Atlas Journal of Biology 2017, pp. 384–391 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Genetic Assessment of Moroccan Tomato (Solanum lycopersicum L.) Genotypes by RAPD and SSR Markers Amraoui Rajae1, Dominique Mingeot2, Mohamed Addi1, Ahmed Elamrani1, Hana Caid Serghi- ni1, Aatika Mihamou1, and Malika Abid1* ¹ Laboratory of Biology of Plants and Microorganisms, Faculté des Sciences Oujda – Université Moham- med Premier, BP-717 Oujda, Morocco; ² Laboratory of Wallonia Center of Agronomics Researchs (CRA-W), Département Sciences du Vivant, Bâtiment Jean-Baptiste de La Quintinie, Chaussée de Charleroi, 234 B-5030 Gembloux, Belgium Received: May 2, 2017 / Accepted: June 1, 2017 __________________________________________________ * Corresponding author: abidmalika@yahoo.fr 384 Abstract For the first time eight local tomato cultivars collected from four different regions of Morocco were assessed with RAPD and SSR methods. Most of RAPD markers give monomorphic banding profiles. Only OPU03 marker showed a total of 4 polymorphic amplicons out of 8 recorded in FIGUIG2 cultivar. The analysis with SSR markers gives more polymorphism. The number of alleles amplified assessed from 2 to 5 alleles among cultivars. The similarity matrix subjected by the un- weighted pairgroup arithmetic method (UPGMA) clustering grouped the cultivars in four groups where FIGUIG2 cultivar formed a separate and more distant cluster. In addition this cultivar holds the very high percentage of uniformity (99%) indicating that is an homogeneous traditional cultivar with high purity. This genotype can be conserved and used in breeding programs. More traditional Moroccan cultivars must be collected in order to determine their genetic structure. Keywords: Moroccan tomato cultivars, RAPD and SSR markers, genetic diversity. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecom- mons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the origi- nal work is properly cited. Introduction Tomato belongs to Solanaceae family and it is originated from the Andean region, more exactly from Ecuador, Bolivia, Colombia and Chile. Tomato seeds were introduced by Span- iards from Mexico to Europe for domestication (Peralta et al. 2006) and it was then referred as a cultivated plant in Italy. At the end of the XIXth century, tomato cultivars were self-pollinat- ed and farmer saved seeds from a year to the other. New geno- types resulted from spontaneous mutations, natural outcrossing or recombination of pre-existing genetic variation (Bauchet and Causse, 2012). Commercial itinerary has contributed to spread the species worldwide (Diez and Nuez, 2008). This has led to collect existing genetic diversity to preserve and to valorise it all over the world through public or private institutes of plant germplasm. The seed sector in Morocco has known since the early 70s, development and sustained growth that enabled the creation of a national plant genetic quality. However, tomato has not yet benefited from this progress and we do not yet have a certified Moroccan tomato cultivar. In Morocco, the most tomato is grown in greenhouses un- der controlled climate. Nevertheless, almost all the seeds were bought from foreigner countries. The use of certified seed by breeders is the means to exploit and disseminate the most effec- tive advances in seed breeding. Characterization of tomato germplasm is of great impor- A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) tance for current and future agronomic and genetic improvement of the crop. Furthermore, if an improvement programme is to be carried out evaluation is imperative, in order to understand the genetic background and the breeding value of the available tomatoes. Morphological, biochemical and molecular characterization are used to specify genetic diversity between tomato varieties (Garcia et al., 2004). Morphological and biochemical charac- terization is used to evaluate many fruit quality traits such as diameter, height, shelf life, weight size, acidity, colour and firm- ness. Those parameters do not always allow the quantification of genetic diversity in plants and are dependent of environmental factors (Cooke, 1994). Molecular markers are an efficient tool to investigate the genetic basis of agronomic traits and to make easier the transfer and accumulation of desirable traits between breeding lines. Many molecular techniques including amplified fragment length polymorphism (AFLP), restricted fragment length polymorphism (RFLP), simple sequence repeats (SSR) and ran- dom amplified polymorphic DNA (RAPD) were used to set up genetic variation in tomato cultivar collections (Bredemeijer et al., 1998; Park et al., 2004; Garcia-Martinez et al., 2006). Local tomato germplasm may provide some natural variation that is present in the species as a whole (Brush, 2000; Feuillet et al., 2008) since local varieties represent the main source of genetic variation in the cultivated species. They can offer big in- terest for their use in scientific studies and in breeding programs (Chable et al., 2009). For this reason, eight local tomato geno- types and one French commercial tomato used as control were assessed using RAPDs and SSRs markers to examine the genetic variability, to establish their relationships and to compare the usefulness of these markers. Materials and methods Plant Materials Nine lots of tomato were studied in this investigation including one commercial variety of French origin “Saint Pierre” from Vita Company used as a control. The other lots were collected from four different regions of Morocco and are listed as follow: From Berkane region (Northeast) two tomato lots noted BERKANE1 and BERKANE2, from Figuig region (South east), two tomato lots noted FIGUIG1 and FIGUIG2, from Rissani region (South west) three batches noted RISSANIB, RISSANIO and RISSANIN in which we found a difference in color between the seeds and finaly, one tomato lot from HOCEIMA region (Northern Morocco). The seeds of each lot of tomato were sown in the green- house for germination and growth. After two weeks of sowing, the seedlings were transplanted and grown in green house at the nursery of the park Lala Aïcha with a regular watering. Genomic DNA Isolation For total genomic DNA extraction, fresh leave of each lot of tomato were grounded in liquid nitrogen and stored at -80°C. Genomic DNA isolation was performed following the procedure of the DNeasy Plant mini kit de Qiagen. RAPD and SSR Primers Ten RAPD random primers and fourteen microsatellite mark- ers were used for molecular characterization of Moroccan to- mato lots. All RAPD and SSR primers were chosen among the highly polymorphic primers published on the literatures (Suli- man-Pollatschek et al., 2002; Areshchenkova and Ganal, 2002) and have been applied successfully for assessing different plant genotypes. The used RAPD primers are OPC09, OPU03, OPA14, OPU14, OPA15, OPB17, OPB18, OPC08, OPG17 and OPV19. The simple sequences repeat (SSR) primers are listed in Table 2. RAPD-PCR Amplification PCR assays was performed in a 25µl final volume, contain- ing 20ng of genomic DNA, 0.2µM of operon random primer, 100µM dNTPs, 2.5mM MgCl2, 1mg /ml BSA, 5 X PCR reaction buffer, and 1 U Taq DNA polymerase (Promega). The amplifcations were conducted with Thermal Cycler (Ap- plied system), with an initial 5 min at 94°C that was followed by 45 cycles of 1 min at 94°C, 1 min 30 s at 36°C, and 2 min 30 s at 72°C, ended by 7 min extension at 72°C. PCR product were electrophoresed on 1.4% agarose gel stained with ethid- ium bromide and observed under UV light and photographed. Size of the amplicons was estimated with 1kb DNA ladder which was resolved along with amplified product. Reproducibility of the results was confirmed by repeating the amplification twice. SSR-PCR Amplification For SSR analysis, among the relatively high number of SSR loci already reported in tomato, 14 SSR markers were selected from the published data (Suliman-Pollatschek et al., 2002; He et al., 2003) or on the website of Solanaceae Genomics Network (http://solgenomics.net). PCR amplification was performed in a 20 µl total volume, containing 20 ng of genomic DNA, 0.25 mM of each primer, 200 µM dNTPs, 1.5 mM MgCl2, 1 mg /ml BSA, 1 X PCR buffer, and 1 U Taq DNA polymerase (Promega). The amplifications were conducted with Thermal Cycler (Ap- plied system), with an initial 5 min at 94°C that was followed by 35 cycles of 30 s at 94°C, 45 s at X°C, and 1min 30 s at 72°C, ended by 7 min final extension cycle at 72°C. The amplification products were separated and analyzed on a Licor sequencer type (Westburg) using a 6.5% acrylamide gel. The lengh of the alleles was determined by comparaison with marker loaded on adjacent gel traks. The raw data were collected and analyzed by the analysis software “Gene ImageIR” (Westburg). Cluster Analysis All 9 varieties were clustered based on the estimated genetic distance. The positions of a consistent RAPD or SSR bands were scored and transformed into a binary character matrix “1” for the presence and “0” for the absence of a RAPD and SSR band at a particular position. Genetic similarities between genotypes 385 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 386 were calculated according to Nei and Li’s Coefficient (1979). The similarity matrix was subjected to cluster analysis by the unweighted pairgroup arithmetic method (UPGMA; Sneath and Sokal 1973) and phylogenetic tree was created using the output data and the graphical module of the MVSP 3.1 software. Results and Discussion For the first time in Morocco we studied the genetic variabil- ity of some local tomato cultivars collected from four different regions. Nine lots of tomato were selected in this study including one commercial French variety “Saint Pierre” obtained by Vita Company used as a control. The other tomato lots were collected from farmers and were sourced from different geographic re- gions which differ greatly in their agro-ecological and ethnic compositions. The farmers were asked specific questions as the local names for each tomato lot and the location where they are cultivated. Tomato genotypes collected from Berkane (North- east) and Hoceima (North) regions are adapted to semi-dry climate but humid. Tomato genotypes from Rissani and Figuig regions are cultivated in a very dry environment. Tomato seeds from these regions are collected from a year to the other to maintain germplasm. These lots are usually grown in small fields and reserved to self-consumption or to local markets. RAPD Markers RAPD markers have great potential to evaluate genetic di- versity within accessions and can provide much informations useful in breeding programs. RAPD analysis is technically easy, simple and can generate polymorphic profile suitable for large scale germplasm characterization (Rafalski and Tingey, 1993). In our study, 10 RAPD markers were chosen for the analysis of tomato cultivars variation from four different regions of Mo- rocco in a mixture of 5 plants per lot. The RAPD fragments were scored for their presence (1) and absence (0) for each sample and genetic diversity among groups was calculated on the ba- sis of Nei & Li’s Coefficient (1979). Out of these 10 primers, two primers (OPG17 and OPG19) did not achieve any molecu- lar polymorphism. The same result was obtained with OPG17 primer in 19 Azerbaijan Tomato genotypes (Sharifova et al., 2013). The Rest of the primers have amplified a total of 41 signals out of which 5 were polymorphic and 36 monomorphic. The number of signals amplified by these markers varied be- tween 2 for OPC08 marker and 8 for OPU03. OPC08 marker gives monomorphic banding patterns among all the cultivated cultivars (Figure1). Only random primer OPU03 showed a to- tal of 4 polymorphic amplicons out of 8 amplicons recorded in FIGUIG2. In other study this percentage was much higher. For example, the same primer OPU03 produced highest number of polymorphic bands (21 bands) in 19 tomato varieties (Thamir et al. 2014). With this primer (OPU03), only cultivated tomato FIGUIG2 and BERKANE2 displayed a polymorphism showing 4 specific alleles for FIGUIG2 and only one allele for BERKANE1. This marker discriminate cultivars FIGUIG1, RISSANIB, BERKANE1 and HOCEIMA who have the same profile from RISSANIN, and RISSANIO who share the same profile with the control cultivar Saint Pierre indicating that they could have common origin. These results indicated the existence of limited genetic variation within the studied tomato Moroccan lots. Many others markers showed highly monomorphic profile in the cultivated S. lycopersicum L. (Labate and Roberts, 2002). The values of pair-wise genetic distances ranged between 0,947 and 1 indicating low diversity in the studied tomato geno- types (Table 2). The highest genetic distance (1) was observed between some cultivars like HOCEIMA and FIGUIG1 whereas the lowest genetic distance (0,947) was detected between BER- KANE2 and FIGUIG2 which is an evidence for a low genetic simi- larity value in the tomato germplasm studied. Low degree of the genetic variability is often correlated to a weak discrimination of RAPD markers. Moreover, Miller and Tanksley (1990) estimated that only 5% of genetic variation exist within S. Lycopersicum. Nevertheless, these primers could dived Moroccan tomato varieties into four groups: the first includes FIGUIG1, RISSANIB, BERKANE1 and HOCEIMA lots. The second includes RISSANIN, Saint Pierre and RISSANIO. The third includes only BERKANE2 Figure 1. RAPD electrophoretic pattern of tomato cultivars obtained by the primers OPU 03 and OPC 08 (from left to right) (A: FIGUIG1 B: FIGUIG2, C: RISSANIB, D: RISSANIN, E: Saint Pierre, F: RISSANIO, G: BERKANE1, H: Berkane2, I: HOCEIMA). Right extreme lane represents 1kb DNA. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 387 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Ta bl e 2. S et o f sim pl e se qu en ce r ep ea ts (S SR ) p rim er s us ed in th is in ve st ig at io n. M ar ke r R éf ér en ce M ot if Fo rw ar d R ev er se C hr om os om e T° H yb rid at io n SS R1 4 ht tp : / /s ol ge no m ic s.n et / (A TA )9 TC TG C A TC TG G TG A A G C A A G C TG G A TT G C C TG G TT G A TT T 3 55 ° SS R 2 2 ht tp :/ /s ol ge no m ic s.n et / (A T) 11 G A TC G G C A G TA G G TG C TC TC C A A G A A A C A C C C A TA TC C G C 3 50 ° SS R2 6 ht tp :/ /s ol ge no m ic s.n et / (C G G ) 7 C G C C TA TC G A TA C C A C C A C T A TT G A TC C G TT TG G TT C TG C 2 50 ° SS R6 3 ht tp :/ /s ol ge no m ic s.n et / (A T) 39 C C A C A A A C A A TT C C A TC TC A G C TT C C G C C A TA C TG A TA C G 8 55 °C SS R2 48 ht tp :/ /s ol ge no m ic s.n et / (T A )2 1 G C A TT C G C TG TA G C TC G TT T G G G A G C TT C A TC A TA G TA A C G 10 55 ° SS R5 78 ht tp :/ /s ol ge no m ic s.n et / (A A C )6 (A TC )5 A TT C C C A G C A C A A C C A G A C T G TT G G TG G A TG A A A TT TG TG 6 55 ° To m 23 6- 23 7 Su lim an -P ol la tsc he k et al .2 00 2 A T1 6 G TT TT TT C A A C A TC A A A G A G C T G G A TA G G TT TC G TT A G TG A A C T 9 47 °C TO M 18 4 Su lim an -P ol la tsc he k et al .2 00 2 (A TT T) 3 (A TT )7 C A A C C C C TC TC C TA TT C T C TG C TT TG TC G A G TT TG A A 4 45 TO M 19 6- 19 7 Su lim an -P ol la tsc he k et al .2 00 2 (G A )1 4 C C TC C A A A TC C C A A A A C TC T TG TT TC A TC C A C TA TC A C G A 11 45 TO M 21 0- 21 1 Su lim an -P ol la tsc he k et al .2 00 2 (A TA )1 5 C G TT G G A TT A C TG A G A G G TT TA A C A A A A A TT C A C C C A C A TC G 4 45 TM S 5 2 T. A re sh ch en ko va ·M .W . G an al .2 00 2 (A C )1 4 (A T) 18 TT C TA TC TC A TT TG G C TT C TT C TT A C C TT G A G A A TG G C C TT G 12 55 TM S5 6 T. A re sh ch en ko va ·M .W . G an al .2 00 3 (C T) 19 G A TC TC A A A G G A TG A A C A A TA C TC A TT A G G A G A TT C TT TG TA TC A 1 55 TM S6 3 A re sh ch en ko va an d G an al 20 02 (A T) 4( G T) 18 (A T) 9 G C A G G TA C G C A C G C A TA TA T G C TC C G TC A G G A A TT C TC TC 1 60 °C TM S6 5 T. A re sh ch en ko va ·M .W . G an al .2 00 2 (T A )2 5 (G A )2 0 A G C TT C A TC C A TT A C G C C A C G TG C A TC TG G C G TA C C TA C C 12 60 FI G U IG 1 FI G U IG 2 R IS SA N IB R IS SA N IN R IS SA N IO BE R K A N E1 BE R K A N E2 H O C EI M A Sa in t Pi er re FI G U IG 1 1 FI G U IG 2 0, 96 1 R IS SA N IB 1 0, 96 1 R IS SA N IN 0, 98 7 0, 97 3 0, 98 7 1 R IS SA N IO 0, 98 7 0, 97 3 0, 98 7 1 1 BE R K A N E1 0, 98 7 0, 97 4 0, 98 7 0, 97 4 0, 97 4 1 BE R K A N E2 0, 98 7 0, 94 7 0, 98 7 0, 97 4 0, 97 4 0, 97 4 1 H O C EI M A 1 0, 96 1 0, 98 7 0, 98 7 0, 98 7 0, 98 7 1 Sa in t Pi er re 0, 98 7 0, 97 3 0, 98 7 1 1 0, 97 4 0, 97 4 0, 98 7 1 Ta bl e 1. P ai r- w ise g en et ic d ist an ce s f ro m 1 0 RA PD m ar ke rs o f ei gh t t om at o cu lti va rs a nd o ne F re nc h co m m er ci al c ul tiv ar S ai nt P ie rr e. 388 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Primer Name Allelic Size Range (pb) No. of Alleles Polymorphic Band SSR14 166-235 1 - SSR22 208-214 2 2 SSR26 172-178 1 - SSR63 206-248 4 4 SSR248 220-251 3 3 SSR578 290-299 1 - Tom236-237 154-210 5 5 TOM184 163-206 2 2 TOM196-197 206-214 3 3 TOM210-211 216-222 3 3 TMS52 148-178 5 5 TMS56 102-126 3 3 TMS63 154-181 3 3 TMS65 288-298 4 4 Table 3. Total number of polymorphic bands, seize range, number of alleles for 14 SSR markers used on 8 selected Moroccan tomato cultivars and one commercial variety. Figure 2. (A) and (B) Part of a gel obtained with the SSR primes Tom 236-237 and visualized in a LI-COR sys- tem. FIGUIG1, FIGUIG2, RISSANIB, RISSANIN, RISSANIO, BERKANE1, BERKANE2 and HOCEIMA correspond to tomato local cutivars. SP correspond to Saint Pierre commercial cultivar. Right and left extreme lanes represent 1kb DNA. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 389 Figure 3. Dendrogram constructed from RAPD and SSRs data showing relationship among 8 of local Moroccan tomato cultivar and one French commercial tomato based on Nei and Li (1979) distance and the unweighted pairgroup arithmetic method (UPGMA). Figure 4. Rates of non-uniformity for Moroccan tomato cultivars tested. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g)FIGUIG1 FIGUIG2 RISSANIB RISSANIN RISSANIO BERKANE1 BERKANE2 HOCEIMA Saint Pierre FIGUIG1 1 FIGUIG2 0,512 1 RISSANIB 0,857 0,605 1 RISSANIN 0,723 0,588 0,766 1 RISSANIO 0,75 0,629 0,792 0,923 1 BERKANE1 0,638 0,588 0,681 0,579 0,615 1 BERKANE2 0,653 0,5 0,816 0,7 0,683 0,6 1 HOCEIMA 0,741 0,634 0,852 0,622 0,652 0,8 0,723 1 Saint Pierre 0,651 0,6 0,698 0,882 0,857 0,588 0,667 0,585 1 Table 4. Pair-wise genetic distances from 14 SSR markers of eight tomato cultivars and one French commercial cultivar Saint Pierre. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 390 and the fourth group includes only FIGUIG2. The latest geno- type is clearly differentiated from the rest and could be used in tomato breeding program with specified objectives. Simple Sequence Repeats Markers Simple Sequence Repeats (SSR) or microsatellite markers have been successfully used to discriminate varieties which are morphologically similar and genetically close (Bredmeijer et al., 2002; He et al., 2003; Frary et al., 2005; Sarıkamıs et al., 2006, 2010). SSR markers may be adequate because of their high polymorphism, reproducibility, genetic co-dominance, easy detection, and multiallelic variation (Ruiz et al., 2005). Several studies have demonstrated the usefulness of SSR in cultivar iden- tification (Bredmeijer et al., 1998; He et al., 2003). In this inves- tigation, fourteen microsatellite markers were selected from the published data or on the website of Solanaceae Genomics Net- work (Table 1) and used to screen genetic diversity and genetic relationships among nine tomato cultivars. Three primers (21%) like SSR 14, SSR578 and SSR26 showed monomorphic profiles among all the screened tomato batches with only one amplified band (Table 3). In other studies, this percentage is much higher 49% as reported by Todorovska et al. (2014) and 25% by El- Awady et al. (2012). The other markers generated a polymor- phic banding profile. The number of alleles amplified assessed from 2 alleles with primers Tom 184 and SSR 22 to 5 alleles with markers Tom 236-237 and TMS 52 (Figure 2) with a mean of 2.85 allele per locus. The scorable fragment sizes ranged from approximately 102 pb to 299 bp. Limited allelic variation was also observed in a study of eight tomato varieties and lines with an average of 3 alleles per locus after testing 160 SSR loci (Todorovska et al., 2014). El-Awady et al. (2012) showed also a low level of genetic diversity with an average of 2.1 alleles per locus by using 20 SSR markers in ten tested tomato cultivars. To evaluate genetic relationship within the tomato lots, the data scored from the 14 SSR primers were analyzed on the basis of Nei & Li’s Coefficient (1979). The relationship between tomato germplasm collected from different area in Morocco is illustrated by the data in Table 4. The genetic similarity estimat- ed according to SSR data was scaled between 0.5 and 0.923 suggesting the potential of SSR markers in discriminating among plants compared to RAPD markers. In other studies this coeffi- cient is much higher. For example, Archak et al. (2002) founded a similarity coefficients ranging between 0.610 - 0.976, Fan- juan et al. (2010) showed a genetic similarity varying between 0.72 - 1, Singh et al. (2014) reported a gene diversity from 0.65 to 0.97 underlying a weak genetic diversity in the tomato cultivars collection. The highest similarity value of 0.923 was shown between RISSANIN and RISSANIO while the lowest value 0.5 was observed between FIGUIG2 and BERKANE2. FIGUIG1 lot is distantly related to FIGUIG2 (49%) and relatively closed to RISSANIB (85%). HOCEIMA is close to BERKANE1 (80%) de- spite their diverse sources. RISSANIO and RISSANIB are distant- ly related to the certified control Saint Pierre 12% and 14% respectively suggesting an admixture of the control with those cultivated lots through hybridisation. The other lots are distantly related 30 to 42% to the control indicating a reduced germ- plasm diversity among the lot. The distance matrix based on RAPDs and SSRs data was combined and used to construct a dendrogram (Figure 3). The dendrogram obtained can be divided into four main clusters, one contains only FIGUIG2 which formed a separate and more distant cluster. The second main cluster contains two cultivars each one is presented in one branch HOCEIMA and BERKANE1. The Third one includes only BERKANE1. The fourth cluster is divided into two sub-clusters, in one is branched Saint Pierre, RISSANIO and RISSANIN cultivars and the second one contains RISSANIB and FIGUIG1. Traditional tomato cultivars RISSANIO, RISSANIN on the one hand, RISSANIB and FIGUIG1 on the other hand are closely related to each other and seems to have common origin. This could be explained by the geographical situation of these region, RISSANI and FIGUIG are both situated in the south of Morocco and probably the farmers shared the same basis of the seeds. The dendrogram showed the average of at least 5 grouping DNA samples per genotype. By calculating the rate of non-uni- formity (Figure 4) we found considerable heterogeneity within the traditional cultivars. The rate of non-uniformity varies be- tween the largest percentage (35%) in FIGUIG1 and the lowest (1%) in FIGUIG2. The level of heterogeneity found in the most traditional tomato cultivars could be characterized by a higher level of heterozigosity in some loci. On the other hand, FIGUIG2 possess a very high percentage of uniformity (99%) indicating that is a homogeneous traditional cultivar with high purity trans- lating 100% homozygosity. This information could be efficiently used to establish a property rights and a germplasm conserva- tion. Conclusion SSR markers used in this investigation were more suitable in the eight Moroccan local tomato cultivars as RAPD system. SSRs marker are better identification of tomato genotyping because they are codominant (Korir et al., 2014) while RAPD highlight only the dominant alleles. Except for FIGUIG2 cultivar, the most genotype studied are closely related despite their geographic sources. Only the local FIGUIG2 cultivar shows the high degree of polymorphism since it was characterized with the highest num- ber of unique bands (4) with RAPD markers and (5) with SSR markers in comparison with the other genotype. Furthermore, this lot hold the very high percentage of uniformity (99%) indicat- ing that is a homogeneous traditional cultivar with high purity compared to the rest of tomato lots which are probably an ad- mixture of commercial tomato. FIGUIG2 cultivar is a farmer- selected and adapted in area of local subsistence with semi-dry to dry climate and has low but stable annually yield. The poly- morphism recorded in FIGUIG2 cultivar can be exploited in the management of genetic resources collection in Morocco and the establishment of property rights and protection. This genotype can be conserved and used in breeding programs and could of- fer gene combination to ensure adaptability and reproducibility in dried climate since genetic resources including landraces and wild relatives of crop species play an important role in breeding A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 391 programs (McCouch et al., 2013). In addition, A more acces- sion survey will be necessary to evaluate a range of Moroccan tomato germplasm with more informative marker system and to establish a core collection in the gene banks that enabled the creation of a national plant genetic useful in breeding programs Acknowledgments This study was supported by the Moroccan–Belgian cooperation, Wallonia Brussels International project 2.9 and the Ministry of Education, Higher Education and Scientific Research in Morocco. References Archak S, IL Karihaloo, and A Jain (2002) RAPD markers reveal narrow- ing genetic base of Indian tomato cultivars. Cur Sci 82: 1139-1143. Areshchenkova T and M Ganal (2002) Comparative analysis of poly- morphism and chromosomal location of tomato microsatellite mark- ers isolated from different sources. Theor and Appl Genet 104 (2- 3): 229–235. Bauche, G and M Causse (2012) Genetic diversity in tomato (Solanum lycopersicum) and its wild relatives. pp. 133–162. In Genetic Diver- sity in Plants. Edited by M Caliskan. Intech Open. Bredemeijer G, Cooke R, Ganal M, Peeters R, Isaac P, Noordijk Y, Ren- dell S, Jackson J, Röder M, Wendehake K (2002) Construction and testing of a microsatellite database containing more than 500 to- mato varieties. Theoretical and Applied Genetics 105 (6-7): 1019– 1026. Bredemeijer GMM, P Arens, D Wouters, D Visser, and B Vosman (1998) The use of semi-automated fluorescent microsatellite analysis for to- mato cultivar identification. Theoretical and Applied Genetics 97 (4): 584–590. Brush SB (2000) Genes in the field: on-farm conservation of crop di- versity. IDRC. Chable V, I Goldringer, J Dawson, R Bocci, EL Van Bueren, E Serpolay, JM González, T Valero, T Levillain, JW Van der Burg (2009) Farm seed opportunities: a project to promote landrace use and renew biodiversity. In European Landraces On-farm Conservation, Man- agement and Use. Biodiversity Technical Bulletin 15: 266–274. Diez MJ and F Nuez (2008) Tomato. In Vegetables II Fabaceae, Lili- aceae, Solanaceae, and Umbelliferae. Edited by J Prohens and F Nuez. pp. 249–323. El-Awady MAM, AAE El-Tarras, and MM Hassan (2012) Genetic diver- sity and DNA fingerprint study in tomato (Solanum lycopersicum L.) cultivars grown in Egypt using simple sequence repeats (SSR) mark- ers. African Journal of Biotechnology 11 (96): 16233–16240. Feuillet C, P Langridge, and R Waugh (2008) Cereal breeding takes a walk on the wild side. Trends in Genetics 24 (1): 24–32. Frary A, Y Xu, J Liu, S Mitchell, E Tedeschi, and S Tanksley (2005) De- velopment of a set of PCR-based anchor markers encompassing the tomato genome and evaluation of their usefulness for genetics and breeding experiments. Theor and Appl Genet 111 (2): 291–312. Garcia AAF, LL Benchimol, AMM Barbosa, IO Geraldi, and CL Souza (2004) Comparison of García-Martínez S, L Andreani, M Garcia-Gusano, F Geuna, and JJ Ruiz (2006) Evaluation of amplified fragment length polymorphism and simple sequence repeats for tomato germplasm fingerprinting: utility for grouping closely related traditional cultivars. Genome 49 (6): 648–656. He C, V Poysa, and K Yu (2003) Development and characterization of simple sequence repeat (SSR) markers and their use in determining relationships among Lycopersicon esculentum cultivars. Theor and Appl Genet 106 (2): 363–373. Korir NK, W Diao, R Tao, X Li, E Kayesh, A Li, W Zhen, and S Wang (2014) Genetic diversity and relationships among different tomato varieties revealed by EST-SSR markers. Genetics and Molecular Re- search 13 (1): 43–53. Labate JA and LD Roberts (2002) Genetic variation in heir loom ver- sus modern tomato (Lycopersicon esculentum) cultivars. p. 27 In: Program for the 43rd Annual Meeting of the Society for Economic Botany, NY Botanical Garden, NYC, NY. Miller J and S Tanksley (1990) Rflp analysis of phylogenetic relation- ships and genetic variation in the genus Lycopersicon. Theor and Appl Genet 80 (4): 437–448. Nei M and WH Li (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. PNAS 76: 5269– 5273. Park YH, MA West, and DA St Clair (2004) Evaluation of aflps for germplasm fingerprinting and assessment of genetic diversity in cultivars of tomato (Lycopersicon esculentum l.). Genome 47 (3): 510–518. Peralta IE, S Knapp, and DM Spooner (2006) Nomenclature for wild and cultivated tomatoes. TGC Report 56: 6–12. Rafalski JA and SV Tingey (1993) Genetic diagnostics in plant breed- ing: RAPDs, microsatellites and machines Trends Genet 9: 275–280. RAPD, RFLP, AFLP, and SSR markers for diversity studies in tropical maize inbred lines. Genet Mol Biol 27: 579–588. Ruiz JJ, S García-Martínez, B Picó, M Gao, and CF Quiros (2005) Ge- netic variability and relationship of closely related spanish tradi- tional cultivars of tomato as detected by SRAP and SSR markers. Journal of the American Society for Horticultural Science 130 (1): 88–94. Sarıkamış G, J Marquez, R Maccormack, and R Bennett (2006) High glucosinolate broccoli A delivery system for sulforaphane. Mol Breed 18: 219–228. Sarıkamış G, R Yanmaz, S Ermis, and M Bakir (2010) Genetic char- acterization of pea (Pisum sativum) germplasm from Turkey using morphological and SSR markers. Genet Mol Res 9: 591–600. Sharifova S, S Mehdiyeva, K Theodorikas, and K Roubos (2013) Assess- ment of genetic diversity in cultivated tomato (Solanum lycopersicum L.) genotypes using RAPD primers. Journal of Horticultural Research 21 (1): 83–89. Singh M, NP Singh, S Arya, B Singh and Vaishali (2014) Diversity analy- sis of tomato germplasm (Lycopersicom esculentum markers) using SSR. International Journal of Agricultural Science and Research 4 (4): 41–48. Sneath PHA and RR Sokal (1973) Numerical Taxonomy. The principles and practice of numerical classification. pp. 230-234. Freeman WH Company San Francisco California USA. Suliman-Pollatschek S, K Kashkush, H Shats, J Hillel, and U Lavi (2002) Generation and mapping of aflp, ssrs and snps in Lycopersicon es- culentum. Cellular and Molecular Biology Letters 7 (2A): 583–598. Tabassum N, SK Sony, SK Bhajan, and MN Islam (2013) Analysis of genetic diversity in eleven tomato (Lycopersicon esculentum Mill.) va- rieties using RAPD Markers. Plant Tissue Culture and Biotechnology 23 (1): 49–57. Thamir AJ, AH Al-Saadi, and MC Abbass (2014) Genetic diversity of some tomato Lycopersicon esculentum Mill varieties in Iraq using random amplified polymorphism DNA (RAPD) markers. Journal of Babylon University Pure and Applied Sciences 9 (22): 2342–2351.