Elucidating the contribution of wild related species on autochthonous pear germplasm: A case study from Mount Etna
Elucidating the contribution of wild related species on autochthonous pear germplasm: A case study from Mount Etna
Stefania Bennici 0 1
Giuseppina Las Casas 0 1
Gaetano Distefano 0 1
Mario Di Guardo 0 1
Alberto Continella 0 1
Filippo Ferlito 1
Alessandra Gentile 0 1
Stefano La Malfa 0 1
☯ These authors contributed equally to this work. 1
0 Dipartimento di Agricoltura, Alimentazione e Ambiente, University of Catania , Catania, Italy, 2 Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria , Centro di Ricerca Olivicoltura, Frutticoltura e Agrumicoltura (CREA-OFA) , Acireale , Italy
1 Editor: Pilar Hernandez, Instituto Agricultura Sostenible , SPAIN
The pear (genus Pyrus) is one of the most ancient and widely cultivated tree fruit crops in temperate climates. The Mount Etna area claims a large number of pear varieties differentiated due to a long history of cultivation and environmental variability, making this area particularly suitable for genetic studies. Ninety-five pear individuals were genotyped using the simple sequence repeat (SSR) methodology interrogating both the nuclear (nDNA) and chloroplast DNA (cpDNA) to combine an investigation of maternal inheritance of chloroplast SSRs (cpSSRs) with the high informativity of nuclear SSRs (nSSRs). The germplasm was selected ad hoc to include wild genotypes, local varieties, and national and international cultivated varieties. The objectives of this study were as follows: (i) estimate the level of differentiation within local varieties; (ii) elucidate the phylogenetic relationships between the cultivated genotypes and wild accessions; and (iii) estimate the potential genetic flow and the relationship among the germplasms in our analysis. Eight nSSRs detected a total of 136 alleles with an average minor allelic frequency and observed heterozygosity of 0.29 and 0.65, respectively, whereas cpSSRs allowed identification of eight haplotypes (S4 Table). These results shed light on the genetic relatedness between Italian varieties and wild genotypes. Among the wild species, compared with P. amygdaliformis, few P. pyraster genotypes exhibited higher genetic similarity to local pear varieties. Our analysis revealed the presence of genetic stratification with a `wild' subpopulation characterizing the genetic makeup of wild species and the international cultivated varieties exhibiting the predominance of the `cultivated' subpopulation.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The authors declare the study was
supported by internal funding of Department of
Agriculture, Food and Environment of Catania
University, Catania, Italy. The funder had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
The pear (Pyrus spp.) is one of the most cultivated fruit crops in temperate zones. Pyrus species
are traditionally divided into two groups based on domestication area and geographic
distribution. European pears (P. communis) are cultivated mainly in Europe and the U.S., and Asian
pears (P. pyrifolia, P. bretschneideri and P. ussuriensis) grow in East Asian countries. The genus
Pyrus belongs to the family Rosaceae, subtribe Pyrinae and contains at least 22 widely
recognized primary species, all indigenous to Asia, Europe, and the mountainous area of North
America. The worldwide production of European pear relies on a few main cultivars released
from the late 18th century onward (or derived from those cultivars).
Although Sicily is not important in pear production and does not include large areas of
production, it exhibits significant germplasm diversity. In the Mount Etna area, the large amount
of pear biodiversity could be related to ancient practises of cultivation and seed propagation
combined with the variety of favourable microclimates, soils and orographic conditions. In
addition, pear cultivation in Sicily was historically characterized by a wide use of wild pears (P.
amygdaliformis Vill.; P. communis ssp. pyraster L.) as rootstocks, an agronomical practise that
increases the hardiness and longevity of the trees [
]. The most common species of wild pear
(P. pyraster = P. communis ssp. pyraster L.) is a woody plant closely related to the European
pear. This species comes from the western Black Sea region. Its distribution extends from the
British Isles to Latvia [
] and is widespread in Sicily.
The local pear germplasm of Mount Etna could represent an important source of ecological
interest for specific characteristics, such as high drought resistance, low chill unit requirement,
adaptation to hot and dry summer conditions and low pest and disease incidence, all of which
are pivotal characteristics to consider in the establishment of novel breeding programmes. As
such, ascertaining the genetic relationships and phylogeny of the genus Pyrus and the potential
contribution of wild related species to its origin would be advisable, given the promising
results obtained in other Rosaceae tree crops, such as apple, peach and cherry [3±5].
DNA markers have become powerful tools for cultivar identification, evaluation of genetic
diversity and parentage analysis. Studies on the genetic relationships of pear genotypes were
performed using nuclear [
] and chloroplast restriction fragment length polymorphisms
], random amplification of polymorphic DNA (RAPD) [8±13], amplification
fragment length polymorphisms (AFLPs) [
] and inter-simple sequence repeats (ISSR) [
addition to these markers, SSRs, in particular, have been widely used for genetic relationship
and diversity studies given their hyper-variability, co-dominant nature, prevalent
locus-specificity, and random genome-wide distributions. A large set of SSR markers has been developed
from Japanese and European pear [16±18] and subsequently used for genetic characterization
and identification of different Pyrus species [19±22]. In addition, SSR markers present a high
transferability between related species and genera. SSR markers developed in apple (Malus x
domestica Borkh.) present a high level of transferability and polymorphisms in the subtribe
Pyrinae (formerly the Maloideae), allowing their use to assess genetic diversity and cultivar
identification in pear [23±26]. Markers for several intergenic spacer and intron regions in the
chloroplast genome have been applied for phylogenetic analyses in Pyrus [27±30]. The
maternally inherited chloroplast genome is much smaller than the nuclear genome and is more
conserved [31±32]. Thus, this genome could be used effectively to determine the parentage
germplasms of hybrids. In addition, cytoplasmic markers appear to be suitable for overcoming
the multiple gene copy problem in polyploid phylogenetics, as occasionally reported in pear
]. These markers have been widely used in angiosperms for genetic diversity and
phylogenetic relationship studies to investigate the evolution of plants and gene flow in natural
]. Nuclear and cytoplasmic DNA markers have been employed in Pyrus for both
parentage and taxonomy studies [7, 35±36]. Genetic diversity, structure and hybridization
rates were evaluated in several collections of wild P. pyraster, P. communis, P. pyrifolia and P.
ussuriensis using nuclear and chloroplast SSR markers for improving preservation measures
[37±40]. The use of molecular markers could greatly improve the cost-effectiveness of pear
breeding. The traditional breeding of pears is a costly and time-consuming process due to the
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long juvenile period and large size of the plants (requiring great time and space investments)
as well as the genetic complexity of Pyrus resulting from the self-incompatibility of the genus.
The use of molecular markers could have a direct positive implication for the genetic
characterization of the germplasm collection, laying a foundation for use of genetic polymorphisms
to make predictions of phenotype changes through marker-trait association analysis.
In the present study, nuclear (nSSR) and chloroplast (cpSSR) microsatellites were used (i)
to estimate the level of differentiation within the cultivated genotypes and the wild accessions;
(ii) to elucidate phylogenetic relationships between the cultivated genotypes and the wild
accessions; and (iii) to estimate the potential genetic flow between and the relationship among
local Sicilian pear genotypes, native wild species and international varieties.
Materials and methods
Plant material and DNA extraction
Ninety-five pear genotypes were used in this study (Table 1), including 46 local varieties (LV)
and 21 wild related species (RS) collected from Etna district (Italy), 19 nationally cultivated
varieties (NCV) and 9 internationally cultivated varieties (ICV) (Fig 1). Genotypes were
sampled from different sites as specified in S1 Table.
Genomic DNA was extracted from fresh leaves using ISOLATE II Plant DNA Kits (Bioline,
Meridian Life Science, Memphis, TN, USA). The quantities and qualities of the extracted DNA
samples were determined using a Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA)
spectrophotometer and agarose gel electrophoresis. DNA samples were stored at -20ÊC.
SSR analysis by capillary electrophoresis
PCR amplification was performed using four chloroplast SSR primer pairs derived from the
pear genome and eight nuclear SSR primer pairs derived from the pear and apple genomes
(Table 2). PCR reactions were each performed in a 15-μl volume containing 40 ng genomic
DNA, 1x PCR buffer II, 2 mM magnesium chloride, 0.2 mM dNTPs, 0.3 μM each primer,
0.13 μM 5'-fluorescently labelled M13F primer (CAC GAC GTT GTA AAA CGA C) tagged
with 6-FAM, NED, VIC or PET and 1U of MyTaq DNA polymerase (Bioline). Amplifications
were conducted using a programme with an initial denaturation step at 95ÊC for 15 min
followed by 35 cycles at 95ÊC for 30 sec, 52±55ÊC for 30 sec and 72ÊC for 45 min with a final cycle
of 72ÊC for 15 min. A 0.4- to 0.6-μl aliquot of PCR product (depending on the performance of
amplification of each primer pair) was mixed with 13 μl of formamide and 0.3 μl of LIZ-500
size standard and denatured at 95ÊC for 5 min. Up to four PCR products labelled with 6-FAM,
PET, VIC or NED were pooled before separation in the ABI 3130 Genetic Analyser (Applied
Biosystems, Foster City, CA, USA) and subjected to subsequent analysis using GeneMapper
Genetic distance and clustering
Genetic distance was estimated by analysing dissimilarity indices calculated using allelic data
by simple allele matching to obtain the genetic dissimilarity matrix. Dendrogram trees were
obtained using Dissimilarity Analysis and Representation for Windows software version 5.0
(DARwin5) by the neighbour-joining method [
]. The robustness of branches was tested
using 1,000 bootstraps.
The numbers of genotypes, the numbers of alleles, the major allele frequency (MAF),
the expected heterozygosity (exp-het), the observed heterozygosity (obs-het) and the
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Plants were divided into four groups: wild Related Species (RS), Local Varieties (LV), National Commercial Varieties (NCV), International Commercial Varieties (ICV).
Coordinates on the principal component analysis (PC1 and PC2) and assigned cluster on the neighbour-joining tree (NJ Cluster) are also reported.
polymorphism information content (PIC value) for each SSR marker were calculated using
]. Pairwise fixation index (FST) was calculated using GenePop software [
The level of genetic stratification within the germplasms in the analysis was assessed using
STRUCTURE v.2.3.1 [
]. This analysis was performed on 73 genotypes, excluding those
genotypes for which a third allele was observed for one or more loci. Eight nSSRs were used to
compute the posterior probability [Pr(X|K)] given an increasing number of sub-populations
(ranging from K = 1 to K = 8, with five independent runs each). The computation was
performed with five independent runs using a `Length of Burnin Period' and `Number of MCMC
Reps after Burnin' of 1,000,000 under the admixture model. The most likely number of
subpopulations (K) was identified with STRUCTURE HARVESTER [
] using the ΔK described
by Evanno et al. [
]. Samples were assigned to the sub-population when the assignation
probability (qI) was greater than or equal to 0.8 [47±49]. Principal component analysis (PCA) was
performed using the `stat' package in R (R developing team), whereas median-joining network
analyses were performed using Network 126.96.36.199 ([
sharenet.htm) with default settings.
Capillary electrophoresis analysis produced clear profiles for all four cpSSR and eight nSSR
loci for 91 pear genotypes. In contrast, two local (`Savino' and `Angelico') and two wild (P.
pyraster n. 6 and P. amygdaliformis n. 10) genotypes exhibited no PCR amplification and were
excluded from further analysis.
Nuclear SSR markers allowed the identification of nineteen individuals exhibiting three
alleles in at least one of the nSSRs (data not shown).
The nSSRs detected a total of 136 alleles with sizes ranging from 115 to 256 bp with average
values of 17 and 0.29 for the number of alleles and the MAF, respectively (Table 3). The mean
value of the exp-het was 0.82, whereas the obs-het was 0.65. All eight nSSRs were highly
polymorphic, with PIC values ranging from 0.42 to 0.92. The most polymorphic markers were
TsuENH026 with a total of 21 alleles and 48 genotypes detected, obs-het of 0.74 and PIC of
0.92 and BGT23b with a total of 25 alleles and 43 genotypes, obs-het of 0.55 and PIC of 0.91.
The least informative nSSR was CH04e03 demonstrating an obs-het and PIC of 0.18 and 0.42,
The cpSSR analysis detected a total of 11 alleles with an average value of 2.75 alleles and
sizes ranging from 182 to 216 bp (Table 3). The chloroplast marker PCHSSR27 was
monomorphic, detecting an allele of 195 bp. In contrast, the highest number of alleles (5) was detected
for the PCHSSR3 marker. The MAF ranged from 0.69 (PCHSSR19) to 0.92 (PCHSSR31), and
PIC values ranged from 0.13 (PCHSSR31) to 0.39 (PCHSSR3).
Overall, cpSSRs and nSSRs discriminated 81 of the 91 analysed genotypes, detecting a total
of 147 alleles with an average value of 12.25 and an average MAF of 0.47. The mean values of
exp-het, obs-het and PIC were 0.63, 0.43 and 0.61, respectively.
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Fig 1. Comparison of pear germplasm. Section of fruit and leaves of P. amygdaliformis (a), P. pyraster (b) and
widespread cultivated traditional variety (`Spineddu'; c) on Mount Etna (scale bar = 2 cm).
The genetic relationship among analysed genotypes is presented in the neighbour-joining
dendrogram constructed using both nSSR and cpSSR data (Fig 2). The cluster analysis
identified three main clusters (A-B-C). Cluster A includes six internationally common pear varieties,
including `Max Red Bartlett', `William's', `Dr. Guyot', `Harrow Sweet', `Decana del Comizio'
and `Old Home'; seven NCV; 14 LV and four RS (P. pyraster n. 2, 3, 9 and 10). Among these,
`Max Red Bartlett', `William's' and `Virgolese'-'Pergolesi' were undistinguished.
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Wuyun et al. (2015)
Wuyun et al. (2015)
Wuyun et al. (2015)
Wuyun et al. (2015)
Yamamoto et al. (2002b)
Liebhard et al. (2002)
Yamamoto et al. (2002a)
Liebhard et al. (2002)
Nishitani et al. (2009)
Nishitani et al. (2009)
Yamamoto et al. (2002b)
Liebhard et al. (2002)
Cluster B includes three subgroups (B1-B2-B3). The first subgroup B1 includes three
international varieties, `Butirra Hardy', `Abate Fetel' and `Kaiser'; nine NCV; 20 LV; and 2 RS (P.
pyraster n. 1 and 11). Among these, the two `Bianchetto' accessions (1 and 2) presented the
same SSR profile. The second subgroup includes nine local genotypes, among which
`Faccibedda'-'Pauluzzo' and `Moscatello maiolino'-'Franconello' exhibited the same SSR profile. The
third subgroup includes all nine wild genotypes of P. amygdaliformis, four wild genotypes of P.
pyraster (n. 4, 5, 7 and 8) and the genotypes `Spineddu' (NCV) and `Sciaduna' (LV). The local
genotypes `Moscatello' (NCV) and `Chiuzzu' (LV) represented cluster C.
The 73 genotypes exhibiting one or two alleles for each locus were also included in a
population stratification analysis. Unlike the results of the neighbour-joining analysis, the
population stratification analysis identified two sub-populations (K = 2). This analysis was performed
following a plateau criterion [
], a non-parametric Wilcoxon test [
], and the rate of change
(ΔK) method proposed by Evanno et al. [
] (S2 Table).
Fig 2. Neighbour-joining analysis. Nj dendrogram calculated by employing both nSSR and cpSSR. The three major clusters (named A, B and C) are
STRUCTURE analysis allowed the identification of two groups that will be henceforth
named `wild' and `cultivated' sub-populations (Fig 3, S3 Table). Forty-two samples were
characterized by a predominant (QI > 0.8) `wild' genetic configuration, whereas twenty-two
exhibited a predominance of the `cultivated' sub-population. The remaining nine samples exhibited
QI values less than 0.8 for both subpopulations and were therefore considered `admixed' (S3
Within the four groups of pears (Table 1), individuals exhibited different relative
frequencies of the `wild' and `cultivated' sub-populations. In particular, RS and ICV are mostly
characterized by `wild' and `cultivated' genetic configurations, respectively, whereas LV and NCV
presented a more balanced presence of both sub-populations (S3 Table). The LV group is
characterized by a high relative contribution of the `wild' genetic configuration. In total, 54% of the
cultivars within this group exhibited a predominance of `wild' subpopulation, whereas a
notable proportion of individuals (32%) exhibited a clear predominance of the `cultivated'
subgroup. The same pattern registered in the NCV with nine individuals (60%) exhibiting a
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Fig 3. STRUCTURE results. Inferred population structure: Bar plot generated by STRUCTURE according to the K = 2 model based on eight nSSRs.
strong relative contribution of the `wild' subpopulation, five individuals (33%) exhibiting an
opposite trend in favour of the `cultivated' subpopulation, and the remaining sample, `Gentile',
exhibiting a more balanced admixture between the two sub-populations (S3 Table).
The distinction between `wild' and `cultivated' subpopulations was further confirmed by
the analysis of the fixation index (FST), a summary statistic quantifying the variation in allelic
frequencies between groups. The FST between these two subpopulations was 0.096, whereas
the pairwise FST estimates between `admixed' and `wild' or `admixed' and `cultivated' exhibited
considerably reduced values (0.028 and 0.026, respectively).
To examine the presence of additional genetic stratification, the germplasm collection was
divided into two subsets based on the two sub-populations detected (`wild' and `cultivated'),
and an additional round of structure analysis was separately performed on each of the two
subsets following the approach presented by Urrestarazu and colleagues (2012) [
This nested structure analysis allowed for better characterization of each sub-population.
The `wild' subpopulation (S3 Table) exhibited the highest ΔK for K = 5 (43.1) although a
secondary peak was detected for K = 2 (21.2, S1 Fig). For K = 2, all the P. pyraster and P.
amygdaliformis accessions were assigned to the same subpopulation. In contrast, at K = 5, the two
species were assigned to different sub-populations (`pink' for P. pyraster and `yellow' for P.
amygdaliformis, S2 Fig).
The 22 samples unambiguously assigned to the `cultivated' subpopulation exhibited similar
high ΔK values for K varying from 3 to 4 (S1 Fig). In both scenarios, four samples
(`Bruttu_Beddu', `Putiru_D'Inverno', `Azzone di Cassone' and `Iazzuleddu') exhibited a clear
predominance (QI greater than 0.95 and 0.96 for K equal to 3 and 4, respectively, S2 Fig) of one
subpopulation. The same subpopulation was almost absent in the other samples (QI less than 0.07
and 0.08 for K equal to 3 and 4, respectively, S2 Fig). The cpSSRs of the 91 individuals were
employed for a median-joining network analysis to further elucidate genetic distances among
individuals. The results presented in Fig 4 reveal the presence of nine different haplotypes
originating from the combination of the four cpSSRs. The three most common haplotypes (Hap1,
Hap2 and Hap3) represent 85.5% of the individuals, accounting for 39.5%, 30.7% and 15.3% of
the total genotypes, respectively. The root of the tree (mv1) represents a polymorphism
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Fig 4. Median-joining network analysis reveals relationships among individuals. Haplotypes have been calculated based on the four
cpDNA markers. The pie charts highlight the relative frequency of individuals for each haplotype, exhibiting a clear prevalence (>90%)
of one subpopulation (red = wild subpopulation, blue = cultivated subpopulation). Grey represents samples with more evident genetic
occurring at PCHSSR27, in which one cultivar (`Kaiser') exhibiting an allelic size of 194 bp can
be considered the origin of the median-joining network, whereas all other individuals exhibit
an allelic length of 195 bp (S1 Table). Hap 1, 3, 5, 6 and 8 were further distinguished by
different allelic sizes at PCHSSR3, whereas Hap2 originated from Hap1, exhibiting an additional
mutation at PCHSSR19. Hap 4 and 7 contain 2 different mutations at PCHSSR31 compared
with Hap 3.
Both nSSR and cpSSR were employed for PCA. The first two principal components (PC1
and PC2) accounted for 14.5% of total genotypic variability, allowing a distinction of cultivars
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according to their genetic makeup. The four groups of cultivars defined in Table 1 presented a
characteristic pattern on the biplot presented in Fig 5. International cultivars (ICV) were
plotted on the positive PC1 quadrant, whereas all wild species (RS) exhibited negative PC1 values
with the exception of P. pyraster 10 (plotted on the positive PC1 quadrant). Local cultivars
(LV) and national cultivars (NCV) exhibited a similar pattern characterized by a unimodal
Fig 5. PCA analysis. Two-dimensional PCA plot depicting the distribution of accessions over the first two PCs. Different colours represent the four groups of pears:
international cultivar varieties (ICV, red squares), national cultivar varieties (NCV, orange dots), local varieties (LV, green crosses), and wild related species (RS, blue
triangles). RS are further subdivided into P. pyraster (filled triangles) and P. amygdaliformis (empty triangles).
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distribution of the samples centred in the range between -2 and +2 PC1 values. PC2 allowed a
distinction within the RS, NCV and LV groups. The RS group is composed of individuals
belonging to P. amygdaliformis and P. pyraster species. Extreme negative values indicated P.
amygdaliformis individuals, whereas values of PC2 greater than -3.5 indicated the presence of
P. pyraster. Similarly, NCV and LV can be further differentiated according to PC2, whereas
PC3 can efficiently discriminate within the ICV group (data not shown).
The Mount Etna area claims a large number of differentiated pear varieties due to ancient
cultivation practises and the variability of soil and climatic conditions. Local varieties display
significant variability in many agronomic traits, including fruit size, flowering and ripening
periods and harvesting time [
]. Local germplasm could represent an important source of
genetic diversity that can be readily used by breeders to develop novel cultivars with enhanced
agronomic traits, including fruit quality, adaptability to limiting environmental factors and
resistance to biotic stresses. The Pyrus genus includes important cultivated species that have
been widely studied by means of different molecular markers, mostly nuclear markers.
Polyploidy was occasionally observed in several Pyrus species [
]. In this work, the phylogenetic
relationships among the traditional and broadly cultivated genotypes, wild accessions and
related species in Pyrus were inferred by coupling cytoplasmic and nuclear markers. Two
different types of SSR markers were adopted to combine the investigation of the maternal
inheritance property of cpSSRs with the high informativity of nSSRs [
The study presented here employed eight nSSRs (Table 2). Among these nSSRs, three
(CH02 h11a, CH05d04, CH04e03) were originally developed for apple. The high conservation
between the genera Pyrus and Malus is confirmed by the high heterozygosity of CH02h11a
and CH05d04 with the latter exhibiting the highest allelic diversity within the nSSR marker
set. On the other hand, CH04e03 exhibited the lowest heterozygosity but was still efficiently
used in the germplasm characterization analysis [
]. Although SSRs are not the markers of
election for High Throughput Analysis, the use of both nuclear and cytoplasmic microsatellites
allowed a high level of discrimination of the collection. Our analysis identified four
multilocus nSSRs (BGT23b, TsuENH025, TsuENH026, NH015a), which is consistent with data
previously reported in other studies on genetic characterization in pear [25, 56±58]. The most
likely explanation of the high occurrence of multi-locus SSRs can be traced to the allopolyploid
origin of Maloideae that originated from ancestors belonging to the subfamily of Spiraeoideae
[26, 33, 59±60]. The molecular weight of the amplified fragments, including the M13 tail, falls
in the same range of the amplicons obtained in P. communis for nSSRs [16±17, 55] and P.
ussuriensis for cpSSRs . Overall, the average number of alleles (17) among the analysed
genotypes was increased in this study compared with that observed in earlier studies
performed in several collections of P. communis using different sets of SSRs (ranging from 5.9 to
12.8) [21, 25, 54, 56±57, 61±66].
Molecular characterization using SSR markers highlighted high genetic variability among
the analysed accessions. It was possible to characterize most of the local varieties and to
highlight the genetic relatedness of Italian varieties (NCV + LV) with wild genotypes (Fig 2).
As expected, the international varieties `Williams' and `Max Red Bartlett' exhibited the
same SSR profile, confirming the origin of `Max Red Bartlett' as a bud mutation of `Williams'.
Fingerprint analysis confirmed other previously known pedigree records, such `Harrow Sweet'
(`William' x `Purdue 80±50'), for which the observed SSR profiles were consistent with the
known origin. Some local varieties exhibited the same SSR profile (`Virgolese'-'Pergolesi';
`Faccibedda'-'Pauluzzo'; the small-fruit pear `Moscatello maiolino'-'Franconello'; the two
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accessions of `Bianchetto' n. 1 and 2), indicating possible cases of synonymy or, at the very
least, a close genetic relationship. This question could be further investigated through
phenotypization and/or deeper molecular analysis.
Interestingly, most of the local varieties clustered together in B1 and B2 subclusters of the
dendrogram. In structure analysis, most of the local varieties exhibited an increased
contribution of the `wild' subpopulation to their genetic makeup.
Among the wild species, few P. pyraster genotypes were reported in every cluster, exhibiting
increased genetic similarity to the local pear varieties compared with P. amygdaliformis. In
contrast, P. amygdaliformis appeared to be more conserved.
The use of nSSRs in structure analysis (Fig 3) allowed the definition of two sub-populations,
defining wild species on one side with all the accessions of P. amygdaliformis and P. pyraster and
a second group of cultivated pears. The relative contribution of one of the two sub-populations
clearly demonstrates the different genetic structure of the ICV and RS groups characterized by a
predominant contribution of the `cultivated' and `wild' sub-populations, respectively. In contrast,
increased levels of admixture are registered for NCV and LV groups. These results suggest an
increased contribution of the `wild' subpopulation to the genetic makeup of many Italian
varieties, especially local varieties, compared with internationally cultivated varieties. The FST between
the two subpopulations was 0.096, revealing increased population differentiation compared with
other studies on pear germplasm collected in Spain [
] or Bosnia and Herzegovina [
The low polymorphism observed within the cpSSRs reflects an increased level of
conservation of chloroplast DNA, which is consistent with that previously reported in the wild P.
ussuriensis population . Although none of the represented cpSSRs haplotypes can be
unequivocally traced to wild or cultivated pear accessions, Hap2 seems to be more associated
with cultivated accessions (89% of samples are cultivated accessions or admixed). In contrast,
Hap1 and Hap3 are more associated with wild species (83% and 71%, respectively). Hap4 is
exclusively associated with wild or admixture genotypes. However, limited individuals (6) are
represented by this haplotype, thus preventing firm conclusions from being drawn (Fig 4). The
absence of a haplotype unequivocally associated with wild or cultivated pear is congruent with
the high level of admixture within the genus Pyrus. Additionally, the lack of a haplotype
unequivocally representing one of the two sub-populations (Fig 3) clearly testifies to the
occurrence of allelic interchange between wild and cultivated accessions.
Within related species, a different level of genetic admixture was observed. For example, P.
amygdaliformis accessions were characterized by the absence of `cultivated' subpopulation
contribution compared with P. pyraster accessions. In particular, the close genetic proximity of P.
pyraster to most of the local varieties could be explained by its wide employment as rootstock
given its rusticity. These findings were further confirmed by our PCA analysis (Fig 5), in
which P. pyraster accessions were located in the upper portion of the plot in the same region as
several LV and ICV. In contrast, P. amygdaliformis accessions were mostly present in the lower
left portion of the plot. PCA results were consistent with the outcome of the nested-structure
approach. In the first round of structure analysis, both P. pyraster and P. amygdaliformis
exhibited a predominant contribution of the `wild' subpopulation, which is consistent with that
observed in the PCA analysis in which these accessions were characterized by negative PC1
values. P. pyraster and P. amygdaliformis can be fully characterized through a second round of
structure analysis (at K = 5) or considering the second PC.
For pear, the identification of traits of agronomic importance, including adaptability to
different pedoclimatic conditions and resistance to biotic stresses, is crucial for breeding new
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varieties. Most of these traits can be found in local germplasm that to date has been less
exploited for genetic improvement programmes. The Mount Etna area represents an
important biodiversity repository for many woody species due to the presence of many climatic
and pedological conditions and a wide range of altitudinal levels. In the case of cultivated
species, such as pear, this richness has also been increased by the long history of diffusion
and cultivation. In the present work, a set of ninety-five individual trees representing local
varieties, wild genotypes and nationally and internationally cultivated varieties have been
genotyped to obtain novel insights into pear genetic structure and to elucidate the influence
of wild species on the genetic makeup of local germplasm. The molecular insight presented
in this work could also be useful for future association studies at the genomic level, such as
Genome Wide Association Studies (GWAS), in which a prior knowledge of the genetic
diversity of the germplasm collection is a prerequisite of paramount importance.
Overall, our results allow a better understanding of the variability of the cultivars from the
Etna area. We have provided evidence of allelic interchange between wild and cultivated
subpopulations and provided useful information for better management and conservation of
germplasm and the direction of pear breeding programmes.
S1 Fig. Delta_k Log likelihood curve (ΔK) plotted against increasing K value. A: Plot of the
complete pear collection, B: Plot for the substructure analysis of the `wild' accessions, C: Plot
for the substructure analysis of the `cultivated' accessions.
S2 Fig. Nested structure analysis for the `wild' (A, B) and `cultivated' (C, D) groups. For
each analysis, the results according to the two most likely K value are reported: `wild' K = 2
(A), `wild' K = 5 (B), `cultivated' K = 3 (C), `cultivated' K = 4 (D).
S1 Table. Localization of the analysed genotypes used in this study.
S2 Table. Descriptive statistics of structure analysis. Structure descriptive statistics. For
increasing values of K, the number of replications are reported together with the mean and
standard deviation of the estimated Ln probability of data, the mean rate of change of the
likelihood distribution Ln'(K), the mean absolute value of the 2nd order rate of change of the
likelihood distribution |Ln''(K)| and the ΔK (mean (|Ln''(K)|) / sd(L(K)).
S3 Table. Assigned QI values at K = 2. Individuals exhibiting both QI values less than 0.8
were considered `admixed'. All other individuals are grouped as `wild' or `cultivated' according
to the predominant subpopulation. The status of the individual is also reported
(RSA = Relative Species Amygdaliformis, RS = Relative Species, LV = Local Varieties,
NCV = National Cultivated Varieties, ICV = International Cultivated Varieties).
S4 Table. Haplotypes definition. The four cpDNA markers allowed the definition of eight
haplotypes named with progressive numbers from 1 to 8. Genotypes of the single cpDNA
markers are reported in the last four columns.
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The authors gratefully acknowledge the anonymous reviewers for their suggestions and Prof.
Luca Dondini, University of Bologna, Italy, for providing some of the samples.
Conceptualization: Gaetano Distefano.
Data curation: Mario Di Guardo.
Formal analysis: Stefania Bennici, Giuseppina Las Casas, Mario Di Guardo.
Funding acquisition: Alessandra Gentile.
Investigation: Stefania Bennici, Giuseppina Las Casas, Mario Di Guardo, Alberto Continella,
Filippo Ferlito, Stefano La Malfa.
Methodology: Stefania Bennici, Giuseppina Las Casas.
Resources: Alberto Continella, Filippo Ferlito.
Supervision: Gaetano Distefano, Alessandra Gentile, Stefano La Malfa.
Writing ± original draft: Stefania Bennici.
Writing ± review & editing: Gaetano Distefano, Mario Di Guardo, Stefano La Malfa.
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