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Genetic structure of Jatropha curcas L. in Mexico andprobable centre of origin

Victor Pecina-Quintero a,*, Jose Luis Anaya-Lopez a,Alfredo Zamarripa-Colmenero b, Carlos Alberto Nunez-Colın a,Noe Montes-Garcıa c, Jose Luis Solıs-Bonilla b,Marıa Fernanda Jimenez-Becerril d

a INIFAP-Campo Experimental Bajıo, Carr. Celaya-San Miguel de Allende km 6.5, C.P. 38010 Celaya,

Guanajuato, Mexicob INIFAP-Campo Experimental Rosario Izapa, Tuxtla Chico, Chiapas, Mexicoc INIFAP-Campo Experimental Rıo Bravo, Cd. Rıo Bravo, Tamaulipas, MexicodCentro de Biotecnologıa Genomica del Instituto Politecnico Nacional, Cd. Reynosa, Tamaulipas, Mexico

a r t i c l e i n f o

Article history:

Received 15 July 2013

Received in revised form

12 November 2013

Accepted 15 November 2013

Available online 17 December 2013

Keywords:

Genetic diversity

Jatropha curcas

AFLP markers

Toxic and non-toxic genotypes

Biofuels

Abbreviations: AMOVA, Analysis of Molecmation index; Ne, effective number of allele* Corresponding author. Tel.: þ52 461 611532E-mail address: pecina.victor@inifap.gob.

0961-9534/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2013.11.

a b s t r a c t

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

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

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

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

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

states (Chiapas, Veracruz, Oaxaca, Michoacan, Morelos, Yucatan, Guerrero, Hidalgo and

Puebla), including toxic, non-toxic, and contrasting protein and oil content genotypes was

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

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

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

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

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

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.

ª 2013 Elsevier Ltd. All rights reserved.

ular Variance; He, expected heterozygosity or Nei’s genetic diversity; I, Shannon’s infor-s; PCooA, Principal Coordinate Analysis; % P, percentage of polymorphism.3x108; fax: þ52 461 6115431.mx (V. Pecina-Quintero).ier Ltd. All rights reserved.005

Table 1 e List of Jatropha curcas germplasm from nineMexican states.

Origin states Number of locations Collection number

Chiapas 88 CH 1 to CH 88

Veracruz 23 VER 1 to VER 23

Oaxaca 10 OAX 1 to OAX 10

Michoacan 12 MICH 1 to MICH 12

Morelos 6 MOR 1 to MOR 6

Yucatan 8 YUC 1 to YUC 8

Guerrero 12 GUERR 1 to GUERR 12

Hidalgo 4 HGO 1 to HGO 4

Puebla 12 PUE 1 to PUE 12

b i om a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7e1 5 5148

1. Introduction

The agronomic characteristics of Jatropha curcas L., known in

Mexico as “Pinon mexicano” or “pinon manso”, and the high-

quality 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 devel-

opment 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 2n¼ 22 chromosomes, which corresponds to

the diploid level x ¼ 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 inter-

specific hybrids are diploid, but two triploid hybrids

(2n ¼ 3x ¼ 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 profitmargin realised from this crop is

still very small compared with the effort invested in its culti-

vation. 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 themain traits, thus

allowing the selection of different genotypes to produce hy-

brids 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 col-

lections from India [9e11] or China [12,13], and although a high

variability hasbeen reported in somecases [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 thatMexico, specifically the coastal region

along the Gulf of Mexico, is themost likely centre of origin and

domestication. Themost 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 su-

perior 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 4were

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 extractionTotal 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 m�3 Tris, pH 8.0;

150 mol m�3 EDTA, pH 8.0; 2100 mol m�3 NaCl; 30 g dm�3

CTAB; 10 cm3 dm�3 b-mercaptoethanol].

2.2.2. AFLP analysisAmplified Fragment Length Polymorphism analysis was per-

formed essentially as described by Vos et al. [22]. Genomic

b i om a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7e1 5 5 149

DNA (10 ng mm�3) was digested with 5 U each of EcoRI and

MseI (Roche�) at 37 �C for 3 h. Digested samples were incu-

bated at 70 �C for 15min to deactivate the restriction enzymes.

Adaptors [EcoRI (5 pmol) andMseI (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% poly-

acrylamide gels using a sequencing system (Li-Cor IR2 equip-

ped with two infrared lasers capable of reading at two

wavelengths: 700 and 800 nm). Only bright, clearly distin-

guishable bands between 50 and 700 bp were recorded for

analysis. No-template controls were performed for the PCRs.

2.2.3. Statistical analysisEvery 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 diversitywithin each population (state) wasmeasured

by calculating the percentage of polymorphism (% P), the

effective number of alleles (Ne), the Shannon’s information

index (I) and the expected heterozygosity or Nei’s genetic di-

versity (He), using the program GenAlExª version 6.41 [23].

2.2.4. Analysis of varianceThe binarymatrix 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 con-

ducted in GenAlEx v6 using the PHIPT. The P value was calcu-

lated as the number of values � observed value (including

observed value) O (number of permutations þ 1) [23].

2.2.5. Genetic relationsA dendrogram was calculated using the Dice similarity coef-

ficient [24] and the Unweighted Pair Group Method with

Table 2 e AFLP combinations used, polymorphism level,and observed information content.

Primercombination

Number of fragments

Amplified Polymorphic Unique Rare

E-ACA þ M-GGT 106 89 1 10

E-AGA þ M-GGT 99 92 7 17

E-ACG þ M-CAG 119 96 3 24

E-AAC þ M-CAG 115 105 0 24

Total 439 382 11 75

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 deter-

mined [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 three-

dimensional 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 poly-

morphic (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 frag-

ments, present in less than 10% of the accessions, with an

average of 18 per AFLP combination. The combinations E-

ACG þ M-CAG and E-AAC þ M-CAG had the highest numbers

of rare fragments, with 24 each, while the E-ACA þ 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 Shannon’s 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 Mar-

tınez 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).

Table 5 eAnalysis of molecular variance (AMOVA)withinand among Jatropha curcas populations analysed withAFLP markers.

Source ofvariation

d.f. Variancecomponents

% Total Stat Value

Among populations 8 30.133 56 PHIPT 0.559**

Within populations 166 23.795 44

Total 174 53.928 100

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

Table 3 e Gene diversity in nine Jatropha curcaspopulations from Mexico.

Origin states N Na Ne I He % P

Chiapas 88 1.708 1.256 0.267 0.166 74.26

SE 0.000 0.025 0.014 0.011 0.008

Veracruz 23 1.364 1.206 0.199 0.128 50.57

SE 0.000 0.034 0.015 0.012 0.008

Oaxaca 10 0.927 1.146 0.120 0.082 21.64

SE 0.000 0.034 0.015 0.012 0.008

Michoacan 12 0.950 1.123 0.109 0.072 22.55

SE 0.000 0.034 0.013 0.011 0.007

Morelos 6 0.818 1.102 0.084 0.058 14.81

SE 0.000 0.032 0.013 0.010 0.007

Yucatan 8 0.838 1.103 0.086 0.059 15.95

SE 0.000 0.032 0.013 0.010 0.007

Guerrero 12 1.034 1.172 0.146 0.098 28.02

SE 0.000 0.035 0.016 0.012 0.008

Hidalgo 4 0.765 1.069 0.059 0.040 10.93

SE 0.000 0.030 0.011 0.008 0.006

Puebla 12 0.815 1.098 0.081 0.055 15.03

SE 0.000 0.032 0.013 0.010 0.007

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

effective number of alleles. I: Shannon’s information index. He:

expected heterozygosity. % P: percentage of polymorphism. SE:

standard error.

b i om a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7e1 5 5150

Pairwise comparison of the accessions of J. curcas, grouped

by state, showed a clear differentiation of the Chiapas germ-

plasm 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 dif-

ferences existed, the pairwise comparisons were predomi-

nantly highly significant (Table 6).

To rule out the possibility of deviations induced by the

small sample size of Hidalgo accessions (n ¼ 4), we repeated

the AMOVA and pairwise comparisons excluding these ac-

cessions. In the case of AMOVA, a PHIPT value of 0.56 was

obtained, which was slightly higher than that previously

Table 4 e AFLP combinations and accessions withspecific polymorphic fragments and unique fragments.

Primercombination

Accession Uniquefragments

E-ACA þ M-GGT CH 79 from Chiapa de Corzo,

Chiapas

1

E-AGA þ M-GGT VER 22 from Martınez de la Torre,

Veracruz

3

CH 35 from Venustiano Carranza,

Chiapas

1

CH 37 from Acala, Chiapas 1

CH 62 from Frontera Comalapa,

Chiapas

1

GUERR 5 from Mecatepec,

Guerrero

1

E-ACG þ M-CAG VER 22 from Martınez de la Torre,

Veracruz

2

CH 35 from Venustiano Carranza,

Chiapas

1

obtained (PHIPT ¼ 0.559), while all pairwise comparisons,

including comparisons between Morelos and Guerrero, were

significant (p < 0.05).

3.2. Genetic relations

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 dendro-

gram 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; andMICH 3 and 6; followed byMOR 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 fromMichoacan (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 fromYucatan (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 co-

efficient 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 ac-

cessions: 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,

Table 6 e Pairwise population PHIPT values (below diagonal) and probability levels (above diagonal) of variation amongJatropha curcas populations from different states from Mexico.

Origin state Chis Ver Oax Mich Mor Yuc Guerr Hdgo Pueb

Chis ** ** ** ** ** ** ** **

Ver 0.590 ** ** * ** ** ** **

Oax 0.591 0.106 ** ** ** ** ** **

Mich 0.600 0.095 0.111 ** ** ** ** **

Mor 0.588 0.079 0.202 0.157 ** NS ** **

Yuc 0.591 0.149 0.194 0.154 0.181 ** ** **

Guerr 0.586 0.103 0.105 0.126 0.058 0.120 ** **

Hdgo 0.587 0.189 0.243 0.246 0.293 0.242 0.169 NS

Pueb 0.612 0.296 0.320 0.297 0.379 0.287 0.245 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.

b i om a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7e1 5 5 151

CH 63, CH 74 and CH 79. Subcluster B consisted of 44 acces-

sions: 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 thatmost

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 geno-

types (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. Ge-

netic 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, characterisa-

tion 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 poten-

tial, but is at an early stage of domestication [5,31], therefore

identification and maintenance of a high level of genetic di-

versity are essential for the long-term success of the breeding

programs [31], thus defining the centre of origin and domes-

tication 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 consis-

tent with statements such as those from Genomics Seed

Company Biofuels (SGB, Incorporated), an energy crop com-

pany specialised in developing and delivering bioenergy so-

lutions, 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 showeda total of 11unique fragments, eachpresent

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, whichwere clearly separated fromClusters I and II

in the PCooA. This implies that these accessions could be a

species different from J. curcas, which in turnmay 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 intra-

population heterosis [35,41].

Analysis of variance and pairwise comparison by PHIPTvalue were used to determine whether there is a differentia-

tion of accessions according to their origin (state). This esti-

mator is a measure analogous to FST, and it is used to

determine the level of genetic differentiation between pop-

ulations. The results of both measures range from 0 (no

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 Nei’s similarity coefficient.

b i om a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7e1 5 5152

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.

b i om a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7e1 5 5 153

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 struc-

turing 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 sug-

gests 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 re-

gion, 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 pre-

Hispanic cultures, mainly the Olmec and Maya, domesti-

cated 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.

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 vari-

ation. PCoo1 explained 44.9% of the variation, separating the

accessions into two clearly distinguishable groups: the geno-

types 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,whichwere 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 acces-

sions with the highest oil content (56e69%) are found at alti-

tudes 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 ac-

cessions, the greatmajority 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 ana-

lysed 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 di-

versity recorded in Mexican germplasm, such as the occur-

rence 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

b i om a s s a n d b i o e n e r g y 6 0 ( 2 0 1 4 ) 1 4 7e1 5 5154

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 (pistil-

late 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 char-

acteristics 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 statesweremore 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 pro-

duction in Mexico”, PRECI number: 6017012A.

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