Determination of SSR-markers based genetic diversity with genetic parameters
Vernonia (Vernonia galamensis) is a plants species that contains naturally occurring epoxidized oils in its seeds. However, its potential values are neglected, underestimated and underexploited. In addition, it is also exposed to genetic erosion. Therefore, assessment of genetic diversity with SSR markers generally in plants and particularly in V. galamensis is important for cultivation, breeding and genetic resource management. A total of 79 alleles with an average of 3.9 alleles were detected in this study, which was higher than the one reported by Ramalema et al.  (3.14) in V. galamensis. The author also stated that less genetic diversity was observed among different Vernonia lines. According to Aikpokpodion et al. , a total of 29 bands were detected in genus Vernonia. Hence, the large number of alleles detected in the present study indicated the suitability of microsatellites for genetic linkage and QTL mapping of desirable traits applied to marker-assisted selection (MAS) in breeding programmes.
Polymorphic information content (PIC) values reflect the relative allelic polymorphism of a particular marker and their potential to differentiate the genotypes based on their genetic relationships. The PIC values are also a good indication of the usefulness of markers for linkage analysis when determining the inheritance between offspring and parental genotypes . In this study, the PIC values ranged between 0.50 (Vg-005) and 0.96 (Vg-002) with an average of 0.76. Ramalema et al.  reported the mean PIC value 0.40, which was lower than our result. Microsatellite markers such as Vg-002 and Vg-011 showed the highest polymorphism with 0.96 and 0.93, respectively. Aikpokpodion et al.  also reported the PIC value ranged from 0.27 to 0.78 in V. galamensis accessions, again little lower than the present study. This indicates that the majority of markers were able to distinguish differences among the studied V. galamensis accessions. For most of the loci, expected heterozygosity 0.50 values were higher than that of observed heterozygosity (0.16), revealing a high homozygosity at the given loci among the accessions. The diversity parameters showed high level of polymorphism among the 20 SSR markers, favoring the genetic variation within V. galamensis collection.
Genetic Differentiation and Gene Flow
AMOVA demonstrated that V. galamensis had low variation among the populations (11%). On the other hand, 67% of the total variation was attributed to genetic variability within the populations and 22% was due to variation among individuals within the same population. Similarly, Ramalema  reported 40% of genetic variation due to among different vernonia lines. Aikpokpodion  reported 30% of variation due to among the studied samples and 70% of variation due to within the studied samples. Nwakanma  reported that 36% of variation due to among species and 64% were within species variations. According to IPGRI and Cornell University , Fst value ranging from 0 to 0.05 is small in genetic differentiation, from 0.05 to 0.15 is moderate, and from 0.15 to 0.25 is large, and greater than 0.25 is very large genetic differentiation among populations in terms of allele frequencies. In line with this, the extent of genetic differentiation among the ten populations in terms of allele frequencies measured was moderate (Fst = 0.101), the result of STRUCTURE and Fst showed a strong gene flow among populations that collected from different regions. This may be self-pollination in nature, should be carefully considered in future accessions sampling and collection regarding biodiversity evaluation and conservation in V. galamensis.
Genetic distance is the measure of the allelic substitutions per locus that have occurred during the separate evolution of two populations. In this study, the largest genetic distance was observed between Sidama and East Showa (0.59) populations, while the minimum genetic distance was observed between Borena and Konso (0.24). The overall magnitude of the pairwise population matrix of Nei genetic distance was relatively low .
Clustering and principal co-ordinates analysis among Vernonia galamensis accessions
In the present study, a dendrogram tree was constructed based on the 150 accessions of V. galamensis collected from different geographic and agro-ecological regions. V.galamensis accessions were clustered into four (4) major clusters based on the allelic frequency. Cluster 1 was comprised of 39 accessions, the second cluster characterized as the second major clustering, contained 42 accessions, the third cluster composed of 15 accessions, and the fourth groups consisted of the major clustering which comprised 54 accessions that were collected from different regions of origin. The cluster analysis revealed a week clustering pattern. Hence, accessions collected from different geographic regions/zones of origins were clustered together, since clusters did not follow a clear pattern of geographic origins, which may due to the presence of strong gene flow, or epistatic gene interaction which may require more investigation. Similarly, Nwakanma et al.  reported that 49 Vernonia lines fingerprinted were grouped into four major clusters with no clear-cut separation among accessions related to their origin. Aikpokpodion et al.  also reported that genus Vernonia grouped into four major clusters and showed the existence pattern of relationships between geographical origins and genetic diversity.
Principal components (PC) analysis explores complex data sets and transforms a number of associated variables into a smaller number of PCs. In the present investigation, the principal component analysis revealed that the majority of samples were placed at the center of a two-dimensional coordinate plane and roughly forms four groups with a total variation of 30.04%. This, in turn, agrees with the results of the NJ dendrogram in which there was no unique clustering among accessions from the same population/collection areas. The presence of gene flow among populations/collection areas, accompanied by wind dispersal to a long distance due to the hairy pappus at the tip of seed, may be the most probable explanation behind the mixed clustering of accessions. Although UPGMA and PCoA analyses also showed a certain level of population clustering according to their geographical regions, the clustering pattern is weak, not clustered by their regions of origin. Aikpokpodion et al  reported geographical differentiation observed among Vernonia accessions, indicates the plant isolation by distance, and may be due founder effect as a result of genetic drift as well as a local adaptation. Founder effect is the loss of genetic variation due to the establishment of a new population by a very small number of individuals from the original population.
Population’s genetic structure in Vernonia galamensis
The structure analyses of 150 V. galamensis accessions using a Bayesian-model based approach with the highest ΔK Value, according to Gilbert et al.  and Evanno et al.  method. The samples from 10 collection zones were inferred into six (K = 6) structure outputs. The structure analysis revealed a close relationship (weak sub-division) of the population from 10 collection zones, and in general, six inferred groups (K = 6) with potential admixtures have been observed. It is interesting to indicate that all individual plants analyzed have alleles originated from the six clusters, which supports the presence of a strong gene flow that led to low population differentiation. Hence, accessions collected from the same region of origin did not often group entirely together within a given major groups. There was a high level of population admixture in the structuring of V. galamensis populations, and population genetic structure study using SSR markers, which is the first report in V.galamensis. Most of the magnitudes identified for the SSR markers were important information for cultivation, breeding and genetic resource conservation. Nwakanma et al.  reported clustering in Vernonia species may have arisen from the same putative parents or may have diversed from each other in the course of their evolutionary adaptation in different agro-ecology. In addition, huge intra-species relationships were existed within the species due to ecotypes variation. It is believed that the genetic architecture of a population is the result of breeding system, gene flow within and between populations, isolation mechanisms and prolonged selection by various natural and artificial forces .
The genetic variations across the ten populations showed that populations from East Showa and East Hararghe had the highest number of effective alleles, heterozygosity and Shannon’s diversity index. This indicates that East Showa and East Hararghe may be the most important populations with high genetic diversity, and were the potentials accessions in future V. galamensis improvement and genetic conservation endeavor in Ethiopia. Hence, these populations may serve as potential sources of new genetic variation of important traits (hotspots for in-situ and ex-situ conservation), and used in breeding programs as potential parental sources. Since, in plant natural populations, spatial distribution of genetic variation is primarily determined by seed and pollen dispersal, habitat distribution, micro-environmental selection and genetic drift .