ASSESMENT OF GENETIC DIVERSITY IN GARLIC CLONES USING SSR AND ISSR MARKERS

arlic (Allium sativum L.) is one of the most important bulb crops which has been known since at least 5000 years (Panthee et al., 2006) and cultivated 3000 years ago in Egypt (Ipek and Simon, 2001). It belongs to family Liliaceae and genus Allium, which contains more than 600 species (Osman et al., 2007). It is a perennial plant whose bulb is economically important as a food additive. Garlic bulbs and leaves contain very useful compounds for our health such as oligosaccharides, steroidal glycosides, essential oil, flavonoids, anthocyanins, lectins, prostaglandins, fructan, pectin, adenosine, and vitamins (Arzanlou and Bohlooli, 2010). The organosulfur compound (Allicin) is responsible for the medicinal properties of garlic (Arzanlou and Bohlooli, 2010). Moreover, the regular consumption of garlic prevents cardiovascular diseases, diabetes, asthma, and cancer (Rana et al., 2011). Etoh and Simon (2002) summarized the current classification of the Allium sativum species based on morphological, isozymes and molecular markers into four informal subspecies: the rather diverse longicuspis group including most garlic from Central Asia; the subtropical group which developed under climatic conditions of South, South East and East Asia; the Ophioscorodon group from Central and East Europe; and the Mediterranean sativum group. Garlic has a large and complex genome with two pairs of satellite chromosomes in the basic karyotype (Lee et al., 2003). It is an unusual crop in that, despite being exclusively propagated asexually over centuries, it maintains a diverse phenotype amongst different clones. This makes garlic an ideal species for investigating heritage and diversity.

1. Genetics Department, Faculty of Agriculture, Minia University, Egypt 2. Botany Department, Faculty of Science, Minia University, Egypt 3. Horticulture Department, Faculty of Agriculture, Minia University, Egypt arlic (Allium sativum L.) is one of the most important bulb crops which has been known since at least 5000 years (Panthee et al., 2006) and cultivated 3000 years ago in Egypt (Ipek and Simon, 2001).It belongs to family Liliaceae and genus Allium, which contains more than 600 species (Osman et al., 2007).It is a perennial plant whose bulb is economically important as a food additive.Garlic bulbs and leaves contain very useful compounds for our health such as oligosaccharides, steroidal glycosides, essential oil, flavonoids, anthocyanins, lectins, prostaglandins, fructan, pectin, adenosine, and vitamins (Arzanlou and Bohlooli, 2010).The organosulfur compound (Allicin) is responsible for the medicinal properties of garlic (Arzanlou and Bohlooli, 2010).Moreover, the regular consumption of garlic prevents cardiovascular diseases, diabetes, asthma, and cancer (Rana et al., 2011).Etoh and Simon (2002) summarized the current classification of the Allium sativum species based on morphological, isozymes and molecular markers into four informal subspecies: the rather diverse longicuspis group including most garlic from Central Asia; the subtropical group which developed under climatic conditions of South, South East and East Asia; the Ophioscorodon group from Central and East Europe; and the Mediterranean sativum group.Garlic has a large and complex genome with two pairs of satellite chromosomes in the basic karyotype (Lee et al., 2003).It is an unusual crop in that, despite being exclusively propagated asexually over centuries, it maintains a diverse phenotype amongst different clones.This makes garlic an ideal species for investigating heritage and diversity.
Traditional methods for evaluating garlic diversity rely on resolving differences in morphological characters.However, the information provided by this approach is limited since the expression of such characters may differ under varying environmental conditions.Because of phenotypic flexibility and the occurrence of mutations, the identification and systematic classification of garlic is difficult.DNA polymorphisms are the markers of choice for identification and characterization of plants.They may be representative of the whole genome and they are not subject to environmental modification (Bachmann et al., 2001).Different molecular techniques have been developed to G study garlic diversity, mostly Randomly Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP).Simple sequence repeats (SSRs) or microsatellites are a small array of tandem arranged bases (one to six) dispersed throughout the genome.They have been recognized as useful molecular markers in marker-assisted selection, analysis of genetic diversity and population genetic analysis in various species (Li et al., 2003;Agrama et al., 2007;Ma et al., 2009;Moe et al., 2010).SSRs are reported to be more variable than restriction fragment length polymorphism (RFLP) or RAPD, and they have been widely utilized in plant genomic studies (He et al., 2003).Therefore, these markers would be useful for assessing genetic variation among asexually propagated garlic clones.Today, SSRs are the marker of choice for a broad number of genetic studies because of their high polymorphism, co-dominance, genomic abundance, and facility in laboratory usage.The number of available polymorphic SSR markers for garlic in literatures is still few compared to other minor crops.
Inter Simple Sequence Repeat (ISSR) primers target simple sequence repeats (microsatellites) that are abundant throughout the eukaryotic genome and evolve rapidly (Ipek et al., 2003;Volk et al., 2004;Xu et al., 2005).It is a PCRbased method developed by Zietkiewicz et al. (1994).This method has been used to fingerprint the different plant species and cultivars (Nagaraju et al., 2002;Al-Humaid et al., 2004) and successfully to estimate the extent of genetic diversity at inter-and intra-specific level in a wide range of crop species.It can rapidly differentiate closely related individuals and have been successfully used to assess genetic diversity among closely related cultivars which were difficult to distinguish with other molecular markers (Dagani et al., 2003;Salhi-Hannachi et al., 2004;Okpul et al., 2005;Salhi-Hannachi et al., 2005).
The present work aimed to assessment of the genetic relationships of the most common garlic clones cultivated nowadays in Egypt in comparison with some of the foreign garlic genotypes using SSR and ISSR markers.

Plant materials
Twenty one garlic clones were used in the present work.Eight of them called; Balady, EGA 4, EGA 5 (white color), Egaseed 1, EGA 1, Egaseed 2, EGA 2 and EGA 3 (Purple color) were kindly provided by Egyptian Agricultural Company for Seed Production (EGAS) while clones named AZO 1 through AZO 5 (white color) and Sids 40 (purple color) were obtained from Sids Research Station, ARC, Giza, Egypt.Plants derived from bulbils (topsets) of Egassed 2 clone which abbreviated as Egaseed 2 (ft) and Growers clone (purple color) were also included.In addition, five foreign garlic genotypes (California Late, Early Red Italian, Lorz Italian, Mexican and White Brazilian) which were friendly imported to Egypt from Italy, Brazil, Mexico and the United States of America by MUCIA (Midwest Universities Consortium for International Activities) office (Giza, Egypt) were also incorporated.These entries were classified to the Artichoke garlic group, which belongs to Allium sativum subsp.Sativum.

Extraction of DNA
DNA was extracted in 600 µl of Cornel extraction buffer (500 mM NaCl; 100 mM Tris-HCl, pH 8.0; 50 mM EDTA and 0.84% SDS, equilibrated to 65 º C, mixed with 0.38 g sodium bisulfite/100 ml buffer, and then pH of the warm buffers was adjusted to 7.8-8.0with NaOH).DNA concentration and purity were spectrophotometrically estimated according to Sambrook et al. (1989).

SSR and ISSR analyses PCR conditions for SSR analysis
SSR analysis was performed using 16 microsatellite primers developed for Allium sativum (Cunha et al., 2012).The amplification program used is described by Ma et al. (2009), consisting of an initial denaturing step for 3 min/94C; followed by 30 cycles of denaturation (30s/94C), annealing (45 s/the specific annealing temperature of each pair of primers as shown in Table (1), and extension (1 min/72C) afterward; 10 cycles of denaturation (30 s/94C), annealing (45 s/2C below the specific annealing temperature of each pair of primers) and extension (1 min/72C); and a final elongation step for 10 min/72C.

PCR conditions for ISSR analysis
ISSR analysis was performed using D24, HB13 and HB15 primers (Al-Otayk et al., 2008) which include di-and trinucleotide repeat motifs as shown in Table (2).Amplification was performed in a thermal cycler (Thermo Hybaid) programmed for one cycle of predenaturation at 94C for 2 min; and 35 cycles at 94C for 30 s, 44C for 45 s, and 72C for 1.5 min; followed by 20 min of post extension at 72C.Amplification products of SSR and ISSR reactions were confirmed by electrophoresis in 2% agarose gels stained in ethidium bromide.Sizes of the amplified fragments were estimated according to the standard ladder of 100 bp.

Data analysis
Data of SSRs and ISSRs were scored for computer analysis on the basis of the presence (1) or absence (0) of the amplified products for each sample using GelAnalyzer3 (http://www.geocities.com/egygene, Gel Analyzer Version three, 2007).Data of the similarity matrix were used for cluster analysis by using SPSS Ver.11 software.

RESULTS AND DISCUSSION
EGA 3 clone was discarded from the results because of its unclear results of amplification and electrophoresis.Total number of amplified fragments, number of monomorphic, polymorphic, unique bands and percentage of polymorphism obtained by using 16 SSR primers and three ISSR primers are shown in Table (3).In SSR analysis, the number of amplified bands per primer varied between 1 and 7.All of the studied ISSR primers were polymorphic conferring a 100% of polymorphism.The availability of a relatively high number of polymorphic ISSR markers reflects the heterozygosity of the genome.These results reveal the ability of ISSR technique to detect as much polymorphism in a vegetative as in sexually propagated species.However, the percentage of polymorphism which identified by SSR primers were varied between 33.3 and 100% as shown in Table (3).The results demonstrated that Asa14, Asa17, Asa18 and Asa59 primers generated one monomorphic band of 77, 120, 102 and 113 bp, respectively, which are plain in all studied clones (Fig. 1 a, b, c, and d, respectively).Two monomorphic bands of 104 and 177 bp were generated by using primer Asa24 (Fig. 1e).Asa17 and Asa59 SSR primers produced only one unique band of 154 and 646 bp, respectively, (Fig. 1b and d).Two unique bands of 225 and 250 bp were detected from Egaseed 2(ft) by using HB 13 ISSR primer (Fig. 1g).Cunha et al. (2012) used the same 16 SSR markers that were used in the present work, to aid studies of genetic diversity and to define efficient strategies for germplasm conservation of 75 garlic accessions.A total of 44 alleles were identified, using the polymorphic SSR loci, ranging from two to eight alleles per loci with an average of 4.4 alleles per loci.However, using the same 16 SSR markers, the twenty genotypes studied in the present work revealed a total number of 55 alleles, ranging from one to eight alleles per loci with an average of 3.4 alleles.These results reveal the highest effect of the environmental growth on the genetic variability of garlic clones.Therefore, more studies using different molecular markers might be required to determine the polymorphic relationship of garlic clones cultivated in each environmental area.

Cluster analysis of studied garlic clones on the basis of polymorphisms
The main objective of the study was to analyze the genetic similarity/distance between the studied clones of garlic.Similarity coefficient values among these garlic clones based on band polymorphisms generated by using SSR and ISSR primers are illustrated in Table (4).The highest similarity value (0.969) was found between AZO 2 and AZO 3, while the lowest value (0.482) was found between AZO 4 and EGA 5 clones.Table (4) showed that the level of similarity among AZO clones (AZO 1 to AZO 5) ranged from 0.556 to 0.969.The highest similarity value (0.969) among these clones was found between AZO 2 and AZO 3 while the lowest value (0.556) was found between AZO 3 and AZO 4. The results in Table (4) also indicated that similarity among EGA clones (EGA 1, EGA 2, EGA 4 and EGA5) was 88%.The level of similarity among the foreign genotypes (California Late, Early Red Italian, Lorz Italian, Mexican and White Brazil-ian) ranged from 0.917 to 0.835 (Table 4).Foreign genotypes showing about 85% and 90% similarity with Balady clone and EGA 2 clone, respectively.Low level of similarity was observed among AZO 4 clone and other tested clones except Egaseed 1, Egaseed 2 (ft), California Late and White Brazilian (Table 4).Data in Table (4 The high level of genetic variation observed in this study is consistent with the results from previous studies of garlic carried out using different molecular markers (Ipek et al., 2003(Ipek et al., , 2005(Ipek et al., and 2008;;Lampasona et al., 2003;Volk et al., 2004), thereby confirming the great diversity among garlic accessions.

Table ( 1
(Cunha et al., 2012)f the 16 microsatellite primers developed for Allium sativum.Shown for each primer pair are the forward and reverse sequence, repeat type, annealing temperature when run individually, size of the original fragment, and GenBank accession number(Cunha et al., 2012).

Table ( 4
): Similarity coefficient values among studied garlic clones based on bands polymorphism generated by SSR and ISSR-PCR primers.