It is critical for a germplasm repository to provide reliable information on the germplasm it holds and distributes through on-site evaluation. Characterization based on morphological criteria is often variable across years and locations due to the plasticity and susceptibility of these traits to genotype-environmental interactions. Molecular markers offer a stable and reliable alternative for genetic identification and characterization of germplasm collections. Recently, microsatellite, also known as Simple Sequence Repeats , randomly amplified polymorphic DNA , inter-simple sequence repeat , restriction length polymorphism , and mitochondrial DNA RFLP markers have been used in fingerprinting, and assessing genetic diversity, structure and differentiation in fig collections . As part of an ongoing germplasm characterization effort at the USDA germplasm repository, at Davis, California, we analyzed a subset of the fig collection using microsatellite markers and the preliminary results are reported here. The study attempts to assess the genetic diversity and differentiation within the collection and elucidate the genetic relationships within and between different cultivar groups.The marker, MFC5, which amplified two loci, was excluded from the computation of genetic diversity and differentiation parameters due to discrepancy in the assignment of alleles between two loci. However, blueberry pot the binary data from all 17 loci were used to compute the Nei and Li distance based on the proportion of alleles shared between two accessions for all possible pair-wise combinations.

The resultant matrix was subjected to a cluster analysis following the neighbor-joining method to produce a phenogram. The multilocus SSR genotype data were pooled into groups based on the results of NJ cluster analysis and analyzed for various within-group genetic variability measures, such as mean number of alleles per locus and observed and expected levels of heterozygosities. Genetic divergence among groups was expedited using the distance Wagner procedure based on a matrix of pair-wise distances between groups using Prevosti distance . Multivariate relationships among 142 fig accessions possessing unique multilocus genotypes were examined with principal components analysis using the software NTSYSpc . Accessions were projected along the first three principal axes to visualize genetic affinities.Knowledge of genetic diversity, population structure and differentiation significantly contributes to effective conservation, management and utilization of germplasm collections. Genetic characterization of ex situ collections offers insight into the amount and patterns of distribution of genetic diversity and permits classification of germplasm based on genetic similarities and differences. Nowadays, gene banks around the world are focusing on genetic and phenotypic characterization of germplasm collections in order to promote efficient utilization of germplasm in breeding and development of crops. Characterization permits identification of deficiencies in collections and planning for future collection efforts to strategically enrich existing collections. In clonally propagated, perennial species such as fig, germplasm accessions are preserved as unique genotypes, the genetic and phenotypic integrity of which is important for breeders and researchers who look for particular combinations of traits or genes in an accession.

Phenotypic analysis of variation in clonally maintained, perennial crop collections are age and management-dependent and subject to genotype-environmental interactions and consequently not comparable across environments, but biomolecular evaluations offer a comparable measure of genetic diversity and establish the identity for individual accessions. Germplasm collections of most clonally propagated species often contain morphologically similar accessions having different genetic and geographic origins. Further, genetically identical cultivars may have different names in different collections and countries, probably due to lax use of names by growers, nurserymen, and traders, corruption in English transliteration of original names, the presence of variants within cultivars, and lack or poor documentation of passport data. Fig is adapted to a wide range of climates from tropical, subtropical, Mediterranean, and even to temperate conditions, and has a long domestication and cultivation history, which has led to recognition of numerous ecotypes and landraces selected and maintained by indigenous people for their adaptation to local environments and farming systems, and subtle fruit qualities, which generally possess local synonymous names, a problem that plagues germplasm collections of clonal crops . Condit , in his monograph on fig varieties, lists more than 700 cultivars and the majority have large numbers of synonyms. Many of the old and popular cultivars such as ‘Kadota’, ‘Brown Turkey’, ‘Ischia Green’ and ‘Brunswick’ often possess several synonyms and they generally possessed similar tree structure, morphology and fruit characteristics. Deciphering genetic identity and relationships among these cultivars is complicated due to occurrence of extensive synonymy and non availability of authentic source cultivars for comparison.

The genetic and geographic origin of most of these cultivars is unknown and associated passport data are incomplete, inaccurate, or missing in most germplasm collections. The figs from Turkmenistan have allied tightly in group 8 with good bootstrap support, indicating some level of differentiation from the rest of the figs. However, group 8 also contained ‘Zidi’, a dark purple Smyrna fig from Morocco, ‘Calimyrna’, a yellow Smyrna fig commercially grown in California, and two other U.S. cultivars, ‘Snowden’ and ‘Osborne Prolific.’ This group may represent non-Mediterranean type wild figs found in the Hyrcanic regions of the south Caspian Sea, which some botanist treat as a separate species, F. hyrcana . Both Mediterranean and non-Mediterranean wild figs are fully interfertile and produce hybrids that are adapted to a wide range of ecological conditions . Group 9 features cultivars developed in the early California breeding program such as ‘Conadria’, ‘Deanna’, ‘Tena’, ‘Jurupa’, ‘Gulbun’ and ‘Flanders’ and some of the cultivars used in the hybridization program. The cultivar ‘Adriatic’, which has been extensively used in the California fig breeding program, also clusters within this group. The cultivars ‘Brunswick’, ‘Rattlesnake’, and ‘Capitola Long’ showed identical multilocus genotypes, and were shown to be identical in an earlier study based on sequence-related amplified polymorphisms . Overall, the classification of fig cultivars is largely based on skin and pulp color, floral biology, pollination behavior and parthenocarpy, which are probably governed by simple Mendelian genes and may be unrelated to molecular markers.The fig germplasm collection harbors moderate to high levels of genetic polymorphisms across the microsatellite loci assayed with 140 unique multilocus genotypes out of a total of 194 accessions included in the study. The heterogeneity among loci for levels of heterozygosity and fixation index reflects a complex selection history and genetic structure of populations from which the fig cultivars were originally selected. Averaging over loci, the fig collection approaches panmixia, although some loci deviated significantly, indicating differential selection among loci. The mild genetic structure within the fig collection with deeply dissected branches on the phenetic tree suggests that most variation is locked up at the level of individuals as polymorphic, multilocus heterozygotes. The ten groups identi- fied based on the cluster analysis contained an assortment of fig types, Smyrna, Caprifig, San Pedro, and Common, nursery pots indicating shared ancestry or evolutionary background and are connected through gene flow via Caprifig, the main pollen source . However, support for some groups was marginal, indicating the complex multidimensional nature of molecular variation. The weak genetic structure observed in the present study is probably due to the fact that fig circulates genetic variability across different fig types through a dynamic mutation-recombination process facilitated by a complex pollination mechanism involving the symbiotic relationship between the fig and its pollinator. Further, the genetic relationships within and among fig groups observed in the CA should reflect a complex combination of natural evolution, genetic drifts and founder events during domestication, historical migration of cultivars along human migrations from the center of origin and diversity to secondary centers and regions of commercial production, and genetic modifications through modern plant breeding. Although there was marginal evidence for differentiation, there was marked differences among genetic groups with respect to composition and frequency of alleles for different loci as indicated by the Fisher’s Exact Test. Clonally propagated perennial species such as fig are known to carry relatively high genetic load and tend to exhibit an excess of heterozygotes as a mechanism to overcome the deleterious effects of recessive mutations . Prevalence of Common fig in cultivation around the world probably indicates that human selection has historically favored parthenocarpic fig over pollination dependent Smyrna and San Pedro types, especially in regions lacking the pollinator wasp.

Parthenocarpy in Common fig was probably selected early in the domestication history dating back to the early Neolithic period and possibly derived as a point mutation favored by humans. On the contrary, Lev-Yadun et al. points out that Common figs are dioecious, with male trees producing inedible seedless figs that maintain the pollinating wasps. It is known that the parthenocarpic Common fig, if pollinated, does produce better quality figs than parthenocarpically developed figs, and contain viable seeds, the progeny of which segregates into male and female figs. Both Bayesian and distance based approaches used to examine the genetic structure and differentiation revealed weak genetic structure, probably due to inherently narrow genetic base from which the fig was domesticated, combined with historical migration of germplasm and the outcrossing mode of pollination, which have countered human selection in different fig growing regions of the world. Nevertheless, the CA using the neighbor-joining method identified ten, somewhat narrowly differentiated groups, some of which were further confirmed in the PCA analysis. The Bayesian analysis indicated that most fig genotypes have mixed ancestry, which becomes clear as the K value incraeses in the analysis. At K = 5 without prior population information, the simulation attained the highest likelihood value and had the higher clusteredness, while the cluster composition and membership coefficients somewhat reflected within and among group relationships in the CA and PCA. Although members from different clusters revealed by the CA moved around a bit among different Bayesian clusters at K = 5, most members from groups 1, 2 and 3 formed a cluster, members predominantly from groups 4, 5, 6 and 7 formed a second cluster, and most members from groups 9 and 8 came together in a cluster. However, some members from group 9 were found scattered among three different clusters and similarly some of 6 and 7. Most of the members of group 10 formed a cluster, some showing affinity with members of group 7. Overall, it is challenging to infer the genetic structure and differentiation of outcrossing plant species such as fig with long history of domestication, extensive dispersal, and wide range of adaptation. Most of the alleles are widespread and formed gene frequency clines within and among groups and clusters reflecting the biogeographic history of fig. Further, as warned by the authors of STRUCTURE, the method of inferring K is an ad hoc procedure based on a set of uncertain assumptions and that the inferred K may not always have a clear biological interpretation . Even the DK approach based on the rate of change in the log probability of data between successive K values could not accurately predict the K in fig.Organization of genetic diversity in clonally propagated species germplasm collections is reminiscent of historical genetic structure originating from the complex interaction of evolutionary forces and domestication history of the species. The gene pool of fig examined possesses signifi- cant genetic variability and exhibits narrow differentiation among the ten genetic groups identified by the CA and PCA. However, the model based cluster analysis indicated that most fig genotypes had mixed ancestry and molecular variation is clinal without clear differentiation. The geographic or genetic basis for relationships among cultivars within and among groups and clusters is difficult to decipher due to lack or incomplete passport data. However, groups differed for the composition and frequency of alleles for different loci indicating some sort of mild sub-structuring within the collection. Earlier studies have demonstrated clustering of fig genotypes on a geographic basis , but on limited sampling basis. Fig being a functionally dioecious, there is extensive species-wide gene flow within and among different groups and fig types, and the subtle substructure noticed in this study probably reflects a complex combination of effects of historical dispersal of cultivars and human selection. Further, the weak genetic structure of fig is probably suggestive of a single, complex gene pool featuring extensive dispersal of cultivars homogenizing the local populations. Earlier studies in fig generally reported increased effective population size reducing population subdivision with most variation tending to be within populations . However, a study based on mtDNA restriction fragment length variation demonstrated slightly higher levels of differentiation among natural population and among groups of populations of fig from the Mediterranean region .