Genetic Characterization of Brassica oleracea L. var. acephala DC cultivars from Bosnia and Herzegovina

Fifteen Brassica oleracea L. var. acephala DC accessions were collected and evaluated for their genetic diversity and molecular characteristics using 10 previously developed and used codominant SSR markers. The SSR loci used revealed a total of 51 alleles. The domestic kale varieties were collected locally and the hybrids kales were found in markets. The averages of total heterozygosity (H) and polymorphic information content (PIC) was 0.6774 and 0.6201, respectively. The average number of alleles was 4.2 per locus, where 8 of 10 markers showed high polymorphic information content (PIC), being more than 0.5. The genetic relationships among the populations revealed by Unweighted Pair Group Method with Arithmetic mean (UPGMA), showed a clear clustering into three distinct groups. As expected, all local varieties were separated within two separated clusters, whereas only two domestic varieties shared one cluster with the group of hybrids, indicating similar genetic background shared by domestic and hybrid varieties. These results indicate and confirm the regional and physiological differences between the kale varieties and confirm the SSR marker efficiency for genetic characterization assays.

required. The production of kale in Bosnia and Herzegovina, particularly in Herzegovina, is not of great commercial importance, but it is grown as a very common vegetable in almost every family farm (Sefo et al., 2010). Indeed, through governmental, regional, local and agricultural associations, the conservation of local kale verities is partially carried out, but not efficiently. Therefore, there is a need for efficient species management and reliable conservation, which can be achieved and supported through genetic diversity assessment by using molecular DNA markers. The morphological assessment studies for the trait analysis have been already conducted (Quamruzzaman et al., 2007;Yousef et al., 2015;Zhu et al., 2018). However, due to the effects of environmental variation, the morphological approach is limited and it is time-consuming. Molecular markers are able to overcome these limitations and serve as a powerful and reliable tool for assessing genetic diversity and relationships among species (Izzah et al., 2013). Therefore, various molecular markers, such as RAPD, RFLP, ISSR, AFLP, SSR and EST-SSR offer efficient assessment analysis of genetic diversity in diverse plant species (Sun et al., 2001;Kumar et al., 2009). Among all mentioned molecular markers, microsatellites or commonly known as Simple Sequence Repeat (SSR) are most frequently used (Varshney et al., 2005;Kandemir et al., 2012). Simple sequence repeats (SSRs) are motives that occur randomly in the coding and non-coding part of the genome (Varshney et al., 2005). SSR markers result in highly polymorphic, abundant and reliable results. Further, compared to other DNA markers, the utilization of SSR markers represents a cheap procedure for genetic assessment of plant species, being highly reproducible and abundant DNA system marker (X. Zhang et al., 2015;Izzah et al., 2013;Guo-Liang, 2013;Jones et al., 1997;Powell et al., 1996). Several genetic diversity and relationship studies were conducted on Brassica oleracea L. var. acephala species, using different sets of SSR markers, as in Italy, China, Ireland, Spain (El-Esawi et al., 2016;Lotti et al., 2018;Tortosa et al., 2017;Zhu et al., 2018) and one central European based accession study (Christensen et al., 2011). However, no similar studies were done with genotypes from Bosnia and Herzegovina. Therefore, in the present study, the aim is to genetically characterize 15 kale varieties found in Bosnia and Herzegovina, using 10 codominant SSR markers.

DNA extraction
In total 505 kale individuals, from 15 Brassica oleracea L var. acephala varieties, were used in this study (Table 1). All the analyzed populations, except domestic varieties, are currently commercially available, being very specific, very widespread among agricultural producers. Domestic varieties were collected from farmers as seeds or as fresh, fully grown plant, and stored in -20Cº until DNA isolation. The genomic DNA was isolated from dry seeds and fresh leaves. Plant leaves for the DNA extraction were collected from randomly selected plants on each analyzed site. After harvesting, the seeds and leaves were pulverized with liquid nitrogen using mortar and pestle. The DNA material was isolated according to the CTAB (Cetyltrimethyl ammonium bromide) protocol (Porebski et al., 1997), with a slight modification. The DNA quality was analyzed on 1% agarose gel and quantified using a Multiscan GO spectrophotometer (Thermo Fisher Scientific, USA) and stored in 100µl aliquots in 1 X TE buffer at -20°C until PCR amplification.

SSR analysis
The PCR reactions were conducted in the total volume of 25 µl, containing the following mix: 2 mM of MgCl2, 0.2 mM of dNTPs, 1 x PCR buffer, 0.2µl of DNA Polymerase from Thermus aquaticus (5 units/μL) and 30-50 ng of template DNA. The PCR protocol was optimized to include two phases. The first phase included the denaturation step at 95 ˚C for 5 min, following the touchdown (TD) profile for 20 cycles, with 95 ˚C denaturation for 45s, reducing the annealing temperature for each primer by -0.7 ˚C per cycle for 45s, followed by the extension stage at 72 ˚C for 1 min ( Table 2). The final PCR phase included 15 cycles in total, with 95 ˚C denaturation for 45s, with 54˚C annealing temperature for 45s, with extension at 72˚C for 45s (El-Esawi et al., 2016). A final extension step with 72 ˚C for 5 min was introduced to complete the PCR protocol. For the verification of the expected band size, according the M50pz DNA Ladder -MR20 from BLIRT, the PCR fragments were separated on 3% agarose gel for 60 minutes at 80V in 1 x TE buffer, previously stained with Ethidium bromide (10 mg/mL Sigma). Gel DNA fragments were photographed and documented using Gel documentation system (Chemi Doc XRS System) from Biorad.

Data analysis
For the analysis of SSR loci, all the amplifications were scored as either present (1) or ab-sent (0). Further, to analyze the marker banding scheme, we obtained the number of scored bands (NTB), number of polymorphic bands (NPB) and percentage of polymorphic bands (PPB). The polymorphic information content (PIC) and heterozygosity (H) were assessed to discover the discriminatory power of each SSR marker, and all calculations were done using the online Gene-Calc program (Bińkowski and Miks, 2018). The polymorphic information content was calculated based on the following formula: where pi and pj represents the population frequency of the i th and j th allele (Lemos et al. 2019).
For the heterozygosity genetic variation calculation, the following formula was used: The indication of high heterozygosity values means that the crops have evolved through a long-term natural selection for adaptation or through historic mixing of strains of different populations. In contrary, if the populations were isolated over a longer period of time, losing their unexploited genetic potential, a low level of heterozygosis will be indicated (Liu, 1998).
For the analysis of polymorphic band fractions and number of polymorphic bands, the effective multiplex ratio (EMR) was calculated using formula: where, • n is the average number of fragments amplified by accession to a specific marker (multiplex ratio).
• β is estimated from the number of polymorphic loci (NPB) and the number of non-polymorphic loci (NMB); calculated by the following formula: Further, the resolving power (RP), assesses the marker combination discriminatory potential of the marker combination (Souza et al., 2019). The RP  Kong et al.,2011, c Portis et al., 2007, d Tsaballa et al.,2015  was calculated based on the following formula:

SSR primers Sequence of primer ('5-3') Expected (bp)
• Ib represents the informative fragments, with a scale of 0/1, by the following formula; where pi is the proportion of accessions containing the i th band (Prevost and Wilkinson, 1999). For the detection of polymorphic loci among the genotypes, stability and informativeness of each marker, the marker index (MI) was calculated based on the following formula: The phylogenetic tree was constructed by using hierarchical clustering with UPGMA (Unweighted pair group method with arithmetic mean) analysis, where the genetic distance was measured based on Jaccard's dissimilarity index (Real and Vargas, 1996). The resulting tree was bootstrapped with 1000 replicates to obtain the best confidence (Felsenstein, 1985). For the complete data analysis, the DARwin 6.0.15 software was used, as described by Karić et al. (2019).

Results and Discussion
Combining 10 SSR markers in this study, we amplified in a total of 51 bands (Table 3). All the markers showed clear and observable bands, as shown with marker G11 (Figure 1). The resulted, rooted Phylogenetic UPGMA dendogram is made of three groups (A, B and C). Group A shares one common ancestor, made of Stolac Baljici and Mostar city kales. Group B is divided into two sub-clusters. The first sub-cluster in group B is made of varieties from Stolac, Rivine and Gorica, sharing common origin with all hybrid kales, from the second out group, but clearly separated from all remaining accessions. The third group C is made of two subclusters, Dubrave kales (1 and 2) with Blagaj 2 in one sub-cluster, sharing a common ancestor with kales from Mostar, Blagaj 1 and Koćine 2 in the second sub-group ( Figure 2). In addition, for each marker, the Number of scored bands; Polymorphic bands; Monomorphic bands; Percentage of polymorphic band; Effective multiplex ratio; Marker index and Resolving power were calculated, as given in Table 3  All markers resulted in minimum 33.3 % polymorphic bands, while the highest polymorphic behavior was observed in three markers, 11-H02, 10F11 and EPMS790, with 100.0%. The highest effective multiplex ratio (EMR) in this study is shown in marker G11 with a value of 4.0, and the lowest EMR ratio is observed in markers A03, with 0.7, with an average EMR of 1.99 per marker. Further, the assessment of the discriminatory power of the given SSR marker combination, referring to the resolving power -RP, were in the range of 0.6-1.4, with an average of 1.02. Marker A03 showed the highest RP value with 1.4, and the lowest was 0.6 with 11H02 marker. To check the total utility of each marker, the marker index (MI) factor was observed, as a product of polymorphic information content and effective multiplex ratio (Varshney et al., 2007). Highest MI (2.5) is observed in 10-H02 and the lowest was with A03 marker (0.4). A high marker index indicates that the marker system used is useful for the chosen genetic population (Ghanbari et al., 2019).
Principle coordinate analysis (PCoA) was performed in order to further assess the genetic relationships among the kale varieties by using 10 SSR markers (Sun et al., 2001). PCoA shows the hybrid kale populations RR, BB, BR and NT are separated into one cluster, together with domestic kales SR and SG. The remaining domestic kales were separated into two more distinct clusters, where SB and MC domestic kales are clearly apart populations, as seen in Figure 3. This study involved the genetics characterization analysis of 15 different kale varieties, from BiH, evaluated by using 10 different markers. The genetic diversity of 11 domestic kales of known origin was compared to 4 hybrids, showing sufficient SSR dissimilarity characteristics and reflecting significant genetic diversity among kale varieties used in this study. All samples had higher polymorphic information content values than 0.337. The average value of the total heterozygosity (H) was 0.6774 and for PIC 0.601, values that are significantly higher if compared to similar researches (0.577, 0.249, 0.25, 0.338 and 0.294) (El-Esawi et al., 2016;Hintum et al., 2007;Watson-Jones et al., 2006). The PIC value ranged from 0 to 1, with values closer to 0 indicating no allelic variation, and values closer to 1 indicating that the genotype has only new allele (Guo and Elston, 1999). It is reported that high diversity of locus is demonstrated in PIC value greater than 0.5 (Alhasnawi et al., 2019). Two of the SSR markers (10H02 and G11) have high RP values (from 0.6 to 1.4), hereof being the most informative markers for distinguishing these kale genotypes in our study. However, the resolving power provides no information on the ability of a marker to reflect the genetic relationships of a group of genotypes under the study (Prevost and Wilkinson, 1999). Genetic variation using 10 SSRs loci resulted in a total of 51 alleles, out of which 42 bands (80.7%) were polymorphic, with a mean of 4.2 alleles per locus. Further, an average effective multiplex ratio of 1.99 was obtained, as the average number of amplified bands per marker was 35 for all cultivars, ranging from 16 to 54, confirming high polymorphic information potential. These results indicate that most of our markers are sufficient for the analysis intraspecific genetic diversity, especially marker G11, as a potent marker for fine dissection of the intraspecific relationship in kale varieties, as shown in a previous study (El-Esawi et al., 2016). The phylogenetic tree resulted in expected evolution pattern, where most of domestic kale varieties originated from the same region sharing the same ancestor, hereof being genetically very similar. In contrary, all the hybrid kales are placed clearly in the same group, sharing common ancestor with only two domestic cultivars from Stolac (Rivine and Gorica), indicating a slight possibility that a common genetically similar sort, was used as a variety for the creation of these hybrids. The dendrogram generated using UPGMA cluster analysis, grouped 15 genotypes into three clusters with Jaccard's dissimilarity index ranging from 0.259 to 0.83 (Table S1), with threshold equality of 100%. Through the UPGMA hierarchical clustering and PCoA together, the genetic relationships among individual accessions was strongly assessed. All these analyses suggested the occurrence of a distinct group (Stolac Baljići and Mostar City) might be due to close geographical stratification. However, Stolac Baljići and Mostar City kales share common genetic evolution path with all the other accessions but being farthest away from other accessions. For an additional verification, PCoA analysis is in line with the phylogenetic tree. The PCoA is consistent, with two observable clusters, domestic accessions in one and the hybrids in the second cluster (Figure 1 and Figure 2).
This study confirmed that the applied SSR marker set is effectively used to estimate genetic distances among genotypes, as shown in several previous studies (El-Esawi et al., 2016;Raza et al., 2019). Among all markers, only marker EPMS790 showed lower values than 0.5 for H and PIC. We revealed that 9 of 10 SSR markers had a PIC ≥ 0.5, confirming the codominant markers potential for polymorphism detection in Brassica oleracea L. var. acephala cultivars. However, to have a stronger understanding of the presence of genetic variability in Brassica oleracea L. var. acephala cultivars, the number of molecular markers should be higher and hereof lead to more efficient utilization of existing variation of Brassica crops in Bosnia and Herzegovina.

Conclusion
Brassica oleracea L. var. acephala DC cultivars are globally used as one of the essential crops in the human diet, in agriculture production, horticulture, animal fodder, condiments, oil and biofuel production, as being very rich in mustard oils, vitamins and minerals. Bosnia and Herzegovina (B&H) through centuries bears the tradition to grow and use Brassica oleracea L. var. acephala DC cultivars as crops, especially in the country southern region, Herzegovina. Through the evaluation of Polymorphic Information Content and Heterozygosity analysis, by using 10 codominant SSR markers, we concluded that B&H varieties are genetically diverse in comparison to hybrids. The phylogenetic dendogram joined the domestic kales within two clusters, clearly separating them from hybrid kales. The genetic markers were tested for efficiency through several genetic estimators, where 9 of 10 markers showed sufficient polymorphism and discriminatory power.