MOLECULAR CHARACTERIZATION AND GENETIC DIVERSITY IN SOME EGYPTIAN WHEAT ( Triticum aestivum L . ) USING MICROSATELLITE MARKERS

Wheat (Triticum aestivum L.) is the most important and strategic cereal crop in Egypt and has many bread wheat varieties. Seventeen Egyptian bread wheat varieties used in this study with a set of sixteen wheat microsatellite markers to examine their utility in detecting DNA polymorphism, estimating genetic diversity and identifying genotypes. A total of 190 alleles were detected at 16 loci using 16 microsatellite primer pairs. The number of allele per locus ranged from 8 to 20, with an average of 11.875. The polymorphic information content (PIC) and marker index (MI) average values were 0.8669, 0.8530 respectively. The (GA) n microsatellites were the highest polymorphic (20 alleles). The Jaccard's Coefficient for genetic similarity was ranged from 0.524 to 0.109 with average of 0.375. A dendrogram was prepared based on similarity matrix using UPGMA algorithm, divided the cultivars into two major clusters. The results proved the microsatellite markers utility in detecting polymorphism due to the discrimination of cultivars and estimating genetic diversity.


INTRODUCTION
Wheat (Triticum aestivum L.) is an important and strategic grain crop in the majority of the world.In Egypt, wheat is considered the most important and strategic cereal crop.It represents about 10 percent of the total agricultural production value and about 20 percent of all agricultural imports (FAOSTAT, 2008).Wheat is a self-pollinating polypoid crop that has been bred for a wide array of specific end-use quality traits and various adaptive characteristics, resulting in the development of distinct cultivars tailored to specialized end uses and specific production environments.
Botanists have long used morphological characterization to classify and distinguish genotypes within plant species.The emergence of the Plant Variety Protection (PVP) Act in 1970 had an impact on the protection of plant varieties.Hence, it was necessary to develop the critical tools for classifying and distinguishing genotypes within plant varieties (Rongwen et al., 1995).The polymorphisms of DNA provide a powerful tool for determining and discriminating the levels of genetic variation in plant germplasm.Molecular markers have thus become accurate and reliable tools for identifying and characterizing plant varieties and it has become effective tool for efficient selection of desired agronomic traits since it depends on genotype rather than phenotype.The advances in molecular genetics methodology have led to widespread use of co-dominant molecular markers, especially Simple Sequence Repeats (SSRs), Single Nucleotide Polymorphisms (SNPs) and the amplified fragment length polymorphisms (AFLPs) (Röder et al., 1998;Bohn et al., 1999;Prasad et al., 1999;Prasad et al., 2000;Roy et al., 1999;Varshney et al., 2000; for reviews see Gupta et al., 1999;Gupta and Varshney, 2000;Bered et al., 2005).
The first one define microsatellite term was (Litt and Lutty, 1989), as multilocus probes creating complex banding patterns and usually non-species specific occurring ubiquitously.They essentially belong to the repetitive DNA family.Microsatellite are repeated as of only a few bases, like two or three or five, and the whole repetitive region spans less than 150 bp.Therefore, it needs cloning and sequencing for designing the primers (Weissenbach et al., 1992;Morgante and Olivieri, 1993;Powell et al., 1996).Furthermore, these markers have provided high reproducibility and genetic informativeness (Coombs et al., 2004;Garcia et al., 2007).
Microsatellites produced from whole genome sequences, subgenomic sequences, ESTs and gene sequences have also been applied to DNA fingerprinting and the estimation of genetic diversity within a gene pool.Genetic diversity is defining the heritable variation within and between population's organisms (Ramanatha and Hodgkin, 2002)

Scientific hypothesis
It is expected that there will be a significant similarity between the wheat varieties used in this experiment due to their genetic resources.

MATERIAL AND METHODOLOGY Seed Material
In this experiment, some elite Egyptian bread wheat cultivars (17) were selected in different regions in Egypt as shown in Table (1).These cultivars have been obtained from Gene Bank, Egyptian Ministry of Agriculture.

DNA isolation and microsatellite primers
Twenty five seeds of each wheat varieties have been planted in pots in greenhouse.After two weeks of planting, the plants were enough to get fresh leaves and were collected from each sample.The samples were immediately transferred to liquid nitrogen tank to prevent deterioration.Weighed about 1.5 g from plant leaves samples and ground by liquid nitrogen to obtain fine powder.Total genomic DNA was isolated from leaves of each of the seventeen varieties according to the protocol described by Anderson et al. (1992), with a few modifications intended to improve the quality of DNA: two consecutive extractions with phenol: chloroform (1:1) were carried out by an additional wash of 97% (left at -20 °C for one hour) an 70% pre-cooled ethanol, respectively.The yield and quality of DNA were assessed by spectrophotometer and gel electrophoreses.

Statistic analysis
The fragment(s) sizes in 'Chinese Spring' were taken as standard, and the size differences of the fragments in other genotypes were considered to be the result of alterations in the repeat number of the simple sequences at the corresponding site(s).All distinct DNA fragments scored as present (1) or absent (0) were used to compute pair-wise similarity coefficients (Jaccard, 1908) for each of the markers for the purpose of assessing genetic diversity leading to cluster analysis.PowerMarker software (v3.0, 2004) is statistical software for genetic marker data analysis (Liu and Muse, 2005) was used for estimating of allele number, allele frequencies, genotype, heterozygosity and polymorphic information content (PIC).However, Marker index (MI) was calculated according to Powell et al. (1996), MI = Average polymorphic information content (PIC) x Proportion of polymorphic bands x Average number of loci per assay unit.

Cluster analysis
For phylogenetic analysis, data only from the polymorphic SSR loci were subjected to MVSP software statistical software (Kovach Computing Services, Pentraeth, Wales, U.K).All the 17 wheat varieties were clustered based on the estimated genetic distance.The phylogenetic analysis was carried out with the clustering method of the unweighted Pair Group Method Using Arithmetic Average (UPGMA).

Principal component analysis (PCA)
The original 1 -0 data matrix was used for calculating a correlation matrix between pairs of markers.The correlation matrix was employed for the calculation of eigenvalues, which were then used for determining the coordinates for each genotype that were used for PCA.

RESULTS AND DISCUSSION DNA polymorphism and genotype identification
The results of PCR amplification of a number of microsatellite loci in seventeen Egyptian bread wheat cultivars using sixteen microsatellite primer pairs are summarized in (Table 2).Altogether, 190 alleles at 16 loci were obtained with average 11.875 alleles per locus.The maximum number of alleles detected at Xgwm32-3A belonging to (GA) n was 20 alleles with size ranged from 163-179 bp.However, the minimum number of alleles detected at Xgwm156-5A belonging to (GT) n and Xgwm157-2D (CT) n was 8 alleles with size from 270-299 bp and 107-113 bp respectively, Table 2. Allele frequency per locus varied from eight (Xgwm156-5A and Xgwm157-2D) to twenty Xgwm32-3A.It is known that microsatellite primer pairs are locus specific and that is meant to be a single locus marker comparing with other molecular markers as RFLP probes, RAPD, ISSR and SCoT primers which multilocus (Vivodík et al., 2016).In the present study, the 16 loci that were assigned to specific chromosomes were able to distinguish between 17 Egyptian bread wheat and thus useful for detecting polymorphism.

Genetic diversity
Polymorphism Information Content (PIC) was estimated for 16 loci (Table 2).The PIC values ranged from 0.8137 of locus Xgwm157-2D to 0.9342 of locus Xgwm32-3A with an average of 0.8669.As well as, the heterozygosity values were estimated as well, ranging from 0.8235 to 1.00 with average 0.9816.The marker index (MI) value over all 16 microsatellite markers was 0.8530.The heterozygosity was detected in three loci (Xgwm71.2-2A,Xgwm113-4B and Xgwm161-3D) with values 0.9412, 0.9412 and 0.8235 respectively, (Table 3).The genotype number per locus was estimated based on allelic frequency data, where the highest number detected 17 of locus Xgwm32-3A and lowest number founded 10 of locus Xgwm156-5A and Xgwm157-2D with an average 13.6250 (Table 2).The data of microsatellite loci and the corresponding alleles were used to calculate the polymorphic information content (PIC) and heterozygosity (H) to evaluate a marker system for its ability to detect high levels of DNA polymorphism in an analysis of genetic diversity.In earlier studies on bread wheat, the PIC values were ranged from 0.

Genotype
No.

Genetic similarity
In order to investigate, genetic relationships between 17 Egyptian wheat genotypes cluster analysis based on Jaccard's similarity coefficients and UPGMA algorithm were calculated for the 43 durum wheat germplasm.A Jaccard's genetic similarity matrix is presented in Table 4a and 4b.The average similarity among 17 Egyptian bread wheat was 0.357.The nearest neighbor cluster analysis obtained from Jaccard's similarity coefficient (Figure 1) illustrated the variability between 17 Egyptian bread wheat.The detected of DNA polymorphism by 16 microsatellite markers allowed of estimates genetic distance and clustering of 17 Egyptian bread wheat cultivars in two major groups.The first group (Group I) included Misr 1, Misr 2, Gimmasa 7, Gimmasa 7, Gimmasa 9, Gimmasa 10, Gimmasa 11, Gimmasa 12 and Giza 168.The second group (Group II) included the other cultivars Sids 8, Sids 12, Sids 13, Sakha 93, Sakha 94, Sakha 69, Shandaweel 1, Bani Sweef 3 and Bani Sweef 4. The similarities between 17 Egyptian bread wheat based on 16 microsatellite markers were ranged from 0.109 in Shandweel 1, Bani Sweef 3 and Bani Sweef 4 cultivars to 0.524 in Gimmaza 7 and Gimmaza 9 with average of 0.357.A Jaccard's genetic similarity matrix was estimated between pairs of Egyptian wheat cultivars using 16 microsatellite markers through cluster analysis.This study used UPGMA cluster analysis based on genetic similarity values for SSR alleles from all the wheat cultivars to construct a dendrogram (Figure 1).The similarity value between 17 Egyptian wheat cultivars was ranged from 0.109 to 0.524 with average value 0.357.This average of genetic similarity value (0.357) can be compared with other studies, whereas SSR-based genetic similarity coefficient values of 0.31 (Plaschke et al., 1995) and 0.57 (Bohn et al., 1999).However, STS-based genetic similarity coefficient value of 0.81 (Chen et al., 1994) were reported.In these different studies on genetic diversity in bread wheat, undertaken using a variety of molecular markers, the variation in genetic similarity coefficient values may be attributed either to the differences in number of genotypes and the probes/primers used (e.The principle component analysis was used to visualize the genetic relationships among genotypes shown in Figure 2. Of the total polymorphism, only 40.44% was accounted for by the first two components, implying that the used markers possessed a suitable dispersion of markers in the genome.The 17 Egyptian wheat cultivars were clustered into two groups.The principle component analysis thus is largely compatible to those from cluster analysis obtained from UPGMA.

CONCLUSION
Microsatellites or SSR markers are one of the most common genetic markers and used in many genetic applications.Microsatellites are codominant, highly polymorphic, and Mendelian inherited, all these qualities made it very suitable for such study and the ability to accurately identify differences between wheat varieties in this study.The species specific markers identified would be utilized in future introgression breeding programs.

Figure 2
Figure 2 Matrix plot among 17 Egyptian bread wheat cultivars revealed by principle component analysis based on SSR data.

.
Knowledge of the genetic diversity and population structure within germplasm collections is an Progress in plant breeding requires a broad genetic base with a rich and diverse germplasm collection being the backbone of every successful crop improvement program.In this study, we evaluate the potential of 16 microsatellite primer pairs in general and specific SSRs in particular for polymorphism determination, cultivars identification and to evaluate the level of microsatellite based genetic diversity between 17 Egyptian bread wheat cultivars that were potentially useful in wheat breeding programs.

(Pavel and Vasile, 2012). The
fragment size in "Chinese Spring" was taken as standard Röder

Table 1
List of the elite Egyptian wheat cultivars and region of cultivation.
(Röder et al., 1998;Stephenson et al., 1998) the uniform distribution of the microsatellite primers of this set across the three genomes of bread wheat since in several studies, including the present study, microsatellites have been shown to be more frequent in the A and B genomes, than in the D genome(Röder et al., 1998;Stephenson et al., 1998).

Table 2
Primers of SSR, locus, repeat motif and annealing temperature (Ann.Temp.) were used.
The SSRs relative with them, more alleles detected at (CA) n and (AC) n one loci each, however, (GA) n loci 4 loci, (CT) n and (GT) n loci5 loci each.Röder et al.(1995)reported that (GT) n repeats to be more polymorphic than other simple repeats such as (GA) n in wheat.However, in barley more alleles were detected for (GA) n repeats than for (GT) n repeats (Struss and Plieske 1998).The results were obtained agreement with (

Table 4a
The matrix of Jaccard's similarity to 17 Egyptian bread wheat is indicated in Table1.

Table 4b
The matrix of Jaccard's similarity to 17 Egyptian bread wheat is indicated in Table1.Dendrogram of 17 Egyptian bread wheat based on data on allelic profiles generated using 16 microsatellite primer pairs.

Powell et al., 1996).
technique was sensitive and critical in differentiating between the different varieties of Egyptian wheat under study, as well as in determining the polymorphism, genotype identification, genetic similarity and estimation of genetic diversity. microsatellites