GENETIC DIVERSITY AND POPULATION STRUCTURE IN TUNISIAN CASTOR GENOTYPES (RICINUS COMMUNIS L.) DETECTED USING SCOT MARKERS

Due to the chemical and physical properties of castor oil (Ricinus communis L.) that make it a valuable raw material for numerous industrial applications, including the production of biofuel, interest to develop more and better varieties has been increased. In the present study, the representatives of the genus castor collected from 12 different parts of Tunisia were differentiated by the DNA fingerprinting patterns using 37 SCoT primers. PCR amplification of DNA using 37 primers for SCoT analysis produced 268 DNA fragments that could be scored in all 56 genotypes of Tunisian castor. The number of amplified fragments varied from 4 (SCoT 45, SCoT 31 and ScoT 17) to 10 (SCoT 3, SCoT 11, SCoT 14, SCoT 18 and SCoT 12). Of the 268 amplified bands 230 were polymorphic, with an average of 6.22 polymorphic bands per primer. To determine the level of polymorphism in the analysed group of Tunisian castor genotypes polymorphic information content (PIC) was calculated. The lowest values of polymorphic information content were recorded for SCoT 17 (0.411) and the the highest PIC values were detected for SCoT 14 (0.868) with an average of 0.751. A dendrogram was constructed from a genetic distance matrix based on profiles of the 37 SCoT primers using the unweighted pair-group method with the arithmetic average (UPGMA). According to analysis, the collection of 56 Tunisian castor genotypes were clustered into two main clusters (1 and 2). Of the 56 genotypes of Tunisian castor, 2 unique genotypes were separated (BA-5 and K-4). Genetically the closest were two genotypes from Tunisian region Souassi (S-2 and S-5) in subclaster 2bc. Results showed the utility of SCoT markers for estimation of genetic diversity of castor genotypes leading to genotype identification.


INTRODUCTION
The castor-oil plant (Ricinus communis L.), a member of the spurge family (Euphorbiaceae), is a versatile industrial oil crop that is cultivated in many tropical and subtropical regions of the world (Anjani, 2012).Due to the distinctive characteristics of castor oil, such as its high percentage of ricinoleic acid, unusual chemical structure and low freezing point, castor oil is widely utilised in paints, nylon, aviation oil, lubricants, soaps, inks, dyes, cosmetics, adhesives, biodiesel, and other novel castor-bean-derived products (Scholz and Silva, 2008).Castor is cultivated on commercial scale in an area of 1,525,000 ha in 30 countries with 1,581,000 MT seed production.India, China, Brazil, USSR, Thailand, Ethiopia and Philippines are the major castor growing countries in the world (Damodaram and Hegde, 2010).
Knowledge of genetic variability is important for breeding programs to provide the basis for developing desirable genotypes.Genetic variability in castor bean has been studied using molecular techniques, including random amplified polymorphism DNA (RAPD) (Vivodík et al., 2015) (2017).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.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)

Scientific hypothesis
The present study is focused on estimation of genetic distance between 56 Tunisian castor genotypes, based on 37 SCoT markers.Although the information gathered here would be helpful in future for genomic mapping studies leading to development of castor cultivars with broader genetic background to obtain improved crop productivity.

Statisic analysis
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 the present study, the representatives of the genus Ricinus communis collected from 12 different parts of Tunisia were differentiated by the DNA fingerprinting patterns using 37 SCoT primers.The efficacy of the SCoT technique in this study is further supported by the obtained PIC values of the primers used in the analysis.The PIC value of the SCoT marker system was found to be 0.751 which are at par with the optimal PIC.PCR amplification of DNA using 37 primers (Table 1) for SCoT analysis produced 268 DNA fragments that could be scored in all 56 genotypes of Tunisian castor (Figure 1).The number of amplified fragments varied from 4 (SCoT 45, SCoT 31 and ScoT 17) to 10 (SCoT 3, SCoT 11, SCoT 14, SCoT 18 and SCoT 12), and the amplicon size ranged from 200 to 2500 bp.Of the 268 amplified bands 230 were polymorphic, with an average of 6.22 polymorphic bands per primer.Results indicated the presence of wide genetic variability among different genotypes of Tunisian castor.From these 37 primers, primers SCoT 3, SCoT 14 and SCoT 15 were the most polymorphic, where 9 polymorphic amplification products were detected.The lowest number of amplified polymorphic fragments (3) were detected by primers SCoT 2, SCoT 17, SCoT 31 and SCoT 45.The percentage of polymorphism ranged from 50.00% (SCoT 2) to 100% (SCoT 13, SCoT 15, SCoT 20, SCoT 30, SCoT 44, SCoT 65 and SCoT 66).To determine the level of polymorphism in the analysed group of Tunisian castor genotypes polymorphic information content (PIC) was calculated.The lowest values of polymorphic information content were recorded for SCoT 17 (0.411) and the the highest PIC values were detected for SCoT 14 (0.868) with an average of 0.751.

Figure 1
Figure 1 PCR amplification products of 25 genotypes of Tunisian castor produced with primer SCoT-12.Lane M is Quick-Load® 100 bp DNA ladder and lanes 1-25 are Tunisian castor genotypes.

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Suitability of SCoT markers system has been successfully employed in genetic diversity analysis and fingerprinting of a number of agricultural and horticultural crop species, such as oat (

Table 1
Statistical characteristics of the SCoT markers used in Tunisian castor genotypes Note: TNoB-Total number of bands, NoPB-Number of polymorphic bands, PoPB-Percentage of polymorphic bands (%), PIC-Polymorphic information content.