Determination of Genetic Relations among Tomato Accessions in Sulaymaniyah Region through ISSRs Markers Genetic Relations in Tomato Accessions

: The goal of this study was to examine the genetic difference among thirty two tomato accessions ( Solanum lycopersicum L.) grown in Sulaymaniyah using ISSR molecular markers technique. The results of ISSR markers revealed 65 amplified fragments, 50 of them were polymorphic from using 15 primers. Fifteen ISSR markers used to detect DNA polymorphism gave polymorphism percentage for each primer range between 50–100% with an average polymorphism percentage reaching 75.61%. ISSR6 and Sola 11 gave the highest polymorphism percentage was 100%, while 3F, Sola 5 and Sola 12 did not give any amplification. The mean of PIC (Polymorphic Information Content) value was 0.50 for ISSR markers. The similarity matrix was obtained by using Jaccard’s coefficients, based on polymorphic bands and dendrogram constituted through UPGMA cluster analysis. The dendrogram revealed 4 main groups. Genetic similarity (GS) ranged from 0.261 to 0.941 within studied accessions. The highest


Introduction
Tomato cultivation has always been a very important part of agriculture in most countries of the world. It is an important source of vitamin. The tomato genetic resources are vital for conventional and molecular breeding, and determination of their genetic differences has valuable potential in the tomato industry. Tomato also has been used as model plant species in studies such as the physiology and biochemistry of seed development, germination and dormancy (Suhartanto, 2002). There have been carried out many studies to evaluate the genetic variety of tomato by morphological, biochemical and molecular markers (Abak et al., 1996;Powell et al., 1996;Tam et al., 2005;Terzopoulos and Bebeli, 2008;Bayram et al., 2011;Vishwanath et al., 2011).
The application of molecular markers in plant breeding programs facilitates the improvement of many crop species (Williams, et al., 1990). Nowadays, several molecular markers are developed, of which Inter Simple Sequence Repeats (ISSRs) are one of the widely used types. ISSRs molecular markers that enhance regions between microsatellite loci does not need any information about the sequences to be amplified and shows high polymorphism in the material, being exceptionally helpful in investigations of genetic variation, phylogeny, and genomics (Reddy et al., 2002). This marker (ISSR) technique can handle the above limitations (Pharmawati et al., 2004;Reddy et al., 2002). The ISSR markers have been studied by several researchers for molecular characterization of many plant species such as tomato (Kamel et al., 2010), rice bean (Muthusamy et al., 2008), common bean (Erdinc et al., 2017;Ekincialp and Sensoy, 2018), melon (Erdinç et al., 2013) and coffee (Masumbuko and Bryngelsson, 2006).
The evaluation of genetic diversity within and between populations of tomato landraces is estimated by using morphological, biochemical and molecular characterization (Antonio et al., 2004). In recent years, an important decrease of genetic variability was observed in especially several fruit quality traits of the cultivated tomato (Foolad, 2007). Tomato is one of the economic important crops in Iraq and there are local tomato accessions. It is important that bringing out genetic relations among these accessions for next breeding studies. Therefore, this study was aimed to assess the genetic variety and relationship of 32 tomato accessions locally collected from different regions in from Sulaymaniyah governorate as revealed by ISSR markers.

Material
Thirty two tomato landraces (Solanum lycopersicum L.) accessions collected from different regions province of Sulaymaniyah-Iraq was used in this study (Table 1).

Methods
For genomic DNA isolation, seedlings were grown in a growth chamber at a temperature of 24°C with a 16/8 h day/night photoperiod. Each replicate included 10 seeds per accession. Genomic DNA was extracted in bulk from young fresh leaves employing the CTAB procedure with minor modifications (Doyle and Doyle, 1987). DNA was quantified by Nano Drop, ND 100 spectrophotometer (Nano Drop Technologies, Inc.). DNA were diluted in water to a final concentration of 50 ng and stored at -20°C.
Fifteen primers of ISSR were utilized in our study is shown in Table 2. The PCR amplification was performed using a 25μl mixture contained the following components: 8.7μl of sterile ddH2O, 1X Taq buffer, 2.5 mM of MgCl2 (25mM), 0.6 mM of primer , 0.1 mM of dNTPs, 1 unit of Taq polymerase and 50 ng of template DNA. PCR amplification included a denaturing stage at 94 ˚C for 3 minutes, followed by 35 cycles denaturing step at 94 ˚C for 20 s, an annealing step at a temperature according the melting temperature of each primer (Table 2) for 40 s and an extension step at 72 ˚C for 1 minute. After the last cycle the samples were kept for 10 min at 72 ˚C (Terzopoulos and Bebeli, 2008).
PCR products were electrophoresed on 1.5% agarose gel in 1X TAE buffer at 120 V for 2.5 h. Amplified products were photographed using an Imager Gel Doc XR system (Vilber Lourmat, QUANTUM ST4).

Data analysis
Reproducible and legible bands were scored as 1 (existence) and 0 (absence) according to binary system. Genetic distance among tomato accessions was calculated by using Jaccard similarity coefficients and UPGMA method was used with the similarity coefficient to construct the dendrograms showing genetic diversity with software PAST3. Also, basic coordinate analysis (PCoA) was performed with the same program. The PIC (polymorphic information content) was calculated using the following standard formula (Powell et al., 1996;Smith et al., 1997). According to the "fi" explain the markers frequency in data set.

Results and Discussion
In this study, fifteen of the eighteen ISSR primers produced legible band. Total 65 DNA fragments were detected from ISSR primers ( Table 2) and 50 of them were polymorphic. While the mean of polymorphic band number was 3.33, the highest polymorphic band was obtained from primer 816 (7.00). Primer 10F gave the lowest polymorphic band number (1.00). Polymorphism level was calculated for the fifteen ISSR primers and polymorphism was range between 50-100% and the average polymorphism was 75.61%. Primer 10F gave the lowest polymorphism with 50% while the highest polymorphism was 100% in ISSR6 and Sola 11. The mean of PIC was 0.50 and PIC value of ISSR primers was ranged 0.06-0.95. While PHV6 had the lowest PIC (0.06), primer 10F showed the highest PIC value with 0.95. The high degree of polymorphism and the number of polymorphic bands obtained per assay showed that ISSR is one of the most informative marker systems for tomato genotyping as well as other crop species (Erdinç et al., 2013;Erdinc et al, 2017;Ekincialp et al., 2019;Topaklı and Hepaksoy, 2019) . The highest percentage of polymorphism was obtained from primer ISSR6 and Sola 11 that is 100%, while the lowest percentage of polymorphism was from primer 10F. All of the ISSR primers gave a good percentage of polymorphism. ISSR markers used in this study were mostly polymorphic with a total 50 numbers polymorphic, while the total polymorphic was 81 in another experiment (Todorovska et al., 2014). (Terzopoulos and Bebeli, 2008) obtained 57.8% polymorphism and 59 polymorphic bands from 12 ISSR primers in Greek tomato landraces. (Tikunov et al., 2003) found a polymorphism level between 60 to 89%, while (Suliman-Pollatschek et al., 2002) reproduced the polymorphism as 65%. Alternatively, (Meng et al., 2010) found more polymorphic bands ranged from 4 to 18 with an average of 7.91. There are some studies in tomato which gave lower average number of bands and polymorphism level. (Figueiredo et al., 2016) found in 8 ISSR primers 2.62 numbers of polymorphic band and 27.62% polymorphism level. (Aguilera et al., 2011) obtained 34.02% polymorphism level, while (Shahlaei et al., 2014) found 2.5 numbers of polymorphic band and 8.84% polymorphism level. Most of ISSR primers gave high degree polymorphism (over 70%) and were successful to amplify the accessions of tomato visibly. Some of ISSR primers used to this research gave same percentage of polymorphism in other studies. (Kochieva et al., 2002;Korir et al., 2014), while (Vargas-Ponce et al., 2011;Henareh et al., 2016) obtained 100% polymorphism level from all primers they used. Generally polymorphic information content (PIC) of ISSR primers was high and 7 primers had over 60% PIC. There are some studies which ranged from 0.06 to 0.64 and 0.09 to 0.67, respectively (Bredemeijer et al., 1998;He et al., 2003). Furan and Geboloğlu, (2017) and Erdinc et al., (2017) found PIC values of ISSR primers between 0.28-0.44 and 0.15-0.50, respectively.
The similarity index matrix was obtained to compare genetic variation among the 32 tomato accessions using ISSR primers (Table 3). Jaccard similarity coefficient of accessions based on fifteen ISSR markers ranged from 0.261 to 0.941. The most different tomato accessions were G15 (0.370), G28 (0.538) and G9 (0.630), while the most similar tomato accessions were G11 (0.768), G17 (0.767) and G20 (0.766). The lowest genetic distance was observed among pair of accessions G15-G24 and G15-G26 (0.261) and G15-G30 (0.269), while the highest genetic distance was noted in G7-G16 (0.941), G27-G29 (0.933) and G20-G27 (0.930). Cluster analysis was performed to determine genetic relationships among 32 tomato accessions using pair-wise genetic similarity values by UPGMA based on Jaccard coefficient. According to genetic distance matrix dendogram (Figure 1) divided into four YYU J AGR SCI 30 (4): 810-820 Ibrahım and Erdınc / Determination of Genetic Relations among Tomato Accessions in Sulaymaniyah Region through ISSRs Markers Genetic Relations in Tomato Accessions 814 main groups: First group (Branch I) and the second group (Branch II) included only one accession (respectively G15 and G28). Branch III had G1 and G9 accessions. Branch IV contained 28 accessions and this group was also divided into subgroups. (Thamir et al., 2014), found that genetic similarity ranged from 0.229 to 0.946 among 19 Iraq tomato accessions and they divide into two main groups. Similarly, SSR markers were used to determine genetic relationships among tomato accessions in Iraq and genetic similarity coefficient was between 0.324-0.917 (Al-Tamimi et al., 2015). While (García-Martínez et al., 2006) had close similarity indices among their tomato accessions in another study the values of pair-wise genetic distances ranged from 0.1838 -0.9049, indicating the attendance of extensive genetic diversity (Tabassum et al., 2013). (Sharifova et al., 2013) had also obtained dendrogram for discrimination among tomato accessions with 0.188-1.000 similarity coefficient. (Terzopoulos and Bebeli., 2008) found genetic similarity values ranged from 0.56 to 0.95 with an average of 0.797 among 41 tomato accessions. (Aguilera et al., 2011) studied 96 tomato accessions by ISSR markers and genetic distance ranged from 0 to 0.25. In addition, they pointed out that this marker system has a high efficiency to distinguish accessions. In another different study, 12 modern tomato varieties were used to assign genetic relation. There was obtained Jaccard's coefficient between 0.12-0.88 and varieties were distinguished to 4 main clusters in UPGMA based dendrogram (Kiani and Siahchehreh, 2018). PCoA cluster analysis discriminated 32 tomato accessions into 4 different groups (Group A, Group B, Group C and Group D) (Figure 2). Similarly, UPGMA-based analysis, G15 and G 28 accessions generated a single group in the PCoA analysis (Group A and Group B). While G1 and G9 took part in Group C the other accessions constituted Group D. When two cluster analysis methods were compared the results indicate that groups in PCoA and branches in UPGMA consist of the same accessions (Figure 1 and 2). It was observed that the results obtained from PCoA appropriate to the results with the UPGMA-based cluster method.
815 Table 3. The similarity index by using Jaccard similarity coefficients   G1  G2  G3  G4  G5  G6  G7  G8  G9  G10  G11  G12  G13  G14  G15  G16  G17  G18  G19  G20  G21  G22  G23  G24  G25  G26  G27  G28  G29  G30  G31  G32  G1 1 STRUCTURE analysis performed to examine population structure the most probable K value was 2 (Figure 4). According to this K value, the tomato accessions were separated into 2 subpopulations (Figure 3). The first subpopulation included 18 accessions, while the second subpopulation comprised 14 accessions. (Henareh et al., 2016) assessed the genetic variation of Turkey and Iran tomato accessions. They determined two sub-populations by STRUCTURE analysis and genetic variation in sub-population 2 was higher than sub-population 1. When membership coefficient in individual of a subpopulation is 0.8 or higher, this individual is accepted as pure. Lower membership coefficients are accepted as mixed of at least two different subpopulations (Fukunaga et al., 2005). In our study, 28 accessions had 0.8 or higher membership coefficient; therefore, these accessions can be said to be pure.

Conclusions
In the present study, the ISSR marker system was used to put forward the genetic diversity and population structure that can conceivably be applied for selection of proper parents to present greater genetic variation in tomato breeding programs and help the breeders in the tomato. ISSR markers gave high polymorphism rate and genetic variation was determined in tomato accessions. This study reveals that the ISSR marker system is a helpful and easy method for fingerprinting and distinguishing tomato accessions because of its easy using and reliability. It was thought that analysis of these accessions will lead to the collection of information about the genetic diversity at the genome level; also it is believed that this studied tomato gene pool will be a significant genetic resource in next breeding studies and will contribute the facility of germplasm management. For subsequent studies, the detailed identification of these genotypes in terms of phenotypic and molecular properties becomes important for future breeding studies.