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With 3 figures and 3 tables The genetic relationships between 40 wild hazelnuts collected in northern Spain and cultivated hazelnuts, including 62 local selections and 14 reference cultivars, were investigated using 13 microsatellite loci. Microsatellite analysis revealed considerable diversity; 91 different alleles were identified with a mean of 7 per locus, and polymorphic information content values ranged from 0.43 to 0.83 with a mean of 0.69. The plot obtained from principal coordinate analysis, the unrooted neighbour-joining tree constructed, and the population structure analysis revealed a high level of differentiation between the locally cultivated forms and the remaining materials. Introgressions within these groups were detected in the three analyses. The results indicate that hazelnuts in northern Spain contain (i) a group of accessions clearly differentiated within the Spanish–Italian gene pool, (ii) a group with intermediate forms probably derived from hybridization and (iii) accessions probably derived through exchange with other geographical areas, especially north-eastern Spain. The presence of these different groups within the local cultivated hazelnut germplasm has consequences for its preservation and use.
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Genetic relationship between cultivated and wild hazelnuts (Corylus avellana L.)
collected in northern Spain
A. C
AMPA
1
,N. T
RABANCO
1
,E. P
E
´REZ
-V
EGA
1
,M. R
OVIRA
2
and J. J. F
ERREIRA
1,3
1
A
´rea de Cultivos Hortofrutı
´colas y Forestales, Servicio Regional de Investigacio
´n y Desarrollo Agroalimentario (SERIDA),
33300 Villaviciosa, Asturias, Spain;
2
IRTA – Mas de Bover, Departament Olivicultura, Elaiote
`cnia i Fruita seca, Crta. Reus–El
Morell. Km, 3,8, 43120 Constantı
´, Tarragona, Spain;
3
Corresponding author, E-mail: jjferreira@serida.org
With 3 figures and 3 tables
Received March 24, 2010/Accepted October 8, 2010
Communicated by J. Le
´on
Abstract
The genetic relationships between 40 wild hazelnuts collected in
northern Spain and cultivated hazelnuts, including 62 local selections
and 14 reference cultivars, were investigated using 13 microsatellite
loci. Microsatellite analysis revealed considerable diversity; 91 different
alleles were identified with a mean of 7 per locus, and polymorphic
information content values ranged from 0.43 to 0.83 with a mean of
0.69. The plot obtained from principal coordinate analysis, the
unrooted neighbour-joining tree constructed, and the population
structure analysis revealed a high level of differentiation between the
locally cultivated forms and the remaining materials. Introgressions
within these groups were detected in the three analyses. The results
indicate that hazelnuts in northern Spain contain (i) a group of
accessions clearly differentiated within the Spanish–Italian gene pool,
(ii) a group with intermediate forms probably derived from hybrid-
ization and (iii) accessions probably derived through exchange with
other geographical areas, especially north-eastern Spain. The presence
of these different groups within the local cultivated hazelnut germ-
plasm has consequences for its preservation and use.
Key words: Corylus avellana L. — microsatellite — genetic
diversity — principal coordinate analysis — domestication
European hazelnut (Corylus avellana L.) is one of the main nut
crops in the world. Its native distribution extends from Asia
Minor, the Caucasus Mountains and Ural Mountains to
western Europe (British Isles, Scandinavian islands, and
Central and Southern Europe) and the Mediterranean coast
of North Africa (Thompson et al. 1996). However, in recent
years, the main production areas have been in Turkey, Italy,
the United States and Spain (FAOstat 2008). In Spain,
hazelnut is cultivated mainly in the north-east (Catalonia),
although there are other minor hazelnut-growing areas in the
north, such as Asturias (Tous et al. 2001).
The traditional characterization of many plants such as
hazelnut was based on morpho-agronomic traits (UPOV 1979,
Bioversity International 2008), which can be influenced by
environmental factors. Microsatellite markers or simple
sequence repeats (SSR) are considered robust markers, highly
reproducible and generally inherited as co-dominant loci not
affected by environmental factors. A set of 51 SSR markers
was recently developed in this species (Bassil et al. 2005a,b,
Boccacci et al. 2005) of which 30 were placed on a genetic map
(Mehlenbacher et al. 2006). SSR markers were used to analyse
genetic relationships among cultivars, cultivar fingerprinting
and the identification of synonyms in collections (Boccacci
et al. 2006, 2008, Go
¨kirmak et al. 2009, Gu
¨rcan et al. 2010).
The variation detected by SSR markers has also been used to
investigate the structure and organization of the genetic
diversity in this species. The variation detected at 21 SSR loci
in 270 European hazelnut accessions showed four major
geographical groups or gene pools (Go
¨kirmak et al. 2009):
Central European, Black Sea, English and Spanish–Italian.
Chloroplast microsatellite analysis indicated that hazelnut
genetic diversity was organized in three main groups with
different chloroplast type frequencies, suggesting that hazelnut
was domesticated in three main areas: the Mediterranean,
Turkey and Iran (Boccacci and Botta 2009). In many plant
species, microsatellite markers have been used to investigate
the structure and genetic relationships between wild and
cultivated forms (Grassi et al. 2003, Kwak and Gepts 2009).
The study of the relationship between wild and cultivated
forms in a geographical area can supply information about the
putative domestication events, the evolutionary relationships
or the gene flow between them (Gepts et al. 1986, Papa and
Gepts 2003, Kwak and Gepts 2009). Information about wild
forms may also be useful to breeders wishing to enlarge the
genetic base of their breeding programmes. However, there
is limited information concerning the relationship between
cultivated and wild forms in hazelnut.
In the past, hazelnut was an important crop in Asturias
(Alvarez Requejo 1965); however, at the end of the twentieth
century, the exodus of people from rural areas, together with
problems with hazelnut commercialization, led to abandon-
ment of local crops. In Asturias, wild and cultivated forms of
hazelnut coexist. Wild forms can be found along the banks of
streams or forming small woods in isolated areas. The
cultivated forms can be found in small orchards, gardens
and hedgerows, where they delimit the fields and produce fruit
and wood. A hazelnut germplasm exploration was carried out
in northern Spain (Asturias) over three consecutive years
(2003–2005), and as a result of this exploration, 90 cultivated
trees were selected from the region. The morphological
characterization of this local germplasm revealed a wide
phenotypic variation in nut and husk traits, and many
selections had characteristics appreciated by the market
(Ferreira et al. 2010). The genetic relationships among 50
local selections derived from the survey and 17 well-known
cultivars, including seven cultivars from north-eastern Spain,
were investigated using inter-simple sequence repeat (ISSR)
markers. The results indicated that the local accessions were
Plant Breeding doi:10.1111/j.1439-0523.2010.01835.x
2011 Blackwell Verlag GmbH
wileyonlinelibrary.com
closely related, but relatively distant from the standard
cultivars of north-eastern Spain, Italy, Turkey and the United
States (Ferreira et al. 2010). These results could indicate local
domestication. In many regions, cultivated forms coexist with
wild forms, and it has been suggested that some local cultivars
were selected from local wild populations (Lagerstedt 1975,
Tasias Valls 1975, Thompson et al. 1996, Go
¨kirmak et al.
2009). In this study, the variation detected by SSR markers is
used to (i) verify the differentiation of hazelnuts cultivated in
Asturias from other hazelnut gene pools and (ii) investigate the
genetic relationships between cultivated and wild local hazel-
nuts. This information will be useful to identify local materials
for preservation in collections and to improve knowledge
concerning domestication events of this species.
Materials and Methods
Plant material: A total of 116 trees were analysed in this study
(Table 1): (i) 62 local forms cultivated by farmers, of which 58 were
collected in Asturias (Ferreira et al. 2010) and the remaining four are
local cultivars believed to have originated in Asturias (ÔCasinaÕ,
ÔAmandiÕ,ÔEspinaredoÕand ÔQuiro
´sÕ); (ii) 40 wild trees collected in
autumn of 2008 in the main areas of Asturias in which hazelnut wild
populations are present; and (iii) 14 well-known reference cultivars
representing the worldÕs most important production areas.
The local cultivars (ÔCasinaÕ,ÔAmandiÕ,ÔEspinaredoÕand ÔQuiro
´sÕ)
and the fourteen reference cultivars are maintained in the hazelnut
collection of the Servicio Regional de Investigacio
´n y Desarrollo
Agroalimentario (SERIDA, Villaviciosa, Spain); part of the local
cultivated materials are also being incorporated into this collection.
The corresponding gene pool of the 14 reference cultivars, according to
Go
¨kirmak et al. (2009), is shown in Table 1.
DNA extraction and microsatellite analysis: Genomic DNA was
extracted from young leaves or from immature catkins (only in wild
materials) according to Boccacci et al. (2006). DNA was quantified
using a spectrophotometer and maintained at )20C.
A total of 13 SSR markers were analysed: CAC-B029b, CAC-B109,
CAC-B101, CAC-A014b, CAC-C115, CAC-B010, Cat-B507, Cat-
B107, Cat-B50, Cat-B502, Cat-C504, Cat-B106 and Cat-A114 (Bassil
et al. 2005a, Boccacci et al. 2005). Of these markers, 10 have been
placed on the genetic map of hazelnut (Mehlenbacher et al. 2006). This
SSR set was selected after considering the pattern of bands produced
by the SSRs and the level of polymorphism previously reported (Bassil
et al. 2005b, Boccacci et al. 2005, 2006, Go
¨kirmak et al. 2009). PCR
amplification was performed in a volume of 20 ll containing 50 ng
DNA; 0.5 U Taq-DNA polymerase (BIOTAQTM DNA Polymerase;
BIOLINE, London, UK); 1·buffer; 2.5 m
M
MgCl
2
/l; 200 l
M
each of
dATP, dCTP, dGTP and dTTP; and 0.5 l
M
each of forward and
reverse primers. The amplifications were carried out using the
programs described by the respective authors. PCR products were
resolved on 8% polyacrylamide gels, stained with SYBR Safe DNA gel
stain (Invitrogen, Eugene, OR, USA) and visualized under UV light. A
100-bp ladder (G.E. Healthcare Life Science, Fairfield, CT, USA) and
the software G
ENE
T
OOLS
V4.01 (Syngene, Cambridge, UK) were used
to measure the size of the amplification products.
Data analysis: Alleles with molecular sizes in the range previously
described for each microsatellite were considered in this study.
Accessions with the same fingerprint were considered synonyms, and
only one accession in the group was used in subsequent analysis. The
software P
OWER
M
ARKER
3.25 (Liu and Muse 2005) was used to
calculate the following parameters of genetic diversity: the number of
alleles per locus, observed heterozygosity (Ho), gene diversity (synon-
ymous with expected heterozygosity, He), estimated frequency of null
alleles (r) and the polymorphic information content (PIC). Ho is
calculated as the number of heterozygous genotypes, divided by the
number of total genotypes observed at the locus. He estimates the
probability that two alleles at any locus are different from each other.
The PIC value measures the polymorphism observed in a group of
genotypes at a specified locus.
From the variation provided by the 13 SSR markers, three types of
analysis were performed to group the accessions and investigate the
relationships and structure of the genetic diversity. Using the data
matrix for the presence or absence of each band (allele), a principal
coordinate analysis (PCoA) was performed with the software
NTSYS
PC
V.2.1 (Rohlf 2002), and the two principal coordinates were
used to visualize the dispersion of the accessions in a graph. From a
data matrix that listed the alleles at each SSR marker locus, an
unrooted neighbour-joining tree (NJ) was constructed with the
software PowerMarker using the C.S. Chord distance (Cavalli-Sforza
and Edwards 1967), and the tree was visualized with the software
T
REE
V
IEW
1.6.6 (Page 1996). Data were also analysed with
STRUCTURE
V2.2 software (Pritchard et al. 2000) to determine the number of
Table 1: Hazelnut materials inclu-
ded in the SSR markers analysis
Type of material Geographical origin
Material (accession
or cultivar) Gene pool
1
Local cultivated selections (58) Spain (Northern) As1 to As69
Local cultivars (4) Spain (Northern) Casina
Amandi
Espinaredo
Quiro
´s
Spanish–Italian
Local wild germplasm (40) Spain (Northern) Astw1 to Astw40
Reference cultivars (14) Spain (North-eastern) Gironell
Grifoll
Morell
Negret
Ribet
Segorbe
Spanish–Italian
Italy Camponica
Tonda di Giffoni
Tonda Romana
Santa Maria del Gesu
Spanish–Italian
Turkey Tombul Black Sea
England Daviana English
USA Butler
Ennis
English
SSR, simple sequence repeats.
1
According to Go
¨kirmak et al. (2009).
2A. Campa, N. Trabanco, E. Pe
´rez-Vega et al.
divergent groups (K). Runs were carried out ten times for each K
value ranging from 2 to 4. Each run was performed using the
admixture model and 20 000 MCMC (Markov Chain Monte Carlo)
repetitions following a burn-in of 20 000 iterations during the
analysis, and the run showing the lowest likelihood value was
selected. The statistic DK (Evanno et al. 2005) was used to choose the
optimal Kvalue. The results supplied by this program were visualized
in a graphical bar plot of membership coefficients for each Kvalue.
P
OWER
M
ARKER
software was also used to calculate F
ST
values in the
populations that were eventually selected. F
ST
measures genetic
differences among groups.
Results
Genetic diversity
PCR products were obtained at all SSR loci for all cultivars.
Fingerprint data revealed that 82 of the 116 cultivars had
unique genotypes. Thirty-six trees had two fingerprints: a
group of 30 accessions named the Casina group (Casina,
Amandi, Quiro
´s, Espinaredo, As1, As2, As3, As4, As6, As7,
As8, As9, As17, As19, As20, As28, As32, As38, As43, As46,
As47, As51, As52, As53, As61, As64, As65, As67, As68 and
As69) and another group of six accessions named the As16
group (As16, As31, As41, As48, As49, As50). These two
groups only differed at the microsatellite Cat B502. Table 2
shows five genetic diversity parameters (number of alleles, He,
Ho, r and PIC) obtained per SSR locus in the analyses of the
82 unique accessions. The number of alleles totalled 91 and
ranged from four (Cat-A114) to 10 (CAC-B101) alleles per
locus, with an average of seven. He ranged from 0.47 (for
CAC-B010) to 0.85 (for CAC-B101), with an average of 0.73.
PIC values ranged from 0.43 (CAC-B010) to 0.83 (for CAC-
B101 and CAC-A014b), with an average of 0.69. The most
polymorphic loci were CAC-B101, CAC-A014b, CAC-C115
and CAC-B109, while the least were CAC-B010 and Cat-B106.
Four parameters of genetic diversity for each SSR locus in
the three groups of materials considered in this work (local
cultivated germplasm, local wild materials and reference
cultivars) are shown in Table 3. The group of local cultivated
germplasm generally had lower values for parameters He and
PIC. The values for the group of reference cultivars were
within the range of the local wild materials.
PCoA and cluster analysis
In the two-dimensional PCoA plot (Fig. 1), the first coordinate
explained 14.85% of the variation and the second coordinate
an additional 7.9%. The figure shows two main groups. The
majority of the local cultivated materials appear at the left
(Casina group, As16 group and 12 trees), and the remaining
accessions appear at the right. The reference cultivars appear
at the top right, along with several local cultivated accessions,
and at the top right appear several local accessions (As22, As
35, As36, As57, As58, As60 and As66) and four local wild trees
(AstW8, AstW13 and AstW14). At the bottom right appear
the majority of local wild materials and the locally cultivated
accession As55. Finally, four local cultivated accessions (As12,
As15, As30, and As63) and three local wild accessions
(AstW12, AstW17 and AstW25) had an intermediate position
between the three main groups described previously.
Figure 2 shows the NJ tree obtained. Three distinct main
branches can be observed. One branch (A in Fig. 2) was
formed by 53 locally cultivated hazelnuts (17 accessions and
the groups Casina and As16), four local wild accessions
Table 2: Genetic diversity parameters obtained per SSR locus in the
analyses of 82 unique genotypes
Locus No. of alleles He Ho rPIC
CAC-B029b 7 0.75 0.80 )0.03 0.72
Cat-B507 6 0.70 0.96 )0.16 0.65
Cat-B107 9 0.74 0.55 0.11 0.70
Cat-B504 6 0.73 0.94 )0.12 0.69
Cat-B502 6 0.76 0.92 )0.09 0.73
CAC-B109 8 0.82 0.78 0.02 0.80
CAC-B101 10 0.85 0.58 0.15 0.83
Cat-C504 6 0.70 0.44 0.16 0.67
Cat-B106 7 0.60 0.63 )0.01 0.56
CAC-A014b 8 0.84 0.88 )0.02 0.83
CAC-C115 9 0.83 0.80 0.01 0.80
CAC-B010 5 0.47 0.02 0.30 0.43
Cat-A114 4 0.72 0.63 0.05 0.66
Mean 7 0.73 0.68 0.05 0.69
SSR, simple sequence repeats; He, expected heterozygosity; Ho,
observed heterozygosity; r, estimated frequency of null alleles; PIC,
polymorphic information content.
Table 3: Summary statistics of microsatellites diversity in the three populations analysed in this study considering unique genotypes: reference
cultivars (Ref.), locally cultivated material (Cult.) and wild materials (Wild)
Locus
N. of Alleles He Ho PIC
Ref. Cult. Wild Ref. Cult. Wild Ref. Cult. Wild Ref. Cult. Wild
CAC-B029b 6 6 6 0.68 0.60 0.79 0.64 0.78 0.87 0.64 0.55 0.76
Cat-B507 5 3 6 0.75 0.55 0.71 0.93 1.00 0.95 0.71 0.45 0.67
Cat-B107 7 5 5 0.81 0.72 0.59 0.43 0.81 0.43 0.79 0.67 0.54
Cat-B504 6 6 6 0.74 0.64 0.69 0.64 1.00 1.00 0.71 0.58 0.64
Cat-B502 5 4 6 0.69 0.56 0.80 0.92 0.90 0.94 0.64 0.47 0.77
CAC-B109 7 7 8 0.76 0.78 0.71 0.93 0.93 0.63 0.72 0.75 0.68
CAC-B101 9 9 8 0.82 0.85 0.83 0.50 0.65 0.58 0.80 0.83 0.82
Cat-C504 6 5 5 0.69 0.49 0.77 0.43 0.31 0.54 0.66 0.47 0.74
Cat-B106 4 6 5 0.62 0.58 0.56 0.62 0.63 0.62 0.56 0.52 0.50
CAC-A014b 7 7 8 0.79 0.78 0.84 0.86 0.89 0.87 0.75 0.74 0.82
CAC-C115 4 6 9 0.67 0.70 0.79 0.86 1.00 0.68 0.60 0.65 0.76
CAC-B010 4 5 3 0.54 0.38 0.19 0.00 0.07 0.00 0.50 0.36 0.18
Cat-A114 4 4 4 0.72 0.62 0.66 0.79 0.78 0.5 0.67 0.55 0.60
Mean 5.7 5.6 6.08 0.71 0.63 0.69 0.66 0.75 0.66 0.67 0.58 0.65
He, expected heterozygosity; Ho, observed heterozygosity; PIC, polymorphic information content.
Genetic relationship of C. avellana 3
(AstW12, AstW17, AstW19 and AstW25), and the reference
cultivar ÔRibetÕ; a second branch (B) was constituted of 35
accessions, all classified as local wild material; and a third
branch (C) included 13 reference cultivars, nine local cultivated
hazelnuts (As30, As33, As35, As36, As57, As58, As60, As62
and As66) and the local wild accession AstW13. The local
accessions (wild or cultivated) were not grouped by collection
site (passport data are not shown).
Analysis of the population structure
The organization of genetic diversity was further analysed with
the
STRUCTURE
program in a stepwise fashion from 2 to 4
populations. The estimation of the statistic DK revealed the
highest value for K=2 (DK = 126.5) indicating two main
clusters were present: locally cultivated forms and the remain-
ing materials (Fig. 3). The F
ST
value was 0.068 for these two
groups. Shown in the bar graph for K= 2 (Fig. 3 top) are
several accessions that are not clearly placed in a single group
(e.g. As35, As36, As58 and As55 in the local cultivated group
and AstW12, AstW17, AstW24 and AstW34 in the other
group), but rather contain genes from both groups. At K=3
(DK = 13.8),
STRUCTURE
revealed less differentiation between
the three groups (local cultivated germplasm, local wild
germplasm and reference cultivars). The F
ST
values were
0.094 for local cultivated vs. reference cultivars, 0.091 for local
cultivars vs. local wild accessions and 0.107 for reference
cultivars vs. local wild accessions. Again, some local accessions
showed introgression from other groups. In the local cultivated
forms, As12 and As55 showed introgression from the local
wild germplasm, while trees As15, As22, As30, As58, As33,
As35, As36, As60, As62 and As66 showed a high similarity to
the reference cultivars. In the local wild population, individ-
uals AstW8, AstW13, AstW14, AstW26, AstW31 and AstW40
showed a high similarity to the reference cultivars, while
AstW12, AstW17, AstW25 and AstW34 showed a high
similarity to the local cultivars.
Discussion
Microsatellite analysis showed considerable genetic diversity in
116 analysed hazelnuts, of which 82 had unique genotypes
(different patterns of bands). A total of 91 alleles were
identified, with high values for PIC and number of alleles.
The 13 SSR loci analysed in this study were previously used in
genetic diversity analysis (Bassil et al. 2005a, Boccacci et al.
2005, 2006, Go
¨kirmak et al. 2009) supplying high levels of
polymorphism (PIC > 0.70; number of alleles per locus >7).
In general, the values obtained in this study for these two
parameters were slightly lower than previously reported.
Among the three groups of materials included in this study,
the highest genetic diversity was in the well-known reference
cultivars and the local wild germplasm. The lower values for
the estimated parameters were generally in the group of local
cultivated germplasm. These results are consistent with the
different geographical origin of each group of materials and
with sexual or asexual reproduction. The group of reference
hazelnut cultivars is a diverse set representing the worldÕs most
important production areas (north-eastern Spain, Italy,
Turkey and USA), while the local cultivated and wild materials
included in this analysis were collected in a limited area from
northern Spain (10 602 km
2
). Locally cultivated materials are
commonly propagated using vegetative methods (grafting or
rooted suckers), while local wild materials typically use sexual
reproduction (seeds derived from cross-pollination) that gen-
erates high levels of diversity.
Results revealed that a total of 36 locally cultivated
accessions had two profiles of bands; the Casina group
comprised 30 accessions and the As16 group comprised six
accessions. Go
¨kirmak et al. (2009) used 21 SSR markers, eight
Fig. 1: Two-dimensional plot obtain-
ed from principal coordinate anal-
ysis for 82 unique genotypes.
Locally cultivated materials, wild
hazelnuts and reference cultivars
are indicated using squares, stars
and circles, respectively
4A. Campa, N. Trabanco, E. Pe
´rez-Vega et al.
of them common to the present study, and they also found an
identical profile of alleles in the local cultivars ÔCasinaÕ,
ÔAmandiÕ,ÔEspinaredoÕand ÔQuiro
´sÕ. However, within the
locally cultivated germplasm, a high level of differentiation
among accessions was found using morphological traits and
ISSR markers, as described by Ferreira et al. (2010). The
majority of locally cultivated hazelnuts used in this work were
also analysed using 11 ISSR primers (48 trees), which
generated 66 polymorphic bands. Of the 50 local trees analysed
by Ferreira et al. (2010), only two (As42 and As43) showed an
identical pattern of bands. The difference in levels of genetic
diversity between these two studies is probably due to the
different number of loci examined and perhaps the nature of
the markers. If each ISSR fragment is considered a putative
locus, Ferreira et al. (2010) analysed variation at 66 ISSR loci,
while in the present study, we analysed 13 SSR loci. ISSRs can
identify polymorphisms at both ends, manifested as length
polymorphisms, as well as insertion/deletion events within the
ISSR region.
The PCoA, the NJ tree and the population structure analysis
revealed a high level of differentiation between the locally
cultivated forms and the remaining materials. In the PCoA
plot (Fig. 1), the reference and wild materials could easily be
considered one group rather than two, especially considering
the higher weight of the first axis. In the NJ tree (Fig. 2), the
very short-arm distances for B (local wild hazelnuts) and C
(mainly reference cultivars) are similar to bifurcations within
those groups. Most of the polymorphism is among individuals
rather than among groups. The very low F
ST
values for the
contrast of reference and wild populations, and the structure
analyses also suggest that most of the variation is among
individuals and not subpopulations. The differentiation
between most of the locally cultivated accessions and the
group of reference cultivars is in agreement with the differen-
tiation between the major part of locally cultivated forms and
a set of 17 standard cultivars revealed by the analysis of 66
ISSR markers (Ferreira et al. 2010). Four major geographical
groups or gene pools were described in European hazelnut:
Fig. 2: Neighbour-joining tree of microsatellite diversity based on the C. S. Chord distance implemented in the P
OWER
M
ARKER
software. Three
main branches are indicated: A, group of the locally cultivated hazelnuts; B, group of the local wild hazelnuts; C, group of the reference cultivars
Genetic relationship of C. avellana 5
Central European, Black Sea, English and Spanish–Italian
(Go
¨kirmak et al. 2009, Boccacci and Botta 2010). Cultivars
from the Spanish–Italian, English and Black Sea gene pools
were included in the present analysis (Table 1), and the locally
cultivated hazelnuts were clearly differentiated from them.
Go
¨kirmak et al. (2009) included the local cultivars ÔCasinaÕ,
ÔEspinaredoÕ,ÔAmandiÕand ÔQuiro
´sÕin their analysis, and these
were clustered into a separate subgroup within the Spanish–
Italian group. In addition, chloroplast microsatellite variation
revealed that Casina had the same haplotype as the majority of
Spanish–Italian germplasm (Boccacci and Botta 2009). All
these results suggest that locally cultivated hazelnuts belong to
the Spanish–Italian gene pool but constitute a separate
subgroup.
These findings raise an interesting question about the origin
of locally cultivated forms of hazelnut. Differentiation between
wild and most locally cultivated hazelnuts, and the strong
relationship among the locally cultivated accessions suggest
that locally cultivated forms may have been derived from the
introduction of a few primitive cultivars, followed by a
relatively local evolution that could include crosses among
them and with local wild hazelnut. Evidence of cultivated trees
derived from crosses between natural and cultivated popula-
tions was found in the present study (Fig. 1). In addition, the
close relationship between most of the local cultivated germ-
plasm (Casina group) suggests that the introduction of
cultivated forms may have been recent and has not led to a
broad diversification.
The plot generated from PCoA indicated putative intermedi-
ate forms and introgressions, mainly of some locally cultivated
accessions in the group of reference cultivars. The analysis of
population structure also revealed some introgressions and
admixtures of genotypes, particularly in the locally cultivated
and wild populations. The NJ tree also showed some introgres-
sion in the reference cultivar and the local cultivar groups. The
intermediate forms identified may be derived from natural
crosses between trees belonging to different groups. Hazelnut is
monoecious, dichogamous, wind-pollinated and has sporo-
phytic incompatibility controlled by a single S-locus with
multiple alleles (Thompson 1979, Mehlenbacher 1997, Pomper
et al. 1998). Introgressions in the locally cultivated population of
genotypes close to reference cultivars (e.g. accessions As60, As58
or As57) could be because of the introduction of new cultivars in
northern Spain. Exchange of hazelnut materials between Cata-
lonia and Asturias was reported beginning in the twentieth
century, and several campaigns to promote the use of cultivars
from Catalonia were conducted in the mid-twentieth century
(Alvarez Requejo 1965). This could explain (i) the position in the
group of reference cultivars of some local cultivated accessions in
the plot generated from PCoA and (ii) the presence in the
graphical output of the
STRUCTURE
software of accessions with
similar genotypes to reference cultivars. Finally, the inclusion of
some materials classified as cultivated forms in the group of wild
materials (accessions As30 or As55) could be a mistake in the
classification or the use of wild trees. The exodus of people from
rural areas in the twentieth century led to abandonment of local
hazelnut cultivation in many areas. Many orchards or gardens
with hazelnuts are now forest in which it is difficult to distinguish
the trees originally cultivated from those which arose from
hybridization with local wild hazelnuts.
Fig. 3: Hierarchical organization of genetic relatedness of 82 unique genotypes based on 13 microsatellite markers and analysed by the
STRUCTURE
program considering two and three populations (K=2 and K=3)
6A. Campa, N. Trabanco, E. Pe
´rez-Vega et al.
In summary, this study differentiated between the locally
cultivated accessions and remaining materials analysed in this
study. The local cultivated forms contained (i) a group of
accessions clearly differentiated within the Spanish–Italian
gene pool, (ii) a group with some intermediate forms probably
derived from hybridization and (iii) a group of accessions
probably derived from exchange with other geographical
origins (mainly Catalonia). The differentiation of these groups
within the local material cultivated is of great interest for the
preservation and use of the local genetic diversity.
Acknowledgement
This work was supported by grants RF2008-0014-CO3-02 from the
Ministerio de Ciencia y Tecnologı
´a, Spain. Elena Pe
´rez-Vega was
recipient of a salary fellowship from the Instituto Nacional de
Investigacio
´n y Tecnologı
´a Agraria y Alimentaria (INIA, Spain).
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Genetic relationship of C. avellana 7
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In this work, 18 microsatellite loci were developed in the European hazelnut (Corylus avellana L.) using three enriched genomic libraries. They were evaluated on a set of 20 accessions of this species on the basis of number of alleles (mean: 7.1), expected heterozygosity (mean: 0.67), power of discrimination (mean: 0.77) and polymorphism information content (mean: 0.64). Cross-species transferability was evaluated using seven other Corylus species. All primer pairs amplified in all species, except for CaT-C505 in Corylus ferox and CaT-A114 in Corylus californica.
Thesis
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World hazelnut production is based primarily on selections from the wild. In this study, we used 21 pairs of simple sequence repeat (SSR) primers to investigate genetic diversity in 270 clonal accessions of European hazelnut (Corylus avellana) representing a wide geographic range. Of the 270 accessions, 198 had unique fingerprints while 72 were duplicates. Based on the 198 unique accessions, the number of detected alleles per locus averaged 9.81 and observed heterozygosity (Ho) averaged 0.67. Of the 206 total alleles amplified, 20 were unique to a single accession. A genetic similarity matrix was constructed and the resulting dendrogram revealed four major geographical groups: Central European, Black Sea, English and Spanish-Italian. SSR alleles indicated the parentage of 31 accessions. The fingerprints are publicly available through the Germplasm Resources Information Network (GRIN) database. The identification of duplicate and mislabeled accessions will improve management of hazelnut genebanks, and information on genetic variation in hazelnut will assist the international research community.
Article
We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from http://www.stats.ox.ac.uk/~pritch/home.html.
Conference Paper
Sixteen microsatellite-containing sequences were identified in enriched genomic libraries of the European hazelnut, Corylus avellana L. Of these, seven amplified multiple loci, one was difficult to score and eight amplified polymorphic single loci. Amplification and polymorphism of these eight primer pairs in 20 cultivars of C. avellana were initially determined by electrophoresis using 3% agarose gels. Forward primers were then fluorescently labelled and allele sizes determined by capillary electrophoresis. The addition of PIG-tails to five reverse primers was necessary to eliminate split peaks. Three microsatellite markers (CAC-AC115, CAC-B005 and CAC-B014) reliably distinguished among the cultivars. Microsatellite markers will be used to fingerprint the hazelnut genotypes in our collection and identify duplicate accessions.
Article
Diallele crosses of sibs and parents of 3 parental combinations demonstrated sporophytic type of incompatibility inCorylus avellana L. One gene with multiple alleles was indicated. All 6 alleles present in the 4 parental cultivars and in their progeny exhibited dominance in pollen and independent action in the pistil. Individuals homozygous for S-alleles appeared in progeny of parents having one allele in common. Reciprocal differences occurred in some crosses. The stigmatic surface is the site of the incompatibility reaction. Incompatible pollen germinated abundantly but failed to penetrate into the stigma cells.
Article
Pollen-stigma compatibility was studied in cultivars and more than 1800 seedlings of the European hazelnut (Corylus avellana L). Four new S-alleles were identified, bringing the total to 25 unique alleles within C. avellana. The new alleles are the recessive alleles in ‘Tonda di Giffoni’ and ‘Segorbe’ (S23), in ‘Neue Riesennuss’ (S25), in ‘Gasaway’ (S26), and a dominant allele in a seedling of Turkish origin (S24). Dominance relationships in 233 of the possible 300 pairs of alleles were determined in both pistil and pollen. All alleles exhibited independent action in the pistil, whereas in the pollen either dominance or codominance was exhibited. The dominance hierarchy of alleles in the pollen was revised in light of the new information obtained. All 25 alleles have been assigned to a level in the hierarchy that is linear and now has eight levels. S6 and S9 were reassigned to lower levels in the hierarchy. Thirteen of the alleles are on the level of S1, while S4, S6, S11, and S23 occupy unique positions in the hierarchy. Improved pollen tester clones were identified for several S-alleles. The alleles in 55 cultivars were determined. The alleles identified in ‘DuChilly’ (S10 S14) did not agree with previous reports. Four cultivars have the same alleles as ‘Rmische Nuss’ (S10 S18) and are morphologically indistinguishable from it: ‘Frutto-grosso’, ‘Istarski Okrogloplodna’, ‘Payrone’, and ‘Romai’. ‘Belle di Giubilino’ and ‘Tonda di Biglini’ are both S1 S10 and appear to be synonyms for the same cultivar.
Article
With 3 figures and 4 tablesAbstractHazelnut (Corylus avellana L.) has been a traditional crop in northern Spain. As a result of germplasm exploration over 3 years (2003–05), 90 trees were selected in this region. This study describes phenotypic variation in nut and husk traits and investigates genetic relationships among selections and cultivars using inter simple sequences repeat (ISSR) markers. The local selections were phenotypically diverse and many had characteristics appreciated by the market. Eleven ISSR primers, which generated 66 polymorphic bands, were used in the analysis. The graph from principal coordinates analysis of the molecular marker data showed two main groups, one for the local selections and the other for the standard cultivars. The dendrogram generated from UPGMA cluster analysis showed the same two main groups. The results suggest that the local accessions are closely related to each other, but are relatively distant from the standard cultivars of eastern Spain, Italy and the USA. Selections from northern Spain may be directly useful as new cultivars or alternatively as parents in breeding programmes. The collection and preservation of this genetic diversity is important.
Article
With 6 figures and 6 tablesAbstractEuropean hazelnut (Corylus avellana L.) is an important crop in Turkey, Georgia and Azerbaijan, where cultivars were selected from the native vegetation. Accessions from Turkey have been assigned to the Black Sea group, and cultivars from Georgia and Azerbaijan have a similar phenotype. Genetic diversity was investigated in 88 accessions from these three countries and compared with cultivars from Spain and Italy using 12 microsatellite loci. A high level of genetic diversity (He = 0.71, Ho = 0.70) was observed in the Black Sea accessions. Six Turkish accessions in the US hazelnut collections were found to be synonyms of cultivars in the Turkish collection in Giresun. An unweighted pair-group method using arithmetic average dendrogram and principal component analysis of 109 unique accessions showed a tendency to form subgroups by country of origin, and high diversity within each subgroup. A moderate shift in allelic frequencies (FST = 0.114–0.131) was seen between accessions from the Black Sea and the Spanish-Italian accessions. Simple sequence repeat analysis identified the putative parents of two Turkish cultivars.