Download Morphometry of the organs of cherimoya (Annona cherimola Mill

Scientia Horticulturae 107 (2006) 337–346
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Morphometry of the organs of cherimoya (Annona cherimola Mill.)
and analysis of fruit parameters for the characterization of cultivars,
and Mexican germplasm selections
J. Andrés-Agustı́n a, F. González-Andrés b,*, R. Nieto-Ángel c, A.F. Barrientos-Priego c
b
a
CRUCO-Universidad Autónoma Chapingo, Morelia, Michoacán, Mexico
Departamento de Ingenierı́a Agraria, ESTIA, Universidad de León, Avda de Portugal 41, 24071 León, Spain
c
Departamento de Fitotecnia, Universidad Autónoma Chapingo, Chapingo, Estado de México, Mexico
Received 15 November 2004; received in revised form 22 September 2005; accepted 16 November 2005
Abstract
Three commercially available cherimoya cultivars (Campas, Burtons and White), one cultivar recently obtained and registered in Mexico
(Cortés-II-31) and seven Mexican germplasm selections (S-196, S-256, S-260, S-266, S-9651, S-Selene and S-Carapan) were characterized by
using the morphometric traits of various organs of the adult plant, together with several agronomical and chemical characteristics. The objectives
were: (i) to seek an alternative approach to the definition of cherimoya cultivars through multivariate analysis, using commercial varieties and
Mexican germplasm selections and (ii) to elucidate the grouping of cultivars and selections obtained by multivariate analysis on the basis of their
origin and geographical distribution. Plant material was collected in 2002 from adult plants 3 years after grafting. Twenty-one morphometric
characteristics (seven of leaves, nine of flowers, two of fruits and three of seeds), plus five fruit characteristics of agronomical importance and three
chemical parameters of the fruit were selected for characterizing accessions. The intra-accession variability recorded for the traits selected made
them suitable for identifying cultivars. All of the traits but one were capable of showing up differences between accessions at a significance level of
0.001. Principal Component Analysis (PCA) showed that the traits which yielded the maximum separation between accessions were: leaf blade
form factor, angle of the fifth vein of leaves, upper angle of leaves, area of the cross-section of petals, sepal maximum projected area, weight of
fruits, total soluble solids in fruits, resistance of the skin of fruits to a penetrometer and width-to-length ratio of the maximum projected area of
seeds. Consequently, all of these characteristics may be of interest as descriptors for cherimoya varieties. Furthermore, four consistent groups of
accessions were defined by the three-dimensional plot obtained through projecting the accessions onto the first three principal components and the
tree-diagram, or dendrogram, obtained through Cluster Analysis (CA). Two of these groups were made up of accessions with a well-defined
common origin. The first consisted of selections S-196, S-256, S-260 and S-266 obtained from Coatepec Harinas in Mexico State, while the second
comprised selections S-Selene and S-Carapan from Purépecha native Indian communities in Michoacán State. For the other two groups the origin
of the accessions forming them is not fully known. Hence, further studies, based on molecular markers, might be carried out in order to ascertain if
these accessions are genetically related.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Cherimoya germplasm; Multivariate analysis; Principal component analysis; Cluster analysis; Diversity analysis; Descriptors; Phenotypic
characterization; Cultivar identification; Genetic resources for fruit
1. Introduction
The cherimoya (Annona cherimola Mill.) is a subtropical
fruit tree, indigenous to Andean South America (Lee and
Ellstrand, 1987), most probably originating in Ecuador and the
neighbouring part of Peru (DeCandolle cited by Popenoe
* Corresponding author. Tel.: +34 987291833; fax: +34 987291810.
E-mail address: [email protected] (F. González-Andrés).
0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2005.11.003
(1921)). Nevertheless, the presence of the fruit in Mexico and
Central America has led botanists to assume that it might also
be indigenous to the latter countries (Popenoe, 1921).
As for most of cultivated fruit–tree species, the identification
of cherimoya cultivars is convoluted. Sometimes more than one
genotype has been accidentally given the same cultivar name.
Conversely, some cultivars have several synonymous names.
The problem of identifying them is worsened by variation
within cultivars, even when their name represents a single
genotype (Ellstrand, 1997). In addition, the parentage of most
338
J. Andrés-Agustı́n et al. / Scientia Horticulturae 107 (2006) 337–346
cultivars is unknown and their phylogenetic relationships are
not clear (Rahman et al., 1998). Moreover, the diversity of
cherimoya germplasm, estimated from genetic parameters, is
high in comparison with the average diversity of cultivated
plants (Pascual et al., 1993b). The germplasm diversity in a
cultivated crop depends on the pollination system, the method
of propagation and the process of domestication. The mating
system of cherimoya exhibits the phenomenon of protogynous
dichogamy, a condition in which the receptivity of stigmas
occurs prior to the release of pollen from a given flower. This
increases diversity within natural populations because of cross
mating (Ellstrand, 1997; George, 1984). A very large number of
cherimoya cultivars trace their origin to open pollination
populations (Richardson and Anderson, 1996) and have
thereafter been sexually and asexually propagated. Furthermore, very few breeding programmes have been carried out on
the cherimoya (Pascual et al., 1993b; Andrés-Agustı́n, 1997),
with the development of cultivars based chiefly on selection.
One particular problem is that classification of cherimoya
cultivars has traditionally been based on characteristics of the
surface of the fruit. These are in accordance with five botanical
varieties, smooth, fingerprint, tuberculate, mamillate and
umbonate, recorded long ago (Irazazabal, 1985), together with
other pomological characteristics (Thomson, 1970). However,
the surface and shape may vary substantially from fruit to fruit
even on the same tree (Ellstrand and Lee, 1987). Thus,
classification of cherimoya cultivars on the basis of skin type
has been difficult (Perfectti et al., 1993; Rahman et al., 1998). In
the light of this, there is a need for a reliable classification of
cherimoya cultivars. Several approaches have been tried in
recent years. These have involved isoenzymatic (Ellstrand and
Lee, 1987; Perfectti et al., 1993; Pascual et al., 1993a,b;
Perfectti and Pascual, 1998) or DNA-based molecular
characterization: AFLP (Rahman et al., 1998), PCR-RFLP
(Rahman et al., 1997), and RAPD (Ronning et al., 1995).
Isoenzymatic and molecular characterization have the advantage that they are expressed in young material, making cultivar
identification easy and reliable (Ellstrand and Lee, 1987).
However, morphological characters are the most evident
features. Hence, they are the basis for the description and
identification of cultivars (Perrier, 1998), with crop descriptors
mainly based upon morphology (i.e. IPGRI, 2004). In spite of
the increasing interest in biochemical and molecular techniques
for characterizing and identifying cultivars (Christie, 2001), it
remains crucial to have adequate morphological traits.
Morphological features have well-known problems for germplasm characterization. When used for assessing variability,
they may lead to systematic errors. Several factors explain this.
Chief among them are the polygenic inheritance of morphological features, their generally unknown hereditability
(Perrier, 1998) and the influence of domestication on some
of the morphological and agronomical parameters, which is a
generalized phenomenon in cultivated species (Casas et al.,
1999). In order to overcome these problems, morphological
traits must fulfil certain requirements in order to be adequate for
characterization (Perrier, 1998; González-Andrés, 2001). In
principle, the set of traits must be as extensive as possible and
drawn from various different organs of the plant. Other major
considerations are (i) objectivity: qualitative characteristics
need to have unambiguous and objective alternative expressions, whereas, quantitative characters derived from measurements are more objective in themselves; (ii) consistency: it is
preferable for traits to be related to organs either unaffected or
only slightly affected by selection and the environment. For
metrical (or measurement-based) traits, ratios between measurements remain stabler than absolute measurements. In the
present work morphometric characteristics of several organs of
the adult plant (leaf, flower, fruit and seed) have been used,
together with several agronomical and chemical traits of fruits,
in order to characterize four commercial cherimoya cultivars,
one of them obtained and registered in Mexico, and seven
Mexican selections. The objectives were (i) to look for an
alternative approach to the definition of cherimoya cultivars
through multivariate analysis using commercial varieties and
Mexican germplasm selections, and (ii) to explain the grouping
of cultivars and selections obtained from multivariate analysis
on the basis of their origin and geographical distribution.
2. Materials and methods
The cherimoya cultivars and selections under investigation
(Table 1) were three foreign cultivars widely cultivated in
Mexico, one cultivar obtained and registered in Mexico in 2003
and seven germplasm selections developed in this same
country. Mexican germplasm came from landraces. The plant
material, leaves, flowers, fruits and seeds, was obtained in part
from open-air experimental plots with a randomized complete
block design at the Fundación Salvador Sánchez Colı́n
CICTAMEX, S.C., in Coatepec Harinas in Mexico State. This
is located at latitude 188460 3800 N, longitude 998460 3800 W and at
a height of 2240 m above sea level. It has an average annual
rainfall of 1100 mm and an average temperature of 16 8C.
Other locations yielding material were Jujacato, Tingambato
and Carapan in Michoacán State, Mexico, lying at latitudes
198260 –198510 N, longitudes 1018490 –1028220 W and heights of
1900–2198 m above sea level. The average rainfall for these
sites was between 1300 and 1800 mm yearly and their average
temperature 19.9 8C.
Plant material was collected in 2002 from adult plants 3
years after grafting. Ten different plants were selected per
accession and for each specimen one single leaf and flower, plus
three fruits, were selected on the basis of the criteria given
below. For leaves, a healthy and fully developed organ located
between the eighth and ninth node was collected. This was
scanned and the digital photograph used for morphometric
measurements. One healthy fully developed flower was
randomly selected per plant. It was dissected into the following
parts: petals, receptacle, pedicle and sepals. The polar
projection (maximum projected area) of each of these pieces,
plus the cross-section of the petal, were scanned and the digital
photograph used for measurements. For fruits, three healthy
well-developed fruits were collected per specimen at the
ripening stage, from three different places on the plant: upper,
intermediate and lower. After collection they were weighed,
J. Andrés-Agustı́n et al. / Scientia Horticulturae 107 (2006) 337–346
339
Table 1
Selections and cultivars of cherimoya (Annona cherimola Mill.) studied
Accession
Typea
County of registration for
cultivars and reference
for selections
Origin
Localization of the
experimental plot
in Mexico
Fruits surface
characteristics
Campas
Burtons
White
S-196
S-256
S-260
S-266
S-9651
Cortés II 31
S-Selene
S-Carapan
CC
CC
CC
GS
GS
GS
GS
GS
CC
GS
GS
Spain
New Zealand
USA
Nicolás-Cruz et
Nicolás-Cruz et
Nicolás-Cruz et
Nicolás-Cruz et
Andrés-Agustı́n
Mexico
Andrés-Agustı́n
Andrés-Agustı́n
Unknown
Unknown
Unknown
Mexico: Coatepec Harinas, Mexico
Mexico: Coatepec Harinas, Mexico
Mexico: Coatepec Harinas, Mexico
Mexico: Coatepec Harinas, Mexico
Mexico: Tingambato, Michoacan
Mexico: Tancı́taro, Michoacan
Mexico: Tingambato, Michoacan
Mexico: Carapan, Michoacan
CICTAMEXb
CICTAMEX
CICTAMEX
CICTAMEX
CICTAMEX
CICTAMEX
CICTAMEX
CICTAMEX
Jujacato, Michoacan
Tingambato, Michoacan
Carapan, Michoacan
Umbonate
Umbonate
Fingerprint-tuberculate
Mamillate
Smooth
Mamillate
Umbonate
Umbonate
Mamillate
Fingerprint
Fingerprint
a
b
al. (1996)
al. (1996)
al. (1996)
al. (1996)
(1997)
(1997)
(1997)
CC: Commercial cultivar; GS: germplasm selection.
Fundación Salvador Sánchez Colı́n-CICTAMEX. Coatepec Harinas, Mexico.
morphometric measurements were performed with a vernier
calliper and skin resistance was estimated with a multipurpose
penetrometer. Skin, pulp and seeds were then separated, the two
former were weighed and the seeds were counted. Total soluble
solids in the juice were estimated in terms of Brix degrees with
a refractometer and the pH of the pulp was measured with a pHmeter. To determine the malic acid content (milliequivalent per
100 g), juice was extracted, appropriately diluted with distilled
water, then titrated with 0.1 N NaOH solution, phenolphthalein
being used as an indicator. Twenty seeds were randomly
selected from each accession. They were scanned and the
digital photograph used for measurements. The traits scored
(Table 2 and Fig. 1) were selected in accordance with the
general recommendations of Sneath and Sokal (1973), Perrier
Table 2
List of characters evaluated
Organ
Variable
Abbreviation
2
Leaf
Blade area (cm )
Blade form factora
Petiole length (cm)
Fifth vein angle (8)
Upper angle (8)
Width length1 of the blade
Number of secondary veins in the right side of the blade
LA
LFF
LPL
LFVA
LUA
LW/L
LNV
Flower
Petal maximum projected area (cm2)
Width length1 of the petal maximum projected area
Area of the petal cross-section (cm2)
Form factor of the petal cross-sectiona
Receptacle maximum projected area (cm2)
Form factor of the receptacle maximum projected areaa
Pedicele length (mm)
Pedicele diameter (mm)
Sepal maximum projected area (cm2)
PA
PW/L
PCS
PCSFF
RA
RFF
PdL
PdD
SpA
Fruit
Length of the polar axis (cm)
Length of the polar axis (length of the ecuatorial axis)1
Fruit weight (g)
Skin weight (g)
Pulp percentage as: pulp weight (fruit weight)1 100 (%)
Seed index: number of seeds (fruit weight)1 100
Skin resistance to penetrometer (kg cm2)
Total soluble solids (8Brix)
pH of the pulp
Malic acid content (meq 100 g1)
FPA
FPA/EA
FW
FSW
FPP
FSI
FSR
FTSS
FpH
FMA
Seed
Width length1 of the seed maximum projected area
Maximum projected area form factora
Weight of a single seed obtained from weight of 100 seeds (g)
SW/L
SFF
SW
a
[(4 p area) (perimeter)2].
340
J. Andrés-Agustı́n et al. / Scientia Horticulturae 107 (2006) 337–346
Fig. 1. Characters measured in leaves, flowers and seeds of cherimoya (Annona cherimola Mill.). Character codes according to Table 2.
(1998) and González-Andrés (2001) concerning independence,
objectivity and consistency. All the measurements in digital
photographs were obtained with the image analysis software
Image Tool 3.00 (UTHSCSA, 2000).
Prior to the multivariate analysis of results, some
preliminary statistical analyses were performed. In order to
estimate diversity within accessions, the coefficient of variation
(CV, as a percentage), defined as the standard deviation mean
value1 100, was calculated for each trait in each accession.
Moreover, ANOVA was carried out for each character, so as to
detect which traits showed up significant differences between
accessions. For the multivariate analysis, a data matrix was
prepared with mean values (Table 3) for every accession. Two
different multivariate analyses were carried out: Principal
Component Analysis (PCA) and Cluster Analysis (CA). The
data matrix was standardized by subtracting the mean value of
each trait and dividing the result by the standard deviation. For
the PCA, a similarity matrix among characteristics was
calculated based on the Pearson product-moment correlation.
From this correlation matrix eigenvalue and eigenvector
matrices were obtained, the accessions then being projected
onto the first three principal components. For CA, the similarity
matrix among accessions was calculated using the average
taxonomic distance. The unweighted pair-group method
arithmetic average (UPGMA) was used for clustering. The
numerical analyses were carried out with the assistance of the
software package NTSYS-pc version 2.1 (Rholf, 2000).
3. Results
Table 3 shows the average values obtained after measurement of the parameters listed above in ten leaves and flowers,
30 fruits and 20 seeds per accession. The mean coefficient of
variation (CV) for each characteristic (the mean of the CV for
each of the 11 accessions studied) ranged from 2.5% for the pH
of the fruit pulp (FpH) to 25.3% for the ratio between the length
of the polar axis and length of the equatorial axis in fruits (FPA/
EA). The resistance of the skin of fruits to a penetrometer (FSR)
showed a CV of 20.1%, the seed index (FSI) scored 17.3%,
while the remainder of the traits showed values under 15%.
Twenty-one out of the 29 traits showed a CV below 12% and 14
of less than 10%. The mean CV for each accession (the mean of
the CVs for each of the 29 characteristics measured) ranged
from 8.9% to 14.6%. Selections S-196, S-256, S-260 S-256, SSelene and S-Carapan showed the lowest intra-accession
variability, with a mean CVof under 10%. Conversely, selection
S-9651 and the commercial cultivars showed the highest values
for mean CV. For all traits other than the form factor of the
maximum projected area of the flower receptacle (RA), there
were significant differences between accessions at a significance level of 0.001 (ANOVA analysis, data not shown). The
PCA (Tables 4 and 5 and Fig. 2) showed that the first three
components accounted for 25.33%, 22.97% and 16.59% of the
variance respectively, their cumulative variance being 64.91%.
On the basis of the eigenvector values for traits along the first
three components (Table 4), the three attributes responsible for
maximum separation along the first component (with values in
parentheses) were the maximum projected area of the sepal,
SpA (0.905), total soluble solids in fruit, FTSS (0.878), and
weight of fruits, FW (0.705). Along the second component they
were the width-to-length ratio for seeds, SW/L (0.793), the area
of the petal cross-section, PCS (0.764), and the angle of the
fifth vein of the leaf, LFVA (0.759), while along the third
component they were the upper angle of leaf, LUA (0.808),
Table 3
Mean values for characters scored in leaves flowers, fruits and seeds of cherimoya (Annona cherimola Mill.)
PW/L
PCS
PCSFF
RA
RFF
PdL
PdD
SpA
Seeds
Fruits
FSW
FPP
FSI
FSR
FTSS
FpH
FMA
SW/L
SFF
SW
0.76
0.68
0.68
0.70
0.74
0.73
0.78
0.71
0.74
1.77
2.13
1.66
1.49
1.76
1.38
1.51
2.06
1.91
67.60
55.86
64.27
66.53
68.97
63.88
66.60
59.13
65.12
92.98
85.19
87.02
99.62
105.25
94.92
105.58
83.65
99.87
0.59
0.58
0.66
0.66
0.69
0.62
0.70
0.64
0.59
13
13
14
14
13
15
13
13
14
2.51
1.05
1.58
1.55
1.25
2.21
1.68
1.35
2.02
0.29
0.29
0.25
0.29
0.33
0.25
0.28
0.30
0.25
23.5
8.1
11.1
17.6
14.6
21.7
16.7
10.8
19.3
0.54
0.69
0.58
0.67
0.69
0.66
0.69
0.69
0.69
0.24
0.17
0.21
0.23
0.21
0.22
0.26
0.20
0.22
0.63
0.70
0.79
0.76
0.73
0.73
0.65
0.77
0.66
1.38
1.00
1.32
1.02
1.13
0.92
1.08
1.06
1.60
0.25
0.19
0.22
0.26
0.21
0.24
0.22
0.23
0.27
0.65
0.35
0.42
0.37
0.34
0.45
0.39
0.31
0.51
14.5
12.5
12.5
11.7
12.5
11.2
14.0
12.6
14.3
1.21
1.20
1.21
1.10
1.17
1.04
1.19
1.29
1.38
1002.50
664.40
672.10
614.60
648.00
634.60
823.00
508.30
695.30
120.0
120.0
94.3
140.0
109.0
144.0
140.0
81.3
63.9
85.41
76.95
80.63
68.27
73.38
71.40
74.22
75.74
87.24
4.04
7.97
3.94
8.64
12.56
9.45
13.99
14.04
5.05
3.15
3.34
4.67
7.04
6.11
3.51
3.44
3.80
4.20
21.82
12.19
18.20
18.70
18.00
17.15
24.76
18.10
17.67
5.11
4.82
4.76
5.18
4.78
5.12
4.78
4.68
5.28
0.20
0.20
0.19
0.20
0.20
0.20
0.20
0.21
0.19
0.62
0.54
0.63
0.60
0.56
0.53
0.54
0.54
0.62
0.69
0.68
0.71
0.67
0.58
0.65
0.67
0.68
0.71
0.43
0.37
0.49
0.81
0.58
0.49
0.48
0.45
0.53
174.87
154.17
0.77
0.78
2.53
1.98
62.12
54.50
99.68
98.27
0.59
0.60
15
14
1.45
1.63
0.24
0.28
9.3
10.8
0.69
0.75
0.33
0.35
0.77
0.79
0.92
1.20
0.28
0.27
0.44
0.54
12.5
12.3
1.07
1.13
807.40
700.10
71.1
80.7
87.11
83.00
7.2
9.39
0.53
0.99
19.20
26.40
4.92
4.88
0.25
0.34
0.54
0.54
0.60
0.65
0.47
0.45
Character codes according to Table 2.
341
Fig. 3. Dendrogram obtained after cluster analysis of leaves, flowers, fruits and
seeds of cherimoya (Annona cherimola Mill.) (UPGMA method).
leaf blade form factor, LFF (0.709), and resistance of the fruit
skin to a penetrometer, FSR (0.619). The three-dimensional
grouping obtained after projection of the accessions onto the
first three principal components (Fig. 2) defined four groups.
The first was composed of cultivar Campas and cultivar CortesII-31, the second group, of the germplasm selections S-196, S256, S-260 and S-266. The third comprised the Burtons cultivar
together with selection S-9653 and the fourth and last was made
up of selections S-Selene and S-Carapan. The White cultivar
showed an intermediate position between the second and the
third group. On the plane defined by the first two principal
components, White was closer to the second group. However, it
differed from the rest of the accessions in this group on the basis
of the third axis, consisting mainly of the upper angle of the leaf
and the leaf blade form factor traits.
The dendrogram obtained after CA (Fig. 3) defined four
groups of accessions at a dissimilarity level of 1.23. These
groups were consistent with the groups defined in the PCA
analysis and White was included in the third group. Table 5
shows the mean values for every character in each of the four
groups. Group 1 (Campas and Cortes-II-31) had the highest
values for maximum projected area of petals (PA), area of the
petal cross-section (PCS), pedicle length (PdL), maximum
projected area of sepals (SpA), weight of fruits (FW), pulp
percentage (FPP), length of the polar axis of fruits (FPA), the
Fig. 2. Scatter diagram obtained from the first three principal components after
Principal Component Analysis (PCA) of leaves, flowers, fruits and seeds of
cherimoya (Annona cherimola Mill.).
J. Andrés-Agustı́n et al. / Scientia Horticulturae 107 (2006) 337–346
128.19
153.11
191.83
104.80
103.46
88.10
115.41
117.64
148.96
Campas
Burtons
White
S-196
S-256
S-260
S-266
S-9651
Cortés
II 31
S-Selene
S-Carapan
PA
FW
Sepal
FPA/EA
Pedicel
FPA
Receptacle
Petal
LNV
LW/L
LUA
FFVA
LPL
LFF
LA
Flowers
Leaves
Accession
342
J. Andrés-Agustı́n et al. / Scientia Horticulturae 107 (2006) 337–346
Table 4
Eigenvector values on the first three axes after Principal Component Analysis (PCA) of leaves, flowers, fruits and seeds of cherimoya (Annona cherimola Mill.) data
Organ
Character
Code
1st Component
2nd Component
3rd Component
Leaf
Blade area
Blade form factora
Petiole length
Fifth vein angle
Upper angle
Width length1 of the blade
Number of veins in the right side of the blade
LA
LFF
LPL
FFVA
LUA
LW/L
LNV
0.475
0.493
0.263
0.013
0.088
0.588
0.168
0.432
0.244
0.713
0.759
0.083
0.338
0.392
0.564
0.709
0.359
0.368
0.808
0.330
0.200
Flower
Petal maximum projected area
Width length1 of the petal maximum projected area
Area of the petal cross-section
Form factor of the petal cross-sectiona
Receptacle maximum projected area
Form factor of the receptacle maximum projected area
Pedicele length
Pedicele diameter
Sepal maximum projected area
PA
PW/L
PCS
PCSFF
RA
RFF
PdL
PdD
SpA
0.664
0.552
0.307
0.314
0.477
0.351
0.640
0.609
0.905
0.495
0.198
0.764
0.634
0.619
0.628
0.427
0.298
0.139
0.320
0.078
0.452
0.251
0.563
0.082
0.297
0.434
0.194
Fruit
Length of the polar axis
Length of the polar axis (length of the ecuatorial axis)1
Fruit weight
Skin weight
Pulp percentage as: pulp weight (fruit weight)1 100
Seed index: number of seeds (fruit weight)1 100
Skin resistance to penetrometer
Total soluble solids
pH of the pulp
Malic acid content
FPA
FPA/EA
FW
FSW
FPP
FSI
FSR
FTSS
FpH
FMA
0.543
0.613
0.705
0.686
0.446
0.687
0.392
0.878
0.242
0.477
0.715
0.391
0.208
0.199
0.521
0.199
0.145
0.283
0.318
0.419
0.052
0.125
0.260
0.374
0.383
0.374
0.619
0.272
0.585
0.268
Seed
Width length1 of the seed maximum projected area
Maximum projected area form factora
Weight of a single seed obtained from weight of 100 seeds
SW/L
SFF
SW
0.268
0.464
0.312
0.793
0.613
0.438
0.310
0.333
0.551
a
[(4 p area) (perimeter)2].
ratio between the length of the polar axis and length of the
equatorial axis in fruits (FPA/EA), fruit pulp pH, the width-tolength ratio of the maximum projected area of seeds (SW/L)
and the form factor of the maximum projected area of seeds
(SFF). In contrast, Group 1 presented the lowest values for the
width-to-length ratio of the leaf blade (LW/L), the form factor
of the petal cross-section (PCSFF), the form factor of the
maximum projected area of receptacles (RFF), the seed index
(FSI) and malic acid content (FMA). Group 2 (selections S-266,
S-256, S-260 and S-196) showed the highest values for the
following characteristics: angle of the fifth vein of the leaf
(LFVA), upper angle of leaf (LUA), width-to-length ratio of the
leaf blade (LW/L), width-to-length ratio of the maximum
projected area of petals (PW/L), seed index (FSI), fruit skin
weight (FSW), fruit skin resistance to penetrometer (FSR) and
seed weight (SW). The traits that yielded the lowest values for
Group 2 were: leaf blade area (LA), petiole length (LPL),
pedicle length (PdL), fruit pulp percentage (FPP) and length of
the polar axis (FPA). Group 3 (the Burtons and White cultivars
and selection S-9651) did not show maximum values for any of
the characteristics, but conversely showed the lowest values for
leaf blade form factor (LFF), upper angle of leaf (LUA),
maximum projected area of petals (PA), area of the petal crosssection (PCS), maximum projected area of receptacles (RA),
pedicle diameter (PdD), maximum projected area of sepals
(SpA), fruit weight (FW), total soluble solids in fruit pulp
(FTSS), pulp pH and seed weight (SW). Group 4 (selections SSelene and S-Carapan) showed the highest values for leaf blade
area (LA), leaf blade form factor (LFF), petiole length (LPL),
number of veins on the right-hand side of the leaf blade (LNV),
form factor of the petal cross-section (PCSFF), maximum
projected area of receptacles (RA), form factor of the maximum
projected area of receptacles (RFF), pedicle diameter (PdD),
total soluble solids in fruit pulp (FTSS) and malic acid content
(FMA). Conversely it showed the lowest values for the angle of
the fifth vein of the leaf (LFVA), width-to-length ratio of the
maximum projected area of petals (PW/L), fruit skin weight
(FSW), fruit skin resistance to penetrometer (FSR), the ratio
between the length of the polar axis and the length of the
equatorial axis of fruits (FPA/EA), the width-to-length ratio of
the maximum projected area of seeds (SW/L) and the
maximum projected area form factor of seeds (SFF).
4. Discussion
The grouping obtained through multivariate analysis is not
consistent with the long-standing classification of cultivars into
five botanical varieties based on characteristics of the fruit
surface: smooth, fingerprint, tuberculate, mamillate and
umbonate (Popenoe, 1921). In the scatter diagram obtained
0.48
0.59
0.44
0.46
0.70
0.64
0.69
0.63
0.62
0.56
0.57
0.54
0.19
0.20
0.20
0.29
5.20
4.97
4.75
4.90
19.75
19.65
16.16
22.80
3.68
5.03
3.94
0.76
4.55
11.16
8.65
8.30
86.33
71.82
77.77
85.06
92.17
133.26
98.44
75.91
848.90
680.05
614.93
753.75
1.30
1.13
1.23
1.10
14.37
12.35
12.53
12.38
0.58
0.39
0.36
0.49
0.26
0.23
0.21
0.27
1.49
1.04
1.13
1.06
0.65
0.72
0.75
0.78
0.23
0.23
0.19
0.34
0.62
0.68
0.65
0.72
21.40
17.66
10.02
10.06
0.27
0.29
0.28
0.26
2.26
1.67
1.32
1.54
13.33
13.67
13.33
14.50
0.59
0.67
0.63
0.60
Character codes according to Table 2.
96.42
101.34
85.29
98.98
66.36
66.49
59.75
58.31
1.84
1.54
1.95
2.26
138.58
102.94
154.20
164.52
1
2
3
4
0.75
0.74
0.69
0.78
SW/L
FMA
FpH
FTSS
FSR
FSI
FPP
FSW
FW
FPA/EA
FPA
SpA
PdD
PdL
RFF
RA
PCSFF
PCS
PW/L
PA
LNV
LW/L
LUA
FFVA
LPL
LFF
LA
Group
according
to Fig. 3
Table 5
Mean values for every character, in each one of the groups of accessions defined after multivariate analysis (Figs. 2 and 3) of leaves, flowers, fruits and seeds of cherimoya (Annona cherimola Mill.)
SFF
SW
J. Andrés-Agustı́n et al. / Scientia Horticulturae 107 (2006) 337–346
343
after PCA analysis (Fig. 2) and in the dendrogram (Fig. 3),
Group 1 included one accession with umbonate skin and
another with mamillate skin, while Group 2 had two mamillate
accessions, one umbonate and one smooth. Group 3 had two
umbonate and one fingerprint-tuberculate, whereas Group 4
had two accessions with fingerprint skin. The grouping of
accessions obtained by other authors after isoenzymatic
(Perfectti et al., 1993) or DNA-based molecular characterization (Rahman et al., 1998) was similarly not consistent with
botanical varieties. A classification of cultivars based on
surface characteristics of the fruit has been extensively
criticized (Irazazabal, 1985; Ellstrand and Lee, 1987; Perfectti
et al., 1993; Rahman et al., 1998), because it is not natural and
because of the lack of consistency of these characteristics
within a cultivar or even a single plant. Currently, this approach
has been rejected (Scheldeman et al., 1999) in favour of a
classification based on multiple genetic and morphological
traits, as proposed in this work.
The characteristics chosen here fulfil the requirements
recommended by Perrier (1998) and González-Andrés (2001).
Firstly, they relate to several different organs of the plants:
seven to leaves, nine to flowers, 10 to fruits and three to seeds.
They are objective, since all of them are quantitative and are
determined by measurement. In respect of their consistency,
two points should be considered. The first is stability when
subjected to selection processes or environmental influences.
More than half of the morphometric characteristics measured
relate to flowers and seeds. In general, seeds (Eames, 1961) and
flowers (Eames, 1961; Sherry and Lord, 1996) are the stablest
organs within a given taxon, for example, in comparison with
leaves. This has been specifically reported for the cherimoya
(Perfectti and Camacho, 1999). The second important aspect
about consistency is intra-accession variability. In spite of the
great genetic variability noted in the cherimoya (Pascual et al.,
1993b), bringing with it considerable variation within cultivars
(Ellstrand, 1997), the intra-accession variability of the traits
studied makes them suitable for cultivar identification. A
possible exception would be of the trait of the ratio between the
length of the polar axis and the equatorial axis in fruits, as this
showed a mean variation coefficient of more than 25%. From a
general point of view coefficients of variation of 12% or lower
are acceptable in plant organ characterization. If this coefficient
is higher, then it would be advisable to increase the sample size.
According to Hidalgo (2003) a sample size of 25 should be
enough to keep experimental error below 10% when the
coefficient of variation is 25%. In this work, the sample size for
fruits, the organs with the highest coefficient of variation for
most of the traits, was 30.
On the basis of the ANOVA results, 28 of the characteristics
considered, in other words all except for the form factor of the
maximum projected area of the flower receptacle, may be
useful for identifying cultivars. However, PCA showed that the
traits, which yielded the maximum separation between the
accessions studied were three relating to leaves, two to flowers,
three to fruits and one to seeds. The leaf traits were leaf blade
form factor, angle of the fifth vein of leaves and upper angle
of leaves. For flowers, the characteristics were area of the
344
J. Andrés-Agustı́n et al. / Scientia Horticulturae 107 (2006) 337–346
cross-section of petals and maximum projected area of sepals.
In the case of fruits, the traits were weight of fruits, total soluble
solids in fruits and resistance of the skin of fruits to a
penetrometer. The seed trait was the width-to-length ratio of the
maximum projected area of seeds. Of these more discriminatory characteristics, leaf blade area and total soluble solids of
fruit pulp are the two most likely to be affected by the
environment. Cultivation of the material being characterized in
homogeneous conditions of soil and climate would reduce this
problem. Skin resistance to a penetrometer is the most variable
trait within accessions (with a mean coefficient of variation of
20.1%). In order to use this characteristic for identifying
cultivars, the number of fruits tested should be at least 16, in
order to keep the error of the sample below 10% (Hidalgo,
2003). For the remainder of the characters, the coefficients of
variation observed were at most 14.8% (weight of fruits), all the
others being less.
Eight out of the 28 traits in which ANOVA detected
significant differences between accessions are included in the
list of characteristics in the ‘‘Guidelines for the Conduct of
Tests for Distinctness, Uniformity and Stability’’ for the
cherimoya issued by the International Union for the Protection
of New Varieties of Plants or UPOV (UPOV, 2003). The
inclusion of the other characteristics into the UPOV’s guidelines, especially those classified as stronger discriminants in the
PCA analysis, might be proposed. The characteristics in the
UPOV guide are treated from a qualitative viewpoint, while in
the present work they have been treated from a quantitative
viewpoint. However, most of the UPOV traits are easily
transformable into quantitative ones. Utilization of an
inexpensive and easy-to-use modern image-analysis system,
for instance Image Tool, would simplify the data collection
process.
The grouping of accessions emerging from the work
reported here (Figs. 2 and 3) was based on a large number
of characteristics from several different organs. Except for the
well-known cultivars Campas, Burtons and White, the
accessions were local selections and a new Mexican cultivar,
which were being studied in depth for the first time. Pascual
et al. (1993a) and Perfectti et al. (1993) analyzed isoenzymes
from the cultivars Campas and White. The isoenzymatic results
showed a great distance between the two varieties, which is
consistent with the morphology-based results being reported in
this paper. Group 2 consisted of selections S-196, S-256, S-260
and S-266. These are outstanding genotypes selected from a
single segregated population of 420 individuals located at the
Fundación Salvador Sánchez Colı́n CICTAMEX, S.C., situated
at Coatepec Harinas, in the State of Mexico (Nicolás-Cruz
et al., 1996). This population was grown from the seeds of fruits
collected in the area surrounding Coatepec Harinas. These four
genotypes were selected on the basis of local farmers’ criteria
concerning fruit size and productivity (Rubı́-Arriaga et al.,
1992; Nicolás-Cruz et al., 1996). The main fruit characteristics
of this group contrasting with the other groups (Table 5) are a
high seed index, low pulp content and a relatively resistant skin.
The first two of these characteristics are not much liked by
consumers.
Group 3 consisted of the cultivars Burtons and White and
selection S-9651. This latter was a garden tree grown from seed.
It was picked out in Tingambato, a community located in the
Michoacán State of Mexico. White is a cultivar bred and
registered in the United States of America, while Burtons is a
New Zealand cultivar. One possible hypothesis to explain this
grouping would be that the three accessions have some
common origin. The grounds for proposing such a hypothesis
would be that the cherimoya was introduced into the United
States from Mexico by Judge Robert B. Ord in 1871, with some
of the finest selections, such as White, being sent to New
Zealand and other countries (Schroeder, 1997). However, S9651 has the same geographical origin as S-Selene, but S-9651
is clearly different from S-Selene. It would be possible to use
microsatellite data (Sefc et al., 2000) to discover whether the
morphological, agronomical and chemical similarities of
Burtons, White and S-9651 are due to a genetic relationship.
If that were so, two alternative hypotheses could be considered.
The first would be that White and Burtons came from Mexico
and thus had the same origin as selection S-9651. The
alternative hypothesis would be that S-9651 was introduced
into Mexico from White or Burtons material. From the point of
view of traits of the fruits, this group of accessions showed the
lowest fruit pulp pH and total soluble solid content. Moreover,
they have small flowers, which was reported by Richardson and
Anderson (1996) as hindering hand pollination of the Burtons
cultivar.
The selections S-Selene and S-Carapan, both in Group 4,
come from the Purépecha ethnic communities of Tingambato
and Carapan in Michoacán State, these places being located at a
distance of 30 km one from the other. Both selections are
garden trees. On the basis of their phenotypical similarity and
the strong cultural links between the two communities, the
material could be related. From point of view of fruit
characteristics, these two selections showed high total soluble
solids and malic acid content, which is a positive feature for
taste, and they are highly appreciated by consumers. However,
they have the disadvantage of having the least resistant skin on
their fruits, which leads to difficulties with handling after
harvesting.
The cultivar Cortes-II-31 comes from Michoacán State, like
S-Selene and S-Carapan, but multivariate analysis located it in
Group 1, alongside the Campas cultivar, grown in Spain. The
separation between S-Selene and S-Carapan, on the one hand,
and Cortés-II-31, on the other, that can be observed in the
scatter diagram from PCA is based on the second and third
principal components. The characteristics responsible for the
maximum separation along these components were the widthto-length ratio of the maximum projected area of seeds, the
petal cross-section, the angle of the fifth vein of the leaf, the
upper angle of the leaf, the leaf blade form factor and the
resistance to a penetrometer of the skin of the fruit. Differences
are very clear in petal cross-section, in skin resistance to the
penetrometer and in the width-to-length ratio of the maximum
projected area of seeds, these being much greater in CortesII-31 than in S-Selene and S-Carapan. Although the
three accessions come from Michoacán state, S-Selene and
J. Andrés-Agustı́n et al. / Scientia Horticulturae 107 (2006) 337–346
S-Carapan are from the Purépecha indigenous communities,
whereas, the cultivar Cortes-II-31 comes from Tancı́taro, which
is not an indigenous community but mixed race, with more
outside influences. With respect to the similarity between
Cortés-II-31 and Campas, since the parentage of Campas is
unknown, molecular markers might help ascertain whether
there is a relationship between the two genotypes. Both
cultivars share the fruit traits of high pulp percentage and pH
content, with a low malic acid content.
In conclusion, 20 of the 21 morphometric characteristics
chosen from several different organs of the plants, together with
the five fruit traits of agronomical importance and the three
chemical parameters for fruits, were all found useful for
classification of cherimoya germplasm. The characteristics
yielding the maximum separation between accessions were
three relating to leaves, two to flowers, three to fruits and one to
seeds. Consequently, all these traits might be of interest as
cherimoya descriptors, so that their inclusion in UPOV might
be considered. Moreover, four consistent groups of accessions
were defined. Two of them were made up of accessions with a
well-defined common origin. The origin of the accessions in the
other two groups is unknown, hence further DNA-based studies
might be carried out in the future in order to ascertain the
reasons for this grouping.
Acknowledgements
This work was financially supported by the Servicio Nacional
de Inspección y Certificación de Semillas (SNICS), the Consejo
Nacional de Ciencia y Tecnologı́a (CONACYT), the Centro de
Investigación y Desarrollo del Estado de Michoacán (CIDEM)
and the Dirección General de Investigación y Posgrado of the
Universidad Autónoma Chapingo in Mexico.
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