GENETIC VARIATION AND PHYLOGENETIC RELATIONSHIPS AMONG MAIZE TYPES AND TEOSINTE AS REVEALED BY ISOZYMES AND RAPD MARKERS

aize or corn (Zea mays L., 2n = 20) is one of the most diverse grain crops found in nature. Selection pressure by both humans and nature has resulted in many different types of corn that vary in physical and chemical characteristics. The most common types of corn include dent, flint, flour, sweet, pop, and waxy corn (Knott et al., 1995). The physical appearance of each kernel type is determined by its pattern of endosperm composition as well as quantity and quality of endosperm (Zilic et al., 2011).

Teosinte is the wild relative of maize. Both teosinte and maize belong to the genus Zea, which has four species: Z. luxurians, Z. diploperennis, Z. perennis and Z. mays. Zea mays is in turn divided into four subspecies; mexicana huehuetenangensis, parviglumis and mays. Zea mays ssp. mays is the only cultivated species (maize), the other species and subspecies are wild grasses, referred to as teosintes (Doebley, 1990).
Analysis of genetic divergence in maize can provide some interesting information about differentiation, adaptability and interrelationships of genotypes and giving graphical assessment of genetic variability. A number of methods are currently available for analysis of genetic diversity in germplasm accessions, breeding lines and populations. These methods have relied on pedigree data, morphological data, agronomic performance data, biochemical and molecular (DNA-based) data. Isozymes are good biochemical markers used as a powerful tool both in characterization of cultivar and in genetic and phylogenetic studies for many plant species (Orasmo et al., 2007;Frigo et al., 2009). Isozymes were developed as first co-dominant markers (Ivy et al., 2010). Although, nowadays new DNA-based molecular techniques are used, isozymes still represent a powerful tool for evaluation of genetic variability within and between populations of plants (Zeidler, 2000). Several recent studies have confirmed that the use of isozymes analysis as a biochemical marker can be useful for genomic assessment in Zea mays L. breeding programs (Pereira et al., 2008;Dowd et al., 2010).
Applications of molecular markers, particularly in the last two decades, have led to new insights into domestication events in maize (Matsuoka et al., 2002b), assessing the genetic diversity in the M maize gene pool, understanding phylogenetic relationships and gene flow between maize landraces and the wild relative, teosinte (Van-Heerwaarden et al., 2011;Warburton et al., 2011), helping in tracking the migration routes of maize from the centers of origin (Vigouroux et al., 2008) and understanding the fate of genetic diversity during maize domestication. Molecular markers are stable and detectable in all plant tissues regardless of the growth, differentiation, development or status of cells. They have also many advantages such as abundance in polymorphism, no pleiotrophic effect; less affected by environment and subjected to rapid detection (Agarwal et al., 2008). Among molecular markers, random amplified polymorphic DNAs (RAPDs) which have been extensively used in genetic research owing to their speed and simplicity (Williams et al., 1990). This technique has potential to differentiate and detect differences among genotypes (Schulman, 2007;Bernardo, 2008).
The present work was undertaken to highlight the genetic variation and phylogenetic relationships among maize types (dent, flint, sweet and pop corn) and their wild relative (teosinte) using peroxidase and esterase isozymes in addition to RAPD markers.

Plant materials
A local ecotype of teosinte (Zea mexicana) and four different types of yellow maize (Zea may) including types of the dent, flint, sweet and pop corn were chosen for this study (Table 1). The grains of these genotypes were provided by germplasm bank of the Department of Maize Research, Agricultural Research Center, Giza, Egypt.

Germination and harvest
Twenty grains of each genotype were planted in pots at the Department of Genetics, Faculty of Agriculture, Kafr El-Sheikh University, Kafr El-Sheikh, Egypt. Four weeks after planting, the second and the third leaf were harvested and stored at -20C until performing the isozymes and RAPD assays.

Electrophoretic analysis of isozymes
Two isozymes systems (peroxidase and esterase) were applied in nondenaturing polyacrylamide gel electrophoresis for detecting isozymes variation among the five studied genotypes. Leaf samples (0.250 gm) were frozen with liquid nitrogen and ground in a mortar and pestle with 1 ml of 20% sucrose to extract peroxidase and esterase isozymes. Peroxidase isozymes were localized on the gel using the staining solution composed of 250 mg of benzidine dihydrochloride (moistened with 4 drops of glacial acetic acid) in 100 ml H 2 O and 10 drops of 1% H 2 O 2 was added immediately before being used according to Scandalios (1964). However, esterase isozymes patterns were detected on the gel as described by Vallejos (1983) using α and β-naphthyl acetate (40 mg of each) and 250 mg Fast blue RR dissolved in 98 ml of 0.1 M phosphate buffer (pH 6.5).

DNA extraction
For isolating genomic DNA, equal quantities of leaf tissue harvested from 20 seedlings per genotype at 2-3 leaf stage were bulked and used in DNA isolation using CTAB-chloroform based method according to Saghai-Maroof et al. (1984).

Random amplified polymorphic DNA (RAPD) analysis
RAPD analysis was carried out using seven decamer random primers (OP-A9, OP-B5, OP-B6, OP-B7, OP-B8, OP-B10 and OP-B14) which were purchased from Bio Basic Inc, Canada. The list of primers and their sequences are presented in Table (

Data analysis
The data generated from isozymes (native gel) banding patterns in addition to the banding patterns of the seven RAPD primers were introduced to SPSS package program according to binary values of (1) and (0) for the presence and absence of bands, respectively. The genetic distances among the genotypes were assessed based on Jaccard's similarity coefficient (Jaccard, 1901) using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) analysis (Nei, 1973).

Genetic polymorphism among the studied genotypes a. Isozymes polymorphism
Genetic variations at the biochemical level were studied using nativepolyacrylamide gel electrophoresis (Native-PAGE). Considerable variations in both isozymes systems; peroxidase and esterase, were found among all the studied genotypes ( Fig. 1). A total of 51 bands with 80.39% polymorphism were detected using the two isozymes systems, ten bands were monomorphic and the other 41 bands were polymorphic (Table 3). This polymorphism level was higher than the polymorphism data recorded by Stuber and Goodman (1983) from 23 isozymes loci which provide unique patterns for 73% of the 406 maize inbred lines they surveyed. In the current study, the PAGE migration pattern of peroxidase gave a total of 24 bands for the studied maize types and their ancestor teosinte, 16 bands were polymorphic (66.67% polymorphism) while eight bands were found to be common in all genotypes. Native-PAGE analysis for esterase isozymes exhibited a total of 27 bands, 25 polymorphic bands (92.59% polymorphism) and two monomorphic bands. This result indicated that esterase isozymes exhibited higher polymorphic patterns than peroxidase among the studied genotypes. Esterases in plants have been intensively explored as biochemical markers due its high allele polymorphism (Carvalho et al., 2003;Resende et al., 2004) and its high number of loci by using the polyacrilamide gel electrophoresis system (Carvalho and Machado, 2004). On the other hand, the electrophoretic bands; for both peroxidase and esterase isozymes, showed wide variation in their intensities ranging from faint to dark, reflecting different activities in the seedling tissues of these genotypes. These results might reflect a case of variations in isozymes banding patterns among tested genotypes. The observed isozymes patterns with both peroxidase and esterase in selected genotypes can be explained in terms of allelic differences. Isozymes are all functionally similar forms of enzymes including all polymers of subunits produced by different gene loci or by different alleles at the same locus. Their electrophoretic mobilities are the result of different size and shapes of enzyme mole-cules and their variation is a good indicator of genetic diversity (Shannon, 1968). So, electrophoresis separation of isozymes has been widely used both in taxonomic and genetic studies in maize. In this regard, Pereira et al. (2008) utilized isozymes markers for assess the changes in the genetic variability and distance in a Brazilian composite population of popcorn following four cycles of recurrent selection for yield. They found that the variability detected in this population was less than the breeds of the Northern Flint or Southern Dent (Labate et al., 2003). Many of the Mexican and South American landraces of maize have already been characterized using isozymes (Sanchez et al., 2000(Sanchez et al., & 2007. Furthermore, Gimenes and Lopes (2000) used isozymes analysis in studying 15 maize populations derived from three indigenous maize races.

b. RAPD Polymorphism
Using RAPD analysis with the five genotypes, a total of 116 bands were obtained from seven decamer primers (Fig.  2). The number of bands ranged from 6 bands for the primer OP-B10, to 24 bands for the primers OP-B6 and OP-B7 (Table  3). The primer OP-B6 produced the highest number of polymorphic bands (24 bands). On the other hand, the primer OP-B10 generated only four polymorphic bands. The percentage of polymorphic loci was 87.07% which indicates a high level of polymorphism. This level of polymorphism was higher than the values observed in some maize studies, such as Bruel et al. (2006) who obtained 84.44% of polymorphism in case of studying genetic divergence between maize inbred lines using RAPD markers. In addition, Carvalho et al. (2002) had reported 75.8% polymorphism with ISSR markers in maize. The obtained level of polymorphism depends on the degree of divergence between the genotypes under study. Molecular characterization of maize landraces of India Sharma et al., 2010) and more recently of America and Europe (Warburton et al., 2011), led to significant insights with regard to the genetic diversity and population structure. Teosinte became one of the best-characterized systems in plant molecular population genetics, including studies based on DNA samples recovered from archeological specimens (Jaenicke-Despres et al., 2003).

Genetic relationships among the studied genotypes a. Genetic relationships based on isozymes markers
Similarity coefficient between each pair of genotypes for peroxidases as well as esterases is presented in Table (4). The maximum similarity coefficient of 0.842 and 0.792 for peroxidases and esterases, respectively, was found between dent and flint types indicating a high degree of genetic similarity between them. In addition, the study showed that there is an association between the dendrograms obtained by peroxidases and esterases markers (Fig.  3), which represent the phylogenetic relationships of teosinte with maize types. The dendrograms of both isozymes systems revealed two main clusters. The first cluster has most distant which include teosinte, whereas the second cluster included the four maize types, which were divided into two sub-clusters. The first sub-cluster included dent and flint types since they associated with the highest genetic similarity coefficient (0.842 and 0.792 for peroxidases and esterases, respectively) and the second sub-cluster included the types of sweet and pop corn with similarity coefficient of 0.722 and 0.667 for peroxidases and esterases, respectively. This result is in agreement with Reddy et al. (2012), who reported that today's dent corns originally came from crosses between late-flowering Southern dent corns (Gourdseed) and early-flowering Northern flints. The dendrogram of peroxidases (Fig. 3a) revealed that the two main clusters were less distant than the dendrogram of esterases (Fig. 3b). These results showed high diversity between teosinte and each type of maize.
This result is in consistent with archaeological data and previous studies on maize domestication (Koo and Jiang, 2008;Fu et al., 2010). The theory that maize originated from teosinte has also been considered in detail by Kato (1984). His study showed a very similar chromosome knob configuration in teosinte and maize, and supported the idea that gene flow from teosinte to maize occurred since the beginning of maize cultivation. This gene flow is considered to be responsible for the large genetic and phenotypic vari-ability in maize today. According to Matsuoka et al. (2002a), the diversification of maize races was a result of continuous hybridizations between diverse populations of maize with teosinte in many including novel environments. This resulted in the introgression of a large diversity into maize and might have established the maize races known today. This allowed the introgression of new traits into the different types of maize and selection within them, creating many different morphological and physiological phenotypes in maize. This confirms earlier evidence of allozyme alleles that occurred in ssp. mexicana and in sympatric maize populations, suggesting that these alleles had probably introgressed into maize from mexicana ssp. (Doebley, 1990). These results are in accordance with the findings of Abdel-Tawab et al. (1982), who reported a close relationship between maize and teosinte (Zea mexicana) based on protein electrophoretic patterns.

b. Genetic relationships based on RAPD markers
The RAPD data were used to estimate the genetic similarities and the phylogenetic relationship among the five genotypes. A similarity coefficient matrix among all genotypes is presented in Table (5). The similarity values among the five genotypes ranged from 0.322 to 0.588. The highest similarity index (0.588) was observed between the flint and sweet types, followed by the similarity index between sweet and pop corn (0.532), whereas the lowest one (0.322) was rec-orded between dent and teosinte. Moeller and Schaal (1999), using RAPD markers in Native American maize collections of Great Plains, showed a similarity index that varied from 0.44 to 0.80.
The UPGMA dendrogram based on the similarity indix separated the five genotypes into two main clusters (Fig. 4). Teosinte was the most divergent in the first cluster and the second cluster has been less distance, which consisted of all maize types. These results were in agreement with the results of isozymes which revealed high diversity among maize types and teosinte. There were close relationship among the types of flint and sweet corn, which clustered with a similarity coefficient of 0.588. This association is consistent with their common origin since both of them contain the flint gene pool. Historical records indicated that sweet corn germplasm is largely derived from the Northern Flint race of corn (Gerdes and Tracy, 1994). On the other hand, the highest estimated distance was observed between the ancestor germplasm teosinte and dent corn type. Many studies assessed the degree of genetic relatedness between maize and teosinte. Doebley and Iltis (1980) reported that the genus Zea was divided into sections Luxuriantes (teosinte) and Zea. These subgeneric boundaries of Zea are defined on UPGMA-based dendrogram derived from RAPD analysis. Gene flow among maize and teosinte populations has occurred readily since maize's domestication 9,000 years ago (Ellstrand et al., 2007;Dyer et al., 2009). Van-Heerwaarden et al. (2011) explained the genetic evidence for an apparent highland origin of modern maize by gene flow from Z. mays ssp. mexicana (teosinte).
On the other hand, many studies explained the genetic diversity in maize types. Studies comprising 28 open pollinated varieties of maize (Parentoni et al., 2001) showed that flint and semi-flint genotypes as well as the dent and semident germplasm were placed in different groups by RAPD markers. Recently studies of Munhoz et al. (2009) and Leal et al. (2010) reported that investigation the genetic diversity of popcorn is extremely important especially in comparison with wild forms of popcorn, common maize, sweet maize and ancestral species. Hence, in popcorn, the genetic basis is narrow as it is originated only from the dent type of common maize (Kantety et al., 1995).
In conclusion, these results demonstrate the efficiency of isozymes and RAPD markers in separation and identification the variability of maize types evaluated in present work with their ancestor teosinte. By characterizing these genotypes, it is possible to identify the most genetically distinct genotypes and to use them in breeding programs to maximize the use of genetic resources.

SUMMARY
Genetic variation and phylogenetic relationships among four types of yellow maize (Zea mays L.); dent, flint, sweet and pop corn, and their wild relative; teosinte (Zea mexicana), were assessed using isozymes and random amplified polymorphic DNA (RAPD) markers. The results indicated that the percentage of polymorphic loci; for peroxidase and esterase isozymes, were 66.67% and 92.59%, respectively. By applying RAPD analysis, 116 bands were obtained from seven primers with 87.07% polymorphism. The UPGMA dendrogram based on genetic distance segregated the five genotypes into two main clusters. Both isozymes and RAPD markers separated teosinte into the first cluster, whereas the four maize types were grouped together in the seconds cluster. Dent and flint types were much close to each other with high similarity indices; 0.842 and 0.792 based on peroxidase and esterase isozymes, respectively. Furthermore, the flint type closely related to sweet type in RAPD cluster (similarity index of 0.588). This high variability detected among maize types and teosinte can be used in breeding programs to maximize the use of genetic resources.  Hard endosperm is present on the sides and base of the kernel. The remainder of the kernel is filled with soft starch; when the grain starts drying the soft starch at the top of the kernel contracts, producing the depression for which it is named.

SCSk142 Flint corn
Zea mays indurate Gz653 x Sk6057/7-2 Kernels are characterized by their high percentage of hard endosperm around a small soft centre.

SCSk-sw1
Sweet corn Zea mays rugosa ZPL620/121 x ZPKŜ 8/1-131 The developing grain of sweet maize is higher in sugar content due to one or more recessive mutations blocking conversion of sugar to starch.

SCSk-pop1
Pop corn Zea mays everta HP6208 x HP6252 Kernels are characterized by a high proportion of hard endosperm, which is much higher than in any other maize kernel.