Publicado

2006-07-01

Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD

Analysis of genetic variation in clones of rubber (Hevea brasiliensis) from Asían, South and Central American origin using RAPDs markers

Palabras clave:

cultivar identification, genetic distance, genetic diversity, Hevea brasiliensis, RAPD marker, identificación de cultivos, distancia genética, diversidad genética, marcadores RAPD (es)

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Autores/as

  • César Augusto Hernández R Biólogo
  • Lucía Afanador Kafuri Bióloga, candidata a PhD Facultad de Ciencias Agropecuarias
  • Rafael Arango Isaza Médico PhD Departamento de Biología
  • Mario Lobo Arias Agrónomo, PhD Programa Nacional de Recursos Genéticos

El caucho natural (Hevea brasiliensis) representa a especies potenciales para reforestación y programas de explo­tación comercial en ciudades tropicales como Colombia. La variabilidad genética de una colección de caucho que se encuentra en la Estación experimental de Paraguaycito en Buenavista, departamento del Quindio en Colom­bia fue estudiada para aumentar el conocimiento en cuanto a las especies y realizar un mejor uso de los árboles disponibles. Un total de 25 clones, seis de Sur América, 17 de Asia y 2 de América Central fueron seleccionados y analizados usando RAPDs. Las muestras aisladas de ADN de los árboles fueron con 102 primers, 23 de los cuales mostraron polimorfismos. Aunque se encontró un alto grado de similaridad, los análisis grupales de datos llevaron a diferenciar los árboles de de caucho en términos de su origen geográfico. Por lo tanto, las relaciones genéticas que se encontraron entre los clones podrían ayudar a seleccionar parentales para uso en programas de reproducción y diseño de estrategias para la conservación de los clones que tengan características agronómicas deseables.

Palabras clave: identificación de cultivos, distancia genética, diversidad genética, Hevea brasiliensis, marcadores RAPD.

Rubber (Hevea brasiliensis) represents a potential species for reforestation and commercial exploitation programmes in tropical countries such as Colombia. The genetic variability of a rubber collection kept at the Paraguaicito Experimental Station in Buenavista in the Quindio department of Colombia was studied to improve knowledge regarding this species and make better use of the trees available. A total of 25 clones, six from South-America, 17 from Asia and two from Central-América were selected and analysed using RAPDs. DNA samples isolated from the trees were screened with 102 primers, 23 of which revealed polymorphism. Although a high degree of similarity was found, clustering analysis of the data led to differentiating the rubber trees in terms of their geographical origin. Furthermore, genetic relationships were found amongst the clones which could help in selecting parents for use in breeding programmes and designing strategies for conserving clones having desirable agronomic traits.

Key words: cultivar identification, genetic distance, genetic diversity, Hevea brasiliensis, RAPD marker

vol_viii_No_2_2006_diciembre-29-34.htm
ANALYSIS OF GENETIC VARIATION IN CLONES OF RUBBER (Hevea brasiliensis) FROM ASÍAN, SOUTH AND CENTRAL AMERICAN
Analysis of genetic variation in clones of rubber (Hevea brasiliensis) from Asían, South and Central American origin using RAPDs markers
Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD
César Augusto Hernández R.*, Lucía Afanador Kafuri** Rafael Arango Isaza ***,
Mario Lobo Arias ****
ABSTRACT
Rubber (Hevea brasiliensis) represents a potential species for reforestation and commercial exploitation programmes in tropical countries such as Colombia. The genetic variability of a rubber collection kept at the Paraguaicito Experimental Station in Buenavista in the Quindio department of Colombia was studied to improve knowledge regarding this species and make better use of the trees available. A total of 25 clones, six from South-America, 17 from Asia and two from Central-América were selected and analysed using RAPDs. DNA samples isolated from the trees were screened with 102 primers, 23 of which revealed polymorphism. Although a high degree of similarity was found, clustering analysis of the data led to differentiating the rubber trees in terms of their geographical origin. Furthermore, genetic relationships were found amongst the clones which could help in selecting parents for use in breeding programmes and designing strategies for conserving clones having desirable agronomic traits.
Key words: cultivar identification, genetic distance, genetic diversity, Hevea brasiliensis, RAPD marker.
RESUMEN
El caucho natural (Hevea brasiliensis) representa a especies potenciales para reforestación y programas de explotación comercial en ciudades tropicales como Colombia. La variabilidad genética de una colección de caucho que se encuentra en la Estación experimental de Paraguaycito en Buenavista, departamento del Quindio en Colombia fue estudiada para aumentar el conocimiento en cuanto a las especies y realizar un mejor uso de los árboles disponibles. Un total de 25 clones, seis de Sur América, 17 de Asia y 2 de América Central fueron seleccionados y analizados usando RAPDs. Las muestras aisladas de ADN de los árboles fueron con 102 primers, 23 de los cuales mostraron polimorfismos. Aunque se encontró un alto grado de similaridad, los análisis grupales de datos llevaron a diferenciar los árboles de de caucho en términos de su origen geográfico. Por lo tanto, las relaciones genéticas que se encontraron entre los clones podrían ayudar a seleccionar parentales para uso en programas de reproducción y diseño de estrategias para la conservación de los clones que tengan características agronómicas deseables.
Palabras clave: identificación de cultivos, distancia genética, diversidad genética, Hevea brasiliensis, marcadores RAPD.
Recibido: junio 07 de 2006 Aceptado: agosto 22 de 2006
* Biólogo, MS. c Facultad de Ciencias Agrarias. Politécnico Colombiano Jaime Isaza Cadavid. Tel. (4) 466 37 34, (4) 452 09 99
ext. 102. Correo electrónico: cesaraugu@une.net.co
** Bióloga, candidata a Ph. D Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia sede Medellín. Tel. (4) 430
90 00. Correo electrónico: lafanado@unalmed.edu.co
*** Médico, Ph. D Departamento de Biología, Escuela de Biociencias, Universidad Nacional de Colombia, sede Medellín.
Tel. (4) 441 08 55 ext. 235. Correo electrónico: rafaelarango@une.net.co
**** Agrónomo, Ph. D Programa Nacional de Recursos Genéticos, Corporación Colombiana de Investigación Agropecuaria,
CORPOICA, La Selva, Rionegro, Antioquia, Colombia. Tel. (4) 537 11 33. Correo electrónico: pnrgv@une.net.co
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Rev. Colomb. Biotecnol. Vol. VIII N° 2 Diciembre 2006 29-34_____________________________________________
INTRODUCTION
Rubber (Hevea brasiliensis) represents a potential species for agricultural production and reforestation programmes because it protects water resources and provides the community with long-term profitability (Rincón, 1996). However, the appropriate use of clonally propagated trees is hindered by a lack of knowledge regarding their genetic variability. Cultivated rubber's genetic basis is very constricted since it has been derived from a limited number of trees which were taken to England from the Amazonian rain-forests by Sir Henry Wickham in 1876 (Carrón et ál., 1989). Rubber trees were introduced to Asia, África and back to the Americas from this original collection. The trees were probably underwent different breeding strategies in these locations, involving selecting intra- and inter-specific cross-derived offspring.
Several methods have been used for studying rubber's genetic variability, including isoenzyme analysis and other molecular techniques such as RFLPs, RAPDs and micro-satellites (Besse et ál., 1993,1994;Seguinetál, 1995). These studies have revealed a degree of clustering according to geographical origin in wild and clonally selected populations, the latter offering the advantage of not being affected by environmental factors and leading to determining the degree of divergence amongst different trees. RAPDs markers have been used for determining rubber's genetic variability and (although rather high variability was found) the markers used did not lead to discriminating the trees' geographical origin (Staubetál., 1996; Vargheseetál., 1997).
The molecular characterisation of 25 rubber clones from Asia, South- and Central-América origins was carried out in this study using several RAPDs markers. This approach led to evaluating the potential of the trees to be used in programmes aimed at introducing the species into the central Colombian coffee-growing área.
MATERIALS AND METHODS Plant Materials
Samples of vegetatively propagated clones were collected from Paraguaicito Experimental Station
nursery stock (CENICAFE), Buenavista, in the Quindio department of Colombia. Young leaves were collected from one-year-old trees from 25 South-American (6), Asian (17) and Central-American (2) clones (Table 1).
DNA extraction
DNA was extracted following the methodology described by Varghese et ál., (1997) with some modifications. Young leaves from each previously stored sample were macerated to a fine powder in liquid nitrogen; 50-100 mg of macerated tissue was then mixed with 4 vol 1X CTAB extraction buffer (2% hexadecyltrimethyl-ammonium bromide (CTAB), 20 mM EDTA, 100 mM Tris HCl pH 8.0, 1.4 M NaCl). Samples were placed in a water bath at 65 °C for 30 min followed by two chlorophorm/isoamil (24:1) extractions. DNA was precipitated by adding 1 vol cold isopropanol with gentle mixing until DNA strands were observed. The samples were then centrifuged at 13,000 rpm and the precipítate was washed with cold 70% ethanol at room temperature. The resulting DNA pellet was then dried and suspended in 50-100 uL TE buffer (1 mM Tris HCl and 0.1 mM EDTA, pH 8.0). DNA concentration was determined by BioRad VersaFluor Fluoremeter and then diluted to final 10 ng/u.L concentration.
RAPD assay
DNA amplification conditions using RAPD primers were similar to those described by Varghese et ál., (1997). However, the primers used were from Operon Technologies series OPA, OPAN, OPBA, OPE, OPM plus OPK-19 and OPD-17 previously reported by Seguin, et ál., (1995). A total of 102 primers were tested for choosing those giving polymorphic patterns. Each 25 u.L reaction mixture consisted of 0.1 mM dNTPs (Promega, Madison, WI, USA), 1.9 mM MgCI2, 1X buffer, 0.2 uM primer, 1.4U Taq DNApolymerase (Promega®, Madison, WI, USA) and 50 ng genomic DNA. Amplifications were performed on an MJ Research Inc. Thermal Cycler PTC-100, with an initial cycle of 30s at 94 °C, 45 cycles of 1 min at 92 °C, 1 min at 35 °C and 2 min at 72 °C, followed by a final cycle of 5 min at 72 °C. Amplification products were then separated by electrophoresis at 100V (0.05 Amp) for 3h on 1.4% agarose gels in 1X TBE buffer with 3 mg/mL ethidium
30

ANALYSIS OF GENETIC VARIATION IN CLONES OF RUBBER (Hevea brasiliensis) FROM ASÍAN, SOUTH AND CENTRAL AMERICAN
Table 1. Identification and geographical origin of the 25 Hevea brasiliensis clones used and analysed in this study
Id
Clone
Geographical Origin
1
IAN-713
Brazil (they carne to Paraguaicito from Caquetá)
2
IAN-717
Brazil
3
IAN-2878
Brazil
4
IAN-3087
Brazil
5
IAN-710
Brazil
6
IAN-873
Brazil
7
RRIC-121
Sri Lanka (they carne to Paraguaicito from Guadalupe Island in the Antillas)
8
RRIC-130
Sri Lanka
9
RRIC-132
Sri Lanka
10
RRIC- 42
Sri Lanka
11
RRIC-102
Sri Lanka
12
PR- 255
Indonesia
13
PR- 261
Indonesia
14
93114
Indonesia
15
RRIM- 228
Malaysia
16
RRIM 600
Malaysia
17
RRIM- 703
Malaysia
18
PB 28/59
Malaysia
19
PB- 254
Malaysia
20
PB- 235
Malaysia
21
IR- 22
Vietnam
22
IR- 42
Vietnam
23
AVROS- 2037
Sumatra
24
GT-1
Guatemala
25
GU- 198
Guatemala
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Rev. Colomb. Biotecnol. Vol. VIII N° 2 Diciembre 2006 29-34___________________________________________________________________
bromide.AI kbDNAIadder(Promega, Madison, WI, USA) was used as molecular weight standard. The gels were photographed under ultraviolet light using a Kodak Digital Science® camera system and Adobe Photo de Luxe software.
Statistical analysis
Data was scored as discrete variables using 1 to indícate the presence and 0 the absence of a band. All amplifications were repeated at least three times and only bands which were 100% reproducible in repeated amplifications were considered for estimating genetic distances. Genetic distance was estimated by using Dice's coefficient of similarity (Dice, 1945). A pair-wise distance matrix between genotypes was the basis for cluster analysis by un-weighted pair-group mathematical average (UPGMA) using numerical taxonomy and multivariate analysis system (NTSYS) software, versión 2.0 (Rohlf, 1998).
RESULTS AND DISCUSSION
A total of 102 primers were tested using a mixture of DNA from three clones from different geographical origins (IAN-713, RRIC-121 and GT-1). From these, 65 (23%) presented polymorphism amongst the clones and 42 (65%) produced monomorphic fragments. Figure 1 shows amplification products with primers. The number of polymorphic DNA fragments for each evaluated pri-
mer ranged from 1 to 12, having an average of 6 bands per primer.
Amplified product size varied from 350 bp (primers BA-7 and AN-17) to 3,500 bp (primer AN-15), agreeing with the range reported by Nurhaimi-Haris et ál., (1998) who found fragments ranging from 300 to 2,000 bp. A 1,250 bp fragment was frequently present on Asian clones (PR-261 and RRIC-121) but absent on South-American and Cen-tral-American ones; this marker was thus able to differentiate American clones.
Analysing genetic similarity revealed clones clustering into two well-differentiated groups, A and B (Figure 2) differing by 43% from the markers. Other studies have shown small genetic differences ranging from 20% to 30% (Seguin et ál., 1995; Nurhaimi-Haris etál., 1998). Thisindicatesthat(asexpected) there was an important genetic relationship amongst the individuáis from this collection but that there is still a source of genetic variation which can be used in breeding programmes. The analysis also revealed an evident geographical correlation, since all South-American clones were in group A together with a few Asian clones (RRIC-130, RRIC-121, RRIC-132 and PB-254). This differs from that found by several authors (Varghese et ál., (1997); Seguin et ál., (1995); Besse et ál., (1993) and Nurhaimi-Haris et ál., (1998)) where no correlation was found between genetic diversity and geographical
vol_viii_No_2_2006_diciembre-29-34-1.jpg
Figure 1 . Agarose gel electrophoresis of the amplified products from 25 Hevea clones using the OPBA9 (a) and OPAN15 (b) primers. M corresponds to the 1 Kb ladder (Promega). Lañes 1-25 correspond to the amplification products from the different rubber clones in the same order as indicated in Table 1. Samples were run on 1.4% agarose gel following PCR reactions.
32

ANALYSIS OF GENETIC VARIATION IN CLONES OF RUBBER (Hevea brasiliensis) FROM ASÍAN, SOUTH AND CENTRAL AMERICAN
vol_viii_No_2_2006_diciembre-29-34-2.jpg
Figure 2. Dendogram of 25 Hevea clones based on RAPD analysis. Genetic distance were estimated according to Dice coefficient of similarity and clustering was done using the UPGMA method.
origin. The difference observed here may correspond to the fact that a different set of primers was used in this study.
Group B included most of the Asian clones plus two Central-American ones (GT-1 and GU-198). This tendency was also observed when the markers from individual primers were analysed independently (data not shown), indicating that the Central American clones genetic basis was more closely related to the Asian ones, sharing 70% of their markers. This may indícate that Guatemala germplasm was directly introduced from Asian clones.
Figure 2 also indicates that the Asian PB-254 clone forms a subgroup with South-American IAN-713 and IAN-873 clones, being different from the subgroup to which all the other Asian clones belonged. These findings could be useful for breeders since it could be a good idea to cross clone PB-254 with the Asian clones in the other subgroup to increase their genetic base, especially for programmes aiming to improve quantitative agronomic characteristics, such as látex production.
South-American clones IAN-713 and IAN- 873 belonged to same subgroup and may not be good candidates for being crossed in a breeding programme, given their more than 75% Similarity. Clone IAN-710 and clones RRIC-121 and RRIC-132 shared 73% similarity and clone RRIC-130 shared 62% similarity with all South-American clones, being more closely related to clones IAN-717 and IAN-2878.
The present study has shown that the RAPD markers used were able to differentiate South-American Hevea clones from most of the others which have been reintroduced from Asia. Analysing the markers obtained led to visualising relationships amongst the clones which may be useful in breeding programmes. This fact underlines the importance of carrying out genetic similarity studies since they may give a better idea of the genetic relationships of plant materials used for genetic improvement.
ACKNOWLEDGMENTS
This work was financed by the Universidad Nacional de Colombia gradúate Biotechnology
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Rev. Colomb. Biotecnol. Vol. VIII N° 2 Diciembre 2006 29-34_________________________________________________________
Programme in Medellín and Politécnico Colombiano Jaime Isaza Cadavid also in Medellín.
(Hevea brasiliensis Muell, Arg.) clones. Menara Perkebunan, 66: 9-19.
Rincón, O. 1996. Manual del cultivo del caucho. CORDICAFÉ Publishers, Santafe de Bogotá. Pág. 194.
Rohlf, F.J. 1998. NTSYS, Numerical Taxonomy And Multivariate Analysis System. Versión 2.0. User guide. Department of Ecology and Evolution State University of New York. Publisher, Stony Brook, NY. P. 31
Seguin, M.; Besse, P; Lespinasse, D.; Lebrun, P; Rodier-Goud, M.; Nicolás, D. 1995 Characte-rization of genetic diversity and Hevea genome mapping by biochemical and molecular markers. Proc. of the Symposium on Physiological Molecular and Aspect of the breeding of Hevea. Penag, Malaysia. 19-30.
Staub, J.E.; Serquen, F.C. 1996. Genetic markers, map construction, and their application in plant breeding. Hort. Sci. 31(5): 729-741.
Varghese, YA.; Knaak, C; Sethuraj, M.R.; Ecke, W. 1997. Evaluation of Randon Amplified Polymorphic DNA (RAPD) markers in Hevea brasiliensis. Plant Breeding. 116: 47-52.
BIBLIOGRAPHY
Besse, R; Seguin, M.; Lebrun, R; Lanaud, C. 1993.
Ribosomal DNA variations in wild and cultivated
tree (Hevea brasiliensis). Genome. 36: 1049-
1057. Besse, R; Seguin, M.; Lebrun, R; Chevallier, M.H.;
Nicolás, D.; Lanaud, C. 1994. Genetic diversity
among wild and cultivated populations of Hevea
brasiliensis assessed by nuclear RFLP
analysis. Theor Appl Genet. 88: 199-207. Carrón, M.P.; Enjalric, F.; Lardet, L.; Deschamps,
A. 1989. Rubber (Hevea brasiliensis Müll. Arg.j.
Biotechnology in Agriculture and Forestry. Vol.
5, 222-245. Dice, L.R. 1945. Measures of the amount of
ecological association between species.
Ecology. 26: 297-302. Nurhaimi - Haris, W.S.; Darussamin, A. 1998. RAPD
Analysis of genetic variability in plant rubber

34

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APA

Hernández R, C. A., Afanador Kafuri, L., Arango Isaza, R. y Lobo Arias, M. (2006). Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD. Revista Colombiana de Biotecnología, 8(2), 29–34. https://revistas.unal.edu.co/index.php/biotecnologia/article/view/512

ACM

[1]
Hernández R, C.A., Afanador Kafuri, L., Arango Isaza, R. y Lobo Arias, M. 2006. Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD. Revista Colombiana de Biotecnología. 8, 2 (jul. 2006), 29–34.

ACS

(1)
Hernández R, C. A.; Afanador Kafuri, L.; Arango Isaza, R.; Lobo Arias, M. Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD. Rev. colomb. biotecnol. 2006, 8, 29-34.

ABNT

HERNÁNDEZ R, C. A.; AFANADOR KAFURI, L.; ARANGO ISAZA, R.; LOBO ARIAS, M. Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD. Revista Colombiana de Biotecnología, [S. l.], v. 8, n. 2, p. 29–34, 2006. Disponível em: https://revistas.unal.edu.co/index.php/biotecnologia/article/view/512. Acesso em: 28 mar. 2024.

Chicago

Hernández R, César Augusto, Lucía Afanador Kafuri, Rafael Arango Isaza, y Mario Lobo Arias. 2006. «Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD». Revista Colombiana De Biotecnología 8 (2):29-34. https://revistas.unal.edu.co/index.php/biotecnologia/article/view/512.

Harvard

Hernández R, C. A., Afanador Kafuri, L., Arango Isaza, R. y Lobo Arias, M. (2006) «Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD», Revista Colombiana de Biotecnología, 8(2), pp. 29–34. Disponible en: https://revistas.unal.edu.co/index.php/biotecnologia/article/view/512 (Accedido: 28 marzo 2024).

IEEE

[1]
C. A. Hernández R, L. Afanador Kafuri, R. Arango Isaza, y M. Lobo Arias, «Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD», Rev. colomb. biotecnol., vol. 8, n.º 2, pp. 29–34, jul. 2006.

MLA

Hernández R, C. A., L. Afanador Kafuri, R. Arango Isaza, y M. Lobo Arias. «Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD». Revista Colombiana de Biotecnología, vol. 8, n.º 2, julio de 2006, pp. 29-34, https://revistas.unal.edu.co/index.php/biotecnologia/article/view/512.

Turabian

Hernández R, César Augusto, Lucía Afanador Kafuri, Rafael Arango Isaza, y Mario Lobo Arias. «Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD». Revista Colombiana de Biotecnología 8, no. 2 (julio 1, 2006): 29–34. Accedido marzo 28, 2024. https://revistas.unal.edu.co/index.php/biotecnologia/article/view/512.

Vancouver

1.
Hernández R CA, Afanador Kafuri L, Arango Isaza R, Lobo Arias M. Análisis de la variación genética en clones de caucho (Hevea brasiliensis) de Asia, Suramérica y Centroamérica usando marcadores RAPD. Rev. colomb. biotecnol. [Internet]. 1 de julio de 2006 [citado 28 de marzo de 2024];8(2):29-34. Disponible en: https://revistas.unal.edu.co/index.php/biotecnologia/article/view/512

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