MOLECULAR VARIABILITY OF OAT BASED ON GENE SPECIFIC MARKERS

Oat (Avena sativa L.) is a grass planted as a cereal crop. Cultivation of oat is increasing in the recent years because of its good nutrition value. The aim of our study was to analyze genetic variability of oat accessions based on SCoT markers. Eighteen primers were used to study polymorfism of 8 oat genotypes. All 18 primers produced polymorphic and reproducible data. Altogether 153 different fragments were amplified of which 67 were polymorphic with an average number of 3.72 polymorphic fragments per genotype. The number of polymorphic fragments ranged from one (SCoT9, SCoT62) to nine (SCoT40). The percentage of polymorphic bands ranged from 14.29% (SCoT9) to 60% (SCoT59) with an average of 41.62%. Genetic polymorphism was characterized based on diversity index (DI), probability of identity (PI) and polymorphic information content (PIC). The diversity index of the tested SCoT markers ranged from 0 (SCoT9, SCoT62) to 0.878 (SCoT40) with an average of 0.574. The polymorphic information content ranged from 0 (SCoT9, SCoT62) to 0.876 (SCoT40) with an average of 0.524. Dendrogram based on hierarchical cluster analysis using UPGMA algorithm grouped genotypes into two main clusters. Two genotypes, Taiko and Vok were genetically the closest. Results showed the utility of SCoT markers for estimation of genetic diversity of oat genotypes leading to genotype identification.


INTRODUCTION
Cereals belong to a group of key foods of plant production.Oat together with corn and barley is the most used for feed but for human nutrition is used only a little.Cultivated oats are hexaploid cereals belonging to the genus Avena L., which is found worldwide in almost all agricultural environments.Recently, oats have been receiving increasing interest as human food, mainly because the cereal could be suitable for consumptions by celiac patients (Gálová et.al., 2012).In the Nordic countries and Northern Europe it became a wellestablished crop both for food and feed.Oat belongs to alternative cereals which are used mainly as a supplement to traditional species of cereals (Daou and Zhang, 2012).
Recently, the studies of genetic diversity based mainly on the molecular analysis.Worldwide collections of oats were described by several types of dominant molecular markers, for example AFLP (Fu et al., 2003), RAPD (Baohong et al., 2003) and ISSR (Boczkowska and Tarczyk, 2013).With initiating a trend away from random DNA markers towards gene-targeted markers, a novel marker system called SCOT (Collard and Mackill, 2009) was developed based on the short conserved region flanking the ATG start codon in plant genes.SCOT markers are generally reproducible, and it is suggested that primer length and annealing temperature are not the sole factors determining reproducibility.They are dominant markers like RAPDs and could be used for genetic analysis, quantitative trait loci (QTL) mapping and bulk segregation analysis (Collard and Mackill, 2009).In principle, SCOT is similar to RAPD and ISSR because the same single primer is used as the forward and reverse primer (Collard and Mackill, 2009;Gupta et al. 1994).SCoT marker system has gained popularity for its superiority over other dominant DNA marker systems like RAPD and ISSR for higher polymorphism and better marker resolvability The aim of our study was to detect genetic variability among the set of 8 oat genotypes using 18 SCoT markers and to testify the usefulness of a used set of SCoT primers for the identification and differentiation of oat genotypes.

MATERIAL AND METHODOLOGY
Eight oat (Avena sativa L.) genotypes were used in the present study.Seeds of oat were obtained from the Gene Bank of the Slovak Republic of the Plant Production Research Center in Piešťany.Genomic DNA of rye cultivars was isolated from 100 mg freshly-collected leaf tissue according to GeneJETTM protocol (Fermentas, USA).The concentration and quality of DNA was checked up on 1.0% agarose gel coloured by ethidium bromide and detecting by comparing to λ-DNA with known concentration.SCoT analysis: For analysis 18 SCoT primers were chosen (Table 2) according to the literature (Collard a Mackill, 2009).Amplification of SCoT fragments was performed according to (Collard a Mackill, 2009) (Table 2.).Polymerase chain reaction (PCR) was performed in 15 μL mixture in a programmed thermocycler (Biometra, Germany).Amplified products were separated in 1% agarose gels in 1 × TBE buffer.The gels were stained with ethidium bromide and documented using gel documentation system UVP PhotoDoc-t®.Size of amplified fragments was determined by comparing with 100 bp standard lenght marker (Promega).
Data analysis: For the assessment of the polymorphism between castor genotypes and usability of SSR markers in their differentiation diversity index (DI) (Weir, 1990), the probability of identity (PI) (Paetkau et al., 1995) and polymorphic information content (PIC) (Weber, 1990) were used.The SCoT bands were scored as present (1) or absent (0), each of which was treated as an independent character regardless of its intensity.The binary data generated were used to estimate levels of polymorphism by dividing the polymorphic bands by the total number of scored bands and to prepare a dendrogram.A dendrogram based on hierarchical cluster analysis using the unweighted pair group method with arithmetic average (UPGMA) with the SPSS professional statistics version 17 software package was constructed.

RESULTS AND DISCUSSION
The development of molecular markers has opened up numerous possibilities for their application in plant breeding.For detecting polymorphisms new molecular marker system called SCoT (Collard a Mackill, 2009) was developed which tag coding sequences of the genome.SCoT marker system had initially been validated in the model species rice (Oryza sativa) (Collard and Mackill 2009).
For the molecular analysis of 8 oat genotypes 18 SCoT primers were used.PCR amplifications using 18 SCoT primers produced total 153 DNA fragments that could be scored in all genotypes.The selected primers amplified DNA fragments across the 8 genotypes studied with the number of amplified fragments varying from 4 (SCoT62)  3).The percentage of polymorphic bands ranged from 14.29% (SCoT9) to 60% (SCoT59) with an average of 41.62%.The polymorphic information content (PIC) values varied from 0 (SCoT9, SCoT62) to 0.876 (SCoT40) with an average of 0.524 and index diversity (DI) value ranged from 0 (SCoT9, SCoT62) to 0.878 (SCoT40) with an average of 0.574 (Tab.3).The most polymorphic SCoT40 marker is showed on Figure 2. A dendrogram was constructed from a genetic distance matrix based on profiles of the 18 SCoT primers using the unweighted pair-group method with the arithmetic average (UPGMA).According to analysis, the collection of 8 diverse accessions of oat was clustered into two main clusters (Figure 1).The first cluster contained unique genotype Azur coming from the Czech Republik.Second cluster contained 7 genotypes of oat which were further subdivided into two subclusters (2a, 2b).Subcluster 2a contained unique Austrian genotype Euro and rest of genotypes (6) were included in the subcluster 2b.Genetically the closest were two genotypes, Vok (coming from the Czech Republik) and Taiko (coming from Netherland).
Lower average polymorphism (21%) obtained by SCoT technique was detected by Kallamadi et al. (2015) who analysed molecular diversity of castor (Ricinus communis L.).Out of 36 SCoT primers tested, all primers produced amplification products but only 10 primers resulted in polymorphic fingerprint patterns.Out of a total of 108 bands, 23 (21%) were polymorphic with an average of 2.1 polymorphic bands per primer.The total number of bands per primer varied from 5 and 20 in the molecular size range of 100 -3000 bp.The PIC/DI varied from 0.06 for SCoT28 to 0.45 for SCoT12 with an average of 0.24.On the other side, higher polymorphism with SCoT primers has been reported in crops like peanut (Xiong et  --------+---------+---------+---------+--------- Level of polymorphism in analysed oat genotypes was also determined by calculated polymorphic information content (PIC) (Table 3).Lower PIC values compare to our analysis (0.   2009), which is based on the short conserved region flanking the ATG translation start codon in plant genes.The technique is similar to RAPD or ISSR in that a single primer acts as the forward and the reverse primer, amplicons can be visualized by standard agarose gel electrophoresis, without the need for costly automated electrophoresis systems (Collard and Mackill, 2009).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 reported utilization of SCoT markers for the detection of genetic variability of oat genotypes.In summary, SCoT marker analysis was successfully developed to evaluate the genetic relationships among the genus of oat accessions originated from various regions.The hierarchical cluster analysis divided oat genotypes into 2 main clusters.SCoT markers are generated from the functional region of the genome; the genetic analyses using these markers would be more useful for crop improvement programs.Polymorphism revealed by SCoT technique was abundant and could be used for molecular genetics study of the oat accessions, providing high-valued information for the management of germplasm, improvement of the current breeding strategies, construction of linkage maps, conservation of the genetic resources of oat species and QTL mapping.

Table 1
List of analyzed genotypes of oat.

Table 2
List of used SCoT markers.

Table 3
Statistical characteristics of the SCoT markers used in oat.Dendrogram of 8 oat genotypes prepared based on 18 SCoT markers.

Poczai et al., 2013).
Functional markers developed from the transcribed region of the genome have the ability to reveal polymorphism, which might be directly related to gene function (Start codon targeted polymorphism (SCoT) is a simple and novel marker system first described by Collard and Mackill (