Evaluation of diversity in selected populations of Iranian date palm (Phoenix dactylifera L.)  using ISSR markers

doi:10.21203/rs.3.rs-2881780/v1

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Evaluation of diversity in selected populations of Iranian date palm (Phoenix dactylifera L.) using ISSR markers

https://doi.org/10.21203/rs.3.rs-2881780/v1

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Date palm (Phoenix dactylifera L.) is cultivated mainly in arid and semiarid regions of the south and southwest of Iran. Sixty-nine Iranian genotypes were selected from 13 populations. One Moroccan genotype was also included in the experiment. The genotypes were sampled in Azizabad agro-research station in Kerman province, Iran. The number of alleles per locus ranged from 5 to 10, totaling 65. Polymorphism information content (PIC) varied from 6 to 30%, averaging at 19.4%. The highest and lowest values of effective alleles were found in Kerman (Ne = 1.49) and Bushehr (Ne = 1.15), respectively. The highest value of the Shannon index (I = 40%) and polymorphic loci percentage (PL = 67.69%) were detected in the ‘Kerman’ population. A high degree of genetic diversity was found in the ‘Hormozgan’ population (He = 0.26). The highest correlation coefficient (r = 0.983) was observed between Kerman and Hormozgan. Based on Nei’s genetic identity, the first two principal coordinates (PCoA) showed that the populations of Fars, Morocco, and Bushehr were classified as three separate groups, unlike the Kerman, Hormozgan, Khuzestan, and Persian Gulf Basin (PGB) populations. In addition, bilateral charts and genetic relatedness among the genotypes were illustrated by STRUCTURE software, which divided the 70 date palm genotypes into seven groups. The grouping of genotypes did not correlate with their geographical distribution. Thus, the Q-values of the genotypes grouped most of them as mixed. It seems that a lack of clear separation among the genotypes was because of their shared genetic background. To assist in future crossing programs, our findings showed that ISSR markers prioritized several male stocks of date palm.

Allele

date palm

ISSR

PIC

population

Date palm or date (Phoenix dactylifera L.) is a diploid perennial species (2n = 2x = 36) of the Arecaceae family. Due to its economic importance (Ahmed et al. 2021; Flowers et al. 2019), this tree species has significant value regarding industrial, commercial, medicinal, and ornamental aspects (El-Far et al. 2016; Kurup et al. 2014). Date palms are sometimes cultivated through methods of sustainable agriculture in many arid and semiarid regions, such as the Middle East, North Africa, areas of East and South Africa, and some parts of Europe and the USA (Amom and Nongdam 2017; Flowers et al. 2019). In this respect, Iran is one of the world’s most productive countries, with over 1 million tons of date production, amounting to 13.5% of the total global production (Hussain et al. 2021). The export of dates from Iran can become a significant source of revenue for the country and can reduce the country’s reliance on crude oil exports in the future. In Iran, date palm varieties are more than 400 (Zaid and de Wet 2002). Different date palm populations grow in Iran, which shows the tremendous diversity of this plant in terms of size, growth habit, fruit size, fruit shape, and many other morphological characteristics (Ebrahimi, 2022). Anthropogenic selection, clonal propagation, and movement of germplasm by migration have been the primary sources of genetic diversity in date palm trees (Khanam et al. 2012). Scientific findings reportedly showed that human activity has affected biological mechanisms, geographical isolation, and environmental effects on the date palm genome (Chao and Krueger 2007). Estimating the amount of genetic diversity is one of the basic steps in maintaining and protecting genetic material in gene pools and implementing multi-breeding programs (Laurentin 2009). Date palm breeding is difficult due to the long cycle of the juvenile period and high heterozygosity using traditional breeding methods (Bekheet et al. 2008). Therefore, molecular markers can be used as a practical tool closely related to genetic recognition, parentage, strain or cultivars identification, and taxonomic analysis (Alqaisi et al. 2022). Some DNA-based molecular markers have been used in the genetic diversity of date palm. Randomly amplified polymorphic DNA (RAPD) fingerprints were used for identifying date palm accessions in Tunisia (Trifi et al. 2000), Saudi Arabia (Al-Khalifah and Askari 2003), and Egypt (Adawy et al. 2005). Amplified fragment length polymorphic (AFLP) markers were applied to study the genetic diversity of date palm cultivars in Egypt and USA (California) (Adawy et al. 2005; Battesti et al. 2018; Chao and Krueger 2007). Simple Sequence Repeat markers or microsatellites (SSRs) have evaluated genetic diversity and population structures of date palms in Ethiopia (Ahmed et al. 2021). Among the range of molecular markers, Inter Simple Sequence Repeat (ISSR) consists of the amplification of DNA fragments located in the gaps between the two microsatellite repeat regions on either side of the fragment (Ghasemi et al. 2010). Several studies on genetic diversity used ISSR markers on date palms (Ebrahimi 2022; Karim et al. 2010; Khanam et al. 2012; Srivastava et al. 2013; Zehdi et al. 2004). ISSR markers could be highly prioritized in studying genetic diversity and molecular characterization of the date palm germplasm (Abdulla and Gamal 2010; Munshi Osman 2010). This research aimed to assess genetic relationships among sixty-nine date palm genotypes native to Iran and one exotic genotype. The plants were clonally propagated in Iran and their genetic relationships were evaluated by 10 ISSR markers.

Sampling and DNA extraction and qualification

The date palm collection at Azizabad agro-research station (AAS) is a tree collection with a 50-hectare area in the Kerman province of southeastern Iran, located at 58° 54' to 58° 45' E and 28° 56' to 28° 55' N. The altitude of the agro-research station is 710 m above the sea. Date palm genotypes in the AAS are significantly diverse in terms of tree size, growth habit, fruit size, shape, and many other morphological traits. Samples were collected from 14 populations of Phoenix dactylifera from AAS in Kerman Province, Iran (Table 1). They were divided into (a.) cultivars produced by tissue culture, (b.) cultivars with dry and semi-dry fruits, (c.) male rootstocks (stocks), (d.) commercial cultivars, and (e.) native genotypes (Table 1). Young date palm leaf samples were collected from offshoots, cleaned carefully with distilled water to remove the waxy layer, cut into pieces, and stored at - 80°C until DNA extraction. Genomic DNA was extracted from 45 mg of dried leaf tissue using the DNeasy Plant Minikit (Qiagen) following the manufacturer’s instructions. DNA was analyzed by running one μl DNA in 0.8% (w/v) gels in 0.5X TBE buffer (45 mM Tris base, 45 mM boric acid, one mM EDTA pH 8.0). A 100-bp DNA ladder was used for estimating the approximate molecular weight of DNA bands for each PCR product. Electrophoresis was carried out at 100 volts for 1.30 hours. The gels were visualized under UV light by a NanoDrop spectrophotometer. DNA samples that showed a smear in the gel were discarded.

DNA fingerprinting by ISSR markers

A total of 10 ISSR primers from Bioneer Oligi were used for amplifying the isolated DNA in the current research (Table 2). The markers were highly polymorphic and proved effective in fingerprinting Iranian date palm genotypes (Ebrahimi 2022; Hassanzadeh Khankahdani and Bagheri 2019). Polymerase chain reaction (PCR) amplifications were carried out according to Akçali Giachino (2020). Amplification reactions were subjected to a cycle of 94 °C for 5 min, followed by 34 cycles, each of which consisted of denaturation at 94 °C for 50 s, annealing temperature depending on the GC content of the primer (Table 2) for 45 s, and an extension step at 72 °C for 2 min. A final extension was done at 72ºC for 10 min. SSR fragments were separated by electrophoresis using 3% agarose gel in 0.5X TBE buffer. The fragments were visualized with ethidium bromide (1.0μg ml-1) under UV light.

Data analysis

Banding profiles generated by ISSR assays were compiled separately into a data matrix based on the presence of bands (1) or their absence (0). The average number of alleles per locus (Na), adequate allele number (Ne), and allele frequency were also used (Wright, 1984). The Shannon index (I), polymorphic loci (PL%), polymorphism information content (PIC), and expected heterozygosity (He) were estimated using the program software GenAlEx version 6.503. The Bayesian approach analyzed the population structure (Pritchard et al. 2000). Five independent runs were performed, setting the subpopulations (K) from 1 to 10, burn-in time and the Markov Chain Monte Carlo (MCMC) replication number were set to 500,000, as well as a model for admixture and correlated allele frequencies. Delta K (ΔK) represented the K value based on the second-order rate of change in the likelihood (Evanno et al. 2005). Inferred ancestry estimates of individuals (Q matrix) were derived for the selected population (Pritchard et al. 2000). Trait-marker association analysis was performed using TASSEL 2.1, accounting for population structure and kinship relatedness (Q+K model). Kinship coefficients and linkage disequilibrium (LD) were calculated via TASSEL 2.1.

The thirteen native populations of Phoenix dactylifera were considered diverse. They included 69 native Iranian date palm genotypes from the southernmost provinces of Iran. A commercial cultivar was regarded as the fourteenth population, the ‘Madjool’ from Morocco (Table 1). After analysis via ten polymorphic ISSR markers, the primers produced reproducible and scorable patterns. ISSR fingerprints of the 70 Iranian date palm genotypes were produced by the R06 locus (Fig. 1). The number of alleles (NL) varied from 10 at the R06 locus to 5 at DP3, C826, and C842 loci. The highest polymorphism loci percentage (PL = 100%) was observed in the C887 primer, whereas the lowest (PL = 12.5%) was in C826 (Table 2). In a previous study, the genetic diversity of eight Indian date palm genotypes was evaluated via 2 ISSR markers. The highest and lowest PL values were reportedly 40 and 12.5%, respectively (Srivastava et al. 2013). In our study, the polymorphism information content (PIC) varied from 0.30 at the C877 locus to 0.6 at the C842 locus (Table 2). The highest and the lowest PL (84.61-41.66%), PIC (0.43-0.32), and NL (13-8) were reported in a study of 68 Iranian date palm genotypes from various germplasm repositories using 10 ISSR markers (Ebrahimi 2022). An evaluation of genetic relations among 34 cultivars of Iranian date palms by 12 ISSR markers revealed 112 alleles with an average of PL= 95.4 and PIC= 44.9. In the case of the DP3 locus, the NL, PL%, and PIC were 18, 100, and 42.2, respectively. Regarding the DP6 locus, the values were 16, 94, and 49.9, respectively (Hassanzadeh Khankahdani and Bagheri 2019). These results differed from the current results of this research (Table 2). The lower values obtained in the present study could be due to the use of agarose gel electrophoresis for ISSR screening. Agarose gel is non-toxic and safe to handle. However, an automated detection system and polyacrylamide identified allelic variations at a finer scale, compared to the analysis of agarose gel electrophoresis. In previous research on ISSRs, the reason for a more significant number of alleles was the duplication of some loci, resulting in more alleles (Hormaza 2002). The primers showed a maximum number of amplified loci that effectively evaluated date palm genetic variation (Hassanzadeh Khankahdani and Bagheri 2019). Fifteen ISSR primers were used for screening the presence of polymorphisms among 23 Syrian genotypes. The research showed the highest (130) and the lowest number of alleles (40), as well as the average of PL (53%) (Haider et al. 2012). Seven ISSR markers were used for fingerprinting twenty-five date palm cultivars in different regions of Pakistan. The results showed that NL=36 and PL% (50-100), with an average of 83.57% (Mirbahar et al. 2016). Younis et al. (2008) observed that PL=87% using 20 ISSR primers on seven Egyptian date palm cultivars. Genetic diversity and relatedness among six Egyptian date palm genotypes showed that PL=73% (Moghaieb et al. 2010). Adawy et al. (2005) observed that PL=80.4%. Karim et al. (2010) observed that PL=82% in Egyptian and Tunisian date palm cultivars, respectively. Using seven primers, Hamza et al. (2012) observed that PL=80% in date palm cultivars. The genetic diversity of Ethiopian date palm cultivars was evaluated using 10 ISSR markers, indicating PL values (85.7–100%) and PIC (0.3–0.37) (Takele et al. 2021). However, these results are not in harmony with the present study. The average PL in our ISSR analysis was lower than that observed in the ISSR of previous studies (Adawy et al. 2005; Younis et al., 2008; Haider et al. 2012; Hamza et al. 2012; Karim et al. 2010; Mirbahar et al. 2016; Moghaieb et al. 2010; Takele et al. 2021). Differences in the results might be due to a higher number of primers in their studies. Another reason is that all date palm genotypes in our research may be assumed to have a shared genetic base. Nevertheless, cultivars move away from each other by reciprocal crosses that occur in different generations.

The solution is to use specific markers in the separation of genotypes and sexual propagation instead of the common use of offsets. In the genetic characterization of populations in this research, the mean values of Shannon’s information index (I), number of different alleles (Na), number of effective alleles (Ne), expected heterozygosity (He), and percentage of polymorphic loci (PL) were 1.29, 1.25, 0.21, 0.14, and 34.73%, respectively (Table 3). The data pertaining to the single Moroccan genotype was not included in Table 2 because only the data that have a high level of reproducibility should be used in the STRUCTURE software. This genotype was left out because of its non-repetition. The minimum value defined for (I) is zero, which indicates the absence of diversity, but in values less than 1, the genetic diversity is considered relatively low (Shannon 1948). It indicates the low genetic richness and the shared genetic background of the date genotypes in this research. Constructing similarity matrices and dendrograms is a useful tool for studying genetic diversity. Among the different methods, the estimation of genetic distances among the populations, according to Nei (1973), was calculated mostly for this purpose (Ahmadi et al., 2013; Ershadi & Farrokhi Toolir, 2022). The identification of genetic distances between the studied populations showed the lowest correlation between Bushehr and Fars (r = 0.799) and Bushehr and Morocco (r = 0.799) genotypes (Table 4). The highest correlations between Hormozgan and Kerman (r = 0.983, p < 0.05) was followed by Hormozgan and PGB (r = 0.927, p < 0.05), and then Kerman and PGB (r = 0.921, p < 0.05) (Table 4). The Hormozgan and PGB populations exist in the Hormozgan province, the southern extremity of Iran. This province is influenced by dense fog and high humidity from the Oman Sea. Its climatic conditions are more diverse than the other provinces. Significant climatic differences were evident along an altitudinal gradient, resulting in a shortened plant lifespan and delayed pollination, which affected gene flow and led to genetic differentiation among plant populations (Ershadi and Farrokhi Toolir 2022). Interspecific hybridization may also affect the genetic structure along altitudinal gradients (Wong et al. 2022). Overall, alleles of some genotypes may introgress into other genotypes, causing a genetic cline at specific loci along elevational gradients. Such phenomena may also affect the distribution of genetic diversity within populations, as the inflow of new alleles may increase allelic richness. According to the dimensional equations obtained from principal coordinate analysis (PCoA), 14 populations from seven origins were divided into four main groups (Table 1, Fig. 2). A high level of genetic similarity was observed among Kerman, Hormozgan, Khuzestan, and PGB populations, whereas a low similarity was observed among them, the Fars population and the Moroccan cultivar. The Fars, Morroco and Bushehr populations were classified as three separate groups (Fig. 2). The accumulation of populations at one point of the plot indicated their genetic similarity among the genotypes (Ebrahimi 2022). In PCoA, an optimal condition is when the markers have a uniform and proper distribution in the genome and can be said to represent the entire genome (Hassanzadeh Khankahdani and Bagheri 2019). The obtained information indicated that the ISSR marker primers in this research have good dispersion at the genome level. Genotypes of populations with the highest inter-cluster distance may be used in hybridization programs. In contrast, lower inter-cluster distances indicate close relationships and similarity among the genotypes. Selecting parents from these clusters should be avoided (Srivastava et al. 2013). Grouping the genotypes based on ISSR characteristics showed that genotypes in the same region could be grouped separately, regardless of their geographic origin. This finding has been confirmed in previous studies on Iranian date palms (Ebrahimi 2022; Hassanzadeh Khankahdani and Bagheri 2019; Taghinejad et al. 2018). Similar to our findings, five geographically distant populations were in the same group in a study on the genetic variation of desert date (Balanites aegyptiaca Del.) (Mint Abdelaziz et al. 2020). Hamza et al. (2012) reported a grouping of date palm cultivars according to their fruit characteristics. The PCoA led to the grouping of soft or semi-soft fruits and early or mid-season maturity. From the PCoA analysis, we concluded that male rootstocks from the Bam region of Kerman province and tissue culture stocks from Khuzestan and PGB were more distinct from other genotypes. Thus, they were placed in a separate group (Table 1, Fig. 2). The genetic diversity of 15 date palm cultivars were evaluated in Qatar using the Jacquard similarity coefficient. The results showed that the highest similarity was between Shishai and Gahsab (Zahidi), as well as between Khamin and Abu Ma’an cultivars (Ahmed and Al-Qaradawi 2009). The ‘Abu Ma’an’ from PGB and the ‘Gahsab’ from Kerman were grouped in one separate group, according to our findings (Table 1, Fig. 2). However, these results may further be confirmed with more genotypes and primers. According to Evanno’s method, seven optimal K values pertaining to the populations were selected (Evanno et al. 2005). Therefore, the genotypes were divided into seven subpopulations based on the threshold of 0.75 (Fig. 3 a). The numbers on the vertical axis showed the membership coefficient of each individual in the subpopulations. Genotypes with the same color were attributed to the same subpopulation (Fig. 3 b). According to Q matrices, sixty genotypes (85.71%) had a probability lower than the threshold. Thus, they were regarded as mixed populations (Table 1, Fig. 3 b). One of the reasons for the formation of mixed masses is the occurrence of gene flow between accessions of different regions (Ebrahimi 2022). Some factors, such as the transfer of pollen grains by wind and human activities, as well as migration or common ancestry, can be the reasons for the creation of different accessions in these populations (Mousavi Deraz Mahalle et al. 2014). According to the model-based clustering for the genetic structure of date palm genotypes, seven genetically distinct subpopulations were presented. However, they were not presented in line with their geographical location. Our results displayed that the Iranian populations have a common genetic background and shared alleles. The most differentiated genotypes were observed in the Kerman region, which showed fewer admixtures than the other regions. Generally, the structure showed a shared ancestry between date palm genotypes and early adopted cultivars (Ahmed et al. 2021). Elshibli and Korpelainen (2011) observed a high genetic diversity within the Sudan date palm resource groups. The weak clustering of the cultivars suggested that they did not result from a full cloning process. The ease and rapidity of seed reproduction, coupled with their large availability, support the maintenance of this practice among farmers. Thus, date palm plantations are a mixture of plants, either clonally propagated or seed propagated, with a high genetic variability within the cultivars (Elmeer et al. 2019; Elshibli and Korpelainen 2011). In our study, clonal propagation was mainly done by offshoots that guaranteed genetic uniformity among the individuals of a population. This also led to the lower genetic diversity of the genotypes. Seventy genotypes were analyzed based on 4 ABCDs from 14 populations in 7 regions. However, the ISSR marker can be useful as a suitable marker for the differentiation and analysis of the population structure for date palm genome studies. According to genetic structure population indices, most genotypes had mixed genetic backgrounds, implying a common genetic background and shared alleles between them. However, in this study, three male stocks, no. 22, 28, 27, had values above 0.75 and they were grouped together, whereas the other genotypes were dispersed (Table 1). Since date palm is a dioecious plant, regions where the stocks combine indicate the mixing of genomes. In this study, the clustering pattern of genotypes was independent of their geographical origin. In summary, the analysis of ISSR markers of the date palm genotypes documented the significant variation in situ among the regions. It confirmed the need to conserve this valuable genetic resource. Since the 70 genotypes were genetically close, it is suggested that additional research be aimed at using specific markers such as SSRs or SNPs to better differentiate between the genotypes.

Seventy genotypes were analyzed based on 4 ABCDs from 14 populations in 7 regions. However, the ISSR marker can be helpful as a suitable marker for the differentiation and analysis of population structures. According to the genetic structure indices, most genotypes had mixed genetic backgrounds, implying a common ancestry and shared alleles. However, in this study, three male stocks, no. 22, 28, 27, had values of more than 0.75. These three genotypes were grouped together, whereas the other genotypes were dispersed. Since date palm is a dioecious plant, the mixing of genomes probably occurred where the stocks had similarities. In this study, the clustering pattern of genotypes was independent of their geographical origin. In summary, the analysis of ISSR markers of the date palm genotypes documented a significant variation in situ and confirmed the need to conserve this valuable genetic resource. Since the 70 genotypes were genetically close, it is suggested that additional research be aimed at using specific markers such as SSRs or SNPs to better differentiate between the genotypes.

Acknowledgments

The current research was part of the Ph.D. thesis of Fatemeh Khorshidi. The research was carried out at Kerman Islamic Azad University. The authors thank this university for providing financial support in this study. Also, we thank AREEO, Kerman branch, for providing laboratory facilities, Dr. Hamid Hatami Maleki, University of Maragheh, for sharing his knowledge and experience.

Conflict of interest

The authors acknowledge that there are no financial or competing interests in this research.

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Table 1 Name, origin, and pertinent Q-values of the date palm genotypes

				Q matrix
Pop.	No.	Name	Region (location)	Q1	Q2	Q3	Q4	Q5	Q6	Q7	Group
A	1	Berhi	Khuzestan	0.663	0.09	0.085	0.039	0.047	0.03	0.046	Mixed
	2	Ashrasi	Khuzestan	0.458	0.128	0.122	0.105	0.077	0.062	0.048	Mixed
	3	Kush zabad	Persian Gulf Basin	0.03	0.072	0.112	0.23	0.164	0.081	0.311	Mixed
	4	Abu Maan	Persian Gulf Basin	0.175	0.05	0.289	0.097	0.239	0.048	0.103	Mixed
B	5	Abu narenja	Persian Gulf Basin	0.042	0.069	0.294	0.027	0.413	0.021	0.134	Mixed
	6	Nabat seif	Persian Gulf Basin	0.134	0.04	0.263	0.04	0.333	0.061	0.128	Mixed
	7	Khalas	Persian Gulf Basin	0.017	0.26	0.195	0.051	0.289	0.018	0.169	Mixed
	8	Fard	Persian Gulf Basin	0.023	0.071	0.204	0.093	0.365	0.063	0.181	Mixed
	9	Ghasb	Kerman (Shahdad)	0.29	0.035	0.156	0.2	0.168	0.048	0.101	Mixed
	10	Touri	Hormozgan (Hajjiabad)	0.11	0.031	0.072	0.067	0.041	0.632	0.047	Mixed
	11	Sangshekan	Kerman (Bam)	0.331	0.047	0.064	0.25	0.119	0.027	0.163	Mixed
	12	Diri	Hormozgan (Minab)	0.905	0.021	0.016	0.014	0.017	0.011	0.016	Red
	13	Deglet nour	Persian Gulf Basin	0.912	0.017	0.018	0.011	0.017	0.014	0.011	Red
C	14	Estameran	Khuzestan	0.068	0.039	0.082	0.41	0.145	0.02	0.237	Mixed
	15	Zahedi	Bushehr	0.11	0.122	0.097	0.31	0.131	0.054	0.176	Mixed
	16	Piyarom	Hormozgan (Hajjiabad)	0.214	0.029	0.183	0.222	0.209	0.023	0.12	Mixed
	17	Shahabi	Hormozgan	0.059	0.197	0.076	0.314	0.098	0.032	0.225	Mixed
	18	Medjool	Morocco	0.052	0.034	0.048	0.035	0.037	0.765	0.029	Mixed
	19	Male stock	Kerman (Bam)	0.055	0.425	0.219	0.033	0.151	0.022	0.095	Mixed
	20	Male stock	Kerman (Bam)	0.047	0.642	0.112	0.037	0.051	0.058	0.053	Mixed
	21	Male stock	Kerman (Bam)	0.016	0.846	0.029	0.022	0.022	0.016	0.049	Green
	22	Male stock	Kerman (Bam)	0.023	0.784	0.036	0.023	0.047	0.02	0.066	Green
	23	Male stock	Kerman (Bam)	0.038	0.554	0.112	0.064	0.112	0.034	0.086	Mixed
	24	Male stock	Kerman (Bam)	0.021	0.889	0.017	0.019	0.018	0.016	0.02	Green
D	25	Male stock	Kerman (Bam)	0.103	0.292	0.199	0.125	0.103	0.083	0.096	Mixed
	26	Male stock	Kerman (Bam)	0.014	0.892	0.021	0.016	0.017	0.019	0.021	Green
	27	Male stock	Kerman (Bam)	0.033	0.113	0.023	0.015	0.022	0.778	0.016	Pale blue
	28	Male stock	Kerman (Bam)	0.034	0.79	0.03	0.065	0.038	0.013	0.03	Green
	29	Zardooni	Kerman (Bam)	0.026	0.042	0.041	0.42	0.078	0.036	0.356	Mixed
	30	Kelk sorkh	Kerman (Kahnuj)	0.528	0.041	0.063	0.142	0.081	0.048	0.097	Mixed
	31	Mehmooni	Kerman (Kahnuj)	0.089	0.026	0.234	0.146	0.17	0.13	0.205	Mixed
	32	Fareh kam	Hormozgan (Hajjiabad)	0.036	0.079	0.295	0.181	0.246	0.056	0.106	Mixed
	33	Al mehtari	Kerman (Jiroft)	0.223	0.026	0.105	0.037	0.057	0.523	0.03	Mixed
	34	Halileie	Fars (Lar)	0.037	0.024	0.051	0.022	0.024	0.822	0.02	Pale blue
	35	Kharak	Kerman (Kahnuj)	0.015	0.064	0.131	0.355	0.168	0.026	0.24	Mixed

Table 1 Continued

Pop.	No.	Name	Region (location)	Q matrix							Group
Pop.	No.	Name	Region (location)	Q1	Q2	Q3	Q4	Q5	Q6	Q7	Group
D	36	Amme shanbe	Kerman (Kahnuj)	0.014	0.057	0.269	0.066	0.092	0.412	0.089	Mixed
	37	Gerd daneh	Hormozgan (Minab)	0.027	0.161	0.216	0.25	0.18	0.02	0.148	Mixed
	38	Negar	Kerman (Jiroft)	0.047	0.71	0.05	0.056	0.039	0.034	0.064	Mixed
	39	Zardak	Hormozgan (Roodan)	0.025	0.035	0.211	0.214	0.303	0.044	0.167	Mixed
	40	Nozohur	Kerman (Jiroft)	0.036	0.022	0.156	0.059	0.112	0.555	0.06	Mixed
	41	Mazafati	Kerman (Bam)	0.036	0.027	0.094	0.332	0.201	0.026	0.284	Mixed
	42	Tokhmmorghi	Hormozgan (Hajjiabad)	0.147	0.215	0.114	0.133	0.134	0.105	0.151	Mixed
	43	Seloni	Kerman (Kahnuj)	0.05	0.03	0.102	0.404	0.195	0.031	0.186	Mixed
	44	Lasht	Kerman (Kahnuj)	0.019	0.021	0.324	0.15	0.205	0.056	0.224	Mixed
	45	Nobar	Hormozgan (Fin)	0.018	0.062	0.124	0.242	0.21	0.027	0.316	Mixed
	46	Posht nabi	Hormozgan (Fin)	0.022	0.026	0.126	0.32	0.123	0.033	0.35	Mixed
	47	Shahani	Kerman (Orzuiyeh)	0.039	0.446	0.082	0.125	0.134	0.046	0.129	Mixed
	48	Najmeh	Kerman (Kahnuj)	0.072	0.015	0.212	0.079	0.151	0.33	0.14	Mixed
	49	Karout	Kerman (Bam)	0.608	0.02	0.061	0.125	0.041	0.083	0.062	Mixed
	50	Bazmani	Kerman (Shahdad)	0.192	0.056	0.183	0.145	0.177	0.05	0.196	Mixed
	51	Zeinabi	Kerman (Kahnuj)	0.081	0.031	0.13	0.166	0.085	0.284	0.223	Mixed
	52	Lilangouni	Kerman (Kahnuj)	0.027	0.046	0.235	0.138	0.337	0.059	0.158	Mixed
	53	Peirizi	Fars (Lar)	0.026	0.027	0.148	0.309	0.163	0.03	0.297	Mixed
	54	Gorbehie	Kerman (Jiroft)	0.472	0.05	0.095	0.146	0.076	0.049	0.111	Mixed
	55	Farz	Kerman (Kahnuj)	0.023	0.057	0.188	0.063	0.124	0.458	0.086	Mixed
	56	Maktoub	Hormozgan (Roodan)	0.165	0.018	0.123	0.054	0.063	0.538	0.039	Mixed
	57	Souzdan	Kerman (Jiroft)	0.123	0.009	0.151	0.247	0.197	0.115	0.158	Mixed
	58	Yaghuti	Kerman (Bam)	0.395	0.023	0.082	0.197	0.099	0.055	0.149	Mixed
	59	Khosh kang	Kerman (Kahnuj)	0.028	0.024	0.091	0.387	0.251	0.017	0.202	Mixed
	60	Nesfei	Hormozgan (Roodan)	0.019	0.013	0.272	0.076	0.147	0.343	0.13	Mixed
	61	Halileie	Kerman (Shahdad)	0.058	0.034	0.321	0.082	0.276	0.024	0.204	Mixed
	62	Gorbani	Kerman (Kahnuj)	0.032	0.019	0.094	0.342	0.108	0.034	0.371	Mixed
E	63	Kabkab	Bushehr	0.05	0.022	0.178	0.196	0.296	0.017	0.241	Mixed
	64	Mordaseng	Kerman (Orzuiyeh)	0.047	0.294	0.096	0.16	0.159	0.031	0.214	Mixed
	65	Golami	Hormozgan (Roodan)	0.037	0.023	0.106	0.18	0.096	0.308	0.249	Mixed
	66	Sahlaki	Kerman (Kahnuj)	0.024	0.017	0.075	0.33	0.134	0.014	0.407	Mixed
	67	Zohrei	Kerman (Kahnuj)	0.017	0.025	0.073	0.267	0.069	0.252	0.297	Mixed
	68	Makou	Kerman (Kahnuj)	0.016	0.039	0.089	0.29	0.106	0.144	0.317	Mixed
	69	Khasouei	Kerman (Kahnuj)	0.017	0.041	0.04	0.036	0.026	0.795	0.044	Pale blue
	70	Kheli get	Kerman (Kahnuj)	0.063	0.033	0.081	0.309	0.107	0.079	0.328	Mixed
Pop. = Population, A= cultivars produced by tissue culture, B= cultivars from dry and semi-dry fruits, C= male rootstocks (Stocks), D= commercial cultivars and E= native genotypes)

Table 2 List of the primers and the complementary information of the ISSR assay

Primer	Sequence** (5′--->3′)	At¹ (°C)	GC ratio (%)	NL²	PL³ (%)	ML⁴ (%)	PIC⁵ (%)
C840	5 ́ -GAG AGA GAG AGA GAG AYT-3 ́	47	44.47	8	87.50	12.50	26
DP3	5 ́ - AGA GAG AGA GAG AGA CYA-3 ́	53	52.38	5	80.0	20.0	29
C826	5 ́ - ACA CAC ACA CAC ACA CC-3 ́	51	52.94	5	80.0	20.0	10
R06	5 ́ -GAG AGA GAG AGA GAG AC-3 ́	48	52.94	10	80.0	20.0	17
C842	5 - GAG AGA GAG AGA GAG AYG-3 ́	48	44.44	5	60.0	20.0	16
C888	5 ́ - * BDB CAC ACA CAC ACA CA-3 ́	53	41.18	6	66.6	34.4	20
C835	5 ́ - DVD TCT CTC TCT CTC TC-3 ́	51	44.45	6	100	00.0	20
DP6	5 ́ -VDV CTC TCT CTC TCT CT-3 ́	54	52.38	7	42.85	57.15	20
C842	5 ́ - GAG AGA GAG AGA GAG AYC-3 ́	48	50	6	16.66	83.34	6
C877	5 ́ -TCT CTC TCT CTC TCT CG-3 ́	48	41.18	7	85.71	14.29	30
Total				65	-	-	-
Mean				-	69.93	23.78	19.4
¹Annealing temperature, ²Number of alleles per locus, ³Polymorphic loci, ⁴Monomorphic loci, ⁵Polymorphism information content. Bioneer Oligi synthetic report B= T, G or C; Y= T or C;V= G, C or A; D= T, G or A *(Haider et al., 2012; Hassanzadeh Khankahdani & Bagheri, 2019; Marsafari & Mehrabi, 2013)

Table 3 Genetic population characterization in date palm accessions from different populations produced by 10 ISSR loci

Region	Na¹	Ne²	I ³	He⁴	PL ⁵(%)
Bushehr	1.15	1.15	0.13	0.08	21.54%
Fars	1.24	1.19	0.16	0.11	27.69%
Hormozgan	1.60	1.47	0.37	0.26	60.00%
Kerman	1.67	1.49	0.40	0.27	67.69%
Khuzestan	1.30	1.23	0.19	0.13	32.31%
Persian Gulf Basin	1.32	1.25	0.20	0.13	33.85%
Grand Mean over Loci and regions	1.299	1.258	0.210	0.145	34.73%
¹Number of alleles, ²Number of effective alleles, ³Shannon’s information index, ⁴Expected Heterozygosity, ⁵Percentage of polymorphic loci

Table 4. Estimation of genetic distances among the populations using the correlation matrix (Nei 1973)

	Bushehr	Fars	Hormozgan	Kerman	Khuzestan	Morocco	¹PGB
Bushehr	1.000
Fars	0.799	1.000
Hormozgan	0.890	0.899	1.000
Kerman	0.888	0.900	0.983*	1.000
Khuzestan	0.855	0.846	0.908	0.918	1.000
Morocco	0.799	0.867	0.853	0.864	0.815	1.000
PGB	0.864	0.869	0.927*	0.921*	0.890	0.828	1.000
¹PGB = Persian Gulf Basin = Correlations significant at P<0.05*

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Evaluation of diversity in selected populations of Iranian date palm (Phoenix dactylifera L.) using ISSR markers

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