Integration of Molecular and Metric Traits in the Analysis of Genetic Structure and Differentiation in Cultivated Fig (Ficus carica L.)

1
Integration of Molecular and Metric Traits in the
Analysis of Genetic Structure and Differentiation
in Cultivated Fig (Ficus carica L.)
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
?2 analysis was performed to determine the
heterogeneity among groups. Genetic
differentiation within and among fig groups was
computed using the Wrights 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 Candits 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 ?2 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
Wrights 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
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