Tree Genetics & Genomes (2015) 11:821

DOI 10.1007/s11295-014-0821-2

ORIGINAL PAPER

Genetic diversity and structure of Pyrus accessions of Indian

Himalayan region based on morphological and SSR markers
Jai C. Rana & Rakesh K. Chahota & Vikas Sharma &
Maneet Rana & Nidhi Verma & Bhawna Verma &
Tilak R. Sharma

Received: 12 December 2013 / Revised: 20 November 2014 / Accepted: 24 November 2014

# Springer-Verlag Berlin Heidelberg 2014

J. C. Rana (*)
National Bureau of Plant Genetic Resources, Regional Station,
Phagli Shimla 171004, India
e-mail: ranajc2003@yahoo.com

model-based STRUCTURE analysis assigned genotypes into

five clusters and also showed the extent of admixture within
individuals. Genotypes belonging to Pyrus pyrifolia were
clustered into two different clusters indicating broad genetic
base for this species while Pyrus communis genotypes appeared to have conserved genetic makeup. Dendrogram-based
on Jaccards similarity coefficient and neighbor-joining tree
showed two major groups of Asiatic (P. pyrifolia, Pyrus
pashia, Pyrus serotina and Pyrus jacquemontiana) and European (P. communis) pears. However, accessions belonging to
P. pyrifolia were further divided into subgroups. Cluster analysis based on morphological traits was in agreement with the
dendrogram and structure clusters obtained with SSR data.
The Mantel matrix correspondence test was used to further
compare the molecular and morphological similarity matrices
and positive correlation coefficient (r=0.164; P=0.001) was
observed. Results of this study present important information
about genetic structure of Indian pear accessions which can
contribute significantly to future breeding and improvement
programmes in this species.

R. K. Chahota : V. Sharma : M. Rana : B. Verma : T. R. Sharma (*)

Department of Agricultural Biotechnology, CSK Himachal Pradesh
Agricultural University, Palampur 176062, India
e-mail: sharmat88@yahoo.com

Keywords Pear . Pyrus . Simple sequence repeat (SSR) .

Genetic diversity . Genetic structure . Phylogenetic
relationship

Abstract Genetic diversity and phylogenetic relationships

among 48 pear accessions belonging to six species were
determined using 23 morphological traits and 20 simple sequence repeat (SSR) primers. To assess the genetic diversity,
data on morphological (both quantitative and qualitative) traits
were recorded during 20102012 using standard pear descriptors. It was observed that the traits ratio of fruit length to
diameter, persistency of calyx on mature fruits and fruit surface and pulp texture were important in detecting variation
across pear species. At molecular level, 20 SSR primers
amplified 190 polymorphic alleles with an average of 9.5
alleles per primer. Size of amplified alleles ranged from 85
to 450 bp. Mean polymorphic information content (PIC) was
0.80 showing high level of SSR polymorphism. Bayesian
Communicated by D. Chagn

R. K. Chahota
e-mail: rkchahota@yahoo.com
V. Sharma
e-mail: vikasam@gmail.com

Introduction

M. Rana
e-mail: rana.maneet@gmail.com

Pear, belonging to family Rosaceae, is one of the economically important tree fruit crops produced in the temperate and
sub-temperate regions of more than 80 countries, occupying
an area of 16.23 million ha. World pear production has been
recorded as 23.6 million metric tons during 2012 (FAOSTAT
2012). Pear ranks second to apple in production of temperate
fruits worldwide (Oliveira et al. 1999; Wnsch and Hormaza
2007; Sharma and Rana 2010). The genus Pyrus contains 23

B. Verma
e-mail: vbhawna42@yahoo.com
N. Verma
National Bureau of Plant Genetic Resources, Pusa campus, New
Delhi 110012, India
e-mail: nidhi@nbpgr.ernet.in

821, Page 2 of 14

widely recognized primary species, mainly distributed in temperate Asia, Europe and the mountainous area of North Africa
(Silva et al. 2014). Of the two major cultivated species, Pyrus
communis L. is a commonly cultivated pear species in Europe,
North America, South America, Australia and Africa, while
Pyrus pyrifolia Burm. (Asiatic/Japanese pear) is mainly cultivated in Asia (Wolko et al. 2010). As edible fruits, Asiatic
pears are being cultivated for >3000 years (Lombard and
Westwood 1987; Cao et al. 2012) while in Europe, pears have
been cultivated as early as 1000 B.C. (Hedrick et al. 1921). In
India, Pyrus germplasm includes native (Asiatic), introduced
(European pears), cultivated and wild pears species.
The genetic diversity among different pear species has
accumulated over the years as they cross easily with one
another and the resulting introgressions are allocated various
taxonomic positions (Watkins 1986; Dhillon and Rana 2004;
Sharma and Rana 2010). In India, the diversity is mainly
prevalent in cultivated Asiatic pear (P. pyrifolia) while other
species like Pyrus pashia, Pyrus serotina, Pyrus
jacquemontiana, Pyrus polycarpa, and Pyrus khasiana grow
in wild to semi wild state throughout the temperate and subtemperate regions (Rana et al. 2012; Trivedi et al. 2012; Bhat
et al. 2013). In P. communis, diversity is limited to few
varieties like Babubosha and its ecotypes; however, it is
greatly enriched with introduced varieties from Europe.
Among wild species, P. pashia and P. serotina, locally known
as kainth and zarainth, respectively, and known to have originated in the western Himalayan region of India (Bailey 1953;
Rana et al. 2010), are extensively used as rootstock for both
P. communis and P. pyrifolia in the region (Rana et al. 2007).
A wide range of genetic diversity among Asiatic and European pears occurs in the breeders collections, wild forms/
species and among landraces grown in the villages or on small
farms throughout the world. Yet, there are a limited number of
cultivars that dominate the markets globally (Fowler and
Mooney 1990; Wnsch and Hormaza 2007). In the temperate
Himalayan region of India, different forms/varieties of pear
grow either in the cultivated orchards or in the wild and the
taxonomic status and genetic diversity of these forms is less
studied. Therefore, there is a need to assess the genetic diversity and establish the relationship between species and accessions based on the data generated through morphological
evaluation and simple sequence repeat (SSR) analysis. Several
studies have been carried out in pear to assess the genetic
diversity using a combination of morphological and molecular
tools. This includes morphological evaluation (Asanidze et al.
2011; Santiago-Pereira et al. 2012; Trivedi et al. 2012; Bhat
et al. 2013; Said et al. 2013); RFLPs (Iketani et al. 1998;
Katayama and Uematsu 2003), RAPD (Kim et al. 2005) and
AFLP (Dolatowski et al. 2004; Bao et al. 2008). Recently,
SSR markers have been effectively used in Pyrus species (Yao
et al. 2010; Baraket et al. 2011; Sehic et al. 2012; Cao et al.
2012; Erfani et al. 2012; Akcay et al. 2014).

Tree Genetics & Genomes (2015) 11:821

Using morphological and SSR markers, we have

analysed the genetic diversity of Pyrus germplasm conserved in our temperate fruit Field Gene Bank (FGB).
Morphological and phenological traits have historically
been important in the diversity analysis and establishing
phylogenetic relationship between species. However, they
are susceptible to phenotypic plasticity allowing the assessment of diversity in the presence of environmental
variation (Mondini et al. 2009). In contrast, molecular
markers are useful for such purposes as they are not
influenced by variable environmental conditions or plant
phenology and can discriminate among cultivars and species (Belaj et al. 2007). SSR markers, in particular, are
more useful due to their reproducibility, codominance,
polymorphism and transferability among related species
and genera. They help in resolving the differences that
arise due to widespread crossability and susceptibility of
characters to phenotypic plasticity in the presence of
environmental variation (Nybom and Weising 2010).
The present study was thus designed to evaluate and
assess the genetic diversity and structure of Pyrus germplasm conserved in the FGB and to reveal phylogeny
between different species and cultivars using both morphological evaluation and SSR analysis in 48 accessions
belonging to six species and hybrids of Pyrus. This information will be useful to optimize the management of pear
germplasm and also in the selection of elite and diverse
parental genotypes for maximizing variability and enhancing productivity in pear breeding programmes being run
at different horticultural universities and institutes in the
Indian Himalayan region.

Materials and method

Plant material
The plant materials comprising 48 accessions are being conserved in the FGB of the Regional Station of National Bureau
of Plant Genetic Resources (NBPGR) Shimla, Himachal
Pradesh, India. The FGB is located at 31 05 53.89 N and
77 09 34.92 E at an elevation of 1924 m above sea level.
The accessions belong to six pear species namely P. communis
(15), P. pyrifolia (21), P. pashia (3), P. jacquemontiana (1),
Pyrus ussuriensis (1), P. serotina (3) and four hybrids of
P. communis P. pyrifolia (Table 1). This germplasm has
been collected from different parts of India, particularly the
Himalayan region and also introduced from abroad. The accessions collected from India have been assigned national
identity number and designated as indigenous collection
(IC), whereas the exotic (EC) introductions were assigned
EC numbers.

Tree Genetics & Genomes (2015) 11:821

Table 1

Detail of the plant material used

Species/hybrid

Accessions

Pyrus pyrifolia
(Burm.) Nak.

IC566140, IC555282, IC558066, IC557988,

IC557985, IC552678, IC538512, IC20804,
IC566145, IC557989, IC019392, IC447940,
IC447993, IC20813, IC538508, IC566190,
EC436204, EC552669, EC38740, EC552676

P. pashia Buch.-Ham.
ex D. Don
P. serotina Rehd
P. jacquemontiana
Decne
P. communis L

IC558110, IC538506, IC209705

IC20818, IC20814, IC20808, IC020821
IC538507
IC566142, IC557984, IC484159, EC168557,
EC315334, EC552675, EC126287,
EC57511, EC57516, EC27809, EC27810,
EC200365, EC552668, EC38388, EC329057
EC25781
EC552674, EC552673, EC126286, EC552672

Page 3 of 14, 821

(Mahajan et al. 2002) on randomly selected 10 fruits from

each accession. Mean values of 3-year observations were
taken to analyse data and to draw the conclusions (Table 2).
The data were analysed for mean, variances, correlations,
regression and genetic diversity to deduce the genetic
similarity/dissimilarity and principal component analysis
(PCA) was performed using the statistical software SYST
AT-12 and StatistiXL version 1.10.
Molecular analysis
DNA extraction

Pyrus pyrifolia (Ppy), P. pashia (Ppa), P. serotina (Ps), P. jacquimontiana

(Pj), P. communis (Pc), P. ussuriensis (Pu), P. communis P. pyrifolia
(Ph)

Young leaves of each accession were used for DNA extraction

following CTAB method (Doyle and Doyle 1990) with some
modifications. DNA stocks were prepared in T10E1 buffer and
quantification of DNA was done on 0.8 % agarose gel by
comparing with lambda DNA (Fermentas, Lithuania). Working stock of each sample was prepared by diluting it to make a
concentration of 13 ng/l. These dilutions were further
checked on 0.8 % agarose before being used in PCR reactions.

Morphological evaluation

SSR genotyping

We recorded data on 13 qualitatively assessed and 10 quantitatively measured traits for 3 years (20102012). The qualitatively assessed traits (Fig. 1) were leaf size, leaf margin,
flower size, persistency of calyx, fruit shape, fruit over colour,
fruit surface texture, pulp texture, grittiness, pulp colour, pulp
juiciness, pulp taste and productivity status; while quantitatively measured traits were flower buds/inflorescence, flower
stalk length, peduncle length, fruit length, fruit diameter, fruit
weight, ratio of fruit length to diameter, ratio of fruit length to
peduncle, days to maturity and total soluble solids (TSS).
Traits were measured based on pear descriptors developed
by the National Bureau of Plant Genetic Resources

PCR reactions were carried out using 20 microsatellite

primers developed in pear and apple (Yamamoto et al.
2002a, b, c; Liebhard et al. 2002; Hemmat et al. 2003;
Nishitani et al. 2009). PCR amplification was carried out in
2720 Thermal Cycler (Applied Biosystems, CA, USA), in
12.5-l reaction mixture containing 2-l genomic DNA
(13 ng/l), 1.25 l 10X PCR Buffer (10 mM TrisHCl,
50 mM KCl, pH 8.3), 1.0 l MgCl2 (25 mM), 1.0 l dNTP
mix (0.2 mM each of dATP, dGTP, dTTP, dCTP), 0.5 l of
each of two primers and 0.1 l Taq DNA polymerase (5 U/l).
PCR reactions were performed at 1 cycle of 4 min at 94 C as
initial denaturation, followed by 35 cycles with a denaturation

P. ussuriensis Rehder
P. communis
P. pyrifolia

Fig. 1 Frequency distribution of 48 pear accessions for 13 qualitatively measured traits. Y axis is no. of accessions and X axis is traits

Flower
buds/inflorescence

Flower stalk length

(cm)

Range
Mean+SE
CV %

3.69.0
6.91
41.1

P. communis P. pyrifolia (4)

1.32.2
1.91
21.6

14.321.6
18.43
22.7

22.624.6
23.42
18.6

Range
Mean+SE
CV %

2.64.8
3.41
31.5

10.720.2
16.91
29.6

Range
2.67.8
2.06.9
Mean+SE
4.40.4
3.40.4
CV%
31.9
37.2
P. pashia (3) and P. jacquemontiana (1)

6.09.0
7.01
22.8

21.137.4
26.70.9
19.1

Range
1.59.5
2.25.7
Mean+SE
5.70.4
3.00.2
CV %
42.5
31.2
P. communis (15) and P. ussuriensis (1)

10.737.4
22.50.8
19.5

Peduncle length
(mm)

54.768.9
61.23
11.4

14.168.0
31.42
19.3

39.774.8
61.03
21.8

36.685.1
62.03
23.3

14.185.1
59.12
28.6

Fruit length
(mm)

52.166.5
56.96
16.2

13.172.1
32.63
21.8

36.368.6
56.03.0
19.3

35.491.5
65.43
21.6

13.191.5
58.83
27.9

Fruit diameter
(mm)

77.8102.0
89.06
11.2

18.8130.5
51.43
14.6

45.5167.1
104.99
41.7

54.2275.8
139.213
43.5

18.8275.8
116.38
47.3

Fruit weight
(g)

Range, mean, variance and coefficient of variation among quantitative traits measured in 48 pear accessions

Across the species

Range
1.59.5
1.36.9
Mean+SE
5.50.3
3.10.2
CV %
40.1
38.4
Pyrus pyrifolia (20) and P. serotina (4)

Variability
parameter

Table 2

1.01.1
1.00.06
18.9

0.81.0
0.90.05
17.4

1.01.17
1.00.05
26.8

0.81.1
0.90.04
18.4

0.851.17
1.00.06
24.6

Fruit length/fruit
diameter ratio

2.84.8
3.40.3
28.2

0.62.8
1.30.1
20.3

2.15.3
3.70.4
23.1

1.34.0
2.30.2
22.9

0.65.3
2.80.1
29.2

Fruit length/peduncle
length

148174
161.55
9.8

158235
198.719
18.35

92144
116.84
12.3

147233
186.25
11.3

92235
162.06.0
18.3

Days to
mature

8.911.8
10.81
10.7

11.718.5
15.52
17.5

10.114.7
12.70.4
11.8

8.714.3
11.50.4
10.2

8.718.5
12.23.0
20.4

TSS (%)

821, Page 4 of 14
Tree Genetics & Genomes (2015) 11:821

Tree Genetics & Genomes (2015) 11:821

step at 94 C for 1 min, an annealing step for 1 min at

respective annealing temperature of each primer in a range
of 4957 C and an extension step at 72 C for 1 min,
followed by last cycle of extension at 72 C for 7 min. PCR
products were first checked on 3 % agarose gel and then
resolved in 6 % polyacrylamide gel at a constant power supply
of 65 W at room temperature for 90 min. Gels were prepared
and run in 1X TBE buffer and visualization of fragments was
done using silver-staining procedure. Sizing of alleles was
done with the help of 50-bp DNA ladder (Fermentas,
Lithuania).
Data analysis
All fragments were scored manually and converted into binary
data, i.e. 1 for presence of band and 0 for absence of band. The
polymorphism information content (PIC) of each primer pair
was calculated according to the following formula given by
Botstein et al. (1980) and implemented in Cervus version 3.0:
n
PICi 1  P2 i j
ji

Where Pij is the frequency of the jth pattern for marker i and
summation extends over n patterns. Various genetic diversity
estimates such as expected heterozygosity (He), observed
heterozygosity (Ho), Shannon information index (I), etc. were
calculated with the help of POPGENE version 1.32 (Yeh and
Boyle 1997). Distance-based cluster analysis was performed
and dendrogram based on the unweighted pair group method
of arithmetic mean (UPGMA) was constructed using
Jaccards similarity coefficient with the help of NTSYSpc
2.0 (Rohlf 1998). Neighbor-joining (N-J) tree was constructed
using Dice coefficient with the help of DARwin (Perrier and
Jacquemoud-Collet 2006). Bootstrapping with 1000 replicates
was also performed with DARwin. Bayesian model-based
clustering method implemented in STRUCTURE software,
version: 2.3.3 (Pritchard et al. 2000; Falush et al. 2007) was
utilized to assess the genetic structure at population level as
well as to detect genetic stocks contributing to this germplasm
collection. Ancestry model with admixture and correlated
allele frequency model was set to get the estimates of posterior
probability of data. Ten independent runs were given setting
the value of K from 1 to 10 with three iterations for each value
of K. Length of burn-in period was set at 100,000 and number
of Markov chain Monte Carlo (MCMC) repeats after burn-in
were set at 100,000. Evannos method (Evanno et al. 2005)based program, STRUCTURE HARVESTER, developed by
Earl and Vonholdt (2011) was used to determine the value of
estimated Ln probability of data-LnP(K) and to get the best fit

Page 5 of 14, 821

value of K for the data. STRUCTURE was run for all genotypes collectively and also separately for accessions of
P. pyrifolia and P. communis to assess their genetic structure
at species level. Genetic differentiation (Fst) estimates for
each cluster were also detected by STRUCTURE. Genetic
relationships among the genotypes were also analysed by
principal coordinate analysis (PCA) using the GenAlex 6.4
program (Peakall and Smouse 2006). All the genotypes were
plotted on the first two principal axes. The mantel matrix
correspondence test (Mantel 1967) was used to compare the
molecular and morphological similarity matrices. Analyses of
molecular variance (AMOVA) was performed using GenAlex
6.4 program (Peakall and Smouse 2006).

Results
Morphological traits
Qualitatively assessed traits
Wide range of variation was observed for all the qualitatively
assessed traits on different scales (Fig. 1). Leaf size of half of
the accessions (24) was medium while flower size was small
and medium in most of the accessions (36). Leaf margin was
serrate for 18 accessions of P. communis, P. ussuriensis and 2
hybrid accessions, while it was spinulose serrate for 26 accessions of P. pyrifolia, P. serotina and 2 hybrid accessions. The
fruit shape of accessions of P. communis and P. pyrifolia was
largely ovate-oblong and pyriform while it was round and
globose for wild species P. pashia, P. serotina and
P. jacquemontiana. Fruit over colour was predominantly ranging from yellow to yellow green for 34 accessions. The nature
of calyx was persistent for 18 accessions of P. communis,
P. ussuriensis and two hybrid accessions, while it was deciduous for remaining 30 accessions of P. pyrifolia and other wild
species. The accessions of P. pyrifolia and wild species had
rough to medium rough fruit surface, medium to hard pulp
texture and more grittiness in the pulp compared to
P. communis accessions. Fruit juiciness was higher in the
accessions of P. pyrifolia and P. serotina than in European
accessions. The fruit pulp was sweet to highly sweet for
P. communis, while it was sub-acidic to mild sweet for accessions of other species.
Quantitatively measured traits
Variability was observed among accessions for all the quantitatively measured traits (Table 2). The number of flower
buds/inflorescence was higher in P. pashia and
P. jacquemontiana and hybrid accessions as compared to
P. communis and P. pyrifolia. The range and mean of fruit

821, Page 6 of 14
Table 3

Tree Genetics & Genomes (2015) 11:821

Correlation coefficients among quantitatively measured traits in 48 pear accessions

Flower stalk length

Peduncle length
Fruit length
Fruit diameter
Fruit weight
Ratio of fruit length
to diameter
Ratio of fruit length
to peduncle
Days to maturity
Total soluble solids

Flower
buds/infl.

Flower
stalk length

Peduncle
length

0.02
0.04
0.05
0.04
0.09
0.17

0.03
0.06
0.08
0.04
0.04

0.02
0.18
0.26
0.63**

0.17

0.01
0.13
0.22

0.36**
0.03

Fruit
length

Fruit
diameter

Fruit
weight

Flower length
width ratio

0.95**
0.81**
0.06

0.85**
0.25

0.19

0.71**

0.68**

0.50**

0.32*

0.48**

0.62**
0.22

0.13
0.53**

0.07
0.49**

0.14
0.43**

0.66**
0.05

Flower length
peduncle length
ratio

0.55**
0.21

Days to
mature

0.07

*Significant at p<0.05
**Significant at p<0.01

accessions also took less time for fruit maturity. Although,

wild species were sub-acidic and medium sweet in taste but
they showed high total soluble solids (TSS). The bivariate
correlation coefficients among 10 quantitatively measured
traits (Table 3) showed significant positive correlation among
fruit length, fruit diameter and fruit weight. Principal component analysis (PCA) used to identify the most significant traits
(Fig. 2), showed that the first principal component (PC1),
explained 34.24 % of the total variance, which was mainly
contributed by fruit length, fruit diameter, fruit weight, ratio of
fruit length to peduncle length and TSS. PC2 accounted for
29.34 % variation through peduncle length, ratio of fruit
length to diameter, and days to maturity, whereas flower stalk
length and flower buds/inflorescence contributed 10.98 and

weight was highest in P. pyrifolia accessions (54.2275.8 and

143.7 g), and lowest in P. pashia and P. jacquemontiana
(18.830.5 and 24.4 g), respectively. Wild species with higher
number of flower buds/inflorescence produced higher number
of fruits with small fruit size and reduced fruit weight. The
ratio of fruit length to diameter varied from 0.851.17 for all
the species of Pyrus, but it was higher (1.11.17) for European
pears as compared to Asiatic pears (0.81.0). The ratio of fruit
length to peduncle length was 0.6 to 5.3 across species, while
it was 2.1 to 5.3 for European pears and 0.6 to 4.3 for Asiatic
pear and wild species. The accessions of Asiatic pears were
late in maturity and took as long as 235 days for P. pashia and
228 days for P. pyrifolia to mature compared to a maximum of
144 days for European pears. Interestingly, the hybrid

Fig. 2 Biplot of different

variables loaded on PC1 and PC2

0.50

Fruit_length_fruit_width _ratio
Fruit_length_peduncle_length_ratio

Second Component

0.25
Total_soluble_solids
Flower_stalk_length
Fruit_length

0.00

Fruit_width

Flower_buds_per_infloresence

Fruit_weight

-0.25

Days_to_mature

-0.50

Peduncle_length

-0.4

-0.3

-0.2

-0.1

0.0
0.1
0.2
First Component

0.3

0.4

0.5

Tree Genetics & Genomes (2015) 11:821

Page 7 of 14, 821

Table 4 Eigenvectors for the first four components of quantitatively

measured traits in 48 pear accessions
Traits

PC1

PC2

PC3

PC4

Flower buds/inflor.
Flower stalk length
Peduncle length
Fruit length

0.06
0.09
0.07
0.98

0.32
0.12
0.87
0.03

0.18
0.90
0.03
0.06

0.89
0.23
0.35
0.00

Fruit diameter
Fruit weight
Ratio of fruit length to diameter
Ratio of fruit length to peduncle
Days to maturity
Total soluble solids
Total variance explained

0.93
0.83
0.08
0.71
0.16
0.61
34.24

0.29
0.35
0.83
0.63
0.85
0.21
29.34

0.10
0.21
0.14
0.04
0.04
0.42
10.98

0.01
0.01
0.06
0.15
0.19
0.10
10.26

10.26 % variation to PC3 and PC4, respectively (Table 4).

Dendrogram constructed on the basis of morphological traits
using Euclidean distance and nearest neighbor method did not
show clear groups but largely separated the accessions at
species level with few exceptions (Fig. 3).
Molecular analysis
SSR polymorphism and diversity studies
Twenty SSR primer pairs used in this study amplified 190
alleles with an average of 9.5 alleles per locus. Size of alleles

varied from 85 to 450 bp. Primer pair TsuENH026 amplified a

maximum of 15 alleles, whereas NB103a amplified only 4
alleles. PIC values ranged from 0.58 for primer NB103A to
0.90 for primer TsuENH026 with an average of 0.79 (Table 5).
Highest (0.92) and lowest (0.65) expected heterozygosity (He)
values were obtained for TsuENH026 and NB103a, respectively, with an average of 0.82. Whereas highest (0.98) observed heterozygosity (H O ) value was obtained for
TsuENH044 and lowest (0.47) for NH4013a, with an average
of 0.79. Shannons Information index (I) was recorded maximum (2.51) for TsuENH026, whereas the lowest value (1.16)
of I was recorded for primer NB103a with an average of 1.90.
Bayesian genetic structure
Delta K, which is used to determine the best fit value of
K, was computed by STRUCTURE harvester for the
given range, i.e. 110 and highest value was shown at
K=5. Therefore, STRUCTURE analysis was conducted
for K=5, which largely clustered accessions within species range, except for P. pyrifolia species. Accessions
belonging to P. pyrifolia were grouped in two different
clusters (cluster II & III in Fig. 4a); one of them only
containing accessions of Indian origin (Cluster III). All
P. communis accessions were clustered in a single group
with some admixtures (cluster V). Hybrids of P. communis
P. pyrifolia formed separate cluster (cluster IV). Wild
species P. pashia, P. jacquemontiana and P. serotina were
grouped in a single cluster (cluster I). As accessions of
P. pyrifolia were mainly distributed in two clusters, with
few accessions dispersed across other clusters, further
assessment of the population structure within P. pyrifolia
was done to understand the species complexity. STRU
CTURE analysis confirmed the existence of two gene
pools in the species (Fig. 4b) as indicated by the combined analysis. AMOVA (Table 6) revealed that a major
portion (95 %) of genetic variation resided within different species of Pyrus rather than among species (5 %).
Genetic differentiation (Fst) was highest (0.64) in cluster
V (European cultivated pears) while lowest (0.20) in
cluster IV (hybrids). Cluster I (wild pears) showed a Fst
value of 0.48 while clusters II and III (Asian cultivated
pears) detected values of 0.46 and 0.22, respectively.
Cluster and PCA analyses

0.8

Fig. 3 Dendrogram based on morphological traits constructed using

squared Euclidean distance and group average clustering method

The Mantel matrix correspondence test (Mantel 1967) was

used to compare the molecular and morphological similarity
matrices. Although overall correlations between the distance
coefficients was rather low (r=0.164; P=0.001) but with few
exceptions, both morphological and molecular dendrograms
clustered the genotypes according to their taxonomic status.
Dendrogram based on Jaccards similarity coefficient and

821, Page 8 of 14
Table 5 Diversity statistics for
20 SSR loci studied in 48 pear
accessions

Tree Genetics & Genomes (2015) 11:821

Locus

Ta (C)

Allele Size range (bp)

na

NH4013A
BGT23B
TsuENH029
NB113A
NB103A

55
55
55
55
53

225275
235350
137200
140190
145180

7
11
7
7
4

CH05d04
CH01d08
NH004A
NH015A
CH04e03
TsuENH026
CH01F07A
EMPc11
CH03d12FF
EMPc117
NH029
TsuENH044
GD147
CH01d09
NH007b
Mean

57
55
53
53
45
51
55
51
54
53
53
54
55
51
53

175220
300450
85190
100145
175225
160250
190350
175250
100155
95160
220290
85145
170300
120135
137165

9
10
10
10
7
15
14
11
8
14
9
12
6
10
9
9.5

UPGMA method showed two major groups as shown in

Fig. 5. Group I consisted of P. pyrifolia, P. serotina, P. pashia
and four hybrids of P. communis P. pyrifolia. Further, this
main group was divided into two subgroups: Ia and Ib. Subgroup Ia had accessions of P. pyrifolia, P. pashia and
P. jacquemontiana and subgroup Ib comprised of accessions
of P. pyrifolia, P. serotina and P. communis P. pyrifolia
hybrids and one accession of P. communis. Group II included
accessions of P. communis and P. ussuriensis. The minimum
genetic similarity value of 0.015 between IC552678 and
EC38740 indicated them to be most distant accessions, whereas IC447940 and IC447993 (with the similarity value of
0.745) were found to be genetically most similar. These four
accessions belonged to P. pyrifolia. Neighbor-joining tree
based on Dice coefficient also separated accessions on the
basis of taxonomic status (Fig. 6). This phylogenetic tree also
showed divergence of accessions which could be attributed to
their cultivated and wild nature. In general, the grouping
pattern of accessions showed species delimitations in the tree
with few exceptions and groups were more distinct than in the
phylogenetic tree based on morphological traits. PCA analysis
clearly showed the diverse nature of P. pyrifolia and other
species. Percentages of variation captured by the first three
axes in PCA plot was 30.05, 19.10 and 15.10. Clustering
patterns in PCA was in correspondence with clustering of both
UPGMA tree and the STRUCTURE.

ne

Ho

He

PIC

5.73
7.10
4.05
6.62
2.82

1.80
2.07
1.59
1.99
1.16

0.47
0.89
0.82
0.76
0.78

0.84
0.87
0.76
0.86
0.65

0.80
0.84
0.70
0.83
0.58

7.04
7.20
4.89
6.90
6.00
10.91
10.05
6.82
6.61
7.51
6.25
7.17
4.54
7.34
6.27
6.59

2.02
2.11
1.84
2.07
1.87
2.51
2.43
2.08
1.97
2.27
1.98
2.16
1.65
2.10
1.93
1.98

0.62
0.82
0.82
0.87
0.96
0.96
0.89
0.96
0.77
0.83
0.76
0.98
0.94
0.78
0.60
0.81

0.87
0.87
0.80
0.86
0.84
0.92
0.91
0.86
0.86
0.88
0.85
0.87
0.79
0.87
0.85
0.84

0.84
0.84
0.76
0.83
0.81
0.90
0.89
0.83
0.83
0.85
0.80
0.82
0.84
0.75
0.84
0.80

Discussion
Morphological variation
Diversity in fruit shape has been observed in wild and cultivated varieties of pear (Paganov 2003). Characters like reduced ratio of fruit length to diameter, rough fruit surface, hard
pulp texture and more grittiness were key characters in our
data set, which differentiated the European pears from Asiatic
pears and their wild relatives. The Asiatic pear accessions
were found to be better adapted in the Indian conditions than
accessions of European origin, introduced from Europe and
North America to our FGB. Fruit size has been considered an
important parameter for selection of superior genotypes
through breeding programmes (Westwood and Blaney
1963). However, mechanism of fruit development is influenced by cultural and genetic factors (Harada et al. 2005);
therefore, we optimised the number of fruits on the trees to
record accurate observation of fruit length, fruit diameter and
fruit weight. Earlier studies conducted in India have revealed
the existence of genetic diversity for vegetative, reproductive
and yield parameters in pear germplasm of Asiatic and European origin (Verma et al. 2006; Dhillon and Dhillon 2008;
Kumar et al. 2010). It is also assumed that many species of
Pyrus are inter-crossable and there are no major incompatibility barriers to interspecific hybridization, which may lead to

Tree Genetics & Genomes (2015) 11:821

Page 9 of 14, 821

Fig. 4 a Genetic structure of 48 pear accessions as inferred by STRU

CTURE v2.3.3 with 20 SSR markers data set. Single vertical line
represents an individual genotype and different colours represent

genetic stocks/gene pools. Segments of each vertical line show extent of

admixture in an individual. b Population structure of 20 Pyrus pyrifolia
accessions showing two genetic stocks

greater genetic diversity in spite of the wide geographic distribution of the genus (Westwood and Bjornstad 1971). The
number of flower buds/inflorescence, flower stalk length and
peduncle length did not show any significant correlation
among themselves and also with fruit length and fruit diameter. The peduncle length showed significant negative correlation with ratio of fruit length to diameter and ratio of fruit
length to peduncle length while positive correlation with days
to maturity. This indicates that accessions with long peduncles
have reduced fruit length with late maturity. Fruit length, fruit
diameter, fruit weight and ratio of fruit length to diameter
showed significant positive correlation among themselves,
indicating that weight of fruits depends on the ratio of fruit
length to diameter. TSS showing negative correlation with all

the three fruit traits indicated that the reduced TSS in larger
fruits might be the result of higher water content. Significant
positive correlation among different fruit traits has been observed in sour cherry (Rakonjac et al. 2010) and peach (Verma
et al. 2009).
The scatter plot of residuals versus fitted values drawn
through regressions analysis showed that residuals of majority
of the accessions bounce randomly around 0 line forming
horizontal band (data not shown here). This suggests that the
variances of the error terms are equal and relationship among
accessions is linear. However, few outliers such as EC436204,
EC552669 and IC566145 were observed among the accessions evaluated, which might be due to their extreme values
for a particular trait and in this case, it was higher fruit weight
and more number of flower buds. Principal component analysis identified the most significant traits in the data set and
established genetic relationship among accessions in the evaluated pear germplasm. This determined the number of main
factors to reduce the effective parameters to discriminate
accessions and relationship established through PCA may
correspond to genetic linkage between loci controlling the
traits or pleiotropic effects (Iezzoni and Pritts 1991; Iezzoni
2008; Rakonjac et al. 2010). The first four PCA components

Table 6 Analysis of molecular variance (AMOVA) within and between

Pyrus species
Source

df

SS

MS

Est. Var.

Percent (%)

Among species
Within species
Total

5
42
47

206.529
1257.450
1463.979

41.306
29.939

1.670
29.939
31.609

5
95
100

821, Page 10 of 14

provided a reasonable summary of the data and explained

84.82 % of the total variation. The loading plot drawn for
PC1 and PC2 also indicated the importance of fruit traits
including ratio of fruit length to diameter for explaining the
variance among accessions and species relationship. The relationship between fruit traits and their ratios have been considered very useful characteristics to distinguish variance
among different pear species (Voltas et al. 2007). Dendrogram
showed phenetic relationships among different accessions on
the basis of morphometric analysis and assignment of
individuals to their species groups. While most of the
accessions grouped in accordance to existing taxonomic
hierarchy; however, exceptions could be attributed to low
resolution achieved due to less numbers of morphological
traits/markers used to analyse the present accessions.
SSR diversity and structure
The genetic diversity revealed by SSR loci was supported by
the observed high values of mean allele numbers, heterozygosity and PIC. An average of 9.5 alleles per primer pair
observed was higher than previous studies in pear by Nishitani
et al. (2009) and Wolko et al. (2010) while it was lower than
the study conducted by Volk et al. (2006) in wild P. communis.
Similarly, mean heterozygosity and PIC of 0.81 and 0.80,
respectively, were higher than in the studies detected by other

Tree Genetics & Genomes (2015) 11:821

workers (Bao et al. 2007; Wnsch and Hormaza 2007;

Nishitani et al. 2009; Wolko et al. 2010) in different pear
varieties. Average Shannons Information Index (I) value of
1.98 was also higher than observed by Erfani et al. (2012) who
reported an average I of 1.37 in 48 pear accessions. High
values of all the measures of diversity indicated allelic richness in the analysed germplasm which can be utilized in
breeding programmes to get desired plant types for commercial cultivation. However, the allelic richness in the Pyrus
accessions could be attributed to the multiple species analysed
and also due to the wide crossability and self-incompatibility
of species (Franceschi et al. 2011). Thus, nature of pollination
has a remarkable effect on distribution, diversity and richness
of alleles within and among populations of a given species.
Low level of genetic diversity observed in peach was thought
to be due to its self-pollinating nature (Sosinski et al. 2000).
Presence of rich allelic diversity in pear accessions, as recorded in the present study is perhaps due to the availability of wild
genotypes which grow locally, providing opportunity of crosspollination with cultivated species.
Bayesian genetic structure and admixture analysis performed using software STRUCTURE assigned accessions
into five genetic clusters. In addition to assigning individuals
to different clusters on the basis of allele frequencies, it also
detected the extent of admixture within accessions. Of the five
genetic clusters obtained with STRUCTURE, clusters II and

Fig. 5 Dendrogram of 48 pear accessions based on 190 alleles and constructed using Jaccards similarity coefficient using UPGMA method

Tree Genetics & Genomes (2015) 11:821

Page 11 of 14, 821

Fig. 6 Radial neighbor-joining tree based on 190 alleles from 20 SSR

loci among 48 pear accessions belonging to six species. Two major
clusters representing Asiatic and European pears are shown by purple
and orange arcs, respectively, while sub clusters are labelled and

highlighted according to nature of accessions. Cultivated accessions are

highlighted with blue, wild with orange and hybrids with purple colour.
Bootstrap values60 % are shown

III corresponded to P. pyrifolia, and showed two gene pools

for this species. Interestingly, one of the clusters (cluster III)
was formed by the accessions of Indian origin, while few
Indian accessions of this species were dispersed with some
exotic accessions in cluster II. Further, the STRUCTURE
analysis of 20 accessions of this species again inferred two
genetic clusters which supported the previous view of Iketani
et al. (2012) who also detected two genetic clusters for native
Japanese pear, P. pyrifolia. Cluster I represented all the wild
species of Asiatic origin viz., P. pashia, P. serotina and
P. jacquemontiana, indicating a common genetic background
for these species. Cluster IV corresponded to accessions of
hybrid origin, i.e. P. communis P. pyrifolia. Separate clustering of hybrid cultivars from parental species showed the
intermixing possibilities and high recombining ability of alleles in the reconstituted species which resulted in unique sets
of alleles in the hybrids. Cluster V comprised of accessions of
P. communis. Although low admixture was recorded in few

accessions of this cluster, majority of accessions showed pure

ancestry, conferring this group with a conservative genetic
background. For all the six species, structure analysis showed
five major gene pools and most of the accessions in each
cluster were represented by pure ancestry as shown by membership values. From the structure analysis, it appeared that
P. pyrifolia has a history with highest out crossing rates with
other species which resulted in accumulation of different gene
combinations, whereas P. communis seems to have a more
conservative genetic background. Genetic differentiation
values of the inferred clusters indicated that European cultivated pears were highly differentiated (Fst=0.64) as these
were the most distantly and isolated accessions. On the other
hand, hybrid pear accessions showed lowest genetic differentiation (Fst=0.20) as these were having alleles from various
diverse parental taxa of Pyrus. Further, genetic structure predicted by AMOVA suggested that a greater part (95 %) of
genetic variance resided within species while a lesser part

821, Page 12 of 14

(5 %) of genetic variance resided among the species. These

estimates showed that various allelic combinations were present within species and exchange rates of alleles were very high
within species than among species. This also indicated existence of less natural interspecific hybridization events in Pyrus
species.
Phylogenetic analysis
SSR markers were used for identification of Chinese white
pear cultivars and they were found ideal marker systems for
phylogenetic reconstruction in pear species (Tian et al. 2012).
On the basis of allele sharing and Jaccards similarity matrix,
UPGMA dendrogram inferred two major groups of all pear
accessions. Group I mainly containing P. pyrifolia accessions
also showed affinity with other Asiatic species and formed
one group. Further subgroups of Cluster have shown that 10
P. pyrifolia accessions exhibited close proximity to wild Asiatic species of P. pashia, P. serotina and P. jacquemontiana
(Fig. 5). Moreover, four accessions of hybrid (P. communis
P. pyrifolia) origin were also grouped within the P. pyrifolia
major group. In the second group, accessions of P. communis
and one accession belonging to P. ussuriensis were also included. P. ussuriensis is an Asiatic pear species but this
accession was procured from Hungary and this relatedness
can be a result of geographic adaptation. However, further
investigations are needed to rule out any inadvertent
mislabelling of this accession in the germplasm block. Dice
coefficient-based neighbor-joining (N-J) tree also distinguished different accessions as per species boundaries which
were also supported by robust branching as indicated by
bootstrap values. N-J tree also defined almost all accessions
on the basis of their nature of occurrence as wild or cultivated.
In addition, results also indicated correspondence between
geographic affiliation and genetic relationship for many accessions. Barring exceptions, NJ tree largely formed two
groups of cultivated pear species (P. communis, P. ussuriensis
and P. pyrifolia) and two groups of wild species (P. pashia,
P. jacquemontiana and P. serotina). Accessions with hybrid
parentage clustered in a separate group. N-J tree and UPGMA
results were complemented with PCA analysis. Phylogenetic
analysis revealed that accessions IC566190 and IC538512,
belonging to P. pyrifolia, which grouped with wild species of
Asiatic origin, might be the manifestation of natural interspecific breeding behaviour of pear species. Interestingly, the
accession IC168557 of P. communis clustered with Asiatic
accessions. These observations, however, need further studies
to understand the possible reasons of this deviation. Dendrogram based on morphological traits was also in accordance
with the N-J tree but showed poor resolution of accessions
probably due to less numbers of quantitatively measured
traits. Overall, interrelationships of accessions were in agreement with existing taxonomic ranks and were influenced by

Tree Genetics & Genomes (2015) 11:821

geographic proximity of the species with one another, therefore, forming two major groupsAsiatic and European pears.
The study has relevance because the genetic diversity analysis
of Indian pear germplasm using molecular markers has not
been carried out. On the basis of our evaluation data, we
identified some promising accessions for single and multiple
traits such as EC436204, EC552669, IC536508, IC447993
(fruit weight >190.0 g); EC27809, EC552668, EC200365,
IC484159 (mature in <100 days) and EC126286, EC27810,
EC200365, EC552668 (over all fruit quality), which could be
used as potential donors in the hybridisation programme involving different accessions of P. communis and P. pyrifolia.
Based on the vigour and hardiness, accessions of wild species
can be used as rootstocks for their cultivated allies.

Acknowledgments The authors acknowledge the anonymous reviewers for their critical comments and suggestions.
Data archiving statement The information on accessions used and the
phenotypic data files are being submitted to Genomic Database to
Rosaceae (GDR) at www.rosaceae.org.

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