GENETIC DIVERSITY AMONG SOME SPECIES OF THE GENUS ALLIUM L . USING SSR AND ISSR MARKERS

Genus Allium includes some economically important species like common onion, garlic, chives, and leek under worldwide cultivation. In this study, genetic diversity of some cultivars belonging to three species (Allium sativum L., Allium cepa L. and Allium currat L.) of this genus was investigated using sixteen SSR and three ISSR primers. All primers generated a total of 100 fragments, distributed as, 39% monomorphic, 33% polymorphic and 28% unique. The percentage of polymorphism identified by SSR and ISSR primers varied between 25 and 92.9%. With the highest variability detected for SSR and ISSR regions, the results of similarity demonstrated the presence of a concealable relationship amongst Allium species with a significant degree of similarity among clones belonging to the same species of this genus. DNA sequence of the monomorphic band generated from the five clones and cultivars used in the present work by one of the SSR primers (Asa20) were aligned together and with the earlier obtained sequences belonging to different genera and species. Although all of these sequences were obtained from one monomorphic band using the same primer, the pairwise alignment of the two onion clones showed only 75.49% of homology and nearly the same percentage of homology (73.76%) was obtained between the sequences generated from the two garlic clones. The dendrogram based on genetic similarities between cultivars showed three major clusters. Cluster 1 included only the Egyptian Leek cultivar; cluster 2 comprised the two garlic clones and cluster 3 composed of the two onion cultivars. Generally, the present results corroborate the idea that SSR and ISSR techniques seems to be a convenient tool for genetic diversity of the genus Allium.

enus Allium L. comprises more than 800 species (Fritsch et al., 2010), making it one of the largest monocotyledonous genera.It is a variable genus that is spread widely across the Holarctic region from the dry subtropics to the boreal zone.Allium includes some economically important species like common onion, garlic, chives, and leek under worldwide cultivation, and also species with medicinal properties and others of horticultural merit (Fritsch and Friesen, 2002).Several morphological and anatomical studies on Allium were carried out, and numerous data have been helpful in establishing and assessing the evolutionary lineages in the genus and dealing with newly described taxa (Cheremushkina, 1992;Hanelt and Fritsch, 1994;Friesen, 1995;Mathew, 1996;Khassanov, 1997;Xu and Kamelin, 2000;Dale et al., 2002;Fritsch, 2009;Fritsch and Friesen, 2009).Cytological diversification is also well known in species belonging to genus Allium (El-Mamlouk et al., 2002;Ata, 2005;Osman et al., 2007;Ata and Osman, 2009;Ata et al., 2010).The most common basic chromosome number is x = 8, but other numbers (x = 7, 9, 10, 11) and variation in ploidy also occurs (Xu et al., 1998;Zhou et al., 2007).DNA-based molecular mark-ers were used in studies of genetic diversity and phylogenetic analysis in plants (Savolainen and Chase, 2003;Nybom, 2004).A first approach to characterize the genus Allium by molecular markers was published by Linne von Berg et al. (1996) who conducted a chloroplast DNA RFLP analysis.Among the different molecular markers, Inter-simple sequence repeats (ISSRs) is a PCR-based method developed by Zietkiewicz et al. (1994).ISSRs have been successfully used to estimate the extent of genetic diversity at inter-and intra-specific levels in a wide range of crop species (Ajibade et al., 2000;Huang and Sun, 2000).ISSRs would be a better tool than RAPD for phylogenetic studies (Nagaoka and Ogihara 1997).For instance, ISSR primers produce more reliable and reproducible bands than RAPD primers.Few works have been done in different species, varieties and cultivars of Allium with these markers (Hao et al., 2002;Son et al., 2012).In the genus Allium, Fischer and Bachmann (2000) reported the development of 30 SSR markers on onion bulbs.Otherwise, Wako et al. (2002) and Song et al. (2004) also succeeded in isolating 118 SSRs from a genomic library of bunching onion.Conversely, for the garlic genome, a limited G number of SSR markers have been developed and few are available to generate high density genetic maps in a crop with such a large genome size (Cunha et al., 2012;Ipek et al., 2012).However, there are still many gaps in our knowledge of infra-generic taxonomy, differentiation and evolution in the genus Allium.Thus, the goal of the present study was to study and clarify the diversity and phylogenetic relationships among some species of the genus Allium (Allium sativum L., Allium cepa L. and Allium currat L.) cultivated in Egypt, using SSR and ISSR markers.

Plant materials
Two garlic (Allium sativum L.) clones called Balady or Egyptian (white bulbs) and EGA 3 (purple bulbs), one Egyptian leak (Allium currat L.) cultivar and two onion (Allium cepa L.) cultivars called Giza Red and Giza 6 Mohassan were used in the present study.All of these cultivars were kindly provided by the vegetable Branch, Horticulture Department, Faculty of Agriculture, Minia University.

Extraction of DNA
Total genomic DNA was extracted from young leaves of the aforementioned genotypes following the procedure described by Anwar et al. (2016).The concentrations and purities were spectrophotometrically estimated according to the procedure described by Sambrook et al. (1989).

PCR conditions for SSR analyses
Sixteen microsatellite primers (Table 1) developed in garlic (Allium sativum L.) by Cunha et al. (2012) were used in the present work.The PCR program was as follows: 3 min/94C; followed by 30 cycles of denaturation (30s/94C), annealing (45s/the specific annealing temperature of each pair of primers), and extension (1 min/72C) afterward; 10 cycles of denaturation (30 s/94C), annealing (45s/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 in accordance with the protocol of Ma et al. (2009).

PCR conditions for ISSR analyses
Three ISSR primers (D24, HB13 and HB15) were designed by Al-Otayk et al. (2008).The complete list of the primers and their sequences are shown in Table (2).Amplification was performed in a thermal cycler (Thermo Hybaid) programmed following the protocol of Al-Otayk et al. (2008).PCR products of SSR and ISSR reactions were separated using 2% (w/v) agarose stained in ethidium bromide.Sizes of the amplified fragments were estimated according to the standard ladder of 100 bp.

Data analyses
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).

Sequence comparisons
Purified PCR product of SSR pri- DNA sequences were aligned with Allium sativum microsatellite Asa20 sequence (Cunha et al., 2012), that was retrieved from the NCBI Database (accession no.JN084094) using the Clustal Omega (Multiple Sequence Alignment) program (http://www.ebi.ac.uk/Tools/msa/clustalo) .Gaps were positioned to minimize nucleotide mismatches and treated as missing data in phylogenetic analysis.

RESULTS AND DISCUSSION
In this study, genetic diversity of some cultivars of the genus Allium L. using two different marker systems (SSR and ISSR markers) was investigated.Total numbers and profiles of monomorphic, polymorphic and unique bands along with the percentage of polymorphism in plants of Balady and EGA3 garlic (Allium sativum L) clones, Egyptian leak (Allium currat L.) cultivar and Giza Red and Giza 6 Mohassan onion (Allium cepa L.) cultivars using sixteen SSR and three ISSR primers are shown in Table (3).The used 19 primers generated a total of 100 fragments, distributed as, 39 monomorphic (39%) and 33 polymorphic (33%).The remaining 28 bands were identified as unique, 27 of them from SSR and one from ISSR.However, it may be converted into cultivar-specific markers following further studies based on the present observations (Table 3).The highest number of polymorphic fragments was detected by Asa10 (92.9%), then HB13 (85.7%),Asa59 (83.3%),Asa31 (77.8%) and HB15 (75%) primers.However, the percentage of polymorphism (66.7%) was observed by applying Asa16, Asa18, Asa24, Asa25 and Asa27 primers (Table 3).The lowest percentage of polymorphism (25%) was detected by Asa07 SSR primer.
A total of 33 polymorphic bands without unique have been detected amongst the studied clones and cultivars (Table 3).Fourteen bands of 441 and 1186 bp (Asa18), 162 and 222 bp (Asa24), 490 bp (Asa27), 140 and 580 bp (Asa31), 253, 357 and 433 bp (Asa59), 1104 and 1439 bp (HB13 ISSR) and 311 and 1254 bp (HB15 ISSR) were observed in the two garlic (Allium sativum L.) clones namely Balady and EGA 3 (Fig. 1) and absent in the remaining cultivars.Such observations might suggest the use of these primers as molecular genetic marker for identifying garlic clones.Eight polymorphic bands (228 and 300bp by using Asa31; 208, 489 and 649 bp by using HB13 ISSR primer and 328, 415 and 524 bp using HB15 ISSR primer) could distinguish the two onion (Allium cepa L.) cultivars (Giza Red and Giza 6 Mohassan).Data analysis using primer HB15 illustrated one polymorphic band of 240 bp presented in Egyptian Leek and the two onion cultivars (Allium cepa L.) (Fig. 1).These results display the significant degree of similarity among clones belonging to each species of the genus Allium.
From the 28 unique fragments, data in Fig.
(1) showed the highest numbers of unique bands in the cultivar Egyptian Leek (17 bands for SSR primers).Only one unique band of 578 bp was appeared in onion Giza 6 Mohassan cultivar by using Asa10 SSR marker, while three unique bands were detected in onion Giza Red cultivar.Two (806 and 1210 bp) and five (226,293,296, 984 and 1226 bp) unique bands were generated in Balady and EGA3 clones, respectively (Fig. 1).
The used 16 SSR primers in the present work may support the conclusion of Cunha et al. (2012) who developed these primers.The polymorphic markers developed in their study were useful for further studies of genetic diversity, allele mining, mapping and associative studies, in the management and conservation of garlic collections, and potentially for interspecific genetic studies within the genus Allium.The genetic variability within germplasm collections, assessed by those SSR markers, was a source of information for breeding programs to obtain new varieties with higher nutritional value.Anwar et al. (2016) used the same 16 SSR and 3 ISSR markers that were used in the present work, to aid studies of genetic diversity among garlic clones.In agreement with our present data, they found a high level of polymorphism among the studied clones with both SSR and ISSR markers.The total number of bands that were detected by all used primers was 75 including 6 monomorphic, 5 unique and 64 polymorphic.The percentage of polymorphism identified by SSR primers was varied between 33.3 and 100.However, all of the studied ISSR primers were polymorphic conferring a 100% of polymorphism.

Sequence comparisons
DNA sequence of the monomorphic band generated from the five clones and cultivars used in the present work (2 garlic clones; 1-Egyptian leak and 2-onion cultivars) by SSR primer Asa20 were aligned together and with the earlier obtained sequences belonging to different genera and species of the BLAST database on NCBI.Although all of these sequences were obtained from one monomorphic band using the same primer, the pairwise alignment of the two garlic clones showed only 73.76% of homology (Table 4 & Fig. 2a).Furthermore, nearly the same percentage of homology (75.49%) was obtained between the sequences generated from the two onion cultivars (Table 4 & Fig. 2b).In general, the multiple alignments of the five se-quences did not reveal a considerable degree of homology (Table 4).The dendrogram based on genetic similarities among cultivars showed that there are three major clusters (Fig. 3).Cluster 1 included only the Egyptian leek cultivar; cluster 2 comprised the two garlic clones and cluster 3 composed of the two onion cultivars.Data demonstrate the significant differences of the monomorphic SSR bands amongst the three Allium species and even between the cultivars belonging to the same species.Therefore, for classification and studying the phylogenetic relationships amongst and between species, much more primers and/or molecular markers should be carried out.Thus, construction of a phylogenetic tree according to the banding pattern of the PCR products using a limited number of primers might produce misleading data.
All of these sequences were aligned with Allium sativum microsatellite Asa20 sequence (Cunha et al., 2012), that was retrieved from the NCBI Database (accession no.JN084094).According to the percentages of homology, garlic (Allium sativum L.) clones called Balady was the closest one to Allium sativum microsatellite Asa20 with 85% of homology.Likewise, the second garlic clone (EGA3) showed a considerable degree (81%) of homology to that reference sequence.However, Egyptian Leek as well as the two onion cultivars (Giza Red and Giza 6 Mohassan) showed low significant degree of homology with Allium sativum microsatellite Asa20 (Table 4).These results, also, represent the highest variability of SSR regions between species although the significant conservation of the outside regions used for designing the used primers.

SUMMARY
Genus Allium includes some economically important species like common onion, garlic, chives, and leek under worldwide cultivation.In this study, genetic diversity of some cultivars belonging to three species (Allium sativum L., Allium cepa L. and Allium currat L.) of this genus was investigated using sixteen SSR and three ISSR primers.All primers generated a total of 100 fragments, distributed as, 39% monomorphic, 33% polymorphic and 28% unique.The percentage of polymorphism identified by SSR and ISSR primers varied between 25 and 92.9%.With the highest variability detected for SSR and ISSR regions, the results of similarity demonstrated the presence of a concealable relationship amongst Allium species with a significant degree of similarity among clones belonging to the same species of this genus.DNA sequence of the monomorphic band generated from the five clones and cultivars used in the present work by one of the SSR primers (Asa20) were aligned together and with the earlier obtained sequences belonging to different genera and species.Although all of these sequences were obtained from one monomorphic band using the same primer, the pairwise alignment of the two onion clones showed only 75.49% of homology and nearly the same percentage of homology (73.76%) was obtained between the sequences generated from the two garlic clones.The dendrogram based on genetic similarities between cultivars showed three major clusters.Cluster 1 included only the Egyptian Leek cultivar; cluster 2 comprised the two garlic clones and cluster 3 composed of the two onion cultivars.Generally, the present results corroborate the idea that SSR and ISSR techniques seems to be a convenient tool for genetic diversity of the genus Allium.
mer Asa20 (one monomorphic fragment) was sequenced directly in both directions by Lab.Technology laboratory as sequencing reactions were performed in a MJ Research PTC-225 Peltier Thermal Cycler using a ABI PRISM BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq DNA polymerase (FS enzyme) (Applied Biosystems).In order to find homologous sequences, the sequences earlier obtained were used to retrieve the sequences belonging to different genera from the BLAST database on NCBI (http://www.ncbi.nlm.nih.gov/BLAST).

Table ( 1
(Cunha et al., 2012)f the 16 SSR microsatellite primers developed for Allium sativum, 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 ( 3
): Total number of monomorphic, polymorphic and unique PCR fragments as well as percentage of polymorphism obtained from garlic (Balady and EGA3) clones, Egyptian leak cultivar and Giza Red and Giza 6 Mohassan onion cultivars by using 16 SSR and three ISSR primers.