the main reasons of the existing genetic variation is the process of self­incompatibility which is controlled by genes (Gouta et al., 2010; Szikriszt et al., 2011).The high number of generated alleles in our study may be due to use of several different genotypes that pos­ sessed high levels of genetic diversity. The bands generated by each primer rests on the primer, sequence and the diversity size in special genotype (Shiran et al., 2007). So, the number of bands differed in various genotypes. The private bands show the existence of special genes or sequences in native populations. The common bands show alleles which are shared among the cultivars studied. Thus, the private bands can be used in almond genetic fingerprinting and cultivar recogni­ tion. Genetic similarity and cluster analysis among popula‐ tions Cluster analysis is widely used to study the genetic relationships among germplasms (Li et al., 2010). The UPGMA dendrogram obtained in this study clearly distinguished species from each other and the clades were in accordance with morphological traits. Moreover, all populations were divided into their related taxa. Prunus scoparia (Shiraz population)which seems to be mainly an artificial (cultivated) population and P. scoparia (MianJangal­e­Fasa population) both were separated into the same group. Therefore, we assume that some of the P.scoparia stands in Shiraz region were developed artificially through seed that may have originated from MianJangal­e­Fasa. Lordegan population lay in group IV, being closely related to Eqlid populations. It may be due to the geographic proximity and climatic resemblance between these two geographical locations. Prunus eburnean was grouped in cluster V, close to P. sco‐ paria (Lordegan) populations. This close relationship is logical because both of them belong to Spartioides section within the genus Prunus (Kester and Gradziel, 1996). Moreover, close relationship between domes­ ticated population of almond (P. dulcis) and P. elaeagnifolia could be explained in the same way since they both belong to Eu amygdalus section with­ in the genus Prunus (Kester and Gradziel, 1996). Genetic proximity between the genotypes or pop­ ulations from different regions, for example Nourabad and Firuzabad (Table 5), could be explained by the geographical proximity of the regions, the exchange of plant material between sites and by the probable existence of common ancestors (El Hamzaoui et al., 2014). Also, Noormohammadial et al. (2013) reported the gene exchange among Prunus scoparia populations which is similar to our results. Molecular phylogeny results obtained in this study were similar to our findings using nut and kernel morphological characteristics to cluster the same subset of plant materials with some exceptions (Rahimi Dvin et al., 2017). In that work, we found that P. eburnea and P. scoparia were placed close to each other and P. elaeagnifolia and P. dulcis formed the same clade. The genetic distance among the studied almonds in this experiment is short, indicating that a high capacity for hybridization exists between genotypes and populations. The mating system can greatly affect genetic diversity both within and among popu­ lations. Generally, most of the genetic diversity in self­pollinated plants is distributed among popula­ tions, while in out crossed plants such as almond species, most of the genetic diversity is distributed within populations (Hamrick, 1989). Population structure Gene flow is defined as the gene movement with­ in and between populations (Lowe et al., 2009). The estimate of gene flow (Nm) has been categorized as low (Nm<1), moderate (Nm>1) and extensive (Nm>4) (Kumar et al., 2014). The estimate of Nm (2.7607) was higher than 1, which indicates that the number of migrants per generation can prevent population differentiation caused by genetic drift. Moreover, we conclude that high genetic diversity and lack of differ­ entiation is due to high amount of gene flow. Almond is an important food source for both human and ani­ mals and its seeds can be easily transported by birds and nomads (which is very common in the region) increasing the amount of Nm. Genetic differentiation coefficient is an indicator of genetic diversity and structure of species (Zia et al., 2014). It should be noted that Rosaceae species usually show low levels of genetic differentiation (Fineschi et al., 2005). Based on Slatkin (1985), Nm >1 shows no significant genetic differentiation among populations. In this study, genetic differentiation Rahimi‐Dvin et al. ‐ Almond germoplasm identification by ISSR markers 297 between populations had an average value of 0.15. Similar results were reported by Li et al. (2013) who obtained Gst of 0.18 with apricot. Many factors can influence on genetic differentiation which may occur independently in a population. For example, high dis­ persal rate of seeds must be involved in low genetic differentiation (Fanciulli et al., 2000). Moreover, gametophytic incompatibility, prevents self­fertiliza­ tion and encourages cross­pollination (Weinbaum, 1985) which retains high levels of genetic variability within seedling populations (Arulsekar et al., 1986). As a consequence, populations of almonds which possess gametophytic incompatibilityshow low levels of genetic differentiation. The genetic structure shows the history of popula­ tions with respect to their long­term evolution, muta­ tion, recombination, genetic drift, gene flow, and nat­ ural selection (Slatkin, 1987; Schaal et al., 1998). Therefore, providing information on the genetic diversity and structure of a crop is a prerequisite for the conservation and effective use of germplasms available for breeding (Laidò et al., 2013). Structure results demonstrated a high degree of admixture among individuals across 10 populations, consistent with moderate to high levels of gene flow across populations. Our results are similar to those of Mendigholi et al. (2013), who showed that the plots of structure exhibited the admixture of population and gene exchange which showed the existence of ancestral gene among Prunus scoparia. The lack of population structure and moderate to high gene flow among the species in this study sug­ gests the potential interbreeding among the popula­ tions. Nevertheless, a high individual genetic diversity purveys an optimistic prospect for the survival of the declining population with proper management inter­ position. Results signified that ISSR primers which had been used herein had a significant distinctive power for the evaluation of the polymorphism in various almond populations. The obtained results present Iranian native almond species as a precious source of genetic diversity and recommends that they are an auspicious source of new genes for rootstock and cul­ tivar breeding programs. Results also offer a contri­ bution to the management and conservation of this valuable almond germplasm. Since the Iranian almond species and genotypes have not been select­ ed for breeding programs, they are more probable to have a further diverse genetic background and may be employed in the selection of various genotypes so as to create new cultivars. 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