Molecular characterization of Cuban endemism Carica cubensis Solms using random amplified polymorphic DNA (RAPD) markers

doi:10.4236/as.2010.13012

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Vol.1, No.3, 95-101 (2010) Agricultural Sciences

doi:10.4236/as.2010.13012

Molecular characterization of Cuban endemism Carica

cubensis Solms using random amplified polymorphic

DNA (RAPD) markers

Jesús Rodríguez1, Pedro Rodríguez1, María E. González1, Pedro Martínez-Gómez2*

1Departamento de Fisología y Bioquímica Vegetal, Departamento de Genética, Instituto Nacional de Ciencias Agrícolas INCA,

San José de las Lajas, Cuba

2Departamento de Mejora Vegetal, CEBAS-CSIC, Espinardo, Spain; *Corresponding Author: pmartinez@cebas.csic.es

Received 28 July 2010; revised 30 August 2010; accepted 5 September 2010.

ABSTRACT

The objective of this work is to present an ap-

propriate set of RAPD (random amplified poly-

morphic DNA) markers using single and multi-

plex PCR analysis suitable for the characteriza-

tion of the endemic Cuban species Carica cu-

bensis and the establishment of genetic rela-

tionships with the cultivated species Carica pa-

paya. RAPD markers presented a high level of

polymorphism. In addition, the incorporation of

more than one RAPD primer in the PCR analysis

increased the number of obtained bands and

the polymorphism of these bands. A total of 73

RAPD bands were detected (45 of them poly-

morphic) with the nine RAPD markers assayed

using single and multiplex PCR analysis. Re-

sults demonstrated a reduced genetic variability

within the tested Carica cubensis accessions.

The observed clustering in this species could

be better explained according to geographic

proximity and can indicate the similar prece-

dence of the isolated studied populations. C.

cubensis seem to be subspecies of C. papaya

adapted to the environmental conditions of the

mountains of Cuba or a endemic species close

to C. papaya. The implications of these results

in the creation of effective germplasm core col-

lection in Carica species have been also dis-

cussed.

Keywords: Carica Species; Germplasm; Molecular

Markers; RAPDs; Breeding

1. INTRODUCTION

Carica is a genus of family Caricaceae originating

from Central and South America including more than

forty different species being Carica papaya L. the most

important species from the economically and agronomy

point of view and the most cultivated species in these

areas of Central and South America [1,2]. Cuba can be

considered an area of putative endemism [3,4] of these

species according to the origin and dispersion of the dif-

ferent accessions analyzed. In this sense, one of these

examples of related species is Carica cubensis Solms

syn. Carica prosoposa L., an endemic fruit tree species

from Cuba considered a papaya endemism in prelimi-

nary studies [5]. This specie was described at the first

time by Solms and Grafen in 1889 [6] indicating the

presence of the specie in forested areas of Cuba since

Baracoa to Portero of St. Andre.

Carica cubensis is a shrub from 2 to 4 meters with a

thick trunk and branches and leaves spongy no alternated

and terminals. As well as Carica papaya, this species is

at the same time dioecious with female and male plants,

monocious with female and male flowers in the same

plant, and polygamous with hermaphrodite flowers [7,8].

Flowers are white to yellow-green and can have three

sex forms male (in the case of male plants) and female

and hermaphrodite (in the case of female plants) Figure

1. Only the two last types give fruits. The fruit with a

size around 80-100 grams is smaller than the cultivated

papaya and contains an enzyme (papaine) with intensive

digestive action that can be used for medicinal purpose

against dyspepsia disease and with antitumoral proper-

ties [5,9]. Carica cubensis is a wild related species of the

cultivated papaya species (Carica papaya) with a lot of

potential from the agronomic and breeding point of view.

Some authors, however, included this Carica cubensis

species inside the Carica papaya species. This species

can be particularly useful in marginal lands, where they

have been selected to withstand stress conditions and

where they contribute to sustainable production with few

inputs.

J. Rodríguez et al. / Agricultural Sciences 1 (2010) 95-101

Figure 1. General overview of the Carica cubensis shrub (a)

and detail of racine male flower, female or hermaphrodite

flower and globose fruit in C. cubensis (b, c, and d respectively)

and C. papaya (e, f, and g respectively).

Traditionally, characterization and identification of

variability in the fruit species such as Carica cubensis

has been based on morphological descriptors [5]. Mole-

cular marker technology offers several advantages over

the sole use of traditional descriptors and has become an

essential tool for the study and conservation of the fruit

species. DNA marker technology offers great advantages

for the characterization of these rare fruit germplasm

[10]. The more recent utilization of PCR-based markers

has increased the opportunities for DNA characterization

of populations in a wider range of species [11]. In this

sense, random amplified polymorphic DNA (RAPD)

markers based on the PCR amplification of random lo-

cations in the genome [12,13] can be a suitable marker

to the first molecular analysis of species such as Carica

cubensis [14]. RAPD markers utilize random primers

that amplify random DNA sequences in the genome.

This results in differential amplification of regions that

vary in primer site sequence resulting in polymorphic

amplification products usually analyzed as presence/

absence. These markers have been previously assayed in

the molecular characterization of Carica papaya culti-

vars [15-18] and some wild related species [14,19,20],

the identification of flower sex types [8,21-25] and de-

velopment of genetic linkage maps [26]. In addition, to

improve the capacity of the molecular characterization

assays using these PCR-based markers, multiplex PCR,

a variant of the PCR in which more than one target se-

quence is amplified using more than one pair of primers

are being assayed [27-29].

The objective of this work is to present an appropriate

set of RAPD markers suitable for the molecular charac-

terization and establishing of genetic relationships in the

endemic Cuban species Carica cubensis using single and

multiplex PCR analysis and to establish the genetic rela-

tionships with the cultivated species Carica papaya.

2. MATERIAL AND METHODS

2.1. Plant Material

Young well expanded leaf samples from 18 individual

Carica cubensis trees growing in isolated clusters in

natural populations were collected at the tropical moun-

tains of Cordillera Habana Matanza, around 260 m alti-

tude, in three different locations (Lomas Escaleras de

Jaruco, Recria and Lomas Francisco Javier) between the

cities of Jaruco and San José de las Lajas in the province

of La Habana (Northwest of Cuba) Figure 2. In addition,

three known genotypes of Carica papaya were included

in the study Table 1. Leaf samples were lyophilized be-

fore DNA extraction in a Pharma Lyophilizer (Pharma

Biotek, Chennai, India) and stored at room temperature.

2.2. DNA Extraction

Lyophilized leaves were used for DNA isolation using

the procedure described by Doyle and Doyle [30] with

some modifications. 40 mg of healthy leaf blade was

ground, without use of liquid nitrogen, in 2 ml tube con-

taining 750 μl of worm (65ºC) CTAB extraction buffer.

Homogenate was incubated in a water bath at 65ºC for

15 min, mixed with an equal volume of 24:1 chloroform:

isoamyl alcohol and centrifuged at 6,000 g (20 min). The

upper phase was recovered and mixed with an equal vo-

Figure 2. Location of natural populations of Carica cubensis

collected in Northwest of Cuba and general overview of the

region.

J. Rodríguez et al. / Agricultural Sciences 1 (2010) 95-101

Table 1. Carica cubensis and Carica papaya accessions assayed.

Accession Species Origin Sex of the plant

“Lomas Escaleras-1” Carica cubensis Seedling collected in Lomas Escaleras Plant without flower

“Lomas Escaleras-2” Carica cubensis Seedling collected in Lomas Escaleras Plant without flower

“Lomas Escaleras-3” Carica cubensis Seedling collected in Lomas Escaleras Female

“Lomas Escaleras-4” Carica cubensis Seedling collected in Lomas Escaleras Plant without flower

“Lomas Escaleras-5” Carica cubensis Seedling collected in Lomas Escaleras Plant without flower

“Lomas Escaleras-6” Carica cubensis Seedling collected in Lomas Escaleras Plant without flower

“Recria-1” Carica cubensis Seedling collected in Recria area Plant without flower

“Recria-2” Carica cubensis Seedling collected in Recria area Plant without flower

“Recria-3” Carica cubensis Seedling collected in Recria area Plant without flower

“Recria-4” Carica cubensis Seedling collected in Recria area Plant without flower

“Recria-5” Carica cubensis Seedling collected in Recria area Plant without flower

“Recria-6” Carica cubensis Seedling collected in Recria area Plant without flower

“Lomas Francisco-1” Carica cubensis Seedling collected in Lomas Fco Javier Male

“Lomas Francisco-2” Carica cubensis Seedling collected in Lomas Fco Javier Male

“Lomas Francisco-3” Carica cubensis Seedling collected in Lomas Fco Javier Female

“Lomas Francisco-4” Carica cubensis Seedling collected in Lomas Fco Javier Plant without flower

“Lomas Francisco-5” Carica cubensis Seedling collected in Lomas Fco Javier Female

“Lomas Francisco-6” Carica cubensis Seedling collected in Lomas Fco Javier Male

“Papaya” Carica papaya Open pollination of cultivar “Papaya” Female

“Maradol Roja” Carica papaya Cuban commercial cultivar Plant without flower

“1500” Carica papaya Cuban commercial cultivar Plant without flower

lume of isopropanol at –20°C. The nucleic acid pellet

was washed in 400 μl of 10 mM NH4Ac in 76% ethanol,

dried, resuspended in 50 μl of TE, incubated with 0.5 μg

of RNase-A at 37ºC for 30 min to digest RNA, and

quantified using a Biophotometer (Eppendorf, Barcelona,

Spain).

2.3. Random Amplified Polymorphic DNA

(RAPD) Marker Application

Nine RAPD universal primers (OPA-07, OPB-07,

OPN-14, OPR-15, OPR-16; OPW-12, OPW-13, OPY-13

and OPZ-17) purchased from Operon Biotechnologies

(Huntsville, USA) were assayed performing single (one

primers) and multiplex (combination of two or three

primers) PCR analysis Table 1. Amplifications were

carried out in 20 µL total volume containing 1 × Buffer,

1 mM MgCl2, 0.16 mM of dNTP, 0.4 µmol of each

primer, 1.0 unit of Taq DNA polymerase (New England

Biolabs, Ipswich, USA), and 4 ng templates DNA. The

amplification program consisted of a step of DNA melt-

ing of 4 min at 94°C, followed by 35 cycles of 94°C for

1 min, 35°C for 1 min, and 72°C for 1 min, and a final

elongation step of 72°C for 10 min. Amplified products

were resolved in 2% agarose gels stained using Gel Red

Nucleic Acid Gel Sating® (Biotium, Hatwad, CA, USA)

and visualized with UV transmitted light. A 1 Kb DNA

Ladder (Invitrogen Life Technologies, Barcelona, Spain)

was used as molecular size standard. RAPD amplifica-

tions were repeated at least twice in order to check the

reproducibility of bands.

2.4. Data Analysis

Polymorphic alleles were scored as present or absent

(1/0). DNA band scoring was analyzed using GeneTools

gel analysis software of SYNGENE (Beacon House,

Nuffield Road, Cambridge, UK). The average polymor-

phic information content (PIC) was calculated for RAPD

markers across assay units by applying the formula

given by Powell et al. (1996). Mean character difference

distances were calculated for all pairwise comparisons

with the MEGA4 test (http://www.megasoftware.net/)

[31], which was used to construct UPGMA dendograms

[32] depicting the phenetic relationship among the diff-

erent accessions. Relative support for the branches in

J. Rodríguez et al. / Agricultural Sciences 1 (2010) 95-101

each dendrogram was assessed with 2000 replicates of

UPGMA bootstrap.

3. RESULTS AND DISCUSSION

The nine RAPD primers (OPA-07, OPB-07, OPN-14,

OPR-15, OPR-16; OPW-12, OPW-13, OPY-13 and

OPZ-17) assayed generated polymorphic and reproduci-

ble patterns in the Carica papaya and C. cubensis as-

sayed genotypes. In addition, to improve the capacity

and polymorphism of the molecular characterization

assays using RAPD markers different multiplex PCR

using more two or three RAPD primers have been as-

sayed. Results showed that the incorporation of more

than one RAPD primer in the PCR analysis increased the

number of obtained bands and the polymorphism of

these bands. The number of RAPD bands detected by

each primer depended on species, primer and the single

or multiple PCR analysis performed. A total of 73 RAPD

bands were detected (45 of them polymorphic) with the

nine RAPD markers assayed in C. cubensis using single

and multiplex PCR analysis with a size range between

105 and 3600 bp.

The total number of bands varied from 3 (for OPN-14)

to 7 (for OPW-12 and OPZ-13/OPW-12/OPN-14 primer

combination), with an average of 4.8 bands per analysis.

In addition, the mean PIC score over all loci was 0.70,

ranging from 0.33 in OPN-14 to 0.85 in OPZ-13/OPW-

12/OPN-14 primer combination Table 2. Even using

lyophilized leaf samples results showed a high yield and

good quality DNA with good results after the PCR

analysis Figure 3. This fact is very important taking in

account that the most crucial factor for the application of

RAPD technology is the DNA quality and concentration

[33,34].

In the case of C. papaya polymorphism was lower due

to the reduced number of accessions assayed (only three)

although proportionally higher than the results obtained

in C. cubensis in which we assayed 18 accessions. A

total of 64 RAPD bands were detected (28 of them po-

lymorphic) with the nine RAPD markers assayed in C.

cubensis using single and multiplex PCR analysis with a

size range between 105 and 3600 bp. The total number

of bands varied from 0 (for OPN-14, OPW-13, OPY-13)

to 3 (for OPB-07, OPY-13/OPB-07/OPA-07 and OPW-

13/OPR-16/OPR-15 primer combination, with an aver-

age of 4.2 bands per analysis. In addition, the mean PIC

score over all loci was 0.33, ranging from 0.00 in OPN-

14, OPW-13 and OPY-13 to 0.63 in OPW-13/OPR-16/

OPR-15 primer combination Table 2.

RAPD analysis showed higher number of bands (a-

bundance) and higher polymorphism in comparison with

other markers as has been described in papaya by Jo-

bin-Décor [19] and other tropical species such as cacao

[35] or coffee [36]. The low heterozygosities values

showed in this study can be a consequence of the low

number of Carica cubensis accessions assayed which

can also be genetically close according with the geo-

graphic proximity of the samples collected. For all the

loci studied the expected heterozygosity was greater than

the observed heterozygosity, implying the presence of

null alleles or a deficit of heterozygotes due to non-

random mating. The presence of common RAPD alleles

indicated the closer genetic distance between the culti-

vated papaya and C. cubensis in comparison with other

wild papaya species before studied including C. cauli-

flora, C. pubescens, C. parviflora or C. quercifolia [14,

19,20]. C. cubensis seem to be subspecies of C. papaya

adapted to the environmental conditions of the moun-

tains of Cuba or a Cuban endemic species close to C.

papaya as was indicated previously by Leon and Alain

[5].

Phenetic (taxonomy) relationships among Carica

cubensis accessions were analyzed with several UPGMA

dendograms Figure 4. Relationships moderately sup

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 2 11 12

λ1 2 3 4 5 6 7 8 9 10 11 12 13 14

DNA extraction

RAPD screening

RAPD analysis by single PCR

OPA-07 OPX-03

OPA-10 OPG-13

OPZ-17 OPA-08

OPR-15 OPB-11

OPW-12 OPB-07

OPN-15 OPA-11

1 2 3 4 5 6

OPY-13

RAPD analysis by multiplex PCR

1 2 3 4 5 6

OPY-13 + OPB-7

OPY-13 + OPB-7 + OPA-7

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 2 11 12

λ1 2 3 4 5 6 7 8 9 10 11 12 13 14

DNA extraction

RAPD screening

RAPD analysis by single PCR

OPA-07 OPX-03

OPA-10 OPG-13

OPZ-17 OPA-08

OPR-15 OPB-11

OPW-12 OPB-07

OPN-15 OPA-11

1 2 3 4 5 6

OPY-13

RAPD analysis by multiplex PCR

1 2 3 4 5 6

OPY-13 + OPB-7

OPY-13 + OPB-7 + OPA-7

Figure 3. Agarose gel showing the DNA extraction of

Carica samples (a); the first screening of RAPD markers

in some genotypes (b); and the application of RAPD

markers in all the genotypes using simple (c) and multi-

plex (d) PCR analysis. Λ DNA quantification marker

HindII from Invitrogen. M DNA ladder 1 Kb plus from

Invitrogen.

J. Rodríguez et al. / Agricultural Sciences 1 (2010) 95-101

Table 2. Universal primers (decamers) purchased from Operon Biotechnologies used as RAPD markers molecular characterization

of the Carica cubensis accessions assayed.

Carica cubensis

Marker Number of bandsPolymorphic bandsSize (bp) PIC

OPA-07 4 2 500-800 0.58

OPB-07 5 3 490-5490 0.83

OPN-14 3 1 970-2170 0.33

OPR-15 4 3 560-1110 0.83

OPR-16 5 3 750-3200 0.71

OPW-12 7 4 700-3250 0.75

OPW-13 4 2 500-1400 0.55

OPY-13 3 1 376-755 0.31

OPZ-17 6 4 400-915 0.76

OPY-13/OPB-07 4 3 95-755 0.79

OPY-13/OPB-07/OPA-07 5 3 95-1046 0.82

OPZ-13/OPW-12 6 4 500-2500 0.83

OPZ-13/OPW-12/OPN-14 7 5 300-3600 0.85

OPW-13/OPR-16 5 4 750-3500 0.80

OPW-13/OPR-16/OPR-15 5 3 750-3500 0.80

Figure 4. Dendograms obtained by UPGMA cluster analysis

based on mean character differences among the Carica

cubensis and Carica papaya accessions assayed using RAPD

markers by single and multiplex PCR anlaysis. Numbers in

the branches represent bootstrap values.

ported established two groups in relation to the two dif-

ferent studied species. In addition, bootstrap values of

UPGMA dendogram obtained with the utilization of

RAPDs were slightly higher. In the case of C. cubensis

in the most of cases accessions studied from a location

are closer than accessions from other locations. This

clustering is explained by geographic proximity and can

indicate the similar precedence of the isolated studied

populations. However, no clustering and RAPD markers

association was observed in our samples according to the

sex characterization as has been before observed in C.

papaya species [8,21-23]. These results also can support

both establish hypothesis C. cubensis as a subspecies of

C. papaya or a Cuban endemic species close to C. pa-

paya.

One of the goals of conservation programs in these

rare fruit species such as Carica cubensis is to charac-

terize and maintain existing level of variation and ge-

netic resources [10]. Genetic resources not only provide

the required raw material for suitable genetic crop im-

provement, but offer a unique gene combination to en-

sure adaptability and productivity [37,38]. Designing of

core collection using suitable DNA markers involves an

appropriate use of diversity, offering breeders an oppor-

tunity to work with a manageable number of accessions.

C. cubensis is a better adapted species to the Cuban con-

ditions and could also be a source of new genes for low

temperature resistance or disease resistance including the

Papaya Ring Spot Virus (PRSV) [39,40]. Other impor-

tant characteristic of this species is the smaller fruit

(around 100 grams) which will be a new good commer-

cial trait.

J. Rodríguez et al. / Agricultural Sciences 1 (2010) 95-101

100

Designing of core collection involves an appropriate

use of diversity, offering breeders an opportunity to work

with a manageable number of accessions. Therefore, the

availability of an optimized protocol for DNA charac-

terization to estimate the genetic diversity and to ensure

genetically representative non-redundant samples is of

great interest [41]. In this sense, universal RAPD mark-

ers can be a most suitable in the molecular characteriza-

tion of C. cubensis accessions and to construct efficient

core collections as has been described by Bortolini [42]

in white clover complementary to the application of

more recent developed markers in C. papaya of domi-

nant SPAR (Single primer amplification reaction) [17]

and SCAR (sequence characterized amplified region)

markers [25], and codominant SSR (Single sequence

repeat) markers [43,44].

Other additional advantages of RAPD markers are

that do not require labour intensive and expensive, no

genomic/cDNA libraries are need for development of

probes and very small quantity of DNA is need in the

PCR reactions even using lyophilized leaf samples.

4. ACKNOWLEDGEMENTS

This work is part of an International Collaboration between the

Instituto Nacional de Ciencias Agrícolas (INCA) of La Habana (Cuba)

and the CEBAS-CSIC of Murcia (Spain). Authors want to thanks the

help of the Herbario Nacional de Cuba in the identification of the

Carica cubensis accessions assayed.

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