Use of seed protein polymorphism for discrimination of improvement level and geographic origin of upland rice cultivars

Use of seed protein polymorphism for discrimination of improvement level and geographic origin of upland rice cultivars^*

Ricardo Montalván¹, Akihiko Ando² and Sérgio Echeverrigaray³

¹Departamento de Agronomia,Universidade Estadual de Londrina, Campus Universitário, Caixa Postal 6001, CCA, 86051-970 Londrina, PR, Brasil.

²Departamento de Genética, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Caixa Postal 83, 13400-970 Piracicaba, SP, Brasil.

³Instituto de Biotecnologia, Universidade de Caxias do Sul, Caixa Postal 1352, 95001 Caxias do Sul, RS, Brasil. Send correspondence to R.M., Departamento de Agronomia,Universidade Estadual do Oeste de Paraná, Rua Pernambuco, 1777, Caixa Postal 91, 85960-000 Marechal Cândido Rondon, PR, Brasil.

ABSTRACT

Grain proteins from 58 Brazilian and nine Japanese upland rice cultivars (Oryza sativa L.) were electrophoretically separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric scanning of the electrophoretic profiles permitted the estimation of the relative concentration of 16 protein fractions, which were used as variables for the calculation of Fisher's canonical discriminating functions. Significant differences between mean values of protein fractions were useful in distinguishing Brazilian and Japanese cultivars, as well as improved and non-improved Brazilian rice cultivars in scattered plots. Electrophoretically detectable protein polymorphism in rice grain can indicate geographic origin as well as breeding improvement level of a cultivar. Improved cultivars were those released by plant breeding institutes.

INTRODUCTION

During the past 30 years, various techniques have been used for analysis of genetic variability at the molecular level in plants: isozymes (Glaszman, 1986; Kochko, 1987), DNA restriction fragment length polymorphism (RFLPs) (Helentjaris et al., 1985) and random amplified polymorphic DNA (RAPDs) (Halward et al., 1992). However, seed protein profiles (Ladizinsky and Hymowitz, 1979) are still powerful tools for determining genetic homology at the molecular level and for solving problems in systematic methodology.

Numerous seed protein profile studies have been done with various plant species, such as rice (Gramineae) (Aliaga-Morel et al., 1987), Capsicum sp. (Solanaceae) (Panda et al., 1986), Ricinus communis (Euphorbiaceae) (Sathaiah and Reddy, 1985), Manihot sp. (Euphorbiaceae) (Grattapaglia et al., 1987), and Arachis sp. (Leguminosae) (Bianchi-Hall et al., 1993; Lanham et al., 1994). However, the phenotypic or adaptive significance of molecular polymorphisms was not clear in most cases (Damerval et al., 1987), since molecular variability did not appear to be related to morphological variation, interspecifically or intraspecifically. This strongly supports the hypothesis that molecular polymorphisms are neutral in natural selection (Kimura, 1968, 1993; Kimura and Ohta, 1971).

Damerval et al. (1987) hypothesized that the quantitative variations in gene product levels revealed by electrophoretic techniques is a more important basis for detection of morphological and adaptive change than classical variability (presence/absence). They found that genetic variation in proteins (more or less intense spots), revealed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), in five maize lines was significantly correlated to morphological distances.

Montalván et al. (1995) compared the SDS-PAGE electrophoretic profiles of the seed storage protein of 58 Brazilian rice varieties. The results suggest that data from seed storage protein profiles may be suitable for exploratory analysis of genetic relationship and geographical distribution of rice varieties in Brazil.

The present work was conducted to determine if protein polymorphism in seed protein fractions is related to phenotypic variation in upland rice.

MATERIAL AND METHODS

Fifty-eight Brazilian and nine Japanese upland rice varieties (Oryza sativa L.) (Table I) were cultivated under identical conditions during the 88/89 season at the Experimental Station of AGROCERES Seed Co. Ltd. in Santa Cruz das Palmeiras, São Paulo State, Brazil. All the varieties were obtained from the rice germplasm collection of Department of Genetics, College of Agriculture "Luiz de Queiroz"(ESALQ), São Paulo University (USP).

Grain extracts were electrophoretically separated by 12% SDS-PAGE (Laemmli, 1970; Montalván, 1990, Montalván et al., 1995). Improved cultivars were those released by plant breeding institutes, and non-improved were old, local lines or those with minimal breeding improvement. Densitometric scanning of the electrophoretic profiles was used to calculate the relative concentration of 16 protein fractions, which were used as variables for the Fisher's canonical discriminating functions between Brazilian and Japanese and improved and non-improved cultivars (Manly, 1986).

RESULTS AND DISCUSSION

Discrimination between Brazilian and Japanese cultivars

The profiles of seed storage protein fractions in rice were useful for discriminating Brazilian vs. Japanese cultivars. The Hotelling T²-test (Manly, 1986) showed highly significant differences between the two groups (P < 0.001).

The canonical discriminating function for the Brazilian and Japanese rice cultivars was:

Z₁ = 0.2667B₁ - 0.4119B₂ + 0.3201B₃ + 0.1458B₄ - 0.5780B₅+0.1481B₆ - 0.1241B₇ - 0.1073B₈ + 0.1894B₉ - 0.0598B₁₀-0.0201B₁₁ + 0.0947B₁₂ + 0.3714B₁₃ + 0.2223B₁₄ - 0.1140B₁₅+0.0567B₁₆,where B₁ - B₁₆ are the relative concentrations of the 16 protein fraction bands.

The potential of this function to differentiate Brazilian and Japanese cultivars was proven when the scores given by the canonical discriminating function were plotted (Figure 1). The Japanese cultivars were distributed in the upper part of the scatter diagram and the Brazilian cultivars in the lower part.

Figure 1
- Scattering of Brazilian and Japanese rice genotypes according to the scores given by the canonical discriminating function Z1. (Genotype numbers are defined in Table I).

The large differences found between Brazilian and Japanese rice cultivars may be related to differences in morphological and other traits of their grains. According to Vairavan et al. (1973), the absolute value of the canonical discriminating function coefficient reflects the importance of the assessed traits for primary and secondary differentiation between the cultivars. We found relatively high coefficient bands in function Z₁ such as 5 (B₅), 2 (B₂) and 13 (B₁₃), which make important contributions to the discriminating process. However, they were insufficient, when used individually or together, to satisfactorily discriminate the groups. Discriminant functions combining three or four metric trait values give a good classification that differentiates the indica-japonica rice groups (Morishima and Oka, 1981). As Kochert et al. (1991) suggested, very obvious gross morphological polymorphisms can be the result of a small number of subtle genotypic differences.

Distinction between Brazilian and Japanese rice cultivar groups resulted from the discriminating function of 16 bands. This supports the generally accepted theory that differentiation between Brazilian and Japanese rice cultivars is due to multiple genes, as suggested in the heterosis approach studied by Carbonera (1990). A possible explanation would be that different selective pressures in Brazilian and Japanese environments influenced the seed storage protein fractions studied. This influence was of a greater magnitude in the protein groups that make up bands 2, 5 and 13.

Discrimination between improved and non-improved Brazilian cultivars

Groups of 30 improved and 28 non-improved cultivars were used to calculate the following canonical discriminating function:

Z₂ = 0.3630B₁ + 0.1537B₂ - 0.1969B₃ + 0.2667B₄ + 0.4870B₅ - 0.2318B₆ + 0.2379B₇ - 0.0367B₈ - 0.0122B₉ - 0.0214B₁₀ + 0.2472B₁₁ + 0.0253B₁₂ - 0.0200B₁₃ + 0.3527B₁₄ + 0.2801B₁₅ + 0.2836B₁₆

The Hotelling T²-test confirmed that there were significant differences between improved and non-improved cultivar mean values (P = 0.002). The differences between the groups suggest that artificial selection pressures modified seed protein fraction composition. The coefficients corresponding to bands 5, 1 and 14 showed the highest magnitude in the discrimination of the two groups.

Scatter plots of the whole band discriminating function differentiated between improved and non-improved cultivars (Figure 2). Improved cultivars were mostly found in the top half of the graph, whereas non-improved cultivars were located in the lower half. The graph was divided into strips of similar cultivars, so that the delineation between groups would be more defined: the upper strip contained 78% of the improved cultivars and the lower strip 76% of the non-improved cultivars. The transition strip had an almost identical number of improved and non-improved cultivars.

Figure 2
- Scattering of improved (M) and non-improved (O) Brazilian rice genotypes according to the scores given by the canonical discriminating function Z2.

Demarcation of the three strips is not clearly defined since a gradient of differentiation in the improved materials is likely to exist. Bebyakin and Martynov (1991), in a study of 38 gliadin components in 44 wheat cultivars bred over a period of 65 years, found that the cultivars changed relatively little, as suggested by the presence of the same cluster in cultivars from the 1920s and the 1980s.

Polymorphic seed proteins were useful in estimating the variation among the 58 Brazilian cultivars and in distinguishing between Brazilian and Japanese groups, as well as improved and non-improved Brazilian rice cultivars. One reason would be the "founder population" effects. It may be that the Brazilian rice came from India while the Japanese rice came from Japan and the protein differences represent different evolutionary paths. Neutral theory (Kimura, 1993, and others) claims that much of the intraspecific genetic variability at the molecular level is selectively neutral or nearly neutral and is maintained in the species by the balance between mutational input and random extinction or allele fixation.

ACKNOWLEDGMENTS

The authors wish to thank Dr. C.R.M. Godoi, Department of Mathematics and Statistics, "Luiz de Queiroz" School of Agriculture, University of São Paulo, for his help with statistical analyses and AGROCERES Seed Co. Ltd. for multiplying seed samples and for providing facilities at the experimental station of Santa Cruz das Palmeiras, São Paulo, for the electrophoretic analyses. Publication supported by FAPESP.

RESUMO

Proteínas de grão de 58 genótipos de arroz brasileiro e nove japoneses foram separadas por meio de eletroforese (SDS-PAGE). A observação densitométrica dos perfis eletroforéticos permitiu avaliar as concentrações relativas de 16 frações protéicas que foram usadas como variáveis para a estimativa de funções discriminantes de Fisher. Diferenças significantes foram encontradas entre as frações protéicas dos grupos brasileiros e japoneses, assim como entre os genótipos melhorados e não melhorados. O polimorfismo protéico detectável eletroforetica-mente nos grãos de arroz pode indicar a origem geográfica e o nível de melhoramento dos cultivares.

(Received August 5, 1997)

References

• Aliaga-Morel, J.R., Culiańez-Macia, F.A., Clemente Marin, G. and Primo-Yúfera, E. (1987). Differentiation of rice cultivars by electrophoresis of embryo protein. Theor. Appl. Genet. 74: 224-232.
• Bebyakin, V.M. and Martynov, S.P. (1991). Cluster analysis of gliadin component composition in spring bred wheat cultivars bred in Saratov. Nauchnge Doklady Vysskei Shkoly, Biologicheskie Nauki. No. 5, 110-120 Summary of Plant Breeding Abstract, April, 1992.
• Bianchi-Hall, C.M., Keys, R.D., Stalker, H.T. and Murphy, J.P. (1993). Diversity of seed storage protein patterns in wild peanut (Arachis, Fabaceae) species. Plant Syst. Evol 186: 1-15.
• Carbonera, R. (1990). Avaliaçăo de heterose e divergęncia genética em genótipos de arroz de sequeiro (Oryza sativa L.). Master's thesis, ESALQ/USP, Piracicaba, SP, Brasil.
• Damerval, C., Hebert, Y. and Vienne, D. (1987). Is the polymorphism of protein amounts related to phenotypic variability? A comparison of two-dimensional electrophoresis data with morphological traits in maize. Theor. Appl. Genet 74: 194-202.
• Glaszman, J.C. (1986). A varietal classification of Asian cultivated rice (Oryza sativa L.) based on isozyme polymorphism. In: Rice Genetics (International Rice Research Institute, ed.). Proceedings on International Rice Genetics Symposium, Manila, Philippines, 83-90.
• Grattapaglia, D., Nassar, N.M.A. and Dianese, J.C. (1987). Biossistemática de espécies brasileiras do gęnero Manihot baseada em padrőes de proteína de semente. Cięnc. Cult. 39: 294-300.
• Halward, T., Stalker, T., LaRue, E. and Kochert, G. (1992). Use of single-primer DNA amplification in genetic studies of peanut (Arachis hypogea L.). Plant Mol. Biol 18: 315-325.
• Helentjaris, T., King, G., Slocum, M., Siedenstrang, C. and Wegman, S. (1985). Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding. Plant Mol. Biol. 5: 109-118.
• Kimura, M. (1968). Evolutionary rate at the molecular level. Nature 217: 624-626.
• Kimura, M. (1993). Retrospective of the last quarter century of the neutral theory. Jpn. J. Genet 68: 521-528.
• Kimura, M. and Ohta, T. (1971). Protein polymorphism as a phase of molecular evolution. Nature 229: 467-469.
• Kochert, G., Halward, T., Branch, W.D. and Simpson, C.E. (1991). RFLP variability in peanut (Arachys hipogea L.) cultivars and wild species. Theor. Appl. Genet. 81: 565-570.
• Kochko, A. de. (1987). Isozymic variability of traditional rice (Orysa sativa L.) in Africa. Theor. Appl. Genet. 73: 675-682.
• Ladizinsky, G. and Hymowitz, T. (1979). Seed protein electrophoresis in taxonomic and evolutionary studies. Theor. Appl. Genet. 54: 145-151.
• Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature 227: 680-685.
• Lanham, P.G., Foster, B.P. and McNicol, P. (1994). Seed storage protein variation in Arachis species. Genome 37: 487-496.
• Manly, B.F.J. (1986). Multivariate Statistical Methods: A Primer Chapman and Hall, New York.
• Montalván, R.A. (1990). Determinaçăo de divergęncia genética em germoplasmas de arroz (Oryza sativa L.) através de análisis eletroforéticas de proteína de grăo. Master's thesis, ESALQ/USP, Piracicaba, SP, Brasil.
• Montalván, R., Ando, A. and Echeverrigaray, S. (1995). Phenetic distance and geographical distribution of Brazilian upland rice cultivars using seed protein polymorphism. Breed. Sci. 45: 275-280.
• Morishima, H. and Oka, H. (1981). Phylogenetic differentiation of cultivated rice; XXII. Numerical evaluation of the indica-japonica differentiation. Jpn. J. Breed 31: 402-413.
• Panda, R.C., Kumar, O.A. and Rao, K.G.R. (1986). The use of seed protein electrophoresis in the study of phylogenetic relationship in Chile pepper (Capsicum L.). Theor. Appl. Genet 72: 665-670.
• Sathaiah, V. and Reddy, T.P. (1985). Seed protein profile of castor (Ricinus communis L.) and some Jatropa species. Genet. Agric 39: 35-43.
• Vairavan, S., Siddiq, E.A., Arunachalam, V. and Swaminatham, M.S. (1973). A study on the genetic divergence in rice from North-East Himalaya. Theor. Appl. Genet 43: 213-221.

*

Part of a thesis presented by R.M. to the Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo in partial fulfillment of the requirements for the Master's degree.

Publication Dates

• Publication in this collection
  01 Mar 1999
• Date of issue
  Dec 1998

History

• Received
  05 Aug 1997