- Email: [email protected]
Selection of sugarcane plants resistant to SCMV
Plant Science 165 (2003) 221 /225 www.elsevier.com/locate/plantsci
Selection of sugarcane plants resistant to SCMV A.Y. Zambrano *, J.R. Demey, M. Fuchs, V. Gonza´lez, R. Rea, O. De Sousa, Z. Gutie´rrez Instituto Nacional de Investigaciones Agropecuarias (INIA), Apartado 4521 Maracay 2101, Venezuela Received 15 July 2002; received in revised form 12 March 2003; accepted 12 March 2003
Abstract Sugarcane (Saccharum spp.) is susceptible to several major diseases transmitted by insects such as Sugarcane Mosaic Virus (SCMV). In very susceptible plants, SCMV causes severe dwarfing, leaf and stem necrosis and serious production losses. We obtained sugarcane clones resistant to SCMV using induction mutation by irradiating. Calli from the susceptible cultivar B6749 with 2 krads of gamma rays. The regenerated plantlets were tested for resistance to strain B of SCMV in the greenhouse and the resistant clones were transferred to the field. They have shown stable resistance to viral infection for eight generations of asexual reproduction. To detect genetic changes related to the acquired resistance, 15 resistant clones and a clone from the original cultivar B6749 were analyzed using Random Amplified Polymorphic DNAs (RAPD). The RAPD patterns obtained with the primer OPM14 detected a 854 bp fragment in the 15 resistant clones, which was not present in cultivar B6749. The study of genetic relations between the genotypes resistant to SCMV and its maternal B6749, using all RAPD primers, defined the existence of an associated polymorphic pattern to the resistance to SCMV. Existence of specific patterns for each genotype in the study was also observed. Analysis using RAPDs efficiently differentiated sugarcane clones resistant to SCMV by their genetic changes due to induced mutation. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calli; Mutation; RAPDs; Saccharum spp.; Sugarcane Mosaic Virus
1. Introduction Sugarcane (Saccharum spp.) is a large and highly heterozygous and heterogenous clonally propagated grass of the gramineae family, characterized by a high degree of polyploidy and is a crop of major importance providing about 65% of the sugar produced in the world. Cultivated sugarcane varieties are derived from complex interspecific hybridization between the species S. spontaneum (2n/40/128) and S. officinarum (2n / 60 or 80) [1]. Sugarcane is susceptible to several major viral diseases transmitted by insects. At the present time the most severe viral disease is caused by the Sugarcane Mosaic Virus (SCMV). In very susceptible plants it causes severe dwarfing, leaf and stem necrosis and serious production losses. Development of disease resistant varieties by conventional breeding takes 10/ * Corresponding author. Tel./fax: /58-243-2474111. E-mail address: [email protected] (A.Y. Zambrano).
15 years for one cycle of complete selection [2]. Although mutation breeding of sugarcane is not substitute for conventional breeding, mutation breeding may be considered as a means to improve the products of conventional breeding. Mutations can be induced in calli that primarily change one or a few characters of an outstanding cultivar without altering the remaining phenotypes. Induction of resistance to diseases through tissue culture has provided potentially useful in plants with a specific capability to endure the test compounds. Some attempts to induce tolerance using somaclonal variation and in vitro selection of induced mutations have already been reported. Induced mutations have allowed introduction of stable, desirable traits in different crops species, such as wheat [3,4], rice [5 /7], barley [8], sesame [9], and soybean [10]. In sugarcane, promising results during several cycles of vegetative propagation have been shown in many reports [11 /13]. Various molecular marker types could be used to detect genetic changes related to the induced resistance.
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00162-6
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We chose Random Amplified Polymorphic DNA (RAPD) [14] because (a) it allows random amplification of DNA sequences throughout the entire genome, (b) RAPD polymorphisms result from a nucleotide base change that alters the primer binding site, or from an insertion or deletion within the amplified region [15], (c) polymorphisms usually result in the presence or absence of an amplification product from a single locus [16], (d) these products of amplification can be polymorphic and are used as genetic markers [17]. In sugarcane, RAPD technology has been used to study genetic stability in plants obtained by in vitro cultures [18], to identify somaclonal variants resistant to SCMV [19], to detect the genetic variability among cultivars [20], to analyze the diversity and phylogeny in the genera Saccharum [21] and to identify pre-existing variability selected in vitro for tolerance to glyphosate [22]. Here, we report production of sugarcane clones resistant to SCMV, strain B, using induction mutation, the differences detected in the RAPD patterns of the clones resistant to SCMV compared with the maternal plant.
2. Materials and methods 2.1. Plant material Calli from cultivated sugarcane variety B6749, susceptible to SCMV were irradiated with 2 krad of gamma rays using a 60Co source. After regeneration, the plantlets were tested in the greenhouse using manual inoculation with strain B of SCMV. Then 15 selected plants, free of mosaic symptoms, were transferred to the field station at CIAE-Yaritagua, Yaracuy, Venezuela. The plants were tested for eight generations of asexual reproduction. The 15 resistant clones (2-21-19, 2-23-08, 2-25-28, 2-25-62, 2-29-04, 2-31-06, 2-32-06, 2-32-23, 232-28, 2-32-46, 2-32-49, 2-32-67, 2-34-06, 2-34-31 and 234-36) were selected and compared with the maternal plant (cv B6749) using the RAPD technique. 2.2. DNA analysis Genomic DNA was isolated from fresh, young tissue of sugarcane leaves according to Hoisington et al. [23]. The quality and concentration of genomic DNA were determined by 1% agarose gel electrophoresis. For PCR amplification, the genomic DNA concentration was adjusted to 100 ng/ml and stored at /20 8C. PCR was performed according to Zambrano [22] in a total volume of 25 ml containing 10 mM Tris /HCl (pH 9.0), 50 mM KCl, 0.1% Triton† X-100, 2.5 mM MgCl2, 200 mM of each dNTPs, 0.2 mM primer, 25 ng of genomic DNA and 1 unit Taq DNA polymerase (Promega). The reaction mixture was overlaid with 20 ml of mineral oil. For amplification, twelve primers from Operon
technologies were used: OPA01, OPA04, OPA07, OPB04, OPB07, OPK03, OPK05, OPK15, OPM04, OPM14, OPM18 and OPM20. RAPDs were amplified in a Techne GeneE thermalcycler. An initial denaturation cycle was done at 94 8C for 5 min, followed by 45 cycles of amplification by denaturing at 94 8C for 1 min, annealing at 36 8C for 30 s, and extension at 72 8C for 2 min. The final step was a single extension cycle at 72 8C for 7 min. Amplification products were analyzed by electrophoresis in 1.4% agarose gels using Tris /borate buffer, stained with ethidium bromide and photographed under UV light. l Phage DNA double digested with Hin dIII and Eco RI endonucleases was used as a molecular weight marker. The gels were digitized using the 1D Image Software Analysis [24].
2.3. Data analysis The percentage of polymorphic loci P , the number of alleles A and the average number of alleles per polymorphic locus Ap were calculated according to the following formulae [25]: Percentage of polymorphic loci P: P /(K /n )/100% where k is the number of polymorphic loci, n is the total number of loci investigated. Average number of alleles per locus A: A /aAi / n , where Ai is the number of alleles at the i th locus, n is the total number of loci investigated. Average number of alleles per polymorphic loci Ap: Ap /aApi/np, where Api is the number of alleles at a certain polymorphic locus, np is the total number of genotypes per loci investigated. All polymorphic primers data were included in the analysis. The amplified fragments were scored as present or absent. Genetic relationships among the maternal genotype and the 15 mutants were investigated using cluster analysis of the similarity data and were depicted in a dendrogram. The coefficients of Jaccard, Dice, Simple matching and Rogers and Tanimoto’s distance [26] and three methods of aggregation: single linkage, complete linkage and unweighted pair-group were studied [27]. The co-phenetic value based on genetic similarity was calculated. This is a quantitative indication of the grouping analysis performance and evaluates the stability of the constructed relationship trees [28]. Bootstrapping methodology was used to estimate standard error and confidence intervals of the mean distance in each node. Ordination analysis was performed to study the relationship between the maternal genotype and the 15 mutants, the double-centered similarity matrix was factored and a plot was made showing the genotypes in a 2-dimensional space. All the analyses were computed with the NTSYS-pc version 2.10t program [29].
A.Y. Zambrano et al. / Plant Science 165 (2003) 221 /225
3. Result and discussion The method of genomic DNA extraction was quick, simple and efficient. It produced DNA of good quality with little degradation, and the concentration was between 100 and 250 ng/ml. We tested the sensitivity of the RAPD technique for detecting polymorphisms among the cv B6749 (susceptible to SCMV) and the clones 2-21-19, 2-23-08, 2-25-28, 2-25-62, 2-29-04, 2-3106, 2-32-06, 2-32-23, 2-32-28, 2-32-46, 2-32-49, 2-32-67, 2-34-06, 2-34-31 and 2-34-36 (resistant to SCMV) generated from gamma ray irradiation. Amplification products were obtained in the sixteen DNA samples with the twelve primers tested. High polymorphism was observed with all primers, but of the twelve primers, only OPM-14 was useful for differentiating the mutant resistant clones. The percentage of polymorphic loci P was 90, the abundance of genes in a population the number of alleles A was 2 and the average number of alleles per polymorphic locus Ap was 1.98. The RAPD patterns obtained with OPM-14 detected DNA fragment smaller than 1250 bp in the sixteen samples (Fig. 1). These amplification products revealed a fragment of 854 bp that was present the 15 resistant clones. This fragment was not present in the original cultivar susceptible to SCMV (B6749). Fig. 2a shows the distribution of the co-phenetic values for the different combinations from similarity coefficients and the distance and methods of aggregation, the highest (r /0.98) value is displayed by the dendrogram generated from the UPGMA cluster analysis based on Jaccard’s coefficient that indicated the method that defines the best grouping of RAPD patterns.
223
The study of genetic relations between the maternal genotype (B6749) and the resistant clones using the OPM-14 primer (Fig. 2b) indicates that little or no genetic diversity exists among the majority of the clones analyzed. Nevertheless, the analysis defined that the maternal genotype is separated from the resistant clones. The genetic-similarity estimate among resistant genotypes varied from 0.17 with mutant 2-32-49 placed outside the second main cluster, 0.33 with 2-32-23, 0.37 with 2-21-19 and 2-29-04, 0.43 with 2-23-08, 2-2528, 2-31-06, 2-32-06, 2-32-28, 2-32-46, 2-32-67, 2-34-06, 2-34-31 and 2-34-36 and 0.50 with 2-25-62. The confidence intervals of the mean distance in each node (IC /55%) calculated by using the Bootstrapping methodology revealed a high consistency in the clusters. Principal coordinate analysis (PCoA) was also performed to display the relationship between the maternal genotype and the clones in a two-dimensional UPGMA clustering dendrogram. The PCoA analysis placed the 16 genotypes in two different groups (Fig. 3). The scatter plot shows great variability between groups; the proximity of the clones suggests an equal molecular response to radiation. This analysis detected the existence of an associated polymorphic pattern to conditions of resistance to SCMV, indicating a favorable genetic change product of the induced mutations. Also, the existence of specific patterns for each genotype in the study was observed. The differences detected between maternal genotype (B6749) and the mutants that originated from it through gamma irradiation corroborate the differences detected in the field with respect to resistance to SCMV of the clones. The amplification products with OPM-14 revealed a 854 bp fragment that can be used to characterize 15
Fig. 1. RAPD polymorphisms of sugarcane maternal plant (B6749) and its resistant SCMV clones using primer OPM14. Molecular marker: l DNA/ Hin dIII /Eco RI.
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Fig. 2. (a) Distribution of the co-phenetic values. (b) Genetic similarity between the maternal genotype (B6749 susceptible to SCVM) and the mutants revealed by UPGMA cluster analysis based on the Jaccard’s coefficient.
generations in the field, shows a stable genetic difference not present in the maternal genotype due to induced mutation. This clear differentiation studied through amplification products with OPM-14 opens the way for a more detailed molecular analysis of the basis of the resistance character.
Acknowledgements This research was carried out in CENIAP-INIA, Maracay, Venezuela and was supported by CINAGRI-AIEA VEN/5/12 and Fundacite Aragua.
References
Fig. 3. PCoA of genetic similarities between the maternal genotype (B6749 susceptible to SCVM) and the mutants.
resistant clones. These results are in accordance with Oropeza et al. [19] who found using OPA-04, a 874 bp RAPD fragment associated with SCMV in somaclones generated through somatic embryogenesis by increasing successive subcultures of cultivar PR62258. These similar results reveal the presence of genes associated to these resistant to the virus. The results show stable fingerprints for sixteen genotypes analyzed and allowed us to differentiate the susceptible cultivar to SCMV (B6749) from the resistant clones. The molecular pattern and the stability of the SCMV resistance phenotype over eight consecutive
[1] G.M. Cordeiro, G.O. Taylor, R.J. Henry, Characterisation of microsatellite markers from sugarcane (Saccharum sp.), a highly polyploid species, Plant Sci. 155 (2000) 161 /168. [2] V. Gonza´lez, El mejoramiento gene´tico de la can˜a de azu´car en Venezuela (1961 /1981) I. Seleccio´n de variedades Venezolanas, Can˜a de Azu´car 1 (1983) (1961) 41 /56. [3] C. Ceoloni, G. Del Signore, L. Ercoli, P. Donini, Localitin the alien chromatid segment in common wheat */Aegilops longissima mildew resistant transfer, Hereditas 116 (1992) 239 /245. [4] C. Ceoloni, P. Donini, Combining mutations for the two homoeologous pairing suppressor genes Ph1 and Ph2 in common wheat and in hybrids with alien Triticeae , Genome 36 (1993) 377 /386. [5] T. Nishio, S. Iida, Mutants having a low content of 16-kDa allergenic protein in rice (Oriza sativa L.), Theor. Appl. Genet. 86 (1992) 317 /321. [6] M. Ichii, T. Katagiri, H. Hasegawa, Mutants with low nitrate reductase activity selected from seedling expressing nitrogen deficiency symptoms in rice (Oriza sativa L.), Jpn J. Breed. 43 (1993) 123 /127.
A.Y. Zambrano et al. / Plant Science 165 (2003) 221 /225 [7] M. Yakoo, K. Okumo, Genetic analysis of earliness mutations induced in rice cultivar Nirin 8, Jpn J. Breed. 43 (1993) 1 /11. [8] U. Lundqvist, A. Lundqvist, Dominant resistance to barley mildew race D1, isolated after mutagenic treatments in four highbred barley varieties, Hereditas 115 (1991) 241 /253. [9] R. Pathirama, Gamma ray-induced field tolerance to Phytophtora blight in sesame, Plant Breed. 108 (1992) 314 /319. [10] Y. Takagi, S.M. Raham, Inheritance of high oleic acid content in the seed oil of soybean mutant M23, Theor. Appl. Genet. 92 (1996) 179 /182. [11] T.J. Rao, K.V. Srinivassan, K.G. Alexander, A red resistant mutant of sugarcane induced by gamma irradiation, Proc. Indian Acad. Sci. 64 (1971) 402 /407. [12] R. Urata, D.J. Heinz, Gamma irradiation-induced mutations in sugarcane, Proc. Inter. Soc. Sugarcane Technol. 14 (1971) 402 / 407. [13] S.H. Siddiqui, M. Javed. Mutation breeding in sugarcane (Saccharum sp., hybrid) by gamma irradiation. Proceedings of the Final Research Coordination Meeting, AIEA, 11 /15 Febrero 1982, Coimbatore. [14] J.G.K. Williams, A.R. Kubelik, K.J. Livak, J.A. Rafalski, S.V. Tingey, Polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acid Res. 18 (1990) 6531 /6535. [15] J.G.K. Williams, M.K. Hanafey, J.A. Rafalski, S.V. Tingey, Genetic analysis using random amplified polymorphic DNA markers, Meth. Enzymol. 218 (1993) 704 /740. [16] S.V. Tingey, J.P. del Tufo, Genetic analysis with random amplified polymorphic DNA markers, Plant Physiol. 101 (1993) 349 /352. [17] J. Hu, C.F. Quiros, Identification of broccoli and cauliflower cultivars with RAPD markers, Plant Cell Rep. 10 (1991) 505 /511. [18] P.W. Taylor, J.R. Ko, T.A. Fraser, R.J. Henry, R.G. Bircg, Sensitivity of random amplified polymorphic DNA analysis to
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detect genetic chance in sugarcane during tissue culture, Theor. Appl. Genet. 90 (1995) 1169 /1173. M. Oropeza, P. Guevara, E. de Garcı´a, J.L. Ramı´rez, Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers, Plant Mol. Biol. Rep. 13 (1995) 182 /191. M. Harvey, F.C. Botha, Use of PCR-based methodologies for determination of DNA diversity between Saccharum varieties, Euphytica 89 (1996) 257 /265. N. Vijayan, S. Nair, T.V. Screenivasan, M. Mohan, Analysis of genetic diversity and phylogeny in Saccharum and related genera using RAPD markers, Genet Resour. Crop Evo. 46 (1999) 73 /79. A.Y. Zambrano. Generacio´n de variabilidad para resistencia a dosis crecientes del herbicida glifosato y seleccio´n in vitro en cultivo de ce´lulas en suspensio´n en tres cultivares de can˜a de azu´car. Tesis de Doctorado en Ciencias Agrı´colas. Facultad de agronomı´a. Universidad Central de Venezuela. Maracay, 1999, p. 80. D.A. Hoisington, M. Khairallah, D. Gonza´lez-de-Leo´n. Laboratory Protocols CIMMYT. Applied Molecular Genetics Laboratory. Second Edition. Me´xico, CIMMYT. 1994, p. 51. Eastman Kodak Company. 1D Image Analysis Software, 1997, p. 230. M. Nei, Molecular Evolutionary Genetics, Columbia University Press, New York, USA, 1987, p. 512. R.R. Sokal, P.H.A. Sneath, Principles of Numerical Taxonomy, Freeman, San Francisco, 1963, p. 359. P.H.A. Sneath, R.R. Sokal, Numerical Taxonomy, Freeman, San Francisco, 1973, p. 573. F.J. Rohlf, R.R. Sokal, Comparing numerical taxonomic studies, Systematic Zool. 30 (1981) 459 /490. Exeter Software. NTSYS-pc Version 2.10t. Applied Biostatistics Inc, 2000.
Selection of sugarcane plants resistant to SCMV A.Y. Zambrano *, J.R. Demey, M. Fuchs, V. Gonza´lez, R. Rea, O. De Sousa, Z. Gutie´rrez Instituto Nacional de Investigaciones Agropecuarias (INIA), Apartado 4521 Maracay 2101, Venezuela Received 15 July 2002; received in revised form 12 March 2003; accepted 12 March 2003
Abstract Sugarcane (Saccharum spp.) is susceptible to several major diseases transmitted by insects such as Sugarcane Mosaic Virus (SCMV). In very susceptible plants, SCMV causes severe dwarfing, leaf and stem necrosis and serious production losses. We obtained sugarcane clones resistant to SCMV using induction mutation by irradiating. Calli from the susceptible cultivar B6749 with 2 krads of gamma rays. The regenerated plantlets were tested for resistance to strain B of SCMV in the greenhouse and the resistant clones were transferred to the field. They have shown stable resistance to viral infection for eight generations of asexual reproduction. To detect genetic changes related to the acquired resistance, 15 resistant clones and a clone from the original cultivar B6749 were analyzed using Random Amplified Polymorphic DNAs (RAPD). The RAPD patterns obtained with the primer OPM14 detected a 854 bp fragment in the 15 resistant clones, which was not present in cultivar B6749. The study of genetic relations between the genotypes resistant to SCMV and its maternal B6749, using all RAPD primers, defined the existence of an associated polymorphic pattern to the resistance to SCMV. Existence of specific patterns for each genotype in the study was also observed. Analysis using RAPDs efficiently differentiated sugarcane clones resistant to SCMV by their genetic changes due to induced mutation. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calli; Mutation; RAPDs; Saccharum spp.; Sugarcane Mosaic Virus
1. Introduction Sugarcane (Saccharum spp.) is a large and highly heterozygous and heterogenous clonally propagated grass of the gramineae family, characterized by a high degree of polyploidy and is a crop of major importance providing about 65% of the sugar produced in the world. Cultivated sugarcane varieties are derived from complex interspecific hybridization between the species S. spontaneum (2n/40/128) and S. officinarum (2n / 60 or 80) [1]. Sugarcane is susceptible to several major viral diseases transmitted by insects. At the present time the most severe viral disease is caused by the Sugarcane Mosaic Virus (SCMV). In very susceptible plants it causes severe dwarfing, leaf and stem necrosis and serious production losses. Development of disease resistant varieties by conventional breeding takes 10/ * Corresponding author. Tel./fax: /58-243-2474111. E-mail address: [email protected] (A.Y. Zambrano).
15 years for one cycle of complete selection [2]. Although mutation breeding of sugarcane is not substitute for conventional breeding, mutation breeding may be considered as a means to improve the products of conventional breeding. Mutations can be induced in calli that primarily change one or a few characters of an outstanding cultivar without altering the remaining phenotypes. Induction of resistance to diseases through tissue culture has provided potentially useful in plants with a specific capability to endure the test compounds. Some attempts to induce tolerance using somaclonal variation and in vitro selection of induced mutations have already been reported. Induced mutations have allowed introduction of stable, desirable traits in different crops species, such as wheat [3,4], rice [5 /7], barley [8], sesame [9], and soybean [10]. In sugarcane, promising results during several cycles of vegetative propagation have been shown in many reports [11 /13]. Various molecular marker types could be used to detect genetic changes related to the induced resistance.
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00162-6
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We chose Random Amplified Polymorphic DNA (RAPD) [14] because (a) it allows random amplification of DNA sequences throughout the entire genome, (b) RAPD polymorphisms result from a nucleotide base change that alters the primer binding site, or from an insertion or deletion within the amplified region [15], (c) polymorphisms usually result in the presence or absence of an amplification product from a single locus [16], (d) these products of amplification can be polymorphic and are used as genetic markers [17]. In sugarcane, RAPD technology has been used to study genetic stability in plants obtained by in vitro cultures [18], to identify somaclonal variants resistant to SCMV [19], to detect the genetic variability among cultivars [20], to analyze the diversity and phylogeny in the genera Saccharum [21] and to identify pre-existing variability selected in vitro for tolerance to glyphosate [22]. Here, we report production of sugarcane clones resistant to SCMV, strain B, using induction mutation, the differences detected in the RAPD patterns of the clones resistant to SCMV compared with the maternal plant.
2. Materials and methods 2.1. Plant material Calli from cultivated sugarcane variety B6749, susceptible to SCMV were irradiated with 2 krad of gamma rays using a 60Co source. After regeneration, the plantlets were tested in the greenhouse using manual inoculation with strain B of SCMV. Then 15 selected plants, free of mosaic symptoms, were transferred to the field station at CIAE-Yaritagua, Yaracuy, Venezuela. The plants were tested for eight generations of asexual reproduction. The 15 resistant clones (2-21-19, 2-23-08, 2-25-28, 2-25-62, 2-29-04, 2-31-06, 2-32-06, 2-32-23, 232-28, 2-32-46, 2-32-49, 2-32-67, 2-34-06, 2-34-31 and 234-36) were selected and compared with the maternal plant (cv B6749) using the RAPD technique. 2.2. DNA analysis Genomic DNA was isolated from fresh, young tissue of sugarcane leaves according to Hoisington et al. [23]. The quality and concentration of genomic DNA were determined by 1% agarose gel electrophoresis. For PCR amplification, the genomic DNA concentration was adjusted to 100 ng/ml and stored at /20 8C. PCR was performed according to Zambrano [22] in a total volume of 25 ml containing 10 mM Tris /HCl (pH 9.0), 50 mM KCl, 0.1% Triton† X-100, 2.5 mM MgCl2, 200 mM of each dNTPs, 0.2 mM primer, 25 ng of genomic DNA and 1 unit Taq DNA polymerase (Promega). The reaction mixture was overlaid with 20 ml of mineral oil. For amplification, twelve primers from Operon
technologies were used: OPA01, OPA04, OPA07, OPB04, OPB07, OPK03, OPK05, OPK15, OPM04, OPM14, OPM18 and OPM20. RAPDs were amplified in a Techne GeneE thermalcycler. An initial denaturation cycle was done at 94 8C for 5 min, followed by 45 cycles of amplification by denaturing at 94 8C for 1 min, annealing at 36 8C for 30 s, and extension at 72 8C for 2 min. The final step was a single extension cycle at 72 8C for 7 min. Amplification products were analyzed by electrophoresis in 1.4% agarose gels using Tris /borate buffer, stained with ethidium bromide and photographed under UV light. l Phage DNA double digested with Hin dIII and Eco RI endonucleases was used as a molecular weight marker. The gels were digitized using the 1D Image Software Analysis [24].
2.3. Data analysis The percentage of polymorphic loci P , the number of alleles A and the average number of alleles per polymorphic locus Ap were calculated according to the following formulae [25]: Percentage of polymorphic loci P: P /(K /n )/100% where k is the number of polymorphic loci, n is the total number of loci investigated. Average number of alleles per locus A: A /aAi / n , where Ai is the number of alleles at the i th locus, n is the total number of loci investigated. Average number of alleles per polymorphic loci Ap: Ap /aApi/np, where Api is the number of alleles at a certain polymorphic locus, np is the total number of genotypes per loci investigated. All polymorphic primers data were included in the analysis. The amplified fragments were scored as present or absent. Genetic relationships among the maternal genotype and the 15 mutants were investigated using cluster analysis of the similarity data and were depicted in a dendrogram. The coefficients of Jaccard, Dice, Simple matching and Rogers and Tanimoto’s distance [26] and three methods of aggregation: single linkage, complete linkage and unweighted pair-group were studied [27]. The co-phenetic value based on genetic similarity was calculated. This is a quantitative indication of the grouping analysis performance and evaluates the stability of the constructed relationship trees [28]. Bootstrapping methodology was used to estimate standard error and confidence intervals of the mean distance in each node. Ordination analysis was performed to study the relationship between the maternal genotype and the 15 mutants, the double-centered similarity matrix was factored and a plot was made showing the genotypes in a 2-dimensional space. All the analyses were computed with the NTSYS-pc version 2.10t program [29].
A.Y. Zambrano et al. / Plant Science 165 (2003) 221 /225
3. Result and discussion The method of genomic DNA extraction was quick, simple and efficient. It produced DNA of good quality with little degradation, and the concentration was between 100 and 250 ng/ml. We tested the sensitivity of the RAPD technique for detecting polymorphisms among the cv B6749 (susceptible to SCMV) and the clones 2-21-19, 2-23-08, 2-25-28, 2-25-62, 2-29-04, 2-3106, 2-32-06, 2-32-23, 2-32-28, 2-32-46, 2-32-49, 2-32-67, 2-34-06, 2-34-31 and 2-34-36 (resistant to SCMV) generated from gamma ray irradiation. Amplification products were obtained in the sixteen DNA samples with the twelve primers tested. High polymorphism was observed with all primers, but of the twelve primers, only OPM-14 was useful for differentiating the mutant resistant clones. The percentage of polymorphic loci P was 90, the abundance of genes in a population the number of alleles A was 2 and the average number of alleles per polymorphic locus Ap was 1.98. The RAPD patterns obtained with OPM-14 detected DNA fragment smaller than 1250 bp in the sixteen samples (Fig. 1). These amplification products revealed a fragment of 854 bp that was present the 15 resistant clones. This fragment was not present in the original cultivar susceptible to SCMV (B6749). Fig. 2a shows the distribution of the co-phenetic values for the different combinations from similarity coefficients and the distance and methods of aggregation, the highest (r /0.98) value is displayed by the dendrogram generated from the UPGMA cluster analysis based on Jaccard’s coefficient that indicated the method that defines the best grouping of RAPD patterns.
223
The study of genetic relations between the maternal genotype (B6749) and the resistant clones using the OPM-14 primer (Fig. 2b) indicates that little or no genetic diversity exists among the majority of the clones analyzed. Nevertheless, the analysis defined that the maternal genotype is separated from the resistant clones. The genetic-similarity estimate among resistant genotypes varied from 0.17 with mutant 2-32-49 placed outside the second main cluster, 0.33 with 2-32-23, 0.37 with 2-21-19 and 2-29-04, 0.43 with 2-23-08, 2-2528, 2-31-06, 2-32-06, 2-32-28, 2-32-46, 2-32-67, 2-34-06, 2-34-31 and 2-34-36 and 0.50 with 2-25-62. The confidence intervals of the mean distance in each node (IC /55%) calculated by using the Bootstrapping methodology revealed a high consistency in the clusters. Principal coordinate analysis (PCoA) was also performed to display the relationship between the maternal genotype and the clones in a two-dimensional UPGMA clustering dendrogram. The PCoA analysis placed the 16 genotypes in two different groups (Fig. 3). The scatter plot shows great variability between groups; the proximity of the clones suggests an equal molecular response to radiation. This analysis detected the existence of an associated polymorphic pattern to conditions of resistance to SCMV, indicating a favorable genetic change product of the induced mutations. Also, the existence of specific patterns for each genotype in the study was observed. The differences detected between maternal genotype (B6749) and the mutants that originated from it through gamma irradiation corroborate the differences detected in the field with respect to resistance to SCMV of the clones. The amplification products with OPM-14 revealed a 854 bp fragment that can be used to characterize 15
Fig. 1. RAPD polymorphisms of sugarcane maternal plant (B6749) and its resistant SCMV clones using primer OPM14. Molecular marker: l DNA/ Hin dIII /Eco RI.
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Fig. 2. (a) Distribution of the co-phenetic values. (b) Genetic similarity between the maternal genotype (B6749 susceptible to SCVM) and the mutants revealed by UPGMA cluster analysis based on the Jaccard’s coefficient.
generations in the field, shows a stable genetic difference not present in the maternal genotype due to induced mutation. This clear differentiation studied through amplification products with OPM-14 opens the way for a more detailed molecular analysis of the basis of the resistance character.
Acknowledgements This research was carried out in CENIAP-INIA, Maracay, Venezuela and was supported by CINAGRI-AIEA VEN/5/12 and Fundacite Aragua.
References
Fig. 3. PCoA of genetic similarities between the maternal genotype (B6749 susceptible to SCVM) and the mutants.
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