Isoenzymatic variability in wild potatoes

Variabilidade isoenzimática em batata silvestre

Abstracts

Two species of wild potato Solanum commersonii, subspecies commersonii and malmeanum, and S. chacoense, subspecies muelleri occur in southern Brazil. Their rusticity and easy adaptation to extreme climatic conditions make them valuable for breeding programs. The objective of this work was to analyze the isoenzymatic variability of 113 clones of wild potato subspecies. They were collected and maintained at Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado, at Pelotas, RS, Brazil. Enzymes involved in energetic (group I) or in peripherical (group II) metabolism constituted the material used. Polyacrylamide horizontal gel electrophoresis was used to analyze peroxidase, aspartate transaminase, phosphoglucomutase and isocitrate dehydrogenase isoenzymes. Solanum spp. has considerable genetic variability for isoenzymatic patterns. Cluster analysis classified the clones into 51 subgroups, based on electrophoretic variants of group I enzymes, and into 89, when group II enzyme variants were added. Genotypic differentiation of S. chacoense muelleri in relation to S. commersonii commersonii and S. commersonii malmeanum is evident when expressed through similarity and cluster analysis.

peroxidases; aminotransferases; isomerases; isocitrate dehydrogenase; genetic variation


No sul do Brasil ocorrem apenas duas espécies silvestres de batata, Solanum commersonii, com as subespécies commersonii e malmeanum, e S. chacoense, com a subespécie muelleri, de interesse aos programas de melhoramento, pela rusticidade e fácil adaptação a condições climáticas extremas. O objetivo deste trabalho foi analisar a variabilidade isoenzimática de 113 clones de batata silvestre. O material foi coletado e mantido na Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado, em Pelotas, RS. Foram usadas enzimas envolvidas nos metabolismos energético (grupo I) e periférico (grupo II). Eletroforese horizontal em gel de poliacrilamida foi empregada para análise de isoenzimas de peroxidase, aspartato transaminase, fosfoglucomutase e isocitrato desidrogenase de folhas. Existe grande variabilidade isoenzimática em clones de Solanum spp. A análise de agrupamento permitiu a classificação dos clones em 51 subgrupos, quando baseada em variantes eletroforéticas de enzimas do grupo I, e em 89 subgrupos, quando acrescida de enzimas do grupo II; é evidente a diferenciação genotípica entre S. chacoense muelleri em relação ao S. commersonii malmeanum e ao S. commersonii commersonii, expressa pelas análises de similaridade e agrupamento.

peroxidase; aminotransferase; isomerase; isocitrato desidrogenase; variação genética


Isoenzymatic variability in wild potatoes(1 (1 ) Accepted for publication on July 24, 2000. Part of the M.Sc. thesis presented by the first author to the Universidade Federal de Pelotas (UFPel), RS. )

Beatriz Helena Gomes Rocha(2 (2 ) UFPel, Dep. de Ciência e Tecnologia de Sementes, Caixa Postal 354, CEP 96010900 Capão do Leão, RS. CAPES scholar. Email: biagr@bol.com.br, jbsilva@ufpel.tche.br, juviegas@zaz.com.br ), Eliane Augustin(3 (3 ) Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado, Caixa Postal 403, CEP 96001970 Pelotas, RS . Email: augustin@cpact.embrapa.br ), João Baptista da Silva(2 (2 ) UFPel, Dep. de Ciência e Tecnologia de Sementes, Caixa Postal 354, CEP 96010900 Capão do Leão, RS. CAPES scholar. Email: biagr@bol.com.br, jbsilva@ufpel.tche.br, juviegas@zaz.com.br ) and Judith Viégas(2 (2 ) UFPel, Dep. de Ciência e Tecnologia de Sementes, Caixa Postal 354, CEP 96010900 Capão do Leão, RS. CAPES scholar. Email: biagr@bol.com.br, jbsilva@ufpel.tche.br, juviegas@zaz.com.br )

Abstract ¾ Two species of wild potato Solanum commersonii, subspecies commersonii and malmeanum, and S. chacoense, subspecies muelleri occur in southern Brazil. Their rusticity and easy adaptation to extreme climatic conditions make them valuable for breeding programs. The objective of this work was to analyze the isoenzymatic variability of 113 clones of wild potato subspecies. They were collected and maintained at Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado, at Pelotas, RS, Brazil. Enzymes involved in energetic (group I) or in peripherical (group II) metabolism constituted the material used. Polyacrylamide horizontal gel electrophoresis was used to analyze peroxidase, aspartate transaminase, phosphoglucomutase and isocitrate dehydrogenase isoenzymes. Solanum spp. has considerable genetic variability for isoenzymatic patterns. Cluster analysis classified the clones into 51 subgroups, based on electrophoretic variants of group I enzymes, and into 89, when group II enzyme variants were added. Genotypic differentiation of S. chacoense muelleri in relation to S. commersonii commersonii and S. commersonii malmeanum is evident when expressed through similarity and cluster analysis.

Index terms: peroxidases, aminotransferases, isomerases, isocitrate dehydrogenase, genetic variation.

Variabilidade isoenzimática em batata silvestre

Resumo ¾ No sul do Brasil ocorrem apenas duas espécies silvestres de batata, Solanum commersonii, com as subespécies commersonii e malmeanum, e S. chacoense, com a subespécie muelleri, de interesse aos programas de melhoramento, pela rusticidade e fácil adaptação a condições climáticas extremas. O objetivo deste trabalho foi analisar a variabilidade isoenzimática de 113 clones de batata silvestre. O material foi coletado e mantido na Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado, em Pelotas, RS. Foram usadas enzimas envolvidas nos metabolismos energético (grupo I) e periférico (grupo II). Eletroforese horizontal em gel de poliacrilamida foi empregada para análise de isoenzimas de peroxidase, aspartato transaminase, fosfoglucomutase e isocitrato desidrogenase de folhas. Existe grande variabilidade isoenzimática em clones de Solanum spp. A análise de agrupamento permitiu a classificação dos clones em 51 subgrupos, quando baseada em variantes eletroforéticas de enzimas do grupo I, e em 89 subgrupos, quando acrescida de enzimas do grupo II; é evidente a diferenciação genotípica entre S. chacoense muelleri em relação ao S. commersonii malmeanum e ao S. commersonii commersonii, expressa pelas análises de similaridade e agrupamento.

Termos para indexação: peroxidase, aminotransferase, isomerase, isocitrato desidrogenase, variação genética.

Introduction

Wild species are an important source of germplasm for disease and environmental stress resistance. Two wild potato species, series Commersoniana Buk., occur in southern Brazil: Solanum commersonii (2n = 2x = 24, 36) and Solanum chacoense (2n = 2x = 24, 36). Two subspecies of S. commersonii (commersonii and malmeanum) and S. chacoense (chacoense and muelleri) are known. These species have similar botanical characteristics. Resistance to common scab, bacterial wilt, late blight, drought, heat and frost were observed in S. commersonii. S. chacoense, however, has more useful characteristics, such as, resistance to common scab, powdery scab, bacterial wilt, black leg, virus (A, X, Y, F and leaf roll), aphids, nematodes, drought, heat and frost (Hawkes & Hjerting, 1969; Centro Internacional de La Papa, 1979, 1981; Montaldo, 1984).

A relatively large number of alleles occur naturally in potatoes, as demonstrated by isoenzymatic markers (Douches & Ludlam, 1991). A mean of 5.7 alleles/locus have been identified in 92 accessions of 40 species of Solanum from North and South America, indicating a considerable genetic variability in wild species (Douches et al., 1989). Martinez-Zapater & Oliver (1984) refer to the utilization of isoenzymes in the identification of 67 cultivars of S. tuberosum, including those most used in Europe and North America, and to the analysis of phylogenetic relationships among S. tuberosum and other species, based on aloenzyme variability.

Polymorphism level varies according to the enzyme function, that is, enzymes involved in energetic metabolism, glycolysis and the citric acid cycle, present lower polymorphism levels (group I) than those involved in peripherical metabolism (group II) (Gillespie & Kojima, 1968). Both monomorphic and polymorphic systems should be investigated, involving the highest possible number of metabolic routes, to obtain a random sample of populations and genomes, in order to avoid over or under estimates.

The objective of this work was to analyze the isoenzymatic variability of 113 wild potato clones.

Material and Methods

Solanum commersoniimalmeanum (SCM), S. commersonii commersonii (SCC), S. chacoense muelleri (SChM) and non-identified (NI) clones were cultivated under greenhouse, screenhouse and/or field conditions at Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado (Figure 1). They were collected in the Brazilian states of Rio Grande do Sul (Table 1) and Santa Catarina (clones 110 NI, 119 and 120 SCC), and in Uruguay (clones 07 SCC and 195 NI).

Group I (EC 5.4.2.2 phosphoglucomutase (PGM), EC 1.1.1.41 isocitrate dehydrogenase (IDH) and EC 2.6.1.1 aspartate transaminase (AT)) and group II enzymes (EC 1.11.1.7 peroxidase-PRX) from leaves of, at least, two plants per clone, were analyzed. Samples were taken from leaflets of the third or fourth leaf, before blooming (AT); of the fourth or fifth leaf, at the beginning of blooming (PGM); and of the fifth or sixth leaf, at full blooming (PRX and IDH). Ten milligrams of each sample were squeezed directly (AT) or in 0.01 mL gel buffer and 0.15% 2mercaptoethanol (PRX, PGM and IDH). Clone number 186 was used as a control.

Horizontal electrophoresis, with 5% (PRX, PGM and IDH) and 6% (AT) polyacrylamide gels was used with discontinuous buffer systems described by Scandalios (1969) (PRX and AT) and Shields et al. (1983) (PGM and IDH). Staining methods are referred in Scandalios (1969) (PRX); Ayala et al. (1972) (AT); Vallejos (1983) (IDH and PGM).

Genetic similarity and cluster analysis, using Jaccard coefficient unweighted pair group method, arithmetic average (UPGMA) were calculated using the NTSYS ¾ pc version 1.7.

Results and Discussion

No variation was observed in the peroxidase, aspartate transaminase phosphoglucomutase and isocitrate dehydrogenase isoenzymatic patterns of clones grown under different environmental conditions (greenhouse, screenhouse or field).

Number and concentrations of multiple molecular forms of peroxidase vary with plant ontogenesis (Chen et al., 1970). Thirty-four anodic peroxidase polymorphic bands, in sixty-six patterns, were detected at full blooming (Table 2). There were three to eight bands per pattern (Figure 2). According to Bassiri & Adams (1978), systems with higher number of polymorphic bands, such as peroxidase, are the most adequate to identify cultivars. These enzymes (group II) act over a class of molecules which, normally, come from the environment and that can have qualitatively and quantitatively more variables than enzymes (group I) which act on in vivo substrates, generally restricted to molecules that are products of a previous internal enzymatic reaction (Kojima et al., 1970).

Peroxidase electrophoretic patterns showed high intraspecific variability. Thirteen S. commersonii malmeanum clones and 30 non-identified clones reflected the highest number of individual patterns: twelve and 25, respectively. In 69 clones of S. commersonii commersonii, 44 patterns were observed. Juned et al. (1988) found various peroxidase, aspartate transaminase and phosphoglucoisomerase polymorphic bands, indicating great variability in S. chacoense from Paraguay and Argentina.

Fifteen anodic bands of aspartate transaminase were found in the fourteen patterns detected (Table 3). One to five bands per pattern were observed (Figure 2). As expected, the intraspecific variability found was lower than for peroxidase. Thirteen S. chacoense malmeanum clones and 30 non-identified clones showed, respectively, four and eleven patterns, while the S. commersonii commersonii clones presented eight patterns. All bands were polymorphic.

Seven phosphoglucomutase isoenzyme patterns were observed based on five anodic and polymorphic bands (Table 4). One to four bands were found in each pattern (Figure 2). No enzymatic activity was detected in some clones. Desborough (1983) stated that three distinct forms of phosphoglucomutase were found in potatoes. Suurs et al. (1989), however, observed two to five molecular forms in species of Solanum and Lycopersicon. Using locus Pgm-2, present only in the used as male parent species, Chien-An & Douches (1993) identified 50% of S. tuberosum tuberosum x S. phureja hybrids. This system, added to the other 12, was also used by Douches & Ludlam (1991) to characterize 112 potato cultivars and advanced lines obtained through hybridization, from North America. They concluded that electrophoresis is an efficient method to distinguish sexually originated genotypes.

The isocitrate dehydrogenase zimograms revealed eight anodic and polymorphic bands. One to three bands were detected in each pattern (Figure 2). This reduced number allowed the detection of only 12 patterns (Table 5). Douches et al. (1991) used this enzymatic system, as well as nine others, to examine phylogenetic relationships in 10 cultivars from North America, of historical importance in the last century. Twenty-seven alleles were found in 13 loci. Eleven heterozygotic loci were detected. Locus Idh-1, with two alleles, was monomorphic in just one of the analyzed genotypes, presenting a frequency of 63% for Idh-11.

Cluster analysis of the 113 wild potato clones, based on group I enzymatic systems, allowed their classification into 51 subgroups (Figure 3). Two groups, one consisting of just one clone, number 68 (S. chacoense muelleri) and another large group composed of two subgroups were observed. All but one access of S. commersonii malmeanum , seven of S. commersonii commersonii and 13 of the non-identified clones formed the smaller subgroup. The other subgroup was subdivided into two. One consisted of clone number 107 of S. commersonii commersonii and the other had 79 clones, including 61 of S. commersonii commersonii, 17 of the non-identified group and clone 108 (S. commersonii malmeanum). Various clones formed subgroups which showed maximum similarity. The largest subgroup was composed of 15 accesses of S. commersonii commersonii and seven non-identified clones. Jaccard coefficients varied from 0.05 to 1.00.

Cluster analysis, based on all enzymatic systems studied, allowed the classification of the 113 clones into 89 subgroups (Figure 4). Two groups were found. The first one was composed of the majority of the S. commersonii malmeanum, eight S. commersonii commersonii and 13 non-identified clones. The other was subdivided into two, a small one composed of clone numbers 108 (S. commersonii malmeanum), 105, 110, 127 (non-identified), and 158 (S. commersonii commersonii) and a larger one which grouped 65.5% of total clones analyzed. The second group was consisted of only clone numbers 64 and 109 (S. commersonii malmeanum), and 68 (S. chacoense muelleri). Sixteen subgroups presented maximum similarity. Jaccard coefficients varied from 0.08 to 1.00.

Analysis of genetic similarity revealed a great variability in the potato clones, even when based only on group I enzymes. Jaccard coefficients varied from 0.49 to 0.97 when Samec & Nasinec (1996), using RAPD technique, identified and classified 42 wild and cultivated Pisum sativum genotypes. The authors concluded that some cultivars have common ancestors. In the wild potato clones analyzed in this work, coefficients varied from 0.05 to 1.00. Furthermore, the number of loci studied was lower, since isoenzymic analysis was used.

Douches & Ludlam (1991) were able to separate 112 potato cultivars into 95 groups, based on electrophoretic patterns. However, they used 13 enzymatic systems, including the ones used in this work.

As expected, a higher number of subgroups were formed when enzymes of group II were included in the cluster analysis (Figures 3 and 4). In addition, when peroxidase isoenzymes were considered in the analysis, clone numbers 64, 109 (S. commersonii malmeanum) and 68 (S. chacoense muelleri) were all included in the same group. Preferential clustering was maintained, with most of the S. commersonii malmeanum and S. commersonii commersonii clones included, respectively, in the upper and intermediate parts of the dendograms. S. chacoense muelleri clone remained in the lower part.

Conclusions

1.Solanum spp. has considerable genetic variability for isoenzymatic patterns.

2.Enzymes involved in peripherical metabolism present higher polymorphism level in wild potatoes than enzymes involved in energetic metabolism, glycolisis and citric acid cycle.

3.There is a great genotypic differentiation of S. chacoense muelleri in relation to S. commersonii commersonii and S. commersonii malmeanum.

Acknowledgements

To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), for fellowships; to Delorge Mota da Costa, for clones and information provided; to Ema Gladis Schultz Correa, for technical assistance.

  • AYALA, F. J.; POWELL, J. R.; TRACEY, M. L.; MOURĂO, C. A.; PÉREZ-SALAS, S. Enzyme variability in the Drosophila willistoni group. IV. Genic variation in natural populations of Drosophila willistoni Genetics, Bethesda, v. 70, p. 113-139, 1972.
  • BASSIRI, A.; ADAMS, M. W. An electrophoretic survey of seedling isozymes in several Phaseolus vulgaris Euphytica, Dordrecht, v. 27, p. 447-459, 1978.
  • CENTRO INTERNACIONAL DE LA PAPA (Lima, Peru). Informe anual 1978 Lima, 1979. 124 p.
  • CENTRO INTERNACIONAL DE LA PAPA (Lima, Peru). Informe anual 1980 Lima, 1981. 144 p.
  • CHEN, S.; TOWILL, L. R.; LOEWENBERG, L. Isozyme patterns in developing Xanthium leaves. Physiologia Plantarum, Copenhagen, v. 23, p. 434-444, 1970.
  • CHIEN-AN, L.; DOUCHES, D. S. Production of haploids of potato (Solanum tuberosum subsp. tuberosum) and their identification with electrophoretic analysis. Euphytica, Dordrecht, v. 70, p. 113-126, 1993.
  • DESBOROUGH, S. Potato (Solanum tuberosum L.). In: TANKSLEY, S. D.; ORTON, T. J. (Ed.). Isozymes in plant genetics and breeding Amsterdam : Elsevier, 1983. part B, p. 167-188.
  • DOUCHES, D. S.; LUDLAM, K. Electrophoretic characterization of North American potato cultivars. American Potato Journal, Orono, v. 68, p. 767-780, 1991.
  • DOUCHES, D. S.; LUDLAM, K.; FREYRE, R. Isozyme and plastid DNA assessment of pedigrees of nineteenth century potato cultivars. Theoretical and Applied Genetics, Berlin, v. 82, p. 195-200, 1991.
  • DOUCHES, D.; SCHROETER, B.; LUDLAM, K.; HICKS, K. Allelic diversity among the Solanum species, sect Petota. American Potato Journal, Orono, v. 66, n. 8, p. 517, 1989.
  • GILLESPIE, J. H.; KOJIMA, K. The degree of polymorphisms in enzymes involved in energy production compared to that in nonspecific enzymes in two Drosophila ananassae populations. National Academy of Sciences of the United States of America Proceedings, Washington, v. 61, p. 582-585, 1968.
  • HAWKES, J. G.; HJERTING, J. P. The potatoes of Argentina, Brazil, Paraguay and Uruguay Oxford : Claredon, 1969. 525 p.
  • IBGE (Rio de Janeiro, RJ). Cidades e vilas ž 1998 Disponível: site IBGE. URL: http//www.ibge.gov.br Consultado em 01 jun. 2000.
  • JUNED, S. A.; JACKSON, M. T.; CATTY, J. P. Diversity in the wild potato species Solanum chacoense Bilt. Euphytica, Dordrecht, v. 3, p. 149-156, 1988.
  • KOJIMA, K.; GILLESPIE, J.; TOBARI, Y. N. A profile of Drosophila species enzymes assayed by electrophoresis. I. Number of alleles, heterozygosites, and linkage desequilibrium in glucose-metabolizing systems and some other enzymes. Biochemical Genetics, New York, v. 4, p. 627-637, 1970.
  • MARTINEZ-ZAPATER, J. M.; OLIVER, J. L. Genetic analysis of isozyme loci in tetraploid potatoes (Solanum tuberosum L.). Genetics, Bethesda, v. 5, p. 669-679, 1984.
  • MONTALDO, A. Cultivo y mejoramiento de la papa San José : Instituto Interamericano de Cooperación para la Agricultura, 1984. 706 p.
  • SAMEC, P.; NASINEC, V. The use of RAPD technique for the identification and classification of Pisum sativum L. genotypes. Euphytica, Dordrecht, v. 89, p. 229-234, 1996.
  • SCANDALIOS, J. G. Genetic control of multiple molecular forms of enzymes in plants: a review. Biochemical Genetics, New York, v. 3, p. 37-79, 1969.
  • SHIELDS, C. R.; ORTON, T. J.; STUBER, C. W. An outline of general resource needs and procedures for the electrophoretic separation of active enzymes from plant tissue. In: TANKSLEY, S. D.; ORTON, T. J. (Ed.). Isozymes in plant genetics and breeding Amsterdam : Elsevier, 1983. part. A, p. 443-468.
  • SUURS, L. C. J. M.; JONGEDIJK, E.; TAN, M. M. C. Polyacrylamide gradient-gel electrophoresis: a routine method for high resolution isozyme electrophoresis of Solanum and Lycopersicon species. Euphytica, Dordrecht, v. 40, p. 181-186, 1989.
  • VALLEJOS, C. E. Enzyme activity staining. In: TANKSLEY, S. D.; ORTON, T. J. (Ed.). Isozymes in plant genetics and breeding Amsterdam : Elsevier, 1983. part A, p. 469-515.

  • (1
    ) Accepted for publication on July 24, 2000.
    Part of the M.Sc. thesis presented by the first author to the Universidade Federal de Pelotas (UFPel), RS.
  • (2
    ) UFPel, Dep. de Ciência e Tecnologia de Sementes, Caixa Postal 354, CEP 96010900 Capão do Leão, RS. CAPES scholar. Email:
  • (3
    ) Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado, Caixa Postal 403, CEP 96001970 Pelotas, RS
    . Email:

Publication Dates

  • Publication in this collection
    13 Sept 2001
  • Date of issue
    May 2001

History

  • Accepted
    24 July 2000
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