b i o m a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7 e1 5 5

Available online at www.sciencedirect.com

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Genetic structure of Jatropha curcas L. in Mexico and

probable centre of origin
Victor Pecina-Quintero a,*, Jose Luis Anaya-Lopez a,
Alfredo Zamarripa-Colmenero b, Carlos Alberto Nunez-Coln a,
Noe Montes-Garca c, Jose Luis Sols-Bonilla b,
Mara Fernanda Jimenez-Becerril d
a

INIFAP-Campo Experimental Bajo, Carr. Celaya-San Miguel de Allende km 6.5, C.P. 38010 Celaya,
Guanajuato, Mexico
b
INIFAP-Campo Experimental Rosario Izapa, Tuxtla Chico, Chiapas, Mexico
c
INIFAP-Campo Experimental Ro Bravo, Cd. Ro Bravo, Tamaulipas, Mexico
d
Centro de Biotecnologa Genomica del Instituto Politecnico Nacional, Cd. Reynosa, Tamaulipas, Mexico

article info

abstract

Article history:

The understanding of the genetic diversity and structure of populations of Jatropha curcas in

Received 15 July 2013

their postulated centre of origin will permit to identify genetic material useful for future

Received in revised form

improvement of the species. Although it is estimated that Mexico is the likely centre of

12 November 2013

origin and domestication of J. curcas, so far the true centre of origin still has to be found. A

Accepted 15 November 2013

representative set of 175 accessions of J. curcas from nine central and southeastern Mexican

Available online 17 December 2013

states (Chiapas, Veracruz, Oaxaca, Michoacan, Morelos, Yucatan, Guerrero, Hidalgo and
Puebla), including toxic, non-toxic, and contrasting protein and oil content genotypes was

Keywords:

used for diversity analysis by AFLP markers. The results indicate that Mexico has a high

Genetic diversity

genetic diversity of J. curcas; molecular analysis suggests population structuring in the

Jatropha curcas

different states where this species is spread. The germplasm from Chiapas, where con-

AFLP markers

trasting protein and oil content genotypes were detected, showed the highest genetic di-

Toxic and non-toxic genotypes

versity and clearly varies from the accessions from the other states. This is probably the

Biofuels

most comprehensive study of diversity of germplasm of J. curcas from Mexico, and together
with previous reports on the genetic diversity, biochemistry, morphology and germplasm
agronomics of J. curcas from Chiapas, suggest the likelihood that this area is the centre of
origin for this species and that domestication took place in the states bordering the Gulf of
Mexico, such as Veracruz, Hidalgo, Puebla and Yucatan, where genotypes with low or no
phorbol ester content exist.
2013 Elsevier Ltd. All rights reserved.

Abbreviations: AMOVA, Analysis of Molecular Variance; He, expected heterozygosity or Neis genetic diversity; I, Shannons information index; Ne, effective number of alleles; PCooA, Principal Coordinate Analysis; % P, percentage of polymorphism.
* Corresponding author. Tel.: 52 461 6115323x108; fax: 52 461 6115431.
E-mail address: pecina.victor@inifap.gob.mx (V. Pecina-Quintero).
0961-9534/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biombioe.2013.11.005

148

1.

b i o m a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7 e1 5 5

Introduction

The agronomic characteristics of Jatropha curcas L., known in

Mexico as Pinon mexicano or pinon manso, and the highquality oil yielded by the seeds has made this plant one of the
most attractive species for the commercial production of
biodiesel.
Because of its potential, there has been a rapid expansion
of J. curcas cultivation areas and new demand for the development of improved cultivars in recent years, with India
leading the cultivation and domestication of J. curcas, followed
by China, Brazil and many others [1].
The chromosomes of J. curcas are approximately 1e2 mm
and the plant has 2n 22 chromosomes, which corresponds to
the diploid level x 11 [2]. The lack of chromosome number
variability may be an advantage when crosses are planned
due to regular meiosis. Most of the reported artificial interspecific hybrids are diploid, but two triploid hybrids
(2n 3x 33) have been recovered from crosses between the
diploid J. curcas  Jatropha cathartica Teran & Berlan and J.
curcas  Jatropha podagrica Hook [3].
However, Jatropha can still be considered as a semi-wild,
undomesticated plant showing considerable performance
variability [4,5], and the profit margin realised from this crop is
still very small compared with the effort invested in its cultivation. Therefore, a better understanding of the optimum
agronomical requirements and genetic makeup of J. curcas is
vital for further application of the species [6,7].
Breeding programs with Jatropha are rare compared with
the programs for other oil species, and the initial phase of a
breeding program usually requires an adequate germplasm
bank with a collection of planting material from different
agro-vegetational zones and the presence of genetic diversity
in the population.
Knowing the genetic variability of J. curcas is important for
preserving its genetic diversity and for developing improved
varieties through breeding programs and marker-assisted
selection. In fact, the success of a breeding program depends
largely on knowledge of the available genetic variability and
on estimates of the genetic parameters of the main traits, thus
allowing the selection of different genotypes to produce hybrids and similar genotypes to produce lines [8].
Evaluations of the genetic variability of J. curcas worldwide
are scarce; almost all studies have been performed with collections from India [9e11] or China [12,13], and although a high
variability has been reported in some cases [14e16], most agree
that germplasm from these regions has low genetic variability.
The centre of origin of J. curcas likely exhibits wider genetic
diversity, and although it remains controversial, several lines
of evidence suggest that Mexico, specifically the coastal region
along the Gulf of Mexico, is the most likely centre of origin and
domestication. The most ancient record of the knowledge and
use of J. curcas comes from the Olmecas, a people who lived in
Mexico 3500e5000 years ago and were the first to colonise
Mesoamerica, preceding the Mayas and Aztecs [17].
Mexico does not have improved varieties of J. curcas, and
the degree of genetic diversity among and within natural
populations in this country is poorly studied. The most recent
studies were performed with accessions from the state of

Chiapas, Mexico, where broad genetic diversity was reported

[18,19]. However, the diversity of the germplasm from other
states near the Gulf of Mexico has not been evaluated.
As part of breeding strategies for the identification of superior accessions, which will be used in hybridisation to aid
the development of new varieties, the main objective of this
study was to determine the levels of diversity and genetic
relationships in J. curcas germplasm from nine states in
Mexico, including some states near the Gulf of Mexico, by
Amplified Fragment Length Polymorphisms (AFLP). This
technique has been used to successfully identify genetic
variability in J. curcas and to differentiate between toxic and
non-toxic varieties [11,19,20].

2.

Materials and methods

2.1.

Plant material

A representative set of 175 accessions of J. curcas, where 4 were

from Hidalgo, 6 from Morelos, 8 from Yucatan, 10 from
Oaxaca, 12 from each Michoacan, Guerrero and Puebla, 23
from Veracruz and 88 from Chiapas, was used for diversity
analysis. The accessions were selected from 400 accessions
comprising the J. curcas germplasm bank established at the
National Institute for Forestry, Agriculture and Livestock
Research (INIFAP, Mexico). The germplasm selection was
based on geographical distribution (Table 1) and variations in
qualitative and quantitative characters. The germplasm from
Chiapas was selected among accessions with 20e60% of oil
and 20e70% of protein content (data not shown).

2.2.

Methods

2.2.1.

DNA extraction

Total genomic DNA was extracted from young leaves of five

plants for each accession, following the standard CTAB
method with minor modifications [21]. Two grams of leaves
were ground in liquid nitrogen and then homogenised in
800 mm3 of extraction buffer [100 mol m3 Tris, pH 8.0;
150 mol m3 EDTA, pH 8.0; 2100 mol m3 NaCl; 30 g dm3
CTAB; 10 cm3 dm3 b-mercaptoethanol].

2.2.2.

AFLP analysis

Amplified Fragment Length Polymorphism analysis was performed essentially as described by Vos et al. [22]. Genomic

Table 1 e List of Jatropha curcas germplasm from nine

Mexican states.
Origin states
Chiapas
Veracruz
Oaxaca
Michoacan
Morelos
Yucatan
Guerrero
Hidalgo
Puebla

Number of locations

Collection number

88
23
10
12
6
8
12
4
12

CH 1 to CH 88
VER 1 to VER 23
OAX 1 to OAX 10
MICH 1 to MICH 12
MOR 1 to MOR 6
YUC 1 to YUC 8
GUERR 1 to GUERR 12
HGO 1 to HGO 4
PUE 1 to PUE 12

b i o m a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7 e1 5 5

DNA (10 ng mm3) was digested with 5 U each of EcoRI and

MseI (Roche) at 37  C for 3 h. Digested samples were incubated at 70  C for 15 min to deactivate the restriction enzymes.
Adaptors [EcoRI (5 pmol) and MseI (50 pmol)] were ligated to
restricted DNA fragments in ligation buffer (1 T4 DNA ligase)
with 1 U T4 DNA ligase, and incubated at 37  C overnight. Taq
DNA polymerase (Roche) was used in all PCR reactions. The
selective amplification for AFLP was performed using four
primer-pair combinations (Table 2). Preamplification of the
diluted (10-fold), ligated DNA was carried out with primers
that were complementary to the EcoRI and MseI adaptors and
contained adenine and cytosine, respectively, as the selective
nucleotide. Amplification was performed in a thermal cycler
(Px2 Thermo Electron Corporation, Milford, MA, USA.) using
the following cycling parameters: 20 cycles set at 94  C for 30 s,
56  C for 30 s and 72  C for 60 s. The second amplification was
carried out using four combinations of selective primers for
EcoRI (700 and 800 nm) and MseI, each with three selective
nucleotides (Table 2). All PCR products were independently
resolved via denaturing electrophoresis using 6% polyacrylamide gels using a sequencing system (Li-Cor IR2 equipped with two infrared lasers capable of reading at two
wavelengths: 700 and 800 nm). Only bright, clearly distinguishable bands between 50 and 700 bp were recorded for
analysis. No-template controls were performed for the PCRs.

2.2.3.

Statistical analysis

Every AFLP primer combination was assigned a score of 1 or

0 for the presence or absence of bands, respectively, for each
of the 175 accessions, and a binary matrix was generated.
Genetic diversity within each population (state) was measured
by calculating the percentage of polymorphism (% P), the
effective number of alleles (Ne), the Shannons information
index (I) and the expected heterozygosity or Neis genetic diversity (He), using the program GenAlEx version 6.41 [23].

2.2.4.

Analysis of variance

The binary matrix was used to calculate Analysis of Molecular

Variance (AMOVA), with which the variance components and
their significance levels for variation among populations
(states) and within population were obtained. It was conducted in GenAlEx v6 using the PHIPT. The P value was calculated as the number of values  observed value (including
observed value) O (number of permutations 1) [23].

2.2.5.

Genetic relations

A dendrogram was calculated using the Dice similarity coefficient [24] and the Unweighted Pair Group Method with

Table 2 e AFLP combinations used, polymorphism level,

and observed information content.
Primer
combination
E-ACA M-GGT
E-AGA M-GGT
E-ACG M-CAG
E-AAC M-CAG
Total

Number of fragments
Amplified

Polymorphic

Unique

Rare

106
99
119
115
439

89
92
96
105
382

1
7
3
0
11

10
17
24
24
75

149

Arithmetic Mean (UPGMA) [25], with the Jackknifing Method

used to corroborate it. This bootstrap resampling method was
performed to determine the robustness of the dendrogram.
One thousand bootstrap replicates were obtained from the
original data of 175 accessions. From these 1000 matrices,
confidence limits for each pair-wise comparison were determined [26]. All calculations were performed using FreeTree
0.9.1.50 [27]; the dendrogram was drawn using TreeView 1.6.6
[28]. Moreover, a Principal Coordinate Analysis (PCooA) by the
Dice similarity coefficient [24] was conducted using NTSYS PC
version 2.2 [29]. This analysis was performed to generate a
three-dimensional representation of the accessions and to
corroborate the similarity among them [30]. The threedimensional graphic was drawn using SigmaPlot v10.

3.

Results

3.1.

AFLP analysis

In this study, we used four different AFLP primer pairs (Table

2) to assess the genetic diversity of 175 accessions of J. curcas
from nine Mexican states (Chiapas, Veracruz, Oaxaca,
Michoacan, Morelos, Yucatan, Guerrero, Hidalgo and Puebla;
Table 1). Fragment sizes were between 50 and 500 bp. In total,
439 different fragments were obtained with an average of 109
fragments per combination, of which 382 (87%) were polymorphic (Table 2).
In the frequency and distribution analysis of polymorphic
fragments, we found that 11 fragments were specific or
unique to one accession, with an average of 2.75 fragments
per combination. Additionally, we identified 75 rare fragments, present in less than 10% of the accessions, with an
average of 18 per AFLP combination. The combinations EACG M-CAG and E-AAC M-CAG had the highest numbers
of rare fragments, with 24 each, while the E-ACA M-GGT
combination had the lowest, with 10 rare alleles (Table 2).
The number of effective alleles among populations ranged
from 1.069 to 1.256, while the Shannons information index
and the heterozygosity index ranged from 0.059 to 0.267 and
0.040 to 0.166, respectively. Likewise, the lowest percentage of
polymorphisms belonged to Hidalgo state accessions (10.93%)
and the highest to Chiapas (74.26%). These results showed a
high genetic diversity in the tested populations (Table 3).
Meanwhile, Chiapas state accessions had the highest
number of unique fragments with 42, Veracruz state had five
and Guerrero state had one. The accession VER 22 from Martnez de la Torre, Veracruz, showed the highest number of
unique fragments with a total of five; CH 35 from Venustiano
Carranza, Chiapas had two, while accessions CH 37 from
Acala, CH 62 from Frontera Comalapa, and CH 79 from Chiapa
de Corzo, all from Chiapas, and accession GUERR 5 from
Mecatepec, Guerrero had one fragment each (Table 4).
The analysis of molecular variance (AMOVA) of AFLP data
for populations showed significant differentiation (P < 0.001),
with 56% of the differentiation attributed to variation among
populations (states) and 44% attributed to variation within
populations, as shown in Table 5. The PHIPT estimates were
high (0.559) and highly significant (P < 0.001).

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Table 3 e Gene diversity in nine Jatropha curcas

populations from Mexico.
Origin states
Chiapas
SE
Veracruz
SE
Oaxaca
SE
Michoacan
SE
Morelos
SE
Yucatan
SE
Guerrero
SE
Hidalgo
SE
Puebla
SE

Na

Ne

He

%P

88
0.000
23
0.000
10
0.000
12
0.000
6
0.000
8
0.000
12
0.000
4
0.000
12
0.000

1.708
0.025
1.364
0.034
0.927
0.034
0.950
0.034
0.818
0.032
0.838
0.032
1.034
0.035
0.765
0.030
0.815
0.032

1.256
0.014
1.206
0.015
1.146
0.015
1.123
0.013
1.102
0.013
1.103
0.013
1.172
0.016
1.069
0.011
1.098
0.013

0.267
0.011
0.199
0.012
0.120
0.012
0.109
0.011
0.084
0.010
0.086
0.010
0.146
0.012
0.059
0.008
0.081
0.010

0.166
0.008
0.128
0.008
0.082
0.008
0.072
0.007
0.058
0.007
0.059
0.007
0.098
0.008
0.040
0.006
0.055
0.007

74.26

E-ACG M-CAG

Variance
% Total Stat Value
components

22.55

Levels of significance are based on 999 iterations. **P  0.010

30.133
23.795
53.928

56
44
100

PHIPT 0.559**

14.81
15.95
28.02

3.2.

15.03

The dendrogram constructed from the similarity matrices

showed the formation of two main accession groups (Fig. 1).
The Dice similarity coefficient ranged between 0.54 and 0.99.
Despite the fact that the accessions showed a clear trend of
grouping by geographical origin in Cluster I, the dispersion of
some accessions with similar origin throughout the dendrogram suggests the existence of a broad genetic base.
Cluster II was the most homogeneous group, the similarity
coefficient ranged between 0.69 and 0.99, and consisted of 86
accessions belonging to the states of Veracruz, Oaxaca,
Michoacan, Morelos, Yucatan, Guerrero, Hidalgo and Puebla.
While it showed small groups of genotypes with the same
geographical origin, there were various accessions which
mingled among them. Cluster II begins with a node of five
accessions from the states of Hidalgo, Guerrero, Morelos and
Michoacan (HGO 1, GUERR 6, MOR 5, GUERR 7 and MICH 2;
Fig. 1). In the following nodes, small accession groups were
observed with the same geographic origin as with GUERR 1, 3
and 4; OAX 9 and 10; and MICH 3 and 6; followed by MOR 1, 2, 3
and 4; GUERR 8, 9, 10 and 11; VER 9 and 20; and MICH 5. The
next node is composed of 10 accessions of Veracruz, mixed
with an accession of Oaxaca (VER 21 and 23; OAX 2 and VER 3,
4, 5, 7, 8, 10, 13 and 19), plus seven from Michoacan (MICH 4, 7,
8, 9, 10, 11 and 12) and two from Guerrero (GUERR 2 and 12).
The next node was composed of seven accessions from
Veracruz (VER 1, 2, 12, 14, 15, 16 and 17); two from Oaxaca
(OAX 6 and 7); VER 18; OAX 1 and 4; MICH 1; and OAX 3, 5 and 8,
while the last node from Cluster II included five accessions
from Puebla (PUE 1, 2, 3, 4 and 5), eight from Yucatan (YUC 1, 2,
3, 4, 5, 6, 7 and 8), MOR 6; PUE 6, 10 and 11; HGO 2 and 3; PUE 7,
8, 9 and 12; HGO 4; GUERR 5; and VER 6 and 11.
Cluster I was the most heterogeneous, with similarity coefficient values from 0.54 to 0.96, and included accessions only
from Chiapas. Cluster I was divided into two subclusters (A
and B), and three accessions that diverged from the remaining
genotypes (CH 57, CH 58 and CH 23) and showed the highest
number of rare fragments. Subcluster A consisted of 40 accessions: CH 75, CH 70, CH 48, CH 47 and CH 50, which also
showed rare fragments, and CH 88, CH 86, CH 87, CH 83, CH 85,
CH 60, CH 59, CH 82, CH 81, CH 80, CH 76, CH 56, CH 55, CH 54,
CH 53, CH 51, CH 49, CH 65, CH 62, CH 61, CH 84, CH 64, CH 73,
CH 72, CH 66, CH 71, CH 67, CH 68, CH 69, CH78, CH 77, CH 52,

Accession

Unique
fragments

CH 79 from Chiapa de Corzo,

Chiapas
VER 22 from Martnez de la Torre,
Veracruz
CH 35 from Venustiano Carranza,
Chiapas
CH 37 from Acala, Chiapas
CH 62 from Frontera Comalapa,
Chiapas
GUERR 5 from Mecatepec,
Guerrero
VER 22 from Martnez de la Torre,
Veracruz
CH 35 from Venustiano Carranza,
Chiapas

1
3
1
1
1
1
2
1

obtained (PHIPT 0.559), while all pairwise comparisons,

including comparisons between Morelos and Guerrero, were
significant (p < 0.05).

10.93

Table 4 e AFLP combinations and accessions with

specific polymorphic fragments and unique fragments.

E-AGA M-GGT

d.f.

21.64

Pairwise comparison of the accessions of J. curcas, grouped

by state, showed a clear differentiation of the Chiapas germplasm accessions compared with accessions from Veracruz,
Oaxaca, Michoacan, Morelos, Yucatan, Guerrero, Hidalgo and
Puebla, with PHIPT values higher than 0.58. With the exception
of the comparisons between the germplasm of Morelos and
Guerrero, and Puebla and Hidalgo, where no significant differences existed, the pairwise comparisons were predominantly highly significant (Table 6).
To rule out the possibility of deviations induced by the
small sample size of Hidalgo accessions (n 4), we repeated
the AMOVA and pairwise comparisons excluding these accessions. In the case of AMOVA, a PHIPT value of 0.56 was
obtained, which was slightly higher than that previously

E-ACA M-GGT

Source of
variation

Among populations
8
Within populations 166
Total
174

50.57

N: number of individuals. Na: number of different alleles. Ne:

effective number of alleles. I: Shannons information index. He:
expected heterozygosity. % P: percentage of polymorphism. SE:
standard error.

Primer
combination

Table 5 e Analysis of molecular variance (AMOVA) within

and among Jatropha curcas populations analysed with
AFLP markers.

Genetic relations

151

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Table 6 e Pairwise population PHIPT values (below diagonal) and probability levels (above diagonal) of variation among
Jatropha curcas populations from different states from Mexico.
Origin state
Chis
Ver
Oax
Mich
Mor
Yuc
Guerr
Hdgo
Pueb

Chis
0.590
0.591
0.600
0.588
0.591
0.586
0.587
0.612

Ver

Oax

Mich

Mor

Yuc

**

**
**

**
**
**

**
*
**
**

**
**
**
**
**

0.106
0.095
0.079
0.149
0.103
0.189
0.296

0.111
0.202
0.194
0.105
0.243
0.320

0.157
0.154
0.126
0.246
0.297

0.181
0.058
0.293
0.379

0.120
0.242
0.287

Guerr
**
**
**
**
NS
**
0.169
0.245

Hdgo
**
**
**
**
**
**
**

Pueb
**
**
**
**
**
**
**
NS

0.104

Chis: Chiapas. Ver: Veracruz. Oax: Oaxaca. Mich: Michoacan. Mor: Morelos. Yuc: Yucatan. Guerr: Guerrero. Hdgo: Hidalgo. Pueb: Puebla. Levels of
significance are based on 999 permutations. *P  0.050; **P  0.010; NS: Not significant.

CH 63, CH 74 and CH 79. Subcluster B consisted of 44 accessions: CH 37, CH 31, CH 26, CH 25, CH 24, CH 46, CH 45, CH 42,
CH 41, CH 40, CH 39, CH 33, CH 32, CH 34, CH 11, CH 44, CH 15,
CH 14, CH 3, CH 2, CH 17, CH 13, CH 28, CH 27, CH 30, CH 4, CH
29, CH 12, CH 1, CH 7, CH 5, CH 21, CH 8, CH 6, CH 18, CH 16, CH
20, CH 19, CH 9, CH 36, CH 22, CH 10, CH 43 and CH 38.
The accessions most divergent from each other and with
the accessions of the two Clusters were VER 22, CH 23 and CH
35 (Outliers). Felsenstein confidence indices showed that most
of the groups in the nodes were robust because their values
rise above 0.5.
Principal Coordinate Analysis (PCooA) indicated that the
first three principal coordinates (PCoo) accounted for 52.9% of
the total variance observed, with a percentage of 44.9, 4.7 and
3.3% for PCoo1, PCoo2 and PCoo3, respectively. The principal
coordinate chart (Fig. 2) confirmed the observations made
using the dendrogram, and clearly shows the formation of two
major Clusters (Clusters I and II), the Cluster I subdivision into
A and B Subclusters, and the presence of three outlier genotypes (VER 22, CH 23 and CH 35).

4.

Discussion

The key for success of any genetic improvement program lies

in the availability of genetic variability for desired traits. Genetic resources through global exploration, introduction,
characterisation and evaluation will provide strong base for
development of elite varieties by various improvement
methods. Comprehensive work on collection, characterisation and evaluation of germplasm for growth, morphology,
seed characteristics and yield traits is still in its infancy [31].
Internationally, J. curcas is a crop with high industrial potential, but is at an early stage of domestication [5,31], therefore
identification and maintenance of a high level of genetic diversity are essential for the long-term success of the breeding
programs [31], thus defining the centre of origin and domestication is important to know the regions and accessions with
the highest possible genetic diversity.
The AFLP markers used in this study confirm the existence
of a high genetic diversity of J. curcas in Mexico, they detect a
high level of polymorphism (87%) in the 175 J. curcas accessions
analysed, which allowed discrimination between accessions,

and confirm the usefulness of this marker system [11,32], the

selected primers [32,33], and the sampling strategy [33].
The levels of genetic diversity in accessions identified in
the state of Chiapas, Mexico were comparatively similar to
those of Guatemala and Central America [34] and are consistent with statements such as those from Genomics Seed
Company Biofuels (SGB, Incorporated), an energy crop company specialised in developing and delivering bioenergy solutions, who recently reported that genetic diversity of J. curcas
is comparable to that of maize when analysing germplasm
only from Guatemala and Central America using Single
Nucleotide Polymorphism markers (SNPs) [35].
The frequency and distribution analysis of polymorphic
fragments showed a total of 11 unique fragments, each present
in one individual, and 75 rare fragments present in up to 10% of
the genotypes. Foster et al. [36] indicate that the presence of
such fragments in the germplasm is related to loss of genetic
diversity, possibly due to selection; however in Mexico, there
are still no breeding programs or improved varieties that were
related to diversity loss. Meanwhile, Agrama and Tuinstra [37]
mention that unique and rare alleles are of particular interest
because they may be linked to a particular genotype, thereby
serving as a diagnostic to differentiate a genotype [38] or a
specific region of the genome. The genotypes with the highest
number of unique and rare fragments were accessions VER 22
from the state of Veracruz, and CH 23 and CH 35 from the state
of Chiapas, which were clearly separated from Clusters I and II
in the PCooA. This implies that these accessions could be a
species different from J. curcas, which in turn may confirm the
findings reported by Agrama and Tuinstra [37].
Unique fragments can be useful for identifying unknown
germplasm. In this study, accessions from the state of Chiapas
had 42 unique fragments, the most for a single population,
while Veracruz presented five, and Guerrero one (data not
shown). Such fragments are useful in breeding programs
because they allow selection of diverse genotypes [39,40] to
increase the variability or to exploit inter- and intrapopulation heterosis [35,41].
Analysis of variance and pairwise comparison by PHIPT
value were used to determine whether there is a differentiation of accessions according to their origin (state). This estimator is a measure analogous to FST, and it is used to
determine the level of genetic differentiation between populations. The results of both measures range from 0 (no

152

b i o m a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7 e1 5 5

Fig. 1 e UPGMA dendrogram of 175 accessions of J. curcas from nine states in Mexico, based on the genotypic data from 439
AFLP fragments obtained from four primer combinations. Values at the nodes indicate bootstrap values from 1000
replicates. The scale represents Neis similarity coefficient.

b i o m a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7 e1 5 5

Fig. 2 e Dispersion of J. curcas germplasm from nine states

in Mexico, based on the first three principal coordinates.
PCoo1 separated the populations into two groups that
reflect geographical location of Chiapas germplasm with
the others states.

differentiation) to 1 (no shared alleles). The analysis of all

populations in this study resulted in a PHIPT value of 0.559,
which was highly significant (P < 0.001), suggesting a structuring of the J. curcas populations. Additionally, the genetic
variance estimated by AMOVA (P < 0.001) showed that 60% of
the variance was due to differences among populations, while
40% was within populations. In this regard, Bessega et al. [42]
argue that some species populations are structured because
pollen and seed dispersion is limited, causing population
substructure. In this case, the dispersion of J. curcas in Mexico
has been mainly through asexual reproduction (branches or
cuttings), so no cross-pollination between genotypes is likely
to have occurred from one state to another. Furthermore, the
results of this study suggest that geographical proximity is not
necessarily indicative of genetic similarity and is therefore not
a guide to understanding the genetic structure of this species.
Although the UPGMA analysis showed a clustering trend of
populations by geographical origin, in the dendrogram, there
is considerable overlap in some of the accessions from
different states, except for the state of Chiapas, which suggests the likelihood of J. curcas germplasm exchange among
the eight other states. This genetic flow could be the result of
interest in non-toxic genotypes from the Gulf of Mexico region, which could have been favoured due to the proximity
between the states of Veracruz, Hidalgo, Puebla and Morelos,
and some may have been carried to the state of Guerrero.
Chiapas genotypes have not suffered a recurrent selection
process, most likely because of their toxicity, which leads to
decreased interest in this source of germplasm. Meanwhile,
the genotypes from the Yucatan Peninsula could have been
selected by the Mayan culture, who domesticated and used
this plant. This situation favours the hypothesis that preHispanic cultures, mainly the Olmec and Maya, domesticated and exchanged J. curcas germplasm [17], as our results
clearly show that the genotypes from Yucatan have more
similarity with those in the centre of the country, rather than
those from Chiapas, despite being geographically closer.

153

The clusters observed in the dendrogram agreed with the

dispersion of the accessions of J. curcas identified in the PCooA,
whose three principal coordinates explained 52% of the variation. PCoo1 explained 44.9% of the variation, separating the
accessions into two clearly distinguishable groups: the genotypes from the state of Chiapas (Cluster I) and the rest of the
states, which formed a single tight cluster (Cluster II). PCoo2
explained 4.7% of the variation and separated Cluster I into
Subcluster A and Subcluster B, indicating that the variation
between genotypes of Chiapas was even greater than that
found in the accessions from the other eight states, grouped
mainly in Cluster II, whose variation in PCoo3 was 3.3%. In
relation to the most divergent accessions, accessions VER 22
from the state of Veracruz and CH 23 and CH 35 from the state
of Chiapas, which were grouped as outliers in the dendrogram,
were clearly separated from Clusters I and II in the PCooA,
while accessions 57 and 58, which formed a small node in the
dendrogram, were located very close to Subcluster A.
Previous studies indicate that Chiapas germplasm accessions with the highest oil content (56e69%) are found at altitudes above 500 m [43]. It is likely that the separation of the
accessions from Chiapas into two Subclusters is due to
genotypic and not to environmental variation; there is no
evidence that their formation is influenced by environmental
factors because in each Subcluster accessions from different
regions of Chiapas intermingle. Therefore, further studies are
needed to explain the genetic distribution of germplasm in
this state. The understanding of the genetic diversity and
structure of populations of J. curcas in their postulated centre
of origin will permit to identify genetic material useful for
future improvement of the species. For example, it will be
possible to design crosses between plants from groups that
are genetically distanced [44].
Internationally the majority of genetic diversity studies
with Mexican germplasm of J. curcas are based on a few accessions, the great majority of these diversity studies analysed
between one and 28 Mexican accessions [10,45e47], more
comprehensive analyzes used 134 and 88 accessions, but all
from Chiapas, Mexico [18,19], and the diversity study with
more diverse accession from distinct regions of Mexico analysed 11 accessions from Puebla, and one for each state:
Veracruz, Quintana Roo and Chiapas [48], in contrast in our
study included 175 representative accessions from nine states
of Mexico (Chiapas, Veracruz, Morelos, Yucatan, Guerrero,
Michoacan, Hidalgo, Oaxaca and Puebla), so this is probably
the most comprehensive study of diversity of germplasm of J.
curcas from Mexico.
This study helps to confirm that Mexico has a wide genetic
diversity of J. curcas and to explain the wide phenotypic diversity recorded in Mexican germplasm, such as the occurrence of non-toxic genotypes with little or no phorbol ester
content in some states like Veracruz, Puebla and Hidalgo
[49,50]. In addition, this diversity is shown by the variation in
the protein and oil contents of some accessions from the
National Germplasm Bank at INIFAP, which has genotypes
with oil and protein contents ranging from 21 to 67% and 23 to
59%, respectively, that also show significant differences in
their oil physicochemical properties and in their fatty acid
profiles [51]. The existence of non-toxic genotypes in states
bordering the Gulf of Mexico (e.g., Veracruz, Puebla, Hidalgo

154

b i o m a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7 e1 5 5

and Yucatan) indicates that domestication of J. curcas most

likely occurred in this area by selection of genotypes with low
or zero phorbol ester content, allowing the residual oil paste
extract to be used for human and livestock consumption [50].
Additionally, the wide genetic diversity detected in the
Mexican germplasm, using molecular markers, biochemical
analysis of seeds, morphological and agronomic tests (pistillate flower, male flower, hermaphrodite flowers, inflorescence
number per plant, seed yield) of accessions [18,19,34,51,52],
helps substantiate the theory that particularly Chiapas and
most likely Guatemala are the centre of origin of this species.
The characterisation of some agronomical traits for selected
accession will also be developed.

5.

Conclusion

Mexico has a wide genetic diversity of J. curcas, mainly in the

state of Chiapas, and its germplasm possesses special characteristics not shared by the rest of the country. However, the
most domesticated germplasm is found outside of Chiapas,
suggesting that the centres of origin and domestication are
different.
These results indicate that Chiapas is the most likely
centre of origin, whereas the states which are closer to the
Gulf of Mexico watershed, such as Veracruz, Puebla, Hidalgo
and Yucatan, are the likely sites of domestication, as they
present individuals with low or zero phorbol ester content.
The genotypes found in these states were more similar to each
other than to the germplasm of Chiapas; there no significant
difference in the AFLP profiles between germplasm in Hidalgo
and Puebla. Thus, we might argue that domestication most
likely took place in at least two pre-Hispanic cultures that
inhabited these areas, and support the theory of germplasm
exchange between them.

Acknowledgements
The authors are very grateful to SAGARPA for their financial
support of the project Study of materials for biofuels production in Mexico, PRECI number: 6017012A.

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