GENETIC DIVERGENCE IN TUNISIAN CASTOR BEAN GENOTYPES BASED ON TRAP MARKERS

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 30 TRAP primers. The efficacy of the TRAP technique in this study is further supported by the obtained PIC values of the primers used in the analysis. PCR amplification of DNA using 30 primers for TRAP analysis produced 490 DNA fragments that could be scored in all 56 genotypes of Tunisian castor. The number of amplified fragments varied from 3 (TRAP 04 x arb 1, TRAP 22 x arb 3 and TRAP 23 x arb 3) to 13 (TRAP 56 x arb 2), and the amplicon size ranged from 100 to 1600 bp. Of the 490 amplified bands, 377 were polymorphic, with an average of 5.71 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 TRAP 10 x arb 1 (0.555) and the highest PIC values were detected for TRAP 44 x arb 2 (0.961) with an average of 0.770. A dendrogram was constructed from a genetic distance matrix based on profiles of the 30 TRAP 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 five main clusters. Moreover, functional TRAP markers would be efficiently useful in genetic studies for castor genetic improvement.


INTRODUCTION
Castor bean (syn. castorbean, castor, castor-oil-plant), Ricinus communis L. (2n = 20, X = 10), is a species of flowering plant in the spurge family, Euphorbiaceae. It is an oilseed crop cultivated mainly in India, Mozambique, Brazil, and China (FAOSTAT, 2014). The seeds of castor bean have around 35 -55% oil, and the commercial standard is 44%. The oil percentage of the seeds varies depending on the cultivation environment and the cultivar (Costa and Ramos, 2004). The hydroxylated fatty acid ricinoleic is approximately 80 -90% of the total fatty acids, which gives castor bean oil (ricin oil) unique chemical and physical properties. Ricin oil is a renewable resource and raw material with various industrial applications (e.g., to manufacture paints, lubricants, cosmetics, pharmaceutical drugs, dyes, anilines, disinfectants, germicides, low-temperature lubricating oils, glues and adhesives, fungicide and insecticide bases, printing inks and varnishes, nylon and plastic), and more recently its use as biodiesel has been explored ( Mutlu and Meier, 2010). Now that some of these lineages have been developed, there is a need to study the genetic divergence among them. The genetic divergence among genotypes of any species can be evaluated with molecular markers, for example, target region amplification polymorphism (TRAP) markers. TRAP markers are functional markers that allow combining fixed and specific primers with arbitrary primers (Hu and Vick, 2003). These markers have high levels of polymorphism, which makes them a promising option for the genotypification of germplasm and identification of genes related to desirable agronomic characteristics. Besides, TRAP markers optimize the genetic gains in genetic improvement programs and are a valuable tool used by these programs to study genetic divergence (Agarwal et al., 2008).
Genetic diversity in castor bean was assessed by using both dominant and codominant molecular markers

Scientific hypothesis
TRAP markers are polymorphic enough to distinguish individual genotypes of Tunisian castor germplasm.
Amplification of TRAP fragments was performed according to (Simões et al., 2017a) using decamer arbitrary primers (Table 1 and Table 2). Amplifications were performed in a 15 μL reaction volume containing 1.5 μL of DNA, 7.5 μL of Master Mix (Genei, Bangalore, India), 1.5 μL of primer, and 4.5 μL H 2 O. Amplification was performed in a programmed thermocycler (Biometra, Germany) with the following cycle: 94 °C for 2 min; 5 cycles at 94 °C for 45 s, 35 °C for 45 s and 72 °C for 1 min; followed by 30 cycles at 94 °C for 45 s, 40 °C for 45 s, 72 °C for 1 min; and a final extension of 72 °C for 7 min. Amplified products were electrophoresed in 1.5% agarose in 1× TBE buffer. The gels were stained with ethidium bromide and documented using gel documentation system.

Statistical analysis
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. For the assessment of the polymorphism between genotypes ricin and usability RAPD 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 30 TRAP primers. The efficacy of the TRAP technique in this study is further supported by the obtained PIC values of the primers used in the analysis. PCR amplification of DNA using 30 primers (Table 1 and  Table 2) for TRAP analysis produced 490 DNA fragments that could be scored in all 56 genotypes of Tunisian castor ( Figure 2 and Table 3). The number of amplified fragments varied from 3 (TRAP 04 x arb 1, TRAP 22 x arb 3 and TRAP 23 x arb 3) to 13 (TRAP 56 x arb 2), and the amplicon size ranged from 100 to 1600 bp. Of the 490 amplified bands, 377 were polymorphic, with an average of 5.71 polymorphic bands per primer. Results indicated the presence of wide genetic variability among different genotypes of Tunisian castor. 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 TRAP 10 x arb 1 (0.555) and the highest PIC values were detected for TRAP 44 x arb 2 (0.961) with an average of 0.770. A dendrogram was constructed from a genetic distance matrix based on profiles of the 30 TRAP 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 five main clusters ( Figure 1). Cluster 1 contained 2 unique genotypes of Tunisian castor (K-4 and BA-5) from different regions of Tunisia and cluster 2 was divided into two subclusters (2a and 2b). Subcluster 2a contained 7 genotypes of castor and subcluster 2b contained 4 genotypes of Tunisian castor. Cluster 3 contained 6 genotypes of Tunisian castor and cluster 4 was divided into 3 subclusters (4a, 4b, and 4c). Subcluster 4a contained 2 genotypes of Tunisian castor and subcluster 4b contained 6 genotypes of Tunisian castor and subcluster 4c contained 29 genotypes of Tunisian castor. (Figure 1). Sixteen pairs of TRAP primers generated a total of 398 fragments, of which 396 (99.50%) were polymorphic; fourteen pairs of SSR primers generated a total of 60 fragments, of which 59 (98.33%) were polymorphic. Al-Murish et al. (2013) study efficiency of SRAP, TRAP, and SSR primers in detecting genetic variation among 17 C. arabica genotypes collected from the different valleys of Yafea City, Yemen, and estimate genetic similarity coefficients among these genotypes and classify them according to genetic relationships. The results of the present study demonstrated the presence of genetic variation among coffee genotypes within and between valleys.
In this study, Liu et al. (2015) estimated the genetic relationships within P. aibuhitensis using Target Region Amplified Polymorphisms (TRAP) and Amplified Fragment Length Polymorphisms (AFLP) that were derived from related populations on the coasts of China.  Note: PIC = polymorphism information content Among the TRAPs, 449 bands were observed in total, 439 of which (97.77%) were polymorphic between the P. aibuhitensis populations and were shared between at least four individuals. The Qinzhou (QZ) population had the highest PPB (78.62%), and the Dalian (DL) population had the lowest (60.13%). The objective of study Farias da Silva et al. (2016) was to analyze the genetic diversity of the clonal germplasm of the guarana plant using Target Region Amplification Polymorphism (TRAP) and Sequence-Related Amplification Polymorphism (SRAP) markers. Sixty clones of the guarana plant were analyzed; 18 were cultivars, eight were similar clones according to morpho-agronomic traits, and 34 were clones of a different origin. Singh et al. (2017) study the genetic variations among the twenty-five sugarcane genotypes employing functional molecular (TRAP) markers. Genetic diversity exists among sugarcane germplasm was exploited to identify promising genotypes bearing enviable agronomic traits (sucrose content and multiple disease resistance). Genetically diversified genotype could be exploited as proven parents in sugarcane hybridization programs to establish a promising cross. Mirajkar et al. (2017) study molecular marker profile using 57 markers, comprising of 27 TRAP and 30 SRAP markers in the gamma ray-induced sugarcane mutants. Collectively these markers produced 260 PCR amplicons among which 147 were polymorphic (56.54%). The TRAP marker-based analysis showed that the mutants AKTS-01 and AKTS-16 were more diverse (GS = 94 and 92%, respectively) than the rest of the mutants. In the study of Fabriki-Ourang and Yousefi-Azarkhanian (2018) target region amplification polymorphism (TRAP) and conserved region amplification polymorphism (CoRAP) markers were used for genetic diversity and relationship analysis of 25 Salvia ecotypes/species. Twelve TRAP and CoRAP primer combinations (four arbitrary primers and three fixed primers from Salvia miltiorrhiza expressed sequence tag sequences) amplified 180 loci, of which all were polymorphic. Srivong et al.
(2019) study 17 sugarcane genotypes from Hawaii and Thailand using 12 target region amplification polymorphism (TRAP) markers and partial Sai nucleotide polymorphism. A total of 275 fragments were produced, of which 273 (99.27%) were polymorphic. The polymorphic information content (PIC) ranged from 0.912 -0.959 with an average value of 0.938. Genetic similarity (GS) by Dice's similarity coefficient ranged from 0.19 -0.81 with a mean of 0.44.

CONCLUSION
PCR amplification of DNA using 30 primers for TRAP analysis produced 490 DNA fragments that could be scored in all 56 genotypes of Tunisian castor. The number of amplified fragments varied from 3 (TRAP 04 x arb 1, TRAP 22 x arb 3 and TRAP 23 x arb 3) to 13 (TRAP 56 x arb 2), and the amplicon size ranged from 100 to 1600 bp. Of the 490 amplified bands, 377 were polymorphic, with an average of 5.71 polymorphic bands per primer. To determine the level of polymorphism in the analysed group of Tunisian castor genotypes polymorphic information content (PIC) was calculated. A dendrogram was constructed from a genetic distance matrix based on profiles of the 30 TRAP primers using the unweighted pair-group method with the arithmetic average (UPGMA). TRAP markers could be used to select elite parent genotypes, analysing genetic variation, utilization of genotype potential for trait improvement for adaptation to stress environment. It is therefore suggested that a focused breeding scheme should be adopted while analyzing genome diversity for parent selection to gain maximum value and practical impact on breeding program. TRAP markers exhibited remarkable discriminatory power for genetic diversity analysis.