Molecular Evaluation of Genetic Variability in Tomato ( Lycopersicon esculentum Mill . ) Genotypes by Microsatellite Markers

The objective of this research was to assess genetic diversity using eight microsatellite markers in 30 tomato genotypes from the collection of the Institute of Field and Vegetable Crops in Novi Sad. The SSR markers were selected from publicly available data and Solanaceae Genome Network database. Genotypes were grouped into three clusters, using Ward`s hierarchical clustering method and Euclidean distance measure. Markers SSR248, TMS9, TMS42 and SSR111 had very high PIC (Polymorphism information content) values and can be recommended for the future studies.


Introduction
Harvested areas, quantity production and human consumption provided an important place for tomato (Lycopersicon esculentum Mill.) among other vegetable plants.According to FAO statistical data (Food and Agriculture Organization of the United Nations 2011), the global harvested area of tomato was 4,751,530 ha in 2011.Although derived from highly variable natural habitat, a huge part of genetic variability was lost due to inbreeding during tomato domestication and intensive artificial selection.A huge number of tomato varieties and hybrids were developed by tomato breeders with the aim to overcome existing genotypes in terms of yield, resistance to biotic and abiotic stress and fruit quality.Access to divergent breeding material is necessary for achieving these objectives.Despite the highly diverse natural habitat of the genus Lycopersicon that resulted in great variability of its species, cultivated tomato has a very narrow genetic base.Assessment of genetic diversity in any crop species provides a basis for devising future strategies for crop improvement, conservation and sustainable use (Yi et al. 2008).Molecular markers represent useful tool in these types of studies.Simple sequence repeats (SSRs), also known as microsatellites, have very important role in molecular research having high reproducibility, multi-allelic nature, co-dominant inheritance, high abundance and wide genome coverage (El-Awady et al. 2012).Microsatellites are widely used in studies in different plant species (Stich et al. 2006, Cadalen et al. 2010, Brbaklić et al. 2010, Trkulja et al. 2011, Shah et al. 2013).Numerous studies have pointed to the efficacy of SSR markers for determination of genetic diversity in the genus Solanum (Alvarez et al. 2001, He, Poysa, & Yu 2003, Frary et al. 2005, Garcia-Martinez et al. 2006, Hu et al. 2012).As a consequence of current breeding strategies, diverse local populations and old cultivars are suppressed by modern, high yielding cultivars, uniform with a narrow allelic variability at agronomically important loci.Since the loss of specific alleles means loss of opportunities to develop new and improve existing varieties and hybrids, the aim of this research was to evaluate genetic variability of 30 tomato genotypes using eight SSR markers.

Materials and Methods
Thirty genotypes from tomato collection of the Institute of Field and Vegetable Crops (Novi Sad, Serbia) were chosen in order to assess genetic diversity using eight SSR markers (Tab.1).Plant material originating from different parts of the world included 4 wild Lycopersicon species, 4 local populations, 16 old cultivars, 5 commercial cultivars and 1 breeding line.From the Sol Genomics Network (SGN; available at http://solgenomics.net/), after Bombarely et al. (2011) and publicly available data (Frary et al. 2005, Chen et al. 2009) the following microsatellite markers were selected: SSR248, SSR111, SSR9, SSR66, SSR304, SSR80, TMS9 and TMS42 (Tab.2).The seedlings were grown in wooden boxes in greenhouse for two weeks.Genomic DNA was extracted from leaf tissue using modified CTAB isolation method (Doyle where pi is the frequency of the i-th allele out of the total number of alleles at an SSR locus, in the set of thirty tomato genotypes, and k is a total number of different alleles of a given locus.Statistical software Statistica 9 (StatSoft Inc.Corporation, Tulsa, USA) was used for genotype clustering using Ward`s hierarchical clustering method and Euclidean distance measure (d E ).

Results and Discussion
The genotypes were scored for the presence or the absence of the different SSR alleles.Eight loci were detected having total of 31 alleles, resulting in an average allele number of 3.9 per locus.Number of alleles per locus ranged from 3 to 5, while PIC values of selected markers varied from 0.12 (SSR9) to 0.73 (SSR248).Markers SSR248, TMS9, TMS42 and SSR111 were highly informative having PIC values over 0.5.Kwon, Parkl, & Yi (2009) also found a high level of polymorphism for markers SSR248 and SSR111 with PIC values 0.75 and 0.88, respectively.High PIC value for marker TMS9 (0.62) and low value for marker TMS42 (0.23) were reported by He et al. (2003).Genotypes were grouped into three clusters, but it was not possible to distinguish among genotypes with different origins (Fig. 1).The first group consisted of 10 genotypes and included 5 old cultivars, 2 wild species, 2 local populations and 1 commercial cultivar.In this group, no difference was revealed between cultivar S122 from Serbia and old cultivar S319 from Italy.The most distinct genotypes in this group were old cultivar S3 from the USA and old cultivar  No difference was revealed between two old cultivars originating from Germany (S545 and S30), which were grouped separately from the rest of the analysed genotypes.

Conclusions
Four SSR markers (SSR248, TMS9, TMS42 and SSR111) had very high PIC values and can be recommended for the future studies.Most of the analysed genotypes could be distinguished using eight chosen SSR markers.For better germplasm characterization and development of potential markers for agronomically important traits, more SSR markers should be included in the analysis.This type of research, coupled with the results of field observations, can present a starting point for MAS (Marker Assisted Selection) in tomato breeding.

Conclusions
Four SSR markers (SSR248, TMS9, TMS42 and SSR111) had very high PIC values and can be recommended for the future studies.Most of the analysed genotypes could be distinguished using eight chosen SSR markers.For better germplasm characterization and Tomato genotypes

Figure 1 .
Figure 1.Dendrogram of 30 tomato genotypes based on 8 SSR markers data

Table 1 .
Analyzed tomato genotypes from the collection of the Institute of Field and Vegetable Crops,

Table 2 .
SSR markers used for molecular evaluation of 30 tomato genotypes