Preliminary Study of Diversity of Plukenetia volubilis Based on the Morphological and Genetic Characteristics

Abstract

The aim of this study was to make an initial step in clarification of morphological diversity of sacha inchi (Plukenetia volubilis L.), a traditional oilseed crop, together with use of ISSR primers for a preliminary assessment of genetic variability in the species. Quantitave and qualitative morphological data together with leaf samples for twenty plant individuals of sacha inchi from five distinct populations were gathered in three different communities neighbouring the city of Pucallpa in Peru. Morphological data were processed into PCA analysis graph while for DNA analysis the method of fingerprinting processed into PCO graph and dendrogram was used. Visible morphological dissimilarities in the plant appearance were confirmed and observable differences in phenotypical characteristics among different types of associated cultivations were recorded. For genetic variability twelve from eighteen ISSR primers used and tested in the study could be used directly for further molecular pursuit on this species. Dendrogram with six main clusters was created and thus confirmed polymorphism as allogamy plants shows. Obtained results contributed to the identification of suitable method for further assessment of genetic variability within this species. New horizons for the focus of further research and improvements in onward development and genetic conservation of sacha inchi cultivation were identified, thus linking the observation of morphological features with DNA analysis proved as effective.

Full text

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DOI: 10.2478/v10295-012-0023-6 AGRICULTURA TROPICA ET SUBTROPICA, 45/3, 140-146, 2012
INTRODUCTION
Highly nutritious traditional food crop of the Peruvian
Amazon, sacha inchi (Plukenetia volubilis L.), gained
world’s attention since the oil derived from the sacha
inchi seed won the gold medal at the “World Edible Oil”
competition in Paris in 2004 (Agroindustrias Amazónicas,
2006). This “peanut of the Incas”, or sacha inchi is a
native plant whose origins lie in the Peruvian Amazon
and its potential revenue from cultivation could aid poor
indigenous and mestizo communities to move out of
poverty and improve the diets in the same time (Hamaker
et al., 1992; Manco, 2005).
Sacha inchi is a wild, climbing, semiwoody, perennial
oleaginous plant of the Euphorbiaceae family that grows
in the tropical jungles of America at altitude of between
200 and 1 500 m (Gillespie, 1993; Arévalo, 1996). The
plant provides seeds of a lenticular shape, which are rich
in oil (49%) and proteins (33%) and contain heat-labile
substances with a bitter taste (Hamaker et al., 1992; Guillén
et al., 2003).
Although the composition and properties of sacha inchi
seeds are relatively well known, genetic improvement is
just at the beginning (Arévalo, 1996; Manco, 2005). To
our knowledge, no publication on this issue is available
until today (Juarez, 2007). However, genetic data could
provide useful information and bring details on taxonomy
description, whereas the actual taxonomy description shows
some ambiguous identication and is so far derived only
from morphological characteristics (Gillepsie 1993, 2007;
Rodriguez 2010). Knowledge of genetic relationship and
variability at inter- and intra-species level are important
tools for improvement of plant selection and necessary basis
for collection, conservation and evaluation of plant material
(Gupta, 2008).
Within a preview study in Euphorbiaceae family, the
assessment of genetic variability was done for the species
Jatropha curcas L. The Inter Simple Sequence Repeats
technique (ISSR) showed that polymorphic fragments
are established among different varieties where 4 groups
were designed (Gupta, 2008). For the genus of Plukenetia,
Corazon-Guivin et al. (2008) observed a great genetic
diversity among four populations due to their cross-
pollinated system. ISSR thus enabled to distinguish the
different species of genus Plukenetia (Rodrigez, 2010).
Nevertheless, more analyzes for particular species of
Plukenetia volubilis are needed.
The aim of this work was to make an initial step in
clarication of diversity of sacha inchi by use of ISSR
primers for a preliminary assessment of genetic variability
in this species together with evaluation of morphological
characteristics.
Original Research Article
Preliminary Study of Diversity of Plukenetia volubilis Based on the Morphological
and Genetic Characteristics
Blanka Krivankova1, Petra Hlasna Cepkova1, Martin Ocelak1, Gaelle Juton2, Miroslav Bechyne1,
Bohdan Lojka1
1
Department of Crop Sciences and Agroforestry, Institute of Tropics and Subtropics,
Czech University of Life Sciences Prague, Czech Republic
2
Agrocampus Ouest Centre d’Angers, Angers, France
Abstract
The aim of this study was to make an initial step in clarication of morphological diversity of sacha inchi (Plukenetia volubilis
L.), a traditional oilseed crop, together with use of ISSR primers for a preliminary assessment of genetic variability in the species.
Quantitave and qualitative morphological data together with leaf samples for twenty plant individuals of sacha inchi from ve
distinct populations were gathered in three different communities neighbouring the city of Pucallpa in Peru. Morphological data
were processed into PCA analysis graph while for DNA analysis the method of ngerprinting processed into PCO graph and
dendrogram was used. Visible morphological dissimilarities in the plant appearance were conrmed and observable differences
in phenotypical characteristics among different types of associated cultivations were recorded. For genetic variability twelve from
eighteen ISSR primers used and tested in the study could be used directly for further molecular pursuit on this species. Dendrogram
with six main clusters was created and thus conrmed polymorphism as allogamy plants shows. Obtained results contributed to
the identication of suitable method for further assessment of genetic variability within this species. New horizons for the focus of
further research and improvements in onward development and genetic conservation of sacha inchi cultivation were identied, thus
linking the observation of morphological features with DNA analysis proved as effective.
Keywords: diversity; ISSR markers; morphological characters; Peruvian Amazon; Plukenetia volubilis; sacha inchi.
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AGRICULTURA TROPICA ET SUBTROPICA VOL. 45 (3) 2012
MATERIALS AND METHODS
Sample and data collection
In the period from June 2008 until March 2010, the
data about twenty plant individuals of Plukenetia volubilis
were gathered from ve distinct plots in three different
communities neighbouring the city of Pucallpa in Peru.
Antonio Raimondi (25 km from Pucallpa), Tres de deciembre
(22 km of Pucallpa) and Pimental (35 km from Pucallpa).
Each plot comprised a population of deliberately grown
individuals in varying types of cultivations designed by
farmers based on their knowledge and nancial possibilities.
The random sampling method was used in choosing
particular individuals according to Kindt and Coe (2005)
and 4 plants in each plot were marked. The exact location,
number of evaluated plants and type of cultivation are
presented in Table 1.
Data about morphological characteristics and samples for
DNA analyses were collected from each individual plant.
For DNA extraction young leaves were collected and packed
in plastic tubes with silicagel.
Morphological traits of plants from two more locations
named Antonio Raimondi 4 (AR 4, latitude 08° 21’ 52.3“,
longitude 74° 42’ 21.2”, altitude 157 m AMSL, multiple
cropping) and Pimental 2 (PI 2, latitude 08° 31’ 43.4”,
longitude 74° 51‘ 22,6“, altitude 204 m AMSL, mixed
intercropping) were recorded and evaluated by statistical
analysis (described below). Nevertheless, material for DNA
extraction had not been collected from these locations
due to the unhealthy status of the plants at the time of leaf
collections.
Morphological description
As far as there is no P. volubilis descriptor available
up to now, the appropriate one was created according to
the guidelines “Developing crop descriptor list” from
Bioversity International (2007) using the data from botanical
description of Gillespie as mentioned in 1993. The scale of
possible variance was designed using the only materials of
P. volubilis morphology (Gillespie 1993; Gillespie 1994)
and modied according to own observation. Each evaluated
plant was sampled and conrmed to be P. volubilis and not
any other species.
From each plant, a phenotypic description was established
from the quantitative characteristics (height of a plant,
basal diameter, branching height, length of petiole, blade
length, blade width, leaf length, inorescence length, length
of stylar column, number of seeds per capsule, width of
capsule, diameter of seeds, weight of 10 seeds) and the
qualitative characteristics (crown shape, stem shape, stem
colour, stem structure, leaf shape, shape of leaf margins,
presence of leaf hair, colour of leaf, pointed top of leaf,
colour of inorescence, single glandular knob at petiole
apex, presence of smell, shape of capsule, colour of capsule,
seed shape, colour of seeds) as indicated by classication
of morphological traits by Bioversity International (2007).
Table 1: List of Plukenetia volubilis samples and locations of collection
Sample
number Location Sample code Latitude Longitude Altitude Type of cultivation
1 AR 1.1
2 Antonio Raimondi 1 AR 1.2 08° 22’ 27.8” 74° 43’57.4” 154 m AMSL mixed intercropping
3 AR 1.3
4 AR 1.4
5 AR 2.1
6 Antonio Raimondi 2 AR 2.2 08° 22‘ 01.4“ 74° 42’ 18.7” 155 m AMSL agroforestry
7 AR 2.3
8 AR 2.4
9 AR 3.1
10 Antonio Raimondi 3 AR 3.2 08° 21’ 57.7“ 74° 42’ 20.5” 157 m AMSL agroforestry
11 AR 3.3
12 AR 3.4
13 DE 1.1
14 Tres de deciembre DE 1.2 08° 23’ 02.7” 74° 41’ 26.7” 147 m AMSL monoculture
15 DE 1.3
16 DE 1.4
17 PI 1.1
18 Pimental 1 PI 1.2 08° 31’ 22.2” 74° 49‘ 19.6“ 211 m AMSL monoculture
19 PI 1.3
20 PI 1.4
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AGRICULTURA TROPICA ET SUBTROPICA VOL. 45 (3) 2012
All measurements were taken ten times and from this
value le an arithmetic mean was calculated to obtain the
most probable value of the measurement and to reduce a
measurement error.
DNA ngerprinting
Genomic DNA was isolated from plant material frozen
with liquid nitrogen with Invisorb®Spin Plant Mini Kit
(Stratec Molecular, Germany). Samples were placed in
liquid nitrogen and then grounded to a ne powder. From
these powders, the kit helped to process to a lysis, DNA
binding, two washings and then elution of the DNA.
A set of 18 ISSR primers (Integrate DNA Technologies
- IDT, USA) from the University of British Columbia
Biotechnology Laboratory (UBC Set#9) were used for
screening (Table 2). Polymerase chain reaction (PCR)
amplications were performed in a 20 μl reaction volume
containing, 2 μl of template DNA, 0.5 μl of primer (IDT,
USA), 10 μl PPP Master Mix (Top-Bio, Czech Repulic), 0.2
μl Bovine Serum Albumin (BSA, Fermentas, USA), 7.3μl
PCR H2O (Top-Bio, Czech Repulic). Amplications were
run in a Gradient Thermal Cycler (QB96 Server; Quanta
Biotech, United Kingdom). The PCR was carried out with
modications of the annealing temperature to optimize the
reaction for individual primers.
The cycling conditions were as follows: initial denaturation
step at 95 ºC for 4 min, followed by 45 cycles of denaturation
at 94 ºC for 30 s, primer annealing
at 45 ºC for 45 s, and extension at
72 ºC for 2 min, followed by a nal
extension at 72 ºC for 10 min.
Amplied products were mixed
with loading dye (ThermoScientic,
USA). Electrophoretic separation
was performed with 6 μl of
amplied products on 2% agarose
gel in 1× Tris-borate-EDTA (TBE)
buffer. The size (base pair - bp) of
most intensely amplied band for
each ISSR marker was determined
based on its migration relative to
molecular weight size 100 bp Plus
DNA ladder (ThermoScientic,
USA). Gels were run for about
2.5 – 3 h at 4 V.cm-1. Gels were
stained with SYBR® Safe DNA
Gel Stain (Stratec Molecular,
Germany) and visualized with a
UV transilluminator. Gel pictures
were recorded using the CSL-
MICRODOC System (CLEAVER,
United Kingdom).
Statistical analysis
The statistical analysis of the qualitative and quantitative
morphological characteristics was performed by Basic
Statistics within the software Statistica 7.0 CZ. The
variability among plants in the tested population was
represented by the Principal Component Analysis (PCA)
graphs (software Statistica 7.0 CZ).
Evaluation of ISSR proles was performed in duplicates
and those with stable and clear bands were analyzed. ISSR
fragments were scored as a presence (1) or absence (0) of
bands in the gel prole by statistical software Darwin 5.0
(Perrier and Jacquemoud-Collet, 2006). The dendrogram
was generated using the Neighbour-joining algorithm.
Genetic data were also projected by Principal Coordinates
Analysis (PCO) to the PCO graph according the searched
similarities between cases.
RESULTS
Score evaluation of morphological characters
Data collected for both quantitative and qualitative
characteristics were used to create the score plot by using
PCA (Fig. 1). The variability among tested samples reected
by PCA indicated the total morphological and phenological
variability of 58% accounted by the rst two axes.
Figure 1. Score plot of particular recorded plant samples (PCA graph, software
Statistica 7.0 CZ)
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AGRICULTURA TROPICA ET SUBTROPICA VOL. 45 (3) 2012
According to the score plot several interesting facts
became obvious. Only one plot showed the large uniformity
and formed close group, two other plots showed lower rate of
variation and formed more close groups, whereas the other
plots were more spread but very often following certain
level of factor 1 or factor 2 crossing the both quadrants.
The plot with the lowest rate of variation and high rate
of similarity among particular plants was the plot PI 1 with
monoculture cultivation from the location of Pimental.
Another two plots with a lower rate of variation and higher
rate of similarity were plots AR 1 and AR 4 both of intercrop
cultivation from different locations of Antonio Raimondi.
Plants from the AR 1 plot were localized in the same
quadrant as the samples of PI 1 close to the axe of factor 2
whereas the samples of AR 4 are crossing the both minus
and plus quadrants alongside the axe of factor 2.
Wider expansion and higher variation of single evaluated
plants was present on the plot AR 3 with agroforestry system
of cultivation in Antonio Raimondi where all the plants were
localized within one sector and largest group in the graph
was created.
The other situation was observed in the case of DE 1 plot
of monoculture cultivation from Tres de deciembre where
two of the samples showed almost any variation and were
localized on the similar spot while the other individuals
were in bigger distance and thus possessed greater variation
reaching wide expansion within the both sectors behind the
axe of factor 2. The widest expansion and high variation was
present among the samples from plot AR 2 with agroforestry
type of cultivation in Antonio Raimondi and PI 2 intercrop
cultivation of Pimental. Nevertheless all plants are spread
on the same level alongside the axe of factor 1 crossing from
one sector to another.
DNA analysis
All 18 tested primers provided well visible fragments,
12 primers with very clear and satisfactory intensity, good
reproducible and clear prole. The number of polymorphic
bands per locus ranged from 2 to 16 with an average of
7 bands (Table 2). The size of the amplication fragment
was 200-3000 bp. The highest number of bands was found
for primers UBC824 and UBC834 and in their prole
was observed the high level of polymorphism. Following
primers (UBC 807, UBC810, UBC835, UBC845, UBC846
and UBC847) produced more than eight bands which
were almost polymorphic. The presence of polymorphic
bands ranged between 50 to 100%, the average percentage
of polymorphic bands was 70% from these 12 primers.
Only six primers UBC809, UBC812, UBC813, UBC823,
UBC829 and UBC843 amplied less than ve band and the
bands did not show lower or any polymorphism.
Both PCO (Fig. 2) and cluster analysis grouped together
the tested samples according their similarity based on
their DNA patterns. The dendrogram calculated by using
Neighbour-joining method based on Unweighted Neighbour-
joining algorithm showed six main clusters (Fig. 3). Tested
samples showed lower or more signicant similarity in
frame of tested populations.
Cluster 1 contained three samples from population of
Table 2: List of primers, total number of bands per primer, percentage of polymorphic bands generated per primer
No. Primer Sequence 5´- 3´ Total number of bands Polymorphic fragments Polymorphic bands (%)
1 UBC807 AG AG AG AG AG AG AG AG T 12 8 67
2 UBC809 AG AG AG AG AG AG AG AG G 2 0 0
3 UBC810 GA GA GA GA GA GA GA GA T 8 4 50
4 UBC812 GA GA GA GA GA GA GA GA A 5 0 0
5 UBC813 CT CT CT CT CT CT CT CT T 2 0 0
6 UBC814 CT CT CT CT CT CT CT CT A 5 5 100
7 UBC823 TC TC TC TC TC TC TC TC C 4 0 0
8 UBC824 AG AG AG AG AG AG AG AG YT 16 14 87.5
9 UBC826 AC AC AC AC AC AC AC AC C 2 1 50
10 UBC829 TG TG TG TG TG TG TG TG C 3 0 0
11 UBC834 AG AG AG AG AG AG AG AG YT 16 16 100
12 UBC835 AG AG AG AG AG AG AG AG YC 14 14 100
13 UBC843 CT CT CT CT CT CT CT CT RA 5 0 0
14 UBC845 CT CT CT CT CT CT CT CT RG 8 8 100
15 UBC846 CA CA CA CA CA CA CA CA RT 7 7 100
16 UBC847 CA CA CA CA CA CA CA CA RC 9 7 78
17 UBC848 CA CA CA CA CA CA CA CA RG 5 5 100
18 UBC851 GT GT GT GT GT GT GT GT CT G 5 3 60
Mean 7 5 55
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AGRICULTURA TROPICA ET SUBTROPICA VOL. 45 (3) 2012
Pimental 1 location (PI 1.2, PI 1.3 and PI 1.1). Fourth sample
from area of Pimental PI 1.4 composed together with one
sample of population DE 1.4 and one from DE 1.3 Cluster 2.
Third cluster was presented by populations from two different
locations of Antonio Raimondi where two samples were from
location entitled Antonio Raimondi 1 (AR 1.4 and AR 1.3)
and one was from location named Antonio Raimondi 2 (AR
2.3). Cluster 4 was created mainly by samples collected in
the location of Antonio Raimondi 2 (AR 2.2, AR 2.1, AR
2.4) with subgroups of samples found at the site of Antonio
Raimondi 3 (AR 3.3 and AR 3.2). Two samples of location
Tres de deciembre DE 1.1 and DE 1.2 were included to the
subgroup of cluster 5 together with the sample AR 1.1 and
third samples of population Tres de deciembre DE 1.3. Last
cluster was formed by AR 3.4 and A R1.2.
DISCUSSION
According to Gillespie (1993) P. volubilis can be
distinguished from other species of Plukenetia by the adaxial
presence of single glandular knob at the petiole apex, length
of stylar column varying between 15-30 mm long and the
presence of 4 staminate sepals. We have thus conrmed the
presence of all mentioned characteristics, none of them was
in dispute; thus we made sure that all of the observed plants
called sacha inchi were P. volubilis and not any other.
According to Arévalo (1996), noticeable visible
dissimilarities of morphological traits among the
individual plants can be observed. Considering
the quantitative characteristics, our results were
in complete accordance with this statement.
Nevertheless, for particular clarication of this fact
based on DNA extraction further research would still
be required.
This fact is underlined by the finding mentioned by
Gillespie (1994) and Arévalo (1996) that the type of
pollination of P. volubilis has not been clearly stated
so far. According to the results arising from PCO
analysis and dendrogram noticeable genetic variability
was observed. Thus polymorphism as for allogamy
plants was identified. This finding is in agreement with
preliminary assumptions of other authors (e.g. Arévalo,
1996; Manco 2005) who presumed this fact on the basis
of visible dissimilarities.
According to Ward (2011), plant populations under
different environmental selection pressures generally
show phenotypic differences. Such phenotypic
differences could be the result of phenotypic plasticity
and/or genetic diversification. This fact was taken into
account. Nevertheless, there were no sufficient data
Figure 2. PCO graph of estimated samples of Plukenetia volubilis (software DARwin 5.0)
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AGRICULTURA TROPICA ET SUBTROPICA VOL. 45 (3) 2012
available that would help to orientate in assessing the
results. Thus further long-term research of this aspect
would be needed.
According to the distribution of evaluated plants in
the score plot large uniformity among particular plants
was observed within the plot cultivated as monoculture.
Signicant uniformity within the plots where there was
more space for the development of the plant or fewer plants
in associations was also recorded. There seems to be neither
literature available nor research conducted on this topic
but certain links to the diversity of sacha inchi in different
associations were observed.
Despite the fact that there have been no published results
of previous research conducted on the issue of DNA analysis
the preliminary analysis of the 18 ISSR primers showed
satisfactory results of visualized band. Lower score of
total average percentage of polymorphic bands (47%), was
reached due to six primers which showed very low level of
polymorphism. Thus using ISSR primers in P. volubilis seemed
to be suitable and adequately sensitive for polymorphism
detection. Relatively high percentage of polymorphic bands
(50-100%) indicated that ISSR markers were polymorphic
and informative, thus advisable for estimating genetic
relationships within this species in the future studies.
In this analysis of ISSR markers high level of diversity
was conrmed similarly to Corazón-Guivin et al. (2008) in
four populations of sacha inchi in which the polymorphism
was detected by the technique of direct amplication of
length of polymorpism (DALP).
Likewise the estimation of the genetic variability of P.
volubilis species by the ngerprinting method based on
using ISSR markers as already tested by Rodrigez (2010)
for genus of Plukenetia, showed to be applicable also for
polymorphism description of P. volubilis populations.
ACKNOWLEDGMENTS
The authors express their gratitude and thanks to all
participants of this research from Pimental, Antonio
Raimondi and Tres de deciembre villages who provided us
access to their sacha inchi plots.
The research in Peru was conducted in the framework of
the Czech Republic Development Cooperation in Peru No.
23/MZe/B/07-10 and was also supported by the Internal
Grant Agency of the Institute of Tropics and Subtropics,
Czech University of Life Sciences Prague (IGA ITS) by IGA
511110/1312/3105 and IGA 51110/1312/3115.
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Figure 3. The dendrogram with six main clusters of Plukenetia volubilis samples using Neighbour-joining algorithm based
on Unweighted Neighbour-joining (software DARwin 5.0)
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AGRICULTURA TROPICA ET SUBTROPICA VOL. 45 (3) 2012
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Received for publication: August 22, 2012
Accepted for publication: September 3, 2012
Corresponding author:
Blanka Krivankova
Czech University of Life Sciences Prague
Institute of Tropics and Subtropics
Kamýcká 129, 165 21 Prague 6
Czech Republic
Tel: 00420 776 868 468
E-mail: b.krivankova@email.cz
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