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.
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Results and Discussion
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1
Genetic relationships among fig cultivars
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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.
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Genetic variation within and between clusters
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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.
-0.16
A
-0.16
Snowden
Calimyrna
Osborne_Prolific
UCR291
Kadota
B
Marabout
Marabout_smyrnay
Adriatic
St._Jean
Hative_De_Argentù
Marabout
Marabout_smyrnay
Adriatic
St._Jean
Hative_De_Argentù
-4.38
-4.38
Orphan
Orphan
Dauphine
Dauphine
Axis III (13.3%) -8.60
Axis III (13.3%) -8.60
King
King
Brown_T urkey
Brown_T urkey
Mission
Mission
-12.82
-12.82
Zidi
Zidi
20.64
Axis II (18.3%) 10.88
20.64
15.76
Violette_de_Bordù
Axis II (18.3%) 10.88
6.00
1.12
-17.04
-7.97
15.76
Violette_de_Bordù
6.00
-4.96
Axis I (19.6%)
-1.94
-17.04
1.12
-7.97
1.07
4.08
-4.96
-4.96
Axis I (19.6%)
-1.94
1.07
4.08
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.
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 mandirected evolutionary forces.
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Integration of metric and molecular data
Snowden
Calimyrna
Osborne_Prolific
UCR291
Kadota
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0.005
Nei and Li distance
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. Genetic Resources and Crop Evolution 51: 269-275.
Condit, I. J. 1955. Fig varieties: a monograph. Hilgardia 23: 323-538.
Condit, I.J. 1947. The fig. Waltham, Mass, USA.
Khadari, B., Grout, C., Santoni, S. and Kjellberg, F. 2005. Contrasted genetic diversity and differentiation among Mediterranean populations of Ficus carica L.: A study using mtDNA RFLP. Genetic Resources and Crop Evolution 52: 97-109.
Khadari, B., Hochu, I., Santoni, S. and Kjellberg, F. 2001. Identification and characterization of microsatellite loci in the common fig (Ficus carica L.) and representatives species of the genus Ficus. Molecular Ecology Notes 1: 191-193.
Nei, M., Li, W., 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Acadamy of Sciences, USA. 76, 5269-5273.
Papdopoulou, K., Ehaliotis, C., Tourna, M., Kastanis, P., Karydis, I. and Zervakis, G. 2002. Genetic relatedness among dioecious Ficus carica L. cultivars by randomly amplified polymorphic DNA analysis, and evaluation of agronomic and morphological characters. Genetica 114: 183-194.
Wright, S. 1965. The interpretation of population structure by F-statistics with special regard to systems of mating. Evolution 19: 395-420.
Zohary, D. & Hopf, M. 1993. Domestication of plants in the Old World. Clarendon Press, Oxford.
Cluster 1c
27
Cluster 1b
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.
Abruzzi
Sals
Dark Portuguese
Hearty Chicago
Pied De Boeuf
Dauphine
Italian 372
White San Pedro
Dokkar
Kadota
Trojano
Lemon
LSU Everbearing
Ficotto
King
Italian 253
Brunswick
Red Italian
Capitola Long
Rattlesnake
Doree
LSU Gold
Genoa
Genoa White
San Pietro
Yougo #7
Green Germany
Italian 215
Alma
Drap Dor
Encanto Brown Turkey
Malcoms Super Giant
Archipel
Beers Black
Vista
Violette De Bordeaux
Bourjassotte Grise
Negro Largo
Violet Sepor
Black Madeira
Col De Dame
Maho
Gazir
Fico Verde
Ischia Green
Paradiso
Monstrueuse
Italian 320
Encanto
Barbillone
Hative de Argntteuil
Ischia White
Italian 395
Kukurchinskii
Bosnat
Calvert
Verte
Ak inzhyr Koinekashirskii
Beall
Early Violet
Zheltoplodnyi Okruglyi
Charles Allen
Cuello Dama Negro
Noire de Caromb
Mission
Igo
Persistent Capri1
Verdal Longue
Black Jack
Brown Turkey
Braun Turkey
Walker
Blue Giant
St Jean
Moissoniere
Adriatic
Lampeira
Capri W
Capri Q
Gulbun Selection
Jurupa
Conadria
Persistent Capri2
Marabout smyrnay
Ucr291 4
Hurricane
Tena
Calimyrna
Vina 4
Osborn Prolific
Snowden
Capri X
Maslin 150
Maslin Edible
Dawalki
Calabacita
Catarulla
Green Italian 06
Double Header
Green Panachee
Panachee
Pastiliere
Rouge de Bordeaux
Roscoff
San Joao Branco
Santa Cruz Light
Santa Cruz White
Diredo
Flanders
Sierra
Sierra6 38
Bourjassotte Blanche
Chater Green
Chater Green
Karayaprak
Oregon Little Red
Stanford
Honigal
Renans Strawberry
Roeding #2
Algerian Watts
Orphan
Deanna
Ucr278 128
T30E
Persistent Capri3
Long Fellow Furtado
Long Yellow
Vernino
Capri A
Roeding #3
Armenian
Milco
Bournabat
Marylane
Excel
Italian 169
Italian 358
Yellow Neches
Asisi
hybrid1
Marabout
Marabout
Monaco
Italian 88
Aked
Blanquette
Celeste
Nero Cesar
Becane
LSU
Kop
Fiomi
Hollier
Barada
Ucr291
Afghan
Caucasus 2b
Caucasus 1a
Caucasus 6c
Ak inzhyr Kuruzhdeiskii
Kugitangskii Chernyi
Kury Gol
Shevlan 1
Shih Berdy2
Inzhyr from Sopyev
Zheltyi from Seidov
Nuhurskii
Chikishlyarskii
Shevlan 2
Shevlan 3
Hacin
Zidi
Barnissotte
Bianco
Yede Vern
Cluster 4
Ischia Black
Nero
Giant Amber
Santa Cruz Dark
pseudocarica
hybrid2
Cluster 2
100
30
Abruzzi
Dark Portuguese
Sals
Hearty Chicago
Pied De Boeuf
Adriatic
Lampeira
Capri W
Afghan
Caucasus 2b
Caucasus 1a
Caucasus 6c
Aked
Blanquette
Calimyrna
Vina 4
Osborn Prolific
Snowden
Capri X
Maslin 150
Maslin Edible
Ak inzhyr Koinekashirskii
Kop Fiomi
Black Jack
Braun Turkey
Brown Turkey
Walker
Blue Giant
Persistent Capri1
Ak inzhyr Kuruzhdeiskii
Kugitangskii Chernyi
Kury Gol
Shevlan 1
Shih Berdy2
Inzhyr from Sopyev
Zheltyi from Seidov
Nuhurskii
Armenian
Milco
Algerian Watts
Orphan
Ucr278 128
Deanna
T30E
Long Fellow Furtado
Long Yellow
Persistent Capri3
Alma
Drap Dor
Encanto Brown Turkey
Malcoms Super Giant
Archipel
Italian 320
Dauphine
Italian 372
White San Pedro
Dokkar
LSU Everbearing
Kadota
Trojano
Lemon
Ficotto
King
Ischia White
Asisi
Honigal
Barada
Ucr291
Barbillone
Hative de Argntteuil
Black Madeira
Col De Dame
Maho
Gazir
Fico Verde
Monstrueuse
Ischia Green
Paradiso
Barnissotte
Karayaprak
Oregon Little Red
Stanford
Bourjassotte Blanche
Chater Green
Chater Green
Beall
Early Violet
Charles Allen
Cuello Dama Negro
Noire de Caromb
Mission
Igo
Giant Amber
Santa Cruz Dark
Becane
Celeste
Nero Cesar
LSU Hollier
Beers Black
Violette De Bordeaux
Vista
Bourjassotte Grise
Negro Largo
Violet Sepor
Italian 395
Kukurchinskii
Bianco
Yede Vern
Ischia Black
Nero
Italian 88
St Jean
Bosnat
Calvert
Verte
Bournabat
Marylane
Pastiliere
Rouge de Bordeaux
Yellow Neches
Brunswick
Red Italian
Doree
Capitola Long
Rattlesnake
LSU Gold
Genoa
Genoa White
San Pietro
Yougo #7
Italian 253
Calabacita
Catarulla
Green Italian 06
Double Header
Green Panachee
Panachee
Roscoff
San Joao Branco
Santa Cruz Light
Santa Cruz White
Capri A
Roeding #3
Vernino
Renans Strawberry
Roeding #2
Capri Q
Gulbun Selection
Jurupa
Conadria
Marabout smyrnay
Ucr291 4
Dawalki
Chikishlyarskii
Shevlan 2
Shevlan 3
Encanto
Moissoniere
Diredo
Flanders
Sierra
Sierra6 38
Excel
Italian 169
Italian 358
Green Germany
Italian 215
Hurricane
Persistent Capri2
Tena
hybrid1
Marabout
Marabout
Monaco
Verdal Longue
Zheltoplodnyi Okruglyi
Hacin
Zidi
pseudocarica
hybrid2
Cluster 3
Materials and Methods
34
Cluster 1a
99
30
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