Comparative analysis of genetic diversity of 8 millet genera revealed by ISSR markers

R E G U L A R A R T I C L E Dvořáková, et al.: Genetic diversity of millets 618 Emir. J. Food Agric ● Vol 27 ● Issue 8 ● 2015 agricultural and food security of poor farmers inhabiting arid, infertile, and marginal lands (Garí, 2002). Traditionally, the genetic resources of the millet species were evaluated by: descriptions of their morphological characteristics (Andrews and Kumar, 2006; Brink and Belay, 2006; Kaume, 2006; Jansen and Ong, 1996; Jansen, 2006), by health-impact traits (Kalinová and Moudrý, 2006), and by molecular data. Current studies, based on DNA fingerprinting in millet species, have mainly been carried out for the identification of unknown accessions or genotypes within a single millet species (Supriya et al., 2011; Le Thierry d’Ennequin et al., 2000; Hu et al., 2009; Arya et al., 2013; Qin et al., 2005; Zeid et al., 2012, Nozawa et al., 2006; Adoukonou-Sagbadja et al., 2010). Knowledge about the genetic diversity, and revelations of the genetic relationships among millet species is essential for the suitable conservation and increased use of millet genetic resources, and it also plays an important role in millet breeding. The application of methods using DNA analysis is pivotal for the description of genetic variability within different millet species. One of the alternatives is the use of Inter Simple Sequence Repeats (ISSR) markers, which is known to be a highly variable, reproducible, and cost effective method (Wolfe and Liston, 1998; Yang et al., 1996). Comparing ISSR markers with Random Amplified Polymorphic DNA (RAPD) analysis, there are many advantages on the side of ISSR, which can reveal a greater level of genetic variability. Using longer primers and higher annealing temperatures, they provide results that are more reliable and reproducible (Wolfe and Liston, 1998). ISSR has been widely used in studies of the genetic structure of plants (Li and Jin, 2008), genetic diversity (Sheeja et al., 2009), genetic relationships (Li et al., 2009), phylogeny and evolution (Zamani et al., 2011). They have also been successfully applied to a number of monocotyledonous plants (Ben El Maati et al., 2004; Mondini et al., 2014; Bahieldin et al., 2012; Virk et al., 2000). Findings of genetic similarities in millet species have been performed using ISSR markers in Pennisetum, Setaria, Eleusine and Eragrostis genera (Pedraza-Garcia et al., 2010; Lin et al., 2012; Salimath et al., 1995; Assefa et al., 2003). On the other hand, ISSR markers have never been applied to studies focusing on the genetic diversity of the various other species of millet genera (such as Panicum, Echinochloa, Coix, and Digitaria). There is a deficit of information about the levels of genetic variability among different millet species, and there is also no information available about the genetic relationships among the different genera. The motivation for this study was to uncover the linkages within the group of millet species by the use of ISSR markers. MATERIALS AND METHODS Plant materials A set of 69 accessions, belonging to 8 millet genera, was used (Table 1). Selected millet samples were obtained from the Czech Gene Bank of the Crop Research Institute (CRI), Prague, Czech Republic; from the Botanical Garden of Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Czech Republic; as well as from the United States Department of Agriculture (USDA), Iowa State University. DNA extraction, ISSR amplification, and scoring Young leaves were obtained from plants grown in the greenhouses at the Botanical Garden of Faculty of Tropical AgriSciences CULS Prague, Czech Republic. The fresh leaves were frozen using liquid nitrogen to be ground into a fine powder. Total genomic DNA was isolated using an Invisorb® Spin Plant Mini Kit (Stratec Molecular, Berlin, Germany). The DNA concentration was determined using a Micro-spectrophotometer, UVS-99 (ACT Gene, Piscataway, NJ, USA). A portion of the DNA was diluted to 50 ng/μl, for use in the ISSR analysis, and both the stock and diluted portions were stored at -20°C. A set of 30 ISSR primers (University of British Columbia, Vancouver, Canada) were tested. A set of testing samples, which consisted of every millet species, was used to screen for suitable primers. Twelve reproducible ISSR primers were selected for the final analysis (Table 2). Every 20 μl of PCR reaction mixture was composed of: 10 μl of 2x PPP Master Mix [150 mM Tris-HCl, pH 8.8 (25°C), 40 mM (NH4)2SO4, 0.02% Tween, 20.5 mM MgCl2, 400 μM dATP, 400 μM dCTP, 400 μM dGTP, 400 μM dTTP, 100 U/ml Taq-Purple DNA polymerase, monoclonal antibody anti-Taq (38 nM), stabilizers, and additives (Tob-Bio, Czech Republic)], 10 μM of respective ISSR primer (Integrated DNA Technologies, Belgium), 2 μl of DNA (50 ng/μl), 0.2 μl of BSA (Thermo Scientific, USA), and 7.3 μl PCR H2O (Top-Bio, Czech Republic). The ISSR analysis was carried out using a QB96 Server Gradient Thermal Cycler (Quanta Biotech, UK). The PCR was carried out with modifications of the annealing temperature to optimize the reaction for individual primers. The cycling conditions were as follows: initial denaturation step at 95oC for 4 min, followed by 45 cycles of denaturation at 94oC for 30 s, primer annealing at 45 58oC for 45 s (Table 2), and extension at 72oC for 2 min, followed by a final extension at 72oC for 10 min. Amplified products were mixed with loading dye (Thermo Scientific, USA) and loaded onto the gel. Electrophoretic separation was performed on 2% agarose Dvořáková, et al.: Genetic diversity of millets Emir. J. Food Agric ● Vol 27 ● Issue 8 ● 2015 619 Name Donor Code of donor Origin 1 Pennisetum ciliare (L.) Link USDA PI 161631 South Africa 2 Pennisetum ciliare (L.) Link USDA PI 229662 02 Madagascar 3 Pennisetum glaucum (L.) R. Br. USDA PI 288801 India 4 Pennisetum glaucum (L.) R. Br. USDA PI 337492 Brazil 5 Pennisetum glaucum (L.) R. Br. USDA PI 331353 01 Uganda 6 Pennisetum glaucum (L.) R. Br. USDA PI 343842 01 Senegal 7 Pennisetum glaucum (L.) R. Br. USDA PI 527413 01 Algeria 8 Pennisetum glaucum (L.) R. Br. USDA PI 532176 01 Oman 9 Pennisetum purpureum Schumach. USDA PI 316421 02 Mexico 10 Pennisetum sieberianum (Schltdl.) Stapf & C. E. Hubb. USDA PI 532675 Mali 11 Setaria italica L. subs. italica USDA PI 464525 India 12 Setaria italica L. subs. italica USDA PI 212626 Afghanistan 13 Setaria italica L. subs. italica USDA PI 433391 Taiwan 14 Setaria incrassata (Hochst.) Hack. USDA PI 209210 South Africa 15 Setaria pumila (Poir.) Roem. & Schult. USDA PI 206460 Turkey 16 Panicum bergii Arechav. USDA PI 310031 01 Brazil 17 Panicum coloratum L. USDA PI 224991 01 South Africa 18 Panicum coloratum L. USDA PI 225580 01 South Africa 19 Panicum coloratum L. USDA PI 225582 01 Zambia 20 Panicum coloratum L. var. coloratum USDA PI 226085 01 Kenya 21 Panicum deustum Thunb. USDA PI 364955 01 South Africa 22 Panicum dregeanum Nees USDA PI 364956 01 South Africa 23 Panicum lanipes Mez USDA PI 238346 01 Zaire 24 Panicum miliaceum L. CULS not known 25 Panicum miliaceum L. CULS not known 26 Panicum schinzii Hack. USDA PI 284153 01 Cyprus 27 Panicum sumatrense Roth USDA Ames 14464 India 28 Panicum virgatum L. USDA PI 421901 01 Florida, USA 29 Eleusine coracana (L.) Gaertn. USDA PI 214059 India 30 Eleusine floccifolia (Forssk.) Spreng. USDA PI 196853 Ethiopia 31 Eleusine indica (L.) GAERTN. CRI 14Z2500001 Belgium 32 Eleusine indica (L.) Gaertner. USDA PI 226270 01 Zimbabwe 33 Eleusine multiflora Hochst. ex A. Rich. USDA PI 226067 01 Kenya 34 Eleusine tristachya KUNTH. CRI 14Z2500002 Sweden 35 Coix lacryma‐jobi L. USDA PI 324509 Japan 36 Coix lacryma‐jobi L. USDA PI 320865 India 37 Eragrostis bahiensis Schrad. ex Schult. USDA PI 203648 Brazil 38 Eragrostis capensis (Thunb.) Trin. USDA PI 364803 Brazil 39 Eragrostis cilianensis subs. starosselskyi (Grossh.) Tzvelev USDA PI 212297 Afghanistan 40 Eragrostis curvula (Schrad.) Nees USDA PI 156818 South Africa 41 Eragrostis cylindriflora Hochst. USDA PI 364817 South Africa 42 Eragrostis lappula Nees USDA PI 364260 Brazil 43 Eragrostis lugens Nees USDA PI 203862 Brazil 44 Eragrostis obtusa Munro ex Ficalho & Hiern USDA PI 344546 South Africa 45 Eragrostis pilosa (L.) P. Beauv. USDA PI 223259 Afghanistan 46 Eragrostis plana Nees USDA PI 364340 South Africa 47 Eragrostis racemosa (Thunb.) Steud. USDA PI 192959 Kenya 48 Eragrostis rigidior Pilg. USDA PI 364824 Afghanistan 49 Eragrostis rotifer Rendle USDA PI 208131 South Africa 50 Eragrostis rotifer Rendle USDA PI 364825 Afghanistan 51 Eragrostis secundiflora subsp. oxylepis (Torr.) S. D. Koch USDA PI 295692 South Africa 52 Eragrostis superba Peyr. USDA PI 442111 Japan 53 Eragrostis tef (Zuccagni) Trotter USDA PI 494388 Ethiopia 54 Eragrostis tef (Zuccagni) Trotter USDA PI 442115 Japan 55 Echinochloa crus‐galli (L.) BEAUV. CRI 01Z3010001 Czech Republic 56 Echinochloa crus‐galli (L.) BEAUV. CRI 01Z3010002 Czech Republic Table 1: Millet accessions


INTRODUCTION
Millets represent a diverse group of cereal crops, comprising about a dozen crop species.They belong to different genera, which originated in Africa and Asia, were then subsequently domesticated, and are still cultivated there (McKevith, 2004;Baltensperger and Cai, 2004;FAO, 1995).Millets are small-grain cereals from the grass family (Poaceae) (Baltensperger and Cai, 2004).The millet group is split into two tribes.The tribe Paniceae comprises a number of different species such as Pennisetum glaucum (L.) R. Br., Setaria italica (L.) P. Beauv., Panicum miliaceum L., Coix lacryma-jobi L., Eragrostis tef (Zuccagni) Trotter, Echinochloa crus-galli (L.) P. Beauv., Digitaria exilis (Kippist) Stapf (Belton and Taylor, 2003).Finger millet (Eleusine coracana Gaertn.) is the only species of millet belonging to the second tribe, Chlorideae (Desai, 2004).A distinctive attribute of the millets are their adaptability to adverse agroecological conditions, minimal input requirements, and good nutritional properties.Millets represent a unique biodiversity component in agriculture, and play a significant role in food security for the developing countries in Asia and Africa.They also play a growing role in the processing, and new alternative products for the developed world (Obilana and Manyasa, 2002).From the nutritional point of view, millets are equivalent (or even superior to) other cereals (Lasztity, 1996;Obilana and Manyasa, 2002); moreover, they do not contain glutenforming proteins, making them important in a celiac diet (Amadou et al., 2013;Taylor et al., 2006).Compared to other cereals, millets are mainly suited to less fertile soils and poorer growing conditions, such as intense heat and low rainfall, where other cereal crops may likely fail (National Research Council, 1996;Winch, 2006).Beyond these indisputable qualities, many millet species have an important cultural significance, and play an irreplaceable role in social events and celebrations of the local people.Millets represent crucial plant genetic resources for the agricultural and food security of poor farmers inhabiting arid, infertile, and marginal lands (Garí, 2002).
Knowledge about the genetic diversity, and revelations of the genetic relationships among millet species is essential for the suitable conservation and increased use of millet genetic resources, and it also plays an important role in millet breeding.The application of methods using DNA analysis is pivotal for the description of genetic variability within different millet species.One of the alternatives is the use of Inter Simple Sequence Repeats (ISSR) markers, which is known to be a highly variable, reproducible, and cost effective method (Wolfe and Liston, 1998;Yang et al., 1996).Comparing ISSR markers with Random Amplified Polymorphic DNA (RAPD) analysis, there are many advantages on the side of ISSR, which can reveal a greater level of genetic variability.Using longer primers and higher annealing temperatures, they provide results that are more reliable and reproducible (Wolfe and Liston, 1998).
ISSR has been widely used in studies of the genetic structure of plants (Li and Jin, 2008), genetic diversity (Sheeja et al., 2009), genetic relationships (Li et al., 2009), phylogeny and evolution (Zamani et al., 2011).They have also been successfully applied to a number of monocotyledonous plants (Ben El Maati et al., 2004;Mondini et al., 2014;Bahieldin et al., 2012;Virk et al., 2000).Findings of genetic similarities in millet species have been performed using ISSR markers in Pennisetum, Setaria, Eleusine and Eragrostis genera (Pedraza-Garcia et al., 2010;Lin et al., 2012;Salimath et al., 1995;Assefa et al., 2003).On the other hand, ISSR markers have never been applied to studies focusing on the genetic diversity of the various other species of millet genera (such as Panicum, Echinochloa, Coix, and Digitaria).
There is a deficit of information about the levels of genetic variability among different millet species, and there is also no information available about the genetic relationships among the different genera.The motivation for this study was to uncover the linkages within the group of millet species by the use of ISSR markers.

Plant materials
A set of 69 accessions, belonging to 8 millet genera, was used (Table 1).Selected millet samples were obtained from the Czech Gene Bank of the Crop Research Institute (CRI), Prague, Czech Republic; from the Botanical Garden of Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Czech Republic; as well as from the United States Department of Agriculture (USDA), Iowa State University.

DNA extraction, ISSR amplification, and scoring
Young leaves were obtained from plants grown in the greenhouses at the Botanical Garden of Faculty of Tropical AgriSciences CULS Prague, Czech Republic.The fresh leaves were frozen using liquid nitrogen to be ground into a fine powder.Total genomic DNA was isolated using an Invisorb ® Spin Plant Mini Kit (Stratec Molecular, Berlin, Germany).The DNA concentration was determined using a Micro-spectrophotometer, UVS-99 (ACT Gene, Piscataway, NJ, USA).A portion of the DNA was diluted to 50 ng/μl, for use in the ISSR analysis, and both the stock and diluted portions were stored at -20°C.
A set of 30 ISSR primers (University of British Columbia, Vancouver, Canada) were tested.A set of testing samples, which consisted of every millet species, was used to screen for suitable primers.Twelve reproducible ISSR primers were selected for the final analysis (Table 2).
The PCR was carried out with modifications of the annealing temperature to optimize the reaction for individual primers.The cycling conditions were as follows: initial denaturation step at 95ºC for 4 min, followed by 45 cycles of denaturation at 94ºC for 30 s, primer annealing at 45 -58ºC for 45 s (Table 2), and extension at 72ºC for 2 min, followed by a final extension at 72ºC for 10 min.Amplified products were mixed with loading dye (Thermo Scientific, USA) and loaded onto the gel.Electrophoretic separation was performed on 2% agarose gel in 1x TBE buffer.Gels were run for about 2.5 -3 h at 4 V.cm -1 .Gels were stained with SYBR ® Safe DNA Gel Stain (Life technologies, USA), and visualized with a UV transilluminator.The banding pattern was recorded using a CSL-MICRODOC System (CLEAVER, United Kingdom).
PCR amplification of the samples with each primer was carried out in duplicate to ensure the consistency and reproducibility of the results.

Data analysis
ISSR fragments were scored for the presence (1) or absence (0) of bands in the gel profile.Only strong and clear bands were used to construct a binary matrix.The binary matrix was used to calculate a dissimilarity matrix using Dice's coefficient (Dice, 1945).Data were analyzed using DARwin5 software (Perrier and Jacquemoud-Collet, 2006), and then a final Neighbour joining (NJ) dendrogram (Saitou and Nei, 1987) was constructed by means of the UnWeighted Neighbor-Joining method.Shannon's information index (I, LogBase = e) was estimated by fingerprinting analysis with missing data (FAMD) software, version 1.31 (Schlüter and Harris, 2006) for all accessions according to Hutchenson (1970) and normalised according to Ramezani (2012).The percentage of polymorphic bands and Nei's genetic distance (Nei, 1972;Nei and Takezaki, 1983) were calculated by using FAMD.The Principal Coordinates Analysis (PCoA) was performed by software DARwin 5.0 using the data obtained from the calculation of the Dice's coefficient.
The number of bands generated by the ISSR primers within a single genus varied from 31 to 230.The level of polymorphism within a single genus varied from 12.06% in the Coix genus to 89.88% in the Eragrostis genus, with a mean of 65.81%.

Genetic diversity within and among genera
Shannon's index among all 69 samples was estimated at 0.9689.The Nei's genetic distance matrix among all millet accessions was found to be in the range of 0. At the genus level, the values of Nei's genetic distance indicated a high level of variation (Table 3).The greatest distance was found between the Coix and Setaria genera (0.1476), while the lowest genetic distance was between the Eragrostis and Panicum genera (0.0163).

Cluster analyses based on the ISSR genotyping profile
A dendrogram based on Neighbour joining analysis of the ISSR data was constructed in order to be able to infer the phylogenetic relationships among 69 millet accessions belonging to eight genera.The dendrogram showed that, in most cases, accessions of the same millet genus clustered together (Fig. 1).

DISCUSSION
Despite the importance of millets, the available information of both their phylogenetic relationships and genetic diversity, using molecular markers, is still rather limited.In most of the studies, which were focused on genetic diversity and the relationships of millets, only one millet species or genus (Li et al., 2012;Assefa et al., 2003;Yu et al., 2006;Salimath et al., 1995, Arya et al., 2013, Kim et al., 2014) was often involved; whereas, the current study was aimed at revealing the relationship among and within different millet genera, simultaneously.
In the present study, a Neighbour joining dendrogram offers a new perspective towards an understanding about the relationships at the inter-specific/intra-generic levels.
The accessions were divided into eight clusters (Fig. 1), where a single genus for the most part grouped together, but did not form separate clusters, contrary to our expectations.
The Eragrostis accessions were split into three different clusters, which affirmed the fact that Eragrostis is a large and taxonomically complex genus (Tefera et al., 2006).The level of polymorphism among the evaluated Eragrostis accessions was 89.88%, which corresponds with the findings of Bai et al. (2000), who found a high level of polymorphism in wild Eragrostis species by using RAPD analysis.At the same time, they revealed a relatively low polymorphism in the Eragrostis tef accessions.ISSR analysis was also used to uncover the genetic diversity in Eragrostis tef (Assefa et al., 2003).They obtained much lower estimates for genetic similarity among the Eragrostis accessions (0.26 -0.86).Bai et al. (2000) reported that Eragrostis tef is very close to Eragrostis pilosa, supporting the hypothesis of Ebba (1975) that Eragrostis tef originated from this species.Ayele and Ngyuen (2000) reported that E. pilosa was more closely related to E. tef than E. curvula.Our data are at variance with those findings, because Nei's genetic distance between Eragrostis tef (accession Nos.53 and 54) and Eragrostis pilosa (accession No. 45) were observed at 0.1532 and 0.1725, respectively.Our findings also showed that E. curvula was more closely related to E. tef.
In order to enhance an understanding of the diversity and relationships in the Pennisetum genus, an ISSR analysis incorporated cultivated, wild, and weed Pennisetum species -P.glaucum, P. purpureum, P. ciliare, and P. sieberianum, respectively.Unfortunately, revealing the relationships among Pennisetum species is rather complicated because Pennisetum is a highly cross-pollinated crop, with large numbers of wild relatives, including those that can be inter-crossed (Jauhar, 1968(Jauhar, , 1981;;Jauhar and Hanna, 1998).
The results of present study may also support this fact, since Pennisetum purpureum was highly differentiated from another Pennisetum accession.The Pennisetum purpureum accession was even present in another/different cluster, and could be detected as an admixed individual, as was similarly revealed by Oumar et al. (2008).Regarding to the genetic relationships of another Pennisetum accession, the clustering showed a close relatedness among the domesticated species P. sieberianum and P. glaucum.Donadío et al. (2009) obtained similar results; however, they also uncovered a close relatedness of these Pennisetum species to P. purpureum, which is in disagreement with the data obtained in current study.In all likelihood, these differences might be attributed to variations in the type and number of genotypes, as well as to the techniques employed.Additionally, it should be noted that the grouping of P. glaucum and P. ciliare (syn.Cenchrus ciliaris) accessions strongly supports the finding that Pennisetum and Cenchrus are closely related genera (Clayton and Renvoize, 1986;Crins, 1991).According to Clayton and Renvoize (1986), Cenchrus ciliaris is even considered to be on the boundary between Cenchrus and Pennisetum; and findings of present study support this fact.Although P. ciliare accessions were present in the Pennisetum clusters, they were rather distant from other Pennisetum accessions.Regarding the clustering of all Pennisetum accessions, the clustering reflects both the complicated taxonomy of the Pennisetum genus and of clustering according to geographical origin, which is evident in one of the Pennisetum clusters.
Surprisingly, the Coix lacryma jobi accessions occurred in the Pennisetum cluster, as well (Cluster III).The reason for the clustering of these two Coix accessions with the Pennisetum accessions might have been caused by cross-pollination, which is predominant in Coix sp.(Jansen, 2006).Similarly, the Digitaria exilis accession was present in the same cluster, which supports the view that Pennisetum and Digitaria are considered being a distantly related (Hacker 1995).
Additionally, results of the current study demonstrate the complexity of the Digitaria species.These accessions are scattered throughout the entire dendrogram, which reflects considerable variability in the Digitaria genus.Hayward and Hacker (1980) attributed this large specific diversity within the Digitaria genus to its great antiquity, but also to its significant rate of speciation.Findings of present study showed high genetic divergences between the cultivated Digitaria exilis and the other taxonomically distant Digitaria species (Table 1), which is in concordance with the results obtained by Adoukonou-Sagbadja et al. (2010).Among the wild species investigated, D. eriantha and D. sanguinalis, there were observed as the most distant, genetically, from the cultivated Digitaria exilis.Nevertheless, it should be noted that different hypotheses exist on the reproductive system of Digitaria species, ranging from inbreeding (Watson and Dallwitz, 1992;Sarker et al., 1993) to out-crossing (Hilu et al., 1997).
Although the Setaria genus is also a complex genus containing crop, wild, and weedy species, with different breeding systems at the life cycle and ploidy levels (Wanous, 1990), all Setaria accessions investigated in the present study grouped together (Fig 1).Thus, our findings do not support the results of the phylogenetic studies performed by Doust et al. (2007) or Kellogg et al. (2009), which indicated that the Setaria genus is a collection of unrelated groups.Although Setaria accessions formed a distinct branch in the present study, no clear geographic structure within the genus was found, contrary to the findings of Li et al. (2012), in which a clear geographic structure was revealed by using ISSR markers.In general, the geographic center from which Setaria originated is still controversial.Single and multiple centers of origin for Setaria have been suggested in Eurasia.A center in northern China was first suggested by Vavilov (1926), and confirmed by many archaeologists and archaeobotanists (Smith, 1998;Lu, 1999;Shelach, 2000); now with some genetic studies having confirmed the existence of this center (Hirano et al., 2011;Li et al., 2012).Nevertheless, the multiple domestication theory is widely accepted (Kawase and Sakamoto 1987;Li et al., 1998;Benabdelmouna et al., 2001;Kawase et al., 2005;Fukunaga et al., 2005Fukunaga et al., , 2006)).Despite the limited number of accessions used in the present study, the clustering of the Setaria accessions, and relatively high level of similarity, might suggest a hypothesis of a single center of domestication.Unfortunately, the origin of Setaria still has remained unresolved, and further detailed analysis using large numbers of accessions is required.
The Echinochloa genus is also a taxonomically complicated genus, because clear-cut boundaries between species seldom exist, and the species are very variable.Introgression between species is also common (Brink and Belay, 2006).Last but not least, its great diversity is also caused by its easy adaptation to a wide range of aquatic and ruderal habitats, combined with self-pollination (Partohardjono and Jansen, 1996).These genetic and morphological differences in the Echinochloa species lead to taxonomic problems.Thus, many studies have attempted to understand the population genetic structures of some Echinochloa species, and have revealed their genetic diversity by using molecular markers (Asins et al., 1999;Roy et al., 2000;Rutledge et al., 2000;Tasrif et al., 2004;Altop and Mennan, 2011;Nozawa et al., 2006;Danquah et al., 2002).As with previous studies, the Echinochloa species in the present study were differentiated, and might confirm the theory of Yabuno (1966) and Scholz (1992), that the weedy Echinochloa crus-galli has its cultivated counterpart Echinochloa esculenta.Altop and Mennan (2011) mentioned that the variability among Echinochloa accessions from various locations might be due to its adaptability to the geographic locations, as well as differences in weed management practices.
ISSR analysis revealed a high level of polymorphism in the Panicum genus (89.49%).In the present study, the scattering of Panicum accessions throughout the NJ dendrogram (Fig. 1) and PCoA diagram (Fig. 2) clearly confirmed the fact that the Panicum genus is extremely variable (Hacker, 1995).
The most variable Panicum species in the current study is Panicum coloratum, which is a polymorphic species native to tropical Africa.Furthermore, attributes such as predominant cross-pollination or the development of ecotypes adapted to a wide range of soils (Hacker 1995)  In contrast to Panicum, the Eleusine genus belongs to a relatively small genus (Phillips, 1972;Hilu and de Wet, 1976), but the classification of the genus has been notoriously difficult, not only at the intra-generic level, where considerable disagreements on species delimitation and their relationships have persisted (Bisht andMukai, 2001, 2002;Lye, 1999;Phillips, 1972Phillips, , 1995)).This is also true at the supra-generic level, where its closest allies are disputed (Clayton and Renvoize, 1986).Therefore, some studies have used a comparative analysis of DNA markers in order to get greater knowledge of the interrelatedness of the Eleusine species (Gupta et al., 2010;Hiremath and Salimath, 1992;Salimath et al., 1995).Hiremath and Salimath (1992) used molecular markers to uncover the genetic affinities between Eleusine coracana and three diploid species (viz.Eleusine indica, Eleusine floccifolia, and Eleusine tristachya), which are believed to form a close genetic assemblage within the genus.These results are inconsistent with those obtained in the present study.The Eleusine species were clearly separated and scattered throughout the dendrogram, which might indicate an implemented sorting of the Eleusine species.Hence, these findings could be of value towards a better characterization of the genus.Another interesting finding is that the ISSR technique used in the present study revealed a high level of polymorphism (74%), which is suggestive of the ISSR technique being a most promising tool for uncovering of plant diversity.These findings are in agreement with a comparative study performed by Salimath et al. (1995).
Regarding the level of polymorphism at the genera level, the findings revealed in the present study a broad range of polymorphism.These unique findings confirmed that millets are more or less related.The determination of relatedness might help to resolve the difficult interrelationships among the different millet genera.Despite the fact that Eragrostis and Panicum are complex and variable genera (Tefera and Belay, 2006;Hacker, 1995), the data demonstrated that these genera are the most similar.Further, a close relatedness was also observed between the Eragrostis and Pennisetum genera (Table 3).The genus Pennisetum is also considered to be distantly related to Digitaria (Hacker 1995), which relatively corresponds with our findings.Furthermore, according to our data, the Digitaria genus is close to the Eleusine genus.On the other hand, the Eleusine genus is most distant from the Setaria genus.There has been a hypothesis that Setaria had evolved from Panicum (Brink and Belay, 2006).Nevertheless, the results of this study could not unambiguously confirm this hypothesis due to the relatively high genetic distance.However, the most distant genus from other millet genera implemented in the present study was Coix; with knowledge about this species still being limited.

CONCLUSIONS
To the best of our knowledge, this is the first report where different millet genera were compared simultaneously using ISSR markers.These markers confirmed the presence of a high level of genetic variability among and within the different millet genera.Further, the ISSR cluster analysis, revealed that the majority of accessions of a given genera tend to group together.On the other hand, it must be noted that in some cases the genus boundaries were not very rigid, and accessions of a given genera were scattered throughout the dendrogram.This clustering is probably due to the variation in the types and number of genotypes, species admixtures, different origins of the accessions, different propagation systems, and their ability to adapt to different geographic conditions.Additionally, the results of the present study clearly confirmed the concept that millets are a complex group.Although the present investigation shed some light on a better understanding of the genetic diversity in millets, further studies are required to improve our understanding of the phylogenetic relationships as well as the genetic diversity in millets at the genera/species level.
might be responsible for the segregation of P. coloratum accessions from other Panicum accessions.Also, the Panicum sumatrense accession was separated from other Panicum accessions, which reflected a high variability (van der Hoek andJansen, 1996).Further, the clustering of P. sumatrense with Setaria accessions is quite in agreement with data obtained byLakshmi et al. (2002), where P. sumatrense and Setaria italica accessions were present in the same cluster.In addition,Aliscioni et al. (2003) mentioned that the genus shows a wide range of variation, and relationships within the Panicum genus are not completely clear, which was also evident in our study.Also, M'Ribu andHilu (1994) studied the variation among Panicum species, and their findings revealed the differentiation of individual millet species, which is in accordance with the data obtained in this study.Although most Panicum accessions employed in the present analysis originated in Africa, there were no clear separations of the accessions according to their geographic origins, contrary to the data obtained byHu et al. (2008) andHunt et al. (2011).

Table 1 : (Continued...)
Similar to Cluster I, Cluster II was also mostly formed by Eragrostis accessions.Also present were accessions of other millet genera, viz.Eleusine, Pennisetum, Panicum, and Digitaria.Cluster III primarily contained Pennisetum accessions, followed by Coix, Digitaria, and Eragrostis accessions.Pennisetum accessions of this cluster grouped together; another five Pennisetum accessions did not fall within the same group, but fell into a different cluster (Cluster 7); meaning that the intraspecific genetic diversity among those accessions was large.Although Coix accessions (Nos.36 and 35) clustered with other millet genera, these two accessions formed a distinct branch.Both accessions originated in Asia, specifically in India and Japan.
According to the dendrogram, all 69 accessions were separated into eight clusters.Cluster I consists of 11 millet accessions belonging to four millet genera, viz.Eragrostis, Eleusine, Panicum, and Digitaria.The Eragrostis accessions formed a distinct branch consisting of four accessions; whereas, Eragrostis cylindriflora -No.41 (South Africa origin) and Eragrostis racemosa -No.47 (Kenyan origin) showed the highest similarity to all millet accessions.Three Digitaria accessions (Nos.62, 67, and 68) also comprised a distinct branch.

Table 3 : Genetic distance matrix among 8 millet genera
In Cluster IV, Setaria, Digitaria, Echinochloa, and Panicum accessions clustered together.The Setaria accessions formed a distinct branch consisting of five Setaria accessions(Nos.11,12,13, 14, and 15), with one accession of Panicum sumatrense -No.27.The other two accessions of Panicum, viz.Panicum coloratum -No.17 and Panicum bergii -No.16 showed quite high dissimilarities.Panicum dregeanum -No.22, and Panicum coloratum -No.19.Other Panicum accessions appeared scattered in the dendrogram, possibly due to the large intraspecific genetic diversity among the Panicum accessions.Cluster VI showed a high degree of admixtures of millet genera; the cluster was formed by Panicum, Eleusine, and Digitaria accessions.The accessions Panicum coloratum -No.18 and Eleusine indica -No.32 were quite similar.
Fig 1. NJ dendrogram showing relationships among and wihtin different millet genera.Dendrogram constructed on the basis of ISSR markers.Cluster VII was formed almost completely by accessions of one millet genus, specifically by the Pennisetum genus.Four of these five Pennisetum accessions belong to the same species -Pennisetum glaucum, specifically Nos. 5, 6, 7, and 8.The fifth Pennisetum accession of that cluster was accession No. 2 -Pennisetum ciliare.From these five accessions, two accessions of Pennisetum glaucum were the most similar, specifically, Nos.6 and 5. Cluster VIII primarily included Eragrostis accessions and one accession of Digitaria and Eleusine, Nos.65 and 31, respectively.Digitaria gazensis -No.65 showed a high similarity with Eragrostis rotifer -No.49.The most similar were Eragrostis rotifer -No.50 and Eragrostis lappula -No.42.