START CODON TARGETED (SCOT) POLYMORPHISM REVEALS GENETIC DIVERSITY IN EUROPEAN OLD MAIZE (ZEA MAYS L.) GENOTYPES

Maize ( Zea mays L.) is one of the world’s most important crop plants following wheat and rice, which provides staple food to large number of human population in the world. It is cultivated in a wider range of environments than wheat and rice because of its greater adaptability. Molecular characterization is frequently used by maize breeders as an alternative method for selecting more promising genotypes and reducing the cost and time needed to develop hybrid combinations. In the present investigation 40 genotypes of maize from Czechoslovakia, Hungary, Poland, Union of Soviet Socialist Republics, Slovakia and Yugoslavia were analysed using 20 Start codon targeted (SCoT) markers. These primers produced total 114 fragments across 40 maize genotypes, of which 86 (76.43%) were polymorphic with an average of 4.30 polymorphic fragments per primer and number of amplified fragments ranged from 2 (SCoT 45) to 8 (SCoT 28 and SCoT 63). The polymorphic information content (PIC) value ranged from 0.374 (ScoT 45) to 0.846 (SCoT 28) with an average of 0.739. The dendrogram based on hierarchical cluster analysis using UPGMA algorithm was prepared. The hierarchical cluster analysis showed that the maize genotypes were divided into two main clusters. Unique maize genotype (cluster 1), Zuta Brzica, originating from Yugoslavia separated from others. Cluster 2 was divided into two main clusters (2a and 2b). Subcluster 2a contained one Yugoslavian genotype Juhoslavanska and subcluster 2b was divided in two subclusters 2ba and 2bb. The present study shows effectiveness of employing SCoT markers in analysis of maize, and would be useful for further studies in population genetics, conservation genetics and genotypes improvement.


INTRODUCTION
Maize (Zea mays L.) is one of the world's most important crop plants following wheat and rice, which provides staple food to large number of human population in the world (Ahmad et al., 2011;Iqbal, et al., 2015). Determining genetic diversity can be based on agronomic, morphological, biochemical, and molecular types of information, among others ( Recently, a simple novel DNA marker technique namely start codon targeted (SCoT) polymorphism, was developed by Collard and Mackill (2009). Primers for SCoT marker analysis were designed from the conserved region surrounding the translation initiation codon, ATG (Joshi et al., 1997; Sawant et al., 1999). Single 18-mer oligonucleotides were used as both forward and reverse primer for PCR, and the annealing temperature was set at 50 °C. The amplicons were resolved using standard agarose gel electrophoresis. Suitability of SCoT markers for the construction of genetic maps, fingerprinting and phylogenetic studies has been proved by many authors in many crops, such as tomato (Shahlaei et al., 2014), citrus (Mahjbi et al., 2015), date palm (Al-qurainy et al., 2015), castor (Kallamadi et al., 2015) and mango (Gajera et al., 2014).
The goals of this study were to examine the effectiveness of scot markers for analysis of genetic diversity of maize and to study genetic relationships among 40 maize accessions originating from various geographic regions of europe.

MATERIAL AND METHODOLOGY
Plant material: Forty genotypes of old maize lines originating from six different geographical areas (Table 1) (Table 2). Each 15-μL amplification reaction consisted of 1.5 μL (100 ng) template DNA, 7.5 μL Master Mix (Genei, Bangalore, India), 1.5 μL 10 pmol primer, and 4.5 μL distilled water. Amplification was performed in a programmed thermocycler (Biometra, Germany) using the following program: 94 °C for 3 min; 35 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2 min; a final extension at 72 °C for 5 min. Amplified products were separated in 1.5% agarose in 1 × TBE buffer. The gels were stained with ethidium bromide and documented using gel documentation system UVP PhotoDoc-t ® camera system. A dendrogram was constructed based on hierarchical cluster analysis using the unweighted pair group method with arithmetic average (UPGMA). For the assessment of the polymorphism between genotypes maize and usability SCoT markers in their differentiation we used polymorphic information content (PIC) (Weber, 1990).

RESULTS AND DISCUSSION
In this work, all 20 SCoT primers used for analysis of 40 European old maize genotypes produced amplification products and all resulted in polymorphic fingerprint patterns. Twenty primers produced 114 DNA fragments ( Figure 1) with an average of 5.7 bands per primer ( Table  2). Out of the total of 114 amplified fragments, 86 (76.43 %) were polymorphic, with an average of 4.30 polymorphic bands per primer. From these twenty primers, primers SCoT 28 and SCoT 63, respectively, were the most polymorphic, where 8 polymorphic amplification products were detected. The lowest number of amplified polymorphic fragments (2) was detected by primer SCoT 45. To determine the level of polymorphism in the analysed group of maize genotypes, polymorphic information content (PIC) was calculated (Table 2).   The polymorphic information content (PIC) value ranged from 0.374 (SCoT 45) to 0.846 (SCoT 28) with an average of 0.739. The dendrogram of 40 maize genotypes based on SCoT markers using UGMA algorithm was constructed ( Figure 2). The hierarchical cluster analysis divided maize genotypes into two main clusters. Unique maize genotype Zuta Brzica, originated from Yugoslavia (cluster 1), separated from others. Cluster 2 containing 39 genotypes was divided into two main subclusters (2a and 2b). Subcluster 2a contained one Yugoslavian genotype Juhoslavanska and subcluster 2b was divided in two subclusters 2ba and 2bb. In the subcluster 2ba were grouped 7 genotypes from Hungary (42.87%), Poland (14.29%), Czechoslovakia (14.29%) and Union of Soviet Socialist Republics (28.58%). Subcluster 2bb of 31 genotypes included genotypes of Polish origin (16.15%), Union of Soviet Socialist Republics origin (22.61%), Slovakia origin (19.38%), Czechoslovak origin (25.84%) and Hungarian origin (16.15%). Two genotypes of 2bb subcluster (Czechnicka and Wielkopolanka) from Poland and two genotypes (Voroneskaja and Kocovska Skora) from Union of Soviet Socialist Republics and Slovakia, respectively, were genetically the closest. We can assume that they have close genetic background.
Level of polymorphism in analysed maize genotypes was determined by calculated polymorphic information content (PIC) ( Table 2) (2013) pointed that in general, a higher number of investigated accessions and more varied genetic background result in a higher expected polymorphic rate. Start codon targeted polymorphism (SCoT) is a simple and novel marker system first described by Collard and Mackill (2009), which is based on the short conserved region flanking the ATG translation start codon in plant genes. The higher primer lengths and subsequently higher annealing temperatures ensure higher reproducibility of SCoT markers, compared to RAPD markers (Rajesh et al., 2015). Gorji et al., (2011) presented that SCoTs markers were more informative and effective, followed by ISSRs and AFLP marker system in in fingerprinting of potato varieties.

CONCLUSION
The present work is the first report on genetic variability of maize using SCoT markers. In summary, SCoT marker analysis was successfully developed to evaluate the genetic relationships among the genus maize accessions originated from various regions. The hierarchical cluster analysis showed that the maize genotypes were divided into 2 main clusters. One maize genotype Zuta Brzica, origin from Yugoslavia (cluster 1), was separated from others. Cluster 2 was divided into two main clusters (2a and 2b). Four genotypes of 2bb subcluster (Czechnicka and Wielkopolanka) from Poland and two genotypes (Voroneskaja and Kocovska Skora) from Union of Soviet Socialist Republics and Slovakia, respectively, were genetically the closest. Polymorphism revealed by SCoT technique was so abundant and could be used for molecular genetics study of the maize accessions, providing highvalued information for the management of germplasm, improvement of the current breeding strategies, and conservation of the genetic resources of maize species.