25. New storage protein genes detected by Acidic Formate-PAGE method

H.W.CAI and H. MORISHIMA

National Institute of Genetics, Mishima, 411 Japan

Acidic-PAGE was widely used to detect variations of salt-soluble proteins of wheat and barley (Draper 1987). We applied this method to analyze variation in seed storage protein of rice. A total of 98 rice cultivars and 25 strains of wild rice O. rufipogon including both annual and perennial types were studied. The Acidic Formate-PAGE was carried out as described by Wang et al. (1994) with some modifications. Protein was extracted from the individual ground seeds using 0.05M NaCl and 20% sucrose as extraction buffer.

Salt-soluble proteins were separated into more than ten bands and variability among strains was observed. At least three loci tentatively named APAGE 1, 2 and 3 could be identified. APAGE1 appeared at around 60kD as either of four bands (allele 1 -4) as shown in Fig. 1. Interestingly, allele 1 and 2 were frequently found in Japonica and Indica cultivars, respectively. Allele 3 was frequent in perennial types of O. rufipogon but rarely found in cultivars (two Japonica-like deepwater rice cultivars from Bangladesh proved to carry this allele). On the other hand, the annual types of O. rufipogon were found to carry allele 2 as Indica cultivars (Table 1).

Segregation of 1: 1 ratio for APAGE1 was confirmed in recombinant inbred lines obtained from two crosses. To determine the chromosomal location of APAGE1, we analyzed co-segregation between APAGE1 and 110 RFLP and isozyme markers in 118 recombinant inbred lines (F7) derived from a cross of an Indica cultivar and O. rufipogon. As shown in Fig. 2, this locus was mapped on chromosome 5.

The procedure used in the present study probably separated albumin and globulin fractions of rice seeds, though extracted proteins were not yet determined. Many isozymes and RFLP markers are known to be useful in distinguishing Indica and Japonica cultivars (Morishima and Glaszmann 1990; Qian et al. 1995). But few markers can be used to distinguish cultivated and wild rice as well as annual and perennial types of wild rice. In this study, we found that allelic variation at APAGE1 could distinguish not only Indica and Japonica types but also annual and perennial types of O. rufipogon. Thus APAGE1 could be a useful marker for the evolutionary studies in rice.

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Table 1. Distribution of different alleles of APAGE1 in O. sativa and O. rufipogon
Strain group 
Allele
No. of strains
tested
1 2 3 4
sativa (Indica)   78    
78
saliva (Japonica)  18   2  
20
rufipogon (annual)    16    
16
rufipogon (perennial)  2   5 1
8

References

Draper, S.R., 1987. ISTA variety committee report of the working group for biochemical tests for  cultivar identification 1983-1986. Seed Sci. & Technol. 15: 431-434.

Morishima, H. and J.C. Glaszmann, 1990. Current status of isozyme gene symbols. Rice Genetics Newsletter 7:50-57.

Qian, H.R., J.Y. Zhuang, H.X. Lin, J. Lu and K.L. Zheng., 1995. Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L. Theor Appl Genet 90: 878-884.

Wang C., K. Bian, H.X. Zhang, Z.M. Zhou and J. Wang, 1994. Polyacrylamide gel electrophoresis of salt-soluble proteins for maize variety identification and genetic purity assessment. Seed Sci. & Technol. 22:51-57.