Molecular characterization of Cuban endemism Carica cubensis Solms using random amplified polymorphic DNA (RAPD) markers
ABSTRACT
The objective of this work is to present an appropriate set of RAPD (random amplified polymorphic DNA) markers using single and multiplex PCR analysis suitable for the characterization of the endemic Cuban species Carica cubensis and the establishment of genetic relationships with the cultivated species Carica papaya. 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 polymorphic) with the nine RAPD markers assayed using single and multiplex PCR analysis. Results 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 precedence 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 collection in Carica species have been also discussed.
The objective of this work is to present an appropriate set of RAPD (random amplified polymorphic DNA) markers using single and multiplex PCR analysis suitable for the characterization of the endemic Cuban species Carica cubensis and the establishment of genetic relationships with the cultivated species Carica papaya. 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 polymorphic) with the nine RAPD markers assayed using single and multiplex PCR analysis. Results 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 precedence 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 collection in Carica species have been also discussed.
Cite this paper
Rodríguez, J. , Rodríguez, P. , González, M. and Martínez-Gómez, P. (2010) Molecular characterization of Cuban endemism Carica cubensis Solms using random amplified polymorphic DNA (RAPD) markers. Agricultural Sciences, 1, 95-101. doi: 10.4236/as.2010.13012.
Rodríguez, J. , Rodríguez, P. , González, M. and Martínez-Gómez, P. (2010) Molecular characterization of Cuban endemism Carica cubensis Solms using random amplified polymorphic DNA (RAPD) markers. Agricultural Sciences, 1, 95-101. doi: 10.4236/as.2010.13012.
References
[1] Badillo, V. (1971) Monographia de la familia Caricaceae. Maracay, Venezuela. [2] Manshardt, R.M. (1992) Papaya. In: Biotechnology of Perennial Fruit Crops, F. A. Hammerschlag and R. E. Litz, Eds., Cambridge University Press, Oxford, 489-511. [3] Heinrichs, J., Lindner, M., Groth, H., Hentschel, J., Feldberg, K., Renker, C., Engel, J.J., von Konrat, M., Long, D.G. and Schneider, H. (2006) Goodbye or welcome Gondwana? Insights into the phylogenetic biogeography of the leafy liverwort Plagiochila with a description of Proskauera, gen. nov. (Plagiochilaceae, Jungermanniales). Plant Systematic and Evolution, 258, 227-250. [4] Feldberg, K., Hentschel, J. and Wilson, R. (2007) Phylogenetic biogeography of the leafy liverwort Herbertus based on nuclear and chloroplast DNA sequence data: Correlation between genetic variation and geo-graphical distribution. Journal of Biogeography, 34, 688-698. [5] Leon, H. and Alain, H. (1953) Familia Caricaceae. In: Flora de Cuba, Ed., Museo de Historia Natural del Colegio de La sale, La Habana, 352-354. [6] Solms, L. and Grafen, H. (1889) Die heimath un der ursprung des culti-virten melonebaumes, Carica papaya L. Botanische Zeitung, 49, 791-798. [7] Sing, I. and Sirohi, S.C. (1977) Sex expression studies in papaya. Plant Journal Research, 2, 150-152. [8] Reddy, G.M. (2006) Seedling leaf morphology in identification of sex types and confirmation through RAPD markers in Carica papaya L. Journal of Genetic and Breeding, 60, 1-10. [9] Oviedo, R. and Ventosa, I. (2006) Fichas del Herbario Nacional de Cuba. Academia Nacional de Ciencias de Cuba. [10] Martínez-Gómez, P., Majourhat, K., Zeinalabedini, M., Erogul, D., Khayam-Nekoui, M., Hafidi, A., Piqueras, A. and Gradziel, T.M. (2007) Use of biotechnology for preserving rare fruit germplasm. Bioremediation, Biodiversity and Bio-availability, 1, 31-40. [11] Powell, W., Morgante, M., Andre, C., Hanafey, M., Vogel, J., Tingey, S. and Rafalski, A. (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatel-lite) markers for germplasm analysis. Molecular Breeding, 2, 225-238. [12] Welsh, J. and McClelland, M. (1990) Finger printing genomes using PCR with arbitraries primers. Nucleic Acid Research, 18, 7213-7218. [13] Messaoud, C., Afif, M., Boulila, A., Rejeb, M.N. and Boussaid, M. (2007) Genetic variation of Tunisian Myrtus communis L. (Myrtaceae) populations assessed by isozymes and RAPDs. Annals of Forest Science, 63, 845-853. [14] Sharon, D., Hillel, J., Vainstein, A. and Lavi, U. (1992) Application of DNA finger-printings for identification and genetic analysis of Carica pa-paya and other Carica species. Euphytica, 62, 119-126. [15] Stiles, J.I., Lemine, C., Sondur, S., Morshidi, M.B. and Manshardt, M. (1993) Randomly amplified poly-morphic DNA for evaluating genetic relationships among pa-paya cultivars. Theoretical and Applied Genetics, 85, 697-701. [16] Vitoria, A.P., de Souza, G.A., Bressan-Smith, R.E., Pinto, F.D., Pascal, B., Guimaraes, P.S., Daben, R.F. and Gonzaga, M. (2004) DNA fingerprint of Carica papaya L. genotypes by RAPD markers. Journal of New Seed, 6, 51-65. [17] Saxena, S., Chandra, R., Srivastava, A.P., Mishra, M. and Ranade, S.A. (2005) Analysis of genetic diversity among papaya cultivars using single primer amplification reac-tion (SPAR). Journal of Horticultural Science & Biotechnology, 80, 291-296. [18] da Silva, F.F., Pereira, G.M., Campos, W., Damasceo- Junior, P.C., Santana-Pereira, T.M., Cancela-Ramos, H.G., Pio-Viana, A. and Ferregeti, G.A. (2007a) Monitoring of the genetic variability in papaya parent “Formosa” of “UENF/CALIMAN 01” hybrid via RAPD. Crop Breed- ing and Applied Biotechnology, 7, 36-42. [19] Jobin-Décor, M.P., Graham, G.C., Henry, R.J. and Drew, R.A. (1997) RAPD and isozyme analysis of relationships between Carica papaya and wild species. Genetic Resources and Crop Evolution, 44, 471-477. [20] Magdalita, P.M., Drew, R.A., Adkins, S.W and Godwin, I.D. (1997) Morphological, molecular and cytological analysis of Carica papaya L. and C. cauliflora interspecific hybrids. Theoretical and Applied Genetics, 95, 224-229. [21] Parasmis, A.S., Gupta, V.S., Tambankar, S.A. and Ranjekar, P.K. (2000) A highly reliable sex diagnosis PCR assay for mass screaning of papaya seedlings. Molecular Breeding, 6, 337-344. [22] Lemos, E.G.M., Silva, C.L. and Zcaidad, H.A. (2002) Identification of sex in Carica papaya L. using RAPD markers. Euphytica, 127, 179-184. [23] Urasaki, N., Tokumoto, M., Tanora, K., Kayano, T., Tamaka, H., Oku, H. and Terauchi, P. (2002) A male and hermaphrodite specific RAPD markers for papaya. Theoretical and Applied Genetics, 104, 281-285. [24] da Silva, F.F., Pereira, G.M., Ferrerira-Campos, W., Damasceo-Junior, P.C., Santana-Pereira, T.M., Souza- Filho, G.A., Cancela-Ramos, H.G., Pio-Viana, A. and Ferregeti, G.A. (2007b) DNA marker-assisted sex conver-sion in elite papaya genotype (Carica papaya L.). Crop Breeding and Applied Biotechnology, 7, 52-58. [25] Niroshini, E., Everard, J.M., Karunahayane, E.M. and Tirimanne, M.C.S. (2008) Detection of sequence characterized amplified region (SCAR) markers linked to sex expression in Carica papaya L. Journal of National Science Foundation of Sri Lanka, 36, 145-150. [26] Sondur, S.N., Manshard, R.M. and Stiles, J.I. (1996) A genetic linkage map of papaya based on RAPD markers. Theoretical and Applied Genetics, 93, 542-553. [27] Narvel, J.M., Chu, W.C., Fehr, W.R., Cregan, P.B. and Shoemaker, R.C. (2000) Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome. Molecular Breeding, 6, 175-183. [28] Sánchez-Pérez, R., Dicenta, F. and Martínez-Gómez, P. (2004) Identification of S-alleles in almond using multiplex PCR. Euphytica, 138, 263-269. [29] Hayden, M.J., Nguyen, T.M., Waterman, A. and Chal- mers, K.J. (2008) Multiplex-ready PCR: A new method for multiplexed SSR and SNP genotyping. BMC genomics, 9, 80 [30] Doyle, J.J. and Doyle, J.L. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemestry Bulletin, 19, 11-15. [31] Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596-1599. [32] Nei, M. and Li, W.H. (1979) Mathematical model for studying genetic variation in terms of restriction. Proceedings of the National Academy of Science of USA, 76, 5269-5273. [33] Dax, E., Livneh, O., Edelbaum, O., Kedar, N., Gavish, N., Karchi, H., Milo, J., Sela, I. and Rabinowit, H.D. (1993) A random amplified polymorphic DNA (RAPD) molecular marker for the Tm-2agene in tomato. Euphytica, 1-2, 159-163. [34] Gérard, P.R., Fernández-Manjarrés, J.F., Bertolino, P., Dufour, J., Raquin, C. and Frascaria-Lacostem, F. (2006) New insights in the recognition of the European ash species Fraxinus excelsior and Fraxinus augustifolia as useful tools for forest management. Annals of Forest Science, 63, 733-738. [35] Yamada, M.M., Faleiro, F.G., Lopes, U.V., Bahia, R.C., Pires, J.L., Gomes, L.M.C. and Melo, G.R.P. (2001) Genetic variability in cultivated cacao populations in Bahia, Brazil, detected by isozymes and RAPD markers. Crop Breeding and Applied Biotechnology, 1, 377-384. [36] Teixeira-Cabral, T.A., Sakiyama, N.S., Zambolim, L., Pereira, A.A., Gon?alves-Barros, E. and Sakiyama, C.C.H. (2002) Reproducibility of the RAPD marker and its efficiency in coffee tree genotype grouping analysis. Crop Breeding and Applied Biotechnology, 2, 121-129. [37] Wang, J.C., Hu, J., Liu, N.N., Xu, H.M. and Zhang, S. (2006) Investigation of combining plant genotypic values and molecular marker information for constructing core subsets. Journal of Integrated Plant Biology, 48, 1371- 1378. [38] Lachenaud, P. and Zhang, D. (2008) Genetic diversity and population structure in wild stands of cacao trees (Theobroma cacao L.) in French Guiana. Annals of Forest Science, 65, 310. [39] Magdalita, P.M., Villegas, V.M., Pimentel, R.B. and Bayot, R.G. (1988) Reaction of papaya and related species to ringspot virus Phillipp. Journal of Crop Science, 3, 232-234. [40] Persley, D.M. and Thomas, J.E. (1994) Screening for PRSV resistance. Annual Meeting of Control of Ringspot in papaya, Hawaii, 41-44. [41] Chavarriaga-Aguirre, P., Maya, M.M., Tohme, J., Duque, M.C., Iglesias, C., Bonierbale, M.W., Kresovich, S. and Kochert, G. (1999) Using microsatellites, isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core collection and to assess the usefulness of DNA-based markers to maintain germplasm collections. Molecular Breeding, 5, 263-273. [42] Bortolini, F., Dall’Agnol, M. and Schifino-Wittmann, M.T. (2006) Molecular characterization of the USDA white clover (Trifolium repens L.) core collection by RAPD markers. Genetic Resources and Crop Evolution, 53, 1081-1087. [43] Pérez, J.O., Dambier, D., Ollitrault, P., D′Eeckenbrugge, G.O., Brotiers, P., Froelicher, Y. and Risterucci, A.M. (2006) Microsatellite markers in Carica papaya L.: Isolation, characterization and transferability to Vascon- cellea species. Molecular Ecology Research, 6, 212-217. [44] Eustice, M., Yu, Q., WanLai, C., Thimmapuran, J., Liu, L., Afan, M., Presting, G. and Ming, R. (2008) Development and application of microsatellite markers for ge-nomics analysis in papaya. Tree Genetic and Genomic, 4, 333-341.
[1] Badillo, V. (1971) Monographia de la familia Caricaceae. Maracay, Venezuela. [2] Manshardt, R.M. (1992) Papaya. In: Biotechnology of Perennial Fruit Crops, F. A. Hammerschlag and R. E. Litz, Eds., Cambridge University Press, Oxford, 489-511. [3] Heinrichs, J., Lindner, M., Groth, H., Hentschel, J., Feldberg, K., Renker, C., Engel, J.J., von Konrat, M., Long, D.G. and Schneider, H. (2006) Goodbye or welcome Gondwana? Insights into the phylogenetic biogeography of the leafy liverwort Plagiochila with a description of Proskauera, gen. nov. (Plagiochilaceae, Jungermanniales). Plant Systematic and Evolution, 258, 227-250. [4] Feldberg, K., Hentschel, J. and Wilson, R. (2007) Phylogenetic biogeography of the leafy liverwort Herbertus based on nuclear and chloroplast DNA sequence data: Correlation between genetic variation and geo-graphical distribution. Journal of Biogeography, 34, 688-698. [5] Leon, H. and Alain, H. (1953) Familia Caricaceae. In: Flora de Cuba, Ed., Museo de Historia Natural del Colegio de La sale, La Habana, 352-354. [6] Solms, L. and Grafen, H. (1889) Die heimath un der ursprung des culti-virten melonebaumes, Carica papaya L. Botanische Zeitung, 49, 791-798. [7] Sing, I. and Sirohi, S.C. (1977) Sex expression studies in papaya. Plant Journal Research, 2, 150-152. [8] Reddy, G.M. (2006) Seedling leaf morphology in identification of sex types and confirmation through RAPD markers in Carica papaya L. Journal of Genetic and Breeding, 60, 1-10. [9] Oviedo, R. and Ventosa, I. (2006) Fichas del Herbario Nacional de Cuba. Academia Nacional de Ciencias de Cuba. [10] Martínez-Gómez, P., Majourhat, K., Zeinalabedini, M., Erogul, D., Khayam-Nekoui, M., Hafidi, A., Piqueras, A. and Gradziel, T.M. (2007) Use of biotechnology for preserving rare fruit germplasm. Bioremediation, Biodiversity and Bio-availability, 1, 31-40. [11] Powell, W., Morgante, M., Andre, C., Hanafey, M., Vogel, J., Tingey, S. and Rafalski, A. (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatel-lite) markers for germplasm analysis. Molecular Breeding, 2, 225-238. [12] Welsh, J. and McClelland, M. (1990) Finger printing genomes using PCR with arbitraries primers. Nucleic Acid Research, 18, 7213-7218. [13] Messaoud, C., Afif, M., Boulila, A., Rejeb, M.N. and Boussaid, M. (2007) Genetic variation of Tunisian Myrtus communis L. (Myrtaceae) populations assessed by isozymes and RAPDs. Annals of Forest Science, 63, 845-853. [14] Sharon, D., Hillel, J., Vainstein, A. and Lavi, U. (1992) Application of DNA finger-printings for identification and genetic analysis of Carica pa-paya and other Carica species. Euphytica, 62, 119-126. [15] Stiles, J.I., Lemine, C., Sondur, S., Morshidi, M.B. and Manshardt, M. (1993) Randomly amplified poly-morphic DNA for evaluating genetic relationships among pa-paya cultivars. Theoretical and Applied Genetics, 85, 697-701. [16] Vitoria, A.P., de Souza, G.A., Bressan-Smith, R.E., Pinto, F.D., Pascal, B., Guimaraes, P.S., Daben, R.F. and Gonzaga, M. (2004) DNA fingerprint of Carica papaya L. genotypes by RAPD markers. Journal of New Seed, 6, 51-65. [17] Saxena, S., Chandra, R., Srivastava, A.P., Mishra, M. and Ranade, S.A. (2005) Analysis of genetic diversity among papaya cultivars using single primer amplification reac-tion (SPAR). Journal of Horticultural Science & Biotechnology, 80, 291-296. [18] da Silva, F.F., Pereira, G.M., Campos, W., Damasceo- Junior, P.C., Santana-Pereira, T.M., Cancela-Ramos, H.G., Pio-Viana, A. and Ferregeti, G.A. (2007a) Monitoring of the genetic variability in papaya parent “Formosa” of “UENF/CALIMAN 01” hybrid via RAPD. Crop Breed- ing and Applied Biotechnology, 7, 36-42. [19] Jobin-Décor, M.P., Graham, G.C., Henry, R.J. and Drew, R.A. (1997) RAPD and isozyme analysis of relationships between Carica papaya and wild species. Genetic Resources and Crop Evolution, 44, 471-477. [20] Magdalita, P.M., Drew, R.A., Adkins, S.W and Godwin, I.D. (1997) Morphological, molecular and cytological analysis of Carica papaya L. and C. cauliflora interspecific hybrids. Theoretical and Applied Genetics, 95, 224-229. [21] Parasmis, A.S., Gupta, V.S., Tambankar, S.A. and Ranjekar, P.K. (2000) A highly reliable sex diagnosis PCR assay for mass screaning of papaya seedlings. Molecular Breeding, 6, 337-344. [22] Lemos, E.G.M., Silva, C.L. and Zcaidad, H.A. (2002) Identification of sex in Carica papaya L. using RAPD markers. Euphytica, 127, 179-184. [23] Urasaki, N., Tokumoto, M., Tanora, K., Kayano, T., Tamaka, H., Oku, H. and Terauchi, P. (2002) A male and hermaphrodite specific RAPD markers for papaya. Theoretical and Applied Genetics, 104, 281-285. [24] da Silva, F.F., Pereira, G.M., Ferrerira-Campos, W., Damasceo-Junior, P.C., Santana-Pereira, T.M., Souza- Filho, G.A., Cancela-Ramos, H.G., Pio-Viana, A. and Ferregeti, G.A. (2007b) DNA marker-assisted sex conver-sion in elite papaya genotype (Carica papaya L.). Crop Breeding and Applied Biotechnology, 7, 52-58. [25] Niroshini, E., Everard, J.M., Karunahayane, E.M. and Tirimanne, M.C.S. (2008) Detection of sequence characterized amplified region (SCAR) markers linked to sex expression in Carica papaya L. Journal of National Science Foundation of Sri Lanka, 36, 145-150. [26] Sondur, S.N., Manshard, R.M. and Stiles, J.I. (1996) A genetic linkage map of papaya based on RAPD markers. Theoretical and Applied Genetics, 93, 542-553. [27] Narvel, J.M., Chu, W.C., Fehr, W.R., Cregan, P.B. and Shoemaker, R.C. (2000) Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome. Molecular Breeding, 6, 175-183. [28] Sánchez-Pérez, R., Dicenta, F. and Martínez-Gómez, P. (2004) Identification of S-alleles in almond using multiplex PCR. Euphytica, 138, 263-269. [29] Hayden, M.J., Nguyen, T.M., Waterman, A. and Chal- mers, K.J. (2008) Multiplex-ready PCR: A new method for multiplexed SSR and SNP genotyping. BMC genomics, 9, 80 [30] Doyle, J.J. and Doyle, J.L. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemestry Bulletin, 19, 11-15. [31] Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596-1599. [32] Nei, M. and Li, W.H. (1979) Mathematical model for studying genetic variation in terms of restriction. Proceedings of the National Academy of Science of USA, 76, 5269-5273. [33] Dax, E., Livneh, O., Edelbaum, O., Kedar, N., Gavish, N., Karchi, H., Milo, J., Sela, I. and Rabinowit, H.D. (1993) A random amplified polymorphic DNA (RAPD) molecular marker for the Tm-2agene in tomato. Euphytica, 1-2, 159-163. [34] Gérard, P.R., Fernández-Manjarrés, J.F., Bertolino, P., Dufour, J., Raquin, C. and Frascaria-Lacostem, F. (2006) New insights in the recognition of the European ash species Fraxinus excelsior and Fraxinus augustifolia as useful tools for forest management. Annals of Forest Science, 63, 733-738. [35] Yamada, M.M., Faleiro, F.G., Lopes, U.V., Bahia, R.C., Pires, J.L., Gomes, L.M.C. and Melo, G.R.P. (2001) Genetic variability in cultivated cacao populations in Bahia, Brazil, detected by isozymes and RAPD markers. Crop Breeding and Applied Biotechnology, 1, 377-384. [36] Teixeira-Cabral, T.A., Sakiyama, N.S., Zambolim, L., Pereira, A.A., Gon?alves-Barros, E. and Sakiyama, C.C.H. (2002) Reproducibility of the RAPD marker and its efficiency in coffee tree genotype grouping analysis. Crop Breeding and Applied Biotechnology, 2, 121-129. [37] Wang, J.C., Hu, J., Liu, N.N., Xu, H.M. and Zhang, S. (2006) Investigation of combining plant genotypic values and molecular marker information for constructing core subsets. Journal of Integrated Plant Biology, 48, 1371- 1378. [38] Lachenaud, P. and Zhang, D. (2008) Genetic diversity and population structure in wild stands of cacao trees (Theobroma cacao L.) in French Guiana. Annals of Forest Science, 65, 310. [39] Magdalita, P.M., Villegas, V.M., Pimentel, R.B. and Bayot, R.G. (1988) Reaction of papaya and related species to ringspot virus Phillipp. Journal of Crop Science, 3, 232-234. [40] Persley, D.M. and Thomas, J.E. (1994) Screening for PRSV resistance. Annual Meeting of Control of Ringspot in papaya, Hawaii, 41-44. [41] Chavarriaga-Aguirre, P., Maya, M.M., Tohme, J., Duque, M.C., Iglesias, C., Bonierbale, M.W., Kresovich, S. and Kochert, G. (1999) Using microsatellites, isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core collection and to assess the usefulness of DNA-based markers to maintain germplasm collections. Molecular Breeding, 5, 263-273. [42] Bortolini, F., Dall’Agnol, M. and Schifino-Wittmann, M.T. (2006) Molecular characterization of the USDA white clover (Trifolium repens L.) core collection by RAPD markers. Genetic Resources and Crop Evolution, 53, 1081-1087. [43] Pérez, J.O., Dambier, D., Ollitrault, P., D′Eeckenbrugge, G.O., Brotiers, P., Froelicher, Y. and Risterucci, A.M. (2006) Microsatellite markers in Carica papaya L.: Isolation, characterization and transferability to Vascon- cellea species. Molecular Ecology Research, 6, 212-217. [44] Eustice, M., Yu, Q., WanLai, C., Thimmapuran, J., Liu, L., Afan, M., Presting, G. and Ming, R. (2008) Development and application of microsatellite markers for ge-nomics analysis in papaya. Tree Genetic and Genomic, 4, 333-341.