Integration of Molecular and Metric Traits in the Analysis of Genetic Structure and Differentiation in Cultivated Fig (

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Malli Aradhya and Ed Stover* USDA Germplasm Repository, University of California, Davis, CA 95616, USA. Introduction The fig, Ficus carica L., (Moraceae) is a classical fruit tree of antiquity associated with the beginning of horticulture in the Mediterranean basin. The fig is known to have been domesticated from a group of diverse, spontaneous figs occurring in the Mediterranean region sometime in the Early Neolithic period (Zohary and Hopf, 1993). Although the cultivated fig is gynodioecious, it is functionally dioecious, with pollination facilitated by the mutualistic interaction of pollinator wasps (Balstophagous psenes L.) between two separate fig types, Caprifig and edible fig. Figs are generally classified into four types, mainly based on the floral biology and pollination behavior: Common, Smyrna, Caprifig, and San Pedro. Of the four types, Caprifig, bearing both male and female flowers within the same receptacle or fruit called syconium, is regarded as primitive, and the Common-type, with only pistillate flowers developing into parthenocarpic fruits, is considered highly developed and includes most commercial cultivars (Condit, 1947, 1955). Smyrna and San Pedro types represent intermediate forms. Fig has a long history of domestication and selection in the diverse Mediterranean and surrounding Near Eastern regions, and numerous cultivars have been recognized. Further spread of fig selections into other growing regions has resulted in ambiguity in the description and nomenclature of cultivars. Recently, microsatellite markers, randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), restriction length polymorphism (RFLP), and mitochondrial DNA RFLP markers have been used in fingerprinting, assessing genetic diversity, structure and differentiation in fig collections (Khadari et al., 2001; Papadopoulou et al., 2002; Amel et al., 2004; Khadari et al., 2005). We are present here the results of an analysis of genetic diversity and structure within cultivated fig using a combination of mirosatellite and metric data. Materials and Methods One-hundred eighty one cutivated fig (F. carica) accessions including one gentotype of F. palmata were sampled from the USDA germplasm. Sixteen microsatellite loci (Table 1) were PCR amplified and products resolved using capillary electrophoresis on an ABI Prism 3100 genetic analyzer. The data was analyzed using Genescan, Version 3.1 and Genotyper, Version 2.5 and data assembled as genotypes as well as in binary format. The binary data were used to compute a distance matrix using Nei and Li distance (Nei and LI, 1979) based on the proportion of alleles shared between two accessions for all possible pair-wise combinations. The resultant matrices were subjected to cluster analyses using UPGMA (Unweighted Pair Group Method using Arithmetic means) method to produce a phenogram. The multilocus SSR genotype data were pooled into groups based on the results of NJ and UPGMA cluster analyses and analyzed for various within-group genetic variability measures such as mean number of alleles per locus and observed and expected levels of heterozygosities. Contingency χ2 analysis was performed to determine the heterogeneity among groups. Genetic differentiation within and among fig groups was computed using the Wright’s F-statistics (Wright, 1965). Metric data on twenty-seven horticulturally valuable traits (Table 1) were collected for seventeen representative genotypes from the collection. The variance-covariance matrix computed from the metric data was subjected to a principal components analysis (PCA) to elucidate the relationships among the genotypes. In an attempt to integrate metric and molecular data, minimum spanning trees (MST) generated separately from metric and microsatellite data were superimposed on 3D projection based on the metric data. Results and Discussion Genetic relationships among fig cultivars Although most fig genotypes possessed unique multilocus fingerprints indicating a significant amount genetic variability in the collection, there was no obvious evidence for any significant genetic structure. Fig being functionally dioecious and insect pollinated naturally maintains and circulates high levels of genetic variation within and among cultivar from Caprifig, Smyrna, San Pedro, and Common fig types. However, the cluster analysis revealed a total of six clusters with three major and three minor ones within cluster 1 (Fig 1). The cluster 1 contained mostly Common and San Pedro types with a concentration of San Pedro types (‘Pied de Boeuf’, ‘Dauphine’, ‘King’, ‘White San Pedro’) in subcluster 1a. There were several instances of identical genotypes with different cultivar names. For example the cultivars ‘Brunswick’, ‘Capital Long’, ;Red Italian’, ‘Doree’, and ‘Rattlesnake’, all had identical fingerprints. The popular Common type cultivars such as ‘Brown Turkey’, ‘Walker’, and Black Jack’ were genetically identical. Overall the subclusters 1b and 1c contained mostly Common type figs. Cluster 2 is the biggest cluster contained Common, Smyrna, and Caprifig clutivars. The Smyrna types (eg. ‘Calimyrna’, Marabout Smyrna’, ‘Snowden’, ‘Karayaprak’) are basically confined to this cluster. Cluster 3 and 4 contained some of the less known Common type figs except for the cultivars ‘zidi’, which is Smyrna type fig and ‘Ischia Black’, which is a Common type. Cultivars and selections from Candit’s breeding program such as ‘Gulbun selection’, ‘Jurupa’, ‘Deanna’, and many UCR selections are scattered in different groups suggesting the diversity of material included in his program. Genetic variation within and between clusters Contingency χ2 analysis indicated significant differences in the allele frequencies among clusters with some cluster specific low to moderate frequency alleles. The number of alleles/locus ranged form two for LMFC36 to 10 for LMFC30 with an average of 4.75 alleles/locus. There was excess of heterozygotes in all clusters suggesting heterozygote superiority (Table 1). Genetic differentiation based on Wright’s fixation index FIS indicated that there was significant excess of heterozygotes within clusters (Mean FIS = -0.200) for all loci except one locus (Table 2). FIT, which is a measure of inbreeding coefficient in total population indicated marginal reduction in heterogygosity (0.003), except for four loci showing low to moderate levels of excess of heterozygotes. FST, measure of genetic differentiation among clusters showed moderate reduction in heterozygosity (0.170) suggesting somewhat a weak differentiation among clusters. Integration of metric and molecular data Superimposing of MSTs generated from metric and molecular data on to the 3D projection of 17 accessions along the first three principal axes accounting for 51.2 % of total variation in metric traits indicated significant incongruence in the pair-wise relationships among the 17 accessions included in the PC analysis (Fig. 2). The incongruence between metric data and molecular data in depicting the pair-wise relationships among the 17 fig accessions suggests significant differences in the variance-covariance structures between the metric and molecular traits as a complex response to either natural or man-directed evolutionary forces. Fig. 2. 3D projection of 17 selected genotypes of figs along the first three principal axes from a PCA based on metric data. MSTs are superimposed to show the pair-wise relationships. A, MST generated from the same metric data as used in the PCA; B, MST generated from 16 microsatellite loci. Fig. 1. Genetic relationships among the fig cultivars based on UPGMA cluster analysis. Bootstrap tree is shown on the left References Amel, S.-H., Mokhtar, T., Salwa, Z., Jihene, H., Messaoud, M., Abdel,ajid, R. and Mohamed, M. 2004. Inter-simple sequence repeat fingerprints to assess genetic diversity in Tunisian fig (Ficus carica L.) germplasm. 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