GORTERIA JOURNAL ISSN: 0017-2294

Evaluation of genetic diversity of domestic and wild pistachio genotypes in (Khorasan Razavi) using ISSR markers

Esmail Mirahmadi1, Iman Yousefi Javan2*, Ahmad Ahmadian3, Masoud Alipanah4

1 Ms.d. Department of Plant Production, Faculty of Agriculture, University of Torbat Heydarieh, Torbat Heydarieh, Iran.

2* Assistant Professor, Department of Plant Production, Faculty of Agriculture, University of Torbat Heydarieh, Torbat Heydarieh, Iran (ORCID ID: 0000-0001-6906-8626; phone: +98-51- 51240177; fax: +98-51-52299625; postal address: 9516168595).

3 Assistant Professor, Department of Plant Production, Faculty of Agriculture, University of Torbat Heydarieh, Torbat Heydarieh, Iran.

4 Associate Professor, Department of Plant Production, Faculty of Agriculture, University of Torbat Heydarieh, Torbat Heydarieh, Iran.

Abstract Pistacia vera L. is one of the most important horticultural products of Iran which has a large share of non-oil export earnings. Optimal use of any product requires a rational understanding of its genetic diversity status in nature. Genetic sources of pistachio, identification and identification of vegetative and reproductive characteristics of cultivars and phenotypes of this plant are among the most important pistachio breeding measures. In this study, genetic diversity of 20 pistachio genotypes collected from Khorasan Razavi pistachio, Pistacia atlantica and wild pistachio (Pistacia vera) genotypes were evaluated using 20 ISSR primers. that yielding 74 scaffold bands in total. Among these, 35 bands (47.30%) showed polymorphism. Genotypes were divided into 4 groups based on cluster analysis by UPGMA method and Jaccard similarity coefficient. According to the results, ISSR was recognized as a repeatable method for identifying genotypes in pistachio genus. Ultimately, this will save considerable time and expedite corrective programs. Keywords: Cluster analysis; Genetic distance; Molecular markers; Pistachio genotypes Introduction The genus Pistacia is a member of the Anacardiaceae family and consists of 11 species. Pistacia vera L. (2n = 2x = 30) is a commercially-grown species in the genus (Zohary, 1995), native to north Afghanistan, northeast Iran and central Asian republics (Kafkas et al., 2006). It has been cultivated widely especially in Iran, which allocates the main part of the countries non-petroleum export product. There are three other species in Iran, including P. atlantica subsp. mutica, P. terebinthus and P. khinjuk, which are mainly used as rootstock for P. vera (Hosseinifard et al., 2010). The trend of climate change and the spread of pathogens in the old regions leads to reduced production and the disappearance of global product markets for pistachio farmers. Accurate identification and

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evaluation of pistachio hereditary reserves is essential for proper breeding programs and ensuring the preparation of modified cultivars suitable for these conditions. Extremely diverse ecological conditions in pistachio cultivation areas and genetic cross-breeding between different pistachio genotypes due to the non-pollinating nature of this product, has led to significant genetic diversity in pistachio genotypes in different regions of Iran (Zeng et al., 2019). At present, molecular markers are one of the suitable methods for studying the amount of genetic diversity in the intra and inter- population levels of plant species (Vinson et al., 2018). DNA markers have several advantages over typical phenotype makers. (Teama, 2018). Like the ISSR marker. Despite the extensive genetic diversity of Iranian pistachio germplasm, few studies have addressed this important crop, and only some of its cultivars are commercially used. Thus, the germplasm of this species in Iran is endangered. Therefore, in order to manage and protect the Iranian pistachio, identification and registration of this valuable nut crop is essential for breeding programmes as well as commercial production. Thus, in the current study, nuclear ISSR markers have been used to investigate the extent of diversity in the cultivars of P. vera, P. atlantica and P. terebinthus gathered across Khorasan Razavi State in Iran.

Material and Methods Plant materials and DNA extraction Young leaf samples were kept in liquid nitrogen tanks for the purpose of DNA extraction. collection of leaf samples in May 2019, from fresh, healthy branches and the sunny side of the tree done. Total genomic DNA was extracted from freez-dried leaf tissue using DENAzist Genomic DNA isolation kit 50. Quality and quantity of DNA were determined by a PG Instruments spectrophotometer (T80) and the concentration of DNA was also confirmed by electrophoresis using 0.8% (v/v) agarose for 45 min at 80 V in 0.5X TAE buffer, followed by visualization under UV light after staining with ethidium bromide. One of the objectives of the above research is to compare the genetic diversity between wild and domestic pistachio genotypes and also between domestic cultivars, cultivated in Khorasan Razavi state using ISSR markers through of Statistical software that group the wild and domestic pistachio genotypes in Khorasan Razavi (Table 1, Figure 1). Table1. Collected cultivars and genotypes of pistachio

Ge Cultivar and Region name Sub- notyp code Species Genotype and GPS location provincial e N. name (Sampling location) Region Garmab Torbat 1 AMG Pistacia atlantica Mutica Baneh 35°42′22″N 59°14′29″E Heydarieh Khakhk 2 AMK Pistacia atlantica Mutica Baneh 34°08′59″N 58°38′21″E Torbat 3 TBB Pistacia terebinthus Baneh 34°30′59″N 58°11′04″E Heydarieh Saadatabad 4 AMS Pistacia atlantica Mutica Baneh 35°12′04″N 57°47′22″E Kuhsorkh 5 AMR Pistacia atlantica Mutica Baneh 35°28′N 58°27′E -Khazayii 6 VAK Pistacia vera Akbari 34°42′12″N 59°13′22″E Gonabad 7 VAG Pistacia vera Akbari Gonabad 34°21′10″N 58°41′01″E Ahangaran-Khazayi 8 VHK Pistacia vera Ahmadaghaei 36°06′26″N 58°45′28″E

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Jangal-Bizan 9 VHB Pistacia vera Ahmadaghaei Roshtkhar 34°42′12″N 59°13′22″E Jangal 10 VKK Pistacia vera Kallehghuci Roshtkhar 34°42′12″N 59°13′22″E Jangal 11 VMB Pistacia vera Momtaz Roshtkhar 34°42′12″N 59°13′22″E Feizabad-Sareban Torbat 12 Pistacia vera Fandoghi VFS 35°00′52″N 58°46′48″E Heydarieh Gonabad 13 Pistacia vera Fandoghi Gonabad VFG 34°21′10″N 58°41′01″E Jangal-Tavasoli 14 VGT Pistacia vera Garmeh Roshtkhar 34°42′12″N 59°13′22″E Torbat Jam 15 TKT Pistacia terebinthus Khodru Torbat jam 35°14′38″N 60°37′21″E Feiz Abad Torbat 16 VBS Pistacia vera Bargsiah 35°00′52″N 58°46′48″E Heydarieh Feiz Abad-Sareban Torbat 17 VSS Pistacia vera Sefid 35°00′52″N 58°46′48″E Heydarieh Jangal-Purmohamadi 18 VSP Pistacia vera Sefid Roshtkhar 34°42′12″N 59°13′22″E Jangal-Salehi 19 Pistacia vera Sefid Roshtkhar VSH 34°42′12″N 59°13′22″E Feiz Abad-Zarand Torbat 20 VZS Pistacia vera Badami 35°00′52″N 58°46′48″E Heydarieh

Figure 1. Scattering of collected pistachios from different regions of Khorasan Razavi province. PCR primers, materials and conditions In the present research, which wa s conducted in laboratory of Torbat Heydarieh University in 2019, 20 available ISSR primers w ere assayed for initial screening. Of the 20 primers, 13 primers which gave the most informative patterns (in terms of repeatability & scorability) were selected for identification. Finally, the same 13 ISSR primers were used for fingerprinting. Amplifications of ISSR primers were done in a 25 μL reaction volume containing 3 μL DNA (50 ng), 0.7 μL of 1 mM dNTPmix, 0.2 μL Taq DNA Polymerase, 2.5 μL 1x Master Mix Red Ampliqon, 1.25 μL MgCl2 final concentration, 2.5 μL of 1 μM primer, and 14.8 μL of distilled water. The set of ISSR primers used in our investigation is shown in Table 2.

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Table 2. Name and sequence of ISSR primers used to produce bands from 20 pistachio genotypes.

Nu Nu polymo Poly mber of mber of rphism Pri Primer morphic Sequence created polymo informatio Ta °C mer N. Name percenta 5 '→3' ban rphic n content ge ds bands (PIC)

1 UBC807 7 4 57 0.71 5´(GA)8RC3´ 54°C

2 UBC807 5 0 0 0.80 5´(AG)8YT3´ 52°C

3 K10 5 5 100 0.80 5´(AC)8YG3´ 50°C

4 ISSR10 4 2 50 0.75 5´(TG)8RT3´ 52°C

5 ISSR5 9 1 11 0.85 5´CCA(CT)83´ 52°C

6 UBC873 6 3 50 0.82 5´(GACA)53´ 54°C

7 ISSR7 3 3 100 0.66 5´(GAA)63´ 50°C

8 ISSR8 5 5 100 0.56 5´(CA)8GT3´ 52°C

9 ISSR9 4 3 75 0.68 5´(TG)8G3´ 50°C

10 UBC808 7 1 14 0.18 5´(AG)8T3´ 54°C

11 UBC816 7 0 0 0.86 5´(CA)8RT3´ 54°C

12 UBC845 5 1 20 0.79 5´(CT)8RG3´ 54°C

13 ISSR13 7 7 100 0.84 5´(AC)8T3´ 52°C

Data Analysis The presence or absence of each of the observed bands for each of 13 primers was scored 1 and 0, respectively and then a matrix of 0 and 1 digits in Excel software was provided. Genetic association amongst samples was evaluated by the Jaccard’s similarity coefficient (Jaccard, 1908). Band scoring was based on zero and one. The obtained data were entered into excel software. In order to analyze the obtained data, NTSYS pc (version 2.02) software was used. The similarity matrix was subjected to cluster analysis of UPGMA method, and a dendrogram was generated also using NTSYS software (Rohlf, 1998). Principal Coordinate Analysis (PCoA) was also done. Polymorphic Information Content (PIC) of each of the analyzed ISSR was measured using the formula, PIC = 1- Σpi2 (Tiwari et al., 2013).

Result and Discution Nonanchored ISSR amplification reproducibility and polymorphism ISSR profiling of 20 pistachio genotypes with 20 primer combinations revealed a large number scorable bands ranging in size from 200-2500 nucleotides. The 20 ISSR primer pairs gave reproducible amplification products. A total of 35 alleles were generated using the 13 primer pairs across the studied cultivars (Table 2). And only 13 ISSR primers, produced amplified polymorphic bands, a gel image of ISSR5 primer is shown as an example (Figure 2).

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Figure 2. Bond pattern of ISSR5 primer on 20 collected pistachio genotypes.

The number of different alleles (Na) per primer pairs ranged from 1 (by ISSR5, UBC808 and UBC845) to 7 (ISSR13) with an average of 2.7 alleles per primer pairs (Table 2). Depending on the PIC, Number of effective alleles (Ne) ranged from 0.18 (by UBC806) to 0.86 (by UBC816), with a mean of 0.71 (by UBC807). This study showed that ISSR primers used with AC, CA, GA repeats show higher polymorphisms than other di and tri nucleotide replicates. The ISSR7 primer produced the fewest gene loci or fragments (three bands), and the ISSR5 primer produced the largest number of gene loci (nine bands), (Table 2). Genetic similarity coefficients were obtained with using Cophenetic Correlation Coefficient (CCC). The use of different similarity coefficients for dominant or concurrent markers depends on the ploidy level of the study population. The method that uses the highest correlation coefficient is the best analytical method (Mukaka, 2012). In this study, the quantitative correlation coefficient based on the UPGMA method and three similarity coefficients, dice similarity, jaccard similarity and simple matching were calculated and had a correlation coefficient of 0.79, 0.81 and 0.78, respectively. Clustering and genetic relationships among pistachio cultivars Simple matching coefficient (SMC) are recommended for haploid populations that have been closely kinship and Jacquard similarity coefficients are suggested for haploids popolation that distance kinship. Finally, jaccard similarity coefficient and UPGMA algorithm were selected as the best clustering pattern, which indicates the fit of the dendrogram with the similarity matrix.

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Table 3. Genetic similarity matrix among the studied cultivars and wild pistachio, based on ISSR data estimated by jaccard coefficient.

Genotyp AMG AMK VFG AMS VAK VAG VHK VHB VKK VMB VFS TBB AMR VGT TKT VBS VSS VSP VSH VZS AMG 1.000 AMK 0.772 1.000 VFG 0.767 0.836 1.000 AMS 0.759 0.732 0.821 1.000 VAK 0.742 0.642 0.716 0.682 1.000 VAG 0.652 0.625 0.703 0.750 0.815 1.000 VHK 0.687 0.662 0.738 0.730 0.768 0.785 1.000 VHB 0.692 0.721 0.774 0.710 0.776 0.738 0.857 1.000 VKK 0.662 0.609 0.662 0.705 0.721 0.734 0.855 0.836 1.000 VMB 0.662 0.661 0.662 0.677 0.721 0.734 0.691 0.723 0.746 1.000 VFS 0.672 0.621 0.672 0.662 0.779 0.769 0.776 0.813 0.781 0.781 1.000 TBB 0.770 0.717 0.742 0.733 0.721 0.682 0.716 0.723 0.692 0.719 0.781 1.000 AMR 0.635 0.607 0.661 0.678 0.697 0.683 0.642 0.672 0.667 0.721 0.787 0.750 1.000 VGT 0.646 0.645 0.726 0.717 0.706 0.719 0.676 0.682 0.703 0.730 0.712 0.730 0.763 1.000 TKT 0.590 0.559 0.644 0.691 0.606 0.724 0.677 0.629 0.737 0.678 0.635 0.650 0.679 0.782 1.000 VBS 0.692 0.694 0.803 0.738 0.750 0.766 0.721 0.754 0.672 0.723 0.758 0.778 0.814 0.762 0.741 1.000 VSS 0.662 0.662 0.738 0.730 0.768 0.813 0.818 0.828 0.769 0.716 0.803 0.742 0.746 0.727 0.705 0.887 1.000 VSP 0.687 0.662 0.738 0.703 0.794 0.785 0.791 0.828 0.742 0.691 0.776 0.769 0.719 0.701 0.677 0.887 0.967 1.000 VSH 0.657 0.631 0.708 0.726 0.765 0.839 0.815 0.825 0.766 0.662 0.800 0.738 0.742 0.697 0.729 0.885 0.934 0.934 1.000 VZS 0.588 0.609 0.662 0.677 0.721 0.790 0.769 0.806 0.774 0.667 0.754 0.667 0.694 0.758 0.737 0.806 0.855 0.855 0.915 1.000

The CCC indicated high correlations between the similarity matrix of each marker and the cophenetic matrix obtained from the UPGMA dendrogram. CCC value calculated ISSR was 0.93. And also the CCC is considered to be a very good representative of the data matrix in the dendrogram if it is 0.90 or greater (Arjmand Ghahestani et al., 2016). The mean of genetic similarity for all pistachio genotypes was 0.47 (47 %), which is lower than report given by Other researchers (Kamangar and Farsam, 2006; Aliakbarkhani et al., 2015). They studied genetic diversity among Kerman genotypes with 0.63 and Feizabad region with 0.51, for the mean of genetic similarity. It shows that pistachios of Torbat Heydarieh region have higher genetic diversity than Kerman and Southern Khorasan region pistachios. According to the results of the similarity matrix based on the ISSR marker Sefid Sarban (VSS) genotypes with Sefid Pourmohamad (VSP) had the highest similarity of 0.97. Badami Zarand (VZS) cultivar is one of the most grown and productive cultivars which has shown better adaptation to different commercial and known pistachio cultivars in different climatic conditions and unsuitable soil and water. The Genetic Similarities (GS) ranged from 0.559 to 0.967 (Table 3, Figure 3). This low similarity shows that although wild pistachio should be more similar to Pistacia atlantica (Mutica Baneh), and also, it is closer to some domestic species. Unlike other studies NOROOZI et al. (2009), to identify differences among Ahmadaghaei cultivars and Akbari cultivars in the present study, the percentage of similarity between the genotypes of Ahmadaghaei cultivars was very close (0.857), and also between Akbari cultivars was 0.815. High proportion of similarity between genotypes same region, such as three Sefid genotypes, indicates the appropriateness of the number of primers used, which can show even slight differences between different genotypes of the cultivars. In this study, the mean PIC was estimated to be 0.715. High PIC values for ISSR5, UBC873, UBC816 and ISSR13 markers indicate higher efficiency than other markers in differentiating the genotypes used. Which can be suggested for similar researches. The genotypes in the ISSR data dendrogram were divided into four groups. According to the results, the genotypes of white cultivars in different regions had a high similarity coefficient (Figure 3). All pistachio cultivars of groups 2 and 4 were domestic cultivars.

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This division, can be a compelling reason for considering Khorasan Razavi as one of the pistachios origins. That could be investigated for finding more wild genotypes and gene pools in the future.

Figure 3. Dendrogram of data from ISSR primers based on UPGMA mean algorithm for 20 samples of pistachio genotypes, using the complete linkage method. Pistachio of each sub-branch has its own morphological characteristics. Principal coordinate analysis (PCA) based on genetic similarity matrices were used to visualize the genetic relationships among species. The first two eigenvectors accounted for 44.03% of the total molecular variation. Therefore, PCA results confirmed the results of cluster analysis. Based on PCA, cultivars and wild species were almost separated from each other (Figure 4). Dendrogram of data from ISSR primers, 3D display of main components well shows that the genetic distance between Baneh (wild pistachio) and domestic genotypes is greater than the genetic distance between Torbat Jam and domestic pistachio (Figure 4). The results of the similarity matrix table and Three- dimensional diagram of the principal components can also be used to select genotypes with the longest genetic distance to produce strong hybrids in breeding studies. The same results are obtained from matrix tables in maize and canola plants (Tomkowiak et al., 2019; Wolko et al., 2019).

Figure 4. Three plots derived from the principal component analysis in the pistachio population studied.

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Domesticated cultivars have been maintained by single tree selection and vegetative propagation, which has narrowed the genetic range of pistachio cultivars, especially female cultivars (Arjmand Ghahestani et al., 2016). As a result, in our study, the similarity percentage of domestic genotypes due to vegetative propagation was higher than the similarity percentage between wild genotypes, which confirms the results of the similarity matrix.It is debatable that, the percentage of similarity of domestic genotypes due to vegetative propagation is higher than the percentage of similarity between wild genotypes, which confirms the results of the similarity matrix (Tomkowiak et al., 2019). Similarity matrix analysis showed that the differences between wild species are large compared to domestic species. Therefore, in pistachio breeding programs, wild pistachio population can be used to select various traits such as creating resistance to environmental stresses appeared. Also the results of similarity matrix, PCO analysis and cluster analysis showed pistachio samples collected from pistachio genotypes of Khorasan Razavi contrary to popular belief, it has little similarity and a large genetic distance. Other studies have offered a similar theory (Aliakbarkhani et al., 2015; Farzad Amirebrahimi et al., 2017). Long-distance genetic hybridization can be a good strategy for cross-breeding programs. Genetic distance between populations are a valuable parameter for germplasm protection as well as for plant breeding programs. It is well established that crossbreeding between unrelated and genetically distant parents has more hybrid power and shows the ratio of intersections between close genotypes. The findings of this study indicate that the ISSR markers used in this study have the ability to show polymorphism in the genome of the studied genotypes. And the average of these ISSRs polymorphisms is equal to 52.07%. The high rate of polymorphism obtained from ISSR marker indicates the high strength of this marker in the study of molecular diversity and also indicates the high diversity of pistachio genotypes. Also a small number of ISSR primers with high polymorphic information, they can differentiate a large number of samples and different populations of pistachios. Previous research has shown the high efficiency of ISSR markers in studies related to the determination of genetic diversity and evolutionary relationships in different plant species (Verma et al., 2017; Abdollahi Mandoulakani and Azizi, 2014). High PIC values for some markers indicated their high efficiency in differentiating the genotypes used. Which can be suggested for similar researches. In this study, we tried to show the genetic diversity of pistachios more clearly than other similar studies using ISSR markers (Aliakbarkhani et al., 2015; Afzadi et al., 2007). The information presented here could be useful in selecting suitable rootstocks and scions for improving the existing orchards. Moreover, because of the diversity of weather conditions all over the world, paying attention to the similarities of the genotypes can help us to select suitable genotypes for establishing new orchards in a specific geographical area. Conclusion The native genotypes it has taken a long time to evolve under the prevailing soil and climatic conditions of the condition, can be suitable candidates to be selected for improving programs for pistachio varieties and also could be considered as valuable gene pools. Another result, showed that ISSR-PCR analysis is quick, reliable, produces sufficient polymorphisms for large-scale DNA fingerprinting purposes, and also showed, ISSR markers are able to reveal variability between pistachio cultivars. This report also showed that despite the similarity between species, and the similarity of regional growth, but molecularly there are many differences, and these differences help to identify the native species and genealogy of each plant. The close relationships, and sometimes high genetic diversity, among pistacio accessions revealed in this study can be due to grafting, human selection of superior genotypes, and existence of narrow genetic base in some species of pistachios. This research of the genetic diversity and population structure of pistachio, using ISSR markers is needed to identify the origin and wild relatives of this valuable species. Also, it would be useful to breeders to determine how genetic diversity, as analyzed with molecular markers, relates to phenotypic variability and, more importantly, how it reflects the variability of the important traits.

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